ADP-ribosylation factor compositions and methods of use thereof

Abstract

Described herein are therapeutic compositions comprising a homologous ADP-ribosylation factor (ARF) protein sequence useful for the treatment of obesity, obesity-related disorders, and lipedema, and also for achieving weight loss. Described herein are methods for treatment of obesity, an obesity-related disorder, or lipedema comprising administering a composition comprising an ARF protein or an engineered ARF1 protein to a subject in need thereof. Also described herein are methods to prevent reversion of weight loss achieved from incretin therapy following cessation of incretin treatment.

Description

ADP-RIBOSYLATION FACTOR COMPOSITIONS AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 604,120, filed on November 29, 2023 which is incorporated by reference herein in its entirety.BACKGROUND

[0002] Obesity and excessive weight remain a health concern in large segments of the population. An individual that is overweight or obese is subjected to increased risk for a variety of conditions including hypertension, type 2 diabetes, coronary heart disease, stroke, gallbladder disease, osteoarthritis, sleep apnea, many types of cancer, body pain, and obesity-related mortality. Additionally, an individual that is overweight or obese may subjected to reduced physical functioning and reduced accomplishment of activities of daily living. Obesity and excessive weight are conditions in need of improved treatments and therapeutic interventions to improve quality of life and reduce risk of related health disorders. Lipedema, characterized by an abnormal fat buildup typically on both sides of the lower body, also remains a health concern in numerous individuals. Lipedema often occurs in conjunction with other conditions such as obesity, lymphedema, venous diseases such as chronic venous insufficiency, hypermobility disorders, and hypothyroidism, significantly impacting health and quality of life. Improved treatments for lipedema are needed.SUMMARY

[0003] In certain aspects, disclosed herein are engineered polypeptides, the engineered polypeptides comprising a homologous ADP-ribosylation factor 1 (ARF1) sequence, and a covalently-coupled protein trafficking domain sequence. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 1. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 93% identical to SEQ ID NO: 23. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 95% identical to SEQ ID NO: 1. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 1-23. In some embodiments, the homologous ARF1 sequence comprises one, two, three, four, five, six, seven, eight, or nine amino acid substitutions relative to an amino acid sequence selected from SEQ ID NOs: 1-23. In some embodiments, the homologous ARF1 sequence comprises one or more deletions of one, two, or three amino acids relative to an amino acid sequence selected from SEQ ID NOs: 1-23. In some embodiments, the homologous ARF1 sequence comprises one or more insertions of one, two, three, four, five, six, seven, eight, or nine amino acids relative to an aminoacid sequence selected from SEQ ID NOs: 1-23. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence selected from SEQ ID NOs: 1-23. In some embodiments, the covalently-coupled protein trafficking domain sequence is located N-terminal to the homologous ARF1 sequence. In some embodiments, the covalently-coupled protein trafficking domain sequence is located C-terminal to the homologous ARF1 sequence. In some embodiments, the protein trafficking domain sequence comprises a signal peptide sequence. In some embodiments, the signal peptide sequence comprises an amino acid sequence selected from SEQ ID NOs: 77-86. In some embodiments, the protein trafficking domain sequence comprises a site for myristoylation. In some embodiments, the site for myristoylation is located at or near the N- terminus of the engineered polypeptide. In some embodiments, the site for myristoylation comprises a glycine residue. In some embodiments, the site for myristoylation comprises the peptide sequence MGSS. In some embodiments, the engineered ARF1 polypeptide comprises a peptide tag comprising a peptide length of 4 to 22 amino acids wherein the peptide tag is inserted within the engineered polypeptide. In some embodiments, the peptide tag is positioned between the homologous ARF1 sequence and the covalently-coupled protein trafficking domain sequence. In some embodiments, the engineered ARF1 polypeptide comprises a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is positioned at the N-terminus of the engineered polypeptide. In some embodiments, the engineered ARF1 polypeptide comprises a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is positioned at the C-terminus of the engineered polypeptide. In some embodiments, the protein trafficking domain sequence comprises an Annexin A10 peptide sequence. In some embodiments, the Annexin A10 peptide sequence comprises an amino acid sequence at least about 95% identical to SEQ ID NO: 53. In some embodiments, the Annexin A10 peptide sequence comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the engineered ARF1 polypeptide comprises a cleavage site. In some embodiments, the cleavage site is inserted between the homologous ARF1 sequence and the covalently-coupled protein trafficking domain sequence. In some embodiments, the cleavage site is inserted adjacent to the peptide tag. In some embodiments, the cleavage site comprises a TEV protease recognition motif. In some embodiments, the TEV protease recognition motif comprises the amino acid sequence of SEQ ID NO: 26.

[0004] In an aspect described herein, are engineered polypeptides comprising: a homologous ADP-ribosylation factor 1 (ARF1) sequence, and a half-life extending moiety. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 1. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 93% identical to SEQ ID NO: 23. In some embodiments, thehomologous ARF1 sequence comprises an amino acid sequence at least about 95% identical to SEQ ID NO: 1. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 1-23. In some embodiments, the homologous ARF1 sequence comprises one, two, three, four, five, six, seven, eight, or nine amino acid substitutions relative to an amino acid sequence selected from SEQ ID NOs: 1-23. In some embodiments, the homologous ARF1 sequence comprises one or more deletions of one, two, or three amino acids relative to an amino acid sequence selected from SEQ ID NOs: 1-23. In some embodiments, the homologous ARF1 sequence comprises one or more insertions of one, two, three, four, five, six, seven, eight, or nine amino acids relative to an amino acid sequence selected from SEQ ID NOs: 1-23. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence selected from SEQ ID NOs: 1-23. In some embodiments, the half-life extending moiety is located N-terminal to the homologous ARF1 sequence. In some embodiments, the half-life extending moiety is located C-terminal to the homologous ARF1 sequence. In some embodiments, the half-life extending moiety comprises a fatty acid ester. In some embodiments, the half-life extending moiety comprises a heterologous polypeptide sequence. In some embodiments, the heterologous polypeptide sequence comprises a human serum albumin (HSA) sequence. In some embodiments, the HSA sequence is at least about 95% identical to SEQ ID NO: 51. In some embodiments, the HSA sequence is at least about 96%, 97%, 98%, or 99% identical to SEQ ID NO: 51. In some embodiments, the HSA sequence comprises an amino acid sequence identical to SEQ ID NO: 51. In some embodiments, the heterologous polypeptide sequence comprises an Fc portion of a human immunoglobulin. In some embodiments, the Fc portion of the human immunoglobulin comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 50. In some embodiments, the Fc portion of the human immunoglobulin comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, the engineered polypeptide comprises a protein trafficking domain sequence. In some embodiments, the protein trafficking domain sequence comprises a signal peptide sequence. In some embodiments, the signal peptide sequence comprises an amino acid sequence selected from SEQ ID NOs: 77-86. In some embodiments, the protein trafficking domain sequence comprises a site for myristoylation. In some embodiments, the site for myristoylation is located at or near the N-terminus of the engineered polypeptide. In some embodiments, the site for myristoylation comprises a glycine residue. In some embodiments, the site for myristoylation comprises the peptide sequence MGSS. In some embodiments, the engineered protein comprises a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is inserted within the engineered polypeptide. In some embodiments, thepeptide tag is positioned between the homologous ARF1 sequence and the protein trafficking domain sequence. In some embodiments, the engineered protein comprises a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is positioned at the N- terminus of the engineered polypeptide. In some embodiments, the engineered protein comprises a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is positioned at the C-terminus of the engineered polypeptide. In some embodiments, the engineered protein comprises a cleavage site. In some embodiments, the cleavage site is inserted between the homologous ARF1 sequence and protein trafficking domain sequence. In some embodiments, the cleavage site is inserted adjacent to the peptide tag. In some embodiments, the cleavage site comprises a TEV protease recognition motif. In some embodiments, the TEV protease recognition motif comprises the amino acid sequence of SEQ ID NO: 26.

[0005] In an aspect described herein are methods of treating a subject having obesity, the methods comprising administering an effective amount of an ADP-ribosylation factor 1 (ARF1) protein an engineered ARF1 protein to the subject, In some embodiments, the ARF1 protein or the engineered ARF1 protein comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the ARF1 protein or the engineered ARF1 protein is administered systemically to the subject. In some embodiments, the ARF1 protein or the engineered ARF1 protein is administered by parenteral administration. In some embodiments, parenteral administration comprises injection of the ARF1 protein or the engineered ARF1 protein in the subject. In some embodiments, the injection is intravenous injection. In some embodiments, the injection is intramuscular injection. In some embodiments, the injection is intradermal injection. In some embodiments, the injection is subcutaneous injection. In some embodiments, parenteral administration comprises infusion of the ARF1 protein or the engineered ARF1 protein in the subject.

[0006] In an aspect described herein are methods of treating a subject having lipedema, the methods comprising administering an effective amount of an ADP-ribosylation factor 1 (ARF1) protein or an engineered ARF1 protein to the subject, In some embodiments, the ARF1 protein or the engineered ARF1 protein comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the ARF1 protein or the engineered ARF1 protein is administered systemically to the subject. In some embodiments, the ARF1 protein or engineered ARF1 protein is administered by parenteral administration. In some embodiments, parenteral administration comprises injection of the ARF1 protein or the engineered ARF1 protein in the subject. In some embodiments, the injection is intravenous injection. In some embodiments, the injection is intramuscular injection. In someembodiments, the injection is intradermal injection. In some embodiments, the injection is subcutaneous injection. In some embodiments, parenteral administration comprises infusion of the ARF1 protein or the engineered ARF1 protein in the subject.

[0007] In an aspect described herein are methods of treating a subject having obesity, the methods comprising administering an effective amount of any one of the engineered ARF polypeptides described to the subject. In an aspect described herein are methods of treating a subject having obesity, the methods comprising administering an effective amount of any one of the engineered ARF1 polypeptides described to the subject. In some embodiments, the ARF1 protein is administered systemically to the subject. In some embodiments, the ARF1 protein is administered by parenteral administration. In some embodiments, parenteral administration comprises injection of the ARF1 protein in the subject. In some embodiments, the injection is intravenous injection. In some embodiments, the injection is intramuscular injection. In some embodiments, the injection is intradermal injection. In some embodiments, the injection is subcutaneous injection. In some embodiments, parenteral administration comprises infusion of the ARF1 protein in the subject. In some embodiments, the subject is treated for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 17 days, 21 days, 24 days, 28 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. In some embodiments, the subject is treated for at least 3 weeks. In some embodiments, the subject achieves a percentage of weight loss of at least 2.5%, 5%, 7.5%, 10%, 12.5%, or 15%. In some embodiments, the subject maintains a percentage of lean muscle mass of at least 85%, 90%, 95%, 98%, 99%, or 100% of following a period of treatment. In some embodiments, fasting blood glucose level in the subject is reduced following the administering. In some embodiments, insulin sensitivity in the subject is increased following the administering. In some embodiments, serum insulin level is decreased in the subject following the administering. In some embodiments, an extent of lipolysis is increased in the subject following the administering. In some embodiments, serum levels of triglycerides are decreased in the subject following the administering. In some embodiments, fatty acid update in the subject is not significantly changed following the administering. In some embodiments, a rate of triglyceride hydrolysis is increased in a plurality of adipocytes from the subject following the administering. In some embodiments, the increased rate of triglyceride hydrolysis produces elevated levels of glycerol and fatty acids in a plurality of adipocytes of the subject following the administering.

[0008] In an aspect described herein are methods of weight loss, the methods comprising administering an effective amount of an ADP-ribosylation factor 1 (ARF1) protein to a subject, In some embodiments, the ARF1 protein comprises an amino acid sequence at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, and In some embodiments, the subject achieves a weight loss of at least about 1% of body weight at a time point following the administering. In some embodiments, the time point following the administering is at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 17 days, 21 days, 24 days, 28 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. In some embodiments, the time point following the administering is less than about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 17 days, 21 days, 24 days, 28 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. In some embodiments, the subject is overweight at the start of the administering. In some embodiments, the subject is clinically obese at the start of the administering. In some embodiments, the subject has been diagnosed with an obesity-related disorder. In some embodiments, the obesity-related disorder is lipedema. In some embodiments, the subject has been diagnosed with lipedema. In some embodiments, the subject has been diagnosed with lipedema and is overweight or obese at the start of treatment. In some embodiments, the obesity-related disorder is selected from the group consisting of pediatric obesity, type II diabetes, obesity, an obesity syndrome, clinical obesity, lipedema, metabolic syndrome / pre-diabetes, cardiovascular disease, nonalcoholic steatohepatitis (NASH) or nonalcoholic fatty liver disease (NAFLD), type I diabetes, Cushing’s disease, tumor-induced Cushing’s disease, Prader-Willi syndrome, Bardet-Biedl syndrome, thyroid disorder, thyroid removal, Adrenoleukodystrophy, Niemann-Pick disease, Tangier disease, Polycystic ovary syndrome, Congenital leptin deficiency, Cohen syndrome, Alstrbm syndrome, or Froehlich syndrome, or any combination thereof. In some embodiments, the subject has been diagnosed with obesity. In some embodiments, the subject achieves a weight loss of at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, or 40% of body weight at a time point following the administering. In some embodiments, the ARF1 protein is administered systemically to the subject. In some embodiments, the ARF1 protein is administered by parenteral administration. In some embodiments, parenteral administration comprises injection of the ARF1 protein in the subject. In some embodiments, the injection is intravenous injection. In some embodiments, the injection is intramuscular injection. In some embodiments, the injection is intradermal injection. In some embodiments, the injection is subcutaneous injection. In some embodiments, parenteral administration comprises infusion of the ARF1 protein in the subject. In some embodiments, the ARF1 protein comprises an amino acid sequence selected from SEQ ID NOS: 1-23. In some embodiments, the ARF1 protein is contained with a single polypeptide of an engineered ARF1polypeptide comprising one or more heterologous peptide sequences. In some embodiments, the engineered ARF1 polypeptide has increased stability compared to a wild-type ARF1 protein consisting essentially of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the engineered ARF1 polypeptide has increased in vivo half-life compared to a wild-type ARF1 protein consisting essentially of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the engineered ARF1 polypeptide has improved in vitro expression compared to a wild-type ARF1 protein consisting essentially of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the engineered ARF1 polypeptide has improved in vitro secretion compared to a wild-type ARF1 protein consisting essentially of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the engineered ARF1 polypeptide has improved in vitro purification compared to a wild-type ARF1 protein consisting essentially of the amino acid sequence of SEQ ID NO: 1.

[0009] In an aspect described herein are engineered polypeptides comprising: a homologous ADP-ribosylation factor (ARF) sequence, and a covalently-coupled protein trafficking domain sequence. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 104. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO:105. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 106. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOS: 87-106. In some embodiments, the covalently-coupled protein trafficking domain sequence comprises a signal peptide sequence. In some embodiments, the covalently -coupled protein trafficking domain sequence comprises a site for myristoylation. In some embodiments, the covalently-coupled protein trafficking domain sequence comprises an Annexin A10 peptide sequence. In some embodiments, engineered polypeptide comprises a peptide tag.

[0010] In an aspect described herein are engineered polypeptides comprising: a homologous ADP-ribosylation factor (ARF) sequence, and a half-life extending moiety. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 104. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 105. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO:106. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOS: 87- 106. In some embodiments, the half-life extending moiety comprises a fatty acid ester. In someembodiments, the half-life extending moiety comprises a human serum albumin (HSA) sequence. In some embodiments, the half-life extending moiety comprises an Fc portion of a human immunoglobulin.

[0011] In an aspect described herein are engineered polypeptides comprising: a homologous ADP-ribosylation factor 1 (ARF1) sequence, and a covalently-coupled protein trafficking domain sequence. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 1. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 93% identical to SEQ ID NO: 23. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 95% identical to SEQ ID NO: 1. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 1-23. In some embodiments, the homologous ARF1 sequence comprises one, two, three, four, five, six, seven, eight, or nine amino acid substitutions relative to an amino acid sequence selected from SEQ ID NOs: 1-23. In some embodiments, the homologous ARF1 sequence comprises one or more deletions of one, two, or three amino acids relative to an amino acid sequence selected from SEQ ID NOs: 1-23. In some embodiments, the homologous ARF1 sequence comprises one or more insertions of one, two, three, four, five, six, seven, eight, or nine amino acids relative to an amino acid sequence selected from SEQ ID NOs: 1- 23. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence selected from SEQ ID NOs: 1-23, 123-174, 286-289, or 297-311. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence selected from any one of SEQ ID NOs: 123, 286-287, 289, or 294-296. In some embodiments, the covalently-coupled protein trafficking domain sequence is located N-terminal to the homologous ARF1 sequence. In some embodiments, the covalently-coupled protein trafficking domain sequence is located C-terminal to the homologous ARF1 sequence. In some embodiments, the covalently-coupled protein trafficking domain sequence comprises a signal peptide sequence. In some embodiments, the covalently- coupled protein trafficking domain sequence comprises a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is inserted within the engineered polypeptide. In some embodiments, the peptide tag prevents myristoylation of a glycine residue near the N- terminal portion of the homologous ARF1 sequence. In some embodiments, the homologous ARF1 sequence does not comprise a myristoyl group attached to a glycine reside. In some embodiments, the engineered polypeptide further comprises a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is inserted within the engineered polypeptide. In some embodiments, the engineered polypeptide further comprises a cleavage site. In some embodiments,the cleavage site is inserted between the homologous ARF1 sequence and the covalently-coupled protein trafficking domain sequence.

[0012] In an aspect described herein are pharmaceutical compositions comprising an engineered polypeptide described herein, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition is formulated for systemic administration in a subject in need thereof. In some embodiments, the pharmaceutical composition is formulated for i.v. or s.c. administration.

[0013] In an aspect described herein are methods of treating a subject having obesity, the methods comprising administering an engineered ADP-ribosylation factor 1 (ARF1) protein to the subject, wherein the ARF1 protein comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the engineered ARF1 protein is administered systemically to the subject. In some embodiments, the engineered ARF1 protein is administered by parenteral administration. In some embodiments, administration comprises ongoing administration of the engineered polypeptide according to an administration schedule for a period of at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5, months, 6 months, or more than 6 months. In some embodiments, the subject achieves a weight loss of at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, or 40% of body weight at a time point following the administering.

[0014] In an aspect described herein are methods of treating a subject having obesity, the method comprising administering the engineered polypeptide of described herein to the subject. In some embodiments, administration comprises ongoing administration of the engineered polypeptide according to an administration schedule for a period of at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5, months, 6 months, or more than 6 months. In some embodiments, the subject achieves a weight loss of at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, or 40% of body weight at a time point following the administering.

[0015] In an aspect described herein are methods of promoting weight loss in a subject in need thereof, the methods comprising administering the engineered polypeptide described herein to the subject. In some embodiments, administration comprises ongoing administration of the engineered polypeptide according to an administration schedule for a period of at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5, months, 6 months, or more than 6 months. In some embodiments, the subject achieves aweight loss of at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, or 40% of body weight at a time point following the administering.

[0016] In an aspect described herein are methods of promoting weight loss in a subject in need thereof, the methods comprising administering the engineered polypeptide described herein to the subject. In some embodiments, administration comprises ongoing administration of the engineered polypeptide according to an administration schedule for a period of at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5, months, 6 months, or more than 6 months. In some embodiments, the subject achieves a weight loss of at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, or 40% of body weight at a time point following the administering.

[0017] In an aspect described herein are methods of maintaining weight loss in a subject in need thereof, the methods comprising administering the engineered polypeptide described herein to the subject following the cessation of incretin therapy, wherein the subject maintains an extent of previous incretin-induced weight loss. In some embodiments, the subject is treated for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 17 days, 21 days, 24 days, 28 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. In some embodiments, the subject is treated for at least 3 weeks. In some embodiments, the subject achieves a percentage of weight loss of at least 2.5%, 5%, 7.5%, 10%, 12.5%, or 15%. In some embodiments, the subject maintains a percentage of lean muscle mass of at least 85%, 90%, 95%, 98%, 99%, or 100% of following a period of treatment. In some embodiments, the subject is overweight or clinically obese at the start of the administering. In some embodiments, the subject has been diagnosed with an obesity-related disorder selected from the group consisting of: pediatric obesity, type II diabetes, obesity, an obesity syndrome, clinical obesity, lipedema, metabolic syndrome / pre-diabetes, cardiovascular disease, nonalcoholic steatohepatitis (NASH) or nonalcoholic fatty liver disease (NAFLD), type I diabetes, Cushing’s disease, tumor-induced Cushing’s disease, Prader-Willi syndrome, Bardet- Biedl syndrome, thyroid disorder, thyroid removal, Adrenoleukodystrophy, Niemann-Pick disease, Tangier disease, Polycystic ovary syndrome, Congenital leptin deficiency, Cohen syndrome, Alstrbm syndrome, and Froehlich syndrome. In some embodiments of methods described herein, a homologous ARF1 sequence within the engineered polypeptide comprises an amino acid sequence at least about 78%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 1-23. In some embodiments, the homologous ARF1 sequence comprises an amino acidsequence selected from SEQ ID NOs: 1-23, 123-174, 286-289, or 297-311. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence selected from SEQ ID NO: 123, 286, 287, or 289. In some embodiments of methods described herein, a homologous ARF1 sequence within the engineered polypeptide comprises an amino acid sequence at least about 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 61- 64, 176, 184-186, 194, 217-218, or 315-317. In some embodiments, the homologous ARF1 sequence within the engineered polypeptide comprises an amino acid sequence identical to a sequence selected from SEQ ID NOs: 61-64, 176, 184-186, 194, 217-218, or 315-317. In some embodiments of methods described herein, a homologous ARF1 sequence within the engineered polypeptide comprises an amino acid sequence at least about 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 59-60, 181-182, 192, 196, 215- 216, or 245-284. In some embodiments, the homologous ARF1 sequence of the engineered polypeptide comprises an amino acid sequence identical to a sequence selected from SEQ ID NOs: 59-60, 181-182, 192, 196, 215-216, or 245-284.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The novel features described herein are set forth with particularity in the appended claims. A better understanding of the features and advantages of the features described herein will be obtained by reference to the following detailed description that sets forth illustrative examples, in which the principles of the features described herein are utilized, and the accompanying drawings of which:

[0019] FIG. 1A-FIG. IB show results of engineered ARF1 protein production in Expi239F™ cells (ThermoFisher). FIG. 1A shows a Western blot of MGSS HIS TEV ARFl wt protein produced three days following transfection with an ARF1 expression construct detected using an anti-ARFl antibody. Protein samples from supernatant and cell pellet were assayed. FIG. IB shows a chart measuring the amount of engineered ARF1 protein produced from the respective lanes shown in the Western blot of FIG. 1A.

[0020] FIG. 2A-FIG. 2B show results of His-tagged engineered ARF1 protein production. FIG. 2A shows a Western blot of using anti -His antibody to assess production of His-tagged ARF1 polypeptides from the supernatants of CHO cells at Day 5 post-transfection or in Expi293F cells at Day 4 post-transfection to assess if His-tagged engineered ARF1 polypeptides are secreted. FIG. 2B shows a Western blot using anti-His antibody to assess production of His-tagged ARF1 polypeptides from the cell lysates of CHO cells at Day 5 post-transfection or in Expi293F cells at Day 4 post-transfection to assess if His-tagged engineered ARF1 polypeptides are produced andmaintained intracellularly.

[0021] FIG. 3A-FIG. 3B show results of His-tagged engineered ARF1 protein production in CHO cells. FIG. 3A shows an image of a Western blot using anti-His antibody for detection in samples from supernatant. FIG. 3B shows an image of a Western blot using anti-His antibody for detection in samples from cell lysate.

[0022] FIG. 4A-FIG. 4B show results of engineered ARFl-Fc fusion protein and Flag-tagged ARF1 polypeptide production in Expi293F cells in supernatant and in cell lysates. FIG. 4A shows an image of a Western blot using an anti-His antibody to assay His-tagged engineered ARF1 polypeptides, non-tagged engineered ARF1 polypeptides, and FL AG-tagged engineered ARF1 polypeptides found in the supernatant (sup) or cell pellet (pellet) from Day 3 post-transfection. FIG. 4B shows an image of a Western blot using an anti-ARFl antibody for detection of His- tagged engineered ARF1 polypeptides, ARFl-Fc fusions, and FLAG-tagged engineered ARF1 polypeptides.

[0023] FIG. 5 shows results of Western blot (left) of completed medium extracted protein samples and Western blot (right) of cell pellet extracted protein samples using an anti-ARFl antibody for detection of engineered ARF1 polypeptides with HSA fusions and engineered ARF1 polypeptides with amino acid substitutions testing production and secretion in Expi293F cells in Day 4 post-transfection.

[0024] FIG. 6 shows results of Western blot 2 days post-transfection (left) and Western blot 3 days post-transfection (right) for engineered ARF1 polypeptides extracted from supernatant testing secretion in Expi293F cells.

[0025] FIG. 7 shows results of Western blot (left) and charting of results (right) for engineered ARF1 polypeptides testing intracellular expression and secretion in Expi293F cells on Day 3 posttransfection.

[0026] FIG. 8 shows results of Western blot (left) and charting of results (right) for engineered ARF1 polypeptides testing intracellular expression and secretion in Expi293F cells on Day 3 posttransfection.

[0027] FIG. 9 shows results of Western blot (left) and charting of results (right) for engineered ARF1 polypeptides testing intracellular expression and secretion in Expi293F cells on Day 3 posttransfection.

[0028] FIG. 10 shows results of Western blot (left) and charting of results (right) for engineered ARF1 polypeptides testing secretion in various replicates in Expi293F cells on Day 3 post-transfection compared to expression levels in cell lysates.

[0029] FIG. 11A-FIG. 11B show an in vitro lipolysis study design and test results. FIG. HAshows a study design in which various aspects of lipolysis are assessed in adipocytes. FIG. 11B shows a graph of the dose-response in fold change in lipolysis following contacting of adipocytes in vitro with an ARF1 therapeutic polypeptide.

[0030] FIG. 12A shows a graph of the dose-response on triglyceride content following contacting of adipocytes in vitro with an ARF1 therapeutic polypeptide.

[0031] FIG. 12B shows a graph of the dose-response on lipid droplet intensity following contacting of adipocytes in vitro with an ARF1 therapeutic polypeptide.

[0032] FIG. 13 shows an experimental study design to test metabolic regulation, endocrine function, weight regulation, and tissue pathology in vivo in an obese diabetic model.

[0033] FIG. 14A-FIG. 14B show graphs of results of body weight change over time with ARF1 therapeutic compared to tirzepatide treatment and vehicle treatment in an obese diabetic model. FIG. 14A shows a graph of results of treatment on body weight over time in the diet- induced obesity (DIO) model shown in FIG. 13. FIG. 14B shows a bar chart and statistical analysis of body weight changes by week in the DIO model.

[0034] FIG. 14C-FIG. 14D show charts of body weight change over time with ARF1 therapeutic compared to tirzepatide treatment and vehicle treatment in the DIO model shown in FIG. 13. FIG. 14C shows a chart of baseline body weight and results at Week 3 in the DIO model. FIG. 14D shows a chart of body weight results at Week 1, Week 2, and Week 3 in the DIO model and results of statistical analysis.

[0035] FIG. 15A-FIG. 15B show blood glucose changes following chronic administration of an ARF1 therapeutic polypeptide. FIG. 15A shows a chart of baseline and Week 3 blood glucose (BG) results of ARF1 monotherapy compared to TZP monotherapy and control animals in a HF- DIO mouse model. FIG. 15B shows a chart percentage change in BG following chronic ARF1 monotherapy compared to chronic TZP monotherapy and control animals in a HF -DIO mouse model.

