Herboxidiene splicing modulator antibody-drug conjugates and methods of use

Abstract

To provide linker-drug compounds and antibody-drug conjugates that bind to human oncology targets.SOLUTION: The linker-drug compounds and antibody-drug conjugates comprise a herboxidiene splicing drug moiety. The disclosure further relates to methods and compositions for use in the treatment of neoplastic disorders by administering the antibody-drug conjugates provided herein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This disclosure claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 779,400, filed December 13, 2018; U.S. Provisional Patent Application No. 62 / 779,406, filed December 13, 2018; and U.S. Provisional Patent Application No. 62 / 941,220, filed November 27, 2019. All of the foregoing applications are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to antibody-drug conjugates (ADCs) comprising a herboxidian splicing modulator and an antibody or antigen-binding fragment thereof, such as one that binds to a human oncology antigen target. The disclosure further relates to methods and compositions useful for treating or diagnosing cancers that express the target antigen and / or cancers amenable to treatment by disrupting RNA splicing, as well as methods for making such compositions. [Background technology]

[0003] The majority of protein-coding genes in the human genome consist of multiple exons (coding regions) separated by introns (non-coding regions). Gene expression results in a single precursor messenger RNA (pre-mRNA). Intron sequences are subsequently removed from the pre-mRNA through a process called splicing, resulting in the mature messenger RNA (mRNA). Alternative splicing results in the inclusion of various combinations of exons, resulting in mRNAs that encode distinct protein isoforms.

[0004] RNA splicing is catalyzed by the spliceosome, a dynamic multiprotein-RNA complex composed of five small nuclear RNAs (snRNAs U1, U2, U4, U5, and U6) and associated proteins. The spliceosome assembles on pre-mRNAs and orchestrates a dynamic cascade of multiple RNA and protein interactions that catalyze intron excision and exon ligation (Matera and Wang (2014) Nat Rev Mol Cell Biol. 15(2):108-21). Increasing evidence implicates human diseases in aberrant regulation of RNA splicing, affecting many genes (Scotti and Swanson (2016) Nat Rev Genet. 17(1):19-32).

[0005] The spliceosome is an important target in cancer biology. Several studies have now demonstrated significant changes in the splicing profile of cancer cells, as well as in the splicing factors themselves (Agrawal et al. (2018) Curr Opin Genet Dev. 48:67-74). Alternative splicing can lead to exon inclusion / exclusion, intron retention, or differential utilization of cryptic splice sites (Seiler et al. (2018) Cell Rep. 23(1):282-296). Collectively, these events describe functional changes that may contribute to tumorigenesis or therapeutic resistance (Siegfried and Karni (2018) Curr Opin Genet Dev. 48:16-21).

[0006] Certain natural products can bind to the SF3b spliceosome complex. These small molecules regulate splicing by promoting intron retention and / or exon skipping (Teng et al. (2017) Nat Commun. 8:15522). For example, herboxidiene (Isaac et al. (1992) J. Org. Chem. 57:7220-26), a naturally occurring polyketide isolated from Streptomyces sp. A7847, and its derivatives have been shown to regulate splicing. See, for example, Imaizumi et al. (2017) J. Antibiot. 70:675-79. A significant proportion of resulting transcripts contain premature stop codons that trigger nonsense-mediated mRNA decay (NMD). Furthermore, impaired canonical splicing leads to a significant reduction in canonical transcripts, which can negatively impact cell function and viability, making splicing modulators a promising drug class for the treatment of cancer (Puthenveetil et al. (2016) Bioconjugate Chem. 27:1880-8).

[0007] The proto-oncogene human epidermal growth factor receptor 2 (HER2) encodes a transmembrane tyrosine kinase receptor belonging to the human epidermal growth factor receptor (EGFR) family (King et al. (1985) Science 229:974-6). Overexpression of HER2 allows constitutive activation of growth factor signaling pathways, such as the PI3K-AKT-mTOR pathway, and thus serves as an oncogenic driver in several cancer types, including approximately 20% of invasive breast cancers (Slamon et al. (1989) Science 244:707-12; Gajria and Chandarlapaty (2011) Expert Rev Anticancer Ther. 11:263-75). Given that HER2 amplification mediates a transformed phenotype, and because HER2 expression is largely restricted to malignant cells, HER2 is a promising antigen for targeting certain cancers and / or delivering novel cancer therapies (Parakh et al. (2017) Cancer Treat Rev. 59:1-21). Additional antigens for targeted cancer therapy delivery include, but are not limited to, CD138 (also known as syndecan-1) and ephrin type-A receptor 2 (EPHA2).

[0008] CD138 is a cell surface heparan sulfate proteoglycan essential for maintaining cell morphology and interacting with the surrounding microenvironment (Akl et al. (2015) Oncotarget 6(30):28693-715; Szatmari et al. (2015) Dis Markers 2015:796052). Overall, loss of CD138 expression in cancer cells reduces cell adhesion to the extracellular matrix and enhances cell motility and invasion (Teng et al. (2012) Matrix Biol. 31:3-16). Increased stromal CD138 expression also alters fibronectin production and extracellular matrix organization (Yang et al. (2011) Am J Pathol. 178:325-35). Additionally, increased CD138 expression on stromal fibroblasts is associated with angiogenesis and cancer progression (Maeda et al. (2006) Oncogene 25:1408-12). CD138 expression increases during B cell development, and its presence is a hallmark of plasma cells (Ribatti (2017) Immunol Lett. 188:64-7). CD138 expression is maintained in multiple myeloma, a plasma cell malignancy. Therefore, CD138 is an attractive antigen for targeted therapy of several cancers and other hematological malignancies (Sherbenou et al. (2015) Blood Rev. 29(2):81-91; Wijdenes et al. (1996) Br J Haematol. 94(2):318-23).

[0009] EPHA2 is a transmembrane glycoprotein that is highly overexpressed in several malignant cancer-derived cell lines and advanced cancers (Wykosky and Debinski (2008) Mol Cancer Ref. 6(12):1795-1806). For example, EPHA2 is strongly overexpressed in approximately 61% of GBM patient tumors (Wykosky et al. (2008) Clin Cancer Res. 14:199-208), 76% of ovarian cancers (Thaker et al. (2004) Clin Cancer Res. 10:5145-50), and 85% of prostate adenocarcinomas (Zeng et al. (2003) Am J Pathol. 163:2271-6). The EPHA2 protein is highly overexpressed in a proportion of patient tumors and in a proportion of cells within tumors and is a cell membrane-localized receptor that can internalize upon ligand binding (Walker-Daniels et al. (2002) Mol Cancer Res. 1:79-87). Furthermore, EPHA2 expression is associated with poor prognosis, increased metastasis, and reduced survival. Therefore, because of its expression pattern, localization, and functional importance in cancer patient outcome, EPHA2 is another attractive antigen for the targeted delivery of novel anticancer therapies. Summary of the Invention [Means for solving the problem]

[0010] In various embodiments, the present disclosure provides, in part, novel herboxidiene splicing modulators with anti-neoplastic cellular biological activity. The herboxidiene splicing modulators may be used alone or as part of an ADC to slow, inhibit, and / or reverse tumor growth in mammals, and may be useful in treating human cancer patients. In various embodiments, the present disclosure provides novel antibody-drug conjugates that employ herboxidiene splicing modulators.

[0011] More specifically, in various embodiments, the present disclosure relates to antibody-drug conjugate (ADC) compounds capable of binding to and killing neoplastic cells. In various embodiments, the ADC compounds disclosed herein include a linker connecting a herboxidiene splicing modulator to a full-length antibody or antigen-binding fragment. In various embodiments, the ADC compounds also have the ability to internalize into target cells after binding.

[0012] In some embodiments, the antibody-drug conjugate has the formula (I): Ab-(LH) p wherein Ab is an antibody or antigen-binding fragment that targets a neoplastic cell; H is a herboxidian splicing modulator; L is a linker that covalently links Ab to H; and p is an integer from 1 to 15.

[0013] In some embodiments, H, a herboxidiene splicing modulator, is covalently linked to L through any atom in a compound of formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein: Y is O, S, NR 6 , and CR 6 R 7 Selected from; R 1 , R 2 , and R 3 are each independently selected from hydrogen, hydroxyl, an -O-(C1-C6 alkyl) group, an -O-C(=O)-(C1-C6 alkyl) group, an -C(=O)-O-(C1-C6 alkyl) group, and a C1-C6 alkyl group; R 4 is hydrogen, a C1-C6 alkyl group, a -C(=O)-(C1-C6 alkyl) group, a -C(=O)-(C3-C8 carbocyclyl) group, a -C(=O)-(C3-C8 heterocyclyl) group, and a -C(=O)-NR 6 R 7 Selected from; R 5 is hydrogen, hydroxyl, -CH2-OH, -CO2H, -C(=O)-O-(C1-C6 alkyl) group, -C(=O)-NR 6 R 7 , -NR 6 -C(=O)-R 8 , -OC(=O)-NR 6 R 7 , -NR 6 -C(=O)-R 8 , and -NR 6 -C(=O)-NR 6 R 7 Selected from; R 6 and R 7 are each independently hydrogen, -R 8 , -C(=O)-R 8 , and -C(=O)-OR 8 selected from; and R 8 is selected from a C1-C6 alkyl group, a C3-C8 carbocyclyl group, and a C3-C8 heterocyclyl group; where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 each independently represents a halogen, hydroxyl, a C1-C6 alkyl group, -O-(C1-C6 alkyl) group, -CO2H, -C(=O)-(C1-C6 alkyl) group, -C(=O)-(C3-C8 carbocyclyl) group, -C(=O)-(C3-C8 heterocyclyl) group, -NR 6 R 7 , substituted by 0 to 3 groups independently selected from a C3 to C8 carbocyclyl group, a C1 to C6 alkylhydroxy group, a C1 to C6 alkylalkoxy group, a benzyl group, and a C3 to C8 heterocyclyl group (each of which is optionally independently substituted with 0 or 1 group selected from a halogen, a hydroxyl, a C1 to C3 alkyl group, a C1 to C3 alkoxy group, a C1 to C3 haloalkyl group, -NH-C(=O)(C1 to C3 alkyl), and -NH-C(=O)-O-(C1 to C3 alkyl)); and wherein the valence of the atom covalently bonded to L is not exceeded.

[0014] In some embodiments, H, a herboxidiene splicing modulator, is covalently linked to L through any atom in a compound of Formula (Ia): [ka] or a pharmaceutically acceptable salt thereof, wherein: R 9 is selected from C3-C8 heterocyclyl groups; R 10 is selected from H and C1-C6 alkyl groups; where R 9 and R 10 are each independently substituted with 0 to 3 groups independently selected from halogen, hydroxyl, a C1-C3 alkyl group, a C1-C3 alkoxy group, -NH2, -NH-(C1-C3 alkyl), and -N-(C1-C3 alkyl)2, and wherein the valence of the atom covalently bonded to L is not exceeded.

[0015] In some embodiments, H, a herboxidiene splicing modulator, is a compound of Formula (Ib) covalently linked to L through any atom: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 11 teeth, [ka] (In the formula, * is R 11 represents the point of attachment to the remainder of the compound; R 12 and R 13 are each independently selected from H and methyl; and wherein the valence of the atom covalently bonded to L is not exceeded.

[0016] In some embodiments, H, the herboxidiene splicing modulator, is a compound of formula (II) covalently linked to L through any atom: [ka] or a pharmaceutically acceptable salt thereof, wherein: X is hydroxyl or NR 6 R 7 and; R 6 and R 7 are each independently hydrogen, -R 8 , -C(=O)-R 8 , -C(=O)-OR 8 , -(C1-C6 alkyl)-OC(=O)-R 8 and -(C1-C6 alkyl)-NH-C(=O)-R 8 selected from; and R 8 is selected from a C1-C6 alkyl group, a C3-C8 carbocyclyl group, and a C3-C8 heterocyclyl group; where R 6 , R 7 , and R 8 each independently represents a halogen, hydroxyl, a C1-C6 alkyl group, -O-(C1-C6 alkyl) group, -CO2H, -C(=O)-O-(C1-C6 alkyl) group, -C(=O)-(C1-C6 alkyl) group, -C(=O)-(C3-C8 carbocyclyl) group, -C(=O)-(C3-C8 heterocyclyl) group, -NR 6 R 7, substituted by 0 to 3 groups independently selected from a C3 to C8 carbocyclyl group, a C1 to C6 alkylhydroxy group, a C1 to C6 alkylalkoxy group, a benzyl group, and a C3 to C8 heterocyclyl group (each of which is optionally independently substituted with 0 or 1 group selected from a halogen, a hydroxyl, a C1 to C3 alkyl group, a C1 to C3 alkoxy group, a C1 to C3 haloalkyl group, -NH-C(=O)(C1 to C3 alkyl), and -NH-C(=O)-O-(C1 to C3 alkyl)); and wherein the valence of the atom covalently bonded to L is not exceeded.

[0017] In some embodiments, H, the herboxidiene splicing modulator, is a compound of formula (IIa) covalently linked to L through any atom: [ka] or a pharmaceutically acceptable salt thereof, wherein: Z is NR 9 and O; R 9 is selected from hydrogen and C1-C6 alkyl groups; R 10 and R 11 are each independently selected from hydrogen, halogen, hydroxyl, a C1-C6 alkyl group, a -O-(C1-C6 alkyl) group, -COH, a -C(=O)-O-(C1-C6 alkyl) group, a -C(=O)-(C1-C6 alkyl) group, a -C(=O)-(C3-C8 carbocyclyl) group, a -C(=O)-(C3-C8 heterocyclyl) group, a C3-C8 carbocyclyl group, a C1-C6 alkylhydroxy group, a C1-C6 alkylalkoxy group, a benzyl group, and a C3-C8 heterocyclyl group; R 12 is selected from a C1-C6 alkyl group, a C3-C8 carbocyclyl group, and a C3-C8 heterocyclyl group; where R 9 , R 10 , R 11 , and R 12are each independently substituted with 0 or 1 group selected from halogen, hydroxyl, a C1-C3 alkyl group, a C1-C3 alkoxy group, and a C1-C3 haloalkyl group; t is an integer selected from 1, 2, 3, 4, 5, and 6; and wherein the valence of the atom covalently bonded to L is not exceeded.

[0018] In some embodiments, H, a herboxidiene splicing modulator, is a compound of formula (IIb) covalently linked to L through any atom: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 13 teeth, [ka] (In the formula, * is R 13 represents the point of attachment to the remainder of the compound; R 14 and R 15 are each independently selected from hydrogen and methyl; and wherein the valence of the atom covalently bonded to L is not exceeded.

[0019] In some embodiments, H, the herboxidiene splicing modulator, is a compound of formula (III) covalently linked to L through any atom: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1 , R 2 , and R 3are each independently selected from hydrogen, hydroxyl, an -O-(C1-C6 alkyl) group, an -O-C(=O)-(C1-C6 alkyl) group, an -C(=O)-O-(C1-C6 alkyl) group, and a C1-C6 alkyl group; R 6 and R 7 are each independently hydrogen, -R 8 , -C(=O)-R 8 , and -C(=O)-OR 8 Selected from; R 8 is selected from a C1-C6 alkyl group, a C3-C8 carbocyclyl group, and a C3-C8 heterocyclyl group; and R 9 H, [ka] Selected from; where R 1 , R 2 , R 3 , R 6 , R 7 , and R 8 each independently represents a halogen, hydroxyl, a C1-C6 alkyl group, -O-(C1-C6 alkyl) group, -CO2H, -C(=O)-(C1-C6 alkyl) group, -C(=O)-(C3-C8 carbocyclyl) group, -C(=O)-(C3-C8 heterocyclyl) group, -NR 6 R 7 , substituted by 0 to 3 groups independently selected from a C3 to C8 carbocyclyl group, a C1 to C6 alkylhydroxy group, a C1 to C6 alkylalkoxy group, a benzyl group, and a C3 to C8 heterocyclyl group (each of which is optionally independently substituted by 0 or 1 group selected from a halogen, hydroxyl, a C1 to C3 alkyl group, a C1 to C3 alkoxy group, a C1 to C3 haloalkyl group, -NH-C(=O)(C1 to C3 alkyl), and -NH-C(=O)-O-(C1 to C3 alkyl); wherein the valence of the atom covalently bonded to L is not exceeded; and During the ceremony, * is R 9represents the point of attachment to the remainder of the compound.

[0020] In some embodiments, the linker comprises a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety is cleavable by an enzyme. In some embodiments, the cleavable peptide moiety or linker comprises an amino acid unit. In some embodiments, the amino acid unit comprises valine-citrulline ("Val-Cit" or "VC"). In some other embodiments, the amino acid unit comprises valine-alanine ("Val-Ala" or "VA"). In some other embodiments, the amino acid unit comprises glutamic acid-valine-citrulline ("Glu-Val-Cit" or "EVC"). In some other embodiments, the amino acid unit comprises alanine-alanine-asparagine ("Ala-Ala-Asn" or "AAN").

[0021] In some embodiments, the linker comprises a cleavable glucuronide moiety. In some embodiments, the cleavable glucuronide moiety is cleavable by an enzyme. In some embodiments, the cleavable glucuronide moiety is cleavable by a glucuronidase. In some embodiments, the cleavable glucuronide moiety is cleavable by a β-glucuronidase.

[0022] In some embodiments, the linker comprises at least one spacer unit. In some embodiments, the spacer unit or linker comprises a polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety is -(PEG) m -, where m is an integer from 1 to 10. In some embodiments, m is 2. In some other embodiments, the spacer unit or linker comprises an alkyl moiety. In some embodiments, the alkyl moiety is -(CH2) n -, wherein n is an integer from 1 to 10. In some embodiments, n is 2. In some embodiments, n is 5. In some embodiments, n is 6.

[0023] In some embodiments, the spacer unit is linked to the antibody or antigen-binding fragment via a maleimide (Mal) moiety ("Mal-spacer unit"). In some embodiments, the Mal-spacer unit is reactive with cysteine ​​residues on the antibody or antigen-binding fragment. In some embodiments, the Mal-spacer unit is coupled to the antibody or antigen-binding fragment via a cysteine ​​residue on the antibody or antigen-binding fragment.

[0024] In some embodiments, the linker comprises a Mal-spacer unit and a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Val-Cit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Val-Ala. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Glu-Val-Cit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Ala-Ala-Asn. In some embodiments, the Mal-spacer unit comprises an alkyl moiety. In some embodiments, the Mal-spacer unit comprises a PEG moiety. In some embodiments, the Mal-spacer unit comprises maleimidocaproyl (MC).

[0025] In some embodiments, the Mal-spacer unit links the antibody or antigen-binding fragment to the cleavable portion of the linker. In some embodiments, the cleavable portion of the linker comprises a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. In some embodiments, the linker comprises MC-Val-Cit. In some embodiments, the linker comprises MC-Val-Ala. In some embodiments, the linker comprises MC-Glu-Val-Cit. In some embodiments, the linker comprises MC-Ala-Ala-Asn. In some embodiments, the Mal-spacer unit comprises an alkyl moiety. In some embodiments, the Mal-spacer unit comprises a PEG moiety. In some embodiments, the Mal-spacer unit comprises maleimidocaproyl (MC).

[0026] In some embodiments, the cleavable portion of the linker is directly tethered to the herboxidienes splicing modulator, or a spacer unit connects the cleavable portion of the linker to the herboxidienes splicing modulator. In some embodiments, cleavage of the conjugate releases the herboxidienes splicing modulator from the antibody or antigen-binding fragment and the linker. In some embodiments, the spacer unit connecting the cleavable portion of the linker to the herboxidienes splicing modulator is self-immolative.

[0027] In some embodiments, the spacer unit connecting the cleavable portion of the linker to the herboxidien splicing modulator comprises p-aminobenzyloxycarbonyl (pABC). In some embodiments, pABC connects the cleavable portion of the linker to the herboxidien splicing modulator. In some embodiments, the cleavable portion of the linker comprises a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. In some embodiments, the linker comprises Val-Cit-pABC. In some other embodiments, the linker comprises Val-Ala-pABC. In some embodiments, the linker comprises Glu-Val-Cit-pABC. In some embodiments, the linker comprises Ala-Ala-Asn-pABC.

[0028] In some embodiments, the spacer unit connecting the cleavable portion of the linker to the herboxidien splicing modulator comprises p-aminobenzyl (pAB). In some embodiments, pAB connects the cleavable portion of the linker to the herboxidien splicing modulator. In some embodiments, the cleavable portion of the linker comprises a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. In some embodiments, the linker comprises Val-Cit-pAB. In some other embodiments, the linker comprises Val-Ala-pAB. In some other embodiments, the linker comprises Glu-Val-Cit-pAB. In some other embodiments, the linker comprises Ala-Ala-Asn-pAB.

[0029] In various embodiments, the linker is a non-cleavable linker. In some embodiments, the herboxidiane splicing modulator of the ADC is released upon degradation of the antibody or antigen-binding fragment. In some embodiments, the linker remains covalently associated with at least one amino acid of the antibody and drug upon internalization by and degradation within the target cell.

[0030] In some embodiments, the linker is a non-cleavable linker comprising at least one spacer unit. In some embodiments, the spacer unit or linker comprises a polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety is -(PEG) m -, where m is an integer from 1 to 10. In some embodiments, m is 2. In some other embodiments, the spacer unit or linker comprises an alkyl moiety. In some embodiments, the alkyl moiety is -(CH2) n -or-(CH2) n -O-(CH2) n wherein n is an integer from 1 to 10. In some embodiments, n is 2. In some embodiments, n is 5. In some embodiments, n is 6.

[0031] In some embodiments, the spacer unit of the non-cleavable linker is attached to the antibody or antigen-binding fragment via a maleimide (Mal) moiety ("Mal-spacer unit"). In some embodiments, the Mal-spacer unit is reactive with a cysteine ​​residue on the antibody or antigen-binding fragment. In some embodiments, the Mal-spacer unit is coupled to the antibody or antigen-binding fragment via a cysteine ​​residue on the antibody or antigen-binding fragment. In some embodiments, the Mal-spacer unit comprises an alkyl moiety. In some embodiments, the Mal-spacer unit comprises a PEG moiety. In some embodiments, the linker or Mal-spacer unit comprises maleimidocaproyl (MC). In some embodiments, the linker or Mal-spacer unit comprises maleimidocaproyl (MC) and at least one additional spacer unit. In some embodiments, the linker or Mal-spacer unit comprises MC-(PEG)2. In some embodiments, the linker or Mal-spacer unit comprises MC-(PEG)2 and at least one additional spacer unit. In some embodiments, the linker or Mal-spacer unit comprises Mal-Hex. In some embodiments, the linker or Mal-spacer unit comprises Mal-Hex and at least one additional spacer unit. In some embodiments, the linker or Mal-spacer unit comprises Mal-Et. In some embodiments, the linker or Mal-spacer unit comprises Mal-Et and at least one additional spacer unit. In some embodiments, the linker or Mal-spacer unit comprises Mal-Et-O-Et. In some embodiments, the linker or Mal-spacer unit comprises Mal-Et-O-Et and at least one additional spacer unit. In some embodiments, the Mal-spacer unit links the antibody or antigen-binding fragment to the herboxidien splicing modulator.

[0032] In some embodiments, the Ab is selected from any of the antibodies or binding domain sequences disclosed herein. In some embodiments, the Ab is an antibody or binding domain sequence that targets HER2 and / or neoplastic cells expressing HER2. In some embodiments, the Ab is an antibody or binding domain sequence that targets CD138 and / or neoplastic cells expressing CD138. In some embodiments, the Ab is an antibody or binding domain sequence that targets EPHA2 and / or neoplastic cells expressing EPHA2. In some embodiments, the Ab is an antibody or binding domain sequence that targets MSLN and / or neoplastic cells expressing MSLN. In some embodiments, the Ab is an antibody or binding domain sequence that targets FOLH1 and / or neoplastic cells expressing FOLH1. In some embodiments, the Ab is an antibody or binding domain sequence that targets CDH6 and / or neoplastic cells expressing CDH6. In some embodiments, the Ab is an antibody or binding domain sequence that targets CEACAM5 and / or neoplastic cells expressing CEACAM5. In some embodiments, the Ab is an antibody or binding domain sequence that targets CFClB and / or neoplastic cells expressing CFClB. In some embodiments, the Ab is an antibody or binding domain sequence that targets ENPP3 and / or neoplastic cells expressing ENPP3. In some embodiments, the Ab is an antibody or binding domain sequence that targets FOLR1 and / or neoplastic cells expressing FOLR1. In some embodiments, the Ab is an antibody or binding domain sequence that targets HAVCR1 and / or neoplastic cells expressing HAVCR1. In some embodiments, the Ab is an antibody or binding domain sequence that targets KIT and / or neoplastic cells expressing KIT. In some embodiments, the Ab is an antibody or binding domain sequence that targets MET and / or neoplastic cells expressing MET. In some embodiments, the Ab is an antibody or binding domain sequence that targets MUC16 and / or neoplastic cells expressing MUC16. In some embodiments, the Ab is an antibody or binding domain sequence that targets SLC39A6 and / or neoplastic cells expressing SLC39A6.In some embodiments, the Ab is an antibody or binding domain sequence that targets SLC44A4 and / or neoplastic cells expressing SLC44A4. In some embodiments, the Ab is an antibody or binding domain sequence that targets STEAP1 and / or neoplastic cells expressing STEAP1. In some embodiments, the Ab is an antibody or binding domain sequence that targets another cancer antigen.

[0033] In some embodiments, L is selected from any of the linkers disclosed herein or any combination of linker components disclosed herein. In some embodiments, L is a linker comprising MC-Val-Cit-pABC, Mal-(PEG)2-CO, MC-Val-Ala-pAB, MC-Val-Ala-pABC, MC-Val-Cit-pAB, Mal-Hex, Mal-Et, or Mal-Et-O-Et. In some embodiments, the linker may also comprise one or more additional spacer units. In some embodiments, L is an ADL1, ADL2, ADL5, ADL6, ADL7, ADL10, ADL12, ADL13, ADL14, ADL15, ADL21, ADL22, or ADL23 linker. In some embodiments, L is an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker. In some embodiments, L is an ADL12, ADL14, or ADL15 linker. In some embodiments, an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker may also include one or more additional spacer units. In some embodiments, L is an ADL1 linker, which may optionally include one or more additional spacer units. In some embodiments, L is an ADL2 linker, which may optionally include one or more additional spacer units. In some embodiments, L is an ADL5 linker, which may optionally include one or more additional spacer units. In some embodiments, L is an ADL6 linker, which may optionally include one or more additional spacer units. In some embodiments, L is an ADL7 linker, which may optionally include one or more additional spacer units. In some embodiments, L is an ADL12 linker, which may optionally include one or more additional spacer units. In some embodiments, L is an ADL14 linker, which may optionally include one or more additional spacer units. In some embodiments, L is an ADL15 linker, which may optionally include one or more additional spacer units.In various embodiments of the ADCs described herein, p is 1 to 10. In various embodiments, p is 2 to 8. In various embodiments, p is 4 to 8. In some embodiments, p is 4. In some embodiments, p is 8.

[0034] In some embodiments, a pool of ADCs is provided in which random conjugation occurs, where the average p in the pool is about 2 to about 8. In some embodiments, a pool of ADCs is provided in which random conjugation occurs, where the average p in the pool is about 4 to about 8. In some embodiments, a pool of ADCs is provided in which random conjugation occurs, where the average p in the pool is about 4. In some embodiments, a pool of ADCs is provided in which random conjugation occurs, where the average p in the pool is about 8. Provided herein are compositions (e.g., pharmaceutical compositions) comprising multiple copies of any of the described ADCs, wherein the average drug loading (average p) of the ADCs in the composition is about 3.5 to about 5.5 (e.g., about 4), or about 7 to about 9 (e.g., about 8).

[0035] In some embodiments, the antibody or antigen-binding fragment (Ab) of the ADC targets neoplastic cells derived from a hematological malignancy or a solid tumor. In some embodiments, the antibody or antigen-binding fragment targets neoplastic cells derived from a hematological malignancy. In some embodiments, the hematological malignancy is selected from a B-cell malignancy, leukemia (e.g., acute myeloid leukemia), lymphoma, and myeloma (e.g., multiple myeloma). In some embodiments, the hematological malignancy is selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the antibody or antigen-binding fragment targets neoplastic cells derived from a solid tumor. In some embodiments, the solid tumor is selected from breast cancer (e.g., HER2-positive breast cancer), gastric cancer (e.g., gastric adenocarcinoma), prostate cancer, ovarian cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., serous endometrial carcinoma), salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colorectal cancer, and esophageal cancer. In some embodiments, the solid tumor is selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

[0036] In various embodiments, the antibody or antigen-binding fragment (Ab) of the ADC is an anti-HER2 antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment binds to HER2 and targets neoplastic cells that express HER2 (i.e., the ADC targets neoplastic cells that express HER2). In some embodiments, the antibody or antigen-binding fragment of the ADC is an internalizing anti-HER2 antibody or internalizing antigen-binding fragment thereof.

[0037] In some embodiments, the anti-HER2 antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising the amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO:6 (LCDR3). In some embodiments, the anti-HER2 antibody or antigen-binding fragment is an internalizing antibody or internalizing antigen-binding fragment. In some embodiments, the anti-HER2 antibody or antigen-binding fragment comprises human framework sequences. In some embodiments, the anti-HER2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the anti-HER2 antibody or antigen-binding fragment comprises a human IgG heavy chain constant region. In some embodiments, the anti-HER2 antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the anti-HER2 antibody or antigen-binding fragment comprises a human Ig kappa or lambda light chain constant region. In some embodiments, the anti-HER2 antibody or antigen-binding fragment competes for binding to and / or binds to the same epitope as an antibody comprising a heavy chain variable domain of SEQ ID NO: 19 and a light chain variable domain of SEQ ID NO: 20.

[0038] In various embodiments, the antibody or antigen-binding fragment (Ab) of the ADC is an anti-CD138 antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment binds to CD138 and targets neoplastic cells that express CD138 (i.e., the ADC targets neoplastic cells that express CD138). In some embodiments, the antibody or antigen-binding fragment of the ADC is an internalizing anti-CD138 antibody or internalizing antigen-binding fragment thereof.

[0039] In some embodiments, the anti-CD138 antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising the amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2), and SEQ ID NO:12 (LCDR3). In some embodiments, the anti-CD138 antibody or antigen-binding fragment is an internalizing antibody or internalizing antigen-binding fragment. In some embodiments, the anti-CD138 antibody or antigen-binding fragment comprises human framework sequences. In some embodiments, the anti-CD138 antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:22. In some embodiments, the anti-CD138 antibody or antigen-binding fragment comprises a murine IgG2a heavy chain constant region. In some embodiments, the anti-CD138 antibody or antigen-binding fragment comprises a murine Ig kappa light chain constant region. In some embodiments, the anti-CD138 antibody or antigen-binding fragment comprises a human IgG heavy chain constant region. In some embodiments, the anti-CD138 antibody or antigen-binding fragment comprises a human IgG2a heavy chain constant region. In some embodiments, the anti-CD138 antibody or antigen-binding fragment comprises a human Ig kappa or lambda light chain constant region. In some embodiments, the anti-CD138 antibody or antigen-binding fragment competes for binding to and / or binds to the same epitope as an antibody comprising a heavy chain variable domain of SEQ ID NO:21 and a light chain variable domain of SEQ ID NO:22.

[0040] In various embodiments, the antibody or antigen-binding fragment (Ab) of the ADC is an anti-EPHA2 antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment binds to EPHA2 and targets neoplastic cells that express EPHA2 (i.e., the ADC targets neoplastic cells that express EPHA2). In some embodiments, the antibody or antigen-binding fragment of the ADC is an internalizing anti-EPHA2 antibody or internalizing antigen-binding fragment thereof.

[0041] In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequences of SEQ ID NO: 13 (HCDR1), SEQ ID NO: 14 (HCDR2), and SEQ ID NO: 15 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising the amino acid sequences of SEQ ID NO: 16 (LCDR1), SEQ ID NO: 17 (LCDR2), and SEQ ID NO: 18 (LCDR3). In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment is an internalizing antibody or internalizing antigen-binding fragment. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment comprises human framework sequences. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment comprises a human IgG heavy chain constant region. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment comprises a human Ig kappa or lambda light chain constant region. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment competes for binding to and / or binds to the same epitope as an antibody comprising a heavy chain variable domain of SEQ ID NO:23 and a light chain variable domain of SEQ ID NO:24.

[0042] In some embodiments, provided herein are compounds of formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein: Y is O, S, NR 6 , and CR 6 R 7 Selected from; R 1 , R 2 , and R 3are each independently selected from hydrogen, hydroxyl, an -O-(C1-C6 alkyl) group, an -O-C(=O)-(C1-C6 alkyl) group, an -C(=O)-O-(C1-C6 alkyl) group, and a C1-C6 alkyl group; R 4 is hydrogen, a C1-C6 alkyl group, a -C(=O)-(C1-C6 alkyl) group, a -C(=O)-(C3-C8 carbocyclyl) group, a -C(=O)-(C3-C8 heterocyclyl) group, and a -C(=O)-NR 6 R 7 Selected from; R 5 is hydrogen, hydroxyl, -CH2-OH, -CO2H, -C(=O)-O-(C1-C6 alkyl) group, -C(=O)-NR 6 R 7 , -NR 6 -C(=O)-R 8 , -OC(=O)-NR 6 R 7 , -NR 6 -C(=O)-R 8 , and -NR 6 -C(=O)-NR 6 R 7 Selected from; R 6 and R 7 are each independently hydrogen, -R 8 , -C(=O)-R 8 , and -C(=O)-OR 8 selected from; and R 8 is selected from a C1-C6 alkyl group, a C3-C8 carbocyclyl group, and a C3-C8 heterocyclyl group; where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 each independently represents a halogen, hydroxyl, a C1-C6 alkyl group, -O-(C1-C6 alkyl) group, -CO2H, -C(=O)-(C1-C6 alkyl) group, -C(=O)-(C3-C8 carbocyclyl) group, -C(=O)-(C3-C8 heterocyclyl) group, -NR6 R 7 , a C3-C8 carbocyclyl group, a C1-C6 alkylhydroxy group, a C1-C6 alkylalkoxy group, a benzyl group, and a C3-C8 heterocyclyl group (each of which is optionally independently substituted with 0 or 1 group selected from halogen, hydroxyl, a C1-C3 alkyl group, a C1-C3 alkoxy group, a C1-C3 haloalkyl group, —NH—C(═O)(C1-C3 alkyl), and —NH—C(═O)—O—(C1-C3 alkyl)).

[0043] In some embodiments, provided herein are compounds of formula (Ia): [ka] or a pharmaceutically acceptable salt thereof, wherein: R 9 is selected from a C3-C8 heterocyclyl group; and R 10 is selected from H and C1-C6 alkyl groups, where R 9 and R 10 are each independently substituted with 0 to 3 groups independently selected from halogen, hydroxyl, a C1-C3 alkyl group, a C1-C3 alkoxy group, —NH2, —NH-(C1-C3 alkyl), and —N-(C1-C3 alkyl)2.

[0044] In some embodiments, provided herein are compounds of formula (Ib): [ka] or a pharmaceutically acceptable salt thereof, wherein: R 11 teeth, [ka] (In the formula, * is R 11represents the point of attachment to the remainder of the compound; and R 12 and R 13 are each independently selected from H and methyl.

[0045] In some embodiments, provided herein are compounds of formula (II): [ka] or a pharmaceutically acceptable salt thereof, wherein: X is NR 6 R 7 and; R 6 and R 7 are each independently hydrogen, -R 8 , -C(=O)-R 8 , -C(=O)-OR 8 , -(C1-C6 alkyl)-OC(=O)-R 8 and -(C1-C6 alkyl)-NH-C(=O)-R 8 selected from; and R 8 is selected from a C1-C6 alkyl group, a C3-C8 carbocyclyl group, and a C3-C8 heterocyclyl group; where R 6 , R 7 , and R 8 each independently represents a halogen, hydroxyl, a C1-C6 alkyl group, -O-(C1-C6 alkyl) group, -CO2H, -C(=O)-(C1-C6 alkyl) group, -C(=O)-(C3-C8 carbocyclyl) group, -C(=O)-(C3-C8 heterocyclyl) group, -NR 6 R 7, a C3-C8 carbocyclyl group, a C1-C6 alkylhydroxy group, a C1-C6 alkylalkoxy group, a benzyl group, and a C3-C8 heterocyclyl group (each of which is optionally independently substituted with 0 or 1 group selected from halogen, hydroxyl, a C1-C3 alkyl group, a C1-C3 alkoxy group, a C1-C3 haloalkyl group, —NH—C(═O)(C1-C3 alkyl), and —NH—C(═O)—O—(C1-C3 alkyl)).

[0046] In some embodiments, provided herein are compounds of formula (IIa): [ka] or a pharmaceutically acceptable salt thereof, wherein: Z is NR 9 and O; R 9 is selected from hydrogen and C1-C6 alkyl groups; R 10 and R 11 are each independently selected from hydrogen, halogen, hydroxyl, a C1-C6 alkyl group, a -O-(C1-C6 alkyl) group, -COH, a -C(=O)-O-(C1-C6 alkyl) group, a -C(=O)-(C1-C6 alkyl) group, a -C(=O)-(C3-C8 carbocyclyl) group, a -C(=O)-(C3-C8 heterocyclyl) group, a C3-C8 carbocyclyl group, a C1-C6 alkylhydroxy group, a C1-C6 alkylalkoxy group, a benzyl group, and a C3-C8 heterocyclyl group; R 12 is selected from a C1-C6 alkyl group, a C3-C8 carbocyclyl group, and a C3-C8 heterocyclyl group; where R 9 , R 10 , R 11 , and R 12 are each independently substituted with 0 or 1 group selected from halogen, hydroxyl, a C1-C3 alkyl group, a C1-C3 alkoxy group, and a C1-C3 haloalkyl group; and and t is an integer selected from 1, 2, 3, 4, 5, and 6.

[0047] In some embodiments, provided herein are compounds of formula (IIb): [ka] or a pharmaceutically acceptable salt thereof, wherein: R 13 teeth, [ka] (In the formula, * is R 13 represents the point of attachment to the remainder of the compound; and R 14 and R 15 are each independently selected from hydrogen and methyl.

[0048] In some embodiments, provided herein are compounds of formula (III): [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1 , R 2 , and R 3 are each independently selected from hydrogen, hydroxyl, an -O-(C1-C6 alkyl) group, an -O-C(=O)-(C1-C6 alkyl) group, an -C(=O)-O-(C1-C6 alkyl) group, and a C1-C6 alkyl group; R 6 and R 7 are each independently hydrogen, -R 8 , -C(=O)-R 8 , and -C(=O)-OR 8 Selected from; R 8 is selected from a C1-C6 alkyl group, a C3-C8 carbocyclyl group, and a C3-C8 heterocyclyl group; and R 9H, [ka] Selected from; where R 1 , R 2 , R 3 , R 6 , R 7 , and R 8 each independently represents a halogen, hydroxyl, a C1-C6 alkyl group, -O-(C1-C6 alkyl) group, -CO2H, -C(=O)-(C1-C6 alkyl) group, -C(=O)-(C3-C8 carbocyclyl) group, -C(=O)-(C3-C8 heterocyclyl) group, -NR 6 R 7 , substituted by 0-3 groups independently selected from a C3-C8 carbocyclyl group, a C1-C6 alkylhydroxy group, a C1-C6 alkylalkoxy group, a benzyl group, and a C3-C8 heterocyclyl group (each of which is optionally independently substituted with 0 or 1 group selected from a halogen, hydroxyl, a C1-C3 alkyl group, a C1-C3 alkoxy group, a C1-C3 haloalkyl group, -NH-C(=O)(C1-C3 alkyl), and -NH-C(=O)-O-(C1-C3 alkyl); and During the ceremony, * is R 9 represents the point of attachment to the remainder of the compound] is provided.

[0049] In some embodiments, the present disclosure provides: [ka] [ka] [ka] [ka] [ka] and a pharmaceutically acceptable salt thereof. wherein L is a linker that covalently attaches to the antibody.

[0050] Also provided herein, in various embodiments, are therapeutic uses for the described ADC compounds, herboxidiene compounds, and compositions, e.g., in the treatment of neoplastic disorders, e.g., cancer. In certain aspects, the disclosure provides methods of treating neoplastic disorders, e.g., cancer, that express an antigen targeted by the antibody or antigen-binding fragment of the ADC, such as HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFClB, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1.

[0051] In certain aspects, the disclosure provides methods of treating a subject having or suspected of having a neoplastic disorder by administering to the subject a therapeutically effective amount and / or therapeutically effective regimen of any one of the described ADCs or compositions. In some embodiments, the neoplastic disorder is a hematological malignancy or a solid tumor. In some embodiments, the neoplastic disorder is a hematological malignancy. In some embodiments, the hematological malignancy is selected from a B-cell malignancy, leukemia, lymphoma, and myeloma. In some embodiments, the hematological malignancy is selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the neoplastic disorder is a solid tumor. In some embodiments, the solid tumor is selected from breast cancer (e.g., HER2-positive breast cancer), gastric cancer (e.g., gastric adenocarcinoma), prostate cancer, ovarian cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., serous endometrial cancer), salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colorectal cancer, and esophageal cancer. In some embodiments, the solid tumor is selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

[0052] In some embodiments, treatment with the antibody-drug conjugate or composition induces bystander killing of neoplastic cells that do not express the target antigen but are adjacent to neoplastic cells that do express the target antigen, hi some embodiments, a subject has one or more neoplastic cells that express the target antigen.

[0053] In some embodiments, the target antigen is HER2. In some embodiments, the one or more neoplastic cells are in HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., serous endometrial carcinoma), osteosarcoma, or salivary duct cancer. In some embodiments, the subject is refractory or unresponsive to treatment with (a) an anti-HER2 antibody administered alone and / or (b) a herboxidien splicing modulator administered alone. In some embodiments, the subject is intolerant, unresponsive, or unresponsive to treatment with a herboxidien splicing modulator administered alone.

[0054] In some embodiments, the target antigen is CD138. In some embodiments, the one or more neoplastic cells are in multiple myeloma that express CD138. In some embodiments, the subject is refractory or non-responsive to treatment with (a) an anti-CD138 antibody when administered alone and / or (b) a herboxidiene splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or non-responsive to treatment with a herboxidiene splicing modulator when administered alone.

[0055] In some embodiments, the target antigen is EPHA2. In some embodiments, the one or more neoplastic cells are in EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer. In some embodiments, the subject is refractory or unresponsive to treatment with (a) an anti-EPHA2 antibody when administered alone and / or (b) a herboxidien splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or unresponsive to treatment with a herboxidien splicing modulator when administered alone.

[0056] In some embodiments, the target antigen is MSLN. In some embodiments, the one or more neoplastic cells are in ovarian cancer, cervical cancer, pancreatic cancer, or lung cancer (e.g., lung adenocarcinoma) that expresses MSLN. In some embodiments, the subject is refractory or unresponsive to treatment with (a) an anti-MSLN antibody when administered alone and / or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or unresponsive to treatment with a splicing modulator when administered alone.

[0057] In some embodiments, the target antigen is FOLH1. In some embodiments, the one or more neoplastic cells are in a prostate cancer that expresses FOLH1. In some embodiments, the subject is refractory or non-responsive to treatment with (a) an anti-FOLH1 antibody when administered alone and / or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or non-responsive to treatment with a splicing modulator when administered alone.

