Herboxidiene antibody-drug conjugates and methods of use

Abstract

Linker-drug compounds and antibody-drug conjugates that bind to human oncology targets are disclosed. The linker-drug compounds and antibody-drug conjugates comprise a herboxidiene splicing modulator drug moiety. The disclosure further relates to methods and compositions for use in the treatment of neoplastic disorders by administering the antibody-drug conjugates provided herein. The herboxidiene itself is also claimed. Further claims are directed to its use, and to the use of a neoantigen, generated by the herboxidiene or its ADC, or a vaccine against this neoantigen.

Description

OR10 (la), or a pharmaceutically acceptable salt thereof, which covalently attaches to L through any atom, wherein: R9 is chosen from C3-C8 heterocyclyl groups; R10 is chosen from H and Ci-Ce alkyl groups, wherein R9 and R10 are each independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl, C1-C3 alkyl groups, C1-C3 alkoxy groups, -NH2, -NH-(Ci-C3 alkyl), and -N-(Ci-C3 alkyl)2, and wherein the valency of the atom that is covalently attached to L is not exceeded.

[337] In some embodiments, H, the herboxidiene splicing modulator, comprises a compound O O^R11 of Formula (lb): OR12 (lb), or a pharmaceutically

[338] acceptable salt thereof, which covalently attaches to L through any atom, wherein: 7  \                        H -N N-R- R11 is chosen from     \— /     ,               , and     \— /       , wherein * denotes the point of connectivity of R11 to the remainder of the compound; R12 and R13 are each independently chosen from H and methyl; and wherein the valency of the atom that is covalently attached to L is not exceeded.

[339] In some embodiments, H, the herboxidiene splicing modulator, comprises a compound r o             ° of Formula (II): oh =    =    *         ।                (II), or a pharmaceutically acceptable salt thereof, which covalently attaches through any atom to L, wherein: X is hydroxyl or NR6R7; R6 and R7 are each independently chosen from hydrogen, -R8, -C(=O)-R8, -C(=O)-O-R8, -(Ci-C6 alkyl)-O-C(=O)-R8, and -(Ci-C6 alkyl)-NH-C(=O)-R8; and R8 is chosen from Ci-Ce alkyl groups, Cs-Cs carbocyclyl groups, and Cs-Cs heterocyclyl groups, wherein R6, R7, and R8 are each independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl, Ci-Ce alkyl groups, -O-(Ci-C6 alkyl) groups, -CO2H, -C(=O)-O-(Ci-C6 alkyl) groups, -C(=O)-(Ci-C6 alkyl) groups, -C(=O)-(C3-C8 carbocyclyl) groups, -C(=O)-(C3-Cs heterocyclyl) groups, -NR6R7, C3-C8carbocyclyl groups, Ci-Ce alkylhydroxy groups, Ci-Ce alkylalkoxy groups, benzyl groups, and C3-C8 heterocyclyl groups, each of which may be independently substituted with 0 or 1 group chosen from halogens, hydroxyl, C1-C3 alkyl groups, C1-C3 alkoxy groups, C1-C3 haloalkyl groups, -NH-C(=O)(Ci-C3 alkyl), and -NH-C(=O)-O-(Ci-C3 alkyl), and wherein the valency of the atom that is covalently attached to L is not exceeded.

[340] In some embodiments, H, the herboxidiene splicing modulator, comprises a compound ____R10 R11 „ z >»°                         ii 2¼1 Ji of Formula (Ila): $H =    =              I              r9                (Ila), ora pharmaceutically acceptable salt thereof, which covalently attaches through any atom to L, wherein: Z is chosen from NR9 and O; R9 is chosen from hydrogen and Ci-Ce alkyl groups; R10 and R11 are each independently chosen from hydrogen, halogens, hydroxyl, Ci-Ce alkyl groups, -O-(Ci-C6 alkyl) groups, -CO2H, -C(=O)-O-(Ci-C6 alkyl) groups, -C(=O)-(Ci-C6 alkyl) groups, -C(=O)-(C3-C8 carbocyclyl) groups, -C(=O)-(C3-C8 heterocyclyl) groups, C3-C8 carbocyclyl groups, Ci-Ce alkylhydroxy groups, Ci-Ce alkylalkoxy groups, benzyl groups, and C3-C8 heterocyclyl groups; R12 is chosen from Ci-Ce alkyl groups, C3-C8 carbocyclyl groups, C3-C8 heterocyclyl groups, wherein R9, R10, R11, and R12 are each independently substituted with 0 or 1 group chosen from halogens, hydroxyl, C1-C3 alkyl groups, C1-C3 alkoxy groups, and C1-C3 haloalkyl groups; t is an integer chosen from 1, 2, 3, 4, 5, and 6; and wherein the valency of the atom that is covalently attached to L is not exceeded.

[341] In some embodiments, H, the herboxidiene splicing modulator, comprises a compound acceptable salt thereof, which covalently attaches through any atom to L, wherein: wherein * denotes the point of connectivity of R13 to the remainder of the compound; R14 and R15 are each independently chosen from hydrogen and methyl; and wherein the valency of the atom that is covalently attached to L is not exceeded.

[342] In some embodiments, H, the herboxidiene splicing modulator, comprises a compound of Formula (III): H N (III), ora pharmaceutically acceptable salt thereof, which covalently attaches through any atom to L, wherein: R1, R2, and R3 are each independently chosen from hydrogen, hydroxyl, -O-(Ci-C6 alkyl) groups, -O-C(=O)-(Ci-C6 alkyl) groups, -C(=O)-O-(Ci-C6 alkyl) groups, and Ci-Ce alkyl groups; R6 and R7 are each independently chosen from hydrogen, -R8, -C(=O)-R8, and -C(=O)-O-R8; R8 is chosen from Ci-Ce alkyl groups, Cs-Cs carbocyclyl groups, and Cs-Cs heterocyclyl wherein R1, R2, R3, R6, R7, and R8 are each independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl, Ci-Ce alkyl groups, -O-(Ci-C6 alkyl) groups, -CO2H, -C(=O)-(Ci-C6 alkyl) groups, -C(=O)-(C3-C8 carbocyclyl) groups, -C(=O)-(C3-C8 heterocyclyl) groups, -NR6R7, C3-C8 carbocyclyl groups, Ci-Ce alkylhydroxy groups, Ci-Ce alkylalkoxy groups, benzyl groups, and C3-C8 heterocyclyl groups, each of which may be independently substituted with 0 or 1 group chosen from halogens, hydroxyl, C1-C3 alkyl groups, C1-C3 alkoxy groups, C1-C3 haloalkyl groups, -NH-C(=O)(Ci-C3 alkyl), and -NH-C(=O)-O-(Ci-C3 alkyl), wherein the valency of the atom that is covalently attached to L is not exceeded; and wherein * denotes the point of connectivity of R9 to the remainder of the compound.

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

[344] In some embodiments, the antibody or antigen binding fragment targets a HER2-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-HER2 antibody or antigen binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising 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 an amino acid sequence of SEQ ID NO: 19, and a light chain variable region comprising an amino acid sequence of SEQ ID NQ:20. In some embodiments, the antibody or antigen binding fragment comprises a human IgG 1 heavy chain constant 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 targets a CD138-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-CD138 antibody or antigen binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising 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 an amino acid sequence of SEQ ID NO:21, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:22. In some embodiments, the antibody or antigen binding fragment comprises a murine lgG2a heavy chain constant region. In some embodiments, the antibody or antigen binding fragment comprises a murine Ig kappa light chain constant region. In some embodiments, the antibody or antigen binding fragment comprises a human lgG2a 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 targets an EPHA2-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-EPHA2 antibody or antigen binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising 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 an amino acid sequence of SEQ ID NO:23, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:24. In some embodiments, the antibody or antigen binding fragment comprises a human IgG 1 heavy chain constant 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 targets a MSLN-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-MSLN antibody or antigen binding fragment.

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

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

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

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

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

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

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

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

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

[357] In some other embodiments, the antibody or antigen binding fragment targets a MLIC16-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-MLIC16 antibody or antigen binding fragment.

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

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

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

[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 comprise one or more additional spacer units. In some embodiments, L is an ADL1, ADL2, ADL5, ADL6, 116 ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker. In some embodiments, L is an ADL12, ADL14, or ADL15 linker. In some embodiments, the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL15, ADL21, or ADL23 linker may also comprise one or more additional spacer units.

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

[363] A number of different reactions are available for covalent attachment of the herboxidiene splicing modulator moiety and / or linker moiety to the antibody or antigen binding fragment. This is often accomplished by reaction of one or more amino acid residues of the antibody or antigen binding fragment, including the amine groups of lysine, the free carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl groups of cysteine, and the various moieties of the aromatic amino acids. For instance, non-specific covalent attachment may be undertaken using a carbodiimide reaction to link a carboxy (or amino) group on a herboxidiene splicing modulator moiety to an amino (or carboxy) group on an antibody or antigen binding fragment. Additionally, bifunctional agents such as dialdehydes or imidoesters may also be used to link the amino group on a herboxidiene splicing modulator moiety to an amino group on an antibody or antigen binding fragment. Also available for attachment of drugs (e.g., a herboxidiene splicing modulator) to binding agents is the Schiff base reaction. This method involves the periodate oxidation of a drug that contains glycol or hydroxy groups, thus forming an aldehyde which is then reacted with the binding agent. Attachment occurs via formation of a Schiff base with amino groups of the binding agent. Isothiocyanates may also be used as coupling agents for covalently attaching drugs to binding agents. Other techniques are known to the skilled artisan and within the scope of the present disclosure. Examples of drug moieties that can be generated and linked to an antibody or antigen binding fragment using various chemistries known to in the art include herboxidiene splicing modulators, e.g., the herboxidiene splicing modulators described and exemplified herein. Linker-Herboxidiene Splicing Modulator / Herboxidiene Splicing Modulator Compounds

[364] Further disclosed herein are exemplary linker-herboxidiene splicing modulator (L-H) compounds, as well as compositions comprising multiple copies of such compounds. In various embodiments, the linker-herboxidiene splicing modulator compounds disclosed herein can be defined by the generic formula: L-H, wherein L= a linker moiety, and H = a herboxidiene splicing 117 modulator. In certain embodiments, the disclosed L-H compounds are suitable for use in the ADCs described herein.

[365] In some embodiments, disclosed herein, are compounds of Formula (I): R5 wherein: (I), or a pharmaceutically acceptable salt thereof, Y is chosen from O, S, NR6, and CR6R7; R1, R2, and R3 are each independently chosen from hydrogen, hydroxyl, -O-(Ci-C6 alkyl) groups, -O-C(=O)-(Ci-C6 alkyl) groups, -C(=O)-O-(Ci-C6 alkyl) groups, and Ci-Ce alkyl groups; R4 is chosen from hydrogen, Ci-Ce alkyl groups, -C(=O)-(Ci-C6 alkyl) groups, -C(=O)-(Cs-Cs carbocyclyl) groups, -C(=O)-(C3-Cs heterocyclyl) groups, and -C(=O)-NR6R7; R5 is chosen from hydrogen, hydroxyl, -CH2-OH, -CO2H, -C(=O)-O-(Ci-C6 alkyl) groups, -C(=O)-NR6R7, -NR6-C(=O)-R8, -O-C(=O)-NR6R7, -NR6-C(=O)-R8, and -NR6-C(=O)-NR6R7; R6 and R7 are each independently chosen from hydrogen, -R8, -C(=O)-R8, and -C(=O)-O-R8; and R8 is chosen from Ci-Ce alkyl groups, C3-C8 carbocyclyl groups, and C3-C8 heterocyclyl groups, wherein R1, R2, R3, R4, R5, R6, R7, and R8 are each independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl, Ci-Ce alkyl groups, -O-(Ci-C6 alkyl) groups, -CO2H, -C(=O)-(Ci-C6 alkyl) groups, -C(=O)-(C3-C8 carbocyclyl) groups, -C(=O)-(C3-C8 heterocyclyl) groups, -NR6R7, C3-C8 carbocyclyl groups, Ci-Ce alkylhydroxy groups, Ci-Ce alkylalkoxy groups, benzyl groups, and C3-C8 heterocyclyl groups, each of which may be independently substituted with 0 or 1 group chosen from halogens, hydroxyl, C1-C3 alkyl groups, C1-C3 alkoxy groups, C1-C3 haloalkyl groups, -NH-C(=O)(Ci-C3 alkyl), and -NH-C(=O)-O-(Ci-C3 alkyl).

[366] In some embodiments, provided herein are compounds of Formula (la): O R9 .0 VS ; "                                   (la), or a pharmaceutically acceptable salt thereof, which covalently attaches to L through any atom, wherein: R9 is chosen from C3-C8 heterocyclyl groups; and R10 is chosen from H and Ci-Ce alkyl groups, wherein R9 and R10 are each independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl, C1-C3 alkyl groups, C1-C3 alkoxy groups, -NH2, -NH-(Ci-C3 alkyl), and -N-(Ci-C3 alkyl)2.

[367] In some embodiments, provided herein are compounds of Formula (lb): O O^R11 thereof, wherein: /    \                                     LJ *    / --\   13 —N    N-R13      * KI / \ N —N N-R13   1    /       Xy'Vpn R11 is chosen from     \— /      ,               , and \' .wherein denotes the point of connectivity of R11 to the remainder of the compound; R12 and R13 are each independently chosen from H and methyl.

[368] In some embodiments, provided herein are compounds of Formula (II): r 0            ° 0H =    =    1                         (II), or a pharmaceutically acceptable salt thereof, wherein: X is hydroxyl or NR6R7; R6 and R7 are each independently chosen from hydrogen, -R8, -C(=O)-R8, -C(=O)-O-R8, -(Ci-C6 alkyl)-O-C(=O)-R8, and -(Ci-C6 alkyl)-NH-C(=O)-R8; and R8 is chosen from Ci-Ce alkyl groups, C3-C8 carbocyclyl groups, and C3-C8 heterocyclyl groups, wherein R6, R7, and R8 are each independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl, Ci-Ce alkyl groups, -O-(Ci-C6 alkyl) groups, -CO2H, -C(=O)-O-(Ci-C6 alkyl) groups, -C(=O)-(Ci-C6 alkyl) groups, -C(=O)-(C3-C8 carbocyclyl) groups, -C(=O)-(C3-C8 heterocyclyl) groups, -NR6R7, C3-C8carbocyclyl groups, Ci-Ce alkylhydroxy groups, Ci-Ce alkylalkoxy groups, benzyl groups, and C3-C8 heterocyclyl groups, each of which may be independently substituted with 0 or 1 group chosen from halogens, hydroxyl, C1-C3 alkyl groups, C1-C3 alkoxy groups, C1-C3 haloalkyl groups, -NH-C(=O)(Ci-C3 alkyl), and -NH-C(=O)-O-(Ci-C3 alkyl).

[369] In some embodiments, provided herein are compounds of Formula (Ila): salt thereof, wherein: (Ila), or a pharmaceutically acceptable Z is chosen from NR9 and O; R9 is chosen from hydrogen and Ci-Ce alkyl groups; R10 and R11 are each independently chosen from hydrogen, halogens, hydroxyl, Ci-Ce alkyl groups, -O-(Ci-C6 alkyl) groups, -CO2H, -C(=O)-O-(Ci-C6 alkyl) groups, -C(=O)-(Ci-C6 alkyl) groups, -C(=O)-(C3-Cs carbocyclyl) groups, -C(=O)-(C3-Cs heterocyclyl) groups, C3-C8 carbocyclyl groups, Ci-Ce alkylhydroxy groups, Ci-Ce alkylalkoxy groups, benzyl groups, and C3-C8 heterocyclyl groups; R12 is chosen from Ci-Ce alkyl groups, C3-C8 carbocyclyl groups, C3-C8 heterocyclyl groups, wherein R9, R10, R11, and R12 are each independently substituted with 0 or 1 group chosen from halogens, hydroxyl, C1-C3 alkyl groups, C1-C3 alkoxy groups, and C1-C3 haloalkyl groups; and t is an integer chosen from 1, 2, 3, 4, 5, and 6.

[370] In some embodiments, provided herein are compounds of Formula (lib): ' >*°                I J OH £ = i          I wherein: O. *       z _d, ■ .         r           O'14 R13 is chosen from    K O. <3H    ~ O *         5               11                        * R14 H L^N.d15 R15 CL OH         ____,kR15 *   I        II Mo *         “II Y     H <Ar1 0 R13 (lib), or a pharmaceutically acceptable salt thereof, OH          R15              0 ____,O.___N.___J 0            °^,OH        ~ Ris :           II                       'r                 \ N' ^4 h CM ^4 x O. OH V' 0 *                             -       II R14           H xR15 M 0 5   R14        H M>Ll5 5                                   R15 point of connectivity of R13 to the remainder of the compound; and R14 and R15 are each independently chosen from hydrogen and methyl.

[371] In some embodiments, provided herein are compounds of Formula (III): (III), or a pharmaceutically acceptable salt thereof, wherein: R1, R2, and R3 are each independently chosen from hydrogen, hydroxyl, -O-(Ci-C6 alkyl) groups, -O-C(=O)-(Ci-C6 alkyl) groups, -C(=O)-O-(Ci-C6 alkyl) groups, and Ci-Ce alkyl groups; R6 and R7 are each independently chosen from hydrogen, -R8, -C(=O)-R8, and -C(=O)- O-R8; R8 is chosen from Ci-Ce alkyl groups, Cs-Cs carbocyclyl groups, and Cs-Cs heterocyclyl wherein R1, R2, R3, R6, R7, and R8 are each independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl, Ci-Ce alkyl groups, -O-(Ci-C6 alkyl) groups, -CO2H, -C(=O)-(Ci-C6 alkyl) groups, -C(=O)-(C3-C8 carbocyclyl) groups, -C(=O)-(C3-C8 heterocyclyl) groups, -NR6R7, C3-C8 carbocyclyl groups, Ci-Ce alkylhydroxy groups, Ci-Ce alkylalkoxy groups, benzyl groups, and C3-C8 heterocyclyl groups, each of which may be independently substituted with 0 or 1 group chosen from halogens, hydroxyl, C1-C3 alkyl groups, C1-C3 alkoxy groups, C1-C3 haloalkyl groups, -NH-C(=O)(Ci-C3 alkyl), and -NH-C(=O)-O-(Ci-C3 alkyl); and wherein * denotes the point of connectivity of R9 to the remainder of the compound.

[372] In some embodiments, provided herein is the compound H1, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H4, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H5, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H6, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H7, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H8, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H9, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H10, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H2, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H3, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H12, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H13, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H14, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H15, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H16, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H17, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H18, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H19, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H20, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H21, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H22, or a pharmaceutically 122 acceptable salt thereof. In some embodiments, provided herein is the compound H23, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H24, or a pharmaceutically acceptable salt thereof. In some embodiments, provided herein is the compound H25, or a pharmaceutically acceptable salt thereof.

[373] In some embodiments, provided herein is a compound chosen from: L thereof, wherein L is a linker which covalently attaches to an antibody.

[374] In some embodiments, the linker comprises at least one cleavable peptide moiety. In some embodiments, the at least one cleavable peptide moiety is cleavable by an enzyme. In some embodiments, the linker or cleavable peptide moiety comprises at least one amino acid unit. In some embodiments, the at least one amino acid unit is chosen 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 chosen from alanine, citrulline, and valine. In some embodiments, the linker comprises citrulline and valine. In some embodiments, the linker comprises alanine and valine.

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

[376] In some embodiments, the linker comprises a spacer unit. In some embodiments, the spacer unit is chosen from alkyl groups and polyethylene glycol (PEG) moieties. In some embodiments, the alkyl group is a C1-C12 alkyl group. In some embodiments, the alkyl group is a C1-C6 alkyl group. In some embodiments, the alkyl group is methylene. In some embodiments, the alkyl group is ethylene. In some embodiments, the alkyl group is n-propylene. In some embodiments, the alkyl group is n-butylene. In some embodiments, the alkyl group is n-pentylene. In some embodiments, the alkyl group is n-hexylene. In some embodiments, the PEG moiety comprises -(PEG)m-, wherein m is an integer from 1 to 10. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6.

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

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

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

[380] Drug loading is represented by p, and is also referred to herein as the herboxidiene splicing modulator-to-antibody ratio (HAR). Drug loading may range from 1 to 10 drug moieties per antibody or antigen binding fragment. In some embodiments, p is an integer 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 fragment. In some embodiments, the linker moiety (L) of the ADC attaches to the antibody or antigen binding fragment through a chemically active group on one or more amino acid residues on the antibody or antigen binding fragment. For example, the linker may be attached to the antibody or antigen binding fragment via a free amino, imino, hydroxyl, thiol, or carboxyl group (e.g., to the N- or C-terminus, to the epsilon amino group of one or more lysine residues, to the free carboxylic acid group of one or more 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 can be a natural residue in the amino acid sequence of the antibody or antigen binding fragment, or it can be introduced into the antibody or antigen binding fragment, e.g., by DNA recombinant technology (e.g., by introducing a cysteine residue into the amino acid sequence) or by protein biochemistry (e.g., by reduction, pH adjustment, or hydrolysis).

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

[383] In some embodiments, an antibody or antigen binding fragment is exposed to reducing conditions prior to conjugation in order to generate one or more free cysteine residues. An antibody, in some embodiments, may be reduced with a reducing agent such as dithiothreitol 129 (DTT) or tris(2-carboxyethyl)phosphine (TCEP), under partial or total reducing conditions, to generate reactive cysteine thiol groups. Unpaired cysteines may be generated through partial reduction with limited molar equivalents of TCEP, which can reduce the interchain disulfide bonds which link the light chain and heavy chain (one pair per H-L pairing) and the two heavy chains in the hinge region (two pairs per H-H pairing in the case of human IgG 1) while leaving the intrachain disulfide bonds intact (Stefano et al. (2013) Methods Mol Biol. 1045:145-71). In embodiments, disulfide bonds within the antibodies are reduced electrochemically, e.g., by employing a working electrode that applies an alternating reducing and oxidizing voltage. This approach can allow for on-line coupling of disulfide bond reduction to an analytical device (e.g., an electrochemical detection device, an NMR spectrometer, or a mass spectrometer) or a chemical separation device (e.g., a liquid chromatograph (e.g., an HPLC) or an electrophoresis device (see, e.g., U.S. Publ. No. 20140069822)). In certain embodiments, an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups on amino acid residues, such as cysteine.

