Antibody protac conjugates

Abstract

Branched antibody-PROTAC conjugates (APCs) are provided.SOLUTION: An immunoconjugate having the formula Ab - [L1 - (A-AB-B) m] n, wherein (a) Ab is L2 or a binding fragment thereof, (b) L1 and L2 are each independently a linker, L1 and L2 are the same or different, and L1 binds to L2, (c) A is a target-protein ligand / binder, (d) B is a ubiquitin ligase ligand / binder, and (e) n and m are independently an integer from 1 to 8. Target proteins include kinases, G-protein coupled receptors, transcription factors, phosphatases and RAS superfamily members.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to novel therapeutic agents based on ADC and PROTAC technologies. [Background technology]

[0002] Antibodies have long been essential tools in basic research and medical applications due to their high specificity and affinity for target antigens. A key feature of antibodies is their high specificity and ability to bind to target antigens, thereby marking them for elimination by complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC). Antibodies can also provide therapeutic effects by binding and inhibiting the function of target antigens. However, many unmodified antibodies against tumor-specific antigens often lack therapeutic activity. While some antibodies can instead be successfully applied as guided missiles to deliver potent cytotoxic drugs in the form of antibody-drug conjugates (ADCs), many ADCs have limited therapeutic potential and may require additional improvement.

[0003] Proteolysis targeting chimeras (PROTACs) are two-headed molecules that can eliminate unwanted proteins by inducing selective intracellular protein degradation. PROTACs consist of two protein-binding moieties: one that binds to an E3 ubiquitin ligase and the other that binds to a target protein. Binding of both proteins allows the PROTAC to deliver the target protein to the E3 ligase, resulting in tagging (i.e., ubiquitination) of the target protein for subsequent degradation by the proteasome.

[0004] Ubiquitination involves three major steps: activation, conjugation, and ligation, which are carried out by ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). This cascade results in the covalent attachment of ubiquitin to target proteins. Ubiquitinated proteins are ultimately degraded by the proteasome.

[0005] PROTAC technology was first described in 2001 (Sakamoto et al., "Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation," Proceedings of the National Academy of Sciences of the United States of America. 98(15):8554-9). Since then, this technology has been used to design several drugs, namely, pVHL, MDM2, β-TrCP1, cereblon, and c-IAP1. While these prior art PROTAC drugs are very useful, there is still a need for better PROTAC drugs. Summary of the Invention

[0006] Embodiments of the present invention relate to branched antibody-PROTAC conjugates (APCs). The branched antibody-PROTAC conjugates of the present invention combine the advantages of both the ADC and PROTAC approaches, and the branched form of APCs has several advantages in either development or processing compared to linear forms of APCs. In the branched antibody-PROTAC conjugates of the present invention, the payload (drug) in a conventional ADC is replaced by a PROTAC and attached to the linker portion of the PROTAC molecule. These new therapeutic agents are highly selective, have low toxicity, are safe to use, and have a relatively long in vivo half-life.

[0007] One aspect of the present invention relates to an immunoconjugate. The immunoconjugate according to one embodiment of the present invention has the formula (I): Ab-[L 2 -(AL 1 -B) m ] n wherein (a) Ab is an antibody or binding fragment thereof; and (b) L 1 and L 2 are each independently a linker, and L 1 and L 2 may be the same or different, (c) A is a target protein ligand / binder, (d) B is a ubiquitin ligase ligand / binder, and (e) n and m are each independently an integer from 1 to 8.

[0008] According to some embodiments of the present invention, the target protein may be a kinase, a G protein-coupled receptor, a transcription factor, a phosphatase, and a RAS superfamily member.

[0009] Other aspects of the present invention will become apparent from the following description and the included drawings. APC and Branched Structures [Brief explanation of the drawings]

[0010] [Figure 1] Schematic diagrams showing the structure of linear (unbranched) APC and the structure of branched APC are shown.

[0011] [Figure 2]This figure shows the promotion of BRD4 degradation by various concentrations of ARV-825 and compound 5 (but not compound 7) of the present invention in BT-474 breast cancer cell cultures as analyzed by SDS-PAGE electrophoresis and Western blotting. ARV-825 is a known small molecule BRD4-targeting PROTAC. Compound 5 of the present invention is a branched form of ARV-825. The results indicate that compound 5 has comparable degradation activity against BRD4 to ARV-825. In contrast, compound 7 of the present invention, which contains an additional lysosomal-cleavable dipeptide (valine-citrulline) in compound 5, is unable to degrade BRD4 protein even with treatment at up to 1 μM of compound 7. This result is likely due to the fact that compound 7, which has relatively low permeability due to the valine-citrulline dipeptide, cannot be efficiently internalized into cells. BRD4 and AKT indicate the positions of the BRD4 and AKT bands, and actin was used as a loading control.

[0012] [Figure 3] Example 1 demonstrates specific BRD4 proteolytic activity in HER2-positive BT-474 breast cancer cells, but not in HER2-negative MDA-MB-231 breast cancer cells. Example 1 is a branched trastuzumab-compound 7 immunoconjugate (i.e., a branched APC having trastuzumab as the antibody and compound 7 as the PROTAC). Furthermore, this branched APC does not degrade AKT or actin proteins.