[0058] In some embodiments, the target antigen is CDH6. In some embodiments, the one or more neoplastic cells are in a renal cancer that expresses CDH6. In some embodiments, the subject is refractory or non-responsive to treatment with (a) an anti-CDH6 antibody when administered alone and / or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or non-responsive to treatment with a splicing modulator when administered alone.

[0059] In some embodiments, the target antigen is CEACAM5. In some embodiments, the one or more neoplastic cells are in a colorectal cancer that expresses CEACAM5. In some embodiments, the subject is refractory or non-responsive to treatment with (a) an anti-CEACAM5 antibody when administered alone and / or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or non-responsive to treatment with a splicing modulator when administered alone.

[0060] In some embodiments, the target antigen is CFC1B. In some embodiments, the one or more neoplastic cells are in a pancreatic cancer that expresses CFC1B. In some embodiments, the subject is refractory or non-responsive to treatment with (a) an anti-CFC1B antibody when administered alone and / or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or non-responsive to treatment with a splicing modulator when administered alone.

[0061] In some embodiments, the target antigen is ENPP3. In some embodiments, the one or more neoplastic cells are in a renal cancer that expresses ENPP3. In some embodiments, the subject is refractory or non-responsive to treatment with (a) an anti-ENPP3 antibody when administered alone and / or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or non-responsive to treatment with a splicing modulator when administered alone.

[0062] In some embodiments, the target antigen is FOLR1. In some embodiments, the one or more neoplastic cells are in an ovarian cancer that expresses FOLR1. In some embodiments, the subject is refractory or non-responsive to treatment with (a) an anti-FOLR1 antibody when administered alone and / or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or non-responsive to treatment with a splicing modulator when administered alone.

[0063] In some embodiments, the target antigen is HAVCR1. In some embodiments, the one or more neoplastic cells are in a renal cancer or esophageal cancer that expresses HAVCR1. In some embodiments, the subject is refractory or poorly responsive to treatment with (a) an anti-HAVCR1 antibody when administered alone and / or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, refractory, or poorly responsive to treatment with a splicing modulator when administered alone.

[0064] In some embodiments, the target antigen is KIT. In some embodiments, the one or more neoplastic cells are in a KIT-expressing renal cancer. In some embodiments, the subject is refractory or unresponsive to treatment with (a) an anti-KIT antibody when administered alone and / or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or unresponsive to treatment with a splicing modulator when administered alone.

[0065] In some embodiments, the target antigen is MET. In some embodiments, the one or more neoplastic cells are in a MET-expressing renal or esophageal cancer. In some embodiments, the subject is refractory or non-responsive to treatment with (a) an anti-MET antibody when administered alone and / or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or non-responsive to treatment with a splicing modulator when administered alone.

[0066] In some embodiments, the target antigen is MUC16. In some embodiments, the one or more neoplastic cells are in ovarian, cervical, or breast cancer that express MUC16. In some embodiments, the subject is refractory or non-responsive to treatment with (a) an anti-MUC16 antibody when administered alone and / or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or non-responsive to treatment with a splicing modulator when administered alone.

[0067] In some embodiments, the target antigen is SLC39A6. In some embodiments, the one or more neoplastic cells are in breast cancer or prostate cancer that expresses SLC39A6. In some embodiments, the subject is refractory or non-responsive to treatment with (a) an anti-SLC39A6 antibody when administered alone and / or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or non-responsive to treatment with a splicing modulator when administered alone.

[0068] In some embodiments, the target antigen is SLC44A4. In some embodiments, the one or more neoplastic cells are in a prostate cancer that expresses SLC44A4. In some embodiments, the subject is refractory or non-responsive to treatment with (a) an anti-SLC44A4 antibody when administered alone and / or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or non-responsive to treatment with a splicing modulator when administered alone.

[0069] In some embodiments, the target antigen is STEAP1. In some embodiments, the one or more neoplastic cells are in a prostate cancer that expresses STEAP1. In some embodiments, the subject is refractory or non-responsive to treatment with (a) an anti-STEAP1 antibody when administered alone and / or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or non-responsive to treatment with a splicing modulator when administered alone.

[0070] In certain other aspects, the disclosure provides methods of reducing or inhibiting tumor growth in a subject having or suspected of having a neoplastic disorder by administering to the subject a therapeutically effective amount and / or therapeutically effective regimen of any one of the described ADCs or compositions.

[0071] In some embodiments, treatment with the antibody-drug conjugate or composition induces bystander killing of neoplastic tumor cells that do not express the target antigen but that are adjacent to neoplastic tumor cells that do express the target antigen, hi some embodiments, a tumor comprises one or more neoplastic cells that express the target antigen.

[0072] In some embodiments, the target antigen is HER2. In some embodiments, the one or more neoplastic cells are derived from HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., serous endometrial carcinoma), osteosarcoma, or salivary duct cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-HER2 antibody when administered alone and / or (b) a herboxidian splicing modulator when administered alone.

[0073] In some embodiments, the target antigen is CD138. In some embodiments, the one or more neoplastic cells are derived from multiple myeloma that express CD138. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-CD138 antibody when administered alone and / or (b) a herboxidiene splicing modulator when administered alone.

[0074] In some embodiments, the target antigen is EPHA2. In some embodiments, the one or more neoplastic cells are derived from EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-EPHA2 antibody when administered alone and / or (b) a herboxidiene splicing modulator when administered alone.

[0075] In some embodiments, the target antigen is MSLN. In some embodiments, the one or more neoplastic cells are derived from MSLN-expressing ovarian cancer, cervical cancer, pancreatic cancer, or lung cancer (e.g., lung adenocarcinoma). In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-MSLN antibody administered alone and / or (b) a splicing modulator administered alone.

[0076] In some embodiments, the target antigen is FOLH1. In some embodiments, the one or more neoplastic cells are derived from a prostate cancer that expresses FOLH1. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-FOLH1 antibody when administered alone and / or (b) a splicing modulator when administered alone.

[0077] In some embodiments, the target antigen is CDH6. In some embodiments, the one or more neoplastic cells are derived from a CDH6-expressing renal carcinoma. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-CDH6 antibody when administered alone and / or (b) a splicing modulator when administered alone.

[0078] In some embodiments, the target antigen is CEACAM5. In some embodiments, the one or more neoplastic cells are derived from a colorectal cancer that expresses CEACAM5. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-CEACAM5 antibody when administered alone and / or (b) a splicing modulator when administered alone.

[0079] In some embodiments, the target antigen is CFC1B. In some embodiments, the one or more neoplastic cells are derived from a pancreatic cancer that expresses CFC1B. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-CFC1B antibody when administered alone and / or (b) a splicing modulator when administered alone.

[0080] In some embodiments, the target antigen is ENPP3. In some embodiments, the one or more neoplastic cells are derived from a renal carcinoma that expresses ENPP3. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-ENPP3 antibody when administered alone and / or (b) a splicing modulator when administered alone.

[0081] In some embodiments, the target antigen is FOLR1. In some embodiments, the one or more neoplastic cells are derived from an ovarian cancer that expresses FOLR1. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-FOLR1 antibody when administered alone and / or (b) a splicing modulator when administered alone.

[0082] In some embodiments, the target antigen is HAVCR1. In some embodiments, the one or more neoplastic cells are derived from a renal cancer or an esophageal cancer that expresses HAVCR1. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-HAVCR1 antibody when administered alone and / or (b) a splicing modulator when administered alone.

[0083] In some embodiments, the target antigen is KIT. In some embodiments, the one or more neoplastic cells are derived from a KIT-expressing renal carcinoma. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-KIT antibody administered alone and / or (b) a splicing modulator administered alone.

[0084] In some embodiments, the target antigen is MET. In some embodiments, the one or more neoplastic cells are derived from a MET-expressing renal or esophageal cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-MET antibody when administered alone and / or (b) a splicing modulator when administered alone.

[0085] In some embodiments, the target antigen is MUC16. In some embodiments, the one or more neoplastic cells are derived from ovarian, cervical, or breast cancer that expresses MUC16. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-MUC16 antibody when administered alone and / or (b) a splicing modulator when administered alone.

[0086] In some embodiments, the target antigen is SLC39A6. In some embodiments, the one or more neoplastic cells are derived from breast or prostate cancer that expresses SLC39A6. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-SLC39A6 antibody when administered alone and / or (b) a splicing modulator when administered alone.

[0087] In some embodiments, the target antigen is SLC44A4. In some embodiments, the one or more neoplastic cells are derived from a prostate cancer that expresses SLC44A4. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-SLC44A4 antibody when administered alone and / or (b) a splicing modulator when administered alone.

[0088] In some embodiments, the target antigen is STEAP1. In some embodiments, the one or more neoplastic cells are derived from a prostate cancer that expresses STEAP1. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-STEAP1 antibody when administered alone and / or (b) a splicing modulator when administered alone.

[0089] In yet another aspect, the disclosure provides methods for determining whether a subject having or suspected of having a neoplastic disorder will respond to treatment with any one of the described ADCs or compositions by providing a biological sample from the subject and contacting the biological sample with the ADC or composition. In some embodiments, the biological sample is a tumor sample. In some embodiments, the tumor sample is a tumor biopsy or a blood sample. In some embodiments, the blood sample is selected from blood, a blood fraction, or cells obtained from blood or a blood fraction. In some embodiments, the subject has one or more neoplastic cells that express a target antigen. In some embodiments, the target antigen is HER2. In some embodiments, the one or more neoplastic cells are derived from HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., serous endometrial carcinoma), osteosarcoma, or salivary duct cancer. In some embodiments, the target antigen is CD138. In some embodiments, the one or more neoplastic cells are derived from CD138-expressing multiple myeloma. In some embodiments, the target antigen is EPHA2. In some embodiments, the one or more neoplastic cells are derived from breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer that expresses EPHA2. In some embodiments, the target antigen is MSLN. In some embodiments, the one or more neoplastic cells are derived from ovarian cancer, cervical cancer, pancreatic cancer, or lung cancer (e.g., lung adenocarcinoma) that expresses MSLN. In some embodiments, the target antigen is FOLH1. In some embodiments, the one or more neoplastic cells are derived from prostate cancer that expresses FOLH1. In some embodiments, the target antigen is CDH6. In some embodiments, the one or more neoplastic cells are derived from renal cancer that expresses CDH6. In some embodiments, the target antigen is CEACAM5. In some embodiments, the one or more neoplastic cells are derived from colorectal cancer that expresses CEACAM5. In some embodiments, the target antigen is CFC1B. In some embodiments, the one or more neoplastic cells are derived from pancreatic cancer that expresses CFC1B. In some embodiments, the target antigen is ENPP3. In some embodiments, the one or more neoplastic cells are derived from a renal carcinoma that expresses ENPP3.In some embodiments, the target antigen is FOLR1. In some embodiments, the one or more neoplastic cells are derived from an ovarian cancer that expresses FOLR1. In some embodiments, the target antigen is HAVCR1. In some embodiments, the one or more neoplastic cells are derived from a renal cancer or esophageal cancer that expresses HAVCR1. In some embodiments, the target antigen is KIT. In some embodiments, the one or more neoplastic cells are derived from a renal cancer that expresses KIT. In some embodiments, the target antigen is MET. In some embodiments, the one or more neoplastic cells are derived from a renal cancer or esophageal cancer that expresses MET. In some embodiments, the target antigen is MUC16. In some embodiments, the one or more neoplastic cells are derived from an ovarian cancer, cervical cancer, or breast cancer that expresses MUC16. In some embodiments, the target antigen is SLC39A6. In some embodiments, the one or more neoplastic cells are derived from a breast cancer or prostate cancer that expresses SLC39A6. In some embodiments, the target antigen is SLC44A4. In some embodiments, the one or more neoplastic cells are derived from a prostate cancer that expresses SLC44A4. In some embodiments, the target antigen is STEAP 1. In some embodiments, the one or more neoplastic cells are derived from a prostate cancer that expresses STEAP 1.

[0090] Further provided herein, in various embodiments, are pharmaceutical compositions comprising an ADC and a pharmaceutically acceptable diluent, carrier, and / or excipient. Methods of making the described ADC compounds and compositions are also disclosed. [Brief explanation of the drawings]

[0091] [Figure 1A]Figures 1A-1D show the viability dose response of exemplary payload compounds in HER2-amplified breast cancer cells (HCC1954) and gastric cancer cells (NCI-N87). Cells were incubated with the compounds for 72 hours (3 days) or 144 hours (6 days), and viability was read using CellTiter-Glo® 2.0 reagent. Figure 1A shows the viability dose response of HCC1954 cells after 72 hours of incubation. Figure 1B shows the viability dose response of NCI-N87 cells after 72 hours of incubation. Figure 1C shows the viability dose response of HCC1954 cells after 144 hours of incubation. Figure 1D shows the viability dose response of NCI-N87 cells after 144 hours of incubation. Data are presented as mean ± SD. [Figure 1B] Figures 1A-1D show the viability dose response of exemplary payload compounds in HER2-amplified breast cancer cells (HCC1954) and gastric cancer cells (NCI-N87). Cells were incubated with the compounds for 72 hours (3 days) or 144 hours (6 days), and viability was read using CellTiter-Glo® 2.0 reagent. Figure 1A shows the viability dose response of HCC1954 cells after 72 hours of incubation. Figure 1B shows the viability dose response of NCI-N87 cells after 72 hours of incubation. Figure 1C shows the viability dose response of HCC1954 cells after 144 hours of incubation. Figure 1D shows the viability dose response of NCI-N87 cells after 144 hours of incubation. Data are presented as mean ± SD. [Figure 1C]Figures 1A-1D show the viability dose response of exemplary payload compounds in HER2-amplified breast cancer cells (HCC1954) and gastric cancer cells (NCI-N87). Cells were incubated with the compounds for 72 hours (3 days) or 144 hours (6 days), and viability was read using CellTiter-Glo® 2.0 reagent. Figure 1A shows the viability dose response of HCC1954 cells after 72 hours of incubation. Figure 1B shows the viability dose response of NCI-N87 cells after 72 hours of incubation. Figure 1C shows the viability dose response of HCC1954 cells after 144 hours of incubation. Figure 1D shows the viability dose response of NCI-N87 cells after 144 hours of incubation. Data are presented as mean ± SD. [Figure 1D] Figures 1A-1D show the viability dose response of exemplary payload compounds in HER2-amplified breast cancer cells (HCC1954) and gastric cancer cells (NCI-N87). Cells were incubated with the compounds for 72 hours (3 days) or 144 hours (6 days), and viability was read using CellTiter-Glo® 2.0 reagent. Figure 1A shows the viability dose response of HCC1954 cells after 72 hours of incubation. Figure 1B shows the viability dose response of NCI-N87 cells after 72 hours of incubation. Figure 1C shows the viability dose response of HCC1954 cells after 144 hours of incubation. Figure 1D shows the viability dose response of NCI-N87 cells after 144 hours of incubation. Data are presented as mean ± SD. [Figure 2A] Figures 2A and 2B show the results of an SLC25A19 splicing assay in HER2-amplified breast cancer cells (HCC1954) (Figure 2A) and gastric cancer cells (NCI-N87) (Figure 2B). Cells were incubated with compounds for 6 hours, and splicing of the SLC25A19 transcript was measured in real-time qPCR reactions using specific Taqman primer-probe sets. The y-axis represents the percent (%) response relative to the DMSO control (0.1%). Data are presented as mean ± SD. [Figure 2B]Figures 2A and 2B show the results of an SLC25A19 splicing assay in HER2-amplified breast cancer cells (HCC1954) (Figure 2A) and gastric cancer cells (NCI-N87) (Figure 2B). Cells were incubated with compounds for 6 hours, and splicing of the SLC25A19 transcript was measured in real-time qPCR reactions using specific Taqman primer-probe sets. The y-axis represents the percent (%) response relative to the DMSO control (0.1%). Data are presented as mean ± SD. DETAILED DESCRIPTION OF THE INVENTION

[0092] The disclosed compositions and methods may be more readily understood by reference to the following detailed description.

[0093] Throughout this document, descriptions refer to compositions and methods of using such compositions. Where this disclosure describes or claims features or embodiments relating to a composition, such features or embodiments are equally applicable to methods of using that composition. Similarly, where this disclosure describes or claims features or embodiments relating to methods of using a composition, such features or embodiments are equally applicable to that composition.

[0094] When a range of values ​​is expressed, it includes embodiments using any specific value within that range. Furthermore, reference to values ​​stated in ranges includes each and every value within that range. All ranges are inclusive of their endpoints and are combinable. When values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. The use of "or" is intended to mean "and / or" unless the specific context in which it is used dictates otherwise.

[0095] It is to be understood that certain features of the compositions and methods of the present disclosure, which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the compositions and methods of the present disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

[0096] All references cited herein are incorporated by reference for all purposes. In the event of a conflict between a reference and the present specification, the present specification shall control.

[0097] definition Throughout this specification and claims, various terms are used in connection with the described embodiments. Unless otherwise indicated, such terms should be given their ordinary meaning in the art. Other specifically defined terms should be construed consistent with the definition provided herein.

[0098] As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0099] The term "about" or "approximately" in the context of numerical values ​​and ranges refers to a value or range that approximates or is close to the stated value or range such that the embodiment will operate as intended, such as having a desired amount of nucleic acid or polypeptide in a reaction mixture, as will be apparent to one of skill in the art from the teachings contained herein. In some embodiments, about means ±10% of a numerical quantity.

[0100] The terms "antibody-drug conjugate," "antibody conjugate," "conjugate," "immunoconjugate," and "ADC" are used interchangeably and refer to one or more therapeutic compounds (e.g., herboxidian splicing modulators) linked to one or more antibodies or antigen-binding fragments, and have the general formula: Ab-(LH) pADCs are defined by Formula I, where Ab = antibody or antigen-binding fragment, L = linker moiety, H = herboxidiene splicing modulator (e.g., herboxidiene or a derivative thereof), and p = number of drug moieties per antibody or antigen-binding fragment. ADCs containing herboxidiene splicing modulators may also be more specifically referred to herein as "herboxidiene splicing modulator-loaded antibodies" or "SMLAs." In ADCs containing herboxidiene splicing modulators, "p" refers to the number of herboxidiene splicing modulators linked to the antibody or antigen-binding fragment. In some embodiments, the linker L may comprise a cleavable moiety between the antibody or antigen-binding fragment and the herboxidiene splicing modulator. In some embodiments, the linker L may comprise a cleavable moiety that can be attached to either or both of the antibody or antigen-binding fragment and the herboxidiene splicing modulator via one or more spacer units. In some embodiments, when a spacer unit links a cleavable moiety to a herboxidienes splicing modulator, it is a self-immolative spacer unit. In other embodiments, the linker L does not include a cleavable moiety and is a non-cleavable linker. In some embodiments, the linker L may include at least one spacer unit that can be directly linked to an antibody or antigen-binding fragment and a herboxidienes splicing modulator. Exemplary cleavable and non-cleavable linkers are described and exemplified herein.

[0101] The term "antibody" is used in its broadest sense to refer to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing, through at least one antigen recognition site within the variable region of the immunoglobulin molecule. The heavy chain of an antibody is composed of a heavy chain variable domain (V H ) and heavy chain constant region (C H The light chain consists of a light chain variable domain (V L ) and the light chain constant domain (C L). For purposes of this application, mature heavy and light chain variable domains each comprise three complementarity-determining regions (CDR1, CDR2, and CDR3) within four framework regions (FR1, FR2, FR3, and FR4), flanked from N-terminus to C-terminus by FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. An "antibody" may be naturally occurring or artificial, such as a monoclonal antibody produced by conventional hybridoma technology. The term "antibody" includes full-length monoclonal and polyclonal antibodies, as well as antibody fragments such as Fab, Fab', F(ab')2, Fv, and single-chain antibodies. Antibodies may belong to any one of the five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, or their subclasses (e.g., isotypes IgG1, IgG2, IgG3, IgG4). The term also encompasses human antibodies, chimeric antibodies, humanized antibodies, and any modified immunoglobulin molecule containing an antigen recognition site, so long as it exhibits the desired biological activity (e.g., binds to the target antigen, is internalized into cells expressing the target antigen).

[0102] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies; i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and should not be construed as requiring that the antibody have been produced by any particular method. For example, monoclonal antibodies to be used in the present disclosure may be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be produced by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al. (1991) Nature 352:624-8 and Marks et al. (1991) J Mol Biol. 222:581-97.

[0103] The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of one or more chains is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they specifically bind to the target antigen and / or exhibit the desired biological activity.

[0104] The term "human antibody," as used herein, refers to an antibody produced by a human or an antibody having the amino acid sequence of an antibody produced by a human.

[0105] The term "chimeric antibody," as used herein, refers to an antibody in which the amino acid sequences of the immunoglobulin molecules are derived from two or more species. In some instances, the variable regions of both the heavy and light chains correspond to the variable regions of antibodies derived from one species having the desired specificity, affinity, and activity, while the constant regions are homologous to antibodies derived from another species (e.g., human), thereby minimizing the immune response in the latter species.

[0106] As used herein, the term "humanized antibody" refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Generally, a humanized antibody will comprise substantially all 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 (FR) regions are those of a human immunoglobulin. A humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Humanized antibodies may be further modified by substitution of residues within either the Fv framework regions and / or replaced non-human residues to refine and optimize antibody specificity, affinity, and / or activity.

[0107] The term "antigen-binding fragment" or "antigen-binding portion" of an antibody, as used herein, refers to one or more fragments of an antibody or protein that retain the ability to specifically bind to an antigen (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1). Antigen-binding fragments may also retain the ability to internalize into antigen-expressing cells. In some embodiments, antigen-binding fragments also retain immune effector activity. It has been shown that fragments of full-length antibodies can perform the antigen-binding function of the full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" or "antigen-binding portion" of an antibody include (i) V L , V H , C L , and C H1 (ii) a F(ab')2 fragment, a bivalent fragment containing 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 fragment of an antibody; L and V H (v) a single variable domain, e.g., a V H and (vi) isolated complementarity-determining regions (CDRs). In addition, two domains of an Fv fragment, V, V, and VD, are included. L and V H are encoded by separate genes, but using recombinant methods they can be joined together with synthetic linkers to form V L and V HThese regions can be paired to form a single protein chain (known as a single-chain Fv (scFv)) that forms a monovalent molecule. See, e.g., Bird et al. (1988) Science 242:423-6; and Huston et al. (1988) Proc Natl Acad Sci. USA 85:5879-83. Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" or "antigen-binding portion" of an antibody and are known in the art as an exemplary type of binding fragment that can be internalized into cells upon binding (see, e.g., Zhu et al. (2010) 9:2131-41; He et al. (2010) J Nucl Med. 51:427-32; and Fitting et al. (2015) MAbs 7:390-402). In certain embodiments, scFv molecules may be incorporated into fusion proteins. Other forms of single chain antibodies, such as diabodies, are also encompassed. H and V L A bivalent, bispecific antibody is one in which the domains are expressed on a single polypeptide chain, but use a linker that is short enough to not allow pairing between the two domains on the same chain, thereby forcing them to pair with complementary domains on another chain to create two antigen-binding sites (see, e.g., Holliger et al. (1993) Proc Natl Acad Sci. USA 90:6444-8; and Poljak et al. (1994) Structure 2:1121-3). Antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and such binding fragments are screened for utility (e.g., binding affinity, internalization) in the same manner as intact antibodies. Antigen-binding fragments may also be prepared by cleavage of the intact protein, for example, by protease or chemical cleavage.

[0108] "Internalizing," as used herein in reference to an antibody or antigen-binding fragment, refers to an antibody or antigen-binding fragment that, upon binding to a cell, has the ability to penetrate the lipid bilayer membrane of the cell and be incorporated into an internal compartment, preferably a degradable compartment of the cell (i.e., be "internalized"). For example, an internalizing anti-HER2 antibody is one that has the ability to be incorporated into the cell after binding to HER2 on the cell membrane. In some embodiments, the antibody or antigen-binding fragment used in the ADCs disclosed herein targets a cell surface antigen (e.g., HER2) and is an internalizing antibody or internalizing antigen-binding fragment (i.e., the ADC translocates through the cell membrane after antigen binding). In some embodiments, the internalizing antibody or antigen-binding fragment binds to a receptor on the cell surface. An internalizing antibody or internalizing antigen-binding fragment that targets a receptor on the cell membrane can induce receptor-mediated endocytosis. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment is incorporated into the cell by receptor-mediated endocytosis.

[0109] "Non-internalizing," as used herein in reference to an antibody or antigen-binding fragment, refers to an antibody or antigen-binding fragment that remains on the cell surface upon binding to a cell. In some embodiments, the antibody or antigen-binding fragment used in the ADCs disclosed herein targets a cell surface antigen and is a non-internalizing antibody or non-internalizing antigen-binding fragment (i.e., the ADC remains on the cell surface after antigen binding and does not translocate through the cell membrane). In some embodiments, the non-internalizing antibody or antigen-binding fragment binds to a non-internalizing receptor or other cell surface antigen. Exemplary non-internalizing cell surface antigens include, but are not limited to, CA125 and CEA, and antibodies that bind to non-internalizing antigen targets are also known in the art (see, e.g., Bast et al. (1981) J Clin Invest. 68(5):1331-7; Scholler and Urban (2007) Biomark Med. 1(4):513-23; and Boudousq et al. (2013) PLoS One 8(7):e69613).

[0110] The term "human epidermal growth factor receptor 2," "HER2," or "HER2 / NEU," as used herein, refers to any naturally occurring form of human HER2. The term encompasses full-length HER2 (e.g., UniProt Reference Sequence: P04626; SEQ ID NO: 31) as well as any form of human HER2 that may result from cellular processing. The term also encompasses functional variants or fragments of human HER2, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human HER2 (i.e., variants and fragments are included unless the context dictates that the term is used to refer only to the wild-type protein). HER2 can be isolated from humans or produced recombinantly or by synthetic methods.

[0111] The term "anti-HER2 antibody" or "antibody that binds to HER2" refers to any form of antibody or fragment thereof that binds, e.g., specifically, to HER2, and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and antibody fragments that are biologically functional so long as they bind, e.g., specifically, to HER2. U.S. Pat. No. 5,821,337 provides exemplary HER2-binding sequences, including exemplary anti-HER2 antibody sequences, and is incorporated herein by reference in this regard. In some embodiments, the anti-HER2 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. Trastuzumab (U.S. Pat. No. 5,821,337; Molina et al. (2001) Cancer Res. 61(12):4744-9) is an exemplary anti-human HER2 antibody.

[0112] The terms "syndecan-1," "SDC1," or "CD138," as used herein, refer to any naturally occurring form of human CD138. The term encompasses full-length CD138 (e.g., UniProt Reference Sequence: P18827; SEQ ID NO: 32) as well as any form of human CD138 that may result from cellular processing. The term also encompasses functional variants or fragments of human CD138, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human CD138 (i.e., variants and fragments are included unless the context dictates that the term is used to refer only to the wild-type protein). CD138 can be isolated from humans or produced recombinantly or by synthetic methods.

[0113] The term "anti-CD138 antibody" or "antibody that binds to CD138" refers to any form of antibody or fragment thereof that binds, e.g., specifically, to CD138, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and antibody fragments that are biologically functional so long as they bind, e.g., specifically, to CD138. In some embodiments, the anti-CD138 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment. B-B4 (Tassone et al. (2004) Blood 104:3688-96) is an exemplary anti-human CD138 antibody.

[0114] The term "ephrin type-A receptor 2" or "EPHA2," as used herein, refers to any naturally occurring form of human EPHA2. This term encompasses full-length EPHA2 (e.g., UniProt Reference Sequence: P29317; SEQ ID NO: 33) as well as any form of human EPHA2 that may result from cellular processing. This term also encompasses functional variants or fragments of human EPHA2, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human EPHA2 (i.e., variants and fragments are included unless the context dictates that the term is used to refer only to the wild-type protein). EPHA2 can be isolated from humans or produced recombinantly or by synthetic methods.

[0115] The term "anti-EPHA2 antibody" or "antibody that binds to EPHA2" refers to any form of antibody or fragment thereof that binds, e.g., specifically, to EPHA2, and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and antibody fragments that are biologically functional so long as they bind, e.g., specifically, to EPHA2. WO 2007 / 030642 provides exemplary EPHA2-binding sequences, including exemplary anti-EPHA2 antibody sequences, and is incorporated herein by reference in this regard. In some embodiments, the anti-EPHA2 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment. 1C1 (WO 2007 / 030642; Jackson et al. (2008) Cancer Res. 68(22):9367-74) is an exemplary anti-human EPHA2 antibody.

[0116] The term "mesothelin" or "MSLN," as used herein, refers to any naturally occurring form of human MSLN. This term encompasses full-length MSLN (e.g., UniProt Reference Sequence: Q13421; SEQ ID NO: 94) as well as any form of human MSLN that may result from cellular processing. This term also encompasses functional variants or fragments of human MSLN, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human MSLN (i.e., variants and fragments are included unless the context dictates that the term is used to refer only to the wild-type protein). MSLN can be isolated from humans or produced recombinantly or by synthetic methods.

[0117] The term "anti-MSLN antibody" or "antibody that binds to MSLN" refers to any form of antibody or fragment thereof that binds, e.g., specifically, to MSLN, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and antibody fragments that are biologically functional so long as they bind, e.g., specifically, to MSLN. WO 2011 / 074621 provides exemplary MSLN-binding sequences, including exemplary anti-MSLN antibody sequences, and is incorporated herein by reference. In some embodiments, the anti-MSLN antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment. IC11-25, IC14-30, IC7-4, IC17-35, and 2-9 are exemplary anti-human MSLN antibodies.

[0118] The term "glutamate carboxypeptidase 2" or "FOLH1," as used herein, refers to any naturally occurring form of human FOLH1. The term encompasses full-length FOLH1 (e.g., UniProt Reference Sequence: Q04609; SEQ ID NO: 95) as well as any form of human FOLH1 that may result from cellular processing. The term also encompasses functional variants or fragments of human FOLH1, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human FOLH1 (i.e., variants and fragments are included unless the context dictates that the term is used to refer only to the wild-type protein). FOLH1 can be isolated from humans or produced recombinantly or by synthetic methods.

[0119] The term "anti-FOLH1 antibody" or "antibody that binds to FOLH1" refers to any form of antibody or fragment thereof that binds, e.g., specifically, to FOLH1, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and antibody fragments that are biologically functional so long as they bind, e.g., specifically, to FOLH1. WO 2019 / 012260 and WO 2017 / 212250 provide exemplary FOLH1-binding sequences, including exemplary anti-FOLH1 antibody sequences, and are incorporated herein by reference in their entirety. In some embodiments, the anti-FOLH1 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. J591 (deimmunized) is an exemplary anti-human FOLH1 antibody.

[0120] The term "cadherin-6" or "CDH6," as used herein, refers to any naturally occurring form of human CDH6. The term encompasses full-length CDH6 (e.g., UniProt Reference Sequence: P55285; SEQ ID NO: 96) as well as any form of human CDH6 that may result from cellular processing. The term also encompasses functional variants or fragments of human CDH6, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human CDH6 (i.e., variants and fragments are included unless the context dictates that the term is used to refer only to the wild-type protein). CDH6 can be isolated from humans or produced recombinantly or by synthetic methods.

[0121] The terms "anti-CDH6 antibody" or "antibody that binds to CDH6" refer to any form of antibody or fragment thereof that binds, e.g., specifically, to CDH6, and encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and antibody fragments that are biologically functional so long as they bind, e.g., specifically, to CDH6. WO 2018 / 185618 provides exemplary CDH6-binding sequences, including exemplary anti-CDH6 antibody sequences, and is incorporated herein by reference in this regard. In some embodiments, the anti-CDH6 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0122] The term "carcinoembryonic antigen-related cell adhesion molecule 5" or "CEACAM5," as used herein, refers to any naturally occurring form of human CEACAM5. This term encompasses full-length CEACAM5 (e.g., UniProt Reference Sequence: P06731; SEQ ID NO: 97) as well as any form of human CEACAM5 that may result from cellular processing. This term also encompasses functional variants or fragments of human CEACAM5, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human CEACAM5 (i.e., variants and fragments are included unless the context dictates that the term is used to refer only to the wild-type protein). CEACAM5 can be isolated from humans or produced recombinantly or by synthetic methods.

[0123] The terms "anti-CEACAM5 antibody" or "antibody that binds to CEACAM5" refer to any form of antibody or fragment thereof that binds, e.g., specifically, to CEACAM5, and include monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and antibody fragments that are biologically functional so long as they bind, e.g., specifically, to CEACAM5. U.S. Patent Application Publication No. 2015 / 0125386 provides exemplary CEACAM5 binding sequences, including exemplary anti-CEACAM5 antibody sequences, and is incorporated herein by reference in this regard. In some embodiments, the anti-CEACAM5 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment. hMN14 is an exemplary anti-human CEACAM5 antibody.

[0124] The term "cryptic family protein 1B" or "CFClB" as used herein refers to any naturally occurring form of human CFClB. This term encompasses full-length CFClB (e.g., UniProt Reference Sequence: P0CG36; SEQ ID NO: 98) as well as any form of human CFClB that may result from cellular processing. This term also encompasses functional variants or fragments of human CFClB, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human CFClB (i.e., variants and fragments are included unless the context dictates that the term is used to refer only to the wild-type protein). CFClB can be isolated from humans or produced recombinantly or by synthetic methods.

[0125] The terms "anti-CFC1B antibody" or "antibody that binds to CFC1B" refer to any form of antibody or fragment thereof that binds, e.g., specifically, to CFC1B, and encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and antibody fragments that are biologically functional so long as they bind, e.g., specifically, to CFC1B. WO 2002 / 088170 provides exemplary CFC1B-binding sequences, including exemplary anti-CFC1B antibody sequences, and is incorporated herein by reference in this regard. In some embodiments, the anti-CFC1B antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0126] The term "ectonucleotide pyrophosphatase / phosphodiesterase family member 3" or "ENPP3," as used herein, refers to any naturally occurring form of human ENPP3. This term encompasses full-length ENPP3 (e.g., UniProt Reference Sequence: O14638; SEQ ID NO: 99) as well as any form of human ENPP3 that may result from cellular processing. This term also encompasses functional variants or fragments of human ENPP3, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human ENPP3 (i.e., variants and fragments are included unless the context dictates that the term is used to refer only to the wild-type protein). ENPP3 can be isolated from humans or produced recombinantly or by synthetic methods.

[0127] The terms "anti-ENPP3 antibody" or "antibody that binds to ENPP3" refer to any form of antibody or fragment thereof that binds, e.g., specifically, to ENPP3, and encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and antibody fragments that are biologically functional so long as they bind, e.g., specifically, to ENPP3. Donate et al. ((2016) Clin Cancer Res. 22(8):1989-99) provide exemplary ENPP3-binding sequences, including exemplary anti-ENPP3 antibody sequences, and are incorporated herein by reference in their entirety. In some embodiments, the anti-ENPP3 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0128] The term "folate receptor alpha" or "FOLR1," as used herein, refers to any naturally occurring form of human FOLR1. The term encompasses full-length FOLR1 (e.g., UniProt Reference Sequence: P15328; SEQ ID NO: 100) as well as any form of human FOLR1 that may result from cellular processing. The term also encompasses functional variants or fragments of human FOLR1, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human FOLR1 (i.e., variants and fragments are included unless the context dictates that the term is used to refer only to the wild-type protein). FOLR1 can be isolated from humans or produced recombinantly or by synthetic methods.

[0129] The term "anti-FOLR1 antibody" or "antibody that binds to FOLR1" refers to any form of antibody or fragment thereof that binds, e.g., specifically, to FOLR1, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and antibody fragments that are biologically functional so long as they bind, e.g., specifically, to FOLR1. WO 2005 / 080431 and Coney et al. ((1991) Cancer Res. 51(22):6125-32) provide exemplary FOLR1-binding sequences, including exemplary anti-FOLR1 antibody sequences, and are incorporated herein by reference in their entirety. In some embodiments, the anti-FOLR1 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. Farletuzumab and MOv19 are exemplary anti-human FOLR1 antibodies.

[0130] The term "hepatitis A virus cellular receptor 1" or "HAVCR1," as used herein, refers to any naturally occurring form of human HAVCR1. This term encompasses full-length HAVCR1 (e.g., UniProt Reference Sequence: Q96D42; SEQ ID NO: 101) as well as any form of human HAVCR1 that may result from cellular processing. This term also encompasses functional variants or fragments of human HAVCR1, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human HAVCR1 (i.e., variants and fragments are included unless the context dictates that the term is used to refer only to the wild-type protein). HAVCR1 can be isolated from humans or produced recombinantly or by synthetic methods.

[0131] The terms "anti-HAVCR1 antibody" or "antibody that binds to HAVCR1" refer to any form of antibody or fragment thereof that binds, e.g., specifically, to HAVCR1, and encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and antibody fragments that are biologically functional so long as they bind, e.g., specifically, to HAVCR1. Thomas et al. ((2016) Mol Cancer Ther. 15(12):2946-54) provides exemplary HAVCR1-binding sequences, including exemplary anti-HAVCR1 antibody sequences, and is incorporated herein by reference in its entirety. In some embodiments, the anti-HAVCR1 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0132] The term "mast / stem cell growth factor receptor Kit" or "KIT," as used herein, refers to any naturally occurring form of human KIT. The term encompasses full-length KIT (e.g., UniProt Reference Sequence: P10721; SEQ ID NO: 102) as well as any form of human KIT that may arise by cellular processing. The term also encompasses functional variants or fragments of human KIT, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human KIT (i.e., variants and fragments are included unless the context dictates that the term is used to refer only to the wild-type protein). KIT can be isolated from humans or produced recombinantly or by synthetic methods.

[0133] The term "anti-KIT antibody" or "antibody that binds to KIT" refers to any form of antibody or fragment thereof that binds, e.g., specifically, to KIT, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and antibody fragments that are biologically functional so long as they bind, e.g., specifically, to KIT. Shi et al. ((2016) Proc Natl Acad Sci USA 113(33):E4784-93) and Abrams et al. ((2018) Clin Cancer Res. 24(17):4297-308) provide exemplary KIT-binding sequences, including exemplary anti-KIT antibody sequences, and are incorporated herein by reference in their entirety. In some embodiments, the anti-KIT antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0134] The term "hepatocyte growth factor receptor" or "MET," as used herein, refers to any naturally occurring form of human MET. The term encompasses full-length MET (e.g., UniProt Reference Sequence: P08581; SEQ ID NO: 103) as well as any form of human MET that may arise by cellular processing. The term also encompasses functional variants or fragments of human MET, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human MET (i.e., variants and fragments are included unless the context dictates that the term is used to refer only to the wild-type protein). MET can be isolated from humans or produced recombinantly or by synthetic methods.

[0135] The term "anti-MET antibody" or "antibody that binds to MET" refers to any form of antibody or fragment thereof that binds, e.g., specifically, to MET, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and antibody fragments that are biologically functional so long as they bind, e.g., specifically, to MET. Yang et al. ((2019) Acta Pharmacol Sin.) provides exemplary MET-binding sequences, including exemplary anti-MET antibody sequences, and is incorporated herein by reference in this regard. In some embodiments, the anti-MET antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0136] The term "mucin-16" or "MUC16," as used herein, refers to any naturally occurring form of human MUC16. This term encompasses full-length MUC16 (e.g., UniProt Reference Sequence: Q8WXI7; SEQ ID NO: 104) as well as any form of human MUC16 that may result from cellular processing. This term also encompasses functional variants or fragments of human MUC16, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human MUC16 (i.e., variants and fragments are included unless the context dictates that the term is used to refer only to the wild-type protein). MUC16 can be isolated from humans or produced recombinantly or by synthetic methods.

[0137] The terms "anti-MUC16 antibody" or "antibody that binds to MUC16" refer to any form of antibody or fragment thereof that binds, e.g., specifically, to MUC16, and encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and antibody fragments that are biologically functional so long as they bind, e.g., specifically, to MUC16. Liu et al. ((2016) Ann Oncol. 27(11):2124-30) provide exemplary MUC16-binding sequences, including exemplary anti-MUC16 antibody sequences, and are incorporated herein by reference in their entirety. In some embodiments, the anti-MUC16 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment.

[0138] The term "zinc transporter ZIP6" or "SLC39A6," as used herein, refers to any naturally occurring form of human SLC39A6. This term encompasses full-length SLC39A6 (e.g., UniProt Reference Sequence: Q13433; SEQ ID NO: 105) as well as any form of human SLC39A6 that may result from cellular processing. This term also encompasses functional variants or fragments of human SLC39A6, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human SLC39A6 (i.e., variants and fragments are included unless the context dictates that the term is used to refer only to the wild-type protein). SLC39A6 can be isolated from humans or produced recombinantly or by synthetic methods.

[0139] The term "anti-SLC39A6 antibody" or "antibody that binds to SLC39A6" refers to any form of antibody or fragment thereof that binds, e.g., specifically, to SLC39A6, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and antibody fragments that are biologically functional so long as they bind, e.g., specifically, to SLC39A6. Sussman et al. ((2014) Mol Cancer Ther. 13(12):2991-3000) provides exemplary SLC39A6-binding sequences, including exemplary anti-SLC39A6 antibody sequences, and is incorporated herein by reference in its entirety. In some embodiments, the anti-SLC39A6 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0140] The term "choline transporter-like protein 4" or "SLC44A4," as used herein, refers to any naturally occurring form of human SLC44A4. This term encompasses full-length SLC44A4 (e.g., UniProt Reference Sequence: Q53GD3; SEQ ID NO: 106) as well as any form of human SLC44A4 that may result from cellular processing. This term also encompasses functional variants or fragments of human SLC44A4, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human SLC44A4 (i.e., variants and fragments are included unless the context dictates that the term is used to refer only to the wild-type protein). SLC44A4 can be isolated from humans or produced recombinantly or by synthetic methods.

[0141] The terms "anti-SLC44A4 antibody" or "antibody that binds to SLC44A4" refer to any form of antibody or fragment thereof that binds, e.g., specifically, to SLC44A4, and encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and antibody fragments that are biologically functional so long as they bind, e.g., specifically, to SLC44A4. Mattie et al. ((2016) Mol Cancer Ther. 15(11):2679-87) provide exemplary SLC44A4 binding sequences, including exemplary anti-SLC44A4 antibody sequences, and are incorporated herein by reference in their entirety. In some embodiments, the anti-SLC44A4 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0142] The term "metalloreductase STEAP1" or "STEAP1," as used herein, refers to any naturally occurring form of human STEAP1. The term encompasses full-length STEAP1 (e.g., UniProt Reference Sequence: Q9UHE8; SEQ ID NO: 107) as well as any form of human STEAP1 that may result from cellular processing. The term also encompasses functional variants or fragments of human STEAP1, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human STEAP1 (i.e., variants and fragments are included unless the context dictates that the term is used to refer only to the wild-type protein). STEAP1 can be isolated from humans or produced recombinantly or by synthetic methods.

