Herboxidiene splicing modulator antibody-drug conjugates and methods of use

Abstract

This invention discloses linker-drug compounds and antibody-drug conjugates that bind to human oncology targets. These linker-drug compounds and antibody-drug conjugates include a heptahydrate splicing modulatory drug moiety. The invention further relates to methods and compositions for treating neoplastic conditions by administering the antibody-drug conjugates provided herein.

Description

Technical Field

[0001] This invention relates to antibody-drug conjugates (ADCs) comprising a herboxidiene splicing regulator and an antibody or an antigen-binding fragment thereof, such as antibody-drug conjugates that bind to human oncology antigen targets. The invention further relates to methods and compositions for treating or diagnosing cancers that express target antigens and / or are suitable for treatment by RNA splicing disruption, and methods for manufacturing such compositions. Prior Technology

[0002] Most protein-coding genes in the human genome consist of multiple exons (coding regions) separated by introns (non-coding regions). Gene expression produces a single precursor messenger RNA (pre-mRNA). Subsequently, intron sequences are removed from the pre-mRNA through a process called splicing, producing mature messenger RNA (mRNA). By using alternating splicing of different exon combinations, mRNAs encoding unique protein isoforms are produced.

[0003] RNA splicing is catalyzed by the spliceosome, a dynamic multi-protein-RNA complex composed of five small nuclear RNAs (snRNAs U1, U2, U4, U5, and U6) and associated proteins. The spliceosome assembles on pre-mRNA to establish a dynamic cascade of multiple RNA and protein interactions that catalyze intron excision and exon conjugation (Matera and Wang (2014) Nat Rev Mol Cell Biol. 15(2):108-21). Increasing evidence links human diseases to dysregulation of RNA splicing affecting many genes (Scotti and Swanson (2016) Nat Rev Genet. 17(1):19-32).

[0004] The spliceosome is an important target in cancer biology. Several studies have documented the splicing profile of cancer cells and numerous alterations in their splicing factors (Agrawal et al. (2018) Curr Opin Genet Dev. 48:67-74). Alternating splicing can cause differential exon inclusion / exclusion, intron retention, or hidden splice site use (Seiler et al. (2018) Cell Rep. 23(1):282-296). In summary, these events result in functional alterations that may contribute to tumorigenesis or therapy resistance (Siegfried and Karni (2018) Curr Opin Genet Dev. 48:16-21).

[0005] Certain natural products can bind to the SF3b spliceosome complex. These small molecules regulate splicing by promoting intron retention and / or exon jumping (Teng et al. (2017) Nat Commun. 8:15522). For example, the naturally occurring polyketide hoprene (Isaac et al. (1992) J. Org. Chem. 57:7220-26) and its derivatives have been shown to regulate splicing. See, for example, Imaizumi et al. (2017) J. Antibiot. 70:675-79. A significant portion of the resulting transcripts contain premature stop codons, triggering nonsense-mediated mRNA decay (NMD). Furthermore, due to impaired typical splicing, typical transcripts are significantly reduced, which may adversely affect cell function and viability. For this reason, splice regulators have become a promising class of drugs for the treatment of cancer (Puthenveetil et al. (2016) Bioconjugate Chem. 27:1880-8).

[0006] 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 enables constitutive activation of growth factor signaling pathways such as the PI3K-AKT-mTOR pathway, thereby acting as an oncogenic driver in several types of cancer, 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 amplifies and mediates transformed phenotypes and because HER2 expression is largely limited 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 used for targeted delivery of cancer therapies include, but are not limited to, CD138 (also known as multiligand proteoglycan-1) and pterin A receptor 2 (EPHA2).

[0007] CD138 is a cell surface heparin sulfate proteoglycan essential for maintaining cell morphology and interaction with the surrounding microenvironment (Akl et al. (2015) Oncotarget 6(30):28693-715; Szatmári et al. (2015) Dis Markers 2015:796052). Generally, loss of CD138 expression in cancer cells reduces cell adhesion to the extracellular matrix and enhances cell motility and invasiveness (Teng et al. (2012) Matrix Biol. 31:3-16). Increased matrix CD138 expression also alters fibronectin production and extracellular matrix organization (Yang et al. (2011) Am J Pathol. 178:325-35). In addition, increased CD138 expression in stromal fibroblasts is associated with angiogenesis and cancer development (Maeda et al. (2006) Oncogene 25:1408-12). CD138 expression increases during B cell development and its presence is a marker of plasma cells (Ribatti (2017) Immunol Lett. 188:64-7). CD138 expression is maintained in multiple myeloma, a plasma cell malignancy. Therefore, CD138 is a promising antigen for targeted therapy of several cancers and other hematologic malignancies (Sherbenou et al. (2015) Blood Rev. 29(2):81-91; Wijdenes et al. (1996) Br J Haematol. 94(2):318-23).

[0008] EPHA2 is a transmembrane glycoprotein that is fully overexpressed in several malignant cancer-derived cell lines and in advanced cancer forms (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). EPHA2 protein is highly overexpressed in a certain percentage of patient tumors and a certain percentage of tumor cells, and is a plasma membrane-localized receptor that can be internalized 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 decreased survival. Therefore, EPHA2, due to its expression pattern, localization, and functional importance in cancer patient outcomes, is another promising antigen for targeted delivery of novel anticancer therapies. Summary of the Invention

[0009] In various embodiments, the present invention provides, in part, novel hoprene splice regulators having biological activity against neoplastic cells. Hoprene splice regulators can be used alone or as part of an ADC to slow, inhibit, and / or reverse tumor growth in mammals, and are applicable for the treatment of human cancer patients. In various embodiments, the present invention provides novel antibody-drug conjugates employing hoprene splice regulators.

[0010] More specifically, in various embodiments, the present invention relates to antibody-drug conjugate (ADC) compounds capable of binding to and killing proliferative cells. In various embodiments, the ADC compounds disclosed herein comprise a linker connecting a heptacysteine ​​splicing regulator to a full-length antibody or antigen-binding fragment. In various embodiments, the ADC compounds are also capable of internalization into target cells after binding.

[0011] In some embodiments, the antibody-drug conjugate is of formula (I): Ab-(LH)p, where Ab is an antibody or antigen-binding fragment targeting proliferative cells; H is a Hobstein splicing regulator; L is a linker that covalently links Ab to H; and p is an integer from 1 to 15.

[0012] In some embodiments, H-containing compounds of formula (I) are used as Hobschdiene splice regulators: , or a medically acceptable salt thereof, which is covalently linked to L via any atom, wherein: Y is selected from O, S, NR 6, and CR 6R 7; R1, R2, and R3 are each independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl), -OC(=O)-(C1-C6 alkyl), -C(=O)-O-(C1-C6 alkyl), and C1-C6 alkyl; R4 is selected from hydrogen, C1-C6 alkyl, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbon cycloyl), -C(=O)-(C3-C8 heterocyclic), and -C(=O)-NR6R7; R5 is selected from hydrogen, hydroxyl group, -CH2-OH, -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-NR6R7, -NR6-C(=O)-R8, -OC(=O)-NR6R7, -NR6-C(=O)-R8, and -NR6-C(=O)-NR6R7; R6 and R7 are each independently selected from hydrogen, -R8, -C(=O)-R8, and -C(=O)-OR8; and R8 is selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups. R1, R2, R3, R4, R5, R6, R7, and R8 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR6R7, C3-C8 carbocyclic, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic, each of which may be independently substituted by 0 or 1 group selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C6 alkylhydroxyl, C1-C6 alkyl ... 3-Hydroalkyl, -NH-C(=O)(C1-C3alkyl) and -NH-C(=O)-O-(C1-C3alkyl), and The valence of the atoms covalently attached to L did not exceed the limit.

[0013] In some embodiments, H-containing compounds (Ia) are used as Hobschdiene splice regulators: , or a medically acceptable salt thereof, which is covalently linked to L via any atom, wherein: R9 is selected from C3-C8 heterocyclic groups; R 10 is selected from H and C1-C6 alkyl groups. R9 and R10 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, -NH2, -NH-(C1-C3 alkyl) and -N-(C1-C3 alkyl)2, and The valence of the atoms covalently attached to L did not exceed the limit.

[0014] In some embodiments, H-containing (Ib) compounds that are Hobschdiene splice regulators: , or a medically acceptable salt thereof, which is covalently linked to L via any atom, wherein: R 11 is selected from * indicates the connection point between R 11 and the rest of the compound; R12 and R13 are each independently selected from H and methyl; and The valence of the atoms covalently attached to L did not exceed the limit.

[0015] In some embodiments, H-containing compounds of formula (II) are used as Hobschdiene splice regulators: , or a medically acceptable salt thereof, which is covalently linked to L via any atom, wherein: X represents a hydroxyl group or NR 6R 7; R6 and R7 are each independently selected from hydrogen, -R8, -C(=O)-R8, -C(=O)-OR8, -(C1-C6 alkyl)-OC(=O)-R8, and -(C1-C6 alkyl)-NH-C(=O)-R8; and R8 is selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups. R6, R7, and R8 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR6R7, C3-C8 carbocyclic, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic, each of which may be independently substituted by 0 or 1 group selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C6 alkylhydroxyl, C1-C6 alkylalkoxy, C3 ... 3-Hydroalkyl, -NH-C(=O)(C1-C3alkyl) and -NH-C(=O)-O-(C1-C3alkyl), and The valence of the atoms covalently attached to L did not exceed the limit.

[0016] In some embodiments, H-containing compounds of formula (IIa) are used as Hobschdiene splice regulators: , or a medically acceptable salt thereof, which is covalently linked to L via any atom, wherein: Z is selected from NR 9 and O; R9 is selected from hydrogen and C1-C6 alkyl groups; R10 and R11 are each independently selected from hydrogen, halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbon cycloyl), -C(=O)-(C3-C8 heterocyclic), C3-C8 carbon cycloyl, C1-C6 alkyl hydroxyl, C1-C6 alkyl alkoxy, benzyl, and C3-C8 heterocyclic; R 12 is selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups. R9, R10, R11, and R12 are each independently substituted by one or more groups selected from the following: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, and C1-C3 haloalkyl; t is an integer selected from 1, 2, 3, 4, 5, and 6; and The valence of the atoms covalently attached to L did not exceed the limit.

[0017] In some embodiments, H-containing compounds of formula (IIb) that are Hobschdiene splice regulators: , or a medically acceptable salt thereof, which is covalently linked to L via any atom, wherein: R 13 is selected from * indicates the connection point between R 13 and the rest of the compound; R14 and R15 are each independently selected from hydrogen and methyl; and The valence of the atoms covalently attached to L did not exceed the limit.

[0018] In some embodiments, H-containing compounds of formula (III) are used as Hobschdiene splice regulators: , or a medically acceptable salt thereof, which is covalently linked to L via any atom, wherein: R1, R2, and R3 are each independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl), -OC(=O)-(C1-C6 alkyl), -C(=O)-O-(C1-C6 alkyl), and C1-C6 alkyl; R6 and R7 are each independently selected from hydrogen, -R8, -C(=O)-R8, and -C(=O)-OR8; R8 is selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups; and R 9 is selected from H, ; R1, R2, R3, R6, R7, and R8 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR6R7, C3-C8 carbocyclic, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic, each of which may be independently substituted by 0 or 1 group selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, -NH-C(=O)(C 1-C 3 alkyl) and -NH-C(=O)-O-(C 1-C 3 alkyl), The valence of the atoms covalently connected to L does not exceed the limit; and The asterisk (*) indicates the connection point between R 9 and the rest of the compound.

[0019] In some embodiments, the linker comprises a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety may be cleaved 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 glutamate-valine-citrulline (“Glu-Val-Cit” or “EVC”). In some other embodiments, the amino acid unit comprises alanine-alanine-aspartic acid (“Ala-Ala-Asn” or “AAN”).

[0020] In some embodiments, the linker includes a cleavable glucuronic acid moiety. In some embodiments, the cleavable glucuronic acid moiety may be cleaved by an enzyme. In some embodiments, the cleavable glucuronic acid moiety may be cleaved by a glucuronidase. In some embodiments, the cleavable glucuronic acid moiety may be cleaved by a β-glucuronidase.

[0021] In some embodiments, the linker comprises at least one spacer unit. In some embodiments, the spacer unit or linker comprises a polyethylene glycol (PEG) portion. In some embodiments, the PEG portion comprises -(PEG)m- and 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 portion. In some embodiments, the alkyl portion comprises -(CH2)n- and 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.

[0022] In some embodiments, the spacer unit is linked to an antibody or antigen-binding fragment via a maleic diamide (Mal) portion ("Mal-spacer unit"). In some embodiments, the Mal-spacer unit may react with cysteine ​​residues on the antibody or antigen-binding fragment. In some embodiments, the Mal-spacer unit is bound to the antibody or antigen-binding fragment via cysteine ​​residues on the antibody or antigen-binding fragment.

[0023] In some embodiments, the linker comprises a Mal-spacer unit and a cleavable peptide portion. In some embodiments, the cleavable peptide portion comprises an amino acid unit. In some embodiments, the cleavable peptide portion or amino acid unit comprises Val-Cit. In some embodiments, the cleavable peptide portion or amino acid unit comprises Val-Ala. In some embodiments, the cleavable peptide portion or amino acid unit comprises Glu-Val-Cit. In some embodiments, the cleavable peptide portion or amino acid unit comprises Ala-Ala-Asn. In some embodiments, the Mal-spacer unit comprises an alkyl portion. In some embodiments, the Mal-spacer unit comprises a PEG portion. In some embodiments, the Mal-spacer unit comprises maleic anisodiaminohexyl (MC).

[0024] In some embodiments, the Mal-spacer unit links an antibody or antigen-binding fragment to a cleavable portion in a linker. In some embodiments, the cleavable portion in the linker comprises a cleavable peptide portion. In some embodiments, the cleavable peptide portion comprises an amino acid unit. In some embodiments, the cleavable peptide portion 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 portion. In some embodiments, the Mal-spacer unit comprises a PEG portion. In some embodiments, the Mal-spacer unit comprises maleic anisodiaminohexyl (MC).

[0025] In some embodiments, the cleavable portion of the linker directly binds to the Hopschier splice regulator, or a spacer unit connects the cleavable portion of the linker to the Hopschier splice regulator. In some embodiments, cleavage of the conjugate from the antibody or antigen-binding fragment and the linker releases the Hopschier splice regulator. In some embodiments, the spacer unit that connects the cleavable portion of the linker to the Hopschier splice regulator is a self-degrading spacer unit.

[0026] In some embodiments, the spacer unit connecting the cleavable portion of the linker to the Hobstein splice regulator comprises a p-aminobenzyloxycarbonyl group (pABC). In some embodiments, the pABC connects the cleavable portion of the linker to the Hobstein splice regulator. In some embodiments, the cleavable portion of the linker comprises a cleavable peptide portion. In some embodiments, the cleavable peptide portion comprises an amino acid unit. In some embodiments, the cleavable peptide portion 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.

[0027] In some embodiments, the spacer unit connecting the cleavable portion of the linker to the Hobstein splice regulator comprises p-aminobenzyl (pAB). In some embodiments, pAB connects the cleavable portion of the linker to the Hobstein splice regulator. In some embodiments, the cleavable portion of the linker comprises a cleavable peptide portion. In some embodiments, the cleavable peptide portion comprises an amino acid unit. In some embodiments, the cleavable peptide portion 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.

[0028] In various embodiments, the linker is a non-cleavable linker. In some embodiments, the hoprene splicing regulator of the ADC is released by degrading the antibody or antigen-binding fragment. In some embodiments, after internalization and degradation within the target cell, the linker remains covalently associated with at least one amino acid of the antibody and the drug.

[0029] In some embodiments, the linker is a non-disintegrable linker comprising at least one spacer unit. In some embodiments, the spacer unit or linker comprises a polyethylene glycol (PEG) portion. In some embodiments, the PEG portion comprises -(PEG)m- and 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 portion. In some embodiments, the alkyl portion comprises -(CH2)n- or -(CH2)n-O-(CH2)n and 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.

[0030] In some embodiments, a spacer unit in an indegradable linker is linked to an antibody or antigen-binding fragment via a maleic diimidimide (Mal) portion (“Mal-spacer unit”). In some embodiments, the Mal-spacer unit may react with cysteine ​​residues on the antibody or antigen-binding fragment. In some embodiments, the Mal-spacer unit is attached to the antibody or antigen-binding fragment via cysteine ​​residues on the antibody or antigen-binding fragment. In some embodiments, the Mal-spacer unit comprises an alkyl portion. In some embodiments, the Mal-spacer unit comprises a PEG portion. In some embodiments, the linker or Mal-spacer unit comprises maleic diimidimide hexanoyl (MC). In some embodiments, the linker or Mal-spacer unit comprises maleic diimidimide hexanoyl (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 an antibody or antigen-binding fragment to a Hobschdiene splice regulator.

[0031] In some embodiments, Ab is selected from any of the antibody or binding domain sequences disclosed herein. In some embodiments, Ab is an antibody or binding domain sequence targeting HER2 and / or HER2-expressing hypertrophic cells. In some embodiments, Ab is an antibody or binding domain sequence targeting CD138 and / or CD138-expressing hypertrophic cells. In some embodiments, Ab is an antibody or binding domain sequence targeting EPHA2 and / or EPHA2-expressing hypertrophic cells. In some embodiments, Ab is an antibody or binding domain sequence targeting MSLN and / or MSLN-expressing hypertrophic cells. In some embodiments, Ab is an antibody or binding domain sequence targeting FOLH1 and / or FOLH1-expressing hypertrophic cells. In some embodiments, Ab is an antibody or binding domain sequence targeting CDH6 and / or CDH6-expressing hypertrophic cells. In some embodiments, Ab is an antibody or binding domain sequence targeting CEACAM5 and / or CEACAM5-expressing hypertrophic cells. In some embodiments, Ab is an antibody or binding domain sequence targeting CFC1B and / or CFC1B-expressing hypertrophic cells. In some embodiments, Ab is an antibody or binding domain sequence targeting ENPP3 and / or ENPP3-expressing hypertrophic cells. In some embodiments, Ab is an antibody or binding domain sequence targeting FOLR1 and / or FOLR1-expressing hypertrophic cells. In some embodiments, Ab is an antibody or binding domain sequence targeting HAVCR1 and / or HAVCR1-expressing hypertrophic cells. In some embodiments, Ab is an antibody or binding domain sequence targeting KIT and / or KIT-expressing hypertrophic cells. In some embodiments, Ab is an antibody or binding domain sequence targeting MET and / or MET-expressing hypertrophic cells. In some embodiments, Ab is an antibody or binding domain sequence targeting MUC16 and / or MUC16-expressing hypertrophic cells. In some embodiments, Ab is an antibody or binding domain sequence targeting SLC39A6 and / or SLC39A6-expressing hypertrophic cells. In some embodiments, Ab is an antibody or binding domain sequence targeting SLC44A4 and / or SLC44A4-expressing hypertrophic cells. In some embodiments, Ab is an antibody or binding domain sequence that targets STEAP1 and / or STEAP1-expressing neoplasms. In some embodiments, Ab is an antibody or binding domain sequence that targets another cancer antigen.

[0032] In some embodiments, L is selected from any of the linkers disclosed herein or any combination of the 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 subunits. In some embodiments, L is a linker ADL1, ADL2, ADL5, ADL6, ADL7, ADL10, ADL12, ADL13, ADL14, ADL15, ADL21, ADL22, or ADL23. In some embodiments, L is a connector of ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23. In some embodiments, L is a connector of ADL12, ADL14, or ADL15. In some embodiments, a connector of ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 may also include one or more additional spacer sub-units. In some embodiments, L is an ADL1 connector and may include one or more additional spacer sub-units. In some embodiments, L is an ADL2 connector and may include one or more additional spacer sub-units. In some embodiments, L is an ADL5 connector and may include one or more additional spacer sub-units. In some embodiments, L is an ADL6 connector and may include one or more additional spacer sub-units. In some embodiments, L is an ADL7 connector and may include one or more additional spacing sub-units. In some embodiments, L is an ADL12 connector and may include one or more additional spacing sub-units. In some embodiments, L is an ADL14 connector and may include one or more additional spacing sub-units. In some embodiments, L is an ADL15 connector and may include one or more additional spacing sub-units. In various embodiments of the ADC 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.

[0033] In some embodiments, a set of ADCs is provided, thereby causing random binding, and the mean p in the set is between about 2 and about 8. In some embodiments, a set of ADCs is provided, thereby causing random binding, and the mean p in the set is between about 4 and about 8. In some embodiments, a set of ADCs is provided, thereby causing random binding, and the mean p in the set is about 4. In some embodiments, a set of ADCs is provided, thereby causing random binding, and the mean p in the set is about 8. This document provides compositions (e.g., pharmaceutical compositions) comprising multiple copies of any of the described ADCs, wherein the mean drug loading (mean p) of the ADCs in the composition is between about 3.5 and about 5.5 (e.g., about 4) or between about 7 and about 9 (e.g., about 8).

[0034] In some embodiments, the antibody or antigen-binding fragment (Ab) of the ADC targets neoplastic cells derived from hematologic malignancies or solid tumors. In some embodiments, the antibody or antigen-binding fragment targets neoplastic cells derived from hematologic malignancies. In some embodiments, hematologic malignancies are selected from B-cell malignancies, leukemia (e.g., acute myeloid leukemia), lymphoma, and myeloma (e.g., multiple myeloma). In some embodiments, hematologic malignancies are selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the antibody or antigen-binding fragment targets neoplastic cells derived from solid tumors. In some embodiments, solid tumors are 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 gland carcinoma, melanoma, colorectal cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer, and esophageal cancer. In some embodiments, solid tumors are selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

[0035] In various embodiments, the antibody or antigen-binding fragment (Ab) of the ADC is an anti-HER2 antibody or its antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment binds to HER2 and targets HER2-expressing neoplasms (i.e., the ADC targets HER2-expressing neoplasms). In some embodiments, the antibody or antigen-binding fragment of the ADC is an internalized anti-HER2 antibody or its internalized antigen-binding fragment.

[0036] 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 an internalizing antigen-binding fragment. In some embodiments, the anti-HER2 antibody or antigen-binding fragment comprises a human scaffold sequence. 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 includes a human IgG1 heavy chain constant region. In some embodiments, the anti-HER2 antibody or antigen-binding fragment includes a human Igκ or λ light chain constant region. In some embodiments, the anti-HER2 antibody or antigen-binding fragment competes with an antibody containing the heavy chain variable domain of SEQ ID NO: 19 and the light chain variable domain of SEQ ID NO: 20 for binding to the same antigenic determinant and / or binding to the same antigenic determinant.

[0037] In various embodiments, the antibody or antigen-binding fragment (Ab) of the ADC is an anti-CD138 antibody or its antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment binds to CD138 and targets CD138-expressing neoplasms (i.e., the ADC targets CD138-expressing neoplasms). In some embodiments, the antibody or antigen-binding fragment of the ADC is an internalized anti-CD138 antibody or its internalized antigen-binding fragment.

[0038] 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 an internalizing antigen-binding fragment. In some embodiments, the anti-CD138 antibody or antigen-binding fragment comprises a human framework sequence. 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 mouse IgG2a heavy chain constant region. In some embodiments, the anti-CD138 antibody or antigen-binding fragment comprises a mouse Igκ 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 Igκ or λ light chain constant region. In some embodiments, the anti-CD138 antibody or antigen-binding fragment competes with an antibody comprising the heavy chain variable domain of SEQ ID NO: 21 and the light chain variable domain of SEQ ID NO: 22 for binding to the same antigenic determinant and / or binding to the same antigenic determinant.

[0039] In various embodiments, the antibody or antigen-binding fragment (Ab) of the ADC is an anti-EPHA2 antibody or its antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment binds to EPHA2 and targets EPHA2-expressing neoplasms (i.e., the ADC targets EPHA2-expressing neoplasms). In some embodiments, the antibody or antigen-binding fragment of the ADC is an internalized anti-EPHA2 antibody or its internalized antigen-binding fragment.

[0040] 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 an internalizing antigen-binding fragment. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment comprises a human framework sequence. 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 Igκ or λ light chain constant region. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment competes with an antibody comprising the heavy chain variable domain of SEQ ID NO: 23 and the light chain variable domain of SEQ ID NO: 24 for binding to the same antigenic determinant and / or binding to the same antigenic determinant.

[0041] In some embodiments, the compound of formula (I) disclosed herein: or a medically acceptable salt thereof, wherein: Y is selected from O, S, NR 6, and CR 6R 7; R1, R2, and R3 are each independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl), -OC(=O)-(C1-C6 alkyl), -C(=O)-O-(C1-C6 alkyl), and C1-C6 alkyl; R4 is selected from hydrogen, C1-C6 alkyl, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbon cycloyl), -C(=O)-(C3-C8 heterocyclic), and -C(=O)-NR6R7; R5 is selected from hydrogen, hydroxyl group, -CH2-OH, -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-NR6R7, -NR6-C(=O)-R8, -OC(=O)-NR6R7, -NR6-C(=O)-R8, and -NR6-C(=O)-NR6R7; R6 and R7 are each independently selected from hydrogen, -R8, -C(=O)-R8, and -C(=O)-OR8; and R8 is selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups. R1, R2, R3, R4, R5, R6, R7, and R8 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR6R7, C3-C8 carbocyclic, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic, each of which may be independently substituted by 0 or 1 group selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C6 alkylhydroxyl, C1-C6 alkyl ... 3-Hydroalkyl, -NH-C(=O)(C1-C3alkyl) and -NH-C(=O)-O-(C1-C3alkyl).

[0042] In some embodiments, compounds of formula (Ia) are provided herein: or a medically acceptable salt thereof, wherein: R9 is selected from C3-C8 heterocyclic groups; and R 10 is selected from H and C1-C6 alkyl groups. R9 and R10 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, -NH2, -NH-(C1-C3 alkyl) and -N-(C1-C3 alkyl)2.

[0043] In some embodiments, compounds of formula (Ib) are provided herein:: or its medically acceptable salt, in which R 11 is selected from * indicates the connection point between R 11 and the rest of the compound; and R12 and R13 are each independently selected from H and methyl.

[0044] In some embodiments, compounds of formula (II) are provided herein: or a medically acceptable salt thereof, wherein: X is NR 6R 7; R6 and R7 are each independently selected from hydrogen, -R8, -C(=O)-R8, -C(=O)-OR8, -(C1-C6 alkyl)-OC(=O)-R8, and -(C1-C6 alkyl)-NH-C(=O)-R8; and R8 is selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups. R6, R7, and R8 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR6R7, C3-C8 carbocyclic, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic, each of which may be independently substituted by 0 or 1 group selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, -NH-C(=O)(C1-C 3-alkyl) and -NH-C(=O)-O-(C1-C3alkyl).

[0045] In some embodiments, compounds of formula (IIa) are provided herein: or a medically acceptable salt thereof, wherein: Z is selected from NR 9 and O; R9 is selected from hydrogen and C1-C6 alkyl groups; R10 and R11 are each independently selected from hydrogen, halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbon cycloyl), -C(=O)-(C3-C8 heterocyclic), C3-C8 carbon cycloyl, C1-C6 alkyl hydroxyl, C1-C6 alkyl alkoxy, benzyl, and C3-C8 heterocyclic; R 12 is selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups. R9, R10, R11, and R12 are each independently substituted by one or more groups selected from: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, and C1-C3 haloalkyl; and t is an integer selected from 1, 2, 3, 4, 5 and 6.

[0046] In some embodiments, compounds of formula (IIb) are provided herein: or a medically acceptable salt thereof, wherein: R 13 is selected from * indicates the connection point between R 13 and the rest of the compound; and R14 and R15 are each independently selected from hydrogen and methyl.

