Splicing modulator antibody-drug conjugates and methods of use

Abstract

This invention discloses linker-drug compounds and antibody-drug conjugates that bind to human tumor targets. These linker-drug compounds and antibody-drug conjugates include a splicing modulator 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 splicing modulator and an antibody or antigen-binding fragment thereof that binds to a target human tumor antigen. Further, this invention relates to methods and compositions that can be used to treat or diagnose cancers exhibiting target antigens and / or suitable for treatment by disrupting RNA splicing, as well as methods for preparing 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 different combinations of exons, alternative splicing produces mRNAs encoding different protein isoforms.

[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. This spliceosome assembles on pre-mRNA to establish a dynamic cascade of RNA and protein interactions that catalyze intron excision and exon ligation (Matera and Wang (2014) Nat Rev Mol Cell Biol. 15(2):108-21). Increasing evidence links human diseases to RNA splicing dysregulation affecting many genes (Scotti and Swanson (2016) Nat Rev Genet. 17(1):19-32).

[0004] Splicing systems are a key target in cancer biology. Several studies have demonstrated significant changes in cancer cell splicing profiles and splicing factors themselves (Agrawal et al. (2018) Curr Opin Genet Dev. 48:67-74). Alternative splicing can lead to different exon inclusion / exclusion, intron retention, or cryptic splicing site usage (Seiler et al. (2018) Cell Rep. 23(1):282-296). In summary, these events result in functional changes, which can lead to tumorigenesis or resistance to therapy (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 skipping (Teng et al. (2017) Nat Commun. 8:15522). A significant portion of the resulting transcripts contain premature stop codons, thereby triggering nonsense-mediated mRNA decay (NMD). In addition, due to impaired typical splicing, typical transcripts are significantly reduced, which may adversely affect cell function and viability. For this reason, splicing regulators have become a promising class of drugs for cancer treatment (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, which belongs to the human epidermal growth factor receptor (EGFR) family (King et al. (1985) Science 229:974-6). Overexpression of HER2 can constitutively activate growth factor signaling pathways, such as the PI3K-AKT-mTOR pathway, and thus serve as a carcinogenic driver for 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 the phenotype mediated by HER2 amplification and the fact that HER2 expression is primarily confined to malignant cells, HER2 has become a promising antigen for targeting certain cancers and / or delivering novel cancer therapies (Parakh et al. (2017) Cancer Treat Rev. 59:1-21). Additional antigens for targeted 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 that is 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 increases cell activity and invasion (Teng et al. (2012) Matrix Biol. 31:3-16). Increased matrix CD138 expression also alters the production of fibronectin and the organization of the extracellular matrix (Yang et al. (2011) Am J Pathol. 178:325-35). In addition, increased CD138 expression in stromal fibroblasts is associated with angiogenesis and cancer progression (Maeda et al. (2006) Oncogene 25:1408-12). CD138 expression increases during B cell development and is a marker of plasma cell lineage (Ribatti (2017) Immunol Lett. 188:64-7). CD138 expression is maintained in multiple myeloma (a plasma cell malignancy). Therefore, CD138 is a compelling 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 highly overexpressed in several malignant cancer cell lines and advanced forms of cancer (Wykosky and Debinski (2008) Mol Cancer Ref. 6(12):1795-1806). For example, EPHA2 is highly 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 terms of both the percentage of tumors in patients and the percentage of cells within the tumor, and it 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 performance is associated with poor prognosis, increased metastasis, and decreased survival. Therefore, EPHA2 stands as another compelling antigen for targeted delivery of novel anticancer therapies due to its presentation patterns, localization, and functional importance in cancer patient outcomes. Summary of the Invention

[0009] In various embodiments, the present invention partially provides novel compounds that are biologically active against neoplastic cells. These compounds can slow, inhibit, and / or reverse tumor growth in mammals and can be used to treat human cancer patients. In various embodiments, the present invention provides novel antibody-drug conjugates employing these novel compounds or other functional splicing inhibitor molecules.

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

[0011] In various embodiments, the ADC complex can be represented by formula (I): Ab-(LD)p (I) Ab refers to antibodies or antigen-binding fragments that target neoplastic cells or another tumor-related target; D-series splice modifier; L-systems covalently connect Ab to the connector of D; and p is an integer from 1 to 15.

[0012] In various embodiments, the ADC complex can be represented by formula (I): Ab-(LD)p (I) Among them, Ab is an antibody or its antigen-binding fragment that targets neoplastic cells; D-series (II) splice modifier: or a medically acceptable salt thereof, wherein: R1 is selected from the following groups: non-existent, hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic, -OC(=O)-(C1-C6 alkyl) group, and -CD3; R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic, and -OC(=O)-(C1-C6 alkyl) groups; and R4, R5, and R8 are each independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl) group, -OC(=O)-(C1-C6 alkyl) group, and C1-C6 alkyl; R6 and R7 are each independently selected from hydrogen, -O-R17, -OC(=O)-R17, -OC(=O)-NR15R16, C1-C6 alkyl groups, and -NR15R16; R15 and R16 are each independently selected from hydrogen, R17, -C(=O)-R17, and -C(=O)-O-R17; R17 is selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, benzyl, and C3-C8 heterocyclic groups; and The Z series is selected from... R1, R3, R4, R5, R6, R7, R8, R15, R16, and R17 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl) group, -NR15 / R16, C3-C8 cycloalkyl, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic group. At least one of R6 and R7 is hydrogen; Furthermore, the L series covalently connects Ab to the connector of D; and p is an integer from 1 to 15.

[0013] In some embodiments, the antibody or antigen-binding fragment can be internalized into target cells. In some embodiments, the linker is covalently linked to a splicing regulator ("LD") of formula (II), and the LD has the structure of formula (II-A): or a medically acceptable salt, Z' is selected from; and All other variable systems are defined with respect to equation (II).

[0014] In various other embodiments, the ADC complex can be represented by formula (I): Ab-(LD)p (I) Among them, Ab is an antibody or its antigen-binding fragment that targets neoplastic cells; D-series (IV) splice modifiers: or a medically acceptable salt thereof, wherein: R1 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic, -OC(=O)-(C1-C6 alkyl) group, and -CD3; R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic, and -OC(=O)-(C1-C6 alkyl) groups; and R4, R5, and R8 are each independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl) group, -OC(=O)-(C1-C6 alkyl) group, and C1-C6 alkyl; R6 and R7 are each independently selected from hydrogen, -O-R17, -OC(=O)-R17, -OC(=O)-NR15R16, C1-C6 alkyl groups, and -NR15R16; R15 and R16 are each independently selected from hydrogen, R17, -C(=O)-R17, and -C(=O)-O-R17; and R17 is selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, benzyl, and C3-C8 heterocyclic groups; R1, R3, R4, R5, R6, R7, R8, R15, R16, and R17 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl) group, -NR15 / R16, C3-C8 cycloalkyl, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic group. At least one of R6 and R7 is hydrogen; Furthermore, the L series covalently connects Ab to the connector of D; and p is an integer from 1 to 15.

[0015] In some embodiments, the antibody or antigen-binding fragment is internalized into target cells. In some embodiments, the linker is covalently linked to a splicing regulator (“LD”), and the LD has the structure of formula (IV-A): or a medically acceptable salt thereof.

[0016] In various other embodiments, the ADC complex can be represented by formula (I): Ab-(LD)p (I) Among them, Ab is an antibody or its antigen-binding fragment that targets neoplastic cells; D-series (VI) splice modifier: or a medically acceptable salt thereof, wherein: R1 and R9 are each independently selected from hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic, -OC(=O)-(C1-C6 alkyl) group, and -CD3; R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic and -OC(=O)-(C1-C6 alkyl) groups; R4, R5, and R8 are each independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl) group, -OC(=O)-(C1-C6 alkyl) group, and C1-C6 alkyl; R6 and R7 are each independently selected from hydrogen, -O-R17, -OC(=O)-R17, -OC(=O)-NR15R16, C1-C6 alkyl, -NR15R16, and linkers; R10 is selected from hydrogen, C1-C6 alkyl, -C(=O)-(C1-C6 alkyl) groups, and -CD3; R15 and R16 are each independently selected from hydrogen, R17, -C(=O)-R1, and -C(=O)-O-R17; R17 is selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, benzyl, and C3-C8 heterocyclic groups; and Series a: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; R1, R3, R4, R5, R6, R7, R8, R9, R10, R15, R16, and R17 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl) group, -NR15 R16, C3-C8 cycloalkyl, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic group; At least one of R6 and R7 is hydrogen; and R1 and R9 cannot both be absent; Furthermore, the L series covalently connects Ab to the connector of D; and p is an integer from 1 to 15.

[0017] In some embodiments, the antibody or antigen-binding fragment is internalized into target cells. In some embodiments, the linker is covalently linked to a splicing regulator (“LD”), and the LD has the structure of formula (VI-A): or a medically acceptable salt thereof.

[0018] In various other embodiments, the ADC complex can be represented by formula (I): Ab-(LD)p (I) Among them, Ab is an antibody or its antigen-binding fragment that targets neoplastic cells; D-series (VIII) splice modifiers: or a medically acceptable salt thereof, wherein: R1 is selected from the following groups: non-existent, hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic, -OC(=O)-(C1-C6 alkyl) group, and -CD3; R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic and -OC(=O)-(C1-C6 alkyl) groups; R4 is selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl) group, -OC(=O)-(C1-C6 alkyl) group, and C1-C6 alkyl; and R10 is selected from 3 to 10-membered carbon rings and 3 to 10-membered heterocycles, each substituted by 0 to 3 Ra, wherein each Ra is independently selected from halogens, C1-C6 alkyl, -O-(C1-C6)alkyl, C1-C6 alkylalkoxy, C1-C6 alkylhydroxy, -S(=O)w-(4 to 7-membered heterocycles), 4 to 7-membered carbon rings and 4 to 7-membered heterocycles; R15 and R16 are each independently selected from hydrogen, R17, -C(=O)-R17, and -C(=O)-O-R17; and R17 is selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, benzyl, and C3-C8 heterocyclic groups; R1, R3, R4, R10, R15, R16, and R17 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl) group, -NR15 R16, C3-C8 cycloalkyl, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic group; and Each Ra is independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, -NR15 R16, C1-C6 alkyl, -(C=O)-(C1-C6 alkyl) group, -(C=O)-(C1-C6 alkyl)-(C3-C10 heterocyclic), -S(=O)w-(C3-C8 heterocyclic) group, and C1-C6 alkylcarboxylic acid group, each of which is substituted by 0, 1, or 2 groups independently selected from the following groups: halogen, hydroxyl, -NR15 R16, and C1-C3 alkyl; and w is 0, 1, or 2; Furthermore, the L series covalently connects Ab to the connector of D; and p is an integer from 1 to 15.

[0019] In some embodiments, the antibody or antigen-binding fragment is internalized into target cells. In some embodiments, the linker is covalently linked to a splicing regulator (“LD”), and the LD has the structure of formula (VIII-A): or a medically acceptable salt thereof.

[0020] In some embodiments, the splice modifier comprises a modifier of the SF3b complex. In some embodiments, the splicing modifier comprises pladienolide or a pladienolide derivative. In some embodiments, the splicing modifier comprises pladienolide D or a pladienolide D derivative. In some embodiments, pladienolide D or its derivative comprises D2, D1, D4, D8, D10, D11 (E7107), D20, D21, D22, D12, or D25. In some embodiments, pladienolide D or its derivative comprises D2. In some embodiments, pladienolide D or its derivative comprises D1. In some embodiments, pladienolide D or its derivative comprises D4. In some embodiments, pladienolide D or its derivative comprises D12.

[0021] In some embodiments, prasadiene lactone D or its derivatives are zwitterionic prasadiene lactone D or its derivatives. In some embodiments, zwitterionic prasadiene lactone D or its derivatives comprise D22 or D25.

[0022] In some other embodiments, the splice modulator comprises prasadiene lactone B or a derivative thereof. In some embodiments, prasadiene lactone B or its derivative comprises D9, D18, D19, or D13. In some embodiments, the splice modifier comprises arylpradoadiene lactone. In some embodiments, the arylpradoadiene lactone comprises D15, D14, D16, D17, D26, or D33. In some embodiments, the arylpradoadiene lactone comprises D15. In some embodiments, the arylpradoadiene lactone is a zwitterionic arylpradoadiene lactone. In some embodiments, the zwitterionic arylpradoadiene lactone comprises D33.

[0023] In some embodiments, the splice modulator comprises D1: .

[0024] In some embodiments, the splice modifier comprises D2: .

[0025] In some embodiments, the splice modulator comprises D3: .

[0026] In some embodiments, the splice modifier comprises D4: .

[0027] In some embodiments, the splice modulator comprises D4': .

[0028] In some embodiments, the splice modifier comprises D5: (D5)

[0029] In some embodiments, the splice modifier comprises D6: .

[0030] In some embodiments, the splice modifier comprises D7: .

[0031] In some embodiments, the splice modifier comprises D8: .

[0032] In some embodiments, the splice modifier comprises D9: .

[0033] In some embodiments, the splice modifier comprises D10: .

[0034] In some embodiments, the splice modulator comprises D11: .

[0035] In some embodiments, the splice modifier comprises D12: .

[0036] In some embodiments, the splice modifier comprises D13: .

[0037] In some embodiments, the splice modulator comprises D14: .

[0038] In some embodiments, the splice modifier comprises D15: .

[0039] In some embodiments, the splice modifier comprises D16: .

[0040] In some embodiments, the splice modifier comprises D17: .

[0041] In some embodiments, the splice modifier comprises D18: .

[0042] In some embodiments, the splice modulator comprises D19: .

[0043] In some embodiments, the splice modifier comprises D20: .

[0044] In some embodiments, the splice modulator comprises D21: .

[0045] In some embodiments, the splice modulator comprises D22: .

[0046] In some embodiments, the splice modifier comprises D23: .

[0047] In some embodiments, the splice modulator comprises D24: .

[0048] In some embodiments, the splice modifier comprises D25: .

[0049] In some embodiments, the splice modifier comprises D26: .

[0050] In some embodiments, the splice modulator comprises D27: .

[0051] In some embodiments, the splice modifier comprises D28: .

[0052] In some embodiments, the splice modifier comprises D29: .

[0053] In some embodiments, the splice modifier comprises D30: .

[0054] In some embodiments, the splice modulator comprises D31: .

[0055] In some embodiments, the splice modifier comprises D32: .

[0056] In some embodiments, the splice modulator comprises D33: .

[0057] In some embodiments, the splice modulator comprises D34: .

[0058] In some embodiments, the splice modifier comprises D35: .

[0059] In some embodiments, the splice modulator comprises one of the pharmaceutical portions listed in Table 7. In some embodiments, the splice modulator comprises D1, D2, D3, D4, D4', D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20, D21, D22, D23, D24, D25, D26, D27, D28, D29, D30, D31, D32, D33, D34 and / or D35.

[0060] In some embodiments, a splice modulator is disclosed, and its use as a therapeutic agent, alone or as part of an ADC. In some embodiments, the splice modulator comprises D4, D4', D12, D15, D8, D9, D10, D13, D18, D19, D20, D21, D22, D25, or D33.

[0061] In some embodiments, the splice modulator comprises D4 and the linker comprises MC-Val-Cit-pABC. In some embodiments, the splice modulator comprises D4 and the linker comprises MC-β-glucuronide. In some embodiments, the splice modulator comprises D12 and the linker comprises MC-Val-Cit-pABC. In some embodiments, the splice modulator comprises D12 and the linker comprises MC-β-glucuronide. In some embodiments, the splice modulator comprises D15 and the linker comprises MC-Val-Ala-pAB.

[0062] In various embodiments, the linkers used in the ADCs disclosed herein are stable outside the cell, such that the ADC remains intact when present in extracellular conditions but can cleave upon internalization into cells, such as tumor or cancer cells. In some embodiments, the splicing regulator cleaves from the antibody or antigen-binding fragment when the ADC enters a cell that targets an antigen expressed by an antibody or antigen-binding fragment of the ADC. In some embodiments, the linker is a cleavable linker.

[0063] 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”).

[0064] In some embodiments, the linker includes a cleavable glucuronide moiety. In some embodiments, the cleavable glucuronide moiety can be cleaved by an enzyme. In some embodiments, the cleavable glucuronide moiety can be cleaved by a glucuronidase. In some embodiments, the cleavable glucuronide moiety can be cleaved by a β-glucuronidase.

[0065] 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-, where m is an integer from 1 to 10. In some embodiments, m is a unit of 2. In some other embodiments, the spacer unit or linker comprises an alkyl portion. In some embodiments, the alkyl portion comprises -(CH2)n-, where n is an integer from 1 to 10. In some embodiments, n is a unit of 2. In some embodiments, n is a unit of 5. In some embodiments, n is a unit of 6.

[0066] In some embodiments, the spacer unit is linked to the 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.

[0067] In some embodiments, the linker comprises a Mal-spacer unit and a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Val-Cit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Val-Ala. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Glu-Val-Cit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Ala-Ala-Asn. In some embodiments, the Mal-spacer unit comprises an alkyl moiety. In some embodiments, the Mal-spacer unit comprises a PEG moiety. In some embodiments, the Mal-spacer unit comprises maleic anhydride-hexyl (MC).

[0068] In some embodiments, the Mal-spacer unit links the antibody or antigen-binding fragment to a cleavable portion in the 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).

[0069] In some embodiments, the cleavable portion of the linker is directly bound to the splicing modulator, or a spacer unit connects the cleavable portion of the linker to the splicing modulator. In some embodiments, cleavage of the conjugate releases the splicing modulator from the antibody or antigen-binding fragment and the linker. In some embodiments, the spacer unit that connects the cleavable portion of the linker to the splicing modulator is self-degrading.

[0070] In some embodiments, the spacer unit connecting the cleavable portion of the linker to the splicing regulator comprises a p-aminobenzyloxycarbonyl group (pABC). In some embodiments, the pABC connects the cleavable portion of the linker to the splicing 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.

[0071] In some embodiments, the spacer unit connecting the cleavable portion of the linker to the splicing regulator comprises p-aminobenzyl (pAB). In some embodiments, pAB connects the cleavable portion of the linker to the splicing 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.

[0072] In various embodiments, the linker is a non-cleavable linker. In some embodiments, the splicing regulator in the ADC is released by the degradation of the antibody or antigen-binding fragment. In some embodiments, the linker remains covalently associated with at least one amino acid of the antibody and the drug when internalized and degraded within the target cell.

[0073] In some embodiments, the linker system comprises a non-disintegrable linker of 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-, where m is an integer from 1 to 10. In some embodiments, m is 2. In some other embodiments, the spacer unit or linker comprises an alkyl portion. In some embodiments, the alkyl portion comprises -(CH2)n- or -(CH2)n-O-(CH2)n, where 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.

[0074] In some embodiments, a spacer unit in an insoluble linker is linked to the antibody or antigen-binding fragment via a maleic diimidyl (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 diimidylhexyl (MC). In some embodiments, the linker or Mal-spacer unit comprises maleic diimidylhexyl (MC) and at least one additional spacer unit. In some embodiments, the linker or Mal-spacer unit comprises MC-(PEG)2. In some embodiments, the linker or Mal-spacer unit comprises MC-(PEG)2 and at least one additional spacer unit. In some embodiments, the linker or Mal-spacer unit comprises Mal-Hex. In some embodiments, the linker or Mal-spacer unit comprises Mal-Hex and at least one additional spacer unit. In some embodiments, the linker or Mal-spacer unit comprises Mal-Et. In some embodiments, the linker or Mal-spacer unit comprises Mal-Et and at least one additional spacer unit. In some embodiments, the linker or Mal-spacer unit comprises Mal-Et-O-Et. In some embodiments, the linker or Mal-spacer unit comprises Mal-Et-O-Et and at least one additional spacer unit. In some embodiments, the Mal-spacer unit links the antibody or antigen-binding fragment to the splicing regulator.

[0075] In various embodiments, the ADC complex can be represented by formula (I): Ab-(LD)p (I) Wherein Ab is an antibody or antigen-binding fragment thereof that targets proliferative cells or another tumor-associated target such as a cancer antigen (e.g., any of the antibody or binding domain sequences disclosed herein); D is any small molecule suitable for treating cancer (e.g., a splice modulator, such as any of the splice modulators disclosed herein); L is a linker that covalently links Ab to D (e.g., any of the linkers disclosed herein); and p is an integer from 1 to 15.

[0076] 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 neoplastic cells. In some embodiments, Ab is an antibody or binding domain sequence targeting CD138 and / or CD138-expressing neoplastic cells. In some embodiments, Ab is an antibody or binding domain sequence targeting EPHA2 and / or EPHA2-expressing neoplastic cells. In some embodiments, Ab is an antibody or binding domain sequence targeting another cancer antigen.

[0077] In some embodiments, D is a splice modulator. In some embodiments, D is selected from any of the splice modulators disclosed herein. In some embodiments, D is selected from the splice modulators D2, D1, D4, D8, D10, D11 (E7107), D20, D21, D22, D12, D25, D9, D18, D19, D13, D15, D14, D16, D17, D26, and D33, or any derivative thereof. In some embodiments, D is selected from the splice modulators D4, D12, D15, D8, D9, D10, D13, D18, D19, D20, D21, D22, D25, and D33, or any derivative thereof. In some embodiments, D is a splice modulator comprising D2 or any derivative thereof. In some embodiments, D is a splice modulator comprising D1 or any derivative thereof.

[0078] 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 comprises the following linkers: 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 of ADL1, ADL2, ADL5, ADL6, ADL7, ADL10, ADL12, ADL13, ADL14, ADL15, ADL21, ADL22, or ADL23. In some embodiments, L-series ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 connectors are used. In some embodiments, L-series ADL12, ADL14, or ADL15 connectors are used. In some embodiments, ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 connectors may also include one or more additional spacer sub-units. In some embodiments, L-series ADL1 connectors may include one or more additional spacer sub-units. In some embodiments, L-series ADL2 connectors may include one or more additional spacer sub-units. In some embodiments, L-series ADL5 connectors may include one or more additional spacer sub-units. In some embodiments, L-series ADL6 connectors may include one or more additional spacer sub-units. In some embodiments, the L-series ADL7 connector may include one or more additional spacer sub-units. In some embodiments, the L-series ADL12 connector may include one or more additional spacer sub-units. In some embodiments, the L-series ADL14 connector may include one or more additional spacer sub-units. In some embodiments, the L-series ADL15 connector may include one or more additional spacer sub-units. In various embodiments of the ADC described herein, p-series 1 to 10. In various embodiments, p-series 2 to 8. In various embodiments, p-series 4 to 8. In various embodiments, p-series 4. In some embodiments, p-series 8.

[0079] In some embodiments, the LD of formula (I) is ADL1-D1. In some embodiments, the LD of formula (I) is ADL6-D1. In some embodiments, the LD of formula (I) is ADL5-D2. In some embodiments, the LD of formula (I) is ADL1-D18. In some embodiments, the LD of formula (I) is ADL5-D19. In some embodiments, the LD of formula (I) is ADL14-D1. In some embodiments, the LD of formula (I) is ADL12-D1. In some embodiments, the LD of formula (I) is ADL15-D1. In some embodiments, the LD of formula (I) is ADL12-D20. In some embodiments, the LD of formula (I) is ADL10-D1. In some embodiments, the LD of formula (I) is ADL12-D2. In some embodiments, the LD of formula (I) is ADL15-D2. In some embodiments, the LD of formula (I) is ADL12-D21. In some embodiments, the LD of formula (I) is ADL6-D9. In some embodiments, the LD series of formula (I) is ADL1-D4. In some embodiments, the LD series of formula (I) is ADL1-D3. In some embodiments, the LD series of formula (I) is ADL1-D12. In some embodiments, the LD series of formula (I) is ADL1-D7. In some embodiments, the LD series of formula (I) is ADL1-D6. In some embodiments, the LD series of formula (I) is ADL1-D5. In some embodiments, the LD series of formula (I) is ADL22-D4. In some embodiments, the LD series of formula (I) is ADL5-D10. In some embodiments, the LD series of formula (I) is ADL5-D11. In some embodiments, the LD series of formula (I) is ADL1-D13. In some embodiments, the LD series of formula (I) is ADL1-D8. In some embodiments, the LD series of formula (I) is ADL1-D22. In some embodiments, the LD series of formula (I) is ADL5-D25. In some embodiments, the LD series of formula (I) is ADL12-D22. In some embodiments, the LD series of formula (I) is ADL5-D15. In some embodiments, the LD series of formula (I) is ADL1-D14. In some embodiments, the LD series of formula (I) is ADL5-D26. In some embodiments, the LD series of formula (I) is ADL1-D16. In some embodiments, the LD series of formula (I) is ADL5-D17. In some embodiments, the LD series of formula (I) is ADL1-D33. In some embodiments, the LD series of formula (I) is ADL1-D28. In some embodiments, the LD series of formula (I) is ADL1-D31. In some embodiments, the LD series of formula (I) is ADL1-D29. In some embodiments, the LD series of formula (I) is ADL1-D35.In some embodiments, the LD of formula (I) is ADL5-D32. In some embodiments, the LD of formula (I) is ADL5-D27. In some embodiments, the LD of formula (I) is ADL12-D35. In some embodiments, the LD of formula (I) is ADL12-D28. In some embodiments, the LD of formula (I) is ADL1-D23. In some embodiments, the LD of formula (I) is ADL1-D24.