[0143] The terms "anti-STEAP1 antibody" or "antibody that binds to STEAP1" refer to any form of antibody or fragment thereof that binds, e.g., specifically, to STEAP1, and encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and antibody fragments that are biologically functional so long as they bind, e.g., specifically, to STEAP1. WO 2008 / 052187 provides exemplary STEAP1-binding sequences, including exemplary anti-STEAP1 antibody sequences, and is incorporated herein by reference in this regard. In some embodiments, the anti-STEAP1 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0144] As used herein, the terms "specific," "specifically binds," and "binds specifically" refer to the binding reaction between an antibody or antigen-binding fragment (e.g., an anti-HER2 antibody) and a target antigen (e.g., HER2) in a heterogeneous population of proteins and other biological substances. An antibody can be tested for binding specificity by comparing binding to the appropriate antigen with binding to an irrelevant antigen or antigen mixture under a given set of conditions. An antibody is considered specific if it binds to the appropriate antigen with at least 2-, 5-, 7-, and preferably 10-fold or more higher affinity than the irrelevant antigen or antigen mixture. A "specific antibody" or "target-specific antibody" is one that binds only to a target antigen (e.g., HER2) but does not bind (or exhibits minimal binding to) other antigens. In certain embodiments, an antibody or antigen-binding fragment that specifically binds to a target antigen (e.g., HER2) has a specificity of 1×10 -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M, 1 x 10 -10 Less than M, 1 x 10 -11 Less than M, 1 x 10 -12 Less than M or 1 x 10 -13 K less than M D In certain embodiments, KD In some embodiments, K D is 500 pM to 1 μM, 1 μM to 100 nM, or 100 mM to 10 nM.

[0145] The term "epitope" refers to a portion of an antigen that has the ability to be recognized and specifically bound by an antibody. When the antigen is a polypeptide, the epitope may be formed by contiguous amino acids, or by non-contiguous amino acids that are adjacent to each other when the polypeptide folds into a tertiary structure. The epitope bound by an antibody may be identified using any epitope mapping technique known in the art, including X-ray crystallography for epitope identification by direct visualization of the antigen-antibody complex, and monitoring the binding of antibody fragments or mutant variants of the antigen, or monitoring the solvent accessibility of various portions of the antibody and antigen. Exemplary strategies used to map antibody epitopes include, but are not limited to, array-based oligopeptide scanning, limited proteolysis, site-directed mutagenesis, high-throughput mutagenesis mapping, hydrogen-deuterium exchange, and mass spectrometry (see, e.g., Gershoni et al. (2007) 21:145-56; and Hager-Braun and Tomer (2005) Expert Rev Proteomics 2:745-56).

[0146] Competitive binding and epitope binning can also be used to determine antibodies that share the same or overlapping epitopes. Competitive binding is described in "Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory, Harlow and Lane (1999). st Edition 1988,2 ndCompetitive binding can be assessed using a cross-blocking assay, such as that described in the Journal of Molecular Biology, Vol. 1, pp. 2014 (2014). In some embodiments, competitive binding is identified when binding to a target antigen, such as HER2, by a reference antibody or binding protein (e.g., a binding protein comprising a CDR and / or variable domain selected from those identified in Tables 2-4) is reduced by the test antibody or binding protein by at least about 50% (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or more, or any percentage in between) in a cross-blocking assay. In some embodiments, competitive binding can be due to shared or similar (e.g., partially overlapping) epitopes, or due to steric hindrance of antibodies or binding proteins binding to nearby epitopes (see, e.g., Tzartos, Methods in Molecular Biology (Morris, ed. (1998) vol. 66, pp. 55-66)). In some embodiments, competitive binding can be used to sort binding proteins that share similar epitopes, for example, binding proteins that compete for binding can be "binned" into a group of binding proteins with overlapping or nearby epitopes, while those that do not compete are separated into another group of binding proteins that do not have overlapping or nearby epitopes.

[0147] The term "k" on " or "k a " refers to the on-rate constant for the association of an antibody to an antigen to form an antibody / antigen complex. This rate can be determined using standard assays such as surface plasmon resonance, biolayer interferometry, or ELISA assays.

[0148] The term "k" off " or "k d " refers to the off-rate constant for dissociation of an antibody from the antibody / antigen complex. This rate can be determined using a standard assay such as surface plasmon resonance, biolayer interferometry, or an ELISA assay.

[0149] The term “K D " refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. D is k a / k d This rate can be determined using standard assays such as surface plasmon resonance, biolayer interferometry, or ELISA assays.

[0150] The term "p" or "herboxidian splicing regulator loading" or "herboxidian splicing regulator:antibody ratio" or "herboxidian splicing regulator to antibody ratio" or "HAR" refers to the number of herboxidian splicing regulators per antibody or antigen-binding fragment, i.e., the herboxidian splicing regulator loading, or the number of -LH moieties per antibody or antigen-binding fragment (Ab) in the ADCs disclosed herein. In ADCs that include a herboxidian splicing regulator, "p" refers to the number of herboxidian splicing regulators linked to the antibody or antigen-binding fragment. For example, if two herboxidian splicing regulators (e.g., two compounds each having the structure of H3) are linked to the antibody or antigen-binding fragment, then p=2. In compositions comprising multiple copies of an ADC as described herein, the "average p" refers to the average number of -LH moieties per each antibody or antigen-binding fragment, also referred to as the "average herboxidian splicing modulator loading."

[0151] "Linker" or "linker moiety" is used herein to refer to any chemical moiety capable of covalently tethering a compound, typically a drug moiety such as a herboxidiene splicing modulator drug moiety, to another moiety, such as an antibody or antigen-binding fragment. The linker may be susceptible to or substantially resistant to acid-induced cleavage, peptidase-induced cleavage, light-based cleavage, esterase-induced cleavage, and / or disulfide bond cleavage, under conditions where the compound or antibody remains active.

[0152] The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials. The term "therapeutic agent" or "drug" refers to an agent capable of modulating a biological process and / or having biological activity. The herboxidian splicing modulators described herein are exemplary therapeutic agents.

[0153] The terms "chemotherapeutic agent" or "anti-cancer agent" are used herein to refer to any agent effective in treating cancer, regardless of mechanism of action. Inhibition of metastasis or angiogenesis is frequently a property of chemotherapeutic agents. Chemotherapeutic agents include antibodies, biomolecules, and small molecules, including the herboxidienes splicing modulators described herein. Chemotherapeutic agents can be cytotoxic or cytostatic. The term "cytostatic agent" refers to an agent that inhibits or suppresses cell growth and / or cell proliferation. The term "cytotoxic agent" refers to a substance that causes cell death primarily by interfering with cellular expression activity and / or function.

[0154] As used herein, the terms "herboxidienes splicing modulators," "herboxidienes spliceosome modulators," or "herboxidienes splice modulators" refer to compounds structurally related to herboxidienes that have anti-cancer activity by interacting with components of the spliceosome. In some embodiments, herboxidienes splicing modulators alter the rate or type of splicing in target cells. Herboxidienes splicing modulators that function as inhibitory agents have the ability, for example, to reduce uncontrolled cell proliferation. In some embodiments, herboxidienes splicing modulators may act by binding to the SF3b spliceosome complex. Such modulators may be naturally occurring or may be synthetic derivatives or analogs of herboxidienes. As used herein, the terms "derivative" and "analog," when referring to herboxidiene splicing modulators and the like, refer to any such compound that retains essentially the same, similar, or enhanced biological function or activity as herboxidiene, but has an altered chemical or biological structure. In some embodiments, the herboxidiene splicing modulator is a herboxidiene derivative.

[0155] As used herein, "herboxidienes splicing modulator drug moiety" refers to the component of an ADC or composition that provides the structure of the herboxidienes splicing modulator compound, e.g., the herboxidienes splicing modulator (H) component in an ADC of Formula (I) or in a composition that includes -LH.

[0156] As used herein, "spliceosome" refers to a ribonucleoprotein complex that removes introns from one or more RNA segments, such as pre-mRNA segments.

[0157] The term "homologue" refers to a molecule that exhibits homology to another molecule, for example, by having sequences of chemical residues that are the same or similar at corresponding positions.

[0158] The terms "inhibit" or "inhibition of," as used herein, mean to reduce by a measurable amount and can include, but need not necessarily, complete prevention or inhibition.

[0159] The terms "target negative," "target antigen negative," or "antigen negative" refer to the absence of target antigen expression by a cell or tissue. The terms "target positive," "target antigen positive," or "antigen positive" refer to the presence of target antigen expression. For example, a cell or cell line that does not express a target antigen may be described as target negative, while a cell or cell line that expresses the target antigen may be described as target positive.

[0160] The term "bystander killing" or "bystander effect" refers to the killing of target-negative cells in the presence of target-positive cells, where killing of target-negative cells is not observed in the absence of target-positive cells. Cell-to-cell contact, or at least proximity, between target-positive and target-negative cells allows for bystander killing. This type of killing is distinguishable from "off-target killing," which refers to the indiscriminate killing of target-negative cells. "Off-target killing" can be observed even in the absence of target-positive cells.

[0161] The terms "neoplastic disorder" and "cancer" are used interchangeably herein to refer to the presence of cells with characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and / or certain morphological characteristics. While cancer cells often may be in the form of a tumor or mass, such cells may exist alone within a subject or circulate in the bloodstream as independent cells, such as leukemia or lymphoma cells. The terms "neoplastic disorder" and "cancer" include all types of cancer and cancer metastases, including hematological malignancies, solid tumors, sarcomas, carcinomas, and other solid and non-solid tumor cancers. Hematological malignancies may include B-cell malignancies, cancers of the blood (leukemia), cancers of the plasma cells (myeloma, e.g., multiple myeloma), or cancers of the lymph nodes (lymphoma). Exemplary B-cell malignancies include chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, and diffuse large B-cell lymphoma. Leukemias may include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelocytic leukemia (CML), chronic myelomonocytic leukemia (CMML), acute monocytic leukemia (AMoL), and the like. Lymphomas may include Hodgkin's lymphoma and non-Hodgkin's lymphoma. Other hematological malignancies may include myelodysplastic syndromes (MDS). Solid tumors may include carcinomas such as adenocarcinomas, e.g., breast cancer, pancreatic cancer, prostate cancer, colon or colorectal cancer, lung cancer, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, cholangiocarcinoma, glioma, melanoma, and the like.

[0162] The terms "tumor" and "neoplasm" refer to any mass of tissue resulting from excessive cell growth or proliferation, either benign or malignant, including precancerous lesions.

[0163] The terms "tumor cell" and "neoplastic cell" are used interchangeably and refer to individual cells or the entire population of cells derived from a tumor or neoplasm, including both non-tumorigenic cells and cancer stem cells. As used herein, the term "tumor cell" is modified by the term "non-tumorigenic" to refer simply to tumor cells that lack the capacity for regeneration and differentiation, distinguishing such tumor cells from cancer stem cells.

[0164] The terms "subject" and "patient" are used interchangeably herein to refer to any animal, such as any mammal, including, but not limited to, humans, non-human primates, rodents, etc. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human. In some embodiments, the subject is a mouse. In some embodiments, the subject is a human.

[0165] The term "co-administration" or administration "in combination with" one or more therapeutic agents includes simultaneous and consecutive administration in any order.

[0166] A "pharmaceutical composition" refers to a preparation in a form that allows for administration and subsequently provides the intended biological activity of one or more active ingredients and / or achieves a therapeutic effect, and that does not contain additional components that are unacceptably toxic to the subject to whom the formulation will be administered. The pharmaceutical composition may be sterile.

[0167] "Pharmaceutical excipients" include materials such as adjuvants, carriers, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like.

[0168] "Pharmaceutically acceptable" means approved or expected to be approved by a federal or state regulatory agency for use in animals, and more particularly in humans, or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia.

[0169] A "pharmaceutically acceptable salt" is a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects. Examples of such salts are (a) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like; and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (b) salts formed from elemental anions such as chlorine, bromine, and iodine. See, e.g., Haynes et al., "Commentary: Occurrence of Pharmaceutically Acceptable Anions and Cations in the Cambridge Structural Database," J Pharmaceutical Sciences, vol. 94, no. 10 (2005), and Berge et al., "Pharmaceutical Salts," J Pharmaceutical Sciences, vol. 66, no. 1 (1977), which are incorporated herein by reference.

[0170] The term "effective amount," as used herein, refers to an amount of a compound, ADC, or composition (e.g., a herboxidien splicing modulator or ADC) described herein that is sufficient to achieve a specifically described purpose, e.g., to produce a therapeutic effect after administration, such as a reduction in tumor growth rate or tumor volume, a reduction in cancer symptoms, or some other indication of therapeutic efficacy. An effective amount can be determined by routine methods related to the described purpose. The term "therapeutically effective amount" refers to an amount of a compound, ADC, or composition described herein that is effective to detectably kill, reduce, and / or inhibit tumor cell growth or spread, tumor size or number, and / or other measures of the level, stage, progression, and / or severity of cancer. A therapeutically effective amount may vary depending on the intended application (in vitro or in vivo), or the subject and disease state under treatment, e.g., the subject's weight and age, the severity of the disease state, the method of administration, etc., and can be readily determined by one of ordinary skill in the art. The term also applies to a dose that will induce a specific response in target cells, e.g., inhibition of cell growth. The specific dose may vary depending, for example, on the particular pharmaceutical composition, the subject and their age and pre-existing health condition or risk for health conditions, the dosing regimen to be followed, the severity of the disease, whether it is administered in combination with other agents, the timing of administration, the tissue to which it is administered, and the physical delivery system it is delivered in. In the case of cancer, a therapeutically effective amount of an ADC can reduce the number of cancer cells, reduce tumor size, inhibit (e.g., slow or stop) tumor metastasis, inhibit (e.g., slow or stop) tumor growth, and / or alleviate one or more symptoms.

[0171] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used prior to or at an earlier stage of disease in a subject, the prophylactically effective amount will be less than the therapeutically effective amount.

[0172] As used herein, "treating" or "therapeutic" and grammatically related terms refer to any improvement in any outcome of a disease, such as prolonged survival, reduced morbidity, and / or reduced side effects resulting from an alternative treatment modality. As is readily understood in the art, complete eradication of the disease is encompassed, but is not required for therapeutic action. "Treatment" or "treating," as used herein, refers to the administration of a described ADC or composition to a subject, e.g., a patient. Treatment can be curing, healing, alleviating, mitigating, altering, ameliorating, palliating, improving, or affecting a disorder, such as cancer, a symptom of a disorder, or a predisposition to a disorder. In some embodiments, in addition to treating a subject with a condition, the compositions disclosed herein can also be provided prophylactically to prevent or reduce the likelihood of the condition developing.

[0173] In some embodiments, a labeled ADC is used. Suitable "labels" include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like.

[0174] As used herein, "protein" refers to at least two covalently linked amino acids. This term encompasses polypeptides, oligopeptides, and peptides. In some embodiments, the two or more covalently linked amino acids are linked by a peptide bond. For example, when a protein is recombinantly produced using an expression system and a host cell, the protein may be composed of naturally occurring amino acids and peptide bonds. Alternatively, the protein may contain "peptide or protein analogs," such as synthetic amino acids (e.g., homophenylalanine, citrulline, ornithine, and norleucine) or peptidomimetic structures, i.e., peptoids. Peptoids are an exemplary class of peptidomimetics whose side chains are attached to nitrogen atoms of the peptide backbone rather than the alpha carbon (as in amino acids), and have different hydrogen bonding and conformational characteristics compared to peptides (see, e.g., Simon et al. (1992) Proc Natl Acad Sci. USA 89:9367). As such, peptoids may be resistant to proteolysis or other physiological or storage conditions and may be effective at permeating cell membranes. Such synthetic amino acids may be incorporated by conventional methods well known in the art, particularly during in vitro antibody synthesis. In addition, any combination of peptidomimetics, synthetic, and naturally occurring residues / structures may be used. "Amino acid" also includes imino acid residues, such as proline and hydroxyproline. The amino acid "R group" or "side chain" may be in either the (L) or (S) configuration. In specific embodiments, the amino acid is in the (L) or (S) configuration.

[0175] A "recombinant protein" is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid, using any techniques and methods known in the art. Methods and techniques for making recombinant proteins are well known in the art.

[0176] An "isolated" protein is one that is free from at least some of the materials that normally accompany it in its natural state, e.g., comprising at least about 5% by weight, or at least about 50% by weight, of the total protein in a given sample. It is understood that, in some circumstances, an isolated protein may comprise between 5% and 99.9% by weight of the total protein content. For example, the concentration of the protein may be significantly increased using an inducible promoter or a high-expression promoter, resulting in increased concentration levels of the protein. This definition includes the production of antibodies in a wide variety of organisms and / or host cells known in the art.

[0177] For amino acid sequences, sequence identity and / or similarity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith and Waterman (1981) Adv Appl Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch (1970) J Mol Biol. 48:443, the search for similarity method of Pearson and Lipman (1988) Proc Nat Acad Sci. USA 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the BestFit sequence program described by Devereux et al. (1984) Nucl Acid Res. 12:387-95, preferably using default settings, or by visual inspection. Preferably, percent identity is calculated by FastDB based on the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30 ("Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149 (1988), Alan R. Liss, Inc.).

[0178] An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle (1987) J Mol Evol. 35:351-60; this method is similar to that described by Higgins and Sharp (1989) CABIOS 5:151-3. Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

[0179] Another example of a useful algorithm is the BLAST algorithm described in Altschul et al. (1990) J Mol Biol. 215:403-10; Altschul et al. (1997) Nucl Acid Res. 25:3389-402; and Karin et al. (1993) Proc Natl Acad Sci. USA 90:5873-87. A particularly useful BLAST program is the WU-BLAST-2 program, which was derived from Altschul et al. (1996) Methods in Enzymology 266:460-80. WU-BLAST-2 uses several search parameters, most of which are set to default values. Adjustable parameters are set using the following values: overlap width = 1, overlap ratio = 0.125, and word threshold (T) = 11. The HSP S and HSP S2 parameters are dynamic values ​​that are established by the program itself depending on the composition of the particular sequence and the composition of the particular database in which the sequence of interest is being searched; however, these values ​​may be adjusted to increase sensitivity.

[0180] Another useful algorithm is Gapped BLAST as reported by Altschul et al. (1997) Nucl Acid Res. 25:3389-402. Gapped BLAST uses a BLOSUM-62 substitution score; a threshold T parameter set to 9; ungapped extension initiation by the 2-hit method, imposing a cost of 10+k for gap lengths of k; Xu set to 16, and Xg set to 40 for the database search step and 67 for the output step of the algorithm. Gapped alignments are initiated by scores corresponding to approximately 22 bits.

[0181] Generally, the amino acid homology, similarity, or identity between the proteins disclosed herein and variants thereof, for example, variants of target antigens (such as HER2, CD138, or EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1) and variants of antibody variable domains (including individual variant CDRs) is at least 80% to the sequences set forth herein, for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, nearly 100%, or 100% homology or identity.

[0182] Similarly, "percent (%) nucleic acid sequence identity" with respect to nucleic acid sequences of antibodies and other proteins identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with nucleotide residues in the coding sequence of the antigen-binding protein. An exemplary method utilizes the BLASTN module of WU-BLAST-2 set to default parameters, with overlap width and overlap rate set to 1 and 0.125, respectively.

[0183] Although the site or region where the amino acid sequence variation is introduced is predetermined, the mutation itself need not be predetermined. For example, to optimize the performance of a mutation at a given site, random mutagenesis may be performed at the target codon or region, and the expressed antigen-binding protein CDR variants may be screened for the optimal combination of desired activity. Techniques for creating substitution mutations at predetermined sites in DNA with a known sequence are well known, such as MI3 primer mutagenesis and PCR mutagenesis.

[0184] "Alkyl" or "alkyl group," as used herein, means a straight, branched, or cyclic hydrocarbon chain that is completely saturated. In certain embodiments, an alkyl group can contain 1 to 8 carbon atoms ("C1-C8 alkyl"). In certain embodiments, an alkyl group can contain 1 to 6 carbon atoms ("C1-C6 alkyl"). In certain embodiments, an alkyl group contains 1 to 3 carbon atoms. In still other embodiments, an alkyl group contains 2 to 3 carbon atoms, and in yet other embodiments, an alkyl group contains 1 to 2 carbon atoms.

[0185] "Alkylalkoxy," as used herein, refers to an alkyl group substituted with an alkoxy group.

[0186] "Alkoxy," as used herein, refers to an alkyl group, as defined above, attached to the main carbon chain through an oxygen ("alkoxy") atom.

[0187] "Alkylhydroxy," as used herein, refers to an alkyl group substituted with a hydroxyl group.

[0188] "Hydroxy" or "hydroxyl" as used herein refers to --OH.

[0189] "Carbocycle" or "carbocyclyl," as used herein, includes both aromatic (e.g., aryl) and non-aromatic (e.g., cycloalkyl) groups. In certain embodiments, a carbocyclic group contains 3 to 10 carbon atoms (a "3- to 10-membered carbocyclic ring"). In certain embodiments, a carbocyclic group contains 3 to 8 carbon atoms (a "3- to 8-membered carbocyclic ring"). In certain embodiments, a carbocyclic group contains 3 to 6 carbon atoms (a "3- to 6-membered carbocyclic ring"). In certain embodiments, a carbocyclic group contains 3 to 5 carbon atoms (a "3- to 5-membered carbocyclic ring").

[0190] "Haloalkyl," as used herein, refers to an alkyl group substituted with one or more halogen atoms.

[0191] "Halogen" refers to any halogen radical, for example, -F, -Cl, -Br, or -I.

[0192] The terms "heterocycle," "heterocyclyl," and "heterocyclic," as used herein, mean a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle containing at least one heteroatom in the ring.

[0193] Monocyclic heterocycles are 3-, 4-, 5-, 6-, 7-, or 8-membered rings containing at least one heteroatom independently selected from O, N, and S. In some embodiments, heterocycles are 3- or 4-membered rings containing one heteroatom selected from O, N, and S. In some embodiments, heterocycles are 5-membered rings containing zero or one double bond and one, two, or three heteroatoms selected from O, N, and S. In some embodiments, heterocycles are 6-, 7-, or 8-membered rings containing zero, one, or two double bonds and one, two, or three heteroatoms selected from O, N, and S. Representative examples of monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, dihydropyranyl (including 3,4-dihydro-2H-pyran-6-yl), 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl , oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl (including tetrahydro-2H-pyran-4-yl), tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidethiomorpholinyl (thiomorpholinesulfone), thiopyranyl, and trithianyl.

[0194] Bicyclic heterocycles of the present disclosure can include monocyclic heterocycles fused to an aryl group having a total of 5 to 12 ring atoms, or monocyclic heterocycles fused to a monocyclic cycloalkyl, or monocyclic heterocycles fused to a monocyclic cycloalkenyl, or monocyclic heterocycles fused to a monocyclic heterocycle. Examples of bicyclic heterocycles include, but are not limited to, 3,4-dihydro-2H-pyranyl, 1,3-benzodioxolyl, 1,3-benzodithiolyl, 2,3-dihydro-1,4-benzodioxinyl, 2,3-dihydro-1-benzofuranyl, 2,3-dihydro-1-benzothienyl, 2,3-dihydro-1H-indolyl, and 1,2,3,4-tetrahydroquinolinyl.

[0195] The terms "heterocycle," "heterocyclyl," and "heterocyclic" encompass heteroaryl. "Heteroaryl" refers to a cyclic moiety having one or more closed rings, at least one of which contains one or more heteroatoms (oxygen, nitrogen, or sulfur), wherein at least one of the rings is aromatic, and one or more rings may be independently fused and / or bridged. Examples include, without limitation, phenyl, thiophenyl, triazolyl, pyridinyl, pyrimidinyl, pyridazinyl, and pyrazinyl.

[0196] As described herein, compounds of the present disclosure may contain "optionally substituted" moieties. In general, the term "substituted," whether preceded by the term "optionally," means that one or more hydrogens of the specified moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents may be either the same or different at each position. Combinations of substituents envisioned by the present disclosure are preferably those that result in the formation of stable or chemically feasible compounds.

[0197] Those of ordinary skill in the art will understand that "substituted" or "substituted with" or "absence" includes the implicit proviso that such substitution or absence is subject to the allowed valences of the substituted atom and substituents, and that the substitution or absence results in a stable compound, e.g., a compound that does not spontaneously undergo transformation, such as by rearrangement, cyclization, elimination, etc. For purposes of this disclosure, heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valences of the heteroatoms.

[0198] "Stable" refers to a compound that does not substantially change chemically and / or physically when subjected to conditions that allow for its production, detection, and, in certain embodiments, its recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, a stable compound or chemically feasible compound is one that does not substantially change when placed at a temperature of 40° C. or less in the absence of moisture or other chemically reactive conditions for at least one week. In some embodiments, the compounds disclosed herein are stable.

[0199] Enantiomers as taught herein may include "enantiopure" isomers that contain substantially a single enantiomer at a particular asymmetric center or centers, for example, greater than 90%, 92%, 95%, 98%, or 99%, or even 100%, of a single enantiomer. An "asymmetric center" or "chiral center" refers to a tetrahedral carbon atom containing four different substituents.

[0200] The compounds described herein may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may contain, for example, deuterium ( 2 H), tritium ( 3 H), carbon-13 ( 13 C), or carbon-14 ( 14The compounds may be radiolabeled with radioactive isotopes such as C). All isotopic variations of the compounds disclosed herein, whether radioactive or not, are intended to be encompassed within the scope of the disclosure. Additionally, all tautomeric forms of the compounds described herein are intended to be within the scope of the claimed disclosure.

[0201] Antibody-drug conjugates The antibody-drug conjugate (ADC) compounds of the present disclosure include those with anti-cancer activity. Specifically, the ADC compounds include antibodies or antigen-binding fragments (including antigen-binding fragments thereof) conjugated (i.e., covalently linked by a linker) to herboxidienes splicing modulators, for example, where the herboxidienes splicing modulators have cytotoxic or cytostatic effects when not conjugated to an antibody or antigen-binding fragment. In various embodiments, the herboxidienes splicing modulators have the ability to bind to and / or interact with the SF3b spliceosome complex when not conjugated to an antibody or antigen-binding fragment. In various embodiments, the herboxidienes splicing modulators have the ability to modulate in vitro and / or in vivo RNA splicing when not conjugated to an antibody or antigen-binding fragment. By targeting RNA splicing, in various embodiments, the herboxidienes splicing modulators and ADCs disclosed herein are potent anti-proliferative agents. In various embodiments, the herboxidienes splicing modulators and ADCs disclosed herein can target both actively dividing and quiescent cells.

[0202] In various embodiments, the present disclosure is based, at least in part, on the discovery that certain biologically active herboxidian splicing modulators can confer improved properties when used in ADCs. While herboxidian splicing modulators may exhibit desirable and improved characteristics (e.g., robust SF3b spliceosome complex binding, potent RNA splicing modulation) when used alone, in various embodiments, herboxidian splicing modulators may exhibit reduced desirable and improved characteristics when conjugated to an antibody or antigen-binding fragment. Thus, the development and production of ADCs for use as human therapeutics, e.g., oncology agents, may require more than identifying an antibody that binds to a desired target or targets and has the ability to link to a drug used alone to treat cancer. Linking an antibody to a herboxidian splicing modulator can have a significant effect on the activity of one or both of the antibody and the herboxidian splicing modulator, which effect will vary depending on the type of linker and / or herboxidian splicing modulator selected.Thus, in some embodiments, the components of the ADC (i) retain one or more therapeutic properties exhibited by the antibody and herboxidian splicing modulator moieties alone; (ii) maintain the specific binding properties of the antibody or antigen-binding fragment; (iii) optimize the herboxidian splicing modulator loading and herboxidian splicing modulator to antibody ratio; (iv) enable delivery of the herboxidian splicing modulator moiety by stable association with the antibody or antigen-binding fragment, e.g., intracellular delivery; and (v) maintain the stability of the ADC as an intact conjugate until transported or delivered to the target site. (vi) minimize aggregation of the ADC before or after administration; (vii) achieve the therapeutic effect, e.g., cytotoxic effect, of the herboxidiene splicing modulator moiety after cleavage in the cellular environment or other release mechanism; (viii) exhibit in vivo anti-cancer therapeutic efficacy that is comparable to or superior to that of the antibody and herboxidiene splicing modulator moiety alone; (ix) minimize off-target killing by the herboxidiene splicing modulator moiety; and / or (x) be selected to exhibit desirable pharmacokinetic and pharmacodynamic properties, formulation potential, and toxicological / immunological profile. Each of these properties may be required for the identification of improved ADCs for therapeutic use (Ab et al. (2015) Mol Cancer Ther. 14:1605-13).

[0203] In various embodiments, the ADCs disclosed herein unexpectedly exhibit advantageous properties in some or each of the above-listed categories. For example, in some embodiments, the ADC constructs disclosed herein unexpectedly exhibit advantageous herboxidian splicing modulator loading, aggregation, and / or stability profiles, and / or maintain antibody binding function, drug activity, and / or improved bystander killing while reducing off-target killing, when compared to ADCs comprising alternative linkers and / or drug moieties (e.g., alternative herboxidian splicing modulators). In some embodiments, the ADC constructs disclosed herein demonstrate superior stability, activity, potency, or other efficacy (measured in vivo or in vitro) when compared to ADCs using alternative linkers and / or herboxidian splicing modulator moieties. In some embodiments, the ADC constructs disclosed herein exhibit in vivo therapeutic efficacy when administered as a single dose. In some embodiments, the ADC constructs disclosed herein are unexpectedly stable when compared to ADCs that use alternative linkers and / or herboxidiene splicing modulator moieties.

[0204] The disclosed ADC compounds can selectively deliver effective doses of cytotoxic or cytostatic agents to cancer cells or tumor tissues. The disclosed ADCs have been found to have potent cytotoxic and / or cytostatic activity against cells expressing their respective target antigens (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1). In some embodiments, the cytotoxic and / or cytostatic activity of the ADC depends on the target antigen expression of the cells. In some embodiments, the disclosed ADCs are particularly effective at killing cancer cells that express the target antigen while minimizing off-target killing. In some embodiments, the disclosed ADCs do not exhibit cytotoxic and / or cytostatic effects against cancer cells that do not express the target antigen.

[0205] Exemplary cancers that express HER2 include, but are not limited to, breast cancer, gastric cancer, bladder cancer, urothelial cell carcinoma, esophageal cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., serous endometrial carcinoma), salivary duct cancer, cervical cancer, endometrial cancer, and ovarian cancer (English et al. (2013) Mol Diagn Ther. 17:85-99).

[0206] Exemplary cancers that express CD138 include, but are not limited to, intrathoracic cancers (e.g., lung cancer, mesothelioma), skin cancers (e.g., basal cell carcinoma, squamous cell carcinoma), head and neck cancers (e.g., larynx, hypopharynx, nasopharynx), breast cancer, genitourinary cancers (e.g., cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, urothelial carcinoma), hematological malignancies (e.g., myeloma such as multiple myeloma, B-cell malignancies, Hodgkin's lymphoma), and thyroid cancer (Szatmari et al. (2015) Dis Markers 2015:796052).

[0207] Exemplary cancers that express EPHA2 include breast cancer, brain cancer, ovarian cancer, bladder cancer, pancreatic cancer, esophageal cancer, lung cancer, prostate cancer, melanoma, esophageal cancer, and gastric cancer (Tandon et al. (2011) Expert Opin Ther Targets 15(1):31-51).

[0208] In some embodiments, cleavage of the ADC releases the herboxidien splicing modulator from the antibody or antigen-binding fragment and linker. In some embodiments, the linker and / or herboxidien splicing modulator are designed to promote bystander killing (killing of neighboring cells). In some embodiments, the linker and / or herboxidien splicing modulator are designed to promote bystander killing through cleavage after cellular internalization and diffusion of the linker-herboxidien splicing modulator moiety and / or the herboxidien splicing modulator moiety alone to neighboring cells. In some embodiments, the linker enhances cellular internalization. In some embodiments, the linker is designed to minimize cleavage in the extracellular environment, thereby reducing toxicity to off-target tissues (e.g., non-cancerous tissues), while maintaining ADC binding to target tissues and bystander killing of cancerous tissues surrounding the target cancerous tissue that do not express the antigen targeted by the antibody or antigen-binding fragment of the ADC, but do express the antigen. In some embodiments, the herboxidian splicing modulator moiety, or the catabolic product of the herboxidian splicing modulator moiety produced by cleavage of the ADC, is designed to facilitate uptake by target cells or neighboring cells (i.e., cell permeability). Such herboxidian splicing modulator moieties and catabolic products may be referred to herein as "bystander active," while drug moieties or catabolic products with reduced cell permeability may be referred to as "bystander inactive."

[0209] In some embodiments, the disclosed ADCs also demonstrate bystander killing activity but with reduced off-target cytotoxicity. Without being bound by theory, the bystander killing activity of an ADC may be particularly beneficial when its penetration into solid tumors is limited and / or when target antigen expression among tumor cells is heterogeneous. In some embodiments, ADCs comprising cleavable linkers are particularly effective at bystander killing and / or demonstrate improved bystander killing activity compared to comparable treatment with ADCs comprising non-cleavable linkers. In some embodiments, the ADCs disclosed herein exhibit improved solubility and target cell permeability compared to the drug moiety itself. In some embodiments, the ADCs disclosed herein exhibit improved cytotoxicity compared to the herboxidian splicing modulator moiety itself. In some embodiments, the ADCs disclosed herein use drug moieties that exhibit low cytotoxicity when evaluated as herboxidiene splicing modulators alone, but unexpectedly outperform ADCs containing other herboxidiene splicing modulator moieties that have high cytotoxicity when evaluated as herboxidiene splicing modulators alone. In some embodiments, cleavage and release of the herboxidiene splicing modulator improves the cytotoxicity of the ADC compared to comparable treatment with an ADC containing a non-cleavable linker. In other embodiments, cleavage and release of the herboxidiene splicing modulator is not required for the ADC to possess the desired biological activity. In some embodiments, ADCs containing non-cleavable linkers with increased spacer length (e.g., ADL12) result in the same or comparable cytotoxicity compared to comparable treatment with ADCs containing cleavable linkers (e.g., ADL1, ADL5), and unexpectedly result in superior cytotoxicity compared to comparable treatment with ADCs containing shorter non-cleavable linkers.In some embodiments, ADCs comprising non-cleavable linkers of increased spacer length that do not contain a carbonyl group (e.g., ADL12) result in the same or comparable cytotoxicity compared to equivalent treatment with ADCs comprising cleavable linkers (e.g., ADL1, ADL5), and unexpectedly, result in superior cytotoxicity compared to equivalent treatment with ADCs comprising non-cleavable linkers of the same or comparable spacer length that contain a carbonyl group (e.g., ADL10). In some embodiments, removal of the carbonyl group from a non-cleavable MC linker (e.g., ADL12) can result in a greater than 50-fold, greater than 75-fold, greater than 100-fold, greater than 150-fold, or greater than 200-fold increase in cytotoxicity compared to equivalent treatment with an ADC comprising an unmodified non-cleavable MC linker (e.g., ADL10). In some embodiments, removing the carbonyl group from a non-cleavable MC linker (e.g., ADL12) and increasing the spacer length (e.g., adding at least one spacer unit) can result in an increase in cytotoxicity of greater than 50-fold, greater than 75-fold, greater than 100-fold, greater than 150-fold, or greater than 200-fold relative to comparable treatment with an ADC comprising an unmodified non-cleavable MC linker (e.g., ADL10).

[0210] Provided herein are ADC compounds comprising an antibody or antigen-binding fragment thereof (Ab) that targets tumor cells, a splicing modulator drug moiety (D), and a linker moiety (L) that covalently links the Ab to D. In certain aspects, the antibody or antigen-binding fragment is capable of binding to a tumor-associated antigen (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1) with high specificity and high affinity. In certain embodiments, the antibody or antigen-binding fragment is internalized within the target cell upon binding, e.g., into a degradable compartment within the cell. In various embodiments, an ADC may be used that internalizes upon binding to a target cell, undergoes degradation, and releases the herboxidian splicing modulator moiety to kill the cancer cell. The herboxidienes splicing modulator moiety may be released from the antibody and / or linker moiety of the ADC by enzymatic action, hydrolysis, oxidation, or any other mechanism.

[0211] An exemplary ADC has formula (I): Ab-(LH) p (I) where Ab = antibody or antigen-binding fragment, L = linker moiety, H = herboxidian splicing modulator drug moiety, and p = number of splicing modulator drug moieties per each antibody or antigen-binding fragment.

[0212] In certain embodiments, the herboxidien splicing modulator targeting moiety for use in the described ADCs and compositions is an antibody or antigen-binding fragment. Other exemplary drug targeting moieties for use in the described ADCs and compositions are also provided and described herein. In some embodiments, the herboxidien splicing modulator targeting moiety can be any one of a variety of cell-binding agents and non-antibody scaffolds. In some embodiments, the herboxidien splicing modulator targeting moiety is a cell-binding agent. As used herein, the term "cell-binding agent" refers to any agent capable of binding to an animal (e.g., human) cell and delivering a herboxidien splicing modulator moiety (e.g., a herboxidien splicing modulator drug moiety as disclosed herein). This term encompasses the exemplary antibodies and antigen-binding fragments (e.g., monoclonal antibodies and fragments thereof, such as Fab and scFV) disclosed herein. The term further encompasses exemplary cell-binding agents, such as DARPins, duobodies, bicyclic peptides, nanobodies, centilins, MSH (melanocyte-stimulating hormone), receptor-Fc fusion molecules, T cell receptor structures, steroid hormones such as androgens and estrogens, growth factors, colony-stimulating factors such as EGF, and other non-antibody scaffolds. In various embodiments, non-antibody scaffolds can be broadly divided into two structural classes: domain-sized compounds (about 6-20 kDa) and constrained peptides (about 2-4 kDa). Exemplary domain-sized scaffolds include, but are not limited to, affibodies, affilins, anticalins, atrimers, DARPins, FN3 scaffolds (e.g., adnectins and sentinels), fynomers, Kunitz domains, pronectins, O-bodies, and receptor-Fc fusion proteins, while exemplary constrained peptides include avimers, bicyclic peptides, and Cys knots.In some embodiments, the drug targeting moiety used in the described ADCs and compositions is selected from an affibody, affilin, an anticalin, an atrimer, a DARPin, an FN3 scaffold such as an adnectin or sentinel, a fynomer, a Kunitz domain, a pronectin, an O-body, an avimer, a bicyclic peptide, and a Cys knot. In some embodiments, the drug targeting moiety used in the described ADCs and compositions is a receptor-Fc fusion protein, e.g., a HER2-Fc chimeric fusion protein. Non-antibody scaffolds are reviewed, for example, in Vazquez-Lombardi et al. (2015) Drug Dis Today 20(10):1271-83.

[0213] antibody The antibody or antigen-binding fragment (Ab) of formula (I) includes within its scope any antibody or antigen-binding fragment that specifically binds to a target antigen on a cancer cell. The antibody or antigen-binding fragment may have a dissociation constant (K) of ≦1 mM, ≦100 nM, or ≦10 nM, or any magnitude in between, as measured, for example, by BIAcore® analysis. D ) can bind to the target antigen. In certain embodiments, K D In some embodiments, K D is 500 pM to 1 μM, 1 μM to 100 nM, or 100 mM to 10 nM.

[0214] In some embodiments, the antibody or antigen-binding fragment is a four-chain antibody (also called an immunoglobulin or full-length antibody or intact antibody), comprising two heavy chains and two light chains. In some embodiments, the antibody or antigen-binding fragment is a two-chain half body (one light chain and one heavy chain) or antigen-binding fragment of an immunoglobulin. In some embodiments, the antibody or antigen-binding fragment is an antigen-binding fragment of an immunoglobulin that retains the ability to bind to a target cancer antigen and / or provide immunoglobulin function.

[0215] In some embodiments, the antibody or antigen-binding fragment is an antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is an internalizing antibody or internalizing antigen-binding fragment thereof. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment thereof binds to a target cancer antigen expressed on the surface of a cell and, upon binding, enters the cell. In some embodiments, the herboxidiene splicing modulator drug moiety of the ADC is released from the antibody or antigen-binding fragment of the ADC after the ADC has entered and is present in a cell expressing the target cancer antigen (i.e., after the ADC has been internalized), for example, by cleavage, degradation of the antibody or antigen-binding fragment, or any other suitable release mechanism.

[0216] The amino acid sequences of exemplary antibodies of the present disclosure are shown in Tables 2-4.

[0217] [Table 1]

[0218] [Table 2]

[0219] [Table 3]

[0220] [Table 4]

[0221] [Table 5]

[0222] [Table 6]

[0223]

Table 7

[0224]

Table 8

[0225]

Table 9

[0226]

Table 10

[0227]

Table 11

[0228]

Table 12

[0229]

Table 13

[0230]

Table 14

[0231]

Table 15

[0232] Table 16

[0233] [Table 17]

[0234] [Table 18]

[0235] [Table 19]

[0236] [Table 20]

[0237] [Table 21]

[0238] [Table 22]

[0239] [Table 23]

[0240] [Table 24]

[0241] In various embodiments, the ADCs disclosed herein may comprise any set of heavy and light chain variable domains listed in the table above, or may comprise a set of six CDR sequences from a set of heavy and light chains, for example, by grafting those six CDRs onto a human donor antibody framework of choice. In various embodiments, the ADCs disclosed herein may comprise a set of six CDR sequences from a set of heavy and light chains, for example, by grafting those six CDRs onto a human donor antibody framework of choice, such that the ADC can bind to its target cancer antigen (e.g., 1×10 -8 K less than MD The ADCs may include amino acid sequences that are homologous to those listed in the table above, so long as they retain the ability to bind to the target polypeptide (e.g., at 200 .ANG.) and retain one or more functional properties of the ADCs disclosed herein (e.g., the ability to internalize, modulate RNA splicing, inhibit cell growth, etc.).

[0242] In some embodiments, the ADC further comprises human heavy and light chain constant domains or fragments thereof. For example, the ADC may comprise a human IgG heavy chain constant domain (such as an IgG1) and a human kappa or lambda light chain constant domain. In various embodiments, the antibody or antigen-binding fragment of the described ADC comprises a human immunoglobulin G subtype 1 (IgG1) heavy chain constant domain together with a human Ig kappa light chain constant domain.

[0243] In various other embodiments, the target cancer antigen of the ADC is human epidermal growth factor receptor 2 (HER2).

[0244] In various embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 1, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 2, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 3; a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 4, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 5, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 6, as defined by the Kabat numbering system.

[0245] In various embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO: 19 and the light chain variable region amino acid sequence of SEQ ID NO: 20, or a sequence at least 95% identical to the disclosed sequences. In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19 and / or a light chain variable region amino acid sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 20.

[0246] In various embodiments, the anti-HER2 antibody or antigen-binding fragment thereof is an internalizing antibody or internalizing antigen-binding fragment. In various embodiments, the anti-HER2 antibody comprises a human IgG1 heavy chain constant domain and a human Ig kappa light chain constant domain.

[0247] In various embodiments, the anti-HER2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 19, or a sequence at least 95% identical to SEQ ID NO: 19, and a light chain amino acid sequence of SEQ ID NO: 20, or a sequence at least 95% identical to SEQ ID NO: 20. In particular embodiments, the anti-HER2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 19 and a light chain amino acid sequence of SEQ ID NO: 20, or a sequence at least 95% identical to the disclosed sequences. In some embodiments, the anti-HER2 antibody has a heavy chain amino acid sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19, and a light chain amino acid sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 20. In various embodiments, the anti-HER2 antibody is trastuzumab, or an antigen-binding fragment thereof.