[0047] In some embodiments, compounds of formula (III) are provided herein: or a medically acceptable salt thereof, wherein: R1, R2, and R3 are each independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl), -OC(=O)-(C1-C6 alkyl), -C(=O)-O-(C1-C6 alkyl), and C1-C6 alkyl; R6 and R7 are each independently selected from hydrogen, -R8, -C(=O)-R8, and -C(=O)-OR8; R8 is selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups; and R 9 is selected from H, ; R1, R2, R3, R6, R7, and R8 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR6R7, C3-C8 carbocyclic, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic, each of which may be independently substituted by 0 or 1 group selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, -NH-C(=O)(C 1-C 3 alkyl) and -NH-C(=O)-O-(C 1-C 3 alkyl); and The asterisk (*) indicates the connection point between R 9 and the rest of the compound.

[0048] In some embodiments, this document provides compounds selected from the following: , and medically acceptable salts, Where L is a linker covalently linked to the antibody.

[0049] Furthermore, in various embodiments, this document provides the therapeutic use of the described ADC compounds, heptahydrate compounds, and compositions, for example, in treating neoplastic conditions such as cancer. In some embodiments, the invention provides a method for treating neoplastic conditions, such as cancers manifested by antigens targeted by antibodies or antigen-binding fragments of ADCs, such antigens as HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1.

[0050] In some embodiments, the present invention provides a method for treating an individual with or suspected of having a neoplastic condition, which is carried out by administering to the individual a therapeutically effective amount and / or a therapeutically effective regimen of any of the described ADCs or compositions. In some embodiments, the neoplastic condition is a hematologic malignancy or a solid tumor. In some embodiments, the neoplastic condition is a hematologic malignancy. In some embodiments, the hematologic malignancy is selected from B-cell malignancies, leukemia, lymphoma, and myeloma. In some embodiments, the hematologic malignancy is selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the neoplastic condition 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, colorectal cancer, cervical cancer, pancreatic cancer, kidney 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.

[0051] In some embodiments, treatment with an antibody-drug conjugate or composition induces bystander killing of vesicular cells that do not express the target antigen but are adjacent to vesicular cells that express the target antigen. In some embodiments, an individual has one or more vesicular cells that express the target antigen.

[0052] In some embodiments, the target antigen is HER2. In some embodiments, one or more neoplastic cells are present in HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., serous endometrial cancer), osteosarcoma, or salivary gland carcinoma. In some embodiments, the individual is unresponsive or poorly responsive to treatment with (a) an anti-HER2 antibody administered alone and / or (b) a hoprene splice regulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with a hoprene splice regulator administered alone.

[0053] In some embodiments, the target antigen is CD138. In some embodiments, one or more neoplastic cells are present in CD138-expressing multiple myeloma. In some embodiments, the individual is unresponsive or poorly responsive to treatment with (a) an anti-CD138 antibody administered alone and / or (b) a hoprene splice regulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with a hoprene splice regulator administered alone.

[0054] In some embodiments, the target antigen is EPHA2. In some embodiments, one or more neoplastic cells are present in EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colorectal cancer, or esophageal cancer. In some embodiments, the individual is unresponsive or poorly responsive to treatment with (a) an anti-EPHA2 antibody administered alone and / or (b) a hoprene splice regulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with a hoprene splice regulator administered alone.

[0055] In some embodiments, the target antigen is MSLN. In some embodiments, one or more neoplastic cells are present in MSLN-expressing ovarian cancer, cervical cancer, pancreatic cancer, or lung cancer (e.g., lung adenocarcinoma). In some embodiments, the individual is unresponsive or poorly responsive to treatment with (a) an anti-MSLN antibody administered alone and / or (b) a splicing regulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator administered alone.

[0056] In some embodiments, the target antigen is FOLH1. In some embodiments, one or more neoplastic cells are present in FOLH1-expressing prostate cancer. In some embodiments, the individual is unresponsive or poorly responsive to treatment with (a) an anti-FOLH1 antibody administered alone and / or (b) a splicing regulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator administered alone.

[0057] In some embodiments, the target antigen is CDH6. In some embodiments, one or more proliferative cells are in CDH6-expressing renal cell carcinoma. In some embodiments, the individual is unresponsive or poorly responsive to treatment with (a) an anti-CDH6 antibody administered alone and / or (b) a splicing regulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator administered alone.

[0058] In some embodiments, the target antigen is CEACAM5. In some embodiments, one or more proliferative cells are present in CEACAM5-expressing colorectal cancer. In some embodiments, the individual is unresponsive or poorly responsive to treatment with (a) an anti-CEACAM5 antibody administered alone and / or (b) a splicing regulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator administered alone.

[0059] In some embodiments, the target antigen is CFC1B. In some embodiments, one or more proliferative cells are present in CFC1B-expressing pancreatic cancer. In some embodiments, the individual is unresponsive or poorly responsive to treatment with (a) an anti-CFC1B antibody administered alone and / or (b) a splicing regulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator administered alone.

[0060] In some embodiments, the target antigen is ENPP3. In some embodiments, one or more proliferative cells are in ENPP3-expressing renal cell carcinoma. In some embodiments, the individual is unresponsive or poorly responsive to treatment with (a) an anti-ENPP3 antibody administered alone and / or (b) a splicing regulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator administered alone.

[0061] In some embodiments, the target antigen is FOLR1. In some embodiments, one or more neoplastic cells are in FOLR1-expressing ovarian cancer. In some embodiments, the individual is unresponsive or poorly responsive to treatment with (a) an anti-FOLR1 antibody administered alone and / or (b) a splicing regulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator administered alone.

[0062] In some embodiments, the target antigen is HAVCR1. In some embodiments, one or more proliferative cells are present in HAVCR1-expressing renal cell carcinoma or esophageal cancer. In some embodiments, the individual is unresponsive or poorly responsive to treatment with (a) an anti-HAVCR1 antibody administered alone and / or (b) a splicing regulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator administered alone.

[0063] In some embodiments, the target antigen is KIT. In some embodiments, one or more proliferative cells are in KIT-expressing renal cell carcinoma. In some embodiments, the individual is unresponsive or poorly responsive to treatment with (a) an anti-KIT antibody administered alone and / or (b) a splicing regulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator administered alone.

[0064] In some embodiments, the target antigen is MET. In some embodiments, one or more proliferative cells are in MET-expressing renal cell carcinoma or esophageal cancer. In some embodiments, the individual is unresponsive or poorly responsive to treatment with (a) an anti-MET antibody administered alone and / or (b) a splicing regulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator administered alone.

[0065] In some embodiments, the target antigen is MUC16. In some embodiments, one or more neoplastic cells are present in MUC16-expressing ovarian cancer, cervical cancer, or breast cancer. In some embodiments, the individual is unresponsive or poorly responsive to treatment with (a) an anti-MUC16 antibody administered alone and / or (b) a splicing regulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator administered alone.

[0066] In some embodiments, the target antigen is SLC39A6. In some embodiments, one or more neoplastic cells are present in SLC39A6-expressing breast or prostate cancer. In some embodiments, the individual is unresponsive or poorly responsive to treatment with (a) an anti-SLC39A6 antibody administered alone and / or (b) a splicing regulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator administered alone.

[0067] In some embodiments, the target antigen is SLC44A4. In some embodiments, one or more neoplastic cells are present in SLC44A4-expressing prostate cancer. In some embodiments, the individual is unresponsive or poorly responsive to treatment with (a) an anti-SLC44A4 antibody administered alone and / or (b) a splicing regulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator administered alone.

[0068] In some embodiments, the target antigen is STEAP1. In some embodiments, one or more neoplastic cells are present in STEAP1-expressing prostate cancer. In some embodiments, the individual is unresponsive or poorly responsive to treatment with (a) an anti-STEAP1 antibody administered alone and / or (b) a splicing regulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator administered alone.

[0069] In some other forms, the present invention provides a method for reducing or inhibiting tumor growth in an individual who has or is suspected of having a neoplastic condition, which is carried out by administering to the individual a therapeutically effective amount and / or a therapeutically effective regimen of any of the described ADCs or compositions.

[0070] In some embodiments, treatment with an antibody-drug conjugate or composition induces bystander killing of neoplastic tumor cells that do not express the target antigen but are adjacent to neoplastic tumor cells that express the target antigen. In some embodiments, the tumor comprises one or more neoplastic cells expressing the target antigen.

[0071] In some embodiments, the target antigen is HER2. In some embodiments, 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 cancer), osteosarcoma, or salivary gland carcinoma. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-HER2 antibody administered alone and / or (b) a hoprene splice regulator administered alone.

[0072] In some embodiments, the target antigen is CD138. In some embodiments, one or more proliferative cells are derived from CD138-expressing multiple myeloma. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-CD138 antibody administered alone and / or (b) a hoprene splice regulator administered alone.

[0073] In some embodiments, the target antigen is EPHA2. In some embodiments, one or more neoplastic cells are derived from EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colorectal cancer, or esophageal cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-EPHA2 antibody administered alone and / or (b) a hoprene splice regulator administered alone.

[0074] In some embodiments, the target antigen is MSLN. In some embodiments, 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 regulator administered alone.

[0075] In some embodiments, the target antigen is FOLH1. In some embodiments, one or more neoplasms are derived from FOLH1-expressing prostate cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-FOLH1 antibody administered alone and / or (b) a splicing regulator administered alone.

[0076] In some embodiments, the target antigen is CDH6. In some embodiments, one or more neoplasms are derived from CDH6-expressing renal cell carcinoma. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-CDH6 antibody administered alone and / or (b) a splicing regulator administered alone.

[0077] In some embodiments, the target antigen is CEACAM5. In some embodiments, one or more neoplastic cells are derived from CEACAM5-expressing colorectal cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-CEACAM5 antibody administered alone and / or (b) a splicing regulator administered alone.

[0078] In some embodiments, the target antigen is CFC1B. In some embodiments, one or more proliferative cells are derived from CFC1B-expressing pancreatic cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-CFC1B antibody administered alone and / or (b) a splicing regulator administered alone.

[0079] In some embodiments, the target antigen is ENPP3. In some embodiments, one or more proliferative cells are derived from ENPP3-expressing renal cell carcinoma. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-ENPP3 antibody administered alone and / or (b) a splicing regulator administered alone.

[0080] In some embodiments, the target antigen is FOLR1. In some embodiments, one or more neoplastic cells are derived from FOLR1-expressing ovarian cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-FOLR1 antibody administered alone and / or (b) a splicing regulator administered alone.

[0081] In some embodiments, the target antigen is HAVCR1. In some embodiments, one or more neoplasms are derived from HAVCR1-expressing renal cell carcinoma or esophageal cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-HAVCR1 antibody administered alone and / or (b) a splicing regulator administered alone.

[0082] In some embodiments, the target antigen is KIT. In some embodiments, one or more proliferative cells are derived from KIT-expressing renal cell 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 regulator administered alone.

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

[0084] In some embodiments, the target antigen is MUC16. In some embodiments, one or more neoplastic cells are derived from MUC16-expressing ovarian cancer, cervical cancer, or breast cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-MUC16 antibody administered alone and / or (b) a splicing regulator administered alone.

[0085] In some embodiments, the target antigen is SLC39A6. In some embodiments, one or more proliferative cells are derived from SLC39A6-expressing breast or prostate cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-SLC39A6 antibody administered alone and / or (b) a splicing regulator administered alone.

[0086] In some embodiments, the target antigen is SLC44A4. In some embodiments, one or more neoplastic cells are derived from SLC44A4-expressing prostate cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-SLC44A4 antibody administered alone and / or (b) a splicing regulator administered alone.

[0087] In some embodiments, the target antigen is STEAP1. In some embodiments, one or more neoplastic cells are derived from STEAP1-expressing prostate cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-STEAP1 antibody administered alone and / or (b) a splicing regulator administered alone.

[0088] In other embodiments, the present invention provides a method for determining whether an individual with or suspected of having a neoplastic condition responds to treatment with any of the described ADCs or compositions, by providing a biological sample from the individual 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, blood fractions, or cells obtained from blood or blood fractions. In some embodiments, the individual has one or more neoplastic cells expressing a target antigen. In some embodiments, the target antigen is HER2. In some embodiments, 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 cancer), osteosarcoma, or salivary gland carcinoma. In some embodiments, the target antigen is CD138. In some embodiments, one or more neoplastic cells are derived from CD138-expressing multiple myeloma. In some embodiments, the target antigen is EPHA2. In some embodiments, one or more neoplasm cells are derived from EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colorectal cancer, or esophageal cancer. In some embodiments, the target antigen is MSLN. In some embodiments, one or more neoplasm cells are derived from MSLN-expressing ovarian cancer, cervical cancer, pancreatic cancer, or lung cancer (e.g., lung adenocarcinoma). In some embodiments, the target antigen is FOLH1. In some embodiments, one or more neoplasm cells are derived from FOLH1-expressing prostate cancer. In some embodiments, the target antigen is CDH6. In some embodiments, one or more neoplasm cells are derived from CDH6-expressing renal cell carcinoma. In some embodiments, the target antigen is CEACAM5. In some embodiments, one or more neoplasm cells are derived from CEACAM5-expressing colorectal cancer. In some embodiments, the target antigen is CFC1B. In some embodiments, one or more neoplasm cells are derived from CFC1B-expressing pancreatic cancer. In some embodiments, the target antigen is ENPP3. In some embodiments, one or more neoplasm cells are derived from ENPP3-expressing renal cell carcinoma. In some embodiments, the target antigen is FOLR1. In some embodiments, one or more neoplasm cells are derived from FOLR1-expressing ovarian cancer. In some embodiments, the target antigen is HAVCR1. In some embodiments, one or more neoplasm cells are derived from HAVCR1-expressing renal cell carcinoma or esophageal cancer. In some embodiments, the target antigen is KIT. In some embodiments, one or more neoplasm cells are derived from KIT-expressing renal cell carcinoma. In some embodiments, the target antigen is MET. In some embodiments, one or more neoplasm cells are derived from MET-expressing renal cell carcinoma or esophageal cancer. In some embodiments, the target antigen is MUC16.In some embodiments, one or more neoplasm cells are derived from MUC16-presenting ovarian cancer, cervical cancer, or breast cancer. In some embodiments, the target antigen is SLC39A6. In some embodiments, one or more neoplasm cells are derived from SLC39A6-presenting breast cancer or prostate cancer. In some embodiments, the target antigen is SLC44A4. In some embodiments, one or more neoplasm cells are derived from SLC44A4-presenting prostate cancer. In some embodiments, the target antigen is STEAP1. In some embodiments, one or more neoplasm cells are derived from STEAP1-presenting prostate cancer.

[0089] In various embodiments, this document further provides pharmaceutical compositions comprising an ADC and a pharmaceutically acceptable diluent, carrier, and / or excipient. Methods for producing the described ADC compounds and compositions are also disclosed. Simple Explanation of the Diagram

[0090] [picture] [1A]- [1D] Viability dose-response of exemplary loaded 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 in CellTiter-Glo® 2.0 reagent. [picture] [1A] shows the viability dose-response in HCC1954 cells after 72 hours of culture. [picture] [1B] Viability dose-response in NCI-N87 cells after 72 hours of culture. [picture] [1C] shows the viability dose-response in HCC1954 cells after 144 hours of culture. [picture] [1D] Viability-dose response in NCI-N87 cells after 144 hours of culture. Data are presented as mean ± SD.

[0091] [picture] [2A] and [picture] [2B] Showing breast cancer cells (HCC1954) magnified by HER2. [picture] [2A]) and gastric cancer cells (NCI-N87) [picture] Results of SLC25A19 splicing analysis in [2B]). Cells were incubated with the compound for 6 hours, and SLC25A19 transcript splicing was measured using a specific Taqman primer-probe set in a real-time qPCR reaction. The y-axis represents the percentage of response relative to the DMSO control (0.1%). Data are expressed as mean ± SD. Implementation

[0092] This invention claims 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 aforementioned applications are incorporated herein by reference in their entirety.

[0093] The disclosed compositions and methods can be more easily understood by referring to the following detailed description.

[0094] Throughout this document, descriptions relate to compositions and methods of using such compositions. When the invention describes or claims features or embodiments associated with a composition, such features or embodiments equally apply to methods of using the composition. Similarly, when the invention describes or claims features or embodiments associated with methods of using a composition, such features or embodiments equally apply to the composition.

[0095] When a range of values ​​is expressed, it includes embodiments using any particular value within that range. Furthermore, references to values ​​stated by range include every value within that range. All ranges include their endpoints and are composable. When a value is expressed as an approximation by the preceding use of "about," it should be understood that the particular value forms another embodiment. Unless the context explicitly indicates otherwise, references to a particular numerical value include at least that particular value. Unless otherwise indicated in its specific usage, the use of "or" means "and / or."

[0096] It should be understood that, for clarity, certain features of the compositions and methods disclosed herein described in the case of individual embodiments may also be provided in combination of individual embodiments. Conversely, for simplicity, various features of the disclosed compositions and methods described in the case of individual embodiments may also be provided individually or in any sub-combination.

[0097] All references cited herein are incorporated by way of reference for any purpose. In the event of any discrepancy between the references and this specification, this specification shall prevail. [definition] []

[0098] Throughout this specification and the claims, various terms related to the described states are used. Unless otherwise indicated, these terms will be given their ordinary meaning in the art. Other terms specifically defined are to be interpreted in a manner consistent with the definitions provided herein.

[0099] Unless the context clearly indicates otherwise, the singular forms "a / an" and "the" as used herein include the plural forms.

[0100] As will be readily apparent to those skilled in the art from the teachings contained herein, in the context of numerical values ​​and ranges, the terms "about" or "approximately" refer to values ​​or ranges that are close to or approximately equal to the stated values ​​or ranges so that embodiments can be performed as intended, such as values ​​or ranges for having the desired amount of nucleic acid or polypeptide in a reaction mixture. In some embodiments, about means a numerical value ± 10%.

[0101] The terms "antibody-drug conjugate," "antibody conjugate," "conjugate," "immunoconjugate," and "ADC" are used interchangeably and refer to a therapeutic compound (e.g., a hoprene splice regulator) linked to one or more antibody or antigen-binding fragments, and are defined by the general formula: Ab-(LH)p (Formula I), where Ab = antibody or antigen-binding fragment, L = linker portion, H = hoprene splice regulator (e.g., hoprene or a derivative thereof), and p = number of drug portions of each antibody or antigen-binding fragment. ADCs containing hoprene splice regulators may also be more specifically referred to herein as "antibodies loaded with hoprene splice regulators" or "SMLAs." In ADCs containing hoprene splice regulators, "p" refers to the number of hoprene splice regulators linked to the antibody or antigen-binding fragment. In some embodiments, the linker L may include a cleavable portion between the antibody or antigen-binding fragment and the hoprene splice regulator. In some embodiments, linker L may include a cleavable portion that can be connected via a spacer unit to either or both of an antibody or antigen-binding fragment and a Hobstein splice regulator. In some embodiments, when the spacer unit connects a cleavable portion to the Hobstein splice regulator, it is a self-degrading spacer unit. In other embodiments, linker L does not include a cleavable portion and is a non-cleavable linker. In some embodiments, linker L may include at least one spacer unit that can be directly connected to an antibody or antigen-binding fragment and a Hobstein splice regulator. Illustrative cleavable and non-cleavable linkers are described and illustrated herein.

[0102] The term "antibody" in its broadest sense refers to an immunoglobulin molecule that specifically binds to a target such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or combination thereof via at least one antigen recognition site within the variable region of an immunoglobulin molecule. The heavy chain of an antibody consists of a heavy chain variable domain (VH) and a heavy chain constant domain (CH). The light chain consists of a light chain variable domain (VL) and a light chain constant domain (CL). For the purposes of this application, the mature heavy chain variable domain and light chain variable domain each contain three complementarity-determining regions (CDR1, CDR2, and CDR3) within four framework regions (FR1, FR2, FR3, and FR4) arranged from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. "Antibody" can be naturally occurring or artificial, such as monoclonal antibodies produced using conventional fusion tumor technology. The term "antibody" includes full-length monoclonal antibodies and full-length polyclonal antibodies, as well as antibody fragments and single-chain antibodies such as Fab, Fab', F(ab')2, and Fv. An antibody can be any of the five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (e.g., isotypes IgG1, IgG2, IgG3, and IgG4). The term further encompasses human antibodies, chimeric antibodies, humanized antibodies, and any modified immunoglobulin molecule containing an antigen recognition site, provided it exhibits the desired biological activity (e.g., binding to a target antigen, internalization within a cell expressing the target antigen).

[0103] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting that population are identical, except for a possible small number of naturally occurring mutations. Monoclonal antibodies exhibit high specificity against a single antigenic determinant. In contrast, conventional (multiclonal) antibody formulations typically comprise multiple antibodies targeting different antigenic determinants (or specific to different antigenic determinants). The modifier "monoclonal" indicates that the antibody system is derived from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody used according to the present invention may be manufactured by the fusion tumor method first described by Kohler et al. (1975) Nature 256:495, or by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using techniques described, for example, those described in Clackson et al. (1991) Nature 352:624-8 and Marks et al. (1991) J Mol Biol. 222:581-97.

[0104] The monoclonal antibodies described herein specifically include "chimeric" antibodies, wherein a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remaining portion of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass; and fragments of such antibodies, provided that they specifically bind to the target antigen and / or exhibit the desired biological activity.

[0105] As used herein, the term "human antibody" refers to an antibody produced by humans or an antibody having the amino acid sequence of an antibody produced by humans.

[0106] As used herein, "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. In some cases, the variable regions of both the heavy and light chains correspond to the variable regions of an antibody derived from one species with the desired specificity, affinity, and activity, while the constant regions are homologous to antibodies derived from another species (e.g., humans) to minimize the immune response in the latter species.

[0107] As used herein, the term "humanized antibody" refers to an antibody form containing sequences derived from both non-human (e.g., mouse) and human antibodies. These antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. Generally, humanized antibodies contain substantially all at least one and typically two variable domains, wherein all or substantially all hypervariable loops correspond to variable domains of non-human immunoglobulins and all or substantially all framework (FR) regions are variable domains of human immunoglobulin sequences. Humanized antibodies may also, where applicable, contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the constant region of human immunoglobulins. Humanized antibodies may be further modified to improve and optimize antibody specificity, affinity, and / or activity by substitution of residues within the Fv framework region and / or substituted non-human residues.

[0108] As used herein, the term "antigen-binding fragment" or "antigen-binding moiety" of an antibody refers to one or more fragments of an antibody or protein that retain the ability to specifically bind to antigens (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 be internalized into cells that express the antigen. 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 full-length antibodies. Examples of binding fragments covered by the term "antigen-binding fragment" or "antigen-binding part" of antibody include (i) Fab fragments, i.e., monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, i.e., bivalent fragments comprising two Fab fragments connected by disulfide bridging at the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of antibody; (v) dAb fragments containing a single variable domain, such as VH domain (see, for example, Ward et al. (1989) Nature 341:544-6; and International Publication No. WO 1990 / 005144); and (vi) separated complementarity-determining regions (CDRs). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be conjugated using recombinant methods via synthetic linkers that enable them to be manufactured as a single protein chain, where the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv)). See, for example, Bird et al. (1988) Science 242:423-6; and Huston et al. (1988) Proc Natl Acad Sci. USA 85:5879-83. These single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" or "antigen-binding portion" of antibody, and in this technique, are referred to as exemplary types of binding fragments that can be internalized into the cell after binding (see, for example, 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 some embodiments, the scFv molecule may be incorporated into the fusion protein. Other forms of single-chain antibodies, such as bifunctional antibodies, are also covered.Bifunctional antibodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain. This forces the domains to pair with the complementary domain of the other chain, creating two antigen-binding sites (see, for example, Holliger et al. (1993) Proc Natl Acad Sci. USA 90:6444-8; and Poljak et al. (1994) Structure 2:1121-3). The antigen-binding fragments are obtained using known techniques to those skilled in the art, and the fragments are screened for efficacy (e.g., binding affinity, internalization) in the same manner as the intact antibody. Antigen-binding fragments can be prepared by cleaving the intact protein, for example, by protease or chemical cleavage.

[0109] As used herein with respect to antibody or antigen-binding fragments, "internalization" means that after binding to a cell, the antibody or antigen-binding fragment is absorbed through the cell's lipid bilayer membrane into the internal compartments (i.e., "internalized"), preferably into the cell's degradation compartments. For example, an internalized anti-HER2 antibody is an antibody that is absorbed 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 internalized antibody or internalized antigen-binding fragment (i.e., the ADC is transferred across the cell membrane after antigen binding). In some embodiments, the internalized antibody or antigen-binding fragment binds to a receptor on the cell surface. Internalized antibodies or internalized antigen-binding fragments targeting receptors on the cell membrane can induce receptor-mediated endocytosis. In some embodiments, the internalized antibody or internalized antigen-binding fragment is absorbed into the cell via receptor-mediated endocytosis.

[0110] As used herein with respect to antibody or antigen-binding fragments, “non-internalizing” means that the antibody or antigen-binding fragment remains on the cell surface after binding to the cell. In some embodiments, the antibody or antigen-binding fragments used in the ADCs disclosed herein target cell surface antigens and are non-internalizing antibodies or non-internalizing antigen-binding fragments (i.e., the ADC remains on the cell surface after antigen binding and does not migrate across the cell membrane). In some embodiments, non-internalizing antibodies or antigen-binding fragments bind to non-internalizing receptors or other cell surface antigens. Exemplary non-internalizing cell surface antigens include, but are not limited to, CA125 and CEA, and the antibodies binding to non-internalizing antigen targets are also known in this art (see, for example, 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).

[0111] As used herein, the terms "human epidermal growth factor receptor 2," "HER2," or "HER2 / NEU" refer to any naturally occurring form of human HER2. This term encompasses full-length HER2 (e.g., UniProt reference sequence: P04626; SEQ ID NO: 31) and any form of human HER2 that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human HER2, including but not limited to splice variants, paired gene variants, and isoforms that retain one or more of the biological functions of human HER2 (i.e., unless the context indicates that the term is used only to refer to the wild-type protein, it encompasses variants and fragments). HER2 can be isolated from humans or produced recombinantly or synthetically.

[0112] The term "anti-HER2 antibody" or "antibody bound to HER2" refers to any form of antibody or fragment thereof that binds (e.g., specifically binds) to HER2, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragment binds (e.g., specifically binds) to HER2. U.S. Patent No. 5,821,337 provides exemplary HER2 binding sequences, including exemplary anti-HER2 antibody sequences, and that document is incorporated herein by reference. 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. Patent No. 5,821,337; Molina et al. (2001) Cancer Res. 61(12):4744-9) is an exemplary anti-human HER2 antibody.

[0113] As used herein, the terms "multiligand proteoglycan-1," "SDC1," or "CD138" refer to any naturally occurring form of human CD138. This term encompasses full-length CD138 (e.g., UniProt reference sequence: P18827; SEQ ID NO: 32) and any form of human CD138 that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human CD138, including but not limited to splice variants, paired gene variants, and isoforms that retain one or more of the biological functions of human CD138 (i.e., unless the context indicates that the term is used only to refer to the wild-type protein, it encompasses variants and fragments). CD138 can be isolated from humans or produced recombinantly or synthetically.

[0114] The terms "anti-CD138 antibody" or "antibody bound to CD138" refer to any form of antibody or fragment thereof that binds (e.g., specifically binds) to CD138, and encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragment binds (e.g., specifically binds) to CD138. In some embodiments, the anti-CD138 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. B-B4 (Tassone et al. (2004) Blood 104:3688-96) is an illustrative anti-human CD138 antibody.

[0115] As used herein, the terms "pterin A receptor 2" or "EPHA2" refer to any naturally occurring form of human EPHA2. This term encompasses full-length EPHA2 (e.g., UniProt reference sequence: P29317; SEQ ID NO: 33) and any form of human EPHA2 that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human EPHA2, including but not limited to splice variants, paired gene variants, and isoforms that retain one or more of the biological functions of human EPHA2 (i.e., unless the context indicates that the term is used only to refer to the wild-type protein, it encompasses variants and fragments). EPHA2 can be isolated from humans or produced recombinantly or synthetically.

