Myostatin and activin a immunogens and methods of use

Abstract

This disclosure provides myostatin and activin A peptide immunogen constructs, compositions and methods for their use in, for example, maintaining and improving muscle physiology (e.g., increasing muscle mass, strength, function, tone or endurance), preventing and treating muscular or musculoskeletal injury, preventing and treating a disorder associated with muscle loss or insufficient muscle growth, and / or preventing and treating muscle wasting diseases.

Description

[0001] MYOSTATIN AND ACTIVIN A IMMUNOGENS AND METHODS OF USE FIELD OF THE DISCLOSURE

[0002] This disclosure relates to myostatin and activin A peptide immunogen constructs, compositions and methods of their use (e.g., for maintaining and improving muscle physiology (e.g., increasing muscle mass, strength, tone or endurance), preventing and treating muscular or musculoskeletal injury, preventing and treating a disorder associated with muscle loss or insufficient muscle growth, and / or preventing and treating muscle wasting diseases).

[0003] BACKGROUND

[0004] Muscle wasting is a loss of muscle mass characterized by muscles weakening and shrinking. It can occur due to a number of different causes including, for example, age, genetics, disuse, malnutrition, injury, and certain medical conditions affecting the musculoskeletal or nervous systems. Muscle wasting can cause mobility issues, pain, and discomfort, which in turn may lead to substantially reduced quality of life.

[0005] There is a need for new approaches for the prevention and treatment of muscle wasting.

[0006] SUMMARY

[0007] In a first aspect, the invention provides a peptide immunogen construct comprising a myostatin B cell epitope, a heterologous T cell (Th) epitope, and an optional heterologous spacer, wherein the B cell epitope is covalently linked to the Th epitope directly or through the optional heterologous spacer.

[0008] In some embodiments, the myostatin B cell epitope comprises about 7 to about 30 contiguous amino acids from myostatin or a variant thereof.

[0009] In some embodiments, the myostatin B cell epitope comprises about 7 to about 30 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2 or a variant thereof. In some embodiments, the myostatin B cell epitope comprises up to 3, up to 4, up to 5, or up to 6, amino acid substitutions as compared to the corresponding sequence of the 7-30 amino acid fragment of SEQ ID NO: 1 or SEQ ID NO: 2.

[0010] In some embodiments, the myostatin B cell epitope comprises a variant of a 7-30 amino acid fragment of SEQ ID NO: 1 or SEQ ID NO: 2, and the variant comprises 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions, deletions, or insertions as compared to the corresponding sequence of the 7-30 amino acid fragment of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the myostatin B cell epitope comprises a variant of a 7-30 amino acid fragment of SEQ ID NO: 1 or SEQ ID NO: 2, and the variant comprises 1, 2, or 3 amino acid substitutions as compared to the corresponding sequence of the 7-30 amino acid fragment of SEQ ID NO: 1 or SEQ ID NO: 2.

[0011] In some embodiments, the myostatin B cell epitope comprises one or more amino acid substitutions as compared to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 to facilitate cyclization of the sequence comprising the myostatin B cell epitope.

[0012] In some embodiments, the myostatin B cell epitope comprises 1, 2, or 3 amino acid substitutions as compared to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 to facilitate cyclization of the sequence comprising the myostatin B cell epitope.

[0013] In some embodiments, the myostatin B cell epitope is cyclic or cyclized. In some embodiments, the modifications allowing for cyclization described herein were made to make the B cell epitope structure resemble the tertiary structure of the naturally occurring protein fragment.

[0014] In some embodiments, the invention provides a peptide immunogen construct comprising a myostatin B cell epitope, a heterologous T cell (Th) epitope, and an optional heterologous spacer, wherein the B cell epitope is covalently linked to the Th epitope directly or through the optional heterologous spacer; wherein the myostatin B cell epitope comprises about 7 to about 30 contiguous amino acids from myostatin, or a variant thereof, and / or wherein the myostatin B cell epitope is cyclized.

[0015] In some embodiments, the myostatin B cell epitope comprises or consists of an amino acid sequence within loop 1, loop 2, loop 3, or loop 4 of myostatin, or a variant thereof. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 3-10, 95-109, 145-164, and 166 or a variant thereof. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 5, 97, 98, 101, 105, 146, 147, 151-155, 157, 159, 161, 163, 164, or 166, or a variant thereof. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 5, 97, 98, 101, or 105, or a variant thereof. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 5. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 97. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 98. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 101. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 105. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 146, 147, 151, 152, 154, 155, 157, 159, 161, 163, 164, 166, or a variant thereof. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 146. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 147. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 151, or a variant thereof. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 152, or a variant thereof. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 153, or a variant thereof. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 154, or a variant thereof. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 155, or a variant thereof. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 157, or a variant thereof. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 159, or a variant thereof. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 161, or a variant thereof. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 163, or a variant thereof. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 164, or a variant thereof. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 166, or a variant thereof.

[0016] In some embodiments, the myostatin B cell epitope peptide is acetylated on the N-terminus. In some embodiments, the myostatin B cell epitope peptide comprises an amide group on the C-terminus. In some embodiments, the myostatin B cell epitope peptide is acetylated on the N-terminus and comprises an amide group on the C-terminus. In some embodiments, the myostatin B cell epitope peptide is cyclized using the N-terminus amino group and not acetylated on the N-terminus.

[0017] In some embodiments, the myostatin B cell epitope peptide is cyclized (or cyclic). The myostatin B cell epitope peptide can be cyclized using any bridge, linkage or methodology known in the art or described herein. In some embodiments, the myostatin B cell epitope peptide is cyclic due to the presence of amino acids naturally occurring in the myostatin B cell epitope peptide fragment. In some embodiments, the myostatin B cell epitope peptide is cyclic due to the presence of one or more (e.g., two) amino acid substitutions relative to the naturally occurring amino acid sequence of the myostatin B cell epitope peptide fragment. In some embodiments, the myostatin B cell epitope peptides are cyclic due to, e.g., the presence of cysteine residues, whether naturally occurring in the sequences or inserted, e.g., by substitution. In some embodiments, the myostatin B cell epitope peptide comprises, or is cyclized with, a disulfide (S-S) linkage. In some embodiments, the myostatin B cell epitope peptide comprises, or is cyclized with, a lactam linkage.

[0018] In some embodiments, the Th epitope is derived from a pathogenic protein. In some embodiments, the Th epitope comprises or consists of a sequence selected from any one of SEQ ID NOs: 19-47 or a variant thereof.

[0019] In some embodiments, the Th epitope comprises or consists of the sequence of SEQ ID NO: 30, 32, or 33. In some embodiments, the Th epitope comprises or consists of the sequence of SEQ ID NO: 30. In some embodiments, the Th epitope comprises or consists of the sequence of SEQ ID NO: 32. In some embodiments, the Th epitope comprises or consists of the sequence of SEQ ID NO: 33.

[0020] In some embodiments, the myostatin B cell epitope and the Th epitope are linked to one another by direct covalent linkage. In other embodiments, the myostatin B cell epitope and the Th epitope are linked to one another by the heterologous spacer.

[0021] In some embodiments, the heterologous spacer comprises or consists of one or more amino acids, which are optionally selected from: Lys-, Gly-, Lys-Lys-Lys-, (a, e-N)Lys, Lys-Lys-Lys-eLys, s-N-Lys-Lys-Lys-Lys (SEQ ID NO: 91), and Pro-Pro-Xaa-Pro-Xaa-Pro, wherein Xaa is any amino acid or is aspartic acid (SEQ ID NO: 90). In some embodiments, the heterologous spacer comprises or consists of Lys-Lys-Lys-sLys. In some embodiments, the heterologous spacer comprises or consists of s-N-Lys-Lys-Lys-Lys (SEQ ID NO: 91). In some embodiments, the heterologous spacer comprises or consists of (a, s-N)Lys.

[0022] In some embodiments, (a) the myostatin B cell epitope is located N-terminal to the Th epitope; (b) the Th epitope is located N-terminal to the myostatin B cell epitope; (c) the myostatin B cell epitope is flanked with a Th epitope on both N-terminal and C-terminal ends; (d) any of (a)-(c) wherein the myostatin B cell epitope(s) is linked to the Th epitope(s) by direct covalent linkage; or (e) any one of (a)-(c) wherein the myostatin B cell epitope(s) is linked to the Th epitope(s) by the heterologous spacer. In some embodiments, the myostatin B cell epitope is located N-terminal to the Th epitope. In some embodiments, the Th epitope is located N-terminal to the myostatin B cell epitope.

[0023] In some embodiments, the peptide immunogen construct is of following formula: (Th)m-(A)„-(myostatin B cell epitope), or (myostatin B cell epitope)-(A)n-(Th)m, or (Th)m-(A)n-(myostatin B cell epitope)-(A)n-(Th)m, wherein Th is the T helper epitope; A is the heterologous spacer, which optionally is present and optionally comprises an amino acid; m is from 1 to about 4; and n is from 0 to about 10, wherein optionally the C-terminus of the peptide immunogen construct comprises a-COOH or a-CONH2of an amino acid. In some embodiments, the peptide immunogen construct is of following formula: (Th)m-(A)n--(myostatin B cell epitope), wherein Th is the T helper epitope; A is the heterologous spacer, which optionally is present and optionally comprises an amino acid; m is from 1 to about 4; and n is from 0 to about 10, and wherein the C-terminus of the peptide immunogen construct comprises a-COOH or a-CONH2of an amino acid. In some embodiments, the peptide immunogen construct is of following formula: (myostatin B cell epitope)-(A)n-(Th)m, wherein Th is the T helper epitope; A is the heterologous spacer, which optionally is present and optionally comprises an amino acid; m is from 1 to about 4; and n is from 0 to about 10, and wherein the C-terminus of the peptide immunogen construct comprises a-COOH or a-CONH2of an amino acid. In some embodiments of the formulas provided herein, m is 1 and n is 1. In some embodiments, more than one myostatin B cell epitope is present in the peptide immunogen constructs described herein, e.g., 1-4 myostatin B cell epitopes. In some embodiments, two or three myostatin B cell epitopes are present in the peptide immunogen constructs described herein. In other embodiments, one (1) myostatin B cell epitope is present in the peptide immunogen constructs described herein.

[0024] In some embodiments, the peptide immunogen construct comprises or consists of the sequence of any one of SEQ ID NOs: 48-71, 120-134, 167-186 and 188 or a variant thereof. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 54, 55, 56, 122, 123, 126, 130, 168, 169, 173, 174, 175, 176, 177, 178, 179, 181, 183, 185, or 186, or a variant thereof. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 54, 55, 56, 122, 123, 126 or 130, or a variant thereof. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 54, 55, or 56. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 54. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 55. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 56. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 122. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 123. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 126. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 130. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 168, 169, 173, 174, 175, 176, 177, 178, 179, 181, 183, 185, 186 or a variant thereof. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 168. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 169. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 173. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 174. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 176. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 177. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 179. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 181. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 183. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 185. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 186.

[0025] In another aspect, the invention provides a peptide immunogen construct comprising an activin A B cell epitope, a heterologous T cell (Th) epitope, and an optional heterologous spacer, wherein the B cell epitope is covalently linked to the Th epitope directly or through the optional heterologous spacer.

[0026] In some embodiments, the activin A B cell epitope comprises about 7 to about 30 contiguous amino acids from activin A or a variant thereof.

[0027] In some embodiments, the activin A B cell epitope comprises about 7 to about 30 contiguous amino acids of SEQ ID NO: 12 or a variant thereof. In some embodiments, the activin A B cell epitope comprises up to 3, up to 4, up to 5, or up to 6, amino acid substitutions as compared to the corresponding sequence of the 7-30 amino acid fragment of SEQ ID NO: 12. In some embodiments, the activin A B cell epitope comprises a variant of a 7-30 amino acid fragment of SEQ ID NO: 12, and the variant comprises 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions, deletions, or insertions as compared to the corresponding sequence of the 7-30 amino acid fragment of SEQ ID NO: 12.

[0028] In some embodiments, the activin A B cell epitope comprises a variant of a 7-30 amino acid fragment of SEQ ID NO: 12, and the variant comprises 1, 2, or 3 amino acid substitutions as compared to the corresponding sequence of the 7-30 amino acid fragment of SEQ ID NO: 12.

[0029] In some embodiments, the activin A B cell epitope comprises one or more amino acid substitutions as compared to the sequence of SEQ ID NO: 12 to facilitate cyclization of the sequence comprising the activin A B cell epitope.

[0030] In some embodiments, the activin AB cell epitope comprises 1, 2, or 3 amino acid substitutions as compared to the sequence of SEQ ID NO: 12 to facilitate cyclization of the sequence comprising the activin A B cell epitope.

[0031] In some embodiments, the activin A B cell epitope is cyclic or cyclized.

[0032] In some embodiments, the invention provides a peptide immunogen construct comprising an activin A B cell epitope, a heterologous T cell (Th) epitope, and an optional heterologous spacer, wherein the B cell epitope is covalently linked to the Th epitope directly or through the optional heterologous spacer; wherein the activin A B cell epitope comprises about 7 to about 30 contiguous amino acids from myostatin, or a variant thereof, and / or wherein the activin A B cell epitope is cyclized.

[0033] In some embodiments, the activin A B cell epitope comprises or consists of an amino acid sequence within loop 1, loop 2, loop 3, or loop 4 of activin A, or a variant thereof. In some embodiments, the activin A B cell epitope comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 13-18, 110-119, 187, and 189 or a variant thereof. In some embodiments, the activin A B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 14-18, 111, 116, 118, 119, 187, or a variant thereof. In some embodiments, the activin A B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 14, 15, 17, 116, 119, 187, or a variant thereof. In some embodiments, the activin A B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 111, 116, 118, 119, or 187 or a variant thereof. In some embodiments, the activin A B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 111. In some embodiments, the activin A B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 116. In some embodiments, the activin A B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 118. In some embodiments, the activin A B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 119. In some embodiments, the activin A B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 187. In some embodiments, the activin A B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 14. In some embodiments, the activin A B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 15. In some embodiments, the activin A B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 16. In some embodiments, the activin A B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 17. In some embodiments, the activin A B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 18.

[0034] In some embodiments, the activin A B cell epitope peptide is acetylated on the N-terminus. In some embodiments, the activin A B cell epitope peptide comprises an amide group on the C-terminus. In some embodiments, the activin A B cell epitope peptide is acetylated on the N-terminus and comprises an amide group on the C-terminus. In some embodiments, the activin A B cell epitope peptide is cyclized using the N-terminus amino group and not acetylated on the N-terminus.

[0035] In some embodiments, the activin A B cell epitope peptide is cyclized (or cyclic). The activin A B cell epitope peptide can be cyclized using any bridge, linkage or methodology known in the art or described herein. In some embodiments, the activin A B cell epitope peptide is cyclic due to the presence of amino acids naturally occurring in the activin A B cell epitope peptide fragment. In some embodiments, the activin A B cell epitope peptide is cyclic due to the presence of one or more (e.g., two) amino acid substitutions relative to the naturally occurring amino acid sequence of the activin A B cell epitope peptide fragment. In some embodiments, the activin A B cell epitope peptides are cyclic due to, e.g., the presence of cysteine residues, whether naturally occurring in the sequences or inserted, e.g., by substitution. In some embodiments, the activin A B cell epitope peptide comprises, or is cyclized with, a disulfide (S-S) linkage. In some embodiments, the activin A B cell epitope peptide comprises, or is cyclized with, a lactam linkage.

[0036] In some embodiments, the Th epitope is derived from a pathogenic protein. In some embodiments, the Th epitope comprises or consists of a sequence selected from any one of SEQ ID NOs: 19-47 or a variant thereof.

[0037] In some embodiments, the Th epitope comprises or consists of the sequence of SEQ ID NO: 30, 32, or 33. In some embodiments, the Th epitope comprises or consists of the sequence of SEQ ID NO: 30. In some embodiments, the Th epitope comprises or consists of the sequence of SEQ ID NO: 32. In some embodiments, the Th epitope comprises or consists of the sequence of SEQ ID NO: 33.

[0038] In some embodiments, the myostatin B cell epitope and the Th epitope are linked to one another by direct covalent linkage.

[0039] In some embodiments, the activin A B cell epitope and the Th epitope are linked to one another by the heterologous spacer.

[0040] In some embodiments, the heterologous spacer comprises or consists of one or more amino acids, which are optionally selected from: Lys-, Gly-, Lys-Lys-Lys-, (a, s-N)Lys, Lys-Lys-Lys-sLys, s-N-Lys-Lys-Lys-Lys (SEQ ID NO: 91), and Pro-Pro-Xaa-Pro-Xaa-Pro, wherein Xaa is any amino acid or is aspartic acid (SEQ ID NO: 90). In some embodiments, the heterologous spacer comprises or consists of Lys-Lys-Lys-eLys. In some embodiments, the heterologous spacer comprises or consists of s-N-Lys-Lys-Lys-Lys (SEQ ID NO: 91). In some embodiments, the heterologous spacer comprises or consists of (a, s-N)Lys.

[0041] In some embodiments, (a) the activin A B cell epitope is located N-terminal to the Th epitope; (b) the Th epitope is located N-terminal to the activin A B cell epitope; (c) the activin A B cell epitope is flanked with a Th epitope on both N-terminal and C-terminal ends; (d) any of (a)-(c) wherein the activin A B cell epitope(s) is linked to the Th epitope(s) by direct covalent linkage; or (e) any one of (a)-(c) wherein the activin A B cell epitope(s) is linked to the Th epitope(s) by the heterologous spacer. In some embodiments, the activin A B cell epitope is located N-terminal to the Th epitope. In some embodiments, the Th epitope is located N-terminal to the activin A B cell epitope.

[0042] In some embodiments, the peptide immunogen construct is of following formula: (Th)m-(A)n“(activiii A B cell epitope) or (activin A B cell epitope)-(A)n-(Th)mor (Th)m-(A)n-(activin A B cell epitope)-(A)n-(Th)m, wherein Th is the T helper epitope; A is the heterologous spacer, which optionally is present and optionally comprises an amino acid; m is from 1 to about 4; and n is from 0 to about 10, wherein optionally the C-terminus of the peptide immunogen construct comprises a-COOH or a-CONH2of an amino acid. In some embodiments, the peptide immunogen construct is of following formula: (Th)m-(A)n-(activin A B cell epitope), wherein Th is the T helper epitope; A is the heterologous spacer, which optionally is present and optionally comprises an amino acid; m is from 1 to about 4; and n is from 0 to about 10, and wherein the C-terminus of the peptide immunogen construct comprises a-COOH or a-CONH2of an amino acid. In some embodiments, the peptide immunogen construct is of following formula: (activin A B cell epitope)-(A)n-(Th)m, wherein Th is the T helper epitope; A is the heterologous spacer, which optionally is present and optionally comprises an amino acid; m is from 1 to about 4; and n is from 0 to about 10, and wherein the C-terminus of the peptide immunogen construct comprises a-COOH or a-CONH2of an amino acid. In some embodiments of the formulas provided herein, m is 1 and n is 1. In some embodiments, more than one activin A B cell epitope is present in the peptide immunogen constructs described herein, e.g., 1-4 activin A B cell epitopes. In some embodiments, two or three activin A B cell epitopes are present in the peptide immunogen constructs described herein. In other embodiments, one (1) activin A B cell epitope is present in the peptide immunogen constructs described herein.

[0043] In some embodiments, the peptide immunogen construct comprises or consists of the sequence of any one of SEQ ID NOs: 72-89, 135-144, and 190-191 or a variant thereof. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 75, 76, 78, 79, 85, 136, 138, 139, 140, 141, 142, 143, 144, or 190, or a variant thereof. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 75, 78, 79, 85, 141, 143, 144, or 190, or a variant thereof. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 136, 141, 143, 144, or 190, or a variant thereof. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 136. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 141. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 143. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 144. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 190. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 75. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 76. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 78. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 79. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 84. In some embodiments, the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 85. In another aspect, the invention provides a combination peptide immunogen construct comprising two or more B cell epitopes, a heterologous T cell (Th) epitope, an optional heterologous linker, and an optional heterologous spacer, wherein the two or more B cell epitopes are covalently linked to the Th epitope directly or through the optional heterologous linker and / or optional heterologous spacer. In some embodiments, the two or more B cell epitopes are one to four (1-4) B cell epitopes. In some embodiments, the two or more B cell epitopes are two (2) or three (3) B cell epitopes. In some embodiments, the two or more B cell epitopes are two (2) B cell epitopes. In some embodiments, the two or more B cell epitopes are two or more (e.g., two) identical B cell epitopes, such as any B cell epitope described herein. In some embodiments, the two or more B cell epitopes are two or more (e.g., two) non-identical, different B cell epitopes, such as any two or more B cell epitopes described herein. In some embodiments, the two or more B cell epitopes are two or more (e.g., two) myostatin B cell epitopes (e.g., any myostatin B cell epitopes described herein, identical or different). In some embodiments, the two or more B cell epitopes are two or more (e.g., two) activin A B cell epitopes (e.g., any activin A B cell epitopes described herein, identical or different). In some embodiments, the two or more (e.g., two) B cell epitopes comprise at least one myostatin B cell epitope (e.g., any myostatin B cell epitope described herein) and at least one activin A B cell epitope (e.g., any activin A B cell epitope described herein). In some embodiments, the Th epitope is any Th epitope described herein. In some embodiments, the heterologous spacer is any heterologous spacer described herein. In some embodiments, the heterologous linker is any linker described herein. In some embodiments, the heterologous linker comprises one or more amino acids (e.g., one or more glycines). In some embodiments, the heterologous linker is an amino acid peg derivative. In some embodiments, the heterologous linker is AeeA (i.e., 8-amino-3,6-dioxaoctanoic acid or 2-[2-(2-aminoethoxy)ethoxy]acetic acid (CAS: 134978-97-5)). In other embodiments, other heterologous linkers can be used, for example, and without limitation, a linker comprising 3 to 6 pegs (e.g., l-Amino-3,6,9,12,15,18-hexaoxahenicosan-21-oic acid (6pe), l-amino-4,7,10,13-tetraoxapentadecanoic acid (3peg)), 6-aminohexanoic acid, or one or more epsilon-lysine. In some embodiments, the two or more B cell epitopes can be positioned in linear and / or sequential manner. In some embodiments, the two or more B cell epitopes are positioned in a non-linear and / or nonsequential manner. In some embodiments, the two or more B cell epitopes are branched off from a Th epitope, e.g., as described herein.

[0044] In some embodiments, provided herein is a combination myostatin - activin A peptide immunogen construct comprising a myostatin B cell epitope (e.g., any myostatin B cell epitope described herein), an activin A B cell epitope (e.g., any activin A B cell epitope described herein), and a T helper cell epitope (such as any T helper cell epitope described herein). In some embodiments, the B cell epitopes and the Th epitope are linked by a heterologous spacer (such as any heterologous spacer described herein). In some embodiments, the Th epitope comprises or consists of the sequence of SEQ ID NO: 30, 32, or 33. In some embodiments, the heterologous spacer comprises or consists of Lys-Lys-Lys-eLys, E-N-Lys-Lys-Lys-Lys (SEQ ID NO: 91), or (a, E-N)Lys. In some embodiments of the combination myostatin - activin A peptide immunogen construct, the myostatin B cell epitope comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 3-10, 95-109, 145-164, and 166 or a variant thereof. In some embodiments, the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 5, 97, 146, 147, 152-155, 157, 159, 161, 163, or 166. In some embodiments, the myostatin B cell epitope is of SEQ ID NO: 97. In some embodiments of the combination myostatin - activin A peptide immunogen construct, the activin A B cell epitope comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 13-18, 110-119, 187, and 189 or a variant thereof. In some embodiments, the activin A B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 14-18, 111, 116, 118, 119, or 187. In some embodiments, the activin A B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 14, 15, 17, 116, 119, or 187. In some embodiments, the activin A B cell epitope of SEQ ID NO: 116. In some embodiments, the myostatin B cell epitope is of SEQ ID NO: 97 and the activin A B cell epitope of SEQ ID NO: 116.

