RU2820607C2 - Antibody-drug conjugates containing herboxydiene-based splicing modulator, and methods of use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a group of inventions comprising a splicing modulator or a pharmaceutically acceptable salt thereof, a compound for treating a neoplastic disorder, conjugate of an antibody and a drug for treating a neoplastic disorder, a pharmaceutical composition for treating a neoplastic disorder, use of a splicing modulator or a pharmaceutically acceptable salt thereof, a compound or a conjugate of an antibody and a drug for treating a neoplastic disorder, use of a splicing modulator or a pharmaceutically acceptable salt thereof, a compound or a conjugate of an antibody and a drug for preparing a drug for treating a neoplastic disorder and a method of treating a subject having a neoplastic disorder or suspected presence thereof.

EFFECT: invention extends the range of splicing modulation means.

27 cl, 6 dwg, 14 tbl, 4 ex

Description

[01] The present invention claims the benefit of priority to U.S. Provisional Patent Application No. 62/779,400, filed December 13, 2018; US Provisional Application No. 62/779406, filed December 13, 2018, and US Provisional Application No. 62/941220, filed November 27, 2019. Each of the foregoing applications is incorporated herein by reference in its entirety.

[02] The present invention relates to antibody-drug conjugates (ADCs) containing a herboxidiene splicing modulator and an antibody or antigen-binding fragment thereof, for example, that binds a human tumor antigen target. The present invention further relates to methods and compositions useful in the treatment or diagnosis of cancers that express a target antigen and/or are treatable by disrupting RNA splicing, as well as methods for preparing such compositions.

[03] Most protein-coding genes in the human genome consist of several exons (coding regions) that are separated by introns (non-coding regions). Expression of the gene results in the formation of a single precursor messenger RNA (pre-mRNA). Intronic sequences are subsequently removed from the pre-mRNA through a process called splicing, which results in the formation of mature messenger RNA (mRNA). By including different combinations of exons, alternative splicing produces mRNAs encoding different protein isoforms.

[04] RNA splicing is catalyzed by the spliceosome, a dynamic multiprotein RNA complex consisting of five small nuclear RNAs (snRNA U1, U2, U4, U5, and U6) and associated proteins. The spliceosome assembles onto pre-mRNA to mediate a dynamic cascade of multiple RNA-protein interactions that catalyze intron excision and exon ligation (Matera and Wang (2014) Nat Rev Mol Cell Biol. 15(2):108–21). Accumulating evidence has linked human diseases to dysregulated RNA splicing, which affects many genes (Scotti and Swanson (2016) Nat Rev Genet. 17(1):19–32).

[05] The spliceosome is an important target in oncology biology. Currently, several studies have documented significant changes in the splicing profile of cancer cells, as well as in the splicing factors themselves (Agrawal et al. (2018) Curr Opin Genet Dev. 48:67–74). Alternative splicing can result in differential inclusion/exclusion of exons, intron retention, or use of cryptic splice sites (Seiler et al. (2018) Cell Rep. 23(1):282–296). Collectively, these events underlie functional changes that may contribute to tumorigenesis or therapy resistance (Siegfried and Karni (2018) Curr Opin Genet Dev. 48:16–21).

[06] Certain natural products can bind the SF3b spliceosome complex. These small molecules modulate splicing by promoting intron retention and/or exon skipping (Teng et al. (2017) Nat Commun. 8:15522). For example, it has been shown that herboxidiene, a naturally occurring polyketide isolated from Streptomyces sp. A7847 (Isaac et al. (1992) J. Org. Chem. 57:7220-26), and its derivatives modulate splicing. See for example Imaizumi et al. (2017) J. Antibiot. 70:675-79. A significant portion of the resulting transcripts contain premature stop codons that trigger nonsense-mediated mRNA degradation (NMD). In addition, because canonical splicing is disrupted, the number of canonical transcripts is significantly reduced, which can negatively impact cell function and viability. In this regard, splicing modulators are emerging as a promising class of drugs for the treatment of cancer (Puthenveetil et al (2016) Bioconjugate Chem. 27:1880-8).

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

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

[09] EPHA2 is a transmembrane glycoprotein that is abundantly overexpressed in several cell lines derived from malignant tumors and in advanced cancers (Wykosky and Debinski (2008) Mol Cancer Ref. 6(12):1795-1806). For example, EPHA2 is highly overexpressed in approximately 61% of tumors from patients with GBM (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 adenocarcinoma cases (Zeng et al. (2003) Am J Pathol. 163:2271-6). The EPHA2 protein is overexpressed to a high degree in the percentage of patient tumors and the percentage of cells within the tumor, and is a plasma membrane-localized receptor that can be internalized upon ligand binding (Walker-Daniels et al. (2002) Mol Cancer Res 1:79-87). Moreover, EPHA2 expression is associated with poor prognosis, increased metastasis, and decreased survival. Thus, due to its expression pattern, localization, and functional importance in determining the outcome of cancer patients, EPHA2 represents another attractive antigen for targeted delivery of new anticancer therapies.

[10] In various embodiments, the present invention provides, in particular, novel herboxydiene-based splicing modulators with biological activity against neoplastic cells. Herboxydiene-based splicing modulators can be used alone or as part of an ADC to slow, suppress and/or reverse tumor growth in mammals and may be useful in the treatment of human patients suffering from cancer. In various embodiments, the present invention provides novel antibody-drug conjugates that utilize herboxidiene-based splicing modulators.

[11] In various embodiments, the present invention more specifically relates to antibody-drug conjugate (ADC) compounds that are capable of binding to and killing neoplastic cells. In various embodiments, the ADC compounds disclosed herein comprise a linker that attaches a herboxydiene-based splicing modulator to a full-length antibody or antigen-binding moiety. In various embodiments, the ADC compounds are also capable of internalization into the target cell upon binding.

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

[13] In some embodiments, H, a herboxydiene-based splicing modulator, provides a compound of formula (I): (I), or a pharmaceutically acceptable salt thereof, which are covalently attached to L through any atom where

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

each of R ¹ , R ² and R ³ is independently selected from hydrogen, hydroxyl, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups, -C groups (=O)-O-(C ₁ -C ₆ alkyl) and C ₁ -C ₆ alkyl groups;

R ⁴ is selected from hydrogen, C ₁ -C ₆ alkyl groups, -C(=O)-(C ₁ -C ₆ alkyl) groups, -C(=O)-(C ₃ -C ₈ carbocyclyl) groups, - C(=O)-(C ₃ -C ₈ heterocyclyl) and -C(=O)-NR ⁶ R ⁷ ;

R ⁵ is selected from hydrogen, hydroxyl, -CH ₂ -OH, -CO ₂ H, -C(=O)-O-(C ₁ -C ₆ alkyl), -C(=O)-NR ⁶ R ⁷ groups, -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 ;

R ⁶ and R ⁷ are each independently selected from hydrogen, -R ⁸ , -C(=O)-R ⁸ and -C(=O)-OR ⁸ ; And

R ⁸ is selected from C ₁ -C ₆ alkyl groups, C ₃ -C ₈ carbocyclyl groups and C ₃ -C ₈ heterocyclyl groups,

where each of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ is independently substituted with 0-3 groups independently selected from halogens, hydroxyl, C ₁ -C ₆ alkyl groups, groups - O-(C ₁ -C ₆ alkyl), -CO ₂ H, groups -C(=O)-(C ₁ -C ₆ alkyl), groups -C(=O)-(C ₃ -C ₈ carbocyclyl), groups -C(=O)-(C ₃ -C ₈ heterocyclyl), -NR ⁶ R ⁷ , C ₃ -C ₈ carbocyclyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkyl alkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups, each of which may be independently substituted with 0 or 1 group selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups, C ₁ -C ₃ haloalkyl groups, -NH- C(=O)(C ₁ -C ₃ alkyl) and -NH-C(=O)-O-(C ₁ -C ₃ alkyl), and

where the valency of the atom that is covalently attached to L is not exceeded.

[14] In some embodiments, H, a herboxydiene-based splicing modulator, provides a compound of formula (Ia): (Ia), or a pharmaceutically acceptable salt thereof, which are covalently attached to L through any atom where

R ⁹ is selected from C ₃ -C ₈ heterocyclyl groups;

R ¹⁰ is selected from H and C ₁ -C ₆ alkyl groups,

where each of R ⁹ and R ¹⁰ is independently substituted with 0-3 groups independently selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups, -NH ₂ , -NH-(C ₁ -C ₃ alkyl) and -N-(C ₁ -C ₃ alkyl) ₂ , and

where the valency of the atom that is covalently attached to L is not exceeded.

[15] In some embodiments, H, a herboxydiene-based splicing modulator, provides a compound of formula (Ib): (Ib), or a pharmaceutically acceptable salt thereof, which are covalently attached to L through any atom where

R ¹¹ selected from , And , where * denotes the point of attachment of R ¹¹ to the rest of the connection;

R ¹² and R ¹³ are each independently selected from H and methyl; And

where the valency of the atom that is covalently attached to L is not exceeded.

[16] In some embodiments, H, a herboxydiene-based splicing modulator, provides a compound of formula (II): (II), or a pharmaceutically acceptable salt thereof, which are covalently attached to L through any atom where

X represents hydroxyl or NR ⁶ R ⁷ ;

R ⁶ and R ⁷ are each independently selected from hydrogen, -R ⁸ , -C(=O)-R ⁸ , -C(=O)-OR ⁸ , -(C ₁ -C ₆ alkyl)-OC(=O )-R ⁸ and -(C ₁ -C ₆ alkyl)-NH-C(=O)-R ⁸ ; And

R ⁸ is selected from C ₁ -C ₆ alkyl groups, C ₃ -C ₈ carbocyclyl groups and C ₃ -C ₈ heterocyclyl groups,

wherein each of R ⁶ , R ⁷ and R ⁸ is independently substituted with 0-3 groups independently selected from halogens, hydroxyl, C ₁ -C ₆ alkyl groups, -O-(C ₁ -C ₆ alkyl), -CO ₂ H groups , groups -C(=O)-O-(C ₁ -C ₆ alkyl), groups -C(=O)-(C ₁ -C ₆ alkyl), groups -C(=O)-(C ₃ -C ₈ carbocyclyl), -C(=O)-(C ₃ -C ₈ heterocyclyl) groups, -NR ⁶ R ⁷ , C ₃ -C ₈ carbocyclyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkylalkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups, each of which may be independently substituted with 0-1 group selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups, C ₁ -C ₃ haloalkyl groups , -NH-C(=O)(C ₁ -C ₃ alkyl) and -NH-C(=O)-O-(C ₁ -C ₃ alkyl), and

where the valency of the atom that is covalently attached to L is not exceeded.

[17] In some embodiments, H, a herboxydiene-based splicing modulator, provides a compound of formula (IIa): (II), or a pharmaceutically acceptable salt thereof, which are covalently attached to L through any atom where

Z is selected from NR ⁹ and O;

R ⁹ is selected from hydrogen and C ₁ -C ₆ alkyl groups;

each of R ¹⁰ and R ¹¹ is independently selected from hydrogen, halogens, hydroxyl, C ₁ -C ₆ alkyl groups, -O-(C ₁ -C ₆ alkyl) groups, -CO ₂ H, -C(=O)- groups O-(C ₁ -C ₆ alkyl), -C(=O)-(C ₁ -C ₆ alkyl) groups, -C(=O)-(C ₃ -C ₈ carbocyclyl) groups, -C(= O)-(C ₃ -C ₈ heterocyclyl), C ₃ -C ₈ carbocyclyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkyl alkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups;

R ¹² is selected from C ₁ -C ₆ alkyl groups, C ₃ -C ₈ carbocyclyl groups, C ₃ -C ₈ heterocyclyl groups,

wherein each of R ⁹ , R ¹⁰ , R ¹¹ and R ¹² is independently substituted with 0 or 1 group selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups and C ₁ -C ₃ haloalkyl groups;

t is an integer selected from 1, 2, 3, 4, 5 and 6; And

where the valency of the atom that is covalently attached to L is not exceeded.

[18] In some embodiments, H, a herboxydiene-based splicing modulator, provides a compound of formula (IIb): (IIb), or a pharmaceutically acceptable salt thereof, which are covalently attached to L through any atom where

R ¹³ selected from , , , , , , , , , And , where * denotes the point of attachment of R ¹³ to the rest of the connection;

R ¹⁴ and R ¹⁵ are each independently selected from hydrogen and methyl; And

where the valency of the atom that is covalently attached to L is not exceeded.

[19] In some embodiments, H, a herboxydiene-based splicing modulator, provides a compound of formula (III): (III), or a pharmaceutically acceptable salt thereof, which are covalently attached to L through any atom where

each of R ¹ , R ² and R ³ is independently selected from hydrogen, hydroxyl, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups, -C groups (=O)-O-(C ₁ -C ₆ alkyl) and C ₁ -C ₆ alkyl groups;

R ⁶ and R ⁷ are each independently selected from hydrogen, -R ⁸ , -C(=O)-R ⁸ and -C(=O)-OR ⁸ ;

R ⁸ is selected from C ₁ -C ₆ alkyl groups, C ₃ -C ₈ carbocyclyl groups and C ₃ -C ₈ heterocyclyl groups; And

R ⁹ selected from H, , , , , , , , , And ;

wherein each of R ¹ , R ² , R ³ , R ⁶ , R ⁷ and R ⁸ is independently substituted with 0-3 groups independently selected from halogens, hydroxyl, C ₁ -C ₆ alkyl groups, -O-(C ₁ - C ₆ alkyl), -CO ₂ H, -C(=O)-(C ₁ -C ₆ alkyl) groups, -C(=O)-(C ₃ -C ₈ carbocyclyl) groups, -C(=O) groups )-(C ₃ -C ₈ heterocyclyl), -NR ⁶ R ⁷ , C ₃ -C ₈ carbocyclyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkyl alkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups, each of which may be independently substituted with 0 or 1 group selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups, C ₁ -C ₃ haloalkyl groups, -NH-C(=O)( C ₁ -C ₃ alkyl) and -NH-C(=O)-O-(C ₁ -C ₃ alkyl),

where the valency of the atom that is covalently attached to L is not exceeded; And

where * denotes the point of attachment of R ⁹ to the rest of the connection.

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

[21] In some embodiments, the linker includes a cleavable glucuronide moiety. In some embodiments, the cleavable glucuronide moiety is enzyme cleavable. In some embodiments, the cleavable glucuronide moiety is cleavable by glucuronidase. In some embodiments, the cleavable glucuronide moiety is cleavable by β-glucuronidase.

[22] In some embodiments, the linker includes at least one spacer unit. In some embodiments, the spacer unit or linker includes a polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety comprises -(PEG) _m -, and m is an integer from 1 to 10. In some embodiments, m is 2. In some other embodiments, the spacer unit or linker comprises an alkyl moiety. In some embodiments, the alkyl moiety provides -(CH ₂ ) _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.

[23] In some embodiments, the spacer unit is attached to the antibody or antigen binding moiety via a maleimide (Mal) moiety ("Mal spacer unit"). In some embodiments, the Mal spacer unit is reactive to a cysteine residue in the antibody or antigen binding moiety. In some embodiments, the Mal spacer unit is attached to the antibody or antigen binding fragment via a cysteine residue in the antibody or antigen binding fragment.

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

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

[26] In some embodiments, the cleavable moiety in the linker is directly attached to the herboxydiene splicing modulator, or the spacer unit attaches the cleavable moiety in the linker to the herboxydiene splicing modulator. In some embodiments, cleavage of the conjugate releases the herboxydiene splicing modulator from the antibody or antigen binding moiety and linker. In some embodiments, the spacer unit connecting the cleavable moiety in the linker to the herboxydiene splicing modulator is self-cleavable.

[27] In some embodiments, the spacer unit connecting the cleavable moiety in the linker to the herboxydiene splicing modulator comprises p-aminobenzyloxycarbonyl (pABC). In some embodiments, pABC attaches a cleavable moiety in a linker to a herboxydiene-based splicing modulator. In some embodiments, the cleavable moiety in the linker includes a cleavable peptide moiety. In some embodiments, the cleavable peptide fragment includes an amino acid unit. In some embodiments, the cleavable peptide fragment or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. In some embodiments, the linker comprises Val-Cit-pABC. In some other embodiments, the linker comprises Val-Ala-pABC. In some embodiments, the linker comprises Glu-Val-Cit-pABC. In some embodiments, the linker provides Ala-Ala-Asn-pABC.

[28] In some embodiments, the spacer unit connecting the cleavable moiety in the linker to the herboxydiene splicing modulator comprises p-aminobenzyl (pAB). In some embodiments, pAB attaches a cleavable moiety in a linker to a herboxydiene-based splicing modulator. In some embodiments, the cleavable moiety in the linker includes a cleavable peptide moiety. In some embodiments, the cleavable peptide fragment includes an amino acid unit. In some embodiments, the cleavable peptide fragment or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. In some embodiments, the linker comprises Val-Cit-pAB. In some other embodiments, the linker comprises Val-Ala-pAB. In some other embodiments, the linker comprises Glu-Val-Cit-pAB. In some other embodiments, the linker comprises Ala-Ala-Asn-pAB.

[29] In various embodiments, the linker is a non-cleavable linker. In some embodiments, the herboxydiene splicing modulator from the ADC is released as a result of degradation of the antibody or antigen binding fragment. In some embodiments, the linker remains covalently associated with at least one amino acid of the antibody and the drug after internalization into and degradation in the target cell.

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

[31] In some embodiments, the spacer unit in the non-cleavable linker is attached to the antibody or antigen binding moiety via a maleimide (Mal) moiety ("Mal spacer unit"). In some embodiments, the Mal spacer unit is reactive to a cysteine residue in the antibody or antigen binding moiety. In some embodiments, the Mal spacer unit is attached to the antibody or antigen binding fragment via a cysteine residue in the antibody or antigen binding fragment. In some embodiments, the Mal spacer unit includes an alkyl moiety. In some embodiments, the Mal spacer unit includes a PEG moiety. In some embodiments, the linker or Mal spacer unit comprises maleimidocaproyl (MC). In some embodiments, the linker or Mal spacer unit comprises maleimidocaproyl (MC) and at least one additional spacer unit. In some embodiments, the linker or Mal spacer unit comprises MC-(PEG) ₂ . In some embodiments, the linker or Mal spacer unit comprises MC-(PEG) ₂ and at least one additional spacer unit. In some embodiments, the linker or Mal spacer unit comprises Mal-Hex. In some embodiments, the linker or Mal spacer unit comprises Mal-Hex and at least one additional spacer unit. In some embodiments, the linker or Mal spacer unit comprises Mal-Et. In some embodiments, the linker or Mal spacer unit comprises Mal-Et and at least one additional spacer unit. In some embodiments, the linker or Mal spacer unit comprises Mal-Et-O-Et. In some embodiments, the linker or Mal spacer unit comprises Mal-Et-O-Et and at least one additional spacer unit. In some embodiments, the Mal spacer unit attaches an antibody or antigen binding fragment to a herboxydiene splicing modulator.

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

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

[34] In some embodiments, a pool of ADCs in which random conjugation occurs is presented, and the average p value in the pool is from about 2 to about 8. In some embodiments, a pool of ADCs in which random conjugation occurs is presented, and the average p value in pool is from about 4 to about 8. In some embodiments, the ADC pool in which random conjugation occurs is presented, and the average p value in the pool is approximately 4. In some embodiments, the ADC pool in which random conjugation occurs is presented, and the average value p in the pool is approximately 8. Provided herein are compositions (e.g., pharmaceutical compositions) containing multiple copies of any of the described ADCs, wherein the average drug loading (average p value) of the ADC in the composition is from about 3.5 to about 5. 5 (eg, about 4) or from about 7 to about 9 (eg, about 8).

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

[36] In various embodiments, the antibody or antigen binding fragment (Ab) from the ADC is an anti-HER2 antibody or antigen binding fragment thereof. In some embodiments, the antibody or antigen binding fragment binds to HER2 and targets neoplastic cells expressing HER2 (ie, the ADC targets neoplastic cells expressing HER2). In some embodiments, the antibody or antigen binding fragment from the ADC is an internalizing anti-HER2 antibody or internalizing antigen binding fragment thereof.

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

[38] In various embodiments, the antibody or antigen binding fragment (Ab) from the ADC is an anti-CD138 antibody or antigen binding fragment thereof. In some embodiments, the antibody or antigen binding fragment binds to CD138 and targets neoplastic cells expressing CD138 (ie, the ADC targets neoplastic cells expressing CD138). In some embodiments, the antibody or antigen binding fragment from the ADC is an internalizing anti-CD138 antibody or internalizing antigen binding fragment thereof.

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

[40] In various embodiments, the antibody or antigen binding fragment (Ab) from the ADC is an anti-EPHA2 antibody or antigen binding fragment thereof. In some embodiments, the antibody or antigen binding fragment binds to EPHA2 and targets neoplastic cells expressing EPHA2 (ie, the ADC targets neoplastic cells expressing EPHA2). In some embodiments, the antibody or antigen binding fragment from the ADC is an internalizing anti-EPHA2 antibody or internalizing antigen binding fragment thereof.

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

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

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

each of R ¹ , R ² and R ³ is independently selected from hydrogen, hydroxyl, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups, -C groups (=O)-O-(C ₁ -C ₆ alkyl) and C ₁ -C ₆ alkyl groups;

R ⁴ is selected from hydrogen, C ₁ -C ₆ alkyl groups, -C(=O)-(C ₁ -C ₆ alkyl) groups, -C(=O)-(C ₃ -C ₈ carbocyclyl) groups, - C(=O)-(C ₃ -C ₈ heterocyclyl) and -C(=O)-NR ⁶ R ⁷ ;

R ⁵ is selected from hydrogen, hydroxyl, -CH ₂ -OH, -CO ₂ H, -C(=O)-O-(C ₁ -C ₆ alkyl), -C(=O)-NR ⁶ R ⁷ groups, -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 ;

R ⁶ and R ⁷ are each independently selected from hydrogen, -R ⁸ , -C(=O)-R ⁸ and -C(=O)-OR ⁸ ; And

R ⁸ is selected from C ₁ -C ₆ alkyl groups, C ₃ -C ₈ carbocyclyl groups and C ₃ -C ₈ heterocyclyl groups,

where each of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ is independently substituted with 0-3 groups independently selected from halogens, hydroxyl, C ₁ -C ₆ alkyl groups, groups - O-(C ₁ -C ₆ alkyl), -CO ₂ H, groups -C(=O)-(C ₁ -C ₆ alkyl), groups -C(=O)-(C ₃ -C ₈ carbocyclyl), groups -C(=O)-(C ₃ -C ₈ heterocyclyl), -NR ⁶ R ⁷ , C ₃ -C ₈ carbocyclyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkyl alkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups, each of which may be independently substituted with 0 or 1 group selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups, C ₁ -C ₃ haloalkyl groups, -NH- C(=O)(C ₁ -C ₃ alkyl) and -NH-C(=O)-O-(C ₁ -C ₃ alkyl).

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

R ⁹ is selected from C ₃ -C ₈ heterocyclyl groups; And

R ¹⁰ is selected from H and C ₁ -C ₆ alkyl groups,

where each of R ⁹ and R ¹⁰ is independently substituted with 0-3 groups independently selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups, -NH ₂ , -NH-(C ₁ -C ₃ alkyl) and -N-(C ₁ -C ₃ alkyl) ₂ .

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

R ¹¹ selected from , And , where * denotes the point of attachment of R ¹¹ to the rest of the connection; And

R ¹² and R ¹³ are each independently selected from H and methyl.

[45] In some embodiments, compounds of formula (II) are provided herein: (II), or a pharmaceutically acceptable salt thereof, where

X represents NR ⁶ R ⁷ ;

R ⁶ and R ⁷ are each independently selected from hydrogen, -R ⁸ , -C(=O)-R ⁸ , -C(=O)-OR ⁸ , -(C ₁ -C ₆ alkyl)-OC(=O )-R ⁸ and -(C ₁ -C ₆ alkyl)-NH-C(=O)-R ⁸ ; And

R ⁸ is selected from C ₁ -C ₆ alkyl groups, C ₃ -C ₈ carbocyclyl groups and C ₃ -C ₈ heterocyclyl groups,

wherein each of R ⁶ , R ⁷ and R ⁸ is independently substituted with 0-3 groups independently selected from halogens, hydroxyl, C ₁ -C ₆ alkyl groups, -O-(C ₁ -C ₆ alkyl), -CO ₂ H groups , groups -C(=O)-(C ₁ -C ₆ alkyl), groups -C(=O)-(C ₃ -C ₈ carbocyclyl), groups -C(=O)-(C ₃ -C ₈ heterocyclyl ), -NR ⁶ R ⁷ , C ₃ -C ₈ carbocyclyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkylalkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups, each of which can be independently substituted with 0 or 1 group selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups, C ₁ -C ₃ haloalkyl groups, -NH-C(=O)(C ₁ -C ₃ alkyl) and - NH-C(=O)-O-(C ₁ -C ₃ alkyl).

[46] In some embodiments, compounds of formula (IIa) are provided herein: (IIa), or a pharmaceutically acceptable salt thereof, where

Z is selected from NR ⁹ and O;

R ⁹ is selected from hydrogen and C ₁ -C ₆ alkyl groups;

each of R ¹⁰ and R ¹¹ is independently selected from hydrogen, halogens, hydroxyl, C ₁ -C ₆ alkyl groups, -O-(C ₁ -C ₆ alkyl) groups, -CO ₂ H, -C(=O)- groups O-(C ₁ -C ₆ alkyl), -C(=O)-(C ₁ -C ₆ alkyl) groups, -C(=O)-(C ₃ -C ₈ carbocyclyl) groups, -C(= O)-(C ₃ -C ₈ heterocyclyl), C ₃ -C ₈ carbocyclyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkyl alkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups;

R ¹² is selected from C ₁ -C ₆ alkyl groups, C ₃ -C ₈ carbocyclyl groups, C ₃ -C ₈ heterocyclyl groups,

wherein each of R ⁹ , R ¹⁰ , R ¹¹ and R ¹² is independently substituted with 0 or 1 group selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups and C ₁ -C ₃ haloalkyl groups; And

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

[47] In some embodiments, compounds of formula (IIb) are provided herein: (IIb), or a pharmaceutically acceptable salt thereof, where

R ¹³ selected from , , , , , , , , , And , where * denotes the point of attachment of R ¹³ to the rest of the connection; And

R ¹⁴ and R ¹⁵ are each independently selected from hydrogen and methyl.

[48] In some embodiments, compounds of formula (III) are provided herein: (III), or a pharmaceutically acceptable salt thereof, where

each of R ¹ , R ² and R ³ is independently selected from hydrogen, hydroxyl, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups, -C groups (=O)-O-(C ₁ -C ₆ alkyl) and C ₁ -C ₆ alkyl groups;

R ⁶ and R ⁷ are each independently selected from hydrogen, -R ⁸ , -C(=O)-R ⁸ and -C(=O)-OR ⁸ ;

R ⁸ is selected from C ₁ -C ₆ alkyl groups, C ₃ -C ₈ carbocyclyl groups and C ₃ -C ₈ heterocyclyl groups; And

R ⁹ selected from H, , , , , , , , , And ;

wherein each of R ¹ , R ² , R ³ , R ⁶ , R ⁷ and R ⁸ is independently substituted with 0-3 groups independently selected from halogens, hydroxyl, C ₁ -C ₆ alkyl groups, -O-(C ₁ - C ₆ alkyl), -CO ₂ H, -C(=O)-(C ₁ -C ₆ alkyl) groups, -C(=O)-(C ₃ -C ₈ carbocyclyl) groups, -C(=O) groups )-(C ₃ -C ₈ heterocyclyl), -NR ⁶ R ⁷ , C ₃ -C ₈ carbocyclyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkyl alkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups, each of which may be independently substituted with 0 or 1 group selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups, C ₁ -C ₃ haloalkyl groups, -NH-C(=O)( C ₁ -C ₃ alkyl) and -NH-C(=O)-O-(C ₁ -C ₃ alkyl); And

where * denotes the point of attachment of R ⁹ to the rest of the connection.

[49] In some embodiments, provided herein is a compound selected from:

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

,

and its pharmaceutically acceptable salts,

where L represents a linker that is covalently attached to the antibody.

[50] Also provided herein in various embodiments are routes for the therapeutic use of the disclosed ADC compounds, herboxidiene compounds and compositions, for example, for the treatment of a neoplastic disorder, such as cancer. In certain aspects, the present invention provides methods for treating a neoplastic disorder, such as cancer, that expresses an antigen that is targeted by an antibody or antigen binding fragment from an ADC, such as HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4 or STEAP1.

[51] In certain aspects of the present invention, methods are provided for treating a subject who has or is suspected of having a neoplastic disorder by administering to the subject a therapeutically effective amount of any of the disclosed ADCs or compositions thereof and/or administering an appropriate treatment regimen. In some embodiments, the neoplastic disorder is a hematologic malignancy or solid tumor. In some embodiments, the neoplastic disorder is a hematologic malignancy. In some embodiments, the hematologic malignancy is selected from B-cell malignancy, leukemia, lymphoma, and myeloma. In some embodiments, the hematologic malignancy is selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the neoplastic disorder is a solid tumor. In some embodiments, the solid tumor is selected from breast cancer (eg, HER2-positive breast cancer), gastric cancer (eg, gastric adenocarcinoma), prostate cancer, ovarian cancer, lung cancer (eg, lung adenocarcinoma), uterine cancer ( for example, serous endometrial carcinoma of the uterus), salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer and esophageal cancer. In some embodiments, the solid tumor is selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

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

[53] In some embodiments, the target antigen is HER2. In some embodiments, the one or more neoplastic cells are derived from breast cancer, ovarian cancer, gastric cancer, lung cancer (eg, lung adenocarcinoma), uterine cancer (eg, endometrial serous carcinoma of the uterus), osteosarcoma, or salivary duct carcinoma expressing HER2 . In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-HER2 antibody when administered alone and/or (b) a herboxydiene-based splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or unresponsive to treatment with a herboxidiene-based splicing modulator when administered alone.

[54] In some embodiments, the target antigen is CD138. In some embodiments, the one or more neoplastic cells are derived from multiple myeloma cells expressing CD138. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-CD138 antibody when administered alone and/or (b) a herboxydiene-based splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or unresponsive to treatment with a herboxidiene-based splicing modulator when administered alone.

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

[56] In some embodiments, the target antigen is MSLN. In some embodiments, the one or more neoplastic cells are derived from ovarian cancer, cervical cancer, pancreatic cancer, or lung cancer (eg, lung adenocarcinoma) cells expressing MSLN. In some embodiments, the subject is unresponsive or underresponsive to treatment with (a) an anti-MSLN antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or unresponsive to treatment with a splicing modulator when administered alone.

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

[58] In some embodiments, the target antigen is CDH6. In some embodiments, the one or more neoplastic cells are derived from kidney cancer cells expressing CDH6. In some embodiments, the subject is unresponsive or underresponsive to treatment with (a) an anti-CDH6 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or unresponsive to treatment with a splicing modulator when administered alone.

[59] In some embodiments, the target antigen is CEACAM5. In some embodiments, the one or more neoplastic cells are derived from colorectal cancer cells expressing CEACAM5. In some embodiments, the subject is unresponsive or underresponsive to treatment with (a) an anti-CEACAM5 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or unresponsive to treatment with a splicing modulator when administered alone.

[60] In some embodiments, the target antigen is CFC1B. In some embodiments, the one or more neoplastic cells are derived from pancreatic cancer cells expressing CFC1B. In some embodiments, the subject is unresponsive or underresponsive to treatment with (a) an anti-CFC1B antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or unresponsive to treatment with a splicing modulator when administered alone.

[61] In some embodiments, the target antigen is ENPP3. In some embodiments, the one or more neoplastic cells are derived from kidney cancer cells expressing ENPP3. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-ENPP3 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or unresponsive to treatment with a splicing modulator when administered alone.

[62] In some embodiments, the target antigen is FOLR1. In some embodiments, the one or more neoplastic cells are derived from ovarian cancer cells expressing FOLR1. In some embodiments, the subject is unresponsive or underresponsive to treatment with (a) an anti-FOLR1 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or unresponsive to treatment with a splicing modulator when administered alone.

[63] In some embodiments, the target antigen is HAVCR1. In some embodiments, the one or more neoplastic cells are derived from renal cancer or esophageal cancer cells expressing HAVCR1. In some embodiments, the subject is unresponsive or underresponsive to treatment with (a) an anti-HAVCR1 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or unresponsive to treatment with a splicing modulator when administered alone.

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

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

[66] In some embodiments, the target antigen is MUC16. In some embodiments, the one or more neoplastic cells are derived from ovarian cancer, cervical cancer, or breast cancer cells expressing MUC16. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-MUC16 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or unresponsive to treatment with a splicing modulator when administered alone.

[67] In some embodiments, the target antigen is SLC39A6. In some embodiments, the one or more neoplastic cells are derived from breast cancer or prostate cancer cells expressing SLC39A6. In some embodiments, the subject is unresponsive or underresponsive to treatment with (a) an anti-SLC39A6 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or unresponsive to treatment with a splicing modulator when administered alone.

[68] In some embodiments, the target antigen is SLC44A4. In some embodiments, the one or more neoplastic cells are derived from prostate cancer cells expressing SLC44A4. In some embodiments, the subject is unresponsive or insufficiently responsive to treatment with (a) an anti-SLC44A4 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or unresponsive to treatment with the splicing modulator when administered alone.

[69] In some embodiments, the target antigen is STEAP1. In some embodiments, the one or more neoplastic cells are derived from prostate cancer cells expressing STEAP1. In some embodiments, the subject is unresponsive or underresponsive to treatment with (a) an anti-STEAP1 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, unresponsive, or unresponsive to treatment with a splicing modulator when administered alone.

[70] In other certain aspects of the present invention, methods are provided for reducing or inhibiting tumor growth in a subject who has or is suspected of having a neoplastic disorder by administering to the subject a therapeutically effective amount of any of the disclosed ADCs or compositions thereof and/or administering an appropriate treatment regimens.

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

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

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

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

[75] In some embodiments, the target antigen is MSLN. In some embodiments, the one or more neoplastic cells are derived from ovarian cancer, cervical cancer, pancreatic cancer, or lung cancer (eg, lung adenocarcinoma) cells expressing MSLN. 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 modulator when administered alone.

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

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

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

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

[80] In some embodiments, the target antigen is ENPP3. In some embodiments, the one or more neoplastic cells are derived from kidney cancer cells expressing ENPP3. In some embodiments, the tumor is resistant or refractory to treatment with (a) anti-ENPP3 antibodies when administered alone and/or (b) a splicing modulator when administered alone.

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

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

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

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

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

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

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

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

[89] In still other aspects of the present invention, methods are provided for determining whether a subject who has or is suspected of having a neoplastic disorder will be receptive to treatment with any of the described ADCs or compositions by obtaining a biological sample from the subject and bringing the biological sample into contact with the ADC or composition. In some embodiments, the biological sample is a tumor sample. In some embodiments, the tumor sample is a tumor biopsy or a blood sample. In some embodiments, the blood sample is selected from blood, a blood fraction, or a cell derived from blood or a blood fraction. In some embodiments, the subject has one or more neoplastic cells that express the target antigen. In some embodiments, the target antigen is HER2. In some embodiments, the one or more neoplastic cells are derived from breast cancer, ovarian cancer, gastric cancer, lung cancer (eg, lung adenocarcinoma), uterine cancer (eg, endometrial serous carcinoma of the uterus), osteosarcoma, or salivary duct carcinoma expressing HER2 . In some embodiments, the target antigen is CD138. In some embodiments, the one or more neoplastic cells are derived from multiple myeloma cells expressing CD138. In some embodiments, the target antigen is EPHA2. In some embodiments, the one or more neoplastic cells are derived from breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer cells expressing EPHA2. In some embodiments, the target antigen is MSLN. In some embodiments, the one or more neoplastic cells are derived from ovarian cancer, cervical cancer, pancreatic cancer, or lung cancer (eg, lung adenocarcinoma) cells expressing MSLN. In some embodiments, the target antigen is FOLH1. In some embodiments, the one or more neoplastic cells are derived from prostate cancer cells expressing FOLH1. In some embodiments, the target antigen is CDH6. In some embodiments, the one or more neoplastic cells are derived from kidney cancer cells expressing CDH6. In some embodiments, the target antigen is CEACAM5. In some embodiments, the one or more neoplastic cells are derived from colorectal cancer cells expressing CEACAM5. In some embodiments, the target antigen is CFC1B. In some embodiments, the one or more neoplastic cells are derived from pancreatic cancer cells expressing CFC1B. In some embodiments, the target antigen is ENPP3. In some embodiments, the one or more neoplastic cells are derived from kidney cancer cells expressing ENPP3. In some embodiments, the target antigen is FOLR1. In some embodiments, the one or more neoplastic cells are derived from ovarian cancer cells expressing FOLR1. In some embodiments, the target antigen is HAVCR1. In some embodiments, the one or more neoplastic cells are derived from renal cancer or esophageal cancer cells expressing HAVCR1. In some embodiments, the target antigen is KIT. In some embodiments, the one or more neoplastic cells are derived from kidney cancer cells expressing KIT. In some embodiments, the target antigen is MET. In some embodiments, the one or more neoplastic cells are derived from renal cancer or esophageal cancer cells expressing MET. In some embodiments, the target antigen is MUC16. In some embodiments, the one or more neoplastic cells are derived from ovarian cancer, cervical cancer, or breast cancer cells expressing MUC16. In some embodiments, the target antigen is SLC39A6. In some embodiments, the one or more neoplastic cells are derived from breast cancer or prostate cancer cells expressing SLC39A6. In some embodiments, the target antigen is SLC44A4. In some embodiments, the one or more neoplastic cells are derived from prostate cancer cells expressing SLC44A4. In some embodiments, the target antigen is STEAP1. In some embodiments, the one or more neoplastic cells are derived from prostate cancer cells expressing STEAP1.

[90] In various embodiments, further provided herein are pharmaceutical compositions comprising an ADC and a pharmaceutically acceptable diluent, carrier and/or excipient. Methods for preparing the described ADC compounds and compositions based thereon are also disclosed.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[91] In FIG. 1A-1D show the dose-dependent effects of exemplary payload compounds on the viability of HER2-amplified breast cancer cells (HCC1954) and gastric cancer cells (NCI-N87). Cells were incubated with the compound for 72 hours (3 days) or 144 hours (6 days) and viability was determined using CellTiter-Glo® 2.0 reagent. In fig. Figure 1A shows the dose-dependent effect on the viability of HCC1954 cells after 72 hours of incubation. In fig. 1B shows the dose-dependent effect on the viability of NCI-N87 cells after 72 hours of incubation. In fig. Figure 1C shows the dose-dependent effect on the viability of HCC1954 cells after 144 hours of incubation. In fig. 1D shows the dose-dependent effect on the viability of NCI-N87 cells after 144 hours of incubation. Data are presented as mean ± SD.

[92] In FIG. 2A and FIG. Figure 2B shows the results of splicing analysis of SLC25A19 in breast cancer cells (HCC1954) (Figure 2A) and gastric cancer cells (NCI-N87) with HER2 amplification (Figure 2B). Cells were incubated with compounds for 6 hours and splicing of the SLC25A19 transcript was measured by real-time qPCR reaction using a set of specific Taqman primer probes. The y-axis represents the percentage (%) response compared to DMSO control (0.1%). Data are presented as mean ± SD.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

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

[94] Throughout this text, descriptions refer to the compositions and methods of using the compositions. If the present invention describes or claims a feature or embodiment associated with a composition, such feature or embodiment is equally applicable to methods of using the composition. Likewise, if the present invention describes or claims a feature or embodiment associated with a method of using a composition, such feature or embodiment is equally applicable to the composition.

[95] If a range of values is presented, it includes embodiments using any particular value within the range. In addition, reference to values set forth in a range includes any and all values within that range. All ranges include their own limit values and are combinable. If values are expressed as approximate values using the preceding word "about", it will be understood that the particular value constitutes another embodiment. A reference to a specific numerical value includes at least that specific value unless the context clearly requires otherwise. The use of “or” will mean “and/or” unless the specific context of its application requires otherwise.

[96] It should be understood that certain features of the disclosed compositions and methods, which for clarity are described herein in the context of individual embodiments, may also be presented in combination in a single embodiment. Conversely, various features of the disclosed compositions and methods, which for brevity are described in the context of a single embodiment, may also be presented individually or in any subcombination.

[97] All references cited herein are incorporated by reference for all purposes. If the link and description conflict, the description will control.

Definitions

[98] Throughout this application and claims, various terms related to aspects of the present description are used. Unless otherwise noted, such terms are given their ordinary meaning in the art. Other specifically defined terms should be construed as defined herein.

[99] When used herein, the singular forms include the plural forms unless the context clearly requires otherwise.

[100] The terms "about" or "about" in the context of numerical values and ranges mean values or ranges that are approximately equal to or close to the described values or ranges so that the embodiment can be carried out as intended, such as the required the amount of nucleic acids or polypeptides in the reaction mixture as is apparent to one skilled in the art based on the teachings contained herein. In some embodiments, "about" means plus or minus 10% of the numerical amount.

[101] The terms “antibody-drug conjugate,” “antibody-based conjugate,” “conjugate,” “immunoconjugate,” and “ADC” are used interchangeably and mean one or more therapeutic compounds (eg, herboxydiene-based splicing modulator) that are linked with one or more antibodies or antigen-binding fragments and are defined by the general formula: Ab-(LH) _p (Formula I), where Ab = antibody or antigen-binding fragment, L = linker fragment, H = herboxidiene-based splicing modulator (e.g., herboxidiene or its derivative), and p = number of drug fragments per antibody or antigen-binding fragment. An ADC containing a herboxidiene splicing modulator may also be more specifically referred to herein as a “herboxidiene splicing modulator loaded antibody” or “SMLA.” In ADCs containing a herboxydiene splicing modulator, " p " indicates the number of herboxydiene splicing modulators bound to the antibody or antigen binding moiety. In some embodiments, linker L may include a cleavable moiety between the antibody or antigen binding moiety and the herboxydiene splicing modulator. In some embodiments, linker L may include a cleavable moiety that can be attached to either an antibody or antigen binding moiety or a herboxydiene splicing modulator or both via spacer unit(s). In some embodiments, if the spacer unit attaches a cleavable moiety to a herboxydiene-based splicing modulator, it is a self-cleavable spacer unit. In other embodiments, linker L does not include a cleavable moiety, but is a non-cleavable linker. In some embodiments, linker L may include at least one spacer unit that can be directly attached to the antibody or antigen binding moiety and to the herboxydiene splicing modulator. Exemplary cleavable and non-cleavable linkers are described herein and examples thereof are provided.

[102] The term "antibody" is used in the broadest sense to refer to an immunoglobulin molecule that recognizes a target, such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or combinations thereof, and specifically binds thereto through at least one antigen recognition site in within the variable region of the immunoglobulin molecule. The heavy chain of an antibody consists of a heavy chain variable domain ( _VH ) and a heavy chain constant region ( _CH ). The light chain consists of a light chain variable domain ( _VL ) and a light chain constant domain ( _CL ). For the purposes of this application, each mature heavy chain and light chain variable domain contains three complementarity determining regions (CDR1, CDR2 and CDR3) within four framework regions (FR1, FR2, FR3 and FR4) located from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The "antibody" may be naturally occurring or man-made, such as monoclonal antibodies produced using traditional hybridoma technology. The term "antibody" includes full-length monoclonal antibodies and full-length polyclonal antibodies, as well as antibody fragments such as Fab, Fab', F(ab') ₂ , Fv and single chain antibodies. The antibody may belong to any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, or to subclasses thereof (eg isotypes IgG1, IgG2, IgG3, IgG4). The term further covers human antibodies, chimeric antibodies, humanized antibodies, and any modified immunoglobulin molecule containing an antigen recognition site, provided that it exhibits the desired biological activity (eg, binds the target antigen, is internalized into a cell expressing the target antigen).

[103] As used herein, the term “monoclonal antibody” means an antibody derived from a population of substantially homogeneous antibodies, i.e. The individual antibodies that make up the population are identical, except for possible naturally occurring mutations that may be present in minute quantities. Monoclonal antibodies are highly specific, directed against a single antigenic epitope. In contrast, traditional (polyclonal) antibody preparations typically include a wide variety of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the characteristic of the antibody as being derived from a substantially uniform population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be obtained through the hybridoma method first described in Kohler et al. (1975) Nature 256:495, or can be obtained by recombinant DNA methods (see, for example, US patent No. 4816567). Monoclonal antibodies can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al. (1991) Nature 352:624-8, and Marks et al. (1991) J Mol Biol. 222:581-97.

[104] Monoclonal antibodies described herein particularly include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain(s) is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, provided that they specifically bind the target antigen and/or exhibit the required biological activity.

[105] As used herein, the term “human antibody” means an antibody produced by a human, or an antibody having the amino acid sequence of an antibody produced by a human.

[106] As used herein, the term “chimeric antibody” means antibodies in which the amino acid sequence of an 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 antibodies obtained from one species with the desired specificity, affinity and activity, while the constant regions are homologous to antibodies obtained from another species (for example, human) to minimize the immune response in the latter species.

[107] As used herein, the term “humanized antibody” refers to antibody forms that contain sequences from non-human (eg, mouse) antibodies as well as human antibodies. Such antibodies are chimeric antibodies that contain a minimal sequence derived from a non-human immunoglobulin. In general, a humanized antibody will contain substantially all of at least one and typically two variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions ( FR) are framework regions from the human immunoglobulin sequence. The humanized antibody will also optionally contain at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin constant region. The humanized antibody may be further modified by replacing residues in the Fv framework region and/or within the replaced non-human residues to improve and optimize the specificity, affinity and/or activity of the antibody.

[108] As used herein, the term "antigen binding fragment" or "antigen binding moiety" of an antibody means one or more antibody or protein fragments that retain the ability to specifically bind an antigen (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5 , CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4 or STEAP1). Antigen-binding fragments may also retain the ability to be internalized into a cell expressing the antigen. In some embodiments, the antigen binding fragments also retain immune effector activity. It has been shown that full-length antibody fragments are capable of performing the antigen-binding function of a full-length antibody. Examples of binding fragments encompassed by the term "antigen binding fragment" or "antigen binding portion" of an antibody include: (i) Fab fragment, a monovalent fragment consisting of V_L-, V_H-, C_L- and C_H1-domains; (ii) F(ab')₂-fragment, a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of V_H- and C_H1-domains; (iv) Fv fragment consisting of V_L- and V_H-domains of one antibody arm; (v) dAb fragment which contains one variable domain, for example V_H-domain (see, for example, Ward et al. (1989) Nature 341:544-6; and International Application Publication No. WO 1990/005144); and (vi) a dedicated complementarity determining region (CDR). In addition, although the two domains of the Fv fragment, V_L and V_H, are encoded by separate genes, they can be connected using recombinant methods using a synthetic linker, which allows them to be obtained in the form of a single protein chain in which V_L- and V_H-regions pair to form monovalent molecules (known as 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 by the term “antigen binding fragment” or “antigen binding moiety” of an antibody, and are known in the art as an exemplary type of binding fragment that can be internalized into cells upon binding (see, for example, Zhu et al. (2010) 9:2131-41; He et al. (2010) J Nucl Med 51:427-32; and Fitting et al. In certain embodiments, scFv molecules may be included in the fusion protein. Other forms of single chain antibodies, such as diabodies, are also covered. Diabodies are bivalent bispecific antibodies in which V_H- and V_L-domains are expressed as a single polypeptide chain, but using a linker that is too short to allow pairing between two domains on the same chain, thereby causing the domains to pair with complementary domains of the other chain and create two antigen-binding sites (see, for example, Holliger et al. (1993) Proc Natl Acad Sci. USA 90:6444-8; and Poljak et al. (1994) Structure 2:1121-3). Antigen binding fragments are prepared using conventional techniques known to those skilled in the art, and the binding fragments are screened for utility (eg, binding affinity, internalization) in the same manner as intact antibodies. Antigen-binding fragments can be obtained by cleavage of the intact protein, for example, using a protease or chemical cleavage.

[109] “Internalizable,” as used herein in reference to an antibody or antigen-binding fragment, means an antibody or antigen-binding fragment that can be taken up across the lipid bilayer membrane of a cell into an internal compartment (i.e., “be internalized”) upon binding to the cell , preferably into the cell compartment in which decomposition occurs. For example, an internalizing anti-HER2 antibody is an antibody that can be taken up into a cell after binding to HER2 on the cell membrane. In some embodiments, the antibody or antigen binding fragment used in the ADCs disclosed herein specifically targets a cell surface antigen (e.g., HER2) and is an internalized antibody or internalized antigen binding fragment (i.e., the ADC is transported across the cell membrane upon binding antigen). In some embodiments, the internalized antibody or antigen-binding fragment binds a receptor on a cell surface. An internalized antibody or internalized antigen-binding fragment that specifically targets a receptor on the cell membrane is capable of inducing receptor-mediated endocytosis. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment is taken up into the cell via receptor-mediated endocytosis.

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

[111] As used herein, the term "human epidermal growth factor receptor 2", "HER2" or "HER2/NEU", means any native form of human HER2. The term covers the full-length HER2 sequence (eg, UniProt reference sequence: P04626; SEQ ID NO: 31), as well as any form of human HER2 that may arise from cellular processing. The term also covers functional variants or fragments of human HER2, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more biological functions of human HER2 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to wild-type protein only). HER2 can be isolated from human cells or can be produced recombinantly or using synthetic methods.

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

[113] As used herein, the term "syndecan-1", "SDC1" or "CD138" means any native form of human CD138. The term includes the full-length CD138 sequence (eg, UniProt reference sequence: P18827; SEQ ID NO: 32), as well as any form of human CD138 that may arise from cellular processing. The term also covers functional variants or fragments of human CD138, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more biological functions of human CD138 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to wild-type protein only). CD138 can be isolated from human cells or can be produced recombinantly or using synthetic methods.

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

[115] As used herein, the term “ephrin receptor 2 type-A” or “EPHA2” means any native form of human EPHA2. The term covers the full-length EPHA2 sequence (eg, UniProt reference sequence: P29317; SEQ ID NO: 33), as well as any form of human EPHA2 that may arise from cellular processing. The term also covers functional variants or fragments of human EPHA2, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more biological functions of human EPHA2 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to wild-type protein only). EPHA2 can be isolated from human cells or can be produced recombinantly or using synthetic methods.

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

[117] As used herein, the term “mesothelin” or “MSLN” means any native form of human MSLN. The term covers the full-length MSLN sequence (eg, UniProt reference sequence: Q13421; SEQ ID NO: 94), as well as any form of human MSLN that may arise from cellular processing. The term also covers functional variants or fragments of human MSLN, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more biological functions of human MSLN (i.e., variants and fragments are covered unless the context indicates that the term used to refer to wild-type protein only). MSLN can be isolated from human cells or can be produced recombinantly or using synthetic methods.

[118] The term “anti-MSLN antibody” or “antibody that binds to MSLN” means any form of antibody or fragment thereof that binds, for example specifically binds, to MSLN, and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, for example specifically bind, to MSLN. WO 2011/074621, incorporated herein by reference, provides exemplary MSLN binding sequences, including exemplary anti-MSLN antibody sequences. In some embodiments, the anti-MSLN antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment. 11-25, IC14-30, IC7-4, IC17-35 and 2-9 are exemplary antibodies to human MSLN.

[119] As used herein, the term “glutamate carboxypeptidase 2” or “FOLH1” means any native form of human FOLH1. The term covers the full-length FOLH1 sequence (eg, UniProt reference sequence: Q04609; SEQ ID NO: 95), as well as any form of human FOLH1 that may arise from cellular processing. The term also covers functional variants or fragments of human FOLH1, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more biological functions of human FOLH1 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to wild-type protein only). FOLH1 can be isolated from human cells or can be produced recombinantly or using synthetic methods.

[120] The term “anti-FOLH1 antibody” or “antibody that binds to FOLH1” means any form of antibody or fragment thereof that binds, e.g., specifically binds, FOLH1, and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, for example specifically bind, to FOLH1. WO 2019/012260 and WO 2017/212250, incorporated herein by reference, provide exemplary FOLH1-binding sequences, including exemplary anti-FOLH1 antibody sequences. In some embodiments, the anti-FOLH1 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment. J591 (deimmunized) is an exemplary antibody to human FOLH1.

[121] As used herein, the term “cadherin-6” or “CDH6” means any native form of human CDH6. The term covers the full-length CDH6 sequence (eg, UniProt reference sequence: P55285; SEQ ID NO: 96), as well as any form of human CDH6 that may arise from cellular processing. The term also covers functional variants or fragments of human CDH6, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more biological functions of human CDH6 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to wild-type protein only). CDH6 can be isolated from human cells or can be produced recombinantly or using synthetic methods.

[122] The term “anti-CDH6 antibody” or “antibody that binds to CDH6” means any form of antibody or fragment thereof that binds, e.g., specifically binds, CDH6, and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, for example specifically bind, to CDH6. WO 2018/185618, incorporated herein by reference, provides exemplary CDH6 binding sequences, including exemplary anti-CDH6 antibody sequences. In some embodiments, the anti-CDH6 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment.

[123] As used herein, the term "carcinoembryonic antigen-related cell adhesion molecule 5" or "CEACAM5" means any native form of human CEACAM5. The term covers the full-length CEACAM5 sequence (eg, UniProt reference sequence: P06731; SEQ ID NO: 97), as well as any form of human CEACAM5 that may arise from cellular processing. The term also covers functional variants or fragments of human CEACAM5, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more biological functions of human CEACAM5 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to wild-type protein only). CEACAM5 can be isolated from human cells or can be produced recombinantly or using synthetic methods.

[124] The term “anti-CEACAM5 antibody” or “antibody that binds to CEACAM5” means any form of antibody or fragment thereof that binds, for example specifically binds, to CEACAM5, and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, for example specifically bind, to CEACAM5. US 2015/0125386, incorporated herein by reference, provides exemplary CEACAM5 binding sequences, including exemplary anti-CEACAM5 antibody sequences. In some embodiments, the anti-CEACAM5 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment. hMN14 is an exemplary antibody to human CEACAM5.

[125] As used herein, the term “cryptic protein family protein 1B” or “CFC1B” means any native form of human CFC1B. The term covers the full-length CFC1B sequence (eg, UniProt reference sequence: P0CG36; SEQ ID NO: 98), as well as any form of human CFC1B that may arise from cellular processing. The term also covers functional variants or fragments of human CFC1B, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more biological functions of human CFC1B (i.e., variants and fragments are covered unless the context indicates that the term used to refer to wild-type protein only). CFC1B can be isolated from human cells or can be produced recombinantly or using synthetic methods.

[126] The term “anti-CFC1B antibody” or “antibody that binds to CFC1B” means any form of antibody or fragment thereof that binds, e.g., specifically binds, CFC1B, and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, for example specifically bind, to CFC1B. WO 2002/088170, incorporated herein by reference, provides exemplary CFC1B binding sequences, including exemplary anti-CFC1B antibody sequences. In some embodiments, the anti-CFC1B antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment.

[127] As used herein, the term “ectonucleotide pyrophosphatase/phosphodiesterase family member 3” or “ENPP3” means any native form of human ENPP3. The term covers the full-length ENPP3 sequence (eg, UniProt reference sequence: O14638; SEQ ID NO: 99), as well as any form of human ENPP3 that may arise from cellular processing. The term also covers functional variants or fragments of human ENPP3, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more biological functions of human ENPP3 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to wild-type protein only). ENPP3 can be isolated from human cells or can be produced recombinantly or using synthetic methods.

[128] The term “anti-ENPP3 antibody” or “antibody that binds to ENPP3” means any form of antibody or fragment thereof that binds, for example specifically binds, to ENPP3, and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, for example specifically bind, to ENPP3. In Donate et al. ((2016) Clin Cancer Res. 22(8):1989-99), incorporated herein by reference, provides exemplary ENPP3 binding sequences, including exemplary anti-ENPP3 antibody sequences. In some embodiments, the anti-ENPP3 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment.

[129] As used herein, the term “folate receptor alpha” or “FOLR1” means any native form of human FOLR1. The term covers the full-length FOLR1 sequence (eg, UniProt reference sequence: P15328; SEQ ID NO: 100), as well as any form of human FOLR1 that may arise from cellular processing. The term also covers functional variants or fragments of human FOLR1, including, but not limited to, splice variants, allelic variants, and isoforms that retain one or more biological functions of human FOLR1 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to wild-type protein only). FOLR1 can be isolated from human cells or can be produced recombinantly or using synthetic methods.

[130] The term “anti-FOLR1 antibody” or “antibody that binds to FOLR1” means any form of antibody or fragment thereof that binds, for example specifically binds, to FOLR1, and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, for example specifically bind, to FOLR1. In WO 2005/080431 and Coney et al. ((1991) Cancer Res. 51(22):6125-32), incorporated herein by reference, provides exemplary FOLR1-binding sequences, including exemplary anti-FOLR1 antibody sequences. In some embodiments, the anti-FOLR1 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment. Farletuzumab and MOv19 are exemplary antibodies to human FOLR1.

[131] As used herein, the term “hepatitis A virus cellular receptor 1” or “HAVCR1” means any native form of human HAVCR1. The term covers the full-length HAVCR1 sequence (eg, UniProt reference sequence: Q96D42; SEQ ID NO: 101), as well as any form of human HAVCR1 that may arise from cellular processing. The term also covers functional variants or fragments of human HAVCR1, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more biological functions of human HAVCR1 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to wild-type protein only). HAVCR1 can be isolated from human cells or can be produced recombinantly or using synthetic methods.

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

[133] As used herein, the term “mast/stem cell growth factor receptor Kit” or “KIT” refers to any native form of human KIT. The term covers the full-length KIT sequence (eg, UniProt reference sequence: P10721; SEQ ID NO: 102), as well as any form of human KIT that may arise from cellular processing. The term also covers functional variants or fragments of human KIT, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more biological functions of human KIT (i.e., variants and fragments are covered unless the context indicates that the term used to refer to wild-type protein only). KIT can be isolated from human cells or can be produced recombinantly or using synthetic methods.

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

[135] As used herein, the term “hepatocyte growth factor receptor” or “MET” refers to any native form of human MET. The term covers the full-length MET sequence (eg, UniProt reference sequence: P08581; SEQ ID NO: 103), as well as any form of human MET that may arise from cellular processing. The term also covers functional variants or fragments of human MET, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more biological functions of human MET (i.e., variants and fragments are covered unless the context indicates that the term used to refer to wild-type protein only). MET can be isolated from human cells or can be produced recombinantly or by synthetic methods.

[136] The term "anti-MET antibody" or "antibody that binds to MET" means any form of antibody or fragment thereof that binds, for example specifically binds, to MET, and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, for example specifically bind, to MET. In Yang et al. ((2019) Acta Pharmacol Sin.), incorporated herein by reference, provides exemplary MET binding sequences, including exemplary anti-MET antibody sequences. In some embodiments, the anti-MET antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment.

[137] As used herein, the term “mucin-16” or “MUC16” means any native form of human MUC16. The term covers the full-length MUC16 sequence (eg, UniProt reference sequence: Q8WXI7; SEQ ID NO: 104), as well as any form of human MUC16 that may arise from cellular processing. The term also covers functional variants or fragments of human MUC16, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more biological functions of human MUC16 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to wild-type protein only). MUC16 can be isolated from human cells or can be produced recombinantly or using synthetic methods.

[138] The term “anti-MUC16 antibody” or “antibody that binds to MUC16” means any form of antibody or fragment thereof that binds, for example specifically binds, to MUC16, and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, for example specifically bind, to MUC16. In Liu et al. ((2016) Ann Oncol. 27(11):2124-30), incorporated herein by reference, provides exemplary MUC16-binding sequences, including exemplary anti-MUC16 antibody sequences. In some embodiments, the anti-MUC16 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment.

[139] As used herein, the term “zinc transporter ZIP6” or “SLC39A6” means any native form of human SLC39A6. The term covers the full-length sequence of SLC39A6 (eg, the reference sequence in UniProt:Q13433; SEQ ID NO: 105), as well as any form of human SLC39A6 that may arise from cellular processing. The term also covers functional variants or fragments of human SLC39A6, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more biological functions of human SLC39A6 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to wild-type protein only). SLC39A6 can be isolated from human cells or can be produced recombinantly or using synthetic methods.

[140] The term “anti-SLC39A6 antibody” or “antibody that binds to SLC39A6” means any form of antibody or fragment thereof that binds, e.g., specifically binds, SLC39A6, and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, for example specifically bind, to SLC39A6. In Sussman et al. ((2014) Mol Cancer Ther. 13(12):2991-3000), incorporated herein by reference, provides exemplary SLC39A6-binding sequences, including exemplary anti-SLC39A6 antibody sequences. In some embodiments, the anti-SLC39A6 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment.

[141] As used herein, the term “choline transporter-like protein 4” or “SLC44A4” means any native form of human SLC44A4. The term covers the full-length sequence of SLC44A4 (eg, UniProt reference sequence: Q53GD3; SEQ ID NO: 106), as well as any form of human SLC44A4 that may arise from cellular processing. The term also covers functional variants or fragments of human SLC44A4, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more biological functions of human SLC44A4 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to wild-type protein only). SLC44A4 can be isolated from human cells or can be produced recombinantly or using synthetic methods.

[142] The term “anti-SLC44A4 antibody” or “antibody that binds to SLC44A4” means any form of antibody or fragment thereof that binds, e.g., specifically binds, SLC44A4, and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, for example specifically bind, to SLC44A4. In Mattie et al. ((2016) Mol Cancer Ther. 15(11):2679-87), incorporated herein by reference, provides exemplary SLC44A4-binding sequences, including exemplary anti-SLC44A4 antibody sequences. In some embodiments, the anti-SLC44A4 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment.

[143] As used herein, the term “STEAP1 metal reductase” or “STEAP1” means any native form of human STEAP1. The term covers the full-length sequence of STEAP1 (eg, UniProt reference sequence: Q9UHE8; SEQ ID NO: 107), as well as any form of human STEAP1 that may arise from cellular processing. The term also covers functional variants or fragments of human STEAP1, including, without limitation, splice variants, allelic variants, and isoforms that retain one or more biological functions of human STEAP1 (i.e., variants and fragments are covered unless the context indicates that the term used to refer to wild-type protein only). STEAP1 can be isolated from human cells or can be produced recombinantly or using synthetic methods.

[144] The term “anti-STEAP1 antibody” or “antibody that binds to STEAP1” means any form of antibody or fragment thereof that binds, e.g., specifically binds, STEAP1, and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and biologically functional antibody fragments, provided that they bind, for example specifically bind, to STEAP1. WO 2008/052187, incorporated herein by reference, provides exemplary STEAP1 binding sequences, including exemplary anti-STEAP1 antibody sequences. In some embodiments, the anti-STEAP1 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment.

[145] As used herein, the term "specific""specificallybinds" or "binds in a specific manner" means a binding reaction between an antibody or antigen-binding fragment (eg, an anti-HER2 antibody) and a target antigen (eg, HER2) in a heterogeneous population of proteins and other biological molecules. Antibodies can be tested for binding specificity by comparing binding to a relevant antigen with binding to an irrelevant antigen or mixture of antigens under a specified set of conditions. If an antibody binds to a relevant antigen with an affinity that is at least 2, 5, 7, and preferably 10 or more times greater than the affinity for an irrelevant antigen or a mixture of antigens, it is said to be specific. A "specific antibody" or "target-specific antibody" is an antibody that binds only to a target antigen (eg, HER2) but does not bind (or exhibits minimal binding) to other antigens. In certain embodiments, an antibody or antigen-binding fragment that specifically binds a target antigen (e.g., HER2) has a K _D of less than 1 x 10 ⁻⁶ M, less than 1 x 10 ⁻⁷ M, less than 1 x 10 ⁻⁸ M, less than 1 x 10 ⁻⁹ M, less than 1 x 10 ⁻¹⁰ M, less than 1 x 10 ⁻¹¹ M, less than 1 x 10 ⁻¹² M, or less than 1 x 10 ⁻¹³ M. In some embodiments, K _D is from 1 pM up to 500 pM. In some embodiments, the K _D is from 500 pM to 1 μM, from 1 μM to 100 nM, or from 100 mM to 10 nM.

[146] The term "epitope" refers to the portion of an antigen that an antibody can recognize and specifically bind to. If the antigen is a polypeptide, epitopes can be formed from contiguous amino acids or non-contiguous amino acids brought into contact by tertiary folding of the polypeptide. The epitope bound by an antibody can be identified using any epitope mapping technique known in the art, including X-ray crystallography to identify epitopes by directly visualizing the antigen-antibody complex, as well as monitoring the binding of the antibody to fragments or mutated variants of the antigen or monitoring solvent access to different parts of antibodies and antigens. Exemplary strategies used to map antibody epitopes include, but are not limited to, array-based oligopeptide scanning, limited proteolysis, site-directed mutagenesis, high-throughput mutagenesis-based mapping, hydrogen-deuterium exchange, and mass spectrometry (see, e.g., Gershoni et al. (2007) 21:145-56; and Hager-Braun and Tomer (2005) Expert Rev Proteomics 2:745-56).

[147] Competitive binding and epitope-specific sorting can also be used to identify antibodies that co-bind identical or overlapping epitopes. Competitive binding can be assessed using a cross-blocking assay, such as the assay 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 if the test antibody or binding protein reduces the extent of binding of the reference antibody or binding protein to a target antigen, such as HER2 (e.g., a binding protein containing CDRs and/or variable domains selected from those identified in Tables 2 -4), by at least about 50% in a cross-blocking assay (e.g., by 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5% or greater, or by any percentage between them) and/or vice versa. In some embodiments, competitive binding may be due to shared or similar (e.g., partially overlapping) epitopes or due to steric mismatches in which antibodies or binding proteins bind at nearby epitopes (see, e.g., Tzartos, Methods in Molecular Biology (Morris, ed. . (1998) vol. 66, pp. 55-66)). In some embodiments, competitive binding can be used to sort groups of binding proteins that have similar epitopes. For example, binding proteins that compete for binding may be "sorted" as a group of binding proteins that contain overlapping or closely located epitopes, while proteins that do not compete are placed in a separate group of binding proteins that do not contain overlapping or nearby epitopes.

[148] The term "k _on " or "k _a " refers to the association rate constant when an antibody associates with an antigen to form an antibody/antigen complex. Velocity can be determined using standard assays such as surface plasmon resonance, biolayer inferometry or ELISA analysis.

[149] The term "k _off " or "k _d " refers to the dissociation rate constant when an antibody dissociates from an antibody/antigen complex. Velocity can be determined using standard assays such as surface plasmon resonance, biolayer inferometry or ELISA analysis.

[150] The term "K _D " means the equilibrium dissociation constant for a particular antibody-antigen interaction. K _D is calculated as k _a /k _d . Velocity can be determined using standard assays such as surface plasmon resonance, biolayer inferometry or ELISA analysis.

[151] The term " p " or "herboxydiene splicing modulator load" or "herboxydiene splicing modulator:antibody ratio" or "herboxydiene splicing modulator to antibody ratio" or "HAR" refers to the number of splicing modulators based on herboxydiene on an antibody or antigen-binding fragment, i.e. herboxydiene splicing modulator load or the number of -LH fragments per antibody or antigen binding fragment (Ab) in the ADC disclosed herein. In ADCs containing a herboxydiene splicing modulator, " p " indicates the number of herboxydiene splicing modulators bound to the antibody or antigen binding moiety. For example, if two herboxydiene-based splicing modulators (eg, two compounds each characterized by an H3 structure) are coupled to an antibody or antigen-binding moiety, then p =2. In compositions containing multiple copies of an ADC as described herein, the "average p value" means the average number of -LH fragments per antibody or antigen binding fragment, also referred to as the "average herboxidiene splicing modulator load".

[152] "Linker" or "linker moiety" is used herein to mean any chemical moiety that is capable of covalently attaching a compound, typically a drug moiety, such as a drug moiety that is a splicing modulator based on herboxydiene, to another fragment, such as an antibody or antigen-binding fragment. Linkers may be susceptible or substantially resistant to acid-induced cleavage, peptidase-induced cleavage, light-induced cleavage, esterase-induced cleavage, and/or disulfide bond cleavage under conditions under which the compound or antibody remains active.

[153] The term “agent” is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract obtained from biological materials. The term "therapeutic agent" or "drug" means an agent that is capable of modulating a biological process and/or has biological activity. The herboxydiene-based splicing modulators described herein are exemplary therapeutic agents.

[154] The term "chemotherapeutic agent" or "anticancer agent" is used herein to refer to all agents that are effective in the treatment of cancer, regardless of the mechanism of action. Inhibition of metastasis or angiogenesis is often a characteristic feature of a chemotherapeutic agent. Chemotherapeutic agents include antibodies, biological molecules and small molecules and include the herboxydiene-based splicing modulator compounds described herein. The chemotherapeutic agent may be a cytotoxic or cytostatic agent. The term "cytostatic agent" means an agent that inhibits or inhibits cell growth and/or cell proliferation. The term "cytotoxic agent" means a substance that causes cell death primarily by interfering with the expression activity and/or function of the cell.

[155] As used herein, the terms “herboxydiene splicing modulator”, “herboxydiene spliceosome modulator” or “herboxydiene splicing modulator” mean compounds that exhibit anticancer activity through interaction with spliceosomal components and are structurally related to herboxydiene . In some embodiments, the herboxydiene-based splicing modulator changes the rate or form of splicing in a target cell. Herboxydiene-based splicing modulators that function as inhibitory agents, for example, are capable of reducing uncontrolled cell proliferation. In some embodiments, herboxydiene-based splicing modulators may act by binding to the SF3b spliceosome complex. Such modulators may be naturally occurring or synthetic herboxydiene derivatives or analogues. As used herein, the terms "derivative" and "analog" with respect to a herboxidiene-based splicing modulator or the like mean any such compound that retains substantially the same, similar or improved biological function or activity as the herboxidiene, but is characterized by altered chemical or biological structure. In some embodiments, the herboxydiene-based splicing modulator is a herboxydiene derivative.

[156] As used herein, the term “herboxydiene splicing modulator drug moiety” means a component of an ADC or composition that provides the structure of a herboxydiene splicing modulator compound, e.g. a splicing modulator (H) based on herboxydiene, in an ADC of formula (I) or in a composition containing -L-H.

[157] As used herein, the term "spliceosome" means a ribonucleoprotein complex that removes introns from one or more RNA segments, such as pre-mRNA segments.

[158] The term "homolog" means a molecule that exhibits homology with another molecule, for example, having sequences of chemical residues that are the same or similar at corresponding positions.

[159] As used herein, the term “suppress” or “suppression” means a reduction in the quantity being measured and may include, but does not necessarily include, complete prevention or suppression.

[160] The term “target negative,” “target antigen negative,” or “antigen negative” means the absence of expression of a target antigen in a cell or tissue. The term “target positive,” “target antigen positive,” or “antigen positive” means the presence of target antigen expression. For example, a cell or cell line that does not express a target antigen may be described as target negative, while a cell or cell line that expresses a target antigen may be described as target positive.

[161] The term “non-specific killing” or “non-specific effect” means the killing of target-negative cells in the presence of target-positive cells, but the killing of target-negative cells is not observed in the absence of target-positive cells. Cell-cell contact, or at least proximity, between target-positive and target-negative cells allows nonspecific killing. This type of killing differs from “off-target killing,” which means indiscriminate killing of target-negative cells. “Off-target killing” can be observed in the absence of target-positive cells.

[162] The terms "neoplastic disorder" and "cancer" are used interchangeably herein to refer to the presence of cells exhibiting characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and/or certain morphological characteristics. Often, cancer cells may be in the form of a tumor or mass, but such cells may exist alone in the subject's body or may circulate in the bloodstream as independent cells, such as leukemia or lymphoma cells. The terms “neoplastic disorder” and “cancer” include all types of cancer and cancer metastases, including hematologic malignancies, solid tumors, sarcoma types, carcinoma types, and other solid or non-solid cancers. Hematologic malignancies may include B-cell malignancies, blood cancers (leukemias), plasma cell cancers (myelomas, such as multiple myeloma), or cancers of the lymph nodes (lymphomas). Exemplary B-cell malignancies include chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, and diffuse large B-cell lymphoma. Leukemias may include acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), acute monocytic leukemia (AMoL), etc. Lymphomas may include Hodgkin lymphoma and non-Hodgkin lymphoma. Other hematologic malignancies may include myelodysplastic syndrome (MDS). Solid tumors may include carcinomas such as adenocarcinoma, e.g. breast cancer, pancreatic cancer, prostate cancer, colon or colorectal cancer, lung cancer, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, cholangiocarcinoma, glioma , melanoma, etc.

[163] The terms "tumor" and "neoplasm" mean any mass of tissue that results from excessive growth or proliferation of cells, either benign or malignant, including precancerous lesions.

[164] The terms "tumor cell" and "neoplastic cell" are used interchangeably and mean individual cells or a general population of cells derived from a tumor or neoplasm, including both non-tumorigenic cells and cancer stem cells. As used herein, the term “tumor cell” will be modified by the term “non-tumor cell” if it refers solely to those tumor cells that lack the ability to renew and differentiate to distinguish such tumor cells from cancer stem cells.

[165] The terms “subject” and “patient” are used interchangeably herein to refer to any animal, such as any mammal, including without limitation humans, non-human primates, rodents, and the like. 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.

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

[167] "Pharmaceutical composition" means a preparation that is in a form that allows administration and subsequently provides the intended biological activity of the active ingredient(s) and/or the achievement of a therapeutic effect and which does not contain additional components that are unacceptably toxic for the subject to whom the composition will be administered. The pharmaceutical composition may be sterile.

[168] "Pharmaceutical excipient" includes a material such as an adjuvant, a carrier, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, a preservative, and the like.

[169] "Pharmaceutically acceptable" means approved, or likely to be approved, by a Federal or State government regulatory agency, or listed in the United States Pharmacopoeia or other generally accepted pharmacopoeia for use in animals and, more particularly, in humans.

[170] A "pharmaceutically acceptable salt" is a salt that retains the desired biological activity of the parent compound and does not cause undesirable toxicological effects. Examples of such salts are: (a) acid addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids, for example 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 acid, polyglutamic acid, naphthalene sulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid, polygalacturonic acid and the like; and (b) salts formed from elemental anions such as chlorine, bromine and iodine. Cm. 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.

[171] As used herein, the term "effective amount" means an amount of a compound, ADC, or composition disclosed herein (e.g., a splicing modulator or herboxidiene ADC) that is sufficient to achieve the intended purpose, e.g., to achieve a therapeutic effect after administration, such as a decrease in tumor growth rate or tumor volume, improvement in cancer symptoms, or some other indication of treatment effectiveness. The effective amount can be determined in a conventional manner relative to the intended purpose. The term “therapeutically effective amount” means an amount of a compound, ADC or composition described herein that is effective to detectably kill, reduce and/or inhibit the growth or spread of tumor cells, the size or number of tumors and/or other indicator of level, stage, progression and/or severity of cancer. The therapeutically effective amount may vary depending on the intended use (in vitro or in vivo) or the subject and disease state being treated, e.g., the weight and age of the subject, the severity of the disease state, the route of administration, and the like, as may be readily determined by one skilled in the art. in this field of technology. The term also applies to a dose that will induce a specific response in target cells, such as inhibition of cell growth. The specific dosage may vary, for example, depending on the particular pharmaceutical composition, the subject and his age and current health status or risks associated with the health condition, the schedule of administration to be followed, the severity of the disease, whether it is administered in combination with other means, the time of administration, the tissue into which it is administered, and the physical delivery system in which it is carried. In the case of cancer, a therapeutically effective amount of an ADC may reduce the number of cancer cells, reduce tumor size, suppress (eg, slow or stop) tumor metastasis, suppress (eg, slow or stop) tumor growth, and/or alleviate one or more symptoms.

[172] “Prophylactically effective amount” means an amount that, at the required dosages and for the required periods of time, is effective in achieving the desired prophylactic result. Generally, since the prophylactic dose is administered to subjects prior to the onset of the disease or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[173] As used herein, “treat” or “therapeutic” and related terms mean any improvement in any consequence of a disease, such as prolongation of survival, less clinical manifestations and/or reduction of side effects that result from an alternative therapeutic modality of intervention. As is easily understood from the prior art, complete elimination of the disease is covered by the therapeutic effect, but is not required for it. As used herein, the terms “treating” or “treating” mean administering the described ADC or composition to a subject, such as a patient. Treatment may be curing, healing, mitigating, mitigating, modifying, eliminating, alleviating, temporarily attenuating, ameliorating, or affecting a disorder, symptoms of a disorder, or a predisposition to a disorder, such as cancer. In some embodiments, in addition to treating a subject who has a condition, a composition disclosed herein can also be used prophylactically to prevent or reduce the likelihood of developing the condition.

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

[175] By "protein" as used herein is meant at least two covalently linked amino acids. The term covers polypeptides, oligopeptides and peptides. In some embodiments, two or more covalently linked amino acids are linked through a peptide bond. The protein may be composed of naturally occurring amino acids and peptide bonds, for example when the protein is produced recombinantly using expression systems and host cells. Alternatively, the protein may contain synthetic amino acids (eg homophenylalanine, citrulline, ornithine and norleucine) or peptidomimetic structures, i.e. "peptide or protein analogs" such as peptoids. Peptoids are an illustrative class of peptidomimetics whose side chains are attached to the nitrogen atom of the peptide backbone rather than to the α-carbon atoms (as they are in amino acids), and are characterized by different hydrogen bonding arrangements and conformational characteristics compared to peptides (see e.g. , Simon et al. (1992) Proc Natl Acad Sci USA 89:9367). In this regard, peptoids may be resistant to proteolysis or other physiological or storage conditions and be effective at permeating cell membranes. Such synthetic amino acids can be included, in particular, when the antibody is synthesized in vitro using conventional methods well known in the art. In addition, any combination of peptidomimetic, synthetic and naturally occurring residues/structures can be used. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The "R group" or "side chain" of an amino acid can be in one of the (L) or (S) configurations. In a specific embodiment, the amino acids are in the (L) or (S) configuration.

[176] "Recombinant protein" is a protein produced using recombinant techniques using any techniques and methods known in the art, i.e. due to the expression of recombinant nucleic acid. Methods and techniques for producing recombinant proteins are widely known in the art.

[177] The "isolated" protein is not accompanied by at least some of the material with which it is normally associated in its natural state, e.g., constitutes at least about 5% or at least about 50% by weight of the total protein in said sample . It is known that the isolated protein can range from 5% to 99.9% by weight of the total protein content depending on the circumstances. For example, the protein can be produced at a significantly higher concentration by using an inducible promoter or a promoter that allows for high expression levels, whereby the protein is produced at increased concentration levels. The definition includes the production of an antibody in a wide variety of organisms and/or host cells that are known in the art.

[178] In the case of amino acid sequences, sequence identity and/or similarity can be determined using standard techniques known in the art, including without limitation the local sequence identity algorithm from Smith and Waterman (1981) Adv Appl Math. 2:482, alignment algorithm for determining sequence identity from Needleman and Wunsch (1970) J Mol Biol. 48:443, similarity search method from Pearson and Lipman (1988) Proc Nat Acad Sci. USA 85:2444, computerized implementations of such algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Drive, Madison, WI, USA), the Best Fit sequence analysis program described in Devereux et al. (1984) Nucl Acid Res. 12:387-95, preferably using default settings, or by visual inspection. Preferably, the percent identity is calculated using FastDB based on the following parameters: mismatch penalty - 1; gap penalty - 1; the gap extension penalty is 0.33 and the join penalty is 30 ("Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149 (1988), Alan R. Liss, Inc. ).

[179] An example of a useful algorithm is PILEUP. PILEUP generates multiple sequence alignments from a group of related sequences using progressive pairwise alignments. It can also build a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method from Feng & Doolittle (1987) J Mol Evol. 35:351-60; the method being similar to that described in Higgins and Sharp (1989) CABIOS 5:151-3. Applicable parameters in PILEUP include the default gap insertion penalty of 3.00, the default gap extension penalty of 0.10, and the end gaps that are evaluated.

[180] Another example of a useful algorithm is the BLAST algorithm described in Altschul et al. (1990) J Mol Biol. 215:403-10; Altschul et al. (1997) Nucl Acid Res. 25:3389-402; and Karin et al. (1993) Proc Natl Acad Sci. USA 90:5873-87. A particularly useful BLAST program is the WU-BLAST-2 program, which is derived from Altschul et al. (1996) Methods in Enzymology 266:460-80. WU-BLAST-2 applies several search parameters, most of which are set as default values. The adjustable parameters are set to the following values: overlap length=l, overlap fraction=0.125, threshold word length (T)=II. The HSP S and HSP S2 parameters are dynamic values and are set independently by the program depending on the composition of a specific sequence and the composition of a specific database against which the sequence of interest is being searched; however, the values can be adjusted to improve sensitivity.

[181] An additional applicable algorithm is gap injection BLAST, reported in Altschul et al. (1997) Nucl Acid Res. 25:3389-402. BLAST with the introduction of gaps uses the BLOSUM-62 substitution matrix; threshold parameter T set to 9; "two matches" method for triggering extensions without gaps, the penalty for extending gaps by k is 10+k; Xu is set to 16 and Xg is set to 40 for the database search stage and 67 for the output stage of the algorithms. Gap alignments start at approximately 22 bits.

[182] Generally, amino acid homology, similarity or identity between the proteins disclosed herein and variants thereof, 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 CDR variants) are at least 80% relative to the sequences depicted herein, e.g., homology or identity is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, almost 100% or 100%.

[183] Similarly, "percentage (%) nucleic acid sequence identity" with respect to the nucleic acid sequence of antibodies and other proteins identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical to nucleotide residues in the coding sequence of an antigen binding protein. The specific method uses the BLASTN module from WU-BLAST-2 set to default parameters, with overlap length and overlap fraction set to 1 and 0.125, respectively.

[184] Since the site or region of introduction of the amino acid sequence variation is predetermined, the mutation itself does not need to be predetermined. For example, to optimize the properties of a mutation at a given site, random mutagenesis can be performed at a target codon or region and the expressed CDR variants of antigen binding proteins screened for the optimal combination of desired activity. Methods for obtaining mutations that are substitutions at predetermined sites in DNA with a known sequence are widely known, for example, mutagenesis with the MI3 primer and PCR mutagenesis.

[185] As used herein, the terms "alkyl" or "alkyl group" mean a straight-chain, branched-chain or cyclic hydrocarbon that is fully saturated. In certain embodiments, alkyl groups may contain 1-8 carbon atoms ("C ₁ -C ₈ alkyl"). In certain embodiments, alkyl groups may contain 1-6 carbon atoms ("C ₁ -C ₆ alkyl"). In certain embodiments, alkyl groups contain 1-3 carbon atoms. In yet other embodiments, alkyl groups contain 2-3 carbon atoms, and in other embodiments, alkyl groups contain 1-2 carbon atoms.

[186] As used herein, the term “alkylalkoxy” means an alkyl group substituted with an alkoxy group.

[187] As used herein, the term “alkoxy” means an alkyl group, as previously defined, attached to the carbon backbone via an oxygen atom (“alkoxy”).

[188] As used herein, the term “alkylhydroxy” means an alkyl group substituted with a hydroxyl group.

[189] As used herein, the terms “hydroxy” or “hydroxyl” mean -OH.

[190] As used herein, "carbocycle" or "carbocyclyl" includes both aromatic (eg, aryl) and non-aromatic (eg, cycloalkyl) groups. In certain embodiments, carbocyclic groups contain 3-10 carbon atoms (“3-10 membered carbocycle”). In certain embodiments, carbocyclic groups contain 3-8 carbon atoms (“3-8 membered carbocycle”). In certain embodiments, carbocyclic groups contain 3-6 carbon atoms (“3-6 membered carbocycle”). In certain embodiments, carbocyclic groups contain 3-5 carbon atoms (“3-5 membered carbocycle”).

[191] As used herein, the term “haloalkyl” means an alkyl group substituted with one or more halogen atoms.

[192] "Halogen" means any halogen radical, for example -F, -Cl, -Br or -I.

[193] As used herein, the terms “heterocycle,” “heterocyclyl,” and “heterocyclic” mean a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle containing at least one heteroatom on the ring.

[194] 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 no double bonds or containing 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 no double bonds, containing one or two double bonds, and one, two, or three heteroatoms selected from O, N and S. Illustrative examples of monocyclic heterocycle include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, dihydropyranyl (including 3,4-dihydro-2H-pyran-6-yl), 1 ,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, rolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl (including tetrahydro-2H-pyran-4-yl), tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl and trithianyl.

[195] The bicyclic heterocycles of the present invention may include a monocyclic heterocycle fused with an aryl group, or a monocyclic heterocycle fused with a monocyclic cycloalkyl, or a monocyclic heterocycle fused with a monocyclic cycloalkenyl, or a monocyclic heterocycle fused with a monocyclic heterocycle, having a total of 5 -12 atoms in the ring. Examples of bicyclic heterocycles include, but are not limited to, 3,4-dihydro-2H-pyranyl, 1,3-benzodioxolyl, 1,3-benzodithiolyl, 2,3-dihydro-1,4-benzodioxynyl, 2,3-dihydro-1-benzofuranyl, 2,3-dihydro-1-benzothienyl, 2,3-dihydro-1H-indolyl and 1,2,3,4-tetrahydroquinolinyl.

[196] The terms “heterocycle,” “heterocyclyl,” and “heterocyclic” include heteroaryls. "Heteroaryl" means a cyclic moiety containing one or more closed rings with one or more heteroatoms (oxygen, nitrogen or sulfur) on at least one of the rings, wherein at least one of the rings is aromatic, and wherein the ring or rings can independently be condensed and/or bridged. Examples include, but are not limited to, phenyl, thiophenyl, triazolyl, pyridinyl, pyrimidinyl, pyridazinyl, and pyrazinyl.

[197] As described herein, the compounds of the present invention may contain "optionally substituted" moieties. In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogen atoms in the designated moiety have been replaced by a suitable substituent. Unless otherwise specified, an "optionally substituted" group may contain a suitable substituent at each substitutable position of the group, and if more than one position in any given structure can be substituted by more than one substituent selected from the specified group, then at each position the substituent may be either the same or different. The combinations of substituents provided in the present invention are preferably those that lead to the formation of stable or chemically feasible compounds.

[198] One skilled in the art will appreciate that "substitution" or "substituted" or "absent" includes the implicit condition that such substitution or absence thereof is consistent with the permissible valency of the atom being replaced and the substituent, and that the substitution or its absence results in a stable compound, for example, which does not undergo spontaneous transformation, for example, due to rearrangement, cyclization, elimination, etc. For purposes of this invention, heteroatoms, such as nitrogen, may have hydrogen substituents and/or any possible organic substituents described herein that correspond to the valences of the heteroatoms.

[199] “Stable” means compounds that do not substantially change chemically and/or physically when exposed to conditions allowing them to be produced, detected, and, in certain embodiments, recovered, purified, and used to serve one or more of the purposes disclosed in this document. In some embodiments, a stable compound or chemically feasible compound is a compound that is substantially unchanged during storage at a temperature of 40° C. or less in the absence of moisture or other chemically active conditions for at least a week. In some embodiments, the compounds disclosed herein are stable.

[200] The enantiomers described herein may include "enantiomerically pure" isomers that contain substantially one enantiomer, for example, in an amount equal to 90%, 92%, 95%, 98% or 99% or greater than or equal to 100% of one enantiomer at a particular asymmetric center or centers. "Asymmetric center" or "chiral center" means a tetrahedral carbon atom that contains four different substituents.

[201] The compounds described herein may also contain non-naturally occurring fractions of atomic isotopes at one or more of the atoms that make up the compounds. For example, compounds can be radiolabeled with radioactive isotopes such as, for example, deuterium ( ^2H ), tritium ( ^3H ), carbon-13 ( ^13C ), or carbon-14 ( ^14C ). It is intended that all isotopic variants of the compounds disclosed herein, whether radioactive or not, are within the scope of the present invention. In addition, it is intended that all tautomeric forms of the compounds described herein are within the scope of the claimed invention.

Antibody-drug conjugates

[202] The antibody-drug conjugate (ADC) compounds of the present invention include compounds with anticancer activity. In particular, ADC compounds comprise an antibody or antigen binding fragment (including an antigen binding fragment thereof) conjugated (i.e., covalently linked via a linker) to a herboxidiene splicing modulator, wherein the herboxidiene splicing modulator, if not conjugated with an antibody or antigen-binding fragment, characterized by a cytotoxic or cytostatic effect. In various embodiments, the herboxydiene-based splicing modulator, if not conjugated to an antibody or antigen-binding moiety, is capable of binding and/or interacting with the SF3b spliceosome complex. In various embodiments, a herboxydiene-based splicing modulator, when not conjugated to an antibody or antigen binding moiety, is capable of modulating RNA splicing in vitro and/or in vivo. In various embodiments, by targeting RNA splicing, the splicing modulator and herboxydiene-based ADCs disclosed herein are effective antiproliferative agents. In various embodiments, the herboxydiene splicing modulator and ADC disclosed herein are capable of targeting both actively dividing and quiescent cells.

[203] In various embodiments, the present invention is based at least in part on the discovery that a certain biologically active herboxydiene-based splicing modulator is capable of providing improved properties when used in an ADC. Although the herboxydiene splicing modulator is capable of exhibiting improved features in a desired manner (e.g., reliable binding of the SF3b spliceosome complex, efficient modulation of RNA splicing) when used as such, in various embodiments, the herboxydiene splicing modulator is capable of exhibiting fewer of the same in the desired manner improved features when conjugated to an antibody or antigen-binding fragment. Thus, the development and production of an ADC for use as a human therapeutic agent, such as an oncology therapy, may require more than identifying an antibody capable of binding to the desired target or targets and attaching to the drug used as such for cancer treatment. Binding of an antibody to a herboxidiene splicing modulator is capable of having significant effects on the activity of one or both of the antibody and the herboxidiene splicing modulator, and the effects will vary depending on the type of linker and/or herboxidiene splicing modulator selected. Therefore, in some embodiments, the ADC components are selected to (i) maintain one or more of the therapeutic properties exhibited by the antibody and herboxydiene splicing modulator moieties when used alone, (ii) maintain the specific binding properties of the antibody, or antigen binding fragment; (iii) optimize the herboxidiene splicing modulator load and the herboxidiene splicing modulator to antibody ratio; (iv) provide delivery, eg intracellular delivery, of the herboxydiene splicing modulator moiety by stably attaching to an antibody or antigen binding moiety; (v) maintain the stability of the ADC as an intact conjugate until transported or delivered to the target site; (vi) minimize ADC aggregation before or after administration; (vii) provide a therapeutic effect, eg, a cytotoxic effect of the herboxydiene-based splicing modulator moiety following cleavage or other release mechanism in the cellular environment; (viii) provide in vivo anticancer treatment efficacy comparable to or superior to that of the herboxydiene splicing modulator antibody and fragments when administered alone; (ix) minimize off-target killing by the herboxydiene splicing modulator moiety, and/or (x) provide the desired pharmacokinetic and pharmacodynamic properties, formulation capabilities, and toxicological/immunological profiles. To exhibit each of these properties, it may be necessary to identify an improved ADC for therapeutic use (Ab et al. (2015) Mol Cancer Ther. 14:1605-13).

[204] In various embodiments, the ADCs disclosed herein exhibit unexpectedly beneficial properties in some or each of the categories listed above. For example, in some embodiments, the ADC constructs disclosed herein exhibit unexpectedly favorable herboxydiene-based splicing modulator loading profiles, aggregation and/or stability and/or retain antibody binding function, drug activity and/or improved nonspecific killing , while reducing off-target killing compared to ADCs containing an alternative linker and/or drug moiety (eg, herboxydiene-based alternative splicing modulator). In some embodiments, ADC constructs disclosed herein exhibit superior stability, activity, potency, or other effect (measured in vivo or in vitro) compared to ADCs that utilize an alternative linker and/or moiety that is splicing modulator based on herboxidiene. In some embodiments, the ADC constructs disclosed herein exhibit treatment efficacy in vivo when administered as a single dose. In some embodiments, the ADC constructs disclosed herein are surprisingly stable compared to ADCs that employ an alternative linker and/or herboxydiene splicing modulator moiety.

