DSG2 Compositions and Methods

Abstract

The present disclosure relates primarily to compositions and methods for treating diseases by administering the compositions disclosed herein. Provided herein are DSG2 fusion polypeptides and methods related thereto, including methods for treating and improving cardiomyocyte function.

Description

[Technical field]

[0001] Sequence Listing This application has been submitted with a Sequence Listing in XML format in accordance with WIPO Standard ST26. The Sequence Listing file entitled 10383-108749-04.xml was created on October 24, 2022 and is 48,369 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. [Background technology]

[0002] Among the factors regulating disease progression, dysregulation of the immune system is thought to play a central role. The immune system is composed of a highly regulated, multicellular, complex defense system characterized by high individual variability in responses to injury and antigens. In physiological conditions, it is programmed to distinguish between self and foreign components, interacting with and eliminating structures recognized as foreign. This process can be transformed into a pathological situation in which self-tissues are attacked, which can lead to autoimmune diseases.

[0003] Circulating autoantibodies are said to be significantly associated with cardiac disease. Their prevalence, mechanism of action, and potential therapeutic modulation have been thoroughly investigated. Although myocardial damage is believed to be the key initiating event, genetic predisposition, environmental and epigenetic regulators, and other as yet unknown mechanisms are important for the development of pathological antibody titers observed in peripheral blood and the intensity of inflammation in myocardial structures. In a prospective study, Caforio et al. showed that circulating anti-cardiac autoantibodies precede the manifestation of disease and may be an independent predictor of disease onset (Non-Patent Document 1; the contents of which are incorporated herein by reference in their entirety).

[0004] Currently, there is a lack of therapeutic strategies for treating and / or managing autoantibodies associated with diseases, particularly cardiac disease. The present disclosure provides DSG2 fusion polypeptide-based compositions and methods for treating diseases, including but not limited to cardiac disease, infectious diseases, and the like. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Caforio et al.Circulation.2007;115:76-83 Summary of the Invention

[0006] The present disclosure provides compositions comprising isolated polypeptides. The polypeptides of the present disclosure may comprise all or a portion of a DSG2 protein. In some embodiments, the isolated polypeptide is a desmoglein 2 (DSG2) fusion polypeptide. The DSG2 fusion polypeptide may comprise (a) all or a portion of a DSG2 protein, and / or (b) all or a portion of an immunoglobulin protein. The DSG2 protein may be a Homo sapiens DSG2 protein, a Mus musculus DSG2 protein, a Rattus norvegicus DSG2 protein, a Macaca mulatta DSG2 protein, a Canis lupus familiaris DSG2 protein, or a Danio rerio DSG2 protein. The immunoglobulin protein may be a human or canine immunoglobulin protein.

[0007] In one embodiment, the DSG2 protein may be Homo sapiens DSG2 protein (SEQ ID NO: 1). In one embodiment, the DSG2 protein may be House mouse DSG2 protein (SEQ ID NO: 14). In one embodiment, the DSG2 protein may be Norway rat DSG2 protein (SEQ ID NO: 15). In one embodiment, the DSG2 protein may be Rhesus monkey DSG2 protein (SEQ ID NO: 16). In one embodiment, the DSG2 protein may be dog DSG2 protein (SEQ ID NO: 17). In one embodiment, the DSG2 protein may be zebrafish DSG2 protein (SEQ ID NO: 18).

[0008] In one embodiment, the DSG2 polypeptide may comprise a portion of the DSG2 protein. The portion of the DSG2 protein may comprise an extracellular region of the DSG2 protein. In some aspects, the entire extracellular region of DSG2 may be included in the fusion polypeptide. In one embodiment, the entire extracellular region of DSG2 comprises the amino acid sequence of SEQ ID NO:3. Embodiments of the present disclosure may also comprise a portion of the extracellular region of DSG2. For example, the 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). In some aspects, the DSG2 fusion polypeptide comprises two domains of the extracellular region. For example, the two domains may be EC4EA, EC1EC2, EC2EC3, EC3EC4, EC1EA, EC1EC3, EC2EC4, and / or EC3EA. In some embodiments, the DSG2 fusion polypeptide comprises three domains of the extracellular region. For example, the three domains can be EC1EC3EA, EC1EC4EA, EC1EC3EA, EC3EC4EA, EC1EC2EC3, EC2EC3EC4, and / or EC2EC4EA. In some embodiments, the DSG2 fusion polypeptide comprises four domains of the extracellular region. For example, the three domains can be EC1EC2EC4EA, EC2EC3EC4EA, EC1EC2EC3EC4EA, EC1EC2EC3EC4, and / or EC1EC2EC3EA.

[0009] The DSG2 fusion polypeptide may comprise a portion of an immunoglobulin. The portion may be an Fc region, a 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. In one embodiment, the portion of an immunoglobulin may be an Fc region. The immunoglobulin may be IgG, IgM, IgA, IgD, and / or IgE. As a non-limiting example, the immunoglobulin may be IgG. The composition may include IgG, such as IgG1, IgG2, IgG3, and / or IgG4. The Fc region may be a human Fc region or a canine Fc region. Some non-limiting examples of immunoglobulins useful in the present disclosure include a human IgG1 Fc region (SEQ ID NO:5), a human IgG2 Fc region (SEQ ID NO:7), a human IgG3 Fc region (SEQ ID NO:9), or a human IgG4 Fc region (SEQ ID NO:11), a canine IgG heavy chain DFc region (SEQ ID NO:20), a canine IgG heavy chain AFc region (SEQ ID NO:22), a canine IgG heavy chain B Fc region (SEQ ID NO:24), or a canine IgG heavy chain CFc region (SEQ ID NO:26), a human IgG1 heavy chain constant domain (SEQ ID NO:4), a human IgG2 heavy chain constant domain (SEQ ID NO:6), a human IgG3 heavy chain constant domain (SEQ ID NO:8), or a human IgG4 heavy chain constant domain (SEQ ID NO:10), a canine IgG heavy chain constant domain chain D (SEQ ID NO:19), a canine IgG heavy chain constant domain chain A (SEQ ID NO:21), a canine IgG heavy chain constant domain chain B (SEQ ID NO:23), or a canine IgG heavy chain constant domain chain C (SEQ ID NO:25).

[0010] The polypeptide of the present disclosure may further comprise a linker sequence. The length of the linker may be from about 5 amino acids to about 50 amino acids. In one embodiment, the linker may be GGGGS (SEQ ID NO: 12). In another aspect, the linker may be EAAAK (SEQ ID NO: 13), GGGGS (SEQ ID NO: 27), or IEGRM (SEQ ID NO: 28).

[0011] One embodiment is a pharmaceutical composition for use in treating a disease or disorder caused by anti-desmoglein 2 (DSG2) autoantibodies. The composition comprises a fusion polypeptide that inhibits binding of an anti-DSG2 antibody to a DSG2 extracellular domain. The fusion polypeptide comprises an extracellular region of a human DSG2 protein having at least about 85% sequence identity with amino acid residues 50-609 of SEQ ID NO: 1 or a portion thereof, and an affinity tag. The portion of the extracellular region of the human DSG2 protein may comprise any one or combination of DSG2 domains selected from the group consisting of extracellular cadherin domain 1 (EC1), extracellular cadherin domain 2 (EC2), extracellular cadherin domain 3 (EC3), extracellular cadherin domain 4 (EC4), and extracellular anchor domain (EA). The affinity tag may be an Fc region of an immunoglobulin such as IgG1 or a variant thereof. The variant may have the sequence of SEQ ID NO: 31. The affinity tag may be an Fc region of an IgG4 or a variant thereof. The variant may have the sequence of SEQ ID NO: 32. Alternatively, the affinity tag may be provided by a smaller protein or peptide, such as a polyhistidine peptide. Some embodiments of the fusion polypeptide include a linker sequence located between the extracellular domain of the human DSG2 protein or a portion thereof and the affinity tag. In some embodiments, the linker sequence is SEQ ID NO: 12, 13, 27, or 28. In some embodiments, the disease or disorder is arrhythmia, such as arrhythmogenic right ventricular cardiomyopathy (ARVC), sarcoidosis, or dilated cardiomyopathy. In some embodiments, the disease or disorder is cardiomyopathy, such as arrhythmogenic right ventricular cardiomyopathy (ARVC), sarcoidosis, or dilated cardiomyopathy. The arrhythmia or cardiomyopathy may be caused by a virus, such as SARS-CoV2, adenovirus, hepatitis virus, hepatitis C virus, parvovirus, herpes simplex virus, echovirus, Epstein-Barr virus, rubella, cytomegalovirus, or HIV.

[0012] The present disclosure provides a method of reducing arrhythmogenic phenotype in cardiomyocytes. Such a method may include contacting cardiomyocytes with a composition of the present disclosure. In some embodiments, the arrhythmogenic phenotype may be associated with or caused by an anti-DSG2 antibody. The present disclosure also provides a method of reducing or correcting the arrhythmogenic phenotype by contacting cardiomyocytes with a composition of the present disclosure. The reduction or correction of the arrhythmogenic phenotype may be measured using cardiomyocyte sodium spikes (μV / m). The present disclosure also provides a method of treatment using the compositions described herein.

[0013] The present disclosure also provides a method of treating a condition associated with serum DSG2 autoantibodies. Such a method may include administering to a subject a composition described herein, or a cell expressing a composition described herein. In some embodiments, the condition may be arrhythmia. In some embodiments, the condition may be cardiomyopathy. In some aspects, the condition may be an autoimmune disease.

[0014] The present disclosure provides a method for treating arrhythmia and / or cardiomyopathy in a subject. Such a method may include contacting a subject with an isolated polypeptide or cell of the present disclosure, followed by measuring one or more symptoms or clinical signs associated with arrhythmia, such as electrocardiogram abnormal beats, dizziness, lightheadedness, palpitations, chest pain, shortness of breath, reduced ejection fraction, reduced cardiac output, and / or heart failure. Such a method may include contacting a subject with an isolated polypeptide or cell of the present disclosure, followed by measuring one or more symptoms associated with cardiomyopathy, such as arrhythmia, palpitations, myocarditis, heart failure, reduced cardiac output, and / or reduced ejection fraction. As non-limiting examples, cardiomyopathy may be arrhythmogenic right ventricular cardiomyopathy (ARVC), sarcoidosis, dilated cardiomyopathy. Cardiomyopathy may also be caused by viruses (e.g., SARS-CoV2, adenovirus, hepatitis virus, hepatitis C virus, parvovirus, herpes simplex virus, echovirus, Epstein-Barr virus, rubella, cytomegalovirus, or HIV), bacteria (Staphylococcus, Streptococcus, Borrelia), parasites (Trypanosoma or Toxoplasma), or fungi (Candida, Aspergillus, or Histoplasma). In some embodiments, the subject may have detectable levels of anti-DSG2 antibodies in the serum.

[0015] The present disclosure also provides a method of treating a cardiac abnormality in a subject using the DSG2 fusion polypeptides described herein. In some embodiments, the subject may have serum anti-DSG2 antibodies. The present disclosure also provides a method of reducing anti-DSG2 antibodies in a subject using the DSG2 fusion polypeptides described herein. In some embodiments, the anti-DSG2 antibody level may be reduced by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0016] Provided herein are compositions comprising DSG2 fusion polypeptides. Also provided herein are compositions comprising at least one therapeutic agent. In some embodiments, the compositions of the present disclosure may comprise a combination of a DSG2 fusion polypeptide and at least one therapeutic agent described herein. In some embodiments, the compositions comprise only a therapeutic agent. Non-limiting examples of therapeutic agents include anti-CD20 antibodies, FcRn inhibitory antibodies, intravenous immunoglobulin (IVIG), and / or complement inhibitors such as eculizumab. Also provided herein are methods of treating conditions associated with serum anti-DSG2 autoantibodies using DSG2 fusion polypeptides and / or therapeutic agents described herein. In some embodiments, the condition associated with serum anti-DSG2 autoantibodies may be a cardiac disease or an infectious disease. The cardiac disease may be ARVC, sarcoidosis, dilated cardiomyopathy, or any cardiac disease associated with anti-DSG2 antibodies. [Brief description of the drawings]

[0017] The foregoing and other objects, features and advantages will become apparent from the following description of specific embodiments of the present disclosure, as illustrated in the accompanying drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the present disclosure. [Figure 1A] The inhibition rates of anti-DSG2 antibodies and ARVC serum using DSG2FP#1 are shown. [Figure 1B] The inhibition rates of anti-DSG2 antibodies and ARVC serum using DSG2FP#2 are shown. [Figure 1C] The inhibition rates of anti-DSG2 antibodies and ARVC serum using DSG2FP#3 are shown. [Figure 1D] Figure 1B shows the inhibition rate of anti-DSG2 antibodies and ARVC serum using DSG2FP#4. The DSG2 fusion polypeptide was subjected to size exclusion chromatography and various fractions were examined. Fraction "C" had a predicted MW of approximately 350 kDa and was used for further experiments. In Figure 1B, Figure 1C, and Figure 1D, the whole protein preparation ("whole preparation") was compared to "fraction C". [Figure 2A]2A is a first series of voltage (μV) versus time (ms) graphs showing that lidocaine and anti-DSG2 antibodies attenuate sodium spikes in human iPSC cardiomyocytes in a multielectrode array (MEA) assay. The attenuation of sodium spikes is reversed by the addition of DSG2FP#1 (labeled "DSG2 fusion polypeptide" in FIG. 2A), indicating that DSG2FP#1 completely inhibits the effect of anti-DSG2 antibodies on sodium spikes. [Figure 2B] FIG. 13 is a second series of voltage (μV) versus time (ms) graphs showing that control tests of vehicle, rabbit IgG, and anti-VCAM-1 antibody have no effect on sodium spikes in the MEA assay. [Diagram 3] The cell index after treatment of human iPSC cardiomyocytes with anti-DSG2 antibody and anti-DSG2 antibody + DSG2FP#1 (labeled as "decoy" in Figure 3) in a multi-electrode array (MEA) assay is shown, demonstrating that DSG2FP#1 completely inhibits the effect of anti-DSG2 antibody. [Figure 4A] 1 is a graph of anti-DSG2 antibody signal in a detection assay. [Figure 4B] Figure 1 shows the correlation between inhibition rate and signal-to-background ratio in various groups. Data points for the control group and the ARVC poor clinical data group are clustered on the y-axis, indicating that there is little difference in signal ratio between these two groups. [Diagram 5] 1 depicts a series of bar graphs showing inhibition of binding of ARVC patient sera to the DSG2 extracellular domain by a series of recombinant DSG2 fusion proteins as assessed by electrochemical immunoassay. [Figure 6] FIG. 1 is a bar graph showing inhibition of binding of anti-DSG2 antibody to the DSG2 extracellular domain by recombinant DSG2(50-609)-IgG4Fc fusion protein with or without a linker, as assessed by electrochemical immunoassay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description I. Introduction The production of autoantibodies against self-proteins, called autoantigens, is a hallmark of many autoimmune diseases. The immunoreactivity of autoantibodies in a patient's body fluids provides important diagnostic information when an autoimmune disease is suspected. The spectrum of autoantibodies is often clinically informative for a given autoimmune disease. In some autoimmune diseases, autoantibodies against only one or a few target autoantigens are present, whereas in other conditions autoantibodies against multiple targets may coexist. Among the 70 most common autoimmune diseases, about 100 of the estimated 20,000 human proteins encoded in the genome are thought to constitute the most common antigen targets. However, an increasing number of autoantibodies are being discovered in rare diseases, suggesting that autoantibody-associated autoimmunity is likely to emerge in additional diseases.