[0036] FIG. 16A-FIG. 16B show results of Insulin Tolerance Test (ITT) testing at Week 2 of the study design listed in FIG. 13. Results are graphed in FIG. 16A in 15 minute or 30 minute intervals showing time points of significant differences between test groups. FIG. 16B shows a chart of area under the curve (AUC) calculations by test group and significant differences.

[0037] FIG. 17 shows a chart of serum insulin levels tested at Week 3 of the study design listed in FIG. 13 for Group 1, Group 2, and Group 3 test animals.

[0038] FIG. 18 shows an in vitro study design to test a variety of homologous ARF proteins and engineered ARF proteins for their effects on regulating adipocyte lipid content.

[0039] FIG. 19 shows representative cell labeling on Day 9 of adipocytes treated with varioustest factors according to the study design shown in FIG. 18. Bright staining is Bodipy and darker staining in Hoechst.

[0040] FIG. 20 shows a graph of results of effect on lipid area change following treatment according to the study design shown in FIG. 18.

[0041] FIG. 21 shows alignment of human ARF1, ARF3, and ARF5 protein sequences indicating homologous and divergent amino acids residues at positions within the proteins.

[0042] FIG. 22A-FIG. 22B show graphs of results of body weight change over time with ARF1 therapeutic compared to vehicle treatment in an obese diabetic model. FIG. 22A shows a graph of results of treatment on body weight over time in the diet-induced obesity (DIO) model shown in FIG. 13. FIG. 22B shows a graph of percentage change in fat mass by week in the DIO model.

[0043] FIG. 23A-FIG. 23F show improved lipid handling in adipose and liver in response to ARF1 therapeutic treatment. FIG. 23 A, shows a graph of normalized liver weight. FIG. 23B shows a graph of liver triglyceride level. FIG. 23C shows stained Oil red O staining from liver crosssections were used to measure lipid droplet (LD) size. FIG. 23D shows a graph of average LD area from the Oil red O stained liver cross-sections. FIG. 23E shows a graph of liver fibrotic index. FIG. 23F shows a graph of adipocyte size.

[0044] FIG. 24A-FIG. 24E show the glucoregulatory effect of HIS6-TEV-ARF 1 -T3 IN treatment. FIG. 24A is a chart of non-fasting blood glucose levels in DIO mice. FIG. 24B is a chart of serum insulin levels in DIO mice. FIG. 24C is a chart of fasting blood glucose levels in DIO mice. FIG. 24D is a graph of blood glucose level in an oral glucose tolerance test. FIG. 24E is a graph of blood glucose level from an insulin tolerance test.

[0045] FIG. 25A-FIG. 25D show that HIS6-TEV-ARF1-T3 IN treatment is non-appetite suppressing in a DIO model in contrast to other weight loss therapeutics. FIG. 25A shows a chart of measured food intake. FIG. 25B shows a chart of measured fecal triglyceride levels. FIG. 25C- FIG. 25D are charts showing effect of HIS6-TEV-ARF1-T31N treatment on food intact 24 hours (in FIG. 25C) or 6 hours (in FIG. 25D) following administration.

[0046] FIG. 26A-FIG. 26B show that HIS6-TEV-ARF1-T3 IN treatment maintains body weight loss after semaglutide treatment cessation. FIG. 26A is a graph of percentage change in body weight on Maintenance treatment using an ARF1 therapeutic after stopping semaglutide treatment. FIG. 26B is a chart showing significant differences of percentage change in body weight on Maintenance treatment using an ARF1 therapeutic after stopping semaglutide treatment from FIG. 26A

[0047] FIG. 27A-FIG. 27D show that HIS6-TEV-ARF1-T3 IN treatment promotes weight lossand improves body composition in 45% High fat fed mice. FIG. 27A is a chart showing percentage change in body weight. FIG. 27B is a chart showing percentage change in fat mass. FIG. 27C is a chart showing reduction in normalized liver weight. FIG. 27D shows a graph of blood glucose levels from an oral glucose tolerance test.

[0048] FIG. 28A-FIG. 28B demonstrate that HIS6-TEV-ARF1-T3 IN increases oxygen consumption activity in murine 3T3L1 adipocytes grown in vitro. FIG. 28A is a graph showing effects on oxygen consumption rate (OCR) with two different doses of HIS6-TEV-ARF1-T31N. FIG. 28B is a graph showing effects on oxygen consumption rate (OCR) with three different doses of HIS6-TEV-ARF 1 -T3 IN.

[0049] FIG. 29A-FIG. 29C demonstrate increased energy expenditure (EE) following HIS6- TEV-ARF1-T31N treatment. EE measurements were graphed during the light cycle (FIG. 29A), during the dark cycle (FIG. 29B), and shown as averages between light and dark cycle (FIG. 29C) in DIO mice over a 14 day period.

[0050] FIG. 30A-FIG. 30B demonstrate cellular uptake activity of various engineered ARF1 proteins. FIG. 30A shows human HeLa cells stained to quantitate ARF1 protein signal per cell. FIG. 30B shows a chart of average ARF1 signal / cell from the immunofluorescent staining in FIG.30A

[0051] FIG. 31A-FIG. 31B demonstrate that multiple ARF1 mutations with different tags affect lipid droplet volume in mature 3T3L1 adipocytes. FIG. 31A and FIG. 31B each show charts of fold change in LD intensity after treatment with a variety of engineered ARF1 proteins.

[0052] FIG. 32 demonstrates dose dependent LD reduction after HIS6-TEV-ARF1-T3 IN treatment but not with HSA-ARF1-T3 IN treatment as a chart of fold change in LD intensity in adipocytes.

[0053] FIG. 33 shows a graph of lipolysis fold change comparing the effects of two engineered ARF1 proteins.DETAILED DESCRIPTION

[0054] ADP-ribosylation factor 1 (ARF1) is a member of the human ARF gene family. The ARF family members encode small guanine nucleotide-binding proteins that stimulate the ADP- ribosyltransferase activity. ARF1 protein is produced from the ARF1 gene locus (official full name: ADP ribosylation factor 1; also known an PVNH8). The ARF1 protein was known to be localized to the Golgi apparatus and has been determined to have a central role in intra-Golgi transport. ARF1 protein is herein identified as a molecule that can be secreted from stem cells.

[0055] In mammals, there are typically at least 5 ARF proteins and 11 ARF-like proteins, which together constitute a family of the RAS superfamily. Class 1 ARF proteins include ARF1,and ARF3. Class 2 ARF proteins include ARF4 (formerly known as ARF2) and ARF5. Class 3 ARF proteins include ARF6. The function of endogenous secreted ARF1 protein is unknown and was herein investigated for a role in regulating body weight. As described herein, administration of an ARF1 protein or an engineered ARF1 polypeptide can be used to decrease body weight of a subject. As described herein, administration of an ARF1 protein or an engineered ARF1 polypeptide can be used to maintain body weight of a subject while the subject is consuming a high fat diet or a high caloric diet that may otherwise result in unwanted weight gain.

[0056] In one aspect, described herein is a composition comprising a metabolic restorationpromoting engineered polypeptide, an anti-inflammatory engineered polypeptide, an adipogenesisregulating engineered polypeptide, a weight loss-promoting engineered polypeptide, or any combination thereof. In certain aspects, the composition is for use in a method for weight loss. In certain aspects, the composition is for use in a method for treating a subject having a metabolic disorder. In certain aspects, the composition is for use in a method for treating a subject having an obesity-related disorder. In certain aspects, the composition is for use in a method for treating a subject having obesity. In certain aspects, the composition is for use in a method for treating a subject having a metabolic disorder with pathological regulation of lipolysis. In certain aspects, the obesity or obesity -related disorder is associated with white adipocyte dysfunction. In certain aspects, the obesity or obesity-related disorder is associated with beige adipocyte dysfunction. In certain aspects, the obesity or obesity-related disorder is associated with white adipocyte dysfunction and beige adipocyte dysfunction. In certain aspects, the obesity-related disorder includes the subject having lipedema.

[0057] In certain aspects, the obesity or obesity-related disorder is associated insulin resistance, increased inflammation, increase appetite, or any combination thereof. In certain aspects, the composition comprises a homologous ADP-ribosylation factor (ARF) protein sequence. In certain aspects, the composition comprises a homologous ADP-ribosylation factor 1 (ARF1) protein sequence. In some embodiments, a subject has reduced endogenous levels of secreted ARF1 protein. In some embodiments, the subject having obesity has reduced endogenous levels of secreted ARF1 protein. In some embodiments, the subject having an obesity-related disorder has reduced endogenous levels of secreted ARF1 protein. In some embodiments, reduced endogenous levels of secreted ARF1 protein in the subject are compared to endogenous levels of secreted ARF1 protein in a healthy subject. In certain aspects, the obesity-related disorder includes the subject having lipedema.

[0058] In an aspect, described herein is a composition comprising an engineered polypeptide, wherein the engineered polypeptide comprises a homologous ADP-ribosylation factor 1 (ARF1)protein sequence, and a covalently-coupled protein trafficking domain sequence. In an aspect, described herein is a composition comprising an engineered polypeptide, wherein the engineered polypeptide comprises a homologous ADP-ribosylation factor 1 (ARF1) protein sequence, and a half-life extending moiety. In some embodiments, the engineered polypeptide comprises a signal peptide sequence. In some embodiments, the engineered polypeptide does not comprise a site for myristoylation. In some embodiments, the engineered polypeptide comprises a site for myristoylation. In some embodiments, the engineered polypeptide comprises a peptide tag. In some embodiments, the engineered polypeptide comprises a heterologous peptide sequence. In some embodiments, the heterologous peptide sequence comprises an Annexin A10 peptide sequence. In some embodiments, the engineered polypeptide comprises a heterologous protein sequence. In some embodiments, the heterologous protein sequence comprises a serum albumin sequence or variant thereof. In some embodiments, the heterologous protein sequence comprises a human serum albumin protein sequence. In some embodiments, the heterologous protein sequence comprises an Fc region of an IgG molecule. In some embodiments, the heterologous protein sequence comprises a fluorescent protein sequence. In some embodiments, the fluorescent protein sequence comprises a green fluorescent protein (GFP) or an enhanced green fluorescent protein (EGFP) sequence. In some embodiments, the engineered polypeptide comprises a cleavage site. In some embodiments, the cleavage site comprises a protease recognition motif. In some embodiments, the engineered polypeptide comprises an N-terminal alpha helix deletion.

[0059] In some embodiments, the covalently-coupled protein trafficking domain sequence inserted within or next to the homologous ARF1 protein sequence increases at least one of stability, biological function, or in vivo half-life of the engineered protein comprising the homologous ARF1 protein sequence. In some embodiments, the heterologous polypeptide amino acid sequence inserted within or next to the homologous ARF1 protein sequence increases stability of the modified protein. In some embodiments, the heterologous polypeptide amino acid sequence inserted within or next to the homologous ARF1 protein sequence prevents myristoylation of a glycine residue. In some embodiments, the heterologous polypeptide amino acid sequence inserted N-terminal to the homologous ARF1 protein sequence prevents myristoylation of a glycine residue. In some embodiments, the heterologous polypeptide amino acid sequence inserted within or next to the homologous ARF1 protein sequence increases biological function of the modified protein. In some embodiments, the heterologous polypeptide amino acid sequence inserted within or next to the homologous ARF1 protein sequence increases in vivo half-life of the modified protein. In some embodiments, a heterologous peptide amino acid sequence inserted within or next to the homologous ARF1 protein sequence improves in vitro expression of the modified protein. In someembodiments, a heterologous peptide amino acid sequence inserted within or next to the homologous ARF1 protein sequence improves in vitro purification of the modified protein. In some embodiments, a heterologous polypeptide amino acid sequence inserted within or next to the homologous ARF1 protein sequence improves in vitro expression of the modified protein. In some embodiments, a heterologous polypeptide amino acid sequence inserted within or next to the homologous ARF1 protein sequence improves in vitro purification of the modified protein. In some embodiments, a heterologous peptide amino acid sequence inserted within or next to the homologous ARF1 protein sequence improves in vitro secretion of the modified protein. In some embodiments, a heterologous peptide amino acid sequence inserted within close proximity to the homologous ARF1 protein sequence improves in vitro secretion of the modified protein. In some embodiments, a heterologous polypeptide amino acid sequence inserted within or next to the homologous ARF1 protein sequence improves in vitro secretion of the modified protein. In some embodiments, a heterologous polypeptide amino acid sequence inserted within close proximity to the homologous ARF1 protein sequence improves in vitro secretion of the modified protein. In some embodiments, the engineered protein comprises one or more chemical modifications. In some embodiments, the one or more chemical modifications comprise one or more site-specific covalent attachments of a fatty acid to amino acid residues of the modified protein. In some embodiments, a form of the engineered protein comprising one or more fatty acids maintains a longer serum halflife. In some embodiments, the longer serum half-life allows for a subject to be administered a lower effective dose of a composition comprising the engineered protein. In some embodiments, the lower effective dose is sufficient for treatment of a subject having obesity or an obesity-related disorder. In some embodiments, the lower effective dose is sufficient for treatment of a subject having lipedema. In some embodiments, the lower effective dose is sufficient to achieve weight loss. In some embodiments, the lower effective dose is sufficient to maintain weight loss. In some embodiments, the lower effective dose is sufficient to improve body composition by lowering a relative percentage of fat mass as total body weight.DEFINITIONS

[0060] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the embodiments provided may be practiced without these details. Unless the context requires otherwise, throughout the specification and claims that follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictatesotherwise. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0061] As used herein a composition that is “consisting essentially” of the recited components is a composition that only has the recited elements as active ingredients, but can comprise other non-active components that do not appreciably modify the function or activity of the recited components. Any list disclosed herein that is recited as “comprising” can be recited as “consisting essentially,” to exclude non-recited polypeptide or protein components. Additionally, any list disclosed herein that is recited as “comprising” can be recited as “consisting essentially,” to exclude non-recited method steps.

[0062] As used herein “adipocyte” means a cell also known as a lipocyte or a fat cell that primarily composes adipose tissue. Adipocytes in homeostatic conditions maintain specialized functions in storing energy as fat and metabolizing fat into useable cellular energy sources as needed. Adipocytes are derived from mesenchymal stem cells that give rise to adipocytes through adipogenesis. Pre-adipocytes can be stimulated to form adipocytes. Adipocytes may be from any organism of interest, including, primate; human; canine; feline; murine; equine; porcine; avian; camel; bovine; ovine, and so on.

[0063] As used herein “white adipose tissue” and “brown adipose tissue” are also known respectively as white fat and brown fat. White fat cells reside in white adipose tissue and typically contain a single large lipid droplet surrounded by a layer of cytoplasm. The fat stored in white fat cells is typically in a semi-liquid state, and is comprised primarily of triglycerides and cholesteryl ester. If a subject gains sufficient excess weight up until early adulthood, white fat cells may increase in absolute number. If a subject gains sufficient excess weight, fat cells may generally increase in size. If a subject gains sufficient excess weight, fat cells may generally increase in mass. Brown fat cells reside in brown adipose tissue and are polyhedral in shape. Brown fat cells or brown adipocytes are located in dedicated deposit in a subject’s body. Brown adipose tissue is a site of heat production, or thermogenesis, in mammals. Brown fat cells are typically derived from dermatomyocyte cells, including My f5 -expressing embryonic precursors. In contrast to white fat cells, brown fat cells typically contain considerable relative amounts of cytoplasm with several lipid droplets scattered throughout. Brown fat cells typically maintain a larger quantity of mitochondria than white fat cells. Beige cells or beige adipocytes are inducible and can develop in white fat in response to various activators. Clusters of beige adipocytes (which can be identified as UCP1 -expressing cells) may function in a thermogenic capacity and can develop in white adipose tissue in response to various stimuli. Beige adipocytes may be defined by their location in whiteadipose tissue and by possessing a multiocular lipid droplet morphology, high mitochondrial content, or the expression of known brown fat genes such as Ucpl, Cidea, and Pgcla, or any combination thereof.

[0064] As used herein “overweight” and “obesity” are defined exhibiting an abnormal or excessive fat accumulation that may impair health. In human subjects, a body mass index (BMI) greater than or equal to 25 is used to define a subject as overweight. In human subjects, a BMI greater than or equal to 30 is used to define a subject as obese. In subjects such as children under 5 years of age, overweight can be defined as weight-for-height greater than 2 standard deviations above WHO Child Growth Standards median. In subjects such as children under 5 years of age, obesity can be defined as weight-for-height greater than 3 standard deviations above WHO Child Growth Standards median. In subjects such as children between the ages of 5-19, overweight can be defined as BMI-for-age greater than 1 standard deviation above the WHO Growth Reference median. In subjects such as children between the ages of 5-19, obesity can be defined as greater than 2 standard deviations above the WHO Growth Reference median.

[0065] As used herein the term “about” refers to an amount that is near the stated amount by 10% or less.

[0066] As used herein the terms “individual” “subject,” and “patient” are interchangeable. The subject can be mammal such as a horse, cow, pig, chicken, goat, rabbit, mouse, rat, dog, or cat. In certain embodiments, the subject is a human person.

[0067] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. Once translated, the proteins may be found in a monomeric state or a multimeric state such as a dimeric form. Polypeptides, including the provided polypeptide chains and other peptides, e.g., linkers and binding peptides, may include amino acid residues including natural and / or non-natural amino acid residues. The terms also include post-translational modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, myristoylation, and the like. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts that produce the proteins, errors due to PCR amplification, or errors in protein translation.

[0068] In some embodiments, a recombinant protein is a protein expressed in a system other than a human, e.g., the protein is expressed from bacteria, yeast, or mammalian cells in culture. In some cases, the protein is expressed from Chinese Hamster Ovary cells (CHO cells). In some cases, the protein is expressed from mouse myeloma cells, e.g., (NS0) cells. In some cases, the protein isexpressed from E. colt.

[0069] Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0070] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0071] Heterologous polypeptide amino acid sequences contain amino acid residue sequence not present in a polypeptide sequence originating from a different source (e.g., a different gene or a different species) in which the heterologous polypeptide amino acid sequence is now part of as asingle polypeptide chain. A heterologous polypeptide amino acid sequence may comprise a short peptide tag. A heterologous polypeptide amino acid sequence may consist essentially of a short peptide tag. A heterologous polypeptide amino acid sequence may comprise part of a fusion protein or an engineered modified protein described herein that may comprise, consist, or consist essentially of a fragment of an immunoglobulin molecule, an albumin molecule, an Annexin A10 peptide molecule, a transferrin molecule, an XTEN sequence, a proline-alanine-serine polymer, a homo-amino acid polymer, a glycine-rich sequence, a gelatin-like polymer, an elastin-like peptide, a carboxy -terminal peptide, or combinations thereof.

[0072] As described herein a “ homologous ADP-ribosylation factor 1 (ARF1) protein sequence” is a polypeptide sequence sharing a certain extent of sequence identity with various ARF1 proteins derived from a vertebrate species. The extent of shared sequence identity must be sufficient to use a protein alignment software program to align at least a portion of the homologous ARF1 sequence to a known ARF1 protein derived from a vertebrate species (e.g., at least 50% identity of aligned portions of a polypeptide sequence over a minimum length of 40 amino acids). Exemplary ARF1 proteins derived from various vertebrate species to be used in an assessment of polypeptide alignment with a homologous ARF1 protein sequence can be identified through the following NCBI Accession numbers: NP_001649.1, JAA08426.1, OCT75402.1, BAE31170.1, 8SDW A, DAA31858.1, EHH50487.1, CAI9572194.1, NP_958888.1, or XP_039540413.1.

[0073] In some embodiments, reference to a conjugate, polypeptide conjugate, or protein conjugate refers to a synthetically and / or recombinantly produced molecule comprising a chemical entity covalently bound to one or more amino acids of an amino acid sequence. In some cases, the conjugation is selective such that the chemical entity is connected to a specific amino acid of the amino acid sequence. In some embodiments, the amino acid sequence comprises a polypeptide described herein. For example, the polypeptide described herein is a polypeptide comprising a homologous ARF1 protein sequence.

[0074] In some embodiments, a polypeptide described herein is a proteoform of a protein comprising a homologous ARF1 protein sequence. In some aspects, as used herein a proteoform describes a molecular form of a protein product arising from a gene encoding a protein, such as a protein including, for example, ARF1. In some cases, a proteoform includes proteins that arise from the same gene as a result of genetic variation, alternatively spliced RNA transcripts, post- translational modifications, or polypeptide cleavage event. In some cases, an engineered ARF1 proteins comprises one or more amino adds additions which produce a proteoform that is cleaved at a later time after synthesis to yield a mature engineered ARF1 isoform with desired therapeutic properties.Engineered Polypeptides

[0075] In some aspects, polypeptides described herein that are useful for treating obesity or an obesity-related disorder comprise an engineered polypeptide comprising a homologous ADP- ribosylation factor 1 (ARF1) protein sequence. In some aspects, polypeptides described herein that are useful for treating lipedema comprise an engineered polypeptide comprising a homologous ADP-ribosylation factor 1 (ARF1) protein sequence

[0076] In one aspect, polypeptides described herein that are useful for treating obesity or an obesity-related disorder comprise a modified protein comprising a homologous ADP-ribosylation factor 1 (ARF1) protein sequence. In one aspect, polypeptides described herein that are useful for treating lipedema comprise a modified protein comprising a homologous ADP-ribosylation factor 1 (ARF1) protein sequence. In one aspect, polypeptides described herein that are useful for treating obesity comprise an engineered protein comprising a homologous ADP-ribosylation factor 1 (ARF1) protein sequence. In one aspect, polypeptides described herein that are useful for treating lipedema comprise an engineered protein comprising a homologous ADP-ribosylation factor 1 (ARF1) protein sequence. In one aspect, polypeptides described herein that are useful for treating obesity comprise a modified protein comprising a homologous ADP-ribosylation factor 1 (ARF1) protein sequence. In one aspect, polypeptides described herein that are useful for promoting metabolic restoration comprise an engineered protein comprising a homologous ARF1 protein sequence. In one aspect, polypeptides described herein that are useful for reducing cellular and tissue inflammation comprise an engineered protein comprising a homologous ARF1 protein sequence. In one aspect, polypeptides described herein that are useful for regulating adipogenesis comprise an engineered protein comprising a homologous ARF1 protein sequence. In one aspect, polypeptides described herein that are useful for reducing tissue fibrosis (e.g., liver fibrosis) comprise an engineered protein comprising a homologous ARF1 protein sequence. In one aspect, polypeptides described herein that are useful for promoting weight loss comprise an engineered protein comprising a homologous ARF1 protein sequence. In one aspect, polypeptides described herein that are useful for treating a metabolic disorders comprise an engineered protein comprising a homologous ARF1 protein sequence. In one aspect, polypeptides described herein that are useful for treating a metabolic disorder with pathological regulation of lipolysis comprise an engineered protein comprising a homologous ARF1 protein sequence. In one aspect, polypeptides described herein that are useful for treating obesity, an obesity-related disorder, or lipedema associated with white adipocyte dysfunction comprise an engineered protein comprising a homologous ARF1 protein sequence. In one aspect, polypeptides described herein that are useful for treating obesity, an obesity-related disorder, or lipedema associated with white adipocyte metabolism dysfunctionand excess fat storage comprise an engineered protein comprising a homologous ARF1 protein sequence. In one aspect, polypeptides described herein that are useful for treating obesity or an obesity-related disorder associated with beige adipocyte dysfunction comprise an engineered protein comprising a homologous ARF1 protein sequence. In one aspect, polypeptides described herein that are useful for treating lipedema associated with beige adipocyte dysfunction comprise an engineered protein comprising a homologous ARF1 protein sequence. In one aspect, polypeptides described herein that are useful for treating obesity, an obesity-related disorder, or lipedema is associated with insulin resistance comprise an engineered protein comprising a homologous ARF1 protein sequence. In one aspect, polypeptides described herein that are useful for treating obesity or an obesity-related disorder is associated with increased appetite comprise an engineered protein comprising a homologous ARF1 protein sequence. In one aspect, polypeptides described herein that are useful for treating a subject that is overweight comprise an engineered protein comprising a homologous ADP-ribosylation factor 1 (ARF1) protein sequence. In one aspect, polypeptides described herein that are useful for maintaining weight loss in a subject comprise an engineered protein comprising a homologous ADP-ribosylation factor 1 (ARF1) protein sequence. In one aspect, polypeptides described herein that are useful for treating an inflammatory disorder comprise a modified protein comprising a homologous ADP-ribosylation factor 1 (ARF1) protein sequence.

[0077] In an aspect, polypeptides described herein comprising a homologous ARF1 protein sequence are formulated in a pharmaceutical composition. In certain embodiments, polypeptides described herein comprising a homologous ARF1 protein sequence are included in a composition comprising a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, polypeptides described herein comprising a homologous ARF1 protein sequence are produced by in vitro techniques and included in a composition to be administered to a subject having obesity, an obesity-related disorder. In some embodiments, polypeptides described herein comprising a homologous ARF1 protein sequence are produced by in vitro techniques and included in a composition to be administered to a subject having obesity. In some embodiments, polypeptides described herein comprising a homologous ARF1 protein sequence are produced by in vitro techniques and included in a composition to be administered to a subject that is overweight.

[0078] In an aspect, polypeptides described herein comprising a homologous ARF1 protein sequence comprise a covalently -coupled protein trafficking domain sequence. In some embodiments, the covalently-coupled protein trafficking domain sequence improves a biological function of an engineered polypeptide described herein. In some embodiments, the improved biological function comprises improved protein-protein interaction between the engineered ARF1polypeptide and an endogenous ARF1 binding molecule. In some embodiments, the improved biological function comprises a modification in cell surface binding between the engineered ARF1 polypeptide and an endogenous ARF1 binding molecule. In some embodiments, the improved biological function comprises a modification in cell surface binding between the engineered ARF1 polypeptide cellular trafficking components. In some embodiments, the improved biological function comprises a modification in interaction between the engineered ARF1 polypeptide and cellular components involved in caveolin-mediated endocytosis. In some embodiments, a covalently-coupled protein trafficking domain sequence alters a site for myristoylation. In some embodiments, a covalently-coupled protein trafficking domain sequence is positioned so that a glycine residue that would typically be myristoylated following translation of a endogenously expressed ARF1 protein (e.g., G2 in wild-type human ARF1 listed in SEQ ID NO: 1) does not have a myristoyl group added to the glycine residue of the glycine residue of the homologous ARF1 protein sequence and is thereby not myristoylated. In some embodiments, the covalently-coupled protein trafficking domain sequence or a portion thereof is cleaved off from the engineered ARF1 protein to yield a mature form of engineered ARF1 protein. In some embodiments, a covalently- coupled protein trafficking domain sequence increases stability of the engineered polypeptide. _In some embodiments, a covalently -coupled protein trafficking domain sequence increases in vivo half-life of the engineered polypeptide.Jn some embodiments, a covalently -coupled protein trafficking domain sequence improves in vitro expression of the engineered polypeptide.Jn some embodiments, a covalently-coupled protein trafficking domain sequence improves in vitro secretion of the engineered polypeptide. In some embodiments, a covalently-coupled protein trafficking domain sequence improves in vitro purification of the engineered polypeptide.

[0079] In some embodiments, the engineered ARF1 polypeptide comprise a covalently-linked modification comprising attachment of a sugar moiety to one or more amino acid residues of the engineered polypeptide. In some embodiments, polypeptides comprising a homologous ARF1 protein sequence comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or N-linked glycans. In some embodiments, one or more glycosylated residues of the engineered polypeptide increase at least one of stability, biological function, or in vivo half-life of the homologous ARF1 protein sequence. In some embodiments, one or more glycosylated residues of the engineered polypeptide improve at least one of in vitro expression, in vitro secretion, or in vitro purification of the homologous ARF1 protein sequence.

