Preparation of trifunctionally-linked heterobifunctional compounds and uses thereof

Abstract

Disclosed herein are antibody conjugate compounds for killing cancer cells, comprising components that target a) a tumor-associated extracellular target; and b) at least one tumor associated intracellular target. In one embodiment, the compound further comprises an AND logic gate, wherein cell killing activity requires the presence of a tumor-associated extracellular target and one or more tumor associated intracellular targets. In one embodiment, cell killing activity is only active in the presence of a tumor-associated extracellular target and a tumor associated intracellular target.

Description

y.. o. -PREPARATION OF TRIFUNCTIONALLY-LINKED HETEROBIFUNCTIONAL COMPOUNDS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U. S. C. § 119 of U. S. Provisional Application No. 63 / 746,606 filed January 17, 2025, the contents of which are incorporated herein by reference in their entirety.GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No. R01CA296810 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0003] Compounds useful as linker-payloads for therapeutic purposes are disclosed herein. The present invention describes a linker chemistry wherein heterobifunctional molecules are endowed with an additional chemical handle, e.g., through the addition of a trifunctional linker. Said linker allows for the conjugation of said heterobifunctional molecules to diverse molecular entities without impeding the heterobifunctional molecule’s headgroups binding functionality. Additional compounds, conjugates, methods of preparation, pharmaceutical compositions, and methods of treatment related to conjugates of compounds disclosed herein and a targeting moiety, or binding fragment thereof, are also provided.BACKGROUND

[0004] Heterobifunctional small molecules which induce proximity between pre-defined biological targets causing ternary complex formation and therapeutic protein modulation are among the fastest growing class of drugs in chemical biology. These compounds are composed of two distinct small molecule binders (“warheads”) which are then fused together with a linker. The first demonstration of said heterobifunctional compounds was proteolysis-targeting chimeras (PROTACs) in 2001 (Sakamoto et al., 2001). Small molecule PROTACs have since been developed which employ a small molecule binder for a target protein fused to a small molecule binder which recruits ubiquitin ligase (“E3 ligase”) complexes (Bondeson et al., 2015; Chirnomas et al., 2023; Deshaies, 2015; Gough et al., 2024; Hickey et al., 2024; Hines et al., 2013; Hines et al., 2019; Neklesa et al., 2017; Schneekloth et al., 2008;Schneekloth et al., 2004). Following ternary complex formation of the target protein and ubiquitin ligase complexes, the E3 ligase tags the target protein for degradation through the covalent attachment of a poly-ubiquitin chain.

[0005] Similar to PROTACs, autophagy-targeting chimeras (AUTACs), lysosome-targeting chimeras (LYTACs), and specific and non-genetic inhibitor of apoptosis protein (IAP)-dependent protein erasers (SNIPERs) induce target protein degradation. However, unlike PROTACs, AUTACs induce degradationy.. o. -through selective autophagy (Takahashi et al., 2019; Takahashi et al., 2023) and LYTACs engage asialoglycoprotein receptor to induce degradation of extracellular and membrane proteins (Ahn et al., 2021). SNIPERs similarly recruit ubiquitin ligases, however unlike traditional PROTACs they induce simultaneous degradation of the target protein and LAPs such as cIAP1 and XIAP (Naito et al., 2019; Wang et al., 2022). Conversely, deubiquitinase-targeting chimeras (DUBTACs) have been reported which recruit deubiquitinase enzymes to ubiquitinated target proteins, leading to targeted deubiquitination and subsequent stabilization of proteins (Henning et al., 2022; Liu et al., 2024).

[0006] Inspired by PROTACs and other degrader-based molecules, diverse heterobifunctional molecules have been designed which induce a diverse portfolio of biologic effects beyond protein degradation. Phosphorylation-inducing chimeras (PHICs) recruit cellular kinases to induce phosphorylation (native or neo) of target proteins (Siriwardena et al., 2020), and inversely, phosphatase-recruiting chimeras (PHORCs) recruit phosphatases to induce dephosphorylation of target proteins (Chen et al., 2021; Yamazoe et al., 2019; Zhang et al., 2022; Zheng et al., 2021). Ribonuclease targeting chimeras (RIBOTACs) leverage selective RNA-binding small molecules and RNase binders to induce selective degradation of target RNA, rather than protein (Dey & Jaffrey, 2019; Tong et al., 2023). The acetylation tagging system (AceTAG) recruits lysine acetyltransferases to induce acetylation of target proteins (Wang et al., 2021). Most recently, Raina et. al have created regulated induced proximity targeting chimeras (RIPTACs) which form enduring ternary complexes between target proteins and pan-essential effector proteins preventing pan-essential proteins from interacting with cognate downstream targets (Raina et al., 2024; Sadagopan et al., 2024).

[0007] Because of their chimeric nature, these heterobifunctional molecules often exhibit unfavorable physical-chemical properties that can lead to poor solubility and cell-permeability (Hofmann et al., 2024; Zhao & Dekker, 2022). Furthermore, these compounds typically display poor drug metabolism and pharmacokinetic (DMPK) properties such as limited oral bioavailability and / or rapid in vivo clearance (Edmondson et al., 2019; Pike et al., 2020). To overcome these boundaries, numerous antibody-drug conjugates (ADCs) incorporating heterobifunctional compounds as the active payload have been proposed to improve the clinical relevancy of these compounds. By linking a heterobifunctional molecule to monoclonal antibodies (mAbs), this approach may overcome several existing challenges regarding solubility and DMPK properties and impart favorable tissue / cell-specific targeting capacity. However, many of the disclosed (ADCs) incorporating heterobifunctional compounds leverage PROTACs containing the von Hippel-Lindau tumor suppressor (VHL) ligand (CAS: 1448189-80-7), as it is known that antibody linker attachment via the hydroxyproline fragment present in the compound's VHL-binding region retains the PROTACs’ ability to bind VHL (Dragovich et al., 2020; Dragovich, Pillow, Blake, Sadowsky, Adaligil, Adhikari, Bhakta, et al., 2021; Dragovich, Pillow, Blake, Sadowsky, Adaligil, Adhikari, Chen, et al., 2021; Maneiro et al., 2020; Pillow et al., 2019). However, this linker chemistry is not extensible to other PROTAC systems which employ a Cereblon (CRBN) ligand. Importantly, beyond PROTACs, it is generally poorly understood how toleranty.. o. -heterobifunctional molecule warheads are to chemical modification. This limits the ability to add chemical linkers to one of the two warheads, as conjugating a linker to one or the other warhead may abolish the compound’s ability to bind its cognate protein target.

[0008] Critically, it is known that the overall efficiency of heterobifunctional molecules is dependent not only on the characteristics of the two warheads, but also by the suitability of their linker. For PROTACs and RIPTACs, it has been shown that both the length and composition of the linker are crucial in determining the biological activity and physiochemical properties of the molecules (Sadagopan et al., 2024; Troup et al., 2020). Therefore, there exists a need for a modular and generalizable chemical linker for heterobifunctional molecules which enables conjugation of said heterobifunctional molecule to additional chemical or biological entities while maintaining the biological activity of the molecules. The present disclosure fulfills this need and provides further related advantages.SUMMARY

[0009] One aspect provided herein discloses a targeting compound for killing cancer cells, comprising components that target a) a tumor-associated extracellular target; and b) at least one tumor associated intracellular target.

[0010] In one embodiment of this or any other aspect herein, compound further comprising an AND logic gate, wherein cell killing activity requires the presence of each of the targets.

[0011] In one embodiment of this or any other aspect herein, the compound further comprises an AND logic gate, wherein cell killing activity requires the presence of a tumor-associated extracellular target and one or more tumor associated intracellular targets.

[0012] Another aspect provided herein discloses an antibody conjugate compound for killing cancer cells, comprising components that target a) a tumor-associated extracellular target; and b) at least one tumor associated intracellular target; and c) at least one intracellular effector target.

[0013] In one embodiment of this or any other aspect herein, cell killing activity is only active in the presence of a tumor-associated extracellular target and a tumor associated intracellular target.

[0014] In one embodiment of this or any other aspect herein, the compound further comprises an AND logic gate, wherein cell killing activity requires the presence of a tumor-associated extracellular target, one or more tumor associated intracellular targets, and one or more intracellular effector target.

[0015] In one embodiment of this or any other aspect herein, the compound further comprises an AND logic gate, wherein cell killing activity requires the presence of one or more tumor-associated extracellular targets, one or more tumor associated intracellular targets, and one or more intracellular effector target.

[0016] In one embodiment of this or any other aspect herein, cell killing activity is only active in the presence of one or more tumor-associated extracellular targets, one or more tumor associated intracellular targets, and one or more intracellular effector target.y.. o. -

[0017] In one embodiment of this or any other aspect herein, wherein said payload moiety is chemically stable and membrane-permeable such that, upon intracellular processing and killing of a first tumor cell, the payload moiety: a) diffuses out of said first tumor cell and enters an adjacent second tumor cell; b) induces cell death in said second tumor cell contingent upon the presence of both said tumor-associated intracellular target(s) and said intracellular effector target(s) in said second tumor cell; and c) wherein said induction of cell death in the second tumor cell occurs independently of the presence of the tumor-associated extracellular target on said second tumor cell.

[0018] Another aspect provided herein discloses an antibody conjugate compound comprising: a) an antibody that specifically binds a tumor-associated extracellular antigen; b) a regulated induced-proximity targeting chimera (RIPTAC) comprising: i) a first small-molecule binding moiety that selectively binds an intracellular tumor-associated target protein; and ii) a second small-molecule binding moiety that selectively binds a pan-essential intracellular effector protein; and iii) a RIPTAC linker covalently joining the two small-molecule binding moieties; and c) an antibody linker covalently joining the antibody to the RIPTAC.

[0019] In one embodiment of this or any other aspect herein, the intracellular tumor-associated target is selected from the group consisting of: androgen receptor (AR), enhancer of zeste homolog 2 (EZH2), induced myeloid leukemia cell differentiation protein Mcl-1 (MCL1), Wilms tumor protein (WT33), tumor protein p53 (TP53), and mouse double minute 2 homolog (MDM2).

[0020] In one embodiment of this or any other aspect herein, the pan-essential intracellular effector protein is selected from the group consisting of polo-like-kinase-1 (PLK1), Bromodomain-containing protein 2 (BRD2), Bromodomain-containing protein 3 (BRD3), Bromodomain-containing protein 4 (BRD4), cyclin-dependent kinase 1 (CDK1), cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 5 (CDK5), cyclin-dependent kinase 6 (CDK6), and cyclin-dependent kinase 9 (CDK9).

[0021] In one embodiment of this or any other aspect herein, the antibody linker is attached to the RIPTAC through a chemical handle positioned within the internal linker of the RIPTAC, or via a cleavable linker conjugated to one of the small-molecule binding moieties and being chemically discrete from both small-molecule binding moieties, such that the native binding affinity of each moiety for the respective protein is retained following linker cleavage and payload release.

[0022] In one embodiment of this or any other aspect herein, the RIPTAC linker comprises a scaffold selected from the group consisting of a lysine-derived scaffold selected from Nα-L-lysine, Nε-L-lysine, Nα-D-lysine, ornithine, and diaminobutyric acid, a poly(ethylene glycol) (PEG) scaffold selected from two to twenty-four oxyethylene units, a piperazine scaffold, a piperidine scaffold, a cyclobutene scaffold, an alkyl chain, an alkyne, a morpholine scaffold, a triazole scaffold, an alkyl ether scaffold, a branched 3-arm PEG scaffold, and combinations thereof; and / or wherein the linker is joined to the RIPTAC small-molecule binding moieties via a linkage selected from the group consisting of an azide–alkyne cycloaddition product, an amide bond, and a carbamate bond.y.. o. -

[0023] In one embodiment of this or any other aspect herein, the antibody linker further comprises a self-immolative valine–citrulline–para-aminobenzyl carbamate cleavage motif that is cleavable by cathepsin B after intracellular internalization, the para-aminobenzyl being connected to the RIPTAC through a carbamate linkage to a primary or secondary amine located within the internal linker of the RIPTAC, and wherein the valine–citrulline dipeptide is flanked by a PEG spacer selected from PEG2, PEG4, PEG6, PEG8, or PEG10 or a n-Alkane spacer selected from C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, or C15 to modulate solubility, prevent aggregation, or alter cleavage kinetics, and the para-aminobenzyl group is optionally substituted with methoxy or halo substituents to tune self-immolation rate while effecting scarless release of the RIPTAC such that both small-molecule binding moieties retain their native affinity.

[0024] In one embodiment of this or any other aspect herein, the antibody is a human or humanized IgGl, IgG2 or IgG4 that binds a tumor-associated extracellular antigen selected from the group consisting of: STEAP1, PSMA, CD19, CD20, CD33, BCMA, FOLR1, NaPi-2b, MUC16, CLEC12A, HER2, GPNMB, B7-H3, Trop-2, EGFR and EpCAM.

[0025] In one embodiment of this or any other aspect herein, the antibody is selected from trastuzumab, pertuzumab, vandortuzumab, rituximab, obinutuzumab, loncastuximab, belantamab, cetuximab, or a PSMA×STEAP1 bispecific antibody.

[0026] In one embodiment of this or any other aspect herein, the antibody comprises Fc-engineering for enhanced internalization or reduced effector function.

[0027] In one embodiment of this or any other aspect herein, the first small-molecule binding moiety binds androgen receptor and the second small-molecule binding moiety binds BRD4 or PLK1.

[0028] Another aspect provided herein discloses an antibody conjugate comprising: a) an antibody that specifically binds a tumor-associated extracellular antigen; b) a transcriptional / epigenetic chemical inducer of proximity (TOP) comprising: i) a first small-molecule binding moiety that selectively binds an intracellular transcription factor; and ii) a second small-molecule binding moiety that selectively binds a transcriptional activator or transcriptional repressor; and iii) a TOP linker covalently joining the two small-molecule binding moieties; and c) a tri-functional linker covalently joining the antibody to the TOP, the linker being attached to the TCIP through a chemical handle positioned within the internal linker of the TCIP or via a cleavable linker conjugated to one of the small-molecule binding moieties and being chemically discrete from both small-molecule binding moieties, such that the native binding affinity of each moiety for the respective protein is retained.

[0029] In one embodiment of this or any other aspect herein, the intracellular transcription factor selected from B-cell lymphoma 6 (BCL6), Forkhead-box Protein Pl (FOXPl), Poly(ADP-ribose) Polymerase 1 (PARP1), and Poly(ADP-ribose) Polymerase 2 (PARP2).

[0030] In one embodiment of this or any other aspect herein, the transcriptional activator or transcriptional repressor is selected from the group consisting of: Bromodomain-containing protein 4y.. o. -(BRD4), cyclin-dependent kinase 9 (CDK9), El A-associated protein p300 (p300), and CREB-binding protein (CBP).

[0031] In one embodiment of this or any other aspect herein, the TCIP linker comprises a scaffold selected from the group consisting of a lysine-derived scaffold selected from Nα-L-lysine, Nε-L-lysine, Nα-D-lysine, ornithine, and diaminobutyric acid, a poly(ethylene glycol) (PEG) scaffold selected from two to twenty-four oxyethylene units, a piperazine scaffold, a piperidine scaffold, a cyclobutene scaffold, an alkyl chain, an alkyne, a morpholine scaffold, a triazole scaffold, an alkyl ether scaffold, a branched 3-arm PEG scaffold, and combinations thereof; and / or wherein the linker is joined to the TCIP small-molecule binding moieties via a linkage selected from the group consisting of an azide–alkyne cycloaddition product, an amide bond, and a carbamate bond.

[0032] In one embodiment of this or any other aspect herein, the antibody linker further comprises a self-immolative valine–citrulline–para-aminobenzyl carbamate cleavage motif that is cleavable by cathepsin B after intracellular internalization, the para-aminobenzyl being connected to the TCIP through a carbamate linkage to a primary or secondary amine located within the internal linker of the TCIP, and wherein the valine–citrulline dipeptide is flanked by a PEG spacer selected from PEG2, PEG4, PEG6, PEG8, or PEG10 or a n-Alkane spacer selected from C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, or C15 to modulate solubility, prevent aggregation, or alter cleavage kinetics, and the para-aminobenzyl group is optionally substituted with methoxy or halo substituents to tune self-immolation rate while effecting scarless release of the TCIP such that both small-molecule binding moieties retain their native affinity.

[0033] In one embodiment of this or any other aspect herein, the antibody is a human or humanized IgGl, IgG2 or IgG4 that binds a tumor-associated extracellular antigen selected from the group consisting of: STEAP1, PSMA, CD19, CD20, CD33, BCMA, FOLR1, NaPi-2b, MUC16, CLEC12A, HER2, GPNMB, B7-H3, Trop-2, EGFR and EpCAM.

[0034] In one embodiment of this or any other aspect herein, the antibody is selected from trastuzumab, pertuzumab, vandortuzumab, rituximab, obinutuzumab, loncastuximab, belantamab, cetuximab, CD19×BCMA bispecific antibody, CD19×CD20 bispecific antibody, or a BCMA×CD20 bispecific antibody.

[0035] In one embodiment of this or any other aspect herein, the antibody comprises Fc-engineering for enhanced internalization or reduced effector function.

[0036] Another aspect provided herein discloses a compound having the structure represented by Structures la, lb or 1c:y.. o. -(Structure la)s(Structure lb)(Structure 1c)

[0037] or a pharmaceutically acceptable salt thereof; wherein: A1and A2independently represent small molecule binder (“warhead”) pairings which bind to two distinct biological targets (i.e., protein, RNA, DNA); L1comprises a linker composed of any amino acid, non-proteinogenic amino acid, PEG, n-Alkane, piperidine, cyclobutene, piperazine, or modified combination thereof; L2comprises a linker composed of any cleavable or non-cleavable element which serves to join A1-L1-A2to a targeting compound (TC); wherein the TC comprises a targeting compound consisting of an antibody, singledomain antibody, fusion protein, ligand, or nanoparticle.

[0038] In one embodiment of this or any other aspect herein, L1has the following structure:y.. o. -(Structure 2)

[0039] or a pharmaceutically acceptable salt thereof; wherein:

[0040] R1represents the side chain an amino acid or non-proteinogenic amino acid selected from: i) arginine, histidine, lysine, aspartate, glutamate, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan;ii) norvaline, norleucine, alloisoleucine, isoserine, t-leucine, pipecolate, 2,3-diaminopropionate, 2,4-diaminobutyrate, ornithine, allothreonine, homocysteine, homoserine, ci-amino-n-heptanoate, ci-aminobutyrate, P-aminobutyrate, y-aminobutyrate, ci-aminoisobutyrate; or any chemically modified derivates thereof.

[0041] In one embodiment of this or any other aspect herein, L1has the following structure:(Structure 3) (Structure 4)

[0042] or a pharmaceutically acceptable salt thereof; wherein:

[0043] R2and R3represents the side chain of one or two of the amino acids or non-proteinogenic amino acids selected from:iii) arginine, histidine, lysine, aspartate, glutamate, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan;iv) norvaline, norleucine, alloisoleucine, isoserine, t-leucine, pipecolate, 2,3-diaminopropionate, 2,4-diaminobutyrate, ornithine, allothreonine, homocysteine, homoserine, ci-amino-n-heptanoate, ci-aminobutyrate, P-aminobutyrate, y-aminobutyrate, ci-aminoisobutyrate; or any chemically modified derivates of the aforementioned;

[0044] n1and n2represent the number of monomeric subunits of said amino acids or non-proteinogenic amino acids where n1and n2independently equal a value between 1-10.y.. o. -

[0045] In one embodiment of this or any other aspect herein, L1has the following structure:(Structure 5) (Structure 6)(Structure 7)

[0046] or a pharmaceutically acceptable salt thereof; wherein:

[0047] R4, R5, and R6represents the side chain of 1-3 of the amino acids or non-proteinogenic amino acids selected from:v) arginine, histidine, lysine, aspartate, glutamate, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan;vi) norvaline, norleucine, alloisoleucine, isoserine, t-leucine, pipecolate, 2,3-diaminopropionate, 2,4-diaminobutyrate, ornithine, allothreonine, homocysteine, homoserine, a-amino-n-heptanoate, a-aminobutyrate, P-aminobutyrate, y-aminobutyrate, a-aminoisobutyrate;

[0048] or any chemically modified derivates of the aforementioned;

[0049] n3, n4, and n5represent the number of monomeric subunits of said amino acids or non-proteinogenic amino acids where n3, n4, and n5independently equal a value between 1-10.

[0050] In one embodiment of this or any other aspect herein, L1has the following structure:y.. o. -(Structure 8) (Structure 9)(Structure 10)

[0051] or a pharmaceutically acceptable salt thereof; wherein S1and S2represent spacers selected from the group consisting of: poly(ethylene glycol) (PEG), poly(glycol) chains, alkyl chains, alkynes, triazoles, piperazines, piperidines, and any combination thereof.

[0052] In one embodiment of this or any other aspect herein, L1has the following structure:Atty. Dkt. No. 701586-000161WOPT(Structure 11)(Structure 12)

[0053] or a pharmaceutically acceptable salt thereof; wherein S3, S4, and S5represent optional spacers selected from the group consisting of: poly(ethylene glycol) (PEG), poly(glycol) chains, alkyl chains, alkynes, triazoles, piperazines, piperidines, and any combination thereof.y.. o. -

[0054] In one embodiment of this or any other aspect herein, L1has the following structure:(Structure 13)(Structure 15)

[0055] or a pharmaceutically acceptable salt thereof; wherein S6, S7, S8, and S9represent optional spacers selected from the group consisting of: poly(ethylene glycol) (PEG), poly(glycol) chains, alkyl chains, alkynes, triazoles, piperazines, piperidines, and any combination thereof.

[0056] In one embodiment of this or any other aspect herein, L1has the following structure:y.. o. -(Structure 16)

[0057] or a pharmaceutically acceptable salt thereof; wherein n6, n7, and n8represent the number of monomeric subunits where n6, n7, and n8independently equal a value between 1-20.

[0058] In one embodiment of this or any other aspect herein, L2contains a structure selected from the group consisting of Structure 17, Structure 18, Structure 19, Structure 20, Structure 21, Structure 22, and Structure 23, or a combination thereof,

[0059] or a pharmaceutically acceptable salt, solvate or prodrug thereof,

[0060] wherein n9- n14represent the number of monomeric subunits where n9- n14independently equal a value between 1-20.

[0061] In one embodiment of this or any other aspect herein, A1and A2represent a biologically active pairing of small molecules which functions as a regulated induced proximity targeting chimera (RIPTAC).

[0062] In one embodiment of this or any other aspect herein, A1and A2represent a biologically active pairing of small molecules which functions as a proteolysis-targeting chimera (PROTAC).

[0063] In one embodiment of this or any other aspect herein, A1and A2represent a biologically active pairing of small molecules which functions as an autophagy-targeting chimera (AUTAC).

[0064] In one embodiment of this or any other aspect herein, A1and A2represent a biologically active pairing of small molecules which functions as a specific and non-genetic inhibitor of apoptosis protein (IAP)-dependent protein eraser (SNIPER).

[0065] In one embodiment of this or any other aspect herein, A1and A2represent a biologically active pairing of small molecules which functions as a deubiquitinase-targeting chimera (DUBTAC).

[0066] In one embodiment of this or any other aspect herein, A1and A2represent a biologically active pairing of small molecules which functions as a phosphorylation-inducing chimeras (PHICs).

[0067] In one embodiment of this or any other aspect herein, A1and A2represent a biologically active pairing of small molecules which functions as a phosphatase-recruiting chimera (PHORCs).

[0068] In one embodiment of this or any other aspect herein, A1and A2represent a biologically active pairing of small molecules which functions as a ribonuclease targeting chimera (RIBOTAC).

[0069] In one embodiment of this or any other aspect herein, A1and A2represent a biologically active pairing of small molecules which functions as an acetylation tagging system (AceTAG).y.. o. -

[0070] In one embodiment of this or any other aspect herein, the compound is a stereoisomer, enantiomer, or tautomer thereof or a mixture thereof, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0071] Another aspect provided herein discloses a composition comprising any compound disclosed herein.

[0072] In one embodiment of this or any other aspect herein, the compound further comprising at least a second compound disclosed herein.

[0073] Another aspect provided herein discloses a pharmaceutical composition comprising any compound disclosed herein and a pharmaceutically acceptable carrier.

[0074] In one embodiment of this or any other aspect herein, the compound further comprising at least a second compound disclosed herein.

[0075] Another aspect provided herein discloses a method of treating cancer, the method comprising administering to a patient in need thereof a therapeutically effective amount of any compound, composition, or pharmaceutical composition disclosed herein.

[0076] In one embodiment of this or any other aspect herein, the cancer is selected from the group consisting of: a carcinoma, a primary central nervous system tumor, a melanocytic tumor, a germ cell tumor, a sarcoma, and a hematological malignancy.

[0077] In one embodiment of this or any other aspect herein, the carcinoma is selected from the group consisting of: ovarian cancer, uterine cancer, endometrial cancer, cervical cancer, prostate cancer, breast cancer, lung cancer, oral cancer, esophageal cancer, head and neck cancer, stomach cancer, colon cancer, rectal cancer, basal cell carcinoma, squamous cell carcinoma, sebaceous gland carcinoma, bile duct cancer, gallbladder cancer, liver cancer, pancreatic cancer, bladder cancer, urinary tract cancer, renal cell carcinoma and thyroid cancer.

[0078] In one embodiment of this or any other aspect herein, the primary central nervous system tumor is a brain cancer.

[0079] In one embodiment of this or any other aspect herein, the melanocytic tumor is eye cancer or cutaneous melanoma.

[0080] In one embodiment of this or any other aspect herein, the germ cell tumor is testicular cancer or ovarian cancer.

[0081] In one embodiment of this or any other aspect herein, the sarcoma is a uterine sarcoma.

[0082] In one embodiment of this or any other aspect herein, the hematological malignancy is lymphoma or leukemia.

[0083] Another aspect provided herein discloses an antibody conjugate composition for treatment of prostate cancer, comprising: (a) an internalizing antibody moiety that selectively binds to STEAP1, PSMA, or a bispecific antibody moiety that binds both STEAP1 and PSMA; and (b) a RIPTAC moiety that selectively binds to an intracellular cancer-overexpressed protein, androgen receptory.. o. -(AR), and additionally binds at least one pan-essential effector protein selected from the group consisting of BRD4, PLK1, BRD2, BRD3, CDK1, CDK2, CDK4, CDK5, CDK6, and CDK9.

[0084] In one embodiment of this or any other aspect herein, the antibody moiety is conjugated to the RIPTAC moiety via a cathepsin B cleavable valine-citrulline self-immolative linker, enabling intracellular release of the RIPTAC upon internalization.

[0085] In one embodiment of this or any other aspect herein, the antibody moiety is derived from or comprises vandortuzumab for STEAP1 targeting.

[0086] In one embodiment of this or any other aspect herein, the bispecific antibody moiety binds simultaneously or separately to STEAP1 and / or PSMA cell-surface antigens expressed on prostate cancer cells.

[0087] Another aspect provided herein discloses a method of treating metastatic castration-resistant prostate cancer in a subject comprising administering to the subject a therapeutically effective amount of any antibody conjugate composition disclosed herein.

[0088] In one embodiment of this or any other aspect herein, the treatment selectively accumulates in prostate cancer cells due to antibody targeting and kills prostate cancer cells by inducing stable ternary complexes between AR, RIPTAC, and the pan-essential effector protein, resulting in steric inhibition of effector protein function and selective tumor cell death.

[0089] In one embodiment of this or any other aspect herein, the subject has a cancer comprising a mutation found in human prostate cancer cell lines VCaP, LNCaP, or 22Rv1 with differential or engineered STEAP1 expression, including STEAP1 knockout (STEAPlko), STEAP1 low expression (STEAPllow), and STEAP1 overexpressing (STEAPlhi / amp) variants.

[0090] In one embodiment of this or any other aspect herein, the composition provides superior in vivo anti-tumor efficacy as compared to androgen receptor RIPTACs, androgen receptor antagonists, or androgen receptor degraders.

[0091] Another aspect provided herein discloses a pharmaceutical composition comprising any antibody conjugate composition disclosed herein formulated for intravenous administration.

[0092] Another aspect provided herein discloses an antibody conjugate composition for the treatment of Diffuse Large B-Cell Lymphoma (DLBCL), comprising: (a) an internalizing antibody moiety that specifically binds to one or more cell surface antigens selected from CD 19, CD20, BCMA, or a bispecific antibody moiety that binds two antigens thereof; and (b) a transcriptional / epigenetic chemical inducer of proximity (TOP), with a target moiety that selectively binds to BCL6 and an effector moiety that binds BRD4 or an alternative transcriptional activator selected from cyclin-dependent kinase 9 (CDK9), histone acetyltransferases (HATs), or p300 / CBP complexes.

[0093] In one embodiment of this or any other aspect herein, the antibody is conjugated to the TCIP via a cleavable maleimide-PEG4-Val-Cit-PAB linker, enabling intracellular release of the TOP moiety upon cathepsin B cleavage following internalization.y.. o. -

[0094] In one embodiment of this or any other aspect herein, the antibody moiety is derived from or comprises an anti-CD19 monoclonal antibody (e.g., Loncastuximab), an anti-CD20 monoclonal antibody (e.g., Rituximab), or an anti-BCMA monoclonal antibody (e.g., Belantamab) suitable for internalization into DLBCL cells.

[0095] In one embodiment of this or any other aspect herein, the bispecific antibody moiety binds simultaneously or separately two of the three antigens CD 19, CD20, and BCMA to increase selective delivery and internalization into target cells.

[0096] In one embodiment of this or any other aspect herein, the TCIP moiety mediates ternary complex formation between intracellular BCL6 and BRD4 / CDK9 / HAT / p300 / CBP transcriptional regulators, thereby inducing transcriptional activation and expression of BCL6 downstream genes in DLBCL cells.

[0097] Another aspect provided herein discloses a method of selectively killing DLBCL cells in a subject comprising administering a therapeutically effective amount of any antibody conjugate of disclosed herein, wherein cell killing is mediated by activation of previously repressed transcriptional regulatory pathways critical for lymphoma survival and proliferation.

[0098] In one embodiment of this or any other aspect herein, the subject has a cancer bearing a mutation found in human DLBCL cell lines selected from SUDHL5 or KARPAS422.

[0099] In one embodiment of this or any other aspect herein, the antibody conjugate exhibits improved tumor selectivity and reduced systemic toxicity due to the dual mechanism of targeted antibody internalization and transcriptional / epigenetic chemical inducer of proximity induction.

