Hoplite antibody-drug conjugates and methods of use

Abstract

This disclosure relates to linker-drug compounds and antibody-drug conjugates that bind to human oncology targets. These linker-drug compounds and antibody-drug conjugates comprise a hoprene splice regulator drug moiety. This disclosure further relates to methods and compositions for treating neoplastic conditions by administering such antibody-drug conjugates provided herein. Hoprene itself is also claimed. Further claims are made to its use, and to the use of neoantigens generated from hoprene or its ADCs, or vaccines against such neoantigens.

Description

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

[0002] This disclosure relates to antibody-drug conjugates (ADCs) comprising a herboxidiene splicing regulator and an antibody or an antigen-binding fragment thereof, such as antibody-drug conjugates binding to human oncology antigen targets. This disclosure further relates to methods and compositions that can be used to treat or diagnose cancers expressing target antigens and / or those suitable for treatment via RNA splicing disruption, and methods for manufacturing those compositions.

[0003] 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 alternating splicing with different combinations of exons, mRNAs encoding unique protein variants are produced.

[0004] RNA splicing is catalyzed by the spliceosome, a dynamic multi-protein-RNA complex composed of five small nuclear RNAs (snRNAs U1, U2, U4, U5, and U6) and associated proteins. The spliceosome assembles on pre-mRNA to establish a dynamic cascade of multiple RNA and protein interactions that catalyze intron excision and exon conjugation (Matera and Wang (2014) Nat Rev Mol Cell Biol. [Nature Review of Molecular and Cell Biology] 15(2):108-21). Increasing evidence links human diseases to dysregulation of RNA splicing affecting many genes (Scotti and Swanson (2016) Nat Rev Genet. [Nature Reviews Genetics] 17(1):19-32).

[0005] The spliceosome is an important target in cancer biology. Several studies have documented splicing profiles in cancer cells and numerous alterations in their splicing factors (Agrawal et al. (2018) Curr Opin Genet Dev. [Recent Views in Genetics and Development] 48:67-74). Alternating splicing can cause differential exon inclusion / exclusion, intron retention, or hidden splice site use (Seiler et al. (2018) Cell Rep. [Cell Reports] 23(1):282-296). In summary, these events result in functional alterations that may contribute to tumorigenesis or therapy resistance (Siegfried and Karni (2018) Curr Opin Genet Dev. [Recent Views in Genetics and Development] 48:16-21).

[0006] Certain natural products can bind to the SF3b spliceosome complex. These small molecules regulate splicing by promoting intron retention and / or exon skipping (Teng et al. (2017) Nat Commun. [Nature Communications] 8:15522). For example, naturally occurring polyketides of Hopschiene (Isaac et al. (1992) J. Org. Chem. [Journal of Organic Chemistry] 57:7220-26) and their derivatives have been shown to regulate splicing. See, for example, Imaizumi et al. (2017) J. Antibiot. [Journal of Antibiotics] 70:675-79. A significant portion of the resulting transcripts contain premature stop codons, triggering nonsense-mediated mRNA decay (NMD). Furthermore, due to impaired typical splicing, typical transcripts are significantly reduced, which may adversely affect cell function and viability. For this reason, splice regulators have become a promising class of drugs for the treatment of cancer (Puthenveetil et al. (2016) Bioconjugate Chem. [Bioconjugate Chemistry] 27:1880-8).

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

[0008] CD138 is a cell surface heparan sulfate proteoglycan essential for maintaining cell morphology and interaction with the surrounding microenvironment (Akl et al. (2015) Oncotarget 6(30):28693-715; Szatmári et al. (2015) Dis Markers 2015:796052). Generally, loss of CD138 expression in cancer cells reduces cell adhesion to the extracellular matrix and enhances cell motility and invasiveness (Teng et al. (2012) Matrix Biol 31:3-16). Increased matrix CD138 expression also alters fibronectin production and extracellular matrix organization (Yang et al. (2011) Am J Pathol 178:325-35). Furthermore, increased CD138 expression in stromal fibroblasts is associated with angiogenesis and cancer development (Maeda et al. (2006) Oncogene 25:1408-12). CD138 expression increases during B cell development and its presence is a marker of plasma cells (Ribatti (2017) Immunol Lett. 188:64-7). CD138 expression is maintained in multiple myeloma, a plasma cell malignancy. Therefore, CD138 is a 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).

[0009] EPHA2 is a transmembrane glycoprotein that is adequately overexpressed in several malignant cancer-derived cell lines and in advanced cancer forms (Wykosky and Debinski (2008) Mol Cancer Ref. 6(12):1795-1806). For example, EPHA2 is strongly overexpressed in approximately 61% of GBM patient tumors (Wykosky et al. (2008) Clin Cancer Res. 14:199-208), 76% of ovarian cancers (Thaker et al. (2004) Clin Cancer Res. 10:5145-50), and 85% of prostate adenocarcinomas (Zeng et al. (2003) Am J Pathol. 163:2271-6). EPHA2 protein is highly overexpressed in a certain percentage of patient tumors and a certain percentage of tumor cells, and is a plasma membrane-localized receptor that can be internalized upon ligand binding (Walker-Daniels et al. (2002) Mol Cancer Res. [Molecular Cancer Research] 1:79-87). Furthermore, EPHA2 expression is associated with poor prognosis, increased metastasis, and decreased survival. Therefore, EPHA2, due to its expression pattern, localization, and functional importance in cancer patient outcomes, is another promising antigen for targeted delivery of novel anticancer therapies.

[0010] In various embodiments, this disclosure provides novel hoprene splice regulators having biological activity against neoplastic cells. Hoprene splice regulators can be used alone or as part of an ADC to slow, inhibit, and / or reverse tumor growth in mammals and are suitable for treating human cancer patients. In various embodiments, this disclosure provides novel antibody-drug conjugates employing hoprene splice regulators.

[0011] More specifically, in various embodiments, this disclosure relates to antibody-drug conjugate (ADC) complexes capable of binding to and killing cytoplasmic cells. In various embodiments, the ADC complexes disclosed herein include a linker that connects a splicing regulator to a full-length antibody or antigen-binding fragment. In various embodiments, these ADC complexes are also capable of internalization into target cells after binding.

[0012] In some embodiments, the antibody-drug conjugate is an antibody-drug conjugate having formula (I): Ab-(LH) p Where Ab is an antibody or antigen-binding fragment targeting the proliferative cells; H is a Hobschdiene splicing regulator; L is a linker that covalently links Ab to H; and p is an integer from 1 to 15.

[0013] In some embodiments, H, as a Hobschdiene splice regulator, comprises a compound having formula (I): Or a pharmaceutically acceptable salt thereof, which is covalently linked to L via any atom, wherein:

[0014] Y is selected from O, S, NR 6 and CR 6 R 7 ;

[0015] R 1 、R 2 and R 3 Each is independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl), -OC(=O)-(C1-C6 alkyl), -C(=O)-O-(C1-C6 alkyl), and C1-C6 alkyl;

[0016] R 4 Selected from hydrogen, C1-C6 alkyl, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), and -C(=O)-NR 6 R 7 ;

[0017] R 5 Selected from hydrogen, hydroxyl, -CH2-OH, -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-NR 6 R 7 -NR 6 -C(=O)-R 8 -OC(=O)-NR 6 R 7 -NR 6 -C(=O)-R 8 and -NR 6 -C(=O)-NR 6 R 7 ;

[0018] R 6 and R 7 Each is independently selected from hydrogen, -R 8 -C(=O)-R 8 and -C(=O)-OR 8 ;and

[0019] R 8 Selected from C1-C6 alkyl, C3-C8 carbocyclic and C3-C8 heterocyclic groups.

[0020] where R 1 、R 2 、R 3 、R4 、R 5 、R 6 、R 7 and R 8 Each is independently substituted by 0 to 3 groups independently selected from the following: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR 6 R 7 The group may contain C3-C8 carbocyclic groups, C1-C6 alkylhydroxy groups, C1-C6 alkylalkoxy groups, benzyl groups, and C3-C8 heterocyclic groups, each of which may be independently substituted by 0 or 1 groups selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, -NH-C(=O)(C1-C3 alkyl), and -NH-C(=O)-O-(C1-C3 alkyl).

[0021] The valence of the atoms covalently bonded to L did not exceed the limit.

[0022] In some embodiments, H, as a Hobschdiene splice regulator, comprises a compound having formula (Ia): Or a pharmaceutically acceptable salt thereof, which is covalently linked to L via any atom, wherein:

[0023] R 9 Selected from C3-C8 heterocyclic groups;

[0024] R 10 Selected from H and C1-C6 alkyl groups,

[0025] where R 9 and R 10 Each is independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, -NH2, -NH-(C1-C3 alkyl) and -N-(C1-C3 alkyl)2, and

[0026] The valence of the atoms covalently bonded to L did not exceed the limit.

[0027] In some embodiments, H, as a Hobschdiene splice regulator, comprises a compound having formula (Ib): Or a pharmaceutically acceptable salt thereof, which is covalently linked to L via any atom, wherein:

[0028] R 11 Selected from and Where * indicates R 11 The connection point with the rest of the compound;

[0029] R 12 and R 13 Each is independently selected from H and methyl; and

[0030] The valence of the atoms covalently bonded to L did not exceed the limit.

[0031] In some embodiments, H, as a Hobschdiene splice regulator, comprises a compound having formula (II): Or a pharmaceutically acceptable salt thereof, which is covalently linked to L via any atom, wherein:

[0032] X represents a hydroxyl group or NR. 6 R 7 ;

[0033] R 6 and R 7 Each is independently selected from hydrogen, -R 8 -C(=O)-R 8 -C(=O)-OR 8 -(C1-C6 alkyl)-OC(=O)-R 8 and -(C1-C6 alkyl)-NH-C(=O)-R 8 ;and

[0034] R 8 Selected from C1-C6 alkyl, C3-C8 carbocyclic and C3-C8 heterocyclic groups.

[0035] where R 6 、R 7 and R 8 Each is independently substituted by 0 to 3 groups independently selected from the following: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR 6 R 7 The group may contain C3-C8 carbocyclic groups, C1-C6 alkylhydroxy groups, C1-C6 alkylalkoxy groups, benzyl groups, and C3-C8 heterocyclic groups, each of which may be independently substituted by 0 or 1 groups selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, -NH-C(=O)(C1-C3 alkyl), and -NH-C(=O)-O-(C1-C3 alkyl).

[0036] The valence of the atoms covalently bonded to L did not exceed the limit.

[0037] In some embodiments, H, as a Hobschdiene splice regulator, comprises a compound having formula (IIa): Or a pharmaceutically acceptable salt thereof, which is covalently linked to L via any atom, wherein:

[0038] Z is selected from NR 9 and O;

[0039] R 9 Selected from hydrogen and C1-C6 alkyl groups;

[0040] R 10 and R 11 Each is independently selected from hydrogen, halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), C3-C8 carbocyclic, C1-C6 alkyl hydroxyl, C1-C6 alkyl alkoxy, benzyl and C3-C8 heterocyclic;

[0041] R 12 Selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups.

[0042] where R 9 、R 10 、R 11 and R 12 Each is independently substituted with 0 or 1 of the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy and C1-C3 haloalkyl;

[0043] t is an integer selected from 1, 2, 3, 4, 5, and 6; and

[0044] The valence of the atoms covalently bonded to L did not exceed the limit.

[0045] In some embodiments, H, as a Hobschdiene splice regulator, comprises a compound having formula (IIb): Or a pharmaceutically acceptable salt thereof, which is covalently linked to L via any atom, wherein:

[0046] R 13 Selected from

[0047]

[0048] Where * indicates R 13 The connection point with the rest of the compound;

[0049] R 14 and R15 Each is independently selected from hydrogen and methyl; and

[0050] The valence of the atoms covalently bonded to L did not exceed the limit.

[0051] In some embodiments, H, as a Hobschdiene splice regulator, comprises a compound having formula (III): Or a pharmaceutically acceptable salt thereof, which is covalently linked to L via any atom, wherein:

[0052] R 1 、R 2 and R 3 Each is independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl), -OC(=O)-(C1-C6 alkyl), -C(=O)-O-(C1-C6 alkyl), and C1-C6 alkyl;

[0053] R 6 and R 7 Each is independently selected from hydrogen, -R 8 -C(=O)-R 8 and -C(=O)-OR 8 ;

[0054] R 8 Selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups; and

[0055] R 9 Selected from H,

[0056] and

[0057] where R 1 、R 2 、R 3 、R 6 、R 7 and R 8 Each is independently substituted by 0 to 3 groups independently selected from the following: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR 6 R 7The group may contain C3-C8 carbocyclic, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic groups, each of which may be independently substituted by 0 or 1 of the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, -NH-C(=O)(C1-C3 alkyl), and -NH-C(=O)-O-(C1-C3 alkyl).

[0058] The valence of the atoms covalently bonded to L does not exceed the limit; and

[0059] Where * indicates R 9 The connection point with the rest of the compound.

[0060] In some embodiments, the linker comprises a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety can 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 glutamic acid-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”).

[0061] In some embodiments, the connector includes a cleavable glucuronic acid portion. In some embodiments, the cleavable glucuronic acid portion can be cleaved by an enzyme. In some embodiments, the cleavable glucuronic acid portion can be cleaved by glucuronidase. In some embodiments, the cleavable glucuronic acid portion can be cleaved by β-glucuronidase.

[0062] In some embodiments, the connector includes at least one spacer subunit. In some embodiments, the spacer subunit or connector includes a polyethylene glycol (PEG) portion. In some embodiments, the PEG portion includes -(PEG). m - And m is an integer from 1 to 10. In some embodiments, m is 2. In some other embodiments, the spacer subunit or connector comprises an alkyl portion. In some embodiments, the alkyl portion comprises -(CH2). n - And n is an integer from 1 to 10. In some embodiments, n is 2. In some embodiments, n is 5. In some embodiments, n is 6.

[0063] In some embodiments, the spacer unit is linked to an antibody or antigen-binding fragment via a maleic diamidimide (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.

[0064] In some embodiments, the connector includes a Mal-spacer subunit and a cleavable peptide portion. In some embodiments, the cleavable peptide portion includes an amino acid unit. In some embodiments, the cleavable peptide portion or amino acid unit includes Val-Cit. In some embodiments, the cleavable peptide portion or amino acid unit includes Val-Ala. In some embodiments, the cleavable peptide portion or amino acid unit includes Glu-Val-Cit. In some embodiments, the cleavable peptide portion or amino acid unit includes Ala-Ala-Asn. In some embodiments, the Mal-spacer subunit includes an alkyl portion. In some embodiments, the Mal-spacer subunit includes a PEG portion. In some embodiments, the Mal-spacer subunit includes maleic anhydride hexanoyl (MC).

[0065] In some embodiments, the Mal-spacer unit links an antibody or antigen-binding fragment to a cleavable portion in the adapter. In some embodiments, the cleavable portion in the adapter 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 adapter comprises MC-Val-Cit. In some embodiments, the adapter comprises MC-Val-Ala. In some embodiments, the adapter comprises MC-Glu-Val-Cit. In some embodiments, the adapter 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 anhydride hexanoyl (MC).

[0066] In some embodiments, the cleavable portion of the connector directly binds to the Hopschier splice regulator, or the spacer unit connects the cleavable portion of the connector to the Hopschier splice regulator. In some embodiments, cleavage of the conjugate releases the Hopschier splice regulator from the antibody or antigen-binding fragment and the connector. In some embodiments, the spacer unit that connects the cleavable portion of the connector to the Hopschier splice regulator is a self-ablating spacer unit.

[0067] In some embodiments, the spacer unit connecting the cleavable portion of the connector to the Hobstein splice regulator comprises a p-aminobenzyloxycarbonyl group (pABC). In some embodiments, the pABC connects the cleavable portion of the connector to the Hobstein splice regulator. In some embodiments, the cleavable portion of the connector 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 connector comprises Val-Cit-pABC. In some other embodiments, the connector comprises Val-Ala-pABC. In some embodiments, the connector comprises Glu-Val-Cit-pABC. In some embodiments, the connector comprises Ala-Ala-Asn-pABC.

[0068] In some embodiments, the spacer subunit connecting the cleavable portion of the connector to the Hobstein splice regulator comprises p-aminobenzyl (pAB). In some embodiments, pAB connects the cleavable portion of the connector to the Hobstein splice regulator. In some embodiments, the cleavable portion of the connector 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 connector comprises Val-Cit-pAB. In some other embodiments, the connector comprises Val-Ala-pAB. In some other embodiments, the connector comprises Glu-Val-Cit-pAB. In some other embodiments, the connector comprises Ala-Ala-Asn-pAB.

[0069] In various embodiments, the linker is a non-cleavable linker. In some embodiments, the ADC's hippodelid splicing regulator is released by degrading the antibody or antigen-binding fragment. In some embodiments, after internalization and degradation within target cells, the linker remains covalently associated with at least one amino acid of the antibody and drug.

[0070] In some embodiments, the connector is a non-disintegrable connector comprising at least one spacer subunit. In some embodiments, the spacer subunit or connector comprises a polyethylene glycol (PEG) portion. In some embodiments, the PEG portion comprises -(PEG). m - And m is an integer from 1 to 10. In some embodiments, m is 2. In some other embodiments, the spacer subunit or connector comprises an alkyl portion. In some embodiments, the alkyl portion comprises -(CH2). n -or-(CH2) n-O-(CH2) n And n is an integer from 1 to 10. In some embodiments, n is 2. In some embodiments, n is 5. In some embodiments, n is 6.

[0071] In some embodiments, the spacer subunit in the cleavable linker is linked to an antibody or antigen-binding fragment via a maleic diamidimide (Mal) portion (“Mal-spacer subunit”). In some embodiments, the Mal-spacer subunit may react with cysteine ​​residues on the antibody or antigen-binding fragment. In some embodiments, the Mal-spacer subunit 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 subunit comprises an alkyl portion. In some embodiments, the Mal-spacer subunit comprises a PEG portion. In some embodiments, the linker or Mal-spacer subunit comprises a maleic diamidimide hexanoyl (MC). In some embodiments, the linker or Mal-spacer subunit comprises a maleic diamidimide hexanoyl (MC) and at least one additional spacer subunit. In some embodiments, the linker or Mal-spacer subunit comprises MC-(PEG)2. In some embodiments, the linker or Mal-spacer subunit comprises MC-(PEG)2 and at least one additional spacer unit. In some embodiments, the linker or Mal-spacer subunit comprises Mal-Hex. In some embodiments, the connector or Mal-spacer unit comprises Mal-Hex and at least one additional spacer unit. In some embodiments, the connector or Mal-spacer unit comprises Mal-Et. In some embodiments, the connector or Mal-spacer unit comprises Mal-Et and at least one additional spacer unit. In some embodiments, the connector or Mal-spacer unit comprises Mal-Et-O-Et. In some embodiments, the connector or Mal-spacer unit comprises Mal-Et-O-Et and at least one additional spacer unit. In some embodiments, the Mal-spacer unit links an antibody or antigen-binding fragment to a Hobschdiene splice regulator.

[0072] 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 blast cells expressing HER2 and / or HER2. In some embodiments, Ab is an antibody or binding domain sequence targeting blast cells expressing CD138 and / or CD138. In some embodiments, Ab is an antibody or binding domain sequence targeting blast cells expressing EPHA2 and / or EPHA2. In some embodiments, Ab is an antibody or binding domain sequence targeting blast cells expressing MSLN and / or MSLN. In some embodiments, Ab is an antibody or binding domain sequence targeting blast cells expressing FOLH1 and / or FOLH1. In some embodiments, Ab is an antibody or binding domain sequence targeting blast cells expressing CDH6 and / or CDH6. In some embodiments, Ab is an antibody or binding domain sequence targeting blast cells expressing CEACAM5 and / or CEACAM5. In some embodiments, Ab is an antibody or binding domain sequence targeting blast cells expressing CFC1B and / or CFC1B. In some embodiments, Ab is an antibody or binding domain sequence targeting vesicular cells expressing ENPP3 and / or ENPP3. In some embodiments, Ab is an antibody or binding domain sequence targeting vesicular cells expressing FOLR1 and / or FOLR1. In some embodiments, Ab is an antibody or binding domain sequence targeting vesicular cells expressing HAVCR1 and / or HAVCR1. In some embodiments, Ab is an antibody or binding domain sequence targeting vesicular cells expressing KIT and / or KIT. In some embodiments, Ab is an antibody or binding domain sequence targeting vesicular cells expressing MET and / or MET. In some embodiments, Ab is an antibody or binding domain sequence targeting vesicular cells expressing MUC16 and / or MUC16. In some embodiments, Ab is an antibody or binding domain sequence targeting vesicular cells expressing SLC39A6 and / or SLC39A6. In some embodiments, Ab is an antibody or binding domain sequence targeting vesicular cells expressing SLC44A4 and / or SLC44A4. In some embodiments, Ab is an antibody or binding domain sequence that targets STEAP1 and / or STEAP1-expressing proliferative cells. In some embodiments, Ab is an antibody or binding domain sequence that targets another cancer antigen.

[0073] In some embodiments, L is selected from any of the connectors disclosed herein or any combination of the connector components disclosed herein. In some embodiments, L is a connector comprising MC-Val-Cit-pABC, Mal-(PEG)2-CO, MC-Val-Ala-pAB, MC-Val-Ala-pABC, MC-Val-Cit-pAB, Mal-Hex, Mal-Et, or Mal-Et-O-Et. In some embodiments, the connector may also include one or more additional spacer subunits. In some embodiments, L is a connector ADL1, ADL2, ADL5, ADL6, ADL7, ADL10, ADL12, ADL13, ADL14, ADL15, ADL21, ADL22, or ADL23. In some embodiments, L is a connector ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23. In some embodiments, L is an ADL12, ADL14, or ADL15 connector. In some embodiments, the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 connector may also include one or more additional spacer sub-units. In some embodiments, L is an ADL1 connector and may include one or more additional spacer sub-units. In some embodiments, L is an ADL2 connector and may include one or more additional spacer sub-units. In some embodiments, L is an ADL5 connector and may include one or more additional spacer sub-units. In some embodiments, L is an ADL6 connector and may include one or more additional spacer sub-units. In some embodiments, L is an ADL7 connector and may include one or more additional spacer sub-units. In some embodiments, L is an ADL12 connector and may include one or more additional spacer sub-units. In some embodiments, L is an ADL14 connector and may include one or more additional spacer sub-units. In some embodiments, L is an ADL15 connector and may include one or more additional spacer sub-units. In different embodiments of the ADC described herein, p is 1 to 10. In different embodiments, p is 2 to 8. In different embodiments, p is 4 to 8. In some embodiments, p is 4. In some embodiments, p is 8.

[0074] In some embodiments, a set of ADCs is provided, thereby resulting in random conjugation, and the average p in the set is between about 2 and about 8. In some embodiments, a set of ADCs is provided, thereby resulting in random conjugation, and the average p in the set is between about 4 and about 8. In some embodiments, a set of ADCs is provided, thereby resulting in random conjugation, and the average p in the set is about 4. In some embodiments, a set of ADCs is provided, thereby resulting in random conjugation, 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 described 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).

[0075] In some embodiments, the antibody or antigen-binding fragment (Ab) of the ADC targets proliferative cells derived from hematologic malignancies or solid tumors. In some embodiments, the antibody or antigen-binding fragment targets proliferative cells derived from hematologic malignancies. In some embodiments, hematologic malignancies are selected from B-cell malignancies, leukemia (e.g., acute myeloid leukemia), lymphoma, and myeloma (e.g., multiple myeloma). In some embodiments, hematologic malignancies are selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the antibody or antigen-binding fragment targets proliferative cells derived from solid tumors. In some embodiments, solid tumors are selected from breast cancer (e.g., HER2-positive breast cancer), gastric cancer (e.g., gastric adenocarcinoma), prostate cancer, ovarian cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., serous endometrial cancer), salivary gland carcinoma, melanoma, colorectal cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer, and esophageal cancer. In some embodiments, solid tumors are selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

[0076] In various embodiments, the antibody or antigen-binding fragment (Ab) of the ADC is an anti-HER2 antibody or its antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment binds to HER2 and targets HER2-expressing neoplasms (i.e., the ADC targets HER2-expressing neoplasms). In some embodiments, the antibody or antigen-binding fragment of the ADC is an internalized anti-HER2 antibody or its internalized antigen-binding fragment.

[0077] In some embodiments, the anti-HER2 antibody or antigen-binding fragment comprises 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 framework sequence. In some embodiments, the anti-HER2 antibody or antigen-binding fragment comprises 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 includes a human Igκ or λ light chain constant region. In some embodiments, the anti-HER2 antibody or antigen-binding fragment competes with and / or binds to the same epitope with an antibody containing the heavy chain variable domain of SEQ ID NO:19 and the light chain variable domain of SEQ ID NO:20.

[0078] In various embodiments, the antibody or antigen-binding fragment (Ab) of the ADC is an anti-CD138 antibody or its antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment binds to CD138 and targets CD138-expressing neoplasms (i.e., the ADC targets CD138-expressing neoplasms). In some embodiments, the antibody or antigen-binding fragment of the ADC is an internalized anti-CD138 antibody or its internalized antigen-binding fragment.

[0079] In some embodiments, the anti-CD138 antibody or antigen-binding fragment comprises 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 framework sequence. In some embodiments, the anti-CD138 antibody or antigen-binding fragment comprises 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 competes with and / or binds to the same epitope with an antibody comprising the heavy chain variable domain of SEQ ID NO:21 and the light chain variable domain of SEQ ID NO:22.

[0080] In various embodiments, the antibody or antigen-binding fragment (Ab) of the ADC is an anti-EPHA2 antibody or its antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment binds to EPHA2 and targets EPHA2-expressing neoplasms (i.e., the ADC targets EPHA2-expressing neoplasms). In some embodiments, the antibody or antigen-binding fragment of the ADC is an internalized anti-EPHA2 antibody or its internalized antigen-binding fragment.

[0081] In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment comprises 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 comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:23 and a light chain variable region containing the amino acid sequence of SEQ ID NO:24. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment comprises a human IgG heavy chain constant region. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment comprises a human Igκ or λ light chain constant region. In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment competes with and / or binds to the same epitope with an antibody comprising the heavy chain variable domain of SEQ ID NO:23 and the light chain variable domain of SEQ ID NO:24.

[0082] In some embodiments, compounds having formula (I) are disclosed herein: or a pharmaceutically acceptable salt thereof, wherein:

[0083] Y is selected from O, S, NR 6 and CR 6 R 7 ;

[0084] R 1 、R 2 and R 3 Each is independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl), -OC(=O)-(C1-C6 alkyl), -C(=O)-O-(C1-C6 alkyl), and C1-C6 alkyl;

[0085] R 4 Selected from hydrogen, C1-C6 alkyl, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), and -C(=O)-NR 6 R 7;

[0086] R 5 Selected from hydrogen, hydroxyl, -CH2-OH, -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-NR 6 R 7 -NR 6 -C(=O)-R 8 -OC(=O)-NR 6 R 7 -NR 6 -C(=O)-R 8 and -NR 6 -C(=O)-NR 6 R 7 ;

[0087] R 6 and R 7 Each is independently selected from hydrogen, -R 8 -C(=O)-R 8 and -C(=O)-OR 8 ;and

[0088] R 8 Selected from C1-C6 alkyl, C3-C8 carbocyclic and C3-C8 heterocyclic groups.

[0089] where R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 and R 8 Each is independently substituted by 0 to 3 groups independently selected from the following: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR 6 R 7 C3-C8 carbocyclic, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl and C3-C8 heterocyclic, each of which may be independently substituted by 0 or 1 of the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, -NH-C(=O)(C1-C3 alkyl) and -NH-C(=O)-O-(C1-C3 alkyl).

[0090] In some embodiments, compounds having formula (Ia) are provided herein: or a pharmaceutically acceptable salt thereof, wherein:

[0091] R 9 Selected from C3-C8 heterocyclic groups; and

[0092] R 10 Selected from H and C1-C6 alkyl groups,

[0093] where R 9 and R 10 Each is independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, -NH2, -NH-(C1-C3 alkyl) and -N-(C1-C3 alkyl)2.

[0094] In some embodiments, compounds having formula (Ib) are provided herein: or a pharmaceutically acceptable salt thereof, wherein:

[0095] R 11 Selected from and Where * indicates R 11 The connection point with the rest of the compound; and

[0096] R 12 and R 13 Each is independently selected from H and methyl.