[0248] In various embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises the three heavy chain CDRs and three light chain CDRs of trastuzumab, or wherein the CDRs comprise no more than 1, 2, 3, 4, 5, or 6 amino acid additions, deletions, or substitutions in HCDR1 (SEQ ID NO: 1), HCDR2 (SEQ ID NO: 2), HCDR3 (SEQ ID NO: 3); LCDR1 (SEQ ID NO: 4), LCDR2 (SEQ ID NO: 5), and LCDR3 (SEQ ID NO: 6).

[0249] In various other embodiments, the target cancer antigen of the ADC is human syndecan-1 (CD138).

[0250] In various embodiments, the anti-CD138 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 7, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 8, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 9, a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 10, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 11, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 12, as defined by the Kabat numbering system.

[0251] In various embodiments, the anti-CD138 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the anti-CD138 antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO: 21 and the light chain variable region amino acid sequence of SEQ ID NO: 22, or a sequence at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD138 antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21 and / or a light chain variable region amino acid sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22.

[0252] In various embodiments, the anti-CD138 antibody or antigen-binding fragment thereof is an internalizing antibody or internalizing antigen-binding fragment. In various embodiments, the anti-CD138 antibody comprises a murine IgG2a heavy chain constant domain and a murine Ig kappa light chain constant domain. In various embodiments, the anti-CD138 antibody comprises a human IgG2a heavy chain constant domain and a human Ig kappa light chain constant domain.

[0253] In various embodiments, the anti-CD138 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 21, or a sequence at least 95% identical to SEQ ID NO: 21, and a light chain amino acid sequence of SEQ ID NO: 22, or a sequence at least 95% identical to SEQ ID NO: 22. In particular embodiments, the anti-CD138 antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 21 and a light chain amino acid sequence of SEQ ID NO: 22, or a sequence at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD138 antibody has a heavy chain amino acid sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21 and a light chain amino acid sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22. In various embodiments, the anti-CD138 antibody is B-B4, or an antigen-binding fragment thereof.

[0254] In various embodiments, the anti-CD138 antibody or antigen-binding fragment thereof comprises the three heavy chain CDRs and three light chain CDRs of B-B4, or wherein the CDRs comprise no more than 1, 2, 3, 4, 5, or 6 amino acid additions, deletions, or substitutions in HCDR1 (SEQ ID NO: 7), HCDR2 (SEQ ID NO: 8), HCDR3 (SEQ ID NO: 9); LCDR1 (SEQ ID NO: 10), LCDR2 (SEQ ID NO: 11), and LCDR3 (SEQ ID NO: 12).

[0255] In various other embodiments, the target cancer antibody of the ADC is human ephrin type-A receptor 2 (EPHA2).

[0256] In various embodiments, the anti-EPHA2 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: a heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 13, a heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 14, a heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 15, a light chain CDR1 (LCDR1) consisting of SEQ ID NO: 16, a light chain CDR2 (LCDR2) consisting of SEQ ID NO: 17, and a light chain CDR3 (LCDR3) consisting of SEQ ID NO: 18, as defined by the Kabat numbering system.

[0257] In various embodiments, the anti-EPHA2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO: 23 and the light chain variable region amino acid sequence of SEQ ID NO: 24, or a sequence at least 95% identical to a disclosed sequence. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment thereof has a heavy chain variable region amino acid sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 23 and / or a light chain variable region amino acid sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 24.

[0258] In various embodiments, the anti-EPHA2 antibody or antigen-binding fragment thereof is an internalizing antibody or internalizing antigen-binding fragment. In various embodiments, the anti-EPHA2 antibody comprises a human IgG1 heavy chain constant domain and a human Ig kappa light chain constant domain.

[0259] In various embodiments, the anti-EPHA2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO:23, or a sequence at least 95% identical to SEQ ID NO:23, and a light chain amino acid sequence of SEQ ID NO:24, or a sequence at least 95% identical to SEQ ID NO:24. In particular embodiments, the anti-EPHA2 antibody comprises a heavy chain amino acid sequence of SEQ ID NO:23 and a light chain amino acid sequence of SEQ ID NO:24, or a sequence at least 95% identical to the disclosed sequences. In some embodiments, the anti-EPHA2 antibody has a heavy chain amino acid sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:23 and a light chain amino acid sequence at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:24. In some embodiments, the anti-EPHA2 antibody comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO:23; and a light chain encoded by the nucleotide sequence of SEQ ID NO:24. In various embodiments, the anti-EPHA2 antibody is 1C1, or an antigen-binding fragment thereof.

[0260] In various embodiments, the anti-EPHA2 antibody or antigen-binding fragment thereof comprises the three heavy chain CDRs and three light chain CDRs of 1C1, or wherein the CDRs comprise no more than 1, 2, 3, 4, 5, or 6 amino acid additions, deletions, or substitutions in HCDR1 (SEQ ID NO: 13), HCDR2 (SEQ ID NO: 14), HCDR3 (SEQ ID NO: 15); LCDR1 (SEQ ID NO: 16), LCDR2 (SEQ ID NO: 17), and LCDR3 (SEQ ID NO: 18).

[0261] In various embodiments, amino acid substitutions are of single residues. Insertions can typically be on the order of about 1 to about 20 amino acid residues, although significantly larger insertions can be tolerated as long as biological function (e.g., binding to a target antigen) is maintained. Deletions typically range from about 1 to about 20 amino acid residues, although in some cases deletions can be much larger. Substitutions, deletions, insertions, or any combination thereof can be used to arrive at the final derivative or variant. Generally, these changes are made to a small number of amino acids to minimize changes to the molecule, particularly the immunogenicity and specificity of the antigen-binding protein. However, larger changes can be tolerated in certain circumstances. Conservative substitutions are generally made according to the following chart, shown as Table 6.

[0262] [Table 25]

[0263] Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those shown in Table 6. For example, substitutions may be made that more significantly affect the structure of the polypeptide backbone, e.g., α-helical or β-sheet structure, in the altered region; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain. In general, the substitutions that are likely to produce the greatest changes in the properties of a polypeptide are those in which a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) cysteine ​​or proline is substituted for (or by) any other residue; (c) a residue with an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue with a bulky side chain, e.g., phenylalanine, is substituted for (or by) one without a side chain, e.g., glycine.

[0264] In various embodiments in which variant antibody sequences are used in ADCs, the variants will typically exhibit the same qualitative biological activity and elicit the same immune response, although variants may also be selected to modify the characteristics of the antigen-binding protein as needed. Alternatively, variants may be designed to alter the biological activity of the antigen-binding protein. For example, glycosylation sites may be altered or removed.

[0265] Various antibodies may be used with the ADCs used herein to target cancer cells. As shown below, the linker-payloads of the ADCs disclosed herein are surprisingly effective with various tumor antigen-targeting antibodies. Suitable antigens expressed in tumor cells but not in healthy cells, or highly expressed in tumor cells compared to healthy cells, are known in the art, as are antibodies directed thereto. These antibodies may be used with the linkers and herboxidian splicing modulator payloads disclosed herein. In some embodiments, the antibody or antigen-binding fragment targets HER2, and the HER2-targeting antibody or antigen-binding fragment is trastuzumab. In some embodiments, the antibody or antigen-binding fragment targets CD138, and the CD138-targeting antibody or antigen-binding fragment is B-B4. In some embodiments, the antibody or antigen-binding fragment targets EPHA2, and the EPHA2-targeting antibody or antigen-binding fragment is 1C1. In some embodiments, the disclosed linkers and herboxidian splicing modulator payloads are surprisingly effective with several different tumor-targeting antibodies, with HER2-targeting antibodies such as trastuzumab, CD138-targeting antibodies such as B-B4, and EPHA2-targeting antibodies such as 1C1 providing particularly improved drug:antibody ratios, aggregation levels, stability (i.e., in vitro and in vivo stability), tumor targeting (i.e., cytotoxicity, efficacy), and / or therapeutic efficacy. Improved therapeutic efficacy can be measured in vitro or in vivo and may include a reduction in tumor growth rate and / or a reduction in tumor volume.

[0266] In certain embodiments, another antibody against the same target or an antibody against a different antigen target is used, resulting in at least some of the advantageous functional properties described above (e.g., improved stability, improved tumor targeting, improved therapeutic efficacy, etc.). In some embodiments, some or all of these advantageous functional properties are observed when the disclosed linkers and herboxidian splicing modulator payloads are conjugated to another HER2-targeting, CD138-targeting, or EPHA2-targeting antibody or antigen-binding fragment. In some other embodiments, some or all of these advantageous functional properties are observed when the disclosed linkers and herboxidian splicing modulator payloads are conjugated to a HER2-targeting antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment targets HER2. In some embodiments, the HER2-targeting antibody or antigen-binding fragment is trastuzumab. In some other embodiments, some or all of these advantageous functional properties are observed when the disclosed linkers and herboxidian splicing modulator payloads are conjugated to a CD138-targeting antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment targets CD138. In some embodiments, the CD138-targeting antibody or antigen-binding fragment is B-B4. In some other embodiments, some or all of these advantageous functional properties are observed when the disclosed linkers and herboxidiene splicing modulator payloads are conjugated to an EPHA2-targeting antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment targets EPHA2. In some embodiments, the EPHA2-targeting antibody or antigen-binding fragment is 1C1.

[0267] Linker In various embodiments, the linker of an ADC is sufficiently stable extracellularly to be therapeutically effective. In some embodiments, the linker is stable outside of a cell, such that the ADC remains intact when present in extracellular conditions (e.g., before transport or delivery to a cell). The term "intact," as used in the context of an ADC, means that the antibody or antigen-binding fragment remains attached to the drug moiety (e.g., a herboxidian splicing modulator). As used herein, "stable," in the context of a linker or an ADC that includes a linker, means that no more than about 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 3%, or no more than about 1% (or any percentage therebetween) of the linkers present in a sample of the ADC are cleaved (or otherwise not intact, in the case of the entire ADC) when the ADC is present under extracellular conditions. In some embodiments, the linkers and / or ADCs disclosed herein are surprisingly stable compared to ADCs with alternative linkers and / or alternative linkers and / or herboxidiene splicing modulator payloads. In some embodiments, the ADCs disclosed herein can remain intact for more than about 48 hours, more than 60 hours, more than about 72 hours, more than about 84 hours, or more than about 96 hours.

[0268] Whether a linker is stable extracellularly can be determined, for example, by placing the ADC in plasma for a predetermined period of time (e.g., 2, 4, 6, 8, 16, 24, 48, or 72 hours) and then quantifying the amount of free drug moiety present in the plasma. Stability may allow sufficient time for the ADC to localize to target tumor cells and prevent premature release of the drug moiety, which could reduce the therapeutic index of the ADC by indiscriminately damaging both normal and tumor tissue. In some embodiments, the linker is stable outside the target cell and, once inside the cell, releases the drug moiety from the ADC, allowing the drug to bind to its target (e.g., to the SF3b spliceosome complex). Thus, an effective linker will (i) maintain the specific binding properties of the antibody or antigen-binding fragment; (ii) enable delivery of the drug moiety, e.g., intracellular delivery, through stable association with the antibody or antigen-binding fragment; (iii) remain stable and intact until the ADC has been transported or delivered to its target site; and (iv) achieve the therapeutic effect, e.g., cytotoxic effect, of the drug moiety after cleavage or another release mechanism.

[0269] Linkers can affect the physicochemical properties of ADCs. Many cytotoxic agents are inherently hydrophobic, and linking them to antibodies with additional hydrophobic moieties can lead to aggregation. ADC aggregates are insoluble and often limit the drug loading achievable on the antibody, which can adversely affect ADC efficacy. Protein aggregates in biologics are also commonly associated with increased immunogenicity. As shown below, the linkers disclosed herein result in ADCs with low levels of aggregation and desirable drug loading levels.

[0270] Linkers may be "cleavable" or "non-cleavable" (Ducry and Stump (2010) Bioconjugate Chem. 21:5-13). Cleavable linkers are designed to release the drug moiety (e.g., a herboxidien splicing modulator) when subjected to certain environmental factors, such as when internalized within a target cell, whereas non-cleavable linkers generally rely on degradation of the antibody or antigen-binding fragment itself.

[0271] In some embodiments, the linker is a non-cleavable linker. In some embodiments, the herboxidiene splicing modulator drug moiety of the ADC is released by degradation of the antibody or antigen-binding fragment. Non-cleavable linkers tend to remain covalently associated with at least one amino acid of the antibody and drug, even after internalization by and degradation within the target cell. Numerous exemplary non-cleavable linkers are described herein, and others are known in the art. Exemplary non-cleavable linkers may include thioether, cyclohexyl, N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), or N-hydroxysuccinimide (NHS), one or more polyethylene glycol (PEG) moieties, e.g., 1, 2, 3, 4, 5, or 6 PEG moieties, or one or more alkyl moieties.

[0272] In some embodiments, the linker is a cleavable linker. A cleavable linker refers to any linker that contains a cleavable moiety. As used herein, the term "cleavable moiety" refers to any chemical bond that can be cleaved. Suitable cleavable chemical bonds are well known in the art and include, but are not limited to, acid-labile bonds, protease / peptidase-labile bonds, photolabile bonds, disulfide bonds, and esterase-labile bonds. A linker containing a cleavable moiety can allow for the release of the herboxidiene splicing modulator drug moiety from the ADC by cleavage at a specific site in the linker.

[0273] In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker is sufficient in the intracellular environment to release the herboxidienes splicing modulator drug moiety from the antibody or antigen-binding fragment, activating the drug and / or rendering the drug therapeutically effective. In some embodiments, the herboxidienes splicing modulator drug moiety is not cleaved from the antibody or antigen-binding fragment until the ADC enters a cell expressing an antigen specific for the antibody or antigen-binding fragment of the ADC, upon which the herboxidienes splicing modulator drug moiety is cleaved from the antibody or antigen-binding fragment. In some embodiments, the linker comprises a cleavable moiety positioned such that upon cleavage, no portion of the linker or antibody or antigen-binding fragment remains attached to the herboxidienes splicing modulator drug moiety. Exemplary cleavable linkers include acid-labile linkers, protease / peptidase-sensitive linkers, photolabile linkers, dimethyl-containing, disulfide-containing, or sulfonamide-containing linkers.

[0274] In some embodiments, the linker is a pH-sensitive linker, which is sensitive to hydrolysis at a specific pH value. Typically, pH-sensitive linkers are cleavable under acidic conditions. This cleavage strategy generally takes advantage of the lower pH of intracellular compartments, such as endosomes (pH approximately 5-6) and lysosomes (pH approximately 4.8), compared to the cytosol (pH approximately 7.4), to induce hydrolysis of an acid-labile group in the linker, such as a hydrazone (Jain et al. (2015) Pharm Res 32:3526-40). In some embodiments, the linker is an acid-labile and / or hydrolyzable linker. For example, an acid-labile linker that is hydrolyzable in the lysosome and contains an acid-labile group (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic acid amide, orthoester, acetal, ketal, etc.) can be used. See, e.g., U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker (1999) Pharm Therapeutics 83:67-123; Neville et al. (1989) Biol Chem. 264:14653-61. Such linkers are relatively stable under neutral pH conditions, such as those found in blood, but are unstable below pH 5.5 or 5.0, which approximates the pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to a therapeutic agent via an acylhydrazone bond) (see, e.g., U.S. Patent No. 5,622,929).

[0275] In some embodiments, the linker is cleavable under reducing conditions. In some embodiments, the linker is cleavable in the presence of a reducing agent such as glutathione or dithiothreitol. In some embodiments, the linker is a cleavable disulfide linker or a cleavable sulfonamide linker.

[0276] In some embodiments, the linker is a cleavable disulfide linker. A variety of disulfide linkers are known in the art, including, for example, SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene), including those that can be formed using SPDB and SMPT. See, e.g., Thorpe et al. (1987) Cancer Res. 47:5924-31; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel ed., Oxford U. Press, 1987). See also U.S. Pat. No. 4,880,935. Disulfide linkers are typically used to take advantage of the abundance of intracellular thiols that can facilitate cleavage of the disulfide bond. The intracellular concentration of the most abundant intracellular thiol, reduced glutathione, is generally in the 1-10 nM range, approximately 1,000-fold higher than the most abundant small thiol (i.e., cysteine) in blood, which is approximately 5 μM (Goldmacher et al., In Cancer Drug Discovery and Development: Antibody-Drug Conjugates and Immunotoxins (GLP Hillips ed., Springer, 2013)). Intracellular enzymes of the protein disulfide isomerase family can also contribute to the intracellular cleavage of disulfide linkers. As used herein, a cleavable disulfide linker refers to any linker that contains a cleavable disulfide moiety. The term "cleavable disulfide moiety" refers to a disulfide bond that can be cleaved and / or reduced, for example, by a thiol or an enzyme.

[0277] In some embodiments, the linker is a cleavable sulfonamide linker. As used herein, a cleavable sulfonamide linker refers to any linker that includes a cleavable sulfonamide moiety. The term "cleavable sulfonamide moiety" refers to a sulfonamide group, i.e., a sulfonyl group connected to an amine group, where the sulfur-nitrogen bond can be cleaved.

[0278] In some embodiments, the linker may be a dendritic linker for covalently linking two or more drug moieties to an antibody or antigen-binding fragment via a branched, multifunctional linker moiety. See, e.g., Sun et al. (2002) Bioorg Med Chem Lett. 12:2213-5; Sun et al. (2003) Bioorg Med Chem. 11:1761-8. Dendritic linkers can increase the drug-to-antibody molar ratio, i.e., drug loading, which is related to the efficacy of the ADC. Thus, for example, if an antibody or antigen-binding fragment bears only one reactive cysteine ​​thiol group, multiple herboxidian splicing modulator drug moieties may be attached via a dendritic linker. In some embodiments, linker moieties or linker-drug moieties may be attached to the antibody or antigen-binding fragment via reduced disulfide crosslinking chemistry or lysine-specific techniques. See, for example, WO 2013 / 173391 and WO 2013 / 173393.

[0279] In some embodiments, the linker is cleavable by a cleaving agent, e.g., an enzyme, present in the intracellular environment (e.g., within a lysosome or endosome or caveolae). The linker may be, for example, a peptide linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease.

[0280] In some embodiments, the linker is a cleavable peptide linker. As used herein, a cleavable peptide linker refers to any linker comprising a cleavable peptide moiety. The term "cleavable peptide moiety" refers to any chemically linked amino acids (natural or synthetic amino acid derivatives) that can be cleaved by an agent present in the intracellular environment. For example, the linker may comprise a valine-alanine (Val-Ala) sequence or a valine-citrulline (Val-Cit) sequence that is cleavable by a peptidase such as a cathepsin, e.g., cathepsin B. In some embodiments, the linker may comprise a glutamic acid-valine-citrulline sequence (Glu-Val-Cit). In some embodiments, the linker is an enzyme-cleavable linker, where the cleavable peptide moiety in the linker is cleavable by an enzyme. In some embodiments, the cleavable peptide moiety is cleavable by a lysosomal enzyme, e.g., a cathepsin. In some embodiments, the linker is a cathepsin-cleavable linker. In some embodiments, the cleavable peptide moiety in the linker is cleavable by a lysosomal cysteine ​​cathepsin, such as cathepsin B, C, F, H, K, L, O, S, V, X, or W. In some embodiments, the cleavable peptide moiety is cleavable by cathepsin B. An exemplary dipeptide that can be cleaved by cathepsin B is valine-citrulline (Val-Cit) (Dubowchik et al. (2002) Bioconjugate Chem. 13:855-69).

[0281] In some embodiments, the linker or cleavable peptide moiety in the linker comprises an amino acid unit. In some embodiments, the amino acid unit allows for protease cleavage of the linker, thereby facilitating release of the herboxidiene splicing modulator drug moiety from the ADC upon exposure to one or more intracellular proteases, such as one or more lysosomal enzymes (Doronina et al. (2003) Nat Biotechnol. 21:778-84; Dubowchik and Walker (1999) Pharm Therapeutics 83:67-123). Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include, but are not limited to, valine-alanine (Val-Ala), valine-citrulline (Val-Cit), alanine-asparagine (Ala-Asn), alanine-phenylalanine (Ala-Phe), phenylalanine-lysine (Phe-Lys), alanine-lysine (Ala-Lys), alanine-valine (Ala-Val), valine-lysine (Val-Lys), lysine-lysine (Lys-Lys), phenylalanine-citrulline (Phe-Cit), leucine-citrulline (Leu-Cit), isoleucine-citrulline (Ile-Cit), tryptophan-citrulline (Trp-Cit), and phenylalanine-alanine (Phe-Ala). Exemplary tripeptides include, but are not limited to, alanine-alanine-asparagine (Ala-Ala-Asn), glycine-valine-citrulline (Gly-Val-Cit), glycine-glycine-glycine (Gly-Gly-Gly), phenylalanine-phenylalanine-lysine (Phe-Phe-Lys), glutamic acid-valine-citrulline (Glu-Val-Cit) (see, e.g., Anami et al. (2018) Nat Comm. 9:2512), and glycine-phenylalanine-lysine (Gly-Phe-Lys).Other exemplary amino acid units include, but are not limited to, Gly-Phe-Gly-Gly (SEQ ID NO: 34), Gly-Phe-Leu-Gly (SEQ ID NO: 35), Ala-Leu-Ala-Leu (SEQ ID NO: 36), Phe-N, as described, for example, in U.S. Pat. No. 6,214,345. 9 -Tosyl-Arg, and Phe-N 9 In some embodiments, the amino acid unit in the linker comprises Val-Ala. In some embodiments, the amino acid unit in the linker comprises Val-Cit. In some embodiments, the amino acid unit in the linker comprises Glu-Val-Cit. The amino acid unit may comprise naturally occurring amino acid residues and / or minor amino acids and / or non-naturally occurring amino acid analogs, such as citrulline. The amino acid unit may be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, lysosomal proteases, such as cathepsin B, C, D, or S, or plasmin proteases.

[0282] In some embodiments, the linker is a cleavable β-glucuronide linker. As used herein, a cleavable β-glucuronide linker refers to any linker that includes a cleavable β-glucuronide moiety. An exemplary cleavable β-glucuronide linker has the structure: [ka] Includes:

[0283] The term "cleavable β-glucuronide moiety" refers to a glycosidic bond that can be cleaved by an agent having β-glucuronidase activity. In some embodiments, the linker comprises a glycosidic bond that can be cleaved by β-glucuronidase. β-glucuronidase is a UDP-glucuronosyltransferase that catalyzes the hydrolysis of glycosidic bonds in glucuronides having a β-configuration.

[0284] In some embodiments, the ADCs disclosed herein include a cleavable β-glucuronide moiety in the linker that is cleavable by an enzyme. In some embodiments, the cleavable β-glucuronide moiety in the linker is cleavable by a lysosomal enzyme, such as β-glucuronidase. In some embodiments, the linker is a β-glucuronidase-cleavable linker. In some embodiments, the cleavable β-glucuronide moiety in the linker allows cleavage of the linker by β-glucuronidase after internalization of the ADC, thereby facilitating release of the drug moiety from the ADC in the cellular environment.

[0285] In some embodiments, the linker in any ADC disclosed herein may comprise at least one spacer unit that links the antibody or antigen-binding fragment to the drug moiety (e.g., a herboxidian splicing modulator drug moiety). In some embodiments, the spacer unit between the antibody or antigen-binding fragment and the cleavable moiety, if present, links the cleavage site (e.g., a cleavable peptide moiety) in the linker to the antibody or antigen-binding fragment. In some embodiments, the spacer unit between the drug moiety and the cleavable moiety, if present, links the cleavage site (e.g., a cleavable peptide moiety) in the linker to the drug moiety. In some embodiments, the cleavage site is not present and a spacer unit is used to link the antibody or antigen-binding fragment to the drug moiety.

[0286] In some embodiments, the linker and / or the spacer unit in the linker are substantially hydrophilic. The use of a hydrophilic linker can reduce the extent to which the drug can be extruded from resistant cancer cells by multidrug resistance (MDR) or functionally similar transporters. In some embodiments, the hydrophilic linker can include one or more polyethylene glycol (PEG) moieties, for example, 1, 2, 3, 4, 5, or 6 PEG moieties. In some embodiments, the linker includes two PEG moieties.

[0287] In some embodiments, the spacer unit in the linker comprises one or more PEG moieties. In some embodiments, the spacer unit comprises one or more -(PEG) m -, and m is an integer from 1 to 10 (i.e., m can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, m ranges from 1 to 10; 2 to 8; 2 to 6; 2 to 5; 2 to 4; or 2 to 3. In some embodiments, m is 2. In some embodiments, the spacer unit is (PEG)2, (PEG)3, (PEG)4, (PEG)5, (PEG)6, (PEG)7, (PEG)8, (PEG)9, or (PEG) 10 In some embodiments, the spacer unit comprises (PEG).

[0288] In some embodiments, the spacer unit in the linker comprises an alkyl moiety. In some embodiments, the spacer unit comprises one or more -(CH) n -, where n is an integer between 1 and 10 (i.e., n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, n ranges from 1 to 10; 2 to 8; 2 to 6; 2 to 5; 2 to 4; or 2 to 3. In some embodiments, n is 2. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, the spacer unit is (CH2)2, (CH2)3, (CH2)4, (CH2)5, (CH2)6, (CH2)7, (CH2)8, (CH2)9, or (CH2) 10 In some embodiments, the spacer unit comprises (CH2)2 ("Et"). In some embodiments, the spacer unit comprises (CH2)6 ("Hex"). In some embodiments, the spacer unit comprises (CH2)2-O-(CH2)2 ("Et-O-Et").

[0289] The spacer unit can be used, for example, to link an antibody or antigen-binding fragment to a drug moiety directly or indirectly. In some embodiments, the spacer unit directly links the antibody or antigen-binding fragment to the herboxidien splicing modulator drug moiety. In some embodiments, the antibody or antigen-binding fragment and the herboxidien splicing modulator drug moiety are linked via a spacer unit comprising one or more PEG moieties (e.g., (PEG)2) or one or more alkyl moieties (e.g., (CH2)2, (CH2)6, or (CH2)2-O-(CH2)2). In some embodiments, the spacer unit indirectly links the antibody or antigen-binding fragment to the herboxidien splicing modulator drug moiety. In some embodiments, the spacer unit indirectly links the antibody or antigen-binding fragment to the herboxidien splicing modulator drug moiety through a cleavable moiety (e.g., a cleavable peptide or a cleavable β-glucuronide) and / or a linking moiety, such as a maleimide moiety, for joining the spacer unit to the antibody or antigen-binding fragment.

[0290] The spacer unit, in various embodiments, is attached to the antibody or antigen-binding fragment (ie, antibody or antigen-binding fragment) via a maleimide (Mal) moiety.

[0291] A spacer unit that is attached to an antibody or antigen-binding fragment via a Mal is referred to herein as a "Mal-spacer unit." The term "Mal" or "maleimide moiety," as used herein, refers to a compound that contains a maleimide group and is reactive with sulfhydryl groups, such as the sulfhydryl groups of cysteine ​​residues on an antibody or antigen-binding fragment. Other functional groups reactive with sulfhydryl groups (thiols) include, but are not limited to, iodoacetamide, bromoacetamide, vinylpyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate. In some embodiments, a Mal-spacer unit is reactive with cysteine ​​residues on an antibody or antigen-binding fragment. In some embodiments, a Mal-spacer unit is attached to an antibody or antigen-binding fragment via a cysteine ​​residue. In some embodiments, a Mal-spacer unit comprises a PEG moiety. In some embodiments, a Mal-spacer unit comprises an alkyl moiety.

[0292] In certain embodiments, the linker comprises a Mal-spacer unit and a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the amino acid unit comprises Val-Cit. In some embodiments, the amino acid unit comprises Val-Ala. In some embodiments, the amino acid unit comprises Glu-Val-Cit. In some embodiments, the linker comprises a Mal-spacer unit and Val-Cit. In some embodiments, the linker comprises a Mal-spacer unit and Val-Ala. In some embodiments, the linker comprises a Mal-spacer unit and Val-Cit, wherein the Mal-spacer unit comprises maleimidocaproyl (MC). In some embodiments, the linker comprises a Mal-spacer unit and Val-Ala, wherein the Mal-spacer unit comprises maleimidocaproyl (MC). In some embodiments, the linker comprises a Mal-spacer unit and a cleavable β-glucuronide moiety.

[0293] In some embodiments, the linker comprises the structure: Mal-spacer unit. In some embodiments, the Mal-spacer unit comprises maleimidocaproyl (MC). In some embodiments, the linker comprises the structure: MC. In some embodiments, the linker comprises the structure: Mal-(CH2)2 ("Mal-Et"). In some embodiments, the linker comprises the structure: Mal-(CH2)6 ("Mal-Hex"). In some embodiments, the linker comprises the structure: Mal-(CH2)2-O-(CH2)2 ("Mal-Et-O-Et"). In some embodiments, the linker comprises the structure: Mal-(PEG)2. In some embodiments, the linker comprises the structure: Mal-(PEG)2-CO.

[0294] In various embodiments, a Mal-spacer unit links the antibody or antigen-binding fragment to the cleavable peptide moiety. In some embodiments, the linker comprises Mal-spacer unit-peptide. In some embodiments, the linker comprises the structure: Mal-spacer unit-Val-Cit. In some embodiments, the Mal-spacer unit comprises maleimidocaproyl (MC). In some embodiments, the linker comprises the structure: MC-Val-Cit.

[0295] In some embodiments, the linker comprises the structure: Mal-spacer unit-Val-Ala. In some embodiments, the Mal-spacer unit comprises maleimidocaproyl (MC). In some embodiments, the linker comprises the structure: MC-Val-Ala.

[0296] In various embodiments, a Mal-spacer unit links the antibody or antigen-binding fragment to a cleavable β-glucuronide moiety. In some embodiments, the linker comprises a Mal-spacer unit-β-glucuronide. In some embodiments, the linker comprises an MC-β-glucuronide.

[0297] In various embodiments, the cleavable portion of the linker is directly spliced ​​to the herboxidian splicing modulator drug moiety. In other embodiments, the cleavable portion of the linker is linked to the herboxidian splicing modulator drug moiety using a spacer unit. In various embodiments, the herboxidian splicing modulator is linked to the cleavable portion of the linker by a spacer unit.

[0298] A spacer unit may be "self-immolative" or "non-self-immolative." A "non-self-immolative" spacer unit is one in which some or all of the spacer unit remains attached to the herboxidiene splicing modulator drug moiety upon cleavage of the linker. Examples of non-self-immolative spacer units include, but are not limited to, a glycine spacer unit and a glycine-glycine spacer unit. A non-self-immolative spacer unit may eventually degrade over time, but does not immediately release the entire linked native drug moiety, even under cellular conditions. A "self-immolative" spacer unit allows for the release of the native drug moiety under intracellular conditions. A "native drug" or "native drug moiety" is one in which no part of the spacer unit or other chemical modification remains after cleavage / degradation of the spacer unit.

[0299] Self-immolative chemistries are known in the art and can be readily selected for the disclosed ADCs. In various embodiments, the spacer unit connecting the cleavable portion of the linker to the herboxidienes splicing modulator drug moiety is self-immolative and undergoes self-immolation simultaneously with, or immediately before or after, cleavage of the cleavable portion under intracellular conditions. In some embodiments, the herboxidienes splicing modulator is connected to the cleavable portion of the linker by a self-immolative spacer unit. In certain embodiments, the herboxidienes splicing modulator is connected to the cleavable portion of the linker by a self-immolative spacer unit, the cleavable portion comprises Val-Cit, and maleimidocaproyl (MC) joins the cleavable portion to the antibody or antigen-binding fragment. In certain embodiments, the herboxidienes splicing modulator is connected to the cleavable portion of the linker by a self-immolative spacer unit, the cleavable portion comprises Val-Ala, and maleimidocaproyl (MC) joins the cleavable portion to the antibody or antigen-binding fragment. In certain embodiments, the herboxidienes splicing modulator is linked to the cleavable portion of the linker by a self-immolative spacer unit, where the cleavable portion comprises Glu-Val-Cit, and maleimidocaproyl (MC) joins the cleavable portion to the antibody or antigen-binding fragment. In certain embodiments, the herboxidienes splicing modulator is joined to the antibody or antigen-binding fragment via a Mal-spacer unit (e.g., MC) in the linker joined to a pABC or pAB self-immolative spacer unit and a Val-Cit cleavable portion. In certain other embodiments, the herboxidienes splicing modulator is joined to the antibody or antigen-binding fragment via a Mal-spacer unit (e.g., MC) in the linker joined to a pABC or pAB self-immolative spacer unit and a Val-Ala cleavable portion. In certain other embodiments, the herboxidienes splicing modulator is conjugated to the antibody or antigen-binding fragment via a Mal-spacer unit (e.g., MC) in a linker joined to a pABC or pAB self-immolative spacer unit and a Glu-Val-Cit cleavable moiety.

[0300] In certain embodiments, the self-immolative spacer unit in the linker comprises a p-aminobenzyl unit. In some embodiments, p-aminobenzyl alcohol (pABOH) is attached to the amino acid unit or other cleavable moiety in the linker via an amide bond, creating a carbamate, methylcarbamate, or carbonate between pABOH and the drug moiety (Hamann et al. (2005) Expert Opin Ther Patents 15:1087-103). In some embodiments, the self-immolative spacer unit is or includes p-aminobenzyloxycarbonyl (pABC). Without being bound by theory, the self-immolation of pABC is believed to involve a spontaneous 1,6-elimination reaction (Jain et al. (2015) Pharm Res. 32:3526-40).

[0301] In various embodiments, the structure of p-aminobenzyloxycarbonyl (pABC) used in the disclosed ADCs is shown below: [ka]

[0302] In various embodiments, a self-immolative spacer unit links the cleavable portion of the linker to the herboxidien splicing modulator. In some embodiments, the self-immolative spacer unit is pABC. In some embodiments, pABC links the cleavable portion of the linker to the herboxidien splicing modulator. In some embodiments, pABC undergoes self-immolation upon cleavage of the cleavable portion, and the herboxidien splicing modulator is released from the ADC in its native, active form.

[0303] In some embodiments, the anti-HER2 antibody or antigen-binding fragment is tethered to the herboxidian splicing modulator by a linker comprising MC-Val-Cit-pABC, hi other embodiments, the anti-HER2 antibody or antigen-binding fragment is tethered to the herboxidian splicing modulator by a linker comprising MC-Val-Ala-pABC.

[0304] In some embodiments, the anti-CD138 antibody or antigen-binding fragment is tethered to the herboxidien splicing modulator by a linker comprising MC-Val-Cit-pABC, hi other embodiments, the anti-CD138 antibody or antigen-binding fragment is tethered to the herboxidien splicing modulator by a linker comprising MC-Val-Ala-pABC.

[0305] In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment is tethered to the herboxidien splicing modulator by a linker comprising MC-Val-Cit-pABC, hi other embodiments, the anti-EPHA2 antibody or antigen-binding fragment is tethered to the herboxidien splicing modulator by a linker comprising MC-Val-Ala-pABC.

[0306] In some embodiments, pABC undergoes self-immolation upon cleavage of the cleavable peptide moiety in the linker. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the linker comprises the amino acid unit-pABC. In some embodiments, the amino acid unit is Val-Cit. In some embodiments, the linker comprises Val-Cit-pABC. In some embodiments, the amino acid unit is Glu-Val-Cit. In some embodiments, the linker comprises Glu-Val-Cit-pABC. In some embodiments, the amino acid unit is Val-Ala. In some embodiments, the linker comprises Val-Ala-pABC. In some embodiments, the amino acid unit is Ala-Ala-Asn. In some embodiments, the linker comprises Ala-Ala-Asn-pABC.

[0307] In some embodiments, pABC undergoes self-immolation upon cleavage of the cleavable β-glucuronide moiety in the linker, hi some embodiments, the linker comprises β-glucuronide-pABC.

[0308] In certain embodiments, the self-immolative spacer unit in the linker comprises a p-aminobenzyl unit. In some embodiments, the self-immolative spacer unit in the linker comprises p-aminobenzyl (pAB). In some embodiments, the self-immolation of pAB involves a spontaneous 1,6-elimination reaction.

[0309] In various embodiments, the structure of p-aminobenzyl (pAB) used in the disclosed ADCs is shown below: [ka]

[0310] In various embodiments, a self-immolative spacer unit links the cleavable portion of the linker to the herboxidien splicing modulator. In some embodiments, the self-immolative spacer unit is pAB. In some embodiments, pAB links the cleavable portion of the linker to the herboxidien splicing modulator. In some embodiments, pAB undergoes self-immolation upon cleavage of the cleavable portion, releasing the herboxidien splicing modulator from the ADC in its native, active form.

[0311] In some embodiments, the anti-HER2 antibody or antigen-binding fragment is tethered to the herboxidian splicing modulator by a linker comprising MC-Val-Cit-pAB, hi other embodiments, the anti-HER2 antibody or antigen-binding fragment is tethered to the herboxidian splicing modulator by a linker comprising MC-Val-Ala-pAB.

[0312] In some embodiments, the anti-CD138 antibody or antigen-binding fragment is tethered to the herboxidian splicing modulator by a linker comprising MC-Val-Cit-pAB, hi other embodiments, the anti-CD138 antibody or antigen-binding fragment is tethered to the herboxidian splicing modulator by a linker comprising MC-Val-Ala-pAB.

[0313] In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment is tethered to the herboxidien splicing modulator by a linker comprising MC-Val-Cit-pAB, hi other embodiments, the anti-EPHA2 antibody or antigen-binding fragment is tethered to the herboxidien splicing modulator by a linker comprising MC-Val-Ala-pAB.

[0314] In some embodiments, pAB undergoes self-immolation upon cleavage of the cleavable peptide moiety in the linker. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the linker comprises the amino acid unit -pAB. In some embodiments, the amino acid unit is Val-Cit. In some embodiments, the linker comprises Val-Cit-pAB. In some embodiments, the amino acid unit is Val-Ala. In some embodiments, the linker comprises Val-Ala-pAB. In some embodiments, the amino acid unit is Glu-Val-Cit. In some embodiments, the linker comprises Glu-Val-Cit-pAB. In some embodiments, the amino acid unit is Ala-Ala-Asn. In some embodiments, the linker comprises Ala-Ala-Asn-pAB.

[0315] In some embodiments, pAB undergoes self-immolation upon cleavage of the cleavable β-glucuronide moiety in the linker, hi some embodiments, the linker comprises β-glucuronide-pAB.

[0316] In some other embodiments, the herboxidienes splicing modulator is linked to the cleavable portion of the linker by a non-self-immolative spacer unit. In certain embodiments, the herboxidienes splicing modulator is linked to the cleavable portion of the linker by a non-self-immolative spacer unit, the cleavable portion comprises Val-Cit, and maleimidocaproyl (MC) joins the cleavable portion to the antibody or antigen-binding fragment. In certain embodiments, the herboxidienes splicing modulator is linked to the cleavable portion of the linker by a non-self-immolative spacer unit, the cleavable portion comprises Val-Ala, and maleimidocaproyl (MC) joins the cleavable portion to the antibody or antigen-binding fragment.

[0317] In various aspects, the antibody or antigen-binding fragment of the ADC is conjugated to the herboxidian splicing modulator drug moiety via a linker, wherein the linker comprises a Mal-spacer unit (e.g., MC), a cleavable amino acid unit, and pABC. In some embodiments, the spacer unit comprises an alkyl moiety. In some embodiments, the Mal-spacer unit comprises maleimidocaproyl (MC). In some embodiments, the linker comprises Mal-spacer unit-amino acid unit-pABC. In some embodiments, the linker comprises MC-amino acid unit-pABC. In some embodiments, the linker comprises MC-Val-Cit-pABC. In some embodiments, the linker comprises MC-Val-Ala-pABC. In some embodiments, the linker comprises MC-Glu-Val-Cit-pABC. In some embodiments, the linker comprises MC-Ala-Ala-Asn-pABC.

[0318] In various other aspects, the antibody or antigen-binding fragment of the ADC is conjugated to the herboxidian splicing modulator drug moiety via a linker, wherein the linker comprises a Mal-spacer unit (e.g., MC), a cleavable amino acid unit, and pAB. In some embodiments, the spacer unit comprises an alkyl moiety. In some embodiments, the Mal-spacer unit comprises maleimidocaproyl (MC). In some embodiments, the linker comprises Mal-spacer unit-amino acid unit-pAB. In some embodiments, the linker comprises MC-amino acid unit-pAB. In some embodiments, the linker comprises MC-Val-Cit-pAB. In some embodiments, the linker comprises MC-Val-Ala-pAB. In some embodiments, the linker comprises MC-Glu-Val-Cit-pAB. In some embodiments, the linker comprises MC-Ala-Ala-Asn-pAB.

[0319] In various other aspects, the antibody or antigen-binding fragment of the ADC is conjugated to the herboxidian splicing modulator drug moiety via a linker, where the linker comprises a Mal-spacer unit (e.g., MC), a cleavable β-glucuronide, and pABC. In some embodiments, the linker comprises Mal-spacer unit-β-glucuronide-pABC. In some embodiments, the linker comprises MC-β-glucuronide-pABC.

[0320] In yet other aspects, the antibody or antigen-binding fragment of the ADC is conjugated to the herboxidian splicing modulator drug moiety via a linker, where the linker comprises a Mal-spacer unit (e.g., MC), a cleavable β-glucuronide, and pAB. In some embodiments, the linker comprises Mal-spacer unit-β-glucuronide-pAB. In some embodiments, the linker comprises MC-β-glucuronide-pAB.

[0321] In various embodiments, the ADC compound has formula (I): Ab-(LH) p (I) wherein Ab is an antibody or antigen-binding fragment that targets a neoplastic cell; H is a herboxidian splicing modulator; L is a linker covalently linking Ab to D; and and p is an integer from 1 to 15.

[0322] In some embodiments, the antibody or antigen-binding fragment (Ab) of the ADC is conjugated to the herboxidian splicing modulator drug moiety via a linker, where the linker is any of the linkers disclosed herein or incorporated by reference, or includes one or more components of any of the linkers disclosed herein or incorporated by reference.

[0323] In some embodiments, the linker comprises a cleavable moiety positioned such that after cleavage, no portion of the linker or the antibody or antigen-binding fragment remains bound to the herboxidiene splicing modulator. In some embodiments, the cleavable moiety is a cleavable peptide moiety, e.g., an amino acid unit such as Val-Cit or Val-Ala. In some embodiments, the amino acid unit or linker comprises Val-Cit. In some embodiments, the amino acid unit or linker comprises Val-Ala. In some embodiments, the amino acid unit or linker comprises Glu-Val-Cit.

[0324] In some embodiments, the linker comprises at least one spacer unit that joins the antibody or antigen-binding fragment to the cleavable moiety. In some embodiments, the linker comprises at least one spacer unit that joins the antibody or antigen-binding fragment to the drug moiety. In some embodiments, the spacer unit or linker comprises at least one alkyl moiety.

[0325] In some embodiments, the spacer unit in the linker is attached to the antibody or antigen-binding fragment via a Mal moiety ("Mal-spacer unit"). In some embodiments, the Mal-spacer unit comprises at least one alkyl moiety. In some embodiments, the linker comprises maleimidocaproyl (MC). In some embodiments, the linker comprises Mal-(CH2)2 ("Mal-Et"). In some embodiments, the linker comprises Mal-(CH2)6 ("Mal-Hex"). In some embodiments, the linker comprises Mal-(CH2)2-O-(CH2)2 ("Mal-Et-O-Et"). In some embodiments, the linker comprises Mal-(PEG)2-CO. In some embodiments, the Mal-spacer unit attaches the antibody or antigen-binding fragment to the drug moiety.