[0100] Another aspect provided herein discloses an antibody conjugate composition for treatment of Diffuse Large B-Cell Lymphoma (DLBCL), comprising: (a) an internalizing antibody moiety that specifically binds to at least one cell surface antigen chosen from CD19, CD20, BCMA, or a bispecific antibody moiety that binds two of said antigens; and (b) a regulated induced proximity targeting chimera (RIPTAC) moiety designed to form a ternary complex between the intracellular cancer-associated protein enhancer of zeste homolog 2 (EZH2) and a pan-essential effector protein selected from the group consisting of PLK1, BRD2, BRD3, BRD4, CDK1, CDK2, CDK4, CDK5, CDK6, and CDK9.

[0101] In one embodiment of this or any other aspect herein, the antibody moiety is conjugated to the RIPTAC moiety via a cathepsin B cleavable linker selected from valine-citrulline-p-aminobenzyl carbamate (Val-Cit-PAB) or a maleimide-based self-immolative linker, facilitating intracellular release of the RIPTAC payload upon endocytosis and lysosomal processing.

[0102] In one embodiment of this or any other aspect herein, the antibody moiety is derived from an anti-CD19, anti-CD20, or anti-BCMA monoclonal antibody, each exhibiting internalization upon antigen binding on DLBCL cells.y.. o. -

[0103] In one embodiment of this or any other aspect herein, the bispecific antibody moiety simultaneously or separately binds two of the antigens CD 19, CD20, and BCMA to enhance selective delivery and cellular internalization in lymphoma cells.

[0104] Another aspect provided herein discloses a method of treating DLBCL in a subject, comprising administering a therapeutically effective amount of any antibody conjugate disclosed herein, thereby inducing selective tumor cell death by the formation of a ternary complex between intracellular RIPTAC, EZH2, and a pan-essential effector protein, leading to functional inhibition of essential proteins in lymphoma cells.

[0105] In one embodiment of this or any other aspect herein, the subject has a cancer comprising a mutation found in human DLBCL cell lines selected from SUDHL5 or KARPAS422.

[0106] In one embodiment of this or any other aspect herein, administration of the antibody conjugate results in significant tumor growth inhibition, induction of apoptosis, and improved survival relative to control treatments.

[0107] In one embodiment of this or any other aspect herein, the antibody conjugate exhibits superior therapeutic efficacy and reduced systemic toxicity compared to unconjugated TOP.Definitions

[0108] As used in the specification and claims, the singular form “a,” “an,” and “the” includes plural references unless the context clearly dictates otherwise. It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components.

[0109] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

[0110] The term “about” as used herein in the context of a number refers to a range centered on that number and spanning 15% less than that number and 15% more than that number. The term “about” used in the context of a range refers to an extended range spanning 15% less than that the lowest number listed in the range and 15% more than the greatest number listed in the range. In some embodiments, a given value refers to a range of values (i.e., “about” the given value). For example, in some embodiments, pH 7.4 refers to about pH 7.4 (i.e., a range from 6.3 to 8.5).

[0111] As used herein, a “variant” protein or polypeptide comprises one or more non-natural amino acids, one or more amino acid substitutions, one or more amino acid insertions, one or more amino acid deletions, or any combination thereof, which may occur at one or more sites relative to a reference polypeptide of this disclosure, and wherein the variant protein or polypeptide has substantially similar activity (e.g., enzymatic function, immunogenicity) relative to a reference polypeptide. A variant protein or polypeptide of this disclosure may have at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the amino acid sequence for a reference polypeptide of this disclosure as determined by sequence alignment programs and parameters disclosed herein. A variant polypeptide can result from, for example, a genetic polymorphism or by human manipulation. Conservative substitutions of amino acids are well known and may occur naturally or mayy.. o. -be engineered when a protein is recombinantly produced. Amino acid substitutions, deletions, and additions may be introduced into a protein using mutagenesis methods known in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, NY, 2001). Oligonucleotide-directed site-specific (or segment specific) mutagenesis procedures may be employed to produce polynucleotides having altered codons that provide the desired substitution, deletion, or insertion. Alternatively, random or saturation mutagenesis techniques, such as alanine scanning mutagenesis, error prone polymerase chain reaction mutagenesis or oligonucleotide-directed mutagenesis, may be used to prepare polypeptide variants (see, e.g., Sambrook et al., supra).

[0112] The term “tumor-associated extracellular target” refers to any molecule, moiety, or structural assembly located on, anchored to, or associated with the extracellular surface of the cell membrane of a cancer cell, which is accessible to circulating binding agents. This includes, but is not limited to, cell surface receptors, transmembrane proteins, glycoproteins, extracellular matrix components, lipids, and carbohydrates that are displayed, enriched, or mutated on tumor cells relative to normal cells.

[0113] The term “tumor-associated intracellular target” refers to any endogenous molecule residing within the intracellular compartment of a target cell, including but not limited to the cytosol, nucleus, mitochondria, lysosomes, endoplasmic reticulum, or the inner leaflet of the plasma membrane. Said target may be a polypeptide, protein, nucleic acid (including DNA, mRNA, miRNA), lipid, or metabolite. The tumor-associated intracellular target need not be a driver of tumorigenesis, nor does it require mutation.

[0114] The term “intracellular effector target” refers to an intracellular molecule, distinct from the tumor-associated intracellular target, capable of modulating a biological pathway, enzymatic activity, or cellular state upon interaction with, or proximity to, the tumor-associated intracellular target. Such modulation includes, but is not limited to, the induction of apoptosis, cell cycle arrest, mitotic disruption, genomic instability, DNA damage response, necroptosis, pyroptosis, transcriptional activation or repression, protein degradation (e.g., via ubiquitin-proteasome pathways), or disruption of cellular metabolism.

[0115] As used herein, “binding domain” or a “binding region” or “targeting moiety”refers to a protein, polypeptide, oligopeptide, peptide, carbohydrate, nucleic acid, or combination thereof that is capable of specifically binding to a target or multiple targets (e.g., KRAS, HER2, BTK, EGFR, androgen receptor protein, estrogen receptor protein, PLK1, BRD2, BRD3, BRD4, CDK1, CDK2, CDK4, CDK6, ALK, IDH1, FLT3, FGFR1, FGFR4, HCV-NS3, FGFR2, FGFR3, ERK1, ERK2, FGR, HER3, HER4, or PI3Ka). A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule or another target of interest.Exemplary binding domains of this disclosure include, for example, a Fab', F(ab') 2, Fab, Fv, rlgG, scFv, hcAbs (heavy chain antibodies), a single domain antibody, VHH, VNAR, sdAbs, nanobody, receptor ectodomains or ligand-binding portions thereof, or ligands (e.g., cytokines, chemokines). A “Fab” (fragment antigen binding) is the part of an antibody that binds to antigens and includes the variable region and CHI of the heavy chain linked to the light chain via an inter-chain disulfide bond. A variety of assays are known for identifying binding domains of the present disclosure that specifically bind ay.. o. -particular target, including Western blot, ELISA, and Biacore® analysis. Particularly preferred binding domains comprise immunoglobulin light and heavy chain variable domains (e.g., scFv, Fab) and are herein referred to as “immunoglobulin binding domains” or “immunoglobulin binding proteins.” Immunoglobulin binding domains can be incorporated into a variety of protein scaffolds or structures as described herein, such as an antibody or an antigen binding fragment thereof, a scFv-Fc fusion protein, or a fusion protein comprising two or more of such immunoglobulin binding domains.

[0116] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not. In other words, except where the context requires otherwise, the term “comprise” and variations of the term, such as “comprising”, “comprises” and “comprised”, are not intended to exclude further additives, components, integers or steps.

[0117] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0118] As used herein, the term “subject” or “patient” refers to any organism to which a compound or composition disclosed herein can be administered, e.g., for experimental, diagnostic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “patient” and “subject” are used interchangeably herein. A subject can be male or female.

[0119] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of human diseases and disorders. In addition, compounds, compositions and methods described herein can be used to with domesticated animals and / or pets.

[0120] A subject can be one who has been previously diagnosed with or identified as suffering from or having a disease or disorder, e.g., cancer cancer. Alternatively, a subject can also be one who has not been previously diagnosed. A “subject in need” of treatment for cancer or a disease or disorder associated with cancer can be a subject having cancer or a disease or disorder associated with cancer,y.. o. -diagnosed as having that condition, or at risk of developing that condition.

[0121] In some embodiments, the subject is human. In another embodiment, the subject is an experimental animal or animal substitute as a disease model. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. Examples of subjects include humans, dogs, cats, cows, goats, and mice. The term subject is further intended to include transgenic species. In some embodiments, the subject can be of European ancestry. In some embodiments, the subject can be of African American ancestry. In some embodiments, the subject can be of Asian ancestry.

[0122] In jurisdictions that forbid the patenting of methods that are practiced on the human body, the meaning of “administering” of a composition to a human subject shall be restricted to prescribing a controlled substance that a human subject will self-administer by any technique (e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation that is consistent with laws or regulations defining patentable subject matter is intended. In jurisdictions that do not forbid the patenting of methods that are practiced on the human body, the “administering” of compositions includes both methods practiced on the human body and also the foregoing activities.

[0123] As used herein, the term “parenteral administration,” refers to administration through injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.

[0124] As used herein, the term “subcutaneous administration” refers to administration just below the skin. “Intravenous administration” means administration into a vein.

[0125] As used herein, the term “dose” refers to a specified quantity of a pharmaceutical agent provided in a single administration. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reaction in an individual.

[0126] As used herein, the term “dosage unit” refers to a form in which a pharmaceutical agent is provided. In certain embodiments, a dosage unit is a vial comprising lyophilized composition or compound described herein. In certain embodiments, a dosage unit is a vial comprising reconstituted composition or compound descried herein.

[0127] By the terms “treat,” “treating” or “treatment of’ (and grammatical variations thereof) it is meant that the severity of the subject’s condition is reduced, at least partially improved or stabilized and / or that some alleviation, mitigation, decrease or stabilization in at least one clinical symptom is achieved and / or there is a delay in the progression of cancer or a disease or disorder associated with cancer.

[0128] The terms “prevent,” “preventing” and “prevention” (and grammatical variations thereof) refer to prevention and / or delay of the onset of a disease, disorder and / or a clinical symptom(s) in a subjecty.. o. -and / or a reduction in the severity of the onset of the disease, disorder and / or clinical symptom(s) relative to what would occur in the absence of the methods of the invention. The prevention can be complete, e.g., the total absence of the disease, disorder and / or clinical symptom(s). The prevention can also be partial, such that the occurrence of the disease, disorder and / or clinical symptom(s) in the subject and / or the severity of onset is less than what would occur in the absence of the present invention.

[0129] As used herein, the term “aliphatic” means a saturated or unsaturated and straight, branched, and / or cyclic hydrocarbon having the defined number of carbon atom. Examples include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkylalkenyl, and cycloalkylalkynyl, having the defined number of carbon atoms.

[0130] As used herein, the term “alkyl” refers to an aliphatic hydrocarbon group which can be straight or branched having 1 to about 60 carbon atoms in the chain, and which preferably have about 6 to about 50 carbons in the chain. “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms. “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms. The alkyl group can be optionally substituted with one or more alkyl group substituents which can be the same or different, where “alkyl group substituent” includes halo, amino, aryl, hydroxyl, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy, alkoxycarbonyl, oxo and cycloalkyl. “Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, propyl, i-propyl, n-butyl, t-butyl, n-pentyl, hexyl, heptyl, octyl, decyl, dodecyl, tridecyl, tetradecyl, pentadecyl and hexadecyl. Useful alkyl groups include branched or straight chain alkyl groups of 6 to 50 carbon, and also include the lower alkyl groups of 1 to about 4 carbons and the higher alkyl groups of about 12 to about 16 carbons.

[0131] A “heteroalkyl” group substitutes any one of the carbons of the alkyl group with a heteroatom having the appropriate number of hydrogen atoms attached (e.g., a CH2group to an NH group or an O group). The term “heteroalkyl” include optionally substituted alkyl, alkenyl and alkynyl radicals which have one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus, silicon, or combinations thereof. In certain embodiments, the heteroatom(s) is placed at any interior position of the heteroalkyl group. Examples include, but are not limited to, -CH2-O-CH3, -CH2-CH2-O-CH3, -CH2-NH-CH3, -CH2-CH2-NH-CH3, -CH2-N(CH3)-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and-CH=CH-N(CH3)-CH3. In some embodiments, up to two heteroatoms are consecutive, such as, by way of example, -CH2-NH-OCH3and -CH2-O-Si(CH3)3

[0132] As used herein, the term “alkenyl” refers to an alkyl group containing at least one carbon-carbon double bond. The alkenyl group can be optionally substituted with one or more “alkyl group substituents.” Exemplary alkenyl groups include vinyl, allyl, n-pentenyl, decenyl, dodecenyl, tetradecadienyl, heptadec-8-en-l-yl and heptadec-8,11-dien-l-yl.

[0133] As used herein, the term “alkynyl” refers to an alkyl group containing a carbon-carbon triple bond. The alkynyl group can be optionally substituted with one or more “alkyl group substituents.”y.. o. -Exemplary alkynyl groups include ethynyl, propargyl, n-pentynyl, decynyl and dodecynyl. Useful alkynyl groups include the lower alkynyl groups.

[0134] As used herein, the term “cycloalkyl” refers to a non-aromatic mono- or multicyclic ring system of about 3 to about 12 carbon atoms. The cycloalkyl group can be optionally partially unsaturated. The cycloalkyl group can be also optionally substituted with an aryl group substituent, oxo and / or alkylene. Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl and cycloheptyl. Useful multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl.

[0135] “Heterocyclyl” refers to a nonaromatic 3-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively). Cxheterocyclyl and Cx-Cyheterocyclyl are typically used where X and Y indicate the number of carbon atoms in the ring system. In some embodiments, 1, 2 or 3 hydrogen atoms of each ring can be substituted by a substituent. Exemplary heterocyclyl groups include, but are not limited to piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4-morpholyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolizinyl, 1,4-diazaperhydroepinyl, 1,3-dioxanyl, 1,4-dioxanyland the like.

[0136] “Aryl” refers to an aromatic carbocyclic radical containing about 3 to about 13 carbon atoms. The aryl group can be optionally substituted with one or more aryl group substituents, which can be the same or different, where “aryl group substituent” includes alkyl, alkenyl, alkynyl, aryl, aralkyl, hydroxyl, alkoxy, aryloxy, aralkoxy, carboxy, aroyl, halo, nitro, trihalomethyl, cyano, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxy, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, rylthio, alkylthio, alkylene and — NRR', where R and R' are each independently hydrogen, alkyl, aryl and aralkyl. Exemplary aryl groups include substituted or unsubstituted phenyl and substituted or unsubstituted naphthyl.

[0137] “Heteroaryl” refers to an aromatic 3-8 membered monocyclic, 8-12 membered fused bicyclic, or 11-14 membered fused tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively.

[0138] Exemplary aryl and heteroaryls include, but are not limited to, phenyl, pyridinyl, pyrimidinyl, furanyl, thienyl, imidazolyl, thiazolyl, pyrazolyl, pyridazinyl, pyrazinyl, triazinyl, tetrazolyl, indolyl, benzyl, naphthyl, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl, naphthyl, tetrahydronaphthyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-l,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, IH-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl,y.. o. -isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-l,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl, and the like. In some embodiments, 1, 2, 3, or 4 hydrogen atoms of each ring can be substituted by a substituent.

[0139] As used herein, the term “halogen” or “halo” refers to an atom selected from fluorine, chlorine, bromine and iodine. The term “halogen radioisotope” or “halo isotope” refers to a radionuclide of an atom selected from fluorine, chlorine, bromine and iodine.

[0140] A “halogen-substituted moiety” or “halo-substituted moiety”, as an isolated group or part of a larger group, means an aliphatic, alicyclic, or aromatic moiety, as described herein, substituted by one or more “halo” atoms, as such terms are defined in this application.

[0141] The term “haloalkyl” as used herein refers to alkyl and alkoxy structures structure with at least one substituent of fluorine, chorine, bromine or iodine, or with combinations thereof. In embodiments, where more than one halogen is included in the group, the halogens are the same or they are different. The terms “fluoroalkyl” and “fluoroalkoxy” include haloalkyl and haloalkoxy groups, respectively, in which the halo is fluorine. Exemplary halo-substituted alkyl includes haloalkyl, dihaloalkyl, trihaloalkyl, perhaloalkyl and the like (e.g. halosubstituted (Ci-C3)alkyl includes chloromethyl, dichloromethyl, difluoromethyl, trifluoromethyl (CF3), perfluoroethyl, 2,2,2-trifluoroethyl, 2,2,2-trifluoro-1,1-dichloroethyl, and the like).

[0142] As used herein, the term “amino” means -NH2. The term “alkylamino” means a nitrogen moiety having one straight or branched unsaturated aliphatic, cyclyl, or heterocyclyl radicals attached to the nitrogen, e.g., -NH(alkyl). The term “dialkylamino” means a nitrogen moiety having at two straight or branched unsaturated aliphatic, cyclyl, or heterocyclyl radicals attached to the nitrogen, e.g., -N(alkyl)(alkyl). The term “alkylamino” includes “alkenylamino,” “alkynylamino,” “cyclylamino,” and “heterocyclylamino.” The term “arylamino” means a nitrogen moiety having at least one aryl radical attached to the nitrogen. For example, -NHaryl, and — N(aryl)2. The term “heteroarylamino” means a nitrogen moiety having at least one heteroaryl radical attached to the nitrogen. For example — NHheteroaryl, and — N(heteroaryl)2. Optionally, two substituents together with the nitrogen can also form a ring. Unless indicated otherwise, the compounds described herein containing amino moieties can include protected derivatives thereof. Suitable protecting groups for amino moieties include acetyl,y.. o. -tertbutoxycarbonyl, benzyloxycarbonyl, and the like. Exemplary alkylamino includes, but is not limited to, NH(C1-C10alkyl), such as — NHCH3, — NHCH2CH3, — NHCH2CH2CH3, and — NHCH(CH3)2. Exemplary dialkylamino includes, but is not limited to, — N(C1-C10alkyl)2, such as N(CH3)2, — N(CH2CH3)2, — N(CH2CH2CH3)2, and— N(CH(CH3)2)2.

[0143] The term “aminoalkyl” means an alkyl, alkenyl, and alkynyl as defined above, except where one or more substituted or unsubstituted nitrogen atoms ( — N — ) are positioned between carbon atoms of the alkyl, alkenyl, or alkynyl. For example, an (C2-C6) aminoalkyl refers to a chain comprising between 2 and 6 carbons and one or more nitrogen atoms positioned between the carbon atoms.

[0144] The terms “hydroxyl” and “hydroxyl” mean the radical — OH.

[0145] The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto, and can be represented by one of -O-alkyl, -O-alkenyl, and -O-alkynyl. Aroxy can be represented by -O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined herein. The alkoxy and aroxy groups can be substituted as described above for alkyl. Exemplary alkoxy groups include, but are not limited to O-methyl, O-ethyl, O-n-propyl, O-isopropyl, O-n-butyl, O-isobutyl, O-sec-butyl, O-tert-butyl, O-pentyl, O- hexyl, O-cyclopropyl, O-cyclobutyl, O-cyclopentyl, O-cyclohexyl and the like.

[0146] As used herein, the term “carbonyl” means the radical — C(O) —. It is noted that the carbonyl radical can be further substituted with a variety of substituents to form different carbonyl groups including acids, acid halides, amides, esters, ketones, and the like.

[0147] As used herein, the term “oxo” means double bonded oxygen, i.e., =0.

[0148] The term “carboxy” means the radical — C(O)O —. It is noted that compounds described herein containing carboxy moieties can include protected derivatives thereof, i.e., where the oxygen is substituted with a protecting group. Suitable protecting groups for carboxy moieties include benzyl, tert-butyl, and the like. As used herein, a carboxy group includes -COOH, i.e., carboxyl group.

[0149] The term “ester” refers to a chemical moiety with formula -C(=O)OR, where R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl and heterocycloalkyl.

[0150] The term “cyano” means the radical — CN.

[0151] The term “nitro” means the radical — NO2.

[0152] The term, “heteroatom” refers to an atom that is not a carbon atom. Particular examples of heteroatoms include, but are not limited to nitrogen, oxygen, sulfur and halogens. A “heteroatom moiety” includes a moiety where the atom by which the moiety is attached is not a carbon. Examples of heteroatom moieties include — N=, — NRN—, — N+(O )=, — O —, — S — or — S(O)2—, — OS(O)2—, and — SS —, wherein RNis H or a further substituent.

[0153] The terms “alkylthio” and “thioalkoxy” refer to an alkoxy group, as defined above, where the oxygen atom is replaced with a sulfur. In preferred embodiments, the “alkylthio” moiety is represented by one of -S-alkyl, -S-alkenyl, and -S-alkynyl. Representative alkylthio groups include methylthio,y.. o. -ethylthio, and the like. The term “alkylthio” also encompasses cycloalkyl groups, alkene and cycloalkene groups, and alkyne groups. “Arylthio” refers to aryl or heteroaryl groups.

[0154] The term “sulfinyl” means the radical — SO —. It is noted that the sulfinyl radical can be further substituted with a variety of substituents to form different sulfinyl groups including sulfinic acids, sulfinamides, sulfinyl esters, sulfoxides, and the like.

[0155] The term “sulfonyl” means the radical — SO2 —. It is noted that the sulfonyl radical can be further substituted with a variety of substituents to form different sulfonyl groups including sulfonic acids (-SO3H), sulfonamides, sulfonate esters, sulfones, and the like.

[0156] The term “thiocarbonyl” means the radical — C(S) —. It is noted that the thiocarbonyl radical can be further substituted with a variety of substituents to form different thiocarbonyl groups including thioacids, thioamides, thioesters, thioketones, and the like.

[0157] “Acyl” refers to an alkyl-CO — group, wherein alkyl is as previously described. Exemplary acyl groups comprise alkyl of 1 to about 30 carbon atoms. Exemplary acyl groups also include acetyl, propanoyl, 2-methylpropanoyl, butanoyl and palmitoyl.

[0158] “Aroyl” means an aryl-CO — group, wherein aryl is as previously described. Exemplary aroyl groups include benzoyl and 1- and 2-naphthoyl.

[0159] “Arylthio” refers to an aryl-S — group, wherein the aryl group is as previously described. Exemplary arylthio groups include phenylthio and naphthylthio.

[0160] “Aralkyl” refers to an aryl-alkyl — group, wherein aryl and alkyl are as previously described. Exemplary aralkyl groups include benzyl, phenylethyl and naphthylmethyl.

[0161] “Aralkyloxy” refers to an aralkyl-0 — group, wherein the aralkyl group is as previously described. An exemplary aralkyloxy group is benzyloxy.

[0162] “Aralkylthio” refers to an aralkyl-S — group, wherein the aralkyl group is as previously described. An exemplary aralkylthio group is benzylthio.

[0163] “Alkoxycarbonyl” refers to an alkyl-0 — CO — group. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and t-butyloxycarbonyl.

[0164] “Aryloxycarbonyl” refers to an aryl-0 — CO — group. Exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.

[0165] “Aralkoxycarbonyl” refers to an aralkyl-0 — CO — group. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.

[0166] “Carbamoyl” refers to an H2N — CO — group.

[0167] “Alkylcarbamoyl” refers to a R'RN — CO — group, wherein one of R and R' is hydrogen and the other of R and R' is alkyl as previously described.

[0168] “Dialkylcarbamoyl” refers to R'RN — CO — group, wherein each of R and R' is independently alkyl as previously described.y.. o. -

[0169] “Acyloxy” refers to an acyl-0 — group, wherein acyl is as previously described. “Acylamino” refers to an acyl-NH — group, wherein acyl is as previously described. “Aroylamino” refers to an aroyl-NH — group, wherein aroyl is as previously described.

[0170] The term “optionally substituted” means that the specified group or moiety is unsubstituted or is substituted with one or more (typically 1, 2, 3, 4, 5 or 6 substituents) independently selected from the group of substituents listed below in the definition for “substituents” or otherwise specified. The term “substituents” refers to a group “substituted” on a substituted group at any atom of the substituted group. Suitable substituents include, without limitation, halogen, hydroxyl, caboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxylalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano or ureido. In some cases, two substituents, together with the carbons to which they are attached to can form a ring.

[0171] For example, any alkyl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl or aryl is optionally substituted with 1, 2, 3, 4 or 5 groups selected from OH, CN, -SC(O)Ph, oxo (=0), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl, O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2 — C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2 — C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2— [CH(OH)]m— (CH2)p— OH, CH2— [CH(OH)]m— (CH2)P— NH2or CH2-aryl-alkoxy; “m” and “p” are independently 1, 2, 3, 4, 5 or 6.

[0172] In some embodiments, an optionally substituted group is substituted with 1 substituent. In some other embodiments, an optionally substituted group is substituted with 2 independently selected substituents, which can be same or different. In some other embodiments, an optionally substituted group is substituted with 3 independently selected substituents, which can be same, different or any combination of same and different. In still some other embodiments, an optionally substituted group is substituted with 4 independently selected substituents, which can be same, different or any combination of same and different. In yet some other embodiments, an optionally substituted group is substituted with 5 independently selected substituents, which can be same, different or any combination of same and different.

[0173] An “isocyanato” group refers to a NCO group.

[0174] A “thiocyanato” group refers to a CNS group.

[0175] An “isothiocyanato” group refers to a NCS group.

[0176] “Alkoyloxy” refers to a RC(=O)O- group.

[0177] “Alkoyl” refers to a RC(=O)- group.

[0178] As used herein, the words "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA, genomic DNA),y.. o. -RNA molecules (e.g., mRNA, saRNA, siRNA), natural occurring, mutated, synthetic DNA or RNA molecules, and analogs of the DNA or RNA generated using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products.

[0179] These terms also encompass a gene. The term "gene" or “gene sequence" is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence or may comprise only a coding sequence as in cDNAs, and / or may include cDNAs in combination with regulatory sequences.

[0180] The terms "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.BRIEF DESCRIPTION OF THE DRAWINGS

[0181] FIG. 1 shows schematic of traditional ADCs exhibiting significant on-target, off-tumor toxicity due to indiscriminate payload toxicity and / or premature payload release. Antibody-RIPTAC conjugates (ARCs) differentially kill tumor cells based on intracellular target protein (TP), mitigating off-tumor toxicity and improving therapeutic index.

[0182] FIGs 2A and 2B show schematic of proposed AR / PLK1 RIPTACs (n = 2, 4, or 6).

[0183] FIGs 2C and 2D show schematic of proposed AR / BRD4 RIPTACs (n = 2, 4, or 6). R-group consists of proposed antibody linker.

[0184] FIG. 3 presents schematic of proposed antibody linkers conjugated to targeting compound (TC): Structure 17, Structure 18, Structure 19, Structure 20, Structure 21, Structure 22, and Structure 23.

[0185] FIGs 4A-4C present exemplary data showing design of antibody-RIPTAC conjugates (Fig. 4A) Proposed mechanism of action of ARCs demonstrating dual-logic cytotoxicity in cells dependent on expression of extracellular and intracellular proteins. Dual overexpression (TP+ / TAA+) leads to specific cell lysis, and liberation of free drug due to the bystander effect allows for killing of heterogeneous cancer cells lacking TAA expression (TPVTAA ). Healthy tissue lacking overexpression of intracellular TP is spared regardless of extracellular target expression (TP / TAA+and TP7TAA ) due to RIPTAC logic. (Fig. 4B) Structures of tri-functional RIPTAC compounds 1-5. (Fig. 4C) Proposed overall structure of ARC 1 with cathepsin B cleavable linkage.

[0186] FIG. 5 presents exemplary data showing Differential anti-proliferative effects of free drug in HaloTag model system. Differential cytotoxicity of PLK1 inhibitor, BI-2536, compared to bifunctional (BI-2PEG-CA) and tri-functional RIPTACs (Compounds 1-5) in model cell lines in a 7-day CyQUANT proliferation assay normalized to untreated (UT) controls (N=l-3, mean ± SD).

[0187] FIGs 6A-6E present exemplary data showing mechanisms underlying RIPTAC differential cytotoxicity. (FIG. 6A) Vybrant DyeCycle analysis following 24 h treatment with BI-2536, biy.. o. -functional and tri-functional RIPTACs in 293 HFL and 293 GFPL model cell lines. Representative intensity histogram shown (N=3). (FIG. 6B) Competition assay with TAMRA-CA mitigates RIPTAC-induced cytotoxicity in a 7-day CyQUANT proliferation assay. Values are normalized relative to untreated (UT) controls or media supplemented with 300 nM TAMRA-CA (n=3, mean ± SD). (FIG.6C) 5-day CyQUANT cytotoxicity of bi-functional BI-2PEG-CA RIPTAC in dox-inducible TREx-293 where expression level of HaloTag-FKBP is titrated using doxycycline. Values are normalized relative to dox-treated controls at the corresponding concentration (n=3, mean ± SD). (FIG. 6D) Western blot of 293 HFL cells treated with corresponding concentrations of Compound 5 or BI-2536. (FIG. 6E) Nanoluciferase (NanoLuc) signal 24 h after dose titration of 3arm-PEG RIPTAC in 293T cells co-transfected with PLKl-SmBiT and HaloTag7-FKBPF36V-LgBiT. Luminescence values are normalized relative to un-transfected, untreated (background) controls (n=3, mean ± SD), non-linear Bell-shaped fit is shown (R2 = 0.88).

[0188] FIGs 7A-7C present exemplary data showing preliminary ARC bioconjugation and cytotoxicity. (FIG. 7A) Copper-catalyzed coupling of P4 propargyl and compound 5 to synthesize coil-drug, P4-compound 5. (FIG. 7B) Supramolecular assembly of Trastuzumab-Coil-Compound 5 using recombinantly expressed Trastuzumab-P3 and coil-drug. (FIG. 7C) Differential cytotoxicity of Trastuzumab-Coil-Compound 5 in model cell lines and HER2+ model cell line variants in a 5-day CyQUANT proliferation assay normalized to untreated (UT) controls (n=3, mean ± SD).

[0189] FIGs 8A-8C present exemplary data showing mCRPC protein expression and dependency patterns as an ARC Druggable Axis. (FIG. 8A) STEAP1 protein expression (MS, Gygi) vs. AR protein expression (RPPA) for all cell lines across DepMap. (FIG. 8B) STEAP1 mRNA expression vs. AR protein expression (RPPA) for all primary cancer samples across TCGA. (FIG. 8C) Protein expression (MS, Gygi) vs. CRISPRi dependency score (Chronos) in VCaP cells.

