Antibody conjugates and fusion proteins

Abstract

The present application relates to immunoglobulins, such as immunoconjugates, comprising an ActRII receptor antibody and an agonistic GLP-1 molecule, such as an agonistic GLP-1 peptide. Such constructs are useful for treating a variety of diseases and conditions, including metabolic conditions, such as obesity, diabetes, and the like.

Description

Technical Field

[0001] This invention relates to immunoglobulins, such as immunoconjugates, comprising an ActRII receptor antibody and an agonist GLP-1 molecule, such as an agonist GLP-1 peptide. Such constructs can be used to treat a variety of diseases and conditions, including metabolic disorders such as obesity and diabetes. Background Technology

[0002] Activin type 2 receptors belong to the large TGF-β receptor family and regulate transforming growth factor β ligand signaling. These receptors are involved in many physiological processes, including growth, cell differentiation, homeostasis, bone formation, apoptosis, and many other functions. Two types of activin type 2 receptors exist: ActRIIA (ACVR2A) and ActRIIB (ACVR2B).

[0003] Although these ligands regulate a wide range of processes, they all function through essentially the same pathway: the ligand binds to the type 2 receptor, which recruits and transphosphorylates the type 1 receptor. The type 1 receptor recruits its phosphorylated receptor-regulated SMAD (R-SMAD). The R-SMAD then translocates to the nucleus, where it functions as a transcription factor.

[0004] It has been reported that several ligands that signal through activin type 2 receptors regulate muscle growth (Proc.Natl.Acad.Sci.USA102 (50): 18117-22).

[0005] Glucagon-like peptide-1 (GLP-1) is a tissue-specific post-translational peptide hormone derived from proglucagon peptide. It is produced and secreted after eating by certain neurons in the L-cells of the enterocrine system and the nucleus of the solitary tract in the brainstem. The initial product, GLP-1(1-37), is readily amidated and proteolytically cleaved, yielding two truncated and isokinetic bioactive forms: GLP-1(7-36)amide and GLP-1(7-37). The secondary structure of the active GLP-1 protein comprises two α-helices separated by a linker from amino acid positions 13-20 and 24-35.

[0006] In addition to glucose-dependent insulinotropic peptide (GIP), GLP-1 is an intestinal hypoglycemic agent; therefore, it has the ability to lower blood glucose levels in a glucose-dependent manner by enhancing insulin secretion. Besides its insulin-stimulating effect, GLP-1 is also involved in many regulatory and protective effects. Unlike GIP, the effects of GLP-1 are preserved in patients with type 2 diabetes. Glucagon-like peptide-1 receptor agonists have been approved as drugs for the treatment of diabetes and obesity since the 2000s.

[0007] Combination therapies comprising an ActRII receptor antibody and a glucagon-like peptide-1 receptor (GLP-1) agonist have been reported (WO2023 / 028606), but the individual components (i.e., the ActRII receptor antibody and the GLP-1 agonist) are separate, unconjugated molecules. This invention provides conjugates comprising an ActRII receptor antibody and a GLP-1 agonist, i.e., as single molecules. Therefore, only one single molecule must be developed compared to two molecules in a combination therapy. Pharmaceutical formulations are fine-tuned for single compounds and cannot be universally applied. Furthermore, single molecules do have very different ADME distributions, which need to be considered individually for combination therapies. Moreover, pharmacological synergies are generally only achieved when molecules act simultaneously at the same concentration in the same tissue. This can only be achieved by designing multiple pharmacologies into a single entity. Furthermore, the immunoconjugates described herein allow the GLP-1 peptide to target tissues expressing the ActR2 receptor, such as muscle and heart. Meanwhile, the conjugation of GLP-1 agonists to ActRII receptor antibodies significantly improved the adsorption-reduction mechanism (ADME) of the peptides and, for example, increased the duration of action. Furthermore, surprisingly, unlike semaglutide, the immunoconjugates resulted in weight loss while fully maintaining lean body mass. Attached Figure Description

[0008] Figure 1 Capillary electrophoresis gel images showing the final products of antibody "E272C" (lanes 1 and 4) and "wild-type" (lanes 2 and 5) under non-reducing (lanes 1 and 2) and reducing (lanes 4 and 5) conditions. Lane 3 shows size markers. Use LabChip according to the manufacturer's instructions. ® GXII Touch ™ CE-SDS was performed on the LabChip GXII Touch HT protein characterization system (PerkinElmer) using the HT chip (PerkinElmer) and the ProteinEXact assay kit (PerkinElmer). 2.5 μg of the final product was taken for analysis, and the sample was heated at 70°C for 10 minutes prior to injection.

[0009] Figure 2 The conjugation of peptides to antibodies does not affect the binding of antibodies to ActRIIA (Figure A) and ActRIIB (Figure B).

[0010] Figure 3 The conjugation of peptides to antibodies does not affect activity when measured against activin A (Figure A) or myostatin (Figure B).

[0011] Figure 4 The results showed that the conjugation of peptides to antibodies did not affect the activity of the conjugates in the GLP-1 cAMP assay.

[0012] Figure 5 DIO mice were treated for 17 days with a mediator, smegglutinin, E272C mAb-peptide 5, or E272C mAb-peptide 6. Body weight (Figure A), food intake (Figure B), fat mass (Figure C), and lean body mass (Figure D) were measured.

[0013] Figure 6 The fusion construct tested in Example 6 is shown. Summary of the Invention

[0014] This disclosure relates to an immunoconjugate comprising an ActRII receptor antibody and an agonist GLP-1 peptide.

[0015] The ActRII receptor antibody contained in the immunoconjugate is specific for the polypeptide of SEQ ID No. 12. The ActRII receptor antibody is cross-reactive with the polypeptide of SEQ ID No. 11. The ActRII receptor antibody may contain the CDR amino acid sequences of SEQ ID NO: 1 (CDRH1), SEQ ID NO: 2 (CDRH2), and SEQ ID NO: 3 (CDRH3); and may contain a variable light chain containing the CDR amino acid sequences of SEQ ID NO: 4 (CDRL1), SEQ ID NO: 5 (CDRL2), and SEQ ID NO: 6 (CDRL3). The ActRII receptor antibody can bind to the same epitope as an antibody containing a variable heavy chain and a variable light chain, wherein the variable heavy chain contains the CDR amino acid sequences of SEQ ID NO: 1 (CDRH1), SEQ ID NO: 2 (CDRH2), and SEQ ID NO: 3 (CDRH3); and the variable light chain contains the CDR amino acid sequences of SEQ ID NO: 4 (CDRL1), SEQ ID NO: 5 (CDRL2), and SEQ ID NO: 6 (CDRL3). The ActRII receptor antibody may have a silencing mutation. The ActRII receptor antibody may have an LALA silencing mutation. The ActRII receptor antibody may have a PA-LALA silencing mutation.

[0016] The agonistic GLP-1 peptide contained in the immunoconjugate may be selected from:

[0017] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG-K(MiniPEGAcBr) (SEQ ID No. 14),

[0018] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG-KKgGluC16(MiniPEGAcBr) (SEQ ID No. 15),

[0019] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG-K(GGGGS)3AcBr (SEQ ID No. 16),

[0020] HAibEGTFTSDVSSYLEGQAA-K(MiniPEGAcBr)-EFIAWLVRGRG (SEQ ID No. 17),

[0021] HAibEGTFTSDVSSYLEGQAA-KKgGluC16(MiniPEGAcBr)-EFIAWLVRGRG (SEQ ID No. 18),

[0022] HAibEGTFTSDVSSYLEGQAA-K(GGGGS)3AcBr-EFIAWLVRGRG (SEQ ID No. 19),

[0023] HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-K(MiniPEGAcBr) (SEQ ID No. 20),

[0024] HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-KKgGluC16(MiniPEGAcBr) (SEQ ID No. 21), and

[0025] HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-K(GGGGS)3AcBr (SEQ ID No. 22).

[0026] The agonistic GLP-1 peptide can also be a dual GLP-1 agonist or a triagonist GLP-1 agonist. The GLP-1 peptide may also be selected from:

[0027] YAibEGTFTSDYSIYLDKQAAAibEFVNWLLAGGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ IDNo. ​​23),

[0028] YAibEGTFTSDYSIYLDKQAAAibEFV-K(MiniPEGAcBr)-WLLAGGPSSGAPPPSK-NH2 (SEQID No. 24)

[0029] YAibEGTFTSDYSIYLDKQAAAibEFVNWLLAG-K(MiniPEGAcBr)-NH2(SEQ ID No. 25),

[0030] YAibEGTFTSDYSIYLDKQAAAibEFV-K(MiniPEGAcBr)-WLLAG-NH2(SEQ ID No. 26),

[0031] Ac-hAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLbAlaGGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ ID No. 27)

[0032] Ac-hAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLbAlaG-K(MiniPEGAcBr)-NH2 (SEQID No. 28)),

[0033] YAibAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLbAlaGGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ ID No. 29)

[0034] YAibQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLbAlaG-K(MiniPEGAcBr)-NH2(SEQ IDNo. ​​30)),

[0035] Ac-hAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLisoAspSarGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ ID No. 31)

[0036] Ac-hAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLisoAspSar-K(MiniPEGAcBr)-NH2 (SEQ ID No. 32)

[0037] YAibAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLisoAspSarGPSSGAPPPSK(MiniPEGAcBr)-NH2 (SEQ ID No. 33),

[0038] YAibQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLisoAspSar-K(MiniPEGAcBr)-NH2 (SEQ ID No. 34),

[0039] Ac-hAQGTFTSD-K(MiniPEGAcBr)-SKYLDERAAQDFVQWLLEGGPSSGAPPPS-NH2 (SEQ ID No. 35),

[0040] Ac-hAQGTFTSDKSKYLDERAAQDFVQWLLEGGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ ID No. 36),

[0041] Ac-hAQGTFTSD-K(MiniPEGAcBr)-SKYLDERAAQDFVQWLLEGGPSSGAPPPS-K(gGluC16)-NH2 (SEQ ID No. 37), and

[0042] Ac-hAQGTFTSD-K(gGluC16)-SKYLDERAAQDFVQWLLEGGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ ID No. 38).

