EP4263588A1 - Dsg2-zusammensetzungen und verfahren zur behandlung von covid-19 - Google Patents

Dsg2-zusammensetzungen und verfahren zur behandlung von covid-19

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Publication number
EP4263588A1
EP4263588A1 EP21841114.8A EP21841114A EP4263588A1 EP 4263588 A1 EP4263588 A1 EP 4263588A1 EP 21841114 A EP21841114 A EP 21841114A EP 4263588 A1 EP4263588 A1 EP 4263588A1
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EP
European Patent Office
Prior art keywords
dsg2
isolated polypeptide
covid
protein
domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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EP21841114.8A
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English (en)
French (fr)
Inventor
Shi Yin Foo
Ryan Edward TYLER
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Arvada Therapeutics Inc
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Arvada Therapeutics Inc
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Publication date
Application filed by Arvada Therapeutics Inc filed Critical Arvada Therapeutics Inc
Publication of EP4263588A1 publication Critical patent/EP4263588A1/de
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the disclosure generally relates to DSG2 based methods for treating COVID-19 by administering the compositions disclosed herein.
  • the methods also include the treatment of therapeutic indications associated with the long-term effects of COVID-19 (such as, but not limited to post COVID-19 syndrome or post-COVID-19 cardiac syndrome), including, inflammation in the myocardium and/or reduced ejection fraction/heart failure/cardiomyopathy, as well as the treatment of diseases associated DSG2 autoantibodies e.g., arrhythmogenic right ventricular cardiomyopathy (ARVC), sarcoidosis.
  • ARVC arrhythmogenic right ventricular cardiomyopathy
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • COVID-19 coronavirus disease
  • post-COVID-19 syndrome This condition has been called post-COVID-19 syndrome or "long COVID-19.”
  • patients may also develop a reduced ejection fraction or cardiomyopathy, even after the acute infection of COVID-19 has resolved.
  • Post-COVID-19 cardiac signs and symptoms may coexist with effects on other organ systems, but may also present alone.
  • Patients with post-COVID cardiomyopathy range from those who are asymptomatic to those with fulminant heart failure, arrhythmia and/or sudden cardiac death.
  • COVID-19 virus SARS-CoV-2 affects multiple organ systems, especially lungs and heart. Elevation of cardiac biomarkers, particularly high-sensitivity troponin and/or creatine kinase MB have been commonly observed in patients in COVID-19 infection.
  • the plausible mechanisms of myocardial injury associated with COVID-19 include but are not limited to, 1) hyperinflammation and cytokine storm mediated through pathologic T-cells and monocytes leading to myocarditis, 2) respiratory failure and hypoxemia resulting in damage to cardiac myocytes, 3) down regulation of ACE2 expression and subsequent protective signaling pathways in cardiac myocytes, 4) hypercoagulability and development of coronary microvascular thrombosis, 5) diffuse endothelial injury, and/or, 6) inflammation and/or stress causing coronary plaque rupture or supply-demand mismatch leading to myocardial ischemia/infarction.
  • the post-COVID- 19 syndrome has also been associated with multiple organ damage, including cardiovascular damage. Imaging tests taken months after recovery from COVID-19 have shown lasting damage to the heart muscle, even in people who experienced only mild COVID-19 symptoms. Post-COVID-19 syndrome also appears to be associated with myocarditi s and/or cardiomyopathy with increased risk of arrhythm ia.
  • compositions comprising isolated polypeptides.
  • the polypeptides of the disclosure may include a whole or a portion of the DSG2 protein.
  • the isolated polypeptide is a Desmoglein 2 (DSG2) fusion polypeptide.
  • the DSG2 fusion polypeptide may include (a) a whole or a portion of a DSG2 protein (SEQ ID NO: 1); and/or (b) a whole or a portion of an immunoglobulin protein.
  • the DSG2 polypeptide may include a portion of DSG2 protein.
  • the portion of The DSG2 protein may include the extracellular region of DSG2 protein. In some aspects, the entire extracellular region of DSG2 may be included in the fusion polypeptide.
  • the entire extracellular region of DSG2 includes the amino acid sequence of SEQ ID NO: 3.
  • Embodiments of the disclosure may also include a portion of the extracellular region of DSG2.
  • a portion of the extracellular region may be extracellular cadherin domain 1 (EC1), extracellular cadherin domain 2 (EC2), extracellular cadherin domain 3 (EC3), extracellular cadherin domain 4 (EC4), and/or extracellular anchor domain (EA).
  • the DSG2 fusion polypeptides include 2 domains of the extracellular region.
  • the two domains may be EC4EA, EC1EC2, EC2EC3, EC3EC4, EC1EA, EC1EC3, EC2EC4, and/or EC3EA.
  • the DSG2 fusion polypeptides include three domains of the extracellular region.
  • the three domains may be EC1EC3EA, EC1EC4EA, EC1EC3EA, EC3EC4EA, EC1EC2EC3, EC2EC3EC4, and/or EC2EC4EA.
  • the DSG2 fusion polypeptides may include four domains of the extracellular region.
  • the three domains may be EC1EC2EC4EA, EC2EC3EC4EA, EC1EC2EC3EC4EA, EC1EC2EC3EC4, and/or EC1EC2EC3EA.
  • DSG2 fusion polypeptides may include a portion of an immunoglobulin.
  • the portion may be Fc region, an Fab region, a heavy chain variable (VH) domain, a heavy chain constant domain, a light chain variable (VL) domain, and/or a light chain constant domain.
  • the portion of the immunoglobulin may be an Fc region.
  • the immunoglobulin may be an IgG, an IgM, an IgA, an IgD and/or an IgE.
  • the immunoglobulin may be IgG.
  • the compositions may include an IgG such as IgG1, IgG2, IgG3, and/or IgG4.
  • Non-limiting examples of portions of immunoglobulin useful in the present disclosure include IgG1 Fc region (SEQ ID NO: 5), IgG2 Fc region (SEQ ID NO: 7), IgG3 Fc region (SEQ ID NO: 9), IgG4 Fc region (SEQ ID NO: 1 1), IgG1 heavy chain constant domain (SEQ ID NO: 4), IgG2 heavy chain constant domain (SEQ ID NO: 6), IgG3 heavy chain constant domain (SEQ ID NO: 8), IgG4 or heavy chain constant domain (SEQ ID NO: 10).
  • Polypeptides of the disclosure may further include a linker and/or a signal sequence.
  • the linker may be from about 5 amino acids to about 50 amino acids in length.
  • the linker may be GGGGS (SEQ ID NO: 12).
  • the linker may be EAAAK (SEQ ID NO: 13).
  • the present disclosure al so provides method s of treatment using the compositions described herein.
  • the disclosure provides methods of treating post- COVID-19 syndrome.
  • Such methods may include, i) contacting the subject with the isolated polypeptide of the disclosure and (ii) measuring one or more symptoms associated with post- COVID-19 syndrome selected from the group consisting of arrhythmia, myocarditis, heart failure, shortness of breath, fatigue, edema, orthopnea, limi tations to exertion, impai red cognitive abilities, palpitations, dizziness, syncope, and/or lightheadedness.
  • Treatment with the polypeptides of the disclosure may be effective in ameliorating one or more symptoms associated with post-COVID-19 cardiac syndrome.
  • the subjects with post- COVID-19 have been previously diagnosed with COVID-19 using methods known in the art.
  • the serum of the subject has detectable levels of anti-SARS-Co V-2 antibodies.
  • the serum of the subject has no detectable levels of anti- SARS- CoV- 2 antibodies.
  • methods of treating a subject with COVID-19 by administering the compositions described herein.
  • the serum of the subject with COV1D-19 or post-COVID-19 may have anti- DSG2 antibodies.
  • the present discl osure al so provides a m ethod of treating a condition associated with serum DSG2 autoantibodies.
  • Such methods may include administering the compositions described herein or cells expressing the compositions described herein to a subject.
  • the condition may be a cardiomyopathy.
  • the condition may be an autoimmune disorder.
  • the present disclosure provides methods of treating cardiomyopathy in a subject. Such methods may include contacting the subject with the isolated polypeptides or the cells of the disclosure followed by measuring one or more symptoms associated with cardiomyopathy such as arrhythmia, palpitations, myocarditis, heart failure, poor cardiac output, and/or reduced ejection fraction.