[205] The ADC compounds of the present invention are capable of selectively delivering an effective dose of a cytotoxic or cytostatic agent to cancer cells or tumor tissue. The disclosed ADCs have been found to have potent cytotoxic and/or cytostatic activity against cells expressing the corresponding target antigen (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4 or STEAP1). In some embodiments, the cytotoxic and/or cytostatic activity of the ADC depends on the expression of the target antigen in the cell. In some embodiments, the disclosed ADCs are particularly effective in killing cancer cells expressing a target antigen while minimizing off-target killing. In some embodiments, the disclosed ADCs do not exhibit cytotoxic and/or cytostatic effects on cancer cells that do not express the target antigen.

[206] Exemplary cancers in which HER2 is expressed include, but are not limited to, breast cancer, gastric cancer, bladder cancer, urothelial cell carcinoma, esophageal cancer, lung cancer (eg, lung adenocarcinoma), uterine cancer (eg, endometrial serous carcinoma). uterus), salivary duct carcinoma, cervical cancer, endometrial cancer and ovarian cancer (English et al. (2013) Mol Diagn Ther. 17:85-99).

[207] Exemplary cancers in which CD138 is expressed include, but are not limited to, intrathoracic cancers (eg, lung cancer, mesothelioma), skin cancers (eg, basal cell carcinoma, squamous cell carcinoma), head and neck cancers (eg, larynx, laryngeal pharynx, nasopharynx), breast cancer, urogenital cancer (eg, cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, urothelial cancer), hematological malignancies (eg, myeloma, such as multiple myeloma, B -cell malignancies, Hodgkin's lymphoma) and thyroid cancer (Szatmári et al. (2015) Dis Markers 2015:796052).

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

[209] In some embodiments, cleavage of the ADC releases the herboxydiene splicing modulator from the antibody or antigen binding moiety and linker. In some embodiments, the herboxydiene-based linker and/or splicing modulator is designed to facilitate nonspecific killing (killing neighboring cells). In some embodiments, the herboxydiene-based linker and/or splicing modulator is designed to facilitate nonspecific killing by cleavage following internalization into the cell and diffusion of the herboxydiene-based splicing linker modulator moiety and/or the herboxydiene-based splicing modulator moiety. herboxydiene as such, into neighboring cells. In some embodiments, the linker facilitates internalization into the cell. In some embodiments, a linker is designed to minimize degradation in the extracellular environment and thereby reduce toxicity to non-target tissue (e.g., non-cancerous tissue) while retaining the ability to bind the ADC to the target tissue and non-specifically kill cancerous tissue , which does not express the antigen targeted by the antibody or antigen-binding fragment of the ADC, but which surrounds the target cancer tissue expressing the antigen. In some embodiments, the herboxydiene splicing modulator fragment, or a catabolite of the herboxydiene splicing modulator moiety produced by cleavage of an ADC, is designed to facilitate uptake into target cells or neighboring cells (i.e., capable of entering cell). Such herboxydiene splicing modulator moieties and catabolites may be referred to herein as "non-specifically active", and drug moieties or catabolites with reduced cell penetration may be referred to as "non-specifically inactive".

[210] In some embodiments, the disclosed ADCs also exhibit nonspecific killing activity but low off-target cytotoxicity. Without being limited by theory, the nonspecific killing activity of an ADC may be particularly beneficial if its penetration into a solid tumor is limited and/or the expression of the target antigen is heterogeneous among tumor cells. In some embodiments, an ADC containing a cleavable linker is particularly effective at non-specific killing and/or exhibits improved non-specific killing activity compared to a comparable treatment with an ADC containing a non-cleavable linker. In some embodiments, the ADCs disclosed herein exhibit improved solubility and penetration into target cells compared to drug moieties when administered alone. In some embodiments, the ADCs disclosed herein exhibit improved cytotoxicity compared to the cytotoxicity of the herboxidiene splicing modulator moiety when used alone. In some embodiments, the ADCs disclosed herein utilize drug moieties that exhibit lower cytotoxicity when evaluated as a stand-alone herboxydiene-based splicing modulator while being unexpectedly better than ADCs containing other moieties , which are a herboxidiene-based splicing modulator, which exhibit higher cytotoxicity when evaluated as a separate herboxidiene-based splicing modulator. In some embodiments, cleavage and release of the herboxydiene-based splicing modulator improves the cytotoxicity of the ADC relative to a comparable treatment with an ADC containing a non-cleavable linker. In other embodiments, cleavage and release of the herboxydiene splicing modulator is not required for the ADC to have the desired biological activity. In some embodiments, an ADC containing a non-cleavable linker characterized by an extended spacer length (e.g., ADL12) provides the same or similar cytotoxicity relative to a comparable treatment with an ADC containing a cleavable linker (e.g., ADL1, ADL5) and surprisingly superior cytotoxicity relative to a comparable treatment with an ADC containing a shorter non-cleavable linker. In some embodiments, an ADC containing a non-cleavable linker characterized by an extended spacer length without a carbonyl group (e.g., ADL12) provides the same or similar cytotoxicity relative to a comparable treatment with an ADC containing a cleavable linker (e.g., ADL1, ADL5) and is surprisingly superior cytotoxicity relative to comparable treatment with an ADC containing a non-cleavable linker providing a spacer of the same or similar length to the carbonyl group (eg, ADL10). In some embodiments, removal of a carbonyl group from a non-cleavable MC linker (e.g., ADL12) may result in greater than 50-fold, greater than 75-fold, greater than 100-fold, greater than 150-fold, or greater than 200-fold increased cytotoxicity relative to a comparable treatment using an ADC containing an unmodified, non-cleavable MC linker (eg, ADL10). In some embodiments, removing a carbonyl group from a non-cleavable MC linker (e.g., ADL12) and increasing the length of the spacer (e.g., adding at least one spacer unit) can result in increased cytotoxicity greater than 50-fold, greater than 75-fold, greater than 100 times, more than 150 times, or more than 200 times relative to a comparable treatment with an ADC containing an unmodified non-cleavable MC linker (eg, ADL10).

[211] Presented herein are compounds that are ADCs containing an antibody or antigen binding fragment (Ab) that specifically targets a tumor cell, a splicing modulator drug fragment (D), and a linker fragment (L ), which covalently attaches Ab to D. In certain aspects, the antibody or antigen-binding fragment is capable of binding to a tumor-associated antigen (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT , MET, MUC16, SLC39A6, SLC44A4, STEAP1), with high specificity and high affinity. In certain embodiments, the antibody or antigen-binding fragment undergoes internalization into a target cell upon binding, for example, into a compartment of the cell in which degradation occurs. In various embodiments, ADCs that are internalized upon binding to a target cell, undergo degradation, and release a herboxydiene splicing modulator moiety may be used to kill cancer cells. The herboxydiene splicing modulator moiety may be released from the antibody and/or linker moiety from the ADC by enzymatic action, hydrolysis, oxidation, or any other mechanism.

[212] An exemplary ADC has formula (I):

Ab-(LH) _p (I),

where Ab = antibody or antigen binding fragment, L = linker fragment, H = herboxidiene splicing modulator drug fragment, and p = number of splicing modulator drug fragments per antibody or antigen binding fragment.

[213] In certain embodiments, the herboxydiene-based splicing modulator targeting moiety for use in the disclosed ADCs and compositions is an antibody or antigen binding moiety. other exemplary drug targeting moieties for use in the disclosed ADCs and compositions are also provided and described herein. In some embodiments, the herboxydiene splicing modulator targeting moiety may be any of a variety of cell binding agents and molecular backbones other than antibodies. In some embodiments, the herboxydiene-based splicing modulator targeting moiety is a cell binding agent. As used herein, the term “cell-binding agent” means any agent that is capable of binding to an animal (e.g., human) cell and delivering a herboxidiene splicing modulator moiety (e.g., a drug moiety which is a herboxydiene-based splicing modulator disclosed herein). The term includes exemplary antibodies and antigen binding fragments disclosed herein (eg, monoclonal antibodies and fragments thereof, such as Fab and scFV). The term further covers exemplary cell binding agents such as DARPins, duobodies, bicyclic peptides, nanobodies, centirins, MSH (melanocyte stimulating hormone), receptor-Fc fusion molecules, T cell receptor structures, steroid hormones such as androgens and estrogens, growth factors, colony-stimulating factors such as EGF, and other molecular backbones other than antibodies. In various embodiments, the backbones of molecules other than antibodies can be broadly divided into two structural classes, namely domain-sized compounds (about 6-20 kDa) and restricted peptides (about 2-4 kDa). Exemplary domain-size backbones include, but are not limited to, affibodies, affins, anticalins, atrimers, DARPins, FN3 backbones (eg, adnectins and centirins), finomeres, Kunitz domains, pronectins, O-bodies, and receptor-Fc fusion proteins, with exemplary restricted peptides include avimers, bicyclic peptides and Cys-knots. In some embodiments, the drug targeting moiety used in the disclosed ADCs and compositions is selected from affibody, affilin, anticalin, atrimer, DARPin, an FN3 backbone such as adnectin or centirin, finomer, Kunitz domain, pronectin, O-body , avimer, bicyclic peptide and Cys-unit. In some embodiments, the drug targeting moiety used in the disclosed ADCs and compositions is a receptor-Fc fusion protein, such as a chimeric HER2-Fc fusion protein. A review of the scaffolds of molecules other than antibodies is given, for example, in Vazquez-Lombardi et al. (2015) Drug Dis Today 20(10):1271–83.

Antibodies

[214] An antibody or antigen binding fragment (Ab) of formula (I) includes within its scope any antibody or antigen binding fragment that specifically binds to a target antigen on a cancer cell. An antibody or antigen binding fragment may bind to a target antigen with a dissociation constant (K _D ) of ≤1 mM, ≤100 nM, or ≤10 nM, or any value in between, as measured, for example, by a BIAcore® assay. In certain embodiments, the K _D is from 1 pM to 500 pM. In some embodiments, the K _D is from 500 pM to 1 μM, from 1 μM to 100 nM, or from 100 mM to 10 nM.

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

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

[217] The amino acid sequences of exemplary antibodies of the present invention are presented in Tables 2-4.

Table 1. Antibodies mAb Type Target Trastuzumab (AB185) humanized HER2/NEU B-B4 (AB205) mouse CD138 (syndecan-1) 1C1 (AB206) humanized EPHA2

Table 3. Amino acid sequences of CDR mAbs mAb IgG chain SEQ ID NO Amino acid sequence Trastuzumab (AB185) HCDR1 1 GFNIKDTYIH Trastuzumab (AB185) HCDR2 2 RIYPTNGYTRYADSVKG Trastuzumab (AB185) HCDR3 3 WGGDGFYAMDV Trastuzumab (AB185) LCDR1 4 RASQDVNTAVAW Trastuzumab (AB185) LCDR2 5 SASFLES Trastuzumab (AB185) LCDR3 6 QQHYTTPPT B-B4 (AB205) HCDR1 7 NYWIE B-B4 (AB205) HCDR2 8 ILPGTGRTIYNEKFKGKA B-B4 (AB205) HCDR3 9 RDYYGNFYYAMDY B-B4 (AB205) LCDR1 10 ASQGINNYLN B-B4 (AB205) LCDR2 eleven TSTLQS B-B4 (AB205) LCDR3 12 QQYSKLPRT 1C1 (AB206) HCDR1 13 HYMMA 1C1 (AB206) HCDR2 14 RIGPSGGPTHYADSVKG 1C1 (AB206) HCDR3 15 YDSGYDYVAVAGPAE-YFQH 1C1 (AB206) LCDR1 16 RASWSISTWLA 1C1 (AB206) LCDR2 17 KASNLHT 1C1 (AB206) LCDR3 18 QQYNSYS-RT

Table 4. Amino acid sequences of Ig chains representing a full-length mAb mAb IgG chain Class SEQ ID NO Amino acid sequence Trastuzumab
(AB185) Heavy chain IgG1 25 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGG DGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Trastuzumab
(AB185) Light chain Kappa chain 26 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC B-B4
(AB205) Heavy chain IgG2a 27 QVQLQQSGSELMMPGASVKISCKATGYTFSNYWIQRPGHGLEWIGEILPGTGRTIYNEKFKGKATFTADISSNTVQMQLSSLTSEDSAVYYCARRDYYG NFYYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETV TCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPA ENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG B-B4
(AB205) Light chain Kappa chain 28 DIQMTQSTSSSLSASLGDRVTISCSASQGINNYLNWYQQKPDGTVELLIYYTSTLQSGVPSRFSGSGSGTDYSLTISNLEPEDIGTYYCQQYSKLPRTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSY TCEATHKTSSTSPIVKSFNRNEC 1C1
(AB206) Heavy chain IgG1 29 EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYMMAWVRQAPGKGLEWVSRIGPSGGPTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGYDSGYDYVAVAGPAEYFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 1C1
(AB206) Light chain Kappa chain thirty DIQMTQSPSSLSASVGDRVTITCRASQSISTWLAWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTEFSLTISGLQPDDFATYYCQQYNSYSRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC

Table 5. Exemplary Target Antigen Amino Acid Sequences Antigen SEQ ID NO Amino acid sequence HER2/NEU 31 MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCW GESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWP DSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARG HCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEG ACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRL GSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAK PYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVP QQGFFCPDAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLT CSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKG TPTAENPEYLGLDVPV CD138 32 MRRAALWLWLCALALSLQPALPQIVATNLPPEDQDGSGDDSDNFSGSGAGALQDITLSQQTPSTWKDTQLLTAIPTSPEPTGLEATAASTSTLPAGEGPKEGEAVVLPEVEPGLTAREQEATPRPRETTQLPTTHLASTTTATTAQEPATSHPHRDMQPGHHETSTPAGPSQADLHTPHTEDGGPSATERAAEDGASSQLPAAEGSGEQDFTFETSG ENTAVVAVEPDRRNQSPVDQGATGASQGLLDRKEVLGGVIAGGLVGLIFAVCLVGFMLYRMKKKDEGSYSLEEPKQANGGAYQKPTKQEEFYA EPHA2 33 MELQAARACFALLWGCALAAAAAAQGKEVVLLDFAAAGGELGWLTHPYGKGWDLMQNIMNDMPIYMYSVCNVMSGDQDNWLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRLFTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYKKCP ELLQGLAHFPETIAGSDAPSLATVAGTCVDHAVVPPGGEEPRMHCAVDGEWLVPIGQCLCQAGYEKVEDACQACSPGFFKFEASESPCLECPEHTLPSPEGATSCECEEGFFRAPQDPASMPCTRPPSAPHYLTAVGMGAKVELRWTPPQDSGGREDIVYSVTCEQCWPESGECGPCEASVRYSEPPHGLTRTSVTVSDLEPHMNYTFTVEARNGVSGLV TSRSFRTASVSINQTEPPKVRLEGRSTTSLSVSWSIPPPQQSRVWKYEVTYRKKGDSNSYNVRRTEGFSVTLDDLAPDTTYLVQVQALTQEGQGAG SKVHEFQTLSPEGSGNLAVIGGVAVGVVLLLVLAGVGFFIHRRRKNQRARQSPEDVYFSKSEQLKPLKTYVDPHTYEDPNQAVLKFTTEIHPSCVTR QKVIGAGEFGEVYKGMLKTSSGKKEVPVAIKTLKAGYTEKQRVDFLGEAGIMGQFSHHNIIRLEGVISKYKPMMIITEYMENGALDKFLREKDGEFSVL QLVGMLRGIAAGMKYLANMNYVHRDLAARNILVNSNLVCKVSDFGLSRVLEDDPEATYTTSGGKIPIRWTAPEAISYRKFTSASDVWSFGIVMWEVMTY GERPYWELSNHEVMKAINDGFRLPTPMDCPSAIYQLMMQCWQQERARRPKFADIVSILDKLIRAPDSLKTLADFDPRVSIRLPSTSGSEGVPFRTVSEWLESIKMQQYTEHFMAAGYTAIEKVVQMTNDDIKRIGVRLPGHQKRIAYSLLGLKDQVNTVGIPI MSLN 94 MALPTARPLLGSCGTPALGSLLFLFSLFSLGWVQPSRTGQEAPLDGVLANPNSPRQLLGFPCAEVSGLAVKNVKNVKLSEPPEDLLPLLLLNPDAFSGP QactRFFSRITKANVDLLLLLLPRAPERQRLPALALPALALPALLSEADVRALGGGGGLPRFVASAEVLPRLVSCPPLQEAALALAKGGPSVSTMDMDMDMDMDMDMDMDMDMDMDMDMDMDVLGQPIRSIPIPQGIVAA WrQRSSSRDPSWRQPERTILRPRRPRREVEKTACPSGKAREICKWEACVDAALLATALLATRVNAIPFTYEKLDYPESVIQHLGYLGYLGYLGYLGYLGYLGYLGYLGYLGYLGELKWNVTSLETLKALLEVKGHEMS SPQAPRPRPLPLPVATLIDRFVKGRGQLDTLDTLTLCLSPELSSVPSVPSSIWAVRPPRPDPRQLDVLDVLAPALAPALAFQNMNGAPSFLGAPTEDLSQNVSMDLATFMDLATFMDLATFMKLATFMKLAT DAVLPLTVAEVQKLLGPHPHVEGLKARPVRPVILRQRQDLDLGLGLGPNGIPLVLSMQEALSGPVLTVLASTLA FOLH1 95 MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIA RYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDA QKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSF GTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDDCRDYAVV LRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA CDH6 96 MRTYRYFLLLFWVGQPYPTLSTPLSKRTSGFPAKKRALELSGNSKNELNRSKRSWMWNQFFLLEEYTGSDYQYVGKLHSDQDRGDGSLKYILSGDGAGDLFIINENTGDIQATKRLDREEKPVYILRAQAINRRTGRPVEPESEFIIKIHDINDNEPIFTKEVYTATVPEMSDVGTFVVQVTATDADDPTY GNSAKVVYSILQGQPYFSVESETGIIKTALLNMDRENREQYQVVIQAKDMGGQMGGLSGTTTVNITLTDVNDNPPRFPQSTYQFKTPESSPPGTPIGRIKASDADVGENAEIEYSITDGEGLDMFDVITDQETQEGIITVKKLLDFEKKKVYTLKVEASNPYVEPRFLYLGPFKDSATVRIVVEDVDEPPVFSKLAY ILQIREDAQINTTIGSVTAQDPDAARNPVKYSVDRHTDMDRIFNIDSGNGSIFTSKLLDRETLLWHNITVIATEINNPKQSSRVPLYIKVLDVNDNAPEFAEFYETFVCEKAKADQLIQTLHAVDKDDPYSGHQFSFSLAPEAASGSNFTIQDNKDNTAGILTRKNGYNRHEMSTYLLPVVISDNDYPVQSSTGTVVRVCACDH HGNMQSCHAEALIHPTGLSTGALVAILLCIVILLVTVVLFAALRRQRKKEPLIISKEDIRDNIVSYNDEGGGEEDTQAFDIGTLRNPEAIEDNKLRRDIVPEALFLPRRTPTARDNTDVRDFINQRLKENDTDPTAPPYDSLATYAYEGTGSVADSLSSLESVTTDADQDYDYLSDWGPRFKKLADMYGGVDSDKDS CEACAM5 97 MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLS NGNRTLLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAH NSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNKLSVDHSDPVILNV LYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDA VAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYS WRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAGATVGIMIGVLVGVALI CFC1B 98 MTWRHHVRLLFTVSLALQIINLGNSYQREKHNGGREEVTKVATQKHRQSPLNWTSSHFGEVTGSAEGWGPEEPLPYSWAFGEGASARPRCCRNGGTCVLGSFCVCPAHFTGRYCEHDQRRSECGALEHGAWTLRACHLCRCIFGALHCLPLQTPDRCDPKDFLASHAHGPSAGGAPSLLLLLPCALLHRLLRPDAPAHPRSLVPSVLQ RERRPCGRPGLGHRL ENPP3 99 MESTLTLATEQPVKKNTLKKYKIACIVLLALLVIMSLGLGLGLGLRKLEKQGSCRKKCFDASFRGLENCRCDVACKDRGDCCWDFEDTCVESTRIWMCNKFRCGETRLEASLCSCSDDCLQRKDCCADYKSVCQGETSWLEENCDTAQQSQCPEGFDLPPVILFSMDGFRAEYLYTWDTLMPNINKLKTCGIHSKYMRAMYPTKTFPNHY TIVTGLYPESHGIIDNNMYDVNLNKNFSLSSKEQNNPAWWHGQPMWLTAMYQGLKAATYFWPGSEVAINGSFPSIYMPYNGSVPFEERISTLLKWLDLPKAERPRFYTMYFEEPDSSGHAGGPVSARVIKALQVVDHAFGMLMEGLKQRNLHNCVNIILLADHGMDQTYCNKMEYMTDYFPRINFFYMYEGPAPRIRAHNIPH DFFSFNSEEIVRNLSCRKPDQHFKPYLTPDLPKRLHYAKNVRIDKVHLFVDQQWLAVRSKSNTNCGGGNHGYNNEFRSMEAIFLAHGPSFKEKTEVEPFENIEVYNLMCDLLRIQPAPNNGTHGSLNHLLKVPFYEPSHAEEVSKFSVCGFANPLPTESLDCFCPHLQNSTQLEQVNQMLNLTQEEITATVK VNLPFGRPRVLQKNVDHCLLYHREYVSGFGKAMRMPMWSSYTVPQLGDTSPLPPTVPDCLRADVRVPPSESQKCSFYLADKNITHGFLYPPASNRTSDSQYDALITSNLVPMYEEFRKMWDYFHSVLLIKHATERNGVNVVSGPIFDYNYDGHFDAPDEITKHLANTDVPIPTHYFVVLTSCKNKSHTPENCPGWLDVLPFI IPHRPTNVESCPEGKPEALWVEERFTAHIARVRDVELLTGLDFYQDKVQPVSEILQLKTYLPTFETTI FOLR1 100 MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIW THSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWAAWPFLLSLALMLLWLLS HAVCR1 101 MHPQVVILSLILHLADSVAGSVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTCQNGIVWTNGTHVTYRKDTRYKLLGDLSRRDVSLTIENTAVSDSGVYCCRVEHRGWFNDMKITVSLEIVPPKVTTTPIVTTVPTVTTVRTSTTVPTTTTVPMTTVPTTTVPTTMSIPTTTTVLTTMTVSTTTSVPTTTSIPTTTSVPVTTTVSTFVPPMPLPR QNHEPVATSPSSPQPAETHPTTLQGAIRREPTSSPLYSYTTDGNDTVTESSDGLWNNNQTQLFLEHSLLTANTTKGIYAGVCISVLVLLALLGVIIAKKYFFKKEVQQLSVSFSSLQIKALQNAVEKEVQAEDNIYIENSLYATD KIT 102 MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRLCLHCSVDQEGKSVLSEKFILK VRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNA AIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPRNRLSFGKTLGAGA FGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRIT KICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHDDV MET 103 MKAPAVLAPGILVLLFTLVQRSNGECKEALAKSEMNVNMKYQLPNFTAETPIQNVILHEHHIFLGATNYIYVLNEEDLQKVAEYKTGPVLEHPDCFPCQDCSSKANLSGGVWKDNINMALVVDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCVVSALGAKVLSSVKDRFINFF VGNTINSSYFPDHPLHSISVRRLKETKDGFMFLTDQSYIDVLPEFRDSYPIKYVHAFESNNFIYFLTVQRETLDAQTFHTRIIRFCSINSGLHSYMEMP LECILTEKRKKRSTKKEVFNILQAAYVSKPGAQLARQIGASLNDDILFGVFAQSKPDSAEPMDRSAMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHE HCFNRTLLRNSSGCEARRDEYRTEFTTALQRVDLFMGQFSEVLLTSISTFIKGDLTIANLGTSEGRFMQVVVSRSGPSTPHVNFLLDSHPVSPEVIVEHTLNQNGYTLVITGKKITKIPLNGLGCRHFQSCSQCLSAPPFVQCGWCHDKCVRSEECLSGTWTQQICLPAIYKVFPNSAPLEGGTRLTICGWDFGFRRNNKFDL KKTRVLLGNESCTLTLSESTMNTLKCTVGPAMNKHFNMSIIISNGHGTTQYSTFSYVDPVITSISPKYGPMAGGTLTLTTGNYLNSGNSRHISIGGKTCTLKSVSNSILECYTPAQTISTEFAVKLKIDLANRETSIFSYREDPIVYEIHPTKSFISGGSTITGVGKNLNSVSVPRMVINVHEAGRNNFTVACQHRSNSEIICCTTPSL QQLNLQLPLKTKAFFMLDGILSKYFDLIYVHNPVFKPFEKPVMISMGNENVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLCTVPNDLLKLNSELNIEWKQAISSTVLGKVIVQPDQNFTGLIAGVVSISTALLLLLLGFFLWLKKRKQIKDLGSELVRYDARVHTPHLDRLVSARSVSPTTE MVSNESVDYRATFPEDQFPNSSQNGSCRQVQYPLTDMSPILTSGDSDISSPLLQNTVHIDLSALNPELVQAVQHVVIGPSSLIVHFNEVIGRGHFGCVY HGTLLDNDGKKIHCAVKSLNRITDIGEVSQFLTEGIIMKDFSHPNVLSLLGICLRSEGSPLVVLPYMKHGDLRNFIRNETHNPTVKDLIGFGLQVAKGMK YLASKKFVHRDLAARNCMLDEKFTVKVADFGLARDMYDKEYYSVHNKTGAKLPVKWMALESLQTQKFTTKSDVWSFGVLLWELMTRGAPPYPDVNTFDITVYLLQGRRLLQPEYCPDPLYEVMLKCWHPKAEMRPSFSELVSRISAIFSTFIGEHYVHVNATYVNVKCVAPYPSLLSSEDNADDEVDTRPASFWETS MUC16 104 MLKPSGLPGSSSPTRSLMTGSRSTKATPEMDSGLTGATLSPKTSTGAIVVTEHTLPFTSPDKTLASPTSSVVGRTTQSLGVMSSALPESTSRGMTHSEQRTSPSLSPQVNGTPSRNYPATSMVSGLSSPRTRTSSTEGNFTKEASTYTLTVETTSGPVTEKYTVPTETSTTEGDSTETPWDTRYIPVKITSPMKTFADSTASKENAPVSMTPAETTVTDSHTPGRTNPSFGTLYSSFLDLSPKGTPNSRGETSLELILSTTGYPFSSPEPGSAGHSRISTSAPLSSSASVLDNKISETSIFSGQSLTSPLSPGVPEARASTMPNSAIPFSMTLSNAETSAERVRSTISSLGTPSISTKQTAETILTFHAFAETMDIPSTHIAKTLASEWLGSPGTLGGTSTSALTTTSPSTTLVSEETNTHHSTSGKETEGTLNTSMTPLETSAPGEESEMTATLVPTLGFTTLDSKIRSPSQVSSSHPTRELRTTGSTSGRQSSSTAAHGSSDILRATTSSTSKASSWTSESTAQQFSEPQHTQWVETSPSMKTERPPASTSVAAPITTSVPSVVSGFTTLKTSSTKGIWLEETSADTLIGESTAGPTTHQFAVPTGISMTGGSSTRGSQGTTHLLTRATASSETSADLTLATNGVPVSVSPAVSKTAAGSSPPGGTKPSYTMVSSVIPETSSLQSSAFREGTSLGLTPLNTRHPFSSPEPDSAGHTKISTSIPLLSSASVLEDKVSATSTFSHHKATSSITTGTPEISTKTKPSSAVLSSMTLSNAATSPERVRNATSPLTHPSPSGEETAGSVLTLSTSAETTDSPNIHPTGTLTSESSESPSTLSLPSVSGVKTTFSSSTPSTHLFTSGEETEETSNPSVSQPETSVSRVRTTLASTSVPTPVFPTMDTWPTRSAQFSSSHLVSELRATSSTSVTNSTGSALPKISHLTGTATMSQTNRDTFNDSAAPQSTTWPETSPRFKTGLPSATTTVSTSATSLSATVMVSKFTSPATSSMEATSIREPSTTILTTETTNGPGSMAVASTNIPIGKGYITEGRLDTSHLPIGTTASSETSMDFTMAKESVSMSVSPSQSMDAAGSSTPGRTSQFVDTFSDDVYHLTSREITIPRDGTSSALTPQMTATHPPSPDPGSARSTWLGILSSSPSSPTPKVTMSSTFSTQRVTTSMIMDTVETSRWNMPNLPSTTSLTPSNIPTSGAIGKSTLVPLDTPSPATSLEASEGGLPTLSTYPESTNTPSIHLGAHASSESPSTIKLTMASVVKPGSYTPLTFPSIETHIHVSTARMAYSSGSSPEMTAPGETNTGSTWDPTTYITTTDPKDTSSAQVSTPHSVRTLRTTENHPKTESATPAAYSGSPKISSSPNLTSPATKAWTITDTTEHSTQLHYTKLAEKSSGFETQSAPGPVSVVIPTSPTIGSSTLELTSDVPGEPLVLAPSEQTTITLPMATWLSTSLTEEMASTDLDISSPSSPMSTFAIFPPMSTPSHELSKSEADTSAIRNTDSTTLDQHLGIRSLGRTGDLTTVPITPLTTTWTSVIEHSTQAQDTLSATMSPTHVTQSLKDQTSIPASASPSHLTEVYPELGTQGRSSSEATTFWKPSTDTLSREIETGPTNIQSTPPMDNTTTGSSSSGVTLGIAHLPIGTSSPAETSTNMALERRSSTATVSMAGTMGLLVTSAPGRSISQSLGRVSSVLSESTTEGVTDSSKGSSPRLNTQGNTALSSSLEPSYAEGSQMSTSIPLTSSPTTPDVEFIGGSTFWTKEVTTVMTSDISKSSARTESSSATLMSTALGSTENTGKEKLRTASMDLPSPTPSMEVTPWISLTLSNAPNTTDSLDLSHGVHTSSAGTLATDRSLNTGVTRASRLENGSDTSSKSLSMGNSTHTSMTYTEKSEVSSSIHPRPETSAPGAETTLTSTPGNRAISLTLPFSSIPVEEVISTGITSGPDINSAPMTHSPITPPTIVWTSTGTIEQSTQPLHAVSSEKVSVQTQSTPYVNSVAVSASPTHENSVSSGSSTSSPYSSASLESLDSTISRRNAITSWLWDLTTSLPTTTWPSTSLSEALSSGHSGVSNPSSTTTEFPLFSAASTSAAKQRNPETETHGPQNTAASTLNTDASSVTGLSETPVGASISSEVPLPMAITSRSDVSGLTSESTANPSLGTASSAGTKLTRTISLPTSESLVSFRMNKDPWTVSIPLGSHPTTNTETSIPVNSAGPPGLSTVASDVIDTPSDGAESIPTVSFSPSPDTEVTTISHFPEKTTHSFRTISSLTHELTSRVTPIPGDWMSSAMSTKPTGASPSITLGERRTITSAAPTTSPIVLTASFTETSTVSLDNETTVKTSDILDARKTNELPSDSSSSSDLINTSIASSTMDVTKTASISPTSISGMTASSSPSLFSSDRPQVPTSTTETNTATSPSVSSNTYSLDGGSNVGGTPSTLPPFTITHPVETSSALLAWSRPVRTFSTMVSTDTASGENPTSSNSVVTSVPAPGTWTSVGSTTDLPAMGFLKTSPAGEAHSLLASTIEPATAFTPHLSAAVVTGSSATSEASLLTTSESKAIHSSPQTPTTPTSGANWETSATPESLLVVTETSDTTLTSKILVTDTILFSTVSTPPSKFPSTGTLSGASFPTLLPDTPAIPLTATEPTSSLATSFDSTPLVTIASDSLGTVPETTLTMSETSNGDALVLKTVSNPDRSIPGITIQGVTESPLHPSSTSPSKIVAPRNTTYEGSITVALSTLPAGTTGSLVFSQSSENSETTALVDSSAGLERASVMPLTTGSQGMASSGGIRSGSTHSTGTKTFSSLPLTMNPGEVTAMSEITTNRLTATQSTAPKGIPVKPTSAESGLLTPVSASSSPSKAFASLTTAPPTWGIPQSTLTFEFSEVPSLDTKSASLPTPGQSLNTIPDSDASTASSSLSKSPEKNPRARMMTSTKAISASSFQSTGFTETPEGSASPSMAGHEPRVPTSGTGDPRYASESMSYPDPSKASSAMTSTSLASKLTTLFSTGQAARSGSSSSPISLSTEKETSFLSPTASTSRKTSLFLGPSMARQPNILVHLQTSALTLSPTSTLNMSQEEPPELTSSQTIAEEEGTTAETQTLTFTPSETPTSLLPVSSPTEPTARRKSSPETWASSISVPAKTSLVETTDGTLVTTIKMSSQAAQGNSTWPAPAEETGSSPAGTSPGSPEMSTTLKIMSSKEPSISPEIRSTVRNSPWKTPETTVPMETTVEPVTLQSTALGSGSTSISHLPTGTTSPTKSPTENMLATERVSLSPSPPEAWTNLYSGTPGGTRQSLATMSSVSLESPTARSITGTGQQSSPELVSKTTGMEFSMWHGSTGGTTGDTHVSLSTSSNILEDPVTSPNSVSSLTDKSKHKTETWVSTTAIPSTVLNNKIMAAEQQTSRSVDEAYSSTSSWSDQTSGSDITLGASPDVTNTLYITSTAQTTSLVSLPSGDQGITSLTNPSGGKTSSASSVTSPSIGLETLRANVSAVKSDIAPTAGHLSQTSSPAEVSILDVTTAPTPGISTTITTMGTNSISTTTPNPEVGMSTMDSTPATERRTTSTEHPSTWSSTAASDSWTVTDMTSNLKVARSPGTISTMHTTSFLASSTELDSMSTPHGRITVIGTSLVTPSSDASAVKTETSTSERTLSPSDTTASTPISTFSRVQRMSISVPDILSTSWTPSSTEAEDVPVSMVSTDHASTKTDPNTPLSTFLFDSLSTLDWDTGRSLSSATATTSAPQGATTPQELTLETMISPATSQLPFSIGHITSAVTPAAMARSSGVTFSRPDPTSKKAEQTSTQLPTTTSAHPGQVPRSAATTLDVIPHTAKTPDATFQRQGQTALTTEARATSDSWNEKEKSTPSAPWITEMMNSVSEDTIKEVTSSSSVLRTLNTLDINLESGTTSSPSWKSSPYERIAPSESTTDKEAIHPSTNTVETTGWVTSSEHASHSTIPAHSASSKLTSPVVTTSTREQAIVSMSTTTWPESTRARTEPNSFLTIELRDVSPYMDTSSTTQTSIISSPGSTAITKGPRTEITSSKRISSSFLAQSMRSSDSPSEAITRLSNFPAMTESGGMILAMQTSPPGATSLSAPTLDTSATASWTGTPLATTQRFTYSEKTTLFSKGPEDTSQPSPPSVEETSSSSSLVPIHATTSPSNILLTSQGHSPSSTPPVTSVFLSETSGLGKTTDMSRISLEPGTSLPPNLSSTAGEALSTYEASRDTKAIHHSADTAVTNMEATSSEYSPIPGHTKPSKATSPLVTSHIMGDITSSTSVFGSSETTEIETVSSVNQGLQERSTSQVASSATETSTVITHVSSGDATTHVTKTQATFSSGTSISSPHQFITSTNTFTDVSTNPSTSLIMTESSGVTITTQTGPTGAATQGPYLLDTSTMPYLTETPLAVTPDFMQSEKTTLISKGPKDVSWTSPPSVAETSYPSSLTPFLVTTIPPATSTLQGQHTSSPVSATSVLTSGLVKTTDMLNTSMEPVTNSPQNLNNPSNEILATLAATTDIETIHPSINKAVTNMGTASSAHVLHSTLPVSSEPSTATSPMVPASSMGDALASISIPGSETTDIEGEPTSSLTAGRKENSTLQEMNSTTESNIILSNVSVGAITEATKMEVPSFDATFIPTPAQSTKFPDIFSVASSRLSNSPPMTISTHMTTTQTGSSGATSKIPLALDTSTLETSAGTPSVVTEGFAHSKITTAMNNDVKDVSQTNPPFQDEASSPSSQAPVLVTTLPSSVAFTPQWHSTSSPVSMSSVLTSSLVKTAGKVDTSLETVTSSPQSMSNTLDDISVTSAATTDIETTHPSINTVVTNVGTTGSAFESHSTVSAYPEPSKVTSPNVTTSTMEDTTISRSIPKSSKTTRTETETTSSLTPKLRETSISQEITSSTETSTVPYKELTGATTEVSRTDVTSSSSTSFPGPDQSTVSLDISTETNTRLSTSPIMTESAEITITTQTGPHGATSQDTFTMDPSNTTPQAGIHSAMTHGFSQLDVTTLMSRIPQDVSWTSPPSVDKTSSPSSFLSSPAMTTPSLISSTLPEDKLSSPMTSLLTSGLVKITDILRTRLEPVTSSLPNFSSTSDKILATSKDSKDTKEIFPSINTEETNVKANNSGHESHSPALADSETPKATTQMVITTTVGDPAPSTSMPVHGSSETTNIKREPTYFLTPRLRETSTSQESSFPTDTSFLLSKVPTGTITEVSSTGVNSSSKISTPDHDKSTVPPDTFTGEIPRVFTSSIKTKSAEMTITTQASPPESASHSTLPLDTSTTLSQGGTHSTVTQGFPYSEVTTLMGMGPGNVSWMTTPPVEETSSVSSLMSSPAMTSPSPVSSTSPQSIPSSPLPVTALPTSVLVTTTDVLGTTSPESVTSSPPNLSSITHERPATYKDTAHTEAAMHHSTNTAVTNVGTSGSGHKSQSSVLADSETSKATPLMSTTSTLGDTSVSTSTPNISQTNQIQTEPTASLSPRLRESSTSEKTSSTTETNTAFSYVPTGAITQASRTEISSSRTSISDLDRPTIAPDISTGMITRLFTSPIMTKSAEMTVTTQTTTPGATSQGILPWDTSTTLFQGGTHSTVSQGFPHSEITTLRSRTPGDVSWMTTPPVEETSSGFSLMSPSMTSPSPVSSTSPESIPSSPLPVTALLTSVLVTTTNVLGTTSPEPVTSSPPNLSSPTQERLTTYKDTAHTEAMHASMHTNTAVANVGTSISGHESQSSVPADSHTSKATSPMGITFAMGDTSVSTSTPAFFETRIQTESTSSLIPGLRDTRTSEEINTVTETSTVLSEVPTTTTTEVSRTEVITSSRTTISGPDHSKMSPYISTETITRLSTFPFVTGSTEMAITNQTGPIGTISQATLTLDTSSTASWEGTHSPVTQRFPHSEETTTMSRSTKGVSWQSPPSVEETSSPSSPVPLPAITSHSSLYSAVSGSSPTSALPVTSLLTSGRRKTIDMLDTHSELVTSSLPSASSFSGEILTSEASTNTETIHFSENTAETNMGTTNSMHKLHSSVSIHSQPSGHTPPKVTGSMMEDAIVSTSTPGSPETKNVDRDSTSPLTPELKEDSTALVMNSTTESNTVFSSVSLDAATEVSRAEVTYYDPTFMPASAQSTKSPDISPEASSSHSNSPPLTISTHKTIATQTGPSGVTSLGQLTLDTSTIATSAGTPSARTQDFVDSETTSVMNNDLNDVLKTSPFSAEEANSLSSQAPLLVTTSPSPVTSTLQEHSTSSLVSVTSVPTPTLAKITDMDTNLEPVTRSPQNLRNTLATSEATTDTHTMHPSINTAVANVGTTSSPNEFYFTVSPDSDPYKATSAVVITSTSGDSIVSTSMPRSSAMKKIESETTFSLIFRLRETSTSQKIGSSSDTSTVFDKAFTAATTEVSRTELTSSSRTSIQGTEKPTMSPDTSTRSVTMLSTFAGLTKSEERTIATQTGPHRATSQGTLTWDTSITTSQAGTHSAMTHGFSQLDLSTLTSRVPEYISGTSPPSVEKTSSSSSLLSLPAITSPSPVPTTLPESRPSSPVHLTSLPTSGLVKTTDMLASVASLPPNLGSTSHKIPTTSEDIKDTEKMYPSTNIAVTNVGTTTSEKESYSSVPAYSEPPKVTSPMVTSFNIRDTIVSTSMPGSSEITRIEMESTFSLAHGLKGTSTSQDPIVSTEKSAVLHKLTTGATETSRTEVASSRRTSIPGPDHSTESPDISTEVIPSLPISLGITESSNMTIITRTGPPLGSTSQGTFTLDTPTTSSRAGTHSMATQEFPHSEMTTVMNKDPEILSWTIPPSIEKTSFSSSLMPSPAMTSPPVSSTLPKTIHTTPSPMTSLLTPSLVMTTDTLGTSPEPTTSSPPNLSSTSHEILTTDEDTTAIEAMHPSTSTAATNVETTSSGHGSQSSVLADSEKTKATAPMDTTSTMGHTTVSTSMSVSSETTKIKRESTYSLTPGLRETSISQNASFSTDTSIVLSEVPTGTTAEVSRTEVTSSGRTSIPGPSQSTVLPEISTRTMTRLFASPTMTESAEMTIPTQTGPSGSTSQDTLTLDTSTTKSQAKTHSTLTQRFPHSEMTTLMSRGPGDMSWQSSPSLENPSSLPSLLSLPATTSPPPISSTLPVTISSSPLPVTSLLTSSPVTTTDMLHTSPELVTSSPPKLSHTSDERLTTGKDTTNTEAVHPSTNTAASNVEIPSSGHESPSSALADSETSKATSPMFITSTQEDTTVAISTPHFLETSRIQKESISSLSPKLRETGSSVETSSAIETSAVLSEVSIGATTEISRTEVTSSSRTSISGSAESTMLPEISTTRKIIKFPTSPILAESSEMTIKTQTSPPGSTSESTFTLDTSTTPSLVITHSTMTQRLPHSEITTLVSRGAGDVPRPSSLPVEETSPPSSQLSLSAMISPSPVSSTLPASSHSSSASVTSLLTPGQVKTTEVLDASAEPETSSPPSLSSTSVEILATSEVTTDTEKIHPFSNTAVTKVGTSSSGHESPSSVLPDSETTKATSAMGTISIMGDTSVSTLTPALSNTRKIQSEPASSLTTRLRETSTSEETSLATEANTVLSKVSTGATTEVSRTEAISFSRTSMSGPEQSTMSQDISIGTIPRISASSVLTESAKMTITTQTGPSESTLESTLNLNTATTPSWVETHSIVIQGFPHPEMTTSMGRGPGGVSWPSPPFVKETSPPSSPLSLPAVTSPHPVSTTFLAHIPPSPLPVTSLLTSGPATTTDILGTSTEPGTSSSSSLSTTSHERLTTYKDTAHTEAVHPSTNTGGTNVATTSSGYKSQSSVLADSSPMCTTSTMGDTSVLTSTPAFLETRRIQTELASSLTPGLRESSGSEGTSSGTKMSTVLSKVPTGATTEISKEDVTSIPGPAQSTISPDISTRTVSWFSTSPVMTESAEITMNTHTSPLGATTQGTSTLDTSSTTSLTMTHSTISQGFSHSQMSTLMRRGPEDVSWMSPPLLEKTRPSFSLMSSPATTSPSPVSSTLPESISSSPLPVTSLLTSGLAKTTDMLHKSSEPVTNSPANLSSTSVEILATSEVTTDTEKTHPSSNRTVTDVGTSSSGHESTSFVLADSQTSKVTSPMVITSTMEDTSVSTSTPGFFETSRIQTEPTSSLTLGLRKTSSSEGTSLATEMSTVLSGVPTGATAEVSRTEVTSSSRTSISGFAQLTVSPETSTETITRLPTSSIMTESAEMMIKTQTDPPGSTPESTHTVDISTTPNWVETHSTVTQRFSHSEMTTLVSRSPGDMLWPSQSSVEETSSASSLLSLPATTSPSPVSSTLVEDFPSASLPVTSLLNPGLVITTDRMGISREPGTSSTSNLSSTSHERLTTLEDTVDTEDMQPSTHTAVTNVRTSISGHESQSSVLSDSETPKATSPMGTTYTMGETSVSISTSDFFETSRIQIEPTSSLTSGLRETSSSERISSATEGSTVLSEVPSGATTEVSRTEVISSRGTSMSGPDQFTISPDISTEAITRLSTSPIMTESAESAITIETGSPGATSEGTLTLDTSTTTFWSGTHSTASPGFSHSEMTTLMSRTPGDVPWPSLPSVEEASSVSSSLSSPAMTSTSFFSTLPESISSSPHPVTALLTLGPVKTTDMLRTSSEPETSSPPNLSSTSAEILATSEVTKDREKIHPSSNTPVVNVGTVIYKHLSPSSVLADLVTTKPTSPMATTSTLGNTSVSTSTPAFPETMMTQPTSSLTSGLREISTSQETSSATERSASLSGMPTGATTKVSRTEALSLGRTSTPGPAQSTISPEISTETITRISTPLTTTGSAEMTITPKTGHSGASSQGTFTLDTSSRASWPGTHSAATHRSPHSGMTTPMSRGPEDVSWPSRPSVEKTSPPSSLVSLSAVTSPSPLYSTPSESSHSSPLRVTSLFTPVMMKTTDMLDTSLEPVTTSPPSMNITSDESLATSKATMETEAIQLSENTAVTQMGTISARQEFYSSYPGLPEPSKVTSPVVTSSTIKDIVSTTIPASSEITRIEMESTSTLTPTPRETSTSQEIHSATKPSTVPYKALTSATIEDSMTQVMSSSRGPSPDQSTMSQDISTEVITRLSTSPIKTESTEMTITTQTGSPGATSRGTLTLDTSTTFMSGTHSTASQGFSHSQMTALMSRTPGDVPWLSHPSVEEASSASFSLSSPVMTSSSPVSSTLPDSIHSSSLPVTSLLTSGLVKTTELLGTSSEPETSSPPNLSSTSAEILAITEVTTDTEKLEMTNVVTSGYTHESPSSVLADSVTTKATSSMGITYPTGDTNVLTSTPAFSDTSRIQTKSKLSLTPGLMETSISEETSSATEKSTVLSSVPTGATTEVSRTEAISSSRTSIPGPAQSTMSSDTSMETITRISTPLTRKESTDMAITPKTGPSGATSQGTFTLDSSSTASWPGTHSATTQRFPQSVVTTPMSRGPEDVSWPSPLSVEKNSPPSSLVSSSSVTSPSPLYSTPSGSSHSSPVPVTSLFTSIMMKATDMLDASLEPETTSAPNMNITSDESLAASKATTETEAIHVFENTAASHVETTSATEELYSSSPGFSEPTKVISPVVTSSSIRDNMVSTTMPGSSGITRIEIESMSSLTPGLRETRTSQDITSSTETSTVLYKMPSGATPEVSRTEVMPSSRTSIPGPAQSTMSLDISDEVVTRLSTSPIMTESAEITITTQTGYSLATSQVTLPLGTSMTFLSGTHSTMSQGLSHSEMTNLMSRGPESLSWTSPRFVETTRSSSSLTSLPLTTSLSPVSSTLLDSSPSSPLPVTSLILPGLVKTTEVLDTSSEPKTSSSPNLSSTSVEIPATSEIMTDTEKIHPSSNTAVAKVRTSSSVHESHSSVLADSETTITIPSMGITSAVDDTTVFTSNPAFSETRRIPTEPTFSLTPGFRETSTSEETTSITETSAVLYGVPTSATTEVSMTEIMSSNRIHIPDSDQSTMSPDIITEVITRLSSSSMMSESTQMTITTQKSSPGATAQSTLTLATTTAPLARTHSTVPPRFLHSEMTTLMSRSPENPSWKSSLFVEKTSSSSSLLSLPVTTSPSVSSTLPQSIPSSSFSVTSLLTPGMVKTTDTSTEPGTSLSPNLSGTSVEILAASEVTTDTEKIHPSSSMAVTNVGTTSSGHELYSSVSIHSEPSKATYPVGTPSSMAETSISTSMPANFETTGFEAEPFSHLTSGFRKTNMSLDTSSVTPTNTPSSPGSTHLLQSSKTDFTSSAKTSSPDWPPASQYTEIPVDIITPFNASPSITESTGITSFPESRFTMSVTESTHHLSTDLLPSAETISTGTVMPSLSEAMTSFATTGVPRAISGSGSPFSRTESGPGDATLSTIAESLPSSTPVPFSSSTFTTTDSSTIPALHEITSSSATPYRVDTSLGTESSTTEGRLVMVSTLDTSSQPGRTSSSPILDTRMTESVELGTVTSAYQVPSLSTRLTRTDGIMEHITKIPNEAAHRGTIRPVKGPQTSTSPASPKGLHTGGTKRMETTTTALKTTTTALKTTSRATLTTSVYTPTLGTLTPLNASMQMASTIPTEMMITTPYVFPDVPETTSSLATSLGAETSTALPRTTPSVFNRESETTASLVSRSGAERSPVIQTLDVSSSEPDTTASWVIHPAETIPTVSKTTPNFFHSELDTVSSTATSHGADVSSAIPTNISPSELDALTPLVTISGTDTSTTFPTLTKSPHETETRTTWLTHPAETSSTIPRTIPNFSHHESDATPSIATSPGAETSSAIPIMTVSPGAEDLVTSQVTSSGTDRNMTIPTLTLSPGEPKTIASLVTHPEAQTSSAIPTSTISPAVSRLVTSMVTSLAAKTSTTNRALTNSPGEPATTVSLVTHPAQTSPTVPWTTSIFFHSKSDTTPSMTTSHGAESSSAVPTPTVSTEVPGVVTPLVTSSRAVISTTIPILTLSPGEPETTPSMATSHGEEASSAIPTPTVSPGVPGVVTSLVTSSRAVTSTTIPILTFSLGEPETTPSMATSHGTEAGSAVPTVLPEVPGMVTSLVASSRAVTSTTLPTLTLSPGEPETTPSMATSHGAEASSTVPTVSPEVPGVVTSLVTSSSGVNSTSIPTLILSPGELETTPSMATSHGAEASSAVPTPTVSPGVSGVVTPLVTSSRAVTSTTIPILTLSSSEPETTPSMATSHGVEASSAVLTVSPEVPGMVTSLVTSSRAVTSTTIPTLTISSDEPETTTSLVTHSEAKMISAIPTLAVSPTVQGLVTSLVTSSGSETSAFSNLTVASSQPETIDSWVAHPGTEASSVVPTLTVSTGEPFTNISLVTHPAESSSTLPRTTSRFSHSELDTMPSTVTSPEAESSSAISTTISPGIPGVLTSLVTSSGRDISATFPTVPESPHESEATASWVTHPAVTSTTVPRTTPNYSHSEPDTTPSIATSPGAEATSDFPTITVSPDVPDMVTSQVTSSGTDTSITIPTLTLSSGEPETTTSFITYSETHTSSAIPTLPVSPGASKMLTSLVISSGTDSTTTFPTLTETPYEPETTAIQLIHPAETNTMVPRTTPKFSHSKSDTTLPVAITSPGPEASSAVSTTTISPDMSDLVTSLVPSSGTDTSTTFPTLSETPYEPETTATWLTHPAETSTTVSGTIPNFSHRGSDTAPSMVTSPGVDTRSGVPTTTIPPSIPGVVTSQVTSSATDTSTAIPTLTPSPGEPETTASSATHPGTQTGFTVPIRTVPSSEPDTMASWVTHPPQTSTPVSRTTSSFSHSSPDATPVMATSPRTEASSAVLTTISPGAPEMVTSQITSSGAATSTTVPTLTHSPGMPETTALLSTHPRTETSKTFPASTVFPQVSETTASLTIRPGAETSTALPTQTTSSLFTLLVTGTSRVDLSPTASPGVSAKTAPLSTHPGTETSTMIPTSTLSLGLLETTGLLATSSSAETSTSTLTLTVSPAVSGLSSASITTDKPQTVTSWNTETSPSVTSVGPPEFSRTVTGTTMTLIPSEMPTPPKTSHGEGVSPTTILRTTMVEATNLATTGSSPTVAKTTTTFNTLAGSLFTPLTTPGMSTLASESVTSRTSYNHRSWISTTSSYNRRYWTPATSTPVTSTFSPGISTSSIPSSTAATVPFMVPFTLNFTITNLQYEEDMRHPGSRKFNATERELQGLLKPLFRNSSLEYLYSGCRLASLRPEKDSSATAVDAICTHRPDPEDLGLDRERLYWELSNLTNGIQELGPYTLDRNSLYVNGFTHRSSMPTTSTPGTSTVDVGTSGTPSSSPSPTTAGPLLMPFTLNFTITNLQYEEDMRRTGSRKFNTMESVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKDGAATGVDAICTHRLDPKSPGLNREQLYWELSKLTNDIEELGPYTLDRNSLYVNGFTHQSSVSTTSTPGTSTVDLRTSGTPSSLSSPTIMAAGPLLVPFTLNFTITNLQYGEDMGHPGSRKFNTTERVLQGLLGPIFKNTSVGPLYSGCRLTSLRSEKDGAATGVDAICIHHLDPKSPGLNRERLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHRTSVPTSSTPGTSTVDLGTSGTPFSLPSPATAGPLLVLFTLNFTITNLKYEEDMHRPGSRKFNTTERVLQTLLGPMFKNTSVGLLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHWIPVPTSSTPGTSTVDLGSGTPSSLPSPTTAGPLLVPFTLNFTITNLKYEEDMHCPGSRKFNTTERVLQSLLGPMFKNTSVGPLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHQTSAPNTSTPGTSTVDLGTSGTPSSLPSPTSAGPLLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLTLLRPEKNGAATGMDAICSHRLDPKSPGLNREQLYWELSQLTHGIKELGPYTLDRNSLYVNGFTHRSSVAPTSTPGTSTVDLGTSGTPSSLPSPTTAVPLLVPFTLNFTITNLQYGEDMRHPGSRKFNTTERVLQGLLGPLFKNSSVGPLYSGCRLISLRSEKDGAATGVDAICTHHLNPQSPGLDREQLYWQLSQMTNGIKELGPYTLDRNSLYVNGFTHRSSGLTTSTPWTSTVDLGTSGTPSPVPSPTTTGPLLVPFTLNFTITNLQYEENMGHPGSRKFNITESVLQGLLKPLFKSTSVGPLYSGCRLTLLRPEKDGVATRVDAICTHRPDPKIPGLDRQQLYWELSQLTHSITELGPYTLDRDSLYVNGFTQRSSVPTTSTPGTFTVQPETSETPSSLPGPTATGPVLLPFTLNFTITNLQYEEDMRRPGSRKFNTTERVLQGLLMPLFKNTSVSSLYSGCRLTLLRPEKDGAATRVDAVCTHRPDPKSPGLDRERLYWKLSQLTHGITELGPYTLDRHSLYVNGFTHQSSMTTTRTPDTSTMHLATSRTPASLSGPMTASPLLVLFTINFTITNLRYEENMHHPGSRKFNTTERVLQGLLRPVFKNTSVGPLYSGCRLTLLRPKKDGAATKVDAICTYRPDPKSPGLDREQLYWELSQLTHSITELGPYTLDRDSLYVNGFTQRSSVPTTSIPGTPTVDLGTSGTPVSKPGPSAASPLLVLFTLNFTITNLRYEENMQHPGSRKFNTTERVLQGLLRSLFKSTSVGPLYSGCRLTLLRPEKDGTATGVDAICTHHPDPKSPRLDREQLYWELSQLTHNITELGPYALDNDSLFVNGFTHRSSVSTTSTPGTPTVYLGASKTPASIFGPSAASHLLILFTLNFTITNLRYEENMWPGSRKFNTTERVLQGLLRPLFKNTSVGPLYSGCRLTLLRPEKDGEATGVDAICTHRPDPTGPGLDREQLYLELSQLTHSITELGPYTLDRDSLYVNGFTHRSSVPTTSTGVVSEEPFTLNFTINNLRYMADMGQPGSLKFNITDNVMQHLLSPLFQRSSLGARYTGCRVIALRSVKNGAETRVDLLCTYLQPLSGPGLPIKQVFHELSQQTHGITRLGPYSLDKDSLYLNGYNEPGPDEPPTTPKPATTFLPPLSEATTAMGYHLKTLTLNFTISNLQYSPDMGKGSATFNSTEGVLQHLLRPLFQKSSMGPFYLGCQLISLRPEKDGAATGVDTTCTYHPDPVGPGLDIQQLYWELSQLTHGVTQLGFYVLDRDSLFINGYAPQNLSIRGEYQINFHIVNWNLSNPDPTSSEYITLLRDIQDKVTTLYKGSQLHDTFRFCLVTNLTMDSVLVTVKALFSSNLDPSLVEQVFLDKTLNASFHWLGSTYQLVDIHVTEMESSVYQPTSSSSTQHFYLNFTITNLPYSQDKAQPGTTNYQRNKRNIEDALNQLFRNSSIKSYFSDCQVSTFRSVPNRHHTGVDSLCNFSPLARRVDRVAIYEEFLRMTRNGTQLQNFTLDRSSVLVDGYSPNRNEPLTGNSDLPFWAVILIGLAGLLGVITCLICGVLVTTRRRKKEGEYNVQQQCPGYYQSHLDLEDLQ SLC39A6 105 MARKLSVILILTFALSVTNPLHELKAAAFPQTTEKISPNWESGINVDLAISTRQYHLQQLFYRYGENNLSSVEGFRKLLQNIGIDKIKRIHIHHDHDHHSDHEHHSDHERHSDHEHHSEHEHHSDHDHHSHHNHAASGKNKRKALCPDHDSSGKDPRNSQGKGAHRPEHASGRRNVKDSVSASEVTSTVYNTVSEGTH FLETIETPRPGKLFPKDVSSSTPPSVTSKSRVSRLAGRKTNESVSEPRKGFMYSRNTNENPQECFNASKLLTSHGMGIQVPLNATEFNYLCPAIINQIDARSCLIHTSEKKAEIPPKTYSLQIAWVGGFIAISIISFLSLLGVILVPLMNRVFFKFLLSFLVALAVGTLSGDAFLHLLPHSHASHHHSHSHEEPAMEMKRGPLFSHLSSQ NIEESAYFDSTWKGLTALGGLYFMFLVEHVLTLIKQFKDKKKKNQKKPENDDDVEIKKQLSKYESQLSTNEEKVDTDDRTEGYLRADSQEPSHFDSQQPAVLEEEEVMIAHAHPQEVYNEYVPRGCKNKCHSHFHDTLGQSDDLIHHHHDYHHILHHHHHQNHHPHSHSQRYSREELKDAGVATL AWMVIMGDGLHNFSDGLAIGAAFTEGLSSGLSTSVAVFCHELPHELGDFAVLLKAGMTVKQAVLYNALSAMLAYLGMATGIFIGHYAENVSMWIFALTAGLFMYVALVDMVPEMLHNDASDHGCSRWGYFFLQNAGMLLGFGIMLLISIFEHKIVFRINF SLC44A4 106 MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGYIVVGIVAWLYGDPRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCILSSNIISVAENGLQCPTPQVCVSSCPEDPWTVGKNEFSQTVGEVFYTKNRNFCLPGVPWNMTVITSLQQELCPSFLLPSAPALGRCFPWTNVTPPALPGITNDTTIQQGISGLIDSLNARDISVKIFEDFAQSWYWILVALGVALVLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQLGFTTNLSAYQSVQETWLAALIVLAVLEAILLLMLIFLRQRIRIAIALLKEASKAVGQMMSTMFYPLVTFVLLLICIAYWAMTALYLATSGQPQYVLWASNISSPGCEKVPINTSCNPTAHLVNSSCPGLMCVFQGYSSKGLIQRSVFNLQIYGVLGLFWTLNWVLALGQCVLAGAFASFYWAFHKPQDIPTFPLISAFIRTLRYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCIMCCFKCCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNIVRVVVLDKVTDLLLFFGKLLVVGGVGVLSFFFFSGRIPGLGKDFKSPHLNYYWLPIMTSILGAYVIASGFFSVFGMCVDTLFLCFLEDLERNNGSLDRPYYMSKSLLKILGKKNEAPPDNKKRKK STEAP1 107 MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYK LLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPIVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEICSQL

[218] In various embodiments, the ADC disclosed herein may comprise any set of heavy and light chain variable domains listed in the tables above, or a set of six CDR sequences from a set of heavy and light chains, for example, introduced by transfer of six CDR into the selected human donor antibody framework. In various embodiments, the ADC disclosed herein may contain amino acid sequences that are homologous to the sequences listed in the tables above, provided that the ADC retains the ability to bind to its target cancer antigen (e.g., a K _D of less than 1× ¹⁰⁻⁸ M) and retains one or more of the functional properties of the ADC disclosed herein (eg, ability to internalize, modulate RNA splicing, inhibit cell growth, etc.).

[219] 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 kappa or lambda light chain constant domain. In various embodiments, the antibody or antigen binding fragment of the disclosed ADCs comprises a human immunoglobulin G subtype 1 (IgG1) heavy chain constant domain and a human Ig kappa light chain constant domain.

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

[221] In various embodiments, an anti-HER2 antibody or antigen binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1), consisting of SEQ ID NO: 1, heavy chain CDR2 (HCDR2), consisting of SEQ ID NO: 2, 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, as defined by the system numbering according to Kabat.

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

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

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

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

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

[227] In various embodiments, the anti-CD138 antibody or antigen binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1), consisting of SEQ ID NO: 7, heavy chain CDR2 (HCDR2), consisting of SEQ ID NO: 8, 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 system numbering according to Kabat.

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

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

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

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

[232] In various other embodiments, the target cancer antigen for the ADC is human ephrin type A receptor 2 (EPHA2).

[233] In various embodiments, an anti-EPHA2 antibody or antigen binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1), consisting of SEQ ID NO: 13, heavy chain CDR2 (HCDR2), consisting of SEQ ID NO: 14, 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 system numbering according to Kabat.

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

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

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

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

[238] In various embodiments, the amino acid substitutions are single residue substitutions. Inserts will typically be on the order of about 1 to about 20 amino acid residues, although significantly longer inserts may be permitted as long as the biological function (eg, binding to a target antigen) is preserved. Deletions typically range from about 1 to about 20 amino acid residues, although in some cases deletions can be much longer. Substitutions, deletions, insertions, or any combination thereof may be used to achieve the final derivative or variant. Typically such changes are made on a small number of amino acids to minimize changes to the molecule, particularly the immunogenicity and specificity of the antigen binding protein. However, in some circumstances larger changes may be permitted. Conservative substitutions are usually performed according to the following scheme, depicted as Table 6.

Table 6 Original balance Illustrative substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

[239] Significant changes in function or immunological identity are made by selecting substitutions that are less conservative than those shown in Table 6. For example, substitutions can be made that more significantly affect the structure of the polypeptide backbone in the region of change, for example, an alpha type structure -helix or beta sheet; the charge or hydrophobicity of the molecule at the target site or the volume of the side chain. The substitutions that can generally result in the greatest changes in the properties of a polypeptide are those in which (a) a hydrophilic residue, such as seryl or threonyl, is replaced (or replaced) by a hydrophobic residue, such as leucyl, isoleucyl, phenylalanyl, valyl, or allyl; (b) cysteine or proline is substituted (or replaced) by any other residue; (c) a residue with an electropositive side chain, such as lysyl, arginyl or histidyl, is replaced (or replaced) by an electronegative residue, such as glutamyl or aspartyl; or (d) a residue with a bulky side chain, such as phenylalanine, is replaced (or replaced) by a residue without a side chain, such as glycine.

[240] In various embodiments where variant antibody sequences are used in the ADC, the variants will generally exhibit the same qualitative biological activity and will elicit the same immune response, although variants may also be selected to modify the characteristics of the antigen binding proteins if desired. Alternatively, the variant may be designed such that the biological activity of the antigen binding protein is altered. For example, glycosylation sites may be changed or removed.

[241] Various antibodies can be used with the ADCs used herein to target cancer cells. As shown below, the linker-payload constructs in the ADCs disclosed herein are surprisingly effective with antibodies targeting different tumor antigens. Suitable antigens expressed on tumor cells but not on healthy cells, or expressed on tumor cells at a higher level than on healthy cells, are known in the art, as are antibodies directed against them. These antibodies can be used with the herboxydiene-based splicing modulator linkers and payloads disclosed herein. In some embodiments, the antibody or antigen-binding fragment targets HER2, and the antibody or antigen-binding fragment that targets 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, in addition to the disclosed linkers and herboxydiene splicing modulator payloads being unexpectedly effective with multiple antibodies targeting different tumors, HER2-targeting antibodies such as trastuzumab, antibodies, CD138-targeting antibodies, such as B-B4, and EPHA2-targeting antibodies, such as 1C1, provided particularly improved drug-to-antibody ratio, aggregation level, stability (i.e., in vitro and in vivo stability), targeting tumor effects (i.e., cytotoxicity, activity) and/or treatment efficacy. Improved treatment efficacy can be measured in vitro or in vivo and may include a reduction in tumor growth rate and/or a reduction in tumor volume.

[242] In certain embodiments, alternative antibodies to the same targets or antibodies to different target antigens are used and provide at least some of the beneficial functional properties described above (e.g., improved stability, improved tumor targeting, improved treatment effectiveness, etc.). In some embodiments, some or all of these beneficial functional properties are observed when the disclosed linkers and herboxydiene splicing modulator payloads are conjugated to an alternative antibody or antigen binding moiety that specifically targets HER2, CD138, or EPHA2. In some other embodiments, some or all of these beneficial functional properties are observed when the disclosed linkers and herboxydiene splicing modulator payloads are conjugated to an antibody or antigen binding moiety that specifically targets HER2. In some embodiments, the antibody or antigen binding fragment specifically targets HER2. In some embodiments, the antibody or antigen binding fragment that specifically targets HER2 is trastuzumab. In some other embodiments, some or all of these beneficial functional properties are observed when the disclosed linkers and herboxydiene splicing modulator payloads are conjugated to an antibody or antigen binding moiety that targets CD138. In some embodiments, the antibody or antigen binding fragment specifically 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 beneficial functional properties are observed when the disclosed linkers and herboxydiene splicing modulator payloads are conjugated to an antibody or antigen binding moiety that specifically targets EPHA2. In some embodiments, the antibody or antigen binding fragment specifically targets EPHA2. In some embodiments, the antibody or antigen binding fragment targeting EPHA2 is 1C1.

Linkers

[243] In various embodiments, the linker in the ADC is stable outside the cell sufficiently to be therapeutically effective. In some embodiments, the linker is stable outside the cell such that the ADC remains intact when exposed to extracellular conditions (eg, prior to transport or delivery into the cell). The term "intact" as used in the context of an ADC means that the antibody or antigen binding fragment remains attached to the drug moiety (eg, a herboxidiene splicing modulator). As used herein, the term "stable" in the context of a linker or ADC containing a linker means that no more than 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than approximately 3% or no more than approximately 1% of the linkers (or any percentage therein) in a sample ADC are cleaved (or in the case of all ADCs are otherwise not intact) if the ADC is exposed to extracellular conditions. In some embodiments, the linkers and/or ADCs disclosed herein are unexpectedly stable compared to alternative linkers and/or ADCs with alternative linkers and/or herboxydiene splicing modulator payloads. In some embodiments, the ADCs 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.

[244] Whether the linker is stable outside the cell can be determined, for example, by placing the ADC in blood plasma for a predetermined period of time (for example, 2, 4, 6, 8, 16, 24, 48 or 72 hours) and then quantitating the amount of free drug fragment present in the blood plasma. Stability may provide time for the ADC to localize tumor cells for targeting and prevent premature release of the drug moiety, which may reduce the therapeutic index of the ADC by indiscriminately damaging both normal and tumor tissues. In some embodiments, the linker is stable outside the target cell and causes the drug moiety to be released from the ADC upon entry into the cell so that the drug can bind to its target (eg, the SF3b spliceosome complex). Thus, an effective linker will: (i) support the specific binding properties of the antibody or antigen binding fragment; (ii) provide delivery, for example, intracellular delivery of a drug moiety by stably attaching to an antibody or antigen-binding moiety; (iii) remain stable and intact until the ADC is transported or delivered to its target site; and (iv) provide a therapeutic effect, for example, a cytotoxic effect of the drug fragment after cleavage or release by an alternative mechanism.

[245] Linkers can influence the physicochemical properties of ADCs. Since many cytotoxic agents are hydrophobic in nature, binding to an antibody with an additional hydrophobic moiety may result in aggregation. ADC aggregates are insoluble and often limit the achievable drug load on the antibody, which can negatively impact ADC activity. Protein aggregates of biological substances, in general, have also been associated with increased immunogenicity. As shown below, the linkers disclosed herein result in ADCs with low levels of aggregation and desired drug loading levels.

[246] The linker may be “cleavable” or “non-cleavable” (Ducry and Stump (2010) Bioconjugate Chem. 21:5-13). Cleavable linkers are designed to release a drug moiety (eg, a herboxydiene splicing modulator) upon exposure to certain environmental factors, such as internalization into a target cell, while non-cleavable linkers typically rely on degradation of the antibody or antigen-binding moiety itself .

[247] In some embodiments, the linker is a non-cleavable linker. In some embodiments, the ADC fragment, which is a herboxydiene splicing modulator drug, is released upon degradation of the antibody or antigen binding fragment. Non-cleavable linkers typically remain covalently associated with at least one amino acid of the antibody and the drug after internalization into the target cell and undergo degradation within it. Numerous exemplary non-cleavable linkers are described herein, and others are known in the art. Exemplary non-cleavable linkers may contain a thioether, cyclohexyl, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or N-hydroxysuccinimide (NHS), one or more polyethylene glycol (PEG) moieties, for example 1, 2 , 3, 4, 5 or 6 PEG moieties, or one or more alkyl moieties.

[248] In some embodiments, the linker is a cleavable linker. A cleavable linker means any linker that contains a cleavable moiety. As used herein, the term “cleavable moiety” means any chemical bond that can be cleaved. Suitable cleavable chemical bonds are widely known in the art and include, but are not limited to, acid-labile bonds, protease/peptidase-labile bonds, photolabile bonds, disulfide bonds, and esterase-labile bonds. Linkers containing a cleavable moiety can release the herboxydiene splicing modulator drug moiety from the ADC by cleavage at a specific site in the linker.

[249] In some embodiments, the linker is cleaved in an intracellular environment such that cleavage of the linker sufficiently releases the herboxydiene splicing modulator drug moiety from the antibody or antigen binding moiety in the intracellular environment to activate the drug and/or make the drug therapeutically effective. In some embodiments, the herboxydiene splicing modulator drug fragment is not cleaved from the antibody or antigen binding fragment until the ADC enters a cell that expresses an antigen specific to the antibody or antigen binding fragment from the ADC, and the herboxydiene splicing modulator drug fragment is cleaved from the antibody or antigen binding fragment after entering the cell. In some embodiments, the linker includes a cleavable moiety that is positioned such that no portion of the linker or antibody or antigen binding moiety remains associated with the herboxydiene splicing modulator drug moiety after cleavage. Exemplary cleavable linkers include acid-labile linkers, protease/peptidase-sensitive linkers, photolabile linkers, dimethyl-, disulfide-, or sulfonamide-containing linkers.

[250] In some embodiments, the linker is a pH-sensitive linker that is sensitive to hydrolysis at certain pH values. Typically, the pH-sensitive linker is cleaved under acidic conditions. This cleavage strategy typically takes advantage of the lower pH in the endosomal (pH~5-6) and lysosomal (pH~4.8) intracellular compartments compared to the cytosol (pH~7.4) to drive hydrolysis of an acid-labile group in a linker such as hydrazone (Jain et al. (2015) Pharm Res 32:3526-40). In some embodiments, the linker is an acid-labile and/or hydrolyzable linker. For example, an acid-labile linker that is hydrolyzable in the lysosome and contains an acid-labile group (eg, a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic acid amide, orthoester, acetal, ketal, or the like) may be used. See , for example , US patents No. 5122368; 5824805; 5622929; Dubowchik and Walker (1999) Pharm Therapeutics 83:67-123; Neville et al. (1989) Biol Chem. 264:14653-61. Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable below pH 5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (such as, for example, a thioester attached to a therapeutic agent via an acylhydrazone linkage) (see, for example, US Pat. No. 5,622,929).

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

[252] In some embodiments, the linker is a cleavable disulfide linker. A variety of disulfide linkers are known in the art, including, for example, linkers that can be 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-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), SPDB and SMPT. See , for example , Thorpe et al. (1987) Cancer Res. 47:5924-31; Wawrzynczak et al., in Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987). See also US Patent No. 4880935. Disulfide linkers are typically used to take advantage of the abundance of intracellular thiols, which can facilitate the cleavage of their disulfide bonds. Intracellular concentrations of the most abundant intracellular thiol, reduced glutathione, are typically in the range of 1–10 nM, which is approximately 1000 times higher than the blood concentration of the most abundant low molecular weight thiol (i.e., cysteine) at approximately 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 may also be involved in intracellular cleavage of the disulfide linker. As used herein, a cleavable disulfide linker means any linker that contains a cleavable disulfide moiety. The term "cleavable disulfide moiety" means a disulfide bond that can be cleaved and/or reduced, for example, by a thiol or an enzyme.

[253] In some embodiments, the linker is a cleavable sulfonamide linker. As used herein, a cleavable sulfonamide linker means any linker that contains a cleavable sulfonamide moiety. The term "cleavable sulfonamide moiety" means a sulfonamide group, i.e. a sulfonyl group attached to an amino group where the sulfur-nitrogen bond can be cleaved.

[254] In some embodiments, the linker may be a dendritic type linker for covalently attaching more than one drug moiety to an antibody or antigen binding moiety using a branched multifunctional linker moiety. See , for example , Sun et al. (2002) Bioorg Med Chem Lett. 12:2213-5; Sun et al. (2003) Bioorg Med Chem. 11:1761-8. Dendritic linkers can increase the molar ratio of drug to antibody, i.e. drug load, which is associated with ADC activity. Thus, if the antibody or antigen binding fragment contains only one reactive cysteine thiol group, then, for example, multiple herboxydiene splicing modulator drug moieties can be attached via a dendritic linker. In some embodiments, a linker moiety or linker moiety that is a drug can be attached to an antibody or antigen-binding moiety by disulfide bridge reduction chemistry or limited lysine technology. See , for example , publications of international applications Nos. WO 2013/173391 and WO 2013/173393.

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

[256] In some embodiments, the linker is a cleavable peptide linker. As used herein, a cleavable peptide linker means any linker that contains a cleavable peptide fragment. The term "cleavable peptide fragment" means any chemical bond linking amino acids (natural amino acids or synthetic amino acid derivatives) that can be cleaved by an agent that is 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 is cleavable by a peptidase such as a cathepsin, such as cathepsin B. In some embodiments, the linker may contain a glutamine sequence acid-valine-citrulline (Glu-Val-Cit). In some embodiments, the linker is an enzyme-cleavable linker, and the cleavable peptide moiety in the linker is enzyme-cleavable. In some embodiments, the cleavable peptide fragment is cleavable 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 is cleavable by a lysosomal cysteine cathepsin, such as cathepsin B, C, F, H, K, L, O, S, V, X, or W. In some embodiments, the cleavable peptide moiety is 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. 13:855-69).

[257] In some embodiments, the linker or cleavable peptide fragment in the linker comprises an amino acid unit. In some embodiments, the amino acid unit allows for cleavage of the linker by a protease, thereby facilitating release of the herboxydiene splicing modulator drug moiety from the ADC following exposure to one or more intracellular proteases, such as one or more lysosomal enzymes (Doronina et al. (2003) Nat Biotechnol. 21:778-84; Dubowchik and Walker (1999) Pharm Therapeutics 83:67-123). Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include, but are not limited to, valine-alanine (Val-Ala), valine-citrulline (Val-Cit), alanine-asparagine (Ala-Asn), alanine-phenylalanine (Ala-Phe), phenylalanine-lysine (Phe-Lys) , alanine-lysine (Ala-Lys), alanine-valine (Ala-Val), valine-lysine (Val-Lys), lysine-lysine (Lys-Lys), phenylalanine-citrulline (Phe-Cit), leucine-citrulline ( Leu-Cit), isoleucine-citrulline (Ile-Cit), tryptophan-citrulline (Trp-Cit) and phenylalanine-alanine (Phe-Ala). Exemplary tripeptides include, but are not limited to, alanine-alanine-asparagine (Ala-Ala-Asn), glycine-valine-citrulline (Gly-Val-Cit), glycine-glycine-glycine (Gly-Gly-Gly), phenylalanine-phenylalanine-lysine ( Phe-Phe-Lys), glutamic acid-valine-citrulline (Glu-Val-Cit) (see Anami et al. (2018) Nat Comm. 9:2512) and glycine-phenylalanine-lysine (Gly-Phe-Lys) . Other exemplary amino acid units include, but are not limited to, Gly-Phe-Gly-Gly (SEQ ID NO: 34), Gly-Phe-Leu-Gly (SEQ ID NO: 35), Ala-Leu-Ala-Leu (SEQ ID NO: 36 ), Phe-N ⁹ -tosyl-Arg and Phe-N ⁹ -nitro-Arg, described, for example, in US patent No. 6214345. In some embodiments, the amino acid unit in the linker contains 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 contain naturally occurring amino acid residues and/or minor amino acids and/or non-naturally occurring amino acid analogues such as citrulline. The amino acid units can be designed and optimized for enzymatic degradation by a specific enzyme, for example, a tumor associated protease, a lysosomal protease such as cathepsin B, C, D or S, or plasmin protease.

[258] In some embodiments, the linker is a cleavable β-glucuronide linker. As used herein, a cleavable β-glucuronide linker means any linker that contains a cleavable β-glucuronide moiety. An exemplary cleavable β-glucuronide linker provides the structure:

[259] The term "cleavable β-glucuronide moiety" means a glycosidic bond that can be cleaved by an agent having β-glucuronidase activity. In some embodiments, the linker provides a glycosidic bond that can be cleaved by β-glucuronidase. β-glucuronidase is a UDP-glucuronosyltransferase that catalyzes the hydrolysis of the glycosidic bond of β-configured glucuronides.

[260] In some embodiments, the ADC disclosed herein contains a cleavable β-glucuronide moiety in a linker that is enzymatically cleavable. In some embodiments, the cleavable β-glucuronide moiety in the linker is cleavable by a lysosomal enzyme, such as β-glucuronidase. In some embodiments, the linker is a β-glucuronidase cleavable linker. In some embodiments, a cleavable β-glucuronide moiety in the linker allows for cleavage of the linker by β-glucuronidase following internalization of the ADC, thereby facilitating release of the drug moiety from the ADC in the cellular environment.

[261] In some embodiments, a linker in any of the ADCs disclosed herein may comprise at least one spacer unit that attaches an antibody or antigen binding moiety to a drug moiety (e.g., a drug moiety which is a splicing modulator based on herboxydiene). In some embodiments, the spacer link between the antibody or antigen binding fragment and the cleavable fragment, if present, connects the cleavage site (eg, the cleavable peptide fragment) in the linker to the antibody or antigen binding fragment. In some embodiments, the drug-cleavage moiety spacer link, if present, connects a cleavage site (eg, a cleavable peptide moiety) in the linker to the drug moiety. In some embodiments, there is no cleavage site and a spacer unit is used to link the antibody or antigen binding fragment to the drug moiety.

[262] In some embodiments, the linker and/or the spacer unit in the linker are substantially hydrophilic. A hydrophilic linker can be used to reduce the extent to which a drug can be cleared from resistant cancer cells by multidrug resistance (MDR) protein or functionally similar transporters. In some embodiments, the hydrophilic linker may contain one or more polyethylene glycol (PEG) moieties, such as 1, 2, 3, 4, 5 or 6 PEG moieties. In some embodiments, the linker includes 2 PEG moieties.

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

[264] In some embodiments, the spacer unit in the linker contains an alkyl moiety. In some embodiments, the spacer unit includes one or more -(CH ₂ ) _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 from 1 to 10; from 2 to 8; from 2 to 6; from 2 to 5; from 2 to 4 or from 2 to 3. In some embodiments, n is 2. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, the spacer unit comprises (CH ₂ ) ₂ , (CH ₂ ) ₃ , (CH ₂ ) ₄ , (CH ₂ ) ₅ , (CH ₂ ) ₆ , (CH ₂ ) ₇ , (CH ₂ ) ₈ , (CH ₂ ) ₉ or (CH ₂ ) ₁₀ . In some embodiments, the spacer unit comprises (CH ₂ ) ₂ (“Et”). In some embodiments, the spacer unit comprises (CH ₂ ) ₆ (“Hex”). In some embodiments, the spacer unit comprises (CH ₂ ) ₂ -O-(CH ₂ ) ₂ ("Et-O-Et").

[265] The spacer unit can be used, for example, to link an antibody or antigen binding fragment to a drug moiety, either directly or indirectly. In some embodiments, the spacer unit directly links the antibody or antigen binding moiety to the herboxidiene splicing modulator drug moiety. In some embodiments, the antibody or antigen binding moiety and the herboxydiene splicing modulator drug moiety are joined by a spacer unit containing one or more PEG moieties (e.g., (PEG) ₂ ) or one or more alkyl moieties (for example, (CH ₂ ) ₂ , (CH ₂ ) ₆ or (CH ₂ ) ₂ -O-(CH ₂ ) ₂ ). In some embodiments, the spacer unit indirectly links the antibody or antigen binding moiety to the herboxidiene splicing modulator drug moiety. In some embodiments, the spacer unit links the antibody or antigen-binding moiety to the herboxydiene splicing modulator drug moiety indirectly through a cleavable moiety (e.g., a cleavable peptide or a cleavable β-glucuronide) and/or a coupling moiety to attach a spacer unit to an antibody or antigen-binding moiety, such as a maleimide moiety.

[266] In various embodiments, the spacer unit is attached to an antibody or antigen-binding moiety (ie, an antibody or antigen-binding moiety) via a maleimide (Mal) moiety.

[267] A spacer unit that is attached to an antibody or antigen binding fragment by Mal is referred to herein as a “Mal spacer unit.” As used herein, the term “Mal” or “maleimide moiety” means a compound that contains a maleimide group and which is reactive with a sulfhydryl group, for example, the sulfhydryl group of a cysteine residue in an antibody or antigen-binding moiety. Other functional groups that are reactive with sulfhydryl groups (thiols) include, but are not limited to, iodoacetamide, bromoacetamide, vinylpyridine, disulfide, pyridyl disulfide, isocyanate and isothiocyanate. In some embodiments, the Mal spacer unit is reactive to a cysteine residue in the antibody or antigen binding moiety. In some embodiments, the Mal spacer unit is attached to the antibody or antigen binding moiety via a cysteine residue. In some embodiments, the Mal spacer unit includes a PEG moiety. In some embodiments, the Mal spacer unit includes an alkyl moiety.

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

[269] In some embodiments, the linker comprises a Mal spacer unit structure. In some embodiments, the Mal spacer unit comprises maleimidocaproyl (MC). In some embodiments, the linker provides an MC structure. In some embodiments, the linker has the structure Mal-(CH ₂ ) ₂ (“Mal-Et”). In some embodiments, the linker has the structure Mal-(CH ₂ ) ₆ ("Mal-Hex"). In some embodiments, the linker has the structure Mal-(CH ₂ ) ₂ -O-(CH ₂ ) ₂ ("Mal-Et-O-Et"). In some embodiments, the linker comprises a Mal-(PEG) ₂ structure. In some embodiments, the linker provides the Mal-(PEG) ₂ -CO structure.

[270] In various embodiments, the Mal spacer unit attaches an antibody or antigen binding fragment to a cleavable peptide fragment. In some embodiments, the linker includes a Mal spacer peptide unit. In some embodiments, the linker comprises a Mal-spacer unit-Val-Cit structure. In some embodiments, the Mal spacer unit comprises maleimidocaproyl (MC). In some embodiments, the linker provides the structure MC-Val-Cit.

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

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

[273] In various embodiments, the cleavable moiety in the linker is directly attached to the herboxydiene splicing modulator drug moiety. In other embodiments, the spacer unit is used to attach a cleavable moiety in the linker to a herboxydiene splicing modulator drug moiety. In various embodiments, the herboxydiene-based splicing modulator is attached to the cleavable moiety in the linker via a spacer unit.

[274] The spacer unit may be "self-cleavable" or "non-self-cleavable". A “non-self-cleaving” spacer unit is a spacer unit in which part or all of the spacer unit remains associated with the herboxydiene splicing modulator drug moiety after cleavage of the linker. Examples of non-self-cleaving spacer units include, but are not limited to, a glycine spacer unit and a glycine-glycine spacer unit. Non-self-cleaving spacer units can sometimes degrade over time, but this does not readily release the bound native drug moiety in its entirety under cellular conditions. The “self-destructive” spacer unit ensures the release of the native fragment, which is the drug, under intracellular conditions. "Native drug" or "native drug fragment" is a drug in which no portion of the spacer unit or other chemical modification remains after cleavage/decomposition of the spacer unit.

[275] Self-destructive chemical structures are known in the art and can be readily selected for the disclosed ADCs. In various embodiments, the spacer unit connecting the cleavable moiety in the linker to the herboxydiene splicing modulator drug moiety is self-cleavable and undergoes self-cleavage shortly before or after cleavage of the cleavable moiety under intracellular conditions. In some embodiments, the herboxydiene splicing modulator is attached to the cleavable moiety in the linker via a self-cleavable spacer unit. In certain embodiments, the herboxydiene-based splicing modulator is attached to the cleavable moiety in the linker by a self-cleavable spacer unit, wherein the cleavable moiety comprises Val-Cit and maleimidocaproyl (MC) attaches the cleavable moiety to the antibody or antigen-binding moiety. In certain embodiments, the herboxydiene-based splicing modulator is attached to a cleavable moiety in the linker by a self-cleavable spacer unit, wherein the cleavable moiety comprises Val-Ala and maleimidocaproyl (MC) attaches the cleavable moiety to the antibody or antigen-binding moiety. In certain embodiments, the herboxydiene-based splicing modulator is attached to the cleavable moiety in the linker by a self-cleavable spacer unit, wherein the cleavable moiety comprises Glu-Val-Cit and maleimidocaproyl (MC) attaches the cleavable moiety to the antibody or antigen-binding moiety. In certain embodiments, the herboxydiene splicing modulator is attached to the antibody or antigen binding moiety via a Mal spacer unit (eg, MC) in a linker attached to a Val-Cit cleavable moiety and a pABC or pAB self-cleavable spacer unit. In certain other embodiments, the herboxydiene splicing modulator is attached to the antibody or antigen binding moiety via a Mal spacer unit (eg, MC) in a linker attached to a Val-Ala cleavable moiety and a pABC or pAB self-cleavable spacer unit. In certain other embodiments, the herboxydiene splicing modulator is attached to the antibody or antigen binding moiety via a Mal spacer unit (eg, MC) in a linker attached to a Glu-Val-Cit cleavable moiety and a pABC or pAB self-cleavable spacer unit.

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

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

[278] In various embodiments, the self-cleavable spacer unit attaches a cleavable moiety in a linker to a herboxidiene-based splicing modulator. In some embodiments, the self-cleaving spacer unit is pABC. In some embodiments, pABC attaches a cleavable moiety in a linker to a herboxydiene-based splicing modulator. In some embodiments, the pABC undergoes self-cleavage upon cleavage of the cleavage moiety and the herboxydiene splicing modulator is released from the ADC in its native active form.

[279] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof is linked to a herboxydiene-based splicing modulator via a linker comprising MC-Val-Cit-pABC. In other embodiments, an anti-HER2 antibody or antigen-binding fragment thereof is linked to a herboxydiene-based splicing modulator via a linker comprising MC-Val-Ala-pABC.

[280] In some embodiments, the anti-CD138 antibody or antigen-binding fragment thereof is linked to a herboxydiene-based splicing modulator via a linker comprising MC-Val-Cit-pABC. In other embodiments, the anti-CD138 antibody or antigen-binding fragment thereof is linked to a herboxydiene-based splicing modulator via a linker comprising MC-Val-Ala-pABC.

[281] In some embodiments, the anti-EPHA2 antibody or antigen binding fragment thereof is linked to a herboxydiene splicing modulator via a linker comprising MC-Val-Cit-pABC. In other embodiments, the anti-EPHA2 antibody or antigen-binding fragment thereof is linked to a herboxydiene-based splicing modulator via a linker comprising MC-Val-Ala-pABC.

[282] In some embodiments, pABC undergoes self-cleavage following cleavage of the cleavable peptide moiety in the linker. In some embodiments, the cleavable peptide fragment includes an amino acid unit. In some embodiments, the linker comprises a pABC amino acid unit. In some embodiments, the amino acid unit is Val-Cit. In some embodiments, the linker comprises Val-Cit-pABC. In some embodiments, the amino acid unit is Glu-Val-Cit. In some embodiments, the linker comprises Glu-Val-Cit-pABC. In some embodiments, the amino acid unit is Val-Ala. In some embodiments, the linker comprises Val-Ala-pABC. In some embodiments, the amino acid unit is Ala-Ala-Asn. In some embodiments, the linker provides Ala-Ala-Asn-pABC.

[283] In some embodiments, pABC undergoes self-cleavage following cleavage of the cleavable β-glucuronide moiety in the linker. In some embodiments, the linker comprises β-glucuronide-pABC.

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

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

[286] In various embodiments, the self-cleavable spacer unit attaches a cleavable moiety in a linker to a herboxidiene-based splicing modulator. In some embodiments, the self-cleaving spacer unit is pAB. In some embodiments, pAB attaches a cleavable moiety in a linker to a herboxydiene-based splicing modulator. In some embodiments, the pAB undergoes self-cleavage upon cleavage of the cleavage moiety and the herboxydiene-based splicing modulator is released from the ADC in its native active form.

[287] In some embodiments, the anti-HER2 antibody or antigen-binding fragment thereof is linked to a herboxydiene-based splicing modulator via a linker comprising MC-Val-Cit-pAB. In other embodiments, an anti-HER2 antibody or antigen binding fragment thereof is linked to a herboxydiene splicing modulator via a linker comprising MC-Val-Ala-pAB.

[288] In some embodiments, the anti-CD138 antibody or antigen-binding fragment thereof is linked to a herboxydiene-based splicing modulator via a linker comprising MC-Val-Cit-pAB. In other embodiments, an anti-CD138 antibody or antigen-binding fragment thereof is linked to a herboxydiene-based splicing modulator via a linker comprising MC-Val-Ala-pAB.

[289] In some embodiments, the anti-EPHA2 antibody or antigen-binding fragment thereof is linked to a herboxydiene-based splicing modulator via a linker comprising MC-Val-Cit-pAB. In other embodiments, an anti-EPHA2 antibody or an antigen-binding fragment thereof is linked to a herboxydiene-based splicing modulator via a linker comprising MC-Val-Ala-pAB.

[290] In some embodiments, pAB undergoes self-cleavage following cleavage of the cleavable peptide moiety in the linker. In some embodiments, the cleavable peptide fragment includes an amino acid unit. In some embodiments, the linker comprises a pAB amino acid unit. 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 provides Ala-Ala-Asn-pAB.

[291] In some embodiments, pAB undergoes self-cleavage following cleavage of the cleavable β-glucuronide moiety in the linker. In some embodiments, the linker comprises a β-glucuronide-pAB.

[292] In some other embodiments, the herboxydiene splicing modulator is attached to the cleavable moiety in the linker via a non-self-cleavable spacer unit. In certain embodiments, the herboxydiene-based splicing modulator is attached to the cleavable moiety in the linker by a non-self-cleavable spacer unit, wherein the cleavable moiety comprises Val-Cit and maleimidocaproyl (MC) attaches the cleavable moiety to the antibody or antigen-binding moiety. In certain embodiments, the herboxydiene-based splicing modulator is attached to a cleavable moiety in a linker by a non-self-cleavable spacer unit, wherein the cleavable moiety comprises Val-Ala and maleimidocaproyl (MC) attaches the cleavable moiety to the antibody or antigen-binding moiety.

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

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

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

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

[297] In various embodiments, the ADC compound has formula (I):

Ab-(LH) _p (I),

wherein Ab is an antibody or antigen-binding fragment that specifically targets a neoplastic cell;

H is a herboxidiene-based splicing modulator;

L is a linker that covalently attaches Ab to D; And

p represents an integer between 1 and 15.

[298] In some embodiments, an antibody or antigen binding fragment (Ab) from an ADC is conjugated to a herboxydiene splicing modulator drug moiety via a linker, wherein the linker is any of the linkers disclosed herein or included therein by reference, or contains one or more components of any of the linkers disclosed or incorporated herein by reference.