[0097] In some embodiments, compounds having formula (II) are provided herein: or a pharmaceutically acceptable salt thereof, wherein:

[0098] X is NR 6 R 7 ;

[0099] R 6 and R 7 Each is independently selected from hydrogen, -R 8 -C(=O)-R 8 -C(=O)-OR 8 -(C1-C6 alkyl)-OC(=O)-R 8 and -(C1-C6 alkyl)-NH-C(=O)-R 8 ;and

[0100] R 8 Selected from C1-C6 alkyl, C3-C8 carbocyclic and C3-C8 heterocyclic groups.

[0101] where R 6 、R 7 and R 8Each is independently substituted by 0 to 3 groups independently selected from the following: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR 6 R 7 C3-C8 carbocyclic, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl and C3-C8 heterocyclic, each of which may be independently substituted by 0 or 1 of the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, -NH-C(=O)(C1-C3 alkyl) and -NH-C(=O)-O-(C1-C3 alkyl).

[0102] In some embodiments, compounds having formula (IIa) are provided herein: or a pharmaceutically acceptable salt thereof, wherein:

[0103] Z is selected from NR 9 and O;

[0104] R 9 Selected from hydrogen and C1-C6 alkyl groups;

[0105] R 10 and R 11 Each is independently selected from hydrogen, halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), C3-C8 carbocyclic, C1-C6 alkyl hydroxyl, C1-C6 alkyl alkoxy, benzyl and C3-C8 heterocyclic;

[0106] R 12 Selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups.

[0107] where R 9 、R 10 、R 11 and R 12 Each is independently substituted with one or more groups selected from the following: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, and C1-C3 haloalkyl; and

[0108] t is an integer selected from 1, 2, 3, 4, 5 and 6.

[0109] In some embodiments, compounds having formula (IIb) are provided herein: or a pharmaceutically acceptable salt thereof, wherein:

[0110] R 13 Selected from

[0111]

[0112] and Where * indicates R 13 The connection point with the rest of the compound; and

[0113] R 14 and R 15 Each is independently selected from hydrogen and methyl.

[0114] In some embodiments, compounds having formula (III) are provided herein: or a pharmaceutically acceptable salt thereof, wherein:

[0115] R 1 、R 2 and R 3 Each is independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl), -OC(=O)-(C1-C6 alkyl), -C(=O)-O-(C1-C6 alkyl), and C1-C6 alkyl;

[0116] R 6 and R 7 Each is independently selected from hydrogen, -R 8 -C(=O)-R 8 and -C(=O)-OR 8 ;

[0117] R 8 Selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups; and

[0118] R 9 Selected from H,

[0119] and

[0120] where R 1 、R 2 、R 3 、R 6 、R 7 and R 8Each is independently substituted by 0 to 3 groups independently selected from the following: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR 6 R 7 The group may contain C3-C8 carbocyclic groups, C1-C6 alkylhydroxy groups, C1-C6 alkylalkoxy groups, benzyl groups, and C3-C8 heterocyclic groups, each of which may be independently substituted by 0 or 1 groups selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, -NH-C(=O)(C1-C3 alkyl), and -NH-C(=O)-O-(C1-C3 alkyl); and

[0121] Where * indicates R 9 The connection point with the rest of the compound.

[0122] In some embodiments, this document provides compounds selected from the following:

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129] and its pharmaceutically acceptable salts,

[0130] Where L is a linker covalently attached to the antibody.

[0131] Furthermore, in various embodiments, this document provides the therapeutic use of the described ADC compounds, heptahydrate compounds, and compositions, for example, in treating neoplastic conditions such as cancer. In some aspects, this disclosure provides a method for treating neoplastic conditions, such as cancers expressing antigens targeted by an antibody or antigen-binding fragment of an ADC, such antigens as HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1.

[0132] In some aspects, this disclosure provides a method of treating a subject with or suspected of having a neoplastic condition, which is carried out by administering to the subject any of the described ADC or composition of a therapeutically effective dose and / or therapeutically effective regimen. 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, colorectal cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer, and esophageal cancer. In some embodiments, the solid tumor is selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

[0133] In some embodiments, treatment with an antibody-drug conjugate or composition induces bystander killing of vesicular cells that do not express the target antigen but are adjacent to vesicular cells that express the target antigen. In some embodiments, the subject has one or more vesicular cells expressing the target antigen.

[0134] In some embodiments, the target antigen is HER2. In some embodiments, one or more neoplastic cells are present in HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., serous endometrial cancer), osteosarcoma, or salivary gland carcinoma. In some embodiments, the subject is unresponsive or poorly responsive to treatment with (a) an anti-HER2 antibody alone and / or (b) a hoprene splice regulator alone. In some embodiments, the subject is intolerant, unresponsive, or poorly responsive to treatment with the hoprene splice regulator alone.

[0135] In some embodiments, the target antigen is CD138. In some embodiments, one or more proliferative cells are present in CD138-expressing multiple myeloma. In some embodiments, the subject is unresponsive or poorly responsive to treatment with (a) an anti-CD138 antibody as administered alone and / or (b) a hoprene splice regulator as administered alone. In some embodiments, the subject is intolerant, unresponsive, or poorly responsive to treatment with the hoprene splice regulator as administered alone.

[0136] In some embodiments, the target antigen is EPHA2. In some embodiments, one or more neoplastic cells are present in EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colorectal cancer, or esophageal cancer. In some embodiments, the subject is unresponsive or poorly responsive to treatment with (a) an anti-EPHA2 antibody as administered alone and / or (b) a Hormone splice regulator as administered alone. In some embodiments, the subject is intolerant, unresponsive, or poorly responsive to treatment with a Hormone splice regulator as administered alone.

[0137] In some embodiments, the target antigen is MSLN. In some embodiments, one or more neoplastic cells are in MSLN-expressing ovarian cancer, cervical cancer, pancreatic cancer, or lung cancer (e.g., lung adenocarcinoma). In some embodiments, the subject is unresponsive or poorly responsive to treatment with (a) an anti-MSLN antibody when administered alone and / or (b) a splicing regulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator when administered alone.

[0138] In some embodiments, the target antigen is FOLH1. In some embodiments, one or more neoplastic cells are in FOLH1-expressing prostate cancer. In some embodiments, the subject is unresponsive or poorly responsive to treatment with (a) an anti-FOLH1 antibody administered alone and / or (b) a splicing regulator administered alone. In some embodiments, the subject is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator administered alone.

[0139] In some embodiments, the target antigen is CDH6. In some embodiments, one or more proliferative cells are in CDH6-expressing renal cell carcinoma. In some embodiments, the subject is unresponsive or poorly responsive to treatment with (a) an anti-CDH6 antibody as administered alone and / or (b) a splicing regulator as administered alone. In some embodiments, the subject is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator as administered alone.

[0140] In some embodiments, the target antigen is CEACAM5. In some embodiments, one or more neoplastic cells are in CEACAM5-expressing colorectal cancer. In some embodiments, the subject is unresponsive or poorly responsive to treatment with (a) an anti-CEACAM5 antibody as administered alone and / or (b) a splicing regulator as administered alone. In some embodiments, the subject is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator as administered alone.

[0141] In some embodiments, the target antigen is CFC1B. In some embodiments, one or more proliferative cells are in CFC1B-expressing pancreatic cancer. In some embodiments, the subject is unresponsive or poorly responsive to treatment with (a) an anti-CFC1B antibody administered alone and / or (b) a splicing regulator administered alone. In some embodiments, the subject is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator administered alone.

[0142] In some embodiments, the target antigen is ENPP3. In some embodiments, one or more proliferative cells are in ENPP3-expressing renal cell carcinoma. In some embodiments, the subject is unresponsive or poorly responsive to treatment with (a) an anti-ENPP3 antibody administered alone and / or (b) a splicing regulator administered alone. In some embodiments, the subject is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator administered alone.

[0143] In some embodiments, the target antigen is FOLR1. In some embodiments, one or more neoplastic cells are in FOLR1-expressing ovarian cancer. In some embodiments, the subject is unresponsive or poorly responsive to treatment with (a) an anti-FOLR1 antibody as administered alone and / or (b) a splicing regulator as administered alone. In some embodiments, the subject is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator as administered alone.

[0144] In some embodiments, the target antigen is HAVCR1. In some embodiments, one or more proliferative cells are in HAVCR1-expressing renal cell carcinoma or esophageal cancer. In some embodiments, the subject is unresponsive or poorly responsive to treatment with (a) an anti-HAVCR1 antibody as administered alone and / or (b) a splicing regulator as administered alone. In some embodiments, the subject is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator as administered alone.

[0145] In some embodiments, the target antigen is KIT. In some embodiments, one or more proliferative cells are in KIT-expressing renal cell carcinoma. In some embodiments, the subject is unresponsive or poorly responsive to treatment with (a) an anti-KIT antibody administered alone and / or (b) a splicing regulator administered alone. In some embodiments, the subject is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator administered alone.

[0146] In some embodiments, the target antigen is MET. In some embodiments, one or more proliferative cells are in MET-expressing renal cell carcinoma or esophageal cancer. In some embodiments, the subject is unresponsive or poorly responsive to treatment with (a) an anti-MET antibody administered alone and / or (b) a splicing regulator administered alone. In some embodiments, the subject is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator administered alone.

[0147] In some embodiments, the target antigen is MUC16. In some embodiments, one or more neoplastic cells are present in MUC16-expressing ovarian cancer, cervical cancer, or breast cancer. In some embodiments, the subject is unresponsive or poorly responsive to treatment with (a) an anti-MUC16 antibody administered alone and / or (b) a splicing regulator administered alone. In some embodiments, the subject is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator administered alone.

[0148] In some embodiments, the target antigen is SLC39A6. In some embodiments, one or more proliferative cells are in SLC39A6-expressing breast or prostate cancer. In some embodiments, the subject is unresponsive or poorly responsive to treatment with (a) an anti-SLC39A6 antibody as administered alone and / or (b) a splicing regulator as administered alone. In some embodiments, the subject is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator as administered alone.

[0149] In some embodiments, the target antigen is SLC44A4. In some embodiments, one or more neoplastic cells are in SLC44A4-expressing prostate cancer. In some embodiments, the subject is unresponsive or poorly responsive to treatment with (a) an anti-SLC44A4 antibody as administered alone and / or (b) a splicing regulator as administered alone. In some embodiments, the subject is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator as administered alone.

[0150] In some embodiments, the target antigen is STEAP1. In some embodiments, one or more neoplastic cells are in STEAP1-expressing prostate cancer. In some embodiments, the subject is unresponsive or poorly responsive to treatment with (a) an anti-STEAP1 antibody as administered alone and / or (b) a splicing regulator as administered alone. In some embodiments, the subject is intolerant, unresponsive, or poorly responsive to treatment with the splicing regulator as administered alone.

[0151] In some other aspects, this disclosure provides a method for reducing or inhibiting tumor growth in a subject who has or is suspected of having a neoplastic condition, which is carried out by administering to the subject any of the described ADC or composition of a therapeutically effective dose and / or therapeutically effective regimen.

[0152] In some embodiments, treatment with an antibody-drug conjugate or composition induces bystander killing of neoplastic tumor cells that do not express the target antigen but are adjacent to neoplastic tumor cells that express the target antigen. In some embodiments, the tumor comprises one or more neoplastic cells expressing the target antigen.

[0153] In some embodiments, the target antigen is HER2. In some embodiments, one or more proliferative cells are derived from HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., serous endometrial cancer), osteosarcoma, or salivary gland carcinoma. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-HER2 antibody alone and / or (b) a Hobstein-diene splice regulator alone.

[0154] In some embodiments, the target antigen is CD138. In some embodiments, one or more proliferative cells are derived from CD138-expressing multiple myeloma. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-CD138 antibody alone and / or (b) a Hobschdiene splice regulator alone.

[0155] In some embodiments, the target antigen is EPHA2. In some embodiments, one or more neoplastic cells are derived from EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colorectal cancer, or esophageal cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-EPHA2 antibody when administered alone and / or (b) a Hobstein-diene splice regulator when administered alone.

[0156] In some embodiments, the target antigen is MSLN. In some embodiments, one or more neoplastic cells are derived from MSLN-expressing ovarian cancer, cervical cancer, pancreatic cancer, or lung cancer (e.g., lung adenocarcinoma). In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-MSLN antibody when administered alone and / or (b) a splicing regulator when administered alone.

[0157] In some embodiments, the target antigen is FOLH1. In some embodiments, one or more neoplastic cells are derived from FOLH1-expressing prostate cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-FOLH1 antibody when administered alone and / or (b) a splicing regulator when administered alone.

[0158] In some embodiments, the target antigen is CDH6. In some embodiments, one or more proliferative cells are derived from CDH6-expressing renal cell carcinoma. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-CDH6 antibody administered alone and / or (b) a splicing regulator administered alone.

[0159] In some embodiments, the target antigen is CEACAM5. In some embodiments, one or more neoplastic cells are derived from CEACAM5-expressing colorectal cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-CEACAM5 antibody when administered alone and / or (b) a splicing regulator when administered alone.

[0160] In some embodiments, the target antigen is CFC1B. In some embodiments, one or more proliferative cells are derived from CFC1B-expressing pancreatic cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-CFC1B antibody when administered alone and / or (b) a splicing regulator when administered alone.

[0161] In some embodiments, the target antigen is ENPP3. In some embodiments, one or more proliferative cells are derived from ENPP3-expressing renal cell carcinoma. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-ENPP3 antibody when administered alone and / or (b) a splicing regulator when administered alone.

[0162] In some embodiments, the target antigen is FOLR1. In some embodiments, one or more neoplastic cells are derived from FOLR1-expressing ovarian cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-FOLR1 antibody when administered alone and / or (b) a splicing regulator when administered alone.

[0163] In some embodiments, the target antigen is HAVCR1. In some embodiments, one or more proliferative cells are derived from HAVCR1-expressing renal cell carcinoma or esophageal cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-HAVCR1 antibody when administered alone and / or (b) a splicing regulator when administered alone.

[0164] In some embodiments, the target antigen is KIT. In some embodiments, one or more proliferative cells are derived from KIT-expressing renal cell carcinoma. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-KIT antibody administered alone and / or (b) a splicing regulator administered alone.

[0165] In some embodiments, the target antigen is MET. In some embodiments, one or more proliferative cells are derived from MET-expressing renal cell carcinoma or esophageal cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-MET antibody administered alone and / or (b) a splicing regulator administered alone.

[0166] In some embodiments, the target antigen is MUC16. In some embodiments, one or more proliferative cells are derived from MUC16-expressing ovarian cancer, cervical cancer, or breast cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-MUC16 antibody when administered alone and / or (b) a splicing regulator when administered alone.

[0167] In some embodiments, the target antigen is SLC39A6. In some embodiments, one or more proliferative cells are derived from SLC39A6-expressing breast or prostate cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-SLC39A6 antibody when administered alone and / or (b) a splicing regulator when administered alone.

[0168] In some embodiments, the target antigen is SLC44A4. In some embodiments, one or more neoplastic cells are derived from SLC44A4-expressing prostate cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-SLC44A4 antibody when administered alone and / or (b) a splicing regulator when administered alone.

[0169] In some embodiments, the target antigen is STEAP1. In some embodiments, one or more neoplastic cells are derived from STEAP1-expressing prostate cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-STEAP1 antibody when administered alone and / or (b) a splicing regulator when administered alone.

[0170] In other aspects, this disclosure provides a method for determining whether a subject with or suspected of having a neoplastic condition responds to treatment with any of the described ADCs or compositions, which is carried out by providing a biological sample from the subject and contacting the biological sample with the ADC or composition. In some embodiments, the biological sample is a tumor sample. In some embodiments, the tumor sample is a tumor biopsy or a blood sample. In some embodiments, the blood sample is selected from blood, blood portions, or cells obtained from blood or blood portions. In some embodiments, the subject 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), osteosarcoma, or salivary duct cancer. In some embodiments, the target antigen is CD138. In some embodiments, the one or more neoplastic cells are derived from CD138-expressing multiple myeloma. In some embodiments, the target antigen is EPHA2. In some embodiments, the one or more neoplastic cells are derived from EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colorectal cancer, or esophageal cancer. In some embodiments, the target antigen is MSLN. In some embodiments, one or more neoplasm cells are derived from MSLN-expressing ovarian cancer, cervical cancer, pancreatic cancer, or lung cancer (e.g., lung adenocarcinoma). In some embodiments, the target antigen is FOLH1. In some embodiments, one or more neoplasm cells are derived from FOLH1-expressing prostate cancer. In some embodiments, the target antigen is CDH6. In some embodiments, one or more neoplasm cells are derived from CDH6-expressing renal cell carcinoma. In some embodiments, the target antigen is CEACAM5. In some embodiments, one or more neoplasm cells are derived from CEACAM5-expressing colorectal cancer. In some embodiments, the target antigen is CFC1B. In some embodiments, one or more neoplasm cells are derived from CFC1B-expressing pancreatic cancer. In some embodiments, the target antigen is ENPP3. In some embodiments, one or more neoplasm cells are derived from ENPP3-expressing renal cell carcinoma. In some embodiments, the target antigen is FOLR1. In some embodiments, one or more neoplasm cells are derived from FOLR1-expressing ovarian cancer. In some embodiments, the target antigen is HAVCR1. In some embodiments, one or more neoplasm cells are derived from HAVCR1-expressing renal cell carcinoma or esophageal cancer. In some embodiments, the target antigen is KIT. In some embodiments, one or more proliferative cells are derived from KIT-expressing renal cell carcinoma. In some embodiments, the target antigen is MET. In some embodiments, one or more proliferative cells are derived from MET-expressing renal cell carcinoma or esophageal cancer. In some embodiments, the target antigen is MUC16.In some embodiments, one or more neoplastic cells are derived from MUC16-expressing ovarian cancer, cervical cancer, or breast cancer. In some embodiments, the target antigen is SLC39A6. In some embodiments, one or more neoplastic cells are derived from SLC39A6-expressing breast cancer or prostate cancer. In some embodiments, the target antigen is SLC44A4. In some embodiments, one or more neoplastic cells are derived from SLC44A4-expressing prostate cancer. In some embodiments, the target antigen is STEAP1. In some embodiments, one or more neoplastic cells are derived from STEAP1-expressing prostate cancer.

[0171] In various embodiments, this document further provides pharmaceutical compositions comprising an ADC and a pharmaceutically acceptable diluent, carrier, and / or excipient. Methods for generating the described ADC compounds and compositions are also disclosed. BRIEF DESCRIPTION OF THE DRAWINGS

[0172] Figure 1A-1D This demonstrates the viability-dose response of exemplary loaded compounds in HER2-amplified breast cancer cells (HCC1954) and gastric cancer cells (NCI-N87). Cells were incubated with the compounds for 72 hours (3 days) or 144 hours (6 days) and the activity was measured in CellTiter. 2.0 Read the activity from the reagent. Figure 1A Viability dose-response in HCC1954 cells after 72 hours of culture. Figure 1B Viability dose-response in NCI-N87 cells after 72 hours of culture. Figure 1C Viability dose-response in HCC1954 cells after 144 hours of culture. Figure 1D Viability-dose response in NCI-N87 cells after 144 hours of culture. Data are presented as mean ± SD.

[0173] Figure 2A and Figure 2B Showing breast cancer cells amplified by HER2 (HCC1954) ( Figure 2A ) and gastric cancer cells (NCI-N87) Figure 2B Results of SLC25A19 splicing analysis in [the study]. Cells were incubated with the compound for 6 hours, and SLC25A19 transcript splicing was measured using a specific Taqman primer-probe set in a real-time qPCR reaction. The y-axis represents the percentage of response (%) relative to the DMSO control (0.1%). Data are expressed as mean ± SD. Detailed Implementation

[0174] The disclosed compositions and methods can be more easily understood with reference to the following detailed description.

[0175] Throughout this document, descriptions relate to compositions and methods of using such compositions. When this disclosure describes or claims features or embodiments associated with a composition, such features or embodiments equally apply to methods of using the composition. Similarly, when this disclosure describes or claims features or embodiments associated with methods of using the composition, such features or embodiments equally apply to the composition.

[0176] When a range of values ​​is indicated, it includes embodiments using any particular value within that range. Furthermore, references to values ​​stated by range include every value within that range. All ranges include their endpoints and are composable. When a value is 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 otherwise indicated regarding its specific use, the use of "or" means "and / or."

[0177] It should be understood that some features of the compositions and methods disclosed herein, described in the case of individual embodiments for clarity, may also be provided in combination with individual embodiments. Conversely, various features of the disclosed compositions and methods, described in the case of individual embodiments for simplicity, may also be provided individually or in any sub-combination.

[0178] All references cited herein are incorporated by way of reference for any purpose. In the event of any discrepancy between the references and this specification, this specification shall prevail.

[0179] definition

[0180] Throughout this specification and the claims, various terms related to the described aspects are used. Unless otherwise indicated, these terms will be given their ordinary meaning in the art. Other terms specifically defined are to be interpreted in a manner consistent with the definitions provided herein.

[0181] Unless the context clearly indicates otherwise, the singular forms “a / an” and “the” as used herein include the plural forms.

[0182] As will be apparent to those skilled in the art from the teachings contained herein, in the context of numerical values ​​and ranges, the terms "about" or "approximately" mean approximately or close to the stated value or range so that the embodiments can be performed as intended, such as values ​​or ranges of nucleic acids or polypeptides having the desired amount in the reaction mixture. In some embodiments, about means a numerical value ±10%.

[0183] The terms “antibody-drug conjugate,” “antibody conjugate,” “conjugate,” “immunoconjugate,” and “ADC” are used interchangeably and refer to one or more therapeutic compounds (e.g., splicing regulators) linked to one or more antibodies or antigen-binding fragments and defined by the general formula: Ab-(LH). p (Formula I), where Ab = antibody or antigen-binding fragment, L = linker portion, H = hoprene splice regulator (e.g., hoprene or a derivative thereof), and p = number of pharmaceutical portions per antibody or antigen-binding fragment. An ADC containing a hoprene splice regulator may also be more specifically referred to herein as an "antibody loaded with a hoprene splice regulator" or "SMLA". In an ADC containing a hoprene splice regulator, "p" refers to the number of hoprene splice regulators linked to the antibody or antigen-binding fragment. In some embodiments, the linker L may include a cleavable portion between the antibody or antigen-binding fragment and the hoprene splice regulator. In some embodiments, the linker L may include a cleavable portion that can be linked to either or both of the antibody or antigen-binding fragment and the hoprene splice regulator via a spacer unit. In some embodiments, the spacer unit is a self-ablating spacer unit when it links the cleavable portion to the hoprene splice regulator. In other embodiments, the linker L does not include a cleavable portion and is a non-cleavable linker. In some embodiments, the connector L may include at least one spacer unit that can be directly connected to an antibody or antigen-binding fragment and a Hobstein splicing regulator. Exemplary cleavable and non-cleavable connectors are described and illustrated herein.

[0184] The term "antibody" in its broadest sense refers to an immunoglobulin molecule that recognizes and specifically binds to a target such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing via at least one antigen recognition site within the variable region of the immunoglobulin molecule. The heavy chain of an antibody consists of a heavy chain variable domain (V... H ) and heavy chain constant region (C H The light chain is composed of light chain variable domains (V). L ) and light chain constant domain (C LThe mature heavy chain variable domain and the light chain variable domain each comprise three complementarity-determining regions (CDR1, CDR2, and CDR3) within four framework regions (FR1, FR2, FR3, and FR4) arranged from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. An "antibody" can be naturally occurring or artificial, such as a monoclonal antibody produced by conventional fusion tumor technology. The term "antibody" includes full-length monoclonal antibodies and full-length polyclonal antibodies, as well as antibody fragments and single-chain antibodies such as Fab, Fab', F(ab')2, and Fv. An antibody can be any of the five 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 that it exhibits the desired biological activity (e.g., binding to a target antigen, internalization within a cell expressing the target antigen).

[0185] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting that population are identical except for the possible small number of naturally occurring mutations. Monoclonal antibodies are highly specific for a single antigenic epitope. In contrast, conventional (polyclonal) antibody formulations typically comprise multiple antibodies that target or are specific to different epitopes. The modifier "monoclonal" indicates the characteristic of an antibody derived from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies used according to this disclosure may be manufactured by a 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 MolBiol. 222:581-97.

[0186] The monoclonal antibodies described herein specifically include “chimeric” antibodies, wherein a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass; provided that they specifically bind to the target antigen and / or exhibit the desired biological activity.

[0187] 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.

[0188] As used herein, the term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. In some cases, the variable regions of both the heavy and light chains correspond to the variable regions of an antibody derived from one species with the desired specificity, affinity, and activity, while the constant regions are homologous to antibodies derived from another species (e.g., humans) to minimize the immune response in the latter species.

[0189] As used herein, the term "humanized antibody" refers to an antibody form containing sequences derived from both non-human (e.g., mouse) and human antibodies. These antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. Generally, humanized antibodies contain substantially all at least one and typically two variable domains, wherein all or substantially all hypervariable loops correspond to the variable domains of non-human immunoglobulins and all or substantially all frame (FR) regions are variable domains of human immunoglobulin sequences. Humanized antibodies may also, where appropriate, contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the constant region of human immunoglobulins. Humanized antibodies can be further modified by substitution of residues within the Fv frame regions and / or by substitution of replaced non-human residues to improve and optimize antibody specificity, affinity, and / or activity.

[0190] As used herein, the term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more fragments of an antibody or protein that maintain the ability to specifically bind to an antigen (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1). The antigen-binding fragment may also maintain the ability to be internalized into cells expressing the antigen. In some embodiments, the antigen-binding fragment also maintains immune effector activity. It has been shown that fragments of a full-length antibody can perform the antigen-binding function of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" or "antigen-binding portion" of an antibody include (i) Fab fragments, i.e., fragments composed of V... L Domain, V H Domain, C L Domain and C H1 (ii) a monovalent fragment composed of domains; (iii) a F(ab')2 fragment, i.e., a divalent fragment containing two Fab fragments connected by disulfide bridges at the hinge region; H Domain and C H1(iv) Fd fragments composed of domains; V fragments consisting of a single arm with an antibody. L Domain and V H Fv segments composed of domains; (v) containing, for example, V H The dAb fragment of a single variable domain of the domain (see, for example, Ward et al. (1989) Nature 341:544-6; and International Publication No. WO 1990 / 005144); and (vi) the separated complementarity-determining region (CDR). Furthermore, although the two structural domains V of the Fv fragment... L and V H Encoded by a single gene, but which can be conjugated using recombination methods via synthetic linkers that enable it to be manufactured as a single protein chain, wherein V L District and V H 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 the term antibody, and in this art, are referred to as exemplary types of binding fragments that can be internalized into the cell after binding (see, for example, Zhu et al. (2010) 9:2131-41; He et al. (2010) J Nucl Med. 51:427-32; and Fitting et al. (2015) MAbs 7:390-402). In some embodiments, the scFv molecule may be incorporated into a fusion protein. Other forms of single-chain antibodies, such as bifunctional antibodies, are also covered. Bifunctional antibodies are bivalent bispecific antibodies, in which V H Domain and V L The domain is expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between two domains on the same chain, thereby forcing the domain to pair with a complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger et al. (1993) Proc Natl Acad Sci. USA [Proceedings of the National Academy of Sciences] 90:6444-8; and Poljak et al. (1994) Structure [Structure] 2:1121-3). The antigen-binding fragment is obtained using conventional techniques known to those skilled in the art, and the fragment is screened for utility (e.g., binding affinity, internalization) in the same manner as the intact antibody. The antigen-binding fragment can be prepared by cleaving the intact protein, for example by protease or chemical cleavage.

[0191] As used herein with respect to antibody or antigen-binding fragments, "internalization" means that the antibody or antigen-binding fragment, after binding to a cell, is absorbed through the cell's lipid bilayer into the internal compartments (i.e., "internalized"), preferably into the degradation compartments within the cell. For example, an internalized anti-HER2 antibody is an antibody that is absorbed into the cell after binding to HER2 on the cell membrane. In some embodiments, the antibody or antigen-binding fragment used in the ADCs disclosed herein targets a cell surface antigen (e.g., HER2) and is an internalized antibody or internalized antigen-binding fragment (i.e., the ADC is transferred across the cell membrane after antigen binding). In some embodiments, the internalized antibody or antigen-binding fragment binds to a receptor on the cell surface. Internalized antibodies or internalized antigen-binding fragments targeting receptors on the cell membrane can induce receptor-mediated endocytosis. In some embodiments, the internalized antibody or internalized antigen-binding fragment is absorbed into the cell via receptor-mediated endocytosis.