[0080] In some aspects, polypeptides described herein comprising a homologous ARF1 protein sequence are engineered ARF1 polypeptides. In some embodiments, the engineered ARF1 polypeptides are therapeutic polypeptides. In some embodiments, the engineered polypeptidecomprises one or more amino acid modifications that promote protein stability. In some embodiments, the engineered polypeptide comprises one or more amino acid modifications that increases protein stability compared to an unmodified protein. In some embodiments, the engineered polypeptide comprises one or more amino acid modifications that increases a biological function. In some embodiments, the engineered polypeptide comprises one or more amino acid modifications that increases a biological function compared to an unmodified protein. In some embodiments, the biological function comprises improved binding to an endogenous ARF1 binding protein. In some embodiments, the biological function comprises improved extracellular binding to an endogenous ARF1 binding protein. In some embodiments, the engineered ARF1 polypeptides comprises one or more amino acid modifications that increases in vivo half-life. In some embodiments, the modified protein comprises one or more amino acid modifications that increases in vivo half-life of a component of the engineered polypeptide comprising a homologous ARF1 protein sequence. In some embodiments, the engineered polypeptide comprises one or more amino acid modifications that improves in vitro expression. In some embodiments, the engineered polypeptide comprises one or more amino acid modifications that facilitate protein production. In some embodiments, the engineered polypeptide comprises one or more amino acid modifications that improves in vitro purification. In some embodiments, the engineered polypeptide comprises one or more amino acid modifications that improves in vitro secretion.

[0081] Additional modifications to the engineered ARF1 polypeptides described herein comprise deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, or more amino acids from the N- terminal region of the homologous ARF1 protein sequence. Additional modifications to the engineered ARF1 polypeptides described herein comprise deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, or more amino acids from the C-terminal region of the homologous ARF1 protein sequence. Additional modifications to the engineered ARF1 polypeptides described herein comprise deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, or more amino acids from a region internal to the N-terminus and C-terminus of the homologous ARF1 protein sequence. In some embodiments, the deletion is a deletion of a known inhibitory domain or domains of a homologous ARF1 protein sequence. In some embodiments, the deletion is a deletion of one or more aminoacids known to be required for a GTP-GDP exchange reaction. In some embodiments, the deletion is a deletion of one or more amino acids known to be required for dissociation of GDP from the GDP -bound form of the small GTPase. In some embodiments, the deletion is a deletion of one or more amino acids known to be required for association of GTP to a guanine-nucleotide free form of the small GTPase. In some embodiments, the deletion is a deletion of one or more amino acids known to be required for the catalytic hydrolysis of GTP. In some embodiments, the deletion is a deletion of one or more amino acids found in an alpha helix structure. In some embodiments, the deletion is a deletion of one or more amino acids comprising an alpha helix structure located near the N-terminus of the homologous ARF1 protein sequence. In some embodiments, the deletion is a deletion of amino acids comprising an alpha helix structure located near the N-terminus of the homologous ARF1 protein sequence. A non-limiting example of deletion is a deletion of one or more amino acids comprising an alpha helix structure located near the N-terminus of the homologous ARF1 protein sequence is listed in SEQ ID NO: 209. In some embodiments, the deletion is a deletion of 13 amino acids from the N-terminal region of the homologous ARF1 protein sequence corresponding to the sequence of SEQ ID NO: 1. In some embodiments, the deletion is a deletion of one or more amino acids comprising an alpha helix structure located near the C-terminus of the homologous ARF1 protein sequence. In some embodiments, the deletion is a deletion of amino acids comprising an alpha helix structure located near the C-terminus of the homologous ARF1 protein sequence. A non-limiting example of deletion is a deletion of one or more amino acids comprising an alpha helix structure located near the C-terminus of the homologous ARF1 protein sequence is listed in SEQ ID NO: 210. In some embodiments, the deletion is a deletion of 23 amino acids from the C-terminal region of the homologous ARF1 protein sequence corresponding to the sequence of SEQ ID NO: 1.

[0082] Additional modifications to the engineered ARF1 polypeptides described herein comprise insertions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180 or more amino acids into or proximal to the N-terminal region of the homologous ARF1 protein sequence. Additional modifications to the Engineered ARF1 polypeptides described herein comprise insertions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, or more amino acids into or distal to the C-terminal region of the homologous ARF1 protein sequence. Additional modifications to the engineered ARF1 polypeptides described herein comprise insertions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, or more amino acids into a region internal to the N-terminus and C-terminus of the homologous ARF1 protein sequence.

[0083] Additional modifications to the engineered ARF1 polypeptides described herein comprise substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, or more amino acids in the N-terminal region of the homologous ARF1 protein sequence. Additional modifications to the engineered ARF1 polypeptides described herein comprise substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, or more amino acids in the C-terminal region of the homologous ARF1 protein sequence. Additional modifications to the Engineered ARF1 polypeptides described herein comprise substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, or more amino acids in a region internal to the N-terminus and C-terminus of the homologous ARF1 protein sequence. In some embodiments, the amino acid substitution or substitutions are conservative substitutions. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Exemplary conservative amino acid substitutions are listed in Table 3. In some embodiments, the amino acid substitution or substitutions are not conservative substitutions. In some embodiments, the one or more single amino acid substitutions are constructed in the engineered ARF1 protein within the homologous ARF1 protein sequence. In some embodiments, the one or more single amino acid substitutions within the homologous ARF1 protein sequence are included within a single amino acid polypeptide chain which forms the engineered ARF1 protein. In some embodiments, a single amino acid substitutions within the homologous ARF1 protein sequence is included within a single amino acid polypeptide chain which forms the engineered ARF1 protein. Non-limited examples of single amino acid substitutions within the homologous ARF1 protein sequence that are included either individually within the engineered ARF1 protein or combined within the engineered ARF1 protein are selected from: T31N, Q71L, R99H, Y35H, K127E, T48I, F51L, P131R, P131L, R19C, N126I, D129N, N3A, I4A, F5A, N7A, L8A, F9A, K10A, L12A, F13A, K15A, K16A, E17A, P47A, T48A, I49A, G50A, W66A, D67A, V68A, G69A, G70A, Q71A, H80A, N126A, T158A, C159A, L25F, L25M, L25W, L25Y, D26E, K30H, K30R, T32N, T32Q, T32S, Q71F, Q71I, Q71V, Q71W, K127H, K127R, Q128N, Q128S, Q128T, D129E, C159S, F9L, FBI, N60G, N60Q, T31Q, T31S,T31H, T31R, T31K, T31E, T31D, T31A, T31L, T31I, T31M, T31F, T31W, T31Y, T31V, T31G, T31P, or T31C. In some embodiments, the single amino acid substitutions within the homologous ARF1 protein sequence comprises a mutation at position T31. In some embodiments, the single amino acid substitution within the homologous ARF1 protein sequence comprises a mutation at position T3 IX, wherein X is N, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, E, D, S, or T. In some embodiments, the single amino acid substitution within the homologous ARF1 protein sequence comprises T31N substitution.

[0084] In some aspects, polypeptides described herein comprising a homologous ARF protein sequence are engineered ARF polypeptides comprising sequence derived from an ARF protein family member. In some embodiments, engineered ARF polypeptides are derived from human ARF3 sequence. In some embodiments, engineered ARF polypeptides are derived from human ARF4 sequence. In some embodiments, engineered ARF polypeptides are derived from human ARF5 sequence. In some embodiments, engineered ARF polypeptides are derived from human ARF6 sequence. In some embodiments, engineered ARF polypeptides are derived from human ARF7 sequence. In some embodiments, engineered ARF polypeptides are derived from human ALF-like 1 sequence. In some embodiments, engineered ARF polypeptides are derived from human ALF-like 2 sequence. In some embodiments, engineered ARF polypeptides are derived from human ALF-like 3 sequence. In some embodiments, engineered ARF polypeptides are derived from human ALF-like 4 sequence. In some embodiments, engineered ARF polypeptides are derived from human ALF-like 5 sequence. In some embodiments, engineered ARF polypeptides are derived from human ALF-like 5a sequence. In some embodiments, engineered ARF polypeptides are derived from human ALF-like 7 sequence. In some embodiments, engineered ARF polypeptides are derived from human ALF-like 4c sequence. In some embodiments, engineered ARF polypeptides are derived from human ALF-like 5b sequence. In some embodiments, engineered ARF polypeptides are derived from human ALF-like 10B sequence. In some embodiments, engineered ARF polypeptides are derived from a sequence listed in Table 4. In some embodiments, engineered ARF polypeptides comprise one or more amino acid deletions, additions, or substitutions within the homologous ARF protein sequence protein region. In some embodiments, engineered ARF polypeptides comprise one or more amino acid substitutions within the homologous ARF protein sequence protein region at positions that align with human ARF1 sequence and incorporate one or more of the selected substitutions: T31N, Q71L, R99H, Y35H, K127E, T48I, F51L, P131R, P131L, R19C, N126I, D129N, N3A, I4A, F5A, N7A, L8A, F9A, K10A, L12A, F13A, K15A, K16A, E17A, P47A, T48A, I49A, G50A, W66A, D67A, V68A, G69A, G70A, Q71A, H80A, N126A, T158A, C159A, L25F, L25M, L25W, L25Y,D26E, K30H, K30R, T32N, T32Q, T32S, Q71F, Q71I, Q71V, Q71W, K127H, K127R, Q128N, Q128S, Q128T, D129E, C159S, F9L, FBI, N60G, N60Q, T31Q, T31S, T31H, T31R, T31K, T3 IE, T3 ID, T31 A, T3 IL, T3 II, T3 IM, T3 IF, T31W, T31 Y, T3 IV, T31G, T3 IP, or T31C. In non-limiting examples, the engineered ARF polypeptide comprises sequence derived from any one of SEQ ID NOs: 87-106, wherein one or more of the following substitutions is selected for inclusion in the engineered ARF polypeptide: T31N, Q71L, R99H, Y35H, K127E, T48I, F51L, P131R, P131L, R19C, N126I, D129N, N3A, I4A, F5A, N7A, L8A, F9A, K10A, L12A, F13A, K15A, K16A, E17A, P47A, T48A, I49A, G50A, W66A, D67A, V68A, G69A, G70A, Q71A, H80A, N126A, T158A, C159A, L25F, L25M, L25W, L25Y, D26E, K30H, K30R, T32N, T32Q, T32S, Q71F, Q71I, Q71V, Q71W, K127H, K127R, Q128N, Q128S, Q128T, D129E, C159S, F9L, FBI, N60G, N60Q, T31Q, T31S, T31H, T31R, T31K, T31E, T31D, T31A, T31L, T31I, T31M, T3 IF, T31W, T31 Y, T3 IV, T31G, T3 IP, or T31C.

[0085] Additional modifications to the Engineered ARF1 polypeptides described herein comprise deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, or more amino acids from an inserted heterologous polypeptide sequence, that has been inserted into the engineered polypeptide to increase at least one of stability, biological function, or in vivo half-life of the engineered polypeptide or of a component of the engineered polypeptide comprising the homologous ARF1 protein sequence. Additional modifications to the engineered ARF1 polypeptides comprising a homologous ARF1 protein sequence comprise insertions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, or more amino acids into an inserted heterologous polypeptide sequence, that has been inserted into the modified protein to increase at least one of stability, biological function, or in vivo half-life of the modified protein or of a component of the engineered polypeptide comprising the homologous ARF1 protein sequence. Additional modifications to the engineered ARF1 polypeptides comprising a homologous ARF1 protein sequence comprise substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, or more amino acids in an inserted heterologous polypeptide sequence, that has been inserted into the engineered polypeptide to increase at least one of stability, biological function, or in vivo half-life of the engineered polypeptide or of a component of the engineered polypeptide comprising the homologous ARF1 protein sequence. In some embodiments, the amino acid substitution orsubstitutions are conservative substitutions listed in Table 3. In some embodiments, the amino acid substitution or substitutions are not conservative substitutions.

[0086] In some embodiments, the engineered polypeptide or a component of the engineered polypeptide comprising the homologous ARF1 protein sequence can comprise cleavage products of a pro-protein. Cleavage of a pro-protein can result in activation or higher activity of said proprotein. In certain aspects, the polypeptides comprise only the active domain of a homologous ARF1 pro-protein (e.g., a minimal portion sufficient to create a biological effect). In some embodiments, a component of the modified protein comprising the homologous ARF1 protein sequence is a product or a portion corresponding to a larger modified protein (e.g., a cleavage product). In some embodiments, a covalently-coupled protein trafficking domain sequence is cleaved off of engineered polypeptide. In non-limiting examples described herein, the engineered ARF1 protein comprises a TEV protease recognition motif that may be cleaved off during a step of protein isolation or purification to yield a cleavage product of the pro-protein that comprises homologous ARF1 protein sequence. In some embodiments, the cleavage product comprising the ARF1 protein sequence is formulated in a pharmaceutical composition for systemic delivery to a subject in need thereof.

[0087] In certain aspects, a composition consists essentially of an engineered polypeptide described herein or of a component of the engineered polypeptide comprising the homologous ARF1 protein sequence, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the engineered polypeptide or the component of the engineered polypeptide comprising the homologous ARF1 protein sequence comprises an amino acid sequence listed in Table 1A or Table IB. In certain aspects, a composition consists essentially of an engineered polypeptide described herein or of a component of the engineered polypeptide comprising the homologous ARF protein sequence, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the engineered polypeptide or the component of the engineered polypeptide comprising the homologous ARF1 protein sequence comprises an amino acid sequence listed in Table 4.

[0088] In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 1, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 2, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, thecomposition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 3, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:3. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 4, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 5, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:5. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 6, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:6. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 7, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:7. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 8, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:8. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 9, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:9. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 10, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:10. In certain aspects, described herein is a composition comprising a polypeptide comprising atleast about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 11, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:11. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 12, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:12. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 13, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:13. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 14, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:14. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 15, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:15. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 16, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:16. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 17, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 18, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 19, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, thecomposition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:19. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 20, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:20. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 21, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:21. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 22, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:22. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 23, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:23. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 28, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:28. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 29, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:29. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 30, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:30. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 31, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:31. In certain aspects, described herein is a composition comprising a polypeptide comprising atleast about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 32, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:32. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 33, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:33. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 34, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:34. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 35, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:35. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 36, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:36. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 37, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:37. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 38, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 39. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 39, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 39. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 40, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, thecomposition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 40. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 54, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:54. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 55, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 56, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:56. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 57, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:57. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 58, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:58. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 59, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:59. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 60, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:60. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 61, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:61. In certain aspects, described herein is a composition comprising a polypeptide comprising atleast about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 62, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:62. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 63, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:63. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 64, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:64. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 65, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:65. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 66, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:66. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 67, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:67. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 68, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:68. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 69, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:69. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 70, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, thecomposition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 70. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 73, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:73. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 74, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:74.

[0089] In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 123, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 123. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 124, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 124. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to any one of SEQ ID NOs: 125-171, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in a sequence selected from SEQ ID NOs: 125-171. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to any one of SEQ ID NOs: 172-174, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in a sequence selected from SEQ ID NOs: 172-174. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 175, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 175. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 176, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, thecomposition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 176. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to any one of SEQ ID NOs: 177-180, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in a sequence selected from SEQ ID NOs: 177-180. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to any one of SEQ ID NOs: 181-182, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in a sequence selected from SEQ ID NOs: 181-182. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 183, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 183. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to any one of SEQ ID NOs: 184-186, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in a sequence selected from SEQ ID NOs: 184-186. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to any one of SEQ ID NOs: 187-189, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in a sequence selected from SEQ ID NOs: 187-189. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to any one of SEQ ID NOs: 190-196, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in a sequence selected from SEQ ID NOs: 190-196.b In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to any one of SEQ ID NOs: 197-200, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in a sequence selected from SEQ ID NOs: 197-200. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to anyone of SEQ ID NOs: 201-214, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in a sequence selected from SEQ ID NOs: 201-214. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to any one of SEQ ID NOs: 215-218, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in a sequence selected from SEQ ID NOs: 215-218. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to any one of SEQ ID NOs: 209-244, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in a sequence selected from SEQ ID NOs: 209-244. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to any one of SEQ ID NOs: 245-284 or 291-292, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in a sequence selected from SEQ ID NOs: 245-284 or 291-292. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 285, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 285. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 286, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 286. In certain aspects, described herein is a composition consisting essentially of a polypeptide of SEQ ID NO: 286, and a pharmaceutically acceptable excipient, carrier, or diluent. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 287, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 287. In certain aspects, described herein is a composition consisting essentially of a polypeptide of SEQ ID NO: 287, and a pharmaceutically acceptable excipient, carrier, or diluent. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 288, and apharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 288. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 289, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 289. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 293, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 293. In certain aspects, described herein is a composition consisting essentially of a polypeptide of SEQ ID NO: 293, and a pharmaceutically acceptable excipient, carrier, or diluent. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 294, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 294. In certain aspects, described herein is a composition consisting essentially of a polypeptide of SEQ ID NO: 294, and a pharmaceutically acceptable excipient, carrier, or diluent. . In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 295, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 295. . In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 296, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 296. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to any one of SEQ ID NOs: 297-311, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the amino acid sequence set forth in a sequence selected from SEQ ID NOs: 297-311. In certain aspects, described herein is a composition comprising a polypeptide comprising at least about 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity to any one of SEQ ID NOs: 315-317, and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the composition comprises a polypeptide comprising the aminoacid sequence set forth in a sequence selected from SEQ ID NOs: 315-317. In some embodiments, the composition comprises the peptide tag of SEQ ID NO: 24. In some embodiments, the composition comprises the peptide tag of SEQ ID NO: 25. In some embodiments, the composition comprises the TEV protease recognition motif of SEQ ID NO: 26 or SEQ ID NO; 290. In some embodiments, the composition comprises the myristoylation sequence, peptide tag, and TEV protease recognition motif of SEQ ID NO: 27. In some embodiments, the composition comprises a peptide tag sequence selected from SEQ ID NOs: 41-49. In some embodiments, the composition comprises the HSA domain sequence of SEQ ID NO: 51. In some embodiments, the composition comprises the HSA domain sequence of SEQ ID NO: 52. In some embodiments, the composition comprises the human IgGl Fc domain sequence of SEQ ID NO: 50. In some embodiments, the composition comprises the Annexion A10 peptide sequence of SEQ ID NO: 53. In some embodiments, the composition has a portion of sequence corresponding to SEQ ID NO: 285 cleaved from the mature engineered ARF1 protein following TEV protease digestion. In some embodiments, the composition comprises a peptide linker comprising of a sequence selected from SEQ ID NOs: 75-76 or 312-314.

[0090] In certain aspects, compositions of engineered ARF1 polypeptides described herein comprise polypeptides that decrease the proliferation of adipocytes cells. In certain aspects, the polypeptides decrease proliferation of an adipocyte or an adipocyte progenitor cell by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to an adipocyte or an adipocyte progenitor cell not treated with the polypeptide. Proliferation can be measured by BrdU or EdU incorporation, which can be quantified using suitable methods such as, by way of nonlimiting embodiment, microscopy, flow cytometry, or ELISA. In some embodiments, contacting a plurality of target cells with a composition described herein decreases adipocyte proliferation. In some embodiments, contacting a plurality of target cells with a composition described herein decreases adipocyte proliferation in cells from a subject having obesity or an obesity-related disorder. In some embodiments, contacting a plurality of target cells with a composition described herein decreases adipocyte proliferation in cells from a subject having lipedema.

[0091] In certain aspects, compositions of engineered ARF1 polypeptides described herein comprise polypeptides that modulate differentiation of adipocyte stem cells. In certain aspects, the polypeptides reduce differentiation of adipocyte stem cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to adipocytes not treated with the polypeptide. Differentiation of adipocyte stem cells can be measured by BrdU or EdU incorporation, which can be quantified using suitable methods such as, by way of non-limiting embodiment, microscopy, flow cytometry, or ELISA; and by induction of, or expression level of, one or more marker genesassociated with adipocyte differentiation into adipocytes. An example of a marker gene associated with differentiation of adipocyte stem cells is KLF4. KLF4 expression can be quantified using suitable methods such as, by way of non-limiting embodiment, fluorescence microscopy using an anti-KLF4 antibody, flow cytometry, ELISA, RNAseq, or in situ hybridization. In some embodiments, contacting a plurality of target cells with a composition described herein reduces differentiation of adipocyte stem cells. In some embodiments, contacting a plurality of target cells with a composition described herein reduces differentiation of adipocyte stem cells from a subject having obesity or an obesity-related disorder. In some embodiments, contacting a plurality of target cells with a composition described herein reduces differentiation of adipocyte stem cells from an overweight subject. In some embodiments, contacting a plurality of target cells with a composition described herein reduces differentiation of adipocyte stem cells from a subject having lipedema.

[0092] In certain aspects, compositions of engineered ARF1 polypeptides described herein comprise polypeptides that increase lipolysis in adipocytes. In certain aspects, the polypeptides increase lipolysis by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to adipocytes not treated with the polypeptide. Lipolysis can be measured by taking a measurement of lipolytic products released from adipocytes or fat explants. In some embodiments, measuring fatty acids and glycerol released into incubation media is used as a measurement of lipolysis in vitro in cultured adipocytes.

[0093] In some aspects, the engineered polypeptide comprises a homologous ADP-ribosylation factor 1 (ARF1) protein sequence, and a heterologous polypeptide amino acid sequence inserted within or adjacent to the homologous ARF1 protein sequence. In some embodiments, the engineered polypeptide comprises a ARF1 protein sequence, and a heterologous polypeptide amino acid sequence inserted within the ARF1 protein sequence. In some embodiments, the engineered polypeptide comprises a ARF1 protein sequence, and a heterologous polypeptide amino acid sequence inserted N-terminal to the ARF1 protein sequence. In some embodiments, the engineered polypeptide comprises a ARF1 protein sequence, and a heterologous polypeptide amino acid sequence inserted C-terminal to the ARF1 protein sequence. In some embodiments, the inserted heterologous polypeptide amino acid sequence increases at least one of stability, biological function, or in vivo half-life of the engineered ARF1 polypeptide or of a component of the engineered polypeptide comprising the homologous ARF1 protein sequence. In some embodiments, the inserted heterologous polypeptide amino acid sequence improves at least one of in vitro expression, in vitro secretion, or in vitro purification of the engineered polypeptide or of a component of the engineered polypeptide comprising the homologous ARF1 protein sequence. In some embodiments, the homologous ARF1 protein sequence comprises an amino acid sequence atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 1-23. In some embodiments, the homologous ARF1 protein sequence comprises the amino acid sequence set forth in any one of SEQ ID NOS: 1-23. In some embodiments, amino acid positions listed as X in SEQ ID NO: 23 can be any natural amino acid. In some embodiments, the heterologous polypeptide amino acid sequence comprises a covalently-coupled protein trafficking domain sequence. In some embodiments, the covalently- coupled protein trafficking domain sequence comprises a signal peptide sequence. In some embodiments, the signal peptide sequence comprises an amino acid sequence selected from SEQ ID NOs: 77-86. In some embodiments, the covalently-coupled protein trafficking domain sequence comprises a site for myristoylation. In some embodiments, the site for myristoylation is located at or near the N-terminus of the engineered polypeptide. In some embodiments, the site for myristoylation comprises a glycine residue. In some embodiments, the site for myristoylation comprises the peptide sequence MGSS. In some embodiments, the site for myristoylation comprises a glycine residue. In some embodiments, the site for myristoylation is specifically blocked or removed. In some embodiments, a site typically myristoylated in wild-type ARF1 protein is configured in the engineered ARF1 protein to prevent addition of a myristoyl group to a glycine residue. Position G2 from SEQ ID NO: 1 is typically myristoylated in endogenously produced wild-type ARF1 protein. In some embodiments, the sequence corresponding to G2 in the homologous ARF1 protein sequence is mutated to a natural amino acid residue other than glycine. In some embodiments, the sequence corresponding to G2 in the homologous ARF1 protein sequence is positioned within the engineered ARF1 protein at a location in a single polypeptide chain other than the second amino acid following an initiation methionine, thereby preventing myristoylation. In some embodiments, a site typically myristoylated in wild-type ARF family member protein is configured in the engineered ARF protein to prevent addition of a myristoyl group to a glycine residue. Engineered ARF1 protein designed to prevent myristoylation of an N- terminal glycine residue has modified protein trafficking dynamics compared to endogenously produced ARF1 protein. In some embodiments, the modified protein trafficking dynamics of the engineered ARF1 protein improves function of an ARF1 therapeutic protein. In some embodiments, engineered ARF1 protein form predominantly monomeric structures. In some instances, engineered ARF1 protein existing predominantly in an conformation that preferentially binds GDP form predominantly monomeric structures. In some instances, engineered ARF1 protein existing predominantly in an GDP-locked conformation form predominantly monomeric structures. In some embodiments, engineered ARF1 protein form predominantly dimeric structures. Nonlimiting examples of ARF1 substitutions that produce engineered ARF1 protein existingpredominantly in an GDP -locked conformation comprise single amino acid substitution at position T31 of wild-type human ARF1 of SEQ ID NO: 1. In some embodiments, the T31 substitution is a T31N substitution.

[0094] In some embodiments, the engineered polypeptide comprises a peptide tag. In some embodiments, the peptide tag is positioned N-terminal to the homologous ARF1 protein sequence. In some embodiments, the peptide tag is positioned C-terminal to the homologous ARF1 protein sequence. In some embodiments, the peptide tag can be used to aid in purification of the homologous ARF1 protein sequence. In some embodiments, the peptide tag can be used to aid in production of the homologous ARF1 protein sequence. In some embodiments, the peptide tag can be used to aid in purification of secreted engineered ARF1 polypeptide. In some embodiments, the peptide tag can be used to aid in purification of a secreted component of engineered ARF1 polypeptide comprising the homologous ARF1 protein sequence. In some embodiments, the peptide tag comprises a length of between 4 to 22 amino acids. In some embodiments, the peptide tag is recognized as an epitope and is capable of being bound by a specific antibody. In some embodiments, the peptide tag is positioned between the homologous ARF1 sequence and the covalently-coupled protein trafficking domain sequence. In some embodiments, the peptide tag comprises a peptide length of 4 to 22 amino acids, wherein the peptide tag is positioned at the N- terminus of the engineered polypeptide. In some embodiments, the peptide tag comprises a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is positioned at the C-terminus of the engineered polypeptide. In some embodiments, the peptide tag is selected from a six histidine short peptide, a serine-six histidine short peptide, a Strep-tag, a Ser-Strep tag, an hemagglutinin tag, a Myc-tag, a synthetic NE-tag, a FLAG octapeptide tag, a 3xFLAG peptide tag, and a Spot-tag recognized by nanobody. In some embodiments, the peptide tag comprises an amino acid sequence selected from SEQ ID NOS: 24 or 41-49. In some embodiments, the myristoylation sequence MGSS is positioned adjacent to and N-terminal to the peptide tag sequence. In some embodiments, the protein trafficking domain sequence comprises an Annexin A10 peptide sequence. In some embodiments, the Annexin A10 peptide sequence is a vertebrate Annexin A10 peptide sequence. In some embodiments, the Annexin A10 peptide sequence is a mammalian Annexin A10 peptide sequence. In some embodiments, the Annexin A10 peptide sequence is a human Annexin A10 peptide sequence. In some embodiments, the Annexin A10 peptide sequence comprises an amino acid sequence at least about 95% identical to SEQ ID NO: 53. In some embodiments, the Annexin A10 peptide sequence comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, a peptide protein sequence positioned N-terminal to the homologous ARF1 protein sequence in the engineered ARF1 polypeptide can occlude anamphipathic helix in the N-terminal region of the homologous ARF1 protein sequence. In some embodiments, a peptide protein sequence positioned N-terminal to the homologous ARF1 protein sequence in the engineered ARF1 polypeptide can alter the 3-D shape of an amphipathic helix in the N-terminal region of the homologous ARF1 protein sequence. In some embodiments, the peptide protein sequence positioned N-terminal to the homologous ARF1 protein sequence may share a collective property of altering membrane association kinetics of engineered ARF1 polypeptides that are contacted with the extracellular surface of cells. In some embodiments, the peptide protein sequence positioned N-terminal to the homologous ARF1 protein sequence prevents myristoylation of a glycine residue. In some embodiments, the peptide tag is cleaved away from the homologous ARF1 protein sequence during isolation and / or purification of the mature engineered ARF1 protein. In some embodiments, the peptide tag serves as a covalently-coupled protein trafficking domain sequence by preventing myristoylation of a glycine residue in the homologous ARF1 protein sequence of the engineered ARF1 protein. In some embodiments, a covalently-coupled protein trafficking domain is added to the N-terminus of the homologous ARF sequence. In some embodiments, covalently-coupled protein trafficking domain comprises a peptide tag, a signal sequence, a protease recognition motif, a site for myristoylation, or any combination thereof. In some embodiments, covalently-coupled protein trafficking domain comprises a peptide tag, a signal sequence, a protease recognition motif, or any combination thereof designed such that a glycine residue within the homologous ARF sequence is not myristoylated. In some embodiments, a glycine residue near the N-terminus of the homologous ARF sequence is mutated to a natural amino acid other than glycine. In some embodiments, a glycine residue near the N-terminus of the homologous ARF sequence is deleted from the engineered protein. In some embodiments, a glycine residue near the N-terminus of the homologous ARF sequence has an insertion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 or more amino acids N-terminal to the position of the glycine residue in the homologous ARF sequence which prevents myristoylation of the glycine.