[0116] The term "anti-EPHA2 antibody" or "antibody bound to EPHA2" refers to any form of antibody or fragment thereof that binds (e.g., specifically binds) to EPHA2, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to EPHA2. WO 2007 / 030642 provides exemplary EPHA2 binding sequences, including exemplary anti-EPHA2 antibody sequences, and that document is incorporated herein by reference. In some embodiments, the anti-EPHA2 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. 1C1 (WO 2007 / 030642; Jackson et al. (2008) Cancer Res. 68(22): 9367-74) is an exemplary anti-human EPHA2 antibody.

[0117] As used herein, the terms "mesothelin" or "MSLN" refer to any naturally occurring form of human MSLN. This term encompasses full-length MSLNs (e.g., UniProt reference sequence: Q13421; SEQ ID NO: 94) and any form of human MSLN that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human MSLNs, including but not limited to splice variants, paired gene variants, and isoforms that retain one or more of the biological functions of human MSLNs (i.e., unless the context indicates that the term is used only to refer to wild-type proteins, it encompasses variants and fragments). MSLNs can be isolated from humans or produced recombinantly or synthetically.

[0118] The terms "anti-MSLN antibody" or "antibody bound to MSLN" refer to any form of antibody or fragment thereof that binds (e.g., specifically binds) to MSLN, and encompass monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragment binds (e.g., specifically binds) to MSLN. WO 2011 / 074621 provides exemplary MSLN binding sequences, including exemplary anti-MSLN antibody sequences, and that document is incorporated herein by reference. In some embodiments, the anti-MSLN antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. IC11-25, IC14-30, IC7-4, IC17-35, and 2-9 are exemplary anti-human MSLN antibodies.

[0119] As used herein, the terms "glutamic acid carboxypeptidase 2" or "FOLH1" refer to any naturally occurring form of human FOLH1. This term encompasses full-length FOLH1 (e.g., UniProt reference sequence: Q04609; SEQ ID NO: 95) and any form of human FOLH1 that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human FOLH1, including but not limited to splice variants, paired gene variants, and isoforms that retain one or more of the biological functions of human FOLH1 (i.e., unless the context indicates that the term is used only to refer to the wild-type protein, it encompasses variants and fragments). FOLH1 can be isolated from humans or produced recombinantly or synthetically.

[0120] The terms "anti-FOLH1 antibody" or "antibody bound to FOLH1" refer to any form of antibody or fragment thereof that binds (e.g., specifically binds) to FOLH1, and encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to FOLH1. WO 2019 / 012260 and WO 2017 / 212250 provide exemplary FOLH1 binding sequences, including exemplary anti-FOLH1 antibody sequences, and these documents are incorporated herein by reference. 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.

[0121] As used herein, the terms "cadherin-6" or "CDH6" refer to any naturally occurring form of human CDH6. This term encompasses full-length CDH6 (e.g., UniProt reference sequence: P55285; SEQ ID NO: 96) and any form of human CDH6 that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human CDH6, including but not limited to splice variants, paired gene variants, and isoforms that retain one or more of the biological functions of human CDH6 (i.e., unless the context indicates that the term is used only to refer to the wild-type protein, it encompasses variants and fragments). CDH6 can be isolated from humans or produced recombinantly or synthetically.

[0122] The terms "anti-CDH6 antibody" or "antibody bound to CDH6" refer to any form of antibody or fragment thereof that binds (e.g., specifically binds) to CDH6, and encompass monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragment binds (e.g., specifically binds) to CDH6. WO 2018 / 185618 provides exemplary CDH6 binding sequences, including exemplary anti-CDH6 antibody sequences, and that document is incorporated herein by reference. In some embodiments, the anti-CDH6 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0123] As used herein, the terms "carcinoembryonic antigen-associated cell adhesion molecule 5" or "CEACAM5" refer to any naturally occurring form of human CEACAM5. This term encompasses full-length CEACAM5 (e.g., UniProt reference sequence: P06731; SEQ ID NO: 97) and any form of human CEACAM5 that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human CEACAM5, including but not limited to splice variants, paired gene variants, and isoforms that retain one or more of the biological functions of human CEACAM5 (i.e., unless the context indicates that the term is used only to refer to the wild-type protein, it encompasses variants and fragments). CEACAM5 can be isolated from humans or produced recombinantly or synthetically.

[0124] The terms "anti-CEACAM5 antibody" or "antibody bound to CEACAM5" refer to any form of antibody or fragment thereof that binds (e.g., specifically binds) to CEACAM5, and encompass monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to CEACAM5. US 2015 / 0125386 provides exemplary CEACAM5 binding sequences, including exemplary anti-CEACAM5 antibody sequences, and that document is incorporated herein by reference. In some embodiments, the anti-CEACAM5 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. hMN14 is an exemplary anti-human CEACAM5 antibody.

[0125] As used herein, the terms "hidden family protein 1B" or "CFC1B" refer to any naturally occurring form of human CFC1B. This term encompasses full-length CFC1B (e.g., UniProt reference sequence: P0CG36; SEQ ID NO: 98) and any form of human CFC1B that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human CFC1B, including but not limited to splice variants, paired gene variants, and isoforms that retain one or more of the biological functions of human CFC1B (i.e., unless the context indicates that the term is used only to refer to wild-type protein, it encompasses variants and fragments). CFC1B can be isolated from humans or produced recombinantly or synthetically.

[0126] The terms "anti-CFC1B antibody" or "antibody bound to CFC1B" refer to any form of antibody or fragment thereof that binds (e.g., specifically binds) to CFC1B, and encompass monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragment binds (e.g., specifically binds) to CFC1B. WO 2002 / 088170 provides exemplary CFC1B binding sequences, including exemplary anti-CFC1B antibody sequences, and that document is incorporated herein by reference. In some embodiments, the anti-CFC1B antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0127] As used herein, the terms "exonucleotide pyrophosphatase / phosphodiesterase family member 3" or "ENPP3" refer to any naturally occurring form of human ENPP3. This term encompasses full-length ENPP3 (e.g., UniProt reference sequence: O14638; SEQ ID NO: 99) and any form of human ENPP3 that can be processed by cells. The term also encompasses functional variants or fragments of human ENPP3, including but not limited to splice variants, paired gene variants, and isoforms that retain one or more of the biological functions of human ENPP3 (i.e., unless the context indicates that the term is used only to refer to wild-type proteins, it encompasses variants and fragments). ENPP3 can be isolated from humans or produced recombinantly or synthetically.

[0128] The terms "anti-ENPP3 antibody" or "antibody bound to ENPP3" refer to any form of antibody or fragment thereof that binds (e.g., specifically binds) to ENPP3, and encompass monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to ENPP3. Donate et al. ((2016) Clin Cancer Res. 22(8):1989-99) provided exemplary ENPP3 binding sequences, including exemplary anti-ENPP3 antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-ENPP3 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0129] As used herein, the terms "folate receptor α" or "FOLR1" refer to any naturally occurring form of human FOLR1. This term encompasses full-length FOLR1 (e.g., UniProt reference sequence: P15328; SEQ ID NO: 100) and any form of human FOLR1 that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human FOLR1, including but not limited to splice variants, paired gene variants, and isoforms that retain one or more of the biological functions of human FOLR1 (i.e., unless the context indicates that the term is used only to refer to the wild-type protein, it encompasses variants and fragments). FOLR1 can be isolated from humans or produced recombinantly or synthetically.

[0130] The term "anti-FOLR1 antibody" or "antibody bound to FOLR1" refers to any form of antibody or fragment thereof that binds (e.g., specifically binds) to FOLR1, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragment binds (e.g., specifically binds) 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 these cases and references are incorporated herein by reference. 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.

[0131] As used herein, the terms "hepatitis A virus cell receptor 1" or "HAVCR1" refer to any naturally occurring form of human HAVCR1. This term encompasses full-length HAVCR1 (e.g., UniProt reference sequence: Q96D42; SEQ ID NO: 101) and any form of human HAVCR1 that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human HAVCR1, including but not limited to splice variants, paired gene variants, and isoforms that retain one or more of the biological functions of human HAVCR1 (i.e., unless the context indicates that the term is used only to refer to the wild-type protein, it encompasses variants and fragments). HAVCR1 can be isolated from humans or produced recombinantly or synthetically.

[0132] The terms "anti-HAVCR1 antibody" or "antibody bound to HAVCR1" refer to any form of antibody or fragment thereof that binds (e.g., specifically binds) to HAVCR1, and encompass monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to HAVCR1. Thomas et al. ((2016) Mol Cancer Ther. 15(12):2946-54) provide exemplary HAVCR1 binding sequences, including exemplary anti-HAVCR1 antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-HAVCR1 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0133] As used herein, the terms "mast cell / stem cell growth factor receptor Kit" or "KIT" refer to any naturally occurring form of human KIT. This term encompasses full-length KIT (e.g., UniProt reference sequence: P10721; SEQ ID NO: 102) and any form of human KIT that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human KIT, including but not limited to splice variants, paired gene variants, and isoforms that retain one or more of the biological functions of human KIT (i.e., unless the context indicates that the term is used only to refer to wild-type proteins, it encompasses variants and fragments). KIT can be isolated from humans or produced recombinantly or synthetically.

[0134] The terms "anti-KIT antibody" or "antibody bound to KIT" refer to any form of antibody or fragment thereof that binds (e.g., specifically binds) to KIT, and encompass monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) 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 these documents are incorporated herein by reference. In some embodiments, the anti-KIT antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0135] As used herein, the terms "hepatocyte growth factor receptor" or "MET" refer to any naturally occurring form of human MET. This term encompasses full-length MET (e.g., UniProt reference sequence: P08581; SEQ ID NO: 103) and any form of human MET that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human MET, including but not limited to splice variants, paired gene variants, and isoforms that retain one or more of the biological functions of human MET (i.e., unless the context indicates that the term is used only to refer to wild-type protein, it encompasses variants and fragments). MET can be isolated from humans or produced recombinantly or synthetically.

[0136] The terms "anti-MET antibody" or "antibody bound to MET" refer to any form of antibody or fragment thereof that binds (e.g., specifically binds) to MET, and encompass monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to MET. Yang et al. ((2019) Acta Pharmacol Sin.) provide exemplary MET binding sequences, including exemplary anti-MET antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-MET antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0137] As used herein, the term "mucin-16" or "MUC16" 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) and any form of human MUC16 that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human MUC16, including but not limited to splice variants, paired gene variants, and isoforms that retain one or more of the biological functions of human MUC16 (i.e., unless the context indicates that the term is used only to refer to wild-type protein, it encompasses variants and fragments). MUC16 can be isolated from humans or produced recombinantly or synthetically.

[0138] The term "anti-MUC16 antibody" or "antibody bound to MUC16" refers to any form of antibody or fragment thereof that binds (e.g., specifically binds) to MUC16, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to MUC16. Liu et al. ((2016) Ann Oncol. 27(11):2124-30) provide exemplary MUC16 binding sequences, including exemplary anti-MUC16 antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-MUC16 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0139] As used herein, the terms "zinc transporter ZIP6" or "SLC39A6" refer to any naturally occurring form of human SLC39A6. This term encompasses full-length SLC39A6 (e.g., UniProt reference sequence: Q13433; SEQ ID NO: 105) and any form of human SLC39A6 that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human SLC39A6, including but not limited to splice variants, paired gene variants, and isoforms that retain one or more of the biological functions of human SLC39A6 (i.e., unless the context indicates that the term is used only to refer to wild-type protein, it encompasses variants and fragments). SLC39A6 can be isolated from humans or produced recombinantly or synthetically.

[0140] The terms "anti-SLC39A6 antibody" or "antibody bound to SLC39A6" refer to any form of antibody or fragment thereof that binds (e.g., specifically binds) to SLC39A6, and encompass monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to SLC39A6. Sussman et al. ((2014) Mol Cancer Ther. 13(12):2991-3000) provided exemplary SLC39A6 binding sequences, including exemplary anti-SLC39A6 antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-SLC39A6 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0141] As used herein, the terms "choline transporter-like protein 4" or "SLC44A4" refer to any naturally occurring form of human SLC44A4. This term encompasses full-length SLC44A4 (e.g., UniProt reference sequence: Q53GD3; SEQ ID NO: 106) and any form of human SLC44A4 that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human SLC44A4, including but not limited to splice variants, paired gene variants, and isoforms that retain one or more of the biological functions of human SLC44A4 (i.e., unless the context indicates that the term is used only to refer to the wild-type protein, it encompasses variants and fragments). SLC44A4 can be isolated from humans or produced recombinantly or synthetically.

[0142] The terms "anti-SLC44A4 antibody" or "antibody bound to SLC44A4" refer to any form of antibody or fragment thereof that binds (e.g., specifically binds) to SLC44A4, and encompass monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to SLC44A4. Mattie et al. ((2016) Mol Cancer Ther. 15(11):2679-87) provide exemplary SLC44A4 binding sequences, including exemplary anti-SLC44A4 antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-SLC44A4 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0143] As used herein, the term "metal reductase STEAP1" or "STEAP1" refers to any naturally occurring form of human STEAP1. This term encompasses full-length STEAP1 (e.g., UniProt reference sequence: Q9UHE8; SEQ ID NO: 107) and any form of human STEAP1 that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human STEAP1, including but not limited to splice variants, paired gene variants, and isoforms that retain one or more of the biological functions of human STEAP1 (i.e., unless the context indicates that the term is used only to refer to the wild-type protein, it encompasses variants and fragments). STEAP1 can be isolated from humans or produced recombinantly or synthetically.

[0144] The terms "anti-STEAP1 antibody" or "antibody bound to STEAP1" refer to any form of antibody or fragment thereof that binds (e.g., specifically binds) to STEAP1, and encompass monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to STEAP1. WO 2008 / 052187 provides exemplary STEAP1 binding sequences, including exemplary anti-STEAP1 antibody sequences, and that document is incorporated herein by reference. In some embodiments, the anti-STEAP1 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0145] As used herein, the terms "specific" and "specifically binds / 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) within a heterogeneous population of proteins and other biological products. The binding specificity of an antibody can be tested by comparing binding to an appropriate antigen with binding to unrelated antigens or mixtures of antigens under a given set of conditions. An antibody is considered specific if it binds to an appropriate antigen with an affinity at least 2, 5, 7, and preferably 10 or more times greater than that with unrelated antigens or mixtures of antigens. A "specific antibody" or "target-specific antibody" is an antibody that binds only to the target antigen (e.g., HER2) but not to other antigens (or exhibits minimal binding to other antigens). In some embodiments, the KD of the antibody or antigen-binding fragment that specifically binds to the target antigen (e.g., HER2) is less than 1×10⁻⁶ M, less than 1×10⁻⁷ M, less than 1×10⁻⁸ M, less than 1×10⁻⁹ M, less than 1×10⁻¹⁰ M, less than 1×10⁻¹¹ M, less than 1×10⁻¹² M, or less than 1×10⁻¹³ M. In some embodiments, the KD is from 1 pM to 500 pM. In some embodiments, the KD is between 500 pM and 1 µM, 1 µM and 100 nM, or 100 mM and 10 nM.

[0146] The term "antigenic determinant" refers to a portion of an antigen that can be recognized and specifically bound by an antibody. When the antigen is a polypeptide, the antigenic determinant can be formed from continuous amino acids or discontinuous amino acids adjacent to each other through the tertiary folding of the polypeptide. Antigenic determinants bound by antibodies can be identified using any antigenic determinant localization technique known in this art, including X-ray crystallography for antigenic determinant recognition, which involves direct observation of the antigen-antibody complex, monitoring the binding of antibody to fragments or mutant variants of the antigen, or monitoring the solvent accessibility of different portions of the antibody and antigen. Exemplary strategies for localizing antibody antigenic determinants include, but are not limited to, array-based oligopeptide scanning, restricted proteolysis, site-directed mutagenesis, high-throughput mutagenesis localization, hydrogen-deuterium exchange, and mass spectrometry (see, for example, Gershoni et al. (2007) 21:145-56; and Hager-Braun and Tomer (2005) Expert Rev Proteomics 2:745-56).

[0147] Competitive binding and epitope binning can also be used to identify antibodies that share the same or overlapping antigenic determinants. Competitive binding can be evaluated using cross-blocking analyses such as those described in "Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory, Harlow, and Lane (1st edition 1988, 2nd edition 2014). In some embodiments, competitive binding is identified when, in a cross-blocking analysis, the antibody or binding protein tested reduces the binding of a reference antibody or binding protein (e.g., binding proteins containing CDR and / or variable domains selected from those identified in Tables 2-4) to a target antigen (such as HER2) by at least about 50% (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or higher, or any percentage between thereof), and / or vice versa. In some embodiments, competitive binding may be attributed to shared or similar (e.g., partially overlapping) antigenic determinants, or to steric hindrance in the binding of antibodies or binding proteins at adjacent antigenic determinants (see, for example, Tzartos, Methods in Molecular Biology (Morris, ed. (1998) Vol. 66, pp. 55-66)). In some embodiments, competitive binding may be used to sort groups of binding proteins that share similar antigenic determinants. For example, competitively binding proteins may be “boxed” into groups of binding proteins with overlapping or adjacent antigenic determinants, while non-competitive binding proteins may be grouped into groups of individual binding proteins that do not have overlapping or adjacent antigenic determinants.

[0148] The term "k on" or "ka" refers to the association rate constant of an antibody and an antigen to form an antibody / antigen complex. This rate can be determined using standard analyses such as surface plasma resonance, biolayer interferometry, or ELISA.

[0149] The terms "koff" or "kd" refer to the dissociation rate constant of an antibody from its antibody / antigen complex. This rate can be determined using standard analyses such as surface plasma resonance, biolayer interferometry, or ELISA.

[0150] The term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction. KD is calculated as ka / kd. This rate can be determined using standard analyses such as surface plasma resonance, biolayer interferometry, or ELISA.

[0151] In the ADCs disclosed herein, the term "p" or "Hobstein splice regulator loading," "Hobstein splice regulator:antibody ratio," or "Hobstein splice regulator to antibody ratio," or "HAR," refers to the number of Hobstein splice regulators per antibody or antigen-binding fragment, i.e., the Hobstein splice regulator loading, or the number of -LH portions of each antibody or antigen-binding fragment (Ab). In ADCs containing Hobstein splice regulators, "p" refers to the number of Hobstein splice regulators linked to the antibody or antigen-binding fragment. For example, if two Hobstein splice regulators (e.g., two compounds each having an H3 structure) are both linked to the antibody or antigen-binding fragment, then p = 2. In a composition containing multiple copies of an ADC as described herein, "average p" refers to the average number of -LH portions of each antibody or antigen-binding fragment, also known as "average hobschierne splice regulator loading".

[0152] "Linker" or "linker moiety" is used herein to refer to any chemical moiety capable of covalently attaching a compound (typically a pharmaceutical moiety, such as a Hobstein splice regulator pharmaceutical moiety) to another moiety (such as an antibody or antigen-binding fragment). Linkers may be readily cleaved or substantially resistant to acid-induced cleavage, peptidase-induced cleavage, light-based cleavage, esterase-induced cleavage, and / or disulfide bond cleavage, under conditions that preserve the activity of the compound or antibody.

[0153] The term "pharmaceutical" as used herein refers to a compound, a mixture of compounds, a biomacromolecule, or an extract derived from biological material. The terms "therapeutic agent" or "drug" refer to a pharmaceutical agent that can modulate biological processes and / or has biological activity. The Hobschdiene splice regulator described herein is an illustrative therapeutic agent.

[0154] The terms "chemotherapy agent" or "anticancer agent" are used herein to refer to all agents that are effective in treating cancer regardless of their mechanism of action. Inhibition of metastasis or angiogenesis is often a characteristic of chemotherapy agents. Chemotherapy agents include antibodies, biomolecules, and small molecules, and encompass the Hopschidene splice regulator compounds described herein. Chemotherapy agents can be cytotoxic agents or cell growth inhibitors. The term "cell growth inhibitor" 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 the expression and / or function of cells.

[0155] As used herein, the terms "herboxidiene splicing modulator" and "herboxidiene splice modulator" refer to compounds that possess anticancer activity through interaction with components of the splice body and are structurally related to herboxidiene. In some embodiments, herboxidiene splicing modulators alter the splicing rate or form in target cells. Herboxidiene splicing modulators acting as inhibitors, for example, can reduce uncontrolled cell proliferation. In some embodiments, herboxidiene splicing modulators may act by binding to the SF3b splice body complex. These modulators may be naturally occurring or synthetic herboxidiene derivatives or analogs. When referring to herboxidiene splicing modulators or their analogs, the terms "derivative" and "analyte" as used herein mean any compound that has substantially the same, similar, or enhanced biological function or activity as herboxidiene but has a modified chemical or biological structure. In some embodiments, the Hopschadiene splice regulator is a Hopschadiene derivative.

[0156] As used herein, "Hobstein splice regulator drug portion" refers to a component of an ADC or composition that provides the structure of a hobstein splice regulator compound, such as the hobstein splice regulator (H) component in an ADC of formula (I) or a composition containing -LH.

[0157] As used in this article, "splicing complex" refers to a ribonucleoprotein complex that removes introns from one or more RNA segments, such as pre-mRNA segments.

[0158] The term "homology" refers to a molecule that exhibits homology with another molecule by having, for example, the same or similar sequence of chemical residues at corresponding positions.

[0159] As used in this article, "inhibit / inhibition of" means reducing a measurable quantity, and may include, but does not require, complete prevention or inhibition.

[0160] The terms "target-negative," "target-antigen-negative," or "antigen-negative" refer to cells or tissues that do not express the target antigen. The terms "target-positive," "target-antigen-positive," or "antigen-positive" refer to cells or tissues that express the target antigen. For example, cells or cell lines that do not express the target antigen can be described as target-negative, while cells or cell lines that express the target antigen can be described as target-positive.

[0161] The term "bystander killing" or "bystander effect" refers to the killing of target negative cells in the presence of target positive cells, where the killing of target negative cells is not observed in the absence of target positive cells. Cell-to-cell contact, or at least the proximity between target positive and target negative cells, enables bystander killing. This type of killing can be distinguished from "off-target killing," which refers to the indiscriminate killing of target negative cells. Off-target killing can be observed in the absence of target positive cells.

[0162] The terms "hypertrophic disease" and "cancer" are used interchangeably in this document to refer to cells possessing characteristics characteristic of cancerous cells, such as uncontrolled proliferation, non-destructiveness, metastatic potential, rapid growth and proliferation rates, and / or certain morphological features. Typically, cancer cells may present as tumors or masses, but these cells can exist independently within an individual or circulate independently in the bloodstream as cells such as leukemia cells or lymphoma cells. The terms "hypertrophic disease" and "cancer" encompass all types of cancer and cancer metastases, including hematologic malignancies, solid tumors, sarcomas, carcinomas and other solid tumor cancers, and non-solid tumor cancers. Hematologic malignancies may include B-cell malignancies, blood cancers (leukemia), plasma cell carcinomas (myeloma, such as multiple myeloma), or lymph node carcinomas (lymphoma). Exemplary B-cell malignancies include chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, and diffuse large B-cell lymphoma. Leukemia can include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloid monocytic leukemia (CMML), and acute monocytic leukemia (AMoL). Lymphoma can include Hodgkin's lymphoma and non-Hodgkin's lymphoma. Other hematologic malignancies can include myelomectomy syndrome (MDS). Solid tumors can include carcinomas such as adenocarcinoma (e.g., breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, stomach cancer, cervical cancer, endometrial cancer, ovarian cancer, bile duct cancer, glioma, and melanoma.

[0163] The terms "tumor" and "necrotic tumor" refer to any benign or malignant mass of tissue caused by excessive cell growth or proliferation, including precancerous lesions.

[0164] The terms "tumor cell" and "proliferative cell" are used interchangeably and refer to individual cells or a total cell population derived from a tumor or neoplasm, including both non-tumorigenic cells and cancer stem cells. When referring only to tumor cells that lack the capacity for renewal and differentiation, the term "tumor cell" as used herein is modified by the term "non-tumorigenic" to distinguish them from cancer stem cells.

[0165] The terms "individual" 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, and similar animals. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human. In some embodiments, the individual is a mouse. In some embodiments, the individual is a human.

[0166] The terms "co-administration" or "combination" refer to the administration of one or more therapeutic agents, including simultaneous administration and sequential administration in any order.

[0167] "Pharmaceutical composition" means a formulation which is permitted to be administered with an active ingredient and subsequently provides the intended biological activity and / or achieves a therapeutic effect, and which does not contain any additional components that would have unacceptable toxicity to the individual to which the corresponding compound is administered. Pharmaceutical compositions may be sterile.

[0168] "Pharmaceutical excipients" include substances such as adjuvants, carriers, pH adjusters and buffers, tension modifiers, wetting agents, preservatives and similar reagents.

[0169] "Medically acceptable" means approved or permitted by a federal regulatory agency or state government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals and more specifically in humans.

[0170] "Pharmaceutical acceptable salts" are salts that retain the desired biological activity of the parent compound without imparting unwanted toxicological effects. Examples of such salts include: (a) acid addition salts formed from inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and similar inorganic acids; and salts formed from 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 similar organic acids; and (b) salts formed from elemental anions such as chlorine, bromine and iodine. See, for example, 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.

[0171] As used herein, the term "effective amount" means the amount of a compound, ADC, or composition (e.g., a heptacysteine ​​splice regulator or ADC) described herein that is sufficient to perform the specifically stated purpose, such as being sufficient to produce a therapeutic effect after administration, such as a reduction in tumor growth rate or tumor volume, relief of cancer symptoms, or some other marker of therapeutic efficacy. Effective amounts can be determined in a conventional manner in relation to the stated purpose. The term "therapeutic effective amount" means the amount of a compound, ADC, or composition described herein that effectively and detectably kills tumor cells, reduces and / or inhibits the growth or spread of tumor cells, the size or number of tumors, and / or other measures of the degree, stage, development, and / or severity of cancer. Therapeutic effective amounts can vary depending on the intended application (in vitro or in vivo) or the individual and disease condition being treated, such as the individual's weight and age, the severity of the disease condition, the method of administration, and similar factors that can be readily determined by someone generally skilled in the art. This term also applies to doses that induce a specific response in target cells, such as inhibiting cell growth. The specific dosage may vary depending on factors such as the particular pharmaceutical composition, the individual and their age, existing health conditions or risks associated with those conditions, the dosing regimen to be followed, the severity of the disease, the timing of administration (whether or not it is administered in combination with other agents), the tissue to which it is administered, and the physical delivery system that carries it. In cancer cases, therapeutically effective doses of ADCs can reduce the number of cancer cells, decrease 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.

[0172] "Prophylactic effective dose" refers to the amount that effectively achieves the desired preventive outcome at the necessary dosage and time period. Generally, because the preventive dose is administered to an individual before or in the early stages of the disease, the preventive effective dose is less than the therapeutic effective dose.

[0173] As used herein, the terms "treatment" or "therapeutic" and related grammatical terms refer to any improvement in any outcome of the disease, such as prolonged survival, lower morbidity, and / or reduction of side effects caused by alternative treatment modalities. As is readily understood in this art, treatment procedures encompass, but do not require, the complete eradication of the disease. As used herein, "treatment" means administering the described ADC or composition to an individual, such as a patient. Treatment may be used to cure, heal, alleviate, relieve, alter, remedy, improve, mitigate, improve, or influence a condition (e.g., cancer), its symptoms, or its predisposition. In some embodiments, in addition to treating an individual with the condition, the compositions disclosed herein may be provided prophylactically to prevent or reduce the likelihood of developing the condition.

[0174] In some embodiments, a labeled ADC is used. Suitable "labels" include radioactive nuclei, enzymes, acceptors, cofactors, inhibitors, fluorescent portions, chemiluminescent portions, magnetic particles, and the like.