[0190] FIG 9 presents exemplary data showing DLBCL protein expression patterns as an antibody-TCIP conjugate Druggable Axis. BCL6 mRNA expression vs. CD 19 mRNA expression for all primary cancer samples across TCGA.DETAILED DESCRIPTION OF THE INVENTION

[0191] In one aspect provided herein, the technological disclosure provides modified amino acids and modified non-proteinogenic amino acids to facilitate loading biologically active molecules (also referred to herein as payloads) onto targeting compounds, such as antibodies, ligands, or nanoparticles.

[0192] In another aspect provided the, the technological disclosure provides modified heterobifunctional payloads and linkers incorporating hydrolytically stable tertiary or secondary amides to facilitate loading biologically active molecules onto targeting compounds, such as antibodies, ligands, or nanoparticles. This disclosure further provides use of linker compounds to make a linker-payload for conjugation to a targeting moiety (e.g., antibody, fusion protein, ligand, nanoparticle). An advantage of using amino acids and non-proteinogenic amino acids as a linking group for heterobifunctional small molecules is the addition of ay.. o. -chemical handle that lies between the heterobifunctional molecule’s headgroups (warheads). Alternatively, in one embodiment the use of secondary or tertiary amides as linking groups introduces a reactive handle that may be coupled to a self-immolative spacer, including but not limited to p-aminobenzyl (PAB) carbamates or ethers. Upon activation of the self-immolative moiety (e.g., via enzymatic cleavage or reduction), the linker undergoes a 1,6-elimination or cyclization reaction. An advantage of this mechanism is the resulting scarless (unmodified) release of the heterobifunctional small molecule. A technical effect of this release mechanism is the preservation of the warhead’s steric and electronic environment, ensuring that warhead binding to its target is not compromised by residual linker atoms.

[0193] The addition of said chemical handle enables coupling of the heterobifunctional small molecule onto targeting compounds at the chemical handle as opposed to utilizing a reactive group present on one of the two warheads. Traditional heterobifunctional small molecule linkers employ poly(ethylene glycol) (PEG), poly(glycol) chains, alkyl chains, alkynes, triazoles, piperazines, piperidines, and any combination thereof. However, these linkers impart minimally or non-reactive chemical entities between the two warheads. As a result, to load heterobifunctional compounds onto targeting compounds using said linkers, one must utilize reactive groups present on one of the two warheads, which may abolish the compounds biological activity.

[0194] As therapeutics, heterobifunctional compounds typically display poor drug metabolism and pharmacokinetic (DMPK) properties such as limited oral bioavailability and / or rapid in vivo clearance. The advantage of using modified amino acids, modified non-proteinogenic amino acids, or secondary / tertiary amides to link heterobifunctional molecules to antibodies, fusion proteins, ligands, or nanoparticles may not only improve these properties, but also impart a targeting capability to the heterobifunctional compounds.

[0195] Descriptions of the present invention are not intended to detail each disclosed embodiment or every implementation of the present invention. The description and examples that follow exemplify illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0196] Descriptions of the present invention are not intended to detail each disclosed embodiment or every implementation of the present invention. The description and examples that follow exemplify illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.Antibody Conjugate Compounds

[0197] Provided herein is an antibody drug conjugate compound that targets both a tumor-associated extracellular target and one or more tumor associated- intracellular targets and all targets are required for activity.y.. o. -

[0198] Further provided herein is an antibody drug conjugate compound comprising an AND logic gate where cell killing requires the presence of both a tumor-associated extracellular target and a tumor associated intracellular target.

[0199] Further provided herein is an antibody drug conjugate compound comprising an AND logic gate where cell killing requires the presence of a tumor-associated extracellular target and one or more tumor associated intracellular targets.

[0200] Further provided herein is an antibody drug conjugate compound that only kill cells in the presence of a tumor-associated extracellular target and a tumor associated intracellular target.

[0201] Further provided herein is an antibody drug conjugate compound where cell killing in the presence of both a tumor-associated extracellular target and a tumor associated intracellular target (or targets) leads to release of small-molecule payload that kills adjacent cells expressing the tumor associated intracellular target (or targets).

[0202] Further provided herein is a targeting compound for killing cancer cells, comprising components that target a) a tumor-associated extracellular target; and b) at least one tumor associated intracellular target.

[0203] In one embodiment of this or any other aspect herein, compound further comprising an AND logic gate, wherein cell killing activity requires the presence of each of the targets.

[0204] In one embodiment of this or any other aspect herein, the compound further comprises an AND logic gate, wherein cell killing activity requires the presence of a tumor-associated extracellular target and one or more tumor associated intracellular targets.

[0205] Further provided herein is an antibody conjugate compound for killing cancer cells, comprising components that target a) a tumor-associated extracellular target; and b) at least one tumor associated intracellular target; and c) at least one intracellular effector target.

[0206] In one embodiment of this or any other aspect herein, cell killing activity is only active in the presence of a tumor-associated extracellular target and a tumor associated intracellular target.

[0207] In one embodiment of this or any other aspect herein, the compound further comprises an AND logic gate, wherein cell killing activity requires the presence of a tumor-associated extracellular target, one or more tumor associated intracellular targets, and one or more intracellular effector target.

[0208] In one embodiment of this or any other aspect herein, the compound further comprises an AND logic gate, wherein cell killing activity requires the presence of one or more tumor-associated extracellular targets, one or more tumor associated intracellular targets, and one or more intracellular effector target.

[0209] In one embodiment of this or any other aspect herein, cell killing activity is only active in the presence of one or more tumor-associated extracellular targets, one or more tumor associated intracellular targets, and one or more intracellular effector target.

[0210] In one embodiment, the payload moiety is chemically stable and membrane-permeable such that, upon intracellular processing and killing of a first tumor cell, the payload moiety: a) diffuses out of saidy.. o. -first tumor cell and enters an adjacent second tumor cell; b) induces cell death in said second tumor cell contingent upon the presence of both said tumor-associated intracellular target(s) and said intracellular effector target(s) in said second tumor cell; and c) wherein said induction of cell death in the second tumor cell occurs independently of the presence of the tumor-associated extracellular target on said second tumor cell.

[0211] Further provided herein is an antibody conjugate comprising a) an antibody that specifically binds a tumor-associated extracellular antigen; b) a regulated induced-proximity targeting chimera (RIPTAC) comprising: i) a first small-molecule binding moiety that selectively binds an intracellular tumor-associated target protein; ii) a second small-molecule binding moiety that selectively binds a panessential intracellular effector protein; and iii) a linker covalently joining the two small-molecule binding moieties; and c) a linker covalently joining the antibody to the RIPTAC.

[0212] As use herein, a “regulated induced proximity targeting chimera” or “RIPTAC” refers to a heterobifunctional molecule comprising: (i) a target binding moiety that specifically binds to a target protein; (ii) a pan-essential protein targeting moiety that specifically binds to an essential protein required for cell survival; and (iii) a linker covalently connecting the target binding moiety and the pan-essential protein targeting moiety, wherein the RIPTAC enables regulated control of protein function through induced proximity and protein-protein interactions between the target protein and pan-essential protein.

[0213] In one embodiment, the intracellular tumor-associated target is any known target in the art. In one embodiment, the intracellular tumor-associated target is selected from the group consisting of: androgen receptor (AR), enhancer of zeste homolog 2 (EZH2), induced myeloid leukemia cell differentiation protein Mcl-1 (MCL1), Wilms tumor protein (WT33), tumor protein p53 (TP53), and mouse double minute 2 homolog (MDM2).

[0214] In one embodiment, the pan-essential intracellular effector protein is any effector protein known in the art. In one embodiment, the pan-essential intracellular effector protein is selected from the group consisting ofpolo-like-kinase-1 (PLK1), Bromodomain-containing protein 2 (BRD2), Bromodomain-containing protein 3 (BRD3), Bromodomain-containing protein 4 (BRD4), cyclin-dependent kinase 1 (CDK1), cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 5 (CDK5), cyclin-dependent kinase 6 (CDK6), and cyclin-dependent kinase 9 (CDK9).

[0215] In one embodiment, the linker is attached to the RIPTAC through a chemical handle positioned within the internal linker of the RIPTAC, or via a cleavable linker conjugated to one of the smallmolecule binding moieties and being chemically discrete from both small-molecule binding moieties, such that the native binding affinity of each moiety for the respective protein is retained, e.g., following self-immolative linker cleavage and scarless payload release.

[0216] In one embodiment, the linker comprises a lysine-derived scaffold selected from Nα-L-lysine, Nε-L-lysine, Nα-D-lysine, ornithine, and diaminobutyric acid, or comprises a poly(ethylene glycol)y.. o. -scaffold having a spacer segment of two to twenty-four atoms, including PEG2, PEG4, PEG6, PEG8, PEG12 and PEG24, and optionally a branched architecture selected from 3-arm PEG and 4-arm PEG, and wherein the tri-functional linker further optionally includes one or more heteroatom-containing spacer segments selected from piperazine, piperidine, morpholine, triazole, and alkyl ether, and is joined to the RIPTAC internal linker via an azide–alkyne cycloaddition, an amide coupling, or a carbamate formation, and presents a coupling handle selected from maleimide, N-hydroxysuccinimide (NHS) ester, and para-nitrophenyl carbonate (PNP).

[0217] In one embodiment, the linker further comprises a self-immolative valine–citrulline–para-aminobenzyl carbamate cleavage motif that is cleavable by cathepsin B after intracellular internalization, the para-aminobenzyl carbamate being connected to the RIPTAC through a carbamate linkage to a primary or secondary amine located within the internal linker of the RIPTAC, and wherein the valine–citrulline dipeptide is flanked by a PEG spacer selected from PEG2, PEG4, PEG6 or PEG8 to modulate solubility, prevent aggregation, or alter cleavage kinetics, and the para-aminobenzyl group is optionally substituted with methoxy or halo substituents to tune self-immolation rate while effecting scarless release of the RIPTAC such that both small-molecule binding moieties retain their native affinity. In one embodiment, the flanking spacers can also be n-Alkanes, e.g., C2-C10 chain length.

[0218] In one embodiment, the antibody is a human or humanized IgGl, IgG2 or IgG4 that binds a tumor-associated extracellular antigen selected from the group consisting of: STEAP1, PSMA, CD 19, CD20, CD33, BCMA, FOLR1, NaPi-2b, MUC16, CLEC12A, HER2, GPNMB, B7-H3, Trop-2, EGFR and EpCAM.

[0219] In one embodiment, the antibody is selected from trastuzumab, pertuzumab, vandortuzumab, rituximab, obinutuzumab, loncastuximab, belantamab, cetuximab, or a PSMA×STEAP1 bispecific antibody,

[0220] In one embodiment, the antibody comprises Fc-engineering for enhanced internalization or reduced effector function, e.g., as compared to an non-engineered Fc. One skilled in the art can determine if internalization is enhanced or effector function is reduced as compared to non-engineered Fc, e.g., using appropriate assays known in the art.

[0221] In one embodiment, the first small-molecule binding moiety binds an androgen receptor and the second small-molecule binding moiety binds PLK1.

[0222] In one embodiment, the first small-molecule binding moiety binds an androgen receptor and the second small-molecule binding moiety binds BRD4, e.g., compound 1 disclosed in Table 3 herein.

[0223] In one embodiment, the drug-to-antibody ratio (DAR) is from 1 to 8. In one embodiment, the DAR is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or more. In one embodiment, the DAR is between 1 and 7, 1 and 6, 1 and 5, 1 and 4, 1 and 3, 1 and 2, 2 and 8, 3 and 8, 4 and 8, 5 and 8, 6 and 8, 7 and 8, 2 and 7, 3 and 6, 3 and 5, or any variation therein.y.. o. -

[0224] Also provided herein is an antibody conjugate composition for treatment of prostate cancer, comprising: (a) an internalizing antibody moiety that selectively binds to STEAP1, PSMA, or a bispecific antibody moiety that binds both STEAP1 and PSMA; and (b) a RIPTAC moiety that selectively binds to an intracellular cancer-overexpressed protein, androgen receptor (AR), and additionally binds at least one pan-essential effector protein selected from the group consisting of BRD4, PLK1, BRD2, BRD3, CDK1, CDK2, CDK4, CDK5, CDK6, and CDK9.

[0225] In one embodiment, the antibody moiety is conjugated to the RIPTAC moiety via a cathepsin B cleavable valine-citrulline self-immolative linker, enabling intracellular release of the RIPTAC upon internalization.

[0226] In one embodiment, the antibody moiety is derived from or comprises vandortuzumab for STEAP1 targeting.

[0227] In one embodiment, the bispecific antibody moiety binds simultaneously or separately to STEAP1 and / or PSMA cell-surface antigens expressed on prostate cancer cells.

[0228] Another aspect herein provides an antibody conjugate composition for treatment of Diffuse Large B-Cell Lymphoma (DLBCL), comprising (a) an internalizing antibody moiety that specifically binds to at least one cell surface antigen chosen from CD 19, CD20, BCMA, or a bispecific antibody moiety that binds two of said antigens; and (b) a regulated induced proximity targeting chimera (RIPTAC) moiety designed to form a ternary complex between the intracellular cancer-associated protein enhancer of zeste homolog 2 (EZH2) and a pan-essential effector protein selected from the group consisting of PLK1, BRD2, BRD3, BRD4, CDK1, CDK2, CDK4, CDK5, CDK6, and CDK9. RIPTAC can further be described in, e.g., International Application Publication No. W02024054603, which is incorporated herein by reference.

[0229] In one embodiment, the antibody moiety is conjugated to the RIPTAC moiety via a cathepsin B cleavable linker selected from valine-citrulline-p-aminobenzyl carbamate (Val-Cit-PAB) or a maleimide-based self-immolative linker, facilitating intracellular release of the RIPTAC payload upon endocytosis and lysosomal processing.

[0230] In one embodiment, the antibody moiety is derived from an anti-CD19, anti-CD20, or anti-BCMA monoclonal antibody, each exhibiting internalization upon antigen binding on DLBCL cells.

[0231] In one embodiment, the bispecific antibody moiety simultaneously or separately binds two of the antigens CD 19, CD20, and BCMA to enhance selective delivery and cellular internalization in lymphoma cells.

[0232] Another aspect provided herein is an antibody conjugate comprising: a) an antibody that specifically binds a tumor-associated extracellular antigen; b) a transcriptional / epigenetic chemical inducer of proximity (TOP) comprising: i) a first small-molecule binding moiety that selectively binds an intracellular transcription factor; ii) a second small-molecule binding moiety that selectively binds a transcriptional activator or transcriptional repressor; and iii) a linker covalently joining the two small-molecule binding moieties; and c) a linker covalently joining the antibody to the TOP, the linkery.. o. -being attached to the TCIP through a chemical handle positioned within the internal linker of the TCIP or via a cleavable linker conjugated to one of the small-molecule binding moieties and being chemically discrete from both small-molecule binding moieties, such that the native binding affinity of each moiety for the respective protein is retained following self-immolative linker cleavage and scarless payload release. A transcriptional / epigenetic chemical inducer of proximity (TCIP) is further described in, e.g., US Publication No. US20250127909, which is incorporated herein by reference in its entirety.

[0233] In one embodiment, the intracellular transcription factor is selected from B-cell lymphoma 6 (BCL6), Forkhead-box Protein Pl (FOXP1), Poly(ADP-ribose) Polymerase (PARP) 1, and PARP2.

[0234] In one embodiment, the transcriptional activator or transcriptional repressor is selected from the group consisting of: Bromodomain-containing protein 4 (BRD4), cyclin-dependent kinase 9 (CDK9), ElA-associated protein p300 (p300), and CREB-binding protein (CBP).

[0235] In one embodiment, the linker is atached to the TCIP through a chemical handle positioned within the internal linker of the TCIP or via a cleavable linker conjugated to one of the small-molecule binding moieties and being chemically discrete from both small-molecule binding moieties, such that the native binding affinity of each moiety for the respective protein is retained, e.g., following self-immolative linker cleavage and scarless payload release.

[0236] Another aspect provided herein is an antibody conjugate composition for the treatment of Diffuse Large B-Cell Lymphoma (DLBCL), comprising: (a) an internalizing antibody moiety that specifically binds to one or more cell surface antigens selected from CD 19, CD20, BCMA, or a bispecific antibody moiety that binds two antigens thereof; and (b) a transcriptional / epigenetic chemical inducer of proximity (TCIP), with a target moiety that selectively binds to BCL6 and an effector moiety that binds BRD4 or an alternative transcriptional activator selected from cyclin-dependent kinase 9 (CDK9), histone acetyltransferases (HATs), or p300 / CBP complexes.

[0237] In one embodiment, the antibody is conjugated to the TCIP via a cleavable maleimide-PEG4-Val-Cit-PAB linker, enabling intracellular release of the TCIP moiety upon cathepsin B cleavage following internalization.

[0238] In one embodiment, the antibody moiety is derived from or comprises an anti-CD19 monoclonal antibody (e.g., Loncastuximab), an anti-CD20 monoclonal antibody (e.g., Rituximab), or an anti-BCMA monoclonal antibody (e.g., Belantamab) suitable for internalization into DLBCL cells.

[0239] In one embodiment, the bispecific antibody moiety binds simultaneously or separately two of the three antigens CD 19, CD20, and BCMA to increase selective delivery and internalization into target cells.

[0240] In one embodiment, the TCIP moiety mediates ternary complex formation between intracellular BCL6 and BRD4 / CDK9 / HAT / p300 / CBP transcriptional regulators, thereby inducing transcriptional activation and expression of BCL6 downstream genes in DLBCL cells.

[0241] In one embodiment, the antibody conjugate has a structure disclosed in Table 3.y.. o. -Table 3Antibody Conjugate Structure Notes'"1 AR (intracellular tumor) / BRD4 (intracellular essential) RIPTAC RIPTAC conjugated to Maleimide- r PEG4-Val-Cit-PAB linker iO’*Intended coupling to Vandortuzumab \ * (Anti-STEAP 1 ) or ABBV-969 i y\.~ (STEAP 1 / PSMA bispecific) < & Intended for prostate cancer and p mCRPC treatmentIVy g y yV W"*"■" M y <1 r- X X G Mvs?K BCL6 (intracellular TF) / BRD4 Z" X 0 (intracellular transcriptional.«••••' >•x—'' 8 activator) TCIPx™8’TCIP1 conjugated to Maleimide-,■ «., W'PEG4-Val-Cit-PAB linker z-< 8ft >0 Intended coupling to Anti-CD19 mAb > SK MS (e.g., Loncastuximab)Kr / « fIntended for Diffuse Large B-Cell ™t 1 > -x >;r v » Lymphoma (DLBCL)<;6<....., £...\ li x; —.. MK "•< X " x y-M ■;! / t— N-> Xsy— ( My '■■•s^'e> ■■""••■' K™- o ■■.& '■y.. o. -BCL6 (intracellular TF) / BRD4 (intracellular transcriptional '" X >“®< & y — '.?“«;! "“■< activator) TCIPs t—ii '• —4«“■■■ a- x— 4 r \*ti r'\ >'*** r" ) W / A o ' TCIP1 conjugated to Maleimide- ' J 0 —7i <?% % H5S PEG4-Val-Cit-PAB linker««Ci -■.... / 9Intended coupling to Anti-CD19 mAb HS-4,i •■•$»<• (e.g., Loncastuximab)~~x / "~yIntended for Diffuse Large B-Cell Lymphoma (DLBCL)V»? $ BCL6 (intracellular TF) / CDK9 (intracellular transcriptional 1-4 AJU^A^i> T Y T & activator) TCIP&■ 'ismA CDK-TCIP1 conjugated to oY ci Maleimide-PEG4-Val-Cit-PAB linker I AJIntended coupling to Anti-CD19 mAb J " LI Il l S rVCJ Y K YJ (e.g., Loncastuximab) J I $Intended for Diffuse Large B-Cell ‘XUuMLymphoma (DLBCL)d iW |,c" 'y:..BCL6 (intracellular TF) / y, p300 / CBP(intracellular«3 I«3 transcriptional activator) TCIP <kTCIP3 conjugated to Maleimide- k PEG4-Val-Cit-PAB linker k^■*- Intended coupling to Anti-CD19 mAb _zl,, N>t (e.g., Loncastuximab)O5^ sstMIntended for Diffuse Large B-Cell:#••» 5 1Lymphoma (DLBCL) £ C £:,-Af f f Yz„ 1W t:«kw JMA^OZn ^K-(, Y'U- / #»'AW< W WxSM 4 ^4*~ X lf T j:■• r «Ox„y.. o. -Compounds

[0242] Provided herein is a compound having any of Structures la, lb or 1c, wherein A1and A2independently represent small molecule binder (i.e., a “warhead”) pairings which bind to two distinct biological targets (e.g., protein, RNA, DNA); L1comprises a linker composed of any amino acid, non-proteinogenic amino acid, PEG, n- Alkane, piperidine, cyclobutene, piperazine, or modified combination thereof; L2comprises a linker composed of any cleavable or non-cleavable element which serves to join A1-L1-A2to a targeting compound (TC); wherein TC comprises a targeting compound consisting of an antibody, single-domain antibody, fusion protein, ligand, or nanoparticle.(Structure la)(Structure lb)(Structure 1c)

[0243] In various embodiments, the compound is a stereoisomer, enantiomer, or tautomer thereof or a mixture thereof, or a pharmaceutically acceptable salt, solvate or prodrug thereof. Embodiments of the compounds of this disclosure (e.g., compounds of Structures la, lb or 1c), or their salts, tautomers, or solvates may contain one or more stereocenters and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- ory.. o. -(S)- or, as (D)- or (L)- for amino acids. Embodiments of the present disclosure are meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other features giving rise to geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

[0244] In one embodiment, the compound is a enantiomer. The term “enantiomer” is used to describe one of a pair of molecular isomers which are mirror images of each other and non-superimposable.

[0245] In one embodiment, the compound is a stereoisomer. As used herein, a “stereoisomer” refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. The present disclosure contemplates various stereoisomers and mixtures thereof and includes “enantiomers,” which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0246] In one embodiment, the compound is a tautomer. As used herein, a “tautomer” refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any said compounds. Various tautomeric forms of the compounds are easily derivable by those of ordinary skill in the art.

[0247] L1

[0248] In one embodiment, L1has or comprising the structure of Structure 2.(Structure 2)

[0249] In one embodiment, L1can any linker selected from " L" as disclosed in International Application Publication No. W02024054603, the contents of which is incorporated herein by its entirety.

[0250] In one embodiment, R1of Structure 2 represents the side chain an amino acid or non-proteinogenic amino acid selected from: i) arginine, histidine, lysine, aspartate, glutamate,y.. o. -serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan; or ii) norvaline, norleucine, alloisoleucine, isoserine, t-leucine, pipecolate, 2,3-diaminopropionate, 2,4-diaminobutyrate, ornithine, allothreonine, homocysteine, homoserine, ci-amino-n-heptanoate, ci-aminobutyrate, P-aminobutyrate, y-aminobutyrate, ci-aminoisobutyrate.

[0251] In one embodiment, L1has or comprising the structure of Structure 3 or Structure 4.(Structure 3)(Structure 4)

[0252] In one embodiment, R2and R3of Structure 3 or 4 the side chain of one or two of the amino acids or non-proteinogenic amino acids selected from: i) arginine, histidine, lysine, aspartate, glutamate, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan; or ii) norvaline, norleucine, alloisoleucine, isoserine, t-leucine, pipecolate, 2,3-diaminopropionate, 2,4-diaminobutyrate, ornithine, allothreonine, homocysteine, homoserine, ci-amino-n-heptanoate, ci-aminobutyrate, P-aminobutyrate, y-aminobutyrate, ci-aminoisobutyrate.

[0253] In one embodiment, n1and n2of structures 3 or 4 represent the number of monomeric subunits of said amino acids or non-proteinogenic amino acids, where n1and n2independently equal a value between 1-10.y.. o. -

[0254] In one embodiment, L1has or comprising the structure of Structure 5, Structure 6, or Structure 7.(Structure 5)(Structure 7)

[0255] In one embodiment, R4, R5, and R6of Structure 5, 6, or 7 represents the side chain of 1-3 of the amino acids or non-proteinogenic amino acids selected from: i) arginine, histidine, lysine, aspartate, glutamate, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan; ii) norvaline, norleucine, alloisoleucine, isoserine, t-leucine, pipecolate, 2,3-diaminopropionate, 2,4-diaminobutyrate, ornithine, allothreonine, homocysteine, homoserine, α-amino-n-heptanoate, α-aminobutyrate, β-aminobutyrate, γ-aminobutyrate, α-aminoisobutyrate.

[0256] In one embodiment, n3, n4, and n5of Structure 5, 6, or 7 represent the number of monomeric subunits of said amino acids or non-proteinogenic amino acids where n3, n4, and n5independently equal a value between 1-10.y.. o. -

[0257] In one embodiment, L1has or comprising the structure of Structure 8, Structure 9, or Structure 10.(Structure 8)(Structure 9)(Structure 10)

[0258] In one embodiment, S1and S2of Structure 8, Structure 9, or Structure 10 represent spacers selected from the group consisting of: poly(ethylene glycol) (PEG), poly (glycol) chains, alkyl chains, alkyne, triazole, piperazine, piperidine, and a combination thereof.

[0259] In one embodiment, L1has or comprising the structure of Structure 11 or Structure 12.y.. o. -(Structure 11)(Structure 11)(Structure 12)

[0260] In one embodiment, S3, S4, and S5of Structure 11 or Structure 12 represent optional spacers selected from the group consisting of: poly(ethylene glycol) (PEG), poly (glycol) chains, alkyl chains, alkyne, triazole, piperazine, piperidine, and a combination thereof.

[0261] In one embodiment, L1has or comprising the structure of Structure 13, Structure 14, or Structure 15.(Structure 13)y.. o. -(Structure 14)(Structure 15)

[0262] In one embodiment, S6, S7, S8, and S9of Structure 13, Structure 14, or Structure 15 represent optional spacers selected from the group consisting of: poly(ethylene glycol) (PEG), poly (glycol) chains, alkyl chains, alkyne, triazole, piperazine, piperidine, and a combination thereof.

[0263] In one embodiment, L1has or comprising the structure of Structure 16.(Structure 16)

[0264] In one embodiment, n6, n7, and n8of Structure 16 represent the number of monomeric subunits where n6, n7, and n8independently equal a value between 1-20

[0265] L2y.. o. -

[0266] In one embodiment, L2comprises a structure listed in Figure 3. That is, in various embodiment, L2comprises a structure of Structure 17, Structure 18, Structure 19, Structure 20, Structure 21, Structure 22, or Structure 23,

[0267] In one embodiment, n9- n14of Structure 17, Structure 18, Structure 19, Structure 20, Structure 21, Structure 22, or Structure 23 represent the number of monomeric subunits where n9- n14independently equal a value between 1-20.

[0268] A1and A2

[0269] In one embodiment, A1and A2represent a biologically active pairing of small molecules which functions as a regulated induced proximity targeting chimera (RIPTAC).

[0270] In one embodiment, A1and A2represent a biologically active pairing of small molecules which functions as a proteolysis-targeting chimera (PROTAC). A “proteolysis targeting chimera” or “PROTAC” refers to a heterobifunctional molecule comprising: (i) a targeting moiety that specifically binds to a target protein; (ii) an E3 ligase binding moiety that specifically binds to an E3 ubiquitin ligase; and (iii) a linker covalently connecting the targeting moiety and the E3 ligase binding moiety, wherein the PROTAC induces ubiquitination and proteasomal degradation of the target protein.

[0271] In one embodiment, A1and A2represent a biologically active pairing of small molecules which functions as an autophagy-targeting chimera (AUTAC). An “autophagy-targeting chimera”, “AUTAC” or “AUTOTAC” refers to a heterobifunctional molecule comprising: (i) a targeting moiety that specifically binds to a cellular target; (ii) an autophagy-enhancing moiety capable of promoting the recruitment of autophagy machinery; and (iii) a linker covalently connecting the targeting moiety and the autophagy-enhancing moiety, wherein the AUTAC induces autophagic degradation of the cellular target.

[0272] In one embodiment, A1and A2represent a biologically active pairing of small molecules which functions as a specific and non-genetic inhibitor of apoptosis protein (LAP)-dependent protein eraser (SNIPER). A “specific and non-genetic LAP-dependent protein eraser” or “SNIPER” refers to a heterobifunctional molecule comprising: (i) a targeting ligand that specifically binds to a target protein; (ii) an IAP-binding moiety that binds to an inhibitor of apoptosis protein; and (iii) a linker covalently connecting the targeting ligand and the IAP-binding moiety, wherein the SNIPER facilitates the ubiquitination and proteasomal degradation of the target protein.

[0273] In one embodiment, A1and A2represent a biologically active pairing of small molecules which functions as a deubiquitinase-targeting chimera (DUBTAC). A “deubiquitinase-targeting chimera” or “DUBTAC” refers to a heterobifunctional molecule comprising: (i) a targeting moiety that specifically binds to a ubiquitinated target protein; (ii) a DUB-binding moiety that specifically binds to a deubiquitinase enzyme; and (iii) a linker covalently connecting the targeting moiety and the DUB-binding moiety, wherein the DUBTAC facilitates the removal of ubiquitin from the target protein, thereby stabilizing the target protein.y.. o. -

[0274] In one embodiment, A1and A2represent a biologically active pairing of small molecules which functions as a phosphorylation-inducing chimeras (PHICs). A “phosphorylation-inducing chimeric small molecule” or “PHIC” refers to a heterobifunctional molecule comprising: (i) a targeting moiety that specifically binds to a target protein; (ii) a kinase-binding moiety that specifically binds to a kinase enzyme; and (iii) a linker covalently connecting the targeting moiety and the kinase-binding moiety, wherein the PHIC facilitates phosphorylation of the target protein by the kinase enzyme.

[0275] In one embodiment, A1and A2represent a biologically active pairing of small molecules which functions as a phosphatase-recruiting chimera (PHORCs). A “phosphatase recruitment chimera” or “PHORC” or “phosphorylation-targeting chimera” or “PhosTAC” refers to a heterobifunctional molecule comprising: (i) a targeting moiety that specifically binds to a phosphorylated target protein; (ii) a phosphatase-binding moiety that specifically binds to a phosphatase enzyme; and (iii) a linker covalently connecting the targeting moiety and the phosphatase-binding moiety, wherein the PHORC / PhosTAC facilitates the dephosphorylation of the target protein by the phosphatase enzyme.

[0276] In one embodiment, A1and A2represent a biologically active pairing of small molecules which functions as a ribonuclease targeting chimera (RIBOTAC). A “ribonuclease-targeting chimera” or “RIBOTAC” refers to a heterobifunctional molecule comprising: (i) an RNA-targeting moiety that specifically binds to a target RNA molecule; (ii) a ribonuclease-binding moiety that specifically binds to a ribonuclease enzyme; and (iii) a linker covalently connecting the RNA-targeting moiety and the ribonuclease-binding moiety, wherein the RIBOTAC facilitates the enzymatic degradation of the target RNA by the ribonuclease enzyme.