[0080] In some embodiments, a set of ADCs is provided, thereby causing random binding, and the average 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 average 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 average p in the set is about 4. In some embodiments, a set of ADCs is provided, thereby causing random binding, and the average p in the set is about 8. This document provides compositions (e.g., pharmaceutical compositions) comprising multiple copies of any of the ADCs, wherein the average drug loading (average 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).

[0081] In some embodiments, the antibody or antigen-binding fragment (Ab) in the ADC targets vegetative cells derived from hematologic malignancies or solid tumors. In some embodiments, the antibody or antigen-binding fragment targets vegetative cells derived from hematologic malignancies. In some embodiments, the hematologic malignancy is selected from B-cell malignancies, leukemia (e.g., acute myeloid leukemia), lymphoma, and myeloma (e.g., multiple myeloma). In some embodiments, the hematologic malignancy is selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the antibody or antigen-binding fragment targets vegetative cells derived from solid tumors. 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 gland carcinoma, melanoma, colon 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, and prostate cancer.

[0082] In various embodiments, the antibody or antigen-binding fragment (Ab) in 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 neoplastic cells (i.e., the ADC targets HER2-expressing neoplastic cells). In some embodiments, the antibody or antigen-binding fragment in the ADC is an internalized anti-HER2 antibody or its internalized antigen-binding fragment.

[0083] In some embodiments, the anti-HER2 antibody or antigen-binding fragment includes three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) containing 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) containing 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 includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO:19 and a light chain variable region containing the amino acid sequence of SEQ ID NO:20. In some embodiments, the anti-HER2 antibody or antigen-binding fragment comprises a human IgG heavy chain constant region. In some embodiments, the anti-HER2 antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the anti-HER2 antibody or antigen-binding fragment comprises a human Igκ or λ light chain constant region. In some embodiments, the anti-HER2 antibody or antigen-binding fragment competitively binds to and / or binds to the same antigenic determinant as an antibody comprising the heavy chain variable domain of SEQ ID NO: 19 and the light chain variable domain of SEQ ID NO: 20.

[0084] In various embodiments, the antibody or antigen-binding fragment (Ab) in 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 neoplastic cells (i.e., the ADC targets CD138-expressing neoplastic cells). In some embodiments, the antibody or antigen-binding fragment in the ADC is an internalized anti-CD138 antibody or its internalized antigen-binding fragment.

[0085] In some embodiments, the anti-CD138 antibody or antigen-binding fragment includes three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) containing 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) containing 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 scaffold sequence. In some embodiments, the anti-CD138 antibody or antigen-binding fragment includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 21 and a light chain variable region containing 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 competitively binds to and / or binds to the same antigenic determinant as an antibody comprising the heavy chain variable domain of SEQ ID NO: 21 and the light chain variable domain of SEQ ID NO: 22.

[0086] In various embodiments, the antibody or antigen-binding fragment (Ab) in 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 neoplastic cells (i.e., the ADC targets EPHA2-expressing neoplastic cells). In some embodiments, the antibody or antigen-binding fragment in the ADC is an internalized anti-EPHA2 antibody or its internalized antigen-binding fragment.

[0087] In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment includes three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) containing 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) containing 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 includes 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 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 competitively binds to and / or binds to the same antigenic determinant as an antibody comprising the heavy chain variable domain of SEQ ID NO: 23 and the light chain variable domain of SEQ ID NO: 24.

[0088] In various embodiments, compounds comprising a linker-drug as defined by the following general formula are also provided herein: LD, where L = linker portion and D = drug portion (e.g., splice modulator drug portion). In various embodiments, the linker-drug (LD) compounds disclosed herein may be linked to antibody or antigen-binding fragments and / or adapted to the ADCs disclosed herein, such as the ADC of formula (I).

[0089] In various embodiments, the linker-drug (LD) compounds disclosed herein comprise a linker-drug structure according to formula (III). In various embodiments, the present invention provides a linker-drug (LD) compound of formula (III): or a medically acceptable salt thereof, wherein: R1 is selected from the following groups: non-existent, hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic, -OC(=O)-(C1-C6 alkyl) group, and -CD3; R2 either does not exist or is a connector; R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic, and -OC(=O)-(C1-C6 alkyl) groups; and R4, R5, and R8 are each independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl) group, -OC(=O)-(C1-C6 alkyl) group, and C1-C6 alkyl; R6 and R7 are each independently selected from hydrogen, -O-R17, -OC(=O)-R17, -OC(=O)-NR15R16, C1-C6 alkyl, -NR15R16, and linkers; R15 and R16 are each independently selected from hydrogen, R17, -C(=O)-R17, and -C(=O)-O-R17; R17 is selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, benzyl, and C3-C8 heterocyclic groups; and Z'' series is selected from... R1, R2, R3, R4, R5, R6, R7, R8, R15, R16, and R17 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl) group, -NR15 R16, C3-C8 cycloalkyl, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic group; At least one of R6 and R7 is hydrogen; and If R2 is a connector, then R6 and R7 are not connectors, and if R6 or R7 is a connector, then R2 does not exist.

[0090] In various other embodiments, the linker-drug (LD) compounds disclosed herein comprise a linker-drug structure according to formula (V). In various embodiments, the present invention provides a linker-drug (LD) compound of formula (V): or a medically acceptable salt thereof, wherein: R1 is selected from the following groups: non-existent, hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic, -OC(=O)-(C1-C6 alkyl) group, and -CD3; R2 either does not exist or is a connector; R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic, and -OC(=O)-(C1-C6 alkyl) groups; and R4, R5, and R8 are each independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl) group, -OC(=O)-(C1-C6 alkyl) group, and C1-C6 alkyl; R6 and R7 are each independently selected from hydrogen, -O-R17, -OC(=O)-R17, -OC(=O)-NR15R16, C1-C6 alkyl, -NR15R16, and linkers; R15 and R16 are each independently selected from hydrogen, R17, -C(=O)-R17, and -C(=O)-O-R17; and R17 is selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, benzyl, and C3-C8 heterocyclic groups; R1, R2, R3, R4, R5, R6, R7, R8, R15, R16, and R17 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl) group, -NR15 R16, C3-C8 cycloalkyl, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic group; At least one of R6 and R7 is hydrogen; and If R2 is a connector, then R6 and R7 are not connectors, and if R6 or R7 is a connector, then R2 does not exist.

[0091] In various other embodiments, the linker-drug (LD) compounds disclosed herein comprise a linker-drug structure according to formula (VII). In various embodiments, the present invention provides a linker-drug (LD) compound of formula (VII): or a medically acceptable salt thereof, wherein: R1 and R9 are each independently selected from the absence of, hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic, -OC(=O)-(C1-C6 alkyl) group, and -CD3; R2 either does not exist or is a connector; R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic and -OC(=O)-(C1-C6 alkyl) groups; R4, R5, and R8 are each independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl) group, -OC(=O)-(C1-C6 alkyl) group, and C1-C6 alkyl; R6 and R7 are each independently selected from hydrogen, -O-R17, -OC(=O)-R17, -OC(=O)-NR15R16, C1-C6 alkyl, -NR15R16, and linkers; R10 is selected from hydrogen, C1-C6 alkyl, -C(=O)-(C1-C6 alkyl) groups, and -CD3; R15 and R16 are each independently selected from hydrogen, R17, -C(=O)-R17, and -C(=O)-O-R17; R17 is selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, benzyl, and C3-C8 heterocyclic groups; and Series a: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R15, R16, and R17 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl) group, -NR15 R16, C3-C8 cycloalkyl, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic group; At least one of R6 and R7 is hydrogen; If R2 is a connector, then neither R6 nor R7 is a connector; and if either R6 or R7 is a connector, then R2 does not exist. R1 and R9 cannot both be non-existent.

[0092] In various other embodiments, the linker-drug (LD) compounds disclosed herein comprise a linker-drug structure according to formula (IX). In various embodiments, the present invention provides a linker-drug (LD) compound of formula (IX): or a medically acceptable salt thereof, wherein: R1 is selected from the following groups: non-existent, hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic, -OC(=O)-(C1-C6 alkyl) group, and -CD3; R2 series connector; R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic and -OC(=O)-(C1-C6 alkyl) groups; R4 is selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl) group, -OC(=O)-(C1-C6 alkyl) group, and C1-C6 alkyl; R10 is selected from 3 to 10-membered carbon rings and 3 to 10-membered heterocycles, each substituted by 0 to 3 Ra, wherein each Ra is independently selected from halogens, C1-C6 alkyl, -O-(C1-C6)alkyl, C1-C6 alkylalkoxy, C1-C6 alkylhydroxy, -S(=O)w-(4 to 7-membered heterocycles), 4 to 7-membered carbon rings and 4 to 7-membered heterocycles; R15 and R16 are each independently selected from hydrogen, R17, -C(=O)-R17, and -C(=O)-O-R17; and R17 is selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, benzyl, and C3-C8 heterocyclic groups; R1, R2, R3, R4, R10, R15, R16, and R17 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl) group, -NR15 R16, C3-C8 cycloalkyl, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic group; and Each Ra is independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, -NR15 R16, C1-C6 alkyl, -(C=O)-(C1-C6 alkyl) group, -(C=O)-(C1-C6 alkyl)-(C3-C10 heterocyclic) group, and C1-C6 alkylcarboxylic acid group, each of which is substituted by 0, 1, or 2 groups independently selected from the following groups: halogen, hydroxyl, -NR15 R16, and C1-C3 alkyl; and w is 0, 1, or 2.

[0093] Additionally, in various embodiments, this document provides the therapeutic use of the ADC complex and composition, for example, in treating neoplastic conditions, such as cancer. In some embodiments, the invention provides a method for treating neoplastic conditions, such as cancers, that target antigens expressed by an antibody or antigen-binding fragment in the ADC, the antigen being such as HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1.

[0094] In some embodiments, the present invention provides a method for treating an individual with or suspected of having a neoplastic condition, which is achieved by administering to the individual a therapeutically effective amount and / or regimen of any of the ADC or composition. 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 gland carcinoma, melanoma, colon 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, and prostate cancer.

[0095] In some embodiments, treatment with the 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, the individual has one or more vesicular cells that express the target antigen.

[0096] In some embodiments, the target antigen is HER2. In some embodiments, the one or more proliferative cell lines are derived from HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., serous endometrial cancer), or salivary gland cancer. In some embodiments, the individual is unresponsive or poorly responsive to treatment with (a) an anti-HER2 antibody administered alone and / or (b) a splice modulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with a splice modulator administered alone.

[0097] In some embodiments, the target antigen is CD138. In some embodiments, the one or more proliferative cell lines are derived from 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 splice modulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with a splice modulator administered alone.

[0098] In some embodiments, the target antigen is EPHA2. In some embodiments, the one or more proliferative cell lines are derived from EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon 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 splice modulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with a splice modulator administered alone.

[0099] In some embodiments, the target antigen is MSLN. In some embodiments, the one or more proliferative cell lines are derived from 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 splice modulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with a splice modulator administered alone.

[0100] In some embodiments, the target antigen is FOLH1. In some embodiments, the one or more proliferative cell lines are derived from 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 splice modulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with a splice modulator administered alone.

[0101] In some embodiments, the target antigen is CDH6. In some embodiments, the one or more proliferative cell lines are derived from 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 splice modulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with a splice modulator administered alone.

[0102] In some embodiments, the target antigen is CEACAM5. In some embodiments, the one or more proliferative cell lines are derived from 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 splice modulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with a splice modulator administered alone.

[0103] In some embodiments, the target antigen is CFC1B. In some embodiments, the one or more proliferative cell lines are derived from 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 splice modulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with a splice modulator administered alone.

[0104] In some embodiments, the target antigen is ENPP3. In some embodiments, the one or more proliferative cell lines are derived from 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 splice modulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with a splice modulator administered alone.

[0105] In some embodiments, the target antigen is FOLR1. In some embodiments, the one or more proliferative cell lines are derived from 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 splice modulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with a splice modulator administered alone.

[0106] In some embodiments, the target antigen is HAVCR1. In some embodiments, the one or more proliferative cell lines are derived from 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 splice modulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with a splice modulator administered alone.

[0107] In some embodiments, the target antigen is KIT. In some embodiments, the one or more proliferative cell lines are derived from 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 splice modulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with a splice modulator administered alone.

[0108] In some embodiments, the target antigen is MET. In some embodiments, the one or more proliferative cell lines are derived from 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 splice modulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with a splice modulator administered alone.

[0109] In some embodiments, the target antigen is MUC16. In some embodiments, the one or more proliferative cell lines are derived from 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 splice modulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with a splice modulator administered alone.

[0110] In some embodiments, the target antigen is SLC39A6. In some embodiments, the one or more proliferative cell lines are derived from 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 splice modulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with a splice modulator administered alone.

[0111] In some embodiments, the target antigen is SLC44A4. In some embodiments, the one or more proliferative cell lines are derived from SLC44A4 phenotypical 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 splice modulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with a splice modulator administered alone.

[0112] In some embodiments, the target antigen is STEAP1. In some embodiments, the one or more proliferative cell lines are derived from 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 splice modulator administered alone. In some embodiments, the individual is intolerant, unresponsive, or poorly responsive to treatment with a splice modulator administered alone.

[0113] 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 achieved by administering to the individual a therapeutically effective amount and / or regimen of any of the ADC or composition.

[0114] In some embodiments, treatment with the antibody-drug conjugate or composition induces bystander killing of neoplastic tumor cells that do not express the target antigen but 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.

[0115] In some embodiments, the target antigen is HER2. In some embodiments, the one or more proliferative cell lines are derived from HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., serous endometrial cancer), or salivary gland cancer. In some embodiments, the tumor is resistant to or difficult to treat with (a) an anti-HER2 antibody administered alone and / or (b) a splice modulator administered alone.

[0116] In some embodiments, the target antigen is CD138. In some embodiments, the one or more proliferative cell lines are derived from CD138-expressing multiple myeloma. In some embodiments, the tumor is resistant to or difficult to treat with (a) an anti-CD138 antibody administered alone and / or (b) a splice modulator administered alone.

[0117] In some embodiments, the target antigen is EPHA2. In some embodiments, the one or more proliferative cell lines are derived from EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer. In some embodiments, the tumor is resistant to or difficult to treat with (a) an anti-EPHA2 antibody administered alone and / or (b) a splice modulator administered alone.

[0118] In some embodiments, the target antigen is MSLN. In some embodiments, the one or more proliferative cell lines 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 to or difficult to treat with (a) an anti-MSLN antibody administered alone and / or (b) a splice modulator administered alone.

[0119] In some embodiments, the target antigen is FOLH1. In some embodiments, the one or more proliferative cell lines are derived from FOLH1-expressing prostate cancer. In some embodiments, the tumor is resistant to or difficult to treat with (a) an anti-FOLH1 antibody administered alone and / or (b) a splice modulator administered alone.

[0120] In some embodiments, the target antigen is CDH6. In some embodiments, the one or more proliferative cell lines are derived from CDH6-expressing renal cell carcinoma. In some embodiments, the tumor is resistant to or difficult to treat with (a) an anti-CDH6 antibody administered alone and / or (b) a splice modulator administered alone.

[0121] In some embodiments, the target antigen is CEACAM5. In some embodiments, the one or more proliferative cell lines are derived from CEACAM5-expressing colorectal cancer. In some embodiments, the tumor is resistant to or difficult to treat with (a) an anti-CEACAM5 antibody administered alone and / or (b) a splice modulator administered alone.

[0122] In some embodiments, the target antigen is CFC1B. In some embodiments, the one or more proliferative cell lines are derived from CFC1B-expressing pancreatic cancer. In some embodiments, the tumor is resistant to or difficult to treat with (a) an anti-CFC1B antibody administered alone and / or (b) a splice modulator administered alone.

[0123] In some embodiments, the target antigen is ENPP3. In some embodiments, the one or more proliferative cell lines are derived from ENPP3-expressing renal cell carcinoma. In some embodiments, the tumor is resistant to or difficult to treat with (a) an anti-ENPP3 antibody administered alone and / or (b) a splice modulator administered alone.

[0124] In some embodiments, the target antigen is FOLR1. In some embodiments, the one or more proliferative cell lines are derived from FOLR1-expressing ovarian cancer. In some embodiments, the tumor is resistant to or difficult to treat with (a) an anti-FOLR1 antibody administered alone and / or (b) a splice modulator administered alone.

[0125] In some embodiments, the target antigen is HAVCR1. In some embodiments, the one or more proliferative cell lines are derived from HAVCR1-expressing renal cell carcinoma or esophageal cancer. In some embodiments, the tumor is resistant to or difficult to treat with (a) an anti-HAVCR1 antibody administered alone and / or (b) a splice modulator administered alone.

[0126] In some embodiments, the target antigen is KIT. In some embodiments, the one or more proliferative cell lines are derived from KIT-expressing renal cell carcinoma. In some embodiments, the tumor is resistant to or difficult to treat with (a) anti-KIT antibodies administered alone and / or (b) splice modulators administered alone.

[0127] In some embodiments, the target antigen is MET. In some embodiments, the one or more proliferative cell lines are derived from MET-expressing renal cell carcinoma or esophageal cancer. In some embodiments, the tumor is resistant to or difficult to treat with (a) anti-MET antibodies administered alone and / or (b) splice modulators administered alone.

[0128] In some embodiments, the target antigen is MUC16. In some embodiments, the one or more proliferative cell lines are derived from MUC16-expressing ovarian cancer, cervical cancer, or breast cancer. In some embodiments, the tumor is resistant to or difficult to treat with (a) an anti-MUC16 antibody administered alone and / or (b) a splice modulator administered alone.

[0129] In some embodiments, the target antigen is SLC39A6. In some embodiments, the one or more proliferative cell lines are derived from SLC39A6-expressing breast or prostate cancer. In some embodiments, the tumor is resistant to or difficult to treat with (a) an anti-SLC39A6 antibody administered alone and / or (b) a splice modulator administered alone.

[0130] In some embodiments, the target antigen is SLC44A4. In some embodiments, the one or more proliferative cell lines are derived from SLC44A4 phenotypical prostate cancer. In some embodiments, the tumor is resistant to or difficult to treat with (a) an anti-SLC44A4 antibody administered alone and / or (b) a splice modulator administered alone.

[0131] In some embodiments, the target antigen is STEAP1. In some embodiments, the one or more proliferative cell lines are derived from STEAP1-expressing prostate cancer. In some embodiments, the tumor is resistant to or difficult to treat with (a) an anti-STEAP1 antibody administered alone and / or (b) a splice modulator administered alone.

[0132] In other embodiments, the present invention provides a method for determining whether an individual with or suspected of having a neoplastic condition will respond to treatment with any of the 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 section or a blood sample. In some embodiments, the blood sample is selected from blood, blood fractions, or cells obtained from the 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, the one or more neoplastic cells are derived from HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., serous endometrial cancer), or salivary gland carcinoma. In some embodiments, the target antigen is CD138. In some embodiments, the one or more neoplastic cells are derived from CD138-expressing multiple myeloma. In some embodiments, the target antigen is EPHA2. In some embodiments, the one or more proliferative cell lines are derived from EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer. In some embodiments, the target antigen is MSLN. In some embodiments, the one or more proliferative cell lines 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, the one or more proliferative cell lines are derived from FOLH1-expressing prostate cancer. In some embodiments, the target antigen is CDH6. In some embodiments, the one or more proliferative cell lines are derived from CDH6-expressing renal cell carcinoma. In some embodiments, the target antigen is CEACAM5. In some embodiments, the one or more proliferative cell lines are derived from CEACAM5-expressing colorectal cancer. In some embodiments, the target antigen is CFC1B. In some embodiments, the one or more proliferative cell lines are derived from CFC1B-expressing pancreatic cancer. In some embodiments, the target antigen is ENPP3. In some embodiments, the one or more proliferative cell lines are derived from ENPP3-expressing renal cell carcinoma. In some embodiments, the target antigen is FOLR1. In some embodiments, the one or more proliferative cell lines are derived from FOLR1-expressing ovarian cancer. In some embodiments, the target antigen is HAVCR1. In some embodiments, the one or more proliferative cell lines are derived from HAVCR1-expressing renal cell carcinoma or esophageal cancer. In some embodiments, the target antigen is KIT. In some embodiments, the one or more proliferative cell lines are derived from KIT-expressing renal cell carcinoma. In some embodiments, the target antigen is MET. In some embodiments, the one or more proliferative cell lines are derived from MET-expressing renal cell carcinoma or esophageal cancer. In some embodiments, the target antigen is MUC16.In some embodiments, the one or more proliferative cell lines are derived from MUC16-expressing ovarian cancer, cervical cancer, or breast cancer. In some embodiments, the target antigen is SLC39A6. In some embodiments, the one or more proliferative cell lines are derived from SLC39A6-expressing breast cancer or prostate cancer. In some embodiments, the target antigen is SLC44A4. In some embodiments, the one or more proliferative cell lines are derived from SLC44A4-expressing prostate cancer. In some embodiments, the target antigen is STEAP1. In some embodiments, the one or more proliferative cell lines are derived from STEAP1-expressing prostate cancer.

[0133] In various embodiments, this document further provides pharmaceutical compositions comprising an ADC and a pharmaceutically acceptable diluent, carrier, and / or excipient. Methods for manufacturing the said ADC complex and composition are also disclosed. Simple Explanation of the Diagram

[0134] [picture] [1] Dose-response of illustrative payload compounds in competitive binding assays. Nuclear extracts from 293F cells overexpressing flag-labeled wild-type SF3B1 were immunoprecipitated with anti-SF3B1 antibody and a scintillation proximity assay (SPA) bead mixture. Binding reactions contained the antibody-bead mixture and increments in concentration of the compound, which were then competitive with a 3H-labeled praldene lactone B (PB) probe. The y-axis represents the percentage change in specific binding (response %) relative to the DMSO control (0%). Data are presented as mean ± standard deviation (SD).

[0135] [picture] [2] Splicing regulation by an exemplary payload compound is shown in an in vivo splicing assay. Nuclear extracts from HeLa S3 cells were incubated with Ad2.2 pre-mRNA and increasing concentrations of the compound, and splicing regulation was quantified by RT-PCR. The Ad2.2 sequence was derived from an adenovirus Ad2 pre-mRNA receptor with modifications around the branching point sequence. The y-axis represents the percentage change in splicing (response %) relative to the DMSO control (0%). Data are presented as mean ± SD.

[0136] [picture] [3] Viability dose-response of an exemplary loaded compound in HER2-amplified breast cancer cells (HCC1954). Cells were incubated with the compound for 144 hours (6 days) and viability was read in CellTiter-Glo® reagent. Data are presented as mean ± SD.

[0137] [picture] [4] Results of cell binding analysis are shown. The binding of exemplary HER2-ADC to JIMT1 cells was assessed by flow cytometry. Mean fluorescence intensity was measured to determine the binding of the conjugate, followed by the binding of the PE-labeled secondary antibody. Data are presented as mean ± SD.

[0138] [picture] [5A] Demonstrates the activity-dose response of an exemplary HER2-ADC in HER2-amplified breast cancer cells (HCC1954). [picture] [5B] Demonstrates the dose-response activity of an exemplary HER2-ADC in HER2-amplified gastric cancer cells (N87). [picture] [5C] Viability dose-response of an exemplary HER2-ADC in HER2-amplified breast cancer cells (SKBR3). Cells were incubated with the conjugate for 144 hours (6 days) and viability was read using CellTiter-Glo® reagent. Data are presented as mean ± SD.