[299] In some embodiments, the linker includes a cleavable moiety that is positioned such that no portion of the linker or antibody or antigen binding moiety remains associated with the herboxydiene splicing modulator after cleavage. In some embodiments, the cleavable fragment is a cleavable peptide fragment, for example, 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.

[300] In some embodiments, the linker includes at least one spacer unit connecting the antibody or antigen binding fragment to the cleavable fragment. In some embodiments, the linker includes at least one spacer unit connecting the antibody or antigen binding fragment to the drug moiety. In some embodiments, the spacer unit or linker includes at least one alkyl moiety.

[301] In some embodiments, the spacer unit in the linker is attached to the antibody or antigen binding fragment by a Mal fragment ("Mal spacer unit"). In some embodiments, the Mal spacer unit includes at least one alkyl moiety. In some embodiments, the linker includes maleimidocaproyl (MC). In some embodiments, the linker comprises Mal-(CH ₂ ) ₂ (“Mal-Et”). In some embodiments, the linker comprises Mal-(CH ₂ ) ₆ (“Mal-Hex”). In some embodiments, the linker provides Mal-(CH ₂ ) ₂ -O-(CH ₂ ) ₂ (“Mal-Et-O-Et”). In some embodiments, the linker comprises Mal-(PEG) ₂ -CO. In some embodiments, the Mal spacer unit attaches an antibody or antigen binding fragment to a drug moiety.

[302] In some embodiments, the Mal spacer unit or linker comprises Mal-(PEG)₂, Mal-(PEG)₃, Mal-(PEG)₄, Mal-(PEG)₅, Mal-(PEG)₆, Mal-(PEG)₇ or Mal-(PEG)₈. In some embodiments, the Mal spacer unit or linker comprises Mal-(PEG)₂. In some embodiments, the Mal spacer unit or linker comprises Mal-(PEG)₂-CO, Mal-(PEG)₃-CO, Mal-(PEG)₄-CO, Mal-(PEG)₅-CO, Mal-(PEG)₆-CO, Mal-(PEG)₇-CO or Mal-(PEG)₈-CO. In some embodiments, the Mal spacer unit or linker comprises Mal-(PEG)₂-CO. In some embodiments, the Mal spacer unit or linker comprises Mal-(PEG)₂-CO and at least one additional spacer unit. In some embodiments, Mal-(PEG)₂-CO attaches the antibody or antigen-binding moiety to the drug moiety. In some embodiments, the linker comprises Mal-(PEG)₂-CO or consists of it. Example of a Mal-(PEG) linker₂-CO" is also referred to herein as "ADL2" or the "ADL2" linker.

[303] In some embodiments, the Mal spacer unit or linker includes an MC. In some embodiments, the Mal spacer unit or linker includes an MC and at least one additional spacer unit. In some embodiments, the MC attaches an antibody or antigen-binding fragment to a drug moiety. In some embodiments, the linker includes or consists of an MC. An example "MC" linker is also referred to herein as "ADL10" or "ADL10" linker.

[304] In some embodiments, the Mal spacer unit or linker comprises Mal-(CH ₂ ) ₆ (“Mal-Hex”). In some embodiments, the Mal spacer unit or linker comprises Mal-Hex and at least one additional spacer unit. In some embodiments, Mal-Hex attaches an antibody or antigen-binding fragment to a drug moiety. In some embodiments, the linker comprises Mal-Hex. An example of a "Mal-Hex" linker is also referred to herein as "ADL12" or "ADL12" linker.

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

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

[307] In some other embodiments, the Mal spacer unit attaches an antibody or antigen binding fragment to a cleavable moiety in the linker. In some embodiments, the cleavable moiety in the linker is a cleavable peptide moiety, such as an amino acid unit. In some embodiments, the cleavable peptide fragment is Val-Cit or Val-Ala. In some embodiments, the Mal spacer unit or linker includes an MC. In some embodiments, the linker includes MC-Val-Cit. In some embodiments, the linker includes MC-Val-Ala. In some embodiments, the linker includes MC-Glu-Val-Cit. In some embodiments, the linker provides MC-Ala-Ala-Asn.

[308] In some embodiments, the spacer unit attaches a cleavable moiety in a linker to a herboxydiene-based splicing modulator. In some embodiments, the spacer unit that attaches the cleavage moiety to the herboxydiene splicing modulator is self-cleavable.

[309] In some embodiments, the spacer unit comprises pABC. In some embodiments, pABC attaches the cleavage moiety to a herboxydiene-based splicing modulator. In some embodiments, the cleavable fragment is a cleavable peptide fragment, such as an amino acid unit. In some embodiments, the linker comprises a pABC amino acid unit.

[310] In some embodiments, the linker comprises Val-Cit-pABC. In some embodiments, the linker includes a Val-Cit-pABC and an MC-Mal spacer unit attaching the linker to the antibody or antigen binding moiety. In some embodiments, the linker comprises MC-Val-Cit-pABC. In some embodiments, the linker includes MC-Val-Cit-pABC and at least one additional spacer unit. The example linker MC-Val-Cit-pABC is also referred to herein as “ADL1” or “ADL1” linker.

[311] In some embodiments, the linker comprises Val-Ala-pABC. In some embodiments, the linker includes a Val-Ala-pABC and an MC-Mal spacer unit attaching the linker to the antibody or antigen binding moiety. In some embodiments, the linker comprises MC-Val-Ala-pABC. In some embodiments, the linker includes MC-Val-Ala-pABC and at least one additional spacer unit. An example linker MC-Val-Ala-pABC is also referred to herein as “ADL6” or “ADL6” linker.

[312] In some embodiments, the linker comprises Glu-Val-Cit-pABC. In some embodiments, the linker includes a Glu-Val-Cit-pABC and an MC-Mal spacer unit connecting the linker to the antibody or antigen binding moiety. In some embodiments, the linker comprises MC-Glu-Val-Cit-pABC. In some embodiments, the linker includes MC-Glu-Val-Cit-pABC and at least one additional spacer unit. An example linker MC-Glu-Val-Cit-pABC is also referred to herein as “ADL23” or “ADL23” linker.

[313] In some embodiments, the linker comprises Ala-Ala-Asn-pABC. In some embodiments, the linker includes an Ala-Ala-Asn-pABC and an MC-Mal spacer unit connecting the linker to the antibody or antigen binding moiety. In some embodiments, the linker comprises MC-Ala-Ala-Asn-pABC. In some embodiments, the linker includes MC-Ala-Ala-Asn-pABC and at least one additional spacer unit. An example linker MC-Ala-Ala-Asn-pABC is also referred to herein as “ADL21” or “ADL21” linker.

[314] In some other embodiments, the spacer unit is pAB. In some embodiments, the pAB attaches the cleavable moiety to a herboxydiene-based splicing modulator. In some embodiments, the cleavable fragment is a cleavable peptide fragment, such as an amino acid unit. In some embodiments, the linker comprises a pAB amino acid unit.

[315] In some embodiments, the linker comprises Val-Ala-pAB. In some embodiments, the linker includes a Val-Ala-pAB and an MC-Mal spacer unit attaching the linker to the antibody or antigen binding moiety. In some embodiments, the linker comprises MC-Val-Ala-pAB. In some embodiments, the linker includes MC-Val-Ala-pAB and at least one additional spacer unit. An example linker MC-Val-Ala-pAB is also referred to herein as "ADL5" or "ADL5" linker.

[316] In some embodiments, the linker comprises Val-Cit-pAB. In some embodiments, the linker includes a Val-Cit-pAB and an MC-Mal spacer unit attaching the linker to the antibody or antigen binding moiety. In some embodiments, the linker comprises MC-Val-Cit-pAB. In some embodiments, the linker includes MC-Val-Cit-pAB and at least one additional spacer unit. An example linker MC-Val-Cit-pAB is also referred to herein as “ADL7” or “ADL7” linker.

[317] In some embodiments, the linker comprises β-glucuronide-pABC. In some embodiments, the linker includes a β-glucuronide-pABC and an MC-Mal spacer unit attaching the linker to the antibody or antigen binding moiety. In some embodiments, the linker comprises MC-β-glucuronide-pABC. In some embodiments, the linker comprises MC-β-glucuronide-pABC and at least one additional spacer unit. Example MC-β-glucuronide-pABC is also referred to herein as "ADL13" or the linker "ADL13".

[318] In some embodiments, the linker comprises a β-glucuronide-pAB. In some embodiments, the linker includes a β-glucuronide-pAB and an MC-Mal spacer unit attaching the linker to the antibody or antigen binding moiety. In some embodiments, the linker comprises MC-β-glucuronide-pAB.

[319] In some embodiments, the antibody or antigen binding moiety is conjugated to a herboxydiene splicing modulator drug moiety via a linker ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23. In various embodiments, it has been discovered that ADCs comprising an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker and a splicing modulator drug moiety disclosed herein based on herboxydiene, exhibit properties required for therapeutic ADC. In various embodiments, these properties include, but are not limited to, effective drug loading levels, low levels of aggregation, stability under storage conditions or when in the body's bloodstream (e.g., stability in serum), maintaining affinity for cells expressing the target comparable to affinity of the unconjugated antibody, potent cytotoxicity to cells expressing the target, low levels of off-target cell killing, high levels of non-specific killing and/or effective anti-cancer activity in vivo, all of which are compared to ADCs that use other linker-payload constructs. For example, in various embodiments, ADCs comprising an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker and a herboxidiene-based splicing modulator drug moiety disclosed herein , exhibit increased ability to inhibit the growth and/or proliferation of target-expressing cells compared to ADCs that utilize other linker-payload constructs (e.g., ADL10 linker and herboxydiene-based splicing modulator drug moiety ). In various embodiments, ADCs comprising an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker and a herboxidiene-based splicing modulator drug moiety disclosed herein exhibit unexpectedly increased in vivo stability (e.g., stability in blood plasma) compared to another splicing modulator ADC (e.g., thailanstatin A ADC, for example, as reported in Puthenveetil et al. Bioconjugate Chem. (2016) 27:1880- 8).

[320] In some embodiments, the adequate or superior functionality provided by a particular combination of an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker and a drug moiety disclosed herein representing a splicing modulator at herboxydiene can be observed if the linker-payload construct is conjugated, for example, to an anti-HER2 antibody such as trastuzumab; an anti-CD138 antibody such as B-B4; or an anti-EPHA2 antibody such as 1C1.

[321] In some embodiments, the ADC comprises an ADL1 herboxydiene splicing modulator and an antibody or antigen binding fragment comprising an antibody or antigen binding fragment thereof that retains the ability to be targeted to and internalized into a neoplastic cell. In some embodiments, the ADC comprises an ADL2 herboxydiene splicing modulator and an antibody or antigen binding fragment comprising an antibody or antigen binding fragment thereof that retains the ability to be targeted to and internalized into a neoplastic cell. In some embodiments, the ADC comprises an ADL5 herboxydiene splicing modulator and an antibody or antigen binding fragment comprising an antibody or antigen binding fragment thereof that retains the ability to be targeted to and internalized into a neoplastic cell. In some embodiments, the ADC comprises an ADL6 herboxydiene splicing modulator and an antibody or antigen binding fragment comprising an antibody or antigen binding fragment thereof that retains the ability to be targeted to and internalized into a neoplastic cell. In some embodiments, the ADC comprises an ADL7 herboxydiene splicing modulator and an antibody or antigen binding fragment comprising an antibody or antigen binding fragment thereof that retains the ability to be targeted to and internalized into a neoplastic cell. In some embodiments, the ADC comprises an ADL12 herboxydiene splicing modulator and an antibody or antigen binding fragment comprising an antibody or antigen binding fragment thereof that retains the ability to be targeted to and internalized into a neoplastic cell. In some embodiments, the ADC comprises an ADL13 herboxydiene splicing modulator and an antibody or antigen binding fragment comprising an antibody or antigen binding fragment thereof that retains the ability to be targeted to and internalized into a neoplastic cell. In some embodiments, the ADC comprises an ADL14 herboxydiene splicing modulator and an antibody or antigen binding fragment comprising an antibody or antigen binding fragment thereof that retains the ability to be targeted to and internalized into a neoplastic cell. In some embodiments, the ADC comprises an ADL15 herboxydiene splicing modulator and an antibody or antigen binding fragment comprising an antibody or antigen binding fragment thereof that retains the ability to be targeted to and internalized into a neoplastic cell.

[322] In some embodiments, the ADC comprises an ADL1 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing HER2. In some embodiments, the ADC comprises an ADL2 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing HER2. In some embodiments, the ADC comprises an ADL5 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing HER2. In some embodiments, the ADC comprises an ADL6 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing HER2. In some embodiments, the ADC comprises an ADL7 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing HER2. In some embodiments, the ADC comprises an ADL12 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing HER2. In some embodiments, the ADC comprises an ADL13 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing HER2. In some embodiments, the ADC comprises an ADL14 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing HER2. In some embodiments, the ADC comprises an ADL15 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing HER2.

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

[324] In some embodiments, the ADC has formula (I):

Ab-(LH) _p (I),

Where:

(i) The Ab is an anti-HER2 antibody or an antigen-binding fragment thereof containing 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 light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2) and SEQ ID NO: 6 (LCDR3);

(ii) H is a herboxidiene-based splicing modulator;

(iii) L is a linker comprising ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21 or ADL23; And

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

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

[326] In some embodiments, the ADC comprises an ADL1 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing CD138. In some embodiments, the ADC comprises an ADL2 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing CD138. In some embodiments, the ADC comprises an ADL5 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing CD138. In some embodiments, the ADC comprises an ADL6 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing CD138. In some embodiments, the ADC comprises an ADL7 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing CD138. In some embodiments, the ADC comprises an ADL12 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing CD138. In some embodiments, the ADC comprises an ADL13 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing CD138. In some embodiments, the ADC comprises an ADL14 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing CD138. In some embodiments, the ADC comprises an ADL15 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing CD138.

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

[328] In some embodiments, the ADC has formula (I):

Ab-(LH) _p (I),

Where:

(i) The Ab is an anti-CD138 antibody or antigen binding fragment thereof containing 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 light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 10 (LCDR1), SEQ ID NO: 11 (LCDR2) and SEQ ID NO: 12 (LCDR3);

(ii) H is a herboxidiene-based splicing modulator;

(iii) L is a linker comprising ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21 or ADL23; And

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

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

[330] In some embodiments, the ADC comprises an ADL1 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing EPHA2. In some embodiments, the ADC comprises an ADL2 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing EPHA2. In some embodiments, the ADC comprises an ADL5 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing EPHA2. In some embodiments, the ADC comprises an ADL6 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing EPHA2. In some embodiments, the ADC comprises an ADL7 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing EPHA2. In some embodiments, the ADC comprises an ADL12 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing EPHA2. In some embodiments, the ADC comprises an ADL13 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing EPHA2. In some embodiments, the ADC comprises an ADL14 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing EPHA2. In some embodiments, the ADC comprises an ADL15 herboxydiene splicing modulator and an antibody or antigen binding fragment thereof that specifically targets a neoplastic cell expressing EPHA2.

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

[332] In some embodiments, the ADC has formula (I):

Ab-(LH) _p (I),

Where:

(i) The Ab is an anti-EPHA2 antibody or an antigen-binding fragment thereof containing 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 light chain complementarity determining regions (LCDRs) comprising the amino acid sequences of SEQ ID NO: 16 (LCDR1), SEQ ID NO: 17 (LCDR2) and SEQ ID NO: 18 (LCDR3);

(ii) H is a herboxidiene-based splicing modulator;

(iii) L is a linker comprising ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21 or ADL23; And

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

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

Herboxidiene-based splicing modulators

[334] In some embodiments, the antibody-drug conjugate is an antibody-drug conjugate of formula (I): Ab-(LH) _p , wherein Ab is an antibody or antigen-binding moiety that specifically targets a neoplastic cell; H is a herboxidiene-based splicing modulator; L is a linker that covalently attaches Ab to H; and p is an integer from 1 to 15.

[335] In some embodiments, H, a herboxydiene-based splicing modulator, provides a compound of formula (I): (I), or a pharmaceutically acceptable salt thereof, which are covalently attached to L through any atom where

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

each of R ¹ , R ² and R ³ is independently selected from hydrogen, hydroxyl, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups, -C groups (=O)-O-(C ₁ -C ₆ alkyl) and C ₁ -C ₆ alkyl groups;

R ⁴ is selected from hydrogen, C ₁ -C ₆ alkyl groups, -C(=O)-(C ₁ -C ₆ alkyl) groups, -C(=O)-(C ₃ -C ₈ carbocyclyl) groups, - C(=O)-(C ₃ -C ₈ heterocyclyl) and -C(=O)-NR ⁶ R ⁷ ;

R ⁵ is selected from hydrogen, hydroxyl, -CH ₂ -OH, -CO ₂ H, -C(=O)-O-(C ₁ -C ₆ alkyl), -C(=O)-NR ⁶ R ⁷ groups, -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 ;

R ⁶ and R ⁷ are each independently selected from hydrogen, -R ⁸ , -C(=O)-R ⁸ and -C(=O)-OR ⁸ ; And

R ⁸ is selected from C ₁ -C ₆ alkyl groups, C ₃ -C ₈ carbocyclyl groups and C ₃ -C ₈ heterocyclyl groups,

where each of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ is independently substituted with 0-3 groups independently selected from halogens, hydroxyl, C ₁ -C ₆ alkyl groups, groups - O-(C ₁ -C ₆ alkyl), -CO ₂ H, groups -C(=O)-(C ₁ -C ₆ alkyl), groups -C(=O)-(C ₃ -C ₈ carbocyclyl), groups -C(=O)-(C ₃ -C ₈ heterocyclyl), -NR ⁶ R ⁷ , C ₃ -C ₈ carbocyclyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkyl alkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups, each of which may be independently substituted with 0 or 1 group selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups, C ₁ -C ₃ haloalkyl groups, -NH- C(=O)(C ₁ -C ₃ alkyl) and -NH-C(=O)-O-(C ₁ -C ₃ alkyl), and

where the valency of the atom that is covalently attached to L is not exceeded.

[336] In some embodiments, H, a herboxydiene-based splicing modulator, provides a compound of formula (Ia): (Ia), or a pharmaceutically acceptable salt thereof, which are covalently attached to L through any atom where

R ⁹ is selected from C ₃ -C ₈ heterocyclyl groups;

R ¹⁰ is selected from H and C ₁ -C ₆ alkyl groups,

where each of R ⁹ and R ¹⁰ is independently substituted with 0-3 groups independently selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups, -NH ₂ , -NH-(C ₁ -C ₃ alkyl) and -N-(C ₁ -C ₃ alkyl) ₂ , and

where the valency of the atom that is covalently attached to L is not exceeded.

[337] In some embodiments, H, a herboxydiene-based splicing modulator, provides a compound of formula (Ib): (Ib), or its pharmaceutical

[338] acceptable salt, which are covalently attached to L through any atom where

R ¹¹ selected from , And , where * denotes the point of attachment of R ¹¹ to the rest of the connection;

R ¹² and R ¹³ are each independently selected from H and methyl; And

where the valency of the atom that is covalently attached to L is not exceeded.

[339] In some embodiments, H, a herboxydiene-based splicing modulator, provides a compound of formula (II): (II), or a pharmaceutically acceptable salt thereof, which are covalently attached to L through any atom where

X represents hydroxyl or NR ⁶ R ⁷ ;

R ⁶ and R ⁷ are each independently selected from hydrogen, -R ⁸ , -C(=O)-R ⁸ , -C(=O)-OR ⁸ , -(C ₁ -C ₆ alkyl)-OC(=O )-R ⁸ and -(C ₁ -C ₆ alkyl)-NH-C(=O)-R ⁸ ; And

R ⁸ is selected from C ₁ -C ₆ alkyl groups, C ₃ -C ₈ carbocyclyl groups and C ₃ -C ₈ heterocyclyl groups,

wherein each of R ⁶ , R ⁷ and R ⁸ is independently substituted with 0-3 groups independently selected from halogens, hydroxyl, C ₁ -C ₆ alkyl groups, -O-(C ₁ -C ₆ alkyl), -CO ₂ H groups , groups -C(=O)-O-(C ₁ -C ₆ alkyl), groups -C(=O)-(C ₁ -C ₆ alkyl), groups -C(=O)-(C ₃ -C ₈ carbocyclyl), -C(=O)-(C ₃ -C ₈ heterocyclyl) groups, -NR ⁶ R ⁷ , C ₃ -C ₈ carbocyclyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkylalkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups, each of which may be independently substituted with 0-1 group selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups, C ₁ -C ₃ haloalkyl groups , -NH-C(=O)(C ₁ -C ₃ alkyl) and -NH-C(=O)-O-(C ₁ -C ₃ alkyl), and

where the valency of the atom that is covalently attached to L is not exceeded.

[340] In some embodiments, H, a herboxydiene-based splicing modulator, provides a compound of formula (IIa): (II), or a pharmaceutically acceptable salt thereof, which are covalently attached to L through any atom where

Z is selected from NR ⁹ and O;

R ⁹ is selected from hydrogen and C ₁ -C ₆ alkyl groups;

each of R ¹⁰ and R ¹¹ is independently selected from hydrogen, halogens, hydroxyl, C ₁ -C ₆ alkyl groups, -O-(C ₁ -C ₆ alkyl) groups, -CO ₂ H, -C(=O)- groups O-(C ₁ -C ₆ alkyl), -C(=O)-(C ₁ -C ₆ alkyl) groups, -C(=O)-(C ₃ -C ₈ carbocyclyl) groups, -C(= O)-(C ₃ -C ₈ heterocyclyl), C ₃ -C ₈ carbocyclyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkyl alkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups;

R ¹² is selected from C ₁ -C ₆ alkyl groups, C ₃ -C ₈ carbocyclyl groups, C ₃ -C ₈ heterocyclyl groups,

wherein each of R ⁹ , R ¹⁰ , R ¹¹ and R ¹² is independently substituted with 0 or 1 group selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups and C ₁ -C ₃ haloalkyl groups;

t is an integer selected from 1, 2, 3, 4, 5 and 6; And

where the valency of the atom that is covalently attached to L is not exceeded.

[341] In some embodiments, H, a herboxydiene-based splicing modulator, provides a compound of formula (IIb): (IIb), or a pharmaceutically acceptable salt thereof, which are covalently attached to L through any atom where

R ¹³ selected from , , , , , , , , , And , where * denotes the point of attachment of R ¹³ to the rest of the connection;

R ¹⁴ and R ¹⁵ are each independently selected from hydrogen and methyl; And

where the valency of the atom that is covalently attached to L is not exceeded.

[342] In some embodiments, H, a herboxydiene-based splicing modulator, provides a compound of formula (III): (III), or a pharmaceutically acceptable salt thereof, which are covalently attached to L through any atom where

each of R ¹ , R ² and R ³ is independently selected from hydrogen, hydroxyl, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups, -C groups (=O)-O-(C ₁ -C ₆ alkyl) and C ₁ -C ₆ alkyl groups;

R ⁶ and R ⁷ are each independently selected from hydrogen, -R ⁸ , -C(=O)-R ⁸ and -C(=O)-OR ⁸ ;

R ⁸ is selected from C ₁ -C ₆ alkyl groups, C ₃ -C ₈ carbocyclyl groups and C ₃ -C ₈ heterocyclyl groups; And

R ⁹ selected from H, , , , , , , , , And ;

wherein each of R ¹ , R ² , R ³ , R ⁶ , R ⁷ and R ⁸ is independently substituted with 0-3 groups independently selected from halogens, hydroxyl, C ₁ -C ₆ alkyl groups, -O-(C ₁ - C ₆ alkyl), -CO ₂ H, -C(=O)-(C ₁ -C ₆ alkyl) groups, -C(=O)-(C ₃ -C ₈ carbocyclyl) groups, -C(=O) groups )-(C ₃ -C ₈ heterocyclyl), -NR ⁶ R ⁷ , C ₃ -C ₈ carbocyclyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkyl alkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups, each of which may be independently substituted with 0 or 1 group selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups, C ₁ -C ₃ haloalkyl groups, -NH-C(=O)( C ₁ -C ₃ alkyl) and -NH-C(=O)-O-(C ₁ -C ₃ alkyl),

where the valency of the atom that is covalently attached to L is not exceeded; And

where * denotes the point of attachment of R ⁹ to the rest of the connection.

[343] In some embodiments, the antibody or antigen binding fragment specifically targets a cell expressing HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, and/or or STEAP1.

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

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

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

[347] In some other embodiments, the antibody or antigen binding fragment specifically targets a cell expressing MSLN. In some embodiments, the antibody or antigen binding fragment is an anti-MSLN antibody or antigen binding fragment thereof.

[348] In some other embodiments, the antibody or antigen binding fragment specifically targets a cell expressing FOLH1. In some embodiments, the antibody or antigen binding fragment is an anti-FOLH1 antibody or antigen binding fragment thereof.

[349] In some other embodiments, the antibody or antigen binding fragment specifically targets a cell expressing CDH6. In some embodiments, the antibody or antigen binding fragment is an anti-CDH6 antibody or antigen binding fragment thereof.

[350] In some other embodiments, the antibody or antigen binding fragment specifically targets a cell expressing CEACAM5. In some embodiments, the antibody or antigen binding fragment is an anti-CEACAM5 antibody or antigen binding fragment thereof.

[351] In some other embodiments, the antibody or antigen binding fragment specifically targets a cell expressing CFC1B. In some embodiments, the antibody or antigen binding fragment is an anti-CFC1B antibody or antigen binding fragment thereof.

[352] In some other embodiments, the antibody or antigen binding fragment specifically targets a cell expressing ENPP3. In some embodiments, the antibody or antigen binding fragment is an anti-ENPP3 antibody or antigen binding fragment thereof.

[353] In some other embodiments, the antibody or antigen binding fragment specifically targets a cell expressing FOLR1. In some embodiments, the antibody or antigen binding fragment is an anti-FOLR1 antibody or antigen binding fragment thereof.

[354] In some other embodiments, the antibody or antigen binding fragment specifically targets a cell expressing HAVCR1. In some embodiments, the antibody or antigen binding fragment is an anti-HAVCR1 antibody or antigen binding fragment thereof.

[355] In some other embodiments, the antibody or antigen binding fragment specifically targets a cell expressing KIT. In some embodiments, the antibody or antigen binding fragment is an anti-KIT antibody or antigen binding fragment thereof.

[356] In some other embodiments, the antibody or antigen binding fragment specifically targets a cell expressing MET. In some embodiments, the antibody or antigen binding fragment is an anti-MET antibody or antigen binding fragment thereof.

[357] In some other embodiments, the antibody or antigen binding fragment specifically targets a cell expressing MUC16. In some embodiments, the antibody or antigen binding fragment is an anti-MUC16 antibody or antigen binding fragment thereof.

[358] In some other embodiments, the antibody or antigen binding fragment specifically targets a cell expressing SLC39A6. In some embodiments, the antibody or antigen binding fragment is an anti-SLC39A6 antibody or antigen binding fragment thereof.

[359] In some other embodiments, the antibody or antigen binding fragment specifically targets a cell expressing SLC44A4. In some embodiments, the antibody or antigen binding fragment is an anti-SLC44A4 antibody or antigen binding fragment thereof.

[360] In some other embodiments, the antibody or antigen binding fragment specifically targets a cell expressing STEAP1. In some embodiments, the antibody or antigen binding fragment is an anti-STEAP1 antibody or antigen binding fragment thereof.

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

[362] In certain embodiments, the intermediate that is the precursor of the linker moiety is reacted with a herboxydiene splicing modulator moiety under suitable conditions. In certain embodiments, reactive groups are used in the herboxydiene-based splicing modulator and/or intermediate or linker. The reaction product between the herboxidiene splicing modulator and the intermediate, or a derivatized herboxidiene splicing modulator (herboxidiene splicing modulator plus linker), is subsequently reacted with an antibody or antigen-binding moiety under suitable conditions. Alternatively, the intermediate or linker can be first reacted with an antibody or antigen binding fragment or a derivatized antibody or antigen binding fragment and then reacted with a drug or derivatized drug.

[363] A number of different reactions are available to covalently attach a herboxydiene splicing modulator moiety and/or linker moiety to an antibody or antigen binding moiety. This is often accomplished by the reaction of one or more amino acid residues of the antibody or antigen binding moiety, including lysine amino groups, free carboxylic acid groups of glutamic acid and aspartic acid, cysteine sulfhydryl groups, and various aromatic amino acid moieties. For example, nonspecific covalent coupling can be accomplished using a carbodiimide reaction to couple the carboxy (or amino) group on the herboxydiene splicing modulator moiety to the amino (or carboxy) group on the antibody or antigen binding moiety. Additionally, bifunctional agents such as dialdehydes or imidoesters can also be used to link the amino group in the herboxydiene splicing modulator moiety to the amino group in the antibody or antigen binding moiety. A Schiff base reaction is also suitable for coupling drugs (eg herboxydiene splicing modulator) to binding agents. This method involves the periodate oxidation of a drug that contains glycol or hydroxy groups, resulting in the formation of an aldehyde, which is then reacted with a binder. The attachment occurs by forming a Schiff base with the amino groups of the coupling agent. Isothiocyanates can also be used as coupling agents to covalently attach drugs to binding agents. Other techniques are known to one skilled in the art and are within the scope of the present invention. Examples of drug moieties that can be prepared and linked to an antibody or antigen-binding moiety using various chemical structures known in the art include herboxidiene-based splicing modulators, for example, the herboxydiene-based splicing modulators described and cited as examples in this document.

Compounds comprising a herboxidiene-based splicing modulator linker/herboxidiene-based splicing modulator construct

[364] Further disclosed herein are exemplary compounds comprising a herboxidiene (L-H) splicing linker modulator construct, as well as compositions containing multiple copies of such compounds. In various embodiments, the herboxydiene-based linker-splicing modulator construct compounds disclosed herein may be defined by the general formula L-H, where L = linker moiety and H = herboxydiene-based splicing modulator. In some embodiments, the disclosed L-H compounds are suitable for use in the ADCs described herein.

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

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

each of R ¹ , R ² and R ³ is independently selected from hydrogen, hydroxyl, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups, -C groups (=O)-O-(C ₁ -C ₆ alkyl) and C ₁ -C ₆ alkyl groups;

R ⁴ is selected from hydrogen, C ₁ -C ₆ alkyl groups, -C(=O)-(C ₁ -C ₆ alkyl) groups, -C(=O)-(C ₃ -C ₈ carbocyclyl) groups, - C(=O)-(C ₃ -C ₈ heterocyclyl) and -C(=O)-NR ⁶ R ⁷ ;

R ⁵ is selected from hydrogen, hydroxyl, -CH ₂ -OH, -CO ₂ H, -C(=O)-O-(C ₁ -C ₆ alkyl), -C(=O)-NR ⁶ R ⁷ groups, -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 ;

R ⁶ and R ⁷ are each independently selected from hydrogen, -R ⁸ , -C(=O)-R ⁸ and -C(=O)-OR ⁸ ; And

R ⁸ is selected from C ₁ -C ₆ alkyl groups, C ₃ -C ₈ carbocyclyl groups and C ₃ -C ₈ heterocyclyl groups,

where each of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ is independently substituted with 0-3 groups independently selected from halogens, hydroxyl, C ₁ -C ₆ alkyl groups, groups - O-(C ₁ -C ₆ alkyl), -CO ₂ H, groups -C(=O)-(C ₁ -C ₆ alkyl), groups -C(=O)-(C ₃ -C ₈ carbocyclyl), groups -C(=O)-(C ₃ -C ₈ heterocyclyl), -NR ⁶ R ⁷ , C ₃ -C ₈ carbocyclyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkyl alkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups, each of which may be independently substituted with 0 or 1 group selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups, C ₁ -C ₃ haloalkyl groups, -NH- C(=O)(C ₁ -C ₃ alkyl) and -NH-C(=O)-O-(C ₁ -C ₃ alkyl).

[366] In some embodiments, compounds of formula (Ia) are provided herein: (Ia), or a pharmaceutically acceptable salt thereof, which are covalently attached to L through any atom where

R ⁹ is selected from C ₃ -C ₈ heterocyclyl groups; And

R ¹⁰ is selected from H and C ₁ -C ₆ alkyl groups,

where each of R ⁹ and R ¹⁰ is independently substituted with 0-3 groups independently selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups, -NH ₂ , -NH-(C ₁ -C ₃ alkyl) and -N-(C ₁ -C ₃ alkyl) ₂ .

[367] In some embodiments, compounds of formula (Ib) are provided herein: (Ib), or a pharmaceutically acceptable salt thereof, where

R ¹¹ selected from , And , where * denotes the point of attachment of R ¹¹ to the rest of the connection;

R ¹² and R ¹³ are each independently selected from H and methyl.

[368] In some embodiments, compounds of formula (II) are provided herein: (II), or a pharmaceutically acceptable salt thereof, where

X represents hydroxyl or NR ⁶ R ⁷ ;

R ⁶ and R ⁷ are each independently selected from hydrogen, -R ⁸ , -C(=O)-R ⁸ , -C(=O)-OR ⁸ , -(C ₁ -C ₆ alkyl)-OC(=O )-R ⁸ and -(C ₁ -C ₆ alkyl)-NH-C(=O)-R ⁸ ; And

R ⁸ is selected from C ₁ -C ₆ alkyl groups, C ₃ -C ₈ carbocyclyl groups and C ₃ -C ₈ heterocyclyl groups,

wherein each of R ⁶ , R ⁷ and R ⁸ is independently substituted with 0-3 groups independently selected from halogens, hydroxyl, C ₁ -C ₆ alkyl groups, -O-(C ₁ -C ₆ alkyl), -CO ₂ H groups , groups -C(=O)-O-(C ₁ -C ₆ alkyl), groups -C(=O)-(C ₁ -C ₆ alkyl), groups -C(=O)-(C ₃ -C ₈ carbocyclyl), -C(=O)-(C ₃ -C ₈ heterocyclyl) groups, -NR ⁶ R ⁷ , C ₃ -C ₈ carbocyclyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkylalkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups, each of which may be independently substituted with 0-1 group selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups, C ₁ -C ₃ haloalkyl groups , -NH-C(=O)(C ₁ -C ₃ alkyl) and -NH-C(=O)-O-(C ₁ -C ₃ alkyl).

[369] In some embodiments, compounds of formula (IIa) are provided herein: (IIa), or a pharmaceutically acceptable salt thereof, where

Z is selected from NR ⁹ and O;

R ⁹ is selected from hydrogen and C ₁ -C ₆ alkyl groups;

each of R ¹⁰ and R ¹¹ is independently selected from hydrogen, halogens, hydroxyl, C ₁ -C ₆ alkyl groups, -O-(C ₁ -C ₆ alkyl) groups, -CO ₂ H, -C(=O)- groups O-(C ₁ -C ₆ alkyl), -C(=O)-(C ₁ -C ₆ alkyl) groups, -C(=O)-(C ₃ -C ₈ carbocyclyl) groups, -C(= O)-(C ₃ -C ₈ heterocyclyl), C ₃ -C ₈ carbocyclyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkyl alkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups;

R ¹² is selected from C ₁ -C ₆ alkyl groups, C ₃ -C ₈ carbocyclyl groups, C ₃ -C ₈ heterocyclyl groups,

wherein each of R ⁹ , R ¹⁰ , R ¹¹ and R ¹² is independently substituted with 0 or 1 group selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups and C ₁ -C ₃ haloalkyl groups; And

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

[370] In some embodiments, compounds of formula (IIb) are provided herein: (IIb), or a pharmaceutically acceptable salt thereof, where

R ¹³ selected from , , , , , , , , , And , where * denotes the point of attachment of R ¹³ to the rest of the connection; And

R ¹⁴ and R ¹⁵ are each independently selected from hydrogen and methyl.

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

each of R ¹ , R ² and R ³ is independently selected from hydrogen, hydroxyl, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups, -C groups (=O)-O-(C ₁ -C ₆ alkyl) and C ₁ -C ₆ alkyl groups;

R ⁶ and R ⁷ are each independently selected from hydrogen, -R ⁸ , -C(=O)-R ⁸ and -C(=O)-OR ⁸ ;

R ⁸ is selected from C ₁ -C ₆ alkyl groups, C ₃ -C ₈ carbocyclyl groups and C ₃ -C ₈ heterocyclyl groups; And

R ⁹ selected from H, , , , , , , , And

,

wherein each of R ¹ , R ² , R ³ , R ⁶ , R ⁷ and R ⁸ is independently substituted with 0-3 groups independently selected from halogens, hydroxyl, C ₁ -C ₆ alkyl groups, -O-(C ₁ - C ₆ alkyl), -CO ₂ H, -C(=O)-(C ₁ -C ₆ alkyl) groups, -C(=O)-(C ₃ -C ₈ carbocyclyl) groups, -C(=O) groups )-(C ₃ -C ₈ heterocyclyl), -NR ⁶ R ⁷ , C ₃ -C ₈ carbocyclyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkyl alkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups, each of which may be independently substituted with 0 or 1 group selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups, C ₁ -C ₃ haloalkyl groups, -NH-C(=O)( C ₁ -C ₃ alkyl) and -NH-C(=O)-O-(C ₁ -C ₃ alkyl); And

where * denotes the point of attachment of R ⁹ to the rest of the connection.

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

[373] In some embodiments, provided herein is a compound selected from:

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

, and its pharmaceutically acceptable salts,

where L represents a linker that is covalently attached to the antibody.

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

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

[376] In some embodiments, the linker includes a spacer unit. In some embodiments, the spacer unit is selected from alkyl groups and polyethylene glycol (PEG) moieties. In some embodiments, the alkyl group is a C ₁ -C ₁₂ alkyl group. In some embodiments, the alkyl group is a C ₁ -C ₆ alkyl group. In some embodiments, the alkyl group is methylene. In some embodiments, the alkyl group is ethylene. In some embodiments, the alkyl group is n-propylene. In some embodiments, the alkyl group is n-butylene. In some embodiments, the alkyl group is n-pentylene. In some embodiments, the alkyl group is n-hexylene. In some embodiments, the PEG portion comprises -(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.

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

[378] In some embodiments, the linker includes a Mal spacer unit, an alkyl group, at least one amino acid unit, and a self-cleaving spacer. In some embodiments, the at least one amino acid unit is selected from alanine, citrulline, and valine. In some embodiments, at least one amino acid unit comprises alanine and valine. In some embodiments, at least one amino acid unit comprises citrulline and valine. In some embodiments, the self-cleaving spacer is selected from pAB and pABC. In some embodiments, the self-cleaving spacer comprises pAB. In some embodiments, the self-cleaving spacer comprises pABC. In some embodiments, the alkyl group includes a C ₁ -C ₆ alkyl group.

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

Drug load

[380] Drug load is expressed in p and is also referred to herein as the herboxidiene-antibody splicing modulator ratio (HAR). The drug load can range from 1 to 10 drug fragments 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.

[381] Drug loading may be limited by the number of attachment sites on the antibody or antigen binding moiety. In some embodiments, the linker moiety (L) of the ADC is attached to the antibody or antigen-binding moiety via a reactive group on one or more amino acid residues on the antibody or antigen-binding moiety. For example, a linker may be attached to an antibody or antigen-binding moiety via a free amino, imino, hydroxyl, thiol, or carboxyl group (e.g., the N- or C-terminus, the epsilon-amino group of one or more lysine residues, the free carboxyl group of one or several glutamic acid or aspartic acid residues or to the sulfhydryl group of one or more cysteine residues). The site to which the linker is attached may be a naturally occurring residue in the amino acid sequence of the antibody or antigen binding fragment, or it may be inserted into the antibody or antigen binding fragment, for example, through recombinant DNA technology (for example, by introducing a cysteine residue into the amino acid sequence) or by altering biochemical properties of the protein (for example, by reduction, pH change or hydrolysis).

[382] In some embodiments, the number of drug moieties that can be conjugated to an antibody or antigen-binding moiety is limited by the number of cysteine residues. For example, if the attachment is via a thiol group of a cysteine residue, the antibody may contain only one or more thiol groups of cysteine residues or may contain one or more sufficiently reactive thiol groups through which a linker can be attached. Typically, antibodies do not contain many free and reactive thiol groups on cysteine residues that can be associated with the drug moiety. In fact, most thiol groups of cysteine residues in an antibody are involved in the formation of either interchain or intrachain disulfide bonds. Therefore, in some embodiments, conjugation to cysteines may require at least partial reduction of the antibody. Excessive attachment of a toxin linker to an antibody can destabilize the antibody by reducing accessible cysteine residues to form disulfide bonds. Therefore, the optimal drug:antibody ratio should increase ADC activity (by increasing the number of drug moieties attached to the antibody) without destabilizing the antibody or antigen-binding moiety. In some embodiments, the optimal ratio may be 2, 4, 6, or 8.

[383] In some embodiments, the antibody or antigen binding fragment is subjected to reducing conditions prior to conjugation to generate one or more free cysteine residues. In some embodiments, the antibody may be reduced with a reducing agent, such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP), under partial or complete reduction conditions to form reactive cysteine thiol groups. Unpaired cysteines can be produced through partial reduction with limited molar equivalents of TCEP, which can reduce the intrachain disulfide bonds that connect the light chain and the heavy chain (one pair per H-L pairing) and the two heavy chains at the hinge region (two pairs per H-H pairing in the case human IgG1), while intrachain disulfide bonds remain intact (Stefano et al. (2013) Methods Mol Biol. 1045:145-71). In one embodiment, the disulfide bonds in the antibody are reduced electrochemically, for example, through the use of a working electrode that applies a reducing and oxidizing electrical voltage. This approach may provide the ability to sequentially couple disulfide bond reduction with an analytical device (e.g., an electrochemical detector, NMR spectrometer, or mass spectrometer), or a chemical separation device (e.g., a liquid chromatograph (e.g., HPLC), or an electrophoresis device (see ., for example, publication of US patent application No. 20140069822)). In some embodiments, the antibody is subjected to denaturing conditions to identify reactive nucleophilic groups on amino acid residues, such as cysteine.

[384] The drug loading of an ADC can be controlled in various ways, for example, by: (i) limiting the molar excess of the drug linker intermediate or linker reagent relative to the antibody; (ii) limitations on conjugation reaction time or temperature; (iii) partial or limiting reducing conditions for modification of cysteine thiol groups; and/or (iv) designing, by recombinant techniques, the amino acid sequence of the antibody so that the number and position of cysteine residues is modified to control the number and/or position of attachment sites for the drug linker.

[385] In some embodiments, free cysteine residues are introduced into the amino acid sequence of the antibody or antigen binding fragment. For example, cysteine modified antibodies can be produced where one or more amino acids in the parent antibody are replaced with the amino acid cysteine. Any form of antibody can be engineered in this way, that is, mutated. For example, a Fab fragment of a parent antibody can be engineered to produce a cysteine-modified Fab, referred to as "ThioFab". Similarly, the parent monoclonal antibody can be engineered to produce "ThioMab". A single site mutation produces one modified cysteine residue in ThioFab, whereas a single site mutation produces two modified cysteine residues in ThioMab due to the dimeric nature of the IgG antibody. DNA encoding a variant amino acid sequence of the parent polypeptide can be obtained by a variety of methods known in the art (see, for example, the methods described in International Publication No. WO 2006/034488). Such methods include, but are not limited to, producing, through site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis, preformed DNA encoding a polypeptide. Recombinant antibody variants can also be constructed by restriction enzyme manipulation or by PCR with overlapping primers using synthetic oligonucleotides. ADCs of formula (I) include, but are not limited to, antibodies that have 1, 2, 3 or 4 cysteine amino acids modified (Lyon et al. (2012) Methods Enzymol. 502:123-38). In some embodiments, one or more free cysteine residues are already present in the antibody or antigen binding fragment without the use of genetic engineering, in which case the existing cysteine residues can be used to conjugate the antibody or antigen binding fragment to the drug moiety.

[386] In this process, more than one nucleophilic group reacts with a drug-linker intermediate or linker fragment reagent and then with a drug-fragment reagent in a reaction mixture containing multiple copies an antibody or antigen binding fragment and a linker fragment, the resulting product may then be a mixture of ADC compounds with a distribution of one or more drug moieties attached to each copy of the antibody or antigen binding fragment in the mixture. In some embodiments, the drug load in the ADC mixture resulting from the conjugation reaction ranges from 1 to 10 drug moieties attached to the antibody or antigen binding moiety. The average number of drug fragments per antibody or antigen binding fragment (i.e., average drug loading or average p ) can be calculated by any conventional method known in the art, for example, by mass spectrometry (e.g., LC-MS with reverse phase), and/or high performance liquid chromatography (eg HIC-HPLC). In some embodiments, the average number of drug fragments 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 fragments per antibody or antigen binding fragment is determined by reverse phase liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is from about 1.5 to about 3.5, from about 2.5 to about 4.5, from about 3.5 to about 5.5, from 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 fragments per antibody or antigen binding fragment is from about 2 to about 4, from about 3 to about 5, from about 4 to about 6, from about 5 to about 7, from about 6 to about 8 , from about 7 to about 9, from about 2 to about 8, or from about 4 to about 8.

[387] In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is about 2. In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is about 1.5, about 1.6, about 1 .7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5. In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is 2.

[388] In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is about 4. In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is about 3.5, about 3.6, about 3 .7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, or about 4.5. In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is 4.

[389] In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is about 8. In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is about 7.5, about 7.6, about 7 .7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, or about 8.5. In some embodiments, the average number of drug fragments per antibody or antigen binding fragment is 8.

[390] In various embodiments, the term "about" when used to refer to the average number of drug fragments per antibody or antigen binding fragment means plus or minus 10%.

[391] Individual ADC compounds or "molecules" can be identified in a mixture using mass spectrometry and separated using UPLC or HPLC, such as hydrophobic interaction chromatography (HIC-HPLC). In some embodiments, a uniform or substantially uniform ADC product with a single loading value may be isolated from the conjugation mixture, for example, by electrophoresis or chromatography.

[392] In some embodiments, higher drug loading (eg, p > 8) may result in aggregation, insolubility, toxicity, and loss of cell permeability of certain antibody-drug conjugates. Higher drug load may also negatively impact the pharmacokinetics (eg, clearance) of certain ADCs. In some embodiments, a lower drug load (eg, p < 2) may reduce the activity of certain ADCs on target expressing cells and/or non-target cells. In some embodiments, the drug loading for the ADC of the present invention is in the range of 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.

[393] In some embodiments, a drug load and/or an average drug load of about 2 is provided, for example, by partially reducing intrachain disulfides on the antibody or antigen-binding moiety, which provides beneficial properties. In some embodiments, a drug load and/or an average drug load of about 4 is provided, for example, by partially reducing intrachain disulfides on the antibody or antigen-binding moiety, which provides beneficial properties. In some embodiments, a drug load and/or an average drug load of approximately 8 is provided, for example, by partially reducing intrachain disulfides on the antibody or antigen-binding moiety, which provides beneficial properties. In some embodiments, a drug load and/or an average drug load of less than about 2 may produce unacceptably high levels of unconjugated antibody fragments that may compete with the ADC for binding to the target antigen and/or cause poor treatment efficacy. In some embodiments, a drug load and/or average drug load greater than about 8 may result in unacceptably high levels of product heterogeneity and/or ADC aggregation. Drug loading and/or average drug loading greater than about 8 may also affect the stability of the ADC due to the loss of one or more chemical bonds required to stabilize the antibody or antigen binding moiety.

[394] The present invention includes methods for preparing the described ADCs. Briefly, ADCs contain an antibody or antigen-binding fragment as an antibody or antigen-binding fragment, a drug moiety (eg, a herboxidiene-based splicing modulator), and a linker that connects the drug moiety and the antibody or antigen-binding moiety. In some embodiments, ADCs can be prepared using a linker containing reactive functional groups for covalent attachment to the drug moiety and the antibody or antigen binding moiety. For example, in some embodiments, a cysteine thiol group on an antibody or antigen binding moiety is capable of forming a bond with a reactive functional group of a linker or drug linker intermediate (eg, a maleimide moiety) to form an ADC. The preparation of ADC can be accomplished by any technique known to one skilled in the art.

[395] In some embodiments, an ADC is prepared by sequentially contacting an antibody or antigen-binding moiety with a linker and a drug moiety (e.g., a herboxidiene splicing modulator) such that the antibody or antigen-binding moiety is first covalently bound to the linker , and then the preformed antibody linker intermediate was reacted with the drug moiety. The antibody linker intermediate may or may not be subjected to a purification step before contacting the drug moiety. In other embodiments, an ADC is prepared by contacting an antibody or antigen binding moiety with a linker-drug compound previously formed by reacting the linker with the drug moiety. The preformed linker-drug compound may or may not be subjected to a purification step prior to contacting the antibody or antigen binding moiety. In other embodiments, the antibody or antigen-binding moiety is contacted with a linker and the drug moiety in the same reaction mixture to simultaneously form covalent bonds between the antibody or antigen-binding moiety and the linker and between the linker and the drug moiety. This method of producing an ADC may involve a reaction in which an antibody or antigen binding fragment is contacted with an antibody or antigen binding fragment before adding the linker to the reaction mixture and vice versa. In some embodiments, an ADC is prepared by reacting an antibody or antigen-binding fragment with a linker attached to a drug moiety, such as an ADL1 herboxidiene splicing modulator (e.g., ADL1-79392) or an ADL5 herboxidiene splicing modulator ( eg ADL5-0349) under conditions that allow conjugation.

[396] ADCs prepared in accordance with the methods described above can be subjected to a purification step. The purification step may include any biochemical methods known in the art for purifying proteins, or any combination of these methods. Such methods include, but are not limited to, tangential flow filtration (TFF), affinity chromatography, ion exchange chromatography, any charge transfer or isoelectric focusing chromatography, mixed mode chromatography, such as using CHT (ceramic hydroxyapatite), hydrophobic interaction chromatography, size exclusion chromatography, dialysis, filtration, selective precipitation or any combination thereof.

Therapeutic Uses and Compositions

[397] Disclosed herein are methods of using ADCs and compositions in treating a subject for a disorder, such as a neoplastic disorder. The ADC may be administered alone or in combination with a second therapeutic agent, and may be administered in any pharmaceutically acceptable composition, dosage, and schedule of administration. The effectiveness of treatment using ADCs can be assessed for toxicity as well as efficacy rates and adjusted accordingly. Efficacy measures include, but are not limited to, cytostatic and/or cytotoxic effects observed in vitro or in vivo, tumor volume reduction, tumor growth inhibition, and/or long-term survival.

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

[399] A thymidine incorporation assay can be used to determine whether an ADC has a cytostatic effect. For example, cancer cells expressing the target antigen at a density of 5000 cells/well in a 96-well plate can be cultured for a 72 hour period and exposed to 0.5 μCi ³ H-thymidine for the last 8 hours of the 72 hour period. The incorporation of ³ H-thymidine into culture cells is measured in the presence and absence of ADC.

[400] Necrosis or apoptosis (programmed cell death) can be measured to determine cytotoxicity. Necrosis is usually accompanied by increased permeability of the plasma membrane; swelling of the cell and rupture of the plasma membrane. Apoptosis can be quantified, for example, by measuring DNA fragmentation. Commercial photometric methods are available to quantify DNA fragmentation in vitro. Examples of such assays, including TUNEL (which detects the incorporation of labeled nucleotides into fragmented DNA) and ELISA-based assays, are described in Biochemica (1999) No. 2, pp. 34-37 (Roche Molecular Biochemicals).

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

[402] Cell viability can be measured, for example, by determining the cellular uptake of a dye such as neutral red, trypan blue, crystal violet or ALAMAR™ blue (see, for example, Page et al. (1993) Intl J Oncology 3:473 -6). In this assay, cells are incubated in media containing dye, the cells are washed, and the remaining dye, which reflects the uptake of the dye by the cells, is measured spectrophotometrically. Cell viability can also be measured, for example, by quantifying ATP, an indicator of metabolically active cells. In certain embodiments, the in vitro activity and/or effect of the resulting ADCs or herboxydiene splicing modulator compounds on cell viability can be assessed using the CellTiter-Glo® luminescent cell viability assay, as described in the examples presented herein. In this assay, in certain embodiments, one reagent (CellTiter-Glo® reagent) is added directly to cells cultured in serum-supplemented media. Addition of the reagent results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in the culture. The dye-binding protein sulforhodamine B (SRB) can also be used to measure cytotoxicity (Skehan et al. (1990) J Natl Cancer Inst. 82:1107-12).

[403] Disclosed ADCs can also be evaluated for killing activity. Non-target cell killing activity can be determined, for example, by an assay using two cell lines, one positive for the target antigen and one negative for the target antigen. In certain embodiments, the assay design allows only target-negative cells to be detected. In certain embodiments, cells are seeded under three conditions: (i) target-negative cells (marked or tagged) only; (ii) only target-positive cells; and (iii) co-culture of target-negative cells and target-positive cells. Cells are then treated with ADCs followed by cytotoxicity monitoring. If plate reaction assays are performed by reading using CellTiter-Glo® reagent, the viability of all cell populations can be monitored. When plates are read using OneGlo® reagent, only labeled or target-negative cells generate a signal. Killing of target-negative cells when mixed with target-positive cells indicates killing of non-target cells, while killing of target-negative cells in the absence of target-positive cells indicates non-target killing.

[404] In certain aspects of the present invention, there is provided a method of killing, inhibiting or modulating the growth or interfering with the metabolism of a cancer cell or tissue by disrupting RNA splicing. The method can be used in any subject in which an RNA splicing disorder provides a therapeutic benefit. Subjects who may benefit from disruption of RNA splicing include, but are not limited to, those who have or are at risk for developing a neoplastic disorder, such as a hematologic malignancy or solid tumor. In certain embodiments, the hematologic malignancy is a B-cell malignancy, a blood cancer (leukemia), a plasma cell cancer (myeloma, e.g., multiple myeloma), or a lymph node cancer (lymphoma). In certain embodiments, the hematologic malignancy is selected from myelogenous leukemia or multiple myeloma. In some embodiments, the leukemia is acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), or acute monocytic leukemia (AMO). In some embodiments, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma. In certain embodiments, the hematologic malignancy is myelodysplastic syndrome (MDS). In certain embodiments, the solid tumor is a carcinoma, such as breast cancer, pancreatic cancer, prostate cancer, colon or colorectal cancer, lung cancer, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, cholangiocarcinoma, glioma or melanoma. In certain embodiments, the solid tumor is breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer (eg, lung adenocarcinoma), uterine cancer (eg, uterine endometrial serous carcinoma), salivary duct carcinoma, melanoma, colon cancer colon, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer or esophageal cancer. In some embodiments, the lung cancer is an adenocarcinoma. In some embodiments, the uterine cancer is a serous carcinoma of the endometrium of the uterus.

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

[406] In various embodiments, the disclosed ADCs can be administered to any cell or tissue that expresses CD138, such as a neoplastic cell or tissue that expresses CD138. An exemplary embodiment includes a method of inhibiting CD138-mediated cell signaling or a method of killing a cell. The method can be used with any cell or tissue that expresses CD138, such as a cancer cell or metastatic lesion. Non-limiting examples of cancers that express CD138 include intrathoracic cancers (eg, lung cancer, mesothelioma), skin cancers (eg, basal cell carcinoma, squamous cell carcinoma), head and neck cancers (eg, larynx, hypopharynx, nasopharynx) , breast cancer, urogenital cancer (eg, cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, urothelial cancer), hematologic malignancies (eg, myeloma, such as multiple myeloma, Hodgkin's lymphoma) and cancer thyroid gland (Szatmári et al. (2015) Dis Markers 2015:796052). Non-limiting examples of cells expressing CD138 include MOLP8, human multiple myeloma cells, and cells containing recombinant nucleic acid encoding CD138 or a portion thereof.

[407] In various embodiments, the disclosed ADCs can be administered to any cell or tissue that expresses EPHA2, such as a neoplastic cell or tissue that expresses EPHA2. An exemplary embodiment includes a method of inhibiting EPHA2-mediated cell signaling or a method of killing a cell. The method can be used with any cell or tissue that expresses EPHA2, such as a cancer cell or metastatic lesion. Non-limiting examples of cancers that express EPHA2 include breast cancer, brain cancer, ovarian cancer, bladder cancer, pancreatic cancer, esophageal cancer, lung cancer, prostate cancer, melanoma, esophageal cancer, and gastric cancer (Tandon et al. (2011) Expert Opin Ther Targets 15(1):31-51. Non-limiting examples of cells expressing EPHA2 include PC3, human prostate cancer cells, and cells containing recombinant nucleic acid encoding EPHA2 or a portion thereof.

[408] Exemplary methods include the steps of contacting a cell with an ADC described herein in an effective amount, i.e. quantity sufficient to destroy the cell. The method can be used in relation to cells in culture, for example in vitro, in vivo, ex vivo or in situ.For example, cells that express HER2 (eg, cells obtained from a biopsy of a tumor or metastatic lesion, cells from an established cancer cell line, or recombinant cells) can be cultured in vitro in the culture medium, and the contacting step may be influenced by the addition of ADC to the culture medium. This method will result in the destruction of HER2 expressing cells, including in particular HER2 expressing tumor cells. Alternatively, the ADC can be administered to a subject via any suitable route of administration (eg, intravenous, subcutaneous, or direct contact with tumor tissue) to produce effects in vivo. This approach can be used for antibodies that specifically target other cell surface antigens (eg, CD138, EPHA2).

[409] The in vivo effect of a disclosed ADC therapeutic composition can be assessed in a suitable animal model. For example, xenogeneic cancer models can be used in which cancer explants or passaged xenograft tissues are administered to immunocompromised animals such as nude or SCID mice (Klein et al. (1997) Nature Med. 3:402-8). Efficacy can be predicted using assays that measure suppression of tumorigenesis, tumor regression or metastasis, etc.

[410] In vivo assays that assess stimulation of tumor death through mechanisms such as apoptosis can also be used. In one embodiment, xenografts from tumor-bearing mice treated with a therapeutic composition can be assessed for the presence of apoptotic lesions and compared with untreated xenograft-bearing control mice. The extent to which apoptotic foci are detected in tumors of treated mice is an indicator of the therapeutic efficacy of the composition.

[411] In addition, methods for treating a neoplastic disorder, such as cancer, are provided herein. The ADCs described herein can be administered to a non-human mammal or human for the purpose of therapy. The therapeutic methods involve administering to a subject who has or is suspected of having a neoplastic disorder a therapeutically effective amount of an ADC or composition comprising a herboxidiene-based splicing modulator linked to a targeted antibody that binds to an expressed antigen that is available for binding or localized on the surface. cancer cells. In some embodiments, treatment with an antibody-drug conjugate or composition induces nonspecific killing of neoplastic cells that do not express the target antigen but are adjacent to neoplastic cells that express the target antigen.

[412] An exemplary embodiment is a method of delivering a splicing modulator to a cell expressing HER2, comprising conjugating the herboxydiene-based splicing modulator to an antibody that immunospecifically binds to an epitope of HER2 and exposes the cell to an ADC. Exemplary tumor cells that express HER2 for which the ADCs of the present invention are provided include gastric carcinoma cells and ductal breast carcinoma cells.

[413] Another exemplary embodiment is a method of delivering a herboxydiene splicing modulator to a cell expressing CD138, comprising conjugating the herboxydiene splicing modulator to an antibody that immunospecifically binds to an epitope of CD138 and exposes the cell to an ADC. Exemplary tumor cells that express CD138 for which the ADCs of the present invention are provided include multiple myeloma cells.

[414] Another exemplary embodiment is a method of delivering a herboxidiene splicing modulator to a cell expressing EPHA2, comprising conjugating the herboxidiene splicing modulator to an antibody that immunospecifically binds to an EPHA2 epitope and exposes the cell to an ADC. Exemplary tumor cells that express EPHA2 for which the ADCs of the present invention are provided include prostate cancer cells.

[415] Another illustrative embodiment is a method of reducing or inhibiting the growth of a tumor (eg, a HER2-expressing tumor, a CD138-expressing tumor, an EPHA2-expressing tumor) comprising administering a therapeutically effective amount of an ADC or a composition comprising an ADC. In some embodiments, the treatment is sufficient to reduce or suppress tumor growth in a patient, reduce the number or size of metastatic lesions, reduce tumor burden, reduce primary tumor burden, reduce invasiveness, prolong survival time, and/or maintain or improve quality of life. In some embodiments, the tumor is resistant or refractory to treatment with an antibody or antigen binding fragment of an ADC (e.g., anti-HER2 antibody, anti-CD138 antibody, anti-EPHA2 antibody) when administered alone, and/or the tumor is resistant or refractory to treatment with fragment representing a herboxydiene-based splicing modulator drug when administered individually.

[416] In certain aspects of the present invention, a method is provided for reducing or inhibiting the growth of a tumor expressing HER2. In certain embodiments, treatment with an antibody-drug conjugate or composition induces nonspecific killing of tumor cells that do not express HER2 but are adjacent to neoplastic tumor cells that express HER2. Exemplary cancer types expressing HER2 include, but are not limited to, tumors derived from breast cancer, gastric cancer, bladder cancer, urothelial cell carcinoma, esophageal cancer, lung cancer (eg, lung adenocarcinoma), uterine cancer (eg, uterine endometrial serous carcinoma). ), salivary duct carcinoma, cervical cancer, endometrial cancer and ovarian cancer expressing HER2. In some embodiments, the tumor expressing HER2 is a tumor derived from breast cancer, ovarian cancer, gastric cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., uterine endometrial serous carcinoma), osteosarcoma, or salivary duct carcinoma , expressing HER2. In some embodiments, the HER2-expressing tumor is a lung adenocarcinoma or a uterine endometrial serous carcinoma.

[417] In certain aspects of the present invention, a method is provided for reducing or inhibiting the growth of a tumor expressing CD138. In certain embodiments, treatment with an antibody-drug conjugate or composition induces nonspecific killing of tumor cells that do not express CD138 but are adjacent to neoplastic tumor cells that express CD138. Exemplary types of tumors expressing CD138 include, but are not limited to, tumors derived from intrathoracic cancers (eg, lung cancer, mesothelioma), skin cancers (eg, basal cell carcinoma, squamous cell carcinoma), head and neck cancers (eg, larynx, hypopharynx, nasopharynx ), breast cancer, urogenital cancer (eg, cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, urothelial cancer) and thyroid cancer expressing CD138.

[418] In certain aspects of the present invention, a method is provided for reducing or inhibiting the growth of a tumor expressing EPHA2. In certain embodiments, treatment with an antibody-drug conjugate or composition induces nonspecific killing of tumor cells that do not express EPHA2 but are adjacent to neoplastic tumor cells that express EPHA2. Exemplary tumor types expressing EPHA2 include, but are not limited to, tumors derived from breast cancer, brain cancer, ovarian cancer, bladder cancer, pancreatic cancer, esophageal cancer, lung cancer, prostate cancer, melanoma, esophageal cancer, and gastric cancer. , expressing EPHA2. In certain embodiments, the EPHA2-expressing tumor is a tumor derived from breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer expressing EPHA2.

[419] Moreover, the antibodies of the present invention can be administered to a non-human mammal that expresses an antigen to which the ADC is capable of binding, for veterinary purposes or for use as an animal model of human disease. With regard to the latter, such animal models may be useful for assessing the therapeutic efficacy of the disclosed ADCs (eg, testing dosages and timing of administration).

[420] Additionally, therapeutic uses of the disclosed ADCs and compositions are provided herein. One illustrative embodiment provides the use of an ADC in the treatment of a neoplastic disorder (eg, HER2-expressing cancer, CD138-expressing cancer, EPHA2-expressing cancer). In another illustrative embodiment, an ADC is provided for use in the treatment of a neoplastic disorder (eg, HER2-expressing cancer, CD138-expressing cancer, EPHA2-expressing cancer). Methods for identifying subjects with cancers that express a target antigen (eg, HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1 ) are known in the art and can be used to identify suitable patients for treatment with the disclosed ADC.

[421] Another illustrative embodiment provides the use of an ADC in a method of making a medicament for treating a neoplastic disorder (eg, HER2-expressing cancer, CD138-expressing cancer, EPHA2-expressing cancer).

[422] Therapeutic compositions used in the practice of the above methods can be formulated into pharmaceutical compositions containing a pharmaceutically acceptable carrier suitable for the desired mode of delivery. An illustrative embodiment provides a pharmaceutical composition comprising an ADC of the present invention and a pharmaceutically acceptable carrier. Suitable carriers include any material that, when combined with a therapeutic composition, retains the antitumor function of the therapeutic composition and generally does not interfere with the patient's immune system. Pharmaceutically acceptable carriers include any and all solvents, dispersing media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of the following: water, physiological saline, phosphate buffered saline, dextrose, glycerol, ethanol, mesylate salt and the like, as well as combinations thereof. In many cases, isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride are included in the composition. Pharmaceutically acceptable carriers may additionally contain small amounts of auxiliary agents, such as wetting or emulsifying agents, preservatives or buffers, which increase the shelf life or effectiveness of the ADC.

[423] Therapeutic compositions can be solubilized and administered via any route that provides delivery of the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumoral, intradermal, intra-organ, orthotopic, and the like. Protein-based therapeutics can be lyophilized and stored as sterile powders, for example under vacuum, and then reconstituted in bacteriostatic water (containing, for example, the preservative benzyl alcohol) or sterile water before injection. Therapeutic compositions may contain an ADC or a pharmaceutically acceptable salt thereof, for example a mesylate salt.

[424] In some embodiments, the ADC is administered to the patient daily, bimonthly, or any time in between. Dosages and administration protocols for treating cancer using the above methods will vary depending on the method and the target cancer and will generally depend on a number of other factors considered in the art.

[425] Various delivery systems are known that can be used to administer one or more ADCs of the present invention. Routes of administration of ADCs include, but are not limited to, parenteral administration (eg, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, intratumoral, and transmucosal (eg, intranasal and oral routes). In addition, pulmonary administration may be used, for example using an inhaler or nebulizer, or an aerosol formulation. See for example compositions and methods for pulmonary administration described in US patents No. 6019968, 5985320, 5985309, 5934272, 5874064, 5855913, 5290540 and 4880078; and publications of international applications Nos. WO 1992/019244, WO 1997/032572, WO 1997/044013, WO 1998/031346 and WO 1999/066903. The ADC can be administered by any convenient route, for example, by infusion or bolus injection, or by absorption through epithelial or mucocutaneous membranes (eg, oral mucosa, rectal mucosa and intestinal mucosa, etc.). Administration may be systemic or local.

[426] The therapeutic compositions disclosed herein may be sterile and stable under the conditions of manufacture and storage. In some embodiments, one or more ADCs or one or more pharmaceutical compositions are provided as a dry, sterilized, lyophilized powder or anhydrous concentrate in a hermetically sealed container and may be reconstituted (eg, with water or physiological saline) to a concentration suitable for administration to the subject. In some embodiments, one or more prophylactic or therapeutic agents or one or more pharmaceutical compositions are provided as a dry, sterile lyophilized powder in a sealed container in a dosage form of at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg or at least 100 mg or any amount in between. In some embodiments, the lyophilized ADC or pharmaceutical compositions are stored at a temperature of 2°C to 8°C in the original container. In some embodiments, one or more ADCs or one or more pharmaceutical compositions described herein are provided in liquid form in a sealed container, such as a container labeled with the amount and concentration of the agent. In some embodiments, the liquid form of the administration composition is provided in a sealed container containing at least 0.25 mg/ml, at least 0.5 mg/ml, at least 1 mg/ml, at least 2.5 mg /ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 50 mg/ml , at least 75 mg/ml or at least 100 mg/ml ADC. The liquid form can be stored at 2°C to 8°C in the original container.

[427] In some embodiments, the disclosed ADCs may be included in a pharmaceutical composition suitable for parenteral administration. The injection solution may consist of either a liquid or lyophilized dosage form in a colorless or amber yellow vial, ampoule or pre-filled syringe or other known delivery or storage device.

[428] The compositions described herein can be in various forms. These include, for example, liquid, semi-solid and solid dosage forms such as liquid solutions (eg injections and infusions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended route of administration and therapeutic use.

[429] In various embodiments, treatment comprises a single bolus or repeat administration of an ADC-based drug via a suitable route of administration.

[430] Patients can be assessed regarding the levels of target antigen in a given sample (eg, levels of cells expressing the target antigen) to help determine the most effective dosing regimen, etc. An exemplary embodiment is a method of determining whether a patient will respond to treatment with an ADC of the present invention, comprising obtaining a biological sample from the patient and contacting the biological sample with the ADC. Exemplary biological samples include tissue or biological fluid such as inflammatory exudate, blood, serum, bowel fluid, stool sample, or tumor biopsy (e.g., a tumor biopsy obtained from a patient who has a cancer expressing a target antigen, e.g., cancer expressing HER2, cancer expressing CD138, cancer expressing EPHA2 or at risk of developing it). In some embodiments, a sample (e.g., tissue and/or biological fluid) can be obtained from a subject, and a suitable immunological method can be used to detect and/or measure the expression level of a protein representing the target antigen (e.g., HER2, CD138, EPHA2 , MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4 or STEAP1). Such assessments are also used for monitoring purposes throughout therapy and are useful for assessing therapeutic success in conjunction with assessment of other parameters.

[431] In some embodiments, the effectiveness of an ADC can be assessed by contacting a tumor sample from a subject with the ADC and assessing the growth rate or volume of the tumor. In some embodiments, if the ADC has been determined to be effective, it can be administered to the subject.

[432] The above therapeutic approaches can be combined with any of a variety of additional regimens of surgery, chemotherapy, or radiation therapy. In some embodiments, the ADCs or compositions disclosed herein are formulated and/or administered in conjunction with one or more additional therapeutic agents, such as one or more chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, for example, nitrogen mustards, ethyleneimine compounds and alkyl sulfonates; antimetabolites, such as folic acid, purine or pyrimidine antagonists; antimitotic agents, such as tubulin inhibitors such as eribulin or eribulin mesylate (Halaven™), vinca alkaloids and auristatins; cytotoxic antibiotics; compounds that damage or interfere with DNA expression or replication, for example, DNA minor groove binding agents; and growth factor receptor antagonists. In some embodiments, the chemotherapeutic agent may be a cytotoxic or cytostatic agent. Examples of cytotoxic agents include, but are not limited to, antimitotic agents such as eribulin or eribulin mesylate (Halaven™), auristatins (e.g., monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF)), maytansinoids (e.g., maytansine), dolastatins, duostatins, cryptophycins, vinca alkaloids (eg, vincristine, vinblastine), taxanes, taxols and colchicines; anthracyclines (eg daunorubicin, doxorubicin, dihydroxyanthracinedione); cytotoxic antibiotics (eg, mitomycins, actinomycins, duocarmycins (eg, CC-1065), auromycins, duomycins, calicheamicins, endomycins, phenomycins); alkylating agents (eg cisplatin); intercalating agents (eg ethidium bromide); topoisomerase inhibitors (eg etoposide, tenoposide); radioactive isotopes such as At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² or ²¹³ , P ³² and radioactive isotopes of lutetium (for example, Lu ¹⁷⁷ ); and toxins of bacterial, fungal, plant or animal origin (eg, ricin (eg, ricin A chain), diphtheria toxin, Pseudomonas exotoxin A (eg, PE40), endotoxin, mitogellin, combrestatin, restricttocin, gelonin, alpha-sarcin, abrin (eg abrin A-chain), modeccin (eg modeccin A-chain), chickencin, crotin, inhibitor from Sapaonaria officinalis , glucocorticoid).

[433] This document also discloses uses of one or more of the disclosed ADCs in the manufacture of a drug for the treatment of cancer, for example, in accordance with the methods described above. In some embodiments, the ADCs disclosed herein are used to treat cancer, for example, in accordance with the methods described above.

[434] In various embodiments, kits for the research and therapeutic uses described herein are within the scope of the present invention. Such kits may contain a carrier, package or container that is divided into compartments to accommodate one or more containers such as vials, tubes and the like, each of the containers containing one of the individual elements for use in the method disclosed herein , together with a label or package insert containing instructions for use, such as those described in this document. The kits may contain a container containing a drug moiety. The present invention also provides one or more ADCs or pharmaceutical compositions based thereon, sealed in a container such as an ampoule or sachet, indicating the amount of the agent.

[435] Kits may contain the container described above and one or more other containers associated therewith, which contain materials required from a commercial and user perspective, including buffers, diluents, filters, needles, syringes; carrier, packaging, container, bottle and/or tube with contents and/or instructions for use on the label, as well as package inserts with instructions for use.

[436] A label may be present on or with the container to indicate that the composition is for a particular therapeutic or non-therapeutic use, such as prognostic, prophylactic, diagnostic, or laboratory use. The label may also include a prescription for in vivo or in vitro use, such as those described herein. Prescriptions and other information may also be included in the package insert(s) or label(s) included with or affixed to the kit. The label may be on or associated with the container. The label may be on the container itself if the letters, numbers or other symbols that form the label are pressed or engraved on the container itself. The label may be associated with the container if it is present in a container or carrier that also holds the container, such as a package insert. The label may state that the composition is used to diagnose or treat a condition, such as cancer, described herein.

Neoantigens and methods of their use

[437] In various embodiments, also disclosed herein are methods of treating a patient by inducing the formation of neoantigens in tumor cells that can be targeted by the patient's immune system for clearance. Without being limited by theory, in various embodiments, administration of a herboxydiene-based splicing modulator alone and/or as part of an ADC or composition can produce neoantigens that induce an immune response, induce an immune response to double-stranded RNA, for example, as a result of re-expression of endogenous retroviruses , present in the intron, and/or the production of neoantigens that induce immunogenic cell death.

[438] As used herein, the term “neoantigen” means any antigen to which the immune system has not previously been exposed, which is generated as a result of one or more tumor-specific mutations and/or as a result of tumor exposure to a herboxydiene-based splicing modulator (for example, any one or more than one of the herboxydiene-based splicing modulators disclosed herein, individually and/or as part of an ADC or composition). Tumor-specific mutations may include missense mutations, frameshifts, translocations and splice variants of mRNA, as well as mutations that affect post-translational processing such as phosphorylation and glycosylation. In various embodiments, these exemplary mutations may result from nonsynonymous changes in the coding sequence and/or mutations that alter mRNA processing (eg, splicing). In various embodiments, each of these illustrative mutations may result in molecular changes that can be recognized by the corresponding T cell receptor. In various embodiments, an exemplary neoantigen is a neoantigen whose formation is induced by delivery of a herboxydiene-based splicing modulator alone and/or as part of an ADC or composition. In various embodiments, delivery of a herboxydiene splicing modulator (e.g., any one or more herboxydiene splicing modulators disclosed herein) can induce the formation of a new mRNA splice variant that results in the translation of proteins containing one or more new peptide domains , to which the immune system has not previously been exposed. In various embodiments, the tumor-specific mutations may be mRNA splice variants resulting from the delivery or administration of a herboxidiene splicing modulator, ADC, or a composition comprising a herboxidiene splicing modulator, or ADC.

[439] Without being limited by theory, in various embodiments, delivery of herboxydiene-based splicing modulators alone and as part of an ADC or composition can induce the formation of a new mRNA splice variant (eg, exon skipping, intron retention), resulting in an open frame change reading and/or coding sequences of various genes. In various embodiments, these altered genes are translated into proteins containing one or more new peptide domains that are recognized as foreign by the immune system. In various embodiments, the one or more novel peptide domains do not exist in proteins or in any other part of the human proteome in the absence of treatment with a herboxidiene-based splicing modulator. In various embodiments, proteins containing one or more novel peptide domains can be degraded by the proteasome to generate new peptide fragments that act as substrates for an immunopeptide presentation mechanism, such as through MHC presentation. In various embodiments, novel peptide fragments representing neoantigens can be presented in the MHC1-associated peptidome, for example, on tumor cells.

[440] In various embodiments, delivery of herboxydiene-based splicing modulators alone and as part of an ADC or composition can result in one or more tumor cell-specific events (eg, cell growth arrest). In various embodiments, tumor cell-specific event(s) may result in (1) increased contact with phagocytes (Bracci et al. (2014) Cell Death Differ. 21(1):15-25); (2) transport of new peptide fragments to the tumor-draining lymph node for contact with antigen-presenting cells; (3) processing by antigen presenting cells of new peptide fragments from the phagocytosed tumor cell and presentation of the fragments as neoantigens to populations of circulating naïve T cells; (4) the interaction of new peptide fragments with T cells expressing receptors that recognize the fragments as neoantigens; (5) maturation and activation of effector T cells that mediate responses (eg, CD4+ and/or CD8+ T cells); and/or (6) contact of T cells with additional tumor cells that have been treated with a herboxydiene-based splicing modulator and present new peptide fragments representing neoantigens on their surface in complexes with MHC1 molecules. In various embodiments, tumor cell-specific event(s) may result either directly or indirectly in the recruitment of effector T cells and/or killing of neoantigen-presenting tumor cells.

[441] Also, without being limited by theory, in various embodiments, delivery of herboxydiene-based splicing modulators alone and as part of an ADC or composition can induce re-expression of endogenous retroviruses present in the intron, resulting in an immune response to the double-stranded RNA.

[442] Additionally, without being limited by theory, in various embodiments, delivery of herboxydiene-based splicing modulators alone and as part of an ADC or composition can result in immunogenic cell death caused by the splicing modulator-induced release of neoantigens generated by mutations. In various embodiments, delivery of herboxydiene-based splicing modulators alone and as part of an ADC or composition can induce an immune response to the double-stranded RNA. In various embodiments, an immune response to double-stranded RNA may result from re-expression of endogenous retroviruses present in the intron. In various embodiments, the immune response to the double-stranded RNA may result in the death of tumor cells. In various embodiments, delivery of splicing modulators alone and as part of an ADC or composition can induce immunogenic cell death. In various embodiments, immunogenic cell death may be caused by the release of mutation-derived neoantigens and/or the host immune response to tumor cells.

[443] Accordingly, in various embodiments, methods of treatment are disclosed that involve inducing neoantigens by administering one or more herboxydiene splicing modulators and/or ADCs, and/or compositions containing a herboxydiene splicing modulator or ADC, such as any modulator herboxydiene-based splicing, ADC or composition disclosed herein. In various embodiments, the method comprises administering a herboxydiene splicing modulator, ADC, or composition at a reduced dosage compared to the dosage that would be required in the absence of neoantigen induction. In some embodiments, the method involves administering one or more initial inducing doses to produce neoantigens and induce an immune response (e.g., converting naïve T cells into memory cells), followed by a reduction in dosage or frequency of administration (i.e., due to the combined effect of the splicing modulator on based on herboxydiene, ADC or composition and immune targeting of neoantigens). In some embodiments, treatment may comprise administration of a combination of a herboxydiene splicing modulator, an ADC, or a neoantigen-based immune response inducing composition, and at least one additional therapy (eg, a second anticancer therapy). For example, in some embodiments, treatment may involve administration of a combination of a herboxydiene splicing modulator, an ADC, or a neoantigen-based immune response composition, and one or more immune checkpoint inhibitors. In some embodiments, treatment may involve administering a combination of a herboxydiene-based splicing modulator, an ADC, or a neoantigen-based immune response composition and one or more cytokines or cytokine analogs. In some embodiments, the treatment may involve administration of a combination of a herboxydiene splicing modulator, an ADC, or a composition for inducing a neoantigen-based immune response, and one or more neoantigen vaccines. In some other embodiments, treatment may involve administration of a combination of a herboxydiene splicing modulator, an ADC, or a neoantigen-based immune response composition, and one or more engineered tumor-targeting T cells (eg, CAR-T).

[444] In some embodiments, neoantigens can be used to monitor the effectiveness of treatment with a herboxydiene-based splicing modulator, ADC, or composition. For example, following administration of a herboxydiene-based splicing modulator, ADC, or composition, a sample from a patient (eg, a tumor biopsy) can be obtained and screened for neoantigens or markers of an immune or inflammatory response. If a neoantigen and/or immune response is detected, additional treatment may be given, for example at a reduced dosage.

[445] In various embodiments, methods of treatment are disclosed that involve inducing an immune response to double-stranded RNA by administering one or more herboxidiene splicing modulators and/or ADCs, and/or compositions containing a herboxidiene splicing modulator or ADC, e.g. any herboxydiene splicing modulator, ADC or composition disclosed herein.

[446] In various embodiments, methods of treatment are disclosed that involve inducing immunogenic cell death by administering one or more herboxidiene splicing modulators and/or ADCs, and/or compositions containing a herboxidiene splicing modulator or ADC, such as any modulator herboxydiene-based splicing, ADC or composition disclosed herein.

[447] In various embodiments, administration of a herboxidiene splicing modulator, an ADC, or a composition comprising a herboxidiene splicing modulator can be combined with any known anticancer therapy. Examples of current immune activation strategies available for the treatment of cancer include, but are not limited to, treatment with immune checkpoint inhibitor (ICI) molecules, treatment with cytokines or cytokine analogues, vaccination with tumor vaccines, and engineering T- cells that specifically target the tumor (for example, proliferation of tumor-infiltrating lymphocytes or CAR-T). These technologies are primarily aimed at enhancing or inducing an immune response to pre-existing tumor antigens (either mutations or aberrant expression of cell surface proteins). One or more of these strategies may involve one or more mutations that are capable of inducing the development of a T cell response to the antigen. For example, patient responses to checkpoint inhibition may correlate with mutational load with nonsynonymous changes. In addition, cancer vaccine approaches that rely on pre-existing mutations and the antigenicity of those mutations can be used.

[448] Herboxydiene-based splicing modulators and/or ADCs containing such modulators are capable of inducing broad changes in the transcriptome that occur across multiple lineages. Translation of these altered mRNAs can produce stable and reproducible altered proteins that produce MHC1-binding neopeptides with high affinity for various isotypes of HLA molecules. Without being limited by theory, due to the large number of changes in the transcriptome and proteome, treatment with herboxydiene-based splicing modulators and/or ADCs can increase the number of potentially reactive neoantigens for improved recruitment into the adaptive immune response.

[449] As used herein, the terms “herboxydiene-based splicing modulator,” “splicing modulator,” “spliceosome modulator,” or “spliceosome modulator” refer to compounds that exhibit anticancer activity by interacting with components of the spliceosome. In some embodiments, the splicing modulator changes the rate or form of splicing in a target cell. Herboxydiene-based splicing modulators that function as inhibitory agents, for example, are capable of reducing uncontrolled cell proliferation. Specifically, in some embodiments, herboxydiene-based splicing modulators may act by inhibiting the SF3b spliceosome complex. In some embodiments, the herboxydiene-based splicing modulator is selected from any one or more herboxydiene-based splicing modulators disclosed herein. In some embodiments, the herboxydiene-based splicing modulator used is delivered into the cell and/or administered to the subject as a separate agent. In some other embodiments, the herboxydiene-based splicing modulator employed is delivered into a cell and/or administered to a subject as part of an ADC (eg, an ADC selected from any of the exemplary ADCs disclosed herein). In some other embodiments, the herboxidiene splicing modulator employed is delivered into a cell and/or administered to a subject as part of a composition containing multiple copies of the herboxidiene splicing modulator or multiple copies of an ADC carrying the herboxidiene splicing modulator. Such compositions are disclosed herein.

[450] In some embodiments, a herboxydiene-based splicing modulator that is delivered into a cell and/or administered to a subject as part of an ADC (e.g., an ADC selected from any of the exemplary ADCs disclosed herein) provides additional therapeutic benefits over using a herboxydiene-based splicing modulator that is delivered into the cell and/or administered to the subject as a separate agent. For example, in some embodiments, the herboxidiene splicing modulator used, which is delivered to a cell and/or administered to a subject as part of an ADC, provides targeted delivery of the herboxidiene splicing modulator to a neoplastic cell expressing a target antigen (i.e., an antigen on which specifically targets an antibody fragment from an ADC). In some embodiments, such targeted delivery of a herboxydiene-based splicing modulator reduces off-target treatment and/or off-target cytotoxicity. In some embodiments, such targeted delivery promotes tumor-selective presentation of the neoantigen on neoplastic cells, but not on healthy cells that do not express the target antigen. In some embodiments, such targeted delivery results, for example, in at least 75%, 80%, 85%, 90%, 95%, or 99% alternative splicing and the induction of new mRNAs and peptides associated with MHC molecules re-presenting neoantigens in target neoplastic cells but not in non-target cells. Thus, without being limited by theory, in some embodiments, after priming and/or expansion of effector T cells (e.g., using a neoantigen vaccine), the immune system may preferentially attack neoplastic cells presenting the neoantigen over healthy cells due to preferential expression of neoantigens on tumor cells after treatment with the ADC described herein.

Induction of an immune response and treatment regimen:

[451] In various embodiments, the present invention provides a method of inducing the formation of at least one neoantigen by contacting a neoplastic cell with an effective amount of a herboxidiene splicing modulator, an antibody-drug conjugate (ADC) of a herboxidiene splicing modulator, or a composition , containing a herboxidiene-based splicing modulator or ADC. In various embodiments, the present invention provides a method of inducing an immune response to double-stranded RNA by contacting a neoplastic cell with an effective amount of a herboxidiene splicing modulator, an antibody-drug conjugate of a herboxidiene splicing modulator (ADC), or a composition comprising a splicing modulator based on herboxydiene or ADC. In various embodiments, the present invention provides a method of inducing immunogenic cell death by contacting a neoplastic cell with an effective amount of a herboxydiene splicing modulator, an antibody-drug conjugate (ADC) of a herboxydiene splicing modulator, or a composition comprising a herboxydiene splicing modulator. herboxydiene or ADC.

[452] In some embodiments, the neoplastic cell is in in vitro cell culture. In some embodiments, the neoplastic cell is obtained from a subject. In some embodiments, the neoplastic cell is located in the body of the subject. In some embodiments, the neoplastic cell is derived from a hematologic malignancy or solid tumor. In some embodiments, the hematologic malignancy is selected from B-cell malignancy, leukemia, lymphoma, and myeloma. In some embodiments, the hematologic malignancy is selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the solid tumor is selected from breast cancer (eg, HER2-positive breast cancer), gastric cancer (eg, gastric adenocarcinoma), prostate cancer, ovarian cancer, lung cancer (eg, lung adenocarcinoma), uterine cancer ( for example, serous endometrial carcinoma of the uterus), salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer and esophageal cancer. In some embodiments, the solid tumor is selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

[453] In some embodiments, the present invention further provides a method of inducing the formation of at least one neoantigen and/or the development of a T cell response in a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a splicing modulator based on herboxydiene, ADC, or a composition containing a herboxydiene-based splicing modulator or ADC. Also provided herein, in various embodiments, is a method of treating a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a herboxidiene splicing modulator, ADC, or a composition comprising a herboxidiene splicing modulator, or ADC, wherein administration a herboxydiene splicing modulator, antibody-drug conjugate or composition induces the formation of at least one neoantigen and/or T cell response.

[454] In various embodiments, the present invention provides a method of inducing an immune response to double-stranded RNA in a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a herboxydiene splicing modulator, ADC, or a composition comprising a splicing modulator on based on herboxydiene or ADC. Also provided herein, in various embodiments, is a method of treating a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a herboxidiene splicing modulator, ADC, or a composition comprising a herboxidiene splicing modulator, or ADC, wherein administration a herboxydiene-based splicing modulator, antibody-drug conjugate or composition induces an immune response to double-stranded RNA.

[455] In still other embodiments, the present invention provides a method of inducing immunogenic cell death in a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a herboxydiene-based splicing modulator, an ADC, or a composition comprising a herboxydiene-based splicing modulator. herboxydiene or ADC. Additionally, in various embodiments, provided herein is a method of treating a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a herboxidiene splicing modulator, ADC, or a composition comprising a herboxidiene splicing modulator, or ADC, wherein administration a herboxidiene-based splicing modulator, antibody-drug conjugate or composition induces immunogenic cell death.

[456] In some embodiments, the present invention further provides a method of treating a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a herboxidiene splicing modulator, ADC, or a composition comprising a herboxidiene splicing modulator, or ADC, wherein administration of a herboxidiene splicing modulator, antibody-drug conjugate, or composition in combination with one or more additional therapies comprising a second agent induces immunogenic cell death.

[457] In some embodiments of the therapeutic methods described herein, the administered amount of a herboxydiene splicing modulator, antibody-drug conjugate, composition, or second agent is reduced due to inducing the formation of at least one neoantigen and/or T cell response by compared to a standard dosage of a herboxydiene splicing modulator, antibody-drug conjugate, composition or second agent. In some embodiments, the administered amount of the herboxydiene splicing modulator, antibody-drug conjugate, composition, or second agent is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% , 75% or 90% compared to a standard dosage of a herboxydiene splicing modulator, antibody-drug conjugate, composition or second agent. In some embodiments, the herboxydiene splicing modulator, antibody-drug conjugate, composition, or second agent is administered at at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50 %, 75%, or 90% less frequently than the standard regimen of a herboxydiene splicing modulator, antibody-drug conjugate, composition, or second agent. In some embodiments, the administered amount and/or dosage of the herboxydiene splicing modulator, antibody-drug conjugate, composition, or second agent results in lower systemic toxicity and/or improved tolerability.

[458] As used herein, the term “standard dosage” or “standard dosage regimen” means any usual or generally accepted regimen for administering a therapeutic agent, such as that proposed by a manufacturer, approved by regulatory authorities, or otherwise tested in human subjects to meet the needs of the average patient. In some embodiments, the therapeutic agent is a herboxidiene splicing modulator, an antibody, or an antibody-drug conjugate with anticancer activity.

[459] For example, a standard dosage regimen for trastuzumab, an exemplary anti-HER2 antibody disclosed herein, may be 8 mg/kg administered intravenously over 90 minutes. (week 1), followed by intravenous administration of 6 mg/kg over 30-90 minutes. every 3 weeks (from week 4 until the end of the treatment cycle) (Herceptin® (trastuzumab), FDA Compliant Labeling Supplement, 2017).

[460] As another example, a standard dosing regimen for ipilimumab, an exemplary anti-CTLA4 checkpoint inhibitor antibody, may be 3 mg/kg administered intravenously over 90 minutes. every 3 weeks for 4 doses (Yervoy® (ipilimumab), FDA Compliant Labeling Supplement, 2018). Another standard dosing regimen for ipilimumab may be 10 mg/kg administered intravenously over 90 minutes. every 3 weeks for 4 doses, then 10 mg/kg every 12 weeks for up to 3 years (Yervoy® (ipilimumab), FDA Compliant Labeling Supplement, 2018).

[461] As another example, a standard dosing regimen for nivolumab, an exemplary anti-PD1 checkpoint inhibitor antibody, may be 3 mg/kg IV over 60 minutes. every 2 weeks (Opdivo® (nivolumab), FDA-compliant labeling, 2015).

[462] As another example, a standard dosing regimen for atezolizumab, an exemplary anti-PDL1 checkpoint inhibitor antibody, may be 1200 mg administered intravenously over 60 minutes. every 3 weeks (Tecentriq® (atezolizumab), 2018 FDA Compliant Labeling Supplement).

[463] As another example, a standard dosing regimen for T-DM1, an exemplary anti-HER2 antibody-drug conjugate, may be 3.6 mg/kg administered intravenously over 90 minutes. every 3 weeks (Kadcyla® (T-DM1), 2016 FDA Compliant Labeling Supplement).

[464] In some embodiments, the methods described herein may further include administration of at least one additional therapy (eg, checkpoint inhibitor, neoantigen vaccine, cytokine or cytokine analog, CAR-T, etc.). In some embodiments, the administered amount of the herboxydiene splicing modulator, antibody-drug conjugate, composition, and/or at least one additional therapy is reduced due to the induction of at least one neoantigen and/or T cell response compared to a standard dosage a herboxydiene-based splicing modulator, an antibody-drug conjugate, a composition and/or at least one additional therapy. In some embodiments, the administered amount of the herboxidiene splicing modulator antibody-drug conjugate composition and/or at least one additional therapy is reduced due to inducing an immune response to the double-stranded RNA compared to a standard dosage of the herboxidiene splicing modulator antibody conjugate and a drug, composition and/or at least one additional therapy. In some embodiments, the administered amount of the herboxydiene splicing modulator, antibody-drug conjugate, composition, and/or at least one additional therapy is reduced due to the induction of immunogenic cell death compared to a standard dosage of the herboxydiene splicing modulator, antibody-drug conjugate agents, compositions and/or at least one additional therapy. In some embodiments, the administered amount of the herboxydiene splicing modulator, antibody-drug conjugate, composition, and/or at least one additional therapy is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40 %, 45%, 50%, 75% or 90% compared to a standard dosage of a herboxydiene splicing modulator, antibody-drug conjugate, composition and/or at least one additional therapy. In some embodiments, the herboxydiene splicing modulator, antibody-drug conjugate, composition, and/or at least one additional therapy is administered at at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% less frequently than a standard regimen of administration of a herboxidiene splicing modulator, antibody-drug conjugate, composition, and/or at least one additional therapy. In some embodiments, the administered amount and/or dosage of the herboxydiene splicing modulator, antibody-drug conjugate, composition, and/or at least one additional therapy results in lower systemic toxicity and/or improved tolerability.