[384] The drug loading of an ADC may be controlled in different ways, e.g., by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody; (ii) limiting the conjugation reaction time or temperature; (iii) partial or limiting reductive conditions for cysteine thiol modification; and / or (iv) engineering by recombinant techniques the amino acid sequence of the antibody such that the number and position of cysteine residues is modified for control of the number and / or position of linker-drug attachments.

[385] In some embodiments, free cysteine residues are introduced into the amino acid sequence of the antibody or antigen binding fragment. For example, cysteine engineered antibodies can be prepared wherein one or more amino acids of a parent antibody are replaced with a cysteine amino acid. Any form of antibody may be so engineered, i.e. mutated. For example, a parent Fab antibody fragment may be engineered to form a cysteine engineered Fab referred to as a "ThioFab." Similarly, a parent monoclonal antibody may be engineered to form a "ThioMab." A single site mutation yields a single engineered cysteine residue in a ThioFab, whereas a single site mutation yields two engineered cysteine residues in a ThioMab, due to the dimeric nature of the IgG antibody. DNA encoding an amino acid sequence variant of the parent polypeptide can be prepared by a variety of methods known in the art (see, e.g., the methods described in Inti. Pub. No. WO 2006 / 034488). These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA encoding the polypeptide. Variants of recombinant antibodies may also be constructed by restriction fragment manipulation or by overlap extension PCR with synthetic oligonucleotides. ADCs of Formula (I) include, but are not limited to, antibodies that have 1,2, 3, or 4 engineered cysteine amino acids (Lyon et al. (2012) Methods Enzymol. 502:123-38). In some embodiments, one or more free cysteine residues are already present in an antibody or antigen binding fragment, without the 130 use of engineering, in which case the existing free cysteine residues may be used to conjugate the antibody or antigen binding fragment to a drug moiety.

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

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

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

[389] In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is about 8. In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, or about 8.5. In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is 8. 131

[390] In various embodiments, the term “about,” as used with respect to the average number of drug moieties per antibody or antigen binding fragment, means plus or minus 10%.

[391] Individual ADC compounds, or “species,” may be identified in the mixture by mass spectroscopy and separated by LIPLC or HPLC, e.g. hydrophobic interaction chromatography (HIC-HPLC). In certain embodiments, a homogeneous or nearly homogenous ADC product with a single loading value may be isolated from the conjugation mixture, e.g., by electrophoresis or chromatography.

[392] In some embodiments, higher drug loading (e.g., p > 8) may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody-drug conjugates. Higher drug loading may also negatively affect the pharmacokinetics (e.g., clearance) of certain ADCs. In some embodiments, lower drug loading (e.g., p < 2) may reduce the potency of certain ADCs against target-expressing cells and / or bystander cells. In some embodiments, the drug loading for an ADC of the present disclosure ranges from about 2 to about 8; from about 2 to about 6; from about 2 to about 5; from about 3 to about 5; from about 2 to about 4; or from about 4 to about 8.

[393] In some embodiments, a drug loading and / or an average drug loading of about 2 is achieved, e.g., using partial reduction of intrachain disulfides on the antibody or antigen binding fragment, and provides beneficial properties. In some embodiments, a drug loading and / or an average drug loading of about 4 is achieved, e.g., using partial reduction of intrachain disulfides on the antibody or antigen binding fragment, and provides beneficial properties. In some embodiments, a drug loading and / or an average drug loading of about 8 is achieved, e.g., using partial reduction of intrachain disulfides on the antibody or antigen binding fragment, and provides beneficial properties. In some embodiments, a drug loading and / or an average drug loading of less than about 2 may result in an unacceptably high level of unconjugated antibody species, which can compete with the ADC for binding to a target antigen and / or provide for reduced treatment efficacy. In some embodiments, a drug loading and / or average drug loading of more than about 8 may result in an unacceptably high level of product heterogeneity and / or ADC aggregation. A drug loading and / or an average drug loading of more than about 8 may also affect stability of the ADC, due to loss of one or more chemical bonds required to stabilize the antibody or antigen binding fragment.

[394] The present disclosure includes methods of producing the described ADCs. Briefly, the ADCs comprise an antibody or antigen binding fragment as the antibody or antigen binding fragment, a drug moiety (e.g., a herboxidiene splicing modulator), and a linker that joins the drug moiety and the antibody or antigen binding fragment. In some embodiments, the ADCs can be prepared using a linker having reactive functionalities for covalently attaching to the drug moiety and to the antibody or antigen binding fragment. For example, in some embodiments, a cysteine thiol of an antibody or antigen binding fragment can form a bond with a reactive functional group of a linker or a drug-linker intermediate (e.g., a maleimide moiety) to make an 132 ADC. The generation of the ADCs can be accomplished by any technique known to the skilled artisan.

[395] In some embodiments, an ADC is produced by contacting an antibody or antigen binding fragment with a linker and a drug moiety (e.g., a herboxidiene splicing modulator) in a sequential manner, such that the antibody or antigen binding fragment is covalently linked to the linker first, and then the pre-formed antibody-linker intermediate reacts with the drug moiety. The antibody-linker intermediate may or may not be subjected to a purification step prior to contacting the drug moiety. In other embodiments, an ADC is produced by contacting an antibody or antigen binding fragment with a linker-drug compound pre-formed by reacting a linker with a drug moiety. The pre-formed linker-drug compound may or may not be subjected to a purification step prior to contacting the antibody or antigen binding fragment. In other embodiments, the antibody or antigen binding fragment contacts the linker and the drug moiety in one reaction mixture, allowing simultaneous formation of the covalent bonds between the antibody or antigen binding fragment and the linker, and between the linker and the drug moiety. This method of producing ADCs may include a reaction, wherein the antibody or antigen binding fragment contacts the antibody or antigen binding fragment prior to the addition of the linker to the reaction mixture, and vice versa. In certain embodiments, an ADC is produced by reacting an antibody or antigen binding fragment with a linker joined to a drug moiety, such as ADL1-herboxidiene splicing modulator (e.g., ADL1-79392) or ADL5-herboxidiene splicing modulator (e.g., ADL5-0349), under conditions that allow conjugation.

[396] The ADCs prepared according to the methods described above may be subjected to a purification step. The purification step may involve any biochemical methods known in the art for purifying proteins, or any combination of methods thereof. These include, but are not limited to, tangential flow filtration (TFF), affinity chromatography, ion exchange chromatography, any charge or isoelectric point-based chromatography, mixed mode chromatography, e.g., CHT (ceramic hydroxyapatite), hydrophobic interaction chromatography, size exclusion chromatography, dialysis, filtration, selective precipitation, or any combination thereof. Therapeutic Uses and Compositions

[397] Disclosed herein are methods of using the disclosed ADCs and compositions in treating a subject for a disorder, e.g., a neoplastic disorder. ADCs may be administered alone or in combination with a second therapeutic agent, and may be administered in any pharmaceutically acceptable formulation, dosage, and dosing regimen. ADC treatment efficacy may be evaluated for toxicity as well as indicators of efficacy and adjusted accordingly. Efficacy measures include, but are not limited to, a cytostatic and / or cytotoxic effect observed in vitro or in vivo, reduced tumor volume, tumor growth inhibition, and / or prolonged survival.

[398] Methods of determining whether an ADC exerts a cytostatic and / or cytotoxic effect on a cell are known. For example, the cytotoxic or cytostatic activity of an ADC can be measured by: exposing mammalian cells expressing a target protein of the ADC in a cell culture medium; 133 culturing the cells for a period from about 6 hours to about 6 days; and measuring cell viability. Cell-based in vitro assays may also be used to measure viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of the ADC.

[399] For determining whether an ADC exerts a cytostatic effect, a thymidine incorporation assay may be used. For example, cancer cells expressing a target antigen at a density of 5,000 cells / well of a 96-well plated can be cultured for a 72-hour period and exposed to 0.5 pCi of 3H-thymidine during the final 8 hours of the 72-hour period. The incorporation of 3H-thymidine into cells of the culture is measured in the presence and absence of the ADC.

[400] For determining cytotoxicity, necrosis or apoptosis (programmed cell death) may be measured. Necrosis is typically accompanied by increased permeability of the plasma membrane; swelling of the cell, and rupture of the plasma membrane. Apoptosis can be quantitated, for example, by measuring DNA fragmentation. Commercial photometric methods for the quantitative in vitro determination of DNA fragmentation are available. Examples of such assays, including TUN EL (which detects incorporation of labeled nucleotides in fragmented DNA) and ELISA-based assays, are described in Biochemica (1999) No. 2, pp. 34-37 (Roche Molecular Biochemicals).

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

[402] Cell viability may be measured, e.g., by determining in a cell the uptake of a dye such as neutral red, trypan blue, Crystal Violet, or ALAMAR™ blue (see, e.g., Page etal. (1993) Inti J Oncology 3:473-6). In such an assay, the cells are incubated in media containing the dye, the 134 cells are washed, and the remaining dye, reflecting cellular uptake of the dye, is measured spectrophotometrically. Cell viability may also be measured, e.g., by quantifying ATP, an indicator of metabolically active cells. In certain embodiments, in vitro potency and / or cell viability of prepared ADCs or herboxidiene splicing modulator compounds may be assessed using a CellTiter-Glo® Luminescent Cell Viability Assay, as described in the examples provided herein. In this assay, in certain embodiments, the single reagent (CellTiter-Glo® Reagent) is added directly to cells cultured in serum-supplemented medium. The addition of 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 culture. The protein-binding dye sulforhodamine B (SRB) can also be used to measure cytotoxicity (Skehan et al. (1990) J Natl Cancer Inst. 82:1107-12).

[403] The disclosed ADCs may also be evaluated for bystander killing activity. Bystander killing activity may be determined, e.g., by an assay employing two cell lines, one positive for a target antigen and one negative for a target antigen. In certain embodiments, the design of the assay allows tracking of only target negative cells. In certain embodiments, cells are plated under three conditions: (i) target negative cells alone (tagged or labeled); (ii) target positive cells alone; and (iii) co-culture of target negative cells and target positive cells. Cells are then treated with an ADC followed by monitoring of cytotoxicity. When plates are read with CellTiter-Glo® Reagent, viability of all cell populations can be monitored. When plates are read with OneGlo® Reagent, only the tagged or labeled target negative cells produce a signal. Killing of the targetnegative cells when mixed with target-positive cells is indicative of bystander killing, whereas killing of the target-negative cells in the absence of the target-positive cells is indicative of off-target killing.

[404] In certain aspects, the present disclosure features a method of killing, inhibiting or modulating the growth of, or interfering with the metabolism of, a cancer cell or tissue by disrupting RNA splicing. The method may be used with any subject where disruption of RNA splicing provides a therapeutic benefit. Subjects that may benefit from disrupting RNA splicing include, but are not limited to, those having or at risk of having a neoplastic disorder such as a hematological malignancy or a solid tumor. In certain embodiments, the hematological malignancy is a B-cell malignancy, a cancer of the blood (leukemia), a cancer of plasma cells (myeloma, e.g., multiple myeloma), or a cancer of the lymph nodes (lymphoma). In certain embodiments, the hematological malignancy is acute myelogenous leukemia or multiple myeloma. In certain 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 (AMoL). In certain embodiments, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma. In certain embodiments, the hematological malignancy is myelodysplasia syndrome (MDS). In certain embodiments, the solid tumor is a carcinoma such as breast cancer, pancreatic cancer, prostate cancer, colon or colorectal cancer, lung cancer, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, cholangiocarcinoma, glioma, or melanoma. In certain embodiments, the solid tumor is breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., uterine serous endometrial carcinoma), salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer, or esophageal cancer. In certain embodiments, the lung cancer is lung adenocarcinoma. In certain embodiments, the uterine cancer is uterine serous endometrial carcinoma.

[405] In various embodiments, the disclosed ADCs may be administered in any cell or tissue that expresses HER2, such as a HER2-expressing neoplastic cell or tissue. An exemplary embodiment includes a method of inhibiting HER2-mediated cell signaling or a method of killing a cell. The method may be used with any cell or tissue that expresses HER2, such as a cancerous cell or a metastatic lesion. Non-limiting examples of HER2-expressing cancers include breast cancer, gastric cancer, bladder cancer, urothelial cell carcinoma, esophageal cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., uterine serous endometrial 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 HER2-expressing cells include HCC1954 and SKBR3 human breast ductal carcinoma cells, N87 human gastric carcinoma cells, and cells comprising a recombinant nucleic acid encoding HER2 or a portion thereof.

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

[407] In various embodiments, the disclosed ADCs may be administered in any cell or tissue that expresses EPHA2, such as an EPHA2-expressing neoplastic cell or tissue. An exemplary embodiment includes a method of inhibiting EPHA2-mediated cell signaling or a method of killing a cell. The method may be used with any cell or tissue that expresses EPHA2, such as a cancerous cell or a metastatic lesion. Non-limiting examples of EPHA2-expressing cancers 136 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 EPHA2-expressing cells include PC3 human prostate cancer cells, and cells comprising a recombinant nucleic acid encoding EPHA2 or a portion thereof.

[408] Exemplary methods include the steps of contacting a cell with an ADC, as described herein, in an effective amount, i.e., amount sufficient to kill the cell. The method can be used on cells in culture, e.g. in vitro, in vivo, ex vivo, or in situ. For example, cells that express HER2 (e.g., cells collected by biopsy of a tumor or metastatic lesion; cells from an established cancer cell line; or recombinant cells), can be cultured in vitro in culture medium and the contacting step can be affected by adding the ADC to the culture medium. The method will result in killing of cells expressing HER2, including in particular tumor cells expressing HER2. Alternatively, the ADC can be administered to a subject by any suitable administration route (e.g., intravenous, subcutaneous, or direct contact with a tumor tissue) to have an effect in vivo. This approach can be used for antibodies targeting other cell surface antigens (e.g., CD138, EPHA2).

[409] The in vivo effect of a disclosed ADC therapeutic composition can be evaluated in a suitable animal model. For example, xenogeneic cancer models can be used, wherein cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice (Klein et al. (1997) Nature Med. 3:402-8). Efficacy may be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.

[410] In vivo assays that evaluate the promotion of tumor death by mechanisms such as apoptosis may also be used. In one embodiment, xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.

[411] Further provided herein are methods of treating a neoplastic disorder, e.g., a cancer. The ADCs disclosed herein can be administered to a non-human mammal or human subject for therapeutic purposes. The therapeutic methods entail administering to a subject having or suspected of having a neoplastic disorder a therapeutically effective amount of an ADC or composition comprising a herboxidiene splicing modulator linked to a targeting antibody that binds to an antigen expressed, is accessible to binding, or is localized on a cancer cell surface. In some embodiments, treatment with the antibody-drug conjugate or composition induces bystander killing of neoplastic cells which do not express a target antigen but are adjacent to neoplastic cells which express a target antigen.

[412] An exemplary embodiment is a method of delivering a herboxidiene splicing modulator to a cell expressing HER2, comprising conjugating the herboxidiene splicing modulator to an 137 antibody that immunospecifically binds to a HER2 epitope and exposing the cell to the ADC. Exemplary tumor cells that express HER2 for which the ADCs of the present disclosure are indicated include gastric carcinoma cells and breast ductal carcinoma cells.

[413] Another exemplary embodiment is a method of delivering a herboxidiene splicing modulator to a cell expressing CD138, comprising conjugating the herboxidiene splicing modulator to an antibody that immunospecifically binds to a CD138 epitope and exposing the cell to the ADC. Exemplary tumor cells that express CD138 for which the ADCs of the present disclosure are indicated 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 exposing the cell to the ADC. Exemplary tumor cells that express EPHA2 for which the ADCs of the present disclosure are indicated include prostate cancer cells.

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

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

[417] In certain aspects, the present disclosure provides a method of reducing or inhibiting growth of a CD138-expressing tumor. In certain embodiments, treatment with the antibody-drug conjugate or composition induces bystander killing of tumor cells which do not express CD138 but that are adjacent to neoplastic tumor cells which do express CD138. Exemplary CD138-expressing tumor types include but are not limited to tumors derived from a CD138-expressing intrathoracic cancer (e.g., lung cancer, mesothelioma), skin cancer (e.g., basal cell carcinoma, squamous cell carcinoma), head and neck cancer (e.g., laryngeal, hypopharynx, nasopharyngeal), breast cancer, urogenital cancer (e.g., cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, urothelial cancer), and thyroid cancer.

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

[419] Moreover, antibodies of the present disclosure may be administered to a non-human mammal expressing an antigen with which the ADC is capable of binding for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of the disclosed ADCs (e.g., testing of dosages and time courses of administration).

[420] Further provided herein are therapeutic uses of the disclosed ADCs and compositions. An exemplary embodiment is the use of an ADC in the treatment of a neoplastic disorder (e.g., a HER2-expressing cancer, a CD138-expressing cancer, an EPHA2-expressing cancer). Another exemplary embodiment is an ADC for use in the treatment of a neoplastic disorder (e.g., a HER2-expressing cancer, a CD138-expressing cancer, an EPHA2-expressing cancer). Methods for identifying subjects having cancers that express a target antigen (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MLIC16, SLC39A6, SLC44A4, or STEAP1) are known in the art and may be used to identify suitable patients for treatment with a disclosed ADC.

[421] Another exemplary embodiment is the use of an ADC in a method of manufacturing a medicament for the treatment of a neoplastic disorder (e.g., a HER2-expressing cancer, a CD138-expressing cancer, an EPHA2-expressing cancer).

[422] The therapeutic compositions used in the practice of the foregoing methods may be formulated into pharmaceutical compositions comprising a pharmaceutically acceptable carrier 139 suitable for the desired delivery method. An exemplary embodiment is a pharmaceutical composition comprising an ADC of the present disclosure and a pharmaceutically acceptable carrier. Suitable carriers include any material that, when combined with the therapeutic composition, retains the anti-tumor function of the therapeutic composition and is generally nonreactive with the patient's immune system. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, mesylate salt, and the like, as well as combinations thereof. In many cases, isotonic agents are included, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the ADC.

[423] Therapeutic formulations may be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like. Therapeutic protein preparations can be lyophilized and stored as sterile powders, e.g., under vacuum, and then reconstituted in bacteriostatic water (containing for example, benzyl alcohol preservative) or in sterile water prior to injection. Therapeutic formulations may comprise an ADC or a pharmaceutically acceptable salt thereof, e.g., a mesylate salt.

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

[425] Various delivery systems are known and may be used to administer one or more ADCs of the present disclosure. Methods of administering the ADCs include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural administration, intratumoral administration, and mucosal administration (e.g., intranasal and oral routes). In addition, pulmonary administration may be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., the compositions and methods for pulmonary administration described in U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and Inti. Publ. Nos. WO 1992 / 019244, WO 1997 / 032572, WO 1997 / 044013, WO 1998 / 031346, and WO 1999 / 066903. The ADCs may be administered by any convenient route, for example, by infusion or bolus injection, or by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). Administration can be either systemic or local.

[426] Therapeutic compositions disclosed herein may be sterile and stable under the conditions of manufacture and storage. In some embodiments, one or more of the ADCs, or pharmaceutical compositions, is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. In some embodiments, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions is supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage 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 ADCs or pharmaceutical compositions is stored at between 2°C and 8°C in the original container. In some embodiments, one or more of the ADCs or pharmaceutical compositions described herein is supplied in liquid form in a hermetically sealed container, e.g., a container indicating the quantity and concentration of the agent. In some embodiments, the liquid form of the administered composition is supplied in a hermetically sealed container of 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 may be stored at between 2°C and 8°C in the original container.

[427] In some embodiments, the disclosed ADCs can be incorporated into a pharmaceutical composition suitable for parenteral administration. The injectable solution may be composed of either a liquid or lyophilized dosage form in a flint or amber vial, ampule, or pre-filled syringe, or other known delivery or storage device.

[428] The compositions described herein may be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic application.

[429] In various embodiments, treatment involves single bolus or repeated administration of the ADC preparation via an acceptable route of administration.

[430] Patients may be evaluated for the levels of target antigen in a given sample (e.g. the levels of target antigen expressing cells) in order to assist in determining the most effective dosing regimen, etc. An exemplary embodiment is a method of determining whether a patient will be responsive to treatment with an ADC of the present disclosure, comprising providing a biological sample from the patient and contacting the biological sample with the ADC. Exemplary biological samples include tissue or body fluid, such as an inflammatory exudate, blood, serum, bowel fluid, stool sample, or tumor biopsy (e.g., a tumor biopsy derived from a patient having or at risk of a target antigen-expressing cancer, e.g., a HER2-expressing cancer, a CD138-expressing cancer, an EPHA2-expressing cancer). In some embodiments, a sample 141 (e.g., a tissue and / or body fluid) can be obtained from a subject, and a suitable immunological method can be used to detect and / or measure protein expression of the target antigen (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MLIC16, SLC39A6, SLC44A4, or STEAP1). Such evaluations are also used for monitoring purposes throughout therapy, and are useful to gauge therapeutic success in combination with the evaluation of other parameters.

[431] In some embodiments, the efficacy of an ADC may be evaluated by contacting a tumor sample from a subject with the ADC and evaluating tumor growth rate or volume. In some embodiments, when an ADC has been determined to be effective, it may be administered to the subject.