[0019] Treatment of many autoimmune diseases is suboptimal due to the varying degrees of efficacy and side effects of available interventions. Advances in the treatment of autoimmune diseases require disease-specific information, including how autoantibodies are involved in pathogenesis.

[0020] Desmoglein 2 is one of the cardiac cadherin proteins. Cadherins are calcium-dependent adhesion molecules that provide mechanical adhesion between cells in many tissues. Typically, three calcium ions (12 total) enter pockets in the binding motif that exists between each pair of five consecutive extracellular cadherin (EC) domains, providing the crescent shape necessary to bring the cadherin domains of opposing cells into a 90-degree conformation for trans-binding. This 90-degree conformation is important for binding because it allows tryptophan residues on each EC1 domain to simultaneously insert into the hydrophobic pocket of the alternative cadherin. Interfering with this orientation, such as through missense mutations or binding of antibodies to the proximal extracellular DSG2 domain, can reduce binding and adhesion. Chatterjee et al. identified autoantibodies against cardiac desmoglein 2 (DSG2) protein as a common feature in sera from ARVC patients (Chatterjee D, et al., Eur. Heart J. 2018;39(44):3932-3944; the contents of which are incorporated herein by reference in their entirety). These autoantibodies were specific for ARVC, as they were essentially absent from two independent sets of control sera, as well as from sera from subjects with other forms of inherited cardiomyopathy. Anti-DSG2 antibodies can also be found in some cases of sarcoidosis, a systemic inflammatory disease that causes granulomas in organs, including but not limited to the heart. Anti-DSG2 antibodies are also found in sarcoidosis patients with cardiac involvement (Suna et al. 2020, Eur. Heart Journal, Vol. 41, Supplement 2, November 2020, ehaa946.2127; the contents of which are incorporated herein by reference in their entirety). Patients diagnosed with dilated cardiomyopathy may have mutations in the same desmosomal proteins associated with ARVC. These observations suggest that some patients with dilated cardiomyopathy may actually have an ARVC-like disease mediated by anti-DSG2 autoantibodies, but are diagnosed with dilated cardiomyopathy because they do not fit the typical age or symptoms associated with ARVC.Anti-DSG2 autoantibodies are believed to arise when there is a combination of cardiac cell damage and an activated immune system, for example in the context of infectious diseases known to directly affect the myocardium. Thus, strategies targeting anti-DSG2 antibodies (e.g., DSG2 autoantibodies) may be beneficial in treating symptoms and diseases resulting from the presence of anti-DSG2 antibodies, including, but not limited to, COVID-19, post-COVID-19 syndrome, and / or cardiac disease (e.g., ARVC).

[0021] The present disclosure provides compositions and methods related to DSG2 fusion polypeptides that target anti-DSG2 antibodies. Thus, the DSG2 fusion polypeptides of the present disclosure may be a viable therapeutic strategy in the treatment of diseases associated with anti-DSG2 antibodies and / or diseases associated with autoimmune reactions, including but not limited to COVID-19, post-COVID-19 cardiac syndrome, ARVC, etc. DSG2 fusion polypeptides may also be used to treat other diseases associated with cardiac cell 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 coxsackievirus, adenovirus, echovirus, parvovirus, rubella, and / or cytomegalovirus.

[0022] II. Composition In some embodiments, the present disclosure provides compositions comprising DSG2 fusion polypeptides. The compositions described herein can bind to or interact with anti-DSG2 antibodies. In one embodiment, the compositions of the present disclosure can modulate the activity of anti-DSG2 antibodies. In one embodiment, the compositions of the present disclosure can inhibit the activity of anti-DSG2 antibodies.

[0023] In some embodiments, the present disclosure includes a DSG2 protein. In some aspects, the DSG2 protein may be the entire DSG2 protein or a portion of the DSG2 protein. In some embodiments, the DSG2 protein may be fused with any other protein or protein fragment.

[0024] The DSG2 fusion polypeptides of the present disclosure may include a protein tag. A protein tag is a protein or peptide sequence that is included in a recombinant protein provided for various purposes, usually located at the N-terminus or C-terminus. In this embodiment, the protein tag functions primarily as an affinity tag, but can also provide other benefits such as improved expression, solubility, bioactivity, and pharmacokinetic properties. Affinity tags are added to proteins to allow the proteins to be purified from crude biological sources using affinity techniques. Examples of affinity tags include chitin-binding protein (CBP), maltose-binding protein (MBP), streptavidin, and glutathione S-transferase (GST). A common polyhistidine, known as 6xHis or hexahistidine, is a widely used protein tag that binds to matrices containing immobilized metal ions.

[0025] Solubilization tags are used for recombinant proteins, especially those expressed in species such as E. coli, to aid in proper folding of the protein and prevent aggregation within inclusion bodies. These tags include thioredoxin (TRX) and poly(NANP). Some affinity tags have a dual role as a solubilizer, such as MBP and GST. The Fc region of immunoglobulins is also useful, acting as both an affinity tag and a dimerizer / solubilizer, and also providing a means to detect protein expression using commercially available ELISA kits. The addition of an IgG-Fc tag can also increase protein expression yields and affect the pharmacokinetics of recombinant proteins in vivo.

[0026] In some of the exemplary embodiments described below, the protein tag of the fusion protein is a polyhistidine tag having six consecutive histidine residues, designated 6xHis. In other examples, the protein tag is an Fc region of IgG, including IgG1 (SEQ ID NO:5) or IgG4 (SEQ ID NO:11). These fusion polypeptide embodiments exhibit activity with respect to binding to anti-DSG2 antibodies.

[0027] A DSG2 fusion polypeptide may comprise the entire DSG2 protein or a portion of the DSG2 protein and an affinity tag comprising the entire or a portion of an immunoglobulin protein. In some embodiments, a DSG2 fusion polypeptide may further comprise a linker peptide. In some embodiments, the entire or a portion of the 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, such as an affinity tag, such as TRX, poly(NANP), MBP, GST, or polyhistidine, which may improve expression and purification of the DSG2 protein in vitro or in vivo.

[0028] In some embodiments, the protein tag of the fusion polypeptide is a PAS polypeptide tag. PAS sequences are hydrophilic, uncharged biological polymers with biophysical properties very similar to polyethylene glycol (PEG), and chemically conjugating them to drugs is an established method to extend plasma half-life. In contrast, PAS polypeptides provide fusion with therapeutic proteins at the genetic level, allowing for the generation of fully active proteins and eliminating the need for in vitro coupling or modification steps (Schlapschy et al., Protein Eng. Des. Sei., 2013, 26(8), 489-501, incorporated herein by reference in its entirety). The process of adding a PAS polypeptide to a fusion protein is known as "PASylation". In some embodiments, polyethylene glycol (PEG) is used as a non-protein tag instead of incorporating a PAS polypeptide. The process of adding a PEG moiety is known as "PEGylation". PASylation or PEGylation of certain embodiments of fusion proteins may provide the ability to purify the fusion protein by size exclusion chromatography instead of affinity chromatography.

[0029] DSG2 mutations in the intercalated discs of cardiac cells are believed to be associated with cardiac diseases such as arrhythmias, dilated cardiomyopathy, and especially ARVC (arrhythmogenic right ventricular cardiomyopathy). Chatterjee et al. identified autoantibodies against cardiac DSG2 protein as a common feature in the serum of ARVC patients (Chatterjee D, et al., Eur Heart J. 2018; 39(44): 3932-3944; the contents of which are incorporated herein by reference in their entirety). These autoantibodies were specific to ARVC, as they were essentially absent from two independent sets of control sera, as well as from sera from subjects with other forms of inherited cardiomyopathies. The present disclosure provides DSG2 fusion polypeptides as a therapeutic strategy to target DSG2 autoantibodies. In some embodiments, the DSG2 fusion polypeptides of the present disclosure can bind to DSG2 autoantibodies. In some embodiments, binding of the DSG2 fusion polypeptides of the present disclosure to DSG2 autoantibodies prevents binding of the autoantibodies to endogenous DSG2 in the subject. In this aspect of the disclosure, the DSG2 fusion polypeptide functions as a decoy protein or ligand trap.

[0030] Chatterjee et al. propose that DSG2 protein may contain epitopes that are exposed or released into the extracellular space and / or circulation as a result of cardiomyocyte injury or desmosome mutation. Exposure of these epitopes may occur due to any cardiac injury (such as, but not limited to, infectious myocarditis and / or cardiac trauma). In some embodiments, the compositions of the present disclosure may not contain any mutations. Such released DSG2 protein may bind to antigen-presenting cells to stimulate T cell responses and generate the observed autoantibodies. Exposure of cryptic epitopes due to genetic mutations may contribute to other forms of autoimmunity. In some embodiments, the DSG2 fusion polypeptides of the present disclosure may contain epitopes that include one or more mutations in DSG2.

[0031] A DSG2 fusion polypeptide may be a soluble and / or recombinant polypeptide. The arrangement of the components of a DSG2 fusion polypeptide may be optimized to achieve proper protein expression and / or the intended therapeutic effect. In some embodiments, a DSG2 fusion polypeptide may comprise a format described herein. The formats provided herein include components from N-terminus to C-terminus separated by a ";" between the components. Non-limiting examples of formats for DSG2 fusion polypeptides include: (i) all or a portion of a DSG2 protein; an Fc region; (ii) an Fc region; all or a portion of a DSG2 protein; (iii) all or a portion of a DSG2 protein; a linker; an Fc region; (iv) an Fc region; a linker; all or a portion of a DSG2 protein; (v) an affinity tag; all or a portion of a DSG2 protein; (vi) all or a portion of a DSG2 protein; an affinity tag; (vii) all or a portion of a DSG2 protein; a linker; an affinity tag; (viii) an affinity tag; a linker; all or a portion of a DSG2 protein.

[0032] DSG2 protein In some embodiments, the DSG2 fusion polypeptide of the present disclosure may comprise the entire DSG2 protein. Desmosomal cadherin desmoglein 2 (DSG2) is a transmembrane cell adhesion protein expressed in epithelial and non-epithelial tissues, such as the heart and the gastrointestinal tract. DSG2 is an integral part of the desmosomal unit, a major structure supporting cell-cell adhesion and structural integrity. DSG2 has been shown to regulate numerous cellular processes, including proliferation and apoptosis. In some embodiments, the DSG2 protein is the human DSG2 protein (UniProt ID: Q14126; ENSEMBL ID: ENSP00000261590.8), which consists of 1,118 amino acids and comprises the amino acid sequence of SEQ ID NO: 1. In one embodiment, the DSG2 protein may be encoded by the nucleic acid sequence of SEQ ID NO: 2 (NCBI Reference Sequence: NM_001943.5.; ENSMBL ID: ENST00000261590.13).

[0033] In some embodiments, a DSG2 fusion polypeptide of the disclosure can be a fully processed DSG2 protein comprising amino acids 50-1118 of SEQ ID NO:1. The DSG2 protein may also comprise one or more mutations with respect to the sequence of SEQ ID NO:1. In some embodiments, the mutation in the DSG2 protein may be a mutation associated with a disease state. In one embodiment, the disease state may be arrhythmogenic right ventricular dysplasia / cardiomyopathy. In some embodiments, the DSG2 fusion polypeptide of the present disclosure may comprise an epitope comprising one or more mutations in DSG2. As a non-limiting example, the DSG2 fusion polypeptide may comprise one or more mutations in the region of amino acids 485-531 and / or amino acids 586-610 of SEQ ID NO:1.

[0034] DSG2 belongs to the cadherin superfamily of cell adhesion proteins and shares three distinct regions in common: an extracellular region, a transmembrane domain, and an intracellular signaling region. In some embodiments, the extracellular region of DSG2 can have 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 proteins contains variable numbers of repeats of calcium-binding motifs known as cadherin motifs or EC domains. DSG2 contains four EC domains, referred to herein as EC1, EC2, EC3, and EC4. DSG2 also contains a membrane-proximal extracellular anchor (EA) domain. In some embodiments, a DSG2 fusion polypeptide of the present disclosure can include the entire extracellular region of DSG2. In some aspects, a DSG2 fusion polypeptide can include at least one domain, such as, but not limited to, EC1, EC2, EC3, EC4, and / or EA. In some embodiments, the EC1 domain can be amino acids 50-155 of SEQ ID NO: 1. In some embodiments, the EC2 domain can be amino acids 151-268 of SEQ ID NO: 1. In some embodiments, the EC3 domain can be amino acids 264-384 of SEQ ID NO:1. In some embodiments, the EC4 domain can be amino acids 382-495 of SEQ ID NO:1. In some embodiments, the EA domain can be amino acids 491-609 of SEQ ID NO:1. Table 1 shows the amino acid sequence of the DSG2 protein and the amino acid sequence of the extracellular region of DSG2. In some embodiments, the DSG2 protein of the disclosure can 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.