[0095] In some embodiments, the engineered polypeptide described herein comprises a cleavage site within the amino acid sequence of a single polypeptide chain. In some embodiments, recognition of the cleavage site by a cellular protease can produce site-specific cleavage within the single polypeptide chain of the engineered polypeptide. In some embodiments, the signal peptide is cleaved from the remainder of the engineered polypeptide resulting in a portion of the single polypeptide chain comprising the homologous ARF1 protein sequence being separated from the signal peptide. In some embodiments, cleavage site is inserted between the homologous ARF1 sequence and the covalently-coupled protein trafficking domain sequence. In some embodiments,the cleavage site is inserted adjacent to the peptide tag. In some embodiments, the cleavage site comprises a Tobacco etch virus (TEV) protease recognition motif. In some embodiments, the TEV protease recognition motif comprises the amino acid sequence of SEQ ID NO: 26 or 290. In some embodiments, the engineered polypeptide is exposed to TEV protease. In some embodiments, the TEV protease recognizes the specific amino acid sequence ENLYFQG / S and cleaves between Q and G / S. In some embodiments, cleavage of the site-specific protease recognition sequence results in removal of N-terminal amino acid sequences adjacent to the homologous ARF1 sequence. In some embodiments, cleavage of the site-specific protease recognition sequence results in removal of C-terminal amino acid sequences adjacent to the homologous ARF1 sequence. In some embodiments, a heterologous peptide sequence adjacent to the homologous ARF1 sequence is removed from the single polypeptide chain. In some embodiments, a heterologous protein sequence adjacent to the homologous ARF1 sequence is removed from the single polypeptide chain. In some embodiments, the removed sequence adjacent to the homologous ARF1 sequence comprise a peptide tag. In some embodiments, a covalently-coupled protein trafficking domain sequence adjacent to the homologous ARF1 sequence is removed from the single polypeptide chain.

[0096] In some embodiments, the engineered polypeptide described herein comprises one or more protein linker sequences. In some embodiments, the engineered polypeptide comprises a first linker peptide comprising an amino acid sequence set forth in SEQ ID NO: 75-76 or 312-314. In some embodiments, the first linker peptide comprises SEQ ID NO: 75 and is positioned N-terminal to the amino acid sequence of selected from SEQ ID NOS: 1-23. In some embodiments, the first linker peptide comprises SEQ ID NO: 76 and is positioned N-terminal to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first linker peptide comprises SEQ ID NO: 312 and is positioned N-terminal to the amino acid sequence of selected from SEQ ID NOS: 1-23. In some embodiments, the first linker peptide comprises SEQ ID NO: 313 and is positioned N-terminal to the amino acid sequence of selected from SEQ ID NOS: 1-23. In some embodiments, the first linker peptide comprises SEQ ID NO: 314 and is positioned N-terminal to the amino acid sequence of selected from SEQ ID NOS: 1-23. In some embodiments, the first linker peptide is positioned at the C-terminal portion of the heterologous polypeptide amino acid sequence comprising a sequence selected from SEQ ID NOS: 1-23. In some embodiments, the engineered polypeptide comprises a second linker peptide. In some embodiments, the second linker peptide comprises an amino acid sequence set forth in SEQ ID NO: 75-76 or 312-314. In some embodiments, the second linker peptide is positioned at the N-terminal portion of the heterologous polypeptide amino acid sequence comprising a sequence selected from SEQ ID NOS: 1-23.

[0097] In some aspects described herein, an engineered polypeptide comprises a homologousADP-ribosylation factor 1 (ARF1) sequence, and a half-life extending moiety. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 1. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 1-23. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 93% identical to SEQ ID NO: 23. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 95% identical to SEQ ID NO: 1. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOS: 1-22. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 123. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 123. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence selected from SEQ ID NO: 124-171. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 286. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 287. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 288. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 289. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 293. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 294. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 295. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 296. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence selected from SEQ ID NO: 297-311.

[0098] In some embodiments, the engineered ARF1 polypeptide comprises one or more mutations within the homologous ARF1 sequence relative to an amino acid sequence selected from SEQ ID NOS: 1-23. In some embodiments, the homologous ARF1 sequence comprises one, two, three, four, five, six, seven, eight, or nine amino acid substitutions relative to an amino acid sequence selected from SEQ ID NOs: 1-22. In some embodiments, the homologous ARF1 sequence comprises one or more deletions of one, two, three, four, five, six, seven, eight, nine, ten, or more amino acids relative to an amino acid sequence selected from SEQ ID NOs: 1-22. In someembodiments, the homologous ARF1 sequence comprises one or more deletions of one, two, or three amino acids relative to an amino acid sequence selected from SEQ ID NOs: 1-22. In some embodiments, the homologous ARF1 sequence comprises one or more insertions of one, two, three, four, five, six, seven, eight, or nine amino acids relative to an amino acid sequence selected from SEQ ID NOs: 1-22. In some embodiments, the amino acid substitutions are for any amino acid. In some embodiments, the amino acid substitutions comprise a conservative amino acid sequence. In some embodiments, the conservative amino acid substitution is listed in Table 3. In some embodiments, the homologous ARF1 sequence comprises an amino acid sequence selected from SEQ ID NOs: 1-22. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the homologous ARF1 sequence comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the homologous ARF1sequence comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the initiation methionine of the homologous ARF1 sequence is deleted from the engineered polypeptide. In non-limiting examples, the engineered ARF polypeptide comprises sequence derived from any one of sequences listed in Table 1A or Table IB, wherein one or more of the following substitutions is selected for inclusion in the engineered ARF polypeptide: T31N, Q71L, R99H, Y35H, K127E, T48I, F51L, P131R, P131L, R19C, N126I, D129N, N3A, I4A, F5A, N7A, L8A, F9A, K10A, L12A, F13A, K15A, K16A, E17A, P47A, T48A, I49A, G50A, W66A, D67A, V68A, G69A, G70A, Q71A, H80A, N126A, T158A, C159A, L25F, L25M, L25W, L25Y, D26E, K30H, K30R, T32N, T32Q, T32S, Q71F, Q71I, Q71V, Q71W, K127H, K127R, Q128N, Q128S, Q128T, D129E, C159S, F9L, FBI, N60G, N60Q, T31Q, T31S, T31H, T31R, T31K, T31E, T31D, T31 A, T3 IL, T3 II, T3 IM, T3 IF, T31W, T31 Y, T3 IV, T31G, T3 IP, or T31C based the amino acid positioning of SEQ ID NO: 1.

[0099] In some embodiments, the engineered ARF1 polypeptide comprising the half-life extending moiety has the half-life extending moiety positioned adjacent to the homologous ARF1 protein sequence. In some embodiments, the engineered ARF1 polypeptide comprising the half-life extending moiety has the half-life extending moiety positioned within to the homologous ARF1 protein sequence. In some embodiments, the half-life extending moiety is located N-terminal to the homologous ARF1 sequence. In some embodiments, the half-life extending moiety is located C- terminal to the homologous ARF1 sequence. In some embodiments, the half-life extending moiety comprises a fatty acid ester. In some embodiments, the half-life extending moiety does not comprise a fatty acid ester. In some embodiments, the half-life extending moiety comprises a heterologous polypeptide sequence. In some embodiments, the heterologous polypeptide sequence comprises a serum albumin sequence. In some embodiments, the heterologous polypeptide sequence comprises a mammalian serum albumin sequence. In some embodiments, the heterologous polypeptide sequence comprises a human serum albumin (HSA) sequence, or variant thereof. In some embodiments, the heterologous polypeptide sequence comprises a human serum albumin (HSA) sequence. In some embodiments, the HSA sequence is at least about 95% identical to SEQ ID NO: 51. In some embodiments, the HSA sequence is at least about 96%, 97%, 98%, or 99% identical to SEQ ID NO: 51. In some embodiments, the HSA sequence comprises an amino acid sequence identical to SEQ ID NO: 51. In some embodiments, the HSA sequence consists essentially of the amino acid sequence of SEQ ID NO: 51. In some embodiments, the half-life extending moiety comprises a protein sequence homologous to a mammalian immunoglobulin molecule. In some embodiments, the half-life extending moiety comprises a protein sequence homologous to one or more constant domains within a mammalian immunoglobulin molecule. Insome embodiments, the half-life extending moiety comprises a protein sequence homologous to one or more mammalian IgGl constant region domains. In some embodiments, the half-life extending moiety comprises a protein sequence homologous to one or more human IgGl constant region domains. In some embodiments, the heterologous polypeptide sequence comprises an Fc portion of a human immunoglobulin. In some embodiments, the heterologous polypeptide sequence comprises an Fc portion of a modified human immunoglobulin. In some embodiments, the Fc portion of the human immunoglobulin or of the modified human immunoglobulin comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 50. In some embodiments, the Fc portion of the human immunoglobulin comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, the Fc portion of the human immunoglobulin comprises one or more substitutions that modify effector function. In some embodiments, the one or more substitutions that modify effector function are selected from L234A, L235A, P329G, M252Y, S254T and T256E. In some embodiments, the one or more substitutions comprise L234A / L235A. In some embodiments, the one or more substitutions comprise L234A / L235A / P329G. In some embodiments, the one or more substitutions comprise M252Y, S254T and T256E. In some embodiments, the one or more substitutions comprise L234A, L235A, P329G, M252Y, S254T and T256E.

[0100] In some embodiments, the engineered ARF1 polypeptide comprising the half-life extending moiety comprises a protein trafficking domain sequence. In some embodiments, the protein trafficking domain sequence comprises a signal peptide sequence. In some embodiments, the signal peptide sequence comprises an amino acid sequence selected from SEQ ID NOs: 77-86. In some embodiments, the protein trafficking domain sequence comprises a site for myristoylation. In some embodiments, the site for myristoylation is located at or near the N-terminus of the engineered polypeptide. In some embodiments, the site for myristoylation comprises a glycine residue. In some embodiments, the site for myristoylation comprises the peptide sequence MGSS. In some embodiments, the protein trafficking domain sequence comprises a heterologous protein domain sequence that prevents myristoylation of a glycine residue in a homologous ARF1 protein sequence. In some embodiments, the engineered ARF1 polypeptide exhibits extended serum halflife in part due to a lack a myristoyl group attached to a glycine residue in the homologous ARF1 protein sequence.

[0101] In some embodiments, the engineered ARF1 polypeptide comprising the half-life extending moiety comprises a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is inserted within the engineered polypeptide. In some embodiments, the peptide tag is positioned between the homologous ARF1 sequence and the protein traffickingdomain sequence. In some embodiments, the peptide tag serves as the protein trafficking domain sequence by preventing myristoylation of a glycine residue in a homologous ARF1 protein sequence. In some embodiments, the engineered ARF1 polypeptide comprising the half-life extending moiety comprises a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is positioned at the N-terminus of the engineered polypeptide. In some embodiments, the engineered ARF1 polypeptide comprising the half-life extending moiety comprises a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is positioned at the C-terminus of the engineered polypeptide. In some embodiments, the engineered ARF1 polypeptide comprising the half-life extending moiety comprises a cleavage site. In some embodiments, the cleavage site is inserted between the homologous ARF1 sequence and protein trafficking domain sequence. In some embodiments, the cleavage site is adjacent to the homologous ARF1 sequence. In some embodiments, the cleavage site is positioned N-terminal to the homologous ARF1 sequence. In some embodiments, the cleavage site is positioned C-terminal to the homologous ARF1 sequence. In some embodiments, wherein the cleavage site is inserted adjacent to the peptide tag. In some embodiments, wherein the cleavage site comprises a TEV protease recognition motif. In some embodiments, the TEV protease recognition motif comprises the amino acid sequence of SEQ ID NO: 26 or 290.

[0102] Table 1A and Table IB list amino acid sequences for exemplary peptide and polypeptide components of engineered ARF1 polypeptides and exemplary version of full length engineered ARF1 polypeptides. In some embodiments, the engineered ARF1 polypeptide comprises a six histidine peptide tag of SEQ ID NO: 24. In some embodiments, the engineered ARF1 polypeptide comprises a myristoylation sequence-Six histidine short peptide of SEQ ID NO: 25. In some embodiments, the engineered ARF1 polypeptide comprises a TEV protease recognition motif of SEQ ID NO: 26 or 290. In some embodiments, the engineered ARF1 polypeptide comprises a Myristoylation sequence-Six histidine short peptide + TEV protease recognition motif of SEQ ID NO: 27. In some embodiments, the engineered ARF1 polypeptide comprises a peptide tag selected from SEQ ID NOS: 41-49. In some embodiments, the engineered ARF1 polypeptide comprises a Human IgGl Fc mutant of SEQ ID NO: 50. In some embodiments, the engineered ARF1 polypeptide comprises an HSA protein sequence of SEQ ID NO: 51. In some embodiments, the engineered ARF1 polypeptide comprises an HSA protein sequence of SEQ ID NO: 52. In some embodiments, the engineered ARF1 polypeptide comprises an Annexin A10 peptide sequence of SEQ ID NO: 53. In some embodiments, the engineered ARF1 polypeptide comprises a green fluorescent protein sequence of SEQ ID NO: 71. In some embodiments, the engineered ARF1 polypeptide comprises an enhanced green fluorescent protein sequence of SEQ ID NO: 72. In someembodiments, the engineered ARF1 polypeptide comprises a short flexible linker sequence of SEQ ID NO: 75. In some embodiments, the engineered ARF1 polypeptide comprises a serine-glycine linker sequence of GGGGS(n) wherein n is between 1-5. In some embodiments, the engineered ARF1 polypeptide comprises a long flexible linker sequence of SEQ ID NO: 76. In some embodiments, the engineered ARF1 polypeptide comprises a serine-glycine linker sequence of SEQ ID NO: 312. In some embodiments, the engineered ARF1 polypeptide comprises a serine- glycine linker sequence of SEQ ID NO: 313. In some embodiments, the engineered ARF1 polypeptide comprises a serine-glycine linker sequence of SEQ ID NO: 314. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 38. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 39. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 54. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 57. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 58. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 59. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 60. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 61.In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 62. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 63. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 64. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 65. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 66. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 67. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 68. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 69. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 70. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 73. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 74. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 123. In some embodiments, the engineered ARF1 polypeptide consists essentially of the amino acid sequence set forth in SEQ ID NO: 123. In some embodiments, the engineered ARF1 polypeptide consists essentially of the amino acid sequence set forth in SEQ ID NO: 123 and does not comprise a myristoylated glycine residue. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 124. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 125-284. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 285-289, 293-311, or 315-316. In some embodiments, the engineered ARF1 polypeptide consists essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 285-289, 293-311, or 315-316. In some embodiments, the engineered ARF1 polypeptide consists essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 285-289, 293-311, or 315-316 and does not comprise a myristoylated glycine residue. In some embodiments, the engineered ARF1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 317.Table 1A: Sequences for components of engineered polypeptides and full length Engineered ARF1 polypeptidesTable IB: Sequences for components of engineered polypeptides and full length EngineeredARF1 polypeptides

[0103] In some embodiments, the engineered ARF1 polypeptide comprises one or more post- translational modifications. In some embodiments, the one or more post-translational modifications comprise site-specific glycosylation of species amino acid residues of the modified protein.

[0104] In some embodiments, the engineered ARF1 polypeptide comprises one or more chemical modifications to the modified protein. In some embodiments, the one or more chemical modifications comprises a site-specific attachment of one or more fatty acid residues. In some embodiments, the one or more fatty acid residues are attached via covalent attachment to one or more cysteine residues of the engineered ARF1 polypeptide.Fusion Proteins

[0105] In some aspects, engineered ARF1 polypeptides described herein are fusion proteins. In some embodiments, the fusion protein comprises a homologous ARF1 protein sequence and a heterologous polypeptide amino acid sequence positioned N-terminal to the homologous ARF1 protein sequence. In some embodiments, the fusion protein comprises a ARF1 proprotein sequence and a heterologous polypeptide amino acid sequence positioned N-terminal to the ARF1 proprotein sequence. In some embodiments, the homologous ARF1 protein sequence comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence selected from in SEQ ID NO: 1-23. In some embodiments, the fusion protein comprises a ARF1 proprotein sequence and a heterologous polypeptide amino acid sequence positioned N-terminal to the ARF1 proprotein sequence. In some embodiments, the homologous ARF1 protein sequence comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the fusion protein comprises an HSA protein domain or variant thereof. In some embodiments, the fusion protein comprises a human IgGl constant domain region or variant thereof. In some embodiments, the fusion protein comprises a full-length HSA protein domain or functional portion thereof, followed by a linker sequence, fused directly to the N- terminus of a homologous ARF1 protein. In some embodiments, the fusion protein comprises a homologous ARF1 protein sequence, followed by a linker sequence, fused directly to a full-length HSA protein domain or functional portion thereof. In some embodiments, the fusion protein comprises an Fc portion of a human IgGl molecule or functional fragment thereof, followed by a linker sequence, fused directly to the N-terminus of a homologous ARF1 protein. In some embodiments, the fusion protein comprises a homologous ARF1 protein sequence, followed by alinker sequence, fused directly to an Fc portion of a human IgGl molecule or functional fragment thereof. In some embodiments, the linker sequence comprises SEQ ID NO: 75. In some embodiments, the linker sequence comprises a sequence selected from SEQ ID NO: 76 or 312-314. In some embodiments, the fusion protein yields improved processing of the homologous ARF1 protein contained within the fusion protein to produce mature engineered ARF1. In some embodiments, the fusion protein yields improved processing of the homologous ARF1 protein contained within the fusion protein to increase yield of mature engineered ARF1. In some embodiments, the fusion protein yields improved processing of the ARF1 proprotein contained within the fusion protein to produce mature engineered ARF1. In some embodiments, the fusion protein yields improved processing of the ARF1 proprotein contained within the fusion protein to increase yield of mature engineered ARF1. In some embodiments, the engineered ARF1 fusion protein comprising an HSA domain comprises an amino acid sequence at least about 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 61-64, 176, 184-186, 194, 217-218, or 315-317. In some embodiments, the engineered ARF1 fusion protein comprising an HSA domain comprises an amino acid sequence 100% identical to a sequence selected from SEQ ID NOs: 61-64, 176, 184-186, 194, 217-218, or 315-317.

[0106] In some embodiments, the engineered ARF1 fusion protein comprising an IgG Fc domain comprises an amino acid sequence at least about 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 59-60, 181-182, 192, 196, 215-216, or 245-284. In some embodiments, the engineered ARF1 fusion protein comprising an IgG Fc domain comprises an amino acid sequence 100% identical to a sequence selected from SEQ ID NOs: 59- 60, 181-182, 192, 196, 215-216, or 245-284.

[0107] In some aspects, engineered ARF1 polypeptides described herein are designed to enhance biological function of the polypeptide chain of the engineered polypeptide or a component of the engineered polypeptide comprising the homologous ARF1 protein sequence. In some embodiments, biological function of the engineered ARF1 polypeptide comprising the homologous ARF1 protein sequence is increased compared with an unmodified ARF1 protein.

[0108] In some aspects, engineered ARF1 polypeptides described herein are designed to enhance in vivo half-life of the polypeptide chain comprising the homologous ARF1 protein sequence. In some embodiments, in vivo half-life of the engineered polypeptide or half-life of the component of the engineered polypeptide comprising the homologous ARF1 protein sequence is increased compared with an unmodified ARF1 protein.

[0109] In some aspects, engineered ARF1 polypeptides described herein are designed to enhance in vitro expression of the polypeptide chain or a component of the engineered polypeptidecomprising the homologous ARF1 protein sequence. In some embodiments, in vitro expression of the engineered polypeptide or in vitro expression of the component of the engineered polypeptide comprising the homologous ARF1 protein sequence is improved compared with an unmodified ARF1 protein.

[0110] In some aspects, engineered ARF1 polypeptides described herein are designed to enhance in vitro expression of the polypeptide chain or a component of the engineered polypeptide comprising the homologous ARF1 protein sequence. In some embodiments, in vitro secretion of the engineered polypeptide or in vitro secretion of the component of the engineered polypeptide comprising the homologous ARF1 protein sequence is improved compared with an unmodified ARF1 protein.

[0111] In some aspects, engineered ARF1 polypeptides described herein are designed to enhance in vitro purification of the polypeptide chain or a component of the engineered polypeptide comprising the homologous ARF1 protein sequence. In some embodiments, in vitro purification of the engineered polypeptide or in vitro purification of the component of the engineered polypeptide comprising the homologous ARF1 protein sequence is improved compared with an unmodified ARF1 protein.

[0112] In some aspects, a engineered polypeptide described herein comprises a signal peptide located at the N-terminus of a polypeptide chain. In some embodiments, the signal peptide comprises an amino acid sequence listed in Table 2. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 77. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 78. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 79. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 80. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 81. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 82. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 85. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 86. In some embodiments, the signal peptide is cleaved from the engineered ARF1 polypeptide upon or immediately after translocation of the engineered polypeptide to the plasma membrane. In some embodiments, cleavage of the signal peptide releases the engineered polypeptide from a cell in which it was produced. In some embodiments, secreted engineered ARF1 polypeptides are produced in vitro and purified for use ina therapeutic composition. Table 2 lists exemplary signal peptide sequences for use in expression and production of Engineered ARF1 polypeptides described herein.Table 2: Exemplary Signal Peptide Sequences for Engineered ARF1 polypeptides

[0113] In some aspects an engineered polypeptide described herein does not comprise a signal peptide located at the N-terminus of a polypeptide chain.

[0114] In certain aspects, a composition comprising an engineered polypeptide comprising a homologous ARF1 protein sequence and a heterologous polypeptide amino acid sequence inserted within or adjacent to the homologous ARF1 protein sequence comprises one of more conservative amino acid substitutions. In certain aspects, a composition comprising a fusion protein comprising a homologous ARF1 protein sequence and a heterologous polypeptide amino acid sequence comprises one of more conservative amino acid substitutions. In certain aspects, a composition comprising an engineered polypeptide comprising a homologous ADP-ribosylation factor (ARF) protein sequence and a heterologous polypeptide amino acid sequence comprises one of moreconservative amino acid substitutions. In certain aspects, a composition comprising a fusion protein comprising a homologous ADP-ribosylation factor (ARF) protein sequence and a heterologous polypeptide amino acid sequence comprises one of more conservative amino acid substitutions. In some embodiments, the one of more conservative amino acid substitutions do not significantly decrease the ability of the engineered polypeptide or a component of the engineered polypeptide comprising the homologous ARF1 protein sequence from functioning as a weight loss promoting protein upon administration to a subject in need of weight loss. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., arginine, lysine and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., asparagine, cysteine, glutamine, glycine, serine, threonine, tyrosine, and tryptophan), nonpolar side chains (e.g., alanine, isoleucine, leucine, methionine, phenylalanine, proline, and valine), beta-branched side chains (e.g., isoleucine, threonine, and valine), and aromatic side chains (e.g., histidine, phenylalanine, tryptophan, and tyrosine), and aromatic side chains (e.g., histidine, phenylalanine, tryptophan, and tyrosine). Exemplary conservative amino acid substitutions are listed in Table 3.Table 3: Exemplary Amino Acid Modifications

[0115] In some aspects described herein are compositions comprising a homologous ADP- ribosylation factor (ARF) protein sequence useful for the treatment of obesity and obesity-related disorders and also for achieving weight loss. In one aspect described herein are compositions comprising a homologous ADP-ribosylation factor (ARF) protein sequence useful for the treatment of lipedema and also for achieving weight loss. In some aspects, the homologous ADP-ribosylation factor (ARF) protein sequence shares at least about 75%, 78%, 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a human ARF1 protein sequence. In some aspects, the homologous ADP-ribosylation factor (ARF) protein sequence shares at least about 75%, 78%, 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity to a human ARF2 protein sequence. In some aspects, the homologous ADP-ribosylation factor (ARF) protein sequence shares at least about 75%, 78%, 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a human ARF4 protein sequence. In some aspects, the homologous ADP-ribosylation factor (ARF) protein sequence shares at least about 75%, 78%, 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a human ARF5 protein sequence. In some aspects, the homologous ADP-ribosylation factor (ARF) protein sequence shares at least about 75%, 78%, 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a human ARF6 protein sequence. In some aspects, the homologous ADP-ribosylation factor (ARF) protein sequence shares at least about 75%, 78%, 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a human ARF-like protein sequence listed in Table 4. In some embodiments, homologous ADP-ribosylation factor (ARF) protein sequence comprises an amino acid sequence listed in Table 1A, Table IB, or Table 4. In some embodiments, homologous ADP-ribosylation factor (ARF) protein sequence comprises an amino acid sequence listed in Table 1A. In some embodiments, homologous ADP-ribosylation factor (ARF) protein sequence comprises an amino acid sequence listed in Table IB. In some embodiments, homologous ADP-ribosylation factor (ARF) protein sequence comprises an amino acid sequence listed in Table 4. In some embodiments, the homologous ARF protein sequence comprises a covalently -coupled protein trafficking domain sequence. In some embodiments, the homologous ARF protein sequence comprises one, two, three, four, five, six, seven, eight, or nine amino acid substitutions relative to an amino acid sequence selected from SEQ ID NOs: 87-106. In some embodiments, the homologous ARF protein sequence comprises one or more deletions of one, two, or three amino acids relative to an amino acid sequence selected from SEQ ID NOs: 87-106. In some embodiments, the homologous ARF protein sequence comprises one or more insertions of one, two, three, four, five, six, seven, eight, or nine amino acids relative to an amino acid sequence selected from SEQ ID NOs: 87-106. In some embodiments, the protein trafficking domain sequence comprises a signal peptide sequence. In some embodiments, the protein trafficking domain sequence comprises a site for myristoylation. In some embodiments, the site for myristoylation is located at or near the N-terminus of the engineered polypeptide. In some embodiments, the site for myristoylation comprises a glycine residue. In some embodiments, the site for myristoylation comprises the peptide sequence MGSS. In some embodiments, the homologous ARF protein sequence comprises a peptide tag. In some embodiments, the peptide tag comprises a peptide length of 4 to 22 amino acids and is tag is inserted within engineered ARFpolypeptide. In some embodiments, the peptide tag is positioned between the homologous ARF1 sequence and the covalently-coupled protein trafficking domain sequence. In some embodiments, the protein trafficking domain sequence comprises an Annexin A10 peptide sequence. In some embodiments, an engineered ARF polypeptide comprises a cleavage site. In some embodiments, the cleavage site is inserted between the homologous ARF sequence and the covalently-coupled protein trafficking domain sequence. In some embodiments, the cleavage site comprises a TEV protease recognition motif. In some embodiments, the TEV protease recognition motif comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the homologous ADP- ribosylation factor (ARF) protein sequence is included in a single polypeptide chain further comprising a half-life extending moiety and is referred to as an engineered ARF polypeptide. In some embodiments, the half-life extending moiety is located N-terminal to the homologous ARF1 sequence. In some embodiments, the half-life extending moiety is located C-terminal to the homologous ARF1 sequence. In some embodiments, the half-life extending moiety comprises a fatty acid ester. In some embodiments, the half-life extending moiety comprises a heterologous polypeptide sequence. In some embodiments, heterologous polypeptide sequence comprises a human serum albumin (HSA) sequence. In some embodiments, the HSA sequence is at least about 95% identical to SEQ ID NO: 51. In some embodiments, the HSA sequence is at least about 96%, 97%, 98%, or 99% identical to SEQ ID NO: 51. In some embodiments, the HSA sequence comprises an amino acid sequence identical to SEQ ID NO: 51. In some embodiments, the heterologous polypeptide sequence comprises an Fc portion of a human immunoglobulin. In some embodiments, the Fc portion of the human immunoglobulin comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 50. In some embodiments, the Fc portion of the human immunoglobulin comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, the engineered ARF polypeptide comprises a protein trafficking domain sequence listed in Table 1A. In some embodiments, the engineered ARF polypeptide comprises a protein trafficking domain sequence listed in Table IB.