[432] The above therapeutic approaches can be combined with any one of a wide variety of additional surgical, chemotherapy, or radiation therapy regimens. In some embodiments, the ADCs or compositions disclosed herein are co-formulated and / or co-administered with one or more additional therapeutic agents, e.g., one or more chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, for example, nitrogen mustards, ethyleneimine compounds, and alkyl sulphonates; antimetabolites, for example, folic acid, purine or pyrimidine antagonists; anti-mitotic agents, for example, anti-tubulin agents 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 binders; and growth factor receptor antagonists. In some embodiments, a chemotherapeutic agent may be a cytotoxic or cytostatic agent. Examples of cytotoxic agents include, but are not limited to, anti-mitotic 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 (e.g., vincristine, vinblastine), taxanes, taxols, and colchicines; anthracyclines (e.g., daunorubicin, doxorubicin, dihydroxyanthracindione); cytotoxic antibiotics (e.g., mitomycins, actinomycins, duocarmycins (e.g., CC-1065), auromycins, duomycins, calicheamicins, endomycins, phenomycins); alkylating agents (e.g., cisplatin); intercalating agents (e.g., ethidium bromide); topoisomerase inhibitors (e.g., etoposide, tenoposide); radioisotopes, such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or213, P32, and radioactive isotopes of lutetium (e.g., Lu177); and toxins of bacterial, fungal, plant or animal origin (e.g., ricin (e.g., ricin A-chain), diphtheria toxin, Pseudomonas exotoxin A (e.g., PE40), endotoxin, mitogellin, combrestatin, restrictocin, gelonin, alpha-sarcin, abrin (e.g., abrin A-chain), modeccin (e.g., modeccin A-chain), curicin, crotin, Sapaonaria officinalis inhibitor, glucocorticoid).

[433] Also disclosed herein are uses of one or more of the disclosed ADCs in the manufacture of a medicament for treating cancer, e.g., according to the methods described above. In some embodiments, the ADCs disclosed herein are used for treating cancer, e.g., according to the methods described above.

[434] In various embodiments, kits for use in the laboratory and therapeutic applications described herein are within the scope of the present disclosure. Such kits may comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method disclosed herein, along with a label or insert comprising instructions for use, such as a use described herein. Kits may comprise a container comprising a drug moiety. The present disclosure also provides one or more of the ADCs, or pharmaceutical compositions thereof, packaged in a hermetically sealed container, such as an ampoule or sachette, indicating the quantity of the agent.

[435] Kits may comprise the container described above, and one or more other containers associated therewith that comprise materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes; carrier, package, container, vial and / or tube labels listing contents and / or instructions for use, and package inserts with instructions for use.

[436] A label may be present on or with the container to indicate that the composition is used for a specific therapy or non-therapeutic application, such as a prognostic, prophylactic, diagnostic, or laboratory application. A label may also indicate directions for either in vivo or in vitro use, such as those described herein. Directions and or other information may also be included on an insert(s) or label(s), which is included with or on the kit. The label may be on or associated with the container. A label may be on a container when letters, numbers, or other characters forming the label are molded or etched into the container itself. A label may be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. The label may indicate that the composition is used for diagnosing or treating a condition, such as a cancer a described herein. Neoantiqens and Methods of Use

[437] Also disclosed herein, in various embodiments, are methods of treating a patient by inducing neoantigens in tumor cells that can be targeted by the patient’s immune system for clearance. Without being bound by theory, in various embodiments, administering a herboxidiene splicing modulator, alone and / or as part of an ADC or composition, may produce neoantigens that induce an immune response, induce a double-stranded RNA immune response, e.g., as a result of re-expressed intron-resident endogenous retroviruses, and / or produce neoantigens that induce immunogenic cell death.

[438] As used herein, the term “neoantigen” refers to any antigen to which the immune system has not previously been exposed that arises from one or more tumor-specific mutations and / or from exposing a tumor to a herboxidiene splicing modulator (e.g., any one or more of the herboxidiene splicing modulators disclosed herein, alone and / or as part of an ADC or composition). Tumor-specific mutations can include missense mutations, frameshifts, translocations, and mRNA splicing variants, as well as mutations that influence posttranslational processing, such as phosphorylation and glycosylation. These exemplary mutations, in various 143 embodiments, can be derived from non-synonymous coding changes and / or mutations that alter mRNA processing (e.g., splicing). All of these exemplary mutations, in various embodiments, can result in molecular changes that can be discriminated by an appropriate T-cell receptor. In various embodiments, an exemplary neoantigen is a neoantigen induced by delivery of a herboxidiene splicing modulator, alone and / or as part of an ADC or composition. In various embodiments, delivery of a herboxidiene splicing modulator (e.g., any one or more of the herboxidiene splicing modulators disclosed herein) can induce novel mRNA splicing that results in the translation of proteins containing one or more novel peptide domains to which the immune system has not previously been exposed. In various embodiments, tumor-specific mutations may be mRNA splicing variants resulting from delivery or administration of a herboxidiene splicing modulator, ADC, or composition comprising a herboxidiene splicing modulator or ADC.

[439] Without being bound by theory, in various embodiments, the delivery of herboxidiene splicing modulators, alone and as part of an ADC or composition, may induce novel mRNA splicing (e.g., exon skipping, intron retention) that results in the alteration of the open reading frames and / or coding sequences of various genes. In various embodiments, these altered genes are translated into proteins containing one or more novel peptide domains recognized by the immune system as foreign. In various embodiments, the one or more novel peptide domains do not exist in the proteins or in any other part of the human proteome in the absence of herboxidiene splicing modulator treatment. In various embodiments, the proteins containing the one or more novel peptide domains can be degraded by the proteasome to create novel peptide fragments that act as substrates for the immunopeptide presentation machinery, e.g., via MHC presentation. In various embodiments, the novel peptide fragments representing neoantigens can be presented in the MHC1-bound peptidome, e.g., on tumor cells.

[440] In various embodiments, the delivery of herboxidiene splicing modulators, alone and as part of an ADC or composition, may lead to one or more tumor cell-intrinsic events (e.g., cell growth arrest). In various embodiments, the tumor cell-intrinsic event(s) may lead to (1) enhanced engagement by phagocytic cells (Bracci et al. (2014) Cell Death Differ. 21 (1): 15-25); (2) the transport of novel peptide fragments to a tumor draining lymph node to engage with antigen-presenting cells; (3) antigen-presenting cells processing novel peptide fragments from a phagocytosed tumor cell and presenting the fragments as neoantigens to circulating naive T-cell populations; (4) novel peptide fragments interacting with T-cells expressing receptors that recognize the fragments as neoantigens; (5) maturation and activation of effector T-cell responses (e.g., CD4+ and / or CD8+ T-cells; and / or (6) engagement of T-cells with additional tumor cells exposed to the herboxidiene splicing modulator treatment and presenting novel peptide fragments representing neoantigens on their surface MHC1 complexes. In various embodiments, the tumor cell-intrinsic event(s) may result, either directly or indirectly, in T-cell engagement of effector function and / or killing of neoantigen-presenting tumor cells.

[441] Also, without being bound by theory, in various embodiments, the delivery of herboxidiene splicing modulators, alone and as part of an ADC or composition, may cause the re-expression of intron-resident endogenous retroviruses, leading to a double-stranded RNA immune response.

[442] Further, without being bound by theory, in various embodiments, the delivery of herboxidiene splicing modulators, alone and as part of an ADC or composition, may lead to immunogenic cell death triggered by splice modulator-induced release of mutationally-derived neoantigens. In various embodiments, the delivery of herboxidiene splicing modulators, alone and as part of an ADC or composition, may induce a double-stranded RNA immune response. In various embodiments, the double-stranded RNA immune response can result from the reexpression of intron-resident endogenous retroviruses. In various embodiments, the doublestranded RNA immune response can result in tumor cell death. In various embodiments, the delivery of herboxidiene splicing modulators, alone and as part of an ADC or composition, may induce immunogenic cell death. In various embodiments, the immunogenic cell death can result from release of mutational-derived neoantigens and / or a host immune response against tumor cells.

[443] Accordingly, in various embodiments, methods of treatment are disclosed comprising inducing neoantigens by administering one or more herboxidiene splicing modulators and / or ADCs and / or compositions comprising a herboxidiene splicing modulator or ADC, e.g., any herboxidiene splicing modulator, ADC, or composition disclosed herein. In various embodiments, the method comprises administering a reduced dosage of the herboxidiene splicing modulator, ADC, or composition than would be needed absent the induction of neoantigens. In some embodiments, the method comprises administering one or more initial induction doses to produce neoantigens and induce an immune response (e.g., converting naive T-cells to memory cells), followed by a reduced dosage or administration frequency (i.e., because of the combinatorial effect of the herboxidiene splicing modulator, ADC, or composition and of immune targeting of the neoantigens). In some embodiments, treatment can comprise a combination of administering the herboxidiene splicing modulator, ADC, or composition to induce a neoantigen-based immune response and at least one additional therapy (e.g., a second anti-cancer therapy). For example, in some embodiments, treatment can comprise a combination of administering the herboxidiene splicing modulator, ADC, or composition to induce a neoantigen-based immune response and one or more checkpoint inhibitors. In some embodiments, treatment can comprise a combination of administering the herboxidiene splicing modulator, ADC, or composition to induce a neoantigen-based immune response and one or more cytokines or cytokine analogs. In some embodiments, treatment can comprise a combination of administering the herboxidiene splicing modulator, ADC, or composition to induce a neoantigen-based immune response and one or more neoantigen vaccines. In some other embodiments, treatment can comprise a combination of administering the herboxidiene splicing modulator, ADC, or composition to induce a neoantigen-based immune response and one or more engineered tumor-targeting T-cells (e.g., CAR-T).

[444] In some embodiments, neoantigens can be used to monitor the effectiveness of treatment with a herboxidiene splicing modulator, ADC, or composition. For instance, after administration of a herboxidiene splicing modulator, ADC, or composition, a patient sample (e.g., a tumor biopsy) can be obtained and screened for neoantigens or for identifiers of an immune or inflammatory response. Further treatment can be provided, e.g., at reduced dosage, if a neoantigen and / or immune response is detected.

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

[446] In various embodiments, methods of treatment are disclosed comprising inducing immunogenic cell death by administering one or more herboxidiene splicing modulators and / or ADCs and / or compositions comprising a herboxidiene splicing modulator or ADC, e.g., any herboxidiene splicing modulator, ADC, or composition disclosed herein.

[447] In various embodiments, administration of a herboxidiene splicing modulator, ADC, or composition comprising a herboxidiene splicing modulator can be combined with any known anti-cancer therapy. Examples of current immune activating strategies available for oncology treatment include, but are not limited to, treatment with immune checkpoint inhibitor (ICI) molecules, treatment with cytokines or cytokine analogs, vaccination with tumor-associated vaccines, and engineering tumor-targeting T-cells (e.g., expansion of tumor-infiltrating lymphocytes or CAR-T). These technologies are predominantly focused on enhancing or inducing an immune response to already 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 an antigenic T-cell response. For example, patient responses to checkpoint inhibition may correlate with non-synonymous mutational burden. In addition, cancer vaccine approaches may be used that rely on pre-existing mutations and the antigenicity of these mutations.

[448] Herboxidiene splicing modulators and / or ADCs comprising such modulators may induce broad-ranging changes in the transcriptome that occur in multiple lineages. Translation of these mRNA changes may produce robust and reproducible protein changes that produce MHC1-bound neopeptides with high affinity across multiple HLA isotypes. Without being bound by theory, due to the large number of changes to the transcriptome and proteome, treatment with herboxidiene splicing modulators and / or ADCs may enrich the number of potentially reactive neoantigens for enhanced engagement of the adaptive immune response.

[449] As described herein, the terms “herboxidiene splicing modulator,” “splicing modulator,” “spliceosome modulator,” or “splice modulator” refer to compounds that have anti-cancer activity by interacting with components of the spliceosome. In some embodiments, a splicing modulator alters the rate or form of splicing in a target cell. Herboxidiene splicing modulators that function as inhibitory agents, for example, are capable of decreasing uncontrolled cellular proliferation. In particular, in some embodiments, the herboxidiene splicing modulators may act by inhibiting the SF3b spliceosome complex. In some embodiments, a herboxidiene splicing modulator is chosen from any one or more of the herboxidiene splicing modulators disclosed herein. In some embodiments, a herboxidiene splicing modulator is used, delivered to a cell, and / or administered to a subject as a stand-alone agent. In some other embodiments, a herboxidiene splicing modulator is used, delivered to a cell, and / or administered to a subject as part of an ADC (e.g., an ADC chosen from any of the exemplary ADCs disclosed herein). In some other embodiments, a herboxidiene splicing modulator is used, delivered to a cell, and / or administered to a subject as part of a composition comprising 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 herboxidiene splicing modulator used, delivered to a cell, and / or administered to a subject as part of an ADC (e.g., an ADC chosen from any of the exemplary ADCs disclosed herein) provides added therapeutic benefits over a herboxidiene splicing modulator used, delivered to a cell, and / or administered to a subject as a stand-alone agent. For example, in some embodiments, a herboxidiene splicing modulator used, 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 the target antigen (i.e., the antigen targeted by the antibody moiety of the ADC). In some embodiments, such targeted delivery of the herboxidiene splicing modulator reduces off-target treatment and / or off-target cytotoxicity. In some embodiments, such targeted delivery promotes tumor-selective neoantigen presentation on neoplastic cells, but not on healthy cells that do not express the target antigen. In some embodiments, such targeted delivery leads to, e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%, of the alternative splicing and induction of novel mRNAs and MHC-associated peptides representing neoantigens in targeted neoplastic cells rather than off-target cells. Thus, without being bound by theory, in some embodiments, following effector T-cell priming and / or expansion (e.g., using a neoantigen vaccine), the immune system may preferentially attack neoantigen-presenting neoplastic cells rather than healthy cells due to the preferential expression of neoantigens on tumor cells after treatment with an ADC as disclosed herein. Immune Induction and Treatment Regimen:

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

[452] In some embodiments, the neoplastic cell is present in an in vitro cell culture. In some embodiments, the neoplastic cell is obtained from a subject. In some embodiments, the neoplastic cell is present in a subject. In some embodiments, the neoplastic cell is derived from a hematological malignancy or a solid tumor. In some embodiments, the hematological malignancy is selected from a B-cell malignancy, a leukemia, a lymphoma, and a myeloma. In some embodiments, the hematological malignancy is selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the solid tumor is selected from breast cancer (e.g., HER2-positive breast cancer), gastric cancer (e.g., gastric adenocarcinoma), prostate cancer, ovarian cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., uterine serous endometrial carcinoma), 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 various embodiments, the present disclosure further provides a method of inducing at least one neoantigen and / or a T-cell response in a subject having or suspected of having a neoplastic disorder by administering to the subject an effective amount of a herboxidiene splicing modulator, an ADC, or a composition comprising a herboxidiene splicing modulator or ADC. Also provided herein, in various embodiments, is a method of treating a subject having or suspected of having a neoplastic disorder by administering to the subject an effective amount of a herboxidiene splicing modulator, an ADC, or a composition comprising a herboxidiene splicing modulator or ADC, wherein administration of the herboxidiene splicing modulator, antibody-drug conjugate, or composition induces at least one neoantigen and / or a T-cell response.

[454] In various other embodiments, the present disclosure provides a method of inducing a double-stranded RNA immune response in a subject having or suspected of having a neoplastic disorder by administering to the subject an effective amount of a herboxidiene splicing 148 modulator, an ADC, or a composition comprising a herboxidiene splicing modulator or ADC. Also provided herein, in various embodiments, is a method of treating a subject having or suspected of having a neoplastic disorder by administering to the subject an effective amount of a herboxidiene splicing modulator, an ADC, or a composition comprising a herboxidiene splicing modulator or ADC, wherein administration of the herboxidiene splicing modulator, antibody-drug conjugate, or composition induces a double-stranded RNA immune response.

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

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

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

[458] As used herein, the term “standard dosage” or “standard dosing regimen” refers to any usual or routine dosing regimen for a therapeutic agent, e.g., a regimen proposed by the 149 manufacturer, approved by regulatory authorities, or otherwise tested in human subjects to meet the average patient’s needs. In some embodiments, the therapeutic agent is a herboxidiene splicing modulator, an antibody, or an antibody-drug conjugate with anti-cancer activity.

[459] For instance, a standard dosing regimen for trastuzumab, an exemplary anti-HER2 antibody disclosed herein, may be 8 mg / kg administered intravenously over 90 min (week 1) followed by 6 mg / kg administered intravenously over 30-90 min every 3 weeks (week 4 through the end of the therapy cycle) (Herceptin® (trastuzumab) FDA Label 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 min every 3 weeks for 4 doses (Yervoy® (ipilimumab) FDA Label Supplement, 2018). Another standard dosing regimen for ipilimumab may be 10 mg / kg administered intravenously over 90 min every 3 weeks for 4 doses, followed by 10 mg / kg every 12 weeks for up to 3 years (Yervoy® (ipilimumab) FDA Label Supplement, 2018).

[461] As another example, a standard dosing regimen for nivolumab, an exemplary anti-PD1 checkpoint inhibitor antibody, may be 3 mg / kg administered intravenously over 60 min every 2 weeks (Opdivo® (nivolumab) FDA Label, 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 min every 3 weeks (Tecentriq® (atezolizumab) FDA Label Supplement, 2018).

[463] As yet 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 min every 3 weeks (Kadcyla® (T-DM1) FDA Label Supplement, 2016).

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

[465] In some embodiments, administration of the herboxidiene splicing modulator, antibodydrug conjugate, or composition is initiated before administration of the at least one additional therapy. In other embodiments, administration of the herboxidiene splicing modulator, antibodydrug conjugate, or composition is initiated after administration of the at least one additional therapy. In still other embodiments, administration of the herboxidiene splicing modulator, antibody-drug conjugate, or composition is initiated concurrently with administration of the at least one additional therapy.

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

[468] In some embodiments, repeated administration of the herboxidiene splicing modulator, antibody-drug conjugate, or composition is concurrent with repeated administration of the at least one additional therapy. In some embodiments, administration of the herboxidiene splicing modulator, antibody-drug conjugate, or composition is sequential or staggered with repeated administration of the at least one additional therapy.

[469] In some embodiments, the at least one additional therapy comprises administering a checkpoint inhibitor, e.g., any checkpoint inhibitor disclosed herein. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to the checkpoint inhibitor when administered alone. In some embodiments, the checkpoint inhibitor is targeted at PD1 / PDL1, CTLA4, 0X40, CD40, LAG3, TIM3, GITR, and / or KIR. In some embodiments, the checkpoint inhibitor is targeted at CTLA4, 0X40, CD40, and / or GITR. In some embodiments, the checkpoint inhibitor is an antibody having inhibitory or agonist activity to its target. In some embodiments, a checkpoint inhibitor is targeted with an inhibitory antibody or other similar inhibitory molecule. In other embodiments, a checkpoint inhibitor is targeted with an agonist antibody or other similar agonist molecule.

[470] In some other embodiments, the at least one additional therapy comprises administering a neoantigen vaccine, e.g., any neoantigen vaccine disclosed herein. In some embodiments, the herboxidiene splicing modulator, ADC, or composition is administered before administration of the neoantigen vaccine. In some embodiments, the herboxidiene splicing modulator, ADC, or composition is administered after administration of the neoantigen vaccine. In some embodiments, the herboxidiene splicing modulator, ADC, or composition is administered concurrently with administration of the neoantigen vaccine. In some embodiments, administration of the herboxidiene splicing modulator, ADC, or composition is repeated at least once after initial administration. In some embodiments, the amount of the herboxidiene splicing modulator, ADC, or composition used for repeated administration is reduced as 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 at least one neoantigen peptide ranges from about 10 to about 50 amino acids in length. In some embodiments, the at least one neoantigen peptide ranges from about 10 to about 35 amino acids in length. In some embodiments, the at least one neoantigen peptide ranges from about 15 to about 25 amino acids in length. In some embodiments, the at least one neoantigen peptide comprises one or more than one neoantigen sequence.

[472] In some embodiments, the neoantigen sequence and / or antigenic portion ranges from about 10 to about 50 amino acids in length. In some embodiments, the at least one neoantigen peptide ranges from about 10 to about 35 amino acids in length. In some embodiments, the 152 neoantigen sequence and / or antigenic portion ranges from about 15 to about 25 amino acids in length. In some embodiments, the neoantigen sequence and / or antigenic portion ranges from about 10 to about 20 amino acids in length. In some embodiments, the neoantigen sequence and / or antigenic portion does not exclusively overlap or consist of the canonical peptide sequence (e.g., any of the exemplary canonical peptide sequences underlined in Table 8).

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

[474] An exemplary embodiment of an antigenic portion is the region(s) flanking amino acids 45-53 of SEQ ID NO: 66. Another exemplary embodiment of an antigenic portion is the region(s) flanking 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 a subject (e.g., HLA-A*02:01). In some other embodiments, the antigenic portion 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 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 a neoplastic disorder.

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

[476] In some embodiments, when a herboxidiene splicing modulator is administered (e.g., alone and / or as part of an ADC or composition), a canonical peptide sequence may be derived from and / or encoded by the immediate 5’ in-frame 24 nucleotides preceding an aberrant splicing event induced by the herboxidiene splicing modulator. Thus, in some embodiments, the canonical peptide sequence comprises or consists of the 8 amino acids immediately N-terminal to the neoantigen sequence induced by the herboxidiene splicing modulator. In some embodiments, when a 5’ exon sequence terminates with a terminal nucleotide of a codon, the canonical peptide sequence terminates at the end of the exon. In some other embodiments, when a 5’ exon sequence terminates with one or two of the three nucleotides of a codon, the canonical peptide sequence is derived from and / or encoded by the 24 nucleotides preceding the incomplete codon. In some embodiments, mRNA sequences 3’ of the aberrant splicing event may be translated in the same open reading frame derived from the 5’ exon until reaching a stop codon, whereupon translation may terminate. In some embodiments, when the aberrant splicing event (e.g., exon skipping) results in a conservation of the canonical transcript open reading frame, the C-terminal sequence may be translated for an additional 24 nucleotides, encoding 8 C-terminal amino acids. In this context, in some embodiments, only the region across the aberrant exon junction may encode a neoantigen sequence. In some embodiments, when the open reading frame is shifted (e.g., intron retention), the complete C-terminal sequence (encoded by the 3’ mRNA) may encode a neoantigen sequence.