[0139] [picture] [6] Viability dose-response of an exemplary HER2-ADC in non-HER2-expressing breast cancer cells (MCF7). Cells were incubated with the conjugate for 144 hours (6 days) and viability was read in CellTiter-Glo® reagent. Data are presented as mean ± SD.

[0140] [picture] [7] Results of SLC25A19 splicing analysis in HER2-amplified breast cancer cells (HCC1954). Cells were incubated with the conjugate for 24 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%). Data are expressed as mean ± SD.

[0141] [picture] [8] Results of the bystander kill analysis are shown. H1568 cells (target-positive) or H1568 cells (target-negative) that overexpressed HER2 on luciferase were treated with a demonstrative HER2-ADC, and these cell lines were plated alone or co-cultured together for 144 hours (6 days). The discs were read using OneGlo® reagents. The y-axis represents the percentage of response relative to the PBS control (100%). Data are presented as mean ± SD.

[0142] [picture] [9] This study demonstrates tumor growth kinetics in CB17-SCID mice (6–10 mice / group) implanted with HCC1954 treated with a single intravenous dose of an exemplary HER2-ADC or a corresponding dose-matched payload. Tumor volume was measured twice weekly after treatment. Data are presented as mean ± standard error of mean (SEM).

[0143] [picture]

[10] Viability dose-response of an exemplary CD138-ADC in a CD138-expressing multiple myeloma cell line. MOLP8 cells were incubated with the conjugate for 144 hours (6 days) and viability was read in CellTiter-Glo® reagent. Data are presented as mean ± SD.

[0144] [picture]

[11] Viability dose-response of exemplary EPH2A-ADC in EPHA2 phenotypical prostate cancer cell lines. PC3 cells were incubated with the conjugate for 144 hours (6 days) and viability was read in CellTiter-Glo® reagent. Data are presented as mean ± SD.

[0145] [picture] [12A] and [picture] [12B] Shows the results of an in vitro stability analysis of an exemplary anti-HER2 ADC, AB185-ADL1-D1. The y-axis represents the total antibody ( [picture] [12A]) and the combined (complete) payload ( [picture] [12B] ) concentration; the x-axis represents time measured in hours at 37°C.

[0146] [picture]

[13] Plasma concentration of an exemplary anti-HER2 ADC, AB185-ADL1-D1, following a single intravenous dose in CD17-SCID mice with N87 tumors.

[0147] [picture]

[14] A schematic diagram showing an exemplary RNA sequencing and ligandome experiment.

[0148] [picture]

[15] A schematic diagram showing an exemplary T-cell activation experiment.

[0149] [picture]

[16] Results of FACS analysis are shown. Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) and induced to differentiate into dendritic cells (DCs) by culturing in a cytokine mixture. FACS was performed to verify the maturation of DCs derived from monocytes.

[0150] [picture] [17A] - [D] Displays the results of the ELISpot analysis. [picture] [17A] The ELISpot disc is shown, indicating that neoantigen 1 induces CD8+ T cell activation. CD8+ T cell stimulation is monitored by IFNγ secretion. [picture] [17B] Displaying neoantigen 1 on an ELISpot disc ( [picture] Quantification of IFNγ spots (number of spots) in [17A] ). [picture] [17C] The ELISpot disc is shown, indicating that neoantigen 3 induces CD8+ T cell activation. CD8+ T cell stimulation is monitored by IFNγ secretion. [picture] [17D] Displaying neoantigen 3 ELISpot disc ( [picture] Quantitative analysis (fold change) of IFNγ spots in [17C] ).

[0151] [picture]

[18] A plot showing the splicing power (IC50 qPCR) and cell power (GI50 CTG) of comparative exemplary anti-HER2 ADCs in HCC1954 breast cancer cells. The values ​​shown are ranked according to cell lethality and shaded according to the depth of the selective splicing reaction.

[0152] [picture]

[19] A plot showing the splicing power (IC50 qPCR) and cell power (GI50 CTG) of comparative exemplary anti-HER2 ADCs in N87 gastric cancer cells. The values ​​shown are ranked according to cell lethality and shaded according to the depth of the selective splicing response.

[0153] [picture]

[20] A plot showing the efficacy and lethality (in HCC1954 breast cancer cells) of a comparative example of an anti-HER2 ADC versus the stability and permeability of the corresponding payload. The values ​​shown are ranked according to payload stability and shaded according to payload permeability.

[0154] [picture]

[21] Tumor growth kinetics were shown in CB17-SCID mice (N=8 mice / group) implanted with N87 after two cycles of intravenous treatment with a mediator or 10 mg / kg trastuzumab, TDM1 or exemplary HER2-ADC Q7D. Data are presented as mean ± standard error of mean (SEM) (mm3).

[0155] [picture]

[22] Changes in body weight of CB17-SCID mice (N=8 mice / group) implanted with N87 were shown in each group after two cycles of intravenous treatment with a catalytic agent or 10 mg / kg trastuzumab, TDM1 or exemplary HER2-ADC Q7D. Data are presented as mean ± standard error of mean (SEM) (%).

[0156] [picture]

[23] Tumor growth kinetics (left panel) and body weight changes (right panel) in CB17-SCID mice (N=8 mice / group) implanted with N87 after two cycles of intravenous treatment with a catalyst or 10 mg / kg trastuzumab, TDM1, or the exemplary HER2-ADC Q7D. Data are presented as mean ± SEM (tumor volume, mm3) or mean ± SEM (body weight, %).

[0157] [picture] [24A] - [24D] Demonstrates the pharmacodynamic (PD) regulation of mRNA binding sites in N87-implanted CB17-SCID mice treated intravenously for two cycles with a mediator or 10 mg / kg trastuzumab, TDM1, or an exemplary HER2-ADC Q7D. RT-qPCR was used to monitor FBXW5 (mature mRNA transcript) and the results were displayed on [data / images]. [picture] [24A] and [picture] [24C] RT-qPCR monitoring of TAOK1 (new junctional transcript) and its manifestation in [picture] [24B] and [picture] [24D] In. Within 48 hours ( [picture] [24A] and [picture] [24B]) or at the specified time ( [picture] [24C] and [picture] Animals were collected at [24D] (N=4 animals / group). Tumors were isolated for RNA extraction and RT-qPCR.

[0158] [picture]

[25] A schematic diagram showing an example of target indication analysis. Implementation

[0159] This invention claims priority to U.S. Provisional Patent Application No. 62 / 679,672, filed June 1, 2018; U.S. Provisional Patent Application No. 62 / 679,631, filed June 1, 2018; and U.S. Provisional Patent Application No. 62 / 779,324, filed December 13, 2018. All of the foregoing applications are incorporated herein by reference in their entirety.

[0160] The disclosed compositions and methods can be more readily understood by referring to the following detailed description in conjunction with the accompanying drawings, which form part of this invention.

[0161] 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 also 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 also apply to the composition.

[0162] When a range of values ​​is indicated, it includes embodiments using any particular value within that range. Additionally, references to values ​​stated as a range include every value within that range. All ranges include their endpoints and are composable. When a value is indicated 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 the specific context in which it is used is otherwise specified, the use of "or" means "and / or." All references cited herein are incorporated by way of reference for any purpose. In the event of any conflict between the references and this specification, this specification shall prevail.

[0163] It should be understood that certain features of the disclosed compositions and methods described herein in the case of individual embodiments for clarity may also be provided in combination of individual embodiments. Conversely, various features of the disclosed compositions and methods described herein in the case of individual embodiments for brevity may also be provided individually or in any sub-combination. [definition] []

[0164] Throughout this specification and the claims, various terms related to the various aspects of this specification are used. Unless otherwise indicated, such terms shall have their ordinary meaning in the art. Other specifically defined terms shall be interpreted in accordance with the definitions provided herein.

[0165] Unless the context clearly specifies otherwise, as used herein, the singular forms "a / an" and "the" include the plural forms.

[0166] Those skilled in the art will readily recognize from the teachings contained herein that, in the context of numerical values ​​and ranges, the terms "about" or "approximately" mean an approximate value or range, or a value or range close to said value or range, such that the embodiment can be performed as intended, such as having the desired amount of nucleic acid or polypeptide in the reaction mixture. In some embodiments, about means a numerical amount ±10%.

[0167] The terms "antibody-drug conjugate," "antibody conjugate," "conjugate," "immunoconjugate," and "ADC" are used interchangeably and refer to a conjugate linked to one or more antibody or antigen-binding fragments and one or more therapeutic compounds (e.g., splice modulators), defined by the following general formula: Ab-(LD)p (Formula I), where Ab = antibody or antigen-binding fragment, L = linker portion, D = drug portion (e.g., splice modulator drug portion), and p = number of drug portions for each antibody or antigen-binding fragment. ADCs containing splice modulator drug portions may also be more specifically referred to herein as "antibody loaded with splice modulator" or "SMLA." In ADCs containing splice modulator drug portions, "p" refers to the number of splice modulator compounds 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 therapeutic compound. In some embodiments, the linker L may include a cleavable portion that can be linked to one or both of the antibody or antigen-binding fragment and the therapeutic compound via spacer units. In some embodiments, when a spacer unit links a cleavable portion to a therapeutic compound, it is a self-degrading spacer unit. In other embodiments, the linker L does not include a cleavable portion and is a non-cleavable linker. In some embodiments, the linker L may include at least one spacer unit that can be directly linked to the antibody or antigen-binding fragment and the therapeutic compound. Illustrative cleavable and non-cleavable linkers are described and illustrated herein.

[0168] The term "antibody" is used in its broadest sense, referring to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or a 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 and light chain variable domain each contain three complementarity-determining regions (CDR1, CDR2, and CDR3) arranged from the N-terminus to the C-terminus within four framework regions (FR1, FR2, FR3, and FR4): FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. "Antibody" can be naturally occurring or artificial, such as a monoclonal antibody manufactured using conventional fusion tumor technology. The term "antibody" includes full-length monoclonal antibodies and full-length polyclonal antibodies, as well as antibody fragments such as Fab, Fab', F(ab')2, Fv, and single-chain antibodies. Antibodies can be any of the five major 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 into cells expressing the target antigen).

[0169] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that, apart from a possible small number of naturally occurring mutations, the individual antibody systems constituting that population are identical. Monoclonal antibodies exhibit high specificity against a single antigenic determinant. In contrast, conventional (multiclonal) antibody formulations typically comprise multiple antibodies targeting (or specific to) different antigenic determinants. The modifier "monoclonal" indicates that the antibody is derived from a substantially homogeneous population of antibodies and should not be construed as requiring any special method to manufacture the antibody. For example, the monoclonal antibody intended for use according to the present invention may be prepared 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.

[0170] The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody species or subclass, while the remaining portion of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody species or subclass; and fragments of such antibodies, provided that they specifically bind to the target antigen and / or exhibit the desired biological activity.

[0171] 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.

[0172] As used herein, the term "chimeric antibody" refers to an antibody whose amino acid sequence of an immunoglobulin molecule is derived from two or more species. In some cases, the variable regions of the heavy and light chains correspond to the variable regions of antibodies 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.

[0173] 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. Such antibody systems contain chimeric antibodies derived from minimal sequences of non-human immunoglobulins. Generally, humanized antibodies will contain substantially all of at least one and typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to such regions of the non-human immunoglobulin and all or substantially all of the framework (FR) regions correspond to such regions of the human immunoglobulin sequence. Humanized antibodies may also, where applicable, contain immunoglobulin constant regions (Fc), typically at least a portion of the human immunoglobulin constant region. Humanized antibodies can be further modified by substitution of residues within and / or replaced non-human residues in the Fv framework region to improve and optimize antibody specificity, affinity, and / or activity.

[0174] 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 maintain 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, STEAP1). Antigen-binding fragments may also maintain the ability to be internalized into antigen-expressing cells. In some embodiments, antigen-binding fragments also maintain 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, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, a bivalent fragment comprising two Fab fragments linked 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 the antibody; (v) dAb fragments containing a single variable domain, such as the 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 independent genes, they can be used recombinant methods by conjugating synthetic linkers that enable them to be produced 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. Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" or "antigen-binding portion" of antibody, and exemplary types of binding fragments known in this technique that can be internalized into the cell upon binding are (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.Bivalent bispecific antibodies, where the VH and VL domains are represented on a single polypeptide chain, but with a linker too short to allow pairing between the two domains on the same chain. This forces these domains to pair with complementary domains on another 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 these 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.

[0175] As used herein with respect to antibody or antigen-binding fragments, "internalization" means that when an antibody or antigen-binding fragment binds to a cell, it is absorbed across 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 system can absorb antibodies that have entered 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.

[0176] As used herein with respect to antibody or antigen-binding fragments, “non-internalized” means that the antibody or antigen-binding fragment remains on the cell surface when it binds to a cell. In some embodiments, the antibody or antigen-binding fragments used in the ADCs disclosed herein target cell surface antigens and are non-internalized antibodies or non-internalized 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, the non-internalized antibody or antigen-binding fragment binds to a non-internalized receptor or other cell surface antigen. Exemplary non-internalized cell surface antigens include (but are not limited to) CA125 and CEA, and antibodies that bind to non-internalized 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).

[0177] 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 from 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 can be manufactured recombinantly or synthetically.

[0178] The term "anti-HER2 antibody" or "antibody bound to HER2" refers to any form of antibody or fragment thereof that binds, for example, specifically to HER2, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, as long as they bind, for example, specifically to HER2. U.S. Patent No. 5,821,337 provides exemplary HER2-binding sequences, including exemplary anti-HER2 antibody sequences, and that application is incorporated herein by reference. In some embodiments, the anti-HER2 antibody or internalizing antibody fragment used in the ADCs disclosed herein is an internalizing antibody or 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.

[0179] 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 processed by cells. 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 can be produced recombinantly or synthetically.

[0180] The term "anti-CD138 antibody" or "antibody bound to CD138" refers to any form of antibody or fragment thereof that binds, for example, specifically to CD138, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, as long as they bind, for example, specifically to CD138. In some embodiments, the anti-CD138 anti-systemic internalizing antibody or internalizing antibody fragment used in the ADCs disclosed herein. B-B4 (Tassone et al. (2004) Blood 104:3688-96) is an exemplary anti-human CD138 antibody.

[0181] 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 can be produced recombinantly or synthetically.

[0182] The term "anti-EPHA2 antibody" or "antibody bound to EPHA2" refers to any form of antibody or fragment thereof that binds, for example, specifically to EPHA2, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, as long as they bind, for example, specifically 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 anti-internal antibody or internalizing antibody fragment used in the ADCs disclosed herein. 1C1 (WO 2007 / 030642; Jackson et al. (2008) Cancer Res. 68(22): 9367-74) is an exemplary anti-human EPHA2 antibody.

[0183] 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: 43) and any form of human MSLN that can be obtained from cellular processing. The term also encompasses functional variants or fragments of human MSLN, including (but not limited to) splice variants, paired gene variants, and isoforms that retain one or more of the biological functions of human MSLN (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 can be manufactured recombinantly or synthetically.

[0184] The term "anti-MSLN antibody" or "antibody bound to MSLN" refers to any form of antibody or fragment thereof that binds, for example, specifically to MSLN, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, as long as they bind, for example, specifically 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 anti-internalizing antibody or internalizing antibody fragment used in the ADCs disclosed herein. 11-25, IC14-30, IC7-4, IC17-35, and 2-9 are exemplary anti-human MSLN antibodies.

[0185] 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: 44) and any form of human FOLH1 that can be processed by cells. 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 can be manufactured recombinantly or synthetically.

[0186] The term "anti-FOLH1 antibody" or "antibody bound to FOLH1" refers to any form of antibody or fragment thereof that binds, for example, specifically to FOLH1, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, as long as they bind, for example, specifically to FOLH1. WO 2019 / 012260 and WO 2017 / 212250 provide exemplary FOLH1 binding sequences, including exemplary anti-FOLH1 antibody sequences, and these are incorporated herein by reference. In some embodiments, the anti-FOLH1 anti-systemic internalizing antibody or internalizing antibody fragment used in the ADCs disclosed herein. J591 (deimmunized type) is an exemplary anti-human FOLH1 antibody.

[0187] 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: 45) 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 can be produced recombinantly or synthetically.

[0188] The term "anti-CDH6 antibody" or "antibody bound to CDH6" refers to any form of antibody or fragment thereof that binds, for example, specifically to CDH6, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, as long as they bind, for example, specifically 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 anti-systemic internalizing antibody or internalizing antibody fragment used in the ADCs disclosed herein.

[0189] 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: 46) and any form of human CEACAM5 that can be processed by cells. 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 can be manufactured recombinantly or synthetically.

[0190] The term "anti-CEACAM5 antibody" or "antibody bound to CEACAM5" refers to any form of antibody or fragment thereof that binds, for example, specifically to CEACAM5, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, as long as they bind, for example, specifically 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 anti-systemic internalizing antibody or internalizing antibody fragment used in the ADCs disclosed herein. hMN14 is an exemplary anti-human CEACAM5 antibody.

[0191] As used herein, the term "cryptic family protein 1B" or "CFC1B" refers to any naturally occurring form of human CFC1B. This term encompasses full-length CFC1B (e.g., UniProt reference sequence: P0CG36; SEQ ID NO: 47) and any form of human CFC1B that can be processed by cells. 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 can be manufactured recombinantly or synthetically.

[0192] The terms "anti-CFC1B antibody" or "antibody bound to CFC1B" refer to any form of antibody or fragment thereof that binds, for example, specifically to CFC1B, and encompass monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, as long as they bind, for example, specifically 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 anti-systemic internalizing antibody or internalizing antibody fragment used in the ADCs disclosed herein.

[0193] As used herein, the term "exonucleotide pyrophosphatase / phosphodiesterase family member 3" or "ENPP3" refers to any naturally occurring form of human ENPP3. This term encompasses full-length ENPP3 (e.g., UniProt reference sequence: O14638; SEQ ID NO: 48) 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 can be produced recombinantly or synthetically.

[0194] The term "anti-ENPP3 antibody" or "antibody bound to ENPP3" refers to any form of antibody or fragment thereof that binds, for example, specifically to ENPP3, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, as long as they bind, for example, specifically 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 anti-systemic internalizing antibody or internalizing antibody fragment used in the ADCs disclosed herein.

[0195] 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: 49) and any form of human FOLR1 that can be processed by cells. 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 can be manufactured recombinantly or synthetically.

[0196] The term "anti-FOLR1 antibody" or "antibody bound to FOLR1" refers to any form of antibody or fragment thereof that binds, for example, specifically to FOLR1, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, as long as they bind, for example, specifically to FOLR1. WO 2005 / 080431 and Coney et al. ((1991) Cancer Res. 51(22):6125-32) provide exemplary FOLR1 binding sequences, including exemplary anti-FOLR1 antibody sequences, and these are incorporated herein by reference. In some embodiments, the anti-FOLR1 anti-systemic internalizing antibody or internalizing antibody fragment used in the ADCs disclosed herein are examples of anti-human FOLR1 antibodies. Farletuzumab and MOv19 are exemplary anti-human FOLR1 antibodies.

[0197] 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: 50) and any form of human HAVCR1 that can be processed by cells. 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 can be manufactured recombinantly or synthetically.

[0198] The term "anti-HAVCR1 antibody" or "antibody bound to HAVCR1" refers to any form of antibody or fragment thereof that binds, for example, specifically to HAVCR1, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, as long as they bind, for example, specifically to HAVCR1. Thomas et al. ((2016) Mol Cancer Ther. 15(12):2946-54) provided exemplary HAVCR1 binding sequences, including exemplary anti-HAVCR1 antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-HAVCR1 anti-systemic internalizing antibody or internalizing antibody fragment used in the ADCs disclosed herein.

[0199] 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: 51) and any form of human KIT that can be processed by cells. 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 can be manufactured recombinantly or synthetically.

[0200] The term "anti-KIT antibody" or "antibody bound to KIT" refers to any form of antibody or fragment thereof that binds, for example, specifically to KIT, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, as long as they bind, for example, specifically to KIT. Shi et al. ((2016) Proc Natl Acad Sci USA 113(33):E4784-93) and Abrams et al. ((2018) Clin Cancer Res. 24(17):4297-308) provide exemplary KIT binding sequences, including exemplary anti-KIT antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-KIT antibody or internalizing antibody fragment used in the ADCs disclosed herein is an anti-KIT antibody.

[0201] 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: 52) and any form of human MET that can be processed by cells. 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 can be manufactured recombinantly or synthetically.

[0202] The term "anti-MET antibody" or "antibody bound to MET" refers to any form of antibody or fragment thereof that binds, for example, specifically to MET, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, as long as they bind, for example, specifically 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 anti-systemic internalizing antibody or internalizing antibody fragment used in the ADCs disclosed herein.

[0203] As used herein, the terms "mucin-16" or "MUC16" refer to any naturally occurring form of human MUC16. This term encompasses full-length MUC16 (e.g., UniProt reference sequence: Q8WXI7; SEQ ID NO: 53) 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 the wild-type protein, it encompasses variants and fragments). MUC16 can be isolated from humans or can be manufactured recombinantly or synthetically.

[0204] The term "anti-MUC16 antibody" or "antibody bound to MUC16" refers to any form of antibody or fragment thereof that binds, for example, specifically to MUC16, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, as long as they bind, for example, specifically to MUC16. Liu et al. ((2016) Ann Oncol. 27(11):2124-30) provided exemplary MUC16 binding sequences, including exemplary anti-MUC16 antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-MUC16 anti-systemic internalizing antibody or internalizing antibody fragment used in the ADCs disclosed herein.

[0205] 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: 54) 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 the wild-type protein, it encompasses variants and fragments). SLC39A6 can be isolated from humans or can be manufactured recombinantly or synthetically.

[0206] The terms "anti-SLC39A6 antibody" or "antibody bound to SLC39A6" refer to any form of antibody or fragment thereof that binds, for example, specifically to SLC39A6, and encompass monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, as long as they bind, for example, specifically 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 anti-systemic internalizing antibody or internalizing antibody fragment used in the ADCs disclosed herein.

[0207] 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: 55) 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 can be manufactured recombinantly or synthetically.

[0208] The terms "anti-SLC44A4 antibody" or "antibody bound to SLC44A4" refer to any form of antibody or fragment thereof that binds, for example, specifically to SLC44A4, and encompass monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, as long as they bind, for example, specifically to SLC44A4. Mattie et al. ((2016) Mol Cancer Ther. 15(11):2679-87) provided exemplary SLC44A4 binding sequences, including exemplary anti-SLC44A4 antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-SLC44A4 anti-systemic internalizing antibody or internalizing antibody fragment used in the ADCs disclosed herein.

[0209] 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: 56) and any form of human STEAP1 that can be processed by cells. 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 can be produced recombinantly or synthetically.

[0210] The term "anti-STEAP1 antibody" or "antibody bound to STEAP1" refers to any form of antibody or fragment thereof that binds, for example, specifically to STEAP1, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), multiclonal antibodies, and biologically functional antibody fragments, as long as they bind, for example, specifically 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 anti-systemic internalizing antibody or internalizing antibody fragment used in the ADCs disclosed herein.

[0211] As used herein, the terms "specific," "specifically binds," and "binds specifically" refer to the binding reaction between an antibody or antigen-binding fragment (e.g., an anti-HER2 antibody) and a target antigen (e.g., HER2) in a heterogeneous population of proteins and other biological products. The binding specificity of an antibody can be tested by comparing its binding to an appropriate antigen with its binding to unrelated antigens or mixtures of antigens under a given set of conditions. An antibody is considered specific if its affinity for the appropriate antigen is at least 2, 5, 7, and preferably 10 or higher than its affinity for 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 does not bind to other antigens (or exhibits minimal binding to them). 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 between 1 pM and 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.

[0212] The term "antigenic determinant" refers to the 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 juxtaposed by the tertiary folding of the polypeptide. Antibody-bound antigenic determinants can be identified using any antigenic determinant localization technique known in this art, including X-ray crystallography, by direct observation of the antigen-antibody complex, and by monitoring the binding of antibody to fragments or mutant variants of the antigen, or by 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, restriction 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).