[0013] [Figure 4] 1 shows a synthesis scheme of linear APC by binding via the A binder in ARV-825. DETAILED DESCRIPTION OF THE INVENTION

[0014] An embodiment of the present invention relates to a branched APC. The branched APC binds an antibody to a PROTAC via the linker moiety of the PROTAC. The branched APC of the present invention can be considered an analog of an antibody-drug conjugate (ADC), in which the payload (drug) in a conventional ADC is replaced by a PROTAC that is conjugated to the antibody via the linker in the PROTAC. That is, in the branched APC of the present invention, the antibody (or binding fragment thereof) is not bound via a target protein binding moiety (A) or a ubiquitin ligase binder (B), but rather via a linker (L 2 ) via the linker portion of the PROTAC (L 1 The covalent bond on the antibody can be on a protein moiety (e.g., a constant or variable region) or on a carbohydrate (sugar moiety).

[0015] Figure 1 shows a schematic diagram illustrating the differences between linear and branched APCs. The advantages of branched APCs over linear APCs may include: 1. The antibody is attached to the linker (L) within the PROTAC moiety, rather than to either end (A or B) of the PROTAC moiety. 1 ), maintaining the original structures of the target ligand (A) and E3 ligase ligand (B) within the PROTAC moiety so that the binding affinity of A and B is not altered. 2. Structural modifications to the linker for the attachment of groups are much easier than modifications to the ligand (A or B), and the two linker moieties (L 1 and L 2 ) are highly flexible. 3. Modifications to the linker are compatible with most APCs. Therefore, it is possible to design linkers to use common coupling functional groups so that different antibody moieties can be easily attached to the same PROTAC, or the same antibody can be easily attached to different PROTACs.

[0016] Branched APCs according to embodiments of the present invention may be represented by the following formula (I): [ka] During the ceremony, (a) Ab is an antibody or a binding fragment thereof; (b)L 1 and L 2 are independently linkers, and L 1 is the linker within the PROTAC moiety (i.e., PROTAC=AL 1 -B) and L 2 is the linker part of the PROTAC (L 1 ) is a linker that connects the antibody to the PROTAC, (c) A is a target ligand / binder (i.e., a binder for a target protein, which may be a kinase, a G protein-coupled receptor, a transcription factor, a phosphatase, a RAS superfamily member, etc.); (d) B is a ubiquitin ligase ligand / binder, and the ubiquitin ligase can be an E2 ubiquitin ligase or an E3 ubiquitin ligase; (e) n and m are independently integers of 1 to 8.

[0017] The branched APCs of the present invention combine the advantages of both ADCs and PROTACs and represent a new class of therapeutics. 2 The linker is the L 1 Antibody-L refers to the structure shown in formula (I) above attached to a linker. 2 Other types of APCs, in which a linker is attached to either end (A or B) of the PROTAC, are called "unbranched" or "linear." These new branched APCs have higher selectivity, longer in vivo half-lives, a larger therapeutic window, broader applicability, and are safer to use. Furthermore, these branched APCs are easier to synthesize than unbranched (linear) APCs.

[0018] Antibody-drug conjugates (ADCs) are a class of therapeutic agents in which a drug (or payload) is attached to an antibody or its antigen-binding fragment. The antibody in the ADC binds to a selected target (typically a cellular target), thereby bringing the drug close to the target and providing a highly selective therapeutic effect. An example of an ADC would be an antibody that targets a protein expressed on cancer cells, and the payload would be a cytotoxic drug (e.g., taxol).

[0019] ADCs are large molecules due to the presence of antibodies, typically with a molecular weight of approximately 150 kDa or greater. Therefore, ADCs are not eliminated by renal filtration. Furthermore, antibody constant regions contain sites for interaction with receptors in the kidney, allowing antibodies to be transported back into the circulation. Therefore, antibodies typically have a long in vivo half-life of several weeks. Furthermore, antibodies can be easily internalized by cells, making the delivery of payloads to cells highly efficient. ADCs are promising therapeutic agents because they are specific and long-acting. However, ADC action is payload-dependent and does not function like a catalyst. Therefore, a sufficient amount of payload is required to kill or inhibit the target protein or target cell. Excessive payload can cause toxicity.

[0020] PROTACs consist of two protein-binding moieties, one that binds to an E3 ubiquitin ligase and the other that binds to a target protein. The PROTAC can bind to its target protein and deliver it to the E3 ubiquitin ligase. Once the tertiary complex is formed, the E3 ubiquitin ligase transfers ubiquitin to the surface lysines of the target protein, resulting in a ubiquitinated target protein that is targeted for degradation by the proteasome machinery. After ubiquitination, the PROTAC is released and continues to search for target proteins for ubiquitination and degradation. Thus, PROTACs function like catalysts, and large results can be achieved with small amounts of PROTAC.

[0021] In the above formula (I), the A component is a group that binds to a target protein targeted for degradation. The A component can include any moiety that specifically binds to a target protein. The following are non-limiting examples of small molecule target protein binding moieties: Hsp90 inhibitors, kinase inhibitors, MDM2 inhibitors, compounds targeting human BET bromodomain-containing proteins, HDAC inhibitors, human lysine methyltransferase inhibitors, angiogenesis inhibitors, immunosuppressive compounds, and compounds targeting the aryl hydrocarbon receptor (AHR), among others. The compositions described below exemplify some members of these types of small molecule target protein binding moieties. Such small molecule target protein binding moieties also include pharmaceutically acceptable salts, enantiomers, solvates, and polymorphs of these compositions, as well as other small molecules that can target proteins of interest.