[465] In some embodiments, administration of the herboxydiene splicing modulator, antibody-drug conjugate, or composition is initiated prior to administration of the at least one additional therapy. In other embodiments, administration of the herboxydiene splicing modulator, antibody-drug conjugate, or composition is initiated after administration of at least one additional therapy. In yet other embodiments, administration of the herboxydiene splicing modulator, antibody-drug conjugate, or composition is initiated simultaneously with administration of at least one additional therapy.

[466] In some embodiments, administration of the herboxydiene splicing modulator, antibody-drug conjugate, or composition is repeated at least once after the initial administration. In some embodiments, the amount of herboxydiene splicing modulator, antibody-drug conjugate or composition used for repeated administration is reduced compared to the amount used for initial administration. In some embodiments, the amount of herboxydiene splicing modulator, antibody-drug conjugate or composition used for repeated administration is reduced compared to a standard dosage of herboxydiene splicing modulator, antibody-drug conjugate or composition. In some embodiments, the amount of herboxydiene splicing modulator, antibody-drug conjugate or composition used for re-administration is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75% or 90% compared to the standard dosage or initial dosage of the herboxydiene splicing modulator, antibody-drug conjugate or composition.

[467] In some embodiments, administration of the at least one additional therapy is repeated at least once after the initial administration. In some embodiments, the amount of at least one additional therapy used for repeated administration is reduced compared to the amount used for initial administration. In some embodiments, the amount of at least one additional therapy used for repeated administration is reduced compared to the standard dosage of the at least one additional therapy. In some embodiments, the amount of at least one additional therapy used for reintroduction is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% compared to the standard dosage or the initial dosage of at least one additional therapy.

[468] In some embodiments, reintroduction of the herboxydiene splicing modulator, antibody-drug conjugate, or composition occurs simultaneously with reintroduction of at least one additional therapy. In some embodiments, administration of the herboxydiene splicing modulator, antibody-drug conjugate, or composition is administered sequentially or spaced in time with repeated administration of at least one additional therapy.

[469] In some embodiments, the at least one additional therapy comprises administration of a checkpoint inhibitor, for example, any checkpoint inhibitor disclosed herein. In some embodiments, the subject is intolerant, unresponsive, or deficient in responsiveness to the checkpoint inhibitor when administered alone. In some embodiments, the checkpoint inhibitor specifically targets PD1/PDL1, CTLA4, OX40, CD40, LAG3, TIM3, GITR, and/or KIR. In some embodiments, the checkpoint inhibitor specifically targets CTLA4, OX40, CD40, and/or GITR. In some embodiments, a checkpoint inhibitor is an antibody having inhibitor or agonist activity against its target. In some embodiments, targeting of the checkpoint inhibitor is provided by an inhibitory antibody or other such inhibitory molecule. In other embodiments, targeting of the checkpoint inhibitor is provided by an agonist antibody or other similar agonist molecule.

[470] In some other embodiments, the at least one additional therapy comprises the administration of a neoantigen vaccine, for example, any neoantigen vaccine disclosed herein. In some embodiments, the herboxydiene splicing modulator, ADC, or composition is administered prior to administration of the neoantigen vaccine. In some embodiments, the herboxydiene splicing modulator, ADC, or composition is administered following administration of the neoantigen vaccine. In some embodiments, the herboxydiene splicing modulator, ADC, or composition is administered concomitantly with administration of the neoantigen vaccine. In some embodiments, administration of the herboxydiene splicing modulator, ADC, or composition is repeated at least once after the initial administration. In some embodiments, the amount of the herboxydiene splicing modulator, ADC, or composition used for repeated administration is reduced compared to the amount used for initial administration.

[471] In some embodiments, the neoantigen vaccine comprises at least one neoantigen peptide. In some embodiments, the length of the at least one neoantigenic peptide ranges from about 10 to about 50 amino acids. In some embodiments, the length of the at least one neoantigen peptide ranges from about 10 to about 35 amino acids. In some embodiments, the length of the at least one neoantigenic peptide ranges from about 15 to about 25 amino acids. In some embodiments, the at least one neoantigenic peptide contains one or more neoantigenic sequences.

[472] In some embodiments, the length of the neoantigenic sequence and/or antigenic portion ranges from about 10 to about 50 amino acids. In some embodiments, the length of the at least one neoantigen peptide ranges from about 10 to about 35 amino acids. In some embodiments, the length of the neoantigenic sequence and/or antigenic portion ranges from about 15 to about 25 amino acids. In some embodiments, the length of the neoantigenic sequence and/or antigenic portion ranges from about 10 to about 20 amino acids. In some embodiments, the neoantigenic sequence and/or antigenic moiety does not directly overlap or consist of a canonical peptide sequence (eg, any of the exemplary canonical peptide sequences highlighted in Table 8).

[473] As used herein, the term “antigenic portion” or “antigenic fragment” of a neoantigenic sequence means one or more fragments of a neoantigenic sequence that retain the ability to induce a T cell response (e.g., antigen-specific expansion and/or maturation of T effector population(s) -cells). In some embodiments, the antigenic portion may also retain the ability to be internalized, processed, and/or presented by antigen-presenting cells (eg, dendritic cells). In some embodiments, the antigenic portion also retains T cell priming function. In some embodiments, the length of the antigenic portion of the neoantigenic sequence ranges from about 10 to about 50 amino acids. In some embodiments, the length of the antigenic portion of the neoantigenic sequence ranges from about 10 to about 35 amino acids. In some embodiments, the length of the antigenic portion of the neoantigenic sequence ranges from about 15 to about 25 amino acids. In some embodiments, the length of the antigenic portion of the neoantigenic sequence ranges from about 10 to about 20 amino acids. In some embodiments, the antigenic portion of a neoantigen sequence (eg, the antigenic portion of any of SEQ ID NOs: 66-93) or the mRNA encoding it is formulated into a neoantigen vaccine.

[474] An exemplary embodiment of the antigenic portion is the region(s) flanking amino acids 45-53 of SEQ ID NO: 66. Another exemplary embodiment of the antigenic portion is the region(s) flanking the amino acids 82-90 of SEQ ID NO: 66. In some embodiments, the antigenic portion is capable of binding to at least one HLA allele expressed in the subject (eg, HLA-A*02:01). In some other embodiments, the antigenic moiety is capable of binding to at least one HLA allele expressed in at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, of at least 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from a neoplastic disorder. In some embodiments, the antigenic portion is capable of eliciting a T cell response against a tumor present in at least 1%, at least 5%, or at least 10% of a population of subjects suffering from the neoplastic disorder.

[475] In some embodiments, the antigenic portion does not directly overlap with or consist of a canonical peptide sequence. As used herein, the term “canonical peptide sequence” means any contiguous peptide sequence present in the human proteome in the absence of contact with a herboxydiene splicing modulator (e.g., in the absence of contact with a herboxydiene splicing modulator alone and/or as part of an ADC or composition) and/or to which the immune system has previously been exposed. In some embodiments, the canonical peptide sequence is derived from and/or encoded by the open reading frame of the canonical transcript. Exemplary canonical peptide sequences are highlighted in Table 8.

[476] In some embodiments, if a herboxydiene-based splicing modulator is administered (e.g., alone and/or as part of an ADC or composition), the canonical peptide sequence can be derived from the 24 nucleotides located immediately at the 5' end of the frame before the event aberrant splicing induced by a splicing modulator and/or may be encoded by them. Thus, in some embodiments, the canonical peptide sequence contains or consists of 8 amino acids immediately N-terminal to the neoantigen sequence induced by a herboxidiene splicing modulator. In some embodiments, if the 5' exon sequence ends at the terminal nucleotide of the codon, then the canonical peptide sequence ends at the end of the exon. In some other embodiments, if the 5' exon sequence ends with one or two of the three nucleotides of the codon, then the canonical peptide sequence is derived from and/or encoded by the 24 nucleotides preceding the partial codon. In some embodiments, 3' mRNA sequences in an aberrant splicing event may be translated in the same open reading frame derived from the 5' exon until reaching a stop codon, at which point translation may be terminated. In some embodiments, if an aberrant splicing event (eg, exon skipping) results in retention of the open reading frame of the canonical transcript, the C-terminal sequence may be translated into an additional 24 nucleotides encoding the 8 C-terminal amino acids. In some embodiments, in this context, only the region along the aberrant exon junction may encode a neoantigen sequence. In some embodiments, if the open reading frame is shifted (eg, by retaining an intron), then the entire C-terminal sequence (encoded by the 3'-terminal mRNA) may encode a neoantigen sequence.

[477] In some embodiments, the antigenic portion of the neoantigenic sequence is selected by comparing the neoantigenic sequence to a canonical peptide sequence and selecting a portion of the neoantigenic sequence that does not directly overlap with, consist of, and/or align with the canonical peptide sequence. In some embodiments, the antigenic portion of a neoantigenic sequence can be screened for antigenicity and/or T cell priming function in the same manner as full-length neoantigenic sequences (eg, the neoantigenic sequence from which the antigenic portion is derived). In some embodiments, the antigenic portion of the neoantigen sequence is assessed for antigenicity and/or T cell priming function using a T cell priming assay, such as the exemplary T cell priming experiments described herein.

[478] In some embodiments, the neoantigenic sequence is a subject-specific neoantigenic sequence. In some embodiments, the neoantigen sequence is a personalized neoantigen vaccine for the subject. In some embodiments, the neoantigen sequence used to create a personalized neoantigen vaccine for a subject is capable of binding to at least one HLA allele expressed in the subject. In some embodiments, a personalized neoantigen vaccine is selected by identifying neoantigens expressed in the subject's tumor, for example, after administering a herboxidiene-based splicing modulator or ADC and selecting a vaccine containing the neoantigen sequence found in the patient's tumor.

[479] The term “personalized,” when used to describe a neoantigen vaccine, means a vaccine created by identifying one or more neoantigens produced in a patient, preferably a neoantigen identified in the patient after exposure to a herboxydiene-based splicing modulator, ADC, or composition, and then using one or more of these neoantigens as the basis for a vaccine for the same patient. Accordingly, in some embodiments, the patient is administered a herboxydiene splicing modulator, ADC, or composition and is then screened for the presence of neoantigens produced as a result of the treatment. In some embodiments, the selected neoantigen vaccine comprises a neoantigen peptide or mRNA disclosed herein that has been confirmed to be present in a patient following exposure to a herboxydiene-based splicing modulator, ADC, or composition. In some embodiments, the herboxydiene splicing modulator, ADC or composition and/or peptide or mRNA vaccine can be administered to a patient once or repeatedly. Therefore, in some embodiments, one or more of these neoantigens are used to create a personalized vaccine that is administered to a patient. In some embodiments, one or more neoantigens used to create a personalized vaccine have binding affinity for one or more patient-specific HLA alleles. In some embodiments, the patient expresses one or more MHC1 alleles that bind to one or more neoantigens. Prediction of whether a neoantigen will bind to a specific MHC1 allele can be accomplished using any computational prediction method known in the art. Exemplary computational forecasting methods are disclosed, for example, in Meydan et al. (2013) BMC Bioinformatics 14(Suppl. 2):S13, which is incorporated herein by reference for such methods.

[480] In some other embodiments, the neoantigen sequence is a generic neoantigen sequence. In some embodiments, the neoantigen sequence is a generic neoantigen vaccine.

[481] The term “universal,” when used to describe a neoantigen vaccine, means a vaccine containing a peptide sequence or an mRNA sequence that is based on a common or known neoantigen(s) detectable by neoantigen sequencing, produced in many patients and/or in tissue samples from patients, preferably after exposure to a herboxydiene-based splicing modulator, ADC or composition. The peptide sequence or mRNA sequence used in the vaccine does not need to be present in every patient, but is found in at least a few patients or tissue samples from patients. In some embodiments, the herboxydiene splicing modulator, ADC or composition and/or peptide or mRNA vaccine can be administered to a patient once or repeatedly. Therefore, in some embodiments, the peptide sequence or mRNA sequence is used to vaccinate additional patients. In some embodiments, the patient is administered a herboxidiene splicing modulator, ADC, or composition, and then is administered a peptide or mRNA vaccine based on a known neoantigen to enhance the immune response to neoantigens produced by the herboxidiene splicing modulator, ADC, or composition. In some embodiments, the patient is administered a generic peptide or mRNA vaccine and then administered a single or repeated dose of a herboxidiene splicing modulator, ADC, or composition. In some embodiments, the neoantigen sequence (or sequences) used to create a universal neoantigen vaccine is selected based on the overall occurrence of the MHC1 allele in a given patient population (Maiers et al. (2007) Hum. Immunol. 68(9):779-88) .

[482] In some embodiments, the neoantigen sequence (eg, a universal neoantigen) is capable of binding to at least one HLA allele expressed in at least 10%, at least 15%, at least 20%, at least 25% , at least 30%, at least 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from a neoplastic disorder. In some embodiments, the neoantigen sequence is capable of inducing a T cell response to a tumor present in at least 1%, at least 5%, or at least 10% of a population of subjects suffering from a neoplastic disorder.

[483] In some embodiments, the neoantigenic sequence has been identified by sequencing at least one neoantigenic peptide or mRNA encoding it, the production of which is induced in a subject by administration of an effective amount of a herboxidiene splicing modulator, antibody-drug conjugate or composition. In some embodiments, the at least one neoantigenic peptide comprises a neoantigenic sequence the formation of which is induced by contacting a neoplastic cell with an effective amount of a herboxidiene splicing modulator, an antibody-drug conjugate or composition. In some embodiments, the neoplastic cell is in in vitro cell culture. In some embodiments, the neoplastic cell is obtained from a subject. In some embodiments, the neoplastic cell is located in the body of the subject.

[484] In some embodiments, the neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable carrier (eg, any of the exemplary carriers described herein). In some embodiments, the at least one neoantigenic peptide is associated with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from a peptide, serum albumin, fisurella hemocyanin, immunoglobulin, thyroglobulin, ovalbumin, toxoid or attenuated toxoid derivative, cytokine, and chemokine. In some embodiments, the neoantigenic peptide and the pharmaceutically acceptable carrier are covalently linked through a linker. In some embodiments, the neoantigen peptide and the pharmaceutically acceptable carrier are expressed as a fusion protein. In some embodiments, the neoantigen vaccine contains at least one neoantigen peptide and a pharmaceutically acceptable diluent. In some embodiments, the neoantigen vaccine contains at least one neoantigen peptide and a pharmaceutically acceptable adjuvant.

[485] In some embodiments, the neoantigen vaccine comprises at least one neoantigen mRNA. In some embodiments, the at least one neoantigen mRNA encodes one or more neoantigen sequences.

[486] In some embodiments, the neoantigenic sequence is a subject-specific neoantigenic sequence. In some embodiments, the neoantigen sequence is a personalized neoantigen vaccine for the subject. In some embodiments, the neoantigen sequence is capable of binding to at least one HLA allele expressed in the subject.

[487] In some other embodiments, the neoantigen sequence is a generic neoantigen sequence. In some embodiments, the neoantigen sequence is a generic neoantigen vaccine. In some embodiments, the neoantigen sequence is capable of binding to at least one HLA allele expressed in at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least at least 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from a neoplastic disorder. In some embodiments, the neoantigen sequence is capable of inducing a T cell response to a tumor present in at least 1%, at least 5%, or at least 10% of a population of subjects suffering from a neoplastic disorder.

[488] In some embodiments, a neoantigen sequence has been identified by sequencing the protein sequence of at least one neoantigen. In some embodiments, a neoantigen sequence has been identified by sequencing at least one neoantigen-encoding mRNA that is induced in a subject by administration of an effective amount of a herboxydiene splicing modulator, antibody-drug conjugate, or composition. In some embodiments, the at least one neoantigenic mRNA encodes a neoantigenic sequence, the formation of which is induced by contacting the neoplastic cell with an effective amount of a herboxidiene splicing modulator, antibody-drug conjugate, or composition. In some embodiments, the neoplastic cell is in in vitro cell culture. In some embodiments, the neoplastic cell is obtained from a subject. In some embodiments, the neoplastic cell is located in the body of the subject.

[489] In some embodiments, the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable carrier (eg, any of the exemplary carriers described herein). In some embodiments, the at least one neoantigen mRNA is associated with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from a peptide, serum albumin, fisurella hemocyanin, immunoglobulin, thyroglobulin, ovalbumin, toxoid or attenuated toxoid derivative, cytokine, and chemokine. In some embodiments, the neoantigen vaccine contains at least one neoantigen mRNA and a pharmaceutically acceptable diluent. In some embodiments, the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable adjuvant. In some embodiments, the neoantigen mRNA is encapsulated using an encapsulating agent. In some embodiments, the encapsulating agent is a liposome. In some embodiments, the encapsulating agent is a nanoparticle.

[490] In some embodiments, the at least one additional therapy comprises administering a cytokine or cytokine analog, such as any cytokine or cytokine analog disclosed herein. In some embodiments, the subject is intolerant, unresponsive, or deficient in responsiveness to a cytokine or cytokine analog when administered alone. In some embodiments, the cytokine or cytokine analog provides a T cell enhancer. In some embodiments, the cytokine or cytokine analogue includes IL-2, IL-10, IL-12, IL-15, IFNγ and/or TNFα. In some embodiments, the cytokine or cytokine analogue includes IL-2, IL-10, IL-12 and/or IL-15. In some embodiments, administration of a cytokine or cytokine analog enhances T cell priming following administration of a herboxidiene splicing modulator, antibody-drug conjugate, or composition due to inducing the formation and presentation of neoantigens.

[491] In some embodiments, the at least one additional therapy comprises the administration of engineered tumor-targeting T cells (i.e., CAR-T), such as any CAR-T therapy disclosed herein .

[492] In some embodiments, the methods described herein may further comprise detecting one or more neoantigens and/or T cell response in a subject following administration of a herboxydiene splicing modulator, antibody-drug conjugate or composition, and optionally continuing administration a herboxydiene-based splicing modulator, antibody-drug conjugate or composition if one or more neoantigens and/or T-cell response are detected. In some embodiments, detection of one or more neoantigens and/or T cell response in a subject indicates the effectiveness of treatment with a herboxidiene splicing modulator, antibody-drug conjugate, or composition. In some embodiments, treatment with an additional therapy along with a herboxidiene splicing modulator, antibody-drug conjugate, or composition is continued if one or more neoantigens and/or T-cell responses are detected. In some embodiments, treatment is continued at a reduced dosage and/or frequency if one or more neoantigens and/or T cell response are detected.

[493] In some embodiments, the methods described herein may further comprise detecting an immune response to the double-stranded RNA in a subject following administration of a herboxidiene splicing modulator, antibody-drug conjugate or composition, and optionally continuing administration of the herboxidiene splicing modulator, antibody-drug conjugate or composition if an immune response to double-stranded RNA is detected. In some embodiments, detection of an immune response to double-stranded RNA in a subject indicates the effectiveness of treatment with a herboxidiene splicing modulator, antibody-drug conjugate, or composition. In some embodiments, treatment with an additional therapy along with a herboxidiene splicing modulator, antibody-drug conjugate, or composition is continued if an immune response to the double-stranded RNA is detected. In some embodiments, treatment is continued at a reduced dosage and/or frequency if an immune response to the double-stranded RNA is detected.

[494] In some embodiments, the methods described herein may further include detecting immunogenic cell death in a subject following administration of a herboxidiene splicing modulator, antibody-drug conjugate or composition, and optionally continuing administration of the herboxidiene splicing modulator, antibody conjugate. and the drug or composition if immunogenic cell death is detected. In some embodiments, detection of immunogenic cell death in a subject indicates the effectiveness of treatment with a herboxidiene splicing modulator, antibody-drug conjugate, or composition. In some embodiments, treatment with an additional therapy along with a herboxidiene splicing modulator, antibody-drug conjugate, or composition is continued if immunogenic cell death is detected. In some embodiments, treatment is continued at a reduced dosage and/or frequency if immunogenic cell death is detected.

[495] In some embodiments, the subject has a mutational load with non-synonymous changes of approximately 150 mutations or less. In some embodiments, the subject has a mutational load with nonsynonymous changes of approximately 100 mutations or less. In some embodiments, the subject has a mutational load with nonsynonymous changes of approximately 50 mutations or less. In some embodiments, the subject has or is suspected of having a neoplastic disorder, such as a hematologic malignancy or solid tumor. In some embodiments, the hematologic malignancy is selected from B-cell malignancy, leukemia, lymphoma, and myeloma. In some embodiments, the hematologic malignancy is selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer cancer and esophageal cancer. In some embodiments, the solid tumor is selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

[496] In various embodiments, the present invention further provides a method of treating a subject who has or is suspected of having a neoplastic disorder, comprising: (a) administering to the subject an effective amount of a herboxydiene splicing modulator, an ADC, or a composition comprising a splicing modulator on a herboxydiene-based or ADC, wherein administration of a herboxydiene-based splicing modulator, antibody-drug conjugate or composition induces the formation of at least one neoantigen and/or T cell response; (b) detecting one or more neoantigens and/or T cell response in a subject following administration of a herboxidiene splicing modulator, antibody-drug conjugate or composition; and (c) continuing to administer the herboxydiene splicing modulator, antibody-drug conjugate, or composition if one or more neoantigens and/or T cell responses are detected. In some embodiments, detection of one or more neoantigens and/or T cell response in a subject indicates the effectiveness of treatment with a herboxidiene splicing modulator, antibody-drug conjugate, or composition. In some embodiments, the one or more neoantigens comprise the amino acid sequence of any one of SEQ ID NO: 37-65. In some embodiments, the one or more neoantigens comprise the amino acid sequence of SEQ ID NO: 37. In some embodiments, the one or more neoantigens comprise the amino acid sequence of SEQ ID NO: 39. In some embodiments, the one or more neoantigens comprise the amino acid sequence of any of SEQ ID NO: 46-49.

Combination of herboxydiene/ADC splicing modulator and immune checkpoint inhibitor

[497] In various embodiments, a patient who has a cancer described herein may be treated with a combination of a herboxidiene splicing modulator, an ADC, or a composition and an immune checkpoint inhibitor therapy. Immune checkpoints are inhibitory pathways that slow or stop immune responses and prevent excess tissue damage due to uncontrolled immune cell activity. As used herein, the term “checkpoint inhibitor” means any therapeutic agent, including any small molecule chemical compound, antibody, nucleic acid molecule or polypeptide, or any fragments thereof, that inhibits one or more inhibitory pathways thereby providing a more functional immune system. activity.

[498] Treatment of patients with immune checkpoint inhibition has been shown to have good efficacy in certain clinical indications. The FDA recently approved the use of a checkpoint inhibitor in patients with tumors exhibiting high microsatellite instability, regardless of tissue origin. This registration was based in part on the observation that response rates correlate positively with mutational load (Rizvi et al. (2015) Science 348(6230):124-8; Hellmann et al. (2018) Cancer Cell 33(5): 853-861). Estimates from the literature vary in absolute numbers and by origin, but generally confirm that above a threshold of ~150–250 mutations, the likelihood of response increases. Analysis of TCGA data demonstrates that a large percentage of adult-onset tumors of various origins have a relatively low mutational load with nonsynonymous changes (Vogelstein et al. (2013) Science 339:1549-58). Most lineages have average nonsynonymous mutation rates of ~30–80 per patient, well below the thresholds required to increase the likelihood of response to checkpoint inhibitors.

[499] For example, HER2-positive breast cancer has been shown to have an average of ~60 nonsynonymous mutations present in a patient sample. However, the threshold for efficacy of checkpoint inhibitor treatment, as mentioned above, is estimated to be in the range of ~150-250 non-synonymous mutations, i.e. patients with a value above this threshold are more likely to demonstrate complete remission, partial remission and/or stable disease, while patients with a value below this threshold are more likely to exhibit disease progression. Therefore, strategies are required to increase the observed number of nonsynonymous mutations and/or neoantigens present on tumor cells and may increase the overall likelihood of response to, for example, a checkpoint inhibitor-based therapy. Because cytokines (and their analogues) act through a similar mechanism of action, such strategies may also increase the overall likelihood of response to cytokine-based therapies.

[500] Current response rates for HER2-positive breast cancer are ~15–25% (CTI NCT02129556). In various embodiments disclosed herein, treatment with a herboxydiene splicing modulator, ADC, or composition in combination with an immune checkpoint inhibitor and/or cytokine therapy may improve such response rates. In various embodiments, treatment with a herboxydiene splicing modulator, ADC, or composition in combination with an immune checkpoint inhibitor and/or cytokine therapy can be used against any tumor occurring in adults, particularly those with the median frequency of mutations with nonsynonymous changes is below the calculated threshold of ~150 mutations. In some embodiments, exemplary cancer types suitable for treatment with a herboxydiene splicing modulator, ADC, or composition of the present invention alone or in combination with an additional therapy (e.g., an immune checkpoint inhibitor therapy, an cytokine-based therapies) include, but are not limited to, esophageal cancer, non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer, gastric cancer, endometrial cancer, pancreatic adenocarcinoma, ovarian cancer, prostate cancer, hepatocellular cancer, glioblastoma, breast cancer (eg , HER2-positive breast cancer), lung cancer (eg, non-small cell lung cancer), chronic lymphocytic leukemia, and acute myeloid leukemia. Other illustrative suitable cancer types are defined, for example, in Vogelstein et al. (2013) Science 339:1549–58, which is incorporated herein by reference in its entirety.

[501] Because many immune checkpoint inhibitor therapies rely on persistent expression of tumor-associated antigens, regular boosters of the treatments are required for effectiveness and to “re-stimulate” reactive T cell populations. The inducible nature of the herboxydiene-based splicing modulator or neoantigens generated by the ADCs described herein provides therapeutic regimens that can be designed to enhance the immune response of neoantigen-reactive T cells while limiting T cell exhaustion. often caused by chronic antigen stimulation. For example, in some embodiments, an initial dose of a herboxydiene splicing modulator, ADC, or composition is administered to a subject to trigger aberrant splicing and the production of neoantigen peptides. In some embodiments, after a period of time necessary for protein production and antigen presentation, the subject is then administered an initial dose of a checkpoint inhibitor to stimulate and/or enhance the priming and expansion of effector T cells. In some embodiments, the waiting period between dosing of the herboxydiene splicing modulator, ADC or composition and the immune checkpoint inhibitor is about 2, about 3, about 4, about 5, about 6, or about 7 days. In some embodiments, the waiting period is from about 3 days to about 5 days. In some embodiments, the checkpoint inhibitor specifically targets CTLA4, OX40, CD40, and/or GITR. In some embodiments, the combined therapeutic benefit of the herboxydiene splicing modulator, ADC or composition and the immune checkpoint inhibitor may be additive or superadditive.

[502] In some embodiments, after a period to allow T cell priming and expansion, the subject is administered a second or subsequent dose of a herboxydiene splicing modulator, ADC, or composition to trigger re-presentation of neoantigen peptides. In some embodiments, the waiting period between administration of the initial dose of the immune checkpoint inhibitor and the second or subsequent dose of the herboxydiene splicing modulator, ADC, or composition is about 2, about 3, about 4, or about 5 weeks. In some embodiments, the waiting period is approximately 3 weeks. Upon administration of a second or subsequent dose of the herboxydiene splicing modulator, ADC, or composition, in some embodiments, the immune system can contact tumor cells presenting neoantigens and/or cause tumor cell destruction. In some embodiments, the subject is then administered a second or subsequent dose of the checkpoint inhibitor to further expand the effector memory T cell population after secondary priming and expansion of the T cells has been achieved.

[503] In some embodiments, dosing of the herboxydiene splicing modulator, ADC, or composition following a given exemplary initial treatment regimen may be pulsatile, i.e. The herboxydiene splicing modulator, ADC, or composition may be administered at dosages at long intervals (eg, approximately every 4 weeks, approximately every 5 weeks, approximately every 6 weeks) to promote antigen presentation, T cell recruitment, and/or tumor cell killing , and/or restoration of the memory T cell population. At later time points, in some embodiments, treatment with a herboxydiene-based splicing modulator, ADC, or composition can be combined with one or more immune checkpoint inhibitors aimed at restoring effector functionality in exhausted T cell populations. For example, in some embodiments, at later time points, treatment with a herboxydiene-based splicing modulator, ADC, or composition can be combined with one or more immune checkpoint inhibitors targeting PD1/PDL1, LAG3, and/or TIM3. In some embodiments, the pulsatile nature of neoantigen presentation and priming may allow for less frequent and/or lower dosage administration of the herboxydiene-based immune checkpoint inhibitor and/or splicing modulator, ADC, or composition to be administered. In some embodiments, the pulsatile nature of neoantigen presentation may provide one or more beneficial effects of treatment with an immune checkpoint inhibitor (e.g., an anti-CTLA4 antibody such as ipilimumab) compared to an immune checkpoint inhibitor when administered without concomitant administration of a splicing modulator on herboxydiene-based ADC or composition, for example, by reducing the potential risk of adverse reactions often observed with standard immune checkpoint inhibitor dosing regimens.

[504] In certain embodiments, the checkpoint inhibitor is an inhibitor of the cytotoxic T lymphocyte associated antigen (CTLA4) pathway. CTLA4, also known as CD152, is a protein receptor that negatively regulates immune responses. CTLA4 is constitutively expressed on regulatory T cells, but its expression is increased in conventional T cells only after activation. As used herein, the term “CTLA4 inhibitor” is intended to mean any CTLA4 inhibitor and/or CTLA4 pathway. Exemplary CTLA4 inhibitors include, but are not limited to, anti-CTLA4 antibodies. CTLA4 blocking antibodies for use in humans were developed based on preclinical activity observed in mouse models of antitumor immunity. Exemplary anti-CTLA4 antibodies include, but are not limited to, ipilimumab (MDX-010) and tremelimumab (CP-675,206), both of which are fully human. Ipilimumab is an IgG1 with a plasma half-life of approximately 12-14 days; Tremelimumab is an IgG2 with a plasma half-life of approximately 22 days. Cm., for example, Phan et al. (2003) Proc Natl Acad Sci USA. 100:8372-7; Ribas et al. (2005) J Clin Oncol. 23:8968-77; Weber et al. (2008) J Clin Oncol. 26:5950-6. In some embodiments, the anti-CTLA4 antibody is ipilimumab.

[505] In certain embodiments, the checkpoint inhibitor is an inhibitor of the programmed cell death protein 1 (PD1) pathway. The programmed cell death protein 1 (PD1) pathway represents a master immune control switch that can be recruited by tumor cells to overcome active T cell immune surveillance. Ligands for PD1 (PDL1 and PDL2) are constitutively expressed or can be induced in various tumors. High levels of PDL1 (and to a lesser extent PDL2) expression on tumor cells have been found to correlate with poor prognosis and poor survival in various other types of solid tumors. Additionally, PD1 has been suggested to control the proliferation of tumor-specific T cells in patients with malignant melanoma. These observations suggest that the PD1/PDL1 pathway plays a critical role in tumor evasion from immune recognition and may be an attractive target for therapeutic intervention. The term “PD1 inhibitor” as used herein is intended to mean any PD1 inhibitor and/or PD1 pathway. Exemplary PD1 inhibitors include, but are not limited to, anti-PD1 and anti-PDL1 antibodies. In certain embodiments, the checkpoint inhibitor is an anti-PD1 antibody. Exemplary anti-PD1 antibodies include, but are not limited to, nivolumab and pemprolizumab (MK-3475). Nivolumab, for example, is a fully human immunoglobulin G4 (IgG4)-based anti-PD1 immune checkpoint inhibitor antibody that disrupts the interaction of the PD1 receptor with its ligands PDL1 and PDL2, thereby allowing inhibition of the cellular immune response (Guo et al. (2017) ) J Cancer 8(3):410-6). In some embodiments, the anti-PD1 antibody is nivolumab. Pembrolizumab, for example, is a potent and highly selective humanized IgG4/kappa mAb designed to directly block the interaction between PD1 and its ligands: PDL1 and PDL2. Pembrolizumab significantly increases T cell-mediated immune responses in cultured blood cells from healthy human donors, cancer patients, and nonhuman primates. Pembrolizumab has also been reported to modulate the levels of interleukin-2 (IL-2), tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), and other cytokines. Exemplary anti-PDL1 antibodies include, but are not limited to, atezolizumab, avelumab, and durvalumab. Atezolizumab, for example, is a humanized IgG1 mAb that has been reported to block the PD1/PDL1 interaction by targeting expressed PDL1 on various types of malignant cells. This blockage of the PD1/PDL1 pathway may stimulate immune defense mechanisms against tumors (Abdin et al. (2018) Cancers (Basel) 10(2):32). In some embodiments, the anti-PDL1 antibody is atezolizumab.

[506] In certain embodiments, the checkpoint inhibitor specifically targets PD1/PDL1, CTLA4, OX40, CD40, LAG3, TIM3, GITR, and/or KIR. In certain embodiments, the checkpoint inhibitor specifically targets CTLA4, OX40, CD40, and/or GITR. In certain embodiments, the checkpoint inhibitor is targeted as an inhibitory antibody or other such inhibitory molecule (eg, an anti-CTLA4 or anti-PD1/PDL1 inhibitory antibody). In other certain embodiments, the checkpoint inhibitor has a targeted effect as an agonist for a target; examples from this class include molecules that stimulate OX40, CD40 and/or GITR targets. In some embodiments, a checkpoint inhibitor that targets OX40, CD40, and/or GITR is an agonist antibody. Agonist antibodies directed against OX40 may serve a dual role: inhibit suppression by regulatory T cells while enhancing the functions of effector T cells. Agonist antibodies to GITR have also been shown to render effector T cells more resistant to inhibition induced by regulatory T cells (Karaki et al. (2016) Vaccines (Basel) 4(4):37). Similarly, agonist antibodies to CD40 demonstrate T cell-dependent antitumor activity. Activation of CD40 on dendritic cells increases the cross-presentation of tumor antigens and therefore the number of activated effector T cells directed to the tumor (Ellmark et al. (2015) Oncoimmunol. 4(7):e1011484).

[507] In certain embodiments, the checkpoint inhibitor specifically targets CTLA4 (eg, is an anti-CTLA4 antibody). In certain embodiments, targeting CTLA4 facilitates the priming and activation of naïve T cells. In certain embodiments, the checkpoint inhibitor specifically targets OX40 (eg, is an anti-OX40 antibody). In certain embodiments, targeting OX40 enhances the expansion of effector T cells. In certain embodiments, the checkpoint inhibitor specifically targets CD40 (eg, is an anti-CD40 antibody). In certain embodiments, targeting CD40 suppresses “tolerogenic” T cell priming and/or generation of regulatory T cells. In certain embodiments, the checkpoint inhibitor specifically targets GITR (eg, is an anti-GITR antibody). In certain embodiments, targeting GITR suppresses the activity of regulatory T cells. In certain embodiments, the beneficial effect of a combination therapy (e.g., an effect on at least one symptom or risk/rate of disease progression) using a herboxidiene-based splicing modulator, an ADC, or a composition and agent targeting CTLA4, OX40, CD40, and/or GITR is additive. In some embodiments, the beneficial effect of a combination therapy using a herboxydiene-based splicing modulator, an ADC, or a composition and agent targeting CTLA4, OX40, CD40, and/or GITR is superadditive (ie, synergistic).

[508] Immune checkpoint inhibitor treatment strategies are based on the hypothesis that the treatment facilitates and/or enhances T cell priming in tumor responses that are poorly or deficient in antigenicity (eg, CTLA4), or that the treatment restores and/or or enhances T cells that respond to tumor antigens but are exhausted due to the persistent nature of antigen presentation (eg, PD1, PDL1) (Chen and Mellman (2013) Immunity 39(1):1-10). Examples of suitable therapies and agents for checkpoint inhibition, for example, antibodies to PD1, PDL1 or CTLA4, are known in the art. Cm., For example, WO 2001/014424, WO 2013/173223, WO 2016/007235.

[509] Combining these responses from primed T cells following checkpoint inhibitor therapy with treatment to induce neoantigens in tumor cells to which the primed immune system can respond may provide beneficial synergy. Since neoantigens generated by a herboxydiene-based splicing modulator or ADC have not yet been introduced for T cell priming, combination with a CTLA4 inhibitor may be particularly beneficial. In some embodiments, the treatment comprises the administration of one or more herboxydiene splicing modulators, ADCs, or compositions to induce neoantigen production, and an initial administration of a CTLA4 inhibitor to stimulate CD8 T cell priming, administered before, concurrently, or subsequent to this administration. In some embodiments, the patient is provided with additional administrations of a CTLA4 inhibitor, for example, to further stimulate the priming and/or activation of neoantigen-reactive CD8 populations. In some embodiments, additional administration of a herboxydiene-based splicing modulator, ADC, or composition may be administered to a patient to enhance neoantigen presentation by the tumor. Repeated administrations of the herboxydiene splicing modulator, ADC, or immune checkpoint inhibitor composition and therapy can be administered simultaneously or at spaced intervals. In some embodiments, treatment further includes co-treatment with a PD1/PDL1 inhibitor, for example, to restore effector function of exhausted T cells targeting neoantigen within the tumor microenvironment.

[510] As used herein, the terms “combination” or “combination therapy” mean the administration of one or more of a herboxidiene splicing modulator, ADC, or composition together with an additional agent or therapy (eg, an immune checkpoint inhibitor, a cytokine or a cytokine analog, with a neoantigen vaccine, with a CAR-T) as part of a treatment regimen designed to provide a beneficial (i.e., additive or synergistic) effect from the combined action of one or more administered agents. In some embodiments, the combination may also include one or more additional agents, including, but not limited to, chemotherapeutic agents, anti-angiogenesis agents, and agents that reduce immune suppression (eg, a second checkpoint inhibitor). The beneficial effect of the combination includes, but is not limited to, the pharmacokinetic or pharmacodynamic synergies provided by the combination of therapeutic agents. Administration of such therapeutic agents in combination is typically carried out over a period of time (eg, minutes, hours, days or weeks depending on the combination chosen).

[511] As used herein, the terms “administered in combination” or “co-administered” mean that two or more different treatments are delivered to a subject over a period of time during which the subject suffers from a condition (eg, a neoplastic disorder). For example, in some embodiments, two or more treatments are delivered after a subject has been diagnosed with a disease or disorder and before the disease or disorder has been treated or eliminated, or when the subject has been identified as being at risk for developing it, but before how the subject developed symptoms of the disease. In some embodiments, delivery of one treatment is still in progress when delivery of the second treatment begins, so that overlap is present. In some embodiments, delivery of the first and second treatments begins at the same time. These types of delivery are sometimes referred to herein as “simultaneous,” “joint,” or “concurrent” delivery. In other embodiments, delivery of one treatment ends before delivery of the second treatment begins. This type of delivery is sometimes referred to herein as “rolling” or “sequential” delivery.

[512] In some embodiments, two treatments (eg, a herboxydiene splicing modulator, ADC, or composition and an immune checkpoint inhibitor) are contained in the same composition. Such compositions can be administered in any suitable form and via any suitable route. In other embodiments, two treatments (eg, a herboxydiene splicing modulator, ADC or composition, and an immune checkpoint inhibitor) are administered in separate compositions, in any suitable form, and via any suitable route. For example, in some embodiments, a composition comprising a herboxidiene splicing modulator or ADC and a composition comprising an immune checkpoint inhibitor may be administered simultaneously or sequentially in any order at different time points; in any case, they should be administered close enough in time to provide the desired therapeutic or prophylactic effect.

[513] In embodiments involving either simultaneous or sequential delivery, treatment may be more effective due to combined administration. In some embodiments, the first treatment is more effective, eg, an equivalent effect is observed with a smaller amount of the first treatment (eg, at a lower dose) than would be observed if the first treatment was administered in the absence of the second treatment. In some embodiments, the first treatment is more effective such that the reduction in the severity of a symptom or other parameter associated with the disease or disorder is greater than would be observed if the first treatment were delivered in the absence of the second treatment. In other embodiments, a similar situation is observed for the second treatment. In some embodiments, the benefit of the combination therapy (eg, the effect on at least one symptom or risk/rate of disease progression) is additive. In some embodiments, the benefit of the combination therapy is superadditive.

[514] In various embodiments, the present invention provides a method of treating a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a herboxidiene splicing modulator, ADC, or a composition comprising a herboxidiene splicing modulator, or ADC; and at least one additional therapy (ie, an immune checkpoint inhibitor therapy, a cytokine or cytokine analogue, a neoantigen vaccine, a CAR-T). In some embodiments, administration of a herboxydiene splicing modulator, ADC, or composition induces the formation of at least one neoantigen and/or T cell response. In some embodiments, administration of a herboxydiene-based splicing modulator, ADC, or composition induces an immune response to the double-stranded RNA. In some embodiments, administration of a herboxydiene-based splicing modulator, ADC, or composition enables the induction of immunogenic cell death. In some embodiments, the at least one additional therapy may include at least one, at least two, at least three, at least four, or at least five additional therapies. For example, in some embodiments, a herboxydiene splicing modulator, ADC, or composition may be administered in combination with two immune checkpoint inhibitor therapies, i.e. using two different immune checkpoint inhibitors. In some other embodiments, a herboxydiene splicing modulator, ADC, or composition may be administered in combination with an immune checkpoint inhibitor therapy and a neoantigen vaccine.

[515] In some embodiments of the combination therapy, the amount of herboxydiene splicing modulator, antibody-drug conjugate or composition, and/or at least one additional therapy administered is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75% or 90% compared to a standard dosage of a herboxidiene splicing modulator, antibody-drug conjugate, or composition, and/or at least one additional therapy . In some embodiments, the herboxydiene splicing modulator, antibody-drug conjugate or composition, and/or at least one additional therapy is administered at at least 10%, 15%, 20%, 25%, 30%, 35 %, 40%, 45%, 50%, 75%, or 90% less frequently than a standard regimen of administering a herboxidiene splicing modulator, an antibody-drug conjugate, or composition, and/or at least one additional therapy. In some embodiments, the administered amount and/or dosage of a herboxydiene splicing modulator, antibody-drug conjugate or composition, and/or at least one additional therapy results in lower systemic toxicity and/or improved tolerability.

[516] In some embodiments, administration of the herboxydiene splicing modulator, antibody-drug conjugate, or composition is initiated prior to administration of the at least one additional therapy. In some embodiments, administration of the herboxydiene splicing modulator, antibody-drug conjugate, or composition is initiated after administration of at least one additional therapy. In some embodiments, administration of the herboxydiene splicing modulator, antibody-drug conjugate, or composition is initiated concurrently with administration of at least one additional therapy.

[517] In some embodiments, administration of the herboxydiene splicing modulator, antibody-drug conjugate, or composition is repeated at least once after the initial administration. In some embodiments, the amount of herboxydiene splicing modulator, antibody-drug conjugate or composition used for repeated administration is reduced compared to the amount used for initial administration. In some embodiments, the amount of herboxydiene splicing modulator, antibody-drug conjugate or composition used for repeated administration is reduced compared to a standard dosage of herboxydiene splicing modulator, antibody-drug conjugate or composition. In some embodiments, the amount of herboxydiene splicing modulator, antibody-drug conjugate or composition used for re-administration is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75% or 90% compared to a standard dosage of a herboxydiene splicing modulator, antibody-drug conjugate or composition.

[518] In some embodiments, administration of at least one additional therapy is repeated at least once after the initial administration. In some embodiments, the amount of at least one additional therapy used for repeated administration is reduced compared to the amount used for initial administration. In some embodiments, the amount of at least one additional therapy used for repeated administration is reduced compared to the standard dosage of the at least one additional therapy. In some embodiments, the amount of at least one additional therapy used for reintroduction is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% compared to the standard dosage of at least one additional therapy.

[519] In some embodiments, reintroduction of the herboxydiene splicing modulator, antibody-drug conjugate, or composition occurs simultaneously with reintroduction of at least one additional therapy. In some embodiments, reintroduction of the herboxydiene splicing modulator, antibody-drug conjugate, or composition is performed sequentially or spaced in time with reintroduction of at least one additional therapy.

[520] In various embodiments, the present invention provides a method of treating a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a herboxidiene splicing modulator, ADC, or a composition comprising a herboxidiene splicing modulator, or ADC; and immune checkpoint inhibitor-based therapies. In some embodiments, the checkpoint inhibitor therapy comprises administering at least one checkpoint inhibitor. In some embodiments, a subject is intolerant, unresponsive, or deficient in responsiveness to at least one checkpoint inhibitor when administered alone. In some embodiments, a subject may be considered to be unresponsive or deficient in responsiveness to at least one checkpoint inhibitor, as determined using, for example, immune-related immunotherapy response criteria (irRC) and/or immune-related response criteria for immunotherapy in solid tumors ( irRECIST). See , for example , Wolchok et al. (2009) Clin Cancer Res. 15(23):7412-20; Bohnsack et al. "Adaptation of the Immune-Related Response Criteria:irRECIST" (Abstract 4958) ESMO 2014. Illustrative criteria may include criteria used in the art to determine when tumors in patients suffering from cancer improve ("response") , remains the same (“stabilization”) or worsens (“progression”) over the course of treatment if the treatment being evaluated is an immuno-oncology drug (eg, an immune checkpoint inhibitor). In some embodiments, a subject may be considered intolerant to at least one checkpoint inhibitor if the subject experiences one or more than one adverse (grade 2+) event identified for the corresponding checkpoint inhibitor (eg, ipilimumab). For example, in some embodiments, a subject may be considered intolerant to an ipilimumab-based treatment if the subject experiences one or more adverse events selected from enterocolitis, hepatitis, dermatitis (including toxic epidermal necrolysis), neuropathy, and endocrinopathy (Yervoy ® (ipilimumab) FDA Compliant Labeling Supplement, 2018).

[521] In some embodiments, the checkpoint inhibitor targets PD1/PDL1, CTLA4, OX40, CD40, LAG3, TIM3, GITR, and/or KIR. In some embodiments, the checkpoint inhibitor specifically targets CTLA4, OX40, CD40, and/or GITR. In some embodiments, targeting of the checkpoint inhibitor is provided by an inhibitory antibody or other such inhibitory molecule. In some other embodiments, the checkpoint inhibitor is targeted as an agonist antibody or other such agonist molecule. In some embodiments, the checkpoint inhibitor comprises an inhibitor of the cytotoxic T lymphocyte-associated antigen 4 (CTLA4) pathway. In some embodiments, the CTLA4 inhibitor is an anti-CTLA4 antibody. In some embodiments, the anti-CTLA4 antibody is ipilimumab. In some embodiments, the checkpoint inhibitor comprises an inhibitor of the programmed cell death protein 1 (PD1) pathway. In some embodiments, the PD1 inhibitor is an anti-PD1 antibody. In some embodiments, the anti-PD1 antibody is nivolumab. In some embodiments, the PD1 inhibitor is an anti-PDL1 antibody. In some embodiments, the anti-PDL1 antibody is atezolizumab. In some embodiments, the checkpoint inhibitor includes a CTLA4 inhibitor and a PD1 inhibitor. In some embodiments, the checkpoint inhibitor specifically targets OX40. In some embodiments, the checkpoint inhibitor specifically targets CD40. In some embodiments, the checkpoint inhibitor specifically targets GITR. In some embodiments, the beneficial effect of a combination therapy (e.g., an effect on at least one symptom or risk/rate of disease progression) using a herboxidiene-based splicing modulator, ADC, or composition and an immune checkpoint inhibitor (e.g., an antibody or molecule targeted affecting CTLA4, PD1/PDL1, OX40, CD40 and/or GITR) is additive. In some embodiments, the beneficial effect of combination therapy using a herboxydiene-based splicing modulator, ADC, or composition and an immune checkpoint inhibitor (e.g., an antibody or molecule targeting CTLA4, PD1/PDL1, OX40, CD40, and/or GITR) is superadditive (i.e. synergistic).

[522] In various embodiments, the present invention provides a method of treating a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a herboxidiene splicing modulator, ADC, or a composition comprising a herboxidiene splicing modulator, or ADC; and cytokine or cytokine analogue therapies. In some embodiments, the cytokine or cytokine analogue therapy comprises administering at least one cytokine or cytokine analogue. In some embodiments, a subject is intolerant, unresponsive, or deficient in responsiveness to at least one cytokine or cytokine analog when administered individually.

[523] In some embodiments, the cytokine or cytokine analog provides a T cell enhancer. In some embodiments, the cytokine or cytokine analogue includes IL-2, IL-10, IL-12, IL-15, IFNγ and/or TNFα. In some embodiments, the cytokine or cytokine analogue includes IL-2, IL-10, IL-12 and/or IL-15. In some embodiments, administration of a cytokine or cytokine analog enhances T cell priming following administration of a herboxidiene splicing modulator, antibody-drug conjugate, or composition due to inducing the formation and presentation of neoantigens.

[524] In some embodiments, the cytokine or cytokine analogue comprises IL-2. In some embodiments, IL-2 enhances signals in effector cells to promote their proliferation (Rosenberg (2014) J Immunol. 192(12):5451-8). In some embodiments, the cytokine or cytokine analogue comprises IL-10. In some embodiments, IL-10 enhances the priming and activation of CD8+ T cells (Mumm et al. (2011) Cancer Cell 20(6):781-96). In some embodiments, the cytokine or cytokine analogue comprises IL-12. In some embodiments, IL-12 couples the innate and adaptive immune responses to enhance antigen-specific priming and targeting (Tugues et al. (2015) Cell Death Differ. 22(2):237-46). In some embodiments, the cytokine or cytokine analogue comprises IL-15. In some embodiments, IL-15 enhances the priming and/or activation of effector T cells (CD8). In some embodiments, the cytokine or cytokine analogue comprises IFNγ. In some embodiments, IFNγ complements the secretion of IFNγ by effector T cells. In some embodiments, the cytokine or cytokine analogue comprises TNFα. In some embodiments, TNFα complements the secretion of TNFα by effector T cells.

[525] In some embodiments, an initial dose of a herboxydiene splicing modulator, ADC, or composition is administered to a subject to trigger aberrant splicing and the production of neoantigen peptides. In some embodiments, after a period of time necessary for protein production and antigen presentation, the subject is then administered an initial dose of a cytokine or cytokine analog to stimulate and/or enhance the priming and expansion of effector T cells. In some embodiments, the wait period between dosing of the herboxydiene splicing modulator, ADC or composition and the cytokine or cytokine analog is about 2, about 3, about 4, about 5, about 6, or about 7 days. In some embodiments, the waiting period is from about 3 days to about 5 days. In some embodiments, the cytokine or cytokine analogue is IL-2, IL-10, IL-12, IL-15, IFNγ and/or TNFα. In some embodiments, the combined therapeutic benefit of the herboxydiene splicing modulator, ADC or composition and the cytokine or cytokine analogue may be additive or superadditive.

[526] In some other embodiments, a priming dose of a cytokine or a cytokine analogue is administered to a subject to stimulate and/or enhance the priming and expansion of effector T cells. After a waiting period, in some embodiments, the subject is then administered an initial dose of a herboxydiene-based splicing modulator, ADC, or composition to trigger aberrant splicing and production of neoantigen peptides. In some embodiments, the waiting period between dosing the cytokine or cytokine analog and the herboxydiene splicing modulator, ADC, or composition is about 2, about 3, about 4, about 5, about 6, or about 7 days. In some embodiments, the waiting period is from about 3 days to about 5 days. In some embodiments, the cytokine or cytokine analogue is IL-2, IL-10, IL-12, IL-15, IFNγ and/or TNFα. In some embodiments, the combined therapeutic benefit of a cytokine or cytokine analog and a herboxydiene splicing modulator, ADC, or composition may be additive or superadditive.

[527] In some embodiments, after a period to allow T cell priming and expansion, the subject is administered a second or subsequent dose of a herboxydiene splicing modulator, ADC, or composition to trigger re-presentation of neoantigen peptides. In some embodiments, the wait period between administration of the initial dose of the cytokine or cytokine analog and the second or subsequent dose of the herboxydiene splicing modulator, ADC, or composition is about 2, about 3, about 4, or about 5 weeks. In some embodiments, the waiting period is approximately 3 weeks. In some embodiments, subsequent doses of the cytokine or cytokine analog may be administered, for example, between subsequent doses of the herboxydiene splicing modulator, ADC, or composition. Upon administration of a second or subsequent dose of the herboxydiene splicing modulator, ADC, or composition, in some embodiments, the immune system can contact tumor cells presenting neoantigens and/or cause tumor cell destruction. In some embodiments, dosing of the herboxydiene splicing modulator, ADC, or composition following a given exemplary initial treatment regimen may be pulsatile, i.e. The herboxydiene splicing modulator, ADC, or composition may be administered at dosages at long intervals (eg, approximately every 4 weeks, approximately every 5 weeks, approximately every 6 weeks) to promote antigen presentation, T cell recruitment, and/or tumor cell killing , and/or restoration of the memory T cell population.

[528] In some embodiments, the subject has a mutational load with non-synonymous changes of approximately 150 mutations or less. In some embodiments, the subject has a mutational load with nonsynonymous changes of approximately 100 mutations or less. In some embodiments, the subject has a mutational load with nonsynonymous changes of approximately 50 mutations or less. In some embodiments, the subject has or is suspected of having a neoplastic disorder, such as a hematologic malignancy or solid tumor. In some embodiments, the hematologic malignancy is selected from B-cell malignancy, leukemia, lymphoma, and myeloma. In some embodiments, the hematologic malignancy is selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer cancer and esophageal cancer. In some embodiments, the solid tumor is selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

Combination of herboxydiene/ADC splicing modulator and neoantigen vaccine:

[529] In various embodiments, a patient who has a cancer described herein can be treated with a combination of a herboxidiene splicing modulator, an ADC or composition, and a neoantigen vaccine. Without being limited by theory, vaccines used alone or in combination with immune checkpoint inhibitor (ICI) molecules have been shown to be promising in previous trials (Ott et al. (2017) Nature 547(7662): 217-21; Sahin et al. (2017) Nature 547(7662):222-6), but usually requires sequencing of mutations in the patient's tumor (Ott et al. (2017) Nature 547(7662):217-21; Aldous and Dong (2018) Bioorg. Chem. 26(10):2842-9. Thus, the effectiveness of vaccines often depends on sufficient numbers of nonsynonymous mutations that are antigenic. In general, tumors with a very low mutational load have a small number of candidate antigens, and in the case of tumors with rapid growth, there is limited time to identify and obtain patient-specific vaccines.

[530] To date, attempts to develop vaccines that have broad immunogenicity coverage for a large percentage of patients have focused on proteins that are either frequently mutated, ectopically overexpressed or amplified, and/or that exist as “self” proteins in the body . In addition, these proteins are often expressed in tissues that are not affected by the immune system (eg, neural markers expressed in neuroendocrine tumor types), while other proteins may be normally expressed during embryogenesis (eg, cancer embryonic antigens) . Thus, the utility of vaccines using such proteins as antigens is often limited to specific tumor lineages or subgroups in which one or more antigens are present. The vaccine's usefulness will also need to be confirmed by sequencing patient tumor samples, which can be time-consuming.

[531] Moreover, if these antigens exist as “self” proteins, the immune system will likely be primed to recognize these proteins as “self” and thus will not respond. Or, alternatively, if the immune system is able to mount an effector response to such antigens, it may result in target-related side effects in tissues where the antigen may be expressed. In both of these cases, one of the key problems is that most antigenic peptides are derived from “passenger genes” (i.e. genes that are mutated or amplified during tumorigenesis, but do not play an important role in ensuring long-term survival or proliferation of the tumor as such). For this reason, these genes can be silenced without significant consequences for tumor progression and thus may allow the tumor to “escape” the immune response to these antigens. Without being limited by theory, this mechanism may play a role in tumor evolution, in which random mutations that are highly antigenic are often subject to tumor selection during the early stages of tumorigenesis (Dunn et al. (2004) Annu. Rev. Immunol. 22 :329-60).

[532] In addition, some evidence also indicates that constant antigen presentation and immune stimulation can lead to anergy and exhaustion of immune cells (Pardoll (2012) Nat. Rev. Cancer 12(4):252-64). Such phenotypes underlie the therapeutic rationale for currently used ICI-based therapies, as ICIs have been shown to either curb the immune cell exhaustion phenotype (α-PD1/PD-L1) or facilitate additional immune cell responses (α-CTLA4 ). Notably, for the α-CTLA4-based therapy, it was reported that a subset of patients exhibited serious immune-related adverse events, which may be due to the promotion of T-cell activation and disruption of immune tolerance mechanisms that limit autoreactive immune responses.

[533] Both such approaches (i.e., triggering or enhancing de novo immune responses to neoantigens or reactivation when existing immune responses become anergic or exhausted) involve persistent activation of the immune system. In this regard, these approaches are sensitive to anergy, editing and other tumor-mediated mechanisms aimed at suppressing immune system involvement.

[534] In contrast, treatment with a herboxydiene-based splicing modulator, ADC, or composition disclosed herein can induce an immune response to neoantigen sequences. In some embodiments, the presentation of neoantigens provides the adaptive immune system with more diverse targets with which it contacts and is activated. In some embodiments, the ability of a herboxydiene-based splicing modulator, ADC, or composition to rapidly induce alternative splicing and neoantigen formation may reduce the risk of immune system exhaustion due to chronic exposure to neoantigens generated by mutations and/or limit the ability of tumor cells to adapt to evade therapy. In some embodiments, administration of a herboxydiene splicing modulator, ADC, or composition in combination with a neoantigen vaccine enhances the immune response to neoantigens produced by the herboxydiene splicing modulator, ADC, or composition. In some embodiments, the herboxydiene splicing modulator, ADC, or composition is administered before, during, or after vaccination. In some embodiments, the herboxydiene splicing modulator, ADC or composition and/or vaccine can be administered once or more than once during a course of treatment. In some embodiments, the vaccine is administered once and then the herboxydiene splicing modulator, ADC, or composition is administered multiple times over the course of treatment. In some embodiments, the vaccine is administered once, followed by one or more booster doses over the course of treatment.

[535] As used herein, the term “neoantigen vaccine” means a combined sample of one or more immunogenic neoantigen peptides or mRNA, eg, at least two, at least three, at least four, at least five, or more neoantigen peptides. The term “vaccine” means a composition for developing immunity for the prevention and/or treatment of a disease (eg, a neoplastic disorder, such as a hematologic malignancy or solid tumor). Accordingly, vaccines are medicinal preparations that contain immunogenic agents and are intended for use in humans or animals for the development of specific immune defense mechanisms and the formation of protective substances after vaccination. The neoantigen vaccine may further include a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.

[536] As used herein, the term “immunogenic” means any agent or composition that can induce an immune response, such as a T cell response. The immune response may be mediated by an antibody, or a cell, or both.

[537] In some embodiments, the patient is administered a herboxidiene splicing modulator, ADC, or composition, and then is administered a peptide or mRNA vaccine based on a known neoantigen to enhance the immune response to neoantigens produced by the herboxidiene splicing modulator, ADC, or composition . In some other embodiments, the patient is administered a herboxydiene splicing modulator, ADC, or composition and then screened for the presence of neoantigens produced by the treatment. Therefore, one or more of these neoantigens are used to create a personalized vaccine that is administered to the patient. In any of these embodiments, the herboxydiene splicing modulator, ADC or composition and/or peptide or mRNA vaccine can be administered to a patient once or repeatedly.

[538] In various embodiments, a suitable neoantigen for a vaccine can be identified by screening a panel of altered splicing and persistently expressed transcripts obtained from one or more patient tissue samples (eg, a tumor biopsy). In some embodiments, protein variant sequences are identified in a screened sample based on translation along a junction in the aberrantly spliced mRNA, while retaining portions of the protein sequence (no more than 12 amino acids) flanking amino acid changes spanning the junction. In some embodiments, these peptide fragments spanning splice boundaries are screened for high affinity binding to MHC1 alleles, for example, using an agent such as NetMHC1 (Nielsen et al. (2003) Protein Sci 12(5):1007-17; Andreatta and Neilsen (2016) Bioinformatics 32(4):511-7). These results allow the filtering of neopeptides to neopeptides predicted to bind with high affinity to the HLA allele composition unique to the patient, as well as the collection of pools of neopeptides predicted to bind to a wide range of HLA alleles present at high frequency in different populations (Maiers et al. (2007) Hum Immunol 68(9):779-88). In various embodiments, the identified neopeptides are then formulated into a vaccine, for example by conjugation with a suitable carrier or adjuvant (Ott et al. (2017) Nature 547(7662):217-21), or for delivery as mRNA (Sahin et al (2017) Nature 547(7662):222-6).

[539] In some embodiments, the selection of neoantigen is based on screening the individual patient's tumor response to a herboxydiene splicing modulator, ADC, or composition, identifying one or more neoantigens generated by the treatment for use in subsequent vaccination. In other embodiments, a neoantigen is selected, for example, based on screening a panel of samples obtained from different patients to identify common neoantigens produced by a herboxidiene splicing modulator, ADC, or composition, and then used as a universal vaccine for future patients.

[540] Without being limited by theory, in some embodiments, the use of a universal neoantigen vaccine avoids the need to sequence and analyze the unique mutational status of each patient's tumor, since the selected neoantigens are not dependent on the mutation in the tumor, but rather mimic the neoantigen produced by the action of a splicing modulator on based on herboxydiene, ADC or composition, and generally recognized by the body as foreign. Additionally, in some embodiments, the use of a neoantigen vaccine may be particularly effective because the patient's tumor cells may be more likely to mutate without producing one or more neoantigens that are dependent on the mutation in the tumor, compared to cells that mimic the neoantigen produced under by the action of a herboxydiene-based splicing modulator, ADC or composition. This may provide the opportunity to formulate a core vaccine that has broad immunogenicity coverage for a large percentage of patients, speeding up initiation of treatment regimens. Patients may be vaccinated according to the schedules set forth herein and, before further completion of vaccination, may be further treated with a herboxidiene-based splicing modulator, ADC, or composition, for example, to induce expression of neoantigen peptides. In some embodiments, patients may be administered a herboxydiene splicing modulator, ADC, or composition prior to, concurrently with, or subsequent to vaccination. In some embodiments, patients are administered a herboxydiene splicing modulator, ADC, or composition, are screened for one or more neoantigens identified in a panel of universal neoantigens, and are vaccinated with a universal neoantigen vaccine containing at least one universal neoantigen identified in subject. In some embodiments, patients may be administered the herboxydiene splicing modulator, ADC, or composition once or more than once after vaccination. The herboxidiene splicing modulator, or ADC, or composition and/or vaccine can be administered once or more than once during a course of treatment.

[541] In some embodiments, the vaccine may contain one or more neoantigen peptides or mRNA. In various embodiments, the vaccine may contain one or more long neoantigen peptides. In various embodiments, such "long" neoantigenic peptides undergo efficient internalization, processing, and cross-presentation into specialized antigen-presenting cells, such as dendritic cells. Likewise, long vaccine peptides have been shown in other contexts to induce cytotoxic T cells in humans (Melief and van der Burg (2008) Nat Rev Cancer 8(5):351-60). In various embodiments, the neoantigenic peptide is extended to include the neoantigenic peptide sequence itself in addition to flanking amino acid sequences. In various embodiments, the extended peptide sequence facilitates uptake of the protein by antigen presenting cells, such as dendritic cells. In various embodiments, the extended peptide sequence allows for efficient antigen presentation and T cell priming in different HLA isotype models. In various embodiments, a longer neoantigen peptide and/or an extended peptide sequence exhibits increased uptake by antigen presenting cells (e.g., dendritic cells), increased antigen presentation, and/or increased T cell priming compared to a shorter neoantigen peptide and/or a shorter peptide sequence. sequence (eg, a peptide sequence that is less than about 10 or less than about 5 amino acids in length). In some embodiments, the length of the long neoantigen peptide ranges from about 5 to about 50 amino acids. In some embodiments, the length of the long neoantigen peptide ranges from about 10 to about 50 amino acids. In some embodiments, the length of the at least one neoantigen peptide ranges from about 10 to about 35 amino acids. In some embodiments, the length of the long neoantigen peptide ranges from about 15 to about 25 amino acids.

[542] In some embodiments, the length of the neoantigenic sequence and/or antigenic portion ranges from about 10 to about 35 amino acids. In some embodiments, the length of the neoantigenic sequence and/or antigenic portion ranges from about 15 to about 25 amino acids. In some embodiments, the length of the neoantigenic sequence and/or antigenic portion ranges from about 10 to about 20 amino acids. In some embodiments, the neoantigenic sequence and/or antigenic moiety does not directly overlap or consist of a canonical peptide sequence (eg, any of the exemplary canonical peptide sequences highlighted in Table 8).

[543] The amino acid sequences of exemplary long neoantigenic peptides are presented in Table 8.

[544] These illustrative neoantigenic peptides are generated following administration of ADCs containing pladienolide-based splicing modulators, however, given the similar mechanism of action (ie, similar splicing modulation mechanisms), similar neoantigenic peptides may be generated by herboxidiene-based splicing modulators.

Table 7. Neopeptides Neopeptide SEQ ID NO Connection (HG19) Gene Event type Observed in 1 SPTLPPRSL 37 chr12:49663470-49663610:+ TUBA1C Intron retention H1568 2 HPSIKRGLSSL 38 chr12:42729776-42781257:+ PPHLN1 Exon skipping H1568 3 LLLPHHVL 39 chr12:49663470-49663610:+ TUBA1C Intron retention H1568 4 RTAPGVRPPF 40 chr14:35182767-35183743:- CFL2 Intron retention H1568 5 RPQKSIQAL 41 chr10:28822963-28823162:+ WAC Intron retention H1568 6 APAPPPLPA 42 chr17:80009840-80011149:+ GPS1 Intron retention H1568 7 RPRPSFPVSL 43 chr7:55087058-55134942:+ EGFR Intron retention H1568 8 RPKHGDGFSL 44 chr11:57472287-57472444:- MED19 Intron retention H1568 9 GPAPGKTGL 45 chr7:75932393-75933118:+ HSBP1 Intron retention H1568 10 EAARKGNSL 46 chr1:53480715-53504588:+ SCP2 Exon skipping H1568 eleven RIKEKIEEL 47 chr9:72897499-72912881:+ SMC5 Exon skipping H1568 12 EIKKRFRQF 48 chr1:28531860-28541450:- DNAJC8 Exon skipping H1568 13 HESAAMAET 49 chr11:102272937-102323254:- TMEM123 Exon skipping HCC1954 14 ALKLKQVGV 50 chr1:153610924-153617539:+ CHTOP Exon skipping H1568 15 DLKKRHITF 51 chr13:41323417-41331008:- MRPS31 Exon skipping H1568 16 DVKRNDIAM 52 chr1:41213277-41218822:+ N.F.Y.C. Exon skipping H1568 17 IPSDHILTPA 53 chr6:149718900-149720239:+ TAB2 Exon skipping H1568 18 TVFSTSSLK 54 chr11:61197654-61213412:+ SDHAF2 Exon skipping H1568 19 ITSCLLNF 55 chr5:137892555-137893090:- HSPA9 Intron retention H1568 20 RASPVRGQL 56 chr7:75677544-75677893:+ MDH2 Intron retention H1568 21 VVRKPVIAL 57 chr1:36923582-36929406:- MRPS15 Exon skipping H1568 22 LLSEKKKIS 58 chr6:31750622-31750872:- VARS Intron retention H1568 23 APASKPRPRL 59 chr19:3573798-3574380:+ HMG20B Intron retention H1568 24 RYGQLSEKF 60 chr19:33076813-33078158:+ PDCD5 Exon skipping HCC1954 25 VYISNVSKL 61 chr3:53920961-53925796:- SELK Exon skipping HCC1954 26 LPTKETPSF 62 chr2:85133241-85133394:+ TMSB10 Alternative splice site at the 3' end HCC1954 27 GEAPPPPPA 63 chr17:80223672-80231181:- CSNK1D Intron retention HCC1954 28 LEEISKQEI 64 chr17:27804724-27807385:+ TAOK1 Exon skipping HCC1954 29 IYNHITVKI 65 chr4:2886393-2896308:+ ADD1 Exon skipping HCC1954

[545] The protein sequences of the twenty-nine neopeptides listed in Table 7 may be extended. The extended protein sequence simultaneously includes the neopeptide sequence itself in addition to flanking amino acid sequences. An extended protein sequence better facilitates protein uptake by dendritic cells and promotes antigen presentation and T cell priming in different HLA isotype models. The amino acid sequences of the twenty-nine extended neopeptides are presented in Table 8.

Table 8. Amino acid sequences of extended neopeptides Gene SEQ ID NO Amino acid sequence of extended neopeptide* TUBA1C 66 VDLEPTVIGELTSVTQVRSQGAGTGGLSWGGSAGHSPTLPPRSLSLLLLPHHVLQMKFALALTASSSTLSNSSQARKMLPITMPEGTTPLARRSLTSCWTEFASWLTSAPVFRASWFSTALVGELVLGSPRCSWNVSQLIMARSPSWSSSPFTRRPRFPQL PPHLN1 67 APPRSHPSIKRGLSSL CFL2 68 MVRRARWPGGRGEARKAPRTAPGVRPPF WAC 69 WVNCLFVSGRAAAGGGGGGAVPPYLELAGPPFLLLTLIRIGLGRRSGRAGGRAGTQCGGERGPGFAAFRPLRPFRRLRVCAVCVRGSALGRSVGLPRGGAAGAPFSSSPAPHPRRVLCRCLLFLFFSCHDRRGDSQPYQVPAEAGVEGLEGAGGGREGLLLERRPQKSIQALRCNTSETSTADPLKIPGLVPLALSSKV GPS1 70 MPLPVQVFNLQVTSRGRPGPPRPRAPRHWGRAEVEQGRGACARSRSGTLRAGPPRAARVGGCRAEGASPPWLRAAIGGRRAAPAPPPLPAAHGRGSRPPPRR EGFR 71 QPAQPRTGAPARRPRPRPSFPVSLRSAAPPTGTAGGTGRFVLRPGESGAGGGGDAWDTGLQARRGTAAGTSGAPNRSQLSLTFPAQLRRIGVSGRKPGAGGRLGPGSRTCAPRCLPRARRGPGAHPRGGRCPPAETALFREAEEGTQKYSLPSDPAGQAAF MED19 72 FRLHTGPVSPVGGRRQMGRPKHGDGFSLQVCSFIMEQNG HSBP1 73 GVVEITGEPPCSCRGEEEASRAGRAGGVRLKRGSRGPGELNVGPAPGKTGLLIPLLRNWECGSLLRALSAL SCP2 74 KMGFPEAARKGNSL SMC5 75 LEARIKEKIEELQQALI DNAJC8 76 EIKKRFRQFKQAVYKQ TMEM123 77 AHESAAMETLQHVPS CHTOP 78 NRPSVQAALKLKQVGV MRPS31 79 KTDDLKKRHITFTLGCGIC N.F.Y.C. 80 MKLDEDVKRNDIAMAI TAB2 81 NSISQIPSDHILTPALFITFMTILDL SDHAF2 82 TVFSTSSLKLNQPQKYLKMKSWPC HSPA9 83 AEEDRRKKVITSCLLNFNLSKAQS MDH2 84 RSFSTSAQVGQTRGGLQAEAPRPGPRASPVRGQL MRPS15 85 RGYVVRKPVIALSVKI VARS 86 VDMDFGTGGQGAGPVGRGKDWSCTLAVHLLSEKKKISFSQIDRAWGGSQGTVLDKWGPGVVSELHPSAKEVSVGRNSVESLMTWAS HMG20B 87 EKGSHEEEVRVPALSWGRPRAPAPASKPRPRLDLNCLWLRPQPIFLWKLRPRPVPAATPLTGPLPL PDCD5 88 RYGQLSEKFNRRKVMDS SELK 89 MVYISNVSKLCFSKM TMSB10 90 NTLPTKETPSFLLNPHTSWVPRPHREAPRLRVGVAAPLQRPLPALHSH CSNK1D 91 FGDIYLGEAPPPPPAARRPGPCGCQDQARSRKEVVAPAGSPRKSRHRRIVARTQRPLG TAOK1 92 GSASDLLEEISKQEISF ADD1 93 QLIYNHITVKINLQGD *Underlines indicate amino acids derived from the open reading frame of the canonical transcript (i.e., the canonical peptide sequence).

[546] As used herein, “neoantigenic peptide” or “mRNA vaccine” includes the use of a fragment of a neoantigenic peptide or the mRNA encoding it, provided that the fragment retains immunogenic potential.

[547] In some embodiments, the neoantigen vaccine contains at least one neoantigen peptide. In some embodiments, the neoantigen vaccine contains at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15 or at least 20 neoantigenic peptides. In some embodiments, the length of the neoantigen peptide(s) ranges from about 5 to about 50 amino acids. In some embodiments, the length of the neoantigen peptide(s) ranges from about 10 to about 50 amino acids. In some embodiments, the length of the at least one neoantigen peptide ranges from about 10 to about 35 amino acids. In some embodiments, the length of the neoantigen peptide(s) ranges from about 15 to about 25 amino acids.

[548] In various embodiments, the present invention provides a method of treating a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a herboxidiene splicing modulator, ADC, or a composition comprising a herboxidiene splicing modulator, or ADC; and neoantigen vaccine. The neoantigen vaccine may be, for example, a peptide neoantigen vaccine or a neoantigen mRNA vaccine. In some embodiments, the herboxydiene splicing modulator, ADC, or composition is administered prior to administration of the neoantigen vaccine. In some embodiments, the herboxydiene splicing modulator, ADC, or composition is administered following administration of the neoantigen vaccine. In some embodiments, the herboxydiene splicing modulator, ADC, or composition is administered concomitantly with administration of the neoantigen vaccine. In some embodiments, administration of the herboxydiene splicing modulator, ADC, or composition is repeated at least once after the initial administration. In some embodiments, the amount of the herboxydiene splicing modulator, ADC, or composition used for repeated administration is reduced compared to the amount used for initial administration.

[549] In various embodiments, the present invention further provides a combination comprising a herboxidiene splicing modulator, ADC, or a composition comprising a herboxidiene splicing modulator, or ADC; and a neoantigen vaccine (eg, a universal neoantigen vaccine) for use in treating a subject who has or is suspected of having a neoplastic disorder. In some embodiments, the neoantigen vaccine is a peptide neoantigen vaccine or a neoantigen mRNA vaccine. In some embodiments, the combination further comprises at least one additional therapy. In some embodiments, the at least one additional therapy comprises at least one, at least two, at least three, at least four, or at least five additional therapies.

[550] In various embodiments, the present invention further provides a method of treating a subject who has or is suspected of having a neoplastic disorder by (a) administering to the subject an effective amount of a herboxydiene-based splicing modulator, an ADC, or a composition comprising a herboxydiene-based splicing modulator. herboxydiene or ADC; (b) detecting one or more neoantigens in a subject following administration of a herboxydiene-based splicing modulator, ADC, or composition; (c) comparing one or more neoantigens to a panel of universal neoantigens; and (d) administering to the subject a universal neoantigen vaccine comprising at least one universal neoantigen present in the subject. In some embodiments, a generic neoantigen vaccine is administered alone or in combination with at least one additional therapy. In some embodiments, the at least one additional therapy comprises at least one, at least two, at least three, at least four, or at least five additional therapies.

[551] In some embodiments, the at least one additional therapy comprises administration of a herboxydiene-based splicing modulator, ADC, or composition. In some embodiments, reintroduction of the herboxydiene splicing modulator, ADC, or composition is initiated prior to administration of the universal neoantigen vaccine. In some embodiments, re-splicing modulator, ADC, or composition is initiated after administration of a universal neoantigen vaccine. In some embodiments, reintroduction of the herboxydiene splicing modulator, ADC, or composition is initiated simultaneously with administration of the universal neoantigen vaccine. In some embodiments, the amount of the herboxydiene splicing modulator, ADC, or composition used for repeated administration is reduced compared to the amount used for initial administration. In some embodiments, the amount of herboxydiene splicing modulator, ADC, or composition used for initial and/or repeated administration is reduced compared to a standard dosage of herboxydiene splicing modulator, ADC, or composition when administered in the absence of treatment with a vaccine. In some embodiments, the amount of herboxydiene splicing modulator, ADC, or composition used for initial and/or repeated administration is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75% or 90% compared to a standard dosage of a herboxydiene splicing modulator, ADC or composition.

[552] In some embodiments, the at least one additional therapy comprises administration of a checkpoint inhibitor (eg, any of the exemplary checkpoint inhibitors described herein). In some embodiments, administration of the immune checkpoint inhibitor is initiated prior to administration of the universal neoantigen vaccine and/or reintroduction of the herboxydiene splicing modulator, ADC, or composition. In some embodiments, administration of the immune checkpoint inhibitor is initiated following administration of a universal neoantigen vaccine and/or herboxydiene-based re-splicing modulator, ADC, or composition. In some embodiments, administration of the immune checkpoint inhibitor is initiated concurrently with administration of a universal neoantigen vaccine and/or with reintroduction of a herboxydiene-based splicing modulator, ADC, or composition. In some embodiments, administration of the checkpoint inhibitor is repeated at least once after the initial administration. In some embodiments, the amount of checkpoint inhibitor used for reintroduction is reduced compared to the amount used for initial administration. In some embodiments, the amount of checkpoint inhibitor used for re-administration is reduced compared to the standard dosage of the checkpoint inhibitor. In some embodiments, the amount of immune checkpoint inhibitor used for rechallenge is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% compared to a standard dosage of an immune checkpoint inhibitor. In some embodiments, the subject is intolerant, unresponsive, or deficient in responsiveness to the checkpoint inhibitor when administered alone.

[553] In various embodiments, also provided herein are neoantigen vaccines comprising at least one neoantigen peptide or at least one neoantigen mRNA. In some embodiments, the neoantigen vaccine contains at least one neoantigen peptide. In some other embodiments, the neoantigen vaccine comprises at least one neoantigen mRNA.

[554] Also provided herein in various embodiments are kits comprising a herboxydiene splicing modulator, ADC, or a composition comprising a herboxydiene splicing modulator, or ADC; and a neoantigen vaccine (eg, a universal neoantigen vaccine). In some embodiments, the neoantigen vaccine is a peptide neoantigen vaccine or a neoantigen mRNA vaccine. In some embodiments, the kit further comprises one or more additional components including, but not limited to, instructions for use; other agents, for example, one or more additional therapeutic agents; devices, containers or other materials for preparing a herboxydiene-based splicing modulator, ADC, composition and/or neoantigen vaccine for therapeutic administration; pharmaceutically acceptable carriers and devices, containers or other materials for administering a herboxydiene splicing modulator, ADC, composition and/or neoantigen vaccine to a patient. The instructions for use may include guidance for therapeutic applications, including suggested dosages and/or schedules of administration, for example, for a patient who has or is suspected of having a neoplastic disorder. In various embodiments, the kit further contains instructions for therapeutic use, for example, use of a herboxydiene splicing modulator, ADC or composition, and a neoantigen vaccine to treat or prevent a neoplastic disorder in a patient. In various embodiments, the kit further contains at least one additional therapeutic agent (eg, for administration together with a herboxidiene splicing modulator, ADC or composition and a neoantigen vaccine, eg, an immune checkpoint inhibitor). In various embodiments, the herboxydiene splicing modulator, ADC, composition, and/or neoantigen vaccine are formulated into a pharmaceutical composition.

[555] In some embodiments of the methods and compositions disclosed herein, the neoantigen vaccine contains at least one neoantigen peptide. In some embodiments, the length of the at least one neoantigenic peptide ranges from about 10 to about 50 amino acids. In some embodiments, the length of the at least one neoantigen peptide ranges from about 10 to about 35 amino acids. In some embodiments, the length of the at least one neoantigenic peptide ranges from about 15 to about 25 amino acids.

[556] In some embodiments, the at least one neoantigenic peptide comprises one or more neoantigenic sequences disclosed herein.

[557] In some embodiments, the length of the neoantigenic sequence and/or antigenic portion ranges from about 10 to about 35 amino acids. In some embodiments, the length of the neoantigenic sequence and/or antigenic portion ranges from about 15 to about 25 amino acids. In some embodiments, the length of the neoantigenic sequence and/or antigenic portion ranges from about 10 to about 20 amino acids. In some embodiments, the neoantigenic sequence and/or antigenic moiety does not directly overlap or consist of a canonical peptide sequence (eg, any of the exemplary canonical peptide sequences highlighted in Table 8).

[558] In some embodiments, the neoantigenic sequence is a subject-specific neoantigenic sequence. In some embodiments, the neoantigen sequence is a personalized neoantigen vaccine for the subject. In some embodiments, the neoantigen sequence is capable of binding to at least one HLA allele expressed in the subject.

[559] In some other embodiments, the neoantigen sequence is a generic neoantigen sequence. In some embodiments, the neoantigen sequence is a generic neoantigen vaccine. In some embodiments, the neoantigen sequence is capable of binding to at least one HLA allele expressed in at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least at least 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from a neoplastic disorder. In some embodiments, the neoantigen sequence is capable of inducing a T cell response to a tumor present in at least 1%, at least 5%, or at least 10% of a population of subjects suffering from a neoplastic disorder.

[560] In some embodiments, a neoantigenic sequence has been identified by sequencing at least one neoantigenic peptide that is induced in a subject by administration of an effective amount of a herboxidiene splicing modulator, antibody-drug conjugate, or composition. In some embodiments, the at least one neoantigenic peptide comprises a neoantigenic sequence the formation of which is induced by contacting a neoplastic cell with an effective amount of a herboxidiene splicing modulator, an antibody-drug conjugate or composition. In some embodiments, the neoplastic cell is in in vitro cell culture. In some embodiments, the neoplastic cell is obtained from a subject. In some embodiments, the neoplastic cell is located in the body of the subject.

[561] In some embodiments, the neoantigen vaccine comprises at least one neoantigen peptide or mRNA and a pharmaceutically acceptable carrier. In some embodiments, the neoantigen peptide or mRNA can be attached to a suitable carrier to promote an immune response. Exemplary carriers for binding to immunogenic agents (eg, neoantigenic peptide or mRNA) include serum albumins, fisurella hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, or toxoid from other pathogenic bacteria such as diphtheria bacteria, E. coli bacteria that cause cholera, or H. pylori , or an attenuated derivative of the toxin. Other carriers for stimulating or enhancing the immune response include cytokines such as IL-1, α- and β-peptides IL-1, IL-2, γINF, IL-10, GM-CSF and chemokines such as M1P1α and β and RANTES . Immunogenic agents can also be attached to peptides that enhance tissue transport, as described, for example, in WO 97/17613 and WO 97/17614. In some embodiments, the pharmaceutically acceptable carrier is selected from a peptide, serum albumin, fisurella hemocyanin, immunoglobulin, thyroglobulin, ovalbumin, toxoid or attenuated toxoid derivative, cytokine, and chemokine.

[562] In some embodiments, the neoantigen peptide or mRNA may be attached to a pharmaceutically acceptable carrier. Immunogenic agents can be attached to carriers by chemical crosslinking. Techniques for coupling an immunogenic peptide to a carrier include the formation of disulfide bonds using N-succinimidyl-3-(2-pyridylthio)propionate (SPDP) and succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (if the peptide does not contain sulfhydryl group, this can be done by adding a cysteine residue). These reagents form a disulfide bond between themselves and cysteine residues in the peptide on one protein and an amide bond through the epsilon amino group on lysine or another free amino group on other amino acids. A number of such disulfide/amide bond forming agents are described in Jansen et al. ((1982) Immun Rev. 62:185). Other bifunctional coupling agents form a thioether bond instead of a disulfide bond. A variety of these thioester linkage forming agents are commercially available and include reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid and 2-iodoacetic acid, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid. Carboxyl groups can be activated by combining them with succinimide or sodium salt of 1-hydroxyl-2-nitro-4-sulfonic acid. In some embodiments, the neoantigenic peptide and the pharmaceutically acceptable carrier are covalently linked through a linker.

[563] Neoantigen and other such immunogenic peptides can also be expressed as carrier fusion proteins. The immunogenic peptide may be attached at the amino terminus, the carboxy terminus, or at any location within the peptide (internal) to the carrier. In some embodiments, multiple repeats of an immunogenic peptide may be present in the fusion protein. In some embodiments, the neoantigen peptide and the pharmaceutically acceptable carrier are expressed as a fusion protein.

[564] In some embodiments, the neoantigen vaccine contains at least one neoantigen peptide or mRNA encoding the same and a pharmaceutically acceptable diluent. In some embodiments, the neoantigen vaccine comprises at least one neoantigen peptide or mRNA encoding it and a pharmaceutically acceptable adjuvant (eg, the adjuvant described herein).

[565] In some embodiments of the methods and compositions disclosed herein, the neoantigen vaccine comprises at least one neoantigen mRNA. In some embodiments, the at least one neoantigen mRNA encodes one or more neoantigen sequences.

[566] In some embodiments, the length of the neoantigenic sequence and/or antigenic portion ranges from about 10 to about 50 amino acids. In some embodiments, the length of the at least one neoantigen peptide ranges from about 10 to about 35 amino acids. In some embodiments, the length of the neoantigenic sequence and/or antigenic portion ranges from about 15 to about 25 amino acids. In some embodiments, the length of the neoantigenic sequence and/or antigenic portion ranges from about 10 to about 20 amino acids. In some embodiments, the neoantigenic sequence and/or antigenic moiety does not directly overlap or consist of a canonical peptide sequence (eg, any of the exemplary canonical peptide sequences highlighted in Table 8).

[567] In some embodiments, the neoantigenic sequence is a subject-specific neoantigenic sequence. In some embodiments, the neoantigen sequence is a personalized neoantigen vaccine for the subject. In some embodiments, the neoantigen sequence is capable of binding to at least one HLA allele expressed in the subject.

[568] In some other embodiments, the neoantigen sequence is a generic neoantigen sequence. In some embodiments, the neoantigen sequence is a generic neoantigen vaccine. In some embodiments, the neoantigen sequence is capable of binding to at least one HLA allele expressed in at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least at least 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from a neoplastic disorder. In some embodiments, the neoantigen sequence is capable of inducing a T cell response to a tumor present in at least 1%, at least 5%, or at least 10% of a population of subjects suffering from a neoplastic disorder.

[569] In some embodiments, a neoantigenic sequence has been identified by sequencing at least one neoantigenic mRNA that is induced in a subject by administering an effective amount of a herboxidiene splicing modulator, antibody-drug conjugate, or composition. In some embodiments, the at least one neoantigenic mRNA encodes a neoantigenic sequence, the formation of which is induced by contacting the neoplastic cell with an effective amount of a herboxidiene splicing modulator, antibody-drug conjugate, or composition. In some embodiments, the neoplastic cell is in in vitro cell culture. In some embodiments, the neoplastic cell is obtained from a subject. In some embodiments, the neoplastic cell is located in the body of the subject.

[570] In some embodiments, the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable carrier. In some embodiments, the at least one neoantigen mRNA is associated with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from a peptide, serum albumin, fisurella hemocyanin, immunoglobulin, thyroglobulin, ovalbumin, toxoid or attenuated toxoid derivative, cytokine, and chemokine.

[571] In some embodiments, the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable diluent. In some embodiments, the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable adjuvant (eg, the adjuvant described herein).

[572] In some embodiments, the neoantigen mRNA is encapsulated using an encapsulating agent. In some embodiments, the encapsulating agent protects the neoantigen mRNA from degradation and improves vaccine delivery (McNamara et al. (2015) J Immunol Res. 2015:794528). In some embodiments, the encapsulating agent is a liposome. In some embodiments, the liposome is a cationic liposome, such as N-[1-(2,3-dioleoloxy)propyl]-N,N,N-trimethylammonium chloride 1 (DOTAP). In some embodiments, the encapsulating agent is a nanoparticle. In some embodiments, the nanoparticle protects neoantigenic mRNA from nuclease degradation and/or improves cellular uptake and/or delivery efficiency. In some embodiments, the nanoparticle may be designed such that it is completely biodegradable. In some embodiments, the nanoparticle is a biodegradable core-shell nanoparticle comprising a pH-sensitive poly(b-amino ester) (PBAE) core embedded in a phospholipid shell (Su et al. (2011) Mol Pharm. 8(3) ):774-87). In some embodiments, such nanoparticles are particularly effective in delivering mRNA in vivo and the development of an antitumor immune response.

[573] In some embodiments, the subject has a mutational load with nonsynonymous changes of approximately 150 mutations or less. In some embodiments, the subject has a mutational load with nonsynonymous changes of approximately 100 mutations or less. In some embodiments, the subject has a mutational load with nonsynonymous changes of approximately 50 mutations or less. In some embodiments, the subject has or is suspected of having a neoplastic disorder, such as a hematologic malignancy or solid tumor. In some embodiments, the hematologic malignancy is selected from B-cell malignancy, leukemia, lymphoma, and myeloma. In some embodiments, the hematologic malignancy is selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer cancer and esophageal cancer. In some embodiments, the solid tumor is selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

[574] As used herein, the term “adjuvant” means a substance that is capable of enhancing, enhancing, or modulating the immune response to a concomitant immunogenic agent, such as a neoantigenic peptide or mRNA. In certain embodiments, the neoantigen of the present invention can be administered in combination with adjuvants, i.e. substances that do not themselves induce adaptive immune responses, but enhance or modulate the response to an accompanying neoantigen. A number of adjuvants can be used in combination with the disclosed neoantigens to promote the development of an immune response. In some embodiments, the adjuvant(s) are selected to enhance the intrinsic response to the neoantigen without causing conformational changes in the neoantigen that would affect the qualitative form of the response. In some embodiments, the adjuvant(s) are selected to enhance the priming and/or activation of effector T cells (eg, CD8).

[575] In certain embodiments, the adjuvant is an aluminum salt (alum), such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate. Such adjuvants can be used with or without other specific immunostimulatory agents such as 3-O-deacylated monophosphoryl lipid A (MPL) or 3-DMP, polymeric or monomeric amino acids such as polyglutamic acid or polylysine. Such adjuvants may be used with other specific immunostimulatory agents, such as muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetylnormuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), N-acetylglucosaminyl-N-acetylmuramyl -L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP)), or other components of the bacterial cell wall, or without them. Other adjuvants are oil-in-water emulsions and include (a) MF59 (WO 90/14837) containing 5% squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally containing varying amounts of MTP-PE ), formulated into submicron particles using a microfluidizer such as a Model 110Y microfluidizer (Microfluidics), (b) SAF containing 10% squalene, 0.4% Tween 80, 5% Pluronic L121 block polymer and thr-MDP, or microfluidized into a submicron emulsion, or vortexed to form an emulsion with larger particle sizes, and (c) Ribi™ Adjuvant System (RAS), (Ribi ImmunoChem) containing 2% squalene, 0.2% Tween 80 and one or more bacterial components cell wall from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM) and cell wall scaffold (CWS), for example MPL-FCWS (Detox™). In some embodiments, the adjuvant is a saponin, such as Stimulon™ (QS21), or particles derived therefrom, such as ISCOM (immune stimulating complexes) and ISCOMATRIX. Other adjuvants include complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA), cytokines such as interleukins (IL-1, IL-2 and IL-12), macrophage colony stimulating factor (M-CSF) and tumor necrosis factor (TNF). ).

[576] The adjuvant can be administered with the immunogenic agent (eg, neoantigenic peptide or mRNA) as a single composition, or it can be administered before, concurrently with, or after the administration of the immunogenic agent. In some embodiments, the immunogenic agent and the adjuvant may be packaged and provided in the same vial or may be packaged in separate vials and mixed prior to use. In some embodiments, the immunogenic agent and adjuvant may be packaged with labeling indicating the intended therapeutic use. In some embodiments, if the immunogenic agent and adjuvant are packaged separately, the package may include instructions for mixing prior to use. The choice of adjuvant and/or carrier depends on the stability of the immunogenic composition containing the adjuvant, the route of administration, the dosage schedule, the effectiveness of the adjuvant for the animal species to be vaccinated and for humans, a pharmaceutically acceptable adjuvant being an adjuvant that has been approved or may be approved by appropriate regulatory authorities for administration to humans. For example, Freund's complete adjuvant is not suitable for administration to humans. However, alum, MPL or Freund's incomplete adjuvant (Chang et al. (1998) Adv Drug Deliv Rev. 32:173-186) alone or optionally in combination with any of alum, QS21 and MPL and all combinations thereof are suitable for administration to humans.