[0035] In some embodiments, the DSG2 protein is a non-human DSG2 protein. In some embodiments, the DSG2 protein is a Mus musculus (mouse) DSG2 protein. In one embodiment, the DSG2 protein may comprise 1,122 amino acids and may comprise the amino acid sequence of SEQ ID NO: 14. In some embodiments, the DSG2 protein is a Rattus norvegicus (rat) DSG2 protein. In one embodiment, the rat DSG2 protein may comprise 1,128 amino acids and may comprise the amino acid sequence of SEQ ID NO: 15. In some embodiments, the DSG2 protein is a non-human primate (NHP) DSG2 protein. In some aspects, the NHP DSG2 protein is a Macaca mulatta (rhesus monkey) DSG2 protein, which may comprise 1,115 amino acids and may comprise the amino acid sequence of SEQ ID NO: 16. In some embodiments, the DSG2 protein is a Canis lupus familiaris (dog) DSG2 protein. In some embodiments, the dog DSG2 protein may comprise 1,119 amino acids and may comprise the amino acid sequence of SEQ ID NO: 17. In some embodiments, the DSG2 protein is a Danio rerio (zebrafish) DSG2 protein. In some embodiments, the zebrafish DSG2 protein can include 1,142 amino acids and can include the amino acid sequence of SEQ ID NO:18.

[0036] [Table 1-1]

[0037] [Table 1-2]

[0038] [Table 1-3]

[0039] [Table 1-4]

[0040] The DSG2 fusion polypeptide of the present disclosure may comprise one or more domains of the extracellular region of DSG2. The domain of the extracellular region of DSG2 may comprise one or more repeats of the EC domain or the EA domain, in tandem or in a mixed order. For example, the DSG2 fusion polypeptide may comprise two, three or more repeats of the EC1, EC2, EC3, EC4 or EA domain. When two or more domains and / or two or more repeats of a domain of the extracellular region of DSG2 are present, these domains may be operably linked via a linker as described herein.

[0041] In some embodiments, a DSG2 fusion polypeptide may comprise two domains of the extracellular region of DSG2. Non-limiting examples of domains of the extracellular region of DSG2 present in a fusion polypeptide 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.

[0042] In some embodiments, a DSG2 fusion polypeptide may comprise three domains of the extracellular region of DSG2. Non-limiting examples of domains of the 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, EC3EC1EC4, EC3EC1EA, EC3EC2EC 1, EC3EC2EC4, EC3EC2EA, EC3EC4EC1, EC3EC4EC2, EC3EC4EA, EC3EAEC1, EC3EAEC2, EC3EAEC4, EC4EC1EC2, EC4EC1EC3, EC4EC1EA, EC4EC2EC1, EC4EC2EC3, EC4EC2EA, EC4EC3EC1, EC4EC3EC2, EC4EC3EA, EC4EAEC1, EC4EAEC2, EC4EAEC3, EAEC1EC2, EAEC1EC3, EAEC1EC4, EAEC2EC1, EAEC2EC3, EAEC2EC4, EAEC3EC1, EAEC3EC2, EAEC3EC4, EAEC4EC1, EAEC4EC2, and / or EAEC4EC3.

[0043] In some embodiments, a DSG2 fusion polypeptide may comprise four domains of the extracellular region of DSG2. Non-limiting examples of domains of the extracellular region of DSG2 present in a fusion polypeptide of the present disclosure include EC1EC2EC3EC4, EC1EC2EC3EA, EC1EC2EC4EC3, EC1EC2EC4EA, EC1EC2EAEC3, EC1EC2EAEC4, EC1EC3EC2EC4, EC1EC3EC2EA, EC1EC3EC4EC2, EC1EC3EC4EA, EC1EC3EAEC2, EC1EC3EAEC4, EC1EC4EC2EC3, EC1EC4EC2EA, EC1EC4EC3EC2, EC1EC4E C3EA, EC1EC4EAEC2, EC1EC4EAEC3, EC1EAEC2EC3, EC1EAEC2EC4, EC1EAEC3EC2, EC1EAEC3EC4, EC1EAEC4EC2, EC1EAEC4EC3, EC2EC1EC3EC4, EC2EC 1EC3EA, EC2EC1EC4EC3, EC2EC1EC4EA, EC2EC1EAEC3, EC2EC1EAEC4, EC2EC3EC1EC4, EC2EC3EC1EA, EC2EC3EC4EC1, EC2EC3EC4EA, EC2EC3EAEC1, E C2EC3EAEC4, EC2EC4EC1EC3, EC2EC4EC1EA, EC2EC4EC3EC1, EC2EC4EC3EA, EC2EC4EAEC1, EC2EC4EAEC3, EC2EAEC1EC3, EC2EAEC1EC4, EC2EAEC3EC 1, EC2EAEC3EC4, EC2EAEC4EC1, EC2EAEC4EC3, EC3EC1EC2EC4, EC3EC1EC2EA, EC3EC1EC4EC2, EC3EC1EC4EA, EC3EC1EAEC2, EC3EC1EAEC4, EC3EC2E C1EC4, EC3EC2EC1EA, EC3EC2EC4EC1, EC3EC2EC4EA, EC3EC2EAEC1, EC3EC2EAEC4, EC3EC4EC1EC2, EC3EC4EC1EA, EC3EC4EC2EC1, EC3EC4EC2EA, EC 3EC4EAEC1, EC3EC4EAEC2, EC3EAEC1EC2, EC3EAEC1EC4, EC3EAEC2EC1, EC3EAEC2EC4, EC3EAEC4EC1, EC3EAEC4EC2, EC4EC1EC2EC3, EC4EC1EC2EA,EC4EC1EC3EC2, EC4EC1EC3EA, EC4EC1EAEC2, EC4EC1EAEC3, EC4EC2EC1EC3, EC4EC2EC1EA, EC4EC2EC3EC1, EC4EC2EC3EA, EC4EC2EAEC1, EC4EC2EAEC3, EC4EC3EC1EC2, EC4EC3 EC1EA, EC4EC3EC2EC1, EC4EC3EC2EA, EC4EC3EAEC1, EC4EC3EAEC2, EC4EAEC1EC2, EC4EAEC1EC3, EC4EAEC2EC1, EC4EAEC2EC3, EC4EAEC3EC1, EC4EAEC3EC2, EAEC1EC2EC3, EAE C1EC2EC4, EAEC1EC3EC2, EAEC1EC3EC4, EAEC1EC4EC2, EAEC1EC4EC3, EAEC2EC1EC3, EAEC2EC1EC4, EAEC2EC3EC1, EAEC2EC3EC4, EAEC2EC4EC1, EAEC2EC4EC3, EAEC3EC1EC2, EAEC3EC1EC4, EAEC3EC2EC1, EAEC3EC2EC4, EAEC3EC4EC1, EAEC3EC4EC2, EAEC4EC1EC2, EAEC4EC1EC3, EAEC4EC2EC1, EAEC4EC2EC3, EAEC4EC3EC1, and / or EAEC4EC3EC2.

[0044] In some embodiments, a DSG2 fusion polypeptide may comprise five domains of the extracellular region of DSG2. Non-limiting examples of domains of the extracellular region of DSG2 present in a fusion polypeptide of the present disclosure include: EC1EC2EC3EC4EA, EC1EC2EC3EAEC4, EC1EC2EC4EC3EA, EC1EC2EC4EAEC3, EC1EC2EAEC3EC4, EC1EC2EAEC4EC3, EC1EC3EC2EC4EA, EC1EC3EC2EAEC4, EC1EC3EC4EC2EA, EC1EC3EC4EAEC2, EC1EC3EAEC2EC4, EC1EC3EAEC4EC2, EC1EC4EC2EC3 EA, EC1EC4EC2EAEC3, EC1EC4EC3EC2EA, EC1EC4EC3EAEC2, EC1EC4EAEC2EC3, EC1EC4EAEC3EC2, EC1EAEC2EC3EC4, EC1EAEC2EC4EC3, EC1EAEC3EC 2EC4, EC1EAEC3EC4EC2, EC1EAEC4EC2EC3, EC1EAEC4EC3EC2, EC2EC1EC3EC4EA, EC2EC1EC3EAEC4, EC2EC1EC4EC3EA, EC2EC1EC4EAEC3, EC2EC1EA EC3EC4, EC2EC1EAEC4EC3, EC2EC3EC1EC4EA, EC2EC3EC1EAEC4, EC2EC3EC4EC1EA, EC2EC3EC4EAEC1, EC2EC3EAEC1EC4, EC2EC3EAEC4EC1, EC2EC4 EC1EC3EA, EC2EC4EC1EAEC3, EC2EC4EC3EC1EA, EC2EC4EC3EAEC1, EC2EC4EAEC1EC3, EC2EC4EAEC3EC1, EC2EAEC1EC3EC4, EC2EAEC1EC4EC3, EC2E AEC3EC1EC4, EC2EAEC3EC4EC1, EC2EAEC4EC1EC3, EC2EAEC4EC3EC1, EC3EC1EC2EC4EA, EC3EC1EC2EAEC4, EC3EC1EC4EC2EA, EC3EC1EC4EAEC2, EC 3EC1EAEC2EC4, EC3EC1EAEC4EC2, EC3EC2EC1EC4EA, EC3EC2EC1EAEC4, EC3EC2EC4EC1EA, EC3EC2EC4EAEC1, EC3EC2EAEC1EC4, EC3EC2EAEC4EC1,EC3EC4EC1EC2EA, EC3EC4EC1EAEC2, EC3EC4EC2EC1EA, EC3EC4EC2EAEC1, EC3EC4EAEC1EC2, EC3EC4EAEC2EC1, EC3EAEC1EC2EC4, EC3EAEC1EC4EC2, EC3EAEC2EC1EC4, EC3EAEC2EC4EC1, EC3EAEC4EC1EC2, EC3EAEC4EC2EC1, EC4EC1EC2EC3EA, EC4EC1EC2EAEC3, EC4EC1EC3EC2EA, EC4EC1EC3EAEC2, EC4EC1EAEC2EC3, EC4EC1EAEC3EC2, EC4EC2EC1EC3EA, EC4EC2EC1EAEC3, EC4EC2EC3EC1EA, EC4EC2EC3EAEC1, EC4EC2EAEC1EC3, EC4EC2EAEC3EC1, EC4EC3EC1EC2EA, EC4EC3EC1EAEC2, EC4EC3EC2EC1EA, EC4EC3EC2EAEC1, EC4EC3EAEC1EC2, EC4EC3EAEC2EC1, EC4EAEC1EC2EC3, EC4EAEC1EC3EC2, EC4EAEC2EC1EC3, EC4EAEC2EC3EC1, EC4EAEC3EC1EC2, EC4EAEC3EC2EC1, EAEC1EC2EC3EC4, EAEC1EC2EC4EC3, EAEC1EC3EC2EC4, EAEC1EC3EC4EC2, EAEC1EC4EC2EC3, EAEC1EC4EC3EC2, EAEC2EC1EC3EC4, EAEC2EC1EC4EC3, EAEC2EC3EC1EC4, EAEC2EC3EC4EC1, EAEC2EC4EC1EC3, EAEC2EC4EC3EC1, EAEC3EC1EC2EC4, EAEC3EC1EC4EC2, EAEC3EC2EC1EC4, EAEC3EC2EC4EC1, EAEC3EC4EC1EC2, EAEC3EC4EC2EC1, EAEC4EC1EC2EC3, EAEC4EC1EC3EC2, EAEC4EC2EC1EC3, EAEC4EC2EC3EC1, EAEC4EC3EC1EC2, and / or EAEC4EC3EC2EC1 are included.

[0045] Non-limiting examples of portions of the DSG2 protein and configurations that may be present in a DSG2 fusion polypeptide are shown in Table 2. Any of the DSG2 domains listed in Table 2 can be operably linked to another domain or to another DSG2 domain in a fusion polypeptide using any of the linkers provided herein. The compositions of the disclosure can include portions or fragments of any of the domains listed in Table 2. The domain or combination of domains of SEQ ID NO: 1 or SEQ ID NO: 3 included in the polypeptide 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 50 amino acids upstream or downstream of the domains defined in Table 2. In some embodiments, a domain or combination of domains of SEQ ID NO:1 or SEQ ID NO:3 included in a polypeptide of the present disclosure may be truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 amino acids at the N-terminus or C-terminus of the domain as defined in Table 2. As a non-limiting example, the extracellular region of the DSG2 protein may extend from amino acids spanning amino acids 50 to 609 of SEQ ID NO:1.

[0046] [Table 2]

[0047] Immunoglobulin Proteins In some embodiments, the DSG2 fusion polypeptides of the present disclosure may comprise all or a portion of an immunoglobulin protein. The immunoglobulin protein may be IgG, IgM, IgA, IgD, or IgE. In one embodiment, the immunoglobulin protein may be IgG. Non-limiting examples of IgG include IgG1, IgG2, IgG3, and / or IgG4. The DSG2 fusion polypeptides may comprise a region or portion of an immunoglobulin. Non-limiting examples of immunoglobulin regions include an Fc region, a 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.

[0048] The DSG2 fusion polypeptide may include one or more Fc regions of an immunoglobulin. In some embodiments, the Fc region may include the first constant region immunoglobulin domain (e.g., CH1) or a portion thereof, and in some cases may also include a portion of the hinge. In other aspects, the Fc region does not include the first constant region immunoglobulin domain. Thus, 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 at the N-terminus of these domains. For IgA and IgM, the Fc region may include the J chain. For IgG, the Fc region includes immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). In some embodiments, the Fc region refers to the truncated CH1 domain of an immunoglobulin, as well as CH2 and CH3. Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is usually defined to include residues E216, C226 or P230 at the carboxyl-terminus, with numbering according to the EU index, as well as the Kabat antibody numbering sequence.

[0049] In some embodiments, the DSG2 fusion polypeptide may comprise a human immunoglobulin protein. In some embodiments, the DSG2 fusion polypeptide may comprise a non-human immunoglobulin protein. The non-human immunoglobulin protein may be a dog, rat, mouse, or primate immunoglobulin protein. Non-limiting examples of immunoglobulin subsequences are shown in Table 3. In some embodiments, the DSG2 fusion polypeptide may comprise 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 a fragment of a sequence in Table 3.

[0050] [Table 3-1]

[0051] [Table 3-2]

[0052] [Table 3-3]

[0053] In some embodiments, a DSG2 fusion polypeptide may comprise amino acids 100-330 of the heavy chain constant region of IgG1 (GenBank Accession No. P01857.1, SEQ ID NO: 4). In some embodiments, a DSG2 fusion polypeptide may comprise amino acids 104-330 of the heavy chain constant region of IgG1 (SEQ ID NO: 4), provided herein as SEQ ID NO: 5, which represents the Fc region of the IgG1 heavy chain constant region. In some embodiments, a DSG2 fusion polypeptide may comprise a variant of SEQ ID NO: 5 (designated P329G LALA) (using the Eu amino acid numbering nomenclature). This variant comprises L234A / L235A / P329G mutations in the IgG1 Fc region, provided herein as SEQ ID NO: 31. The "P329G LALA variant" has been shown to reduce the interaction of immunoglobulins with FcγR and Clq, thus minimizing the immune response caused by expression of the DSG2 fusion polypeptide in vivo (Schlothauer, et al. Protein Eng. Des. Sel. 2016, 29(10), 457-466, the entire text of which is incorporated herein by reference). In some alternative embodiments, the IgG1 protein does not include the L234A, L235A, and P329G mutations.