[0192] As used herein with respect to antibody or antigen-binding fragments, “non-internalizing” means that the antibody or antigen-binding fragment remains on the cell surface after binding to the cell. In some embodiments, the antibody or antigen-binding fragments used in the ADCs disclosed herein target cell surface antigens and are non-internalizing antibodies or non-internalizing antigen-binding fragments (i.e., the ADC remains on the cell surface after antigen binding and does not transfer across the cell membrane). In some embodiments, the non-internalizing antibody or antigen-binding fragment binds to a non-internalizing receptor or other cell surface antigen. Exemplary non-internalizing cell surface antigens include, but are not limited to, CA125 and CEA, and the antibodies binding to the non-internalizing antigen targets are also known in this art (see, for example, Bast et al. (1981) J Clin Invest. [Journal of Clinical Research] 68(5):1331-7; Scholler and Urban (2007) Biomark Med. [Medical Biomarkers] 1(4):513-23; and Boudousq et al. (2013) PLoS One [PLOS ONE] 8(7):e69613).

[0193] 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 the full-length HER (e.g., UniProt reference sequence: P04626; SEQ ID NO: 31) and any form of human HER2 that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human HER2, including but not limited to splice variants, allelic variants, and alloforms that retain one or more 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 produced recombinantly or synthetically.

[0194] The term “anti-HER2 antibody” or “antibody bound to HER2” refers to any form of antibody or fragment thereof that binds (e.g., specifically binds) to HER2, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to HER2. U.S. Patent No. 5,821,337 provides exemplary HER2 binding sequences, including exemplary anti-HER2 antibody sequences, and that case is incorporated herein by reference. In some embodiments, the anti-HER2 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. Trastuzumab (U.S. Patent No. 5,821,337; Molina et al. (2001) Cancer Res. 61(12):4744-9) is an exemplary anti-human HER2 antibody.

[0195] As used herein, the terms “multiligand proteoglycan-1,” “SDC1,” or “CD138” refer to any naturally occurring form of human CD138. This term encompasses full-length CD138 (e.g., UniProt reference sequence: P18827; SEQ ID NO: 32) and any form of human CD138 that can be obtained from cellular processing. The term also encompasses functional variants or fragments of human CD138, including but not limited to splice variants, allelic variants, and allotypes that retain one or more 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.

[0196] The term "anti-CD138 antibody" or "antibody bound to CD138" refers to any form of antibody or fragment thereof that binds (e.g., specifically binds) to CD138, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to CD138. In some embodiments, the anti-CD138 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. B-B4 (Tassone et al. (2004) Blood 104:3688-96) is an exemplary anti-human CD138 antibody.

[0197] As used herein, the terms “pteroin 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, allelic variants, and allotypes that retain one or more 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.

[0198] The term “anti-EPHA2 antibody” or “antibody bound to EPHA2” refers to any form of antibody or fragment thereof that binds (e.g., specifically binds) to EPHA2, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to EPHA2. WO2007 / 030642 provides exemplary EPHA2 binding sequences, including exemplary anti-EPHA2 antibody sequences, and that document is incorporated herein by reference. In some embodiments, the anti-EPHA2 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. 1C1 (WO 2007 / 030642; Jackson et al. (2008) Cancer Res. 68(22):9367-74) is an exemplary anti-human EPHA2 antibody.

[0199] As used herein, the terms “mesothelin” or “MSLN” refer to any naturally occurring form of human MSLN. This term encompasses full-length MSLN (e.g., UniProt reference sequence: Q13421; SEQ ID NO: 94) 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, allelic variants, and alloforms that retain one or more 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 produced recombinantly or synthetically.

[0200] The term "anti-MSLN antibody" or "antibody bound to MSLN" refers to any form of antibody or fragment thereof that binds (e.g., specifically binds) to MSLN, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to MSLN. WO 2011 / 074621 provides exemplary MSLN binding sequences, including exemplary anti-MSLN antibody sequences, and that document is incorporated herein by reference. In some embodiments, the anti-MSLN antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. IC11-25, IC14-30, IC7-4, IC17-35, and 2-9 are exemplary anti-human MSLN antibodies.

[0201] As used herein, the terms “glutamic acid carboxypeptidase 2” or “FOLH1” refer to any naturally occurring form of human FOLH1. This term encompasses full-length FOLH1 (e.g., UniProt reference sequence: Q04609; SEQ ID NO: 95) and any form of human FOLH1 that can be processed by cells. The term also encompasses functional variants or fragments of human FOLH1, including but not limited to splice variants, allelic variants, and alloforms that retain one or more 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 produced recombinantly or synthetically.

[0202] The term "anti-FOLH1 antibody" or "antibody bound to FOLH1" refers to any form of antibody or fragment thereof that binds (e.g., specifically binds) to FOLH1, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to FOLH1. WO2019 / 012260 and WO 2017 / 212250 provide exemplary FOLH1 binding sequences, including exemplary anti-FOLH1 antibody sequences, and such documents are incorporated herein by reference. In some embodiments, the anti-FOLH1 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. J591 (deimmunized) is an exemplary anti-human FOLH1 antibody.

[0203] As used herein, the terms “cadherin-6” or “CDH6” refer to any naturally occurring form of human CDH6. This term encompasses full-length CDH6 (e.g., UniProt reference sequence: P55285; SEQ ID NO: 96) and any form of human CDH6 that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human CDH6, including but not limited to splice variants, allelic variants, and allotypes that retain one or more 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.

[0204] The term "anti-CDH6 antibody" or "antibody bound to CDH6" refers to any form of antibody or fragment thereof that binds (e.g., specifically binds) to CDH6, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to CDH6. WO 2018 / 185618 provides exemplary CDH6 binding sequences, including exemplary anti-CDH6 antibody sequences, and that document is incorporated herein by reference. In some embodiments, the anti-CDH6 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0205] As used herein, the terms “carcinoembryonic antigen-associated cell adhesion molecule 5” or “CEACAM5” refer to any naturally occurring form of human CEACAM5. This term encompasses full-length CEACAM5 (e.g., UniProt reference sequence: P06731; SEQ ID NO: 97) and any form of human CEACAM5 that can be obtained from cellular processing. The term also encompasses functional variants or fragments of human CEACAM5, including but not limited to splice variants, allelic variants, and alloforms that retain one or more 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 produced recombinantly or synthetically.

[0206] The terms "anti-CEACAM5 antibody" or "antibody bound to CEACAM5" refer to any form of antibody or fragment thereof that binds (e.g., specifically binds) to CEACAM5, and encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to CEACAM5. US2015 / 0125386 provides exemplary CEACAM5 binding sequences, including exemplary anti-CEACAM5 antibody sequences, and that case is incorporated herein by reference. In some embodiments, the anti-CEACAM5 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. hMN14 is an exemplary anti-human CEACAM5 antibody.

[0207] As used herein, the terms “hidden family protein 1B” or “CFC1B” refer to any naturally occurring form of human CFC1B. This term encompasses full-length CFC1B (e.g., UniProt reference sequence: P0CG36; SEQ ID NO: 98) and any form of human CFC1B that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human CFC1B, including but not limited to splice variants, allelic variants, and alloforms that retain one or more biological functions of human CFC1B (i.e., unless the context indicates that the term is used only to refer to the wild-type protein, it encompasses variants and fragments). CFC1B can be isolated from humans or can be produced recombinantly or synthetically.

[0208] The terms "anti-CFC1B antibody" or "antibody bound to CFC1B" refer to any form of antibody or fragment thereof that binds (e.g., specifically binds) to CFC1B, and encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to CFC1B. WO2002 / 088170 provides exemplary CFC1B binding sequences, including exemplary anti-CFC1B antibody sequences, and that document is incorporated herein by reference. In some embodiments, the anti-CFC1B antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0209] 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: 99) and any form of human ENPP3 that can be processed by cells. The term also encompasses functional variants or fragments of human ENPP3, including but not limited to splice variants, allelic variants, and allotypes that retain one or more biological functions of human ENPP3 (i.e., unless the context indicates that the term is used only to refer to wild-type protein, it encompasses variants and fragments). ENPP3 can be isolated from humans or can be produced recombinantly or synthetically.

[0210] The term “anti-ENPP3 antibody” or “antibody bound to ENPP3” refers to any form of antibody or fragment thereof that binds (e.g., specifically binds) to ENPP3, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to ENPP3. Donate et al. ((2016) Clin Cancer Res. 22(8):1989-99) provide exemplary ENPP3 binding sequences, including exemplary anti-ENPP3 antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-ENPP3 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0211] As used herein, the terms “folate receptor α” or “FOLR1” refer to any naturally occurring form of human FOLR1. This term encompasses full-length FOLR1 (e.g., UniProt reference sequence: P15328; SEQ ID NO: 100) and any form of human FOLR1 that can be obtained from cellular processing. The term also encompasses functional variants or fragments of human FOLR1, including but not limited to splice variants, allelic variants, and allotypes that retain one or more 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 produced recombinantly or synthetically.

[0212] The term “anti-FOLR1 antibody” or “antibody bound to FOLR1” refers to any form of antibody or fragment thereof that binds (e.g., specifically binds) to FOLR1, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to FOLR1. WO2005 / 080431 and Coney et al. ((1991) Cancer Res. 51(22):6125-32) provide exemplary FOLR1 binding sequences, including exemplary anti-FOLR1 antibody sequences, and these cases and documents are incorporated herein by reference. In some embodiments, the anti-FOLR1 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment. Farletuzumab and MOv19 are exemplary anti-human FOLR1 antibodies.

[0213] As used herein, the terms “hepatitis A virus cell receptor 1” or “HAVCR1” refer to any naturally occurring form of human HAVCR1. This term encompasses full-length HAVCR1 (e.g., UniProt reference sequence: Q96D42; SEQ ID NO: 101) and any form of human HAVCR1 that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human HAVCR1, including but not limited to splice variants, allelic variants, and alloforms that retain one or more 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 produced recombinantly or synthetically.

[0214] The term “anti-HAVCR1 antibody” or “antibody bound to HAVCR1” refers to any form of antibody or fragment thereof that binds (e.g., specifically binds) to HAVCR1, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to HAVCR1. Thomas et al. ((2016) Mol Cancer Ther. [Molecular Cancer Therapeutics] 15(12):2946-54) provide exemplary HAVCR1 binding sequences, including exemplary anti-HAVCR1 antibody sequences, and that literature is incorporated herein by reference. In some embodiments, the anti-HAVCR1 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0215] As used herein, the terms “mast cell / stem cell growth factor receptor Kit” or “KIT” refer to any naturally occurring form of human KIT. This term encompasses full-length KIT (e.g., UniProt reference sequence: P10721; SEQ ID NO: 102) and any form of human KIT that can be processed by cells. The term also encompasses functional variants or fragments of human KIT, including but not limited to splice variants, allelic variants, and alloforms that retain one or more 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 produced recombinantly or synthetically.

[0216] The term “anti-KIT antibody” or “antibody bound to KIT” refers to any form of antibody or fragment thereof that binds (e.g., specifically binds) to KIT, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to KIT. Exemplary KIT binding sequences, including exemplary anti-KIT antibody sequences, are provided by Shi et al. ((2016) Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences] 113(33): E4784-93) and Abrams et al. ((2018) Clin Cancer Res. [Clinical Cancer Research] 24(17): 4297-308), and these references are incorporated herein by reference. In some embodiments, the anti-KIT antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0217] As used herein, the terms “hepatocyte growth factor receptor” or “MET” refer to any naturally occurring form of human MET. This term encompasses full-length MET (e.g., UniProt reference sequence: P08581; SEQ ID NO: 103) and any form of human MET that can be processed by cells. The term also encompasses functional variants or fragments of human MET, including but not limited to splice variants, allelic variants, and alloforms that retain one or more biological functions of human MET (i.e., unless the context indicates that the term is used only to refer to the wild-type protein, it encompasses variants and fragments). MET can be isolated from humans or can be produced recombinantly or synthetically.

[0218] The term "anti-MET antibody" or "antibody bound to MET" refers to any form of antibody or fragment thereof that binds (e.g., specifically binds) to MET, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to MET. Yang et al. ((2019) Acta Pharmacol Sin. [Chinese Journal of Pharmacology]) provide exemplary MET binding sequences, including exemplary anti-MET antibody sequences, which are incorporated herein by reference. In some embodiments, the anti-MET antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0219] As used herein, the term "mucin-16" or "MUC16" refers to any naturally occurring form of human MUC16. This term encompasses full-length MUC16 (e.g., UniProt reference sequence: Q8WXI7; SEQ ID NO: 104) and any form of human MUC16 that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human MUC16, including but not limited to splice variants, allelic variants, and alloforms that retain one or more 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 produced recombinantly or synthetically.

[0220] The term “anti-MUC16 antibody” or “antibody bound to MUC16” refers to any form of antibody or fragment thereof that binds (e.g., specifically binds) to MUC16, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to MUC16. Liu et al. ((2016) Ann Oncol. [Annals of Oncology] 27(11):2124-30) provide exemplary MUC16 binding sequences, including exemplary anti-MUC16 antibody sequences, and that literature is incorporated herein by reference. In some embodiments, the anti-MUC16 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0221] As used herein, the terms “zinc transporter ZIP6” or “SLC39A6” refer to any naturally occurring form of human SLC39A6. This term encompasses full-length SLC39A6 (e.g., UniProt reference sequence: Q13433; SEQ ID NO: 105) and any form of human SLC39A6 that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human SLC39A6, including but not limited to splice variants, allelic variants, and alloforms that retain one or more 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 produced recombinantly or synthetically.

[0222] The terms “anti-SLC39A6 antibody” or “antibody bound to SLC39A6” refer to any form of antibody or fragment thereof that binds (e.g., specifically binds) to SLC39A6, and encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to SLC39A6. Sussman et al. ((2014) Mol Cancer Ther. [Molecular Cancer Therapeutics] 13(12):2991-3000) provide exemplary SLC39A6 binding sequences, including exemplary anti-SLC39A6 antibody sequences, and that literature is incorporated herein by reference. In some embodiments, the anti-SLC39A6 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0223] As used herein, the terms “choline transporter-like protein 4” or “SLC44A4” refer to any naturally occurring form of human SLC44A4. This term encompasses full-length SLC44A4 (e.g., UniProt reference sequence: Q53GD3; SEQ ID NO: 106) and any form of human SLC44A4 that can be obtained from cellular processing. The term also encompasses functional variants or fragments of human SLC44A4, including but not limited to splice variants, allelic variants, and alloforms that retain one or more biological functions of human SLC44A4 (i.e., variants and fragments are included unless the context indicates that the term is used only to refer to the wild-type protein). SLC44A4 can be isolated from humans or can be produced recombinantly or synthetically.

[0224] The terms “anti-SLC44A4 antibody” or “antibody bound to SLC44A4” refer to any form of antibody or fragment thereof that binds (e.g., specifically binds) to SLC44A4, and encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind (e.g., specifically bind) to SLC44A4. Mattie et al. ((2016) Mol Cancer Ther. [Molecular Cancer Therapeutics] 15(11):2679-87) provide exemplary SLC44A4 binding sequences, including exemplary anti-SLC44A4 antibody sequences, and that literature is incorporated herein by reference. In some embodiments, the anti-SLC44A4 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0225] As used herein, the term “metal reductase STEAP1” or “STEAP1” refers to any naturally occurring form of human STEAP1. This term encompasses full-length STEAP1 (e.g., UniProt reference sequence: Q9UHE8; SEQ ID NO: 107) and any form of human STEAP1 that can be obtained through cellular processing. The term also encompasses functional variants or fragments of human STEAP1, including but not limited to splice variants, allelic variants, and alloforms that retain one or more 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.

[0226] The terms "anti-STEAP1 antibody" or "antibody bound to STEAP1" refer to any form of antibody or fragment thereof that binds to (e.g., specifically binds to) STEAP1, and encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments, provided that such biologically functional antibody fragments bind to (e.g., specifically bind to) STEAP1. WO 2008 / 052187 provides exemplary STEAP1 binding sequences, including exemplary anti-STEAP1 antibody sequences, and that document is incorporated herein by reference. In some embodiments, the anti-STEAP1 antibody used in the ADCs disclosed herein is an internalizing antibody or an internalizing antibody fragment.

[0227] As used herein, the terms "specificity," "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 binding to an appropriate antigen with binding to unrelated antigens or mixtures of antigens under a given set of conditions. An antibody is considered specific if it binds to an appropriate antigen with an affinity at least 2, 5, 7, and preferably 10 or more times greater than that with unrelated antigens or mixtures of antigens. A "specific antibody" or "target-specific antibody" is an antibody that binds only to a target antigen (e.g., HER2) but not to other antigens (or exhibits minimal binding to other antigens). In some embodiments, the K of an antibody or antigen-binding fragment that specifically binds to a target antigen (e.g., HER2) is... D 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, KD is 1 pM to 500 pM. In some embodiments, K D Between 500pM and 1μM, 1μM and 100nM, or 100mM and 10nM.

[0228] The term "epitope" refers to a portion of an antigen that can be recognized and specifically bound by an antibody. When the antigen is a polypeptide, the epitope can be formed from consecutive amino acids or from discontinuous amino acids adjacent to each other via tertiary folding of the polypeptide. Epitopes bound by antibodies can be identified using any epitope localization technique known in this art, including X-ray crystallography for epitope identification, which involves direct observation of the antigen-antibody complex, monitoring the binding of the antibody to fragments or mutant variants of the antigen, or monitoring the solvent accessibility of different portions of the antibody and antigen. Exemplary strategies for localizing antibody epitopes include, but are not limited to, array-based oligopeptide scanning, restricted proteolysis, site-directed mutagenesis, high-throughput mutagenesis localization, hydrogen-deuterium exchange, and mass spectrometry (see, for example, Gershoni et al. (2007) 21:145-56; and Hager-Braun and Tomer (2005) Expert Rev Proteomics 2:745-56).

[0229] Competitive binding and epitope binning can also be used to identify antibodies that share the same or overlapping epitopes. Competitive binding can be evaluated using cross-blocking analyses as described in 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 being tested reduces the binding of a reference antibody or binding protein (e.g., binding proteins containing a CDR and / or variable domains selected from those identified in Tables 2-4) to a target antigen (such as HER2) by at least about 50% (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or higher, or any percentage between thereof), and / or vice versa. In some embodiments, competitive binding may be attributed to shared or similar (e.g., partially overlapping) epitopes, or due to steric hindrance where the antibody or binding protein binds at a neighboring epitope (see, for example, Tzartos, Methods in Molecular Biology (Morris, ed., Vol. 66, pp. 55-66)). In some embodiments, competitive binding can be used to sort groups of binding proteins that share similar epitopes. For example, competitively binding proteins may be “boxed” into groups of binding proteins with overlapping or neighboring epitopes, while non-competitive binding proteins may be grouped into separate groups of binding proteins without overlapping or neighboring epitopes.

[0230] The term "k"on "or "k a "" refers to the association rate constant of antibodies and antigens to form antibody / antigen complexes. This rate can be determined using standard analyses such as surface plasma resonance, biolayer interferometry, or ELISA.

[0231] The term "k" off "or "k d "" refers to the dissociation rate constant of the antibody from the antibody / antigen complex. This rate can be determined using standard analyses such as surface plasma resonance, biolayer interferometry, or ELISA.

[0232] Term "K" D "K" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction. D via k a / k d This rate can be calculated using standard analyses such as surface plasma resonance, biolayer interferometry, or ELISA.

[0233] In the ADCs disclosed herein, the term "p" or "Hobstein splice regulator loading" or "Hobstein splice regulator:antibody ratio" or "Hobstein splice regulator to antibody ratio" or "HAR" refers to the number of Hobstein splice regulators per antibody or antigen-binding fragment, i.e., the Hobstein splice regulator loading, or the number of -LH portions of each antibody or antigen-binding fragment (Ab). In ADCs containing Hobstein splice regulators, "p" refers to the number of Hobstein splice regulators linked to the antibody or antigen-binding fragment. For example, if two Hobstein splice regulators (e.g., two compounds each having an H3 structure) are both linked to the antibody or antigen-binding fragment, then p = 2. In compositions containing multiple copies of the ADC as described herein, "average p" refers to the average number of -LH portions of each antibody or antigen-binding fragment, also referred to as "average Hobstein splice regulator loading".

[0234] The term "connector" or "connector part" is used herein to refer to any chemical part capable of covalently attaching a compound (typically a pharmaceutical part, such as a Hobstein splice regulator pharmaceutical part) to another part (such as an antibody or antigen-binding fragment). A connector may be readily available for or substantially resistant to acid-induced cleavage, peptidase-induced cleavage, light-based cleavage, esterase-induced cleavage, and / or disulfide bond cleavage, under conditions that preserve the activity of the compound or antibody.

[0235] The term "pharmaceutical" is used herein to refer to a compound, a mixture of compounds, a biomacromolecule, or an extract made from biological material. The terms "therapeutic agent" or "drug" refer to a pharmaceutical agent capable of modulating biological processes and / or possessing biological activity. The Hobschdiene splice regulator described herein is an exemplary therapeutic agent.

[0236] The terms "chemotherapy agent" or "anticancer agent" are used herein to refer to all agents that are effective in treating cancer regardless of their mechanism of action. Inhibition of metastasis or angiogenesis is often a characteristic of chemotherapy agents. Chemotherapy agents include antibodies, biomolecules, and small molecules, and encompass the Hopschidene splice regulator compounds described herein. Chemotherapy agents can be cytotoxic agents or cell growth inhibitors. The term "cell growth inhibitor" refers to an agent that inhibits or suppresses cell growth and / or cell proliferation. The term "cytotoxic agent" refers to a substance that causes cell death primarily by interfering with the expression activity and / or function of cells.

[0237] As used herein, the terms "herboxidiene splicing modulator" and "herboxidiene splice modulator" refer to compounds that have anticancer activity through interaction with components of the splice body and are structurally related to herboxidiene. In some embodiments, herboxidiene splicing modulators alter the splicing rate or form in target cells. Herboxidiene splicing modulators acting as inhibitors, for example, can reduce uncontrolled cell proliferation. In some embodiments, herboxidiene splicing modulators can function by binding to the SF3b splice body complex. Such modulators can be naturally occurring or synthetic herboxidiene derivatives or analogs. When referring to herboxidiene splicing modulators or their analogs, the terms "derivative" and "analyte" as used herein mean any compound that has substantially the same, similar, or enhanced biological function or activity as herboxidiene but has a modified chemical or biological structure. In some embodiments, herboxidiene splicing modulators are herboxidiene derivatives.

[0238] As used herein, “Hobstein splice regulator drug portion” refers to a component of an ADC or composition that provides the structure of a hobstein splice regulator compound, such as the hobstein splice regulator (H) component in an ADC having formula (I) or a composition containing -LH.

[0239] As used in this article, "splicing complex" refers to a ribonucleoprotein complex that removes introns from one or more RNA segments, such as pre-mRNA segments.

[0240] The term "homology" refers to a molecule that exhibits homology with another molecule by having, for example, the same or similar sequence of chemical residues at corresponding positions.

[0241] As used herein, the term "inhibit" or "inhibition of" means the reduction of a measurable quantity, and may include, but does not require, complete prevention or inhibition.

[0242] 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.

[0243] 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. Cell-to-cell contact, or at least the proximity between target-positive and target-negative cells, enables bystander killing. This type of killing can be distinguished from "off-target killing," which refers to the indiscriminate killing of target-negative cells. Off-target killing can be observed in the absence of target-positive cells.

[0244] The terms “hypertrophic disease” and “cancer” are used interchangeably herein to refer to cells possessing characteristics characteristic of cancerous cells, such as uncontrolled proliferation, non-destructiveness, metastatic potential, rapid growth and proliferation rates, and / or certain morphological features. Typically, cancer cells may present as tumors or masses, but these cells may exist alone within the subject or circulate independently in the bloodstream as cells such as leukemia cells or lymphoma cells. The terms “hypertrophic disease” and “cancer” encompass all types of cancer and cancer metastases, including hematologic malignancies, solid tumors, sarcomas, carcinomas and other solid tumor cancers, and non-solid tumor cancers. Hematologic malignancies may include B-cell malignancies, blood cancers (leukemia), plasma cell carcinomas (myeloma, such as multiple myeloma), or lymph node carcinomas (lymphoma). Exemplary B-cell malignancies include chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, and diffuse large B-cell lymphoma. Leukemia can include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloid monocytic leukemia (CMML), and acute monocytic leukemia (AMoL). Lymphoma can include Hodgkin's lymphoma and non-Hodgkin's lymphoma. Other hematologic malignancies can include myelomectomy syndrome (MDS). Solid tumors can include carcinomas such as adenocarcinoma (e.g., breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, stomach cancer, cervical cancer, endometrial cancer, ovarian cancer, bile duct cancer, glioma, and melanoma.

[0245] The terms “tumor” and “necrotic tumor” refer to any benign or malignant mass of tissue caused by excessive growth or proliferation of cells, including precancerous lesions.

[0246] The terms “tumor cell” and “proliferative cell” are used interchangeably and refer to individual cells or a total cell population derived from a tumor or neoplasm, including both non-tumorigenic cells and cancer stem cells. When referring only to tumor cells that lack the capacity for renewal and differentiation, the term “tumor cell” as used herein is modified by the term “non-tumorigenic” to distinguish those tumor cells from cancer stem cells.

[0247] The terms "subject" and "patient" are used interchangeably herein to refer to any animal, such as any mammal, including but not limited to humans, non-human primates, rodents, and similar animals. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human. In some embodiments, the subject is a mouse. In some embodiments, the subject is a human.

[0248] 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.

[0249] "Pharmaceutical composition" means a formulation which is permitted to administer 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 subject administering the formulation. Pharmaceutical compositions may be sterile.

[0250] "Drug excipients" include substances such as adjuvants, carriers, pH adjusters and buffers, tension modifiers, wetting agents, preservatives and similar reagents.

[0251] "Pharmaceutical acceptable" means approved or permitted by a federal regulatory agency or state government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals and more specifically in humans.

[0252] "Pharmaceutically acceptable salts" are salts that retain the desired biological activity of the parent compound without conferring unwanted toxicological effects. Examples of such salts include: (a) acid addition salts formed from inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and similar inorganic acids; and salts formed from organic acids, such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid and similar organic acids; and (b) salts formed from elemental anions such as chlorine, bromine and iodine. See, for example, Haynes et al., “Commentary: Occurrence of Pharmaceutically Acceptable Anions and Cations in the Cambridge Structural Database,” J Pharmaceutical Sciences, Vol. 94, No. 10 (2005), and Berge et al., “Pharmaceutical Salts,” J Pharmaceutical Sciences, Vol. 66, No. 1 (1977), which are incorporated herein by reference.

[0253] As used herein, the term "effective amount" means an amount of a compound, ADC, or composition (e.g., a heptacysteine ​​splice regulator or ADC) described herein that is sufficient to perform the specifically stated purpose, such as being sufficient to produce a therapeutic effect after administration, such as a reduction in tumor growth rate or tumor volume, relief of cancer symptoms, or some other marker of therapeutic efficacy. An effective amount can be determined in a conventional manner in relation to the stated purpose. The term "therapeutic effective amount" means an 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 extent, stage, development, and / or severity of cancer. A therapeutically effective amount can vary depending on the intended application (in vitro or in vivo) or the subject being treated and the disease condition, such as the subject's weight and age, the severity of the disease condition, the method of administration, and similar factors readily determined by those skilled in the art. This term also applies to doses that induce a specific response in target cells, such as inhibiting cell growth. The specific dosage may vary depending on factors such as the particular drug composition, the subject and their age, existing health conditions or risks associated with the health condition, the dosing regimen to be followed, the severity of the disease, the timing of administration (whether or not it is administered in combination with other agents), the tissue to which it is administered, and the physical delivery system that carries it. In cancer cases, therapeutically effective amounts 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.

[0254] "Effective preventive dose" refers to the amount that effectively achieves the desired preventive outcome at the necessary dose and time period. Typically, because the preventive dose is administered to subjects before or in the early stages of the disease, the effective preventive dose is less than the effective therapeutic dose.