[577] In various embodiments, the present invention further provides methods for screening and identifying at least one neoantigen. More specifically, in various embodiments, the present invention provides a method for identifying at least one neoantigen by (a) contacting a neoplastic cell with an effective amount of a herboxydiene splicing modulator, ADC, or a composition comprising a herboxydiene splicing modulator, or ADC; (b) detecting at least one alternatively spliced mRNA transcript after contacting the neoplastic cell with a herboxydiene-based splicing modulator, ADC, or composition; (c) predicting translation of at least one alternatively spliced mRNA transcript into at least one peptide; and (d) comparing at least one peptide to a reference proteome, wherein at least one neoantigen is identified if at least one peptide is not matches any peptides in the reference proteome. In various embodiments, the method further comprises bringing one or more additional neoplastic cells into contact to identify at least one universal neoantigen. In various embodiments, the method is repeated for one or more additional neoplastic cells or one or more samples (eg, tissue biopsy) to confirm suitable neoantigens (eg, for use in a neoantigen vaccine) and/or to identify one or more universal neoantigens.

[578] In various other embodiments, the present invention provides a method for identifying at least one neoantigen by (a) contacting a neoplastic cell with an effective amount of a herboxidiene splicing modulator, ADC, or a composition comprising a herboxidiene splicing modulator, or ADC; (b) identifying at least one peptide containing a potential neoantigenic sequence after contacting a neoplastic cell with a herboxydiene-based splicing modulator, ADC, or composition and (c) comparing at least one peptide to a reference proteome, wherein at least one a neoantigen is identified if at least one peptide does not match any peptides in the reference proteome. In various embodiments, the method further comprises bringing one or more additional neoplastic cells into contact to identify at least one universal neoantigen. In various embodiments, the method is repeated for one or more additional neoplastic cells or one or more samples (eg, tissue biopsy) to confirm suitable neoantigens (eg, for use in a neoantigen vaccine) and/or to identify one or more universal neoantigens.

[579] In some embodiments of the neoantigen identification methods described herein, detection of at least one alternatively spliced mRNA transcript involves RNAseq. In some embodiments, predicting the translation of at least one alternatively spliced mRNA transcript involves quantifying the change as a percent splicing success rate (dPSI) value for the at least one transcript. In some embodiments, translation prediction of at least one alternatively spliced mRNA transcript involves RiboSeq and/or ribosome profiling.

[580] In some embodiments of the neoantigen identification methods described herein, the methods further include evaluating at least one peptide for predicted binding to major histocompatibility complex (MHC) molecules. In some embodiments, predicted binding to MHC molecules is determined by measuring predicted binding strength based on initial affinity for at least one peptide. In some embodiments, a predicted binding strength based on initial affinity that is approximately 500 nM or greater is indicative of binding to MHC molecules. In some embodiments, predicted binding to MHC molecules is determined by identifying a distribution of predicted binding strength values for a series of random peptides and comparing the predicted binding strength of at least one peptide to this distribution. In some embodiments, the predicted binding strength in the top 2% of the distribution indicates weak binding to MHC molecules. In some embodiments, the predicted binding strength in the top 0.5% of the distribution indicates strong binding to MHC molecules.

[581] In some embodiments of the neoantigen identification methods described herein, the neoplastic cell is in in vitro cell culture. In some embodiments, the neoplastic cell is obtained from a subject. In some embodiments, the neoplastic cell is located in the body of the subject.

[582] In various embodiments, also provided herein are methods of producing a neoantigen vaccine by (a) identifying at least one neoantigen (e.g., at least one neoantigen peptide or mRNA encoding the same) using any of the exemplary identification methods disclosed in herein and (b) formulating at least one neoantigen together with a pharmaceutically acceptable carrier, diluent or adjuvant (eg, any of the pharmaceutically acceptable carriers, diluents or adjuvants described herein).

[583] In some embodiments, the length of the at least one neoantigen and/or antigenic portion ranges from about 10 to about 50 amino acids. In some embodiments, the length of the at least one neoantigen peptide ranges from about 10 to about 35 amino acids. In some embodiments, the length of the at least one neoantigen and/or antigenic portion ranges from about 15 to about 25 amino acids. In some embodiments, the length of the at least one neoantigen and/or antigenic portion ranges from about 10 to about 20 amino acids. In some embodiments, the at least one neoantigen and/or antigenic moiety does not directly overlap or consist of a canonical peptide sequence (eg, any of the exemplary canonical peptide sequences highlighted in Table 8).

[584] In some embodiments, the at least one neoantigen used in the vaccine is attached to a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from a peptide, serum albumin, fisurella hemocyanin, immunoglobulin, thyroglobulin, ovalbumin, toxoid or attenuated toxoid derivative, cytokine, and chemokine.

Combination of herboxydiene/ADC splicing modulator and engineered T cells (CAR-T)

[585] in various embodiments, a patient who has a cancer described herein can be treated using a combination of a herboxidiene splicing modulator, ADC or composition and one or more engineered T cells targeting the tumor (i.e. .e. CAR-T). Thus, in some embodiments, the present invention provides a method of treating a subject who has or is suspected of having a neoplastic disorder by administering to the subject an effective amount of a herboxidiene splicing modulator, ADC, or a composition comprising a herboxidiene splicing modulator, or ADC; and engineered tumor-targeting T cells (i.e., CAR-T). In various embodiments, a chimeric T cell receptor can be constructed using antigen recognition sequences that are reactive to the identified neoantigen.

[586] For example, in various embodiments, to target changes induced by a herboxydiene splicing modulator or ADC in the extracellular domains of cell surface proteins, a chimeric antigen reactive T cell receptor (CAR) can be engineered by initially identifying antibodies that recognize a neoantigen protein domain expressed on the cell surface. The antigen recognition sequences of such antibodies can then be fused to a T cell receptor domain to achieve selective targeting and activation.

[587] Various other embodiments employ the strategy of incorporating the antigen presenting machinery of tumor cells along with neoantigens generated by a herboxidiene-based splicing modulator or ADC. In some embodiments, cells containing known and commonly expressed HLA alleles (eg, HLA-A*02:01) can be treated with a herboxydiene-based splicing modulator, ADC, or composition, and MHC1-bound neoantigens are identified through ligandomics. In some embodiments, these peptides can be used to prime and/or expand T cells from healthy donors expressing the same HLA allele. In some embodiments, such T cells can be isolated and the T cell receptor (TCR) α and β chains sequenced to identify cognate antigen recognition/variable regions. In some embodiments, a cognate CAR can then be constructed.

[588] In some embodiments, the CAR sequences are cloned into a population of patient-derived T cells and the cells are expanded using currently available protocols. In some embodiments, the engineered T cells are then transfused back into the patient's bloodstream followed by treatment with a herboxydiene splicing modulator, ADC, or composition. Following treatment with a herboxydiene-based splicing modulator, ADC, or composition, antigen presentation by tumor cells may begin in some embodiments. In some embodiments, a population of engineered T cells can contact and kill antigen-presenting tumor cells.

[589] Those skilled in the art will appreciate that other suitable modifications and adaptations of the methods of the present invention described herein are obvious and can be made using suitable equivalents without departing from the scope of the present invention or the embodiments disclosed herein . Having described the present invention in detail, the same will be more clearly understood with reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.

EXAMPLES

EXAMPLE 1

[589] Methods are described for synthesizing payloads, linkers, and compounds based on a linker-payload (linker-drug, L-H) conjugate construct characterized by the structure shown in Tables 9-11. Linker-payload conjugate constructs have been used in the preparation of antibody-drug conjugates (ADCs). Exemplary ADCs are described in Examples 3-5.

1.1 Reagents and materials

[590] The starting materials used in the following synthetic methods are either commercially available or can be easily prepared using standard methods from known materials. The disclosed linker-payload conjugated constructs can be prepared using the reactions and techniques described herein. In the description of the synthetic methods described below, it should be understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, experimental duration and work-up procedure, can be selected as standard conditions for a given reaction unless otherwise noted . One skilled in the art of organic synthesis will appreciate that the functional groups present in various parts of the molecule must be compatible with the proposed reagents and reactions. Substituents that are incompatible with the reaction conditions will be obvious to one skilled in the art, and therefore alternative methods are provided herein.

[591] Preparative liquid chromatography-mass spectrometry (LC/MS) was performed using a Waters self-purification system and an XTerra MS C18 column (5 μm, 19 mm x 100 mm) under acidic mobile phase conditions. Nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz using a Varian instrument (Agilent Technologies). Microwave heating was performed using a Biotage Emrys Liberator or Initiator microwave reactor. Column chromatography was performed using a Teledyne Isco Combiflash Rf200d. Solvent removal was carried out using either a rotary evaporator from Büchi or a centrifugal evaporator from Genevac.

[592] Terms/abbreviations. As used herein, the term “under-inert atmosphere” refers to replacing the air in a reactor (eg, reaction vessel, flask, glass reactor) with an inert gas, substantially free of moisture, such as nitrogen or argon. The following abbreviations are used in this document: DCM = dichloromethane, DMF = dimethylformamide, HPLC = high-performance liquid chromatography, KHMDS = potassium bis(trimethylsilyl)amide, LC/MS = liquid chromatography-mass spectrometry, MeOH = methanol, b.t. = room temperature, TBSCl = tert-butyldimethylsilyl chloride, THF = tetrahydrofuran, TLC = thin layer chromatography. Multiplicity is indicated using the following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, sxt = sextet, m = multiplet, dd = doublet of doublets, ddd = doublet of doublets of doublets, dt = doublet of triplets , br s = wide singlet.

[593] LC/MS. Mobile phases = A (0.1% formic acid in H2O) and B (0.1% formic acid in acetonitrile). Gradient = B from 5% to 95% in 1.8 min. Column = Waters Acquity BEH C18 column (1.7 µm, 2.1 x 50 mm).

Table 9. Structure of Illustrative Drug Fragments (Payloads) Payload/ID structure
H1
H2
H3
H4
H5
H6
H7
H8
H9
H10
H11
H12
H13
H14
H15
H16
H17
H18
H19
H20
H21
H22
H23
H24
H25

Table 10. Structure of Exemplary Linkers

Linker/ID structure
(IUPAC name)
ADL1 / “MC-Val-Cit-pABC”
({4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido] -3-methylbutanamido]pentanamido]phenyl}methyl formate)
ADL2 / “MC-(PEG) ₂ -CO”
(3-{2-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}propanal)
ADL5 / “MC-Val-Ala-pAB”
(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-[(1S)-2-methyl-1-{[(1S)-1-[(4- methylphenyl)carbamoyl]ethyl]carbamoyl}propyl]hexanamide)
ADL6 / “MC-Val-Ala-pABC”
({4-[(2S)-2-[(2S)-2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido]-3-methylbutanamido]propanamido ]phenyl}methyl formate)
ADL7 / “MC-Val-Cit-pAB”
(N-[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[(4-methylphenyl)carbamoyl]butyl]carbamoyl}-2-methylpropyl]-6-(2,5-dioxo -2,5-dihydro-1H-pyrrol-1-yl)hexanamide)
ADL10/MC
(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanal)
ADL12/Mal-Hex
(1-hexyl-2,5-dihydro-1H-pyrrole-2,5-dione)
ADL13 / “MC-β-glucuronide”
((2S,3S,4S,5R,6S)-6-(2-{3-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido]propanamido}- 4-[(formyloxy)methyl]phenoxy)-3,4,5-trihydroxyoxane-2-carboxylic acid)
ADL14 / “Mal-Et”
(1-ethyl-2,5-dihydro-1H-pyrrole-2,5-dione)
ADL15 / “Mal-Et-O-Et”
(1-(2-ethoxyethyl)-2,5-dihydro-1H-pyrrole-2,5-dione)
ADL21 / “MC-Ala-Ala-Asp-pABC”
ADL22 / “MC-(PEG) ₂ -Val-Cit-pABC”
ADL23 / “MC-Glu-Val-Cit-pABC”

Table 11. Structures of exemplary compounds based on linker-payload (L-H) conjugated constructs
ADL1-H1
ADL1-H2
ADL1-H3
ADL1-H4
ADL1-H5
ADL1-H6
ADL1-H7
ADL1-H8
ADL1-H9
ADL1-H10
ADL2-H11
ADL2-H1

1.2 Preparation of ADL1-H1, ADL1-H2, ADL1-H3 and ADL1-H4

1.2.1 Brief description. General procedure 1

Scheme 1

[594] Stage 1. Fermentation and biological transformation

[595] Twenty percent stock solution of Saccharothrix sp. EAS-AB4564, isolated from soil in Japan, in glycerol, was inoculated into the first seed culture in a research tube containing 10 ml of SY-32 medium (1% D-glucose, 1% soluble starch, 0.5% Bactosoytone, 0. 5% yeast extract, 0.2% ammonium sulfate, 0.2% NaCl and 2.3% TES, at pH 8.0). The first culture for sowing was shaken for 2 days at 28°C on a reciprocating shaker at 200 rpm. After fermentation, the first seed culture was inoculated into a sterile second seed culture in 1% v/v Erlenmeyer flasks, each containing 100 ml of new SY-32 medium. Second inoculation cultures were shaken for 2 days at 28°C on a rotary shaker (Iwashiya bioscience SC-144-GR) at 200 rpm. After fermentation, 250 mL of the second culture was inoculated into a 15 L fermenter (Sanki seiki MAT-15) containing 10 L of new SY-32 medium and 2 mL of PE-M antifoam. Fermentation was carried out at 28°C under stirring and aeration conditions (450 rpm, 10 l/min). After 48 hours, the broth culture was centrifuged at 3000 rpm. within 10 minutes. After removing the supernatant, 10 L of 20 mM phosphate buffer (pH 7.0) was added to the microbial sediment to wash the microbial cells. The suspension was centrifuged at 3000 rpm. for 10 minutes. After removing the wash buffer, 10 L of reaction buffer (1% D-glucose, 0.2% magnesium chloride hexahydrate, 2.3% TES and 1.3 g herboxydiene, pH 8.0) was added to the microbial sediment and the suspension was transferred to a 15 L fermenter. The biological transformation was carried out for 6 hours at 28°C with stirring and aeration (450 rpm, 10 l/min).

[596] Isolation of 5-hydroxyherboxidiene

[597] XAD-7HP (400 g) was added to the above mixture (10 L) and the mixture was stirred (EYELA MAZELA Z) for 30 minutes at 300 rpm. After shaking, separation of XAD-7HP was carried out using dyeing and a test sieve (0.25 mm opening and 0.16 mm wire diameter). The same operation was repeated. The collected XAD-7HP was extracted with 1 L of acetone and the solvent was removed in vacuo. The extract was purified using reverse phase MPLC (Yamazen EPCLC-W-prep 2XY) with gradient elution (YMC-DispoPack AT ODS-25, 120 g, 25 to 55% acetonitrile in water with the addition of 0.1% formic acid, in for 15 minutes) to obtain 5-OH-herboxydiene (397.47 mg), with a conversion yield of 29.5%. The ¹ H NMR and mass spectrometry data were consistent with those known from the literature. See for example EP0781772 B1 and Ghosh et al. (2014) Org. Lett. 16:3154-57.

[598] Step 2. Synthesis of methyl-2-((2R,4R,5S,6S)-4-hydroxy-6-((S,2E,4E)-7-((2R,3R)-3-(( 2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H- pyran-2-yl)acetate

[599] To a solution of 2-((2R,4R,5S,6S)-4-hydroxy-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R, 4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2- yl)acetic acid (45 mg, 0.099 mmol) in THF (2 ml) and MeOH (0.5 ml) at 0°C, trimethylsilyldiazomethane (2.0 M in hexanes, 0.148 ml, 0.297 mmol) was added dropwise. The resulting mixture was then gradually warmed to room temperature and stirred for 1 hour before cooling to 0°C. Acetic acid (0.017 mL, 0.297 mmol) was then added and stirred for 30 minutes. The mixture was then diluted with AcOEt and saturated aqueous sodium bicarbonate. The organic layer was isolated, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The isolated residue was purified by silica gel chromatography to give methyl 2-((2R,4R,5S,6S)-4-hydroxy-6-((S,2E,4E)-7-((2R,3R)-3 -((2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro -2H-pyran-2-yl)acetate as a colorless oil (36.1 mg, 78% yield).

[600] ¹ H NMR (400 MHz, CHLOROFORM-d) 0.80 (d, J=6.83 Hz, 3 H) 0.86 (d, J=6.83 Hz, 3 H) 1.03 ( d, J=6.83 Hz, 3 H) 1.16 (d, J=6.3 Hz, 3 H) 1.20-1.24 (m, 1H) 1.26 (s, 3H) 1, 31- 1.34 (m, 3 H) 1.40-1.59 (m, 2 H) 1.63 (br. s., 2 H) 1.69 (s, 3 H) 1.87 (dd , J=13.66, 4.88 Hz, 1 H) 2.03 (ddd, J=10.37, 4.02, 2.20 Hz, 1 H) 2.42 (dd, J=15.61 , 6.34 Hz, 2 H) 2.54 (d, J=9.8 Hz, 2 H) 2.62 (dd, J=15.6, 6.3 Hz, 1 H) 2.95 (t , J=5.37 Hz, 1 H) 3.33-3.44 (m, 2 H) 3.52 (s, 3 H) 3.65 (s, 3 H) 3.79-3.90 ( m, 2 H) 5.45 (dd, J=15.12, 8.78 Hz, 1 H) 5.88 (d, J=11.22 Hz, 1 H) 6.21 (dd, J=15 ,12, 10.73 Hz, 1 H).

[601] Step 3: Synthesis of carbamate

[602] To a mixture of methyl 2-((2R,4R,5S,6S)-4-hydroxy-6-((S,2E,4E)-7-((2R,3R)-3-((2R, 3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran- 2-yl)acetate (1.0 eq., 0.075 mmol), 4-nitrophenylcarbonyl chloride (2 eq.), Hunig's base (0.065 ml, 4.5 eq.) in dichloromethane (0.04 M) was added at 0°C DMAP (0.05 eq). The mixture was then warmed to room temperature and stirred for 16 hours. Piperazine (10 eq.) was added to the mixture and the resulting mixture was stirred for an additional 1 hour. The mixture was then diluted with DCM and the organic layer was isolated, washed with water and brine, and dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was purified by silica gel chromatography and then by amino-functionalized silica gel chromatography to obtain the desired product.

[603] Step 4: Synthesis of carboxylic acid

[604] To a mixture of the methyl ester obtained in step 3 (1.0 eq, 0.039 mmol) in MeOH (0.02 M ml) was added an aqueous solution of sodium hydroxide (1.0 eq, 2 N). The mixture was heated to 40°C and stirred at this temperature for 4 hours. The resulting mixture was then cooled to 0°C and neutralized with aqueous hydrochloric acid (200 μl, 2N). The mixture was then concentrated in vacuo and the resulting residue was purified by preparative HPLC (H2O/MeCN/HCOOH=80/20/0.1 to 60/40/0.1) to obtain the desired product.

1.2.2 H1 synthesis

[605] (2S,3S,4R,6R)-2-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-Hydroxy-3- methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-6-(2-methoxy-2-oxoethyl)-3-methyltetrahydro-2H-pyran -4-ylpiperazine-1-carboxylate

[606] The title compound was synthesized according to step 3 of section 1.2.1 to give a pale yellow oil (22.3 mg, 52% yield).

[607] ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.71 (d, J=6.83 Hz, 3 H) 0.85 (d, J=6.8 Hz, 3 H) 1, 03 (d, J=6.83 Hz, 3 H) 1.16 (d, J=6.8 Hz, 3 H) 1.20-1.25 (m, 1H) 1.26 (s, 3H) 1.37 (q, J=11.55 Hz, 1 H) 1.51 (dt, J=8.90, 6.52 Hz, 1 H) 1.63-1.67 (m, 1 H) 1 .69 (s, 3H) 1.83-2.07 (m, 10 H) 2.11-2.17 (m, 1 H) 2.36-2.45 (m, 2 H) 2.51- 2.62 (m, 2 H) 2.81 (s, 4 H) 2.95 (t, J=5.12 Hz, 1 H) 3.40-3.49 (m, 4 H) 3.52 (s, 3 H) 3.65 (s, 3 H) 3.80-3.94 (m, 2 H) 4.56 (td, J=10.98, 4.39 Hz, 1 H) 5, 46 (dd, J=15.12, 8.78 Hz, 1 H) 5.90 (d, J=10.24 Hz, 1 H) 6.21 (dd, J=15.12, 10.73 Hz , 1 H).

[608] 2-((2R,4R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-Hydroxy -3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyl-4-((piperazin-1-carbonyl)oxy) tetrahydro-2H-pyran-2-yl)acetic acid (H1)

[609] The title compound was synthesized according to step 4 of section 1.2.1 to give a colorless oil (16.0 mg, 73% yield). LC/MS (ESI, mass/charge), 567.77 [M+H] ⁺ .

[610] ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ ppm 0.72 (d, J=6.3 Hz, 3 H) 0.80 (d, J=6.83 Hz, 3 H) 1 .03 (d, J=6.3 Hz, 3 H) 1.08 (d, J=6.3 Hz, 3 H) 1.17 (dd, J=13.2, 11.2 Hz, 1 H ) 1.25 (s, 3H) 1.36 (q, J=11.4 Hz, 1 H) 1.45-1.50 (m, 2 H) 1.68 (s, 3 H) 1.90 (dd, J=13.42, 4.15 Hz, 1 H) 2.14 (dd, J=11.71, 3.90 Hz, 1 H) 2.36-2.52 (m, 3 H) 2.63 (d, J=9.27 Hz, 1 H) 2.95 (dd, J=5.85, 4.39 Hz, 1 H) 3.17 (br. s, 4 H) 3.46 -3.53 (m, 4 H) 3.56-3.60 (m, 1 H) 3.69 nr. s, 4 H) 3.74-3.82 (m, 2 H) 3.85-3.92 (m, 1 H) 4.58 (td, J=10.49, 4.39 Hz, 1 H ) 5.49 (dd, J=15.12, 8.78 Hz, 1 H) 5.94 (d, J=10.73 Hz, 1 H) 6.28 (dd, J=14.88, 10 .98 Hz, 1 H).

1.2.3 H2 synthesis

[611] (2S,3S,4R,6R)-2-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-Hydroxy-3- methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-6-(2-methoxy-2-oxoethyl)-3-methyltetrahydro-2H-pyran -4-yl-1,4-diazepane-1-carboxylate

[612] The title compound was synthesized according to step 3 of section 1.2.1 (30.3 mg, 60% yield).

[613] ¹ H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.71 (d, J=6.11 Hz, 3 H) 0.84 (d, J=6.73 Hz, 3 H) 1, 00-1.05 (m, 4 H) 1.16 (d, J=6.11 Hz, 3 H) 1.19-1.24 (m, 1 H) 1.26 (s, 3 H) 1 .32-1.40 (m, 1 H) 1.51 (dt, J=9.17, 6.42 Hz, 1 H) 1.63-1.67 (m, 1 H) 1.69 (s , 3 H) 1.73-1.81 (m, 3 H) 1.83-1.90 (m, 2 H) 2.07-2.18 (m, 1 H) 2.41 (dd, J =15.28, 6.11 Hz, 2 H) 2.51-2.60 (m, 2 H) 2.82-2.95 (m, 5 H) 3.41-3.49 (m, 4 H) 3.51 (s, 3 H) 3.64 (s, 3 H) 3.79-3.95 (m, 2 H) 4.57 (td, J=10.70, 4.28 Hz, 1 H) 5.45 (dd, J=14.98, 8.86 Hz, 1 H) 5.90 (d, J=11.00 Hz, 1 H) 6.20 (dd, J=14.98 , 10.70 Hz, 1 H).

[614] 2-((2R,4R,5S,6S)-4-((1,4-Diazepan-1-carbonyl)oxy)-6-((S,2E,4E)-7-((2R, 3R)-3-((2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl) -5-methyltetrahydro-2H-pyran-2-yl)acetic acid (H2)

[615] The title compound was synthesized according to step 4 of section 1.2.1 to give a colorless oil (20.9 mg, 71% yield). LC/MS (ESI, mass/charge), 581.81 [M+H] ⁺ .

[616] ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ ppm 0.74 (d, J=6.34 Hz, 3 H) 0.80 (d, J=7.32 Hz, 3 H) 1 .03 (d, J=6.34 Hz, 3 H) 1.08 (d, J=6.34 Hz, 3 H) 1.12-1.23 (m, 1 H) 1.25 (s, 3 H) 1.31-1.41 (m, 1 H) 1.41-1.53 (m, 1 H) 1.63-1.74 (m, 4 H) 1.84-1.93 ( m, 1 H) 2.04 (br. s., 2 H) 2.11-2.21 (m, 1 H) 2.34-2.54 (m, 3 H) 2.63 (d, J =9.27 Hz, 1 H) 2.90-2.98 (m, 2 H) 3.21-3.36 (m, 7 H) 3.50 (s, 3 H) 3.53-3, 66 (m, 2 H) 3.69-3.79 (m, 3 H) 3.83-3.95 (m, 1 H) 4.57 (br. s., 1 H) 5.49 (dd , J=14.88, 9.03 Hz, 1 H) 5.94 (d, J=10.73 Hz, 1 H) 6.24-6.36 (m, 1 H).

1.2.4 H3 synthesis

[617] (2S,3S,4R,6R)-2-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-Hydroxy-3- methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-6-(2-methoxy-2-oxoethyl)-3-methyltetrahydro-2H-pyran -4-yl-3-(methylamino)pyrrolidine-1-carboxylate

[618] The title compound was synthesized according to step 3 of section 1.2.1 to give a colorless oil (9.6 mg, 19% yield).

[619] 1H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.71 (d, J=6.3 Hz, 3 H) 0.85 (d, J=6.73 Hz, 3 H) 0.99 -1.05 (m, 4 H) 1.17 (d, J=6.11 Hz, 4 H) 1.27 (s, 3 H) 1.32-1.45 (m, 1 H) 1, 49-1.68 (m, 6 H) 1.70 (s, 3 H) 1.88 (dd, J=13.45, 4.89 Hz, 1 H) 2.00-2.08 (m, 1 H) 2.10-2.17 (m, 1 H) 2.38-2.47 (m, 5 H) 2.52-2.61 (m, 2 H) 2.94 (t, J= 5.50 Hz, 1 H) 3.17-3.26 (m, 1 H) 3.43 (d, J=10.4 Hz, 2 H) 3.52 (s, 4 H) 3.65 ( s, 3 H) 3.78-3.86 (m, 1 H) 3.87-3.95 (m, 1 H) 4.51-4.57 (m, 1 H) 5.45 (dd, J=14.98, 8.86 Hz, 1 H) 5.90 (d, J=10.39 Hz, 1 H) 6.21 (dd, J=14.98, 10.70 Hz, 1 H) .

[620] 2-((2R,4R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-Hydroxy -3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyl-4-((3-(methylamino)pyrrolidin-1 -carbonyl)oxy)tetrahydro-2H-pyran-2-yl)acetic acid (H3)

[621] The title compound was synthesized according to step 4 of section 1.2.1 to give a colorless oil (9.8 mg, quantitative yield). LC/MS (ESI, mass/charge), 581.81 [M+H] ⁺ .

[622] ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ ppm 0.71 (d, J=6.34 Hz, 3 H) 0.85 (d, J=6.83 Hz, 3 H) 1 .02 (d, J=6.34 Hz, 3 H) 1.16 (d, J=6.34 Hz, 3 H) 1.22-1.24 (m, 1 H) 1.26 (s, 3 H) 1.31-1.42 (m, 1 H) 1.45-1.66 (m, 4 H) 1.69 (s, 3 H) 1.87 (dd, J=13.66, 4.88 Hz, 1 H) 2.02-2.20 (m, 2 H) 2.38-2.51 (m, 4 H) 2.51-2.60 (m, 2 H) 2.94 (t, J=5.12 Hz, 1 H) 3.08-3.26 (m, 2 H) 3.33-3.40 (m, 1 H) 3.43 (d, J=9.76 Hz, 2 H) 3.51 (s, 3 H) 3.64 (s, 3 H) 3.83-3.94 (m, 1 H) 4.51-4.59 (m, 1 H) 5 .45 (dd, J=15.12, 8.78 Hz, 1 H) 5.89 (d, J=10.73 Hz, 1 H) 6.20 (dd, J=15.12, 10.73 Hz, 1 H).

1.2.5 H12 synthesis

[623] The title compound was synthesized according to step 3 (13.8 mg, 91% yield) and step 4 of section 1.2.1 to give a white amorphous solid (4.58 mg, 76% yield). LC/MS (ESI, mass/charge), 581.55 [M+H] ⁺ .

[624] 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.71 (d, J=6.83 Hz, 3 H) 0.85 (d, J=6.83 Hz, 3 H) 1.03 (d, J=6.34 Hz, 3 H) 1.16 (d, J=6.34 Hz, 3 H) 1.26 (s, 4 H) 1.48-1.57 (m, 1 H ) 1.61-1.68 (m, 1 H) 1.89 (dd, J=13.66, 4.39 Hz, 1 H) 2.20 (br d, J=8.29 Hz, 1 H ) 2.30 (s, 4 H) 2.33-2.51 (m, 6 H) 2.52-2.57 (m, 2 H) 2.57-2.63 (m, 1 H) 2 .78-3.21 (m, 9 H) 3.35-3.66 (m, 9 H) 3.70-3.99 (m, 2 H) 4.52 (br d, J=3.90 Hz, 1 H) 5.41-5.58 (m, 1 H) 5.92 (br d, J=11.22 Hz, 1 H) 6.13-6.29 (m, 1 H).

1.2.6 H4 synthesis

[625] H4 was synthesized according to Scheme 2.

Scheme 2

[626] Step 1. 2,5-Dioxopyrrolidin-1-yl-2-((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-( (2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H -pyran-2-yl)acetate

[627] To a mixture of 2-((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-hydroxy -3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl)acetic acid (herboxidiene , 300 mg, 0.684 mmol) in DCM (10 ml) at 0°C was added DCC (310 mg, 1.505 mmol) and the resulting mixture was stirred for 15 minutes. N-hydroxysuccinamide (173 mg, 1.505 mmol) was then added to the mixture. The resulting mixture was warmed to room temperature and stirred for 16 hours. The mixture was then filtered through celite and the filter cake washed with DCM. The filtrate was concentrated in vacuo and the resulting residue was purified by silica gel chromatography (12 g, heptane/AcOEt=50/50 to 0/100) to give the title compound as a colorless solid (357 mg, 97% yield).

[628] ¹ H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.66 (d, J=6.73 Hz, 3 H) 0.83-0.89 (m, 3 H) 1.03 (d , J=6.73 Hz, 3 H) 1.17 (d, J=6.1 Hz, 3 H) 1.19-1.25 (m, 4 H) 1.27 (s, 3H) 1, 39-1.56 (m, 3 H) 1.59 (s, 3 H) 1.70 (s, 3 H) 1.79-1.93 (m, 3 H) 2.36-2.43 (m , 1 H) 2.52-2.55 (m, 2 H) 2.70 (dd, J=15.28, 7.34 Hz, 1 H) 2.81 (br. s., 3 H) 2 ,83-2.90 (m, 1 H) 2.95 (t, J=5.50 Hz, 1 H) 3.34 (d, J=9.78 Hz, 1 H) 3.52 (s, 3 H) 3.79-3.87 (m, 2 H) 5.43 (dd, J=15.28, 9.17 Hz, 1 H) 5.88 (d, J=11.00 Hz, 1 H) 6.21-6.26 (m, 1 H).

[629] Step 2. (S)-5-Hydroxy-2-(2-((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3- ((2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro- 2H-pyran-2-yl)acetamido)pentanoic acid

[630] To a mixture of the compound obtained in step 1 (40 mg, 0.075 mmol) and (S)-2-amino-5-hydroxypentanoic acid (19.88 mg, 0.149 mmol) in DMF (2 ml) was added DIPEA ( 0.065 ml, 0.373 mmol). The resulting mixture was stirred at room temperature for 4 hours and then diluted with AcOEt. The organic layer was isolated, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting precipitate was used at stage 3 without additional purification.

[631] ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.66 (d, J=6.3 Hz, 3 H) 0.85 (d, J=6.8 Hz, 3 H) 1, 03 (d, J=6.8 Hz, 3 H) 1.15 (d, J=6.3 Hz, 3 H) 1.19-1.23 (m, 2 H) 1.27 (s, 3H ) 1.40-1.61 (m, 6H) 1.72 (s, 3H) 1.82-1.93 (m, 3 H) 2.37-2.43 (m, 2 H) 2.56 (d, J=9.8 Hz, 1 H) 2.99 (t, J=5.3 Hz, 1 H) 3.33 (d, J=9.8 Hz, 1 H) 3.52 (s , 3H) 3.54-3.70 (m, 4 H) 3.81-3.87 (m, 2 H) 4.56 (d, J=5.4 Hz, 1 H) 5.48 (dd , J=15.12, 8.29 Hz, 1 H) 5.89 (d, J=11.2 Hz, 1 H) 6.22 (dd, J=15.1, 10.7 Hz, 1 H ) 7.30 (d, J=7.3 Hz 1 H).

[632] Step 3. Methyl-(S)-5-hydroxy-2-(2-((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)- 3-((2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5- methyltetrahydro-2H-pyran-2-yl)acetamido)pentanoate

[633] To a mixture of the compound obtained in step 2 (35 mg, 0.063 mmol) in THF (2 ml) and methanol (0.5 ml) at 0°C was added trimethylsilyldiazomethane (2.0 M in hexanes, 0.095 ml, 0.19 mmol). The mixture was then warmed to room temperature and stirred for 1 hour. The mixture was then cooled to 0°C and quenched by adding acetic acid. The resulting mixture was stirred for 30 minutes and then concentrated in vacuo. The resulting residue was purified by silica gel chromatography (12 g, heptane/AcOEt = 90/10 to 30/70) to give the title compound (15.7 mg, 44% yield).

[634] ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.66 (d, J=6.8 Hz, 3 H) 0.85 (d, J=6.8 Hz, 3 H) 1, 03 (d, J=6.3 Hz, 3 H) 1.16 (d, J=6.8 Hz, 3 H) 1.19-1.23 (m, 3 H) 1.27 (s, 3 H) 1.47-1.70 (m, 7 H) 1.73 (s, 3 H) 1.77-1.94 (m, 6 H) 2.34-2.59 (m, 5 H) 2.96 (t, J=5.4 Hz 1 H) 3.29-3.40 (m, 1 H) 3.52 (s, 3 H) 3.56-3.66 (m, 3 H) 3.70 (s, 3 H) 3.77-3.89 (m, 1 H) 4.61 (td, J=8.05, 4.88 Hz, 1 H) 5.46 (dd, J= 15.12, 8.78 Hz, 1 H) 5.89 (d, J=10.73 Hz, 1 H) 6.23 (dd, J=15.12, 10.73 Hz, 1 H) 7, 10 (d, J=8.29 Hz, 1 H).

[635] Stage 4. (S)-4-(2-((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R, 3R,4R)-4-Hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran- 2-yl)acetamido)-5-methoxy-5-oxopentylpiperazine-1-carboxylate

[636] To a mixture of the compound obtained in step 3 (15.7 mg, 0.028 mmol), 4-nitrophenylchloroformate (11.15 mg, 0.055 mmol) and Hunig's base (0.024 ml, 0.138 mmol) in DCM (1 ml) at DMAP (1.689 mg, 0.014 mmol) was added at room temperature. The resulting mixture was stirred at room temperature for 16 hours. Piperazine (23.82 mg, 0.277 mmol) was then added and the mixture was stirred for an additional 1 hour. The mixture was then diluted with DCM and the organic layer was isolated, washed with water and brine, and dried over sodium sulfate, filtered and concentrated in vacuo. The crude residue was used in the next step without purification.

[637] Stage 5. (S)-2-(2-((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R, 3R,4R)-4-Hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran- 2-yl)acetamido)-5-((piperazine-1-carbonyl)oxy)pentanoic acid (H4)

[638] To a mixture of the residue obtained in step 4 (15 mg, 0.022 mmol) and MeOH (1 ml) was added an aqueous solution of sodium hydroxide (2 N, 100 μl, 0.20 mmol). The mixture was heated to 40°C and stirred for 2 hours. The mixture was then cooled to 0°C and an aqueous solution of hydrochloric acid (100 μl, 2N) was added. The resulting mixture was then concentrated in vacuo and the resulting residue was purified by preparative HPLC (H ₂ O/MeCN/NH ₃ aq = 60/40/0.1 to 30/70/0.1) to give the title compound in as a colorless oil (7.0 mg, 48% yield from steps 4 and 5). LC/MS (ESI, mass/charge), 666.90 [M+H] ⁺ .

[639] ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ ppm 0.65 (d, J=6.83 Hz, 3 H) 0.81 (d, J=7.32 Hz, 3 H) 0 ,98-1.04 (m, 3 H) 1.08 (d, J=6.34 Hz, 3 H) 1.17 (d, J=11.71 Hz, 1 H) 1.22-1, 28 (m, 4 H) 1.35 (d, J=6.83 Hz, 1 H) 1.46-1.66 (m, 10 H) 1.70 (s 3 H) 1.82-1, 94 (m, 3 H) 2.25-2.31 (m, 1 H) 2.36-2.45 (m, 2 H) 2.61-2.65 (m, 1 H) 2.95 ( dd, J=6.10, 4.15 Hz, 1 H) 3.14 (br. s., 2 H) 3.50 (s, 3 H) 3.62-3.71 (m, 4 H) 3.76 (t, J=6.3 Hz, 1 H) 4.03 (dd, J=4.15, 1.71 Hz, 1 H) 4.29 (br. s., 1 H) 5, 45 (d, J=6.34 Hz, 1 H) 5.87 (d, J=10.73 Hz, 1 H) 6.27 (dd, J=15.12, 10.73 Hz, 1 H) .

1.2.7 General procedure for the synthesis of the MC-Val-Cit-pABC-payload linker construct

[640] The general procedure for the synthesis of linker-payload constructs MC-Val-Cit-pABC is indicated in Scheme 3.

Scheme 3

[641] To a mixture of the payload (e.g., the compound synthesized according to the above steps; 1.0 eq.) and 4-((S)-2-((S)-2-(6-(2,5- dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate (1.0 eq.) in DMF (0.028 M) added DIPEA (3.03 eq.) at room temperature. The resulting mixture was stirred at room temperature for 16 hours, concentrated in vacuo and the resulting residue purified by preparative reverse phase HPLC (H ₂ O/MeCN/HCOOH = 60/40/0.1 to 40/60/0, 1) obtaining the required connection.

1.2.8 Synthesis of ADL1-H1

[642] ADL1-H1 (2-((2R,4R,5S,6S)-4-((4-(((4-((R)-2-((S)-2-(6-(2 ,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)piperazine-1-carbonyl)oxy)-6-(( S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6 -Methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl)acetic acid) was synthesized according to the general procedure given in section 1.2.7 and obtained as a colorless oil ( 12.7 mg, yield 39%).

[643] ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ ppm 0.71 (d, J=5.85 Hz, 4 H) 0.80 (d, J=6.34 Hz, 3 H) 0 .94 (dd, J= 6.1, 3.2 Hz, 6H) 1.01 (d, J=6.3 Hz, 3 H) 1.08 (d, J=6.6 Hz, 3 H) 1.13-1.19 (m, 1H) 1.25 (s, 4 H) 1.29-1.36 (m, 2 H) 1.46-1.65 (m, 7H) 1.68 ( s, 3H) 1.71-1.76 (m, 1H) 1.84-1.93 (m, 2 H) 2.02-2.15 (m, 2 H) 2.25 (t, J= 7.32 Hz, 2 H) 2.37-2.51 (m, 3 H) 2.58-2.67 (m, 2 H) 2.79 (s, 4 H) 2.82-2.96 (m, 2 H) 3.08-3.23 (m, 8 H) 3.39-3.54 (m, 15 H) 3.76 (t, J=6.34 Hz, 1 H) 3, 85-3.97 (m, 2 H) 4.13 (d, J=7.32 Hz, 1 H) 4.46-4.58 (m, 2 H) 5.07 (s, 2 H) 5 .48 (dd, J=14.88, 9.03 Hz, 1 H) 5.93 (d, J=10.73 Hz, 1 H) 6.28 (dd, J=14.88, 10.98 Hz, 1 H) 7.30 (d, J=7.81 Hz, 2 H) 7.57 (d, J=7.81 Hz, 2 H).

1.2.9 ADL1-H4

[644] ADL1-H4 ((S)-5-((4-(((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5- dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)piperazine-1-carbonyl)oxy)-2-(2-((2R,5S,6S) -6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2 -yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl)acetamido)pentanoic acid) was synthesized according to the general procedure specified in section 1.2.7, and was obtained as a colorless oil (2.4 mg, 44% yield).

[645] ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ ppm 0.64 (d, J=6.50 Hz, 3 H) 0.80 (d, J=6.80 Hz, 3 H) 0 .84-0.89 (m, 1 H) 0.89-0.97 (m, 6 H) 0.97-1.03 (m, 2 H) 1.08 (d, J=6.34 Hz , 3 H) 1.10-1.16 (m, 1 H) 1.22-1.25 (m, 3 H) 1.25-1.32 (m, 6 H) 1.49-1.66 (m, 9 H) 1.69 (s, 3 H) 1.80-1.95 (m, 3 H) 1.95-2.15 (m, 2 H) 2.25 (t, J=7 ,20 Hz, 2 H) 2.33-2.48 (m, 1 H) 2.62 (d, J=9.76 Hz, 1 H) 2.91-2.98 (m, 1 H) 3 .04-3.13 (m, 1 H) 3.39-3.47 (m, 7 H) 3.49 (s, 3 H) 3.63 (s, 1 H) 3.76 (t, J =6.50 Hz, 1 H) 3.99 (br. s, 1 H) 4.14 (d, J=7.20 Hz, 1 H) 4.44-4.51 (m, 1 H) 4 .44-4.51 (m, 1 H) 5.07 (br. s, 2 H) 5.43 (dd, J=15.37, 9.03 Hz, 1 H) 5.82-5.90 (m, 1 H) 6.19-6.36 (m, 1 H) 6.77 (s, 1 H) 7.30 (d, J=8.29 Hz, 2 H) 7.57 (d, J=8.78 Hz, 2 H).

1.2.10 ADL1-H2

[646] ADL1-H2 (2-((2R,4R,5S,6S)-4-((4-(((4-((R)-2-((R)-2-(6-(2 ,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)-1,4-diazepan-1-carbonyl)oxy) -6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2 -yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl)acetic acid) was synthesized according to the general procedure given in section 1.2.7 and obtained as a colorless oil (10.4 mg, 34% yield).

[647] ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ ppm 0.61-0.68 (m, 3 H) 0.79 (d, J=6.83 Hz, 3 H) 0.94 ( t, J=6.10 Hz, 6 H) 1.02 (d, J=6.34 Hz, 3 H) 1.04-1.10 (m, 3 H) 1.10-1.17 (m , 1 H) 1.19-1.31 (m, 8 H) 1.42-1.49 (m, 2 H) 1.51-1.65 (m, 8 H) 1.67 (s, 3 H) 1.69-1.78 (br. s, 3 H) 1.83-1.93 (m, 2 H) 1.94 (s, 1 H) 2.00-2.10 (m, 2 H) 2.25 (t, J=7.32 Hz, 2 H) 2.30-2.38 (m, 1 H) 2.38-2.49 (m, 2 H) 2.63 (d, J=9.27 Hz, 1 H) 2.94 (t, J=5.12 Hz, 1 H) 3.05-3.21 (m, 2 H) 3.39-3.48 (m, 7 H) 3.49 (s, 3 H) 3.52-3.63 (m, 5 H) 3.67-3.81 (m, 2 H) 3.90 (br. s., 1 H) 4 .17 (t, J=7.30 Hz, 1 H) 4.45-4.55 (m, 2 H) 5.00-5.08 (m, 2 H) 5.48 (dd, J=14 ,88, 9.51 Hz, 1 H) 5.87-5.95 (m, 1 H) 6.23-6.32 (m, 1 H) 6.76 (s, J=4.01 Hz, 2 H) 7.28 (q, J=7.81 Hz, 2 H) 7.58 (t, J=7.07 Hz, 2 H).

1.2.11 ADL1-H3

[648] ADL1-H3 (2-((2R,4R,5S,6S)-4-((3-((((4-((R)-2-((R)-2-(6-( 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)pyrrolidin-1-carbonyl)oxy )-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran- 2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl)acetic acid) was synthesized according to the general procedure given in section 1.2.7 and was obtained as a colorless oil (18.8 mg, 37% yield).

[649] ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ ppm 0.72 (d, J=6.83 Hz, 3 H) 0.80 (d, J=6.83 Hz, 3 H) 0 .90-0.97 (m, 6 H) 1.02 (d, J=6.34 Hz, 3 H) 1.08 (d, J=6.34 Hz, 3 H) 1.16 (dd, J=12.93, 11.46 Hz, 1 H) 1.22-1.28 (m, 4 H) 1.28-1.37 (m, 2 H) 1.44-1.67 (m, 8 H) 1.68 (s, 3 H) 1.90 (dd, J=13.42, 4.15 Hz, 2 H) 2.00-2.15 (m, 4 H) 2.25 (t , J=7.32 Hz, 2 H) 2.38-2.52 (m, 3 H) 2.63 (d, J=9.27 Hz, 1 H) 2.84 (s, 3 H) 2 ,93-2.97 (m, 1 H) 3.08 (d, J=6.34 Hz, 1 H) 3.16 (d, J=6.83 Hz, 1 H) 3.42-3, 47 (m, 3 H) 3.49 (s, 3 H) 3.51-3.57 (m, 2 H) 3.76 (t, J=6.34 Hz, 1 H) 3.85-3 .92 (m, 1 H) 4.14-4.19 (m, 1 H) 4.47-4.55 (m, 2 H) 4.71 (d, J=7.32 Hz, 1 H) 5.06 (s, 2 H) 5.48 (dd, J=14.88, 9.03 Hz, 1 H) 5.93 (d, J=11.22 Hz, 1 H) 6.28 (dd , J=14.88, 10.98 Hz, 1 H) 6.76 (s, J=5.13 Hz, 2 H) 7.30 (d, J=8.29 Hz, 2 H) 7.56 (d, J=8.29 Hz, 2 H).

1.3 Preparation of ADL1-H5, ADL1-H6 and ADL1-H7

1.3.1 Brief description. General procedure 2

Scheme 3

[650] Stage 1. 2-((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4- Hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl)acetamide

[651] To a mixture of 2-((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-hydroxy -3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl)acetic acid (herboxidiene , 750 mg, 1.71 mmol) and triethylamine (1.192 ml, 8.55 mmol) in THF (15 ml) was added ethyl chloroformate (0.767 ml, 5.13 mmol) at 0°C. The resulting mixture was stirred at 0°C for 30 minutes. and then ammonia in methanol (7 M, 3.66 mL, 25.65 mmol) was added. The resulting mixture was stirred for an additional 30 minutes at the same temperature before concentrating in vacuo and purifying the resulting residue by silica gel chromatography (heptane/AcOEt = 50/50 to 0/100, then AcOEt = 80/20) to give the above in the title compound as a colorless solid (632 mg, 85% yield).

[652] ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.67 (d, J=6.34 Hz, 3 H) 0.85 (d, J=6.7 Hz, 3 H) 1, 05 (d, J=6.34 Hz, 3 H) 1.16 (d, J=6.7 Hz, 3 H) 1.20-1.25 (m, 3 H) 1.27 (s, 3 H) 1.34-1.45 (m, 1 H) 1.49-1.65 (m, 7 H) 1.71 (s, 3 H) 1.82-1.97 (m, 2 H) 2.01-2.05 (m, 1 H) 2.33-2.48 (m, 3 H) 2.54 (d, J=9.27 Hz, 2 H) 2.95-2.99 ( m, 1 H) 3.34 (d, J=10.0 Hz, 1 H) 3.52 (s, 3 H) 3.63-3.70 (m, 1 H) 3.80-3.87 (m, 1 H) 4.08-4.14 (m, 1 H) 5.24-5.33 (m, 1 H) 5.47 (dd, J=15.12, 9.27 Hz, 1 H) 5.90 (d, J=10.73 Hz, 1 H) 6.22 (dd, J=14.88, 10.98 Hz, 1 H) 6.61 (br. s, 1 H).

[653] Stage 2. 2-((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-3- Methoxy-4-((triethylsilyl)oxy)pentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2 -yl)acetamide

[654] To a mixture of the compound isolated in step 1 (719 mg, 1.446 mmol) in DCM (15 ml) and triethylamine (2.015 ml, 14.458 mmol) at 0°C was added chlorotriethylsilane (1090 mg, 7.229 mmol) and N, N-dimethylpyridin-4-amine (177 mg, 1.446 mmol). The resulting mixture was then warmed to room temperature and stirred for 16 hours. After stirring, the mixture was diluted with DCM (100 ml) and the mixture was washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting precipitate was combined with AcOEt (100 ml) and amino-functionalized silica (50 g). The resulting suspension was stirred at room temperature for 16 hours and then filtered. The filtrate was washed with AcOEt/MeOH (9/1, 150 ml). The combined stock solution was concentrated in vacuo and the isolated residue was purified by silica gel chromatography (40 g, heptane/AcOEt=70/30 to 0/100) to give the title compound as a colorless solid (807 mg, quant.) .

[655] ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.60 (q, J=7.8 Hz, 6 H) 0.67 (d, J=6.3 Hz, 3 H) 0, 76 (d, J=7.32 Hz, 3 H) 0.95 (t, J=7.8 Hz, 9 H) 1.04 (td, J=3.3, 1.7 Hz, 6 H) 1.17-1.20 (m, 1 H) 1.24 (s, 3 H) 1.34-1.46 (m, 2 H) 1.50-1.58 (m, 1 H) 1, 61 (s, 3 H) 1.70 (s, 3 H) 1.82-1.92 (m, 2 H) 2.33-2.45 (m, 3 H) 2.63 (d, J= 9.27 Hz, 1 H) 3.05-3.09 (m, 1 H), 3.34 (d, J=9.76 Hz, 1 H) 3.50 (s, 3 H) 3.62 -3.70 (m, 1 H) 3.85 (t, J=6.59 Hz, 1 H) 5.29 (br. s., 1 H) 5.48 (dd, J=14.88, 8.54 Hz, 1 H) 5.90 (d, J=10.73 Hz, 1 H) 6.21 (dd, J=14.64, 11.22 Hz, 1 H) 6.63 (br. s., 1 H).

[656] Step 3. Allyl-(((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-3 -methoxy-4-((triethylsilyl)oxy)pentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran- 2-yl)methyl)carbamate

[657] A mixture of the compound isolated in step 2 (807 mg, 1.462 mmol) and iodobenzene diacetate (1413 mg, 4.387 mmol) in allyl alcohol (20 ml) was heated to 60°C and stirred for 3 hours. The mixture was then cooled to room temperature and AcOEt and aqueous sodium bicarbonate (100 ml each) were added thereto. The organic phase was isolated and the aqueous phase was extracted with AcOEt. The combined organic extracts were washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was purified by silica gel chromatography (40 g, heptane/AcOEt = 90/10 to 50/50) to give the title compound as a colorless oil (663 mg, 75% yield).

[658] ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.55-0.67 (m, 9 H) 0.77 (d, J=6.7 Hz, 3 H) 0.87 (t , J=6.5 Hz, 2 H) 0.95 (t, J=7.8 Hz, 9 H) 1.04 (t, J=5.5 Hz, 6 H) 1.16-1.32 (m, 9 H) 1.39-1.53 (m, 2 H) 1.58 (s, 3 H) 1.69 (s, 3 H) 1.80-1.91 (m, 2 H) 2.40 (br. s, 1 H) 2.64 (d, J=9.27 Hz, 1 H) 3.01-3.10 (m, 2 H) 3.26 (d, J=10, 24 Hz, 1 H) 3.35-3.44 (m, 2 H) 3.50 (s, 3 H) 3.85 (t, J=6.5 Hz, 1 H) 4.54 (d, J=5.37 Hz, 2 H) 5.12 (br. s., 1 H) 5.19 (dt, J=10.49, 1.10 Hz, 1 H) 5.25-5.33 ( m, 1 H) 5.45 (dd, J=15.12, 8.78 Hz, 1 H) 5.85-5.97 (m, 2 H) 6.22 (dd, J=14.88, 10.98 Hz, 1 H).

[659] Step 4. ((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-3-Methoxy- 4-((triethylsilyl)oxy)pentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl )methanamine

[660] To a mixture of the compound obtained in step 3 (663 mg, 1.091 mmol) and THF (15 ml, 183.065 mmol) was added a borane-dimethylamine complex (643 mg, 10.906 mmol). After purging the container with nitrogen, tetrakis(triphenylphosphine)palladium(0) (37.8 mg, 0.033 mmol) was added and the mixture was stirred at room temperature for 1 hour. The mixture was then diluted with AcOEt and saturated aqueous sodium bicarbonate (100 ml each) and the phases were separated. The aqueous layer was extracted with AcOEt (50 ml x 3) and then the combined organic fractions were washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was purified by chromatography on amino-functionalized silica gel (40 g, heptane/AcOEt = 70/30 to 0/100) to give the title compound as a white solid (525 mg, 92% yield).

[661] ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.60 (q, J=7.80 Hz, 6 H) 0.65 (d, J=6.8 Hz, 3 H) 0, 77 (d, J=6.83 Hz, 3 H) 0.95 (t, J=7.8 Hz, 9 H) 1.03 (dd, J=9.0, 6.6 Hz, 6 H) 1.14-1.21 (m, 1 H) 1.23 (s, 3 H), 1.30-1.34 (m, 1 H) 1.42 (ddd, J=9.4, 7, 2.7 Hz, 1 H) 1.48-1.59 (m, 6 H) 1.70 (s, 3 H) 1.81-1.91 (m, 2 H) 2.34-2.44 (m, 1 H) 2.62-2.74 (m, 3 H) 3.06 (dd, J=6.83, 2.93 Hz, 1 H) 3.25-3.31 (m, 2 H) 3.50 (s, 3 H) 3.81-3.89 (quin, J=6.5 Hz, 1 H) 5.44 (dd, J=15.12, 8.78 Hz, 1 H ) 5.88 (d, J=10.73 Hz, 1 H) 6.22 (dd, J=15.12, 10.73 Hz, 1 H).

[662] Step 5. 6-Hydroxy-N-(((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R, 4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2- yl)methyl)-N-methylhexanamide

[663] To a mixture of the compound isolated in step 5 (133 mg, 0.254 mmol) and 6-methoxy-6-oxohexanoic acid (0.056 ml, 0.381 mmol) in THF (2 ml) was added HATU (145 mg, 0.381 mmol) and the mixture was then stirred at room temperature for 16 hours. AcOEt was then added and the resulting mixture was washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was combined with THF (2 ml) and cooled to 0°C. Lithium borohydride (22.1 mg, 1.016 mmol) was then added and the mixture was stirred at the same temperature for 30 minutes. The mixture was then warmed to room temperature and stirred for an additional 1 hour. A saturated aqueous solution of ammonium chloride was added to the mixture, followed by the addition of AcOEt and water. The organic phase was isolated, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting precipitate was then dissolved in THF (5 mL, 61.02 mmol) and TBAF (1 M solution in THF, 0.508 mL, 0.508 mmol) was added at 0°C. The resulting mixture was stirred at 0°C for 30 minutes, then warmed to room temperature, followed by stirring at room temperature for an additional 2 hours. The mixture was then diluted with AcOEt and the organic phase was isolated, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was purified by silica gel chromatography (40 g silica, 0-100% heptane/EtOAc) to give the title compound as a colorless oil (113 mg, 85% yield).

[664] ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.64 (d, J=6.3 Hz, 3 H) 0.84 (d, J=6.8 Hz, 3 H) 1, 03 (d, J=6.3 H, 3 H) 1.14 (d, J=6.3 H, 3 H) 1.17-1.21 (m, 3 H) 1.25 (s, 3 H) 1.27-1.40 (m, 3 H) 1.46-1.60 (m, 6 H) 1.60-1.67 (m, 2 H) 1.69 (s, 3 H) 1.77-1.92 (m, 3 H) 2.15 (t, J=7.3 Hz, 2 H) 2.40 (br. s, 1 H) 2.53 (d, J=9, 76 Hz, 2 H) 2.92-3.02 (m, 2 H) 3.26 (d, J=9.76 Hz, 1 H) 3.34-3.45 (m, 1 H) 3, 50 (s, 3 H) 3.52-3.62 (m, 3 H) 3.80-3.83 (m, 1 H) 5.45 (dd, J=15.12, 8.78 Hz, 1 H) 5.89 (d, J=10.73 Hz, 1 H) 5.96 (br. s, 1 H) 6.23 (dd, J=14.88, 10.98 Hz, 1 H) .

[665] Step 6. General procedure for carbamate synthesis

[666] DMAP ( 0.5 eq.). The resulting mixture was stirred at room temperature for 16 hours. Amine (2.0 eq) was then added and the mixture was stirred for an additional 1 hour. The mixture was then diluted with dichloromethane and the organic layer was isolated, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was then purified by amino-functionalized silica gel chromatography to give the desired compound.

1.3.2 Synthesis of ADL1-H5

1.3.2.1 H5 synthesis

[667] 6-((((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-Hydroxy -3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl)methyl)amino) -6-oxohexylpiperazine-1-carboxylate (H5)

[668] Steps 1-6 in section 1.3.1 provided the title compound as a colorless oil (24.5 mg, 71% yield). LC/MS (ESI, mass/charge), 636.89 [M+H] ⁺ .

[669] ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.65 (d, J=6.34 Hz, 3 H) 0.85 (d, J=6.83 Hz, 3 H) 0, 95 (dd, J=16.8, 6.6 Hz, 1 H) 1.03 (d, J=6.34 Hz, 3 H) 1.15 (d, J=6.34 Hz, 3 H) 1.18-1.25 (m, 2 H) 1.26 (s, 3 H) 1.29-1.40 (m, 3 H) 1.47-1.69 (m, 8 H) 1, 70 (s, 3 H) 1.79-1.92 (m, 2 H) 1.92-2.07 (m, 4 H) 2.15 (t, J=7.56 Hz, 2 H) 2 ,36-2.45 (m, 1 H) 2.54 (d, J=9.27 Hz, 1 H) 2.80 (br. s., 4 H) 2.89-3.04 (m, 2 H) 3.27 (d, J=10.24 Hz, 1 H) 3.35-3.46 (m, 5 H) 3.51 (s, 3 H) 3.52-3.58 (m , 2 H) 3.83 (t, J=6.34 Hz, 1 H) 4.05 (t, J=6.34 Hz, 2 H) 5.46 (dd, J=15.12, 8, 78 Hz, 1 H) 5.82-5.95 (m, 2 H) 6.24 (dd, J=15.12, 10.73 Hz, 1 H).

1.3.2.2 Synthesis of ADL1-H5

[670] 1-(4-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3 -methylbutanamido)-5-ureidopentanamido)benzyl)-4-(6-((((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-( (2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H -pyran-2-yl)methyl)amino)-6-oxohexyl)piperazine-1,4-dicarboxylate (ADL1-H5)

[671] The procedure specified in section 1.2.7 was performed to obtain the title compound as a colorless oil (25.8 mg, 54% yield).

[672] ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ ppm 0.65 (d, J=6.83 Hz, 3 H) 0.80 (d, J=6.83 Hz, 3 H) 0 .94 (dd, J=6.34, 4.39 Hz, 7 H) 1.01 (d, J=6.34 Hz, 3 H) 1.07 (d, J=6.30 Hz, 4 H ) 1.11-1.38 (m, 14 H) 1.45-1.66 (m, 14 H) 1.69 (s, 3 H) 1.70-1.79 (m, 1 H) 1 .80-1.92 (m, 3 H) 2.00-2.09 (m, 1 H) 2.17 (t, J=7.07 Hz, 2 H) 2.25 (t, J=7 .32 Hz, 3 H) 2.42 (br. s., 1 H) 2.63 (d, J=9.76 Hz, 1 H) 2.95 (dd, J=5.85, 4.39 Hz, 1 H) 3.02-3.11 (m, 2 H) 3.17 (d, J=6.83 Hz, 1 H) 3.37-3.48 (m, 14 H) 3.50 (s, 4 H) 3.53 (t, J=4.50 Hz, 2 H) 3.63-3.67 (m, 2 H) 3.76 (t, J=6.34 Hz, 1 H ) 4.05 (t, J=6.34 Hz, 3 H) 4.14 (d, J=7.32 Hz, 1 H) 4.48 (dd, J=8.78, 4.88 Hz, 1 H) 5.06 (s, 2 H) 5.46 (dd, J=15.12, 8.78 Hz, 1 H) 5.90 (d, J=10.73 Hz, 1 H) 6, 20-6.38 (m, 1 H) 6.76 (s 2 H) 7.30 (d, J=8.29 Hz, 2 H) 7.57 (d, J=8.29 Hz, 2 H ).

1.3.3 Synthesis of ADL1-H6

1.3.3.1 H6 synthesis

[673] 6-((((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-Hydroxy -3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl)methyl)amino) -6-oxohexyl-1,4-diazepane-1-carboxylate (H6)

[674] Steps 1-6 in section 1.3.1 provided the title compound as a colorless oil (15.3 mg, 43% yield). LC/MS (ESI, mass/charge), 650.92[M+H] ⁺ .

[675] ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.65 (d, J=6.34 Hz, 3 H) 0.85 (d, J=6.34 Hz, 3 H) 0, 93-1.00 (m, 1 H) 1.03 (d, J=6.83 Hz, 3 H) 1.16 (d, J=6.83 Hz, 3 H) 1.18-1.24 (m, 2 H) 1.27 (s, 3 H) 1.29-1.41 (m, 3 H) 1.49-1.55 (m, 2 H) 1.59-1.68 (m , 5 H) 1.71 (s, 3 H) 1.73-1.92 (m, 7 H) 2.16 (t, J=7.56 Hz, 2 H) 2.36-2.46 ( m, 1 H) 2.54 (d, J=9.27 Hz, 1 H) 2.81-2.93 (m, 4 H) 2.93-3.04 (m, 2 H) 3.27 (d, J=9.76 Hz, 1 H) 3.37-3.49 (m, 4 H) 3.52 (s, 3 H) 3.54-3.59 (m, 2 H) 3, 79-3.88 (m, 1 H) 4.05 (t, J=6.34 Hz, 2 H) 5.46 (dd, J=15.12, 8.78 Hz, 1 H) 5.82 -5.96 (m, 2 H) 6.24 (dd, J=15.12, 10.73 Hz, 1 H).

1.3.3.2 Synthesis of ADL1-H6

[676] 1-(4-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3 -methylbutanamido)-5-ureidopentanamido)benzyl)-4-(6-((((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-( (2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H -pyran-2-yl)methyl)amino)-6-oxohexyl)-1,4-diazepan-1,4-dicarboxylate (ADL-H6)

[677] The procedure specified in section 1.2.7 was performed to obtain the title compound as a colorless oil (11.6 mg, 52% yield).

[678] ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ ppm 0.65 (d, J=6.34 Hz, 3 H) 0.80 (d, J=6.83 Hz, 2 H) 0 .94 (dd, J=6.83, 4.39 Hz, 6 H) 0.98-1.16 (m, 9 H) 1.19-1.36 (m, 9 H) 1.44-1 .65 (m, 14 H) 1.69 (s, 3 H) 1.71-1.78 (m, 4 H) 1.80-1.92 (m, 3 H) 2.00-2.10 (m, 1 H) 2.11-2.19 (m, 2 H) 2.25 (t, J=7.56 Hz, 3 H) 3.03-3.21 (m, 4 H) 3, 36-3.49 (m, 10 H) 3.49-3.51 (m, 3 H) 3.51-3.57 (m, 12 H) 3.62-3.67 (m, 8 H) 3.70-3.82 (m, 1 H) 3.97-4.05 (m, 2 H) 4.14 (d, J=7.32 Hz, 1 H) 4.46-4.52 ( m, 1 H) 5.00-5.08 (m, 3 H) 5.35-5.52 (m, 1 H) 5.35-5.52 (m, 1 H) 5.87-5, 95 (m, 1 H) 6.24-6.33 (m, 1 H) 6.76 (s, 2 H) 7.26-7.37 (m, 3 H) 7.57 (d, J= 7.32 Hz, 3 H) 8.19-8.24 (m, 1 H).

1.3.4 Synthesis of ADL1-H7

1.3.4.1 H7 synthesis

[679] 6-((((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-Hydroxy -3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl)methyl)amino) -6-oxohexyl-3-(methylamino)pyrrolidine-1-carboxylate (H7)

[680] Steps 1-6 in section 1.3.1 provided the title compound as a colorless oil (26.6 mg, 76% yield). LC/MS (ESI, mass/charge), 650.88[M+H] ⁺ .

[681] ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.65 (d, J=6.34 Hz, 3 H) 0.85 (d, J=6.34 Hz, 3 H) 0, 93-0.99 (m, 1 H) 1.04 (d, J=6.34 Hz, 3 H) 1.16 (dd, J=6.34, 0.98 Hz, 3 H) 1.19 -1.22 (m, 2 H) 1.27 (s, 3 H) 1.31-1.41 (m, 3 H) 1.47-1.67 (m, 8 H) 1.70 (s , 4 H) 1.80-1.87 (m, 2 H) 1.89 (d, J=3.90 Hz, 1 H) 2.03 (dd, J=12.68, 6.34 Hz, 1 H) 2.15 (t, J=7.32 Hz, 2 H) 2.41 (m, 3 H) 2.54 (d, J=9.76 Hz, 1 H) 2.94-3, 13 (m, 2 H) 3.20 (m, 1 H) 3.27 (d, J=9.76 Hz, 1 H) 3.35-3.50 (m, 3 H) 3.51 (s , 3 H) 3.53-3.60 (m, 2 H) 3.79-3.88 (m, 1 H) 4.04 (t, J=6.34 Hz, 2 H) 5.46 ( dd, J=15.12, 8.78 Hz, 1 H) 5.82-5.96 (m, 2 H) 6.24 (dd, J=14.88, 10.98 Hz, 1 H).

1.3.4.2 Synthesis of ADL1-H7

[682] 6-((((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-Hydroxy -3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl)methyl)amino) -6-oxohexyl-3-((((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl )hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)pyrrolidine-1-carboxylate (ADL1-H7)

[683] The procedure specified in section 1.2.7 was used (the mixture was stirred for 16 hours instead of 2 hours) to obtain the title compound as a colorless oil (20.1 mg, 54% yield).

[684] ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ ppm 0.65 (d, J=6.30 Hz, 3 H) 0.80 (d, J=6.83 Hz, 3 H) 0 .94 (dd, J=6.59, 4.63 Hz, 7 H) 1.01 (d, J=6.34 Hz, 3 H) 1.08 (d, J=6.34 Hz, 4 H ) 1.11-1.23 (m, 4 H) 1.23-1.27 (m, 6 H) 1.27-1.38 (m, 5 H) 1.43-1.65 (m, 14 H) 1.69 (s, 3 H) 1.70-1.77 (m, 1 H) 1.80-1.92 (m, 3 H) 2.00-2.09 (m, 3 H ) 2.17 (t, J=7.32 Hz, 2 H) 2.25 (t, J=7.30 Hz, 3 H) 2.38-2.46 (m, 1 H) 2.63 ( d, J=9.27 Hz, 1 H) 2.83 (s, 3 H) 2.84 (s, 3 H) 2.92-2.96 (m, 1 H) 2.97 (s, 3 H) 3.02-3.21 (m, 4 H) 3.30-3.42 (m, 3 H) 3.45 (t, J=7.07 Hz, 3 H) 3.50 (s, 3 H) 3.51-3.58 (m, 3 H) 3.72-3.80 (m, 1 H) 4.03 (t, J=6.34 Hz, 2 H) 4.15 (d , J=7.32 Hz, 1 H) 4.49 (dd, J=9.03, 5.12 Hz, 1 H) 5.06 (s, 2 H) 5.46 (dd, J=14, 88, 9.03 Hz, 1 H) 5.90 (d, J=11.22 Hz, 1 H) 6.28 (dd, J=14.88, 10.98 Hz, 1 H) 6.76 ( s, 2 H) 7.30 (d, J=5.90 Hz, 2 H) 7.57 (d, J=8.29 Hz, 2 H) 7.95 (s, 1 H).

1.4 Synthesis of ADL1-H8, ADL1-H9 and ADL1-H10

1.4.1 Brief description. General procedure 4

Scheme 4

[685] Step 1. tert-Butyl-3-hydroxypropanoate

[686] To a mixture of sodium hydride (269 mg, 6.157 mmol) in DMF (30 ml) was added tert-butyl-3-hydroxypropanoate (0.909 ml, 6.157 mmol) at 0°C. The mixture was stirred at 0°C for 1 hour and then methyl bromoacetate (0.624 ml, 6.157 mmol) was added dropwise. The resulting mixture was warmed to room temperature and stirred at room temperature for 16 hours. Saturated aqueous ammonium chloride was then added and the mixture was extracted with AcOEt. The combined organic extracts were then washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The isolated residue was purified by silica gel chromatography (80 g, heptane/AcOEt = 80/20 to 50/50) to give the title compound (97 mg, 7% yield) as a colorless oil.

[687] ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.42 (s, 9 H) 2.49-2.58 (m, 2 H) 3.41 (s, 3 H) 3.69 -3.79 (m, 2 H) 4.05-4.13 (m, 2 H).

[688] Step 2: 3-(2-Methoxy-2-oxoethoxy)propanoic acid

[689] A mixture of tert-butyl 3-(2-methoxy-2-oxoethoxy)propanoate (66 mg, 0.302 mmol) in dichloromethane (2 ml) and TFA (2 ml) was stirred at room temperature for 3 hours. The mixture was then concentrated to dryness to obtain the title compound as a colorless oil (58 mg, 100%).

[690] ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 2.62-2.78 (m, 2 H) 3.76 (s, 3 H) 3.78-3.85 (m, 2 H ) 4.14 (d, J=1.95 Hz, 2 H).

[691] Stage 3. N-(((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4 -Hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl)methyl) -3-(2-hydroxyethoxy)propenamide

[692] To the mixture ((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-3-methoxy-4 -((triethylsilyl)oxy)pentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl) methanamine (133 mg, 0.254 mmol) and 3-(2-methoxy-2-oxoethoxy)propanoic acid (61.7 mg, 0.381 mmol) in THF (5 ml) added HATU (145 mg, 0.381 mmol) and DIPEA (221 µl, 1.27 mmol) at room temperature. The mixture was stirred for 16 hours and then diluted with AcOEt. The organic layer was isolated, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo.

[693] The isolated residue was dissolved in THF (5 ml) and cooled to 0°C and then lithium borohydride (22.12 mg, 1.016 mmol) was added. The resulting mixture was stirred at the same temperature for 30 minutes and then warmed to room temperature and stirred for another 2 hours. Then, saturated aqueous ammonium chloride and water were added, and the aqueous layer was extracted with AcOEt. The combined organic layers were washed with water and brine, dried over sodium sulfate, and concentrated in vacuo. The isolated residue was then dissolved in THF (5 ml) and TBAF (1 M solution in THF, 0.508 ml, 0.508 mmol) was added at 0°C. After stirring at 0°C for 30 minutes, the mixture was warmed to room temperature and stirred for 2 hours. The mixture was then diluted with AcOEt and the organic layer was isolated, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was purified by silica gel chromatography (24 g, heptane/AcOEt = 70/30 to 0/100, then AcOEt/MeOH = 80/20) to give the title compound (68 mg, 51% yield) as colorless oil.

[694] ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.65 (d, J=6.34 Hz, 3 H) 0.85 (d, J=6.83 Hz, 3 H) 1, 04 (d, J=6.34 Hz, 3 H) 1.16 (d, J=6.8 Hz, 4 H) 1.19-1.22 (m, 2 H) 1.27 (s, 3 H) 1.42-1.65 (m, 4 H) 1.71 (s, 3 H) 1.79-1.92 (m, 2 H) 2.37-2.50 (m, 4 H) 2.54 (d, J=9.27 Hz, 2 H) 2.89-3.05 (m, 2 H) 3.28 (d, J=10.24 Hz, 1 H) 3.41 (td , J=8.54, 2.44 Hz, 1 H) 3.52 (s, 3 H) 3.54-3.59 (m, 2 H) 3.61-3.68 (m, 2 H) 3.69-3.76 (m, 2 H) 3.78-3.91 (m, 2 H) 5.43-5.52 (m, 1 H) 5.92 (d, J=10.73 Hz, 1 H) 6.24 (dd, J=15.12, 10.73 Hz, 1 H) 6.77 (m, 1 H).

[695] Step 4: Synthesis of carbamate

[696] To the mixture N-(((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4- hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl)methyl)- 3-(2-hydroxyethoxy)propanamide (1.0 eq, 0.043 mmol), 4-nitrophenylchloroformate (2.0 eq, 0.086 mmol) and DIPEA (5.0 eq) in DCM (0.04 M) were added DMAP (5.0 eq.) at room temperature. The mixture was stirred for 16 hours at room temperature and then piperazine (10.0 eq.) was added and the mixture was stirred for another 1 hour. The resulting mixture was then diluted with dichloromethane and the organic layer was isolated, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was purified by amino-functionalized silica gel chromatography (heptane/AcOEt=50/50 to 0/100) to give the desired compound.

1.4.2 Synthesis of ADL1-H8

1.4.2.1 H8 synthesis

[697] 2-(3-((((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)- 4-Hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl)methyl )amino)-3-oxopropoxy)ethylpiperazine-1-carboxylate (H8)

[698] Steps 1-4 in section 1.4.1 provided the title compound as a pale yellow oil (19.2 mg, 70% yield). LC/MS (ESI, mass/charge), 638.78[M+H] ⁺ .

[699] ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.65 (d, J=6.83 Hz, 3 H) 0.85 (d, J=6.83 Hz, 3 H) 0, 96 (dd, J=18.54, 6.34 Hz, 1 H) 1.03 (d, J=5.85 Hz, 3 H) 1.16 (d, J=5.85 Hz, 4 H) 1.18-1.25 (m, 2 H) 1.27 (s, 3 H) 1.29-1.39 (m, 1 H) 1.41-1.63 (m, 4 H) 1, 70 (s, 3 H) 1.77-1.97 (m, 7 H) 2.44 (t, J=5.61 Hz, 2 H) 2.47-2.57 (m, 1 H) 2 .80 (br. s., 4 H) 2.85-2.99 (m, 1 H) 2.99-3.09 (m, 1 H) 3.27 (d, J=9.76 Hz, 1 H) 3.36-3.48 (m, 5 H) 3.52 (s, 3 H) 3.54-3.59 (m, 1 H) 3.59-3.68 (m, 2 H ) 3.68-3.74 (m, 2 H) 3.77-3.90 (m, 1 H) 4.15-4.23 (m, 2 H) 4.36 (t, J=6, 10 Hz, 1 H) 5.44 (dd, J=14.88, 8.54 Hz, 1 H) 5.88 (d, J=10.73 Hz, 1 H) 6.23 (dd, J= 14.88, 10.98 Hz, 1 H) 6.41 (br. s., 1 H).

1.4.2.2 H8 synthesis

[700] 1-(4-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3 -methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(3-((((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)- 3-((2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5- methyltetrahydro-2H-pyran-2-yl)methyl)amino)-3-oxopropoxy)ethyl)piperazine-1,4-dicarboxylate (ADL1-H8)

[701] A similar procedure to section 1.2.7 was performed to obtain the title compound as a colorless oil (18.3 mg, 60% yield).

[702] ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ ppm 0.65 (d, J=6.30 Hz, 3 H) 0.80 (d, J=6.83 Hz, 3 H) 0 .94 (dd, J=6.59, 4.15 Hz, 7 H) 1.01 (d, J=6.83 Hz, 3 H) 1.08 (d, J=6.34 Hz, 3 H ) 1.10-1.23 (m, 4 H) 1.25 (s, 3 H) 1.26-1.32 (m, 3 H) 1.44-1.65 (m, 10 H) 1 .68 (s, 3 H) 1.70-1.75 (m, 1 H) 1.80-1.92 (m, 3 H) 2.00-2.09 (m, 1 H) 2.25 (t, J=7.56 Hz, 2 H) 2.41 (t, J=5.85 Hz, 2 H) 2.63 (d, J=9.27 Hz, 1 H) 2.95 (dd , J=6.34, 4.39 Hz, 1 H) 3.08 (dt, J=13.05, 6.40 Hz, 1 H) 3.14-3.21 (m, 1 H) 3, 38-3.48 (m, 14 H) 3.49 (s, 4 H) 3.58-3.65 (m, 2 H) 3.69 (t, J=6.10 Hz, 2 H) 3 .72-3.80 (m, 1 H) 4.13-4.19 (m, 3 H) 4.48 (dd, J=9.02, 5.12 Hz, 1 H) 5.07 (s , 2 H) 5.46 (dd, J=15.12, 9.27 Hz, 1 H) 5.90 (d, J=10.24 Hz, 1 H) 6.28 (dd, J=14, 88, 10.98 Hz, 1 H) 6.76 (s, 2 H) 7.30 (d, J=8.78 Hz, 2 H) 7.57 (d, J=7.25 Hz, 2 H ).

1.4.3 Synthesis of ADL1-H9

1.4.3.1 H9 synthesis

[703] 2-(3-((((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)- 4-Hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl)methyl )amino)-3-oxopropoxy)ethyl-1,4-diazepane-1-carboxylate (H9)

[704] Steps 1-4 in section 1.4.1 provided the title compound as a colorless oil (21.1 mg, 75% yield). LC/MS (ESI, mass/charge), 652.86[M+H] ⁺ .

[705] ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.65 (d, J=6.83 Hz, 3 H) 0.85 (d, J=7.32 Hz, 3 H) 0, 92-1.00 (m, 1 H) 1.03 (d, J=6.8 H, 3 H) 1.16 (d, J=6.8 H, 3 H) 1.20-1.24 (m, 1 H) 1.27 (s, 4 H) 1.47-1.60 (m, 4 H) 1.70 (s, 3 H) 1.73-1.90 (m, 8 H) 2.37-2.47 (m, 3 H) 2.53 (d, J=9.8 Hz, 1 H) 2.81-2.86 (m, 2 H) 2.87-2.92 ( m, 2 H) 2.94-2.97 (m, 1 H) 2.99-3.07 (m, 1 H) 3.27 (d, J=9.76 Hz, 1 H) 3.38 -3.50 (m, 4 H) 3.52 (s, 3 H) 3.55-3.59 (m, 1 H) 3.61-3.68 (m, 2 H) 3.68-3 .74 (m, 2 H) 3.77-3.91 (m, 1 H) 4.19 (br. s., 2 H) 4.37 (t, J=6.10 Hz, 1 H) 5 ,41-5.50 (m, 1 H) 5.89 (d, J=11.22 Hz, 1 H) 6.23 (dd, J=14.88, 10.98 Hz, 1 H) 6, 46 (dd, J=3.17, 1.22 Hz, 1 H).

1.4.3.2 Synthesis of ADL1-H9

[706] 1-(4-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3 -methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(3-((((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)- 3-((2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5- methyltetrahydro-2H-pyran-2-yl)methyl)amino)-3-oxopropoxy)ethyl)-1,4-diazepan-1,4-dicarboxylate (ADL1-H9)

[707] A similar procedure to section 1.2.7 was performed to give the title compound (25.2 mg, 72% yield).

[708] ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ ppm 0.65 (d, J=6.30 Hz, 3 H) 0.80 (d, J=6.83 Hz, 3 H) 0 .93 (t, J=5.85 Hz, 7 H) 1.01 (d, J=6.30 Hz, 3 H) 1.08 (d, J=6.30 Hz, 3 H) 1.11 -1.21 (m, 2 H) 1.21-1.32 (m, 7 H) 1.43-1.65 (m, 10 H) 1.69 (s, 3 H) 1.70-1 .91 (m, 5 H) 2.05 (q, J=6.8 Hz, 1 H) 2.25 (t, J=7.32 Hz, 2 H) 2.36-2.48 (m, 3 H) 2.63 (d, J=9.27 Hz, 1 H) 2.95 (dd, J=5.85, 4.39 Hz, 1 H) 3.03-3.13 (m, 2 H) 3.13-3.21 (m, 1 H) 3.37-3.48 (m, 7 H) 3.48-3.59 (m, 8 H) 3.62-3.70 (m , 2 H) 3.76 (quin, J=6.46 Hz, 1 H) 4.11-4.22 (m, 2 H) 4.47-4.53 (m, 1 H) 5.02- 5.09 (m, 2 H) 5.45 (dd, J=14.88, 9.03 Hz, 1 H) 5.90 (d, J=10.73 Hz, 1 H) 6.28 (dd , J=14.64, 11.22 Hz, 1 H) 6.76 (s, 2 H) 7.26-7.37 (m, 2 H) 7.57 (d, J=7.81 Hz, 2 H).

1.4.4 Synthesis of ADL1-H10

1.4.4.1 H10 synthesis

[709] 2-(3-((((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)- 4-Hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl)methyl )amino)-3-oxopropoxy)ethyl-3-(methylamino)pyrrolidine-1-carboxylate (H10)

[710] Steps 1-4 in section 1.4.1 provided the title compound as a colorless oil (24.8 mg, 88% yield). LC/MS (ESI, mass/charge), 652.91 [M+H] ⁺ .

[711] ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.65 (d, J=6.34 Hz, 3 H) 0.85 (d, J=6.83 Hz, 3 H) 0, 96 (dd, J=18.78, 6.59 Hz, 1 H) 1.03 (d, J=6.8 Hz, 3 H) 1.16 (d, J=6.34 Hz, 3 H) 1.18-1.25 (m, 3 H) 1.27 (s 3 H) 1.20-1.32 (m, 5 H) 1.41-1.63 (m, 4 H) 1.70 (s, 3 H) 1.72-1.90 (m, 4 H) 2.02 (dd, J=12.44, 6.10 Hz, 2 H) 2.41 (s 3 H) 2.43 -2.50 (m, 2 H)2.54 (d, J=9.76 Hz, 1 H) 2.95 (t, J=5.37 Hz, 1 H) 3.01-3.14 ( m, 1 H) 3.18-3.24 (m, 1 H) 3.27 (d, J=9.7 Hz, 1 H) 3.38-3.49 (m, 2 H) 3.52 (s, 6 H) 3.61-3.65 (m, 2 H) 3.69-3.74 (m, 2 H) 3.83 (quin, J=5.98 Hz, 1 H) 4, 15-4.22 (m, 1 H) 4.34 (t, J=6.10 Hz, 1 H) 5.44 (dd, J=15.12, 8.78 Hz, 1 H) 5.88 (d, J=11.22 Hz, 1 H) 6.23 (dd, J=14.88, 10.98 Hz, 1 H) 6.43 -6.51 (m, 1 H).

1.4.4.2 Synthesis of ADL1-H10

[712] A similar procedure to section 1.2.7 was performed to obtain the title compound as a colorless oil (22.1 mg, 57% yield).

[713] 1H NMR (400 MHz, METHANOL-d4) δ ppm 0.65 (d, J=6.34 Hz, 3 H) 0.80 (d, J=6.83 Hz, 3 H) 0.94 (dd, J=6.34, 4.88 Hz, 8 H) 1.01 (dd, J=3.90, 2.93 Hz, 4 H) 1.08 (d, J=6.30 Hz, 3 H) 1.11-1.22 (m, 3 H) 1.22-1.31 (m, 7 H) 1.44-1.65 (m, 10 H) 1.68 (s 3 H) 1.71-1.91 (m, 4 H) 1.99-2.09 (m, 3 H) 2.25 (t, J=7.56 Hz, 2 H) 2.41 (t, J= 5.85 Hz, 2 H) 2.62 (d, J=9.27 Hz, 1 H) 2.84 (m, 4 H) 2.93-2.99 (m, 2 H) 3.04- 3.13 (m, 2 H) 3.13-3.21 (m, 1 H) 3.43 (t, J=7.07 Hz, 2 H) 3.50 (s, 3 H) 3.52 -3.56 (m, 2 H) 3.61 (t, J=4.39 Hz, 2 H) 3.69 (t, J=5.37 Hz, 2 H) 3.73-3.79 ( m, 1 H) 4.15 (d, J=7.32 Hz, 3 H) 4.49 (dd, J=9.03, 5.12 Hz, 1 H) 5.06 (s, 2 H) 5.45 (dd, J=14.88, 9.03 Hz, 1 H) 5.90 (d, J=11.22 Hz, 1 H) 6.28 (dd, J=14.88, 10, 98 Hz, 1 H) 6.76 (s, J=4.56 Hz, 2 H) 7.30 (d, J=8.29 Hz, 2 H) 7.57 (d, J=8.29 Hz , 2H).

1.5 Synthesis of ADL2-H1

[714] 2-((2R,4R,5S,6S)-4-((4-(3-(2-(2-(2,5-Dioxo-2,5-dihydro-1H-pyrrole-1- yl)ethoxy)ethoxy)propanoyl)piperazine-1-carbonyl)oxy)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4- hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl)acetic acid ( ADL2-H1)

[715] To a mixture of H1 (see, for example, section 1.2.2; 22.5 mg, 0.03 mmol) in DMF (1 ml) was added 2,5-dioxopyrrolidin-1-yl-3-{2-[ 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}propanoate (10.55 mg, 0.03 mmol) and DIPEA (0.016 ml, 0.089 mmol). The resulting mixture was stirred at room temperature for 4 hours. The solvent was then removed in vacuo and the resulting residue was purified by reverse phase chromatography (ODS, 24 g, H ₂ O/MeCN = 95/5 to 60/40) to give the title compound as a colorless oil (15.7 mg, yield 65%).

[716] ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ ppm 0.73 (d, J=6.34 Hz, 4 H) 0.97-1.15 (m, 13 H) 1.21 ( s, 2 H) 1.28-1.41 (m, 1 H) 1.42-1.62 (m, 2 H) 1.69 (d, J=4.39 Hz, 4 H) 2.08 -2.20 (m, 1 H) 2.33-2.39 (m, 1 H) 2.42-2.58 (m, 3 H) 2.63 (t, J=5.30 Hz, 2 H) 2.91-2.94 (m, 1 H) 3.33 (d, J=3.41 Hz, 1 H) 3.36 (d, J=1.95 Hz, 2 H) 3.44 -3.60 (m, 18 H) 3.60-3.67 (m, 4 H) 3.69 (t, J=5.37 Hz, 2 H) 3.75-3.78 (m, 1 H) 3.81-4.00 (m, 2 H) 4.22-4.31 (m, 1 H) 4.52-4.60 (m, 1 H) 5.58-5.72 (m , 1 H) 5.89-6.00 (m, 1 H) 6.13-6.31 (m, 1 H) 6.71-6.89 (m, 2 H).

1.6 Synthesis of ADL2-H11

[717] Step 1. Synthesis of (2R,3R,4R)-4-((2R,3R)-3-((S,3E,5E)-6-((2S,3S,6R)-6-(aminomethyl )-3-methyltetrahydro-2H-pyran-2-yl)-2-methylhepta-3,5-dien-1-yl)-3-methyloxiran-2-yl)-3-methoxypentan-2-ol (H11)

[718] To the mixture ((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-3-methoxy-4 -((triethylsilyl)oxy)pentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl) methanamine (32 mg, 0.061 mmol) in THF (1 ml) was added TBAF (1 M solution in THF, 0.183 ml, 0.183 mmol). The resulting mixture was stirred at room temperature for 2 hours and then diluted with AcOEt. The organic phase was isolated, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The isolated residue was purified by chromatography on amino-functionalized silica gel (24 g, heptane/AcOEt = 50/50 to 0/100, then AcOEt/MeOH = 80/20) to give the title compound as a colorless oil (15, 1 mg, yield 60.4%). LC/MS (ESI, mass/charge), 410.07 [M+H] ⁺ .

[719] ¹ H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.65 (d, J=6.80 Hz, 3 H) 0.86 (d, J=6.80 Hz, 3 H) 1, 03 (d, J=6.80 Hz, 3 H) 1.16 (d, J=6.30 Hz, 4 H) 1.19-1.24 (m, 2 H) 1.27 (s, 3 H) 1.29-1.38 (m, 1 H) 1.44-1.57 (m, 3 H) 1.62-1.66 (m, 1 H) 1.71 (s, 5 H) 1.74-1.93 (m, 3 H) 2.34-2.45 (m, 1 H) 2.54 (d, J=9.00 Hz, 1 H) 2.62-2.74 ( m, 2 H) 2.92-2.98 (m, 1 H) 3.24-3.32 (m, 2 H) 3.52 (s, 3 H) 3.79-3.88 (m, 1 H) 5.39-5.48 (m, 1 H) 5.88 (d, J=10.73 Hz, 1 H) 6.23 (ddd, J=14.64, 11.22, 3, 41 Hz, 1 H).

[720] Step 2. Synthesis of 3-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propoxy)ethoxy)-N-(((2R,5S, 6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxirane -2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl)methyl)propanamide (ADL2-H11)

[721] To a mixture of the compound obtained in step 1 (15.1 mg, 0.037 mmol) in DMF (1 ml, 12.915 mmol) was added 2,5-dioxopyrrolidin-1-yl-3-{2-[2-( 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy]ethoxy}propanoate (13.1 mg, 0.037 mmol) and DIPEA (0.019 ml, 0.111 mmol). The resulting mixture was stirred at room temperature for 16 hours. The mixture was then concentrated in vacuo and the resulting residue was purified by reverse phase chromatography (ODS, 24 g, H ₂ O/MeCN = 95/5 to 60/40) to give the title compound as a colorless oil (14.9 mg, yield 62%).

1.7 Synthesis of H18, H20, H23, H16, H15, H24, H13, H14, H17, H19 and H21

[722] H18, H20, H23, H16, H15, H24, H13, H14, H17, H19 and H21 were prepared using the general procedure listed below (general procedure 1.7).

[723] To a solution of 2,5-dioxopyrrolidin-1-yl-2-((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-(( 2R,3R,4R)-4-hydroxy-3-methoxypentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H- pyran-2-yl)acetate (1.0 eq.) in DMF (0.028 M) was added with amine (16 mg, 0.056 mmol) and DIPEA (2.0 eq.). The reaction mixture was stirred at room temperature for 16 hours, then the mixture was purified by HPLC to obtain the desired product.

1.7.1 H20 synthesis

[724] General Procedure 1.7 gave H20 as a colorless amorphous product (15.7 mg, 0.022 mmol, 79% yield). LC/MS (ESI, mass/charge), 707.99 [M+H] ⁺ .

[725] 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.66 (d, J=6.83 Hz, 3 H) 0.85 (d, J=6.83 Hz, 3 H) 1.04 (d, J=6.83 Hz, 3 H) 1.16 (d, J=6.34 Hz, 3 H) 1.18-1.25 (m, 2 H) 1.27 (s, 4 H ) 1.30-1.59 (m, 8 H) 1.71-1.80 (m, 2 H) 1.81-1.89 (m, 2 H) 2.35 (br d, J=3 ,90 Hz, 2 H) 2.38-2.44 (m, 1 H) 2.52-2.60 (m, 4 H) 2.79-2.86 (m, 4 H) 2.96 ( t, J=5.37 Hz, 1 H) 3.06-3.16 (m, 1 H) 3.24-3.30 (m, 1 H) 3.32 (d, J=9.76 Hz , 1 H) 3.52 (s, 3 H) 3.64 (br d, J=4.39 Hz, 5 H) 3.71 (s, 3 H) 3.83 (t, J=5.85 Hz, 1 H) 4.30-4.42 (m, 1 H) 5.40-5.53 (m, 3 H) 5.89 (br d, J=10.73 Hz, 1 H) 6, 16-6.28 (m, 1 H) 6.75 (br t, J=5.85 Hz, 1 H).

1.7.2 H23 synthesis

[726] H23 (6.9 mg, 37% yield) was prepared using general procedure 1.7. LC/MS (ESI, mass/charge), 665.96 [M+H] ⁺ .

