Antibody protac conjugates

Abstract

An immunoconjugate having the formula Ab-[L1-(A-L2-B) m] n, wherein: (a) Ab is an antibody or a binding fragment thereof; (b) L1 and L2 are each independently a linker wherein L1 and L2 are the same or different, and wherein L1 is linked to L2; (c) A is a target protein ligand / conjugate; (d) B is a ubiquitin ligase ligand / conjugate, and (e) n and m are independently integers from 1 to 8. The target proteins include a kinase, a G protein coupled receptor, a transcription factor, a phosphatase, and a RAS superfamily member.

Description

[0001] This application is a divisional application of the patent application with application number 201980018480.5 and application date January 9, 2019. Technical Field

[0002] The present invention relates to novel therapeutic agents based on ADC and PROTAC technology. Background Art

[0003] Antibodies have long been an indispensable tool in basic research and medical use because they have high specificity and affinity for target antigens. A key feature of antibodies is their high specificity and their ability to bind target antigens, which are labeled to remove complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). Antibodies can also confer therapeutic benefits by binding to target antigens and inhibiting the function of target antigens. However, many unmodified antibodies for tumor-specific antigens generally lack therapeutic activity. Although some antibodies can be successfully used as guided missiles to deliver effective cytotoxic drugs in the form of antibody-drug conjugates (ADCs), many ADCs have limited therapeutic potential and may need further improvement.

[0004] Proteolysis targeting chimera (PROTAC) is a two-headed molecule that can remove unwanted proteins by inducing selective intracellular proteolysis. PROTAC consists of two protein binding parts, one for binding to E3 ubiquitin ligase and the other for binding to target protein. By binding to two proteins, PROTAC brings the target protein to the E3 ligase, resulting in the labeling (i.e. ubiquitination) of the target protein, which is then degraded by the proteasome.

[0005] Ubiquitination involves three major steps: activation, conjugation, and ligation, which are performed by ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3), respectively. The result of this continuous cascade is the covalent attachment of ubiquitin to the target protein. Ubiquitinated proteins are ultimately degraded by the proteasome.

[0006] 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, the technology has been used for several drug designs: pVHL, MDM2, β-TrCP1, cereblon, and c-IAP1. Although these prior art PROTAC drugs are very useful, there is still a need for better PROTAC drugs. Summary of the invention

[0007] Embodiments of the present invention are about branched antibody-PROTAC conjugates (APCs). The branched antibody-PROTAC conjugates of the present invention combine the advantages of both ADC and PROTAC approaches, and compared with linear APCs, branched APCs have several benefits in development or processing. In the branched antibody-PROTAC conjugates of the present invention, the load (drug) in the conventional ADC is displaced by PROTAC and connected to the linker portion of the PROTAC molecule. These new therapeutic agents have high selectivity, low toxicity, are safer to use, and have longer half-lives in vivo.

[0008] One aspect of the present invention relates to immunoconjugates. An 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 a binding fragment thereof; (b) L 1 and L 2 Each is independently a linker, wherein L 1 and L 2 can 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.

[0009] 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.

[0010] Other aspects of the invention will become apparent from the following description and the included drawings. Structure of APC and Branched Chains BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Shown are schematics illustrating the structure of a linear (unbranched) APC and the structure of a branched APC.

[0012] Figure 2 As analyzed by SDS-PAGE electrophoresis and western blotting, various concentrations of ARV-825 and compound 5 of the present invention (but not compound 7 of the present invention) promote the degradation of BRD4 in BT-474 breast cancer cell cultures. ARV-825 is a known small molecule PROTAC targeting BRD4. Compound 5 of the present invention is a branched form of ARV-825. The results show that compound 5 has a degradation activity comparable to that of ARV-825 to BRD4. In contrast, compound 7 of the present invention, which is a compound 5 with another dipeptide (valine-citrulline) that can be removed by lysosomes, cannot degrade BRD4 protein when treated with compound 7 up to 1 μM. This result may be due to the fact that compound 7 with relatively low permeability due to valine-citrulline dipeptide cannot be effectively internalized into cells. BRD4 and AKT mark the positions of BRD4 and AKT bands, and actin is used as a loading control.

[0013] Figure 3 Example 1 shows that specific BRD4 protein degradation activity is expressed 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 with trastuzumab as an antibody and compound 7 as a PROTAC). In addition, this branched APC does not cause degradation of AKT protein or actin.