[0022] In general, target proteins may include, for example, structural proteins, receptors, enzymes, cell surface proteins, proteins involved in cellular function, and the like. Therefore, the A component of the ADC-PROTAC can be used to target any of the following proteins, including all variants, mutations, splice variants, indels, and fusions of these target proteins: FoxO1, HDAC, DP-1, E2F, ABL, AMPK, BRK, BRSK I, BRSK2, BTK, CAMKK1, CAMKKα, CAMKKβ, Rb, Suv39HI, SCF, p19INK4D, GSK-3, pi8 INK4, myc, cyclin E, CDK2, CDK9, CDG4 / 6, cyclin D, pl6 INK4A, cdc25A, BMI1, SCF, Akt, CHK1 / 2, C1δ, CK1γ, C2, CLK2, CSK, DDR2, DYRK1A / 2 / 3, EF2K, EPH-A2 / A4 / B1 / B2 / B3 / B4, and EIF2A. 3, Smad2, Smad3, Smad4, Smad7, p53, p21 Cipl, PAX, Fyn, CAS, C3G, SOS, Tal, Raptor, RACK-1, CRK, Rapl, Rac, KRas, NRas, HRas, GRB2, FAK, PI3K, spred, Spry, mTOR, MPK, LKB1, PAK 1 / 2 / 4 / 5 / 6, PDGFRA, PYK2, Src, SRPK1, PLC, PKC, PKA, PKBα / β, PKCα / γ / ζ, PKD, PLK1, PRAK, PRK2, WAVE-2, TSC2, DAPKI, B AD, IMP, C-TAK1, TAK1, TAO1, TBK1, TESK1, TGFBR1, TIE2, TLK1, TrkA, TSSK1, TTBK1 / 2, TTK, Tpl2 / cotl, MEK1, MEK2, PLDL Erk1, Erk2, Erk5, Erk8, p90RSK, PEA-15, SRF, p27 KIP1, TIF 1a, HMGN1, ER81, MKP-3, c-Fos, FGF-R1, GCK, GSK3β, HER4, HIPK1 / 2 / 3 / , IGF-1R, CDC25, UBF, LAMTOR2, Statl, StaO, CREB, JAK, Src, PTEN, NF-kappa B, HECTH9, Bax, HSP70, HSP90, Apaf-1, Cytoc, BCL-2, Bcl-xL, Smac, XIAP, caspase-9, caspase-3, caspase-6, caspase-7, CDC37, TAB, IKK, TRADD, TRAF2, R1P1, FLIP, TAKl, JNK1 / 2 / 3, Lck, A-Raf, B-Raf, C-Raf, MOS, MLK1 / 3, MN 1 / 2, MSK1, MST2 / 3 / 4, MPSK1, MEKKI, ME K4, MEL, ASK1, MINK1, MKK 1 / 2 / 3 / 4 / 6 / 7, NE 2a / 6 / 7, NUAK1, OSR1, SAP, STK33, Syk, Lyn, PDK1, PHK, PIM 1 / 2 / 3, ataxin-1, mTORC1, MDM2, p21, Waf1, cyclin D1, Lamln A, Tpl2, Myc, catenin, Wnt, IKKβ, IKKγ, IKKα, IKKε, ELK, p65RelA, IRAKI, IRA 2, IRAK4, IRR, FADD, TRAF6, TRAF3, MKK3, MKK6, ROCK2, RSK1 / 2, SGK 1, SmMLCK, SIK2 / 3, ULK1 / 2, VEGFR1, WNK 1, YES1, ZAP70, MAP4K3, MAP4K5, MAPKlb, MAPKAP-K2 K3, p38α / β / δ / γMAPK, Aurora A, Aurora B, Aurora C, MCAK, Clip, MAPKAPK, FAK, MARK The peptide or small molecule can be any peptide or small molecule that binds to a protein target such as 1 / 2 / 3 / 4, Muc1, SHC, CXCR4, Gap-1, Myc, β-catenin / TCF, Cb1, BRM, Mcl-1, BRD2, BRD3, BRD4, AR, RAS, ErbB3, EGFR, IRE1, HPK1, RIPK2, and ERct.

[0023] The B component is a group that binds to an E3 ubiquitin ligase. E3 ubiquitin ligases (of which there are over 600 known in humans) confer substrate specificity to ubiquitination. There are known ligands that bind to these ligases. As described herein, the E3 ubiquitin ligase binding group can be a peptide or small molecule that can bind to an E3 ubiquitin ligase. Examples of E3 ubiquitin ligases include von Hippel-Lindau (VHL); cereblon, XIAP, E3A; MDM2; anaphase-promoting complex; UBR5 (EDDI); and SOC. S / BC-box / eloBC / CUL5 / RING;LNXp80;CBX4;CBLL1;HACE1;HECTD1;HECTD2;HECTD3;HECW1;HECW2;HERC1;HERC2;HERC3;HERC4;HUWE1;ITCH;NEDD4;NEDD4L;PPIL2;PRPF19;PIAS1;PIAS2;PIAS3;PIAS4;R ANBP2;RNF4;RBX1;SMURF1;SMURF2;STUB1;TOPORS;TRIP12;UBE3A;UBE3B;UBE3C;UBE4A;UBE4B;UBOX S;UBR5;WWP1;WWP2;Parkin;A20 / TNFAIP3;AMFR / gp78;ARA54;β-TrCP1 / BTRC;BRCA1;CBL;CHIP / STUB 1;E6;E6AP / UBE3A;F-box protein 15 / FBXO15;FBXW7 / Cdc4;GRAIL / RNF128;HOIP / RNF31;cIAP-1 / HIAP-2;cIAP-2 / HIAP-1;cIAP(pan);ITCH / AIP4;KAP1;MARCH8,,Mind Bomb 1 / MIB1;Mind Bomb 2 / MIB2;MuRF1 / TRIM63;NDFIP1;NEDD4;NleL;Parkin;RNF2;RNF4;RNF8;RNF168;RNF43;SART1;Skp2;SM and ZNRF3.