[0175] As used herein, “protein” means at least two covalently linked amino acids. The term encompasses polypeptides, oligopeptides, and peptides. In some embodiments, two or more covalently linked amino acids are linked by peptide bonds. Proteins can consist of naturally occurring amino acids and peptide bonds, for example, when proteins are recombinantly produced using an expression system and host cells. Alternatively, proteins may comprise synthetic amino acids (e.g., homophenylalanine, citrulline, ornithine, and oroleucine) or peptide mimicry structures (i.e., “peptides or protein analogs,” such as peptide-like structures). Peptides-like structures are exemplary classes of peptide mimics whose side chains are attached to the nitrogen atom of the peptide backbone rather than to the α-carbon (as it is in the amino acid), and have different hydrogen bonding and conformational characteristics compared to peptides (see, for example, Simon et al. (1992) Proc Natl Acad Sci. USA 89:9367). Therefore, peptide-like structures may be resistant to protein hydrolysis or other physiological or storage conditions and can effectively permeate cell membranes. These synthetic amino acids can be incorporated, specifically when antibodies are synthesized in vitro using conventional methods well known in this art. Additionally, any combination of peptide mimics, synthetic residues / structures, and naturally occurring residues / structures can be used. "Amino acids" also include imine residues such as proline and hydroxyproline. The amino acid "R group" or "side chain" can be in an (L)- or (S)- configuration. In one specific embodiment, the amino acid is in an (L)- or (S)- configuration.

[0176] "Recombinant protein" is a protein produced using recombinant technology, any technology and method known in this technology, i.e., by expressing recombinant nucleic acids. Methods and techniques for producing recombinant proteins are well known in this technology.

[0177] The "isolated" protein is not accompanied by at least one material that it normally associates with in its native state, for example, comprising at least about 5% by weight or at least about 50% by weight of the total protein in a given sample. It should be understood that the isolated protein may comprise from 5% by weight to 99.9% by weight of the total protein content, depending on the circumstances. For example, proteins can be produced at significantly higher concentrations using inducible or high-performance promoters, thereby enabling the production of proteins at increased concentration levels. This definition includes the production of antibodies in a wide variety of organisms and / or host cells known in this art.

[0178] For amino acid sequences, sequence identity and / or similarity can be determined using, but is 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 similarity retrieval method of Pearson and Lipman (1988) Proc Nat Acad Sci. USA 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Suite, Genetics Computer Group, 575 Science Drive, Madison, Wis.), and the Best Fit sequence program described by Devereux et al. (1984) Nucl Acid Res. 12:387-95, as well as standard techniques known in this technique, preferably using preset settings or by testing. Preferably, the consistency percentage is calculated by FastDB based on the following parameters: mismatch penalty 1; void penalty 1; void size penalty 0.33; and joint penalty 30 ("Current Methods in Sequence Comparison and Analysis", Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149 (1988), Alan R. Liss, Inc).

[0179] An example of an applicable algorithm is PILEUP. PILEUP uses progressive pairwise alignment to generate multiple sequence alignments from a set of related sequences. It can also draw a tree diagram showing the clustering relationships used to generate the alignments. PILEUP uses a simplified form of the progressive alignment method described by Feng and Doolittle (1987) J Mol Evol. 35:351-60; this method is similar to that described by Higgins and Sharp (1989) CABIOS 5:151-3. Applicable PILEUP parameters include a preset gap weight of 3.00, a preset gap length weight of 0.10, and weighted end gaps.

[0180] Another example of a suitable algorithm is the BLAST algorithm described below: 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 suitable BLAST program is the WU-BLAST-2 program obtained 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. The adjustable parameters are set with the following values: overlap interval = 1, overlap score = 0.125, and word threshold (T) = II. The HSP S and HSP S2 parameters are dynamic values ​​and are determined by the program itself based on the composition of a specific sequence and the composition of the specific database against which the sequence of interest is compared; however, these values ​​can be adjusted to improve sensitivity.

[0181] An additional applicable algorithm is the gapped BLAST reported by Altschul et al. (1997) Nucl Acid Res. 25:3389-402. The gapped BLAST uses BLOSUM-62 instead of scoring; the threshold parameter T is set to 9; a two-hit method is used to trigger the gapless extension, adding a gap length k at a cost of 10+k; Xu is set to 16, and Xg is set to 40 during the database retrieval phase and 67 during the algorithm output phase. Gap matching is triggered by a score corresponding to approximately 22.

[0182] Generally, the amino acid homology, similarity, or identity between the proteins and their variants (including 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 antibody variable domain variants (including individual variants CDR)) disclosed in this article and the sequences described herein is at least 80%, for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, approximately 100%, or 100%.

[0183] Similarly, the "nucleic acid sequence identity percentage (%)" for the nucleic acid sequences of antibodies and other proteins identified herein is defined as the percentage of nucleotide residues in candidate sequences that are identical to the nucleotide residues in the coding sequence of the antigen-binding protein. The specific method utilizes a BLASTN module of WU-BLAST-2 with preset parameters, wherein the overlap interval and overlap fraction are set to 1 and 0.125, respectively.

[0184] Although the sites or regions where amino acid sequence changes are introduced are predetermined, the mutations themselves do not need to be predetermined. For example, to optimize the effectiveness of mutations at a given site, the optimal combination of random mutation induction at the target codon or region can be used to screen for the desired activity of expressed antigen-binding protein CDR variants. Techniques for inducing substitution mutations at predetermined sites in DNA with known sequences are well known, such as MI3 primer mutation induction and PCR mutation induction.

[0185] As used herein, "alkyl / alkyl group" means a fully saturated straight-chain, branched-chain, or cyclic hydrocarbon chain. In some embodiments, the alkyl group may contain 1-8 carbon atoms ("C1-C8 alkyl"). In some embodiments, the alkyl group may contain 1-6 carbon atoms ("C1-C6 alkyl"). In some embodiments, the alkyl group contains 1-3 carbon atoms. In still other embodiments, the alkyl group contains 2-3 carbon atoms, and in still other embodiments, the alkyl group contains 1-2 carbon atoms.

[0186] As used in this article, "alkylalkoxy" means an alkyl group substituted with an alkoxy group.

[0187] As used herein, "alkoxy" refers to an alkyl group as previously defined, which is attached to the main carbon chain via an oxygen ("alkoxy") atom.

[0188] As used in this article, "alkylhydroxyl" means an alkyl group that has been substituted with a hydroxyl group.

[0189] As used in this article, "hydroxyl" refers to -OH.

[0190] As used herein, "carbocyclic" or "carbocyclic group" includes both aromatic groups (e.g., aryl) and non-aromatic groups (e.g., cycloalkyl). In some embodiments, the carbocyclic group contains 3-10 carbon atoms ("3- to 10-membered carbocyclic"). In some embodiments, the carbocyclic group contains 3-8 carbon atoms ("3- to 8-membered carbocyclic"). In some embodiments, the carbocyclic group contains 3-6 carbon atoms ("3- to 6-membered carbocyclic"). In some embodiments, the carbocyclic group contains 3-5 carbon atoms ("3- to 5-membered carbocyclic").

[0191] As used in this article, "haloalkyl" refers to an alkyl group that has been substituted with one or more halogen atoms.

[0192] "Halogen" refers to any halogen group, such as -F, -Cl, -Br or -I.

[0193] As used in this article, the terms "heterocycle" or "heterocyclic group" refer to a monocyclic, bicyclic, or tricyclic heterocycle containing at least one heteroatom in the ring.

[0194] A monocyclic heterocycle is a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom independently selected from O, N, and S. In some embodiments, the heterocycle is a 3- or 4-membered ring containing one heteroatom selected from O, N, and S. In some embodiments, the heterocycle is a 5-membered ring containing zero or one double bond and one, two, or three heteroatoms selected from O, N, and S. In some embodiments, the heterocycle is a 6-, 7-, or 8-membered ring 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, aziridine, aziridine-heptyl, aziridine-propyl, diaziridine-heptyl, and 1... 3-Dioxacyclohexyl, 1,3-dioxapentyl, dihydropiperanyl (including 3,4-dihydro-2H-piperan-6-yl), 1,3-dithiocyclopentyl, 1,3-dithiaalkyl, imidazolinyl, imidazodinyl, isothiazolinyl, isothiazolinyl, isoxazolinyl, isoxazolinyl, morpholinyl, oxadiazolinyl, oxadiazolinyl, oxazolinyl, oxazolinyl Piperazinyl, piperidinyl, piperanyl, pyrazolinyl, pyrazolinyl, pyrrololinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropiperanyl (including tetrahydro-2H-piperanyl-4-yl), tetrahydrothiophenyl, thiadiazolinyl, thiadiazolinyl, thiazolinyl, thiazolinyl, thiomorpholinyl, 1,1-dioxo-ionized thiomorpholinyl (thiomorpholinyl), thiopiperanyl and trithiaalkyl.

[0195] The bicyclic heterocycles of this invention may include monocyclic heterocycles fused with aryl groups, monocyclic heterocycles fused with monocyclic cycloalkyl groups, monocyclic heterocycles fused with monocyclic cycloalkenyl groups, or monocyclic heterocycles fused with monocyclic heterocycles having a total of 5 to 12 ring atoms. Examples of bicyclic heterocycles include, but are not limited to, 3,4-dihydro-2H-piperanyl, 1,3-benzo-m-dioxacyclopentenyl, 1,3-benzodithiocyclopentenyl, 2,3-dihydro-1,4-benzo-m-dioxacyclohexenyl, 2,3-dihydro-1-benzofuranyl, 2,3-dihydro-1-benzothiopheneyl, 2,3-dihydro-1H-indolyl, and 1,2,3,4-tetrahydroquinolinyl.

[0196] The terms "heterocycle" and "heterocyclic" encompass heteroaryl groups. A "heteroaryl" group refers to a cyclic moiety having one or more closed rings, with at least one heteroatom (oxygen, nitrogen, or sulfur) in at least one of these rings, wherein at least one of these rings is an aromatic ring, and wherein the ring or the rings may be independently fused and / or bridged. Examples include, but are not limited to, phenyl, thiophene, triazolyl, pyridyl, pyrimidinyl, pyridazinyl, and pyrazinyl.

[0197] As described herein, the compounds of the present invention may contain a "substituted" portion, as appropriate. Generally, the term "substituted," whether preceded by the term "as appropriate," means that one or more hydrogens of the specified portion have been replaced by suitable substituents. Unless otherwise indicated, the "substituted" group may have suitable substituents at each substituted position of the group, and the substituents at each position may be the same or different when more than one position in any given structure is substituted by more than one substituent selected from the specified group. The substituent combinations contemplated according to the present invention are preferably combinations that result in the formation of stable or chemically viable compounds.

[0198] Those skilled in the art will understand that "substitution," "substituted," or "absent" includes the following implicit limitations: the substitution or absence is based on the permissible valence of the substituted atom and the substituent, and the substitution or absence produces a stable compound, for example, a stable compound that does not spontaneously undergo transformations such as rearrangement, cyclization, or elimination. For the purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of the organic compounds described herein that satisfy the heteroatom valence.

[0199] "Stable" means that a compound is substantially unmodified chemically and / or physically when subjected to one or more of the conditions that allow it to be generated, detected, and, in some embodiments, recovered, purified, and used for the purposes disclosed herein. In some embodiments, a stable or chemically feasible compound is one that is substantially unmodified when kept at a temperature of 40°C or lower for at least one week in the absence of moisture or other chemically reactive conditions. In some embodiments, the compounds disclosed herein are stable.

[0200] The enantiomers taught in this article may include "enantiomerically pure" isomers, which substantially contain a single enantiomer at one or more specific asymmetric centers, such as a single enantiomer greater than or equal to 90%, 92%, 95%, 98%, or 99% or equal to 100%. An "asymmetric center" or "chiral center" refers to a tetrahedral carbon atom containing four different substituents.

[0201] The compounds described herein may also contain atomic isotopes in non-natural proportions at one or more sites of the atoms constituting such compounds. For example, the compounds may be radiolabeled with radioactive isotopes such as deuterium (2H), tritium (3H), carbon-13 (13C), or carbon-14 (14C). All isotopic variants of the compounds disclosed herein, whether radioactive or not, are intended to be covered within the scope of this invention. Furthermore, all tautomerisms of the compounds described herein are intended to be within the scope of this invention. Antibody-drug conjugates

[0202] The antibody-drug conjugate (ADC) compounds of this invention include ADC compounds with anticancer activity. Specifically, ADC compounds include antibody or antigen-binding fragments (including their antigen-binding fragments) bound (i.e., covalently linked by a linker) to a Hopschierne splice regulator, for example, wherein the Hopschierne splice regulator has cytotoxic or cell growth-inhibiting effects when not bound to the antibody or antigen-binding fragment. In various embodiments, the Hopschierne splice regulator can bind to and / or interact with the SF3b spliceosome complex when not bound to the antibody or antigen-binding fragment. In various embodiments, the Hopschierne splice regulator can regulate RNA splicing in vitro and / or in vivo when not bound to the antibody or antigen-binding fragment. In various embodiments, by targeting RNA splicing, the Hopschierne splice regulator and ADC disclosed herein are potent antiproliferative agents. In various embodiments, the Hopschierne splice regulator and ADC disclosed herein can target both actively dividing cells and dormant cells.

[0203] In various embodiments, the present invention is at least in part based on the discovery that specific bioactive hoprene splice regulators can provide improved properties when used in ADCs. While hoprene splice regulators can exhibit ideally improved properties when used alone (e.g., robust SF3b spliceosome complex binding, potent regulation of RNA splicing), in various embodiments, hoprene splice regulators may exhibit fewer of these ideally improved properties when bound to antibody or antigen-binding fragments. Therefore, the development and production of ADCs for human therapeutics, such as for oncology, may require more than just identifying antibodies capable of binding to one or more desired targets and being linked to drugs used alone to treat cancer. Linking an antibody to a hoprene splice regulator can have a significant impact on the activity of one or both of the antibody and the hoprene splice regulator, i.e., an effect that varies depending on the type of linker and / or hoprene splice regulator selected. Therefore, in some embodiments, the components of the ADC are selected to (i) maintain one or more therapeutic properties exhibited by the separated antibody and hoprene splice regulator moiety; (ii) maintain the specific binding properties of the antibody or antigen-binding fragment; (iii) optimize the hoprene splice regulator loading and the hoprene splice regulator to antibody ratio; (iv) allow delivery via stable linkage to the antibody or antigen-binding fragment, such as intracellular delivery of the hoprene splice regulator moiety; and (v) maintain the stability of the ADC as a complete conjugate until transported or delivered to the target site. (vi) Minimize aggregation of the ADC before or after administration; (vii) Allow therapeutic effects, such as cytotoxic effects, of the hoprene splice regulator moiety following lysis or other release mechanisms in the cellular environment; (viii) Demonstrate in vivo anticancer efficacy comparable to or superior to that of the isolated antibody and the hoprene splice regulator moiety; (ix) Minimize off-target killing by the hoprene splice regulator moiety; and / or (x) Demonstrate the desired pharmacokinetic and pharmacodynamic properties, reproducibility, and toxicological / immunological profile. These properties may be required to identify modified ADCs for therapeutic use (Ab et al. (2015) Mol Cancer Ther. 14:1605-13).

[0204] In various embodiments, the ADCs disclosed herein exhibit some or all of the unexpectedly advantageous properties of the categories listed above. For example, in some embodiments, the ADC constructs disclosed herein exhibit unexpectedly advantageous profiles of hoprene splice regulator loading, aggregation, and / or stability compared to ADCs containing alternative linkers and / or drug portions (e.g., alternative hoprene splice regulators), and / or retain antibody binding function, drug activity, and / or improved bystander killing while reducing off-target killing. In some embodiments, the ADC constructs disclosed herein exhibit superior stability, activity, potency, or other effects (in vivo or in vitro measurements) compared to ADCs using alternative linkers and / or hoprene splice regulator portions. In some embodiments, the ADC constructs disclosed herein exhibit in vivo therapeutic efficacy with a single dose. In some embodiments, the ADC constructs disclosed herein are unexpectedly stable compared to ADCs using alternative linkers and / or hoprene splice regulator portions.

[0205] The ADC compounds of this invention can selectively deliver effective doses of cytotoxic agents or cell growth inhibitors to cancer cells or tumor tissues. It has been found that the disclosed ADCs exhibit potent cytotoxic and / or cell growth inhibitory activity against cells expressing specific 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 cell growth inhibitory activity of the ADC depends on the expression of the target antigen in the cells. In some embodiments, the disclosed ADCs are particularly effective at killing cancer cells expressing the target antigen while minimizing off-target killing. In some embodiments, the disclosed ADCs do not exhibit cytotoxic and / or cell growth inhibitory effects on cancer cells that do not express the target antigen.

[0206] Exemplary HER2-positive cancers include, but are not limited to, breast cancer, gastric cancer, bladder cancer, urethral epithelial 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).

[0207] Exemplary CD138-related cancers 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., laryngeal cancer, laryngopharyngeal cancer, nasopharyngeal cancer), breast cancer, genitourinary cancers (e.g., cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, urethral cancer), hematologic malignancies (e.g., myeloma (such as multiple myeloma), B-cell malignancies, Hodgkin's lymphoma), and thyroid cancer (Szatmári et al. (2015) Dis Markers 2015:796052).

[0208] Exemplary EPHA2-related cancers 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 Opinion Ther Targets 15(1):31-51).

[0209] In some embodiments, ADC cleavage releases a hobsteine ​​splice regulator from the antibody or antigen-binding fragment and linker. In some embodiments, the linker and / or hobsteine ​​splice regulator is designed to promote bystander killing (neighbor cell killing). In some embodiments, the linker and / or hobsteine ​​splice regulator is designed to promote bystander killing via cleavage after cell internalization and diffusion of the linker-hobsteine ​​splice regulator portion and / or the hobsteine ​​splice regulator portion alone to neighboring cells. In some embodiments, the linker promotes cell internalization. In some embodiments, the linker is designed to minimize cleavage in the extracellular environment and thereby reduce toxicity to off-target tissues (e.g., non-cancerous tissues) while maintaining ADC binding to the target tissue and bystander killing of cancerous tissues that do not express antigens targeted by the antibody or antigen-binding fragment of the ADC but surround target cancerous tissues expressing those antigens. In some embodiments, the hoprene splice regulator moiety or the catabolite of the hoprene splice regulator moiety generated by ADC cleavage is designed to promote uptake by target cells or by adjacent cells (i.e., permeable cells). These hoprene splice regulator moieties and catabolites may be referred to herein as “bystander activity,” while drug moieties or catabolites with reduced cell permeability may be referred to as “bystander inactivity.”

[0210] In some embodiments, the disclosed ADCs also exhibit bystander-killing activity, but with low off-target cytotoxicity. Without being bound by theory, the bystander-killing activity of an ADC can be particularly advantageous when ADC penetration into solid tumors is limited and / or when the target antigen expression in tumor cells is heterogeneous. In some embodiments, ADCs containing cleavable linkers are particularly effective at bystander-killing and / or exhibit improved bystander-killing activity compared to equivalent treatments with ADCs containing non-cleavable linkers. In some embodiments, the ADCs disclosed herein exhibit improved solubility and target cell penetration compared to the drug portion alone. In some embodiments, the ADCs disclosed herein exhibit improved cytotoxicity compared to the cytotoxicity of the hopsediene splice regulator portion alone. In some embodiments, the ADCs disclosed herein use a drug portion that exhibits lower cytotoxicity when evaluated as the hopsediene splice regulator alone, but are unexpectedly superior to ADCs containing other hopsediene splice regulator portions that exhibit higher cytotoxicity when evaluated as the hopsediene splice regulator alone. In some embodiments, the cleavage and release of the hippodelid splice regulator improves the cytotoxicity of the ADC relative to equivalent treatment with an ADC containing a non-cleavable linker. In other embodiments, the ADC does not require the cleavage and release of the hippodelid splice regulator to have the desired biological activity. In some embodiments, an ADC containing a non-cleavable linker with an increased spacer length (e.g., ADL12) provides the same or similar cytotoxicity relative to equivalent treatment with an ADC containing a cleavable linker (e.g., ADL1, ADL5), and unexpectedly superior cytotoxicity relative to equivalent treatment with an ADC containing a shorter non-cleavable linker. In some embodiments, an ADC comprising a non-cleavable linker (e.g., ADL12) having an increased spacer length and no carbonyl group provides the same or similar cytotoxicity relative to equivalent treatments with an ADC comprising a cleavable linker (e.g., ADL1, ADL5), and unexpectedly superior cytotoxicity relative to equivalent treatments with an ADC comprising a non-cleavable linker (e.g., ADL10) having the same or similar spacer length and a carbonyl group. In some embodiments, removal of the carbonyl group from the non-cleavable MC linker (e.g., ADL12) can cause an increase in cytotoxicity greater than 50-fold, greater than 75-fold, greater than 100-fold, greater than 150-fold, or greater than 200-fold relative to equivalent treatments with an ADC comprising an unmodified non-cleavable MC linker (e.g., ADL10).In some embodiments, relative to equivalent treatment with an ADC containing an unmodified, non-cleavable MC linker (e.g., ADL10), removing the carbonyl group from the non-cleavable MC linker (e.g., ADL12) and increasing the spacer length (e.g., adding at least one spacer unit) can cause an increase in cytotoxicity greater than 50-fold, greater than 75-fold, greater than 100-fold, greater than 150-fold, or greater than 200-fold.

[0211] This document provides ADC compounds comprising an antibody or antigen-binding fragment thereof targeting tumor cells (Ab), a splice regulator drug moiety (D), and a linker moiety (L) covalently linking the Ab to the D. In some embodiments, the antibody or antigen-binding fragment can bind to tumor-associated antigens (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 some embodiments, the antibody or antigen-binding fragment is internalized into the target cell after binding, for example, into the degradation compartment of the cell. In various embodiments, ADCs that are internalized after binding to the target cell, undergo degradation, and release the hoprene splice regulator moiety to kill cancer cells can be used. The hobstein splice regulator moiety can be released from the antibody and / or linker portion of the ADC via enzymatic action, hydrolysis, oxidation, or any other mechanism.

[0212] An exemplary ADC has equation (I): Ab-(LH)p (I) Where Ab = antibody or antigen binding fragment, L = linker portion, H = hophecediene splice regulator drug portion, and p = number of splice regulator drug portions for each antibody or antigen binding fragment.

[0213] In some embodiments, the portion used for targeting the hoprene splice regulator in the described ADC and compositions is an antibody or antigen-binding fragment. Other exemplary targeted drug portions for use in the described ADC and compositions are also provided and described herein. In some embodiments, the portion targeting the hoprene splice regulator may be any of a variety of cell binders and non-antibody backbones. In some embodiments, the portion targeting the hoprene splice regulator is a cell binder. As used herein, the term "cell binder" means any agent capable of binding to animal (e.g., human) cells and delivering a hoprene splice regulator portion (e.g., a hoprene splice regulator drug portion as disclosed herein). This term encompasses the exemplary antibodies and antigen-binding fragments disclosed herein (e.g., monoclonal antibodies and fragments such as Fab and scFV). The term further encompasses exemplary cell binding agents such as DARPin, duobody, bicyclic peptide, nanoantibody, centyrin, MSH (melanocyte-stimulating hormone), receptor-Fc fusion molecules, T-cell receptor structures, steroid hormones (such as androgens and estrogens), growth factors, community-stimulating factors (such as EGF), and other non-antibody backbones. In various embodiments, non-antibody backbones can be broadly categorized into two structural classes: compounds with domain-defined sizes (approximately 6-20 kDa) and restricted peptides (approximately 2-4 kDa). The scaffolds of exemplary domain-defined sizes include, but are not limited to, affinity antibodies, affilin, anticarrier protein, atrimer, DARPin, FN3 scaffolds (such as adnectin and sintine), fynomer, Kunitz domain, pronectin, O-body, and receptor-Fc fusion proteins, while exemplary restriction peptides include high-affinity multimers, bicyclic peptides, and Cys-knots. In some embodiments, portions of the ADC and the targeted drug in the composition are selected from affinity antibodies, affilin, anticarrier protein, atrimer, DARPin, FN3 scaffolds (such as adnectin or sintine), fynomer, Kunitz domain, pronectin, O-body, high-affinity multimers, bicyclic peptides, and Cys-knots. In some embodiments, the portion of the ADC described and the targeted drug in the composition is a receptor-Fc fusion protein, such as a HER2-Fc chimeric fusion protein. Non-antibody backbones are reviewed, for example, in Vazquez-Lombardi et al. (2015) Drug Dis Today 20(10):1271-83. [Antibody] []

[0214] Formula (I) includes, within its scope, any antibody or antigen-binding fragment that specifically binds to a target antigen on cancer cells. The antibody or antigen-binding fragment may bind to the target antigen, wherein, as measured by, for example, a BIAcore® assay, the dissociation constant (KD) is ≤1 mM, ≤100 nM, or ≤10 nM, or any amount between these values. In some embodiments, the KD is from 1 pM to 500 pM. In some embodiments, the KD is between 500 pM and 1 µM, 1 µM and 100 nM, or 100 mM and 10 nM.

[0215] In some embodiments, the antibody or antigen-binding fragment is a four-chain antibody (also known as an immunoglobulin, 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 double-chain half-antibody (one light chain and one heavy chain) or an 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 provides immunoglobulin function.

[0216] In some embodiments, the antibody or antigen-binding fragment is an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is an internalizing antibody or an internalizing antigen-binding fragment thereof. In some embodiments, the internalizing antibody or its internalizing antigen-binding fragment binds to the target cancer antigen expressed on the cell surface and enters the cell after binding. In some embodiments, after the ADC enters and is present in the cell expressing the target cancer antigen (i.e., after the ADC has been internalized), the heptahydrate splice regulator drug portion of the ADC is released from the antibody or antigen-binding fragment of the ADC, for example by lysis, degradation by the antibody or antigen-binding fragment, or by any other suitable release mechanism.