[0116] Additional modifications to the engineered ARF polypeptides and compositions comprising a homologous ARF protein sequence described herein comprise substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, or more amino acids in the N-terminal region of the homologous ARF protein sequence. Additional modifications to the engineered ARF1 polypeptides and compositions comprising a homologous ARF protein sequence described herein comprise substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, or more amino acids in the C-terminal region of the homologous ARF protein sequence. Additional modifications to the engineered ARF1 polypeptides and compositions comprising a homologous ARF protein sequence described herein comprise substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, or more amino acids in a region internal to the N-terminus and C-terminus of the homologous ARF protein sequence. In some embodiments, the amino acid substitution or substitutions are conservative substitutions. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Exemplary conservative amino acid substitutions are listed in Table 3. In some embodiments, the amino acid substitution or substitutions are not conservative substitutions. FIG. 21 shows an alignment of human ARF1, ARF3, and ARF5 protein sequences. In some embodiments, non-conserved amino acid positions may be mutated either by insertion, deletion, or substitution in the engineered ARF protein. In some embodiments, the substitution is a substitution representing a conservative amino acid change. In some embodiments, the substitution is not a substitution representing a conservative amino acid change. In some embodiments, non-conserved amino acid positions between ARF1 and ARF3 in FIG. 21 represent positions that when mutated can modulate function of the engineered ARF protein. In some embodiments, non-conserved amino acid positions between ARF1 and ARF5 in FIG. 21 represent positions that when mutated can modulate function of the engineered ARF protein. In some embodiments, non-conserved amino acid positions between ARF3 and ARF5 in FIG. 21 represent positions that when mutated can modulate function of the engineered ARF protein. In some embodiments, non-conserved amino acid positions between ARF1 and both ARF3 and ARF5 in FIG. 21 represent positions that when mutated can modulate function of the engineered ARF protein. In some embodiments, the modulation may comprises an increase in effectiveness for the engineered protein to function in weight loss when administered to a subject in need of weight loss. In some embodiments, the modulation may comprises an decrease in effectiveness for the engineered protein to function in weight loss when administered to a subject in need of weight loss. In some embodiments, a composition comprising a homologous ARF protein sequence to Class 1 and Class 2 ARF proteins is effective for eliciting weight loss. In some embodiments, a composition comprising a homologous ARF protein sequence to Class 1 and Class 3 ARF proteins is effective for eliciting weight loss. In some embodiments, a composition comprising a homologous ARF protein sequence to Class 2 and Class 3 ARF proteins is effective for eliciting weight loss. In some embodiments, a composition comprising a homologous ARF protein sequenceto Class 1, Class 2, and Class 3 ARF proteins is effective for eliciting weight loss.

[0117] In some embodiments, the engineered ARF1 polypeptide or the composition comprising a homologous ARF protein sequence comprises one or more mutations within the homologous ARF sequence relative to an amino acid sequence selected from SEQ ID NOS: 87-106. In some embodiments, the homologous ARF sequence comprises one, two, three, four, five, six, seven, eight, or nine amino acid substitutions relative to an amino acid sequence selected from SEQ ID NOs: 87-106. In some embodiments, the homologous ARF1 sequence comprises one or more deletions of one, two, three, four, five, six, seven, eight, nine, ten, or more amino acids relative to an amino acid sequence selected from SEQ ID NOs: 87-106. In some embodiments, the homologous ARF sequence comprises one or more deletions of one, two, or three amino acids relative to an amino acid sequence selected from SEQ ID NOs: 87-106. In some embodiments, the homologous ARF sequence comprises one or more insertions of one, two, three, four, five, six, seven, eight, or nine amino acids relative to an amino acid sequence selected from SEQ ID NOs: 87-106. In some embodiments, the amino acid substitutions are for any amino acid. In some embodiments, the amino acid substitutions comprise a conservative amino acid sequence. In some embodiments, the conservative amino acid substitution is listed in Table 3. In some embodiments, the homologous ARF sequence comprises an amino acid sequence selected from SEQ ID NOs: 87- 106. In some embodiments, the homologous ARF sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 87. In some embodiments, the homologous ARF sequence comprises the amino acid sequence of SEQ ID NO: 87. In some embodiments, the homologous ARF sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 88. In some embodiments, the homologous ARF sequence comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments, the homologous ARF sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 89. In some embodiments, the homologous ARF sequence comprises the amino acid sequence of SEQ ID NO: 89. In some embodiments, the homologous ARF sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 90. In some embodiments, the homologous ARF sequence comprises the amino acid sequence of SEQ ID NO: 90. In some embodiments, the homologous ARF sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 91. In some embodiments, the homologous ARF sequence comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the homologous ARF sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 92. In some embodiments, the homologous ARF sequence comprises the amino acid sequence of SEQ ID NO: 92. In some embodiments, the homologous ARF sequence comprises an amino acid sequence at least about 90% identical to SEQID NO: 93. In some embodiments, the homologous ARF sequence comprises the amino acid sequence of SEQ ID NO: 93. In some embodiments, the homologous ARF sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 94. In some embodiments, the homologous ARF sequence comprises the amino acid sequence of SEQ ID NO: 94. In some embodiments, the homologous ARF sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 95. In some embodiments, the homologous ARF sequence comprises the amino acid sequence of SEQ ID NO: 95. In some embodiments, the homologous ARF sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 96. In some embodiments, the homologous ARF sequence comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the homologous ARF sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 97. In some embodiments, the homologous ARF sequence comprises the amino acid sequence of SEQ ID NO: 97. In some embodiments, the homologous ARF sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 98. In some embodiments, the homologous ARF sequence comprises the amino acid sequence of SEQ ID NO: 98. In some embodiments, the homologous ARF sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 99. In some embodiments, the homologous ARF sequence comprises the amino acid sequence of SEQ ID NO: 99. In some embodiments, the homologous ARF sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 100. In some embodiments, the homologous ARF sequence comprises the amino acid sequence of SEQ ID NO: 100. In some embodiments, the homologous ARF sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 101. In some embodiments, the homologous ARF sequence comprises the amino acid sequence of SEQ ID NO: 101. In some embodiments, the homologous ARF sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 102. In some embodiments, the homologous ARF sequence comprises the amino acid sequence of SEQ ID NO: 102. In some embodiments, the homologous ARF sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 103. In some embodiments, the homologous ARF sequence comprises the amino acid sequence of SEQ ID NO: 103. In some embodiments, the homologous ARF sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 104. In some embodiments, the homologous ARF sequence comprises the amino acid sequence of SEQ ID NO: 104. In some embodiments, the homologous ARF sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 105. In some embodiments, the homologous ARF sequence comprises the amino acid sequence of SEQ ID NO: 105. In some embodiments, the homologous ARF sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 106.In some embodiments, the homologous ARF sequence comprises the amino acid sequence of SEQ ID NO: 106. In some embodiments, amino acid positions notated as X in SEQ ID NOS: 104-106 can be any amino acid. In some embodiments, a covalently-coupled protein trafficking domain is added to the N-terminus of the homologous ARF sequence. In some embodiments, covalently- coupled protein trafficking domain comprises a peptide tag, a signal sequence, a protease recognition motif, a site for myristoylation, or any combination thereof. In some embodiments, covalently-coupled protein trafficking domain comprises a peptide tag, a signal sequence, a protease recognition motif, or any combination thereof designed such that a glycine residue within the homologous ARF sequence is not myristoyl ated. In some embodiments, a glycine residue near the N-terminus of the homologous ARF sequence is mutated to a natural amino acid other than glycine. In some embodiments, a glycine residue near the N-terminus of the homologous ARF sequence is deleted from the engineered protein. In some embodiments, a glycine residue near the N-terminus of the homologous ARF sequence has an insertion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 or more amino acids N-terminal to the position of the glycine residue in the homologous ARF sequence which prevents myristoylation of the glycine. An exemplary engineered ARF3 protein for therapeutic use is listed in SEQ ID NO: 202. An exemplary engineered ARF4 protein for therapeutic use is listed in SEQ ID NO: 203. An exemplary engineered ARF5 protein for therapeutic use is listed in SEQ ID NO: 204. An exemplary engineered ARF6 protein for therapeutic use is listed in SEQ ID NO: 205. An exemplary engineered ARF7 protein for therapeutic use is listed in SEQ ID NO: 206.

[0118] Table 4 lists amino acid sequences for additional exemplary polypeptide components of homologous ARF protein sequences and engineered ARF protein sequences not listed in Table 1A or Table IB.Therapeutic Indications and Methods of Treatment

[0119] In some aspects described herein are methods of treating an obesity disorder comprising administering a therapeutically effective amount of a composition described herein to a subject having the obesity disorder. In some aspects, the methods comprise administering to the subject an engineered ARF1 polypeptide or functional derivative thereof. In some aspects described herein are methods of treating an obesity disorder comprising administering a therapeutically effective amount of a composition described herein to a subject having the obesity disorder. In some aspects, the methods comprise administering to the subject an effective amount of an engineered ARF1 polypeptide or functional derivative thereof, wherein the effective amount improves one or more symptoms associated with the obesity disorder in the subject. In some embodiments, the obesity disorder comprises lipedema in the subject.

[0120] In some aspects described herein are methods of treating an obesity-related disorder comprising administering a composition described herein to a subject having the obesity-related disorder. In some aspects described herein are methods of treating an obesity-related disorder comprising administering a therapeutically effective amount of a composition described herein to asubject having the obesity-related disorder. In some aspects, the methods comprise administering to the subject an effective amount of a engineered ARF1 polypeptide or functional derivative thereof, wherein the effective amount improves one or more symptoms associated with the obesity-related disorder in the subject. In some embodiments, the obesity-related disorder comprises lipedema in the subject.

[0121] In some aspects described herein are methods of treating an overweight subject comprising administering a composition described herein to the subject. In some aspects described herein are methods of treating an overweight subject comprising administering a therapeutically effective amount of a composition described herein to the subject. In some aspects, the methods comprise administering to the subject an effective amount of a engineered ARF1 polypeptide or functional derivative thereof, wherein the effective amount improves one or more symptoms associated with the subject being overweight.

[0122] In some aspects described herein are methods of promoting weight loss in an overweight subject comprising administering a composition described herein to the subject. In some aspects described herein are methods of promoting weight loss in a subject comprising administering a therapeutically effective amount of a composition described herein to the subject. In some aspects, the methods comprise administering to the subject an effective amount of a engineered ARF1 polypeptide or functional derivative thereof, wherein the effective amount improves one or more symptoms associated with the subject being overweight. In some aspects, the methods comprise administering to the subject an effective amount of a engineered ARF1 polypeptide or functional derivative thereof, wherein the effective amount promotes weight loss. In some aspects, the methods comprise administering to the subject an effective amount of a engineered ARF1 polypeptide or functional derivative thereof, wherein the effective amount improves body composition by reducing adipose tissue percentage weight in the body composition of the subject.

[0123] In some aspects described herein are methods of maintaining weight loss in a subject comprising administering a composition described herein to the subject. In some aspects described herein are methods of maintaining weight loss in a subject comprising administering a therapeutically effective amount of a composition described herein to the subject. In some aspects, the methods comprise administering to the subject an effective amount of a engineered ARF1 polypeptide or functional derivative thereof, wherein the effective amount improves one or more symptoms associated with the subject being overweight. In some aspects, the methods comprise administering to the subject an effective amount of a engineered ARF1 polypeptide or functionalderivative thereof, wherein the effective amount maintains an extent of weight loss in the subject. In some aspects, the methods comprise administering to the subject an effective amount of a engineered ARF1 polypeptide or functional derivative thereof, wherein the effective amount maintains an extent of weight loss in the subject achieved by prior treatment with one or more incretin therapeutics. In some aspects, the methods comprise administering to the subject an effective amount of a engineered ARF1 polypeptide or functional derivative thereof, wherein the effective amount maintains an extent of weight loss in the subject achieved by prior treatment with a GLP-1R agonist. In some aspects, the methods comprise administering to the subject an effective amount of a engineered ARF1 polypeptide or functional derivative thereof, wherein the effective amount maintains an extent of weight loss in the subject achieved by prior treatment with semaglutide. In some aspects, the methods comprise administering to the subject an effective amount of a engineered ARF1 polypeptide or functional derivative thereof, wherein the effective amount maintains an extent of weight loss in the subject achieved by prior treatment with a GLP- 1R / GIP-R dual agonist. In some aspects, the methods comprise administering to the subject an effective amount of a engineered ARF1 polypeptide or functional derivative thereof, wherein the effective amount maintains an extent of weight loss in the subject achieved by prior treatment with tirzepatide. In some embodiments, the extent of weight loss maintained in the subject is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, or greater than 25% of the amount of weigh lost from a previous weight loss regimen. In some embodiments, the extent of weight loss maintained in the subject is at least about 8% of the amount of weigh lost from a previous weight loss regimen. In some embodiments, the previous weight loss regimen comprises previous administration of an incretin. In some embodiments, the previous weight loss regimen comprises previous administration of a GLP-1R agonist. In some embodiments, the previous weight loss regimen comprises previous administration of semaglutide. In some embodiments, the previous weight loss regimen comprises previous administration of a GLP-1R / GIP-R dual agonist. In some embodiments, the previous weight loss regimen comprises previous administration of tirzepatide. In some embodiments, the methods of maintaining weight loss comprises preventing reversion of weight loss achieved from incretin treatment following cessation of incretin administration. In some embodiments, the prevention of reversion of weight loss comprises preventing regain of no more than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or 25% of weight loss achieved from previous incretin treatment. In some embodiments, the prevention of reversion of weight loss comprises preventing regain of no more than about 12% of weight loss achieved from previous incretin treatment. In some embodiments, the prevention of reversion of weight loss comprises maintenance of a stablebody weight for at least about 4 weeks following chronic administration of the engineered ARF1 polypeptide or functional derivative thereof. In some embodiments, the prevention of reversion of weight loss comprises maintenance of a stable body weight for at least about 4 weeks following chronic administration of the engineered ARF1 polypeptide.

[0124] In some aspects described herein are methods of treating a metabolic-related disorder comprising administering a composition described herein to a subject having the metabolic-related disorder. In some aspects described herein are methods of treating a metabolic-related disorder comprising administering a therapeutically effective amount of a composition described herein to a subject having the metabolic-related disorder. In some aspects, the methods comprise administering to the subject an effective amount of a engineered ARF1 polypeptide or functional derivative thereof, wherein the effective amount improves one or more symptoms associated with the metabolic-related disorder in the subject.

[0125] In some aspects described herein are methods of treating an inflammation-related disorder comprising administering a therapeutically effective amount of a composition described herein to a subject having the inflammation-related disorder. In some aspects, the methods comprise administering to the subject an effective amount of a engineered ARF1 polypeptide or functional derivative thereof, wherein the effective amount improves one or more symptoms associated with the inflammation-related disorder in the subject.

[0126] In some aspects described herein are methods of treating an inflammatory disorder resulting in muscle atrophy comprising administering a therapeutically effective amount of a composition described herein to a subject having the inflammatory disorder. In some aspects, the methods comprise administering to the subject an effective amount of a modified protein or functional derivative thereof, wherein the effective amount improves one or more symptoms associated with the inflammatory disorder in the subject.

[0127] In some aspects described herein are methods of treating a disorder of lipolysis dysfunction comprising administering a therapeutically effective amount of a composition described herein to a subject having the lipolysis dysfunction disorder. In some aspects, the methods comprise administering to the subject an effective amount of a engineered ARF1 polypeptide or functional derivative thereof, wherein the effective amount improves one or more symptoms associated with the lipolysis dysfunction disorder in the subject.

[0128] In some aspects described herein are methods of treating a subject by administering a therapeutic composition described herein, wherein the subject has pediatric obesity, type II diabetes, obesity, an obesity syndrome, clinical obesity, lipedema, metabolic syndrome / pre-diabetes, cardiovascular disease, nonalcoholic steatohepatitis (NASH) or nonalcoholic fatty liver disease (NAFLD), type I diabetes, Cushing’s disease, tumor-induced Cushing’s disease, Prader- Willi syndrome, Bardet-Biedl syndrome, thyroid disorder, thyroid removal, Adrenoleukodystrophy, Niemann-Pick disease, Tangier disease, Polycystic ovary syndrome, Congenital leptin deficiency, Cohen syndrome, Alstrbm syndrome, or Froehlich syndrome, or any combination thereof.

[0129] In some aspects described herein are methods of treating a subject having obesity, the methods comprising administering an engineered ADP-ribosylation factor 1 (ARF1) protein to the subject, wherein the engineered ARF1 protein comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some aspects described herein are methods of treating a subject having obesity, the methods comprising administering an effective amount of an engineered ADP-ribosylation factor 1 (ARF1) protein to the subject, wherein the engineered ARF1 protein comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some aspects described herein are methods of promoting weight loss in a subject, the methods comprising administering an engineered ADP-ribosylation factor 1 (ARF1) protein to the subject, wherein the engineered ARF1 protein comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some aspects described herein are methods of promoting weight loss in a subject, the methods comprising administering an effective amount of an engineered ADP-ribosylation factor 1 (ARF1) protein to the subject, wherein the engineered ARF1 protein comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the engineered ARF1 protein comprises an amino acid sequence selected from one of SEQ ID NOs: 123-174. In some embodiments, the engineered ARF1 protein comprises an amino acid sequence of SEQ ID NO: 123. In some embodiments, the engineered ARF1 protein comprises an amino acid sequence selected from one of SEQ ID NOs: 286-289 or 297-311. In some embodiments, the engineered ARF1 protein comprises an amino acid sequence of SEQ ID NO: 286. In some embodiments, the engineered ARF1 protein comprises an amino acid sequence of SEQ ID NO: 287. In some embodiments, the engineered ARF1 protein comprises an amino acid sequence of SEQ ID NO: 288. In some embodiments, the engineered ARF1 protein comprises an amino acid sequence of SEQ ID NO: 289. In some embodiments, the engineered ARF1 protein comprises an amino acid sequence of SEQ ID NO: 293. In some embodiments, the engineered ARF1 protein comprises an amino acid sequence of SEQ ID NO: 294. In some embodiments, the engineered ARF1 protein comprises an amino acid sequence of SEQ ID NO: 295. In someembodiments, the engineered ARF1 protein comprises an amino acid sequence selected from one of SEQ ID NOs: 61-64, 176, 184-186, 194, 217-218, or 315-317. In some embodiments, the engineered ARF1 protein comprises an amino acid sequence selected from one of SEQ ID NOs: 61-64, 176, 184-186, 194, 217-218, or 315-317.In some embodiments, the engineered ARF1 protein is administered systemically to the subject. In some embodiments, the engineered ARF1 protein is administered by parenteral administration. In some embodiments, parenteral administration comprises injection of the engineered ARF1 protein in the subject. In some embodiments, the injection is intravenous injection. In some embodiments, the injection is intramuscular injection. In some embodiments, the injection is intradermal injection. In some embodiments, the injection is subcutaneous injection. In some embodiments, parenteral administration comprises infusion of the engineered ARF1 protein in the subject. In some embodiments, the subject is treated for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 17 days, 21 days, 24 days, 28 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. In some embodiments, the subject is treated for at least 3 weeks. In some embodiments, the subject achieves a percentage of weight loss of at least 2.5%, 5%, 7.5%, 10%, 12.5%, or 15%. In some embodiments, the subject maintains a percentage of weight loss following cessation of incretin therapy of at least 2.5%, 5%, 7.5%, 10%, 12.5%, or 15% of body weight. In some embodiments, the subject maintains a percentage of lean muscle mass of at least 85%, 90%, 95%, 98%, 99%, or 100% compared to the percentage of lean muscle mass prior to start of treatment following a period of treatment with administration of the engineered ARF1 protein. In some embodiments, non-fasting blood glucose level in the subject is reduced following the administering. In some embodiments, fasting blood glucose level in the subject is reduced following the administering. In some embodiments, insulin sensitivity in the subject is increased following the administering. In some embodiments, serum insulin level is decreased in the subject following the administering. In some embodiments, results from a glucose tolerance test are improved in the subject following the administering. In some embodiments, results from an insulin tolerance test are improved in the subject following the administering. In some embodiments, administering the engineered ARF1 protein does not substantially suppress appetite in the subject. In some embodiments, administering the engineered ARF1 protein does not suppress appetite in the subject. In some embodiments, administering the engineered ARF1 protein promotes weight loss in the subject without decreasing an extent of food intake. In some embodiments of methods described herein, weight loss is achieved without a significant reduction in caloric consumption. In some embodiments of methods described herein, previous weight loss is maintained without a significantreduction in caloric consumption. In some embodiments of methods described herein, previous weight loss achieved from incretin therapy is maintained without a significant reduction in caloric consumption following cessation of the incretin therapy. In some embodiments of methods described herein, the subject maintains a stable amount of previous weight loss without regaining more than about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of previously lost body weight following cessation of incretin therapy. In some embodiments of methods described herein, the subject maintains a stable amount of previous weight loss without regaining more than about 12% - 13% of previously lost body weight following cessation of incretin therapy. In some embodiments, caloric uptake is not significantly reduced in the subject following the administration of the engineered ARF1 protein. In some embodiments of methods described herein, the subject demonstrates an increase in oxygen consumption following the administering. In some embodiments of methods described herein, the subject demonstrates an increase in oxygen expenditure following the administering. In some embodiments of methods described herein, lipid droplet size is reduced in the subject following the administering. In some embodiments of methods described herein, volume of lipid droplet area is reduced in the subject following the administering. In some embodiments of methods described herein, glucose homeostasis is improved in the subject following the administering. In some embodiments of methods described herein, glucose homeostasis is improved in the subject having diet-induced obesity following the administering. In some embodiments of methods described herein, lipid handling is improved in the subject following the administering. In some embodiments of methods described herein, liver fibrosis in the subject is reduced following the administering. In some embodiments of methods described herein, adipocyte size in the subject is decreased following the administering. In some embodiments, an extent of lipolysis is increased in the subject following the administering. In some embodiments, serum levels of triglycerides are decreased in the subject following the administering. In some embodiments, serum levels of high-density lipoprotein (HDL) are increased in the subject following the administering. In some embodiments, fatty acid uptake in the subject is not significantly changed following the administering. In some embodiments, a rate of triglyceride hydrolysis is increased in a plurality of adipocytes from the subject following the administering. In some embodiments, the increased rate of triglyceride hydrolysis produces elevated levels of glycerol and fatty acids in a plurality of adipocytes of the subject following the administering. In some embodiments, the subject is overweight at the start of the administering. In some embodiments, the subject is clinically obese at the start of the administering. In some embodiments, the subject has achieved previous weight loss at the start of the administering. In some embodiments, the subject has achieved previous weight loss at the start of the administering and themethod comprises maintenance of previous weight loss. In some embodiments, the subject has been diagnosed with an obesity-related disorder. In some embodiments, the obesity-related disorder is selected from the group consisting of pediatric obesity, type II diabetes, obesity, an obesity syndrome, clinical obesity, metabolic syndrome / pre-diabetes, cardiovascular disease, nonalcoholic steatohepatitis (NASH) or nonalcoholic fatty liver disease (NAFLD), type I diabetes, Cushing’s disease, tumor-induced Cushing’s disease, Prader-Willi syndrome, Bardet-Biedl syndrome, thyroid disorder, thyroid removal, Adrenoleukodystrophy, Niemann-Pick disease, Tangier disease, Polycystic ovary syndrome, Congenital leptin deficiency, Cohen syndrome, Alstrbm syndrome, or Froehlich syndrome, or any combination thereof. In some embodiments of methods described herein, weight loss is promoted in subjects having metabolic syndrome / pre-diabetes, cardiovascular disease, nonalcoholic steatohepatitis (NASH) or nonalcoholic fatty liver disease (NAFLD), type I diabetes, Cushing’s disease, tumor-induced Cushing’s disease, Prader-Willi syndrome, Bardet- Biedl syndrome, thyroid disorder, thyroid removal, Adrenoleukodystrophy, Niemann-Pick disease, Tangier disease, Polycystic ovary syndrome, Congenital leptin deficiency, Cohen syndrome, Alstrbm syndrome, or Froehlich syndrome, or any combination thereof. In some embodiments, the subject has been diagnosed with obesity. In some embodiments, the subject has been diagnosed with lipedema. In some embodiments, the method comprises administering an engineered ARF1 polypeptide described herein to the subject.