[0024] An exemplary E3 ubiquitin ligase is the von Hippel-Lindau (VHL) tumor suppressor, which is the substrate-recognition subunit of the E3 ligase complex VCB-Cul2. The VCB-Cul2 complex consists of VHL, elongins B and C, Cul2, and Rbx1. A major substrate of VHL is hypoxia-inducible factor let (HIF-let), a transcription factor that upregulates genes such as the proangiogenic growth factor VEGF and the erythropoiesis-inducing cytokine erythropoietin in response to low oxygen levels. Compounds that bind to VHL can be hydroxyproline compounds, such as those disclosed in WO 2013 / 106643, as well as other compounds described in U.S. Patent Nos. 2016 / 0045607, 2014187777, 20140356322, and 9,249,153.

[0025] Another exemplary E3 ubiquitin ligase is cereblon. Cereblon is a protein that forms an E3 ubiquitin ligase complex with damaged DNA-binding protein 1 (DDB1), cullin 4A (CUL4A), and cullin regulatory factor 1 (ROC1). This complex ubiquitinates numerous other proteins. Cereblon's ubiquitination of target proteins increases the levels of fibroblast growth factor 8 (FGF8) and fibroblast growth factor 10 (FGF1O). FGF8 then regulates numerous developmental processes, such as the formation of limbs and auditory vesicles. In the absence of cereblon, DDB1 forms a complex with DDB2, which functions as a DNA damage-binding protein. Thalidomide, lenalidomide, pomalidomide, and their analogs are known to bind to cereblon. Other small molecule compounds that bind to cereblon are also known, such as those disclosed in U.S. Patent Nos. 2016 / 0058872 and 2015 / 0291562. Furthermore, phthalimide conjugation with binders, such as BET bromodomain antagonists, can provide PROTACs with highly selective cereblon-dependent BET proteolysis. Winter et al., Science, June 19, 2015, p. 1376. Such PROTACs can be conjugated to antibodies as described herein to form APCs.

[0026] The specificity of a PROTAC depends on its targeting ligand binding to the target protein for degradation. If specificity is not high, this can lead to off-target effects (side effects). Furthermore, PROTACs are generally small molecules (MW approximately 1000). They rely on diffusion to enter cells, which is not very efficient (especially for molecular weights around 1000). Furthermore, because they are small molecules, they typically have a short in vivo half-life due to renal filtration. Therefore, PROTACs may need to be administered at a relatively high dosing frequency.

[0027] The antibody-PROTAC conjugates (APCs) of the present invention are similar in size to antibodies or ADCs, which have long in vivo half-lives. Therefore, the APCs of the present invention also have long in vivo half-lives (e.g., several weeks) and can enter cells by internalization due to the presence of antibodies. Furthermore, the APCs have dual selectivity, one derived from the antibody and the other derived from the target protein binder within the PROTAC. For example, the antibody within the APC of the present invention can bind to a specific antigen on a cancer cell, and then the APC enters the cell via internalization. Once inside the cell, the target protein binder within the PROTAC portion finds the target protein and delivers it to an E3 ubiquitin ligase for ubiquitination. The ubiquitinated target protein is marked for degradation by the proteasome. Therefore, the APCs of the present invention are highly selective and have few adverse effects.

[0028] Furthermore, the APCs of the present invention have the advantage of a catalytic mechanism of action, similar to PROTACs. Therefore, the therapeutically effective dose of APC can be lowered, and because APCs have a long in vivo half-life, they can be administered less frequently. These properties enhance the specificity of the APCs of the present invention and make them safer to use.

[0029] Table 1 below summarizes and compares some properties of ADCs, PROTACs, and APCs. [Table 1]

[0030] Thus, the APC format represents a novel and promising approach to new therapeutics, which can generally be applied to any target protein associated with any disorder (see Crews et al., "Proteolysis-Targeting Chimeras: Induced Protein Degradation as a Therapeutic Strategy," ACS Chem. Biol. 2017, 12(4), 892-898).

[0031] Embodiments of the present invention can be applied to any target protein that causes a disease or disorder by obtaining an antibody and then using the antibody to bind to a PROTAC (i.e., a target protein binder linked to an E3 ubiquitin ligase ligand or inhibitor). The antibody is directed to an antigen expressed on cells containing the target protein. The target protein binder within the PROTAC depends on which protein is being targeted. For example, in the case of a target enzyme (e.g., a kinase), an inhibitor can be designed as the ligand / binder.

[0032] In the case of E3 ubiquitin ligase ligands / binders, several molecules are known to bind to various E3 ubiquitin ligases. Examples include: [ka]

[0033] Nutlin derivatives bind to MDM2 (double minute 2 homolog; also known as E3 ubiquitin-protein ligase Mdm2), a negative regulator of the p53 tumor suppressor. Mdm2 functions as an E3 ubiquitin ligase that recognizes the N-terminal transactivation domain (TAD) of the p53 tumor suppressor and as an inhibitor of p53 transcriptional activation. Bestatin (ubenimex) engages cIAP1 (cellular inhibitor of apoptosis protein-1). The IMiD thalidomide and its derivatives pomalidomide and lenalidomide bind to cereblon.

