Methods and systems for enhanced multispecific metabolic and cellular targeting

Abstract

Materials, methods, and systems are provided for proteins comprising a muscle fortifying agent and metabolic modulatory component. The muscle fortifying agent is, for example, a myostatin modulator or the like, an activin type II receptor modulator or the like, or any and each combination thereof. The metabolic component may be an anti-GIPR antibody or fragment thereof and a metabolic modulator. Exemplary metabolic modulators include GLP-1 receptor agonist. In one aspect, the present invention teaches and describes materials, methods, and systems of metabolic modulation, including modulatory agents. For example, the improved materials, methods, and systems of the present invention include novel agents that regulate metabolism in a host. Included are molecules that specifically bind to one or more than one molecule or target, including multispecific molecules, conjugates of a muscle fortifying agent, and for example an additional active metabolic component directly connected or joined via a linker. In particular, the invention includes conjugates or fusions or the like comprising a muscle fortifying agent in combination with a metabolic modulator, including for example a glucagon-like peptide-1 (GLP-1) receptor binding molecule, such as an agonist, and / or an antagonist of the glucose-dependent insulinotropic peptide receptor (GIPR), etc.; and includes nucleic acids and expression vectors encoding the conjugates, recombinant cells thereof, and compositions comprising the conjugates suitable for development and use in hosts. Materials and methods of producing the modulators and various uses are provided.

Description

METHODS AND SYSTEMS FOR ENHANCED MULTISPECIFIC METABOLIC AND CELLULAR TARGETINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of United States Provisional Patent Application No. 63 / 715,389, filed November 1, 2024 and United States Provisional Patent Application No. 63 / 715,396, filed November 1, 2024, the disclosures of which are incorporated herein by reference in their entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which is being submitted herewith electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 23, 2025, is named JBI6958WOPCTl_SL.xml and is 285,314 bytes in size.FIELD

[0003] The present invention teaches and describes materials, methods, and systems of metabolic modulation, including modulatory agents. For example, the improved materials, methods, and systems of the present invention include novel agents that regulate metabolism in a host. Included are molecules that specifically bind to one or more than one molecule or target, including multispecific molecules, conjugates of a muscle fortifying agent, and for example an additional active metabolic component directly connected or joined via a linker. In particular, the invention includes conjugates or fusions or the like comprising a muscle fortifying agent in combination with a metabolic modulator, including for example a glucagon-like peptide-1 (GLP-1) receptor binding molecule, such as an agonist, and / or an antagonist of the glucose-dependent insulinotropic peptide receptor (GIPR), etc.; and includes nucleic acids and expression vectors encoding the conjugates, recombinant cells thereof, and compositions comprising the conjugates suitable for development and use in hosts. Materials and methods of producing the modulators and various uses are provided.BACKGROUND

[0004] Host metabolism is complex. Metabolic modulation, including limited use of metabolic modulatory agents, has been reported. For example, glucagon-like peptide-1 (GLP- 1) receptor agonists have been explored, yielding varying results. See, for example, Holst, J. J.GLP-1 physiology in obesity and development of incretin-based drugs for chronic weight management. Nat Metab, 2024, 6, 1866-1885.SUMMARY

[0005] The inventors, understanding the unique complexities and challenges of metabolism and metabolic modulation in hosts, and use of metabolic modulatory agents appreciated such challenges and address them herein. Against this backdrop, the present invention addresses unmet needs in the art for, inter alia, novel metabolic modulation achieving enhanced effects, including superior multi-specific approaches and targeting, to improve health and reduce and or eliminate undesired consequences in hosts, tissues, and cells. For example, the present invention addresses the need for more effective solutions to the unwanted loss of muscle and musculature (i.e., providing muscle fortifying agents) associated with the modulation of metabolism (e.g., design and use of GLP-1 receptor agonists, GIPR antagonists, etc.).

[0006] Accordingly, provided are proteins comprising metabolic modulatory agents or metabolic modulatory components. Also included is (i) a muscle fortifying agent and (ii) a metabolic modulatory component. The muscle fortifying agent may be for example a myostatin modulator, an activin type II receptor (i.e., ActRIIA or ActRIIB) modulator, or a combination thereof. The myostatin modulators suitable for use in the present invention can vary, and for example may be a molecule that blocks binding of myostatin to its receptor, inhibits myostatin activity, or a combination thereof. In addition to acting as muscle fortifying agents, the myostatin modulators suitable for use in the present invention may also stabilize or increase bone density.

[0007] In some embodiments, the inhibition of myostatin activity by the myostatin modulator results in an increase in muscle mass, retention of muscle mass or a reduction in loss of muscle mass, an increase in bone density, retention of bone density or a reduction in loss of bone density, or a combination of both an increase, retention or stabilization of muscle mass and bone density. In some embodiments, the activin type II receptor modulator comprises a molecule that blocks binding of ligands such as myostatin to the activin type II receptor. In some embodiments, inhibition of binding to the activin type II receptor results in an increase in muscle mass or a reduction in loss of muscle mass, an increase in bone density or a reduction in loss of bone density, or a combination of both an increase or stabilization of muscle mass and bone density.

[0008] In some embodiments, the muscle fortifying agent is an ActRIIA or ActRIIB modulator, such as an antibody against the ligand binding domain of ActRIIA or ActRIIB, i.e., anti-ActRIIA or anti-ActRIIB antibody, or a bispecific antibody targeted against the ligand binding domains of each of ActRIIA and ActRIIB.

[0009] In some embodiments, the muscle fortifying agent is a myostatin modulator comprising a binding agent that binds to myostatin, a myostatin antagonist, or a combination thereof. The binding agent may comprise a receptor, an antibody, or a fragment thereof. In some embodiments, the myostatin modulator is an anti-myostatin antibody (see Table 4, which lists SEQ ID NOs: 111-158, STS, SEQ ID NOs: 160-164, SAS, and SEQ ID NOs: 166-178).

[0010] In some embodiments, the anti-myostatin antibody comprises a light chain (LC) comprising light chain complementarity determining regions (LC-CDRs) 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 122, SEQ ID NO: 123, and SEQ ID NO: 124, respectively, and a heavy chain (HC) comprising heavy chain complementarity determining regions (HC-CDRs) 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 119, SEQ ID NO: 120, and SEQ ID NO: 121, respectively.

[0011] In some embodiments, the anti-myostatin antibody comprises a LC comprising LC- CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 128, SEQ ID NO: 129, and SEQ ID NO: 130, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 125, SEQ ID NO: 126, and SEQ ID NO: 127, respectively.

[0012] In some embodiments, the anti-myostatin antibody comprises a LC comprising LC- CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 134, SEQ ID NO: 135, and SEQ ID NO: 136, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 131, SEQ ID NO: 132, and SEQ ID NO: 133, respectively.

[0013] In some embodiments, the anti-myostatin antibody comprises a LC comprising LC- CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 140, SEQ ID NO: 141, and SEQ ID NO: 142, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 137, SEQ ID NO: 138, and SEQ ID NO: 139, respectively.

[0014] In some embodiments, the anti-myostatin antibody comprises a LC comprising LC- CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 146, SEQ ID NO: 147, and SEQ ID NO: 148, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 143, SEQ ID NO: 144, and SEQ ID NO: 145, respectively.

[0015] In some embodiments, the anti-myostatin antibody comprises a LC comprising LC- CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 152, SEQ ID NO: 153, and SEQ ID NO: 154, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 149, SEQ ID NO: 150, and SEQ ID NO: 151, respectively.

[0016] In some embodiments, the anti-myostatin antibody comprises a LC comprising LC- CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 158, STS, and SEQ ID NO: 160, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 155, SEQ ID NO: 156, and SEQ ID NO: 157, respectively.

[0017] In some embodiments, the anti-myostatin antibody comprises a LC comprising LC- CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 164, SAS, and SEQ ID NO: 166, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 161, SEQ ID NO: 162, and SEQ ID NO: 163, respectively.

[0018] In some embodiments, the anti-myostatin antibody comprises a LC comprising LC- CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 170, SEQ ID NO: 171, and SEQ ID NO: 172, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 167, SEQ ID NO: 168, and SEQ ID NO: 169, respectively.

[0019] In some embodiments, the anti-myostatin antibody comprises a LC comprising LC- CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 173, SEQ ID NO: 174, and SEQ ID NO: 175, respectively.

[0020] In some embodiments, the anti-myostatin antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 113 and a heavy chainvariable region comprising the amino acid sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 112. In some embodiments, the anti-myostatin antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 117 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 116.

[0021] In some embodiments, the anti-myostatin antibody includes intact antibodies or antibody fragments such as Fv fragments, single chain scFv fragments (scFv), single chain disulfide bond stabilized scFv fragments or stapled scFv fragment (spFv), Fab, F(ab)2, or single chain antibodies.

[0022] In some embodiments, the anti-myostatin antibody comprises a stapled scFv (spFv) comprising the amino acid sequence set forth in SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO: 118.

[0023] In some embodiments, the metabolic modulatory component comprises (a) an anti- GIPR antibody or fragment thereof (see Table 3, which lists SEQ ID NOs: 37-90, YAS, SEQ ID NOs: 92-96, YAS, and SEQ ID NOs: 98-110) and (b) a metabolic modulator.

[0024] In some embodiments, the anti-GIPR antibody comprises a LC comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively.

[0025] In some embodiments, the anti-GIPR antibody comprises a LC comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59, respectively.

[0026] In some embodiments, the anti-GIPR antibody comprises a LC comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively.

[0027] In some embodiments, the anti-GIPR antibody comprises a LC comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, respectively, and a HC comprising HC-CDRs 1 through 3comprising the amino acid sequences set forth in SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively.

[0028] In some embodiments, the anti-GIPR antibody comprises a LC comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively.

[0029] In some embodiments, the anti-GIPR antibody comprises a LC comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: 86, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 81, SEQ ID NO: 82, and SEQ ID NO: 83, respectively.

[0030] Iln some embodiments, the anti-GIPR antibody comprises a LC comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 90, YAS, and SEQ ID NO: 92, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89, respectively.

[0031] In some embodiments, the anti-GIPR antibody comprises a LC comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 96, YAS, and SEQ ID NO: 98, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 93, SEQ ID NO: 94, and SEQ ID NO: 95, respectively.

[0032] In some embodiments, the anti-GIPR antibody comprises a LC comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 102, SEQ ID NO: 103, and SEQ ID NO: 104, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101, respectively.

[0033] In some embodiments, the anti-GIPR antibody comprises a LC comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107, respectively.

[0034] In some embodiments, the anti-GIPR antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 41 or SEQ ID NO: 42, and a heavy chain variable region comprising the amino acid sequence set forth in any one of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40.

[0035] In some embodiments, the anti-GIPR antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 49 or SEQ ID NO: 50, and a heavy chain variable region comprising the amino acid sequence set forth in any one of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48.

[0036] In some embodiments, the anti-GIPR antibody includes intact antibodies or antibody fragments such as Fv fragments, single chain scFv fragments (scFv), single chain disulfide bond stabilized scFv fragments or stapled scFv fragment (spFv), Fab, F(ab)2, or single chain antibodies.

[0037] In some embodiments, any of the antibodies provided herein may be engineered as stapled scFv (spFv) antibodies, such as comprising a light chain variable region linked via a linker to a heavy chain variable region, wherein the linker may comprise the amino acid sequence set forth in SEQ ID NO: 179.

[0038] In some embodiments, any of the antibodies provided herein may be engineered as sweeping antibodies.

[0039] In some embodiments, any of the antibodies provided herein may be engineered to bind neonatal Fc receptor (FcRn) with greater affinity at pH 7.0 to 7.8, such as 7.4. For example, the Fc portion of the sweeping antibody may bind to FcRn at a pH of 7.4 with a KD ranging from 10-3M to 10-9M, such as 10-3M to 10-8M, or 10-3M to 10-7M, or 10-3M to 10-6M. In some embodiments, any of the antibodies provided herein may be engineered to include a mutation in a CDR that decreases binding to an antigen at low pH, such as by inclusion of histidine residues in the binding interface. Thus, in some embodiments, the binding interface of the antibody may be engineered to include is mutation to a histidine. In some embodiments, at least 1, 2, 3, 4, or 5 amino acids are mutated to histidine. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are mutated to histidine.

[0040] In some embodiments, the amino acid residues AA of a sweeping antibody Fc region are required for binding to FcRn. In some embodiments, the amino acid residues AA of a sweeping antibody Fc region affect binding to FcRn.

[0041] In some embodiments, the affinity of sweeping antibodies to FcRn is increased to extend their pharmacokinetic (PK) properties as compared to their conventional counterparts. For example, in some embodiments, sweeping antibodies elicit less adverse reactions due to their efficacy at lower doses. In some embodiments, sweeping antibodies are administered less frequently. In some embodiments, transcytosis of sweeping antibodies to certain tissue types is increased.

[0042] In some embodiments, the metabolic modulator comprises an antibody, a peptide, a fusion protein, a modified peptide, or a compound. The metabolic modulator may comprise a molecule having the activity of a glucagon-like peptide-1 (GLP-1) receptor agonist, a glucose-dependent insulinotropic peptide receptor (GIPR) antagonist (also known as a gastric inhibitory peptide receptor), or a combination thereof. The metabolic modulator may comprise a GLP-1 receptor (GLP-1R) agonist, a GIPR antagonist, or a combination thereof. The metabolic modulator may comprise a peptide comprising the amino acid sequence as set forth in any one of SEQ ID NOs: 1-14, 17 or 190, or a variant thereof. The metabolic modulator may comprise a peptide comprising the amino acid sequence as set forth in SEQ ID NOs: 18 or 19, or a variant thereof. Exemplary variants comprise at least one amino acid substitution. The metabolic modulator may comprise a peptide comprising the amino acid sequence as set forth in SEQ ID NOS: 15 or 16 comprising at least one amino acid substitution (see Table 1, which lists SEQ ID NOs: 1-19 and 190).

[0043] In some embodiments, the anti-GIPR antibody or fragment thereof and the metabolic modulator are connected directly, such as in a fusion protein. In some embodiments, the protein comprises a linker, wherein the anti-GIPR antibody or fragment thereof and the metabolic modulator are connected via the linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a synthetic linker, for example a nonpeptide organic linker. In some embodiments, the linker is a combination of a peptide and a synthetic linker. In some embodiments, the linker may be a peptide linker, such as any of the peptides comprising the amino acid sequence as set forth in SEQ ID NOs: 20-36 (see Table 2).

[0044] In some embodiments, the muscle fortifying agent and the metabolic component are connected directly, such as in a fusion protein. In other embodiments, the muscle fortifying agent and the metabolic component are connected directly, connected via a peptide linker, connected via a synthetic linker, for example a non-peptide organic linker, or any combination thereof. In some embodiments, the protein comprises a linker, wherein themuscle fortifying agent and the metabolic component are connected via the linker. In some embodiments, the linker is a synthetic linker, for example a non-peptide organic linker. In some embodiments, the linker is a combination of a peptide and a synthetic linker, for example a non-peptide organic linker. In some embodiments, the linker may be a peptide linker, such as any of the peptides comprising the amino acid sequence as set forth in SEQ ID NOs: 20-36 (see Table 2).

[0045] Also provided herein is a multi-specific protein comprising (i) a muscle fortifying agent means and (ii) a metabolic modulatory component means.

[0046] The muscle fortifying agent means may be a myostatin modulator and the like, an activin type II receptor modulator and the like, or an agent having the activity of the myostatin modulator or activin type II receptor modulator, or any combination thereof. The myostatin modulator may block, eliminate, or significantly reduce binding of myostatin to its receptor, inhibit myostatin activity, or any combination thereof.

[0047] The muscle fortifying agent means may comprise a binding agent that binds myostatin, a myostatin antagonist, or a combination thereof. The binding agent may comprise a receptor, an antibody, or a fragment thereof, such as any of the antibodies or fragments thereof disclosed herein.

[0048] The metabolic component means may comprise (a) an anti-GIPR binding means or fragment thereof, and (b) a metabolic modulator means. The metabolic component means may comprise (a) any of the anti-GIPR binding agents or fragments thereof disclosed herein, and (b) any of the metabolic modulators disclosed herein.

[0049] The anti-GIPR binding means or fragment thereof and the metabolic modulator means may be connected directly, such as via a linker means. For example, the anti-GIPR binding means or fragment thereof and the metabolic modulator means may be connected via the linker means. The linker means is a peptide linker. The linker means may be a synthetic linker.

[0050] The metabolic modulator means may comprise an antibody, a peptide, a fusion protein, a modified peptide, or a compound.

[0051] Also provided are nucleic acids encoding any of the proteins disclosed herein, or a portion thereof, optionally operably linked to a heterologous promoter.

[0052] Also provided are compositions, such as those suitable for use in pharmaceutical compositions comprising any of the proteins disclosed herein, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0053] Further provided is a method of treating undesired outcomes of metabolic modulation, including treating muscle wasting, a metabolic disorder, or a combination thereof, in a subject. The method generally comprises administering to the subject any of the proteins disclosed herein, or a pharmaceutical composition thereof, wherein the subject is optionally a mammal, for example a human, a simian, a mouse, a rat, a cow, or a llama.

[0054] Further yet provided is a system for treating muscle wasting, a metabolic disorder, or a combination thereof, in a subject. The system comprises any of the proteins disclosed herein, or pharmaceutical compositions thereof, and a delivery system. The delivery system may comprise a syringe, a pump, an inhaler, a patch, or an intranasal delivery device. In some embodiments, the protein is administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, transdermal, or oral route of administration.

[0055] Also provided herein is a protein comprising an anti-GIPR antibody and a linker, comprising (a) a light chain comprising light chain complementarity determining regions (LC-CDRs) 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively, and a heavy chain comprising heavy chain complementarity determining regions (HC-CDRs) 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively; (b) a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively; (c) a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively; (d) a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 90, YAS, and SEQ ID NO: 92, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89, respectively; or (e) a light chain comprising LC-CDRs 1 through 3comprising the amino acid sequences set forth in SEQ ID NO: 102, SEQ ID NO: 103, and SEQ ID NO: 104, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101, respectively.

[0056] Also provided herein is a protein comprising an anti-GIPR antibody, a linker, and a GLP-1R agonist comprising (a) a light chain comprising light chain complementarity determining regions (LC-CDRs) 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively, and a heavy chain comprising heavy chain complementarity determining regions (HC-CDRs) 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively; (b) a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively;(c) a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively; (d) a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 90, YAS, and SEQ ID NO: 92, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89, respectively; or (e) a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 102, SEQ ID NO: 103, and SEQ ID NO: 104, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101, respectively. In some embodiments, the GLP-1R agonist comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14 and 190.

[0057] In some embodiments, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 20-36 and 187-189.

[0058] In some embodiments, the protein comprises a light chain variable region comprising the amino acid of SEQ ID NO: 42, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 40.

[0059] In some embodiments, the anti-GIPR antibody or a fragment thereof is an IgGl isotype.

[0060] In some embodiments, the anti-GIPR antibody or a fragment thereof comprises at least one Fc mutation.

[0061] In some embodiments, the anti-GIPR antibody or a fragment thereof comprises Fc mutations L234A, L235A, P329G, E272C, M428L, and N434S, wherein residue numbering is according to the EU index.

[0062] In some embodiments, the anti-GIPR antibody or a fragment thereof comprises a light chain of SEQ ID NO: 186 and a heavy chain of SEQ ID NO: 185.

[0063] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 187-189.

[0064] In some embodiments, the protein or antibody of the present invention comprises the light chain variable region comprising at least 95% identity to any one of the sequences set forth in SEQ ID NOs: 41-42 or 49-50 and the heavy chain variable region comprising at least 95% identity to any one of the sequences set forth in SEQ ID NOs: 37-40 or 43-48.BRIEF DESCRIPTION OF THE DRAWINGS

[0065] FIG. 1 illustrates exemplary methods to block activity of undesired changes in muscle or musculature, etc. by applying a muscle fortifying agent according to the present disclosure.

[0066] FIG. 2 illustrates exemplary metabolic modulator peptide-linker complexes according to the present disclosure. Figure discloses SEQ ID NOs: 201-202, respectively, in order of appearance.

[0067] FIGs. 3A-3F illustrate several possible formats for the invention proteins and conjugates, or portions thereof, of the present disclosure. FIG. 3A illustrates bispecific antibody formats, comprising binding domains specific to GIPR and a muscle fortifying agent, such as myostatin, and multispecific appended antibody formats, comprising binding domains specific to GIPR and a muscle fortifying agent, such as myostatin, wherein the bispecific and multispecific formats are conjugable to a GLP-1R modulator; FIG. 3B illustrates bispecific and multispecific antibody fragments, fusion, and conjugate formats, comprising binding domains specific to GIPR and a muscle fortifying agent, such as myostatin, wherein the bispecific and multispecific formats are conjugable to a GLP-1Rmodulator, wherein the heavy chains are shown as solid black, dark grey, or vertically striped regions, the light chains are shown as diagonal striped, white, or horizontally striped regions, and the white dots on a black background and black dots on a white background are nanobodies . FIG. 3C illustrates multispecific Fab x Fab antibody formats, wherein the GLP- 1R agonist is conjugated to the Fc CH3 domain; FIG. 3D illustrates multispecific Fab x scFv antibody formats, wherein the GLP-1R agonist is conjugated to the Fc CH3 domain; FIG. 3E illustrates multispecific Fab x Fab antibody formats, wherein the GLP-1R agonist is conjugated to the Fc CH2 domain; FIG. 3F illustrates multispecific Fab x scFv antibody formats, wherein the GLP-1R agonist is conjugated to the Fc CH2 domain, wherein “a.” - anti-myostatin binding domains, “b ” - anti-GIPR binding domains, and “c ” - a GLP-1R agonist.