[0213] Competitive binding and antigenic determinant grouping can also be used to identify antibodies sharing consistent 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 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 percentages, or any percentage between thereof), and / or vice versa. In some embodiments, competitive binding may be caused by shared or similar (e.g., partially overlapping) antigenic determinants, or by steric hindrance when an antibody or binding protein binds to a neighboring antigenic determinant (see, for example, Tzartos, Methods in Molecular Biology (Morris ed. (1998) Vol. 66, pp. 55-66)). In some embodiments, competitive binding can be used to sort groups of binding proteins that share similar antigenic determinants. For example, competitively binding proteins may be “grouped” into groups of binding proteins with overlapping or neighboring antigenic determinants, while non-competitive binding proteins may be grouped into groups of independent binding proteins without overlapping or neighboring antigenic determinants.

[0214] The term "kon" or "ka" refers to the association rate constant of antibody-antigen association to form antibody / antigen complexes. This rate can be determined using standard analyses such as surface plasma resonance, biolayer interferometry, or ELISA.

[0215] 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 interference, or ELISA.

[0216] 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 interference, or ELISA.

[0217] The terms "p" or "drug load," "drug:antibody ratio," "drug to antibody ratio," or "DAR" refer to the number of drug portions of each antibody or antigen-binding fragment, i.e., the drug load, or the number of -LD portions of each antibody or antigen-binding fragment (Ab) in the ADC of formula (I). In an ADC containing splice modulator drug portions, "p" refers to the number of splice modulator compounds linked to the antibody or antigen-binding fragment. For example, if two splice modulator compounds (e.g., two compounds each having a D1 structure) are linked to the antibody or antigen-binding fragment, then p = 2. In a composition containing multiple copies of the ADC of formula (I), "average p" refers to the average number of -LD portions of each antibody or antigen-binding fragment, also known as "average drug load."

[0218] "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 splice modulator pharmaceutical moiety, to another moiety such as an antibody or antigen-binding fragment. Linkers are readily cleaved or substantially resistant to acid-induced cleavage, peptidase-induced cleavage, light-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, under conditions that preserve the activity of the compound or antibody.

[0219] The term "reagent" as used herein refers to a compound, a mixture of compounds, a biological macromolecule, or an extract made from biological material. The terms "therapeutic agent" or "drug" refer to a reagent capable of modulating biological processes and / or possessing biological activity. The splice modulator compounds described herein are exemplary therapeutic agents.

[0220] 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 splicing 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 induces cell death primarily by interfering with the expression and / or function of cells.

[0221] As used herein, the terms "splicing modulator," "splicing body modulator," or "splice modulator" refer to compounds that exhibit anticancer activity through interaction with components of the spliceosome. In some embodiments, splicing modulators alter the rate or form of splicing in target cells. Splicing modulators acting as inhibitors, for example, can reduce uncontrolled cell proliferation. In some embodiments, splicing modulators may act by binding to the SF3b spliceosome complex. Such modulators may be natural or synthetic compounds. Non-limiting examples of splicing modulators and classes of such modulators include pralidoxetine (e.g., pralidoxetine D or pralidoxetine B), pralidoxetine derivatives (e.g., pralidoxetine D or pralidoxetine B derivatives), herboxidiene, herboxidiene derivatives, spliceostatin, spliceostatin derivatives, sudemycin, or sudemycin derivatives. As used herein, when referring to splice modulators or analogues, the terms "derivative" and "analogue" mean any such compound that retains substantially the same, similar, or enhanced biological function or activity as the original compound, but has a modified chemical or biological structure. In some embodiments, the splice modulator comprises prasadiene lactone or a prasadiene lactone derivative.

[0222] As used herein, "pradoadienolide derivatives" refers to compounds whose structure is related to members of the natural product family called pradoadienolides and retain one or more of the biological functions of the starting compound. Pradoadienolides were first identified in the bacterium *Streptomyces platensis* (Mizui et al. (2004) J Antibiot. 57:188-96) as having potent cytotoxicity and causing cell cycle arrest at the G1 and G2 / M phases (e.g., Bonnal et al. (2012) Nat Rev Drug Dis 11:847-59). There are seven naturally occurring pradoadienolides, namely pradoadienolide AG (Mizui et al. (2004) J Antibiot. 57:188-96; Sakai et al. (2004) J Antibiotics 57:180-7). U.S. Patent Nos. 7,884,128 and 7,816,401 describe exemplary methods for the synthesis of prasadiene lactones B and D, and each of these methods is incorporated herein by reference. The synthesis of prasadiene lactones B and D can also be performed using the exemplary methods described in Kanada et al. ((2007) Angew Chem Int Ed. 46:4350-5). Kanada et al. and International Publication No. WO 2003 / 099813 describe exemplary methods for the synthesis of E7107(D11) (compound 45 of WO 2003 / 099813) from prasadiene lactone D (WO 2003 / 099813, 11107D). The corresponding U.S. Patent No. is 7,550,503 by Kotake et al. The synthetic methods described in these references are each incorporated herein by reference.

[0223] As used herein, "splicing modulator drug portion" refers to the component in an ADC or composition that provides the structure of a splicing modulator compound, such as the splicing modulator (D) component in an ADC of formula (I) or in a composition containing -LD.

[0224] As used in this article, "splicing body" refers to a ribonucleoprotein complex that removes introns from one or more RNA segments, such as pre-mRNA segments.

[0225] The term "homology" refers to a molecule that exhibits homology with another molecule because, for example, it has the same or similar chemical residue sequence at the corresponding position.

[0226] As used herein, the terms "inhibit" or "inhibition of" mean the reduction of a measurable quantity, and may include, but do not require, complete prevention or inhibition.

[0227] The terms "target-negative," "target-antigen-negative," or "antigen-negative" refer to the absence of target antigen expression in cells or tissues. The terms "target-positive," "target-antigen-positive," or "antigen-positive" refer to the presence of target antigen expression. 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.

[0228] The term "bystander killing" or "bystander effect" refers to the killing of target negative cells in the presence of target positive cells, where no killing of target negative cells is observed in the absence of target positive cells. Bystander killing can occur through intercellular contact, or at least between target positive and target negative cells in close proximity. 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.

[0229] The terms "hypertrophic disease" and "cancer" are used interchangeably in this document, referring 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. Cancer cells typically present as tumors or masses, but these cells can exist alone within an individual or circulate independently in the bloodstream, such as leukemia 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 and non-solid tumor cancers. Hematologic malignancies can include B-cell malignancies, blood cancers (leukemia), plasma cell cancers (myeloma, such as multiple myeloma), or lymph node cancers (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 myelodysplastic syndrome (MDS). Solid tumors can include carcinomas such as adenocarcinoma (e.g., breast cancer, pancreatic cancer, prostate cancer, colon or colorectal cancer, lung cancer, stomach cancer, cervical cancer, endometrial cancer, ovarian cancer, bile duct carcinoma, glioma, melanoma, etc.

[0230] The terms "tumor" and "necrotic tumor" refer to any tissue mass caused by excessive cell growth or proliferation, which can be benign or malignant, including precancerous lesions.

[0231] The terms "tumor cell" and "proliferative cell" are used interchangeably and refer to individual cells or a total cell population originating from a tumor or neoplasm, including both non-tumorigenic cells and cancer stem cells. As used herein, when referring only to tumor cells lacking the capacity for renewal and differentiation, the term "tumor cell" will be modified by the term "non-tumorigenic" to distinguish it from cancer stem cells.

[0232] The terms "individual" and "patient" are used interchangeably herein and 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 mammalian is a mouse. In some embodiments, the mammalian is a human. In some embodiments, the individual is a mouse. In some embodiments, the individual is a human.

[0233] 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.

[0234] "Pharmaceutical composition" means a formulation which is permitted to be administered to an active ingredient and subsequently provides the intended biological activity and / or achieves a therapeutic effect of the active ingredient, and which does not contain any additional components that would have unacceptable toxicity to the individual to whom the formulation is administered. The pharmaceutical composition may be sterile.

[0235] "Pharmaceutical excipients" include materials such as adjuvants, carriers, pH adjusters and buffers, tension modifiers, wetting agents, preservatives and the like.

[0236] "Medically acceptable" means approved or permitted by federal or state regulatory agencies, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals and more specifically in humans.

[0237] "Medically acceptable salts" refers to 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 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 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.

[0238] As used herein, the term "effective amount" refers to the amount of a compound, ADC, or composition (e.g., a splicing modulator or ADC) described herein sufficient to perform the purposes specifically stated herein, such as producing a therapeutic effect after administration, such as reducing tumor growth rate or tumor volume, reducing cancer symptoms, or other indicators of therapeutic efficacy. Effective amounts can be determined in a conventional manner in relation to the stated purpose. The term "therapeutic effective amount" refers to 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 level, stage, progression, and / or severity of cancer. Therapeutic effective amounts can vary depending on the intended application (in vitro or in vivo) or the individual being treated and the disease condition, such as the individual's weight and age, the severity of the disease condition, the method of administration, and similar factors, and can be readily determined by someone generally skilled in the art. This term also applies to doses that will induce a specific response in target cells, such as inhibiting cell growth. The specific dosage can 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 followed, the severity of the disease, whether it is administered in combination with other agents, the timing of administration, the tissue to which it is administered, and the physical delivery system 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.

[0239] "Prophylactic effective dose" refers to the amount that effectively achieves the desired preventive result at the necessary dosage and within the necessary 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 will be less than the therapeutic effective dose.

[0240] As used herein, "treatment" or "therapeutic" and grammatically related terms refer to any improvement of any consequence of a disease, such as prolonged survival, lower morbidity, and / or reduction of side effects caused by alternative treatment modalities. As readily understood in this art, treatment procedures encompass but do not require the complete eradication of the disease. As used herein, "treatment" or "treatment" means administering the ADC or composition to an individual, such as a patient. Treatment can be curative, healing, relief, mitigation, alteration, remedy, improvement, amelioration, modification, or influence of a condition, symptoms of a condition, or a predisposition to the condition (e.g., cancer). In some embodiments, in addition to treating an individual with a condition, the compositions disclosed herein may be provided prophylactically to prevent or reduce the likelihood of developing the condition.

[0241] In some embodiments, a labeled ADC is used. Suitable for "labeling" includes radioactive nuclei, enzymes, acceptors, cofactors, inhibitors, fluorescent portions, chemiluminescent portions, magnetic particles, and the like.

[0242] As used herein, "protein" means at least two covalently linked amino acids. This term encompasses polypeptides, oligopeptides, and peptides. In some embodiments, the two or more covalently linked amino acids are linked by peptide bonds. Proteins can be composed of naturally occurring amino acids and peptide bonds, for example, when the protein is prepared recombinantly using an expression system and host cells. Alternatively, the protein may include 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. Peptide-like structures are a class of exemplary peptide mimics whose side chains are linked to the nitrogen atom of the peptide backbone, rather than to an α-carbon (as in amino acids), and have hydrogen bonding and conformational characteristics different from peptides (see, for example, Simon et al. (1992) Proc Natl Acad Sci. USA 89:9367). Therefore, peptide-like structures can be resistant to proteolysis or other physiological or storage conditions and are effective in penetrating cell membranes. In particular, when antibodies are synthesized in vivo using conventional methods well-known in this technology, such synthetic amino acids can be incorporated. Furthermore, any combination of peptide mimics, synthetic and naturally occurring residues / structures can also be used. "Amino acids" also include imine residues, such as proline and hydroxyproline. The "R group" or "side chain" of an amino acid can be in an (L)- or (S)- configuration. In a particular embodiment, the amino acid is in an (L)- or (S)- configuration.

[0243] "Recombinant protein" refers to a protein prepared using recombinant technology, using any techniques and methods known in this technology, that is, by expressing recombinant nucleic acids. The methods and techniques used to manufacture recombinant proteins are well known in this technology.

[0244] "Isolated" proteins are not accompanied by at least one material that they normally associate with in their native state, such as 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 isolated proteins may constitute from 5% to 99.9% by weight of the total protein, depending on the circumstances. For example, proteins can be prepared at significantly higher concentrations by using inducible or high-performance promoters, resulting in the production of the protein at increased concentration levels. This definition includes the production of antibodies in a variety of organisms and / or host cells known in this art.

[0245] For amino acid sequences, sequence identity and / or similarity can be determined using standard techniques known in this field, including (but not limited to) the local sequence identity algorithm of Smith and Waterman (1981) Adv Appl Math. 2:482; the sequence identity alignment algorithm of Needleman and Wunsch (1970) J Mol Biol. 48:443; the similarity retrieval method of Pearson and Lipman (1988) Proc Nat Acad Sci. USA 85:2444; computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFAST in the 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, preferably using preset settings or by checking. Preferably, the consistency percentage is calculated using FastDB based on the following parameters: mismatch penalty 1; void penalty 1; void size penalty 0.33; and bonding 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.).

[0246] A useful example of an algorithm is PILEUP. PILEUP uses progressive, step-by-step alignment to generate a set of related sequence alignments. 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. Useful PILEUP parameters include a preset gap weight of 3.00, a preset gap length weight of 0.10, and a weighted terminal gap.

[0247] Another example of a useful 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 useful BLAST program is the WU-BLAST-2 program, which is derived from Altschul et al. (1996) Methods in Enzymology 266:460-80. WU-BLAST-2 uses several retrieval parameters, most of which are set to default values. Adjustable parameters are set using the following values: overlap interval = 1, overlap score = 0.125, and word threshold (T) = 11. 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 a specific database for retrieving the sequence of interest; however, these values ​​can be adjusted to increase sensitivity.

[0248] An additional useful algorithm is the gap-filled BLAST reported by Altschul et al. (1997) Nucl Acid Res. 25:3389-402. The gap-filled BLAST uses BLOSUM-62 instead of scoring; the threshold parameter T is set to 9; a two-hit method is used to trigger gap-free extensions, 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-filled alignments are triggered by scores corresponding to approximately 22 bits.

[0249] Generally, the amino acid homology, similarity, or identity between the proteins and their variants disclosed herein, including target antigen variants (such as HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1) and antibody variable domain variants (including each variant CDR), 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%.

[0250] Similarly, the "percentage of nucleic acid sequence identity (%)" for nucleic acid sequences of antibodies and other proteins identified herein is defined as the percentage of nucleotide residues in the candidate sequence that are identical to 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.

[0251] The sites or regions where amino acid sequence changes are introduced are predetermined, while the mutations themselves do not need to be predetermined. For example, to optimize the efficacy of mutations at a given site, random mutations can be induced at the target codon or region, and the optimal combination of antigen-binding protein CDR variants can be screened for the desired activity. Techniques for substitution mutations at predetermined sites in DNA with known sequences are well-known, such as MI3 primer mutation induction and PCR mutation induction.

[0252] As used herein, "alkyl (alkyl / alkyl group)" means a fully saturated straight-chain, branched-chain, or cyclic hydrocarbon chain. In some embodiments, the alkyl group contains 1-8 carbon atoms ("C1-C8 alkyl"). In some embodiments, the alkyl group contains 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.

[0253] As used herein, "alkylalkoxy" means an alkyl group substituted with an alkoxy group. 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.

[0254] As used herein, "alkylamino" means an alkyl group substituted with an amino group. As used herein, "amino" refers to -NH2, -NH (alkyl), or -N (alkyl) (alkyl).

[0255] As used herein, "alkylhydroxy" means an alkyl group substituted with an amino group. As used herein, "hydroxyl" refers to -OH.

[0256] "Alkyl group" refers to a divalent group of an alkyl group. For example, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2- and -CH2CH2CH2CH2CH2CH2- refer to methylene, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl, respectively.

[0257] As used herein, "carbocyclic" 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").

[0258] "Halogen" refers to any halogen group, such as -F, -Cl, -Br or -I.

[0259] As used herein, "heterocyclic", "heterocyclic group" and "heterocyclic" mean a monocyclic, bicyclic or tricyclic heterocycle containing at least one heteroatom in the ring.

[0260] A monocyclic heterocyclic system contains at least one 3-, 4-, 5-, 6-, 7-, or 8-membered ring independently selected from O, N, and S heteroatoms. In some embodiments, the heterocyclic system contains one 3- or 4-membered ring selected from O, N, and S heteroatoms. In some embodiments, the heterocyclic system contains zero or one double bond and one, two, or three 5-membered rings selected from O, N, and S heteroatoms. In some embodiments, the heterocyclic system contains zero, one, or two double bonds and one, two, or three 6-, 7-, or 8-membered rings selected from O, N, and S heteroatoms. Representative examples of monocyclic heterocycles include (but are not limited to) azacyclobutane, azacycloheptane, azacyclopropane, diazacycloheptane, 1,3-dioxane, 1,3-dioxane, dioxanepentane, dihydropiperanyl (including 3,4-dihydro-2H-piperan-6-yl), 1,3-dithioalkyl, 1,3-dithiaalkyl, imidazolinyl, imidazodinyl, isothiazolinyl, isothiazolinyl, isoxazolinyl, isoxazolinyl, morpholinyl Oxadiazolinyl, oxadiazinyl, oxadiazolinyl, oxadiazinyl, piperazinyl, piperidinyl, piperanyl, pyrazolinyl, pyrazinyl, pyrrololinyl, pyrrolinyl, tetrahydrofuranyl, tetrahydropiperanyl (including tetrahydro-2H-piperanyl-4-yl), tetrahydrothiophenyl, thiadiazolinyl, thiadiazinyl, thiazolinyl, thiazolinyl, thiazolinyl, thiomorpholinyl, 1,1-dioxo-ionylthiomorpholinyl (thiomorpholinyl), thiopiperanyl and trithiaalkyl.

[0261] The bicyclic heterocycles of this invention 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-benzodioxanepentenyl, 1,3-benzodithiocyclopentenyl, 2,3-dihydro-1,4-benzodioxanehexenyl, 2,3-dihydro-1-benzofuranyl, 2,3-dihydro-1-benzothiopheneyl, 2,3-dihydro-1H-indolyl, and 1,2,3,4-tetrahydroquinolinyl.

[0262] The terms "heterocyclic," "heterocyclic group," and "heterocyclic" encompass heteroaryl groups. A "heteroaryl group" refers to a cyclic moiety having one or more closed rings and having one or more heteroatoms (oxygen, nitrogen, or sulfur) in at least one ring, wherein at least one of these rings is an aromatic ring, and wherein the one or more rings may be independently fused and / or bridged. Examples include (but are not limited to) phenyl, thiophene, triazolyl, pyridyl, pyrimidinyl, pyridazinyl, and pyrazinyl.

[0263] As described herein, the compounds of the present invention may contain a "substituted" portion, as appropriate. Generally, the term "substituted," whether or not preceded by the term "as appropriate," means that one or more hydrogens of the specified portion have been replaced by a suitable substituent. Unless otherwise indicated, the "substituted" group may have a suitable substituent 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 preferred combination of substituents according to the present invention is one that results in a stable or chemically viable compound.

[0264] Those skilled in the art will understand that "substitution," "substituted with," or "absent" includes implicit limitations: such substitution or absence is based on the permissible valence states of the substituted atom and substituent, and the substitution or absence produces a stable compound, for example, one 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.

[0265] "Stable" means that a compound is not substantially altered chemically and / or physically when subjected to conditions permissible for its manufacture, detection, and in some embodiments, its recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, a stable or chemically viable compound is one that is not substantially altered when kept at 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.

[0266] 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 "enantiomerically pure center" refers to a tetrahedral carbon atom containing four different substituents.

[0267] The compounds described herein may also contain atomic isotopes in non-natural proportions at one or more atoms constituting such compounds. For example, these 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 covered within the scope of the claimed disclosures. Antibody-Drug Conjugates

[0268] The antibody-drug conjugate (ADC) complexes of this invention comprise complexes with anticancer activity. Specifically, these ADC complexes comprise an antibody or antigen-binding fragment (including its antigen-binding fragment) bound (i.e., covalently linked by a linker) to a drug moiety (e.g., a splicing modulator), wherein the drug moiety, when not bound to the antibody or antigen-binding fragment, exhibits cytotoxic or cell growth-inhibiting effects. In various embodiments, the drug moiety, when not bound to the antibody or antigen-binding fragment, is capable of binding to and / or interacting with the SF3b spliceosome complex. In various embodiments, the drug moiety, when not bound to the antibody or antigen-binding fragment, is capable of modulating RNA splicing in vitro and / or in vivo. In various embodiments, by targeting RNA splicing, the drug moiety and ADC disclosed herein are potent antiproliferative agents. In various embodiments, the drug moiety and ADC disclosed herein can target actively dividing cells and dormant cells.

[0269] In various embodiments, the present invention is at least in part based on the discovery that certain bioactive splicing modulators, when used in ADCs, can provide improved properties. Splicing modulators, when used alone, can exhibit desirablely improved features (e.g., robust SF3b spliceosome complex binding, potent regulation of RNA splicing), and in various embodiments, the splicing modulator can exhibit a small amount of these desirable improved features when bound to an antibody or antigen-binding fragment. Therefore, the development and manufacture of ADCs for human therapeutic purposes, such as as oncology agents, 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 drug can significantly affect the activity of one or both of the antibody and the drug, and this effect will vary depending on the type of linker and / or drug selected. Therefore, in some embodiments, the selected ADC components should (i) retain one or more therapeutic properties exhibited by the isolated antibody and drug portions; (ii) maintain the specific binding properties of the antibody or antigen-binding fragment; (iii) optimize the drug load and drug-to-antibody ratio; (iv) allow delivery via stable linkage to the antibody or antigen-binding fragment, such as intracellular delivery of the drug portion; (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) achieve therapeutic effects, such as cytotoxicity, after the drug portion undergoes lysis or other release mechanisms in the cellular environment; (viii) exhibit in vivo anticancer therapeutic efficacy similar to or superior to that of the isolated antibody and drug portions; (ix) minimize off-target killing caused by the drug portion; and / or (x) exhibit the desired pharmacokinetic and pharmacodynamic properties, adaptability, and toxicological / immunological profile. Each of these properties may be required to identify an improved ADC for therapeutic use (Ab et al. (2015) Mol Cancer Ther. 14:1605-13).

[0270] In various embodiments, the ADCs disclosed herein exhibit some or every one of the characteristics unexpectedly advantageous in the categories listed above. For example, in some embodiments, the ADC constructs disclosed herein exhibit unexpectedly advantageous drug loading, aggregation, and / or stability morphologies, and / or maintain antibody binding function, drug activity, and / or improve bystander killing while reducing off-target killing, compared to ADCs that include alternative linkers and / or drug portions (e.g., alternative splicing modulators). In some embodiments, the ADC constructs disclosed herein exhibit superior stability, activity, potency, or other effects (measured in vivo or in vitro) compared to ADCs using alternative linkers and / or drug portions (e.g., alternative splicing modulators). In some embodiments, the ADC constructs disclosed herein exhibit in vivo therapeutic efficacy when administered as a single dose. In some embodiments, the ADC constructs disclosed herein are unexpectedly stable compared to ADCs using alternative linkers and / or drug portions (e.g., alternative splicing modulators).

[0271] The ADC complex of this invention can selectively deliver effective doses of cytotoxic 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, 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 in 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.

[0272] Examples of HER2-positive cancers include (but are not limited to) breast cancer, gastric cancer, bladder cancer, urothelial 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).

[0273] 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, laryngopharyngeal, nasopharyngeal carcinoma), 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 malignancy, Hodgkin's lymphoma), and thyroid cancer (Szatmári et al. (2015) Dis Markers 2015:796052).

[0274] 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).

[0275] In some embodiments, lysis of the ADC releases a splicing modulator from the antibody or antigen-binding fragment and the linker. In some embodiments, the linker and / or splicing modulator is designed to promote bystander killing (killing of neighboring cells). In some embodiments, the linker and / or splicing modulator is designed to promote bystander killing via lysis after the linker-drug fraction and / or the drug fraction alone have been internalized and diffused into neighboring cells. In other embodiments, the linker promotes internalization. In some embodiments, the linker is designed to minimize lysis 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 tissue that does not express antigens that are targets of the antibody or antigen-binding fragment in the ADC but surrounds the target cancerous tissue expressing those antigens. In some embodiments, the drug fraction or its catabolic metabolites generated by ADC lysis are designed to promote uptake by target cells or neighboring cells (i.e., cell permeability). In this paper, drug fractions and their catabolites are referred to as "bystander active," while drug fractions or catabolites with low cell permeability are referred to as "bystander inactive."