[0034] In the following description, specific examples are used to illustrate embodiments of the present invention. In the examples, the bromodomain and extraterminal domain (BET) family of proteins is used as the target protein, and trastuzumab is used as the antibody. However, any specific BET inhibitor, ubiquitin ligase 3 inhibitor, and trastuzumab used in these examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention. Those skilled in the art will understand that other modifications and variations are possible without departing from the scope of the present invention.

[0035] The bromodomain and extra-terminal domain (BET) family of proteins, including BRD2, BRD3, and BRD4, plays an important role in many cellular processes, including inflammatory gene expression, mitosis, and virus / host interactions, by controlling histone acetylation-dependent chromatin complex assembly. BET protein inhibitors reversibly bind to bromodomains or BET proteins, namely, BRD2, BRD3, BRD4, and BRDT. They can prevent protein-protein interactions between BET proteins and acetylated histones and transcription factors. Therefore, BET inhibitors have anticancer, immunosuppressive, and other effects. The use of BET inhibitors in APCs targets BET family proteins for ubiquitination, thereby eliminating them via the proteasome.

[0036] Details of certain embodiments of the present invention are set forth below, but these details are given by way of example only, and those skilled in the art will recognize that other modifications and variations are possible without departing from the scope of the present invention. Example 1 Preparation of Trastuzumab-BRD4-PROTAC-1 Synthesis of compound 2 [ka]

[0037] In this example, the BET inhibitor is OTX015, which is an orally bioavailable small molecule inhibitor of BRD2, BRD3, and BRD4 (EC 50 OTX015 downregulates c-Myc expression and induces cell cycle arrest and apoptosis. Therefore, OTX015 has antiproliferative effects against various solid tumors and leukemias.

[0038] Compound 2: To a mixture of OTX015 (1) (0.2 mmol) and 1-bromo-2-(2-bromoethoxy)ethane (1 mmol) in dimethylformamide (5 mL) was added potassium carbonate (0.6 mmol). The mixture was stirred at 50 °C for 24 hours. After the reaction was complete, the reaction mixture was extracted with dichloromethane and water. The organic layer was then washed with brine and dried over MgSO 4 . The organic solvent was removed under reduced pressure. The residue was purified by column chromatography using methanol:dichloromethane (1:19) to give a yellow solid compound 2 (58% yield). 1 H NMR (600 MHz, chloroform-d): δ 7.45(d,J=9.1Hz,2H),7.38(d,J=8.6Hz,2H),7.28(d,J=8.6Hz,2H),6.78(d, J=9.1Hz,2H),4.71(dd,J=8.0,6.2Hz,1H),4.06(dd,J=5.4,4.5Hz,2H),3.86- 3.82(m,4H),3.78(dd,J=14.5,8.0Hz,1H),3.57(dd,J=14.5,6.2Hz,1H),3.4 7(t,J=6.2Hz,2H),2.66(s,3H),2.38(d,J=0.6Hz,3H),1.65(d,J=0.6Hz,3H). LCMS(ESI):m / z[C 33 H 37 ClN6O4S+H] + Calculated value 642.08, Measured value 642.52 [M+H] + . Synthesis of compound 4 [ka]

[0039] Compound 4: To a solution of pomalidomide (3) (0.2 mmol) and tert-butyl (2-(2-aminoethoxy)ethyl)carbamate (0.22 mmol) in DMF (5 mL) was added N,N-diisopropylethylamine (0.4 mmol). The reaction mixture was stirred at 90° C. for 12 hours. After the reaction was complete, the reaction mixture was extracted with dichloromethane and water. The organic layer was then washed with brine and dried over MgSO. The organic solvent was removed under reduced pressure. After purification on a flash column using ethyl acetate:hexane (2:3), the yellow residue was dissolved in dichloromethane and trifluoroacetic acid (1 mL) was added. The mixture was stirred at room temperature for 0.5 hours. The reaction mixture was then extracted with dichloromethane and water. The organic layer was then washed with brine and dried over MgSO. The organic solvent was removed under reduced pressure. The residue was purified by column chromatography using methanol:dichloromethane (1:9) to give compound 4 as a yellow solid. 1 H NMR(600MHz,chloroform-d)δ 7.35(t,J=7.8Hz,1H),6.93(d,J=7.0Hz,1H),6.78(d,J=8.6Hz,1H),6.38(d,J=5.1Hz,1H),4.81(dd,J=11.6,5.1Hz,1H),3. 62(d,J=4.0Hz,2H),3.56(d,J=4.5Hz,2H),3.33(d,J=4.1Hz,2H),3.05-3.04(m,2H),2.71-2.53(m,3H),2.01-1.93(m,1H). LCMS(ESI):m / z[C 33 H 37 ClN6O4S+H] + Calculated value 361.14, Found value 361.22 [M+H] + . Synthesis of compound 5 [ka]