[0217] The amino acid sequences of the exemplary antibodies of this invention are described in Tables 2-4. [] Table 1. Antibodies [mAb] [type] [Target] Trastuzumab (AB185) humanization HER2 / NEU B-B4 (AB205) mouse CD138 (Multiligand Proteoglycan-1) 1C1 (AB206) humanization EPHA2 Table 2. Amino acid sequences in the mAb variable region [mAb] [IgG] [chain] [SEQ ID NO] [Amino acid sequence] Trastuzumab (AB185) Heavy chain 19 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS Trastuzumab (AB185) Light chain 20 DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT B-B4 (AB205) Heavy chain twenty one QVQLQQSGSELMMPGASVKISCKATGYTFSNYWIQRPGHGLEWIGEILPGTGRTIYNEKFKGKATFTADISSNTVQMQLSSLTSEDSAVYYCARRDYYGNFYYAMDYWGQGTSVTVSS B-B4 (AB205) Light chain twenty two DIQMTQSTSSLSASLGDRVTISCSASQGINNYLNWYQQKPDGTVELLIYYTSTLQSGVPSRFSGSGSGTDYSLTISNLEPEDIGTYYCQQYSKLPRTFGGGTKLEIK 1C1 (AB206) Heavy chain twenty three EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYMMAWVRQAPGKGLEWVSRIGPSGGPTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGYDSGYDYVAVAGPAE-YFQHWGQGTLVTVSS 1C1 (AB206) Light chain twenty four DIQMTQSPSSSLSASVGDRVTITCRASQSISTWLAWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTEFSLTISGLQPDDFATYYCQQYNSYS-RTFGQGTKVEIK [] [, , ] [] [surface] [3. mAb CDR] [Of] [Amino acid sequence] [] [mAb] [IgG] [chain] [SEQ ID NO] [Amino acid sequence] Trastuzumab (AB185) HCDR1 1 GFNIKDTYIH Trastuzumab (AB185) HCDR2 2 RIYPTNGYTRYADSVKG Trastuzumab (AB185) HCDR3 3 WGGDGFYAMDV Trastuzumab (AB185) LCDR1 4 RASQDVNTAVAW Trastuzumab (AB185) LCDR2 5 SASFLES Trastuzumab (AB185) LCDR3 6 QQHYTTPPT B-B4 (AB205) HCDR1 7 NYWIE B-B4 (AB205) HCDR2 8 ILPGTGRTIYNEKFKGKA B-B4 (AB205) HCDR3 9 RDYYGNFYYAMDY B-B4 (AB205) LCDR1 10 ASQGINNYLN B-B4 (AB205) LCDR2 11 TSTLQS B-B4 (AB205) LCDR3 12 QQYSKLPRT 1C1 (AB206) HCDR1 13 HYMMA 1C1 (AB206) HCDR2 14 RIGPSGGPTHYADSVKG 1C1 (AB206) HCDR3 15 YDSGYDYVAVAGPAE-YFQH 1C1 (AB206) LCDR1 16 RASWSISTWLA 1C1 (AB206) LCDR2 17 KASNLHT 1C1 (AB206) LCDR3 18 QQYNSYS-RT [] [surface] [4.] [Full Length] [mAb Ig] [Chain amino acid sequence] [mAb] [IgG] [Chain] [Category] [SEQ ID NO] [Amino acid sequence] Trastuzumab (AB185) Heavy chain IgG1 25 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Trastuzumab 〔 (AB185) Light chain κ 26 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC B - B4 [[ID= Heavy chain IgG2a 27 QVQLQQSGSELMMPGASVKISCKATGYTFSNYWIQRPGHGLEWIGEILPGTGRTIYNEKFKGKATFTADISSNTVQMQLSSLTSEDSAVYYCARRDYYGNFYYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG B - B4 (AB205) Light chain κ 28 DIQMTQSTSSLSASLGDRVTISCSASQGINNYLNWYQQKPDGTVELLIYYTSTLQSGVPSRFSGSGSGTDYSLTISNLEPEDIGTYYCQQYSKLPRTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 1C1 (AB206) Heavy chain IgG1 29 EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYMMAWVRQAPGKGLEWVSRIGPSGGPTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGYDSGYDYVAVAGPAEYFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 1C1 (AB206) Light chain κ 30 DIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTEFSLTISGLQPDDFATYYCQQYNSYSRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [Table] [5.] [Exemplary target antigen amino acid sequence] [] [Antigen] [SEQ ID NO] [Amino acid sequence] HER2 / NEU 31 CD138 32 MRRAALWLWLCALALSLQPALPQIVATNLPPEDQDGSGDDSDNFSGSGAGALQDITLSQQTPSTWKDTQLLTAIPTSPEPTGLEATAASTSTLPAGEGPKEGEAVVLPEVEPGLTAREQEATPRPRETTQLPTTHLASTTTATTAQEPATSHPHRDMQPGHHETSTPAGPSQADLHTPHTEDGGPSATERAAEDGASSQLPAAEGSGEQDFTFETSGENTAVVAVEPDRRNQSPVDQGATGASQGLLDRKEVLGGVIAGGLVGLIFAVCLVGFMLYRMKKKDEGSYSLEEPKQANGGAYQKPTKQEEFYA EPHA2 33 MELQAARACFALLWGCALAAAAAAQGKEVVLLDFAAAGGELGWLTHPYGKGWDLMQNIMMPIYMYSVCNVMSGDQDNWLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRLFTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYKKCPELLQGLAHFPETIAGSDAPSLATVAGTCVDHAVVPPGGEEPR MHCAVDGEWLVPIGQCLCQAGYEKVEDACQACSPGFFKFEASEPCLECPEHTLPSPEGATSCECEEGFFRAPQDPASMPCTRPPSAPHYLTAVGMGAKVELRWTPPQDSGGREDIVYSVTCEQCWPESGECGPCEASVRYSEPPHGLTRTSVTVSDLEPHMNYTFTVEARNGVSGLVTRSFRTASVSINGTEPPKVRLEGRSTTSLSVSWSPIPQQSRVWKYEVTYRKKGDSNSYNVRRTE GFSVTLDDLAPDTTYLVQVQALTQEGQGAGSKVHEFQTLSPEGSGNLAVIGGVAVGVVLLLVLAGVGFFIHRRRKNQRARQSPEDVYFSKSEQLKPLKTYVDPHTYEDPNQAVLKFTTEIHPSCVTRQKVIGAGEGFGEVYKGMLKTSSGKKEVVAIKTLKAGYTEKQRVDFLGEAGIMGQFSHHNIIRLEGVISKYKPMMIITEYMENGALDKFLREKDGEFSVLQLVGMLRGIAAGMKYLAN MNYVHRDLAARNILVNSNLVCKVSDFGLSRVLEDDPEATYTTSGGKIPIRWTAPEAISYRKFTSASDVWSFGIVMWEVMTYGERPYWELSNHEVMKAINDGFRLPTMDCPSAIYQLMMQCWQQERARRPKFADIVSILDKLIRAPDSLKTLADFDPRVSIRLPSTSGSEGVPFRTVSEWLESIKMQQYTEHFMAAGYTAIEKVVQMTNDDIKRIGVRLPGHQKRIAYSLLGLKDQVNTVGIPI MSLN 94 MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGTPCLLGPGPVLTVLALLLASTLA FOLH1 95 MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILG GHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELV EKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA CDH6 96 MRTYRYFLLLFWVGQPYPTLSTPLSKRTSGFPAKKRALELSGNSKNELNRSKRSWMNQFFLLEEYTGSDYQYVGKLHSDQDRGDGSLKYILSGDGAGDLFIINENTGDIQATKRLDREEKPVYILRAQAINRRTGRPVEPESEFIIKIHDINDNEPIFTKEVYTATVPEMSDVGTFVVQVTATDADDPTYGNSAKVVYSILQGQPYFSVESETGIIKTALNMDRENREQYQVVIQAKDMGGQMGGLSGTTTVNITLTDVNDNPPRPQSTYQFKTPESSPPGTPIGRIKASDADVGENAEIEYSITDGEGLDMFDIVITDQETQEGIITVKKLLDFEKKKVYTLKVEASNPYVEPRFLYLGPFKDSATVRIVVEDVDEPPVFSKLAYILQIRED AQINTTIGSVTAQDPDAARNPVKYSVDRHTDMDRIFNIDSGNGSIFTSKLLDRETLLWHNITVIATEINNPKQSSRVPLYIKVLDVNDNAPEFAEFYETFVCEKAKADQLIQTLHAVDKDDPYSGHQFSFSLAPEAASGSNFTIQDNKDNTAGILTRKNGYNRHEMSTYLLPVVISDNDYPVQSSTGTVTVRVCACD HHGNMQSCHAEALIHPTGLSTGALVAILLCIVILLVTVVLFAALRRQRKKEPLIISKEDIRDNIVSYNDEGGGEEDTQAFDIGTLRNPEAIEDNKLRRDIVPEALPLPRRTPTARDNTDVRDFINQRLKENDTTDPTAPPYDSLATYAYEGTGSVADSLSSLESVTTDADQDYDYLSDWGPRFKKLADMYGGVDSKDS CEACAM5 97 MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNKLSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAGATVGIMIGVLVGVALI CFC1B 98 MTWRHHVRLLFTVSLALQIINLGNSYQREKHNGGREEVTKVATQKHRQSPLNWTSSHFGEVTGSAEGWGPEEPLPYSWAFGEGASARPRCCRNGGTCVLGSFCVCPAHFTGRYCEHDQRRSECGALEHGAWTLRACHLCRCIFGALHCLPLQTPDRCDPKDFLASHAHGPSAGGAPSLLLLLPCALLHRLLRPDAPAHPRSLVPSVLQRERRPCGRPGLGHRL ENPP3 99 MESTLTLATEQPVKKNTLKKYKIACIVLLALLVIMSLGLGLGLGLRKLEKQGSCRKKCFDASFRGLENCRCDVACKDRGDCCWDFEDTCVESTRIWMCNKFRCGETRLEASLCSCSDDCLQRKDCCADYKSVCQGETSWLEENCDTAQQSQCPEGFDLPPVILFSMDGFRAEYLYTWDTLMPNINKLKTCGIHSKYMRAMYPTKTFPNHYTIVTGLYPESHGIIDNNMYDVNLNKNFSLSSKEQNNPAWWHGQPMWLTAMYQGLKAATYFWPGSEVAINGSFPSIYMPYNGSVPFEERISTLLKWLDLPKAERPRFYTMYFEEPDSSGHAGGPVSARVIKALQVVDHAFGMLMEGLKQRNLHNCVNIILLADHGMDQTYCNKMEYMTDYFPRINFFYMYEGPAPRIRAHNIPHDFFSFNSEEIVRNLSCRKPDQHFKPYLTPDLPKRLHYAKNVRIDKVHLFVDQQWLAVRSKSNTNCGGGNHGYNNEFRSMEAIFLAHGPSFKEKTEVEPFENIEVYNLMCDLLRIQPAPNNGTHGSLNHLLKVPFYEPSHAEEVSKFSVCGFANPLPTESLDCFCPHLQNSTQLEQVNQMLNLTQEEITATVKVNLPFGRPRVLQKNVDHCLLYHREYVSGFGKAMRMPMWSSYTVPQLGDTSPLPPTVPDCLRADVRVPPSESQKCSFYLADKNITHGFLYPPASNRTSDSQYDALITSNLVPMYEEFRKMWDYFHSVLLIKHATERNGVNVVSGPIFDYNYDGHFDAPDEITKHLANTDVPIPTHYFVVLTSCKNKSHTPENCPGWLDVLPFIIPHRPTNVESCPEGKPEALWVEERFTAHIARVRDVELLTGLDFYQDKVQPVSEILQLKTYLPTFETTI FOLR1 100 MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWAAWPFLLSLALMLLWLLS HAVCR1 101 MHPQVVILSLILHLADSVAGSVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTCQNGIVWTNGTHVTYRKDTRYKLLGDLSRRDVSLTIENTAVSDSGVYCCRVEHRGWFNDMKITVSLEIVPPKVTTTPIVTTVPTVTTVRTSTTVPTTTTVPMTTVPTTTVPTTMSIPTTTTVLTTMTVSTTTSVPTTTSIPTTTSVPVTTTVSTFVPPMPLPRQNHEPVATSPSSPQPAETHPTTLQGAIRREPTSSPLYSYTTDGNDTVTESSDGLWNNNQTQLFLEHSLLTANTTKGIYAGVCISVLVLLALLGVIIAKKYFFKKEVQQLSVSFSSLQIKALQNAVEKEVQAEDNIYIENSLYATD KIT 102 MRGARGAWDFLCVLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAPVVSKASYLLREGEEFTVCTIKDFVSSVYS TWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFKPPEHQQWIYMNRTFDTKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASLVPVDQTLNSSGPPFGKLVVQSSIDSSAFKHNGT VECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPNRLSFGKTLGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLNLNFLRKRDSFICSKQEDHAEAALYKNLLHSKSCSDSTNEYMDMKPGVSYVV PTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFFLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHDDV WITH 103 MUC16 104 SLC39A6 105 MARKLSVILILTFALSVTNPLHELKAAAFPQTTEKISPNWESGINVDLAISTRQYHLQQLFYRYGENNSLSVEGFRKLLQNIGIDKIKRIHIHHDHDHHSDHEHHSDHERHSDHEHHSEHEHHSDHDHHSHHNHAASGKNKRKALCPDHDSDSSGKDPRNSQGKGAHRPEHASGRRNVKDSVSASEVTSTVYNTVSEGTHFLETIETPRPGKLFPKDVSSSTPPSVTSKSRVSRLAGRKTNESVSEPRKGFMYSRNTNENPQECFNASKLLTSHGMGIQVPLNATEFNYLCPAIINQIDARSCLIHTSEKKAEIPPKTYSLQIAWVGGFIAISIISFLSLLGVILVPLMNRVFFKFLLSFLVALAVGTLSGDAFLHLLPHSHASHHHSHSHEEPAMEMKRGPLFSHLSSQNIEESAYFDSTWKGLTALGGLYFMFLVEHVLTLIKQFKDKKKKNQKKPENDDDVEIKKQLSKYESQLSTNEEKVDTDDRTEGYLRADSQEPSHFDSQQPAVLEEEEVMIAHAHPQEVYNEYVPRGCKNKCHSHFHDTLGQSDDLIHHHHDYHHILHHHHHQNHHPHSHSQRYSREELKDAGVATLAWMVIMGDGLHNFSDGLAIGAAFTEGLSSGLSTSVAVFCHELPHELGDFAVLLKAGMTVKQAVLYNALSAMLAYLGMATGIFIGHYAENVSMWIFALTAGLFMYVALVDMVPEMLHNDASDHGCSRWGYFFLQNAGMLLGFGIMLLISIFEHKIVFRINF SLC44A4 106 MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGYIVVGIVAWLYGDPRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCILSSNIISVAENGLQCPTPQVCVSSCPEDPWTVGKNEFSQTVGEVFYTKNRNFCLPGVPWNMTVITSLQQELCPSFLLPSAPALGRCFPWTNVTPPALPGITNDTTIQQGISGLIDSLNARDISVKIFEDFAQSWYWILVALGVALVLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQLGFTTNLSAYQSVQETWLAALIVLAVLEAILLLMLIFLRQRIRIAIALLKEASKAVGQMMSTM FYPLVTFVLLLICIAYWAMTALYLATSGQPQYVLWASNISSPGCEKVPINTSCNPTAHLVNSSCPGLMCVFQGYSSKGLIQRSVFNLQIYGVLGLFWTLNWVLALGQCVLAGAFASFYWAFHKPQDIPTFPLISAFIRTLRYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCIMCCFKCCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNIVRVVVLDKVTDLLLFFGKLLVVGGVGVLSFFFSGRIPGLGKDFKSPHLNYYWLPIMTSILGAYVIASGFFSVFGMCVDTLFLCFLEDLERNNGSLDRPYYMSKSLLKILGKKNEAPPDNKKRKK STEAP1 107 MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSF FFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPIVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEICSQL

[0218] In various embodiments, the ADC disclosed herein may comprise any set of heavy and light chain variable domains listed in the table above, or, for example, a set of six CDR sequences derived from the heavy and light chain sets obtained by transplanting six CDRs into a selected human donor antibody framework. In various embodiments, the ADC disclosed herein may comprise amino acid sequences homologous to the sequences listed in the table above, provided that the ADC retains its ability to bind to its target cancer antigen (e.g., where the KD is less than 1 × 10⁻⁸ M) and retains one or more functional properties of the ADC disclosed herein (e.g., the ability to internalize, regulate RNA splicing, inhibit cell growth, etc.).

[0219] 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 IgG1) and a human κ or λ 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 and a human Ig κ light chain constant domain.

[0220] In various other embodiments, the target cancer antigen of the ADC is human epidermal growth factor receptor 2 (HER2).

[0221] In various embodiments, the anti-HER2 antibody or its antigen-binding fragment comprises the following three heavy chain CDRs and three light chain CDRs: as defined by the Kabat numbering system, heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:1, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:2, and heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; light chain CDR1 (LCDR1) consisting of SEQ ID NO:4, light chain CDR2 (LCDR2) consisting of SEQ ID NO:5, and light chain CDR3 (LCDR3) consisting of SEQ ID NO:6.

[0222] In various embodiments, the anti-HER2 antibody or its 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 its antigen-binding fragment 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 sequences having at least 95% identity with the disclosed sequences. In some embodiments, the anti-HER2 antibody or its antigen-binding fragment comprises a heavy chain variable region amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:19 and / or a light chain variable region amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:20.

[0223] In various embodiments, the anti-HER2 antibody or its antigen-binding fragment is an internalizing antibody or an internalizing antigen-binding fragment. In various embodiments, the anti-HER2 antibody comprises a human IgG1 heavy chain constant domain and a human Igκ light chain constant domain.

[0224] In various embodiments, the anti-HER2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:19 or a sequence having at least 95% identity with SEQ ID NO:19 and the light chain amino acid sequence of SEQ ID NO:20 or a sequence having at least 95% identity with SEQ ID NO:20. In certain embodiments, the anti-HER2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:19 and the light chain amino acid sequence of SEQ ID NO:20 or a sequence having at least 95% identity with the disclosed sequence. In some embodiments, the anti-HER2 antibody has a heavy chain amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:19 and a light chain amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:20. In various embodiments, the anti-HER2 antibody is trastuzumab or its antigen-binding fragment.

[0225] In various embodiments, the anti-HER2 antibody or its antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs of trastuzumab, or wherein the CDRs include no more than one, two, three, four, five or six amino acids added, deleted or substituted from 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).

[0226] In various other embodiments, the target cancer antigen of the ADC is human multiligand proteoglycan-1 (CD138).

[0227] In various embodiments, the anti-CD138 antibody or its antigen-binding fragment comprises the following three heavy chain CDRs and three light chain CDRs: as defined by the Kabat numbering system, heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:7, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:8, and heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:9; light chain CDR1 (LCDR1) consisting of SEQ ID NO:10, light chain CDR2 (LCDR2) consisting of SEQ ID NO:11, and light chain CDR3 (LCDR3) consisting of SEQ ID NO:12.

[0228] In various embodiments, the anti-CD138 antibody or its 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 its antigen-binding fragment 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 having at least 95% identity with the disclosed sequence. In some embodiments, the anti-CD138 antibody or its antigen-binding fragment comprises a heavy chain variable region amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:21 and / or a light chain variable region amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:22.

[0229] In various embodiments, the anti-CD138 antibody or its antigen-binding fragment is an internalizing antibody or an internalizing antigen-binding fragment. In various embodiments, the anti-CD138 antibody comprises a mouse IgG2a heavy chain constant domain and a mouse Igκ light chain constant domain. In various embodiments, the anti-CD138 antibody comprises a human IgG2a heavy chain constant domain and a human Igκ light chain constant domain.

[0230] In various embodiments, the anti-CD138 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:21 or a sequence having at least 95% identity with SEQ ID NO:21 and the light chain amino acid sequence of SEQ ID NO:22 or a sequence having at least 95% identity with SEQ ID NO:22. In certain embodiments, the anti-CD138 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:21 and the light chain amino acid sequence of SEQ ID NO:22 or a sequence having at least 95% identity with the disclosed sequence. In some embodiments, the anti-CD138 antibody has a heavy chain amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:21 and a light chain amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:22. In various embodiments, the anti-CD138 antibody is B-B4 or an antigen-binding fragment thereof.

[0231] In various embodiments, the anti-CD138 antibody or its antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs of B-B4, or wherein the CDRs include no more than one, two, three, four, five or six amino acids of 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), with addition, deletion or substitution.

[0232] In various other embodiments, the target cancer antigen of the ADC is human pterin A receptor 2 (EPHA2).

[0233] In various embodiments, the anti-EPHA2 antibody or its antigen-binding fragment comprises the following three heavy chain CDRs and three light chain CDRs: as defined by the Kabat numbering system, heavy chain CDR1 (HCDR1) consisting of SEQ ID NO: 13, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO: 14, and heavy chain CDR3 (HCDR3) consisting of SEQ ID NO: 15; light chain CDR1 (LCDR1) consisting of SEQ ID NO: 16, light chain CDR2 (LCDR2) consisting of SEQ ID NO: 17, and light chain CDR3 (LCDR3) consisting of SEQ ID NO: 18.

[0234] In various embodiments, the anti-EPHA2 antibody or its antigen-binding fragment comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:23 and a light chain variable region containing the amino acid sequence of SEQ ID NO:24. In some embodiments, the anti-EPHA2 antibody or its antigen-binding fragment 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 sequences having at least 95% identity with the disclosed sequences. In some embodiments, the anti-EPHA2 antibody or its antigen-binding fragment comprises a heavy chain variable region amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:23 and / or a light chain variable region amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:24.

[0235] In various embodiments, the anti-EPHA2 antibody or its antigen-binding fragment is an internalizing antibody or an internalizing antigen-binding fragment. In various embodiments, the anti-EPHA2 antibody comprises a human IgG1 heavy chain constant domain and a human Igκ light chain constant domain.

[0236] In various embodiments, the anti-EPHA2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:23 or a sequence having at least 95% identity with SEQ ID NO:23 and the light chain amino acid sequence of SEQ ID NO:24 or a sequence having at least 95% identity with SEQ ID NO:24. In a particular embodiment, the anti-EPHA2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:23 and the light chain amino acid sequence of SEQ ID NO:24 or a sequence having at least 95% identity with the disclosed sequence. In some embodiments, the anti-EPHA2 antibody has a heavy chain amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:23 and a light chain amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with 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 its antigen-binding fragment.

[0237] In various embodiments, the anti-EPHA2 antibody or its antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs of 1C1, or wherein the CDRs include no more than one, two, three, four, five or six amino acids added, deleted or substituted, of 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).

[0238] In various embodiments, amino acid substitutions have individual residues. Insertions are typically on the order of about 1 to about 20 amino acid residues, but significantly larger insertions are permissible as long as biological function (e.g., binding to a target antigen) is maintained. Deletions are typically in the range of about 1 to about 20 amino acid residues, but in some cases, deletions may be much larger. Substitution, deletion, insertion, or any combination thereof can be used to obtain the final derivative or variant. Generally, such changes to several amino acids are made to minimize alterations to the immunogenicity and specificity of the molecule, specifically the antigen-binding protein. However, in some cases, more variations are permissible. Conservative substitutions are generally performed according to the diagrams depicted below, as shown in Table 6. [surface] [6] Exemplary substitution of original residues Ala Ser Arg Lys Asn Gln、His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn、Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu、Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

[0239] Substantial changes in functional or immune properties can be made by selecting substitutions with lower conservation than those shown in Table 6. For example, substitutions can have a more significant impact on: altering the structure of the polypeptide backbone in the region, such as α-helical or β-sheet structures; the charge or hydrophobicity of the molecule at the target site; or the volume of the side chains. Substitutions that typically produce the most significant alterations to the properties of a polypeptide are those that exhibit the following characteristics: (a) substitution of hydrophilic residues, such as serine or threonine, for hydrophobic residues, such as leucine, isoleucine, phenylalanine, valine, or propylalanine (or substitution by hydrophobic residues); (b) substitution of any other residue, such as cysteine ​​or proline (or substitution by any other residue); (c) substitution of positively charged residues, such as lysine, spermine, or histidine, for negatively charged residues, such as glutamine or aspartic acid (or substitution by negatively charged residues); or (d) substitution of residues with bulky side chains, such as phenylalanine, for residues without side chains, such as glycine (or substitution by residues without side chains).

[0240] In various embodiments where variant antibody sequences are used in ADCs, the variants typically exhibit the same qualitative biological activity and elicit the same immune response, but variants can also be selected to modify the characteristics of the antigen-binding protein as needed. Alternatively, variants can be designed to alter the biological activity of the antigen-binding protein. For example, glycosylation sites can be modified or removed.

[0241] Various antibodies can be used in conjunction with the ADCs used herein to target cancer cells. As shown below, the linker-capacitors in the ADCs disclosed herein are unexpectedly effective in targeting antibodies against different tumor antigens. Suitable antigens expressed on tumor cells rather than healthy cells, or expressed on tumor cells at higher levels than on healthy cells, and antibodies against such antigens are known in this art. These antibodies can be used in conjunction with the linker and Hopsonide splice regulator capacitors disclosed herein. In some embodiments, the antibody or antigen-binding fragment targets HER2, and the antibody or antigen-binding fragment targeting HER2 is trastuzumab. In some embodiments, the antibody or antigen-binding fragment targets CD138, and the antibody or antigen-binding fragment targeting CD138 is B-B4. In some embodiments, the antibody or antigen-binding fragment targets EPHA2, and the antibody or antigen-binding fragment targeting EPHA2 is 1C1. In some embodiments, while the disclosed linker and hoprene splice regulator payloads have been unexpectedly effective with several different tumor-targeting antibodies, antibodies targeting HER2 (such as trastuzumab), CD138 (such as B-B4), and EPHA2 (such as 1C1) offer specifically improved therapeutic effects in terms of antibody ratio, aggregation, stability (i.e., in vitro and in vivo stability), tumor targeting (i.e., cytotoxicity, potency), and / or therapeutic efficacy. Improved therapeutic efficacy can be measured in vitro or in vivo and may include a slowed tumor growth rate and / or a reduced tumor volume.

[0242] In some embodiments, alternative antibodies targeting the same target or antibody against different antigens are used, providing 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 linker and hoprene splice regulator payload binds to an alternative antibody or antigen-binding fragment targeting HER2, CD138, or EPHA2. In some other embodiments, some or all of these advantageous functional properties are observed when the disclosed linker and hoprene splice regulator payload binds to an antibody or antigen-binding fragment targeting HER2. In some embodiments, the antibody or antigen-binding fragment targets HER2. In some embodiments, the antibody or antigen-binding fragment targeting HER2 is trastuzumab. In some other embodiments, some or all of these advantageous functional properties are observed when the disclosed linker and hoprene splice regulator payload binds to an antibody or antigen-binding fragment targeting CD138. In some embodiments, the antibody or antigen-binding fragment targets CD138. In some embodiments, the antibody or antigen-binding fragment targeting CD138 is B-B4. In some other embodiments, some or all of these advantageous functional properties are observed when the disclosed linker and Hopschier diene splicing regulator payload binds to the antibody or antigen-binding fragment targeting EPHA2. In some embodiments, the antibody or antigen-binding fragment targets EPHA2. In some embodiments, the antibody or antigen-binding fragment targeting EPHA2 is 1C1. [Connector] []

[0243] In various embodiments, the linkers in the ADC are extracellularly stable in a manner sufficient to achieve therapeutic efficacy. In some embodiments, the linkers are stable outside the cell such that the ADC remains intact when present in extracellular conditions (e.g., prior to transport or delivery into cells). The term "intact" as used in the context of ADCs means that the antibody or antigen-binding fragment remains linked to the drug moiety (e.g., a hobsteine ​​splice regulator). In the context of linkers or ADCs containing linkers, "stable" as used herein means that no more than 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 in the ADC sample are cleaved (or the overall ADC is otherwise incomplete) when the ADC is present in extracellular conditions. In some embodiments, the linkers and / or ADCs disclosed herein are unexpectedly stable compared to ADCs with alternative linkers and / or ADCs having alternative linkers and / or hobsteine ​​splice regulator payloads. In some embodiments, the ADC 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.

[0244] Extracellular stability of the linker can be determined, for example, by including the ADC in plasma for a predetermined time period (e.g., 2, 4, 6, 8, 16, 24, 48, or 72 hours) and subsequently quantifying the amount of free drug fraction present in the plasma. Stability allows the ADC to target tumor cells at the appropriate time and prevents premature release of the drug fraction, which could reduce the therapeutic index of the ADC by indiscriminately damaging both normal and tumor tissues. In some embodiments, the linker is stable outside the target cell and releases the drug fraction from the ADC once inside the cell, allowing the drug to bind to its target (e.g., to the SF3b splice complex). Thus, an effective linker will: (i) maintain the specific binding properties of the antibody or antigen-binding fragment; (ii) allow the drug fraction to be delivered (e.g., intracellularly) via stable linking to 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) allow the therapeutic effects of the drug fraction, such as cytotoxicity, following cleavage or alternative release mechanisms.

[0245] Linkers can influence the physicochemical properties of ADCs. Since many cytotoxic agents are inherently hydrophobic, linking them to antibodies with an additional hydrophobic moiety can lead to aggregation. ADC aggregates are often insoluble and typically limit achievable drug loading onto the antibody, which can adversely affect the efficacy of the ADC. Generally, protein aggregates in biologics are also associated with increased immunogenicity. As shown below, the linkers disclosed in this article enable ADCs to exhibit low aggregation levels and the desired drug loading.

[0246] Linkers can be "cleavable" or "non-cleavable" (Ducry and Stump (2010) Bioconjugate Chem. 21:5-13). Cleavable linkers are designed to release drug moieties upon exposure to certain environmental factors, such as upon internalization into target cells (e.g., Hobstein splicing regulators), while non-cleavable linkers generally rely on the degradation of the antibody or antigen-binding fragment itself.