[0276] In some embodiments, the disclosed ADCs also exhibit bystander-killing activity, but with lower off-target cytotoxicity. Not bound by theory, bystander-killing activity of ADCs can be particularly beneficial when ADC penetration into solid tumors is limited and / or when target antigen expression in tumor cells is heterogeneous. In some embodiments, ADCs containing cleavable linkers exhibit particularly potent bystander-killing activity and / or demonstrate improved bystander-killing activity compared to similar treatments utilizing ADCs containing non-cleavable linkers. In some embodiments, the ADCs disclosed herein exhibit improved solubility and target cell penetration relative to the drug portion alone. In some embodiments, the ADCs disclosed herein exhibit improved cytotoxicity relative to the drug portion alone. In some embodiments, the ADCs disclosed herein use a drug portion that exhibits lower cytotoxicity when evaluated as a drug alone, and unexpectedly outperform ADCs containing other drug portions that exhibit higher cytotoxicity when evaluated as a drug alone. In some embodiments, the cleavage and release of splice modulators improve the cytotoxicity of ADCs compared to similar treatments utilizing ADCs containing non-cleavable linkers. In other embodiments, the cleavage and release of splice modulators are not required for the ADC to have the desired biological activity. In some embodiments, an ADC comprising a non-cleavable linker with an increased spacer length (e.g., ADL12) provides the same or similar cytotoxicity relative to similar treatments using ADCs comprising cleavable linkers (e.g., ADL1, ADL5) and unexpectedly superior cytotoxicity relative to similar treatments using ADCs comprising shorter non-cleavable linkers. In some embodiments, an ADC comprising a non-cleavable linker with an increased spacer length and no carbonyl group (e.g., ADL12) provides the same or similar cytotoxicity relative to similar treatments using ADCs comprising cleavable linkers (e.g., ADL1, ADL5) and unexpectedly superior cytotoxicity relative to similar treatments using ADCs comprising non-cleavable linkers with the same or similar spacer length and containing a carbonyl group (e.g., ADL10). In some embodiments, removing the carbonyl group from the non-cleavable MC linker (e.g., ADL12) can increase cytotoxicity by more than 50-fold, more than 75-fold, more than 100-fold, more than 150-fold, or more than 200-fold relative to similar treatments using ADCs comprising unmodified non-cleavable MC linkers (e.g., ADL10). In some embodiments, 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 increase cytotoxicity by more than 50-fold, more than 75-fold, more than 100-fold, more than 150-fold, or more than 200-fold relative to similar treatments using an ADC containing an unmodified non-cleavable MC linker (e.g., ADL10).

[0277] This document provides an ADC complex comprising an antibody or antigen-binding fragment thereof targeting tumor cells (Ab), a splicing modulator 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, STEAP1) with high specificity and high affinity. In some embodiments, the antibody or antigen-binding fragment is internalized into the target cell upon binding, for example, into the degradation compartment of the cell. In various embodiments, ADCs that are internalized upon binding to the target cell, undergo degradation, and release the splicing modulator drug moiety to kill cancer cells can be used. The splicing modulator drug moiety can be released from the antibody and / or linker moiety in the ADC by enzymatic action, hydrolysis, oxidation, or any other mechanism.

[0278] An exemplary ADC has equation (I): Ab-(LD)p (I) Where Ab = antibody or antigen binding fragment, L = linker portion, D = splice modulator drug portion, and p = number of splice modulator drug portions for each antibody or antigen binding fragment.

[0279] In some preferred embodiments, the drug-targeting portion used in the ADC and composition is an antibody or antigen-binding fragment. Other exemplary drug-targeting portions used in the ADC and composition are also provided and described herein. In some embodiments, the drug-targeting portion can be any of a variety of cell binders and non-antibody scaffolds. In some embodiments, the drug-targeting portion 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 drug portion (e.g., a splice modulator drug portion as disclosed herein). The term encompasses 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 binders such as DARPin, duobody, bicyclic peptide, nanoantibody, centyrin, melanocyte-stimulating hormone (MSH), receptor-Fc fusion molecules, T-cell receptor structures, steroid hormones such as androgens and estrogens, growth factors, colony-stimulating factors such as EGF, and other non-antibody scaffolds. In various embodiments, non-antibody scaffolds can be broadly classified into two structural types: compounds with defined domain sizes (approximately 6-20 kDa) and restriction peptides (approximately 2-4 kDa). Exemplary scaffolds with defined domain sizes include (but are not limited to) affinity antibodies, affilin, anticarrier proteins, atrimer, DARPin, FN3 scaffolds (e.g., adnectin and sintin), fynomer, Kunitz domain, pronectin, O antibodies, and receptor-Fc fusion proteins, while exemplary restriction peptides include high-affinity multimers, bicyclic peptides, and Cys-knots. In some embodiments, the drug-targeting portion used in the ADC and the composition is selected from affibody, avidin, anticarrier protein, atrimer, DARPin, FN3 scaffolds such as fibronectin or sintine, phenotype, Konnitz domain, fibronectin connexin, O antibody, high-affinity multimer, bicyclic peptide, and Cys knot. In some embodiments, the drug-targeting portion used in the ADC and the composition is a receptor-Fc fusion protein, such as HER2-Fc chimeric fusion protein. Non-antibody scaffolds are reviewed, for example, in Vazquez-Lombardi et al. (2015) Drug Dis Today 20(10):1271-83. [Antibody] []

[0280] The antibody or antigen-binding fragment (Ab) of Formula (I) includes, within its scope, any antibody or antigen-binding fragment that specifically binds to a target antigen on cancer cells. This antibody or antigen-binding fragment may bind to the target antigen, wherein, as measured by, for example, BIAcore® analysis, the dissociation constant (KD) is ≤1 mM, ≤100 nM, or ≤10 nM, or any amount in between. In some embodiments, KD is from 1 pM to 500 pM. In some embodiments, KD is between 500 pM and 1 µM, 1 µM and 100 nM, or 100 mM and 10 nM.

[0281] In some embodiments, the antibody or antigen-binding fragment is a quadruple-chain antibody (also known as an immunoglobulin or a full-length 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 the function of an immunoglobulin.

[0282] 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 internalizing antigen-binding fragment thereof binds to a target cancer antigen expressed on the cell surface and enters the cell upon binding. In some embodiments, after the ADC enters and is present in the cell expressing the target cancer antigen (i.e., after ADC internalization), the splice modulator drug portion in the ADC is released from the antibody or antigen-binding fragment of the ADC, for example by lysis, by degradation by the antibody or antigen-binding fragment, or by any other suitable release mechanism.

[0283] The amino acid sequences of the antibodies of the present invention are illustrated in Tables 2-4. Table 1. Antibodies [surface] [2. mAb] [Amino acid sequence in the variable region] [surface] [3. mAb CDR] [Amino acid sequence] [surface] [4.] [full length] [mAb Ig] [Amino acid sequence of the chain] [surface] [5.] [Example target antigen amino acid sequence] []

[0284] In various embodiments, the ADC disclosed herein may comprise any set of the 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 grafting 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., its KD is less than 1 × 10⁻⁸ M) and retains one or more functional properties of the ADC disclosed herein (e.g., internalization ability, regulation of RNA splicing, inhibition of cell growth, etc.).

[0285] 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 ADC comprises a human immunoglobulin G subtype 1 (IgG1) heavy chain constant domain and a human Igκ light chain constant domain.

[0286] In various other embodiments, the target cancer antigen of the ADC is human epidermal growth factor receptor 2 (HER2).

[0287] 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.

[0288] In various embodiments, the anti-HER2 antibody or its antigen-binding fragment includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 19 and a light chain variable region containing 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 a sequence that is at least 95% identical to the disclosed sequence. In some embodiments, the anti-HER2 antibody or its antigen-binding fragment has a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the heavy chain variable region amino acid sequence of SEQ ID NO: 19 and / or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable region amino acid sequence of SEQ ID NO: 20.

[0289] 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.

[0290] In various embodiments, the anti-HER2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 19 or a sequence that is at least 95% identical to SEQ ID NO: 19, and the light chain amino acid sequence of SEQ ID NO: 20 or a sequence that is at least 95% identical to 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 that is at least 95% identical to the disclosed sequence. In some embodiments, the anti-HER2 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 20. In various embodiments, the anti-HER2 antibody is trastuzumab or its antigen-binding fragment.

[0291] 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 such CDRs include no more than one, two, three, four, five or six amino acid additions, deletions or substitutions of 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).

[0292] In various other embodiments, the target cancer antigen of the ADC is human multiligand proteoglycan-1 (CD138).

[0293] 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.

[0294] In various embodiments, the anti-CD138 antibody or its antigen-binding fragment includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 21 and a light chain variable region containing 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 that is at least 95% identical to the disclosed sequence. In some embodiments, the anti-CD138 antibody or its antigen-binding fragment has a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the heavy chain variable region amino acid sequence of SEQ ID NO: 21 and / or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable region amino acid sequence of SEQ ID NO: 22.

[0295] 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 murine IgG2a heavy chain constant domain and a murine 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.

[0296] In various embodiments, the anti-CD138 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 21 or a sequence that is at least 95% identical to SEQ ID NO: 21, and the light chain amino acid sequence of SEQ ID NO: 22 or a sequence that is at least 95% identical to 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 that is at least 95% identical to the disclosed sequence. In some embodiments, the anti-CD138 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 21 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 22. In various embodiments, the anti-CD138 anti-system B-B4 or its antigen-binding fragment.

[0297] 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 such CDRs include no more than one, two, three, four, five or six amino acids added, deleted or substituted for 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).

[0298] In various other embodiments, the target cancer antigen of the ADC is human pteroin type A receptor 2 (EPHA2).

[0299] 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.

[0300] In various embodiments, the anti-EPHA2 antibody or its antigen-binding fragment includes 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 a sequence that is at least 95% identical to the disclosed sequence. In some embodiments, the anti-EPHA2 antibody or its antigen-binding fragment has a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the heavy chain variable region amino acid sequence of SEQ ID NO: 23 and / or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to the light chain variable region amino acid sequence of SEQ ID NO: 24.

[0301] 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.

[0302] In various embodiments, the anti-EPHA2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 23 or a sequence that is at least 95% identical to SEQ ID NO: 23, and the light chain amino acid sequence of SEQ ID NO: 24 or a sequence that is at least 95% identical to 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 that is at least 95% identical to the disclosed sequence. In some embodiments, the anti-EPHA2 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 23 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 24. In some embodiments, the anti-EPHA2 antibody comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 23 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 24. In various embodiments, the anti-EPHA2 anti-system 1C1 or its antigen-binding fragment.

[0303] 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 such CDRs include 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) with no more than one, two, three, four, five, or six amino acids added, deleted, or substituted.

[0304] In various embodiments, amino acid substitution is a single-residue substitution. Insertion is typically from about 1 to about 20 amino acid residues, but more insertions are permissible as long as biological function (e.g., binding to the target antigen) is preserved. Deletion is 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, particularly antigen-binding proteins. However, in some cases, larger changes are permissible. Conservative substitutions are generally performed according to the diagrams depicted in Table 6 below. [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

[0305] Significant changes in functional or immune properties can be achieved by selecting substitutions with lower conservation than those shown in Table 6. For example, substitutions can be made that more significantly affect the following: 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 generally cause the greatest changes in peptide properties are those in which (a) a hydrophilic residue, such as serinexyl or threoninexyl, replaces a hydrophobic residue, such as leucinexyl, isoleucinexyl, phenylalaninexyl, valinexyl, or apropylalanine (or is substituted by a hydrophobic residue); (b) cysteine ​​or proline replaces any other residue (or is substituted by any other residue); (c) a residue with a positively charged side chain, such as lysinexyl, sperminexyl, or histidinexyl, replaces a negatively charged residue, such as glutaminexyl or aspartic acid (or is substituted by a negatively charged residue); or (d) a residue with a bulky side chain, such as phenylalanine, replaces a residue without a side chain, such as glycine (or is substituted by that residue).

[0306] In various embodiments of using variant antibody sequences in ADCs, these variants typically exhibit the same qualitative biological activity and elicit the same immune response; however, variants may be selected as needed to alter the characteristics of the antigen-binding protein. Alternatively, the variant may be designed to change the biological activity of the antigen-binding protein. For example, glycosylation sites may be altered or removed.

[0307] Various antibodies can be used in the ADCs used herein to target cancer cells. As shown below, the linker-load in the ADCs disclosed herein is unexpectedly effective in the presence of antibodies targeting different tumor antigens. Suitable antigens that are expressed on tumor cells rather than healthy cells, or expressed in higher amounts on tumor cells than on healthy cells, and antibody systems against such antigens are known in this art. These antibodies can be used with the linkers and splicing modulator loads disclosed herein. In some embodiments, the antibody or antigen-binding fragment targets HER2, and the HER2-targeting antibody or antigen-binding fragment is trastuzumab. In some embodiments, the antibody or antigen-binding fragment targets CD138, and the CD138-targeting antibody or antigen-binding fragment is B-B4. In some embodiments, the antibody or antigen-binding fragment targets EPHA2, and the EPHA2-targeting antibody or antigen-binding fragment is 1C1. In some embodiments, the disclosed linker and splicing regulator payloads have been unexpectedly effective in the presence of several different tumor-targeting antibodies, with antibodies targeting HER2 such as trastuzumab, antibodies targeting CD138 such as B-B4, and antibodies targeting EPHA2 such as 1C1 providing significantly improved drug efficacy in terms of antibody ratio, aggregation level, 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.

[0308] In some embodiments, alternative antibodies targeting the same target or antibodies targeting different antigens are used and provide 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 splicing modulator are loaded and bound to an alternative HER2, CD138, or EPHA2-targeting antibody or antigen-binding fragment. In some other embodiments, some or all of these advantageous functional properties are observed when the disclosed linker and splicing modulator are loaded and bound to a HER2-targeting antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment targets HER2. In some embodiments, the HER2-targeting antibody or antigen-binding fragment is trastuzumab. In some other embodiments, some or all of these advantageous functional properties are observed when the disclosed linker and splicing modulator are loaded and bound to a CD138-targeting antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment targets CD138. In some embodiments, the CD138-targeting antibody or antigen-binding fragment is B-B4. In some other embodiments, some or all of these advantageous functional properties are observed when the disclosed linker and splicing regulator are loaded and bound to the EPHA2-targeting antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment targets EPHA2. In some embodiments, the EPHA2-targeting antibody or antigen-binding fragment is 1C1. [Connector] []

[0309] In various embodiments, the linkers in the ADC are stabilized extracellularly in a manner sufficient to be therapeutically effective. In some embodiments, the linkers are stabilized extracellularly 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 splicing modulator). As used herein, in the context of linkers or ADCs containing linkers, "stable" 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% of the linkers (or any percentage therebetween) in the ADC sample are cleaved (or otherwise incomplete in the case of the overall ADC) when the ADC is present in extracellular conditions. In some embodiments, the linkers and / or ADCs disclosed herein are unexpectedly stable compared to alternative linkers and / or ADCs with alternative linker and / or splicing modulator 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.

[0310] 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 then quantifying the amount of free drug fraction present in that plasma. Stability allows the ADC to target tumor cells at the appropriate time and prevents premature release of the drug fraction, which could otherwise reduce the therapeutic index of the ADC by indiscriminately damaging normal and tumor tissues. In some embodiments, the linker is stable outside the target cell and releases the drug fraction from the ADC upon entering the cell, thereby allowing the drug to bind to its target (e.g., the SF3b spliceosome complex). Thus, an effective linker will: (i) maintain the specific binding properties of the antibody or antigen-binding fragment; (ii) allow delivery via stable linking to the antibody or antigen-binding fragment, such as intracellular delivery of the drug fraction; (iii) maintain stability and integrity until the ADC is transported or delivered to the target site; and (iv) achieve the therapeutic effect of the drug fraction, such as cytotoxicity, after lysis or alternative release mechanisms.

[0311] 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 insoluble and typically limit the drug load available on the antibody, potentially adversely affecting the ADC's efficacy. Generally, protein aggregates in biologics are also associated with increased immunogenicity. As shown below, the linkers disclosed in this article enable ADCs to exhibit lower aggregation levels and the desired drug load.

[0312] Linkers can be "cleavable" or "non-cleavable" (Ducry and Stump (2010) Bioconjugate Chem. 21:5-13). Cleavable linkers are designed to release drug moieties (e.g., splicing regulators) when subjected to certain environmental factors, such as when internalized into target cells, while non-cleavable linkers generally rely on the degradation of the antibody or antigen-binding fragment itself.

[0313] In some embodiments, the linker is a non-cleavable linker. In some embodiments, the splicing modulator drug portion of the ADC is released by degradation of the antibody or antigen-binding fragment. When internalized and degraded 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 others 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, such as 1, 2, 3, 4, 5, or 6 PEG moieties; or one or more alkyl moieties.

[0314] In some embodiments, the linker is a cleavable linker. A cleavable linker refers to any linker containing a cleavable portion. As used herein, the term "cleavable portion" refers to any chemical bond that can be cleaved. Suitable cleavable chemical bonds are those well known in the 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 cleavable portions allow the release of the splice modulator drug portion from the ADC via cleavage at specific sites within the linker.

[0315] In some embodiments, the linker may cleave under intracellular conditions, thereby releasing the splice modulator drug portion sufficiently from the antibody or antigen-binding fragment in the intracellular environment to activate the drug and / or make the drug therapeutically effective. In some embodiments, the splice modulator drug portion 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 upon entry into the cell, the splice modulator drug portion cleaves from the antibody or antigen-binding fragment. In some embodiments, the linker includes a cleavable portion positioned such that neither the linker nor the antibody or antigen-binding fragment remains bound to the splice modulator drug portion upon cleavage. Exemplary cleavable linkers include acid-instable linkers, protease / peptidase-sensitive linkers, photostable linkers, dimethyl-containing linkers, disulfide-containing linkers, or sulfonamide-containing linkers.

[0316] 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 pH lower than that of the cytosol (approximately pH 7.4) in intracellular compartments of the endosome (approximately pH 5-6) and lysosome (approximately pH 4.8) to trigger the hydrolysis of acid-instable groups in the linker, such as hydrazones (Jain et al. (2015) Pharm Res 32:3526-40). In some embodiments, the linker is an acid-instable and / or hydrolyzable linker. For example, acid-instable linkers that can be hydrolyzed in lysosomes and contain acid-instable groups (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitine, orthoesters, acetals, ketals, or the like) can be used. See, for example, U.S. Patents 5,122,368, 5,824,805, and 5,622,929; Dubowchik and Walker (1999) Pharm Therapeutics 83:67-123; Neville et al. (1989) Biol Chem. 264:14653-61. Such linkers are relatively stable under neutral pH conditions, such as those found in blood, but unstable below pH 5.5 or 5.0 (approximate to lysosomal pH). In some embodiments, hydrolyzable linkers are thioether linkers (such as thioethers linked to therapeutic agents via acetohydrazone bonds) (see, for example, U.S. Patent 5,622,929).

[0317] In some embodiments, the linker can be cleaved under reducing conditions. In some embodiments, the linker can be cleaved in the presence of a reducing agent such as glutathione or dithiothreitol. In some embodiments, the linker is a disulfide linker or a sulfonamide linker.

[0318] In some embodiments, the linker system can be cleaved disulfide linkers. Various disulfide linker systems are known in this art, including, for example, linkers formed using N-succinimido-5-acetylthioacetate (SATA), N-succinimido-3-(2-pyridyldithio)propionate (SPDP), N-succinimido-3-(2-pyridyldithio)butyrate (SPDB), and N-succinimidooxycarbonyl-α-methyl-α-(2-pyridyl-dithio)toluene (SMPT), SPDB, and SMPT. See, for example, Thorpe et al. (1987) Cancer Res. 47:5924-31; Wawrzynczak et al., In 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 utilize the abundance of intracellular thiols, which can facilitate the cleavage of their disulfide bonds. The most abundant intracellular thiol, reduced glutathione, typically has an intracellular concentration in the range of 1–10 nM, which is approximately 1,000 times higher than the approximately 5 µM intracellular concentration of the most abundant low-molecular-weight thiol in the blood (i.e., cysteine) (Goldmacher et al., In 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 thiols or enzymes.

[0319] In some embodiments, the linker is a cleavable sulfonylurea linker. As used herein, a cleavable sulfonylurea linker means 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 can be cleaved.

[0320] In some embodiments, the linker may be a dendritic linker that covalently links more than one drug moiety to an antibody or antigen-binding fragment via branched, multifunctional linker portions. 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 drug-to-antibody molar ratio, i.e., drug loading, which is related to the efficacy of the ADC. Therefore, in cases where the antibody or antigen-binding fragment carries only one reactive cysteine ​​thiol group, multiple splice modulator drug moieties, for example, can be linked via dendritic linkers. In some embodiments, the linker portion or linker-drug portion can 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.

[0321] In some embodiments, the linker may be cleaved by a lysing agent, such as an enzyme, present in the intracellular environment (e.g., lysosomes, nucleosomal bodies, or cell membrane pits). The linker may be a peptide linker cleaved, for example, by intracellular peptidases or proteases, including (but not limited to) lysosomal or nucleosomal proteases.

[0322] 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 a reagent present in the intracellular environment. For example, a linker may contain a valine-alanine (Val-Ala) sequence or a valine-citrulline (Val-Cit) sequence, which can be cleaved by a peptidase such as cathepsin, for example, cathepsin B. In some embodiments, the linker may contain a glutamate-valine-citrulline (Glu-Val-Cit) sequence. In some embodiments, the linker is enzyme-cleavable, 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 lysosomal cysteine ​​cathepsins, such as cathepsins 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 example dipeptide that may be cleaved by cathepsin B is valine-citrulline (Val-Cit) (Dubowchik et al. (2002) Bioconjugate Chem. 13:855-69).

[0323] In some embodiments, the linker or the cleavable peptide portion of the linker comprises an amino acid unit. In some embodiments, the amino acid unit enables the linker to be cleaved by a protease, thereby promoting the release of the splicing regulator drug portion from the ADC upon exposure to one or more intracellular proteases, such as one or more lysosomal enzymes (Doronina et al. (2003) Nat Biotechnol. 21:778-84; Dubowchik and Walker (1999) Pharm Therapeutics 83:67-123). Exemplary amino acid units include (but are not limited to) dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include (but are not limited to) valine-alanine (Val-Ala), valine-citrulline (Val-Cit), alanine-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), leucine-citrulline (Leu-Cit), isoleucine-citrulline (Ile-Cit), tryptophan-citrulline (Trp-Cit), and phenylalanine-alanine (Phe-Ala). Exemplary tripeptides include (but are not limited to) alanine-alanine-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, for example, Anami et al. (2018) Nat Comm. 9:2512, with regard to exemplary linkers containing Glu-Val-Cit, which are incorporated herein by reference) 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-N9-toluenesulfonyl-Arg, and Phe-N9-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 naturally occurring amino acid residues and / or trace amounts of amino acids and / or non-naturally occurring amino acid analogs, such as citrulline. The amino acid unit can be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, lysosomal proteases such as cathepsin B, C, D, or S, or fibrinolytic proteases.

[0324] In some embodiments, the linker is a cleavable β-glucuronide linker. As used herein, a cleavable β-glucuronide linker refers to any linker containing a cleavable β-glucuronide moiety. An exemplary cleavable β-glucuronide linker comprises the following structure: .

[0325] The term "cleavable β-glucuronide moiety" refers to a glycosidic bond that can be cleaved by a reagent 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 bonds of β-configured glucuronides.

[0326] In some embodiments, the ADC disclosed herein includes a cleavable β-glucuronide moiety in a linker, which can be cleaved by an enzyme. In some embodiments, the cleavable β-glucuronide 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-glucuronide moiety in the linker allows the linker to be cleaved by β-glucuronidase after ADC internalization, thereby promoting the release of the drug moiety from the ADC in the cellular environment.

[0327] In some embodiments, the linker in any ADC disclosed herein may include at least one spacer unit for attaching an antibody or antigen-binding fragment to a drug portion (e.g., a splicing modulator drug portion). In some embodiments, when a spacer unit is present between the antibody or antigen-binding fragment and the cleavable portion, a cleavage site (e.g., a cleavable peptide portion) in the linker is attached to the antibody or antigen-binding fragment. In some embodiments, when a spacer unit is present between the drug portion and the cleavable portion, a cleavage site (e.g., a cleavable peptide portion) in the linker is attached to the drug portion. In some embodiments, no cleavage site is present, and the spacer unit is used to attach the antibody or antigen-binding fragment to the drug portion.

[0328] 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 out of 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) moieties, such as 1, 2, 3, 4, 5, or 6 PEG moieties. In some embodiments, the linker comprises two PEG moieties.