[0040] Compound 5: To a solution of compound 2 (0.2 mmol), compound 4 (0.6 mmol), and potassium iodide (0.2 mmol) in acetonitrile / dimethylformamide (3:1, 4 mL) was added potassium carbonate (0.6 mmol). The reaction mixture was stirred at 50° C. for 72 hours. After the reaction was complete, the reaction mixture was extracted with dichloromethane and water. The organic layer was then washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure. The residue was purified by column chromatography using methanol:dichloromethane (1:12) to give compound 5 as a yellow solid (19.7% yield). 1 H NMR(600MHz,chloroform-d)δ 7.53-7.36(m,5H),7.36-7.29(m,2H),7.09(dd,J=7.0,3.7Hz,1H),6.88(d,J=8.7Hz,1H) ,6.82(dd,J=8.7,1.4Hz,2H),4.89(dt,J=12.5,6.1Hz,1H),4.66(ddd,J=7.9,6.0,3.5Hz, 1H),4.10-4.02(m,2H),3.84-3.58(m,9H),3.55-3.41(m,2H),3.41-3.30(m,2H),3.02-2 .91(m,3H),2.87-2.68(m,3H),2.67(s,3H),2.40(s,3H),2.10-2.07(m,1H),1.67(s,3H). LCMS(ESI):m / z[C 33 H 37 ClN6O4S+H] + Calculated value 922.30, Found value 922.49 [M+H] + Synthesis of compound 7 [ka]

[0041] Compound 7: To a solution of compound 5 (0.2 mmol) and Mal-C5-VC-PAB-PNP compound 6 (0.2 mmol) in dimethylformamide (5 mL), hydroxybenzotriazole (0.4 mmol) and pyridine (0.4 mmol) were added. The reaction mixture was stirred at room temperature for 72 hours. After the reaction was complete, the reaction mixture was extracted with dichloromethane and water. The organic layer was then washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure. The residue was purified by column chromatography using methanol:dichloromethane (1:16) to give compound 7 as a yellow solid (46.3% yield). 1 H NMR(600MHz,chloroform-d)δ 7.55-7.40(m,7H),7.33(d,J=7.2Hz,2H),7.25-7.19(m,2H),7.08(d,J=7.0Hz,1H),6.92-6.83(m,1H),6.79-6.68(m,2H), 6.68-6.65(m,2H),5.07-5.00(m,2H),4.77-4.71(m,1H),4.71-4.60(m,1H),4.37-4.29(m,1H),4.04-3.96(m,1H),3.93-3 .85(m,1H),3.77-3.34(m,21H),3.28-3.14(m,1H),3.11-3.02(m,1H),2.93-2.71(m,3H),2.69(s,3H),2.42(s,3H),2.04- 2.01(m,1H),1.91-1.81(m,2H),1.77-1.70(m,1H),1.69(s,3H),1.62-1.54(m,4H),1.33-1.25(m,5H),0.91-0.87(m,6H). LCMS(ESI):m / z[C 33 H 37 ClN6O4S+H] + Calculated value + 1520.58, measured value 1521.14 [M+H] + . Conjugation of trastuzumab-BRD4-PROTAC 1 [ka]

[0042] A solution of 1 mg (5.0 mg / mL) of trastuzumab in a buffer solution (25 mM sodium borate pH 8, 0.025 M NaCl, 1 mM diethylenetriaminepentaacetic acid (DTPA)) was treated with tris(2-carboxyethyl)phosphine (TCEP, 4.0 molar equivalents) at 37 °C for 2 hours. Excess TCEP in the buffer solution (25 mM sodium borate pH 8, 0.025 M NaCl, 1 mM DTPA) was removed using an Amicon Ultra-15 centrifugal filter device with a NMWL of 30 kDa, followed by treatment with compound 7 (20 molar equivalents) at 25 °C for 4 hours. The reaction mixture was cut off and concentrated using an Amicon Ultra-15 centrifugal filter device with a NMWL of 30 kDa in pH 7.4 PBS buffer to obtain trastuzumab-BRD4-PROTAC 1. Example 2 Preparation of Trastuzumab-BRD4-PROTAC-2 Synthesis of compound 8 [ka]

[0043] Compound 8: To a solution of succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (0.75 mmol) in acetonitrile (7 mL) was added 1,2-ethanedithiol (0.82 mmol). The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction mixture was extracted with dichloromethane and water. The organic layer was then washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure. The residue was purified by column chromatography using ethyl acetate:hexane (3:2) to give compound 8 (34.8% yield) as a white solid. Synthesis of compound 9 [ka]

[0044] Compound 9: To a solution of compound 7 (0.02 mmol) in acetonitrile / dimethylformamide (1:1, 6 mL) was added compound 8 (0.04 mmol). The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the reaction mixture was extracted with dichloromethane and water. The organic layer was then washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure. The residue was purified by column chromatography using ethyl acetate:hexane (3:2) to give compound 9 (86.2% yield) as a yellow solid. Conjugation of trastuzumab-BRD4-PROTAC 2 [ka]

[0045] To a solution of 1 mg (5.0 mg / mL) of trastuzumab in a buffer solution (50 mM potassium phosphate, 50 mM sodium chloride, 2 mM EDTA; pH 6.5), 30 equivalents of 9 (5 mM in DMSO) were slowly added. The reaction mixture was stirred at 37 °C for 18 hours. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifugal filter device with a NMWL of 30 kDa in pH 7.4 PBS buffer to obtain trastuzumab-BRD4-PROTAC 2.