[0014] Figure 4 Shown is the synthetic scheme of linear APC linked via the A conjugate in ARV-825. DETAILED DESCRIPTION

[0015] Embodiments of the present invention relate to branched APCs. Branched APCs couple antibodies to PROTACs via the linker portion of PROTACs. The branched APCs of the present invention can be viewed as analogs of antibody-drug conjugates (ADCs), wherein the payload (drug) in conventional ADCs is replaced by PROTACs, and PROTACs are coupled to antibodies via the linkers in PROTACs. That is, in the branched APCs of the present invention, antibodies (or binding fragments thereof) are coupled to PROTACs via the linkers (L 2 ) and PROTAC via the linker portion of PROTAC (L1 ) is covalently linked to the antibody rather than being linked via a target protein binding moiety (A) or a ubiquitin ligase conjugate (B). The covalent link on the antibody can be on the protein portion (e.g., constant region or variable region) or on the carbohydrate (sugar portion).

[0016] Figure 1 A schematic diagram illustrating the differences between linear and branched APCs is shown. Advantages of branched APCs over linear APCs may include the following:

[0017] 1. Maintain the original structure of the target ligand (A) and the E3 ligase ligand (B) in the PROTAC part so that the binding affinity of A and B will not change because the antibody is connected to the linker (L) in the PROTAC part. 1 ), rather than being attached to either end of the PROTAC moiety (A or B).

[0018] 2. The structural modification of the linker group on the linker is much easier than the modification on the ligand (A or B), and the two linkers (L 1 and L 2 ) are more flexible.

[0019] 3. Modification of linkers is applicable to most APCs. Therefore, linkers can be designed to use common coupling functional groups so that different antibody moieties can be easily coupled to the same PROTAC, or the same antibody can be easily coupled to different PROTACs.

[0020] The branched APC according to an embodiment of the present invention can be represented by the following formula (I):

[0021]

[0022] in:

[0023] (a) Ab is an antibody or a binding fragment thereof;

[0024] (b)L 1 and L 2 are independently linkers, wherein L 1 is a linker within the PROTAC moiety (i.e., PROTAC = AL 1 -B), and L 2 is through the linker portion of PROTAC (L 1 ) A linker connecting the antibody to the PROTAC;

[0025] (c) A is a target ligand / binder (i.e., a binder of a target protein, which may be a kinase, a G protein-coupled receptor, a transcription factor, a phosphatase, a RAS superfamily member, etc.);

[0026] (d) B is a ubiquitin ligase ligand / binder, wherein the ubiquitin ligase can be an E2 or E3 ubiquitin ligase, and

[0027] (e) n and m are independently integers of 1 to 8.

[0028] The branched APC of the present invention has the advantages of both ADC and PROTAC and represents a new class of therapeutic agents. The term "branched" refers to the structure shown in formula (I) above, wherein the antibody-L 2 Linkers and L in PROTAC 1 Linker coupling. 2 Other types of APCs with linkers attached to either end (A or B) of PROTAC will be referred to as "unbranched" or "linear". These new branched APCs have high selectivity, long in vivo half-life, large therapeutic window, wide applicability, and are safer to use. In addition, these branched APCs are easier to synthesize than unbranched (linear) APCs.

[0029] Antibody-drug conjugates (ADCs) are a class of therapeutic agents in which a drug (or payload) is linked to an antibody or its antigen-binding fragment. The antibody in the ADC binds to a selected target (usually a target on a cell), thereby bringing the drug to the vicinity of the target, thereby producing a highly selective therapeutic effect. An example of an ADC can be an antibody targeting a protein expressed on a cancer cell, and the payload can be a cytotoxic agent (e.g., paclitaxel).

[0030] Due to the presence of antibodies, ADC is a macromolecule, with a molecular weight of generally about 150KDa or higher. Therefore, kidney filtration does not eliminate ADC. In addition, the antibody constant region includes sites that interact with receptors in the kidney, which can transport and recycle the antibody back into the circulation. Therefore, the half-life of the antibody in vivo is long, generally several weeks. In addition, the antibody can be easily internalized by cells, so that the load is very effectively delivered to the cell. Since ADC has specificity and long-term effect, it is a promising therapeutic agent. However, the effect of ADC depends on the load, which does not work like a catalyst. Therefore, a sufficient amount of load is needed to kill or inhibit the target protein or cell. Overloading may cause toxicity.

[0031] PROTAC consists of two protein binding parts, one for binding to E3 ubiquitin ligase and the other for binding to target protein. PROTAC can bind to its target protein and bring it to E3 ubiquitin ligase. After the formation of the tertiary complex, E3 ubiquitin ligase transfers ubiquitin to the surface lysine of the target protein, resulting in ubiquitinated target protein, which is destined to be degraded by the proteasome machinery. After ubiquitination, PROTAC is released and continues to search for target proteins for ubiquitination and degradation. Therefore, PROTAC acts like a catalyst, and a small amount of PROTAC can achieve substantial results.