[0054] In some embodiments, a DSG2 fusion polypeptide can comprise amino acids 99-327 of IgG4 (GenBank Accession No. P01861.1, SEQ ID NO: 10), which is provided herein as SEQ ID NO: 11 and represents the Fc region of the IgG4 heavy chain constant region. In some embodiments, a DSG2 fusion polypeptide can comprise a variant of SEQ ID NO: 11 that contains three mutations designated S228P, L235E, and P329G (using Eu amino acid numbering nomenclature). The sequence of the variant containing these three mutations is provided herein as SEQ ID NO: 32.

[0055] The human IgG4 S228P / L235E / P329G variant of IgG4 (also referred to herein as "SPLE P329G") is a variant of IgG4 that previously demonstrated minimal FcγR binding activity (see Newman et al. 2001, Clin. Immunol., 98, 164-174; the contents of which are incorporated herein by reference in their entirety). In some embodiments, the IgG4 protein does not comprise the above mutations.

[0056] Signal sequence Signal sequences (sometimes called signal peptides, targeting signals, targeting peptides, localization sequences, transit peptides, leader sequences, or leader peptides) direct proteins (e.g., polypeptides of the present disclosure) to designated intracellular and / or extracellular locations. Signal sequences can be short (5-50 amino acids long) peptides present at the N-terminus of most newly synthesized proteins that are destined for a specific location. Signal sequences are recognized by the signal recognition particle (SRP) and cleaved using type I and type II signal peptide peptidases. Incorporation of signal sequences from human proteins as DSG2 fusion polypeptides of the present disclosure can direct polypeptides of the present disclosure to specific intracellular and / or extracellular locations. These signal sequences have been experimentally verified and are cleavable (Zhang et al., Protein Sci. 2004, 13:2819-2824).

[0057] In some embodiments, a signal sequence may be located at the N-terminus or C-terminus of a polypeptide of the present disclosure and may, but need not necessarily, be cleaved from the polypeptide to generate a "mature" polypeptide, as described herein.

[0058] In some examples, the signal sequence can be a secretory signal sequence derived from a naturally secreted protein, and variants thereof. In some cases, a signal sequence can be used to direct the polypeptide of the present disclosure to the surface membrane of target cells.Expressing the polypeptide of the present disclosure on the surface of target cells can be useful for limiting the diffusion of the polypeptide of the present disclosure to non-target in vivo environments, which can improve the safety profile of the polypeptide of the present disclosure.In addition, the membrane presentation of the polypeptide of the present disclosure can enable physiological and qualitative signaling, as well as stabilization and recycling of the polypeptide, which can increase its half-life.

[0059] The signal sequence may be a heterologous signal sequence from another organism, such as a virus, yeast, or bacteria, which can direct a polypeptide of the disclosure to a specific intracellular location, such as the nucleus (e.g., EP1209450). Other examples include the aspartic protease (NSP24) signal sequence from Trichoderma (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), the E. coli enterotoxin II signal peptide (e.g., U.S. Pat. No. 6,605,697 to Kwon et al.), the E. coli secretion signal sequence (e.g., U.S. Patent Application Publication No. 2016 / 090404 to Malley et al.), the lipase signal sequence from methylotrophic yeast (e.g., U.S. Pat. No. 8,975,041), and the signal peptide of DNase from Corynebacterium bacteria (e.g., U.S. Pat. No. 4,965,197), the contents of each of which are incorporated herein by reference in their entirety.

[0060] In some embodiments, the signal peptide can be an IgG1 signal peptide or an IgG2 signal peptide. In some embodiments, the signal peptide can be a mouse IgGκ kappa light chain signal peptide having the amino acid sequence METDTLLLWVLLLWVPGSTG (SEQ ID NO: 29). This signal peptide is one of the best characterized signal peptides used to improve transgene expression (Fonseca et al., Vaccine, 2018, 36(20):2799-2808, incorporated herein by reference in its entirety). Exemplary embodiments of the fusion proteins described below (except DSG2FP#1) contained the signal peptide of SEQ ID NO: 29 upon expression. The signal peptide was then cleaved during protein production from each fusion protein before testing for inhibition of anti-DSG2 antibodies.

[0061] Linker In some embodiments, the DSG2 fusion polypeptide of the present disclosure may include at least one linker. The linker may be disposed between one or more regions of the polypeptide of the present disclosure. In one embodiment, the linker may be disposed between all or a portion of the DSG2 protein and all or a portion of the immunoglobulin protein. In one aspect, the linker may be disposed between one or more domains of the DSG2 protein.

[0062] In some embodiments, the linker can be a polypeptide. In some embodiments, the linker can include a combination of amino acid residues. In some embodiments, the linker can include about 1-50 amino acid residues. In some embodiments, the linker can include about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues.

[0063] The linker of the present disclosure is about 1-100 amino acids long and may link any domain / region of the effector module (also known as a peptide linker). The linker may be 1-40 amino acids long, or 2-30 amino acids long, or 20-80 amino acids long, or 50-100 amino acids long. The length of the linker may be optimized depending on the type of polypeptide configuration and based on the crystal structure of the polypeptide. In some cases, it may be preferable to select a shorter linker length. In some embodiments, the peptide linker may be composed of amino acids linked by peptide bonds, preferably 1 to 20 amino acids linked by peptide bonds, 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), aspartic acid (D), glutamic acid (E), asparagine (N), and glutamine (Q). One or more of these amino acids may be glycosylated, as will be appreciated by those of skill in the art. In some aspects, the amino acids of the peptide linker may be selected from alanine (A), glycine (G), proline (P), asparagine (R), serine (S), glutamine (Q), and lysine (K).

[0064] In some embodiments, the linker can be a flexible linker or a rigid linker. Flexible linkers can be composed of small non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. These amino acids are flexible due to their small size, allowing movement of the functional domains to which they are attached. The most commonly used flexible linkers have a sequence consisting of a stretch of mainly glycine and serine residues ("GS" linker). An example of the most widely used flexible linker has the sequence (Gly-Gly-Gly-Gly-Ser)n. By adjusting the copy number "n", the length of this GS linker can be optimized to achieve proper separation of the functional domains or to maintain the necessary inter-domain interactions. In some embodiments, the linker can include additional amino acids such as Thr or Ala to maintain flexibility, as well as polar amino acids such as Lys or Glu to improve solubility. In some embodiments, the DSG2 fusion polypeptide can include a flexible linker such as (Gly)8, which is composed purely of glycine residues. The linker sequence avoided large hydrophobic residues to maintain good solubility in aqueous solution.

[0065] In some embodiments, the linker may be a rigid linker. Non-limiting examples of rigid linkers include linkers having a sequence of (EAAAK)n (n=1-5). In some embodiments, the rigid linker may have a proline-rich sequence (XP)n, where X represents any amino acid, preferably Ala, Lys, or Glu.

[0066] In some embodiments, the linker can be GGGGGS (SEQ ID NO: 12) or EAAAK (SEQ ID NO: 13). In some embodiments, the linker can be GGGGS (SEQ ID NO: 27). In some embodiments, the linker can be IEGRMD (SEQ ID NO: 28).

[0067] DSG2 fusion polypeptides Particular embodiments of fusion polypeptides were investigated in the examples described below. In the following description, the components are represented in N-terminal to C-terminal format, and the components are separated by semicolons. One of the DSG2 fusion polypeptides investigated was obtained from R&D Systems (rndsystems.com) and is named "Recombinant Human Desmoglein 2 Fc Chimeric Protein, CF". This fusion polypeptide is a research tool developed to adhere fibroblasts to plate wells, and has not been studied in the context of inhibiting anti-DSG2 antibodies for therapeutic purposes. The structure of this fusion polypeptide, interchangeably named herein as "tool decoy" and "DSG2FP#1", includes an N-terminus including residues Ala49 to Gly608 of GenBank Accession No. CAA81226, which represents the entire sequence of the extracellular domain of DSG2 (included herein as SEQ ID NO: 30); a linker with the sequence IEGRMD (SEQ ID NO: 28); and residues Pro100 to Lys330 of human IgG1 (SEQ ID NO: 4). In some exemplary embodiments of Table 4, the DSG2 extracellular domain comprises amino acids 50-609 of the human DSG2 sequence (SEQ ID NO: 1). In some exemplary embodiments of Table 4, the Fc region of the immunoglobulin is an IgG1 Fc domain or an IgG4 Fc domain. In some exemplary embodiments of Table 4, the IgG1 sequence comprises amino acids 100-330 or 104-330 of SEQ ID NO: 4. In some exemplary embodiments of Table 4, the IgG4 sequence comprises amino acids 99-327 of SEQ ID NO: 10. In some exemplary embodiments of Table 4, the IgG1 sequence is SEQ ID NO: 31, which may be referred to herein as the "P329G LALA variant". In some embodiments, the immunoglobulin protein may be the human IgG4 S228P / L235E / P3229G variant of IgG4 (SEQ ID NO: 32). For greater clarity, the names of the mutations of the variants described above refer to the Eu antibody numbering system and not to the positions within the sequences identified in this paragraph. In some embodiments, a DSG2 fusion polypeptide may be synthesized as a disulfide-bonded dimer.

[0068] In some embodiments, the DSG2 fusion polypeptide may include at least one linker. The linker may be present between any two components of the fusion protein.

[0069] Table 4 provides examples of DSG2 fusion polypeptides.

[0070] [Table 4]

[0071] Treatment In some embodiments, the present disclosure provides a therapeutic agent that targets a condition, disease, disorder, or symptom associated with serum anti-DSG2 antibodies. In some embodiments, the therapeutic agent may include an immunoglobulin protein. In some embodiments, the therapeutic agent may be an anti-CD20 antibody, such as, but not limited to, rituximab. In some embodiments, the therapeutic agent described herein may result in inhibition of autoantibody function by targeting either the Fab or Fc fragment. In some embodiments, the therapeutic agent may be intravenously administered immunoglobulin (IVIG). Administration of excess IgG may saturate FcRn, resulting in more rapid clearance of all IgG molecules (including autoantibodies). In some embodiments, the therapeutic agent may be an FcRn-inhibiting monoclonal antibody. In one embodiment, the FcRn-inhibitory monoclonal antibody can be SYNT001 (Blumberg LJ, et al. Sci Adv. 2019 Dec. 18;5(12):eaax9586.doi:10.1126 / sciadv.aax9586; the contents of which are incorporated herein by reference in their entirety). In some embodiments, the therapeutic agent can be an anti-C5 antibody, such as eculizumab. Activation of the complement system in autoimmune diseases can lead to binding of immune cells to their immune complexes, and the subsequent intracellular signaling events are important for pathogenesis.

[0072] The therapeutic agent of the present disclosure may be used alone or in combination with the DSG2 fusion polypeptide described herein.When used in combination with DSG2 fusion polypeptide, the therapeutic agent can be administered before, at the same time, or after the subject is provided with DSG2 fusion polypeptide.In one embodiment, the therapeutic agent is a CAAR for treating the condition associated with anti-DSG2 antibody that is not ARVC.

[0073] In one embodiment, the compositions of the present disclosure include a combination of therapeutic agents, such as CAAR and DSG2 fusion polypeptides, and can be used to treat diseases, such as, but not limited to, ARVC, COVID-19, post-COVID-19 syndrome, sarcoidosis, dilated cardiomyopathy, or any disease associated with anti-DSG2 antibodies.

[0074] Polynucleotides In some embodiments, the polypeptides of the present disclosure are encoded by a polynucleotide or variants thereof described herein. Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with a β-D-ribo configuration, α-LNA (a diastereomer of LNA) with an aL-ribo configuration, 2′-amino-LNA with a 2′-amino functional group, and 2′-amino-a-LNA with a 2′-amino functional group), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or hybrids or combinations thereof.

[0075] Thus, disclosed herein are polynucleotides encoding peptides or polypeptides that contain substitutions, insertions and / or additions, deletions, and covalent modifications relative to a reference sequence, particularly a polypeptide sequence. For example, sequence tags or amino acids, such as one or more lysines, can be added to the peptide sequences described herein (e.g., at the N-terminus or C-terminus). Sequence tags can be used for peptide purification or localization. Lysines can be used to increase the solubility of the peptide or to allow biotinylation. Alternatively, amino acid residues located at the carboxy- and amino-terminal regions of the amino acid sequence of a peptide or protein can be optionally deleted to provide a truncated sequence. Depending on the use of the sequence, specific amino acids (e.g., C- or N-terminal residues) can instead be deleted, for example, to express the sequence as part of a larger sequence that is soluble or bound to a solid support.

[0076] Once any of the features have been identified or defined as desired components of the polypeptide to be encoded by the polynucleotides described herein, any of the manipulations and / or alterations of these features can be performed by moving, swapping, inverting, deleting, randomizing, or duplicating these features. It is further understood that the manipulation of features can produce the same results as the modification of the molecules described herein. For example, manipulation to delete a domain will result in a change in the length of the molecule, as would modifying a nucleic acid to encode a molecule of less than full length.

[0077] III. Pharmaceutical Compositions and Delivery The fusion polypeptides described herein can be used as therapeutic agents. In some embodiments, the present disclosure provides pharmaceutical compositions comprising at least one pharma- ceutically acceptable carrier and a fusion polypeptide.

[0078] In some embodiments, the composition is administered to a human, human patient, or subject. The description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, but those skilled in the art will understand that such compositions are generally also suitable for administration to other animals, such as non-human animals, such as non-human mammals. Modifications to make pharmaceutical compositions suitable for administration to humans suitable for administration to a variety of animals are well understood, and an ordinary skilled veterinary pharmacologist can design and / or perform such modifications with only routine experimentation, if necessary. Subjects to which the pharmaceutical composition is contemplated include, but are not limited to, humans and / or other primates; mammals (including commercially relevant mammals, such as dogs, cows, pigs, horses, sheep, cats, mice, and / or rats); and / or birds (including commercially relevant birds, such as poultry, chickens, ducks, geese, and / or turkeys). As a non-limiting example, the composition of the present disclosure can be administered to dogs to treat ARVC.