[0277] In one embodiment, A1and A2represent a biologically active pairing of small molecules which functions as an acetylation tagging system (AceTAG). An “acetylation tagging system” or “AceTAG” refers to a heterobifunctional molecule comprising: (i) a targeting moiety that specifically binds to a target protein; (ii) an acetyltransferase-binding moiety that specifically binds to an acetyltransferase enzyme; and (iii) a linker covalently connecting the targeting moiety and the acetyltransferase-binding moiety, wherein the AceTAG facilitates the acetylation of the target protein by the acetyltransferase enzyme.

[0278] Targeting Compound (TC)

[0279] In various embodiments, the TC comprises an antibody, single-domain antibody, fusion protein, ligand, or nanoparticle. Throughout this disclosure, the term “antibody” refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive toward, an antigen. The portion of the antibody that binds the antigen may be referred to as an “antigen binding domain.” In certain embodiments, an antibody is an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as an antigen-binding portion of an intact antibody that has or retains the capacity to bind a target molecule. An antibody or an antigen binding fragment thereof of this disclosure can include, for example, polyclonal, monoclonal, and genetically engineered antibodies. A monoclonal antibody or antigen-binding portion thereof of this disclosure can be, fory.. o. -example, non-human (e.g., murine, rabbit), chimeric, humanized, or human. In certain embodiments, an antibody of this disclosure is a heteroconjugate, bispecific, multi-specific, diabody, triabody, or tetrabody. Immunoglobulin structure and function are reviewed, for example, in Greenfield, Ed., Antibodies: A Laboratory Manual, Chapters 2 and 3 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 2014).

[0280] For example, the terms “VL” and “VH” refer to the variable binding region from an antibody light and heavy chain, respectively. The variable binding regions are made up of discrete, well-defined sub-regions known as “complementarity determining regions” (CDRs) and “framework regions” (FRs). The term “CL” refers to an “immunoglobulin light chain constant region” or a “light chain constant region,” i.e., a constant region from an antibody light chain. The term “CH” refers to an “immunoglobulin heavy chain constant region” or a “heavy chain constant region,” which is further divisible, depending on the antibody isotype into CH1, CH2, and CH3 (IgA, IgD, IgG), or CH1, CH2, CH3, and CH4 domains (IgE, IgM).

[0281] The term “target”, “target protein”, or “biological target” as used herein refers to a biological substance, compound, or molecule, which after activation, inhibition, degradation, or binding has a specific and intended downstream therapeutic effect. The term “target” encompasses cytosolic proteins (e.g., Bruton’s tyrosine kinase (BTK), Kirsten rat sarcoma viral oncogene homolog (KRAS)), nuclear proteins (e.g., c-myc, P-catenin, MDM2), membrane bound proteins (e.g., G protein-coupled receptors, tyrosine kinase transmembrane receptors, hormone receptors, chemokine receptors, ligand gated ion channels, programmed death-ligand 1) and extracellular proteins (e.g., tumor necrosis factor alpha (TNF-a), transforming growth factor beta (TGF-P)). The term “target” or “biological target” may also encompass enzymes, voltage gated ion channels, structural proteins, nucleic acids, transporters, signaling proteins, or another ligand. The term “target” may refer to a single entity or a set of entities comprising multiple components of a shared signaling pathway. Additional target proteins may include HER2, EGFR, androgen receptor protein, estrogen receptor protein, ALK, IDH1, FLT3, FGFR1, FGFR4, HCV-NS3, FGFR2, FGFR3, ERK1, ERK2, FGR, HER3, HER4, or PI3Ka.

[0282] In one embodiment, the compound comprises at least one linker. As used herein, a “linker” refers to a contiguous chain of at least one atom, such as carbon, oxygen, silicon, nitrogen, sulfur, phosphorous, and combinations thereof, which connects a portion of a molecule to another portion of the same molecule or to a different molecule or fragment thereof via a covalent bond (e.g., a single bond, double bond, or triple bond). In some embodiments, the linker is a proteinogenic amino acid, a non-proteinogenic amino acid, poly(ethylene glycol), piperazine, piperidine, cyclobutene, alkyl chain, alkyne, triazole, or combinations thereof. Unless otherwise stated specifically in the specification, a linker may be optionally substituted.

[0283] In one embodiment, the compound comprises at least one immolative element. As used herein, “immolative element” or “self-immolative spacer” refers to chemically labile group that facilitates release or cleavage between portions of a molecule (e.g., between an antibody-linker and a payload).y.. o. -An immolative element typically a divalent linker motif that is amenable to one or more chemical reactions that ultimately results in cleavage of the covalent bonds between the two terminal ends of the group. The reactions occur as a result some type of stimuli to the system (e.g., a protease catalyzing the reaction during its recognition of a trigger element or decrease in pH when the antibody-drug conjugate is subjected to an intracellular environment) which then results in controlled release of a payload. Commonly used immolative elements are the para-aminobenzyl carbamate (PABC), para-aminobenzyl ether (PAB), diaminoethyl / ethylene diamine, P-glucuronide, o-hydroxydihydrocinnamic acid derivatives, and hemiaminal.

[0284] The foregoing terms include all systems that comprise effective cleavage elements in the endo-lysosomal processing of antibody-drug conjugates. Exemplary systems include cathepsin-based enzymatic peptide sequences, such as Val-Cit, Vai- Ala, Ala- Ala, Gly-Gly-Phe-Gly, or the like. Also included are trigger elements that are sensitive to the enzyme P-glucuronidase, which contain a glucuronic acid as a trigger element. In some embodiments, a trigger element is directly and covalently bound to an immolative element. In some embodiments, an immolative element is directly and covalently bound to a payload and once cleavage occurs, a payload is then irreversibly released.

[0285] In one embodiment, the compound comprises at least one non-cleavable element. As used herein, “non-cleavable element” refers to a linker that does not include either a trigger element or an immolative element and is not cleaved under normal physiological conditions. Depending on the target, many conjugates can be quite effective without a formal cleavage release element.Accordingly, in some embodiments a heterobifunctional payload may be directly attached to a proteinogenic or non-proteinogenic amino acid which is then conjugated to a targeting compound via a non-cleavable linker.

[0286] In one embodiment, the compound comprises at least one trigger element. As used herein, “trigger element” refers to a molecular motif that is recognized by a biological molecule (e.g., a protease such as cathepsin) or susceptible to a chemical reaction in a biological environment (e.g., acid labile). Typically an enzymatic recognition of the trigger element results in a catalyzed cleavage reaction that results in release of a payload from the antibody+linker(s).

[0287] In one embodiment, the compound comprises at least one heteroalkylene element. As used herein, “heteroalkylene element” refers to a linker comprising one or more heteroalkylene, as defined above. In some embodiments, a heteroalkylene element can be used to optimize characteristics of the linker-payload. In some embodiments, a heteroalkylene elements) increases a linear connection between other elements (e.g., charged elements, hydrophilic elements, etc.). In some embodiments, a heteroalkylene element increases the distance between a polar cap or a payload of the phenyl-maleimide portion of Structure (1). Heteroalkylene elements vary in length, structure, polarity, degree of branching, or the like. The aforementioned variables allow the linker-payload and, in turn, the antibody-linker-drug conjugate to have the best overall properties (e.g., solubility, to be monodisperse, etc.).y.. o. -

[0288] In some embodiments, a heteroalkylene element comprises a linear polyethylene glycol with two ethylene glycol units (i.e., PEG2) and one or more amino acid(s). In another embodiment, a heteroalkylene element comprises a Gly-Gly dipeptide. In some embodiments, a heteroalkylene element includes a PEG 1-24, di-, tri- and tetra-peptide, or combinations thereof. In other embodiments, a heteroalkylene element comprises amino acids. In another embodiment, a heteroalkylene element comprises PEG1-24. In another embodiment, a heteroalkylene element comprises PEG1-12. In another embodiment, a heteroalkylene element comprises PEG2-6. In some embodiments, both PEGx and amino acids combine to form a heteroalkylene element. Exemplary linear PEG moieties can be found, e.g., in PCT Publication No. WO 2021 / 207701, which PEG moieties are hereby incorporated by reference in its entirety.

[0289] In one embodiment, the compound comprises at least one hydrophilic element. As used herein, “hydrophilic element” refers to a portion of Structure (1) that effectively promotes solubility in aqueous solvents (e.g., water, phosphate buffered saline) for the entirety of the molecule. In effect, a hydrophilic element conceals or counteracts hydrophobic portions of the linker-payload such that the resultant molecule is stable in an aqueous environment (e.g., pH 7.4 buffered water). In some embodiments, a hydrophilic element produces a stable and soluble linker-payload with improved overall physicochemical properties. A hydrophilic element provides a suitable chemical functionality that enables stable conjugation to a protein. In some embodiments, incorporation of an appropriate hydrophilic elements) leads to the resultant protein conjugate to possess an ADME / DMPK profile that is more protein-like.

[0290] In some embodiments, a hydrophilic element includes PEG linkers of medium length (1014 units or PEG10-14). In some embodiments, a hydrophilic element includes PEG lengths from 224 units (i.e., PEG2-24). In some embodiments, the length and amount of branching included in a hydrophilic element can be adjusted based on other elements present in Structure (1). In some embodiments, the hydrophilic element is a C1-C6 alkoxy (e.g., methoxy). In some embodiments, a hydrophilic element comprises poly-sarcosine (PSAR). One aspect of a hydrophilic element imparts polarity and hydrophilicity to the overall conjugate. In some embodiments, a hydrophilic element is large enough in size and flexible enough in structure to mask hydrophobicity with or without a trigger element. In some embodiments, a hydrophilic element is divalent. In some embodiments, a hydrophilic element is a monovalent radical. In some embodiments, a hydrophilic element is multivalent. In some embodiments, a hydrophilic element is branched. In some embodiments, a hydrophilic element is linear.

[0291] Hydrophilic groups (e.g., moieties that make up a hydrophilic element) are known to those of ordinary skill in the art. For example, hydrophilic groups can be found in PCT Publication Nos. WO 2019 / 217591, WO 2018 / 089373 and the hydrophilic groups from each are hereby incorporated by reference in their entirety.

[0292] In one embodiment, the compound comprises the elements as disclosed in Table 4. In one embodiment, the compound comprises the elements and for the indications as disclosed in Table 4.y.. o. -Table 4SmallIndication Extracellular Target IntracellularMolecule Effector Protein Modality TargetMetastaticCastration AR (WT / LBDSTEAP1 / PSMA BRD4 RIPTACResistant Prostate mutations)Cancer (mCRPC)Diffuse Large B- CD19 / CD20 / BCMA BRD4 Cell Lymphoma TCIP BCL6(DLBCL)Diffuse Large B- CD19 / CD20 / BCMA EZH2 BRD4 / PLK1 Cell Lymphoma RIPTAC(DLBCL)Acute Myeloid CD33 / CLEC12A FOXP1 TCIP BRD4 / CDK9 Leukemia

[0293] Derivatives and prodrugs

[0294] In various embodiments, compounds disclosed herein include enantiomers, derivatives, prodrugs, and pharmaceutically acceptable salts thereof. The term “derivative” as used herein refers to a chemical substance related structurally to another, i.e., an “original” substance, which can be referred to as a “parent” compound. A “derivative” can be made from the structurally-related parent compound in one or more steps. The general physical and chemical properties of a derivative are also similar to the parent compound.Preparation

[0295] Preparation of heterobifunctional regulated induced proximity targeting chimeras (RIPTACs) employing amino acids and / or non-proteinogenic amino acids facilitating conjugation to additional chemical or biological entities.

[0296] The synthetic route illustrated in Scheme 1 is a general method for preparing compound (6). Reaction of amine on the compound (1) and carboxylic acid on compound (2) and amine under amide coupling conditions provides amide (3). Removal of the protection group (PG1) from compound 3 provides compound (4). The PG1 may be, for example, a Boc protecting group that can be removed by treating the compound with trifluoroacetic acid. Coupling of compound (4) with compound (5) under amide coupling conditions provides the final compound (6).y.. o. -Scheme 1

[0297] The synthetic route illustrated in Scheme 2 is a general method for preparing compounds 6b.Reaction of amine on the compound (1) and carboxylic acid on compound (2) and amine under amide coupling conditions provides amide (3b). Removal of the protection group (PG2) from compound (3b) provides compound (4b). The PG2 may be, for example, a Fmoc protecting group that can be removed by treating the compound with piperidine. Coupling of compound (4b) with compound (5) under amide coupling conditions provides the final compound (6b).Scheme 2

[0298] The modular synthetic route illustrated in Scheme 1 and Scheme 2 can be readily modified to provide additional compounds by conducting functional group transformations on the intermediate and / or final compounds. Such functional group transformations are well known in the art, asdescribed in, for example, Comprehensive Organic Synthesis (BM Trost & I. Fleming, eds., 1991- 1992); Organic Synthesis, 3rd Ed. (Michael B. Smith, Wavefunction, Inc., Irvine: 2010); Moderny.. o. -Methods of Organic Synthesis, 4 th Ed. (William Carruthers and Iain Coldham Cambridge University Press, Cambridge: 2004); March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 8th Ed., (Michael B. Smith, John Wiley & Sons, New York: 2020); and Comprehensive Organic Transformations: A Guide to Functional Group Preparations, 3rd Ed. (Richard C. Larock, ed., John Wiley & Sons, New York: 2018).

[0299] Protecting group strategies may be deployed as appropriate to accommodate differing functional groups in the molecules used in the synthetic route. Protecting group chemistry and strategy is described in, for example, Protecting Groups in Organic Synthesis, 3rd Edition, TW Greene and PGM Wuts, John Wiley & Sons, 1999 and Greene's Protective Groups in Organic Synthesis, 5th Ed., (Peter GM Wuts, John Wiley & Sons: 2014).

[0300] In certain embodiments, the compound is a compound in Table 1, or a pharmaceutically acceptable salt thereof.

[0301] In certain embodiments, the compound is a compound in Table 1 with a R-group consisting of a compound from Table 2, or a pharmaceutically acceptable salt thereof. In one embodiment, n is equal to a value between 1-20.vV s & CiPEA, HOA1. OMFScheme 3

[0302] The synthetic route illustrated in Scheme 3 is a general method for preparing compound (9). Removal of the protection group (PG2) from compound (6) provides compound (7). The PG2 may be,y.. o. -for example, a Fmoc protecting group that can be removed by treating the compound with piperidine. Coupling of compound (7) with compound (8) (such as a nucleophilic substitution reaction) provides the final compound (9).Scheme 4

[0303] The synthetic route illustrated in Scheme 4 is a general method for preparing compounds of formula (12). Solution of targeting compound is mixed with compound (10) and stirred at room temperature to provide compound (12). Targeting compound may be an antibody prepared with TCEP reduction.

[0304] Preparation of diverse heterobifunctional compounds

[0305] In various embodiments, the diverse heterobifunctional compound is a transcriptional / epigenetic chemical inducer of proximity (TCIP), regulated induced proteolysistargeting chimera (PROTAC), autophagy-targeting chimera (AUTAC), specific and non-genetic inhibitor of apoptosis protein (IAP)-dependent protein eraser (SNIPER), deubiquitinase-targeting chimera (DUBTAC), phosphorylation-inducing chimera (PHIC), phosphatase-recruiting chimera (PHORC), ribonuclease targeting chimera (RIBOTAC), and acetylation tagging system (AceTAG) employing amino acid and / or non-proteinogenic amino acids facilitating conjugation to additional chemical or biological entities.

[0306] Employing a similar synthetic route as illustrated in Scheme 1, one can produce diverse heterobifunctional compounds simply by using diverse warheads which achieve a biologically active pairing for the purposes of protein degradation (e.g., PROTAC, AUTAC, SNIPER), protein stabilization (DUBTAC), protein phosphorylation (PHIC), protein dephosphorylation (PHORC), RNA degradation (RIBOTAC), protein acetylation (AceTAG), or transcriptional activation / suppression (TOP).Compositionsy.. o. -

[0307] One aspect disclosed herein is a composition comprising a compound disclosed herein, e.g., a compound having a structure of Structures la, lb or 1c.

[0308] Another aspect disclosed herein is a pharmaceutical composition comprising a compound disclosed herein, e.g., a compound having a structure of Structures la, lb or 1c.

[0309] The compositions described herein can be in form of a pharmaceutical composition. For example, the composition comprising a compound disclosed herein can further comprise with one or more pharmaceutically acceptable carriers (additives), excipient and / or diluents.

[0310] The compositions can be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; or (3) topical application, for example, as a film, sheet, dressing, cream, ointment, liquid, gel, hydrogel, emulsion, suspension or a controlled-release patch or spray applied to the skin. Delivery using topical, oral or intravenous methods can be particularly advantageous. Accordingly, in some embodiments, the composition is formulated for oral or intravenous administration.

[0311] In some embodiments, the composition is formulated for topical administration. For topical administration, the composition can be in the form of a film, sheet, dressing, cream, ointment, liquid, gel, hydrogel, emulsion, suspension or a controlled-release patch or spray applied. In some embodiments, the composition formulated for topical administration is in the form of an adhesive.

[0312] In one embodiment, the composition is formulated for and delivered via subcutaneous injections. In one embodiment, the composition is formulated for and delivered via microneedle patches

[0313] In some embodiments, the composition is formulated for oral or intravenous administration.

[0314] Generally, the composition, e.g., pharmaceutical composition comprises an effective or therapeutically effective amount of the compound. The phrase “therapeutically effective amount” as used herein means that amount of a compound, material, or composition comprising a compound described herein which is effective for producing some desired therapeutic effect in a subject at a reasonable benefit / risk ratio applicable to any medical treatment, e.g., treatment of cancer or a disease or disorder due to cancer.

[0315] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0316] Embodiments of the compositions described herein include pharmaceutically acceptable carriers (additives), excipient and / or diluents. The phrase “pharmaceutically acceptable carrier” as used hereiny.. o. -means a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer’s solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

[0317] In some embodiments, the composition is formulated as a unit dosage formulation.

[0318] Techniques and formulations generally can be found in Remington’s Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, anti-tumoral compounds described herein can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank’s solution or Ringer’s solution. In addition, the anti-tumoral compound described herein can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.

[0319] For oral administration, the pharmaceutical composition can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueousy.. o. -vehicles (e.g., pharmaceutically acceptable oils, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate.

[0320] Preparations for oral administration can be suitably formulated to give controlled release of the active compound. For buccal administration the compositions can take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use as described herein are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0321] The compound of the present invention can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0322] In addition to the formulations described previously, the anti-tumoral compound can also be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the antibodies can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0323] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be through nasal sprays or using suppositories. For topical administration, the anti-tumoral compound can be formulated into ointments, salves, gels, or creams as generally known in the art. A wash solution can be used locally to treat an injury or inflammation to accelerate healing.

[0324] The compositions can, if desired, be presented in a pack or dispenser device which can contain one or more-unit dosage forms containing the active ingredient. The pack can for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration.y.. o. -

[0325] In some embodiments, the composition further comprises one or more of binders, viscosity modifiers, preservatives, humectants, emollients, pH stabilizing agents, chelating agents, gelling agents, thickening agents, emulsifiers, buffers, and carriers.

[0326] As used here, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0327] As used here, the term "pharmaceutically-acceptable carrier" means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, binding agents, fillers, lubricants, coloring agents, disintegrants, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative, water, salt solutions, alcohols, antioxidants, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like can also be present in the formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein.Methods of treatmenty.. o. -

[0328] One aspect provided herein is a methods of treating a subject in need thereof, e.g., having cancer, the method comprising administering to the subject an effective amount of any compound disclosed herein, or composition thereof.

[0329] In one embodiment, the method further comprises administering at least a second therapeutic. For example, the subject is further administered at least a second anti-cancer therapeutic.

[0330] In one embodiment, the cancer is a solid tumor.

[0331] In one embodiment, the cancer is a hematologic tumor.

[0332] In one embodiment, the cancer is a metastatic. In one embodiment, the cancer is not metastatic.

[0333] In one embodiment, the cancer is a carcinoma, a primary central nervous system tumor, a melanocytic tumor, a germ cell tumor, a sarcoma, and a hematological malignancy.

[0334] As used herein, a “carcinoma” refers to any malignant neoplasm originating from epithelial cells, including but not limited to cells lining glandular structures, ducts, or mucosal surfaces.Carcinomas may arise in a wide variety of tissues, including reproductive, gastrointestinal, respiratory, endocrine, and integumentary systems. Exemplary carcinomas include, without limitation, ovarian carcinoma, endometrial carcinoma, cervical carcinoma, prostate carcinoma, breast carcinoma, lung carcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, gastric carcinoma, colorectal carcinoma, renal cell carcinoma, urothelial carcinoma, hepatocellular carcinoma, cholangiocarcinoma, pancreatic ductal adenocarcinoma, thyroid carcinoma, cutaneous squamous cell carcinoma, basal cell carcinoma, and sebaceous gland carcinoma.

[0335] As used herein, a “primary central nervous system (CNS) tumor” refers to any neoplastic growth arising from neural or glial cell lineages, meningeal tissues, or other intracranial or spinal structures. Primary CNS tumors include, without limitation, gliomas (e.g., astrocytoma, oligodendroglioma, glioblastoma), medulloblastoma, ependymoma, meningioma, schwannoma, and other tumors of neuronal or glial origin.

[0336] As used herein, a “melanocytic tumor” refers to any benign or malignant neoplasm derived from melanocytes, including but not limited to cutaneous melanoma, mucosal melanoma, uveal or ocular melanoma, acral melanoma, and desmoplastic melanoma. Melanocytic tumors may exhibit mutations in BRAF, NRAS, KIT, GNAQ, or GNA11, among other genes, and are characterized by dysregulated pigment cell proliferation and invasive potential.

[0337] As used herein, a “germ cell tumor” refers to any tumor arising from germline precursor cells, including testicular, ovarian, extragonadal, or mediastinal germ cell tumors. Germ cell tumors encompass seminomas, dysgerminomas, embryonal carcinomas, choriocarcinomas, yolk sac tumors, teratomas, and mixed germ cell tumors. These neoplasms may exhibit characteristic markers (e.g., AFP, P-hCG) and originate from totipotent or pluripotent cell populations.

[0338] As used herein, a “sarcoma” refers to a malignant neoplasm arising from mesenchymal or connective tissue lineages, including but not limited to bone, muscle, fat, vascular tissue, cartilage, ory.. o. -fibroblasts. Representative sarcomas include osteosarcoma, chondrosarcoma, Ewing sarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, synovial sarcoma, and undifferentiated pleomorphic sarcoma. Sarcomas are characterized by high cellular heterogeneity and may exhibit chromosomal translocations, fusion genes (e.g., EWSR1-FLI1), or dysregulated developmental pathways.

[0339] As used herein, a “hematological malignancy” refers to any neoplastic disorder of blood-forming tissues, including malignancies of lymphoid or myeloid origin. Hematological malignancies include, without limitation, lymphomas (e.g., Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, T-cell lymphoma), leukemias (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia), plasma cell neoplasms (e.g., multiple myeloma), and myeloproliferative neoplasms.

[0340] In one embodiment, the carcinoma is selected from the group consisting of: ovarian cancer, uterine cancer, endometrial cancer, cervical cancer, prostate cancer, breast cancer, lung cancer, oral cancer, esophageal cancer, head and neck cancer, stomach cancer, colon cancer, rectal cancer, basal cell carcinoma, squamous cell carcinoma, sebaceous gland carcinoma, bile duct cancer, gallbladder cancer, liver cancer, pancreatic cancer, bladder cancer, urinary tract cancer, renal cell carcinoma and thyroid cancer. In one embodiment, the primary central nervous system tumor is a brain cancer. In one embodiment, the melanocytic tumor is eye cancer or cutaneous melanoma. In one embodiment, the germ cell tumor is testicular cancer or ovarian cancer. In one embodiment, the sarcoma is a uterine sarcoma. In one embodiment, the hematological malignancy is lymphoma or leukemia.

[0341] One aspect herein provides a method of treating metastatic castration-resistant prostate cancer in a subject comprising administering to the subject a therapeutically effective amount of the antibody conjugate composition disclosed herein, e.g., an antibody conjugate composition for treatment of prostate cancer, comprising: (a) an internalizing antibody moiety that selectively binds to STEAP1, PSMA, or a bispecific antibody moiety that binds both STEAP1 and PSMA; and (b) a RIPTAC moiety that selectively binds to an intracellular cancer-overexpressed protein, androgen receptor (AR), and additionally binds at least one pan-essential effector protein selected from the group consisting of BRD4, PLK1, BRD2, BRD3, CDK1, CDK2, CDK4, CDK5, CDK6, and CDK9.

[0342] In one embodiment, the treatment selectively accumulates in prostate cancer cells due to antibody targeting and kills prostate cancer cells by inducing stable ternary complexes between AR, RIPTAC, and the pan-essential effector protein, resulting in steric inhibition of effector protein function and selective tumor cell death.

[0343] In one embodiment, the subject has or is at risk at having a cancer comprising a mutation found in human prostate cancer cell lines VCaP, LNCaP, or 22Rv1 with differential or engineered STEAP1 expression, including STEAP1 knockout (STEAPlko), STEAP1 low expression (STEAPllow), and STEAP1 overexpressing (STEAPlhi / amp) variants.y.. o. -

[0344] In one embodiment, the composition provides superior in vivo anti-tumor efficacy as compared to androgen receptor RIPTACs, androgen receptor antagonists, or androgen receptor degraders, e.g., it is superior by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or more as compared to androgen receptor RIPTACs, androgen receptor antagonists, or androgen receptor degraders.

[0345] Also provided herein is a method of selectively killing DLBCL cells in a subject comprising administering a therapeutically effective amount of the antibody conjugate disclosed herein, wherein cell killing is mediated by activation of previously repressed transcriptional regulatory pathways critical for lymphoma survival and proliferation. For example, administering the antibody conjugate composition for the treatment of Diffuse Large B-Cell Lymphoma (DLBCL), comprising: (a) an internalizing antibody moiety that specifically binds to one or more cell surface antigens selected from CD 19, CD20, BCMA, or a bispecific antibody moiety that binds two antigens thereof; and (b) a transcriptional / epigenetic chemical inducer of proximity (TCIP), with a target moiety that selectively binds to BCL6 and an effector moiety that binds BRD4 or an alternative transcriptional activator selected from cyclin-dependent kinase 9 (CDK9), histone acetyltransferases (HATs), or p300 / CBP complexes.

[0346] Also provided herein is a method of treating DLBCL in a subject, comprising administering a therapeutically effective amount of the antibody conjugate disclosed herein, thereby inducing selective tumor cell death by the formation of a ternary complex between intracellular RIPTAC, EZH2, and a pan-essential effector protein, leading to functional inhibition of essential proteins in lymphoma cells.

[0347] In one embodiment, the subject has or is at risk of having DLBCL. In one embodiment, the subject has a cancer bearing a mutation found in human DLBCL cell lines selected from SUDHL5 or KARPAS422.

[0348] In one embodiment, the antibody conjugate exhibits improved tumor selectivity and reduced systemic toxicity due to the dual mechanism of targeted antibody internalization and transcriptional / epigenetic chemical inducer of proximity induction, e.g., it is improved by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or more as compared to an appropriate control.

[0349] In one embodiment, administration of the antibody conjugate results in significant tumor growth inhibition, induction of apoptosis, and improved survival relative to control treatments, e.g., e.g., it is improved by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or more as compared to an appropriate control.

[0350] In one embodiment, the antibody conjugate exhibits superior therapeutic efficacy and reduced systemic toxicity compared to unconjugated RIPTAC or antibody monotherapy, e.g., it is improved by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or more as compared to an appropriate control.

[0351] Administrationy.. o. -

[0352] The compounds described herein can be administered to a subject having or diagnosed as having a disease or disorder, e.g., cancer. In some embodiments, the methods described herein comprise administering an effective amount of at least one compound disclosed herein to a subject in order alleviate at least one symptom associated with the disease or disorder. As used herein, "alleviating at least one symptom " is ameliorating any condition or symptom associated with the disease or disorder. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compounds described herein to subjects are known to those of skill in the art. In one embodiment, the compounds are administered systemically or locally. In one embodiment, the compounds are administered intravenously. In one embodiment, the compounds are administered continuously, in intervals, or sporadically. The route of administration of the compounds will be optimized by a skilled practitioner.

[0353] The term “effective amount" as used herein refers to the amount of the compounds that can be administered to a subject having or the disease or disorder, e.g., cancer, needed to alleviate at least one or more symptom. The term "therapeutically effective amount" therefore refers to an amount of the compounds that is sufficient to provide a particular effect when administered to a typical subject. It is not generally practicable to specify an exact “effective amount". However, for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0354] In one embodiment, the subject is administered more than one compound in a single composition. In one embodiment, the subject is administered two separate compositions, each comprising a single compound. When the subject is administered two separate compositions, the two separate compositions can be administered at substantially the same time. Alternatively, the two separate compositions can be administered sequentially. For example, the second separate composition is administered within at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 96 hours, or more following administration of the first separate composition.

[0355] Effective amounts, toxicity, and therapeutic efficacy can be evaluated by standard pharmaceutical procedures in cell cultures or experimental animals. The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50.Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated iny.. o. -animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the compounds, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0356] Dosage

[0357] " Unit dosage form" as the term is used herein refers to a dosage for suitable one administration. By way of example a unit dosage form can be an amount of therapeutic disposed in a delivery device, e.g., a syringe or intravenous drip bag. In one embodiment, a unit dosage form is administered in a single administration. In another, embodiment more than one unit dosage form can be administered simultaneously.

[0358] The dosage of the compounds as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to administer further cells, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosage should not be so large as to cause adverse side effects, such as cytokine release syndrome. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

[0359] Combinational therapy

[0360] In one embodiment, the compounds described herein is used as a monotherapy. In one embodiment, the compounds described herein can be used in combination with other known agents and therapies, for example, for a disease or disorder, e.g., cancer. Administered "in combination," as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous", “at substantially the same time” or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disease or disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect ofy.. o. -the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered. The compounds described herein and the at least one additional therapy can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the compounds described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed. The compounds can be administered before another treatment, concurrently with the treatment, post-treatment, or during remission of the disorder.

[0361] Parenteral Dosage Forms

[0362] Parenteral dosage forms of the compounds described herein can be administered to a subject by various routes, including, but not limited to, epidural injection, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, controlled-release parenteral dosage forms, and emulsions.