[0043] The agonistic GLP-1 peptide can be conjugated to the ActRII receptor antibody via cysteine ​​engineering. The agonistic GLP-1 peptide can be conjugated to the ActRII receptor antibody at positions 272, 361, 384, 389, or 434 via cysteine ​​engineering. The agonistic GLP-1 peptide can also be conjugated to the ActRII receptor antibody via a cysteine ​​residue located in the hinge region.

[0044] This disclosure also relates to a pharmaceutical composition comprising the aforementioned immunoconjugate.

[0045] This disclosure also relates to the aforementioned immunoconjugates or pharmaceutical compositions for use in a medicament. The use in a medicament may be for the treatment of metabolic disorders. The metabolic disorders may be selected from the group consisting of: obesity, diabetes, metabolic syndrome, antipsychotic-associated obesity, glucocorticoid-induced obesity, hypothalamic obesity associated with craniopharyngioma, Prader-Willi syndrome, and monogenic disorders associated with obesity. The use in a medicament may also be for the treatment of obesity-related comorbidities, wherein the symptoms are selected from the group consisting of: glucose intolerance, prediabetes, insulin resistance, hypertriglyceridemia, overweight-related physical damage, osteoporosis, kidney disease, obstructive sleep apnea, sex hormone-induced damage, endocrine-reproductive disorders, osteoarthritis, gastrointestinal cancer, dyslipidemia, hypertension, heart failure, coronary arthritis, stroke, and / or gallstones.

[0046] This disclosure also relates to an ActRII receptor antibody, wherein the ActRII receptor antibody is fused to an agonistic GLP-1 peptide.

[0047] Embodiments of the present invention

[0048] Antibody

[0049] As used herein, the term "antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds that interacts with an antigen. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) and scattered with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelified antibodies, and chimeric antibodies. Antibodies can be any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgD, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Both the light and heavy chains are divided into structurally and functionally homologous regions.

[0050] The terms "specifically binds to," "specifically to," or "specifically recognizes" in the context of antibodies refer to antibodies that are specific to an antigen and capable of distinguishing the target antigen from one or more reference antigens. Specificity is not absolute but a relative property. For example, it can be determined by standard ELISA assays or standard flow cytometry assays. Scoring can be performed by standard colorimetric methods (e.g., secondary antibodies with horseradish peroxide and tetramethylbenzidine with hydrogen peroxide) or by binding a secondary antibody labeled with PE or another dye or marker. Reactions in some wells are scored by, for example, optical density (OD) at 450 nm or by mean fluorescence intensity (MFI) in flow cytometry. Typical background (=negative reaction) can be 0.1 OD; typical positive reaction can be 1 OD. Background and positive reaction MFI are highly dependent on instrument settings. Positive / negative differences can exceed 10-fold. Typically, binding specificity is determined not by using a single reference antigen, but by using a group of about three to five unrelated antigens (such as milk powder, BSA, transferrin, etc.). For flow cytometry, a variety of antigen-negative cells can be used. However, antibodies that specifically bind to antigens may be cross-reactive with corresponding orthologous antigens from other species (e.g., species homologs). In some embodiments, this cross-reactivity with orthologous antigens is even preferred.

[0051] As used herein, an antibody is considered "cross-reactive" or "cross-reactive" if it binds to a closely related antigen or the same antigen from another species. In this disclosure, the term is used for antibodies that are specific to ActRIIB but also bind to ActRIIA.

[0052] As used herein, the term "ActRII receptor antibody" refers to an antibody that is specific to ActRIIA, an antibody that is specific to ActRIIB, and an antibody that is specific to both ActRIIA and ActRIIB, i.e., an antibody that is cross-reactive between ActRIIA and ActRIIB.

[0053] As used herein, the terms “ActRIIA” and “ACVR2A” refer to the human protein having UniProt ID P27037. ActRIIA has the following amino acid sequence:

[0054] MGAAAKLAFAVFLISCSSGAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPYYNILLYSLVPLMLIAGIVICAFWVYRHHKMAYPPVLVPTQDPGPPPPSPLLGLKPLQLLEVKARGRFGCVWKAQLLNEYVAVKIFPIQDKQSWQNEYEVYSLPGMKHENILQFIGAEKRGTSVDVDLWLITAFHEKGSLSDFLKANVVSWNELCHIAETMARGLAYLHEDIPGLKDGHKPAISHRDIKSKNVLLKNNLTACIADFGLALKFEAGKSAGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELASRCTAADGPVDEYMLPFEEEIGQHPSLEDMQEVVVHKKKRPVLRDYWQKHAGMAMLCETIEECWDHDAEARLSAGCVGERITQMQRLTNIITTEDIVTVVTMVTNVDFPPKESSL (SEQ ID No.11)

[0055] As used herein, the terms "ActRIIB" and "ACVR2B" refer to the human protein having UniProt ID Q13705.

[0056] MTAPWVALALLWGSLCAGSGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPP PTAPTLLTVLAYSLLPIGGLSLIVLLAFWMYRHRKPPYGHVDIHEDPGPPPPSPLVGLKPLQLLEIKARGRFGCVWKAQLMNDFVAVKIFPLQDKQSWQSEREIFSTPGMKHENLLQFIAAEKRGSNLE VELWLITAFHDKGSLTDYLKGNIITWNELCHVAETMSRGLSYLHEDVPWCRGEGHKPSIAHRDFKSKNVLLKSDLTAVLADFGLAVRFEPGKPPGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDMY AMGLVLWELVSRCKAADGPVDEYMLPFEEEIGQHPSLEELQEVVVHKKMRPTIKDHWLKHPGLAQLCVTIECWDHDAEARLSAGCVEERVSLIRRSVNGTTSDCLVSLVTSVTNVDLPPKESSI (SEQ ID No.12)

[0057] Activin receptor II B (ActRIIB) is the receptor for myostatin, activin, and bone morphogenetic protein (BMP). The interaction between myostatin and this receptor regulates the inhibition of skeletal muscle differentiation via a Smad-dependent pathway. It is believed that skeletal muscle formation can be induced by inhibiting or preventing myostatin from binding to ActRIIB (e.g., via ActRII receptor antibodies). Regulation of activin receptor II A (ActRIIA) also plays a role in the regulation of muscle growth (Morvan et al. 2017).

[0058] In human clinical studies, exemplary ActRII receptor antibodies binding to ActRIIA and ActRIIB have shown that they not only increase lean muscle mass but also reduce fat mass and improve glycemic control (WO2010 / 125003; WO2018 / 116201; Heymsfield et al. 2021;4(l):e2033457, JAMA). Exemplary ActRII receptor antibodies that may be used in the context of this disclosure include the antibodies described in WO02010 / 125003, WO2013 / 063536, WO2013 / 188448, WO2014 / 172448, WO2017 / 156488, WO2020 / 243448, and WO2021 / 174198.

[0059] In a specific embodiment, this disclosure relates to the antibody bimagrumab (BYM338; WO2010 / 125003). Bimagrumab has the following amino acid sequence.

[0060] Table 1 :

[0061]

[0062] In some embodiments, the ActRII receptor antibody of this disclosure comprises a variable heavy chain containing the CDR amino acid sequences of SEQ ID NO: 1 (CDRH1), SEQ ID NO: 2 (CDRH2), and SEQ ID NO: 3 (CDRH3).

[0063] In some embodiments, the ActRII receptor antibody of this disclosure comprises a variable light chain comprising the CDR amino acid sequences of SEQ ID NO: 4 (CDRL1), SEQ ID NO: 5 (CDRL2), and SEQ ID NO: 6 (CDRL3).

[0064] In some embodiments, the ActRII receptor antibody of this disclosure comprises a variable heavy chain containing the CDR amino acid sequences of SEQ ID NO: 1 (CDRH1), SEQ ID NO: 2 (CDRH2), and SEQ ID NO: 3 (CDRH3); and a variable light chain containing the CDR amino acid sequences of SEQ ID NO: 4 (CDRL1), SEQ ID NO: 5 (CDRL2), and SEQ ID NO: 6 (CDRL3).

[0065] In some embodiments, the ActRII receptor antibody of this disclosure comprises: a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; and / or a variable light chain comprising the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith.

[0066] In some embodiments, the ActRII receptor antibody of this disclosure comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO:9, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith; and / or a light chain comprising the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith.

[0067] In some embodiments, the ActRII receptor antibody of this disclosure has the same binding specificity as antibodies comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain comprises the CDR amino acid sequences of SEQ ID NO: 1 (CDRH1), SEQ ID NO: 2 (CDRH2), and SEQ ID NO: 3 (CDRH3); and the variable light chain comprises the CDR amino acid sequences of SEQ ID NO: 4 (CDRL1), SEQ ID NO: 5 (CDRL2), and SEQ ID NO: 6 (CDRL3).

[0068] In some embodiments, the ActRII receptor antibody of this disclosure binds to the same epitope as an antibody comprising a variable heavy chain and a variable light chain, the variable heavy chain comprising the CDR amino acid sequences of SEQ ID NO: 1 (CDRH1), SEQ ID NO: 2 (CDRH2), and SEQ ID NO: 3 (CDRH3); the variable light chain comprising the CDR amino acid sequences of SEQ ID NO: 4 (CDRL1), SEQ ID NO: 5 (CDRL2), and SEQ ID NO: 6 (CDRL3).

[0069] In some embodiments, the ActRII receptor antibody of this disclosure binds to the same epitope as an antibody comprising a variable heavy chain and a variable light chain, the variable heavy chain comprising the CDR amino acid sequences of SEQ ID NO: 1 (CDRH1), SEQ ID NO: 2 (CDRH2), and SEQ ID NO: 3 (CDRH3); the variable light chain comprising the CDR amino acid sequences of SEQ ID NO: 4 (CDRL1), SEQ ID NO: 5 (CDRL2), and SEQ ID NO: 6 (CDRL3), and wherein the ActRII receptor antibody contains engineered cysteine.

[0070] In some embodiments, the ActRII receptor antibody of this disclosure binds to the same epitopes as an antibody comprising a variable heavy chain and a variable light chain, the variable heavy chain comprising the CDR amino acid sequences of SEQ ID NO: 1 (CDRH1), SEQ ID NO: 2 (CDRH2), and SEQ ID NO: 3 (CDRH3); the variable light chain comprising the CDR amino acid sequences of SEQ ID NO: 4 (CDRL1), SEQ ID NO: 5 (CDRL2), and SEQ ID NO: 6 (CDRL3), and wherein the ActRII receptor antibody contains engineered cysteine ​​residues at positions 272, 361, 384, 389, or 434.