[0329] In some embodiments, the spacer subunits in the connectors include one or more PEG portions. In some embodiments, the spacer subunits include 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 subunits include (PEG)2, (PEG)3, (PEG)4, (PEG)5, (PEG)6, (PEG)7, (PEG)8, (PEG)9, or (PEG)10. In some embodiments, the spacer subunits include (PEG)2.

[0330] 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 (CH2)2-O-(CH2)2 ("Et-O-Et").

[0331] Spacer units can be used to directly or indirectly connect antibody or antigen-binding fragments to a drug portion, for example. In some embodiments, the spacer unit directly connects the antibody or antigen-binding fragment to the splice modulator drug portion. In some embodiments, the antibody or antigen-binding fragment and the splice modulator drug portion are connected 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 connects the antibody or antigen-binding fragment to the splice modulator drug portion. In some embodiments, the spacer unit indirectly connects the antibody or antigen-binding fragment to the splice modulator drug portion via a cleavable portion (e.g., a cleavable peptide-cleavable β-glucuronide) and / or a linking portion, such as a maleic anhydride portion, for attaching the spacer unit to the antibody or antigen-binding fragment.

[0332] 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).

[0333] A 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 a thiosulfate group on a cysteine ​​residue of 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 the antibody or antigen-binding fragment. In some embodiments, the Mal-spacer unit is attached to the 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.

[0334] 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-glucuronide portion.

[0335] In some embodiments, the linker comprises the structure: Mal-spacer unit. In some embodiments, the Mal-spacer unit comprises maleic anisodiaminohexyl (MC). In some embodiments, the linker comprises the structure: MC. In some embodiments, the linker comprises the structure: Mal-(CH2)2 (“Mal-Et”). In some embodiments, the linker comprises the structure: Mal-(CH2)6 (“Mal-Hex”). In some embodiments, the linker comprises the structure: Mal-(CH2)2-O-(CH2)2 (“Mal-Et-O-Et”). In some embodiments, the linker comprises the structure: Mal-(PEG)2. In some embodiments, the linker comprises the structure: Mal-(PEG)2-CO.

[0336] 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 the structure: Mal-spacer unit-Val-Cit. In some embodiments, the Mal-spacer unit comprises maleic anhydride-iminohexyl (MC). In some embodiments, the linker comprises the structure: MC-Val-Cit.

[0337] In some embodiments, the linker comprises the structure: Mal-spacer unit-Val-Ala. In some embodiments, the Mal-spacer unit comprises maleic anisodiaminohexyl (MC). In some embodiments, the linker comprises the structure: MC-Val-Ala.

[0338] In various embodiments, the Mal-spacer unit links an antibody or antigen-binding fragment to a cleavable β-glucuronide moiety. In some embodiments, the linker comprises the Mal-spacer unit-β-glucuronide. In some embodiments, the linker comprises MC-β-glucuronide.

[0339] In various embodiments, the cleavable portion of the connector is directly coupled to the splice modulator drug portion. In other embodiments, a spacer subunit is used to connect the cleavable portion of the connector to the splice modulator drug portion. In various embodiments, the splice modulator is connected to the cleavable portion of the connector via a spacer subunit.

[0340] Spacer units can be either "self-degrading" or "non-self-degrading." "Non-self-degrading" spacer units are those in which some or all of the spacer units remain bound to the splice regulator drug portion during 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 they do not readily and completely release the attached native drug portion under cellular conditions. "Self-degrading" spacer units allow the release of the native drug portion under intracellular conditions. The "native drug" or "native drug portion" refers to the portion of the spacer unit that does not retain the spacer unit or other chemically modified portions after spacer unit cleavage / degradation.

[0341] 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 of the linker to the splice modulator drug portion is self-degrading and undergoes self-degradation simultaneously with or shortly before / after cleavage of the cleavable portion under intracellular conditions. In some embodiments, the splice modulator is connected to the cleavable portion of the linker via a self-degrading spacer unit. In some embodiments, the splice modulator is connected to the cleavable portion of 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 splice modulator is connected to the cleavable portion of the linker via a self-degrading spacer unit, the cleavable portion comprising Val-Ala, and a maleic anhydride hexyl group (MC) binds the cleavable portion to an antibody or antigen-binding fragment. In some embodiments, the splicing modulator is connected to a cleavable portion in a linker via a self-degradable spacer unit, the cleavable portion comprising Glu-Val-Cit, and a maleic-diaminohexyl (MC) conjugates the cleavable portion to an antibody or antigen-binding fragment. In some embodiments, the splicing modulator is conjugated to an antibody or antigen-binding fragment via a Mal-spacer unit (e.g., MC) in a linker, the linker conjugating the Val-Cit cleavable portion and a pABC or pAB self-degradable spacer unit. In some other embodiments, the splicing modulator is conjugated to an antibody or antigen-binding fragment via a Mal-spacer unit (e.g., MC) in a linker, the linker conjugating the Val-Ala cleavable portion and a pABC or pAB self-degradable spacer unit. In some other embodiments, the splicing modulator is conjugated to an antibody or antigen-binding fragment via a Mal-spacer unit (e.g., MC) in a linker, the linker conjugating the Glu-Val-Cit cleavable portion and a pABC or pAB self-degradable spacer unit.

[0342] 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 the cleavable portion of the amino acid unit in the linker, and a carbamate, methyl carbamate, or carbonate is formed between pABOH and the drug moiety (Hamann et al. (2005) Expert Opinion Ther Patents 15:1087-103). In some embodiments, the self-degrading spacer unit may comprise a p-aminobenzyloxycarbonyl group (pABC). Without being bound by theory, it is considered that the self-degradation of pABC involves a spontaneous 1,6-elimination reaction (Jain et al. (2015) Pharm Res. 32:3526-40).

[0343] In various embodiments, the structure of the p-aminobenzooxycarbonyl (pABC) used in the disclosed ADC is shown below: .

[0344] In various embodiments, a self-decomposing spacer unit connects the pyrolytic portion of the connector to the splice modulator. In some embodiments, the self-decomposing spacer unit is a pABC. In some embodiments, the pABC connects the pyrolytic portion of the connector to the splice modulator. In some embodiments, the pABC undergoes self-decomposition after the pyrolytic portion pyrolyzes, and the splice modulator is released from the ADC in its native, active form.

[0345] In some embodiments, the anti-HER2 antibody or antigen-binding fragment is attached to the splicing modulator via a linker comprising MC-Val-Cit-pABC. In other embodiments, the anti-HER2 antibody or antigen-binding fragment is attached to the splicing modulator via a linker comprising MC-Val-Ala-pABC.

[0346] In some embodiments, the anti-CD138 antibody or antigen-binding fragment is bound to the splicing regulator via a linker comprising MC-Val-Cit-pABC. In other embodiments, the anti-CD138 antibody or antigen-binding fragment is bound to the splicing regulator via a linker comprising MC-Val-Ala-pABC.

[0347] In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment is bound to the splicing regulator via a linker comprising MC-Val-Cit-pABC. In other embodiments, the anti-EPHA2 antibody or antigen-binding fragment is bound to the splicing regulator via a linker comprising MC-Val-Ala-pABC.

[0348] In some embodiments, pABC undergoes self-decomposition 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 Val-Ala. In some embodiments, the linker comprises Val-Ala-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 Ala-Ala-Asn. In some embodiments, the linker comprises Ala-Ala-Asn-pABC.

[0349] In some embodiments, pABC undergoes self-decomposition after the cleavable β-glucuronide portion in the linker is partially cleaved. In some embodiments, the linker comprises β-glucuronide-pABC.

[0350] 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.

[0351] In various embodiments, the structure of p-aminobenzyl (pAB) used in the disclosed ADC is shown below: .

[0352] In various embodiments, a self-decomposing spacer unit connects the pyrolytic portion of the connector to the splice modulator. In some embodiments, the self-decomposing spacer unit is a pAB. In some embodiments, the pAB connects the pyrolytic portion of the connector to the splice modulator. In some embodiments, the pAB undergoes self-decomposition upon pyrolysis of the pyrolytic portion, and the splice modulator is released from the ADC in its native, active form.

[0353] In some embodiments, the anti-HER2 antibody or antigen-binding fragment is attached to the splicing modulator via a linker comprising MC-Val-Cit-pAB. In other embodiments, the anti-HER2 antibody or antigen-binding fragment is attached to the splicing modulator via a linker comprising MC-Val-Ala-pAB.

[0354] In some embodiments, the anti-CD138 antibody or antigen-binding fragment is bound to the splicing modulator via a linker comprising MC-Val-Cit-pAB. In other embodiments, the anti-CD138 antibody or antigen-binding fragment is bound to the splicing modulator via a linker comprising MC-Val-Ala-pAB.

[0355] In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment is bound to the splicing modulator via a linker comprising MC-Val-Cit-pAB. In other embodiments, the anti-EPHA2 antibody or antigen-binding fragment is bound to the splicing modulator via a linker comprising MC-Val-Ala-pAB.

[0356] In some embodiments, pAB undergoes self-decomposition 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.

[0357] In some embodiments, pAB undergoes self-decomposition after the cleavable β-glucuronide portion in the linker is partially cleaved. In some embodiments, the linker comprises β-glucuronide-pAB.

[0358] In some other embodiments, the splicing modulator is connected to a cleavable portion in the linker via a non-self-degrading spacer unit. In some embodiments, the splicing modulator is connected to a cleavable portion in the linker via a non-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 splicing modulator is connected to a cleavable portion in the linker via a non-self-degrading spacer unit, the cleavable portion comprising Val-Ala, and a maleic anhydride hexyl group (MC) binds the cleavable portion to an antibody or antigen-binding fragment.

[0359] In various forms, the antibody or antigen-binding fragment of the ADC binds to the splicing modulator drug moiety via a linker, wherein the linker comprises a Mal-spacer unit (e.g., MC), a cleavable amino acid unit, and pABC. In some embodiments, the spacer unit comprises an alkyl 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.

[0360] In various other forms, the antibody or antigen-binding fragment of the ADC is bound to the splicing modulator drug moiety via a linker, wherein the linker comprises a Mal-spacer unit (e.g., MC), a cleavable amino acid unit, and pAB. In some embodiments, the spacer unit comprises an alkyl 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.

[0361] In various other configurations, the antibody or antigen-binding fragment of the ADC binds to the splicing modulator drug moiety via a linker, wherein the linker comprises a Mal-spacer unit (e.g., MC), a cleavable β-glucuronide, and pABC. In some embodiments, the linker comprises a Mal-spacer unit-β-glucuronide-pABC. In some embodiments, the linker comprises MC-β-glucuronide-pABC.

[0362] In other embodiments, the antibody or antigen-binding fragment of the ADC binds to the splicing modulator drug moiety via a linker, wherein the linker comprises a Mal-spacer unit (e.g., MC), a cleavable β-glucuronide, and pAB. In some embodiments, the linker comprises a Mal-spacer unit-β-glucuronide-pAB. In some embodiments, the linker comprises MC-β-glucuronide-pAB.

[0363] In various embodiments, the ADC complex has formula (I): Ab-(LD)p (I) Among them, Ab is an antibody or antigen-binding fragment that targets neoplastic cells; D-series splice modifier; L-systems covalently connect Ab to the connector of D; and p is an integer from 1 to 15.

[0364] In some embodiments, the antibody or antigen-binding fragment (Ab) of the ADC binds to the splice modulator 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.

[0365] In some embodiments, the linker includes a cleavable portion arranged such that, after cleavage, neither the linker nor any portion of the antibody or antigen-binding fragment retains binding to the splicing regulator. In some embodiments, the cleavable portion is a cleavable peptide moiety, 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.

[0366] In some embodiments, the linker includes at least one spacer unit for attaching an antibody or antigen-binding fragment to a cleavable portion. In some embodiments, the linker includes at least one spacer unit for attaching an antibody or antigen-binding fragment to a drug portion. In some embodiments, the spacer unit or linker includes at least one alkyl portion.

[0367] 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.

[0368] In some embodiments, the Mal-spacer unit or connector 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 connector comprises Mal-(PEG)2. In some embodiments, the Mal-spacer unit or connector 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 connector comprises Mal-(PEG)2-CO. In some embodiments, the Mal-spacer unit or connector 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".

[0369] 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".

[0370] 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 “ADL12 linker”.

[0371] 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”.

[0372] 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.

[0373] 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.

[0374] In some embodiments, the spacer subunit connects the detachable portion of the connector to the shear modulator. In some embodiments, the spacer subunit connecting the detachable portion to the shear modulator is self-decomposing.

[0375] In some embodiments, the spacer subunit comprises a pABC. In some embodiments, the pABC connects a cleavable portion to a splicing 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.

[0376] In some embodiments, the linker comprises Val-Cit-pABC. In some embodiments, the linker comprises Val-Cit-pABC and an MC Mal-spacer unit for attaching 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".

[0377] In some embodiments, the linker comprises Val-Ala-pABC. In some embodiments, the linker comprises Val-Ala-pABC and an MC Mal-spacer unit for attaching 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.

[0378] In some embodiments, the linker comprises Glu-Val-Cit-pABC. In some embodiments, the linker comprises Glu-Val-Cit-pABC and an MC-Mal-spacer unit 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 the MC-Glu-Val-Cit-pABC linker are also referred to herein as "ADL23" or the "ADL23" linker.

[0379] In some embodiments, the linker comprises Ala-Ala-Asn-pABC. In some embodiments, the linker comprises Ala-Ala-Asn-pABC and an MC-Ala-Ala-Asn-pABC spacer unit for binding 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 "ADL21" linker.

[0380] In some other embodiments, the spacer subunit comprises a pAB. In some embodiments, the pAB connects a cleavable portion to a splicing 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.

[0381] In some embodiments, the linker comprises Val-Ala-pAB. In some embodiments, the linker comprises Val-Ala-pAB and an MC Mal-spacer unit for attaching 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.

[0382] In some embodiments, the linker comprises Val-Cit-pAB. In some embodiments, the linker comprises Val-Cit-pAB and an MC Mal-spacer unit for attaching 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.

[0383] In some embodiments, the linker comprises β-glucuronide-pABC. In some embodiments, the linker comprises β-glucuronide-pABC and an MC-Mal spacer unit for attaching 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 linkers are also referred to herein as "ADL13" or "ADL13" linkers.

[0384] In some embodiments, the linker comprises β-glucuronide-pAB. In some embodiments, the linker comprises β-glucuronide-pAB and an MC Mal-spacer unit for attaching the linker to an antibody or antigen-binding fragment. In some embodiments, the linker comprises MC-β-glucuronide-pAB.

[0385] In some embodiments, the antibody or antigen-binding fragment binds to the splice modulator drug portion via a linker of ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15. It has been found that, in various embodiments, ADCs comprising linkers of ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 and the splice modulator drug portion disclosed herein exhibit the desired characteristics of 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 compared to ADCs using other linker-loads. For example, in various embodiments, compared to ADCs using other linker-loads (e.g., the ADL10 linker and splice modulator drug portion), ADCs containing ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linkers and the splice modulator drug portion disclosed herein exhibit increased ability to inhibit the growth and / or proliferation of target cells. In various embodiments, compared to ADCs based on other splice modulators (e.g., thailanstatin A-based ADCs reported in Puthenveetil et al. Bioconjugate Chem. (2016) 27:1880-8), ADCs containing ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linkers and the splice modulator drug portion disclosed herein exhibit unexpectedly increased in vivo stability (e.g., plasma stability).

[0386] In some embodiments, by utilizing the linker-load conjugate with, for example, an anti-HER2 antibody such as trastuzumab; an anti-CD138 antibody such as B-B4; or an anti-EPHA2 antibody such as 1C1, good or superior functional properties can be observed provided by specific combinations of the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23 or ADL15 linker and the splice modulator drug portion disclosed herein.

[0387] In some embodiments, the ADC comprises an ADL1-splicing modulator and an antibody or antigen-binding fragment thereof that has the ability to maintain targeting and internalization of the vesicular cells. In some embodiments, the ADC comprises an ADL2-splicing modulator and an antibody or antigen-binding fragment thereof that has the ability to maintain targeting and internalization of the vesicular cells. In some embodiments, the ADC comprises an ADL5-splicing modulator and an antibody or antigen-binding fragment thereof that has the ability to maintain targeting and internalization of the vesicular cells. In some embodiments, the ADC comprises an ADL6-splicing modulator and an antibody or antigen-binding fragment thereof that has the ability to maintain targeting and internalization of the vesicular cells. In some embodiments, the ADC comprises an ADL7-splicing modulator and an antibody or antigen-binding fragment thereof that has the ability to maintain targeting and internalization of the vesicular cells. In some embodiments, the ADC comprises an ADL12-splicing modulator and an antibody or antigen-binding fragment thereof that has the ability to maintain targeting and internalization of the vesicular cells. In some embodiments, the ADC comprises an ADL13-splicing modulator and an antibody or antigen-binding fragment thereof that has the ability to maintain targeting and internalization of the vesicular cells. In some embodiments, the ADC comprises an ADL14-splicing modulator and an antibody or antigen-binding fragment thereof that has the ability to maintain targeting and internalization of the vesicular cells. In some embodiments, the ADC comprises an ADL15-splicing modulator and an antibody or antigen-binding fragment thereof that has the ability to maintain targeting and internalization of the vesicular cells.

[0388] In some embodiments, the ADC comprises an ADL1 splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing neoplasms. In some embodiments, the ADC comprises an ADL2 splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing neoplasms. In some embodiments, the ADC comprises an ADL5 splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing neoplasms. In some embodiments, the ADC comprises an ADL6 splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing neoplasms. In some embodiments, the ADC comprises an ADL7 splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing neoplasms. In some embodiments, the ADC comprises an ADL12 splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing neoplasms. In some embodiments, the ADC comprises an ADL13 splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing neoplasms. In some embodiments, the ADC comprises an ADL14 splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing neoplasms. In some embodiments, the ADC comprises an ADL15-splicing regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing neoplasms.

[0389] In some embodiments, the antibody or antigen-binding fragment targeting HER2-expressing neoplasms is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment targeting HER2-expressing neoplasms includes three heavy chain complementarity-determining regions (HCDRs) containing 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) containing the amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO:6 (LCDR3).

[0390] In some embodiments, the ADC has equation (I): Ab-(LD)p (I) in: (i) Ab is an anti-HER2 antibody or its antigen-binding fragment, comprising three heavy chain complementarity-determining regions (HCDRs) containing 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) containing the amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO:6 (LCDR3). (ii) D-series splice modifiers; (iii) The L series includes connectors of ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15; and (iv)p refers to integers from 1 to 15.

[0391] In some embodiments, the antibody or antigen-binding fragment thereof targeting HER2-expressing neoplasms includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 19 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody or antigen-binding fragment thereof targeting HER2-expressing neoplasms includes 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.

[0392] In some embodiments, the ADC comprises an ADL1 splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing neoplasms. In some embodiments, the ADC comprises an ADL2 splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing neoplasms. In some embodiments, the ADC comprises an ADL5 splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing neoplasms. In some embodiments, the ADC comprises an ADL6 splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing neoplasms. In some embodiments, the ADC comprises an ADL7 splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing neoplasms. In some embodiments, the ADC comprises an ADL12 splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing neoplasms. In some embodiments, the ADC comprises an ADL13 splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing neoplasms. In some embodiments, the ADC comprises an ADL14 splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing neoplasms. In some embodiments, the ADC comprises an ADL15-splicing regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing proliferative cells.

[0393] In some embodiments, the antibody or antigen-binding fragment targeting CD138-expressing neoplasms is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment targeting CD138-expressing neoplasms includes three heavy chain complementarity-determining regions (HCDRs) containing 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) containing the amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3).

[0394] In some embodiments, the ADC has equation (I): Ab-(LD)p (I) in: (i) Ab is an anti-CD138 antibody or its antigen-binding fragment, comprising three heavy chain complementarity-determining regions (HCDRs) containing 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) containing the amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3). (ii) D-series splice modifiers; (iii) The L series includes connectors of ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15; and (iv)p refers to integers from 1 to 15.

[0395] In some embodiments, an antibody or antigen-binding fragment thereof targeting CD138-expressing neoplasms includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 21 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 22. In some embodiments, an antibody or antigen-binding fragment thereof targeting CD138-expressing neoplasms includes a murine IgG2a heavy chain constant domain and a murine Igκ light chain constant domain. In some embodiments, an antibody or antigen-binding fragment thereof targeting CD138-expressing neoplasms includes a human IgG2a heavy chain constant domain and a human Igκ light chain constant domain. In some embodiments, the antibody system 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.

[0396] In some embodiments, the ADC comprises an ADL1 splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing hypertrophic cells. In some embodiments, the ADC comprises an ADL2 splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing hypertrophic cells. In some embodiments, the ADC comprises an ADL5 splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing hypertrophic cells. In some embodiments, the ADC comprises an ADL6 splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing hypertrophic cells. In some embodiments, the ADC comprises an ADL7 splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing hypertrophic cells. In some embodiments, the ADC comprises an ADL12 splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing hypertrophic cells. In some embodiments, the ADC comprises an ADL13 splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing hypertrophic cells. In some embodiments, the ADC comprises an ADL14 splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing hypertrophic cells. In some embodiments, the ADC comprises an ADL15-splicing regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing neoplasmic cells.

[0397] In some embodiments, the antibody or antigen-binding fragment targeting EPHA2-expressing neoplasms is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment targeting EPHA2-expressing neoplasms includes three heavy chain complementarity-determining regions (HCDRs) containing the amino acid sequences of SEQ ID NO: 13 (HCDR1), SEQ ID NO: 14 (HCDR2), and SEQ ID NO: 15 (HCDR3); and three light chain complementarity-determining regions (LCDRs) containing the amino acid sequences of SEQ ID NO: 16 (LCDR1), SEQ ID NO: 17 (LCDR2), and SEQ ID NO: 18 (LCDR3).

[0398] In some embodiments, the ADC has equation (I): Ab-(LD)p (I) in: (i) Ab is an anti-EPHA2 antibody or its antigen-binding fragment, comprising three heavy chain complementarity-determining regions (HCDRs) containing the amino acid sequences of SEQ ID NO: 13 (HCDR1), SEQ ID NO: 14 (HCDR2), and SEQ ID NO: 15 (HCDR3); and three light chain complementarity-determining regions (LCDRs) containing the amino acid sequences of SEQ ID NO: 16 (LCDR1), SEQ ID NO: 17 (LCDR2), and SEQ ID NO: 18 (LCDR3). (ii) D-series splice modifiers; (iii) The L series includes connectors of ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15; and (iv)p refers to integers from 1 to 15.

[0399] In some embodiments, the antibody or antigen-binding fragment thereof targeting EPHA2-expressing neoplasms includes 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 antibody or antigen-binding fragment thereof targeting EPHA2-expressing neoplasms includes a human IgG1 heavy chain constant domain and a human Igκ light chain constant domain. In some embodiments, the antibody system 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. [Drugs Section] []

[0400] The pharmaceutical portion (D) of the ADC described herein can be any chemotherapeutic agent. Useful types of chemotherapeutic agents include, for example, RNA splicing regulators. In some preferred embodiments, the pharmaceutical portion is a splicing regulator. Exemplary splicing regulator compounds will be described and illustrated herein.

[0401] In various embodiments, the pharmaceutical component is a splice modulator compound of formula (II): or a medically acceptable salt thereof, wherein: R1 is selected from the following groups: non-existent, hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic, -OC(=O)-(C1-C6 alkyl) group, and -CD3; R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic, and -OC(=O)-(C1-C6 alkyl) groups; and R4, R5, and R8 are each independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl) group, -OC(=O)-(C1-C6 alkyl) group, and C1-C6 alkyl; R6 and R7 are each independently selected from hydrogen, -O-R17, -OC(=O)-R17, -OC(=O)-NR15R16, C1-C6 alkyl groups, and -NR15R16; R15 and R16 are each independently selected from hydrogen, R17, -C(=O)-R17, and -C(=O)-O-R17; R17 is selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, benzyl, and C3-C8 heterocyclic groups; and The Z series is selected from... R1, R3, R4, R5, R6, R7, R8, R15, R16, and R17 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl) group, -NR15 / R16, C3-C8 cycloalkyl, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic group. At least one of R6 and R7 is a hydrogen atom.