  • symptoms associated with cardiomyopathy such as arrhythmia, palpitations, myocarditis, heart failure, poor cardiac output, and/or reduced ejection fraction.
  • the cardiomyopathy may be arrhythmogenic right ventricular cardiomyopathy (AR VC) .
  • the cardiomyopathy may also be caused by a virus (e.g., SARS- CoV2, adenovirus, hepatitis virus, hepatitis C virus, parvovirus, herpes simplex virus, echovirus, Epstein-Barr virus, rubella, cytomegalovirus, or HIV), a bacterium (Staphylococcus, Streptococcus, or Borrelia) , a parasite (Trypanosoma or Toxoplasma) or a fungus (Candida, Aspergillus, or Histoplasma).
  • the subject may have detectable levels of anti-DSG2 antibodies in their serum. DESCRIPTION OF THE FIGURES
  • HC healthy controls: PC, post-COVID-19: ARVC, arrhythmogenic right ventricular cardiomyopathy; S/NC, signal/negative control; individual diamonds represent a single serum sample each; box and whisker limits represent 25th-75th and 10th-90th percentiles respectively.
  • P-values are based on non-parametric rank-based Wilcoxon-Mann-Whitney 2 -sided test.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • ICTV International Committee of Taxonomy of Viruses
  • the SARS-CoV-2 particles use a special surface glycoprotein (spike protein) to bind to angiotensin converting enzyme 2 (ACE2) which is most abundant in the type II alveolar cells of the lungs, and thus enters the host cell.
  • ACE2 angiotensin converting enzyme 2
  • the genome of coronavirus is then replicated in the host cell.
  • the density of ACE2 receptors in each tissue correlates with the severity of the COVID-19 disease in that tissue.
  • ACE2 receptors are also expressed on the outer surface of cells in the arteries, heart, kidney, and intestines. As a result, COVID-19 may cause multi-organ failure in extremely severe cases.
  • COVID-19 The symptoms of COVID-19 range from mild (e.g., fever, cough, shortness of breath), to severe, such as pneumonia and acute respiratory distress syndrome (ARDS), sepsis and septic shock, multi organ failure, including acute kidney injury , and cardiac injury .
  • respiratory illness is the dominant clinal manifestation of COVID-19 infection, multi organ failure may also occur.
  • myocardial injury was observed to be the cause of death in 40% of the cases (Ruan Q.et al. Intensive Care Med. 2020;46(5):846-848).
  • Various cardiac complications have been associated with active COVID-19 infection, including arrhythmia, myocarditis, and acute myocardial injury .
  • Arrhythmias associated with COVID-19 can also be attributed to the treatment with azithromycin, hydroxychloroquine, and some antivirals that can cause QT prolongation.
  • Acute myocardial injury in COVID-19 can range from asymptomatic elevation of cardiac troponins to fulminant myocarditi s and circulatory shock.
  • Myocardial injury can manifest either alone or can occur in combination with arrythmia based on the clinical course of the infection.
  • the proinflammatory milieu and increased sympathetic stimulation in COVID-19 may further increase the risk for cardiovascular complications, such as cardiac arrythmias, worsening of existing heart failure (HF), or development of new-onset HF.
  • cardiovascular complications such as cardiac arrythmias, worsening of existing heart failure (HF), or development of new-onset HF.
  • hypoxia and electrolyte disturbances can further potentiate the risk for arrythmias.
  • post-COVID-19 syndrome may also be associated with cardiac symptoms and is herein referred to as Post-COVID-19 cardiac syndrome.
  • Many patients who have recovered from COVID-19 show persistent inflammation in the myocardium as measured by MRI.
  • up to 60% of both symptomatic and asymptomatic COVID-19 patients had MRI evidence of ongoing myocardial inflammation, an average of 71 days after recovery from the acute phase of COVID-19 (Puntmann et al. JAMA Cardiol.
  • post-COVID-19 syndrome patients with post-COVID-19 cardiac manifestations may herein be referred to as post-COVID-19 cardiac syndrome. Patients with post-COVID-19 syndrome also experience chronic fatigue syndrome which may be driven by undiagnosed cardiac output reductions.
  • Symptoms may include shortness of breath, fatigue, edema, orthopnea, limitations to exertion and impaired cognitive abilities due to poor cardiac output (“brain fog”), arrhythmia, palpitations, dizziness, syncope, lightheadedness, heart failure, hospitalizations due to heart failure and/or arrhythmia. In some cases, death may occur as a resul t of heart fai lure and/or arrhythmia. In some embodiments, the post-COVID- 19 syndrome may not be associated with cardiac manifestations.
  • arrhythmogenic right ventricular cardiomyopathy Similar to ARVC, patients with COVID-19 and/or post-COVID-19 syndrome also display worsened cardiac function upon physical exertion.
  • ARVC arrhythmogenic right ventricular cardiomyopathy
  • DSG2 Desmoglein 2
  • DSG2 antibodies can also be found in some cases of sarcoidosis, a systemic inflammatory disease which results in granulomas in organs, such as, but not limited to the heart. Anti-DSG2 antibodies are also found in sarcoid patients with cardiac involvement (Suna et al. 2020, Eur. Heart Journal, Volume 41, Issue Supplement_2, November 2020, ehaa946.2127; the contents of which are herein incorporated by reference in its entirety).
  • dilated cardiomyopathy Patients with a diagnosis of dilated cardiomyopathy may have mutations in the same desmosome proteins that are associated with ARVC. These observations suggest that some dilated cardiomyopathy patients may actually have ARVC-like disease, mediated by DSG2 antibodies, but have been diagnosed as dilated cardiomyopathy because they do not fit the typical age and/or presentation associated with ARVC.
  • Anti-DSG2 autoantibodies are considered to arise when there is a combination of cardiac cellular damage and an activated immune system - typically in the setting of an infection known to affect the myocardium directly. From a pathogenesis standpoint, viral infections such as COVID-19, generally trigger a vigorous immune response that is crucial for viral clearance, with a cascade of events involving both the innate and adaptive immune arms.
  • Direct and indirect myocardial damage is also caused by COVID-19 infection, allowing for cardiac proteins to be exposed to the activated immune system.
  • Immunological alterations are also observed in patients with COVID-19 condition. These range from a maladaptive immune response and abnormal cytokine/chemokine production, to hyperactivation of T cells and increased number of activated monocytes, macrophages and neutrophils (Chang, S.E., et al. Nature Communications 2021; 12:5417; Liu, Y., et al. Curr. Opin. Rheumatol. 2021; 33: 155-162; Lee, C. C. E., et al. Diseases 2021; 9:47; the contents of each of which are herein incorporated by reference in their entirety).
  • COVID-19 has infected at least 200 million people worldwide with approximately 4.5 million deaths attributable to COVID 19 disease to date. There is growing recognition that COVID-19 infections can cause a variety of long-term sequelae, of which, cardiac involvement may be the most under-recognized as its symptoms may be attributed to other organ systems.
  • COVID 19 infections have been associated with MRI evidence of myocardial in volvement and arrhythmias well into recovery, independent of preexisting conditions, severity and overall course of the acute illness, and the time from the original diagnosis. The percentage of patients who will develop a depressed ejection fraction subsequently is currently not well-understood, although frank cardiomyopathy has been described in post- COVID-19 patients.
  • Viral infections including COVID- 19, have been hypothesized to contribute to autoimmune responses, e.g., by exposing previously bidden cryptic epitopes on damaged cells to an activated immune system (Ehrenfeld M., et al. Autoimmunity Reviews 2020 102597; the contents of which are herein incorporated by reference in its entirety).
  • the high incidence of cardiac involvement seen in COVID-19 infections indicates that anti-DSG2 autoantibodies may be generated as a result.
  • the present disclosure provides compositions and methods related to DSG2 fusion polypeptides for targeting anti-DSG2 antibodies.
  • the DSG2 fusion polypeptides of the disclosure may therefore be a viable therapeutic strategy in the treatment of COVID-19, post- COVID-19 cardiac syndrome, and/or ARVC.