[0079] Provided herein are fusion polypeptides and pharmaceutical compositions thereof that can be used in combination with one or more pharma- ceutically acceptable excipients. In some embodiments, the fusion polypeptides and pharmaceutical compositions of the present disclosure may be delivered via subcutaneous or intravenous routes.

[0080] In some embodiments, pharma- ceutically acceptable excipients include, but are not limited to, any solvent, dispersion medium, diluent, or other liquid vehicle, dispersion or suspension aid, surfactant, isotonicity agent, thickener or emulsifier, preservative, solid binder, lubricant, flavoring agent, stabilizer, antioxidant, osmolality adjuster, pH adjuster, etc., suitable for the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety). The use of conventional excipient vehicles may be considered within the scope of the present disclosure, unless the conventional excipient vehicle is incompatible with the substance or its derivatives, such as producing undesirable biological effects or interacting in a deleterious manner with other components of the pharmaceutical composition.

[0081] In some embodiments, a pharma- ceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient may be approved by the U.S. Food and Drug Administration. In some embodiments, the excipient may be pharmaceutical grade. In some embodiments, the excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0082] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surfactants and / or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants, and / or oils. Such excipients may be optionally included in the pharmaceutical composition. The composition may also include excipients such as cocoa butter or suppository wax, colorants, coating agents, sweeteners, flavoring agents, and / or perfuming agents.

[0083] 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, corn starch, powdered sugar, and the like, and / or combinations thereof.

[0084] Examples of granulating and / or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinylpyrrolidone (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and the like, and / or combinations thereof.

[0085] Examples of surfactants and / or emulsifiers include 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., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., For example, 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), glyceryl monooleate, sorbitan monooleate (SPAN® 80), polyoxyethylene esters (e.g. ... monolaurate (TWEEN® 60), polyoxyethylene sorbitan monooleate (TWEEN® 80), polyoxyethylene sorbitan monolaurate (TWEEN® 80), polyoxyethylene sorbitan monolaurate (TWEEN® 80), polyoxyethylene sorbitan monolaurate (TWEEN® 80), polyoxyethylene sorbitan monolaurate (TWEEN® 80), polyoxyethylene sorbitan monolaurate (TWEEN® 80), polyoxyethylene sorbitan monolaurate (TWEEN® 80), polyoxyethylene sorbitan monolaurate (TWEEN® 80), polyoxyethylene sorbitan monolaurate (TWEEN® 80), polyoxyethylene sorbitan monolaurate (TWEEN® 80), polyoxyethylene sorbit Polyoxyethylene monostearate (MYRJ® 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL™), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether (BRIJ® 30), poly(vinylpyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate,Examples of suitable oleic acid salts include, but are not limited to, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLUORINC™ F68, POLOXAMER™ 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or combinations thereof.

[0086] Exemplary binders include, but are not limited to, starches (such as, for example, corn starch and starch paste); gelatin; sugars (such as, for example, sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); amino acids (such as, for example, glycine); natural and synthetic gums (such as, for example, acacia, sodium alginate, Irish moss extract, panwar gum, ghatti gum, isapol shell mucilage, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinylpyrrolidone), magnesium aluminum silicate (VEEGUM®), and larch arabogalactan); alginates, polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohols; and the like; and combinations thereof.

[0087] Exemplary preservatives include, but are not limited to, antioxidants, chelating agents, antibacterial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Oxidation is a potential degradation pathway for mRNA, especially for liquid mRNA formulations. To prevent oxidation, antioxidants can be added to the formulation. Exemplary 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. Exemplary 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, butylparaben, methylparaben, ethylparaben, propylparaben, 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, hydroxybenzoic acid, and / or phenylethyl 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. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANTPLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN™, NEOLONE®, KATHON®, and / or EUXYL®.

[0088] In some embodiments, the pH of the pharmaceutical solution is maintained between pH 5 and pH 8 to improve stability. Exemplary buffers for controlling pH include, but are not limited to, sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium carbonate, and / or sodium malate. In another embodiment, the above exemplary buffers may be used with additional monovalent counterions (including, but not limited to, potassium). Divalent cations can also be used as counterions in buffers, but are not preferred due to complex formation and mRNA degradation.

[0089] Examples of buffers include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, 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, hydroxide calcium phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixture, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and the like, and / or combinations thereof.

[0090] Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and the like, and combinations thereof.

[0091] Exemplary oils include almond, apricot kernel, avocado, babassu, bergamot, blackcurrant seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, evening primrose, fish, linseed, geraniol, gourd, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea Exemplary oils include, but are not limited to, cubeba, macadamia 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, savory, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, camellia, vetiver, walnut, and wheat germ oil. Exemplary 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.

[0092] At the discretion of the formulator, excipients such as cocoa butter or a suppository wax, coloring agents, coating agents, sweetening, flavoring, and / or perfuming agents can also be present in the composition. Examples of additives include the addition of physiologically biocompatible buffers (e.g., trimethylamine hydrochloride), chelating agents (e.g., DTPA or DTPA bisamide, etc.) or calcium chelate complexes (e.g., calcium DTPA, CaNaDTPA-bisamide), or, optionally, calcium or sodium salts (e.g., calcium chloride, calcium ascorbate, calcium gluconate, or calcium lactate). Additionally, antioxidants and suspending agents may be used.

[0093] In some embodiments, compositions of the present disclosure may be administered by any route that produces a therapeutically effective result, including enteral (into the intestine), gastrointestinal, epidural (into the dura), oral (through the mouth), transdermal, peridural, intracerebral (into the brain), intraventricular (into the ventricles), epithelial (application to the skin), intradermal, (into the skin itself), subcutaneous (under the skin), intranasal 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 injection (into the bone marrow), intraspinal (into the spinal canal), intraperitoneal (injection or injection into the peritoneum), intravesical infusion, intravitreal (through the eye), intracavernous injection (into a pathological cavity). Intracavitary (to the base of the penis), intravaginal, intrauterine, extra-amniotic, transdermal (diffusion through intact skin for systemic distribution), transmucosal (diffusion through mucous membranes), vaginal, inhalation (inhaled through the nose), sublingual, sublabial, enema, ophthalmic (to the conjunctiva), ear, auricular (in or through the ear), buccal (towards the cheek), conjunctival, cutaneous, dental (to one or more teeth), electroosmosis, intracervical, intranasal, tracheal internal, extracorporeal, hemodialysis, infiltrating, interstitial, intra-abdominal, intra-amniotic, intra-articular, intra-bile duct, intra-bronchus, intra-synovial, intrachondral (inside cartilage), intra-cauda equina (inside the cauda equina), intra-cistern (inside the cisterna magna of the cerebellar medulla), intra-corneal (inside the cornea), intra-dental keratinum, intra-coronary (inside the coronary artery), intracavernosus (inside the expandable space of the corpus cavernosum of the penis), intra-discal (inside the intervertebral disc), intraductal (inside the duct of a gland), intraduodenal (inside the duodenum), intradural (in or under the dura), intraepidermal (to the epidermis), intraesophageal (into the esophagus), intragastric (in the stomach), intragingival (in the gums), intraileal (in the distal part of the small intestine), intralesional (into or directly introduced into a localized lesion), intraluminal (in the lumen), intralymphatic (in the lymphatic vessels), intramedullary (in the bone marrow cavity), intrameningeal (in the meninges), intraocular (in the eye), intraovarian (in the ovary), intrapericardial (in the pericardium), intrapleural (in the pleura), intraprostatic (in the prostate), intrapulmonary (in the lungs or their bronchi), intrasinus (in the nasal or periorbital sinuses), intraspinal (in the spinal column), intrasynovial (in the synovial cavities of a joint), intratendon (in tendons), intratesticular (in the testes), intrathecal (in the cerebrospinal fluid at any level of the neuraxis), intrathoracic (in the thorax), intracanal (in the canals of an organ), intratumoral (in a tumor), intratympanic (in the middle ear), intravascular (in blood vessels), intraventricular (in the ventricles of the heart),These include, but are not limited to, iontophoresis (using an electric current to infuse ions of soluble salts into body tissues), irrigation (cleansing or flushing an open wound or body cavity), laryngeal (directly into the larynx), nasogastric (from the nose to the stomach), occlusive dressing, ophthalmic (into the external eye), oropharyngeal (directly into the mouth and pharynx), parenteral, transdermal, periarticular, peridural, perineural, periodontal, rectal, respiratory (into the respiratory tract by oral or nasal inhalation for a local or systemic effect), retrobulbar (behind the pons or behind the eye), intramyocardial (entering the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, transplacental (across the placenta), transtracheal (through the wall of the trachea), transtympanic (across the tympanic cavity), ureteral, urethral, ​​vaginal, sacral block, diagnostic, nerve block, bile perfusion, cardiac perfusion, phototherapy, or spinal administration. In certain embodiments, the composition may be administered in a manner that allows it to cross the blood-brain barrier, the vascular barrier, or other epithelial barrier.

[0094] Therapeutically effective doses may be readily determined by those skilled 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. IV.How to use Methods of using the DSG2 fusion polypeptide compositions of the present disclosure are provided herein. In one embodiment, the compositions of the present disclosure can be used to treat diseases associated with DSG2 autoantibodies. In one embodiment, the compositions of the present disclosure reduce cardiotoxicity associated with DSG2 antibodies of any etiology. In some embodiments, the DSG2 fusion polypeptide compositions can be used to reduce anti-DSG2 antibodies in a subject. The subject may not have a condition, disease, or disorder that is known to generate anti-DSG2 antibodies. In some embodiments, the DSG2 fusion polypeptide compositions can be used to reduce serum DSG2 antibody levels. The fusion polypeptides of the present disclosure can reduce DSG2 antibody levels by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0095] In some embodiments, any disease associated with the arrhythmogenic and / or cardiomyopathic effects of anti-DSG2 antibodies in myocardium can be treated with the DSG2 fusion polypeptides described herein. Non-limiting examples of such indications include arrhythmogenic right ventricular cardiomyopathy (ARVC), sarcoidosis, dilated cardiomyopathy, post-infectious cardiomyopathy, reduced cardiac function, reduced ejection fraction, heart failure, arrhythmia, and myocarditis.

[0096] The efficacy of treatment or improvement of a disease can be assessed, for example, by measuring disease progression, disease remission, severity of symptoms, pain relief, quality of life, reduction in abnormal findings in cardiac tests, dosage of drug required to maintain therapeutic effect, levels of disease markers, or other measurable parameters appropriate for a given disease being treated or prevented. It is well within the capabilities of one of ordinary skill in the art to monitor the efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In the context of administration of a fusion polypeptide or pharmaceutical composition thereof, "effective against" a disease or disorder indicates that administration in a clinically relevant manner results in a beneficial effect in at least some patients, such as amelioration of symptoms, cure, reduction in disease burden, extension of life, improvement in quality of life, reduction in the need for blood transfusions, or other effects generally recognized as positive by physicians familiar with the treatment of a particular type of disease or disorder.

[0097] Therapeutic or prophylactic efficacy is evident when one or more parameters indicative of the disease state show substantial, often statistically significant, improvements, or when symptoms do not worsen or appear as would otherwise be expected. As an example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more, in a measurable parameter of the disease may be indicative of effective treatment. The efficacy of a given compound or composition may also be determined using an experimental animal model of a given disease known in the art. When an experimental animal model is used, the efficacy of the treatment is demonstrated when a statistically significant change in a marker or symptom is observed.

[0098] In some embodiments, the DSG2 fusion polypeptides of the present disclosure can be used to treat cardiac abnormalities in a subject. In some embodiments, the subject to be treated can have serum anti-DSG2 antibodies.

[0099] Cardiomyopathy and arrhythmias In some embodiments, the compositions of the present disclosure may be used to treat arrhythmias. Arrhythmia refers to an inappropriate or abnormal pattern of electrical activity in the heart. The compositions of the present disclosure may be used to treat one or more types of arrhythmias, such as those found in non-limiting examples of diseases such as arrhythmogenic right ventricular cardiomyopathy, sarcoidosis, acute sequelae of COVID-19, dilated cardiomyopathy, hypertrophic cardiomyopathy, and / or restrictive cardiomyopathy. In one embodiment, patients with cardiomyopathy may express serum DSG2 autoantibodies in their serum.

[0100] In some embodiments, the compositions of the present disclosure may be used to treat cardiomyopathy. Cardiomyopathy refers to the impairment of the structure and function of the muscular wall of the ventricle. The compositions of the present disclosure may be used to treat one or more types of cardiomyopathy, including but not limited to arrhythmogenic right ventricular cardiomyopathy, sarcoidosis, acute sequelae of COVID-19 (PASC), dilated cardiomyopathy, hypertrophic cardiomyopathy, and / or restrictive cardiomyopathy. In one embodiment, patients with cardiomyopathy may express serum DSG2 autoantibodies in their serum.

[0101] In one embodiment, the compositions of the present disclosure may be used to treat arrhythmogenic right ventricular cardiomyopathy (ARVC). Arrhythmogenic right ventricular cardiomyopathy / dysplasia (ARVC / ARVD) is a myocardial disorder associated with ventricular arrhythmias, heart failure, and sudden death. ARVC is a cardiac disease characterized by fulminant and recurrent arrhythmias, with the development of cardiomyopathy and associated fibrofatty replacement of the myocardium in the later stages. In addition to genetic mutations in structural and signaling proteins of cardiomyocyte desmosomes, the patient's immune system is also thought to be involved in ARVC. Mutations in the DSG2 protein have been associated with ARVC, and autoantibodies targeting DSG2 have been identified in patients with this disease. Although approximately 50% of ARVC patients do not have known desmosomal mutations, these patients may still develop DSG2 autoantibodies. In some embodiments, DSG2 fusion proteins can be used to treat ARVC patients with one or more mutations in the DSG2 protein. In some aspects, DSG2 fusion proteins can be used to treat ARVC patients with no known mutations in the DSG2 protein. In some embodiments, the DSG2 fusion polypeptides of the present disclosure can target DSG2 autoantibodies associated with ARVC.