[477] In some embodiments, an antigenic portion of a neoantigen sequence is chosen by comparing the neoantigen sequence to the canonical peptide sequence; and selecting a portion of the neoantigen sequence that does not exclusively overlap, consist of, and / or align with the canonical peptide sequence. An antigenic portion of a neoantigen sequence, in some embodiments, can be screened for antigenicity and / or T-cell priming function in the same manner as are full-length neoantigen sequences (e.g., the neoantigen sequence from which the antigenic portion is derived). In some embodiments, an antigenic portion of a neoantigen sequence is evaluated 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 neoantigen sequence is a neoantigen sequence specific to the subject. 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 a subject’s tumor, e.g., after administration of a herboxidiene splicing modulator or ADC, and selecting a vaccine comprising a neoantigen sequence observed in the patient’s tumor.

[479] The term “personalized” when used to describe a neoantigen vaccine refers to a vaccine created by identifying one or more neoantigens produced in a patient, preferably one identified 154 in the patient after an exposure to a herboxidiene splicing modulator, ADC, or composition, and then using one or more of those neoantigens as the basis of the vaccine for the same patient. Accordingly, in some embodiments, a patient is given a herboxidiene splicing modulator, ADC, or composition and screened for neoantigens produced by the treatment. In some embodiments, the selected neoantigen vaccine comprises a neoantigen peptide or mRNA disclosed herein and confirmed to be present in the patient after exposure to the herboxidiene splicing modulator, ADC, or composition. In some embodiments, the herboxidiene splicing modulator, ADC, or composition and / or peptide or mRNA vaccine may be administered to the patient once or repeatedly. Subsequently, in some embodiments, one or more of those neoantigens are used to create a personalized vaccine that is given to the patient. In some embodiments, the one or more neoantigens used to create a personalized vaccine possess binding affinity for one or more patient-specific HLA alleles. In some embodiments, the patient expresses one or more MHC1 alleles that bind to the one or more neoantigens. The prediction of whether a given neoantigen will bind to a specific MHC1 allele can be determined using any computational prediction method known in the art. Exemplary computational prediction methods are disclosed, e.g., 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 universal neoantigen sequence. In some embodiments, the neoantigen sequence is a universal neoantigen vaccine.

[481] The term “universal” when used to describe a neoantigen vaccine refers to a vaccine having a peptide or mRNA sequence that is based on common or known neoantigen(s) observed by sequencing neoantigens produced in multiple patients and / or patient tissue samples, preferably after an exposure to a herboxidiene splicing modulator, ADC, or composition. The peptide or mRNA sequence used in the vaccine need not be present in every patient but rather be observed in at least several patients or patient tissue samples. In some embodiments, the herboxidiene splicing modulator, ADC, or composition and / or peptide or mRNA vaccine may be administered to the patient once or repeatedly. Subsequently, in some embodiments, that peptide or mRNA sequence is used for vaccinating further patients. In some embodiments, a patient is given a herboxidiene splicing modulator, ADC, or composition, and then given a peptide or mRNA vaccine of known neoantigen to enhance immune response to the neoantigens produced by the herboxidiene splicing modulator, ADC, or composition. In some embodiments, a patient is given a universal peptide or mRNA vaccine and then given a herboxidiene splicing modulator, ADC, or composition once or repeatedly. In some embodiments, the neoantigen sequence (or sequences) used to create a universal neoantigen vaccine is selected based on overall MHC1 allele frequency in a given patient population (Maiers etal. (2007) Hum. Immunol. 68(9):779-88).

[482] In some embodiments, the neoantigen (e.g., a universal neoantigen) sequence is capable of binding to at least one HLA allele expressed in at least 10%, at least 15%, at least 155 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from the neoplastic disorder. In some embodiments, the neoantigen sequence 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.

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

[484] In some embodiments, the neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable carrier (e.g., any of the exemplary carriers described herein). In some embodiments, the at least one neoantigen peptide is linked to the pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from a peptide, a serum albumin, a keyhole limpet hemocyanin, an immunoglobulin, a thyroglobulin, an ovalbumin, a toxoid or an attenuated toxoid derivative, a cytokine, and a chemokine. In some embodiments, the neoantigen peptide and the pharmaceutically acceptable carrier are covalently attached via 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 comprises at least one neoantigen peptide and a pharmaceutically acceptable diluent. In some embodiments, the neoantigen vaccine comprises 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 than one neoantigen sequence.

[486] In some embodiments, the neoantigen sequence is a neoantigen sequence specific to the subject. 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 universal neoantigen sequence. In some embodiments, the neoantigen sequence is a universal 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 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from the neoplastic 156 disorder. In some embodiments, the neoantigen sequence 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.

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

[489] In some embodiments, the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable carrier (e.g., any of the exemplary carriers described herein). In some embodiments, the at least one neoantigen mRNA is linked to the pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from a peptide, a serum albumin, a keyhole limpet hemocyanin, an immunoglobulin, a thyroglobulin, an ovalbumin, a toxoid or an attenuated toxoid derivative, a cytokine, and a chemokine. 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. In some embodiments, the neoantigen mRNA is encapsulated by 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, e.g., any cytokine or cytokine analog disclosed herein. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to the cytokine or cytokine analog when administered alone. In some embodiments, the cytokine or cytokine analog comprises a T-cell enhancer. In some embodiments, the cytokine or cytokine analog comprises IL-2, IL-10, IL-12, IL-15, IFNy, and / or TNFa. In some embodiments, the cytokine or cytokine analog comprises IL-2, IL-10, IL-12, and / or IL-15. In some embodiments, administering the cytokine or cytokine analog enhances T-cell priming following administration of a herboxidiene splicing modulator, antibody-drug conjugate, or composition due to the induction and presentation of neoantigens.

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

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

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

[494] In some embodiments, the methods described herein may further comprise detecting immunogenic cell death in the subject after administration of the herboxidiene splicing modulator, antibody-drug conjugate, or composition, and, optionally, continuing administration of the herboxidiene splicing modulator, antibody-drug conjugate, or composition if immunogenic cell death is detected. In some embodiments, detecting immunogenic cell death in the subject indicates efficacy of treatment with the herboxidiene splicing modulator, antibody-drug conjugate, or composition. In some embodiments, treatment with the additional therapy, along with 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 non-synonymous mutational burden of about 150 mutations or less. In some embodiments, the subject has a non-synonymous mutational burden of about 100 mutations or less. In some embodiments, the subject has a non-synonymous mutational burden of about 50 mutations or less. In some embodiments, the subject has or is suspected of having a neoplastic disorder, e.g., a hematological malignancy or 158 a solid tumor. In some embodiments, the hematological malignancy is selected from a B-cell malignancy, a leukemia, a lymphoma, and a myeloma. In some embodiments, the hematological 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, 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 disclosure further provides a method of treating a subject having or suspected of having a neoplastic disorder, comprising: (a) administering to the subject an effective amount of a herboxidiene splicing modulator, an ADC, or composition comprising a herboxidiene splicing modulator or ADC, wherein administration of the herboxidiene splicing modulator, antibody-drug conjugate, or composition induces at least one neoantigen and / or a T-cell response; (b) detecting one or more neoantigens and / or a T-cell response in the subject after administration of the herboxidiene splicing modulator, antibodydrug conjugate, or composition; and (c) continuing administration of the herboxidiene splicing modulator, antibody-drug conjugate, or composition if one or more neoantigens and / or a T-cell response is detected. In some embodiments, detecting one or more neoantigens and / or a T-cell response in the subject indicates efficacy of treatment with the herboxidiene splicing modulator, antibody-drug conjugate, or composition. In some embodiments, the one or more neoantigens comprise an amino acid sequence of any one of SEQ ID NOs: 37-65. In some embodiments, the one or more neoantigens comprise an amino acid sequence of SEQ ID NO: 37. In some embodiments, the one or more neoantigens comprise an amino acid sequence of SEQ ID NO: 39. In some embodiments, the one or more neoantigens comprise an amino acid sequence of any one of SEQ ID NOs: 46-49. Combination of Herboxidiene Splicing Modulator / ADC and Immune Checkpoint Inhibition:

[497] In various embodiments, a patient having a cancer as described herein can be treated with a combination of a herboxidiene splicing modulator, ADC, or composition and a checkpoint inhibitor therapy. Immune checkpoints are inhibitory pathways that slow down or stop immune reactions and prevent excessive tissue damage from uncontrolled activity of immune cells. As used herein, the term "checkpoint inhibitor" is meant to refer to any therapeutic agent, including any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or any fragments thereof, that inhibits one or more of the inhibitory pathways, thereby allowing more extensive immune activity.

[498] Treatment of patients with immune checkpoint inhibition has been shown to have robust efficacy in certain clinical indications. Recently, the FDA approved use of a checkpoint inhibitor in patients with tumors exhibiting high microsatellite instability, agnostic to the tissue lineage. 159 This approval was based, in part, on the observation that response rates correlate positively with mutational burden (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 lineage, but generally support that above a threshold of -150-250 mutations, the probability of response rises. Analysis of TCGA data shows that a large percentage of adult-onset tumor lineages have comparatively low non-synonymous mutational burden (Vogelstein et al. (2013) Science 339:1549-58). Most lineages have median non-synonymous mutational rates of -SOSO per patient, well below the thresholds for improved odds of response to checkpoint inhibitors.

[499] For instance, HER2-positive breast cancer has been shown to have a median of -60 non-synonymous mutations present per patient sample. However, the threshold for checkpoint inhibitor treatment efficacy, as mentioned above, is estimated to be in the range of -150-250 non-synonymous mutations, i.e., patients above this threshold are more likely to show complete remission, partial remission, and / or stable disease, whereas patients below this threshold are more likely to exhibit progressive disease. Strategies to enhance the apparent number of non-synonymous mutations and / or neoantigens being presented on tumor cells are therefore desirable, and may enhance the overall probability of response, e.g., to checkpoint inhibitor therapies. As cytokines (and analogs thereof) act via a similar mechanism of action, such strategies may also enhance the overall probability of response to cytokine-based therapies.

[500] Current response rates in HER2-positive breast cancer are -15-25% (CTI NCT02129556). In various embodiments disclosed herein, treatment with a herboxidiene splicing modulator, ADC, or composition in combination with a checkpoint inhibitor and / or cytokine therapy may improve such response rates. In various embodiments, treatment with a herboxidiene splicing modulator, ADC, or composition in combination with a checkpoint inhibitor and / or cytokine therapy may apply to any adult-onset tumor, particularly those in which the median non-synonymous mutational rate is below the estimated -150 mutations threshold. In various embodiments, exemplary cancer types suitable for treatment with a herboxidiene splicing modulator, ADC, or composition of the present disclosure, alone or in combination with an additional therapy (e.g., a checkpoint inhibitor therapy, a cytokine therapy) 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 (e.g., HER2-positive breast cancer), lung cancer (e.g., non-small cell lung cancer), chronic lymphocytic leukemia, and acute myeloid leukemia. Other exemplary suitable cancer types are identified, e.g., in Vogelstein et al. (2013) Science 339:1549-58, which is incorporated herein by reference in its entirety.

[501] As many checkpoint inhibitor therapies are based on chronic expression of tumor-associated antigens, regular treatment boosts are required for efficacy and for “re-boosting” reactive T-cell populations. The inducible nature of herboxidiene splicing modulator or ADC-derived neoantigens described herein provide for therapeutic dosing regimens that may be 160 designed to enhance the immune response of neoantigen-reactive T-cells, while limiting T-cell exhaustion often caused by chronic antigen stimulation. For instance, in some embodiments, an initial dose of a herboxidiene splicing modulator, ADC, or composition is administered to a subject to trigger aberrant splicing and production of neoantigen peptides. After a period of time to allow for protein production and antigen presentation, in some embodiments, the subject is then administered an initial dose of a checkpoint inhibitor to boost and / or enhance effector T-cell priming and expansion. In some embodiments, the wait period between doses of herboxidiene splicing modulator, ADC, or composition and checkpoint inhibitor is about 2, about 3, about 4, about 5, about 6, or about 7 days. In some embodiments, the wait period is between about 3 days and about 5 days. In some embodiments, the checkpoint inhibitor is targeted at CTLA4, 0X40, CD40, and / or GITR. In some embodiments, the combination therapeutic benefit of a herboxidiene splicing modulator, ADC, or composition and a checkpoint inhibitor may be additive or superadditive.

[502] In some embodiments, after a period to allow for T-cell priming and expansion, the subject is then administered a second or subsequent dose of the herboxidiene splicing modulator, ADC, or composition to trigger re-presentation of neoantigen peptides. In some embodiments, the wait period between an initial dose of a checkpoint inhibitor and a second or subsequent dose of a herboxidiene splicing modulator, ADC, or composition is about 2, about 3, about 4, or about 5 weeks. In some embodiments, the wait period is about 3 weeks. Following a second or subsequent dose of the herboxidiene splicing modulator, ADC, or composition, in some embodiments, the immune system may engage with the neoantigen-presenting tumor cells and / or elicit tumor cell killing. In some embodiments, the subject is then administered a second or subsequent dose of the checkpoint inhibitor to further expand the memory effector T-cell population, after allowing for secondary T-cell priming and expansion.

[503] In some embodiments, dosing of the herboxidiene splicing modulator, ADC, or composition following this exemplary initial treatment regimen can be pulsatile, i.e., the herboxidiene splicing modulator, ADC, or composition may be dosed at prolonged intervals (e.g., about every 4 weeks, about every 5 weeks, about every 6 weeks) to allow for antigen presentation, T-cell engagement and / or tumor cell killing, and / or recovery of the memory T-cell population. At later timepoints, in some embodiments, the herboxidiene splicing modulator, ADC, or composition treatment may be combined with one or more checkpoint inhibitors targeted to restore effector functionality to exhausted T-cell populations. For example, in some embodiments, at later timepoints, the herboxidiene splicing modulator, ADC, or composition treatment may be combined with one or more checkpoint inhibitors targeted at PD1 / PDL1, LAG3, and / or TIM3. In some embodiments, the pulsed nature of neoantigen presentation and priming may allow a checkpoint inhibitor and / or a herboxidiene splicing modulator, ADC, or composition to be administered less frequently and / or at lower doses. In some embodiments, the pulsed nature of neoantigen presentation may provide one or more treatment benefits for a 161 checkpoint inhibitor (e.g., an anti-CTLA4 antibody such as ipilimumab), as compared to the checkpoint inhibitor when administered without concurrent herboxidiene splicing modulator, ADC, or composition treatment, for example, by lowering the potential risk of adverse reactions often observed with the checkpoint inhibitor’s standard dosing regimen.

[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 downregulates immune responses. CTLA4 is constitutively expressed in regulatory T-cells, but only upregulated in conventional T-cells after activation. As used herein, the term “CTLA4 inhibitor” is meant to refer to any inhibitor of CTLA4 and / or the 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 the pre-clinical activity seen in mouse models of anti-tumor 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 IgG 1 with a plasma half-life of approximately 12-14 days; tremelimumab is an lgG2 with a plasma half-life of approximately 22 days. See, e.g., 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 death-1 (PD1) pathway. The programmed cell death 1 (PD1) pathway represents a major immune control switch which may be engaged by tumor cells to overcome active T-cell immune surveillance. The ligands for PD1 (PDL1 and PDL2) are constitutively expressed or can be induced in various tumors. High expression of PDL1 on tumor cells (and to a lesser extent of PDL2) has been found to correlate with poor prognosis and survival in various other solid tumor types. Furthermore, PD1 has been suggested to regulate tumor-specific T-cell expansion in patients with malignant melanoma. These observations suggest that the PD1 / PDL1 pathway plays a critical role in the tumor immune evasion and may be considered an attractive target for therapeutic intervention. As used herein, the term “PD1 inhibitor” is meant to refer to any inhibitor of PD1 and / or the 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 pembrolizumab (MK-3475). Nivolumab, for example, is a fully human immunoglobulin G4 (lgG4) PD1 immune checkpoint inhibitor antibody that disrupts the interaction of the PD1 receptor with its ligands PDL1 and PDL2, thereby inhibiting 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 mAb of the lgG4 / kappa isotype designed to directly block the interaction between PD1 and its ligands, PDL1 and PDL2. Pembrolizumab strongly enhances T lymphocyte immune responses in cultured blood cells from healthy human donors, cancer patients, and primates. Pembrolizumab has also been reported to modulate the level of interleukin-2 (IL-2), tumor necrosis factor alpha (TNFa), interferon gamma (IFNy), and other cytokines. Exemplary anti-PDL1 antibodies include but are not limited to atezolizumab, avelumab, and durvalumab. Atezolizumab, for example, is an IgG 1 humanized mAb that is reported to block the PD1 / PDL1 interaction, by targeting the expressed PDL1 on numerous kinds of malignant cells. This blockage of the PD1 / PDL1 pathway may stimulate the 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 is targeted at PD1 / PDL1, CTLA4, OX40, CD40, LAG3, TIM3, GITR, and / or KIR. In certain embodiments, the checkpoint inhibitor is targeted at CTLA4, OX40, CD40, and / or GITR. In certain embodiments, a checkpoint inhibitor is targeted with an inhibitory antibody or other similar inhibitory molecule (e.g., an inhibitory anti-CTLA4 or anti-PD1 / PDL1 antibody). In certain other embodiments, a checkpoint inhibitor is targeted with an agonist for the target; examples of this class include the stimulatory targets OX40, CD40, and / or GITR. In some embodiments, the checkpoint inhibitor targeted at 0X40, CD40, and / or GITR is an agonist antibody. Agonist antibodies directed against 0X40 may have a dual role, inhibiting regulatory T-cell suppression, while enhancing effector T-cell functions. Agonist anti-GITR antibodies have also been shown to make effector T-cells more resistant to the inhibition induced by regulatory T-cells (Karaki et al. (2016) Vaccines (Basel) 4(4):37). Likewise, agonist CD40 antibodies demonstrate T-cell-dependent anti-tumor activity. Activation of CD40 on dendritic cells increases cross-presentation of tumor antigens and consequently the number of activated tumor-directed effector T-cells (Ellmark et al. (2015) Oncoimmunol. 4(7):e1011484).

[507] In certain embodiments, the checkpoint inhibitor is targeted at CTLA4 (e.g., an anti-CTLA4 antibody). In certain embodiments, targeting CTLA4 facilitates priming and activation of naive T-cells. In certain embodiments, the checkpoint inhibitor is targeted at 0X40 (e.g., an anti-OX40 antibody). In certain embodiments, targeting 0X40 enhances expansion of effector T-cells. In certain embodiments, the checkpoint inhibitor is targeted at CD40 (e.g., an anti-CD40 antibody). In certain embodiments, targeting CD40 inhibits “tolerogenic” priming of T-cells and / or formation of regulatory T-cells. In certain embodiments, the checkpoint inhibitor is targeted at GITR (e.g., an anti-GITR antibody). In certain embodiments, targeting GITR inhibits activity of regulatory T-cells. In certain embodiments, the benefit of combination therapy (e.g., the effect on at least one symptom or the risk / rate of disease progression) with a herboxidiene splicing modulator, ADC, or composition and a CTLA4-, OX40-, CD40-, and / or GITR-targeted agent is additive. In some embodiments, the benefit of combination therapy with a herboxidiene splicing modulator, ADC, or composition and a CTLA4-, OX40-, CD40-, and / or GITR-targeted agent is superadditive (i.e., synergistic).

[508] Checkpoint inhibitor treatment strategies are based on the hypothesis that treatment facilitates and / or enhances priming of T-cell responses to weakly or poorly antigenic tumors (e.g., CTLA4) or that treatment restores and / or reinvigorates T-cells that respond to tumor antigens, but have become “exhausted” due to the chronic nature of the antigen presentation (e.g., PD1, PDL1) (Chen and Mellman (2013) Immunity 39(1):1-10). Examples of suitable checkpoint inhibition therapies and agents, e.g., anti-PD1, anti-PDL1, oranti-CTLA4 antibodies, are known in the art. See, e.g., WO 2001 / 014424 WO 2013 / 173223, WO 2016 / 007235.

[509] Combining these primed T-cell responses following checkpoint inhibitor therapy with treatment to induce neoantigens in tumor cells to which the primed immune system can react may provide beneficial synergy. As the herboxidiene splicing modulator or ADC-derived neoantigens have not yet been presented for T-cell priming, combination with a CTLA4 inhibitor may be particularly beneficial. In some embodiments, treatment comprises administering one or more herboxidiene splicing modulator, ADC, or composition to induce the production of neoantigens, followed before, concurrently, or thereafter by an initial administration of a CTLA4 inhibitor to stimulate CD8 T-cell priming. In some embodiments, additional administrations of an CTLA4 inhibitor are provided to the patient, e.g., to further stimulate priming and / or activation of neoantigen-reactive CD8 populations. In some embodiments, additional administrations of herboxidiene splicing modulator, ADC, or composition can be given to the patient to increase neoantigen presentation by the tumor. Repeat administrations of herboxidiene splicing modulator, ADC, or composition and checkpoint inhibitor therapy can occur concurrently or in staggered intervals. In some embodiments, treatment further comprises administering a PD11 PDL1 inhibitor co-treatment, e.g., to restore effector function of exhausted neoantigen-targeted T-cells within the tumor microenvironment.

[510] The terms “combination” or “combination therapy,” as used herein, refer to the administration of one or more herboxidiene splicing modulator, ADC, or composition together with an additional agent or therapy (e.g., a checkpoint inhibitor, a cytokine or cytokine analog, a neoantigen vaccine, CAR-T), as part of a treatment regimen intended to provide a beneficial (i.e., additive or synergistic) effect from the co-action of one or more of the 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 (e.g., a second checkpoint inhibitor). The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents. Administration of these therapeutic agents in combination typically is carried out over a defined time period (for example, minutes, hours, days, or weeks, depending upon the combination selected).