[0045] In some embodiments, the combination peptide immunogen construct comprises B cell epitopes sequentially positioned in a linear peptide. In other embodiments, the combination peptide immunogen comprises B cell epitopes that are positioned in a non-linear or non-sequential manner or branched off from a Th epitope. In some embodiments, the B cell epitope peptides are attached to the same side (N-terminal or C-terminal) of the Th epitope using a branched amino acid, which can be: (i) a diamino acid if the B-cell epitopes are attached to the N-terminus of a Th epitope, or (ii) a dicarboxylic acid if the B-cell epitopes are attached to the C-terminus of a Th epitope. Any diamino acid and dicarboxylic acid known in the art or described herein can be used. In some embodiments, the diamino acid is lysine. Other diamino acids include, without limitation, ornitine, 2,4-Diaminobutyric acid, and 2, 3 -diaminopropionic acid. In some embodiments, the dicarboxylic acid is glutamic acid. Other dicarboxylic acids include, without limitation, aspartic acid and a-amino adipic acid. Between the branched amino acid and the B cell epitope peptide, a heterologous linker can be used. In some embodiments, the heterologous linker comprises one or more amino acids (e.g., one or more Glycines). In some embodiments, the heterologous linker is an amino acid peg derivative. In some embodiments, the heterologous linker is AeeA (i.e., 8-amino-3,6-dioxaoctanoic acid or 2-[2-(2-aminoethoxy)ethoxy]acetic acid (CAS: 134978-97-5)). In other embodiments, other heterologous linkers can be used, for example, and without limitation, a linker comprising 3 to 6 pegs (e.g., l-Amino-3,6,9,12,15,18-hexaoxahenicosan-21-oic acid (6pe), l-amino-4,7,10,13-tetraoxapentadecanoic acid (3peg)), 6-aminohexanoic acid, or one or more epsilon-lysine. The combination peptide immunogen construct described herein can comprise the B cell epitope peptides described herein covalently linked to any Th epitope using any heterologous spacer described herein or known in the art, wherein the heterologous spacer connects the branched amino acid to the Th epitope. In some embodiments, the heterologous spacer comprises an amino acid (e.g., 2, 3, 4 or 5 Lysine residues). In some embodiments, the heterologous spacer comprises or consists of Lys-Lys-Lys-sLys, s-N-Lys-Lys-Lys-Lys (SEQ ID NO: 91), or (a, s-N)Lys. In some embodiments, the combination myostatin - activin A peptide immunogen construct is acetylated on its terminal NH2.

[0046] In some embodiments, the combination myostatin - activin A peptide immunogen construct is of SEQ ID NO: 165 (i.e., Ac-STVINHYRMRGHSPFANLKSSAeeA(Ac-QKYPHTHLVHQANPRGSAGPAeeA)K-KKKrKUbithl (SEQ ID NO: 165), where the peptide between ( ) indicates that it is off the side chain of the Lys and not the alpha amino group, AeeA is a peg spacer, and the Ac indicates that the construct is acetylated on its terminal NH2). In other embodiments, another myostatin B cell epitope (e.g., any one described herein), another activin A B cell epitope (e.g., any one described herein), another linker (e.g., any one described herein), and / or another T helper cell epitope (e.g., any one described herein) can be used in the combination myostatin-activin peptide immunogen constructs of the invention (instead of those used in the construct of SEQ ID NO: 165).

[0047] The invention further provides a nucleic acid molecule encoding a peptide immunogen construct described herein.

[0048] The invention further provides a vector comprising a nucleic acid molecule described herein. The invention further provides an isolated antibody or an epitope-binding fragment thereof that specifically binds to the myostatin B cell epitope of the peptide immunogen construct or an activin A B cell epitope of a peptide immunogen construct described herein.

[0049] The invention further provides a composition comprising one or more of: (a) a peptide immunogen construct described herein, a nucleic acid described herein, a vector described herein, an isolated antibody described herein, or a peptide immunogen construct described herein and a peptide immunogen construct described herein, and (b) a pharmaceutically acceptable carrier, diluent, or adjuvant.

[0050] The invention further provides a composition comprising: (a) a peptide immunogen construct comprising a myostatin B cell epitope, a heterologous T cell (Th) epitope, and an optional heterologous spacer, wherein the B cell epitope is covalently linked to the Th epitope directly or through the optional heterologous spacer; optionally wherein the myostatin B cell epitope comprises about 7 to about 30 contiguous amino acids from myostatin, or a variant thereof, and optionally wherein the myostatin B cell epitope is cyclized, (b) a peptide immunogen construct comprising an activin A B cell epitope, a heterologous T cell (Th) epitope, and an optional heterologous spacer, wherein the B cell epitope is covalently linked to the Th epitope directly or through the optional heterologous spacer; optionally wherein the activin A B cell epitope comprises about 7 to about 30 contiguous amino acids from myostatin, or a variant thereof, and optionally wherein the activin A B cell epitope is cyclized, and (c) a pharmaceutically acceptable carrier, diluent, or adjuvant.

[0051] In some embodiments, the composition comprises an adjuvant that is a mineral salt of aluminum, which optionally is selected from Al(OH)3and AlPO4.

[0052] In some embodiments, the composition comprises a CpG oligonucleotide (e.g., CpGl). In some embodiments, administration of the peptide immunogen constructs comprising a myostatin B cell epitope described herein stimulate generation of one or more antibodies to myostatin, wherein such one or more antibodies inhibit myostatin- induced signaling, e.g., in a cellbased assay.

[0053] In some embodiments, administration of the peptide immunogen constructs comprising an activin A B cell epitope described herein stimulate generation of one or more antibodies to activin A, wherein such one or more antibodies inhibit activin A-induced signaling, e.g., in a cell-based assay.

[0054] The invention further provides a method of maintaining or improving muscle physiology in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct described herein, a nucleic acid molecule described herein, a vector described herein, an isolated antibody described herein, or a composition described herein, or a combination of any thereof. The invention further provides a method of maintaining or improving muscle mass or growth in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct described herein, a nucleic acid molecule described herein, a vector described herein, an isolated antibody described herein, or a composition described herein, or a combination of any thereof.

[0055] The invention further provides a method of maintaining or improving muscle strength in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct described herein, a nucleic acid molecule described herein, a vector described herein, an isolated antibody described herein, or a composition described herein, or a combination of any thereof.

[0056] The invention further provides a method of maintaining or improving muscle tone in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct described herein, a nucleic acid molecule described herein, a vector described herein, an isolated antibody described herein, or a composition described herein, or a combination of any thereof.

[0057] The invention further provides a method of maintaining or improving muscle endurance in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct described herein, a nucleic acid molecule described herein, a vector described herein, an isolated antibody described herein, or a composition described herein, or a combination of any thereof.

[0058] The invention further provides a method of maintaining or improving muscle function or performance in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct described herein, a nucleic acid molecule described herein, a vector described herein, an isolated antibody described herein, or a composition described herein, or a combination of any thereof.

[0059] The invention further provides a method of preventing or treating muscular injury in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct described herein, a nucleic acid molecule described herein, a vector described herein, an isolated antibody described herein, or a composition described herein, or a combination of any thereof.

[0060] The invention further provides a method of preventing or treating musculoskeletal injury in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct described herein, a nucleic acid molecule described herein, a vector described herein, an isolated antibody described herein, or a composition described herein, or a combination of any thereof.

[0061] The invention further provides a method of preventing and treating a disorder associated with muscle loss or insufficient muscle growth in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct described herein, a nucleic acid molecule described herein, a vector described herein, an isolated antibody described herein, or a composition described herein, or a combination of any thereof.

[0062] The invention further provides a method of treating or preventing the development of muscle wasting in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct described herein, a nucleic acid molecule described herein, a vector described herein, an isolated antibody described herein, or a composition described herein, or a combination of any thereof.

[0063] The invention further provides a method of treating, preventing, reducing, inhibiting, or slowing the development of one or more symptom of a muscle wasting disease in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct described herein, a nucleic acid molecule described herein, a vector described herein, an isolated antibody described herein, a composition described herein, or a combination thereof.

[0064] The invention further provides a method of treating, preventing, reducing, inhibiting, or slowing age- or disease-related muscle loss in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct described herein, a nucleic acid molecule described herein, a vector described herein, an isolated antibody described herein, a composition described herein, or a combination thereof.

[0065] The invention further provides a method of treating, preventing, reducing, inhibiting, or slowing the development of sarcopenia in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct described herein, a nucleic acid molecule described herein, a vector described herein, an isolated antibody described herein, a composition described herein, or a combination thereof.

[0066] The invention further provides a method of preventing loss or increasing lean body mass (and optionally reducing fat mass) in a subject in need thereof, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct described herein, a nucleic acid molecule described herein, a vector described herein, an isolated antibody described herein, a composition described herein, or a combination thereof. In some embodiments, the subject has or is at risk of loss of lean body mass (such as muscle wasting). In some embodiments, the subject is obese.

[0067] The invention further provides a method of preventing loss of muscle, or increasing muscle strength and / or performance, in a subject in need thereof, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct described herein, a nucleic acid molecule described herein, a vector described herein, an isolated antibody described herein, a composition described herein, or a combination thereof. In some embodiments, the subject has sarcopenia or cachexia.

[0068] The invention further provides a method of maintaining or improving bone physiology in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct described herein, a nucleic acid molecule described herein, a vector described herein, an isolated antibody described herein, or a composition described herein, or a combination of any thereof.

[0069] The invention further provides a method of inducing an immune response to myostatin and / or activin A in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct described herein, a nucleic acid molecule described herein, a vector described herein, an isolated antibody described herein, a composition described herein, or a combination thereof.

[0070] The invention further provides a method of treating or preventing insulin resistance in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct described herein, a nucleic acid molecule described herein, a vector described herein, an isolated antibody described herein, a composition described herein, or a combination thereof.

[0071] The invention further provides a method of maintaining or improving glucose metabolism in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct described herein, a nucleic acid molecule described herein, a vector described herein, an isolated antibody described herein, a composition described herein, or a combination thereof.

[0072] The invention further provides a method of treating or preventing metabolic disease, disorder or syndrome in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct described herein, a nucleic acid molecule described herein, a vector described herein, an isolated antibody described herein, a composition described herein, or a combination thereof.

[0073] In some embodiments, the subject has or is at risk of developing muscle wasting due to an inherited or genetic cause, inflammation, infection, injury, systemic disease, toxin, or other environmental exposure. In some embodiments, the subject has or is at risk of developing muscle wasting due to immobilization, prolonged bed rest, and / or injury. In some embodiments, the subject has or is at risk of developing muscle wasting due to sarcopenia or progressive loss of muscle strength due to aging.

[0074] In some embodiments, the subject has an inherited or genetic myopathy, which is optionally selected from muscular dystrophy (e.g., Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD), myotonic dystrophy (e.g., DM1 and DM2), facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, and limb girdle muscular dystrophy), congenital myopathy (e.g., nemaline myopathy and central core myopathy), metabolic myopathy (e.g., acid maltase or acid alpha- 1, 4-glucosidase deficiency (Pompe’s disease), glycogen storage disorders 3-11, carnitine deficiency, fatty acid oxidation defects, and carnitine palmitoyl transferase deficiency), and mitochondrial myopathy.

[0075] In some embodiments, the subject has or is at risk of developing an autoimmune or inflammatory myopathy (i.e., myositis), which is optionally selected from dermatomyositis, polymyositis, inclusion body myositis, and juvenile myositis, or another autoimmune disorder (e.g., myasthenia gravis).

[0076] In some embodiments, the subject has or is at risk of developing an infection, which optionally is selected from an infection by a virus (e.g., HIV, influenza virus, and Epstein-Barr virus), a bacterium (e.g., pyomyositis, S. aureus, and streptococci), a spirochete (e.g., Lyme disease), and a parasite (e.g., trichinosis).

[0077] In some embodiments, the subject has or is at risk of exposure to a toxin, which optionally is selected from a medication (e.g., a cholesterol-lowering medication (e.g., a statin), propofol, amiodaron, colchicine, chloroquine, an antiviral, a protease inhibitor, omeprazole, and tryptophan), alcohol, and toluene.

[0078] In some embodiments, the subject has or is at risk of developing a systemic disease, which optionally is selected from an endocrine disorder (e.g., thyroid, parathyroid, pituitary, and adrenal disorder), systemic inflammatory disease (e.g., systemic lupus erythematosus, rheumatoid arthritis, scleroderma, Sjogren’s syndrome, mixed connective disease, and sarcoidosis), electrolyte imbalance, critical illness myopathy, and amyloid myopathy.

[0079] In some embodiments, the subject has or is at risk of developing a neurological disease or condition, which optionally is selected from amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), spinal muscular atrophy (SMA), Guillain-Barre syndrome, carpel tunnel syndrome, Charcot-Marie-Tooth disease, Parkinson’s disease, Lewy body disease, polio, spinal cord injury, peripheral nerve injury, stroke, and nerve damage caused by diabetes, toxins, or alcohol.

[0080] In some embodiments, the subject is or is expected to be present in an environment with decreased or no gravity, e.g., during a space flight or being at a space station or the like.

[0081] In some embodiments, the subject has or experiences prolonged inactivity (e.g., is bedridden, has a seated job or a sedentary lifestyle, or cannot move limbs due to stroke or other brain disease), burn, long-term corticosteroid therapy, malnutrition, anorexia, cachexia, osteoarthritis, advanced age, cancer, chronic kidney disease, heart failure, or chronic obstructive pulmonary disease. In some embodiments, the subject has or is at risk of developing one or more symptom selected from muscle loss, movement issues, balance problems, trouble walking, trouble using arms or legs, contractures, muscle rigidity, muscle twitching, fasciculations, muscle cramps, numbness, tingling, painful sensations, breathing problems, curved spine, heart problems, swallowing problems (which may lead to, e.g., nutritional problems and aspiration pneumonia), reduced muscle mass (e.g., in general or in one limb), weakness (e.g., in general or in one limb), pain, discomfort, one arm or leg being smaller than the other, weakness in one arm or leg, numbness or tingling in an arm or leg, trouble walking, balance problems, facia weakness, gradual memory loss, reduced muscle strength, impaired ability to perform physical activities, decrease in muscle size, cramps, stiffness, spasms, fatigue, motor delay, respiratory impairment, and bulbar muscle dysfunction.

[0082] In some embodiments, the subject has or is at risk of developing insulin resistance, type II diabetes, elevated A1C levels, pre-diabetes, metabolic syndrome, hypertension, hyperglycemia, hypercholesterolemia, high triglycerides, overweight, or obesity.

[0083] In some embodiments, the subject is a mammal, e.g., a human.

[0084] The invention also includes use of any of the peptide immunogen constructs, nucleic acids, vectors, compositions, and combinations in methods such as any of those described herein, and use of these materials in the preparation of medicaments for carrying out such methods.

[0085] Other features and advantages of the invention will be apparent from the following description and the claims.

[0086] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing antibody titers against myostatin resulting from immunization of Guinea pigs with the indicated myostatin peptide immunogen constructs.

[0087] FIG. 2 is a graph showing immunogenicity of the peptide immunogen construct of SEQ ID NO: 55 injected into Guinea pigs.

[0088] FIG. 3 is a graph showing the results of a neutralization assay testing peptide (SEQ ID NO: 55)-induced antibodies in HEK293 cells.

[0089] FIG. 4 is a set of graphs showing the effects of peptide (SEQ ID NO: 55)-induced antibodies on fiber width and number of nuclei in C2C12 cells.

[0090] FIG. 5 is a schematic showing a treatment regimen used for in vivo studies.

[0091] FIG. 6 is a series of graphs showing immunogenicity at week 11 of mice vaccinated with the indicated doses of peptide immunogen construct of SEQ ID NO: 55. FIG. 7 is a graph showing body weights of peptide construct (SEQ ID NO: 55) -vaccinated mice at the indicated times.

[0092] FIG. 8 is a set of graphs showing twitch tension and tetanic tension in tibialis anterior isolated from peptide construct (SEQ ID NO: 55)-vaccinated mice.

[0093] FIG. 9 is a table and a set of graphs showing immunogenicity of the indicated activin A peptide constructs injected into Guinea pigs.

[0094] FIG. 10 is a set of graphs showing the results of an assay of the effects of purified activin A IgGs on activin A-induced SMAD2 / 3 signaling.

[0095] FIG. 11 is a graph showing immunogenicity of the indicated activin A peptide constructs in rats at week 6 post-vaccination.

[0096] FIG. 12 is a graph showing immunogenicity of the indicated myostatin peptide constructs in rats at week 6 post-vaccination.

[0097] FIG. 13 is a graph showing immunogenicity of the indicated myostatin or activin A peptide constructs in Guinea pigs at 15 weeks post- vaccination.

[0098] FIG. 14 is a graph showing the change in body weights over time of Guinea pigs immunized with the indicated myostatin or activin A peptide construct.

[0099] FIG. 15A is a graph showing the immunogenicity of the indicated latent myostatin peptide constructs in rats at week 15 post- vaccination and FIG. 15B shows antibody titers against latent myostatin resulting from the immunization.

[0100] FIG. 16A is a graph showing the immunogenicity of the indicated promyostatin peptide constructs in rats at week 15 post-vaccination and FIG. 16B shows antibody titers against promyostatin resulting from the immunization.

[0101] FIG. 17A is a graph showing the immunogenicity of the indicated mature myostatin peptide constructs in rats at week 15 post- vaccination and FIG. 17B shows antibody titers against mature myostatin resulting from the immunization.

[0102] FIG. 18 is a graph showing antibody titers against mature myostatin or GDF-11 at 15 weeks after the immunization of rats with the myostatin construct of SEQ ID NO: 168.

[0103] FIG. 19A is a graph showing the immunogenicity of the indicated activin A peptide constructs in rats at week 15 post-vaccination and FIG. 19B shows antibody titers against mature activin A resulting from the immunization.

[0104] FIG. 20 is a graph showing the immunogenicity of the indicated myostatin peptide constructs in rats at week 15 post-immunization. FIG. 21 is a graph showing the immunogencity of the indicated activin A peptide constructs in rats at week 15 post- immunization.

[0105] FIG. 22 depicts an exemplary structure of a bivalent construct (containing two B cell epitopes), specifically the construct of SEQ ID NO: 165.

[0106] DETAILED DESCRIPTION

[0107] The present disclosure is based, in part, on the discovery that certain myostatin and activin A peptides can be used to induce an immune response to myostatin and activin A, respectively, leading to improvements in muscle physiology and, thus, improvements in certain features of muscle wasting.

[0108] Accordingly, the disclosure is directed to certain myostatin and activin A peptides, as well as peptide immunogen constructs and compositions that include these peptides.

[0109] The disclosure additionally is directed to methods of maintaining or improving muscle physiology (e.g., maintaining or increasing one, two, three, four or more of: muscle mass, growth, strength, tone, endurance, function or performance) in a subject in need thereof by administration of a peptide immunogen construct or a related molecule or composition described herein.

[0110] The disclosure additionally is directed to methods of preventing or treating a muscle wasting disease or condition in a subject in need thereof by administration of a peptide immunogen construct or a related molecule or composition described herein. The present disclosure is also directed to methods of treating, preventing, reducing, or inhibiting one or more symptoms of a muscle wasting disease. Further, the disclosure is directed to methods of preventing and treating insulin resistance and related diseases and conditions. Advantageously, the compositions and methods of the disclosure can be used to provide substantial benefits for inhibiting, slowing the development of, or treating the diseases and conditions described herein (or one or more symptoms thereof), leading to improved quality of life and prolonged health span.

[0111] The compositions and methods of the disclosure are described in an exemplary manner below.

[0112] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0113] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including explanations of terms, will control.

[0114] 1 In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0115] The singular terms “a,” “an,” and “the” include the respective plural terms, unless context indicates otherwise. Similarly, the word "or" is intended to include “and” unless the context clearly indicates otherwise. Hence “comprising A or B” means including A or B, or A and B. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed methods, suitable methods and materials are described below.

[0116] The term “about,” when used to modify a numeric value herein, indicate that deviations of up to 10% above and belo w the numeric value remain within the intended meaning of the recited value. In some embodiments, the deviation is up to about 7.5%, 6%, 5%, 4%, 3%, 2% or 1% above and below the recited value.

[0117] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference herein in their entirety. All references or portions of references cited in this application are expressly incorporated herein by reference in their entirety for any purpose as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

[0118] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0119] Immunotherapeutic Agents and Compositions

[0120] The compositions and methods of the disclosure can include or use, for example, one or more peptide or peptide fusion (e.g., a peptide immunogen construct as described herein), a protein (e.g., an antibody), or a nucleic acid molecule (e.g., mRNA or nucleic acid in a viral vector) encoding one of said molecules, wherein the molecule is directed against myostatin or activin A. In some embodiments, the compositions and methods include or employ one or more molecules directed against myostatin and one or more molecules directed against activin A.

[0121] B cell epitope Peptides

[0122] In some embodiments, the compositions and methods of the disclosure employ a peptide immunogen construct including a B cell epitope from myostatin linked to a heterologous T helper cell (Th) epitope, directly or through an optional heterologous linker or spacer.

[0123] The B cell epitope portion of the myostatin peptide immunogen constructs can optionally include about 7 to about 30 (e.g., 7-29, 7-28, 7-27, 7-26, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7- 18, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, or 7-10, or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) contiguous amino acid residues from mature myostatin, which is located at the C-terminal end of myostatin preproprotein, and corresponds, e.g., to the sequence from about the aspartic acid at ammo acid position 267 (D267) to about the serine at ammo acid position 375 (S375) of full-length myostatin preproprotein (SEQ ID NO: 1). The sequence of human mature myostatin is set forth in SEQ ID NO: 2. In some embodiments, the B cell epitope portion of the myostatin peptide immunogen constructs can optionally include about 7 to about 30 (e.g., 7-29, 7-28, 7-27, 7-26, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, or 7-10, or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) contiguous ammo acid residues from full-length myostatin preproprotein (SEQ ID NO: 1). In some embodiments, the B cell epitope portion of the myostatin peptide immunogen constructs can optionally include about 7 to about 30 (e.g., 7-29, 7-28, 7-27, 7-26, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, or 7-10, or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) contiguous ammo acid residues from about the leucine at ammo acid position 80 (L80) to about the leucine at amino acid position 270 (L270) of full-length myostatin preproprotein (SEQ ID NO: 1). The heterologous Th epitope portion of the peptide immunogen constructs can optionally be derived from pathogenic proteins. The B cell epitope and Th epitope portions of the peptide immunogen constructs act together when administered to a subject to stimulate the generation of antibodies that specifically recognize and bind to the myostatin B cell epitope portion of the constructs.

[0124] Accordingly, the phrase “myostatin peptide immunogen construct,” as used herein, refers to a peptide containing (a) a B cell epitope having about 7 to about 30 (e.g., 7-29, 7-28, 7-27, 7-26, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, or 7-10, or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) contiguous ammo acid residues from myostatin (e.g., mature myostatin or myostatin preprotein),; (b) a heterologous Th epitope; and (c) an optional heterologous spacer.