[727] 1H NMR (400 MHz, METHANOL-d4) δ ppm 0.66 (d, J=6.34 Hz, 3 H) 0.80 (d, J=6.83 Hz, 3 H) 1.02 (d, J=6.34 Hz, 3 H) 1.08 (d, J=6.34 Hz, 3 H) 1.12-1.23 (m, 2 H) 1.25 (s, 4 H ) 1.31-1.37 (m, 1 H) 1.43-1.55 (m, 2 H) 1.63 (br d, J=13.17 Hz, 1 H) 1.67 (s, 3 H) 1.81-1.93 (m, 3 H) 2.21-2.40 (m, 1 H) 2.63 (d, J=9.76 Hz, 1 H) 2.74 (s , 2 H) 2.92-2.97 (m, 1 H) 3.05 (br s, 3 H) 3.12-3.22 (m, 1 H) 3.31-3.38 (m, 2 H) 3.50 (s, 3 H) 3.63 (br s, 4 H) 3.68-3.78 (m, 2 H) 4.23 (br d, J=4.39 Hz, 1 H) 5.45 (dd, J=15.12, 8.78 Hz, 1 H) 5.89 (br d, J=10.73 Hz, 1 H) 6.27 (dd, J=14.88 , 10.98 Hz, 1 H).

1.7.3 H16 synthesis

[728] General Procedure 1.7 gave H16 as a colorless amorphous product (6.4 mg, 34% yield). LC/MS (ESI, mass/charge), 679.86[M+H] ⁺ .

[729] 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.67 (br d, J=6.34 Hz, 3 H) 0.85 (br d, J=6.83 Hz, 3 H) 1 .01-1.06 (m, 3 H) 1.14-1.19 (m, 3 H) 1.19-1.28 (m, 5 H) 1.43 (br dd, J=12.20 , 9.76 Hz, 1 H) 1.47-1.55 (m, 1 H) 1.61-1.64 (m, 1 H) 1.71 (br d, J=7.32 Hz, 3 H) 1.83-1.90 (m, 2 H) 2.03-2.20 (m, 1 H) 2.37-2.44 (m, 3 H) 2.49-2.56 (m , 5 H) 2.75 (br s, 1 H) 2.78-2.84 (m, 3 H) 2.87-2.94 (m, 1 H) 2.94-2.98 (m, 1 H) 3.35 (br dd, J=13.41, 10.00 Hz, 2 H) 3.52 (s, 4 H) 3.60 (br s, 4 H) 3.69 (br s, 3 H) 3.71 (s, 4 H) 3.83 (td, J=12.56, 6.59 Hz, 2 H) 4.06-4.20 (m, 1 H) 4.57-4 .68 (m, 1 H) 5.47 (td, J=15.98, 8.54 Hz, 1 H) 5.81-5.96 (m, 1 H) 6.14-6.36 (m , 1 H).

1.7.4 H15 synthesis

[730] General procedure 1.7 provided H15 as a by-product during the synthesis of H16 (6.6 mg, 35% yield). LC/MS (ESI, mass/charge), 665.46 [M+H] ⁺ .

[731] 1H NMR (400 MHz, METHANOL-d4) δ ppm 0.66 (br d, J=6.34 Hz, 4 H) 0.81 (br d, J=5.85 Hz, 3 H) 0 .99-1.04 (m, 4 H) 1.07-1.10 (m, 4 H) 1.17 (br s, 1 H) 1.19-1.27 (m, 5 H) 1. 46-1.55 (m, 2 H) 1.65 (br s, 1 H) 1.68 (s, 4 H) 1.82-1.95 (m, 2 H) 2.00-2.18 (m, 1 H) 2.29-2.49 (m, 4 H) 2.61-2.67 (m, 4 H) 2.92-3.03 (m, 5 H) 3.34 (br d, J=10.24 Hz, 1 H) 3.50 (s, 3 H) 3.58-3.77 (m, 8 H) 4.28 (t, J=9.76 Hz, 1 H) 5.40-5.53 (m, 1 H) 5.83-5.99 (m, 1 H) 6.28 (dd, J=14.63, 10.73 Hz, 1 H).

1.7.5 H24 synthesis

[732] General Procedure 1.7 gave H24 as a colorless amorphous product (12.1 mg, 94% yield). LC/MS (ESI, mass/charge), 693.72 [M+H] ⁺ .

[733] 1H NMR (400 MHz, METHANOL-d4) δ ppm 0.66 (d, J=6.34 Hz, 3 H) 0.80 (d, J=6.83 Hz, 3 H) 1.02 (d, J=6.83 Hz, 4 H) 1.08 (d, J=6.34 Hz, 4 H) 1.32-1.37 (m, 8 H) 1.44-1.50 ( m, 4 H) 1.65-1.68 (m, 5 H) 1.75-1.96 (m, 4 H) 2.19-2.39 (m, 3 H) 2.40-2, 52 (m, 2 H) 2.63 (d, J=9.27 Hz, 1 H) 2.80 (s, 3 H) 2.95 (dd, J=6.34, 4.39 Hz, 1 H) 3.06-3.15 (m, 1 H) 3.31-3.40 (m, 2 H) 3.50 (s, 3 H) 3.65-3.72 (m, 7 H) 3.73-3.80 (m, 2 H) 4.10-4.24 (m, 1 H) 5.47 (dd, J=15.12, 9.27 Hz, 1 H) 5.89 ( d, J=10.24 Hz, 1 H) 6.28 (dd, J=15.12, 10.73 Hz, 1 H).

1.7.6 H18 synthesis

[734] General Procedure 1.7 gave H18 as a colorless amorphous product (8.1 mg, 53% yield). LC/MS (ESI, mass/charge), 678.68[MH] ⁺ .

[735] 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.67 (br d, J=6.34 Hz, 3 H) 0.87 (d, J=6.83 Hz, 3 H) 1, 03 (br d, J=6.83 Hz, 3 H) 1.15-1.24 (m, 5 H) 1.28 (s, 3 H) 1.50-1.67 (m, 5 H) 1.75 (br s, 1 H) 1.82-1.94 (m, 3 H) 2.37-2.44 (m, 3 H) 2.56-2.60 (m, 4 H) 2 .71-2.93 (m, 3 H) 3.00 (br t, J=5.12 Hz, 1 H) 3.37 (br d, J=10.24 Hz, 1 H) 3.53 ( s, 3 H) 3.55-3.63 (m, 1 H) 3.65-3.76 (m, 3 H) 3.83-3.91 (m, 1 H) 4.04 (br d , J=4.39 Hz, 1 H) 4.08-4.16 (m, 1 H) 4.41-4.53 (m, 2 H) 5.42-5.55 (m, 1 H) 5.91 (br d, J=10.73 Hz, 1 H) 6.14-6.28 (m, 1 H) 7.21 (br d, J=7.32 Hz, 1 H).

1.7.7 H13 synthesis

[736] General Procedure 1.7 gave H13 as a colorless amorphous product (6.7 mg, 36.7% yield). LC/MS (ESI, mass/charge), 652.86 [M+H] ⁺ .

[737] 1H NMR (400 MHz, METHANOL-d4) δ ppm 0.66 (d, J=6.34 Hz, 3 H) 0.81 (d, J=6.34 Hz, 3 H) 1.02 (d, J=6.34 Hz, 3 H) 1.05-1.10 (m, 3 H) 1.14-1.23 (m, 2 H) 1.26 (s, 4 H) 1, 34-1.52 (m, 2 H) 1.52-1.59 (m, 1 H) 1.65 (br s, 1 H) 1.70 (s, 4 H) 1.82-1.92 (m, 2 H) 2.41 (qd, J=14.39, 6.10 Hz, 3 H) 2.63 (d, J=9.76 Hz, 1 H) 2.73 (br d, J =4.39 Hz, 3 H) 2.95 (br t, J=5.12 Hz, 2 H) 2.99-3.09 (m, 3 H) 3.30-3.36 (m, 1 H) 3.50 (s, 3 H) 3.53-3.65 (m, 2 H) 3.65-3.78 (m, 4 H) 4.24-4.42 (m, 2 H) 4.55 (br s, 1 H) 5.36-5.55 (m, 1 H) 5.89 (br d, J=10.73 Hz, 1 H) 6.21-6.43 (m, 1 H).

1.7.8 H14 synthesis

[738] General Procedure 1.7 gave H14 as a colorless amorphous solid (15.5 mg, 83% yield). LC/MS (ESI, mass/charge), 666.90 [M+H] ⁺ .

[739] 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.65 (br d, J=6.34 Hz, 3 H) 0.85 (br d, J=6.83 Hz, 3 H) 1 .02 (br d, J=6.34 Hz, 3 H) 1.14-1.28 (m, 8 H) 1.38-1.57 (m, 2 H) 1.57-1.65 ( m, 2 H) 1.81-1.89 (m, 2 H) 2.04-2.19 (m, 2 H) 2.41 (br d, J=4.88 Hz, 3 H) 2, 54 (d, J=9.76 Hz, 1 H) 2.65 (s, 3 H) 2.91-3.03 (m, 5 H) 3.33 (br d, J=9.76 Hz, 1 H) 3.52 (s, 3 H) 3.70 (br s, 5 H) 3.81-3.87 (m, 1 H) 4.08-4.18 (m, 2 H) 4, 52 (br d, J=6.34 Hz, 1 H) 5.45 (br dd, J=15.12, 8.78 Hz, 1 H) 5.89 (br d, J=10.73 Hz, 1 H) 6.21 (br dd, J=14.88, 10.98 Hz, 2 H) 7.15 (br d, J=7.32 Hz, 1 H).

1.7.9 H17 synthesis

[740] General Procedure 1.7 gave H17 as a colorless amorphous solid (12.94 mg, 68% yield). LC/MS (ESI, mass/charge), 680.66 [M+H] ⁺ .

[741] 1H NMR (400 MHz, METHANOL-d4) δ ppm 0.66 (d, J=6.83 Hz, 4 H) 0.81 (d, J=7.32 Hz, 3 H) 0.97 -1.04 (m, 5 H) 1.08 (d, J=6.34 Hz, 5 H) 1.10-1.16 (m, 2 H) 1.20-1.23 (m, 1 H) 1.25 (s, 4 H) 1.69 (d, J=0.98 Hz, 4 H) 1.83 (br d, J=3.41 Hz, 1 H) 1.87 (br d , J=4.39 Hz, 1 H) 1.90 (br d, J=4.39 Hz, 1 H) 2.24-2.30 (m, 1 H) 2.40 (br dd, J= 13.90, 9.02 Hz, 2 H) 2.58-2.62 (m, 5 H) 2.64 (s, 1 H) 2.84 (br t, J=4.88 Hz, 5 H ) 2.95 (dd, J=6.34, 4.39 Hz, 1 H) 3.30-3.32 (m, 1 H) 3.50 (s, 3 H) 3.56-3.64 (m, 6 H) 3.74-3.79 (m, 1 H) 4.02 (br t, J=5.37 Hz, 3 H) 4.32-4.35 (m, 1 H) 4 .35-4.37 (m, 1 H) 5.45 (dd, J=15.12, 8.78 Hz, 1 H) 5.85-5.91 (m, 1 H) 6.27 (dd , J=15.12, 10.73 Hz, 1 H).

1.7.10 Synthesis of H19

[742] General Procedure 1.7 gave H19 as a colorless amorphous solid (5.53 mg, 56.4% yield). LC/MS (ESI, mass/charge), 694.08 [M+H] ⁺ .

[743] 1H NMR (400 MHz, METHANOL-d4) δ ppm 0.66 (d, J=6.83 Hz, 3 H) 0.81 (d, J=6.83 Hz, 3 H) 0.95 -0.95 (m, 1 H) 1.02 (d, J=6.83 Hz, 3 H) 1.08 (d, J=6.34 Hz, 3 H) 1.12-1.22 ( m, 2 H) 1.26 (s, 4 H) 1.34-1.52 (m, 7 H) 1.62 (br d, J=13.66 Hz, 2 H) 1.68 (s, 4 H) 1.81-1.93 (m, 3 H) 2.08-2.08 (m, 1 H) 2.20-2.39 (m, 2 H) 2.39-2.50 ( m, 1 H) 2.61-2.68 (m, 4 H) 2.90-2.97 (m, 5 H) 3.11-3.15 (m, 2 H) 3.30-3, 34 (m, 1 H) 3.50 (s, 3 H) 3.56-3.62 (m, 4 H) 3.76 (t, J=6.34 Hz, 1 H) 4.11-4 .22 (m, 1 H) 5.45 (dd, J=15.12, 8.78 Hz, 1 H) 5.90 (br d, J=11.22 Hz, 1 H) 6.29 (dd , J=14.88, 10.98 Hz, 1 H).

1.7.11 H21 synthesis

[744] General procedure 1.7 was carried out to obtain H21 as a colorless amorphous solid (12.3 mg, 62.1% yield). LC/MS (ESI, mass/charge), 708.03 [M+H] ⁺ .

[745] 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.67 (br d, J=6.34 Hz, 3 H) 0.85 (br d, J=6.83 Hz, 3 H) 1 .02 (br d, J=6.83 Hz, 3 H) 1.15-1.29 (m, 9 H) 1.29-1.42 (m, 3 H) 1.47-1.63 ( m, 6 H) 1.74-1.79 (m, 1 H) 1.86 (br dd, J=13.17, 3.90 Hz, 2 H) 2.40 (br d, J=5, 37 Hz, 3 H) 2.51-2.55 (m, 4 H) 2.78 (br s, 4 H) 2.96 (br t, J=5.12 Hz, 1 H) 3.18 ( td, J=12.07, 6.10 Hz, 2 H) 3.37 (br d, J=9.76 Hz, 1 H) 3.52 (s, 3 H) 3.57 (br s, 4 H) 3.69 (s, 4 H) 3.84 (br t, J=5.85 Hz, 1 H) 4.50-4.57 (m, 1 H) 4.85-4.93 (m , 2 H) 5.44 (br dd, J=15.12, 8.78 Hz, 1 H) 5.92 (br d, J=10.24 Hz, 1 H) 6.15-6.38 ( m, 1 H) 7.24-7.29 (m, 2 H) 8.24 (br s, 1 H).

1.8 Synthesis of H22 and H25

[746] H22 and H25 were prepared using the general procedure described below.

[747] Step 1. To a solution of ((2R,5S,6S)-6-((S,2E,4E)-7-((2R,3R)-3-((2R,3R,4R)-3- methoxy-4-((triethylsilyl)oxy)pentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2 -l)methanamine (16.6 mg, 0.027 mmol) and 4-carboxy-1-cyclohexanemethanol (mixture of cis and trans isomers, 5.11 mg, 0.032 mmol) in dichloromethane (2 ml) was added EDC (6.20 mg, 0.032 mmol) and HOBT (4.54 mg, 0.03 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 hours. The mixture was then diluted with dichloromethane, and the organic layer was washed with water and brine, then dried over anhydrous sodium sulfate. The solid was filtered and the filtrate was concentrated and purified by silica gel chromatography to give the desired product as a colorless oil (8.3 mg, 47% yield).

[748] Step 2. To a solution of a mixture of cis-, trans-isomers 4-(hydroxymethyl)-N-(((2R,5S,6S)-6-((S,2E,4E)-7-((2R, 3R)-3-((2R,3R,4R)-3-methoxy-4-((triethylsilyl)oxy)pentan-2-yl)-2-methyloxiran-2-yl)-6-methylhepta-2,4- dien-2-yl)-5-methyltetrahydro-2H-pyran-2-yl)methyl)cyclohexanecarboxamide (21 mg, 0.032 mmol) and DIPEA (0.028 ml, 0.158 mmol) in dichloromethane (1 ml) at room temperature were added in portions of 4 -nitrophenylcarbonyl chloride (12.75 mg, 0.063 mmol) and DMAP (1.932 mg, 0.016 mmol). The resulting mixture was stirred at the same temperature for 16 hours. 1-Methylpiperazine (31.7 mg, 0.316 mmol) was then added and stirred for an additional 1 hour. The reaction mixture was diluted with dichloromethane and the organic layer was washed with water and brine, then dried over Na ₂ SO ₄ . The solid was filtered and the filtrate was concentrated to dryness. The resulting residue was dissolved in 1.0 ml MeOH and 10 mg p-TsOH and stirred for 2 hours at room temperature. The reaction mixture was quenched by adding 100 μl of DIPEA and then the solvent was removed in vacuo. The resulting residue was purified by NH-silica gel chromatography (heptane/AcOEt = 50/50 to 0/100) to give H25 and H22.

1.8.1 H25 synthesis

[749] H25 was obtained as a colorless amorphous product (7.0 mg, 32.7% yield). LC/MS (ESI, mass/charge), 676.94 [M+H] ⁺ .

[750] 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.68 (d, J=6.83 Hz, 3 H) 1.02 (d, J=7.32 Hz, 3 H) 1.10 (d, J=6.83 Hz, 3 H) 1.17 (d, J=6.34 Hz, 4 H) 1.25 (s, 3 H) 1.47-1.62 (m, 11 H ) 1.71 (s, 3 H) 1.80-1.86 (m, 4 H) 2.13 (s, 1 H) 2.27-2.36 (m, 10 H) 2.49-2 .65 (m, 1 H) 2.99-3.12 (m, 1 H) 3.29 (d, J=10.20 Hz, 1 H) 3.32 (s, 4 H) 3.47 ( br t, J=4.88 Hz, 4 H) 3.72-3.73 (m, 1 H) 3.73-3.74 (m, 1 H) 3.99 (d, J=6.83 Hz, 2 H) 4.21-4.36 (m, 1 H) 5.56-5.71 (m, 1 H) 5.92 (s, 1 H) 6.18-6.31 (m, 1 H).

1.8.2 H22 synthesis

[751] H22 was obtained as a colorless amorphous product (5.6 mg, 26.2% yield). LC/MS (ESI, mass/charge), 676.89[M+H] ⁺ .

[752] 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.68 (d, J=6.83 Hz, 3 H) 1.02 (d, J=6.83 Hz, 3 H) 1.10 (d, J=7.32 Hz, 3 H) 1.14 (d, J=6.34 Hz, 3 H) 1.17 (s, 3 H) 1.21-1.41 (m, 2 H ) 1.49-1.55 (m, 5 H) 1.58-1.62 (m, 6 H) 1.70 (s, 3 H) 1.80-1.86 (m, 4 H) 2 ,26-2.30 (m, 4 H) 2.35 (br s, 4 H) 2.46-2.57 (m, 1 H) 2.89 (dd, J=3.66, 2.20 Hz, 1 H) 3.01-3.11 (m, 1 H) 3.27 (d, J=9.76 Hz, 1 H) 3.38 (s, 3 H) 3.47 (br t, J=4.88 Hz, 4 H) 3.53 (ddd, J=10.24, 6.83, 3.41 Hz, 1 H) 3.76 (d, J=5.37 Hz, 1 H) 3.89 (dd, J=6.34, 1.95 Hz, 1 H) 3.99 (d, J=7.32 Hz, 2 H) 5.68-5.77 (m, 1 H) 5 .91 (d, J=11.22 Hz, 1 H) 6.18 (dd, J=14.88, 10.98 Hz, 1 H).

EXAMPLE 2

[753] Exemplary herboxydiene-based spliceosome modulator payloads used in the production of ADCs were profiled. The payloads were evaluated for binding to the SF3b complex, in vitro splicing activity, and cell growth inhibition ability.

2.1 In Vitro splicing (IVS)

[754] An in vitro splicing assay was performed to evaluate payload activity in a cell-free system. Payloads were incubated with nuclear extracts and a pre-mRNA construct as substrate and minigenes.

[755] Preparation of nuclear extract from HeLa: pellets containing HeLa S3 cells were resuspended in hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl ₂ , 10 mM KCl, 0.2 mM PMSF and 0.5 mM DTT) and the suspension was adjusted to a total of 5 pelleted cell volumes (PCV). After centrifugation, the supernatant was discarded and the cells were adjusted to 3 PCV with hypotonic buffer and incubated on ice for 10 minutes. Cells were lysed using a Dounce homogenizer and then centrifuged. The supernatant was discarded and the pellet was resuspended with ½ volume of pelleted nuclei (PNV) low-salt buffer (20 mM HEPES, pH 7.9, 1.5 mM MgCl ₂ , 20 mM KCl, 0.2 mM EDTA, 25% glycerol, 0.2 mM PMSF, 0.5 mM DTT), then ½ PNV high salt buffer (same as low salt buffer except 1.4 M KCl was used). The kernels were stirred gently for 30 min. before centrifugation. The supernatant (nuclear extract) was then dialyzed into storage buffer (20 mM HEPES, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 0.2 mM PMSF, 0.5 mM DTT). Protein concentration was determined using a NanoDrop 8000 UV-Vis spectrophotometer (ThermoFisher Scientific).

[756] IVS: All sequences derived from Ad2 (Pellizzoni et al. (1998) Cell 95(5):615-24) were cloned into the pcDNA3.1(+) vector (Promega) using restriction sites at the 5' end for EcoRI and 3' end for XbaI. Plasmids were linearized using XbaI and used as DNA templates in in vitro transcription reactions. The intronless plasmid FtzΔi (Luo and Reed (1999) 96(26):14937-42) was linearized using EcoRI. All RNAs were transcribed in vitro and then purified using MEGAScript T7 (Invitrogen) and MegaClear (Invitrogen) kits, respectively. For splicing reactions using Ad2 pre-mRNA variants, 1 μl reaction mixture was prepared using 8 μg of nuclear extracts obtained from HeLa S3 cells, 2 ng pre-mRNA, 0.2 ng FTZΔi, and varying concentrations of compounds or DMSO. After 15 min. preincubation at 30°C, 1 μl of splicing activation buffer (0.5 mM ATP, 20 mM creatine phosphate, 1.6 mM MgCl) was added₂) and the reaction mixtures were incubated for 90 min. at 30°C. Reaction mixtures were then quenched using 13 μL of DMSO, and 25 nL were used for RT-qPCR. RT-qPCR reaction mixtures were prepared using the TaqMan RNA-to-C 1-step kit_T (Life Technologies), RNA from splicing reactions, primer-probe sets for Ad2 mRNA (forward: ACTCTCTTCCGCATCGCTGT; reverse: CCGACGGGTTTCCGATCCAA; probe: CTGTTGGGCTCGCGGTTG) and Ftz (forward: TGGCATCAGATTGCAAAGAC; reverse: ACGCCGGGTGATGTATCTAT; probe: CGAAACGCACCCGTCAGACG). Prism 7 (Graphpad) was used to fit the spliced product data to a nonlinear regression curve and normalized to a control sample (DMSO).

[757] Given that all payloads tested specifically bound to the SF3b complex and exhibited similar binding profiles, it was hypothesized that all would also modulate splicing to a comparable extent. All payloads significantly modulated Ad2.2 pre-mRNA splicing (see Table 13). In the presence of payload, a decrease in the amount of spliced product was observed.

2.2 Cell viability

[758] The HCC1954 (American Type Culture Collection (ATCC)) ductal breast carcinoma cell line was seeded at 2000 cells/well in flat-bottomed 96-well tissue culture plates (Corning) in a total volume of 90 μl tissue culture medium supplemented with 10 % fetal bovine serum (ThermoFisher Scientific). Cells were treated using a 3-fold serial dilution of the compound from 200 nM to 0.03 nM. Each concentration was tested in triplicate. At the treatment time point, a plate of untreated cells was assessed using the CellTiter-Glo®2.0 Luminescent Cell Viability Assay according to the manufacturer's recommendations (Promega; #G9241). CellTiter-Glo® 2.0 reagent was added to the medium, incubated, and analyzed on an EnVision multichannel reader (PerkinElmer). Values are presented at time zero (T0). The number of viable cells after 144 hours (T144) of compound treatment was also determined using the CellTiter-Glo®2.0 luminescent cell viability assay. The luminescence value at time zero (T0), DMSO growth control (C), and growth in the presence of test compound (T144) were used to calculate percent growth for each compound concentration level. The percentage of growth inhibition was calculated as: [(T144-T0)/(C-T0)] x 100 - for concentrations for which T144>/=T0, or [(T144-T0)/T0] x 100 for concentrations for which T144<T0. Dose-response curves were plotted using Prism 7 (Graphpad) and fitted using nonlinear regression analysis and log (inhibitor) versus response variable slope (four parameters).

[759] Dose-response relationships for cell viability were determined for all payloads in HER2-amplified HCC1954 breast cancer cells. Most of the payloads tested exhibited GI ₅₀ values (ie, the concentration of a compound that causes a 50% reduction in cell proliferation) in the low nanomolar range (see Table 13).

EXAMPLE 3

[760] Exemplary payload compounds evaluated in Example 2 were conjugated to an exemplary anti-HER2 antibody (trastuzumab) via cysteine residues on the antibody. The preparation and evaluation of exemplary anti-HER2 antibody ADCs are described below.

3.1 Antibody

[761] The antibody trastuzumab (“AB185”) (Molina et al. (2001) Cancer Res. 61(12):4744-9) was used to generate an anti-HER2 antibody-based ADC (also referred to herein as SMLA).

3.2 Bioconjugation

[762] Antibody (trastuzumab) at 10 mg/ml in PBS buffer (pH 7.0) was mixed with 5 mM TCEP (2-4 molar equivalents) (ThermoFisher Scientific; #77720) to cleave interchain disulfide bonds. The reaction mixture was stirred gently at 22 ^° C for 3 hours. Propylene glycol (15% v/v) was then added followed by 8 molar equivalents of the linker-payload construct (6 mM stock solution in DMSO) and the solution was thoroughly mixed. The reaction mixture was placed on a rotating rack in an incubator at 22 ^° C. After 2 hours of conjugation, the reaction mixture was purified to remove unconjugated payload using AKTA GE M150 (HiTrapTM 26/10 desalting columns; flow rate: 3 ml/min) ( GE Healthcare Bio-Sciences) in DPBS (pH 7.5). The resulting conjugate was concentrated by ultrafiltration through Amicon (30 kDa, Ultra-4) (EMD Millipore) and sterile filtered through a 0.22-μm disposable PVDF filter (EMD Millipore). The resulting clear solution was assessed by UV-VIS to determine the antibody concentration ([mAb]; mol/L) and conjugated payload concentration ([LD]; mol/L) in accordance with the Beer-Lambert law (A= E * c * l) and according to the following equations:

A _{280 nm=} E ^mAb _{280 nm} * [mAb] * l+ E ^LD _{280 nm} * [LD] * l

A _{252 nm=} E ^mAb _{252 nm} * [mAb] * l+ E ^LD _{252 nm} * [LD] * l

E ^mAb _{280 nm} : trastuzumab=213.380 cm ^-1 M ^-1

E ^mAb _{252 nm} : trastuzumab=79.112 cm ^-1 M ^-1

E ^LD _{280 nm} ⁼ 800 cm^-1M^-1

E ^LD _{252 nm} ⁼ 31,000 cm^-1M^-1

Abbreviations: c - molar concentration; l - optical path length (Nanodrop: 0.1 cm); E - molar light absorption coefficient; A - absorption.

3.3 Determination of biophysical characteristics

[763] Herboxydiene-antibody splicing modulator ratio (HAR), percent aggregation, and percent unconjugated payload were determined for exemplary anti-HER2 antibody ADCs by liquid chromatography-mass spectrometry (LC/MS), size exclusion chromatography (SEC), reverse phase high performance liquid chromatography (HPLC), respectively. In general, conjugates contain less than 2% drug in free form and contain less than 10% aggregates.

3.3.1 LC/MS - HAR analysis

[764] LC/MS analysis was performed using an Agilent 1290 UPLC system coupled to an Agilent G6224A Accurate Mass TOF mass spectrometer. The conjugate was deglycosylated using PNGase F (New England Biolabs; #P0705L) for 4 hours at 37 ^° C, denatured using 8 M Gdn-HCl (Sigma; #G9284), and finally separated into light and heavy chain domains with using DTT (5 mM final concentration) (Promega; no. V3151). The resulting sample was loaded onto an Agilent PLRP-S column (2.1 x 150 mm, 8 µm) and eluted with a gradient of 25% B to 50% B over 28 min. at room temperature (RT). Mobile phase A was water with 0.05% TFA, mobile phase B was acetonitrile with 0.04% TFA, and the flow rate was 1 ml/min. HAR was calculated from the deconvoluted mass spectrum using weighted averages of unconjugated and conjugated drug peak intensities for the light chain (L0 or L1) and heavy chain (H0, H1, H2, and H3). The total HAR for the intact conjugate was calculated using the equation: (HAR _LC * 2) + (HAR _HC *2) = total HAR. HAR values for exemplary anti-HER2 antibody ADCs are shown in Table 12.

3.3.2 SEC analysis - aggregation

[765] Size exclusion chromatography was performed using a TOSON-G3000SWXL column (#008541) in 0.2 M potassium phosphate (pH 7) with 0.25 mM potassium chloride and 15% (v/v) IPA at a flow rate of 0.75 ml /min. The area of the absorption peaks at 280 nm was determined for high molecular weight and monomeric components of the conjugate by integrating the area under the curve. The percentage of monomers for exemplary anti-HER2 antibody ADCs is shown in Table 12.

3.3.3 HPLC Analysis - Free Form Herboxidiene Splicing Modulator

[766] The conjugate of interest was precipitated using 10 volumes of acetonitrile on ice for 2 hours and pelleted by centrifugation. Supernatants containing residual unconjugated payload were then loaded onto an Agilent Poroshell 120 SB-C18 120A column (4.6 x 100 mm, 2.7 μm) and eluted with a gradient of 45% B to 70% B over 10 min. at RT, mobile phase A was 100% water, mobile phase B was 100% acetonitrile, and the flow rate was 0.6 ml/min. with detection at 252 nm. The amount of residual herboxydiene splicing modulator in free form was quantified by UV detection by comparison with an external standard curve for the unconjugated linker-payload construct. The percentage of free form herboxydiene splicing modulator for exemplary anti-HER2 antibody ADCs is shown in Table 12.

3.4 Determination of binding characteristics

3.4.1 Determination of binding characteristics of target positive cells by FACS

[767] The binding of unconjugated anti-HER2 antibody and anti-HER2 antibody-based ADC to target-positive cells was assessed by flow cytometry using indirect immunofluorescence. JIMT1 (DSMZ) cells, a breast cancer cell line that endogenously expresses HER2, were seeded (5 x 10 ⁴ cells/well) in a 96-well v-bottom plate (Greiner Bio-One) and incubated for 2 hours at 4°C with test compounds diluted to various concentrations in assay medium (RPMI-1640 supplemented with 10% (w/v) fetal bovine serum albumin (Thermo Fisher Scientific)). Cells were then washed with PBS+2% FBS (FACS buffer) and stained with phycoerythrin (PE)-labeled goat anti-immunoglobulin G (IgG) antibody (Invitrogen) for 40 min. at 4°C in the dark. Cells were washed with cold FACS buffer and fixed using FluroFix buffer (Biolegend) for 30 min. at room temperature. Fixed cells were washed using FACS buffer. Fixed cells were analyzed by geometric mean PE fluorescence intensity using an LSRFortessa flow cytometer (BD Bioscience). Trastuzumab and T-DM1 (DM1 conjugated to trastuzumab) were included as controls.

3.5 In vitro analysis

3.5.1 Cell viability

[768] Anti-HER2 ADCs were tested in several HER2-amplified cell lines for their ability to inhibit cell growth. The cell line HCC1954 (ATCC) was used (2000 cells/well). Cell viability assays were performed as described in section 2.3.

[769] Surprisingly, not all ADCs were active on HCC1954 cells, despite having similar binding profiles (see Table 13) and payloads with similar biochemical properties. For example, ADCs with the ADL2 linker were less capable of inhibiting cell growth (eg, comparing the activity of ADL1-H1 and ADL2-H1).

Table 12. Determination of physical characteristics of herboxydiene splicing modulator payloads SMLA Herboxidiene-based splicing modulator Linker Medicine in free form, % Monomer percentage Concentration (mg/ml) Average value
penetrating ability,
penetrated AB against Caco-2 (10 ^-6 cm/s) Average value
penetrating power ER Average value
stability, stability
stability, t1/2, pH 5.5 (days) Average value
stability, stability
stability, %, pH 5.5, t24 hours (%) AB185-ADL1-H3 H3 ADL1 n. O. 99 1,380 n. O. n. O. n. O. n. O. n. O. AB185-ADL1-H1 H1 ADL1 0.300 97 2,550 0.263 0.334 1,270 3.5 79 AB185-ADL1-H8 H8 ADL1 n. O. 98 0.320 n. O. n. O. n. O. n. O. n. O. AB185-ADL1-H5 H5 ADL1 n. O. 98 2,490 1,040 23.6 22,692 >7 >100 AB185-ADL1-H7 H7 ADL1 n. O. 97 2,570 n. O. n. O. n. O. n. O. n. O. AB185-ADL1-H10 H10 ADL1 n. O. 98 2,380 n. O. n. O. n. O. n. O. n. O. AB185-ADL1-H9 H9 ADL1 n. O. 98 2,550 11,596 n. O. n. O. n. O. n. O. AB185-ADL1-H4 H4 ADL1 0.400 91 2,740 19,263 n. O. n. O. n. O. n. O. AB185-ADL1-H6 H6 ADL1 n. O. 99 2,050 5.725 n. O. n. O. n. O. n. O. AB185-ADL2-H1 H1 ADL2 n. O. n. O. 2,090 0.263 0.334 1,270 3.5 79 AB185-ADL2-H11 H11 ADL2 n. O. n. O. 3,060 n. O. n. O. n. O. n. O. n. O.

Table 13. Characterization of Exemplary Anti-HER2 Antibody ADCs and Corresponding Herboxidiene Splicing Modulator Payloads SMLA activity Payload activity in binding SF3B1 and modulating mRNA splicing Cellular activity of herboxidiene-based splicing modulator SMLA Herboxidiene-based splicing modulator Linker HAR CTGlo,
avg. geom.
GI50 (nM),
HER2 SMLA screening,
HCC
1954.1 CTGlo,
avg. geom.
LD50 (nM),
HER2 SMLA screening,
HCC
1954.1 SPA-ATS,
avg. geom.
IC50 (nM),
SF3B1 (WT),
HE
LA.2 qPCR-IVS-ATS,
avg. geom.
IC50 (nM),
Ad2.1,
HE
LA.2 qPCR-IVS-ATS,
avg. geom.
IC50 (nM),
Ad2.2,
HE
LA.2 CTGlo
-ATS,
avg. geom.
GI50 (nM),
72 hours
THP1.1 CTGlo-ATS,
avg. geom.
LD50 (nM),
72 hours
THP1.1 CTGlo-ATS,
average value
minimum answer, %
72 hours
THP1.1 CTGlo-ATS,
avg. geom.
GI50 (nM),
72 hours
NCIH
1650.1 CTGlo-ATS,
avg. geom.
LD50 (nM),
72 hours
NCIH
1650.1 CTGlo-ATS,
average value
minimum answer, %
72 hours
NCIH
1650.1 CTGlo-ATS,
average value
minimum answer, %
72 hours
OPM2.1 AB185-ADL1-H3 H3 ADL1 4,520 0.067 0.246 n. O. 562,476 35.185 54,563 478.332 -92.943 114.915 >10000 19,182 -98.873 AB185-ADL1-H1 H1 ADL1 4,800 0.048 0.310 11,671 72,619 33,667 114.194 697.273 -84.238 182.271 >10000 10,179 -83.666 AB185-ADL1-H8 H8 ADL1 5,920 0.030 0.980 n. O. 12,902 12,916 1.218 11,851 -102,030 1.105 >10000 17,053 -103.148 AB185-ADL1-H5 H5 ADL1 4,920 0.180 1,040 12,903 30,268 17,764 0.433 3.415 -103.352 0.407 >10000 -25.308 -103.609 AB185-ADL1-H7 H7 ADL1 5,160 0.305 1,388 n. O. 14,452 12,059 0.650 6,756 -99.328 0.772 >10000 5,754 -100.376 AB185-ADL1-H10 H10 ADL1 4,710 0.162 3,963 n. O. 21,704 16,500 2,058 21,021 -98.789 3.036 >10000 9,194 -100.672 AB185-ADL1-H9 H9 ADL1 4,770 0.323 36.1 n. O. 9,679 8,659 1.337 14,441 -101.645 0.481 2.681 2.327 >10000 AB185-ADL1-H4 H4 ADL1 4,010 0.516 200 14,497 31,583 29,555 649.406 5337.399 -40.902 463,968 2575.18 728,583 >10000 AB185-ADL1-H6 H6 ADL1 6,090 3.952 400 n. O. 9,514 7,188 0.701 6,522 -101.275 0.219 1.336 0.978 >10000 AB185-ADL2-H1 H1 ADL2 3,200 355 400 11,671 72,619 33,667 114.194 697.273 -84.238 182.271 >10000 10,179 -83.666 AB185-ADL2-H11 H11 ADL2 2,600 235 400 n. O. n. O. n. O. n. O. n. O. n. O. n. O. n. O. n. O. n. O.

n. O. = undefined

3.5.2 Penetration ability of payloaded ADCs towards Caco-2 cells

[770] Caco-2 cells were cultured for 21 days in 24-well plates with Transwell inserts at 37°C, 95% humidity, 5% CO ₂ . The integrity of the cell monolayer was confirmed by TEER (transepithelial electrical resistance) and Lucifer yellow dye. Payloads were applied in duplicate at 10 μm separately on each side of the cell monolayer. Apical to basolateral (AB) and basolateral to apical (BA) penetration rates were determined by aliquoting both chambers immediately after treatment (t=0) and after incubation for 2 hours. The samples were protein precipitated with an organic solvent containing an internal standard and analyzed by LC-MS/MS (SCIEX; API 5500). The ratio of payload peak area/internal standard values versus time in both directions was used to obtain penetration values (cm/sec). The efflux ratio was calculated by dividing BA/AB. Control compounds for low and high penetration and efflux performed as expected. Penetration values are given in Table 12.

3.5.3 Chemical stability of ADC with payload

[771] Herboxydiene splicing modulators were incubated in McIlvaine (citrate phosphate) buffer, pH 5.5 (Boston Bioproducts; #BB-2466) at a final concentration of 20 μM (less than 0.5% DMSO from the stock solution). A solution of a splicing modulator based on herboxydiene and an internal standard was pipetted into 96-well plates, run by UPLC (Waters Aquity H-class) and analyzed using the original chromatographic signal (t=0). The column was a UPLC HSS T3, 1.8 μm, 2.1 x 50 mm column (#186003538). A 95% to 10% gradient of mobile phase A was used over 1 min, where A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile (flow rate 0.9 ml/min.). Place the rest of the herboxydiene splicing modulator solution in a plate shaker at 37°C (Eppendorf ThermoMixer). UPLC analyzes of samples were repeated at time points 24, 72 and 96 hours after incubation at 37°C. The ratio of peak area values for the herboxydiene splicing modulator and the internal standard was determined for three time points: time 0, day 1, and either day 3 or day 4. Time point 0 was defined as 100. The ratio of peak area values for later time points time was compared to time 0. The percentage of modulator remaining was calculated as follows: (ratio of peak area values at day X/ratio of peak area values at time 0) * 100 = % modulator remaining. The slope of the straight line was calculated in Excel and compared with the logarithm of the % of the remaining modulator and the time point. Half-life values were calculated in Excel using natural log(2)/slope and are shown in Table 12.

EXAMPLE 4

[772] Exemplary payload compounds were evaluated as described below.

4.1 In Vitro analysis

4.1.1 Cell viability

[773] Dose-response relationships for cell viability were determined for exemplary payload compounds in HCC1954 breast cancer cells and HER2-amplified NCI-N87 gastric cancer cells.

[774] HCC1954 ductal breast carcinoma cells (American Type Culture Collection (ATCC)) or NCI-N87 gastric carcinoma cells (ATCC) were seeded at 500 cells/well in flat-bottomed 384-well tissue culture plates (Corning) at a total volume of 30 µl of tissue culture medium supplemented with 10% fetal bovine serum (ThermoFisher Scientific). Cells were treated using a 4-fold serial dilution of the compound from 10,000 nM to 0.01 nM. Each concentration was tested in triplicate. At the treatment time point, the plate containing untreated cells was assessed using the CellTiter-Glo® 2.0 Luminescent Cell Viability Assay according to the manufacturer's recommendations (Promega; #G9241). CellTiter-Glo® 2.0 reagent was added to the medium, incubated, and analyzed on an EnVision multichannel reader (PerkinElmer). Values are presented at time zero (T0). The number of viable cells 72 (T72) or 144 hours (T144) after treatment with the compound was also determined using the CellTiter-Glo®2.0 luminescent cell viability assay. The luminescence value at time zero (T0), DMSO growth control (C), and growth in the presence of test compound (T72 or T144) were used to calculate percent growth for each compound concentration level. The percentage of growth inhibition was calculated as (for example): [(T144-T0)/(C-T0)] x 100 - for concentrations for which T144>/=T0, or [(T144-T0)/T0] x 100 for concentrations , for which T144<T0. Dose-response curves were plotted using Prism 8 (Graphpad) and fitted using nonlinear regression analysis and log (inhibitor) versus response variable slope (four parameters).

[775] In FIG. 1A-1D show the dose-dependent effects of exemplary payload compounds on the viability of HER2-amplified breast cancer cells (HCC1954) and gastric cancer cells (NCI-N87). In fig. Figure 1A shows the dose-dependent effect on the viability of HCC1954 cells after 72 hours of incubation. In fig. 1B shows the dose-dependent effect on the viability of NCI-N87 cells after 72 hours of incubation. In fig. Figure 1C shows the dose-dependent effect on the viability of HCC1954 cells after 144 hours of incubation. In fig. 1D shows the dose-dependent effect on the viability of NCI-N87 cells after 144 hours of incubation. Table 14 shows the GI50, LD50 and Rmin values for all compounds tested.

4.1.2 PD analysis of cell splicing

[776] Splicing of the mature SLC25A19 transcript was also examined in HCC1954 and NCI-N87 cells treated with increasing concentrations of exemplary payload compounds.

[777] HCC1954 or NCI-N87 (ATCC) cells were seeded in RPMI+10%FBS (ATCC) at 1000 cells per well at 20 μl per well. Cells were treated with compounds in a dose-dependent manner at 4-fold dilutions. After 6 hours, cells were washed with PBS and lysed using 30 μl of CL buffer (IgePal CA-630, 5 M NaCl, 1 M Tris-HCl 1 M, pH 7.4 in water) containing 25 μl/ml RNasin® ( Promega) and incubated for 20 min. at k.t. on a shaker-rocker. The resulting mixture (1 μl) was used to assess splicing modulation in a reverse transcription-PCR reaction using Taqman Fast Virus 1-Step MasterMix (Applied Biosystems) and the following Taqman primers according to the manufacturer's recommendations: SLC25A19 (Invitrogen, Hs00222265_m1); RPLPO (Invitrogen, Hs99999902_m1).

[778] In FIG. 2A and FIG. Figure 2B shows the results of splicing analysis in breast cancer cells (HCC1954) (Figure 2A) and gastric cancer cells (NCI-N87) with HER2 amplification (Figure 2B). Table 14 shows the IC50 values for all compounds tested.

--->

LIST OF SEQUENCES

<110> EISAI R&D MANAGEMENT CO., LTD.

<120> ANTIBODY-DRUG CONJUGATES CONTAINING

SPLICING MODULATOR BASED ON HERBOXIDIENE, AND METHODS OF THEIR APPLICATION

<130> 12636.0011-00304

<140> PCT/US2019/066029

<141> 2019-12-12

<150> 62/941,220

<151> 2019-11-27

<150> 62/779,406

<151> 2018-12-13

<150> 62/779,400

<151> 2018-12-13

<160> 113

<170> PatentIn version 3.5

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Gly Phe Asn Ile Lys Asp Thr Tyr Ile His

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<223> /note="Description of the artificial sequence:

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Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys

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Gly

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<223> /note="Description of the artificial sequence:

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Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Val

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<212> PROTEIN

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Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp

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Ser Ala Ser Phe Leu Glu Ser

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Gln Gln His Tyr Thr Thr Pro Pro Thr

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Asn Tyr Trp Ile Glu

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Ile Leu Pro Gly Thr Gly Arg Thr Ile Tyr Asn Glu Lys Phe Lys Gly

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Lys Ala

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Arg Asp Tyr Tyr Gly Asn Phe Tyr Tyr Ala Met Asp Tyr

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<223> /note="Description of the artificial sequence:

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Ala Ser Gln Gly Ile Asn Asn Tyr Leu Asn

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<223> /note="Description of the artificial sequence:

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Thr Ser Thr Leu Gln Ser

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Gln Gln Tyr Ser Lys Leu Pro Arg Thr

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<212> PROTEIN

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His Tyr Met Met Ala

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<223> /note="Description of the artificial sequence:

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Arg Ile Gly Pro Ser Gly Gly Pro Thr His Tyr Ala Asp Ser Val Lys

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Gly

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<400> 15

Tyr Asp Ser Gly Tyr Asp Tyr Val Ala Val Ala Gly Pro Ala Glu Tyr

1 5 10 15

Phe Gln His

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<213> Artificial sequence

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<223> /note="Description of the artificial sequence:

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Arg Ala Ser Trp Ser Ile Ser Thr Trp Leu Ala

1 5 10

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<212> PROTEIN

<213> Artificial sequence

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<400> 17

Lys Ala Ser Asn Leu His Thr

15

<210> 18

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

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<223> /note="Description of the artificial sequence:

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<400> 18

Gln Gln Tyr Asn Ser Tyr Ser Arg Thr

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<210> 19

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

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<400> 19

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 20

<211> 109

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

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<400> 20

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr

100 105

<210> 21

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

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<400> 21

Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Met Met Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Asn Tyr

20 25 30

Trp Ile Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly Glu Ile Leu

35 40 45

Pro Gly Thr Gly Arg Thr Ile Tyr Asn Glu Lys Phe Lys Gly Lys Ala

50 55 60

Thr Phe Thr Ala Asp Ile Ser Ser Asn Thr Val Gln Met Gln Leu Ser

65 70 75 80

Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Arg Asp

85 90 95

Tyr Tyr Gly Asn Phe Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 22

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

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<400> 22

Asp Ile Gln Met Thr Gln Ser Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Asn Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Glu Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Gly Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 23

<211> 128

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

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<400> 23

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr

20 25 30

Met Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Gly Pro Ser Gly Gly Pro Thr His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Tyr Asp Ser Gly Tyr Asp Tyr Val Ala Val Ala Gly Pro Ala

100 105 110

Glu Tyr Phe Gln His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 24

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

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<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Ser Leu Thr Ile Ser Gly Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 25

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 25

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 26

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

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<400> 26

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 27

<211> 441

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 27

Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Met Met Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Asn Tyr

20 25 30

Trp Ile Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly Glu Ile Leu

35 40 45

Pro Gly Thr Gly Arg Thr Ile Tyr Asn Glu Lys Phe Lys Gly Lys Ala

50 55 60

Thr Phe Thr Ala Asp Ile Ser Ser Asn Thr Val Gln Met Gln Leu Ser

65 70 75 80

Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Arg Asp

85 90 95

Tyr Tyr Gly Asn Phe Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro

115 120 125

Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly

130 135 140

Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn

145 150 155 160

Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr

180 185 190

Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser

195 200 205

Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro

210 215 220

Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys

245 250 255

Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp

260 265 270

Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser

305 310 315 320

Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly

325 330 335

Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln

340 345 350

Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe

355 360 365

Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu

370 375 380

Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe

385 390 395 400

Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn

405 410 415

Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr

420 425 430

Glu Lys Ser Leu Ser His Ser Pro Gly

435 440

<210> 28

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 28

Asp Ile Gln Met Thr Gln Ser Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Asn Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Glu Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Gly Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala

100 105 110

Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly

115 120 125

Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

130 135 140

Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

<210> 29

<211> 457

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 29

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr

20 25 30

Met Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Gly Pro Ser Gly Gly Pro Thr His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Gly Tyr Asp Ser Gly Tyr Asp Tyr Val Ala Val Ala Gly Pro Ala

100 105 110

Glu Tyr Phe Gln His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

130 135 140

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

145 150 155 160

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170 175

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

180 185 190

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

195 200 205

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

210 215 220

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

225 230 235 240

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

245 250 255

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

260 265 270

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

275 280 285

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

290 295 300

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

305 310 315 320

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

325 330 335

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

340 345 350

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

355 360 365

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

370 375 380

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

385 390 395 400

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

405 410 415

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

420 425 430

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

435 440 445

Gln Lys Ser Leu Ser Leu Ser Pro Gly

450 455

<210> 30

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 30

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Ser Leu Thr Ile Ser Gly Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 31

<211> 1255

<212> PROTEIN

<213> Homo sapiens

<400> 31

Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu

1 5 10 15

Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys

20 25 30

Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His

35 40 45

Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr

50 55 60

Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val

65 70 75 80

Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu

85 90 95

Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr

100 105 110

Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro

115 120 125

Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser

130 135 140

Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln

145 150 155 160

Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn

165 170 175

Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys

180 185 190

His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser

195 200 205

Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys

210 215 220

Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys

225 230 235 240

Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu

245 250 255

His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val

260 265 270

Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg

275 280 285

Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu

290 295 300

Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln

305 310 315 320

Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys

325 330 335

Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu

340 345 350

Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys

355 360 365

Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp

370 375 380

Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe

385 390 395 400

Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro

405 410 415

Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg

420 425 430

Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu

435 440 445

Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly

450 455 460

Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val

465 470 475 480

Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr

485 490 495

Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His

500 505 510

Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys

515 520 525

Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys

530 535 540

Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys

545 550 555 560

Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys

565 570 575

Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp

580 585 590

Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu

595 600 605

Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln

610 615 620

Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys

625 630 635 640

Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser

645 650 655

Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly

660 665 670

Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg

675 680 685

Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly

690 695 700

Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu

705 710 715 720

Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys

725 730 735

Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile

740 745 750

Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu

755 760 765

Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg

770 775 780

Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu

785 790 795 800

Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg

805 810 815

Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly

820 825 830

Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala

835 840 845

Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe

850 855 860

Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp

865 870 875 880

Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg

885 890 895

Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val

900 905 910

Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala

915 920 925

Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro

930 935 940

Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met

945 950 955 960

Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe

965 970 975

Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu

980 985 990

Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu

995 1000 1005

Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr

1010 1015 1020

Leu Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly

1025 1030 1035

Ala Gly Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg

1040 1045 1050

Ser Gly Gly Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu

1055 1060 1065

Glu Ala Pro Arg Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser

1070 1075 1080

Asp Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu

1085 1090 1095

Gln Ser Leu Pro Thr His Asp Pro Ser Pro Leu Gln Arg Tyr Ser

1100 1105 1110

Glu Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp Gly Tyr Val

1115 1120 1125

Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu Tyr Val Asn Gln Pro

1130 1135 1140

Asp Val Arg Pro Gln Pro Pro Ser Pro Arg Glu Gly Pro Leu Pro

1145 1150 1155

Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu Arg Pro Lys Thr Leu

1160 1165 1170

Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala Phe Gly

1175 1180 1185

Gly Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly Ala

1190 1195 1200

Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp

1205 1210 1215

Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala Pro

1220 1225 1230

Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr

1235 1240 1245

Leu Gly Leu Asp Val Pro Val

1250 1255

<210> 32

<211> 310

<212> PROTEIN

<213> Homo sapiens

<400> 32

Met Arg Arg Ala Ala Leu Trp Leu Trp Leu Cys Ala Leu Ala Leu Ser

1 5 10 15

Leu Gln Pro Ala Leu Pro Gln Ile Val Ala Thr Asn Leu Pro Pro Glu

20 25 30

Asp Gln Asp Gly Ser Gly Asp Asp Ser Asp Asn Phe Ser Gly Ser Gly

35 40 45

Ala Gly Ala Leu Gln Asp Ile Thr Leu Ser Gln Gln Thr Pro Ser Thr

50 55 60

Trp Lys Asp Thr Gln Leu Leu Thr Ala Ile Pro Thr Ser Pro Glu Pro

65 70 75 80

Thr Gly Leu Glu Ala Thr Ala Ala Ser Thr Ser Thr Leu Pro Ala Gly

85 90 95

Glu Gly Pro Lys Glu Gly Glu Ala Val Val Leu Pro Glu Val Glu Pro

100 105 110

Gly Leu Thr Ala Arg Glu Gln Glu Ala Thr Pro Arg Pro Arg Glu Thr

115 120 125

Thr Gln Leu Pro Thr Thr His Leu Ala Ser Thr Thr Thr Ala Thr Thr

130 135 140

Ala Gln Glu Pro Ala Thr Ser His Pro His Arg Asp Met Gln Pro Gly

145 150 155 160

His His Glu Thr Ser Thr Pro Ala Gly Pro Ser Gln Ala Asp Leu His

165 170 175

Thr Pro His Thr Glu Asp Gly Gly Pro Ser Ala Thr Glu Arg Ala Ala

180 185 190

Glu Asp Gly Ala Ser Ser Gln Leu Pro Ala Ala Glu Gly Ser Gly Glu

195 200 205

Gln Asp Phe Thr Phe Glu Thr Ser Gly Glu Asn Thr Ala Val Val Ala

210 215 220

Val Glu Pro Asp Arg Arg Asn Gln Ser Pro Val Asp Gln Gly Ala Thr

225 230 235 240

Gly Ala Ser Gln Gly Leu Leu Asp Arg Lys Glu Val Leu Gly Gly Val

245 250 255

Ile Ala Gly Gly Leu Val Gly Leu Ile Phe Ala Val Cys Leu Val Gly

260 265 270

Phe Met Leu Tyr Arg Met Lys Lys Lys Asp Glu Gly Ser Tyr Ser Leu

275 280 285

Glu Glu Pro Lys Gln Ala Asn Gly Gly Ala Tyr Gln Lys Pro Thr Lys

290 295 300

Gln Glu Glu Phe Tyr Ala

305 310

<210> 33

<211> 976

<212> PROTEIN

<213> Homo sapiens

<400> 33

Met Glu Leu Gln Ala Ala Arg Ala Cys Phe Ala Leu Leu Trp Gly Cys

1 5 10 15

Ala Leu Ala Ala Ala Ala Ala Ala Gln Gly Lys Glu Val Val Leu Leu

20 25 30

Asp Phe Ala Ala Ala Gly Gly Glu Leu Gly Trp Leu Thr His Pro Tyr

35 40 45

Gly Lys Gly Trp Asp Leu Met Gln Asn Ile Met Asn Asp Met Pro Ile

50 55 60

Tyr Met Tyr Ser Val Cys Asn Val Met Ser Gly Asp Gln Asp Asn Trp

65 70 75 80

Leu Arg Thr Asn Trp Val Tyr Arg Gly Glu Ala Glu Arg Ile Phe Ile

85 90 95

Glu Leu Lys Phe Thr Val Arg Asp Cys Asn Ser Phe Pro Gly Gly Ala

100 105 110

Ser Ser Cys Lys Glu Thr Phe Asn Leu Tyr Tyr Ala Glu Ser Asp Leu

115 120 125

Asp Tyr Gly Thr Asn Phe Gln Lys Arg Leu Phe Thr Lys Ile Asp Thr

130 135 140

Ile Ala Pro Asp Glu Ile Thr Val Ser Ser Asp Phe Glu Ala Arg His

145 150 155 160

Val Lys Leu Asn Val Glu Glu Arg Ser Val Gly Pro Leu Thr Arg Lys

165 170 175

Gly Phe Tyr Leu Ala Phe Gln Asp Ile Gly Ala Cys Val Ala Leu Leu

180 185 190

Ser Val Arg Val Tyr Tyr Lys Lys Cys Pro Glu Leu Leu Gln Gly Leu

195 200 205

Ala His Phe Pro Glu Thr Ile Ala Gly Ser Asp Ala Pro Ser Leu Ala

210 215 220

Thr Val Ala Gly Thr Cys Val Asp His Ala Val Val Pro Pro Gly Gly

225 230 235 240

Glu Glu Pro Arg Met His Cys Ala Val Asp Gly Glu Trp Leu Val Pro

245 250 255

Ile Gly Gln Cys Leu Cys Gln Ala Gly Tyr Glu Lys Val Glu Asp Ala

260 265 270

Cys Gln Ala Cys Ser Pro Gly Phe Phe Lys Phe Glu Ala Ser Glu Ser

275 280 285

Pro Cys Leu Glu Cys Pro Glu His Thr Leu Pro Ser Pro Glu Gly Ala

290 295 300

Thr Ser Cys Glu Cys Glu Glu Gly Phe Phe Arg Ala Pro Gln Asp Pro

305 310 315 320

Ala Ser Met Pro Cys Thr Arg Pro Pro Ser Ala Pro His Tyr Leu Thr

325 330 335

Ala Val Gly Met Gly Ala Lys Val Glu Leu Arg Trp Thr Pro Pro Gln

340 345 350

Asp Ser Gly Gly Arg Glu Asp Ile Val Tyr Ser Val Thr Cys Glu Gln

355 360 365

Cys Trp Pro Glu Ser Gly Glu Cys Gly Pro Cys Glu Ala Ser Val Arg

370 375 380

Tyr Ser Glu Pro Pro His Gly Leu Thr Arg Thr Ser Val Thr Val Ser

385 390 395 400

Asp Leu Glu Pro His Met Asn Tyr Thr Phe Thr Val Glu Ala Arg Asn

405 410 415

Gly Val Ser Gly Leu Val Thr Ser Arg Ser Phe Arg Thr Ala Ser Val

420 425 430

Ser Ile Asn Gln Thr Glu Pro Pro Lys Val Arg Leu Glu Gly Arg Ser

435 440 445

Thr Thr Ser Leu Ser Val Ser Trp Ser Ile Pro Pro Pro Gln Gln Ser

450 455 460

Arg Val Trp Lys Tyr Glu Val Thr Tyr Arg Lys Lys Gly Asp Ser Asn

465 470 475 480

Ser Tyr Asn Val Arg Arg Thr Glu Gly Phe Ser Val Thr Leu Asp Asp

485 490 495

Leu Ala Pro Asp Thr Thr Tyr Leu Val Gln Val Gln Ala Leu Thr Gln

500 505 510

Glu Gly Gln Gly Ala Gly Ser Lys Val His Glu Phe Gln Thr Leu Ser

515 520 525

Pro Glu Gly Ser Gly Asn Leu Ala Val Ile Gly Gly Val Ala Val Gly

530 535 540

Val Val Leu Leu Leu Val Leu Ala Gly Val Gly Phe Phe Ile His Arg

545 550 555 560

Arg Arg Lys Asn Gln Arg Ala Arg Gln Ser Pro Glu Asp Val Tyr Phe

565 570 575

Ser Lys Ser Glu Gln Leu Lys Pro Leu Lys Thr Tyr Val Asp Pro His

580 585 590

Thr Tyr Glu Asp Pro Asn Gln Ala Val Leu Lys Phe Thr Thr Glu Ile

595 600 605

His Pro Ser Cys Val Thr Arg Gln Lys Val Ile Gly Ala Gly Glu Phe

610 615 620

Gly Glu Val Tyr Lys Gly Met Leu Lys Thr Ser Ser Gly Lys Lys Glu

625 630 635 640

Val Pro Val Ala Ile Lys Thr Leu Lys Ala Gly Tyr Thr Glu Lys Gln

645 650 655

Arg Val Asp Phe Leu Gly Glu Ala Gly Ile Met Gly Gln Phe Ser His

660 665 670

His Asn Ile Ile Arg Leu Glu Gly Val Ile Ser Lys Tyr Lys Pro Met

675 680 685

Met Ile Ile Thr Glu Tyr Met Glu Asn Gly Ala Leu Asp Lys Phe Leu

690 695 700

Arg Glu Lys Asp Gly Glu Phe Ser Val Leu Gln Leu Val Gly Met Leu

705 710 715 720

Arg Gly Ile Ala Ala Gly Met Lys Tyr Leu Ala Asn Met Asn Tyr Val

725 730 735

His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Asn Ser Asn Leu Val

740 745 750

Cys Lys Val Ser Asp Phe Gly Leu Ser Arg Val Leu Glu Asp Asp Pro

755 760 765

Glu Ala Thr Tyr Thr Thr Ser Gly Gly Lys Ile Pro Ile Arg Trp Thr

770 775 780

Ala Pro Glu Ala Ile Ser Tyr Arg Lys Phe Thr Ser Ala Ser Asp Val

785 790 795 800

Trp Ser Phe Gly Ile Val Met Trp Glu Val Met Thr Tyr Gly Glu Arg

805 810 815

Pro Tyr Trp Glu Leu Ser Asn His Glu Val Met Lys Ala Ile Asn Asp

820 825 830

Gly Phe Arg Leu Pro Thr Pro Met Asp Cys Pro Ser Ala Ile Tyr Gln

835 840 845

Leu Met Met Gln Cys Trp Gln Gln Glu Arg Ala Arg Arg Pro Lys Phe

850 855 860

Ala Asp Ile Val Ser Ile Leu Asp Lys Leu Ile Arg Ala Pro Asp Ser

865 870 875 880

Leu Lys Thr Leu Ala Asp Phe Asp Pro Arg Val Ser Ile Arg Leu Pro

885 890 895

Ser Thr Ser Gly Ser Glu Gly Val Pro Phe Arg Thr Val Ser Glu Trp

900 905 910

Leu Glu Ser Ile Lys Met Gln Gln Tyr Thr Glu His Phe Met Ala Ala

915 920 925

Gly Tyr Thr Ala Ile Glu Lys Val Val Gln Met Thr Asn Asp Asp Ile

930 935 940

Lys Arg Ile Gly Val Arg Leu Pro Gly His Gln Lys Arg Ile Ala Tyr

945 950 955 960

Ser Leu Leu Gly Leu Lys Asp Gln Val Asn Thr Val Gly Ile Pro Ile

965 970 975

<210> 34

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 34

Gly Phe Gly Gly

1

<210> 35

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 35

Gly Phe Leu Gly

1

<210> 36

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 36

Ala Leu Ala Leu

1

<210> 37

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 37

Ser Pro Thr Leu Pro Pro Arg Ser Leu

15

<210> 38

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 38

His Pro Ser Ile Lys Arg Gly Leu Ser Ser Leu

1 5 10

<210> 39

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 39

Leu Leu Leu Pro His His Val Leu

15

<210> 40

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 40

Arg Thr Ala Pro Gly Val Arg Pro Pro Phe

1 5 10

<210> 41

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 41

Arg Pro Gln Lys Ser Ile Gln Ala Leu

15

<210> 42

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 42

Ala Pro Ala Pro Pro Pro Leu Pro Ala

15

<210> 43

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 43

Arg Pro Arg Pro Ser Phe Pro Val Ser Leu

1 5 10

<210> 44

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 44

Arg Pro Lys His Gly Asp Gly Phe Ser Leu

1 5 10

<210> 45

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 45

Gly Pro Ala Pro Gly Lys Thr Gly Leu

15

<210> 46

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 46

Glu Ala Ala Arg Lys Gly Asn Ser Leu

15

<210> 47

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 47

Arg Ile Lys Glu Lys Ile Glu Glu Leu

15

<210> 48

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 48

Glu Ile Lys Lys Arg Phe Arg Gln Phe

15

<210> 49

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 49

His Glu Ser Ala Ala Met Ala Glu Thr

15

<210> 50

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 50

Ala Leu Lys Leu Lys Gln Val Gly Val

15

<210> 51

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 51

Asp Leu Lys Lys Arg His Ile Thr Phe

15

<210> 52

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 52

Asp Val Lys Arg Asn Asp Ile Ala Met

15

<210> 53

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 53

Ile Pro Ser Asp His Ile Leu Thr Pro Ala

1 5 10

<210> 54

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 54

Thr Val Phe Ser Thr Ser Ser Leu Lys

15

<210> 55

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 55

Ile Thr Ser Cys Leu Leu Asn Phe

15

<210> 56

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 56

Arg Ala Ser Pro Val Arg Gly Gln Leu

15

<210> 57

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 57

Val Val Arg Lys Pro Val Ile Ala Leu

15

<210> 58

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 58

Leu Leu Ser Glu Lys Lys Lys Ile Ser

15

<210> 59

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 59

Ala Pro Ala Ser Lys Pro Arg Pro Arg Leu

1 5 10

<210> 60

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 60

Arg Tyr Gly Gln Leu Ser Glu Lys Phe

15

<210> 61

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 61

Val Tyr Ile Ser Asn Val Ser Lys Leu

15

<210> 62

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 62

Leu Pro Thr Lys Glu Thr Pro Ser Phe

15

<210> 63

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 63

Gly Glu Ala Pro Pro Pro Pro Pro Ala

15

<210> 64

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 64

Leu Glu Glu Ile Ser Lys Gln Glu Ile

15

<210> 65

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 65

Ile Tyr Asn His Ile Thr Val Lys Ile

15

<210> 66

<211> 160

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 66

Val Asp Leu Glu Pro Thr Val Ile Gly Glu Leu Thr Ser Val Thr Gln

1 5 10 15

Val Arg Ser Gln Gly Ala Gly Thr Gly Gly Leu Ser Trp Gly Gly Ser

20 25 30

Ala Gly His Ser Pro Thr Leu Pro Pro Arg Ser Leu Ser Leu Leu Leu

35 40 45

Leu Pro His His Val Leu Gln Met Lys Phe Ala Leu Ala Leu Thr Ala

50 55 60

Ser Ser Ser Thr Leu Ser Asn Ser Ser Gln Ala Arg Lys Met Leu Pro

65 70 75 80

Ile Thr Met Pro Glu Gly Thr Thr Pro Leu Ala Arg Arg Ser Leu Thr

85 90 95

Ser Cys Trp Thr Glu Phe Ala Ser Trp Leu Thr Ser Ala Pro Val Phe

100 105 110

Arg Ala Ser Trp Phe Ser Thr Ala Leu Val Gly Glu Leu Val Leu Gly

115 120 125

Ser Pro Arg Cys Ser Trp Asn Val Ser Gln Leu Ile Met Ala Arg Ser

130 135 140

Pro Ser Trp Ser Ser Pro Phe Thr Arg Arg Pro Arg Phe Pro Gln Leu

145 150 155 160

<210> 67

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 67

Ala Pro Pro Arg Ser His Pro Ser Ile Lys Arg Gly Leu Ser Ser Leu

1 5 10 15

<210> 68

<211> 28

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 68

Met Val Arg Arg Ala Arg Trp Pro Gly Gly Arg Gly Glu Ala Arg Lys

1 5 10 15

Ala Pro Arg Thr Ala Pro Gly Val Arg Pro Pro Phe

20 25

<210> 69

<211> 199

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 69

Trp Val Asn Cys Leu Phe Val Ser Gly Arg Ala Ala Ala Gly Gly Gly

1 5 10 15

Gly Gly Gly Ala Val Pro Pro Tyr Leu Glu Leu Ala Gly Pro Pro Phe

20 25 30

Leu Leu Leu Thr Leu Ile Arg Ile Gly Leu Gly Arg Arg Ser Gly Arg

35 40 45

Ala Gly Gly Arg Ala Gly Thr Gln Cys Gly Gly Glu Arg Gly Pro Gly

50 55 60

Phe Ala Ala Phe Arg Pro Leu Arg Pro Phe Arg Arg Leu Arg Val Cys

65 70 75 80

Ala Val Cys Val Arg Gly Ser Ala Leu Gly Arg Ser Val Gly Leu Pro

85 90 95

Arg Gly Gly Ala Ala Gly Ala Pro Phe Ser Ser Ser Pro Ala Pro His

100 105 110

Pro Arg Arg Val Leu Cys Arg Cys Leu Leu Phe Leu Phe Phe Ser Cys

115 120 125

His Asp Arg Arg Gly Asp Ser Gln Pro Tyr Gln Val Pro Ala Glu Ala

130 135 140

Gly Val Glu Gly Leu Glu Gly Ala Gly Gly Gly Arg Glu Gly Leu Leu

145 150 155 160

Leu Glu Arg Arg Pro Gln Lys Ser Ile Gln Ala Leu Arg Cys Asn Thr

165 170 175

Ser Glu Thr Ser Thr Ala Asp Pro Leu Lys Ile Pro Gly Leu Val Pro

180 185 190

Leu Ala Leu Ser Ser Lys Val

195

<210> 70

<211> 101

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 70

Met Pro Leu Pro Val Gln Val Phe Asn Leu Gln Val Thr Ser Arg Gly

1 5 10 15

Arg Pro Gly Pro Pro Arg Pro Arg Ala Pro Arg His Trp Gly Arg Ala

20 25 30

Glu Val Glu Gln Gly Arg Gly Ala Cys Ala Arg Ser Arg Ser Gly Thr

35 40 45

Leu Arg Ala Gly Pro Pro Arg Ala Ala Arg Val Gly Gly Cys Arg Ala

50 55 60

Glu Gly Ala Ser Pro Pro Trp Leu Arg Ala Ala Ile Gly Gly Arg Arg

65 70 75 80

Ala Ala Pro Ala Pro Pro Pro Leu Pro Ala Ala His Gly Arg Gly Ser

85 90 95

Arg Pro Pro Arg Arg

100

<210> 71

<211> 162

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 71

Gln Pro Ala Gln Pro Arg Thr Gly Ala Pro Ala Arg Arg Pro Arg Pro

1 5 10 15

Arg Pro Ser Phe Pro Val Ser Leu Arg Ser Ala Ala Pro Pro Thr Gly

20 25 30

Thr Ala Gly Gly Thr Gly Arg Phe Val Leu Arg Pro Gly Glu Ser Gly

35 40 45

Ala Gly Gly Gly Gly Asp Ala Trp Asp Thr Gly Leu Gln Ala Arg Arg

50 55 60

Gly Thr Ala Ala Gly Thr Ser Gly Ala Pro Asn Arg Ser Gln Leu Ser

65 70 75 80

Ser Leu Thr Phe Pro Ala Gln Leu Arg Arg Ile Gly Val Ser Gly Arg

85 90 95

Lys Pro Gly Ala Gly Gly Arg Leu Gly Pro Gly Ser Arg Thr Cys Ala

100 105 110

Pro Arg Cys Leu Pro Arg Ala Arg Arg Gly Pro Gly Ala His Pro Arg

115 120 125

Gly Gly Arg Cys Pro Pro Ala Glu Thr Ala Leu Phe Arg Glu Ala Glu

130 135 140

Glu Gly Thr Gln Lys Tyr Ser Leu Pro Ser Asp Pro Ala Gly Gln Ala

145 150 155 160

Ala Phe

<210> 72

<211> 39

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 72

Phe Arg Leu His Thr Gly Pro Val Ser Pro Val Gly Gly Arg Arg Gln

1 5 10 15

Met Gly Arg Pro Lys His Gly Asp Gly Phe Ser Leu Gln Val Cys Ser

20 25 30

Phe Ile Met Glu Gln Asn Gly

35

<210> 73

<211> 71

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 73

Gly Val Val Glu Ile Thr Gly Glu Pro Pro Cys Ser Cys Arg Gly Glu

1 5 10 15

Glu Glu Ala Ser Arg Ala Gly Arg Ala Gly Gly Val Arg Leu Lys Arg

20 25 30

Gly Ser Arg Gly Pro Gly Glu Leu Asn Val Gly Pro Ala Pro Gly Lys

35 40 45

Thr Gly Leu Leu Ile Pro Leu Leu Arg Asn Trp Glu Cys Gly Ser Leu

50 55 60

Leu Arg Ala Leu Ser Ala Leu

65 70

<210> 74

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 74

Lys Met Gly Phe Pro Glu Ala Ala Arg Lys Gly Asn Ser Leu

1 5 10

<210> 75

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 75

Leu Glu Ala Arg Ile Lys Glu Lys Ile Glu Glu Leu Gln Gln Ala Leu

1 5 10 15

Ile

<210> 76

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 76

Glu Ile Lys Lys Arg Phe Arg Gln Phe Lys Gln Ala Val Tyr Lys Gln

1 5 10 15

<210> 77

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 77

Ala His Glu Ser Ala Ala Met Ala Glu Thr Leu Gln His Val Pro Ser

1 5 10 15

<210> 78

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 78

Asn Arg Pro Ser Val Gln Ala Ala Leu Lys Leu Lys Gln Val Gly Val

1 5 10 15

<210> 79

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 79

Lys Thr Asp Asp Leu Lys Lys Arg His Ile Thr Phe Thr Leu Gly Cys

1 5 10 15

Gly Ile Cys

<210> 80

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 80

Met Lys Leu Asp Glu Asp Val Lys Arg Asn Asp Ile Ala Met Ala Ile

1 5 10 15

<210> 81

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 81

Asn Ser Ile Ser Gln Ile Pro Ser Asp His Ile Leu Thr Pro Ala Leu

1 5 10 15

Phe Ile Thr Phe Met Thr Ile Leu Asp Leu

20 25

<210> 82

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 82

Thr Val Phe Ser Thr Ser Ser Leu Lys Leu Asn Gln Pro Gln Lys Tyr

1 5 10 15

Leu Lys Met Lys Ser Trp Pro Cys

20

<210> 83

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 83

Ala Glu Glu Asp Arg Arg Lys Lys Val Ile Thr Ser Cys Leu Leu Asn

1 5 10 15

Phe Asn Leu Ser Lys Ala Gln Ser

20

<210> 84

<211> 34

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 84

Arg Ser Phe Ser Thr Ser Ala Gln Val Gly Gln Thr Arg Gly Gly Leu

1 5 10 15

Gln Ala Glu Ala Pro Arg Pro Gly Pro Arg Ala Ser Pro Val Arg Gly

20 25 30

Gln Leu

<210> 85

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 85

Arg Gly Tyr Val Val Arg Lys Pro Val Ile Ala Leu Ser Val Lys Ile

1 5 10 15

<210> 86

<211> 86

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 86

Val Asp Met Asp Phe Gly Thr Gly Gly Gln Gly Ala Gly Pro Val Gly

1 5 10 15

Arg Gly Lys Asp Trp Ser Cys Thr Leu Ala Val His Leu Leu Ser Glu

20 25 30

Lys Lys Lys Ile Ser Phe Ser Gln Ile Asp Arg Ala Trp Gly Gly Ser

35 40 45

Gln Gly Thr Val Leu Asp Lys Trp Gly Pro Gly Val Val Ser Glu Leu

50 55 60

His Pro Ser Ala Lys Glu Val Ser Val Gly Arg Asn Ser Val Glu Ser

65 70 75 80

Leu Met Thr Trp Ala Ser

85

<210> 87

<211> 66

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 87

Glu Lys Gly Ser His Glu Glu Glu Val Arg Val Pro Ala Leu Ser Trp

1 5 10 15

Gly Arg Pro Arg Ala Pro Ala Pro Ala Ser Lys Pro Arg Pro Arg Leu

20 25 30

Asp Leu Asn Cys Leu Trp Leu Arg Pro Gln Pro Ile Phe Leu Trp Lys

35 40 45

Leu Arg Pro Arg Pro Val Pro Ala Ala Thr Pro Leu Thr Gly Pro Leu

50 55 60

ProLeu

65

<210> 88

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 88

Arg Tyr Gly Gln Leu Ser Glu Lys Phe Asn Arg Arg Lys Val Met Asp

1 5 10 15

Ser

<210> 89

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 89

Met Val Tyr Ile Ser Asn Val Ser Lys Leu Cys Phe Ser Lys Met

1 5 10 15

<210> 90

<211> 48

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 90

Asn Thr Leu Pro Thr Lys Glu Thr Pro Ser Phe Leu Leu Asn Pro His

1 5 10 15

Thr Ser Trp Val Pro Arg Pro His Arg Glu Ala Pro Arg Leu Arg Val

20 25 30

Gly Val Ala Ala Pro Leu Gln Arg Pro Leu Pro Ala Leu His Ser His

35 40 45

<210> 91

<211> 58

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

polypeptide"

<400> 91

Phe Gly Asp Ile Tyr Leu Gly Glu Ala Pro Pro Pro Pro Ala Ala

1 5 10 15

Arg Arg Pro Gly Pro Cys Gly Cys Gln Asp Gln Ala Arg Ser Arg Lys

20 25 30

Glu Val Val Ala Pro Ala Gly Ser Pro Arg Lys Ser Arg His Arg Arg

35 40 45

Ile Val Ala Arg Thr Gln Arg Pro Leu Gly

50 55

<210> 92

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 92

Gly Ser Ala Ser Asp Leu Leu Glu Glu Ile Ser Lys Gln Glu Ile Ser

1 5 10 15

Phe

<210> 93

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

peptide"