[511] Administered “in combination” or “co-administration,” as used herein, means that two or more different treatments are delivered to a subject during the subject’s affliction with a medical condition (e.g., a neoplastic disorder). For example, in some embodiments, the two or more 164 treatments are delivered after the subject has been diagnosed with a disease or disorder, and before the disease or disorder has been cured or eliminated, or when a subject is identified as being at risk but before the subject has developed symptoms of the disease. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second treatment begins, so that there is overlap. In some embodiments, the first and second treatment are initiated at the same time. These types of delivery are sometimes referred to herein as “simultaneous,” “concurrent,” or “concomitant” delivery. In other embodiments, the delivery of one treatment ends before delivery of the second treatment begins. This type of delivery is sometimes referred to herein as “successive” or “sequential” delivery.

[512] In some embodiments, the two treatments (e.g., a herboxidiene splicing modulator, ADC, or composition and a checkpoint inhibitor) are comprised in the same composition. Such compositions may be administered in any appropriate form and by any suitable route. In other embodiments, the two treatments (e.g., a herboxidiene splicing modulator, ADC, or composition and a checkpoint inhibitor) are administered in separate compositions, in any appropriate form and by any suitable route. For example, in some embodiments, a composition comprising a herboxidiene splicing modulator or ADC and a composition comprising a checkpoint inhibitor may be administered concurrently or sequentially, in any order at different points in time; in either case, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect.

[513] In embodiments of either simultaneous or sequential delivery, treatment may be more effective because of combined administration. In some embodiments, the first treatment is more effective, e.g., an equivalent effect is seen with less of the first treatment (e.g., with a lower dose), than would be seen if the first treatment were administered in the absence of the second treatment. In some embodiments, the first treatment is more effective such that the reduction in a symptom, or other parameter associated with the disease or disorder, is greater than what would be observed with the first treatment delivered in the absence of the second treatment. In other embodiments, an analogous situation is observed with the second treatment. In some embodiments, the benefit of combination therapy (e.g., the effect on at least one symptom or the risk / rate of disease progression) is additive. In some embodiments, the benefit of combination therapy is superadditive.

[514] In various embodiments, the present disclosure provides a method of treating a subject having or suspected of having a neoplastic disorder by administering to the subject an effective amount of a herboxidiene splicing modulator, an ADC, or a composition comprising a herboxidiene splicing modulator or ADC; and at least one additional therapy (e.g., a checkpoint inhibitor therapy, a cytokine or cytokine analog, a neoantigen vaccine, CAR-T). In some embodiments, administration of the herboxidiene splicing modulator, ADC, or composition induces at least one neoantigen and / or a T-cell response. In some embodiments, administration of the herboxidiene splicing modulator, ADC, or composition induces a double-165 stranded RNA immune response. In some embodiments, administration of the herboxidiene splicing modulator, ADC, or composition induces immunogenic cell death. In some embodiments, the at least one additional therapy may comprise at least one, at least two, at least three, at least four, or at least five additional therapies. For example, in some embodiments, a herboxidiene splicing modulator, ADC, or composition may be administered in combination with two checkpoint therapies, i.e., using two different checkpoint inhibitors. In some other embodiments, a herboxidiene splicing modulator, ADC, or composition may be administered in combination with a checkpoint inhibitor therapy and a neoantigen vaccine.

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

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

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

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

[519] In some embodiments, repeated administration of the herboxidiene splicing modulator, antibody-drug conjugate, or composition is concurrent with repeated administration of the at least one additional therapy. In some embodiments, repeated administration of the herboxidiene splicing modulator, antibody-drug conjugate, or composition is sequential or staggered with repeated administration of the at least one additional therapy.

[520] In various embodiments, the present disclosure provides a method of treating a subject having or suspected of having a neoplastic disorder by administering to the subject an effective amount of a herboxidiene splicing modulator, an ADC, or a composition comprising a herboxidiene splicing modulator or ADC; and a checkpoint inhibitor therapy. In some embodiments, the checkpoint inhibitor therapy comprises administering at least one checkpoint inhibitor. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to the at least one checkpoint inhibitor when administered alone. In some embodiments, a subject may be considered non-responsive or poorly responsive to the at least one checkpoint inhibitor as determined using, e.g., the immune-related Response Criteria (irRC) and / or the immune-related Response Evaluation Criteria in Solid Tumors (irRECIST). See, e.g., 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. Exemplary criteria may include those used in the art to define when tumors in cancer patients improve (“respond”), remain the same (“stabilize”), or worsen (“progress”) during treatment, when the treatment being evaluated is an immune-oncology drug (e.g., a checkpoint inhibitor). In some embodiments, a subject may be considered intolerant to the at least one checkpoint inhibitor if the subject presents with one or more than one adverse (grade 2+) event identified for the respective checkpoint inhibitor (e.g., ipilimumab). In some embodiments, for example, a subject may be considered intolerant to ipilimumab treatment if the subject presents with one or more adverse events selected from enterocolitis, hepatitis, dermatitis (including toxic epidermal necrolysis), neuropathy, and endocrinopathy (Yervoy® (ipilimumab) FDA Label Supplement, 2018).

[521] In some embodiments, the checkpoint inhibitor is targeted at PD1 / PDL1, CTLA4, 0X40, CD40, LAG3, TIM3, GITR, and / or KIR. In some embodiments, the checkpoint inhibitor is targeted at CTLA4, 0X40, CD40, and / or GITR. In some embodiments, the checkpoint inhibitor is targeted with an inhibitory antibody or other similar inhibitory molecule. In some other embodiments, the checkpoint inhibitor is targeted with an agonist antibody or other similar agonist molecule. In some embodiments, the checkpoint inhibitor comprises a cytotoxic T-lymphocyte-associated antigen 4 pathway (CTLA4) inhibitor. 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 a programmed death-1 pathway (PD1) inhibitor. 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 comprises a CTLA4 inhibitor and a PD1 inhibitor. In some embodiments, the checkpoint inhibitor is targeted at 0X40. In some embodiments, the checkpoint inhibitor is targeted at CD40. In some embodiments, the checkpoint inhibitor is targeted at GITR. In some embodiments, the benefit of combination therapy (e.g., the effect on at least one symptom or the risk / rate of disease progression) with a herboxidiene splicing modulator, ADC, or composition and a checkpoint inhibitor (e.g., a CTLA4-, PD1 / PDL1-, OX40-, CD40-, and / or GITR-targeted antibody or molecule) is additive. In some embodiments, the benefit of combination therapy with a herboxidiene splicing modulator, ADC, or composition and a checkpoint inhibitor (e.g., a CTLA4-, PD1 / PDL1, OX40-, CD40-, and / or GITR-targeted antibody or molecule) is superadditive (i.e., synergistic).

[522] In various embodiments, the present disclosure provides a method of treating a subject having or suspected of having a neoplastic disorder by administering to the subject an effective amount of a herboxidiene splicing modulator, an ADC, or a composition comprising a herboxidiene splicing modulator or ADC; and a cytokine or cytokine analog therapy. In some embodiments, the cytokine or cytokine analog therapy comprises administering at least one cytokine or cytokine analog. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to the at least one cytokine or cytokine analog when administered alone.

[523] In some embodiments, the cytokine or cytokine analog comprises a T-cell enhancer. In some embodiments, the cytokine or cytokine analog comprises IL-2, IL-10, IL-12, IL-15, IFNy, and / or TN Fa. In some embodiments, the cytokine or cytokine analog comprises IL-2, IL-10, IL-12, and / or IL-15. In some embodiments, administering the cytokine or cytokine analog enhances T-cell priming following administration of a herboxidiene splicing modulator, antibodydrug conjugate, or composition due to induction and presentation of neoantigens.

[524] In some embodiments, the cytokine or cytokine analog comprises IL-2. In some embodiments, IL-2 boosts signals to effector cells promoting their expansion (Rosenberg (2014) J Immunol. 192(12):5451-8). In some embodiments, the cytokine or cytokine analog comprises 168 IL-10. In some embodiments, IL-10 boosts CD8+ T-cell priming and activation (Mumm et al. (2011) Cancer Cell 20(6):781-96). In some embodiments, the cytokine or cytokine analog comprises IL-12. In some embodiments, IL-12 links the innate and adaptive immune responses to boost antigen-specific priming and targeting (Tugues et al. (2015) Cell Death Differ. 22(2):237-46). In some embodiments, the cytokine or cytokine analog comprises IL-15. In some embodiments, IL-15 boosts T-effector (CD8) cell priming and / or activation. In some embodiments, the cytokine or cytokine analog comprises IFNy. In some embodiments, IFNy supplements T-effector cell secretion of IFNy. In some embodiments, the cytokine or cytokine analog comprises TNFa. In some embodiments, TNFa supplements T-effector cell secretion of TNFa.

[525] In some embodiments, an initial dose of a herboxidiene splicing modulator, ADC, or composition is administered to a subject to trigger aberrant splicing and production of neoantigen peptides. After a period to allow for protein production and antigen presentation, in some embodiments, the subject is then administered an initial dose of a cytokine or cytokine analog to boost and / or enhance effector T-cell priming and expansion. In some embodiments, the wait period between doses of herboxidiene splicing modulator, ADC, or composition and cytokine or cytokine analog is about 2, about 3, about 4, about 5, about 6, or about 7 days. In some embodiments, the wait period is between about 3 days and about 5 days. In some embodiments, the cytokine or cytokine analog is IL-2, IL-10, IL-12, IL-15, IFNy, and / or TNFa. In some embodiments, the combination therapeutic benefit of a herboxidiene splicing modulator, ADC, or composition and a cytokine or cytokine analog may be additive or superadditive.

[526] In some other embodiments, an initial dose of a cytokine or cytokine analog is administered to a subject to boost and / or enhance effector T-cell priming and expansion. After a wait period, in some embodiments, the subject is then administered an initial dose of a herboxidiene splicing modulator, ADC, or composition to trigger aberrant splicing and production of neoantigen peptides. In some embodiments, the wait period between doses of cytokine or cytokine analog and herboxidiene splicing modulator, ADC, or composition is about 2, about 3, about 4, about 5, about 6, or about 7 days. In some embodiments, the wait period is between about 3 days and about 5 days. In some embodiments, the cytokine or cytokine analog is IL-2, IL-10, IL-12, IL-15, IFNy, and / or TNFa. In some embodiments, the combination therapeutic benefit of a cytokine or cytokine analog and a herboxidiene splicing modulator, ADC, or composition may be additive or superadditive.

[527] In some embodiments, after a period to allow for T-cell priming and expansion, the subject is then administered a second or subsequent dose of the herboxidiene splicing modulator, ADC, or composition to trigger re-presentation of neoantigen peptides. In some embodiments, the wait period between an initial dose of a cytokine or cytokine analog and a second or subsequent dose of a herboxidiene splicing modulator, ADC, or composition is about 2, about 3, about 4, or about 5 weeks. In some embodiments, the wait period is about 3 weeks. 169 In some embodiments, subsequent doses of the cytokine or cytokine analog may be administered, e.g., interspersed between subsequent doses of the herboxidiene splicing modulator, ADC, or composition. Following a second or subsequent dose of the herboxidiene splicing modulator, ADC, or composition, in some embodiments, the immune system may engage with the neoantigen-presenting tumor cells and / or elicit tumor cell killing. In some embodiments, dosing of the herboxidiene splicing modulator, ADC, or composition following this exemplary initial treatment regimen can be pulsatile, i.e., the herboxidiene splicing modulator, ADC, or composition may be dosed at prolonged intervals (e.g., about every 4 weeks, about every 5 weeks, about every 6 weeks) to allow for antigen presentation, T-cell engagement and / or tumor cell killing, and / or recovery of the memory T-cell population.

[528] In some embodiments, the subject has a non-synonymous mutational burden of about 150 mutations or less. In some embodiments, the subject has a non-synonymous mutational burden of about 100 mutations or less. In some embodiments, the subject has a non-synonymous mutational burden of about 50 mutations or less. In some embodiments, the subject has or is suspected of having a neoplastic disorder, e.g., a hematological malignancy or a solid tumor. In some embodiments, the hematological malignancy is selected from a B-cell malignancy, a leukemia, a lymphoma, and a myeloma. In some embodiments, the hematological 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, and esophageal cancer. In some embodiments, the solid tumor is selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma. Combination of Herboxidiene Splicing Modulator / ADC and Neoantigen Vaccine:

[529] In various embodiments, a patient having a cancer as described herein can be treated with a combination of a herboxidiene splicing modulator, ADC, or composition and a neoantigen vaccine.Without being bound by theory, vaccines, used alone or in combination with immune checkpoint inhibitor (ICI) molecules, have shown promise in early trials (Ott et al. (2017) Nature 547(7662):217-21; Sahin et al. (2017) Nature 547(7662):222-6), but generally require sequencing of patient tumor mutations (Ott et al. (2017) Nature 547(7662):217-21; Aldous and Dong (2018) Bioorg. Med. Chern. 26(10):2842-9). As such, vaccines are often dependent on sufficient numbers of non-synonymous mutations that are antigenic. In general, tumors with very low mutation burden provide few candidate antigens, and those with rapid growth provide limited time to identify and produce patient-specific vaccines.

[530] To date, attempts to develop vaccines that would be broadly immunogenic across 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 within the organism. In addition, these proteins are often expressed in immunologically restricted tissues (e.g., 170 neuronal markers expressed in neuroendocrine tumor types), while others may be normally expressed during embryogenesis (e.g., oncofetal antigens). Thus, utility of vaccines using such proteins as antigens is often limited to specific tumor lineages or subsets where one or more of the antigens are presented. Vaccine utility would also need to be confirmed by sequencing of patient tumor samples, which can be time-consuming.

[531] Moreover, if these antigens exist as “self” proteins, the immune system would likely be primed to recognize these as “self’ and thus, not respond. Or, alternatively, if the immune system is able to mount an effector response to these antigens, it may lead to on-target side effects in tissues where the antigen may be expressed. In both of these cases, one of the key challenges is that most antigenic peptides are derived from “passenger” genes (i.e., genes that are mutated or amplified in the course of tumorigenesis, but that do not play a critical role in the continued survival or proliferation of the tumor itself). As such, these genes may be silenced without significant consequence to the tumor progression, and thus would allow a tumor to “escape” an immune response against these antigens. Without wishing to be bound by theory, this mechanism may play a role in tumor evolution, where random mutations that are strongly antigenic are often “selected against” by the tumor during the early stages of tumorigenesis (Dunn etal. (2004) Annu. Rev. Immunol. 22:329-60).

[532] In addition, certain evidence also indicates that chronic antigen presentation and immune stimulation may lead to immune cell anergy and exhaustion (Pardoll (2012) Nat. Rev. Cancer 12(4):252-64). These phenotypes underlie the therapeutic rationale behind current ICI treatments, as ICI has been shown to either repress the exhausted immune cell phenotype (a-PD1 / PD-L1) or to facilitate additional immune cell responses (O-CTLA4). Notably, with O-CTLA4 therapy, a certain subset of patients have been reported to exhibit severe immune-related adverse events that may be ascribed to the promotion of T-cell activation and a break of the immune tolerance mechanisms that restrain self-reactive immune responses.

[533] Both of these approaches (i.e., triggering or enhancing de novo immune responses to neoantigens or derepressing the anergy or exhaustion of existing immune responses) are linked to a chronic immune activation. As such, these approaches are sensitive to anergy, editing, and other tumor-mediated mechanisms designed to suppress immune engagement.

[534] In contrast, treatment with a herboxidiene splicing modulator, ADC, or composition disclosed herein may induce an immune response to sequences representing neoantigens. In some embodiments, presentation of neoantigens provides the adaptive immune system with more divergent targets with which to engage and activate. In some embodiments, the ability of a herboxidiene splicing modulator, ADC, or composition to acutely induce alternative splicing and the resulting neoantigens may reduce the risk of immune system fatigue due to chronic exposure to mutation-driven neoantigens and / or limit the ability of tumor cells to adapt to evade therapy. In some embodiments, administering a herboxidiene splicing modulator, ADC, or composition in combination with a neoantigen vaccine enhances the immune response to the 171 neoantigens produced by the herboxidiene splicing modulator, ADC, or composition. In some embodiments, the herboxidiene splicing modulator, ADC, or composition is administered before, during, or after vaccination. In some embodiments, the herboxidiene splicing modulator, ADC, or composition and / or vaccine may be administered once or more than once during the course of treatment. In some embodiments, the vaccine is administered once and the herboxidiene splicing modulator, ADC, or composition is administered more than once during the course of treatment. In some embodiments, the vaccine is administered once and then one or more boosters are administered during the course of treatment.

[535] As used herein, the term “neoantigen vaccine” refers to a pooled sample of one or more immunogenic neoantigen peptides or mRNAs, for example at least two, at least three, at least four, at least five, or more neoantigen peptides. The term "vaccine" refers to a composition for generating immunity for the prophylaxis and / or treatment of a disease (e.g., a neoplastic disorder, e.g., a hematological malignancy or solid tumor). Accordingly, vaccines are medicaments which comprise immunogenic agents and are intended to be used in humans or animals for generating specific immune defenses and protective substances after vaccination. A neoantigen vaccine can additionally include a pharmaceutically acceptable carrier, diluent, excipient, and / or adjuvant.

[536] As used herein, the term “immunogenic” refers to any agent or composition that can elicit an immune response, e.g., a T-cell response. The immune response can be antibody- or cell-mediated, or both.

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

[538] In various embodiments, a suitable neoantigen for a vaccine can be identified by screening a panel of transcripts with altered splicing and robust expression from one or more tissue samples in a patient (e.g., from a tumor biopsy). In some embodiments, variant protein sequences are identified in the screened sample based on translation across the aberrantly spliced mRNA junction while retaining portions of the protein sequence (up to 12 amino acids) flanking the junction-spanning amino acid changes. In some embodiments, these junctionspanning peptide fragments are scanned for high affinity binding to MHC1 alleles, e.g., using a tool such as NetMHCI (Nielsen et al. (2003) Protein Sci 12(5): 1007-17; Andreatta and Neilsen (2016) Bioinformatics 32(4):511-7). These results allow for filtering of the neopeptides to those 172 that are predicted high affinity binders for a unique patient HLA allele makeup as well as assembly of pools of neopeptides predicted to be broadly binding to HLA alleles that are present with high frequencies in different populations (Maiers et al. (2007) Hum Immunol 68(9):779-88). In various embodiments, the identified neopeptides are then formulated as a vaccine, e.g., by conjugation to a suitable carrier or adjuvant (Ott et al. (2017) Nature 547(7662):217-21), or for delivery as an mRNA (Sahin et al. (2017) Nature 547(7662):222-6).

[539] In some embodiments, the selected neoantigen is based on a screen of an individual patent’s tumor response to the herboxidiene splicing modulator, ADC, or composition to identify one or more neoantigens resulting from treatment to use in subsequent vaccination. In other embodiments, a neoantigen is chosen, e.g., based on screening a panel of samples from different patients to identify common neoantigens produced by the herboxidiene splicing modulator, ADC, or composition and then used as a universal vaccine for future patients.

[540] Without being bound by theory, in some embodiments, use of a universal neoantigen vaccine would avoid the need to sequence and analyze the unique mutation status of each patient’s tumor because the chosen neoantigens are not dependent on tumor mutation but rather mimic a neoantigen produced by a herboxidiene splicing modulator, ADC, or composition and generally recognized by the body as foreign. In addition, in some embodiments, use of a neoantigen vaccine may be particularly effective since a patient’s tumor cells may be more likely to mutate away from producing one or more neoantigens that are dependent on tumor mutation, as compared to those that mimic a neoantigen produced by a herboxidiene splicing modulator, ADC, or composition. This may allow for the formulation of a bulk vaccine that would be broadly immunogenic across a large percentage of patients, expediting the initiation of a treatment regime. Patients may be vaccinated according to the schedules outlined herein and, prior to following completion of the vaccination, could be further treated with a herboxidiene splicing modulator, ADC, or composition, e.g., to induce expression of the neoantigen peptides. In some embodiments, patients may be administered a herboxidiene splicing modulator, ADC, or composition before, at the same time as, or after vaccination. In some embodiments, patients are administered a herboxidiene splicing modulator, ADC, or composition, screened for one or more neoantigens found in a panel of universal neoantigens, and vaccinated with a universal neoantigen vaccine comprising at least one universal neoantigen identified in the subject. In some embodiments, patients may be administered a herboxidiene splicing modulator, ADC, or composition once or more than once after vaccination. Herboxidiene splicing modulator or ADC or composition and / or vaccine may be administered once or more than once during the course of treatment.

[541] In various embodiments, a vaccine may comprise one or more than one neoantigen peptide or mRNA. In various embodiments, a vaccine may comprise one or more than one long neoantigen peptide. Such “long” neoantigen peptides, in various embodiments, undergo efficient internalization, processing, and cross-presentation in professional antigen-presenting 173 cells such as dendritic cells. Similarly, 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, a neoantigen peptide is extended to comprise the neoantigen peptide sequence itself in addition to flanking amino acid sequences. In various embodiments, the extended peptide sequence facilitates the uptake of protein by antigenpresenting cells, e.g., dendritic cells. In various embodiments, the extended peptide sequence enables efficient antigen presentation and T-cell priming in models with different HLA isotypes. In various embodiments, a longer neoantigen peptide and / or extended peptide sequence exhibits increased uptake by antigen-presenting cells (e.g., dendritic cells), increased antigen presentation, and / or increased T-cell priming, as compared to a shorter neoantigen peptide and / or shorter peptide sequence (e.g., a peptide sequence less than about 10 or less than about 5 amino acids in length). In some embodiments, a long neoantigen peptide ranges from about 5 to about 50 amino acids in length. In some embodiments, a long neoantigen peptide ranges from about 10 to about 50 amino acids in length. In some embodiments, the at least one neoantigen peptide ranges from about 10 to about 35 amino acids in length. In some embodiments, a long neoantigen peptide ranges from about 15 to about 25 amino acids in length.