[0255] As used herein, the terms “therapeutic” or “therapeutic” and grammatically relevant terms refer to any improvement in any outcome of a disease, such as prolonged survival, lower morbidity, and / or reduction of side effects resulting from alternative treatment modalities. As is readily understood in this art, a treatment procedure encompasses, but does not require, the complete eradication of the disease. As used herein, “treatment” means the administration of the described ADC or composition to a subject, such as a patient. Treatment may be used to cure, heal, alleviate, relieve, alter, remedy, improve, mitigate, improve, or influence a condition (e.g., cancer), its symptoms, or predisposition. In some embodiments, in addition to treating a subject with a condition, the compositions disclosed herein may be provided prophylactically to prevent or reduce the likelihood of developing that condition.

[0256] In some embodiments, labeled ADCs are used. Suitable “labels” include radioactive nuclei, enzymes, acceptors, cofactors, inhibitors, fluorescent portions, chemiluminescent portions, magnetic particles, and the like.

[0257] As used herein, “protein” means at least two covalently linked amino acids. The term encompasses polypeptides, oligopeptides, and peptides. In some embodiments, two or more covalently linked amino acids are linked by peptide bonds. Proteins can be composed of naturally occurring amino acids and peptide bonds, for example, when proteins are recombinantly produced using an expression system and host cells. Alternatively, proteins can comprise synthetic amino acids (e.g., homophenylalanine, citrulline, ornithine, and oroleucine) or peptide analog structures (i.e., “peptide or protein analogs,” such as peptide-like structures). Peptides-like structures are exemplary categories of peptide analogs whose side chains are attached to the nitrogen atoms of the peptide backbone rather than to the α-carbon (as it is in the amino acid group), and have different hydrogen bonding and conformational features compared to peptides (see, for example, Simon et al. (1992) Proc Natl Acad Sci. USA [Proceedings of the National Academy of Sciences] 89:9367). Therefore, peptide-like structures may be resistant to protein hydrolysis or other physiological or storage conditions and can efficiently penetrate cell membranes. Such synthetic amino acids can be incorporated, particularly when antibodies are synthesized in vitro using conventional methods well known in this art. Furthermore, any combination of peptide-mimicking, synthetic, and naturally occurring residues / structures can be used. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The amino acid "R group" or "side chain" can be in (L)- or (S)- configuration. In one specific embodiment, the amino acid is in (L)- or (S)- configuration.

[0258] "Recombinant protein" is a protein produced using recombinant technology, any technology and method known in this technology, i.e., by expressing recombinant nucleic acids. Methods and techniques for producing recombinant proteins are well known in this technology.

[0259] The “isolated” protein is not accompanied by at least one material to which it normally associates in its 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 the isolated protein may, depending on the circumstances, comprise from 5% by weight to 99.9% by weight of the total protein content. For example, the protein can be produced at a significantly higher concentration using an inducible promoter or a highly expressed promoter, thereby enabling the production of the protein at increased concentration levels. This definition includes the production of antibodies in a wide variety of organisms and / or host cells known in this art.

[0260] For amino acid sequences, sequence identity and / or similarity can be determined using, but not limited to, the local sequence identity algorithm of Smith and Waterman (1981) Adv. Appl. Math. [Advances in Applied Mathematics] 2:482, the sequence identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. [Journal of Molecular Biology] 48:443, the similarity method retrieval of Pearson and Lipman (1988) Proc. Nat. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package of the Genetics Computer Group at 575 Science Drive, Madison, Wisconsin), and Devereux et al. (1984) Nucl. Acid. The best-fit sequencing procedure described in Res. [Nucleic Acid Research] 12:387-95 is a known standard technique, preferably using preset settings or determined by testing. Preferably, the percentage of consistency is calculated by FastDB based on the following parameters: mismatch penalty 1; gap penalty 1; gap size penalty 0.33; and conjugation 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.).

[0261] An example of an applicable algorithm is PILEUP. PILEUP uses progressive pairwise alignment to generate multiple sequence alignments from a set of related sequences. It can also draw a tree diagram showing the clustering relationships used to generate the alignments. PILEUP uses a simplified form of the progressive alignment method described by Feng and Doolittle (1987) J Mol Evol. [Journal of Molecular Evolution] 35:351-60; this method is similar to that described by Higgins and Sharp (1989) CABIOS 5:151-3. Applicable PILEUP parameters include a preset gap weight of 3.00, a preset gap length weight of 0.10, and a weighted end gap.

[0262] Another example of a suitable algorithm is the BLAST algorithm described below: Altschul et al. (1990) J MolBiol. [Journal of Molecular Biology] 215:403-10; Altschul et al. (1997) Nucl Acid Res. [Nucleic Acid Research] 25:3389-402; and Karin et al. (1993) Proc Natl Acad Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 90:5873-87. A particularly suitable BLAST program is the WU-BLAST-2 program obtained from Altschul et al. (1996) Methods in Enzymology [Enzymological Methods] 266:460-80. WU-BLAST-2 uses several search parameters, most of which are set to default values. The adjustable parameters are set with the following values: overlap interval = I, overlap score = 0.125, and word threshold (T) = II. The HSP S and HSP S2 parameters are dynamic values ​​and are determined by the program itself based on the composition of the specific sequence and the composition of the specific database against which the target sequence is compared; however, these values ​​can be adjusted to improve sensitivity.

[0263] An additional applicable algorithm is the gapped BLAST reported by Altschul et al. (1997) Nucl Acid Res. 25:3389-402. The gapped BLAST uses BLOSUM-62 instead of a scoring system; the threshold parameter T is set to 9; a two-hit method is used to trigger the gapless extension, adding a gap length k at a cost of 10+k; Xu is set to 16, and Xg is set to 40 during the database retrieval phase and 67 during the algorithm output phase. Gap alignment is triggered by a scoring system corresponding to approximately 22.

[0264] Generally, the amino acid homology, similarity, or identity between the proteins and their variants disclosed herein (including variants of target antigens such as HER2, CD138 or EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1, and antibody variable domain variants (including individual variants CDR)) 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%.

[0265] 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 candidate sequences that are identical to nucleotide residues in the coding sequence of the antigen-binding protein. The specific method utilizes the BLASTN module of WU-BLAST-2 with preset parameters, where the overlap interval and overlap fraction are set to 1 and 0.125, respectively.

[0266] Although the sites or regions where amino acid sequence changes are introduced are predetermined, the mutations themselves do not need to be predetermined. For example, to optimize the efficacy of mutations at a given site, the optimal combination of random mutation induction at target codons or regions can be used to screen for the desired activity of expressed antigen-binding protein CDR variants. 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.

[0267] As used herein, "alkyl" or "alkyl group" means a fully saturated straight-chain, branched-chain, or cyclic hydrocarbon chain. In some embodiments, the alkyl group may contain 1-8 carbon atoms ("C1-C8 alkyl"). In some embodiments, the alkyl group may contain 1-6 carbon atoms ("C1-C6 alkyl"). In some embodiments, the alkyl group contains 1-3 carbon atoms. In still other embodiments, the alkyl group contains 2-3 carbon atoms, and in still other embodiments, the alkyl group contains 1-2 carbon atoms.

[0268] As used in this article, "alkylalkoxy" means an alkyl group substituted with an alkoxy group.

[0269] 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.

[0270] As used in this article, "alkylhydroxyl" refers to an alkyl group that has been substituted with a hydroxyl group.

[0271] As used in this article, "hydroxyl (hydroxy or hydroxyl)" refers to -OH.

[0272] As used herein, "carbocyclic" or "carbocyclic group" includes both aromatic groups (e.g., aryl) and non-aromatic groups (e.g., cycloalkyl). In some embodiments, the carbocyclic group contains 3-10 carbon atoms ("3- to 10-membered carbocyclic"). In some embodiments, the carbocyclic group contains 3-8 carbon atoms ("3- to 8-membered carbocyclic"). In some embodiments, the carbocyclic group contains 3-6 carbon atoms ("3- to 6-membered carbocyclic"). In some embodiments, the carbocyclic group contains 3-5 carbon atoms ("3- to 5-membered carbocyclic").

[0273] As used in this article, “haloalkyl” refers to an alkyl group that has been substituted with one or more halogen atoms.

[0274] "Halogen" refers to any halogen group, such as -F, -Cl, -Br, or -I.

[0275] As used herein, the terms “heterocycle” or “heterocyclyl” and “heterocyclic group” refer to monocyclic, bicyclic, or tricyclic heterocycles that contain at least one heteroatom in the ring.

[0276] A monocyclic heterocycle is a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom independently selected from O, N, and S. In some embodiments, the heterocycle is a 3- or 4-membered ring containing one heteroatom selected from O, N, and S. In some embodiments, the heterocycle is a 5-membered ring containing zero or one double bond and one, two, or three heteroatoms selected from O, N, and S. In some embodiments, the heterocycle is a 6-, 7-, or 8-membered ring containing zero, one, or two double bonds and one, two, or three heteroatoms selected from O, N, and S. Representative examples of monocyclic heterocycles include, but are not limited to, azaheptanyl, azaheptanyl, diazaheptanyl, 1,3-dioxane, 1,3-dioxolane, dihydropyranyl (including 3,4-dihydro-2H-pyran-6-yl), 1,3-dithiocyclopentane, 1,3-dithiaalkyl, imidazolinyl, imidazolinyl, isothiazolinyl, isothiazolinyl, isoxazolinyl, isoxazolinyl, morpholinyl, and oxane. Diazolinyl, oxadiazolinyl, oxazolinyl, oxazolinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolinyl, pyrrololinyl, pyrrolylyl, tetrahydrofuranyl, tetrahydropyranyl (including tetrahydro-2H-pyran-4-yl), tetrahydrothiophenyl, thiadiazolinyl, thiadiazolinyl, thiazolinyl, thiazolinyl, thiomorpholinyl, 1,1-thiomorpholinyl dioxide (thiomorpholinone), thiopyranyl, and trithiaalkyl.

[0277] The bicyclic heterocycles disclosed herein may include monocyclic heterocycles fused to an aryl group, or monocyclic heterocycles fused to a monocyclic cycloalkyl group, or monocyclic heterocycles fused to a monocyclic cycloalkenyl group, or monocyclic heterocycles fused to a monocyclic heterocycle having a total of 5 to 12 ring atoms. Examples of bicyclic heterocycles include, but are not limited to, 3,4-dihydro-2H-piperanyl, 1,3-benzo-m-dioxacyclopentenyl, 1,3-benzodithiocyclopentenyl, 2,3-dihydro-1,4-benzo-m-dioxacyclohexenyl, 2,3-dihydro-1-benzofuranyl, 2,3-dihydro-1-benzothiopheneyl, 2,3-dihydro-1H-indolyl, and 1,2,3,4-tetrahydroquinolinyl.

[0278] The terms "heterocycle" or "heterocyclyl" and "heterocyclic" encompass heteroaryl groups. A "heteroaryl" group refers to a cyclic moiety having one or more closed rings and, in at least one of these rings, one or more heteroatoms (oxygen, nitrogen, or sulfur), wherein at least one of these rings is an aromatic ring, and wherein the ring or such rings may be independently fused and / or bridged. Examples include, but are not limited to, phenyl, thiophene, triazolyl, pyridyl, pyrimidinyl, pyridazinyl, and pyrazinyl.

[0279] As described herein, the compounds disclosed may contain a “substitutable” portion. Generally, the term “substitutable”, whether or not preceded by the term “substitutable,” means that one or more hydrogens of the specified portion are replaced by suitable substituents. Unless otherwise indicated, the “substitutable” group may have suitable substituents at each substituted position of the group, and the substituents at each position may be the same or different when more than one position in any given structure is substituted by more than one substituent selected from the specified group. The substituent combinations contemplated in this disclosure are preferably combinations that result in the formation of stable or chemically viable compounds.

[0280] Those skilled in the art will understand that "substituted," "substituted," or "absent" includes the following implicit limitations: the substitution or absence is based on the permissible valence of the substituted atom and the substituent, and the substitution or absence produces a stable compound, for example, a stable compound that does not spontaneously undergo transformations such as rearrangement, cyclization, elimination, etc. For the purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents in the organic compounds described herein that satisfy the heteroatom valence.

[0281] "Stable" means that a compound is not substantially chemically and / or physically altered when subjected to conditions that permit its generation, detection, and in some embodiments its recovery, purification, and use for the purposes disclosed herein. In some embodiments, a stable or chemically viable compound is one that is substantially unchanged when kept at a temperature of 40°C or lower for at least one week in the absence of moisture or other chemically reactive conditions. In some embodiments, the compounds disclosed herein are stable.

[0282] The enantiomers taught herein may include “enantiomerically pure” isomers, which substantially contain a single enantiomer at one or more specific asymmetric centers, such as a single enantiomer greater than or equal to 90%, 92%, 95%, 98%, or 99% or equal to 100%. An “asymmetric center” or “chiral center” refers to a tetrahedral carbon atom containing four different substituents.

[0283] The compounds described herein may also contain atomic isotopes in non-natural proportions at one or more sites among the atoms constituting such compounds. For example, the compounds may be derived from substances such as deuterium (… 2 H), tritium ( 3 H), carbon-13 ( 13 C) or carbon-14 ( 14 C) Radioactive isotope labeling. All isotopic variants of the compounds disclosed herein, whether radioactive or not, are intended to be included within the scope of this disclosure. Furthermore, all tautomers of the compounds described herein are intended to be included within the scope of this disclosure.

[0284] Antibody-drug conjugates

[0285] The antibody-drug conjugate (ADC) compounds disclosed herein include ADC compounds with anticancer activity. Specifically, ADC compounds include antibodies or antigen-binding fragments (including their antigen-binding fragments) bound (i.e., covalently linked via a linker) to a Hopschdiene splice regulator, for example, wherein the Hopschdiene splice regulator has cytotoxic or cell growth-inhibiting effects when not bound to the antibody or antigen-binding fragment. In various embodiments, the Hopschdiene splice regulator is capable of binding to and / or interacting with the SF3b spliceosome complex when not bound to the antibody or antigen-binding fragment. In various embodiments, the Hopschdiene splice regulator is capable of modulating RNA splicing in vitro and / or in vivo when not bound to the antibody or antigen-binding fragment. In various embodiments, the Hopschdiene splice regulator and ADC disclosed herein are potent antiproliferative agents by targeting RNA splicing. In various embodiments, the Hopschdiene splice regulator and ADC disclosed herein can target both actively dividing cells and dormant cells.

[0286] In various embodiments, this disclosure is based, at least in part, on the discovery that specific bioactive hoprene splice regulators can provide improved properties when used in ADCs. While hoprene splice regulators can exhibit ideally improved properties when used alone (e.g., robust SF3b spliceosome complex binding, potent regulation of RNA splicing), in various embodiments, hoprene splice regulators may exhibit fewer of these ideally improved properties when bound to antibodies or antigen-binding fragments. Therefore, the development and production of ADCs for human therapeutics, such as for oncology, may require more than just identifying antibodies capable of binding to one or more desired targets and being linked to drugs used alone to treat cancer. Linking an antibody to a hoprene splice regulator can have a significant impact on the activity of one or both of the antibody and the hoprene splice regulator, depending on the type of linker and / or hoprene splice regulator selected. Therefore, in some embodiments, the components of the ADC are selected to (i) maintain one or more therapeutic properties exhibited by the separated antibody and Hobstein splice regulator moiety; (ii) maintain the specific binding properties of the antibody or antigen-binding fragment; (iii) optimize the loading of the Hobstein splice regulator and the ratio of the Hobstein splice regulator to the antibody; (iv) allow delivery via stable linkage to the antibody or antigen-binding fragment, such as intracellular delivery of the Hobstein splice regulator moiety; and (v) maintain the stability of the ADC as a complete conjugate until transported or delivered to the target site. (vi) Minimize aggregation of the ADC before or after administration; (vii) Allow therapeutic effects, such as cytotoxic effects, of the hoprene splice regulator moiety following lysis or other release mechanisms in the cellular environment; (viii) Demonstrate in vivo anticancer efficacy comparable to or superior to that of isolated antibodies and the hoprene splice regulator moiety; (ix) Minimize off-target killing via the hoprene splice regulator moiety; and / or (x) Demonstrate the desired pharmacokinetic and pharmacodynamic properties, reproducibility, and toxicological / immunological profile. These properties may be required to identify modified ADCs for therapeutic use (Ab et al. (2015) Mol Cancer Ther. [Molecular Cancer Therapeutics] 14:1605-13).

[0287] In various embodiments, the ADCs disclosed herein exhibit unexpectedly advantageous properties in some or all of the categories listed above. For example, in some embodiments, the ADC constructs disclosed herein exhibit unexpectedly advantageous hopsid splice regulator loading, aggregation and / or stability profiles, and / or retention of antibody binding function, drug activity and / or improved bystander killing, while reducing off-target killing, compared to ADCs containing alternative linkers and / or hopsid splice regulator portions. In some embodiments, the ADC constructs disclosed herein exhibit superior stability, activity, potency or other effects (in vivo or in vitro measurements) compared to ADCs using alternative linkers and / or hopsid splice regulator portions. In some embodiments, the ADC constructs disclosed herein exhibit in vivo therapeutic efficacy upon single-dose administration. In some embodiments, the ADC constructs disclosed herein are unexpectedly stable compared to ADCs using alternative linkers and / or hopsid splice regulator portions.

[0288] The disclosed ADC compounds selectively deliver effective doses of cytotoxic agents or cell growth inhibitors to cancer cells or tumor tissues. The disclosed ADCs have been found to possess potent cytotoxic and / or cell growth inhibitory activity against cells expressing specific target antigens (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1). In some embodiments, the cytotoxic and / or cell growth inhibitory activity of the ADC depends on the expression of the target antigen in the cells. In some embodiments, the disclosed ADCs are particularly effective at killing cancer cells expressing the target antigen while minimizing off-target killing. In some embodiments, the disclosed ADCs do not exhibit cytotoxic and / or cell growth inhibitory effects on cancer cells that do not express the target antigen.

[0289] Exemplary HER2-expressing cancers include, but are not limited to, breast cancer, gastric cancer, bladder cancer, urethral epithelial cell carcinoma, esophageal cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., serous endometrial carcinoma), salivary duct cancer, cervical cancer, endometrial cancer, and ovarian cancer (English et al. (2013) Mol Diagn Ther. [Molecular Diagnostics and Therapy] 17:85-99).

[0290] Exemplary CD138-expressing cancers include, but are not limited to, intrathoracic cancers (e.g., lung cancer, mesothelioma), skin cancers (e.g., basal cell carcinoma, squamous cell carcinoma), head and neck cancers (e.g., laryngeal cancer, laryngopharyngeal cancer, nasopharyngeal cancer), breast cancer, genitourinary cancers (e.g., cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, urethral cancer), hematologic malignancies (e.g., myeloma (such as multiple myeloma), B-cell malignancies, Hodgkin's lymphoma), and thyroid cancer (Szatmári et al. (2015) Dis Markers [Disease Markers] 2015:796052).

[0291] Exemplary EPHA2-expressing 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).

[0292] In some embodiments, ADC cleavage from antibody or antigen-binding fragments and linkers releases a hobsteine ​​splice regulator. In some embodiments, the linker and / or hobsteine ​​splice regulator is designed to promote bystander killing (neighbor cell killing). In some embodiments, the linker and / or hobsteine ​​splice regulator is designed to promote bystander killing via cleavage after internalization and diffusion of the linker-hobsteine ​​splice regulator portion and / or the hobsteine ​​splice regulator portion alone to neighboring cells. In some embodiments, the linker promotes internalization. In some embodiments, the linker is designed to minimize cleavage in the extracellular environment and thereby reduce toxicity to off-target tissues (e.g., non-cancerous tissues) while maintaining ADC binding to target tissues and bystander killing of cancerous tissues that do not express antigens targeted by the antibody or antigen-binding fragment of the ADC but surround target cancerous tissues expressing those antigens. In some embodiments, the catabolite of the hobsteine ​​splice regulator portion or the hobsteine ​​splice regulator portion produced by ADC cleavage is designed to promote uptake by target cells or neighboring cells (i.e., permeable cells). These types of Hobstein splice regulator moieties and catabolites are referred to as "bystander activity" in this paper, while drug moieties or catabolites with reduced cell permeability are referred to as "bystander inactivity".

[0293] In some embodiments, the disclosed ADC also exhibits bystander-killing activity, but with low off-target cytotoxicity. Without being bound by theory, the bystander-killing activity of an ADC can be particularly advantageous when ADC penetration into solid tumors is limited and / or when target antigen expression in tumor cells is heterogeneous. In some embodiments, ADCs containing cleavable linkers are particularly effective at bystander-killing and / or exhibit improved bystander-killing activity compared to comparable treatments with ADCs containing non-cleavable linkers. In some embodiments, the ADCs disclosed herein exhibit improved solubility and target cell penetration compared to the drug portion alone. In some embodiments, the ADCs disclosed herein exhibit improved cytotoxicity compared to the cytotoxicity of the hoprene splice regulator portion alone. In some embodiments, the ADCs disclosed herein use a drug portion that exhibits lower cytotoxicity when evaluated as the hoprene splice regulator alone, but are unexpectedly superior to ADCs containing other hoprene splice regulator portions that exhibit higher cytotoxicity when evaluated as the hoprene splice regulator alone. In some embodiments, the cleavage and release of the hippelin splice regulator improves the cytotoxicity of the ADC relative to equivalent treatments with an ADC containing a non-cleavable linker. In other embodiments, the ADC does not require the cleavage and release of the hippelin splice regulator to have the desired biological activity. In some embodiments, an ADC containing a non-cleavable linker with an increased spacer length (e.g., ADL12) provides the same or similar cytotoxicity relative to equivalent treatments with an ADC containing a cleavable linker (e.g., ADL1, ADL5), and unexpectedly superior cytotoxicity relative to equivalent treatments with an ADC containing a shorter non-cleavable linker. In some embodiments, an ADC containing a non-cleavable linker with an increased spacer length and no carbonyl group (e.g., ADL12) provides the same or similar cytotoxicity relative to equivalent treatments with an ADC containing a cleavable linker (e.g., ADL1, ADL5), and unexpectedly superior cytotoxicity relative to equivalent treatments with an ADC containing a non-cleavable linker with the same or similar spacer length and a carbonyl group (e.g., ADL10). In some embodiments, removal of the carbonyl group from the non-cleavable MC linker (e.g., ADL12) can cause an increase in cytotoxicity greater than 50-fold, greater than 75-fold, greater than 100-fold, greater than 150-fold, or greater than 200-fold relative to equivalent treatment with an ADC containing an unmodified non-cleavable MC linker (e.g., ADL10).In some embodiments, the removal of a carbonyl group from a non-cleavable MC linker (e.g., ADL12) and the increase in spacer length (e.g., the addition of at least one spacer unit) can result in a greater than 50-fold, greater than 75-fold, greater than 100-fold, greater than 150-fold, or greater than 200-fold increase in cytotoxicity compared to equivalent treatment with an ADC containing an unmodified non-cleavable MC linker (e.g., ADL10).

[0294] This document provides ADC compounds comprising an antibody or antigen-binding fragment thereof targeting tumor cells (Ab), a splice regulator drug moiety (D), and a linker moiety (L) covalently linked to the Ab to the D. In some aspects, the antibody or antigen-binding fragment is capable of binding to tumor-associated antigens (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1) with high specificity and high affinity. In some embodiments, the antibody or antigen-binding fragment is internalized into the target cells after binding, for example, into the degradation compartment of the cell. In other embodiments, ADCs that are internalized after binding to target cells, undergo degradation, and release the hoprene splice regulator moiety to kill cancer cells can be used. The hoprene splice regulator moiety can be released from the antibody and / or linker moiety of the ADC through enzymatic action, hydrolysis, oxidation, or any other mechanism.

[0295] An exemplary ADC has equation (I):

[0296] Ab-(LH) p (I)

[0297] Where Ab = antibody or antigen binding fragment, L = linker portion, H = hophecediene splice regulator drug portion, and p = number of splice regulator drug portions for each antibody or antigen binding fragment.

[0298] In some embodiments, the portion targeting the hoprene splice regulator in the described ADC and compositions is an antibody or antigen-binding fragment. Other exemplary targeted pharmaceutical portions for the described ADC and compositions are also provided and described herein. In some embodiments, the portion targeting the hoprene splice regulator may be any of a variety of cell binders and non-antibody backbones. In some embodiments, the portion targeting the hoprene splice regulator is a cell binder. As used herein, the term "cell binder" means any agent capable of binding to animal (e.g., human) cells and delivering a hoprene splice regulator portion (e.g., a hoprene splice regulator pharmaceutical portion as disclosed herein). This term encompasses the exemplary antibodies and antigen-binding fragments disclosed herein (e.g., monoclonal antibodies and fragments such as Fab and scFV). The term further encompasses exemplary cell binders such as DARPin, duobody, bicyclic peptide, nanobody, centyrin, MSH (melanocyte-stimulating hormone), receptor-Fc fusion molecules, T-cell receptor structures, steroid hormones (such as androgens and estrogens), growth factors, community-stimulating factors (such as EGF), and other non-antibody backbones. In various embodiments, non-antibody backbones can be broadly categorized into two structural classes: compounds with domain-defined sizes (approximately 6-20 kDa) and restricted peptides (approximately 2-4 kDa). Exemplary domain-sized backbones include, but are not limited to, affinity antibodies, affilin, anticarrier protein, atrimer, DARPin, FN3 backbones (such as adnectin and sintine), fynomer, Kunitz domain, pronectin, O-body, and receptor-Fc fusion proteins, while exemplary restrictive peptides include high-affinity multimers, bicyclic peptides, and Cys-knots. In some embodiments, the portion of the target drug in the described ADC and composition is selected from affinity antibodies, affilin, anticarrier protein, atrimer, DARPin, FN3 backbones (such as adnectin or sintine), fynomer, Kunitz domain, pronectin, O-body, high-affinity multimer, bicyclic peptide, and Cys-knot. In some embodiments, the portion of the target drug in the described ADC and composition is a receptor-Fc fusion protein, such as a HER2-Fc chimeric fusion protein. Non-antibody backbones are reviewed, for example, in Vazquez-Lombardi et al. (2015) Drug Dis Today 20(10):1271-83.

[0299] Antibody

[0300] Formula (I) antibody or antigen-binding fragment (Ab) within its scope includes any antibody or antigen-binding fragment that specifically binds to a target antigen on cancer cells. The antibody or antigen-binding fragment may bind to the target antigen, wherein, for example... Analysis of the measured dissociation constant (K) D ≤1 mM, ≤100 nM, or ≤10 nM, or any amount in between. In some embodiments, K D The range is from 1 pM to 500 pM. In some embodiments, K D Between 500pM and 1μM, 1μM and 100nM, or 100mM and 10nM.

[0301] In some embodiments, the antibody or antigen-binding fragment is a four-chain antibody (also referred to as an immunoglobulin, full-length antibody, or intact antibody) comprising two heavy chains and two light chains. In some embodiments, the antibody or antigen-binding fragment is a double-chain half-antibody (one light chain and one heavy chain) or an antigen-binding fragment of an immunoglobulin. In some embodiments, the antibody or antigen-binding fragment is an antigen-binding fragment of an immunoglobulin that retains the ability to bind to a target cancer antigen and / or provides immunoglobulin function.

[0302] In some embodiments, the antibody or antigen-binding fragment is an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is an internalizing antibody or an internalizing antigen-binding fragment thereof. In some embodiments, the internalizing antibody or its internalizing antigen-binding fragment binds to the target cancer antigen expressed on the cell surface and enters the cell after binding. In some embodiments, after the ADC enters and is present in the cell expressing the target cancer antigen (i.e., after the ADC has been internalized), the heptahydrate splice regulator drug portion of the ADC is released from the antibody or antigen-binding fragment of the ADC, for example by lysis, degradation by the antibody or antigen-binding fragment, or by any other suitable release mechanism.

[0303] The amino acid sequences of the exemplary antibodies disclosed herein are set forth in Tables 2-4.