[0326] In some embodiments, the Mal-spacer unit or linker comprises Mal-(PEG)2, Mal-(PEG)3, Mal-(PEG)4, Mal-(PEG)5, Mal-(PEG)6, Mal-(PEG)7, or Mal-(PEG)8. In some embodiments, the Mal-spacer unit or linker comprises Mal-(PEG)2. In some embodiments, the Mal-spacer unit or linker comprises Mal-(PEG)2-CO, Mal-(PEG)3-CO, Mal-(PEG)4-CO, Mal-(PEG)5-CO, Mal-(PEG)6-CO, Mal-(PEG)7-CO, or Mal-(PEG)8-CO. In some embodiments, the Mal-spacer unit or linker comprises Mal-(PEG)2-CO. In some embodiments, the Mal-spacer unit or linker comprises Mal-(PEG)2-CO and at least one additional spacer unit. In some embodiments, Mal-(PEG)2-CO links the antibody or antigen-binding fragment to the drug moiety. In some embodiments, the linker comprises or consists of Mal-(PEG)2-CO. An example of a "Mal-(PEG)2-CO" linker is also referred to herein as an "ADL2" or "ADL2" linker.

[0327] In some embodiments, the Mal-spacer unit or linker comprises MC. In some embodiments, the Mal-spacer unit or linker comprises MC and at least one additional spacer unit. In some embodiments, the MC links the antibody or antigen-binding fragment to the drug moiety. In some embodiments, the linker comprises or consists of MC. Examples of "MC" linkers are also referred to herein as "ADL10" or "ADL10" linkers.

[0328] In some embodiments, the Mal-spacer unit or linker comprises Mal-(CH2)6 ("Mal-Hex"). In some embodiments, the Mal-spacer unit or linker comprises Mal-Hex and at least one additional spacer unit. In some embodiments, the Mal-Hex links the antibody or antigen-binding fragment to the drug moiety. In some embodiments, the linker comprises Mal-Hex. Examples of "Mal-Hex" linkers are also referred to herein as "ADL12" or "ADL12" linkers.

[0329] In some embodiments, the Mal-spacer unit or linker comprises Mal-(CH2)2 ("Mal-Et"). In some embodiments, the Mal-spacer unit or linker comprises Mal-Et and at least one additional spacer unit. In some embodiments, Mal-Et links the antibody or antigen-binding fragment to the drug moiety. In some embodiments, the linker comprises Mal-Et. An example of a "Mal-Et" linker is also referred to herein as an "ADL14" or "ADL14" linker.

[0330] In some embodiments, the Mal-spacer unit or linker comprises Mal-(CH2)2-O-(CH2)2 ("Mal-Et-O-Et"). In some embodiments, the Mal-spacer unit or linker comprises Mal-Et-O-Et and at least one additional spacer unit. In some embodiments, Mal-Et-O-Et connects the antibody or antigen-binding fragment to the drug moiety. In some embodiments, the linker comprises Mal-Et-O-Et. An example of a "Mal-Et-O-Et" linker is also referred to herein as an "ADL15" or "ADL15" linker.

[0331] In some other embodiments, a Mal-spacer unit links the antibody or antigen-binding fragment to the cleavable portion of the linker. In some embodiments, the cleavable portion of the linker is a cleavable peptide moiety, such as an amino acid unit. In some embodiments, the cleavable peptide moiety is Val-Cit or Val-Ala. In some embodiments, the Mal-spacer unit or linker comprises MC. In some embodiments, the linker comprises MC-Val-Cit. In some embodiments, the linker comprises MC-Val-Ala. In some embodiments, the linker comprises MC-Glu-Val-Cit. In some embodiments, the linker comprises MC-Ala-Ala-Asn.

[0332] In some embodiments, a spacer unit links the cleavable portion of the linker to the herboxidien splicing modulator. In some embodiments, the spacer unit linking the cleavable portion to the herboxidien splicing modulator is self-immolative.

[0333] In some embodiments, the spacer unit comprises pABC. In some embodiments, pABC links the cleavable moiety to the herboxidien splicing modulator. In some embodiments, the cleavable moiety is a cleavable peptide moiety, such as an amino acid unit. In some embodiments, the linker comprises the amino acid unit-pABC.

[0334] In some embodiments, the linker comprises Val-Cit-pABC. In some embodiments, the linker comprises Val-Cit-pABC and an MC Mal-spacer unit that joins the linker to the antibody or antigen-binding fragment. In some embodiments, the linker comprises MC-Val-Cit-pABC. In some embodiments, the linker comprises MC-Val-Cit-pABC and at least one additional spacer unit. An example of an MC-Val-Cit-pABC linker is also referred to herein as an "ADL1" or "ADL1" linker.

[0335] In some embodiments, the linker comprises Val-Ala-pABC. In some embodiments, the linker comprises Val-Ala-pABC and an MC Mal-spacer unit that joins the linker to the antibody or antigen-binding fragment. In some embodiments, the linker comprises MC-Val-Ala-pABC. In some embodiments, the linker comprises MC-Val-Ala-pABC and at least one additional spacer unit. An example of an MC-Val-Ala-pABC linker is also referred to herein as an "ADL6" or "ADL6" linker.

[0336] In some embodiments, the linker comprises Glu-Val-Cit-pABC. In some embodiments, the linker comprises Glu-Val-Cit-pABC and an MC Mal-spacer unit that joins the linker to the antibody or antigen-binding fragment. In some embodiments, the linker comprises MC-Glu-Val-Cit-pABC. In some embodiments, the linker comprises MC-Glu-Val-Cit-pABC and at least one additional spacer unit. An example of an MC-Glu-Val-Cit-pABC linker is also referred to herein as an "ADL23" or "ADL23" linker.

[0337] In some embodiments, the linker comprises Ala-Ala-Asn-pABC. In some embodiments, the linker comprises Ala-Ala-Asn-pABC and an MC Mal-spacer unit that joins the linker to the antibody or antigen-binding fragment. In some embodiments, the linker comprises MC-Ala-Ala-Asn-pABC. In some embodiments, the linker comprises MC-Ala-Ala-Asn-pABC and at least one additional spacer unit. An example of an MC-Ala-Ala-Asn-pABC linker is also referred to herein as an "ADL21" or "ADL21" linker.

[0338] In some other embodiments, the spacer unit comprises pAB. In some embodiments, pAB links the cleavable moiety to the herboxidien splicing modulator. In some embodiments, the cleavable moiety is a cleavable peptide moiety, such as an amino acid unit. In some embodiments, the linker comprises the amino acid unit -pAB.

[0339] In some embodiments, the linker comprises Val-Ala-pAB. In some embodiments, the linker comprises Val-Ala-pAB and an MC Mal-spacer unit that joins the linker to the antibody or antigen-binding fragment. In some embodiments, the linker comprises MC-Val-Ala-pAB. In some embodiments, the linker comprises MC-Val-Ala-pAB and at least one additional spacer unit. An example of an MC-Val-Ala-pAB linker is also referred to herein as an "ADL5" or "ADL5" linker.

[0340] In some embodiments, the linker comprises Val-Cit-pAB. In some embodiments, the linker comprises Val-Cit-pAB and an MC Mal-spacer unit that joins the linker to the antibody or antigen-binding fragment. In some embodiments, the linker comprises MC-Val-Cit-pAB. In some embodiments, the linker comprises MC-Val-Cit-pAB and at least one additional spacer unit. An example of an MC-Val-Cit-pAB linker is also referred to herein as an "ADL7" or "ADL7" linker.

[0341] In some embodiments, the linker comprises β-glucuronide-pABC. In some embodiments, the linker comprises β-glucuronide-pABC and an MC Mal-spacer unit that joins the linker to the antibody or antigen-binding fragment. In some embodiments, the linker comprises MC-β-glucuronide-pABC. In some embodiments, the linker comprises MC-β-glucuronide-pABC and at least one additional spacer unit. An example of MC-β-glucuronide-pABC is also referred to herein as "ADL13" or the "ADL13" linker.

[0342] In some embodiments, the linker comprises β-glucuronide-pAB. In some embodiments, the linker comprises β-glucuronide-pAB and an MC Mal-spacer unit that joins the linker to the antibody or antigen-binding fragment. In some embodiments, the linker comprises MC-β-glucuronide-pAB.

[0343] In some embodiments, the antibody or antigen-binding fragment is conjugated to a herboxidien splicing modulator drug moiety via an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker. In various embodiments, ADCs comprising an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker disclosed herein and a herboxidien splicing modulator drug moiety have been discovered to demonstrate desirable properties for therapeutic ADCs. In various embodiments, these properties include, but are not limited to, effective drug loading levels, low aggregation levels, stability under storage conditions or while circulating in the body (e.g., serum stability), retention of affinity for target-expressing cells comparable to that of an unconjugated antibody, potent cytotoxicity against target-expressing cells, low levels of off-target cell killing, high levels of bystander killing, and / or effective in vivo anti-cancer activity, all when compared to ADCs that use other linker-payloads. For example, in various embodiments, an ADC comprising an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker disclosed herein and a herboxidian splicing modulator drug moiety exhibits increased ability to inhibit the growth and / or proliferation of target-expressing cells when compared to ADCs that use other linker-payloads (e.g., an ADL10 linker and a herboxidian splicing modulator drug moiety). In various embodiments, ADCs comprising an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker disclosed herein and a herboxidiene splicing modulator drug moiety unexpectedly exhibit increased in vivo stability (e.g., plasma stability) when compared to other splicing modulator-based ADCs (e.g., tylanstatin A-based ADCs, e.g., as reported in Puthenveetil et al. Bioconjugate Chem. (2016) 27:1880-8).

[0344] In some embodiments, the good or excellent functional properties afforded by particular combinations of the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linkers disclosed herein and herboxidiene splicing modulator drug moieties may be observed in linker-payloads conjugated to, for example, anti-HER2 antibodies, such as trastuzumab; anti-CD138 antibodies, such as B-B4; or anti-EPHA2 antibodies, such as 1C1.

[0345] In some embodiments, an ADC comprises an ADL1-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof retaining the ability to target and internalize into neoplastic cells. In some embodiments, an ADC comprises an ADL2-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof retaining the ability to target and internalize into neoplastic cells. In some embodiments, an ADC comprises an ADL5-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof retaining the ability to target and internalize into neoplastic cells. In some embodiments, an ADC comprises an ADL6-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof retaining the ability to target and internalize into neoplastic cells. In some embodiments, an ADC comprises an ADL7-herboxidiene splicing modulator and an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof retaining the ability to target and internalize into neoplastic cells. In some embodiments, an ADC comprises an ADL12-herboxidiene splicing modulator and an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof retaining the ability to target and internalize into neoplastic cells. In some embodiments, an ADC comprises an ADL13-herboxidiene splicing modulator and an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof retaining the ability to target and internalize into neoplastic cells. In some embodiments, an ADC comprises an ADL14-herboxidiene splicing modulator and an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof retaining the ability to target and internalize into neoplastic cells. In some embodiments, the ADC comprises an ADL15-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof, comprising an antibody or antigen-binding fragment thereof that retains the ability to target and internalize into neoplastic cells.

[0346] In some embodiments, an ADC comprises an ADL1-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing HER2. In some embodiments, an ADC comprises an ADL2-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing HER2. In some embodiments, an ADC comprises an ADL5-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing HER2. In some embodiments, an ADC comprises an ADL6-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing HER2. In some embodiments, an ADC comprises an ADL7-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing HER2. In some embodiments, an ADC comprises an ADL12-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing HER2. In some embodiments, an ADC comprises an ADL13-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing HER2. In some embodiments, an ADC comprises an ADL14-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing HER2. In some embodiments, an ADC comprises an ADL15-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing HER2.

[0347] In some embodiments, the antibody or antigen-binding fragment thereof that targets HER2-expressing neoplastic cells is an internalizing antibody or internalizing antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment thereof that targets HER2-expressing neoplastic cells comprises three heavy chain complementarity-determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity-determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO:6 (LCDR3).

[0348] In some embodiments, the ADC has formula (I): Ab-(LH) p (I) [In formula: (i) the Ab is an anti-HER2 antibody or antigen-binding fragment thereof comprising three heavy chain complementarity-determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity-determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO:6 (LCDR3); (ii) H is a herboxidian splicing modulator; (iii) L is a linker comprising ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23; and (iv) p is an integer of 1 to 15.

[0349] In some embodiments, an antibody or antigen-binding fragment thereof that targets HER2-expressing neoplastic cells comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, an antibody or antigen-binding fragment thereof that targets HER2-expressing neoplastic cells comprises a human IgG1 heavy chain constant domain and a human Ig kappa light chain constant domain. In some embodiments, the antibody is trastuzumab. In some embodiments, p is an integer from 1 to 10, 2 to 8, or 4 to 8. In some embodiments, p is 4. In some embodiments, p is 8.

[0350] In some embodiments, an ADC comprises an ADL1-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138. In some embodiments, an ADC comprises an ADL2-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138. In some embodiments, an ADC comprises an ADL5-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138. In some embodiments, an ADC comprises an ADL6-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138. In some embodiments, an ADC comprises an ADL7-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138. In some embodiments, an ADC comprises an ADL12-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138. In some embodiments, an ADC comprises an ADL13-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138. In some embodiments, an ADC comprises an ADL14-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138. In some embodiments, an ADC comprises an ADL15-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138.

[0351] In some embodiments, the antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138 is an internalizing antibody or internalizing antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138 comprises three heavy chain complementarity-determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementarity-determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2), and SEQ ID NO:12 (LCDR3).

[0352] In some embodiments, the ADC has formula (I): Ab-(LH) p (I) [In formula: (i) the Ab is an anti-CD138 antibody or antigen-binding fragment thereof comprising three heavy chain complementarity-determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementarity-determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2), and SEQ ID NO:12 (LCDR3); (ii) H is a herboxidian splicing modulator; (iii) L is a linker comprising ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23; and (iv) p is an integer of 1 to 15.

[0353] In some embodiments, an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138 comprises a mouse IgG2a heavy chain constant domain and a mouse Ig kappa light chain constant domain. In some embodiments, an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing CD138 comprises a human IgG2a heavy chain constant domain and a human Ig kappa light chain constant domain. In some embodiments, the antibody is B-B4. In some embodiments, p is an integer between 1 and 10, between 2 and 8, or between 4 and 8. In some embodiments, p is 4. In some embodiments, p is 8.

[0354] In some embodiments, an ADC comprises an ADL1-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2. In some embodiments, an ADC comprises an ADL2-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2. In some embodiments, an ADC comprises an ADL5-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2. In some embodiments, an ADC comprises an ADL6-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2. In some embodiments, an ADC comprises an ADL7-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2. In some embodiments, an ADC comprises an ADL12-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2. In some embodiments, an ADC comprises an ADL13-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2. In some embodiments, an ADC comprises an ADL14-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2. In some embodiments, an ADC comprises an ADL15-herboxidienes splicing modulator and an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2.

[0355] In some embodiments, the antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2 is an internalizing antibody or internalizing antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2 comprises three heavy chain complementarity-determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO: 13 (HCDR1), SEQ ID NO: 14 (HCDR2), and SEQ ID NO: 15 (HCDR3); and three light chain complementarity-determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 16 (LCDR1), SEQ ID NO: 17 (LCDR2), and SEQ ID NO: 18 (LCDR3).

[0356] In some embodiments, the ADC has formula (I): Ab-(LH) p (I) [In formula: (i) the Ab is an anti-EPHA2 antibody or antigen-binding fragment thereof comprising three heavy chain complementarity-determining regions (HCDRs) comprising the amino acid sequences of SEQ ID NO: 13 (HCDR1), SEQ ID NO: 14 (HCDR2), and SEQ ID NO: 15 (HCDR3); and three light chain complementarity-determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 16 (LCDR1), SEQ ID NO: 17 (LCDR2), and SEQ ID NO: 18 (LCDR3); (ii) H is a herboxidian splicing modulator; (iii) L is a linker comprising ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23; and (iv) p is an integer of 1 to 15.

[0357] In some embodiments, an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, an antibody or antigen-binding fragment thereof that targets neoplastic cells expressing EPHA2 comprises a human IgG1 heavy chain constant domain and a human Ig kappa light chain constant domain. In some embodiments, the antibody is 1C1. In some embodiments, p is an integer from 1 to 10, 2 to 8, or 4 to 8. In some embodiments, p is 4. In some embodiments, p is 8.

[0358] Herboxidienes splicing modulators In some embodiments, the antibody-drug conjugate has the formula (I): Ab-(LH) p wherein Ab is an antibody or antigen-binding fragment that targets a neoplastic cell; H is a herboxidian splicing modulator; L is a linker that covalently links Ab to H; and p is an integer from 1 to 15.

[0359] In some embodiments, H, a herboxidiene splicing modulator, is covalently linked to L through any atom in a compound of formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein: Y is O, S, NR 6 , and CR 6 R 7 Selected from; R 1 , R 2 , and R 3 are each independently selected from hydrogen, hydroxyl, an -O-(C1-C6 alkyl) group, an -O-C(=O)-(C1-C6 alkyl) group, an -C(=O)-O-(C1-C6 alkyl) group, and a C1-C6 alkyl group; R 4is hydrogen, a C1-C6 alkyl group, a -C(=O)-(C1-C6 alkyl) group, a -C(=O)-(C3-C8 carbocyclyl) group, a -C(=O)-(C3-C8 heterocyclyl) group, and a -C(=O)-NR 6 R 7 Selected from; R 5 is hydrogen, hydroxyl, -CH2-OH, -CO2H, -C(=O)-O-(C1-C6 alkyl) group, -C(=O)-NR 6 R 7 , -NR 6 -C(=O)-R 8 , -OC(=O)-NR 6 R 7 , -NR 6 -C(=O)-R 8 , and -NR 6 -C(=O)-NR 6 R 7 Selected from; R 6 and R 7 are each independently hydrogen, -R 8 , -C(=O)-R 8 , and -C(=O)-OR 8 selected from; and R 8 is selected from a C1-C6 alkyl group, a C3-C8 carbocyclyl group, and a C3-C8 heterocyclyl group; where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 each independently represents a halogen, hydroxyl, a C1-C6 alkyl group, -O-(C1-C6 alkyl) group, -CO2H, -C(=O)-(C1-C6 alkyl) group, -C(=O)-(C3-C8 carbocyclyl) group, -C(=O)-(C3-C8 heterocyclyl) group, -NR 6 R 7, substituted by 0 to 3 groups independently selected from a C3 to C8 carbocyclyl group, a C1 to C6 alkylhydroxy group, a C1 to C6 alkylalkoxy group, a benzyl group, and a C3 to C8 heterocyclyl group (each of which is optionally independently substituted with 0 or 1 group selected from a halogen, a hydroxyl, a C1 to C3 alkyl group, a C1 to C3 alkoxy group, a C1 to C3 haloalkyl group, -NH-C(=O)(C1 to C3 alkyl), and -NH-C(=O)-O-(C1 to C3 alkyl)); and wherein the valence of the atom covalently bonded to L is not exceeded.

[0360] In some embodiments, H, a herboxidiene splicing modulator, is covalently linked to L through any atom in a compound of Formula (Ia): [ka] or a pharmaceutically acceptable salt thereof, wherein: R 9 is selected from C3-C8 heterocyclyl groups; R 10 is selected from H and C1-C6 alkyl groups, where R 9 and R 10 are each independently substituted with 0 to 3 groups independently selected from halogen, hydroxyl, a C1-C3 alkyl group, a C1-C3 alkoxy group, -NH2, -NH-(C1-C3 alkyl), and -N-(C1-C3 alkyl)2, and wherein the valence of the atom covalently bonded to L is not exceeded.

[0361] In some embodiments, H, a herboxidiene splicing modulator, is a compound of Formula (Ib) covalently linked to L through any atom: [ka] or a pharmaceutically acceptable salt thereof, wherein: R11 teeth, [ka] (In the formula, * is R 11 represents the point of attachment to the remainder of the compound; R 12 and R 13 are each independently selected from H and methyl; and wherein the valence of the atom covalently bonded to L is not exceeded.

[0362] In some embodiments, H, the herboxidiene splicing modulator, is a compound of formula (II) covalently linked to L through any atom: [ka] or a pharmaceutically acceptable salt thereof, wherein: X is hydroxyl or NR 6 R 7 and; R 6 and R 7 are each independently hydrogen, -R 8 , -C(=O)-R 8 , -C(=O)-OR 8 , -(C1-C6 alkyl)-OC(=O)-R 8 and -(C1-C6 alkyl)-NH-C(=O)-R 8 selected from; and R 8 is selected from a C1-C6 alkyl group, a C3-C8 carbocyclyl group, and a C3-C8 heterocyclyl group; where R 6 , R 7 , and R 8each independently represents a halogen, hydroxyl, a C1-C6 alkyl group, -O-(C1-C6 alkyl) group, -CO2H, -C(=O)-O-(C1-C6 alkyl) group, -C(=O)-(C1-C6 alkyl) group, -C(=O)-(C3-C8 carbocyclyl) group, -C(=O)-(C3-C8 heterocyclyl) group, -NR 6 R 7 , substituted by 0 to 3 groups independently selected from a C3 to C8 carbocyclyl group, a C1 to C6 alkylhydroxy group, a C1 to C6 alkylalkoxy group, a benzyl group, and a C3 to C8 heterocyclyl group (each of which is optionally independently substituted with 0 or 1 group selected from a halogen, a hydroxyl, a C1 to C3 alkyl group, a C1 to C3 alkoxy group, a C1 to C3 haloalkyl group, -NH-C(=O)(C1 to C3 alkyl), and -NH-C(=O)-O-(C1 to C3 alkyl)); and wherein the valence of the atom covalently bonded to L is not exceeded.

[0363] In some embodiments, H, the herboxidiene splicing modulator, is a compound of formula (IIa) covalently linked to L through any atom: [ka] or a pharmaceutically acceptable salt thereof, wherein: Z is NR 9 and O; R 9 is selected from hydrogen and C1-C6 alkyl groups; R 10 and R 11are each independently selected from hydrogen, halogen, hydroxyl, a C1-C6 alkyl group, a -O-(C1-C6 alkyl) group, -COH, a -C(=O)-O-(C1-C6 alkyl) group, a -C(=O)-(C1-C6 alkyl) group, a -C(=O)-(C3-C8 carbocyclyl) group, a -C(=O)-(C3-C8 heterocyclyl) group, a C3-C8 carbocyclyl group, a C1-C6 alkylhydroxy group, a C1-C6 alkylalkoxy group, a benzyl group, and a C3-C8 heterocyclyl group; R 12 is selected from a C1-C6 alkyl group, a C3-C8 carbocyclyl group, and a C3-C8 heterocyclyl group; where R 9 , R 10 , R 11 , and R 12 are each independently substituted with 0 or 1 group selected from halogen, hydroxyl, a C1-C3 alkyl group, a C1-C3 alkoxy group, and a C1-C3 haloalkyl group; t is an integer selected from 1, 2, 3, 4, 5, and 6; and wherein the valence of the atom covalently bonded to L is not exceeded.

[0364] In some embodiments, H, a herboxidiene splicing modulator, is a compound of formula (IIb) covalently linked to L through any atom: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 13 teeth, [ka] (In the formula, * is R 13 represents the point of attachment to the remainder of the compound; R 14 and R 15 are each independently selected from hydrogen and methyl; and wherein the valence of the atom covalently bonded to L is not exceeded.

[0365] In some embodiments, H, the herboxidiene splicing modulator, is a compound of formula (III) covalently linked to L through any atom: [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1 , R 2 , and R 3 are each independently selected from hydrogen, hydroxyl, an -O-(C1-C6 alkyl) group, an -O-C(=O)-(C1-C6 alkyl) group, an -C(=O)-O-(C1-C6 alkyl) group, and a C1-C6 alkyl group; R 6 and R 7 are each independently hydrogen, -R 8 , -C(=O)-R 8 , and -C(=O)-OR 8 Selected from; R 8 is selected from a C1-C6 alkyl group, a C3-C8 carbocyclyl group, and a C3-C8 heterocyclyl group; and R 9 H, [ka] Selected from; where R 1 , R 2 , R 3 , R 6 , R 7 , and R 8 each independently represents a halogen, hydroxyl, a C1-C6 alkyl group, -O-(C1-C6 alkyl) group, -CO2H, -C(=O)-(C1-C6 alkyl) group, -C(=O)-(C3-C8 carbocyclyl) group, -C(=O)-(C3-C8 heterocyclyl) group, -NR 6 R 7, substituted by 0 to 3 groups independently selected from a C3 to C8 carbocyclyl group, a C1 to C6 alkylhydroxy group, a C1 to C6 alkylalkoxy group, a benzyl group, and a C3 to C8 heterocyclyl group (each of which is optionally independently substituted by 0 or 1 group selected from a halogen, hydroxyl, a C1 to C3 alkyl group, a C1 to C3 alkoxy group, a C1 to C3 haloalkyl group, -NH-C(=O)(C1 to C3 alkyl), and -NH-C(=O)-O-(C1 to C3 alkyl); wherein the valence of the atom covalently bonded to L is not exceeded; and During the ceremony, * is R 9 represents the point of attachment to the remainder of the compound.

[0366] In some embodiments, the antibody or antigen-binding fragment targets cells that express HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, and / or STEAP1.

[0367] In some embodiments, the antibody or antigen-binding fragment targets HER2-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-HER2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising the amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO:6 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

[0368] In some other embodiments, the antibody or antigen-binding fragment targets CD138-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-CD138 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) comprising the amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2), and SEQ ID NO:12 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:22. In some embodiments, the antibody or antigen-binding fragment comprises a murine IgG2a heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a murine Ig kappa light chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG2a heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

[0369] In some other embodiments, the antibody or antigen-binding fragment targets EPHA2-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-EPHA2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequences of SEQ ID NO: 13 (HCDR1), SEQ ID NO: 14 (HCDR2), and SEQ ID NO: 15 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising the amino acid sequences of SEQ ID NO: 16 (LCDR1), SEQ ID NO: 17 (LCDR2), and SEQ ID NO: 18 (LCDR3). In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

[0370] In some other embodiments, the antibody or antigen-binding fragment targets MSLN-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-MSLN antibody or antigen-binding fragment.

[0371] In some other embodiments, the antibody or antigen-binding fragment targets FOLH1-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-FOLH1 antibody or antigen-binding fragment.

[0372] In some other embodiments, the antibody or antigen-binding fragment targets CDH6-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-CDH6 antibody or antigen-binding fragment.

[0373] In some other embodiments, the antibody or antigen-binding fragment targets CEACAM5-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-CEACAM5 antibody or antigen-binding fragment.

[0374] In some other embodiments, the antibody or antigen-binding fragment targets CFC1B-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-CFC1B antibody or antigen-binding fragment.

[0375] In some other embodiments, the antibody or antigen-binding fragment targets ENPP3-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-ENPP3 antibody or antigen-binding fragment.

[0376] In some other embodiments, the antibody or antigen-binding fragment targets FOLR1-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-FOLR1 antibody or antigen-binding fragment.

[0377] In some other embodiments, the antibody or antigen-binding fragment targets HAVCR1-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-HAVCR1 antibody or antigen-binding fragment.

[0378] In some other embodiments, the antibody or antigen-binding fragment targets KIT-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-KIT antibody or antigen-binding fragment.

[0379] In some other embodiments, the antibody or antigen-binding fragment targets MET-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-MET antibody or antigen-binding fragment.

[0380] In some other embodiments, the antibody or antigen-binding fragment targets MUC16-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-MUC16 antibody or antigen-binding fragment.

[0381] In some other embodiments, the antibody or antigen-binding fragment targets SLC39A6-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-SLC39A6 antibody or antigen-binding fragment.

[0382] In some other embodiments, the antibody or antigen-binding fragment targets SLC44A4-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-SLC44A4 antibody or antigen-binding fragment.

[0383] In some other embodiments, the antibody or antigen-binding fragment targets STEAP1-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-STEAP1 antibody or antigen-binding fragment.

[0384] In some embodiments, L is selected from any of the linkers disclosed herein or any combination of linker components disclosed herein. In some embodiments, L is a linker comprising MC-Val-Cit-pABC, Mal-(PEG)2-CO, MC-Val-Ala-pAB, MC-Val-Ala-pABC, MC-Val-Cit-pAB, Mal-Hex, Mal-Et, or Mal-Et-O-Et. In some embodiments, the linker may also comprise one or more additional spacer units. In some embodiments, L is an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker. In some embodiments, L is an ADL12, ADL14, or ADL15 linker. In some embodiments, the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker may also include one or more additional spacer units.

[0385] In certain embodiments, an intermediate that is a precursor to the linker moiety is reacted with a herboxidienes splicing modulator moiety under appropriate conditions. In certain embodiments, a reactive group on the herboxidienes splicing modulator and / or intermediate or linker is used. The reaction product between the herboxidienes splicing modulator and the intermediate, or a derivatized herboxidienes splicing modulator (herboxidienes splicing modulator + linker) is then reacted with an antibody or antigen-binding fragment under appropriate conditions. Alternatively, the intermediate or linker may first be reacted with an antibody or antigen-binding fragment, or a derivatized antibody or antigen-binding fragment, and then reacted with a drug or derivatized drug.

[0386] Several different reactions are available for covalently linking the herboxidien splicing modulator moiety and / or linker moiety to an antibody or antigen-binding fragment. This is often achieved by reaction of one or more amino acid residues of the antibody or antigen-binding fragment, including the amine groups of lysine, the free carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl groups of cysteine, and various moieties of aromatic amino acids. For example, nonspecific covalent linking can be performed using a carbodiimide reaction to link a carboxyl group (or amino group) on the herboxidien splicing modulator moiety to an amino group (or carboxyl group) on the antibody or antigen-binding fragment. In addition, bifunctional agents such as dialdehydes or imidoesters can also be used to link an amino group on the herboxidien splicing modulator moiety to an amino group on the antibody or antigen-binding fragment. Schiff base reactions can also be used to link drugs (e.g., herboxidien splicing modulators) to binding agents. This method involves periodate oxidation of a drug containing a glycol or hydroxy group, resulting in the formation of an aldehyde, which then reacts with the binder. Attachment occurs via the formation of a Schiff base with the amino group of the binder. Isothiocyanates can also be used as coupling agents to covalently attach drugs to binders. Other techniques are known to those skilled in the art and are within the scope of the present disclosure. Examples of drug moieties that can be prepared and linked to antibodies or antigen-binding fragments using various chemistries known in the art include herboxidian splicing modulators, such as those described and exemplified herein.

[0387] Linker-Herboxidienes Splicing Modulator / Herboxidienes Splicing Modulator Compound Further disclosed herein are exemplary linker-herboxidienes splicing modulator (LH) compounds, as well as compositions comprising multiple copies of such compounds. In various embodiments, the linker-herboxidienes splicing modulator compounds disclosed herein can be defined by the general formula: LH, where L = linker moiety, and H = herboxidienes splicing modulator. In certain embodiments, the disclosed LH compounds are suitable for use in the ADCs described herein.

[0388] In some embodiments, provided herein are compounds of formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein: Y is O, S, NR 6 , and CR 6 R 7 Selected from; R 1 , R 2 , and R 3 are each independently selected from hydrogen, hydroxyl, an -O-(C1-C6 alkyl) group, an -O-C(=O)-(C1-C6 alkyl) group, an -C(=O)-O-(C1-C6 alkyl) group, and a C1-C6 alkyl group; R 4 is hydrogen, a C1-C6 alkyl group, a -C(=O)-(C1-C6 alkyl) group, a -C(=O)-(C3-C8 carbocyclyl) group, a -C(=O)-(C3-C8 heterocyclyl) group, and a -C(=O)-NR 6 R 7 Selected from; R 5 is hydrogen, hydroxyl, -CH2-OH, -CO2H, -C(=O)-O-(C1-C6 alkyl) group, -C(=O)-NR 6 R 7 , -NR 6 -C(=O)-R 8 , -OC(=O)-NR 6 R 7 , -NR 6 -C(=O)-R8 , and -NR 6 -C(=O)-NR 6 R 7 Selected from; R 6 and R 7 are each independently hydrogen, -R 8 , -C(=O)-R 8 , and -C(=O)-OR 8 selected from; and R 8 is selected from a C1-C6 alkyl group, a C3-C8 carbocyclyl group, and a C3-C8 heterocyclyl group; where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 each independently represents a halogen, hydroxyl, a C1-C6 alkyl group, -O-(C1-C6 alkyl) group, -CO2H, -C(=O)-(C1-C6 alkyl) group, -C(=O)-(C3-C8 carbocyclyl) group, -C(=O)-(C3-C8 heterocyclyl) group, -NR 6 R 7 , a C3-C8 carbocyclyl group, a C1-C6 alkylhydroxy group, a C1-C6 alkylalkoxy group, a benzyl group, and a C3-C8 heterocyclyl group (each of which is optionally independently substituted with 0 or 1 group selected from halogen, hydroxyl, a C1-C3 alkyl group, a C1-C3 alkoxy group, a C1-C3 haloalkyl group, —NH—C(═O)(C1-C3 alkyl), and —NH—C(═O)—O—(C1-C3 alkyl)).

[0389] In some embodiments, provided herein are compounds of formula (Ia) covalently attached to L through any atom: [ka] or a pharmaceutically acceptable salt thereof, wherein: R9 is selected from a C3-C8 heterocyclyl group; and R 10 is selected from H and C1-C6 alkyl groups; where R 9 and R 10 are each independently substituted with 0 to 3 groups independently selected from halogen, hydroxyl, a C1-C3 alkyl group, a C1-C3 alkoxy group, —NH2, —NH-(C1-C3 alkyl), and —N-(C1-C3 alkyl)2.

[0390] In some embodiments, provided herein are compounds of formula (Ib): [ka] or a pharmaceutically acceptable salt thereof, wherein: R 11 teeth, [ka] (In the formula, * is R 11 represents the point of attachment to the remainder of the compound; R 12 and R 13 are each independently selected from H and methyl.

[0391] In some embodiments, provided herein are compounds of formula (II): [ka] or a pharmaceutically acceptable salt thereof, wherein: X is hydroxyl or NR 6 R 7 and; R 6 and R 7 are each independently hydrogen, -R 8 , -C(=O)-R 8 , -C(=O)-OR 8, -(C1-C6 alkyl)-OC(=O)-R 8 and -(C1-C6 alkyl)-NH-C(=O)-R 8 selected from; and R 8 is selected from a C1-C6 alkyl group, a C3-C8 carbocyclyl group, and a C3-C8 heterocyclyl group; where R 6 , R 7 , and R 8 each independently represents a halogen, hydroxyl, a C1-C6 alkyl group, -O-(C1-C6 alkyl) group, -CO2H, -C(=O)-O-(C1-C6 alkyl) group, -C(=O)-(C1-C6 alkyl) group, -C(=O)-(C3-C8 carbocyclyl) group, -C(=O)-(C3-C8 heterocyclyl) group, -NR 6 R 7 , a C3-C8 carbocyclyl group, a C1-C6 alkylhydroxy group, a C1-C6 alkylalkoxy group, a benzyl group, and a C3-C8 heterocyclyl group (each of which is optionally independently substituted with 0 or 1 group selected from halogen, hydroxyl, a C1-C3 alkyl group, a C1-C3 alkoxy group, a C1-C3 haloalkyl group, —NH—C(═O)(C1-C3 alkyl), and —NH—C(═O)—O—(C1-C3 alkyl)).

[0392] In some embodiments, provided herein are compounds of formula (IIa): [ka] or a pharmaceutically acceptable salt thereof, wherein: Z is NR 9 and O; R 9 is selected from hydrogen and C1-C6 alkyl groups; R 10 and R 11are each independently selected from hydrogen, halogen, hydroxyl, a C1-C6 alkyl group, a -O-(C1-C6 alkyl) group, -COH, a -C(=O)-O-(C1-C6 alkyl) group, a -C(=O)-(C1-C6 alkyl) group, a -C(=O)-(C3-C8 carbocyclyl) group, a -C(=O)-(C3-C8 heterocyclyl) group, a C3-C8 carbocyclyl group, a C1-C6 alkylhydroxy group, a C1-C6 alkylalkoxy group, a benzyl group, and a C3-C8 heterocyclyl group; R 12 is selected from a C1-C6 alkyl group, a C3-C8 carbocyclyl group, and a C3-C8 heterocyclyl group; where R 9 , R 10 , R 11 , and R 12 are each independently substituted with 0 or 1 group selected from halogen, hydroxyl, a C1-C3 alkyl group, a C1-C3 alkoxy group, and a C1-C3 haloalkyl group; and and t is an integer selected from 1, 2, 3, 4, 5, and 6.

[0393] In some embodiments, provided herein are compounds of formula (IIb): [ka] or a pharmaceutically acceptable salt thereof, wherein: R 13 teeth, [ka] (In the formula, * is R 13 represents the point of attachment to the remainder of the compound; and R 14 and R 15 are each independently selected from hydrogen and methyl.

[0394] In some embodiments, provided herein are compounds of formula (III): [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1 , R 2 , and R 3 are each independently selected from hydrogen, hydroxyl, an -O-(C1-C6 alkyl) group, an -O-C(=O)-(C1-C6 alkyl) group, an -C(=O)-O-(C1-C6 alkyl) group, and a C1-C6 alkyl group; R 6 and R 7 are each independently hydrogen, -R 8 , -C(=O)-R 8 , and -C(=O)-OR 8 Selected from; R 8 is selected from a C1-C6 alkyl group, a C3-C8 carbocyclyl group, and a C3-C8 heterocyclyl group; and R 9 H, [ka] is selected from where R 1 , R 2 , R 3 , R 6 , R 7 , and R 8 each independently represents a halogen, hydroxyl, a C1-C6 alkyl group, -O-(C1-C6 alkyl) group, -CO2H, -C(=O)-(C1-C6 alkyl) group, -C(=O)-(C3-C8 carbocyclyl) group, -C(=O)-(C3-C8 heterocyclyl) group, -NR 6 R 7, substituted by 0-3 groups independently selected from a C3-C8 carbocyclyl group, a C1-C6 alkylhydroxy group, a C1-C6 alkylalkoxy group, a benzyl group, and a C3-C8 heterocyclyl group (each of which is optionally independently substituted with 0 or 1 group selected from a halogen, hydroxyl, a C1-C3 alkyl group, a C1-C3 alkoxy group, a C1-C3 haloalkyl group, -NH-C(=O)(C1-C3 alkyl), and -NH-C(=O)-O-(C1-C3 alkyl); and During the ceremony, * is R 9 represents the point of attachment to the remainder of the compound] is provided.

[0395] In some embodiments, provided herein is Compound H1, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is Compound H4, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is Compound H5, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is Compound H6, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is Compound H7, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is Compound H8, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is Compound H9, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is Compound H10, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is Compound H2, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is Compound H3, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is Compound H12, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is compound H13, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is compound H14, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is compound H15, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is compound H16, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is compound H17, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is compound H18, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is compound H19, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is compound H20, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is compound H21, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is compound H22, or a pharmaceutically acceptable salt thereof.In some embodiments, provided herein is compound H23, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is compound H24, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is compound H25, or a pharmaceutically acceptable salt thereof.

[0396] In some embodiments, the present disclosure provides: [ka] [ka] [ka] [ka] [ka] and pharmaceutically acceptable salts thereof where L is a linker that covalently attaches to the antibody. A compound selected from:

[0397] In some embodiments, the linker comprises at least one cleavable peptide moiety. In some embodiments, the at least one cleavable peptide moiety is cleavable by an enzyme. In some embodiments, the linker or cleavable peptide moiety comprises at least one amino acid unit. In some embodiments, the at least one amino acid unit is selected from arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, and citrulline. In some embodiments, the at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, the linker comprises citrulline and valine. In some embodiments, the linker comprises alanine and valine.

[0398] In some embodiments, the linker comprises a moiety selected from a sulfonamide, a β-glucuronide, a disulfide, and a carbonyl. In some embodiments, the linker comprises a sulfonamide. In some embodiments, the linker comprises a β-glucuronide. In some embodiments, the linker comprises a disulfide. In some embodiments, the linker comprises a carbonyl.

[0399] In some embodiments, the linker comprises a spacer unit. In some embodiments, the spacer unit is selected from an alkyl group and a polyethylene glycol (PEG) moiety. In some embodiments, the alkyl group is a C1-C 12In some embodiments, the alkyl group is an alkyl group. In some embodiments, the alkyl group is a C1-C6 alkyl group. In some embodiments, the alkyl group is methylene. In some embodiments, the alkyl group is ethylene. In some embodiments, the alkyl group is n-propylene. In some embodiments, the alkyl group is n-butylene. In some embodiments, the alkyl group is n-pentylene. In some embodiments, the alkyl group is n-hexylene. In some embodiments, the PEG moiety is -(PEG) m -, wherein m is an integer from 1 to 10. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6.

[0400] In some embodiments, the linker comprises a maleimide (Mal) moiety ("Mal-spacer unit"). In some embodiments, the linker comprises a self-immolative spacer unit. In some embodiments, the self-immolative spacer unit is selected from p-aminobenzyloxycarbonyl (pABC) and p-aminobenzyl (pAB).

[0401] In some embodiments, the linker comprises a Mal-spacer unit, an alkyl group, at least one amino acid unit, and a self-immolative spacer. In some embodiments, the at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, the at least one amino acid unit comprises alanine and valine. In some embodiments, the at least one amino acid unit comprises citrulline and valine. In some embodiments, the self-immolative spacer is selected from pAB and pABC. In some embodiments, the self-immolative spacer comprises pAB. In some embodiments, the self-immolative spacer comprises pABC. In some embodiments, the alkyl group comprises a C1-C6 alkyl group.

[0402] In some embodiments, the linker comprises a Mal-spacer unit, a PEG moiety, at least one amino acid unit, and a self-immolative spacer. In some embodiments, the at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, the at least one amino acid unit comprises alanine and valine. In some embodiments, the at least one amino acid unit comprises citrulline and valine. In some embodiments, the self-immolative spacer is selected from pAB and pABC. In some embodiments, the self-immolative spacer comprises pAB. In some embodiments, the self-immolative spacer comprises pABC. In some embodiments, the PEG moiety is -(PEG) m -, wherein m is an integer of 1 to 6.

[0403] Drug Loading Drug loading is represented by p, also referred to herein as the herboxidian splicing modulator-to-antibody ratio (HAR). Drug loading may range from 1 to 10 drug moieties per antibody or antigen-binding fragment. In some embodiments, p is an integer between 1 and 10. In some embodiments, p is an integer between 1 and 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is an integer between 2 and 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In some embodiments, p is an integer between 1 and 8. In some embodiments, p is an integer between 2 and 5. In some embodiments, p is an integer between 2 and 4. In some embodiments, p is an integer between 3 and 4. In other embodiments, p is an integer between 4 and 8. In other embodiments, p is 1, 2, 3, 4, 5, 6, 7, or 8, preferably 4 or 8.