[542] In some embodiments, the neoantigen sequence and / or antigenic portion ranges from about 10 to about 35 amino acids in length. In some embodiments, the neoantigen sequence and / or antigenic portion ranges from about 15 to about 25 amino acids in length. In some embodiments, the neoantigen sequence and / or antigenic portion ranges from about 10 to about 20 amino acids in length. In some embodiments, the neoantigen sequence and / or antigenic portion does not exclusively overlap or consist of the canonical peptide sequence (e.g., any of the exemplary canonical peptide sequences underlined in Table 8).

[543] Amino acid sequences of exemplary long neoantigen peptides are set forth in Tables 8.

[544] These exemplary neoantigen peptides are generated after administration of ADCs containing pladienolide splicing modulators, however, given the similar mechanism of action (i.e., similar mechanisms of splicing modulation), similar neoantigen peptides may be produced by herboxidiene splicing modulators. Table 7. Neopeptides Neopeptide SEQ ID NO Junction (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 11 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:+ NFYC 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 Alt 3’ss 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 can be extended. The extended protein sequence incorporates both the neopeptide sequence itself in addition to flanking amino acid sequences. The extended protein sequence better facilitates the uptake of 175 protein by dendritic cells and enables antigen presentation and T-cell priming in models with different HLA isotypes. Amino acid sequences of the twenty nine extended neopeptides are set forth in Table 8. Table 8. Amino acid sequences of extended neopeptides Gene SEQ ID NO Extended neopeptide amino acid sequence* TUBA1C 66 VDLEPTVIGELTSVTQVRSQGAGTGGLSWGGSAGHSPTLPPRSL SLLLLPHHVLQMKFALALTASSSTLSNSSQARKMLPITMPEGTT PLARRSLTSCWTEFASWLTSAPVFRASWFSTALVGELVLGSPRC SWNVSQLIMARSPSWSSPFTRRPRFPQL PPHLN1 67 APPRSHPSIKRGLSSL CFL2 68 MVRRARWPGGRGEARKAPRTAPGVRPPF WAC 69 WVNCLFVSGRAAAGGGGGGAVPPYLELAGPPFLLLTLIRIGLGR RSGRAGGRAGTQCGGERGPGFAAFRPLRPFRRLRVCAVCVRGSA LGRSVGLPRGGAAGAPFSSSPAPHPRRVLCRCLLFLFFSCHDRR GDSQPYQVPAEAGVEGLEGAGGGREGLLLERRPQKSIQALRCNT SETSTADPLKIPGLVPLALSSKV GPS1 70 MPLPVQVFNLQVTSRGRPGPPRPRAPRHWGRAEVEQGRGACARS RSGTLRAGPPRAARVGGCRAEGASPPWLRAAIGGRRAAPAPPPL PAAHGRGSRPPRR EGFR 71 QPAQPRTGAPARRPRPRPSFPVSLRSAAPPTGTAGGTGRFVLRP GE SGAGGGGDAWDTGLQARRGTAAGTSGAPNRSQLS SLT FPAQL RRIGVSGRKPGAGGRLGPGSRTCAPRCLPRARRGPGAHPRGGRC PPAETALFREAEEGTQKYSLPSDPAGQAAF MED19 72 FRLHTGPVSPVGGRRQMGRPKHGDGFSLQVCSFIMEQNG HSBP1 73 GVVEITGEPPCSCRGEEEASRAGRAGGVRLKRGSRGPGELNVGP APGKTGLLIPLLRNWECGSLLRALSAL SCP2 74 KMGFPEAARKGNSL SMC5 75 LEARIKEKIE ELQQALI DNAJC8 76 EIKKRFRQFKQAVYKQ TMEM123 77 AHESAAMAETLQHVPS CHTOP 78 NRPSVQAALKLKQVGV MRPS31 79 KTDDLKKRHIT FTLGCGIC NFYC 80 MKLDEDVKRNDIAMAI TAB2 81 NSISQIPSDHILT PAL FITFMTIL DL SDHAF2 82 TVFSTSSLKLNQPQKYLKMKSWPC HSPA9 83 AEEDRRKKVITSCLLNFNLSKAQS MDH2 84 RSFSTSAQVGQTRGGLQAEAPRPGPRASPVRGQL MRPS15 85 RGYVVRKPVIALSVKI VARS 86 VDMDFGTGGQGAGPVGRGKDWSCTLAVHLLSEKKKISFSQIDRA WGGSQGTVLDKWGPGVVSELHPSAKEVSVGRNSVESLMTWAS HMG20B 87 EKGSHEEEVRVPALSWGRPRAPAPASKPRPRLDLNCLWLRPQPI FLWKLRPRPVPAATPLTGPLPL PDCD5 88 RYGQLSEKFNRRKVMDS SELK 89 MVYISNVSKLCFSKM TMSB10 90 NTLPTKETPSFLLNPHTSWVPRPHREAPRLRVGVAAPLQRPLPA LHSH CSNK1D 91 FGDIYLGEAPPPPPAARRPGPCGCQDQARSRKEVVAPAGSPRKS RHRRIVARTQRPLG TAOK1 92 GSASDLLEEISKQEIS F ADD1 93 QLIYNHITVKINLQGD * Underline indicates amino acids derived from the canonical transcript reading open frame (i.e., the canonical peptide sequence).

[546] As used herein, a neoantigen peptide or mRNA vaccine encompasses using a fragment of a neoantigen peptide or its encoding mRNA, so long as that fragment retains immunogenic potential.

[547] In some embodiments, a neoantigen vaccine comprises at least one neoantigen peptide. In some embodiments, a neoantigen vaccine comprises 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 neoantigen peptides. In some embodiments, the neoantigen peptide(s) range from about 5 to about 50 amino acids in length. In some embodiments, the neoantigen peptide(s) range from about 10 to about 50 amino acids in length. In some embodiments, the at least one neoantigen peptide ranges from about 10 to about 35 amino acids in length. In some embodiments, the neoantigen peptide(s) range from about 15 to about 25 amino acids in length.

[548] In various embodiments, the present disclosure provides a method of treating a subject having or suspected of having a neoplastic disorder by administering to the subject an effective amount of a herboxidiene splicing modulator, an ADC, or a composition comprising a herboxidiene splicing modulator or ADC; and a neoantigen vaccine. A neoantigen vaccine may be, e.g., a peptide or mRNA neoantigen vaccine. In some embodiments, the herboxidiene splicing modulator, ADC, or composition is administered before administration of the neoantigen vaccine. In some embodiments, the herboxidiene splicing modulator, ADC, or composition is administered after administration of the neoantigen vaccine. In some embodiments, the herboxidiene splicing modulator, ADC, or composition is administered concurrently with administration of the neoantigen vaccine. In some embodiments, administration of the herboxidiene splicing modulator, ADC, or composition is repeated at least once after initial administration. In some embodiments, the amount of the herboxidiene splicing modulator, ADC, or composition used for repeated administration is reduced as compared to the amount used for initial administration.

[549] In various embodiments, the present disclosure further provides a combination comprising a herboxidiene splicing modulator, an ADC, or a composition comprising a herboxidiene splicing modulator or ADC; and a neoantigen vaccine (e.g., a universal neoantigen vaccine) for use in treating a subject having or suspected of having a neoplastic disorder. In some embodiments, the neoantigen vaccine is a peptide or mRNA neoantigen 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 disclosure further provides a method of treating a subject having or suspected of having a neoplastic disorder by (a) administering to the subject an effective amount of a herboxidiene splicing modulator, an ADC, or a composition comprising a herboxidiene splicing modulator or ADC; (b) detecting one or more neoantigens in the subject after administration of the herboxidiene splicing modulator, ADC, or composition; (c) comparing the 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, the universal 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 repeated administration of the herboxidiene splicing modulator, ADC, or composition. In some embodiments, repeated administration of the herboxidiene splicing modulator, ADC, or composition is initiated before administration of the universal neoantigen vaccine. In some embodiments, repeated of the herboxidiene splicing modulator, ADC, or composition is initiated after administration of the universal neoantigen vaccine. In some embodiments, repeated administration of the herboxidiene splicing modulator, ADC, or composition is initiated concurrently with administration of the universal neoantigen vaccine. In some embodiments, the amount of the herboxidiene splicing modulator, ADC, or composition used for repeated administration is reduced as compared to the amount used for initial administration. In some embodiments, the amount of the herboxidiene splicing modulator, ADC, or composition used for the initial and / or repeated administration is reduced as compared to a standard dosage of the herboxidiene splicing modulator, ADC, or composition when used without a vaccine treatment. In some embodiments, the amount of the herboxidiene splicing modulator, ADC, or composition used for initial and / or repeated administration is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 178 40%, 45%, 50%, 75%, or 90%, as compared to a standard dosage of the herboxidiene splicing modulator, ADC, or composition.

[552] In some embodiments, the at least one additional therapy comprises administering a checkpoint inhibitor (e.g., any of the exemplary checkpoint inhibitors described herein). In some embodiments, administration of the checkpoint inhibitor is initiated before administration of the universal neoantigen vaccine and / or repeated administration of the herboxidiene splicing modulator, ADC, or composition. In some embodiments, administration of the checkpoint inhibitor is initiated after administration of the universal neoantigen vaccine and / or repeated of the herboxidiene splicing modulator, ADC, or composition. In some embodiments, administration of the checkpoint inhibitor is initiated concurrently with administration of the universal neoantigen vaccine and / or repeated administration of the herboxidiene splicing modulator, ADC, or composition. In some embodiments, administration of the checkpoint inhibitor is repeated at least once after initial administration. In some embodiments, the amount of the checkpoint inhibitor used for repeated administration is reduced as compared to the amount used for initial administration. In some embodiments, the amount of the checkpoint inhibitor used for repeated administration is reduced as compared to a standard dosage of the checkpoint inhibitor. In some embodiments, the amount of the checkpoint inhibitor used for repeated administration is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90%, as compared to a standard dosage of the checkpoint inhibitor. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to the checkpoint inhibitor when administered alone.

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

[554] Also provided herein, in various embodiments, are kits comprising a herboxidiene splicing modulator, an ADC, or a composition comprising a herboxidiene splicing modulator or ADC; and a neoantigen vaccine (e.g., a universal neoantigen vaccine). In some embodiments, the neoantigen vaccine is a peptide or mRNA neoantigen vaccine. In some embodiments, the kit further comprises one or more additional components, including but not limited to: instructions for use; other agents, e.g., one or more additional therapeutic agents; devices, containers, or other materials for preparing the herboxidiene splicing modulator, ADC, composition, and / or neoantigen vaccine for therapeutic administration; pharmaceutically acceptable carriers; and devices, containers, or other materials for administering the herboxidiene splicing modulator, ADC, composition, and / or neoantigen vaccine to a patient. Instructions for use can include guidance for therapeutic applications including suggested dosages and / or modes of administration, e.g., in a patient having or suspected of having a neoplastic disorder. In various embodiments, the kit further contains instructions for therapeutic 179 use, e.g., use of the herboxidiene splicing modulator, ADC, or composition, and the 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 (e.g., for administering together with the herboxidiene splicing modulator, ADC, or composition, and the neoantigen vaccine, e.g., a checkpoint inhibitor). In various embodiments, the herboxidiene splicing modulator, ADC, composition, and / or neoantigen vaccine is formulated as a pharmaceutical composition.

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

[556] In some embodiments, the at least one neoantigen peptide comprises one or more than one neoantigen sequence disclosed herein.

[557] In some embodiments, the neoantigen sequence and / or antigenic portion ranges from about 10 to about 35 amino acids in length. In some embodiments, the neoantigen sequence and / or antigenic portion ranges from about 15 to about 25 amino acids in length. In some embodiments, the neoantigen sequence and / or antigenic portion ranges from about 10 to about 20 amino acids in length. In some embodiments, the neoantigen sequence and / or antigenic portion does not exclusively overlap or consist of the canonical peptide sequence (e.g., any of the exemplary canonical peptide sequences underlined in Table 8).

[558] In some embodiments, the neoantigen sequence is a neoantigen sequence specific to the subject. 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 universal neoantigen sequence. In some embodiments, the neoantigen sequence is a universal 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 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from the neoplastic disorder. In some embodiments, the neoantigen sequence 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.

[560] In some embodiments, the neoantigen sequence has been identified by sequencing at least one neoantigen peptide induced in the subject by administering an effective amount of the herboxidiene splicing modulator, antibody-drug conjugate, or composition. In some embodiments, the at least one neoantigen peptide comprises a neoantigen sequence induced by contacting a neoplastic cell with an effective amount of the herboxidiene splicing modulator, 180 antibody-drug conjugate, or composition. In some embodiments, the neoplastic cell is present in an in vitro cell culture. In some embodiments, the neoplastic cell is obtained from the subject. In some embodiments, the neoplastic cell is present in the subject.

[561] In some embodiments, the neoantigen vaccine comprises at least one neoantigen peptide or mRNA and a pharmaceutically acceptable carrier. In various embodiments, a neoantigen peptide or mRNA can be linked to a suitable carrier to help elicit an immune response. Exemplary carriers for linking to immunogenic agents (e.g., a neoantigen peptide or mRNA) include serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, or a toxoid from other pathogenic bacteria, such as diphtheria, E. coli, cholera, or H. pylori, or an attenuated toxin derivative. Other carriers for stimulating or enhancing an immune response include cytokines such as IL-1, IL-1 a and p peptides, IL-2, ylNF, IL-10, GM-CSF, and chemokines, such as M1P1a and p and RANTES. Immunogenic agents can also be linked to peptides that enhance transport across tissues, as described, e.g., in WO 97 / 17613 and WO 97 / 17614. In some embodiments, the pharmaceutically acceptable carrier is selected from a peptide, a serum albumin, a keyhole limpet hemocyanin, an immunoglobulin, a thyroglobulin, an ovalbumin, a toxoid or an attenuated toxoid derivative, a cytokine, and a chemokine.

[562] In some embodiments, the neoantigen peptide or mRNA may be linked to the pharmaceutically acceptable carrier. Immunogenic agents can be linked to carriers by chemical crosslinking. Techniques for linking an immunogenic peptide to a carrier include the formation of disulfide linkages using N-succinimidyl-3-(2-pyridyl-thio) propionate (SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (if the peptide lacks a sulfhydryl group, this can be provided by addition of a cysteine residue). These reagents create a disulfide linkage between themselves and peptide cysteine resides on one protein and an amide linkage through the epsilon-amino on a lysine, or other free amino group in other amino acids. A variety of such disulfide / amide-forming agents are described in Jansen et al. ((1982) Immun Rev. 62:185). Other bifunctional coupling agents form a thioether rather than a disulfide linkage. Many of these thioether-forming agents are commercially available and include reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, and 2-iodoacetic acid, 4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid. The carboxyl groups can be activated by combining them with succinimide or 1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt. In some embodiments, the neoantigen peptide and the pharmaceutically acceptable carrier are covalently attached via a linker.

[563] Neoantigen and other such immunogenic peptides can also be expressed as fusion proteins with carriers. The immunogenic peptide can be linked at the amino terminus, the carboxyl terminus, or at a site anywhere within the peptide (internally) to the carrier. In some embodiments, multiple repeats of the immunogenic peptide can 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 comprises at least one neoantigen peptide or its encoding mRNA and a pharmaceutically acceptable diluent. In some embodiments, the neoantigen vaccine comprises at least one neoantigen peptide or its encoding mRNA and a pharmaceutically acceptable adjuvant (e.g., an adjuvant as 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 than one neoantigen sequence.

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

[567] In some embodiments, the neoantigen sequence is a neoantigen sequence specific to the subject. 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 universal neoantigen sequence. In some embodiments, the neoantigen sequence is a universal 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 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from the neoplastic disorder. In some embodiments, the neoantigen sequence 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.

[569] In some embodiments, the neoantigen sequence has been identified by sequencing at least one neoantigen mRNA induced in the subject by administering an effective amount of the herboxidiene splicing modulator, antibody-drug conjugate, or composition. In some embodiments, the at least one neoantigen mRNA encodes a neoantigen sequence induced by contacting a neoplastic cell with an effective amount of the herboxidiene splicing modulator, antibody-drug conjugate, or composition. In some embodiments, the neoplastic cell is present in an in vitro cell culture. In some embodiments, the neoplastic cell is obtained from the subject. In some embodiments, the neoplastic cell is present in 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 linked to the pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from a peptide, a serum albumin, a keyhole limpet hemocyanin, an immunoglobulin, a thyroglobulin, an ovalbumin, a toxoid or an attenuated toxoid derivative, a cytokine, and a 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 (e.g., an adjuvant as described herein).

[572] In some embodiments, the neoantigen mRNA is encapsulated by 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-trimethyl ammonium chloride 1 (DOTAP). In some embodiments, the encapsulating agent is a nanoparticle. In some embodiments, the nanoparticle protects the neoantigen mRNA from nuclease degradation and / or enhances cell uptake and / or delivery efficiency. In some embodiments, the nanoparticle may be engineered to be fully degradable. In some embodiments, the nanoparticle is a biodegradable core-shell structured nanoparticle with a pH responsive poly-(b-amino ester) (PBAE) core enveloped by a phospholipid shell (Su et al. (2011) Mol Pharm. 8(3):774-87). In some embodiments, such nanoparticles are particularly efficient in delivering mRNA in vivo and eliciting an anti-tumor immune response.

[573] In some embodiments, the subject has a non-synonymous mutational burden of about 150 mutations or less. In some embodiments, the subject has a non-synonymous mutational burden of about 100 mutations or less. In some embodiments, the subject has a non-synonymous mutational burden of about 50 mutations or less. In some embodiments, the subject has or is suspected of having a neoplastic disorder, e.g., a hematological malignancy or a solid tumor. In some embodiments, the hematological malignancy is selected from a B-cell malignancy, a leukemia, a lymphoma, and a myeloma. In some embodiments, the hematological 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, 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, “adjuvant” refers to a substance that is capable of increasing, amplifying, or modulating an immune response to an accompanying immunogenic agent, e.g., a neoantigen peptide or mRNA. In certain embodiments, a neoantigen of the present disclosure can be administered in combination with adjuvants, i.e., substances that do not themselves cause adaptive immune responses, but amplify or modulate the response to an accompanying neoantigen. A variety of adjuvants can be used in combination with the disclosed neoantigens, in order to elicit an immune response. In some embodiments, the adjuvant(s) are chosen to augment 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 chosen to enhance T-effector (e.g., CD8) cell priming and / or activation.

[575] In certain embodiments, the adjuvant is an aluminum salt (alum), such as aluminum hydroxide, aluminum phosphate, and aluminum sulphate. Such adjuvants can be used with or without other specific immunostimulating agents, such as 3 de-O-acylated monophosphoryl lipid A (MPL) or 3-DMP, polymeric or monomeric amino acids, such as polyglutamic acid or polylysine. Such adjuvants can be used with or without other specific immunostimulating agents, such as muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(T-2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), N-acetylglucsaminyl-N-acetylmuramyl-L-AI-D-isoglu-L-Ala-dipalmitoxy propylamide (DTP-DPP)), or other bacterial cell wall components. 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 various amounts of MTP-PE) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics), (b) SAF, containing 10% Squalene, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi™ adjuvant system (RAS), (Ribi ImmunoChem) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphoryllipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), for example MPL-FCWS (Detox™). In some embodiments, the adjuvant is a saponin, such as Stimulon™ (QS21) or particles generated therefrom such as ISCOMs (immunostimulating 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] An adjuvant can be administered with an immunogenic agent (e.g., a neoantigen peptide or mRNA) as a single composition, or can be administered before, concurrent with, or after administration of the immunogenic agent. In some embodiments, the immunogenic agent and adjuvant can be packaged and supplied in the same vial or can be packaged in separate vials and mixed before use. In some embodiments, the immunogenic agent and adjuvant can be packaged with a label, indicating the intended therapeutic application. In some embodiments, if the immunogenic agent and adjuvant are packaged separately, the packaging can include instructions for mixing before use. The choice of an adjuvant and / or carrier depends on the stability of the immunogenic formulation containing the adjuvant, the route of administration, the dosing schedule, the efficacy of the adjuvant for the species being vaccinated, and, in humans, a pharmaceutically acceptable adjuvant is one that has been approved or is approvable for human administration by pertinent regulatory bodies. For example, Complete Freund's adjuvant is not suitable for human administration. However, alum, MPL or Incomplete Freund's 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 human administration.

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

[578] In various other embodiments, the present disclosure provides a method of identifying at least one neoantigen by (a) contacting a neoplastic cell with an effective amount of a herboxidiene splicing modulator, an ADC, or a composition comprising a herboxidiene splicing modulator or ADC; (b) detecting at least one peptide comprising a potential neoantigen sequence after contacting the neoplastic cell with the herboxidiene splicing modulator, ADC, or composition; and (c) comparing the at least one peptide to a reference proteome, wherein at least one neoantigen is identified if the at least one peptide does not match any peptides in the reference proteome. In various embodiments, the method further comprises contacting one or more additional neoplastic cells to identify at least one universal neoantigen. In various embodiments, the method is repeated on one or more additional neoplastic cells or samples (e.g., a tissue biopsy) to confirm suitable neoantigens (e.g., 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, detecting at least one alternatively-spliced mRNA transcript comprises RNAseq. In some embodiments, predicting translation of the at least one alternatively-spliced mRNA transcript comprises quantifying the change in percent spliced in (dPSI) value for the at least one transcript. In some embodiments, predicting translation of the at least one alternatively-spliced mRNA transcript comprises RiboSeq and / or ribosomal profiling.

[580] In some embodiments of the neoantigen identification methods described herein, the methods further comprise evaluating the at least one peptide for predicted major histocompatibility complex (MHC) binding. In some embodiments, predicted MHC binding is determined by measuring raw affinity predicted binding strength of the at least one peptide. In some embodiments, a raw affinity predicted binding strength of about 500 nM or higher indicates MHC binding. In some embodiments, predicted MHC binding is determined by identifying a distribution of predicted binding strengths for a series of random peptides; and comparing predicted binding strength of the at least one peptide to the distribution. In some embodiments, a predicted binding strength in the top 2% of the distribution indicates weak MHC binding. In some embodiments, a predicted binding strength in the top 0.5% of the distribution indicates strong MHC binding.