[0304] Table 1. Antibodies

[0305] mAb type target Trastuzumab (AB185) Humanization HER2 / NEU B-B4(AB205) mouse CD138 (Multiligand Proteoglycan-1) 1C1(AB206) Humanization EPHA2

[0306] Table 2. Amino acid sequences of the variable region of mAb

[0307]

[0308]

[0309] Table 3. Amino acid sequences of mAb CDR

[0310]

[0311] Table 4. Amino acid sequence of full-length mAb Ig chain

[0312]

[0313]

[0314] Table 5. Exemplary target antigen amino acid sequences

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327] In various embodiments, the ADC disclosed herein may comprise any set of heavy and light chain variable domains listed in the table above, or, for example, a set of six CDR sequences derived from the heavy and light chain sets 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 remains bound to its target cancer antigen (e.g., where K...). D Less than 1×10 -8 The ability to (M) and maintain one or more functional properties of the ADCs disclosed herein (e.g., the ability to internalize, regulate RNA splicing, inhibit cell growth, etc.) is sufficient.

[0328] In some embodiments, the ADC further comprises human heavy and light chain constant domains or fragments thereof. For example, the ADC may comprise a human IgG heavy chain constant domain (such as IgG1) and a human κ or λ light chain constant domain. In various embodiments, the antibody or antigen-binding fragment of the described ADC comprises a human immunoglobulin G subtype 1 (IgG1) heavy chain constant domain and a human Igκ light chain constant domain.

[0329] In various other embodiments, the target cancer antigen of the ADC is human epidermal growth factor receptor 2 (HER2).

[0330] In different 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.

[0331] In various embodiments, the anti-HER2 antibody or its antigen-binding fragment comprises 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 amino acid sequence of the heavy chain variable region of SEQ ID NO:19 and the amino acid sequence of the light chain variable region of SEQ ID NO:20, or a sequence having at least 95% identity with the disclosed sequence. In some embodiments, the anti-HER2 antibody or its antigen-binding fragment has a heavy chain variable region amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:19 and / or a light chain variable region amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:20.

[0332] 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.

[0333] In various embodiments, the anti-HER2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:19 or a sequence having at least 95% identity with SEQ ID NO:19 and the light chain amino acid sequence of SEQ ID NO:20 or a sequence having at least 95% identity with SEQ ID NO:20. In specific embodiments, the anti-HER2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:19 and the light chain amino acid sequence of SEQ ID NO:20 or a sequence having at least 95% identity with the disclosed sequence. In some embodiments, the anti-HER2 antibody has a heavy chain amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:19 and a light chain amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:20. In various embodiments, the anti-HER2 antibody is trastuzumab or its antigen-binding fragment.

[0334] In various embodiments, the anti-HER2 antibody or its antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs of trastuzumab, or wherein the CDRs include no more than one, two, three, four, five or six amino 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).

[0335] In various other embodiments, the target cancer antigen of the ADC is human multiligand proteoglycan-1 (CD138).

[0336] In various embodiments, the anti-CD138 antibody or its antigen-binding fragment comprises the following three heavy chain CDRs and three light chain CDRs: 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, as defined by the Kabat numbering system.

[0337] In various embodiments, the anti-CD138 antibody or its antigen-binding fragment comprises 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 amino acid sequence of the heavy chain variable region of SEQ ID NO:21 and the amino acid sequence of the light chain variable region of SEQ ID NO:22, or a sequence having at least 95% identity with the disclosed sequence. In some embodiments, the anti-CD138 antibody or its antigen-binding fragment has a heavy chain variable region amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:21 and / or a light chain variable region amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:22.

[0338] In various embodiments, the anti-CD138 antibody or its antigen-binding fragment is an internalizing antibody or an internalizing antigen-binding fragment. In various embodiments, the anti-CD138 antibody comprises a mouse IgG2a heavy chain constant domain and a mouse Igκ light chain constant domain. In various embodiments, the anti-CD138 antibody comprises a human IgG2a heavy chain constant domain and a human Igκ light chain constant domain.

[0339] In various embodiments, the anti-CD138 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:21 or a sequence having at least 95% identity with SEQ ID NO:21 and the light chain amino acid sequence of SEQ ID NO:22 or a sequence having at least 95% identity with SEQ ID NO:22. In specific embodiments, the anti-CD138 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:21 and the light chain amino acid sequence of SEQ ID NO:22 or a sequence having at least 95% identity with the disclosed sequence. In some embodiments, the anti-CD138 antibody has a heavy chain amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:21 and a light chain amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:22. In various embodiments, the anti-CD138 antibody is B-B4 or an antigen-binding fragment thereof.

[0340] In various embodiments, the anti-CD138 antibody or its antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs of B-B4, or wherein the CDRs include no more than one, two, three, four, five or six amino acid additions, deletions or substitutions of HCDR1 (SEQ ID NO:7), HCDR2 (SEQ ID NO:8), HCDR3 (SEQ ID NO:9), LCDR1 (SEQ ID NO:10), LCDR2 (SEQ ID NO:11) and LCDR3 (SEQ ID NO:12).

[0341] In various other embodiments, the target cancer antigen of the ADC is human pteroin type A receptor 2 (EPHA2).

[0342] In various embodiments, the anti-EPHA2 antibody or its antigen-binding fragment comprises the following three heavy chain CDRs and three light chain CDRs: 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, as defined by the Kabat numbering system.

[0343] In various embodiments, the anti-EPHA2 antibody or its antigen-binding fragment comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:23 and a light chain variable region containing the amino acid sequence of SEQ ID NO:24. In some embodiments, the anti-EPHA2 antibody or its antigen-binding fragment comprises the amino acid sequence of the heavy chain variable region of SEQ ID NO:23 and the amino acid sequence of the light chain variable region of SEQ ID NO:24, or a sequence having at least 95% identity with the disclosed sequence. In some embodiments, the anti-EPHA2 antibody or its antigen-binding fragment has a heavy chain variable region amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:23 and / or a light chain variable region amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:24.

[0344] 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.

[0345] In various embodiments, the anti-EPHA2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:23 or a sequence having at least 95% identity with SEQ ID NO:23 and the light chain amino acid sequence of SEQ ID NO:24 or a sequence having at least 95% identity with SEQ ID NO:24. In a particular embodiment, the anti-EPHA2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:23 and the light chain amino acid sequence of SEQ ID NO:24 or a sequence having at least 95% identity with the disclosed sequence. In some embodiments, the anti-EPHA2 antibody has a heavy chain amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:23 and a light chain amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:24. In some embodiments, the anti-EPHA2 antibody comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO:23 and a light chain encoded by the nucleotide sequence of SEQ ID NO:24. In different embodiments, the anti-EPHA2 antibody is 1C1 or its antigen-binding fragment.

[0346] In various embodiments, the anti-EPHA2 antibody or its antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs of 1C1, or wherein the CDRs include no more than one, two, three, four, five or six amino acid additions, deletions or substitutions of HCDR1 (SEQ ID NO:13), HCDR2 (SEQ ID NO:14), HCDR3 (SEQ ID NO:15), LCDR1 (SEQ ID NO:16), LCDR2 (SEQ ID NO:17) and LCDR3 (SEQ ID NO:18).

[0347] In different embodiments, amino acid substitutions have individual residues. Insertions are typically on the order of about 1 to about 20 amino acid residues, but significantly larger insertions are permissible as long as biological function (e.g., binding to a target antigen) is maintained. Deletions are typically in the range of about 1 to about 20 amino acid residues, but in some cases, deletions may be much larger. Substitution, deletion, insertion, or any combination thereof can be used to obtain the final derivative or variant. Generally, these changes are made to several amino acids to minimize alterations to the molecule, specifically the immunogenicity and specificity of the antigen-binding protein. However, in some cases, more variations are permissible. Conservative substitutions are generally performed according to the diagrams depicted below, as shown in Table 6.

[0348] Table 6

[0349] Exemplary substitution of original residues

[0350] Ala Ser

[0351] Arg Lys

[0352] Asn Gln,His

[0353] Asp Glu

[0354] Cys Ser

[0355] Gln Asn

[0356] Glu Asp

[0357] Gly Pro

[0358] His Asn,Gln

[0359] Ile Leu,Val

[0360] Leu Ile,Val

[0361] Lys Arg,Gln,Glu

[0362] Met Leu,Ile

[0363] Phe Met,Leu,Tyr

[0364] Ser Thr

[0365] Thr Ser

[0366] Trp Tyr

[0367] Tyr Trp,Phe

[0368] Val Ile,Leu

[0369] Substantial changes in functional or immune properties can be made by selecting substitutions with lower conservation than those shown in Table 6. For example, substitutions can have a more significant impact on: altering the structure of the polypeptide backbone in a region, such as α-helical or β-folded structures; the charge or hydrophobicity of the molecule at the target site; or the volume of the side chains. Substitutions that typically produce the most significant alterations in peptide properties are those that include: (a) substitution of a hydrophilic residue, such as serine or threonine, for a hydrophobic residue, such as leucine, isoleucine, phenylalanine, valine, or alanine (or substitution via a hydrophobic residue); (b) substitution of any other residue, such as cysteine ​​or proline; (c) substitution of a positively charged residue, such as lysine, arginine, or histidine, for a negatively charged residue, such as glutamyl or aspartic (or substitution via a negatively charged residue); or (d) substitution of a bulky residue, such as phenylalanine, for a residue without a side chain, such as glycine (or substitution via a residue without a side chain).

[0370] In different embodiments where the variant antibody sequence is used in an ADC, the variants typically exhibit the same qualitative biological activity and elicit the same immune response, but variants can also be selected as needed to modify the characteristics of the antigen-binding protein. Alternatively, the variant can be designed to alter the biological activity of the antigen-binding protein. For example, glycosylation sites can be altered or removed.

[0371] Various antibodies can be used with the ADCs used herein to target cancer cells. As shown below, the linker-load in the ADCs disclosed herein is surprisingly effective with antibodies targeting different tumor antigens. Suitable antigens expressed on tumor cells rather than healthy cells, or expressed on tumor cells at higher levels than on healthy cells, and antibodies against such antigens are known in this art. These antibodies can be used with the linker and Hobstein splice regulator load disclosed herein. In some embodiments, the antibody or antigen-binding fragment targets HER2, and the antibody or antigen-binding fragment targeting HER2 is trastuzumab. In some embodiments, the antibody or antigen-binding fragment targets CD138, and the antibody or antigen-binding fragment targeting CD138 is B-B4. In some embodiments, the antibody or antigen-binding fragment targets EPHA2, and the antibody or antigen-binding fragment targeting EPHA2 is 1C1. In some embodiments, although the disclosed linker and hoprene splicing regulator payloads are unexpectedly effective with several different tumor-targeting antibodies, antibodies targeting HER2 (such as trastuzumab), antibodies targeting CD138 (such as B-B4), and antibodies targeting EPHA2 (such as 1C1) offer specifically improved therapeutic effects: antibody ratio, aggregation, stability (i.e., in vitro and in vivo stability), tumor targeting (i.e., cytotoxicity, potency), and / or therapeutic efficacy. Improved therapeutic efficacy can be measured in vitro or in vivo and may include a slowed tumor growth rate and / or a reduced tumor volume.

[0372] In some embodiments, alternative antibodies targeting the same target or antibody against different antigen targets are used, providing at least some of the advantageous functional properties described above (e.g., improved stability, improved tumor targeting, improved therapeutic efficacy, etc.). In some embodiments, some or all of these advantageous functional properties are observed when the disclosed linker and hoprene splice regulator payload binds to an alternative antibody or antigen-binding fragment targeting HER2, CD138, or EPHA2. In some other embodiments, some or all of these advantageous functional properties are observed when the disclosed linker and hoprene splice regulator payload binds to an antibody or antigen-binding fragment targeting HER2. In some embodiments, the antibody or antigen-binding fragment targets HER2. In some embodiments, the antibody or antigen-binding fragment targeting HER2 is trastuzumab. In some other embodiments, some or all of these advantageous functional properties are observed when the disclosed linker and hoprene splice regulator payload binds to an antibody or antigen-binding fragment targeting CD138. In some embodiments, the antibody or antigen-binding fragment targets CD138. In some embodiments, the antibody or antigen-binding fragment targeting CD138 is B-B4. In some other embodiments, some or all of these advantageous functional properties are observed when the disclosed linker and Hobstein splicing regulator are effectively loaded and bound to the antibody or antigen-binding fragment targeting EPHA2. In some embodiments, the antibody or antigen-binding fragment targets EPHA2. In some embodiments, the antibody or antigen-binding fragment targeting EPHA2 is 1C1.

[0373] connector

[0374] In various embodiments, the linkers in the ADC are extracellularly stable in a manner sufficient to achieve therapeutic efficacy. In some embodiments, the linkers are stable outside the cells such that the ADC remains intact when present in extracellular conditions (e.g., prior to transport or delivery to cells). The term “intact” as used in the context of ADCs means that the antibody or antigen-binding fragment remains attached to the drug moiety (e.g., a Hopschide splice regulator). In the context of linkers or ADCs containing linkers, “stable” as used herein means that no more than 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 3%, or no more than about 1% (or any percentage between these) of the linker in the ADC sample is cleaved (or the overall ADC is otherwise incomplete) when the ADC is present in extracellular conditions. In some embodiments, the linkers and / or ADCs disclosed herein are unexpectedly stable compared to alternative linkers and / or ADCs with alternative linker and / or Hopschide splice regulator payloads. In some embodiments, the ADC disclosed herein may 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.

[0375] Extracellular stability of the linker can be determined, for example, by including the ADC in plasma for a predetermined time period (e.g., 2, 4, 6, 8, 16, 24, 48, or 72 hours) and subsequently quantifying the amount of free drug fraction present in the plasma. Stability allows the ADC to target tumor cells at the appropriate time and prevents premature release of the drug fraction, which could reduce the therapeutic index of the ADC by indiscriminately damaging both normal and tumor tissues. In some embodiments, the linker is stable outside the target cells and releases the drug fraction from the ADC once inside the cells, allowing the drug to bind to its target (e.g., to the SF3b splice complex). Thus, an effective linker will: (i) maintain the specific binding properties of the antibody or antigen-binding fragment; (ii) allow the drug fraction to be delivered (e.g., intracellularly) via a stable linker to the antibody or antigen-binding fragment; (iii) remain stable and intact until the ADC has been transported or delivered to its target site; and (iv) allow the therapeutic effects of the drug fraction, such as cytotoxicity, following cleavage or alternative release mechanisms.

[0376] Connectors can affect the physicochemical properties of ADCs. Since many cytotoxic agents are inherently hydrophobic, linking them to antibodies with an additional hydrophobic moiety can lead to aggregation. ADC aggregates are often insoluble and typically limit achievable drug loading on the antibody, which can adversely affect the efficacy of the ADC. Generally, protein aggregates in biologics are also associated with increased immunogenicity. As shown below, the connectors disclosed herein enable ADCs to exhibit low aggregation and the desired drug loading.

[0377] Linkers can be “cleavable” or “non-cleavable” (Ducry and Stump (2010) Bioconjugate Chem. [Bioconjugate Chemistry] 21:5-13). Cleavable linkers are designed to release drug moieties (e.g., Hobstein splicing regulators) upon exposure to certain environmental factors, such as internalization into target cells, while non-cleavable linkers generally rely on the degradation of the antibody or antigen-binding fragment itself.

[0378] In some embodiments, the linker is a non-cleavable linker. In some embodiments, the drug portion of the ADC's hobschdiene splice regulator is released by degradation of the antibody or antigen-binding fragment. Upon internalization and degradation within target cells, non-cleavable linkers tend to maintain covalent association with at least one amino acid of the antibody and the drug. Several exemplary non-cleavable linkers are described herein, and other non-cleavable linkers are known in the art. Exemplary non-cleavable linkers may comprise a thioether, a cyclohexyl group, N-succinimide-4-(N-cis-butenedimide-methyl)cyclohexane-1-carboxylate (SMCC) or N-hydroxysuccinimide (NHS), one or more polyethylene glycol (PEG) portions (e.g., 1, 2, 3, 4, 5, or 6 PEG portions), or one or more alkyl portions.

[0379] In some embodiments, the connector is a cleavable connector. A cleavable connector refers to any connector 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 well known in this art and include, but are not limited to, acid-instable bonds, protease / peptidase-instable bonds, light-instable bonds, disulfide bonds, and esterase-instable bonds. A connector containing a cleavable portion allows the release of the Hobstein splice regulator drug moiety from the ADC via cleavage at a specific site within the connector.

[0380] In some embodiments, the linker is cleavable under intracellular conditions such that the cleavage of the linker adequately releases the hoprene splice regulator drug moiety from the antibody or antigen-binding fragment in the intracellular environment to activate the drug and / or confer therapeutic efficacy. In some embodiments, the hoprene splice regulator drug moiety does not cleave from the antibody or antigen-binding fragment until the ADC enters a cell expressing an antigen specific to the antibody or antigen-binding fragment of the ADC, and after entering the cell, the hoprene splice regulator drug moiety cleaves from the antibody or antigen-binding fragment. In some embodiments, the linker includes a cleavable portion positioned such that, after cleavage, a portion of the linker or antibody or antigen-binding fragment does not remain bound to the hoprene splice regulator drug moiety. Exemplary cleavable linkers include acid-instable linkers, protease / peptidase-sensitive linkers, photostable linkers, dimethyl-containing linkers, disulfide-containing linkers, or sulfonamide-containing linkers.

[0381] In some embodiments, the linker is a pH-sensitive linker and is sensitive to hydrolysis at certain pH values. Typically, pH-sensitive linkers can be hydrolyzed under acidic conditions. This cleavage strategy generally utilizes a lower pH in the endosomal intracellular compartment (pH about 5-6) and the lysosomal intracellular compartment (pH about 4.8) compared to the cytosol intracellular compartment (pH about 7.4) to trigger the hydrolysis of acid-instable groups in linkers such as hydrazones (Jain et al. (2015) Pharm Res [Pharmaceutical Research] 32:3526-40). In some embodiments, the linker is an acid-instable linker and / or a hydrolyzable linker. For example, acid-instable linkers that can be hydrolyzed in lysosomes and contain acid-instable groups (e.g., hydrazones, hemicarbazones, thiohemicarbazones, cis-aconitamide, orthoesters, acetals, ketals, or analogs thereof) can be used. See, for example, U.S. Patent Nos. 5,122,368, 5,824,805, and 5,622,929; Dubowchik and Walker (1999) Pharm Therapeutics 83:67-123; Neville et al. (1989) Biol Chem. 264:14653-61. These types of linkers are relatively stable under neutral pH conditions, such as the pH of blood, but unstable below pH 5.5 or 5.0 (the approximate pH of lysosomes). In some embodiments, the hydrolyzable linker is a thioether linker (such as a thioether linked to the therapeutic agent via an acylhydrazone bond) (see, for example, U.S. Patent No. 5,622,929).

[0382] In some embodiments, the connector may be cleaved under reducing conditions. In some embodiments, the connector may be cleaved in the presence of a reducing agent such as glutathione or dithiothreitol. In some embodiments, the connector is a cleavable disulfide connector or a cleavable sulfonamide connector.

[0383] In some embodiments, the connector is a pyrolytic disulfide connector. Various disulfide connectors are known in the art, including, for example, disulfide connectors formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)toluene), SPDB, and SMPT. See, for example, Thorpe et al. (1987) Cancer Res. 47:5924-31; Wawrzynczak et al., Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogel, ed., Oxford University Press, 1987). See also U.S. Patent No. 4,880,935. Disulfide linkers are often used to develop the abundance of intracellular thiols, which can facilitate the cleavage of their disulfide bonds. The intracellular concentrations of the most abundant intracellular thiols and reduced glutathione are generally in the range of 1–10 nM, which is about 1,000 times higher than the intracellular concentration of the most abundant low-molecular-weight thiols (i.e., cysteine) in the blood at about 5 μM (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.

[0384] In some embodiments, the connector is a cleavable sulfonamide connector. As used herein, a cleavable sulfonamide connector refers to any connector containing a cleavable sulfonamide moiety. The term "cleavable sulfonamide moiety" refers to a sulfonamide group, i.e., a sulfonyl group attached to an amine group, wherein the sulfur-nitrogen bond is cleavable.

[0385] In some embodiments, the linker may be a dendritic linker for covalently linking more than one drug moiety to an antibody or antigen-binding fragment via a branched, multifunctional linker portion. See, for example, Sun et al. (2002) Bioorg Med Chem Lett. [Bioorganic and Medicinal Chemistry Letters] 12:2213-5; Sun et al. (2003) Bioorg Med Chem. [Bioorganic and Medicinal Chemistry] 11:1761-8. Dendritic linkers can increase the drug-to-antibody molar ratio, i.e., the drug loading, which is related to the potency of the ADC. Thus, for example, in the case where the antibody or antigen-binding fragment carries only one reactive cysteine ​​thiol group, multiple Hobschdiene splice regulator drug moieties may be linked via a dendritic linker. In some embodiments, the linker portion or linker-drug portion may be linked to the antibody or antigen-binding fragment via a reduced disulfide bridging chemical or a restricted lysine utilization technique. See, for example, International Publication Nos. WO 2013 / 173391 and WO 2013 / 173393.

[0386] In some embodiments, the linker can be cleaved by a cleaving agent, such as an enzyme, present in the intracellular environment (e.g., lysosomes, endosomes, or cell membrane pits). The linker can be a peptide linker cleaved by, for example, intracellular peptidases or proteases, including but not limited to lysosomal or endosomal proteases.

[0387] In some embodiments, the linker is a cleavable peptide linker. As used herein, a cleavable peptide linker refers to any linker containing a cleavable peptide moiety. The term "cleavable peptide moiety" refers to any chemically bonded amino acid (natural or synthetic amino acid derivative) that can be cleaved by an agent present in the intracellular environment. For example, the linker may contain a valine-alanine (Val-Ala) sequence or a valine-citrulline (Val-Cit) sequence that can be cleaved by a peptidase such as cathepsin (e.g., cathepsin B). In some embodiments, the linker may contain a glutamic-valine-citrulline (Glu-Val-Cit) sequence. In some embodiments, the linker is an enzyme-cleavable linker and the cleavable peptide moiety in the linker is cleavable 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 moiety in the linker can be cleaved by a lysosomal cysteine ​​cathepsin such as cathepsin B, C, F, H, K, L, O, S, V, X, or W. In some embodiments, the cleavable peptide moiety can be cleaved by cathepsin B. An exemplary dipeptide that can be cleaved by cathepsin B is valine-citrulline (Val-Cit) (Dubowchik et al. (2002) Bioconjugate Chem. [Bioconjugate Chemistry] 13:855-69).

[0388] In some embodiments, the linker or the cleavable peptide portion within the linker comprises an amino acid unit. In some embodiments, the amino acid unit allows for linker cleavage by a protease, thereby facilitating the release of the heptahydrate splice 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. [Nature Biotechnology] 21:778-84; Dubowchik and Walker (1999) PharmTherapeutics [Pharmacology and 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-aspartic acid (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 Anami et al. (2018) Nat. Comm. [Nature Communications] 9:2512), and glycine-phenylalanine-lysine (Gly-Phe-Lys). Other exemplary amino acid units include, but are not limited to, Gly-Phe-Gly-Gly (SEQ ID NO:34), Gly-Phe-Leu-Gly (SEQ ID NO:35), Ala-Leu-Ala-Leu (SEQ ID NO:36), and Phe-N... as described, for example, in U.S. Patent No. 6,214,345. 9 -tosyl-Arg and Phe-N 9-Nitro-Arg. 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 secondary amino acids and / or non-naturally occurring amino acid analogs (such as citrulline). The amino acid unit may be designed and optimized for enzymatic cleavage by a specific enzyme, such as a tumor-associated protease, a lysosomal protease (such as cathepsin B, C, D, or S), or a plasminogen lysin.

[0389] In some embodiments, the connector is a cleavable β-glucuronic acid connector. As used herein, a cleavable β-glucuronic acid connector refers to any connector containing a cleavable β-glucuronic acid portion. An exemplary cleavable β-glucuronic acid connector comprises the following structure:

[0390]

[0391] The term "cleavable β-glucuronic acid moiety" refers to a glycosidic bond that can be cleaved by an agent having β-glucuronidase activity. In some embodiments, the linker comprises 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 glucuronic acids having a β-configuration.

[0392] In some embodiments, the ADC disclosed herein comprises a cleavable β-glucuronic acid moiety in a linker that can be cleaved by an enzyme. In some embodiments, the cleavable β-glucuronic acid moiety in the linker can be cleaved by a lysosomal enzyme, such as β-glucuronidase. In some embodiments, the linker is a β-glucuronidase-cleavable linker. In some embodiments, the cleavable β-glucuronic acid moiety in the linker allows for linker cleavage by β-glucuronidase after ADC internalization, thereby facilitating the release of the drug moiety from the ADC in the cellular environment.

[0393] In some embodiments, the connector in any of the ADCs disclosed herein may include at least one spacer subunit for attaching an antibody or antigen-binding fragment to a drug portion (e.g., a hoprene splice regulator drug portion). In some embodiments, the spacer subunit between the antibody or antigen-binding fragment and the cleavable portion, when present, attaches a cleavage site (e.g., a cleavable peptide portion) in the connector to the antibody or antigen-binding fragment. In some embodiments, the spacer subunit between the drug portion and the cleavable portion, when present, attaches a cleavage site (e.g., a cleavable peptide portion) in the connector to the drug portion. In some embodiments, a cleavage site is absent, and the spacer subunit is used to attach the antibody or antigen-binding fragment to the drug portion.

[0394] In some embodiments, the connector and / or the spacer subunits within the connector are substantially hydrophilic. Hydrophilic connectors can be used to reduce the extent to which drugs can be pumped out of drug-resistant cancer cells via multidrug resistance (MDR) or functionally similar transporters. In some embodiments, the hydrophilic connector may include one or more polyethylene glycol (PEG) portions, such as 1, 2, 3, 4, 5, or 6 PEG portions. In some embodiments, the connector comprises 2 PEG portions.

[0395] In some embodiments, the spacer subunit in the connector includes one or more PEG portions. In some embodiments, the spacer subunit includes one or more -(PEG) portions. m - and m is an integer from 1 to 10 (i.e., m can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, m 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 subunit comprises (PEG)2.

[0396] In some embodiments, the spacer subunits in the connector comprise alkyl portions. In some embodiments, the spacer subunits comprise one or more -(CH2) portions. 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”).

[0397] Spacer units can be used, for example, to directly or indirectly link antibody or antigen-binding fragments to a drug moiety. In some embodiments, the spacer unit directly links the antibody or antigen-binding fragment to the Hopsonide splice regulator drug moiety. In some embodiments, the antibody or antigen-binding fragment and the Hopsonide splice regulator drug moiety are linked via a spacer unit comprising one or more PEG portions (e.g., (PEG)2) or one or more alkyl portions (e.g., (CH2)2, (CH2)6, or (CH2)2-O-(CH2)2). In some embodiments, the spacer unit indirectly links the antibody or antigen-binding fragment to the Hopsonide splice regulator drug moiety. In some embodiments, the spacer unit indirectly links the antibody or antigen-binding fragment to the Hopsonide splice regulator drug moiety via a cleavable portion (e.g., a cleavable peptide or a cleavable β-glucuronic acid) and / or a linking portion (e.g., a maleic anhydride portion) for attaching the spacer unit to the antibody or antigen-binding fragment.

[0398] In different embodiments, the spacer subunit is partially linked to the antibody or antigen-binding fragment (i.e., the antibody or antigen-binding fragment) via maleic anhydride (Mal).

[0399] 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-butenedimide moiety" means a compound containing a maleimide group and capable of reacting with a thiosulfate group, such as the thiosulfate group of a cysteine ​​residue on the 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.

[0400] In some embodiments, the connector includes a Mal-spacer subunit and a cleavable peptide portion. In some embodiments, the cleavable peptide portion includes an amino acid unit. In some embodiments, the amino acid unit includes Val-Cit. In some embodiments, the amino acid unit includes Val-Ala. In some embodiments, the amino acid unit includes Glu-Val-Cit. In some embodiments, the connector includes a Mal-spacer subunit and Val-Cit. In some embodiments, the connector includes a Mal-spacer subunit and Val-Ala. In some embodiments, the connector includes a Mal-spacer subunit and Val-Cit, wherein the Mal-spacer subunit includes a maleic anhydride hexanoyl (MC). In some embodiments, the connector includes a Mal-spacer subunit and Val-Ala, wherein the Mal-spacer subunit includes a maleic anhydride hexanoyl (MC). In some embodiments, the connector includes a Mal-spacer subunit and a cleavable β-glucuronic acid portion.