[0363] Suitable vehicles that can be used to provide parenteral dosage forms of the disclosure are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0364] Controlled and Delayed Release Dosage Forms

[0365] In some embodiments of the aspects described herein, the compound is administered to a subject by controlled- or delayed-release means. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. (Kim, Chemg-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000)). Controlled-release formulations can be used to control a compound’s onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations cany.. o. -be used to ensure that the maximum effectiveness of the compound is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug.

[0366] A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the compounds described herein. Examples include, but are not limited to, those described in U. S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719;5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185, each of which is incorporated herein by reference in their entireties. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)), multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions. Additionally, ion exchange materials can be used to prepare immobilized, adsorbed salt forms of the disclosed compounds and thus effect controlled delivery of the drug. Examples of specific anion exchangers include, but are not limited to, DUOLITE® A568 and DUOLITE® AP 143 (Rohm& Haas, Spring House, Pa. USA).

[0367] Efficacy

[0368] The efficacy of any compound described herein can be determined by the skilled practitioner. However, a treatment is considered “effective treatment," as the term is used herein, if one or more of the signs or symptoms of the disease or disorder are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and / or the incidence of an injury treated according to the methods described herein or any other measurable parameter appropriate. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions. Methods of measuring these indicators are known to those of skill in the art and / or are described herein.

[0369] Efficacy can be assessed in animal models of a condition described herein, for example, a mouse model, as the case may be. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed.

[0370] All patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to they.. o. -applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0371] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

[0372] The disclosed invention can further be described in the following numbered paragraphs:1. A targeting compound for killing cancer cells, comprising components that target a) a tumor-associated extracellular target; and b) at least one tumor associated intracellular target.2. A antibody conjugate compound for killing cancer cells, comprising components that target a) a tumor-associated extracellular target; and b) at least one tumor associated intracellular target; and c) at least one intracellular effector target.3. In one embodiment of any preceding paragraph, cell killing activity is only active in the presence of a tumor-associated extracellular target and a tumor associated intracellular target.4. An antibody conjugate compound comprising: a) an antibody that specifically binds a tumor-associated extracellular antigen; b) a regulated induced-proximity targeting chimera (RIPTAC) comprising: i) a first small-molecule binding moiety that selectively binds an intracellular tumor-associated target protein; and ii) a second small-molecule binding moiety that selectively binds a panessential intracellular effector protein; and iii) a RIPTAC linker covalently joining the two smallmolecule binding moieties; and c) an antibody linker covalently joining the antibody to the RIPTAC.5. In one embodiment of any preceding paragraph, the intracellular tumor-associated target is selected from the group consisting of: androgen receptor (AR), enhancer of zeste homolog 2 (EZH2), induced myeloid leukemia cell differentiation protein Mcl-1 (MCL1), Wilms tumor protein (WT33), tumor protein p53 (TP53), and mouse double minute 2 homolog (MDM2).6. In one embodiment of any preceding paragraph, the pan-essential intracellular effector protein is selected from the group consisting of polo-like-kinase-1 (PLK1), Bromodomain-containing protein 2 (BRD2), Bromodomain-containing protein 3 (BRD3), Bromodomain-containing protein 4 (BRD4), cyclin-dependent kinase 1 (CDK1), cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 5 (CDK5), cyclin-dependent kinase 6 (CDK6), and cyclin-dependent kinase 9 (CDK9).7. In one embodiment of any preceding paragraph, the antibody linker is attached to the RIPTAC through a chemical handle positioned within the internal linker of the RIPTAC, or via a cleavable linker conjugated to one of the small-molecule binding moieties and being chemically discrete from both small-molecule binding moieties, such that the native binding affinity of each moiety for the respective protein is retained following linker cleavage and payload release.y.. o. -8. In one embodiment of any preceding paragraph, the RIPTAC linker comprises a scaffold selected from the group consisting of a lysine-derived scaffold selected from Nα-L-lysine, Nε-L-lysine, Nα-D-lysine, ornithine, and diaminobutyric acid, a poly(ethylene glycol) (PEG) scaffold selected from two to twenty-four oxyethylene units, a piperazine scaffold, a piperidine scaffold, a cyclobutene scaffold, an alkyl chain, an alkyne, a morpholine scaffold, a triazole scaffold, an alkyl ether scaffold, a branched 3-arm PEG scaffold, and combinations thereof; and / orwherein the linker is joined to the RIPTAC small-molecule binding moieties via a linkage selected from the group consisting of an azide–alkyne cycloaddition product, an amide bond, and a carbamate bond.9. In one embodiment of any preceding paragraph, the antibody linker further comprises a self-immolative valine–citrulline–para-aminobenzyl carbamate cleavage motif that is cleavable by cathepsin B after intracellular internalization, the para-aminobenzyl being connected to the RIPTAC through a carbamate linkage to a primary or secondary amine located within the internal linker of the RIPTAC, and wherein the valine–citrulline dipeptide is flanked by a PEG spacer selected from PEG2, PEG4, PEG6, PEG8, or PEG10 or a n-Alkane spacer selected from C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, or C15 to modulate solubility, prevent aggregation, or alter cleavage kinetics, and the para-aminobenzyl group is optionally substituted with methoxy or halo substituents to tune self-immolation rate while effecting scarless release of the RIPTAC such that both small-molecule binding moieties retain their native affinity.10. In one embodiment of any preceding paragraph, the antibody is a human or humanized IgGl, IgG2 or IgG4 that binds a tumor-associated extracellular antigen selected from the group consisting of: STEAP1, PSMA, CD19, CD20, CD33, BCMA, FOLR1, NaPi-2b, MUC16, CLEC12A, HER2, GPNMB, B7-H3, Trop-2, EGFR and EpCAM.11. In one embodiment of any preceding paragraph, the antibody is selected from trastuzumab, pertuzumab, vandortuzumab, rituximab, obinutuzumab, loncastuximab, belantamab, cetuximab, or a PSMA×STEAP1 bispecific antibody.12. In one embodiment of any preceding paragraph, the antibody comprises Fc-engineering for enhanced internalization or reduced effector function.13. In one embodiment of any preceding paragraph, the first small-molecule binding moiety binds androgen receptor and the second small-molecule binding moiety binds BRD4 or PLK1.14. An antibody conjugate comprising: a) an antibody that specifically binds a tumor-associated extracellular antigen; b) a transcriptional / epigenetic chemical inducer of proximity (TCIP) comprising: i) a first small-molecule binding moiety that selectively binds an intracellular transcription factor; and ii) a second small-molecule binding moiety that selectively binds a transcriptional activator or transcriptional repressor; and iii) a TCIP linker covalently joining the two small-molecule binding moieties; and c) a tri-functional linker covalently joining the antibody to the TCIP, the linker being attached to the TCIP through a chemical handle positioned within the internaly.. o. -linker of the TCIP or via a cleavable linker conjugated to one of the small-molecule binding moieties and being chemically discrete from both small-molecule binding moieties, such that the native binding affinity of each moiety for the respective protein is retained.15. In one embodiment of any preceding paragraph, the intracellular transcription factor selected from B-cell lymphoma 6 (BCL6), Forkhead-box Protein Pl (FOXP1), Poly(ADP-ribose) Polymerase 1 (PARP1), and Poly(ADP-ribose) Polymerase 2 (PARP2).16. In one embodiment of any preceding paragraph, the transcriptional activator or transcriptional repressor is selected from the group consisting of: Bromodomain-containing protein 4 (BRD4), cyclin-dependent kinase 9 (CDK9), El A-associated protein p300 (p300), and CREB-binding protein (CBP).17. In one embodiment of any preceding paragraph, the TOP linker comprises a scaffold selected from the group consisting of a lysine-derived scaffold selected from Nα-L-lysine, Nε-L-lysine, Nα-D-lysine, ornithine, and diaminobutyric acid, a poly(ethylene glycol) (PEG) scaffold selected from two to twenty-four oxyethylene units, a piperazine scaffold, a piperidine scaffold, a cyclobutene scaffold, an alkyl chain, an alkyne, a morpholine scaffold, a triazole scaffold, an alkyl ether scaffold, a branched 3-arm PEG scaffold, and combinations thereof; and / or wherein the linker is joined to the TCIP small-molecule binding moieties via a linkage selected from the group consisting of an azidealkyne cycloaddition product, an amide bond, and a carbamate bond.18. In one embodiment of any preceding paragraph, the antibody linker further comprises a self-immolative valine–citrulline–para-aminobenzyl carbamate cleavage motif that is cleavable by cathepsin B after intracellular internalization, the para-aminobenzyl being connected to the TCIP through a carbamate linkage to a primary or secondary amine located within the internal linker of the TCIP, and wherein the valine–citrulline dipeptide is flanked by a PEG spacer selected from PEG2, PEG4, PEG6, PEG8, or PEG10 or a n-Alkane spacer selected from C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, or C15 to modulate solubility, prevent aggregation, or alter cleavage kinetics, and the para-aminobenzyl group is optionally substituted with methoxy or halo substituents to tune self-immolation rate while effecting scarless release of the TCIP such that both small-molecule binding moieties retain their native affinity.19. In one embodiment of any preceding paragraph, the antibody is a human or humanized IgGl, IgG2 or IgG4 that binds a tumor-associated extracellular antigen selected from the group consisting of: STEAP1, PSMA, CD19, CD20, CD33, BCMA, FOLR1, NaPi-2b, MUC16, CLEC12A, HER2, GPNMB, B7-H3, Trop-2, EGFR and EpCAM.20. In one embodiment of any preceding paragraph, the antibody is selected from trastuzumab, pertuzumab, vandortuzumab, rituximab, obinutuzumab, loncastuximab, belantamab, cetuximab, CD19xBCMA bispecific antibody, CD19xCD20 bispecific antibody, or a BCMAxCD20 bispecific antibody.y.. o. -21. In one embodiment of any preceding paragraph, the antibody comprises Fc-engineering for enhanced internalization or reduced effector function.22. A compound having the structure represented by Structures la, lb or 1c:(Structure la)(Structure lb)(Structure 1c)or a pharmaceutically acceptable salt thereof; wherein: A1and A2independently represent small molecule binder (“warhead”) pairings which bind to two distinct biological targets (i.e., protein, RNA, DNA); L1comprises a linker composed of any amino acid, non-proteinogenic amino acid, PEG, n-Alkane, piperidine, cyclobutene, piperazine, or modified combination thereof; L2comprises a linker composed of any cleavable or non-cleavable element which serves to join A1-L1-A2to a targeting compound (TC); wherein the TC comprises a targeting compound consisting of an antibody, singledomain antibody, fusion protein, ligand, or nanoparticle.23. In one embodiment of any preceding paragraph, L1has the following structure:y.. o. -(Structure 2)or a pharmaceutically acceptable salt thereof; wherein:R1represents the side chain an amino acid or non-proteinogenic amino acid selected from: i) arginine, histidine, lysine, aspartate, glutamate, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan;ii) norvaline, norleucine, alloisoleucine, isoserine, t-leucine, pipecolate, 2,3-diaminopropionate, 2,4-diaminobutyrate, ornithine, allothreonine, homocysteine, homoserine, ci-amino-n-heptanoate, ci-aminobutyrate, P-aminobutyrate, y-aminobutyrate, ci-aminoisobutyrate;or any chemically modified derivates thereof.24. In one embodiment of any preceding paragraph, L1has the following structure:(Structure 3) (Structure 4)or a pharmaceutically acceptable salt thereof; wherein:R2and R3represents the side chain of one or two of the amino acids or non-proteinogenic amino acids selected from:i) arginine, histidine, lysine, aspartate, glutamate, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan;ii) norvaline, norleucine, alloisoleucine, isoserine, t-leucine, pipecolate, 2,3-diaminopropionate, 2,4-diaminobutyrate, ornithine, allothreonine, homocysteine, homoserine, ci-amino-n-heptanoate, ci-aminobutyrate, P-aminobutyrate, y-aminobutyrate, ci-aminoisobutyrate;or any chemically modified derivates of the aforementioned;y.. o. -n1and n2represent the number of monomeric subunits of said amino acids or non-proteinogenic amino acids where n1and n2independently equal a value between 1-10.25. In one embodiment of any preceding paragraph, L1has the following structure:(Structure 7)or a pharmaceutically acceptable salt thereof; wherein:R4, R5, and R6represents the side chain of 1-3 of the amino acids or non-proteinogenic amino acids selected from:i) arginine, histidine, lysine, aspartate, glutamate, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan;ii) norvaline, norleucine, alloisoleucine, isoserine, t-leucine, pipecolate, 2,3-diaminopropionate, 2,4-diaminobutyrate, ornithine, allothreonine, homocysteine, homoserine, a-amino-n-heptanoate, a-aminobutyrate, P-aminobutyrate, y-aminobutyrate, a-aminoisobutyrate;or any chemically modified derivates of the aforementioned;n3, n4, and n5represent the number of monomeric subunits of said amino acids or non-proteinogenic amino acids where n3, n4, and n5independently equal a value between 1-10.26. In one embodiment of any preceding paragraph, L1has the following structure:y.. o. -(Structure 8) (Structure 9)(Structure 10)or a pharmaceutically acceptable salt thereof; wherein S1and S2represent spacers selected from the group consisting of: poly(ethylene glycol) (PEG), poly(glycol) chains, alkyl chains, alkynes, triazoles, piperazines, piperidines, and any combination thereof27. In one embodiment of any preceding paragraph, L1has the following structure:Atty. Dkt. No. 701586-000161WOPT(Structure 11)(Structure 12)or a pharmaceutically acceptable salt thereof; wherein S3, S4, and S5represent optional spacers selected from the group consisting of: poly(ethylene glycol) (PEG), poly(glycol) chains, alkyl chains, alkynes, triazoles, piperazines, piperidines, and any combination thereof.y.. o. -28. In one embodiment of any preceding paragraph, L1has the following structure:(Structure 13)(Structure 14)(Structure 15)or a pharmaceutically acceptable salt thereof; wherein S6, S7, S8, and S9represent optional spacers selected from the group consisting of: poly(ethylene glycol) (PEG), poly(glycol) chains, alkyl chains, alkynes, triazoles, piperazines, piperidines, and any combination thereof.29. In one embodiment of any preceding paragraph, L1has the following structure:4900-3869-3513.2y.. o. -(Structure 16)or a pharmaceutically acceptable salt thereof; wherein n6, n7, and n8represent the number of monomeric subunits where n6, n7, and n8independently equal a value between 1-20.30. In one embodiment of any preceding paragraph, L2contains a structure selected from the group consisting of Structure 17, Structure 18, Structure 19, Structure 20, Structure 21, Structure 22, and Structure 23, or a combination thereof,or a pharmaceutically acceptable salt, solvate or prodrug thereof,wherein n9- n14represent the number of monomeric subunits where n9- n14independently equal a value between 1-20.31. In one embodiment of any preceding paragraph, A1and A2represent a biologically active pairing of small molecules which functions as a regulated induced proximity targeting chimera (RIPTAC).32. In one embodiment of any preceding paragraph, A1and A2represent a biologically active pairing of small molecules which functions as a proteolysis-targeting chimera (PROTAC).33. In one embodiment of any preceding paragraph, A1and A2represent a biologically active pairing of small molecules which functions as an autophagy-targeting chimera (AUTAC).34. In one embodiment of any preceding paragraph, A1and A2represent a biologically active pairing of small molecules which functions as a specific and non-genetic inhibitor of apoptosis protein (IAP)-dependent protein eraser (SNIPER).35. In one embodiment of any preceding paragraph, A1and A2represent a biologically active pairing of small molecules which functions as a deubiquitinase-targeting chimera (DUBTAC).36. In one embodiment of any preceding paragraph, A1and A2represent a biologically active pairing of small molecules which functions as a phosphorylation-inducing chimeras (PHICs).37. In one embodiment of any preceding paragraph, A1and A2represent a biologically active pairing of small molecules which functions as a phosphatase-recruiting chimera (PHORCs).38. In one embodiment of any preceding paragraph, A1and A2represent a biologically active pairing of small molecules which functions as a ribonuclease targeting chimera (RIBOTAC).39. In one embodiment of any preceding paragraph, A1and A2represent a biologically active pairing of small molecules which functions as an acetylation tagging system (AceTAG).4900-3869-3513.2y.. o. -40. In one embodiment of any preceding paragraph, the compound is a stereoisomer, enantiomer, or tautomer thereof or a mixture thereof, or a pharmaceutically acceptable salt, solvate or prodrug thereof.41. A composition comprising any compound disclosed herein.42. In one embodiment of any preceding paragraph, the compound further comprising at least a second compound disclosed herein.43. A pharmaceutical composition comprising any compound disclosed herein and a pharmaceutically acceptable carrier.44. In one embodiment of any preceding paragraph, the compound further comprising at least a second compound disclosed herein.45. A method of treating cancer, the method comprising administering to a patient in need thereof a therapeutically effective amount of any compound, composition, or pharmaceutical composition disclosed herein.46. In one embodiment of any preceding paragraph, the cancer is selected from the group consisting of: a carcinoma, a primary central nervous system tumor, a melanocytic tumor, a germ cell tumor, a sarcoma, and a hematological malignancy.47. In one embodiment of any preceding paragraph, the carcinoma is selected from the group consisting of: ovarian cancer, uterine cancer, endometrial cancer, cervical cancer, prostate cancer, breast cancer, lung cancer, oral cancer, esophageal cancer, head and neck cancer, stomach cancer, colon cancer, rectal cancer, basal cell carcinoma, squamous cell carcinoma, sebaceous gland carcinoma, bile duct cancer, gallbladder cancer, liver cancer, pancreatic cancer, bladder cancer, urinary tract cancer, renal cell carcinoma and thyroid cancer.48. In one embodiment of any preceding paragraph, the primary central nervous system tumor is a brain cancer.49. In one embodiment of any preceding paragraph, the melanocytic tumor is eye cancer or cutaneous melanoma.50. In one embodiment of any preceding paragraph, the germ cell tumor is testicular cancer or ovarian cancer.51. In one embodiment of any preceding paragraph, the sarcoma is a uterine sarcoma.52. In one embodiment of any preceding paragraph, the hematological malignancy is lymphoma or leukemia.52. An antibody conjugate composition for treatment of prostate cancer, comprising: (a) an internalizing antibody moiety that selectively binds to STEAP1, PSMA, or a bispecific antibody moiety that binds both STEAP1 and PSMA; and (b) a RIPTAC moiety that selectively binds to an intracellular cancer-overexpressed protein, androgen receptor (AR), and additionally binds at least one pan-essential effector protein selected from the group consisting of BRD4, PLK1, BRD2, BRD3, CDK1, CDK2, CDK4, CDK5, CDK6, and CDK9.4900-3869-3513.2y.. o. -53. In one embodiment of any preceding paragraph, the antibody moiety is conjugated to the RIPTAC moiety via a cathepsin B cleavable valine-citrulline self-immolative linker, enabling intracellular release of the RIPTAC upon internalization.54. In one embodiment of any preceding paragraph, the antibody moiety is derived from or comprises vandortuzumab for STEAP1 targeting.55. In one embodiment of any preceding paragraph, the bispecific antibody moiety binds simultaneously or separately to STEAP1 and / or PSMA cell-surface antigens expressed on prostate cancer cells.56. A method of treating metastatic castration-resistant prostate cancer in a subject comprising administering to the subject a therapeutically effective amount of any antibody conjugate composition disclosed herien.57. In one embodiment of any preceding paragraph, the treatment selectively accumulates in prostate cancer cells due to antibody targeting and kills prostate cancer cells by inducing stable ternary complexes between AR, RIPTAC, and the pan-essential effector protein, resulting in steric inhibition of effector protein function and selective tumor cell death.58. In one embodiment of any preceding paragraph, the subject has a cancer comprising a mutation found in human prostate cancer cell lines VCaP, LNCaP, or 22Rv1 with differential or engineered STEAP1 expression, including STEAP1 knockout (STEAPlko), STEAP1 low expression (STEAPllow), and STEAP1 overexpressing (STEAPlhi / amp) variants.59. In one embodiment of any preceding paragraph, the composition provides superior in vivo anti-tumor efficacy as compared to androgen receptor RIPTACs, androgen receptor antagonists, or androgen receptor degraders.60. A pharmaceutical composition comprising any antibody conjugate composition disclosed herein formulated for intravenous administration.61. An antibody conjugate composition for the treatment of Diffuse Large B-Cell Lymphoma (DLBCL), comprising: (a) an internalizing antibody moiety that specifically binds to one or more cell surface antigens selected from CD 19, CD20, BCMA, or a bispecific antibody moiety that binds two antigens thereof; and (b) a transcriptional / epigenetic chemical inducer of proximity (TOP), with a target moiety that selectively binds to BCL6 and an effector moiety that binds BRD4 or an alternative transcriptional activator selected from cyclin-dependent kinase 9 (CDK9), histone acetyltransferases (HATs), orp300 / CBP complexes.62. In one embodiment of any preceding paragraph, the antibody is conjugated to the TCIP via a cleavable maleimide-PEG4-Val-Cit-PAB linker, enabling intracellular release of the TCIP moiety upon cathepsin B cleavage following internalization.63. In one embodiment of any preceding paragraph, the antibody moiety is derived from or comprises an anti-CD19 monoclonal antibody (e.g., Loncastuximab), an anti-CD20 monoclonal4900-3869-3513.2y.. o. -antibody (e.g., Rituximab), or an anti-BCMA monoclonal antibody (e.g., Belantamab) suitable for internalization into DLBCL cells.61. In one embodiment of any preceding paragraph, the bispecific antibody moiety binds simultaneously or separately two of the three antigens CD 19, CD20, and BCMA to increase selective delivery and internalization into target cells.62. In one embodiment of any preceding paragraph, the TCIP moiety mediates ternary complex formation between intracellular BCL6 and BRD4 / CDK9 / HAT / p300 / CBP transcriptional regulators, thereby inducing transcriptional activation and expression of BCL6 downstream genes in DLBCL cells.63. A method of selectively killing DLBCL cells in a subject comprising administering a therapeutically effective amount of any antibody conjugate of disclosed herein, wherein cell killing is mediated by activation of previously repressed transcriptional regulatory pathways critical for lymphoma survival and proliferation.64. In one embodiment of any preceding paragraph, the subject has a cancer bearing a mutation found in human DLBCL cell lines selected from SUDHL5 or KARPAS422.65. In one embodiment of any preceding paragraph, the antibody conjugate exhibits improved tumor selectivity and reduced systemic toxicity due to the dual mechanism of targeted antibody internalization and transcriptional / epigenetic chemical inducer of proximity induction.66. An antibody conjugate composition for treatment of Diffuse Large B-Cell Lymphoma (DLBCL), comprising: (a) an internalizing antibody moiety that specifically binds to at least one cell surface antigen chosen from CD 19, CD20, BCMA, or a bispecific antibody moiety that binds two of said antigens; and (b) a regulated induced proximity targeting chimera (RIPTAC) moiety designed to form a ternary complex between the intracellular cancer-associated protein enhancer of zeste homolog 2 (EZH2) and a pan-essential effector protein selected from the group consisting of PLK1, BRD2, BRD3, BRD4, CDK1, CDK2, CDK4, CDK5, CDK6, and CDK9.67. In one embodiment of any preceding paragraph, the antibody moiety is conjugated to the RIPTAC moiety via a cathepsin B cleavable linker selected from valine-citrulline-p-aminobenzyl carbamate (Val-Cit-PAB) or a maleimide-based self-immolative linker, facilitating intracellular release of the RIPTAC payload upon endocytosis and lysosomal processing.68. In one embodiment of any preceding paragraph, the antibody moiety is derived from an anti-CD19, anti-CD20, or anti-BCMA monoclonal antibody, each exhibiting internalization upon antigen binding on DLBCL cells.69. In one embodiment of any preceding paragraph, the bispecific antibody moiety simultaneously or separately binds two of the antigens CD 19, CD20, and BCMA to enhance selective delivery and cellular internalization in lymphoma cells.70. A method of treating DLBCL in a subject, comprising administering a therapeutically effective amount of any antibody conjugate disclosed herein, thereby inducing selective tumor cell death by the4900-3869-3513.2y.. o. -formation of a ternary complex between intracellular RIPTAC, EZH2, and a pan-essential effector protein, leading to functional inhibition of essential proteins in lymphoma cells.71. In one embodiment of any preceding paragraph, the subject has a cancer comprising a mutation found in human DLBCL cell lines selected from SUDHL5 or KARPAS422.72. In one embodiment of any preceding paragraph, administration of the antibody conjugate results in significant tumor growth inhibition, induction of apoptosis, and improved survival relative to control treatments.73. In one embodiment of any preceding paragraph, the antibody conjugate exhibits superior therapeutic efficacy and reduced systemic toxicity compared to unconjugated TOP.74. In one embodiment of any preceding paragraph, further comprising an AND logic gate, wherein cell killing activity requires the presence of a tumor-associated extracellular target and one or more tumor associated intracellular targets.75. In one embodiment of any preceding paragraph, cell killing activity is only active in the presence of one or more tumor-associated extracellular targets and one or more tumor associated intracellular targets.76. In one embodiment of any preceding paragraph, further comprising an AND logic gate, wherein cell killing activity requires the presence of a tumor-associated extracellular target, one or more tumor associated intracellular targets, and one or more intracellular effector target.77. In one embodiment of any preceding paragraph, further comprising an AND logic gate, wherein cell killing activity requires the presence of one or more tumor-associated extracellular targets, one or more tumor associated intracellular targets, and one or more intracellular effector target.78. In one embodiment of any preceding paragraph, cell killing activity is only active in the presence of one or more tumor-associated extracellular targets, one or more tumor associated intracellular targets, and one or more intracellular effector target.79. In one embodiment of any preceding paragraph, wherein said payload moiety is chemically stable and membrane-permeable such that, upon intracellular processing and killing of a first tumor cell, the payload moiety:a) diffuses out of said first tumor cell and enters an adjacent second tumor cell;b) induces cell death in said second tumor cell contingent upon the presence of both said tumor-associated intracellular target(s) and said intracellular effector target(s) in said second tumor cell; and c) wherein said induction of cell death in the second tumor cell occurs independently of the presence of the tumor-associated extracellular target on said second tumor cell4900-3869-3513.2y.. o. -EXAMPLES

[0373] EXAMPLE 1: Antibody-RIPTAC Conjugates Enable Versatile Dual-Logic Precision Therapeutics

[0374] Antibody-drug conjugates (ADCs) represent a transformative approach in precision oncology, combining the specific targeting of monoclonal antibodies with potent cytotoxic payloads. However, their therapeutic potential is constrained by off-tumor toxicities, often stemming from the indiscriminate nature of traditional payloads. We are advancing ADC technology by leveraging Regulated Induced Proximity Targeting Chimeras (RIPTACs), an innovative class of heterobifunctional molecules that induce cancer cell-specific toxicity. RIPTACs achieve selective killing by knocking out essential survival proteins by stabilizing protein-protein interactions between a tumor-specific target protein (TP) and an essential protein (EP), with their activity independent of the target protein's role in disease progression. Our approach involves conjugating these RIPTACs to antibodies targeted to proteins overexpressed on the surface of tumor cells, creating antibody-RIPTAC conjugates (ARCs). ARCs comprised of STEAP1-targeting antibodies and androgen receptor (AR)-targeting RIPTACs serve as a novel treatment strategy tailored for castration-resistant prostate cancer(CRPC).This dual-targeting mechanism enhances therapeutic specificity by integrating extracellular antigen recognition with intracellular TP-driven cytotoxicity, addressing a critical challenge in ADC development. ARCs will redefine the therapeutic index of ADCs, offering a highly precise and safer treatment modality for aggressive cancers like CRPC.

[0375] Significance. Antibody-drug conjugates (ADCs) combine the precise targeting capabilities of monoclonal antibodies with potent cytotoxic payloads, offering substantial therapeutic potential for precision oncology. The vast majority of ADCs leverage microtubule inhibitor payloads, which, while effective, are highly toxic if delivered to non-cancerous tissue. Recent advances in payload development include DNA damaging agents, topoisomerase I inhibitors, and immune agonists.However, these agents similarly exhibit dose limiting toxicity profiles or stimulate uncontrolled immune responses following systemic administration. Largely, this narrow therapeutic window is attributed to: (1) the lack of tumor-specific antigens and (2) undesired, premature release of highly cytotoxic payloads via mechanisms such as retro-Michael addition. Challenges arising from off-tumor toxicities due to payload or linker selection have slowed the development of novel ADCs and led to large number of clinical candidate failures.

[0376] Introducing a “smart” payload that activates only in cancer cells overcomes the current limitations of traditional ADCs and unlock the full potential of this emerging therapeutic modality. Among the fastest growing class of drugs in chemical biology are heterobifunctional small molecules which induce proximity and ternary complex formation between distinct biological targets.Heterobifunctional small molecules, which regulate proteins of interest (POIs), serve as these “smart” payloads, as they selectively affect cancer cells while remaining relatively non-toxic in healthy cells.4900-3869-3513.2y.. o. -We conceived that the ideal heterobifunctional for an ADC system would be newly reported, regulated induced proximity targeting chimeras (RIPTACs).

[0377] RIPTACs offer a unique mechanism in the heterobifunctional space, wherein they selectively kill cells expressing a high abundance of target protein (TP), while sparring those not expressing the TP, in a manner that is independent of TP function. They do this by inducing stable ternary complex formation between a TP selectively overexpressed in tumor cells and a pan-expressed, essential protein (EP) required for cell survival. RIPTAC-induced protein-protein interactions (PPIs) between the TP and EP abrogates the function of the EP, leading to specific killing of cancer cells which highly express the TP.

[0378] Leveraging RIPTACs as a novel payload for ADCs imparts dual “death” switches on the ADC formulation: (1) extracellular antigen targeting and internalization into cancer cells via the antibody, and (2) targeting cancer-specific or mutant proteins within the cell through the payload. This duallogic mechanism of antibody-RIPTAC conjugates (ARCs) will enhance the tumor-specific precision of the therapy, further improving the therapeutic index (TI) of ADCs and establishing a new paradigm in precision oncology (Fig. 1).