[0046] As described above, the APC of the present invention comprises antibody-L 2 The linker is the L 1 As shown in the example above, L 1 L for linker 2 The attachment of the linker does not alter the A or B binder of the PROTAC, and the attachment reaction is relatively easy. 2 Attempts to synthesize linear (unbranched) APCs in which the linker is attached to either the A binder or the B binder are shown. Example 3: Synthesis of linear BRD4-PROTAC (17)

[0047] Figure 4 illustrates a synthetic scheme for the possible synthesis of ARV-825 via A-binder conjugation. Functional group modification of protein ligands or ligase binders is not straightforward. Furthermore, not all protein ligands or ligase binders have functional groups suitable for modification. In this example, the chlorine atom on OTX015, the protein ligand of the PROTAC ARV-825, is difficult to convert to another functional group, such as an amino group. OTX015 or ARV-825 does not exhibit reactivity in the Buchwald reaction (a palladium-catalyzed coupling reaction) or the Ullmann reaction (a copper-catalyzed coupling reaction). Harsh reaction conditions, such as metal halide exchange, lead to compound decomposition. According to the literature (EP 1887008A1), the different functional groups of the BRD4 inhibitor must be introduced first. In other words, directly coupling a linker to the protein ligand or ligase binder further complicates the synthesis.

[0048] In contrast, the branched linker strategy disclosed in this invention provides a new method for connecting any protein ligand or ligase binder to form an APC, with the following advantages: The APC maintains the structure of the target protein ligand and E3 ligase ligand, so binding affinity is not altered. Structural modification for attaching groups to the linker is much easier than that to the ligand. Modifications to the linker are suitable for most PROTACs, and "common" coupling functional groups can be designed for different PROTACs so that the same antibody can be coupled to different PROTACs, or the same PROTAC can be coupled to different antibodies. Example 4 Biological activity

[0049] Various immunoconjugates of formula (I) were tested for specificity and ability to degrade target proteins. A brief description of the various assays is provided below.

[0050] Western blot The cellular efficacy of compounds of formula (I) against BRD4 proteolysis was evaluated. Bromodomain protein 4 (BRD4), a member of the BET (bromodomain and extraterminal) family of proteins, is involved in the tumorigenesis of hematological malignancies and solid tumors. BRD4 recognizes and binds to acetylated histones, playing an important role in the transmission of epigenetic memory through cell division and transcriptional regulation. Potent inhibitors targeting BRD4 have shown antitumor activity and suppress the proliferation and transformation of various cancer cells. This has made BRD4 a promising therapeutic target for cancer treatment. BRD4 proteolysis-inducing chimeric molecules (PROTACs) have been shown to have anticancer activity by inducing BRD4 proteolysis. However, in addition to their anticancer effects, normal cells are also affected by these drugs. This leads to undesirable adverse effects of BET inhibition, such as autism-like syndrome and impaired memory formation.

[0051] In the present invention, we have created a branched BRD4-PROTAC antibody conjugate to maintain the BRD4-PROTAC modality, increase the specificity of cancer cell targeting, and reduce potential off-target effects. "BRD4-PROTAC" refers to a PROTAC containing a target binder for the BRD4 protein. The "BRD4-PROTAC" is conjugated to an antibody that recognizes an antigen on cancer cells. The high specificity of the antibody allows the resulting conjugate (i.e., Ab-BRD4-PROTAC) to target specific cancer cells and reduce toxicity to healthy cells. For example, the antibody can be trastuzumab, and the cancer cells can be HER2-positive BT-474 breast cancer cells. Various compounds of Formula (I) (i.e., with different BRD4 binders, different E3 binders, and / or different antibodies) were tested for cellular BRD4 protein degradation via Western blotting assay. The following uses a trastuzumab-conjugated BRD4-PROTAC to illustrate the advantages of embodiments of the present invention.

[0052] For Western blot experiments, HER2-positive BT-474 breast cancer cells and HER2-negative MDA-MB-231 breast cancer cells were cultured overnight in DMEM and L15 media containing 10% FBS, respectively. On the day of the assay, 200,000 cells were pretreated with each test compound for 24 hours. After 24 hours, whole cell lysates were harvested by adding 2x SDS sample buffer. Proteins were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. Protein expression was detected using immunoblots with various primary and secondary antibodies according to standard protocols. Antibodies against BRD4, as well as anti-rabbit IgG and HRP-conjugated secondary antibodies, were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against actin were purchased from Millipore (Burlington, MA). Immunoblots were revealed by chemiluminescence (SuperSignal™ West Femto Maximum Sensitivity Substrate, Thermo Fisher, Waltham, MA) and detected by a ChemiDoc™ MP Imaging System (Bio-Rad, Hercules, CA). The band intensities of Western blots were also quantified by the ChemiDoc™ MP Imaging System. The relative intensities of bands corresponding to the drug-treated groups were compared with those of the untreated group.

[0053] Figure 2 shows the results of the assay. ARV-825 (CAS No. 1818885-28-7) is a heterobifunctional molecule containing a BRD4-binding moiety linked to the cereblon-binding moiety of an E3 ligase. ARV-825 is a proteolysis-targeting chimeric molecule (PROTAC). (See Lu, J. et al., "Hijacking the E3 ubiquitin ligase cereblon to efficiently target BRD4," Chem. Biol. 22(6), 755-763 (2015)). Compound 5 of the present invention is a branched form of ARV-825. Compound 7 of the present invention is compound 5 with an additional, lysosomal-cleavable dipeptide (valine-citrulline) linker.