[0032] In the above formula (I), component A is a group that binds to the target protein to be degraded. Component A may include any part that specifically binds to the target protein. The following are non-limiting examples of small molecule target protein binding parts: Hsp90 inhibitors, kinase inhibitors, MDM2 inhibitors, compounds targeting proteins containing human BET bromodomains, HDAC inhibitors, human lysine methyltransferase inhibitors, angiogenesis inhibitors, immunosuppressive compounds, and compounds targeting aryl hydrocarbon receptors (AHR), etc. The compositions described below illustrate some members of these types of small molecule target protein binding parts. Such small molecule target protein binding parts also include pharmaceutically acceptable salts, enantiomers, solvates, and polymorphs of these compositions, as well as other small molecules that can target proteins of interest.

[0033] Typically, target proteins may include, for example, structural proteins, receptors, enzymes, cell surface proteins, proteins associated with cell functions, and the like. Therefore, the A component of ADC-PROTAC can be any peptide or small molecule that binds to a protein target, such as FoxOl, 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, cycloprotein D, p16 INK4A, cdc25A, BMI1, SCF, Akt, CHK1 / 2, C 1δ, CK1γ, C 2, CLK2, CSK, DDR2, DYRK1A / 2 / 3, EF2K, EPH-A2 / A4 / B 1 / B2 / B3 / B4, 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, LKBl, PAK 1 / 2 / 4 / 5 / 6, PDGFRA, PYK2, Src, SRPK1, PLC, PKC, PKA, PKBα / β, PKCα / γ / ζ, PKD, PLKl, PRAK, PRK2, WAVE-2, TSC2, DAPKI, B AD, IMP, C-TAK1, TAKl, TAOl, 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 la, 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-κB, 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, TAK1, 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, Taxol-1, mTORCl, MDM2, p21 Wafl, Cyclin Dl, Lamln A, Tpl2, Myc, Catenin, Wnt, IKK-β, IKK-γ, IKK-α, IKK-ε, ELK, p65RelA, IRAKI, IRA2, IRAK4, IRR, FADD, TRAF6, TRAF3, MKK3, MKK6, ROCK2, RSK1 / 2, SGK 1, SmMLCK, SIK2 / 3, ULK1 / 2, VEGFR1, WNK l, YES1, ZAP70, MAP4K3, MAP4K5, MAPKlb, MAPKAP-K2 K3, p38α / β / δ / γMAPK, Aurora A, Aurora B, Aurora C, MCAK, Clip, MAPKAPK, FAK, MARK 1 / 2 / 3 / 4, Mucl, SHC, CXCR4, Gap-1, Myc, β-catenin / TCF, Cbl, BRM, Mcl-1, BRD2, BRD3, BRD4, AR, RAS, ErbB3, EGFR, IRE1, HPK1, RIPK2, and ERct, including all variants, mutations, splice variants, indels, and fusions of these target proteins listed.

[0034] The B component is a group that binds an E3 ubiquitin ligase. E3 ubiquitin ligases (of which over 600 are known in humans) confer specificity to the receptor for ubiquitination. There are known ligands that bind to these ligases. As described herein, an E3 ubiquitin ligase binding group can be a peptide or small molecule that can bind an E3 ubiquitin ligase. Examples of E3 ubiquitin ligases include: vonHippel-Lindau (VHL); cerebellin, XIAP, E3A; MDM2; anaphase-promoting complex; UBR5 (EDDI); SOCS / BC-box / eloBC / CUL5 / RING; LNXp80; CBX4; CBLL1; HACE1; HECTD1; HECTD2; HECTD3; HECW1; HECW2; HERC1; HERC2; HERC3; HERC4; HUWE1; ITCH; NEDD4; NEDD4 L; PPIL2; PRPF19; PIAS1; PIAS2; PIAS3; PIAS4; RANBP2; RNF4; RBX1; SMURF1; SMURF2; STUB1; TOPORS; TRIP12; UBE3A; UBE3B; UB E3C; UBE4A; UBE4B; UBOXS; UBR5; WWP1; WWP2; Parkin; A20 / TNFAIP3; AMFR / gp78; ARA54; β-TrCPl / 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 Bomb1 / MIB1; Mind Bomb 2 / MIB2; MuRF1 / TRIM63; NDFIP1; NEDD4; NleL; Parkin; RNF2; RNF4; RNF8; RNF168; RNF43; SART1; Skp 2; SMURF2; TRAF-1; TRAF-2; TRAF-3; TRAF-4; TRAF-5; TRAF-6; TRIMS; TRIM21; TRIM32; UBR5; and ZNRF3.