[0379] Technology

[0380] RIPTACs demonstrate differential toxicity dependent on target protein expression. As a proof of concept, we used an engineered HEK293 model system, wherein cells where stably transfected with lentivirus overexpressing a FLAG-tagged HaloTag7-FKBPF36V (HaloTag-FKBP) as our TP (hereafter referred to as “293 HFL”). The HaloTag is commonly used as a model target for experimental heterobifunctional compounds as chloroalkane (CA) binders are known to specifically bind to the HaloTag in vitro. As a control, a cell line overexpressing EGFP in replacement of the HaloTag-FKBP protein was generated (hereafter referred to as “293 GFPL”). Based on preliminary work from Raina et al.4, we selected polo-like kinase 1 (PLK1) and the corresponding PLK1 inhibitor, BI-2536, as our EP ligand. Through a two-step amide coupling scheme, we replicated the RIPTAC synthesis using BI-2536 at one end, the HaloTag-binding CA at the other, and a poly(ethylene glycol) (PEG)-linker (hereafter “BI-2PEG-CA”). In order to synthesize an ARC, the RIPTAC must be linked to the antibody via an additional linker, however, conjugating an antibody linker to one of the end-groups may result in abrogation of the binder’s ability to bind either PLK1 or HaloTag. Therefore, we synthesized a novel modified RIPTAC where the PEG2 linker in BI-2PEG-CA is substituted with a 3-arm PEG variant. An additional chemical handle is placed on the linker allowing for downstream conjugation to an antibody, which provides unimpeded access to the RIPTAC headgroup binders (hereafter “BI-3armPEG-CA”). Next, we treated 293 HFL (model “cancer” cells) and 293 GFPL (model “healthy” cells) with synthesized RIPTACs or BI-2536 in a 5-day assay with continuous compound exposure (Fig. 5). Both BI-2PEG-CA (GI50 < 25pM) and BI-3armPEG-CA (GI50 ~ 25pM) demonstrate significant toxicity in the TP expressing 293 HFL cells, however, remain relatively non-toxic in 293 GFPL cells lacking only HaloTag expression (GI50 of4900-3869-3513.2y.. o. -0.37μM and 2.83μM, respectively). Unlike the RIPTACs, the PLK1 inhibitor alone is similarly potent against both 293 GFPL cells (GI50 = 0.51nM) and 293_HFL cells (GI50 = 1.89nM). These data in addition to prior reports demonstrate that RIPTACs exhibit TP-dependent toxicity, where the TP is not required to be a disease driver, and RIPTAC structure is tolerant of novel 3-arm structures allowing for downstream antibody coupling.

[0381] Identification of lead program in castration-resistant prostate cancer (CRPC). Prostate cancer (PC) is the second most common cancer in US men, and is responsible for 1 in 44 male deaths. First-line treatment for PC is androgen-deprivation therapy (ADT), which provides remission of the disease in nearly 90% of patients. However, after only 2-3 years nearly all patients accumulate resistance mutations and progress to CRPC, where metastatic CRPC (mCRPC) is associated with poor prognosis and a mean survival time of 16-18 months.

[0382] In the mCRPC setting, >80% of patients harbor amplifications of the AR gene or the upstream enhancer region of DNA, leading to overexpression of AR in tumor cells. Additionally, transcriptomic and proteomic profiling has identified six transmembrane epithelial antigen of the prostate 1 (STEAP1) as one of the most highly enriched cell surface antigens on mCRPC cells, whereas STEAP1 demonstrates limited expression in normal tissue. This pairing culminates in a novel druggable axis that is uniquely suited towards our ARC approach, whereby an AR RIPTAC would achieve potent toxicity in only mCRPC cells and linking this to a STEAP1 antibody serves to shuttle the RIPTACs directly to both the primary tumor and metastatic sites.

[0383] Synthesis and in vitro characterization of PC ARCs. First, we will express and purify the STEAP1 antibody, vandortuzumab, by co-transfecting Expi293 cells with plasmids containing the heavy and light chain fragments and purifying the folded antibody via protein A chromatography. Vandortuzumab-based ADCs were previously discontinued following Phase I clinical trials due to severe liver toxicities, likely due to the high, indiscriminate toxicity of MMAE payload delivered off-tumor. Therefore, we will prepare a small library of novel AR RIPTAC payloads which induce toxicity only in AR overexpressing PC cells. Specifically we will prepare this RIPTAC library using the AR targeting domain from Bavdegalutamide (i.e., ARV-110) and BI-2536, or selective bromodomain and extra-terminal (BET) protein inhibitor, JQ1. The AR and EP targeting domain (e.g., BI-2536, JQ1) will be linked using an array of poly(ethylene glycol) (PEG) spacers and lysine linkers to create a diverse library of tri-functional RIPTACs (Fig. 2). The use of amino acid linkages between EP and TP, instead of a 3-arm PEG linkage used in the preliminary data, will allow the creation of a library of RIPTACs with different stereochemistry, length, and rigidity that affect the efficiency of ternary complex formation. By creating the library, we will investigate structure-function relationships of the compounds. We will validate RIPTAC synthesis using 1H NMR, 13C NMR and UPLC-MS, and confirm antibody coupling and drug-to-antibody ratio (DAR) using LC / MS.

[0384] To assess cytotoxicity of AR RIPTACs, we will first screen the small molecules in VCaP (AR high), 22Rv1 (AR medium) and DU 145 (AR low) prostate cancer cell lines. We anticipate higher4900-3869-3513.2y.. o. -toxicity in VCaP cells as RIPTAC cytoxicity is highly dependent on both the presence and concentration of TP. Following initial screening, we will conjugate the top two performing AR RIPTACs onto vandortuzumab antibody via cysteine coupling through valine-citrulline cleavable linker (Fig. 2, R-group) attached to the lysine moiety of the RIPTACs creating prostate cancertargeting ARCs. The resulting ARC will be similarly screened in VCaP and VCaP STEAP1 knockout (ko) cells, where we anticipate STEAP1 ko will abolish ARC cytotoxicity experienced in parental VCaP cells. To elucidate the mechanism of action, we will assay cells following treatment using Vybrant DyeCycle stain to evaluate cell cycle (e.g., PLK1 inhibition leads to cell locking in G2 / M Phase), western blot against AR, PLK1 and BET bromodomain protein downstream targets (e.g., TMPRSS2, Phospho-Cyclin Bl, and c-Myc, respectively), and competition assays using unlinked binders to show loss of RIPTAC potency due to competition.

[0385] In vivo proof of concept in CRPC model. To mimic the traditional progression of PC, we will use a castrate VCaP xenograft model in male SCID mice. The VCaP cells will be inoculated subcutaneously into the flank of male SCID mice and tumors will be allowed to grow for 4-5 weeks or until animals reach a serum PSA level of >5 pg / L following tail vein blood collection. Thereafter, all mice with tumors will be castrated and randomly assigned to vehicle, control, or experimental treatment groups. Treatment administration will begin when mice reach pre-castration serum PSA levels. Controls will include enzalutamide (20 mg / kg per day), a second-generation androgen receptor inhibitor which has been previously shown to improve clinical outcomes in CRPC patients in combination with ADT, and vandortuzumab as an antibody alone control. Experimental treatment groups will be composed of the top performing prostate cancer-targeting ARC as well as the corresponding free-drug RIPTAC used in the ARC formulation. Antibody and ARC doses will be experimentally identified as 1 / 2 of maximum tolerated dose. ARC, vandortuzumab, and free RIPTAC will be delivered via tail- vein injection, and enzalutamide will be delivered via oral gavage. Mice will be monitored regularly for tumor burden and will be euthanized according to IACUC guidelines, or after 100 days. After euthanasia, blood and tissue will be collected for evaluation of treatment toxicity and serum PSA levels. Toxicity will be evaluated in a blinded fashion by our pathologist via histopathological evaluation and scoring of primary organs, as well as clinical markers such as weight, red blood cell count, and AST / ALT levels.

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[0387] Abstract

[0388] Antibody-drug conjugates (ADCs) are an innovative cornerstone of precision oncology, yet their clinical utility is constrained by a lack of tumor-selective surface antigens and the indiscriminate toxicity of conventional cytotoxic payloads. To address these limitations, we describe antibody-RIPTAC conjugates (ARCs), a dual-logic therapeutic platform that unites antibody-guided delivery and regulated induced proximity targeting chimeras (RIPTACs), a novel chemically induced proximity modality that triggers intracellular inhibition of pan-essential proteins dependent on tumorspecific protein expression. We designed a small library of RIPTACs that incorporate a ligand against a model target protein (TP), connected by a tri-functional linker to an effector ligand for PLK1. Several tri-functional designs display potent, TP-dependent killing and the additional chemical handle on the linker enables conjugation of the RIPTACs to trastuzumab via a supramolecular coiled-coil bioconjugation strategy. The resulting ARC selectively ablates HER2+ / TP+ cells while sparing HER2- and TP- populations. Analysis of genomic data across DepMap and TCGA uncovers the STEAP1 / androgen receptor (AR) axis in metastatic prostate cancer as an ideal clinically actionable ARC application. Our findings establish ARCs as a modular framework that integrates surface-level tumor targeting with intracellular context-specific synthetic lethality, providing a novel strategy to overcome the therapeutic index limitations of traditional ADCs and the delivery challenges associated with heterobifunctional small molecules such as RIPTACs.

[0389] Introduction4900-3869-3513.2y.. o. -

[0390] Antibody-drug conjugates (ADCs) combine the precise targeting capabilities of monoclonal antibodies with potent cytotoxic payloads, offering substantial therapeutic potential for precision oncology. The choice of payload is crucial for ADC efficacy. The vast majority of ADCs leverage microtubule inhibitor payloads, which, while effective, are highly toxic upon delivery to non-cancerous tissue.1 Recent advances in payload development include DNA damaging agents, topoisomerase I inhibitors and immune agonists. However, these agents similarly exhibit dose limiting toxicity profiles (DNA damaging agents, topoisomerase I inhibitors) or stimulate uncontrolled immune responses following systemic administration (immune agonists).2, 3 Largely this narrow therapeutic window is attributed to: (1) the scarcity of truly tumor-specific antigens3, 4 and (2) undesired, premature release of cytotoxic payloads via mechanisms such as retro-Michael additions. Challenges arising from off-tumor toxicities due to payload or linker selection have slowed the development of novel ADCs and led to ~84% of ADC programs being terminated in phase I or phases I / II clinical trials since 2000.2, 6

[0391] Introducing a “smart” payload that activates its cytotoxic properties only in cancer cells may overcome the current limitations of traditional ADCs and unlock the full potential of this emerging therapeutic modality.7, 8 Among the fastest growing class of drugs in chemical biology are heterobifunctional small molecules which induce proximity and ternary complex formation between distinct biological targets. Heterobifunctional small molecules, which modulate proteins of interest (POIs), may serve as these “smart” payloads, as they selectively affect cancer cells while remaining relatively non-toxic in healthy cells. Specifically, we recognized that the ideal heterobifunctional for an ADC system would be newly reported, regulated induced proximity targeting chimeras (RIPTACs).9

[0392] RIPTACs offer a unique mechanism in the heterobifunctional space, wherein they selectively kill cells expressing a high abundance of target protein (TP), while sparring those not expressing the TP, in a manner that is independent of TP function. They function by inducing stable ternary complex formation between a TP selectively overexpressed in tumor cells and a pan-expressed, essential protein (EP) required for cell survival. RIPTAC-induced protein-protein interactions (PPIs) between the TP and EP abrogates the function of the EP, leading to specific killing of cancer cells which highly express the TP.

[0393] Leveraging RIPTACs as a novel payload for ADCs imparts a dual “death” switch on the ADC formulation: (1) extracellular antigen targeting and internalization into cancer cells via the antibody, and (2) targeting cancer-specific or mutant proteins within the cell through the payload. Thus, the dual-logic mechanism of antibody-RIPTAC conjugates (ARCs) enhances tumor-specific precision, improving the therapeutic index (TI) of ADCs and offering the potential to establish a new paradigm in precision oncology.

[0394] Dual protein expression model system4900-3869-3513.2y.. o. -

[0395] To create a model system to test dual-logic ARCs, we leveraged a previously developed system from Raina et al.9 whereby HEK 293 cells were engineered to stably overexpress FLAG-tagged HaloTag7-FKBPF36V (HaloTag-FKBP) with a C-terminal P2A-EGFP sequence as the TP via lentiviral transduction. A cell line that overexpresses only EGFP was used as a TP negative cell line. We then further engineered the HaloTag-FKBP-Lentivirus cells (“293 HFL”) and GFP-Lentivirus cells (“293 GFPL”) to create variants which stably express full length receptor tyrosine-protein kinase erbB-2 (HER2), HaloTag-FKBP-HER2-Lentivirus (“293 HFL HER2”) and GFP-HER2-Lentivirus (“293 GFPL HER2”) cells, again using lentiviral transduction with a different selection marker. Following selection, we confirmed GFP and HER2 expression by flow cytometry. These four cell lines represent four possible protein profiles to demonstrate antibody-RIPTAC conjugate (ARC) logic: dual positive 293 HFL HER2 cells, dual negative 293 GFPL cells, TP+ / HER2- 293 HFL cells, and HER2+ / TP- 293 GFPL HER2 cells.

[0396] Synthesis and characterization of tri-functional RIPTACs

[0397] Due to their chimeric nature, heterobifunctional molecules often exhibit unfavorable physicalchemical properties that lead to poor solubility and cell-permeability.10, 11 Furthermore, these compounds typically display poor drug metabolism and pharmacokinetic (DMPK) properties such as limited oral bioavailability and / or rapid in vivo clearance, thus, requiring large (e.g., grams / patient) doses to be efficacious.12, 13 To overcome these boundaries, numerous ADCs incorporating heterobifunctional compounds as the active payload are being explored to improve the clinical relevancy of these compounds.14- 18 By linking a heterobifunctional molecule to a monoclonal antibody (mAb), this approach overcomes several existing challenges regarding solubility and DMPK properties and impart favorable tissue / cell-specific targeting capacity. However, many of the disclosed ADCs incorporating heterobifunctional compounds leverage PROTACs containing the von Hippel-Lindau tumor suppressor (VHL) ligand, as it is known that cleavable antibody linker attachment via the hydroxyproline fragment present in the compound's VHL-binding region retains the PROTACs’ ability to bind VHL.14- 18 However, this linker chemistry is not extensible to other PROTAC systems which employ a cereblon (CRBN) ligand. Importantly, beyond PROTACs, it is generally poorly understood how tolerant heterobifunctional molecule warheads are to chemical modification. This limits the ability to add chemical linkers to one of the two warheads, as conjugating a linker to one or the other warhead may abolish the compound’s ability to bind its cognate protein target.

[0398] These limitations led us to explore an alternative strategy centered on the creation of tri-functional linkers, whereby the antibody linker is directly conjugated to the RIPTAC linker rather than one of the two warheads. We employed two strategies to create tri-functional compounds: (1) lysine linkers and (2) oligomeric 3-arm poly(ethylene glycol) (PEG) linkers. Using a chloroalkane (CA) as our target ligand (TL) for the HaloTag and polo-like kinase 1 (PLK1) inhibitor, BI 2536, for our effector ligand (EL) we synthesized compounds 1-5 (FIG. 4A-4C).

[0399] Tri-functional RIPTACs show enhanced anti-proliferative activity in TP expressing cells4900-3869-3513.2y.. o. -

[0400] We synthesized the aforementioned tri-functional RIPTACs and replicated the synthesis of a traditional bi-functional variant using BI-2536 at one end, and CA at the other end, linked together with a short PEG linker (hereafter “BI-2PEG-CA”). Using these compounds, we treated 293 HFL cells (model “cancer” cells) and 293 GFPL cells (model “healthy” cells) with PLK1 inhibitor (BI-2536), bi-functional RIPTAC (BI-2PEG-CA), and each of our tri-functional RIPTACs in a 7-day assay with continuous compound exposure followed by DNA-based proliferation analysis (FIG. 5).

[0401] BI-2PEG-CA, demonstrates significant toxicity (IC50 < 25 pM) in the TP expressing HFL cells, however, remains relatively non-toxic in GFPL cells lacking HaloTag expression (IC50 = 0.37 pM, fold shift > 19,000). Unlike the RIPTAC, BI-2536 is similarly potent against both GFPL cells and HFL cells (IC50 = 1.3 and 2.4 nM, respectively). From the tri-functional library, only compound 4 (BI-N£-L-Lysine-2PEG-CA, fold shift = 12) and 5 (BI-3arm-PEG-CA, fold shift = 2,400) exhibit enhanced cytotoxicity in TP expressing 293 HFL cells compared to 293 GFPL cells, which is anticipated to be further enhanced by antibody conjugation (FIG. 5). Surprisingly, compounds 1 (BI-Na-L-Lysine-4PEG-CA), 2 (BI-N£-L-Lysine-4PEG-CA), and 3 (BI-Na-D-Lysine-4PEG-CA) display no differential cytotoxicity. Compounds 1 and 3 contain the same number of atoms between each headgroup as BI-2PEG-CA, however, demonstrate nearly identical IC50 values between 293 HFL and non-TP expressing 293 GFPL cells. Compound 2 retains similar chemical features to compound 4, however the addition of two PEG units in the linker diminishes activity. These results suggest that compounds 1-3 either do not induce ternary complexes between HaloTag / PLKl, or the resulting ternary complex is not sufficiently stabilized by PPIs to result in robust silencing of PLK1 activity.

[0402] Mechanisms underlying RIPTAC differential cytotoxicity

[0403] We next evaluated the mechanism by which our synthesized tri-functional RIPTACs lead to cytotoxicity, with the goal of functionally comparing them to PLK1 inhibitors and bi-functional RIPTACs. As PLK1 facilitates centrosome maturation and spindle formation in G2 / M phase, promoting entry into M phase, we hypothesized that our RIPTACs would lead to TP-dependent cell cycle arrest in G2 / M phase.19 We treated 293 HFL and 293 GFPL cells with BI-2536, bi-functional RIPTAC (BI-2PEG-CA), or tri-functional compound 5, then stained for DNA content using Vybrant DyeCycle Violet stain prior to analysis on flow cytometry (FIG. 6A). Cultured cells typically show a bimodal distribution: a large G0 / G1 peak (2N), a smaller G2 / M peak (4N) for dividing cells, and an S-phase band (2-4N) as cells are replicating DNA. As anticipated, BI-2536 causes cell cycle arrest in G2 / M phase in a dose-dependent manner, independent of HaloTag expression. Both RIPTACs also induce dose-dependent cell cycle arrest in G2 / M phase in HaloTag expressing 293 HFL cells.However, only at the highest dosage (1 pM) of BI-2PEG-CA do we see 4N accumulation in293 GFPL cells, whereas this occurs at doses as low as 50 nM when treated with PLK1 inhibitor. Tri-functional compound 5 displays no indication of cell cycle arrest in off-target 293 GFPL cells up to the highest dose assessed (1 pM). Together, these data demonstrate that RIPTACs trigger mitotic catastrophe in a context dependent manner based on target protein expression.4900-3869-3513.2y.. o. -

[0404] At a molecular level we aimed to directly demonstrate that the triggering of mitotic catastrophe is target dependent and that the TP and EP indeed form a stable ternary complex. Firstly, we designed a competition experiment, whereby 293 HFL cells were plated in media containing a fluorescent HaloTag binding probe, tetramethylrhodamine chloroalkane (TAMRA-CA), prior to RIPTAC treatment the next day for a 7-day assay with continuous RIPTAC exposure. Pre-treatment with TAMRA-CA would saturate HaloTag7 being expressed in the HFL cells, preventing RIPTAC-induced ternary complex formation while still allowing for RIPTACs to interact with PLK1.Following TAMRA-CA competition, both bi-functional and tri-functional RIPTAC potency against 293 HFL cells was robustly abrogated (FIG. 6B). Encouragingly, TAMRA-CA competition in HaloTag expressing 293 HFL cells leads to similar RIPTAC cytotoxicity as experienced in HaloTag non-expressing 293 GFPL cells.

[0405] To validate that RIPTAC cytotoxicity is linked to TP abundance, we next used TREx-293 cells engineered to express a doxycycline (dox)-inducible HaloTag-FKBP. In the absence of dox, TREx-293 cells lack expression of HaloTag-FKBP, mimicking 293 GFPL cells. Following treatment with bi-functional BI-2PEG-CA, these cells exhibit a similar cytotoxicity profile to 293 GFPL cells (IC50 = ~0.27 pM) (FIG. 6C). However, at doses as low as 0.25 ng / mL, dox-induced expression of HaloTag-FKBP leads to a pronounced downward shift in the cytotoxicity profile of BI-2PEG-CA. At high levels of dox-induction (>10 ng / mL), TREx-293 cells begin to mimic constitutively expressing 293 HFL cells with significant toxicity observed following BI-2PEG-CA treatment (IC50 < 25 pM). These data confirm that RIPTAC-induced cytotoxicity is not only dependent on the presence of TP, but is acutely sensitive to the relative abundance of TP. Moreover, this TREx-293 model demonstrates that increased sensitivity to RIPTACs is not simply a consequence of elevated cell stress due to constitutive expression of HaloTag-FKBP.

[0406] To further corroborate that RIPTAC-induced cell cycle arrest is a consequence of on-target inhibition of PLK1, we evaluated downstream effectors of PLK1. We treated 293 HFL cells for 24 h with increasing concentrations of unmodified inhibitor (BI-2536) or tri-functional compound 5 to measure their effects on Cyclin Bl. PLK1 is known to phosphorylate Cyclin Bl on Seri 33 to promote M phase entry.20 Paradoxically, following treatment and immunoblotting, both BI-2536 and compound 5 lead to an increasing concentration of phospho-cyclin Bl (Serl33) at higher doses. This may be attributed to PLK1 ’s additional roles, including activation of the anaphase-promoting complex / cyclosome (APC / C)-Cdc20 complex which canonically degrades cyclin Bl.20, 21 Therefore, PLK1 inhibition during the metaphase to anaphase transition may prevent degradation of previously phosphorylated cyclin Bl leading to increases in phospho-cyclin Bl. Notably, similar findings have been observed in breast and prostate cancer cell lines.22, 23

[0407] Finally, we next confirmed that compound 5 induces ternary complex formation between HaloTag and PLK1 using a NanoBiT assay in WT HEK 293T cells co-transfected with PLKl-SmBiT and HaloTag7-LgBiT plasmids (FIG. 6D and 6E). Compound 5 exhibits a bell-shaped response curve4900-3869-3513.2y.. o. -characteristic of classical ternary complex equilibria. At low concentrations, insufficient ternary complex formation occurs, while at high concentrations, the equilibrium shifts toward binary complexes, PLK1-RIPTAC and HaloTag-RIPTAC, leading to a “hook effect”.24

[0408] In sum, these data demonstrate that both bi-functional and tri-functional molecules binding to HaloTag and PLK1 can selectively inhibit proliferation of cells, this effect is dependent on HaloTag expression and abundance, and ternary complex formation leads to PLK1 inhibition and cell cycle arrest.

[0409] Preparation of antihody-RIPTAC conjugates (ARCs) and dual-logic cell killing in vitro

[0410] To prepare ARCs we leveraged a novel bioconjugation strategy in development at the Grinstaff lab based on the supramolecular assembly of coiled-coil peptides for uniform, stoichiometric and sitespecific antibody conjugation. Using peptides identified by Gradisar and Jerala,25 we used a pair of coil peptides (P3 and P4) that remain unstructured individually but spontaneously form a heterodimeric coiled-coil at a 1:1 molar ratio, driven by complementary electrostatic (glutamic acid / lysine) and hydrophobic (leucine / isoleucine) interactions across four heptad repeat domains.

[0411] First, we recombinantly produced anti-HER2 mAb, trastuzumab, fused to coil P3 (receiving coil) at the C-terminus of the heavy chain using a short GS linker by co-transfecting Expi293 cells with plasmids encoding light chain and heavy chain-P3 fragments, followed by protein A chromatography purification. Next, we used copper-catalyzed click chemistry to conjugate compound 5 to P4 (docking coil), which was modified with a C-terminal propargyl glycine (FIG. 7A). We then assembled the ARCs by simply mixing trastuzumab-P3 with P4-compound 5 in mild aqueous conditions followed by spin column centrifugation to isolate the desired product (FIG. 7B).

[0412] Following preparation of the trastuzumab-coil-compound 5 conjugate, we treated 293 HFL, 293 GFPL, and their HER2+ variants (293 HFL HER2 and 293 GFPL HER2) with the ARC to assess cytotoxicity (FIG. 7C). Encouragingly, the HER2 / HaloTag targeting ARC only exhibits cytotoxicity in HER2+, HaloTag+ cells (293 HFL HER2), with no discernable cytotoxicity in the other three cell lines. However, the observed viability at a drug dosage of 0.1 pM (~75%) was considerably higher than 293 HFL cells treated with compound 5 as a free small molecule (<10%). The concentration is reported as “approximate”, as the concentration of loaded drug may be significantly lower than reported.

[0413] Identification of ARC-druggable axis in metastatic castration-resistant prostate cancer (mCRPC)

[0414] To identify ideal disease indications for ARC selectivity, we utilized The Cancer Dependency Map (DepMap)26 and The Cancer Genome Atlas (TCGA)27, which are large repositories providing extensive omics, drug screening, and genetic perturbation data from diverse tumor cell lines and primary cancers, respectively. Key criteria in identifying suitable targets include: (1) significantly higher expression of extracellular and intracellular target proteins in cancer vs. non-cancer tissues; (2) strong cancer cell dependence on EP, with EP and intracellular TP colocalized intracellularly; and (3)4900-3869-3513.2y.. o. -a sufficient intracellular TP-to-EP ratio for effective EP silencing. DepMap proteomics analysis across all cell lines shows a strong overexpression of androgen receptor (AR) and six transmembrane epithelial antigen of prostate 1 (STEAP1), distinctly separated from all other cell lines, in the most commonly used prostate cancer cell lines, VCaP and LNCaP clone FGC (8A). TCGA analysis confirms this trend in primary tumor samples and demonstrates that clustering is highly specific to prostate cancer samples (FIG. 8B). This suggests a unique therapeutic opportunity: an AR-dependent RIPTAC linked to a STEAP1 antibody which selectively targets and kills metastatic prostate cancer cells. We analyzed Chronos dependency scores in DepMap across all genes in VCaP cells to identify EP candidates for prostate cancer. Chronos scores offer a normalized measure of gene essentiality, with values < -1 considered pan-essential genes. When comparing Chronos scores to protein expression data, we observed multiple highly essential genes identified in previous screens (BRD4, AURKA / B, CDK1 / 4 / 9, and PLK1) which display a favorable expression ratio of TP-to-EP (e.g., AR-to-BRD4) (FIG. 8C). Notably, this trend is conserved among other common prostate cancer cell lines such as 22Rv1 and LNCaP clone FGC. Importantly, EP candidates such as PLK1 have been recently shown to promote enzalutamide resistance in mCRPC cells through the Hedgehog signaling pathway, therefore RIPTACs are primed to target known resistance mechanisms.28

[0415] Discussion

[0416] Steric blocking of cancer-dependent proteins using chemically induced proximity is an emerging strategy in precision oncology. While this approach historically relied on recruiting ubiquitously expressed scaffolds such as immunophilins to induce proximity-based interference with essential proteins, 29-31 recent work from Raina et al.9 introduced RIPTACs, a generalizable strategy that instead employs a cancer-specific protein as the inhibitory component. By leveraging overexpressed, tumor-specific proteins to inhibit pan-essential proteins, RIPTACs achieve a high therapeutic index, functionally resembling antibody-drug conjugates in small-molecule form.

[0417] Despite this potential, RIPTACs share key limitations with other chimeric modalities such as PROTACs, including poor solubility, limited bioavailabilify, and suboptimal pharmacokinetics. Additionally, because RIPTACs rely on occupancy-based pharmacology rather than catalytic degradation, they face further barriers to therapeutic accumulation and clinical translation. We addressed these limitations by engineering a new class of antibody-RIPTAC conjugates (ARCs), which integrate a generalizable tri-functional linker design and targeting antibodies to specifically deliver RIPTACs to target tissues. As an added benefit to the ARC platform, ARCs employ a duallogic killing mechanism: antibody-mediated delivery targeting extracellular tumor-associated antigens, and RIPTAC-based inhibition of essential effectors in an intracellular tumor proteindependent fashion. Through this combinatorial targeting, ARCs simultaneously address a central challenge of both ADCs and chimeric proximity inducers — the narrow therapeutic index resulting from on-target / off-tumor toxicity and non-specific delivery.4900-3869-3513.2y.. o. -

[0418] Our findings demonstrate that, like bi-functional RIPTACs, tri-functional designs selectively kill cells overexpressing a specific target protein, as demonstrated in the HaloTag-FKBP model system. By embedding a chemical handle within the linker, tri-functional RIPTACs allow for antibody conjugation without altering either warhead. Among the HaloTag / PLKl -targeting compounds tested, only select linkers (compounds 4 and 5) achieve effective proximity-induced killing, with compound 5 exhibiting a therapeutic index comparable to bi-functional RIPTACs. These findings underscore the importance of spatial configuration, linker flexibility, and linker polarity in RIPTAC-mediated ternary complex formation. While the current compounds are not yet pharmacologically optimized and reflect a limited exploration of the linker design space, we anticipate that future iterations will achieve improved potency and selectivity. Our data further establishes that bi-functional (BI-2PEG-CA) and tri-functional (compound 5) RIPTACs induce TP-dependent mitotic arrest through stable ternary complex formation, as confirmed by competition experiments, dox-inducible expression studies, modulation of PLK1 downstream effectors and NanoBiT PPI assays.

[0419] To facilitate tissue-specific delivery, we developed a modular ARC platform using site-specific supramolecular assembly via coiled-coil peptide pairing. This approach avoids the heterogeneity and inefficiencies of traditional ADC bioconjugation and enables tunable loading of chemically complex payloads. The trastuzumab-ARC construct selectively killed HER2+ / HaloTag+ cells but spared HER2- or TP- cells, validating the dual-logic hypothesis. As RIPTACs exhibit lower IC50 values than traditional ADC payloads, such as auristatins, traditional loading techniques such as cysteine-based antibody coupling using a maleimide-terminated antibody linker (FIG. 4C) would allow for accessing higher DARs and traditional ADC characterization workflows.32

[0420] Beyond proof of concept, we applied open-source genomic studies to identify high-priority indications for ARC deployment. Integrating proteomics, dependency scores, and transcriptomic profiles across DepMap and TCGA, we identified the STEAP1 / AR axis in metastatic prostate cancer as a compelling use case. This tumor type is marked by co-overexpression of a druggable extracellular antigen (STEAP1) and cancer-specific intracellular transcription factor (AR), enabling selective ARC activity. Notably, several candidate pan-essential effectors, including PLK1 and AURKB, may contribute to therapeutic resistance and have previously discovered small molecule ligands, making them ideal targets for ARC-mediated inhibition.