<400> 93

Gln Leu Ile Tyr Asn His Ile Thr Val Lys Ile Asn Leu Gln Gly Asp

1 5 10 15

<210> 94

<211> 630

<212> PROTEIN

<213> Homo sapiens

<400> 94

Met Ala Leu Pro Thr Ala Arg Pro Leu Leu Gly Ser Cys Gly Thr Pro

1 5 10 15

Ala Leu Gly Ser Leu Leu Phe Leu Leu Phe Ser Leu Gly Trp Val Gln

20 25 30

Pro Ser Arg Thr Leu Ala Gly Glu Thr Gly Gln Glu Ala Ala Pro Leu

35 40 45

Asp Gly Val Leu Ala Asn Pro Pro Asn Ile Ser Ser Leu Ser Pro Arg

50 55 60

Gln Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu Ser Thr Glu

65 70 75 80

Arg Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys Asn Val Lys Leu

85 90 95

Ser Thr Glu Gln Leu Arg Cys Leu Ala His Arg Leu Ser Glu Pro Pro

100 105 110

Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu Leu Leu Phe Leu Asn Pro

115 120 125

Asp Ala Phe Ser Gly Pro Gln Ala Cys Thr Arg Phe Phe Ser Arg Ile

130 135 140

Thr Lys Ala Asn Val Asp Leu Leu Pro Arg Gly Ala Pro Glu Arg Gln

145 150 155 160

Arg Leu Leu Pro Ala Ala Leu Ala Cys Trp Gly Val Arg Gly Ser Leu

165 170 175

Leu Ser Glu Ala Asp Val Arg Ala Leu Gly Gly Leu Ala Cys Asp Leu

180 185 190

Pro Gly Arg Phe Val Ala Glu Ser Ala Glu Val Leu Leu Pro Arg Leu

195 200 205

Val Ser Cys Pro Gly Pro Leu Asp Gln Asp Gln Gln Glu Ala Ala Arg

210 215 220

Ala Ala Leu Gln Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser Thr Trp

225 230 235 240

Ser Val Ser Thr Met Asp Ala Leu Arg Gly Leu Leu Pro Val Leu Gly

245 250 255

Gln Pro Ile Ile Arg Ser Ile Pro Gln Gly Ile Val Ala Ala Trp Arg

260 265 270

Gln Arg Ser Ser Arg Asp Pro Ser Trp Arg Gln Pro Glu Arg Thr Ile

275 280 285

Leu Arg Pro Arg Phe Arg Arg Glu Val Glu Lys Thr Ala Cys Pro Ser

290 295 300

Gly Lys Lys Ala Arg Glu Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys

305 310 315 320

Trp Glu Leu Glu Ala Cys Val Asp Ala Ala Leu Leu Ala Thr Gln Met

325 330 335

Asp Arg Val Asn Ala Ile Pro Phe Thr Tyr Glu Gln Leu Asp Val Leu

340 345 350

Lys His Lys Leu Asp Glu Leu Tyr Pro Gln Gly Tyr Pro Glu Ser Val

355 360 365

Ile Gln His Leu Gly Tyr Leu Phe Leu Lys Met Ser Pro Glu Asp Ile

370 375 380

Arg Lys Trp Asn Val Thr Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu

385 390 395 400

Val Asn Lys Gly His Glu Met Ser Pro Gln Ala Pro Arg Arg Pro Leu

405 410 415

Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val Lys Gly Arg Gly Gln

420 425 430

Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala Phe Tyr Pro Gly Tyr

435 440 445

Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser Val Pro Pro Ser Ser

450 455 460

Ile Trp Ala Val Arg Pro Gln Asp Leu Asp Thr Cys Asp Pro Arg Gln

465 470 475 480

Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala Phe Gln Asn Met Asn

485 490 495

Gly Ser Glu Tyr Phe Val Lys Ile Gln Ser Phe Leu Gly Gly Ala Pro

500 505 510

Thr Glu Asp Leu Lys Ala Leu Ser Gln Gln Asn Val Ser Met Asp Leu

515 520 525

Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val Leu Pro Leu Thr Val

530 535 540

Ala Glu Val Gln Lys Leu Leu Gly Pro His Val Glu Gly Leu Lys Ala

545 550 555 560

Glu Glu Arg His Arg Pro Val Arg Asp Trp Ile Leu Arg Gln Arg Gln

565 570 575

Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gln Gly Gly Ile Pro Asn

580 585 590

Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu Ala Leu Ser Gly Thr

595 600 605

Pro Cys Leu Leu Gly Pro Gly Pro Val Leu Thr Val Leu Ala Leu Leu

610 615 620

Leu Ala Ser Thr Leu Ala

625 630

<210> 95

<211> 750

<212> PROTEIN

<213> Homo sapiens

<400> 95

Met Trp Asn Leu Leu His Glu Thr Asp Ser Ala Val Ala Thr Ala Arg

1 5 10 15

Arg Pro Arg Trp Leu Cys Ala Gly Ala Leu Val Leu Ala Gly Gly Phe

20 25 30

Phe Leu Leu Gly Phe Leu Phe Gly Trp Phe Ile Lys Ser Ser Asn Glu

35 40 45

Ala Thr Asn Ile Thr Pro Lys His Asn Met Lys Ala Phe Leu Asp Glu

50 55 60

Leu Lys Ala Glu Asn Ile Lys Lys Phe Leu Tyr Asn Phe Thr Gln Ile

65 70 75 80

Pro His Leu Ala Gly Thr Glu Gln Asn Phe Gln Leu Ala Lys Gln Ile

85 90 95

Gln Ser Gln Trp Lys Glu Phe Gly Leu Asp Ser Val Glu Leu Ala His

100 105 110

Tyr Asp Val Leu Leu Ser Tyr Pro Asn Lys Thr His Pro Asn Tyr Ile

115 120 125

Ser Ile Ile Asn Glu Asp Gly Asn Glu Ile Phe Asn Thr Ser Leu Phe

130 135 140

Glu Pro Pro Pro Pro Gly Tyr Glu Asn Val Ser Asp Ile Val Pro Pro

145 150 155 160

Phe Ser Ala Phe Ser Pro Gln Gly Met Pro Glu Gly Asp Leu Val Tyr

165 170 175

Val Asn Tyr Ala Arg Thr Glu Asp Phe Phe Lys Leu Glu Arg Asp Met

180 185 190

Lys Ile Asn Cys Ser Gly Lys Ile Val Ile Ala Arg Tyr Gly Lys Val

195 200 205

Phe Arg Gly Asn Lys Val Lys Asn Ala Gln Leu Ala Gly Ala Lys Gly

210 215 220

Val Ile Leu Tyr Ser Asp Pro Ala Asp Tyr Phe Ala Pro Gly Val Lys

225 230 235 240

Ser Tyr Pro Asp Gly Trp Asn Leu Pro Gly Gly Gly Val Gln Arg Gly

245 250 255

Asn Ile Leu Asn Leu Asn Gly Ala Gly Asp Pro Leu Thr Pro Gly Tyr

260 265 270

Pro Ala Asn Glu Tyr Ala Tyr Arg Arg Gly Ile Ala Glu Ala Val Gly

275 280 285

Leu Pro Ser Ile Pro Val His Pro Ile Gly Tyr Tyr Asp Ala Gln Lys

290 295 300

Leu Leu Glu Lys Met Gly Gly Ser Ala Pro Pro Asp Ser Ser Trp Arg

305 310 315 320

Gly Ser Leu Lys Val Pro Tyr Asn Val Gly Pro Gly Phe Thr Gly Asn

325 330 335

Phe Ser Thr Gln Lys Val Lys Met His Ile His Ser Thr Asn Glu Val

340 345 350

Thr Arg Ile Tyr Asn Val Ile Gly Thr Leu Arg Gly Ala Val Glu Pro

355 360 365

Asp Arg Tyr Val Ile Leu Gly Gly His Arg Asp Ser Trp Val Phe Gly

370 375 380

Gly Ile Asp Pro Gln Ser Gly Ala Ala Val Val His Glu Ile Val Arg

385 390 395 400

Ser Phe Gly Thr Leu Lys Lys Glu Gly Trp Arg Pro Arg Arg Thr Ile

405 410 415

Leu Phe Ala Ser Trp Asp Ala Glu Glu Phe Gly Leu Leu Gly Ser Thr

420 425 430

Glu Trp Ala Glu Glu Asn Ser Arg Leu Leu Gln Glu Arg Gly Val Ala

435 440 445

Tyr Ile Asn Ala Asp Ser Ser Ile Glu Gly Asn Tyr Thr Leu Arg Val

450 455 460

Asp Cys Thr Pro Leu Met Tyr Ser Leu Val His Asn Leu Thr Lys Glu

465 470 475 480

Leu Lys Ser Pro Asp Glu Gly Phe Glu Gly Lys Ser Leu Tyr Glu Ser

485 490 495

Trp Thr Lys Lys Ser Pro Ser Pro Glu Phe Ser Gly Met Pro Arg Ile

500 505 510

Ser Lys Leu Gly Ser Gly Asn Asp Phe Glu Val Phe Phe Gln Arg Leu

515 520 525

Gly Ile Ala Ser Gly Arg Ala Arg Tyr Thr Lys Asn Trp Glu Thr Asn

530 535 540

Lys Phe Ser Gly Tyr Pro Leu Tyr His Ser Val Tyr Glu Thr Tyr Glu

545 550 555 560

Leu Val Glu Lys Phe Tyr Asp Pro Met Phe Lys Tyr His Leu Thr Val

565 570 575

Ala Gln Val Arg Gly Gly Met Val Phe Glu Leu Ala Asn Ser Ile Val

580 585 590

Leu Pro Phe Asp Cys Arg Asp Tyr Ala Val Val Leu Arg Lys Tyr Ala

595 600 605

Asp Lys Ile Tyr Ser Ile Ser Met Lys His Pro Gln Glu Met Lys Thr

610 615 620

Tyr Ser Val Ser Phe Asp Ser Leu Phe Ser Ala Val Lys Asn Phe Thr

625 630 635 640

Glu Ile Ala Ser Lys Phe Ser Glu Arg Leu Gln Asp Phe Asp Lys Ser

645 650 655

Asn Pro Ile Val Leu Arg Met Met Asn Asp Gln Leu Met Phe Leu Glu

660 665 670

Arg Ala Phe Ile Asp Pro Leu Gly Leu Pro Asp Arg Pro Phe Tyr Arg

675 680 685

His Val Ile Tyr Ala Pro Ser Ser His Asn Lys Tyr Ala Gly Glu Ser

690 695 700

Phe Pro Gly Ile Tyr Asp Ala Leu Phe Asp Ile Glu Ser Lys Val Asp

705 710 715 720

Pro Ser Lys Ala Trp Gly Glu Val Lys Arg Gln Ile Tyr Val Ala Ala

725 730 735

Phe Thr Val Gln Ala Ala Ala Glu Thr Leu Ser Glu Val Ala

740 745 750

<210> 96

<211> 790

<212> PROTEIN

<213> Homo sapiens

<400> 96

Met Arg Thr Tyr Arg Tyr Phe Leu Leu Leu Phe Trp Val Gly Gln Pro

1 5 10 15

Tyr Pro Thr Leu Ser Thr Pro Leu Ser Lys Arg Thr Ser Gly Phe Pro

20 25 30

Ala Lys Lys Arg Ala Leu Glu Leu Ser Gly Asn Ser Lys Asn Glu Leu

35 40 45

Asn Arg Ser Lys Arg Ser Trp Met Trp Asn Gln Phe Phe Leu Leu Glu

50 55 60

Glu Tyr Thr Gly Ser Asp Tyr Gln Tyr Val Gly Lys Leu His Ser Asp

65 70 75 80

Gln Asp Arg Gly Asp Gly Ser Leu Lys Tyr Ile Leu Ser Gly Asp Gly

85 90 95

Ala Gly Asp Leu Phe Ile Ile Asn Glu Asn Thr Gly Asp Ile Gln Ala

100 105 110

Thr Lys Arg Leu Asp Arg Glu Glu Lys Pro Val Tyr Ile Leu Arg Ala

115 120 125

Gln Ala Ile Asn Arg Arg Thr Gly Arg Pro Val Glu Pro Glu Ser Glu

130 135 140

Phe Ile Ile Lys Ile His Asp Ile Asn Asp Asn Glu Pro Ile Phe Thr

145 150 155 160

Lys Glu Val Tyr Thr Ala Thr Val Pro Glu Met Ser Asp Val Gly Thr

165 170 175

Phe Val Val Gln Val Thr Ala Thr Asp Ala Asp Asp Pro Thr Tyr Gly

180 185 190

Asn Ser Ala Lys Val Val Tyr Ser Ile Leu Gln Gly Gln Pro Tyr Phe

195 200 205

Ser Val Glu Ser Glu Thr Gly Ile Ile Lys Thr Ala Leu Leu Asn Met

210 215 220

Asp Arg Glu Asn Arg Glu Gln Tyr Gln Val Val Ile Gln Ala Lys Asp

225 230 235 240

Met Gly Gly Gln Met Gly Gly Leu Ser Gly Thr Thr Thr Val Asn Ile

245 250 255

Thr Leu Thr Asp Val Asn Asp Asn Pro Pro Arg Phe Pro Gln Ser Thr

260 265 270

Tyr Gln Phe Lys Thr Pro Glu Ser Ser Pro Pro Gly Thr Pro Ile Gly

275 280 285

Arg Ile Lys Ala Ser Asp Ala Asp Val Gly Glu Asn Ala Glu Ile Glu

290 295 300

Tyr Ser Ile Thr Asp Gly Glu Gly Leu Asp Met Phe Asp Val Ile Thr

305 310 315 320

Asp Gln Glu Thr Gln Glu Gly Ile Ile Thr Val Lys Lys Leu Leu Asp

325 330 335

Phe Glu Lys Lys Lys Val Tyr Thr Leu Lys Val Glu Ala Ser Asn Pro

340 345 350

Tyr Val Glu Pro Arg Phe Leu Tyr Leu Gly Pro Phe Lys Asp Ser Ala

355 360 365

Thr Val Arg Ile Val Val Glu Asp Val Asp Glu Pro Pro Val Phe Ser

370 375 380

Lys Leu Ala Tyr Ile Leu Gln Ile Arg Glu Asp Ala Gln Ile Asn Thr

385 390 395 400

Thr Ile Gly Ser Val Thr Ala Gln Asp Pro Asp Ala Ala Arg Asn Pro

405 410 415

Val Lys Tyr Ser Val Asp Arg His Thr Asp Met Asp Arg Ile Phe Asn

420 425 430

Ile Asp Ser Gly Asn Gly Ser Ile Phe Thr Ser Lys Leu Leu Asp Arg

435 440 445

Glu Thr Leu Leu Trp His Asn Ile Thr Val Ile Ala Thr Glu Ile Asn

450 455 460

Asn Pro Lys Gln Ser Ser Arg Val Pro Leu Tyr Ile Lys Val Leu Asp

465 470 475 480

Val Asn Asp Asn Ala Pro Glu Phe Ala Glu Phe Tyr Glu Thr Phe Val

485 490 495

Cys Glu Lys Ala Lys Ala Asp Gln Leu Ile Gln Thr Leu His Ala Val

500 505 510

Asp Lys Asp Asp Pro Tyr Ser Gly His Gln Phe Ser Phe Ser Leu Ala

515 520 525

Pro Glu Ala Ala Ser Gly Ser Asn Phe Thr Ile Gln Asp Asn Lys Asp

530 535 540

Asn Thr Ala Gly Ile Leu Thr Arg Lys Asn Gly Tyr Asn Arg His Glu

545 550 555 560

Met Ser Thr Tyr Leu Leu Pro Val Val Ile Ser Asp Asn Asp Tyr Pro

565 570 575

Val Gln Ser Ser Thr Gly Thr Val Thr Val Arg Val Cys Ala Cys Asp

580 585 590

His His Gly Asn Met Gln Ser Cys His Ala Glu Ala Leu Ile His Pro

595 600 605

Thr Gly Leu Ser Thr Gly Ala Leu Val Ala Ile Leu Leu Cys Ile Val

610 615 620

Ile Leu Leu Val Thr Val Val Leu Phe Ala Ala Leu Arg Arg Gln Arg

625 630 635 640

Lys Lys Glu Pro Leu Ile Ile Ser Lys Glu Asp Ile Arg Asp Asn Ile

645 650 655

Val Ser Tyr Asn Asp Glu Gly Gly Gly Glu Glu Asp Thr Gln Ala Phe

660 665 670

Asp Ile Gly Thr Leu Arg Asn Pro Glu Ala Ile Glu Asp Asn Lys Leu

675 680 685

Arg Arg Asp Ile Val Pro Glu Ala Leu Phe Leu Pro Arg Arg Thr Pro

690 695 700

Thr Ala Arg Asp Asn Thr Asp Val Arg Asp Phe Ile Asn Gln Arg Leu

705 710 715 720

Lys Glu Asn Asp Thr Asp Pro Thr Ala Pro Pro Tyr Asp Ser Leu Ala

725 730 735

Thr Tyr Ala Tyr Glu Gly Thr Gly Ser Val Ala Asp Ser Leu Ser Ser

740 745 750

Leu Glu Ser Val Thr Thr Asp Ala Asp Gln Asp Tyr Asp Tyr Leu Ser

755 760 765

Asp Trp Gly Pro Arg Phe Lys Lys Leu Ala Asp Met Tyr Gly Gly Val

770 775 780

Asp Ser Asp Lys Asp Ser

785 790

<210> 97

<211> 702

<212> PROTEIN

<213> Homo sapiens

<400> 97

Met Glu Ser Pro Ser Ala Pro Pro His Arg Trp Cys Ile Pro Trp Gln

1 5 10 15

Arg Leu Leu Leu Thr Ala Ser Leu Leu Thr Phe Trp Asn Pro Pro Thr

20 25 30

Thr Ala Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly

35 40 45

Lys Glu Val Leu Leu Leu Val His Asn Leu Pro Gln His Leu Phe Gly

50 55 60

Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Ile

65 70 75 80

Gly Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser

85 90 95

Gly Arg Glu Ile Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Ile

100 105 110

Ile Gln Asn Asp Thr Gly Phe Tyr Thr Leu His Val Ile Lys Ser Asp

115 120 125

Leu Val Asn Glu Glu Ala Thr Gly Gln Phe Arg Val Tyr Pro Glu Leu

130 135 140

Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu Asp Lys

145 150 155 160

Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Ala Thr Tyr

165 170 175

Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln

180 185 190

Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Val Thr Arg Asn

195 200 205

Asp Thr Ala Ser Tyr Lys Cys Glu Thr Gln Asn Pro Val Ser Ala Arg

210 215 220

Arg Ser Asp Ser Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Ala Pro

225 230 235 240

Thr Ile Ser Pro Leu Asn Thr Ser Tyr Arg Ser Gly Glu Asn Leu Asn

245 250 255

Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe

260 265 270

Val Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn

275 280 285

Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr Cys Gln Ala His Asn Ser

290 295 300

Asp Thr Gly Leu Asn Arg Thr Thr Val Thr Thr Ile Thr Val Tyr Ala

305 310 315 320

Glu Pro Pro Lys Pro Phe Ile Thr Ser Asn Asn Ser Asn Pro Val Glu

325 330 335

Asp Glu Asp Ala Val Ala Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr

340 345 350

Thr Tyr Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg

355 360 365

Leu Gln Leu Ser Asn Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr

370 375 380

Arg Asn Asp Val Gly Pro Tyr Glu Cys Gly Ile Gln Asn Lys Leu Ser

385 390 395 400

Val Asp His Ser Asp Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp

405 410 415

Asp Pro Thr Ile Ser Pro Ser Tyr Thr Tyr Tyr Arg Pro Gly Val Asn

420 425 430

Leu Ser Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser

435 440 445

Trp Leu Ile Asp Gly Asn Ile Gln Gln His Thr Gln Glu Leu Phe Ile

450 455 460

Ser Asn Ile Thr Glu Lys Asn Ser Gly Leu Tyr Thr Cys Gln Ala Asn

465 470 475 480

Asn Ser Ala Ser Gly His Ser Arg Thr Thr Val Lys Thr Ile Thr Val

485 490 495

Ser Ala Glu Leu Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro

500 505 510

Val Glu Asp Lys Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Ala Gln

515 520 525

Asn Thr Thr Tyr Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser

530 535 540

Pro Arg Leu Gln Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn

545 550 555 560

Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Cys Gly Ile Gln Asn Ser

565 570 575

Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu Tyr Gly

580 585 590

Pro Asp Thr Pro Ile Ile Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gly

595 600 605

Ala Asn Leu Asn Leu Ser Cys His Ser Ala Ser Asn Pro Ser Pro Gln

610 615 620

Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu

625 630 635 640

Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe

645 650 655

Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile

660 665 670

Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala Gly Ala Thr

675 680 685

Val Gly Ile Met Ile Gly Val Leu Val Gly Val Ala Leu Ile

690 695 700

<210> 98

<211> 223

<212> PROTEIN

<213> Homo sapiens

<400> 98

Met Thr Trp Arg His His Val Arg Leu Leu Phe Thr Val Ser Leu Ala

1 5 10 15

Leu Gln Ile Ile Asn Leu Gly Asn Ser Tyr Gln Arg Glu Lys His Asn

20 25 30

Gly Gly Arg Glu Glu Val Thr Lys Val Ala Thr Gln Lys His Arg Gln

35 40 45

Ser Pro Leu Asn Trp Thr Ser Ser His Phe Gly Glu Val Thr Gly Ser

50 55 60

Ala Glu Gly Trp Gly Pro Glu Glu Pro Leu Pro Tyr Ser Trp Ala Phe

65 70 75 80

Gly Glu Gly Ala Ser Ala Arg Pro Arg Cys Cys Arg Asn Gly Gly Thr

85 90 95

Cys Val Leu Gly Ser Phe Cys Val Cys Pro Ala His Phe Thr Gly Arg

100 105 110

Tyr Cys Glu His Asp Gln Arg Arg Ser Glu Cys Gly Ala Leu Glu His

115 120 125

Gly Ala Trp Thr Leu Arg Ala Cys His Leu Cys Arg Cys Ile Phe Gly

130 135 140

Ala Leu His Cys Leu Pro Leu Gln Thr Pro Asp Arg Cys Asp Pro Lys

145 150 155 160

Asp Phe Leu Ala Ser His Ala His Gly Pro Ser Ala Gly Gly Ala Pro

165 170 175

Ser Leu Leu Leu Leu Leu Pro Cys Ala Leu Leu His Arg Leu Leu Arg

180 185 190

Pro Asp Ala Pro Ala His Pro Arg Ser Leu Val Pro Ser Val Leu Gln

195 200 205

Arg Glu Arg Arg Pro Cys Gly Arg Pro Gly Leu Gly His Arg Leu

210 215 220

<210> 99

<211> 875

<212> PROTEIN

<213> Homo sapiens

<400> 99

Met Glu Ser Thr Leu Thr Leu Ala Thr Glu Gln Pro Val Lys Lys Asn

1 5 10 15

Thr Leu Lys Lys Tyr Lys Ile Ala Cys Ile Val Leu Leu Ala Leu Leu

20 25 30

Val Ile Met Ser Leu Gly Leu Gly Leu Gly Leu Gly Leu Arg Lys Leu

35 40 45

Glu Lys Gln Gly Ser Cys Arg Lys Lys Cys Phe Asp Ala Ser Phe Arg

50 55 60

Gly Leu Glu Asn Cys Arg Cys Asp Val Ala Cys Lys Asp Arg Gly Asp

65 70 75 80

Cys Cys Trp Asp Phe Glu Asp Thr Cys Val Glu Ser Thr Arg Ile Trp

85 90 95

Met Cys Asn Lys Phe Arg Cys Gly Glu Thr Arg Leu Glu Ala Ser Leu

100 105 110

Cys Ser Cys Ser Asp Asp Cys Leu Gln Arg Lys Asp Cys Cys Ala Asp

115 120 125

Tyr Lys Ser Val Cys Gln Gly Glu Thr Ser Trp Leu Glu Glu Asn Cys

130 135 140

Asp Thr Ala Gln Gln Ser Gln Cys Pro Glu Gly Phe Asp Leu Pro Pro

145 150 155 160

Val Ile Leu Phe Ser Met Asp Gly Phe Arg Ala Glu Tyr Leu Tyr Thr

165 170 175

Trp Asp Thr Leu Met Pro Asn Ile Asn Lys Leu Lys Thr Cys Gly Ile

180 185 190

His Ser Lys Tyr Met Arg Ala Met Tyr Pro Thr Lys Thr Phe Pro Asn

195 200 205

His Tyr Thr Ile Val Thr Gly Leu Tyr Pro Glu Ser His Gly Ile Ile

210 215 220

Asp Asn Asn Met Tyr Asp Val Asn Leu Asn Lys Asn Phe Ser Leu Ser

225 230 235 240

Ser Lys Glu Gln Asn Asn Pro Ala Trp Trp His Gly Gln Pro Met Trp

245 250 255

Leu Thr Ala Met Tyr Gln Gly Leu Lys Ala Ala Thr Tyr Phe Trp Pro

260 265 270

Gly Ser Glu Val Ala Ile Asn Gly Ser Phe Pro Ser Ile Tyr Met Pro

275 280 285

Tyr Asn Gly Ser Val Pro Phe Glu Glu Arg Ile Ser Thr Leu Leu Lys

290 295 300

Trp Leu Asp Leu Pro Lys Ala Glu Arg Pro Arg Phe Tyr Thr Met Tyr

305 310 315 320

Phe Glu Glu Pro Asp Ser Ser Gly His Ala Gly Gly Pro Val Ser Ala

325 330 335

Arg Val Ile Lys Ala Leu Gln Val Val Asp His Ala Phe Gly Met Leu

340 345 350

Met Glu Gly Leu Lys Gln Arg Asn Leu His Asn Cys Val Asn Ile Ile

355 360 365

Leu Leu Ala Asp His Gly Met Asp Gln Thr Tyr Cys Asn Lys Met Glu

370 375 380

Tyr Met Thr Asp Tyr Phe Pro Arg Ile Asn Phe Phe Tyr Met Tyr Glu

385 390 395 400

Gly Pro Ala Pro Arg Ile Arg Ala His Asn Ile Pro His Asp Phe Phe

405 410 415

Ser Phe Asn Ser Glu Glu Ile Val Arg Asn Leu Ser Cys Arg Lys Pro

420 425 430

Asp Gln His Phe Lys Pro Tyr Leu Thr Pro Asp Leu Pro Lys Arg Leu

435 440 445

His Tyr Ala Lys Asn Val Arg Ile Asp Lys Val His Leu Phe Val Asp

450 455 460

Gln Gln Trp Leu Ala Val Arg Ser Lys Ser Asn Thr Asn Cys Gly Gly

465 470 475 480

Gly Asn His Gly Tyr Asn Asn Glu Phe Arg Ser Met Glu Ala Ile Phe

485 490 495

Leu Ala His Gly Pro Ser Phe Lys Glu Lys Thr Glu Val Glu Pro Phe

500 505 510

Glu Asn Ile Glu Val Tyr Asn Leu Met Cys Asp Leu Leu Arg Ile Gln

515 520 525

Pro Ala Pro Asn Asn Gly Thr His Gly Ser Leu Asn His Leu Leu Lys

530 535 540

Val Pro Phe Tyr Glu Pro Ser His Ala Glu Glu Val Ser Lys Phe Ser

545 550 555 560

Val Cys Gly Phe Ala Asn Pro Leu Pro Thr Glu Ser Leu Asp Cys Phe

565 570 575

Cys Pro His Leu Gln Asn Ser Thr Gln Leu Glu Gln Val Asn Gln Met

580 585 590

Leu Asn Leu Thr Gln Glu Glu Ile Thr Ala Thr Val Lys Val Asn Leu

595 600 605

Pro Phe Gly Arg Pro Arg Val Leu Gln Lys Asn Val Asp His Cys Leu

610 615 620

Leu Tyr His Arg Glu Tyr Val Ser Gly Phe Gly Lys Ala Met Arg Met

625 630 635 640

Pro Met Trp Ser Ser Tyr Thr Val Pro Gln Leu Gly Asp Thr Ser Pro

645 650 655

Leu Pro Pro Thr Val Pro Asp Cys Leu Arg Ala Asp Val Arg Val Pro

660 665 670

Pro Ser Glu Ser Gln Lys Cys Ser Phe Tyr Leu Ala Asp Lys Asn Ile

675 680 685

Thr His Gly Phe Leu Tyr Pro Pro Ala Ser Asn Arg Thr Ser Asp Ser

690 695 700

Gln Tyr Asp Ala Leu Ile Thr Ser Asn Leu Val Pro Met Tyr Glu Glu

705 710 715 720

Phe Arg Lys Met Trp Asp Tyr Phe His Ser Val Leu Leu Ile Lys His

725 730 735

Ala Thr Glu Arg Asn Gly Val Asn Val Val Ser Gly Pro Ile Phe Asp

740 745 750

Tyr Asn Tyr Asp Gly His Phe Asp Ala Pro Asp Glu Ile Thr Lys His

755 760 765

Leu Ala Asn Thr Asp Val Pro Ile Pro Thr His Tyr Phe Val Val Leu

770 775 780

Thr Ser Cys Lys Asn Lys Ser His Thr Pro Glu Asn Cys Pro Gly Trp

785 790 795 800

Leu Asp Val Leu Pro Phe Ile Ile Pro His Arg Pro Thr Asn Val Glu

805 810 815

Ser Cys Pro Glu Gly Lys Pro Glu Ala Leu Trp Val Glu Glu Arg Phe

820 825 830

Thr Ala His Ile Ala Arg Val Arg Asp Val Glu Leu Leu Thr Gly Leu

835 840 845

Asp Phe Tyr Gln Asp Lys Val Gln Pro Val Ser Glu Ile Leu Gln Leu

850 855 860

Lys Thr Tyr Leu Pro Thr Phe Glu Thr Thr Ile

865 870 875

<210> 100

<211> 257

<212> PROTEIN

<213> Homo sapiens

<400> 100

Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val

1 5 10 15

Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu

20 25 30

Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly

35 40 45

Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala

50 55 60

Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr

65 70 75 80

Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys

85 90 95

Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn

100 105 110

Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg

115 120 125

Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu

130 135 140

Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp

145 150 155 160

Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln

165 170 175

Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile

180 185 190

Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg

195 200 205

Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu

210 215 220

Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala

225 230 235 240

Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu

245 250 255

Ser

<210> 101

<211> 364

<212> PROTEIN

<213> Homo sapiens

<400> 101

Met His Pro Gln Val Val Ile Leu Ser Leu Ile Leu His Leu Ala Asp

1 5 10 15

Ser Val Ala Gly Ser Val Lys Val Gly Gly Glu Ala Gly Pro Ser Val

20 25 30

Thr Leu Pro Cys His Tyr Ser Gly Ala Val Thr Ser Met Cys Trp Asn

35 40 45

Arg Gly Ser Cys Ser Leu Phe Thr Cys Gln Asn Gly Ile Val Trp Thr

50 55 60

Asn Gly Thr His Val Thr Tyr Arg Lys Asp Thr Arg Tyr Lys Leu Leu

65 70 75 80

Gly Asp Leu Ser Arg Arg Asp Val Ser Leu Thr Ile Glu Asn Thr Ala

85 90 95

Val Ser Asp Ser Gly Val Tyr Cys Cys Arg Val Glu His Arg Gly Trp

100 105 110

Phe Asn Asp Met Lys Ile Thr Val Ser Leu Glu Ile Val Pro Pro Lys

115 120 125

Val Thr Thr Thr Pro Ile Val Thr Thr Val Pro Thr Val Thr Thr Val

130 135 140

Arg Thr Ser Thr Thr Val Pro Thr Thr Thr Thr Val Pro Met Thr Thr

145 150 155 160

Val Pro Thr Thr Thr Val Pro Thr Thr Met Ser Ile Pro Thr Thr Thr

165 170 175

Thr Val Leu Thr Thr Met Thr Val Ser Thr Thr Thr Ser Val Pro Thr

180 185 190

Thr Thr Ser Ile Pro Thr Thr Thr Ser Val Pro Val Thr Thr Thr Val

195 200 205

Ser Thr Phe Val Pro Pro Met Pro Leu Pro Arg Gln Asn His Glu Pro

210 215 220

Val Ala Thr Ser Pro Ser Ser Pro Gln Pro Ala Glu Thr His Pro Thr

225 230 235 240

Thr Leu Gln Gly Ala Ile Arg Arg Glu Pro Thr Ser Ser Pro Leu Tyr

245 250 255

Ser Tyr Thr Thr Asp Gly Asn Asp Thr Val Thr Glu Ser Ser Asp Gly

260 265 270

Leu Trp Asn Asn Asn Gln Thr Gln Leu Phe Leu Glu His Ser Leu Leu

275 280 285

Thr Ala Asn Thr Thr Lys Gly Ile Tyr Ala Gly Val Cys Ile Ser Val

290 295 300

Leu Val Leu Leu Ala Leu Leu Gly Val Ile Ile Ala Lys Lys Tyr Phe

305 310 315 320

Phe Lys Lys Glu Val Gln Gln Leu Ser Val Ser Phe Ser Ser Leu Gln

325 330 335

Ile Lys Ala Leu Gln Asn Ala Val Glu Lys Glu Val Gln Ala Glu Asp

340 345 350

Asn Ile Tyr Ile Glu Asn Ser Leu Tyr Ala Thr Asp

355 360

<210> 102

<211> 976

<212> PROTEIN

<213> Homo sapiens

<400> 102

Met Arg Gly Ala Arg Gly Ala Trp Asp Phe Leu Cys Val Leu Leu Leu

1 5 10 15

Leu Leu Arg Val Gln Thr Gly Ser Ser Gln Pro Ser Val Ser Pro Gly

20 25 30

Glu Pro Ser Pro Pro Ser Ile His Pro Gly Lys Ser Asp Leu Ile Val

35 40 45

Arg Val Gly Asp Glu Ile Arg Leu Leu Cys Thr Asp Pro Gly Phe Val

50 55 60

Lys Trp Thr Phe Glu Ile Leu Asp Glu Thr Asn Glu Asn Lys Gln Asn

65 70 75 80

Glu Trp Ile Thr Glu Lys Ala Glu Ala Thr Asn Thr Gly Lys Tyr Thr

85 90 95

Cys Thr Asn Lys His Gly Leu Ser Asn Ser Ile Tyr Val Phe Val Arg

100 105 110

Asp Pro Ala Lys Leu Phe Leu Val Asp Arg Ser Leu Tyr Gly Lys Glu

115 120 125

Asp Asn Asp Thr Leu Val Arg Cys Pro Leu Thr Asp Pro Glu Val Thr

130 135 140

Asn Tyr Ser Leu Lys Gly Cys Gln Gly Lys Pro Leu Pro Lys Asp Leu

145 150 155 160

Arg Phe Ile Pro Asp Pro Lys Ala Gly Ile Met Ile Lys Ser Val Lys

165 170 175

Arg Ala Tyr His Arg Leu Cys Leu His Cys Ser Val Asp Gln Glu Gly

180 185 190

Lys Ser Val Leu Ser Glu Lys Phe Ile Leu Lys Val Arg Pro Ala Phe

195 200 205

Lys Ala Val Pro Val Val Ser Val Ser Lys Ala Ser Tyr Leu Leu Arg

210 215 220

Glu Gly Glu Glu Phe Thr Val Thr Cys Thr Ile Lys Asp Val Ser Ser

225 230 235 240

Ser Val Tyr Ser Thr Trp Lys Arg Glu Asn Ser Gln Thr Lys Leu Gln

245 250 255

Glu Lys Tyr Asn Ser Trp His His Gly Asp Phe Asn Tyr Glu Arg Gln

260 265 270

Ala Thr Leu Thr Ile Ser Ser Ala Arg Val Asn Asp Ser Gly Val Phe

275 280 285

Met Cys Tyr Ala Asn Asn Thr Phe Gly Ser Ala Asn Val Thr Thr Thr

290 295 300

Leu Glu Val Val Asp Lys Gly Phe Ile Asn Ile Phe Pro Met Ile Asn

305 310 315 320

Thr Thr Val Phe Val Asn Asp Gly Glu Asn Val Asp Leu Ile Val Glu

325 330 335

Tyr Glu Ala Phe Pro Lys Pro Glu His Gln Gln Trp Ile Tyr Met Asn

340 345 350

Arg Thr Phe Thr Asp Lys Trp Glu Asp Tyr Pro Lys Ser Glu Asn Glu

355 360 365

Ser Asn Ile Arg Tyr Val Ser Glu Leu His Leu Thr Arg Leu Lys Gly

370 375 380

Thr Glu Gly Gly Thr Tyr Thr Phe Leu Val Ser Asn Ser Asp Val Asn

385 390 395 400

Ala Ala Ile Ala Phe Asn Val Tyr Val Asn Thr Lys Pro Glu Ile Leu

405 410 415

Thr Tyr Asp Arg Leu Val Asn Gly Met Leu Gln Cys Val Ala Ala Gly

420 425 430

Phe Pro Glu Pro Thr Ile Asp Trp Tyr Phe Cys Pro Gly Thr Glu Gln

435 440 445

Arg Cys Ser Ala Ser Val Leu Pro Val Asp Val Gln Thr Leu Asn Ser

450 455 460

Ser Gly Pro Pro Phe Gly Lys Leu Val Val Gln Ser Ser Ile Asp Ser

465 470 475 480

Ser Ala Phe Lys His Asn Gly Thr Val Glu Cys Lys Ala Tyr Asn Asp

485 490 495

Val Gly Lys Thr Ser Ala Tyr Phe Asn Phe Ala Phe Lys Gly Asn Asn

500 505 510

Lys Glu Gln Ile His Pro His Thr Leu Phe Thr Pro Leu Leu Ile Gly

515 520 525

Phe Val Ile Val Ala Gly Met Met Cys Ile Ile Val Met Ile Leu Thr

530 535 540

Tyr Lys Tyr Leu Gln Lys Pro Met Tyr Glu Val Gln Trp Lys Val Val

545 550 555 560

Glu Glu Ile Asn Gly Asn Asn Tyr Val Tyr Ile Asp Pro Thr Gln Leu

565 570 575

Pro Tyr Asp His Lys Trp Glu Phe Pro Arg Asn Arg Leu Ser Phe Gly

580 585 590

Lys Thr Leu Gly Ala Gly Ala Phe Gly Lys Val Val Glu Ala Thr Ala

595 600 605

Tyr Gly Leu Ile Lys Ser Asp Ala Ala Met Thr Val Ala Val Lys Met

610 615 620

Leu Lys Pro Ser Ala His Leu Thr Glu Arg Glu Ala Leu Met Ser Glu

625 630 635 640

Leu Lys Val Leu Ser Tyr Leu Gly Asn His Met Asn Ile Val Asn Leu

645 650 655

Leu Gly Ala Cys Thr Ile Gly Gly Pro Thr Leu Val Ile Thr Glu Tyr

660 665 670

Cys Cys Tyr Gly Asp Leu Leu Asn Phe Leu Arg Arg Lys Arg Asp Ser

675 680 685

Phe Ile Cys Ser Lys Gln Glu Asp His Ala Glu Ala Ala Leu Tyr Lys

690 695 700

Asn Leu Leu His Ser Lys Glu Ser Ser Cys Ser Asp Ser Thr Asn Glu

705 710 715 720

Tyr Met Asp Met Lys Pro Gly Val Ser Tyr Val Val Pro Thr Lys Ala

725 730 735

Asp Lys Arg Arg Ser Val Arg Ile Gly Ser Tyr Ile Glu Arg Asp Val

740 745 750

Thr Pro Ala Ile Met Glu Asp Asp Glu Leu Ala Leu Asp Leu Glu Asp

755 760 765

Leu Leu Ser Phe Ser Tyr Gln Val Ala Lys Gly Met Ala Phe Leu Ala

770 775 780

Ser Lys Asn Cys Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu

785 790 795 800

Thr His Gly Arg Ile Thr Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp

805 810 815

Ile Lys Asn Asp Ser Asn Tyr Val Val Lys Gly Asn Ala Arg Leu Pro

820 825 830

Val Lys Trp Met Ala Pro Glu Ser Ile Phe Asn Cys Val Tyr Thr Phe

835 840 845

Glu Ser Asp Val Trp Ser Tyr Gly Ile Phe Leu Trp Glu Leu Phe Ser

850 855 860

Leu Gly Ser Ser Pro Tyr Pro Gly Met Pro Val Asp Ser Lys Phe Tyr

865 870 875 880

Lys Met Ile Lys Glu Gly Phe Arg Met Leu Ser Pro Glu His Ala Pro

885 890 895

Ala Glu Met Tyr Asp Ile Met Lys Thr Cys Trp Asp Ala Asp Pro Leu

900 905 910

Lys Arg Pro Thr Phe Lys Gln Ile Val Gln Leu Ile Glu Lys Gln Ile

915 920 925

Ser Glu Ser Thr Asn His Ile Tyr Ser Asn Leu Ala Asn Cys Ser Pro

930 935 940

Asn Arg Gln Lys Pro Val Val Asp His Ser Val Arg Ile Asn Ser Val

945 950 955 960

Gly Ser Thr Ala Ser Ser Ser Gln Pro Leu Leu Val His Asp Asp Val

965 970 975

<210> 103

<211> 1390

<212> PROTEIN

<213> Homo sapiens

<400> 103

Met Lys Ala Pro Ala Val Leu Ala Pro Gly Ile Leu Val Leu Leu Phe

1 5 10 15

Thr Leu Val Gln Arg Ser Asn Gly Glu Cys Lys Glu Ala Leu Ala Lys

20 25 30

Ser Glu Met Asn Val Asn Met Lys Tyr Gln Leu Pro Asn Phe Thr Ala

35 40 45

Glu Thr Pro Ile Gln Asn Val Ile Leu His Glu His His Ile Phe Leu

50 55 60

Gly Ala Thr Asn Tyr Ile Tyr Val Leu Asn Glu Glu Asp Leu Gln Lys

65 70 75 80

Val Ala Glu Tyr Lys Thr Gly Pro Val Leu Glu His Pro Asp Cys Phe

85 90 95

Pro Cys Gln Asp Cys Ser Ser Lys Ala Asn Leu Ser Gly Gly Val Trp

100 105 110

Lys Asp Asn Ile Asn Met Ala Leu Val Val Asp Thr Tyr Tyr Asp Asp

115 120 125

Gln Leu Ile Ser Cys Gly Ser Val Asn Arg Gly Thr Cys Gln Arg His

130 135 140

Val Phe Pro His Asn His Thr Ala Asp Ile Gln Ser Glu Val His Cys

145 150 155 160

Ile Phe Ser Pro Gln Ile Glu Glu Pro Ser Gln Cys Pro Asp Cys Val

165 170 175

Val Ser Ala Leu Gly Ala Lys Val Leu Ser Ser Val Lys Asp Arg Phe

180 185 190

Ile Asn Phe Phe Val Gly Asn Thr Ile Asn Ser Ser Tyr Phe Pro Asp

195 200 205

His Pro Leu His Ser Ile Ser Val Arg Arg Leu Lys Glu Thr Lys Asp

210 215 220

Gly Phe Met Phe Leu Thr Asp Gln Ser Tyr Ile Asp Val Leu Pro Glu

225 230 235 240

Phe Arg Asp Ser Tyr Pro Ile Lys Tyr Val His Ala Phe Glu Ser Asn

245 250 255

Asn Phe Ile Tyr Phe Leu Thr Val Gln Arg Glu Thr Leu Asp Ala Gln

260 265 270

Thr Phe His Thr Arg Ile Ile Arg Phe Cys Ser Ile Asn Ser Gly Leu

275 280 285

His Ser Tyr Met Glu Met Pro Leu Glu Cys Ile Leu Thr Glu Lys Arg

290 295 300

Lys Lys Arg Ser Thr Lys Lys Glu Val Phe Asn Ile Leu Gln Ala Ala

305 310 315 320

Tyr Val Ser Lys Pro Gly Ala Gln Leu Ala Arg Gln Ile Gly Ala Ser

325 330 335

Leu Asn Asp Asp Ile Leu Phe Gly Val Phe Ala Gln Ser Lys Pro Asp

340 345 350

Ser Ala Glu Pro Met Asp Arg Ser Ala Met Cys Ala Phe Pro Ile Lys

355 360 365

Tyr Val Asn Asp Phe Phe Asn Lys Ile Val Asn Lys Asn Asn Val Arg

370 375 380

Cys Leu Gln His Phe Tyr Gly Pro Asn His Glu His Cys Phe Asn Arg

385 390 395 400

Thr Leu Leu Arg Asn Ser Ser Gly Cys Glu Ala Arg Arg Asp Glu Tyr

405 410 415

Arg Thr Glu Phe Thr Thr Ala Leu Gln Arg Val Asp Leu Phe Met Gly

420 425 430

Gln Phe Ser Glu Val Leu Leu Thr Ser Ile Ser Thr Phe Ile Lys Gly

435 440 445

Asp Leu Thr Ile Ala Asn Leu Gly Thr Ser Glu Gly Arg Phe Met Gln

450 455 460

Val Val Val Ser Arg Ser Gly Pro Ser Thr Pro His Val Asn Phe Leu

465 470 475 480

Leu Asp Ser His Pro Val Ser Pro Glu Val Ile Val Glu His Thr Leu

485 490 495

Asn Gln Asn Gly Tyr Thr Leu Val Ile Thr Gly Lys Lys Ile Thr Lys

500 505 510

Ile Pro Leu Asn Gly Leu Gly Cys Arg His Phe Gln Ser Cys Ser Gln

515 520 525

Cys Leu Ser Ala Pro Pro Phe Val Gln Cys Gly Trp Cys His Asp Lys

530 535 540

Cys Val Arg Ser Glu Glu Cys Leu Ser Gly Thr Trp Thr Gln Gln Ile

545 550 555 560

Cys Leu Pro Ala Ile Tyr Lys Val Phe Pro Asn Ser Ala Pro Leu Glu

565 570 575

Gly Gly Thr Arg Leu Thr Ile Cys Gly Trp Asp Phe Gly Phe Arg Arg

580 585 590

Asn Asn Lys Phe Asp Leu Lys Lys Thr Arg Val Leu Leu Gly Asn Glu

595 600 605

Ser Cys Thr Leu Thr Leu Ser Glu Ser Thr Met Asn Thr Leu Lys Cys

610 615 620

Thr Val Gly Pro Ala Met Asn Lys His Phe Asn Met Ser Ile Ile Ile

625 630 635 640

Ser Asn Gly His Gly Thr Thr Gln Tyr Ser Thr Phe Ser Tyr Val Asp

645 650 655

Pro Val Ile Thr Ser Ile Ser Pro Lys Tyr Gly Pro Met Ala Gly Gly

660 665 670

Thr Leu Leu Thr Leu Thr Gly Asn Tyr Leu Asn Ser Gly Asn Ser Arg

675 680 685

His Ile Ser Ile Gly Gly Lys Thr Cys Thr Leu Lys Ser Val Ser Asn

690 695 700

Ser Ile Leu Glu Cys Tyr Thr Pro Ala Gln Thr Ile Ser Thr Glu Phe

705 710 715 720

Ala Val Lys Leu Lys Ile Asp Leu Ala Asn Arg Glu Thr Ser Ile Phe

725 730 735

Ser Tyr Arg Glu Asp Pro Ile Val Tyr Glu Ile His Pro Thr Lys Ser

740 745 750

Phe Ile Ser Gly Gly Ser Thr Ile Thr Gly Val Gly Lys Asn Leu Asn

755 760 765

Ser Val Ser Val Pro Arg Met Val Ile Asn Val His Glu Ala Gly Arg

770 775 780

Asn Phe Thr Val Ala Cys Gln His Arg Ser Asn Ser Glu Ile Ile Cys

785 790 795 800

Cys Thr Thr Pro Ser Leu Gln Gln Leu Asn Leu Gln Leu Pro Leu Lys

805 810 815

Thr Lys Ala Phe Phe Met Leu Asp Gly Ile Leu Ser Lys Tyr Phe Asp

820 825 830

Leu Ile Tyr Val His Asn Pro Val Phe Lys Pro Phe Glu Lys Pro Val

835 840 845

Met Ile Ser Met Gly Asn Glu Asn Val Leu Glu Ile Lys Gly Asn Asp

850 855 860

Ile Asp Pro Glu Ala Val Lys Gly Glu Val Leu Lys Val Gly Asn Lys

865 870 875 880

Ser Cys Glu Asn Ile His Leu His Ser Glu Ala Val Leu Cys Thr Val

885 890 895

Pro Asn Asp Leu Leu Lys Leu Asn Ser Glu Leu Asn Ile Glu Trp Lys

900 905 910

Gln Ala Ile Ser Ser Thr Val Leu Gly Lys Val Ile Val Gln Pro Asp

915 920 925

Gln Asn Phe Thr Gly Leu Ile Ala Gly Val Val Ser Ile Ser Thr Ala

930 935 940

Leu Leu Leu Leu Leu Gly Phe Phe Leu Trp Leu Lys Lys Arg Lys Gln

945 950 955 960

Ile Lys Asp Leu Gly Ser Glu Leu Val Arg Tyr Asp Ala Arg Val His

965 970 975

Thr Pro His Leu Asp Arg Leu Val Ser Ala Arg Ser Val Ser Pro Thr

980 985 990

Thr Glu Met Val Ser Asn Glu Ser Val Asp Tyr Arg Ala Thr Phe Pro

995 1000 1005

Glu Asp Gln Phe Pro Asn Ser Ser Gln Asn Gly Ser Cys Arg Gln

1010 1015 1020

Val Gln Tyr Pro Leu Thr Asp Met Ser Pro Ile Leu Thr Ser Gly

1025 1030 1035

Asp Ser Asp Ile Ser Ser Pro Leu Leu Gln Asn Thr Val His Ile

1040 1045 1050

Asp Leu Ser Ala Leu Asn Pro Glu Leu Val Gln Ala Val Gln His

1055 1060 1065

Val Val Ile Gly Pro Ser Ser Leu Ile Val His Phe Asn Glu Val

1070 1075 1080

Ile Gly Arg Gly His Phe Gly Cys Val Tyr His Gly Thr Leu Leu

1085 1090 1095

Asp Asn Asp Gly Lys Lys Ile His Cys Ala Val Lys Ser Leu Asn

1100 1105 1110

Arg Ile Thr Asp Ile Gly Glu Val Ser Gln Phe Leu Thr Glu Gly

1115 1120 1125

Ile Ile Met Lys Asp Phe Ser His Pro Asn Val Leu Ser Leu Leu

1130 1135 1140

Gly Ile Cys Leu Arg Ser Glu Gly Ser Pro Leu Val Val Leu Pro

1145 1150 1155

Tyr Met Lys His Gly Asp Leu Arg Asn Phe Ile Arg Asn Glu Thr

1160 1165 1170

His Asn Pro Thr Val Lys Asp Leu Ile Gly Phe Gly Leu Gln Val

1175 1180 1185

Ala Lys Gly Met Lys Tyr Leu Ala Ser Lys Lys Phe Val His Arg

1190 1195 1200

Asp Leu Ala Ala Arg Asn Cys Met Leu Asp Glu Lys Phe Thr Val

1205 1210 1215

Lys Val Ala Asp Phe Gly Leu Ala Arg Asp Met Tyr Asp Lys Glu

1220 1225 1230

Tyr Tyr Ser Val His Asn Lys Thr Gly Ala Lys Leu Pro Val Lys

1235 1240 1245

Trp Met Ala Leu Glu Ser Leu Gln Thr Gln Lys Phe Thr Thr Lys

1250 1255 1260

Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Leu Met Thr

1265 1270 1275

Arg Gly Ala Pro Pro Tyr Pro Asp Val Asn Thr Phe Asp Ile Thr

1280 1285 1290

Val Tyr Leu Leu Gln Gly Arg Arg Leu Leu Gln Pro Glu Tyr Cys

1295 1300 1305

Pro Asp Pro Leu Tyr Glu Val Met Leu Lys Cys Trp His Pro Lys

1310 1315 1320

Ala Glu Met Arg Pro Ser Phe Ser Glu Leu Val Ser Arg Ile Ser

1325 1330 1335

Ala Ile Phe Ser Thr Phe Ile Gly Glu His Tyr Val His Val Asn

1340 1345 1350

Ala Thr Tyr Val Asn Val Lys Cys Val Ala Pro Tyr Pro Ser Leu

1355 1360 1365

Leu Ser Ser Glu Asp Asn Ala Asp Asp Glu Val Asp Thr Arg Pro

1370 1375 1380

Ala Ser Phe Trp Glu Thr Ser

1385 1390

<210> 104

<211> 14507

<212> PROTEIN

<213> Homo sapiens

<400> 104

Met Leu Lys Pro Ser Gly Leu Pro Gly Ser Ser Ser Pro Thr Arg Ser

1 5 10 15

Leu Met Thr Gly Ser Arg Ser Thr Lys Ala Thr Pro Glu Met Asp Ser

20 25 30

Gly Leu Thr Gly Ala Thr Leu Ser Pro Lys Thr Ser Thr Gly Ala Ile

35 40 45

Val Val Thr Glu His Thr Leu Pro Phe Thr Ser Pro Asp Lys Thr Leu

50 55 60

Ala Ser Pro Thr Ser Ser Val Val Gly Arg Thr Thr Gln Ser Leu Gly

65 70 75 80

Val Met Ser Ser Ala Leu Pro Glu Ser Thr Ser Arg Gly Met Thr His

85 90 95

Ser Glu Gln Arg Thr Ser Pro Ser Leu Ser Pro Gln Val Asn Gly Thr

100 105 110

Pro Ser Arg Asn Tyr Pro Ala Thr Ser Met Val Ser Gly Leu Ser Ser

115 120 125

Pro Arg Thr Arg Thr Ser Ser Thr Glu Gly Asn Phe Thr Lys Glu Ala

130 135 140

Ser Thr Tyr Thr Leu Thr Val Glu Thr Thr Ser Gly Pro Val Thr Glu

145 150 155 160

Lys Tyr Thr Val Pro Thr Glu Thr Ser Thr Thr Glu Gly Asp Ser Thr

165 170 175

Glu Thr Pro Trp Asp Thr Arg Tyr Ile Pro Val Lys Ile Thr Ser Pro

180 185 190

Met Lys Thr Phe Ala Asp Ser Thr Ala Ser Lys Glu Asn Ala Pro Val

195 200 205

Ser Met Thr Pro Ala Glu Thr Thr Val Thr Asp Ser His Thr Pro Gly

210 215 220

Arg Thr Asn Pro Ser Phe Gly Thr Leu Tyr Ser Ser Phe Leu Asp Leu

225 230 235 240

Ser Pro Lys Gly Thr Pro Asn Ser Arg Gly Glu Thr Ser Leu Glu Leu

245 250 255

Ile Leu Ser Thr Thr Gly Tyr Pro Phe Ser Ser Pro Glu Pro Gly Ser

260 265 270

Ala Gly His Ser Arg Ile Ser Thr Ser Ala Pro Leu Ser Ser Ser Ala

275 280 285

Ser Val Leu Asp Asn Lys Ile Ser Glu Thr Ser Ile Phe Ser Gly Gln

290 295 300

Ser Leu Thr Ser Pro Leu Ser Pro Gly Val Pro Glu Ala Arg Ala Ser

305 310 315 320

Thr Met Pro Asn Ser Ala Ile Pro Phe Ser Met Thr Leu Ser Asn Ala

325 330 335

Glu Thr Ser Ala Glu Arg Val Arg Ser Thr Ile Ser Ser Leu Gly Thr

340 345 350

Pro Ser Ile Ser Thr Lys Gln Thr Ala Glu Thr Ile Leu Thr Phe His

355 360 365

Ala Phe Ala Glu Thr Met Asp Ile Pro Ser Thr His Ile Ala Lys Thr

370 375 380

Leu Ala Ser Glu Trp Leu Gly Ser Pro Gly Thr Leu Gly Gly Thr Ser

385 390 395 400

Thr Ser Ala Leu Thr Thr Thr Ser Pro Ser Thr Thr Leu Val Ser Glu

405 410 415

Glu Thr Asn Thr His His Ser Thr Ser Gly Lys Glu Thr Glu Gly Thr

420 425 430

Leu Asn Thr Ser Met Thr Pro Leu Glu Thr Ser Ala Pro Gly Glu Glu

435 440 445

Ser Glu Met Thr Ala Thr Leu Val Pro Thr Leu Gly Phe Thr Thr Leu

450 455 460

Asp Ser Lys Ile Arg Ser Pro Ser Gln Val Ser Ser Ser His Pro Thr

465 470 475 480

Arg Glu Leu Arg Thr Thr Gly Ser Thr Ser Gly Arg Gln Ser Ser Ser

485 490 495

Thr Ala Ala His Gly Ser Ser Asp Ile Leu Arg Ala Thr Thr Ser Ser

500 505 510

Thr Ser Lys Ala Ser Ser Trp Thr Ser Glu Ser Thr Ala Gln Gln Phe

515 520 525

Ser Glu Pro Gln His Thr Gln Trp Val Glu Thr Ser Pro Ser Met Lys

530 535 540

Thr Glu Arg Pro Pro Ala Ser Thr Ser Val Ala Ala Pro Ile Thr Thr

545 550 555 560

Ser Val Pro Ser Val Val Ser Gly Phe Thr Thr Leu Lys Thr Ser Ser

565 570 575

Thr Lys Gly Ile Trp Leu Glu Glu Thr Ser Ala Asp Thr Leu Ile Gly

580 585 590

Glu Ser Thr Ala Gly Pro Thr Thr His Gln Phe Ala Val Pro Thr Gly

595 600 605

Ile Ser Met Thr Gly Gly Ser Ser Thr Arg Gly Ser Gln Gly Thr Thr

610 615 620

His Leu Leu Thr Arg Ala Thr Ala Ser Ser Glu Thr Ser Ala Asp Leu

625 630 635 640

Thr Leu Ala Thr Asn Gly Val Pro Val Ser Val Ser Pro Ala Val Ser

645 650 655

Lys Thr Ala Ala Gly Ser Ser Pro Pro Gly Gly Thr Lys Pro Ser Tyr

660 665 670

Thr Met Val Ser Ser Val Ile Pro Glu Thr Ser Ser Leu Gln Ser Ser

675 680 685

Ala Phe Arg Glu Gly Thr Ser Leu Gly Leu Thr Pro Leu Asn Thr Arg

690 695 700

His Pro Phe Ser Ser Pro Glu Pro Asp Ser Ala Gly His Thr Lys Ile

705 710 715 720

Ser Thr Ser Ile Pro Leu Leu Ser Ser Ala Ser Val Leu Glu Asp Lys

725 730 735

Val Ser Ala Thr Ser Thr Phe Ser His His Lys Ala Thr Ser Ser Ile

740 745 750

Thr Thr Gly Thr Pro Glu Ile Ser Thr Lys Thr Lys Pro Ser Ser Ala

755 760 765

Val Leu Ser Ser Met Thr Leu Ser Asn Ala Ala Thr Ser Pro Glu Arg

770 775 780

Val Arg Asn Ala Thr Ser Pro Leu Thr His Pro Ser Pro Ser Gly Glu

785 790 795 800

Glu Thr Ala Gly Ser Val Leu Thr Leu Ser Thr Ser Ala Glu Thr Thr

805 810 815

Asp Ser Pro Asn Ile His Pro Thr Gly Thr Leu Thr Ser Glu Ser Ser

820 825 830

Glu Ser Pro Ser Thr Leu Ser Leu Pro Ser Val Ser Gly Val Lys Thr

835 840 845

Thr Phe Ser Ser Ser Thr Pro Ser Thr His Leu Phe Thr Ser Gly Glu

850 855 860

Glu Thr Glu Glu Thr Ser Asn Pro Ser Val Ser Gln Pro Glu Thr Ser

865 870 875 880

Val Ser Arg Val Arg Thr Thr Leu Ala Ser Thr Ser Val Pro Thr Pro

885 890 895

Val Phe Pro Thr Met Asp Thr Trp Pro Thr Arg Ser Ala Gln Phe Ser

900 905 910

Ser Ser His Leu Val Ser Glu Leu Arg Ala Thr Ser Ser Thr Ser Val

915 920 925

Thr Asn Ser Thr Gly Ser Ala Leu Pro Lys Ile Ser His Leu Thr Gly

930 935 940

Thr Ala Thr Met Ser Gln Thr Asn Arg Asp Thr Phe Asn Asp Ser Ala

945 950 955 960

Ala Pro Gln Ser Thr Thr Trp Pro Glu Thr Ser Pro Arg Phe Lys Thr

965 970 975

Gly Leu Pro Ser Ala Thr Thr Thr Val Ser Thr Ser Ala Thr Ser Leu

980 985 990

Ser Ala Thr Val Met Val Ser Lys Phe Thr Ser Pro Ala Thr Ser Ser

995 1000 1005

Met Glu Ala Thr Ser Ile Arg Glu Pro Ser Thr Thr Ile Leu Thr

1010 1015 1020

Thr Glu Thr Thr Asn Gly Pro Gly Ser Met Ala Val Ala Ser Thr

1025 1030 1035

Asn Ile Pro Ile Gly Lys Gly Tyr Ile Thr Glu Gly Arg Leu Asp

1040 1045 1050

Thr Ser His Leu Pro Ile Gly Thr Thr Ala Ser Ser Glu Thr Ser

1055 1060 1065

Met Asp Phe Thr Met Ala Lys Glu Ser Val Ser Met Ser Val Ser

1070 1075 1080

Pro Ser Gln Ser Met Asp Ala Ala Gly Ser Ser Thr Pro Gly Arg

1085 1090 1095

Thr Ser Gln Phe Val Asp Thr Phe Ser Asp Asp Val Tyr His Leu

1100 1105 1110

Thr Ser Arg Glu Ile Thr Ile Pro Arg Asp Gly Thr Ser Ser Ala

1115 1120 1125

Leu Thr Pro Gln Met Thr Ala Thr His Pro Pro Ser Pro Asp Pro

1130 1135 1140

Gly Ser Ala Arg Ser Thr Trp Leu Gly Ile Leu Ser Ser Ser Pro

1145 1150 1155

Ser Ser Pro Thr Pro Lys Val Thr Met Ser Ser Thr Phe Ser Thr

1160 1165 1170

Gln Arg Val Thr Thr Ser Met Ile Met Asp Thr Val Glu Thr Ser

1175 1180 1185

Arg Trp Asn Met Pro Asn Leu Pro Ser Thr Thr Ser Leu Thr Pro

1190 1195 1200

Ser Asn Ile Pro Thr Ser Gly Ala Ile Gly Lys Ser Thr Leu Val

1205 1210 1215

Pro Leu Asp Thr Pro Ser Pro Ala Thr Ser Leu Glu Ala Ser Glu

1220 1225 1230

Gly Gly Leu Pro Thr Leu Ser Thr Tyr Pro Glu Ser Thr Asn Thr

1235 1240 1245

Pro Ser Ile His Leu Gly Ala His Ala Ser Ser Glu Ser Pro Ser

1250 1255 1260

Thr Ile Lys Leu Thr Met Ala Ser Val Val Lys Pro Gly Ser Tyr

1265 1270 1275

Thr Pro Leu Thr Phe Pro Ser Ile Glu Thr His Ile His Val Ser

1280 1285 1290

Thr Ala Arg Met Ala Tyr Ser Ser Gly Ser Ser Pro Glu Met Thr

1295 1300 1305

Ala Pro Gly Glu Thr Asn Thr Gly Ser Thr Trp Asp Pro Thr Thr

1310 1315 1320

Tyr Ile Thr Thr Thr Asp Pro Lys Asp Thr Ser Ser Ala Gln Val

1325 1330 1335

Ser Thr Pro His Ser Val Arg Thr Leu Arg Thr Thr Glu Asn His

1340 1345 1350

Pro Lys Thr Glu Ser Ala Thr Pro Ala Ala Tyr Ser Gly Ser Pro

1355 1360 1365

Lys Ile Ser Ser Ser Pro Asn Leu Thr Ser Pro Ala Thr Lys Ala

1370 1375 1380

Trp Thr Ile Thr Asp Thr Thr Glu His Ser Thr Gln Leu His Tyr

1385 1390 1395

Thr Lys Leu Ala Glu Lys Ser Ser Gly Phe Glu Thr Gln Ser Ala

1400 1405 1410

Pro Gly Pro Val Ser Val Val Ile Pro Thr Ser Pro Thr Ile Gly

1415 1420 1425

Ser Ser Thr Leu Glu Leu Thr Ser Asp Val Pro Gly Glu Pro Leu

1430 1435 1440

Val Leu Ala Pro Ser Glu Gln Thr Thr Ile Thr Leu Pro Met Ala

1445 1450 1455

Thr Trp Leu Ser Thr Ser Leu Thr Glu Glu Met Ala Ser Thr Asp

1460 1465 1470

Leu Asp Ile Ser Ser Pro Ser Ser Pro Met Ser Thr Phe Ala Ile

1475 1480 1485

Phe Pro Pro Met Ser Thr Pro Ser His Glu Leu Ser Lys Ser Glu

1490 1495 1500

Ala Asp Thr Ser Ala Ile Arg Asn Thr Asp Ser Thr Thr Leu Asp

1505 1510 1515

Gln His Leu Gly Ile Arg Ser Leu Gly Arg Thr Gly Asp Leu Thr

1520 1525 1530

Thr Val Pro Ile Thr Pro Leu Thr Thr Thr Trp Thr Ser Val Ile

1535 1540 1545

Glu His Ser Thr Gln Ala Gln Asp Thr Leu Ser Ala Thr Met Ser

1550 1555 1560

Pro Thr His Val Thr Gln Ser Leu Lys Asp Gln Thr Ser Ile Pro

1565 1570 1575

Ala Ser Ala Ser Pro Ser His Leu Thr Glu Val Tyr Pro Glu Leu

1580 1585 1590

Gly Thr Gln Gly Arg Ser Ser Ser Glu Ala Thr Thr Phe Trp Lys

1595 1600 1605

Pro Ser Thr Asp Thr Leu Ser Arg Glu Ile Glu Thr Gly Pro Thr

1610 1615 1620

Asn Ile Gln Ser Thr Pro Pro Met Asp Asn Thr Thr Thr Gly Ser

1625 1630 1635

Ser Ser Ser Gly Val Thr Leu Gly Ile Ala His Leu Pro Ile Gly

1640 1645 1650

Thr Ser Ser Pro Ala Glu Thr Ser Thr Asn Met Ala Leu Glu Arg

1655 1660 1665

Arg Ser Ser Thr Ala Thr Val Ser Met Ala Gly Thr Met Gly Leu

1670 1675 1680

Leu Val Thr Ser Ala Pro Gly Arg Ser Ile Ser Gln Ser Leu Gly

1685 1690 1695

Arg Val Ser Ser Val Leu Ser Glu Ser Thr Thr Glu Gly Val Thr

1700 1705 1710

Asp Ser Ser Lys Gly Ser Ser Pro Arg Leu Asn Thr Gln Gly Asn

1715 1720 1725

Thr Ala Leu Ser Ser Ser Leu Glu Pro Ser Tyr Ala Glu Gly Ser

1730 1735 1740

Gln Met Ser Thr Ser Ile Pro Leu Thr Ser Ser Pro Thr Thr Pro

1745 1750 1755

Asp Val Glu Phe Ile Gly Gly Ser Thr Phe Trp Thr Lys Glu Val

1760 1765 1770

Thr Thr Val Met Thr Ser Asp Ile Ser Lys Ser Ser Ala Arg Thr

1775 1780 1785

Glu Ser Ser Ser Ala Thr Leu Met Ser Thr Ala Leu Gly Ser Thr

1790 1795 1800

Glu Asn Thr Gly Lys Glu Lys Leu Arg Thr Ala Ser Met Asp Leu

1805 1810 1815

Pro Ser Pro Thr Pro Ser Met Glu Val Thr Pro Trp Ile Ser Leu

1820 1825 1830

Thr Leu Ser Asn Ala Pro Asn Thr Thr Asp Ser Leu Asp Leu Ser

1835 1840 1845

His Gly Val His Thr Ser Ser Ala Gly Thr Leu Ala Thr Asp Arg

1850 1855 1860

Ser Leu Asn Thr Gly Val Thr Arg Ala Ser Arg Leu Glu Asn Gly

1865 1870 1875

Ser Asp Thr Ser Ser Lys Ser Leu Ser Met Gly Asn Ser Thr His

1880 1885 1890

Thr Ser Met Thr Tyr Thr Glu Lys Ser Glu Val Ser Ser Ser Ile

1895 1900 1905

His Pro Arg Pro Glu Thr Ser Ala Pro Gly Ala Glu Thr Thr Leu

1910 1915 1920

Thr Ser Thr Pro Gly Asn Arg Ala Ile Ser Leu Thr Leu Pro Phe

1925 1930 1935

Ser Ser Ile Pro Val Glu Glu Val Ile Ser Thr Gly Ile Thr Ser

1940 1945 1950

Gly Pro Asp Ile Asn Ser Ala Pro Met Thr His Ser Pro Ile Thr

1955 1960 1965

Pro Pro Thr Ile Val Trp Thr Ser Thr Gly Thr Ile Glu Gln Ser

1970 1975 1980

Thr Gln Pro Leu His Ala Val Ser Ser Glu Lys Val Ser Val Gln

1985 1990 1995

Thr Gln Ser Thr Pro Tyr Val Asn Ser Val Ala Val Ser Ala Ser

2000 2005 2010

Pro Thr His Glu Asn Ser Val Ser Ser Gly Ser Ser Thr Ser Ser

2015 2020 2025

Pro Tyr Ser Ser Ala Ser Leu Glu Ser Leu Asp Ser Thr Ile Ser

2030 2035 2040

Arg Arg Asn Ala Ile Thr Ser Trp Leu Trp Asp Leu Thr Thr Ser

2045 2050 2055

Leu Pro Thr Thr Thr Trp Pro Ser Thr Ser Leu Ser Glu Ala Leu

2060 2065 2070

Ser Ser Gly His Ser Gly Val Ser Asn Pro Ser Ser Thr Thr Thr

2075 2080 2085

Glu Phe Pro Leu Phe Ser Ala Ala Ser Thr Ser Ala Ala Lys Gln

2090 2095 2100

Arg Asn Pro Glu Thr Glu Thr His Gly Pro Gln Asn Thr Ala Ala

2105 2110 2115

Ser Thr Leu Asn Thr Asp Ala Ser Ser Val Thr Gly Leu Ser Glu

2120 2125 2130

Thr Pro Val Gly Ala Ser Ile Ser Ser Glu Val Pro Leu Pro Met

2135 2140 2145

Ala Ile Thr Ser Arg Ser Asp Val Ser Gly Leu Thr Ser Glu Ser

2150 2155 2160

Thr Ala Asn Pro Ser Leu Gly Thr Ala Ser Ser Ala Gly Thr Lys

2165 2170 2175

Leu Thr Arg Thr Ile Ser Leu Pro Thr Ser Glu Ser Leu Val Ser

2180 2185 2190

Phe Arg Met Asn Lys Asp Pro Trp Thr Val Ser Ile Pro Leu Gly

2195 2200 2205

Ser His Pro Thr Thr Asn Thr Glu Thr Ser Ile Pro Val Asn Ser

2210 2215 2220

Ala Gly Pro Pro Gly Leu Ser Thr Val Ala Ser Asp Val Ile Asp

2225 2230 2235

Thr Pro Ser Asp Gly Ala Glu Ser Ile Pro Thr Val Ser Phe Ser

2240 2245 2250

Pro Ser Pro Asp Thr Glu Val Thr Thr Ile Ser His Phe Pro Glu

2255 2260 2265

Lys Thr Thr His Ser Phe Arg Thr Ile Ser Ser Leu Thr His Glu

2270 2275 2280

Leu Thr Ser Arg Val Thr Pro Ile Pro Gly Asp Trp Met Ser Ser

2285 2290 2295

Ala Met Ser Thr Lys Pro Thr Gly Ala Ser Pro Ser Ile Thr Leu

2300 2305 2310

Gly Glu Arg Arg Thr Ile Thr Ser Ala Ala Pro Thr Thr Ser Pro

2315 2320 2325

Ile Val Leu Thr Ala Ser Phe Thr Glu Thr Ser Thr Val Ser Leu

2330 2335 2340

Asp Asn Glu Thr Thr Val Lys Thr Ser Asp Ile Leu Asp Ala Arg

2345 2350 2355

Lys Thr Asn Glu Leu Pro Ser Asp Ser Ser Ser Ser Ser Asp Leu

2360 2365 2370

Ile Asn Thr Ser Ile Ala Ser Ser Thr Met Asp Val Thr Lys Thr

2375 2380 2385

Ala Ser Ile Ser Pro Thr Ser Ile Ser Gly Met Thr Ala Ser Ser

2390 2395 2400

Ser Pro Ser Leu Phe Ser Ser Asp Arg Pro Gln Val Pro Thr Ser

2405 2410 2415

Thr Thr Glu Thr Asn Thr Ala Thr Ser Pro Ser Val Ser Ser Asn

2420 2425 2430

Thr Tyr Ser Leu Asp Gly Gly Ser Asn Val Gly Gly Thr Pro Ser

2435 2440 2445

Thr Leu Pro Pro Phe Thr Ile Thr His Pro Val Glu Thr Ser Ser

2450 2455 2460

Ala Leu Leu Ala Trp Ser Arg Pro Val Arg Thr Phe Ser Thr Met

2465 2470 2475

Val Ser Thr Asp Thr Ala Ser Gly Glu Asn Pro Thr Ser Ser Asn

2480 2485 2490

Ser Val Val Thr Ser Val Pro Ala Pro Gly Thr Trp Thr Ser Val

2495 2500 2505

Gly Ser Thr Thr Asp Leu Pro Ala Met Gly Phe Leu Lys Thr Ser

2510 2515 2520

Pro Ala Gly Glu Ala His Ser Leu Leu Ala Ser Thr Ile Glu Pro

2525 2530 2535

Ala Thr Ala Phe Thr Pro His Leu Ser Ala Ala Val Val Thr Gly

2540 2545 2550

Ser Ser Ala Thr Ser Glu Ala Ser Leu Leu Thr Thr Ser Glu Ser

2555 2560 2565

Lys Ala Ile His Ser Ser Pro Gln Thr Pro Thr Thr Pro Thr Ser

2570 2575 2580

Gly Ala Asn Trp Glu Thr Ser Ala Thr Pro Glu Ser Leu Leu Val

2585 2590 2595

Val Thr Glu Thr Ser Asp Thr Thr Leu Thr Ser Lys Ile Leu Val

2600 2605 2610

Thr Asp Thr Ile Leu Phe Ser Thr Val Ser Thr Pro Pro Ser Lys

2615 2620 2625

Phe Pro Ser Thr Gly Thr Leu Ser Gly Ala Ser Phe Pro Thr Leu

2630 2635 2640

Leu Pro Asp Thr Pro Ala Ile Pro Leu Thr Ala Thr Glu Pro Thr

2645 2650 2655

Ser Ser Leu Ala Thr Ser Phe Asp Ser Thr Pro Leu Val Thr Ile

2660 2665 2670

Ala Ser Asp Ser Leu Gly Thr Val Pro Glu Thr Thr Leu Thr Met

2675 2680 2685

Ser Glu Thr Ser Asn Gly Asp Ala Leu Val Leu Lys Thr Val Ser

2690 2695 2700

Asn Pro Asp Arg Ser Ile Pro Gly Ile Thr Ile Gln Gly Val Thr

2705 2710 2715

Glu Ser Pro Leu His Pro Ser Ser Thr Ser Pro Ser Lys Ile Val

2720 2725 2730

Ala Pro Arg Asn Thr Thr Tyr Glu Gly Ser Ile Thr Val Ala Leu

2735 2740 2745

Ser Thr Leu Pro Ala Gly Thr Thr Gly Ser Leu Val Phe Ser Gln

2750 2755 2760

Ser Ser Glu Asn Ser Glu Thr Thr Ala Leu Val Asp Ser Ser Ala

2765 2770 2775

Gly Leu Glu Arg Ala Ser Val Met Pro Leu Thr Thr Gly Ser Gln

2780 2785 2790

Gly Met Ala Ser Ser Gly Gly Ile Arg Ser Gly Ser Thr His Ser

2795 2800 2805

Thr Gly Thr Lys Thr Phe Ser Ser Leu Pro Leu Thr Met Asn Pro

2810 2815 2820

Gly Glu Val Thr Ala Met Ser Glu Ile Thr Thr Asn Arg Leu Thr

2825 2830 2835

Ala Thr Gln Ser Thr Ala Pro Lys Gly Ile Pro Val Lys Pro Thr

2840 2845 2850

Ser Ala Glu Ser Gly Leu Leu Thr Pro Val Ser Ala Ser Ser Ser

2855 2860 2865

Pro Ser Lys Ala Phe Ala Ser Leu Thr Thr Ala Pro Pro Thr Trp

2870 2875 2880

Gly Ile Pro Gln Ser Thr Leu Thr Phe Glu Phe Ser Glu Val Pro

2885 2890 2895

Ser Leu Asp Thr Lys Ser Ala Ser Leu Pro Thr Pro Gly Gln Ser

2900 2905 2910

Leu Asn Thr Ile Pro Asp Ser Asp Ala Ser Thr Ala Ser Ser Ser

2915 2920 2925

Leu Ser Lys Ser Pro Glu Lys Asn Pro Arg Ala Arg Met Met Thr

2930 2935 2940

Ser Thr Lys Ala Ile Ser Ala Ser Ser Phe Gln Ser Thr Gly Phe

2945 2950 2955

Thr Glu Thr Pro Glu Gly Ser Ala Ser Pro Ser Met Ala Gly His

2960 2965 2970

Glu Pro Arg Val Pro Thr Ser Gly Thr Gly Asp Pro Arg Tyr Ala

2975 2980 2985

Ser Glu Ser Met Ser Tyr Pro Asp Pro Ser Lys Ala Ser Ser Ala

2990 2995 3000

Met Thr Ser Thr Ser Leu Ala Ser Lys Leu Thr Thr Leu Phe Ser

3005 3010 3015

Thr Gly Gln Ala Ala Arg Ser Gly Ser Ser Ser Ser Pro Ile Ser

3020 3025 3030

Leu Ser Thr Glu Lys Glu Thr Ser Phe Leu Ser Pro Thr Ala Ser

3035 3040 3045

Thr Ser Arg Lys Thr Ser Leu Phe Leu Gly Pro Ser Met Ala Arg

3050 3055 3060

Gln Pro Asn Ile Leu Val His Leu Gln Thr Ser Ala Leu Thr Leu

3065 3070 3075

Ser Pro Thr Ser Thr Leu Asn Met Ser Gln Glu Glu Pro Pro Glu

3080 3085 3090

Leu Thr Ser Ser Gln Thr Ile Ala Glu Glu Glu Gly Thr Thr Ala

3095 3100 3105

Glu Thr Gln Thr Leu Thr Phe Thr Pro Ser Glu Thr Pro Thr Ser

3110 3115 3120

Leu Leu Pro Val Ser Ser Pro Thr Glu Pro Thr Ala Arg Arg Lys

3125 3130 3135

Ser Ser Pro Glu Thr Trp Ala Ser Ser Ile Ser Val Pro Ala Lys

3140 3145 3150

Thr Ser Leu Val Glu Thr Thr Asp Gly Thr Leu Val Thr Thr Ile

3155 3160 3165

Lys Met Ser Ser Gln Ala Ala Gln Gly Asn Ser Thr Trp Pro Ala

3170 3175 3180

Pro Ala Glu Glu Thr Gly Ser Ser Pro Ala Gly Thr Ser Pro Gly

3185 3190 3195

Ser Pro Glu Met Ser Thr Thr Leu Lys Ile Met Ser Ser Lys Glu

3200 3205 3210

Pro Ser Ile Ser Pro Glu Ile Arg Ser Thr Val Arg Asn Ser Pro

3215 3220 3225

Trp Lys Thr Pro Glu Thr Thr Val Pro Met Glu Thr Thr Val Glu

3230 3235 3240

Pro Val Thr Leu Gln Ser Thr Ala Leu Gly Ser Gly Ser Thr Ser

3245 3250 3255

Ile Ser His Leu Pro Thr Gly Thr Thr Ser Pro Thr Lys Ser Pro

3260 3265 3270

Thr Glu Asn Met Leu Ala Thr Glu Arg Val Ser Leu Ser Pro Ser

3275 3280 3285

Pro Pro Glu Ala Trp Thr Asn Leu Tyr Ser Gly Thr Pro Gly Gly

3290 3295 3300

Thr Arg Gln Ser Leu Ala Thr Met Ser Ser Val Ser Leu Glu Ser

3305 3310 3315

Pro Thr Ala Arg Ser Ile Thr Gly Thr Gly Gln Gln Ser Ser Pro

3320 3325 3330

Glu Leu Val Ser Lys Thr Thr Gly Met Glu Phe Ser Met Trp His

3335 3340 3345

Gly Ser Thr Gly Gly Thr Thr Gly Asp Thr His Val Ser Leu Ser

3350 3355 3360

Thr Ser Ser Asn Ile Leu Glu Asp Pro Val Thr Ser Pro Asn Ser

3365 3370 3375

Val Ser Ser Leu Thr Asp Lys Ser Lys His Lys Thr Glu Thr Trp

3380 3385 3390

Val Ser Thr Thr Ala Ile Pro Ser Thr Val Leu Asn Asn Lys Ile

3395 3400 3405

Met Ala Ala Glu Gln Gln Thr Ser Arg Ser Val Asp Glu Ala Tyr

3410 3415 3420

Ser Ser Thr Ser Ser Trp Ser Asp Gln Thr Ser Gly Ser Asp Ile

3425 3430 3435

Thr Leu Gly Ala Ser Pro Asp Val Thr Asn Thr Leu Tyr Ile Thr

3440 3445 3450

Ser Thr Ala Gln Thr Thr Ser Leu Val Ser Leu Pro Ser Gly Asp

3455 3460 3465

Gln Gly Ile Thr Ser Leu Thr Asn Pro Ser Gly Gly Lys Thr Ser

3470 3475 3480

Ser Ala Ser Ser Val Thr Ser Pro Ser Ile Gly Leu Glu Thr Leu

3485 3490 3495

Arg Ala Asn Val Ser Ala Val Lys Ser Asp Ile Ala Pro Thr Ala

3500 3505 3510

Gly His Leu Ser Gln Thr Ser Ser Pro Ala Glu Val Ser Ile Leu

3515 3520 3525

Asp Val Thr Thr Ala Pro Thr Pro Gly Ile Ser Thr Thr Ile Thr

3530 3535 3540

Thr Met Gly Thr Asn Ser Ile Ser Thr Thr Thr Pro Asn Pro Glu

3545 3550 3555

Val Gly Met Ser Thr Met Asp Ser Thr Pro Ala Thr Glu Arg Arg

3560 3565 3570

Thr Thr Ser Thr Glu His Pro Ser Thr Trp Ser Ser Thr Ala Ala

3575 3580 3585

Ser Asp Ser Trp Thr Val Thr Asp Met Thr Ser Asn Leu Lys Val

3590 3595 3600

Ala Arg Ser Pro Gly Thr Ile Ser Thr Met His Thr Thr Ser Phe

3605 3610 3615

Leu Ala Ser Ser Thr Glu Leu Asp Ser Met Ser Thr Pro His Gly

3620 3625 3630

Arg Ile Thr Val Ile Gly Thr Ser Leu Val Thr Pro Ser Ser Asp

3635 3640 3645

Ala Ser Ala Val Lys Thr Glu Thr Ser Thr Ser Glu Arg Thr Leu

3650 3655 3660

Ser Pro Ser Asp Thr Thr Ala Ser Thr Pro Ile Ser Thr Phe Ser

3665 3670 3675

Arg Val Gln Arg Met Ser Ile Ser Val Pro Asp Ile Leu Ser Thr

3680 3685 3690

Ser Trp Thr Pro Ser Ser Thr Glu Ala Glu Asp Val Pro Val Ser

3695 3700 3705

Met Val Ser Thr Asp His Ala Ser Thr Lys Thr Asp Pro Asn Thr

3710 3715 3720

Pro Leu Ser Thr Phe Leu Phe Asp Ser Leu Ser Thr Leu Asp Trp

3725 3730 3735

Asp Thr Gly Arg Ser Leu Ser Ser Ala Thr Ala Thr Thr Ser Ala

3740 3745 3750

Pro Gln Gly Ala Thr Thr Pro Gln Glu Leu Thr Leu Glu Thr Met

3755 3760 3765

Ile Ser Pro Ala Thr Ser Gln Leu Pro Phe Ser Ile Gly His Ile

3770 3775 3780

Thr Ser Ala Val Thr Pro Ala Ala Met Ala Arg Ser Ser Gly Val

3785 3790 3795

Thr Phe Ser Arg Pro Asp Pro Thr Ser Lys Lys Ala Glu Gln Thr

3800 3805 3810

Ser Thr Gln Leu Pro Thr Thr Thr Ser Ala His Pro Gly Gln Val

3815 3820 3825

Pro Arg Ser Ala Ala Thr Thr Leu Asp Val Ile Pro His Thr Ala

3830 3835 3840

Lys Thr Pro Asp Ala Thr Phe Gln Arg Gln Gly Gln Thr Ala Leu

3845 3850 3855

Thr Thr Glu Ala Arg Ala Thr Ser Asp Ser Trp Asn Glu Lys Glu

3860 3865 3870

Lys Ser Thr Pro Ser Ala Pro Trp Ile Thr Glu Met Met Asn Ser

3875 3880 3885

Val Ser Glu Asp Thr Ile Lys Glu Val Thr Ser Ser Ser Ser Val

3890 3895 3900

Leu Arg Thr Leu Asn Thr Leu Asp Ile Asn Leu Glu Ser Gly Thr

3905 3910 3915

Thr Ser Ser Pro Ser Trp Lys Ser Ser Pro Tyr Glu Arg Ile Ala

3920 3925 3930

Pro Ser Glu Ser Thr Thr Asp Lys Glu Ala Ile His Pro Ser Thr

3935 3940 3945

Asn Thr Val Glu Thr Thr Gly Trp Val Thr Ser Ser Glu His Ala

3950 3955 3960

Ser His Ser Thr Ile Pro Ala His Ser Ala Ser Ser Lys Leu Thr

3965 3970 3975

Ser Pro Val Val Thr Thr Ser Thr Arg Glu Gln Ala Ile Val Ser

3980 3985 3990

Met Ser Thr Thr Thr Trp Pro Glu Ser Thr Arg Ala Arg Thr Glu

3995 4000 4005

Pro Asn Ser Phe Leu Thr Ile Glu Leu Arg Asp Val Ser Pro Tyr

4010 4015 4020

Met Asp Thr Ser Ser Thr Thr Gln Thr Ser Ile Ile Ser Ser Pro

4025 4030 4035

Gly Ser Thr Ala Ile Thr Lys Gly Pro Arg Thr Glu Ile Thr Ser

4040 4045 4050

Ser Lys Arg Ile Ser Ser Ser Phe Leu Ala Gln Ser Met Arg Ser

4055 4060 4065

Ser Asp Ser Pro Ser Glu Ala Ile Thr Arg Leu Ser Asn Phe Pro

4070 4075 4080

Ala Met Thr Glu Ser Gly Gly Met Ile Leu Ala Met Gln Thr Ser

4085 4090 4095

Pro Pro Gly Ala Thr Ser Leu Ser Ala Pro Thr Leu Asp Thr Ser

4100 4105 4110

Ala Thr Ala Ser Trp Thr Gly Thr Pro Leu Ala Thr Thr Gln Arg

4115 4120 4125

Phe Thr Tyr Ser Glu Lys Thr Thr Leu Phe Ser Lys Gly Pro Glu

4130 4135 4140

Asp Thr Ser Gln Pro Ser Pro Pro Ser Val Glu Glu Thr Ser Ser

4145 4150 4155

Ser Ser Ser Leu Val Pro Ile His Ala Thr Thr Ser Pro Ser Asn

4160 4165 4170

Ile Leu Leu Thr Ser Gln Gly His Ser Pro Ser Ser Thr Pro Pro

4175 4180 4185

Val Thr Ser Val Phe Leu Ser Glu Thr Ser Gly Leu Gly Lys Thr

4190 4195 4200

Thr Asp Met Ser Arg Ile Ser Leu Glu Pro Gly Thr Ser Leu Pro

4205 4210 4215

Pro Asn Leu Ser Ser Thr Ala Gly Glu Ala Leu Ser Thr Tyr Glu

4220 4225 4230

Ala Ser Arg Asp Thr Lys Ala Ile His His Ser Ala Asp Thr Ala

4235 4240 4245

Val Thr Asn Met Glu Ala Thr Ser Ser Glu Tyr Ser Pro Ile Pro

4250 4255 4260

Gly His Thr Lys Pro Ser Lys Ala Thr Ser Pro Leu Val Thr Ser

4265 4270 4275

His Ile Met Gly Asp Ile Thr Ser Ser Thr Ser Val Phe Gly Ser

4280 4285 4290

Ser Glu Thr Thr Glu Ile Glu Thr Val Ser Ser Val Asn Gln Gly

4295 4300 4305

Leu Gln Glu Arg Ser Thr Ser Gln Val Ala Ser Ser Ala Thr Glu

4310 4315 4320

Thr Ser Thr Val Ile Thr His Val Ser Ser Gly Asp Ala Thr Thr

4325 4330 4335

His Val Thr Lys Thr Gln Ala Thr Phe Ser Ser Gly Thr Ser Ile

4340 4345 4350

Ser Ser Pro His Gln Phe Ile Thr Ser Thr Asn Thr Phe Thr Asp

4355 4360 4365

Val Ser Thr Asn Pro Ser Thr Ser Leu Ile Met Thr Glu Ser Ser

4370 4375 4380

Gly Val Thr Ile Thr Thr Gln Thr Gly Pro Thr Gly Ala Ala Thr

4385 4390 4395

Gln Gly Pro Tyr Leu Leu Asp Thr Ser Thr Met Pro Tyr Leu Thr

4400 4405 4410

Glu Thr Pro Leu Ala Val Thr Pro Asp Phe Met Gln Ser Glu Lys

4415 4420 4425

Thr Thr Leu Ile Ser Lys Gly Pro Lys Asp Val Ser Trp Thr Ser

4430 4435 4440

Pro Pro Ser Val Ala Glu Thr Ser Tyr Pro Ser Ser Leu Thr Pro

4445 4450 4455

Phe Leu Val Thr Thr Ile Pro Pro Ala Thr Ser Thr Leu Gln Gly

4460 4465 4470

Gln His Thr Ser Ser Pro Val Ser Ala Thr Ser Val Leu Thr Ser

4475 4480 4485

Gly Leu Val Lys Thr Thr Asp Met Leu Asn Thr Ser Met Glu Pro

4490 4495 4500

Val Thr Asn Ser Pro Gln Asn Leu Asn Asn Pro Ser Asn Glu Ile

4505 4510 4515

Leu Ala Thr Leu Ala Ala Thr Thr Asp Ile Glu Thr Ile His Pro

4520 4525 4530

Ser Ile Asn Lys Ala Val Thr Asn Met Gly Thr Ala Ser Ser Ala

4535 4540 4545

His Val Leu His Ser Thr Leu Pro Val Ser Ser Glu Pro Ser Thr

4550 4555 4560

Ala Thr Ser Pro Met Val Pro Ala Ser Ser Met Gly Asp Ala Leu

4565 4570 4575

Ala Ser Ile Ser Ile Pro Gly Ser Glu Thr Thr Asp Ile Glu Gly

4580 4585 4590

Glu Pro Thr Ser Ser Leu Thr Ala Gly Arg Lys Glu Asn Ser Thr

4595 4600 4605

Leu Gln Glu Met Asn Ser Thr Thr Glu Ser Asn Ile Ile Leu Ser

4610 4615 4620

Asn Val Ser Val Gly Ala Ile Thr Glu Ala Thr Lys Met Glu Val

4625 4630 4635

Pro Ser Phe Asp Ala Thr Phe Ile Pro Thr Pro Ala Gln Ser Thr

4640 4645 4650

Lys Phe Pro Asp Ile Phe Ser Val Ala Ser Ser Arg Leu Ser Asn

4655 4660 4665

Ser Pro Pro Met Thr Ile Ser Thr His Met Thr Thr Thr Gln Thr

4670 4675 4680

Gly Ser Ser Gly Ala Thr Ser Lys Ile Pro Leu Ala Leu Asp Thr

4685 4690 4695

Ser Thr Leu Glu Thr Ser Ala Gly Thr Pro Ser Val Val Thr Glu

4700 4705 4710

Gly Phe Ala His Ser Lys Ile Thr Thr Ala Met Asn Asn Asp Val

4715 4720 4725

Lys Asp Val Ser Gln Thr Asn Pro Pro Phe Gln Asp Glu Ala Ser

4730 4735 4740

Ser Pro Ser Ser Gln Ala Pro Val Leu Val Thr Thr Leu Pro Ser

4745 4750 4755

Ser Val Ala Phe Thr Pro Gln Trp His Ser Thr Ser Ser Pro Val

4760 4765 4770

Ser Met Ser Ser Val Leu Thr Ser Ser Leu Val Lys Thr Ala Gly

4775 4780 4785

Lys Val Asp Thr Ser Leu Glu Thr Val Thr Ser Ser Pro Gln Ser

4790 4795 4800

Met Ser Asn Thr Leu Asp Asp Ile Ser Val Thr Ser Ala Ala Thr

4805 4810 4815

Thr Asp Ile Glu Thr Thr His Pro Ser Ile Asn Thr Val Val Thr

4820 4825 4830

Asn Val Gly Thr Thr Gly Ser Ala Phe Glu Ser His Ser Thr Val

4835 4840 4845

Ser Ala Tyr Pro Glu Pro Ser Lys Val Thr Ser Pro Asn Val Thr

4850 4855 4860

Thr Ser Thr Met Glu Asp Thr Thr Ile Ser Arg Ser Ile Pro Lys

4865 4870 4875

Ser Ser Lys Thr Thr Arg Thr Glu Thr Glu Thr Thr Ser Ser Leu

4880 4885 4890

Thr Pro Lys Leu Arg Glu Thr Ser Ile Ser Gln Glu Ile Thr Ser

4895 4900 4905

Ser Thr Glu Thr Ser Thr Val Pro Tyr Lys Glu Leu Thr Gly Ala

4910 4915 4920

Thr Thr Glu Val Ser Arg Thr Asp Val Thr Ser Ser Ser Ser Thr

4925 4930 4935

Ser Phe Pro Gly Pro Asp Gln Ser Thr Val Ser Leu Asp Ile Ser

4940 4945 4950

Thr Glu Thr Asn Thr Arg Leu Ser Thr Ser Pro Ile Met Thr Glu

4955 4960 4965

Ser Ala Glu Ile Thr Ile Thr Thr Gln Thr Gly Pro His Gly Ala

4970 4975 4980

Thr Ser Gln Asp Thr Phe Thr Met Asp Pro Ser Asn Thr Thr Pro

4985 4990 4995

Gln Ala Gly Ile His Ser Ala Met Thr His Gly Phe Ser Gln Leu

5000 5005 5010

Asp Val Thr Thr Leu Met Ser Arg Ile Pro Gln Asp Val Ser Trp

5015 5020 5025

Thr Ser Pro Pro Ser Val Asp Lys Thr Ser Ser Pro Ser Ser Phe

5030 5035 5040

Leu Ser Ser Pro Ala Met Thr Thr Pro Ser Leu Ile Ser Ser Thr

5045 5050 5055

Leu Pro Glu Asp Lys Leu Ser Ser Pro Met Thr Ser Leu Leu Thr

5060 5065 5070

Ser Gly Leu Val Lys Ile Thr Asp Ile Leu Arg Thr Arg Leu Glu

5075 5080 5085

Pro Val Thr Ser Ser Leu Pro Asn Phe Ser Ser Thr Ser Asp Lys

5090 5095 5100

Ile Leu Ala Thr Ser Lys Asp Ser Lys Asp Thr Lys Glu Ile Phe

5105 5110 5115

Pro Ser Ile Asn Thr Glu Glu Thr Asn Val Lys Ala Asn Asn Ser

5120 5125 5130

Gly His Glu Ser His Ser Pro Ala Leu Ala Asp Ser Glu Thr Pro

5135 5140 5145

Lys Ala Thr Thr Gln Met Val Ile Thr Thr Thr Val Gly Asp Pro

5150 5155 5160

Ala Pro Ser Thr Ser Met Pro Val His Gly Ser Ser Glu Thr Thr

5165 5170 5175

Asn Ile Lys Arg Glu Pro Thr Tyr Phe Leu Thr Pro Arg Leu Arg

5180 5185 5190

Glu Thr Ser Thr Ser Gln Glu Ser Ser Phe Pro Thr Asp Thr Ser

5195 5200 5205

Phe Leu Leu Ser Lys Val Pro Thr Gly Thr Ile Thr Glu Val Ser

5210 5215 5220

Ser Thr Gly Val Asn Ser Ser Ser Lys Ile Ser Thr Pro Asp His

5225 5230 5235

Asp Lys Ser Thr Val Pro Pro Asp Thr Phe Thr Gly Glu Ile Pro

5240 5245 5250

Arg Val Phe Thr Ser Ser Ile Lys Thr Lys Ser Ala Glu Met Thr

5255 5260 5265

Ile Thr Thr Gln Ala Ser Pro Pro Glu Ser Ala Ser His Ser Thr

5270 5275 5280

Leu Pro Leu Asp Thr Ser Thr Thr Leu Ser Gln Gly Gly Thr His

5285 5290 5295

Ser Thr Val Thr Gln Gly Phe Pro Tyr Ser Glu Val Thr Thr Leu

5300 5305 5310

Met Gly Met Gly Pro Gly Asn Val Ser Trp Met Thr Thr Pro Pro

5315 5320 5325

Val Glu Glu Thr Ser Ser Val Ser Ser Leu Met Ser Ser Pro Ala

5330 5335 5340

Met Thr Ser Pro Ser Pro Val Ser Ser Thr Ser Pro Gln Ser Ile

5345 5350 5355

Pro Ser Ser Pro Leu Pro Val Thr Ala Leu Pro Thr Ser Val Leu

5360 5365 5370

Val Thr Thr Thr Asp Val Leu Gly Thr Thr Ser Pro Glu Ser Val

5375 5380 5385

Thr Ser Ser Pro Pro Asn Leu Ser Ser Ile Thr His Glu Arg Pro

5390 5395 5400

Ala Thr Tyr Lys Asp Thr Ala His Thr Glu Ala Ala Met His His

5405 5410 5415

Ser Thr Asn Thr Ala Val Thr Asn Val Gly Thr Ser Gly Ser Gly

5420 5425 5430

His Lys Ser Gln Ser Ser Val Leu Ala Asp Ser Glu Thr Ser Lys

5435 5440 5445

Ala Thr Pro Leu Met Ser Thr Thr Ser Thr Leu Gly Asp Thr Ser

5450 5455 5460

Val Ser Thr Ser Thr Pro Asn Ile Ser Gln Thr Asn Gln Ile Gln

5465 5470 5475

Thr Glu Pro Thr Ala Ser Leu Ser Pro Arg Leu Arg Glu Ser Ser

5480 5485 5490

Thr Ser Glu Lys Thr Ser Ser Thr Thr Glu Thr Asn Thr Ala Phe

5495 5500 5505

Ser Tyr Val Pro Thr Gly Ala Ile Thr Gln Ala Ser Arg Thr Glu

5510 5515 5520

Ile Ser Ser Ser Arg Thr Ser Ile Ser Asp Leu Asp Arg Pro Thr

5525 5530 5535

Ile Ala Pro Asp Ile Ser Thr Gly Met Ile Thr Arg Leu Phe Thr

5540 5545 5550

Ser Pro Ile Met Thr Lys Ser Ala Glu Met Thr Val Thr Thr Gln

5555 5560 5565

Thr Thr Thr Pro Gly Ala Thr Ser Gln Gly Ile Leu Pro Trp Asp

5570 5575 5580

Thr Ser Thr Thr Leu Phe Gln Gly Gly Thr His Ser Thr Val Ser

5585 5590 5595

Gln Gly Phe Pro His Ser Glu Ile Thr Thr Leu Arg Ser Arg Thr

5600 5605 5610

Pro Gly Asp Val Ser Trp Met Thr Thr Pro Pro Val Glu Glu Thr

5615 5620 5625

Ser Ser Gly Phe Ser Leu Met Ser Pro Ser Met Thr Ser Pro Ser

5630 5635 5640

Pro Val Ser Ser Thr Ser Pro Glu Ser Ile Pro Ser Ser Pro Leu

5645 5650 5655

Pro Val Thr Ala Leu Leu Thr Ser Val Leu Val Thr Thr Thr Asn

5660 5665 5670

Val Leu Gly Thr Thr Ser Pro Glu Pro Val Thr Ser Ser Pro Pro

5675 5680 5685

Asn Leu Ser Ser Pro Thr Gln Glu Arg Leu Thr Thr Tyr Lys Asp

5690 5695 5700

Thr Ala His Thr Glu Ala Met His Ala Ser Met His Thr Asn Thr

5705 5710 5715

Ala Val Ala Asn Val Gly Thr Ser Ile Ser Gly His Glu Ser Gln

5720 5725 5730

Ser Ser Val Pro Ala Asp Ser His Thr Ser Lys Ala Thr Ser Pro

5735 5740 5745

Met Gly Ile Thr Phe Ala Met Gly Asp Thr Ser Val Ser Thr Ser

5750 5755 5760

Thr Pro Ala Phe Phe Glu Thr Arg Ile Gln Thr Glu Ser Thr Ser

5765 5770 5775

Ser Leu Ile Pro Gly Leu Arg Asp Thr Arg Thr Ser Glu Glu Ile

5780 5785 5790

Asn Thr Val Thr Glu Thr Ser Thr Val Leu Ser Glu Val Pro Thr

5795 5800 5805

Thr Thr Thr Thr Glu Val Ser Arg Thr Glu Val Ile Thr Ser Ser

5810 5815 5820

Arg Thr Thr Ile Ser Gly Pro Asp His Ser Lys Met Ser Pro Tyr

5825 5830 5835

Ile Ser Thr Glu Thr Ile Thr Arg Leu Ser Thr Phe Pro Phe Val

5840 5845 5850

Thr Gly Ser Thr Glu Met Ala Ile Thr Asn Gln Thr Gly Pro Ile

5855 5860 5865

Gly Thr Ile Ser Gln Ala Thr Leu Thr Leu Asp Thr Ser Ser Thr

5870 5875 5880

Ala Ser Trp Glu Gly Thr His Ser Pro Val Thr Gln Arg Phe Pro

5885 5890 5895

His Ser Glu Glu Thr Thr Thr Met Ser Arg Ser Thr Lys Gly Val

5900 5905 5910

Ser Trp Gln Ser Pro Pro Ser Val Glu Glu Thr Ser Ser Pro Ser

5915 5920 5925

Ser Pro Val Pro Leu Pro Ala Ile Thr Ser His Ser Ser Leu Tyr

5930 5935 5940

Ser Ala Val Ser Gly Ser Ser Pro Thr Ser Ala Leu Pro Val Thr

5945 5950 5955

Ser Leu Leu Thr Ser Gly Arg Arg Lys Thr Ile Asp Met Leu Asp

5960 5965 5970

Thr His Ser Glu Leu Val Thr Ser Ser Leu Pro Ser Ala Ser Ser

5975 5980 5985

Phe Ser Gly Glu Ile Leu Thr Ser Glu Ala Ser Thr Asn Thr Glu

5990 5995 6000

Thr Ile His Phe Ser Glu Asn Thr Ala Glu Thr Asn Met Gly Thr

6005 6010 6015

Thr Asn Ser Met His Lys Leu His Ser Ser Val Ser Ile His Ser

6020 6025 6030

Gln Pro Ser Gly His Thr Pro Pro Lys Val Thr Gly Ser Met Met

6035 6040 6045

Glu Asp Ala Ile Val Ser Thr Ser Thr Pro Gly Ser Pro Glu Thr

6050 6055 6060

Lys Asn Val Asp Arg Asp Ser Thr Ser Pro Leu Thr Pro Glu Leu

6065 6070 6075

Lys Glu Asp Ser Thr Ala Leu Val Met Asn Ser Thr Thr Glu Ser

6080 6085 6090