[0068] FIG. 4 shows the antagonism of exemplary anti-GIPR antibodies of the invention to HEK 293 reporter cells that express GIPR.

[0069] FIG. 5 A and FIG. 5B show dual GIPR antagonism and GLP-1R agonism against reporter cells expressing either GIPR (FIG. 5A) or both GIPR and GLP-1R (FIG. 5B).

[0070] FIG. 6 shows an exemplary reaction scheme for the generation of an anti-GIPR antibody / GLP-1 receptor agonist conjugate molecule of the present disclosure.

[0071] FIG. 7A shows mean serum concentrations of exemplary anti-GIPR / GLP-1 conjugate molecule after a single subcutaneous dose to male Mauritian cynomolgus monkeys.

[0072] FIG. 7B shows mean weight loss after a single subcutaneous dose to male Mauritian cynomolgus monkeys, wherein circles represent anti-GIPR / GLP-1 conjugate molecule, squares represent vehicle control.

[0073] FIG. 8 shows exemplary sequences of the invention, including the amino acid sequences for the heavy and light chains and their respective complementarity determining regions of representative antibodies according to the present disclosure, exemplary GLP-1R agonists peptides, GIPR agonists, and exemplary linkers, wherein K* is K-(CH2)4-NH-CO- CHs-Br or maleimide.DETAILED DESCRIPTION

[0074] In the following description, the present invention is set forth in the context of various alternative implementations of proteins comprising a muscle fortifying agent, such as a myostatin modulator, and a metabolic component, such as a metabolic modulator, andconjugates thereof joined via linkers. However, before providing a detailed description of the proteins, systems, and methods of the present disclosure, certain definitions and abbreviations are provided to enhance the reader’s understanding of the disclosed invention.DEFINITIONSAND ABBREVIATIONS

[0075] While the general inventive concepts are susceptible of embodiment in many forms, there are shown in the drawings, and will be described herein in detail, specific embodiments thereof with the understanding that the present disclosure is to be considered an exemplification of the principles of the general inventive concepts. Accordingly, the general inventive concepts are not intended to be limited to the specific embodiments illustrated herein.

[0076] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0077] Various publications, articles, and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles, or the like, which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

[0078] The articles “a,” “an,” and “the” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “a” linker means one linker or more than one linker.

[0079] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±5%, preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0080] It should also be understood that the term “substantially,” and like terms, used herein when referring to a dimension or characteristic of a component of the invention, indicate thatthe described dimension / characteristic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), would not vary the least significant digit.

[0081] As used herein, the term “polynucleotide,” synonymously referred to as “nucleic acid molecule,” “nucleotides” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.

[0082] The term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0083] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” (or an equivalent phrase) includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version(s) contain an intron(s).

[0084] As used herein, the term “vector” is a replicon in which another nucleic acid segment can be operably inserted to bring about the replication or expression of the segment.

[0085] As used herein, the term “host cell” refers to a cell comprising a nucleic acid molecule of the invention. The “host cell” can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line. In one embodiment, a “host cell” is a cell transfected with a nucleic acid molecule disclosed herein. In another embodiment, a “host cell” is a progeny or potential progeny of such a transfected cell. A progeny of a cell may or may not be identical to the parent cell, e.g., due to mutations or environmental influences that can occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

[0086] The term “expression” as used herein, refers to the biosynthesis of a gene product. The term encompasses the transcription of a gene into RNA. The term also encompasses translation of RNA into one or more polypeptides and further encompasses all naturally occurring post-transcriptional and post-translational modifications. The expressed molecule can be within the cytoplasm of a host cell, into the extracellular milieu such as the growth medium of a cell culture or anchored to the cell membrane.

[0087] As used herein, the terms “peptide,” “polypeptide,” or “protein” can refer to a molecule comprised of amino acids and can be recognized as a protein by those of skill in the art. The conventional one-letter or three-letter code for amino acid residues is used herein.The terms “peptide,” “polypeptide,” and “protein” can be used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. Also included within the definition are, for example,antibodies, antibody-peptide conjugates, and proteins comprising antibody-peptide conjugates.

[0088] The peptide sequences described herein are written according to the usual convention whereby the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Although isomeric forms of the amino acids are known, it is the L-form of the amino acid that is represented unless otherwise expressly indicated.

[0089] In some embodiments, the proteins and peptides may be joined and / or expressed together and may be referred to as fusion proteins. As used herein, the term “fusion protein” refers to a protein or a polypeptide that encompasses two or more peptide segments linked together to create a sequence that is not present in the same naturally occurring polypeptide. In some embodiments, the two or more polypeptides are covalently linked. In further embodiments, the two or more polypeptides are covalently linked by peptide bonds, linkers, or disulfide bonds. Fusion proteins can be produced by a number of methods that are well known to those familiar with the art, most commonly, by introducing into a cell a vector(s) comprising a nucleic acid sequence that encodes or specifies a fusion protein amino acid sequence.

[0090] Additional amino acids or polypeptides can be incorporated into a fusion protein to elicit additional functional properties, e.g., stability, half-life, multimerization, ease of purification. An example of such an element is a polypeptide linker. Other examples include platforms that extend half-life such as lipidation, antibody Fc, or fusion with certain moi eties, such as human serum albumin (HSA) (e.g., see Cheang and Moyle, Chem Med Chem. 2018, 13:662-71). As an example, the half-life extending domain may be an albumin or albumin binding moiety, or an Fc fragment such as selected from the group consisting of an IgGl, IgG2, IgG3, and IgG4 Fc fragments or a variant thereof, or a PEG moiety.

[0091] A “functional variant” of a peptide or protein includes an amino acid sequence that differs from the noted amino acid sequence by at least one amino acid substitution. Exemplary substitutions may be selected from the group consisting of: (i) substitution of an amino acid having a polar side chain for a different amino acid having a polar side chain wherein amino acids having a polar side chain are Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys; (ii) substitution of an amino acid having a non-polar side chain for a different amino acid having a non-polar side chain wherein amino acids having a non-polar side chain are Gly, Ala, Vai, Leu, He, Phe, Trp, Pro, and Met; (iii) substitution of an amino acid havingan aliphatic side chain for a different amino acid having an aliphatic side chain wherein amino acids having a aliphatic side chain are Gly, Ala Vai, Leu, and He; (iv) substitution of an amino acid having a cyclic side chain for a different amino acid having a cyclic side chain wherein amino acids having a cyclic side chain are Phe, Tyr, Trp, His, and Pro; (v) substitution of an amino acid having an aromatic side chain for a different amino acid having an aromatic side chain wherein amino acids having an aromatic side chain are Phe, Tyr, and Trp; (vi) substitution of an amino acid having an acidic side chain for a different amino acid having an acidic side chain wherein amino acids having an acidic side chain are Asp, and Glu; (vii) substitution of an amino acid having a basic side chain for a different amino acid having a basic side chain wherein amino acids having a basic side chain are Lys, Arg, and His; (viii) substitution of an amino acid having an amide side chain for a different amino acid having an amide side chain wherein amino acids having an amide side chain are Asn, and Gin; (ix) substitution of an amino acid having a hydroxy side chain for a different amino acid having a hydroxy side chain wherein amino acids having a hydroxy side chain are Ser, and Thr; (x) substitution of an amino acid having a sulfur-containing side chain for a different amino acid having a sulfur-containing side chain wherein amino acids having a sulfur- containing side chain are Cys, and Met; (xi) substitution of a neutral, weakly hydrophobic amino acid for a different neutral, weakly hydrophobic amino acid wherein neutral, weakly hydrophobic amino acids are Pro, Ala, Gly, Ser, and Thr; (xii) substitution of a hydrophilic, acidic amino acid for a different hydrophilic, acidic amino acid wherein hydrophilic, acidic amino acids are Gin, Asn, Glu, and Asp, and (xiii) substitution of a hydrophobic amino acid for a different hydrophobic amino acid wherein hydrophobic amino acids are Leu, He, and Vai.

[0092] A peptide that comprises or consists of a sequence means that the peptide can comprise the sequence, consist of the sequence, or comprise at least the full sequence. A peptide that “comprises at least” a peptide sequence, such as “comprising at least the sequence of GIP(3-30): GTFISDYSIAMDKIHQQDFVNWLLAQK,” (SEQ ID NO: 19) means that the peptide includes all of the peptide sequence but does not exclude that additional components or amino acids may be present.

[0093] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0094] As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence that is required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances this sequence may also include an enhancer sequence and other regulatory elements that are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

[0095] A “constitutive” promoter is a nucleotide sequence that, when operably linked with a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0096] An “inducible” promoter is a nucleotide sequence that, when operably linked with a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer that corresponds to the promoter is present in the cell.

[0097] A “tissue-specific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0098] A “signal transduction pathway” refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. The phrase “cell surface receptor” includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the plasma membrane of a cell.

[0099] The terms “antibody” and "antibodies" as used herein are meant in a broad sense and include immunoglobulin molecules including polyclonal antibodies, monoclonal antibodies including murine, human, human-adapted, humanized, and chimeric monoclonal antibodies, antibody fragments, bispecific or multispecific antibodies, dimeric, tetrameric, or multimeric antibodies, single chain antibodies, antibody-peptide fusions, and antibody-peptide conjugates. In some embodiments, the term “antibody” includes appended antibody formats.

[0100] The terms “appended antibody” refers to a bispecific or multispecific antibody that has been engineered for bispecificity or multispecificity by appending either the amino or carboxy terminus or both of either light or heavy chains with additional antigen-binding units.

[0101] Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (k) and lambda (1), based on the amino acid sequences of their constant domains.

[0102] The term "antibody fragments" refers to a portion of an immunoglobulin molecule that retains the heavy chain and / or the light chain antigen binding site, such as heavy chain complementarity determining regions (HCDR) 1, 2 and 3; light chain complementarity determining regions (LCDR) 1, 2 and 3; a heavy chain variable region (VH); or a light chain variable region (VL). Antibody fragments include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a domain antibody (dAb) fragment, which consists of a VH domain. VH and VL domains can be engineered and linked together via a synthetic linker to form various types of single chain antibody designs where the VH / VL domains pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in PCT Inti. Publ. Nos. W01998 / 44001, WO1988 / 01649, WO1994 / 13804, and WO 1992 / 01047. These antibody fragments are obtained using well known techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are full length antibodies.

[0103] The term “stapled single chain Fv” or “spFv” refers to a scFv that comprises one or more disulfide bonds between the VH and the linker or the VL and the linker. Typically, the spFv may comprise one disulfide bond between the VH and the linker, one disulfide bond between the VL and the linker, or two disulfide bonds between the VH and the linker and the VL and the linker. scFv molecules that comprise disulfide bonds between the VH and the VL are excluded from the term “spFv.” The “stapling” strategy is widely applicable to all VH / VL domains and pre-existing scFv molecules providing structural identity to scFv and improved stability. The spFv described herein may be conjugated into bispecific or multispecific format.

[0104] The phrase "isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody specifically binding myostatin is substantially free of antibodies that specifically bind antigens other than myostatin or human myostatin). Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals. An isolated antibody that specifically binds human myostatin, however, can have cross-reactivity to other antigens, such as orthologs of human myostatin (e.g., mouse, rat), or other members of the Transforming Growth Factor P (TGFP) family, such as GDF11 or Activin A.

[0105] "Humanized antibody" refers to an antibody in which the antigen binding sites are derived from non-human species and the variable region frameworks are derived from human immunoglobulin sequences. Humanized antibodies may include substitutions in the framework regions so that the framework may not be an exact copy of expressed human immunoglobulin or germline gene sequences.

[0106] "Human antibody" refers to an antibody having heavy and light chain variable regions in which both the framework and the antigen binding sites are derived from sequences of human origin. If the antibody contains a constant region, the constant region also is derived from sequences of human origin. A human antibody comprises heavy or light chain variable regions that are "derived from" sequences of human origin wherein the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such systems include human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals such as mice carrying human immunoglobulin loci as described herein. A human antibody may also contain amino acid differences when compared to the human germline or rearranged immunoglobulin sequences due to for example naturally occurring somatic mutations or intentional introduction of substitutions in the framework or antigen binding sites. Typically, a human antibody is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical in amino acid sequence to an amino acid sequence encoded by a human germline or rearranged immunoglobulin gene.

[0107] Isolated humanized antibodies may be synthetic. Human antibodies, while derived from human immunoglobulin sequences, may be generated using systems such as phage display incorporating synthetic CDRs and / or synthetic frameworks, or can be subjected to in vitro mutagenesis to improve antibody properties, resulting in antibodies that do not naturally exist within the human antibody germline repertoire in vivo.

[0108] The term "recombinant antibody" as used herein, includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the antibody, antibodies isolated from a recombinant, combinatorial antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences, sequences, or antibodies that are generated in vitro using Fab arm exchange such as bispecific antibodies.

[0109] The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope, or in a case of a bispecific monoclonal antibody, a dual binding specificity to two distinct epitopes. Monoclonal antibodies having higher order binding specificities display binding specificity to a greater number of epitopes, e.g., trispecific monoclonal antibodies display binding specificity to three epitopes.

[0110] The term "epitope" as used herein means a portion of an antigen to which an antibody specifically binds. Epitopes usually consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope can be composed of contiguous and / or discontiguous amino acids that form a conformational spatial unit. For a discontiguous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3- dimensional space through the folding of the protein molecule.[oni] In some embodiments, antibodies bind an antigen but cannot effectively eliminate the antigen from the plasma. In other embodiments, the antibodies provided herein have an affinity to an antigen that is sensitive to pH. Such pH-sensitive antibodies may bind to the antigen in plasma at neutral pH and dissociate from the antigen in an acidic endosome, thus reducing antibody-mediated antigen accumulation and / or promoting antigen clearance from the plasma (e.g., sweeping antibodies). This functionality is achieved through modification of the antibody's Fc region to enhance interaction with the neonatal Fc receptor (FcRn), particularly under acidic conditions such as those found in endosomes. The engineered Fc domain allows for pH-dependent binding behavior: the antibody binds its target antigentightly at physiological (neutral) pH in the bloodstream, but releases the antigen at the acidic pH found within endosomal compartments.

[0112] Thus, as used herein, the term “sweeping antibodies” refer to antibodies having both pH-sensitive antigen binding and at least a threshold level of binding to cell surface neonatal Fc receptor (FcRn) at neutral or physiological pH, such as a pH ranging from 7.0 to 7.6. Upon internalization, these sweeping antibodies release the antigen in an acidic endosome, which may be degraded. In some embodiments, a sweeping antibody, no longer bound to the antigen, may then be released (e.g., by exocytosis) by the cell back into the serum.

[0113] In contrast, non-sweeping antibodies represent conventional monoclonal antibodies that lack the engineered Fc modifications required for antigen recycling. These antibodies bind to their specific antigens and are typically cleared from circulation together with the bound antigen molecule. The lack of pH-dependent binding and FcRn-enhanced recycling means that each non-sweeping antibody molecule is generally capable of binding and clearing only a single antigen molecule before being removed from the system.

[0114] As used herein, the terms “determining,” “assessing,” “assaying,” “measuring,” and “detecting” refer to both quantitative and qualitative determinations, and as such, the term “determining” can be used interchangeably herein with “assaying,” “measuring,” and the like. Where a quantitative determination is intended, the phrase “determining an amount” of binding and the like is used. Where a qualitative determination is intended, the phrase “determining a level” of binding or “detecting” binding is used.

[0115] The terms “binds” or “binding” refer to an interaction between molecules including, for example, to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non- covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope. The ratio of dissociation rate (koff) to association rate (kon) of a binding molecule (e.g., an antibody) to a monovalent antigen (koff / kon) is the dissociation constant KD, which is inversely related to affinity. The lower the KD value, the higher the affinity of the antibody. The value of KD varies for different complexes of antibody and antigen and depends on both kon and koff. The dissociation constant KD for an antibodyprovided herein can be determined using any method provided herein or any other method well known to those skilled in the art. The affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen. When complex antigens containing multiple, repeating antigenic determinants, such as a polyvalent antigen, come in contact with antibodies containing multiple binding sites, the interaction of antibody with antigen at one site will increase the probability of a reaction at a second site. The strength of such multiple interactions between a multivalent antibody and antigen is called the avidity.

[0116] The terms "inhibit" or "neutralize," as used herein with respect to the activity of a binding agent, such as an antibody, refers to the ability to substantially antagonize, prohibit, prevent, restrain, slow, disrupt, alter, eliminate, stop, or reverse the progression or severity of, for example, the biological activity of the target antigen protein, or a disease or condition associated with activity of the target antigen protein. For example, an anti-myostatin antibody may inhibit or neutralize the activity of myostatin by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, or even 100%, or a range defined by any two of the foregoing values.

[0117] Regarding conjugates, the present invention includes one or more drug-to-antibody ratios (DARs) for the multispecific antibodies of the novel conjugates described herein, including one to eight, between two and four, between two and three, between three and four, etc. The DAR of the present invention refers to the number of non-antibody molecules attached to the antibody or antibody like molecules. Biodistribution, pharmacokinetics, and tolerability and assayed in accordance with the present invention. For example, in one aspect of the invention a low DAR is obtained wherein reduction in potency is achieved or a high DAR is obtained wherein distinct pharmacokinetics and toxicity are observed and antibody structure and stability are observed. For example, analytical methods for measuring DAR include UV / Vis spectroscopy, hydrophobic interaction chromatography (HIC), RP-HPLC, and LC-MS and the like. The weighted average DAR value can be calculated using the percentage of chromatographic peak area and the number of conjugated drugs, as a person having ordinary skill in the art in view of the teaching of the present invention would be aware.

[0118] An “antigen” is a structure to which an antibody can selectively bind. A target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurringor synthetic compound. In some embodiments, the target antigen is a polypeptide. In certain embodiments, an antigen is associated with a cell, for example, is present on or in a cell.

[0119] By the term “modulating,” as used herein, should be understood to mean mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0120] In some embodiments of any of the compositions or methods described herein, a range is intended to comprise every integer or fraction or value within the range.

[0121] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers and are intended to be non-exclusive or open-ended. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Embodiments described herein as “comprising” one or more features may also be considered as disclosure of the corresponding embodiments “consisting of’ and / or “consisting essentially of’ such features.PROTEINS

[0122] Subjects receiving metabolic modulators such as GLP-1R agonists and / or GIPR antagonists experience a reduction in mass, including muscle mass, such as lean muscle mass, which is generally attributed to the substantial calorie restriction associated with these medications. The present disclosure encompasses enhanced metabolic modulators such as GLP-1R agonists and / or GIPR antagonists and also the recognition that concomitant administration of a muscle fortifying agent with the GLP-1R agonists and / or GIPR antagonists may provide stabilization or increase in the muscle mass, such as lean muscle mass.

[0123] Accordingly, the present disclosure provides molecules, including proteins that comprise metabolic modulatory agents, and provides molecules comprising (i) a muscle fortifying agent, and (ii) a metabolic component.

[0124] The muscle fortifying agent may be a myostatin modulator, an activin type II receptor modulator, or a combination thereof.

[0125] The myostatin modulator may block binding of myostatin to its receptor, inhibit myostatin activity resulting in an increase in muscle mass or a reduction in loss of muscle mass, or a combination thereof. For example, the myostatin modulator may comprise a binding agent that binds myostatin, a myostatin antagonist, or a combination thereof. The binding agent may comprise a receptor, an antibody, or a fragment thereof.

[0126] In some embodiments, inhibition of myostatin activity by the myostatin modulator results in an increase in muscle mass or a reduction in loss of muscle mass.

[0127] The present disclosure further encompasses the recognition that administration of a muscle fortifying agent such as a myostatin modulator and / or an activin type II receptor modulator may result in an increase in bone density and / or a stabilization of bone density.

[0128] Thus, in some embodiments, the inhibition of myostatin activity by the myostatin modulator or inhibition of myostatin binding to the activin type II receptor may result in an increase in bone density, a stabilization of bone density, or a reduction in loss of bone density.I. METABOLIC MODULATORY COMPONENTS

[0129] The metabolic modulatory component may comprise a molecule having an activity that modulates a metabolic function, such as glycemic regulation. The metabolic modulatory component may comprise or may be or may modulate any of amylin, NPY, NPY2, GLP-1, GLP-2, GLP-1 and glucagon, GLIP-1 and GIP, GLP-1 and glucagon and GIP, GLP-1 and amylin, GDF-15, CB1, ACVR2B, and the like. The metabolic modulatory component may comprise or may be a molecule having the activity of a GLP-1R agonist, a GIPR antagonist, or a combination thereof. For example, the metabolic modulatory component may comprise a GLP-1R agonist, a GIPR antagonist, or a combination thereof.