[0125] In certain embodiments, the myostatin peptide immunogen construct can be represented by the formulae: (Th)m~-(A)n~(myostatin peptide)P~-X, (myostatin peptide)P“(A)n-(Th)m-X, or (Th)m-(A)n-(myostatin peptide)p-(A)n-(Th)m-X, wherein Th is a heterologous T helper epitope; A is an optional heterologous spacer; (myostatin peptide) is a myostatin B cell epitope having about 7 to about 30 (e.g., 7-29, 7-28, 7-27, 7-26, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-19, 7-18, 7- 17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, or 7-10, or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) contiguous ammo acid residues from mature myostatin (e.g., a peptide derived from loop 1, loop 2, loop 3, or loop 4 of mature myostatin, e.g., as described herein); X is an a-COOH or a-CONH₂ of an ammo acid; m is an integer from 1 to about 4; p is an integer from 1 to about 4 (e.g., 2); and n is an integer from 0 to about 10. In certain embodiments, the myostatin peptide immunogen construct can be represented by the formulae: (Th)m-(A)n-(myostatin peptide)p-X, (myostatin peptide)p-(A)n-(Th)m~X, or (Th )m-(A)n--{myostatin peptide)p-(A)„-(Th)m-X, wherein Th is a heterologous T helper epitope; A is an optional heterologous spacer; (myostatin peptide) is a myostatin B cell epitope having about 7 to about 30 (e.g., 7-29, 7-28, 7-27, 7-26, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, or 7-10, or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) contiguous ammo acid residues from myostatin preprotein; X is an a-COOH or a-CONH₂ of an amino acid; m is an integer from 1 to about 4; p is an integer from 1 to about 4 (e.g., 2); and n is an integer from 0 to about 10. In some embodiments, A is an amino acid and n indicates the number of amino acids, wherein each A can be identical to one another or one or more of the A’s can be a different ammo acid. It is to be understood that the myostatin peptides described herein can optionally include one or more modifications (e.g., one or more substitution, deletion, or addition), for example, one or more (e.g., 1, 2, 3, 4, or 5) substitutions, which optionally may be designed to facilitate peptide cyclization. In some embodiments, the peptides are linear. In other embodiments, the peptides are cyclic due to, e.g., the presence of, e.g., cysteine residues, whether naturally occurring in the sequences or inserted, e.g., by substitution. The various components of the disclosed myostatin peptide immunogen constructs are described further below, after a description of activin A peptides.

[0126] In some embodiments, the compositions and methods of the disclosure employ a peptide immunogen construct including a B cell epitope from activin A linked to a heterologous T helper cell (Th) epitope, directly or through an optional heterologous linker or spacer.

[0127] The B cell epitope portion of the activin A peptide immunogen constructs can optionally include about 7 to about 30 (e.g., 7-29, 7-28, 7-27, 7-26, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7- 18, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, or 7-10, or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) contiguous amino acid residues from mature activin A, which is located at the C-terminal end of activin A preproprotein. The sequence of human mature activin A is set forth in SEQ ID NO: 12. The heterologous Th epitope portion of the peptide immunogen constructs can optionally be derived from pathogenic proteins. The B cell epitope and Th epitope portions of the peptide immunogen constructs act together when administered to a subject to stimulate the generation of antibodies that specifically recognize and bind to the activin A B cell epitope portion of the constructs.

[0128] Accordingly, the phrase “activin A peptide immunogen construct,” as used herein, refers to a peptide containing (a) a B cell epitope having about 7 to about 30 (e.g., 7-29, 7-28, 7-27, 7-26, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, or 7-10, or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) contiguous ammo acid residues from activin A (e.g., mature activin A); (b) a heterologous Th epitope; and (c) an optional heterologous spacer.

[0129] In certain embodiments, the peptide immunogen construct can be represented by the formulae: (Th)m-(A)n--(activin A peptide)p--X, (activin A peptide)p---(A)n--(Th)m--X, or (Th)m-(A)n-(activin A peptide)p-(A)n-(Th)m-X, wherein Th is a heterologous T helper epitope; A is an optional heterologous spacer; (activin A peptide) is an activin A B cell epitope having about 7 to about 30 (e.g., 7-29, 7-28, 7-27, 7-26, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, or 7-10, or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) contiguous amino acid residues from mature activin A (e.g., a peptide derived from loop 1, loop 2, loop 3, or loop 4 of mature activin A, e.g., as described herein); X is an a-COOH or a-CONH₂ of an amino acid; m is an integer from 1 to about 4; p is an integer from 1 to about 4 (e.g., 2); and n is an integer from 0 to about 10. In some embodiments, A is an amino acid and n indicates the number of amino acids, wherein each A can be identical to one another or one or more of the A’s can be a different ammo acid. It is to be understood that the activin A peptides described herein can optionally include one or more modifications (e.g., one or more substitution, deletion, or addition), for example, one or more (e.g., 1, 2, 3, 4, or 5) substitutions, which optionally may be designed to facilitate peptide cyclization. In some embodiments, the peptides are linear. In other embodiments, the peptides are cyclic due to, e.g., the presence of, e.g., cysteine residues, whether naturally occurring in the sequences or inserted, e.g., by substitution. The various components of the disclosed activin A peptide immunogen constructs are described further below.

[0130] In some embodiments, the B cell epitope peptides described herein are acetylated on the N-terminus (e.g., when the B cell epitope is linked to the N-terminal end of the T helper cell epitope). In some embodiments, the B cell epitope peptides described herein comprise an amide group on the C-terminus (e.g., when the B cell epitope is linked to the C-terminal end of the T helper cell epitope). In some embodiments, the B cell epitope peptide is acetylated on the N-terminus and comprises an amide group on the C-terminus. In some embodiments, the B cell epitope peptide described herein is cyclized using the N-terminus amino group and not acetylated on the N-terminus.

[0131] Myostatin and activin A fragments

[0132] The term “myostatin preproprotein” and the like, as used herein, refers to (a) the full-length myostatin preproprotein from any organism that expresses myostatin. In some embodiments, the myostatin preproprotein is human. In certain embodiments, the full-length human myostatin preproprotein has 375 ammo acids (see, e.g., SEQ ID NO: I). Myostatin preproprotein includes an N-terminal core of hydrophobic amino acids, which act as a signal sequence for secretion.

[0133] Proteolytic processing in the Golgi at an RSRR site by a serine protease, such as furin or another member of the proprotein convertase family, generates an N-terminal latency-associated peptide (LAP) and C-terminal mature myostatin.

[0134] The term “mature my ostatin,” as used herein, refer to the ammo acid sequence of the myostatin preproprotein that is C-terminal from the serine protease cleavage site RSRR. In certain embodiments, myostatin or mature myostatin is human. In certain embodiments, mature myostatin has the amino acid sequence between residues 267-375 (SEQ ID NO: 2) of myostatin preproprotein (SEQ ID NO: 1).

[0135] The phrase “myostatin fragment,” “myostatin peptide,” or “B cell epitope of myostatin,” as used herein, refers to a portion of the full-length myostatin sequence that includes about 7 to about 30 (e.g., 7-15, 7-14, 7-13, 7-12, 7-11, or 7-10, or 7, 8, 9, 10, 11, 12, 13, 14, or 15) contiguous amino acid residues from the myostatin sequence. The various myostatin fragments described herein can be referred to by their amino acid positions in relation to the full-length sequence of prepromyostatin represented by SEQ ID NO: 1. In some embodiments, a myostatin fragment includes one or more ammo acid changes (e.g., substitutions, deletions, or additions), for example, one or more amino acid substitutions (e.g., 1, 2, 3, 4, 5, or 6 amino acid substitutions). In some embodiments, the one or more amino acid changes, for example, the one or more (e.g., 1, 2, 3, 4, or 5) ammo acid substitutions, are designed to facilitate cyclization of the myostatin fragment. In some embodiments, the myostatin fragment has the sequence of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10 (see Table 1). In some embodiments, the myostatin fragment has the sequence of SEQ ID NO: 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, or 109 (see Table 1). In some embodiments, the myostatin fragment has the sequence of SEQ ID NO: 145, 146, 147, 148, 149, 150, 151, 152, 153, or 154 (see Table 1). In some embodiments, tire myostatin fragment has the sequence of SEQ ID NO: 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, or 166 (see Table 1).

[0136] The term “activin preproprotein” and the like, as used herein, refers to (a) the full-length activin A preproprotein from any organism that expresses activin A. In some embodiments, the activin A preproprotein is human. In certain embodiments, the full-length human activin A preproprotein has 426 amino acids (see, e.g., SEQ ID NO: 11). Activin A preproprotein includes an N-terminal core of hydrophobic ammo acids, which act as a signal sequence for secretion.

[0137] Proteolytic processing in the Golgi at an RRRR site by a serine protease, such as furin or another member of the proprotein convertase family, generates C-terminal mature activin A, which will homo- or hetero-dimerize to form a physiologically active molecule.

[0138] The terms “mature activin A,” as used herein, refer to the amino acid sequence of the activin A preproprotein that is C-terminal from the serine protease cleavage site RRRR. In certain embodiments, activin A or mature activin A is human. In certain embodiments, mature activin A has the ammo acid sequence between residues 311 to 426 (SEQ ID NO: 12) of activin A preproprotein (SEQ ID NO: 11).

[0139] The phrase “activin A fragment,” “activin A peptide,” or “B cell epitope of activin A,” as used herein, refers to a portion of the full-length activin A sequence that includes about 7 to about 30 (e.g., 7-15, 7-14, 7-13, 7-12, 7-11, or 7-10, or 7, 8, 9, 10, 11, 12, 13, 14, or 15) contiguous ammo acid residues from the activin A sequence. The various activin A fragments described herein can be referred to by their amino acid positions in relation to the full-length sequence of preproactivin A represented by SEQ ID NO: 11. In some embodiments, an activin A fragment includes one or more amino acid changes (e.g., substitutions, deletions, or additions), for example, one or more amino acid substitutions (e.g., 1, 2, 3, 4, 5, or 6 amino acid substitutions). In some embodiments, the one or more ammo acid changes, for example, the one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, are designed to facilitate cyclization of the activin A fragment. In some embodiments, the activin A fragment has the sequence of SEQ ID NO: 13, 14, 15, 16, 17, or 18 (see Table 2). In some embodiments, the activin A fragment has the sequence of SEQ ID NO: 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 187, or 189 (see Table 2).

[0140] Myostatin and activin A peptide analogues

[0141] The myostatin and activin A fragments of the present disclosure also include immunologically functional analogues or homologues of myostatin or activin A, or fragments thereof. Functional immunological analogues or homologues of myostatin or activin A, or fragments thereof, include variants that retain substantially the same immunogenicity as the original peptide. Immunologically functional analogues can have one or more conservative substitutions in an ammo acid position; a change in overall charge; a covalent attachment to another moiety; or amino acid additions, insertions, or deletions; and / or any combination thereof.

[0142] Conservative substitutions are those in which one amino acid residue is substituted for another ammo acid residue with similar chemical properties. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; the polar neutral ammo acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; the positively charged (basic) ammo acids include arginine, lysine, and histidine; and the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0143] Immunologically functional analogues can include amino acid sequences that comprise conservative substitutions, additions, deletions, or insertions from one to about four amino acid residues that elicit immune responses that are cross-reactive with myostatin or myostatin fragments, or activin A or activin A fragments. The conservative substitutions, additions, and insertions can be accomplished with natural or non-natural amino acids. Non-naturally occurring amino acids include, but are not limited to, s-N Lysine, B-alanine, ornithine, norleucine, norvaline, hydroxyproline, thyroxine, y-amino butyric acid, homoserine, citrulline, aminobenzoic acid, 6-aminocaproic acid (Aca; 6-aminohexanoic acid), hydroxyproline, mercaptopropionic acid (MPA), 3-nitro-tyrosine, pyroglutamic acid, 2, 3 -diaminopropionic acid, 2,4-diaminobutyric acid, ornithine, and the like. Naturally occurring amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, myostatin or activin A B cell epitopes provided herein comprise a non-naturally occurring amino acid, e.g., one or more of the following: 6-aminohexanoic acid, 2, 3 -diaminopropionic acid, 2,4-diaminobutyric acid, and ornithine.

[0144] In some embodiments, the immunologically functional analogues of the myostatin or activin A B cell epitope peptides comprise one or more (e.g., two) amino acid substitutions with a non-naturally occurring amino acid to allow cyclization of the peptide.

[0145] In certain embodiments, the immunologically functional analogue of a particular peptide includes the same amino acid sequence as the original peptide and further includes three lysine residues (Lys-Lys-Lys) added to the amino terminus of the myostatin or activin A peptide (or a fragment thereof). In such embodiments, the addition of three lysine residues to the original peptide sequence changes the overall charge of the original peptide but does not alter the function of the original peptide. In certain embodiments, a functional analogue of a myostatin or activin A peptide has at least 50% sequence identity to the original ammo acid sequence. In other embodiments, the functional analogue has at least 80% identity to the original ammo acid sequence. In yet other embodiments, the functional analogue has at least 85% identity to the original amino acid sequence. In still other embodiments, the functional analogue has at least 90% identity to the original amino acid sequence. In further embodiments, the functional analogue has at least 95% or more (e.g., at least 96%, 97%, 98%, or 99%) identity to the original amino acid sequence. The percent identity between two sequences can be determined manually by inspection of the two optimally aligned sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters, as is known in the art. In some embodiments, the functional analogue includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions or deletions, as compared to the original amino acid sequence. In some embodiments, 1, 2, or 3 substitutions are included.

[0146] Heterologous T helper cell epitopes (Th epitopes)

[0147] Peptide immunogen constructs of disclosure include a B cell epitope from myostatin or activin A (e.g., as described above) covalently linked to a heterologous T helper cell (Th) epitope directly or through an optional heterologous spacer. The heterologous Th epitopes in the myostatin or activin A peptide immunogen constructs enhance the immunogenicity of the myostatin or activin A peptides, which facilitates the production of specific high titer antibodies directed against the optimized target B cell epitopes (i.e., the myostatin peptide or the activin A peptide) through rational design.

[0148] The term “heterologous,” as used herein in reference to myostatin, refers to an amino acid sequence that is derived from an ammo acid sequence that is not part of, or homologous with, the wild-type sequence of myostatin. Thus, a heterologous Th epitope is a Th epitope derived from an amino acid sequence that is not naturally found in myostatin (i.e., the Th epitope is not autologous to myostatin). Since the Th epitope is heterologous to myostatin, the natural amino acid sequence of myostatin is not extended in either the N-terminal or C-terminal directions when the heterologous Th epitope is covalently linked to the myostatin peptide.

[0149] The term “heterologous,” as used herein in reference to activin A, refers to an amino acid sequence that is derived from an amino acid sequence that is not part of, or homologous with, the wild-type sequence of activin A. Thus, a heterologous Th epitope is a Th epitope derived from an ammo acid sequence that is not naturally found in activin A (i.e., the Th epitope is not autologous to activin A). Since the Th epitope is heterologous to activin A, the natural ammo acid sequence of activin A is not extended in either the N-terminal or C-terminal directions when the heterologous Th epitope is covalently linked to the activin A peptide.

[0150] The heterologous Th epitope of the present disclosure can be any Th epitope that does not have an amino acid sequence naturally found in the B cell epitope-containing peptide of the construct in which it occurs (i.e., the myostatin or activin A peptide). The Th epitope can have an amino acid sequence derived from any species (e.g., human, pig, cattle, dog, rat, mouse, guinea pigs, etc.) or from a pathogen (e.g., a measles virus or a hepatis virus (e.g., a hepatitis virus surface protein; see below)). The Th epitope can also have promiscuous binding motifs to MHC class II molecules of multiple species. In certain embodiments, the Th epitope comprises multiple promiscuous MHC class II binding motifs to allow7maximal activation of T helper cells leading to initiation and regulation of immune responses. The Th epitope is preferably immunosilent on its own, i.e., little, if any, of the antibodies generated by the myostatin peptide or the activin A immunogen constructs will be directed towards the Th epitope(s), thus allowing a very focused immune response directed to the targeted B cell epitope of the myostatin or activin A peptide.

[0151] Th epitopes include, but are not limited to, amino acid sequences derived from foreign pathogens, as exemplified in Table 3 (SEQ ID NOs: 19-47). Further, Th epitopes include idealized artificial Th epitopes and combinatorial idealized artificial Th epitopes (e.g., SEQ ID NOs: 20 and 27-33). The heterologous Th epitope peptides presented as a combinatorial sequence (e.g., SEQ ID NOs: 28-31), contain a mixture of amino acid residues represented at specific positions within the peptide framework based on the variable residues of homologues for that particular peptide. An assembly of combinatorial peptides can be synthesized in one process by adding a mixture of the designated protected ammo acids, instead of one particular ammo acid, at a specified position during the synthesis process. Such combinatorial heterologous Th epitope peptide assemblies can allow broad Th epitope coverage for animals having a diverse genetic background. Representative combinatorial sequences of heterologous Th epitope peptides include SEQ ID NOs: 28-31, which are shown in Table 3. Th epitope peptides of the present disclosure provide broad reactivity and immunogenicity to animals and patients from genetically diverse populations. In some embodiments, the sequence is SEQ ID NO: 30. In some embodiments, the sequence is SEQ ID NO: 32. In some embodiments, the sequence is SEQ ID NO: 33. Peptide immunogen constructs comprising Th epitopes can optionally be produced simultaneously in a single solid-phase peptide synthesis in tandem with the B cell epitopecontaining myostatin or activin A peptide.

[0152] Th epitopes also include immunological analogues of Th epitopes (see, e.g., above description of analogs). Immunological Th analogues include immune-enhancing analogs, cross-reactive analogs, and fragments of any of these Th epitopes that are sufficient to enhance or stimulate an immune response to the myostatin or activin A peptides.

[0153] Immunologically functional analogues of the Th epitope peptides are also effective and can be used in the methods of the disclosure. Immunologically functional Th analogues can include conservative substitutions, additions, deletions, and insertions of from one to about five ammo acid residues (e.g., I, 2, 3, 4, or 5) in the Th epitope which do not essentially modify the Th-stimulating function of the Th epitope. The conservative substitutions, additions, and insertions can be accomplished with natural or non-natural amino acids, which can also be the case for myostatin and activin A peptides, as described above.

[0154] Table 3 identifies another variation of a functional analogue for Th epitope peptide. In particular, SEQ ID NOs: 20 and 27 of MvFl and MvF2 Th are functional analogues of SEQ ID NOs: 30 and 32 of MvF4 and MvF5 in that they differ in the amino acid frame by the deletion (SEQ ID NOs: 20 and 27) or the inclusion (SEQ ID NOs: 30 and 32) of two ammo acids each at the N-and C-termini. The differences between these two series of analogous sequences would not affect the function of the Th epitopes contained within these sequences. Therefore, functional immunological Th analogues can, for example, include several versions of the Th epitope derived from Measles Virus Fusion protein MvFl-4 Ths (SEQ ID NOs: 20, 27, 28, 30, and 32) and from Hepatitis Surface protein HBsAg 1-3 Ths (SEQ ID NOs: 29, 31, and 33).

[0155] The Th epitope in the peptide immunogen constructs can be covalently linked at either N- or C-terminal end of the myostatin or activin A peptide. In some embodiments, the Th epitope is covalently linked to the N-terminal end of the myostatin or activin A peptide. In other embodiments, the Th epitope is covalently linked to the C-terminal end of the myostatin or activin A peptide. In certain embodiments, more than one Th epitope is covalently linked to the myostatin or activin A peptide. When more than one Th epitope is linked to the myostatin or activin A peptide, each Th epitope can have the same amino acid sequence or different amino acid sequences. In addition, when more than one Th epitope is linked to the myostatin or activin A peptide, the Th epitopes can be arranged in any order. For example, the Th epitopes can be consecutively linked to the N-terminal end of the myostatin or activin A peptide, or consecutively linked to the C-terminal end of the myostatin or activin A peptide, or a Th epitope can be covalently linked to the N-terminal end of the myostatin or activin A peptide while a separate Th epitope is covalently linked to the C-terminal end of the myostatin or activin A peptide. There is no limitation in the arrangement of the Th epitopes in relation to the myostatin or activin A peptide.

[0156] In some embodiments, the Th epitope is covalently linked to the myostatin or activin A peptide directly. In other embodiments, the Th epitope is covalently linked to the myostatin or activin A peptide through a heterologous spacer, e.g., as described in further detail below.

[0157] Heterologous Spacer or L inker

[0158] The peptide immunogen constructs optionally include a heterologous spacer or linker that covalently links the B cell epitope from myostatin or activin. A to the heterologous T helper cell (Th) epitope.

[0159] As discussed above, the term “heterologous” refers to an ammo acid sequence that is derived from an ammo acid sequence that is not part of, or homologous with, the wild-type sequence of myostatin, in the case of myosin peptide immunogen constructs, or activin A, in the case of activin A peptide immunogen constructs. Thus, the natural ammo acid sequence of myostatin is not extended in either the N-terminal or C-terminal directions when the heterologous spacer or linker is covalently linked to the B cell epitope from myostatin because the spacer is heterologous to the myostatin sequence. Similarly, the natural amino acid sequence of activin A is not extended in either the N-terminal or C-terminal directions when the heterologous spacer or linker is covalently linked to the B ceil epitope from activin A because the spacer is heterologous to the activin A sequence.

[0160] The spacer or linker is any molecule or chemical structure capable of linking two ammo acids and / or peptides together. The spacer can vary in length or polarity depending on the application. The spacer attachment can be through an amide- or carboxyl-linkage but other functionalities are possible as well. The spacer can include a chemical compound, a naturally occurring amino acid, or a non-naturaily occurring amino acid.

[0161] The spacer or linker can provide structural features to the myostatin or activin A peptide immunogen construct. Structurally, the spacer or linker provides a physical separation of the Th epitope from the B cell epitope of the myostatin or activin A fragment. The physical separation by the spacer can disrupt any artificial secondary structures created by joining the Th epitope to the B cell epitope. Additionally, the physical separation of the epitopes by the spacer can eliminate interference between the Th cell and / or B cell responses. Furthermore, the spacer or linker can be designed to create or modify a secondary structure of the peptide immunogen construct. For example, a spacer can be designed to act as a flexible hinge to enhance the separation of the Th epitope and B cell epitope. A flexible hinge spacer can also permit more efficient interactions between the presented peptide immunogen and the appropriate Th cells and B cells to enhance the immune responses to the Th epitope and B cell epitope. Examples of sequences of flexible hinges are found in the immunoglobulin heavy chain hinge region, which are often proline rich. One particularly useful flexible hinge that can be used as a spacer is provided by the sequence Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 90), where Xaa is any amino acid, for example, aspartic acid. In some embodiments, the spacer comprises K-K-K, EK-K-K-K, or K-K-K- sK.

[0162] In some embodiments, the spacer is a polylysine spacer or a spacer comprising or consisting of two or more lysines. In some embodiments, the spacer comprises or consists of 2, 3 or 4 lysines (e.g., 3-4 lysines). In some embodiments, the spacer comprises an epsilon lysine linkage. In some embodiments, the spacer comprises e-Lys (eK) orLys-e-Lys (K-sK). In some embodiments, the sequence of the spacer comprises s-Lys-Lys-Lys-Lys or Lys-Lys-Lys- e-Lys. As is understood in the art, for a construct including e-Lys as or within a spacer, the C-terminus of a first peptide sequence is conjugated to the epsilon amino group of the lysine to form a first amide bond, and the N-terminus of a second peptide sequence is conjugated to the carboxyl group of the lysine to form a second amide bond.