[0404] Drug loading may be limited by the number of attachment sites on the antibody or antigen-binding fragment. In some embodiments, the linker moiety (L) of the ADC is attached to the antibody or antigen-binding fragment through a chemically reactive group on one or more amino acid residues on the antibody or antigen-binding fragment. For example, the linker may be attached to the antibody or antigen-binding fragment via a free amino, imino, hydroxyl, thiol, or carboxyl group (e.g., to the N- or C-terminus, to the epsilon amino group of one or more lysine residues, to the free carboxylic acid groups of one or more glutamic acid or aspartic acid residues, or to the sulfhydryl groups of one or more cysteine ​​residues). The linker attachment site may be a native residue in the amino acid sequence of the antibody or antigen-binding fragment, or it may be introduced into the antibody or antigen-binding fragment by, for example, recombinant DNA techniques (e.g., by introducing a cysteine ​​residue into the amino acid sequence) or protein biochemistry (e.g., by reduction, pH adjustment, or hydrolysis).

[0405] In some embodiments, the number of drug moieties that can be conjugated to an antibody or antigen-binding fragment is limited by the number of free cysteine ​​residues. For example, if the attachment is via a cysteine ​​thiol group, the antibody may have only one or a few cysteine ​​thiol groups, or may have only one or a few sufficiently reactive thiol groups to which a linker can be attached. Generally, antibodies do not contain many free and reactive cysteine ​​thiol groups that can be linked to a drug moiety. In fact, most cysteine ​​thiol residues in antibodies are involved in either interchain or intrachain disulfide bonds. Therefore, conjugation to cysteines may require at least partial reduction of the antibody in some embodiments. Conjugating too many linker-toxins to an antibody can destabilize the antibody by reducing cysteine ​​residues available for disulfide bond formation. Therefore, the optimal drug:antibody ratio should increase the potency of the ADC (by increasing the number of drug moieties attached per antibody) without destabilizing the antibody or antigen-binding fragment. In some embodiments, the optimal ratio may be 2, 4, 6, or 8.

[0406] In some embodiments, the antibody or antigen-binding fragment is exposed to reducing conditions prior to conjugation to generate one or more free cysteine ​​residues. In some embodiments, the antibody may be reduced under partial or complete reducing conditions with a reducing agent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP), which may generate reactive cysteine ​​thiol groups. Unpaired cysteines may also be generated through partial reduction with a limited molar equivalent of TCEP, which can reduce the interchain disulfide bonds connecting the light and heavy chains (one pair for each HL pairing) and the two heavy chains in the hinge region (two pairs for each HH pairing in the case of human IgG1) while leaving the intrachain disulfide bonds intact (Stefano et al. (2013) Methods Mol Biol. 1045:145-71). In embodiments, disulfide bonds in antibodies are electrochemically reduced, for example, by utilizing a working electrode that applies alternating reduction and oxidation voltages. This approach may allow for online coupling of disulfide bond reduction to analytical instruments (e.g., electrochemical detection instruments, NMR spectrometers, or mass spectrometers) or chemical separation instruments (e.g., liquid chromatography (e.g., HPLC) or electrophoresis instruments (see, e.g., U.S. Patent Application Publication No. 20140069822)). In certain embodiments, antibodies are subjected to denaturing conditions to expose reactive nucleophilic groups on amino acid residues, such as cysteines.

[0407] The drug loading of an ADC may alternatively be controlled, for example, by (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody; (ii) limiting the conjugation reaction time or temperature; (iii) partial or limited reducing conditions for cysteine ​​thiol modification; and / or (iv) recombinantly altering the amino acid sequence of the antibody so that the number and position of cysteine ​​residues are modified to control the number and / or position of linker-drug bonds.

[0408] In some embodiments, a free cysteine ​​residue is introduced into the amino acid sequence of an antibody or antigen-binding fragment. For example, cysteine-engineered antibodies can be prepared in which one or more amino acids of a parent antibody are replaced with a cysteine ​​amino acid. Any form of antibody can be modified, i.e., mutated, in this manner. For example, a parent Fab antibody fragment can be modified to form a cysteine-engineered Fab, referred to as a "ThioFab." Similarly, a parent monoclonal antibody can be modified to form a "ThioMab." In a ThioFab, a single-site mutation results in a single engineered cysteine ​​residue, whereas in a ThioMab, due to the dimeric nature of IgG antibodies, a single-site mutation results in two engineered cysteine ​​residues. DNA encoding amino acid sequence variants of a parent polypeptide can be prepared by various methods known in the art (see, for example, the methods described in WO 2006 / 034488). These methods include, but are not limited to, site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously prepared DNA encoding the polypeptide. Recombinant antibody variants may also be constructed by restriction fragment manipulation or overlap extension PCR with synthetic oligonucleotides. ADCs of Formula (I) include, but are not limited to, antibodies with one, two, three, or four engineered cysteine ​​amino acids (Lyon et al. (2012) Methods Enzymol. 502:123-38). In some embodiments, an antibody or antigen-binding fragment already has one or more free cysteine ​​residues without modification, in which case the existing free cysteine ​​residues can be used to conjugate the antibody or antigen-binding fragment to a drug moiety.

[0409] When two or more nucleophilic groups react with a drug-linker intermediate or linker moiety reagent, followed by a drug moiety reagent, in a reaction mixture containing multiple copies of an antibody or antigen-binding fragment and a linker moiety, the resulting product can be a mixture of ADC compounds with a distribution of one or more drug moieties attached to each copy of the antibody or antigen-binding fragment in the mixture. In some embodiments, the drug loading of the ADC mixture resulting from the conjugation reaction ranges from 1 to 10 drug moieties attached to each antibody or antigen-binding fragment. The average number of drug moieties per antibody or antigen-binding fragment (i.e., average drug loading, or average p) can be calculated by any conventional method known in the art, such as mass spectrometry (e.g., reverse-phase LC-MS) and / or high-performance liquid chromatography (e.g., HIC-HPLC). In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is determined by hydrophobic interaction chromatography-high-performance liquid chromatography (HIC-HPLC). In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is determined by reverse-phase liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 1.5 to about 3.5, about 2.5 to about 4.5, about 3.5 to about 5.5, about 4.5 to about 6.5, about 5.5 to about 7.5, about 6.5 to about 8.5, or about 7.5 to about 9.5. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 2 to about 4, about 3 to about 5, about 4 to about 6, about 5 to about 7, about 6 to about 8, about 7 to about 9, about 2 to about 8, or about 4 to about 8.

[0410] In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 2. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is 2.

[0411] In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 4. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, or about 4.5. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is 4.

[0412] In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 8. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, or about 8.5. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is 8.

[0413] In various embodiments, the term "about" when used in reference to the average number of drug moieties per each antibody or antigen-binding fragment means ±10%.

[0414] Individual ADC compounds, or "species," can be identified in the mixture by mass spectrometry and separated by UPLC or HPLC, e.g., hydrophobic interaction chromatography (HIC-HPLC). In certain embodiments, homogeneous or nearly homogeneous ADC products of a single loading level can be separated from the conjugation mixture, e.g., by electrophoresis or chromatography.

[0415] In some embodiments, high drug loading (e.g., p>8) may result in aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates. High drug loading may also adversely affect the pharmacokinetics (e.g., clearance) of certain ADCs. In some embodiments, low drug loading (e.g., p<2) may reduce the potency of certain ADCs against target-expressing cells and / or bystander cells. In some embodiments, the drug loading of ADCs of the present disclosure ranges from about 2 to about 8; about 2 to about 6; about 2 to about 5; about 3 to about 5; about 2 to about 4; or about 4 to about 8.

[0416] In some embodiments, a drug loading and / or average drug loading of about 2 is achieved, e.g., using partial reduction of intrachain disulfides on the antibody or antigen-binding fragment, resulting in beneficial properties. In some embodiments, a drug loading and / or average drug loading of about 4 is achieved, e.g., using partial reduction of intrachain disulfides on the antibody or antigen-binding fragment, resulting in beneficial properties. In some embodiments, a drug loading and / or average drug loading of about 8 is achieved, e.g., using partial reduction of intrachain disulfides on the antibody or antigen-binding fragment, resulting in beneficial properties. In some embodiments, a drug loading and / or average drug loading of less than about 2 may result in unacceptably high levels of unconjugated antibody species that may compete with the ADC for binding to the target antigen and / or result in reduced therapeutic efficacy. In some embodiments, a drug loading and / or average drug loading of more than about 8 may result in unacceptably high levels of product heterogeneity and / or ADC aggregation. Drug loading and / or average drug loading greater than about 8 may also affect the stability of the ADC due to the loss of one or more chemical bonds necessary to stabilize the antibody or antigen-binding fragment.

[0417] The present disclosure includes methods for making the described ADCs. Briefly, an ADC comprises an antibody or antigen-binding fragment, such as the present antibody or antigen-binding fragment, a drug moiety (e.g., a herboxidian splicing modulator), and a linker that connects the drug moiety to the antibody or antigen-binding fragment. In some embodiments, ADCs can be prepared using a linker with a drug moiety and a reactive functional group for covalently attaching to the antibody or antigen-binding fragment. For example, in some embodiments, a cysteine ​​thiol of the antibody or antigen-binding fragment can form a bond with a reactive functional group (e.g., a maleimide moiety) of a linker or drug-linker intermediate to create an ADC. Preparation of an ADC can be accomplished by any technique known to those of skill in the art.

[0418] In some embodiments, ADCs are prepared by sequentially contacting an antibody or antigen-binding fragment with a linker and a drug moiety (e.g., a herboxidiene splicing modulator); i.e., the antibody or antigen-binding fragment is first covalently linked to the linker, and then this preformed antibody-linker intermediate is reacted with the drug moiety. The antibody-linker intermediate may or may not be subjected to a purification step prior to contacting with the drug moiety. In other embodiments, ADCs are prepared by contacting an antibody or antigen-binding fragment with a linker-drug compound that is preformed by reacting the linker with the drug moiety. The preformed linker-drug compound may or may not be subjected to a purification step prior to contacting with the antibody or antigen-binding fragment. In other embodiments, the antibody or antigen-binding fragment is contacted with the linker and drug moiety in a single reaction mixture, achieving simultaneous formation of covalent bonds between the antibody or antigen-binding fragment and the linker and between the linker and the drug moiety. This method of making an ADC can include contacting an antibody or antigen-binding fragment with an antibody or antigen-binding fragment before adding a linker to the reaction mixture, or vice versa. In certain embodiments, the ADC is made by reacting an antibody or antigen-binding fragment with a linker tethered to a drug moiety, such as an ADL1-herboxidienes splicing modulator (e.g., ADL1-79392) or an ADL5-herboxidienes splicing modulator (e.g., ADL5-0349), under conditions that allow conjugation.

[0419] The ADC prepared as described above may be subjected to a purification step. The purification step may include any biochemical method known in the art for purifying proteins, or any combination of such methods. Such methods include, but are not limited to, tangential flow filtration (TFF), affinity chromatography, ion exchange chromatography, any charge- or isoelectric-based chromatography, mixed-mode chromatography, e.g., CHT (ceramic hydroxyapatite), hydrophobic interaction chromatography, size-exclusion chromatography, dialysis, filtration, selective precipitation, or any combination thereof.

[0420] Therapeutic Uses and Compositions Disclosed herein are methods of using the disclosed ADCs and compositions in treating a subject for a disorder, e.g., a neoplastic disorder. The ADCs may be administered alone or in combination with a second therapeutic agent, and may be administered in any pharmaceutically acceptable formulation, dosage, and administration regimen. ADC therapeutic efficacy may be assessed with respect to toxicity and efficacy indicators and adjusted accordingly. Measures of efficacy include, but are not limited to, cytostatic and / or cytotoxic effects observed in vitro or in vivo, reduction in tumor volume, tumor growth inhibition rate, and / or prolonged survival.

[0421] Methods for determining whether an ADC exerts a cytostatic and / or cytotoxic effect on cells are known. For example, the cytotoxic or cytostatic activity of an ADC can be measured by exposing mammalian cells expressing a target protein to the ADC in cell culture medium, culturing the cells for a period of about 6 hours to about 6 days, and measuring cell viability. Cell-based in vitro assays can also be used to measure viability (proliferation), cytotoxicity, and apoptosis induction (caspase activation) of the ADC.

[0422] A thymidine incorporation assay may be used to determine whether an ADC exerts a cytostatic effect. For example, cancer cells expressing a target antigen plated at a density of 5,000 cells per well of a 96-well plate are cultured for a 72-hour period and treated with 0.5 μCi of thymidine for the last 8 hours of the 72-hour period. 3 H-thymidine can be exposed to cells in culture. 3 H-thymidine incorporation is measured in the presence and absence of the ADC.

[0423] Cytotoxicity may be determined by measuring necrosis or apoptosis (programmed cell death). Necrosis is typically accompanied by increased cell membrane permeability, cell swelling, and membrane rupture. Apoptosis can be quantified, for example, by measuring DNA fragmentation. Commercially available photometric methods for quantitatively determining DNA fragmentation in vitro are available. Examples of such assays, including TUNEL (which detects the incorporation of labeled nucleotides into fragmented DNA) and ELISA-based assays, are described in Biochemica (1999) No. 2, pp. 34-37 (Roche Molecular Biochemicals).

[0424] Apoptosis may also be determined by measuring morphological changes in cells. For example, similar to necrosis, loss of cell membrane integrity can be determined by measuring the uptake of certain dyes (e.g., fluorescent dyes such as acridine orange or ethidium bromide). Methods for measuring the number of apoptotic cells are described by Duke and Cohen, *Current Protocols in Immunology* (Coligan et al., eds. (1992) pp. 3.17.1-3.17.16). Cells can also be labeled with DNA dyes (e.g., acridine orange, ethidium bromide, or propidium iodide) and observed for chromatin condensation and margination along the inner nuclear membrane. Apoptosis may also be determined, in some embodiments, by screening for caspase activity. In some embodiments, the Caspase-Glo® assay can be used to measure caspase-3 and caspase-7 activity. In some embodiments, the assay provides a luminescent caspase-3 / 7 substrate in a reagent optimized for caspase activity, luciferase activity, and cell lysis. In some embodiments, addition of the Caspase-Glo® 3 / 7 Reagent in an "add-mix-measure" format can result in cell lysis, followed by caspase cleavage of the substrate and the generation of a "glow-type" luminescent signal by the luciferase. In some embodiments, luminescence can be proportional to the amount of caspase activity present and can serve as an indicator of apoptosis. Other morphological changes that can be measured to determine apoptosis include, for example, cytoplasmic condensation, increased membrane blebbing, and cell shrinkage. Determining any of these effects on cancer cells indicates that the ADC is useful in treating cancer.

[0425] Cell viability may be measured, for example, by determining the uptake of a dye, such as neutral red, trypan blue, crystal violet, or ALAMAR™ blue, into cells (see, e.g., Page et al. (1993) Intl J Oncology 3:473-6). In such assays, cells are incubated in medium containing the dye, the cells are washed, and the residual dye, which reflects cellular uptake of the dye, is measured spectrophotometrically. Cell viability may also be measured, for example, by quantifying ATP, an indicator of metabolically active cells. In certain embodiments, the in vitro potency and / or cell viability of prepared ADC or herboxidiene splicing modulator compounds may be determined using the CellTiter-Glo® luminescent cell viability assay, as described in the examples provided herein. In this assay, in certain embodiments, a single reagent (CellTiter-Glo® Reagent) is added directly to cells cultured in serum-supplemented medium. Addition of the reagent results in cell lysis and the production of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in culture. The protein-binding dye sulforhodamine B (SRB) can also be used to measure cytotoxicity (Skehan et al. (1990) J Natl Cancer Inst. 82:1107-12).

[0426] The disclosed ADCs may also be evaluated for bystander killing activity. Bystander killing activity may be determined, for example, by an assay using two cell lines, one target antigen-positive and one target antigen-negative. In certain embodiments, the assay design allows for tracking of only target-negative cells. In certain embodiments, cells are plated under three conditions: (i) target-negative cells alone (tagged or labeled); (ii) target-positive cells alone; and (iii) coculture of target-negative and target-positive cells. The cells are then treated with the ADC, followed by monitoring cytotoxicity. Reading the plate with CellTiter-Glo® reagent monitors the viability of all cell populations. Reading the plate with OneGlo® reagent provides a signal only from tagged or labeled target-negative cells. Killing of target-negative cells when mixed with target-positive cells is indicative of bystander killing, while killing of target-negative cells in the absence of target-positive cells is indicative of off-target killing.

[0427] In certain aspects, the present disclosure features a method for killing, inhibiting or modulating the growth of, or disrupting the metabolism of cancer cells or tissues by disrupting RNA splicing. The method can be used in any subject who would benefit from disrupted RNA splicing. Subjects who may benefit from disrupted RNA splicing include, but are not limited to, those with or at risk of having a neoplastic disorder, such as a hematological malignancy or solid tumor. In certain embodiments, the hematological malignancy is a B-cell malignancy, a cancer of the blood (leukemia), a cancer of plasma cells (myeloma, e.g., multiple myeloma), or a cancer of the lymph nodes (lymphoma). In certain embodiments, the hematological malignancy is acute myeloid leukemia or multiple myeloma. In certain embodiments, the leukemia is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelocytic leukemia (CML), chronic myelomonocytic leukemia (CMML), or acute monocytic leukemia (AMoL). In certain embodiments, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma. In certain embodiments, the hematological malignancy is myelodysplastic syndrome (MDS). In certain embodiments, the solid tumor is a carcinoma such as breast cancer, pancreatic cancer, prostate cancer, colon or colorectal cancer, lung cancer, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, cholangiocarcinoma, glioma, or melanoma. In certain embodiments, the solid tumor is breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., serous endometrial carcinoma), salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colorectal cancer, or esophageal cancer. In certain embodiments, the lung cancer is lung adenocarcinoma. In certain embodiments, the uterine cancer is serous endometrial carcinoma.

[0428] In various embodiments, the disclosed ADCs may be administered in any cell or tissue that expresses HER2, such as a HER2-expressing neoplastic cell or tissue. Exemplary embodiments include methods of inhibiting HER2-mediated cell signaling or killing cells. The methods can be used in any cell or tissue that expresses HER2, such as cancerous cells or metastatic lesions. Non-limiting examples of cancers that express HER2 include breast cancer, gastric cancer, bladder cancer, urothelial cell carcinoma, esophageal cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., serous endometrial carcinoma), salivary duct cancer, cervical cancer, endometrial cancer, and ovarian cancer (English et al. (2013) Mol Diagn Ther. 17:85-99). Non-limiting examples of HER2-expressing cells include HCC1954 and SKBR3 human breast ductal carcinoma cells, N87 human gastric carcinoma cells, and cells containing a recombinant nucleic acid encoding HER2 or a portion thereof.

[0429] In various embodiments, the disclosed ADCs may be administered in any cell or tissue that expresses CD138, such as a CD138-expressing neoplastic cell or tissue. Exemplary embodiments include methods of inhibiting CD138-mediated cell signaling or killing cells. The methods can be used in any cell or tissue that expresses CD138, such as cancerous cells or metastatic lesions. Non-limiting examples of cancers that express CD138 include intrathoracic cancers (e.g., lung cancer, mesothelioma), skin...

Claims

1. Formula (I): Ab-(L-H) ｐ (I) [In the formula: Ab is an antibody or antigen-binding fragment that targets neoplastic cells; H is a herboxidienes splicing modulator; L is a linker that covalently links Ab to H; and p is an integer from 1 to 15. Antibody-drug conjugates of

2. A compound of formula (I) wherein the herboxidiene splicing modulator is covalently attached to L through any atom: 【Chemical 1】 or a pharmaceutically acceptable salt thereof, wherein: Y is O, S, NR ６ , and C.R. ６ R ７ Selected from: R １ , R ２ , and R ３ are each independently hydrogen, hydroxyl, —O—(C １ ~C ６ alkyl) group, —O—C(═O)—(C １ ~C ６ alkyl) group, —C(═O)—O—(C １ ~C ６ alkyl) groups, and C １ ~C ６ alkyl groups; R ４ is hydrogen, C １ ~C ６ Alkyl group, —C(═O)—(C １ ~C ６ alkyl) group, —C(═O)—(C ３ ~C ８ carbocyclyl group, —C(═O)—(C ３ ~C ８ heterocyclyl group, and —C(═O)—NR ６ R ７ Selected from: R ５ is hydrogen, hydroxyl, -CH ２ —OH, —CO ２ H, -C(=O)-O-(C １ ~C ６ alkyl) group, —C(═O)—NR ６ R ７ , -NR ６ -C(=O)-R ８ , -OC(=O)-NR ６ R ７ , -NR ６ -C(=O)-R ８ , and -NR ６ —C(═O)—NR ６ R ７ Selected from: R ６ and R ７ are each independently hydrogen, —R ８ , —C(═O)—R ８ and —C(═O)—O—R ８ is selected from: R ８ is C １ ~C ６ Alkyl group, C ３ ~C ８ Carbocyclyl groups, and C ３ ~C ８ heterocyclyl groups, Here, R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , and R ８ are each independently a halogen, a hydroxyl, or a C １ ~C ６ alkyl group, —O—(C １ ~C ６ alkyl group, —CO ２ H, -C(=O)-(C １ ~C ６ alkyl) group, —C(═O)—(C ３ ~C ８ carbocyclyl group, —C(═O)—(C ３ ~C ８ heterocyclyl group, —NR ６ R ７ , C ３ ~C ８ Carbocyclyl group, C １ ~C ６ Alkylhydroxy group, C １ ~C ６ Alkylalkoxy group, benzyl group, and C ３ ~C ８ Heterocyclyl groups, each of which may be selected from halogen, hydroxyl, C １ ~C ３ Alkyl group, C １ ~C ３ Alkoxy group, C １ ~C ３ Haloalkyl groups, —NH—C(═O)(C １ ~C ３ alkyl), and —NH—C(═O)—O—(C １ ~C ３ alkyl), and wherein the valence of the atom covalently attached to L does not exceed the valence of the atom.

3. A compound of formula (Ia) wherein the herboxidiene splicing modulator is covalently attached to L through any atom: 【Chemistry 2】 or a pharmaceutically acceptable salt thereof, wherein: R ９ is C ３ ~C ８ heterocyclyl groups; R １０ is H and C １ ~C ６ alkyl groups, Here, R ９ and R １０ are each independently a halogen, a hydroxyl, or C １ ~C ３ Alkyl group, C １ ~C ３ Alkoxy group, —NH ２ , —NH—(C １ ~C ３ alkyl), and —N—(C １ ~C ３ alkyl) ２ and wherein the valence of the atom covalently attached to L does not exceed the valence of the atom.

4. A compound of formula (Ib) wherein the herboxidiene splicing modulator is covalently attached to L through any atom: 【Chemistry 3】 or a pharmaceutically acceptable salt thereof, wherein: R １１ teeth, 【Chemistry 4】 (In the formula, ＊ is R １１ represents the point of attachment to the remainder of the compound; R １２ and R １３ are each independently selected from H and methyl; and wherein the valence of the atom covalently attached to L does not exceed the valence of the atom.

5. the herboxidiene splicing modulator is covalently attached to L via any atom; the herboxidienes splicing modulator is selected from H1, H2, H3, and H12; and The antibody-drug conjugate of any one of claims 1 to 4, wherein the valence of the atom covalently attached to L is not exceeded.

6. L is covalently linked to said herboxidiene splicing modulator ("LH"), and LH has the following structure: 【Chemistry 5】 The antibody-drug conjugate of claim 1 or 2, having a structure selected from:

7. The herboxidiene splicing modulator is covalently attached to L through any atom of formula (II): 【Chemistry 6】 or a pharmaceutically acceptable salt thereof, wherein: X is hydroxyl or NR ６ R ７ and R ６ and R ７ are each independently hydrogen, —R ８ , —C(═O)—R ８ , -C(=O)-OR ８ , -(C １ ~C ６ alkyl)-O-C(=O)-R ８ , and -(C １ ~C ６ alkyl)-NH-C(=O)-R ８ is selected from: R ８ is C １ ~C ６ Alkyl group, C ３ ~C ８ Carbocyclyl groups, and C ３ ~C ８ heterocyclyl groups, Here, R ６ , R ７ , and R ８ are each independently a halogen, a hydroxyl, or C １ ~C ６ alkyl group, —O—(C １ ~C ６ alkyl group, —CO ２ H, -C(=O)-O-(C １ ~C ６ alkyl) group, —C(═O)—(C １ ~C ６ alkyl) group, —C(═O)—(C ３ ~C ８ carbocyclyl group, —C(═O)—(C ３ ~C ８ heterocyclyl group, —NR ６ R ７ , C ３ ~C ８ Carbocyclyl group, C １ ~C ６ Alkylhydroxy group, C １ ~C ６ Alkylalkoxy group, benzyl group, and C ３ ~C ８ Heterocyclyl groups, each of which may be selected from halogen, hydroxyl, C １ ~C ３ Alkyl group, C １ ~C ３ Alkoxy group, C １ ~C ３ Haloalkyl groups, —NH—C(═O)(C １ ~C ３ alkyl), and —NH—C(═O)—O—(C １ ~C ３ alkyl), and wherein the valency of the atom covalently attached to L does not exceed that of the atom covalently attached to L.

8. The herboxidiene splicing modulator of formula (IIa) is covalently attached to L through any atom: 【Chemistry 7】 or a pharmaceutically acceptable salt thereof, wherein: Z is NR ９ and O; R ９ is hydrogen and C １ ~C ６ alkyl groups; R １０ and R １１ are each independently hydrogen, halogen, hydroxyl, C １ ~C ６ alkyl group, —O—(C １ ~C ６ alkyl group, —CO ２ H, -C(=O)-O-(C １ ~C ６ alkyl) group, —C(═O)—(C １ ~C ６ alkyl) group, —C(═O)—(C ３ ~C ８ carbocyclyl group, —C(═O)—(C ３ ~C ８ heterocyclyl group, C ３ ~C ８ Carbocyclyl group, C １ ~C ６ Alkylhydroxy group, C １ ~C ６ Alkylalkoxy group, benzyl group, and C ３ ~C ８ heterocyclyl groups; R １２ is C １ ~C ６ Alkyl group, C ３ ~C ８ Carbocyclyl group, C ３ ~C ８ heterocyclyl groups, Here, R ９ , R １０ , R １１ , and R １２ are each independently a halogen, a hydroxyl, or a C １ ~C ３ Alkyl group, C １ ~C ３ Alkoxy groups, and C １ ~C ３ substituted with 0 or 1 group selected from haloalkyl groups; t is an integer selected from 1, 2, 3, 4, 5, and 6; and wherein the valency of the atom covalently attached to L does not exceed the valency of the atom covalently attached to L.

9. The herboxidiene splicing modulator of formula (IIb) is covalently attached to L through any atom: 【Chemistry 8】 or a pharmaceutically acceptable salt thereof, wherein: R １３ teeth, 【Chemistry 9】 (In the formula, ＊ is R １３ represents the point of attachment to the remainder of the compound; R １４ and R １５ are each independently selected from hydrogen and methyl; and wherein the valency of the atom covalently attached to L does not exceed the valency of the atom covalently attached to L.

10. the herboxidienesplicing modulator is covalently attached to L via any atom; the herboxidienes splicing modulator is selected from H4, H13, H14, H15, H16, H17, H18, H19, H20, H21, H23, and H24; and 10. The antibody-drug conjugate of claim 1, 7, 8, or 9, wherein the valence of the atom covalently attached to L is not exceeded.

11. L is covalently linked to said herboxidiene splicing modulator ("LH"), and LH has the following structure: 【Chemistry 10】 and a pharmaceutically acceptable salt thereof.

12. The herboxidiene splicing modulator is covalently attached to L through any atom of formula (III): 【Chemistry 11】 or a pharmaceutically acceptable salt thereof, wherein: R １ , R ２ , and R ３ are each independently hydrogen, hydroxyl, —O—(C １ ~C ６ alkyl) group, —O—C(═O)—(C １ ~C ６ alkyl) group, —C(═O)—O—(C １ ~C ６ alkyl) groups, and C １ ~C ６ alkyl groups; R ６ and R ７ are each independently hydrogen, —R ８ , —C(═O)—R ８ and —C(═O)—O—R ８ Selected from: R ８ is C １ ~C ６ Alkyl group, C ３ ~C ８ Carbocyclyl groups, and C ３ ~C ８ heterocyclyl groups; and R ９ H, 【Chemistry 12】 Selected from: Here, R １ , R ２ , R ３ , R ６ , R ７ , and R ８ are each independently a halogen, a hydroxyl, or a C １ ~C ６ alkyl group, —O—(C １ ~C ６ alkyl group, —CO ２ H, -C(=O)-(C １ ~C ６ alkyl) group, —C(═O)—(C ３ ~C ８ carbocyclyl group, —C(═O)—(C ３ ~C ８ heterocyclyl group, —NR ６ R ７ , C ３ ~C ８ Carbocyclyl group, C １ ~C ６ Alkylhydroxy group, C １ ~C ６ Alkylalkoxy group, benzyl group, and C ３ ~C ８ Heterocyclyl groups, each of which may be selected from halogen, hydroxyl, C １ ~C ３ Alkyl group, C １ ~C ３ Alkoxy group, C １ ~C ３ Haloalkyl groups, —NH—C(═O)(C １ ~C ３ alkyl), and —NH—C(═O)—O—(C １ ~C ３ substituted with 0 to 3 groups independently selected from (alkyl), wherein the valence of said atom covalently bonded to L is not exceeded; and During the ceremony, ＊ is R ９ represents the point of attachment to the remainder of the compound.

13. the herboxidienesplicing modulator is covalently attached to L via any atom; the herboxidienes splicing modulator is selected from H5, H6, H7, H8, H9, H10, H11, H22, and H25; and The antibody-drug conjugate of claim 1 or 12, wherein the valence of the atom covalently attached to L is not exceeded.

14. The linker is covalently attached to the herboxidiene splicing modulator ("LH"), and LH has the following structure: 【Chemistry 13】 and a pharmaceutically acceptable salt thereof.

15. the herboxidienesplicing modulator is covalently attached to L via any atom; the herboxidienes splicing modulator is selected from H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18, H19, H20, H21, H22, H23, H24, and H25; and The antibody-drug conjugate of claim 1 , wherein the valence of the atom covalently attached to L is not exceeded.

16. The antibody-drug conjugate of claim 15, wherein the herboxidian splicing modulator is H1.

17. The antibody-drug conjugate of claim 15, wherein the herboxidian splicing modulator is H2.

18. The antibody-drug conjugate of claim 15, wherein the herboxidienes splicing modulator is H3.

19. The antibody-drug conjugate of claim 15, wherein the herboxidienes splicing modulator is H4.

20. The antibody-drug conjugate of claim 15, wherein the herboxidienes splicing modulator is H5.

21. The antibody-drug conjugate of claim 15, wherein the herboxidienes splicing modulator is H6.

22. The antibody-drug conjugate of claim 15, wherein the herboxidienes splicing modulator is H7.

23. The antibody-drug conjugate of claim 15, wherein the herboxidienes splicing modulator is H8.

24. The antibody-drug conjugate of claim 15, wherein the herboxidian splicing modulator is H9.

25. The antibody-drug conjugate of claim 15, wherein the herboxidienes splicing modulator is H10.

26. The antibody-drug conjugate of claim 15, wherein the herboxidian splicing modulator is H11.

27. The antibody-drug conjugate of claim 15, wherein the herboxidian splicing modulator is H12.

28. The antibody-drug conjugate of claim 15, wherein the herboxidienes splicing modulator is H13.

29. The antibody-drug conjugate of claim 15, wherein the herboxidienes splicing modulator is H14.

30. The antibody-drug conjugate of claim 15, wherein the herboxidienes splicing modulator is H15.

31. The antibody-drug conjugate of claim 15, wherein the herboxidienes splicing modulator is H16.

32. The antibody-drug conjugate of claim 15, wherein the herboxidienes splicing modulator is H17.

33. The antibody-drug conjugate of claim 15, wherein the herboxidienes splicing modulator is H18.

34. The antibody-drug conjugate of claim 15, wherein the herboxidian splicing modulator is H19.

35. The antibody-drug conjugate of claim 15, wherein the herboxidian splicing modulator is H20.

36. The antibody-drug conjugate of claim 15, wherein the herboxidian splicing modulator is H21.

37. The antibody-drug conjugate of claim 15, wherein the herboxidian splicing modulator is H22.

38. The antibody-drug conjugate of claim 15, wherein the herboxidian splicing modulator is H23.

39. The antibody-drug conjugate of claim 15, wherein the herboxidienes splicing modulator is H24.

40. The antibody-drug conjugate of any one of claims 1 to 39, wherein the linker is a cleavable linker.

41. The antibody-drug conjugate of claim 40, wherein the linker comprises a cleavable peptide moiety.

42. The antibody-drug conjugate of claim 41, wherein the cleavable peptide moiety is enzymatically cleavable.

43. The antibody-drug conjugate of any one of claims 40 to 42, wherein the cleavable peptide moiety or linker comprises an amino acid unit.

44. 44. The antibody-drug conjugate of claim 43, wherein the amino acid unit comprises valine-citrulline (Val-Cit).

45. 44. The antibody-drug conjugate of claim 43, wherein the amino acid unit comprises valine-alanine (Val-Ala).

46. 44. The antibody-drug conjugate of claim 43, wherein the amino acid unit comprises glutamine-valine-citrulline (Glu-Val-Cit).

47. 44. The antibody-drug conjugate of claim 43, wherein the amino acid unit comprises alanine-alanine-asparagine (Ala-Ala-Asn).

48. The antibody-drug conjugate of claim 40, wherein the linker comprises a cleavable glucuronide moiety.

49. 49. The antibody-drug conjugate of claim 48, wherein the cleavable glucuronide moiety is enzymatically cleavable.

50. 50. The antibody-drug conjugate of claim 48 or 49, wherein the cleavable glucuronide moiety is cleavable by a glucuronidase.

51. The antibody-drug conjugate of any one of claims 48 to 50, wherein the cleavable glucuronide moiety is cleavable by β-glucuronidase.

52. The antibody-drug conjugate of any one of claims 1 to 51, wherein the linker comprises at least one spacer unit.

53. 53. The antibody-drug conjugate of claim 52, wherein the spacer unit or linker comprises a polyethylene glycol (PEG) moiety.

54. The PEG moiety is -(PEG) ｍ -, wherein m is an integer from 1 to 10.

55. The antibody-drug conjugate of claim 54, wherein m is 2.

56. 53. The antibody-drug conjugate of claim 52, wherein the spacer unit or linker comprises an alkyl moiety.

57. The alkyl portion is —(CH ２ ) ｎ -, wherein n is an integer from 1 to 10.

58. 58. The antibody-drug conjugate of claim 57, wherein n is 2.

59. 58. The antibody-drug conjugate of claim 57, wherein n is 5.

60. 58. The antibody-drug conjugate of claim 57, wherein n is 6.

61. 61. The antibody-drug conjugate of any one of claims 52 to 60, wherein the spacer unit is attached to the antibody or antigen-binding fragment via a maleimide (Mal) moiety ("Mal-spacer unit").

62. 62. The antibody-drug conjugate of claim 61, wherein the Mal-spacer unit is reactive with a cysteine ​​residue on the antibody or antigen-binding fragment.

63. 63. The antibody-drug conjugate of claim 61 or 62, wherein the Mal-spacer unit is attached to the antibody or antigen-binding fragment via a cysteine ​​residue on the antibody or antigen-binding fragment.

64. 64. The antibody-drug conjugate of any one of claims 61 to 63, wherein the linker comprises the Mal-spacer unit and a cleavable peptide moiety.

65. 65. The antibody-drug conjugate of claim 64, wherein the cleavable peptide moiety comprises an amino acid unit.

66. 66. The antibody-drug conjugate of claim 64 or 65, wherein the cleavable peptide moiety or amino acid unit comprises Val-Cit.

67. 66. The antibody-drug conjugate of claim 64 or 65, wherein the cleavable peptide moiety or amino acid unit comprises Val-Ala.

68. 66. The antibody-drug conjugate of claim 64 or 65, wherein the cleavable peptide moiety or amino acid unit comprises Glu-Val-Cit.

69. 66. The antibody-drug conjugate of claim 64 or 65, wherein the cleavable peptide moiety or amino acid unit comprises Ala-Ala-Asn.

70. 70. The antibody-drug conjugate of any one of claims 61 to 69, wherein the Mal-spacer unit comprises an alkyl moiety.

71. 70. The antibody-drug conjugate of any one of claims 61 to 69, wherein the Mal-spacer unit comprises a PEG moiety.

72. 72. The antibody-drug conjugate of any one of claims 61 to 71, wherein the Mal-spacer unit comprises maleimidocaproyl (MC).

73. 73. The antibody-drug conjugate of any one of claims 61 to 72, wherein the Mal-spacer unit links the antibody or antigen-binding fragment to the cleavable portion of the linker.

74. 74. The antibody-drug conjugate of claim 73, wherein the cleavable portion of the linker comprises a cleavable peptide moiety.

75. 75. The antibody-drug conjugate of claim 74, wherein the cleavable peptide moiety comprises an amino acid unit.

76. 76. The antibody-drug conjugate of claim 74 or 75, wherein the cleavable peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn.

77. The antibody-drug conjugate of any one of claims 73 to 76, wherein the linker comprises MC-Val-Cit.

78. The antibody-drug conjugate of any one of claims 73 to 76, wherein the linker comprises MC-Val-Ala.

79. The antibody-drug conjugate of any one of claims 73 to 76, wherein the linker comprises MC-Glu-Val-Cit.

80. The antibody-drug conjugate of any one of claims 73 to 76, wherein the linker comprises MC-Ala-Ala-Asn.

81. The antibody-drug conjugate of any one of claims 73 to 80, wherein the Mal-spacer unit comprises an alkyl moiety.

82. The antibody-drug conjugate of any one of claims 73 to 80, wherein the Mal-spacer unit comprises a PEG moiety.

83. The antibody-drug conjugate of any one of claims 73 to 82, wherein the Mal-spacer unit comprises maleimidocaproyl (MC).

84. The antibody-drug conjugate of any one of claims 52 to 83, wherein the cleavable portion of the linker is directly joined to the splicing regulator or a spacer unit connects the cleavable portion of the linker to the splicing regulator.

85. The antibody-drug conjugate of claim 84, wherein cleavage of the conjugate releases the splicing modulator from the antibody or antigen-binding fragment and the linker.

86. 86. The antibody-drug conjugate of claim 84 or 85, wherein the spacer unit connecting the cleavable portion of the linker to the splicing modulator is self-immolative.

87. 87. The antibody-drug conjugate of any one of claims 84 to 86, wherein the spacer unit connecting the cleavable portion of the linker to the splicing modulator comprises p-aminobenzyloxycarbonyl (pABC).

88. 88. The antibody-drug conjugate of claim 87, wherein the pABC links the cleavable portion of the linker to the splicing modulator.

89. 89. The antibody-drug conjugate of claim 87 or 88, wherein the cleavable portion of the linker comprises a cleavable peptide moiety.

90. 90. The antibody-drug conjugate of claim 89, wherein the cleavable peptide moiety comprises an amino acid unit.

91. 91. The antibody-drug conjugate of claim 89 or 90, wherein the cleavable peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn.

92. The antibody-drug conjugate of any one of claims 87 to 91, wherein the linker comprises Val-Cit-pABC.

93. The antibody-drug conjugate of any one of claims 87 to 91, wherein the linker comprises Val-Ala-pABC.

94. The antibody-drug conjugate of any one of claims 87 to 91, wherein the linker comprises Glu-Val-Cit-pABC.

95. The antibody-drug conjugate of any one of claims 87 to 91, wherein the linker comprises Ala-Ala-Asn-pABC.

96. 87. The antibody-drug conjugate of any one of claims 84 to 86, wherein the spacer unit connecting the cleavable portion of the linker to the splicing modulator comprises p-aminobenzyl (pAB).

97. 97. The antibody-drug conjugate of claim 96, wherein the pAB links the cleavable portion of the linker to the splicing modulator.

98. 98. The antibody-drug conjugate of claim 96 or 97, wherein the cleavable portion of the linker comprises a cleavable peptide moiety.

99. 99. The antibody-drug conjugate of claim 98, wherein the cleavable peptide moiety comprises an amino acid unit.

100. 100. The antibody-drug conjugate of claim 98 or 99, wherein the cleavable peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn.

101. The antibody-drug conjugate of any one of claims 96 to 100, wherein the linker comprises Val-Cit-pAB.

102. The antibody-drug conjugate of any one of claims 96 to 100, wherein the linker comprises Val-Ala-pAB.

103. The antibody-drug conjugate of any one of claims 96 to 100, wherein the linker comprises Glu-Val-Cit-pAB.

104. The antibody-drug conjugate of any one of claims 96 to 100, wherein the linker comprises Ala-Ala-Asn-pAB.

105. The antibody-drug conjugate of any one of claims 1 to 39, wherein the linker is a non-cleavable linker.

106. The antibody-drug conjugate of claim 105, wherein the linker comprises at least one spacer unit.

107. The antibody-drug conjugate of claim 105 or 106, wherein the spacer unit or linker comprises a polyethylene glycol (PEG) moiety.

108. The PEG moiety is -(PEG) ｍ -, wherein m is an integer from 1 to 10.

109. The antibody-drug conjugate of claim 108, wherein m is 2.

110. The antibody-drug conjugate of claim 105 or 106, wherein the spacer unit or linker comprises an alkyl moiety.

111. The alkyl portion is —(CH ２ ) ｎ -, wherein n is an integer from 1 to 10.

112. The antibody-drug conjugate of claim 111, wherein n is 2.

113. The antibody-drug conjugate of claim 111, wherein n is 5.

114. The antibody-drug conjugate of claim 111, wherein n is 6.

115. 115. The antibody-drug conjugate of any one of claims 106 to 114, wherein the spacer unit is attached to the antibody or antigen-binding fragment via a maleimide (Mal) moiety ("Mal-spacer unit").

116. The antibody-drug conjugate of claim 115, wherein the Mal-spacer unit is reactive with a cysteine ​​residue on the antibody or antigen-binding fragment.

117. 117. The antibody-drug conjugate of claim 115 or 116, wherein the Mal-spacer unit is attached to the antibody or antigen-binding fragment via a cysteine ​​residue on the antibody or antigen-binding fragment.

118. The antibody-drug conjugate of any one of claims 115 to 117, wherein the Mal-spacer unit comprises an alkyl moiety.

119. The antibody-drug conjugate of any one of claims 115 to 117, wherein the Mal-spacer unit comprises a PEG moiety.

120. 120. The antibody-drug conjugate of any one of claims 115 to 119, wherein the linker or Mal-spacer unit comprises maleimidocaproyl (MC).

121. The antibody-drug conjugate of claim 120, wherein the linker or Mal-spacer unit comprises maleimidocaproyl (MC) and at least one additional spacer unit.

122. The linker or Mal-spacer unit is MC-(PEG) ２ 120. The antibody-drug conjugate of any one of claims 115 to 119, comprising:

123. The linker or Mal-spacer unit is MC-(PEG) ２ and at least one additional spacer unit.

124. 120. The antibody-drug conjugate of any one of claims 115 to 119, wherein the linker or Mal-spacer unit comprises Mal-Hex.

125. The antibody-drug conjugate of claim 124, wherein the linker or Mal-spacer unit comprises Mal-Hex and at least one additional spacer unit.

126. 120. The antibody-drug conjugate of any one of claims 115 to 119, wherein the linker or Mal-spacer unit comprises Mal-Et.

127. The antibody-drug conjugate of claim 126, wherein the linker or Mal-spacer unit comprises Mal-Et and at least one additional spacer unit.

128. 120. The antibody-drug conjugate of any one of claims 115 to 119, wherein the linker or Mal-spacer unit comprises Mal-Et-O-Et.