[0421] Collectively, our work provides a platform for combining tumor-selective extracellular targeting with intracellular context-driven synthetic lethality, expanding the chemical diversity of payloads for antibody-based therapies. ARCs offer a compelling solution to the long-standing limitations of ADC payload specificity and bi-functional molecule delivery, and open new avenues for addressing therapeutic resistance in genetically defined cancers. Future efforts will focus on optimizing RIPTAC pharmacology and conjugation methodologies and evaluating in vivo efficacy in tumor models with endogenous expression of validated ARC axes, such as STEAP1 / AR in metastatic prostate cancer.4900-3869-3513.2y.. o. -

[0422] Materials and Methods

[0423] Chemicals

[0424] (R)-4-((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzoic acid was purchased from Ambeed (A666739). N-(Azido-PEG3)-N-(PEG2-NH-Boc)-PEG3-acid was purchased from BroadPharm (BP-23962). N6-(tert-butoxycarbonyl)-L-lysine (00363), (tert-butoxycarbonyl)-L-lysine (02708) and N6-(tert-butoxycarbonyl)-D-lysine (03097) were purchased from Chem Impex. All other starting materials and reagents were purchased from Sigma-Aldrich unless stated otherwise.

[0425] Cell culture

[0426] HEK 293 (CRL-1573) and HEK 293T (CRL-3216) cells were obtained from the American Type Culture Collection (ATCC) and grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% FBS and Penicillin (100 U / mL) / Streptomycin (100 pg / mL) (PenStrep, Gibco). HaloTag7-FKBPF36V-P2A-EGFP expressing HEK 293 cells (293 HFL) and EGFP expressing HEK 293 cells (293 GFPL) were a kind gift from Craig Crews’ lab at Yale University and grown in DMEM supplemented with 10% FBS, PenStrep, and 2 pg / mL puromycin (InvivoGen) to maintain protein overexpression. Following lentiviral transduction with HER2 (FL), 293 HFL HER2 and 293 GFPL HER2 cells were grown in DMEM supplemented with 10% FBS, PenStrep, 2 pg / mL puromycin, and 200 pg / mL hygromycin B gold (InvivoGen) to maintain protein overexpression

[0427] Plasmid design and construction

[0428] Plasmids were constructed using established molecular biology methods and Gibson or Golden Gate assembly. Engineered cassettes were subcloned into vectors containing Ampicillin resistance as a bacterial selection marker. All plasmids were sequence-verified and archived in NEB Stable competent E. coli (New England Biolabs, C3040).

[0429] Trastuzumab light chain and heavy chain-P3 plasmids were constructed by subcloning cassettes into the pcDNA 3.1(-) mammalian expression vector backbone (Invitrogen) digested with BsmI / BamHI. Donor plasmids for HER2 (FL) lentiviral integration were constructed by subcloning cassettes into the pHR’SIN vector backbone digested with EcoRI / NotI;33 HER2 expression is driven by a EFla promoter and these vectors were additionally modified to contain hygromycin B resistance, separated by a P2A sequence following HER2, as mammalian selection markers. PLKl-SmBiT and HaloTag7-LgBiT plasmids were assembled using the Mammalian Toolkit (Addgene, Kit #1000000180) where protein expression is driven by a weaker PGK promoter.

[0430] Lentiviral transduction of HEK 293 cell lines

[0431] Full length HER2 expression vectors were cloned into lentiviral donor plasmids and stably integrated into 293 HFL and 293 GFPL cell lines using lentiviral transduction. Lentivirus was harvested from transfected HEK 293T cells. 600,000 HEK 293T were seeded into 6-well treated plates for 24 hours and subsequently transfected with 4.8 pg total DNA in a NaCl-polyethylenimine (PEI) solution. 2.4 ug of the donor HER2 plasmid was co-transfected with 1.65 pg of pCMVR8.744900-3869-3513.2y.. o. -plasmid (Addgene #22036), 220 ng of pAdVantage plasmid (Promega), and 550 ng of the pMD2. G VSVG plasmid (Addgene #12259). 24 h after transfection, cell media was exchanged for fresh media and cells were incubated for 48 hours prior to harvesting lentiviral supernatant. Lentiviral supernatant was collected and centrifuged for 5 minutes at 2100 g, filtered with 0.45 pm syringe filter, and stored at -80°C until use.

[0432] For transduction, 293 HFL and 293 GFPL cells were seeded into poly-L lysine coated 6-well treated plates (750,000 cells / well). The next day, media was exchanged for fresh media supplemented with 8 pg / mL polybrene and 400 pL of lentiviral media was added to the cells. Infected cells were incubated for 48 hours prior to subculturing and selection with 200 pg / mL of hygromycin B for cells expressing HER2. Cells were continually subcultured under hygromycin B selection to maintain expression of HER2 and used for downstream experiments.

[0433] Flow cytometry protein expression

[0434] To validate EGFP and HER2 expression, WT HEK 293, 293_GFPL, 293_HFL,293 GFPL HER2, and 293 HFL HER2 were plated in poly-L lysine coated 24-well plates (50,000 cells / well) in complete media with a total volume of 500 pL / well. 48 h later, cells were washed once with IX PBS and lifted with Versene solution (Gibco). Cells were then transferred to individual microcentrifuge tubes and pelleted via centrifugation. After pelleting, cells were labelled with 1 pL / pellet Alexa Fluor 647 anti-human CD340 (erbB2 / HER2) antibody (BioLegend, 324412) diluted in FACS buffer (IX PBS, 2% FBS and 1 mM EDTA) for 30 min on ice. After labelling, cells were washed two times with FACS buffer to remove any unbound or non-specifically bound antibody and then subjected to flow cytometry on an Attune NxT Flow Cytometer (Invitrogen). Cell debris was excluded by gating on the forward and side scatter plot. Representative quadrant plots are shown for all included events, and corresponding % of cells in each quadrant is displayed.

[0435] CyQUANT cell proliferation assay

[0436] Cells were plated in poly-L lysine (R& D Systems) coated 96- well plates (1,000 cells / well) in complete media with a total volume of 100 pL / well. For competition experiments, 293 HFL cells were plated in complete media supplemented with 300 nM TAMRA-CA (Promega, G8251) to block available HaloTag. The next day, compounds were titrated in 100% DMSO and diluted in growth medium. 100 pL of the compound / medium mixture was added to cells, bringing the total volume in each well to 200 pL at the listed concentration of compound. DMSO was used as a negative control. Cells were then cultured at 37°C with 5% CO2 for 7 days in a humidified tissue culture incubator.

[0437] On Day 7 of treatment, cell viability was quantified with CyQUANT cell proliferation assay (Invitrogen). Briefly, microplates were gently inverted and blotted onto paper towels to remove medium from the wells. Microplates were then stored at -80°C until samples are to be assayed.Following freezing, plates were thawed at room temperature, and 200 pL / well of the DNA dye / cell-lysis buffer was added to each sample well. Wells were mixed gently with multi-channel pipette and then transferred to a black 96-well plate after which total DNA was quantified with a SpectraMax iD34900-3869-3513.2y.. o. -(Molecular Devices) plate reader (Ex: 480 nm, Em: 520 nm). Viability was normalized relative to untreated control wells, after correcting for background absorbance. A four-parameter logistic (4PL) regression fit was applied to dose-response data in GraphPad Prism data analysis software to determine the half-maximal inhibitory concentration (IC50) for each compound.

[0438] Vybrant DyeCycle assay

[0439] Cells were plated in poly-L lysine coated 96- well plates (15,000 cells / well) in complete media with a total volume of 100 pL / well. The next day, compounds were titrated as above and 100 L of the compound / medium mixture was added to cells, bringing the total volume in each well to 200 pL at the listed concentration of compound. 24 h later, cells were washed once with IX PBS (phosphate buffered saline, Gibco) and trypsinized using Trypsin-EDTA (0.25%) (Gibco). After pelleting, cells were resuspended in complete media supplemented with 1 mM EDTA (ethylenediaminetetraacetic acid, Thermo Scientific) and 5 pM Vybrant DyeCycle Violet stain (Invitrogen) and placed in an incubator at 37°C with 5% CO2 for 30 min. Following incubation, cells were immediately analyzed via flow cytometry. Cell debris was excluded by gating on the forward and side scatter plot.Representative intensity histograms shown are normalized by mode for each condition.

[0440] Immunoblotting

[0441] Cells were plated in poly-L lysine coated 12-well plates (200,000 cells / well) in complete media with a total volume of 1 mL / well. The next day, compounds were titrated as above and 1 mL of the compound / medium mixture was added to cells, bringing the total volume in each well to 2 mL at the listed concentration of compound. To prepare protein lysates, cells were lysed in plate with RIPA buffer (Bio-Rad) and protein concentrations were measured using a Pierce BCA Protein Assay (ThermoFisher). 10 pg of lysate was run on a 12% SDS-PAGE gel and transferred to a PVDF membrane (Bio-Rad). Incubations with primary antibodies, Cyclophilin-B (Cell Signaling Technology, 43603S) and Phospho-Cyclin Bl (Serl33) (Cell Signaling Technology, 4133S) were performed overnight at 4 oC at a concentration of 1: 1000 in 5% BSA in TBS-T. Secondary-HRP incubation (Cell Signaling Technology) was conducted for one hour at room temperature at a concentration of 1:2000. Chemiluminescence was read using SuperSignal West Atto Ultimate Sensitivity substrate (ThermoFisher) on a ChemiDoc XRS+ (Bio-Rad).

[0442] NanoBiT PPI assay

[0443] WT HEK 293T cells were plated in white, poly-L lysine coated 96-well plates (20,000 cells / well) in complete media. The next day, compounds were titrated in 100% DMSO and diluted in growth medium. Media in the wells was exchanged for 100 pL of the compound / medium mixture at the listed concentrations. Immediately following media exchange, cells were co-transfected with HaloTag7-LgBiT and PLK1-SmBiT (1:1, 100 ng total) plasmids using Lipofectamine 3000 (Invitrogen) and incubated for 24 h at 37°C with 5% CO2. The following day, media was exchanged for Opti-MEM (Gibco) and Nano-Glo luciferase substrate (Promega, N2012), and luminescence was monitored using a SpectraMax iD3 plate reader.4900-3869-3513.2y.. o. -

[0444] Antibody production and preparation of ARC

[0445] Anti-HER2 antibody, Trastuzumab, linked to P3 (receiving coil) was expressed by cotransfecting Expi293 cells with plasmids encoding light chain and heavy chain-P3 fragments using the ExpiFectamine 293 Transfection Kit (Gibco). Following manufacturer’s instructions, folded antibody was purified from cell supernatant via Pierce Protein A purification columns (Thermo Scientific). Average yield 5-7 days post-transfection was ~10 mg / L of cell culture.

[0446] Cancer Genomics Analyses

[0447] Methods for quantification of biomarkers are detailed by DepMap and elsewhere.37 Briefly, protein expression data was either quantified via multiplexed tandem mass tag mass spectrometry (i.e., “Relative Protein Expression Proteomics”) or reverse phase protein array (i.e., “RPPA”). Gene expression levels were quantified as log2(TPM+l) and the gene copy number was calculated relative to the ploidy of the rest of the genome for a given cell line. Dependency effect sizes were measured using Chronos scores, which are derived from a population dynamics model that accounts for sgRNA efficiency, screen quality, intrinsic growth rates, and DNA cutting toxicity bias.38 These continuous scores typically reflect gene essentiality, with essential genes often exhibiting Chronos scores below -1, and unexpressed genes showing scores near zero.

[0448] References1. Barnscher SD. The Clinical Landscape Of ADCs In 2023: Diverse Technologies, Narrow Target Clinical Leader2023.2. Dumontet C, Reichert JM, Senter PD, Lambert JM, Beck A. Antibody-drug conjugates come of age in oncology. Nature Reviews Drug Discovery. 2023;22(8):641-61. doi: 10.1038 / s41573-023-00709-2.3. Fu Z, Li S, Han S, Shi C, Zhang Y. Antibody drug conjugate: the “biological missile” for targeted cancer therapy. Signal Transduction and Targeted Therapy. 2022;7(l). doi: 10.1038 / s41392-022-00947-7.4. Criscitiello C, Morganti S, Curigliano G. Antibody-drug conjugates in solid tumors: a look into novel targets. Journal of Hematology & Oncology. 2021; 14(1). doi: 10.1186 / sl3045-021-01035-z. 5. Szijj PA, Bahou C, Chudasama V. Minireview: Addressing the retro-Michael instability of maleimide bioconjugates. Drug Discovery Today: Technologies. 2018;30:27-34. doi: 10.1016 / j.ddtec.2018.07.002.6. Maecker H, Jonnalagadda V, Bhakta S, Jammalamadaka V, Junutula JR. 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[0449] (a) Synthesis of tert-butyl (21-chloro-8-oxo-3,6,12,15-tetraoxa-9-azahenicosyl)carbamate

[0450] To a solution of 2,2-dimethyl-4-oxo-3,8,ll-trioxa-5-azatridecan-13-oic acid (95 mg, 0.36 mmol) in anhydrous N, N-dimethylformamide (DMF, 2.0 mL) were added N, N-diisopropylethylamine (DIPEA, 175 L, 1.0 mmol) and l-[bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 205 mg, 0.54 mmol) and the mixture was stirred at 25 °C for 10 min. Next, 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-l-aminium hydrochloride (88 mg, 0.36 mmol) was and the reaction mixture was stirred at ambient temperature for 12 h. Upon completion, the reaction mixture was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (50 mL). The organic phase was washed sequentially with deionized water (3 x 10 mL) and brine (1 x 10 mL), then dried over anhydrous sodium sulfate. The solution was filtered and concentrated under reduced pressure to yield the desired product (89 mg, 53%).DCM RT, -1 h93%

[0451] (b) Synthesis of 2-(2-(2-aminocthoxy)cthoxy)-A-(2-(2-((6 chlorohexyl)oxy)ethoxy)ethyl)acetamide

[0452] To a stirring solution of tert-butyl (21-chloro-8-oxo-3,6,12,15-tetraoxa-9-azahenicosyl)carbamate (89 mg, 0.19 mmol) in anhydrous dichloromethane (DCM, 1.0 mL) at 0 °C was added trifluoroacetic acid (TFA, 1.0 mL) dropwise. The reaction mixture was allowed to warm to room temperature and stirred for 40 min. The reaction was monitored by TLC. The solvent was4900-3869-3513.2y.. o. -removed under reduced pressure, and the residue was redissolved in DCM (10 mL) and dried under reduced pressure. This redissolution and drying step was repeated two additional times to remove residual TFA and yield the desired product (65 mg, 93%).

[0453] (c) Synthesis of BI-2PEG-CA: (R)-N-(21-chloro-8-oxo-3,6,12,15-tetraoxa-9-azahenicosyl)-4-((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzamide

[0454] To a solution of (R)-4-((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzoic acid (dTAG Targeting Ligand 1, 68 mg, 0.16 mmol) in anhydrous DMF (1.5 mL) was added N, N-diisopropylethylamine (DIPEA, 87 pL, 0.50 mmol), followed by EDCI. HC1 (50 mg, 0.24 mmol). The reaction mixture was stirred at room temperature for 5 min, after which 1-hydroxy-7-azabenzotriazole (HOBt, 32 mg, 0.24 mmol) was added. After an additional 10 min of stirring, 2-(2-(2-aminoethoxy)ethoxy)-A-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)acetamide (59 mg, 0.16 mmol) was added and the reaction mixture was stirred at ambient temperature for 16 h. Upon completion, the reaction was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (50 mL), and the organic layer was washed sequentially with deionized water (3 x 10 mL) and brine (1 x 10 mL), then dried over anhydrous Na2SO4. The solution was filtered and concentrated under reduced pressure. The crude product was redissolved in acetonitrile and purified by preparative reverse-phase HPLC (Cl 8 column; Water(0.225%FA)–ACN(0.225%FA); 25%–95% ACN(0.225%FA)) to afford the desired product as a clear oil (5.8 mg, 4.7% yield).Scheme S2: Synthetic route for Compound 1BOC'N^O^°^O-^°HBOC'N^O^O^O^°^^^^CI THF RT, 16 h40%

[0455] (d) Synthesis of tert-Butyl (18-chloro-3,6,9,12-tetraoxaoctadecyl)carbamate

[0456] To a solution of tert-butyl (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)carbamate (900 mg, 3.06 mmol) in THF (12 mL) were added l-chloro-6-iodohexane (754 mg, 3.06 mmol) and KOH (211 mg, 3.76 mmol) and the mixture was stirred at 25 °C for 16 h. The reaction was quenched with saturated aqueous NaH₂PO₄ solution (10 mL), and the mixture was extracted with EtOAc (30 mL x3). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced4900-3869-3513.2y.. o. -pressure. The residue was purified by silica gel chromatography (PE: EtOAc =40:60) to give the desired product (510 mg, 40%) as a yellow oil.

[0457] ¹H NMR (400 MHz, DMSO-d₆) δ 5.01 (br s, 1H), 3.68 - 3.60 (m, 10H), 3.59 - 3.56 (m, 2H), 3.54 - 3.50 (m, 4H), 3.45 (t, J= 6.4 Hz, 2H), 3.30 (t, J= 5.2 Hz, 2H), 1.82 - 1.72 (m, 2H), 1.62 - 1.55 (m, 2H), 1.47 - 1.42 (m, 11H), 1.41 - 1.33 (m, 2H).TFA DCM RT, 1 h-100%

[0458] (e) Synthesis of 18-Chloro-3,6,9,12-tetraoxaoctadecan-l-amine trifluoroacetate

[0459] To a solution of tert-butyl (18-chloro-3,6,9,12-tetraoxaoctadecyl)carbamate (300 mg, 0.73 mmol) in DCM (4 mL) was added TFA (2 mL) and the mixture was stirred at RT for 1 h. The mixture was concentrated to give the desired product (300 mg, quant) as a yellow solid.

[0460] LCMS: m / z 312.3 [M+H]+.BOC'NHOHDIEA(3 eq), HATU (1.2 eq) DMF0°C-RT, 16 h26%

[0461] (f) Synthesis of N6-(tert-Butoxycarbonyl)-N2-(4-(((R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzoyl)-L-lysine

[0462] To a solution of (R)-4-((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzoic acid (100 mg, 0.24 mmol) in DMF (2 mL) at 0 °C were added DIEA (92.9 mg, 0.72 mmol) and HATU (109 mg, 0.28 mmol). After stirring at RT for 30 min, N6-(tert-butoxycarbonyl)-L-lysine (60 mg, 0.24 mmol) was added and the mixture was stirred at RT for 16 h. The reaction was quenched with water (10 mL) and the mixture extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine, dried over Na₂SO₄, filtered and concentrated under reduced pressure to give the desired product (40 mg, 26%) as a yellow solid, which was used in the next step without further purification.

[0463] LCMS: m / z 654.3 [M+H]+.4900-3869-3513.2y.. o. -

[0464] (g) Synthesis of Compound 1: N-((S)-l-Amino-25-chloro-6-oxo-10, 13,16, 19-tetraoxa-7-azapentacosan-5-yl)-4-(((R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzamide

[0465] To a solution of N6-(tert-butoxycarbonyl)-N2-(4-(((R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzoyl)-L-lysine (70 mg, 0.11 mmol) in DMF (1 mL) at 0 °C were added DIEA (41.4 mg, 0.33 mmol), EDCI. HC1 (24.6 mg, 0.13 mmol) and HOBT (17 mg, 0.13 mmol). After stirring at 0 °C for 1 h, 18-chloro-3,6,9,12-tetraoxaoctadecan-l-amine trifluoroacetate (33.3 mg, 0.11 mmol) was added and the mixture was stirred at RT for 8 h. The reaction was quenched with water (5 mL) and the mixture extracted with DCM (5 mL x 3). The combined organic layers were washed with brine, dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was dissolved in a 4 M HC1 in dioxane solution (2 mL) and stirred at RT for 1 h. The mixture was concentrated and the residue purified by prep-HPLC (GILSON-281, Pntulips BP-C18, 10 pm, 21.2 x 250 mm, eluting with a gradient of ACN in water with 0.1% Formic acid, at a flow rate of 25.0 mL / min, 10%-40% ACN over 18 min) to give desired product (11.6 mg, 8%) as a white solid.

[0466] LCMS: m / z 847.4 [M+H]+.

[0467] ’H NMR (400 MHz, DMSO-fi ) 88.46 - 8.42 (m, 2H), 8.39 (d, J= 8.0 Hz, 1H), 8.05 (t, J= 5.6 Hz, 1H), 7.85 (s, 1H), 7.61 (s, 1H), 7.58 - 7.54 (m, 2H), 4.48 - 4.30 (m, 2H), 4.24 (dd, J= 7.6, 3.6 Hz, 1H), 3.95 (s, 3H), 3.61 (t, J= 6.8 Hz, 2H), 3.51 - 3.46 (m, 10H), 3.45 - 3.40 (m, 5H), 3.35 (t, J= 6.4 Hz, 3H), 3.25 (s, 3H), 3.23 - 3.19 (m, 2H), 2.74 - 2.65 (m, 2H), 2.07 - 1.96 (m, 1H), 1.95 - 1.84 (m, 2H), 1.82 - 1.72 (m, 5H), 1.72 - 1.58 (m, 6H), 1.54 - 1.42 (m, 4H), 1.36 - 1.28 (m, 6H), 0.76 (t, J = 7.2 Hz, 3H).Scheme S3: Synthetic route for Compound 2BOC'NHBOC'NH o (h)

[0468] (h) Synthesis of N2-(tert-Butoxycarbonyl)-N6-(4-(((R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzoyl)-L-lysine4900-3869-3513.2y.. o. -

[0469] To a solution of (R)-4-((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzoic acid (200 mg, 0.47 mmol) in DMF (4 mL) were added DIEA (182 mg, 1.41 mmol) and HATU (214 mg, 0.56 mmol). After stirring at RT for 30 min, (tert-butoxycarbonyl)-L-lysine (116 mg, 0.47 mmol) was added and the mixture was stirred at RT for 16 h. The reaction was quenched with water (10 mL) and the mixture extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine, dried over Na₂SO₄, filtered and concentrated under reduced pressure to give the desired product (400 mg, crude) as a yellow solid, which was used in the next step without further purification.

[0470] LCMS: m / z 654.4 [M+H]+.<5 "

[0471] i) Synthesis of Compound 2: N-((S)-5-Amino-25-chloro-6-oxo-10,13,16,19-tetraoxa-7-azapentacosyl)-4-(((R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzamide

[0472] To a solution of N2-(tert-butoxycarbonyl)-N6-(4-(((R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzoyl)-L-lysine (100 mg) in DMF (1 mL) at 0 °C were added DIEA (58.0 mg, 0.45 mmol), EDCI. HC1 (34.5 mg, 0.18 mmol) and HOBT (24.3 mg, 0.18 mmol). After stirring at 0 °C for 1 h, 18-chloro-3,6,9,12-tetraoxaoctadecan-l-amine trifluoroacetate (47.6 mg, 0.15 mmol) was added and the mixture was stirred at RT for 8 h. The reaction was quenched with water (5 mL) and the mixture extracted with DCM (5 mL x 3). The combined organic layers were washed with brine, dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was dissolved in a 4 M HC1 in dioxane solution (2 mL) and stirred at RT for 1 h. The mixture was concentrated and the residue purified by prep-HPLC (GILSON-281, Pntulips BP-C18, 10 pm, 21.2 × 250 mm, eluting with a gradient of ACN in water with 0.1% Formic acid, at a flow rate of 25.0 mL / min, 10%-40% ACN over 18 min) to give the desired product (11.8 mg, 12% overtwo steps) as a white solid.

[0473] LCMS: m / z 847.5 [M+H]+.

[0474] ’H NMR (400 MHz, DMSO-J6): 88.42 (d, J= 8.9 Hz, 1H), 8.38 (t, J= 5.4 Hz, 1H), 8.30 (t, J = 5.5 Hz, 1H), 7.84 (s, 1H), 7.60 (s, 1H), 7.50 - 7.44 (m, 2H), 4.40 - 4.29 (m, 1H), 4.24 (dd, J= 7.6, 3.6 Hz, 1H), 3.95 (s, 3H), 3.64 - 3.57 (m, 3H), 3.53 - 3.46 (m, 9H), 3.45 - 3.40 (m, 4H), 3.35 (t, J= 6.4 Hz, 3H), 3.25 (s, 3H), 3.30 - 3.18 (m, 4H), 2.07 - 1.96 (m, 1H), 1.94 - 1.84 (m, 2H), 1.82 - 1.72 (m, 4H), 1.72 - 1.58 (m, 7H), 1.54 - 1.42 (m, 4H), 1.36-1.28 (m, 6H), 0.76 (t, J = 7.2 Hz, 3H).Scheme S4: Synthetic route for Compound 34900-3869-3513.2y.. o. -

[0475] j) Synthesis ofN6-(tert-Butoxycarbonyl)-N2-(4-(((R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzoyl)-D-lysine

[0476] To a solution of (R)-4-((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzoic acid (100 mg, 0.24 mmol) in DMF (2.0 mL) at 0 °C were added DIEA (92.9 mg, 0.72 mmol) and HATU (109 mg, 0.28 mmol). After stirring at 0 °C for 2 h, N6-(tert-butoxycarbonyl)-D-lysine (60.0 mg, 0.24 mmol) was added and the mixture was stirred at RT for 16 h. The reaction was quenched with water (10 mL) and the mixture extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine, dried over Na₂SO₄, filtered and concentrated under reduced pressure to give the desired product (95.0 mg) as a yellow solid, which was used in the next step without further purification.

[0477] LCMS: m / z 654.3 [M+H]+.

[0478] k) Synthesis of Compound 3: N-((R)-l-Amino-25-chloro-6-oxo-10, 13,16, 19-tetraoxa-7-azapentacosan-5-yl)-4-(((R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzamide hydrochloride

[0479] To a solution of N6-(tert-butoxycarbonyl)-N2-(4-(((R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzoyl)-D-lysine (90.0 mg) in DMF (2.0 mL) at 0 °C were added DIEA (53.3 mg, 0.41 mmol), EDCI. HC1 (31.7 mg, 0.17 mmol) and HOBT (22.3 mg, 0.17 mmol). After stirring at 0 °C for 30 min, 18-chloro-3,6,9,12-tetraoxaoctadecan-l-amine trifluoroacetate (42.8 mg, 0.14 mmol) was added and the mixture was stirred at RT for 16 h. The reaction was quenched with water (5 mL) and the mixture extracted with EtOAc (5 mL x 3). The combined organic phases were washed with brine, dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was dissolved in a 4 M HC1 in dioxane solution (2 mL) and stirred at RT for 1 h. The mixture was concentrated and the residue purified by prep-HPLC (GILSON-281, Pntulips BP-C18, 10 pm, 21.2 x 250 mm, eluting with a gradient of ACN in water with 0.1% Formic4900-3869-3513.2y.. o. -acid, at a flow rate of 25.0 mL / min, 10%- 40% ACN over 18 min) to give the desired product (10.3 mg, 5% over two steps) as a white solid.

[0480] LCMS: m / z 847.4 [M+H]+.

[0481] ¹H NMR (400 MHz, DMSO-d₆) δ 8.47 - 8.40 (m, 2H), 8.36 (d, J = 7.9 Hz, 1H), 8.06 - 7.98 (m, 1H), 7.85 (s, 1H), 7.61 (s, 1H), 7.59 - 7.51 (m, 2H), 4.49 - 4.30 (m, 2H), 4.28 - 4.21 (m, 1H), 3.95 (s, 3H), 3.61 (t, J = 6.6 Hz, 2H), 3.51 -3.16 (m, 23H, partially obscured by water peak), 2.71 -2.63 (m, 2H), 2.07 - 1.96 (m, 1H), 1.95 - 1.85 (m, 2H), 1.84 - 1.56 (m, 10H), 1.55 - 1.42 (m, 4H), 1.41 - 1.18 (m, 7H), 0.76 (t, J = 7.5 Hz, 3H).Scheme S5: Synthetic route for Compound 4

[0482] 1) Synthesis of Compound 4: N-((S)-5-Amino-6-((2-(2-((6-chlorohexyl)oxy)ethoxy)ethyl)amino)-6-oxohexyl)-4-(((R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo- 5.6.7.8-tetrahydropteridin-2-yl)amino)-3-methoxybenzamide

[0483] To a solution of N2-(tert-butoxycarbonyl)-N6-(4-(((R)-8-cyclopentyl-7-ethyl-5-methyl-6-oxo- 5.6.7.8-tetrahydropteridin-2-yl)amino)-3-methoxybenzoyl)-L-lysine (200 mg, 0.3 mmol) in DMF (2 mL) at 0 °C were added DIEA (116 mg, 0.9 mmol), EDCI. HC1 (69 mg, 0.36 mmol) and HOBT (49 mg, 0.36 mmol). After stirring at 0 °C for 1 h, 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-l -amine hydrochloride (78 mg, 0.3 mmol) was added and the mixture was stirred at RT for 8 h. The reaction was quenched with water (5 mL) and the mixture extracted with EtOAc (5 mL x 3). The combined organic layers were washed with brine, dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was dissolved in a 4 M HCI in dioxane solution (2 mL) and stirred at RT for 1 h. The mixture was concentrated and the residue purified by prep-HPLC (GILSON-281, Pntulips BP-C18, 10 pm, 21.2 x 250 mm, eluting with a gradient of ACN in water with 0.1% Formic acid, at a flow rate of 25.0 mL / min, 10%-40% ACN over 18 min) to give the desired product (15.0 mg, 6%) as a yellow solid.

[0484] LCMS: m / z 759.4 [M+H]+.

[0485] ’H NMR (400 MHz, DMSO-J6): <58.41 (d, J= 8.3 Hz, 1H), 8.31 (t, J= 5.5 Hz, 1H), 8.26 (s, 1H), 8.06 (t, J= 5.6 Hz, 1H), 7.84 (s, 1H), 7.59 (s, 1H), 7.50 - 7.44 (m, 2H), 4.40 - 4.29 (m, 1H), 4.24 (dd, J= 7.6, 3.6 Hz, 1H), 3.95 (s, 3H), 3.60 (t, J= 6.6 Hz, 2H), 3.51 - 3.42 (m, 4H), 3.40 (t, J= 5.9 Hz, 2H), 3.34 (t, J= 6.5 Hz, 2H), 3.30 - 3.13 (m, 4H), 3.25 (s, 3H), 2.06 - 1.96 (m, 1H), 1.95 -1.84 (m, 2H), 1.82 - 1.72 (m, 4H), 1.71 - 1.55 (m, 6H), 1.55 - 1.41 (m, 5H), 1.40 - 1.22 (m, 6H), 0.76 (t, J = 7.2 Hz, 3H).4900-3869-3513.2y.. o. -Scheme S6: Synthetic route for Compound 5

[0486] (m) Synthesis of tert-butyl (10-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-35-chloro-9,22-dioxo-3,6,13,16,19,26,29-heptaoxa-10,23-diazapentatriacontyl)carbamate

[0487] To a solution of 15-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-2,2-dimethyl-4,14-dioxo-3,8, 11, 18,21,24-hexaoxa-5, 15-diazaheptacosan-27-oic acid (N-(Azido-PEG3)-N-(PEG2-NH-Boc)-PEGa-acid, 50 mg, 0.073 mmol) in anhydrous DMF (2.0 mL) was added N, N-diisopropylethylamine (DIPEA, 105 pL, 0.60 mmol), followed by l-[bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 38 mg, 0.10 mmol). The reaction mixture was stirred at room temperature for 10 min before the addition of 2-(2-((6-chlorohexyl)oxy)ethoxy)ethan-1-aminium hydrochloride (19 mg, 0.073 mmol). Stirring was continued for 12 h at ambient temperature. After completion, the reaction mixture was concentrated under reduced pressure. The residue was redissolved in ethyl acetate (50 mL), and the organic phase was washed sequentially with deionized water (3 x 10 mL) and brine (1 x 10 mL), then dried over anhydrous Na₂SO₄. The solution was filtered and concentrated under reduced pressure. The crude residue was redissolved in ethyl acetate and purified by silica gel chromatography using a cyclohexane / ethyl acetate gradient as the eluent to afford the desired product as a clear oil (28.0 mg, 43%).