[0130] In some aspects described herein, methods of treatment of a subject in need thereof comprise administering a composition described herein to the subject, wherein the administering decreased body weight (BW) of the subject. In some embodiments, BW is decreased by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, or 40% of body weight of the subject at a time point following the administering. In some embodiments, decreased body in the subject comprises a dose-dependent decrease in adipocyte cell size of the subject following the administering of the composition. In some embodiments, decreased body in the subject comprises a reduction of adipose tissue in the subject. In some embodiments, the reduction in adipose tissue comprises a reduction in the volume of adipose tissue in at least a part of the subject’s body. In some embodiments, the reduction in adipose tissue comprises a reduction in the mass of adipose tissue in at least a part of the subject’s body. In some embodiments, the reduction of adipose tissue in the subject is measured as a function of change in total body weight of the subject. In some embodiments, a reduction in adipose tissue can be determined by measuring the thickness of skin of the subject with an adipometer or by measuring the circumference of one or more body parts of the subject. In some embodiments, a reduction in adipose tissue can be determined by measuring tissue composition of the subject byusing standard measuring techniques such as EchoMRI. In some embodiments, adipose tissue is reduced by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, or 40% of body weight of the subject at a time point following the administering. In some embodiments, the dose-dependent decrease in adipocyte cell size or reduction of adipose tissue of the subject following the administering of the composition allows for a precise titration of optimal therapeutic dosage range to achieve a sufficient achieve a reduction in body weight. In some embodiments, the effective amount to produce the dose-dependent decrease in adipocyte cell size or reduction of adipose tissue is at least about 0.005, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.070, 0.075, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.75, 3.0, 3.25, 3.50, 3.75, 4.0, 4.5, 5.0, 6.0, 7.0, 7.5, 8.0, 9.0, or 10 pg / mL of the engineered ARF1 polypeptide. In some embodiments, the effective amount to produce the dose-dependent decrease in adipocyte cell size or reduction of adipose tissue is less than about 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.070, 0.075, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, 2.75, 3.0, 3.25, 3.50, 3.75, 4.0, 4.5, 5.0, 6.0, 7.0, 7.5, 8.0, 9.0, 10, 15, 20, 25, 30, 35, 40, 45, or 50 pg / mL of the modified protein or of the engineered ARF1 polypeptide. In some embodiments, the effective amount to produce the dose-dependent decrease in adipocyte cell size or reduction of adipose tissue is at least about 0.005, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.070, 0.075, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.75, 3.0, 3.25, 3.50, 3.75, 4.0, 4.5, 5.0, 6.0, 7.0, 7.5, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 27, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 pg / kg of body weight of the subject of the engineered ARF1 polypeptide. In some embodiments, the effective amount to produce the dose-dependent decrease in adipocyte cell size or reduction of adipose tissue is at least about 4 pg / kg of body weight. In some embodiments, the effective amount to produce the dose-dependent decrease in adipocyte cell size or reduction of adipose tissue is less than about 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.070, 0.075, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.75, 3.0, 3.25, 3.50, 3.75, 4.0, 4.5, 5.0, 6.0, 7.0,7.5, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 27, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 125, 150, 200, 250, 300, 350, 400, 450, or 500 pg / kg of body weight of the subject of the engineered ARF1 polypeptide. In some embodiments, the effectivedosage is determined based on i.v. formulation of the therapeutic composition. In some embodiments, the effective dosage is determined based on s.c. formulation of the therapeutic composition. In some embodiments, the effective dose in a human subject is determined based on studies in an animal model wherein the human equivalent dosage is determined account to a method described in Nair AB et al., A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm. 2016 Mar; 7(2): 27-31 which is hereby incorporated by reference regarding use of methods for determining therapeutic dosage. In some embodiments, the effective amount to produce the dose-dependent decrease in adipocyte cell size is at least about 0.025 pg / mL of the engineered ARF1 polypeptide. In some embodiments, the reduction in adipose tissue comprises a reduction in white adipose tissue by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, or 40% of body weight of the subject at a time point following the administering. In some embodiments, the reduction in adipose tissue comprises a reduction in brown adipose tissue by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, or 40% of body weight of the subject at a time point following the administering. In some embodiments, the reduction in adipose tissue comprises a reduction in combined measurement of white adipose tissue and brown adipose tissue by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, or 40% of body weight of the subject at a time point following the administering. In some embodiments, the reduction in adipose tissue comprises a reduction in white adipose tissue by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, or 40% of body weight of the subject at a time point following the administering without a significant decrease in a measured amount of brown adipose tissue. In some embodiments, the reduction in adipose tissue comprises a reduction in white adipose tissue by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, or 40% of body weight of the subject at a time point following the administering wherein the volume of a plurality of white adipocytes is reduced. In some embodiments, the reduction in adipose tissue comprises a reduction in white adipose tissue by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, or 40% of body weight of the subject at a time point following the administering wherein a proportion of beige adipocytes compared to white adipocytes in one or more locations in the subject in increased. In some embodiments, the reduction of body weight in the subject comprises a reduction in white adipose tissue mass in the subject and an increase in a percentage of beige adipocytes at one or more whitefat locations in the subject.

[0131] In some aspects described herein, methods of treatment of a subject in need thereof comprise administering a composition described herein to the subject, wherein the administering lowers overall body weight, lowers fat / lean muscle mass ratio, increases insulin sensitivity, decreases ectopic fat deposition, increases fat beiging and differentiation of brown adipose tissue, decreases system inflammation, decreases tissue inflammation, improves adiposity markers in blood serum, improves adipocyte and lipid droplet size, reduces adipose tissue fibrosis, decreases an extent of muscle atrophy, or any combination thereof. In some embodiments, an effective amount to produce the beneficial effect in the subject as a dose-dependent effective amount. In some embodiments, the effective amount to produce the dose-dependent effect is at least about 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.070, 0.075, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.75, 3.0, 3.25, 3.50, 3.75, 4.0, 4.5, 5.0, 6.0, 7.0,7.5, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 27, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 125, 150, or 300 pg / kg of body weight of the subject of the engineered ARF1 polypeptide. In some embodiments, the effective amount to produce the dosedependent effect is less than about 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.070, 0.075, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.75, 3.0, 3.25, 3.50, 3.75, 4.0, 4.5, 5.0, 6.0, 7.0, 7.5, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 27, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 125, 150, 200, 250, 300, 350, 400, 450, or 500 pg / kg of body weight of the subject of the engineered ARF1 polypeptide. In some embodiments, the effective amount to produce the dose-dependent effect is between about 1-300 pg / kg of body weight of the subject of a wild-type ARF1 protein or of the engineered ARF1 polypeptide. In some embodiments, the effective amount to produce the dose-dependent effect is between about 30-300 pg / kg of body weight of the subject of a wild-type ARF1 protein or of the engineered ARF1 polypeptide.Ages of subjects

[0132] In some embodiments, of methods described herein, the subject is a human subject. In some embodiments, the subject is aged at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 105 years old. In some embodiments, the subject is aged at least 18 years of age. In some embodiments, the subject is aged at least 21 years of age. In some embodiments, the subject is aged at least 25 years of age. In some embodiments, the subject is aged at least 35 years of age. In some embodiments, the subject is aged at least 40 years of age. In some embodiments, the subject is aged at least 50 years of age. In some embodiments, the subject is aged at least 60 years of age. In some embodiments, the subject is aged at least 70 years of age. In some embodiments, the subject is an infant, toddler, or child between the ages of 0- 18 years old.Scheduled routes of administration and amounts

[0133] In certain aspects, the polypeptide (e.g., a modified protein, a homologous ARF1 protein sequence, a ARF1 protein sequence, or an engineered ARF1 polypeptide) can be administered by any suitable route such as, for example, subcutaneous, intravenous, intramuscular, pulmonary, or intradermal. In certain aspects, the polypeptide (e.g., a modified protein, a homologous ARF1 protein sequence, a ARF1 protein sequence, or a Engineered ARF1 polypeptide) is administered on a suitable dosage schedule, for example, weekly, twice weekly, monthly, twice monthly, once every three weeks, or once every four weeks. The polypeptide (e.g., a modified protein, a homologous ARF1 protein sequence, a ARF1 protein sequence, or a Engineered ARF1 polypeptide) can be administered in any therapeutically effective amount. In certain aspects, the therapeutically acceptable amount is about 0.001 pg / kg to about 1 pg / kg. In certain aspects, the therapeutically acceptable amount is about 0.001 pg / kg to about 0.002 pg / kg, about 0.001 pg / kg to about 0.005 pg / kg, about 0.001 pg / kg to about 0.01 pg / kg, about 0.001 pg / kg to about 0.02 pg / kg, about 0.001 pg / kg to about 0.05 pg / kg, about 0.001 pg / kg to about 0.1 pg / kg, about 0.001 pg / kg to about 0.2 pg / kg, about 0.001 pg / kg to about 0.5 pg / kg, about 0.001 pg / kg to about 1 pg / kg, about 0.002 pg / kg to about 0.005 pg / kg, about 0.002 pg / kg to about 0.01 pg / kg, about 0.002 pg / kg to about 0.02 pg / kg, about 0.002 pg / kg to about 0.05 pg / kg, about 0.002 pg / kg to about 0.1 pg / kg, about 0.002 pg / kg to about 0.2 pg / kg, about 0.002 pg / kg to about 0.5 pg / kg, about 0.002 pg / kg to about 1 pg / kg, about 0.005 pg / kg to about 0.01 pg / kg, about 0.005 pg / kg to about 0.02 pg / kg, about 0.005 pg / kg to about 0.05 pg / kg, about 0.005 pg / kg to about 0.1 pg / kg, about 0.005 pg / kg to about 0.2 pg / kg, about 0.005 pg / kg to about 0.5 pg / kg, about 0.005 pg / kg to about 1 pg / kg, about 0.01 pg / kg to about 0.02 pg / kg, about 0.01 pg / kg to about 0.05 pg / kg, about 0.01 pg / kg to about 0.1 pg / kg, about 0.01 pg / kg to about 0.2 pg / kg, about 0.01 pg / kg to about 0.5 pg / kg, about 0.01 pg / kg to about 1 pg / kg, about 0.02 pg / kg to about 0.05 pg / kg, about 0.02 pg / kg to about 0.1 pg / kg,about 0.02 .g / kg to about 0.2 gg / kg, about 0.02 gg / kg to about 0.5 gg / kg, about 0.02 gg / kg to about 1 gg / kg, about 0.05 gg / kg to about 0.1 gg / kg, about 0.05 gg / kg to about 0.2 gg / kg, about 0.05 gg / kg to about 0.5 gg / kg, about 0.05 gg / kg to about 1 gg / kg, about 0.1 gg / kg to about 0.2 gg / kg, about 0.1 gg / kg to about 0.5 gg / kg, about 0.1 gg / kg to about 1 gg / kg, about 0.2 gg / kg to about 0.5 gg / kg, about 0.2 gg / kg to about 1 gg / kg, or about 0.5 gg / kg to about 1 gg / kg. In certain aspects, the therapeutically acceptable amount is about 0.001 gg / kg, about 0.002 gg / kg, about 0.005 gg / kg, about 0.01 gg / kg, about 0.02 gg / kg, about 0.05 gg / kg, about 0.1 gg / kg, about 0.2 gg / kg, about 0.5 gg / kg, or about 1 gg / kg. In certain aspects, the therapeutically acceptable amount is at least about 0.001 gg / kg, about 0.002 gg / kg, about 0.005 gg / kg, about 0.01 gg / kg, about 0.02 gg / kg, about 0.05 gg / kg, about 0.1 gg / kg, about 0.2 gg / kg, or about 0.5 gg / kg. In certain aspects, the therapeutically acceptable amount is at most about 0.002 gg / kg, about 0.005 gg / kg, about 0.01 gg / kg, about 0.02 gg / kg, about 0.05 gg / kg, about 0.1 gg / kg, about 0.2 gg / kg, about 0.5 gg / kg, or about 1 gg / kg. In certain aspects, the therapeutically acceptable amount is about 0.1 gg / kg to about 50 gg / kg. In certain embodiments, the therapeutically acceptable amount is about 0.1 gg / kg to about 0.2 gg / kg, about 0.1 gg / kg to about 0.5 gg / kg, about 0.1 gg / kg to about 1 gg / kg, about 0.1 gg / kg to about 2 gg / kg, about 0.1 gg / kg to about 5 gg / kg, about 0.1 gg / kg to about 10 gg / kg, about 0.1 gg / kg to about 20 gg / kg, about 0.1 gg / kg to about 50 gg / kg, about 0.2 gg / kg to about 0.5 gg / kg, about 0.2 gg / kg to about 1 gg / kg, about 0.2 gg / kg to about 2 gg / kg, about 0.2 gg / kg to about 5 gg / kg, about 0.2 gg / kg to about 10 gg / kg, about 0.2 gg / kg to about 20 gg / kg, about 0.2 gg / kg to about 50 gg / kg, about 0.5 gg / kg to about 1 gg / kg, about 0.5 gg / kg to about 2 gg / kg, about 0.5 gg / kg to about 5 gg / kg, about 0.5 gg / kg to about 10 gg / kg, about 0.5 gg / kg to about 20 gg / kg, about 0.5 gg / kg to about 50 gg / kg, about 1 gg / kg to about 2 gg / kg, about 1 gg / kg to about 5 gg / kg, about 1 gg / kg to about 10 gg / kg, about 1 gg / kg to about 20 gg / kg, about 1 gg / kg to about 50 gg / kg, about 2 gg / kg to about 5 gg / kg, about 2 gg / kg to about 10 gg / kg, about 2 gg / kg to about 20 gg / kg, about 2 gg / kg to about 50 gg / kg, about 5 gg / kg to about 10 gg / kg, about 5 gg / kg to about 20 gg / kg, about 5 gg / kg to about 50 gg / kg, about 10 gg / kg to about 20 gg / kg, about 10 gg / kg to about 50 gg / kg, or about 20 gg / kg to about 50 gg / kg. In certain aspects, the therapeutically acceptable amount is about 0.1 gg / kg, about 0.2 gg / kg, about 0.5 gg / kg, about 1 gg / kg, about 2 gg / kg, about 5 gg / kg, about 10 gg / kg, about 20 gg / kg, or about 50 gg / kg. In certain aspects, the therapeutically acceptable amount is at least about 0.1 gg / kg, about 0.2 gg / kg, about 0.5 gg / kg, about 1 gg / kg, about 2 gg / kg, about 5 gg / kg, about 10 gg / kg, or about 20 gg / kg. In certain aspects, the therapeutically acceptable amount is at most about 0.2 gg / kg, about 0.5 gg / kg, about 1 gg / kg, about 2 gg / kg, about 5 gg / kg, about 10 gg / kg, about 20 gg / kg, or about 50 gg / kg.Nucleic Acids

[0134] In certain aspects, described herein, are nucleic acids that encode the engineered ARF1 polypeptides described herein. In certain aspects, the nucleic acids are exogenous. In certain aspects, the nucleic acid is a plasmid. In certain aspects, the nucleic acid is a viral vector. In certain aspects, the viral vector is an adenovirus, lentivirus, retrovirus, adeno-associated virus, or vaccinia virus. In certain aspects, the nucleic acid comprises RNA. In certain aspects, the nucleic acid encodes any of the polypeptides disclosed herein (e.g., a homologous ARF1 protein sequence or an engineered ARF1 protein sequence). In certain aspects, the nucleic acid encodes any one or more polypeptide embodiments described herein. Nucleic acids according to this description can comprise additional nucleic acid sequences sufficient to propagate the vector or express a polypeptide encoded by the vector. In certain aspects, the nucleic acid comprises a universal promoter, such as the CMV promoter, or an inducible promoter system such as a TETON, TETOFF or GAL4. In certain aspects, the nucleic acid is expressed via a tissue specific promoter or one compatible with a eukaryotic or prokaryotic cellular expression system, such as a mouse albumin promoter. The nucleic acid can further comprise a sequence encoding a suitable purification tag (e.g., HIS-tag, V5, FLAG, MYC). In some embodiments, a nucleic acid sequence encoding a component of a modified protein, a full length modified protein, or a Engineered ARF1 polypeptide is disclosed in Table 5.Table 5: Nucleic acid sequences encoding polypeptidesProduction of polypeptides

[0135] Once a polypeptide (e.g., a modified protein, a homologous ARF1 protein sequence, a ARF1 protein sequence, or an engineered ARF1 polypeptide) is selected it can be purified or synthesized in any suitable manner. A nucleic acid encoding the polypeptide can be cloned into a suitable vector and expressed in a suitable cellular system. In certain aspects, the cellular system is a prokaryotic cell system. In certain aspects, the cellular system is a eukaryotic cell system. In certain aspects, the cellular system is a mammalian cell system. The supernatants from such an expression system can be subjected to one or more purification steps involving centrifugation, ultracentrifugation, filtration, diafiltration, tangential-flow filtration, dialysis, chromatography (e.g., cation exchange, ion exchange, hydrophobic interaction, reverse phase, affinity, or size exclusion). The polypeptides can be purified to an extent suitable for human administration. Additionally, polypeptides can be synthesized for inclusion in a formulation to be administered to a humansubject. In certain aspects, the polypeptides can be produced by a suitable peptide synthesis method, such as solid-phase synthesis.Master cell bank and transgenic cells

[0136] In a certain aspect, described herein is a master cell bank comprising a cell that comprises a nucleic acid encoding one or more polypeptides (e.g., a modified protein, a homologous ARF1 protein sequence, a ARF1 protein sequence, or a Engineered ARF1 polypeptide) integrated into its genome creating a transgenic cell-line. In some aspects, the master cell bank comprises a plurality of cells that each comprise a nucleic acid encoding a polypeptide (e.g., a modified protein, a homologous ARF1 protein sequence, a ARF1 protein sequence, or an engineered ARF1 polypeptide). In certain aspects, the nucleic acid is maintained extrachromosomally on a plasmid or yeast artificial chromosome. In certain aspects, the nucleic acid is integrated into a chromosomal location. In certain aspects, the cell is a yeast cell. In certain aspects, the yeast is Pichia pastor is or Saccharomyces cerevisiae. In certain aspects, the cell is a mammalian cell. In certain aspects, the mammalian cell is a 293T cell or derivative thereof (e.g., 293T-Rex). In certain aspects, the mammalian cell is a Chinese Hamster Ovary cell or derivative thereof (e.g., CHO-DXB11, CHO-K1, CHO-DG44, or CHO-S). In certain aspects, the mammalian cell is a mouse myeloma cell, e.g., (NSO) cells. In certain aspects, the cell is a bacterial cell. In some embodiments, the bacterial cell is an E. coli cell.

[0137] In certain aspects, the transgenic mammalian, yeast, or bacterial cell is a master cell bank that comprises a cryopreservative suitable for freezing to at least about -80° or below. In certain aspects, the master cell bank comprises glycerol at between about 10 and about 30%, and is suitable for long-term storage at about -80° or below. In certain aspects, the master cell bank can preserve a transgenic mammalian, yeast, or bacterial strain for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years.

[0138] Pharmaceutically acceptable excipients, carriers, and diluents

[0139] The polypeptide (e.g., a modified protein, a homologous ARF1 protein sequence, a ARF1 protein sequence, or an engineered ARF1 polypeptide) described herein can be administered in a pharmaceutical composition that comprises one or more pharmaceutically acceptable excipients, carriers, or diluents. The exact components can differ based upon the preferred route of administration. The excipients used in a pharmaceutical composition can provide additional function to the polypeptide by making the polypeptide suitable for a particular route of administration (e.g., intravenous, topical, subcutaneous, or intramuscular), increasing polypeptide stability, increasing penetration of a desired tissue (e.g., skeletal muscle, vasculature, adipose tissue, or bone), increasing residence time at particular site, increasing solubility, enhancing theefficacy of the polypeptide, and / or reducing inflammatory reactions coincident with administration.

[0140] In certain aspects, the polypeptides (e.g., a modified protein, a homologous ARF1 protein sequence, a ARF1 protein sequence, or an engineered ARF1 polypeptide) described herein are included in a pharmaceutical composition with a solubilizing emulsifying, or dispersing agent. In certain aspects, the solubilizing agent can allow high-concentration solutions of modified protein or engineered ARF1 polypeptide that exceed at least about 2 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, or 20 mg / mL. In certain aspects, the efficacy of the modified protein or Engineered ARF1 polypeptide to treat a desired indication in a subject is sufficient to allow for less-concentrated formulations of the therapeutic molecule to be prepared. Carbomers in an aqueous pharmaceutical composition serve as emulsifying agents and viscosity modifying agents. In certain aspects, the pharmaceutically acceptable excipient comprises or consists of a carbomer. In certain aspects, the carbomer comprises or consists of carbomer 910, carbomer 934, carbomer 934P, carbomer 940, carbomer 941, carbomer 1342, or combinations thereof. Cyclodextrins in an aqueous pharmaceutical composition serve as solubilizing and stabilizing agents. In certain aspects, the pharmaceutically acceptable excipient comprises or consists of a cyclodextrin. In certain aspects, the cyclodextrin comprises or consists of alpha cyclodextrin, beta cyclodextrin, gamma cyclodextrin, or combinations thereof. Lecithin in a pharmaceutical composition may serve as a solubilizing agent. In certain aspects, the solubilizing agent comprises or consists of lecithin. Poloxamers in a pharmaceutical composition serve as emulsifying agents, solubilizing agents, and dispersing agents. In certain aspects, the pharmaceutically acceptable excipient comprises or consists of a poloxamer. In certain aspects, the poloxamer comprises or consists of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407, or combinations thereof. Polyoxyethylene sorbitan fatty acid esters in a pharmaceutical composition serve as emulsifying agents, solubilizing agents, surfactants, and dispersing agents. In certain aspects, the pharmaceutically acceptable excipient comprises or consists of a polyoxyethylene sorbitan fatty acid ester. In certain aspects, the polyoxyethylene sorbitan fatty acid ester comprises or consists of polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, polysorbate 120, or combinations thereof. Polyoxyethylene stearates in a pharmaceutical composition serve as emulsifying agents, solubilizing agents, surfactants, and dispersing agents. In certain aspects, the pharmaceutically acceptable excipient comprises or consists of a polyoxyethylene stearate. In certain aspects, the polyoxyethylene stearate comprises or consists of polyoxyl 2 stearate, polyoxyl 4 stearate, polyoxyl 6 stearate, polyoxyl 8 stearate, polyoxyl 12 stearate, polyoxyl 20 stearate, polyoxyl 30 stearate, polyoxyl 40 stearate, polyoxyl 50 stearate, polyoxyl 100 stearate, polyoxyl 150 stearate, polyoxyl 4distearate, polyoxyl 8 distearate, polyoxyl 12 distearate, polyoxyl 32 distearate, polyoxyl 150 distearate, or combinations thereof. Sorbitan esters in a pharmaceutical composition serve as emulsifying agents, solubilizing agents, and non-ionic surfactants, and dispersing agents. In certain aspects, the pharmaceutically acceptable excipient comprises or consists of a sorbitan ester. In certain aspects, the sorbitan ester comprises or consists of sorbitan laurate, sorbitan oleate, sorbitan palmitate, sorbitan stearate, sorbitan trioleate, sorbitan sesquioleate, or combinations thereof. In certain aspects, solubility can be achieved with a protein carrier. In certain embodiments the protein carrier comprises recombinant human albumin.

[0141] In certain aspects, the polypeptide (e.g., a modified protein, a homologous ARF1 protein sequence, a ARF1 protein sequence, or an engineered ARF1 polypeptide) of the current disclosure are formulated to increase stability. Polypeptides in aqueous formulations may require stabilization to prevent degradation. In certain aspects, the stabilizer comprises pH buffers, salts, amino acids, polyols / disaccharides / polysaccharides, liposomes, surfactants, antioxidants, reducing agents, or chelating agents. In certain aspects, the stabilizer comprises or consists of a polyol / non-reducing sugar. In certain embodiments, the non-reducing sugar comprises or consists of sucrose, mannitol, trehalose, raffinose, stachyose, xylitol, starch, verbascose, or combinations thereof. Polypeptides can be encapsulated in liposomes to increase stability. In certain aspects, the stabilizer comprises or consists of liposomes. In certain aspects, the liposomes comprise or consists of ipalmitoylphosphatidylcholine (DPPC) liposomes, phosphatidylcholine:cholesterol (PC:Chol) (70:30) liposomes, or dipalmitoylphosphatidylcholine: dipalmitoylphosphatidylserine (DPPC:DPPS) liposomes (70:30). Non-ionic surfactants can increase the stability of a polypeptide. In certain aspects, the stabilizer comprises or consists of a non-ionic surfactant. In certain aspects, the non-ionic surfactant comprises or consists of polysorbates (e.g., poly sorbate 80, poly sorbate 20), alkylsaccharides alkyl ethers and alkyl glyceryl ethers, polyoxyethelene (4) lauryl ether; polyoxyethylene cetyl ethers, polyoxyethylene stearyl ethers, sorbitan fatty acid esters, polyoxyethylene fatty acid esters, or combinations thereof. In certain aspects, the polypeptide is formulated with a protein surfactant, such as recombinant human serum albumin as a stabilizer. Antioxidants or reducing agents can increase the stability of a polypeptide. In certain aspects, the stabilizer comprises or consists of an antioxidant or reducing agent. In certain aspects, the reducing agent comprises or consists of dithiothreitol, ethylenediaminetetraacetic acid, 2-Mercaptoethanol, Tris(2-carboxyethyl)phosphine hydrochloride, Tris(hydroxypropyl)phosphine, or combinations thereof. In certain aspects, the antioxidant comprises or consists of methionine, ascorbic acid, citric acid, alpha tocopherol, sodium bisulfite, ascorbyl palmitate, erythorbic acid, or combinations thereof. Chelating agents can stabilize polypeptides by reducing the activity of proteases. In certainaspects, the stabilizer comprises or consists of a chelating agent. In certain aspects, the chelating agent comprises or consists of ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(P- aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), metal complexes (e.g. Zn-protein complexes), or combinations thereof. Buffer agents can stabilize polypeptides by reducing the acid hydrolysis of polypeptides. In certain aspects, the stabilizer comprises or consists of a buffer agent. In certain aspects, the buffer agent comprises or consists of sucrose octa-sulfate, ammonium carbonate, ammonium phosphate, boric acid, sodium citrate, potassium citrate, lactic acid, 3-(N- morpholino)propanesulfonic acid (MOPS), 2-(N-morpholino)ethanesulfonic acid (MES), hydroxymethylaminomethane (Tris), calcium carbonate, calcium phosphate or combinations thereof.

[0142] In certain aspects, the polypeptide (e.g., a modified protein, a homologous ARF1 protein sequence, a ARF1 protein sequence, or an engineered ARF1 polypeptide) of the current disclosure are included in a pharmaceutical composition suitable for intravenous administration comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. In certain aspects, the polypeptides of the current disclosure are administered suspended in a sterile solution. In certain aspects, the solution is one commonly used for administration of biological formulations, and comprises, for example, about 0.9% NaCl or about 5% dextrose. In certain aspects, the solution further comprises one or more of: buffers, for example, acetate, citrate, histidine, succinate, phosphate, potassium phosphate, bicarbonate and hydroxymethylaminomethane (Tris); surfactants, for example, polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and pol oxamer 188; polyol / disaccharide / polysaccharides, for example, glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; amino acids, for example, glycine, histidine, leucine, or arginine; antioxidants, for example, ascorbic acid, methionine; or chelating agents, for example, EDTA, or EGTA.

[0143] In certain aspects, the polypeptide (e.g., a modified protein, a homologous ARF1 protein sequence, a ARF1 protein sequence, or an engineered ARF1 polypeptide) of the current disclosure are included in a pharmaceutical composition suitable for intramuscular or subcutaneous administration comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. Formulations suitable for intramuscular or subcutaneous injection can include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include ethanol, polyols (inositol, propyleneglycol, polyethylene-glycol, glycerol, cremophor and the like) and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such asethyl oleate. Proper fluidity is maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Formulations suitable for subcutaneous injection also contain optional additives such as preserving, wetting, emulsifying, and dispensing agents.

[0144] In certain aspects, the polypeptide (e.g., a modified protein, a homologous ARF1 protein sequence, a ARF1 protein sequence, or an engineered ARF1 polypeptide) of the current disclosure are formulated for topical administration as a cream, gel, paste, ointment, or emulsion. Excipients in a cream, gel, paste, ointment, or emulsion can comprise gelatin, casein, lecithin, gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glyceryl monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyethylene glycols, polyoxyethylene stearates, colloidol silicon dioxide, phosphates, sodium dodecyl sulfate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethy cellulose phthalate, noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, polyvinylpyrrolidone, sugars, and starches.

[0145] The excipient used with the polypeptide (e.g., a modified protein, a homologous ARF1 protein sequence, a ARF1 protein sequence, or an engineered ARF1 polypeptide) described herein will allow for storage, formulation, or administration of highly concentrated formulations. In certain aspects, a highly concentrated polypeptide (e.g., a modified protein, a homologous ARF1 protein sequence, a ARF1 protein sequence, or an engineered ARF1 polypeptide) comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 20, 25, 40, 45, 50 or more milligrams per milliliter.