Asn Thr Val Phe Ser Ser Val Ser Leu Asp Ala Ala Thr Glu Val

6095 6100 6105

Ser Arg Ala Glu Val Thr Tyr Tyr Asp Pro Thr Phe Met Pro Ala

6110 6115 6120

Ser Ala Gln Ser Thr Lys Ser Pro Asp Ile Ser Pro Glu Ala Ser

6125 6130 6135

Ser Ser His Ser Asn Ser Pro Pro Leu Thr Ile Ser Thr His Lys

6140 6145 6150

Thr Ile Ala Thr Gln Thr Gly Pro Ser Gly Val Thr Ser Leu Gly

6155 6160 6165

Gln Leu Thr Leu Asp Thr Ser Thr Ile Ala Thr Ser Ala Gly Thr

6170 6175 6180

Pro Ser Ala Arg Thr Gln Asp Phe Val Asp Ser Glu Thr Thr Ser

6185 6190 6195

Val Met Asn Asn Asp Leu Asn Asp Val Leu Lys Thr Ser Pro Phe

6200 6205 6210

Ser Ala Glu Glu Ala Asn Ser Leu Ser Ser Gln Ala Pro Leu Leu

6215 6220 6225

Val Thr Thr Ser Pro Ser Pro Val Thr Ser Thr Leu Gln Glu His

6230 6235 6240

Ser Thr Ser Ser Leu Val Ser Val Thr Ser Val Pro Thr Pro Thr

6245 6250 6255

Leu Ala Lys Ile Thr Asp Met Asp Thr Asn Leu Glu Pro Val Thr

6260 6265 6270

Arg Ser Pro Gln Asn Leu Arg Asn Thr Leu Ala Thr Ser Glu Ala

6275 6280 6285

Thr Thr Asp Thr His Thr Met His Pro Ser Ile Asn Thr Ala Val

6290 6295 6300

Ala Asn Val Gly Thr Thr Ser Ser Pro Asn Glu Phe Tyr Phe Thr

6305 6310 6315

Val Ser Pro Asp Ser Asp Pro Tyr Lys Ala Thr Ser Ala Val Val

6320 6325 6330

Ile Thr Ser Thr Ser Gly Asp Ser Ile Val Ser Thr Ser Met Pro

6335 6340 6345

Arg Ser Ser Ala Met Lys Lys Ile Glu Ser Glu Thr Thr Phe Ser

6350 6355 6360

Leu Ile Phe Arg Leu Arg Glu Thr Ser Thr Ser Gln Lys Ile Gly

6365 6370 6375

Ser Ser Ser Asp Thr Ser Thr Val Phe Asp Lys Ala Phe Thr Ala

6380 6385 6390

Ala Thr Thr Glu Val Ser Arg Thr Glu Leu Thr Ser Ser Ser Arg

6395 6400 6405

Thr Ser Ile Gln Gly Thr Glu Lys Pro Thr Met Ser Pro Asp Thr

6410 6415 6420

Ser Thr Arg Ser Val Thr Met Leu Ser Thr Phe Ala Gly Leu Thr

6425 6430 6435

Lys Ser Glu Glu Arg Thr Ile Ala Thr Gln Thr Gly Pro His Arg

6440 6445 6450

Ala Thr Ser Gln Gly Thr Leu Thr Trp Asp Thr Ser Ile Thr Thr

6455 6460 6465

Ser Gln Ala Gly Thr His Ser Ala Met Thr His Gly Phe Ser Gln

6470 6475 6480

Leu Asp Leu Ser Thr Leu Thr Ser Arg Val Pro Glu Tyr Ile Ser

6485 6490 6495

Gly Thr Ser Pro Pro Ser Val Glu Lys Thr Ser Ser Ser Ser Ser

6500 6505 6510

Leu Leu Ser Leu Pro Ala Ile Thr Ser Pro Ser Pro Val Pro Thr

6515 6520 6525

Thr Leu Pro Glu Ser Arg Pro Ser Ser Pro Val His Leu Thr Ser

6530 6535 6540

Leu Pro Thr Ser Gly Leu Val Lys Thr Thr Asp Met Leu Ala Ser

6545 6550 6555

Val Ala Ser Leu Pro Pro Asn Leu Gly Ser Thr Ser His Lys Ile

6560 6565 6570

Pro Thr Thr Ser Glu Asp Ile Lys Asp Thr Glu Lys Met Tyr Pro

6575 6580 6585

Ser Thr Asn Ile Ala Val Thr Asn Val Gly Thr Thr Thr Ser Glu

6590 6595 6600

Lys Glu Ser Tyr Ser Ser Val Pro Ala Tyr Ser Glu Pro Pro Lys

6605 6610 6615

Val Thr Ser Pro Met Val Thr Ser Phe Asn Ile Arg Asp Thr Ile

6620 6625 6630

Val Ser Thr Ser Met Pro Gly Ser Ser Glu Ile Thr Arg Ile Glu

6635 6640 6645

Met Glu Ser Thr Phe Ser Leu Ala His Gly Leu Lys Gly Thr Ser

6650 6655 6660

Thr Ser Gln Asp Pro Ile Val Ser Thr Glu Lys Ser Ala Val Leu

6665 6670 6675

His Lys Leu Thr Thr Gly Ala Thr Glu Thr Ser Arg Thr Glu Val

6680 6685 6690

Ala Ser Ser Arg Arg Thr Ser Ile Pro Gly Pro Asp His Ser Thr

6695 6700 6705

Glu Ser Pro Asp Ile Ser Thr Glu Val Ile Pro Ser Leu Pro Ile

6710 6715 6720

Ser Leu Gly Ile Thr Glu Ser Ser Asn Met Thr Ile Ile Thr Arg

6725 6730 6735

Thr Gly Pro Pro Leu Gly Ser Thr Ser Gln Gly Thr Phe Thr Leu

6740 6745 6750

Asp Thr Pro Thr Thr Ser Ser Arg Ala Gly Thr His Ser Met Ala

6755 6760 6765

Thr Gln Glu Phe Pro His Ser Glu Met Thr Thr Val Met Asn Lys

6770 6775 6780

Asp Pro Glu Ile Leu Ser Trp Thr Ile Pro Pro Ser Ile Glu Lys

6785 6790 6795

Thr Ser Phe Ser Ser Ser Leu Met Pro Ser Pro Ala Met Thr Ser

6800 6805 6810

Pro Pro Val Ser Ser Thr Leu Pro Lys Thr Ile His Thr Thr Pro

6815 6820 6825

Ser Pro Met Thr Ser Leu Leu Thr Pro Ser Leu Val Met Thr Thr

6830 6835 6840

Asp Thr Leu Gly Thr Ser Pro Glu Pro Thr Thr Ser Ser Pro Pro

6845 6850 6855

Asn Leu Ser Ser Thr Ser His Glu Ile Leu Thr Thr Asp Glu Asp

6860 6865 6870

Thr Thr Ala Ile Glu Ala Met His Pro Ser Thr Ser Thr Ala Ala

6875 6880 6885

Thr Asn Val Glu Thr Thr Ser Ser Gly His Gly Ser Gln Ser Ser

6890 6895 6900

Val Leu Ala Asp Ser Glu Lys Thr Lys Ala Thr Ala Pro Met Asp

6905 6910 6915

Thr Thr Ser Thr Met Gly His Thr Thr Val Ser Thr Ser Met Ser

6920 6925 6930

Val Ser Ser Glu Thr Thr Lys Ile Lys Arg Glu Ser Thr Tyr Ser

6935 6940 6945

Leu Thr Pro Gly Leu Arg Glu Thr Ser Ile Ser Gln Asn Ala Ser

6950 6955 6960

Phe Ser Thr Asp Thr Ser Ile Val Leu Ser Glu Val Pro Thr Gly

6965 6970 6975

Thr Thr Ala Glu Val Ser Arg Thr Glu Val Thr Ser Ser Gly Arg

6980 6985 6990

Thr Ser Ile Pro Gly Pro Ser Gln Ser Thr Val Leu Pro Glu Ile

6995 7000 7005

Ser Thr Arg Thr Met Thr Arg Leu Phe Ala Ser Pro Thr Met Thr

7010 7015 7020

Glu Ser Ala Glu Met Thr Ile Pro Thr Gln Thr Gly Pro Ser Gly

7025 7030 7035

Ser Thr Ser Gln Asp Thr Leu Thr Leu Asp Thr Ser Thr Thr Lys

7040 7045 7050

Ser Gln Ala Lys Thr His Ser Thr Leu Thr Gln Arg Phe Pro His

7055 7060 7065

Ser Glu Met Thr Thr Leu Met Ser Arg Gly Pro Gly Asp Met Ser

7070 7075 7080

Trp Gln Ser Ser Pro Ser Leu Glu Asn Pro Ser Ser Leu Pro Ser

7085 7090 7095

Leu Leu Ser Leu Pro Ala Thr Thr Ser Pro Pro Pro Ile Ser Ser

7100 7105 7110

Thr Leu Pro Val Thr Ile Ser Ser Ser Pro Leu Pro Val Thr Ser

7115 7120 7125

Leu Leu Thr Ser Ser Pro Val Thr Thr Thr Asp Met Leu His Thr

7130 7135 7140

Ser Pro Glu Leu Val Thr Ser Ser Pro Pro Lys Leu Ser His Thr

7145 7150 7155

Ser Asp Glu Arg Leu Thr Thr Gly Lys Asp Thr Thr Asn Thr Glu

7160 7165 7170

Ala Val His Pro Ser Thr Asn Thr Ala Ala Ser Asn Val Glu Ile

7175 7180 7185

Pro Ser Ser Gly His Glu Ser Pro Ser Ser Ala Leu Ala Asp Ser

7190 7195 7200

Glu Thr Ser Lys Ala Thr Ser Pro Met Phe Ile Thr Ser Thr Gln

7205 7210 7215

Glu Asp Thr Thr Val Ala Ile Ser Thr Pro His Phe Leu Glu Thr

7220 7225 7230

Ser Arg Ile Gln Lys Glu Ser Ile Ser Ser Leu Ser Pro Lys Leu

7235 7240 7245

Arg Glu Thr Gly Ser Ser Val Glu Thr Ser Ser Ala Ile Glu Thr

7250 7255 7260

Ser Ala Val Leu Ser Glu Val Ser Ile Gly Ala Thr Thr Glu Ile

7265 7270 7275

Ser Arg Thr Glu Val Thr Ser Ser Ser Arg Thr Ser Ile Ser Gly

7280 7285 7290

Ser Ala Glu Ser Thr Met Leu Pro Glu Ile Ser Thr Thr Arg Lys

7295 7300 7305

Ile Ile Lys Phe Pro Thr Ser Pro Ile Leu Ala Glu Ser Ser Glu

7310 7315 7320

Met Thr Ile Lys Thr Gln Thr Ser Pro Pro Gly Ser Thr Ser Glu

7325 7330 7335

Ser Thr Phe Thr Leu Asp Thr Ser Thr Thr Pro Ser Leu Val Ile

7340 7345 7350

Thr His Ser Thr Met Thr Gln Arg Leu Pro His Ser Glu Ile Thr

7355 7360 7365

Thr Leu Val Ser Arg Gly Ala Gly Asp Val Pro Arg Pro Ser Ser

7370 7375 7380

Leu Pro Val Glu Glu Thr Ser Pro Pro Ser Ser Gln Leu Ser Leu

7385 7390 7395

Ser Ala Met Ile Ser Pro Ser Pro Val Ser Ser Thr Leu Pro Ala

7400 7405 7410

Ser Ser His Ser Ser Ser Ala Ser Val Thr Ser Leu Leu Thr Pro

7415 7420 7425

Gly Gln Val Lys Thr Thr Glu Val Leu Asp Ala Ser Ala Glu Pro

7430 7435 7440

Glu Thr Ser Ser Pro Pro Ser Leu Ser Ser Thr Ser Val Glu Ile

7445 7450 7455

Leu Ala Thr Ser Glu Val Thr Thr Asp Thr Glu Lys Ile His Pro

7460 7465 7470

Phe Ser Asn Thr Ala Val Thr Lys Val Gly Thr Ser Ser Ser Gly

7475 7480 7485

His Glu Ser Pro Ser Ser Val Leu Pro Asp Ser Glu Thr Thr Lys

7490 7495 7500

Ala Thr Ser Ala Met Gly Thr Ile Ser Ile Met Gly Asp Thr Ser

7505 7510 7515

Val Ser Thr Leu Thr Pro Ala Leu Ser Asn Thr Arg Lys Ile Gln

7520 7525 7530

Ser Glu Pro Ala Ser Ser Leu Thr Thr Arg Leu Arg Glu Thr Ser

7535 7540 7545

Thr Ser Glu Glu Thr Ser Leu Ala Thr Glu Ala Asn Thr Val Leu

7550 7555 7560

Ser Lys Val Ser Thr Gly Ala Thr Thr Glu Val Ser Arg Thr Glu

7565 7570 7575

Ala Ile Ser Phe Ser Arg Thr Ser Met Ser Gly Pro Glu Gln Ser

7580 7585 7590

Thr Met Ser Gln Asp Ile Ser Ile Gly Thr Ile Pro Arg Ile Ser

7595 7600 7605

Ala Ser Ser Val Leu Thr Glu Ser Ala Lys Met Thr Ile Thr Thr

7610 7615 7620

Gln Thr Gly Pro Ser Glu Ser Thr Leu Glu Ser Thr Leu Asn Leu

7625 7630 7635

Asn Thr Ala Thr Thr Pro Ser Trp Val Glu Thr His Ser Ile Val

7640 7645 7650

Ile Gln Gly Phe Pro His Pro Glu Met Thr Thr Ser Met Gly Arg

7655 7660 7665

Gly Pro Gly Gly Val Ser Trp Pro Ser Pro Pro Phe Val Lys Glu

7670 7675 7680

Thr Ser Pro Pro Ser Ser Pro Leu Ser Leu Pro Ala Val Thr Ser

7685 7690 7695

Pro His Pro Val Ser Thr Thr Phe Leu Ala His Ile Pro Pro Ser

7700 7705 7710

Pro Leu Pro Val Thr Ser Leu Leu Thr Ser Gly Pro Ala Thr Thr

7715 7720 7725

Thr Asp Ile Leu Gly Thr Ser Thr Glu Pro Gly Thr Ser Ser Ser

7730 7735 7740

Ser Ser Leu Ser Thr Thr Ser His Glu Arg Leu Thr Thr Tyr Lys

7745 7750 7755

Asp Thr Ala His Thr Glu Ala Val His Pro Ser Thr Asn Thr Gly

7760 7765 7770

Gly Thr Asn Val Ala Thr Thr Ser Ser Gly Tyr Lys Ser Gln Ser

7775 7780 7785

Ser Val Leu Ala Asp Ser Ser Pro Met Cys Thr Thr Ser Thr Met

7790 7795 7800

Gly Asp Thr Ser Val Leu Thr Ser Thr Pro Ala Phe Leu Glu Thr

7805 7810 7815

Arg Arg Ile Gln Thr Glu Leu Ala Ser Ser Leu Thr Pro Gly Leu

7820 7825 7830

Arg Glu Ser Ser Gly Ser Glu Gly Thr Ser Ser Gly Thr Lys Met

7835 7840 7845

Ser Thr Val Leu Ser Lys Val Pro Thr Gly Ala Thr Thr Glu Ile

7850 7855 7860

Ser Lys Glu Asp Val Thr Ser Ile Pro Gly Pro Ala Gln Ser Thr

7865 7870 7875

Ile Ser Pro Asp Ile Ser Thr Arg Thr Val Ser Trp Phe Ser Thr

7880 7885 7890

Ser Pro Val Met Thr Glu Ser Ala Glu Ile Thr Met Asn Thr His

7895 7900 7905

Thr Ser Pro Leu Gly Ala Thr Thr Gln Gly Thr Ser Thr Leu Asp

7910 7915 7920

Thr Ser Ser Thr Thr Ser Leu Thr Met Thr His Ser Thr Ile Ser

7925 7930 7935

Gln Gly Phe Ser His Ser Gln Met Ser Thr Leu Met Arg Arg Gly

7940 7945 7950

Pro Glu Asp Val Ser Trp Met Ser Pro Pro Leu Leu Glu Lys Thr

7955 7960 7965

Arg Pro Ser Phe Ser Leu Met Ser Ser Pro Ala Thr Thr Ser Pro

7970 7975 7980

Ser Pro Val Ser Ser Thr Leu Pro Glu Ser Ile Ser Ser Ser Pro

7985 7990 7995

Leu Pro Val Thr Ser Leu Leu Thr Ser Gly Leu Ala Lys Thr Thr

8000 8005 8010

Asp Met Leu His Lys Ser Ser Glu Pro Val Thr Asn Ser Pro Ala

8015 8020 8025

Asn Leu Ser Ser Thr Ser Val Glu Ile Leu Ala Thr Ser Glu Val

8030 8035 8040

Thr Thr Asp Thr Glu Lys Thr His Pro Ser Ser Asn Arg Thr Val

8045 8050 8055

Thr Asp Val Gly Thr Ser Ser Ser Gly His Glu Ser Thr Ser Phe

8060 8065 8070

Val Leu Ala Asp Ser Gln Thr Ser Lys Val Thr Ser Pro Met Val

8075 8080 8085

Ile Thr Ser Thr Met Glu Asp Thr Ser Val Ser Thr Ser Thr Pro

8090 8095 8100

Gly Phe Phe Glu Thr Ser Arg Ile Gln Thr Glu Pro Thr Ser Ser

8105 8110 8115

Leu Thr Leu Gly Leu Arg Lys Thr Ser Ser Ser Glu Gly Thr Ser

8120 8125 8130

Leu Ala Thr Glu Met Ser Thr Val Leu Ser Gly Val Pro Thr Gly

8135 8140 8145

Ala Thr Ala Glu Val Ser Arg Thr Glu Val Thr Ser Ser Ser Arg

8150 8155 8160

Thr Ser Ile Ser Gly Phe Ala Gln Leu Thr Val Ser Pro Glu Thr

8165 8170 8175

Ser Thr Glu Thr Ile Thr Arg Leu Pro Thr Ser Ser Ile Met Thr

8180 8185 8190

Glu Ser Ala Glu Met Met Ile Lys Thr Gln Thr Asp Pro Pro Gly

8195 8200 8205

Ser Thr Pro Glu Ser Thr His Thr Val Asp Ile Ser Thr Thr Pro

8210 8215 8220

Asn Trp Val Glu Thr His Ser Thr Val Thr Gln Arg Phe Ser His

8225 8230 8235

Ser Glu Met Thr Thr Leu Val Ser Arg Ser Pro Gly Asp Met Leu

8240 8245 8250

Trp Pro Ser Gln Ser Ser Val Glu Glu Thr Ser Ser Ala Ser Ser

8255 8260 8265

Leu Leu Ser Leu Pro Ala Thr Thr Ser Pro Ser Pro Val Ser Ser

8270 8275 8280

Thr Leu Val Glu Asp Phe Pro Ser Ala Ser Leu Pro Val Thr Ser

8285 8290 8295

Leu Leu Asn Pro Gly Leu Val Ile Thr Thr Asp Arg Met Gly Ile

8300 8305 8310

Ser Arg Glu Pro Gly Thr Ser Ser Thr Ser Asn Leu Ser Ser Thr

8315 8320 8325

Ser His Glu Arg Leu Thr Thr Leu Glu Asp Thr Val Asp Thr Glu

8330 8335 8340

Asp Met Gln Pro Ser Thr His Thr Ala Val Thr Asn Val Arg Thr

8345 8350 8355

Ser Ile Ser Gly His Glu Ser Gln Ser Ser Val Leu Ser Asp Ser

8360 8365 8370

Glu Thr Pro Lys Ala Thr Ser Pro Met Gly Thr Thr Tyr Thr Met

8375 8380 8385

Gly Glu Thr Ser Val Ser Ile Ser Thr Ser Asp Phe Phe Glu Thr

8390 8395 8400

Ser Arg Ile Gln Ile Glu Pro Thr Ser Ser Leu Thr Ser Gly Leu

8405 8410 8415

Arg Glu Thr Ser Ser Ser Glu Arg Ile Ser Ser Ala Thr Glu Gly

8420 8425 8430

Ser Thr Val Leu Ser Glu Val Pro Ser Gly Ala Thr Thr Glu Val

8435 8440 8445

Ser Arg Thr Glu Val Ile Ser Ser Arg Gly Thr Ser Met Ser Gly

8450 8455 8460

Pro Asp Gln Phe Thr Ile Ser Pro Asp Ile Ser Thr Glu Ala Ile

8465 8470 8475

Thr Arg Leu Ser Thr Ser Pro Ile Met Thr Glu Ser Ala Glu Ser

8480 8485 8490

Ala Ile Thr Ile Glu Thr Gly Ser Pro Gly Ala Thr Ser Glu Gly

8495 8500 8505

Thr Leu Thr Leu Asp Thr Ser Thr Thr Thr Phe Trp Ser Gly Thr

8510 8515 8520

His Ser Thr Ala Ser Pro Gly Phe Ser His Ser Glu Met Thr Thr

8525 8530 8535

Leu Met Ser Arg Thr Pro Gly Asp Val Pro Trp Pro Ser Leu Pro

8540 8545 8550

Ser Val Glu Glu Ala Ser Ser Val Ser Ser Ser Leu Ser Ser Pro

8555 8560 8565

Ala Met Thr Ser Thr Ser Phe Phe Ser Thr Leu Pro Glu Ser Ile

8570 8575 8580

Ser Ser Ser Pro His Pro Val Thr Ala Leu Leu Thr Leu Gly Pro

8585 8590 8595

Val Lys Thr Thr Asp Met Leu Arg Thr Ser Ser Glu Pro Glu Thr

8600 8605 8610

Ser Ser Pro Pro Asn Leu Ser Ser Thr Ser Ala Glu Ile Leu Ala

8615 8620 8625

Thr Ser Glu Val Thr Lys Asp Arg Glu Lys Ile His Pro Ser Ser

8630 8635 8640

Asn Thr Pro Val Val Asn Val Gly Thr Val Ile Tyr Lys His Leu

8645 8650 8655

Ser Pro Ser Ser Val Leu Ala Asp Leu Val Thr Thr Lys Pro Thr

8660 8665 8670

Ser Pro Met Ala Thr Thr Ser Thr Leu Gly Asn Thr Ser Val Ser

8675 8680 8685

Thr Ser Thr Pro Ala Phe Pro Glu Thr Met Met Thr Gln Pro Thr

8690 8695 8700

Ser Ser Leu Thr Ser Gly Leu Arg Glu Ile Ser Thr Ser Gln Glu

8705 8710 8715

Thr Ser Ser Ala Thr Glu Arg Ser Ala Ser Leu Ser Gly Met Pro

8720 8725 8730

Thr Gly Ala Thr Thr Lys Val Ser Arg Thr Glu Ala Leu Ser Leu

8735 8740 8745

Gly Arg Thr Ser Thr Pro Gly Pro Ala Gln Ser Thr Ile Ser Pro

8750 8755 8760

Glu Ile Ser Thr Glu Thr Ile Thr Arg Ile Ser Thr Pro Leu Thr

8765 8770 8775

Thr Thr Gly Ser Ala Glu Met Thr Ile Thr Pro Lys Thr Gly His

8780 8785 8790

Ser Gly Ala Ser Ser Gln Gly Thr Phe Thr Leu Asp Thr Ser Ser

8795 8800 8805

Arg Ala Ser Trp Pro Gly Thr His Ser Ala Ala Thr His Arg Ser

8810 8815 8820

Pro His Ser Gly Met Thr Thr Pro Met Ser Arg Gly Pro Glu Asp

8825 8830 8835

Val Ser Trp Pro Ser Arg Pro Ser Val Glu Lys Thr Ser Pro Pro

8840 8845 8850

Ser Ser Leu Val Ser Leu Ser Ala Val Thr Ser Pro Ser Pro Leu

8855 8860 8865

Tyr Ser Thr Pro Ser Glu Ser Ser His Ser Ser Pro Leu Arg Val

8870 8875 8880

Thr Ser Leu Phe Thr Pro Val Met Met Lys Thr Thr Asp Met Leu

8885 8890 8895

Asp Thr Ser Leu Glu Pro Val Thr Thr Ser Pro Pro Ser Met Asn

8900 8905 8910

Ile Thr Ser Asp Glu Ser Leu Ala Thr Ser Lys Ala Thr Met Glu

8915 8920 8925

Thr Glu Ala Ile Gln Leu Ser Glu Asn Thr Ala Val Thr Gln Met

8930 8935 8940

Gly Thr Ile Ser Ala Arg Gln Glu Phe Tyr Ser Ser Tyr Pro Gly

8945 8950 8955

Leu Pro Glu Pro Ser Lys Val Thr Ser Pro Val Val Thr Ser Ser

8960 8965 8970

Thr Ile Lys Asp Ile Val Ser Thr Thr Ile Pro Ala Ser Ser Glu

8975 8980 8985

Ile Thr Arg Ile Glu Met Glu Ser Thr Ser Thr Leu Thr Pro Thr

8990 8995 9000

Pro Arg Glu Thr Ser Thr Ser Gln Glu Ile His Ser Ala Thr Lys

9005 9010 9015

Pro Ser Thr Val Pro Tyr Lys Ala Leu Thr Ser Ala Thr Ile Glu

9020 9025 9030

Asp Ser Met Thr Gln Val Met Ser Ser Ser Arg Gly Pro Ser Pro

9035 9040 9045

Asp Gln Ser Thr Met Ser Gln Asp Ile Ser Thr Glu Val Ile Thr

9050 9055 9060

Arg Leu Ser Thr Ser Pro Ile Lys Thr Glu Ser Thr Glu Met Thr

9065 9070 9075

Ile Thr Thr Gln Thr Gly Ser Pro Gly Ala Thr Ser Arg Gly Thr

9080 9085 9090

Leu Thr Leu Asp Thr Ser Thr Thr Phe Met Ser Gly Thr His Ser

9095 9100 9105

Thr Ala Ser Gln Gly Phe Ser His Ser Gln Met Thr Ala Leu Met

9110 9115 9120

Ser Arg Thr Pro Gly Asp Val Pro Trp Leu Ser His Pro Ser Val

9125 9130 9135

Glu Glu Ala Ser Ser Ala Ser Phe Ser Leu Ser Ser Pro Val Met

9140 9145 9150

Thr Ser Ser Ser Pro Val Ser Ser Thr Leu Pro Asp Ser Ile His

9155 9160 9165

Ser Ser Ser Leu Pro Val Thr Ser Leu Leu Thr Ser Gly Leu Val

9170 9175 9180

Lys Thr Thr Glu Leu Leu Gly Thr Ser Ser Glu Pro Glu Thr Ser

9185 9190 9195

Ser Pro Pro Asn Leu Ser Ser Thr Ser Ala Glu Ile Leu Ala Ile

9200 9205 9210

Thr Glu Val Thr Thr Asp Thr Glu Lys Leu Glu Met Thr Asn Val

9215 9220 9225

Val Thr Ser Gly Tyr Thr His Glu Ser Pro Ser Ser Val Leu Ala

9230 9235 9240

Asp Ser Val Thr Thr Lys Ala Thr Ser Ser Met Gly Ile Thr Tyr

9245 9250 9255

Pro Thr Gly Asp Thr Asn Val Leu Thr Ser Thr Pro Ala Phe Ser

9260 9265 9270

Asp Thr Ser Arg Ile Gln Thr Lys Ser Lys Leu Ser Leu Thr Pro

9275 9280 9285

Gly Leu Met Glu Thr Ser Ile Ser Glu Glu Thr Ser Ser Ala Thr

9290 9295 9300

Glu Lys Ser Thr Val Leu Ser Ser Val Pro Thr Gly Ala Thr Thr

9305 9310 9315

Glu Val Ser Arg Thr Glu Ala Ile Ser Ser Ser Arg Thr Ser Ile

9320 9325 9330

Pro Gly Pro Ala Gln Ser Thr Met Ser Ser Asp Thr Ser Met Glu

9335 9340 9345

Thr Ile Thr Arg Ile Ser Thr Pro Leu Thr Arg Lys Glu Ser Thr

9350 9355 9360

Asp Met Ala Ile Thr Pro Lys Thr Gly Pro Ser Gly Ala Thr Ser

9365 9370 9375

Gln Gly Thr Phe Thr Leu Asp Ser Ser Ser Thr Ala Ser Trp Pro

9380 9385 9390

Gly Thr His Ser Ala Thr Thr Gln Arg Phe Pro Gln Ser Val Val

9395 9400 9405

Thr Thr Pro Met Ser Arg Gly Pro Glu Asp Val Ser Trp Pro Ser

9410 9415 9420

Pro Leu Ser Val Glu Lys Asn Ser Pro Pro Ser Ser Leu Val Ser

9425 9430 9435

Ser Ser Ser Val Thr Ser Pro Ser Pro Leu Tyr Ser Thr Pro Ser

9440 9445 9450

Gly Ser Ser His Ser Pro Val Pro Val Thr Ser Leu Phe Thr

9455 9460 9465

Ser Ile Met Met Lys Ala Thr Asp Met Leu Asp Ala Ser Leu Glu

9470 9475 9480

Pro Glu Thr Thr Ser Ala Pro Asn Met Asn Ile Thr Ser Asp Glu

9485 9490 9495

Ser Leu Ala Ala Ser Lys Ala Thr Thr Glu Thr Glu Ala Ile His

9500 9505 9510

Val Phe Glu Asn Thr Ala Ala Ser His Val Glu Thr Thr Ser Ala

9515 9520 9525

Thr Glu Glu Leu Tyr Ser Ser Ser Pro Gly Phe Ser Glu Pro Thr

9530 9535 9540

Lys Val Ile Ser Pro Val Val Thr Ser Ser Ser Ile Arg Asp Asn

9545 9550 9555

Met Val Ser Thr Thr Met Pro Gly Ser Ser Gly Ile Thr Arg Ile

9560 9565 9570

Glu Ile Glu Ser Met Ser Ser Leu Thr Pro Gly Leu Arg Glu Thr

9575 9580 9585

Arg Thr Ser Gln Asp Ile Thr Ser Ser Thr Glu Thr Ser Thr Val

9590 9595 9600

Leu Tyr Lys Met Pro Ser Gly Ala Thr Pro Glu Val Ser Arg Thr

9605 9610 9615

Glu Val Met Pro Ser Ser Arg Thr Ser Ile Pro Gly Pro Ala Gln

9620 9625 9630

Ser Thr Met Ser Leu Asp Ile Ser Asp Glu Val Val Thr Arg Leu

9635 9640 9645

Ser Thr Ser Pro Ile Met Thr Glu Ser Ala Glu Ile Thr Ile Thr

9650 9655 9660

Thr Gln Thr Gly Tyr Ser Leu Ala Thr Ser Gln Val Thr Leu Pro

9665 9670 9675

Leu Gly Thr Ser Met Thr Phe Leu Ser Gly Thr His Ser Thr Met

9680 9685 9690

Ser Gln Gly Leu Ser His Ser Glu Met Thr Asn Leu Met Ser Arg

9695 9700 9705

Gly Pro Glu Ser Leu Ser Trp Thr Ser Pro Arg Phe Val Glu Thr

9710 9715 9720

Thr Arg Ser Ser Ser Ser Leu Thr Ser Leu Pro Leu Thr Thr Ser

9725 9730 9735

Leu Ser Pro Val Ser Ser Thr Leu Leu Asp Ser Ser Pro Ser Ser

9740 9745 9750

Pro Leu Pro Val Thr Ser Leu Ile Leu Pro Gly Leu Val Lys Thr

9755 9760 9765

Thr Glu Val Leu Asp Thr Ser Ser Glu Pro Lys Thr Ser Ser Ser

9770 9775 9780

Pro Asn Leu Ser Ser Thr Ser Val Glu Ile Pro Ala Thr Ser Glu

9785 9790 9795

Ile Met Thr Asp Thr Glu Lys Ile His Pro Ser Ser Asn Thr Ala

9800 9805 9810

Val Ala Lys Val Arg Thr Ser Ser Ser Val His Glu Ser His Ser

9815 9820 9825

Ser Val Leu Ala Asp Ser Glu Thr Thr Ile Thr Ile Pro Ser Met

9830 9835 9840

Gly Ile Thr Ser Ala Val Asp Asp Thr Thr Val Phe Thr Ser Asn

9845 9850 9855

Pro Ala Phe Ser Glu Thr Arg Arg Ile Pro Thr Glu Pro Thr Phe

9860 9865 9870

Ser Leu Thr Pro Gly Phe Arg Glu Thr Ser Thr Ser Glu Glu Thr

9875 9880 9885

Thr Ser Ile Thr Glu Thr Ser Ala Val Leu Tyr Gly Val Pro Thr

9890 9895 9900

Ser Ala Thr Thr Glu Val Ser Met Thr Glu Ile Met Ser Ser Asn

9905 9910 9915

Arg Ile His Ile Pro Asp Ser Asp Gln Ser Thr Met Ser Pro Asp

9920 9925 9930

Ile Ile Thr Glu Val Ile Thr Arg Leu Ser Ser Ser Ser Met Met

9935 9940 9945

Ser Glu Ser Thr Gln Met Thr Ile Thr Thr Gln Lys Ser Ser Pro

9950 9955 9960

Gly Ala Thr Ala Gln Ser Thr Leu Thr Leu Ala Thr Thr Thr Ala

9965 9970 9975

Pro Leu Ala Arg Thr His Ser Thr Val Pro Pro Arg Phe Leu His

9980 9985 9990

Ser Glu Met Thr Thr Leu Met Ser Arg Ser Pro Glu Asn Pro Ser

9995 10000 10005

Trp Lys Ser Ser Leu Phe Val Glu Lys Thr Ser Ser Ser Ser Ser

10010 10015 10020

Leu Leu Ser Leu Pro Val Thr Thr Ser Pro Ser Val Ser Ser Thr

10025 10030 10035

Leu Pro Gln Ser Ile Pro Ser Ser Ser Phe Ser Val Thr Ser Leu

10040 10045 10050

Leu Thr Pro Gly Met Val Lys Thr Thr Asp Thr Ser Thr Glu Pro

10055 10060 10065

Gly Thr Ser Leu Ser Pro Asn Leu Ser Gly Thr Ser Val Glu Ile

10070 10075 10080

Leu Ala Ala Ser Glu Val Thr Thr Asp Thr Glu Lys Ile His Pro

10085 10090 10095

Ser Ser Ser Met Ala Val Thr Asn Val Gly Thr Thr Ser Ser Gly

10100 10105 10110

His Glu Leu Tyr Ser Ser Val Ser Ile His Ser Glu Pro Ser Lys

10115 10120 10125

Ala Thr Tyr Pro Val Gly Thr Pro Ser Ser Met Ala Glu Thr Ser

10130 10135 10140

Ile Ser Thr Ser Met Pro Ala Asn Phe Glu Thr Thr Gly Phe Glu

10145 10150 10155

Ala Glu Pro Phe Ser His Leu Thr Ser Gly Phe Arg Lys Thr Asn

10160 10165 10170

Met Ser Leu Asp Thr Ser Ser Val Thr Pro Thr Asn Thr Pro Ser

10175 10180 10185

Ser Pro Gly Ser Thr His Leu Leu Gln Ser Ser Lys Thr Asp Phe

10190 10195 10200

Thr Ser Ser Ala Lys Thr Ser Ser Pro Asp Trp Pro Pro Ala Ser

10205 10210 10215

Gln Tyr Thr Glu Ile Pro Val Asp Ile Ile Thr Pro Phe Asn Ala

10220 10225 10230

Ser Pro Ser Ile Thr Glu Ser Thr Gly Ile Thr Ser Phe Pro Glu

10235 10240 10245

Ser Arg Phe Thr Met Ser Val Thr Glu Ser Thr His His Leu Ser

10250 10255 10260

Thr Asp Leu Leu Pro Ser Ala Glu Thr Ile Ser Thr Gly Thr Val

10265 10270 10275

Met Pro Ser Leu Ser Glu Ala Met Thr Ser Phe Ala Thr Thr Gly

10280 10285 10290

Val Pro Arg Ala Ile Ser Gly Ser Gly Ser Pro Phe Ser Arg Thr

10295 10300 10305

Glu Ser Gly Pro Gly Asp Ala Thr Leu Ser Thr Ile Ala Glu Ser

10310 10315 10320

Leu Pro Ser Ser Thr Pro Val Pro Phe Ser Ser Ser Thr Phe Thr

10325 10330 10335

Thr Thr Asp Ser Ser Thr Ile Pro Ala Leu His Glu Ile Thr Ser

10340 10345 10350

Ser Ser Ala Thr Pro Tyr Arg Val Asp Thr Ser Leu Gly Thr Glu

10355 10360 10365

Ser Ser Thr Thr Glu Gly Arg Leu Val Met Val Ser Thr Leu Asp

10370 10375 10380

Thr Ser Ser Gln Pro Gly Arg Thr Ser Ser Ser Pro Ile Leu Asp

10385 10390 10395

Thr Arg Met Thr Glu Ser Val Glu Leu Gly Thr Val Thr Ser Ala

10400 10405 10410

Tyr Gln Val Pro Ser Leu Ser Thr Arg Leu Thr Arg Thr Asp Gly

10415 10420 10425

Ile Met Glu His Ile Thr Lys Ile Pro Asn Glu Ala Ala His Arg

10430 10435 10440

Gly Thr Ile Arg Pro Val Lys Gly Pro Gln Thr Ser Thr Ser Pro

10445 10450 10455

Ala Ser Pro Lys Gly Leu His Thr Gly Gly Thr Lys Arg Met Glu

10460 10465 10470

Thr Thr Thr Thr Ala Leu Lys Thr Thr Thr Thr Ala Leu Lys Thr

10475 10480 10485

Thr Ser Arg Ala Thr Leu Thr Thr Ser Val Tyr Thr Pro Thr Leu

10490 10495 10500

Gly Thr Leu Thr Pro Leu Asn Ala Ser Met Gln Met Ala Ser Thr

10505 10510 10515

Ile Pro Thr Glu Met Met Ile Thr Thr Pro Tyr Val Phe Pro Asp

10520 10525 10530

Val Pro Glu Thr Thr Ser Ser Leu Ala Thr Ser Leu Gly Ala Glu

10535 10540 10545

Thr Ser Thr Ala Leu Pro Arg Thr Thr Pro Ser Val Phe Asn Arg

10550 10555 10560

Glu Ser Glu Thr Thr Ala Ser Leu Val Ser Arg Ser Gly Ala Glu

10565 10570 10575

Arg Ser Pro Val Ile Gln Thr Leu Asp Val Ser Ser Ser Glu Pro

10580 10585 10590

Asp Thr Thr Ala Ser Trp Val Ile His Pro Ala Glu Thr Ile Pro

10595 10600 10605

Thr Val Ser Lys Thr Thr Pro Asn Phe Phe His Ser Glu Leu Asp

10610 10615 10620

Thr Val Ser Ser Thr Ala Thr Ser His Gly Ala Asp Val Ser Ser

10625 10630 10635

Ala Ile Pro Thr Asn Ile Ser Pro Ser Glu Leu Asp Ala Leu Thr

10640 10645 10650

Pro Leu Val Thr Ile Ser Gly Thr Asp Thr Ser Thr Thr Phe Pro

10655 10660 10665

Thr Leu Thr Lys Ser Pro His Glu Thr Glu Thr Arg Thr Thr Trp

10670 10675 10680

Leu Thr His Pro Ala Glu Thr Ser Ser Thr Ile Pro Arg Thr Ile

10685 10690 10695

Pro Asn Phe Ser His His Glu Ser Asp Ala Thr Pro Ser Ile Ala

10700 10705 10710

Thr Ser Pro Gly Ala Glu Thr Ser Ser Ala Ile Pro Ile Met Thr

10715 10720 10725

Val Ser Pro Gly Ala Glu Asp Leu Val Thr Ser Gln Val Thr Ser

10730 10735 10740

Ser Gly Thr Asp Arg Asn Met Thr Ile Pro Thr Leu Thr Leu Ser

10745 10750 10755

Pro Gly Glu Pro Lys Thr Ile Ala Ser Leu Val Thr His Pro Glu

10760 10765 10770

Ala Gln Thr Ser Ser Ala Ile Pro Thr Ser Thr Ile Ser Pro Ala

10775 10780 10785

Val Ser Arg Leu Val Thr Ser Met Val Thr Ser Leu Ala Ala Lys

10790 10795 10800

Thr Ser Thr Thr Asn Arg Ala Leu Thr Asn Ser Pro Gly Glu Pro

10805 10810 10815

Ala Thr Thr Val Ser Leu Val Thr His Pro Ala Gln Thr Ser Pro

10820 10825 10830

Thr Val Pro Trp Thr Thr Ser Ile Phe Phe His Ser Lys Ser Asp

10835 10840 10845

Thr Thr Pro Ser Met Thr Thr Ser His Gly Ala Glu Ser Ser Ser

10850 10855 10860

Ala Val Pro Thr Pro Thr Val Ser Thr Glu Val Pro Gly Val Val

10865 10870 10875

Thr Pro Leu Val Thr Ser Ser Arg Ala Val Ile Ser Thr Thr Ile

10880 10885 10890

Pro Ile Leu Thr Leu Ser Pro Gly Glu Pro Glu Thr Thr Pro Ser

10895 10900 10905

Met Ala Thr Ser His Gly Glu Glu Ala Ser Ser Ala Ile Pro Thr

10910 10915 10920

Pro Thr Val Ser Pro Gly Val Pro Gly Val Val Thr Ser Leu Val

10925 10930 10935

Thr Ser Ser Arg Ala Val Thr Ser Thr Thr Ile Pro Ile Leu Thr

10940 10945 10950

Phe Ser Leu Gly Glu Pro Glu Thr Thr Pro Ser Met Ala Thr Ser

10955 10960 10965

His Gly Thr Glu Ala Gly Ser Ala Val Pro Thr Val Leu Pro Glu

10970 10975 10980

Val Pro Gly Met Val Thr Ser Leu Val Ala Ser Ser Arg Ala Val

10985 10990 10995

Thr Ser Thr Thr Leu Pro Thr Leu Thr Leu Ser Pro Gly Glu Pro

11000 11005 11010

Glu Thr Thr Pro Ser Met Ala Thr Ser His Gly Ala Glu Ala Ser

11015 11020 11025

Ser Thr Val Pro Thr Val Ser Pro Glu Val Pro Gly Val Val Thr

11030 11035 11040

Ser Leu Val Thr Ser Ser Ser Gly Val Asn Ser Thr Ser Ile Pro

11045 11050 11055

Thr Leu Ile Leu Ser Pro Gly Glu Leu Glu Thr Thr Pro Ser Met

11060 11065 11070

Ala Thr Ser His Gly Ala Glu Ala Ser Ser Ala Val Pro Thr Pro

11075 11080 11085

Thr Val Ser Pro Gly Val Ser Gly Val Val Thr Pro Leu Val Thr

11090 11095 11100

Ser Ser Arg Ala Val Thr Ser Thr Thr Ile Pro Ile Leu Thr Leu

11105 11110 11115

Ser Ser Ser Glu Pro Glu Thr Thr Pro Ser Met Ala Thr Ser His

11120 11125 11130

Gly Val Glu Ala Ser Ser Ala Val Leu Thr Val Ser Pro Glu Val

11135 11140 11145

Pro Gly Met Val Thr Ser Leu Val Thr Ser Ser Arg Ala Val Thr

11150 11155 11160

Ser Thr Thr Ile Pro Thr Leu Thr Ile Ser Ser Asp Glu Pro Glu

11165 11170 11175

Thr Thr Thr Ser Leu Val Thr His Ser Glu Ala Lys Met Ile Ser

11180 11185 11190

Ala Ile Pro Thr Leu Ala Val Ser Pro Thr Val Gln Gly Leu Val

11195 11200 11205

Thr Ser Leu Val Thr Ser Ser Gly Ser Glu Thr Ser Ala Phe Ser

11210 11215 11220

Asn Leu Thr Val Ala Ser Ser Gln Pro Glu Thr Ile Asp Ser Trp

11225 11230 11235

Val Ala His Pro Gly Thr Glu Ala Ser Ser Val Val Pro Thr Leu

11240 11245 11250

Thr Val Ser Thr Gly Glu Pro Phe Thr Asn Ile Ser Leu Val Thr

11255 11260 11265

His Pro Ala Glu Ser Ser Ser Thr Leu Pro Arg Thr Thr Ser Arg

11270 11275 11280

Phe Ser His Ser Glu Leu Asp Thr Met Pro Ser Thr Val Thr Ser

11285 11290 11295

Pro Glu Ala Glu Ser Ser Ser Ala Ile Ser Thr Thr Ile Ser Pro

11300 11305 11310

Gly Ile Pro Gly Val Leu Thr Ser Leu Val Thr Ser Ser Gly Arg

11315 11320 11325

Asp Ile Ser Ala Thr Phe Pro Thr Val Pro Glu Ser Pro His Glu

11330 11335 11340

Ser Glu Ala Thr Ala Ser Trp Val Thr His Pro Ala Val Thr Ser

11345 11350 11355

Thr Thr Val Pro Arg Thr Thr Pro Asn Tyr Ser His Ser Glu Pro

11360 11365 11370

Asp Thr Thr Pro Ser Ile Ala Thr Ser Pro Gly Ala Glu Ala Thr

11375 11380 11385

Ser Asp Phe Pro Thr Ile Thr Val Ser Pro Asp Val Pro Asp Met

11390 11395 11400

Val Thr Ser Gln Val Thr Ser Ser Gly Thr Asp Thr Ser Ile Thr

11405 11410 11415

Ile Pro Thr Leu Thr Leu Ser Ser Gly Glu Pro Glu Thr Thr Thr

11420 11425 11430

Ser Phe Ile Thr Tyr Ser Glu Thr His Thr Ser Ser Ala Ile Pro

11435 11440 11445

Thr Leu Pro Val Ser Pro Gly Ala Ser Lys Met Leu Thr Ser Leu

11450 11455 11460

Val Ile Ser Ser Gly Thr Asp Ser Thr Thr Thr Phe Pro Thr Leu

11465 11470 11475

Thr Glu Thr Pro Tyr Glu Pro Glu Thr Thr Ala Ile Gln Leu Ile

11480 11485 11490

His Pro Ala Glu Thr Asn Thr Met Val Pro Arg Thr Thr Pro Lys

11495 11500 11505

Phe Ser His Ser Lys Ser Asp Thr Thr Leu Pro Val Ala Ile Thr

11510 11515 11520

Ser Pro Gly Pro Glu Ala Ser Ser Ala Val Ser Thr Thr Thr Ile

11525 11530 11535

Ser Pro Asp Met Ser Asp Leu Val Thr Ser Leu Val Pro Ser Ser

11540 11545 11550

Gly Thr Asp Thr Ser Thr Thr Phe Pro Thr Leu Ser Glu Thr Pro

11555 11560 11565

Tyr Glu Pro Glu Thr Thr Ala Thr Trp Leu Thr His Pro Ala Glu

11570 11575 11580

Thr Ser Thr Thr Val Ser Gly Thr Ile Pro Asn Phe Ser His Arg

11585 11590 11595

Gly Ser Asp Thr Ala Pro Ser Met Val Thr Ser Pro Gly Val Asp

11600 11605 11610

Thr Arg Ser Gly Val Pro Thr Thr Thr Ile Pro Pro Ser Ile Pro

11615 11620 11625

Gly Val Val Thr Ser Gln Val Thr Ser Ser Ala Thr Asp Thr Ser

11630 11635 11640

Thr Ala Ile Pro Thr Leu Thr Pro Ser Pro Gly Glu Pro Glu Thr

11645 11650 11655

Thr Ala Ser Ser Ala Thr His Pro Gly Thr Gln Thr Gly Phe Thr

11660 11665 11670

Val Pro Ile Arg Thr Val Pro Ser Ser Glu Pro Asp Thr Met Ala

11675 11680 11685

Ser Trp Val Thr His Pro Pro Gln Thr Ser Thr Pro Val Ser Arg

11690 11695 11700

Thr Thr Ser Ser Phe Ser His Ser Ser Pro Asp Ala Thr Pro Val

11705 11710 11715

Met Ala Thr Ser Pro Arg Thr Glu Ala Ser Ser Ala Val Leu Thr

11720 11725 11730

Thr Ile Ser Pro Gly Ala Pro Glu Met Val Thr Ser Gln Ile Thr

11735 11740 11745

Ser Ser Gly Ala Ala Thr Ser Thr Thr Val Pro Thr Leu Thr His

11750 11755 11760

Ser Pro Gly Met Pro Glu Thr Thr Ala Leu Leu Ser Thr His Pro

11765 11770 11775

Arg Thr Glu Thr Ser Lys Thr Phe Pro Ala Ser Thr Val Phe Pro

11780 11785 11790

Gln Val Ser Glu Thr Thr Ala Ser Leu Thr Ile Arg Pro Gly Ala

11795 11800 11805

Glu Thr Ser Thr Ala Leu Pro Thr Gln Thr Thr Ser Ser Leu Phe

11810 11815 11820

Thr Leu Leu Val Thr Gly Thr Ser Arg Val Asp Leu Ser Pro Thr

11825 11830 11835

Ala Ser Pro Gly Val Ser Ala Lys Thr Ala Pro Leu Ser Thr His

11840 11845 11850

Pro Gly Thr Glu Thr Ser Thr Met Ile Pro Thr Ser Thr Leu Ser

11855 11860 11865

Leu Gly Leu Leu Glu Thr Thr Gly Leu Leu Ala Thr Ser Ser Ser

11870 11875 11880

Ala Glu Thr Ser Thr Ser Thr Leu Thr Leu Thr Val Ser Pro Ala

11885 11890 11895

Val Ser Gly Leu Ser Ser Ala Ser Ile Thr Thr Asp Lys Pro Gln

11900 11905 11910

Thr Val Thr Ser Trp Asn Thr Glu Thr Ser Pro Ser Val Thr Ser

11915 11920 11925

Val Gly Pro Pro Glu Phe Ser Arg Thr Val Thr Gly Thr Thr Met

11930 11935 11940

Thr Leu Ile Pro Ser Glu Met Pro Thr Pro Pro Lys Thr Ser His

11945 11950 11955

Gly Glu Gly Val Ser Pro Thr Thr Ile Leu Arg Thr Thr Met Val

11960 11965 11970

Glu Ala Thr Asn Leu Ala Thr Thr Gly Ser Ser Pro Thr Val Ala

11975 11980 11985

Lys Thr Thr Thr Thr Phe Asn Thr Leu Ala Gly Ser Leu Phe Thr

11990 11995 12000

Pro Leu Thr Thr Pro Gly Met Ser Thr Leu Ala Ser Glu Ser Val

12005 12010 12015

Thr Ser Arg Thr Ser Tyr Asn His Arg Ser Trp Ile Ser Thr Thr

12020 12025 12030

Ser Ser Tyr Asn Arg Arg Tyr Trp Thr Pro Ala Thr Ser Thr Pro

12035 12040 12045

Val Thr Ser Thr Phe Ser Pro Gly Ile Ser Thr Ser Ser Ile Pro

12050 12055 12060

Ser Ser Thr Ala Ala Thr Val Pro Phe Met Val Pro Phe Thr Leu

12065 12070 12075

Asn Phe Thr Ile Thr Asn Leu Gln Tyr Glu Glu Asp Met Arg His

12080 12085 12090

Pro Gly Ser Arg Lys Phe Asn Ala Thr Glu Arg Glu Leu Gln Gly

12095 12100 12105

Leu Leu Lys Pro Leu Phe Arg Asn Ser Ser Leu Glu Tyr Leu Tyr

12110 12115 12120

Ser Gly Cys Arg Leu Ala Ser Leu Arg Pro Glu Lys Asp Ser Ser

12125 12130 12135

Ala Thr Ala Val Asp Ala Ile Cys Thr His Arg Pro Asp Pro Glu

12140 12145 12150

Asp Leu Gly Leu Asp Arg Glu Arg Leu Tyr Trp Glu Leu Ser Asn

12155 12160 12165

Leu Thr Asn Gly Ile Gln Glu Leu Gly Pro Tyr Thr Leu Asp Arg

12170 12175 12180

Asn Ser Leu Tyr Val Asn Gly Phe Thr His Arg Ser Ser Met Pro

12185 12190 12195

Thr Thr Ser Thr Pro Gly Thr Ser Thr Val Asp Val Gly Thr Ser

12200 12205 12210

Gly Thr Pro Ser Ser Ser Pro Ser Pro Thr Thr Ala Gly Pro Leu

12215 12220 12225

Leu Met Pro Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Gln Tyr

12230 12235 12240

Glu Glu Asp Met Arg Arg Thr Gly Ser Arg Lys Phe Asn Thr Met

12245 12250 12255

Glu Ser Val Leu Gln Gly Leu Leu Lys Pro Leu Phe Lys Asn Thr

12260 12265 12270

Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg

12275 12280 12285

Pro Glu Lys Asp Gly Ala Ala Thr Gly Val Asp Ala Ile Cys Thr

12290 12295 12300

His Arg Leu Asp Pro Lys Ser Pro Gly Leu Asn Arg Glu Gln Leu

12305 12310 12315

Tyr Trp Glu Leu Ser Lys Leu Thr Asn Asp Ile Glu Glu Leu Gly

12320 12325 12330

Pro Tyr Thr Leu Asp Arg Asn Ser Leu Tyr Val Asn Gly Phe Thr

12335 12340 12345

His Gln Ser Ser Val Ser Thr Thr Ser Thr Pro Gly Thr Ser Thr

12350 12355 12360

Val Asp Leu Arg Thr Ser Gly Thr Pro Ser Ser Leu Ser Ser Pro

12365 12370 12375

Thr Ile Met Ala Ala Gly Pro Leu Leu Val Pro Phe Thr Leu Asn

12380 12385 12390

Phe Thr Ile Thr Asn Leu Gln Tyr Gly Glu Asp Met Gly His Pro

12395 12400 12405

Gly Ser Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln Gly Leu

12410 12415 12420

Leu Gly Pro Ile Phe Lys Asn Thr Ser Val Gly Pro Leu Tyr Ser

12425 12430 12435

Gly Cys Arg Leu Thr Ser Leu Arg Ser Glu Lys Asp Gly Ala Ala

12440 12445 12450

Thr Gly Val Asp Ala Ile Cys Ile His His Leu Asp Pro Lys Ser

12455 12460 12465

Pro Gly Leu Asn Arg Glu Arg Leu Tyr Trp Glu Leu Ser Gln Leu

12470 12475 12480

Thr Asn Gly Ile Lys Glu Leu Gly Pro Tyr Thr Leu Asp Arg Asn

12485 12490 12495

Ser Leu Tyr Val Asn Gly Phe Thr His Arg Thr Ser Val Pro Thr

12500 12505 12510

Ser Ser Thr Pro Gly Thr Ser Thr Val Asp Leu Gly Thr Ser Gly

12515 12520 12525

Thr Pro Phe Ser Leu Pro Ser Pro Ala Thr Ala Gly Pro Leu Leu

12530 12535 12540

Val Leu Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Lys Tyr Glu

12545 12550 12555

Glu Asp Met His Arg Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu

12560 12565 12570

Arg Val Leu Gln Thr Leu Leu Gly Pro Met Phe Lys Asn Thr Ser

12575 12580 12585

Val Gly Leu Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg Ser

12590 12595 12600

Glu Lys Asp Gly Ala Ala Thr Gly Val Asp Ala Ile Cys Thr His

12605 12610 12615

Arg Leu Asp Pro Lys Ser Pro Gly Val Asp Arg Glu Gln Leu Tyr

12620 12625 12630

Trp Glu Leu Ser Gln Leu Thr Asn Gly Ile Lys Glu Leu Gly Pro

12635 12640 12645

Tyr Thr Leu Asp Arg Asn Ser Leu Tyr Val Asn Gly Phe Thr His

12650 12655 12660

Trp Ile Pro Val Pro Thr Ser Ser Thr Pro Gly Thr Ser Thr Val

12665 12670 12675

Asp Leu Gly Ser Gly Thr Pro Ser Ser Leu Pro Ser Pro Thr Thr

12680 12685 12690

Ala Gly Pro Leu Leu Val Pro Phe Thr Leu Asn Phe Thr Ile Thr

12695 12700 12705

Asn Leu Lys Tyr Glu Glu Asp Met His Cys Pro Gly Ser Arg Lys

12710 12715 12720

Phe Asn Thr Thr Glu Arg Val Leu Gln Ser Leu Leu Gly Pro Met

12725 12730 12735

Phe Lys Asn Thr Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu

12740 12745 12750

Thr Leu Leu Arg Ser Glu Lys Asp Gly Ala Ala Thr Gly Val Asp

12755 12760 12765

Ala Ile Cys Thr His Arg Leu Asp Pro Lys Ser Pro Gly Val Asp

12770 12775 12780

Arg Glu Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr Asn Gly Ile

12785 12790 12795

Lys Glu Leu Gly Pro Tyr Thr Leu Asp Arg Asn Ser Leu Tyr Val

12800 12805 12810

Asn Gly Phe Thr His Gln Thr Ser Ala Pro Asn Thr Ser Thr Pro

12815 12820 12825

Gly Thr Ser Thr Val Asp Leu Gly Thr Ser Gly Thr Pro Ser Ser

12830 12835 12840

Leu Pro Ser Pro Thr Ser Ala Gly Pro Leu Leu Val Pro Phe Thr

12845 12850 12855

Leu Asn Phe Thr Ile Thr Asn Leu Gln Tyr Glu Glu Asp Met His

12860 12865 12870

His Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln

12875 12880 12885

Gly Leu Leu Gly Pro Met Phe Lys Asn Thr Ser Val Gly Leu Leu

12890 12895 12900

Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro Glu Lys Asn Gly

12905 12910 12915

Ala Ala Thr Gly Met Asp Ala Ile Cys Ser His Arg Leu Asp Pro

12920 12925 12930

Lys Ser Pro Gly Leu Asn Arg Glu Gln Leu Tyr Trp Glu Leu Ser

12935 12940 12945

Gln Leu Thr His Gly Ile Lys Glu Leu Gly Pro Tyr Thr Leu Asp

12950 12955 12960

Arg Asn Ser Leu Tyr Val Asn Gly Phe Thr His Arg Ser Ser Val

12965 12970 12975

Ala Pro Thr Ser Thr Pro Gly Thr Ser Thr Val Asp Leu Gly Thr

12980 12985 12990

Ser Gly Thr Pro Ser Ser Leu Pro Ser Pro Thr Thr Ala Val Pro

12995 13000 13005

Leu Leu Val Pro Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Gln

13010 13015 13020

Tyr Gly Glu Asp Met Arg His Pro Gly Ser Arg Lys Phe Asn Thr

13025 13030 13035

Thr Glu Arg Val Leu Gln Gly Leu Leu Gly Pro Leu Phe Lys Asn

13040 13045 13050

Ser Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu Ile Ser Leu

13055 13060 13065

Arg Ser Glu Lys Asp Gly Ala Ala Thr Gly Val Asp Ala Ile Cys

13070 13075 13080

Thr His His Leu Asn Pro Gln Ser Pro Gly Leu Asp Arg Glu Gln

13085 13090 13095

Leu Tyr Trp Gln Leu Ser Gln Met Thr Asn Gly Ile Lys Glu Leu

13100 13105 13110

Gly Pro Tyr Thr Leu Asp Arg Asn Ser Leu Tyr Val Asn Gly Phe

13115 13120 13125

Thr His Arg Ser Ser Gly Leu Thr Thr Ser Thr Pro Trp Thr Ser

13130 13135 13140

Thr Val Asp Leu Gly Thr Ser Gly Thr Pro Ser Pro Val Pro Ser

13145 13150 13155

Pro Thr Thr Thr Gly Pro Leu Leu Val Pro Phe Thr Leu Asn Phe

13160 13165 13170

Thr Ile Thr Asn Leu Gln Tyr Glu Glu Asn Met Gly His Pro Gly

13175 13180 13185

Ser Arg Lys Phe Asn Ile Thr Glu Ser Val Leu Gln Gly Leu Leu

13190 13195 13200

Lys Pro Leu Phe Lys Ser Thr Ser Val Gly Pro Leu Tyr Ser Gly

13205 13210 13215

Cys Arg Leu Thr Leu Leu Arg Pro Glu Lys Asp Gly Val Ala Thr

13220 13225 13230

Arg Val Asp Ala Ile Cys Thr His Arg Pro Asp Pro Lys Ile Pro

13235 13240 13245

Gly Leu Asp Arg Gln Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr

13250 13255 13260

His Ser Ile Thr Glu Leu Gly Pro Tyr Thr Leu Asp Arg Asp Ser

13265 13270 13275

Leu Tyr Val Asn Gly Phe Thr Gln Arg Ser Ser Val Pro Thr Thr

13280 13285 13290

Ser Thr Pro Gly Thr Phe Thr Val Gln Pro Glu Thr Ser Glu Thr

13295 13300 13305

Pro Ser Ser Leu Pro Gly Pro Thr Ala Thr Gly Pro Val Leu Leu

13310 13315 13320

Pro Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Gln Tyr Glu Glu

13325 13330 13335

Asp Met Arg Arg Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu Arg

13340 13345 13350

Val Leu Gln Gly Leu Leu Met Pro Leu Phe Lys Asn Thr Ser Val

13355 13360 13365

Ser Ser Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro Glu

13370 13375 13380

Lys Asp Gly Ala Ala Thr Arg Val Asp Ala Val Cys Thr His Arg

13385 13390 13395

Pro Asp Pro Lys Ser Pro Gly Leu Asp Arg Glu Arg Leu Tyr Trp

13400 13405 13410

Lys Leu Ser Gln Leu Thr His Gly Ile Thr Glu Leu Gly Pro Tyr

13415 13420 13425

Thr Leu Asp Arg His Ser Leu Tyr Val Asn Gly Phe Thr His Gln

13430 13435 13440

Ser Ser Met Thr Thr Thr Arg Thr Pro Asp Thr Ser Thr Met His

13445 13450 13455

Leu Ala Thr Ser Arg Thr Pro Ala Ser Leu Ser Gly Pro Met Thr

13460 13465 13470

Ala Ser Pro Leu Leu Val Leu Phe Thr Ile Asn Phe Thr Ile Thr

13475 13480 13485

Asn Leu Arg Tyr Glu Glu Asn Met His His Pro Gly Ser Arg Lys

13490 13495 13500

Phe Asn Thr Thr Glu Arg Val Leu Gln Gly Leu Leu Arg Pro Val

13505 13510 13515

Phe Lys Asn Thr Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu

13520 13525 13530

Thr Leu Leu Arg Pro Lys Lys Asp Gly Ala Ala Thr Lys Val Asp

13535 13540 13545

Ala Ile Cys Thr Tyr Arg Pro Asp Pro Lys Ser Pro Gly Leu Asp

13550 13555 13560

Arg Glu Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr His Ser Ile

13565 13570 13575

Thr Glu Leu Gly Pro Tyr Thr Leu Asp Arg Asp Ser Leu Tyr Val

13580 13585 13590

Asn Gly Phe Thr Gln Arg Ser Ser Val Pro Thr Thr Ser Ile Pro

13595 13600 13605

Gly Thr Pro Thr Val Asp Leu Gly Thr Ser Gly Thr Pro Val Ser

13610 13615 13620

Lys Pro Gly Pro Ser Ala Ala Ser Pro Leu Leu Val Leu Phe Thr

13625 13630 13635

Leu Asn Phe Thr Ile Thr Asn Leu Arg Tyr Glu Glu Asn Met Gln

13640 13645 13650

His Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln

13655 13660 13665

Gly Leu Leu Arg Ser Leu Phe Lys Ser Thr Ser Val Gly Pro Leu

13670 13675 13680

Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro Glu Lys Asp Gly

13685 13690 13695

Thr Ala Thr Gly Val Asp Ala Ile Cys Thr His His Pro Asp Pro

13700 13705 13710

Lys Ser Pro Arg Leu Asp Arg Glu Gln Leu Tyr Trp Glu Leu Ser

13715 13720 13725

Gln Leu Thr His Asn Ile Thr Glu Leu Gly Pro Tyr Ala Leu Asp

13730 13735 13740

Asn Asp Ser Leu Phe Val Asn Gly Phe Thr His Arg Ser Ser Val

13745 13750 13755

Ser Thr Thr Ser Thr Pro Gly Thr Pro Thr Val Tyr Leu Gly Ala

13760 13765 13770

Ser Lys Thr Pro Ala Ser Ile Phe Gly Pro Ser Ala Ala Ser His

13775 13780 13785

Leu Leu Ile Leu Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Arg

13790 13795 13800

Tyr Glu Glu Asn Met Trp Pro Gly Ser Arg Lys Phe Asn Thr Thr

13805 13810 13815

Glu Arg Val Leu Gln Gly Leu Leu Arg Pro Leu Phe Lys Asn Thr

13820 13825 13830

Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg

13835 13840 13845

Pro Glu Lys Asp Gly Glu Ala Thr Gly Val Asp Ala Ile Cys Thr

13850 13855 13860

His Arg Pro Asp Pro Thr Gly Pro Gly Leu Asp Arg Glu Gln Leu

13865 13870 13875

Tyr Leu Glu Leu Ser Gln Leu Thr His Ser Ile Thr Glu Leu Gly

13880 13885 13890

Pro Tyr Thr Leu Asp Arg Asp Ser Leu Tyr Val Asn Gly Phe Thr

13895 13900 13905

His Arg Ser Ser Val Pro Thr Thr Ser Thr Gly Val Val Ser Glu

13910 13915 13920

Glu Pro Phe Thr Leu Asn Phe Thr Ile Asn Asn Leu Arg Tyr Met

13925 13930 13935

Ala Asp Met Gly Gln Pro Gly Ser Leu Lys Phe Asn Ile Thr Asp

13940 13945 13950

Asn Val Met Gln His Leu Leu Ser Pro Leu Phe Gln Arg Ser Ser

13955 13960 13965

Leu Gly Ala Arg Tyr Thr Gly Cys Arg Val Ile Ala Leu Arg Ser

13970 13975 13980

Val Lys Asn Gly Ala Glu Thr Arg Val Asp Leu Leu Cys Thr Tyr

13985 13990 13995

Leu Gln Pro Leu Ser Gly Pro Gly Leu Pro Ile Lys Gln Val Phe

14000 14005 14010

His Glu Leu Ser Gln Gln Thr His Gly Ile Thr Arg Leu Gly Pro

14015 14020 14025

Tyr Ser Leu Asp Lys Asp Ser Leu Tyr Leu Asn Gly Tyr Asn Glu

14030 14035 14040

Pro Gly Pro Asp Glu Pro Pro Thr Thr Pro Lys Pro Ala Thr Thr

14045 14050 14055

Phe Leu Pro Pro Leu Ser Glu Ala Thr Thr Ala Met Gly Tyr His

14060 14065 14070

Leu Lys Thr Leu Thr Leu Asn Phe Thr Ile Ser Asn Leu Gln Tyr

14075 14080 14085

Ser Pro Asp Met Gly Lys Gly Ser Ala Thr Phe Asn Ser Thr Glu

14090 14095 14100

Gly Val Leu Gln His Leu Leu Arg Pro Leu Phe Gln Lys Ser Ser

14105 14110 14115

Met Gly Pro Phe Tyr Leu Gly Cys Gln Leu Ile Ser Leu Arg Pro

14120 14125 14130

Glu Lys Asp Gly Ala Ala Thr Gly Val Asp Thr Thr Cys Thr Tyr

14135 14140 14145

His Pro Asp Pro Val Gly Pro Gly Leu Asp Ile Gln Gln Leu Tyr

14150 14155 14160

Trp Glu Leu Ser Gln Leu Thr His Gly Val Thr Gln Leu Gly Phe

14165 14170 14175

Tyr Val Leu Asp Arg Asp Ser Leu Phe Ile Asn Gly Tyr Ala Pro

14180 14185 14190

Gln Asn Leu Ser Ile Arg Gly Glu Tyr Gln Ile Asn Phe His Ile

14195 14200 14205

Val Asn Trp Asn Leu Ser Asn Pro Asp Pro Thr Ser Ser Glu Tyr

14210 14215 14220

Ile Thr Leu Leu Arg Asp Ile Gln Asp Lys Val Thr Thr Leu Tyr

14225 14230 14235

Lys Gly Ser Gln Leu His Asp Thr Phe Arg Phe Cys Leu Val Thr

14240 14245 14250

Asn Leu Thr Met Asp Ser Val Leu Val Thr Val Lys Ala Leu Phe

14255 14260 14265

Ser Ser Asn Leu Asp Pro Ser Leu Val Glu Gln Val Phe Leu Asp

14270 14275 14280

Lys Thr Leu Asn Ala Ser Phe His Trp Leu Gly Ser Thr Tyr Gln

14285 14290 14295

Leu Val Asp Ile His Val Thr Glu Met Glu Ser Ser Val Tyr Gln

14300 14305 14310

Pro Thr Ser Ser Ser Ser Thr Gln His Phe Tyr Leu Asn Phe Thr

14315 14320 14325

Ile Thr Asn Leu Pro Tyr Ser Gln Asp Lys Ala Gln Pro Gly Thr

14330 14335 14340

Thr Asn Tyr Gln Arg Asn Lys Arg Asn Ile Glu Asp Ala Leu Asn

14345 14350 14355

Gln Leu Phe Arg Asn Ser Ser Ile Lys Ser Tyr Phe Ser Asp Cys

14360 14365 14370

Gln Val Ser Thr Phe Arg Ser Val Pro Asn Arg His His Thr Gly

14375 14380 14385

Val Asp Ser Leu Cys Asn Phe Ser Pro Leu Ala Arg Arg Val Asp

14390 14395 14400

Arg Val Ala Ile Tyr Glu Glu Phe Leu Arg Met Thr Arg Asn Gly

14405 14410 14415

Thr Gln Leu Gln Asn Phe Thr Leu Asp Arg Ser Ser Val Leu Val

14420 14425 14430

Asp Gly Tyr Ser Pro Asn Arg Asn Glu Pro Leu Thr Gly Asn Ser

14435 14440 14445

Asp Leu Pro Phe Trp Ala Val Ile Leu Ile Gly Leu Ala Gly Leu

14450 14455 14460

Leu Gly Val Ile Thr Cys Leu Ile Cys Gly Val Leu Val Thr Thr

14465 14470 14475

Arg Arg Arg Lys Lys Glu Gly Glu Tyr Asn Val Gln Gln Gln Cys

14480 14485 14490

Pro Gly Tyr Tyr Gln Ser His Leu Asp Leu Glu Asp Leu Gln

14495 14500 14505

<210> 105

<211> 755

<212> PROTEIN

<213> Homo sapiens

<400> 105

Met Ala Arg Lys Leu Ser Val Ile Leu Ile Leu Thr Phe Ala Leu Ser

1 5 10 15

Val Thr Asn Pro Leu His Glu Leu Lys Ala Ala Ala Phe Pro Gln Thr

20 25 30

Thr Glu Lys Ile Ser Pro Asn Trp Glu Ser Gly Ile Asn Val Asp Leu

35 40 45

Ala Ile Ser Thr Arg Gln Tyr His Leu Gln Gln Leu Phe Tyr Arg Tyr

50 55 60

Gly Glu Asn Asn Ser Leu Ser Val Glu Gly Phe Arg Lys Leu Leu Gln

65 70 75 80

Asn Ile Gly Ile Asp Lys Ile Lys Arg Ile His Ile His His Asp His

85 90 95

Asp His His Ser Asp His Glu His His Ser Asp His Glu Arg His Ser

100 105 110

Asp His Glu His His Ser Glu His Glu His His Ser Asp His Asp His

115 120 125

His Ser His His Asn His Ala Ala Ser Gly Lys Asn Lys Arg Lys Ala

130 135 140

Leu Cys Pro Asp His Asp Ser Asp Ser Ser Gly Lys Asp Pro Arg Asn

145 150 155 160

Ser Gln Gly Lys Gly Ala His Arg Pro Glu His Ala Ser Gly Arg Arg

165 170 175

Asn Val Lys Asp Ser Val Ser Ala Ser Glu Val Thr Ser Thr Val Tyr

180 185 190

Asn Thr Val Ser Glu Gly Thr His Phe Leu Glu Thr Ile Glu Thr Pro

195 200 205

Arg Pro Gly Lys Leu Phe Pro Lys Asp Val Ser Ser Ser Thr Pro Pro

210 215 220

Ser Val Thr Ser Lys Ser Arg Val Ser Arg Leu Ala Gly Arg Lys Thr

225 230 235 240

Asn Glu Ser Val Ser Glu Pro Arg Lys Gly Phe Met Tyr Ser Arg Asn

245 250 255

Thr Asn Glu Asn Pro Gln Glu Cys Phe Asn Ala Ser Lys Leu Leu Thr

260 265 270

Ser His Gly Met Gly Ile Gln Val Pro Leu Asn Ala Thr Glu Phe Asn

275 280 285

Tyr Leu Cys Pro Ala Ile Ile Asn Gln Ile Asp Ala Arg Ser Cys Leu

290 295 300

Ile His Thr Ser Glu Lys Lys Ala Glu Ile Pro Pro Lys Thr Tyr Ser

305 310 315 320

Leu Gln Ile Ala Trp Val Gly Gly Phe Ile Ala Ile Ser Ile Ile Ser

325 330 335

Phe Leu Ser Leu Leu Gly Val Ile Leu Val Pro Leu Met Asn Arg Val

340 345 350

Phe Phe Lys Phe Leu Leu Ser Phe Leu Val Ala Leu Ala Val Gly Thr

355 360 365

Leu Ser Gly Asp Ala Phe Leu His Leu Leu Pro His Ser His Ala Ser

370 375 380

His His His Ser His Ser His Glu Glu Pro Ala Met Glu Met Lys Arg

385 390 395 400

Gly Pro Leu Phe Ser His Leu Ser Ser Gln Asn Ile Glu Glu Ser Ala

405 410 415

Tyr Phe Asp Ser Thr Trp Lys Gly Leu Thr Ala Leu Gly Gly Leu Tyr

420 425 430

Phe Met Phe Leu Val Glu His Val Leu Thr Leu Ile Lys Gln Phe Lys

435 440 445

Asp Lys Lys Lys Lys Asn Gln Lys Lys Pro Glu Asn Asp Asp Asp Val

450 455 460

Glu Ile Lys Lys Gln Leu Ser Lys Tyr Glu Ser Gln Leu Ser Thr Asn

465 470 475 480

Glu Glu Lys Val Asp Thr Asp Asp Arg Thr Glu Gly Tyr Leu Arg Ala

485 490 495

Asp Ser Gln Glu Pro Ser His Phe Asp Ser Gln Gln Pro Ala Val Leu

500 505 510

Glu Glu Glu Glu Val Met Ile Ala His Ala His Pro Gln Glu Val Tyr

515 520 525

Asn Glu Tyr Val Pro Arg Gly Cys Lys Asn Lys Cys His Ser His Phe

530 535 540

His Asp Thr Leu Gly Gln Ser Asp Asp Leu Ile His His His Asp

545 550 555 560

Tyr His His Ile Leu His His His His Gln Asn His His Pro His

565 570 575

Ser His Ser Gln Arg Tyr Ser Arg Glu Glu Leu Lys Asp Ala Gly Val

580 585 590

Ala Thr Leu Ala Trp Met Val Ile Met Gly Asp Gly Leu His Asn Phe

595 600 605

Ser Asp Gly Leu Ala Ile Gly Ala Ala Phe Thr Glu Gly Leu Ser Ser

610 615 620

Gly Leu Ser Thr Ser Val Ala Val Phe Cys His Glu Leu Pro His Glu

625 630 635 640

Leu Gly Asp Phe Ala Val Leu Leu Lys Ala Gly Met Thr Val Lys Gln

645 650 655

Ala Val Leu Tyr Asn Ala Leu Ser Ala Met Leu Ala Tyr Leu Gly Met

660 665 670

Ala Thr Gly Ile Phe Ile Gly His Tyr Ala Glu Asn Val Ser Met Trp

675 680 685

Ile Phe Ala Leu Thr Ala Gly Leu Phe Met Tyr Val Ala Leu Val Asp

690 695 700

Met Val Pro Glu Met Leu His Asn Asp Ala Ser Asp His Gly Cys Ser

705 710 715 720

Arg Trp Gly Tyr Phe Phe Leu Gln Asn Ala Gly Met Leu Leu Gly Phe

725 730 735

Gly Ile Met Leu Leu Ile Ser Ile Phe Glu His Lys Ile Val Phe Arg

740 745 750

Ile Asn Phe

755

<210> 106

<211> 710

<212> PROTEIN

<213> Homo sapiens

<400> 106

Met Gly Gly Lys Gln Arg Asp Glu Asp Asp Glu Ala Tyr Gly Lys Pro

1 5 10 15

Val Lys Tyr Asp Pro Ser Phe Arg Gly Pro Ile Lys Asn Arg Ser Cys

20 25 30

Thr Asp Val Ile Cys Cys Val Leu Phe Leu Leu Phe Ile Leu Gly Tyr

35 40 45

Ile Val Val Gly Ile Val Ala Trp Leu Tyr Gly Asp Pro Arg Gln Val

50 55 60

Leu Tyr Pro Arg Asn Ser Thr Gly Ala Tyr Cys Gly Met Gly Glu Asn

65 70 75 80

Lys Asp Lys Pro Tyr Leu Leu Tyr Phe Asn Ile Phe Ser Cys Ile Leu

85 90 95

Ser Ser Asn Ile Ile Ser Val Ala Glu Asn Gly Leu Gln Cys Pro Thr

100 105 110

Pro Gln Val Cys Val Ser Ser Cys Pro Glu Asp Pro Trp Thr Val Gly

115 120 125

Lys Asn Glu Phe Ser Gln Thr Val Gly Glu Val Phe Tyr Thr Lys Asn

130 135 140

Arg Asn Phe Cys Leu Pro Gly Val Pro Trp Asn Met Thr Val Ile Thr

145 150 155 160

Ser Leu Gln Gln Glu Leu Cys Pro Ser Phe Leu Leu Pro Ser Ala Pro

165 170 175

Ala Leu Gly Arg Cys Phe Pro Trp Thr Asn Val Thr Pro Pro Ala Leu

180 185 190

Pro Gly Ile Thr Asn Asp Thr Thr Ile Gln Gln Gly Ile Ser Gly Leu

195 200 205

Ile Asp Ser Leu Asn Ala Arg Asp Ile Ser Val Lys Ile Phe Glu Asp

210 215 220

Phe Ala Gln Ser Trp Tyr Trp Ile Leu Val Ala Leu Gly Val Ala Leu

225 230 235 240

Val Leu Ser Leu Leu Phe Ile Leu Leu Leu Arg Leu Val Ala Gly Pro

245 250 255

Leu Val Leu Val Leu Ile Leu Gly Val Leu Gly Val Leu Ala Tyr Gly

260 265 270

Ile Tyr Tyr Cys Trp Glu Glu Tyr Arg Val Leu Arg Asp Lys Gly Ala

275 280 285

Ser Ile Ser Gln Leu Gly Phe Thr Thr Asn Leu Ser Ala Tyr Gln Ser

290 295 300

Val Gln Glu Thr Trp Leu Ala Ala Leu Ile Val Leu Ala Val Leu Glu

305 310 315 320

Ala Ile Leu Leu Leu Met Leu Ile Phe Leu Arg Gln Arg Ile Arg Ile

325 330 335

Ala Ile Ala Leu Leu Lys Glu Ala Ser Lys Ala Val Gly Gln Met Met

340 345 350

Ser Thr Met Phe Tyr Pro Leu Val Thr Phe Val Leu Leu Leu Ile Cys

355 360 365

Ile Ala Tyr Trp Ala Met Thr Ala Leu Tyr Leu Ala Thr Ser Gly Gln

370 375 380

Pro Gln Tyr Val Leu Trp Ala Ser Asn Ile Ser Ser Pro Gly Cys Glu

385 390 395 400

Lys Val Pro Ile Asn Thr Ser Cys Asn Pro Thr Ala His Leu Val Asn

405 410 415

Ser Ser Cys Pro Gly Leu Met Cys Val Phe Gln Gly Tyr Ser Ser Lys

420 425 430

Gly Leu Ile Gln Arg Ser Val Phe Asn Leu Gln Ile Tyr Gly Val Leu

435 440 445

Gly Leu Phe Trp Thr Leu Asn Trp Val Leu Ala Leu Gly Gln Cys Val

450 455 460

Leu Ala Gly Ala Phe Ala Ser Phe Tyr Trp Ala Phe His Lys Pro Gln

465 470 475 480

Asp Ile Pro Thr Phe Pro Leu Ile Ser Ala Phe Ile Arg Thr Leu Arg

485 490 495

Tyr His Thr Gly Ser Leu Ala Phe Gly Ala Leu Ile Leu Thr Leu Val

500 505 510

Gln Ile Ala Arg Val Ile Leu Glu Tyr Ile Asp His Lys Leu Arg Gly

515 520 525

Val Gln Asn Pro Val Ala Arg Cys Ile Met Cys Cys Phe Lys Cys Cys

530 535 540

Leu Trp Cys Leu Glu Lys Phe Ile Lys Phe Leu Asn Arg Asn Ala Tyr

545 550 555 560

Ile Met Ile Ala Ile Tyr Gly Lys Asn Phe Cys Val Ser Ala Lys Asn

565 570 575

Ala Phe Met Leu Leu Met Arg Asn Ile Val Arg Val Val Val Leu Asp

580 585 590

Lys Val Thr Asp Leu Leu Leu Phe Phe Gly Lys Leu Leu Val Val Gly

595 600 605

Gly Val Gly Val Leu Ser Phe Phe Phe Phe Ser Gly Arg Ile Pro Gly

610 615 620

Leu Gly Lys Asp Phe Lys Ser Pro His Leu Asn Tyr Tyr Trp Leu Pro

625 630 635 640

Ile Met Thr Ser Ile Leu Gly Ala Tyr Val Ile Ala Ser Gly Phe Phe

645 650 655

Ser Val Phe Gly Met Cys Val Asp Thr Leu Phe Leu Cys Phe Leu Glu

660 665 670

Asp Leu Glu Arg Asn Asn Gly Ser Leu Asp Arg Pro Tyr Tyr Met Ser

675 680 685

Lys Ser Leu Leu Lys Ile Leu Gly Lys Lys Asn Glu Ala Pro Pro Asp

690 695 700

Asn Lys Lys Arg Lys Lys

705 710

<210> 107

<211> 339

<212> PROTEIN

<213> Homo sapiens

<400> 107

Met Glu Ser Arg Lys Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys Met

1 5 10 15

Lys Pro Arg Arg Asn Leu Glu Glu Asp Asp Tyr Leu His Lys Asp Thr

20 25 30

Gly Glu Thr Ser Met Leu Lys Arg Pro Val Leu Leu His Leu His Gln

35 40 45

Thr Ala His Ala Asp Glu Phe Asp Cys Pro Ser Glu Leu Gln His Thr

50 55 60

Gln Glu Leu Phe Pro Gln Trp His Leu Pro Ile Lys Ile Ala Ala Ile

65 70 75 80

Ile Ala Ser Leu Thr Phe Leu Tyr Thr Leu Leu Arg Glu Val Ile His

85 90 95

Pro Leu Ala Thr Ser His Gln Gln Tyr Phe Tyr Lys Ile Pro Ile Leu

100 105 110

Val Ile Asn Lys Val Leu Pro Met Val Ser Ile Thr Leu Leu Ala Leu

115 120 125

Val Tyr Leu Pro Gly Val Ile Ala Ala Ile Val Gln Leu His Asn Gly

130 135 140

Thr Lys Tyr Lys Lys Phe Pro His Trp Leu Asp Lys Trp Met Leu Thr

145 150 155 160

Arg Lys Gln Phe Gly Leu Leu Ser Phe Phe Phe Ala Val Leu His Ala

165 170 175

Ile Tyr Ser Leu Ser Tyr Pro Met Arg Arg Ser Tyr Arg Tyr Lys Leu

180 185 190

Leu Asn Trp Ala Tyr Gln Gln Val Gln Gln Asn Lys Glu Asp Ala Trp

195 200 205

Ile Glu His Asp Val Trp Arg Met Glu Ile Tyr Val Ser Leu Gly Ile

210 215 220

Val Gly Leu Ala Ile Leu Ala Leu Leu Ala Val Thr Ser Ile Pro Ser

225 230 235 240

Val Ser Asp Ser Leu Thr Trp Arg Glu Phe His Tyr Ile Gln Ser Lys

245 250 255

Leu Gly Ile Val Ser Leu Leu Leu Gly Thr Ile His Ala Leu Ile Phe

260 265 270

Ala Trp Asn Lys Trp Ile Asp Ile Lys Gln Phe Val Trp Tyr Thr Pro

275 280 285

Pro Thr Phe Met Ile Ala Val Phe Leu Pro Ile Val Val Leu Ile Phe

290 295 300

Lys Ser Ile Leu Phe Leu Pro Cys Leu Arg Lys Lys Ile Leu Lys Ile

305 310 315 320

Arg His Gly Trp Glu Asp Val Thr Lys Ile Asn Lys Thr Glu Ile Cys

325 330 335

Ser Gln Leu

<210> 108

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

primer"

<400> 108

actctcttcc gcatcgctgt 20

<210> 109

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

primer"

<400> 109

ccgacgggtt tccgatccaa 20

<210> 110

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

probe"

<400> 110

ctgttgggct cgcggttg 18

<210> 111

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

primer"

<400> 111

tggcatcaga ttgcaaagac 20

<210> 112

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

Primer"

<400> 112

acgccgggtg atgtatctat 20

<210> 113

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic

probe"

<400> 113

cgaaacgcac ccgtcagacg 20

<---

Claims (80)

1. Splicing modulator with the following formula: (I) or a pharmaceutically acceptable salt thereof, where Y is selected from O, S, NR ⁶ and CR ⁶ R ⁷ ; each of R ¹ , R ² and R ³ is independently selected from hydrogen, hydroxyl, -O-(C ₁ -C ₆ alkyl) groups, -OC(=O)-(C ₁ -C ₆ alkyl) groups, -C groups (=O)-O-(C ₁ -C ₆ alkyl) and C ₁ -C ₆ alkyl groups; R ⁴ is selected from C ₁ -C ₆ alkyl groups, -C(=O)-(C ₁ -C ₆ alkyl) groups, -C(=O)-(C ₃ -C ₈ carbocyclyl) groups, -C( =O)-(C ₃ -C ₈ heterocyclyl) and -C(=O)-NR ⁶ R ⁷ ; R ⁵ is selected from hydrogen, hydroxyl, -CH ₂ -OH, -CO ₂ H, -C(=O)-O-(C ₁ -C ₆ alkyl), -C(=O)-NR ⁶ R ⁷ groups, -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 ; R ⁶ and R ⁷ are each independently selected from hydrogen, -R ⁸ , -C(=O)-R ⁸ and -C(=O)-OR ⁸ ; And R ⁸ is selected from C ₁ -C ₆ alkyl groups, C ₃ -C ₈ carbocyclyl groups and C ₃ -C ₈ heterocyclyl groups, where each of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ is independently substituted with 0-3 groups independently selected from halogens, hydroxyl, C ₁ -C ₆ alkyl groups, groups - O-(C ₁ -C ₆ alkyl), -CO ₂ H, groups -C(=O)-(C ₁ -C ₆ alkyl), groups -C(=O)-(C ₃ -C ₈ carbocyclyl), groups -C(=O)-(C ₃ -C ₈ heterocyclyl), -NR ⁶ R ⁷ , C ₃ -C ₈ carbocyclyl groups, C ₁ -C ₆ alkylhydroxy groups, C ₁ -C ₆ alkyl alkoxy groups, benzyl groups and C ₃ -C ₈ heterocyclyl groups, each of which may be independently substituted with 0 or 1 group selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups, C ₁ -C ₃ haloalkyl groups, -NH- C(=O)(C ₁ -C ₃ alkyl) and -NH-C(=O)-O-(C ₁ -C ₃ alkyl). 2. Splicing modulator according to claim 1, where the splicing modulator has the following formula: , or a pharmaceutically acceptable salt thereof, where R ⁹ is selected from C ₃ -C ₈ heterocyclyl groups; And R ¹⁰ is selected from H and C ₁ -C ₆ alkyl groups, where each of R ⁹ and R ¹⁰ is independently substituted with 0-3 groups independently selected from halogens, hydroxyl, C ₁ -C ₃ alkyl groups, C ₁ -C ₃ alkoxy groups, -NH ₂ , -NH-(C ₁ -C ₃ alkyl) and -N-(C ₁ -C ₃ alkyl) ₂ . 3. Splicing modulator according to claim 1, wherein the splicing modulator is a compound of formula (Ib) (Ib) or a pharmaceutically acceptable salt thereof, where R ¹¹ is selected from , And , where * denotes the point of attachment of R ¹¹ to the rest of the connection; And R ¹² and R ¹³ are each independently selected from H and methyl. 4. Splicing modulator according to claim 1, where the splicing modulator is selected from (H1), (H2), (H3), (H12) and pharmaceutically acceptable salts thereof, optionally, the splicing modulator is selected from H1, H3 or a pharmaceutically acceptable salt thereof. 5. A compound for treating a neoplastic disorder comprising a splicing modulator linker of the formula L-H, or a pharmaceutically acceptable salt thereof, wherein L is a linker and where H is a splicing modulator according to claim 1. 6. The compound according to claim 5, where H is covalently attached through any atom to L, the valency of the atom that is covalently attached to L is not exceeded; optionally, where L represents a cleavable linker. 7. The connection according to claim 5, selected from: , , , ; , and their pharmaceutically acceptable salts. 8. Connection according to claim 5, selected from: ; ; ; and their pharmaceutically acceptable salts. 9. The compound according to claim 5, where the linker is a cleavable linker containing at least one cleavable fragment; and where the linker contains: (i) cleavable peptide; optionally wherein the cleavage peptide comprises valine-citrulline (Val-Cit) or valine-alanine (Val-Ala); or (ii) a cleavable glucuronide moiety; optionally, wherein the glucuronide moiety to be cleaved is enzyme-cleavable; optionally, wherein the cleavable glucuronide moiety is cleavable by glucuronidase; optionally, wherein the glucuronide moiety to be cleaved is β-glucuronidase-cleavable. 10. The compound of claim 9, wherein the linker contains a maleimide (Mal) moiety, optionally wherein the Mal moiety contains maleimidocaproyl (MC), wherein the linker optionally contains MC-Val-Cit or MC-Val-Ala. 11. The compound according to claim 9, where the linker contains at least one spacer unit, optionally, where or (i) the spacer unit optionally comprises a polyethylene glycol (PEG) moiety, wherein the PEG moiety comprises -(PEG) _m - and m is an integer from 1 to 10, optionally where m is 2; or (ii) the spacer unit comprises a moiety, optionally wherein the alkyl moiety comprises -(CH ₂ ) _n - and n is an integer from 1 to 10, optionally wherein n is 2, 5 or 6; optionally where the spacer unit attaches to the maleimide (Mal) moieties (“Mal spacer unit”). 12. The compound of claim 9, wherein the cleavable fragment is directly attached to the splicing modulator of the compound, or wherein a spacer unit attaches the cleavable fragment to the splicing modulator of the compound, optionally, wherein cleavage of the cleavable fragment releases the splicing modulator from the linker, optionally, wherein a spacer unit attaches the cleavable fragment the fragment in the linker to the splicing modulator is self-cleaving. 13. The compound according to claim 12, where the spacer unit connecting the cleavable fragment in the linker to the splicing modulator contains p-aminobenzyl (pAB) or p-aminobenzyloxycarbonyl (pABC) fragments, optionally, where pAB and pABC fragments attach the cleavable fragment in the linker to a splicing modulator, optionally wherein the cleavage peptide contains Val-Cit or Val-Ala; optionally where the linker contains Val-Cit-pAB, Val-Ala-pAB, Val-Cit-pABC or Val-Ala-pABC; optionally where the linker comprises MC-Val-Cit-pAB, MC-Val-Ala-pAB, MC-Val-Cit-pABC or MC-Val-Ala-pABC. 14. An antibody-drug conjugate for treating a neoplastic disorder, wherein the antibody-drug conjugate has formula (I) Ab-(LH) _p (I), wherein Ab is an antibody or antigen-binding fragment that specifically targets a neoplastic cell; L-H contains a compound according to any one of paragraphs. 5-13; And p represents an integer between 1 and 15. 15. Antibody-drug conjugate according to claim 14, where the neoplastic cell is obtained from: (i) hematological malignancy; optionally, wherein the hematologic malignancy is selected from B-cell malignancy, leukemia, lymphoma, myeloma, acute myeloid leukemia, and multiple myeloma; or (ii) a solid tumor, optionally, wherein the solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer , kidney cancer, colorectal cancer and esophageal cancer. 16. Antibody-drug conjugate according to claim 14, where the neoplastic cell is: (i) a cell expressing HER2, optionally, wherein the antibody or antigen binding fragment is an antibody to HER2 or an antigen binding fragment thereof; and/or wherein 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); and/or wherein the antibody or antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20, optionally wherein the antibody or antigen binding fragment comprises a constant region human IgG1 heavy chain, and/or wherein the antibody or antigen binding fragment comprises a human Ig kappa light chain constant region; (ii) a cell expressing CD138; optionally, wherein the antibody or antigen binding fragment is an anti-CD138 antibody or antigen binding fragment thereof; and/or wherein 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); and/or wherein the antibody or antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22, optionally wherein the antibody or antigen binding fragment comprises a constant region human IgG2a heavy chain, and/or wherein the antibody or antigen binding fragment comprises a human Ig kappa light chain constant region; or (iii) a cell expressing EPHA2; optionally, wherein the antibody or antigen binding fragment is an anti-EPHA2 antibody or antigen binding fragment thereof; and/or wherein 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); and/or wherein the antibody or antigen binding fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24, optionally wherein the antibody or antigen binding fragment comprises a constant region human IgG1 heavy chain, and/or wherein the antibody or antigen binding fragment comprises a human Ig kappa light chain constant region. 17. Pharmaceutical composition for the treatment of a neoplastic disorder, containing: (i) a splicing modulator according to any one of paragraphs. 1-4 or a pharmaceutically acceptable salt thereof; (ii) a connection according to any one of paragraphs. 5-13 or (iii) an antibody-drug conjugate according to any one of paragraphs. 14-16 and a pharmaceutically acceptable carrier. 18. Use of a splicing modulator according to any one of claims. 1-4 or a pharmaceutically acceptable salt thereof, a compound according to any one of paragraphs. 5-13 or an antibody-drug conjugate according to any one of paragraphs. 14-16 for the treatment of neoplastic disorder. 19. Use of a splicing modulator according to any one of claims. 1-4 or a pharmaceutically acceptable salt thereof, a compound according to any one of paragraphs. 5-13 or an antibody-drug conjugate according to any one of paragraphs. 14-16 for the manufacture of a medicament for the treatment of a neoplastic disorder. 20. Use according to claim 18, where the neoplastic disorder is: (i) HER2-positive breast cancer, ovarian cancer, gastric cancer, lung cancer, uterine cancer, osteosarcoma or salivary duct carcinoma; optionally, wherein the HER2-expressing lung cancer is a lung adenocarcinoma and/or the HER2-expressing uterine cancer is a uterine endometrial serous carcinoma; (ii) CD138-expressing multiple myeloma; or (iii) EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer or esophageal cancer. 21. Use according to claim 19, where the neoplastic disorder is: (i) HER2-positive breast cancer, ovarian cancer, gastric cancer, lung cancer, uterine cancer, osteosarcoma or salivary duct carcinoma; optionally, wherein the HER2-expressing lung cancer is a lung adenocarcinoma and/or the HER2-expressing uterine cancer is a uterine endometrial serous carcinoma; (ii) CD138-expressing multiple myeloma; or (iii) EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer or esophageal cancer. 22. The use of claim 18, wherein the splicing modulator, compound or antibody-drug conjugate is for use in the treatment of a neoplastic disorder in combination with a checkpoint inhibitor that specifically targets CTLA4, PD1, PDL1, OX40, CD40, GITR, LAG3 , TIM3 and/or KIR. 23. The use of claim 19, wherein the splicing modulator, compound or antibody-drug conjugate is for use in the manufacture of a medicament for the treatment of a neoplastic disorder in combination with a checkpoint inhibitor that specifically targets CTLA4, PD1, PDL1, OX40, CD40 , GITR, LAG3, TIM3 and/or KIR. 24. The use of claim 20, wherein the splicing modulator, compound or antibody-drug conjugate is for use in the treatment of a neoplastic disorder in combination with a checkpoint inhibitor that specifically targets CTLA4, PD1, PDL1, OX40, CD40, GITR, LAG3 , TIM3 and/or KIR. 25. A method of treating a subject who has or is suspected of having a neoplastic disorder, comprising administering to the subject a therapeutically effective amount of a splicing modulator according to any one of claims. 1-4, or a pharmaceutically acceptable salt thereof, a compound according to any one of paragraphs. 5-13 or an antibody-drug conjugate according to any one of paragraphs. 14-16. 26. The method according to claim 25, where the neoplastic disorder is: (i) HER2-positive breast cancer, ovarian cancer, gastric cancer, lung cancer, uterine cancer, osteosarcoma or salivary duct carcinoma; optionally, wherein the HER2-expressing lung cancer is a lung adenocarcinoma and/or the HER2-expressing uterine cancer is a uterine endometrial serous carcinoma; (ii) CD138-expressing multiple myeloma; or (iii) EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer or esophageal cancer. 27. The method of claim 25, further comprising administering to the subject a checkpoint inhibitor that specifically targets CTLA4, PD1, PDL1, OX40, CD40, GITR, LAG3, TIM3, and/or KIR.

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