[0035] An exemplary E3 ubiquitin ligase is the von Hippel-Lindau (VHL) tumor suppressor, which is a substrate recognition subunit of the E3 ligase complex VCB-Cul2. The VCCB-Cul2 complex consists of VHL, elongin B and C, Cul2, and Rbx1. The main substrate of VHL is hypoxia-inducible factor let (HIF-let), which is a transcription factor that upregulates genes such as angiogenic growth factor VEGF and erythropoietin, a cytokine that induces red blood cells, in response to low oxygen levels. Compounds that bind to VHL can be hydroxyproline compounds, such as those disclosed in WO2013 / 106643, and other compounds described in US2016 / 0045607, WO2014187777, US20140356322, and US 9,249,153.

[0036] 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 1 regulator (ROC 1). This complex ubiquitinates many other proteins. Ubiquitination of cereblon by target proteins leads to increased levels of fibroblast growth factor 8 (FGF8) and fibroblast growth factor 10 (FGF10). FGF8 in turn regulates many developmental processes, such as limb and otic vesicle formation. In the absence of cereblon, DDB1 forms a complex with DDB2, which acts as a DNA damage binding protein. It is known that thalidomide, lenalidomide, pomalidomide and its analogs bind to cereblon. Other small molecule compounds that bind cerebellin are also known, such as compounds disclosed in US2016 / 0058872 and US2015 / 0291562. In addition, coupling of phthalimide to a binder (e.g., an antagonist of a BET bromodomain) can provide PROTACs with highly selective cerebellin-dependent BET protein degradation. Winter et al., Science, June 19, 2015, p. 1376. Such PROTACs can be coupled to antibodies described herein to form APCs.

[0037] The specificity of PROTAC depends on the target ligand that it binds to the target protein for degradation. If the specificity is not high, this may lead to off-target effects (side effects). In addition, PROTAC is usually a small molecule (MW about 1000). It depends on diffusion into cells and has low efficiency (especially when the molecular weight is about 1000). In addition, because it is a small molecule, the half-life in vivo is usually short due to kidney filtration. Therefore, it may be necessary to give PROTAC with a higher dosing frequency.

[0038] The antibody-PROTAC conjugate (APC) of the present invention is similar in size to an antibody or ADC, and has a long half-life in vivo. Therefore, the APC of the present invention also has a long half-life in vivo (e.g., several weeks), and can enter cells by internalization due to the presence of antibodies. In addition, APC has dual selectivity: one from the antibody and the other from the target protein conjugate in PROTAC. For example, the antibody in the APC of the present invention can bind to a specific antigen on a cancer cell, and then the APC enters the cell by internalization. Once in the cell, the target protein conjugate in the PROTAC portion will find the target protein and bring it to the E3 ubiquitin ligase for ubiquitination. The ubiquitinated target protein is marked for degradation by the proteasome. Therefore, the APC of the present invention has high selectivity and few side effects.

[0039] In addition, the APC of the present invention has the advantage of a catalytic mode of action, similar to PROTAC. Therefore, the therapeutically effective dose of APC can be lower, and due to its longer half-life in vivo, it can be administered less frequently. These properties make the APC of the present invention more specific and safer to use.

[0040] Table 1 below summarizes and compares some characteristics of ADC, PROTAC and APC.

[0041] Table 1

[0042]

[0043] Therefore, the APC format is novel and represents a promising new therapeutic approach. This approach 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).

[0044] 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 couple with a PROTAC (such as a target protein conjugate coupled to an E3 ubiquitin ligase ligand or inhibitor). The antibody is directed against an antigen expressed on a cell containing the target protein. The target protein conjugate in a PROTAC will depend on the protein being targeted. For example, for a target enzyme (e.g., a kinase), an inhibitor can be designed as a ligand / conjugate.

[0045] With respect to E3 ubiquitin ligase ligands / binders, several molecules are known to bind various E3 ubiquitin ligases.

[0046] Examples include:

[0047]

[0048] Nutlin derivatives bind MDM2 (double micro 2 homolog; also known as the E3 ubiquitin-protein ligase Mdm2), which is a negative regulator of the p53 tumor suppressor. Mdm2 has 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) binds cIAP1 (inhibitor of apoptosis protein-1). The IMiD thalidomide and its derivatives pomalidomide and lenalidomide bind cerebellum protein.