[0247] In some embodiments, the linker is a non-cleavable linker. In some embodiments, the heptahydrate splice regulator drug portion of the ADC is released by degradation of the antibody or antigen-binding fragment. Upon internalization and degradation within the target cell, the non-cleavable linker tends to maintain covalent association with at least one amino acid of the antibody and the drug. Several exemplary non-cleavable linkers are described herein, and other non-cleavable linkers are known in the art. Exemplary non-cleavable linkers may comprise a thioether, a cyclohexyl group, N-succinimidyl-4-(N-cis-butenediamide-methyl)cyclohexane-1-carboxylate (SMCC) or N-hydroxysuccinimidyl (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.

[0248] In some embodiments, the linker is a cleavable linker. A cleavable linker means any linker containing a cleavable portion. As used herein, the term "cleavable portion" means any chemical bond that can be cleaved. Suitable cleavable chemical bonds are well known in this art and include, but are not limited to, acid-instable bonds, protease / peptidase-instable bonds, light-instable bonds, disulfide bonds, and esterase-instable bonds. Linkers containing a cleavable portion allow the release of the Hobstein splice regulator drug moiety from the ADC via cleavage at a specific site within the linker.

[0249] In some embodiments, the linker is cleavable under intracellular conditions such that the cleavage of the linker adequately releases the hoprene splice regulator drug moiety from the antibody or antigen-binding fragment in the intracellular environment to activate the drug and / or confer therapeutic efficacy. In some embodiments, the hoprene splice regulator drug moiety does not cleave from the antibody or antigen-binding fragment until the ADC enters a cell expressing an antigen specific to the antibody or antigen-binding fragment of the ADC, and after entering the cell, the hoprene splice regulator drug moiety cleaves from the antibody or antigen-binding fragment. In some embodiments, the linker includes a cleavable portion positioned such that, after cleavage, a portion of the linker or antibody or antigen-binding fragment does not remain bound to the hoprene splice regulator drug moiety. Exemplary cleavable linkers include acid-instable linkers, protease / peptidase-sensitive linkers, photostable linkers, dimethyl-containing linkers, disulfide-containing linkers, or sulfonamide-containing linkers.

[0250] In some embodiments, the linker is a pH-sensitive linker and is sensitive to hydrolysis at certain pH values. Typically, pH-sensitive linkers can be hydrolyzed under acidic conditions. This cleavage strategy generally utilizes a lower pH in the endosomal intracellular compartment (pH ~ 5-6) and the lysosomal intracellular compartment (pH ~ 4.8) compared to the cytosol intracellular compartment (pH ~ 7.4) to trigger the hydrolysis of acid-instable groups in linkers such as hydrazones (Jain et al. (2015) Pharm Res 32:3526-40). In some embodiments, the linker is an acid-instable linker and / or a hydrolyzable linker. For example, acid-instable linkers containing acid-instable groups that can be hydrolyzed in lysosomes (e.g., hydrazones, hemicarbazones, thiohemicarbazones, cis-aconitine acetylamine, orthoesters, acetals, ketals, or analogs thereof) can be used. See, for example, U.S. Patent Nos. 5,122,368, 5,824,805, and 5,622,929; Dubowchik and Walker (1999) Pharm Therapeutics 83:67-123; and Neville et al. (1989) Biol Chem. 264:14653-61. These linkers are relatively stable under neutral pH conditions, such as the pH of blood, but unstable below pH 5.5 or 5.0 (the approximate pH of lysosomes). In some embodiments, the linker can be hydrolyzed as a thioether linker (such as a thioether linked to a therapeutic agent via a hydantoin bond) (see, for example, U.S. Patent No. 5,622,929).

[0251] In some embodiments, the linker may be cleaved under reducing conditions. In some embodiments, the linker may be cleaved 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.

[0252] In some embodiments, the linker is a cleavable disulfide linker. Various disulfide linkers are known in this art, including, for example, disulfide linkers formed using SATA (N-succinimido-5-acetylthioacetate), SPDP (N-succinimido-3-(2-pyridyldithio)propionate), SPDB (N-succinimido-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidooxycarbonyl-α-methyl-α-(2-pyridyl-dithio)toluene), SPDB, and SMPT. See, for example, Thorpe et al. (1987) Cancer Res. 47:5924-31; Wawrzynczak et al., Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogel, ed., Oxford U. Press, 1987). See also U.S. Patent No. 4,880,935. Disulfide linkers are typically used to develop the abundance of intracellular thiols, which can facilitate the cleavage of their disulfide bonds. The intracellular concentrations of the most abundant intracellular thiols, and reduced glutathione, are generally in the range of 1–10 nM, approximately 1,000 times higher than the intracellular concentrations of the most abundant low-molecular-weight thiols (i.e., cysteine) in the blood at approximately 5 µM (Goldmacher et al., Cancer Drug Discovery and Development: Antibody-Drug Conjugates and Immunotoxins (GL Phillips ed., Springer, 2013)). Intracellular enzymes of the protein disulfide isomerase family can also promote the intracellular cleavage of disulfide linkers. As used herein, a cleavable disulfide linker refers to any linker containing 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.

[0253] In some embodiments, the linker is a cleavable sulfonylurea linker. As used herein, a cleavable sulfonylurea linker refers to any linker containing a cleavable sulfonylurea moiety. The term "cleavable sulfonylurea moiety" refers to a sulfonylurea group, i.e., a sulfonylurea group attached to an amine group, wherein the sulfur-nitrogen bond is cleavable.

[0254] In some embodiments, the linker may be a dendritic linker for covalently linking more than one drug moiety to an antibody or antigen-binding fragment via a branched, multifunctional linker portion. See, for example, 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 molar ratio of drug to antibody, i.e., the drug loading, which is related to the efficacy of the ADC. Thus, for example, in the case where the antibody or antigen-binding fragment carries only one reactive cysteine ​​thiol group, multiple Hobstein splice regulator drug moieties may be linked via dendritic linkers. In some embodiments, the linker portion or linker-drug portion may be linked to the antibody or antigen-binding fragment via a reduced disulfide bridging chemical or a restricted lysine utilization technique. See, for example, International Publications Nos. WO 2013 / 173391 and WO 2013 / 173393.

[0255] In some embodiments, the linker may be cleaved by a cleavage agent, such as an enzyme, present in the intracellular environment (e.g., lysosomes, endosomes, or cell membrane pits). The linker may be a peptide linker cleaved by, for example, intracellular peptidases or proteases, including but not limited to lysosomal or endosomal proteases.

[0256] In some embodiments, the linker is a cleavable peptide linker. As used herein, a cleavable peptide linker refers to any linker containing a cleavable peptide moiety. The term "cleavable peptide moiety" refers to any chemically bonded amino acid (natural or synthetic amino acid derivative) that can be cleaved by an agent present in the intracellular environment. For example, the linker may contain a valine-alanine (Val-Ala) sequence or a valine-citrulline (Val-Cit) sequence that can be cleaved by a peptidase such as cathepsin (e.g., cathepsin B). In some embodiments, the linker may contain a glutamate-valine-citrulline (Glu-Val-Cit) sequence. In some embodiments, the linker is an enzyme-cleavable linker and the cleavable peptide moiety in the linker can be cleaved by an enzyme. In some embodiments, the cleavable peptide moiety can be cleaved by a lysosomal enzyme such as cathepsin. In some embodiments, the linker is a cathepsin-cleavable linker. In some embodiments, the cleavable peptide portion of the linker may be cleaved 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 portion may be cleaved by cathepsin B. An exemplary dipeptide that may be cleaved by cathepsin B is valine-citrulline (Val-Cit) (Dubowchik et al. (2002) Bioconjugate Chem. 13:855-69).

[0257] In some embodiments, the linker or the cleavable peptide portion within the linker comprises an amino acid unit. In some embodiments, the amino acid unit allows the linker to be cleaved by a protease, thereby facilitating the release of the heptahydrate splice regulator drug portion from the ADC after 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-aspartate (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), leu-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-aspartic acid (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 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 9-tosyl-Arg, and Phe-N 9-Nitro-Arg, as described, for example, in U.S. Patent No. 6,214,345. 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 a naturally occurring amino acid residue and / or a secondary amino acid and / or a non-naturally occurring amino acid analog (such as citrulline).Amino acid units can be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, lysosomal proteases (e.g., cathepsins B, C, D, or S), or plasminogen lysins.

[0258] In some embodiments, the linker is a cleavable β-glucuronic acid linker. As used herein, a cleavable β-glucuronic acid linker refers to any linker containing a cleavable β-glucuronic acid moiety. An exemplary cleavable β-glucuronic acid linker comprises the following structure:

[0259] The term "cleavable β-glucuronic acid moiety" refers to a glycosidic bond that can be cleaved by an agent having β-glucuronidase activity. In some embodiments, the linker contains a glycosidic bond that can be cleaved by β-glucuronidase. β-glucuronidase is a UDP-glucuronyltransferase that catalyzes the hydrolysis of the glycosidic bond of a β-configured glucuronic acid.

[0260] In some embodiments, the ADC disclosed herein includes a cleavable β-glucuronic acid moiety in a linker that can be cleaved by an enzyme. In some embodiments, the cleavable β-glucuronic acid moiety in the linker can be cleaved 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-glucuronic acid moiety in the linker allows for linker cleavage by β-glucuronidase after ADC internalization, thereby promoting the release of the drug moiety from the ADC in the cellular environment.

[0261] In some embodiments, the linker of any of the ADCs disclosed herein may include at least one spacer unit for attaching an antibody or antigen-binding fragment to a drug moiety (e.g., a hoprene splice regulator drug moiety). In some embodiments, the spacer unit between the antibody or antigen-binding fragment and the cleavable moiety, when present, attaches a 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, when present, attaches a cleavage site (e.g., a cleavable peptide moiety) in the linker to the drug moiety. In some embodiments, no cleavage site is present, and the spacer unit is used to link the antibody or antigen-binding fragment to the drug moiety.

[0262] In some embodiments, the linker and / or the spacer units within the linker are substantially hydrophilic. Hydrophilic linkers can be used to reduce the extent to which drugs can be pumped from drug-resistant cancer cells via multidrug resistance (MDR) or functionally similar transporters. In some embodiments, the hydrophilic linker may include one or more polyethylene glycol (PEG) portions, such as 1, 2, 3, 4, 5, or 6 PEG portions. In some embodiments, the linker comprises two PEG portions.

[0263] In some embodiments, the spacer subunits in the connector include one or more PEG portions. In some embodiments, the spacer subunit includes one or more -(PEG)m-, where 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 is in the range of 1 to 10; in the range of 2 to 8; in the range of 2 to 6; in the range of 2 to 5; in the range of 2 to 4; or in the range of 2 to 3. In some embodiments, m is 2. In some embodiments, the spacer subunit includes (PEG)2, (PEG)3, (PEG)4, (PEG)5, (PEG)6, (PEG)7, (PEG)8, (PEG)9, or (PEG)10. In some embodiments, the spacer subunit includes (PEG)2.

[0264] In some embodiments, the spacer subunits in the linker comprise an alkyl portion. In some embodiments, the spacer subunit comprises one or more -(CH2)n-, and n is an integer from 1 to 10 (i.e., n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, n is in the range of 1 to 10; in the range of 2 to 8; in the range of 2 to 6; in the range of 2 to 5; in the range of 2 to 4; or in the range of 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 subunit comprises (CH2)2, (CH2)3, (CH2)4, (CH2)5, (CH2)6, (CH2)7, (CH2)8, (CH2)9, or (CH2)10. In some embodiments, the spacer subunit comprises (CH2)2 ("Et"). In some embodiments, the spacer subunit comprises (CH2)6 ("Hex"). In some embodiments, the spacer subunit comprises (CH 2) 2-O-(CH 2) 2 ("Et-O-Et").

[0265] Spacer units can be used to directly or indirectly link antibody or antigen-binding fragments to a drug moiety. In some embodiments, the spacer unit directly links the antibody or antigen-binding fragment to the hoprene splice regulator drug moiety. In some embodiments, the antibody or antigen-binding fragment and the hoprene splice regulator drug moiety are linked via a spacer unit comprising one or more PEG portions (e.g., (PEG)2) or one or more alkyl portions (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 hoprene splice regulator drug moiety. In some embodiments, the spacer unit indirectly links the antibody or antigen-binding fragment to the hoprene splice regulator drug moiety via a cleavable portion (e.g., a cleavable peptide or a cleavable β-glucuronic acid) and / or a linking portion (e.g., a maleic anhydride portion) for attaching the spacer unit to the antibody or antigen-binding fragment.

[0266] In various embodiments, the spacer subunit is partially linked to the antibody or antigen-binding fragment (i.e., the antibody or antigen-binding fragment) via maleic diacetylimine (Mal).

[0267] The spacer unit linked to an antibody or antigen-binding fragment via a Mal is referred to herein as a "Mal-spacer unit". As used herein, the term "Mal" or "cis-butenedialiimine moiety" means a compound containing a cis-butenedialiimine group and capable of reacting with a thiosulfate group, such as the thiosulfate group of a cysteine ​​residue on an antibody or antigen-binding fragment. Other functional groups capable of reacting with a thiosulfate group (thiol) include, but are not limited to, iodoacetamide, bromoacetamide, vinylpyridine, disulfides, pyridyl disulfides, isocyanates, and isothiocyanates. In some embodiments, the Mal-spacer unit may react with a cysteine ​​residue on an antibody or antigen-binding fragment. In some embodiments, the Mal-spacer unit is attached to an antibody or antigen-binding fragment via a cysteine ​​residue. In some embodiments, the Mal-spacer unit comprises a PEG moiety. In some embodiments, the Mal-spacer unit comprises an alkyl moiety.

[0268] In some embodiments, the linker comprises a Mal-spacer unit and a cleavable peptide portion. In some embodiments, the cleavable peptide portion 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 maleic anhydride-hexyl (MC). In some embodiments, the linker comprises a Mal-spacer unit and Val-Ala, wherein the Mal-spacer unit comprises maleic anhydride-hexyl (MC). In some embodiments, the linker comprises a Mal-spacer unit and a cleavable β-glucuronic acid portion.

[0269] In some embodiments, the linker comprises the following structure: Mal-spacer unit. In some embodiments, the Mal-spacer unit comprises maleic anisodiaminohexyl (MC). In some embodiments, the linker comprises the following structure: MC. In some embodiments, the linker comprises the following structure: Mal-(CH2)2 ("Mal-Et"). In some embodiments, the linker comprises the following structure: Mal-(CH2)6 ("Mal-Hex"). In some embodiments, the linker comprises the following structure: Mal-(CH2)2-O-(CH2)2 ("Mal-Et-O-Et"). In some embodiments, the linker comprises the following structure: Mal-(PEG)2. In some embodiments, the linker comprises the following structure: Mal-(PEG)2-CO.

[0270] In various embodiments, the Mal-spacer unit links an antibody or antigen-binding fragment to a cleavable peptide moiety. In some embodiments, the linker comprises a Mal-spacer unit-peptide. In some embodiments, the linker comprises a Mal-spacer unit-Val-Cit. In some embodiments, the Mal-spacer unit comprises maleic anisodiaminohexyl (MC). In some embodiments, the linker comprises a MC-Val-Cit.

[0271] In some embodiments, the linker comprises the following structure: Mal-spacer unit-Val-Ala. In some embodiments, the Mal-spacer unit comprises maleic anisodiaminohexyl (MC). In some embodiments, the linker comprises the following structure: MC-Val-Ala.

[0272] In various embodiments, the Mal-spacer unit links an antibody or antigen-binding fragment to a cleavable β-glucuronic acid moiety. In some embodiments, the linker comprises the Mal-spacer unit-β-glucuronic acid. In some embodiments, the linker comprises MC-β-glucuronic acid.

[0273] In various embodiments, the cleavable portion of the linker is directly attached to the Hobstein splice regulator drug portion. In other embodiments, a spacer unit is used to connect the cleavable portion of the linker to the Hobstein splice regulator drug portion. In various embodiments, the Hobstein splice regulator is connected to the cleavable portion of the linker via a spacer unit.

[0274] Spacer units can be either "self-degrading" or "non-self-degrading." "Non-self-degrading" spacer units are those in which part or all of the spacer unit remains bound to the Hobschdiene splice regulator drug moiety after linker cleavage. Examples of non-self-degrading spacer units include, but are not limited to, glycine spacer units and glycine-glycine spacer units. Non-self-degrading spacer units may eventually degrade over time, but do not readily and completely release the attached natural drug moiety under cellular conditions. "Self-degrading" spacer units allow for the release of the natural drug moiety under intracellular conditions. "Natural drug" or "natural drug moiety" refers to the portion of the spacer unit that does not retain the spacer unit or any other chemical modifications after spacer unit cleavage / degradation.

[0275] Self-degrading chemicals are known in this art and can be readily selected for the disclosed ADC. In various embodiments, the spacer unit connecting the cleavable portion in the linker to the drug portion of the Hopschadiene splice regulator is a self-degrading spacer unit, and self-degrades simultaneously with, before, or shortly after the cleavage of the cleavable portion under intracellular conditions. In some embodiments, the Hopschadiene splice regulator is connected to the cleavable portion in the linker via a self-degrading spacer unit. In some embodiments, the Hopschadiene splice regulator is connected to the cleavable portion in the linker via a self-degrading spacer unit, the cleavable portion comprising Val-Cit, and a maleic anhydride hexyl group (MC) binds the cleavable portion to an antibody or antigen-binding fragment. In some embodiments, the Hopschadiene splicing regulator system is connected to a cleavable portion in a linker via a self-degrading spacer unit, the cleavable portion comprising Val-Ala, and a cis-butenedylidene-hexylyl group (MC) binds the cleavable portion to an antibody or antigen-binding fragment. In some embodiments, the Hopschadiene splicing regulator system is connected to a cleavable portion in a linker via a self-degrading spacer unit, the cleavable portion comprising Glu-Val-Cit, and a cis-butenedylidene-hexylyl group (MC) binds the cleavable portion to an antibody or antigen-binding fragment. In some embodiments, the Hopschadiene splicing regulator system binds to an antibody or antigen-binding fragment via a Mal-spacer unit (e.g., MC) bound to the Val-Cit cleavable portion in a linker and a pABC or pAB self-degrading spacer unit. In some embodiments, the Hopschadiene splicing regulator system attaches to the antibody or antigen-binding fragment via a Mal-spacer unit (e.g., MC) and a pABC or pAB self-degrading spacer unit in a linker that is bound to the Val-Ala cleavable portion. In other embodiments, the Hopschadiene splicing regulator system attaches to the antibody or antigen-binding fragment via a Mal-spacer unit (e.g., MC) and a pABC or pAB self-degrading spacer unit in a linker that is bound to the Glu-Val-Cit cleavable portion.

[0276] In some embodiments, the self-degrading spacer unit in the linker comprises a p-aminobenzyl unit. In some embodiments, p-aminobenzyl alcohol (pABOH) is linked via a amide bond to an amino acid unit or other cleavable portion in the linker, and a carbamate, methyl carbamate, or carbonate is formed between pABOH and the pharmaceutical portion (Hamann et al. (2005) Expert Opinion Ther Patents 15:1087-103). In some embodiments, the self-degrading spacer unit is or comprises a p-aminobenzyloxycarbonyl group (pABC). Without being bound by theory, the self-degradation of pABC is considered to involve a spontaneous 1,6-elimination reaction (Jain et al. (2015) Pharm Res. 32:3526-40).

[0277] In various embodiments, the structure of the p-aminobenzooxycarbonyl (pABC) used in the disclosed ADC is shown below:

[0278] In various embodiments, a self-decomposing spacer unit connects the cleavable portion of the linker to the Hopschadiene splice regulator. In some embodiments, the self-decomposing spacer unit is pABC. In some embodiments, pABC connects the cleavable portion of the linker to the Hopschadiene splice regulator. In some embodiments, pABC self-decomposes after the cleavable portion cleaves, and the Hopschadiene splice regulator is released from the ADC in its native active form.

[0279] In some embodiments, the anti-HER2 antibody or antigen-binding fragment is attached to the Hobstein splice regulator via a linker comprising MC-Val-Cit-pABC. In other embodiments, the anti-HER2 antibody or antigen-binding fragment is attached to the Hobstein splice regulator via a linker comprising MC-Val-Ala-pABC.

[0280] In some embodiments, the anti-CD138 antibody or antigen-binding fragment is attached to the Hopschier splice regulator via a linker comprising MC-Val-Cit-pABC. In other embodiments, the anti-CD138 antibody or antigen-binding fragment is attached to the Hopschier splice regulator via a linker comprising MC-Val-Ala-pABC.

[0281] In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment is attached to the Hopschier splice regulator via a linker comprising MC-Val-Cit-pABC. In other embodiments, the anti-EPHA2 antibody or antigen-binding fragment is attached to the Hopschier splice regulator via a linker comprising MC-Val-Ala-pABC.

[0282] In some embodiments, pABC self-decomposes after the cleavable peptide portion in the linker is cleaved. In some embodiments, the cleavable peptide portion comprises an amino acid unit. In some embodiments, the linker comprises an 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.

[0283] In some embodiments, pABC undergoes self-decomposition after the cleavable β-glucuronic acid in the linker is partially cleaved. In some embodiments, the linker comprises β-glucuronic acid-pABC.

[0284] In some embodiments, the self-decomposing spacer subunit in the linker comprises a p-aminobenzyl unit. In some embodiments, the self-decomposing spacer subunit in the linker comprises p-aminobenzyl (pAB). In some embodiments, the self-decomposition of pAB involves a spontaneous 1,6-elimination reaction.

[0285] In various embodiments, the structure of p-aminobenzyl (pAB) used in the disclosed ADC is shown below:

[0286] In various embodiments, a self-decomposing spacer unit connects the cleavable portion of the linker to the Hopschadiene splice regulator. In some embodiments, the self-decomposing spacer unit is pAB. In some embodiments, pAB connects the cleavable portion of the linker to the Hopschadiene splice regulator. In some embodiments, pAB self-decomposes after the cleavable portion cleaves, and the Hopschadiene splice regulator is released from the ADC in its native active form.

[0287] In some embodiments, the anti-HER2 antibody or antigen-binding fragment is attached to the Hobstein splice regulator via a linker comprising MC-Val-Cit-pAB. In other embodiments, the anti-HER2 antibody or antigen-binding fragment is attached to the Hobstein splice regulator via a linker comprising MC-Val-Ala-pAB.

[0288] In some embodiments, the anti-CD138 antibody or antigen-binding fragment is attached to the Hopschier splice regulator via a linker comprising MC-Val-Cit-pAB. In other embodiments, the anti-CD138 antibody or antigen-binding fragment is attached to the Hopschier splice regulator via a linker comprising MC-Val-Ala-pAB.

[0289] In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment is attached to the Hopschier splice regulator via a linker comprising MC-Val-Cit-pAB. In other embodiments, the anti-EPHA2 antibody or antigen-binding fragment is attached to the Hopschier splice regulator via a linker comprising MC-Val-Ala-pAB.

[0290] In some embodiments, pAB self-decomposes after the cleavable peptide portion in the linker is cleaved. In some embodiments, the cleavable peptide portion comprises an amino acid unit. In some embodiments, the linker comprises an 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.

[0291] In some embodiments, pAB undergoes self-decomposition after the cleavable β-glucuronic acid in the linker is partially cleaved. In some embodiments, the linker comprises β-glucuronic acid-pAB.

[0292] In some other embodiments, the Hopschadiene splice regulator is connected to a cleavable portion in the linker via a non-self-degrading spacer unit. In some embodiments, the Hopschadiene splice regulator is connected to a cleavable portion in the linker via a non-self-degrading spacer unit, the cleavable portion comprising Val-Cit, and a cis-butenedylidene iminohexanoyl (MC) group binds the cleavable portion to an antibody or antigen-binding fragment. In some embodiments, the Hopschadiene splice regulator is connected to a cleavable portion in the linker via a non-self-degrading spacer unit, the cleavable portion comprising Val-Ala, and a cis-butenedylidene iminohexanoyl (MC) group binds the cleavable portion to an antibody or antigen-binding fragment.

[0293] In various states, the antibody or antigen-binding fragment of the ADC is bound to the drug moiety of the Hobstein splice regulator 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 portion. In some embodiments, the Mal-spacer unit comprises maleic anhydride-iminohexyl (MC). In some embodiments, the linker comprises a Mal-spacer unit-amino acid unit-pABC. In some embodiments, the linker comprises an MC-amino acid unit-pABC. In some embodiments, the linker comprises an MC-Val-Cit-pABC. In some embodiments, the linker comprises an MC-Val-Ala-pABC. In some embodiments, the linker comprises an MC-Glu-Val-Cit-pABC. In some embodiments, the linker comprises an MC-Ala-Ala-Asn-pABC.

[0294] In various other states, the antibody or antigen-binding fragment of the ADC is bound to the drug moiety of the Hobstein diene splicing regulator 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 portion. In some embodiments, the Mal-spacer unit comprises maleic anhydride-iminohexyl (MC). In some embodiments, the linker comprises a Mal-spacer unit-amino acid unit-pAB. In some embodiments, the linker comprises an MC-amino acid unit-pAB. In some embodiments, the linker comprises an MC-Val-Cit-pAB. In some embodiments, the linker comprises an MC-Val-Ala-pAB. In some embodiments, the linker comprises an MC-Glu-Val-Cit-pAB. In some embodiments, the linker comprises an MC-Ala-Ala-Asn-pAB.

[0295] In various other states, the antibody or antigen-binding fragment of the ADC is bound to the drug moiety of the Hobstein splice regulator via a linker, wherein the linker comprises a Mal-spacer unit (e.g., MC), a cleavable β-glucuronic acid, and pABC. In some embodiments, the linker comprises a Mal-spacer unit-β-glucuronic acid-pABC. In some embodiments, the linker comprises MC-β-glucuronic acid-pABC.

[0296] In other embodiments, the antibody or antigen-binding fragment of the ADC is bound to the drug moiety of the Hobstein splice regulator via a linker, wherein the linker comprises a Mal-spacer unit (e.g., MC), cleavable β-glucuronic acid, and pAB. In some embodiments, the linker comprises a Mal-spacer unit-β-glucuronic acid-pAB. In some embodiments, the linker comprises MC-β-glucuronic acid-pAB.

[0297] In various embodiments, the ADC compound has formula (I): Ab-(LH) p (I) Where Ab is an antibody or antigen-binding fragment targeting the proliferative cells; H is the Hobsch diene splicing regulator; L is the connector that covalently links Ab to D; and p is an integer from 1 to 15.

[0298] In some embodiments, the antibody or antigen-binding fragment (Ab) of the ADC is bound to the Hobstein splice regulator drug portion via a linker, wherein the linker is any of the linkers disclosed or incorporated herein by reference, or comprises one or more components of any of the linkers disclosed or incorporated herein by reference.

[0299] In some embodiments, the linker includes a cleavable portion positioned such that a portion of the linker, antibody, or antigen-binding fragment does not remain bound to the Hobschdiene splice regulator after cleavage. In some embodiments, the cleavable portion is a cleavable peptide portion, such as an amino acid unit, like Val-Cit or Val-Ala. In some embodiments, the amino acid unit or linker includes Val-Cit. In some embodiments, the amino acid unit or linker includes Val-Ala. In some embodiments, the amino acid unit or linker includes Glu-Val-Cit.

[0300] In some embodiments, the linker includes at least one spacer unit that attaches an antibody or antigen-binding fragment to a cleavable portion. In some embodiments, the linker includes at least one spacer unit that attaches an antibody or antigen-binding fragment to a drug portion. In some embodiments, the spacer unit or linker includes at least one alkyl portion.

[0301] In some embodiments, a spacer unit in the linker is linked to an antibody or antigen-binding fragment via a Mal portion ("Mal-spacer unit"). In some embodiments, the Mal-spacer unit comprises at least one alkyl portion. In some embodiments, the linker comprises maleic anhydride-iminohexyl (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 links an antibody or antigen-binding fragment to a drug moiety.