  • DSG2 fusion polypeptides may also be used in the treatment of other diseases associated with cardiac cellular damage, such as, but not limited to, arrhythmogenic cardiomyopathy (AC), sarcoidosis, dilated cardiomyopathy with anti-DSG2 autoantibodies and viral infections, including but not limited to those caused by coxsackie virus, adenovirus, echoviruses, parvovirus, rubella and/or cytomegalovirus.
  • AC arrhythmogenic cardiomyopathy
  • sarcoidosis sarcoidosis
  • dilated cardiomyopathy with anti-DSG2 autoantibodies and viral infections including but not limited to those caused by coxsackie virus, adeno
  • compositions that include DSG2 fusion polypeptides.
  • Compositions described herein may be capable of binding to or interacting with anti-DSG2 antibodies.
  • the compositions of the disclosure may modulate the activity of anti-DSG2 antibodies.
  • the compositions of the disclosure may inhibit the activity of the anti-DSG2 antibodies.
  • the present disclosure includes a DSG2 protein.
  • the DSG2 protein may be the whole DSG2 protein or a portion of the DSG2 protein.
  • the DSG2 protein may be fused to any other protein or fragment of a protein.
  • DSG2 fusion polypeptides of the present disclosure may include the whole or a portion of a DSG2 protein and a whole or a portion of an immunoglobulin protein .
  • the DSG2 fusion polypeptides may further include a linker and/or a signal peptide.
  • the whole or a portion of DSG2 protein may be fused to a protein that is not an immunoglobulin.
  • the DSG2 protein may be fused to a protein or a fragment of a protein that is capable of improving the expression of DSG2 protein in vitro or in vivo.
  • DSG2 mutations within intercalated discs in heart cells have been implicated in cardiac diseases including arrhythmia, dilated cardiomyopathy, and particularly ARVC (Arrhythmogenic right ventricular cardiomyopathy).
  • ARVC Arrhythmogenic right ventricular cardiomyopathy.
  • Chatterjee et al. identified autoantibodies to the cardiac DSG2 protein as a comm on feature in the sera of patients with ARVC ( Chatterjee D, et al., Eur Heart J. 2018;39(44):3932-3944; the contents of which are herein incorporated by reference in its entirety). These autoantibodies were specific to ARVC, as they were essentially absent in two independent sets of control sera, as well as sera from subjects with other forms of heritable cardiomyopathy.
  • DSG2 autoantibodies identified by Chatterjee et al. suggests that targeting DSG2 antibodies may represent a therapeutic strategy in the treatment of cardiomyopathies associated with diseases such as, but not limited to ARVC and/or COVID-19.
  • the present disclosure provides DSG2 fusion polypeptides as a therapeutic strategy for targeting DSG2 autoantibodies.
  • DSG2 fusion polypeptides of the present disclosure may bind to DSG2 autoantibodies.
  • binding of the DSG2 fusion polypeptides of the disclosure to the DSG2 autoantibodies precludes the binding of the autoantibodies to the endogenous DSG2 in a subject.
  • the DSG2 fusion polypeptides function as a decoy protein or a ligand trap.
  • the DSG2 protein may include epitopes, which are exposed or released into the intercellular space and/or circulation and as a result of DSG2 mutations. Unmasking of these epitopes may also occur from any cardiac damage (such as, but not limited to, infective myocarditis, and/or cardiac trauma).
  • the compositions of the disclosure may not include any mutations.
  • Such released DSG2 proteins may link with an antigen-presenting cell to stimulate a T-cell response, generating the observed autoantibodies. Unmasking of cryptic epitopes by gene mutations could contribute to other forms of autoimmunity.
  • DSG2 fu sion polypeptides of the disclosure may include epitopes containing one or more mutations in DSG2.
  • DSG2 fusion polypeptide may be a soluble and/or recombinant polypeptides.
  • the arrangement of components in the DSG2 fusion polypeptide may be optimized to achieve the suitable protein expression and/or the intended therapeutic effect.
  • the DSG2 fusion polypeptide may include formats described herein.
  • the formats provided herein include components from N terminus to C terminus delineated by a between the components.
  • Non-limiting examples of the formats of the DSG2 fusion polypeptides include, (i) the whole or a portion of DSG2 protein; Fc region (ii) Fc region; the whole or a portion of the DSG2 protein (iii) signal sequence; the whole or a portion of the DSG2 protein; Fc region (iv) the signal sequence; Fc region; the whole or a portion of the DSG2 protein (v) the whole or a portion of DSG2 protein; linker; Fc region (vi) Fc region; linker; the whole or a portion of the DSG2 protein (vii) signal sequence; the whole or a portion of the DSG2 protein; linker; Fc region (viii) signal sequence; Fc region; linker; whole or a portion of the DSG2 protein.
  • the DSG2 fusion polypeptides of the present disclosure may include the entire DSG2 protein.
  • the desmosomal cadherin desmoglein-2 (DSG2) is a transmembrane cell adhesion protein that is widely expressed in epithelial and non-epithelial tissues, such as the heart, intestine, and epidermis. DSG2 has been shown to regulate numerous cellular processes, including proliferation and apoptosis. In epithelial and myocyte cells, DSG2 is a component of the cell-cell adhesion structure and its cytoplasmic tail interacts with a series of proteins in direct contact with cell adhesion and intercellular junction/cell type regulators.
  • the DSG2 protein is a human DSG2 protein (UniProt ID: Q14126; ENSEMBL Protein ID: ENSP00000261590.8) which consists of 1,118 amino acids, and includes the amino acid sequence of SEQ ID NO: 1.
  • the DSG2 protein may be encoded by nucleic acid sequence of SEQ ID NO: 2 (NCBI Reference Sequence: NM_001943.5; ENSMBL ID: ENST00000261590.13).
  • the DSG2 fusion polypeptide of the present disclosure may be a fully processed DSG2 protein comprising amino acids 50-1118 of SEQ ID NO: 1 .
  • the DSG2 protein may also include one or more mutations with respect to the sequence of SEQ ID NO: 1.
  • DSG2 protein mutation may be a mutation associated with a disease state.
  • the disease state may be Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy.
  • DSG2 fusion polypeptides of the disclosure may include epitopes containing one or more mutations in DSG2.
  • DSG2 fusion polypeptides may include one or more mutations in the region of amino acids 485-531 and/or amino acids 586-610 of SEQ ID NO: 1.
  • DSG2 belongs to the cadherin superfamily of cell adhesion proteins, which commonly feature the three distinct regions: an extracellular region, a transmembrane domain and an intracellular signaling region.
  • the extracellular region of DSG2 may include the amino acid sequence of SEQ ID NO: 3, which is amino acids 50- 609 of SEQ ID NO: 1.
  • the extracellular region of cadherin family of proteins contain a varying number of repeats of calcium-binding motifs, known as cadherin motifs or EC domains.
  • DSG2 contains four EC domains herein referred to as EC1, EC2, EC3 and EC4.
  • DSG2 also includes an extracellular anchor (EA) domain that is proximal to the membrane.
  • EA extracellular anchor
  • the DSG2 fusion polypeptides of the disclosure may include the entire extracellular region of DSG2.
  • the DSG2 fusion polypeptide may include at least one domain, such as, but not limited to, EC1, EC2, EC3, EC4, and/or EA.
  • the EC1 domain may be amino acids 50-155 of SEQ ID NO: 1.
  • the EC2 domain may be amino acids 151-268 of SEQ ID NO: 1.
  • the EC3 domain may be amino acids 264-384 of SEQ ID NO: 1.
  • the EC4 domain may be amino acids 382-495 of SEQ ID NO: 1.
  • the EA domain may be amino acids 491-608 of SEQ ID NO: 1.
  • Table 1 provides the amino acid sequence of the DSG2 protein as well as the amino acid sequence of the extracellular region of DSG2.
  • the DSG2 proteins of the present disclosure may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to any of the sequences in Table 1 or fragments of the sequences in Table 1.
  • the DSG2 fusion polypeptides of the present disclosure may include one or more domains of the extracellular region of DSG2.
  • the domains from the extracellular region of DSG2 may include repeats of one or more of the EC domains or EA domains, in tandem or in a mixed order.