[0049] The following description uses specific examples to illustrate embodiments of the present invention. These examples use the bromodomain and extra terminal domain (BET) protein family as target proteins and use trastuzumab as an antibody. However, any specific BET inhibitor, ubiquitin ligase 3 inhibitor and trastuzumab used in these examples are only for illustration and should not be construed as limiting the scope of the present invention. It will be appreciated by those skilled in the art that other modifications and variations are possible without departing from the scope of the present invention.

[0050] The bromodomain and extra-terminal domain (BET) protein family, including BRD2, BRD3 and BRD4, plays a key role in many cellular processes, including inflammatory gene expression, mitosis and virus / host interactions, by controlling the assembly of histone acetylation-dependent chromatin complexes. Inhibitors of BET proteins reversibly bind to bromodomain or BET proteins: BRD2, BRD3, BRD4, and BRDT. They can prevent protein-protein interactions between BET proteins and acetylated histones and transcription factors. Therefore, BET inhibitors have anti-cancer, immunosuppressive and other effects. Using BET inhibitors in APCs targets BET family proteins for ubiquitination, thereby causing the proteasome to eliminate BET family proteins.

[0051] The following describes the details of some embodiments of the present invention. However, these details are for illustration only, and those skilled in the art will appreciate that other modifications and variations are possible without departing from the scope of the present invention.

[0052] Example 1: Preparation of Trastuzumab-BRD4-PROTAC-1

[0053] Synthesis of compound 2

[0054]

[0055] In this example, the BET inhibitor is OTX015, which is an orally bioavailable small molecule inhibitor of BRD2, BRD3, and BRD4 (EC 50 =10 to 19 nM). OTX015 downregulates c-Myc expression and induces cell cycle arrest and apoptosis. Therefore, it has antiproliferative effects on a variety of solid tumors and leukemias.

[0056] 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 completion of the reaction, the reaction mixture was extracted with dichloromethane and water. Then, the organic layer was washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure. The residue was purified by column chromatography with methanol: dichloromethane (1:19) to give yellow solid compound 2 (58% yield). 1H NMR (600MHz, chloroform-d): δ7.45(d,J=9.1Hz,2H),7.38(d,J=8.6Hz,2H),7.28(d,J=8.6H z,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) ,3.47(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, Found value 642.52 [M+H] + .

[0057] Synthesis of compound 4

[0058]

[0059] 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 completed, the reaction mixture was extracted with dichloromethane and water. Then, the organic layer was washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure. After purification with ethyl acetate: hexane (2: 3) on a flash tower, 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. Thereafter, the reaction mixture was extracted with dichloromethane and water. Then, the organic layer was washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure. The residue was purified by column chromatography with methanol: dichloromethane (1: 9) to obtain yellow solid compound 4. 1H NMR (600MHz, chloroform-d) δ7.35(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=11.6,5.1 Hz,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, experimental value 361.22 [M+H] + .

[0060] Synthesis of compound 5

[0061]

[0062] Compound 5: Potassium carbonate (0.6 mmol) was added 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). The reaction mixture was stirred at 50 °C for 72 hours. After the reaction was completed, the reaction mixture was extracted with dichloromethane and water. Then, the organic layer was washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure. The residue was purified by column chromatography with methanol: dichloromethane (1:12) to give a yellow solid compound 5 (19.7% yield). 1 H NMR (600 MHz, chloroform-d) δ7.53-7.36 (m, 5H), 7.36-7.29 (m, 2H), 7.09 (dd, J = 7.0, 3.7 Hz, 1H), 6.88 (d, J = 8.7 Hz, 1H), 6.82 (dd, J = 8.7, 1.4 Hz, 2H), 4.89 (dt, J = 12.5, 6.1 Hz, 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 37ClN6O4S+H] + Calculated value 922.30, Found value 922.49 [M+H] + .

[0063] Synthesis of compound 7

[0064]

[0065] Compound 7: Hydroxybenzotriazole (0.4 mmol) and pyridine (0.4 mmol) were added to a solution of compound 5 (0.2 mmol) and Mal-C5-VC-PAB-PNP compound 6 (0.2 mmol) in dimethylformamide (5 mL). The reaction mixture was stirred at room temperature for 72 hours. After the reaction was completed, the reaction mixture was extracted with dichloromethane and water. Then, the organic layer was washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure. The residue was purified by column chromatography with methanol: dichloromethane (1:16) to obtain yellow solid compound 7 (46.3% yield). 1 H NMR (600MHz, chloroform-d) δ7.55-7.40(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),6.7 9-6.68(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,1 H),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, experimental value 1521.14 [M+H] + .