[0054] As shown in Figure 2, compound 5 is as effective as ARV-825 in promoting BRD4 degradation in this cell culture assay. In contrast, compound 7 was unable to degrade BRD4 protein at up to 1 μM compound 7 treatment, which was likely due to the valine-citrulline dipeptide that results in low permeability. This result suggests that if the APC is cleaved early before entering the cell, the released PROTAC (e.g., compound 7) will not cause off-target effects. Therefore, the APC of the present invention will have a relatively high safety margin.

[0055] Figure 3 shows that the compound 7 trastuzumab antibody conjugate (i.e., Example 1) exhibits specific BRD4 proteolytic activity in HER2-positive BT-474 breast cancer cells, but not in HER2-negative MDA-MB-231 breast cancer cells. This APC does not degrade AKT or actin proteins. This indicates that the branched coupling of the antibody to the linker within the PROTAC did not impair the activity of the PROTAC. Importantly, in an in vivo setting, the antibody (trastuzumab) directs APC to these cells expressing specific antigens (e.g., HER2), thereby reducing off-target effects. Thus, Example 1 is as effective as, but safer than, AV-825 in clinical use.

[0056] These results demonstrate that the antibody conjugate (branched APC) of the present invention can enhance cancer cell targeting specificity, enhance cellular uptake of PROTAC modalities, and reduce potential off-target effects. Furthermore, when the antibody is released early, the released PROTAC (e.g., compound 7) has relatively low permeability due to the valine-citrulline dipeptide, preventing unwanted off-target effects and providing a high safety margin.

[0057] Antiproliferative activity As described above, the APCs of the present invention can be used to treat diseases or disorders with specific antigens. The diseases can be cancer, autoimmune diseases, infectious diseases, or vascular proliferative disorders. The cancers can be lung cancer, colon cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, bladder cancer, gastric cancer, kidney cancer, salivary gland cancer, ovarian cancer, endometrial cancer, cervical cancer, oral cancer, skin cancer, brain cancer, lymphoma, or leukemia. The inhibition of cell proliferation by the APCs of the present invention was measured using the CellTiter™-96 assay. The cytotoxicity of the APCs was evaluated against breast cancer cell lines with various HER2 expression phenotypes. The results showed that the APCs of the present invention are toxic only to HER2-positive breast cancer cells, and BRD4 protein, Src kinase, or RAS protein can be specifically targeted for proteasomal degradation by using an appropriate PROTAC conjugated to trastuzumab.

[0058] The APCs of the present invention are promising new therapeutic agents because they possess the advantages of ADCs and PROTACs. Furthermore, the branched APCs of the present invention exhibit advantages over linear ADCs (PROTACs) and are useful for treating disorders with specific antigens.

[0059] Some embodiments of the present invention relate to a method for treating a disease or disorder using the APC of the present invention. The disease can be cancer. Specific examples of cancer can include breast cancer, gastric cancer, squamous cell carcinoma, colon cancer, and leukemia that expresses specific antigens. The antibody used in the APC can be trastuzumab, cetuximab, rituximab, brentuximab, gemtuzumab, inotuzumab, sacituzumab, alemtuzumab, or nimotuzumab. A specific example of the APC can be a branched trastuzumab-linked PROTAC for targeting breast cancer or gastric cancer with HER2 expression.

[0060] Branched antibody-linked PROTACs can be synthesized in different formats, such as with different linkers or different antibody conjugation methods. Compounds 18 and 19 are examples showing different linker forms for lysine conjugation. The synthesis of PROTACs in compounds 18 and 19 follows the same process as compound 9 using a PEG linker. Compounds 18 and 19 can be synthesized using the same process as in Example 2 above. Branched PROTACs with PEG linkers have excellent solubility and can be conjugated to antibodies. [ka]

[0061]

[0030] Embodiments of the present invention have been described using a limited number of examples. Those skilled in the art will appreciate that other modifications and variations are possible without departing from the scope of the present invention. Accordingly, the scope of protection should be limited only by the appended claims.

Claims

1. An immunoconjugate having the structure of formula (I): 【Chemistry 1】 During the ceremony, (a) the Ab is trastuzumab, cetuximab, rituximab, brentuximab, gemtuzumab, inotuzumab, sacituzumab, alemtuzumab, or nimotuzumab; (b) L １ and L ２ are each independently a linker, and L １ and L ２ are the same or different, and L １ Is L ２ binds to (c) A is a target protein ligand / binder having the structure: 【Chemistry 2】 Here, (L1) is L １ represents the binding site for (d) B is a ubiquitin ligase ligand / binder having the structure: 【Transformation 3】 Here, (L1) is L １ represents the binding site for (e) n and m are each independently an integer from 1 to 8; Immunoconjugates.

2. 10. A method for the preparation of a pharmaceutical composition comprising administering to a subject the immunoconjugate of claim 1 and one or more pharmaceutically acceptable excipients. Pharmaceutical compositions.

3. A pharmaceutical composition for treating a disease, comprising:

10. A pharmaceutical composition comprising an effective amount of the immunoconjugate of claim 1 or the pharmaceutical composition of claim 2. Pharmaceutical compositions.

4. the disease is cancer; The pharmaceutical composition according to claim 3.

5. the cancer is breast cancer or gastric cancer, and the Ab is trastuzumab; The pharmaceutical composition according to claim 4.

6. The pharmaceutical composition of claim 4, wherein the cancer is colon cancer or squamous cell carcinoma and the Ab is cetuximab.

7. The disease is leukemia and the Ab is rituximab, brentuximab, gemtuzumab, inotuzumab, or alemtuzumab; The pharmaceutical composition according to claim 4.

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