[0130] The metabolic modulatory component may comprise (a) a GIPR-antagonist and (b) a metabolic modulator. In some embodiments, the GIPR-antagonist (a) may a GIPR peptide and / or an anti-GIPR antibody or fragment thereof. In some embodiments, the metabolic modulator (b) may be a GLP-1R agonist.(a) GIPR antagonists

[0131] GIP, a 42 amino acid peptide, binds to the GIP receptor (GIPR) on the surface of islet beta cells. GIP stimulates glucagon secretion, thus exacerbating hyperglycemia. Moreover, there is evidence for a role of GIP in fat metabolism with the demonstration of GIPR expression in adipose tissue and an association between high BMI and increased GIP levels. Under physiological conditions, GIP is degraded by the enzyme dipeptidylpeptidase 4 (DPP- 4), which cleaves at the third position of the GIP molecule to yield GIP(3-42). GIP(l-30- NFE) is produced as a result of post-translational processing and is an agonist on the GIPR. DPP -4 is found to catalyze cleavage of secreted GIP(l-30) at the third position to produce GIP(3-30-NH2), a potent GIPR antagonists.

[0132] In some embodiments, the GIPR antagonist refers to compounds that reduce or inhibit GIP activation of GIPR. Such antagonists include chemically synthesized small molecules (e.g., SKL-14959), peptides, and antigen binding proteins.

[0133] An “antigen binding protein” as used herein means any protein that specifically binds a specified target antigen, such as a GIPR polypeptide (e.g., a GIPR polypeptide such as provided in any of SEQ ID NO: 180-184; Table 5). The term encompasses both peptides and functional variants thereof, as well as intact antibodies that comprise at least two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and mutations thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments. An antigen binding protein also includes domain antibodies such as nanobodies, scFvs and spFvs, and fusion proteins comprising these domain antibodies.

[0134] In general, a GIPR antigen binding protein is said to “specifically bind” its target antigen GIPR when the antigen binding protein exhibits essentially background binding to non-GIPR molecules. An antigen binding protein that specifically binds GIPR may, however, cross-react with GIPR polypeptides from different species. Typically, a GIPR antigen binding protein specifically binds human GIPR when the dissociation constant (KD) is <10-7M as measured via a surface plasma resonance technique (e.g., BIACore, GE-Healthcare Uppsala, Sweden) or Kinetic Exclusion Assay (KinExA, Sapidyne, Boise, Id.). A GIPR antigen binding protein specifically binds human GIPR with “high affinity” when the KD is <5* IO-9M, and with “very high affinity” when the KD is <5* IO-10M, as measured using methods described.

[0135] In some embodiments, that reduction or inhibition of GIP activation of GIPR can be measured using a GIPR activity assay. In some embodiments, GIPR activity assay (alsoreferred to as a “GIPR functional assay”) means an assay that can be used to measure GIP or a GIP binding protein activity in a cellular setting, in vitro or ex vivo or in vivo. In one embodiment, the “activity” (or “functional”) assay” can be a cAMP assay in GIPR expressing cells, in which GIP can induce cAMP signal, and the activity of a GIP / GIPR binding protein could be measured in the presence / absence of GIP ligand, in which IC50 / EC50 and degree of inhibition / activation can be obtained (Gault, et al., Biochemical and Biophysical Research Communications, 2002, 290: 1420-1426). In another embodiment, the “activity” (or “functional”) assay can be an insulin secretion assay in pancreatic beta cells, in which GIP can induce glucose-dependent insulin secretion, and the activity of a GIP / GIPR binding protein could be measured in the presence / absence of GIP ligand, in which IC50 / EC50 and degree of inhibition / activation can be obtained (id).(i) GIPR antagonist: peptides

[0136] In some embodiments, the GIPR antagonists may include peptides and functional variants thereof that are capable of binding to one or more GIPRs, antagonizing one or more GIPRs, displacing the native GIP(l-42) (SEQ ID NO: 15) and / or GIP(l-30) (SEQ ID NO: 16) from one or more GIPRs, or having a higher affinity for a given GIPR than GIP(1- 42) and / or (GIP1-30). GIPR antagonists may include the DPP-4 cleavage product of GIP(1- 42) or GIP(l-30), i.e., GIP(3-42) (SEQ ID NO: 18) or GIP(3-30) (SEQ ID NO: 19).

[0137] In some embodiments, the GIPR antagonists include functional variants of any of SEQ ID NOS: 14-16 or 18 that are antagonists of GIPR. A functional variant of a GIPR antagonist may comprise at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to one of the sequences set forth in SEQ ID NOS: 14-16 or 18.

[0138] In one embodiment the peptide is non-naturally occurring. In another embodiment the peptide is synthetic. In one embodiment the peptide is an isolated peptide.(ii) GIPR antagonist: anti-GIPR antibodies

[0139] In some embodiments, the GIPR antagonists may include an anti-GIPR antibody or fragment thereof that comprises at least one binding domain that binds GIPR. In some embodiments, the GIPR antagonist is the antibody comprising GIPR binding domains. In some embodiments, the anti-GIPR antibody or fragment thereof comprises two binding domains that bind GIPR.

[0140] In some embodiments, the anti-GIPR antibody includes intact antibodies or antibody fragments such as Fv fragments, single chain scFv fragments (scFv), single chain disulfide bond stabilized scFv fragments or stapled scFv fragment (spFv), Fab, F(ab)2, or single chain antibodies.

[0141] In some embodiments, the anti-GIPR antibody or fragment thereof comprises at least one Fab portion that binds GIPR. In some embodiments, the anti-GIPR antibody or fragment thereof comprises two Fab portions that bind GIPR.

[0142] In some embodiments, the anti-GIPR antibody or fragment thereof comprises at least one scFv portion that binds GIPR. In some embodiments, the anti-GIPR antibody or fragment thereof comprises two scFv portions that bind GIPR. In some embodiments the scFv portion is stabilized, i.e., may be an spFv. The spFv formats are known in the art and were described in International Patent Application Publication No. W02021030657, the content of which is herein incorporated by reference in its entirety.

[0143] In some embodiments, the protein comprising (i) a muscle fortifying agent, and (ii) a metabolic component, or a component thereof, comprises a format illustrated in FIGs. 3A- 3F

[0144] In some embodiments, the anti-GIPR antibody or fragment thereof or the antibody comprising GIPR binding domains comprises a format illustrated in FIGs. 3A or 3B, which include BsIgGs, appended IgGs, BsAb fragments, bispecific fusion proteins, and BsAb conjugates. In FIGs. 3A and 3B, the heavy chains are shown as solid black, dark grey, or vertically striped regions, the light chains are shown as diagonal striped, white, or horizontally striped regions, and the white dots on a black background and black dots on a white background are nanobodies. For example, the anti-GIPR antibody or fragment thereof or the antibody comprising GIPR binding domains, and / or the anti-myostatin antibody or fragment thereof or antibody comprising myostatin binding domains, may include a bispecific IgG, such as any of a crossMab, DAF (two-in-one or four-in-one), DutaMab, DT- IgG, knob-in-holes common, knob-in-holes assembly, charge pair, Fab-arm exchange, SEEDbody, Triomab, LUZ-Y. Fcab, x -body, or orthogonal Fab; or a bispecific antibody fragment, such as any of a nanobody, nanobody-HAS, BiTE, diabody, DART, TandAn, scDiabody, scDiabody-CH3, Diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab’)2, F(ab’)2-scFv2, scFv-KIH, Fab-scFv-Fc, tetraval ent HCab, scDiabody -Fc, Diabody -Fc, tandem scFv-Fc, Intrabody; or anappended IgG, such as any of a DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-ScFv, scFv- (L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv- IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG (four-in-one); or a bispecific fusion protein, such as any of a dock and lock, ImmTAC, HASbody, scDiabody-HAS, or tandem scFv- Toxin; or a bispecific antibody conjugate, such as any of an IgG-IgG, Cov-X-body, or scFvl- PEG-scFv2.

[0145] FIG. 3C and FIG. 3E depict exemplary formats for GLP-1 -conjugated, multispecific antibodies whose Fab domains bind to GIPR and to latent myostatin. FIG. 3D and FIG. 3F depict GLP-1 -conjugated, multispecific antibodies whose scFv and Fab domains bind to GIPR and to latent myostatin. Binding domains are indicated in the figure, where the anti- myostatin binding domains are labeled “a,” the anti-GIPR binding domains are labeled “b,” and the GLP-1R agonist is labeled “c ”

[0146] In some embodiments, the anti-GIPR antibody or fragment thereof or the antibody comprising GIPR binding domains comprises an Fc domain. The antibodies or fragments thereof described herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) of immunoglobulin molecule. In some embodiments, the anti-GIPR antibody or fragment thereof comprises an at least one Fc modification. In some embodiments, the Fc modification is an Fc silencing modification. In certain embodiments, the modification to the Fc region of the antibody results in the decrease or elimination of an effector function of the antibody. In certain embodiments, the effector function is ADCC, ADCP, and / or CDC. In some embodiments, the effector function is ADCC. In other embodiments, the effector function is ADCP. In other embodiments, the effector function is CDC. In one embodiment, the effector function is ADCC and ADCP. In one embodiment, the effector function is ADCC and CDC. In one embodiment, the effector function is ADCP and CDC. In one embodiment, the effector function is ADCC, ADCP and CDC. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody. For example, substitutions into human IgGl using IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330, and 331 were shown to greatly reduce ADCC and CDC (see, e.g., Armour et al., 1999, Eur. J. Immunol. 29(8):2613-24; and Shields et al., 2001, J. Biol. Chem. 276(9): 6591-604). Other Fc variants are provided elsewhere herein.

[0147] In some embodiments, the Ig constant region or the fragment of the Ig constant region or the Fc domain comprises at least one mutation that modulates a half-life of the bindingagent of the instant invention. In some embodiments, the at least one mutation that modulates the half-life of the binding agent is selected from the group consisting of H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R, wherein residue numbering is according to the EU index. In some embodiments, the binding agent comprises a first Ig constant region or a fragment thereof and a second Ig constant region or a fragment thereof. In some embodiments, one or both of the first Ig constant region or a fragment thereof and a second Ig constant region or a fragment thereof comprises at least one mutation that modulates a half-life of the binding agent independently selected from the group consisting of H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R, wherein residue numbering is according to the EU index.

[0148] In some embodiments, the anti-GIPR antibody or fragment thereof or the antibody comprising GIPR binding domains is derived from IgGl. In some embodiments, the anti- GIPR antibody or fragment thereof comprises a combination of Fc mutations comprising L234A, L235A, P329G, E272C, M428L, and N434S, wherein residue numbering is according to the EU index.

[0149] In some embodiments, the anti-GIPR antibody or fragment thereof or the antibody comprising GIPR binding domains comprises a modification to facilitate linking to the metabolic modulator, directly, via a peptide linker, via a synthetic linker, for example a nonpeptide organic linker, or otherwise. Linkers are discussed further elsewhere herein. The modification may include a sequence substitution, deletion, or insertion. The modification may be in the Fc domain.

[0150] In some embodiments, the anti-GIPR antibody or fragment thereof comprises a light chain variable region (VL) selected from SEQ ID NOs: 41, 42, 49, and 50 as listed in Table 3

[0151] In some embodiments, the anti-GIPR antibody or fragment thereof comprises a light chain variable region (VL) comprising VL CDRs as shown in Table 3, which include at least SEQ ID Nos: 54-56 and 60-62 (Kabat), SEQ ID Nos: 66-68 and 72-74 (AbM); SEQ ID Nos: 78-80 and 84-86 (Chothia); SEQ ID No: 90, YAS, and SEQ ID No: 92 and SEQ ID No: 96, YAS, and SEQ ID No: 98 (IMGT); SEQ ID Nos: 102-104 and 108-110 (CONTACT).

[0152] In some embodiments, the anti-GIPR antibody or fragment thereof comprises a heavy chain variable region (VH) selected from SEQ ID NOs: 37-40 and 43-48 as listed in Table 3.

[0153] In some embodiments, the anti-GIPR antibody or fragment thereof comprises a heavy chain variable region (VH) comprising VH CDRs as shown in Table 3, which include at least SEQ ID Nos: 51-53 and 57-59 (Kabat), SEQ ID Nos: 63-65 and 69-71 (AbM); SEQ ID Nos: 75-77 and 81-83 (Chothia); SEQ ID Nos: 87-89 and 93-95 (IMGT); SEQ ID Nos: 99-101 and 105-107 (CONTACT).

[0154] In some embodiments, the anti-GIPR antibody comprises a light chain comprising light chain complementarity determining regions 1 through 3 (LC-CDRs 1-3) comprising the amino acid sequences set forth in SEQ ID NO: 54-56, respectively, and a heavy chain comprising heavy chain CDRs 1 through 3 (HC-CDRS 1-3) comprising the amino acid sequences set forth in SEQ ID NO: 51-53, respectively.

[0155] In some embodiments, the anti-GIPR antibody comprises LC-CDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NO: 60-62, respectively, and HC-CDRS 1-3 comprising the amino acid sequences set forth in SEQ ID NO: 57-59, respectively.

[0156] In some embodiments, the anti-GIPR antibody comprises LC-CDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NO: 66-68, respectively, and HC-CDRS 1-3 comprising the amino acid sequences set forth in SEQ ID NO: 63-65, respectively.

[0157] In some embodiments, the anti-GIPR antibody comprises LC-CDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NO: 72-74, respectively, and HC-CDRS 1-3 comprising the amino acid sequences set forth in SEQ ID NO: 69-71, respectively.

[0158] In some embodiments, the anti-GIPR antibody comprises LC-CDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NO: 78-80, respectively, and HC-CDRS 1-3 comprising the amino acid sequences set forth in SEQ ID NO: 75-77, respectively.

[0159] In some embodiments, the anti-GIPR antibody comprises LC-CDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NO: 84-86, respectively, and HC-CDRS 1-3 comprising the amino acid sequences set forth in SEQ ID NO: 81-83, respectively.

[0160] In some embodiments, the anti-GIPR antibody comprises LC-CDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NO: 90, YAS, and SEQ ID NO: 92, respectively, and HC-CDRS 1-3 comprising the amino acid sequences set forth in SEQ ID NO: 87-89, respectively.

[0161] In some embodiments, the anti-GIPR antibody comprises LC-CDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NO: 96, YAS, and SEQ ID NO: 98,respectively, and HC-CDRS 1-3 comprising the amino acid sequences set forth in SEQ ID NO: 93-95, respectively.

[0162] In some embodiments, the anti-GIPR antibody comprises LC-CDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NO: 102-104, respectively, and HC-CDRS 1-3 comprising the amino acid sequences set forth in SEQ ID NO: 99-101, respectively.

[0163] In some embodiments, the anti-GIPR antibody comprises LC-CDRs 1-3 comprising the amino acid sequences set forth in SEQ ID NO: 108-110, respectively, and HC-CDRS 1-3 comprising the amino acid sequences set forth in SEQ ID NO: 105-107, respectively.

[0164] In some embodiments, the anti-GIPR antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 41 or SEQ ID NO: 42, and a heavy chain variable region comprising the amino acid sequence set forth in any one of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40; or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 49 or SEQ ID NO: 50, and a heavy chain variable region comprising the amino acid sequence set forth in any one of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48.

[0165] In some embodiments, the anti-GIPR antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 42 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 40.

[0166] In some embodiments, the anti-GIPR antibody comprises a light chain (LC) comprising the amino acid sequence of SEQ ID NO 186: and a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 185.

[0167] In one embodiment, the anti-GIPR antibody or a fragment thereof or the antibody comprising GIPR binding domains comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-42 or 49-50 and a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-40 or 43-48, wherein the antibody comprises at least one cysteine conjugation site at a position selected from the group consisting of S239C, E272C, or S400C (Eu numbering). In some embodiments, the peptide is conjugated via the side chain of Q295. In some embodiments, the peptide is conjugated via the side chain of E294Q of a mutant Fc having E294Q and Q295A amino acid substitutions. In some embodiments, the peptide is conjugated via transglutaminase chemistry.(b) GLP-1 R agonists

[0168] GLP-1 is a peptide hormone that is synthesized by the enteroendocrine cells of the gut and released in response to the intake of food. It is secreted primarily in two forms, GLP-1(7- 37) and GLP-1 (7-36)NH2, both of which bind to a specific GLP-1 receptor (GLP-1R) found in many tissues including the pancreatic beta-cells where it augments glucose-stimulated insulin secretion and in the brainstem where it controls satiety and meal size (Muller TD, et al., Mol. Metab., 2019, 30:72-130).

[0169] Agonists of GLP-1R have been found to improve glucose homeostasis and drive weight loss in humans. GLP-1R agonists may be GLP-1 analogues, as further discussed below. GLP-1R agonist therapeutics include peptide sequences based on either native human GLP-1 or the exendins, such as exendin-4, a peptide isolated from the saliva of the Gila monster lizard. The exendins have sequence homology to native GLP-1 and can bind the GLP-1R.

[0170] Exemplary GLP-1R agonists include GLP-1 peptides, peptide analogues of GLP-1 (e.g., variant peptides), any of the exendins known to bind to the GLP-1R, and / or GLP-1 peptide fusions or conjugates. The GLP-1R agonists or variant peptide thereof can be chosen based on at least one of the following criteria: (i) expression yield and purity, (ii) in vitro stability, (iii) in vitro potency, (iv) the retention of in vitro potency in combination with the myostatin modulator or variant thereof, and (vi) properties of the myostatin-GLP-lR agonist fusion proteins (e.g., in vivo stability, in vivo potency, etc.).

[0171] Exemplary GLP-1R agonist peptides further include peptides that are configured to have sufficient homology and functionality to the native GLP-1, and are thus capable of binding to the GLP-1R in tissues including the pancreas and brainstem resulting in the same signaling pathway and exhibiting the same or similar physiological activity as when the native GLP-1 binds the GLP-1R in these tissues.

[0172] The GLP-1R agonists may comprise a peptide comprising the amino acid sequence as set forth in any of SEQ ID NOs: 1-14, 17 or 190. In some embodiments, the GLP-1R agonists include functional variants of any of SEQ ID NOs: 1-14, 17 or 190 that are agonists of one or more GLP-lRs. A functional variant of a GLP-1R agonist may comprise at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to one of the sequences set forth in SEQ ID NOs: 1-14, 17 or 190.

[0173] In some embodiments, the peptide is non-naturally occurring. In other embodiments, the peptide is synthetic. In yet other embodiments, the peptide is an isolated peptide.

[0174] In some embodiments, the GLP-1R agonists may comprise "GLP-1 fragments." A GLP-1 fragment is a polypeptide obtained after truncation of one or more amino acids from the N-terminus and / or C-terminus of GLP-1(7-37)OH or an analog or derivative thereof. The nomenclature used to describe GLP-1(7-37)OH is also applicable to GLP-1 fragments. For example, GLP-1 (9-36)OH denotes a GLP-1 fragment obtained by truncating two amino acids from the N-terminus and one amino acid from the C-terminus. The amino acids in the fragment are denoted by the same number as the corresponding amino acid in GLP-1 (7- 37)OH. For example, the N-terminal glutamic acid in GLP-1(9-36)OH is at position 9; position 12 is occupied by phenylalanine; and position 22 is occupied by glycine, as in GLP- 1(7-37)OH. For GLP-1 (7-36)OH, the glycine at position 37 of GLP-1 (7-37)OH is deleted.

[0175] In some embodiments, the GLP-1R agonists may comprise polypeptides in which one or more amino acids have been added to the N-terminus and / or C-terminus of GLP-1 (7- 37)OH, or fragments or analogs thereof. In some embodiments, the GLP-1 compound has up to about thirty-nine amino acids. The amino acids in the "extended" GLP-1 compound are denoted by the same number as the corresponding amino acid in GLP-1 (7-37)OH. For example, the N-terminus amino acid of a GLP-1 compound obtained by adding two amino acids to the N-terminal of GLP-1(7-37)OH is at position 5; and the C-terminus amino acid of a GLP-1 compound obtained by adding one amino acid to the C-terminus of GLP-1(7-37)OH is at position 38. Thus, position 12 is occupied by phenylalanine and position 22 is occupied by glycine in both of these "extended" GLP-1 compounds, as in GLP-1 (7-37)OH. Amino acids 1-6 of an extended GLP-1 compound are preferably the same as or a conservative substitution of the amino acid at the corresponding position of GLP-1(1-37)OH. Amino acids 38-45 of an extended GLP-1 compound are preferably the same as or a conservative substitution of the amino acid at the corresponding position of glucagon or Exendin-4.

[0176] In some embodiments, the GLP-1R agonists may comprise "GLP-1 analogs." A GLP- 1 analog has sufficient homology to GLP-1(7-37)OH or a fragment of GLP-1(7-37)OH such that the analog has insulinotropic activity. Preferably, a GLP-1 analog has the amino acid sequence of GLP-1(7-37)OH or a fragment thereof, modified so that from one, two, three, four or five amino acids differ from the amino acid in the corresponding position of GLP-1(7- 37)OH or a fragment of GLP-1 (7-37)OH. In the nomenclature used herein to designate GLP- 1 compounds, the substituting amino acid and its position is indicated prior to the parentstructure. For example, Glu22-GLP-1(7-37)OH designates a GLP-1 compound in which the glycine normally found at position 22 of GLP-1(7-37)OH has been replaced with glutamic acid; Val8-Glu22-GLP-1(7-37)OH designates a GLP-1 compound in which alanine normally found at position 8 and glycine normally found at position 22 of GLP-1(7-37)OH have been replaced with valine and glutamic acid, respectively.