[0066] Conjugation of trastuzumab-BRD4-PROTAC 1

[0067]

[0068] A solution of 1 mg (5.0 mg / mL) of trastuzumab in buffer (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 was removed using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL in buffer (25 mM sodium borate pH 8, 0.025 M NaCl, 1 mM DTPA), followed by treatment with compound 7 (20 molar equivalents) at 25°C for 4 hours. The reaction mixture was truncated and concentrated using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL in pH 7.4 PBS buffer to obtain trastuzumab-BRD4-PROTAC 1.

[0069] Example 2: Preparation of Trastuzumab-BRD4-PROTAC-2

[0070] Synthesis of compound 8

[0071]

[0072] Compound 8: 1,2-ethanedithiol (0.82 mmol) was added to a solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide (SMCC) (0.75 mmol) in acetonitrile (7 mL). The reaction mixture was stirred at room temperature for 3 hours. After the reaction was completed, the reaction mixture was extracted with dichloromethane and water. Then, the organic layer was washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure. The residue was purified by column chromatography with ethyl acetate: hexane (3:2) to obtain white solid compound 8 (34.8% yield).

[0073] Synthesis of compound 9

[0074]

[0075] Compound 9: Compound 8 (0.04 mmol) was added to a solution of compound 7 (0.02 mmol) in acetonitrile / dimethylformamide (1:1, 6 mL). The reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed, the reaction mixture was extracted with dichloromethane and water. Then, the organic layer was washed with brine and dried over MgSO4. The organic solvent was removed under reduced pressure. The residue was purified by column chromatography with ethyl acetate: hexane (3:2) to obtain yellow solid compound 9 (86.2% yield).

[0076] Conjugation of trastuzumab-BRD4-PROTAC 2

[0077]

[0078] To a solution of trastuzumab 1 mg (5.0 mg / mL) in buffer (50 mM potassium phosphate, 50 mM sodium chloride, 2 mM EDTA; pH 6.5) was slowly added 30 equivalents of 9 (5 mM in DMSO). 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 30 kDa NMWL in pH 7.4 PBS buffer to obtain trastuzumab-BRD4-PROTAC 2.

[0079] As described above, the APC of the present invention has a branched form, wherein the antibody-L 2 Linkers and L in PROTAC 1 As shown in the above embodiment, L 2 Linker and L 1 The attachment of the linker does not change the A or B binder of the PROTAC, and the ligation reaction is relatively easy. As a comparison, the following example illustrates an attempt to synthesize a linear (unbranched) APC, in which L 2 The linker is coupled to the A binder or the B binder.

[0080] Example 3: Synthesis of a linear form of BRD4-PROTAC (17)

[0081] Figure 4 The possible synthesis scheme of the A conjugate connection synthesis via ARV-825 is described. It is not easy to modify the functional groups of protein ligands or ligase conjugates. In addition, not all protein ligands or ligase conjugates have suitable functional groups for modification. In this embodiment, the chlorine atom on OTX015 (protein ligand of PROTAC ARV-825) is difficult to be converted into another functional group, such as amino group. OTX015 or ARV-825 show no reactivity under Buchwald reaction (palladium-catalyzed coupling reaction) and Ullman reaction (copper-catalyzed coupling reaction). Severe reaction conditions, such as metal halide exchange, will cause compound decomposition. According to the literature (EP1887008A1), different BRD4 inhibitor functional groups should be introduced at the beginning. In other words, directly coupling the conjugate to the protein ligand or ligase conjugate will make the synthesis more complicated.

[0082] In contrast, the branched linker strategy disclosed in the present invention provides a new method to connect any protein ligand or ligase conjugate to form an APC with the following advantages. The APC maintains the structure of the target protein ligand and the E3 ligase ligand, and therefore the binding affinity does not change. The structural modification of the linker group on the linker is much easier than on the ligand. Modifications on the linker are applicable to 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.

[0083] Example 4: Biological Activity

[0084] Various immunoconjugates of formula (I) were tested for their specificity and ability to degrade target proteins. A brief description of the different assays is described below.

[0085] Western blot

[0086] The cellular efficacy of the compound of formula (I) in BRD4 protein degradation was evaluated. Bromodomain protein 4 (BRD4) is one of the BET (bromodomain and extra) family proteins and is involved in the tumorigenesis of hematological malignancies and solid tumors. BRD4 recognizes and binds to acetylated histones and plays a key role in epigenetic memory transmission across cell division and transcriptional regulation. Effective inhibitors targeting BRD4 show antitumor activity, inhibiting the proliferation and transformation of various cancer cells. This causes BRD4 to become a promising therapeutic target for cancer treatment. It has been shown that BRD4 proteolysis targeting chimeras (PROTACs) have anticancer activity by inducing BRD4 protein degradation. However, in addition to the anticancer effect, normal cells are also affected by these agents. This leads to the worrying side effects of BET inhibition, such as autism-like syndrome and damage to memory formation.