[0488] (n) Synthesis of 3-(2-(2-aminoethoxy)ethoxy)-N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-N-(25-chloro-12-oxo-3,6,9,16,19-pentaoxa-13-azapentacosyl)propanamide

[0489] To a stirring solution of tert-butyl (10-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-35-chloro-9,22-dioxo-3,6,13,16,19,26,29-heptaoxa-10,23-diazapentatriacontyl)carbamate (28 mg, 0.0315 mmol) in anhydrous dichloromethane (DCM, 1.0 mL) at -20 °C was added trifluoroacetic acid (TFA, 1.0 mL) dropwise. The reaction mixture was allowed to warm to room temperature and stirred for 40 min. Completion of the deprotection was confirmed by TLC. The solvent was removed under reduced pressure, and the residue was redissolved in DCM (10 mL) and concentrated under reduced pressure. This redissolution and drying step was repeated three additional times to ensure complete removal of residual TFA, affording the desired product (24.0 mg, 98%).4900-3869-3513.2y.. o. -

[0490] (o) Synthesis of Compound 5: (R)-N-(10-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-35-chloro-9,22-dioxo-3,6,13,16,19,26,29-heptaoxa-10,23-diazapentatriacontyl)-4-((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzamide

[0491] To a solution of (R)-4-((8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)-3-methoxybenzoic acid (dTAG Targeting Ligand 1, 13 mg, 0.030 mmol) in anhydrousDMF (1.5 mL) was added N, N-diisopropylethylamine (DIPEA, 31 pL, 0.18 mmol), followed byEDCI. HC1 (16 mg, 0.070 mmol). The reaction mixture was stirred at room temperature for 5 min,after which l-hydroxy-7-azabenzotriazole (HOBt, 9.5 mg, 0.070 mmol) was added. After anadditional 10 min of stirring, 3-(2-(2-aminoethoxy)ethoxy)-N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-N-(25-chloro- 12-oxo-3,6,9,16,19-pentaoxa- 13-azapentacosyl)propanamide (24 mg, 0.030 mmol) was added. The reaction mixture was stirred atambient temperature for 22 h. Upon completion, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (50 mL), and the organic phase was washedsequentially with deionized water (3 x 10 mL) and brine (1 x 10 mL), then dried over anhydrousNazSCL. The solution was filtered and concentrated under reduced pressure. The crude product was redissolved in acetonitrile and purified by preparative reverse-phase HPLC (Cl 8 column;Water(0.225%FA)–ACN(0.225%FA); 25%–95% ACN(0.225%FA)) to afford the desired product as aclear oil (4.1 mg, 11.4%).4900-3869-3513.2TABLE 1CompoundStructureNo.1-1Ji ' -H M & o 5 j?l 1 f l j" M L— *!AV T ° u r if H y A,zV A ^ Aw «C V^Y v.HJ OiCl1-2 \ o..s ° r^r'YHxr-.* _ R. V- xk J,sJL Y Y n iHH H r Y K^- T >N A-or v "—A-"ClM0 0cr1-3.so f A- si K. J / Y o MHo uT 'VV, V J- A A Hi r A M A H CiJ 'xAAHo o4900-3869-3513.2Atty. Dkt. No. 701586-000161WOPT4900-3869-3513.2Atty. Dkt. No. 701586-000161WOPT4900-3869-3513.2Atty. Dkt. No. 701586-000161WOPT4900-3869-3513.2Atty. Dkt. No. 701586-000161WOPT4900-3869-3513.2Atty. Dkt. No. 701586-000161WOPT4900-3869-3513.2Atty. Dkt. No. 701586-000161WOPT4900-3869-3513.2Atty. Dkt. No. 701586-000161WOPT4900-3869-3513.2TABLE 2 CompoundStructureNo.4900-3869-3513.2Atty. Dkt. No. 701586-000161WOPT4900-3869-3513.2Atty. Dkt. No. 701586-000161WOPT4900-3869-3513.2Atty. Dkt. No. 701586-000161WOPT4900-3869-3513.2II-7 o e y o nun o || v I r uf o H AA / A A ^x tx A A jr Ai I 1 H J| £ HW " x / o O x@ -HH O^ HHJIII-8 o «fixh H II I H< X N A A N <xY Y T Y Y Y0 ^A^ OH \A\A-*II-9 H2^ Y'.. NH0r / TX H |! H< A, A. X.. XY y x > n a Y | Y j Y Y |Q xAs / ^4900-3869-3513.211-114900-3869-3513.2Atty. Dkt. No. 701586-000161WOPT4900-3869-3513.2Atty. Dkt. No. 701586-000161WOPT4900-3869-3513.2

Claims

y.. o. -CLAIMS1. A targeting compound for killing cancer cells, comprising components that targeta) a tumor-associated extracellular target; andb) at least one tumor associated intracellular target.

2. An antibody conjugate compound for killing cancer cells, comprising components that target a) a tumor-associated extracellular target; andb) at least one tumor associated intracellular target; andc) at least one intracellular effector target.

3. The antibody conjugate compound of claim 2, further comprising an AND logic gate, wherein cell killing activity requires the presence of each of the targets.

4. An antibody conjugate compound comprising:a) an antibody that specifically binds a tumor-associated extracellular antigen;b) a regulated induced-proximity targeting chimera (RIPTAC) comprising:i) a first small-molecule binding moiety that selectively binds an intracellular tumor-associated target protein; andii) a second small-molecule binding moiety that selectively binds a pan-essential intracellular effector protein; andiii) a RIPTAC linker covalently joining the two small-molecule binding moieties; and c) an antibody linker covalently joining the antibody to the RIPTAC.

5. The compound of claim 4, wherein the intracellular tumor-associated target is selected from the group consisting of: androgen receptor (AR), enhancer of zeste homolog 2 (EZH2), induced myeloid leukemia cell differentiation protein Mcl-1 (MCL1), Wilms tumor protein (WT33), tumor protein p53 (TP53), and mouse double minute 2 homolog (MDM2).

6. The compound of claim 4 or 5, wherein the pan-essential intracellular effector protein is selected from the group consisting of polo-like-kinase-1 (PLKl), Bromodomain-containing protein 2 (BRD2), Bromodomain-containing protein 3 (BRD3), Bromodomain-containing protein 4 (BRD4), cyclin-dependent kinase 1 (CDK1), cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 5 (CDK5), cyclin-dependent kinase 6 (CDK6), and cyclin-dependent kinase 9 (CDK9).4900-3869-3513.2y.. o. -7. The compound of any of claims 4-6, wherein the antibody linker is attached to the RIPTAC through a chemical handle positioned within the internal linker of the RIPTAC, or via a cleavable linker conjugated to one of the small-molecule binding moieties and being chemically discrete from both small-molecule binding moieties, such that the native binding affinity of each moiety for the respective protein is retained following linker cleavage and payload release.

8. The compound of any of claims 4-7, wherein the RIPTAC linker comprises a scaffold selected from the group consisting of a lysine-derived scaffold selected from Nα-L-lysine, Nε-L-lysine, Nα-D-lysine, ornithine, and diaminobutyric acid, a poly(ethylene glycol) (PEG) scaffold selected from two to twenty-four oxyethylene units, a piperazine scaffold, a piperidine scaffold, a cyclobutene scaffold, an alkyl chain, an alkyne, a morpholine scaffold, a triazole scaffold, an alkyl ether scaffold, a branched 3-arm PEG scaffold, and combinations thereof; and / orwherein the linker is joined to the RIPTAC small-molecule binding moieties via a linkage selected from the group consisting of an azide–alkyne cycloaddition product, an amide bond, and a carbamate bond.

9. The compound of any of claims 4-8, wherein the antibody linker further comprises a self-immolative valine–citrulline–para-aminobenzyl carbamate cleavage motif that is cleavable by cathepsin B after intracellular internalization, the para-aminobenzyl being connected to the RIPTAC through a carbamate linkage to a primary or secondary amine located within the internal linker of the RIPTAC, and wherein the valine–citrulline dipeptide is flanked by a PEG spacer selected from PEG2, PEG4, PEG6, PEG8, or PEG10 or a n-Alkane spacer selected from C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, or C15 to modulate solubility, prevent aggregation, or alter cleavage kinetics, and the para-aminobenzyl group is optionally substituted with methoxy or halo substituents to tune self-immolation rate while effecting scarless release of the RIPTAC such that both small-molecule binding moieties retain their native affinity.

10. The compound of any of claims 4-9, wherein the antibody is a human or humanized IgGl, IgG2 or IgG4 that binds a tumor-associated extracellular antigen selected from the group consisting of:STEAP1, PSMA, CD19, CD20, CD33, BCMA, FOLR1, NaPi-2b, MUC16, CLEC12A, HER2, GPNMB, B7-H3, Trop-2, EGFR and EpCAM.

11. The compound of any of claims 4-9, wherein the antibody is selected from trastuzumab, pertuzumab, vandortuzumab, rituximab, obinutuzumab, loncastuximab, belantamab, cetuximab, or a PSMA×STEAP1 bispecific antibody.4900-3869-3513.2y.. o. -12. The compound of any of claims 4-11, wherein the antibody comprises Fc-engineering for enhanced internalization or reduced effector function.

13. The compound of any of claims 4-12, wherein the first small-molecule binding moiety binds androgen receptor and the second small-molecule binding moiety binds BRD4 or PLK1.

14. An antibody conjugate comprising:a) an antibody that specifically binds a tumor-associated extracellular antigen;b) a transcriptional / epigenetic chemical inducer of proximity (TCIP) comprising:i) a first small-molecule binding moiety that selectively binds an intracellular transcription factor; andii) a second small-molecule binding moiety that selectively binds a transcriptional activator or transcriptional repressor; andiii) a TOP linker covalently joining the two small-molecule binding moieties; andc) a tri-functional linker covalently joining the antibody to the TCIP, the linker being attached to the TCIP through a chemical handle positioned within the internal linker of the TCIP or via a cleavable linker conjugated to one of the small-molecule binding moieties and being chemically discrete from both small-molecule binding moieties, such that the native binding affinity of each moiety for the respective protein is retained.

15. The compound of claim 14, wherein the intracellular transcription factor selected from B-cell lymphoma 6 (BCL6), Forkhead-box Protein Pl (FOXPl), Poly(ADP-ribose) Polymerase 1 (PARP1), and Poly(ADP-ribose) Polymerase 2 (PARP2).

16. The compound of claim 14 or 15, wherein the transcriptional activator or transcriptional repressor is selected from the group consisting of: Bromodomain-containing protein 4 (BRD4), cyclin-dependent kinase 9 (CDK9), El A-associated protein p300 (p300), and CREB-binding protein (CBP).

17. The compound of any of claims 14-16, wherein the TCIP linker comprises a scaffold selected from the group consisting of a lysine-derived scaffold selected from Nα-L-lysine, Nε-L-lysine, Nα-D-lysine, ornithine, and diaminobutyric acid, a poly(ethylene glycol) (PEG) scaffold selected from two to twenty-four oxyethylene units, a piperazine scaffold, a piperidine scaffold, a cyclobutene scaffold, an alkyl chain, an alkyne, a morpholine scaffold, a triazole scaffold, an alkyl ether scaffold, a branched 3-arm PEG scaffold, and combinations thereof; and / orwherein the linker is joined to the TCIP small-molecule binding moieties via a linkage selected from the group consisting of an azide–alkyne cycloaddition product, an amide bond, and a carbamate bond.4900-3869-3513.2y.. o. -18. The compound of any of claims 14-17, wherein the antibody linker further comprises a self-immolative valine–citrulline–para-aminobenzyl carbamate cleavage motif that is cleavable by cathepsin B after intracellular internalization, the para-aminobenzyl being connected to the TOP through a carbamate linkage to a primary or secondary amine located within the internal linker of the TCIP, and wherein the valine–citrulline dipeptide is flanked by a PEG spacer selected from PEG2, PEG4, PEG6, PEG8, or PEG10 or a n-Alkane spacer selected from C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, or C15 to modulate solubility, prevent aggregation, or alter cleavage kinetics, and the para-aminobenzyl group is optionally substituted with methoxy or halo substituents to tune self-immolation rate while effecting scarless release of the TCIP such that both small-molecule binding moieties retain their native affinity.

19. The compound of any of claims 14-18, wherein the antibody is a human or humanized IgGl, IgG2 or IgG4 that binds a tumor-associated extracellular antigen selected from the group consisting of: STEAP1, PSMA, CD19, CD20, CD33, BCMA, FOLR1, NaPi-2b, MUC16, CLEC12A, HER2, GPNMB, B7-H3, Trop-2, EGFR and EpCAM.

20. The compound of any of claims 14-19, wherein the antibody is selected from trastuzumab, pertuzumab, vandortuzumab, rituximab, obinutuzumab, loncastuximab, belantamab, cetuximab, CD19xBCMA bispecific antibody, CD19xCD20 bispecific antibody, or a BCMAxCD20 bispecific antibody.

21. The compound of any of claims 14-20, wherein the antibody comprises Fc-engineering for enhanced internalization or reduced effector function.

22. A compound having the structure represented by Structures la, lb or 1c:TCIi.(Structure la)4900-3869-3513.2y.. o. -(Structure lb)(Structure 1c)or a pharmaceutically acceptable salt thereof; wherein:A1and A2independently represent small molecule binder (“warhead”) pairings which bind to two distinct biological targets (i.e., protein, RNA, DNA);L1comprises a linker composed of any amino acid, non-proteinogenic amino acid, PEG, n-Alkane, piperidine, cyclobutene, piperazine, or modified combination thereof;L2comprises a linker composed of any cleavable or non-cleavable element which serves to join A1-L1-A2to a targeting compound (TC);wherein the TC comprises a targeting compound consisting of an antibody, single-domain antibody, fusion protein, ligand, or nanoparticle.

23. The compound of claim 22, wherein L1has the following structure:(Structure 2)or a pharmaceutically acceptable salt thereof; wherein:R1represents the side chain an amino acid or non-proteinogenic amino acid selected from: i) arginine, histidine, lysine, aspartate, glutamate, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan;4900-3869-3513.2y.. o. -ii) norvaline, norleucine, alloisoleucine, isoserine, t-leucine, pipecolate, 2,3-diaminopropionate, 2,4-diaminobutyrate, ornithine, allothreonine, homocysteine, homoserine, ci-amino-n-heptanoate, ci-aminobutyrate, P-aminobutyrate, y-aminobutyrate, ci-aminoisobutyrate;or any chemically modified derivates thereof.

24. The compound of claim 22, wherein L1has the following structure:(Structure 3) (Structure 4) or a pharmaceutically acceptable salt thereof; wherein:R2and R3represents the side chain of one or two of the amino acids or non-proteinogenic amino acids selected from:i) arginine, histidine, lysine, aspartate, glutamate, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan;ii) norvaline, norleucine, alloisoleucine, isoserine, t-leucine, pipecolate, 2,3-diaminopropionate, 2,4-diaminobutyrate, ornithine, allothreonine, homocysteine, homoserine, ci-amino-n-heptanoate, ci-aminobutyrate, -aminobutyrate, y-aminobutyrate, ci-aminoisobutyrate;or any chemically modified derivates of the aforementioned;n1and n2represent the number of monomeric subunits of said amino acids or non-proteinogenic amino acids where n1and n2independently equal a value between 1-10.

25. The compound of claim 22, wherein, L1has the following structure:(Structure 5) (Structure 6)4900-3869-3513.2y.. o. -(Structure 7)or a pharmaceutically acceptable salt thereof; wherein:R4, R5, and R6represents the side chain of 1-3 of the amino acids or non-proteinogenic amino acids selected from:i) arginine, histidine, lysine, aspartate, glutamate, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan;ii) norvaline, norleucine, alloisoleucine, isoserine, t-leucine, pipecolate, 2,3-diaminopropionate, 2,4-diaminobutyrate, ornithine, allothreonine, homocysteine, homoserine, a-amino-n-heptanoate, a-aminobutyrate, P-aminobutyrate, y-aminobutyrate, a-aminoisobutyrate;or any chemically modified derivates of the aforementioned;n3, n4, and n5represent the number of monomeric subunits of said amino acids or non-proteinogenic amino acids where n3, n4, and n5independently equal a value between 1-10.

26. The compound of claim 22, wherein L1has the following structure:(Structure 8) (Structure 9)(Structure 10)4900-3869-3513.2y.. o. -or a pharmaceutically acceptable salt thereof; wherein S1and S2represent spacers selected from the group consisting of: poly(ethylene glycol) (PEG), poly(glycol) chains, alkyl chains, alkynes, triazoles, piperazines, piperidines, and any combination thereof27. The compound of claim 22, wherein L1has the following structure:4900-3869-3513.2Atty. Dkt. No. 701586-000161WOPT(Structure 11)(Structure 12)or a pharmaceutically acceptable salt thereof; wherein S3, S4, and S5represent optional spacers selected from the group consisting of: poly(ethylene glycol) (PEG), poly(glycol) chains, alkyl chains, alkynes, triazoles, piperazines, piperidines, and any combination thereof.4900-3869-3513.2Atty. Dkt. No. 701586-000161WOPT28. The compound of claim 22, wherein L1has the following structure:(Structure 14)(Structure 15)or a pharmaceutically acceptable salt thereof; wherein S6, S7, S8, and S9represent optional spacers selected from the group consisting of: poly(ethylene glycol) (PEG), poly(glycol) chains, alkyl chains, alkynes, triazoles, piperazines, piperidines, and any combination thereof.

29. The compound of claim 22, wherein L1has the following structure:Atty. Dkt. No. 701586-000161WOPT(Structure 16)or a pharmaceutically acceptable salt thereof; wherein n6, n7, and n8represent the number of monomeric subunits where n6, n7, and n8independently equal a value between 1-20.

30. The compound of claim 22, wherein L2contains a structure selected from the group consisting of Structure 17, Structure 18, Structure 19, Structure 20, Structure 21, Structure 22, and Structure 23, or a combination thereof,or a pharmaceutically acceptable salt, solvate or prodrug thereof,wherein n9- n14represent the number of monomeric subunits where n9- n14independently equal a value between 1-20.

31. The compound of claim 22, wherein A1and A2represent a biologically active pairing of small molecules which functions as a regulated induced proximity targeting chimera (RIPTAC).

32. The compound of claim 22, wherein A1and A2represent a biologically active pairing of small molecules which functions as a proteolysis-targeting chimera (PROTAC).

33. The compound of claim 22, wherein A1and A2represent a biologically active pairing of small molecules which functions as an autophagy-targeting chimera (AUTAC).

34. The compound of claim 22, wherein A1and A2represent a biologically active pairing of small molecules which functions as a specific and non-genetic inhibitor of apoptosis protein (IAP)-dependent protein eraser (SNIPER).

35. The compound of claim 22, wherein A1and A2represent a biologically active pairing of small molecules which functions as a deubiquitinase-targeting chimera (DUBTAC).Atty. Dkt. No. 701586-000161WOPT36. The compound of claim 22, wherein A1and A2represent a biologically active pairing of small molecules which functions as a phosphorylation-inducing chimeras (PHICs).

37. The compound of claim 22, wherein A1and A2represent a biologically active pairing of small molecules which functions as a phosphatase-recruiting chimera (PHORCs).

38. The compound of claim 22, wherein A1and A2represent a biologically active pairing of small molecules which functions as a ribonuclease targeting chimera (RIBOTAC).

39. The compound of claim 22, wherein A1and A2represent a biologically active pairing of small molecules which functions as an acetylation tagging system (AceTAG).

40. The compound of claim 22, wherein the compound is a stereoisomer, enantiomer, or tautomer thereof or a mixture thereof, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

41. A composition comprising a compound of any of claims 1-40.

42. The composition of claim 41, further comprising at least a second compound of any of claims 1-40.

43. A pharmaceutical composition comprising a compound of any of claims 1-40 and a pharmaceutically acceptable carrier.

44. The pharmaceutical composition of claim 43, further comprising at least a second compound of any of claims 1-40.

45. A method of treating cancer, the method comprising administering to a patient in need thereof a therapeutically effective amount of a compound of any of claims 1-40, a composition of claim 41 or 42, or pharmaceutical composition of claim 43 or 44.

46. The method of claim 45, wherein the cancer is selected from the group consisting of: a carcinoma, a primary central nervous system tumor, a melanocytic tumor, a germ cell tumor, a sarcoma, and a hematological malignancy.Atty. Dkt. No. 701586-000161WOPT47. The method of claim 45, wherein the carcinoma is selected from the group consisting of: ovarian cancer, uterine cancer, endometrial cancer, cervical cancer, prostate cancer, breast cancer, lung cancer, oral cancer, esophageal cancer, head and neck cancer, stomach cancer, colon cancer, rectal cancer, basal cell carcinoma, squamous cell carcinoma, sebaceous gland carcinoma, bile duct cancer, gallbladder cancer, liver cancer, pancreatic cancer, bladder cancer, urinary tract cancer, renal cell carcinoma and thyroid cancer.

48. The method of claim 45, wherein the primary central nervous system tumor is a brain cancer.

49. The method of claim 45, wherein the melanocytic tumor is eye cancer or cutaneous melanoma.

50. The method of claim 45, wherein the germ cell tumor is testicular cancer or ovarian cancer.

51. The method of claim 45, wherein the sarcoma is a uterine sarcoma.

52. The method of claim 45, wherein the hematological malignancy is lymphoma or leukemia.

53. An antibody conjugate composition for treatment of prostate cancer, comprising:(a) an internalizing antibody moiety that selectively binds to STEAP1, PSMA, or a bispecific antibody moiety that binds both STEAP1 and PSMA; and(b) a RIPTAC moiety that selectively binds to an intracellular cancer-overexpressed protein, androgen receptor (AR), and additionally binds at least one pan-essential effector protein selected from the group consisting of BRD4, PLK1, BRD2, BRD3, CDK1, CDK2, CDK4, CDK5, CDK6, and CDK9.

54. The antibody conjugate composition of claim 52, wherein the antibody moiety is conjugated to the RIPTAC moiety via a cathepsin B cleavable valine-citrulline self-immolative linker, enabling intracellular release of the RIPTAC upon internalization.

55. The antibody conjugate composition of claim 52 or 53, wherein the antibody moiety is derived from or comprises vandortuzumab for STEAP1 targeting.

56. The antibody conjugate composition of claim 52, wherein the bispecific antibody moiety binds simultaneously or separately to STEAP1 and / or PSMA cell-surface antigens expressed on prostate cancer cells.Atty. Dkt. No. 701586-000161WOPT57. A method of treating metastatic castration-resistant prostate cancer in a subject comprising administering to the subject a therapeutically effective amount of the antibody conjugate composition of any one of claims 52-55.

58. The method of claim 56, wherein the treatment selectively accumulates in prostate cancer cells due to antibody targeting and kills prostate cancer cells by inducing stable ternary complexes between AR, RIPTAC, and the pan-essential effector protein, resulting in steric inhibition of effector protein function and selective tumor cell death.

59. The method of claim 56 or 57, wherein the subject has a cancer comprising a mutation found in human prostate cancer cell lines VCaP, LNCaP, or 22Rv1 with differential or engineered STEAP1 expression, including STEAP1 knockout (STEAPlko), STEAP1 low expression (STEAP11OW), and STEAP1 overexpressing (STEAPlhi / amp) variants.

60. The method of any one of claims 56-58, wherein the composition provides superior in vivo anti-tumor efficacy as compared to androgen receptor RIPTACs, androgen receptor antagonists, or androgen receptor degraders.

61. A pharmaceutical composition comprising the antibody conjugate composition of any one of claims 52-55 formulated for intravenous administration.

62. An antibody conjugate composition for the treatment of Diffuse Large B-Cell Lymphoma (DLBCL), comprising:(a) an internalizing antibody moiety that specifically binds to one or more cell surface antigens selected from CD 19, CD20, BCMA, or a bispecific antibody moiety that binds two antigens thereof; and(b) a transcriptional / epigenetic chemical inducer of proximity (TCIP), with a target moiety that selectively binds to BCL6 and an effector moiety that binds BRD4 or an alternative transcriptional activator selected from cyclin-dependent kinase 9 (CDK9), histone acetyltransferases (HATs), or p300 / CBP complexes.

63. The antibody conjugate of claim 56, wherein the antibody is conjugated to the TCIP via a cleavable maleimide-PEG4-Val-Cit-PAB linker, enabling intracellular release of the TCIP moiety upon cathepsin B cleavage following internalization.Atty. Dkt. No. 701586-000161WOPT64. The antibody conjugate of claim 56 or 57, wherein the antibody moiety is derived from or comprises an anti-CD19 monoclonal antibody (e.g., Loncastuximab), an anti-CD20 monoclonal antibody (e.g., Rituximab), or an anti-BCMA monoclonal antibody (e.g., Belantamab) suitable for internalization into DLBCL cells.

65. The antibody conjugate of any of claim 56-58, wherein the bispecific antibody moiety binds simultaneously or separately two of the three antigens CD 19, CD20, and BCMA to increase selective delivery and internalization into target cells.

66. The antibody conjugate of any of claim 56-59, wherein the TCIP moiety mediates ternary complex formation between intracellular BCL6 and BRD4 / CDK9 / HAT / p300 / CBP transcriptional regulators, thereby inducing transcriptional activation and expression of BCL6 downstream genes in DLBCL cells.

67. A method of selectively killing DLBCL cells in a subject comprising administering a therapeutically effective amount of the antibody conjugate of any of claim 1-5, wherein cell killing is mediated by activation of previously repressed transcriptional regulatory pathways critical for lymphoma survival and proliferation.

68. The method of claim 66, wherein the subject has a cancer bearing a mutation found in human DLBCL cell lines selected from SUDHL5 or KARPAS422.

69. The method of claim 66, wherein the antibody conjugate exhibits improved tumor selectivity and reduced systemic toxicity due to the dual mechanism of targeted antibody internalization and ranscriptional / epigenetic chemical inducer of proximity induction.

70. An antibody conjugate composition for treatment of Diffuse Large B-Cell Lymphoma (DLBCL), comprising:(a an internalizing antibody moiety that specifically binds to at least one cell surface antigen chosen from CD 19, CD20, BCMA, or a bispecific antibody moiety that binds two of said antigens; and(b a regulated induced proximity targeting chimera (RIPTAC) moiety designed to form a ternary complex etween the intracellular cancer-associated protein enhancer of zeste homolog 2 (EZH2) and a pan-essential effector protein selected from the group consisting of PLK1, BRD2, BRD3, BRD4, CDK1, CDK2, CDK4, CDK5, CDK6, and CDK9.71 The antibody conjugate of claim 69, wherein the antibody moiety is conjugated to the RIPTAC moiety via a cathepsin B cleavable linker selected from valine-citrulline-p-aminobenzyl carbamate (Val-Cit-PAB) or aAtty. Dkt. No. 701586-000161WOPTmaleimide-based self-immolative linker, facilitating intracellular release of the RIPTAC payload upon endocytosis and lysosomal processing.

72. The antibody conjugate of claim 69 or 70, wherein the antibody moiety is derived from an anti-CD19, anti-CD20, or anti-BCMA monoclonal antibody, each exhibiting internalization upon antigen binding on DLBCL cells.

73. The antibody conjugate of any of claims 69-71, wherein the bispecific antibody moiety simultaneously or separately binds two of the antigens CD 19, CD20, and BCMA to enhance selective delivery and cellular internalization in lymphoma cells.

74. A method of treating DLBCL in a subject, comprising administering a therapeutically effective amount of the antibody conjugate of any of claim 69-72, thereby inducing selective tumor cell death by the formation of a ternary complex between intracellular RIPTAC, EZH2, and a pan-essential effector protein, leading to functional inhibition of essential proteins in lymphoma cells.

75. The method of claim 73, wherein the subject has a cancer comprising a mutation found in human DLBCL cell lines selected from SUDHL5 or KARPAS422.

76. The method of claim 73, wherein administration of the antibody conjugate results in significant tumor growth inhibition, induction of apoptosis, and improved survival relative to control treatments.

77. The method of any of claims 73-75, wherein the antibody conjugate exhibits superior therapeutic efficacy and reduced systemic toxicity compared to unconjugated TCIP.

78. The target compound of claim 1, further comprising an AND logic gate, wherein cell killing activity requires the presence of a tumor-associated extracellular target and one or more tumor associated intracellular targets.

79. The target compound of claim 1, wherein cell killing activity is only active in the presence of one or more tumor-associated extracellular targets and one or more tumor associated intracellular targets.

80. The antibody conjugate compound of any preceding claim, further comprising an AND logic gate, wherein cell killing activity requires the presence of a tumor-associated extracellular target, one or more tumor associated intracellular targets, and one or more intracellular effector target.Atty. Dkt. No. 701586-000161WOPT81. The antibody conjugate compound of any preceding claim, further comprising an AND logic gate, wherein cell killing activity requires the presence of one or more tumor-associated extracellular targets, one or more tumor associated intracellular targets, and one or more intracellular effector target.

82. The antibody conjugate compound of any preceding claim, wherein cell killing activity is only active in the presence of one or more tumor-associated extracellular targets, one or more tumor associated intracellular targets, and one or more intracellular effector target.

83. The antibody conjugate compound of any preceding claim, wherein said payload moiety is chemically stable and membrane-permeable such that, upon intracellular processing and killing of a first tumor cell, the payload moiety:a) diffuses out of said first tumor cell and enters an adjacent second tumor cell;b) induces cell death in said second tumor cell contingent upon the presence of both said tumor-associated intracellular target(s) and said intracellular effector target(s) in said second tumor cell; andc) wherein said induction of cell death in the second tumor cell occurs independently of the presence of the tumor-associated extracellular target on said second tumor cell.

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