WO2025072356A2 - Ecl1 beta-1 adrenergic receptor vaccines, proteins, and nucleic acid sequences - Google Patents

Abstract

The present invention relates to systems, kits, and methods for treating a subject that has cardiovascular disease or Systemic Sclerosis with at least one of the following: a) a first antigenic protein that elicits the production of anti-ECL1 b1AR antibodies in said subject, b) a second antigenic protein comprising a portion of human b1AR ECL1, or modified portion thereof (e.g., cyclic peptide) linked to a portion of human b1AR ECL2; c) a mixture of an antigenic protein comprising at least a portion of human b1AR ECL1, and an antigenic protein comprising at least a portion of human b1AR ECL2; d) a vector comprising a nucleic acid sequence encoding such antigenic proteins, and g) a mRNA sequence encoding a portion of ECL1 of human b1-Adrenergic Receptor.

Description

ECL1 BETA-1 ADRENERGIC RECEPTOR VACCINES, PROTEINS, AND NUCLEIC ACID SEQUENCES

The present application claims priority to U.S. Provisional application serial number 63/585,093, filed September 25, 2023, which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to systems, kits, and methods for treating a subject that has cardiovascular disease or Systemic Sclerosis with at least one of the following: a) a first antigenic protein that elicits the production of anti-ECLl [31 AR antibodies in said subject, b) a second antigenic protein comprising a portion of human piAR ECL1, or modified portion thereof (e.g., cyclic peptide) linked to a portion of human [31 AR ECL2; c) a mixture of an antigenic protein comprising at least a portion of human [31 AR ECL1, and an antigenic protein comprising at least a portion of human [31 AR ECL2; d) a vector comprising a nucleic acid sequence encoding such antigenic proteins, and g) a mRNA sequence encoding a portion of ECL1 of human (31 -Adrenergic Receptor.

BACKGROUND

(3-adrenergic receptors (pARs) are one of the most well studied proto-typical 7-trans- membrane receptors that are powerful regulators of cardiac function (Rockman et al., 2002; Vasudevan et al., 2011a). Among the PARs, pi- and P2-ARs are highly expressed in the myocardium (Rockman et al., 2002). Catecholamine binding to the PARs results in G- protein coupling leading to adenylyl cyclase activation, mediating cAMP-protein kinase A (PKA) signal cascade (Wallukat, 2002). Activation of cAMP-PKA cascade alters calcium cycling resulting in increased myocardial contractility. Although pi - and P2-ARs play a key role in myocardial contraction, there is also increasing appreciation that beyond contraction, they may have distinct roles to play in phenotypic outcomes (Dungen et al., 2020; Liaudet et al., 2014). Increasing evidence from in vivo and in vitro studies show functional divergence between piAR and P2ARs. Cellular studies show that chronic catecholamine stimulation leads to cardiomyocyte apoptosis through activation of piARs, while P2ARs may mediate cardioprotective signaling (Milano et al., 1994; Steinberg, 2018). Consistently, cardiomyocyte overexpression of pi ARs leads to maladaptive cardiac remodeling, while overexpression of P2ARs results in hypertrophic response, reflecting their divergent roles (Engelhardt et al., 1999; Steinberg, 2018). However, pi AR downregulation (loss of cell surface receptors) and desensitization (inability to be activated by catecholamine) are one of the key hallmark features of human heart failure (Port and Bristow, 2001 ; Steinberg, 2018).

Dilated cardiomyopathy (DCM) is one the most commonly observed phenotype of heart failure associated with progressive loss in ventricular function, wherein idiopathic DCM represents pathogenesis without a specific known cause (Magnusson et al., 1994; Wynne, 1988). Patients with idiopathic DCM have a diagnosis of diverse etiologies and varied presentations however, dysregulation of the immune system is considered to be one of the central players in cardiac pathogenesis. The human immune system is a complex multicellular regulated defense mechanism that is characterized by inter-individual variability in response to a similar stress/injury. In healthy homeostatic conditions, it is designed to discriminate between self and foreign components, and clear components deemed to be foreign (Crampton et al., 2010; Kaya et al., 2012; Mann, 2011). However, when this regulatory control is lost, it leads to pathological circumstances wherein self-components are attacked, resulting in auto-immune disease (Crampton et al., 2010; Kaya et al., 2012; Mann, 2011). Thus, autoantibodies generated against the self-antigens exacerbate and may accelerate the disease progression (Crampton et al., 2010; Kaya et al., 2012; Mann, 2011).