  • DSG2 fusion polypeptide may include 2, 3, or more repeats of EC1, EC2, EC3, EC4 or EA domains.
  • the domains may be operably linked via a linker described herein.
  • the DSG2 fusion polypeptide may include two domains of the extracellular region of DSG2.
  • domains of extracellular region of DSG2 present in the fusion polypeptides of the present disclosure include, EC1EC2, EC1EC3, EC1EC4, EC1EA, EC2EC1, EC2EC3, EC2EC4, EC2EA, EC3EC1, EC3EC2, EC3EC4, EC3EA, EC4EC1, EC4EC2, EC4EC3, EC4EA, EAEC1, EAEC2, EAEC3, and/or EAEC4.
  • the DSG2 fusion polypeptide may include three domains of the extracellular region of DSG2.
  • domains of extracellular region of DSG2 present in the fusion polypeptides of the present disclosure include, EC1EC2EC3, EC1EC2EC4, EC1EC2EA, EC1EC3EC2, EC1EC3EC4, EC1EC3EA, EC1EC4EC2, EC1EC4EC3, EC1EC4EA, EC1EAEC2, EC1EAEC3, EC1EAEC4, EC2EC1EC3, EC2EC1EC4, EC2EC1EA, EC2EC3EC1, EC2EC3EC4, EC2EC3EA, EC2EC4EC1, EC2EC4EC3, EC2EC4EA, EC2EAEC1, EC2EAEC3, EC2EAEC4, EC3EC1EC2, EC3EC1,EC2, EC3EC4, EC1EC1EA, EC2
  • the DSG2 fusion polypeptide may include four domains of the extracellular region of DSG2.
  • Non limiting examples domains of extracellular region of DSG2 present in the fusion polypeptides of the present disclosure include, EC1EC2EC3EC4, EC1EC2EC3EA, EC1EC2EC4EC3, EC1EC2EC4EA, EC1EC2EAEC3, EC1EC2EAEC4, EC1EC3EC2EC4, EC1EC3EC2EA, EC1EC3EC4EC2, EC1EC3EC4EA, EC1EC3EAEC2, EC1EC3EAEC4, EC1EC4EC2EC3, EC1EC4EC2EA, EC1EC4EC3EC2, EC1EC4EC3EA, EC1EC4EAEC2, EC1EC4EAEC3, EC1EAEC2EC3, EC1EAEC2EC4, EC1EAEC3EC2, EC1EC4
  • the DSG2 fusion polypeptide may include five domains of the extracellular region of DSG2.
  • Non limiting examples domains of extracellular region of DSG2 present in the fusion polypeptides of the present disclosure include EC1EC2EC3EC4EA, EC1EC2EC3EAEC4, EC1EC2EC4EC3EA, EC1EC2EC4EAEC3, EC1EC2EAEC3EC4, EC1EC2EAEC4EC3, EC1EC3EC2EC4EA, EC1EC3EC2EAEC4, EC1EC3EC4EC2EA, EC1EC3EC4EAEC2, EC1EC3EAEC2EC4, EC 1 EC3EAEC4EC2,
  • EC2EC1EAEC3EC4 EC2EC1EAEC4EC3, EC2EC3EC1EC4EA, EC2EC3EC1EAEC4,
  • EC3EC4EAEC1EC2 EC3EC4EAEC2EC1, EC3EAEC1EC2EC4, EC3EAEC1EC4EC2,
  • EC4EC3EAEC1EC2 EC4EC3EAEC2ECL EC4EAEC1EC2EC3, EC4EAEC1EC3EC2,
  • EAEC3EC4EC1EC2 EAEC3EC4EC2EC1, EAEC4EC1EC2EC3, EAEC4EC1EC3EC2,
  • Non-limiting examples of a portion of the DSG2 protein as well as possible configurations present in the DSG2 fusion polypeptide are provided in Table 2. Any of the DSG2 domains described in Table 2 may be operably linked to another domain within the fusion polypeptide or to another DSG2 domain using any of the linker provided herein or any linker known in the art.
  • the compositions of the disclosure may include a portion or a fragment of any of the domains described in Table 2.
  • the domains or combination of domains of SEQ ID NO: 1 or SEQ ID NO: 3 included in the polypeptides of the disclosure may be extended by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 or
  • the domains or combination of domains of SEQ ID NO: 1 or SEQ ID NO: 3 included in the polypeptides of the disclosure may be truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
  • the extracellular region of DSG2 protein may extend from the amino acids spanning from 50-610 of SEQ ID NO:
  • DSG2 fusion polypeptides of the present disclosure may include a whole or a portion of an immunoglobulin protein.
  • the immunoglobulin protein may be an IgG, an IgM, an IgA, an IgD and an IgE.
  • the immunoglobulin protein may be an IgG.
  • Non-limiting examples of IgG may be IgG1, IgG2, IgG3 and/or IgG4.
  • DSG2 fusion polypeptides may include a region or a portion of an immunoglobulin.
  • Non- limiting examples of a region of an immunoglobulin such as, Fc region, an Fab region, a heavy chain variable (VH) domain, a heavy chain constant domain, a light chain variable (VL) domain, and/or a light chain constant domain.
  • DSG2 fusion polypeptides may include one or more Fc regions of an immunoglobulin.
  • the Fc region may include the first constant region immunoglobulin domain (e.g., CH1) or a portion thereof, and in some cases, part of the hinge.
  • the Fc region excludes the first constant region immunoglobulin domain.
  • an Fc may refer to the last two constant region immunoglobulin domains (e.g., CH2 and CH3) of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains.
  • Fc may include the J chain.
  • the Fc domain comprises immunoglobulin domains Cy2 and Cy3 (Cy2 and Cy3) and the lower hinge region between Cyl (Cyl) and Cy2 (Cy2).
  • an Fc refers to a truncated CH1 domain, and CH2 and CH3 of an immunoglobulin.
  • the human IgG heavy chain Fc region are typically usually defined to include residues E216 or C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat Antibody Numbering sequence.
  • the immunoglobulin protein may include additional cell targeting modules (and may herein be referred to as cell targeting antibody CTAB).
  • Non-limiting examples of the sequences of porti on s of immunoglobulin are provided in Table 3.
  • the DSG2 fusion polypeptides may include an immunoglobulin protein or a portion thereof having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to any of the sequences in Table 3 or fragments of the sequences in Table 3.
  • Signal sequences (sometimes referred to as signal peptides, targeting signals, target peptides, localization sequences, transit peptides, leader sequences or leader peptides) direct proteins (e.g., the polypeptides of the present disclosure) to their designated cellular and/or extracellular locations.
  • a signal sequence may be a short (about 5-50 amino acids long) peptide present at the N-terminus of the majority of newly synthesized proteins that are destined towards a particular location.
  • Signal sequences can be recognized by signal recognition particles (SRPs) and cleaved using type I and type II signal peptide peptidases.
  • SRPs signal recognition particles
  • Signal sequences derived from human proteins can be incorporated as a DSG2 fusion polypeptides of the present disclosure to direct the polypeptides of the disclosure to a particular cellular and/or extracellular location. These signal sequences are experimentally verified and can be cleaved (Zhang et al., Protein Sci. 2004, 13:2819-2824).
  • a signal sequence may be located at the N-terminus or C- terminus of the polypeptides of the present disclosure, and may be, although not necessarily, cleaved off the polypeptide to yield a “mature” polypeptide, as discussed herein.
  • a signal sequence may be a secreted signal sequence derived from a naturally secreted protein, and its variant thereof.
  • signal sequences directing the polypepti des of the disclosure to the surface membrane of a target cell may be used.
  • Expression of the polypeptides of the disclosure on the surface of the target cell may be usefill to limit the diffusion of the polypeptides of the disclosure to non-target in vivo envi ronments, thereby potentially improving the safety profile of the polypeptides of the disclosure.
  • the membrane presentation of the polypeptides of the disclosure may allow for physiological and qualitative signaling as well as stabilization and recycling of the polypeptide for a longer half-life.
  • a signal sequence may be a heterogeneous signal sequence from other organisms such as virus, yeast and bacteria, which can direct the polypeptides of the disclosure to a particular cellular site, such as a nucleus (e.g., EP 1209450).