[0071] In some embodiments, the ActRII receptor antibody of this disclosure binds to the same epitope as an antibody comprising a variable heavy chain and a variable light chain, the variable heavy chain comprising the CDR amino acid sequences of SEQ ID NO: 1 (CDRH1), SEQ ID NO: 2 (CDRH2), and SEQ ID NO: 3 (CDRH3); the variable light chain comprising the CDR amino acid sequences of SEQ ID NO: 4 (CDRL1), SEQ ID NO: 5 (CDRL2), and SEQ ID NO: 6 (CDRL3), wherein the antibody comprises a silencing Fc modification. In some embodiments, the silencing mutation is an LALA mutation.

[0072] In some embodiments, the ActRII receptor antibody of this disclosure binds to the same epitope as an antibody comprising a variable heavy chain and a variable light chain, the variable heavy chain comprising the CDR amino acid sequences of SEQ ID NO: 1 (CDRH1), SEQ ID NO: 2 (CDRH2), and SEQ ID NO: 3 (CDRH3); the variable light chain comprising the CDR amino acid sequences of SEQ ID NO: 4 (CDRL1), SEQ ID NO: 5 (CDRL2), and SEQ ID NO: 6 (CDRL3), wherein the antibody comprises a silencing Fc modification. In some embodiments, the silencing mutation is a PA-LALA mutation.

[0073] In some embodiments, the ActRII receptor antibody of this disclosure binds to the same epitope as an antibody comprising a variable heavy chain and a variable light chain, the variable heavy chain comprising the CDR amino acid sequences of SEQ ID NO: 1 (CDRH1), SEQ ID NO: 2 (CDRH2), and SEQ ID NO: 3 (CDRH3); the variable light chain comprising the CDR amino acid sequences of SEQ ID NO: 4 (CDRL1), SEQ ID NO: 5 (CDRL2), and SEQ ID NO: 6 (CDRL3), wherein the antibody comprises a silencing Fc modification. In some embodiments, the silencing mutation is a PG-LALA mutation.

[0074] In some embodiments, the ActRII receptor antibody of this disclosure binds to the same epitope as an antibody comprising a variable heavy chain and a variable light chain, the variable heavy chain comprising the CDR amino acid sequences of SEQ ID NO: 1 (CDRH1), SEQ ID NO: 2 (CDRH2), and SEQ ID NO: 3 (CDRH3); the variable light chain comprising the CDR amino acid sequences of SEQ ID NO: 4 (CDRL1), SEQ ID NO: 5 (CDRL2), and SEQ ID NO: 6 (CDRL3), wherein the antibody comprises a silencing Fc modification. In some embodiments, the silencing mutation is an AEASS mutation.

[0075] In some embodiments, the Fc portion of the ActRII receptor antibody of this disclosure may also be an allotype. A well-known exemplary allotype is R... When the K allotype is introduced into the Fc region of bimalumab, it produces the following amino acid sequence in the full heavy chain:

[0076] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSSYINWVRQAPGQGLEWMGTINPVSGSTSYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYCARGGWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK K VEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID No. 13)

[0077] As used herein in the context of silent antibodies or antibodies containing silent mutations, the term "silent" or "silent" refers to a mutation in the Fc domain of such antibodies that partially or completely reduces binding to one or more cell surface Fcγ receptors, thereby reducing or attenuating, and in some embodiments substantially completely eliminating one or more Fc-mediated antibody effector functions, such as ADCC, ADCP, and CDC complement responses (see, for example, Kang and Jung, Experimental and Molecular Medicine (2019) 51:138). Silent effector functions can be acquired by mutations in the Fc region of the antibody and have been described in the art (e.g., Strohl, Biotechnology 20: 685-91, for LALA and N297A; Baudino et al., J. Immunol.181: 6664-69, for D265A). Other exemplary Fc silencing mutations include amino acid substitutions at one or more of the following positions: E233, L234, L235, G236, N297, P331, and P329 (see, for example, U.S. Patent Nos. 6,737,056 and 7,332,581; WO 2004 / 056312 and WO2021 / 234402; and Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604). Silent mutations also include (according to the EU index) LALA (L234A / L235A), PA-LALA (L234A / L235A / P329A) and PG-LALA (L234A / L235A / P329G) mutations, as well as AEASS mutations (L234A / L235E / G237A / A330S / P331S).

[0078] In some embodiments, the ActRII receptor antibody of this disclosure is a modified or improved form of an antibody comprising a variable heavy chain containing the CDR amino acid sequences of SEQ ID NO: 1 (CDRH1), SEQ ID NO: 2 (CDRH2), and SEQ ID NO: 3 (CDRH3); and a variable light chain containing the CDR amino acid sequences of SEQ ID NO: 4 (CDRL1), SEQ ID NO: 5 (CDRL2), and SEQ ID NO: 6 (CDRL3). Preferably, the modified or improved form of the antibody has a higher affinity for ActRII to achieve equivalent inhibition of both ActRII receptors. It exhibits less nonspecific binding and is designed to improve PK and align with PK and associated PD effects. It has optimized stability for custom formulations.

[0079] In some embodiments, the ActRII receptor antibody of this disclosure has an Fc domain, such as a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, or a human IgG4 Fc domain. In some embodiments, the Fc domain is wild-type. In some embodiments, the ActRII receptor antibody of this disclosure includes embodiments in which the Fc domain is modified, for example, to increase serum half-life. In some embodiments, the ActRII receptor antibody of this disclosure includes embodiments in which the Fc domain is a human IgG1 Fc domain and is modified to increase serum half-life. In some embodiments, the ActRII receptor antibody of this disclosure includes embodiments in which modifications to increase serum half-life relative to the human IgG1 Fc domain according to EU numbering schemes include one or more of 259I, 252Y, 307Q, 308F, 428L, 434H, 434F, 434Y, 434A, 434M, and 434S. In some embodiments, the ActRII receptor antibody of this disclosure includes embodiments in which, relative to the human IgG1 Fc domain according to the EU numbering scheme, these modifications include one or more of 259I / 434S, 308F / 434S, 308F / 428L / 434S, 259I / 308F / 434S, 307Q / 308F / 434S, 250I / 308F / 434S, and 308F1319L / 434S.

[0080] In some embodiments, the Fc domain of this ActRII receptor antibody is modified to include the mutations M252Y, S254T, and T256E relative to the human IgG1 Fc domain according to the EU numbering scheme. These mutations are referred to as "YTE mutations". Antibodies carrying YTE mutations have been reported to have an increased antibody half-life (WO2002 / 060919).

[0081] In some embodiments, the ActRII receptor antibody of this disclosure binds to ActRIIB with a KD of 100 nM or less, 10 nM or less, or 1 nM or less. Preferably, the ActRII receptor antibody binds to ActRIIB with an affinity of 100 pM or less (i.e., 100 pM, 50 pM, 10 pM, 1 pM or less). In some embodiments, the ActRII receptor antibody binds to ActRIIB with an affinity between 10 pM and 20 pM.

[0082] In some embodiments, the ActRII receptor antibody of this disclosure binds to ActRIIA with an affinity 5 times, more preferably 10 times, even more preferably 50 times, and even more preferably 100 times that of ActRIIA. In some embodiments, the ActRII receptor antibody binds to ActRIIA with an affinity of 100 pM or greater (i.e., 250 pM, 500 pM, 1 nM, 5 nM or greater).

[0083] In some embodiments, ActRII pathway agents other than ActRII receptor antibodies are used. These ActRII pathway agents directly bind to ActRII receptor ligands, such as myostatin or activin. Such agents include, but are not limited to, myostatin inhibitors (e.g., myostatin antibodies or myostatin small molecule antagonists), activin inhibitors (e.g., activin antibodies or activin small molecule antagonists), or soluble extracellular fractions of ActRIIB or ActRIIA, which may act as a “ligand sink” optionally further stabilized with Fc. Therefore, in some embodiments, a method for treating the metabolic disorders of this disclosure is provided herein, comprising administering to a subject in need an ActRII pathway agent (other than an ActRII receptor antibody) in combination with a GLP-1 agonist, wherein the agent is selected from the group consisting of myostatin inhibitors, activin inhibitors, or soluble fractions of the ActRII receptor.

[0084] GLP-1 agonists

[0085] The glucagon-like peptide-1 receptor is primarily considered to be the pancreatic β-cell receptor, which stimulates insulin secretion upon binding to the hormone glucagon-like peptide-1 (GLP-1). GLP-1 is an intestinal hypoglycemic hormone that regulates insulin release in response to blood glucose levels. GLP-1 agonists mimic the action of the GLP-1 peptide and activate the GLP-1 receptor upon binding, thereby stimulating insulin secretion. In addition to its glucose-regulating role, GLP-1 has also been identified to function in other tissues such as the brain, stomach, intestines, heart, and muscles.

[0086] However, GLP-1 agonists have exhibited significant side effects and tolerability issues at effective doses. GLP-1 agonists have also been shown to reduce lean body mass in subjects during treatment. Warnings and precautions for GLP-1 agonists (e.g., semaglutide) include, but are not limited to: thyroid C-cell tumor, acute pancreatitis, acute gallbladder disease, hypoglycemia, acute kidney injury, serious adverse gastrointestinal reactions, hypersensitivity reactions (e.g., anaphylactic reactions and angioedema), diabetic retinopathy complications in subjects with type 2 diabetes, increased heart rate, and suicidal behavior and ideation. Adverse reactions reported in at least 5% of subjects treated with GLP-1 agonists (e.g., semaglutide) include nausea, diarrhea, vomiting, constipation, abdominal pain, headache, fatigue, indigestion, dizziness, abdominal distension, hiccups, hypoglycemia in patients with type 2 diabetes, flatulence, gastroenteritis, and gastroesophageal reflux disease. Therefore, optimized dosage and treatment with GLP-1 agonists are necessary.

[0087] As used herein, the term “GLP-1 agonist” refers to a compound that fully or partially activates the human GLP-1 receptor. The term GLP-1 agonist also includes dual GLP-1 agonists that also activate the GIP receptor or the glucagon receptor, respectively, and triple GLP-1 agonists that simultaneously and to varying degrees activate the GLP-1, GIP, and GCG receptors.