[0146] In certain aspects, the polypeptides of the current disclosure are shipped / stored lyophilized and reconstituted before administration. In certain embodiments, lyophilized modified protein or engineered ARF1 polypeptide formulations comprise a bulking agent such as, mannitol, sorbitol, sucrose, trehalose, and dextran 40. The lyophilized formulation can be contained in a vial comprised of glass. The Engineered ARF1 polypeptides or Engineered ARF1 polypeptides when formulated, whether reconstituted or not, can be buffered at a certain pH, generally less than 7.0. In certain embodiments, the pH can be between 4.5 and 6.5, 4.5 and 6.0, 4.5 and 5.5, 4.5 and 5.0, or 5.0 and 6.0.Kits

[0147] Disclosed herein are kits. A kit can comprise a modified protein, salt thereof, formulation, or composition described herein. A kit can comprise a fusion protein, salt thereof, formulation, or composition described herein. A kit can comprise an engineered ARF1 polypeptide, salt thereof,formulation, or composition described herein. In some embodiments, the kit comprises a diluent, an excipient, or a carrier. In some aspects, the modified protein, salt thereof, formulation, or composition can be packaged in a container. In some aspects, the fusion protein, salt thereof, formulation, or composition can be packaged in a container. In some aspects, the engineered ARF1 polypeptide, salt thereof, formulation, or composition can be packaged in a container. In some embodiments, a kit can further comprise instructions that direct administration of the modified protein or of a unit dose of the modified protein or formulation to a subject. In some embodiments, a kit can further comprise instructions that direct administration of the fusion protein or of a unit dose of the fusion protein or formulation to a subject. In some embodiments, a kit can further comprise instructions that direct administration of the engineered ARF1 polypeptide or of a unit dose of the engineered ARF1 polypeptide or formulation to a subject. In some embodiments, a kit can further comprise instructions for the use thereof. In some embodiments, the instructions for use designate one or more indications in a subject in need of treatment, wherein the one or more indications comprise pediatric obesity, type II diabetes, obesity, an obesity syndrome, clinical obesity, lipedema, metabolic syndrome / pre-diabetes, cardiovascular disease, nonalcoholic steatohepatitis (NASH) or nonalcoholic fatty liver disease (NAFLD), type I diabetes, Cushing’s disease, tumor-induced Cushing’s disease, Prader-Willi syndrome, Bardet-Biedl syndrome, thyroid disorder, thyroid removal, Adrenoleukodystrophy, Niemann-Pick disease, Tangier disease, Polycystic ovary syndrome, Congenital leptin deficiency, Cohen syndrome, Alstrbm syndrome, or Froehlich syndrome, or any combination thereof. In some embodiments, the kit further comprises a drug delivery device for administering the pharmaceutical composition to a subject. In some embodiments, the drug delivery device is a syringe or an injector pen.

[0148] Methods of making a kit can include placing a modified protein, salt thereof, formulation, or composition described herein in a container for packaging. Methods of making a kit can include placing a fusion protein, salt thereof, formulation, or composition described herein in a container for packaging. Methods of making a kit can include placing an engineered ARF1 polypeptide, salt thereof, formulation, or composition described herein in a container for packaging. A method can further comprise an inclusion of instructions for use. In some cases, instructions for use can direct administration of a modified protein, or of a unit dose of a modified protein or formulation to a subject. In some cases, instructions for use can direct administration of a fusion protein, or of a unit dose of a fusion protein or formulation to a subject. In some cases, instructions for use can direct administration of an engineered ARF1 polypeptide, or of a unit dose of an engineered ARF1 polypeptide or formulation to a subject.EXAMPLES

[0149] The following illustrative examples are representative of embodiments of the compositions and methods described herein and are not meant to be limiting in any way. Example 1 — Construction and testing of engineered polypeptides

[0150] ARF1 protein sequences were analyzed for structural features relating to biological function of ARF1 and potential modifications that could modulate stability, biological function, or in vivo half-life of an engineered protein. Features were also analyzed with regard to modifications that could improve in vitro expression or in vitro purification of the engineered protein.

[0151] Insertions, deletions, or amino acid substitutions of various peptide sequences are made at positions of interest in homologous ARF1 protein sequences. SEQ ID NOS: 1-10 list various vertebrate ARF1 protein sequences that are selected for engineering. Versions of amino acid substitutions in the protein sequence of SEQ ID NO: 1 were made. As an example, SEQ ID NO: 11 has the initiation Methionine deleted from the protein sequence of SEQ ID NO: 1. An another example, SEQ ID NO: 12 has a T31N amino acid substitution, wherein the amino acid numbering is according to wild-type human ARF1 (SEQ ID NO: 1). As another example, SEQ ID NO: 13 has a Q71L amino acid substitution. As another example, SEQ ID NO: 14 has an R99H amino acid substitution. As another example, SEQ ID NO: 15 has a Y35H amino acid substitution. As another example, SEQ ID NO: 16 has a K127E amino acid substitution. As another example, SEQ ID NO: 17 has a T48I amino acid substitution. As another example, SEQ ID NO: 18 has a F51L amino acid substitution. As another example, SEQ ID NO: 19 has a P131R amino acid substitution. As another example, SEQ ID NO: 20 has a P131L amino acid substitution. As another example, SEQ ID NO: 21 has an R19C amino acid substitution. As another example, SEQ ID NO: 22 has the following amino acid substitutions: T31N, Q71L, N126I, and D129N. As another example, SEQ ID NO: 23 lists in the protein sequence eleven positions relative to SEQ ID NO: 1 marked by an X which can be substituted with any amino acid or retain the wild-type amino acid relative to SEQ ID NO: 1. These engineered versions of ARF1 protein are tested either individually, or incorporated into a further engineered protein construct having additional amino acid sequences in a single polypeptide chain and then tested.

[0152] Various inserts, N-terminal additions, or C-terminal additions to wild-type and engineered ARF1 polypeptide sequence are made. ARF1 fusions proteins produced by insertion, N- terminal addition, and or C-terminal addition of one or more heterologous peptide sequences are engineered for various purposes such as: improved fusion protein production, improved secretion, improved fusion protein purification, improved pharmacokinetics following administration (e.g., serum half-life, Cmax, AUC, Tmax, etc.), improved pharmacodynamics, improved therapeutic efficacy, and any combination thereof. Examples include inserting a peptide tag within anengineered ARF1 polypeptide sequence or positioning a peptide tag N-terminal or C-terminal to the engineered ARF1 polypeptide sequence. Examples include incorporation of a six histidine tag (SEQ ID NO: 24), a serine with a six histidine tag (SEQ ID NO: 41), a Strep-tag (SEQ ID NO: 42), a serine with a Strep tag (SEQ ID NO: 43), a hemagglutinin (HA) tag (SEQ ID NO: 44), a Myc tag (SEQ ID NO: 45), a synthetic NE-tag (SEQ ID NO: 46), a FLAG octapeptide tag (SEQ ID NO: 47), three consecutive copies of a FLAG octapeptide tag (SEQ ID NO: 48), and a Spot-tag (SEQ ID NO: 49).

[0153] Another example is an insertion, N-terminal addition, or C-terminal addition of one or more heterologous peptide sequences. Examples of inserted heterologous peptide sequences include inserting a human serum albumin (HSA) protein sequence or variant thereof N-terminal or C-terminal to the engineered ARF1 polypeptide sequence. In these exemplary instances, an engineered HSA-ARF1 fusion protein is produced. Further examples include inserting a serine residue upstream of an HSA protein sequence or variant thereof inserted N-terminal or C-terminal to the engineered ARF1 polypeptide sequence. Exemplary HSA sequences are listed in SEQ ID NO: 51 and SEQ ID NO: 52. In one example, a Flag-tag is positioned N-terminal to an HSA sequence, wherein the HSA sequence is positioned N-terminal to a ARF1 sequence (e.g., SEQ ID NO: 61). In another example, an HSA sequence is positioned N-terminal to a ARF1 sequence (e.g., SEQ ID NO: 62) and the engineered protein does not comprise a tag. In another example, the HSA- ARF1 fusion protein does not comprise a methionine at the N-terminus. In one example, an Annexin A10 peptide sequence is positioned N-terminal to an HSA sequence, wherein the HSA sequence is positioned N-terminal to a ARF1 sequence (e.g., SEQ ID NO: 63). In one example, a 6XHis peptide tag is positioned N-terminal to a TEV protease recognition sequence, wherein both the 6XHis peptide tag and TEV sequence are positioned N-terminal to an HSA sequence, wherein the HSA sequence is positioned N-terminal to a ARF1 sequence (e.g., SEQ ID NO: 64). In one example, a Flag-tag is positioned N-terminal to an HSA sequence, wherein the HSA sequence is positioned N-terminal to a ARF1 sequence and the HSA and ARF1 domains are separated by a flexible glycine serine linker sequence (e.g., SEQ ID NO: 176). In one example, an N-terminal HSA sequence having an initiation methionine is position upstream of an ARF1 sequence in a single polypeptide chain wherein the HSA and ARF1 domains are separated by a flexible glycineserine linker sequence (e.g., SEQ ID NO: 184). In one example, an N-terminal signal peptide sequence is N-terminal to an HSA sequence followed by a flexible glycine-serine linker sequence followed by a C-terminal ARF1 sequence (e.g., SEQ ID NO: 185). In one example, an N-terminal 6XHis sequence tag is followed by a TEV protease recognition motif sequence, both positioned upstream of an HSA sequence followed by a flexible glycine-serine linker sequence followed by aC-terminal ARF1 sequence (e.g., SEQ ID NO: 186). In one example, a 6XHis-tagged HSA-ARF1 fusion protein is selectively purified by using immobilized metal affinity chromatography (IMAC) to isolated 6XHis-tagged protein molecules. For HSA-ARF1 fusion proteins comprising a TEV protease recognition motif sequence, these fusion proteins may then be cleaved by a TEV protease to separate a peptide tag and the remainder of the TEV protease recognition motif sequence (e.g., SEQ ID NO: 285) from the HSA-ARF1 fusion protein. TEV protease cleaves specifically at the sequence "ENLYFQ / G or S" (where the slash indicates the cleavage site), meaning it cuts between the glutamine (Q) and the glycine or serine (G / S) residues within a protein substrate. The HSA- ARF1 fusion protein may then be further purified from the peptide tag. In one example, a purified HSA-ARF1 fusion protein following TEV cleavage comprises SEQ ID NO: 315. In one example, a purified HSA-ARF1 fusion protein having an T31N substitution within the ARF1 domain following TEV cleavage comprises SEQ ID NO: 316. In one example, an N-terminal signal peptide sequence is N-terminal to an HSA sequence followed by a flexible glycine-serine linker sequence followed by a C-terminal ARF1 sequence (e.g., SEQ ID NO: 194). In one example, an N-terminal signal peptide sequence is N-terminal to an HSA sequence followed by a flexible glycine-serine linker sequence followed by a C-terminal ARF1 sequence comprising a T31N substitution (e.g., SEQ ID NO: 217). In one example, an N-terminal signal peptide sequence is N-terminal to an ARF1 sequence followed by a flexible glycine-serine linker sequence followed by a C-terminal HSA sequence (e.g., SEQ ID NO: 218). In one example, an N-terminal signal peptide sequence is N-terminal to an ARF1-T31N sequence followed by a flexible glycine-serine linker sequence followed by a C-terminal HSA sequence (e.g., SEQ ID NO: 317).

[0154] In some examples of an HSA-ARF1 fusion protein, one or more flexible linker sequences are used to separate functional domains of the fusion protein. Glycine-serine linkers are examples of one or more flexible linker sequences used in engineered ARF1 fusion proteins. In one example, the glycine-serine linker comprises a concatmer of GS(n=i-5). In one example, the glycine- serine linker comprises a concatmer of GGGS(n=i-5). In one example, the glycine-serine linker comprises SEQ ID NO: 75. In one example, the glycine-serine linker comprises SEQ ID NO: 76. In one example, the glycine-serine linker comprises SEQ ID NO: 312. In one example, the glycine- serine linker comprises SEQ ID NO: 313. In one example, the glycine-serine linker comprises SEQ ID NO: 314.

[0155] In another example of ARF1 fusion proteins engineered by insertion, N-terminal addition, or C-terminal addition of one or more heterologous peptide sequences is insertion of a green fluorescent protein (GFP) sequence or variant thereof (e.g., eGFP) N-terminal or C-terminal to the engineered ARF1 polypeptide sequence. Exemplary ARF1-GFP fusion proteins include thoselisted in SEQ ID NOs: 73, 74, 190, 191, and 195.

[0156] In another example ARF1 fusion proteins engineered by insertion, N-terminal addition, or C-terminal addition of one or more heterologous peptide sequences is insertion of an immunoglobulin protein sequence or variant thereof N-terminal or C-terminal to the engineered ARF1 polypeptide sequence. In several examples of Fc-ARFl fusion proteins, an immunoglobulin protein sequence or variant thereof is positioned N-terminal to an ARF1 sequence (e.g., SEQ ID NOs: 196, 215, 249, 250, 283, or 284). In several examples of ARFl-Fc fusion proteins, an immunoglobulin protein sequence or variant thereof is positioned C-terminal to an ARF1 sequence (e.g., SEQ ID NOs: 59, 60, 181, 182, 192, 216, 245-248, or 251-282). In several examples, an Fc- ARFl or ARFl-Fc fusion protein comprises a flexible serine-glycine linker sequence to separate the IgG Fc domain and ARF1 domain. ). In several examples, an Fc-ARFl or ARFl-Fc fusion protein comprises a signal peptide sequence to improve secretion of the fusion protein and aid in substitution fusion protein production and purification. In several examples, an Fc-ARFl or ARFl- Fc fusion protein comprises one or more amino acid substitutions within the ARF1 domain to improve biological efficacy and / or improve fusion protein production and purification. In several examples, an Fc-ARFl or ARFl-Fc fusion protein comprises one or more amino acid substitutions within the Fc domain. In some examples of Fc-ARFl or ARFl-Fc fusion protein, one or more amino acid substitutions within the Fc domain reduced effector response of the immunoglobulin domain. Examples include IgGl L234AZL235A (LALA) substitution within the Fc domain. LALA substitution has the effects of reducing binding of the fusion protein to FcyRs, making these fusion proteins silent or near silent for FcyR-mediated effector functions. LALA substitution has the effect of reducing binding to complement component Clq. LALA substitution has the effect of reducing antibody-dependent cellular cytotoxicity (ADCC). LALA substitution has the effect of reducing complement binding / activation. LALA substitution has the effect of reducing inflammatory cytokine release. In some examples, LALA substitution is further combined with P329G substitution (LALA-PG) as an Fc variant with reduced effector function. LALA-PG substitution has the effect of eliminating complement binding and fixation as well as Fcy-dependent, antibodydependent, cell-mediated cytotoxicity in various IgG molecules. Additional examples include IgGl M252Y, S254T and T256E (YTE) substitution within the Fc domain. When YTE substitutions are included together, an Fc fusion protein exhibits significantly enhance its binding affinity to the neonatal Fc receptor (FcRn), leading to a longer serum half-life for the fusion protein. In some examples, LALA or LALA-PG is combined with YTE substitution. In some examples, human IgG4 or a variant thereof is used for the Fc portion of an Fc-ARFl or ARFl-Fc fusion protein. In some examples, human IgG2 or a variant thereof is used for the Fc portion of an Fc-ARFl orARFl-Fc fusion protein.

[0157] Another example is an insertion, N-terminal addition, or C-terminal addition of an Annexin A10 peptide sequence to the engineered ARF1 polypeptide sequence (e.g., SEQ ID NO: 63).

[0158] Another example of a modification to an engineered ARF1 polypeptide sequence is the N-terminal addition of a peptide sequence recognized for a specific type of post-translational modification. One example used here is that addition of a myristoylation recognition sequence positioned N-terminal to the engineered ARF1 polypeptide sequence. The peptide sequence MGSS was inserted at the N-terminal position of various engineered ARF1 constructs (e.g., SEQ ID NOS: 29-40). In some instances, the myristoylation recognition sequence was positioned next to a peptide tag sequence, or next to a protease recognition sequence, or next to both a peptide tag sequence and a protease recognition sequence. An example protease recognition sequence used was the TEV protease recognition motif as shown in SEQ ID NO: 26. An example myristoylation recognition sequence and peptide tag sequence used was SEQ ID NO: 25. An example myristoylation recognition sequence, peptide tag sequence, and protease recognition sequence used was SEQ ID NO: 27. Myristoylation can occur on penultimate N-terminal glycine residues and is known to require the prior removal of the initial methionine residue. Following recognition of a myristoylation sequence, a lipid modification to the protein occurs involving the addition of a 14- carbon unsaturated fatty acid, myristic acid, to the N-terminal glycine of a subset of selected proteins. This post-translational modification promotes binding of the protein to cell membranes for a variety of biological functions.

[0159] Another example of a modification to an engineered ARF1 polypeptide sequence or an ARF1 fusion protein is a designed amino acid structure to prevent the N-terminal addition of a specific type of post-translational modification. One example used here is the prevention of myristoylation on a glycine residue near the N-terminus of an engineered ARF1 polypeptide or ARF1 fusion protein. Wild-type human ARF1 proteins produced intracellularly exhibit a significant extent of myristoylation on glycine position 2 (G2) of polypeptide corresponding to SEQ ID NO: 1. Engineered ARF1 polypeptide sequences or ARF1 fusion proteins lacking myristoylation on ARF1-G2 were produced and subsequently tested for in vivo efficacy. Constructing the relative position of G2 in the ARF1 region of the engineered ARF1 polypeptide sequence or an ARF1 fusion protein in a position different than following the initiation methionine in a single polypeptide chain prevents addition of this particular lipid modification in the endoplasmic reticulum following protein translation. Engineered ARF1 polypeptide sequences or ARF1 fusion proteins lacking N- terminal myristoylation were produced to alter protein trafficking dynamics. By this means, theengineered ARF1 polypeptide sequences or ARF1 fusion proteins were designed to significantly alter association with specific cellular membrane compartments, modulate protein-protein interactions, and alter cellular membrane anchoring in comparison to myristoylated engineered ARF1 polypeptide sequences or ARF1 fusion proteins. In one example, the 6XHis-tagged-TEV- ARF1-T31N protein listed in SEQ ID NO: 123 and the 6XHis-tagged-TEV-ARFl-wild_type listed in SEQ ID NO: 124 were produced and later tested for in vivo efficacy. In the polypeptide produced from SEQ ID NO: 123 and SEQ ID NO: 124, the ARF1 G2 residue is not significantly myristoylated. Further examples of engineered ARF1 proteins lacking N-terminal myristoylation include polypeptides of SEQ ID NOs: 125-174. SEQ ID NOs: 123-174 were constructed to comprise a 6XHis tag and TEV recognition motif to further aid in protein purification and isolation of cleavage product comprising engineered ARF1 polypeptide sequence if TEV protease treatment is undertaken. Exemplary cleaved ARF1 polypeptide sequences lacking N-terminal myristoylation are listed in SEQ ID NOs: 286-289 and 297-311. Engineered ARF1 polypeptides from SEQ ID NOs: 286-289 and 297-311 comprise an additional glycine residue at position 1 compared to wildtype human ARF1 sequences (SEQ ID NO: 1). This additional N-terminal glycine is due to the remainder of the N-terminal TEV recognition sequence following TEV protease treatment. In another example, an alternative TEV recognition sequence is utilized (SEQ ID NO: 290) to produce engineered ARF1 proteins lacking N-terminal myristoylation. Examples include SEQ ID NOs: 291 and 292. Engineered ARF1 polypeptides from SEQ ID NOs: 293-295 comprise an additional serine residue at position 1 compared to wild-type human ARF1 sequences (SEQ ID NO: 1). This additional N-terminal serine is due to the remainder of the N-terminal alternative TEV recognition sequence following TEV protease treatment. Engineered ARF1 fusion proteins in which the ARF1 G2 residue is not significantly myristoylated are also constructed by positioning one or more heterologous peptide sequences N-terminal to the ARF1 coding sequence in the single polypeptide chain. In some examples, engineered ARF1 proteins or ARF1 fusion proteins lacking N-terminal myristoylation are produced, concentration, and formulated for administration to a subject in need thereof in a pharmaceutical composition further comprising a pharmaceutically acceptable excipient, carrier, or diluent. In one example, the protein of SEQ ID NO: 123 lacking N-terminal myristoylation was formulated for i.p. administration in a pharmaceutical composition further comprising a pharmaceutically acceptable excipient, carrier, or diluent. In another example, the protein of SEQ ID NO: 124 lacking N-terminal myristoylation was formulated for i.p. administration in a pharmaceutical composition further comprising a pharmaceutically acceptable excipient, carrier, or diluent.

[0160] In some examples, linker sequences separate the inserted heterologous protein sequenceor sequences from the homologous ARF1 protein sequence within the engineered polypeptide or within ARF1 fusion proteins. Exemplary linker sequences used in engineered polypeptide constructs include a short flexible linker sequence (SEQ ID NO: 75), a long flexible linker sequence (SEQ ID NO: 76), or other serine-glycine linkers including SEQ ID NOs: 312-314.

[0161] In some examples, the effect of including a signal peptide sequence at the N-terminus of an engineered ARF1 polypeptide on protein production, protein secretion, protein accumulation, or a combination thereof was examined. A signal peptide sequence was selected from exemplary sequences listed as SEQ ID NOS: 77-86. Engineered ARF1 constructs were generated with a selected signal peptide sequence N-terminal to the engineered or wild-type ARF1 protein sequence. Mammalian cells were transfected with engineered ARF1 polypeptide expression constructs and samples were taken from supernatant and cell pellet do assay protein production levels.

[0162] Nucleic acid sequences encoding engineered ARF1 polypeptides are cloned into an expression vector (e.g., pmaxCloning™ vector). Exemplary signal peptide sequences and human ARF1 mRNA coding sequences used as a basis for producing the engineered ARF1 polypeptides are listed in SEQ ID NOs: 107-121. In one example, sequence encoding an engineered ARF1 protein described herein was used for expression of His-TEV-ARF1-T3 IN by a plasmid under control of mouse albumin promoter. Coding sequence from this plasmid is listed in SEQ ID NO: 122.

[0163] Engineered ARF1 constructs are transfected into various cell lines and tested for protein localization and protein production. Three transfection methods were utilized to test the constructs and are described below:

[0164] Method 1: Expi293F™ cells, TransIT-PRO transfection:

[0165] Materials:

[0166] Expi293F™ cells (Gibco Cat A14527)

[0167] Expi293 Expression Medium (Gibco Cat A1435101)

[0168] Opti-MEM I Reduced Serum Medium (Gibco Cat 31985070)

[0169] TransIT-PRO (MirusBio Cat MIR 5750)

[0170] Method from MirusBio for Expi293F™ (25mL volume example):

[0171] Day -1 : Split cells to 2 x 106cell / ml density

[0172] Day 0:

[0173] Check cell viability > 95%.

[0174] Dilute cells to 2.5 x 106cell / ml density and 25mL volume. Incubate in incubator shaker.

[0175] Warm TransIT-PRO to room temperature and vortex gently.

[0176] Add 25 pg plasmid DNA to room temperature 2.5mL Opti-MEM. Mix.

[0177] Add 25 pL TransIT-PRO. Mix.

[0178] Incubate at room temperature for 15 min.

[0179] Add mixture to cells.

[0180] Incubate in incubator shaker and harvest.

[0181] Method 2: Expi293F™ cells, ExpiFectamine 293 transfection:

[0182] Materials:

[0183] Expi293F™ cells (Gibco Cat A14527)

[0184] Expi293 Expression Medium (Gibco Cat A1435101)

[0185] Opti-MEM I Reduced Serum Medium (Gibco Cat 31985070)

[0186] ExpiFectamine 293 Transfection Kit (Gibco Cat A14524)

[0187] Method from Gibco user guide (25mL volume example):

[0188] Day -1 : Split cells to 2.5 x 106cell / ml density

[0189] Day 0:

[0190] Check cell viability > 95%.

[0191] Dilute cells to 3 x 106cell / ml density and 25mL volume. Incubate in incubator shaker.

[0192] Mix ExpiFectamine by inverting 5 times.

[0193] Add 25pg plasmid DNA to 1 ,5mL room temperature Opti-MEM. Mix.

[0194] Add 80pL ExpiFectamine to 1.4mL room temperature Opti-MEM. Mix. Incubate at room temperature for 5 min.

[0195] Combine DNA and ExpiFectamine mixtures. Mix. Incubate at room temperature for 10- 20 min.

[0196] Add mixture to cells. Incubate in incubator shaker.

[0197] Day 1 : Feed 18 to 22 hours post-transfection

[0198] Combine 150pL Enhancer 1 and 1.5mL Enhancer 2. (No need to pre-warm.)

[0199] Add mixture to cells.

[0200] Incubate in incubator shaker and harvest.

[0201] Method 3: ExpiCHO-S cells, ExpiFectamine CHO transfection:

[0202] Materials:

[0203] ExpiCHO-S cells (Gibco Cat A29127)

[0204] ExpiCHO Expression Medium (Gibco Cat A2910001)

[0205] OptiPRO SFM Medium (Gibco Cat 12309019)

[0206] ExpiFectamine CHO Transfection Kit (Gibco Cat A29129)

[0207] Method from Gibco user guide (25mL volume example):

[0208] Day -1 : Split cells to 3 x 106cell / ml density

[0209] Day 0:

[0210] Check cell viability > 95%.

[0211] Dilute cells to 6 x 106cell / ml density and 25mL volume. Incubate in incubator shaker.

[0212] Mix ExpiFectamine by inverting 5 times.

[0213] Add 25 pg plasmid DNA to ImL cold (take from 4°C and use immediately on bench) OptiPRO. Mix.

[0214] Add 80pL ExpiFectamine to 920pL cold OptiPRO. Mix.

[0215] Combine DNA and ExpiFectamine mixtures immediately or up to 5 min. Mix. Incubate at room temperature for 1-5 min.

[0216] Add mixture to cells. Incubate in incubator shaker.

[0217] Day 1 : Feed 18 to 22 hours post-transfection

[0218] Combine 150pL Enhancer and 6mL Feed. (No need to pre-warm.)

[0219] Add mixture to cells.

[0220] Incubate in incubator shaker and harvest.

[0221] The expression construct for the Engineered ARF1 polypeptide named MGSS HIS TEV ARFl wt (SEQ ID NO: 29) was transfected into Expi239F cells using Method 1 or Method 2. Supernatant samples were purified to assay Engineered ARF1 polypeptide secreted from the cells. Cell pellet samples were purified to assay Engineered ARF1 polypeptide produced and maintained within the cells. Purified protein samples were assayed by Western blot using standard techniques with an anti-ARFl antibody used for detection. FIG. 1A shows an image of a Western blot indicating that MGSS HIS TEV ARFl wt was abundantly produced and located with the supernatant and within the cell pellet when cells were harvested 3 days post-transfection. FIG. IB shows a chart of protein production levels of MGSS HIS TEV ARFl wt following transfection and protein purification. As shown in the chart, supernatant samples demonstrated robust and greater protein production than cell pellet samples. This was a surprising result given known roles for ARF1 as proteins localized to the Golgi apparatus.

[0222] Expression of various His-tagged Engineered ARF1 polypeptide constructs was tested and compared in Expi239F cells and CHO cells. As shown via Western blot using an anti-HIS antibody in FIG. 2A, constructs tested with a signal sequence did not produce engineered ARF1 polypeptide in detectable levels in the cell supernatant either at Day 5 (in CHO cells) or at Day 4 (in Expi239F cells) post transfection. Constructs lacking an N-terminal signal peptide sequence did show robust production in the supernatant at Day 5 (in CHO cells) or at Day 4 (in Expi239F cells). As shown via Western blot using an anti-HIS antibody in FIG. 2B, constructs tested with a signal sequence did not produce engineered ARF1 polypeptide in detectable levels in the cell pellet eitherat Day 5 (in CHO cells) or at Day 4 (in Expi239F cells) post transfection. Constructs lacking an N- terminal signal peptide sequence did show robust production in the cell pellet at Day 5 (in CHO cells) or at Day 4 (in Expi239F cells). In comparing expression per cell type, the engineered ARF1 polypeptides showed high expression levels ion Expi293F cells compared to CHO cells. Constructs tested in FIG. 2A-FIG. 2B are listed in Table 6.