[0163] The structure of the Lys-s-Lys (K-sK) linkage is shown below:

[0164]

[0165] The spacer or linker can also provide functional features to the myostatin or activin A peptide immunogen construct. For example, the spacer or linker can be designed to change the overall charge of the myostatin or activin A peptide immunogen construct, which can affect the solubility of the peptide immunogen construct. Additionally, changing the overall charge of the myostatin or activin A peptide immunogen construct can affect the ability of the peptide immunogen construct to associate with other compounds and reagents. As discussed in further detail below, the myostatin or activin A peptide immunogen construct can be formed into a stable immunostimulatory complex with a highly charged oligonucleotide, such as CpG oligomers through electrostatic association. The overall charge of the myostatin or activin A peptide immunogen construct is important for the formation of these stable immunostimulatory complexes.

[0166] Chemical compounds that can be used as a spacer or linker include, but are not limited to (2-aminoethoxy) acetic acid (AEA), 5-aminovaleric acid (AVA), 6-aminocaproic acid (Ahx), 8-amino-3,6-dioxaoctanoic acid (AEEA, mini-PEGl), 12-amino-4,7,10-trioxadodecanoic acid (mini-PEG2), 15-amino-4,7,10,13-tetraoxapenta-decanoic acid (mini-PEG3), trioxatridecan-succinamic acid (Ttds), 12-amino-dodecanoic acid, Fmoc-5-amino-3-oxapentanoic acid (OlPen), and the like.

[0167] Naturally occurring ammo acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0168] Non-naturally occurring amino acids include, but are not limited to, s-N Lysine, P-alanine, ornithine, norleucine, norvaline, hydroxyproline, thyroxine, y-amino butyric acid, homoserine, citrulline, aminobenzoic acid, 6-aminocaproic acid (Aca; 6-aminohexanoic acid), hydroxyproline, mercaptopropionic acid (MPA), 3 -nitro-tyrosine, pyroglutamic acid, and the like.

[0169] The spacer or linker in a myostatin or activin A peptide immunogen construct can be covalently linked at either N- or C- terminal end of the Th epitope and the myostatin or activin A peptide. In some embodiments, the spacer is covalently linked to the C-terminal end of the Th epitope and to the N-terminal end of the myostatin or activin A peptide. In other embodiments, the spacer is covalently linked to the C-terminal end of the myostatin or activin A peptide and to the N-terminal end of the Th epitope. In certain embodiments, more than one spacer or linker can be used, for example, when more than one Th epitope is present in the peptide immunogen construct. When more than one spacer is used, each spacer can be the same as each other or different. Additionally, when more than one Th epitope is present in the peptide immunogen construct, the Th epitopes can be separated with a spacer, which can be the same as, or different from, the spacer used to separate the Th epitope from the B cell epitope. There is no limitation in the arrangement of the spacer in relation to the Th epitope or the myostatin or activin A fragment.

[0170] In certain embodiments, the heterologous spacer or linker is a naturally occurring amino acid or a non-naturally occurring amino acid. In other embodiments, the spacer contains more than one naturally occurring or non-naturally occurring amino acid (e.g., the spacer is a peptide). In specific embodiments, the spacer is Lys-, Gly-, Lys-Lys-Lys-, (a, s-N)Lys, Lys-Lys-Lys-s-Lys, or s-N-Lys- Lys-Lys-Lys (SEQ ID NO: 91) or SEQ ID NO: 91 wherein the e-K is on the C-terminal end of the Lys-Lys-Lys peptide.

[0171] Multivalent or bivalent peptide immunogen constructs

[0172] In some embodiments, provided herein is a multivalent or bivalent peptide immunogen construct comprising two or more B cell epitopes, a heterologous T cell (Th) epitope, an optional heterologous linker, and an optional heterologous spacer, wherein the two or more B cell epitopes are covalently linked to the Th epitope directly or through the optional heterologous linker and / or spacer. In some embodiments, the two or more B cell epitopes are two B cell epitopes. In some embodiments, the two or more B cell epitopes are two or more (e.g., two) identical B cell epitopes, such as any B cell epitope described herein. In some embodiments, the two or more B cell epitopes are two or more (e.g., two) non-identical, different B cell epitopes, such as any two or more B cell epitopes described herein. In some embodiments, the two or more B cell epitopes are two or more (e.g., two) myostatin B cell epitopes (e.g., any myostatin B cell epitopes described herein, identical or different). In some embodiments, the two or more B cell epitopes are two or more (e.g., two) activin A B cell epitopes (e.g., any activin A B cell epitopes described herein, identical or different). In some embodiments, the two or more (e.g., two) B cell epitopes comprise at least one myostatin B cell epitope (e.g., any myostatin B cell epitope described herein) and at least one activin A B cell epitope (e.g., any activin AB cell epitope described herein).

[0173] In some embodiments, a peptide immunogen provided herein comprises two or more (e.g., 2) B cell epitopes sequentially positioned in a linear peptide.

[0174] In some embodiments, a peptide immunogen provided herein comprises two or more (e.g., 2) B cell epitopes that are positioned in a non-linear or non-sequential manner or branched off from a Th epitope. The branched design may improve or simplify peptide synthesis and / or improve or eliminate steric interactions resulting in poor interaction between the peptide and its target.

[0175] In some embodiments, the B cell epitope peptides are attached to the same side (N-terminal or C-terminal) of the Th epitope using a branched amino acid, which can be: (i) a diamino acid if the B-cell epitopes are attached to the N-terminus of a Th epitope, or (ii) a dicarboxylic acid if the B-cell epitopes are attached to the C-terminus of a Th epitope. Any diamino acid and dicarboxylic acid known in the art or described herein can be used. In some embodiments, the diamino acid is lysine. Other diamino acids include, without limitation, ornitine, 2,4-Diaminobutyric acid, and 2,3-diaminopropionic acid. In some embodiments, the dicarboxylic acid is glutamic acid. Other dicarboxylic acids include, without limitation, aspartic acid and a-amino adipic acid. By adding more than one branched amino acid, the number of B cell epitope peptides supported by the same Th epitope can be added to a peptide immunogen provided herein. In some embodiments, the B cell epitope peptides are attached to the same side (N-terminal or C-terminal) of the Th epitope using two or more branched amino acids as described herein or known in the art.

[0176] Between the branched amino acid and the B cell epitope peptide, a heterologous linker can be used, e.g., to (i) to prevent steric interferences between the B cell epitope peptides during synthesis, and / or (ii) to avoid steric interference that would prevent either the binding of B cell receptor to B cell epitope or the binding of MHC receptor to Th epitope. In some embodiments, the linker comprises one or more amino acids (e.g., one or more Glycines). In some embodiments, the linker is an amino acid peg derivative. In some embodiments, the heterologous linker is AeeA (i.e., 8-amino-3,6-dioxaoctanoic acid or 2-[2-(2-aminoethoxy)ethoxy]acetic acid (CAS: 134978-97-5)). In other embodiments, other heterologous linkers can be used, for example, and without limitation, a spacer comprising 3 to 6 pegs (e.g., l-Amino-3,6,9,12,15,18-hexaoxahenicosan-21-oic acid (6pe), 1-amino-4,7,10,13-tetraoxapentadecanoic acid (3peg)), 6-aminohexanoic acid, or one or more epsilon-lysine. Other spacers or linkers described herein can also be used between the branched amino acid and the B cell epitope peptide.

[0177] In some embodiments, B cell epitopes can be synthesized simultaneously using a Fmoc-Lys(Fmoc)-OH on the branched amino acid. In some embodiments, B cell epitopes can be synthetized sequentially by using Lysine with orthogonal protections on the oc -amino group and on s -amino group, for example with Lysine using Fmoc-Lys(Mtt)-OH, Fmoc-Lys(DDe)-OH and Fmoc-Lys(Alloc)-OH, where different B-cell epitopes can be synthesized on the same Th epitope.

[0178] Specific embodiments of the peptide immunogen constructs

[0179] The myostatin peptide immunogen construct can be represented by the formulae:

[0180] (Th)m-(A)n-(myostatin fragment), or (myostatin fragment)-(A)n-(Th)m-, or (Th)m-(A)n-(myostatin fragment) -(A)n-(Th)m-, wherein Th is a heterologous T helper epitope; A is an optional heterologous spacer; (myostatin fragment) is a myostatin B cell epitope having about 7 to about 30 (e.g., 7-29, 7-28, 7-27, 7-26, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, or 7-10, or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) contiguous amino acid residues of myostatin (or a variant thereof, e.g., as described herein); m is an integer from 1 to about 4; and n is an integer from 0 to about 10. In some embodiments, a C-terminal X is included, which is an a-COOH or a-CONH2of an amino acid. In some embodiments, A is an ammo acid and n indicates the number of amino acids, wherein each A can be identical to one another or one or more of the A’s can be a different amino acid.

[0181] In certain embodiments, the heterologous Th epitope in the myostatin peptide immunogen construct has an ammo acid sequence selected from any of SEQ ID NOs: 19-47, or combinations thereof, shown in Table 3. In specific embodiments, the Th epitope has an amino acid sequence selected from any of SEQ ID NOs: 27-33. In some embodiments, the Th epitope has an amino acid sequence of SEQ ID NOs: 30. In some embodiments, the Th epitope has an ammo acid sequence of SEQ ID NOs: 32. In certain embodiments, the myostatin peptide immunogen construct contains more than one Th epitope.

[0182] In certain embodiments, the optional heterologous spacer is selected from any of Lys-, Gly-, Lys-Lys-Lys-, (a, e-N)Lys, e-N-Lys-Lys-Lys-Lys (SEQ ID NO: 91), and combinations thereof. In specific embodiments, the heterologous spacer is s-N-Lys-Lys-Lys-Lys (SEQ ID NO: 91). In some embodiments, the heterologous spacer is Lys-Lys-Lys-sLys.

[0183] In certain embodiments, the myostatin fragment has about 7 to about 30 (e.g., 7-29, 7-28, 7- 27, 7-26, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, or 7-10, or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) amino acid residues of myostatin, corresponding to the sequence from about the D at amino acid position 267 (D267) to about the S at amino acid position 375 (S375) of full-length myostatin. In certain embodiments, the myostatin fragment has about 7 to about 30 (e.g., 7-29, 7-28, 7-27, 7-26, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, or 7-10, or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) ammo acid residues from about the leucine at ammo acid position 80 (L80) to about the leucine at ammo acid position 270 (L270) of full-length myostatin preproprotein (SEQ ID NO: 1). In specific embodiments, the myostatin fragment has an amino acid sequence of a fragment of SEQ ID NO: 2, as shown in Table 1. In specific embodiments, the myostatin fragment has an amino acid sequence of a fragment of SEQ ID NO: 1, as shown in Table 1. In some embodiments, the myostatin peptide has a sequence selected from SEQ ID NOs: 3-10, as shown in Table 1. In some embodiments, the myostatin peptide has a sequence selected from SEQ ID NOs: 95-109, as shown in Table 1. In some embodiments, the myostatin peptide has a sequence selected from SEQ ID NOs: 145-164 and 166, as shown in Table 1. In some embodiments, the myostatin peptide has a sequence selected from any one of: SEQ ID NOs: 5, 97, 98, 101, 105, 146, and 147, as shown in Table 1. In certain embodiments, the myostatin peptide immunogen construct has an amino acid sequence selected from any of SEQ ID NOs: 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, and 71, as shown in Table 4. In certain embodiments, the myostatin peptide immunogen construct has an amino acid sequence selected from any of SEQ ID NOs: 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, and 134, as shown in Table 4. In certain embodiments, the myostatin peptide immunogen construct has an ammo acid sequence selected from any of SEQ ID NOs: 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186 and 188, as shown in Table 4. In certain embodiments, the myostatin peptide immunogen construct has an amino acid sequence selected from any one of: SEQ ID NOs: 54, 55, 56, 122, 123, 126, 130, 168, 169, 173, 174, 176, 177, 179, 181, 183, 185, and 186 as shown m Table 4.

[0184] The activin A peptide immunogen construct can be represented by the formulae:

[0185] (Th)m-(A)n--(activin A fragment), or (activin A fragment)-(A)n-(Th)m-, or (Th)m-(A)n-(activin A fragment)-(A)n-(Th)m- wherein Th is a heterologous T helper epitope; A is an optional heterologous spacer; (activin A fragment) is an activin A B cell epitope having about 7 to about 30 (e.g., 7-29, 7-28, 7-27, 7-26, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, or 7-10, or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) contiguous amino acid residues of activin A (or a variant thereof, e.g., as described herein); m is an integer from 1 to about 4; and n is an integer from 0 to about 10. In some embodiments, a C-terminal X is included, which is an a-COOH or a-CONHz of an amino acid. In some embodiments, A is an amino acid and n indicates the number of amino acids, wherein each A can be identical to one another or one or more of the A’s can be a different amino acid.

[0186] In certain embodiments, the heterologous Th epitope in the activin A peptide immunogen construct has an amino acid sequence selected from any of SEQ ID NOs: 19-47, or combinations thereof, shown in Table 3. In specific embodiments, the Th epitope has an amino acid sequence selected from any of SEQ ID NOs: 27-33. In some embodiments, the Th epitope has an amino acid sequence of SEQ ID NOs: 30. In some embodiments, the Th epitope has an ammo acid sequence of SEQ ID NOs: 32. In certain embodiments, the activin A peptide immunogen construct contains more than one Th epitope.

[0187] In certain embodiments, the optional heterologous spacer is selected from any of Lys-, Gly-, Lys-Lys-Lys-, Lys-Lys-Lys-s-Lys, (a, e-N)Lys, s-N-Lys-Lys-Lys-Lys (SEQ ID NO: 91), and combinations thereof. In specific embodiments, the heterologous spacer is s-N-Lys-Lys-Lys-Lys (SEQ ID NO: 91). In some embodiments, the heterologous spacer is Lys-Lys-Lys-s-Lys. In certain embodiments, the activin A fragment has about 7 to about 30 (e.g., 7-29, 7-28, 7-27, 7-26, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, or 7-10, or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) ammo acid residues of activin A. In specific embodiments, the activin A fragment has an ammo acid sequence represented by SEQ ID NO: 12, as shown in Table 1. In some embodiments, the activin A peptide has a sequence selected from SEQ ID NOs: 13-18, as shown in Table 2. In some embodiments, the activin A peptide has a sequence selected from SEQ ID NOs: 110-119, 187, and 189, as shown in Table 2. In some embodiments, the activin A peptide has a sequence selected from any one of: SEQ ID NOs: 111, 116, 118 and 119, as shown in Table 2. In certain embodiments, the activin A peptide immunogen construct has an ammo acid sequence selected from any of SEQ ID NOs: 72-89, as shown in Table 5. In certain embodiments, the activin A peptide immunogen construct has an amino acid sequence selected from any of SEQ ID NOs: 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 190, and 191 as shown in Table 5. In certain embodiments, the activin A peptide immunogen construct has an ammo acid sequence selected from any one of: SEQ ID NOs: 136, 141, 143, 144, and 190 as shown in Table 5.

[0188] Compositions

[0189] Peptide immunogen constructs and related molecules described herein can be comprised with compositions, including pharmaceutical compositions, which are capable of eliciting an immune response and the production of antibodies against the peptide immunogen constructs in a subject (e.g., a human patient). The disclosed compositions can include one or a mixture of more than one of the peptide immunogen constructs. In some embodiments, the compositions include one or more myostatin peptide immunogen construct described herein. In some embodiments, the compositions include one or more activin A peptide immunogen construct described herein. In some embodiments, the compositions include one or more myostatin peptide immunogen construct described herein and one or more activin A peptide immunogen construct described herein. In some embodiments, the compositions include one myostatin peptide immunogen construct described herein and one activin A peptide immunogen construct described herein. As one example, the compositions can include construct peptide (SEQ ID NO: 55) as described herein. As another example, the composition can include a myostatin peptide immunogen construct selected from Table 4 (optionally wherein the myostatin peptide portion of the construct is 7-30, 7-29, 7-28, 7-27, 7-26, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, or 7-10, or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ammo acids in length), together with an activin A peptide immunogen construct selected from Table 5 (optionally wherein the activin A peptide portion of the construct is 7-30, 7-29, 7-28, 7-27, 7-26, 7-25, 7-24, 7-23, 7-22, 7-21, 7-20, 7-19, 7-18, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, or 7-10, or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ammo acids in length). As another example, the composition can include, a myostatin peptide immunogen construct of SEQ ID NO: 54, 55, 56, 122, 123, 126, 130, 168, 169, 173, 174, 176, 177, 179, 181, 183, 185, or 186 (or another myostatin peptide immunogen construct described herein), and an activin A peptide immunogen construct of SEQ ID NO: 136, 141, 143,144, or 190 (or another activin A peptide immunogen construct described herein).

[0190] Furthermore, in some embodiments, the compositions can include the peptide immunogen construct(s) together with one or more additional component, e.g., carriers, adjuvants, buffers, and / or other suitable reagents. In some embodiments, the compositions include the peptide immunogen constructs in the form of a stabilized immunostimulatory complex with a CpG oligomer that is optionally supplemented with an adjuvant.

[0191] Compositions containing one or more disclosed peptide immunogen construct can be in liquid or solid form. Liquid compositions can include water, buffers, solvents, salts, and / or any other acceptable reagent that does not alter the structural or functional properties of the peptide immunogen constructs.

[0192] Pharmaceutical compositions

[0193] The present disclosure includes pharmaceutical compositions containing the disclosed peptide immunogen constructs).

[0194] Pharmaceutical compositions can contain carriers and / or other additives in a pharmaceutically acceptable delivery system. Accordingly, pharmaceutical compositions can contain a pharmaceutically effective amount of one or more peptide immunogen construct together with pharmaceutically acceptable carrier, adjuvant, and / or other excipients such as diluents, additives, stabilizing agents, preservatives, solubilizing agents, buffers, and the like.

[0195] Pharmaceutical compositions can contain one or more adjuvant that act(s) to accelerate, prolong, or enhance the immune response to the peptide immunogen constructs) without having any specific antigenic effect itself. Adjuvants used in the pharmaceutical composition can include oils, aluminum salts, virosomes, aluminum phosphate (e.g., ADJU-PHOS®), aluminum hydroxide (e.g., ALHYDROGEL®), liposyn, saponin, squalene, L121, Emulsigen®, monophosphoryl lipid A (MPL), QS21, ISA 35, ISA 206, ISA50V, ISA51, ISA 720, as well as the other adjuvants and emulsifiers.

[0196] In some embodiments, the pharmaceutical composition contains Montanide™ ISA 51 (an oil adjuvant composition comprised of vegetable oil and mannide oleate for production of water-in-oil emulsions), Tween® 80 (also known as polysorbate 80 or polyoxyethylene (20) sorbitan monooleate), a CpG oligonucleotide, and / or any combination thereof. In other embodiments, the pharmaceutical composition is a water-in-oil-in-water (i.e., w / o / w) emulsion with Emulsigen or Emulsigen D as the adjuvant.

[0197] Pharmaceutical compositions can be formulated for immediate release or for sustained release. Additionally, the pharmaceutical compositions can be formulated for induction of systemic, or localized mucosal, immunity through immunogen entrapment and co-administration with microparticles. Such delivery systems are readily determined by one of ordinary skill in the art.

[0198] Pharmaceutical compositions can be prepared as injectables, either as liquid solutions or suspensions. Liquid vehicles containing the peptide immunogen constructs can also be prepared prior to injection. The pharmaceutical compositions can be administered by any suitable mode of application, for example, intramuscularly, subcutaneously, intradermally, intravenously, intraperitoneally, intranasally, orally, etc. and by use of any suitable formulation or delivery device.

[0199] Pharmaceutical compositions can also be formulated in a suitable dosage unit form. In some embodiments, the pharmaceutical composition contains from about 0.5 pg to about 1 mg of the peptide immunogen construct(s) per kg body weight. In some embodiments, the pharmaceutical composition contains 10-1000 pg, e.g., 20-500 pg, 50-400 pg, 50-500 pg, 100-400 pg, or 100-300 pg of an immunotherapy as described herein (e.g., a peptide immunogen construct(s)). In some embodiments, the indicated dose is the total dose of one or more peptide immunogen constructs administered. In some embodiments where more than one peptide immunogen construct are administered, each of the peptide immunogen constructs are administered in about equal amount by weight. Effective doses of the pharmaceutical compositions vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human, but non-human mammals, including cattle, pigs, goats, sheep, horses, dogs, cats, and transgenic mammals can also be treated. When delivered in multiple doses, the pharmaceutical compositions may be conveniently divided into an appropriate amount per dosage unit form as determined to be appropriate by one skilled in the art. The administered dosage will depend on the age, weight, and general health of the subject as is well known in the therapeutic arts.

[0200] In some embodiments, the pharmaceutical composition contains more than one peptide immunogen construct and / or antibody. A pharmaceutical composition containing a mixture of more than one peptide immunogen construct (and / or antibody) can allow' for synergistic enhancement of the immunoefficacy of the constructs. Pharmaceutical compositions containing more than one peptide immunogen construct can be more effective in a larger genetic population due to a broad MHC class II coverage thus provide an improved immune response to the peptide immunogen constructs.

[0201] In some embodiments, the pharmaceutical composition contains one or more myostatin peptide immunogen constructs described herein and / or one or more activin A peptide immunogen constructs described herein.

[0202] In some embodiments, the pharmaceutical composition contains a myostatin peptide immunogen construct selected from SEQ ID NOs: 48-71, 120-134, 167-186, and 188, as well as homologues, analogues, fragments, and / or combinations thereof. In specific embodiments, pharmaceutical compositions contain a myostatin peptide immunogen construct selected from SEQ ID NOs: 48-71, 120-134, 167-186, and 188, and any combination thereof. In some embodiments, pharmaceutical compositions contain a myostatin peptide immunogen construct selected from SEQ ID NO: 54, 55, 56, 122, 123, 126, 130, 168, 169, 173, 174, 175, 176, 177, 178, 179, 181, 183, 185, and 186, and any combination thereof. In some embodiments, pharmaceutical compositions contain a myostatin peptide immunogen construct selected from SEQ ID NO: 54, 55, 56, 122, 123, 126 and 130.

[0203] In some embodiments, the pharmaceutical composition contains an activin A peptide immunogen construct selected from SEQ ID NOs: 72-89, 135-144, and 190-191, as well as homologues, analogues, fragments, and / or combinations thereof. In specific embodiments, pharmaceutical compositions contain an activin A peptide immunogen construct selected from SEQ ID NOs: 72-89, 135-144, and 190-191, and any combination thereof. In some embodiments, pharmaceutical compositions contain an activin A peptide immunogen construct selected from SEQ ID NO: 75, 76, 78, 79, 85, 136, 138, 139, 140, 141, 142, 143, 144, and 190, and any combination thereof.

[0204] In some embodiments, the pharmaceutical composition contains a myostatin peptide immunogen construct and an activin A peptide immunogen construct. In some embodiments, the pharmaceutical composition contains a myostatin peptide immunogen construct selected from SEQ ID NOs: 48-71, 120-134, 167-186 and 188, as well as homologues, analogues, fragments, and / or combinations thereof, and an activin A peptide immunogen construct selected from SEQ ID NOs:72-89 and 135-144, as well as homologues, analogues, fragments, and / or combinations thereof. In specific embodiments, pharmaceutical compositions contain a myostatin peptide immunogen construct selected from SEQ ID NOs: 48-71, 120-134, 167-186 and 188, and any combination thereof, and an activin A peptide immunogen construct selected from SEQ ID NOs: 72-89, 135-144, and 190-191. In some embodiments, the pharmaceutical composition contains a myostatin peptide immunogen construct selected from SEQ ID NO: 54, 55, 56, 122, 123, 126, 130, 168, 169, 173, 174, 175, 176, 177, 178, 179, 181, 183, 185, and 186, as well as homologues, analogues, fragments, and / or combinations thereof, and an activin A peptide immunogen construct selected from SEQ ID NO: 75, 76, 78, 79, 85, 136, 138, 139, 140, 141, 142, 143, 144, and 190, as well as homologues, analogues, fragments, and / or combinations thereof.