[0088] As provided herein, the GLP-1 agonists of this disclosure can be peptides, antibodies, small molecules, or aptamers. In some embodiments, the GLP-1 agonist is a peptide or peptide analogue, including but not limited to: exenatide, extended-release exenatide, dulaglutide, liraglutide, lixisenatide, smegglutide, tirzepatide, cotadutide, noiiglutide, oxyntomodulin, or pemvidutide. In some embodiments, the GLP-1 agonist is a small molecule non-peptide agonist, such as danuglipron. GLP-1 agonists are also described and characterized in Obes Sci Pract. March 2017; 3(1): 3-14, Ther Adv Endocrinol Metab 2021, Vol. 12: 1-15 and 2022; 23(3): 521-539.

[0089] Other exemplary GLP-1 agonists include:

[0090] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG-K(MiniPEGAcBr) (SEQ ID No. 14)

[0091] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG-KKgGluC16(MiniPEGAcBr) (SEQ ID No. 15)

[0092] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG-K(GGGGS)3AcBr (SEQ ID No. 16)

[0093] HAibEGTFTSDVSSYLEGQAA-K(MiniPEGAcBr)-EFIAWLVRGRG (SEQ ID No. 17)

[0094] HAibEGTFTSDVSSYLEGQAA-KKgGluC16(MiniPEGAcBr)-EFIAWLVRGRG (SEQ ID No. 18)

[0095] HAibEGTFTSDVSSYLEGQAA-K(GGGGS)3AcBr-EFIAWLVRGRG (SEQ ID No. 19)

[0096] HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-K(MiniPEGAcBr) (SEQ ID No. 20)

[0097] HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-KKgGluC16(MiniPEGAcBr) (SEQ ID No. 21)

[0098] HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-K(GGGGS)3AcBr (SEQ ID No. 22)

[0099] As used herein, the term “GLP-1 dual agonist” refers to a GLP-1 agonist that activates a second receptor other than the GLP-1 receptor, such as the glucose-dependent insulinotropic (GIP) receptor or the glucagon (GCG) receptor.

[0100] In some embodiments, the GLP-1 agonist is a dual agonist that binds to and activates or inactivates a second receptor other than the GLP-1 receptor, wherein the second receptor is a glucose-dependent insulinotropic (GIP) receptor or a glucagon (GCG) receptor. An exemplary dual-acting GLP-1 agonist that binds to both the GLP-1 receptor and the GIP receptor is telpolide. Exemplary dual-acting GLP-1 agonists that bind to both the GLP-1 receptor and the GCG receptor are codalotide, noriglycopeptide, and gastrin. In some embodiments, the GLP-1 agonist is also a GIP antagonist. In some embodiments, the GLP-1 agonist is also a GCG agonist.

[0101] Exemplary GLP-1 / GIP dual agonists include:

[0102] YAibEGTFTSDYSIYLDKQAAAibEFVNWLLAGGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ IDNo. ​​23)

[0103] YAibEGTFTSDYSIYLDKQAAAibEFV-K(MiniPEGAcBr)-WLLAGGGPSSGAPPPSK-NH2 (SEQID No. 24)

[0104] YAibEGTFTSDYSIYLDKQAAAibEFVNWLLAG-K(MiniPEGAcBr)-NH2 (SEQ ID No. 25)

[0105] YAibEGTFTSDYSIYLDKQAAAibEFV-K(MiniPEGAcBr)-WLLAG-NH2 (SEQ ID No. 26)

[0106] GLP-1 / GIP dual agonists have also been described and characterized in Diabetes Metab Syndr Obes 2019; 12: 1973-198, Bioorg Chem. 2021; 106:104492 and Drug Design, Development and Therapy 2022:161547-1559.

[0107] Exemplary GLP-1 / GCG dual agonists include:

[0108] Ac-hAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLbAlaGGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ ID No. 27)

[0109] Ac-hAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLbAlaG-K(MiniPEGAcBr)-NH2 (SEQ ID No. 28)

[0110] YAibAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLbAlaGGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ ID No. 29)

[0111] YAibQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLbAlaG-K(MiniPEGAcBr)-NH2 (SEQ ID No. 30)

[0112] Ac-hAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLisoAspSarGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ ID No. 31)

[0113] Ac-hAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLisoAspSar-K(MiniPEGAcBr)-NH2 (SEQ ID No. 32)

[0114] YAibAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLisoAspSarGPSSGAPPPSK(MiniPEGAcBr)-NH2 (SEQ ID No. 33)

[0115] YAibQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLisoAspSar-K(MiniPEGAcBr)-NH2 (SEQ ID No. 34)

[0116] GLP-1 / GCG dual agonists are also described and characterized in Diabetologia (2017) 60(10):1851 - 61, Bioconjugate Chem. 2020, 31, 4, 1167 - 1176 and Nature Communications (2022) 13:3613.

[0117] As used herein, the term “GLP-1 triple agonist” refers to a GLP-1 agonist that activates two receptors other than the GLP-1 receptor, such as the glucose-dependent insulinotropic (GIP) receptor and the glucagon (GCG) receptor.

[0118] In some embodiments, the GLP-1 agonist is a GLP-1 triple agonist. In some embodiments, the GLP-1 agonist is a GIP / GLP / glucagon receptor triple agonist, such as a GGG triple agonist, for example, LY343794.

[0119] Exemplary GLP-1 / GIP / GCG triple agonists include:

[0120] Ac-hAQGTFTSD-K(MiniPEGAcBr)-SKYLDERAAQDFVQWLLEGPGPSSGAPPPS-NH2 (SEQ ID No. 35)

[0121] Ac-hAQGTFTSDKSKYLDERAAQDFVQWLLEGGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ IDNo. ​​36)

[0122] Ac-hAQGTFTSD-K(MiniPEGAcBr)-SKYLDERAAQDFVQWLLEGGPSSGAPPPS-K(gGluC16)-NH2 (SEQ ID No. 37)

[0123] Ac-hAQGTFTSD-K(gGluC16)-SKYLDERAAQDFVQWLLEGGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ ID No. 38)

[0124] GLP-1 triple agonists are also described and characterized in Nat Med 2015; 21(1):27-36, Endocrine Reviews, 2018, 39(5):719-38 and Cell Metabolism 34, 1234-1247, September 6, 2022.

[0125] Abbreviations and structures used in the context of GLP-1 peptides:

[0126] h: D-histidine

[0127] βAla: β-alanine

[0128] Aib: 2-Aminoisobutyric acid

[0129] isoAsp: isoaspartic acid

[0130] Sar: Creatine

[0131] K(MiniPEGAcBr):

[0132]

[0133] KKγGluC16MiniPEGAcBr:

[0134]

[0135] K(GGGGS)3AcBr:

[0136]

[0137] KγGluC16:

[0138]

[0139] Adhesion

[0140] There are various techniques that allow peptides or modified peptides to be conjugated to antibodies. This includes cysteine ​​engineering of antibodies. In this technique, a peptide is conjugated to a cysteine ​​residue (typically the Fc moiety) artificially introduced into the antibody.

[0141] As used herein in the context of antibodies, the terms "cysteine ​​engineered" or "engineered cysteine" refer to an antibody construct engineered to introduce at least one cysteine ​​insertion mutation. In this context, "cysteine ​​mutation" refers to the introduction of a non-natural cysteine ​​residue into the sequence of a parent antibody construct. The inserted cysteine ​​residue can serve as a conjugation site for one or more active agents. In this disclosure, such an active agent is an agonist GLP-1 peptide.

[0142] Various positions in the antibody have been reported as suitable for cysteine ​​engineering, including but not limited to positions 241, 243, 251, 253, 258, 264, 269, 271, 272, 274, 280, 281, 285, 288, 291, 293, 294, 296, 301, 307, 309, 311, 318, 329, 340, 341, 345, 357, 361, 384, 385, 386, 387, 389, 401, 402, 411, 417, 433, 434, 435, and 439 (from Medimmune_WO2015157595). This article exemplifies antibodies engineered at positions E272, E361, N384, E389, and N434.

[0143] Peptides can be conjugated to cysteine-engineered antibodies via Michael addition, for example, maleimide-modified peptides, or via halogen substitution of the corresponding modified peptide (e.g., bromoacetylated peptides). Chemically modified peptides can be conjugated, for example, to cysteine-engineered antibodies, as described in ACS Chem Biol. 2017 Sep 15; 12(9):2427-2435.

[0144] Other conjugation techniques are also known for generating conjugates containing ActRII receptor antibodies and agonistic GLP-1 peptides. These additional conjugation techniques include conjugation via lysine-linked peptides or acetylene / azide-modified peptides (via click chemistry).

[0145] In some embodiments, this disclosure relates to an immunoconjugate comprising an ActRII receptor antibody and an agonist GLP-1 peptide, wherein the ActRII receptor antibody is conjugated to the agonist GLP-1 peptide via cysteine ​​engineering.

[0146] In some embodiments, this disclosure relates to an immunoconjugate comprising an ActRII receptor antibody and an agonist GLP-1 peptide, wherein the ActRII receptor antibody is conjugated to the agonist GLP-1 peptide via cysteine ​​engineering, and wherein the conjugation technique employs a bromoacetylated peptide.

[0147] In some embodiments, this disclosure relates to an immunoconjugate comprising an ActRII receptor antibody and an agonistic GLP-1 peptide, wherein the agonistic GLP-1 peptide is conjugated to the ActRII receptor antibody at positions 272, 361, 384, 389, or 434. The agonistic GLP-1 peptide may also be conjugated to the ActRII receptor antibody via a cysteine ​​residue located in the hinge region.

[0148] Furthermore, spacer regions of various lengths and rigidities can be introduced to further optimize the immunoconjugates of this disclosure. Commonly used spacer regions include polyamides or polyethylene glycol (PEG).

[0149] ActRII antagonists

[0150] In some embodiments, the ActRII receptor antibody of the immunoconjugate disclosed herein may be replaced by an ActRII antagonist. Such an ActRII antagonist may be a portion different from the ActRII receptor antibody, such as a TGFβ-like ligand, myostatin, AT, propeptide, activin A, or derivatives or analogs thereof. Preferably, the ActRII antagonist is an antagonist of both ActRIIA and ActRIIB.