  • Other examples may include Aspartic Protease (NSP24) signal sequences from Trichoderma that can increase secretion of fused protein such as enzymes (e.g., U. S. Pat. NO. 8,093,016 to Cervin and Kim), bacterial lipoprotein signal sequences (e.g., PCT publication NO. 1991/09952 to Lau and Rioux), E.coli enterotoxin II signal peptides (e.g., U.S. Pat. NO.
  • E.coli secretion signal sequence e.g., U.S. patent publication NO. 2016/090404 to Malley et al.
  • a lipase signal sequence from a methylotrophic yeast e.g., U.S. Pat. NO. 8,975,041
  • signal peptides for DNases derived from Coryneform bacteria e.g., U.S. Pat. NO. 4,965,197
  • the DSG2 fusi on polypeptides of the present di sclosure may include at least one linker.
  • the linker may be positioned between one or more regions of the polypeptides of the disclosure.
  • the linker may be positioned between the whole or a portion of a DSG2 protein and the whole or a portion of an immunoglobulin protein .
  • the linker may be positioned between one or m ore domain s of the DSG2 protein.
  • the linker may be a polypeptide. In some embodiments, the linker may comprise a combination of amino acid residues. In some embodiments, the linker may comprise about 1-50 amino acid residues. In some embodiments, the linker may comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acid residues.
  • Th e linkers of the present disclosure may be from about 1 to 100 amino acids in length, which links together any of the domains/regions of the effector module (also called peptide linker).
  • the linker may be 1-40 amino acids in length, or 2-30 amino acids in length, or 20-80 amino acids in length, or 50-100 amino acids in length.
  • Linker length may also be optimi zed depending on the type of configuration of the polypepti de and based on the crystal structure of the polypeptide. In some instances, a shorter linker length may be preferably selected.
  • the peptide linker may be made up of amino acids linked together by peptide bonds, preferably from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I), Serine (S), Cysteine (C), Threonine (T), Methionine (M), Proline (P), Phenylalanine (F), Tyrosine (Y), Tryptophan (W), Histidine (H), Lysine (K), Arginine (R), Aspartate (D), Glutamic acid (E), Asparagine (N), and Glutamine (Q).
  • amino acids of a peptide linker may be selected from Alanine (A), Glycine (G), Proline (P), Asparagine (R), Serine (S), Glutamine (Q) and Lysine (K).
  • the linker may be a flexible linker or a rigid linker.
  • Flexible linkers may be composed of small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The small size of these amino acids provides flexibility, and allows for mobility of the connecting functional domains.
  • the most commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues (“GS” linker).
  • GS linker Gly-Gly-Gly-Gly-Ser
  • An example of the most widely used flexible linker has the sequence of (Gly-Gly-Gly-Gly-Ser)n. By adjusting the copy number “n”, the length of this GS linker may be optimized to achieve appropriate separation of the functional domains, or to maintain necessary inter-domain interactions.
  • linkers may include additional amino acids such as Thr and Ala to maintain flexibility, as well as polar amino acids such as Lys and Glu to improve solubility.
  • the DSG2 fusion polypeptide may include a flexible linker, such as (Gly)8 (SEQ ID NO: 14), consisting of purely of glycine residues. The linker sequence avoided large hydrophobic residues to maintain good solubility in aqueous solutions.
  • the linker may be a rigid linker.
  • the rigid linker may have a Proline-rich sequence, (XP)n, with X designating any amino acid, preferably Ala, Lys, or Glu.
  • the linker may be GGGGS (SEQ ID NO: 12). In some embodiments, the linker may be GGGGGS (SEQ ID NO: 16) and EAAAK (SEQ ID NO: 13).
  • the polypeptides of the present disclosure are encoded by polynucleotides or variants thereof described herein.
  • Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a ⁇ -D-ribo configuration, ⁇ -LNA having an a-L-ribo configuration (a diastereomer of LN A), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino-a-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or hybrids or combinations thereof.
  • RNAs ribonucle
  • polypeptide sequences encoding peptides or polypeptides containing substitutions, insertions and/or additions, deletions, and covalent modifications with respect to reference sequences, in particular the polypeptide sequences are disclosed herein.
  • sequence tags or amino acids such as one or more lysines
  • Sequence tags can be used for peptide purification or localization.
  • Lysines can be used to increase peptide solubility or to allow for biotinylation.
  • amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences.
  • Certain amino acids e.g., C- terminal or N-terminal residues
  • any of the features have been identified or defined as a desired component of a polypeptide to be encoded by a polynucleotide described herein, any of several manipulations and/or modifications of these features may be performed by moving, swapping, inverting, deleting, randomizing or duplicating. Furthermore, it is understood that manipulation of features may result in the same outcome as a modification to the molecules described herein. For example, a manipulation which involved deleting a domain would result in the alteration of the length of a molecule just as modification of a nucleic acid to encode less than a foil length molecule would.
  • fusion polypeptides described herein may be used as therapeutic agents.
  • the present disclosure provides pharmaceutical compositions comprising at least one pharmaceutically acceptable carrier and a fusion polypeptide.
  • compositions are administered to humans, human patients, or subjects.
  • pharmaceutical compositions are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g., non-human mammal s.
  • Modification of pharmaceutical compositions suitable for adm inistration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation.
  • compositions of the disclosure include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as dogs, cattle, pigs, horses, sheep, cats, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
  • compositions of the disclosure may be administered to dogs to treat ARVC.
  • fusion polypeptides and pharmaceutical composition thereof which may be used in combination with one or more pharmaceutically acceptable excipients.
  • the pharmaceutically acceptable excipients include, but are not limited to, any and all sol vents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, flavoring agents, stabilizers, anti- oxidants, osmolality adjusting agents, pH adjusting agents and the like, as suited to the particular dosage form desired.
  • Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21" Edition, A. R.
  • a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleteri ous manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.
  • a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least. 99%, or 100% pure.
  • an excipient is approved for use for humans and for veterinary use. In some embodiments, an excipient may be approved by United States Food and Drug Administration. In some embodiments, an excipient may be of pharmaceutical grade. In some embodiments, an exci pient may meet the standards of the United S tates Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
  • USP United S tates Pharmacopoeia
  • EP European Pharmacopoeia
  • British Pharmacopoeia the British Pharmacopoeia
  • International Pharmacopoeia International Pharmacopoeia
  • compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical compositions.
  • the composition may also include excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents.
  • Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof.
  • Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, com starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation- exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinylpyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, crosslinked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, etc., and/or combinations thereof.
  • crospovidone cross-linked polyvinylpyrrolidone
  • sodium carboxymethyl starch sodium starch
  • Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chon- drux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite (aluminum silicate) and VEEGUM® (magnesium aluminum silicate), long chain amino acid derivatives, high molecular weight alcohols (e.g.
  • natural emulsifiers e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chon- drux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin
  • colloidal clays e.g. bentonite (alumin
  • stearyl alcohol cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol
  • carbomers e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxy vinyl polymer
  • carrageenan cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g.
  • polyoxyethylene sorbitan monolaurate TWEEN®20
  • polyoxyethylene sorbitan TWEEN®60
  • polyoxyethylene sorbitan monooleate TWEEN®80
  • sorbitan monopalmitate SPAN®40
  • sorbitan monostearate SPAN®60
  • sorbitan tristearate SPAN®65
  • polyoxyethylene esters e.g.
  • polyoxyethylene monostearate MYRJ®45
  • polyoxyethylene hydrogenated castor oil polyethoxydated castor oil
  • polyoxymethylene stearate polyethoxydated castor oil
  • SOLUTOL® sucrose fatty acid esters
  • polyethylene glycol fatty acid esters e.g. CREMOPHOR®
  • polyoxyethylene ethers e.g.
  • polyoxyethylene lauryl ether (BRIJ®30), poly (vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLU0RINC®F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.
  • Exemplary binding agents include, but are not limited to, starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); amino acids (e.g., glycine); natural and synthetic gums (e.g.
  • acacia sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyd cellulose, hydroxypropyl methydcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl -pyrrolidone), magnesium aluminum silicate (VEEGUM®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and combinations thereof.
  • Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives. Oxidation is a potential degradation pathway for mRNA, especially for liquid mRNA formulations. In order to prevent oxidation, antioxidants can be added to the formulation.
  • antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, EDTA, m-cresol, methionine, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, thioglycerol and/or sodium sulfite.
  • Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
  • EDTA ethylenediaminetetraacetic acid
  • citric acid monohydrate disodium edetate
  • dipotassium edetate dipotassium edetate
  • edetic acid fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
  • antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal.
  • Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid.
  • Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenyl ethyl alcohol .
  • Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and/or phytic acid.
  • preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BUT), ethylenediamine, sodium lauryl sulfate (SLS), sodium laury l ether sulfate (SEES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL®115, GERMABEN®, NEOLONETM, KATHONTM, and/or EUXYL®.
  • the pH of the pharmaceutical solutions is maintained between pH 5 and pH 8 to improve stability .
  • exemplary buffers to control pH may include, but are not limited to sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium carbonate, and/or sodium malate.
  • the exemplary buffers listed above may be used with additional monovalent counterions (including, but not limited to potassium). Divalent cations may also be used as buffer counterions; however, these are not preferred due to complex formation and/or mR.NA degradation.
  • Exemplary buffering agents may also include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water
  • Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.
  • oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, com, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana
  • oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinations thereof.
  • Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/ or perfuming agents can be present in the composition, according to the judgment of the formulator.
  • Exemplary additives include physiologically biocompatible buffers (e.g., trimethylamine hydrochloride), addition of chelants (such as, for example, DTPA or DTPA- bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA- bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
  • chelants such as, for example, DTPA or DTPA- bisamide
  • calcium chelate complexes as for example calcium DTPA, CaNaDTPA- bisamide
  • calcium or sodium salts for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate.
  • antioxidants and suspending agents can be used.
  • compositions of the present disclosure may be administered by any route which results in a therapeutically effective outcome.
  • routes include, but are not limited to enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracave
  • Therapeutically effective doses will be easily determined by one of skill in the art and will depend on the severity and course of the disease, the patient's health and response to treatment, and the judgment of the treating physician.
  • the DSG2 fusion polypeptides of the disclosure may be used to treat one or more diseases or conditions described herein, in a subject. Such methods may include contacting the subject with the DSG2 fusion polypeptides. In some embodiments, the contacting the subject may include administering DSG2 fusion polypeptides to the subject. In some embodiments, the contacting the subject may include treating the subject with the DSG2 fusion polypeptides of the disclosure. In one embodiment, compositions of the disclosure may be used to treat diseases associated with DSG2 autoantibodies. In one embodiment, the compositions of the disclosure mitigate cardiotoxicity associated with DSG2 antibodies.
  • any therapeutic disease associated with the inflammation in myocardium may be treated with the DSG2 fusion polypeptides as described herein.
  • Non- limiting examples of such indications include arrhythmogenic right ventricular cardiomyopathy (ARVC), sarcoidosis, dilated cardiomyopathy, post-infectious cardiomyopathy, compromised cardiac function, reduced ejection fraction, heart failure, arrhythmia and myocarditis.
  • ARVC arrhythmogenic right ventricular cardiomyopathy
  • sarcoidosis sarcoidosis
  • dilated cardiomyopathy dilated cardiomyopathy
  • post-infectious cardiomyopathy compromised cardiac function
  • reduced ejection fraction heart failure
  • arrhythmia arrhythmia and myocarditis.
  • Efficacy of treatment or ameliorati on of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters.
  • a disease or disorder in connection with the administration of a fusion polypeptides or pharmaceutical composition thereof, "effective against" a disease or disorder indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a fraction of patients, such as an improvement of symptoms, a cure, a reduction in disease load, extension of life, improvement in quality of life, a reduction in the need for blood transfusions or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disease or disorder.
  • a treatment or preventive effect is evident when there is a significant improvement, often statistically significant, in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated.
  • a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more may be indicati ve of effective treatment.
  • Efficacy for a given compound or composition may also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant modulation in a marker or symptom is observed.
  • DSG2 fusion polypeptides or compositions containing the fusion polypeptides as described herein can be administered to treat COVID-19 or long-tenn effects of COVID- 19.
  • compositions of the disclosure may be usefill in treating COVID-19 and/or individuals infected with SARS-CoV-2.
  • An infected person may be symptomatic, pre-symptomatic, and asymptomatic.
  • COVID-19 transmission may occur from symptomatic, pre- symptomatic, and asymptomatic people infected with SARS-CoV-2.
  • Symptomatic transmission may refer to transmission occurring before a person experiencing symptoms.
  • Pre-symptomatic transmission may refer to transmission occurring prior to onset of symptoms of COVID- 19.
  • COVID-19 may be associated with one or more symptoms such as, but not limited to, fever or chills, cough, shortness of breath or difficulty breathing, fatigue, muscle or body aches, headache, new loss of taste or smell, sore throat, congestion or runny nose, nausea or vomiting, diarrhea, trouble breathing, persistent pain or pressure in the chest.
  • symptoms such as, but not limited to, fever or chills, cough, shortness of breath or difficulty breathing, fatigue, muscle or body aches, headache, new loss of taste or smell, sore throat, congestion or runny nose, nausea or vomiting, diarrhea, trouble breathing, persistent pain or pressure in the chest.
  • DSG2 fusion polypeptides may be used to treat one or more stages of COVID- 19 disease.
  • adults with SARS-CoV-2 infection may be grouped into the following severity of illness categories. However, the criteria for each category may overlap or vary across clinical guidelines and clinical trials, and a patient’s clinical status may change over time (COVID-19 Treatment Guidelines Panel. Coronavirus Disease 2019 (COVID-19) Treatment Guidelines. National Institutes of Health. Available at www.covidl9treatmentguidelines.nih.gov/. Accessed 12/11/2020).
  • the compositions of the disclosure may be used to treat asymptomatic or presymptomatic infection which may include individuals who test positive for SARS-CoV-2 using a virologic test (i.e .
  • compositions of the disclosure may be used to treat mild illness which includes individuals who have any of the various signs and symptoms of COVID-19 (e.g., fever, cough, sore throat, malaise, headache, muscle pain, nausea, vomi ting, diarrhea, loss of taste and sm ell) but who do not have shortness of breath, dyspnea, or abnormal chest imaging.
  • compositions of the disclosure may be used to treat moderate illness which may include individuals who show evidence of lower respiratory disease during clinical assessm ent or imaging and who have saturation of oxygen (SpO2) ⁇ 94% on room air at sea level.
  • compositions of the disclosure may be used to treat severe illness which includes individuals who have SpO2 ⁇ 94% on room air at sea level, a ratio of arterial partial pressure of oxygen to fraction of inspired oxygen (PaO2/FiO2) ⁇ 300 mmHg, respiratory frequency >30 breaths per minute, or lung infiltrates >50%.
  • the compositions of the disclosure may be used to treat critical illness which includes individuals who have respiratory failure, pneumonia, acute respiratory distress syndrome (ARDS), sepsis, septic shock, multiple organ dysfunction, and/or multi organ failure, including acute kidney injury , and cardiac injury .
  • compositions of the disclosure may be provided to the subject prior to the onset of symptoms but after exposure to the virus since there is an incubation period between exposure and symptom onset to the virus.
  • the incubation period of novel coronavirus SARS-CoV-2 is generally between two and fourteen days, with an average of five days (Lombardi et al., J. Hosp. Infect. 2020 doi: 10.1016/j.jhin.2020.03.003; the contents of which are herein incorporated by' reference in its entirety).
  • compositions of the disclosure may also be administered in combination with one or more therapeutic agents recommended for use in the treatment of COVID-19.
  • DSG2 fusion polypeptides described herein may be used in combination with one or more therapeutic agents such as, but not limited to, Remdesivir, chloroquine, hydroxychloroquine, loop diuretics, azithromycin, Lopinavir, Ritonavir, ivermectin, interleukin inhibitors, interferons, kinase inhibitors, glucocorticosteroids, and/or SARS CoV- 2 monoclonal antibodies (e.g., Bamlanivimab, Casirivimab, Imdevimab).