[0205] Pharmaceutical compositions containing a myostatin and / or activin A peptide immunogen construct can be used to elicit an immune response and to produce antibodies in a subject upon administration. In some embodiments, a pharmaceutical composition as described herein is administered to a subject 1, 2, 3, 4, 5, or more times as determined to be appropriate by those of skill in the art. The compositions can be administered, for example, in an initial dose, followed by 1 or more (e.g., 2, 3, 4, 5, or more) booster doses. In some embodiments, an initial dose is administered in week 1 and then is followed by a dose at week 5 and an additional dose at week 13. In some embodiments, the amount of each dose is the same (e.g., 100 pg or 300 pg; also see above). In some embodiments, the amount of each dose varies, as can be determined to be appropriate by those of skill in the art. For example, the initial dose may be lower, e.g., 40 pg, followed by higher doses of 100 pg, 300 pg, 500 pg, or 1000 pg in subsequent administrations (e.g., at weeks 5 and 13).

[0206]

[0207] The compositions of the present disclosure can also contain one or more peptide immunogen construct in the form of an immunostimulatory complex with a CpG oligonucleotide. Such immunostimulatory complexes are specifically adapted to act as an adjuvant and as a peptide immunogen stabilizer. The immunostimulatory complexes are in particulate form, which can efficiently present the peptide immunogen(s) to the cells of the immune system to produce an immune response. The immunostimulatory complexes may be formulated as a suspension for parenteral administration. The immunostimulatory complexes may also be formulated in the form of water in oil emulsions, as a suspension in combination with a mineral salt or with an in-situ gelling polymer for the efficient delivery of the peptide immunogen(s) to the cells of the immune system of a subject following parenteral administration.

[0208] The stabilized immunostimulatory complex can be formed by complexing one or more peptide immunogen construct with an anionic molecule, oligonucleotide, polynucleotide, or combinations thereof via electrostatic association. The stabilized immunostimulatory complexes may be incorporated into a pharmaceutical composition as an immunogen delivery system.

[0209] In certain embodiments, the one or more peptide immunogen construct is designed to contain a cationic portion that is positively charged at a pH in the range of 5.0 to 8.0. The net charge on the cationic portion of the peptide immunogen construct, or mixture of constructs, is calculated by assigning a +1 charge for each lysine (K), arginine (R) or histidine (H), a -1 charge for each aspartic acid (D) or glutamic acid (E) and a charge of 0 for the other ammo acid within the sequence. The charges are summed within the cationic portion of the peptide immunogen construct and expressed as the net average charge. A suitable peptide immunogen has a cationic portion with a net average positive charge of +1. In some embodiments, the peptide immunogen has a net positive charge in a range that is larger than +2. In some embodiments, the cationic portion of the peptide immunogen construct is the heterologous spacer. In certain embodiments, the cationic portion of the peptide immunogen construct has a charge of +4 when the spacer sequence is (a, E-N)Lys, s-N-Lys-Lys-Lys-Lys (SEQ ID NO: 91).

[0210] An “anionic molecule” as described herein refers to any molecule that is negatively charged at a pH in the range of 5.0-8.0. In certain embodiments, the anionic molecule is an oligomer or polymer. The net negative charge on the oligomer or polymer is calculated by assigning a -1 charge for each phosphodiester or phosphorothioate group in the oligomer. A suitable anionic oligonucleotide is a single-stranded DNA molecule with 8 to 64 nucleotide bases, with the number of repeats of the CpG motif in the range of 1 to 10. In some embodiments, the CpG immunostimulatory single-stranded DNA molecules contain 18-48 nucleotide bases, with the number of repeats of CpG motif in the range of 3 to 8.

[0211] In some embodiments, the anionic oligonucleotide is represented by the formula: 5!X1CGX23!wherein C and G are unmethylated; and X1is selected from A (adenine), G (guanine) and T (thymine); and X2is C (cytosine) or T (thymine). In other embodiments, the anionic oligonucleotide is represented by the formula: 5’ (XQ2CG(X4) 3' wherein C and G are unmethylated; and X’ is selected from A, T, or G; and X4is C or T,

[0212] In some embodiments, the CpG oligonucleotide is as described in WO 03 / 068169, the contents of which are incorporated by reference. In some embodiments, the CpG comprises or consists of a sequence of CpGl (tcgtcgtttt gtcgttttgt cgttttgtcg tt; SEQ ID NO: 92), CpG2 (tcgtcgtttt gtcgttttgt cgtt; SEQ ID NO: 93), or CpG3 (tcgtcgtttt gtcgttttgt cgtt; SEQ ID NO: 94). In some embodiments, the CpG oligonucleotide (e.g., CpGl, CpG2, or CpG3) is a phosphorothioate oligonucleotide.

[0213] The resulting immunostimulatory complex is in the form of particles with a size typically in the range from 1-50 microns and is a function of many factors including the relative charge stoichiometry and molecular weight of the interacting species. The particulated immunostimulatory complex has the advantage of providing adjuvantation and upregulation of specific immune responses in vivo. Additionally, the stabilized immunostimulatory complex is suitable for preparing pharmaceutical compositions by various processes including water-in-oil emulsions, mineral salt suspensions, and polymeric gels.

[0214] Methods of making peptide immunogen constructs

[0215] The peptide immunogen constructs of the disclosure can be made using chemical synthesis methods that are well known in the art (see, e.g.. Fields et al., Chapter 3 in Synthetic Peptides: A User’s Guide, ed. Grant, W. H. Freeman & Co., New York, NY, 1992, p.77). For example, the peptide immunogen constructs can be synthesized using the automated Merrifield techniques of solid phase synthesis with the a-NEfc protected by either t~Boc or F-moc chemistry using side chain protected amino acids on, for example, an Applied Biosystems Peptide Synthesizer Model 430A or 431. Preparation of peptide immunogen constructs comprising combinatorial library peptides for Th epitopes can be accomplished by providing a mixture of alternative amino acids for coupling at a given variable position. After complete assembly of a desired peptide immunogen construct, the resin can be treated according to standard procedures to cleave the peptide from the resin and the functional groups on the amino acid side chains can be deblocked. The free peptide can be purified by HPLC and characterized biochemically, for example, by amino acid analysis or by sequencing. Purification and characterization methods for peptides are well known to those of skill in the art. The quality of peptides produced by this chemical process can be controlled and defined and, as a result, reproducibility of peptide immunogen constructs, immunogenicity, and yield can be assured. The range in structural variability that allows for retention of an intended immunological activity has been found to be far more accommodating than the range in structural variability allowed for retention of a specific drug activity by a small molecule drug or the desired activities and undesired toxicides found in large molecules that are co-produced with biologically derived drugs. Thus, peptide analogues, either intentionally designed or inevitably produced by errors of the synthetic process as a mixture of deletion sequence byproducts that have chromatographic and immunologic properties similar to the intended peptide, are frequently as effective as a purified preparation of the desired peptide. Designed analogues and unintended analogue mixtures are effective as long as a discerning QC procedure is developed to monitor both the manufacturing process and the product evaluation process so as to guarantee the reproducibility and efficacy of the final product employing these peptides.

[0216] The peptide immunogen constructs can also be made using recombinant DNA technology including using nucleic acid molecules, vectors, and / or host cells. As such, nucleic acid molecules encoding the peptide immunogen constructs and immunologically functional analogues thereof are also encompassed by the present disclosure as part of the present invention. Similarly, vectors, including expression vectors, comprising nucleic acid molecules as well as host cells containing the vectors are also encompassed by the present disclosure as part of the present invention.

[0217] Various exemplary embodiments also encompass methods of producing the peptide immunogen constructs and immunologically functional analogues of the myostatin or activin A fragment derived peptide immunogen constructs. For example, methods can include a step of incubating a host cell containing an expression vector containing a nucleic acid molecule encoding a peptide immunogen construct and / or immunologically functional analogue thereof under such conditions where the peptide and / or analogue is expressed. The longer synthetic peptide immunogens can be synthesized by well-known recombinant DNA techniques. Such techniques are provided in well-known standard manuals with detailed protocols. To construct a gene encoding a peptide of this invention, the amino acid sequence is reverse translated to obtain a nucleic acid sequence encoding the amino acid sequence, preferably with codons that are optimum for the organism in which the gene is to be expressed. Next, a synthetic gene is made typically by synthesizing oligonucleotides which encode the peptide and any regulatory elements, if necessary. The synthetic gene is inserted in a suitable cloning vector and transfected into a host cell. The peptide is then expressed under suitable conditions appropriate for the selected expression system and host. The peptide is purified and characterized by standard methods. Methods for manufacturing of immimostimulatory complexes

[0218] As noted above, the compositions and methods of the disclosure can further include or employ immunostimulatory complexes comprising peptide immunogen constructs and CpG oligodeoxynucleotide (ODN) molecules. Stabilized immunostimulatory complexes (ISC) are derived from a cationic portion of the peptide immunogen construct and a polyanionic CpG ODN molecule. The self-assembling system is driven by electrostatic neutralization of charge.

[0219] Stoichiometry of the molar charge ratio of cationic portion of the peptide immunogen construct to anionic oligomer determines extent of association. The non-covalent electrostatic association of peptide immunogen construct and CpG ODN is a completely reproducible process. The peptide / CpG ODN immunostimulatory complex aggregates, which facilitate presentation to the “professional” antigen-presenting cells (APC) of the immune system thus further enhancing of the immunogenicity of the complexes. These complexes are easily characterized for quality control during manufacturing. The peptide / CpG ISC are well tolerated in vivo. This particulate system comprising CpG ODN and fragment derived peptide immunogen constructs was designed to take advantage of the generalized B cell mitogemcity associated with CpG ODN use and yet promote balanced Th-l / Th-2 type responses.

[0220] The CpG ODN in the disclosed pharmaceutical compositions can be 100% bound to immunogen in a process mediated by electrostatic neutralization of opposing charge, resulting in the formation of micron-sized particulates. The particulate form allows for a significantly reduced dosage of CpG from the conventional use of CpG adjuvants, less potential for adverse innate immune responses, and facilitates alternative immunogen processing pathways including antigen-presenting cells (APC). Consequently, such formulations offer potential advantages by promoting the stimulation of immune responses by alternative mechanisms.

[0221] Antibodies

[0222] The disclosure also provides antibodies that specifically recognize and bind to myostatin and / or activin A, for example, to a B cell epitope portion of a peptide immunogen construct described herein. In some embodiments, the provided antibodies are elicited in response to vaccination with one or more peptide immunogen constructs described herein. In some embodiments, the antibodies elicited by administration of one or more peptide immunogen constructs described herein to a subject are harvested or isolated from the subject, and optionally purified. Antibodies for use in therapy can be generated using standard methods in the art and include, e.g,, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific and trispecific antibodies), and antibody fragments, provided that the desired antigen-binding activity and specificity is maintained. Antibody fragments include, for example, Fv, single-chain Fv (scFv), Fab, Fab’, di-scFv, sdAb (single domain antibody), and (Fab’)2 (including a chemically linked F(ab’)2). Antibodies also include, e.g., chimeric antibodies, humanized antibodies, and antibodies of various species such as mouse, human, cynomolgus monkey, etc. Furthermore, antibody variants having the sequences from other organisms are also included. Antibody fragments also include either orientation of single chain scFvs, tandem di-scFv, diabodies, tandem tri-sdcFv, minibodies, etc. Antibody fragments further include nanobodies (sdAb, an antibody having a single, monomeric domain, such as a pair of variable domains of heavy chains, without a light chain). An antibody fragment can be referred to as being a specific species in some embodiments (for example, human scFv or a mouse scFv). This denotes the sequences of at least part of the non-CDR regions, rather than the source of the construct.

[0223] Methods of Treatment

[0224] The present disclosure provides methods for treating, delaying, lessening, preventing, and / or improving one or more symptoms of a muscle wasting disease or condition, or insulin resistance, or glucose metabolism, or metabolic disease, using the disclosed immunotherapies (e.g., peptide immunogen constructs (and related compositions and complexes), nucleic acid molecules, vectors, and / or antibodies directed against the B cell epitope of the peptide immunogen constructs). In some embodiments, the methods include administering to a subject a composition containing one or more of the disclosed peptide immunogen constructs. In some embodiments, the methods include administering to a subject a composition containing one or more disclosed antibodies. In certain embodiments, the compositions utilized in the methods contain one or more disclosed peptide immunogen construct in the form of a stable immunostimulatory complex with negatively charged oligonucleotides, such as CpG oligomers, through electrostatic association, which complexes are further supplemented, optionally, with mineral salts or oil as adjuvant, for administration to subjects with muscle wasting diseases or conditions, or insulin resistance. The disclosed methods also include dosing regimens, dosage forms, and routes for administering the peptide immunogen constructs to a subject at risk for, or with, a muscle wasting disease or condition, or insulin resistance. As explained above, use of one or more my ostatin peptide immunogen construct in combination with one or more activin A peptide immunogen construct is included in the disclosure. The present disclosure also provides methods for maintaining or improving muscle physiology using the disclosed immunotherapies (e.g., peptide immunogen constructs (and related compositions and complexes), nucleic acid molecules, vectors, and / or antibodies directed against the B cell epitope of the peptide immunogen constructs). In some embodiments, the methods include administering to a subject a composition containing one or more disclosed peptide immunogen constructs. In some embodiments, provided herein are methods of increasing muscle mass or muscle growth using the peptide immunogen constructs described herein. In some embodiments, provided herein are methods of increasing muscle strength using the peptide immunogen constructs described herein. In some embodiments, provided herein are methods of increasing muscle tone using the peptide immunogen constructs described herein. In some embodiments, provided herein are methods of increasing muscle endurance using the peptide immunogen constructs described herein. In some embodiments, provided herein are methods of improving muscle function or performance using the peptide immunogen constructs described herein.

[0225] The present disclosure also provides methods for treating, delaying, lessening, preventing, and / or improving one or more symptoms of muscular or musculoskeletal inj ury using the disclosed immunotherapies (e.g., peptide immunogen constructs (and related compositions and complexes), nucleic acid molecules, vectors, and / or antibodies directed against the B cell epitope of the peptide immunogen constructs). In some embodiments, the methods include administering to a subject a composition containing one or more disclosed peptide immunogen constructs. In some embodiments, provided herein are methods of preventing or treating muscular injury using the peptide immunogen constructs described herein. In some embodiments, provided herein are methods of preventing or treating musculoskeletal injury using the peptide immunogen constructs described herein. In some embodiments, the muscular or musculoskeletal injury is a muscle tear or muscle strain.

[0226] The present disclosure also provides methods for treating, delaying, lessening, preventing, and / or improving one or more symptoms of muscle loss or insufficient muscle growth using the disclosed immunotherapies (e.g., peptide immunogen constructs (and related compositions and complexes), nucleic acid molecules, vectors, and / or antibodies directed against the B cell epitope of the peptide immunogen constructs). In some embodiments, the methods include administering to a subject a composition containing one or more disclosed peptide immunogen constructs. In some embodiments, provided herein are methods of preventing or treating muscle loss using the peptide immunogen constructs described herein. In some embodiments, provided herein are methods of preventing or treating insufficient muscle growth using the peptide immunogen constructs described herein. In some embodiments, provided herein are methods of preventing or treating muscle atrophy using the peptide immunogen constructs described herein. In some embodiments, provided herein are methods of preventing or treating cachexia or muscle loss associated with a disease or condition (e.g., cancer) using the peptide immunogen constructs described herein. In some embodiments, provided herein are methods of preventing or treating age- or disease-related muscle loss in a subject using the peptide immunogen constructs described herein (e.g., wherein the disease is cancer). In some embodiments, the subject is 65 years of age or older. In some embodiments, provided herein are methods of preventing or treating sarcopenia in a subject using the peptide immunogen constructs described herein (e.g., wherein the disease is cancer). In some embodiments, administering of peptide immunogen constructs described herein prevents loss or increases lean body mass in a subject. In some embodiments, administering of peptide immunogen constructs described herein increases muscle strength and / or performance.

[0227] The present disclosure also provides methods for treating, delaying, lessening, preventing, and / or improving metabolic disease or one or more symptoms of metabolic disease using the disclosed immunotherapies (e.g., peptide immunogen constructs (and related compositions and complexes), nucleic acid molecules, vectors, and / or antibodies directed against the B cell epitope of the peptide immunogen constructs). In some embodiments, the methods include administering to a subject a composition containing one or more disclosed peptide immunogen constructs. In some embodiments, provided herein are methods of preventing or treating metabolic disease using the peptide immunogen constructs described herein.

[0228] In some embodiments, provided herein are methods of maintaining or improving glucose metabolism using the disclosed immunotherapies (e.g., peptide immunogen constructs (and related compositions and complexes), nucleic acid molecules, vectors, and / or antibodies directed against the B cell epitope of the peptide immunogen constructs). In some embodiments, the methods include administering to a subject a composition containing one or more disclosed peptide immunogen constructs. In some embodiments, provided herein are methods of maintaining or improving glucose metabolism using the peptide immunogen constructs described herein.

[0229] In some embodiments, provided herein are methods of treating or preventing insulin resistance using the disclosed immunotherapies (e.g., peptide immunogen constructs (and related compositions and complexes), nucleic acid molecules, vectors, and / or antibodies directed against the B cell epitope of the peptide immunogen constructs). In some embodiments, the methods include administering to a subject a composition containing one or more disclosed peptide immunogen constructs. In some embodiments, provided herein are methods of treating or preventing insulin resistance using the peptide immunogen constructs described herein.

[0230] As used herein, the term "preventing" a disease or condition in a subject refers to administering a medicament (e.g., the peptide immunogen construct described herein) to the subject prior to the onset of the disease or condition, when administration of the medicament to a statistical sample prior to the onset of the disease or condition reduces the occurrence of the disease or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the occurrence or severity of one or more symptoms of the disease or condition relative to the untreated control sample.

[0231] As used herein, the term “treating” a disease or condition in a subject refers to administering a medicament (e.g., the peptide immunogen construct described herein) to the subject having or suspected of having a disease or condition (i.e., after the onset of the disease or condition), such that at least one symptom of the disease or condition is decreased or prevented from worsening.

[0232] As used herein, a “subject” is a human or a non-human animal. This term includes mammals, such as humans, primates, livestock animals, companion animals and animal models of disease.

[0233] As used herein, “administering” or “administration of’ to a subject can be carried out using one of a variety of methods known to those skilled in the art. In some embodiments, the administration is parenteral. In some embodiments, the administration is intravenous, subcutaneous, intramuscular, intradermal, or intraperitoneal. For example, peptide immunogens described herein can be administered intramuscularly. In some embodiments, the pharmaceutical compositions are formulated for any of the administration methods described herein, e.g., for intramuscular (IM) administration. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0234] As used herein, the term an “effective amount” or a “therapeutically effective amount” is an amount of a peptide immunogen that, when administered to a subject will have or expected to have a therapeutic effect. The therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject’s size, health and age, and the nature and extent of the condition being treated. Subjects who can be treated according to the methods of the disclosure include patients, such as human patients, who have or are at risk of developing a muscle wasting disease or condition such as one or more disease or condition described herein. Symptoms that can be improved according to the methods of the disclosure include, for example, one or more symptom of muscle wasting such as one or more of those listed below. The subjects can also include patients, such as human patients, who have or are at risk of developing insulin resistance. The human patients who can be treated according to the methods of the disclosure include pediatric, teen, adult, and elderly patients. Nonhuman animals, e.g., those described elsewhere herein, can also be treated according to the methods of the disclosure. In some embodiments, the subject is a mammal. In some embodiments, the subject is a farm animal (e.g., a cow, a pig, a goat, or a sheep). In some embodiments, the subject is a domestic animal (e.g., a dog or a cat). In some embodiments, the subject is a human.

[0235] Muscle wasting can be caused by diseases and conditions of the muscle, as well as diseases and conditions of the nerves that control muscles. In addition, muscle wasting can be caused by injury (e.g., injury to a muscle or nerve), lack of use of a muscle, exposure to a toxin, and / or a systemic disease. Furthermore, the cause of muscle wasting may be genetic, acquired, direct, or indirect. The methods and compositions of the disclosure can be used in the treatment or prevention of any of these types of diseases or conditions (e.g., as described herein), as well as to prevent, treat, inhibit, or ameliorate one or more symptom thereof (e.g., as described herein). Moreover, it should be understood that, in some embodiments, treatment according to the present disclosure may be effective against one or more symptoms of a muscle wasting disease, while not addressing the underlying cause. It should also be understood that, in some embodiments, treatment according to the present disclosure may be carried out in combination with other therapies directed at treating the underlying cause of the muscle wasting and / or one or more symptoms thereof.

[0236] In more detail, muscle wasting diseases or conditions that can be treated according to the methods of the disclosure include those that are caused, for example, by inherited or genetic causes, inflammation, infection, injury, systemic diseases, toxins, and other environmental exposures.

[0237] Specific, non-limiting examples of muscle wasting diseases or conditions that can be treated according to the methods of the disclosure are provided, as follows.

[0238] Inherited or genetic myopathies include, for example, muscular dystrophy (e.g., Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD), myotonic dystrophy (e.g., DM1 and DM2), facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, and limb girdle muscular dystrophy), congenital myopathy (e.g., nemaline myopathy and central core myopathy), metabolic myopathy (e.g., acid maltase or acid alpha-1, 4-glucosidase deficiency (Pompe’s disease), glycogen storage disorders 3-11, carnitine deficiency, fatty acid oxidation defects, and carnitine palmitoyl transferase deficiency), and mitochondrial myopathy. Autoimmune or inflammatory myopathies (i.e., myositis) include, e.g., dermatomyositis, polymyositis, inclusion body myositis, and juvenile myositis, as well as other autoimmune disorders (e.g., myasthenia gravis). Infections that may lead to muscle wasting include, for example, infections by viruses (e.g., HIV, influenza virus, and Epstein-Barr virus), bacteria (e.g., pyomyositis, S. aureus, and streptococci), spirochetes (e.g., Lyme disease), and parasites (e.g., trichinosis). Toxins that can cause myopathies include, for example, certain medications (e.g., cholesterol-lowering medications (e.g., statins), propofol, amiodaron, colchicine, chloroquine, antivirals, protease inhibitors, omeprazole, and tryptophan), and toxins such as alcohol and toluene. Systemic diseases can also lead to muscle wasting, e.g., endocrine disorders (e.g., thyroid, parathyroid, pituitary, or adrenal disorders), systemic inflammatory disease (e.g., systemic lupus erythematosus, rheumatoid arthritis, scleroderma, Sjogren’s syndrome, mixed connective disease, and sarcoidosis), electrolyte imbalance, critical illness myopathy, and amyloid myopathy.

[0239] Neurological diseases and conditions that may be characterized by muscle wasting include, for example, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), spinal muscular atrophy (SMA), Guillain-Barre syndrome, carpel tunnel syndrome, Char cot-Marie-Tooth disease, Parkinson’s disease, Lewy body disease, polio, spinal cord injury, peripheral nerve injury, stroke, and nerve damage caused by diabetes, toxins, or alcohol.

[0240] In addition to the above, being present in an environment with decreased or no gravity, e.g., during a space flight or being at a space station or the like, can lead to muscle wasting.

[0241] Other diseases or conditions associated with muscle wasting include prolonged inactivity (e.g., in those who are bedridden, have seated jobs or a sedentary lifestyle, or cannot move limbs due to stroke or other brain disease), burns, long-term corticosteroid therapy, malnutrition, anorexia, cachexia, osteoarthritis, age, cancer, chronic kidney disease, heart failure, and chronic obstructive pulmonary disease.