[0402] In some embodiments, R1 is selected from hydrogen, C1-C4 alkyl, C1-C4 alkylcarboxylic acid groups, and C3-C8 cycloalkyl. In some embodiments, R1 is hydrogen. In some embodiments, R1 is C1-C4 alkyl. In some embodiments, R1 is methyl. In some embodiments, R1 is ethyl. In some embodiments, R1 is C1-C4 alkylcarboxylic acid groups. In some embodiments, R1 is -CH2CH2CH2CO2H. In some embodiments, R1 is C3-C8 cycloalkyl. In some embodiments, R1 is cycloheptyl.

[0403] In some embodiments, R3 is selected from hydrogen, C1-C4 alkyl, C1-C4 alkylalkoxy, C1-C4 alkylcarboxylic acid, and C1-C4 alkylhydroxy. In some embodiments, R3 is selected from hydrogen and C1-C4 alkylcarboxylic acid. In some embodiments, R3 is hydrogen. In some embodiments, R3 is a C1-C4 alkylcarboxylic acid. In some embodiments, R3 is -CH2CH2CO2H.

[0404] In some embodiments, R4 is selected from hydrogen, hydroxyl, -O-(C1-C4 alkyl) group, -OC(=O)-(C1-C4 alkyl) group, and C1-C4 alkyl. In some embodiments, R4 is hydrogen. In some embodiments, R4 is hydroxyl. In some embodiments, R4 is -O-(C1-C4 alkyl) group. In some embodiments, R4 is -OCH3. In some embodiments, R4 is -OCH2CH3. In some embodiments, R4 is -OC(=O)-(C1-C4 alkyl) group. In some embodiments, R4 is -OC(=O)-CH3. In some embodiments, R4 is -OC(=O)-CH2CH3. In some embodiments, R4 is C1-C4 alkyl. In some embodiments, R4 is methyl. In some embodiments, R4 is ethyl.

[0405] In some embodiments, R5 is selected from hydrogen, hydroxyl, -O-(C1-C4 alkyl) group, and C1-C4 alkyl. In some embodiments, R5 is hydrogen. In some embodiments, R5 is hydroxyl. In some embodiments, R5 is -O-(C1-C4 alkyl) group. In some embodiments, R5 is C1-C4 alkyl.

[0406] In some embodiments, R6 is hydrogen. In some embodiments, R7 is hydrogen. In some embodiments, R6 is hydrogen and R7 is -O-R17. In some embodiments, R6 is hydrogen and R7 is -OR17, wherein R17 is selected from hydrogen and C1-C4 alkyl groups. In some embodiments, R6 is hydrogen and R7 is -O-R17, wherein R17 is hydrogen. In some embodiments, R6 is -O-R17 and R7 is hydrogen. In some embodiments, R6 is -O-R17 and R7 is hydrogen, wherein R17 is selected from hydrogen and C1-C4 alkyl groups. In some embodiments, R6 is -O-R17 and R7 is hydrogen, wherein R17 is hydrogen. In some embodiments, R6 is hydrogen and R7 is -NR15 R16. In some embodiments, R6 is hydrogen and R7 is -NR15 R16, wherein R15 is H and R16 is selected from hydrogen, R17, -C(=O)-R17, and -C(=O)-O-R17. In some embodiments, R6 is hydrogen and R7 is -NR15 R16, wherein R15 is H and R16 is selected from hydrogen, R17, -C(=O)-R17, and -C(=O)-O-R17, wherein R17 is selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, and C3-C8 heterocyclic. In some embodiments, R6 is -O-R17. In some embodiments, R6 is -OC(=O)-R17. In some embodiments, R6 is C1-C6 alkyl. In some embodiments, R6 is C1-C4 alkyl. In some embodiments, R6 is C1 alkyl. In some embodiments, R6 is NR15 R16. In some embodiments, R7 is O-R17. In some embodiments, R7 is OC (=O)-R17. In some embodiments, R7 is C1-C6 alkyl. In some embodiments, R7 is C1-C4 alkyl. In some embodiments, R7 is C1 alkyl. In some embodiments, R7 is NR15 R16.

[0407] In some embodiments, R8 is selected from hydrogen, hydroxyl, -O-(C1-C4 alkyl) group, and (C1-C4 alkyl) group. In some embodiments, R8 is hydrogen. In some embodiments, R8 is hydroxyl. In some embodiments, R8 is -O-(C1-C4 alkyl) group. In some embodiments, R8 is -O-(C1 alkyl) group.

[0408] In some embodiments, R15 is hydrogen. In some embodiments, R15 is R17. In some embodiments, R15 is -C(=O)-R17. In some embodiments, R15 is -C(=O)-O-R17.

[0409] In some embodiments, R16 is hydrogen. In some embodiments, R16 is R17. In some embodiments, R16 is -C(=O)-R17. In some embodiments, R16 is -C(=O)-O-R17.

[0410] In some embodiments, R17 is selected from hydrogen, C1-C4 alkyl, C3-C6 cycloalkyl, and C3-C8 heterocyclic groups. In some embodiments, R17 is hydrogen. In some embodiments, R17 is C1-C4 alkyl. In some embodiments, R17 is C1 alkyl. In some embodiments, R17 is C3-C6 cycloalkyl. In some embodiments, R17 is C3 cycloalkyl. In some embodiments, R17 is C4 cycloalkyl. In some embodiments, R17 is C5 cycloalkyl. In some embodiments, R17 is C6 cycloalkyl. In some embodiments, R17 is C3-C8 heterocyclic group. In some embodiments, R17 is C3 heterocyclic group. In some embodiments, R17 is C4 heterocyclic group. In some embodiments, R17 is C5 heterocyclic group. In some embodiments, R17 is C6 heterocyclic group. In some embodiments, R17 is C7 heterocyclic group. In some embodiments, R17 is C8 heterocyclic group.

[0411] In some embodiments, the Z series In some embodiments, the Z series In some embodiments, the Z series In some embodiments, the Z series In some embodiments, the Z series .

[0412] In some embodiments, the splice modulator compound of formula (II) is connected to the linker L in an ADC of formula (I), as shown in formula (II-A): , Z' is selected from; and All other variable systems are defined with respect to equation (II).

[0413] In various other embodiments, the pharmaceutical fraction is a splice modulator compound of formula (IV): or a medically acceptable salt thereof, wherein: R1 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic, -OC(=O)-(C1-C6 alkyl) group, and -CD3; R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic, and -OC(=O)-(C1-C6 alkyl) groups; and R4, R5, and R8 are each independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl) group, -OC(=O)-(C1-C6 alkyl) group, and C1-C6 alkyl; R6 and R7 are each independently selected from hydrogen, -O-R17, -OC(=O)-R17, -OC(=O)-NR15R16, C1-C6 alkyl groups, and -NR15R16; R15 and R16 are each independently selected from hydrogen, R17, -C(=O)-R17, and -C(=O)-O-R17; and R17 is selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, benzyl, and C3-C8 heterocyclic groups; R1, R3, R4, R5, R6, R7, R8, R15, R16, and R17 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl) group, -NR15 / R16, C3-C8 cycloalkyl, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic group. At least one of R6 and R7 is a hydrogen atom.

[0414] In some embodiments, R1 is selected from hydrogen, C1-C4 alkyl, C1-C4 alkylcarboxylic acid groups, and C3-C8 cycloalkyl. In some embodiments, R1 is hydrogen. In some embodiments, R1 is C1-C4 alkyl. In some embodiments, R1 is methyl. In some embodiments, R1 is ethyl. In some embodiments, R1 is C1-C4 alkylcarboxylic acid groups. In some embodiments, R1 is -CH2CH2CH2CO2H. In some embodiments, R1 is C3-C8 cycloalkyl. In some embodiments, R1 is cycloheptyl.

[0415] In some embodiments, R3 is selected from hydrogen, C1-C4 alkyl, C1-C4 alkylalkoxy, C1-C4 alkylcarboxylic acid, and C1-C4 alkylhydroxy. In some embodiments, R3 is selected from hydrogen and C1-C4 alkylcarboxylic acid. In some embodiments, R3 is hydrogen. In some embodiments, R3 is a C1-C4 alkylcarboxylic acid. In some embodiments, R3 is -CH2CH2CO2H.

[0416] In some embodiments, R4 is selected from hydrogen, hydroxyl, -O-(C1-C4 alkyl) group, -OC(=O)-(C1-C4 alkyl) group, and C1-C4 alkyl. In some embodiments, R4 is hydrogen. In some embodiments, R4 is hydroxyl. In some embodiments, R4 is -O-(C1-C4 alkyl) group. In some embodiments, R4 is -OCH3. In some embodiments, R4 is -OCH2CH3. In some embodiments, R4 is -OC(=O)-(C1-C4 alkyl) group. In some embodiments, R4 is -OC(=O)-CH3. In some embodiments, R4 is -OC(=O)-CH2CH3. In some embodiments, R4 is C1-C4 alkyl. In some embodiments, R4 is methyl. In some embodiments, R4 is ethyl.

[0417] In some embodiments, R5 is selected from hydrogen, hydroxyl, -O-(C1-C4 alkyl) group, and C1-C4 alkyl. In some embodiments, R5 is hydrogen. In some embodiments, R5 is hydroxyl. In some embodiments, R5 is -O-(C1-C4 alkyl) group. In some embodiments, R5 is C1-C4 alkyl.

[0418] In some embodiments, R6 is hydrogen. In some embodiments, R7 is hydrogen. In some embodiments, R6 is hydrogen and R7 is -O-R17. In some embodiments, R6 is hydrogen and R7 is -OR17, wherein R17 is selected from hydrogen and C1-C4 alkyl groups. In some embodiments, R6 is hydrogen and R7 is -O-R17, wherein R17 is hydrogen. In some embodiments, R6 is -O-R17 and R7 is hydrogen. In some embodiments, R6 is -O-R17 and R7 is hydrogen, wherein R17 is selected from hydrogen and C1-C4 alkyl groups. In some embodiments, R6 is -O-R17 and R7 is hydrogen, wherein R17 is hydrogen. In some embodiments, R6 is hydrogen and R7 is -NR15 R16. In some embodiments, R6 is hydrogen and R7 is -NR15 R16, wherein R15 is H and R16 is selected from hydrogen, R17, -C(=O)-R17, and -C(=O)-O-R17. In some embodiments, R6 is hydrogen and R7 is -NR15 R16, wherein R15 is H and R16 is selected from hydrogen, R17, -C(=O)-R17, and -C(=O)-O-R17, wherein R17 is selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, and C3-C8 heterocyclic. In some embodiments, R6 is -O-R17. In some embodiments, R6 is -OC(=O)-R17. In some embodiments, R6 is C1-C6 alkyl. In some embodiments, R6 is C1-C4 alkyl. In some embodiments, R6 is C1 alkyl. In some embodiments, R6 series - NR15 R16.

[0419] In some embodiments, R7 is O-R17. In some embodiments, R7 is OC(=O)-R17. In some embodiments, R7 is C1-C6 alkyl. In some embodiments, R7 is C1-C4 alkyl. In some embodiments, R7 is C1 alkyl. In some embodiments, R7 is NR15-R16.

[0420] In some embodiments, R8 is selected from hydrogen, hydroxyl, -O-(C1-C4 alkyl) group, and (C1-C4 alkyl) group. In some embodiments, R8 is hydrogen. In some embodiments, R8 is hydroxyl. In some embodiments, R8 is -O-(C1-C4 alkyl) group. In some embodiments, R8 is -O-(C1 alkyl) group.

[0421] In some embodiments, R15 is hydrogen. In some embodiments, R15 is R17. In some embodiments, R15 is -C(=O)-R17. In some embodiments, R15 is -C(=O)-O-R17.

[0422] In some embodiments, R16 is hydrogen. In some embodiments, R16 is R17. In some embodiments, R16 is -C(=O)-R17. In some embodiments, R16 is -C(=O)-O-R17.

[0423] In some embodiments, R17 is selected from hydrogen, C1-C4 alkyl, C3-C6 cycloalkyl, and C3-C8 heterocyclic groups. In some embodiments, R17 is hydrogen. In some embodiments, R17 is C1-C4 alkyl. In some embodiments, R17 is C1 alkyl. In some embodiments, R17 is C3-C6 cycloalkyl. In some embodiments, R17 is C3 cycloalkyl. In some embodiments, R17 is C4 cycloalkyl. In some embodiments, R17 is C5 cycloalkyl. In some embodiments, R17 is C6 cycloalkyl. In some embodiments, R17 is C3-C8 heterocyclic group. In some embodiments, R17 is C3 heterocyclic group. In some embodiments, R17 is C4 heterocyclic group. In some embodiments, R17 is C5 heterocyclic group. In some embodiments, R17 is C6 heterocyclic group. In some embodiments, R17 is C7 heterocyclic group. In some embodiments, R17 is C8 heterocyclic group.

[0424] In some embodiments, the splice modulator compound of formula (IV) is connected to the linker L in an ADC of formula (I), as shown in formula (IV-A): .

[0425] In various other embodiments, the pharmaceutical fraction is a splice modulator compound of formula (VI): or a medically acceptable salt thereof, wherein: R1 and R9 are each independently selected from hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic, -OC(=O)-(C1-C6 alkyl) group, and -CD3; R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic and -OC(=O)-(C1-C6 alkyl) groups; R4, R5, and R8 are each independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl) group, -OC(=O)-(C1-C6 alkyl) group, and C1-C6 alkyl; R6 and R7 are each independently selected from hydrogen, -O-R17, -OC(=O)-R17, -OC(=O)-NR15R16, C1-C6 alkyl, -NR15R16, and linkers; R10 is selected from hydrogen, C1-C6 alkyl, -C(=O)-(C1-C6 alkyl) groups, and -CD3; R15 and R16 are each independently selected from hydrogen, R17, -C(=O)-R17, and -C(=O)-O-R17; R17 is selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, benzyl, and C3-C8 heterocyclic groups; and Series a: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; R1, R3, R4, R5, R6, R7, R8, R9, R10, R15, R16, and R17 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl) group, -NR15 R16, C3-C8 cycloalkyl, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic group; At least one of R6 and R7 is hydrogen; and R1 and R9 cannot both be absent at the same time.

[0426] In some embodiments, R1 is selected from hydrogen, C1-C4 alkyl, C1-C4 alkylcarboxylic acid groups, and C3-C8 cycloalkyl. In some embodiments, R1 is hydrogen. In some embodiments, R1 is C1-C4 alkyl. In some embodiments, R1 is methyl. In some embodiments, R1 is ethyl. In some embodiments, R1 is C1-C4 alkylcarboxylic acid groups. In some embodiments, R1 is -CH2CH2CH2CO2H. In some embodiments, R1 is C3-C8 cycloalkyl. In some embodiments, R1 is cycloheptyl.

[0427] In some embodiments, R3 is selected from hydrogen, C1-C4 alkyl, C1-C4 alkylalkoxy, C1-C4 alkylcarboxylic acid, and C1-C4 alkylhydroxy. In some embodiments, R3 is selected from hydrogen and C1-C4 alkylcarboxylic acid. In some embodiments, R3 is hydrogen. In some embodiments, R3 is a C1-C4 alkylcarboxylic acid. In some embodiments, R3 is -CH2CH2CO2H.

[0428] In some embodiments, R4 is selected from hydrogen, hydroxyl, -O-(C1-C4 alkyl) group, -OC(=O)-(C1-C4 alkyl) group, and C1-C4 alkyl. In some embodiments, R4 is hydrogen. In some embodiments, R4 is hydroxyl. In some embodiments, R4 is -O-(C1-C4 alkyl) group. In some embodiments, R4 is -OCH3. In some embodiments, R4 is -OCH2CH3. In some embodiments, R4 is -OC(=O)-(C1-C4 alkyl) group. In some embodiments, R4 is -OC(=O)-CH3. In some embodiments, R4 is -OC(=O)-CH2CH3. In some embodiments, R4 is C1-C4 alkyl. In some embodiments, R4 is methyl. In some embodiments, R4 is ethyl.

[0429] In some embodiments, R5 is selected from hydrogen, hydroxyl, -O-(C1-C4 alkyl) group, and C1-C4 alkyl. In some embodiments, R5 is hydrogen. In some embodiments, R5 is hydroxyl. In some embodiments, R5 is -O-(C1-C4 alkyl) group. In some embodiments, R5 is C1-C4 alkyl.

[0430] In some embodiments, R9 is selected from the absence of hydrogen, C1-C4 alkyl, -(C=O)-(C1-C4 alkyl) group, and -CD3. In some embodiments, R9 is absent. In some embodiments, R9 is hydrogen. In some embodiments, R9 is C1-C4 alkyl. In some embodiments, the C1-C4 alkyl is methyl. In some embodiments, the C1-C4 alkyl is ethyl. In some embodiments, R9 is -(C=O)-(C1-C4 alkyl) group. In some embodiments, the -(C=O)-(C1-C4 alkyl) group is -(C=O)-methyl. In some embodiments, R9 is -CD3.

[0431] In some embodiments, R10 is selected from hydrogen, C1-C4 alkyl, -(C=O)-(C1-C4 alkyl) group, and -CD3. In some embodiments, R10 is hydrogen. In some embodiments, R10 is C1-C4 alkyl. In some embodiments, the C1-C4 alkyl group is methyl. In some embodiments, the C1-C4 alkyl group is ethyl. In some embodiments, R10 is -(C=O)-(C1-C4 alkyl) group. In some embodiments, the -(C=O)-(C1-C4 alkyl) group is -(C=O)-methyl. In some embodiments, R10 is -CD3.

[0432] In some embodiments, R6 is hydrogen. In some embodiments, R7 is hydrogen. In some embodiments, R6 is hydrogen and R7 is -O-R17. In some embodiments, R6 is hydrogen and R7 is -OR17, wherein R17 is selected from hydrogen and C1-C4 alkyl groups. In some embodiments, R6 is hydrogen and R7 is -O-R17, wherein R17 is hydrogen. In some embodiments, R6 is -O-R17 and R7 is hydrogen. In some embodiments, R6 is -O-R17 and R7 is hydrogen, wherein R17 is selected from hydrogen and C1-C4 alkyl groups. In some embodiments, R6 is -O-R17 and R7 is hydrogen, wherein R17 is hydrogen. In some embodiments, R6 is hydrogen and R7 is -NR15 R16. In some embodiments, R6 is hydrogen and R7 is -NR15 R16, wherein R15 is H and R16 is selected from hydrogen, R17, -C(=O)-R17, and -C(=O)-O-R17. In some embodiments, R6 is hydrogen and R7 is -NR15 R16, wherein R15 is H and R16 is selected from hydrogen, R17, -C(=O)-R17, and -C(=O)-O-R17, wherein R17 is selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, and C3-C8 heterocyclic. In some embodiments, R6 is -O-R17. In some embodiments, R6 is -OC(=O)-R17. In some embodiments, R6 is C1-C6 alkyl. In some embodiments, R6 is C1-C4 alkyl. In some embodiments, R6 is C1 alkyl. In some embodiments, R6 series - NR15 R16.

[0433] In some embodiments, R7 is O-R17. In some embodiments, R7 is OC(=O)-R17. In some embodiments, R7 is C1-C6 alkyl. In some embodiments, R7 is C1-C4 alkyl. In some embodiments, R7 is C1 alkyl. In some embodiments, R7 is NR15-R16.

[0434] In some embodiments, R8 is selected from hydrogen, hydroxyl, -O-(C1-C4 alkyl) group, and (C1-C4 alkyl) group. In some embodiments, R8 is hydrogen. In some embodiments, R8 is hydroxyl. In some embodiments, R8 is -O-(C1-C4 alkyl) group. In some embodiments, R8 is -O-(C1 alkyl) group.

[0435] In some embodiments, R15 is hydrogen. In some embodiments, R15 is R17. In some embodiments, R15 is -C(=O)-R17. In some embodiments, R15 is -C(=O)-O-R17.

[0436] In some embodiments, R16 is hydrogen. In some embodiments, R16 is R17. In some embodiments, R16 is -C(=O)-R17. In some embodiments, R16 is -C(=O)-O-R17.

[0437] In some embodiments, R17 is selected from hydrogen, C1-C4 alkyl, C3-C6 cycloalkyl, and C3-C8 heterocyclic groups. In some embodiments, R17 is hydrogen. In some embodiments, R17 is C1-C4 alkyl. In some embodiments, R17 is C1 alkyl. In some embodiments, R17 is C3-C6 cycloalkyl. In some embodiments, R17 is C3 cycloalkyl. In some embodiments, R17 is C4 cycloalkyl. In some embodiments, R17 is C5 cycloalkyl. In some embodiments, R17 is C6 cycloalkyl. In some embodiments, R17 is C3-C8 heterocyclic group. In some embodiments, R17 is C3 heterocyclic group. In some embodiments, R17 is C4 heterocyclic group. In some embodiments, R17 is C5 heterocyclic group. In some embodiments, R17 is C6 heterocyclic group. In some embodiments, R17 is C7 heterocyclic group. In some embodiments, R17 is C8 heterocyclic group.

[0438] In some embodiments, a is a series 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, a is a series 1, 2, 3, 4, 5, or 6. In some embodiments, a is a series 1, 2, 3, 4, or 5. In some embodiments, a is a series 1, 2, 3, or 4. In some embodiments, a is a series 1, 2, or 3. In some embodiments, a is a series 1 or 2. In some embodiments, a is a series 1. In some embodiments, a is a series 2. In some embodiments, a is a series 3. In some embodiments, a is a series 4. In some embodiments, a is a series 5. In some embodiments, a is a series 6. In some embodiments, a is a series 7. In some embodiments, a is a series 8. In some embodiments, a is a series 9. In some embodiments, a is a series 10.

[0439] In some embodiments, the splice modulator compound of formula (VI) is connected to the linker L in an ADC of formula (I), as shown in formula (VI-A): .

[0440] In various other embodiments, the pharmaceutical fraction (VIII) is a splice modulator compound: or a medically acceptable salt thereof, wherein: R1 is selected from the following groups: non-existent, hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic, -OC(=O)-(C1-C6 alkyl) group, and -CD3; R3 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic and -OC(=O)-(C1-C6 alkyl) groups; R4 is selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl) group, -OC(=O)-(C1-C6 alkyl) group, and C1-C6 alkyl; and R10 is selected from 3 to 10-membered carbon rings and 3 to 10-membered heterocycles, each substituted by 0 to 3 Ra, wherein each Ra is independently selected from halogens, C1-C6 alkyl, -O-(C1-C6)alkyl, C1-C6 alkylalkoxy, C1-C6 alkylhydroxy, -S(=O)w-(4 to 7-membered heterocycles), 4 to 7-membered carbon rings and 4 to 7-membered heterocycles; R15 and R16 are each independently selected from hydrogen, R17, -C(=O)-R17, and -C(=O)-O-R17; and R17 is selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, benzyl, and C3-C8 heterocyclic groups; R1, R3, R4, R10, R15, R16, and R17 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl) group, -NR15 R16, C3-C8 cycloalkyl, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic group; and Each Ra is independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, -NR15 R16, C1-C6 alkyl, -(C=O)-(C1-C6 alkyl) group, -(C=O)-(C1-C6 alkyl)-(C3-C10 heterocyclic) and C1-C6 alkyl carboxylic acid group, each of which is substituted by 0, 1 or 2 groups independently selected from the following groups: halogen, hydroxyl, -NR15 R16 and C1-C3 alkyl; and w is 0, 1, or 2.

[0441] In some embodiments, R1 is selected from the absence of hydrogen, C1-C4 alkyl, C1-C4 alkylcarboxylic acid groups, and C3-C8 cycloalkyl groups. In some embodiments, R1 is hydrogen. In some embodiments, R1 is C1-C4 alkyl. In some embodiments, R1 is methyl. In some embodiments, R1 is ethyl. In some embodiments, R1 is C1-C4 alkylcarboxylic acid groups. In some embodiments, R1 is -CH2CH2CH2CO2H. In some embodiments, R1 is C3-C8 cycloalkyl. In some embodiments, R1 is cycloheptyl.

[0442] In some embodiments, R3 is selected from hydrogen, C1-C4 alkyl, C1-C4 alkylalkoxy, C1-C4 alkylcarboxylic acid, and C1-C4 alkylhydroxy. In some embodiments, R3 is selected from hydrogen and C1-C4 alkylcarboxylic acid. In some embodiments, R3 is hydrogen. In some embodiments, R3 is a C1-C4 alkylcarboxylic acid. In some embodiments, R3 is -CH2CH2CO2H.