[0177] In some embodiments, the GLP-1R agonists may comprise "GLP-1 derivatives." A GLP-1 derivative is defined as a molecule having the amino acid sequence of GLP-1 or of a GLP-1 analog, but additionally having chemical modification of one or more of its amino acid side groups, a-carbon atoms, terminal amino group, or terminal carboxylic acid group. A chemical modification includes, but is not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties. Modifications at amino acid side groups include, without limitation, acylation of lysine s-amino groups, N-alkylation of arginine, histidine, or lysine, alkylation of glutamic or aspartic carboxylic acid groups, and deamidation of glutamine or asparagine. Modifications of the terminal amino group include, without limitation, the des-amino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the terminal carboxy group include, without limitation, the amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications. Lower alkyl is C1-C4 alkyl. Furthermore, one or more side groups, or terminal groups, may be protected by protective groups known to the ordinarily skilled protein chemist. The a-carbon of an amino acid may be mono-or dimethylated.

[0178] Any GLP-1R agonist can be part of the heterologous fusion proteins of the present invention as long as the GLP-1 compound itself is able to bind and induce signaling through the GLP-1 receptor. GLP-1 receptor binding and signal transduction can be assessed using in vitro assays such as those described in EP0619322 and U.S. Pat. No. 5,120,712, respectively, incorporated herein by reference in their entirety.

[0179] Numerous active GLP-1 fragments, analogs and derivatives are known in the art and any of these analogs and derivatives can also be part of the heterologous fusion proteins of the present invention. Some examples of novel GLP-1 analogs as well as GLP-1 analogs and derivatives known in the art are provided herein.

[0180] Some GLP-1 analogs and GLP-1 fragments known in the art include, for example, GLP-l(7-34) and GLP-l(7-35), GLP-l(7-36), Gln9-GLP-l(7-37), D-Gln9-GLP-l(7-37), Thr16-Lys18-GLP-l(7-37), and Lys18-GLP-l(7-37). GLP-1 analogs such as GLP-l(7-34) andGLP-l(7-35) are disclosed in U.S. Pat. No. 5,118,666. Biologically processed forms of GLP- 1 which have insulinotropic properties, such as GLP-l(7-36) are also known. Other known biologically active GLP-1 compounds are disclosed in U.S. Pat. No 5,977,071, U.S. Pat. No. 5,545,618, and Adelhorst, et al., J. Biol. Chem. 1994, 269:6275.

[0181] In some embodiments, the GLP-1R agonists may comprise GLP-1 compounds described in U.S. Pat. No 7,271,149, incorporated by reference herein in its entirety.

[0182] In one embodiment, the GLP-1 receptor agonist polypeptide comprises the amino acid sequence of H[Aib]EGTFTSDYSSYLEEXi7AAX2oEFIX24WLX27X28GGG, where Aib at position 2 is a-amino-isobutyric acid; Xi7is selected from glutamine (Q), lysine (K), glutamic acid (E), or arginine (R), X20 is selected from K, R, Q, or homoarginine (hArg), X24 is selected from alanine (A), E, Q, aspartic acid (D), d-Ala, glycine (G), asparagine (N), K, (lS,2S)-2-Aminocyclopentanecarboxylic acid (ACPC), (3S,4R)-4-Amino-3- pyrrolidinecarboxylic acid (APCA), threonine (T), serine (S), or tyrosine (Y); X27 is selected from valine (V), K, a-amino-isobutyric acid (Aib), N, S, T, Y, Q, R; and X28 is selected from K, E, R, Q, D, N, G, S, T.

[0183] In one embodiment, the GLP-1 receptor agonist polypeptide comprises the amino acid sequence of H[Aib]EGTFTSDYSSYLEEXi7AAX2oEFIX24WLX27X28GGG, where X17 is Q or K; X20 is K or R; X24 is A, E, or Q; X27 is V or K; and X28 is K, E, or R.

[0184] In one embodiment, the GLP-1 receptor agonist polypeptide comprises the amino acid sequence of H[Aib]EGTFTSDYSSYLEEXi7AAREFIX24WLX27X28GGG, where X17 is Q or K; X24 is A, E, or Q; X27 is V or K; and X28 is K, E, or R. Alternatively, the GLP-1 receptor agonist comprises the amino acid sequence of H[Aib]EGTFTSDYSSYLEEXi7AAX2oEFIX24WLKX28GGG, where X17 is Q or K; X20 is K or R; X24 is A, E, or Q; and X28 is K, E, or R.II. MUSCLE FORTIFYING AGENT

[0185] Myostatin, also known as GDF-8, is a member of the TGFP superfamily, i.e., a family of structurally related growth factors, all of which are endowed with physiologically important growth-regulatory and morphogenetic properties. The TGF-P superfamily includes Bone Morphogenetic Proteins (BMPs), activins, inhibins, Mullerian inhibiting substance, glial-derived neurotrophic factor, and a still growing number of Growth and Differentiation Factors (GDFs), such as GDF-8, which belongs to a subfamily including two members: Myostatin and GDF-11.

[0186] Like other members of the TGFP superfamily, myostatin is initially expressed as inactive precursor polypeptides termed pro-myostatin. Myostatin is activated by two separate protease cleavage steps that release the inhibitory “mature” myostatin (see FIG. 1 at left middle, wherein the inactive precursor myostatin is cleaved to release the mature myostatin). The mature myostatin binds with high affinity to the activin type IIB receptor (ActRIIB), and with lesser affinity to the activin receptor (ActRIIA). Intracellular signaling is initiated by binding of dimeric myostatin to ActRIIB followed by recruitment of a low-affinity type I receptor, either activin-like kinase 4 (ALK4) or activin-like kinase 5 (ALK5) (see FIG. 1 at left). Phosphorylation of the type I receptor results in initiation of the intracellular signaling pathway that is responsible for myostatin's biological effects. That is, downstream signaling from these receptors induces muscle atrophy. As such, the activity of myostatin may be blocked by hindering the activation step of myostatin or by blocking the binding of active myostatin to its receptor.(a) Myostatin modulators

[0187] Modulators of myostatin activity include agents capable of binding to myostatin and blocking or inhibiting the activity of myostatin. Such agents include antibodies that block myostatin binding to its receptor (ALK-4, ActRIIB, ActRIIA; see FIG. 1 at right), antibodies that bind to mature myostatin, and / or antibodies that bind to the pro-protein region of myostatin and block the cleavage that releases the mature myostatin. In some embodiments, the modulators of myostatin activity bind to latent myostatin. Exemplary antibodies include any of the antibodies disclosed in Pat. Nos. US 7,261,893, US 7,320,789, US 7,745,583, US 8,063,188, US 8,066,995, US 8,940,874, US 8,999,343, US 9,505,831, US 9,751,937, and US Pat. Publ. No. 2007 / 0178095. In an embodiment, the activity of the mature myostatin protein, when bound by one or more of the presently disclosed antibodies, is neutralized or inhibited by at least 50%, optionally at least 60, 62, 64, 66, 68, 70, 72, 72, 76, 78, 80, 82, 84, 86, or 88%, optionally at least 90, 91, 92, 93, or 94%, and optionally at least 95% to 100% relative to a mature myostatin protein that is not bound by one or more of the presently disclosed antibodies.

[0188] Myostatin also plays a role in bone health, and its neutralization or inhibition can lead to increased bone density. More specifically, myostatin inhibits osteoblast differentiation and stimulates osteoclast activity in a dose-dependent manner. Studies on mice lacking myostatin (myostatin null mice) have shown a significant increase in muscle mass and bone density inthe limbs, spine, and jaw. Furthermore, myostatin inhibition has been linked to increased bone formation and decreased bone resorption.

[0189] In some embodiments, the myostatin antagonists may include an anti-myostatin antibody or fragment thereof that comprises at least one binding domain that binds myostatin. In some embodiments, the anti -myostatin antibody or fragment thereof comprises two binding domains that bind myostatin.

[0190] In some embodiments, the anti-myostatin antibody includes intact antibodies or antibody fragments such as Fv fragments, single chain scFv fragments (scFv), single chain disulfide bond stabilized scFv fragments or stapled scFv fragment (spFv), Fab, F(ab)2, or single chain antibodies.

[0191] In some embodiments, the anti-myostatin antibody or fragment thereof comprises at least one Fab portion that binds myostatin. In some embodiments, the anti-myostatin antibody or fragment thereof comprises two Fab portions that bind myostatin.

[0192] In some embodiments, the anti-myostatin antibody or fragment thereof comprises at least one scFv portion that binds myostatin. In some embodiments, the anti-myostatin antibody or fragment thereof comprises two scFv portions that bind GIPR. In some embodiments the scFv portion is stabilized, i.e., may be an spFv.

[0193] In some embodiments, the anti-myostatin antibody or fragment thereof comprises an Fc modification, for example Fc silencing. Fc silencing may include the process of eliminating the binding of Fc to Fc gamma receptors and / or complement protein Clq, thus abolishing immune effector functions such as antibody dependent cell-mediated cytotoxicity (ADCC).

[0194] In some embodiments, the anti-myostatin antibody or fragment thereof comprises a modification to facilitate linking to the metabolic modulator, directly, via a peptide linker, via a synthetic linker, for example a non-peptide organic linker, or otherwise. Linkers are discussed further elsewhere herein. The modification may include a sequence substitution, deletion, or insertion. The modification may be in the Fc domain.

[0195] In some embodiments, the anti-myostatin antibody comprises a light chain comprising light chain complementarity determining regions 1 through 3 (LC-CDR1-3) comprising the amino acid sequences set forth in SEQ ID NO: 122-124, respectively, and a heavy chain comprising heavy chain CDRs 1 through 3 (HC-CDR1-3) comprising the amino acid sequences set forth in SEQ ID NO: 119-121, respectively.

[0196] In some embodiments, the anti-myostatin antibody comprises LC-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 128-130, respectively, and HC-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 125-127, respectively.

[0197] In some embodiments, the anti-myostatin antibody comprises LC-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 134-136, respectively, and HC-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 131-133, respectively.

[0198] In some embodiments, the anti-myostatin antibody comprises LC-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 140-142, respectively, and HC-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 137-139, respectively.

[0199] In some embodiments, the anti-myostatin antibody comprises LC-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 146-148, respectively, and HC-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 143-145, respectively.

[0200] In some embodiments, the anti-myostatin antibody comprises LC-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 152-154, respectively, and HC-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 149-151, respectively.

[0201] In some embodiments, the anti-myostatin antibody comprises LC-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 158, STS, and SEQ ID NO: 160, respectively, and HC-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 155-157, respectively.

[0202] In some embodiments, the anti-myostatin antibody comprises LC-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 164, SAS, and SEQ ID NO: 166, respectively, and HC-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 161-163, respectively.

[0203] In some embodiments, the anti-myostatin antibody comprises LC-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 170-172, respectively, and HC-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 167-169, respectively.

[0204] In some embodiments, the anti-myostatin antibody comprises LC-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 176-178, respectively, and HC-CDR1-3 comprising the amino acid sequences set forth in SEQ ID NO: 173-175, respectively.

[0205] In some embodiments, the anti-myostatin antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 113 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 112. In some embodiments, the anti-myostatin antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 117 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 116.

[0206] In some embodiments, the anti-myostatin antibody comprises an scFv comprising a light chain variable region defined by a light chain complementarity determining regions 1 through 3 (LC-CDR1-3) linked to a heavy chain variable region defined by a heavy chain complementarity determining regions 1 through 3 (HC-CDR1-3), wherein the linker comprises the amino acid sequence set forth in SEQ ID NO: 179. The specific combinations of LC-CDR1-3 and HC-CDR1-3 that define the light chain variable region and the heavy chain variable region, respectively, may be any of the combinations defined hereinabove.

[0207] In some embodiments, the anti-myostatin antibody comprises a stapled scFv (spFv) comprising a light chain variable region defined by a light chain complementarity determining regions 1 through 3 (LC-CDR1-3) linked to a heavy chain variable region defined by a heavy chain complementarity determining regions 1 through 3 (HC-CDR1-3), wherein the linker comprises the amino acid sequence set forth in SEQ ID NO: 179. The specific combinations of LC-CDR1-3 and HC-CDR1-3 that define the light chain variable region and the heavy chain variable region, respectively, may be any of the combinations defined hereinabove.

[0208] In some embodiments, the anti-myostatin antibody comprises a stapled scFv (spFv) comprising the amino acid sequence set forth in SEQ ID NO: 114, SEQ ID NO: 115, or SEQ ID NO: 118.

[0209] Further agents capable of binding to myostatin and blocking or inhibiting the activity of myostatin include soluble receptor proteins or variants thereof, such as a soluble ActRIIB protein. Modulators of myostatin further include a peptide comprising the amino acidsequence of the myostatin pro-peptide, Follistatin, Follistatin Related Gene (FLRG), and / or Growth and Differentiation Factor- Associated Serum Protein- 1 or 2 (Grasp- 1 or Grasp-2).(b) Activin type II Receptor modulators

[0210] Agents capable of binding to the myostatin receptor ActRIIB and / or ActRIIA (ActRIIA / B) may block or inhibit the activity of myostatin. Activin receptors are transmembrane proteins composed of a ligand-binding extracellular domain with a cysteine- rich region, a transmembrane domain, and a cytoplasmic domain required for cell signaling through serine / threonine phosphorylation events. The interaction between myostatin and ActRIIA / B regulates the inhibition of skeletal muscle differentiation via a SMAD-dependent pathway. Thus, by inhibiting or preventing myostatin from binding to ActRIIA / B, the formation of skeletal muscle can be induced.

[0211] Inhibition of ActRIIA / B has been shown to reverse muscle wasting and leads to increased lean muscle volume and strength in animal models. The inhibition of ActRIIA / B has also been shown to promote increased muscle volume in human subjects, and a reduction in fat mass.

[0212] In some embodiments, the modulator of muscle mass may be an antibody against ActRIIA and / or ActRIIB, which may neutralize or inhibit ActRIIA / B protein activity in vitro or in vivo (e.g., IC50). Exemplary antibodies against ActRIIB include those disclosed in International Patent Application Publication U.S. Pat. No. 7,893,213, U.S. Pat. Publ. 2018 / 0111991, and WO2014 / 172448, the contents of which are incorporated herein by reference. Exemplary antibodies against ActRIIA include those disclosed in U.S. Pat. Pub. No. 2018 / 0008672.III. FUSION PROTEINS AND CONJUGATES

[0213] In some embodiments, the metabolic modulatory component comprises (a) an anti- GIPR antibody or fragment thereof and (b) a metabolic modulator. In some embodiments, the (a) anti-GIPR antibody or fragment thereof and (b) metabolic modulator are connected directly, or via a peptide linker, resulting in a fusion protein. In other embodiments, they are connected via a peptide, synthetic or organic linker, resulting in a conjugate.

[0214] In some embodiments, the muscle fortifying agent and the metabolic modulatory component are connected directly, or via a peptide linker, resulting in a fusion protein. Insome embodiments, the muscle fortifying agent and the metabolic modulatory component are connected via a peptide, synthetic or organic linker, resulting in a conjugate.

[0215] In some embodiments, the metabolic modulator comprises amylin, NPY, NPY2, GLP- 1, GLP-2, GLP-1 and glucagon, GLP-1 and GIP, GLP-1 and glucagon and GIP, GLP-1 and amylin, GDF-15, CB1, ACVR2B, or any combination thereof.

[0216] In some embodiments, the metabolic modulator comprises a molecule having the activity of a GLP-1 receptor agonist, a GIPR antagonist, or a combination thereof. In some embodiments, the metabolic modulator comprises a GLP-1 receptor agonist, a GIPR antagonist, or a combination thereof. In some embodiments, the metabolic modulator comprises a GLP-1 receptor agonist and a GIPR antagonist.

[0217] The GLP-1R agonist may be a peptide comprising the amino acid sequence as set forth in any of SEQ ID NO: 1-14, 17 or 190, or a functional peptide or a fragment thereof. In some embodiments, the GLP-1R agonist comprises SEQ ID NO: 17. In some embodiments, the GLP-1R agonist comprises SEQ ID NO: 190.

[0218] The GIPR antagonist may be a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 18 or 19, or a functional variant thereof. The GIPR antagonist may be a functional variant of a peptide comprising the amino acid sequence as set forth in SEQ ID NO: 15, 16, 18 or 19. Of note, a functional variant of a GIPR antagonist is a peptide capable of inhibiting the activity of the GIPR.

[0219] In some embodiments, the GIPR antagonist is an antibody or a fragment thereof. In some embodiments, the anti-GIPR antibody comprises a LC comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively.

[0220] In some embodiments, the anti-GIPR antibody comprises a LC comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59, respectively.

[0221] In some embodiments, the anti-GIPR antibody comprises a LC comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 66, SEQ ID NO:67, and SEQ ID NO: 68, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively.

[0222] In some embodiments, the anti-GIPR antibody comprises a LC comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively.

[0223] In some embodiments, the anti-GIPR antibody comprises a LC comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively.

[0224] In some embodiments, the anti-GIPR antibody comprises a LC comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: 86, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 81, SEQ ID NO: 82, and SEQ ID NO: 83, respectively.

[0225] In some embodiments, the anti-GIPR antibody comprises a LC comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 90, YAS, and SEQ ID NO: 92, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89, respectively.

[0226] In some embodiments, the anti-GIPR antibody comprises a LC comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 96, YAS, and SEQ ID NO: 98, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 93, SEQ ID NO: 94, and SEQ ID NO: 95, respectively.

[0227] In some embodiments, the anti-GIPR antibody comprises a LC comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 102, SEQ ID NO: 103, and SEQ ID NO: 104, respectively, and a HC comprising HC-CDRs 1 through 3comprising the amino acid sequences set forth in SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101, respectively.

[0228] In some embodiments, the anti-GIPR antibody comprises a LC comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110, respectively, and a HC comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107, respectively.

[0229] In some embodiments, the anti-GIPR antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 41 or SEQ ID NO: 42, and a heavy chain variable region comprising the amino acid sequence set forth in any one of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40.

[0230] In some embodiments, the anti-GIPR antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 49 or SEQ ID NO: 50, and a heavy chain variable region comprising the amino acid sequence set forth in any one of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48.

[0231] In some embodiments, the muscle fortifying agent and the anti-GIPR antibody or fragment thereof are connected directly, or via a peptide linker, resulting in a fusion protein. In some embodiments, the muscle fortifying agent and the anti-GIPR antibody or a fragment thereof are connected via a peptide, synthetic or organic linker, resulting in a conjugate.

[0232] In some embodiments, the muscle fortifying agent, e.g., anti-myostatin antibody or anti-ActRIIB antibody, and the anti-GIPR antibody may be configured as a bi-specific antibody. In some embodiments, the muscle fortifying agent, e.g., anti -myostatin antibody and the anti-GIPR antibody may be configured as a bi-specific antibody. In some embodiments, the anti-myostatin antibody, anti-ActRIIB antibody, and anti-GIPR antibody may be configured as a multi-specific antibody. Exemplary configurations of the multispecific and bispecific antibodies of the current invention are shown in FIGs. 3A-3F. In some embodiments, the GLP-1 receptor agonist, such as any disclosed herein (e.g., SEQ. ID. NOS.: 1-14, 17 or 190) may be linked or conjugated to the bi-specific or multi-specific antibodies, or in other arrangements as disclosed herein (e.g., FIGs. 3A-3F and FIG. 6).

[0233] The present disclosure relates to a multi-specific protein comprising (i) a muscle fortifying agent means or fragment thereof and (ii) a metabolic modulatory means. In someembodiments, the muscle fortifying agent means or fragment thereof and the metabolic modulatory means are connected directly resulting in a fusion protein. In some embodiments, the muscle fortifying agent means and the metabolic modulatory means are connected via a linker means.

[0234] The present disclosure also relates to a multi-specific protein comprising (i) an anti- GIPR binding means or fragment thereof and (ii) a metabolic modulatory means. In some embodiments, the anti-GIPR binding means or fragment thereof and the metabolic modulatory means are connected directly resulting in a fusion protein. In some embodiments, the anti-GIPR binding means or fragment thereof and the metabolic modulatory means are connected via a linker means.

[0235] The present disclosure further yet relates to (i) a muscle fortifying agent means or fragment thereof and an anti-GIPR binding means or fragment thereof and (ii) a metabolic modulatory means. In some embodiments, the muscle fortifying agent means or fragment thereof and the anti-GIPR binding means or fragment thereof (i) comprises a bispecific antibody. In some embodiments, the muscle fortifying agent means or fragment thereof and the anti-GIPR binding means or fragment thereof are connected via a linker means. In some embodiments, the bispecific antibody of (i) is connected to the metabolic modulatory means via a linker means.

[0236] In some embodiments, the muscle fortifying agent means or fragment thereof comprises a binding agent that binds myostatin, a myostatin antagonist, or a combination thereof. The muscle fortifying agent means or fragment thereof may comprise an anti- myostatin antibody, such as any of the anti-myostatin antibodies disclosed herein.