[0151] Therefore, in some embodiments, this disclosure relates to conjugates comprising an ActRII antagonist and an agonist GLP-1 peptide.

[0152] In some embodiments, this disclosure relates to conjugates comprising an ActRII antagonist and an agonist GLP-1 peptide, wherein the ActRII antagonist is specific for the peptides of SEQ ID No. 12 and SEQ ID No. 11.

[0153] In some embodiments, this disclosure relates to conjugates comprising an ActRII antagonist and an agonist GLP-1 peptide, wherein the agonist GLP-1 peptide is selected from:

[0154] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG-K(MiniPEGAcBr) (SEQ ID No. 14),

[0155] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG-KKgGluC16(MiniPEGAcBr) (SEQ ID No. 15),

[0156] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG-K(GGGGS)3AcBr (SEQ ID No. 16),

[0157] HAibEGTFTSDVSSYLEGQAA-K(MiniPEGAcBr)-EFIAWLVRGRG (SEQ ID No. 17),

[0158] HAibEGTFTSDVSSYLEGQAA-KKgGluC16(MiniPEGAcBr)-EFIAWLVRGRG (SEQ ID No. 18),

[0159] HAibEGTFTSDVSSYLEGQAA-K(GGGGS)3AcBr-EFIAWLVRGRG (SEQ ID No. 19),

[0160] HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-K(MiniPEGAcBr) (SEQ ID No. 20),

[0161] HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-KKgGluC16(MiniPEGAcBr) (SEQ ID No. 21), and

[0162] HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-K(GGGGS)3AcBr (SEQ ID No. 22).

[0163] In some embodiments, this disclosure relates to conjugates comprising an ActRII antagonist and an agonist GLP-1 peptide, wherein the agonist GLP-1 peptide is a dual or triple GLP-1 agonist. Preferably, the dual or triple GLP-1 agonist is selected from...

[0164] YAibEGTFTSDYSIYLDKQAAAibEFVNWLLAGGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ IDNo. ​​23),

[0165] YAibEGTFTSDYSIYLDKQAAAibEFV-K(MiniPEGAcBr)-WLLAGGGPSSGAPPPSK-NH2 (SEQID No. 24),

[0166] YAibEGTFTSDYSIYLDKQAAAibEFVNWLLAG-K(MiniPEGAcBr)-NH2 (SEQ ID No. 25),

[0167] YAibEGTFTSDYSIYLDKQAAAibEFV-K(MiniPEGAcBr)-WLLAG-NH2 (SEQ ID No. 26),

[0168] Ac-hAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLbAlaGGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ ID No. 27),

[0169] Ac-hAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLbAlaG-K(MiniPEGAcBr)-NH2 (SEQID No. 28),

[0170] YAibAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLbAlaGGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ ID No. 29),

[0171] YAibQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLbAlaG-K(MiniPEGAcBr)-NH2 (SEQ ID No. 30),

[0172] Ac-hAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLisoAspSarGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ ID No. 31),

[0173] Ac-hAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLisoAspSar-K(MiniPEGAcBr)-NH2 (SEQ ID No. 32),

[0174] YAibAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLisoAspSarGPSSGAPPPSK(MiniPEGAcBr)-NH2 (SEQ ID No. 33),

[0175] YAibQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLisoAspSar-K(MiniPEGAcBr)-NH2 (SEQ ID No. 34),

[0176] Ac-hAQGTFTSD-K(MiniPEGAcBr)-SKYLDERAAQDFVQWLLEGGPSSGAPPPS-NH2 (SEQ IDNo. 35),

[0177] Ac-hAQGTFTSDKSKYLDERAAQDFVQWLLEGGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ IDNo. 36),

[0178] Ac-hAQGTFTSD-K(MiniPEGAcBr)-SKYLDERAAQDFVQWLLEGGPSSGAPPPS-K(gGluC16)-NH2 (SEQ ID No. 37), and

[0179] Ac-hAQGTFTSD-K(gGluC16)-SKYLDERAAQDFVQWLLEGGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ ID No. 38).

[0180] GLP-1 agonists

[0181] In some embodiments, the agonistic GLP-1 peptide of the immunoconjugates disclosed herein may be replaced by an agonistic GLP-1 small molecule. Such small molecules are commonly referred to as GLP-1 secretagogues (Drug Disc Today 25, 2023 (2021); Nat Commun 13, 4633 (2022)). This term refers to an agent that promotes the secretion of GLP-1 from cells (e.g., enteroendocrine cells). Exemplary GLP-1 secretagogues include BDM72881, HD0471042, YH1896, BMS-903452, LY292208, LY2922470, BMs9-86118, SCO-267, and JWU-A021.

[0182] Therefore, in some embodiments, this disclosure relates to conjugates comprising an ActRII receptor antibody and an agonistic GLP-1 small molecule. In some embodiments, this disclosure relates to conjugates comprising an ActRII receptor antibody and a GLP-1 secretagogue. In some embodiments, the agonistic GLP-1 small molecule or GLP-1 secretagogue is selected from BDM72881, HD0471042, YH1896, BMS-903452, LY292208, LY2922470, BMs9-86118, SCO-267, and JWU-A021.

[0183] In some embodiments, this disclosure relates to conjugates comprising an ActRII receptor antibody and an agonist GLP-1 small molecule, wherein the ActRII receptor antibody is specific for the peptide of SEQ ID No. 12 and the peptide of SEQ ID No. 11. In some embodiments, this disclosure relates to conjugates comprising an ActRII receptor antibody and a GLP-1 secretagogue, wherein the ActRII receptor antibody is specific for the peptide of SEQ ID No. 12 and the peptide of SEQ ID No. 11.

[0184] In some embodiments, this disclosure relates to conjugates comprising an ActRII receptor antibody and an agonist GLP-1 small molecule, wherein the ActRII receptor antibody binds to the same epitope as an antibody comprising a variable heavy chain and a variable light chain, the variable heavy chain comprising the CDR amino acid sequences of SEQ ID NO: 1 (CDRH1), SEQ ID NO: 2 (CDRH2), and SEQ ID NO: 3 (CDRH3); and the variable light chain comprising the CDR amino acid sequences of SEQ ID NO: 4 (CDRL1), SEQ ID NO: 5 (CDRL2), and SEQ ID NO: 6 (CDRL3). In some embodiments, this disclosure relates to a conjugate comprising an ActRII receptor antibody and a GLP-1 secretin, wherein the ActRII receptor antibody binds to the same epitope as an antibody comprising a variable heavy chain and a variable light chain, the variable heavy chain comprising the CDR amino acid sequences of SEQ ID NO: 1 (CDRH1), SEQ ID NO: 2 (CDRH2), and SEQ ID NO: 3 (CDRH3); and the variable light chain comprising the CDR amino acid sequences of SEQ ID NO: 4 (CDRL1), SEQ ID NO: 5 (CDRL2), and SEQ ID NO: 6 (CDRL3).

[0185] Antibody fusion

[0186] The agonist GLP-1 peptide disclosed herein can also be genetically fused with an ActRII receptor antibody. Preferably, the agonist GLP-1 peptide is fused to the N-terminus of a variable heavy chain at the N-terminus of a variable light chain. Optionally, the variable heavy chain or variable light chain of the agonist GLP-1 peptide and the ActRII antibody are separated by a linker. The agonist GLP-1 peptide can be fused to one or both variable chains of the antibody. If the agonist GLP-1 peptide is fused to both variable chains of the antibody, then two agonist GLP-1 peptides are fused to the variable heavy chain or two agonist GLP-1 peptides are fused to the variable light chain.

[0187] Therefore, in some embodiments, this disclosure relates to an ActRII receptor antibody, wherein the ActRII receptor antibody is fused to an agonist GLP-1 peptide. In some embodiments, this disclosure relates to an ActRII receptor antibody, wherein the ActRII receptor antibody is genetically fused to an agonist GLP-1 peptide.

[0188] In some embodiments, this disclosure relates to an ActRII receptor antibody, wherein the ActRII receptor antibody is fused to an agonist GLP-1 peptide, and wherein the agonist GLP-1 peptide is fused to the N-terminus of a variable heavy chain of the ActRII receptor antibody. In some embodiments, this disclosure relates to an ActRII receptor antibody, wherein the ActRII receptor antibody is fused to an agonist GLP-1 peptide, and wherein the agonist GLP-1 peptide is fused to the N-terminus of a variable light chain of the ActRII receptor antibody.

[0189] In some embodiments, this disclosure relates to an ActRII receptor antibody fused to an agonist GLP-1 peptide, wherein the agonist GLP-1 peptide is fused to the N-terminus of a variable heavy chain of the ActRII receptor antibody, and wherein the agonist GLP-1 peptide and the variable heavy chain are separated by a linker. In some embodiments, this disclosure relates to an ActRII receptor antibody fused to an agonist GLP-1 peptide, wherein the agonist GLP-1 peptide is fused to the N-terminus of a variable light chain of the ActRII receptor antibody, and wherein the agonist GLP-1 peptide and the variable light chain are separated by a linker. Preferably, the linker is a glycine-serine-rich linker. More preferably, the linker is a (G4S)2 linker. Also preferably, the linker comprises or is composed of the amino acid sequence of SEQ ID No. 48.

[0190] In some embodiments, this disclosure relates to an ActRII receptor antibody, wherein the ActRII receptor antibody is fused to one or both of the variable chains of the ActRII receptor antibody.

[0191] In some embodiments, this disclosure relates to an ActRII receptor antibody, wherein the ActRII receptor antibody is fused to a variable chain of the ActRII receptor antibody. In some embodiments, the variable chain is a variable heavy chain. In other embodiments, the variable chain is a variable light chain.

[0192] In some embodiments, this disclosure relates to an ActRII receptor antibody, wherein the ActRII receptor antibody is fused to two variable heavy chains of an ActRII receptor antibody. In other embodiments, this disclosure relates to an ActRII receptor antibody, wherein the ActRII receptor antibody is fused to two variable light chains of an ActRII receptor antibody.