[0242] Symptoms that can be prevented, treated, inhibited, or ameliorated according to the invention include, for example, muscle loss, movement issues, balance problems, trouble walking, trouble using arms or legs, contractures, muscle rigidity, muscle twitching, fasciculations, muscle cramps, numbness, tingling, painful sensations, breathing problems, curved spine, heart problems, swallowing problems (which may lead to, e.g., nutritional problems and aspiration pneumonia), reduced muscle mass (e.g., in general or in one limb), weakness (e.g., in general or in one limb), pain, discomfort, one arm or leg being smaller than the other, weakness in one arm or leg, numbness or tingling in an arm or leg, trouble walking, balance problems, facia weakness, gradual memory loss, reduced muscle strength, impaired ability to perform physical activities, decrease in muscle size, cramps, stiffness, spasms, fatigue, motor delay, respiratory impairment, and bulbar muscle dysfunction.

[0243] Furthermore, as noted above, the methods and compositions of the disclosure can be used to prevent or treat insulin resistance in a subject, such as a human patient described herein. In some embodiments, the subject has or is at risk of developing type II diabetes or pre-diabetes. In some embodiments, the subject has or is at risk of developing metabolic syndrome. In some embodiments, the subject has or is at risk of developing hypertension, hyperglycemia, hypercholesterolemia, high triglycerides, overweight, and / or obesity. In some embodiments, treatment according to the present disclosure improves one or more symptom in such subjects, e.g., decreases insulin resistance, reduces blood glucose levels, reduces A1C levels, and / or decreases or normalizes blood pressure, cholesterol levels, and / or triglycerides.

[0244] The above-described methods comprise administering a pharmaceutical composition comprising a pharmacologically effective amount of one or more immunotherapy targeting myostatin and / or a pharmacologically effective amount of an immunotherapy targeting activin A (e.g., one or more peptide immunogen construct and / or an antibody, e.g., as described herein) to a subject in need thereof. The amounts and regimens used in the methods can be consistent with the information provided above in the section concerning compositions or as determined to be appropriate by those of skill in the art.

[0245] The following examples illustrate certain features and aspects of the disclosure and are not to be considered as limiting of the scope of the disclosure in any way.

[0246] EXAMPLES

[0247] Example 1

[0248] Myostatin is a secreted growth differentiation factor that is a member of the TGF-β protein family and is a negative regulator of muscle growth. It is synthesized as a 375 amino acid precursor, which includes an N-terminal signal sequence for secretion. The precursor protein is cleaved by a serine protease at an RSRR site to generate a latency-associated peptide (LAP) and mature myostatin, with the latter being characterized by nine conserved cysteine residues that play a role in eventual homo-dimerization. Mature myostatin is comprised of amino acids 267-375 of the proprotein and is characterized by intrachain disulfide bonds between the following sets of amino acids: 281-340, 309-372, and 313-374. The three-dimensional structure of myostatin is characterized by four loops (loops 1-4). We have designed peptide immunogens corresponding to certain myostatin loop sequences. In some examples, the peptides were designed to include certain amino acid substitutions that facilitate cyclization of the peptides, resulting in structures that mimic those of the sequences when present in the context of an intact loop. We have found that the designed peptide immunogens, present in the context of constructs that also include heterologous T cell epitopes, are immunogenic in animal models. The induced antibodies have neutralizing effects in cell culture studies, and the peptide immunogens can provide improvements in features of muscle wasting in an animal model system. Additional information about these studies is provided below.

[0249] Immunogenicity

[0250] The myostatin peptide immunogen constructs listed in FIG. 1 were injected IM into Guinea pigs at 400 ug peptide in montanide (ISA-51) on week 0, and at 100 ug peptide in montanide on weeks 3, 6, 9 and 12. Serum samples obtained at 6, 9, 12, and 15 weeks-post immunization (wpi) were diluted as indicated in the figure and OD 450 readings were obtained. The results show that the peptide immunogen constructs are immunogenic as compared to placebo and pre-immune sera, with SEQ ID NOs: 54-56 showing the highest activity.

[0251] Four Guinea pigs were vaccinated with a peptide of SEQ ID NO: 55 at 400 ug per injection, IM in monatide ISA-51, at 0, 3, 6, 9, and 12 wpi. Sera were collected on weeks 9, 12, and 15 postimmunization. Pooled sera were analyzed on plates coated with full-length myostatin protein. FIG.

[0252] 2 shows that the immunizations resulted in the production of antibodies that recognize myostatin, as compared to pre-immune sera.

[0253] Neutralization - HEK293 Cells

[0254] Peptide (SEQ ID NO: 55)-induced antibodies were tested in a neutralization assay in HEK293 cells. Sera from immunized Guinea pigs were collected on weeks 6, 9, 12, and 15 wpi. IgG fractions were isolated and tested for in vitro activity in a reporter assay. Myostatin can bind to TGFβ receptor, composed of two TβR-I and two TβR-II, activate downstream smad pathway, and then induce expression of a reporter protein, SEAP. Neutralizing effects of peptide (SEQ ID NO: 55)-induced IgGs were evaluated in this assay along with a control monoclonal antibody against myostatin. The results are shown in FIG. 3, which shows concentration and time post-immunization effects of peptide (SEQ ID NO: 55) immunization on neutralization. Methods: Human TGFβ SEAP reporter HEK293 cells (InvivoGen) were grown following the manufacturer’s instructions. All cells were cultured in 5% CO2 at 37°C, and the medium was replaced every 3 days. HEK-blue TGFβ cells allow the detection of bioactive TGFβ by monitoring the activation of the TGFβ / Smad pathway. They were generated by stable transfection of HEK-293 cells with the human TGFBRI, Smad 3, and Smad 4 genes. For neutralization, IgGs diluted with 2% FBS in DMEM mixed with 250 ng / mL myostatin and neutralized for 1 hour. Removed medium of HEK-blue TGFβ and added 100 uL mixed solution in each well of 96 well plates. Incubated the plate at 37°C in a CO2 incubator for 16 hours. Prepared 160 uL of resuspended QUANTI-Blue per well and added 40 uL of induced cells supernatant. After incubating for 1 hour, the determination of SEAP levels by using a spectrophotometer at 620-655 nm.

[0255] Neutralization - C2C12 Cells

[0256] Myostatin inhibits muscle cell differentiation in C2C12 cells. Peptide (SEQ ID NO: 55)-induced antibodies were evaluated in myostatin-treated C2C12 cells. Muscle cell differentiation was determined by fiber width and number of nuclei per cell. The results are shown in FIG. 4. Treatment with peptide (SEQ ID NO: 55) (5 ug / mL) countered the effects of 8 ug / mL myostatin on fiber width. Treatment with peptide (SEQ ID NO: 55) (5 and 10 ug / mL) also countered the effects of 8 ug / mL myostatin on number of nuclei per MHC+ cell.

[0257] Methods: C2C12 mouse skeletal muscle cell line obtained from ATCC were grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, 12800-017, Thermo Fisher) supplemented with 10% fetal bovine serum (10437-208, Thermo Fisher) and penicillin-streptomycin 100 lU / mL in 5% CO2 at 37°C. The growth medium was changed every 3 days. C2C12 cells that had grown to approximately 80% confluence in culture flasks were trypsinized and seeded into culture plates to allow incubation in DMEM containing 10% FBS until reaching about 90% confluence. To induce myoblast differentiation, C2C12 cells were grown in DMEM supplemented with 2% horse serum (16050-122, Thermo Fisher) for different stages14. After the medium was replaced with DMEM containing 2% horse serum (HS), the cells were induced to differentiate into myotubes until fusion of more than 90% cells into myotubes.

[0258] C2C12 myoblasts were seeded onto Nunc Thermanox coverslips or 96 well-plates at a density of 20,000 cells / cm2. After a 16-hour attachment period, DMEM with 2% HS was added to culture. The cells were incubated at 37°C in 5% CO2 for 72 hours, after which the media was replaced with fresh media for a further 72 hours. IgGs were pre-incubated with 8 pg / mL myostatin for 2 hours and then added to the cells at day 6 for a further 72 hours. In Vivo Studies

[0259] In vivo effects of peptide (SEQ ID NO: 55) immunization of mice were analyzed by testing for antibody titers, as well as effects on body weight and muscle force. The treatment regimen is described below and illustrated in FIG. 5.

[0260] Animals. Naive male C57BL / 6 mice (15-16 months old) were obtained from National Laboratory Animal Center (Taiwan). Mice were kept in a humidity and temperature-controlled vivarium on a 12-hour light / dark cycle and were allowed access to food ad libitum. Mice were housed in middle-sized cage and each cage housed up to 8 littermates. Mice were at allowed to acclimate for at least 3 days and were 16-17 months old at study initiation. All animal care and use followed the Institutional Animal Care and Use Committee guidelines set forth by the United Biomedical, Inc. Asia (UBIA). It was noted that animal #33 (no bedding), #22 (vaccinated to blood vessel), #24, and #37 were found dead on 1, 4, 11, and 16 wpi, respectively.

[0261] Vaccination. Mice were first pseudo-randomly assigned to different groups adjusted for matching average body weights. The mean of body weight of each group was approximate. After grouping, mice were immunized with 0.2 ml of high dose of peptide (SEQ ID NO: 55) (200 pg / ml), low dose of peptide (SEQ ID NO: 55) (50 pg / ml), adjuvant (ISA51), or saline on thigh (0.1 ml / thigh). Mice received vaccination at 0, 3, and 6 wpi.

[0262] Sera collection. Whole blood samples were collected from facial vein by lancet every 2-3 weeks. Blood was allowed to clot by keeping at room temperature for at least 30 minutes. Sera were collected from the supernatants after centrifugation at 5,000 g for 10 minutes at 22°C and stored at -80°C until use.

[0263] Antibody titers

[0264] The results of antibody titer analysis at week 11 are shown in FIG. 6. The results show the presence of antibodies in sera in tested in peptide-coated wells from mice immunized with peptide (SEQ ID NO: 55) (10 pg or 40 pg), as compared to saline and adjuvant (ISA-51) only control.

[0265] Methods: Pre- immune and immune sera from animals were collected according to experimental vaccination protocols and heated at 56°C for 30 minutes to inactivate serum complement factors. The peptide-coated wells were incubated with 250 pL of 3% by weight of gelatin in PBS in 37°C for 1 hour to block non-specific protein binding sites, followed by three washes with PBS containing 0.05% by volume of TWEEN® 20 and dried. 100 pL of samples were added to each of the wells and allowed to react for 60 minutes at 37°C. The wells were then washed six times with 0.05% by volume TWEEN® 20 in PBS to remove unbound antibodies. Horseradish peroxidase (HRP) -conjugated species-specific goat anti-IgG was used as a labeled tracer to bind with the antibody / peptide antigen complex formed in positive wells. One hundred microliters of the peroxidase-labeled goat anti-IgG, at a pre-titered optimal dilution and in 1% by volume normal goat serum with 0.05% by volume TWEEN® 20 in PBS, was added to each well and incubated at 37°C for another 30 minutes. The wells were washed six times with 0.05% by volume TWEEN® 20 in PBS to remove unbound antibody and reacted with 100 pL of the substrate mixture containing 0.04% by weight 3’, 3’, 5’, 5 ’-Tetramethylbenzidine (TMB) and 0.12% by volume hydrogen peroxide in sodium citrate buffer for another 15 minutes. This substrate mixture was used to detect the peroxidase label by forming a colored product. Reactions were stopped by the addition of 100 pL of IM H2SO4 and absorbance at 450 nm (A450) determined. For the determination of antibody titers of the vaccinated animals that received the various myostatin derived peptide immunogens, 10-fold serial dilutions of sera from 1: 100 to 1: 10,000 were tested, and the titer of a tested serum, expressed as Log10, was calculated by linear regression analysis of the A450 with the cutoff A450 set at 0.5. Serially diluted sera were tested and positive titers were expressed as Log10 of the reciprocal dilution.

[0266] Body Weight

[0267] The results of body weight analysis are shown in FIG. 7. The results show that the body weights of mice vaccinated with peptide (SEQ ID NO: 55) were higher than those of mice in the saline and adjuvant (ISA-51) only treated controls.

[0268] Body weights were measured on a weekly or bi-weekly basis. Only control mice (administered saline or adjuvant) and mice developing high antibody titers after immunization with peptide (SEQ ID NO: 55) are included in the analysis (4 mice in the peptide (SEQ ID NO: 55) 40 mg / dose group). Body weights are presented as mean percentage change from baseline + SEM. A two-way ANOVA indicated significant treatment x time interaction (F24467 = 2.703; p < 0.001), a significant group effect (F244 = 5.738; p < 0.05) and a significant time effect (F3.679, 51.20 = 7.526; p < 0.001). * p < 0.05, ** p < 0.01 versus ISA51-treated mice.

[0269] Muscle Force

[0270] The results of ex vivo muscle force analysis are shown in FIG. 8. The results show increased twitch tension and tetanic tension in tibialis anterior of muscles from mice immunized with peptide (SEQ ID NO: 55), as compared to adjuvant (ISA-51) only treated mice.

[0271] Muscle extraction. At completion of the 16-week in-life period, mice were sacrificed by cervical dislocation and their legs were ablated. The skin was removed from the leg. The fascia covering the tibialis anterior (TA) muscle was gently removed. After that, a fishhook was tied on the distal TA tendon by inflexible surgical suture. The distal TA tendon was then cut and used to peel off the TA muscle. Another fishhook was tied on the proximal TA tendon. Lastly, the TA muscle was removed by cutting the proximal tendon. Once removing the TA muscle, the extensor digitorum longus (EDL) muscle was exposed. The EDL muscle was extracted the same way as the TA muscle. To remove the GA muscle, Achilles tendon was cut and used to peel off the gastrocnemius (GA) muscle. As the GA muscle was lifted, the SOL muscle becomes apparent on the underside of the GA muscle. The proximal soleus muscles (SOL) tendon was cut and used to pull the SOL muscle away from the GA muscle. After the whole SOL muscle was free from the GA muscle, cutting the distal SOL tendon to extract the SOL muscle. Finally, the GA muscle was removed by cutting the proximal GA tendon. All muscles were extracted within 1 hour after sacrifice. Muscles were either preserved in Krebs-Henseleit (KH) buffer (118 mM NaCl, 4.7 mM KC1, 1.2 mMMgS04, 1.25 mM CaCl2, 1.2 mMKH2PO4, 25 mMNaHC03, 11 mM glucose, pH 7.4) for ex vivo force measurement, or transferred to 4% paraformaldehyde (PFA) for long-term storage.

[0272] Ex vivo force measurement. Muscles were immersed in KH buffer at 25 °C in 4-channel tissue bath system (SINGA #MB04II, Taiwan, accompanied with Xctionview 2.0 software) with air bubbling (O2: CO2= 95%: 5%) for 15 minutes prior to force measurement. For twitch and tetanic tension measurement, muscles were tightly secured at two hooks, and the length was measured. Maximal twitch tension (MTT) was determined by stimulating muscles with a range of voltages from 5 to 30 V. Each stimulation lasted 0.5 millisecond and muscles were allowed to rest for at least 30 seconds between each stimulation. After MTT was determined, muscles were allowed to rest for at least 3 minutes before further stimulation. For twitch tension (Pt), a supramaximal voltage (20% above MTT) was applied to muscles for around 0.5 millisecond, and contractile force was recorded right after stimulation. For tetanic tension (Po), a supramaximal voltage (20% above MTT) at 150 Hz (totally 300 ms) was applied to muscles, contractile force was measured.

[0273] Example 2

[0274] Activin A is a member of the activin family of proteins, which in turn are members of the TGF-β superfamily. Like myostatin, activin A activates the Smad2 / 3 transcription pathway, which, by limiting protein synthesis and increasing protein degradation, results in a reduction in muscle mass. Activin A is synthesized as a 426 amino acid precursor, which includes an N-terminal signal sequence for secretion. The precursor protein is cleaved by a protease at an RRRR site to generate mature Activin A. Mature activin A is comprised of amino acids 311-426 of the proprotein and is characterized by intrachain disulfide bonds. The three-dimensional structure of activin A is characterized by four loops (loops 1-4). We have designed peptide immunogens corresponding to certain activin A loop sequences. In some examples, the peptides were designed to include certain amino acid substitutions that facilitate cyclization of the peptides, resulting in structures that mimic those of the sequences when present in the context of an intact loop. We have found that the designed peptide immunogens, present in the context of constructs that also include heterologous T cell epitopes, are immunogenic an animal model. The induced antibodies have neutralizing effects in cell culture studies. Additional information about these studies is provided below.

[0275] The activin A peptide immunogen constructs listed in FIG. 9 were injected IM into Guinea pigs (400 ug in montanide ISA-51). Serum samples obtained at 9 and 12 weeks-post immunization (wpi) were diluted as indicated in the figure and OD 450 readings were obtained. The results show that the peptide immunogen constructs are immunogenic as compared to placebo, with SEQ ID NOs: 73, 75, 76, 78, 79, and 85 being the most immunogenic.

[0276] Antibodies were generated by immunization of Guinea pigs with the activin A peptide immunogen constructs listed in FIG. 10. IgGs obtained from the Guinea pigs and purified at 9-, 12-, and 15 -weeks post- immunization (wpi) were tested for their ability to inhibit activin A-induced SMAD2 / 3 signaling in a cell-based reporter system at 750, 250, and 75 pg / mL. The results show time and concentration-dependent inhibition of activin A-induced signaling for most constructs, in particular the following constructs: SEQ ID NOs: 75, 78, 79, and 85.

[0277] Further, the activin A peptide immunogen constructs listed in FIG. 11 (SEQ ID NO: 135-144) were injected IM into rats at weeks 0 and 3. Each injection consisted of 300 pg of peptide immunogen construct formulated in Adju-Phos with 100 pg of CpGl. Serum samples obtained at 6 weeks-post immunization (wpi) were diluted as indicated in the figure and analyzed on plates coated with full-length activin A protein. OD 450 readings of sera from individual animals were averaged together. FIG. 11 shows that the immunizations resulted in the production of antibodies that recognize activin A, with SEQ ID NOs: 141, 143, and 144 showing the highest activity.

[0278] Example 3

[0279] The myostatin peptide immunogen constructs listed in FIG. 12 (SEQ ID Nos: 120-134) were injected IM into rats at weeks 0 and 3. Each injection consisted of 300 pg of peptide immunogen construct formulated in Adju-Phos with 100 pg of CpGl. Serum samples obtained at 6 weeks-post immunization (wpi) were diluted as indicated in the figure and analyzed on plates coated with full-length myostatin protein. OD 450 readings of sera from individual animals were averaged together. FIG. 12 shows that the immunizations resulted in the production of antibodies that recognize myostatin, with SEQ ID NOs: 122, 123, 126, and 130 showing the highest activity.

[0280] Example 4

[0281] Guinea pigs were individually injected IM with a myostatin construct (SEQ ID NOs: 122, 167, or 168) or with an activin A construct (SEQ ID NOs: 141 or 144) at weeks 0, 3, 6, 9, and 12. Each injection consisted of 300 pg of peptide immunogen construct formulated in Adju-Phos with 100 pg of CpGl. Serum samples obtained at 15 weeks-post immunization (wpi) were diluted as indicated in the figure and analyzed on plates coated with full-length myostatin or activin A protein. OD 450 readings of sera from individual animals were averaged together. The immunogenicity results are shown in FIG. 13. The body weights of the guinea pigs in this study were also tracked over time and FIG. 14 shows that guinea pigs immunized with constructs of SEQ ID NOs: 141, 122, and 168 gained more weight than those immunized with the other constructs shown.

[0282] Example 5

[0283] Additional myostatin constructs were generated with the goal of reducing or removing crossreactivity with GDF-11. Specifically, constructs were made to target latent myostatin, promyostatin, and mature myostatin. Additional constructs were also made to target activin A. In FIGs. 15, 16, 17, and 18, the immunogenicity and antibody titers were determined as follows. The myostatin or activin A peptide immunogen constructs shown in each figure were injected IM into individual rats at weeks 0, 3, 6, and 12. Each injection consisted of 300 pg of peptide immunogen construct formulated in Adju-Phos with 100 pg of CpGl. For immunogenicity assays, serum samples obtained at 15 weeks-post immunization (wpi) were diluted as indicated in the figure and analyzed on plates coated with either latent myostatin, promyostatin, mature myostatin, or mature activin A. OD 450 readings of sera from individual animals were averaged together. To determine antibody titers, pre- immune sera and sera from immunized rats were collected at week 15 according to experimental vaccination protocols and stored at -80°C. 96-well microtiter plates were coated with 1 pg / mL of protein (mature myostatin, latent myostatin, promyostatin, mature activin A, or GDF-11) diluted in PBS. Coated plates were sealed and incubated at 4°C overnight. Plates were removed from incubation, washed four times with PBS containing 0.05% by volume TWEEN® 20 (PBS-T) and then blocked by adding 250 pL of SuperBlock™ blocking buffer followed by a 2-hour incubation, shaking (500 rpm) at 37°C. Blocking buffer was removed after incubation and plates were washed once with PBS-T before the addition of sera. Pre-immune and immune serum samples were thawed at 4°C and then diluted 1: 20 in ELISA diluent (2% BSA by weight in PBS). Diluted sera were titrated by 8- or 12-point serial 3 -fold dilution in a dilution block and 100 pL aliquots of each serum dilution were added to the 96 well plate in duplicate. Diluted sera were incubated for 1 hour at 25°C and then washed six times with PBS-T. Horseradish peroxidase (HRP)-conjugated species-specific goat anti-IgG was used as a labeled tracer to bind with the antibody / peptide antigen complex formed in positive wells. One hundred microliters of the HRP linked goat anti-species IgG at a pre-titered optimal dilution in ELISA diluent was added to each well and incubated at 25°C, shaking (500 rpm) for 45 minutes. The wells were then washed six times with PBS-T to remove unbound antibody and reacted with 100 pL of the substrate (1-Step™ TMB ELISA Substrate) for 15 minutes. This substrate mixture was used to detect the peroxidase label by forming a colored product. Reactions were stopped by the addition of 100 pL of IM H2SO4 and absorbance at 450 nm (A450) determined. For the determination of antibody titers of the vaccinated animals that received the various myostatin or activin A derived peptide immunogens, 3-fold serial dilutions of sera from 1:20 to 1:3542940 were tested, and the titer of a tested serum was determined as the EC50 from a nonlinear four-parameter curve and expressed as the LoglO EC50 titer.

[0284] FIG. 15 shows the immunogenicity and antibody titers resulting from immunizations with the latent myostatin constructs of SEQ ID Nos: 177, 180, 181, or 182. FIG. 15A shows that, at week 15, immunization with all of the constructs resulted in production of antibodies against latent myostatin, but the immunizations with SEQ ID NO: 177 and SEQ ID NO: 181 resulted in the strongest production of antibodies that recognize latent myostatin. FIG. 15B shows that immunization with all four constructs led to high antibody titers, but immunizations with SEQ ID NO: 177 and SEQ ID NO: 181 resulted in the highest antibody titers.