[0401] In some embodiments, the connector comprises the following structure: Mal-spacer subunit. In some embodiments, the Mal-spacer subunit comprises a maleic anhydride hexanoyl group (MC). In some embodiments, the connector comprises the following structure: MC. In some embodiments, the connector comprises the following structure: Mal-(CH2)2 (“Mal-Et”). In some embodiments, the connector comprises the following structure: Mal-(CH2)6 (“Mal-Hex”). In some embodiments, the connector comprises the following structure: Mal-(CH2)2-O-(CH2)2 (“Mal-Et-O-Et”). In some embodiments, the connector comprises the following structure: Mal-(PEG)2. In some embodiments, the connector comprises the following structure: Mal-(PEG)2-CO.

[0402] In various embodiments, the Mal-spacer unit links an antibody or antigen-binding fragment to a cleavable peptide moiety. In some embodiments, the adapter comprises a Mal-spacer unit-peptide. In some embodiments, the adapter comprises the structure: Mal-spacer unit-Val-Cit. In some embodiments, the Mal-spacer unit comprises a maleic anhydride hexanoyl group (MC). In some embodiments, the adapter comprises the structure: MC-Val-Cit.

[0403] In some embodiments, the connector comprises the following structure: Mal-spacer subunit-Val-Ala. In some embodiments, the Mal-spacer subunit comprises a maleic anhydride hexanoyl group (MC). In some embodiments, the connector comprises the following structure: MC-Val-Ala.

[0404] In various embodiments, the Mal-spacer subunit links an antibody or antigen-binding fragment to a cleavable β-glucuronic acid moiety. In some embodiments, the adapter comprises the Mal-spacer subunit-β-glucuronic acid. In some embodiments, the adapter comprises MC-β-glucuronic acid.

[0405] In different embodiments, the cleavable portion of the connector is directly attached to the Hobstein splice regulator drug portion. In other embodiments, a spacer unit is used to connect the cleavable portion of the connector to the Hobstein splice regulator drug portion. In different embodiments, the Hobstein splice regulator is connected to the cleavable portion of the connector via a spacer unit.

[0406] The spacer unit can be either "self-ablating" or "non-self-ablating." A "non-self-ablating" spacer unit is one in which part or all of the spacer unit remains bound to the Hobstein-diene splice regulator drug moiety after linker cleavage. Examples of non-self-ablating spacer units include, but are not limited to, glycine spacer units and glycine-glycine spacer units. Non-self-ablating spacer units may eventually degrade over time, but do not readily and completely release the attached natural drug moiety under cellular conditions. "Self-ablating" spacer units allow for the release of the natural drug moiety under intracellular conditions. "Natural drug" or "natural drug moiety" refers to the portion of the spacer unit that does not retain the spacer unit after cleavage / degradation, or other chemical modifications.

[0407] Self-degrading chemicals are known in this art and can be readily selected for the disclosed ADC. In various embodiments, the spacer unit connecting the cleavable portion in the connector to the drug portion of the Hopsonide splice regulator is a self-ablating spacer unit, and self-degrades simultaneously with, before, or shortly after the cleavage of the cleavable portion under intracellular conditions. In some embodiments, the Hopsonide splice regulator is connected to the cleavable portion in the connector via a self-ablating spacer unit. In some embodiments, the Hopsonide splice regulator is connected to the cleavable portion in the connector via a self-ablating spacer unit, the cleavable portion comprising Val-Cit, and a maleic anhydride hexanoyl (MC) group binding the cleavable portion to an antibody or antigen-binding fragment. In some embodiments, the Hopsonide splice regulator is connected to the cleavable portion in the connector via a self-ablating spacer unit, the cleavable portion comprising Val-Ala, and a maleic anhydride hexanoyl (MC) group binding the cleavable portion to an antibody or antigen-binding fragment. In some embodiments, the Hopschadiene splicing regulator is connected to a cleavable portion in the linker via a self-ablating spacer unit, the cleavable portion comprising Glu-Val-Cit, and a maleic anhydride hexanoyl group (MC) binds the cleavable portion to an antibody or antigen-binding fragment. In some embodiments, the Hopschadiene splicing regulator binds to an antibody or antigen-binding fragment via a Mal-spacer unit (e.g., MC) bound to the Val-Cit cleavable portion in the linker and a pABC or pAB self-ablating spacer unit. In some embodiments, the Hopschadiene splicing regulator binds to an antibody or antigen-binding fragment via a Mal-spacer unit (e.g., MC) bound to the Val-Ala cleavable portion in the linker and a pABC or pAB self-ablating spacer unit. In some embodiments, the Hopschadiene splicing regulator binds to an antibody or antigen-binding fragment via a Mal-spacer unit (e.g., MC) bound to the Val-Val-Cit cleavable portion in the linker and a pABC or pAB self-ablating spacer unit.

[0408] In some embodiments, the self-ablating spacer subunit in the connector comprises a p-aminobenzyl unit. In some embodiments, p-aminobenzyl alcohol (pABOH) is linked via an amide bond to an amino acid unit or other cleavable portion in the connector, and a carbamate, methyl carbamate, or carbonate is formed between pABOH and the pharmaceutical portion (Hamann et al. (2005) Expert Opin Ther Patents 15:1087-103). In some embodiments, the self-ablating spacer subunit is or comprises a p-aminobenzyloxycarbonyl group (pABC). Without being bound by theory, it is considered that the self-decomposition of pABC involves a spontaneous 1,6-elimination reaction (Jain et al. (2015) Pharm Res. 32:3526-40).

[0409] In different embodiments, the structure of the p-aminobenzyloxycarbonyl (pABC) used in the disclosed ADC is shown below:

[0410]

[0411] In various embodiments, a self-ablating spacer unit connects the cleavable portion of the connector to the Hobstein splice regulator. In some embodiments, the self-ablating spacer unit is pABC. In some embodiments, pABC connects the cleavable portion of the connector to the Hobstein splice regulator. In some embodiments, pABC self-decomposes after the cleavable portion cleaves, and the Hobstein splice regulator is released from the ADC in its native active form.

[0412] In some embodiments, the anti-HER2 antibody or antigen-binding fragment binds to the Hobstein splice regulator via a linker comprising MC-Val-Cit-pABC. In other embodiments, the anti-HER2 antibody or antigen-binding fragment binds to the Hobstein splice regulator via a linker comprising MC-Val-Ala-pABC.

[0413] In some embodiments, the anti-CD138 antibody or antigen-binding fragment binds to the Hobstein splice regulator via a linker comprising MC-Val-Cit-pABC. In other embodiments, the anti-CD138 antibody or antigen-binding fragment binds to the Hobstein splice regulator via a linker comprising MC-Val-Ala-pABC.

[0414] In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment binds to the Hobstein splice regulator via a linker comprising MC-Val-Cit-pABC. In other embodiments, the anti-EPHA2 antibody or antigen-binding fragment binds to the Hobstein splice regulator via a linker comprising MC-Val-Ala-pABC.

[0415] In some embodiments, pABC self-decomposes after the cleavable peptide portion in the linker is cleaved. In some embodiments, the cleavable peptide portion comprises an amino acid unit. In some embodiments, the linker comprises an amino acid unit - pABC. In some embodiments, the amino acid unit is Val-Cit. In some embodiments, the linker comprises Val-Cit-pABC. In some embodiments, the amino acid unit is Glu-Val-Cit. In some embodiments, the connector comprises Glu-Val-Cit-pABC. In some embodiments, the amino acid unit is Val-Ala. In some embodiments, the linker comprises Val-Ala-pABC. In some embodiments, the amino acid unit is Ala-Ala-Asn. In some embodiments, the linker comprises Ala-Ala-Asn-pABC.

[0416] In some embodiments, pABC undergoes self-decomposition after the cleavable β-glucuronic acid in the connector is partially cleaved. In some embodiments, the connector comprises β-glucuronic acid-pABC.

[0417] In some embodiments, the self-denying spacer subunit in the connector comprises a p-aminobenzyl unit. In some embodiments, the self-denying spacer subunit in the connector comprises p-aminobenzyl (pAB). In some embodiments, the self-decomposition of pAB involves a spontaneous 1,6-elimination reaction.

[0418] In different embodiments, the structure of p-aminobenzyl (pAB) used in the disclosed ADC is shown below:

[0419]

[0420] In various embodiments, a self-ablating spacer unit connects the cleavable portion of the connector to the Hopberg diene splice regulator. In some embodiments, the self-ablating spacer unit is pAB. In some embodiments, pAB connects the cleavable portion of the connector to the Hopberg diene splice regulator. In some embodiments, pAB self-decomposes after the cleavable portion cleaves, and the Hopberg diene splice regulator is released from the ADC in its native active form.

[0421] In some embodiments, the anti-HER2 antibody or antigen-binding fragment binds to the Hobstein splice regulator via a linker comprising MC-Val-Cit-pAB. In other embodiments, the anti-HER2 antibody or antigen-binding fragment binds to the Hobstein splice regulator via a linker comprising MC-Val-Ala-pAB.

[0422] In some embodiments, the anti-CD138 antibody or antigen-binding fragment binds to the Hobstein splice regulator via a linker comprising MC-Val-Cit-pAB. In other embodiments, the anti-CD138 antibody or antigen-binding fragment binds to the Hobstein splice regulator via a linker comprising MC-Val-Ala-pAB.

[0423] In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment binds to the Hobstein splice regulator via a linker comprising MC-Val-Cit-pAB. In other embodiments, the anti-EPHA2 antibody or antigen-binding fragment binds to the Hobstein splice regulator via a linker comprising MC-Val-Ala-pAB.

[0424] In some embodiments, pAB self-decomposes after the cleavable peptide portion in the linker is cleaved. In some embodiments, the cleavable peptide portion comprises an amino acid unit. In some embodiments, the linker comprises an amino acid unit - pAB. In some embodiments, the amino acid unit is Val-Cit. In some embodiments, the linker comprises Val-Cit-pAB. In some embodiments, the amino acid unit is Val-Ala. In some embodiments, the linker comprises Val-Ala-pAB. In some embodiments, the amino acid unit is Glu-Val-Cit. In some embodiments, the linker comprises Glu-Val-Cit-pAB. In some embodiments, the amino acid unit is Ala-Ala-Asn. In some embodiments, the linker comprises Ala-Ala-Asn-pAB.

[0425] In some embodiments, pAB undergoes self-decomposition after the cleavable β-glucuronic acid in the connector is partially cleaved. In some embodiments, the connector comprises β-glucuronic acid-pAB.

[0426] In some other embodiments, the Hopschadiene splicing regulator is connected to the cleavable portion in the adapter via a non-ablative spacer unit. In some embodiments, the Hopschadiene splicing regulator is connected to the cleavable portion in the adapter via a non-ablative spacer unit, the cleavable portion comprising Val-Cit, and a maleic anhydride hexanoyl (MC) group binds the cleavable portion to an antibody or antigen-binding fragment. In some embodiments, the Hopschadiene splicing regulator is connected to the cleavable portion in the adapter via a non-ablative spacer unit, the cleavable portion comprising Val-Ala, and a maleic anhydride hexanoyl (MC) group binds the cleavable portion to an antibody or antigen-binding fragment.

[0427] In various aspects, the antibody or antigen-binding fragment of the ADC binds to the drug moiety of the Hobstein splice regulator via a linker, wherein the linker comprises a Mal-spacer unit (e.g., MC), a cleavable amino acid unit, and a pABC. In some embodiments, the spacer unit comprises an alkyl portion. In some embodiments, the Mal-spacer unit comprises a maleic anhydride hexanoyl group (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.

[0428] In various other aspects, the antibody or antigen-binding fragment of the ADC binds to the drug moiety of the Hobstein splice regulator via a linker, wherein the linker comprises a Mal-spacer unit (e.g., MC), a cleavable amino acid unit, and pAB. In some embodiments, the spacer unit comprises an alkyl portion. In some embodiments, the Mal-spacer unit comprises a maleic anhydride hexanoyl group (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.

[0429] In various other aspects, the antibody or antigen-binding fragment of the ADC binds to the drug moiety of the Hobstein splice regulator via a linker, wherein the linker comprises a Mal-spacer unit (e.g., MC), a cleavable β-glucuronic acid, and pABC. In some embodiments, the linker comprises a Mal-spacer unit-β-glucuronic acid-pABC. In some embodiments, the linker comprises MC-β-glucuronic acid-pABC.

[0430] In other aspects, the antibody or antigen-binding fragment of the ADC binds to the drug moiety of the Hobstein splice regulator via a linker, wherein the linker comprises a Mal-spacer unit (e.g., MC), a cleavable β-glucuronic acid, and pAB. In some embodiments, the linker comprises a Mal-spacer unit-β-glucuronic acid-pAB. In some embodiments, the linker comprises MC-β-glucuronic acid-pAB.

[0431] In different embodiments, the ADC compound has formula (I):

[0432] Ab-(LH) p (I)

[0433] Ab represents an antibody or antigen-binding fragment that targets neoplastic cells;

[0434] H is the Hobsch diene splicing regulator;

[0435] L is the connector that covalently links Ab to D; and

[0436] p is an integer from 1 to 15.

[0437] In some embodiments, the antibody or antigen-binding fragment (Ab) of the ADC binds to the Hobstein splice regulator drug moiety via a linker, wherein the linker is any of the linkers disclosed or incorporated herein by reference, or one or more components comprising any of the linkers disclosed or incorporated herein by reference.

[0438] In some embodiments, the linker includes a cleavable portion positioned such that a portion of the linker or antibody or antigen-binding fragment does not remain bound to the Hobschdiene splice regulator after cleavage. In some embodiments, the cleavable portion is a cleavable peptide portion, such as an amino acid unit, such as Val-Cit or Val-Ala. In some embodiments, the amino acid unit or linker comprises Val-Cit. In some embodiments, the amino acid unit or linker comprises Val-Ala. In some embodiments, the amino acid unit or linker comprises Glu-Val-Cit.

[0439] In some embodiments, the connector includes at least one spacer subunit for attaching an antibody or antigen-binding fragment to a cleavable portion. In some embodiments, the connector includes at least one spacer subunit for attaching an antibody or antigen-binding fragment to a drug portion. In some embodiments, the spacer subunit or connector includes at least one alkyl portion.

[0440] In some embodiments, the spacer subunit in the adapter is linked to an antibody or antigen-binding fragment via a Mal portion (“Mal-spacer subunit”). In some embodiments, the Mal-spacer subunit comprises at least one alkyl portion. In some embodiments, the adapter comprises a maleic anhydride hexanoyl (MC). In some embodiments, the adapter comprises Mal-(CH2)2 (“Mal-Et”). In some embodiments, the adapter comprises Mal-(CH2)6 (“Mal-Hex”). In some embodiments, the adapter comprises Mal-(CH2)2-O-(CH2)2 (“Mal-Et-O-Et”). In some embodiments, the adapter comprises Mal-(PEG)2-CO. In some embodiments, the Mal-spacer subunit links an antibody or antigen-binding fragment to a pharmaceutical portion.

[0441] In some embodiments, the Mal-spacer subunit 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 subunit or connector comprises Mal-(PEG)2. In some embodiments, the Mal-spacer subunit 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. 8- CO. In some embodiments, the Mal-spacer subunit or adapter comprises Mal-(PEG)2-CO. In some embodiments, the Mal-spacer subunit or adapter comprises Mal-(PEG)2-CO and at least one additional spacer subunit. In some embodiments, Mal-(PEG)2-CO links an antibody or antigen-binding fragment to a drug moiety. In some embodiments, the adapter comprises or is composed of Mal-(PEG)2-CO. Examples of the “Mal-(PEG)2-CO” adapter are also referred to herein as “ADL2” or “ADL2 adapter”.

[0442] In some embodiments, the Mal-spacer subunit or adapter comprises an MC. In some embodiments, the Mal-spacer subunit or adapter comprises an MC and at least one additional spacer subunit. In some embodiments, the MC links an antibody or antigen-binding fragment to a drug portion. In some embodiments, the adapter comprises or is composed of an MC. Examples of “MC” adapters are also referred to herein as “ADL10” or “ADL10 adapter”.

[0443] In some embodiments, the Mal-spacer subunit or adapter comprises Mal-(CH2)6 (“Mal-Hex”). In some embodiments, the Mal-spacer subunit or adapter comprises Mal-Hex and at least one additional spacer subunit. In some embodiments, Mal-Hex links an antibody or antigen-binding fragment to a drug moiety. In some embodiments, the adapter comprises Mal-Hex. Examples of the “Mal-Hex” adapter are also referred to herein as “ADL12” or the “ADL12 adapter”.

[0444] In some embodiments, the Mal-spacer unit or adapter comprises Mal-(CH2)2 (“Mal-Et”). In some embodiments, the Mal-spacer unit or adapter 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 adapter comprises Mal-Et. Examples of the “Mal-Et” adapter are also referred to herein as “ADL14” or the “ADL14 adapter”.

[0445] In some embodiments, the Mal-spacer unit or adapter comprises Mal-(CH2)2-O-(CH2)2 (“Mal-Et-O-Et”). In some embodiments, the Mal-spacer unit or adapter 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 adapter comprises Mal-Et-O-Et. Examples of the “Mal-Et-O-Et” adapter are also referred to herein as “ADL15” or the “ADL15” adapter.

[0446] In some other embodiments, the Mal-spacer subunit links an antibody or antigen-binding fragment to a cleavable portion in the adapter. In some embodiments, the cleavable portion in the adapter 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 subunit or adapter contains MC. In some embodiments, the adapter contains MC-Val-Cit. In some embodiments, the adapter contains MC-Val-Ala. In some embodiments, the adapter contains MC-Glu-Val-Cit. In some embodiments, the adapter contains MC-Ala-Ala-Asn.

[0447] In some embodiments, the spacer unit connects the pyrolytic portion of the connector to the Hobstein splice regulator. In some embodiments, the spacer unit that connects the pyrolytic portion to the Hobstein splice regulator is a self-ablating spacer unit.

[0448] In some embodiments, the spacer subunit comprises pABC. In some embodiments, the pABC connects a cleavable portion to a Hobschdiene splice regulator. In some embodiments, the cleavable portion is a cleavable peptide portion, such as an amino acid unit. In some embodiments, the linker comprises an amino acid unit - pABC.

[0449] In some embodiments, the connector comprises Val-Cit-pABC. In some embodiments, the connector comprises Val-Cit-pABC and an MC Mal-spacer subunit for attaching the connector to an antibody or antigen-binding fragment. In some embodiments, the connector comprises MC-Val-Cit-pABC. In some embodiments, the connector comprises MC-Val-Cit-pABC and at least one additional spacer subunit. Examples of the MC-Val-Cit-pABC connector are also referred to herein as “ADL1” or “ADL1” connectors.

[0450] In some embodiments, the connector comprises Val-Ala-pABC. In some embodiments, the connector comprises Val-Ala-pABC and an MC Mal-spacer subunit for attaching the connector to an antibody or antigen-binding fragment. In some embodiments, the connector comprises MC-Val-Ala-pABC. In some embodiments, the connector comprises MC-Val-Ala-pABC and at least one additional spacer subunit. Examples of the MC-Val-Ala-pABC connector are also referred to herein as “ADL6” or “ADL6” connectors.

[0451] In some embodiments, the connector comprises Glu-Val-Cit-pABC. In some embodiments, the connector comprises Glu-Val-Cit-pABC and an MC Mal-spacer subunit for attaching the connector to an antibody or antigen-binding fragment. In some embodiments, the connector comprises MC-Glu-Val-Cit-pABC. In some embodiments, the connector comprises MC-Glu-Val-Cit-pABC and at least one additional spacer subunit. Examples of the MC-Glu-Val-Cit-pABC connector are also referred to herein as “ADL23” or the “ADL23” connector.

[0452] In some embodiments, the connector comprises Ala-Ala-Asn-pABC. In some embodiments, the connector comprises an Ala-Ala-Asn-pABC and an MC Mal-spacer subunit for attaching the connector to an antibody or antigen-binding fragment. In some embodiments, the connector comprises MC-Ala-Ala-Asn-pABC. In some embodiments, the connector comprises MC-Ala-Ala-Asn-pABC and at least one additional spacer subunit. Examples of the MC-Ala-Ala-Asn-pABC connector are also referred to herein as “ADL21” or “ADL21” connectors.

[0453] In some other embodiments, the spacer subunit comprises pAB. In some embodiments, the pAB connects a cleavable portion to a Hobschdiene splice regulator. In some embodiments, the cleavable portion is a cleavable peptide portion, such as an amino acid unit. In some embodiments, the linker comprises an amino acid unit - pAB.

[0454] In some embodiments, the connector comprises Val-Ala-pAB. In some embodiments, the connector comprises Val-Ala-pAB and an MC Mal-spacer subunit for attaching the connector to an antibody or antigen-binding fragment. In some embodiments, the connector comprises MC-Val-Ala-pAB. In some embodiments, the connector comprises MC-Val-Ala-pAB and at least one additional spacer subunit. Examples of the MC-Val-Ala-pAB connector are also referred to herein as “ADL5” or the “ADL5” connector.

[0455] In some embodiments, the connector comprises Val-Cit-pAB. In some embodiments, the connector comprises Val-Cit-pAB and an MC Mal-spacer subunit for attaching the connector to an antibody or antigen-binding fragment. In some embodiments, the connector comprises MC-Val-Cit-pAB. In some embodiments, the connector comprises MC-Val-Cit-pAB and at least one additional spacer subunit. Examples of the MC-Val-Cit-pAB connector are also referred to herein as “ADL7” or the “ADL7” connector.

[0456] In some embodiments, the adapter comprises β-glucuronide-pABC. In some embodiments, the adapter comprises β-glucuronide-pABC and an MC Mal-spacer subunit for attaching the adapter to an antibody or antigen-binding fragment. In some embodiments, the adapter comprises MC-β-glucuronide-pABC. In some embodiments, the adapter comprises MC-β-glucuronide-pABC and at least one additional spacer subunit. Examples of MC-β-glucuronide-pABC are also referred to herein as “ADL13” or “ADL13” adapters.

[0457] In some embodiments, the connector comprises β-glucuronide-pAB. In some embodiments, the connector comprises β-glucuronide-pAB and MC Mal-spacer subunits that bind the connector to an antibody or antigen-binding fragment. In some embodiments, the connector comprises MC-β-glucuronide-pAB.

[0458] In some embodiments, the antibody or antigen-binding fragment binds to the Hobstein splice regulator drug moiety via a linker of ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23. It has been found that, in various embodiments, ADCs comprising a linker of ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 and the Hobstein splice regulator drug moiety disclosed herein exhibit the properties required for a therapeutic ADC. In various embodiments, these properties 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-expressing cells comparable to unbound antibodies, potent cytotoxicity against target-expressing cells, lower off-target cell-killing levels, higher bystander-killing levels, and / or effective in vivo anticancer activity, all of which are relative to ADCs using other linker-loads. For example, in various embodiments, compared to ADCs using other linker-loads (e.g., the ADL10 linker and the hoprene splice regulator drug portion), ADCs containing ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linkers and the hoprene splice regulator drug portion disclosed herein exhibit an increased ability to inhibit the growth and / or proliferation of target-expressing cells. In various embodiments, compared to ADCs based on other splicing regulators (such as the thailanstatin A-based ADC reported in Puthenveetil et al. Bioconjugate Chem. [Bioconjugate Chemistry] (2016) 27:1880-8), ADCs containing ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21 or ADL23 linkers and the heptahydrate splicing regulator drug moiety disclosed herein exhibited unexpectedly increased in vivo stability (e.g., plasma stability).

[0459] In some embodiments, favorable or superior functional properties can be observed in the binding to a linker-load of, 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), provided by specific combinations of the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker with the hoprene splice regulator drug moiety disclosed herein.

[0460] In some embodiments, the ADC comprises an ADL1-Hobstein splice regulator and an antibody or antigen-binding fragment thereof that has the ability to maintain targeting and internalization within the vesicular cells. In some embodiments, the ADC comprises an ADL2-Hobstein splice regulator and an antibody or antigen-binding fragment thereof that has the ability to maintain targeting and internalization within the vesicular cells. In some embodiments, the ADC comprises an ADL5-Hobstein splice regulator and an antibody or antigen-binding fragment thereof that has the ability to maintain targeting and internalization within the vesicular cells. In some embodiments, the ADC comprises an ADL6-Hobstein splice regulator and an antibody or antigen-binding fragment thereof that has the ability to maintain targeting and internalization within the vesicular cells. In some embodiments, the ADC comprises an ADL7-Hobstein splice regulator and an antibody or antigen-binding fragment thereof that has the ability to maintain targeting and internalization within the vesicular cells. In some embodiments, the ADC comprises an ADL12-Hobstein splice regulator and an antibody or antigen-binding fragment thereof that has the ability to maintain targeting and internalization within the vesicular cells. In some embodiments, the ADC comprises an ADL13-Hobstein splice regulator and an antibody or antigen-binding fragment thereof that has the ability to maintain targeting and internalization within the vesicular cells. In some embodiments, the ADC comprises an ADL14-Hobstein splice regulator and an antibody or antigen-binding fragment thereof that has the ability to maintain targeting and internalization within the vesicular cells. In some embodiments, the ADC comprises an ADL15-Hobstein splice regulator and an antibody or antigen-binding fragment thereof that has the ability to maintain targeting and internalization within the vesicular cells.

[0461] In some embodiments, the ADC comprises an ADL1-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing blast cells. In some embodiments, the ADC comprises an ADL2-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing blast cells. In some embodiments, the ADC comprises an ADL5-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing blast cells. In some embodiments, the ADC comprises an ADL6-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing blast cells. In some embodiments, the ADC comprises an ADL7-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing blast cells. In some embodiments, the ADC comprises an ADL12-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing blast cells. In some embodiments, the ADC comprises an ADL13-horsepower splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing blast cells. In some embodiments, the ADC comprises an ADL14-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing blast cells. In some embodiments, the ADC comprises an ADL15-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting HER2-expressing blast cells.

[0462] In some embodiments, the antibody or antigen-binding fragment thereof targeting HER2-expressing neoplastic cells is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment thereof targeting HER2-expressing neoplastic cells comprises 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).

[0463] In some embodiments, the ADC has equation (I):

[0464] Ab-(LH) p (I)

[0465] in:

[0466] (i)Ab is an anti-HER2 antibody or its antigen-binding fragment containing three heavy chain complementarity-determining regions (HCDRs) of the amino acid sequences containing SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2) and SEQ ID NO:3 (HCDR3) and three light chain complementarity-determining regions (LCDRs) of the amino acid sequences containing SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2) and SEQ ID NO:6 (LCDR3);

[0467] (ii)H is a Hobsch diene splicing regulator;

[0468] (iii) L is a connector containing ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23; and

[0469] (iv) p is an integer from 1 to 15.

[0470] In some embodiments, the antibody or antigen-binding fragment thereof targeting HER2-expressing neoplastic cells comprises 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 neoplastic cells comprises a human IgG1 heavy chain constant domain and a human Igκ light chain constant domain. In some embodiments, the antibody is trastuzumab. In some embodiments, p is an integer from 1 to 10, 2 to 8, or 4 to 8. In some embodiments, p is 4. In some embodiments, p is 8.

[0471] In some embodiments, the ADC comprises an ADL1-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing vesicular cells. In some embodiments, the ADC comprises an ADL2-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing vesicular cells. In some embodiments, the ADC comprises an ADL5-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing vesicular cells. In some embodiments, the ADC comprises an ADL6-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing vesicular cells. In some embodiments, the ADC comprises an ADL7-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing vesicular cells. In some embodiments, the ADC comprises an ADL12-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting CD138-expressing vesicular cells. In some embodiments, the ADC comprises an ADL13-Hobstein splice regulator and an antibody or an antigen-binding fragment thereof targeting CD138-expressing vesicular cells. In some embodiments, the ADC comprises an ADL14-Hobstein splice regulator and an antibody or an antigen-binding fragment thereof targeting CD138-expressing vesicular cells. In some embodiments, the ADC comprises an ADL15-Hobstein splice regulator and an antibody or an antigen-binding fragment thereof targeting CD138-expressing vesicular cells.

[0472] In some embodiments, the antibody or its antigen-binding fragment targeting CD138-expressing cytoplasmic cells is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment targeting CD138-expressing cytoplasmic cells comprises 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).