[0223] Table 6: Listing of engineered ARF1 polypeptides tested in Expi239F cells and CHO cells

[0224] To further test production of His-tagged Engineered ARF1 polypeptides, CHO cells were used to assay His-tagged Engineered ARF1 polypeptide found in the supernatant from Day 3- 5 post-transfection and in the cell lysate from Day 3-5 post-transfection. As shown in FIG. 3A in an image of a Western blot using anti -His antibody for detection, engineered ARF1 polypeptides listed in Table 6 that were tested including a signal peptide sequence did not show detectable levels of Engineered ARF1 polypeptide in the supernatant. His-TEV-WPl and WP2-His were detected in the supernatant on Days 3 and 5 indicating continued production of secreted Engineered ARF1 polypeptide from these constructs. As shown in FIG. 3B in an image of a Western blot using anti- His antibody for detection, engineered ARF1 polypeptides listed in Table 6 that were tested including a signal peptide sequence did not show detectable levels of Engineered ARF1 polypeptide in the cell lysate. His-TEV-WPl and WP2-His were detected in the cell lysate on Days 3 and 4, and less strongly present by Day 5, indicating continued production of intracellular engineered ARF1 polypeptide from these constructs. These results demonstrate that the signal peptide at the N-terminal region of the His-tagged constructs completely abolished expression of the N-terminal or C-terminal His-tagged engineered ARF1 polypeptides.

[0225] Engineered ARF 1 polypeptides with IgG Fc fusions or with FLAG tags were tested for protein production with and without an N-terminal signal peptide sequence. Expi293F cells were used to assay His-tagged engineered ARF1 polypeptides found in the supernatant or cell pellet from Day 3 post-transfection via Western blot using an anti-His antibody as shown in FIG. 4A. Expi293F cells were used to assay His-tagged engineered ARF1 polypeptides, non-tagged engineered ARF1 polypeptides, and FLAG-tagged engineered ARF1 polypeptides found in thesupernatant (sup) or cell pellet (pellet) from Day 3 post-transfection via Western blot using an anti- ARF1 antibody as shown in FIG. 4B. Engineered ARF1 polypeptides listed in Table 7 were tested.Table 7: Listing of engineered ARF1 polypeptides tested in Expi239F cells

[0226] As shown in FIG. 4A in an image of a Western blot using anti-His antibody for detection, His-tagged engineered ARF1 polypeptides list in Table 7 were detected with the anti-His antibody. ARFl-Fc fusions and FLAG-tagged ARF1 polypeptides were not detected with the anti- His antibody. As shown in FIG. 4B in an image of a Western blot using anti-ARFl antibody for detection, His-tagged engineered ARF1 polypeptides, ARFl-Fc fusions, and FLAG-tagged engineered ARF1 polypeptides were detected. The results indicated the ARFl-Fc fusions with and without a signal peptide showed expression in both supernatant and in cell pellet indicating that ARFl-Fc fusion proteins were both secreted and found intracellularly.

[0227] Engineered ARF1 polypeptides with HSA fusions were tested for production and secretion in Expi293F cells in Day 4 post-transfection. Engineered ARF1 polypeptides with single amino acid substitutions were tested for production and secretion in Expi293F cells. Results of Western blot using anti-ARFl antibody are shown in FIG. 5 (expression in completed medium on left and expression in cell pellet on right). Engineered ARF1 polypeptides tested are listed in Table 8.

[0228] Table 8: Listing of engineered HSA-ARF1 polypeptides and ARF1 single amino acid substitutions tested in Expi239F cells

[0229] As shown in FIG. 5, HSA0-HSA3 were all expressed and found in the cell pellet, whereas HSAO was also robustly expressed in the supernatant. Single substitution mutants were all expressed and found in the cell pellet. The ARF1-T31N construct, a mutant form of ARF1 having low affinity for GTP believed to have dominant negative function, was not detected to be strongly secreted. FLAG-tagged HSA-ARF1 (HSAO) was more strongly secreted than the ARF1-T31N construct.

[0230] As shown in FIG. 6, various engineered ARF1 polypeptide constructs were tested for expression in the supernatant of Expi293F cells on Day 2 and Day 3. FIG. 6 shows results on Western blot using an anti-ARFl antibody for detection on Day 2 (left) and on Day 3 (right) posttransfection. Engineered ARF1 polypeptides tested are listed in Table 9.

[0231] Table 9: Listing of engineered HSA-ARF1 polypeptides tested for secretion in Expi239F cells on Day 2 and Day 3

[0232] The results indicated that the ARF1-T3 IN mutant construct was not detectable as secreted while other HA-tagged constructs showed low expression in the supernatant (WT-HA, Q71L-hyperactive, R99H).

[0233] As shown in FIG. 7, various engineered ARF1 polypeptide constructs were tested for intracellular expression in Expi293F cells on Day 3. FIG. 7 shows results on Western blot using an anti-ARFl antibody for detection on Day 3 post-transfection (left) and a chart of protein levels from the Western blot (right). For these experiments, the following samples were assayed. Lane 1 was ARF1 WT with no tag in supernatant. Lane 2 was ARF1 WT with no tag in cell pellet. Lane 3 was ARF1 WT with HA-tag in supernatant. Lane 4 was ARF1 WT with HA-tag in cell pellet. Lane 5 was ARF1-T31N in supernatant. Lane 6 was ARF1-T31N in cell pellet. 1 pg of WP1 was run in Lane 7 as a control. The results in FIG. 7 indicate that ARF1-WT with no tag, ARF1-WT with HA- tag, and ARF1-T31N did not secrete and have low expression levels intracellularly.

[0234] As shown in FIG. 8, various engineered ARF1 polypeptide constructs were tested for intracellular expression and secreted expression in Expi293F cells on Day 3. FIG. 8 shows results on Western blot using an anti-ARFl antibody for detection on Day 3 post-transfection (left) and a chart of protein levels from the Western blot (right). Engineered ARF1 polypeptides tested are listed in Table 10. Qualitative levels of expression are indicated by WB note.

[0235] Table 10: Listing of engineered HSA-ARF1 polypeptides tested for secretion in Expi239F cells on Day 3

[0236] The results indicated that His TEV ARF 1_T3 IN, SP flag TEV ARF 1_T3 IN, ARFI GFP His, and ARFl_GFP_His_T3 IN were all strongly expressed in the supernatant at Day 3. A4_His_TEV_ARFl was detected in the supernatant but less strongly expressed.

[0237] As shown in FIG. 9, various engineered ARF1 polypeptide constructs were tested for intracellular expression and secreted expression in Expi293F cells on Day 3. FIG. 9 shows results on Western blot using an anti-ARFl antibody for detection on Day 3 post-transfection (left) and a chart of protein levels from the Western blot (right). Engineered ARF1 polypeptides tested are listed in Table 11. Qualitative levels of expression are indicated by WB note to indicate which constructs yielded enhanced secreted expression, enhanced intracellular expression, or both enhanced secreted and intracellular expression.

[0238] Table 11: Listing of engineered HSA-ARF1 polypeptides tested for secretion in Expi239F Day 3

[0239] The results indicated that His_TEV_ARFl_T3 IN, MGSS HIS TEV ARFl, and A4_His_TEV_ARFl were expressed at moderate to low levels in the supernatant whereas ARFI GFP His and ARFl_GFP_His_T31N were expressed at higher levels in the supernatant. All constructs tested show strong intracellular expression.

[0240] As shown in FIG. 10, replicate samples of various engineered ARF1 polypeptide constructs were tested for secreted expression in Expi293F cells on Day 3 and compared to intracellular expression levels. FIG. 10 shows results on Western blot using an anti-ARFl antibody for detection on Day 3 post-transfection (left) and a chart of protein levels from the Western blot (right). Engineered ARF1 polypeptides tested are listed in Table 12. Qualitative levels of expression are indicated by WB note to indicate which constructs yielded enhanced secreted expression, enhanced intracellular expression, or both enhanced secreted and intracellular expression.

[0241] Table 12: Listing of engineered HSA-ARF1 polypeptides tested for secretion in Expi239F Day 3

[0242] The results indicated that His TEV ARF1 T3 IN tested in six replicate samples was expressed at moderate levels in the supernatant whereas WS4-Flag and Flag-T3 IN were expressed at higher levels in the supernatant. WS4-Flag and Flag-T3 IN were expressed more strongly in the supernatant than intracellularly.

[0243] The ExpiCHO™ Expression System Kit (ThermoFisher) is used according to the manufacturer’s instructions for expression and secretion of modified proteins. In the case of a His- tagged engineered polypeptides, protein purification proceeds by the IMAC method to rapidly purify and enrich a polyhistidine affinity-tagged protein. Various other protein purification methods may be used for engineered polypeptides that do not comprise a polyhistidine tag. Next, size exclusion chromatography (SEC), also known as gel filtration, is used as a chemically inert mode of chromatography employed for biomolecule purification to further purify the engineered polypeptide. Next, the purified engineered polypeptide is further concentrated using standard protein concentration techniques.

[0244] Following production, purification, and concentration of the engineered ARF1 polypeptides, testing begins through various in vitro and in vivo assays.Example 2 — Therapeutic dosage effect of ARF1 on lipolysis

[0245] For this example, FIG. 11A shows an in vitro study design using cultured mouse 3T3L1 adipocytes to assess the effects of various ARF1 dosages on lipolysis. Dosages of ARF1 ranging from 0.4 ng / mL to 20 pg / mL were contacted with cultured differentiated adipocytes. Total lipid intensity and LD size was measured by imaging lipid drops. Secreted glycerol in the culture media was assayed as fold change compared to control for the lipolysis assay. Mouse 3T3L1 adipocytes were differentiated for 4-5 days followed by 3 days of treatment with an ARF1 therapeutic in maintenance medium (DMEM / F12 + 1 % FBS) after lipid loading and are assayed on Day 4.

[0246] Results of the lipolysis assay are graphed in FIG. 11B indicating a dose-dependentincrease in lipolysis in response to increasing concentration of His_ARFl_T31N. The His-ARFl- T3 IN protein tested in this example comprises the sequence of SEQ ID NO: 123. In FIG. 11B, n=7-9 independent studies for the lipolysis assay. For statistical analysis in FIG. 11B, one-way ANOVA followed by Bonferroni test, ARF1 treatment groups were compared to the vehicle control (**,p<0.0.01, ***, p<0.001). Lower dashed line on the chart indicates a baseline standard set for lipolysis fold change at a value of 1.0 for the vehicle control. Upper dashed line on the chart indicates Emax (maximal lipolytic response) for the positive control as a value great than 10 fold change from baseline. Results of an additional lipolysis assay from the study design in FIG. 11A are graphed in FIG. 33 comparing lipolysis fold change in response to increasing concentration of His-ARF1-T31N or His-ARF1-T31N. The His-ARF1-T31N protein tested in this example comprises the sequence of SEQ ID NO: 123. The His-ARFl-WT protein tested in this example comprises the sequence of SEQ ID NO: 124. In this example, a dose-dependent increase in lipolysis in response to increasing concentration of His-ARF1-T31N protein (non-myristoylated and preferentially GDP -bound) was evident. Increased lipolysis in response His-ARFl-WT protein (non-myristoylated and comprising wild-type ARF1 protein sequence) was evident, but the dosedependent increase in lipolysis in response to increasing concentration of His-ARFl-WT protein was not demonstrated. These results indicate that non-myristoylated forms of ARF1 protein increased lipolysis compared to vehicle control and that the most pronounced increase in lipolysis (also demonstrating a dose-dependent increase) was with the non-myristoylated, preferentially GDP -bound form of ARF1-T31N.

[0247] As alternative therapeutics to His-ARF1-T3 IN, one or more engineered ARF1 polypeptides, ARF1 fusion proteins, or ARF1 wild-type proteins listed in Table 1A or Table IB are tested using the procedures in this example.

[0248] Using the study design in FIG. 11 A, the effects of various ARF1 dosages on triglyceride content and lipid droplet staining were assayed. In FIG. 12A, dosages of ARF1 (His- ARF1-T31N) ranging from 0.4 ng / mL to 20 pg / mL were contacted with cultured differentiated adipocytes and triglyceride (TG) content was assayed. FIG. 12A shows a dose-dependent decrease in TG content following contacting of cells with His-ARF1-T31N (SEQ ID NO: 123). n=3 independent studies for each of TG content analysis. In FIG. 12B, the results of neutral lipid imaging in response to His-ARF1-T3 IN treated were graphed. Neutral lipid imaging was accomplished using Bodipy dye for labeling neutral lipids, wheat germ agglutinin (WGA) staining to label outer membranes, and DAPI as a nuclear stain for cellular quantitation. FIG. 12B shows a dose-dependent decrease in lipid droplet (LD) intensity following contacting of cells with His- ARF1-T31N (SEQ ID NO: 123). n=3 independent studies for each of LD intensity analysis.Baseline standards were set for vehicle control and Emax for the positive control, both shown in FIG. 12A and FIG. 12B. For statistical analysis in FIG. 12A and FIG. 12B, one-way ANOVA followed by Bonferroni test, ARF1 treatment groups were compared to the vehicle control (*, p<0.05**, p<0.0.01, ***, p<0.001).

[0249] Using the study design in FIG. 11 A, the effects of various ARF1 dosages on fatty acid uptake are assayed.Example 3 — Administration of a therapeutic composition to improve diet induced obesity

[0250] A high fat diet induced obesity (HF-DIO) model is used to test efficacy of therapeutic compositions described here for hyperlipidemia, hyperglycemia, endocrine function, or a combination thereof. Additionally, effects on inflammatory profile are analyzed in this model. One of four mouse strains is selected for use for the HF-DIO model: Kunming, C57B1 / 6J, BALB / c, or ICR. Kunming and ICR are outbred strains of mice allowing the study of high fat diet induced obesity in the context of genetic variation as a possible contributing factor to the phenotypes under study. C57B1 / 6J and BALB / c strains of mice are isogenic (inbred), thereby bypassing potential genetic variation as a contributing factor to the phenotypes under study. All four mouse strains allow for the study of environmental and epigenetic influences as contributing factor to the phenotypes under study.

[0251] HF-DIO Study Design

[0252] For a selected mouse strain, mice obtained at approximately 15 weeks of age and 10 mice were grouped as control and 30 are grouped as test arm. Animals were raised in standard mouse husbandry conditions including 23 ± 2°C, 55 ± 5% relative humidity, 12 h light / dark cycle. DIO-C57BL / 6J mice fed on a high fat diet (HFD) (60% kcal from fat) were used as for diet- induced obesity (DIO) groups. Control C57BL / 6J mice fed on a low fat diet (LFD) (10% kcal from fat) were used as for DIO-control groups.

[0253] After 3 weeks of acclimation, control animals were continued to be fed a standard chow diet (LFD) and test model animals were continued to be fed a high fat diet (HFD). The high fat diet is formulated according to the protocol described in Chen L. et al., A mix of apple pomace polysaccharide improves mitochondrial function and reduces oxidative stress in the liver of high- fat diet-induced obese mice. Mol Nutr Food Res. 2017 Mar;61(3), which is hereby incorporated by reference regarding induction and testing of HF-DIO models. Animals are maintained on respective diets for a period of 8 weeks prior to initiation of therapeutic study.

[0254] Therapeutic Study Design

[0255] Therapeutic study lasted for 24 days and was run according to the experimental study design listed in FIG. 13.

[0256] High fat diet fed animals (60% kcal from fat HFD) were grouped accordingly (10 animals per group): Group 1 - vehicle treated (PBS by IP injection). Group 2 - treated with ARF1 protein (His-ARF1-T31N at 4mg / kg by IP injection). Group 3 - treated with Tirzepatide (TZP; 10 nmol / kg by s.c. injection). Group 4 - DIO-control diet mice (LFD) vehicle treated (PBS by IP injection).

[0257] In certain instances, Group 5 is treated with an engineered ARF1 polypeptide or an ARF1 fusion protein described herein. In one non-limiting example, Group 5 animals are treated with a protein produced from the amino acid sequence listed in SEQ ID NO: 123. In one nonlimiting example, Group 5 animals are treated with a protein comprising the amino acid sequence listed in SEQ ID NO: 287, wherein neither G1 nor G2 in SEQ ID NO: 287 are myristoyl ated. In one non-limiting example, Group 5 animals are treated with a protein comprising the amino acid sequence listed in SEQ ID NO: 287 wherein a TEV recognition motif has not been cleaved. In one non-limiting example, Group 5 animals are treated with a protein comprising the amino acid sequence listed in SEQ ID NO: 287 wherein a TEV recognition motif has been cleaved and the protein according to SEQ ID NO: 287 has been purified for administration to the subject. In certain instances, Group 5 is treated with an engineered ARF polypeptide or an ARF fusion protein described herein sharing a certain percentage of sequence identity (e.g., > 90%) with an ARF protein family member such as ARF3, ARF4, ARF5, or ARF6. In certain instances, Group 5 is treated with an engineered ARF polypeptide or an ARF fusion protein described herein sharing a certain percentage of sequence identity (e.g., > 78%) with an ARF protein family member such as ARF5. In certain instances, Group 5 is treated with an engineered ARF polypeptide or an ARF fusion protein designed starting with the amino acid sequence of human ARF3, which is > 96% identical to human ARF1. In certain instances, engineered ARF3 polypeptide or an ARF3 fusion protein is designed for use in this example. In certain instances, Group 5 is treated with an engineered ARF polypeptide or an ARF fusion protein designed starting with the amino acid sequence of human ARF5, which is > 78% identical to human ARF1. In certain instances, engineered ARF5 polypeptide or an ARF5 fusion protein is designed for use in this example. In certain instances, engineered ARF3 polypeptide or ARF3 fusion protein comprises a ARF protein sequence at least about 96% identical to SEQ ID NO: 1. In certain instances, engineered ARF3 polypeptide or ARF3 fusion protein comprises a sequence identical to wild-type ARF3 sequence listed in SEQ ID NO: 87. In certain instances, engineered ARF3 polypeptide or ARF3 fusion protein comprises a sequence at least about 95% identical to wild-type ARF3 sequence listed in SEQ ID NO: 87. In certain instances, engineered ARF5 polypeptide or ARF5 fusion protein comprises a ARF protein sequence at least about 78% identical to SEQ ID NO: 1. In certaininstances, engineered ARF5 polypeptide or ARF5 fusion protein comprises a sequence identical to wild-type ARF5 sequence listed in SEQ ID NO: 89. In certain instances, engineered ARF5 polypeptide or ARF5 fusion protein comprises a sequence at least about 78% identical to wild-type ARF5 sequence listed in SEQ ID NO: 89. In certain instances, engineered ARF4 polypeptide or ARF4 fusion protein sequences are designed for use in this example. In certain instances, engineered ARF6 polypeptide or ARF6 fusion protein sequences are designed for use in this example. In certain instances, engineered ARF family member proteins or ARF family member fusion proteins are designed such that the homologous ARF domain is not myristoylated at a glycine reside. Such designed include, but at not limited to, an insertion, mutation, or amino acid addition near the N-terminus of the engineered ARF protein or ARF fusion protein such that a glycine residue is not present following an initiation methio...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. An engineered polypeptide comprising: a homologous ADP-ribosylation factor 1 (ARF1) sequence, and a covalently-coupled protein trafficking domain sequence.

2. The engineered polypeptide of claim 1, wherein the homologous ARF1 sequence comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 1.3 The engineered polypeptide of claim 1, wherein the homologous ARF1 sequence comprises an amino acid sequence at least about 93% identical to SEQ ID NO: 23.4 The engineered polypeptide of claim 1, wherein the homologous ARF1 sequence comprises an amino acid sequence at least about 95% identical to SEQ ID NO: 1.5 The engineered polypeptide of claim 1, wherein the homologous ARF1 sequence comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 1-23.6 The engineered polypeptide of claim 5, wherein the homologous ARF1 sequence comprises one, two, three, four, five, six, seven, eight, or nine amino acid substitutions relative to an amino acid sequence selected from SEQ ID NOs: 1-23.7 The engineered polypeptide of claim 5, wherein the homologous ARF1 sequence comprises one or more deletions of one, two, or three amino acids relative to an amino acid sequence selected from SEQ ID NOs: 1-23.8 The engineered polypeptide of claim 5, wherein the homologous ARF1 sequence comprises one or more insertions of one, two, three, four, five, six, seven, eight, or nine amino acids relative to an amino acid sequence selected from SEQ ID NOs: 1-23.9 The engineered polypeptide of claim 1, wherein the homologous ARF1 sequence comprises an amino acid sequence selected from SEQ ID NOs: 1-23, 123-174, 286-289, or 297-311.10 The engineered polypeptide of claim 1, wherein the homologous ARF1 sequence comprises the amino acid sequence selected from any one of SEQ ID NOs: 123, 286-287, 289, or 294-296.11 The engineered polypeptide of claim 1, wherein the covalently-coupled protein trafficking domain sequence is located N-terminal to the homologous ARF1 sequence.12 The engineered polypeptide of claim 1, wherein the covalently-coupled protein trafficking domain sequence is located C-terminal to the homologous ARF1 sequence.13 The engineered polypeptide of claim 11, wherein the covalently-coupled protein trafficking domain sequence comprises a signal peptide sequence.

14. The engineered polypeptide of claim 1, wherein the covalently-coupled protein trafficking domain sequence comprises a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is inserted within the engineered polypeptide.

15. The engineered polypeptide of claim 14, wherein the peptide tag prevents myristoylation of a glycine residue near the N-terminal portion of the homologous ARF1 sequence.

16. The engineered polypeptide of claim 1, wherein the homologous ARF1 sequence does not comprise a myristoyl group attached to a glycine reside.

17. The engineered polypeptide claim 1, further comprising a peptide tag comprising a peptide length of 4 to 22 amino acids, wherein the peptide tag is inserted within the engineered polypeptide.

18. The engineered polypeptide of claim 14, further comprising a cleavage site.

19. The engineered polypeptide of claim 18, wherein the cleavage site is inserted between the homologous ARF1 sequence and the covalently-coupled protein trafficking domain sequence.

20. A pharmaceutical composition comprising the engineered polypeptide of claim 1, and a pharmaceutically acceptable excipient, carrier, or diluent.

21. The pharmaceutical composition of claim 20, wherein the composition is formulated for systemic administration in a subject in need thereof.

22. The pharmaceutical composition of claim 21, wherein the composition is formulated for i.v. or s c administration.23 A method of treating a subject having obesity, the method comprising administering an engineered ADP-ribosylation factor 1 (ARF1) protein to the subject, wherein the ARF1 protein comprises an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,identical to SEQ ID NO: 1.24 The method of claim 23, wherein the engineered ARF1 protein is administered systemically to the subject.25 The method of claim 24, wherein the engineered ARF1 protein is administered by parenteral administration.26 The method of claim 23, wherein administration comprises ongoing administration of the engineered polypeptide according to an administration schedule for a period of at least about 1 week 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks 12 weeks, 4 months, 5, months, 6 months, or more than 6 months.27 The method of claim 26, wherein the subject achieves a weight loss of at least about 2%, 3%,35 , or 40% of body weight at a time point following the administering.

28. A method of treating a subject having obesity, the method comprising administering the engineered polypeptide of claim 16 to the subject.

29. The method of claim 28, wherein administration comprises ongoing administration of the engineered polypeptide according to an administration schedule for a period of at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5, months, 6 months, or more than 6 months.

30. The method of claim 29, wherein the subject achieves a weight loss of at least about 2%, 3%, 4 , 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30 , 35%, or 40% of body weight at a time point following the administering.31 A method of promoting weight loss in a subject in need thereof, the method comprising administering the engineered polypeptide of claim 1 to the subject.32 The method of claim 31, wherein administration comprises ongoing administration of the engineered polypeptide according to an administration schedule for a period of at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5, months, 6 months, or more than 6 months.33 The method of claim 32, wherein the subject achieves a weight loss of at least about 2%, 3%, 4 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 35%, or 40% of body weight at a time point following the administering.34 A method of promoting weight loss in a subject in need thereof, the method comprising administering the engineered polypeptide of claim 16 to the subject.35 The method of claim 34, wherein administration comprises ongoing administration of the engineered polypeptide according to an administration schedule for a period of at least about 1 week 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks 12 weeks, 4 months, 5, months, 6 months, or more than 6 months.36 The method of claim 35, wherein the subject achieves a weight loss of at least about 2%, 3%,35 , or 40% of body weight at a time point following the administering.37 A method of maintaining weight loss in a subject in need thereof, the method comprising administering the engineered polypeptide of claim 1 or claim 16 to the subject following the cessation of incretin therapy, wherein the subject maintains an extent of previous incretin-induced weight loss.38 The method of any one claims 23-37, wherein the subject is treated for at least about 1 day, 2 days 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 17 days, 21 days, 24 days, 28 days,30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months.

39. The method of any one of claims 23-38, wherein the subject is treated for at least 3 weeks.

40. The method of any one of claims 23-39, wherein the subject achieves a percentage of weight loss of at least 2.5%, 5%, 7.5%, 10%, 12.5%, or 15%.

41. The method of any one of claims 23-40, wherein the subject maintains a percentage of lean muscle mass of at least 85%, 90%, 95%, 98%, 99%, or 100% of following a period of treatment.

42. The method of any one of claims 23-41, wherein the subject is overweight or clinically obese at the start of the administering.

43. The method of claim 42, wherein the subject has been diagnosed with an obesity-related disorder selected from the group consisting of: pediatric obesity, type II diabetes, obesity, an obesity syndrome, clinical obesity, lipedema, metabolic syndrome / pre-diabetes, cardiovascular disease, nonalcoholic steatohepatitis (NASH) or nonalcoholic fatty liver disease (NAFLD), type I diabetes, Cushing’s disease, tumor-induced Cushing’s disease, Prader-Willi syndrome, Bardet- Biedl syndrome, thyroid disorder, thyroid removal, Adrenoleukodystrophy, Niemann-Pick disease, Tangier disease, Polycystic ovary syndrome, Congenital leptin deficiency, Cohen syndrome, Alstrbm syndrome, and Froehlich syndrome.

44. The method of any one of claims 23-43, wherein a homologous ARF1 sequence within the engineered polypeptide comprises an amino acid sequence at least about 78%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 1-23.

45. The method of claim 44, wherein the homologous ARF1 sequence comprises an amino acid sequence selected from SEQ ID NOs: 1-23, 123-174, 286-289, or 297-311.

46. The method of claim 44, wherein the homologous ARF1 sequence comprises the amino acid sequence selected from SEQ ID NO: 123, 286, 287, or 289.

47. The method of any one of claims 23-46, wherein a homologous ARF1 sequence within the engineered polypeptide comprises an amino acid sequence at least about 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 61-64, 176, 184-186, 194, 217-218, or 315-317.

48. The method of claim 47, wherein the homologous ARF1 sequence within the engineered polypeptide comprises an amino acid sequence identical to a sequence selected from SEQ ID NOs: 61-64, 176, 184-186, 194, 217-218, or 315-317.

49. The method of any one of claims 23-46, wherein a homologous ARF1 sequence within the engineered polypeptide comprises an amino acid sequence at least about 90%, 92%, 94%, 95%,96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 59-60, 181-182, 192, 196, 215-216, or 245-284.

50. The method of any claim 49, wherein the homologous ARF1 sequence of the engineered polypeptide comprises an amino acid sequence identical to a sequence selected from SEQ ID NOs: 59-60, 181-182, 192, 196, 215-216, or 245-284.

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