[0285] The immunogenicity and antibody titers resulting from immunizations with the promyostatin peptide immunogen constructs of SEQ ID NOs: 183, 184, 185, 186, or 188 are shown in Figure 16. FIG. 16A and FIG. 16B show that the immunizations all constructs resulted in production of antibodies against promyostatin, but immunizations with SEQ ID NO: 183, SEQ ID NO: 185, and SEQ ID NO: 186 resulted in the strongest production of antibodies that recognize promyostatin and the highest antibody titers at week 15. The constructs targeting mature myostatin of SEQ ID NO: 167, 168, 169, 173, 174, 175, 176, 178, or 179 were injected into individual rats at weeks 0, 3, 6, and 9 and immunogenicity and antibody titers against mature myostatin were determined at week 15. FIG. 17A shows that the majority of the constructs resulted in production of antibodies that recognize mature myostatin, but immunizations with SEQ ID NO: 169, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 176, and SEQ ID NO: 179 resulted in the strongest production of antibodies. As shown in FIG. 17B, antibody titers were the highest in rats immunized with SEQ ID NO: 169, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 178, and SEQ ID NO: 179.

[0286] SEQ ID NO: 168 was injected IM into rats at weeks 0, 3, 6, 9, and 12. At week 15, antibody titers against mature myostatin and mature GDF-11 were determined, and the results are shown in FIG. 18. The rats immunized with SEQ ID NO: 168 had high titers of antibodies that recognize mature myostatin but low titers of antibodies that recognize GDF-11.

[0287] Lastly, rats were immunized at weeks 0, 3, 6, 9, and 12 with the activin A constructs of SEQ ID NOs: 141, 190, or 191. Immunization with all three constructs resulted in the production of antibodies (FIG. 19A) and high antibody titers (FIG. 19B) at week 15, with SEQ ID NO: 141 and SEQ ID NO: 190 showing the greatest effect.

[0288] Example 6

[0289] A combo myostatin - activin A peptide immunogen construct was generated using the myostatin B cell epitope of SEQ ID NO: 97 and the activin A B cell epitope of SEQ ID NO: 116. The resulting construct has the following amino acid sequence: Ac-STVINHYRMRGHSPFANLKSSAeeA(Ac-QKYPHTHLVHQANPRGSAGPAeeA)K-KKKrKUbithl (SEQ ID NO: 165), where the peptide between ( ) indicates that it is off the side chain of the Lys and not the alpha amino group, AeeA is a peg spacer, and the Ac indicates that the construct is acetylated on its terminal NH2. SEQ ID NO: 165 was injected IM into rats at weeks 0, 3, 6, 9, and 12. Each injection consisted of 300 pg of peptide immunogen construct formulated in Adju-Phos with 100 pg of CpGl. Serum samples obtained at 15 weeks-post immunization (wpi) were diluted as indicated in the figure and analyzed on plates coated with full-length myostatin protein (FIG. 20) or full-length activin A protein (FIG. 21). OD 450 readings of sera from individual animals were averaged together. In the same experiment, additional rats were individually injected with other myostatin constructs (SEQ ID NOs: 122, 133, 169, 170, 171, or 172) or other activin constructs (SEQ ID NO: 141) at the same dose and timing and the immunogenicity of those constructs was also evaluated. FIGs. 20 and 21 show that the immunizations with SEQ ID NO: 165 resulted in the production of antibodies that recognize both myostatin and activin A. Immunization with SEQ ID NO: 165 generates a higher antibody titer against myostatin than immunization with SEQ ID NO: 122 (construct containing SEQ ID NO: 97) and a similar antibody titer against activin A compared to immunization with SEQ ID NO: 141 (construct containing SEQ ID NO: 116). SEQ ID NO: 169 also generates a high antibody titer at 15 weeks. Table 1 - Amino Acid sequences of myostatin and fragments (myostatin B cell epitopes) thereof*

[0290]

[0291]

[0292]

[0293]

[0294]

[0295] *Note: “Ahx” is 6-aminohexanoic acid. “Dap” is L-2,3 diaminopropionic acid (CAS 1482-97-9). “Dab” is L-2,4 diaminobutyric acid (CAS 1883-09-6). “Om” is L-Omithine (CAS 3184-13-2).

[0296] *Each of the listed B cell epitope peptides is acetylated on the N-terminus and has an amide on the C-terminus (except for the peptides that are cyclized using the N-terminus amino group, such as peptides of SEQ ID NO: 103 (Ahx-KYPHTHLVHQANPRGSAGE) and SEQ ID NO: 106 (PHTHLVHQANPRGSAE)). Table 2 - Amino Acid sequences of activin A and fragments (activin A B cell epitopes) thereof*

[0297]

[0298]

[0299] *Each of the listed B cell epitope peptides is acetylated on the N-terminus and has an amide on the C-terminus (except for the peptides that are cyclized using the N-terminus amino group, such as peptides of SEQ ID NO: 103 (Ahx-KYPHTHLVHQANPRGSAGE) and SEQ ID NO: 106 (PHTHLVHQANPRGSAE)).

[0300] Table 3 - Amino Acid Sequences of Pathogen Protein Derived Th Epitopes Including Idealized Artificial Th Epitopes for Use in Myostatin and Activin A Peptide Immunogen Constructs

[0301]

[0302]

[0303] Table 4 - Amino Acid Sequences of Myostatin Peptide Immunogen Constructs*

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311] *Note: “Ahx” is 6-aminohexanoic acid. “Dap” is L-2,3 diaminopropionic acid (CAS 1482-97-9).

[0312] “Dab” is L-2,4 diaminobutyric acid (CAS 1883-09-6). “Om” is L-Omithine (CAS 3184-13-2).

[0313] *For each peptide immunogen construct listed, B cell epitope peptides are acetylated on the N-terminus and have an amide on the C-terminus (except for the peptides that are cyclized using the N-terminus amino group, such as peptides of SEQ ID NO: 103 (Ahx-KYPHTHLVHQANPRGSAGE) and SEQ ID NO: 106 (PHTHLVHQANPRGSAE)).

[0314] Note: for each peptide immunogen construct listed in this table, the indication of a mixed T

[0315] cell epitope (e.g., UBITh3) is to be considered as including the presence of any form of the T

[0316] cell epitope peptide, including any one or more (e.g., all) of the possible combinations.

[0317] Accordingly, the sequence of UBITh3 can be considered as ISIXEIXXVIVXXIETILF,

[0318] wherein the 1stX is S or T, the 2ndX is K or R, the 3rdX is G or T, the 4thX is H or T, and the 5thX is K or R, and the selections are independent of one another. If more than one mixed T cell epitope (e.g., UBITh3) is present, then each is to be considered as independently including any one or more (e.g., all) of the possible combinations as described herein. The same applies for other mixed T cell epitopes. See Table 3 for details. In some embodiments, the UBITh3 peptide is replaced with the UBIThl or UBITh2 peptide. This note also applies to Table 5.

[0319] Table 5 - Amino Acid Sequences of Activin A Peptide Immunogen Constructs*

[0320]

[0321]

[0322]

[0323]

[0324] * For each peptide immunogen construct listed, B cell epitope peptides are acetylated on the N-terminus and have an amide on the C-terminus (except for the peptides that are cyclized using the N-terminus amino group, such as peptides of SEQ ID NO: 103 (Ahx-KYPHTHLVHQANPRGSAGE) and SEQ ID NO: 106 (PHTHLVHQANPRGSAE)).

[0325] Accordingly, the invention includes each of the constructs listed in Table 4, above, but with any one of the Th epitopes listed in Table 3 inserted in place of UBIThl, UBITh2, or UBITh3.

[0326] Furthermore, any of the linkers described herein can be utilized in place of the linkers set forth in Table 4. The invention also includes each of the constructs listed in Table 5, above, but with any one of the Th epitopes listed in Table 3 inserted in place of UBIThl or UBITh3. Furthermore, any of the linkers described herein can be utilized in place of the linkers set forth in Table 5.

[0327] Exemplary linker sequences are as follows:

[0328] SEQ ID NO: 90 Pro-Pro-Xaa-Pro-Xaa-Pro Xaa = any amino acid, e.g., aspartic acid SEQ ID NO: 91;-N-Lys-Lys-Lys-Lys

[0329] OTHER EMBODIMENTS

[0330] Various modifications and variations of the described invention 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 such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.

[0331] Some embodiments are within the scope of the following numbered paragraphs.

[0332] 1. A peptide immunogen construct comprising a myostatin B cell epitope, a heterologous T cell (Th) epitope, and an optional heterologous spacer, wherein the B cell epitope is covalently linked to the Th epitope directly or through the optional heterologous spacer.

[0333] 2. The peptide immunogen construct of paragraph 1, wherein the myostatin B cell epitope comprises about 7 to about 30 contiguous amino acids from myostatin or a variant thereof.

[0334] 3. The peptide immunogen construct of paragraph 1 or 2, wherein the myostatin B cell epitope comprises about 7 to about 30 contiguous amino acids of SEQ ID NO:1 or SEQ ID NO: 2 or a variant thereof.

[0335] 4. The peptide immunogen construct of paragraph 3, wherein the myostatin B cell epitope comprises a variant of an 7-30 amino acid fragment of SEQ ID NO: 1 or SEQ ID NO: 2, and the variant comprises 1, 2, 3, 4, 5, or 6 amino acid substitutions, deletions, or insertions as compared to the corresponding sequence of the 7-30 amino acid fragment of SEQ ID NO: 1 or SEQ ID NO: 2.

[0336] 5. The peptide immunogen construct of paragraph 4, wherein the myostatin B cell epitope comprises a variant of an 7-30 amino acid fragment of SEQ ID NO: 1 or SEQ ID NO: 2, and the variant comprises 1, 2, or 3 amino acid substitutions as compared to the corresponding sequence of the 7-30 amino acid fragment of SEQ ID NO: 1 or SEQ ID NO: 2.

[0337] 6. The peptide immunogen construct of any one of paragraphs 1 to 5, wherein the myostatin B cell epitope comprises 1, 2, or 3 amino acid substitutions as compared to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 to facilitate cyclization of the sequence comprising the myostatin B cell epitope.

[0338] 7. The peptide immunogen construct of any one of paragraphs 1 to 6, wherein the myostatin B cell epitope comprises or consists of an amino acid sequence within loop 1, loop 2, loop 3, or loop 4 of myostatin, or a variant thereof, and optionally comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 3-10, 95-109, 145-164, and 166 or a variant thereof.

[0339] 8. The peptide immunogen construct of any one of paragraphs 1 to 7, wherein the Th epitope is derived from a pathogenic protein, and optionally comprises or consists of a sequence selected from any one of SEQ ID NOs: 19-47 or a variant thereof.

[0340] 9. The peptide immunogen construct of any one of paragraphs 1 to 8, wherein the Th epitope comprises or consists of the sequence of SEQ ID NO: 30, 32, or 33.

[0341] 10. The peptide immunogen construct of any one of paragraphs 1 to 9, wherein the myostatin B cell epitope and the Th epitope are linked to one another by the heterologous spacer.

[0342] 11. The peptide immunogen construct of paragraph 10, wherein the heterologous spacer comprises or consists of one or more amino acids, which are optionally selected from: Lys-, Gly-, Lys-Lys-Lys-, Lys-Lys-Lys-s-Lys, (a, s-N)Lys, s-N-Lys-Lys-Lys-Lys (SEQ ID NO: 91), and Pro-Pro-Xaa-Pro-Xaa-Pro, wherein Xaa is any amino acid or is aspartic acid (SEQ ID NO: 90).

[0343] 12. The peptide immunogen construct of any one of paragraphs I to 11, wherein: (a) the myostatin B cell epitope is located N-terminal to the Th epitope; (b) the Th epitope is located N-terminal to the myostatin B cell epitope; (c) the myostatin B cell epitope is flanked with a Th epitope on both N-terminal and C-terminal ends; (d) any of (a)-(c) wherein the myostatin B cell epitope(s) is linked to the Th epitope(s) by direct covalent linkage; or (e) any one of (a)-(c) wherein the myostatin B cell epitope(s) is linked to the Th epitope) s) by the heterologous spacer.

[0344] 13. The peptide immunogen construct of any one of paragraphs 1 to 12, wherein the peptide immunogen construct is of following formula: (Th)m~-(A)n-(myostatin B cell epitope) or (myostatin B cell epitope)-(A)n-(Th)m or (Th)m---(A)n---(rnyostatin B cell epitope)--(A)n-(Th)mwherein Th is the T helper epitope; A is the heterologous spacer, which optionally comprises an ammo acid; m is from 1 to about 4; and n is from 0 to about 10, wherein optionally the C-terminus of the peptide immunogen construct comprises a-COOH or a-CONH2of an ammo acid.

[0345] 14. The peptide immunogen construct of any one of paragraphs 1 to 13, wherein the peptide immunogen construct comprises or consists of the sequence of any one of SEQ ID NOs: 48-71, 120-134, 167-186 and 188 or a variant thereof.

[0346] 15. A peptide immunogen construct comprising an activin A B cell epitope, a heterologous T cell (Th) epitope, and an optional heterologous spacer, wherein the B cell epitope is covalently linked to the Th epitope directly or through the optional heterologous spacer. 16. The peptide immunogen construct of paragraph 15, wherein the activin A B cell epitope comprises about 7 to about 30 contiguous amino acids from activin A or a variant thereof.

[0347] 17. The peptide immunogen construct of paragraph 15 or 16, wherein the activin A B cell epitope comprises about 7 to about 30 contiguous amino acids of SEQ ID NO: 12 or a variant thereof.

[0348] 18. The peptide immunogen construct of paragraph 17, wherein the activin A B cell epitope comprises a variant of a 7-30 amino acid fragment of SEQ ID NO: 12, and the variant comprises 1, 2, 3, 4, 5, or 6 amino acid substitutions, deletions, or insertions as compared to the corresponding sequence of the 7-30 amino acid fragment of SEQ ID NO: 12.

[0349] 19. The peptide immunogen construct of paragraph 18, wherein the activin A B cell epitope comprises a variant of a 7-30 amino acid fragment of SEQ ID NO: 12, and the variant comprises 1, 2, or 3 amino acid substitutions as compared to the corresponding sequence of the 7-30 amino acid fragment of SEQ ID NO: 12.

[0350] 20. The peptide immunogen construct of any one of paragraphs 15 to 19, wherein the activin A B cell epitope comprises 1, 2, or 3 amino acid substitutions as compared to the sequence of SEQ ID NO: 12 to facilitate cyclization of the sequence comprising the activin A B cell epitope.

[0351] 21. The peptide immunogen construct of any one of paragraphs 15 to 20, wherein the activin A B cell epitope comprises or consists of an amino acid sequence within loop 1, loop 2, loop 3, or loop 4 of activin A, or a variant thereof, and optionally comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 13-18, 110-119, 187, and 189 or a variant thereof.

[0352] 22. The peptide immunogen construct of any one of paragraphs 15 to 21, wherein the Th epitope is derived from a pathogenic protein, and optionally comprises or consists of a sequence selected from any one of SEQ ID NOs: 19-47 or a variant thereof.

[0353] 23. The peptide immunogen construct of any one of paragraphs 15 to 22, wherein the Th epitope comprises or consists of the sequence of SEQ ID NO: 30, 32, or 33.

[0354] 24. The peptide immunogen construct of any one of paragraphs 15 to 23, wherein the activin A B cell epitope and the Th epitope are linked to one another by the heterologous spacer.

[0355] 25. The peptide immunogen construct of paragraph 24, wherein the heterologous spacer comprises or consists of one or more amino acids, which are optionally selected from: Lys-, Gly-, Lys-Lys-Lys-, Lys-Lys-Lys-e-Lys, (a, e-N)Lys, s-N-Lys-Lys-Lys-Lys (SEQ ID NO: 91), and Pro-Pro-Xaa-Pro-Xaa-Pro, wherein Xaa is any amino acid or is aspartic acid (SEQ ID NO: 90). 26. The peptide immunogen construct of any one of paragraphs 15 to 25, wherein: (a) the activin A B cell epitope is located N-terminal to the Th epitope; (b) the Th epitope is located N-terminal to the activin A B cell epitope; (c) the activin A B cell epitope is flanked with a Th epitope on both N-terminal and C-terminal ends; (d) any of (a)-(c) wherein the activin A B cell epitope(s) is linked to the Th epitope(s) by direct covalent linkage; or (e) any one of (a)-(c) wherein the activin A B cell epitope(s) is linked to the Th epitope(s) by the heterologous spacer.

[0356] 27. The peptide immunogen construct of any one of paragraphs 15 to 26, wherein the peptide immunogen construct is of following formula: (Th)m--(A)n--( activin A B cell epitope) or (activin A B cell epitope)-(A)n-(Th)mor (Th)m---(A)n-(activin A B cell epitope)--(A)n---(Th)m wherein Th is the T helper epitope; A is the heterologous spacer, which optionally comprises an amino acid; m is from 1 to about 4; and n is from 0 to about 10, wherein optionally the C-terminus of the peptide immunogen construct comprises a-COOH or a-CONH₂ of an amino acid.

[0357] 28. The peptide immunogen construct of any one of paragraphs 15 to 27, wherein the peptide immunogen construct comprises or consists of the sequence of any one of SEQ ID NOs: 72-89, 135-144, and 190-191 or a variant thereof.

[0358] 29. A nucleic acid molecule encoding a peptide immunogen construct of any one of paragraphs 1 to 28.

[0359] 30. A vector comprising the nucleic acid molecule of paragraph 29.

[0360] 31. An isolated antibody or an epitope-binding fragment thereof that specifically binds to the myostatin B cell epitope of the peptide immunogen construct of any one of paragraphs 1 to 14 or the activin A B cell epitope of the peptide immunogen construct of any one of paragraphs 15 to 28.

[0361] 32. A composition comprising one or more of: (a) a peptide immunogen construct of any one of paragraphs 1 to 28, a nucleic acid of paragraph 29, a vector of paragraph 30, an isolated antibody of paragraph 31, or a peptide immunogen construct of any one of paragraphs 1 to 14 and a peptide immunogen construct of any one of paragraphs 15 to 28, and (b) a pharmaceutically acceptable carrier, diluent, or adjuvant.

[0362] 33. The composition of paragraph 32, wherein the composition comprises an adjuvant that is a mineral salt of aluminum, which optionally is selected from Al(OH)3and AlPO4.

[0363] 34. The composition of paragraph 32 or 33, wherein the composition comprises a CpG oligonucleotide.

[0364] 35. A method of treating or preventing the development of muscle wasting in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct of any one of paragraphs 1 to 28, a nucleic acid molecule of paragraph 29, a vector of paragraph 30, an isolated antibody of paragraph 31, a composition of any one of paragraphs 32 to 34, or a combination of any thereof.

[0365] 36. A method of treating, preventing, reducing, inhibiting, or slowing the development of one or more symptom of a muscle wasting disease in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct of any one of paragraphs 1 to 28, a nucleic acid molecule of paragraph 29, a vector of paragraph 30, an isolated antibody of paragraph 31, a composition of any one of paragraphs 32 to 34, or a combination thereof.

[0366] 37. A method of inducing an immune response to myostatin and / or activin A in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct of any one of paragraphs 1 to 28, a nucleic acid molecule of paragraph 29, a vector of paragraph 30, an isolated antibody of paragraph 31, a composition of any one of paragraphs 32 to 34, or a combination thereof.

[0367] 38. A method of treating or preventing insulin resistance in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct of any one of paragraphs 1 to 28, a nucleic acid molecule of paragraph 29, a vector of paragraph 30, an isolated antibody of paragraph 31, or a composition of any one of paragraphs 32 to 34, or a combination thereof.

[0368] 39. The method of any one of paragraphs 35 to 38, wherein the subject has or is at risk of developing muscle wasting due to an inherited or genetic cause, inflammation, infection, injury, systemic disease, toxin, or other environmental exposure.

[0369] 40. The method of any one of paragraphs 35 to 39, wherein the subject has an inherited or genetic myopathy, which is optionally selected from muscular dystrophy (e.g., Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD), myotonic dystrophy (e.g., DM1 and DM2), facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, and limb girdle muscular dystrophy), congenital myopathy (e.g., nemaline myopathy and central core myopathy), metabolic myopathy (e.g., acid maltase or acid alpha- 1, 4-glucosidase deficiency (Pompe’s disease), glycogen storage disorders 3-11, carnitine deficiency, fatty acid oxidation defects, and carnitine palmitoyl transferase deficiency), and mitochondrial myopathy.

[0370] 41. The method of any one of paragraphs 35 to 40, wherein the subject has or is at risk of developing an autoimmune or inflammatory myopathy (i.e., myositis), which is optionally selected from dermatomyositis, polymyositis, inclusion body myositis, and juvenile myositis, or another autoimmune disorder (e.g., myasthenia gravis).

[0371] 42. The method of any one of paragraphs 35 to 41, wherein the subject has or is at risk of developing an infection, which optionally is selected from an infection by a virus (e.g., HIV, influenza virus, and Epstein-Barr virus), a bacterium (e.g., pyomyositis, S. aureaus, and streptococci), a spirochete (e.g., Lyme disease), and a parasite (e.g., trichinosis).

[0372] 43. The method of any one of paragraphs 35 to 42, wherein the subject has or is at risk of exposure to a toxin, which optionally is selected from a medication (e.g., a cholesterol-lowering medication (e.g., a statin), propofol, amiodaron, colchicine, chloroquine, an antiviral, a protease inhibitor, omeprazole, and tryptophan), alcohol, and toluene.

[0373] 44. The method of any one of paragraphs 35 to 43, wherein the subject has or is at risk of developing a systemic disease, which optionally is selected from an endocrine disorder (e.g., thyroid, parathyroid, pituitary, and adrenal disorder), systemic inflammatory disease (e.g., systemic lupus erythematosus, rheumatoid arthritis, scleroderma, Sjogren’s syndrome, mixed connective disease, and sarcoidosis), electrolyte imbalance, critical illness myopathy, and amyloid myopathy.

[0374] 45. The method of any one of paragraphs 35 to 44, wherein the subject has or is at risk of developing a neurological disease or condition, which optionally is selected from amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), spinal muscular atrophy (SMA), Guillain-Barre syndrome, carpel tunnel syndrome, Charcot-Marie-Tooth disease, Parkinson’s disease, Lewy body disease, polio, spinal cord injury, peripheral nerve injury, stroke, and nerve damage caused by diabetes, toxins, or alcohol.

[0375] 46. The method of any one of paragraphs 35 to 45, wherein the subject is or is expected to be present in an environment with decreased or no gravity, e.g., during a space flight or being at a space station or the like.

[0376] 47. The method of any one of paragraphs 35 to 46, wherein the subject has or experiences prolonged inactivity (e.g., is bedridden, has a seated job or a sedentary lifestyle, or cannot move limbs due to stroke or other brain disease), burn, long-term corticosteroid therapy, malnutrition, anorexia, cachexia, osteoarthritis, advanced age, cancer, chronic kidney disease, heart failure, or chronic obstructive pulmonary disease.

[0377] 48. The method of any one of paragraphs 35 to 47, wherein the subject has or is at risk of developing one or more symptom selected from muscle loss, movement issues, balance problems, trouble walking, trouble using arms or legs, contractures, muscle rigidity, muscle twitching, fasciculations, muscle cramps, numbness, tingling, painful sensations, breathing problems, curved spine, heart problems, swallowing problems (which may lead to, e.g., nutritional problems and aspiration pneumonia), reduced muscle mass (e.g., in general or in one limb), weakness (e.g., in general or in one limb), pain, discomfort, one arm or leg being smaller than the other, weakness in one arm or leg, numbness or tingling in an arm or leg, trouble walking, balance problems, facia weakness, gradual memory loss, reduced muscle strength, impaired ability to perform physical activities, decrease in muscle size, cramps, stiffness, spasms, fatigue, motor delay, respiratory impairment, and bulbar muscle dysfunction.

[0378] 49. The method of any one of paragraphs 35 to 48, wherein the subject has or is at risk of developing insulin resistance, type II diabetes, elevated A1C levels, pre-diabetes, metabolic syndrome, hypertension, hyperglycemia, hypercholesterolemia, high triglycerides, overweight, or obesity.