[0473] In some embodiments, the ADC has equation (I):

[0474] Ab-(LH) p (I)

[0475] in:

[0476] (i)Ab is an anti-CD138 antibody or its antigen-binding fragment containing three heavy chain complementarity-determining regions (HCDRs) of the amino acid sequences containing SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2) and SEQ ID NO:9 (HCDR3) and three light chain complementarity-determining regions (LCDRs) of the amino acid sequences containing SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2) and SEQ ID NO:12 (LCDR3);

[0477] (ii)H is a Hobsch diene splicing regulator;

[0478] (iii) L is a connector containing ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23; and

[0479] (iv) p is an integer from 1 to 15.

[0480] In some embodiments, the antibody or antigen-binding fragment thereof targeting CD138-expressing vesicular cells comprises 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 thereof targeting CD138-expressing vesicular cells comprises a mouse IgG2a heavy chain constant domain and a mouse Igκ light chain constant domain. In some embodiments, the antibody or antigen-binding fragment thereof targeting CD138-expressing vesicular cells comprises a human IgG2a heavy chain constant domain and a human Igκ light chain constant domain. In some embodiments, the antibody is B-B4. In some embodiments, p is an integer from 1 to 10, 2 to 8, or 4 to 8. In some embodiments, p is 4. In some embodiments, p is 8.

[0481] In some embodiments, the ADC comprises an ADL1-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing vesicular cells. In some embodiments, the ADC comprises an ADL2-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing vesicular cells. In some embodiments, the ADC comprises an ADL5-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing vesicular cells. In some embodiments, the ADC comprises an ADL6-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing vesicular cells. In some embodiments, the ADC comprises an ADL7-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing vesicular cells. In some embodiments, the ADC comprises an ADL12-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing vesicular cells. In some embodiments, the ADC comprises an ADL13-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing vesicular cells. In some embodiments, the ADC comprises an ADL14-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing vesicular cells. In some embodiments, the ADC comprises an ADL15-Hobstein splice regulator and an antibody or antigen-binding fragment thereof targeting EPHA2-expressing vesicular cells.

[0482] In some embodiments, the antibody or antigen-binding fragment thereof targeting EPHA2-expressing cytoplasmic cells is an internalizing antibody or an internalizing antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment thereof targeting EPHA2-expressing cytoplasmic cells comprises 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).

[0483] In some embodiments, the ADC has equation (I):

[0484] Ab-(LH) p (I)

[0485] in:

[0486] (i)Ab is an anti-EPHA2 antibody or its antigen-binding fragment containing three heavy chain complementarity-determining regions (HCDRs) of the amino acid sequences containing SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2) and SEQ ID NO:15 (HCDR3) and three light chain complementarity-determining regions (LCDRs) of the amino acid sequences containing SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2) and SEQ ID NO:18 (LCDR3);

[0487] (ii)H is a Hobsch diene splicing regulator;

[0488] (iii) L is a connector containing ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23; and

[0489] (iv) p is an integer from 1 to 15.

[0490] In some embodiments, the antibody or antigen-binding fragment thereof targeting EPHA2-expressing cytoplasmic cells comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:23 and a light chain variable region containing the amino acid sequence of SEQ ID NO:24. In some embodiments, the antibody or antigen-binding fragment thereof targeting EPHA2-expressing cytoplasmic cells comprises a human IgG1 heavy chain constant domain and a human Igκ light chain constant domain. In some embodiments, the antibody is 1C1. In some embodiments, p is an integer from 1 to 10, 2 to 8, or 4 to 8. In some embodiments, p is 4. In some embodiments, p is 8.

[0491] Hobsch diene splice regulator

[0492] In some embodiments, the antibody-drug conjugate is of formula (I): Ab-(LH) p Where Ab is an antibody or antigen-binding fragment targeting the proliferative cells; H is a Hobschdiene splicing regulator; L is a linker that covalently links Ab to H; and p is an integer from 1 to 15.

[0493] In some embodiments, H, as a Hobschdiene splice regulator, comprises a compound having formula (I): Or a pharmaceutically acceptable salt thereof, which is covalently linked to L via any atom, wherein:

[0494] Y is selected from O, S, NR 6 and CR 6 R 7 ;

[0495] R 1 、R 2 and R 3 Each is independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl), -OC(=O)-(C1-C6 alkyl), -C(=O)-O-(C1-C6 alkyl), and C1-C6 alkyl;

[0496] R 4 Selected from hydrogen, C1-C6 alkyl, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), and -C(=O)-NR 6 R 7 ;

[0497] R 5 Selected from hydrogen, hydroxyl, -CH2-OH, -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-NR 6 R 7 -NR 6 -C(=O)-R 8 -OC(=O)-NR 6 R 7 -NR 6 -C(=O)-R 8 and -NR 6 -C(=O)-NR 6 R 7 ;

[0498] R 6 and R 7 Each is independently selected from hydrogen, -R 8 -C(=O)-R 8 and -C(=O)-OR 8 ;and

[0499] R 8 Selected from C1-C6 alkyl, C3-C8 carbocyclic and C3-C8 heterocyclic groups.

[0500] where R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 and R 8 Each is independently substituted by 0 to 3 groups independently selected from the following: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR6 R 7 The group may contain C3-C8 carbocyclic, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic groups, each of which may be independently substituted by 0 or 1 of the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, -NH-C(=O)(C1-C3 alkyl), and -NH-C(=O)-O-(C1-C3 alkyl).

[0501] The valence of the atoms covalently bonded to L did not exceed the limit.

[0502] In some embodiments, H, as a Hobschdiene splice regulator, comprises a compound having formula (Ia): Or a pharmaceutically acceptable salt thereof, which is covalently linked to L via any atom, wherein:

[0503] R 9 Selected from C3-C8 heterocyclic groups;

[0504] R 10 Selected from H and C1-C6 alkyl groups,

[0505] where R 9 and R 10 Each is independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, -NH2, -NH-(C1-C3 alkyl) and -N-(C1-C3 alkyl)2, and

[0506] The valence of the atoms covalently bonded to L did not exceed the limit.

[0507] In some embodiments, H, as a Hobschdiene splice regulator, comprises a compound having formula (Ib):

[0508] or a pharmaceutically acceptable salt thereof, which is covalently linked to L via any atom, wherein:

[0509] R 11 Selected from and Where * indicates R 11 The connection point with the rest of the compound;

[0510] R 12 and R 13 Each is independently selected from H and methyl; and

[0511] The valence of the atoms covalently bonded to L did not exceed the limit.

[0512] In some embodiments, H, as a Hobschdiene splice regulator, comprises a compound having formula (II): Or a pharmaceutically acceptable salt thereof, which is covalently linked to L via any atom, wherein:

[0513] X represents a hydroxyl group or NR. 6 R 7 ;

[0514] R 6 and R 7 Each is independently selected from hydrogen, -R 8 -C(=O)-R 8 -C(=O)-OR 8 -(C1-C6 alkyl)-OC(=O)-R 8 and -(C1-C6 alkyl)-NH-C(=O)-R 8 ;and

[0515] R 8 Selected from C1-C6 alkyl, C3-C8 carbocyclic and C3-C8 heterocyclic groups.

[0516] where R 6 、R 7 and R 8 Each is independently substituted by 0 to 3 groups independently selected from the following: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR 6 R 7 The group may contain C3-C8 carbocyclic, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic groups, each of which may be independently substituted by 0 or 1 of the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, -NH-C(=O)(C1-C3 alkyl), and -NH-C(=O)-O-(C1-C3 alkyl).

[0517] The valence of the atoms covalently bonded to L did not exceed the limit.

[0518] In some embodiments, H, as a Hobschdiene splice regulator, comprises a compound having formula (IIa): Or a pharmaceutically acceptable salt thereof, which is covalently linked to L via any atom, wherein:

[0519] Z is selected from NR 9 and O;

[0520] R9 Selected from hydrogen and C1-C6 alkyl groups;

[0521] R 10 and R 11 Each is independently selected from hydrogen, halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), C3-C8 carbocyclic, C1-C6 alkyl hydroxyl, C1-C6 alkyl alkoxy, benzyl and C3-C8 heterocyclic;

[0522] R 12 Selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups.

[0523] where R 9 、R 10 、R 11 and R 12 Each is independently substituted with 0 or 1 of the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy and C1-C3 haloalkyl;

[0524] t is an integer selected from 1, 2, 3, 4, 5, and 6; and

[0525] The valence of the atoms covalently bonded to L did not exceed the limit.

[0526] In some embodiments, H, as a Hobschdiene splice regulator, comprises a compound having formula (IIb): Or a pharmaceutically acceptable salt thereof, which is covalently linked to L via any atom, wherein:

[0527] R 13 Selected from

[0528] and Where * indicates R 13 The connection point with the rest of the compound;

[0529] R 14 and R 15 Each is independently selected from hydrogen and methyl; and

[0530] The valence of the atoms covalently bonded to L did not exceed the limit.

[0531] In some embodiments, H, as a Hobschdiene splice regulator, comprises a compound having formula (III): Or a pharmaceutically acceptable salt thereof, which is covalently linked to L via any atom, wherein:

[0532] R 1 、R 2 and R 3 Each is independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl), -OC(=O)-(C1-C6 alkyl), -C(=O)-O-(C1-C6 alkyl), and C1-C6 alkyl;

[0533] R 6 and R 7 Each is independently selected from hydrogen, -R 8 -C(=O)-R 8 and -C(=O)-OR 8 ;

[0534] R 8 Selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups; and

[0535] R 9 Selected from H,

[0536] and

[0537] where R 1 、R 2 、R 3 、R 6 、R 7 and R 8 Each is independently substituted by 0 to 3 groups independently selected from the following: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR 6 R 7 The group may contain C3-C8 carbocyclic, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl, and C3-C8 heterocyclic groups, each of which may be independently substituted by 0 or 1 of the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, -NH-C(=O)(C1-C3 alkyl), and -NH-C(=O)-O-(C1-C3 alkyl).

[0538] The valence of the atoms covalently bonded to L does not exceed the limit; and

[0539] Where * indicates R 9 The connection point with the rest of the compound.

[0540] In some embodiments, the antibody or antigen-binding fragment targets cells expressing the following: HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, and / or STEAP1.

[0541] In some embodiments, the antibody or antigen-binding fragment targets cells expressing HER2. In some embodiments, the antibody or antigen-binding fragment is an anti-HER2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment comprises 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 comprises 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 comprises a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Igκ light chain constant region.

[0542] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing CD138. In some embodiments, the antibody or antigen-binding fragment is an anti-CD138 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment comprises 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 comprises 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 comprises a mouse IgG2a heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a mouse Igκ light chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a constant region of the human IgG2a heavy chain. In some embodiments, the antibody or antigen-binding fragment comprises a constant region of the human Igκ light chain.

[0543] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing EPHA2. In some embodiments, the antibody or antigen-binding fragment is an anti-EPHA2 antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment comprises 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 comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO:23 and a light chain variable region containing the amino acid sequence of SEQ ID NO:24. In some embodiments, the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a human Igκ light chain constant region.

[0544] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing MSLN. In some embodiments, the antibody or antigen-binding fragment is an anti-MSLN antibody or antigen-binding fragment.

[0545] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing FOLH1. In some embodiments, the antibody or antigen-binding fragment is an anti-FOLH1 antibody or antigen-binding fragment.

[0546] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing CDH6. In some embodiments, the antibody or antigen-binding fragment is an anti-CDH6 antibody or antigen-binding fragment.

[0547] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing CEACAM5. In some embodiments, the antibody or antigen-binding fragment is an anti-CEACAM5 antibody or antigen-binding fragment.

[0548] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing CFC1B. In some embodiments, the antibody or antigen-binding fragment is an anti-CFC1B antibody or antigen-binding fragment.

[0549] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing ENPP3. In some embodiments, the antibody or antigen-binding fragment is an anti-ENPP3 antibody or antigen-binding fragment.

[0550] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing FOLR1. In some embodiments, the antibody or antigen-binding fragment is an anti-FOLR1 antibody or antigen-binding fragment.

[0551] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing HAVCR1. In some embodiments, the antibody or antigen-binding fragment is an anti-HAVCR1 antibody or antigen-binding fragment.

[0552] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing KIT. In some embodiments, the antibody or antigen-binding fragment is an anti-KIT antibody or antigen-binding fragment.

[0553] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing MET. In some embodiments, the antibody or antigen-binding fragment is an anti-MET antibody or antigen-binding fragment.

[0554] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing MUC16. In some embodiments, the antibody or antigen-binding fragment is an anti-MUC16 antibody or antigen-binding fragment.

[0555] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing SLC39A6. In some embodiments, the antibody or antigen-binding fragment is an anti-SLC39A6 antibody or antigen-binding fragment.

[0556] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing SLC44A4. In some embodiments, the antibody or antigen-binding fragment is an anti-SLC44A4 antibody or antigen-binding fragment.

[0557] In some other embodiments, the antibody or antigen-binding fragment targets cells expressing STEAP1. In some embodiments, the antibody or antigen-binding fragment is an anti-STEAP1 antibody or antigen-binding fragment.

[0558] In some embodiments, L is selected from any of the connectors disclosed herein or any combination of the connector components disclosed herein. In some embodiments, L is a connector comprising MC-Val-Cit-pABC, Mal-(PEG)2-CO, MC-Val-Ala-pAB, MC-Val-Ala-pABC, MC-Val-Cit-pAB, Mal-Hex, Mal-Et, or Mal-Et-O-Et. In some embodiments, the connector may also include one or more additional spacer subunits. In some embodiments, L is a connector ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23. In some embodiments, L is a connector ADL12, ADL14, or ADL15. In some embodiments, the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21 or ADL23 connector may also include one or more additional spacer subunits.

[0559] In some embodiments, an intermediate serving as a precursor to a linker portion is reacted with a Hobstein splice regulator portion under suitable conditions. In some embodiments, a reactive group is used on the Hobstein splice regulator and / or the intermediate or linker. Subsequently, the reaction product between the Hobstein splice regulator and the intermediate, or the derived Hobstein splice regulator (Hobstein splice regulator plus linker), is reacted with an antibody or antigen-binding fragment under suitable conditions. Alternatively, the intermediate or linker may first be reacted with an antibody or antigen-binding fragment or a derived antibody or antigen-binding fragment, and subsequently with a drug or a derived drug.

[0560] Several different reactions are available for covalently linking the Hopschdiene splice regulator moiety and / or linker moiety to an antibody or antigen-binding fragment. This is typically achieved by reacting one or more amino acid residues of the antibody or antigen-binding fragment, including the amino group of lysine, the free carboxyl group of glutamic acid and aspartic acid, the sulfhydryl group of cysteine, and various moieties of aromatic amino acids. For example, nonspecific covalent linking can be performed using a carbodiimide reaction to link a carboxyl (or amino) group on the Hopschdiene splice regulator moiety to an amino (or carboxyl) group on the antibody or antigen-binding fragment. Alternatively, bifunctional reagents such as dialdehydes or imino esters can be used to link an amino group on the Hopschdiene splice regulator moiety to an amino group on the antibody or antigen-binding fragment. The Schiff base reaction can also be used to link a drug (e.g., a Hopschdiene splice regulator) to a binder. This method involves the oxidation of a drug containing a diol or hydroxyl group by periodate, thereby forming an aldehyde, which is then reacted with a binder. The linkage occurs via the formation of a Schiff base using the amino groups of the binder. Isothiocyanates may also be used as coupling agents to covalently link the drug to the binder. Other techniques are known to those skilled in the art and are within the scope of this disclosure. Examples of drug portions generated and linked to antibody or antigen-binding fragments using various chemical reactions known in this art include Hopschadiene splice regulators, such as those described and exemplified herein.

[0561] Connector-Hobstein splice regulator / Hobstein splice regulator compound

[0562] This document further discloses exemplary joint-Hropshire diene splice regulator (LH) compounds and compositions comprising multiple copies of such compounds. In various embodiments, the joint-Hropshire diene splice regulator compounds disclosed herein can be defined by the general formula: LH, where L = joint portion and H = hiker-Hropshire splice regulator. In some embodiments, the disclosed LH compounds are applicable to the ADCs described herein.

[0563] In some embodiments, compounds having formula (I) are disclosed herein: or a pharmaceutically acceptable salt thereof, wherein:

[0564] Y is selected from O, S, NR 6 and CR 6 R 7 ;

[0565] R 1 、R 2 and R 3Each is independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl), -OC(=O)-(C1-C6 alkyl), -C(=O)-O-(C1-C6 alkyl), and C1-C6 alkyl;

[0566] R 4 Selected from hydrogen, C1-C6 alkyl, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), and -C(=O)-NR 6 R 7 ;

[0567] R 5 Selected from hydrogen, hydroxyl, -CH2-OH, -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-NR 6 R 7 -NR 6 -C(=O)-R 8 -OC(=O)-NR 6 R 7 -NR 6 -C(=O)-R 8 and -NR 6 -C(=O)-NR 6 R 7 ;

[0568] R 6 and R 7 Each is independently selected from hydrogen, -R 8 -C(=O)-R 8 and -C(=O)-OR 8 ;and

[0569] R 8 Selected from C1-C6 alkyl, C3-C8 carbocyclic and C3-C8 heterocyclic groups.

[0570] where R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R 7 and R 8 Each is independently substituted by 0 to 3 groups independently selected from the following: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR 6 R 7C3-C8 carbocyclic, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl and C3-C8 heterocyclic, each of which may be independently substituted by 0 or 1 of the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, -NH-C(=O)(C1-C3 alkyl) and -NH-C(=O)-O-(C1-C3 alkyl).

[0571] In some embodiments, compounds having formula (Ia) are provided herein: Or a pharmaceutically acceptable salt thereof, which is covalently linked to L via any atom, wherein:

[0572] R 9 Selected from C3-C8 heterocyclic groups; and

[0573] R 10 Selected from H and C1-C6 alkyl groups,

[0574] where R 9 and R 10 Each is independently substituted by 0 to 3 groups independently selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, -NH2, -NH-(C1-C3 alkyl) and -N-(C1-C3 alkyl)2.

[0575] In some embodiments, compounds having formula (Ib) are provided herein: or a pharmaceutically acceptable salt thereof, wherein:

[0576] R 11 Selected from and Where * indicates R 11 The connection point with the rest of the compound;

[0577] R 12 and R 13 Each is independently selected from H and methyl.

[0578] In some embodiments, compounds having formula (II) are provided herein: or a pharmaceutically acceptable salt thereof, wherein:

[0579] X represents a hydroxyl group or NR. 6 R 7 ;

[0580] R 6 and R 7 Each is independently selected from hydrogen, -R 8 -C(=O)-R 8 -C(=O)-OR 8-(C1-C6 alkyl)-OC(=O)-R 8 and -(C1-C6 alkyl)-NH-C(=O)-R 8 ;and

[0581] R 8 Selected from C1-C6 alkyl, C3-C8 carbocyclic and C3-C8 heterocyclic groups.

[0582] where R 6 、R 7 and R 8 Each is independently substituted by 0 to 3 groups independently selected from the following: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR 6 R 7 C3-C8 carbocyclic, C1-C6 alkylhydroxy, C1-C6 alkylalkoxy, benzyl and C3-C8 heterocyclic, each of which may be independently substituted by 0 or 1 of the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, -NH-C(=O)(C1-C3 alkyl) and -NH-C(=O)-O-(C1-C3 alkyl).

[0583] In some embodiments, compounds having formula (IIa) are provided herein: or a pharmaceutically acceptable salt thereof, wherein:

[0584] Z is selected from NR 9 and O;

[0585] R 9 Selected from hydrogen and C1-C6 alkyl groups;

[0586] R 10 and R 11 Each is independently selected from hydrogen, halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-O-(C1-C6 alkyl), -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), C3-C8 carbocyclic, C1-C6 alkyl hydroxyl, C1-C6 alkyl alkoxy, benzyl and C3-C8 heterocyclic;

[0587] R 12 Selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups.

[0588] where R9 、R 10 、R 11 and R 12 Each is independently substituted with one or more groups selected from the following: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, and C1-C3 haloalkyl; and

[0589] t is an integer selected from 1, 2, 3, 4, 5 and 6.

[0590] In some embodiments, compounds having formula (IIb) are provided herein: or a pharmaceutically acceptable salt thereof, wherein:

[0591]

[0592]

[0593] Where * indicates R 13 The connection point with the rest of the compound; and

[0594] R 14 and R 15 Each is independently selected from hydrogen and methyl.

[0595] In some embodiments, compounds having formula (III) are provided herein: or a pharmaceutically acceptable salt thereof, wherein:

[0596] R 1 、R 2 and R 3 Each is independently selected from hydrogen, hydroxyl, -O-(C1-C6 alkyl), -OC(=O)-(C1-C6 alkyl), -C(=O)-O-(C1-C6 alkyl), and C1-C6 alkyl;

[0597] R 6 and R 7 Each is independently selected from hydrogen, -R 8 -C(=O)-R 8 and -C(=O)-OR 8 ;

[0598] R 8 Selected from C1-C6 alkyl, C3-C8 carbocyclic, and C3-C8 heterocyclic groups; and

[0599] R 9 Selected from H,

[0600] and

[0601]

[0602] where R 1 、R 2 、R 3 、R 6 、R 7 and R 8 Each is independently substituted by 0 to 3 groups independently selected from the following: halogen, hydroxyl, C1-C6 alkyl, -O-(C1-C6 alkyl), -CO2H, -C(=O)-(C1-C6 alkyl), -C(=O)-(C3-C8 carbocyclic), -C(=O)-(C3-C8 heterocyclic), -NR 6 R 7 The group may contain C3-C8 carbocyclic groups, C1-C6 alkylhydroxy groups, C1-C6 alkylalkoxy groups, benzyl groups, and C3-C8 heterocyclic groups, each of which may be independently substituted by 0 or 1 groups selected from the following groups: halogen, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, -NH-C(=O)(C1-C3 alkyl), and -NH-C(=O)-O-(C1-C3 alkyl); and

[0603] Where * indicates R 9 The connection point with the rest of the compound.

[0604] In some embodiments, this document provides compound H1 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H4 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H5 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H6 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H7 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H8 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H9 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H10 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H2 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H3 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H12 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H13 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H14 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H15 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H16 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H17 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H18 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H19 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H20 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H21 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H22 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H23 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H24 or a pharmaceutically acceptable salt thereof. In some embodiments, this document provides compound H25 or a pharmaceutically acceptable salt thereof.

[0605] In some embodiments, this document provides compounds selected from the following:

[0606]

[0607]

[0608]

[0609]

[0610]

[0611] And its pharmaceutically acceptable salt, wherein L is a linker covalently linked to the antibody.

[0612] In some embodiments, the linker comprises at least one cleavable peptide moiety. In some embodiments, the at least one cleavable peptide moiety is cleavable by an enzyme. In some embodiments, the linker or cleavable peptide moiety comprises at least one amino acid unit. In some embodiments, the at least one amino acid unit is selected from arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, aspartic acid, glutamic acid, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, and citrulline. In some embodiments, the at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, the linker comprises citrulline and valine. In some embodiments, the linker comprises alanine and valine.

[0613] In some embodiments, the connector comprises a portion selected from sulfonamide, β-glucuronic acid, disulfide, and carbonyl. In some embodiments, the connector comprises sulfonamide. In some embodiments, the connector comprises β-glucuronic acid. In some embodiments, the connector comprises disulfide. In some embodiments, the connector comprises carbonyl.

[0614] In some embodiments, the connector includes spacer subunits. In some embodiments, the spacer subunits are selected from alkyl and polyethylene glycol (PEG) portions. In some embodiments, the alkyl group is C1-C. 12 Alkyl group. In some embodiments, the alkyl group is a C1-C6 alkyl group. In some embodiments, the alkyl group is methylene. In some embodiments, the alkyl group is ethyl. In some embodiments, the alkyl group is n-propyl. In some embodiments, the alkyl group is n-butyl. In some embodiments, the alkyl group is n-pentyl. In some embodiments, the alkyl group is n-hexyl. In some embodiments, the PEG portion includes -(PEG). m - where m is an integer from 1 to 10. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6.

[0615] In some embodiments, the connector includes a maleic anhydride (Mal) portion (“Mal-spacer unit”). In some embodiments, the connector includes a self-ablating spacer unit. In some embodiments, the self-ablating spacer unit is selected from p-aminobenzyloxycarbonyl (pABC) and p-aminobenzyl (pAB).

[0616] In some embodiments, the connector comprises a Mal-spacer unit, an alkyl group, at least one amino acid unit, and a self-ablating spacer. In some embodiments, the at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, the at least one amino acid unit comprises alanine and valine. In some embodiments, the at least one amino acid unit comprises citrulline and valine. In some embodiments, the self-ablating spacer is selected from pAB and pABC. In some embodiments, the self-ablating spacer comprises pAB. In some embodiments, the self-ablating spacer comprises pABC. In some embodiments, the alkyl group comprises C1-C6 alkyl groups.

[0617] In some embodiments, the connector comprises a Mal-spacer unit, a PEG portion, at least one amino acid unit, and a self-ablating spacer. In some embodiments, the at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, the at least one amino acid unit comprises alanine and valine. In some embodiments, the at least one amino acid unit comprises citrulline and valine. In some embodiments, the self-ablating spacer is selected from pAB and pABC. In some embodiments, the self-ablating spacer comprises pAB. In some embodiments, the self-ablating spacer comprises pABC. In some embodiments, the PEG portion comprises -(PEG). m -, where m is an integer from 1 to 6.

[0618] Drug loading

[0619] The drug loading is represented by p and is also referred to herein as the Hobstein splice regulator to antibody ratio (HAR). The drug loading can range from 1 to 10 drug fractions per antibody or antigen-binding fragment. In some embodiments, p is an integer from 1 to 10. In some embodiments, p is an integer from 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is an integer from 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In some embodiments, p is an integer from 1 to 8. In some embodiments, p is an integer from 2 to 5. In some embodiments, p is an integer from 2 to 4. In some embodiments, p is an integer from 3 to 4. In other embodiments, p is an integer from 4 to 8. In other embodiments, p is 1, 2, 3, 4, 5, 6, 7, or 8, preferably 4 or 8.

[0620] The drug loading capacity may be limited by the number of linker sites on the antibody or antigen-binding fragment. In some embodiments, the linker portion (L) of the ADC is linked to the antibody or antigen-binding fragment via a chemically active group on one or more amino acid residues. For example, the linker may be linked to the antibody or antigen-binding fragment via a free amino group, imine group, hydroxyl group, thiol group, or carboxyl group (e.g., linked to the N-terminus or C-terminus, an ε-amino group linked to one or more lysine residues, a free carboxyl group linked to one or more glutamic acid or aspartic acid residues, or a sulfhydryl group linked to one or more cysteine ​​residues). The linker site may be a native residue in the amino acid sequence of the antibody or antigen-binding fragment, or may be introduced into the antibody or antigen-binding fragment, for example, through DNA recombination technology (e.g., by introducing cysteine ​​residues into the amino acid sequence) or through protein biochemistry (e.g., by reduction, pH adjustment, or hydrolysis).

[0621] In some embodiments, the number of pharmaceutical moieties that can bind to an antibody or antigen-binding fragment is limited by the number of free cysteine ​​residues. For example, in the case of cysteine ​​thiols, the antibody may have only one or more cysteine ​​thiols, or may have only one or more sufficiently reactive thiols that can be linked via a linker. Generally, antibodies do not contain a large number of free reactive cysteine ​​thiols that can be linked to pharmaceutical moieties. In fact, most cysteine ​​thiols in antibodies are involved in inter- or intra-chain disulfide bonds. Therefore, in some embodiments, conjugation to cysteine ​​may require at least partial reduction of the antibody. Excessive linking of linker-toxins to the antibody can destabilize the antibody by reducing the cysteine ​​residues available for forming disulfide bonds. Therefore, the optimized drug:antibody ratio should increase the potency of the ADC (by increasing the number of linked pharmaceutical moieties per antibody) without destabilizing the antibody or antigen-binding fragment. In some embodiments, the optimal ratio may be 2, 4, 6, or 8.