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

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

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

[584] In some embodiments, the at least one neoantigen used in the vaccine is linked to the pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from a peptide, a serum albumin, a keyhole limpet hemocyanin, an immunoglobulin, a thyroglobulin, an ovalbumin, a toxoid or an attenuated toxoid derivative, a cytokine, and a chemokine. Combination of Herboxidiene Splicing Modulator / ADC and Engineered T-Cells (CAR-T):

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

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

[587] In various other embodiments, a strategy integrating the antigen presentation machinery of tumor cells together with herboxidiene splicing modulator- or ADC-derived neoantigens is employed. In some embodiments, cells containing known and frequently represented HLA alleles (e.g., HLA-A*02:01) can be treated with a herboxidiene splicing modulator, ADC, or composition and MHC1-bound neoantigens are identified by ligandomics. In some embodiments, these peptides can be used to prime and / or expand T-cells from healthy donors expressing the same HLA allele. Such T-cells, in some embodiments, can be isolated and the T-cell receptor (TCR) a and p chains sequenced to identify the cognate antigen recognition / variable regions. In some embodiments, a cognate CAR can then be engineered.

[588] In some embodiments, the CAR sequences are cloned into patient-derived T-cell populations and expanded using currently available protocols. In some embodiments, the engineered T-cells are then transfused back into the patient’s circulation, following treatment with a herboxidiene splicing modulator, ADC, or composition. After treatment with the herboxidiene splicing modulator, ADC, or composition, in some embodiments, the tumor cells may begin to present antigen. In some embodiments, the engineered T-cell population can engage with and kill antigen presenting tumor cells.

[589] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the disclosure described herein are obvious and may be made using suitable equivalents without departing from the scope of the disclosure or the embodiments disclosed herein. Having now described the disclosure in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting. EXAMPLES EXAMPLE 1

[589] Synthesis methods for payloads, linkers, and conjugatable linker-payload (linker-drug, L-H) compounds, having the structures shown in Tables 9-11, are described. Conjugatable linkerpayloads were 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 synthesis methods are either commercially available or can be readily prepared by standard methods from known materials. The disclosed conjugatable linker-payloads can be prepared using the reactions and techniques described herein. In the description of the synthetic methods described below, it is to be understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment, and workup procedures, can be chosen to be the conditions standard for that reaction, unless otherwise indicated. It is understood by one skilled in the art of organic synthesis that the functionality present on various portions of the molecule should be compatible with the reagents and reactions proposed. Substituents not compatible with the reaction conditions are apparent to one skilled in the art, and alternate methods are therefore indicated herein.

[591] Preparative liquid chromatography-mass spectrometry (LC / MS) was conducted using a Waters AutoPurification System and an XTerra MS C18 column (5 pm, 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. Column chromatography was carried out using a Teledyne Isco Combiflash Rf200d. Solvent removal was carried out using either a Buchi rotary evaporator or a Genevac centrifugal evaporator.

[592] Terms / Abbreviations: As used herein, the term “inerted” refers to replacement of the air in a reactor (e.g., a reaction vessel, a flask, a glass reactor) with an essentially moisture-free, inert gas, such as nitrogen or argon. The following abbreviations are used herein: DCM=dichloromethane, DMF= dimethylformamide, HPLC=high performance liquid chromatography, KHMDS=potassium bis(trimethylsilyl)amide, LC / MS=liquid chromatographymass spectrometry, MeOH=methanol, RT=room temperature, TBSCI=tert-butyldimethylsilyl chloride, THF=tetrahydrofuran, TLC=thin-layer chromatography. Multiplicities are 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=a broad singlet.

[593] LC / MS: Mobile phases=A (0.1% formic acid in H2O) and B (0.1% formic acid in acetonitrile). Gradient=B 5% to 95% in 1.8 min. Column= Waters Acquity BEH C18 column (1.7 pm, 2.1 x 50 mm). Table 9. Structures of exemplary drug moieties (payloads) Payload Structure I ID H H1 H2 H3 H4 H5 H6 Table 10. Structures of exemplary linkers Linker Structure I ID (IUPAC Name) O ADL1 I “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) ADL5 I “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 I “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 I “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) O ADL12 / “Mal-Hex” (1-hexyl-2,5-dihydro-1H-pyrrole-2,5-dione) oh o ADL13 / “MC-p-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) O 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 conjugatable linker-payload (L-H) compounds CL NH2 NH ADL1-H3 ADL1-H4 °^NH2 NH ADL1-H5 clnh2 NH ADL1-H6 O^NH2 NH ADL1-H7 O.NH2 NH ADL1-H8 NH ADL1-H9 °^NH2 NH ADL1-H10 O ADL2-H11 ADL2-H1 1.2 Preparation of ADL1-H1, ADL1-H2, ADL1-H3, and ADL1-H4 1.2.1 Overview - General procedure 1 Scheme 1

[594] Step 1: Fermentation and Bioconversion

[595] Twenty percent glycerol stock solution of Saccharothrix sp. EAS-AB4564, isolated from soil in Japan, was inoculated into a first seed culture in a test tube containing 10 mL of SY-32 medium (1% D-glucose, 1% starch soluble, 0.5% bactosoytone, 0.5% yeast extract, 0.2% ammonium sulfate, 0.2% NaCI and 2.3% TES, at pH 8.0). The first seed culture 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 Erlenmeyer flasks with 1% v / v, each containing 100 mL of new SY-32 medium. The second seed 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 antifoam PE-M. The fermentation was conducted at 28°C under the condition of stirring and aeration (450 rpm, 10 L / min). After 48 hours, the culture broth was centrifuged at 3000 rpm for 10 minutes. After removal of supernatant, 10 L of 20 mM phosphate buffer (pH 7.0) was added to microbial pellet for the washing of microbial cells. The suspension was centrifuged at 3000 rpm for 10 minutes. After removal of wash buffer, 10 L of reaction buffer (1% D-glucose, 0.2% magnesium chloride hexahydrate, 2.3% TES and 1.3 g of Herboxidiene, pH 8.0) was added to microbial pellet, and the suspension was transferred into a 15 L fermenter. The bio-conversion was conducted for 6 hours at 28°C while stirring and aerating (450 rpm, 10 L / min).

[596] Isolation of 5-hydroxy herboxidiene

[597] XAD-7HP (400 g) was added to the above-mentioned mixture (10 L), and the mixture was stirred (EYELA MAZELA Z) for 30 minutes at 300 rpm. After the agitation, separation of XAD-7HP was determined using staining and a testing sieve (0.25 mm aperture and 0.16 mm wire diameter). The same operation was repeated. The collected XAD-7HP was extracted by 1 L of acetone, and the solvent was removed in vacuo. The extract was purified by reverse phase MPLC (Yamazen EPCLC-W-prep 2XY) with gradient elution (YMC-DispoPack AT ODS-25 120 g, 25 to 55% acetonitrile in water added with 0.1% formic acid, over 15 minutes) to afford 5-OH herboxidiene (397.47 mg), with a 29.5% conversion yield. 1H-NMR and mass spectrometric data were consistent with the literature. See, e.g., 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 OH

[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 was added trimethylsilyldiazomethane (2.0 M in hexanes, 0.148 mL, 0.297 mmol) dropwise. The resulting mixture was then gradually warmed to room temperature and was stirred for 1 hr 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 afford 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] 1H 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: Carbamate Synthesis

[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., .075 mmol), 4-nitrophenyl carbonochloridate (2 eq.), Hunig’s base (0.065 mL, 4.5 eq.) in dichloromethane (0.04 M) at 0 °C was added DMAP (0.05 eq.). The mixture was then warmed to room temperature and stirred for 16 hr. Piperazine (10 eq.) was added to the mixture and resulting mixture was stirred for an additional 1 hr. The mixture was then diluted with DCM, 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 and then by aminofunctionalized silica gel chromatography to afford the desired product.

[603] Step 4: Carboxylic Acid Synthesis

[604] To a mixture of the methyl ester obtained from Step 3 (1.0 eq., 0.039 mmol) in MeOH (0.02M ml) was added aqueous sodium hydroxide (1.0 eq., 2N). The mixture was warmed to 40°C and was stirred at that temperature for 4 hr. The resulting mixture was then cooled to 0°C and neutralized with aqueous hydrochloric acid (200 pL, 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 afford the desired product. 1.2.2 Synthesis of H1

[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-yl piperazine-1 -carboxylate

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

[607] 1H NMR (400 MHz, CHLOROFORM-d) 6 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- ((piperazine-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 afford a colorless oil (16.0 mg, 73% yield). LC / MS (ESI, m / z), 567.77 [M+H]+.

[610] 1H NMR (400 MHz, METHANOL-d4) 6 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 Synthesis of H2

[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-di azepane-1 -carboxylate

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

[613] 1H NMR (500 MHz, CHLOROFORM-d) 6 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) 1.321.40 (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-diazepane-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 afford a colorless oil (20.9 mg, 71% yield). LC / MS (ESI, m / z), 581.81 [M+H]+.

[616] 1H NMR (400 MHz, METHANOL-d4) 6 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 Synthesis of H3

[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 HN— OMe OH

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

[619] 1H NMR (500 MHz, CHLOROFORM-d) 6 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) 1.491.68 (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)pyrrolidine-1-carbonyl)oxy)tetrahydro-2H-pyran-2-yl)acetic acid (H3) HN 0

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

[622] 1H NMR (400 MHz, METHANOL-d4) 6 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).

[623] The title compound was synthesized according to Step 3 (13.8 mg, 91% yield) and Step 4 of section 1.2.1 to afford a white armophous solid (4.58 mg, 76% yield). LC / MS (ESI, m / z), 581.55 [M+H]+.

[624] 1H NMR (400 MHz, CHLOROFORM-d) 6 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 (brd, J=8.29 Hz, 1 H) 2.30 (s, 4 H) 2.332.51 (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 Synthesis of H4

[625] H4 was synthesized according to Scheme 2: Step 1 DIPEA DMF, rt Step 2 OH OH OH Oh o cr OH Step 4 DCM, DIPEA, DCM then piperazine (2) 2N NaOH 40 °c, MeOH Step 5 Scheme 2 HO ch2n2 o -«------ (1)O2N DCC, DCM 0 °C - RT THF, Methanol 0 °C - RT Step 3 OH

[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. Then, N-hydroxysuccinamide (173 mg, 1.505 mmol) was added to the mixture. The resulting mixtuxe was warmed to room temperature and stirred for 16 hours. Subsequently, the mixture was filtered through celite and the filter cake was 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 afford the title compound as a coloroless solid. (357 mg, 97% yield).

[628] 1H NMR (500 MHz, CHLOROFORM-d) 5 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, 3H) 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 residue was used in Step 3 without further purification.

[631] 1H NMR (400 MHz, CHLOROFORM-d) 5 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 from 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. Subsequently, the mixture was cooled to 0°C and quenched by addition of acetic acid. The resulting mixture was stirred for 30 minutes and was then concentrated in vacuo. The resulting residue was purified by silica gel chromatography (12 g, Heptane / AcOEt = 90 / 10 to 30 / 70) to afford the title compound (15.7 mg, 44% yield).

[634] 1H NMR (400 MHz, CHLOROFORM-d) 5 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] Step 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-oxopentyl piperazine-1-carboxylate

[636] To a mixture of the compound produced 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 room temperature was added DMAP (1.689 mg, 0.014 mmol). The resulting mixture was stirred at room temperature for 16 hr. Subsequently, piperazine (23.82 mg, 0.277 mmol) was added and the mixture was stirred for another 1 hr. Then, the mixture was diluted with DCM, and organic layer was isolated, washed with water and brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The crude residue was used in the next step without purification.

[637] Step 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 aqueous sodium hydroxide (2 N, 100 pl, 0.20 mmol). The mixture was warmed to 40°C and stirred for 2 hr. The mixture was then cooled to 0°C, and aqueous hydrochloric acid (100 pL, 2N) was added. The resulting mixture was then concentrated in vacuo and the resulting residue was purified by preparative HPLC (H2O / MeCN / NH3aq. = 60 / 40 / 0.1 to 30 / 70 / 0.1) to afford the title compound as a colorless oil. (7.0 mg, 48% yield over Steps 4 and 5). LC / MS (ESI, m / z), 666.90 [M+H]+.

[639] 1H NMR (400 MHz, METHANOL-d4) 5 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 Synthesis of MC-Val-Cit-pABC Linker-Payloads

[640] The general procedure for the synthesis of MC-Val-Cit-pABC linker-payloads is outlined in Scheme 3. H Scheme 3

[641] To a mixture of the payload (e.g., a compound synthesized according to the aforementioned 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) was added DI PEA (3.03 eq.) at room temp. The resulting mixture was stirred at room temperature for 16 hr, concentrated in vacuo, and the resulting residue was purified by reverse-phase preparative HPLC (H2O / MeCN / HCOOH = 60 / 40 / 0.1 to 40 / 60 / 0.1) to afford the desired compound. 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-1 H-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 outlined in section 1.2.7 and obtained as a colorless oil (12.7 mg, 39% yield).

[643] 1H NMR (400 MHz, METHANOL-d4) 5 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) 1.841.93 (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).

[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 outlined in section 1.2.7 and obtained as a colorless oil (2.4 mg, 44% yield).

[645] 1H NMR (400 MHz, METHANOL-d4) 5 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) 1.801.95 (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-diazepane-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 outlined in section 1.2.7 and obtained as a colorless oil (10.4 mg, 34% yield).

[647] 1H NMR (400 MHz, METHANOL-d4) 5 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) 1.191.31 (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) 1.831.93 (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-1 H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl) (methyl)amino)pyrrolidine-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 outlined in section 1.2.7 and obtained as a colorless oil (18.8 mg, 37% yield).

[649] 1H NMR (400 MHz, METHANOL-d4) 5 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 Overview - General Procedure 2 Step 3 Scheme 3

[650] Step 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 min, 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 afford the title compound as a colorless solid (632 mg, 85% yield).

[652] 1H NMR (400 MHz, CHLOROFORM-d) 5 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] Step 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 from 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 residue was combined with in 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 mother liquor was concentrated in vacuo and the isolated residue was purified by silica gel chromatography (40 g, Heptane / AcOEt = 70 / 30 to 0 / 100) to afford the title compound as a colorless solid (807 mg, quant.).

[655] 1H NMR (400 MHz, CHLOROFORM-d) 5 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) 1.171.20 (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 to it was added AcOEt and aqueous sodium bicarbonate (100 mL each). 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 (40g, Heptane / AcOEt = 90 / 10 to 50 / 50) to afford the title compound as a colorless oil (663 mg, 75% yield).

[658] 1H NMR (400 MHz, CHLOROFORM-d) 5 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 borane-dimethylamine complex (643 mg, 10.906 mmol). After the purging the vessel with nitrogen, tetrakis(triphenylphosphine) palladium(O) (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 puried by amino-functionalied silica gel chromatography (40g, Heptane / AcOEt = 70 / 30 to 0 / 100) to afford the title compound as a white solid (525 mg, 92% yield).

[661] 1H NMR (400 MHz, CHLOROFORM-d) 5 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 HATLI (145 mg, 0.381 mmol) and the mixture was then stirred at room temperature for 16 hours. Subsequently, AcOEt was added and the resulting mixture was washed with water and brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The obtained residue was combined with THF (2 mL) and cooled to 0 °C. Then, lithium borohydride (22.1 mg, 1.016 mmol) was added and the mixture was stirred at the same temperature for 30 minutes. Subsequently, the mixture was warmed to room temperature and stirred for an additional 1 hour. To the mixture was added saturated aqueous ammonium chloride, followed by AcOEt and water. The organic phase was isolated, washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue 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 afford the title compound as a colorless oil (113 mg, 85% yield).

[664] 1H NMR (400 MHz, CHLOROFORM-d) 5 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) 1.271.40 (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 Synthesis of Carbamate

[666] To a mixture of the compound isolated from Step 5 (28.3 mg, 0.054 mmol), 4-nitrophenyl chloroformate (2.0 eq.) and Hunig’s base (5 eq.) in DCM (0.05 M) was added DMAP (0.5 eq.). The resulting mixture was stirred at room temperature for 16 hours. Then, amine (2.0 eq.) was added, and the mixture was stirred for an additional 1 hour. Then, the mixture was 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 afford the desired compound. 1.3.2 Synthesis of ADL1-H5 1.3.2.1 Synthesis of H5

[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-oxohexyl piperazine-1 -carboxylate (H5)

[668] Steps 1-6 outlined in section 1.3.1 were employed to afford the title compound as a colorless oil (24.5 mg, 71% yield). LC / MS (ESI, m / z), 636.89 [M+H]+.

[669] 1H NMR (400 MHz, CHLOROFORM-d) 6 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 CL NH2 NH

[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 outlined in section 1.2.7 was employed to afford the title compound as a colorless oil (25.8 mg, 54% yield).

[672] 1H NMR (400 MHz, METHANOL-d4) 6 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) 217 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 Synthesis of H6

[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 outlined in section 1.3.1 were employed to afford the title compound as a colorless oil (15.3 mg, 43% yield). LC / MS (ESI, m / z), 650.92[M+H]+.

[675] 1H NMR (400 MHz, CHLOROFORM-d) 5 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 O NH2 NH

[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-di azepa ne-1,4-di carboxy I ate (ADL-H6)

[677] The procedure outlined in section 1.2.7 was employed to afford the title compound as a colorless oil (11.6 mg, 52% yield).

[678] 1H NMR (400 MHz, METHANOL-d4) 5 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 Synthesis of H7

[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 outlined in section 1.3.1 were employed to afford the title compound as a colorless oil (26.6 mg, 76% yield). LC / MS (ESI, m / z), 650.88[M+H]+.

[681] 1H NMR (400 MHz, CHLOROFORM-d) 5 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 O NH2 NH

[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-1 H-pyrrol-1 -yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl) (methyl)amino)pyrrolidine-1-carboxylate (ADL1-H7)

[683] The procedure outlined in section 1.2.7 was employed (the mixture was stirred for 16 hours instead of 2 hours) to afford the title compound as a colorless oil (20.1 mg, 54% yield).

[684] 1H NMR (400 MHz, METHANOL-d4) 6 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) 1.701.77 (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 Overview - General Procedure 4 Step 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 tertbutyl 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 was stirred at room temperature for 16 hours. Subsequently, saturated aqueous ammonium chloride was 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 afford the title compound (97 mg, 7% yield) as a colorless oil.

[687] 1H NMR (400 MHz, CHLOROFORM-d) 5 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 o JI ___ o

[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 afford the title compound as a colorless oil (58 mg, 100%).

[690] 1H NMR (400 MHz, CHLOROFORM-d) 5 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] Step 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 a mixture 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-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) was added HATLI (145 mg, 0.381 mmol) and DIPEA (221 pL, 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 down 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. Subsequently, 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 (1M in THF solution, 0.508 mL, 0.508 mmol) was added at 0°C. After stirring at 0C for 30 minutes, the mixture was warmed to room temperature and stirred for 2 hours. Subsequently, the mixture was 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 afford the title compound (68 mg, 51% yield) as a colorless oil.

[694] 1H NMR (400 MHz, CHLOROFORM-d) 5 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) 1.42 221 - 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 a mixture of 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-nitrophenyl chloroformate (2.0 eq., 0.086 mmol) and DI PEA (5.0 eq.) in DCM (0.04 M) was 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 an additional 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 furnish the desrired compound. 1.4.2 Synthesis of ADL1-H8 1.4.2.1 Synthesis of H8

[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)ethyl piperazine-1-carboxylate (H8)

[698] Steps 1-4 outlined in section 1.4.1 were employed to afford the title compound as a pale yellow oil (19.2 mg, 70% yield). LC / MS (ESI, m / z), 638.78[M+H]+.

[699] 1H NMR (400 MHz, CHLOROFORM-d) 5 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 Synthesis of H8

[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 procedure similar to the one outlined in section 1.2.7 was employed to afford the title compound as a colorless oil (18.3 mg, 60% yield).

[702] 1H NMR (400 MHz, METHANOL-d4) 6 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) 1.101.23 (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 Synthesis of H9

[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 outlined in section 1.4.1 were employed to afford the title compound as a colorless oil (21.1 mg, 75% yield). LC / MS (ESI, m / z), 652.86[M+H]+.

[705] 1H NMR (400 MHz, CHLOROFORM-d) 6 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-diazepane-1,4-di carboxy I ate (ADL1-H9)

[707] A procedure similar to the one outlined in section 1.2.7 was employed to afford the title compound (25.2 mg, 72% yield).

[708] 1H NMR (400 MHz, METHANOL-d4) 5 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 Synthesis of H10

[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-l-carboxylate (H10)

[710] Steps 1-4 outlined in section 1.4.1 were employed to afford the title compound as a colorless oil (24.8 mg, 88% yield). LC / MS (ESI, m / z), 652.91 [M+H]+.

[711] 1H NMR (400 MHz, CHLOROFORM-d) 6 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).

[712] A procedure similar to the one outlined in section 1.2.7 was employed to afford the title compound as a colorless oil (22.1 mg, 57% yield).

[713] 1H NMR (400 MHz, METHANOL-d4) 5 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, 2 H). 1.5 Synthesis of ADL2-H1

[714] 2-((2R,4R,5S,6S)-4-((4-(3-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-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, e.g., 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. Then, the solvent was removed in vacuo and the resulting residue was purified by reverse phase chromatography (ODS, 24 g, H2O / MeCN = 95 / 5 to 60 / 40) to afford the title compound as a colorless oil (15.7 mg, 65% yield).

[716] 1H NMR (400 MHz, METHANOL-d4) 5 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 a mixture 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-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 amino-functionalized silica gel chromatography (24 g, Heptane / AcOEt = 50 / 50 to 0 / 100, then AcOEt / MeOH = 80 / 20) to furnish the title compound as a colorless oil (15.1 mg, 60.4% yield). LC / MS (ESI, m / z), 410.07 [M+H]+.