[0443] In some embodiments, R4 is selected from hydrogen, hydroxyl, -O-(C1-C4 alkyl) group, -OC(=O)-(C1-C4 alkyl) group, and C1-C4 alkyl. In some embodiments, R4 is hydrogen. In some embodiments, R4 is hydroxyl. In some embodiments, R4 is -O-(C1-C4 alkyl) group. In some embodiments, R4 is -OCH3. In some embodiments, R4 is -OCH2CH3. In some embodiments, R4 is -OC(=O)-(C1-C4 alkyl) group. In some embodiments, R4 is -OC(=O)-CH3. In some embodiments, R4 is -OC(=O)-CH2CH3. In some embodiments, R4 is C1-C4 alkyl. In some embodiments, R4 is methyl. In some embodiments, R4 is ethyl.

[0444] In some embodiments, R10 is selected from 6 to 9-membered carbon rings and 6 to 9-membered heterocycles, each of which is substituted by 0 to 2 Ra groups, wherein each Ra group is independently substituted by 0 to 3 groups independently selected from: halogen, hydroxyl, C1-C6 alkyl, -(C=O)-(C1-C6 alkyl) group, -(C=O)-(C1-C6 alkyl)-(3 to 10-membered heterocycle) group and C1-C6 alkyl carboxylic acid group.

[0445] In some embodiments, the carbocyclic system is a phenyl group substituted with 0 to 2 Ra groups, wherein each Ra group is independently substituted with 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -(C=O)-(C1-C6 alkyl) group, -(C=O)-(C1-C6 alkyl)-(3 to 10-membered heterocyclic) group, and C1-C6 alkyl carboxylic acid group. In some embodiments, the phenyl group is substituted with 2 Ra groups, wherein each Ra group is independently substituted with 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -(C=O)-(C1-C6 alkyl) group, -(C=O)-(C1-C6 alkyl)-(3 to 10-membered heterocyclic) group, and C1-C6 alkyl carboxylic acid group. In some embodiments, the phenyl system is... .

[0446] In some embodiments, the heterocycle is a 9-membered heterocycle substituted with 0 to 2 Ra groups, wherein each Ra group is independently substituted with 0 to 3 groups independently selected from: halogen, hydroxyl, C1-C6 alkyl, -(C=O)-(C1-C6 alkyl) group, -(C=O)-(C1-C6 alkyl)-(3 to 10-membered heterocycle) group, and C1-C6 alkyl carboxylic acid group. In some embodiments, the 9-membered heterocycle is... .

[0447] In some embodiments, Ra is selected from halogens, 3 to 10-membered carbon rings, and 3 to 10-membered heterocycles, wherein each Ra is independently substituted by 0 to 3 groups independently selected from: halogens, hydroxyl groups, C1-C6 alkyl groups, -(C=O)-(C1-C6 alkyl) groups, -(C=O)-(C1-C6 alkyl)-(3 to 10-membered heterocycle) groups, and C1-C6 alkyl carboxylic acid groups. In some embodiments, Ra is selected from halogens.

[0448] In some embodiments, R15 is hydrogen. In some embodiments, R15 is R17. In some embodiments, R15 is -C(=O)-R17. In some embodiments, R15 is -C(=O)-O-R17.

[0449] In some embodiments, R16 is hydrogen. In some embodiments, R16 is R17. In some embodiments, R16 is -C(=O)-R17. In some embodiments, R16 is -C(=O)-O-R17.

[0450] In some embodiments, R17 is selected from hydrogen, C1-C4 alkyl, C3-C6 cycloalkyl, and C3-C8 heterocyclic groups. In some embodiments, R17 is hydrogen. In some embodiments, R17 is C1-C4 alkyl. In some embodiments, R17 is C1 alkyl. In some embodiments, R17 is C3-C6 cycloalkyl. In some embodiments, R17 is C3 cycloalkyl. In some embodiments, R17 is C4 cycloalkyl. In some embodiments, R17 is C5 cycloalkyl. In some embodiments, R17 is C6 cycloalkyl. In some embodiments, R17 is C3-C8 heterocyclic group. In some embodiments, R17 is C3 heterocyclic group. In some embodiments, R17 is C4 heterocyclic group. In some embodiments, R17 is C5 heterocyclic group. In some embodiments, R17 is C6 heterocyclic group. In some embodiments, R17 is C7 heterocyclic group. In some embodiments, R17 is C8 heterocyclic group.

[0451] In some embodiments, the splice modulator compound of formula (VIII) is connected to the linker L in an ADC of formula (I), as shown in formula (VIII-A): .

[0452] In various embodiments, the pharmaceutical component is selected from splice modulators of D2 and D1.

[0453] In various embodiments, the drug portion is D2. The structure of the D2 drug portion used in the disclosed ADC is shown below in various embodiments: .

[0454] In various embodiments, the linker in the ADC described herein (e.g., the ADC of formula (I)) is covalently linked to the D2 drug moiety via an amine on a piperazine group. In various embodiments, the drug moiety is a D2 derivative. In various embodiments, the D2 derivative retains at least one biological function or activity of D2 (e.g., SF3b complex binding, in vitro splicing activity, cytotoxicity), but has a modified chemical structure.

[0455] In various embodiments, the pharmaceutical portion is D1 or a pharmaceutically acceptable salt thereof. The structure of the D1 pharmaceutical portion used in the disclosed ADC is shown below in various embodiments: .

[0456] In various embodiments, the linker in the ADC described herein (e.g., the ADC of formula (I)) is covalently linked to the D1 drug moiety via an amine on a piperazine group. In various embodiments, the drug moiety is a D1 derivative. In various embodiments, the D1 derivative retains at least one biological function or activity of D1 (e.g., SF3b complex binding, in vitro splicing activity, cytotoxicity), but has a modified chemical structure.

[0457] In some embodiments, the splice modulator comprises D1: .

[0458] In some embodiments, the splice modifier comprises D2: .

[0459] In some embodiments, the splice modifier includes D3: .

[0460] In some embodiments, the splice modifier includes D4: .

[0461] In some embodiments, the splice modifier comprises D4': .

[0462] In some embodiments, the splice modifier includes D5: (D5)

[0463] In some embodiments, the splice modifier includes D6: .

[0464] In some embodiments, the splice modifier includes D7: .

[0465] In some embodiments, the splice modifier includes D8: .

[0466] In some embodiments, the splice modifier includes D9: .

[0467] In some embodiments, the splice modifier includes D10: .

[0468] In some embodiments, the splice modifier includes D11: .

[0469] In some embodiments, the splice modifier comprises D12: .

[0470] In some embodiments, the splice modifier includes D13: .

[0471] In some embodiments, the splice modifier comprises D14: .

[0472] In some embodiments, the splice modifier includes D15: .

[0473] In some embodiments, the splice modifier comprises D16: .

[0474] In some embodiments, the splice modifier comprises D17: .

[0475] In some embodiments, the splice modifier comprises D18: .

[0476] In some embodiments, the splice modifier comprises D19: .

[0477] In some embodiments, the splice modifier comprises D20: .

[0478] In some embodiments, the splice modifier includes D21: .

[0479] In some embodiments, the splice modifier comprises D22: .

[0480] In some embodiments, the splice modifier includes D23: .

[0481] In some embodiments, the splice modifier comprises D24: .

[0482] In some embodiments, the splice modifier comprises D25: .

[0483] In some embodiments, the splice modifier comprises D26: .

[0484] In some embodiments, the splice modifier comprises D27: .

[0485] In some embodiments, the splice modifier includes D28: .

[0486] In some embodiments, the splice modifier includes D29: .

[0487] In some embodiments, the splice modifier comprises D30: .

[0488] In some embodiments, the splice modifier includes D31: .

[0489] In some embodiments, the splice modifier comprises D32: .

[0490] In some embodiments, the splice modifier includes D33: .

[0491] In some embodiments, the splice modifier includes D34: .

[0492] In some embodiments, the splice modifier comprises D35: .

[0493] An exemplary ADC has the formula (I): Ab-(LD)p (I) where Ab is an antibody or antigen-binding fragment that targets neoplastic cells; D series D2; L-systems covalently connect Ab to the connector of D; and p is an integer from 1 to 15.

[0494] 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.

[0495] In some embodiments, the antibody or antigen-binding fragment targets HER2-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-HER2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment includes three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) containing 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) containing 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 containing the amino acid sequence of SEQ ID NO:19 and a light chain variable region containing 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.

[0496] In some other embodiments, the antibody or antigen-binding fragment targets CD138-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-CD138 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment includes three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) containing 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) containing 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 containing the amino acid sequence of SEQ ID NO: 21 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody or antigen-binding fragment includes a murine IgG2a heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a murine Igκ light chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG2a heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Igκ light chain constant region.

[0497] In some other embodiments, the antibody or antigen-binding fragment targets EPHA2-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-EPHA2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment includes three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) containing 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) containing 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 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 antibody or antigen-binding fragment includes a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a constant region of the human Igκ light chain.

[0498] In some other embodiments, the antibody or antigen-binding fragment targets MSLN-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-MSLN antibody or antigen-binding fragment.

[0499] In some other embodiments, the antibody or antigen-binding fragment targets FOLH1-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-FOLH1 antibody or antigen-binding fragment.

[0500] In some other embodiments, the antibody or antigen-binding fragment targets CDH6-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-CDH6 antibody or antigen-binding fragment.

[0501] In some other embodiments, the antibody or antigen-binding fragment targets CEACAM5-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-CEACAM5 antibody or antigen-binding fragment.

[0502] In some other embodiments, the antibody or antigen-binding fragment targets CFC1B phenotypical cells. In some embodiments, the antibody or antigen-binding fragment is an anti-CFC1B antibody or antigen-binding fragment.

[0503] In some other embodiments, the antibody or antigen-binding fragment targets ENPP3-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-ENPP3 antibody or antigen-binding fragment.

[0504] In some other embodiments, the antibody or antigen-binding fragment targets FOLR1-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-FOLR1 antibody or antigen-binding fragment.

[0505] In some other embodiments, the antibody or antigen-binding fragment targets HAVCR1-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-HAVCR1 antibody or antigen-binding fragment.

[0506] In some other embodiments, the antibody or antigen-binding fragment targets KIT-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-KIT antibody or antigen-binding fragment.

[0507] In some other embodiments, the antibody or antigen-binding fragment targets MET-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-MET antibody or antigen-binding fragment.

[0508] In some other embodiments, the antibody or antigen-binding fragment targets MUC16 phenotypical cells. In some embodiments, the antibody or antigen-binding fragment is an anti-MUC16 antibody or antigen-binding fragment.

[0509] In some other embodiments, the antibody or antigen-binding fragment targets SLC39A6 phenotypical cells. In some embodiments, the antibody or antigen-binding fragment is an anti-SLC39A6 antibody or antigen-binding fragment.

[0510] In some other embodiments, the antibody or antigen-binding fragment targets SLC44A4 phenotypical cells. In some embodiments, the antibody or antigen-binding fragment is an anti-SLC44A4 antibody or antigen-binding fragment.

[0511] In some other embodiments, the antibody or antigen-binding fragment targets STEAP1-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-STEAP1 antibody or antigen-binding fragment.

[0512] 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 comprises the following linkers: 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 of ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15. In some embodiments, L is a linker of ADL12, ADL14, or ADL15. In some embodiments, the connectors ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 may also include one or more additional spacer subunits.

[0513] Another example of an exemplary ADC has equation (I): Ab-(LD)p (I) in Ab refers to antibodies or antigen-binding fragments that target neoplastic cells; D series D1; L-systems covalently connect Ab to the connector of D; and p is an integer from 1 to 15.

[0514] 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.

[0515] In some embodiments, the antibody or antigen-binding fragment targets HER2-expressing cells. In some embodiments, the antibody or antigen-binding fragment is an anti-HER2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment includes three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) containing 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) containing 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 containing the amino acid sequence of SEQ ID NO:19 and a light chain variable region containing the amino acid sequence of SEQ ID NO:20. In some embod...

Claims

1. A compound represented by formula (III) or a pharmaceutically acceptable salt thereof, wherein: Z'' is selected from: R1 is selected from: non-existent, hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic, -OC(=O)-(C1-C6 alkyl) group and -CD3; R2 contains a linker L that can cleave a dipeptide or tripeptide moiety; R3 is selected from: hydrogen, C1-C6 alkyl, C1-C6 alkylalkoxy, C1-C6 alkylamino, C1-C6 alkylcarboxylic acid, C1-C6 alkylhydroxy, C3-C8 cycloalkyl, benzyl, C3-C8 heterocyclic and -OC(=O)-(C1-C6 alkyl) group; R4, R R5 and R8 are each independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl) group, -OC(=O)-(C1-C6 alkyl) group, and C1-C6 alkyl; R6 and R7 are each independently selected from hydrogen, -OR17, -OC(=O)-R17, -OC(=O)-NR15R16, C1-C6 alkyl, and -NR15R16; R15 and R16 are each independently selected from hydrogen, R17, -C(=O)-R17, and -C(=O)-OR17; and R17 is selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, benzyl, and C3-C8 heterocyclic group; wherein R1, R3, R4, R5, R6, R7, and R 8 are each independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl) group, -NR15R16, C3-C8 cycloalkyl, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl and C3-C8 heterocyclic; wherein at least one of R6 and R7 is hydrogen; and wherein if Z'' is hydrogen, R3, R6 and R7 are hydrogen, R4 is methoxy and R5 is hydroxyl, then R1 is neither present nor methyl.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein: Z'' is selected from: R1 is selected from non-existent, hydrogen, C1-C4 alkyl, C1-C4 alkylcarboxylic acid group, C1-C4 alkylhydroxy and C3-C8 cycloalkyl; R3 is selected from hydrogen, C1-C4 alkyl, C1-C4 alkylalkoxy, C1-C4 alkylcarboxylic acid group and C1-C4 alkylhydroxy; R4 is selected from hydrogen, hydroxyl, -O-(C1-C4 alkyl) group, -OC(=O)-(C1-C4 alkyl) group and C1-C4 alkyl; R5 is selected from hydrogen, hydroxyl, -O-(C1-C4 alkyl) group and C1-C4 alkyl; R6 is selected from hydrogen, -OR17, -OC(=O)-R17 and C1-C4 alkyl; R7 is selected from hydrogen, -OR17, -OC(=O)-R17 and C1-C4 alkyl.

17. -OC(=O)-NR 15R 16, C1-C6 alkyl and -NR 15R 16; R8 is selected from hydrogen, hydroxyl, -O-(C1-C4 alkyl) group and C1-C4 alkyl; R17 is selected from hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, benzyl and C3-C8 heterocyclic group; wherein R1, R3, R4, R5, R6, R7 and R8 are each independently substituted by 0 to 3 independently selected from the following groups: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl) group, -NR 15R 16, C3-C8 cycloalkyl, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl and C3-C8 heterocyclic group; wherein R At least one of R6 and R7 is hydrogen; and if Z'' is hydrogen, R3, R6 and R7 are hydrogen, R4 is methoxy and R5 is hydroxyl, then R1 is neither present nor methyl.

3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein: The Z'' series; R1 series is selected from non-existent, hydrogen, methyl, and C1-C4 alkylcarboxylic acid groups; R3 series is selected from hydrogen and C1-C4 alkylcarboxylic acid groups; R4 series is selected from hydrogen, hydroxyl, -O-(C1-C4 alkyl) group, -OC(=O)-(C1-C4 alkyl) group, and C1-C4 alkyl; R5 series is selected from hydrogen and hydroxyl; R6 series is hydrogen; R7 series is hydrogen; R8 series is selected from hydrogen and hydroxyl; and wherein R1, R3, and R4 are each independently substituted by 0 to 3 independently selected groups from the following: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl) group, C3-C8 cycloalkyl, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic group; and wherein if R3, R6, and R If R is a 7-series H, R is a 4-series methoxy group and R is a 5-series hydroxyl group, then R1 is neither present nor methyl.

4. The compound or its pharmaceutically acceptable salt as claimed in claim 1 is selected from compounds and their pharmaceutically acceptable salts according to the following formula.

5. The compound or its pharmaceutically acceptable salt as claimed in claim 1 is selected from compounds and their pharmaceutically acceptable salts according to the following formula.

6. A compound or a pharmaceutically acceptable salt thereof as claimed in any of claims 1 to 5, wherein the cleavable dipeptide portion comprises valine-citrulline (Val-Cit) or valine-alanine (Val-Ala), or wherein the cleavable tripeptide portion comprises glutamate-valine-citrulline (Glu-Val-Cit) or alanine-alanine-aspartic acid (Ala-Ala-Asn).

7. The compound of claim 6 or a pharmaceutically acceptable salt thereof, wherein the linker L comprises a maleic diamide (Mal) moiety.

8. The compound of claim 7 or a pharmaceutically acceptable salt thereof, wherein the Mal moiety comprises maleic anhydride (MC).

9. The compound of claim 8 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.

10. The compound of claim 6 or a pharmaceutically acceptable salt thereof, wherein the linker L comprises at least one spacer unit comprising: (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.

11. The compound of claim 10 or a pharmaceutically acceptable salt thereof, wherein the at least one spacer subunit is connected to the maleic diimidamine (Mal) moiety ("Mal-spacer subunit").

12. The compound of claim 6 or a pharmaceutically acceptable salt thereof, wherein the cleavable dipeptide portion or the cleavable tripeptide portion of the linker L comprises valine (Val) linked to alanine (Ala), Ala being covalently bonded to the compound of formula (III) directly or via any spacer subunit.

13. The compound of claim 6 or a pharmaceutically acceptable salt thereof, wherein the cleavable dipeptide portion or the cleavable tripeptide portion in the linker L comprises valine (Val) linked to citrulline (Cit), Cit being covalently bonded to the compound of formula (III) directly or via any spacer subunit.

14. The compound of claim 6 or a pharmaceutically acceptable salt thereof, wherein the connector L is directly connected to the splice modulator, or the connector L is connected to the splice modulator by a spacer unit.

15. The compound of claim 14 or a pharmaceutically acceptable salt thereof, wherein the spacer unit of the linker L to which the cleavable dipeptide portion or the cleavable tripeptide portion is linked to the splicing regulator is self-degrading.

16. The compound of claim 14 or a pharmaceutically acceptable salt thereof, wherein the spacer unit of the linker L to which the cleavable dipeptide portion or the cleavable tripeptide portion is linked to the splice modulator comprises p-aminobenzoxycarbonyl (pABC) or p-aminobenzyl (pAB).

17. The compound of claim 16 or a pharmaceutically acceptable salt thereof, wherein the pABC or pAB links the cleavable dipeptide portion or the cleavable tripeptide portion of the linker L to the splicing regulator.

18. The compound of claim 17 or a pharmaceutically acceptable salt thereof, wherein the cleavable dipeptide portion comprises Val-Cit or Val-Ala, or the cleavable tripeptide portion comprises Glu-Val-Cit or Ala-Ala-Asn.

19. The compound of claim 18 or a pharmaceutically acceptable salt thereof, wherein the linker L comprises Val-Cit-pABC, Val-Ala-pABC, Glu-Val-Cit-pABC, Ala-Ala-Asn-pABC, Val-Cit-pAB, Val-Ala-pAB, Glu-Val-Cit-pAB, or Ala-Ala-Asn-pAB.

20. The compound of claim 19 or a pharmaceutically acceptable salt thereof, wherein the linker L comprises MC-Val-Cit-pABC, MC-Val-Ala-pABC, MC-Glu-Val-Cit-pABC, MC-Ala-Ala-Asn-pABC, MC-Val-Cit-pAB, MC-Val-Ala-pAB, MC-Glu-Val-Cit-pAB, or MC-Ala-Ala-Asn-pAB.

21. A compound or a pharmaceutically acceptable salt thereof, as claimed in any of claims 1 to 5, wherein the linker L comprises: or.

22. A pharmaceutical composition comprising a compound of any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

23. The composition of claim 22 is for treating an individual who has or is suspected of having a neoplastic disease, wherein the neoplastic disease is: (i) a hematologic malignancy selected from B-cell malignancies, acute myeloid leukemia and multiple myeloma; or (ii) a solid tumor selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer and esophageal cancer.

24. An antibody-drug conjugate of formula (I), Ab-(LD) p (I) wherein Ab is an antibody or antigen-binding fragment targeting proliferative cells; LD comprises a compound as claimed in any one of claims 1 to 21; and p is an integer from 1 to 15.

25. The antibody-drug conjugate of claim 24, wherein the linker L comprises MC-Val-Cit, MC-Val-Ala, MC-Glu-Val-Cit, or MC-Ala-Ala-Asn.

26. The antibody-drug conjugate of claim 24, wherein the linker L comprises at least one spacer unit having a p-aminobenzyloxycarbonyl (pABC) or p-aminobenzyl (pAB), the pABC or pAB being a linker unit to the cleavable dipeptide portion or the cleavable tripeptide portion of the linker L for splicing regulation.

27. The antibody-drug conjugate of claim 26, wherein the linker L comprises Val-Cit-pABC, Val-Ala-pABC, Glu-Val-Cit-pABC, Ala-Ala-Asn-pABC, Val-Cit-pAB, Val-Ala-pAB, Glu-Val-Cit-pAB, or Ala-Ala-Asn-pAB.

28. The antibody-drug conjugate of claim 27, wherein the linker L comprises MC-Val-Cit-pABC, MC-Val-Ala-pABC, MC-Glu-Val-Cit-pABC, MC-Ala-Ala-Asn-pABC, MC-Val-Cit-pAB, MC-Val-Ala-pAB, MC-Glu-Val-Cit-pAB, or MC-Ala-Ala-Asn-pAB.

29. The antibody-drug conjugate as requested in item 24, wherein p is an integer from 1 to 10.

30. The antibody-drug conjugate as requested in item 29, wherein p is an integer from 2 to 8.

31. The antibody-drug conjugate as claimed in claim 29, wherein p is an integer from 4 to 8.

32. The antibody-drug conjugate of claim 24, wherein the antibody or antigen-binding fragment targets proliferative cells derived from hematologic malignancies or solid tumors, wherein the hematologic malignancies are selected from B-cell malignancies, acute myeloid leukemia, and multiple myeloma, and the solid tumors are selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer, and esophageal cancer.

33. The antibody-drug conjugate of claim 24, wherein a) the antibody or antigen-binding fragment targets HER2-expressing cells, wherein the antibody or antigen-binding fragment is an anti-HER2 antibody or antigen-binding fragment; and / or the antibody or antigen-binding fragment includes three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) of 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) of the amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO:6 (LCDR3); or the antibody or antigen-binding fragment includes a heavy chain variable region of the amino acid sequence of SEQ ID NO:19 and a light chain variable region of the amino acid sequence of SEQ ID NO:20; b) The antibody or antigen-binding fragment targets CD138-expressing cells, wherein the antibody or antigen-binding fragment is an anti-CD138 antibody or antigen-binding fragment; and / or the antibody or antigen-binding fragment includes three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) of 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) of the amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2), and SEQ ID NO: 12 (LCDR3); or the antibody or antigen-binding fragment includes a heavy chain variable region of the amino acid sequence of SEQ ID NO: 21 and a light chain variable region of the amino acid sequence of SEQ ID NO: 22; c) The antibody or antigen-binding fragment targets EPHA2-expressing cells, wherein the antibody or antigen-binding fragment is an anti-EPHA2 antibody or antigen-binding fragment; and / or the antibody or antigen-binding fragment includes three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) containing 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) containing 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 includes 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; d) the antibody or antigen-binding fragment targets CEACAM5-expressing cells, wherein the antibody or antigen-binding fragment is an anti-CEACAM5 antibody or antigen-binding fragment; or e) the antibody or antigen-binding fragment targets STEAP1-expressing cells, wherein the antibody or antigen-binding fragment is an anti-STEAP1 antibody or antigen-binding fragment.

34. The antibody-drug conjugate of claim 33, wherein the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region and / or the antibody or antigen-binding fragment comprises a human Igκ light chain constant region.

35. A composition comprising a plurality of copies of an antibody-drug conjugate as claimed in claim 24, wherein the average p-value of the antibody-drug conjugate in the composition is about 3.5 to about 5.5, or about 7 to about 9, or about 4, or about 8.

36. An antibody-drug conjugate as claimed in claim 24, for the treatment of an individual who has or is suspected of having a neoplastic disease, wherein the neoplastic disease is: (i) a hematologic malignancy selected from B-cell malignancies, acute myeloid leukemia, and multiple myeloma; or (ii) a solid tumor selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct cancer, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer, and esophageal cancer.

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