[0087] In the present invention, a BRD4-PROTAC antibody conjugate with a branched form is produced to maintain the integrity of the BRD4-PROTAC modality, enhance the specificity of cancer cell targeting, and reduce potential off-target effects. "BRD4-PROTAC" refers to a PROTAC of a target binder including a BRD4 protein. By coupling "BRD4-PROTAC" with an antibody that recognizes an antigen on a cancer cell. The high specificity of the antibody allows the resulting conjugate (such as Ab-BRD4-PROTAC) to target specific cancer cells and produce less toxicity to healthy cells. For example, the antibody can be trastuzumab, and the cancer cell can be a HER2-positive BT-474 breast cancer cell. Various compounds of formula (I) (e.g., with different BRD4 binders, different E3 binders, and / or different antibodies) are tested by western blotting in cell BRD4 protein degradation. The benefits of embodiments of the present invention are illustrated below using trastuzumab-coupled BRD4-PROTAC.

[0088] For western blotting experiments, HER2-positive BT-474 and HER2-negative MDA-MB-231 breast cancer cells were cultured in DMEM and L15 medium with 10% FBS, respectively, and cultured overnight. On the day of analysis, 200,000 cells were pretreated with each test compound for 24 hours. After 24 hours, whole cell lysates were harvested by adding 2× SDS sample buffer. Proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. Protein expression was detected by immunoblotting using various primary and secondary antibodies according to standard protocols. Anti-BRD4 antibodies and anti-rabbit IgG, HRP-linked secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-actin antibodies were purchased from Millipore (Burlington, MA). Immunoblotting was performed by chemiluminescence (SuperSignal TM West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific, Waltham, MA) and is available through ChemiDoc TM MP imaging system (Bio-Rad, Hercules, CA). Band intensity of Western blots was also measured by ChemiDoc TM MP Imaging System Quantification The relative intensity of the bands corresponding to the drug-treated groups was compared to that of the untreated groups.

[0089] Figure 2The analysis results are shown. ARV-825 (CAS No. 1818885-28-7) is a heterobifunctional molecule comprising a BRD4 binding portion connected to an E3 ligase cereblon binding portion. ARV-825 is a proteolytic targeting chimera (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 a compound 5 with an additional dipeptide (valine-citrulline) linker that can be removed by lysosomes.

[0090] like Figure 2 As shown, in this cell culture analysis, compound 5 is as effective as ARV-825 in promoting the degradation of BRD4. In contrast, compound 7 cannot degrade BRD4 protein when treated with up to 1 μM of compound 7, which may be caused by the lower permeability caused by the valine-citrulline dipeptide. This result shows that if APC is removed prematurely 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 higher safety factor.

[0091] Figure 3 Compound 7 trastuzumab antibody conjugate (such as Example 1) expresses specific BRD4 protein degradation activity in HER2-positive BT-474 breast cancer cells but not in HER2-negative MDA-MB-231 breast cancer cells. This APC does not cause degradation of AKT protein or actin. It shows that the side chain coupling of the antibody and the linker in PROTAC does not impair the activity of PROTAC. Importantly, in vivo, the antibody (trastuzumab) guides APC to those cells expressing specific antigens (such as HER2), thereby reducing off-target effects. Therefore, Example 1 is as effective as AV-825 in clinical applications but safer.

[0092] These results indicate that the antibody conjugates (branched APCs) of the present invention can enhance the specificity of cancer cell targeting, enhance cellular uptake of PROTAC forms, and reduce potential off-target effects. In addition, if the antibody is separated prematurely, the released PROTAC (e.g., compound 7) has relatively low permeability due to the valine-citrulline dipeptide and does not cause undesirable off-target effects, resulting in a high safety margin.

[0093] Antiproliferative activity

[0094] As described above, the APCs of the present invention can be used to treat diseases or disorders that carry specific antigens. These diseases can be cancer, autoimmune diseases, infectious diseases, or angiogenic disorders. The cancer can be lung cancer, colon cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, bladder cancer, stomach cancer, kidney cancer, salivary gland cancer, ovarian cancer, uterine cancer, cervical cancer, oral cancer, skin cancer, brain cancer, lymphoma, or leukemia. TM -96 assay to measure the inhibition of cell growth by the APC of the present invention. The cytotoxicity of APC was evaluated in breast cancer cell lines with different HER2 expression phenotypes. The results showed that the APC of the present invention was toxic only to HER2-positive breast cancer cells, in which BRD4 protein, Src kinase, or RAS protein can be specifically targeted for proteasomal degradation by using a suitable PROTAC coupled to trastuzumab.