[0102] Cardiac arrhythmias and / or cardiomyopathy may be clinical findings associated with acute inflammatory processes caused by infections such as Coxsackieviruses and coronaviruses, such inflammation of the myocardium is called myocarditis. In some embodiments, myocarditis may be caused by viruses, bacteria, parasites, and / or fungi. In some embodiments, the compositions of the present disclosure may be used to treat and / or prevent myocarditis associated with viruses. Non-limiting examples of viruses associated with myocarditis include adenoviruses causing the common cold, COVID-19; hepatitis B and hepatitis C; parvoviruses causing mild rashes (fifth disease) especially in children; and / or herpes simplex viruses, echoviruses causing gastrointestinal infections, Epstein-Barr virus causing mononucleosis, rubella, cytomegalovirus, and HIV.

[0103] In some embodiments, the compositions of the present disclosure may be used to treat and / or prevent myocarditis caused by bacteria. Non-limiting examples of bacteria associated with myocarditis include Staphylococcus, Streptococcus, and / or Borrelia. In some embodiments, the compositions of the present disclosure may be used to treat and / or prevent myocarditis caused by parasites. Non-limiting examples of parasites associated with myocarditis include Trypanosoma cruzi and Toxoplasma, some of which are transmitted by insects and cause a condition called Chagas disease. In some embodiments, the compositions of the present disclosure may be used to treat and / or prevent myocarditis caused by fungi. Non-limiting examples of fungi associated with myocarditis include Candida, Aspergillus, and other fungi such as Histoplasma.

[0104] Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) Arrhythmogenic right ventricular cardiomyopathy (ARVC), also known as arrhythmogenic right ventricular dysplasia / cardiomyopathy (ARVD / C), is a complex and devastating cardiac disease that is common in young people and athletes and shows diversity with regard to clinical features. Typical clinical manifestations of ARVC include palpitations, arrhythmic presyncope / syncope, and sudden cardiac death due to ventricular arrhythmias, suggesting a primary electrical involvement. However, ARVC patients can also show clinical manifestations associated with structural disease, typically in the later stages of the disease, such as myocardial remodeling consisting of myocardial thinning and dilation, and ventricular (right and / or left ventricular) dysfunction, and / or fibrofatty replacement of the myocardium, suggesting a primary structural involvement. The structural nature of the disease is further reinforced as ARVC. Although ARVC has been referred to as a "disease of the desmosomal" and human genetic studies have shown that approximately 40-50% of patients have mutations in genes that code for components of the desmosomal intercellular junctions (e.g., desmoplakin (DSP), plakoglobin (JUP), plakophilin 2 (PKP2), desmoglein 2 (DSG2)), genetic studies strongly suggest that abnormal genes alone are not sufficient to cause ARVC disease.

[0105] Currently, there are no effective treatments for ARVC, and no randomized trials of ARVC-specific therapies, screening regimens, or medications have been performed. As a result, treatment strategies for patients with ARVC are primarily directed at symptomatic palliation of electrophysiological effects and are based on clinical expertise, results of registry-based retrospective studies, and studies in model systems. As a result, existing treatments for patients with ARVC rely on the use of antiarrhythmic medications (sotalol, amniodarone, beta-blockers) and, if patients become resistant or intolerant to antiarrhythmic therapy, transition to more invasive options such as implantable cardioverter defibrillators or cardiac catheter ablation. However, current treatments have limited efficacy in managing the disease, and as many as 40% of patients with ARVC die within 10–11 years of initial diagnosis, highlighting the need for the development of more effective treatments for patients with ARVC.

[0106] COVID-19 The DSG2 fusion polypeptides or compositions comprising the fusion polypeptides described herein can be administered to treat COVID-19 or the long-term effects of COVID-19.

[0107] In some embodiments, the compositions of the present disclosure may be useful for treating individuals infected with COVID-19 and / or SARS-CoV-2. Infected individuals may be symptomatic, pre-symptomatic, and asymptomatic. According to the World Health Organization (WHO), COVID-19 transmission can occur from symptomatic, pre-symptomatic, and asymptomatic individuals infected with SARS-CoV-2. Symptomatic infection may refer to infection that occurs before a person experiences symptoms. Pre-symptomatic infection may refer to infection before COVID-19 symptoms appear.

[0108] COVID-19 may be accompanied by one or more symptoms, including 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, stuffy or runny nose, nausea or vomiting, diarrhea, trouble breathing, persistent pain or pressure in the chest, etc. In some embodiments, COVID-19 infection may be asymptomatic but still give rise to anti-DSG2 antibodies.

[0109] DSG2 fusion polypeptides may be used to treat one or more stages of COVID-19 disease. Typically, adults infected with SARS-CoV-2 are classified into the following categories depending on the severity of the disease: 1) SARS-CoV-2 infection; 2) SARS-CoV-2 infection; 3) SARS-CoV-2 infection; 4) SARS-CoV-2 infection; 5) SARS-CoV-2 infection; 6) SARS-CoV-2 infection; 7) SARS-CoV-2 infection; 8) SARS-CoV-2 infection; 9) SARS-CoV-2 infection; 10) SARS-CoV-2 infection; 11) SARS-CoV-2 infection; 12) SARS-CoV-2 infection; 13) SARS-CoV-2 infection; 14) SARS-CoV-2 infection; 15) SARS-CoV-2 infection; 16) SARS-CoV-2 infection; 17) SARS-CoV-2 infection; 18) SARS-CoV-2 infection; 19) SARS-CoV-2 infection; 19) SARS-CoV-2 infection; 20) SARS-CoV-2 infection; 21) SARS-CoV-2 infection; 22) SARS-CoV-2 infection; 23) SARS-CoV-2 infection; 24) SARS-CoV-2 infection; 25) SARS-CoV-2 infection; 26) SARS-CoV-2 infection; 27) SARS-CoV-2 infection; 28) SARS-CoV-2 infection; 29) SARS-CoV-2 infection; 30) SARS-CoV-2 infection; 31) SARS-CoV-2 infection; 32) SARS-CoV-2 infection; 33) SARS-CoV-2 infection; 34) SARS-CoV-2 infection; 35) SARS-CoV-2 infection; 36 In some embodiments, the compositions of the present disclosure may be for treating mild illness, including individuals with any of the various signs and symptoms of COVID-19 (e.g., fever, cough, sore throat, fatigue, headache, muscle pain, nausea, vomiting, diarrhea, loss of taste and smell), but no shortness of breath, difficulty breathing, or abnormal chest imaging. In some embodiments, the compositions of the present disclosure may be for treating moderate illness, which may include individuals who exhibit evidence of lower respiratory tract disease during clinical evaluation or imaging and have an oxygen saturation (SpO2) of 94% or greater on room air at sea level. In some embodiments, the compositions of the present disclosure may be for treating severe illness, including individuals with an SpO2 of less than 94% on room air at sea level, an arterial blood oxygen pressure to inspired oxygen (PaO2 / FiO2) ratio of less than 300 mmHg, a respiratory rate of more than 30 breaths per minute, or pulmonary infiltrates of more than 50%. In some embodiments, compositions of the present disclosure may be for treating critical illnesses, including individuals with respiratory failure, septic shock, and / or multiple organ failure.

[0110] Also provided herein are methods of preventing one or more symptoms associated with COVID-19. In one embodiment, since there is an incubation period between exposure to the virus and the onset of symptoms, the compositions of the present disclosure may be provided to a subject prior to the onset of symptoms but after exposure to the virus. The incubation period for the novel coronavirus SARS-CoV-2 is typically 2-14 days, with an average of 5 days (Lombardi et al., J. Hosp. Infect. 2020 doi:10.1016 / j.jhin.2020.03.003; the contents of which are incorporated herein by reference in their entirety).

[0111] The compositions of the present disclosure may also be administered in combination with one or more therapeutic agents recommended for use in the treatment of COVID-19. In some embodiments, the 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, azithromycin, lopinavir, ritonavir, ivermectin, interleukin inhibitors, interferons, kinase inhibitors, glucocorticosteroids, and / or SARS CoV-2 monoclonal antibodies (e.g., bamlanivimab, casirivimab, imdevimab).

[0112] Beginning in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic, infecting millions of people with coronavirus disease (referred to as COVID-19) (Wu et al., 2020 Nature 579, 265-269) and resulting in over one million deaths worldwide. Patients infected with SARS-CoV-2 can experience a range of clinical symptoms, from asymptomatic to severe illness. Emerging research suggests that in some cases, even those who have had mild symptoms may continue to have symptoms for a long time after initial recovery. This condition has been termed post-COVID-19 syndrome, or “long COVID-19” or “long COVID-19” or “post-acute sequelae of COVID-19 (PASC).” It is currently estimated that up to 28% of patients infected with COVID-19 continue to have palpitations 3 months after recovery from acute COVID-19 infection (Puntmann, et al., Nature Med. 2022, 28, 2117-2123, the entire text of which is incorporated herein by reference). In addition, even after the acute COVID-19 infection has subsided, patients may develop reduced ejection fraction and cardiomyopathy. Cardiac signs and symptoms after COVID-19 may coexist with effects on other organ systems or may appear alone. Patients with long COVID may present with arrhythmias alone, cardiomyopathy alone, or both. Patients with cardiomyopathy range from asymptomatic to those with fulminant heart failure, arrhythmias, and / or sudden cardiac death.

[0113] The COVID-19 virus, SARS-CoV-2, affects multiple organ systems, especially the lungs and heart. Elevations of cardiac biomarkers, especially high-sensitivity troponin and / or creatine kinase MB, are commonly observed in patients with COVID-19 infection. A review of clinical analyses conducted by Bavishi et al. found that myocardial injury occurred in 20% of patients with COVID-19 infection (Prog Cardiovasc Dis. 2020 September-October issue; 63(5):682-689). Possible mechanisms of myocardial injury associated with COVID-19 include, but are not limited to, 1) hyperinflammation and cytokine storm mediated via pathological T cells and monocytes resulting in myocarditis, 2) respiratory failure and hypoxemia leading to cardiomyocyte damage, 3) ACE2 expression in cardiomyocytes and subsequent downregulation of protective signaling pathways, 4) development of hypercoagulation and coronary microvascular thrombosis, 5) diffuse endothelial dysfunction, and / or 6) myocardial ischemia / infarction in post-COVID-19 syndrome due to inflammation and / or stress-induced coronary plaque rupture or supply-demand mismatch.

[0114] In some embodiments, the compositions of the present disclosure may be used to treat post-COVID-19 syndrome. There are increasing reports of patients with persistent symptoms after recovery from acute COVID-19, referred to herein as "post-COVID-19 syndrome," and individuals suffering from these symptoms are commonly referred to as "long haulers." In some embodiments, a patient may be considered to have post-COVID-19 syndrome if they have suffered from one or more symptoms for up to one month, up to two months, up to three months, up to four months, up to five months, up to six months, or for more than a year.

[0115] Post-COVID-19 syndrome is also associated with multiple organ damage, including cardiovascular damage. Imaging studies performed months after recovery from COVID-19 have shown long-term damage to the heart muscle, even in people who only experienced mild COVID-19 symptoms. Post-COVID-19 syndrome also appears to be associated with an increased risk of arrhythmias and / or myocarditis and / or cardiomyopathy.

[0116] Currently, there is a lack of therapeutic strategies to treat and / or manage COVID-19 and post-COVID-19 syndrome. Cardiac manifestations of COVID-19 place a tremendous burden on the healthcare system due to the extensive resources and potential intensive care support required for these patients. In particular, there is an urgent need to develop therapeutics that suppress the inflammatory response to reduce the incidence and mortality of myocardial injury associated with COVID-19 and post-COVID-19 syndrome. The present disclosure provides DSG2 fusion polypeptide-based compositions and methods for treating diseases, including but not limited to COVID-19 and / or post-COVID-19 syndrome.

[0117] The compositions of the present disclosure may be used to treat one or more symptoms related to the cardiovascular system in post-COVID-19 syndrome (i.e., post-COVID-19 cardiac syndrome). In one study of 100 patients who had recently recovered from COVID-19, cardiac magnetic resonance imaging revealed that 78 (78%) had cardiac involvement and 60 (60%) had ongoing myocarditis, regardless of pre-existing conditions, the severity and overall course of the acute illness, and the time since initial diagnosis (Puntmann et al. JAMA Cardiol. 2020;5(11):1265-1273). In a smaller study, 15% of athletes had evidence of myocardial abnormalities after recovery from acute COVID-19. In one embodiment, the DSG2 fusion polypeptide may be used to treat myocarditis in subjects with post-COVID-19 syndrome.

[0118] In some embodiments, the compositions described herein may be used for the treatment of post-COVID-19 syndrome not associated with any cardiac indication. In some embodiments, the compositions of the present disclosure may be used to treat subjects exhibiting symptoms of COVID-19 up to weeks, months, and / or years after an initial diagnosis of COVID-19. In some embodiments, patients with post-COVID-19 syndrome may exhibit 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, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years, or more after an initial diagnosis of COVID-19. A diagnosis of COVID-19 can be established using methods known in the art (e.g., reverse transcription polymerase chain reaction and / or antibody testing). In some embodiments, subjects suffering from post-COVID-19 syndrome may be treated with the compositions of the present disclosure.

[0119] In some embodiments, the compositions of the present disclosure may be used to treat COVID-19 patients who have developed reduced cardiac function, most notably reduced ejection fraction, with or without overt symptoms of heart failure. In some embodiments, the compositions of the present disclosure may be used to treat arrhythmias.

[0120] The compositions of the present disclosure can alleviate one or more symptoms associated with post-COVID-19 syndrome. In some embodiments, the symptoms of post-COVID-19 syndrome can be the same as acute COVID-19. In some aspects, the symptoms associated with post-COVID-19 syndrome can be shortness of breath, fatigue, edema, orthopnea, limitations to exertion, decreased cognitive ability, palpitations, dizziness, fainting, lightheadedness, heart failure, and / or arrhythmia.

[0121] In some embodiments, compositions of the present disclosure may be used to treat post-COVID-19 syndrome, with symptoms that overlap with post-intensive care syndrome, which has also been reported in non-COVID-19 patients.

[0122] In some embodiments, compositions of the present disclosure may be used to treat subjects with post-COVID-19 syndrome who may have one or more long-term complications related to the cardiovascular system (e.g., inflammation of the heart muscle), respiratory system (pulmonary function abnormalities), renal system (acute kidney injury), skin (rash, hair loss), neurological complications (smell and taste problems, sleep disorders, poor concentration, memory impairment), and / or psychiatric disorders (depression, anxiety, mood changes).

[0123] In some embodiments, the compositions of the present disclosure may be used to treat subjects with post-COVID-19 syndrome who may have one, two, or more associated comorbidities, non-limiting examples of which include, but are not limited to, hypertension, thyroid disease, immune disorders, COPD (chronic obstructive pulmonary disease), high blood pressure, obesity, psychiatric disorders, and diabetes.