[0302] 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 an antibody or antigen-binding fragment to a drug moiety. In some embodiments, the linker comprises or is composed of Mal-(PEG)2-CO. Examples of the "Mal-(PEG)2-CO" linker are also referred to herein as "ADL2" or "ADL2" linker.

[0303] In some embodiments, the Mal-spacer unit or linker comprises an MC. In some embodiments, the Mal-spacer unit or linker comprises an MC and at least one additional spacer unit. In some embodiments, the MC links an antibody or antigen-binding fragment to a drug moiety. In some embodiments, the linker comprises or is composed of an MC. Examples of "MC" linkers are also referred to herein as "ADL10" or "ADL10 linker".

[0304] 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, Mal-Hex links an antibody or antigen-binding fragment to a drug moiety. In some embodiments, the linker comprises Mal-Hex. Examples of the “Mal-Hex” linker are also referred to herein as “ADL12” or the “ADL12 linker”.

[0305] 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 an antibody or antigen-binding fragment to a drug moiety. In some embodiments, the linker comprises Mal-Et. Examples of the “Mal-Et” linker are also referred to herein as “ADL14” or the “ADL14 linker”.

[0306] 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 links an antibody or antigen-binding fragment to a drug moiety. In some embodiments, the linker comprises Mal-Et-O-Et. Examples of the “Mal-Et-O-Et” linker are also referred to herein as “ADL15” or the “ADL15” linker.

[0307] In some other embodiments, the Mal-spacer unit links an antibody or antigen-binding fragment to a cleavable portion in the linker. In some embodiments, the cleavable portion in the linker is a cleavable peptide portion, such as an amino acid unit. In some embodiments, the cleavable peptide portion 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.

[0308] In some embodiments, the spacer unit connects the cleavable portion of the connector to the Hobstein splice regulator. In some embodiments, the spacer unit that connects the cleavable portion to the Hobstein splice regulator is a self-decomposing spacer unit.

[0309] In some embodiments, the spacer subunit comprises pABC. In some embodiments, pABC links a cleavable portion to a Hobschdiene splice regulator. In some embodiments, the cleavable portion is a cleavable peptide portion, such as an amino acid unit. In some embodiments, the linker comprises an amino acid unit - pABC.

[0310] 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 binds the linker to an 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. Examples of MC-Val-Cit-pABC linkers are also referred to herein as "ADL1" or "ADL1 linker".

[0311] 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 binds the linker to an 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. Examples of the MC-Val-Ala-pABC linker are also referred to herein as "ADL6" or the "ADL6" linker.

[0312] In some embodiments, the linker comprises Glu-Val-Cit-pABC. In some embodiments, the linker comprises Glu-Val-Cit-pABC and MC Mal-spacer unit for attaching the linker to an 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. Examples of MC-Glu-Val-Cit-pABC linkers are also referred to herein as "ADL23" or "ADL23" linkers.

[0313] In some embodiments, the linker comprises Ala-Ala-Asn-pABC. In some embodiments, the linker comprises an Ala-Ala-Asn-pABC and an MC Mal-spacer unit that binds the linker to an 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. Examples of the MC-Ala-Ala-Asn-pABC linker are also referred to herein as "ADL21" or the "ADL21" linker.

[0314] In some other embodiments, the spacer subunit comprises pAB. In some embodiments, pAB connects a cleavable portion to a Hobschdiene splice regulator. In some embodiments, the cleavable portion is a cleavable peptide portion, such as an amino acid unit. In some embodiments, the linker comprises an amino acid unit - pAB.

[0315] 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 binds the linker to an 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. Examples of the MC-Val-Ala-pAB linker are also referred to herein as "ADL5" or the "ADL5" linker.

[0316] 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 binds the linker to an 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. Examples of the MC-Val-Cit-pAB linker are also referred to herein as "ADL7" or the "ADL7" linker.

[0317] 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 binds the linker to an 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. Examples of MC-β-glucuronide-pABC are also referred to herein as "ADL13" or the "ADL13" linker.

[0318] In some embodiments, the linker comprises β-glucuronide-pAB. In some embodiments, the linker comprises β-glucuronide-pAB and MC Mal-spacer units that bind the linker to an antibody or antigen-binding fragment. In some embodiments, the linker comprises MC-β-glucuronide-pAB.

[0319] In some embodiments, an antibody or antigen-binding fragment is bound to the hoprene splice regulator drug moiety via a linker of ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23. It has been found that, in various embodiments, ADCs comprising linkers of ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 and the hoprene splice regulator drug moiety disclosed herein exhibit the characteristics desired for a therapeutic ADC. In various embodiments, these characteristics include, but are not limited to, effective drug loading, low aggregation, stability under storage conditions or when circulating in vivo (e.g., serum stability), maintaining affinity for target cells comparable to unbound antibodies, potent cytotoxicity against target cells, lower off-target cell killing levels, higher bystander killing levels, and / or effective in vivo anticancer activity, all of which are relative to ADCs using other linker-loads. For example, in various embodiments, compared to ADCs using other linker-loads (e.g., the ADL10 linker and the Hopsediene splice regulator drug portion), ADCs containing ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linkers and the Hopsediene splice regulator drug portion disclosed herein exhibit an increased ability to inhibit the growth and / or proliferation of target cells. In various embodiments, compared with ADCs based on other splice regulators (such as the thailanstatin A-based ADC reported in Puthenveetil et al. Bioconjugate Chem. (2016) 27:1880-8), ADCs containing ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21 or ADL23 linkers and the heptahydrate splice regulator drug portion disclosed herein exhibit unexpectedly increased in vivo stability (e.g., plasma stability).

[0320] In some embodiments, favorable or superior functional properties can be observed in the case of linker-loads such as anti-HER2 antibodies (e.g., trastuzumab), anti-CD138 antibodies (e.g., B-B4), or anti-EPHA2 antibodies (e.g., 1C1), provided by specific combinations of ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linkers with the heptahydrate splice regulator drug moieties disclosed herein.

[0321] In some embodiments, the ADC comprises an ADL1-Hobstein splice regulator and an antibody or antigen-binding fragment thereof that contains an antibody capable of maintaining targeting and internalization within the vesicular cells. In some embodiments, the ADC comprises an ADL2-Hobstein splice regulator and an antibody or antigen-binding fragment thereof that contains an antibody capable of maintaining targeting and internalization within the vesicular cells. In some embodiments, the ADC comprises an ADL5-Hobstein splice regulator and an antibody or antigen-binding fragment thereof that contains an antibody capable of maintaining targeting and internalization within the vesicular cells. In some embodiments, the ADC comprises an ADL6-Hobstein splice regulator and an antibody or antigen-binding fragment thereof that contains an antibody capable of maintaining targeting and internalization within the vesicular cells. In some embodiments, the ADC comprises an ADL7-Hobstein splice regulator and an antibody or antigen-binding fragment thereof that contains an antibody capable of maintaining targeting and internalization within the vesicular cells. In some embodiments, the ADC comprises an ADL12-Hobstein splice regulator and an antibody or antigen-binding fragment thereof that contains an antibody capable of maintaining targeting and internalization within the vesicular cells. In some embodiments, the ADC comprises an ADL13-Hobstein splice regulator and an antibody or antigen-binding fragment thereof that contains an antibody capable of maintaining targeting and internalization within the vesicular cells. In some embodiments, the ADC comprises an ADL14-Hobstein splice regulator and an antibody or antigen-binding fragment thereof that contains an antibody capable of maintaining targeting and internalization within the vesicular cells. In some embodiments, the ADC comprises an ADL15-Hobstein splice regulator and an antibody or antigen-binding fragment thereof that contains an antibody capable of maintaining targeting and internalization within the vesicular cells.

[0322] In some embodiments, the ADC comprises an ADL1-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing blast cells. In some embodiments, the ADC comprises an ADL2-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing blast cells. In some embodiments, the ADC comprises an ADL5-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing blast cells. In some embodiments, the ADC comprises an ADL6-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing blast cells. In some embodiments, the ADC comprises an ADL7-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing blast cells. In some embodiments, the ADC comprises an ADL12-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing blast cells. In some embodiments, the ADC comprises an ADL13-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing blast cells. In some embodiments, the ADC comprises an ADL14-horbhidiene splicing regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing neoplasms. In some embodiments, the ADC comprises an ADL15-horbhidiene splicing regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing neoplasms.

[0323] In some embodiments, the antibody or its antigen-binding fragment targeting HER2-expressing neoplasms is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment targeting HER2-expressing neoplasms includes 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).

[0324] In some embodiments, the ADC has equation (I): Ab-(LH) p (I) in: (i) Ab is an anti-HER2 antibody or its antigen-binding fragment comprising three heavy chain complementarity-determining regions (HCDRs) of the amino acid sequences comprising SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3) and three light chain complementarity-determining regions (LCDRs) of the amino acid sequences comprising SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO:6 (LCDR3); (ii) H is a Hobsch diene splicing regulator; (iii) L is a connector containing ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23; and (iv) p is an integer from 1 to 15.

[0325] In some embodiments, the antibody or antigen-binding fragment thereof targeting HER2-expressing neoplasms 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 thereof targeting HER2-expressing neoplasms comprises a human IgG1 heavy chain constant domain and a human Igκ 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.

[0326] In some embodiments, the ADC comprises an ADL1-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing vesicular cells. In some embodiments, the ADC comprises an ADL2-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing vesicular cells. In some embodiments, the ADC comprises an ADL5-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing vesicular cells. In some embodiments, the ADC comprises an ADL6-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing vesicular cells. In some embodiments, the ADC comprises an ADL7-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing vesicular cells. In some embodiments, the ADC comprises an ADL12-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing vesicular cells. In some embodiments, the ADC comprises an ADL13-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing vegetative cells. In some embodiments, the ADC comprises an ADL14-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing vegetative cells. In some embodiments, the ADC comprises an ADL15-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing vegetative cells.

[0327] In some embodiments, the antibody or its antigen-binding fragment targeting CD138-expressing neoplasms is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment targeting CD138-expressing neoplasms includes 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).

[0328] In some embodiments, the ADC has equation (I): Ab-(LH) p (I) in: (i) Ab is an anti-CD138 antibody or its antigen-binding fragment comprising three heavy chain complementarity-determining regions (HCDRs) of the amino acid sequences comprising SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3) and three light chain complementarity-determining regions (LCDRs) of the amino acid sequences comprising SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2), and SEQ ID NO:12 (LCDR3); (ii) H is a Hobsch diene splicing regulator; (iii) L is a connector containing ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23; and (iv) p is an integer from 1 to 15.

[0329] In some embodiments, an antibody or antigen-binding fragment thereof targeting CD138-expressing neoplasms 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 targeting CD138-expressing neoplasms comprises a mouse IgG2a heavy chain constant domain and a mouse Igκ light chain constant domain. In some embodiments, an antibody or antigen-binding fragment thereof targeting CD138-expressing neoplasms comprises a human IgG2a heavy chain constant domain and a human Igκ light chain constant domain. In some embodiments, the antibody is B-B4. 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.

[0330] In some embodiments, the ADC comprises an ADL1-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing vesicular cells. In some embodiments, the ADC comprises an ADL2-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing vesicular cells. In some embodiments, the ADC comprises an ADL5-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing vesicular cells. In some embodiments, the ADC comprises an ADL6-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing vesicular cells. In some embodiments, the ADC comprises an ADL7-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing vesicular cells. In some embodiments, the ADC comprises an ADL12-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing vesicular cells. In some embodiments, the ADC comprises an ADL13-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing vesicular cells. In some embodiments, the ADC comprises an ADL14-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing vegetative cells. In some embodiments, the ADC comprises an ADL15-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing vegetative cells.

[0331] In some embodiments, the antibody or its antigen-binding fragment targeting EPHA2-expressing neoplasms is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment targeting EPHA2-expressing neoplasms includes three heavy chain complementarity-determining regions (HCDRs) of the amino acid sequences comprising SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three light chain complementarity-determining regions (LCDRs) of the amino acid sequences comprising SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2), and SEQ ID NO:18 (LCDR3).

[0332] In some embodiments, the ADC has equation (I): Ab-(LH) p (I) in: (i) Ab is an anti-EPHA2 antibody or its antigen-binding fragment comprising three heavy chain complementarity-determining regions (HCDRs) of the amino acid sequences comprising SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3) and three light chain complementarity-determining regions (LCDRs) of the amino acid sequences comprising SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2), and SEQ ID NO:18 (LCDR3); (ii) H is a Hobsch diene splicing regulator; (iii) L is a connector containing ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23; and (iv) p is an integer from 1 to 15.

[0333] In some embodiments, the antibody or antigen-binding fragment thereof targeting EPHA2-expressing neoplasms 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 thereof targeting EPHA2-expressing neoplasms comprises a human IgG1 heavy chain constant domain and a human Igκ 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. [Hobsch diene splicing regulator] []

[0334] In some embodiments, the antibody-drug conjugate is of formula (I): Ab-(LH)p, where Ab is an antibody or antigen-binding fragment targeting proliferative cells; H is a Hobstein splicing regulator; L is a linker that covalently links Ab to H; and p is an integer from 1 to 15.

[0335] In some embodiments, H-containing compounds of formula (I) are used as Hobschdiene splice regulators: , or a medically acceptable salt thereof, which is covalently linked to L via any atom, wherein: Y is selected from O, S, NR 6, and CR 6R 7; R1, R2, and R3 are each independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl), -OC(=O)-(C1-C6 alkyl), -C(=O)-O-(C1-C6 alkyl), and C1-C6 alkyl; R4 is selected from hydrogen, C1-C6 alkyl, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbon cycloyl), -C(=O)-(C3-C8 heterocyclic), and -C(=O)-NR6R7; R5 is selected from hydrogen, hydroxyl group, -CH2-OH, -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-NR6R7, -NR6-C(=O)-R8, -OC(=O)-NR6R7, -NR6-C(=O)-R8, and -NR6-C(=O)-NR6R7; R6 and R7 are each independently selected from hydrogen, -R8, -C(=O)-R8, and -C(=O)-OR8; and R8 is selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups. R1, R2, R3, R4, R5, R6, R7, and R8 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR6R7, C3-C8 carbocyclic, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic, each of which may be independently substituted by 0 or 1 group selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C6 alkylhydroxyl, C1-C6 alkyl ... 3-Hydroalkyl, -NH-C(=O)(C1-C3alkyl) and -NH-C(=O)-O-(C1-C3alkyl), and The valence of the atoms covalently attached to L did not exceed the limit.

[0336] In some embodiments, H-containing compounds (Ia) are used as Hobschdiene splice regulators: , or a medically acceptable salt thereof, which is covalently linked to L via any atom, wherein: R9 is selected from C3-C8 heterocyclic groups; R 10 is selected from H and C1-C6 alkyl groups. R9 and R10 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, -NH2, -NH-(C1-C3 alkyl) and -N-(C1-C3 alkyl)2, and The valence of the atoms covalently attached to L did not exceed the limit.

[0337] In some embodiments, H-containing (Ib) compounds that are Hobschdiene splice regulators: , or a medically acceptable salt thereof, which is covalently linked to L via any atom, wherein: R 11 is selected from * indicates the connection point between R 11 and the rest of the compound; R12 and R13 are each independently selected from H and methyl; and The valence of the atoms covalently attached to L did not exceed the limit.

[0338] In some embodiments, H-containing compounds of formula (II) are used as Hobschdiene splice regulators: , or a medically acceptable salt thereof, which is covalently linked to L via any atom, wherein: X represents a hydroxyl group or NR 6R 7; R6 and R7 are each independently selected from hydrogen, -R8, -C(=O)-R8, -C(=O)-OR8, -(C1-C6 alkyl)-OC(=O)-R8, and -(C1-C6 alkyl)-NH-C(=O)-R8; and R8 is selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups. R6, R7, and R8 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR6R7, C3-C8 carbocyclic, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic, each of which may be independently substituted by 0 or 1 group selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C6 alkylhydroxyl, C1-C6 alkylalkoxy, C3 ... 3-Hydroalkyl, -NH-C(=O)(C1-C3alkyl) and -NH-C(=O)-O-(C1-C3alkyl), and The valence of the atoms covalently attached to L did not exceed the limit.

[0339] In some embodiments, H-containing compounds of formula (IIa) are used as Hobschdiene splice regulators: , or a medically acceptable salt thereof, which is covalently linked to L via any atom, wherein: Z is selected from NR 9 and O; R9 is selected from hydrogen and C1-C6 alkyl groups; R10 and R11 are each independently selected from hydrogen, halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbon cycloyl), -C(=O)-(C3-C8 heterocyclic), C3-C8 carbon cycloyl, C1-C6 alkyl hydroxyl, C1-C6 alkyl alkoxy, benzyl, and C3-C8 heterocyclic; R 12 is selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups. R9, R10, R11, and R12 are each independently substituted by one or more groups selected from the following: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, and C1-C3 haloalkyl; t is an integer selected from 1, 2, 3, 4, 5, and 6; and The valence of the atoms covalently attached to L did not exceed the limit.

[0340] In some embodiments, H-containing compounds of formula (IIb) that are Hobschdiene splice regulators: , or a medically acceptable salt thereof, which is covalently linked to L via any atom, wherein: R 13 is selected from * indicates the connection point between R 13 and the rest of the compound; R14 and R15 are each independently selected from hydrogen and methyl; and The valence of the atoms covalently attached to L did not exceed the limit.

[0341] In some embodiments, H-containing compounds of formula (III) are used as Hobschdiene splice regulators: , or a medically acceptable salt thereof, which is covalently linked to L via any atom, wherein: R1, R2, and R3 are each independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl), -OC(=O)-(C1-C6 alkyl), -C(=O)-O-(C1-C6 alkyl), and C1-C6 alkyl; R6 and R7 are each independently selected from hydrogen, -R8, -C(=O)-R8, and -C(=O)-OR8; R8 is selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups; and R 9 is selected from H, ; R1, R2, R3, R6, R7, and R8 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR6R7, C3-C8 carbocyclic, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic, each of which may be independently substituted by 0 or 1 group selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, -NH-C(=O)(C 1-C 3 alkyl) and -NH-C(=O)-O-(C 1-C 3 alkyl), The valence of the atoms covalently connected to L does not exceed the limit; and The asterisk (*) indicates the connection point between R 9 and the rest of the compound.

[0342] In some embodiments, the antibody or antigen-binding fragment targets cells exhibiting the following characteristics: HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, and / or STEAP1.

[0343] In some embodiments, the antibody or antigen-binding fragment targets cells expressing HER2. 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 includes 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 includes 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 includes a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment includes a human Igκ light chain constant region.

[0344] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing CD138. 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 includes 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 includes 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 includes a mouse IgG2a heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment includes a mouse Igκ light chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a constant region of the human IgG2a heavy chain. In some embodiments, the antibody or antigen-binding fragment comprises a constant region of the human Igκ light chain.

[0345] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing EPHA2. 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 includes 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 includes 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 includes a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment includes a human Igκ light chain constant region.

[0346] In some other embodiments, the antibody or antigen-binding fragment targets cells exhibiting MSLN. In some embodiments, the antibody or antigen-binding fragment is an anti-MSLN antibody or antigen-binding fragment.

[0347] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing FOLH1. In some embodiments, the antibody or antigen-binding fragment is an anti-FOLH1 antibody or antigen-binding fragment.

[0348] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing CDH6. In some embodiments, the antibody or antigen-binding fragment is an anti-CDH6 antibody or antigen-binding fragment.

[0349] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing CEACAM5. In some embodiments, the antibody or antigen-binding fragment is an anti-CEACAM5 antibody or antigen-binding fragment.

[0350] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing CFC1B. In some embodiments, the antibody or antigen-binding fragment is an anti-CFC1B antibody or antigen-binding fragment.

[0351] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing ENPP3. In some embodiments, the antibody or antigen-binding fragment is an anti-ENPP3 antibody or antigen-binding fragment.

[0352] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing FOLR1. In some embodiments, the antibody or antigen-binding fragment is an anti-FOLR1 antibody or antigen-binding fragment.

[0353] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing HAVCR1. In some embodiments, the antibody or antigen-binding fragment is an anti-HAVCR1 antibody or antigen-binding fragment.

[0354] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing KIT. In some embodiments, the antibody or antigen-binding fragment is an anti-KIT antibody or antigen-binding fragment.

[0355] In some other embodiments, the antibody or antigen-binding fragment targets cells exhibiting MET. In some embodiments, the antibody or antigen-binding fragment is an anti-MET antibody or antigen-binding fragment.

[0356] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing MUC16. In some embodiments, the antibody or antigen-binding fragment is an anti-MUC16 antibody or antigen-binding fragment.

[0357] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing SLC39A6. In some embodiments, the antibody or antigen-binding fragment is an anti-SLC39A6 antibody or antigen-binding fragment.

[0358] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing SLC44A4. In some embodiments, the antibody or antigen-binding fragment is an anti-SLC44A4 antibody or antigen-binding fragment.

[0359] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing STEAP1. In some embodiments, the antibody or antigen-binding fragment is an anti-STEAP1 antibody or antigen-binding fragment.

[0360] In some embodiments, L is selected from any of the linkers disclosed herein or any combination of the 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 include one or more additional spacer subunits. In some embodiments, L is a linker ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23. In some embodiments, L is a linker ADL12, ADL14, or ADL15. In some embodiments, the connectors ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 may also include one or more additional spacer subunits.

[0361] In some embodiments, an intermediate serving as a precursor to the linker moiety is reacted with the Hopschierne splice regulator moiety under appropriate conditions. In some embodiments, a reactive group is used on the Hopschierne splice regulator and / or the intermediate or linker. Subsequently, the reaction product between the Hopschierne splice regulator and the intermediate, or the derived Hopschierne splice regulator (Hopschierne splice regulator plus linker), is 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 derived antibody or antigen-binding fragment, and subsequently with a drug or a derived drug.

[0362] Several different reactions are available for covalently linking the Hobstein splice regulator moiety and / or linker moiety to an antibody or antigen-binding fragment. This is typically achieved by reacting one or more amino acid residues of the antibody or antigen-binding fragment, including the amino group of lysine, the free carboxyl group of glutamic acid and aspartic acid, the thio group 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 (or amino) group on the Hobstein splice regulator moiety to an amino (or carboxyl) group on the antibody or antigen-binding fragment. Alternatively, bifunctional reagents such as dialdehydes or imine esters can be used to link an amino group on the Hobstein splice regulator moiety to an amino group on the antibody or antigen-binding fragment. The Schiff base reaction can also be used to link a drug (e.g., the Hobstein splice regulator) to a binder. This method involves the oxidation of a drug containing a diol or hydroxyl group by periodate to form an aldehyde, which is then reacted with a binder. The linkage occurs via the formation of a Schiff base using the amino group of the binder. Isothiocyanates can also be used as coupling agents to covalently link the drug to the binder. Other techniques are known to those skilled in the art and are within the scope of this invention. Examples of drug portions generated and linked to antibody or antigen-binding fragments using various chemical reactions known in this art include Hopschadiene splice regulators, such as those described and exemplified herein. [Connector] [-] [Hobsch diene splicing regulator] [ / ] [Hobsch diene splice regulator compound] []

[0363] This document further discloses exemplary linker-Hobschdiene splice regulator (LH) compounds and compositions comprising multiple copies of such compounds. In various embodiments, the linker-Hobschdiene splice regulator compounds disclosed herein can be defined by the general formula: LH, where L = linker moiety and H = Hobschdiene splice regulator. In some embodiments, the disclosed LH compounds are suitable for the ADCs described herein.

[0364] In some embodiments, the compound of formula (I) disclosed herein: or a medically acceptable salt thereof, wherein: Y is selected from O, S, NR 6, and CR 6R 7; R1, R2, and R3 are each independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl), -OC(=O)-(C1-C6 alkyl), -C(=O)-O-(C1-C6 alkyl), and C1-C6 alkyl; R4 is selected from hydrogen, C1-C6 alkyl, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbon cycloyl), -C(=O)-(C3-C8 heterocyclic), and -C(=O)-NR6R7; R5 is selected from hydrogen, hydroxyl group, -CH2-OH, -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-NR6R7, -NR6-C(=O)-R8, -OC(=O)-NR6R7, -NR6-C(=O)-R8, and -NR6-C(=O)-NR6R7; R6 and R7 are each independently selected from hydrogen, -R8, -C(=O)-R8, and -C(=O)-OR8; and R8 is selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups. R1, R2, R3, R4, R5, R6, R7, and R8 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR6R7, C3-C8 carbocyclic, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic, each of which may be independently substituted by 0 or 1 group selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C6 alkylhydroxyl, C1-C6 alkyl ... 3-Hydroalkyl, -NH-C(=O)(C1-C3alkyl) and -NH-C(=O)-O-(C1-C3alkyl).

[0365] In some embodiments, compounds of formula (Ia) are provided herein: , or a medically acceptable salt thereof, which is covalently linked to L via any atom, wherein: R9 is selected from C3-C8 heterocyclic groups; and R 10 is selected from H and C1-C6 alkyl groups. R9 and R10 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, -NH2, -NH-(C1-C3 alkyl) and -N-(C1-C3 alkyl)2.

[0366] In some embodiments, compounds of formula (Ib) are provided herein: or a medically acceptable salt thereof, wherein: R 11 is selected from * indicates the connection point between R 11 and the rest of the compound; R12 and R13 are each independently selected from H and methyl.

[0367] In some embodiments, compounds of formula (II) are provided herein: or a medically acceptable salt thereof, wherein: X represents a hydroxyl group or NR 6R 7; R6 and R7 are each independently selected from hydrogen, -R8, -C(=O)-R8, -C(=O)-OR8, -(C1-C6 alkyl)-OC(=O)-R8, and -(C1-C6 alkyl)-NH-C(=O)-R8; and R8 is selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups. R6, R7, and R8 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR6R7, C3-C8 carbocyclic, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic, each of which may be independently substituted by 0 or 1 group selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C6 alkylhydroxyl, C1-C6 alkylalkoxy, C3 ... 3-Hydroalkyl, -NH-C(=O)(C1-C3alkyl) and -NH-C(=O)-O-(C1-C3alkyl).

[0368] In some embodiments, compounds of formula (IIa) are provided herein: or a medically acceptable salt thereof, wherein: Z is selected from NR 9 and O; R9 is selected from hydrogen and C1-C6 alkyl groups; R10 and R11 are each independently selected from hydrogen, halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbon cycloyl), -C(=O)-(C3-C8 heterocyclic), C3-C8 carbon cycloyl, C1-C6 alkyl hydroxyl, C1-C6 alkyl alkoxy, benzyl, and C3-C8 heterocyclic; R 12 is selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups. R9, R10, R11, and R12 are each independently substituted by one or more groups selected from: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, and C1-C3 haloalkyl; and t is an integer selected from 1, 2, 3, 4, 5 and 6.

[0369] In some embodiments, compounds of formula (IIb) are provided herein: or a medically acceptable salt thereof, wherein: R 13 is selected from * indicates the connection point between R 13 and the rest of the compound; and R14 and R15 are each independently selected from hydrogen and methyl.

[0370] In some embodiments, compounds of formula (III) are provided herein: or a medically acceptable salt thereof, wherein: R1, R2, and R3 are each independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl), -OC(=O)-(C1-C6 alkyl), -C(=O)-O-(C1-C6 alkyl), and C1-C6 alkyl; R6 and R7 are each independently selected from hydrogen, -R8, -C(=O)-R8, and -C(=O)-OR8; R8 is selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups; and R 9 is selected from H, , R1, R2, R3, R6, R7, and R8 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR6R7, C3-C8 carbocyclic, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic, each of which may be independently substituted by 0 or 1 group selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, -NH-C(=O)(C 1-C 3 alkyl) and -NH-C(=O)-O-(C 1-C 3 alkyl); and The asterisk (*) indicates the connection point between R 9 and the rest of the compound.