[0237] In some embodiments, the anti-GIPR binding means or fragment thereof may comprise an anti-GIPR antibody, such as any of the anti-GIPR antibodies disclosed herein.

[0238] In some embodiments, the metabolic modulatory means may comprise a molecule having the activity of a glucagon-like peptide-1 (GLP-1) receptor agonist, a GIPR antagonist, or a combination thereof. For example, the metabolic modulatory means may comprise a GLP-1 receptor agonist, such as a peptide comprising the amino acid sequence as set forth in any one of SEQ ID NOs: 1-14, 17, or 190, or a variant thereof, wherein the variant comprises at least one amino acid substitution. The metabolic modulatory means may comprise a GIPR antagonist, such as a peptide comprising the amino acid sequence as set forth in SEQ IDNOs: 15, 16, 18, or 19, or a variant thereof, wherein the variant comprises at least one amino acid substitution.

[0239] In some embodiments, the linker means may be a synthetic or organic linker, resulting in a conjugate. In some embodiments, the linker is a peptide, such as any of the peptide linkers disclosed herein.Methods for Conjugation

[0240] An exemplary molecule of the present disclosure comprises a first GLP-1 receptor agonist polypeptide conjugated to an antagonist anti-GIPR human antibody. This molecule is referred to herein as an “anti-GIPR antibody / GLP-1 receptor agonist conjugate molecule”, a “conjugate molecule”, or an “anti-GIPR / GLP-1 conjugate molecule”. Exemplary antagonist anti-GIPR antibodies are disclosed infra.

[0241] According to this embodiment of the disclosure, the antagonist anti-GIPR antibody of this anti-GIPR / GLP-1 conjugate molecule comprises a first cysteine conjugation site within a light chain or a heavy chain of the anti-GIPR antibody. The first GLP-1 receptor agonist polypeptide of the molecule is conjugated, at its C-terminus, to the first cysteine conjugation site of the anti-GIPR antibody.

[0242] In one embodiment, this anti-GIPR / GLP-1 conjugate molecule further comprises a second GLP-1 receptor agonist polypeptide. The second GLP-1 receptor agonist polypeptide is conjugated, at its C-terminus, to a second cysteine conjugation site of the anti-GIPR antibody. The second cysteine conjugation site of the anti-GIPR antibody is also located within a light chain or heavy chain of the anti-GIPR antibody. Preferably the first and second cysteine conjugations sites of the anti-GIPR antibody are located at corresponding positions within each light chain or within each heavy chain.

[0243] In one embodiment, the first and second GLP-1 receptor agonist polypeptides of this conjugate molecule that are conjugated to the anti-GIPR antibody comprise the same amino acid sequence. In one embodiment, the first and second GLP-1 receptor agonist polypeptides of the conjugate molecule comprise different amino acid sequences.

[0244] In one embodiment, this anti-GIPR / GLP-1 conjugate molecule further comprises a third GLP-1 receptor agonist polypeptide. The third GLP-1 receptor agonist polypeptide is conjugated, at its C-terminus, to a third cysteine conjugation site of the anti-GIPR antibody. The third cysteine conjugation site of the anti-GIPR antibody is also located within a light chain or heavy chain of the anti-GIPR antibody.

[0245] In one embodiment, this anti-GIPR / GLP-1 conjugate molecule further comprises a fourth GLP-1 receptor agonist polypeptide. The fourth GLP-1 receptor agonist polypeptide is conjugated, at its C-terminus, to a fourth cysteine conjugation site of the anti-GIPR antibody. The fourth cysteine conjugation site of the anti-GIPR antibody is also located within a light chain or heavy chain of the anti-GIPR antibody.

[0246] In one embodiment, the first GLP-1 receptor agonist polypeptide of the conjugate molecule is directly conjugated, at its C-terminus, to the first cysteine conjugation site of the anti-GIPR antibody. In one embodiment, each of the first and second GLP-1 receptor agonist polypeptides of the molecule is directly conjugated, at its C-terminus, to the first and second cysteine conjugations sites of the anti-GIPR antibody, respectively. The position of the first and second cysteine conjugation sites within the anti-GIPR antibody are described in more detail infra.

[0247] In one embodiment, the first GLP-1 receptor agonist polypeptide of the conjugate molecule is conjugated, at its C-terminus, to the first cysteine conjugation site of the anti- GIPR antibody via a linker moiety. In one embodiment, each of the first and second GLP-1 receptor agonist polypeptides of the conjugate molecule are conjugated, at their C-terminus, to the first and second cysteine conjugation sites of the anti-GIPR antibody, respectively, via first and second linker moieties, respectively.

[0248] In one embodiment a linker moiety couples the C-terminal end of a GLP-1 receptor agonist polypeptide to a conjugation site on an anti-GIPR antibody. The presence of any linker moiety in the GLP-1 receptor agonist conjugate molecules as described herein is optional. In one embodiment, the linker moiety serves primarily as a spacer to positionjoin, connect, or optimize presentation or position of the functional moieties of the molecule (e.g., the GLP-1 receptor agonist polypeptide and / or the anti-GIPR antibody). In another embodiment, a linker moiety is utilized to optimize pharmacological activity or stability of the GLP-1 receptor agonist containing molecules disclosed herein. In a preferred embodiment, the linker moiety of the anti-GIPR antibody / GLP-1 receptor agonist polypeptide conjugate molecule described herein contributes to the enhanced stability of the molecule in terms of resistance to cleavage upon in vivo administration.

[0249] In one embodiment, the linker moiety is a peptide linker moiety. Peptide linkers are made up of amino acids that are typically linked together by peptide bonds and are a length of from 1 to about 40 amino acid residues, preferably, a length of from 10 to about 30 aminoacid residues, and more preferably, a length of from 10 to about 20 amino acid residues. In some embodiments, the peptide linker may comprise amino acid residues where one or more of the amino acid residues are not linked via a peptide bond. In one embodiment, the amino acid residues of the linker are chosen from among the twenty canonical amino acids. In one embodiment, the amino acid residues of the linker are chosen from among glycine, alanine, proline, asparagine, glutamine, and / or serine. Even more preferably, a peptidyl linker is made up of a majority of amino acids that are sterically unhindered, such as glycine, serine, and alanine linked by a peptide bond. It is also desirable that, if present, the peptidyl linker is one that is not subject to rapid proteolytic turnover in circulation.

[0250] Exemplary linker moi eties of the anti-GIPR antibody / GLP-1 receptor agonist conjugate molecules described herein include the linker peptide of SEQ ID NOs: 20-36 and 187-189.

[0251] In one embodiment, the linkage between a GLP-1 receptor polypeptide and a peptide linker is a peptide bond between the C-terminal amino acid residue of the GLP-1R agonist polypeptide and N-terminal amino acid residue of the peptide linker. In one embodiment, the linkage between a GLP-1 receptor polypeptide and a peptide linker is an amide bond between the C-terminal amino acid residue of the GLP-1R agonist polypeptide and the side chain of a lysine residue (i.e., at the a amino group of the lysine) on the peptide linker. In one embodiment, the linkage between a GLP-1 receptor polypeptide and a peptide linker is an amide bond between the C-terminal amino acid residue of the GLP-1R agonist polypeptide and the amino group of a C-terminal lysine residue on the peptide linker.

[0252] In one embodiment, the molecule disclosed herein comprises a GLP-1 receptor agonist polypeptide of SEQ ID NO: 190 covalently linked to the linker peptide of SEQ ID NO: 187, 188, or 189. In one embodiment, the C-terminal lysine of the linker peptide is modified, e.g., with a bromoacetyl handle, for coupling to the cysteine residue of the conjugation site on the anti-GIPR antibody.

[0253] The fusion proteins or conjugates disclosed herein can be characterized or assessed for GLP-1R agonist and / or GIPR antagonist biological activities including, but not limited to effects on food intake, oral glucose tolerance tests, measurements of blood glucose levels, insulin resistance analysis, changes in body weight, pharmacokinetic analysis, toxicokinetic analysis, immunoassays and mass spec analysis of the level and stability of full-length fusion proteins, and human plasma ex vivo stability analysis. The fusion proteins or conjugatesdisclosed herein can be characterized or assessed for changes in muscle mass via methods well known in the art, such as via dual energy X-ray absorptiometry (DXA) scans.

[0254] The fusion proteins or conjugates disclosed herein can be characterized or assessed for myostatin antagonist biological activities including but not limited to effects on muscle mass and bone density. For example, the efficacy of the various proteins disclosed herein (e.g., modulators of muscle mass such as anti-myostatin antibodies) for improvements in bone density may be confirmed using well established models of osteoporosis. For example, ovariectomized mice have been used to test the efficacy of new osteoporosis drug treatments (Alexander et al., J. Bone Min. Res., 2001, 16: 1665-1673; and Anderson et al., J.Endocrinol., 2001, 170: 529-537). Similar to humans, these rodents exhibit a rapid loss of bone following ovariectomy, especially in cancellous bone. Outcome assessments are based on bone mineral density, biochemical markers of bone turnover in serum and urine, bone strength, and histology / histomorphometry.IV. SWEEPING ANTIBODIES

[0255] Any of the disclosed antibodies or conjugates thereof may be engineered as sweeping antibodies. Examples of mutations that increase Fc receptor binding, increase uptake into a cell, increase dissociation at low pH, and / or are useful in generating sweeping antibodies, are well known in the art (Schroter et al., mAbs, 2015, 7(1): 138-51 ; Yang et al., mAbs, 2017, 9(7): 1105-1117; and Igawa et al., Immunological Reviews, 2016, 270(1): 132-51; all of which are incorporated herein by reference in their entirety). Methods of generating such antibodies as well of testing their efficacy are also provided therein.

[0256] In some embodiments, the mutation is of the human Fc domain and selected from mutation of histidine 268 to glutamine, valine 309 to leucine, alanine 330 to serine and proline 331 to serine. The numbering provided is for IgGl, but comparable mutations, such as can be determined by a skilled artisan, may be made in other IgGs. Further, the efficacy of such antibodies can be examined by performing binding / dissociate assays in media of various pHs. Tests in model organisms such as mice and monkeys are also possible, in which serum levels of the antibody are measured before and after addition thereof.

[0257] In some embodiments, the antibody comprises a mutation in a CDR that decreases binding to an antigen at low pH. It has been shown that incorporation of histidine residues in the binding interfaces of antibodies can be used to engineer pH-dependent binding. The basis for the pH-sensitive binding arises from the histidine's sensitivity to protonation as a result oflowered pH-values in various microenvironments, including intracellular microenvironments, more specifically endosomal vesicles, more preferably early endosomes. Protonation of a histidines' side chain in binding-interfaces can alter electrostatic interactions or induce conformational changes that lead to pH-dependent differences in binding affinity. Thus, in some embodiments, the mutation is mutation to a histidine. In some embodiments, at least 1, 2, 3, 4, or 5 amino acids are mutated to histidine. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are mutated to histidine.

[0258] HIS-scanning, by which each amino acid in the CDRs is replaced by a histidine and pH dependent binding is assessed, is a well-established technique known in the art. More than one histidine mutation may be combined to synergistically or linearly increase the effect of the mutations. Combinatorial HIS-scanning library approaches using phage or yeast display are also well known in the art.V. LINKERS

[0259] Linkers suitable to join the muscle fortifying agent and metabolic component, and / or to join the anti-GIPR antibody or fragment thereof and the metabolic modulator, may be synthetic linkers, peptide linkers, non-peptide linkers, and the like. Synthetic linkers may include a non-peptide organic linker.

[0260] Preferably, the linker may be a linker that does not interfere with folding of the components of the proteins disclosed herein, or that helps to ensure correct folding, such as by minimizing steric hindrance and not interfering significantly with the structure of each component of the protein.

[0261] A linker may be covalently bound to a polypeptide component of the fusion protein or conjugate.

[0262] The linker joining the muscle fortifying agent and the metabolic component may be the same type of linker as used to join the anti-GIPR antibody or fragment thereof and the metabolic modulator. In some embodiments, the linkers are different types of linkers.

[0263] In some embodiments, the linker is a peptide linker comprising 2 to 120 amino acids. For example, the peptide linker may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 or 120 amino acids.

[0264] In certain embodiments, the linker peptide can contain an alanine-proline repeat (i.e., an AP repeat), wherein an AP dipeptide can be referred to as an AP unit. The AP repeat may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 AP units (SEQ ID NO: 271). In certain embodiments, the linker peptide can, for example, comprise 2 to 25 AP units (SEQ ID NO: 259), 5 to 25 units (SEQ ID NO: 260), 4 to 23 AP units (SEQ ID NO: 261), 6 to 21 AP units (SEQ ID NO: 262), 8 to 19 AP units (SEQ ID NO: 263), 10 to 17 AP units (SEQ ID NO: 264), 12 to 15 AP units (SEQ ID NO: 265), 5 to 10 AP units (SEQ ID NO: 266), 5 to 15 AP units (SEQ ID NO: 267), 10 to 25 AP units (SEQ ID NO: 268), 15 to 25 AP units (SEQ ID NO: 269), 20 to 25 AP units (SEQ ID NO: 270), or any value in between. In certain embodiments, the AP repeat can be internal to an alanine-serine dipeptide (i.e., an AS unit) and a glycine-threonine dipeptide (i.e., a GT unit). The AS unit can, for example, be at the amino terminal end of the linker peptide. The GT unit can, for example, be at the carboxyl terminal end of the linker peptide.

[0265] In certain embodiments, the linker peptide comprises a glycine-glycine-glycine- glycine-serine repeat (i.e., a G4S repeat (SEQ ID NO: 191)), wherein a G4S pentapeptide (SEQ ID NO: 191) can be referred to as a G4S unit (SEQ ID NO: 191). The G4S repeat may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 G4S units (SEQ ID NO: 192). In certain embodiments, the linker peptide can, for example, comprise 2 to 15 G4S units (SEQ ID NO: 203), 4 to 13 G4S units (SEQ ID NO: 204), 6 to 11 G4S units (SEQ ID NO: 205), 8 to 9 G4S units (SEQ ID NO: 206), 2 to 8 G4S units (SEQ ID NO: 207), 2 to 6 G4S units (SEQ ID NO: 208), 6 to 8 G4S units (SEQ ID NO: 209), 7 to 8 G4S units (SEQ ID NO: 210), 7 to 9 G4S units (SEQ ID NO: 211), 7 to 10 G4S units (SEQ ID NO: 212), or any value in between. In certain embodiments, an AS or a GS unit can, for example, be at the amino terminal end of the linker peptide. In certain embodiments, the G4S repeat (SEQ ID NO: 191) can be internal to an alanine-serine dipeptide (i.e., an AS unit) and a glycine-threonine dipeptide (i.e., a GT unit). The AS unit can, for example, be at the amino terminal end of the linker peptide. The GT unit can, for example, be at the carboxyl terminal end of the linker peptide.

[0266] In certain embodiments, the linker peptide can contain a glycine-glycine-glycine- glycine-alanine repeat (i.e., a G4A repeat (SEQ ID NO: 193)), wherein a G4A pentapeptide (SEQ ID NO: 193) can be referred to as a G4A unit (SEQ ID NO: 193). The G4A repeat may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 G4A units (SEQ ID NO: 194). Incertain embodiments, the linker peptide can, for example, comprise 2 to 15 G4A units (SEQ ID NO: 213), 4 to 13 G4A units (SEQ ID NO: 214), 6 to 11 G4A units (SEQ ID NO: 215), 8 to 9 G4A units (SEQ ID NO: 216), 2 to 8 G4A units (SEQ ID NO: 217), 2 to 6 G4A units (SEQ ID NO: 218), 6 to 8 G4A units (SEQ ID NO: 219), 7 to 8 G4A units (SEQ ID NO: 220), 7 to 9 G4A units (SEQ ID NO: 221), 7 to 10 G4A units (SEQ ID NO: 222), or any value in between. In certain embodiments, a GA unit can, for example be at the amino terminal end of the linker peptide.

[0267] In certain embodiments, the linker peptide can contain a glutamate-alanine-alanine- alanine-lysine repeat (i.e., an EA3K repeat (SEQ ID NO: 195)), wherein an EA3K pentapeptide (SEQ ID NO: 195) can be referred to as an EA3K unit (SEQ ID NO: 195). The EA3K repeat may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 EA3K units (SEQ ID NO: 196). In certain embodiments, the linker peptide can, for example, comprise 2 to 25 EA3K units (SEQ ID NO: 223), 5 to 25 EA3K units (SEQ ID NO: 224), 4 to 23 EA3K units (SEQ ID NO: 225), 6 to 21 EA3K units (SEQ ID NO: 226), 8 to 19 EA3K units (SEQ ID NO: 227), 10 to 17 EA3K units (SEQ ID NO: 228), 12 to 15 EA3K units (SEQ ID NO: 229), 5 to 10 EA3K units (SEQ ID NO: 230), 5 to 15 EA3K units (SEQ ID NO: 231), 10 to 25 EA3K units (SEQ ID NO: 232), 15 to 25 EA3K units (SEQ ID NO: 233), 20 to 25 EA3K units (SEQ ID NO: 234), or any value in between. In certain embodiments, the EA3K repeat (SEQ ID NO: 195) can be internal to an alanine-serine dipeptide (i.e., an AS unit) and a glycine-threonine dipeptide (i.e., a GT unit). The AS unit can, for example, be at the amino terminal end of the linker peptide. The GT unit can, for example, be at the carboxyl terminal end of the linker peptide.

[0268] In certain embodiments, the linker peptide can contain a proline-glycine-glysine- glycine-serine repeat (i.e., an PG3S repeat (SEQ ID NO: 197)), wherein an PG3S pentapeptide (SEQ ID NO: 197) can be referred to as an PG3S unit (SEQ ID NO: 197). The PG3S repeat may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 PG3S units (SEQ ID NO: 198). In certain embodiments, the linker peptide can, for example, comprise 2 to 25 PG3S units (SEQ ID NO: 235), 5 to 25 PG3S units (SEQ ID NO: 236), 4 to 23 PG3S units (SEQ ID NO: 237), 6 to 21 PG3S units (SEQ ID NO: 238), 8 to 19 PG3S units (SEQ ID NO: 239), 10 to 17 PG3S units (SEQ ID NO: 240), 12 to 15 PG3S units (SEQ ID NO: 241), 5 to 10 PG3S units (SEQ ID NO: 242), 5 to 15 PG3S units (SEQ ID NO: 243), 10 to 25 PG3S units (SEQ ID NO: 244), 15 to 25 PG3S units (SEQ ID NO: 245), 20 to 25 PG3S units (SEQ ID NO: 246), or any value in between. In certain embodiments, the PG3Srepeat (SEQ ID NO: 197) can include a glycine-serine dipeptide (i.e., a GS unit) at the amino terminal end of the linker peptide.

[0269] In certain embodiments, the linker peptide can contain an alanine-glycine-glysine- glycine-serine repeat (i.e., an AG3S repeat (SEQ ID NO: 199)), wherein an AG3S (SEQ ID NO: 199) pentapeptide can be referred to as an AG3S unit (SEQ ID NO: 199). The AG3S repeat may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 AG3S units (SEQ ID NO: 200). In certain embodiments, the linker peptide can, for example, comprise 2 to 25 AG3S units (SEQ ID NO: 247), 5 to 25 AG3S units (SEQ ID NO: 248), 4 to 23 AG3S units (SEQ ID NO: 249), 6 to 21 AG3S units (SEQ ID NO: 250), 8 to 19 AG3S units (SEQ ID NO: 251), 10 to 17 AG3S units (SEQ ID NO: 252), 12 to 15 AG3S units (SEQ ID NO: 253), 5 to 10 AG3S units (SEQ ID NO: 254), 5 to 15 AG3S units (SEQ ID NO: 255), 10 to 25 AG3S units (SEQ ID NO: 256), 15 to 25 AG3S units (SEQ ID NO: 257), 20 to 25 AG3S units (SEQ ID NO: 258), or any value in between. In certain embodiments, the AG3S repeat (SEQ ID NO: 199) can include a glycine-serine dipeptide (i.e., a GS unit) at the amino terminal end of the linker peptide.

[0270] In certain embodiments, the linker peptide can contain EGKSSGSGSESKST (SEQ ID NO: 32) or a repeat thereof, such as 2, 3, 4, 5, 6, 7, 8. 9, or 10 repeats of the 14 amino acid peptide. In certain embodiments, the 14 amino acid peptide or repeat thereof can be internal to a glycine-glycine-serine tripeptide (i.e., an GGS unit), such that the GGS unit can be at the amino terminal end of the linker peptide and the carboxyl terminal end of the linker peptide.

[0271] In certain embodiments, the linker peptide can be a poly-glycine peptide. The polyglycine peptide can comprise about 6 to about 50 glycine residues, about 10 to about 45 glycine residues, about 15 to about 40 glycine residues, about 20 to about 35 glycine residues, about 25 to about 30 glycine residues, about 20 to about 25 glycine residues, or any number in between. The poly-glycine first linker peptide can comprise 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 glycine residues.