[719] 1H NMR (400 MHz, CHLOROFORM-d) 5 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) 2.342.45 (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 of3-(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- methyloxi ran-2-yl)-6-methylhepta-2,4-dien-2-yl)-5-methyltetrahydro-2H-pyran-2- yl)methyl)propanamide (ADL2-H11) o

[721] To a mixture of the compound obtained from 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 DI PEA (0.019 ml, 0.111 mmol). The resulting mixture was stirred at room temperature for 16 hours. Subsequently, the mixture was concentrated in vacuo and the resulting residue was purified by reverse phase chromatography (ODS, 24 g, H2O / MeCN = 95 / 5 to 60 / 40) to afford the title compound as a colorless oil (14.9 mg, 62% yield). 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 via the general procedure 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 equiv.) in DMF (0.028M) was added amine (16 mg, 0.056 mmol) and DIPEA (2.0 equiv.). The reaction mixture was stirred at room temperature for 16 hours, then the mixture was purified by HPLC to furnish the desired product.

[724] General procedure 1.7 was employed to afford H20 as a colorless amorphous product (15.7 mg, 0.022 mmol, 79% yield). LC / MS (ESI, m / z), 707.99 [M+H]+.

[725] 1H NMR (400 MHz, CHLOROFORM-d) 6 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 Synthesis of H23 HN^O A .OH

[726] H23 (6.9 mg, 37% yield) was obtained from general procedure 1.7. LC / MS (ESI, m / z), 665.96 [M+H]+.

[727] 1H NMR (400 MHz, METHANOL-d4) 5 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 (brd, 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 Synthesis of H16

[728] General procedure 1.7 was employed to afford H16 as a colorless amorphous product (6.4 mg, 34% yield). LC / MS (ESI, m / z), 679.86[M+H]+.

[729] 1H NMR (400 MHz, CHLOROFORM-d) 5 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 (brdd, 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 Synthesis of H15

[730] General procedure 1.7 afforded H15 as a side product during synthesis of H16 (6.6 mg, 35% yield). LC / MS (ESI, m / z), 665.46 [M+H]+.

[731] 1H NMR (400 MHz, METHANOL-d4) 6 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) 2.292.49 (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 Synthesis of H24 HN^O .OH

[732] General procedure 1.7 was employed to afford H24 as a colorless amorphous product (12.1 mg, 94% yield). LC / MS (ESI, m / z), 693.72 [M+H]+.

[733] 1H NMR (400 MHz, METHANOL-d4) 5 ppm 0.66 (d, J=6.34 Hz, 3 H) 0.80 (d, J=6.83 Hz, 3H) 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 Synthesis of H18

[734] General procedure 1.7 was employed to afford H18 as a colorless amorphous product (8.1 mg, 53% yield). LC / MS (ESI, m / z), 678.68[M-H]+.

[735] 1H NMR (400 MHz, CHLOROFORM-d) 5 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 Synthesis of H13

[736] General procedure 1.7 was employed to afford H13 as a colorless amorphous product (6.7 mg, 36.7 % yield). LC / MS (ESI, m / z), 652.86 [M+H]+.

[737] 1H NMR (400 MHz, METHANOL-d4) 5 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 Synthesis of H14

[738] General procedure 1.7 was employed to afford H14 as a colorless amorphous solid (15.5 mg, 83 % yield). LC / MS (ESI, m / z), 666.90 [M+H]+.

[739] 1H NMR (400 MHz, CHLOROFORM-d) 6 ppm 0.65 (br d, J=6.34 Hz, 3 H) 0.85 (br d, J=6.83 Hz, 3 H) 1.02 (brd, 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 (brd, 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 Synthesis of H17

[740] General procedure 1.7 was employed to afford H17 as a colorless amorphous solid (12.94 mg, 68 % yield). LC / MS (ESI, m / z), 680.66 [M+H]+.

[741] 1H NMR (400 MHz, METHANOL-d4) 5 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 was employed to afford H19 as a colorless amorphous solid (5.53 mg, 56.4 % yield). LC / MS (ESI, m / z), 694.08 [M+H]+.

[743] 1H NMR (400 MHz, METHANOL-d4) 6 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 (brd, 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 (brd, J=11.22 Hz, 1 H) 6.29 (dd, J=14.88, 10.98 Hz, 1 H). 1.7.11 Synthesis of H21

[744] General procedure 1.7 was employed to afford H21 was obtained as a colorless amorphous solid (12.3 mg, 62.1 % yield). LC / MS (ESI, m / z), 708.03 [M+H]+.

[745] 1H NMR (400 MHz, CHLOROFORM-d) 5 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 (brdd, 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 via the general procedure 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, 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 temp for 16 hours. Then, the mixture was 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 obtain the desired product as a colorless oil (8.3 mg, 47% yield)

[748] Step 2: To a solution of cis, trans mixture of 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 DI PEA (0.028 ml, 0.158 mmol) in dichloromethane (1 ml) at room temperature was added 4-nitrophenyl carbonochloridate (12.75 mg, 0.063 mmol) and DMAP (1.932 mg, 0.016 mmol) portion-wise. The resulting mixture was stirred at the same temperature for 16 hours. Then, 1-methylpiperazine (31.7 mg, .316 mmol) was added and was stirred for another 1 hour. The reaction mixture was diluted with dichloromethane and the organic layter was washed with water and brine, then dried over Na2SO4. The solid was filtered and the filtrate was concentrated to dryness. The obtained residue was dissolved in 1.0 mL of MeOH and 10 mg of p-TsOH and stirred for 2 hours at room temperature. The reaction was quenched by addition of 100uL of DI PEA, and the solvent was then removed in vacuo. The obtained residue was purified by NH-silica gel chromatography (Hep / AcOEt = 50 / 50 to 0 / 100) to afford H25 and H22. 1.8.1 Synthesis of H25

[749] H25 was obtained as a colorless amorphous product (7.0 mg, 32.7% yield). LC / MS (ESI, m / z), 676.94 [M+H]+.

[750] 1H NMR (400 MHz, CHLOROFORM-d) 5 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 (brt, 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 Synthesis of H22

[751] H22 was obtained as a colorless amorphous product (5.6 mg, 26.2 % yield). LC / MS (ESI, m / z), 676.89[M+H]+.

[752] 1H NMR (400 MHz, CHLOROFORM-d) 5 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 (brt, 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 herboxidiene spliceosome modulator payloads used in the preparation of ADCs were profiled. Payloads were evaluated for binding to the SF3b complex, in vitro splicing activity, and ability to inhibit cell growth. 2.1 In Vitro Splicing (IVS)

[754] To evaluate payload activity in a cell-free system, an in vitro splicing assay was performed. The payloads were incubated with nuclear extracts and pre-mRNA substrate minigenes.

[755] HeLa nuclear extract preparation: HeLa S3 cell pellets were resuspended in hypotonic buffer (10 mM HEPES pH 7.9, 1.5 mM MgCI2, 10 mM KCI, 0.2 mM PMSF, 0.5 mM DTT) and the suspension was brought up to a total of 5 packed cell volume (PCV). After centrifugation, the supernatant was discarded, and the cells were brought up to 3 PCV with hypotonic buffer and incubated on ice for 10 min. Cells were lysed using a dounce homogenizer and then centrifuged. The supernatant was discarded, and the pellet was resuspended with Vi packed nuclear volume (PNV) of low salt buffer (20 mM HEPES pH 7.9, 1.5 mM MgCI2, 20 mM KCI, 0.2 mM EDTA, 25% glycerol, 0.2 mM PMSF, 0.5 mM DTT), followed by 1 / 2 PNV of high salt buffer (same as low salt buffer except 1.4 M KCI). The nuclei were gently mixed for 30 min before centrifuging. The supernatant (nuclear extract) was then dialyzed into storage buffer (20 mM HEPES pH 7.9, 100 mM KCI, 0.2 mM EDTA, 20% glycerol, 0.2 mM PMSF, 0.5 mM DTT). Protein concentration was determined using NanoDrop 8000 UV-Vis spectrophotometer (ThermoFisher Scientific).

[756] IVS: All Ad2-derived sequences (Pellizzoni et al. (1998) Cell 95(5):615-24) were cloned into pcDNA3.1(+) vector (Promega) using 5' EcoRI and 3' Xbal restriction sites. The plasmids were linearized using Xbal and used as DNA templates in in vitro transcription reactions. The FtzAi intron-less plasmid (Luo and Reed (1999) 96(26):14937-42) was linearized using EcoRI. All RNAs were in vitro transcribed and then purified using MEGAScript T7 (Invitrogen) and MegaClear (Invitrogen) kits, respectively. For splicing reactions using Ad2 variant pre-mRNAs, 1 pL reactions were prepared using 8 pg nuclear extracts prepared from HeLa S3, 2 ng pre-mRNA, 0.2 ng FTZAi, and varying concentrations of compounds or DMSO. After a 15 min preincubation at 30 °C, 1 pL splicing activation buffer (0.5 mM ATP, 20 mM creatine phosphate, 1.6 mM MgCI2) was added, and the reactions were incubated for 90 min at 30 °C. The reactions were then quenched with 13 pL DMSO, and 25 nL was used for RT-qPCR. RT-qPCR reactions were prepared using TaqMan RNA-to-CT 1-step kit (Life Technologies), RNA from splicing reactions, Ad2 (forward: ACTCTCTTCCGCATCGCTGT; reverse: CCGACGGGTTTCCGATCCAA; probe: CTGTTGGGCTCGCGGTTG) and Ftz (forward: TGGCATCAGATTGCAAAGAC; reverse: ACGCCGGGTGATGTATCTAT; probe: CGAAACGCACCCGTCAGACG) mRNA primer-probe sets. Prism 7 (Graphpad) was used for non-linear regression curve fitting of the formed spliced product and normalized to the control (DMSO) sample.

[757] Given that all tested payloads specifically bind to the SF3b complex and demonstrate similar binding profiles, it was hypothesized that all payloads should also modulate splicing to a comparable degree. All payloads significantly modulated splicing of Ad2.2 pre-mRNA (see Table 13). In the presence of payload, a decrease in the amount of spliced product was observed. 2.2 Cell Viability

[758] HCC1954 (American Type Culture Collection (ATCC)) breast ductal carcinoma cells were plated at 2000 cells / well in flat bottom 96-well tissue culture plates (Corning) in a total volume of 90 pL tissue culture medium supplemented with 10% fetal bovine serum (ThermoFisher Scientific). Cells were treated with a 3-fold serial dilution of compound from 200 nM to 0.03 nM. Each concentration was tested in triplicate. At the time of treatment, a plate of untreated cells was evaluated using 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 assayed on an EnVision Multilabel Reader (PerkinElmer). Values represent time zero (TO). The number of viable cells following 144 hours (T144) of compound treatment was also determined using the CellTiter-Glo®2.0 Luminescent Cell Viability Assay. Using the luminescence value at time zero (TO), DMSO control growth (C), and test growth in the presence of compound (T144), the percentage growth was calculated at each of the compound concentrations levels. Percentage 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. The dose response curve plots were generated using Prism 7 (Graphpad) and fit using nonlinear regression analysis and the log(inhibitor) versus response—variable slope (four parameters).

[759] Cell viability dose response was determined for all payloads in HER2-amplified HCC1954 breast cancer cells. Most of the tested payloads exhibited GI50 values (i.e., concentration of compound to cause 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 ADCs is described below. 3.1 Antibody

[761] Trastuzumab antibody (“AB185”) (Molina et al. (2001) Cancer Res. 61(12):4744-9) was used for the preparation of anti-HER2 ADCs (also referred to herein as SMLAs). 3.2 Bioconjugation

[762] Antibody (trastuzumab) at 10 mg / mL in PBS ...

Claims

1. A splicing modulator of Formula (I):R1 -    - R3        1               (|), or a pharmaceutically acceptable saltthereof, wherein:Y is chosen from O, S, NR6, and CR6R7;R1, R2, and R3 are each independently chosen from hydrogen, hydroxyl, -O-(C1-C6 alkyl) groups, -O-C(=O)-(C1-C6 alkyl) groups, -C(=O)-O-(C1-C6 alkyl) groups, and C1-C6 alkyl groups;R4 is chosen from C1-C6 alkyl groups, -C(=O)-(C1-C6 alkyl) groups, -C(=O)-(C3-C8 carbocyclyl) groups, -C(=O)-(C3-C8 heterocyclyl) groups, and -C(=O)-NR6R7;R5 is chosen from hydrogen, hydroxyl, -CH2-OH, -CO2H, -C(=O)-O-(C1-C6 alkyl) groups, -C(=O)-NR6R7, -NR6-C(=O)-R8, -O-C(=O)-NR6R7, -NR6-C(=O)-R8, and -NR6-C(=O)-NR6R7;R6 and R7 are each independently chosen from hydrogen, -R8, -C(=O)-R8, and -C(=O)-O-R8; andR8 is chosen from C1-C6 alkyl groups, C3-C8 carbocyclyl groups, and C3-C8 heterocyclyl groups,wherein R1, R2, R3, R4, R5, R6, R7, and R8 are each independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl, C1-C6 alkyl groups, -O-(C1-C6 alkyl) groups, -CO2H, -C(=O)-(C1-C6 alkyl) groups, -C(=O)-(C3-C8 carbocyclyl) groups, -C(=O)-(C3-C8 heterocyclyl) groups, -NR6R7, C3-C8 carbocyclyl groups, C1-C6 alkylhydroxy groups, C1-C6 alkylalkoxy groups, benzyl groups, and C3-C8 heterocyclyl groups, each of which may be independently substituted with 0 or 1 group chosen from halogens, hydroxyl, C1-C3 alkyl groups, C1-C3 alkoxy groups, C1-C3 haloalkyl groups, -NH-C(=O)(C1-C3 alkyl), and -NH-C(=O)-O-(C1-C3 alkyl).

2. The splicing modulator of claim 1, wherein the splicing modulator is a compoundof Formula (Ia):2019397062   26 Aug 2026(Ia), or a pharmaceutically acceptable saltthereof, wherein:R9 is chosen from C3-C8 heterocyclyl groups; andR10 is chosen from H and C1-C6 alkyl groups,wherein R9 and R10 are each independently substituted with 0 to 3 groupsindependently chosen from halogens, hydroxyl, C1-C3 alkyl groups, C1-C3 alkoxygroups, -NH2, -NH-(C1-C3 alkyl), and -N-(C1-C3 alkyl)2.

3. The splicing modulator of claim 1 or claim 2, wherein the splicing modulator is acompound of Formula (Ib):0(Ib), or a pharmaceutically acceptable saltR11 is chosen from, andHNR1 $, wherein* denotes the point of connectivity of R11 to the remainder of the compound; and R12 and R13 are each independently chosen from H and methyl.

4. The splicing modulator of claim 1, wherein the splicing modulator is H1, or a pharmaceutically acceptable salt thereof.

5. The splicing modulator of claim 1, wherein the splicing modulator is H22019397062   26 Aug 2026or a pharmaceutically acceptable salt thereof.

6. The splicing modulator of claim 1, wherein the splicing modulator is H3, or a pharmaceutically acceptable salt thereof.

7. The splicing modulator of claim 1, wherein the splicing modulator is H12or a pharmaceutically acceptable salt thereof.

8. A compound comprising a linker-payload conjugate of formula L-H, wherein L isa linker comprising a cleavable moiety, and wherein H is the splicing modulator of any one of claims 1-7 or a pharmaceutically acceptable salt thereof.

9. The compound of claim 8, wherein H is covalently attached through a nitrogenatom to L and the valency of the nitrogen atom that is covalently attached to L is not exceeded.

10. The compound of claim 8 or 9, wherein the compound is selected from:2019397062   26 Aug 2026, and, or a pharmaceutically acceptable salt thereof.

11. The compound of any one of claims 8-10, wherein the compound is selectedfrom:2019397062   26 Aug 2026Hor a pharmaceutically acceptable salt thereof.

12. The compound of any one of claims 8-10, wherein the compound is2019397062   26 Aug 2026salt thereof., or a pharmaceutically acceptable13. The compound of any one of claims 8-10, wherein the cleavable moiety comprises a cleavable peptide moiety, wherein the cleavable peptide moiety comprises valine-citrulline (Val-Cit), valine-alanine (Val-Ala), glutamic acid-valine-citrulline (Glu-Val-Cit), or alanine-alanine-asparagine (Ala-Ala-Asn).

14. The compound of any one of claims 8 to 10, wherein the cleavable moiety comprises a cleavable glucuronide moiety, wherein the cleavable glucuronide moiety is cleavable by a glucuronidase.

15. The compound of any one of claims 8 to 10 and 13-14, wherein the linker comprises a maleimide (Mal) moiety.

16. The compound of claim 15, wherein the Mal moiety comprises a maleimidocaproyl (MC) moiety.

17. The compound of claim 16, wherein the linker comprises MC-Val-Cit, MC-Val-Ala, MC-Glu-Val-Cit, or MC-Ala-Ala-Asn.

18. The compound of any one of claims 8 to 17, wherein the linker comprises at least one spacer unit, wherein the spacer unit comprises:(i) a polyethylene glycol (PEG) moiety, wherein the PEG moiety comprises -(PEG)m- and m is an integer from 1 to 10; and / or(ii) an alkyl moiety, wherein the alkyl moiety comprises -(CH2)n- and n is an integer from 1 to 10.2019397062   26 Aug 202619. The compound of any one of claims 8 to 18, wherein the cleavable moiety in the linker is directly attached to the splicing modulator20. The compound of any one of claims 8 to 18, wherein a spacer unit attaches the cleavable moiety in the linker to the splicing modulator.

21. The compound of claim 20, wherein the spacer unit attaching the cleavable moiety in the linker to the splicing modulator is self-immolative.

22. The compound of claim 21, wherein the spacer unit attaching the cleavable moiety in the linker to the splicing modulator comprises a p-aminobenzyloxycarbonyl (pABC) or a p-aminobenzyl (pAB).

23. The compound of claim 22, wherein the linker comprises Val-Cit-pABC, Val-Ala-pABC, Glu-Val-Cit-pABC, Ala-Ala-Asn-pABC, Val-Cit-pAB, Val-Ala-pAB, Glu-Val-Cit-pAB, or Ala-Ala-Asn-pAB.

24. The compound of claim 23, wherein the linker comprises MC-Val-Cit-pABC, MC-Val-Ala-pABC, MC-Glu-Val-Cit-pABC, MC-Ala-Ala-Asn-pABC, MC-Val-Cit-pAB, MC-Val-Ala-pAB, MC-Glu-Val-Cit-pAB, or MC-Ala-Ala-Asn-pAB.

25. An antibody-drug conjugate of Formula (I):Ab-(L-H)p    (I)wherein:Ab is an antibody or antigen binding fragment which targets a neoplastic cell;L-H comprises a compound of any one of claims 8-24, wherein H is the splicing modulator and L is the linker, and wherein L covalently attaches Ab to H; andp is an integer from 1 to 15.

26. The antibody-drug conjugate of claim 25, wherein the neoplastic cell is derived from:(i) a hematological malignancy, wherein the hematological malignancy is selected from a B-cell malignancy, a leukemia, a lymphoma, and a myeloma; or2019397062   26 Aug 2026(ii) a solid tumor, 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.

27. The antibody-drug conjugate of claim 26, wherein the hematological malignancy is selected from acute myeloid leukemia and multiple myeloma.

28. The antibody-drug conjugate of any one of claims 24 to 26, wherein the neoplastic cell is:(i) a HER2-expressing cell, wherein the antibody or antigen binding fragment is an anti-HER2 antibody or antigen binding fragment, wherein the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising 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 an amino acid sequence of SEQ ID NO:19, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:20; or(ii) a CD138-expressing cell, wherein the antibody or antigen binding fragment targets a CD138-expressing cell, wherein the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising 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 an amino acid sequence of SEQ ID NO:21, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:22; or(iii) an EPHA2-expressing cell, wherein the antibody or antigen binding fragment is an anti-EPHA2 antibody or antigen binding fragment, wherein the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three light chain 2532019397062   26 Aug 2026complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising 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 an amino acid sequence of SEQ ID NO:23, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:24.

29. A pharmaceutical composition comprising: the splicing modulator of any one of claims 1-7, the compound of any one of claims 8-24, or the antibody-drug conjugate of any one of claims 25-28; and a pharmaceutically acceptable carrier.

30. Use of the splicing modulator of any one of claims 1-7, the compound of any one of claims 8-24, or the antibody-drug conjugate of any one of claims 25-28, in the manufacture of a medicament for the treatment of a neoplastic disorder.

31. The use of claim 30, wherein the neoplastic disorder is a HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer, uterine cancer, osteosarcoma, or salivary duct carcinoma; a CD138-expressing multiple myeloma; or an EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer.

32. The use of claim 30 or 31, wherein the medicament is for use in the treatment of a neoplastic disorder in combination with a checkpoint inhibitor which targets CTLA4, PD1, PDL1, OX40, CD40, GITR, LAG3, TIM3, and / or KIR.

33. A method of treating a subject having or suspected of having a neoplastic disorder, the method comprising administering to the subject a therapeutically effective amount of the splicing modulator of any one of claims 1-7, the compound of any one of claims 8-24, the antibody-drug conjugate of any one of claims 25-28, or the pharmaceutical composition of claim 29.

34. The method of claim 33, wherein the neoplastic disorder is a HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer, uterine cancer, osteosarcoma, or salivary duct carcinoma; a CD138-expressing multiple myeloma; or an EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer.2019397062   26 Aug 202635. The method of claim 33 or 34, wherein the method further comprises administering at least one additional therapy, wherein the at least one additional therapy comprises a checkpoint inhibitor which targets CTLA4, PD1, PDL1, OX40, CD40, GITR, LAG3, TIM3, and / or KIR.

36. A neoantigen vaccine comprising at least one neoantigen peptide or at least one neoantigen mRNA, wherein the at least one neoantigen peptide comprises or the at least one neoantigen mRNA encodes a neoantigen sequence induced by contacting a neoplastic cell with the splicing modulator of any one of claims 1-7, the compound of any one of claims 8-24, and / or the antibody-drug conjugate of any one of claims 25-28.

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