Circulating autoantibodies have been identified in heart failure and increasing evidence suggests that they may be critically linked to heart failure pathogenesis. Autoantibodies against [MAR have been observed in patients with heart failure (30-40%), and are positively co-related with the phenotype (Baba, 2010; Iwata et al., 2001; Jahns et al., 1999b; Magnusson et al., 1990; Magnusson et al., 1994; Nagatomo et al., 2009; Stork et al., 2006). Studies have shown that (31 AR autoantibody stabilizes the receptor in an active form prolonging its activation mimicking catecholamines (Deubner et al., 2010). Furthermore, Pl AR autoantibodies can elevate the L-type Ca²⁺ current increasing in vitro contractility (Christ et al., 2001). This elevated and prolonged activation reflects the hyper-sympathetic state associated with deleterious cardiac remodeling and DCM. Although P-blockers ameliorate the signaling from the sympathetic overdrive, their role in upregulation of PARs is thought to underlie the worsening outcomes of heart failure due to binding by piAR autoantibodies.

SUMMARY OF THE INVENTION

The present invention relates to systems, kits, and methods for treating a subject that has cardiovascular disease or Systemic Sclerosis with at least one of the following: a) a first antigenic protein that elicits the production of anti-ECLl piAR antibodies in said subject, b) a second antigenic protein comprising a portion of human piAR ECL1, or modified portion thereof (e.g., cyclic peptide) linked to a portion of human piAR ECL2; c) a mixture of an antigenic protein comprising at least a portion of human piAR ECL1, and an antigenic protein comprising at least a portion of human piAR ECL2; d) a vector comprising a nucleic acid sequence encoding such antigenic proteins, and g) a mRNA sequence encoding a portion of ECL1 of human pi-Adrenergic Receptor.

In certain embodiments, provided herein are methods of treating a subject that has cardiovascular disease (CVD), or Systemic Sclerosis (SS), comprising: treating a subject that has CVD or SS with at least one of the following: a) anti-ECLl IgG3 human pi-Adrenergic Receptor antibodies (ECL1 IgG3 piAR antibodies) or antigen-binding portion thereof, optionally wherein ECL1 lgG3 piAR antibodies bind a region of human pi -Adrenergic Receptor in SEQ ID NO:1 ; b) an anti-ECLl pi-Adrenergic Receptor monoclonal antibody or antigen-binding portion thereof, or an anti-ECLl pi-Adrenergic Receptor monoclonal nanobody or antigen-binding portion thereof, optionally wherein the monoclonal antibody or monoclonal antibody binds a region of human pi -Adrenergic Receptor in SEQ ID NO:1; c) a first antigenic protein that elicits the production of anti-ECLl pi AR antibodies in the subject, optionally wherein the anti-ECLl piAR antibodies are IgG3 antibodies; optionally wherein the first antigenic protein comprises a least eight (e.g., 8, 9, 10, or 11) consecutive amino acids from WGRWEYGSFFC (SEQ ID NO: 1) or SEQ ID NOs: 31 or 32, optionally wherein the first antigenic protein is conjugated to a carrier proteins, such as KLH to the C-terminus of Cysteine, a second antigenic protein comprising a portion of human [31 AR ECU or modified version thereof (e.g., cyclic peptide) linked to a portion of human [31 AR ECL2, optionally wherein the second antigenic protein is a carrier protein such as KLH; optionally wherein the portion of [31AR ECL1 comprises at least 8 (e.g., 8, 9, 10, or 11) consecutive amino acids from WGRWEYGSFFC (SEQ ID NO: 1) or SEQ ID NOs: 31 or 32; and optionally wherein the portion of [31AR ECL2 comprises at least 10 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18 ... or 25) consecutive amino acids from HWWRAESDEARRCYNDPKCCDFVTNR (SEQ ID NOG) or from ECL 2 portion of SEQ ID NOs: 33 or 35; e) a mixture of a third antigenic protein comprising at least a portion of human (31AR ECL1 or modified version thereof, and a fourth antigenic protein comprising at least a portion of human (31 AR ECL2; f) a vector comprising a nucleic acid sequence encoding: i) the anti-ECLl IgG3 (31 AR antibodies or antigen-binding portion thereof, ii) the anti-ECLl (31 -Adrenergic Receptor monoclonal antibody or antigen-binding portion thereof, iii) the anti-ECLl (31 -Adrenergic Receptor monoclonal nanobody or antigen-binding portion thereof, iv) the first and/or second antigenic protein, or v) the third and fourth antigen proteins; g) a first mRNA sequence encoding at least an eight amino acid portion of ECL1 of human (31 -Adrenergic Receptor or modified version thereof (e.g., cyclic peptide), optionally wherein the first mRNA sequence comprises at least 24 (e.g., at least 24, 25, 26, 27, 28, 29, 30, or more) consecutive nucleotides from ECL1 mRNA, or modified version thereof (e.g., cyclic peptide) shown in any of SEQ ID NO:2 and 8-12, 33, or 35, optionally wherein the first mRNA sequence comprises modified bases and is present in a liposome nanoparticle; and/or h) the first mRNA sequence and a second mRNA sequence, wherein the second mRNA sequence encodes at least 10 amino acid portion of ECL2 of human (31 -Adrenergic Receptor, or modified version thereof, and wherein the second mRNA sequence comprises at least 24 (e.g., 24 ... 35 ... 50 ... 65 ... or 70) consecutive nucleotides from ECL2 mRNA shown in any of SEQ ID NOs:7, 13-30, 33, and 35), and optionally wherein the first and second mRNA sequences comprise modified bases and are present in a liposome nanoparticle; and/or i) a fifth antigenic protein comprising at least 8 or 10 consecutive amino acids from SEQ ID NOs. 4, 5, or 6, or a vector comprising a nucleic acid sequence encoding said at least 8 or 10 consecutive amino acids from SEQ ID NOs: 4, 5, or 6.