[0095] The APCs of the present invention are promising new therapeutic agents because they have the advantages of both ADCs and PROTACs. In addition, the branched APCs of the present invention show advantages over linear ADC PROTACs and can be used to treat disorders carrying specific antigens.

[0096] Some embodiments of the present invention relate to methods for treating diseases or disorders using the APC of the present invention. The disease may be cancer. Specific examples of cancer may include breast cancer, gastric cancer, squamous cell carcinoma, colon cancer, and leukemia expressing specific antigens. Antibodies for APC may be trastuzumab, cetuximab, rituximab, brentuximab, gemtuzumab, inotuzumab, sacituzumab, alemtuzumab, nimotuzumab. Specific examples of APC may be branched trastuzumab-coupled PROTACs for targeting breast cancer or gastric cancer with HER2 expression.

[0097] Branched antibody-coupled PROTACs can be synthesized in different forms, such as different linkers or different antibody coupling methods. Compounds 18 and 19 are examples showing different linker forms for lysine coupling. The synthesis of PROTACs in compounds 18 and 19 follows the same method as compound 9 with a PEG linker. Compounds 18 and 19 can be synthesized using the same method as in Example 2 above. Branched PROTACs with a PEG linker can exhibit better solubility and coupling to antibodies.

[0098]

[0099] The embodiments of the present invention have been described with a limited number of examples. It will be appreciated by those skilled in the art that other modifications and variations are possible without departing from the scope of the present invention. Therefore, the scope of protection shall be limited only by the appended claims.

Claims

1. An immunoconjugate having a structure of formula (I) in: (a) Ab is an antibody or a binding fragment thereof; (b)L 1 and L 2 Each independently is a linker, wherein L 1 and L 2 the same or different, and wherein L 1 Connect L 2 ; (c) A is the target protein ligand / binder, and its structure is Where (L1) represents the link to L 1 The connection point; (d) B is a ubiquitin ligase ligand / binder, and its structure is Where (L1) represents the link to L 1 connection point, and (e) n and m are independently an integer from 1 to 8.

2. The immunoconjugate according to claim 1, characterized in that Ab is a monoclonal antibody or a variant thereof.

3. The immunoconjugate according to claim 1, characterized in that Ab binds to one or more polypeptides selected from the group consisting of: DLL3, EDAR, CLL1; BMPR1B; E16; STEAP1; 0772P; MPF; NaPi2b; Sema 5b; PSCA hlg; ETBR; MSG783; STEAP2; TrpM4; CRIPTO; CD21; CD79b; FcRH2; B7-H4; HER2; NCA; MDP; IL20Rct; Brevican; EphB2R; ASLG659; PSCA; GEDA; BAFF-R; CD22; CD79a; CXCRS; HLA-DOB; P2X5; CD72; LY64; FcRH1; IRTA2; TENB2; PMEL17; TMEFF1; GDNF-Ra1; Ly6E; TMEM46; Ly6G6D; LGR5; RET; LY6K; GPR19; GPR54; ASPHD1; tyrosinase; TMEM118; GPR172A; MUC16, and CD33.

4. The immunoconjugate according to claim 2, characterized in that Ab is trastuzumab, cetuximab, rituximab, brentuximab, gemtuzumab, inotuzumab, sahikizumab, alemtuzumab, or nimotuzumab.

5. A pharmaceutical composition comprising the immunoconjugate according to claim 1 and one or more pharmaceutically acceptable excipients.

6. A pharmaceutical composition for treating a disease, characterized in that: The pharmaceutical composition comprises an effective amount of the immunoconjugate according to claim 1 or the composition according to claim 5.

7. The pharmaceutical composition according to claim 6, characterized in that The disease is cancer.

8. The pharmaceutical composition according to claim 7, characterized in that The cancer is breast cancer or gastric cancer, and the Ab is trastuzumab.

9. The pharmaceutical composition according to claim 7, characterized in that The cancer is colon cancer or squamous cell carcinoma, and the Ab is cetuximab.

10. The pharmaceutical composition according to claim 7, characterized in that The disease is leukemia, and the Ab is rituximab, brentuximab, gemtuzumab, inotuzumab, or alemtuzumab.

Images