[0124] V. Definition Domain: As used herein with reference to polypeptides, the term "domain" refers to a motif in a polypeptide having one or more distinguishable structural or functional features or characteristics (e.g., binding ability, functioning as a site of protein-protein interaction).

[0125] Expression vector: As used herein, the term "expression vector" refers to a vector that contains a nucleic acid sequence that codes for at least a portion of a transcribable gene product. Expression vectors can contain various control sequences. Control sequences refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that control transcription and translation, vectors and expression vectors can also contain nucleic acid sequences that perform other functions. The term also includes recombinant plasmids or viruses that contain a polynucleotide that is delivered to a host cell either in vitro or in vivo. In some embodiments, the host cell is a transient cell line or a stable cell line. In some embodiments, the host cell is selected from the group consisting of CHO cells, HEK293 cells, and NS0 cells.

[0126] Feature: A "feature" when referring to a polypeptide is defined as a molecular component based on a distinct amino acid sequence. Features of the polypeptides encoded by the polynucleotides described herein include local conformational shapes, folds, loops, half-loops, domains, half-domains, sites, termini, or any combination thereof.

[0127] Fusion protein: As used herein, the term "fusion protein" or chimeric protein refers to a protein or polypeptide that contains two or more amino acid sequences or active fragments thereof that are not naturally occurring in the same polypeptide. In some embodiments, two or more separate polypeptides are operably linked together, chemically, by a covalent bond, such as a peptide bond, or fused together. Recombinant fusion polypeptides are artificially created by recombinant DNA technology.

[0128] Half domain: As used herein in reference to a polypeptide, the term "half domain" refers to a portion of a specified domain that has at least half the number of amino acids present in the domain from which it is derived. It is understood that a domain does not necessarily contain an even number of amino acid residues. Thus, if a domain contains or is identified as containing an odd number of amino acids, a half domain of the odd domain constitutes the integer portion or the next integer portion of the domain (number of amino acids in the domain / 2±0.5 amino acids). For example, a domain identified as a 7 amino acid domain may generate half domains of 3 or 4 amino acids (7 / 2=3.5±0.5 is 3 or 4). It is also understood that subdomains may be identified within a domain or half domain, and that these subdomains may have less than all of the structural or functional characteristics identified in the domain or half domain from which it is derived. It is also understood that the amino acids that constitute any of the domain types described herein need not be contiguous along the backbone of the polypeptide (i.e., non-adjacent amino acids may structurally fold over one another to generate a domain, half domain, or subdomain).

[0129] Immune response: As used herein, the term "immune response" refers to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic diseases. These conditions are characterized by the expression of a variety of factors, such as antibodies, immune cells, cytokines, chemokines, and other signaling molecules, that can affect cellular and systemic defense systems.

[0130] Linker: As used herein, "linker" refers to a functional group (e.g., a chemical or polypeptide) that covalently links two or more polypeptides. As used herein, a "peptide linker" is two or more amino acids used to link two proteins together.

[0131] Modulation: As used herein, the term "modulation" is art-recognized and refers to the upregulation (i.e., activation or stimulation), downregulation (i.e., inhibition or suppression) of a response, or a combination or combination of the two, alone.

[0132] Polynucleotide: As used herein, the term "polynucleotide" refers to a sequence of nucleotides linked by phosphodiester bonds. Polynucleotides are presented herein in a 5' to 3' orientation. A polynucleotide is a deoxyribonucleic acid (DNA) molecule or a ribonucleic acid (RNA) molecule. When the polynucleotide is a DNA molecule, the molecule can be a gene or a cDNA molecule. Nucleotide bases are designated herein by the single letter code adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U). Polynucleotides can be prepared using standard techniques well known to those skilled in the art.

[0133] Polypeptide: In some embodiments, the compositions of the present disclosure are polypeptides or proteins, or variants thereof. According to the present disclosure, any amino acid-based molecule (natural or non-natural) is referred to as a "polypeptide", which term includes "peptide", "peptidomimetic", and "protein". As used herein, "polypeptide" refers to a polymer of amino acid residues (natural or non-natural), most often linked by peptide bonds. The term as used herein refers to proteins, polypeptides, and peptides of any size, structure, or function. A "peptidomimetic" or "polypeptide mimic" is a polypeptide that contains structural elements within the molecule that are not found in natural polypeptides (i.e., a polypeptide that is composed only of the 20 proteinogenic amino acids). In some embodiments, a peptidomimetic can reproduce or mimic the biological action of a natural peptide.

[0134] Polypeptide variant: The term "polypeptide variant" refers to a molecule that differs in amino acid sequence from a native or reference sequence. Amino acid sequence variants may have substitutions, deletions, and / or insertions at specific positions within the amino acid sequence compared to the native or reference sequence. Typically, a variant has at least about 50% identity (homology) to the native or reference sequence, preferably at least about 80%, more preferably at least about 90% identity (homology) to the native or reference sequence.

[0135] Recombinant: As used herein, the term "recombinant" refers to a genetic entity that differs from that typically found in nature. When applied to a polynucleotide or gene, it means that the polynucleotide is the product of various combinations of cloning, restriction and / or ligation steps, as well as other procedures that result in the production of a structure that differs from that of polynucleotides found in nature.

[0136] Sample: As used herein, the term "sample" refers to an aliquot or portion taken from a source and / or provided for analysis or processing. In some embodiments, the sample is from a biological source such as tissue, cells, or components (e.g., bodily fluids including, but not limited to, blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen). In some embodiments, the sample is or can include a homogenate, lysate, or extract prepared from a whole organism or a subset of its tissues, cells, or components, or a fraction or part thereof, including, but not limited to, plasma, serum, spinal fluid, lymphatic fluid, external parts of the skin, respiratory tract, intestinal tract, genitourinary tract, tears, saliva, milk, blood cells, tumors, or organs. In some embodiments, the sample is or includes a medium, such as a nutrient broth or gel, which may contain cellular components such as proteins. In some embodiments, a "primary" sample is an aliquot of a source. In some embodiments, the primary sample is subjected to one or more processing (eg, separation, purification, etc.) steps to prepare the sample for analysis or other uses.

[0137] Sequence identity: The term "sequence identity" refers to the percentage of identical nucleotides or amino acids as demonstrated by sequence alignment. For example, two peptides having 20 amino acid residues each have 18 / 20, or 90%, sequence identity if their amino acid sequences are identical except for differences at two positions.

[0138] Substantially: As used herein, the term "substantially" refers to a qualitative state of exhibiting a complete or nearly complete extent or degree of a property or characteristic of interest. One of ordinary skill in the art of biology will understand that biological and chemical phenomena rarely reach completion, progress toward perfection, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0139] Terminal: As used herein, the term "terminal" when referring to a polypeptide refers to the end of a peptide or polypeptide. Such terminals are not limited to the first or last site of a peptide or polypeptide, but may also include additional amino acids in the terminal region. The polypeptide-based molecules described herein may be characterized as having both an N-terminus (terminated with an amino acid having a free amino group (NH2)) and a C-terminus (terminated with an amino acid having a free carboxyl group (COOH)). Proteins described herein are optionally composed of multiple polypeptide chains (multimers, oligomers) linked by disulfide bonds or non-covalent forces. This type of protein will have multiple N-terminuses and C-terminuses. Alternatively, the termini of a polypeptide may optionally be modified to begin or end with a non-polypeptide-based moiety, such as an organic conjugate.

[0140] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means the amount of agent delivered that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition.

[0141] Treatment: As used herein, the term "treatment" refers to partially or completely alleviating, ameliorating, enhancing, palliating, delaying the onset, inhibiting progression, reducing the severity, and / or reducing the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment may be administered to subjects who do not show signs of the disease, disorder, and / or condition and / or to subjects who show only early signs of the disease, disorder, and / or condition, with the aim of reducing the risk of developing a pathology associated with the disease, disorder, and / or condition.

[0142] Treatment: As used herein, the terms "treatment", "treating" and the like refer to the alleviation or reduction of a pathological process. In the context of this disclosure, in relation to any of the other conditions described herein below, the terms "treatment", "treating" and the like refer to the alleviation or reduction of at least one symptom associated with such condition, or the slowing or reversal of the progression or predicted progression of such condition.

[0143] Therapeutic dose: As used herein, a "therapeutic dose" refers to one or more doses of a therapeutic agent administered in the course of treating or alleviating a therapeutic indication. A therapeutic dose may be adjusted to maintain a desired concentration or activity level of the therapeutic agent in a bodily fluid or biological system.

[0144] VI. Equivalents and Scope While various embodiments have been specifically shown and described in this disclosure, it will be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the embodiments disclosed herein and set forth in the appended claims.

[0145] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the present disclosure is not limited to the above description, but is as set forth in the appended claims.

[0146] In the claims, articles such as "a," "said," and the like may mean one or more, unless specifically indicated to the contrary or clear from the context. A claim or description containing "or" between one or more members of a group is deemed to be satisfied when one, more than one, or all of the group members are present in, used in, or otherwise relevant to a given product or method, unless specifically indicated to the contrary or clear from the context. The present disclosure includes embodiments in which only one member of a group is present in, used in, or otherwise relevant to a given product or method. The present disclosure includes embodiments in which multiple group members or all group members are present in, used in, or otherwise relevant to a given product or method.

[0147] Additionally, the term "comprising" has an open meaning, allowing for, but not requiring, the inclusion of additional elements or steps. Thus, when the term "comprising" is used in this application, the terms "consisting of" and "or including" are also encompassed and disclosed.

[0148] When ranges are specified, the upper and lower limits are inclusive. Furthermore, unless otherwise stated or apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges can be considered in different embodiments of this disclosure to any particular value or subrange within the stated range, up to one-tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0149] Furthermore, it should be understood that certain embodiments of the present disclosure that fall under the prior art may be expressly excluded from any one or more of the claims. Such embodiments are deemed known to those skilled in the art and may be excluded even if the exclusion is not expressly set forth herein. Certain embodiments of the compositions disclosed herein may be excluded from any one or more of the claims for any reason, whether related to the existence of prior art or not.

[0150] All cited sources, for example, references, publications, databases, database entries, and techniques cited herein, are incorporated by reference into this application, even if not expressly stated in the cited sources. In the event of a conflict between the statements of a cited source and this application, the statements of this application shall control.

[0151] The section and table headings are not intended to be limiting. Working Example Example 1. DSG2 fusion polypeptides and their interaction with anti-DSG2 antibodies Candidate DSG2 fusion polypeptides include the extracellular domain (ECD) portion of DSG2 fused to IgGFc or its variants. Binding the ECD to the Fc portion of an immunoglobulin was considered beneficial for DSG2 fusion polypeptides, as it would improve protein stability, enhance PK properties, and allow for efficient purification. Table 4 shows the DSG2 fusion polypeptides obtained or prepared.

[0152] The entire preparation of DSG2 fusion polypeptide was confirmed to be highly heterogeneous as seen by one-step affinity purification. Initial evaluation of the production suggested high aggregation. As shown in Table 5, dynamic light scattering (DLS) showed that all candidates showed an average polymer distribution index (PDI) of >0.25, indicating that the samples likely contained particles of multiple sizes due to advanced aggregation or precipitation. Six candidates were compared to a reference sample. All six candidates showed approximately 4-8 times larger peak 1 mode diameter. Fusion polypeptides containing IgG4 showed approximately 2 times larger peak 1 mode diameter compared to fusion polypeptides containing IgG1.

[0153] [Table 5]

[0154] Table 6 shows the melting temperature Tm values ​​of the candidate DSG2 fusion polypeptides. The presence of three Tm values ​​suggests that the candidate DSG2 fusion polypeptides may contain aggregates prior to analysis. As the temperature increased, the aggregates began to move, unfold, and diffuse. The derivative graphs of the four samples with the third Tm value showed a post-melting transition. The Tm values ​​provided during the analysis showed similar values ​​among all the DSG2 fusion polypeptides. This may suggest that the thermal stability of the structures is similar.

[0155] [Table 6]

[0156] The DSG2 fusion polypeptides were tested for their ability to inhibit anti-DSG2 antibodies by anti-DSG2 antibody mesoscale discovery (MSD) assay. The tested DSG2FP#2, DSG2FP#3, DSG2FP#4, DSG2FP#5, DSG2FP#6, and DSG2FP#7 fusion polypeptides were able to bind and block anti-DSG2 antibodies. The DSG2 fusion polypeptide DSG2FP#1 was able to bind and block anti-DSG2 antibodies and serum obtained from ARVC patients (hereinafter also referred to as ARVC serum) (see FIG. 1A). The DSG2 fusion polypeptides were subjected to size exclusion chromatography, and various elution fractions were examined. Fraction "C" showed a predicted MW of approximately 350 kDa and was used for further experiments. As shown in Table 7 and FIG. 1B, FIG. 1C, and FIG. 1D, enrichment of fraction C improved the ability of the DSG2 fusion polypeptide to inhibit ARVC patient serum. This effect is more pronounced in DSG2FP#2 and DSG2FP#3 than in DSG2FP#4.

[0157] [Table 7]

[0158] Example 2. Effects of anti-DSG2 antibody and DSG2 fusion polypeptide on cardiac function To determine the effect of anti-DSG2 antibodies in cardiomyocytes, a cardiac in vitro proarrhythmia (CiPA) assay was performed (Sager et al. Am Heart J 2014;167:292-300; the contents of which are incorporated herein by reference in their entirety). CiPA is envisioned to consist of four components: (1) evaluation of the effect of test agents on cardiac ion channel assays; (2) computational modeling of cardiac action potentials based on ion channel results and integrating the data with cardiac function; (3) experimentally measuring the effect of test agents in human ventricular myocytes to confirm the modeling results; and (4) scoring of the results. Multi-electrode array (MEA) assay is a component of CiPA. The MEA assay uses human induced pluripotent stem cell (iPSC)-derived cardiomyocytes to assess two-dimensional excitation propagation by recording spontaneous ECG-like field potentials. The sodium spikes quantified below are a measure of specific sodium channel currents in cardiomyocytes and are also described in Blinova K, et al. Cell Rep. 2018 Sep 25;24(13):3582-3592, the contents of which are incorporated herein by reference in their entirety. CiPA is considered a clinically validated cardiomyocyte-based assay by the FDA and EMA. An arrhythmic signal in the assay is considered evidence that a particular drug, such as an anti-DSG2 antibody, can cause arrhythmias in humans. Parameters evaluated include measurements of changes in the spikes of components of electrical signal propagation (e.g., sodium spikes) and the cell index, which is a function of cell impedance and cell number. Changes in sodium spikes may be indicative of arrhythmias, while changes in cell index (CI) may indicate changes in cell junctions.