[0193] treat

[0194] The terms “treatment” and the like are used herein to generally mean obtaining the desired pharmacological and / or physiological effect with a therapeutic agent. This effect may be preventative, such as reducing the likelihood of the disease or its symptoms occurring in a subject, in relation to complete or partial prevention of the disease or its symptoms, and / or therapeutic, in relation to complete or partial relief of symptoms, or a partial or complete cure of the disease and / or an adverse effect attributable to the disease. As used herein, “treatment” covers any treatment of a disease in mammals and includes: (a) preventing the disease from occurring in subjects who may be susceptible to the disease but have not yet been diagnosed with it; (b) suppressing or slowing the onset or development of the disease; or (c) alleviating the disease, such as by the disappearance of symptoms that cause or are associated with the disease. The therapeutic agent may be administered before, during, or after the onset of the disease. Of particular interest may be the treatment of an ongoing disease in which the treatment stabilizes or reduces undesirable clinical symptoms in a patient. In some embodiments, the treatment is administered before complete loss of function of the affected tissue. In some embodiments, the therapeutic agent is administered during the symptomatic phase of the disease, and in some embodiments, after the symptomatic phase of the disease.

[0195] The terms “individual,” “subject,” and “patient” are used interchangeably herein and refer to any subject in need of treatment or therapy. A subject may be a mammalian subject. Mammal subjects include, for example, humans, non-human primates, rodents (e.g., rats, mice), rabbits (e.g., rabbits), ungulates (e.g., cattle, sheep, pigs, horses, goats, etc.). In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate, such as a cynomolgus monkey. In some embodiments, the subject is a companion animal (e.g., a cat, a dog).

[0196] The terms “obesity-related comorbidities,” “obesity-related symptoms,” and “obesity-related conditions” are used interchangeably and refer to conditions dependent on the subject’s obesity-related health status. In some implementations, obesity-related comorbidities or conditions increase the subject’s risk of death. Obesity-related comorbidities include, but are not limited to: high blood pressure / hypertension, high LDL cholesterol, low HDL cholesterol, high triglyceride levels (dyslipidemia), type 2 diabetes, coronary artery disease, stroke, gallbladder disease, osteoarthritis, sleep apnea, breathing problems, cancer, gastroesophageal reflux disease, severe COVID-19, overall mortality, low quality of life, mental illnesses (such as clinical depression, anxiety, and other mental disorders), and physical pain and physical dysfunction.

[0197] As used in this article, “metabolic disorders” refers to disorders that affect the metabolism of mammals, including but not limited to: obesity, diabetes (type I and type II), metabolic syndrome, antipsychotic drug-induced obesity, glucocorticoid-induced obesity, hypothalamic obesity associated with craniopharyngioma, and monogenic disorders associated with obesity. Monogenic disorders associated with obesity in humans may include, but are not limited to, Bardet-Biedl syndrome, and disorders caused by mutations in one or more of the following genes: ADCY3, ALMS1, ARL6, BBS1, BBS2, BBS4, BBS5, BBS7, BBS9, BBS10, BBS12, BDNF, CCDC28B, CEP290, CREBBP, EP300, GNAS, IER3IP1, MC3R, MKKS, MKS1, MRAP2, NTRK2, PCSK1, PHF6, POMC, SH2B1, SIM1, TMEM67, TRIM32, TTC8, and VPS13B, or combinations thereof. Metabolic disorders may also be associated with complex genetic conditions, such as Prader-Willi syndrome.

[0198] As used in this article, "Body Mass Index" or "BMI" is calculated by dividing body weight in kilograms (kg) by square meters (m²). 2 Height is calculated and rounded to one decimal place. As used in this article, "obesity" in adults is defined as a BMI greater than or equal to 30 kg / m². 2 In human youth, "obesity" is defined as a BMI greater than or equal to the 95th percentile of the age- and sex-specific body mass index in the 2000 CDC growth chart. The term "overweight" is defined as a BMI greater than or equal to 25 and less than 30.

[0199] "Lean body mass" is defined as the total weight of a subject minus the subject's fat mass and bone mass. Lean body mass and fat mass can be measured by, for example, bioelectrical impedance analysis (BIA), magnetic resonance imaging (MRI), or dual X-ray absorptiometry (DXA).

[0200] As used herein, the term "pharmaceutical composition" refers to pharmaceutically active compounds, such as the immunoconjugates of this disclosure, and pharmaceutically acceptable excipients.

[0201] In some embodiments, this disclosure relates to pharmaceutical compositions comprising the immunoconjugates of this disclosure.

[0202] In some embodiments, this disclosure relates to the immunoconjugates or pharmaceutical compositions of this disclosure for use in a medicament. In some embodiments, the use in a medicament is for the treatment of metabolic disorders. In some embodiments, the metabolic disorders are selected from the group consisting of: obesity, diabetes, metabolic syndrome, antipsychotic drug-related obesity, glucocorticoid-induced obesity, hypothalamic obesity associated with craniopharyngioma, Prader-Willi syndrome, and monogenic disorders associated with obesity. In some embodiments, the use is for obesity-related comorbidities, wherein the symptoms are selected from the group consisting of: glucose intolerance, prediabetes, insulin resistance, hypertriglyceridemia, overweight-related physical impairment, osteoporosis, kidney disease, obstructive sleep apnea, sex hormone-induced damage, endocrine-reproductive disorders, osteoarthritis, gastrointestinal cancer, dyslipidemia, hypertension, heart failure, coronary arthritis, stroke, and / or gallstones. Example

[0203] Example 1: Antibody Production

[0204] Several ActRII receptor antibodies were generated. The base of the construct is the antibody bismalumab, which is an antibody containing the variable heavy chain of SEQ ID No. 7 and the variable light chain of SEQ ID No. 8. Various derivatives of bismalumab were used in this study, none of which affected antibody specificity.

[0205] To conjugate GLP-1 peptides with antibodies, cysteine-engineered forms of bimalumab are generated by replacing various amino acids in the Fc region with cysteine ​​residues. Specifically, the following cysteine-engineered variants are produced: E272C, N384C, N434C, N361C, and N389C. The corresponding antibodies have the following full-weight chain sequences:

[0206] Table 2 :

[0207]

[0208]

[0209] In principle, antibodies may contain additional mutations, such as LALA, PA-LALA, or PG-LALA silencing mutations or YTE mutations in the Fc region, to increase the antibody's half-life.

[0210] The antibody disclosed herein was produced by Icosagen (Tartumaa, Estonia) using standard techniques. The binder was purified via HiTrap MabSelect PrismA and preparative SEC-HPLC. QC was performed using capillary electrophoresis on a LabChip GXII, measurement of endotoxin content, and analytical SEC-HPLC. An exemplary capillary electrophoresis gel is shown below. Figure 1 Medium. Endotoxin level less than 0.02 EU / mg. Monomer content >95% after one freeze-thaw cycle.

[0211] Example 2: Generation of agonistic GLP-1 peptides

[0212] The Fmoc strategy was used to generate peptides. All peptides were synthesized and purified using Rink resin as a solid support on state-of-the-art instruments, as exemplified for peptide 1.

[0213] Peptide 1

[0214] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG-K(MiniPEG)AcBr (SEQ ID No. 14)

[0215] General procedure for peptide synthesis :

[0216] 1) Resin Preparation: DMF (20 mL) was added to Rink amide MBHA resin (0.50 mmol, 1.00 equivalent, Sub: 0.5 mmol / g), and the mixture was stirred at 25 °C for 0.5 hours under a N2 atmosphere. The reaction mixture was then filtered off, and the resin was treated with DMF (40.0 mL). 5) Washing.

[0217] 2) Deprotection of Fmoc groups: Add 40.0 mL of DMF containing 20% ​​piperidine to the resin and stir for 15 minutes at 25°C under N2 atmosphere. Then, remove the resin with 40.0 mL of DMF. 5) Wash and filter.

[0218] 3) Coupling: A solution of Fmoc-Lys(Dde)-OH (3.0 equivalents) and DIEA (6.0 equivalents) in DMF (20.0 mL) was added to the resin, followed by the addition of HBTU (2.85 equivalents), and the mixture was stirred at 25°C for 30 minutes under a N2 atmosphere. The resin was then coated with DMF (40.0 mL). 5) Washing. The coupling reaction was monitored by the ninhydrin colorimetric reaction.

[0219] 4) Repeat steps 2 to 3 above for coupling the following amino acids: (2-30).

[0220] Table 3 :

[0221]

[0222] 5) Deprotection of Dde groups: Add DMF (40.0 mL) containing 3% hydrazine hydrate, and stir the resin at 25°C for 30 minutes under N2 atmosphere. Then, remove the DMF from the resin and rinse with 40.0 mL of DMF. 5) Wash and filter.

[0223] 6) Coupling: A solution of Fmoc-PEG4-OH (3.0 equivalents) and DIEA (6.00 equivalents) in DMF (20.0 mL) was added to the resin, followed by the addition of HBTU (2.85 equivalents), and the mixture was stirred at 25°C for 30 minutes under a N2 atmosphere. The resin was then coated with DMF (40.0 mL). 5) Washing. The coupling reaction was monitored by the ninhydrin colorimetric reaction.

[0224] 7) Repeat steps 2 to 3 above for coupling the following amino acids: (32).

[0225] Table 4 :

[0226]

[0227] 8) Deprotection of Fmoc groups: Add DMF (40.0 mL) containing 20% ​​piperidine, and stir the resin at 25°C for 15 minutes under N2 atmosphere. Then, remove the resin with DMF (40.0 mL). 5) Wash and filter.

[0228] 9) Coupling AcBr: A solution of bromoacetic acid (6.0 equivalents) and DIC (6.00 equivalents) in DMF (20.0 mL) was added to the resin and stirred at 25 °C for 30 minutes under N2 atmosphere. The resin was then coated with DMF (40.0 mL). 5) Washing. The coupling reaction was monitored by the ninhydrin colorimetric reaction.

[0229] Peptide cleavage and purification :

[0230] 1) The resin was washed twice with DMF (50 mL), three times with MeOH (50 mL), and then vacuum dried to obtain 5.0 g of peptide resin.

[0231] 2) At 25°C, add 50 mL of the lysis mixture (2.5% H2O / 2.5% Tis / 2.5% MPA / 92.5% TFA) to a flask containing peptide resin with side chain protection, and stir the mixture for 120 minutes.

[0232] 3) After filtration, collect the lysate mixture, precipitate the crude peptide with 500 mL of cold isopropyl ether and centrifuge (3000 rpm, 2 min), and wash twice with 500 mL of isopropyl ether. Vacuum dry the crude peptide for 1 hour.