[0379] The disclosure is not limited to the exemplary embodiments and applications presented herein or to the manner in which the exemplary embodiments and applications operate or are described herein. Other embodiments are within the scope of the claims.

Claims

What is claimed is:

1. A peptide immunogen construct comprising a myostatin B cell epitope, a heterologous T cell (Th) epitope, and an optional heterologous spacer, wherein the B cell epitope is covalently linked to the Th epitope directly or through the optional heterologous spacer; wherein the myostatin B cell epitope comprises about 7 to about 30 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2 or a variant thereof.

2. The peptide immunogen construct of claim 1, wherein the myostatin B cell epitope comprises a variant of an 7-30 amino acid fragment of SEQ ID NO: 1 or SEQ ID NO: 2, and the variant comprises 1, 2, 3, 4, 5, or 6 amino acid substitutions, deletions, or insertions as compared to the corresponding sequence of the 7-30 amino acid fragment of SEQ ID NO: 1 or SEQ ID NO: 2.

3. The peptide immunogen construct of claim 1, wherein the myostatin B cell epitope comprises up to 3, or up to 6, amino acid substitutions as compared to the corresponding sequence of the 7-30 amino acid fragment of SEQ ID NO: 1 or SEQ ID NO: 2.

4. The peptide immunogen construct of any one of claims 1-3, wherein the myostatin B cell epitope comprises one or more (optionally 1, 2, or 3) amino acid substitutions as compared to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 to facilitate or enable cyclization of the sequence comprising the myostatin B cell epitope, optionally wherein the myostatin B cell epitope is cyclized.

5. The peptide immunogen construct of any one of claims 1-4, wherein the myostatin B cell epitope comprises or consists of an amino acid sequence within loop 1, loop 2, loop 3, or loop 4 of myostatin, or a variant thereof.

6. The peptide immunogen construct of any one of claims 1-5, wherein the myostatin B cell epitope comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 3-10, 95-109, 145-164, and 166 or a variant thereof.

7. The peptide immunogen construct of claim 6, wherein the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 5, 97, 98, 101, 105, 146, 147, 151-155, 157, 159, 161, 163, 164, or 166.

8. The peptide immunogen construct of claim 6, wherein the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 5, 97, 98, 101, or 105.

9. The peptide immunogen construct of claim 6, wherein the myostatin B cell epitope comprises or consists of the amino acid sequence of SEQ ID NOs: 146, 147, 151, 152, 154, 155, 157, 159, 161, 163, 164, or 166.

10. The peptide immunogen construct of any one of claims 1-9, wherein the Th epitope is selected from any one of SEQ ID NOs: 19-47.

11. The peptide immunogen construct of claim 10, wherein the Th epitope is of SEQ ID NO: 30, 32, or 33.

12. The peptide immunogen construct of any one of claims 1-11, wherein the myostatin B cell epitope and the Th epitope are linked to one another by the heterologous spacer.

13. The peptide immunogen construct of claim 12, wherein the heterologous spacer comprises or consists of one or more amino acids, which are optionally selected from: Lys-, Gly-, Lys-Lys-Lys-, Lys-Lys-Lys-e-Lys, (a, s-N)Lys, s-N-Lys-Lys-Lys-Lys (SEQ ID NO: 91), andPro-Pro-Xaa-Pro-Xaa-Pro, wherein Xaa is any amino acid or is aspartic acid (SEQ ID NO: 90).

14. The peptide immunogen construct of claim 12, wherein the heterologous spacer is Lys-Lys-Lys-, Lys-Lys-Lys-s-Lys, (a, s-N)Lys, or s-N-Lys-Lys-Lys-Lys (SEQ ID NO: 91).

15. The peptide immunogen construct of any one of claims 1-14, wherein:(a) the myostatin B cell epitope is located N-terminal to the Th epitope;(b) the Th epitope is located N-terminal to the myostatin B cell epitope;(c) the myostatin B cell epitope is flanked with a Th epitope on both N-terminal and C-terminal ends;(d) the myostatin B cell epitope is acetylated on the N-terminus or cyclized using the N-terminus amino group, and have an amide on the C-terminus;(e) any of (a)-(d) wherein the myostatin B cell epitope(s) is linked to the Th epitope(s) by direct covalent linkage; or(f) any one of (a)-(d) wherein the myostatin B cell epitope(s) is linked to the Th epitope(s) by the heterologous spacer.

16. The peptide immunogen construct of any one of claims 1-15, wherein the peptide immunogen construct is of following formula:(Th)m-(A)n-(myostatin B cell epitope)por(myostatin B cell epitope)p---(A)n-(Th)mor(Th)m-(A)n---(myostatin B cell epitope)P---(A)!!-(Th)mwhereinTh is the T helper epitope;A is the heterologous spacer, which optionally comprises an ammo acid; m is from 1 to about 4;p is from 1 to about 4;andn is from 0 to about 10, wherein optionally the C-terminus of the peptide immunogen construct comprises a-COOH or a-CONl-fc of an amino acid; optionally wherein m is 1, p is 1 or 2. and n is 1.

17. The peptide immunogen construct of claim 1, wherein the peptide immunogen construct comprises or consists of the sequence of any one of SEQ ID NOs: 48-71, 120-134, 167-186 and 188 or a variant thereof.

18. The peptide immunogen construct of claim 17, wherein the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 54, 55, 56, 122, 123, 126, 130, 168, 169, 173, 174, 175, 176, 177, 178, 179, 181, 183, 185, or 186.

19. The peptide immunogen construct of claim 17, wherein the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 54, 55, 56, 122, 123, 126 or 130.

20. The peptide immunogen construct of claim 17, wherein the peptide immunogen construct comprises or consists of the sequence of SEQ ID NO: 168, 169, 173, 174, 176, 177, 179, 181, 183, 185, or 186.

21. A peptide immunogen construct comprising an activin A B cell epitope, a heterologous T cell (Th) epitope, and an optional heterologous spacer, wherein the B cell epitope is covalently linked to the Th epitope directly or through the optional heterologous spacer, wherein the activin A B cell epitope comprises about 7 to about 30 contiguous amino acids of SEQ ID NO: 12 or a variant thereof.

22. The peptide immunogen construct of claim 21, wherein the activin A B cell epitope comprises a variant of a 7-30 amino acid fragment of SEQ ID NO: 12, and the variant comprises 1, 2, 3, 4, 5, or 6 amino acid substitutions, deletions, or insertions as compared to the corresponding sequence of the 7-30 amino acid fragment of SEQ ID NO: 12.

23. The peptide immunogen construct of claim 21, wherein the activin A B cell epitope comprises up to 3, or up to 6, amino acid substitutions as compared to the corresponding sequence of the 7-30 amino acid fragment of SEQ ID NO: 12.

24. The peptide immunogen construct of any one of claims 21-23, wherein the activin A B cell epitope comprises one or more (optionally 1, 2, or 3) amino acid substitutions as compared to the sequence of SEQ ID NO: 12 to facilitate or enable cyclization of the sequence comprising the activin A B cell epitope, optionally wherein the activin A B cell epitope is cyclized.

25. The peptide immunogen construct of any one of claims 21-24, wherein the activin A B cell epitope comprises or consists of an amino acid sequence within loop 1, loop 2, loop 3, or loop 4 of activin A, or a variant thereof.

26. The peptide immunogen construct of any one of claims 21-25, wherein the activin A B cell epitope comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 13-18, 110-119, 187, and 189 or a variant thereof.

27. The peptide immunogen construct of claim 26, wherein the activin A B cell epitope comprises or consists of the amino acid sequence of SEQ ID NO: 14, 15, 17, 111, 116, 118, 119, or 187.

28. The peptide immunogen construct of any one of claims 21-27, wherein the Th epitope is selected from any one of SEQ ID NOs: 19-47.

29. The peptide immunogen construct of claim 28, wherein the Th epitope comprises or consists of the sequence of SEQ ID NO: 30, 32, or 33.

30. The peptide immunogen construct of any one of claims claim 21-29, wherein the activin A B cell epitope and the Th epitope are linked to one another by the heterologous spacer.

31. The peptide immunogen construct of claim 30, wherein the heterologous spacer comprises or consists of one or more amino acids, which are optionally selected from: Lys-, Gly-, Lys-Lys-Lys-, Lys-Lys-Lys-e-Lys, (a, s-N)Lys, s-N-Lys-Lys-Lys-Lys (SEQ ID NO: 91), andPro-Pro-Xaa-Pro-Xaa-Pro, wherein Xaa is any amino acid or is aspartic acid (SEQ ID NO: 90).

32. The peptide immunogen construct of claim 31, wherein the heterologous spacer is Lys-Lys-Lys-, Lys-Lys-Lys-s-Lys, (a, s-N)Lys, or s-N-Lys-Lys-Lys-Lys (SEQ ID NO: 91).

33. The peptide immunogen construct of any one of claims 21-32, wherein:(a) the activin A B cell epitope is located N-terminal to the Th epitope;(b) the Th epitope is located N-terminal to the activin A B cell epitope;(c) the activin A B cell epitope is flanked with a Th epitope on both N-terminal and C-terminal ends;(d) the activin A B cell epitope is acetylated on the N-terminus or cyclized using the N-terminus amino group, and have an amide on the C-terminus;(e) any of (a)-(d) wherein the activin A B cell epitope(s) is linked to the Th epitope(s) by direct covalent linkage; or(f) any one of (a)-(d) wherein the activin A B cell epitope(s) is linked to the Th epitope(s) by the heterologous spacer.

34. The peptide immunogen construct of any one of claims 21-33, wherein the peptide immunogen construct is of following formula:(Th)m-(A)n~(activin A B cell epitope)por(activin A B cell epitope)p--(A)„-(Th)mor(Th)m-(A)n~(activin A B cell epitope)p~-(A)n--(Th)mwhereinTh is the T helper epitope;A is the heterologous spacer, which optionally comprises an ammo acid; m is from 1 to about 4;p is from 1 to about 4;andn is from 0 to about 10, wherein optionally the C-terminus of the peptide immunogen construct comprises a-COOH or a-CONl-fc of an amino acid; optionally m is 1, p is 1 or 2, and n is 1.

35. The peptide immunogen construct of claim 21, wherein the peptide immunogen construct comprises or consists of the sequence of any one of SEQ ID NOs: 72-89, 135-144, and 190-191 or a variant thereof.

36. The peptide immunogen construct of claim 20, wherein the peptide immunogen construct comprises or consists of the sequence of SEQ ID NOs: 75, 76, 78, 79, 85, 136, 138, 139, 140, 141, 142, 143, 144, or 190; optionally wherein the construct comprises or consists of SEQ ID NOs: 75, 76, 78, 79, 85, 136, 141, 143, 144 or 190.

37. A peptide immunogen construct comprising:(i) two or more B cell epitopes selected from:a myostatin B cell epitope comprising about 7 to about 30 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2 or a variant thereof, andan activin A B cell epitope comprising about 7 to about 30 contiguous amino acids of SEQ ID NO: 12 or a variant thereof;(iii) a heterologous T cell (Th) epitope, and(iv) optionally, one or more heterologous spacers and / or linkers,wherein the myostatin and activin A B cell epitopes are covalently linked to the Th epitope directly or through the one or more heterologous spacers and / or linkers.

38. The peptide immunogen construct of claim 37, wherein the two or more B cell epitopes are two to four B cell epitopes, optionally wherein the two or more B cell epitopes are two or three B cell epitopes.

39. The peptide immunogen construct of claim 37 or 38, wherein the two or more B cell epitopes are two or more of the myostatin B cell epitopes, optionally wherein the two or more B cell epitopes do not comprise an activin A B cell epitope.

40. The peptide immunogen construct of claim 37 or 38, wherein the two or more B cell epitopes are two or more of the activin A B cell epitopes, optionally wherein the two or more B cell epitopes do not comprise a myostatin B cell epitope.

41. The peptide immunogen construct of claim 37 or 38, wherein the two or more B cell epitopes comprise at least one myostatin B cell epitope and at least one activin A B cell epitope.

42. A peptide immunogen construct comprising:(i) a myostatin B cell epitope, wherein the myostatin B cell epitope comprises about 7 to about 30 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2 or a variant thereof;(ii) an activin A B cell epitope, wherein the activin A B cell epitope comprises about 7 to about 30 contiguous amino acids of SEQ ID NO: 12 or a variant thereof;(iii) a heterologous T cell (Th) epitope, and(iv) one or more heterologous spacers and / or linkers,wherein the myostatin and activin A B cell epitopes are covalently linked to the Th epitope through the one or more heterologous spacers and / or linkers.

43. The peptide immunogen construct of any one of claims 37-42, wherein the one or more heterologous spacers and / or linkers comprise a branched amino acid.

44. The peptide immunogen construct of claim 43, wherein the branched amino acid is a diamino acid and the B cell epitopes are attached to the N-terminus of the Th epitope, optionally wherein the diamino acid is lysine.

45. The peptide immunogen construct of claim 43, wherein the branched amino acid is a dicarboxylic acid and the B cell epitopes are attached to the C-terminus of the Th epitope, optionally wherein the dicarboxylic acid is glutamic acid.

46. The peptide immunogen construct of any one of claims 43-45, wherein the branched amino acid is linked to each of the B cell epitopes by a linker.

47. The peptide immunogen construct of claim 46, wherein the linker is an amino acid peg derivative; optionally wherein the peg derivative is 8-amino-3,6-dioxaoctanoic acid or 2-[2-(2-aminoethoxy)ethoxy]acetic acid (“AeeA”).

48. The peptide immunogen construct of any one of claims 43-47, wherein the branched amino acid is covalently linked to the Th epitope by a heterologous spacer.

49. The peptide immunogen construct of claim 48, wherein the heterologous spacer is a polylysine spacer, optionally comprising or consisting of 3 or 4 lysines.

50. The peptide immunogen construct of claim 49, wherein the heterologous spacer is Lys-Lys-Lys-, Lys-Lys-Lys-s-Lys, (a, s-N)Lys, or s-N-Lys-Lys-Lys-Lys (SEQ ID NO: 91).

51. The peptide immunogen construct of any one of claims 37-39 and 41-50, wherein the myostatin B cell epitope comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 3-10, 95-109, 145-164, and 166 or a variant thereof; optionally wherein the myostatin B cell epitope is of the amino acid sequence of SEQ ID NO: 5, 97, 98, 101, 105, 146, 147, 151-155, 157, 159, 161, 163, 164, or 166.

52. The peptide immunogen construct of any one of claims 37-38 and 40-50, wherein the activin A B cell epitope comprises or consists of the amino acid sequence of any one of SEQ IDNOs: 13-18, 110-119, 187, and 189 or a variant thereof; optionally wherein the activin AB cell epitope is of the amino acid sequence of SEQ ID NO: 14, 15, 17, 111, 116, 118, 119, or 187.

53. The peptide immunogen construct of any one of claims 37-52, wherein the myostatin B cell epitope and / or activin A B cell epitope is cyclized.

54. The peptide immunogen construct of any one of claims 37-53, wherein the myostatin B cell epitope is of the amino acid sequence of SEQ ID NO: 97 or a variant thereof, and / or the activin A B cell epitope is of the amino acid sequence of SEQ ID NO: 116 or a variant thereof.

55. The peptide immunogen construct of any one of claims 37-54, wherein the Th epitope is selected from any one of SEQ ID NOs: 19-47, optionally wherein the Th epitope is of the amino acid sequence of SEQ ID NO: 30, 32, or 33, optionally wherein the Th epitope is of the amino acid sequence of SEQ ID NO: 32.

56. The peptide immunogen construct of claim 37 or 42, which comprises or consists of Ac-STVINHYRMRGHSPFANLKSSAeeA(Ac-QKYPHTHLVHQANPRGSAGPAeeA)K-KKKrK-Ubithl (SEQ ID NO: 165).

57. A nucleic acid molecule encoding a peptide immunogen construct of any one of claims 1-56.

58. A vector comprising the nucleic acid molecule of claim 57.

59. An isolated antibody or an epitope-binding fragment thereof that specifically binds to and / or elicited by the myostatin B cell epitope of the peptide immunogen construct of any one of claims 1 -20, 37-39, and 41-56, and / or that specifically binds to and / or elicited by the activin A B cell epitope of the peptide immunogen construct of any one of claims 21-36, 37-38, and 40-56.

60. A composition comprising one or more of: (a) one or more peptide immunogen constructs of any one of claims 1 to 56, a nucleic acid of claim 57, a vector of claim 58, an isolated antibody of claim 59, or a peptide immunogen construct of any one of claims 1 to 20 and a peptideimmunogen construct of any one of claims 21 to 36, and (b) a pharmaceutically acceptable carrier, diluent, or adjuvant.

61. The composition of claim 60, wherein the composition comprises an adjuvant that is a mineral salt of aluminum, which optionally is selected from Al(OH)3 and AIPO4.

62. The composition of claim 60 or 61, wherein the composition comprises a CpG oligonucleotide, optionally a GpGl oligonucleotide.

63. A method of treating or preventing the development of muscle wasting in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct of any one of claims 1 to 56, a nucleic acid molecule of claim 57, a vector of claim 58, an isolated antibody of claim 59, or a composition of any one of claims 60-62.

64. A method of treating, preventing, reducing, inhibiting, or slowing the development of one or more symptom of a muscle wasting disease in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct of any one of claims 1 to 56, a nucleic acid molecule of claim 57, a vector of claim 58, an isolated antibody 59, or a composition of any one of claims 60-62.

65. A method of inducing an immune response to myostatin and / or activin A in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct of any one of claims 1 to 56, a nucleic acid molecule of claim 57, a vector of claim 58, an isolated antibody of claim 59, or a composition of any one of claims 60-62.

66. A method of treating or preventing insulin resistance in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct of any one of claims 1 to 56, a nucleic acid molecule of claim 57, a vector of claim 58, an isolated antibody of claim 59, or a composition of any one of claims 60-62.

67. The method of claim 63 or 64, wherein the subject has or is at risk of developing muscle wasting due to an inherited or genetic cause, inflammation, infection, injury, systemic disease, toxin, or other environmental exposure.

68. The method of claim 63 or 64, wherein the subject has or is at risk of developing muscle wasting due to an inherited or genetic myopathy, which is optionally selected from muscular dystrophy (e.g., Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD), myotonic dystrophy (e.g., DM1 and DM2), facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, and limb girdle muscular dystrophy), congenital myopathy (e.g., nemaline myopathy and central core myopathy), metabolic myopathy (e.g., acid maltase or acid alpha- 1,4-glucosidase deficiency (Pompe’s disease), glycogen storage disorders 3-11, carnitine deficiency, fatty acid oxidation defects, and carnitine palmitoyl transferase deficiency), and mitochondrial myopathy.

69. The method of claim 63 or 64, wherein the subject has or is at risk of developing muscle wasting due to an autoimmune or inflammatory myopathy (i.e., myositis), which is optionally selected from dermatomyositis, polymyositis, inclusion body myositis, and juvenile myositis, or another autoimmune disorder (e.g., myasthenia gravis).

70. The method of claim 63 or 64, wherein the subject has or is at risk of developing muscle wasting due to an infection, which optionally is selected from an infection by a virus (e.g., HIV, influenza virus, and Epstein-Barr virus), a bacterium (e.g., pyomyositis, S. aureus, and streptococci), a spirochete (e.g., Lyme disease), and a parasite (e.g., trichinosis).

71. The method of claim 63 or 64, wherein the subject has or is at risk of developing muscle wasting due to exposure to a toxin, which optionally is selected from a medication (e.g., a cholesterol-lowering medication (e.g., a statin), propofol, amiodaron, colchicine, chloroquine, an antiviral, a protease inhibitor, omeprazole, and tryptophan), alcohol, and toluene.

72. The method of claim 63 or 64, wherein the subject has or is at risk of developing muscle wasting due to a systemic disease, which optionally is selected from an endocrine disorder (e.g., thyroid, parathyroid, pituitary, and adrenal disorder), systemic inflammatory disease (e.g., systemiclupus erythematosus, rheumatoid arthritis, scleroderma, Sjogren’s syndrome, mixed connective disease, and sarcoidosis), electrolyte imbalance, critical illness myopathy, and amyloid myopathy.

73. The method of claim 63 or 64, wherein the subject has or is at risk of developing muscle wasting due to a neurological disease or condition, which optionally is selected from amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), spinal muscular atrophy (SMA), Guillain-Barre syndrome, carpel tunnel syndrome, Charcot-Marie-Tooth disease, Parkinson’s disease, Lewy body disease, polio, spinal cord injury, peripheral nerve injury, stroke, and nerve damage caused by diabetes, toxins, or alcohol.

74. The method of claim 63 or 64, wherein the subject has or is at risk of developing muscle wasting due to presence in an environment with decreased or no gravity, e.g., during a space flight or being at a space station or the like.

75. The method of claim 63 or 64, wherein the subject has or is at risk of developing muscle wasting due to prolonged inactivity (e.g., is bedridden, has a seated job or a sedentary lifestyle, or cannot move limbs due to stroke or other brain disease), burn, long-term corticosteroid therapy, malnutrition, anorexia, cachexia, osteoarthritis, advanced age, cancer, chronic kidney disease, heart failure, or chronic obstructive pulmonary disease.

76. The method of claim 63 or 64, wherein the subject has or is at risk of developing one or more symptom selected from muscle loss, movement issues, balance problems, trouble walking, trouble using arms or legs, contractures, muscle rigidity, muscle twitching, fasciculations, muscle cramps, numbness, tingling, painful sensations, breathing problems, curved spine, heart problems, swallowing problems (which may lead to, e.g., nutritional problems and aspiration pneumonia), reduced muscle mass (e.g., in general or in one limb), weakness (e.g., in general or in one limb), pain, discomfort, one arm or leg being smaller than the other, weakness in one arm or leg, numbness or tingling in an arm or leg, trouble walking, balance problems, facia weakness, gradual memory loss, reduced muscle strength, impaired ability to perform physical activities, decrease in muscle size, cramps, stiffness, spasms, fatigue, motor delay, respiratory impairment, and bulbar muscle dysfunction.

77. The method of claim 66, wherein the subject has or is at risk of developing insulin resistance, type II diabetes, elevated A1C levels, pre-diabetes, metabolic syndrome, hypertension, hyperglycemia, hypercholesterolemia, high triglycerides, overweight, or obesity.

78. A method of maintaining or improving muscle physiology in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct of any one of claims 1 to 56, a nucleic acid molecule of claim 57, a vector of claim 58, an isolated antibody of claim 59, or a composition of any one of claims 60-62.

79. The method of claim 78, wherein the maintaining or improving muscle physiology is maintaining or increasing muscle mass, muscle strength, muscle tone, and / or muscle endurance.

80. A method of treating or preventing muscular or musculoskeletal injury in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct of any one of claims 1 to 56, a nucleic acid molecule of claim 57, a vector of claim 58, an isolated antibody of claim 59, or a composition of any one of claims 60-62.

81. The method of claim 80, wherein the muscular or musculoskeletal injury is muscle tear or muscle strain.

82. A method of treating or preventing a disorder associated with muscle loss or insufficient muscle growth in a subject, the method comprising administering to the subject an effective amount of an immunotherapy targeting myostatin and / or activin A, wherein the immunotherapy is selected from a peptide immunogen construct of any one of claims 1 to 56, a nucleic acid molecule of claim 57, a vector of claim 58, an isolated antibody of claim 59, or a composition of any one of claims 60-62.

83. The method of claim 82, wherein the subject has muscle atrophy.

84. The method of claims 82 or 83, wherein the disorder is cachexia or muscle loss associated with a cancer or tumor.

85. The method of any one of claims 63-84, wherein the subject is a human.

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