[0622] In some embodiments, the antibody or antigen-binding fragment is exposed to reducing conditions prior to conjugation to generate one or more free cysteine ​​residues. In some embodiments, the antibody may be reduced to reactive cysteine ​​thiols under partial or total reducing conditions with a reducing agent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). Unpaired cysteine ​​residues may be generated via partial reduction with a limited molar equivalent of TCEP, which reduces the interchain disulfide bonds linking the light and heavy chains (one pair / HL pair) and the two heavy chains in the hinge region (two pairs / HH pair in the case of human IgG1), but leaves the intrachain disulfide bonds intact (Stefano et al. (2013) Methods Mol Biol. [Molecular Biology Methods] 1045:145-71). In embodiments, disulfide bonds within the antibody are reduced electrochemically, for example, by employing a working electrode with alternating reducing and oxidizing voltages. This method allows for the online coupling of disulfide bond reduction with analytical devices (e.g., electrochemical detection devices, NMR spectrometers, or mass spectrometers) or chemical separation devices (e.g., liquid chromatography (e.g., HPLC) or electrophoresis devices (see, for example, U.S. Publication No. 20140069822)). In some embodiments, the antibody is subjected to denaturing conditions to expose reactive nucleophilic groups on amino acid residues such as cysteine.

[0623] The drug loading of an ADC can be controlled in various ways, such as by: (i) limiting the molar excess of the drug-adaptor intermediate or adaptor reagent relative to the antibody; (ii) limiting the conjugation reaction time or temperature; (iii) using partial or restrictive reduction conditions for cysteine ​​thiol modification; and / or (iv) engineering the amino acid sequence of the antibody through recombinant technology to control the number and / or position of the modified cysteine ​​residues to control the number and / or position of the adaptor-drug linkage.

[0624] In some embodiments, free cysteine ​​residues are introduced into the amino acid sequence of an antibody or antigen-binding fragment. For example, cysteine-engineered antibodies can be prepared in which one or more amino acids of the parent antibody are replaced by cysteine ​​amino acid substitutions. Any form of antibody can be engineered in this way, i.e., mutated. For example, a parent Fab antibody fragment can be engineered to form a cysteine-engineered Fab, referred to as “ThioFab”. Similarly, a parent monoclonal antibody can be engineered to form “ThioMab”. A single point mutation in ThioFab produces a single engineered cysteine ​​residue, while a single point mutation in ThioMab produces two engineered cysteine ​​residues due to the dimer nature of IgG antibodies. DNA encoding amino acid sequence variants of the parent polypeptide can be prepared by various methods known in this art (see, for example, the method described in International Publication No. WO 2006 / 034488). These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutation induction of DNA encoding polypeptides previously prepared, PCR mutation induction, and cartridge mutation induction. Recombinant antibody variants can also be constructed using synthetic oligonucleotides via restriction fragment manipulation or by overlap extension PCR. ADCs having formula (I) include, but are not limited to, antibodies having 1, 2, 3, or 4 engineered cysteine ​​amino acids (Lyon et al. (2012) Methods Enzymol. [Enzymological Methods] 502:123-38). In some embodiments, without engineering, one or more free cysteine ​​residues are already present in the antibody or antigen-binding fragment; in this case, the existing free cysteine ​​residues can be used to bind the antibody or antigen-binding fragment to the drug moiety.

[0625] In cases where more than one nucleophilic group reacts with a drug-linker intermediate or linker moiety reagent, followed by a reaction with a drug moiety reagent, the resulting product in a reaction mixture comprising multiple replicas of an antibody or antigen-binding fragment and a linker moiety may be a mixture of ADC compounds having a distribution of one or more drug moieties linked to replicas of the antibody or antigen-binding fragment in the mixture. In some embodiments, the drug loading in the mixture of ADCs obtained from the conjugation reaction is in the range of 1 to 10 linked drug moieties per antibody or antigen-binding fragment. The average number of drug moieties per antibody or antigen-binding fragment (i.e., average drug loading or average p) can be calculated by any conventional method known in this art, such as by mass spectrometry (e.g., reversed-phase LC-MS) and / or high-performance liquid chromatography (e.g., HIC-HPLC). In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is determined by hydrophobic interaction chromatography-high-performance liquid chromatography (HIC-HPLC). In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is determined by reversed-phase liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the average number of drug portions in each antibody or antigen-binding fragment is about 1.5 to about 3.5, about 2.5 to about 4.5, about 3.5 to about 5.5, about 4.5 to about 6.5, about 5.5 to about 7.5, about 6.5 to about 8.5, or about 7.5 to about 9.5. In some embodiments, the average number of drug portions in each antibody or antigen-binding fragment is about 2 to about 4, about 3 to about 5, about 4 to about 6, about 5 to about 7, about 6 to about 8, about 7 to about 9, about 2 to about 8, or about 4 to about 8.

[0626] In some embodiments, the average number of drug portions of each antibody or antigen-binding fragment is about 2. In some embodiments, the average number of drug portions of each antibody or antigen-binding fragment is about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5. In some embodiments, the average number of drug portions of each antibody or antigen-binding fragment is 2.

[0627] In some embodiments, the average number of drug portions in each antibody or antigen-binding fragment is about 4. In some embodiments, the average number of drug portions in each antibody or antigen-binding fragment is about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, or about 4.5. In some embodiments, the average number of drug portions in each antibody or antigen-binding fragment is 4.

[0628] In some embodiments, the average number of drug portions in each antibody or antigen-binding fragment is about 8. In some embodiments, the average number of drug portions in each antibody or antigen-binding fragment is about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, or about 8.5. In some embodiments, the average number of drug portions in each antibody or antigen-binding fragment is 8.

[0629] In different embodiments, the term "about" as used with respect to the average number of pharmaceutical portions of each antibody or antigen-binding fragment means ±10%.

[0630] Individual ADC compounds or "species" can be identified in the mixture by mass spectrometry and separated by UPLC or HPLC, such as hydrophobic interaction chromatography (HIC-HPLC). In some embodiments, homogeneous or near-homogeneous ADC products with a single loading value can be separated, for example, by electrophoresis or chromatography, from the self-conjugation mixture.

[0631] In some embodiments, higher drug loading (e.g., p>8) can cause aggregation, insolubility, toxicity, or loss of cell permeability in certain antibody-drug conjugates. Higher drug loading may also adversely affect the pharmacokinetics (e.g., clearance) of certain ADCs. In some embodiments, lower drug loading (e.g., p<2) may reduce the potency of certain ADCs against target-expressing cells and / or bystander cells. In some embodiments, the drug loading of the ADCs disclosed herein ranges from about 2 to about 8; from about 2 to about 6; from about 2 to about 5; from about 3 to about 5; from about 2 to about 4; or from about 4 to about 8.

[0632] In some embodiments, a drug loading of about 2 and / or an average drug loading is achieved, for example, by partial reduction of intrachain disulfides on the antibody or antigen-binding fragment, and beneficial properties are provided. In some embodiments, a drug loading of about 4 and / or an average drug loading is achieved, for example, by partial reduction of intrachain disulfides on the antibody or antigen-binding fragment, and beneficial properties are provided. In some embodiments, a drug loading of about 8 and / or an average drug loading is achieved, for example, by partial reduction of intrachain disulfides on the antibody or antigen-binding fragment, and beneficial properties are provided. In some embodiments, a drug loading of less than about 2 and / or an average drug loading may result in unacceptably high levels of unbound antibody species that may compete with the ADC for binding to the target antigen and / or provide reduced therapeutic efficacy. In some embodiments, a drug loading of more than about 8 and / or an average drug loading may result in unacceptably high levels of product heterogeneity and / or ADC aggregation. A drug loading of more than about 8 and / or an average drug loading may also affect the stability of the ADC due to the loss of one or more chemical bonds required to stabilize the antibody or antigen-binding fragment.

[0633] This disclosure includes methods for producing the described ADC. In short, an ADC comprises an antibody or antigen-binding fragment as the antibody or antigen-binding fragment, a pharmaceutical moiety (e.g., a hoprene splice regulator), and a linker that connects the pharmaceutical moiety and the antibody or antigen-binding fragment. In some embodiments, the ADC may be prepared using a linker having reactive functional groups for covalently linking to the pharmaceutical moiety and the antibody or antigen-binding fragment. For example, in some embodiments, a cysteine ​​thiol of the antibody or antigen-binding fragment may form a bond with a reactive functional group (e.g., a maleic anhydride moiety) of the linker or drug-linker intermediate to produce the ADC. ADC generation can be achieved by any technique known to those skilled in the art.

[0634] In some embodiments, an ADC is generated by first sequentially contacting an antibody or antigen-binding fragment with a linker and a pharmaceutical moiety (e.g., a hoprene splice regulator) such that the antibody or antigen-binding fragment is covalently linked to the linker, and then reacting a pre-formed antibody-linker intermediate with the pharmaceutical moiety. The antibody-linker intermediate may or may not undergo a purification step before contacting the pharmaceutical moiety. In other embodiments, an ADC is generated by contacting an antibody or antigen-binding fragment with a linker-drug compound pre-formed by reacting the linker with the pharmaceutical moiety. The pre-formed linker-drug compound may or may not undergo a purification step before contacting the antibody or antigen-binding fragment. In other embodiments, the antibody or antigen-binding fragment contacts a linker and a pharmaceutical moiety in a reaction mixture, allowing for simultaneous covalent bonding between the antibody or antigen-binding fragment and the linker, and between the linker and the pharmaceutical moiety. This method of generating an ADC may include a reaction in which the antibody or antigen-binding fragment is contacted before the linker is added to the reaction mixture, and vice versa. In some embodiments, the ADC is generated by reacting an antibody or antigen-binding fragment with a connector bound to the drug moiety, such as an ADL1-Hopshire splice regulator (e.g., ADL1-79392) or an ADL5-Hopshire splice regulator (e.g., ADL5-0349), under conditions that allow conjugation.

[0635] The ADC prepared according to the methods described above can undergo purification steps. Purification steps may involve any biochemical method or any combination thereof known in this art for purifying proteins. These methods include, but are not limited to, tangential flow filtration (TFF), affinity chromatography, ion exchange chromatography, any charge- or isoelectric point-based chromatography, mixed-mode chromatography (e.g., CHT (ceramic hydroxyapatite)), hydrophobic interaction chromatography, particle size separation chromatography, dialysis, filtration, selective precipitation, or any combination thereof.

[0636] Therapeutic Uses and Compositions

[0637] This document discloses a method for treating a subject's condition (e.g., adenoidosis) using the disclosed ADC and composition. The ADC can be administered alone or in combination with a second therapeutic agent, and can be administered with any pharmaceutically acceptable formulation, dosage, and dosing regimen. The therapeutic efficacy of the ADC can be assessed and adjusted accordingly for toxicity and efficacy measures. Efficacy measures include, but are not limited to, cell growth inhibition and / or cytotoxicity observed in vitro or in vivo, tumor volume reduction, tumor growth inhibition, and / or prolonged survival.

[0638] Methods are known for determining whether ADCs exert cell growth inhibitory and / or cytotoxic effects on cells. For example, the cytotoxic or cell growth inhibitory activity of ADCs can be measured by: exposing mammalian cells expressing the ADC's target protein to cell culture medium; culturing cells for a period of approximately 6 hours to approximately 6 days; and measuring cell viability. Cell-based in vitro analyses can also be used to measure ADC activity (proliferation), cytotoxicity, and apoptosis induction (activation of apoptotic proteases).

[0639] To determine whether an ADC exerts an inhibitory effect on cell growth, thymidine can be incorporated into the analysis. For example, cancer cells expressing the target antigen at a density of 5,000 cells per well of a coated 96-well plate can be cultured for 72 hours and exposed to 0.5 μCi of thymidine during the last 8 hours of the 72-hour period. 3 H-thymidine. Measurements were taken in cells of the culture in both the presence and absence of ADC. 3 The amount of H-thymidine added.

[0640] To determine cytotoxicity, necrosis or apoptosis (planned cell death) can be measured. Necrosis is usually accompanied by increased plasma membrane permeability; cell swelling and plasma membrane rupture. Apoptosis can be quantified, for example, by measuring DNA fragmentation. Commercial photometric methods for quantifying DNA fragmentation in vitro are available. Examples of such assays (including TUNEL, which detects labeled nucleotides incorporated into fragmented DNA, and ELISA-based assays) are described in Biochemica, (1999), Vol. 2, pp. 34-37 (Roche Molecular Biochemicals).

[0641] Apoptosis can also be measured by measuring morphological changes in cells. For example, similar to necrosis, loss of plasma membrane integrity can be measured by measuring the uptake of certain dyes (e.g., fluorescent dyes such as acridine orange or ethidium bromide). Methods for measuring the number of apoptotic cells have been described in Duke and Cohen, Current Protocols in Immunology (Coligan et al., eds. (1992), pp. 3.17.1–3.17.16). Cells can also be labeled with DNA dyes (e.g., acridine orange, ethidium bromide, or propidium iodide) and the aggregation and marginalization of chromosomes along the inner nuclear membrane can be observed. In some embodiments, apoptosis can also be measured by screening for apoptotic protease activity. In some embodiments, Caspase- The analysis measures the activities of apoptotic protease-3 and apoptotic protease-7. In some embodiments, the analysis provides luminescent apoptotic protease-3 / 7 receptors in reagents optimized for apoptotic protease activity, luciferase activity, and cell lysis. In some embodiments, Caspase- is added in an "add-mix-measure" format. The 3 / 7 reagent induces cell lysis, followed by cleavage of the receptor's apoptotic proteases and the generation of a "glow-like" luminescence signal produced by luciferase. In some embodiments, the luminescence may be proportional to the amount of apoptotic protease activity present and may serve as an indicator of apoptosis. Other morphological changes in apoptosis that can be measured include, for example, cytoplasmic condensation, increased membrane vesicle formation, and cell contraction. ADCs can be used to treat cancer by measuring any of these effects on cancer cells.

[0642] Cell viability can be measured, for example, by measuring substances in cells such as neutral red, trypan blue, crystal violet, or ALAMAR. TM The absorption of blue dye is used for measurement (see, for example, Page et al. (1993) Intl J Oncology 3:473-6). In such analyses, cells are cultured in a dye-containing medium, washed, and the remaining dye, reflecting the cell's absorption of the dye, is measured spectrophotometrically. Cell viability can also be measured, for example, by quantifying ATP as an indicator of metabolically active cells. In some embodiments, the in vitro potency and / or cell viability of the prepared ADC or Hobstein splice regulator compound can be measured using CellTiter as described in the examples provided herein. The assay is performed using luminescent cell viability analysis. In some embodiments, a single reagent (CellTiter-) is used in this analysis. The reagent was added directly to cells cultured in a serum-supplemented medium. The addition of the reagent caused cell lysis and generated a luminescent signal proportional to the amount of ATP present. The amount of ATP was directly proportional to the number of cells present in the culture. Cytotoxicity could also be measured using the protein-binding dye sulforhodamine B (SRB) (Skehan et al. (1990) J Natl Cancer Inst. [Journal of the American Institute for Cancer Research] 82:1107-12).

[0643] The bystander-killing activity of the disclosed ADC can also be evaluated. Bystander-killing activity can be determined, for example, by employing an analysis of two cell lines, one positive and one negative for the target antigen. In some embodiments, the analysis is designed to track only target-negative cells. In some embodiments, cells are plated under three conditions: (i) target-negative cells alone (labeled or marked); (ii) target-positive cells alone; and (iii) a co-culture of target-negative and target-positive cells. The cells are then treated with the ADC, followed by monitoring for cytotoxicity. When using CellTiter- When using the reagent reading tray, the viability of all cell populations can be monitored. When the reagent reading disc is activated, only labeled or marked target-negative cells generate a signal. Killing of target-negative cells mixed with target-positive cells indicates bystander killing, while killing of target-negative cells in the absence of target-positive cells indicates off-target killing.

[0644] In some aspects, this disclosure is characterized by methods for killing cancer cells or tissues, inhibiting or regulating the growth of cancer cells or tissues, or interfering with the metabolism of cancer cells or tissues by disrupting RNA splicing. This method can be used by any subject for whom RNA splicing disruption provides therapeutic benefits. Subjects who may benefit from disrupting RNA splicing include, but are not limited to, subjects with or at risk of having a neoplastic condition, such as hematologic malignancies or solid tumors. In some embodiments, hematologic malignancies are B-cell malignancies, blood cancers (leukemia), plasma cell carcinomas (myeloma, such as multiple myeloma), or lymph node carcinomas (lymphoma). In some embodiments, hematologic malignancies are acute myeloid leukemia or multiple myeloma. In some embodiments, leukemia is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloid monocytic leukemia (CMML), or acute monocytic leukemia (AMoL). In some embodiments, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma. In some embodiments, the hematologic malignancy is myelomectomy syndrome (MDS). In some embodiments, the solid tumor is a carcinoma, such as breast cancer, pancreatic cancer, prostate cancer, colorectal cancer or rectal cancer, lung cancer, stomach cancer, cervical cancer, endometrial cancer, ovarian cancer, bile duct carcinoma, glioma, or melanoma. In some embodiments, the solid tumor is breast cancer, stomach cancer, prostate cancer, ovarian cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., serous endometrial carcinoma), salivary gland carcinoma, melanoma, colorectal cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer, or esophageal cancer. In some embodiments, lung cancer is lung adenocarcinoma. In some embodiments, uterine cancer is serous endometrial carcinoma.

[0645] In various embodiments, the disclosed ADC can be administered to any HER2-expressing cell or tissue, such as a HER2-expressing proliferative cell or tissue. Exemplary embodiments include methods for inhibiting HER2-mediated cell signaling or for killing cells. This method can be used in any HER2-expressing cell or tissue, such as cancer cells or metastatic lesions. Non-limiting examples of HER2-expressing cancers include 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. [Molecular Diagnostics and Therapy] 17:85-99). Non-limiting examples of HER2-expressing cells include HCC1954 and SKBR3 human breast ductal carcinoma cells, N87 human gastric cancer cells, and cells containing recombinant nucleic acids encoding HER2 or a portion thereof.

[0646] In various embodiments, the disclosed ADC can be administered to any CD138-expressing cell or tissue, such as a proliferative cell or tissue expressing CD138. Exemplary embodiments include methods for inhibiting CD138-mediated cell signaling or for killing cells. This method can be used in any cell or tissue expressing CD138, such as cancer cells or metastatic lesions. Non-limiting examples of CD138-expressing cancers include intrathoracic cancers (e.g., lung cancer, mesothelioma), skin cancers (e.g., basal cell carcinoma, squamous cell carcinoma), head and neck cancers (e.g., laryngeal cancer, laryngopharyngeal cancer, nasopharyngeal cancer), breast cancer, genitourinary cancers (e.g., cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, urethral cancer), hematologic malignancies (e.g., myeloma, such as multiple myeloma, Hodgkin's lymphoma), and thyroid cancer (Szatmári et al. (2015) Dis Markers [Disease Markers] 2015:796052). Non-limiting examples of cells expressing CD138 include MOLP8 human multiple myeloma cells and cells containing recombinant nucleic acids encoding CD138 or a portion thereof.

[0647] In various embodiments, the disclosed ADC can be administered to any cell or tissue expressing EPHA2, such as a proliferative cell or tissue expressing EPHA2. Exemplary embodiments include methods for inhibiting EPHA2-mediated cell signaling or for killing cells. This method can be used in any cell or tissue expressing EPHA2, such as cancer cells or metastatic lesions. Non-limiting examples of EPHA2-expressing 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 on Ther Targets 15(1):31-51). Non-limiting examples of EPHA2-expressing cells include PC3 human prostate cancer cells and cells containing recombinant nucleic acids encoding EPHA2 or a portion thereof.

[0648] Exemplary methods include the step of contacting cells with an effective amount (i.e., an amount sufficient to kill cells) of an ADC as described herein. This method can be used on cells, for example, in vitro, in vivo, in vitro, or in situ cultures. For example, cells expressing HER2 can be cultured in vitr...

Claims

1. An antibody-drug conjugate having formula (I): Ab-(L-H) p (I) in: Ab is an anti-HER2 antibody or antigen-binding fragment, wherein Ab comprises three heavy chain complementary determining regions (HCDR1, HCDR2 and HCDR3) comprising the amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2 (HCDR2) and SEQ ID NO: 3 (HCDR3); and three light chain complementary determining regions (LCDR1, LCDR2 and LCDR3) comprising the amino acid sequences of SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2) and SEQ ID NO: 6 (LCDR3); L is a linker that covalently links Ab to H, wherein L comprises MC-Val-Cit; H is a Hoprichthyrene splicing regulator, wherein the Hoprichthyrene splicing regulator comprises a compound having the following formula: or a pharmaceutically acceptable salt thereof, wherein: R 11 Selected from and Where * indicates R 11 the point of attachment to the rest of the compound; R 12 and R 13 are each independently selected from hydrogen and methyl; and p is an integer of 1-15.

2. The antibody-drug conjugate of claim 1, wherein the linker L comprises MC-Val-Cit-pAB. The antibody-drug conjugate of claim 1 , wherein the linker L comprises MC-Val-Cit-pABC.

4. The antibody-drug conjugate of claim 1, wherein the Hoppleridiene splicing regulator H is selected from: and pharmaceutically acceptable salts thereof.

5. The antibody-drug conjugate of claim 1, wherein LH is selected from: and pharmaceutically acceptable salts thereof.

6. An antibody-drug conjugate having formula (I): Ab-(L-H) p (I) in: Ab is an anti-HER2 antibody or antigen-binding fragment, wherein Ab comprises three heavy chain complementary determining regions (HCDR1, HCDR2 and HCDR3) comprising the amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2 (HCDR2) and SEQ ID NO: 3 (HCDR3); and three light chain complementary determining regions (LCDR1, LCDR2 and LCDR3) comprising the amino acid sequences of SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2) and SEQ ID NO: 6 (LCDR3); L is a linker that covalently links Ab to H, wherein L comprises MC-Val-Cit; H is a Hoprichthyrene splicing regulator, wherein the Hoprichthyrene splicing regulator comprises a compound having the following formula: or a pharmaceutically acceptable salt thereof, wherein: R 13 Selected from Where * indicates R 13 the point of attachment to the rest of the compound; and R 14 and R 15 are each independently selected from hydrogen and methyl; and p is an integer of 1-15.

7. The antibody-drug conjugate of claim 6, wherein the linker L comprises MC-Val-Cit-pAB.

8. The antibody-drug conjugate of claim 6, wherein the linker L comprises MC-Val-Cit-pABC.

9. The antibody-drug conjugate of claim 6, wherein the Hoppleridiene splicing regulator H is selected from: (H4) and pharmaceutically acceptable salts thereof.

10. The antibody-drug conjugate of claim 6, wherein LH is selected from: and pharmaceutically acceptable salts thereof.

11. An antibody-drug conjugate having formula (I): Ab-(L-H) p (I) in: Ab is an anti-HER2 antibody or antigen-binding fragment, wherein Ab comprises three heavy chain complementary determining regions (HCDR1, HCDR2 and HCDR3) comprising the amino acid sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2 (HCDR2) and SEQ ID NO: 3 (HCDR3); and three light chain complementary determining regions (LCDR1, LCDR2 and LCDR3) comprising the amino acid sequences of SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2) and SEQ ID NO: 6 (LCDR3); L is a linker that covalently links Ab to H, wherein L comprises MC-Val-Cit; H is a Hoprichthyrene splicing regulator, wherein the Hoprichthyrene splicing regulator comprises a compound having the following formula: or a pharmaceutically acceptable salt thereof, wherein: R 1 is hydrogen; R 2 is methoxy; R 3 is methyl; and R 9 Selected from hydrogen, Where * indicates R 9 the point of attachment to the rest of the compound; and p is an integer of 1-15.

12. The antibody-drug conjugate of claim 11, wherein the linker L comprises MC-Val-Cit-pAB.

13. The antibody-drug conjugate of claim 11, wherein the linker L comprises MC-Val-Cit-pABC.

14. The antibody-drug conjugate of claim 11, wherein the Hoppleridiene splicing regulator H is selected from: and pharmaceutically acceptable salts thereof.

15. The antibody-drug conjugate of claim 11, wherein LH is selected from: and pharmaceutically acceptable salts thereof.

16. The antibody-drug conjugate of any one of claims 1 to 15, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

20.

17. The antibody-drug conjugate of claim 16, wherein the antibody or antigen-binding fragment comprises a human IgG1 heavy chain constant region.

18. The antibody-drug conjugate of claim 17, wherein the antibody or antigen-binding fragment comprises a human Ig kappa light chain constant region.

19. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 18 and a pharmaceutically acceptable carrier.

20. Use of the antibody-drug conjugate of any one of claims 1 to 18 in the preparation of a medicament for treating a subject having or suspected of having a neoplastic disorder.

21. The use of claim 20, wherein the neoplastic disorder is a solid tumor.

22. The use of claim 21, wherein the neoplastic disorder is a solid tumor, and wherein the solid tumor is selected from breast cancer and gastric cancer.

23. The use of claim 20, wherein the medicament is formulated to induce: (i) at least one neoantigen, (ii) a T cell response, or (iii) both (i) and (ii).

24. The use of claim 20, wherein the medicament is formulated to contain at least one additional therapeutic agent.

25. The use of claim 24, wherein the at least one additional therapeutic agent comprises a checkpoint inhibitor.

26. The use of claim 25, wherein the checkpoint inhibitor targets CTLA4, PD1, PDL1, OX40, CD40, LAG3, TIM3, KIR and / or GITR.

27. The use of claim 25, wherein the checkpoint inhibitor comprises a cytotoxic T lymphocyte-associated antigen 4 pathway (CTLA4) inhibitor.

28. The use according to claim 27, wherein the CTLA4 inhibitor is an anti-CTLA4 antibody.

29. The use according to claim 28, wherein the anti-CTLA4 antibody is ipilimumab.

30. The use of claim 25, wherein the checkpoint inhibitor comprises a programmed death-1 pathway (PD1) inhibitor.

31. A compound having the formula: or a pharmaceutically acceptable salt thereof, wherein: R 11 Selected from and Where * indicates R 11 the point of attachment to the rest of the compound; and R 12 and R 13 are each independently selected from hydrogen and methyl.

32. The compound of claim 31 selected from: or pharmaceutically acceptable salts thereof.

33. A compound having the formula: or a pharmaceutically acceptable salt thereof, wherein: R 13 Selected from Where * indicates R 13 the point of attachment to the rest of the compound; and R 14 and R 15 are each independently selected from hydrogen and methyl.

34. The compound of claim 33, wherein the compound is selected from: and pharmaceutically acceptable salts thereof.

35. A compound having the formula: or a pharmaceutically acceptable salt thereof, wherein: R 1 is hydroxyl group; R 2 is methoxy; R 3 is methyl; and R 9 Selected from hydrogen, Where * indicates R 9 The point of attachment to the rest of the compound.

36. The compound of claim 35, wherein the compound is selected from: and pharmaceutically acceptable salts thereof.

37. A pharmaceutical composition comprising a compound according to any one of claims 31 to 36 and a pharmaceutically acceptable carrier.

38. Use of a compound as claimed in any one of claims 31 to 36 in the preparation of a medicament for treating a subject suffering from or suspected of suffering from a neoplastic disorder.

39. The use of claim 38, wherein the neoplastic disorder is a hematological malignancy or a solid tumor.

40. The use of claim 39, wherein the hematological malignancy is selected from the group consisting of B-cell malignancies, leukemias, lymphomas, myelomas, acute myeloid leukemias and multiple myeloma.

41. The use of claim 39, wherein the neoplastic disorder is the solid tumor, and wherein the solid tumor is selected from breast cancer and gastric cancer.

42. The use of claim 38, wherein the medicament is formulated to induce: (i) at least one neoantigen, (ii) a T cell response, or (iii) both (i) and (ii).

43. The use of claim 38, wherein the medicament is formulated to contain at least one additional therapeutic agent.

44. The use of claim 43, wherein the at least one additional therapeutic agent comprises a checkpoint inhibitor.

45. The use of claim 44, wherein the checkpoint inhibitor targets CTLA4, PD1, PDL1, OX40, CD40, LAG3, TIM3, KIR and / or GITR.

46. ​​The use of claim 44, wherein the checkpoint inhibitor comprises a cytotoxic T lymphocyte-associated antigen 4 pathway (CTLA4) inhibitor.

47. The use of claim 46, wherein the CTLA4 inhibitor is an anti-CTLA4 antibody.

48. The use of claim 47, wherein the anti-CTLA4 antibody is ipilimumab.

49. The use of claim 44, wherein the checkpoint inhibitor comprises a programmed death-1 pathway (PD1) inhibitor.

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