[0233] 4) The crude peptide (2.0 g) was purified by preparative HPLC (TFA conditions: A: 0.075% TFA / H2O, B: ACN) to obtain the final product (217.8 mg, 9.64% yield, 89.77% purity, TFA) in the form of a white solid, which was confirmed by LCMS.

[0234] Purification conditions

[0235] Table 5 :

[0236]

[0237] Analyze data :

[0238] R t =11.442 minutes, purity: 89.77%, MS calculated value: 4057.32, MS measured value: [M+3H] 3+ =1353.3156

[0239] The following peptides were synthesized accordingly:

[0240] Peptide 2 :

[0241] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG-K(GGGGS)3AcBr (SEQ ID No. 16)

[0242] R t =13.3 minutes, purity: 91.58%, MS calculated value: 4508.6, MS measured value: [M+3H] 3+ =1503.67

[0243] Peptide 3 :

[0244] HAibEGTFTSDVSSYLEGQAA-K(MiniPEG)AcBr-EFIAWLVRGRG (SEQ ID No. 17)

[0245] R t =11.4 minutes, purity: 96.0%, MS calculated value: 4012.29, MS measured value: [M+3H] 3+ =1338.31

[0246] Peptide 4 :

[0247] HAibEGTFTSDVSSYLEGQAA-K(GGGGS)3AcBr-EFIAWLVRGRG (SEQ ID No. 19)

[0248] R t =10.2 minutes, purity: 93.25%, MS calculated value: 4463.57, MS measured value: [M+3H] 3+ =1488.67

[0249] Peptide 5 :

[0250] HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-K(MiniPEG)AcBr (SEQ ID No. 20)

[0251] R t =10.8 minutes, purity: 87.11%, MS calculated value: 4140.47, MS measured value: [M+3H] 3+ =1381.00

[0252] Peptide 6 :

[0253] HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-K(GGGGS)3AcBr (SEQ ID No. 22)

[0254] R t =12.9 minutes, purity: 95.71%, MS calculated value: 4591.74, MS measured value: [M+3H] 3+ =1148.77

[0255] abbreviation :

[0256] Aib: Aminoisobutyric acid

[0257] AcBr: Bromoacetic acid

[0258] MiniPEG: Amino-polyethylene glycol-acid or 1-amino-3,6,9,12-tetraoxapentadecan-15-acid - CAS 581065-95-4

[0259] MBHA resin: 4-methyldiphenylmethylamine hydrochloride

[0260] DDE: 2-Acetyldimethyl ketone

[0261] MPA: 3-Mercaptopropionic acid

[0262] DIC: N,N'-Diisopropylcarbodiimide

[0263] TIS: Triisopropylsilane

[0264] Example 3: Conjugation of peptides with cysteine-engineered antibodies

[0265] The peptide was conjugated to a cysteine-engineered antibody via halogen substitution of the correspondingly modified peptide (i.e., a bromoacetylated peptide). 5 mg of antibody dissolved in histidine / NaCl buffer (5 mg / ml) was buffer-exchanged for an equal volume of reducing buffer (50 mM Na₂SO₄ / 2.0 mM EDTA, pH 7.5) via three cycles (3 × 10 min, 10000 rpm) on an Amicon Ultra-4, Centrifual filter, Ultracel-30K. 100 equivalents of water containing TCEP solution (0.5 M concentration) were added to the final volume, and the reaction vessel was shaken at 20 °C for 22 h. The mixture was desalted into fresh reaction buffer using a Zeba desalting column (MWCO40000, prepared according to the buffer exchange procedure (Thermo Scientific)) using conjugation buffer (centrifugation to remove storage buffer for 2 min, addition of 2 ml reaction buffer, centrifugation for 2 min, repeated twice, centrifugation for 6 min after the third buffer addition – centrifugation speed 700 g). The sample was then added to a desalting column and centrifuged for 4 minutes to collect the desalted sample. The eluent was then transferred to a 2 ml Eppendorf tube and 20 equivalents of PBS (10 mM concentration) containing DHAA solution were added. The solution was gently shaken at RT. After an additional 22 hours, 10 equivalents of BrAc-modified peptide solution (10 mM in water) were added, and the mixture was shaken overnight at RT. Subsequently, the sample was filtered and purified by 3 cycles (3 × 10 min, 10000 rpm) on an Amicon Ultra-4, Centrifual filter, and Ultracel-10K buffer exchanged to PBS buffer. The conjugates obtained in PBS (concentration approximately 5 mg / ml) were then purified by preparative SEC (Superdex 200 Increase, 10 / 300 GL column, PBS as eluent), and the final analysis of the products was performed by HIC chromatography and LCMS after combining the pure fractions. The final concentration was determined by Nanodrop UV. Characterize all immunoconjugates and assess their quality (MS, SDS PAGE, SEC, HIC, heparin-binding column).

[0266] Example 4: ActR2A / B binding and functional activity

[0267] Example 4.1: Affinity of antibodies and conjugates to ActRIIA and ActRIIB

[0268] The affinity interaction profile of immunoglobulins was determined using an immunoassay called Meso Scale Discovery (MSD) solution equilibration titration (MSD-SET). In this assay, an antigen (ActRIIA or ActRIIB) was coated onto an MSD 96-well detection plate. The SET plate was prepared by mixing a constant concentration of immunoglobulin with the titrated antigen and incubating overnight at 4°C to reach equilibration. After blocking the MSD plate, 50 μl of sample solution containing the immunoglobulin-antigen mixture (SET plate) was transferred to the antigen-coated detection MSD plate. These samples were then incubated for 20 minutes to allow binding of any free antibodies without significantly altering the equilibration. To detect the antibodies bound on the plate, a detection antibody (PN: R32AJ-5) labeled with anti-human (goat) Sulfo-TAG was added. Electrochemiluminescence (ECL) readings were then detected by adding read buffer and measuring ECL using an MSD instrument (Sector S 600MM). Data analysis was performed on GraphPad Prism using the IgG equation from Journal of Biomolecular Screening 2015, Vol. 20(10), pp. 1256-1267. Various bimalumab-derived antibodies containing engineered cysteine ​​at position 272 (E272C) were tested in this experiment. Antibodies were conjugated to peptides 5 and 6, and binding to receptors ActRIIA and ActRIIB was compared to unconjugated cysteine-engineered antibodies.

[0269] The results are shown in Table 6 and Figure 2 It can be demonstrated that the binding of antibodies to ActRIIA and ActRIIB is largely unaffected by the conjugated peptides.

[0270] Table 6 :

[0271]

[0272] Example 4.2: Antagonistic activity of ActRIIA and B reporter genes in cell assays

[0273] In addition, antagonistic activity was assessed in ActRIIA and B reporter gene assays. HEK293 cells (BPS BioScience) reporting the SBE gene of the TGFB / SMAD signaling pathway were washed with PBS, separated using 0.05% trypsin, and seeded at 35,000 cells / well in 96-well white plates containing 100 µL of MEM medium (10% FBS, 1% NEAA, 1 mM sodium pyruvate, 1% penicillin / streptomycin). The next day, the medium was replaced with 90 µL of assay medium containing diluted antibody of interest (10 µg / mL–0.00015 µg / mL) or a control (MEM medium, 0.5% FBS, 1% NEAA, 1 mM sodium pyruvate, 1% penicillin / streptomycin). Cells were incubated at 37°C and 5% CO2 for 4 hours. Subsequently, 10 µL of 10 ng / mL activin (catalog number 120-14E, Peprotech) or 500 ng / mL myostatin (catalog number 120-00, Peprotech) was added to the cells and the cells were incubated overnight. The next day, 100 µL of ONE-Step luciferase assay kit (BPSBioscience) was added to the cell solution (both equilibrated to room temperature) and incubated on a plate shaker for 15 minutes, followed by measurement of luminescence using an Envision plate reader.

[0274] This experiment tested various bimalumab-derived antibodies containing an engineered cysteine ​​residue (E272C) at position 272. As shown below, the antibodies were conjugated with various peptides, and... ActRIIA and B reported gene cell assays The activity of the antibody was compared with that of an unconjugated cysteine-engineered antibody.

[0275] The results are shown in Table 7 below. Figure 3 In this study, it was demonstrated that all tested conjugates remained active when measured against activin A or myostatin.

[0276] Table 7 :

[0277]

[0278] Example 4.3: Functional activity in GLP-1 cAMP assay

[0279] Prior to assay setup, the assay buffer was prepared as a 1x stimulation buffer (Cisbio #62AM4PEJ) containing 500 μM IBMX (Sigma, #I5879). The compound was serially diluted 4-fold in the assay buffer for 10 spots using a Bravo V11 instrument (Agilent), and 5 μL of the compound was transferred to an OptiPlate-384 (PerkinElmer, #6007290) to achieve a maximum concentration of 20 nM in the assay plate. The plate was centrifuged at 1000 rpm for 5 seconds.

[0280] HEK293 cells expressing GLP-1R (constructed by WuXi Apptech) were isolated using 0.25% trypsin at 37°C for several minutes. Upon cell isolation, the cell suspension was diluted in 10 mL of HBSS (Invitrogen, #14025) and transferred to a 15 mL tube. The cells were centrifuged at 1000 rpm for 5 minutes at room temperature. The supernatant was removed, and the cell pellet was resuspended in 10 mL of HBSS. The cells were resuspended in assay buffer to a final concentration of 0.2 Mio cells / mL. 10 μL of this cell solution was transferred to an Optiplate-384.

[0281] Incubate the plate at 23°C for 30 minutes, then add 10 μL of cAMP working assay solution (38 parts cAMP lysis buffer, 1 part cAMP-D2, and 1 part anti-cAMP cavitation compound reagent), as described by the supplier (CisBio, #62AM4PEJ). Cover the plate with a TopSeal-A membrane and incubate at room temperature for 60 minutes. Remove the TopSeal-A membrane and analyze the signal using EnVision2015 (PerkinElmer).

[0282] In this assay, peptides 1-6 and their corresponding E272C conjugates were tested. The results are shown in Table 8 below. Figure 4 It can be demonstrated that the conjugate retains high activity and full function in cAMP assays.