  • compositions of the present disclosure may be used to treat post-COVID-19 syndrome.
  • post-COVID-19 syndrome There have been an increasing number of reports of patients who experience persistent symptoms after recovering from acute COVID-19 and is herein referred to as “post-COVID-19 syndrome” and individuals suffering from these symptoms are commonly referred to as “long haulers.”
  • a patient may be considered to have post-COVID-19 syndrome if they suffer from one or more symptoms for up to a month, up to two months, up to three months, up to four months, up to five months, up to six months, up to seven months, up to eight months, up to nine months, up to ten months, up to eleven months, up to a year or more after SARS CoV-2 infection.
  • the subject may have no symptoms after SARS CoV-2 infection.
  • the subject may have no known COVID-19 or SARS CoV2 infection but may have serum anti- DSG2 antibodies.
  • compositions of the present disclosure may be used to treat one or more symptoms associated with the cardiovascular system in post-COVID-19 syndrome (i.e., post COVID-19 cardiac syndrome).
  • post-COVID-19 syndrome i.e., post COVID-19 cardiac syndrome.
  • cardiac magnetic resonance imaging revealed cardiac involvement in 78 patients (78%) and ongoing myocardial inflammation in 60 patients (60%), which was independent of preexisting conditions, severity and overall course of the acute illness, and the time from the original diagnosis (Puntmann et al. JAMA Cardiol. 2020:5(11): 1265-1273).
  • 15% of athletes had evidence for myocarditis after recovery from acute COVID-19.
  • the DSG2 fusion polypeptides may be used to treat myocarditis in a subject with post-COVID-19 syndrome.
  • compositions described herein may be used to treat post-COVID-19 syndrome that is not associated with any cardiac indications.
  • compositions of the present disclosure may be used to treat subjects who show symptoms of COVID-19, for up to weeks, months, and/or years after initial diagnosis of COVID-19.
  • post-COVID-19 syndrome patients may demonstrate symptoms for and/or after 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 1 month, 2 months ,3 months, 4 months, 5 months, 6 months, 7months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years or more after initial diagnosis of COVID- 19.
  • Diagnosis of COVID-19 may be established using methods known in the art (for e.g., reverse transcription polymerase chain reaction and/or antibody tests).
  • subjects affected by post-COVID-19 syndrome may be treated with the compositions of the disclosure for up to 1 week, up to 1 month, and/or up to one year.
  • compositions of the disclosure may be used to treat COVID-19 patients who have or develop compromised cardiac function, most notably a reduced ejection fraction, with or without overt symptoms of heart failure. In some embodiments, the compositions of the disclosure may be used to treat arrhythmia.
  • compositions of the present disclosure may ameliorate one or more symptoms associated with post-COVID-19 syndrome.
  • the symptoms of post- COVID-19 syndrome may be the same as acute COVID-19.
  • the symptoms associated with post-COVID-19 syndrome may be shortness of breath, fatigue, edema, orthopnea, limitations to exertion, impaired cognitive abilities, palpitations, dizziness, syncope, lightheadedness, heart failure, and/or arrhythmia.
  • compositions of the disclosure may be used to treat post COVID-19 syndrome with symptoms that overlap with the post-intensive care syndrome that has also been described in patients without COVID-19.
  • compositions of tire disclosure may be used to treat subjects with post-COVID-19 syndrome who may have one or more long-term complications associated with the cardiovascular system (e.g., inflammation of the heart, muscle), respiratory system (lung function abnormalities), renal systems (acute kidney injury), dermatologic (rash, hair loss), neurological complications (smell and taste problems, sleep issues, difficulty with concentration, memory problems), and/or psychiatric problems (depression, anxiety, changes in mood).
  • cardiovascular system e.g., inflammation of the heart, muscle
  • respiratory system lung function abnormalities
  • renal systems acute kidney injury
  • dermatologic rash, hair loss
  • neurological complications smell and taste problems, sleep issues, difficulty with concentration, memory problems
  • psychiatric problems depression, anxiety, changes in mood
  • composi ti ons of the disclosure may be used to treat post- COVID-19 syndrome subjects who may have one, two or more associated co-morbidities.
  • co-morbidities include, but are not limited to, hypertension, thyroid disease, immune disorders, COPD (chronic obstructive pulmonary disease), high blood pressure, obesity, mental health conditions, and diabetes.
  • compositions of the disclosure may be used to block autoantibodies generated during COVID-19 or SARS CoV2 infection.
  • compositions of the disclosure may be used to block au toantibodies developed following COVID-19 infection.
  • compositions of the disclosure may be used in combination with immunomodulatory drugs and biological agents targeting pro inflammatory cytokines.
  • compositions of the disclosure may be used in combination with corticosteroids, JAK inhibitors, IL-1 blockade and IL-6 receptor antagonists.
  • a high-throughput autoantibody discovery method known as rapid extracellular antigen profiling (REAP) was implemen ted to screen a cohort of 194 individuals infected with COVID-19, comprising 172 patients with COVID-19 and 22 healthcare workers with mild disease or asymptomatic infection, for autoantibodies against 2,770 extracellular and secreted proteins (members of the exoproteome).
  • RRP rapid extracellular antigen profiling
  • compositions of the disclosure may be used to block autoantibodies that predated COVID-19 infection. In some embodiments, compositions of the disclosure may be used to block autoantibodies that may be induced after COVID-19 infection.
  • compositions of the disclosure may be used to treat cardiomyopathies.
  • Cardiomyopathy refers to progressive impairment of the structure and function of the muscular walls of the heart chambers.
  • Compositions of the disclosure may be used to treat one or more types of cardiomyopathies, such as, but not limited to, dilated cardiomyopathy, hypertrophic cardiomyopathy and/or restrictive cardiomyopathy.
  • patients with cardiomyopathies may demonstrate serum DSG2 autoantibodies in their serum .
  • compositions of the disclosure may be used to treat Arrhythmogenic right ventricular cardiomyopathy (ARVC).
  • ARVC Arrhythmogenic right ventricular cardiomyopathy/dysplasia
  • ARVC is a heart muscle disorder associated with ventricular arrhythmia, heart failure, and sudden death.
  • ARVC is a degenerative cardiac disease characterized by the progressi ve loss of ventricular function and arrhythmias.
  • patient immune systems also have been known to play a role in ARVC disease pathology. Mutations in DSG2 protein have been associated with ARVC and autoantibodies targeting DSG2 have been identified in patients with the disease.
  • DSG2 fusion proteins may be used to treat ARVC patients who have one or more mutations in the DSG2 protein. In some aspects, the DSG2 fusion proteins may be used to treat ARVC patients with no known mutations in the DSG2 protein. In some embodiments, DSG2 fusion polypeptides of the disclosure may target DSG2 autoantibodies associated with ARVC.
  • Cardiomyopathy may be associated with inflammation and is herein referred to as myocarditis.
  • myocarditis may be caused by viruses, bacteria, parasites, and/or fungi.
  • compositions of the disclosure may be used to treat and/or prevent myocarditis associated with viruses.
  • viruses associated with myocarditis include, common cold causing adenovirus, COVID-19; hepatitis B and C; parvovirus, which causes a mild rash, usually in children (fifth disease); and/or herpes simplex virus, gastrointestinal infections causing echoviruses, mononucleosis causing Epstein-Barr virus, rubella, cytomegalovirus, and HIV.
  • cardiovascular complications have occurred frequently in association with COVID-19 and even months after the infection. These cardiovascular complications include myocardial injury and myocarditis, acute coronary syndromes, heart failure, arrythmias, and thromboembolic events. In addition, cardiac symptoms, palpitations, chest pain, and dyspnea have been observed in patients, weeks to months after the initial infection. (Lee, C. C. E., et al. Diseases 2021; 9:47; the contents of each of which are herein incorporated by reference in their entirety). In some embodiments, compositions of the disclosure may be used to treat cardiovascular complications such as myocardial injury and myocarditis, acute coronary syndromes, heart failure, arrythmias, and thromboembolic events.
  • compositions of the disclosure may be used to treat and/or prevent myocarditis caused by bacteria.