[0371] In some embodiments, this document provides compound H1 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H4 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H5 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H6 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H7 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H8 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H9 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H10 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H2 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H3 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H12 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H13 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H14 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H15 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H16 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H17 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H18 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H19 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H20 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H21 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H22 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H23 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H24 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H25 or a pharmaceutically acceptable salt thereof.

[0372] In some embodiments, this document provides compounds selected from the following: and medically acceptable salts, Where L is a linker covalently linked to the antibody.

[0373] In some embodiments, the linker comprises at least one cleavable peptide moiety. In some embodiments, the at least one cleavable peptide moiety may be cleaved by an enzyme. In some embodiments, the linker or the 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, aspartic acid, glutamic acid, 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.

[0374] In some embodiments, the linker comprises a portion selected from sulfonamide, β-glucuronic acid, disulfide, and carbonyl. In some embodiments, the linker comprises sulfonamide. In some embodiments, the linker comprises β-glucuronic acid. In some embodiments, the linker comprises disulfide. In some embodiments, the linker comprises carbonyl.

[0375] In some embodiments, the linker includes spacer units. In some embodiments, the spacer units are selected from alkyl and polyethylene glycol (PEG) portions. In some embodiments, the alkyl group is C1-C12 alkyl. In some embodiments, the alkyl group is C1-C6 alkyl. In some embodiments, the alkyl group is methylene. In some embodiments, the alkyl group is ethyl. In some embodiments, the alkyl group is n-propyl. In some embodiments, the alkyl group is n-butyl. In some embodiments, the alkyl group is n-pentyl. In some embodiments, the alkyl group is n-hexyl. In some embodiments, the PEG portion includes -(PEG)m-, where 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.

[0376] In some embodiments, the linker comprises a maleic anhydride (Mal) moiety ("Mal-spacer unit"). In some embodiments, the linker comprises a self-degrading spacer unit. In some embodiments, the self-degrading spacer unit is selected from p-aminobenzoxycarbonyl (pABC) and p-aminobenzyl (pAB).

[0377] In some embodiments, the linker comprises a Mal-spacer unit, an alkyl group, at least one amino acid unit, and a self-degrading 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-degrading spacer is selected from pAB and pABC. In some embodiments, the self-degrading spacer comprises pAB. In some embodiments, the self-degrading spacer comprises pABC. In some embodiments, the alkyl group comprises a C1-C6 alkyl group.

[0378] In some embodiments, the linker comprises a Mal-spacer unit, a PEG portion, at least one amino acid unit, and a self-degrading 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-degrading spacer is selected from pAB and pABC. In some embodiments, the self-degrading spacer comprises pAB. In some embodiments, the self-degrading spacer comprises pABC. In some embodiments, the PEG portion comprises -(PEG)m-, where m is an integer from 1 to 6. [] [Drug Load] []

[0379] The drug loading is represented by p and is also referred to herein as the Hobstein splice regulator to antibody ratio (HAR). The drug loading can range from 1 to 10 drug fractions per antibody or antigen-binding fragment. In some embodiments, p is an integer from 1 to 10. In some embodiments, p is an integer from 1 to 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 from 2 to 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 from 1 to 8. In some embodiments, p is an integer from 2 to 5. In some embodiments, p is an integer from 2 to 4. In some embodiments, p is an integer from 3 to 4. In other embodiments, p is an integer from 4 to 8. In other embodiments, p is 1, 2, 3, 4, 5, 6, 7, or 8, preferably 4 or 8.

[0380] The drug loading can be limited by the number of linking sites on the antibody or antigen-binding fragment. In some embodiments, the linker portion (L) of the ADC is linked to the antibody or antigen-binding fragment via a chemically active group on one or more amino acid residues. For example, the linker can be linked to the antibody or antigen-binding fragment via a free amino group, imine group, hydroxyl group, thiol group, or carboxyl group (e.g., linked to the N-terminus or C-terminus, linked to an ε-amino group on one or more lysine residues, linked to a free carboxyl group on one or more glutamic acid or aspartic acid residues, or linked to a sulfhydryl group on one or more cysteine ​​residues). The linker site can be a native residue in the amino acid sequence of the antibody or antigen-binding fragment, or it can be introduced into the antibody or antigen-binding fragment, for example, by DNA recombination technology (e.g., by introducing cysteine ​​residues into the amino acid sequence) or by protein biochemistry (e.g., by reduction, pH adjustment, or hydrolysis).

[0381] In some embodiments, the number of drug moieties that can bind to an antibody or antigen-binding fragment is limited by the number of free cysteine ​​residues. For example, in the case of cysteine ​​thiol linkages, an antibody may have only one or more cysteine ​​thiol groups, or may have only one or more sufficiently reactive thiol groups that a linker can connect to. Generally, antibodies do not contain a large number of free reactive cysteine ​​thiol groups that can be linked to drug moieties. In fact, most cysteine ​​thiol residues in antibodies are involved in inter- or intra-chain disulfide bonds. Therefore, in some embodiments, binding to cysteine ​​may require at least partial reduction of the antibody. Excessive linking of linker-toxin to antibody can destabilize the antibody by reducing cysteine ​​residues available for forming disulfide bonds. Therefore, an optimized drug:antibody ratio should increase the potency of the ADC (by increasing the number of linked drug moieties per antibody) without destabilizing the antibody or antigen-binding fragment. In some embodiments, the optimal ratio may be 2, 4, 6, or 8.

[0382] In some embodiments, the antibody or antigen-binding fragment is exposed to reducing conditions prior to binding to generate one or more free cysteine ​​residues. In some embodiments, the antibody may be reduced to reactive cysteine ​​thiol groups under partial or total reducing conditions using a reducing agent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). Unpaired cysteine ​​may be generated by partial reduction with a limited molar equivalent of TCEP, which reduces the interchain disulfide bonds connecting the light and heavy chains (one pair / HL pair) and the two heavy chains in the hinge region (two pairs / HH pair in the case of human IgG1), but leaves the intrachain disulfide bonds intact (Stefano et al. (2013) Methods Mol Biol. 1045:145-71). In embodiments, disulfide bonds within the antibody may be reduced electrochemically, for example, by employing a working electrode with alternating reducing and oxidizing voltages. This method allows for the online coupling of disulfide bond reduction with analytical devices (e.g., electrochemical detection devices, NMR spectrometers, or mass spectrometers) or chemical separation devices (e.g., liquid chromatography (e.g., HPLC) or electrophoresis devices (see, for example, U.S. Publication No. 20140069822)). In some embodiments, the antibody is subjected to denaturing conditions to expose reactive nucleophilic groups on amino acid residues such as cysteine.

[0383] The drug loading of an ADC can be controlled in various ways, such as by: (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody; (ii) limiting the binding reaction time or temperature; (iii) using partial or limiting reduction conditions for cysteine ​​thiol modification; and / or (iv) using recombinant technology to engineer the amino acid sequence of the antibody to control the number and / or position of modified cysteine ​​residues to control the number and / or position of linker-drug linkages.

[0384] In some embodiments, free cysteine ​​residues are 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 the parent antibody are replaced by cysteine ​​amino acid substitutions. Any form of antibody can be engineered in this way, i.e., mutated. For example, a parent Fab antibody fragment can be engineered to form a cysteine-engineered Fab, referred to as "ThioFab". Similarly, a parent monoclonal antibody can be engineered to form "ThioMab". A single point mutation in ThioFab produces a single engineered cysteine ​​residue, while a single point mutation in ThioMab, due to the dimer nature of IgG antibodies, produces two engineered cysteine ​​residues. DNA encoding amino acid sequence variants of the parent polypeptide can be prepared by various methods known in this art (see, for example, the method described in International Publication No. WO 2006 / 034488). These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutation induction of DNA encoding a previously prepared polypeptide, PCR mutation induction, and cartridge mutation induction. Recombinant antibody variants can also be constructed by restriction fragment manipulation or by synthetic oligonucleotides used in overlap extension PCR. Formula (I) ADCs include, but are not limited to, antibodies having 1, 2, 3, or 4 engineered cysteine ​​amino acids (Lyon et al. (2012) Methods Enzymol. 502:123-38). In some embodiments, without the use of engineering, one or more free cysteine ​​residues are already present in the antibody or antigen-binding fragment, in which case the existing free cysteine ​​residues can be used to bind the antibody or antigen-binding fragment to the drug moiety.

[0385] In cases where more than one nucleophilic group reacts with a drug-linker intermediate or linker moiety reagent, followed by a reaction with a drug moiety reagent, the resulting product in a reaction mixture comprising multiple replicas of an antibody or antigen-binding fragment and a linker moiety can be a mixture of ADC compounds having one or more drug moieties linked to each replica of the antibody or antigen-binding fragment in the mixture. In some embodiments, the drug loading in the mixture of ADCs obtained from the binding reaction is in the range of 1 to 10 linked drug moieties per 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 this art, such as by mass spectrometry (e.g., reversed-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 in each antibody or antigen-binding fragment is determined by reversed-phase liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the average number of drug moieties in each 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 in each 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.

[0386] In some embodiments, the average number of drug portions of each antibody or antigen-binding fragment is about 2. In some embodiments, the average number of drug portions of each 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 portions of each antibody or antigen-binding fragment is 2.

[0387] In some embodiments, the average number of drug portions of each antibody or antigen-binding fragment is about 4. In some embodiments, the average number of drug portions of each 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 portions of each antibody or antigen-binding fragment is 4.

[0388] In some embodiments, the average number of drug portions of each antibody or antigen-binding fragment is about 8. In some embodiments, the average number of drug portions of each 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 portions of each antibody or antigen-binding fragment is 8.

[0389] In various embodiments, the term "about" as used with respect to the average number of pharmaceutical portions of each antibody or antigen-binding fragment means ± 10%.

[0390] Individual ADC compounds or "species" can be identified in the mixture by mass spectrometry and separated by UPLC or HPLC, such as hydrophobic interaction chromatography (HIC-HPLC). In some embodiments, homogeneous or near-homogeneous ADC products with a single loading value can be separated from the bound mixture, for example, by electrophoresis or chromatography.

[0391] In some embodiments, higher drug loading (e.g., p > 8) can cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates. Higher drug loading may also adversely affect the pharmacokinetics (e.g., clearance) of certain ADCs. In some embodiments, lower drug loading (e.g., p < 2) may reduce the potency of certain ADCs against target cells and / or bystander cells. In some embodiments, the drug loading of the ADCs of the present invention is in the range of 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.

[0392] In some embodiments, a drug loading of about 2 and / or an average drug loading is achieved, for example, by partial reduction of intrachain disulfides on the antibody or antigen-binding fragment, and beneficial properties are provided. In some embodiments, a drug loading of about 4 and / or an average drug loading is achieved, for example, by partial reduction of intrachain disulfides on the antibody or antigen-binding fragment, and beneficial properties are provided. In some embodiments, a drug loading of about 8 and / or an average drug loading is achieved, for example, by partial reduction of intrachain disulfides on the antibody or antigen-binding fragment, and beneficial properties are provided. In some embodiments, a drug loading of less than about 2 and / or an average drug loading may result in unacceptably high levels of unbound antibody species that may compete with the ADC to bind to the target antigen and / or provide reduced therapeutic efficacy. In some embodiments, a drug loading of more than about 8 and / or an average drug loading may result in unacceptably high levels of product heterogeneity and / or ADC aggregation. Drug loadings greater than approximately 8 and / or average drug loadings may also affect the stability of ADCs due to the loss of one or more chemical bonds required to stabilize antibody or antigen-binding fragments.

[0393] This invention includes a method for producing the described ADC. In short, an ADC comprises an antibody or antigen-binding fragment as the antibody or antigen-binding fragment, a pharmaceutical moiety (e.g., a hoprene splice regulator), and a linker that connects the pharmaceutical moiety and the antibody or antigen-binding fragment. In some embodiments, the ADC can be prepared using a linker having a reactive functional group for covalently linking to the pharmaceutical moiety and the antibody or antigen-binding fragment. For example, in some embodiments, the cysteine ​​thiol of the antibody or antigen-binding fragment can form a bond with a reactive functional group (e.g., a maleic anhydride moiety) of the linker or drug-linker intermediate to produce the ADC. ADC generation can be achieved by any technique known to those skilled in the art.

[0394] In some embodiments, an ADC is generated by first sequentially contacting an antibody or antigen-binding fragment with a linker and a pharmaceutical moiety (e.g., a hoprene splice regulator) such that the antibody or antigen-binding fragment is covalently linked to the linker, and then reacting a pre-formed antibody-linker intermediate with the pharmaceutical moiety. The antibody-linker intermediate may or may not undergo a purification step before contacting the pharmaceutical moiety. In other embodiments, an ADC is generated by contacting an antibody or antigen-binding fragment with a linker-pharmaceutical compound, which is pre-formed by reacting the linker with the pharmaceutical moiety. The pre-formed linker-pharmaceutical compound may or may not undergo a purification step before contacting the antibody or antigen-binding fragment. In other embodiments, the antibody or antigen-binding fragment contacts a linker and a pharmaceutical moiety in a reaction mixture, allowing covalent bonds to form simultaneously between the antibody or antigen-binding fragment and the linker and between the linker and the pharmaceutical moiety. This method of generating an ADC may include a reaction in which the antibody or antigen-binding fragment is contacted before the linker is added to the reaction mixture, and vice versa. In some embodiments, the ADC is generated by reacting an antibody or antigen-binding fragment with a linker bound to a drug moiety, such as the ADL1-Hopshire splice regulator (e.g., ADL1-79392) or the ADL5-Hopshire splice regulator (e.g., ADL5-0349), under conditions that allow binding.

[0395] The ADC prepared according to the method described above can undergo purification steps. Purification steps may involve any biochemical method or any combination thereof known in this art for purifying proteins. These methods include, but are not limited to, tangential flow filtration (TFF), affinity chromatography, ion exchange chromatography, any charge- or isoelectric point-based chromatography, mixed-mode chromatography (e.g., CHT (ceramic hydroxyapatite)), hydrophobic interaction chromatography, particle size sieving chromatography, dialysis, filtration, selective precipitation, or any combination thereof. Therapeutic Uses and Compositions

[0396] This article discloses a method for treating individual conditions (e.g., neoplastic conditions) using the disclosed ADC and combinations. The ADC can be administered alone or in combination with a second therapeutic agent, and can be administered with any pharmaceutically acceptable formulation, dosage, and dosing regimen. The therapeutic efficacy of the ADC can be assessed and adjusted accordingly based on toxicity and efficacy metrics. Efficacy measures include, but are not limited to, cell growth inhibition and / or cytotoxicity observed in vitro or in vivo, tumor volume reduction, tumor growth inhibition, and / or prolonged survival.

[0397] Methods are known for determining whether ADCs exert cell growth inhibitory and / or cytotoxic effects on cells. For example, the cytotoxic or cell growth inhibitory activity of ADCs can be measured by: exposing mammalian cells expressing the target protein of the ADC to cell culture medium; culturing the cells for a period of approximately 6 hours to approximately 6 days; and measuring cell viability. In vitro cell-based analyses can also be used to measure ADC activity (proliferation), cytotoxicity, and apoptosis induction (activation of apoptotic proteases).

[0398] To determine whether an ADC exerts an inhibitory effect on cell growth, a thymidine incorporation assay can be used. For example, cancer cells representing the target antigen, cultured at a density of 5,000 cells per well of a 96-well plate, can be exposed to 0.5 μCi of 3H-thymidine during the last 8 hours of the 72-hour period. The incorporation of 3H-thymidine into the culture cells is measured in both the presence and absence of the ADC.

[0399] To determine cytotoxicity, necrosis or apoptosis (planned cell death) can be measured. Necrosis is usually accompanied by increased plasma membrane permeability; cell swelling and plasma membrane rupture. Apoptosis can be quantified, for example, by measuring DNA fragmentation. Commercial photometric me...

Claims

1. A splicing regulator of the following formula: , or a medically acceptable salt thereof, wherein: X is NR 6R 7; R 6 and R 7 are each independently selected from hydrogen, -R 8, -C(=O)-R 8, -C(=O)-OR 8, -(C 1-C 6 alkyl)-OC(=O)-R 8 and -(C 1-C 6 alkyl)-NH-C(=O)-R 8; and R 8 is selected from C 1-C 6 alkyl, C 3-C 8 carbocyclic and C 3-C 8 heterocyclic, wherein R 6, R 7 and R 8 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C 1-C 6 alkyl, -O-(C 1-C 6 alkyl), -CO 2H, -C(=O)-O-(C 1-C 6 alkyl), -C(=O)-(C 1-C 6 alkyl), -C(=O)-(C 3-C -C(=O)-(C3-C8 heterocyclic), C3-C8 carbon cyclic, C1-C6 alkyl hydroxyl, C1-C6 alkyl alkoxy, benzyl and C3-C8 heterocyclic, each of which may be independently substituted by 0 or 1 of the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, -NH-C(=O)(C1-C3 alkyl) and -NH-C(=O)-O-(C1-C3 alkyl).

2. The splicing modulator of claim 1, wherein the splicing modulator is of the following form: , or a medically acceptable salt thereof, wherein: Z is selected from NR 9 and O; R 9 is selected from hydrogen and C1-C6 alkyl; R 10 and R 11 are each independently selected from hydrogen, halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO 2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbon cycloyl), -C(=O)-(C3-C8 heterocyclic), C3-C8 carbon cycloyl, C1-C6 alkylhydroxyl, C1-C6 alkylalkoxy, benzyl and C3-C8 heterocyclic; R 12 is selected from C1-C6 alkyl, C3-C8 carbon cycloyl, C3-C8 heterocyclic, wherein R 9, R 10, R 11 and R 12 are each independently substituted by 0 or 1 of the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy and C1-C3 haloalkyl; and t is an integer selected from 1, 2, 3, 4, 5 and 6.

3. The splice modulator of claim 1, wherein the splice modulator is of the following formula: (IIb), or a medically acceptable salt thereof, wherein: R 13 is selected from , , , , , , , , , , and , where * indicates the connection point of R 13 with the rest of the compound; and R 14 and R 15 are each independently selected from hydrogen and methyl.

4. The splice modulator as claimed in claim 1, wherein the splice modulator is selected from: (H4), (H13), (H14), (H15), (H16), (H17), (H18), (H19), (H20), (H21), (H23), (H24) and their pharmaceutically acceptable salts.

5. A compound or a pharmaceutically acceptable salt thereof, the compound comprising a linker-splicing regulator of the formula LH, wherein L is a linker and H is a splicing regulator as claimed in claim 1.

6. The compound of claim 5 or its pharmaceutically acceptable salt, wherein H is covalently linked to L via any atom, and the valence of the atom covalently linked to L does not exceed the limit.

7. The compound of claim 5 or its pharmaceutically acceptable salt thereof is selected from , ...

8. A splicing regulator of the following formula: , or a medically acceptable salt thereof, wherein: R1, R2, and R3 are each independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl), -OC(=O)-(C1-C6 alkyl), -C(=O)-O-(C1-C6 alkyl), and C1-C6 alkyl; R9 is selected from H; wherein R1, R2, and R3 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbon cycloyl), -C(=O)-(C3-C8 heterocyclic), -NR6R7, C3-C8 carbon cycloyl, C1-C6 alkylhydroxyl, C1-C6 alkylalkoxy, benzyl, and C 3-C 8 heterocyclic groups, each of which may be independently substituted by 0 or 1 of the following groups: halogen, hydroxyl, C 1-C 3 alkyl, C 1-C 3 alkoxy, C 1-C 3 haloalkyl, -NH-C(=O)(C 1-C 3 alkyl), and -NH-C(=O)-O-(C 1-C 3 alkyl); R 6 and R 7 are each independently selected from hydrogen, -R 8, -C(=O)-R 8, and -C(=O)-OR 8; R 8 is selected from C 1-C 6 alkyl, C 3-C 8 carbocyclic, and C 3-C 8 heterocyclic groups; and * indicates the connection point of R 9 to the rest of the compound.

9. The splice modulator of claim 8, wherein the splice modulator is selected from: (H5), (H6), (H7), (H8), (H9), (H10), (H11), (H22), (H25) and their pharmaceutically acceptable salts.

10. A compound or a pharmaceutically acceptable salt thereof, the compound comprising a linker-splicing regulator of the formula LH, wherein L is a linker and H is a splicing regulator as claimed in claim 8.

11. A compound of claim 10 or a pharmaceutically acceptable salt thereof, wherein H is covalently linked to L via any atom, and the valence of the atom covalently linked to L does not exceed the limit.

12. The compound of claim 10 or its pharmaceutically acceptable salt is selected from , , , , , , , , , and its pharmaceutically acceptable salt.

13. A compound or a pharmaceutically acceptable salt thereof, as claimed in any of claims 5 to 7 and 10 to 12, wherein the linker L is a cleavable linker containing a cleavable portion.

14. The compound of claim 13 or a pharmaceutically acceptable salt thereof, wherein the cleavable portion comprises a cleavable peptide portion.

15. The compound of claim 14 or a pharmaceutically acceptable salt thereof, wherein the cleavable peptide portion comprises valine-citrulline (Val-Cit), valine-alanine (Val-Ala), glutamate-valine-citrulline (Glu-Val-Cit), or alanine-alanine-aspartic acid (Ala-Ala-Asn).

16. The compound of claim 10 or a pharmaceutically acceptable salt thereof, wherein the cleavable peptide portion comprises a cleavable glucuronic acid portion.

17. The compound of claim 13 or a pharmaceutically acceptable salt thereof, wherein the linker L comprises at least one spacer unit, wherein the at least one spacer unit comprises: (i) a polyethylene glycol (PEG) portion, wherein the PEG portion comprises -(PEG)m- and m is an integer from 1 to 10; or (ii) an alkyl portion, wherein the alkyl portion comprises -(CH2)n- and n is an integer from 1 to 10.

18. The compound of claim 13 or a pharmaceutically acceptable salt thereof, wherein the linker L comprises a maleic diamide (Mal) moiety.

19. The compound of claim 18 or a pharmaceutically acceptable salt thereof, wherein the Mal portion is linked to Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn.

20. The compound of claim 17 or a pharmaceutically acceptable salt thereof, wherein the at least one spacer subunit is connected to the Mal portion (Mal-spacer subunit).

21. The compound of claim 20 or a pharmaceutically acceptable salt thereof, wherein the Mal-spacer subunit is connected to the cleavable portion of the linker L.

22. The compound of claim 21 or a pharmaceutically acceptable salt thereof, wherein the Mal-spacer unit comprises a maleic-diaminohexyl (MC) moiety.

23. The compound of claim 22 or a pharmaceutically acceptable salt thereof, wherein the linker L comprises MC-Val-Cit, MC-Val-Ala, MC-Glu-Val-Cit, or MC-Ala-Ala-Asn.

24. The compound of claim 20 or a pharmaceutically acceptable salt thereof, wherein the Mal-spacer unit reacts with a cysteine ​​residue on an antibody or antigen-binding fragment.

25. The compound of claim 13 or a pharmaceutically acceptable salt thereof, wherein the cleavable portion of the linker L is directly connected to the splicing regulator H.

26. The compound of claim 13 or a pharmaceutically acceptable salt thereof, wherein the spacer subunit connects the cleavable portion of the linker L to the splicing regulator H.

27. The compound of claim 26 or a pharmaceutically acceptable salt thereof, wherein the spacer unit to which the cleavable portion is attached to the splice regulator H is self-degrading.

28. The compound of claim 26 or a pharmaceutically acceptable salt thereof, wherein the spacer subunit to which the cleavable portion is attached to the splice regulator H comprises p-aminobenzyl (pAB) or p-aminobenzoxycarbonyl (pABC).

29. The compound of claim 28 or a pharmaceutically acceptable salt thereof, wherein the linker L comprises Val-Cit-pAB, Val-Ala-pAB, Glu-Val-Cit-pAB, Ala-Ala-Asn-pAB, Val-Cit-pABC, Val-Ala-pABC, Glu-Val-Cit-pABC, or Ala-Ala-Asn-pABC.

30. The compound of claim 28 or a pharmaceutically acceptable salt thereof, wherein the linker L comprises MC-Val-Cit-pAB, MC-Val-Ala-pAB, MC-Glu-Val-Cit-pAB, MC-Ala-Ala-Asn-pAB, MC-Val-Cit-pABC, MC-Val-Ala-pABC, MC-Glu-Val-Cit-pABC, or MC-Ala-Ala-Asn-pABC.

31. A compound of any one of claims 5 to 7 or a pharmaceutically acceptable salt thereof, wherein the linker is a non-cleavable linker.

32. The compound of claim 31 or a pharmaceutically acceptable salt thereof, wherein the linker L comprises a Mal-spacer unit.

33. The compound of claim 32 or a pharmaceutically acceptable salt thereof, wherein the Mal-spacer subunit comprises Mal-(PEG)2, Mal-Hex, Mal-Et, Mal-Et-O-Et or Mal(PEG)2-CO.

34. The compound or its pharmaceutically acceptable salt as claimed in claim 5 is selected from: and its pharmaceutically acceptable salt.

35. The compound of claim 10 or its pharmaceutically acceptable salt is selected from: , , , , , , and its pharmaceutically acceptable salt.

36. An antibody-drug conjugate of formula (I), Ab-(LH)p(I), wherein: Ab is an antibody or antigen-binding fragment that targets proliferative cells; LH comprises a compound or a pharmaceutically acceptable salt thereof as described in any of claims 5 to 7 and 10 to 35; and p is an integer from 1 to 15.

37. The antibody-drug conjugate of claim 36, wherein the proliferative cell line is derived from: (i) hematologic malignancies selected from B-cell malignancies, leukemia, lymphoma and myeloma; or (ii) solid tumors selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct cancer, melanoma, colorectal cancer, cervical cancer, pancreatic cancer, kidney cancer and esophageal cancer.

38. The antibody-drug conjugate of claim 36, wherein the proliferative cell line is derived from: (i) hematologic malignancies selected from acute myeloid leukemia and multiple myeloma; or (ii) solid tumors, namely colorectal cancer.

39. The antibody-drug conjugate of claim 36, wherein the proliferative cell line is: (i) a HER2-expressing cell, wherein the antibody or antigen-binding fragment is an anti-HER2 antibody or antigen-binding fragment, and / or 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); or 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; (ii) CD138 phenotyped cells, wherein the antibody or antigen-binding fragment is an anti-CD138 antibody or antigen-binding fragment, and / or wherein the antibody or antigen-binding fragment contains three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) of the amino acid sequences comprising 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) of the amino acid sequences comprising SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2), and SEQ ID NO:12 (LCDR3); or wherein the antibody or antigen-binding fragment contains a heavy chain variable region comprising the amino acid sequence comprising SEQ ID NO:21 and a light chain variable region comprising the amino acid sequence comprising SEQ ID NO:22; or (iii) EPHA2 phenotyped cells, wherein the antibody or antigen-binding fragment is an anti-EPHA2 antibody or antigen-binding fragment, and / or wherein the antibody or antigen-binding fragment contains 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);Or, the antibody or antigen-binding fragment may contain 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.

40. An antibody-drug conjugate as requested in item 36, wherein p is an integer from 1 to 10.

41. A pharmaceutical composition comprising (i) a splice regulator or a pharmaceutically acceptable salt thereof of any one of claims 1 to 4, 8 and 9, (ii) a compound or a pharmaceutically acceptable compound thereof of any one of claims 5 to 7 and 10 to 35, or (iii) an antibody-drug conjugate of any one of claims 36 to 40, and a pharmaceutically acceptable carrier.

42. Use of a splice regulator or a pharmaceutically acceptable salt thereof as claimed in any of claims 1 to 4, 8 and 9, a compound or a pharmaceutically acceptable antibody-drug conjugate thereof as claimed in any of claims 5 to 7 and 10 to 35, or an antibody-drug conjugate thereof as claimed in any of claims 36 to 40, for the preparation of a medicament for treating a neoplastic condition.

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