129. 129. The antibody-drug conjugate of claim 128, wherein the linker or Mal-spacer unit comprises Mal-Et-O-Et and at least one additional spacer unit.

130. 130. The antibody-drug conjugate of any one of claims 115 to 129, wherein the Mal-spacer unit links the antibody or antigen-binding fragment to the splicing modulator.

131. The antibody-drug conjugate of any one of claims 115 to 130, wherein the linker comprises MC-Val-Cit-pABC.

132. The linker is MC-(PEG). ２ The antibody-drug conjugate of any one of claims 115 to 130, comprising -CO.

133. 133. The antibody-drug conjugate of any one of claims 1 to 132, wherein the antibody or antigen-binding fragment targets neoplastic cells derived from a hematological malignancy or a solid tumor.

134. 134. The antibody-drug conjugate of any one of claims 1 to 133, wherein the antibody or antigen-binding fragment targets neoplastic cells derived from a hematological malignancy.

135. 135. The antibody-drug conjugate of claim 133 or 134, wherein the hematological malignancy is selected from a B-cell malignancy, leukemia, lymphoma, and myeloma.

136. 136. The antibody-drug conjugate of any one of claims 133 to 135, wherein the hematological malignancy is selected from acute myeloid leukemia and multiple myeloma.

137. The antibody-drug conjugate of any one of claims 1 to 133, wherein the antibody or antigen-binding fragment targets neoplastic cells derived from a solid tumor.

138. 138. The antibody-drug conjugate of claim 133 or 137, wherein the solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colorectal cancer, and esophageal cancer.

139. The antibody-drug conjugate of any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets a HER2-expressing cell.

140. 140. The antibody-drug conjugate of claim 139, wherein the antibody or antigen-binding fragment is an anti-HER2 antibody or antigen-binding fragment.

141. The antibody-drug conjugate of claim 139 or 140, wherein the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2 (HCDR2), and SEQ ID NO: 3 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising the amino acid sequences of SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO: 6 (LCDR3).

142. The antibody-drug conjugate of any one of claims 139 to 141, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

20.

143. The antibody-drug conjugate of any one of claims 139 to 142, wherein the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region.

144. The antibody-drug conjugate of any one of claims 139 to 143, wherein the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

145. The antibody-drug conjugate of any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets CD138-expressing cells.

146. The antibody-drug conjugate of claim 145, wherein the antibody or antigen-binding fragment is an anti-CD138 antibody or antigen-binding fragment.

147. The antibody-drug conjugate of claim 145 or 146, wherein the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising the amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2), and SEQ ID NO:12 (LCDR3).

148. The antibody-drug conjugate of any one of claims 145 to 147, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

22.

149. The antibody-drug conjugate of any one of claims 145 to 148, wherein the antibody or antigen-binding fragment comprises a human IgG2a heavy chain constant region.

150. 150. The antibody-drug conjugate of any one of claims 145 to 149, wherein the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

151. The antibody-drug conjugate of any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets an EPHA2-expressing cell.

152. The antibody-drug conjugate of claim 151, wherein the antibody or antigen-binding fragment is an anti-EPHA2 antibody or antigen-binding fragment.

153. The antibody-drug conjugate of claim 151 or 152, wherein the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising the amino acid sequences of SEQ ID NO: 13 (HCDR1), SEQ ID NO: 14 (HCDR2), and SEQ ID NO: 15 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising the amino acid sequences of SEQ ID NO: 16 (LCDR1), SEQ ID NO: 17 (LCDR2), and SEQ ID NO: 18 (LCDR3).

154. The antibody-drug conjugate of any one of claims 151 to 153, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

24.

155. The antibody-drug conjugate of any one of claims 151 to 154, wherein the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region.

156. The antibody-drug conjugate of any one of claims 151 to 155, wherein the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

157. The antibody-drug conjugate of any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets MSLN-expressing cells.

158. The antibody-drug conjugate of claim 157, wherein the antibody or antigen-binding fragment is an anti-MSLN antibody or antigen-binding fragment.

159. The antibody-drug conjugate of any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets FOLH1-expressing cells.

160. The antibody-drug conjugate of claim 159, wherein the antibody or antigen-binding fragment is an anti-FOLH1 antibody or antigen-binding fragment.

161. The antibody-drug conjugate of any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets a CDH6-expressing cell.

162. The antibody-drug conjugate of claim 161, wherein the antibody or antigen-binding fragment is an anti-CDH6 antibody or antigen-binding fragment.

163. The antibody-drug conjugate of any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets CEACAM5-expressing cells.

164. The antibody-drug conjugate of claim 163, wherein the antibody or antigen-binding fragment is an anti-CEACAM5 antibody or antigen-binding fragment.

165. The antibody-drug conjugate of any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets CFC1B-expressing cells.

166. The antibody-drug conjugate of claim 165, wherein the antibody or antigen-binding fragment is an anti-CFC1B antibody or antigen-binding fragment.

167. The antibody-drug conjugate of any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets an ENPP3-expressing cell.

168. The antibody-drug conjugate of claim 167, wherein the antibody or antigen-binding fragment is an anti-ENPP3 antibody or antigen-binding fragment.

169. The antibody-drug conjugate of any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets FOLR1-expressing cells.

170. The antibody-drug conjugate of claim 169, wherein the antibody or antigen-binding fragment is an anti-FOLR1 antibody or antigen-binding fragment.

171. The antibody-drug conjugate of any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets HAVCR1-expressing cells.

172. The antibody-drug conjugate of claim 171, wherein the antibody or antigen-binding fragment is an anti-HAVCR1 antibody or antigen-binding fragment.

173. The antibody-drug conjugate of any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets KIT-expressing cells.

174. The antibody-drug conjugate of claim 173, wherein the antibody or antigen-binding fragment is an anti-KIT antibody or antigen-binding fragment.

175. The antibody-drug conjugate of any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets a MET-expressing cell.

176. 176. The antibody-drug conjugate of claim 175, wherein the antibody or antigen-binding fragment is an anti-MET antibody or antigen-binding fragment.

177. The antibody-drug conjugate of any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets MUC16-expressing cells.

178. The antibody-drug conjugate of claim 177, wherein the antibody or antigen-binding fragment is an anti-MUC16 antibody or antigen-binding fragment.

179. The antibody-drug conjugate of any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets SLC39A6-expressing cells.

180. 180. The antibody-drug conjugate of claim 179, wherein the antibody or antigen-binding fragment is an anti-SLC39A6 antibody or antigen-binding fragment.

181. The antibody-drug conjugate of any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets SLC44A4-expressing cells.

182. The antibody-drug conjugate of claim 181, wherein the antibody or antigen-binding fragment is an anti-SLC44A4 antibody or antigen-binding fragment.

183. The antibody-drug conjugate of any one of claims 1 to 138, wherein the antibody or antigen-binding fragment targets STEAP1-expressing cells.

184. The antibody-drug conjugate of claim 183, wherein the antibody or antigen-binding fragment is an anti-STEAP1 antibody or antigen-binding fragment.

185. The antibody-drug conjugate of any one of claims 1 to 184, wherein p is 1 to 10.

186. The antibody-drug conjugate of any one of claims 1 to 185, wherein p is 2 to 8.

187. The antibody-drug conjugate of any one of claims 1 to 186, wherein p is 4 to 8.

188. The antibody-drug conjugate of any one of claims 1 to 187, wherein p is 4.

189. The antibody-drug conjugate of any one of claims 1 to 187, wherein p is 8.

190. Compounds of formula (I): 【Chemistry 14】 or a pharmaceutically acceptable salt thereof, wherein: Y is O, S, NR ６ , and C.R. ６ R ７ Selected from: R １ , R ２ , and R ３ are each independently hydrogen, hydroxyl, —O—(C １ ~C ６ alkyl) group, —O—C(═O)—(C １ ~C ６ alkyl) group, —C(═O)—O—(C １ ~C ６ alkyl) groups, and C １ ~C ６ alkyl groups; R ４ is hydrogen, C １ ~C ６ Alkyl group, —C(═O)—(C １ ~C ６ alkyl) group, —C(═O)—(C ３ ~C ８ carbocyclyl group, —C(═O)—(C ３ ~C ８ heterocyclyl group, and —C(═O)—NR ６ R ７ Selected from: R ５ is hydrogen, hydroxyl, -CH ２ —OH, —CO ２ H, -C(=O)-O-(C １ ~C ６ alkyl) group, —C(═O)—NR ６ R ７ , -NR ６ -C(=O)-R ８ , -OC(=O)-NR ６ R ７ , -NR ６ -C(=O)-R ８ , and -NR ６ —C(═O)—NR ６ R ７ Selected from: R ６ and R ７ are each independently hydrogen, —R ８ , —C(═O)—R ８ and —C(═O)—O—R ８ is selected from: R ８ is C １ ~C ６ Alkyl group, C ３ ~C ８ Carbocyclyl groups, and C ３ ~C ８ heterocyclyl groups, Here, R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , and R ８ are each independently a halogen, a hydroxyl, or a C １ ~C ６ alkyl group, —O—(C １ ~C ６ alkyl group, —CO ２ H, -C(=O)-(C １ ~C ６ alkyl) group, —C(═O)—(C ３ ~C ８ carbocyclyl group, —C(═O)—(C ３ ~C ８ heterocyclyl group, —NR ６ R ７ , C ３ ~C ８ Carbocyclyl group, C １ ~C ６ Alkylhydroxy group, C １ ~C ６ Alkylalkoxy group, benzyl group, and C ３ ~C ８ Heterocyclyl groups, each of which may be selected from halogen, hydroxyl, C １ ~C ３ Alkyl group, C １ ~C ３ Alkoxy group, C １ ~C ３ Haloalkyl groups, —NH—C(═O)(C １ ~C ３ alkyl), and —NH—C(═O)—O—(C １ ~C ３ and 0 to 3 groups independently selected from (alkyl), which may be independently substituted with 0 or 1 group selected from (alkyl).

191. Compounds of formula (Ia): 【Chemistry 15】 or a pharmaceutically acceptable salt thereof, wherein: R ９ is C ３ ~C ８ heterocyclyl groups; and R １０ is H and C １ ~C ６ alkyl groups, Here, R ９ and R １０ are each independently a halogen, a hydroxyl, or a C １ ~C ３ Alkyl group, C １ ~C ３ Alkoxy group, —NH ２ , —NH—(C １ ~C ３ alkyl), and —N—(C １ ~C ３ alkyl) ２ 191. The compound of claim 190, wherein:

192. Compound of formula (Ib): 【Chemistry 16】 or a pharmaceutically acceptable salt thereof, wherein: R １１ teeth, 【Chemistry 17】 (In the formula, ＊ is R １１ represents the point of attachment to the remainder of the compound; and R １２ and R １３ are each independently selected from H and methyl.

193. 191. The compound of claim 190, which is H1, or a pharmaceutically acceptable salt thereof.

194. 191. The compound of claim 190, which is H2, or a pharmaceutically acceptable salt thereof.

195. 191. The compound of claim 190, which is H3, or a pharmaceutically acceptable salt thereof.

196. 191. The compound of claim 190, which is H12, or a pharmaceutically acceptable salt thereof.

197. Formula (II): 【Chemistry 18】 or a pharmaceutically acceptable salt thereof, wherein: X is NR ６ R ７ and R ６ and R ７ are each independently hydrogen, —R ８ , —C(═O)—R ８ , -C(=O)-OR ８ , -(C １ ~C ６ alkyl)-O-C(=O)-R ８ , and -(C １ ~C ６ alkyl)-NH-C(=O)-R ８ is selected from: R ８ is C １ ~C ６ Alkyl group, C ３ ~C ８ Carbocyclyl groups, and C ３ ~C ８ heterocyclyl groups, Here, R ６ , R ７ , and R ８ are each independently a halogen, a hydroxyl, or a C １ ~C ６ alkyl group, —O—(C １ ~C ６ alkyl group, —CO ２ H, -C(=O)-O-(C １ ~C ６ alkyl) group, —C(═O)—(C １ ~C ６ alkyl) group, —C(═O)—(C ３ ~C ８ carbocyclyl group, —C(═O)—(C ３ ~C ８ heterocyclyl group, —NR ６ R ７ , C ３ ~C ８ Carbocyclyl group, C １ ~C ６ Alkylhydroxy group, C １ ~C ６ Alkylalkoxy group, benzyl group, and C ３ ~C ８ Heterocyclyl groups, each of which may be selected from halogen, hydroxyl, C １ ~C ３ Alkyl group, C １ ~C ３ Alkoxy group, C １ ~C ３ Haloalkyl groups, —NH—C(═O)(C １ ~C ３ alkyl), and —NH—C(═O)—O—(C １ ~C ３ and 0 to 3 groups independently selected from (alkyl), which may be independently substituted with 0 or 1 group selected from (alkyl).

198. Formula (IIa): 【Chemistry 19】 or a pharmaceutically acceptable salt thereof, wherein: Z is NR ９ and O; R ９ is hydrogen and C １ ~C ６ alkyl groups; R １０ and R １１ are each independently hydrogen, halogen, hydroxyl, C １ ~C ６ alkyl group, —O—(C １ ~C ６ alkyl group, —CO ２ H, -C(=O)-O-(C １ ~C ６ alkyl) group, —C(═O)—(C １ ~C ６ alkyl) group, —C(═O)—(C ３ ~C ８ carbocyclyl group, —C(═O)—(C ３ ~C ８ heterocyclyl group, C ３ ~C ８ Carbocyclyl group, C １ ~C ６ Alkylhydroxy group, C １ ~C ６ Alkylalkoxy group, benzyl group, and C ３ ~C ８ heterocyclyl groups; R １２ is C １ ~C ６ Alkyl group, C ３ ~C ８ Carbocyclyl group, C ３ ~C ８ heterocyclyl groups, Here, R ９ , R １０ , R １１ , and R １２ are each independently a halogen, a hydroxyl, or a C １ ~C ３ Alkyl group, C １ ~C ３ Alkoxy groups, and C １ ~C ３ substituted with 0 or 1 group selected from haloalkyl groups; and t is an integer selected from 1, 2, 3, 4, 5, and 6.

199. Formula (IIb): 【Chemistry 20】 or a pharmaceutically acceptable salt thereof, wherein: R １３ teeth, 【Chemical 21】 (In the formula, ＊ is R １３ represents the point of attachment to the remainder of the compound; and R １４ and R １５ are each independently selected from hydrogen and methyl.

200. 198. The compound of claim 197, which is H4, or a pharmaceutically acceptable salt thereof.

201. 198. The compound of claim 197, which is H13, or a pharmaceutically acceptable salt thereof.

202. 198. The compound of claim 197, which is H14, or a pharmaceutically acceptable salt thereof.

203. 198. The compound of claim 197, which is H15, or a pharmaceutically acceptable salt thereof.

204. 198. The compound of claim 197, which is H16, or a pharmaceutically acceptable salt thereof.

205. 198. The compound of claim 197, which is H17, or a pharmaceutically acceptable salt thereof.

206. 198. The compound of claim 197, which is H18, or a pharmaceutically acceptable salt thereof.

207. 198. The compound of claim 197, which is H19, or a pharmaceutically acceptable salt thereof.

208. 198. The compound of claim 197, which is H20, or a pharmaceutically acceptable salt thereof.

209. 198. The compound of claim 197, which is H21, or a pharmaceutically acceptable salt thereof.

210. 198. The compound of claim 197, which is H23, or a pharmaceutically acceptable salt thereof.

211. 198. The compound of claim 197, which is H24, or a pharmaceutically acceptable salt thereof.

212. Compounds of formula (III): 【Chemical 22】 or a pharmaceutically acceptable salt thereof, wherein: R １ , R ２ , and R ３ are each independently hydrogen, hydroxyl, —O—(C １ ~C ６ alkyl) group, —O—C(═O)—(C １ ~C ６ alkyl) group, —C(═O)—O—(C １ ~C ６ alkyl) groups, and C １ ~C ６ alkyl groups; R ６ and R ７ are each independently hydrogen, —R ８ , —C(═O)—R ８ and —C(═O)—O—R ８ Selected from: R ８ is C １ ~C ６ Alkyl group, C ３ ~C ８ Carbocyclyl groups, and C ３ ~C ８ heterocyclyl groups; and R ９ H, 【Chemical 23】 Selected from: Here, R １ , R ２ , R ３ , R ６ , R ７ , and R ８ are each independently a halogen, a hydroxyl, or C １ ~C ６ alkyl group, —O—(C １ ~C ６ alkyl group, —CO ２ H, -C(=O)-(C １ ~C ６ alkyl) group, —C(═O)—(C ３ ~C ８ carbocyclyl group, —C(═O)—(C ３ ~C ８ heterocyclyl group, —NR ６ R ７ , C ３ ~C ８ Carbocyclyl group, C １ ~C ６ Alkylhydroxy group, C １ ~C ６ Alkylalkoxy group, benzyl group, and C ３ ~C ８ Heterocyclyl groups, each of which may be selected from halogen, hydroxyl, C １ ~C ３ Alkyl group, C １ ~C ３ Alkoxy group, C １ ~C ３ Haloalkyl groups, —NH—C(═O)(C １ ~C ３ alkyl), and —NH—C(═O)—O—(C １ ~C ３ substituted with 0 to 3 groups independently selected from (alkyl); During the ceremony, ＊ is R ９ represents the point of attachment to the remainder of the compound.

213. 213. The compound of claim 212, which is H5, or a pharmaceutically acceptable salt thereof.

214. 213. The compound of claim 212, which is H6, or a pharmaceutically acceptable salt thereof.

215. 213. The compound of claim 212, which is H7, or a pharmaceutically acceptable salt thereof.

216. 213. The compound of claim 212, which is H8, or a pharmaceutically acceptable salt thereof.

217. 213. The compound of claim 212, which is H9, or a pharmaceutically acceptable salt thereof.

218. 213. The compound of claim 212, which is H10, or a pharmaceutically acceptable salt thereof.

219. 213. The compound of claim 212, which is H22, or a pharmaceutically acceptable salt thereof.

220. 220. A pharmaceutical composition comprising a compound according to any one of claims 190 to 219, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

221. 190. A composition comprising multiple copies of the antibody-drug conjugate of any one of claims 1 to 189, wherein the average p of said antibody-drug conjugates in said composition is from about 3.5 to about 5.

5.

222. The composition of claim 221, wherein the average p of the antibody-drug conjugates in the composition is about 4.

223. 190. A composition comprising multiple copies of the antibody-drug conjugate of any one of claims 1 to 189, wherein the average p of said antibody-drug conjugates in said composition is from about 7 to about 9.

224. The composition of claim 223, wherein the average p of the antibody-drug conjugates in the composition is about 8.

225. 226. A method of treating a subject having or suspected of having a neoplastic disorder, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate of any one of claims 1-189, a compound of any one of claims 190-219, or a composition of any one of claims 220-224.

226. 226. The method of claim 225, wherein the neoplastic disorder is a hematological malignancy or a solid tumor.

227. 227. The method of claim 225 or 226, wherein the neoplastic disorder is a hematological malignancy.

228. 228. The method of claim 227, wherein the hematological malignancy is selected from acute myeloid leukemia and multiple myeloma.

229. 227. The method of claim 225 or 226, wherein the neoplastic disorder is a solid tumor.

230. 230. The method of claim 229, wherein the solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colorectal cancer, and esophageal cancer.

231. 231. The method of any one of claims 225-230, wherein the subject has one or more neoplastic cells that express a target antigen.

232. 232. The method of claim 231, wherein the target antigen is HER2.

233. 233. The method of claim 231 or 232, wherein the one or more neoplastic cells are derived from HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer, uterine cancer, osteosarcoma, or salivary duct cancer.

234. The method according to claim 233, wherein the lung cancer is lung adenocarcinoma and / or the uterine cancer is serous endometrial cancer.

235. The method according to any one of claims 232 to 234, wherein the subject is refractory or poorly responsive to treatment with (a) an anti-HER2 antibody alone and / or (b) a splicing regulator alone.

236. The method according to any one of claims 232 to 235, wherein the subject is intolerant, refractory, or poorly responsive to treatment with a splicing regulator alone.

237. The method according to claim 231, wherein the target antigen is CD138.

238. The method according to claim 231 or 237, wherein the one or more neoplastic cells are derived from multiple myeloma expressing CD138.

239. The method according to claim 237 or 238, wherein the subject is refractory or poorly responsive to treatment with (a) an anti-CD138 antibody alone and / or (b) a splicing regulator alone.

240. The method according to any one of claims 237 to 239, wherein the subject is intolerant, refractory, or poorly responsive to treatment with a splicing regulator alone.

241. The method according to claim 231, wherein the target antigen is EPHA2.

242. The method according to claim 231 or 241, wherein the one or more neoplastic cells are derived from breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer expressing EPHA2.

243. The method according to claim 241 or 242, wherein the subject is refractory or poorly responsive to treatment with (a) an anti-EPHA2 antibody alone and / or (b) a splicing regulator alone.

244. The method according to any one of claims 241 to 243, wherein the subject is intolerant, refractory, or poorly responsive to treatment with a splicing regulator alone.

245. A method for reducing or inhibiting the growth of a tumor in a subject having or suspected of having a neoplastic disorder, the method comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate according to any one of claims 1 to 189, a compound according to any one of claims 190 to 219, or a composition according to any one of claims 220 to 224.

246. The method according to claim 245, wherein the tumor comprises one or more neoplastic cells expressing a target antigen.

247. 247. The method of claim 246, wherein the target antigen is HER2.

248. 248. The method of claim 246 or 247, wherein the one or more neoplastic cells are derived from HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer, uterine cancer, osteosarcoma, or salivary duct cancer.

249. The method of claim 248, wherein the lung cancer is lung adenocarcinoma and / or the uterine cancer is serous endometrial carcinoma.

250. 250. The method of any one of claims 247 to 249, wherein the tumor is resistant or refractory to treatment with (a) an anti-HER2 antibody when administered alone and / or (b) a splicing modulator when administered alone.

251. The method of claim 246, wherein the target antigen is CD138.

252. The method of claim 246 or 251, wherein the one or more neoplastic cells are derived from multiple myeloma that expresses CD138.

253. The method of claim 251 or 252, wherein the tumor is resistant or refractory to treatment with (a) an anti-CD138 antibody when administered alone and / or (b) a splicing modulator when administered alone.

254. 247. The method of claim 246, wherein the target antigen is EPHA2.

255. 255. The method of claim 246 or 254, wherein the one or more neoplastic cells are derived from an EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer.

256. The method of claim 254 or 255, wherein the tumor is resistant or refractory to treatment with (a) an anti-EPHA2 antibody when administered alone and / or (b) a splicing modulator when administered alone.

257. 224。 A method of determining whether a subject having or suspected of having a neoplastic disorder will respond to treatment with an antibody-drug conjugate of any one of claims 1 to 189, a compound of any one of claims 190 to 219, or a composition of any one of claims 220 to 224, said method comprising providing a biological sample from said subject, and contacting said biological sample with an antibody-drug conjugate of any one of claims 1 to 189, a compound of any one of claims 190 to 219, or a composition of any one of claims 220 to 224.

258. 258. The method of claim 257, wherein the biological sample is a tumor sample.

259. The method of claim 258, wherein the tumor sample is a tumor biopsy or a blood sample.

260. 260. The method of claim 259, wherein the blood sample is selected from blood, a blood fraction, or cells obtained from the blood or blood fraction.

261. 261. The method of any one of claims 257 to 260, wherein the subject has one or more neoplastic cells that express the target antigen.

262. 262. The method of claim 261, wherein the target antigen is HER2.

263. 263. The method of claim 261 or 262, wherein the one or more neoplastic cells are derived from HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer, uterine cancer, osteosarcoma, or salivary duct cancer.

264. 264. The method of claim 263, wherein the lung cancer is lung adenocarcinoma and / or the uterine cancer is serous endometrial carcinoma.

265. The method of claim 261, wherein the target antigen is CD138.

266. The method of claim 261 or 265, wherein the one or more neoplastic cells are derived from multiple myeloma that expresses CD138.

267. 262. The method of claim 261, wherein the target antigen is EPHA2.

268. 268. The method of claim 261 or 267, wherein the one or more neoplastic cells are derived from an EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer.

269. An antibody-drug conjugate according to any one of claims 1 to 189, a compound according to any one of claims 190 to 219, or a composition according to any one of claims 220 to 224 for use in the treatment of a neoplastic disorder.

270. 270. The antibody-drug conjugate, compound, or composition for use of claim 269, wherein the neoplastic disorder is characterized by having one or more neoplastic cells that express the target antigen.

271. 271. The antibody-drug conjugate, compound, or composition for use according to claim 270, wherein the target antigen is HER2.

272. 272. The antibody-drug conjugate, compound, or composition for use according to claim 270 or 271, wherein the neoplastic disorder is HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer, uterine cancer, osteosarcoma, or salivary duct cancer.

273. 273. The antibody-drug conjugate, compound, or composition of claim 272, wherein said lung cancer is lung adenocarcinoma and / or said uterine cancer is serous endometrial carcinoma.

274. 271. The antibody-drug conjugate, compound, or composition for use according to claim 270, wherein the target antigen is CD138.

275. 275. The antibody-drug conjugate, compound, or composition for use according to claim 270 or 274, wherein said neoplastic disorder is CD138-expressing multiple myeloma.

276. 271. The antibody-drug conjugate, compound, or composition for use according to claim 270, wherein the target antigen is EPHA2.

277. The antibody-drug conjugate, compound, or composition for use according to claim 270 or 276, wherein the neoplastic disorder is EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer.

278. 22. Use of an antibody-drug conjugate according to any one of claims 1 to 189, a compound according to any one of claims 190 to 219, or a composition according to any one of claims 220 to 224 in the treatment of a neoplastic disorder.

279. 279. The use of claim 278, wherein the neoplastic disorder is characterized by having one or more neoplastic cells that express the target antigen.

280. 280. The use of claim 279, wherein the target antigen is HER2.

281. 281. The use of claim 279 or 280, wherein the neoplastic disorder is HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer, uterine cancer, osteosarcoma, or salivary duct cancer.

282. The use of claim 281, wherein the lung cancer is lung adenocarcinoma and / or the uterine cancer is serous endometrial carcinoma.

283. The use described in claim 279, wherein the target antigen is CD138.

284. 284. The use of claim 279 or 283, wherein the neoplastic disorder is CD138-expressing multiple myeloma.

285. 280. The use of claim 279, wherein the target antigen is EPHA2.

286. 286. The use of claim 279 or 285, wherein the neoplastic disorder is EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer.

287. 22. Use of an antibody-drug conjugate of any one of claims 1 to 189, a compound of any one of claims 190 to 219, or a composition of any one of claims 220 to 224 in the manufacture of a medicament for the treatment of a neoplastic disorder.

288. 288. The use of claim 287, wherein the neoplastic disorder is characterized by having one or more neoplastic cells that express the target antigen.

289. The use of claim 288, wherein the target antigen is HER2.

290. 289. The use of claim 288 or 289, wherein the neoplastic disorder is HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer, uterine cancer, osteosarcoma, or salivary duct cancer.

291. 291. The use of claim 290, wherein the lung cancer is lung adenocarcinoma and / or the uterine cancer is serous endometrial carcinoma.

292. The use described in claim 288, wherein the target antigen is CD138.

293. 293. The use of claim 288 or 292, wherein the neoplastic disorder is CD138-expressing multiple myeloma.

294. The use of claim 288, wherein the target antigen is EPHA2.

295. 295. The use of claim 288 or 294, wherein the neoplastic disorder is EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer.

296. A method for making an antibody-drug conjugate of any one of claims 1 to 189, comprising reacting an antibody or antigen-binding fragment with a linker tethered to a splicing modulator under conditions that allow conjugation.

297. 22. A method of inducing at least one neoantigen, comprising contacting a neoplastic cell with an effective amount of an antibody-drug conjugate of any one of claims 1-189, a compound of any one of claims 190-219, or a composition of any one of claims 220-224, thereby inducing the production of at least one neoantigen.

298. 298. The method of claim 297, wherein the neoplastic cells are present in an in vitro cell culture.

299. 299. The method of claim 297 or 298, wherein the neoplastic cells are obtained from a subject.

300. The method of claim 297, wherein the neoplastic cells are present in a subject.

301. 301. The method of any one of claims 297 to 300, wherein the neoplastic cells are derived from a hematological malignancy or a solid tumor.

302. 302. The method of claim 301, wherein the hematological malignancy is selected from a B-cell malignancy, leukemia, lymphoma, and myeloma.

303. 303. The method of claim 301 or 302, wherein the hematological malignancy is selected from acute myeloid leukemia and multiple myeloma.

304. 302. The method of claim 301, wherein the solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colorectal cancer, and esophageal cancer.

305. 226. A method of inducing at least one neoantigen and / or T cell response in a subject having or suspected of having a neoplastic disorder, the method comprising administering to the subject an effective amount of an antibody-drug conjugate of any one of claims 1 to 189, a compound of any one of claims 190 to 219, or a composition of any one of claims 220 to 224.

306. 22. A method of treating a subject having or suspected of having a neoplastic disorder, comprising administering to the subject an effective amount of an antibody-drug conjugate of any one of claims 1 to 189, a compound of any one of claims 190 to 219, or a composition of any one of claims 220 to 224, wherein administration of the compound, antibody-drug conjugate, or composition induces at least one neo-antigen and / or T cell response.

307. 307. The method of claim 305 or 306, wherein the amount of the compound, antibody-drug conjugate, or composition administered is reduced due to induction of at least one neo-antigen and / or T cell response when compared to a standard dosage of the compound, antibody-drug conjugate, or composition.

308. 308. The method of any one of claims 305-307, wherein the dosage of the compound, antibody-drug conjugate, or composition is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% when compared to a standard dosage of the compound, antibody-drug conjugate, or composition.

309. 309. The method of any one of claims 305-308, wherein the compound, antibody-drug conjugate, or composition is administered at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% less frequently when compared to a standard administration regimen for the compound, antibody-drug conjugate, or composition.

310. 310. The method of any one of claims 305-309, wherein the administration amount and / or dosage of said compound, antibody-drug conjugate, or composition results in reduced systemic toxicity and / or improved tolerability.

311. 311. The method of any one of claims 305-310, further comprising administering at least one additional therapy.

312. 312. The method of claim 311, wherein the administered amount of said compound, antibody-drug conjugate, composition, and / or said at least one additional therapy is reduced due to induction of at least one neo-antigen and / or T cell response when compared to standard dosages of said compound, antibody-drug conjugate, composition, and / or said at least one additional therapy.

313. The method of claim 311 or 312, wherein the dosage of the compound, antibody-drug conjugate, composition, and / or the at least one additional therapy is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% when compared to the standard dosage of the splicing modulator, antibody-drug conjugate, composition, and / or the at least one additional therapy.

314. 314. The method of any one of claims 311-313, wherein the compound, antibody-drug conjugate, composition, and / or the at least one additional therapy is administered at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% less frequently when compared to a standard administration regimen of the compound, antibody-drug conjugate, composition, and / or the at least one additional therapy.

315. 315. The method of any one of claims 311-314, wherein the administration amount and / or dosage of said compound, antibody-drug conjugate, composition, and / or said at least one additional therapy results in reduced systemic toxicity and / or improved tolerability.

316. 316. The method of any one of claims 311-315, wherein administration of the compound, antibody-drug conjugate, or composition is initiated prior to administration of the at least one additional therapy.

317. 316. The method of any one of claims 311-315, wherein administration of the compound, antibody-drug conjugate, or composition is initiated after administration of the at least one additional therapy.

318. 316. The method of any one of claims 311-315, wherein administration of the compound, antibody-drug conjugate, or composition is initiated simultaneously with administration of the at least one additional therapy.

319. 319. The method of any one of claims 305-318, wherein the administration of the compound, antibody-drug conjugate, or composition is repeated at least one time after an initial administration.

320. 320. The method of claim 319, wherein the amount used in repeat administrations of the compound, antibody-drug conjugate, or composition is reduced as compared to the amount used in the initial administration.

321. 321. The method of claim 319 or 320, wherein the amount used for repeat administration of the compound, antibody-drug conjugate, or composition is reduced when compared to a standard dosage of the compound, antibody-drug conjugate, or composition.

322. 322. The method of any one of claims 319-321, wherein the amount used for repeat administration of the compound, antibody-drug conjugate, or composition is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% when compared to a standard dosage of the compound, antibody-drug conjugate, or composition.

323. 323. The method of any one of claims 311-322, wherein the administration of the at least one additional therapy is repeated at least once after an initial administration.

324. The method of claim 323, wherein the amount used in repeat administrations of the at least one additional therapy is reduced compared to the amount used in the initial administration.

325. 325. The method of claim 323 or 324, wherein the amount used for repeat administration of the at least one additional therapy is reduced when compared to the standard dosage of the at least one additional therapy.

326. 326. The method of any one of claims 323-325, wherein the amount used for repeat administration of the at least one additional therapy is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% when compared to a standard dosage of the at least one additional therapy.

327. 327. The method of any one of claims 319-326, wherein the repeated administration of said compound, antibody-drug conjugate, or composition is concurrent with the repeated administration of said at least one additional therapy.

328. 327. The method of any one of claims 319-326, wherein the repeat administration of said compound, antibody-drug conjugate, or composition is sequential or staggered with the repeat administration of said at least one additional therapy.

329. 329. The method of any one of claims 311-328, wherein the at least one additional therapy comprises administering a checkpoint inhibitor.

330. 330. The method of claim 329, wherein the subject is intolerant, refractory, or non-responsive to the checkpoint inhibitor when administered alone.

331. 331. The method of claim 329 or 330, wherein the checkpoint inhibitor targets CTLA4, PD1, PDL1, OX40, CD40, GITR, LAG3, TIM3, and / or KIR.

332. 332. The method of any one of claims 329-331, wherein the checkpoint inhibitor targets CTLA4, OX40, CD40, and / or GITR.

333. 333. The method of any one of claims 329-332, wherein the checkpoint inhibitor comprises a cytotoxic T-lymphocyte-associated antigen 4 pathway (CTLA4) inhibitor.

334. The method of claim 333, wherein the CTLA4 inhibitor is an anti-CTLA4 antibody.

335. The method of claim 334, wherein the anti-CTLA4 antibody is ipilimumab.

336. 333. The method of any one of claims 329-332, wherein the checkpoint inhibitor comprises a programmed death-1 pathway (PD1) inhibitor.

337. The method of claim 336, wherein the PD1 inhibitor is an anti-PD1 antibody.

338. The method of claim 337, wherein the anti-PD1 antibody is nivolumab.

339. The method of claim 336, wherein the PD1 inhibitor is an anti-PDL1 antibody.

340. The method of claim 339, wherein the anti-PDL1 antibody is atezolizumab.

341. The method of any one of claims 329 to 332, wherein the checkpoint inhibitors comprise a CTLA4 inhibitor and a PD1 inhibitor.

342. The method of claim 341, wherein the CTLA4 inhibitor is an anti-CTLA4 antibody.

343. The method of claim 342, wherein the anti-CTLA4 antibody is ipilimumab.

344. The method of claim 341, wherein the PD1 inhibitor is an anti-PD1 antibody.

345. The method of claim 344, wherein the anti-PD1 antibody is nivolumab.

346. The method of claim 341, wherein the PD1 inhibitor is an anti-PDL1 antibody.

347. The method of claim 346, wherein the anti-PDL1 antibody is atezolizumab.

348. 329. The method of any one of claims 311-328, wherein the at least one additional therapy comprises administering a neoantigen vaccine.

349. 349. The method of claim 348, wherein said compound, antibody-drug conjugate, or composition is administered prior to administration of said neo-antigen vaccine.

350. 349. The method of claim 348, wherein said compound, antibody-drug conjugate, or composition is administered after administration of said neo-antigen vaccine.

351. 349. The method of claim 348, wherein said compound, antibody-drug conjugate, or composition is administered simultaneously with administration of said neo-antigen vaccine.

352. 352. The method of any one of claims 348-351, wherein administration of the compound, antibody-drug conjugate, or composition is repeated at least one time after an initial administration.

353. The method of claim 352, wherein the amount used in repeat administrations of the compound, antibody-drug conjugate, or composition is reduced as compared to the amount used in the initial administration.

354. 354. The method of any one of claims 348-353, wherein the neo-antigen vaccine comprises at least one neo-antigen peptide.

355. 355. The method of claim 354, wherein said at least one neo-antigenic peptide ranges from about 10 to about 35 amino acids in length.

356. 356. The method of claim 354 or 355, wherein said at least one neo-antigenic peptide ranges from about 15 to about 25 amino acids in length.

357. 357. The method of any one of claims 354 to 356, wherein said at least one neo-antigen peptide comprises one or more neo-antigen sequences.

358. The method of claim 357, wherein the neoantigen sequence is a neoantigen sequence specific to the subject.

359. The method of claim 357, wherein the neoantigen sequence is a universal neoantigen sequence.

360. The method of claim 358, wherein the neoantigen sequence is a neoantigen vaccine personalized for the subject.

361. The method of claim 360, wherein the neoantigen sequence is identified by sequencing at least one neoantigen induced in the subject by administering an effective amount of the compound, antibody-drug conjugate, or composition.

362. 362. The method of claim 360 or 361, wherein the neoantigen sequence is capable of binding to at least one HLA allele expressed in the subject.

363. The method of claim 359, wherein the neoantigen sequence is a universal neoantigen vaccine.

364. The method of claim 363, wherein the neoantigenic sequence is capable of binding to at least one HLA allele expressed in at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from the neoplastic disorder.

365. The method of claim 363 or 364, wherein the neoantigenic sequence is capable of eliciting a T cell response against tumors present in at least 1%, at least 5%, or at least 10% of a population of subjects suffering from the neoplastic disorder.

366. 366. The method of any one of claims 354-365, wherein said at least one neo-antigen peptide comprises a neo-antigen sequence induced by contacting a neoplastic cell with an effective amount of said splicing modulator, antibody-drug conjugate, or composition.

367. 349. The method of claim 348, wherein the neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable carrier.

368. 368. The method of claim 367, wherein said at least one neo-antigenic peptide is linked to said pharmaceutically acceptable carrier.

369. The method of claim 367 or 368, wherein the pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocyanin, immunoglobulins, thyroglobulin, ovalbumin, toxoids or attenuated toxoid derivatives, cytokines, and chemokines.

370. 370. The method of any one of claims 367 to 369, wherein the neo-antigenic peptide and the pharmaceutically acceptable carrier are covalently linked via a linker.

371. 370. The method of any one of claims 367 to 369, wherein the neo-antigenic peptide and the pharmaceutically acceptable carrier are expressed as a fusion protein.

372. 349. The method of claim 348, wherein the neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable diluent.

373. 349. The method of claim 348, wherein the neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable adjuvant.

374. The method of claim 366, wherein the neoplastic cells are present in an in vitro cell culture.

375. The method of claim 366 or 374, wherein the neoplastic cells are obtained from the subject.

376. The method of claim 366, wherein the neoplastic cells are present in the subject.

377. 354. The method of any one of claims 348-353, wherein said neo-antigen vaccine comprises at least one neo-antigen mRNA.

378. The method of claim 377, wherein said at least one neo-antigen mRNA encodes one or more neo-antigen sequences.

379. The method of claim 378, wherein the neoantigen sequence is a neoantigen sequence specific to the subject.

380. The method of claim 378, wherein the neoantigen sequence is a universal neoantigen sequence.

381. The method of claim 379, wherein the neoantigen sequence is a neoantigen vaccine personalized for the subject.

382. The method of claim 381, wherein the neoantigen sequence is identified by sequencing at least one neoantigen induced in the subject by administering an effective amount of the compound, antibody-drug conjugate, or composition.

383. The method of claim 381 or 382, ​​wherein the neoantigen sequence has the ability to bind to at least one HLA allele expressed in the subject.

384. The method of claim 380, wherein the neoantigen sequence is a universal neoantigen vaccine.

385. The method of claim 384, wherein the neoantigenic sequence is capable of binding to at least one HLA allele expressed in at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from the neoplastic disorder.

386. The method of claim 384 or 385, wherein the neoantigenic sequence is capable of eliciting a T cell response against tumors present in at least 1%, at least 5%, or at least 10% of a population of subjects suffering from the neoplastic disorder.

387. 387. The method of any one of claims 378-386, wherein said at least one neo-antigen mRNA encodes a neo-antigen sequence induced by contacting a neoplastic cell with an effective amount of said compound, antibody-drug conjugate, or composition.

388. 378. The method of claim 377, wherein the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable carrier.

389. The method of claim 388, wherein said at least one neo-antigen mRNA is linked to said pharmaceutically acceptable carrier.

390. The method of claim 388 or 389, wherein the pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocyanin, immunoglobulins, thyroglobulin, ovalbumin, toxoids or attenuated toxoid derivatives, cytokines, and chemokines.

391. 378. The method of claim 377, wherein the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable diluent.

392. 378. The method of claim 377, wherein the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable adjuvant.

393. 393. The method of any one of claims 377 to 392, wherein the neo-antigen mRNA is encapsulated in an encapsulating agent.

394. The method of claim 393, wherein the encapsulating agent is a liposome.

395. 394. The method of claim 393, wherein the encapsulating agent is a nanoparticle.

396. The method of claim 387, wherein the neoplastic cells are present in an in vitro cell culture.

397. The method of claim 387 or 396, wherein the neoplastic cells are obtained from the subject.

398. The method of claim 387, wherein the neoplastic cells are present in the subject.

399. 329. The method of any one of claims 311-328, wherein the at least one additional therapy comprises administering a cytokine or cytokine analog.

400. The method of claim 399, wherein the subject is intolerant, refractory, or poorly responsive to the cytokine or cytokine analog when administered alone.

401. 401. The method of claim 399 or 400, wherein the cytokine or cytokine analog comprises a T cell enhancer.

402. 402. The method of any one of claims 399-401, wherein said cytokine or cytokine analog comprises IL-2, IL-10, IL-12, IL-15, IFNγ, and / or TNFα.

403. 329. The method of any one of claims 311-328, wherein said at least one additional therapy comprises administering modified tumor-targeting T cells.

404. 404. The method of any one of claims 305-403, further comprising detecting one or more neo-antigens and / or T cell responses in said subject after administration of said compound, antibody-drug conjugate, or composition.

405. 405. The method of claim 404, further comprising continuing administration of said compound, antibody-drug conjugate, or composition if one or more neo-antigen and / or T cell responses are detected.

406. 406. The method of claim 404 or 405, further comprising continuing administration of said compound, antibody-drug conjugate, or composition less frequently and / or at a reduced dosage if one or more neo-antigen and / or T cell responses are detected.

407. 407. The method of any one of claims 404-406, wherein detection of one or more neo-antigens and / or T cell responses in the subject indicates the efficacy of treatment with the compound, antibody-drug conjugate, or composition.

408. The method of any one of claims 305-407, wherein said subject has a non-synonymous mutation load of about 150 mutations or less.

409. The method of any one of claims 305-408, wherein said subject has a non-synonymous mutation load of about 100 mutations or less.

410. The method of any one of claims 305-409, wherein said subject has a non-synonymous mutation load of about 50 mutations or less.

411. 411. The method of any one of claims 305 to 410, wherein the neoplastic disorder is a hematological malignancy or a solid tumor.

412. 412. The method of claim 411, wherein the hematological malignancy is selected from B-cell malignancies, leukemia, lymphoma, and myeloma.