[0159] Commercially available anti-DSG2 antibodies were tested in the cardiomyocyte CiPA assay. Anti-DSG2 antibodies were found to suppress cardiomyocyte sodium spikes, and this effect was time and dose dependent. Voltage (μV) is measured at time intervals of 0, 1 h, 12 h, 24 h, 48 h, 72 h, and 96 h. As shown in Figure 2, the effect of anti-DSG2 antibodies (0.5 μg / mL) on sodium spikes was comparable to that of 10 μM lidocaine (positive control). The effect of anti-DSG2 antibodies on cardiomyocyte sodium spikes was restored by 1.1 μg / mL of DSG2 fusion polypeptide, DSG2FP#1. As expected, the negative control, vehicle, rabbit IgG, and 0.5 μg / mL of anti-VCAM-1 antibody did not cause any change in sodium spikes over time.

[0160] Various doses of anti-DSG2 antibody affected the cell index. The cell index is inversely proportional to the conductivity of the cell layer to current. An increase in conductivity across the cell layer may indicate an increase in gaps in the cell layer, which suggests weakened cell-cell adhesion. Thus, an increase in conductivity across the cell layer may result in a decrease in the cell index. No dose response was observed. The concentration of anti-DSG2 antibody was varied from 0.00005 μg / mL to 5 μg / mL. Treatment with high doses of lidocaine (positive control) significantly decreased the cell index, likely resulting in cell death. The concentration of lidocaine was varied from 3 μM to 100 μM. Of note, none of the negative controls (vehicle, anti-VCAM-1 antibody, rabbit IgG) showed a decrease in the cell index. The concentration of anti-DSG2 antibody was varied from 0.00005 μg / mL to 5 μg / mL. The effect of anti-DSG2 antibody on the cell index was reversed by the addition of the DSG2 fusion polypeptide, DSG2FP#1 (see FIG. 3 and Table 8).

[0161] [Table 8-1]

[0162] [Table 8-2]

[0163] Example 3. DSG2 antibody levels in ARVC patients ARVC diagnosis is based on a complex tool known as the Task Force Criteria, which provides varying levels of confidence regarding ARVC diagnosis. We investigated the utility of DSG2 antibodies as an indicator of ARVC disease state and severity. Approximately 50 cc of serum was collected from patients diagnosed with ARVC. A total of 10 ARVC patients and 5 healthy controls were used for the analysis. ARVC serum samples were divided into more and less certain diagnoses based on data available in the patients' medical records, and anti-DSG2 antibody levels were compared using an assay based on the anti-DSG2 antibody mesoscale discovery (MSD). The results of the assay showed that the signal intensity of anti-DSG2 antibodies in the assay correlated well with the confidence of the diagnostic clinical data. ARVC serum samples taken from patients with strong clinical data had higher levels of anti-DSG2 antibodies compared to the other two groups (Figures 4A and 4B). The assay signal from serum samples from patients with strong clinical data could also be blocked by adding the competing DSG2 polypeptide DSG2-Fc (DSG2FP#1).

[0164] Example 4. Inhibition of binding of anti-DSG2 antibodies present in sera of ARVC patients to the DSG2 extracellular domain by DSG2 fusion polypeptides with various affinity tags The ability of the DSG2 fusion polypeptide to inhibit the binding of anti-DSG2 antibodies in the serum of ARVC patients to the DSG2 extracellular domain was investigated by electrochemical immunoassay. The fusion polypeptide was added to a final concentration of 5 μg / mL, and the patient serum was used at a final concentration of 10% (v / v). The results are shown in the bar graph of Figure 5, where the numbers 961, 965, 978, 986, and 964 indicate the serum samples of ARVC patients.

[0165] In FIG. 5, the first data series from the left is for DSG2FP#1 (shown above as amino acids 49-608 of SEQ ID NO:30; linker (IEGRMD (SEQ ID NO:28)); amino acids 100-330 of IgG1 (SEQ ID NO:4)). The second data series from the left is for DSG2 (50~602) -6xHis. This is a fusion polypeptide of DSG2 amino acids 50-602 (complete extracellular domain) of SEQ ID NO: 1 with a hexahistidine peptide tag. The third data series from the left is EC1, which is a fusion of DSG2 amino acids 50-155 (EC1) of SEQ ID NO: 1 with a hexahistidine peptide tag. (50~155) -6xHis and EC2, which is a fusion of DSG2 amino acids 151 to 268 (EC2) of SEQ ID NO: 1 with a hexahistidine peptide tag (151~268) The fourth data series from the left shows the combination of fusion polypeptides containing EA (491~602)-6xHis, which is a fusion of DSG2 amino acids 491-602 (EA) of SEQ ID NO: 1 with a hexahistidine peptide tag. In the inhibition results in FIG. 5, 100% inhibition indicates complete inhibition, indicating that DSG2FP#1 provides the best inhibitory activity. Two additional assays show at least 60% inhibitory activity by fusion polypeptides containing a hexahistidine tag instead of the IgG1 Fc region. Thus, it is clear that binding of anti-DSG2 antibodies in the serum of ARVC patients to the DSG2 extracellular domain can be inhibited by DSG2 fusion polypeptides with affinity tags other than the Fc region of IgG. Other affinity tags may be found to be useful in alternative embodiments of DSG2 polypeptides, including but not limited to CBP, MBP, streptavidin, and GST. Additionally, fusion polypeptides with a portion of the extracellular domain (ECD) including the EA region show inhibition of binding of anti-DSG2 antibodies in the serum of ARVC patients to the DSG2 extracellular domain. These findings provide a basis for predicting that fusion polypeptides comprising the DSG2 extracellular domain or parts thereof, in combination with various affinity tags, may be suitable therapeutic agents for use in the treatment of diseases caused by anti-DSG2 autoantibodies, including various cardiac arrhythmias, which function by inhibiting the interaction between anti-DSG2 antibodies and the DSG2 extracellular domain.

[0166] Example 5. Inhibition of anti-DSG2 antibody binding to the DSG2 extracellular domain by DSG2 fusion polypeptides with IgG4 affinity tags Inhibition of the binding of anti-DSG2 antibodies to the DSG2 extracellular domain by two recombinant DSG2 fusion polypeptides was evaluated by electrochemical immunoassay. The fusion polypeptides were added to a final concentration of 5 mg / mL. The anti-DSG2 antibodies were used at a final concentration of 500 ng / mL. The results are shown in FIG. 6. Complete inhibition in the assay is 100% inhibition. DSG2 recombinant proteins: The black bar is for DSG2-(IgG4)FC (DSG2FP#6), which is a fusion protein of the DSG2 extracellular domain represented by amino acids 50-609 of SEQ ID NO: 1 and amino acids 99-332 of the IgG4 Fc domain of SEQ ID NO: 11. The grey bar is for DSG2-GGGGS-(IgG4)FC (DSG2FP#7), which is a fusion protein of the full-length DSG2 extracellular domain represented by amino acids 50-609 of SEQ ID NO: 1 with the GGGGS peptide linker (SEQ ID NO: 12) and the IgG4 Fc domain of amino acids 99-332 of SEQ ID NO: 11.

[0167] Both fusion polypeptides show similar inhibitory activity of more than 80%, indicating that the use of human IgG4 Fc region as affinity tag does not impair the anti-DSG2 antibody inhibitory function of the fusion polypeptide, and the presence of a linker in this case does not significantly affect the inhibitory activity. These findings provide a basis for predicting that fusion polypeptides containing the DSG2 extracellular domain or a part thereof, in combination with various affinity tags including Fc regions of other immunoglobulins, will be suitable therapeutic agents for use in the treatment of diseases caused by anti-DSG2 autoantibodies, including various cardiac arrhythmias.

Claims

1. 1. A desmoglein 2 (DSG2) fusion polypeptide, comprising: a. All or part of the Homo sapiens DSG2 protein; and b. Proteins or protein fragments that can improve the expression and / or stability of the DSG2 protein in vitro or in vivo or that are used to purify the DSG2 fusion polypeptide. The fusion polypeptide comprising:

2. The fusion polypeptide of claim 1 , wherein the DSG2 protein has the sequence of SEQ ID NO:

1.

3. The fusion polypeptide of claim 1 , wherein the fusion polypeptide comprises a portion of the DSG2 protein.

4. The fusion polypeptide according to claim 3 , wherein the portion of the DSG2 protein is the entire extracellular domain of the DSG2 protein or a portion of the extracellular domain of the DSG2 protein.

5. The fusion polypeptide according to claim 3 , wherein the portion of the DSG2 protein is the entire extracellular domain of the DSG2 protein.

6. The fusion polypeptide of claim 1 , wherein the protein fragment comprises a portion of an immunoglobulin protein.

7. The fusion polypeptide of claim 6 , wherein the immunoglobulin protein is IgG, IgM, IgA, IgD, or IgE.

8. The fusion polypeptide of claim 7, wherein the immunoglobulin protein is IgE.

9. The fusion polypeptide of claim 7, wherein the immunoglobulin protein is IgG.

10. The fusion polypeptide of claim 9, wherein the IgG is IgG1, IgG2, IgG3, or IgG4.

11. 7. The fusion polypeptide of claim 6, wherein the portion of the immunoglobulin protein is an Fc region, a Fab region, a heavy chain variable (VH) domain, a heavy chain constant domain, a light chain variable (VL) domain, or a light chain constant domain.

12. The fusion polypeptide of claim 11, wherein the portion of the immunoglobulin protein is an Fc region.

13. The fusion polypeptide of claim 12, wherein the Fc region is an IgG1 Fc region (SEQ ID NO: 5), an IgG2 Fc region (SEQ ID NO: 7), an IgG3 Fc region (SEQ ID NO: 9), or an IgG4 Fc region (SEQ ID NO: 11).

14. The fusion polypeptide of claim 1, wherein the DSG2 fusion polypeptide further comprises a linker.

15. The fusion polypeptide of claim 14, wherein the linker is from about 5 amino acids to about 50 amino acids in length.

16. 16. The fusion polypeptide of claim 15, wherein the linker is GGGGGS (SEQ ID NO: 12), EAAAK (SEQ ID NO: 13), GGGGS (SEQ ID NO: 27), or IEGRMD (SEQ ID NO: 28).

17. The fusion polypeptide of claim 1 , wherein the protein or a fragment of the protein comprises an affinity tag.

18. A fusion polypeptide described in claim 5, wherein the entire extracellular region of the DSG2 protein comprises the amino acid sequence of sequence number 3.

19. The fusion polypeptide of claim 4 , wherein the portion of the DSG2 protein is a portion of the extracellular domain of the DSG2 protein.

20. The fusion polypeptide of claim 19, wherein the portion of the extracellular region of the DSG2 protein comprises at least one domain selected from the group consisting of extracellular cadherin domain 1 (EC1), extracellular cadherin domain 2 (EC2), extracellular cadherin domain 3 (EC3), extracellular cadherin domain 4 (EC4), and extracellular anchor domain (EA).

21. A cell expressing the fusion polypeptide of any one of claims 1 to 20.

22. A composition comprising a fusion polypeptide according to any one of claims 1 to 20 and at least one therapeutic agent.

23. 23. The composition of claim 22, wherein the at least one therapeutic agent is selected from the group consisting of an anti-CD20 antibody, an FcRn-inhibiting antibody, intravenous immunoglobulin (IVIG), and eculizumab.

24. 1. A pharmaceutical composition for use in treating a disease or disorder caused by an anti-desmoglein 2 (DSG2) autoantibody, the pharmaceutical composition comprising: a fusion polypeptide that inhibits binding of an anti-DSG2 antibody to DSG2, the fusion polypeptide comprising: A pharmaceutical composition comprising the extracellular domain of human DSG2 protein, or a portion thereof, having at least about 85% sequence identity with amino acid residues 50 to 609 of SEQ ID NO: 1, and a protein or a fragment of the protein, which is capable of improving the expression and / or stability of said DSG2 protein in vitro or in vivo, or is used to purify said fusion polypeptide.

25. 25. The pharmaceutical composition of claim 24, wherein the portion of the extracellular region of the human DSG2 protein comprises any one or combination of DSG2 domains selected from the group consisting of extracellular cadherin domain 1 (EC1), extracellular cadherin domain 2 (EC2), extracellular cadherin domain 3 (EC3), extracellular cadherin domain 4 (EC4), and extracellular anchor domain (EA).

26. 25. The pharmaceutical composition of claim 24, wherein the protein or fragment of the protein is an Fc region of an immunoglobulin.

27. 27. The pharmaceutical composition of claim 26, wherein the immunoglobulin is IgG1 or IgG4.

28. 27. The pharmaceutical composition of claim 26, wherein the immunoglobulin is a variant of IgG1 having the sequence of SEQ ID NO:

31.

29. 27. The pharmaceutical composition of claim 26, wherein the immunoglobulin is a variant of IgG4 having the sequence of SEQ ID NO:

32.

30. The pharmaceutical composition described in claim 24, wherein the protein or a fragment of the protein is an affinity tag.

31. 31. The pharmaceutical composition of claim 30, wherein the affinity tag is a polyhistidine tag.

32. The pharmaceutical composition described in claim 24, further comprising a linker sequence located between the extracellular domain or a portion thereof of the human DSG2 protein and the protein or a fragment of the protein.

33. 33. The pharmaceutical composition of claim 32, wherein the linker sequence is SEQ ID NO: 12, 13, 27, or 28.

34. The pharmaceutical composition according to any one of claims 24 to 33, wherein the disease or disorder is arrhythmia and / or cardiomyopathy.

35. 35. The pharmaceutical composition of claim 34, wherein the arrhythmia and / or cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy (ARVC), sarcoidosis, acute sequelae of COVID-19, or dilated cardiomyopathy.

36. 35. The pharmaceutical composition of claim 34, wherein the arrhythmia and / or cardiomyopathy is caused proximally or distally by a virus.

37. 37. The pharmaceutical composition of claim 36, wherein the virus is SARS-CoV2, adenovirus, hepatitis virus, hepatitis C virus, parvovirus, herpes simplex virus, echovirus, Epstein-Barr virus, rubella, cytomegalovirus, or HIV.

Images