[0283] Table 8 :

[0284]

[0285] Example 4.4: Conclusions of in vitro studies

[0286] Surprisingly, the conjugation of the agonist GLP-1 peptide to the ActRII antibody essentially does not affect the activity of either part of the molecule. Specifically, the conjugated agonist GLP-1 peptide does not affect the binding and activity of the ActRII antibody and is completely converted into functional ActRII activity. Similarly, when conjugated to the ActRII antibody, the activity of the agonist GLP-1 peptide is essentially equivalent to that of the isolated (unconjugated) agonist GLP-1 peptide. Despite the conjugation of corresponding other parts of the molecule, both parts of the molecule (i.e., the agonist GLP-1 peptide and the ActRII antibody) retain full activity.

[0287] This discovery offers significant advantages for the development of corresponding therapeutic agents. Compared to combination therapies consisting of two separate components, conjugated products require only a single formulation and a single route of administration, and avoid the need to identify compatible dosing regimens for the two individual molecules.

[0288] Example 5: In vivo efficacy study

[0289] The in vitro studies described above were experimentally validated by corresponding in vivo studies. Diet-induced obese (DIO) mice were used as a model to measure weight loss and improvement in body composition after treatment with the anti-ActRIIA / B-GLP-1 conjugate.

[0290] Prior to study recruitment, male C57BL / 6JRj mice obtained from Janvier Labs were subjected to a 60% high-fat diet (HFD) for 21 weeks, starting at 5 weeks of age. Animals were randomized to treatment groups based on body weight and body composition measurements prior to administration. The conjugate (or its corresponding mediator, 0.9% NaCl) was administered twice weekly via intraperitoneal (IP) injection for 2 weeks, concurrently with semaglutide (or its corresponding mediator, phosphate-buffered saline containing 0.1% bovine serum albumin) at 10 nmol / kg daily via subcutaneous (SC) injection for 17 days. Treatment groups consisted of mediator-only (IP and SC routes), semaglutide SC (+IP mediator), E272C mAb-peptide 5 (10 mg / kg), and E272C mAb-peptide 6 (2.5 mg / kg or 10 mg / kg) + SC mediator. HFD continued during treatment. Animal body weight was recorded daily. Food intake was measured daily during the first week of treatment and every two weeks thereafter. Body composition (fat and lean body mass) was analyzed weekly using magnetic resonance imaging (MRI).

[0291] The therapeutic activity of the conjugates disclosed herein can be confirmed in in vivo studies. Importantly, the conjugates of this disclosure result in a significant reduction in fat body mass without loss of lean (muscle) mass, whereas the weight loss of smegglutinin can be attributed to the loss of both fat mass and significant lean (muscle) body mass. Lean body mass is completely preserved after treatment with the conjugates.

[0292] Example 6: Fusion Antibody

[0293] In this experiment, it was tested whether the agonist GLP-1 peptide could also be linked to the ActRII antibody via genetic fusion. To this end, the GLP-1 peptide was fused to the N-terminal gene of the variable heavy chain of the ActRII antibody.

[0294] Two different agonistic GLP-1 peptides were used, both with three amino acid substitutions compared to native GLP-1. The GLP-1 peptides are separated from the variable heavy chain by short, glycine-serine-rich linkers. The amino acid sequences of the tested fusion constructs are shown in the table below.

[0295] Table 9 (Underlined: GLP-1 agonists; Italic: linkers; Bold: initiation of variable heavy chains) :

[0296]

[0297] In some fusion constructs, GLP-1 is fused to the N-terminus of two variable heavy chains (double fusion), while in others, GLP-1 is fused to the N-terminus of only one variable heavy chain (single fusion). Unfused control antibodies and semaglutide were used as controls. The tested constructs are depicted on... Figure 6 The measurement was performed as described in Example 4 above.

[0298] The table below shows the EC50 determined using activin A in the ActRIIA / B-dependent SMAD-activated RGA assay.

[0299] Table 10 :

[0300]

[0301] In this assay, the monofusion constructs showed comparable efficiency to the unfused control molecule, while the difusions showed a slightly higher EC50.

[0302] The table below shows the EC50 determined in the hGLP-1R-dependent cAMP / PKA-activated RGA assay.

[0303] Table 11 :

[0304]

[0305] In this assay, both the single-fusion and dual-fusion constructs were at least as effective as smegglutinin, with the dual-fusion construct showing higher efficiency than the single-fusion construct.

Claims

1. An immunoconjugate comprising an ActRII receptor antibody and an agonist GLP-1 peptide.

2. The immunoconjugate according to claim 1, wherein the ActRII receptor antibody is specific for the polypeptide of SEQ ID No.

12.

3. The immunoconjugate according to claim 1 or 2, wherein the ActRII receptor antibody cross-reacts with the polypeptide of SEQ ID No.

11.

4. The immunoconjugate according to any one of the preceding claims, wherein the ActRII receptor antibody binds to the same epitope as an antibody comprising a variable heavy chain and a variable light chain, the variable heavy chain comprising the CDR amino acid sequences of SEQ ID NO: 1 (CDRH1), SEQ ID NO: 2 (CDRH2), and SEQ ID NO: 3 (CDRH3); and the variable light chain comprising the CDR amino acid sequences of SEQ ID NO: 4 (CDRL1), SEQ ID NO: 5 (CDRL2), and SEQ ID NO: 6 (CDRL3).

5. The immunoconjugate according to any one of the preceding claims, wherein the agonist GLP-1 peptide is selected from: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG-K(MiniPEGAcBr) (SEQ ID No. 14), HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG-KKgGluC16(MiniPEGAcBr) (SEQ ID No. 15), HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG-K(GGGGS)3AcBr (SEQ ID No. 16), HAibEGTFTSDVSSYLEGQAA-K(MiniPEGAcBr)-EFIAWLVRGRG (SEQ ID No. 17), HAibEGTFTSDVSSYLEGQAA-KKgGluC16(MiniPEGAcBr)-EFIAWLVRGRG (SEQ ID No. 18), HAibEGTFTSDVSSYLEGQAA-K(GGGGS)3AcBr-EFIAWLVRGRG (SEQ ID No. 19), HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-K(MiniPEGAcBr) (SEQ ID No. 20), HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-KKgGluC16(MiniPEGAcBr) (SEQ ID No. 21), and HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-K(GGGGS)3AcBr (SEQ ID No. 22).

6. The immunoconjugate according to any one of claims 1 to 4, wherein the agonist GLP-1 peptide is a dual or triple GLP-1 agonist.

7. The immunoconjugate according to claim 6, wherein the agonist GLP-1 peptide is selected from: YAibEGTFTSDYSIYLDKQAAAibEFVNWLLAGGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ ID No. 23), YAibEGTFTSDYSIYLDKQAAAibEFV-K(MiniPEGAcBr)-WLLAGGGPSSGAPPPSK-NH2 (SEQ IDNo. ​​24), YAibEGTFTSDYSIYLDKQAAAibEFVNWLLAG-K(MiniPEGAcBr)-NH2 (SEQ ID No. 25), YAibEGTFTSDYSIYLDKQAAAibEFV-K(MiniPEGAcBr)-WLLAG-NH2 (SEQ ID No. 26), Ac-hAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLbAlaGGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ ID No. 27), Ac-hAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLbAlaG-K(MiniPEGAcBr)-NH2 (SEQ ID No. 28), YAibAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLbAlaGGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ ID No. 29), YAibQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLbAlaG-K(MiniPEGAcBr)-NH2 (SEQ ID No. 30), Ac-hAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLisoAspSarGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ ID No. 31), Ac-hAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLisoAspSar-K(MiniPEGAcBr)-NH2 (SEQID No. 32), YAibAQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLisoAspSarGPSSGAPPPSK(MiniPEGAcBr)-NH2 (SEQ ID No. 33), YAibQGTFTDSK(gGluC16)SKYLDERAAQDFVQWLLisoAspSar-K(MiniPEGAcBr)-NH2 (SEQ ID No. 34), Ac-hAQGTFTSD-K(MiniPEGAcBr)-SKYLDERAAQDFVQWLLEGPGPSSGAPPPS-NH2 (SEQ ID No. 35), Ac-hAQGTFTSDKSKYLDERAAQDFVQWLLEGGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ ID No. 36), Ac-hAQGTFTSD-K(MiniPEGAcBr)-SKYLDERAAQDFVQWLLEGGPSSGAPPPS-K(gGluC16)-NH2 (SEQ ID No. 37), and Ac-hAQGTFTSD-K(gGluC16)-SKYLDERAAQDFVQWLLEGGPSSGAPPPS-K(MiniPEGAcBr)-NH2 (SEQ ID No. 38).

8. The immunoconjugate according to any one of claims 2 to 6, wherein the agonist GLP-1 peptide is conjugated to the ActRII receptor antibody via cysteine ​​engineering.

9. The immunoconjugate of claim 7, wherein the agonist GLP-1 peptide is conjugated to the ActRII receptor antibody at positions 272, 361, 384, 389, or 434 of the ActRII receptor antibody.

10. The immunoconjugate according to any one of the preceding claims, wherein the ActRII receptor antibody has a silent mutation.

11. A pharmaceutical composition comprising an immunoconjugate according to any one of the preceding claims.

12. The immunoconjugate according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11, wherein the immunoconjugate or pharmaceutical composition is used in a medicament.

13. The immunoconjugate or pharmaceutical composition for use according to claim 12, wherein the use in the pharmaceutical is for the treatment of metabolic disorders.

14. The immunoconjugate or pharmaceutical composition for use according to claim 13, wherein the metabolic disorder is selected from the group consisting of: obesity, diabetes, metabolic syndrome, antipsychotic drug-related obesity, glucocorticoid-induced obesity, hypothalamic obesity associated with craniopharyngioma, Prader-Willi syndrome, and monogenic disorders associated with obesity.

15. The immunoconjugate or pharmaceutical composition of claim 14, wherein the treatment is for obesity-related comorbidities, wherein the symptoms are selected from the group consisting of: glucose intolerance, prediabetes, insulin resistance, hypertriglyceridemia, overweight-related physical damage, osteoporosis, kidney disease, obstructive sleep apnea, sex hormone damage, endocrine and reproductive disorders, osteoarthritis, gastrointestinal cancer, dyslipidemia, hypertension, heart failure, coronary arthritis, stroke, and / or gallstones.

16. An ActRII receptor antibody, wherein the ActRII receptor antibody is fused to an agonistic GLP-1 peptide.

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