413. 413. The method of claim 411 or 412, wherein the hematological malignancy is selected from acute myeloid leukemia and multiple myeloma.

414. 412. The method of claim 411, wherein the solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colorectal cancer, and esophageal cancer.

415. 1. A method of treating a subject having or suspected of having a neoplastic disorder, comprising: (a) administering to the subject an effective amount of an antibody-drug conjugate of any one of claims 1 to 189, a compound of any one of claims 190 to 219, or a composition of any one of claims 220 to 224, wherein administration of the compound, antibody-drug conjugate, or composition induces at least one neo-antigen and / or T cell response; (b) detecting one or more neo-antigen and / or T cell responses in the subject after administration of the compound, antibody-drug conjugate, or composition; and (c) continuing the administration of the compound, antibody-drug conjugate, or composition if one or more neo-antigen and / or T cell responses are detected. A method comprising:

416. 416. The method of claim 415, wherein detection of one or more neo-antigens and / or T cell responses in the subject indicates the effectiveness of treatment with the compound, antibody-drug conjugate, or composition.

417. 226. A method of treating a subject having or suspected of having a neoplastic disorder, comprising administering to the subject an effective amount of an antibody-drug conjugate of any one of claims 1-189, a compound of any one of claims 190-219, or a composition of any one of claims 220-224; and at least one additional therapy.

418. 418. The method of claim 417, wherein the at least one additional therapy comprises at least one, at least two, at least three, at least four, or at least five additional therapies.

419. 419. The method of claim 417 or 418, wherein administration of said compound, antibody-drug conjugate, or composition induces at least one neo-antigen and / or T cell response.

420. 420. The method of any one of claims 417-419, wherein the dosage of the compound, antibody-drug conjugate, or composition and / or the at least one additional therapy is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% when compared to standard dosages of the compound, antibody-drug conjugate, or composition and / or the at least one additional therapy.

421. 421. The method of any one of claims 417-420, wherein the compound, antibody-drug conjugate, or composition and / or the at least one additional therapy is administered at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% less frequently when compared to a standard administration regimen for the compound, antibody-drug conjugate, or composition and / or the at least one additional therapy.

422. 422. The method of any one of claims 417-421, wherein the administration amount and / or dosage of said compound, antibody-drug conjugate, or composition and / or said at least one additional therapy results in reduced systemic toxicity and / or improved tolerability.

423. 423. The method of any one of claims 417-422, wherein administration of the compound, antibody-drug conjugate, or composition is initiated prior to administration of the at least one additional therapy.

424. 423. The method of any one of claims 417-422, wherein administration of the compound, antibody-drug conjugate, or composition is initiated after administration of the at least one additional therapy.

425. 423. The method of any one of claims 417-422, wherein administration of the compound, antibody-drug conjugate, or composition is initiated simultaneously with administration of the at least one additional therapy.

426. 426. The method of any one of claims 417 to 425, wherein administration of the compound, antibody-drug conjugate, or composition is repeated at least one time after an initial administration.

427. The method of claim 426, wherein the amount used in repeat administrations of the compound, antibody-drug conjugate, or composition is reduced as compared to the amount used in the initial administration.

428. 428. The method of claim 426 or 427, wherein the amount used for repeat administration of the compound, antibody-drug conjugate, or composition is reduced when compared to a standard dosage of the compound, antibody-drug conjugate, or composition.

429. The method of any one of claims 426 to 428, wherein the amount used for repeat administration of the compound, antibody-drug conjugate, or composition is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% when compared to a standard dosage of the compound, antibody-drug conjugate, or composition.

430. 430. The method of any one of claims 417-429, wherein the administration of the at least one additional therapy is repeated at least once after an initial administration.

431. The method of claim 430, wherein the amount used in repeat administrations of the at least one additional therapy is reduced compared to the amount used in the initial administration.

432. 432. The method of claim 430 or 431, wherein the amount used for repeat administration of the at least one additional therapy is reduced when compared to a standard dosage of the at least one additional therapy.

433. 433. The method of any one of claims 430-432, wherein the amount used for repeat administration of the at least one additional therapy is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% when compared to a standard dosage of the at least one additional therapy.

434. 434. The method of any one of claims 426-433, wherein the repeated administration of said compound, antibody-drug conjugate, or composition is concurrent with the repeated administration of said at least one additional therapy.

435. 434. The method of any one of claims 426-433, wherein the repeated administration of said compound, antibody-drug conjugate, or composition is sequential or staggered with the repeated administration of said at least one additional therapy.

436. 436. The method of any one of claims 417-435, wherein the at least one additional therapy comprises administering a checkpoint inhibitor.

437. The method of claim 436, wherein the subject is intolerant, refractory, or non-responsive to the checkpoint inhibitor when administered alone.

438. The method of claim 436 or 437, wherein the checkpoint inhibitor targets CTLA4, PD1, PDL1, OX40, CD40, GITR, LAG3, TIM3, and / or KIR.

439. The method of any one of claims 436-438, wherein the checkpoint inhibitor targets CTLA4, OX40, CD40, and / or GITR.

440. 440. The method of any one of claims 436-439, wherein the checkpoint inhibitor comprises a cytotoxic T-lymphocyte-associated antigen 4 pathway (CTLA4) inhibitor.

441. The method of claim 440, wherein the CTLA4 inhibitor is an anti-CTLA4 antibody.

442. The method of claim 441, wherein the anti-CTLA4 antibody is ipilimumab.

443. 440. The method of any one of claims 436 to 439, wherein the checkpoint inhibitor comprises a programmed death-1 pathway (PD1) inhibitor.

444. The method of claim 443, wherein the PD1 inhibitor is an anti-PD1 antibody.

445. The method of claim 444, wherein the anti-PD1 antibody is nivolumab.

446. The method of claim 443, wherein the PD1 inhibitor is an anti-PDL1 antibody.

447. The method of claim 446, wherein the anti-PDL1 antibody is atezolizumab.

448. The method of any one of claims 436 to 439, wherein the checkpoint inhibitors comprise a CTLA4 inhibitor and a PD1 inhibitor.

449. The method of claim 448, wherein the CTLA4 inhibitor is an anti-CTLA4 antibody.

450. The method of claim 449, wherein the anti-CTLA4 antibody is ipilimumab.

451. The method of claim 448, wherein the PD1 inhibitor is an anti-PD1 antibody.

452. The method of claim 451, wherein the anti-PD1 antibody is nivolumab.

453. The method of claim 448, wherein the PD1 inhibitor is an anti-PDL1 antibody.

454. The method of claim 453, wherein the anti-PDL1 antibody is atezolizumab.

455. 436. The method of any one of claims 417 to 435, wherein said at least one additional therapy comprises administering a neoantigen vaccine.

456. 456. The method of claim 455, wherein said compound, antibody-drug conjugate, or composition is administered prior to administration of said neo-antigen vaccine.

457. 456. The method of claim 455, wherein said compound, antibody-drug conjugate, or composition is administered after administration of said neo-antigen vaccine.

458. 456. The method of claim 455, wherein said compound, antibody-drug conjugate, or composition is administered simultaneously with administration of said neo-antigen vaccine.

459. The method of any one of claims 455-458, wherein administration of the compound, antibody-drug conjugate, or composition is repeated at least one time after an initial administration.

460. 460. The method of claim 459, wherein the amount used in repeat administrations of the compound, antibody-drug conjugate, or composition is reduced as compared to the amount used in the initial administration.

461. 461. The method of any one of claims 455-460, wherein the neo-antigen vaccine comprises at least one neo-antigen peptide.

462. 462. The method of claim 461, wherein said at least one neo-antigenic peptide ranges from about 10 to about 35 amino acids in length.

463. 463. The method of claim 461 or 462, wherein said at least one neo-antigenic peptide ranges from about 15 to about 25 amino acids in length.

464. 464. The method of any one of claims 461 to 463, wherein said at least one neo-antigen peptide comprises one or more neo-antigen sequences.

465. The method of claim 464, wherein the neoantigen sequence is a neoantigen sequence specific to the subject.

466. The method of claim 464, wherein the neoantigen sequence is a universal neoantigen sequence.

467. The method of claim 465, wherein the neoantigen sequence is a neoantigen vaccine personalized for the subject.

468. The method of claim 467, wherein the neoantigen sequence is identified by sequencing at least one neoantigen induced in the subject by administering an effective amount of the compound, antibody-drug conjugate, or composition.

469. The method of claim 467 or 468, wherein the neoantigen sequence has the ability to bind to at least one HLA allele expressed in the subject.

470. The method of claim 466, wherein the neoantigen sequence is a universal neoantigen vaccine.

471. The method of claim 470, wherein the neoantigenic sequence is capable of binding to at least one HLA allele expressed in at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from the neoplastic disorder.

472. The method of claim 470 or 471, wherein the neoantigenic sequence is capable of eliciting a T cell response against tumors present in at least 1%, at least 5%, or at least 10% of a population of subjects suffering from the neoplastic disorder.

473. 473. The method of any one of claims 464-472, wherein said at least one neo-antigenic peptide comprises a neo-antigenic sequence induced by contacting a neoplastic cell with an effective amount of said compound, antibody-drug conjugate, or composition.

474. 456. The method of claim 455, wherein the neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable carrier.

475. 475. The method of claim 474, wherein said at least one neo-antigenic peptide is linked to said pharmaceutically acceptable carrier.

476. The method of claim 474 or 475, wherein the pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocyanin, immunoglobulins, thyroglobulin, ovalbumin, toxoids or attenuated toxoid derivatives, cytokines, and chemokines.

477. The method of any one of claims 474 to 476, wherein the neo-antigenic peptide and the pharmaceutically acceptable carrier are covalently linked via a linker.

478. 477. The method of any one of claims 474 to 476, wherein the neo-antigenic peptide and the pharmaceutically acceptable carrier are expressed as a fusion protein.

479. 456. The method of claim 455, wherein the neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable diluent.

480. 456. The method of claim 455, wherein the neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable adjuvant.

481. The method of claim 473, wherein the neoplastic cells are present in an in vitro cell culture.

482. The method of claim 473 or 481, wherein the neoplastic cells are obtained from the subject.

483. The method of claim 473, wherein the neoplastic cells are present in the subject.

484. 461. The method of any one of claims 455-460, wherein said neo-antigen vaccine comprises at least one neo-antigen mRNA.

485. The method of claim 484, wherein said at least one neo-antigen mRNA encodes one or more neo-antigen sequences.

486. The method of claim 485, wherein the neoantigen sequence is a neoantigen sequence specific to the subject.

487. The method of claim 485, wherein the neoantigen sequence is a universal neoantigen sequence.

488. The method of claim 486, wherein the neoantigen sequence is a neoantigen vaccine personalized for the subject.

489. The method of claim 488, wherein the neoantigen sequence is identified by sequencing at least one neoantigen induced in the subject by administering an effective amount of the compound, antibody-drug conjugate, or composition.

490. The method of claim 488 or 489, wherein the neoantigen sequence has the ability to bind to at least one HLA allele expressed in the subject.

491. The method of claim 487, wherein the neoantigen sequence is a universal neoantigen vaccine.

492. The method of claim 491, wherein the neoantigenic sequence is capable of binding to at least one HLA allele expressed in at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from the neoplastic disorder.

493. The method of claim 491 or 492, wherein the neoantigenic sequence is capable of eliciting a T cell response against tumors present in at least 1%, at least 5%, or at least 10% of a population of subjects suffering from the neoplastic disorder.

494. 494. The method of any one of claims 484-493, wherein said at least one neo-antigen mRNA encodes a neo-antigen sequence induced by contacting a neoplastic cell with an effective amount of said compound, antibody-drug conjugate, or composition.

495. 485. The method of claim 484, wherein the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable carrier.

496. The method of claim 495, wherein said at least one neo-antigen mRNA is linked to said pharmaceutically acceptable carrier.

497. The method of claim 495 or 496, wherein the pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocyanin, immunoglobulins, thyroglobulin, ovalbumin, toxoids or attenuated toxoid derivatives, cytokines, and chemokines.

498. 485. The method of claim 484, wherein the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable diluent.

499. 485. The method of claim 484, wherein the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable adjuvant.

500. 500. The method of any one of claims 484 to 499, wherein the neo-antigen mRNA is encapsulated in an encapsulating agent.

501. The method of claim 500, wherein the encapsulating agent is a liposome.

502. 501. The method of claim 500, wherein the encapsulating agent is a nanoparticle.

503. The method of claim 494, wherein the neoplastic cells are present in an in vitro cell culture.

504. The method of claim 494 or 503, wherein the neoplastic cells are obtained from the subject.

505. The method of claim 494, wherein the neoplastic cells are present in the subject.

506. 436. The method of any one of claims 417 to 435, wherein the at least one additional therapy comprises administering a cytokine or cytokine analog.

507. The method of claim 506, wherein the subject is intolerant, refractory, or poorly responsive to the cytokine or cytokine analog when administered alone.

508. The method of claim 506 or 507, wherein the cytokine or cytokine analog comprises a T cell enhancer.

509. 509. The method of any one of claims 506 to 508, wherein the cytokine or cytokine analog comprises IL-2, IL-10, IL-12, IL-15, IFNγ, and / or TNFα.

510. 436. The method of any one of claims 417-435, wherein the at least one additional therapy comprises administering modified tumor-targeting T cells.

511. 511. The method of any one of claims 417-510, wherein said subject has a non-synonymous mutation load of about 150 mutations or less.

512. 512. The method of any one of claims 417-511, wherein said subject has a nonsynonymous mutation load of about 100 mutations or less.

513. 513. The method of any one of claims 417-512, wherein said subject has a nonsynonymous mutation load of about 50 mutations or less.

514. 514. The method of any one of claims 417 to 513, wherein the neoplastic disorder is a hematological malignancy or a solid tumor.

515. 515. The method of claim 514, wherein the hematological malignancy is selected from B-cell malignancies, leukemia, lymphoma, and myeloma.

516. 516. The method of claim 514 or 515, wherein the hematological malignancy is selected from acute myeloid leukemia and multiple myeloma.

517. 515. The method of claim 514, wherein the solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colorectal cancer, and esophageal cancer.

518. 1. A method for identifying at least one neoantigen, comprising: (a) contacting a neoplastic cell with an effective amount of an antibody-drug conjugate of any one of claims 1-189, a compound of any one of claims 190-219, or a composition of any one of claims 220-224; (b) detecting at least one alternatively spliced ​​mRNA transcript after contacting said compound, antibody-drug conjugate, or composition with said neoplastic cells; (c) predicting the translation of the at least one alternatively spliced ​​mRNA transcript into at least one peptide; and (d) comparing said at least one peptide with a reference proteome, and if said at least one peptide does not match any peptide in said reference proteome, identifying at least one neoantigen. A method comprising:

519. 519. The method of claim 518, wherein detecting at least one alternatively spliced ​​mRNA transcript comprises RNAseq.

520. 520. The method of claim 518 or 519, wherein predicting the translation of the at least one alternatively spliced ​​mRNA transcript comprises quantifying the change in percent spliced-in (dPSI) value for the at least one transcript.

521. 521. The method of any one of claims 518-520, wherein predicting translation of said at least one alternatively spliced ​​mRNA transcript comprises RiboSeq and / or ribosome profiling.

522. 522. The method of any one of claims 518-521, further comprising evaluating said at least one peptide for predicted major histocompatibility complex (MHC) binding.

523. 523. The method of claim 522, wherein the predicted MHC binding is determined by measuring the uncorrected affinity predicted binding strength of said at least one peptide.

524. The method of claim 523, wherein an uncorrected affinity predicted binding strength of about 500 nM or greater indicates MHC binding.

525. 525. The method of any one of claims 522-524, wherein predicted MHC binding is determined by identifying a distribution of predicted binding strengths for a series of random peptides; and comparing the predicted binding strength of said at least one peptide to said distribution.

526. 526. The method of claim 525, wherein predicted binding strengths in the top 2% of the distribution indicate weak MHC binding.

527. 526. The method of claim 525, wherein predicted binding strengths in the top 0.5% of the distribution indicate strong MHC binding.

528. The method of any one of claims 518 to 527, wherein the neoplastic cells are in in vitro cell culture.

529. The method of claim 528, wherein the neoplastic cells are obtained from the subject.

530. The method of any one of claims 518 to 527, wherein the neoplastic cells are present in the subject.

531. 531. The method of any one of claims 518-530, further comprising contacting with one or more additional neoplastic cells to identify at least one universal neoantigen.

532. 1. A method for identifying at least one neoantigen, comprising: (a) contacting a neoplastic cell with an effective amount of an antibody-drug conjugate of any one of claims 1-189, a compound of any one of claims 190-219, or a composition of any one of claims 220-224; (b) detecting at least one peptide comprising a potential neoantigenic sequence after contacting the compound, antibody-drug conjugate, or composition with the neoplastic cells; and (c) comparing the at least one peptide to a reference proteome, and if the at least one peptide does not match any peptide in the reference proteome, identifying at least one neoantigen. A method comprising:

533. 533. The method of claim 532, further comprising evaluating said at least one peptide for predicted major histocompatibility complex (MHC) binding.

534. 534. The method of claim 533, wherein the predicted MHC binding is determined by measuring the uncorrected affinity predicted binding strength of said at least one peptide.

535. The method of claim 534, wherein an uncorrected affinity predicted binding strength of about 500 nM or greater indicates MHC binding.

536. 536. The method of any one of claims 533-535, wherein predicted MHC binding is determined by identifying a distribution of predicted binding strengths for a series of random peptides; and comparing the predicted binding strength of said at least one peptide to said distribution.

537. 537. The method of claim 536, wherein predicted binding strengths in the top 2% of the distribution indicate weak MHC binding.

538. 537. The method of claim 536, wherein predicted binding strengths in the top 0.5% of the distribution indicate strong MHC binding.

539. The method of any one of claims 532-538, wherein the neoplastic cells are in in vitro cell culture.

540. The method of claim 539, wherein the neoplastic cells are obtained from the subject.

541. The method of any one of claims 532 to 538, wherein the neoplastic cells are present in the subject.

542. 542. The method of any one of claims 532-541, further comprising contacting with one or more additional neoplastic cells to identify at least one universal neoantigen.

543. A method for producing a neo-antigen vaccine, comprising: (a) identifying at least one neoantigen using the method of any one of claims 518 to 542; and (b) formulating said at least one neoantigen with a pharmaceutically acceptable carrier, diluent, or adjuvant; A method comprising:

544. The method of claim 543, wherein said at least one neoantigen is linked to said pharmaceutically acceptable carrier.

545. The method of claim 544, wherein the pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocyanin, immunoglobulins, thyroglobulin, ovalbumin, toxoids or attenuated toxoid derivatives, cytokines, and chemokines.

546. 1. A method of treating a subject having or suspected of having a neoplastic disorder, comprising: (a) administering to the subject an effective amount of an antibody-drug conjugate of any one of claims 1 to 189, a compound of any one of claims 190 to 219, or a composition of any one of claims 220 to 224; (b) detecting one or more neoantigens in the subject after administration of the compound, antibody-drug conjugate, or composition; (c) comparing said one or more neoantigens to a panel of universal neoantigens; and (d) administering to the subject a universal neo-antigen vaccine comprising at least one universal neo-antigen present in the subject. A method comprising:

547. 547. The method of claim 546, wherein the universal neo-antigen vaccine is administered alone or in combination with at least one additional therapy.

548. The method of claim 547, wherein the at least one additional therapy comprises at least one, at least two, at least three, at least four, or at least five additional therapies.

549. The method of claim 547 or 548, wherein said at least one additional therapy comprises repeated administration of said compound, antibody-drug conjugate, or composition.

550. 550. The method of claim 549, wherein the repeat administration of said compound, antibody-drug conjugate, or composition is initiated prior to administration of said universal neo-antigen vaccine.

551. 550. The method of claim 549, wherein repetition of said compound, antibody-drug conjugate, or composition is initiated after administration of said universal neo-antigen vaccine.

552. 550. The method of claim 549, wherein the repeat administration of said compound, antibody-drug conjugate, or composition is initiated simultaneously with administration of said universal neo-antigen vaccine.

553. 553. The method of any one of claims 549-552, wherein the amount used in repeat administrations of the compound, antibody-drug conjugate, or composition is reduced when compared to the amount used in the initial administration.

554. 554. The method of any one of claims 549-553, wherein the amount used for repeat administration of the compound, antibody-drug conjugate, or composition is reduced when compared to a standard dosage of the compound, antibody-drug conjugate, or composition.

555. 555. The method of any one of claims 549-554, wherein the amount used for repeat administration of the compound, antibody-drug conjugate, or composition is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% when compared to a standard dosage of the compound, antibody-drug conjugate, or composition.

556. 556. The method of any one of claims 547-555, wherein said at least one additional therapy comprises administering a checkpoint inhibitor.

557. 557. The method of claim 556, wherein administration of said checkpoint inhibitor is initiated prior to administration of said universal neo-antigen vaccine and / or prior to repeat administration of said compound, antibody-drug conjugate, or composition.

558. 557. The method of claim 556, wherein administration of said checkpoint inhibitor is initiated after administration of said universal neo-antigen vaccine and / or after repeat administration of said compound, antibody-drug conjugate, or composition.

559. 557. The method of claim 556, wherein administration of said checkpoint inhibitor is initiated simultaneously with administration of said universal neo-antigen vaccine and / or repeated administrations of said compound, antibody-drug conjugate, or composition.

560. 560. The method of any one of claims 556-559, wherein administration of the checkpoint inhibitor is repeated at least one time after an initial administration.

561. 561. The method of claim 560, wherein the amount used in repeat administrations of the checkpoint inhibitor is reduced as compared to the amount used in the initial administration.

562. 562. The method of claim 560 or 561, wherein the amount used for repeat administration of the checkpoint inhibitor is reduced as compared to a standard dosage of the checkpoint inhibitor.

563. 563. The method of any one of claims 560-562, wherein the amount used for repeat administration of the checkpoint inhibitor is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% when compared to a standard dosage of the checkpoint inhibitor.

564. The method of any one of claims 556 to 563, wherein the subject is intolerant, refractory, or non-responsive to the checkpoint inhibitor when administered alone.

565. 565. The method of any one of claims 556-564, wherein the checkpoint inhibitor targets CTLA4, PD1, PDL1, OX40, CD40, GITR, LAG3, TIM3, and / or KIR.

566. The method of any one of claims 556-565, wherein the checkpoint inhibitor targets CTLA4, OX40, CD40, and / or GITR.

567. 567. The method of any one of claims 556-566, wherein said checkpoint inhibitor comprises a cytotoxic T-lymphocyte-associated antigen 4 pathway (CTLA4) inhibitor.

568. The method of claim 567, wherein the CTLA4 inhibitor is an anti-CTLA4 antibody.

569. The method of claim 568, wherein the anti-CTLA4 antibody is ipilimumab.

570. 567. The method of any one of claims 556-566, wherein said checkpoint inhibitor comprises a programmed death-1 pathway (PD1) inhibitor.

571. The method of claim 570, wherein the PD1 inhibitor is an anti-PD1 antibody.

572. The method of claim 571, wherein the anti-PD1 antibody is nivolumab.

573. The method of claim 570, wherein the PD1 inhibitor is an anti-PDL1 antibody.

574. The method of claim 573, wherein the anti-PDL1 antibody is atezolizumab.

575. The method of any one of claims 556 to 566, wherein the checkpoint inhibitors comprise a CTLA4 inhibitor and a PD1 inhibitor.

576. The method of claim 575, wherein the CTLA4 inhibitor is an anti-CTLA4 antibody.

577. The method of claim 576, wherein the anti-CTLA4 antibody is ipilimumab.

578. The method of claim 575, wherein the PD1 inhibitor is an anti-PD1 antibody.

579. The method of claim 578, wherein the anti-PD1 antibody is nivolumab.

580. The method of claim 575, wherein the PD1 inhibitor is an anti-PDL1 antibody.

581. The method of claim 580, wherein the anti-PDL1 antibody is atezolizumab.

582. 582. The method of any one of claims 546-581, wherein said universal neo-antigen vaccine comprises at least one neo-antigen peptide.

583. 583. The method of claim 582, wherein said at least one neo-antigenic peptide ranges from about 10 to about 35 amino acids in length.

584. 584. The method of claim 582 or 583, wherein said at least one neo-antigenic peptide ranges from about 15 to about 25 amino acids in length.

585. 585. The method of any one of claims 582-584, wherein said at least one neo-antigen peptide comprises one or more universal neo-antigen sequences.

586. The method of claim 585, wherein the universal neoantigen sequence is capable of binding to at least one HLA allele expressed in at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from the neoplastic disorder.

587. The method of claim 585 or 586, wherein the universal neo-antigen sequence is capable of eliciting a T cell response against tumors present in at least 1%, at least 5%, or at least 10% of a population of subjects suffering from the neoplastic disorder.

588. 583. The method of claim 582, wherein the universal neo-antigen vaccine comprises at least one neo-antigen peptide and a pharmaceutically acceptable carrier.

589. 589. The method of claim 588, wherein said at least one neo-antigenic peptide is linked to said pharmaceutically acceptable carrier.

590. The method of claim 588 or 589, wherein the pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocyanin, immunoglobulins, thyroglobulin, ovalbumin, toxoids or attenuated toxoid derivatives, cytokines, and chemokines.

591. 591. The method of any one of claims 588 to 590, wherein the neo-antigenic peptide and the pharmaceutically acceptable carrier are covalently linked via a linker.

592. 591. The method of any one of claims 588-590, wherein the neo-antigenic peptide and the pharmaceutically acceptable carrier are expressed as a fusion protein.

593. 583. The method of claim 582, wherein the universal neo-antigen vaccine comprises at least one neo-antigen peptide and a pharmaceutically acceptable diluent.

594. 583. The method of claim 582, wherein the universal neo-antigen vaccine comprises at least one neo-antigen peptide and a pharmaceutically acceptable adjuvant.

595. 582. The method of any one of claims 546-581, wherein said universal neo-antigen vaccine comprises at least one neo-antigen mRNA.

596. 596. The method of claim 595, wherein said at least one neo-antigen mRNA encodes one or more universal neo-antigen sequences.

597. The method of claim 596, wherein the universal neoantigen sequence is capable of binding to at least one HLA allele expressed in at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from the neoplastic disorder.

598. The method of claim 596 or 597, wherein the universal neo-antigen sequence is capable of eliciting a T cell response against tumors present in at least 1%, at least 5%, or at least 10% of a population of subjects suffering from the neoplastic disorder.

599. 596. The method of claim 595, wherein the universal neo-antigen vaccine comprises at least one neo-antigen mRNA and a pharmaceutically acceptable carrier.

600. 600. The method of claim 599, wherein said at least one neo-antigen mRNA is linked to said pharmaceutically acceptable carrier.

601. The method of claim 599 or 600, wherein the pharmaceutically acceptable carrier is selected from peptides, serum albumin, keyhole limpet hemocyanin, immunoglobulins, thyroglobulin, ovalbumin, toxoids or attenuated toxoid derivatives, cytokines, and chemokines.

602. 596. The method of claim 595, wherein the universal neo-antigen vaccine comprises at least one neo-antigen mRNA and a pharmaceutically acceptable diluent.

603. 596. The method of claim 595, wherein the universal neo-antigen vaccine comprises at least one neo-antigen mRNA and a pharmaceutically acceptable adjuvant.

604. The method of any one of claims 595-603, wherein the neo-antigen mRNA is encapsulated in an encapsulating agent.

605. The method of claim 604, wherein the encapsulating agent is a liposome.

606. The method of claim 604, wherein the encapsulating agent is a nanoparticle.

607. The method of any one of claims 546-606, wherein said subject has a non-synonymous mutation load of about 150 mutations or less.

608. The method of any one of claims 546-607, wherein said subject has a non-synonymous mutation load of about 100 mutations or less.

609. The method of any one of claims 546-608, wherein said subject has a non-synonymous mutation load of about 50 mutations or less.

610. The method of any one of claims 546 to 609, wherein the neoplastic disorder is a hematological malignancy or a solid tumor.

611. 611. The method of claim 610, wherein the hematological malignancy is selected from B-cell malignancies, leukemia, lymphoma, and myeloma.

612. 612. The method of claim 610 or 611, wherein the hematological malignancy is selected from acute myeloid leukemia and multiple myeloma.

613. 611. The method of claim 610, wherein the solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, renal cancer, colorectal cancer, and esophageal cancer.

614. 226. A neo-antigen vaccine comprising at least one neo-antigen peptide, wherein said at least one neo-antigen peptide comprises a neo-antigen sequence derived by contacting a neoplastic cell with an effective amount of an antibody-drug conjugate of any one of claims 1-189, a compound of any one of claims 190-219, or a composition of any one of claims 220-224.

615. 615. The neoantigen vaccine of claim 614, wherein said at least one neoantigen peptide ranges from about 10 to about 35 amino acids in length.

616. 616. The neo-antigen vaccine of claim 614 or 615, wherein said at least one neo-antigen peptide ranges from about 15 to about 25 amino acids in length.

617. 617. The neo-antigen vaccine of any one of claims 614 to 616, further comprising a pharmaceutically acceptable carrier.

618. The neoantigen vaccine of claim 617, wherein said at least one neoantigen peptide is linked to said pharmaceutically acceptable carrier.

619. 619. The neo-antigen vaccine of claim 617 or 618, wherein the pharmaceutically acceptable carrier is selected from a peptide, serum albumin, keyhole limpet hemocyanin, immunoglobulin, thyroglobulin, ovalbumin, a toxoid or attenuated toxoid derivative, a cytokine, and a chemokine.

620. The neoantigen vaccine of any one of claims 617 to 619, wherein the neoantigen peptide and the pharmaceutically acceptable carrier are covalently linked via a linker.

621. 620. The neoantigen vaccine of any one of claims 617 to 619, wherein the neoantigen peptide and the pharmaceutically acceptable carrier are expressed as a fusion protein.

622. 617. The neo-antigen vaccine of any one of claims 614 to 616, further comprising a pharmaceutically acceptable diluent.

623. 617. The neo-antigen vaccine of any one of claims 614 to 616, further comprising a pharmaceutically acceptable adjuvant.

624. The neoantigen vaccine of any one of claims 614 to 623, wherein the neoplastic cells are present in an in vitro cell culture.

625. The neoantigen vaccine of any one of claims 614 to 624, wherein the neoplastic cells are obtained from a subject.

626. The neoantigen vaccine of any one of claims 614 to 623, wherein the neoplastic cells are present in a subject.

627. 22. A neo-antigen vaccine comprising at least one neo-antigen mRNA, wherein said at least one neo-antigen mRNA encodes a neo-antigen sequence induced by contacting a neoplastic cell with an effective amount of an antibody-drug conjugate of any one of claims 1-189, a compound of any one of claims 190-219, or a composition of any one of claims 220-224.

628. 628. The neo-antigen vaccine of claim 627, further comprising a pharmaceutically acceptable carrier.

629. 629. The neoantigen vaccine of claim 628, wherein said at least one neoantigen mRNA is linked to said pharmaceutically acceptable carrier.

630. 630. The neo-antigen vaccine of claim 628 or 629, wherein the pharmaceutically acceptable carrier is selected from a peptide, serum albumin, keyhole limpet hemocyanin, immunoglobulin, thyroglobulin, ovalbumin, a toxoid or attenuated toxoid derivative, a cytokine, and a chemokine.

631. 628. The neo-antigen vaccine of claim 627, further comprising a pharmaceutically acceptable diluent.

632. 628. The neo-antigen vaccine of claim 627, further comprising a pharmaceutically acceptable adjuvant.

633. The neoantigen vaccine of any one of claims 627-632, wherein the neoantigen mRNA is encapsulated in an encapsulating agent.

634. The neo-antigen vaccine of claim 633, wherein the encapsulating agent is a liposome.

635. The neo-antigen vaccine of claim 633, wherein the encapsulating agent is a nanoparticle.

636. The neoantigen vaccine of any one of claims 627-635, wherein the neoplastic cells are in in vitro cell culture.

637. The neoantigen vaccine of any one of claims 627-636, wherein the neoplastic cells are obtained from a subject.

638. The neoantigen vaccine of any one of claims 627-635, wherein the neoplastic cells are present in a subject.

639. 220. A method of treating a subject having or suspected of having a neoplastic disorder, comprising administering to said subject a therapeutically effective amount of a compound of any one of claims 190 to 219 or a pharmaceutical composition of claim 220.

640. 220. A method of reducing or inhibiting tumor growth in a subject having or suspected of having a neoplastic disorder, comprising administering to said subject a therapeutically effective amount of a compound of any one of claims 190 to 219 or a pharmaceutical composition of claim 220. [Request 641] [Chemical 24] 【Chemistry 25】 【Chemical 26】 【Chemical 27】 【Chemical formula 28】 and pharmaceutically acceptable salts thereof wherein L is a linker that is covalently attached to the antibody. [Request 642] [Chemical 29] or a pharmaceutically acceptable salt thereof. 【Request 643】 【Chemical 30】 or a pharmaceutically acceptable salt thereof. 【Request 644】 【Chemical 31】 or a pharmaceutically acceptable salt thereof. [Request 645] [Chemical 32] or a pharmaceutically acceptable salt thereof. [Request 646] [Chemical 33] or a pharmaceutically acceptable salt thereof. 【Request 647】 【Chemical 34】 or a pharmaceutically acceptable salt thereof. [Request 648] [Chemical 35] or a pharmaceutically acceptable salt thereof. 【Request 649】 【Chemical 36】 or a pharmaceutically acceptable salt thereof.

650. [Catalog 37] or a pharmaceutically acceptable salt thereof.

651. [Catalog 38] or a pharmaceutically acceptable salt thereof.

652. 【Catalog 39】 or a pharmaceutically acceptable salt thereof. [Request 653] [Chemical 40] or a pharmaceutically acceptable salt thereof. [Request 654] [Chemical 41] or a pharmaceutically acceptable salt thereof.

655. [Catalog 42] or a pharmaceutically acceptable salt thereof. [Request 656] [Chemical 43] or a pharmaceutically acceptable salt thereof. 【Request 657】 【Chemical 44】 or a pharmaceutically acceptable salt thereof.

658. 【Catalog 45】 or a pharmaceutically acceptable salt thereof.

659.

46. or a pharmaceutically acceptable salt thereof.

660. [Catalog 47] or a pharmaceutically acceptable salt thereof.

661. [Catalog 48] or a pharmaceutically acceptable salt thereof.

662. [Catalog 49] or a pharmaceutically acceptable salt thereof.

663. [Catalog 50] or a pharmaceutically acceptable salt thereof.

664. [Catalog 51] or a pharmaceutically acceptable salt thereof.

665. 【Catalog 52】 or a pharmaceutically acceptable salt thereof. 【Request 666】 【Chemical 53】 or a pharmaceutically acceptable salt thereof. 【Request 667】 【Chemical 54】 or a pharmaceutically acceptable salt thereof. 【Request 668】 【Chemical 55】 or a pharmaceutically acceptable salt thereof.

669. 【Catalog 56】 or a pharmaceutically acceptable salt thereof.

670. 【Catalog 57】 or a pharmaceutically acceptable salt thereof.

671. 【Catalog 58】 or a pharmaceutically acceptable salt thereof.

672. 【Catalog 59】 or a pharmaceutically acceptable salt thereof. 【Request 673】 【Chemical 60】 or a pharmaceutically acceptable salt thereof. 【Request 674】 【Chemical 61】 or a pharmaceutically acceptable salt thereof. 【Request 675】 【Chemical 62】 or a pharmaceutically acceptable salt thereof. 【Request 676】 【Chemical 63】 or a pharmaceutically acceptable salt thereof. 【Request 677】 【Chemical 64】 or a pharmaceutically acceptable salt thereof. 【Request 678】 【Chemical 65】 or a pharmaceutically acceptable salt thereof.

679. The compound of any one of claims 641 to 678, wherein the linker is a cleavable linker.

680. The compound of claim 679, wherein the linker comprises a cleavable peptide moiety.

681. The compound of claim 680, wherein the cleavable peptide portion is cleavable by an enzyme.

682. 682. The compound of any one of claims 679-681, wherein the cleavable peptide moiety or linker comprises an amino acid unit.

683. The compound of claim 682, wherein the amino acid unit comprises valine-citrulline (Val-Cit).

684. 683. The compound of claim 682, wherein the amino acid unit comprises valine-alanine (Val-Ala).

685. 683. The compound of claim 682, wherein the amino acid unit comprises glutamine-valine-citrulline (Glu-Val-Cit).

686. 683. The compound of claim 682, wherein the amino acid unit comprises alanine-alanine-asparagine (Ala-Ala-Asn).

687. The compound of claim 679, wherein the linker comprises a cleavable glucuronide moiety.

688. The compound of claim 687, wherein the cleavable glucuronide moiety is cleavable by an enzyme.

689. The compound of claim 687 or 688, wherein the cleavable glucuronide moiety is cleavable by a glucuronidase.

690. 689. The compound of any one of claims 687-689, wherein the cleavable glucuronide moiety is cleavable by β-glucuronidase.

691. The compound of any one of claims 641 to 690, wherein the linker comprises at least one spacer unit.

692. The compound of claim 691, wherein the spacer unit or linker comprises a polyethylene glycol (PEG) moiety.

693. The PEG moiety is -(PEG) ｍ -, and m is an integer from 1 to 10.

694. The compound of claim 693, wherein m is 2.

695. The compound of claim 693, wherein the spacer unit or linker comprises an alkyl moiety.

696. The alkyl portion is —(CH ２ ) ｎ -, wherein n is an integer from 1 to 10.

697. The compound of claim 696, wherein n is 2.

698. The compound of claim 696, wherein n is 5.

699. The compound of claim 696, wherein n is 6.

700. 700. The compound of any one of claims 691 to 699, wherein the spacer unit is attached to the antibody or antigen-binding fragment via a maleimide (Mal) moiety ("Mal-spacer unit").

701. The compound of claim 700, wherein the Mal-spacer unit is reactive with a cysteine ​​residue on the antibody or antigen-binding fragment.

702. 702. The compound of claim 700 or 701, wherein the Mal-spacer unit is attached to the antibody or antigen-binding fragment via a cysteine ​​residue on the antibody or antigen-binding fragment.

703. 703. The compound of any one of claims 700-702, wherein the linker comprises the Mal-spacer unit and a cleavable peptide moiety.

704. The compound of claim 703, wherein the cleavable peptide portion comprises an amino acid unit.

705. 705. The compound of claim 703 or 704, wherein the cleavable peptide moiety or amino acid unit comprises Val-Cit.

706. 705. The compound of claim 703 or 704, wherein the cleavable peptide moiety or amino acid unit comprises Val-Ala.

707. 705. The compound of claim 703 or 704, wherein the cleavable peptide moiety or amino acid unit comprises Glu-Val-Cit.

708. 705. The compound of claim 703 or 704, wherein the cleavable peptide moiety or amino acid unit comprises Ala-Ala-Asn.

709. The compound of any one of claims 700 to 708, wherein the Mal-spacer unit comprises an alkyl moiety.

710. The compound of any one of claims 700-708, wherein the Mal-spacer unit comprises a PEG moiety.

711. The compound of any one of claims 700 to 710, wherein the Mal-spacer unit comprises maleimidocaproyl (MC).

712. 712. The compound of any one of claims 700-711, wherein the Mal-spacer unit links the antibody or antigen-binding fragment to the cleavable portion of the linker.

713. The compound of claim 712, wherein the cleavable portion of the linker comprises a cleavable peptide moiety.

714. The compound of claim 713, wherein the cleavable peptide portion comprises an amino acid unit.

715. 715. The compound of claim 713 or 714, wherein the cleavable peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn.

716. The compound of any one of claims 712 to 715, wherein the linker comprises MC-Val-Cit.

717. The compound of any one of claims 712 to 715, wherein the linker comprises MC-Val-Ala.

718. The compound of any one of claims 712 to 715, wherein the linker comprises MC-Glu-Val-Cit.

719. The compound of any one of claims 712 to 715, wherein the linker comprises MC-Ala-Ala-Asn.

720. The compound of any one of claims 712 to 719, wherein the Mal-spacer unit comprises an alkyl moiety.

721. The compound of any one of claims 712 to 719, wherein the Mal-spacer unit comprises a PEG moiety.

722. The compound of any one of claims 712 to 721, wherein the Mal-spacer unit comprises maleimidocaproyl (MC).

723. The compound of any one of claims 691 to 722, wherein the cleavable portion of the linker is directly joined to the splicing regulator or a spacer unit connects the cleavable portion of the linker to the splicing regulator.

724. The compound of claim 723, wherein cleavage of the conjugate releases the splicing modulator from the antibody or antigen-binding fragment and the linker.

725. 725. The compound of claim 723 or 724, wherein the spacer unit connecting the cleavable portion of the linker to the splicing modulator is self-immolative.

726. 726. The compound of any one of claims 723 to 725, wherein the spacer unit connecting the cleavable portion of the linker to the splicing modulator comprises p-aminobenzyloxycarbonyl (pABC).

727. The compound of claim 726, wherein the pABC links the cleavable portion of the linker to the splicing modulator.

728. 728. The compound of claim 726 or 727, wherein the cleavable portion of the linker comprises a cleavable peptide moiety.

729. 729. The compound of claim 728, wherein the cleavable peptide portion comprises an amino acid unit.

730. 730. The compound of claim 728 or 729, wherein the cleavable peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn.

731. The compound of any one of claims 726 to 730, wherein the linker comprises Val-Cit-pABC.

732. The compound of any one of claims 726 to 730, wherein the linker comprises Val-Ala-pABC.

733. The compound of any one of claims 726 to 730, wherein the linker comprises Glu-Val-Cit-pABC.

734. 731. The compound of any one of claims 726-730, wherein the linker comprises Ala-Ala-Asn-pABC.

735. 726. The compound of any one of claims 723 to 725, wherein the spacer unit connecting the cleavable portion of the linker to the splicing modulator comprises p-aminobenzyl (pAB).

736. The compound of claim 735, wherein the pAB links the cleavable portion of the linker to the splicing modulator.

737. 737. The compound of claim 735 or 736, wherein the cleavable portion of the linker comprises a cleavable peptide moiety.

738. 738. The compound of claim 737, wherein the cleavable peptide portion comprises an amino acid unit.

739. 739. The compound of claim 737 or 738, wherein the cleavable peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn.

740. The compound of any one of claims 735 to 739, wherein the linker comprises Val-Cit-pAB.

741. The compound of any one of claims 735 to 739, wherein the linker comprises Val-Ala-pAB.

742. The compound of any one of claims 735 to 739, wherein the linker comprises Glu-Val-Cit-pAB.

743. The compound of any one of claims 735 to 739, wherein the linker comprises Ala-Ala-Asn-pAB.

744. The compound of any one of claims 641 to 678, wherein the linker is a non-cleavable linker.

745. The compound of claim 744, wherein the linker comprises at least one spacer unit.

746. The compound of claim 744 or 745, wherein the spacer unit or linker comprises a polyethylene glycol (PEG) moiety.

747. The PEG moiety is -(PEG) ｍ -, and m is an integer from 1 to 10.

748. The compound of claim 747, wherein m is 2.

749. 746. The compound of claim 744 or 745, wherein the spacer unit or linker comprises an alkyl moiety.

750. The alkyl portion is —(CH ２ ) ｎ -, and n is an integer from 1 to 10.

751. 10. A method of making an antibody-drug conjugate, comprising reacting an antibody or antigen-binding fragment with a compound of any one of claims 641-750 under conditions that allow conjugation.

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