  • bacteria associated with myocarditis include, Staphylococcus, Streptococcus, and/or Borrelia.
  • compositions of the disclosure may be used to treat and/or prevent myocarditis caused by parasites.
  • parasites associated with myocarditis include, Trypanosoma cruzi and Toxoplasma, including some that are transmitted by insects and can cause a condition called Chagas disease.
  • compositions of the disclosure may be used to treat and/or prevent myocarditis caused by fungi.
  • fungi associated with myocarditis include, Candida, Aspergillus; and other fungi, such as Histoplasma.
  • Domain As used herein when referring to polypeptides the term “domain” refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions).
  • Expression vector refers to a vector containing a nucl eic acid sequence coding for at least part, of a gene product capable of being transcribed.
  • Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism, In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.
  • the term also includes a recombinant plasmid or virus that comprises a polynucleotide to be delivered into a host cell, either in vitro or in vivo.
  • the host cell is a transient cell line or a stable cell line. In some embodiments, it is selected from the group consisting of CHO, HEK293 and NSO.
  • Fusion protein refers to protein or polypeptide comprising two or more sequences of amino acids or active fragments thereof that are not naturally present in the same polypeptide.
  • two or more separate polypeptides are operably covalently linked, e.g., chemically linked, or fused together by peptide bonds. Recombinant fusion polypeptides are created artificially by recombinant DNA technology.
  • sub- domains may be identified within domains or half-domains, these subdomains possessing less than all of the structural or functional properties identified in the domains or half domains from which they were derived. It is also understood that the amino acids that comprise any of the domain types herein need not be contiguous along the backbone of the polypeptide (i.e., nonadjacent amino acids may fold structurally to produce a domain, half-domain or subdomain).
  • Immune response refers to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
  • Linker refers to a functional group (e.g., a chemical or polypeptide) in which a covalent bond joins two or more polypeptides.
  • a “peptide linker” is two or more amino acids used to bind two proteins to each other.
  • Modulation As used herein, the term “modulation” is recognized in the art and refers to up regulation (i.e., activation or stimulation), down regulation (i.e., inhibition or suppression) of a response, or the two in combination or apart.
  • Polynucleotide refers to a sequence of nucleotides connected by phosphodiester linkages. Polynucleotides are presented herein in the direction from the 5' to the 3' direction.
  • a polynucleotide can be a deoxyribonucleic acid (DNA) molecule or ribonucleic acid (RNA) molecule. Where a polynucleotide is a DNA molecule, that molecule can be a gene or a cDNA molecule.
  • Nucleotide bases are indicated herein by a single letter code: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I) and uracil (U).
  • a polynucleotide can be prepared using standard techniques well known to one of skill in the art.
  • compositions of the present disclosure are polypeptides or proteins or variants thereof.
  • any amino acid-based molecule may be termed a “polypeptide” and this term embraces “peptides,” “peptidomimetics,” and “proteins.”
  • polypeptide means a polymer of amino acid residues (natural or unnatural) linked together most often by- peptide bonds.
  • the term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function.
  • a “peptidomimetic” or “polypeptide mimetic” is a polypeptide in which the molecule contains structural elements that are not found in natural polypeptides (i.e., polypeptides comprised of only the 20 proteinogenic amino acids). In some embodiments, peptidomimetics are capable of recapitulating or mimicking the biological action(s) of a natural peptide.
  • Polypeptide variant refers to molecules which differ in their amino acid sequence from a native or reference sequence.
  • the amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence.
  • variants will possess at least about 50% identity (homology) to a native or reference sequence, and preferably, they will be at least about 80%, more preferably at least about 90% identical (homologous) to a native or reference sequence.
  • Recombinant refers to a genetic entity distinct from that generally found in nature. As applied to a polynucleotide or gene, this means that the polynucleotide is the product of various combinations of cloning, restriction and/or ligation steps, and other procedures that result in the production of a construct that is distinct from a polynucleotide found in nature.
  • sample refers to an aliquot or portion taken from a source and/or provided for analysis or processing.
  • a sample is from a biological source such as a tissue, cell or component part, (e.g., a body fluid, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).
  • a biological source such as a tissue, cell or component part, (e.g., a body fluid, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).
  • a sample may be or include a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, or organs.
  • a sample is or includes a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins.
  • a “primary” sample is an aliquot of the source.
  • a primary sample is subjected to one or more processing (e.g., separation, purification, etc.) steps to prepare a sample for analysis or other use.
  • the term “substantially” refers to the qualitative conditi on of exhibiting total or near-total extent or degree of a characteri stic or property of interest.
  • One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • the term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
  • Terminus refers to an extremity of a peptide or polypeptide. Such extremity is not limited only to the first or final site of the peptide or polypeptide but may include additional amino acids in the terminal regions.
  • the poly peptide-based molecules described herein may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). Proteins described herein are in some cases made up of multiple poly-peptide chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers).
  • polypeptides may be modified such that they begin or end, as the case may be, with a non- polypeptide based moiety such as an organic conjugate.
  • therapeutically effective amount means an amount of an agent to be delivered that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition.
  • Treating- refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of. reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a di sease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. [0130] Treatment: As used herein the terms “treat,” “treatment,” and the like, refer to relief from or alleviation of pathological processes.
  • the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression or anticipated progression of such condition.
  • treatment dose refers to one or more doses of a therapeutic agent administered in the course of addressing or alleviating a therapeutic indication. Treatment doses may be adjusted to maintain a desired concentration or level of activity of a therapeutic agent in a body fluid or biological system.
  • articles such as “a,” “an,” and ‘the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more m embers of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the disclosure includes embodiments in which exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process.
  • the disclosure includes embodiments in which more than one, or all group members are present in, employed in, or otherwise relevant to a given product or process.
  • DSG2 fusion polypeptides described herein are produced by recombinant DNA techniques by synthesizing DNA encoding the desired polypeptide. Once coding sequences for the desired polypeptides are synthesized or isolated, they are cloned into any suitable vector for expression.
  • the expression vector is inserted into a suitable host cell by transformation, transduction, and/or transfection.
  • the sequences of the DSG2 fusion polypeptides may be optimized to yield maximal expression in a host cell.
  • the host cell is any host cell known in the art for expression of recombinant proteins.
  • a number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, IIEK293, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney (“MDBK”) cells, NOS cells derived from carcinoma cells, such as sarcoma, as well as others.
  • ATCC American Type Culture Collection
  • ATCC American Type Culture Collection
  • CHO Chinese hamster ovary
  • HeLa cells HeLa cells
  • IIEK293 baby hamster kidney
  • BHK baby hamster kidney
  • COS monkey kidney cells
  • human hepatocellular carcinoma cells e.g., Hep G2
  • MDBK Madin-Darby bovine kidney
  • NOS cells derived from carcinoma cells such as sarcoma
  • yeast host cells useful in the present disclosure include inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces Jragilis, Kluyver omyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica.
  • the fusion polypeptides of the present disclosure are produced by growing host cells expressing the expression vector under conditions whereby the protein of interest is expressed. The protein is then isolated from the host cells and purified.
  • the fusion polypeptides of the present disclosure may be synthesized by conventional techniques known in the art, for example, by chemical synthesis such as solid phase peptide synthesis.
  • Viral infections including COVID-19, have been hypothesized to contribute to autoimmune responses, e.g., by exposing previously hidden cryptic epitopes on damaged cells to an activated immune system (Ehrenfeld M, et al.. Autoimmunity Reviews 2020;! 02597; the contents of which are herein incorporated by reference in its entirely).
  • an activated immune system Ehrenfeld M, et al.. Autoimmunity Reviews 2020;! 02597; the contents of which are herein incorporated by reference in its entirely.
  • anti-DSG2 autoantibodies might be generated as a result.
  • the mean signal intensity of anti-DSG2 antibodies in the post COVID-19 samples was significantly higher than that of a healthy control population as shown in Fig. 1 (19 ⁇ 83.2 in the post COVID-19 sample vs. 2.1 ⁇ 6.8 in the healthy control population, p value ⁇ 0.001).
  • 29.3% of the post COVID-19 infection samples demonstrated a signal higher than the

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