[0272] In some embodiments, the linker further comprises a C-terminal amino acid selected from the group consisting of lysine, cysteine, serine, threonine, tyrosine, aspartic acid, glutamic acid, glutamine, and asparagine, to allow for bioconjugation. In some embodiments, the linker further comprises a C-terminal amino acid, such as lysine or a cysteine, to allow for bioconjugation. In some embodiments, the C-terminal amino acid ofthe linker peptide is modified, e.g., with a bromoacetyl handle or maleimide handle on the sidechain, as shown in FIG. 6, for coupling to the cysteine residue of the conjugation site on the antibody, comprising GIPR binding domains.

[0273] In some embodiments, the linker can be a flexible linker comprising any of the sequences of SEQ ID NOs: 20-36 or 187-189.

[0274] In some embodiments, the linker or the method of linking is as described in any of U.S. Patent Application Pub. Nos. 20170151341; 2017 / 0043033; 2014 / 0356385; 2006 / 0104968; and 2023 / 0263904, or a derivative thereof.

[0275] In some embodiments, the linker is attached to an azide-modified amino acid using copper-free click chemistry, for example as described in U.S. Patent No. 12,076,412.

[0276] In some embodiments, the linker is a releasable type, such as a chain of amino acids containing at least one bond that can break under physiological conditions. This includes bonds that are pH-labile, acid-labile, base-labile, oxidatively labile, metabolically labile, biochemically labile, or enzyme-labile. It is important to note that the physiological conditions leading to bond cleavage do not necessarily involve biological or metabolic processes; they may also include standard chemical reactions like hydrolysis or substitution reactions. For instance, an endosome may have a lower pH than cytosolic conditions, or disulfide bond exchange reactions may occur with intracellular thiols.

[0277] The releasable linker can result in a shortened half-life of the conjugate, allowing the myostatin modulator and metabolic modulator to function independently while retaining biological activity. In some embodiments, internalization of the myostatin modulator and / or metabolic modulators cause release of the metabolic modulator from the conjugate and may enhance activity of either or both of the myostatin modulator and metabolic modulator.PHARMACEUTICAL COMPOSITION

[0278] In some embodiments, the fusion protein or conjugate is provided as a pharmaceutical composition comprising the fusion protein or conjugate and a pharmaceutically acceptable carrier. “Pharmaceutically acceptable” means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effects in the subjects to which they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art.

[0279] The pharmaceutically acceptable carrier can include one or more of pharmaceutically acceptable excipient, buffer, stabilizer or other materials known to those skilled in the art.Examples of pharmaceutically acceptable carriers include, but are not limited to, one or more of water, saline, buffer, isotonic agents such as sugars, polyalcohols, auxiliary substances such as wetting or emulsifying agents, preservatives, as well as combinations thereof. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient(s) at the dosages and concentrations employed. The precise nature of the carrier or other material can depend on the route of administration, e.g., intramuscular, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., gut), intranasal or intraperitoneal routes. For example, liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can also be included. Compositions for parenteral administration can be stored in lyophilized form or in a solution and are generally placed into a container having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0280] According to embodiments of the invention, a pharmaceutical composition can comprise one or more additional components, such as another active ingredient.NUCLEIC ACID MOLECULES

[0281] The invention also provides an isolated nucleic acid molecule encoding a fusion protein or conjugate of the invention. In embodiments, the isolated nucleic acid molecule encodes a fusion protein comprising the amino acid sequence of any one of the disclosed anti-myostatin antibody or fragment thereof, or anti-ActRIIB antibody or fragment thereof and any one of the amino acid sequences of an anti-GIPR antibody or fragment thereof. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence encoding a GLP-1R agonist or GIPR antagonist, such as SEQ ID NOs: 1-19.

[0282] In certain embodiments, the isolated nucleic acid molecule encodes a fusion protein comprising (i) the amino acid sequence of any one of the disclosed anti-myostatin antibodies or anti-ActRIIB antibodies, (ii) any one of the amino acid sequences of an GLP-1R agonists or GIPR antagonist, such as SEQ ID NO: 1-19, and (iii) any one of the amino acid sequences of the linkers disclosed herein, such as SEQ ID NOs: 20-36 and 187-189, wherein (i) and (ii) are connected via the linker (iii).

[0283] According to other embodiments, the nucleic acid molecule encoding the fusion protein can be in an expression vector. Expression vectors include, but are not limited to,vectors for recombinant protein expression and vectors for delivery of nucleic acids into a subject for expression in a tissue of the subject, such as viral vectors. Examples of viral vectors suitable for use with the invention include, but are not limited to adenoviral vectors, adeno-associated virus vectors, lentiviral vectors, etc. The vector can also be a non-viral vector. Examples of non-viral vectors include, but are not limited to plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, etc. The vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, or an origin of replication.

[0284] According to other embodiments of the invention, the nucleic acid molecule encoding the fusion protein can be codon optimized for improved recombinant expression from a desired host cell, such as Human Embryonic Kidney (EEK) or Chinese hamster ovary (CHO) cells, using methods known in the art in view of the present disclosure.

[0285] The invention also provides a host cell comprising a nucleic acid molecule encoding a fusion protein of the invention. Host cells include, but are not limited to, host cells for recombinant protein expression and host cells for delivery of the nucleic acid into a subject for expression in a tissue of the subject. Examples of host cells suitable for use with the invention include, but are not limited to, EEK or CHO cells.

[0286] In another general aspect, the invention relates to a method of obtaining a fusion protein of the invention. In a general aspect, the method comprises: (1) culturing a host cell comprising a nucleic acid molecule encoding a fusion protein under a condition that the fusion protein is produced, and (2) recovering the fusion protein produced by the host cell. The fusion protein can be purified further using methods known in the art.

[0287] In some embodiments, the fusion protein is expressed in host cells and purified therefrom using a combination of one or more standard purification techniques, including, but not limited to, affinity chromatography, size exclusion chromatography, ultrafiltration, and dialysis. Preferably, the fusion protein is purified to be free of any proteases.

[0288] The invention further provides a composition comprising a nucleic acid molecule encoding a fusion protein or conjugate of the invention and a pharmaceutically acceptable carrier. Compositions comprising a nucleic acid molecule encoding a fusion protein or conjugate of the invention can comprise a delivery vehicle for introduction of the nucleic acid molecule into a cell for expression of the fusion protein. Examples of nucleic acid deliveryvehicles include liposomes, biocompatible polymers, including natural polymers and synthetic polymers, lipoproteins, polypeptides, polysaccharides, lipopolysaccharides, artificial viral envelopes, metal particles, and bacteria, viruses, such as baculoviruses, adenoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors and other recombination vehicles typically used in the art that have been described for expression in a variety of eukaryotic hosts.METHODS

[0289] The invention relates to use of the proteins (e.g., peptides, fusion proteins or conjugates, antibodies) or pharmaceutical compositions described herein to treat or prevent a metabolic disease, disorder, or condition. The invention also relates to use of the proteins (e.g., peptides, fusion proteins or conjugates, antibodies) or pharmaceutical compositions described herein to treat or prevent a muscle and neuromuscular disorder or bone degenerative disease such as osteoporosis. The antibodies are optionally used to prevent, diagnose, or treat such medical disorders in mammals, optionally in humans.

[0290] According to embodiments of the invention, a method of treating or preventing a metabolic disease, disorder, or condition in a subject in need of the treatment comprises administering to the subject a therapeutically or prophylactically effective amount of a pharmaceutical composition of the invention. Any of the proteins or pharmaceutical compositions described herein can be used in a method of the invention, including pharmaceutical compositions comprising a fusion protein or conjugate of the invention or pharmaceutical compositions comprising a nucleic acid encoding the fusion protein or conjugate.

[0291] According to embodiments of the invention, a method of treating or preventing a muscle and neuromuscular or bone degenerative disease, disorder, or condition in a subject in need of the treatment comprises administering to the subject a therapeutically or prophylactically effective amount of a pharmaceutical composition of the invention. Any of the proteins or pharmaceutical compositions described herein can be used in a method of the invention, including pharmaceutical compositions comprising a fusion protein or conjugate of the invention or pharmaceutical compositions comprising a nucleic acid encoding the fusion protein or conjugate.

[0292] As used herein, “subject” means any animal, particularly a mammal, most particularly a human, who will be or has been treated by a method according to an embodiment of theinvention. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) such as monkeys or apes, humans, etc., more particularly a human.

[0293] A “metabolic disease, disorder or condition” refers to any disorder related to abnormal metabolism. Examples of metabolic diseases, disorders or conditions that can be treated according to a method of the invention include, but are not limited to, type 2 diabetes, elevated glucose levels, elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, rheumatoid arthritis, adipose tissue disorder such as obesity, impaired glucose tolerance, metabolic syndromes (e.g., syndrome X), insulin resistance induced by trauma such as burns.

[0294] A “muscle and neuromuscular disorder” may include muscular dystrophy, muscle atrophy, congestive obstructive pulmonary disease, muscle wasting syndrome, sarcopenia, or cachexia, while a “bone degenerative disease” refers to any disease or disorder associated with a loss of bone, which may include osteoporosis, especially in the elderly and / or postmenopausal women, glucocorticoid-induced osteoporosis, osteopenia, and osteoporosis- related fractures. Other target metabolic bone diseases and disorders amendable to treatment with the proteins and therapies of the invention include low bone mass due to chronic glucocorticoid therapy, premature gonadal failure, androgen suppression, vitamin D deficiency, secondary hyperparathyroidism, nutritional deficiencies, and anorexia nervosa.

[0295] The terms “treat,” “treating,” and “treatment” as used herein refer to administering a composition to a subject to achieve a desired therapeutic or clinical outcome in the subject. In one embodiment, the terms “treat,” “treating,” and “treatment” refer to administering a pharmaceutical composition of the invention to reduce, alleviate or slow the progression or development of a metabolic disorder, such as type 2 diabetes, elevated glucose levels, elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, or rheumatoid arthritis.

[0296] The term “therapeutically effective amount” means an amount of a therapeutically active compound needed to elicit the desired biological or clinical effect. According to embodiments of the invention, “a therapeutically effective amount” is an amount sufficient to effect beneficial or desired results, including clinical results. A therapeutically effective amount can be administered in one or more administrations. In terms of a disease state, aneffective amount is an amount sufficient to ameliorate, stabilize, or delay development of a disease. According to specific embodiments of the invention, a therapeutically effective amount is an amount of a fusion protein needed to treat or prevent a metabolic disease, disorder or condition, such as type 2 diabetes, elevated glucose levels, elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic injury, congestive heart failure, or rheumatoid arthritis.

[0297] According to embodiments of the invention, a pharmaceutical composition of the invention can be administered to a subject by any method known to those skilled in the art in view of the present disclosure, such as by intramuscular, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., gut), intranasal or intraperitoneal route of administration. In particular embodiments, a pharmaceutical composition of the invention is administered to a subject by intravenous injection or subcutaneous injection.

[0298] Parameters such as the dosage amount, frequency of administration, and duration of administration of a pharmaceutical composition to a subject according to an embodiment of the invention are not limited in any particular way. The optimum values of such parameters can depend on a variety of factors, such as the subject to be treated, the particular metabolic disease to be treated, the severity of the disease, the route of administration, etc., and one of ordinary skill in the art will be able to determine the optimum values for such parameters in order to achieve the desired therapeutic or clinical outcome. For example, a pharmaceutical composition can be administered once per day, or more than once per day, such as twice, three times, four times, etc. Atypical dosage can range from about 0.1 pg / kg to up to about 100 mg / kg or more of the fusion protein, depending on the factors such as those mentioned above.SYSTEMS

[0299] The invention also relates to systems for treating muscle wasting, a metabolic disorder, or a combination thereof in a subject comprising administering to the subject any of the fusion proteins or conjugates disclosed herein.

[0300] In some embodiments, the system further comprises a delivery system. In further embodiments, the delivery system comprises a syringe.

[0301] In some embodiments, the immunogenic composition is administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, transdermal, or oral route of administration.KITS

[0302] The invention also relates to kits comprising a pharmaceutical composition of the invention. The kit can contain a first container having a dried protein of the invention (e.g., fusion protein or conjugate) and a second container having an aqueous solution to be mixed with the dried fusion protein prior to administration to a subject.

[0303] The kit can contain a single container containing a liquid pharmaceutical composition of the invention.

[0304] The kit can contain a single-dose administration unit or multiple dose administration units of a pharmaceutical composition of the invention. The kit can also include one or more pre-filled syringes (e.g., liquid syringes and lyosyringes). A kit can also comprise instructions for the use thereof. The instructions can describe the use and nature of the materials provided in the kit and can be tailored to the precise metabolic disorder being treated.

[0305] In some embodiments, the kit further includes additional sterile tubes useful for making dilutions to the fusion proteins, conjugates, or pharmaceutical compositions.

[0306] In further embodiments, any of the fusion proteins, conjugates, pharmaceutical compositions, or dilutions thereof are to be administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, transdermal, or oral route of administration.EXAMPLESExample 1: Production of an anti-GIPR antibody

[0307] (a) Immunization and hybridoma fusion

[0308] Six to twelve week old female mice such as B6129 were immunized with human GIPR-ECD-his protein and cyno GIPR-ECD-his protein, or human GIPR-ECD-hFc.Immunization was performed following the standard lACUC-approved hock immunization method at a Charles River Labs animal facility (825 Industrial Road, Ste 100B, San Carlos, CA).

[0309] After 30-60 days of immunization, B cells from lymph nodes and spleen were harvested and pooled for fusion. Electric fusion was performed with Napagene ECFG21 and manufacturer’s instructions were followed.

[0310] Fused hybridoma cells were plated into microtiter plates and grown for 7-14 days before screening. Multiple fusion and screening campaigns were performed from the same immunization strategy to maximize the number of clones screened.

[0311] (b) Hybridoma screening

[0312] Hybridoma supernatants were screened by human GIPR-ECD binding ELISA to identify clones that can bind to GIPR. Briefly, goat anti -mouse Fc was coated on ELISA plates overnight and then blocked. Hybridoma supernatants were added to the plates. After incubation and washing, 50ng / ml of biotin-human GIPR-ECD-his was then added to the plates.

[0313] Hybridoma clones were initially screened by ELISA to select binders to human GIPR- ECD, and those that demonstrated strong binding were interrogated in huGIPR reporter cells to identify functional blockers of GIP-GIPR interaction (e.g., GIPR antagonists) to identify functional blockers of GIP-GIPR interaction (e.g., GIPR antagonists).

[0314] Hybridoma clones that showed binding to GIPR-ECD were then selected. A 293 reporter cell line with engineered GIPR was used to assess hybridoma activity. Hybridoma clones that showed blocking activities were expanded, purified, and re-tested in the reporter assay to rank order, and compared to reference antibodies.

[0315] Clones that showed blocking activity were sequenced using 5’ SMARTER RACE kit from Takara (Cat #634858) and manufacturer’s instructions were followed. One clone of choice had an EC90 of 3.0 nM to 5.7 nM in a GIPR reporter cell assay depending on the reporter cells. Exemplary assay for antagonism of the exemplary anti-GIPR antibodies of the invention to the reporter cells that express GIPR, is shown in FIG. 4.

[0316] (c) Humanized anti-GIPR antibody

[0317] A clone was humanized by grafting CDRs into human germline sequences and select framework residues were also mutated. Different light chain and heavy chain combinations were transfected, purified, and tested for GIPR blocking activities in the reporter-based assay. Multiple HC and LC humanization constructs were generated, and these were expressed in different combinations to identify the best lead candidate. Humanized leads were selected based on potency in reporter cell assays (below), as well as other attributes including transient expression titer in 293F cells, purification behavior, and biochemical binding on Biacore. An exemplary humanized clone of choice demonstrated an EC50 of 3.1 nM using a GIPR reporter cell assay and a KD of about 2* 10-10 M in a biochemical binding assay (e.g.,measured via a surface plasma resonance technique, e.g., BIACore, GE-Healthcare Uppsala, Sweden).

[0318] (d) Dual GIP / GLP-1 HTRF Reporter Assay

[0319] Lead antibodies were cloned into chimeric antibody format and purified to confirm blocking activities using GIPR reporter cells. A dual GIP / GLP-1 homogeneous time-resolved fluorescence (HTRF) reporter assay measuring cAMP production was performed on the anti- GIPR antibody component or the chimeric antibody component of an inventive proteins (i.e., anti-GIPR as defined by any of SEQ ID Nos: 37-110 linked to GLP-1 as defined by any of SEQ ID Nos: 1-14, 17 or 190, optionally attached via a linker as defined by any of SEQ ID Nos: 20-36 or 187-189). 293F cells stably expressing human GIPR were used to measure anti-GIPR-induced cAMP generation in a HTRF assay (Cisbio, cat #62AM4PEJ). Serial dilutions of the inventive proteins of the present disclosure were incubated with about 30,000 cells in assay buffer (0.1% BSA, 500 pM IBMX in F12 media) for 30 min at 37° C before being treated with GIP at final concentration of 0.05 nM. Cells were incubated for 30 min at 37° C and lysed in lysis buffer containing cAMP-d2 and cAMP cryptate (CISBIO) for 1 hour at room temperature. The fluorescence was measured in Evision plate reader (PerkinElmer) and the cAMP levels were expressed as a ratio of 665 / 620 nm and recorded (see Example 3(a)).

[0320] The clone of choice demonstrated an EC50 of 2.2 pg / ml and an EC90 of 61.2 pg / ml in a GIPR reporter cell assay and an EC50 of 21.5 pg / ml and an EC90 of 128 pg / ml in a GLP-1 reporter cell assay.

[0321] Exemplary assay for GLP-1 / GIPR receptor activities is shown in FIG. 5. Dual GIPR antagonism and GLP-1R agonism against reporter cells expressing either GIPR or both GIPR and GLP-1R was assessed.

[0322] (e) Pharmacokinetics

[0323] The anti-GIPR (representative antibody AB025) GLP-1 conjugate molecule was well tolerated following a single subcutaneous administration in six male Mauritian cynomolgus monkeys, with no significant adverse events observed throughout the study period. The compound demonstrated high and sustained systemic exposure, as indicated by robust and persistent total antibody levels. Importantly, reductions in body weight were recorded on Days 8 and 15 post-dose, suggesting a pharmacodynamic response aligned with weightmodulation. Graphical representation of the observed effects are provided in FIGs. 7A and7BExample 2. Generation of Antibody / GLP-1 Peptide Conjugates.

[0324] The reaction scheme for the generation of conjugates of GLP-1 peptide agonist and antibodies comprising GIPR binding domains, is shown in FIG. 6.

[0325] Antibodies comprising GIPR binding domains with a specific cysteine mutation (2 g) were incubated with 800 mL solution of 2.5 mM cystamine and 1 mM cysteamine in 40 mM HEPES buffer, pH 7.5-8.5 for 15-20 h. The reaction mixture was filtered using a 0.22 pm filter and diluted in 1200 mL of 100 mM sodium acetate buffer pH 5. Cation exchange chromatography was used to purify the bis-cysteamine-capped antibody from the reaction mixture. The 2 L of reaction mixture diluted in 100 mM sodium acetate buffer pH 5 was loaded onto a 240 mL SP HP column at 20 mL / min. The column was washed with 2 column volumes of 100 mM sodium acetate pH 5, followed by a 0-20% gradient of 100 mM sodium acetate with 1 M NaCl pH 5. The main peak containing bis-cysteamine-capped antibodies was collected and buffer exchanged to 10 mM sodium acetate with 9% sucrose pH 5.2 using tangential flow filtration.

[0326] Bis-cysteamine-capped antibodies (6 mg / mL in 10 mM sodium acetate with 9% sucrose) was partially reduced using 3-4 equivalents of triphenylphosphine-3, 3', 3 "-trisulfonic acid trisodium salt (TPPTS) at RT for 60-90 min. TPPTS was removed, and partially reduced cys antibodies were buffer exchanged to 50 mM sodium phosphate buffer containing 2 mM ethylenediaminetetraacetic acid (EDTA) pH 7.5. To the partially reduced cys antibodies were added 6-10 equivalents of dehydroascorbic acid (DHAA), and oxidation was carried out until only trace amount of partially reduced antibody species were observed (30-120 min). Without removing DHAA, 3-8 equivalents of bromoacetyl-GLP-lR agonist peptide was added to the reaction mixture and incubated for 12-16 hrs. Either cation exchange chromatography or hydrophobic interaction chromatography were used to purify the conjugates. For cation exchange, the reaction was diluted in 100 mM sodium acetate buffer pH 5 and loaded on a 5 mL SPHP column at 5 mL / min. The column was then washed with 2 column volumes of 20 mM sodium acetate pH 5, followed by a 0-20% gradient of 20 mM sodium acetate with 1 M NaCl pH 5. For hydrophobic interaction chromatography, the reaction was diluted in a buffer containing 1 M ammonium sulfate and 20 mM sodium acetate pH 5. The sample was then loaded on a 5 mL Butyl HP column at 5 mL / min. The column was washed with 2 columnvolumes of a buffer containing 1 M ammonium sulfate and 20 mM sodium acetate pH 5, followed by a 0-100% gradient of a buffer containing 20 mM sodium acetate and 10% acetonitrile pH 5. The main peak containing the antibody / GLP-1 conjugates comprising a drug-to-antibody ratio of 2: 1 (DAR2) was collected and buffer exchanged into 10 mM sodium acetate with 9% sucrose pH 5.2 using spin concentration.Example 3: Cell Based Assays

[0327] (a) GLP-1 Receptor Agonist Activity.

[0328] cAMP production is measured, indicative of GLP-1R activation. CHOK1 cells stably expressing the human GLP-1R alone or both GLP-1R and GIPR are used to measure cAMP production induced by the peptides / proteins / conjugates of the present disclosure (“inventive proteins”). Measurement of cAMP production may use a homogeneous time-resolved fluorescence (HTRF) assay (CISBIO, cat #62AM4PEJ). Serial diluted inventive proteins are incubated with about 40,000 cells in assay buffer (0.1% BSA, 500 uM IB MX in F12 media) for 15 min at 37° C. Cells are then lysed with lysis buffer containing cAMP-d2 and cAMP cryptate (CISBIO) and incubated for 1 hour at room temperature before measurement in an Evision plate reader (PerkinElmer). The cAMP levels are expressed as a fluorescence ratio of 665 / 620 nm and recorded. cAMP production is assessed and associated with GLP-1R activation of the molecules described herein.

[0329] Alternatively, the insulinotropic activity of the inventive proteins is determined as follows. Pancreatic islets are isolated from pancreatic tissue from normal rats by a modification of the method of Lacy, P.E., et al., Diabetes, 1967, 16:35-39, in which the collagenase digest of pancreatic tissue is separated on a Ficoll gradient (27%, 23%, 20.5% and 11% in Hanks' balanced salt solution, pH 7.4). The islets are collected from the 20.5% / l 1% interface, washed and handpicked free of exocrine and other tissue under a stereomicroscope. The islets are incubated overnight in RPMI 1640 medium supplemented with 10% fetal bovine serum and containing 11 mM glucose at 37° C and 95% air / 5% CO2. The islets are then transferred to RPMI 1640 medium supplemented with 10% fetal bovine serum and containing 5.6 mM glucose. The islets are incubated for 60 minutes at 37° C, 95% air / 5% CO2. An inventive protein of the present disclosure is prepared at test concentrations (e.g., InM and lOnM concentrations) in RPMI medium containing 10% fetal bovine serum and 16.7 mM glucose. About 8 to 10 isolated islets are then transferred by pipette to a total volume of 250 pl of the medium containing the inventive protein in 96 well microtiter dishes.The islets are incubated in the presence of the inventive protein at 37° C, 95% air / 5% CO2 for 90 minutes. Aliquots of islet-free medium are collected and 100 pl or so thereof are assayed for the amount of insulin present by radioimmuno-assay using an Equate Insulin RIA Kit (Binax, Inc., Portland, ME).

[0330] (b) GIPR Antagonist Activity.

[0331] HEK 293T cells stably expressing human GIPR are used to measure inventive protein-induced cAMP generation in a HTRF assay (Cisbio, cat #62AM4PEJ). Serial dilutions of the inventive proteins of the present disclosure are incubated with about 30,000 cells in assay buffer (0.1% BSA, 500 pM IBMX in F12 media) for 30 min at 37° C before being treated with GIP at final concentration of 0.05 nM. Cells are incubated for 30 min at 37° C and lysed in lysis buffer containing cAMP-d2 and cAMP cryptate (CISBIO) for 1 hour at room temperature. The fluorescence is measured in Evision plate reader (PerkinElmer) and the cAMP levels are expressed as a ratio of 665 / 620 nm and recorded.

[0332] (c) Myostatin Inhibitor Activity

[0333] In this assay, a plasmid encoding a reporter gene, i.e., luciferase gene, downstream of a SMAD binding element (“SBE”), more specifically (CAGA)i2 expresses luciferase protein when a molecule such as myostatin, GDF-11, or another TGF-P superfamily member binds its own receptor, thereby triggering SMAD signaling which results in a phosphorylated SMAD complex which is capable of binding the SBE. The amount of active myostatin exposed to the cells is directly proportional to the quantity of luciferase enzyme produced which is directly proportional to the quantity of light produced and measurable. The presence of an inhibitor, such as any of the inventive proteins disclosed herein may reduce the quantity of myostatin able to activate the SBE which ultimately results in a reduced production of light. This assay is described in International Publication No. WO 2004 / 037861 incorporated herein in its entirety.

[0334] 293E cells (Edge Biosystems) are grown in in a T-75 flasks containing DMEM / F12 media (1 : 1) (Gibco 10565-042) and 10% FBS. The cells are transfected with a mix of 100 pl lipofectamine 2000 (Invitrogen 11668-019), 5 ml OptiMEM I (Gibco 51985-034) and 30 pg SB-luciferase DNA for 4 hours at 37° C. The transfection mix is then removed and complete media added for 1 hour at 37° C. The cells are then trypsinized and re-suspended in complete media at 2^ 106 cells / ml and 50 pl plated in each well of a Biocoat 96-well plate (BD 35- 6461) and incubated for 1 hour at 37° C. After the incubation is complete, media is replacedwith 100 pl of each inventive protein to be tested , which is serially diluted 1 :2 and preincubated for 1 hour at 37° C. with a 1 : 1 solution of 40 ng / ml myostatin (R&D Systems 788- G8) or GDF-11 (R&D Systems) in complete media.

[0335] The plate is left overnight at 37° C., 5% CO2 and the following day 100 pl of a 1 : 1 mix of Gio Lysis Buffer and Bright-Glo Luciferase reagent (Promega) is added to each well and mixed by pipetting. From this mix, 150 pl is transferred to a white 96-well plate and luminescence measured using a luminometer. Luminescence is then plotted against Fab concentration and the IC50 for each Fab for myostatin and GDF-11 is calculated.

[0336] Using this assay, the IC50 for an anti-myostatin disclosed is typically about InM.

[0337] While specific conditions for this Myostatin / SBE Reporter Assay are described herein, other growth conditions for the cells, assay conditions including varying amounts of myostatin in the reaction, types of cells (e.g., 293HEK (ATCC)), and / or types of reporters (e.g., CAT, P-gal, GFP, etc.) may be used.

[0338] (d) Glucose Stimulated Insulin Secretion (GSIS) Assay

[0339] Human pancreatic islet microtissues are purchased from a source, such as InSphero and are in 96 well format. Microtissues are incubated for 16 hours in InSphero assay media. Tissue is treated for 1 hour with fresh KREBS buffer (129 mM NaCl, 4.8 mM KC1, 1.2 mM KH2PO4, 1.2 mM MgSO4-7H2O, 10 mM HEPES, 1.3 mM CaCl2, 0.5% BSA, pH 7.4) with 2.8 mM glucose. Following 2 KREBS buffer washes, they are incubated with KREBS buffer alone, 2.8 mM or 11 mM glucose in KREBS buffer with or without GLP-l(Phoenix Pharmaceuticals) and varied amounts (i.e., dose response) of the inventive proteins disclosed herein for 16 hours. Supernatant is collected and may be analyzed for insulin secretion according to manufacturer's protocol (Mercodia). Optical density is read at 450 nm on a TEC AN microplate reader.

[0340] (e) Neutralizing Ability of Anti-Myostatin Antibodies or Conjugates Thereof

[0341] The ability of the inventive proteins to neutralize myostatin mediated signaling are confirmed using, for example a reporter gene assay. A reporter construct is constructed by placing 12 CAGA boxes upstream of the TATA box and transcription initiation site from the adenovirus major later promoter in luciferase reporter vector pGL3 (Promega). The CAGA box, which is found in the promoter of the PALI gene, is a TGFP response element that also responds to myostatin. The human rhabdomyosarcoma cell line A204 (ATCC HTB-82) is transiently transfected with pGL3(CAGA)12 and cultured in 96-well plates in McCoy's 5Amedium supplemented with 2 mM glutamine, 100 U / ml streptomycin, 100 pg / ml penicillin and 10% fetal calf serum for 16 hrs. The inventive proteins disclosed herein are preincubated with myostatin (10 ng / ml) in medium supplemented with 1 mg / ml BSA for 1 hr. at room temperature. Cells are then treated for 6 hrs at 37° C with test samples and controls including no myostatin and myostatin (10 ng / ml) with no antibody added. Luciferase activity is measured using the Luciferase Assay System (Promega),

[0342] An average EC50 value for the anti-myostatin disclosed herein has been found to be about 9 nM.

[0343] (f) Elisa Assay for Myostatin Binding Affinity.

[0344] Myostatin binding by the inventive proteins disclosed herein may also be analyzed using quantitative ELISA. The ability of the inventive proteins to inhibit myostatin binding to its cognate high affinity receptor ActRIIB is determined by calculating IC50 values.

[0345] For ELISA, an ActRIIB-Fc fusion protein (1 pg / ml in 0.2 M sodium carbonate buffer) is coated on 96 well flat-bottom assay plates overnight at 4° C. Coated plates are then blocked with 1 mg / ml BSAin PBS 0.1% Tween (200 pl / well) for 1 hour at room temperature or overnight at 4° C and then washed. Different inventive protein concentrations are combined with 10 ng / ml myostatin conjugated to biotin and incubated for 45 minutes at room temperature. After incubation, the test solution is added to the blocked ELISA plate (100 pl / well) and further incubated for 1 hour at room temperature. After washing the wells, the amount of myostatin bound to the immobilized ActRIIB-Fc relative to control is detected with streptavidin-horseradish peroxidase (30-minute incubation) and TMB. Colorimetric measurements at 450 nm are recorded in a microplate reader.

[0346] IC50 values determined using this assay have demonstrated the anti -myostatin disclosed herein has an IC50 value of 9 nM for myostatin and GDF-11. IC50 value of >1000 nM can be determined for Activin A, Activin B, and BMP9.

[0347] (g) Surface Plasmon Resonance Assay for Myostatin Binding Affinity.

[0348] Surface plasmon resonance (SPR) is performed at 25° C using a BIACORE 3000 (GE Healthcare) machine. Protein A is immobilized on all flow cells of a CM5 sensorchip using amine coupling chemistry. The surface is activated by injecting a solution of 0.2M N-ethyl-N- dimethyl-amino-propyl-carbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS) for 7 minutes. Protein A is diluted to 50 pg / ml in 10 mM Sodium acetate buffer at pH 5.0 and injected for 3 minutes at a flow rate of 10 pl per minute. The surface is then blocked with IMethanolamine (ETH) for 7 minutes. Final immobilization levels of protein A are typically between 1000-1200 Response Units (RU). The immobilization procedure is followed by several washes with running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% P20) to equilibrate the surface.

[0349] Inventive proteins are diluted to 0.25 pg / ml in HBS-EP buffer. Solutions of each inventive protein are injected over the Protein A coated flow cells 2, 3 or 4 at a rate of 10 pl / min, yielding approximately 200 RU of captured antibody. A myostatin titration series (2- fold dilutions from 4.0 nM to 0.125 nM) is prepared in 0.01M Sodium acetate at pH 5.0, 0.15M NaCl, 3 mM EDTA, 0.005% P20. The latter solution is also used as running buffer. Myostatin solution is injected over the inventive protein for 2 minutes at a flow rate of 50 pl / min and allowed to dissociate for 30 min. After each cycle of injection and capture the sensor chip surface is regenerated with 30 pl of 10 mM NaPCU, 0.5 M NaCl at pH 2.5 at a flow rate of 50 pl / min. BlAevaluation software (ver. 4.1.1, GE Healthcare) may be used for data analysis. Data may be double referenced by subtracting the signal contributed by the buffer and the reference surface. A Langmuir 1 : 1 model may be used to globally fit the sensorgram data and calculate KD values.

[0350] Using this assay, an anti -myostatin disclosed herein has a KD of 2.59 pM.Example 4: In Vivo Mouse Assays

[0351] (a) Screening for GLP-1, GIPR, and / or myostatin activity, GIPR.

[0352] Diabetic mice are selected to screen for GLP-1 activity, GIPR inhibition, and / or myostatin inhibition of the inventive proteins. Treatment with the inventive proteins is expected to reduce plasma glucose levels at different time points post injection. In addition, body weight loss or inhibition of body weight gain is measured indicative of GLP-1 activity, GIPR inhibition, or a combination of both, from administration of the inventive proteins.

[0353] Male db / db mice (#642) are obtained from Jackson Laboratories (Bar Harbor, ME) and delivered at 8-9 weeks of age. Mice are group-housed at four per cage and maintained in controlled environmental conditions with 12-hour light (e.g., 6:30 AM-6:30 PM) and dark cycles (e.g., 6:30 PM-6:30 AM). Mice are fed a standard rodent chow diet (2020 Harlan Teklad) with free access to drinking water.

[0354] A glucose tolerance test is used to rank the potency of the inventive proteins. C57B 16 mice are acclimated to environmental conditions for one to two weeks. Prior to dosing, mice are handled and body weight measurements taken. One day prior to dosing, conscious miceare bled for plasma glucose measurement using a handheld glucometer designed for rodent plasma. On the day of dosing, baseline plasma glucose and body weight measurements are taken. The cage mean for plasma glucose and body weight are used to stratify cages into treatment groups. Mice are maintained in their home cages.

[0355] Following administration, plasma glucose is measured at various time points at (e.g., 1, 3, and 6 hr) or (4 hr) and plasma glucose and body weight measurements are taken every 24 hrs thereafter. Plasma glucose levels are measured until levels returned to near baseline levels and body weight measurements are taken until the rate of body weight gain was similar to Vehicle-treated control group.

[0356] Diet-induced obese mice (DIO) are used as a model to measure weight loss effects from treatment with different inventive proteins. Male c57bl6 mice are obtained from Harlan Laboratories and delivered at 26 days of age. Mice are group-housed with littermates consisting between two to four mice per cage. Following one week of acclimation, mice are started on a feeding regimen of high-fat diet (HFD: DI 2492, Research Diets, New Brunswick, N.J.) or are continued on standard rodent diet (2020, Harlan Laboratories). After 12 weeks of feeding, single-housed mice are acclimated to routine handling, food intake measurement, and daily saline injections (IP). Blood is collected from the retro orbital sinus of conscious DIO mice. A small volume of blood is immediately placed on a handheld glucometer designed for rodent plasma and the remaining blood is placed in EDTA filled collection tubes with a protease-inhibitor cocktail (Roche Diagnostics, Indianapolis, Ind.).

[0357] Following centrifugation, plasma is stored at -80° C. Mice are stratified into treatment groups based on body weight, and plasma glucose measurements. Mice are treated with Vehicle or one of the inventive proteins. Body weight is measured daily and cumulative food intake over three days is averaged for a daily food intake value. Food intake is measured between study days 0-3 and days 6-9. A blood collection is taken on study Day 9, 1 hour post injection and a terminal blood collection is taken on study Day 12, 1 or 3 days post the previous test article injection (e.g., vehicle, inventive proteins). Plasma insulin is measured with a mouse ELISA kit following manufacturer's instructions (ALPCO, Salem, N.H.). Plasma glucose, cholesterol, triglyceride, non-esterified fatty acids, AST, and ALT are measured using an Olympus AU400e Chemistry Analyzer (Olympus America, Center Valley, Pa ).

[0358] (b) Determination of body composition

[0359] Before the first treatment and then at week four and week eight, animals are anesthetized and imaged using dual-energy X-ray absorptiometry (DEXA), computerized x- ray tomography (CT), and magnetic resonance imaging (MRI) to detect and measure body composition, including lean mass and fat content. The effect of the disclosed myostatin antibodies and conjugates thereof on the volume of the epaxial muscles lying dorsal to the vertebral column over lumbar vertebrae L3-L5 is measured by CT scan. Full body lean mass may also be determined by small animal NMR imaging before and after treatment.

[0360] (c) Determination of increases in muscle mass

[0361] An in vivo study to determine changes in the muscle mass is conducted with seven- week-old female mice (e.g., female BALBc; male CS7BL). Mice are weighed and evenly distributed with respect to body weight into groups. The disclosed inventive proteins or a control (e.g., isotype matched antibody) is injected into the mice intraperitoneally at, e.g., 50 mg / kg twice weekly for 4 weeks; 60 mg / kg / week for 14 weeks. Animals are assessed for gain in lean body mass by subjecting them to dexascan analysis before and after the treatment period. Muscle mass is assessed by dissecting and weighing the gastrocnemius and quadriceps. The peri-uterine fat pad is also removed and weighed.

[0362] (d) Determination of increases in muscle mass in diabetic mice

[0363] The mice are either wild type at the agouti locus (a) or carry the lethal yellow mutation (Ay) at that locus. The Ay mutation causes adult-onset obesity and diabetes, which allows determination of the effect of the disclosed inventive proteins on muscle, excess fat, and blood glucose in a diabetic background. Total body mass is measured weekly. Muscle mass is assessed by dissecting and weighing the gastrocnemius and quadriceps. The epididymal and inguinal fat pads are also removed and weighed. Twelve weeks into the study, the mice are fasted and blood glucose levels are measured.

[0364] (e) Determination of increases in muscle mass and strength in SCID mice

[0365] In another assay useful to determine whether an antibody of the invention blocks myostatin activity in an animal model, an inventive protein may be tested in adult SCID mice. SCID mice suffer from severe combined immune deficiency and therefore do not generate an immunological reaction following injection of an antibody of the invention.Muscle mass is used as an indicator for myostatin activity in mice treated with an antibody of the invention.

[0366] Female SCID / CB17 mice (Taconic Biotechnology) are weighed and distributed into groups of ten. An inventive protein in PBS buffer is injected subcutaneously into the mice at various doses (10, 5, and 2 mg / kg) on days 0 and 7. In a control group, IgG at 10 mg / kg is injected subcutaneously into the mice on days 0 and 7. On day 14, muscle strength, the strength of the front limb, is measured with a grip strength test meter (e.g., model 1027 csx, Columbus Instruments). The animals are terminated, and muscle mass is assessed by nuclear magnetic resonance (NMR). The gastrocnemius and quadriceps muscle wet weights are also measured as well as body weight. The data may be transformed by a Box Cox transformation method to normalize the data. Outliers for each parameter may be identified by statistical means, such as with JMP 5.1 software (SAS, Inc.) and excluded from the data set. Statistical significance is determined by ANOVA and a Student's t-test, and a p value of less than 0.05 is considered significant.

[0367] Treatment of the SCID / CB17 mice with an anti -myostatin disclosed herein at doses of 5 mg / kg and 10 mg / kg results in statistically significant improvements relative to the control IgG group for all parameters tested, such as about 20% increase in total weight of the quadriceps and about 10% increase in grip strength.

[0368] (f) Determination of increases in muscle strength

[0369] To determine if the increases in muscle size will lead to an increase in muscle strength, a grip strength test is performed with aged and young mice treated with the disclosed inventive proteins using a grip strength meter, such as purchased from Columbia Instruments (Columbus, Ohio; model 1027csx). Mice are allowed to grip and pull on the grid, and the peak force of the pull is recorded. Untrained mice are tested five times in succession without rest. The peak force for each test is recorded.

[0370] Grip strength may also be tested by placing a test animal on a wire grid, allowing it to grip the mesh with all limbs, and then pulling on the tail and measuring maximal peak force as the animal releases its grip. Data per animal is averaged from several trials.

[0371] (g) Determination of increases muscle mass and lean mass in mdx mice

[0372] The mdx mutation of the X-linked dystrophin gene (Dmd) arose spontaneously in C57BL / 10ScSn mice and causes a point mutation within an exon at gene position 3185 converting a glutamine codon to a termination codon and resulting in premature terminationof the dystrophin protein. As a result, mdx mice lack functional dystrophin and serve as a small animal model of human Duchenne muscular dystrophy. Starting around 3 weeks, muscle necrosis develops with some visible muscle weakness. While skeletal limb muscles are characterized by a persistent and progressive degeneration and necrosis, this is offset by a regenerative response activated by satellite cells and muscle hypertrophy. The muscles of mdx mutants have an overall reduction in elasticity, making them more susceptible to injury due to lengthening-activation. Leg muscles in mutant mice initially develop normally, but the differentiation of regenerated myotubes into both fast and slow fiber types is significantly inhibited. The comparatively mild phenotype of the mdx mice can, in part, be attributed to the compensatory function of the dystrophin-related protein utrophin, which is highly upregulated in regenerating muscle fibers in adult mdx mutants. In contrast to limb muscles, the diaphragm muscles of mdx mice do not undergo a significant regeneration phase such that the continuous dystrophy weakens these muscles with age. The specific twitch force, specific tetanic force and maximum power are all reduced in the diaphragm of mdx mutants.

[0373] Eight-week-old male mdx and control C57B1 / 6 mice are dosed intraperitoneally (IP) once per week for eight weeks with the inventive proteins or vehicle control (PBS). In these experiments, mdx mice are treated with inventive protein or vehicle control, and C57B1 / 6 mice (e.g., control) are treated with inventive protein or PBS. At the end of the treatment period, full body lean mass, grip strength, and muscle mass are measured. Full body lean mass may be determined by small animal NMR imaging. Grip strength may be tested by placing a test animal on a wire grid, allowing it to grip the mesh with all limbs, and then pulling on the tail and measuring maximal peak force as the animal released its grip. After measuring lean body mass and grip strength, the mice are euthanized and the quadriceps and gastrocnemius muscles dissected and weighed.

[0374] Lean muscle mass has been found to increase in the mdx mice treated with an anti- myostatin disclosed herein an average of 7.28 ± 0.4 g compared to an average 4.83 ± 0.4 g in mdx mice treated with PBS. Thus, lean muscle mass increased by 50% in the mdx mice that received the incentive proteins.

[0375] The mass of specific muscles - the gastrocnemius and quadriceps muscles - has been found to increase by 12.2% and 12.1%, respectively, in mdx mice treated with an anti- myostatin antibody disclosed herein as compared to vehicle only controls.

[0376] (h) Determination of Musculoskeletal Efficacy

[0377] Male ICR mice (8 weeks old, Taconic NY) are housed in a temperature-controlled room (24° C.) with a reversed 12 hour light / dark cycle and water and food are available ad libitum. Over a 15 week time-frame, an inventive protein is administered every other week by subcutaneous injection to test mice and an isotype-matched IgGl is administered to age matched control mice. At each dosing time point, the dose given is adjusted according to the body weight of each animal. The following measurements are recorded at the beginning and end of the study: body weight, body muscle mass by quantitative magnetic resonance (QMR, Echo Medical Systems, Tex.) analysis, body grip-strength (Columbus Instruments, Ohio), and the bone mass (bone mineral density, BMD, mg / cc) of the femoral bones.Example 5: In Vivo Multi-specific-Cyno Assay

[0378] (a) Weight and Blood Chemistry

[0379] Naive male spontaneously obese cynomolgus monkeys (age 9-14 years) are acclimated / trained to experimental procedures prior to study initiation and sorted into treatment groups with equal distribution among groups for sorting parameters (body weight and blood chemistries). Monkeys are then injected once weekly for 6-8 weeks with vehicle or inventive protein. After the 6-8 week treatment period, monkeys are optionally put through a 4-10 week washout phase. During training / treatment / washout phases of study, total energy intake is monitored daily; body weight, blood chemistries and drug exposure are monitored weekly or every other week. The concentrations of total myostatin in monkey serum were quantified using an enzyme-linked immunoassay (ELISA).

[0380] (b) Lean Body Mass and Muscle volume

[0381] Eight-week trials in which animals are dosed with an inventive protein, such as weekly by IV administration, and provided excess food to ensure a positive nitrogen balance. In a first study, a group of male subjects and a group of female subjects are administered PBS vehicle or an inventive protein, such as at doses of 1.0 mg / kg to 50 mg / kg. Before the first treatment and then at week four and week eight, animals are anesthetized and imaged using dual-energy X-ray absorptiometry (DEXA), computerized x-ray tomography (CT), and magnetic resonance imaging (MRI) to detect and measure body composition, including lean mass and fat content.

[0382] The whole-body dual energy X-ray absorptiometry (DXA) scans (e.g., using Luna iDXA, GE Healthcare, Madison, WI, USA) are conducted by positioning anesthetizedanimals supine and centered on the scanner bed with the spine oriented along the long-axis centerline of the scanner table. Similar positioning is used for the CT scans and MRI.

[0383] Subject animals from the first study are then euthanized and necropsied. In the second study, only male subjects are used and received vehicle alone or an inventive protein at doses of 1.0 mg / kg to 50 mg / kg. The subject animals are imaged at eight weeks as in the first study. Thereafter, the animals are maintained on a supplemented diet and imaged again at 12, 17 and 26 weeks.

[0384] The eight week data from each of the first and second studies has demonstrated that treatment with an anti-myostatin disclosed herein provides a dose dependent improvement in muscle mass. Dosing with an anti-myostatin antibody disclosed herein at 10 mg / ml showed about 10% increase in mean axial muscle volume, while the higher dose of 30 mg / ml showed about 24% increase in mean axial muscle volume (as compared to vehicle only treatment). These gains have been found to persist for weeks after the last injection for the higher dose (i.e., 30 mg / ml) group.Example 6: Anti-Myostatin in Older Patients

[0385] A phase II clinical trial (NCT01604408) conducted across 21 investigator sites in Argentina, Australia, France, Germany, Sweden, and the USA, involved administration of an anti-myostatin disclosed herein to patients aged 75 years or older who had experienced a fall within the previous year. Eligible participants demonstrated low physical performance, as assessed by hand grip strength and chair rise tests. Participants were stratified based on country, age, hand grip strength, and chair rise test results, and were randomly assigned (1 : 1) via a computer-generated sequence to receive either a placebo or 315 mg of the anti- myostatin antibody administered subcutaneously at weeks 0 (randomization), 4, 8, 12, 16, and 20, followed by a 16-week observation period.

[0386] A change in appendicular lean body mass from baseline to 24 weeks was observed: the control group receiving placebo, showed a loss of 0.123 Kg and the study group receiving the anti-myostatin antibody gained 0.303 Kg, and difference of 0.43 Kg. Physical performance measures, such as four-step stair climbing time, usual gait speed, and the time required to rise from a chair five times (with or without the use of arms, depending on the participant’s ability) also showed significant changes: the group receiving the anti-myostatin antibody had a four-step stair climbing time 0.46 seconds faster than control, a twelve-stepstair climbing time 1.28 seconds faster than control, a chair rise with arms time 4.15 seconds faster than control, and a usual gait speed 0.05 m / s faster than control.

[0387] Exemplary sequences referenced in this application are provided in Tables 1-5 below.Table 1. Sequences of Metabolic ModulatorsK is an acylated lysine; X is aminoisobutyric acid; and K is acylated with an yGlu-2xOEG linker and Cl 8 fatty diacid moiety. Aib is 2-aminoisobutyric acid. K* is K-(CH2)4-NH-CO- CTE-Br or maleimide (see FIG. 2).Table 2: Sequences of LinkersK* is K-(CH2)4-NH-CO-CH2-Br or maleimide (see FIG. 2). Table 3. Anti-GIPR Antibody SequencesTable 4. Anti-Myostatin AntibodiesTable 5: GIPR polypeptides

[0388] All publications and patents referred to herein are incorporated by reference in their entirety. Various modifications and variations of the described subject matter will be apparent to those skilled in the art without departing from the scope and spirit of the invention.Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to these embodiments. Indeed, various modifications for carrying out the invention are obvious to those skilled in the art and are intended to be within the scope of the following claims.

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Claims

CLAIMSWhat is claimed is:

1. A multi-specific protein comprising:(i) a muscle fortifying agent; and(ii) a metabolic modulatory component.

2. The protein of claim 1, wherein the muscle fortifying agent is a myostatin modulator, an activin type II receptor modulator and the like, or an agent having the activity of the myostatin modulator or activin type II receptor modulator, or any combination thereof.

3. The protein of claim 2, wherein the myostatin modulator blocks, eliminates, or significantly reduces binding of myostatin to its receptor, inhibits myostatin activity, or a combination thereof.

4. The protein of any one of claims 1-3, wherein the muscle fortifying agent comprises a binding agent that binds myostatin, a myostatin antagonist, or a combination thereof.

5. The protein of claim 4, wherein the binding agent comprises a receptor, an antibody, or a fragment thereof.

6. The protein of any one of claims 1-5, wherein the metabolic modulatory component comprises:(a) an antibody against glucose-dependent insulinotropic peptide receptor (anti-GIPR) or fragment thereof, and(b) a metabolic modulator.

7. The protein of claim 6, wherein the anti-GIPR antibody or fragment thereof and the metabolic modulator are connected directly.

8. The protein of claim 6, comprising a linker, wherein the anti-GIPR antibody or fragment thereof and the metabolic modulator are connected via the linker.

9. The protein of claim 8, wherein the linker is a peptide linker.

10. The protein of claim 8, wherein the linker is a synthetic linker.

11. The protein of claim 6, wherein the metabolic modulator comprises an antibody, a peptide, a fusion protein, a modified peptide, or a compound.

12. The protein of any one of claims 6-11, wherein the anti-GIPR antibody comprises a light chain comprising light chain complementarity determining regions (LC-CDRs) 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively, and a heavy chain comprising heavy chaincomplementarity determining regions (HC-CDRs) 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively.

13. The protein of any one of claims 6-11, wherein the anti-GIPR antibody comprises a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59, respectively.

14. The protein of any one of claims 6-11, wherein the anti-GIPR antibody comprises a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively.

15. The protein of any one of claims 6-11, wherein the anti-GIPR antibody comprises a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively.

16. The protein of any one of claims 6-11, wherein the anti-GIPR antibody comprises a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively.

17. The protein of any one of claims 6-11, wherein the anti-GIPR antibody comprises a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 84, SEQ ID NO: 85, and SEQ ID NO: 86, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 81, SEQ ID NO: 82, and SEQ ID NO: 83, respectively.

18. The protein of any one of claims 6-11, wherein the anti-GIPR antibody comprises a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 90, YAS, and SEQ ID NO: 92, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89, respectively.

19. The protein of any one of claims 6-11, the anti-GIPR antibody comprises a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 96, YAS, and SEQ ID NO: 98, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 93, SEQ ID NO: 94, and SEQ ID NO: 95, respectively.

20. The protein of any one of claims 6-11, wherein the anti-GIPR antibody comprises a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 102, SEQ ID NO: 103, and SEQ ID NO: 104, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101, respectively.

21. The protein of any one of claims 6-11, wherein the anti-GIPR antibody comprises a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 108, SEQ ID NO: 109, and SEQ ID NO: 110, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107, respectively.

22. The protein of any one of claims 6-11, wherein the anti-GIPR antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 41 or SEQ ID NO: 42, and a heavy chain variable region comprising the amino acid sequence set forth in any one of SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO:39, or SEQ ID NO: 40.

23. The protein of any one of claims 6-11, wherein the anti-GIPR antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 49 or SEQ ID NO: 50, and a heavy chain variable region comprising the amino acid sequence set forth in any one of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48.

24. The protein of any one of claims 6-11, wherein the anti-GIPR antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 42 and a heavy chain variable region comprising the amino acid sequence SEQ ID NO:40.

25. The protein of claim 24, wherein the anti-GIPR antibody is an IgGl isotype.

26. The protein of claim 24 or claim 25, wherein the anti-GIPR antibody comprises at least one Fc mutation.

27. The protein of any one of claims 24-26, wherein the anti-GIPR antibody comprises Fc mutations L234A, L235A, P329G, E272C, M428L, and N434S, wherein residue numbering is according to the EU index.

28. The protein of any one of claims 24-27, wherein the anti-GIPR antibody comprises a light chain of SEQ ID NO: 186 and a heavy chain of SEQ ID NO: 185.

29. The protein of any one of claims 6-11, wherein the anti-GIPR antibody includes intact antibodies or antibody fragments such as Fv fragments, single chain scFv fragments (scFv), single chain disulfide bond stabilized scFv fragments or stapled scFv fragment (spFv), Fab, F(ab)2, or single chain antibodies.

30. The protein of any one of claims 6-29, wherein the metabolic modulator comprises a molecule having the activity of a glucagon-like peptide-1 (GLP-1) receptor agonist, a GIPR antagonist, or a combination thereof.

31. The protein of any one of claims 6-30, wherein the metabolic modulator comprises a GLP-1 receptor agonist, a GIPR antagonist, or a combination thereof.

32. The protein of any one of claims 6-31, wherein the metabolic modulator comprises a peptide comprising the amino acid sequence as set forth in any one of SEQ ID NOs: 1-14 or 190, or a variant thereof, wherein the variant comprises at least one amino acid substitution.

33. The protein of any one of claims 6-31, wherein the metabolic modulator comprises a peptide comprising the amino acid sequence as set forth in SEQ ID NO: 18 or 19, or a variant thereof, wherein the variant comprises at least one amino acid substitution.

34. The protein of claim 6-31, wherein the metabolic modulator comprises a peptide comprising the amino acid sequence as set forth in SEQ ID NO: 15 or 16 with at least one amino acid substitution.

35. The protein of any one of claims 1-34, wherein the muscle fortifying agent and the metabolic component are connected directly.

36. The protein of any one of claims 1-34, comprising a linker, wherein the muscle fortifying agent and the metabolic component are connected via the linker.

37. The protein of claim 36, wherein the linker is a peptide linker.

38. The protein of claim 36, wherein the linker is a synthetic linker.

39. The protein of any one of claims 1-34, wherein the muscle fortifying agent and the metabolic component are connected directly, connected via a peptide linker, connected via a synthetic linker, or any combination thereof.

40. The protein of any one of claims 1-39, wherein the muscle fortifying agent is an antibody or a fragment thereof, that specifically binds to myostatin.

41. A nucleic acid encoding the protein of any one of claims 1-40, or a portion thereof, optionally operably linked to a heterologous promoter.

42. A composition of any one of claims 1-40.

43. The composition of claim 42, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.

44. A system for detecting or treating muscle wasting, a metabolic disorder, or a combination thereof in a subject, comprising the protein of any one of claims 1-34 or a composition thereof and, optionally a delivery system.

45. The system of claim 44, wherein the delivery system comprises a syringe, a pump, an inhaler, a patch, or an intranasal delivery device.

46. The system of claim 44 or 45, wherein the protein is administered to the subject by a mucosal, intranasal, intramuscular, intravenous, subcutaneous, intradermal, subdermal, transdermal, or oral route of administration.

47. Abi-specific protein comprising:(i) an anti-GIPR antibody or a fragment thereof; and(ii) a GLP-1R agonist.

48. The protein of claim 47, wherein the anti-GIPR antibody or a fragment thereof comprises a light chain comprising light chain complementarity determining regions (LC-CDRs) 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively, and a heavy chain comprising heavy chain complementarity determining regions (HC-CDRs) 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively.

49. The protein of claim 47, wherein the anti-GIPR antibody or a fragment thereof comprises a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively.

50. The protein of claim 47, wherein the anti-GIPR antibody or a fragment thereof comprises a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80,respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively.

51. The protein of claim 47, wherein the anti-GIPR antibody or a fragment thereof comprises a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 90, YAS, and SEQ ID NO: 92, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89, respectively.

52. The protein of claim 47, wherein the anti-GIPR antibody or a fragment thereof comprises a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 102, SEQ ID NO: 103, and SEQ ID NO: 104, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101, respectively.

53. The protein of any one of claims 47-52, wherein the anti-GIPR antibody or a fragment thereof comprises a light chain variable region comprising the amino acid of SEQ ID NO: 42, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 40.

54. The protein of any one of claims 47-53, wherein the anti-GIPR antibody or a fragment thereof is an IgGl isotype.

55. The protein of any one of claims 47-54, wherein the anti-GIPR antibody or a fragment thereof comprises at least one Fc mutation.

56. The protein of any one of claims 47-55, wherein the anti-GIPR antibody or a fragment thereof comprises Fc mutations L234A, L235A, P329G, E272C, M428L, and N434S, wherein residue numbering is according to the EU index.

57. The protein of any one of claims 47-56, wherein the anti-GIPR antibody or a fragment thereof comprises a light chain of SEQ ID NO: 186 and a heavy chain of SEQ ID NO: 185.

58. The protein of any one of claims 47-57, wherein the anti-GIPR antibody or a fragment thereof and the GLP-1R agonist are connected via a linker.

59. The protein of claim 58, wherein the linker is a peptide linker.

60. The protein of claim 58, wherein the linker is a synthetic linker.

61. The protein of any one of claims 47-60, wherein the anti-GIPR antibody or a fragment thereof and the GLP-1R agonist are connected directly, connected via a peptide linker, connected via a synthetic linker, or any combination thereof.

62. The protein of any one of claims 47-60, wherein the GLP-1R agonist comprises SEQ ID NO: 190.

63. An isolated anti-GIPR antibody or a fragment thereof, comprising(a) a light chain comprising light chain complementarity determining regions (LC- CDRs) 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively, and a heavy chain comprising heavy chain complementarity determining regions (HC-CDRs) 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively;(b) a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively;(c) a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively;(d) a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 90, YAS, and SEQ ID NO: 92, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89, respectively; or(e) a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 102, SEQ ID NO: 103, and SEQ ID NO: 104, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101, respectively.

64. The anti-GIPR antibody or a fragment thereof of claim 63, comprising a light chain variable region comprising the amino acid of SEQ ID NO: 42, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 40.

65. The anti-GIPR antibody or a fragment thereof of claim 63 or 64, wherein the anti- GIPR antibody or a fragment thereof is an IgGl isotype.

66. The anti-GIPR antibody or a fragment thereof of claims 63-65, wherein the anti-GIPR antibody or a fragment thereof comprises at least one Fc mutation.

67. The anti-GIPR antibody or a fragment thereof of claims 63-66, wherein the anti-GIPR antibody or a fragment thereof comprises Fc mutations L234A, L235A, P329G, E272C, M428L, and N434S, wherein residue numbering is according to the EU index.

68. The anti-GIPR antibody or a fragment thereof of claims 63-67, wherein the anti-GIPR antibody or a fragment thereof comprises a light chain of SEQ ID NO: 186 and a heavy chain of SEQ ID NO: 185.

69. A protein comprising an anti-GIPR antibody and a linker, comprising(a) a light chain comprising light chain complementarity determining regions (LC- CDRs) 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively, and a heavy chain comprising heavy chain complementarity determining regions (HC-CDRs) 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively;(b) a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively;(c) a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively;(d) a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 90, YAS, and SEQ ID NO: 92, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89, respectively; or(e) a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 102, SEQ ID NO: 103, and SEQ ID NO: 104, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising theamino acid sequences set forth in SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101, respectively, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 20-36 and 187-189.

70. The protein of claim 69, comprising a light chain variable region comprising the amino acid of SEQ ID NO: 42, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 40.

71. The protein of claim 69 or 70, wherein the anti-GIPR antibody or a fragment thereof is an IgGl isotype.

72. The protein of claims 69-71, wherein the anti-GIPR antibody or a fragment thereof comprises at least one Fc mutation.

73. The protein of claims 69-72, wherein the anti-GIPR antibody or a fragment thereof comprises Fc mutations L234A, L235A, P329G, E272C, M428L, and N434S, wherein residue numbering is according to the EU index.

74. The protein of claims 69-73, wherein the anti-GIPR antibody or a fragment thereof comprises a light chain of SEQ ID NO: 186 and a heavy chain of SEQ ID NO: 185.

75. The protein of claims 69-74, wherein the linker comprises the amino acid sequence of SEQ ID NO: 187 -189.

76. A protein comprising an anti-GIPR antibody, a linker, and a GLP-1R agonist comprising(a) a light chain comprising light chain complementarity determining regions (LC- CDRs) 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively, and a heavy chain comprising heavy chain complementarity determining regions (HC-CDRs) 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively;(b) a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively;(c) a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising theamino acid sequences set forth in SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively;(d) a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 90, YAS, and SEQ ID NO: 92, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89, respectively; or(e) a light chain comprising LC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 102, SEQ ID NO: 103, and SEQ ID NO: 104, respectively, and a heavy chain comprising HC-CDRs 1 through 3 comprising the amino acid sequences set forth in SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101, respectively, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 20-36 and 187-189.

77. The protein of claim 76, comprising a light chain variable region comprising the amino acid of SEQ ID NO: 42, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 40.

78. The protein of claim 76 or 77, wherein the anti-GIPR antibody or a fragment thereof is an IgGl isotype.

79. The protein of claims 76-78, wherein the anti-GIPR antibody or a fragment thereof comprises at least one Fc mutation.

80. The protein of claims 76-79, wherein the anti-GIPR antibody or a fragment thereof comprises Fc mutations L234A, L235A, P329G, E272C, M428L, and N434S, wherein residue numbering is according to the EU index.

81. The protein of claims 76-80, wherein the anti-GIPR antibody or a fragment thereof comprises a light chain of SEQ ID NO: 186 and a heavy chain of SEQ ID NO: 185.

82. The protein of claims 76-81, wherein the linker comprises the amino acid sequence of SEQ ID NO: 187, 188, or 189.

83. The protein of claims 76-82, wherein the GLP-1R agonist comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14 and 190.

84. A multi-specific protein comprising:(i) a muscle fortifying agent means or fragment thereof and / or an anti-GIPR binding means or fragment thereof; and(ii) a metabolic modulatory means.

85. The protein of claim 84, wherein the muscle fortifying agent means or fragment thereof comprises a binding agent that binds myostatin, a myostatin antagonist, or a combination thereof, and wherein the metabolic modulatory means comprises a GLP- 1 receptor agonist, a GIPR antagonist, or a combination thereof.

86. The protein of claim 84 or 85, wherein the anti-GIPR binding means or fragment thereof comprises the anti-GIPR antibody according to any one of claims 63 to 68.

87. The protein of any one of claims 84 to 86, wherein the metabolic modulatory means comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 1-19 or 190.

88. The protein of any one of claims 84 to 87, comprising:(i) an anti-myostatin antibody connected to the metabolic modulatory means directly or via a linker;(ii) an anti-GIPR antibody connected to the metabolic modulatory means directly or via a linker, such as set forth in any one of claims 47 to 62; or(iii) a bispecific antibody comprising an anti-myostatin antibody fragment and an anti-GIPR antibody fragment, the bispecific antibody is connected to the metabolic modulatory means directly or via a linker.

89. The protein or antibody of any one of claims 22-24, 53, 64, 70, or 77, wherein the light chain variable region comprises at least 95% identity to any one of the sequences set forth in SEQ ID NOs: 41-42 or 49-50 and the heavy chain variable region comprises at least 95% identity to any one of the sequences set forth in SEQ ID NOs: 37-40 or 43-48.

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