In further embodiments, the subject is negative, or lower than a control, for IgG3 Pl AR antibodies prior to the treatment. In other embodiments, the CVD comprises dilated cardiomyopathy. In certain embodiments, the methods further comprise: testing a blood, serum, or plasma sample from the subject for the presence of IgG3 iAR antibodies prior to the treatment. In particular embodiments, the subject is a human. In some embodiments, the ECL1 IgG3 pi AR antibodies, or antigen-binding portion thereof, or the monoclonal antibodies or nanobodies, are human or humanized antibodies, and the subject is a human.

In further embodiments, the treating comprises intravenous treatment. In additional embodiments, the vector comprises a plasmid, adeno- associated virus, or adeno-associated virus. In additional embodiments, the subject is on a P-blocker prior to the treatment.

In certain embodiments, the methods further comprise: administering a P-blocker to the subject prior to, during, or after the treatment. In additional embodiments, the P-blocker is selected from the group consisting of: Acebutolol, Atenolol, Bisoprolol, Metoprolol, Nadolol, Nebivolol, and Propranolol. In certain embodiments, the ECL 1 IgG3 piAR antibodies are autoantibodies purified from a blood, serum, or plasma sample of a donor or the subject themselves. In some embodiments, provided herein are kits, systems, or compositions comprising: a) at least one of the following: i) anti-ECLl IgG3 human 01-Adrenergic Receptor antibodies (ECL1 IgG3 01AR antibodies) or antigen-binding portion thereof, optionally wherein ECL1 IgG3 01AR antibodies bind a region of human pi-Adrenergic Receptor in SEQ ID NO:1; ii) an anti-ECLl 01-Adrenergic Receptor monoclonal antibody or antigen-binding portion thereof, or an anti-ECLl pi -Adrenergic Receptor monoclonal nanobody or antigen-binding portion thereof, optionally wherein the monoclonal antibody or monoclonal antibody binds a region of human pi-Adrenergic Receptor in SEQ ID NO: 1; iii) a first antigenic protein that elicits the production of anti-ECLl piAR antibodies in the subject, optionally wherein the anti-ECLl piAR antibodies are IgG3 antibodies; optionally wherein the first antigenic protein comprises a least eight consecutive amino acids from WGRWEYGSFFC (SEQ ID NO: 1) or SEQ ID NOs: 31 or 32, optionally wherein the first antigenic protein is conjugated KLH to the C-terminus of Cysteine) a second antigenic protein comprising a portion of human piAR ECL1, or modified version thereof (e.g., cyclic peptide) linked to a portion of human pi AR ECL2, optionally wherein the second antigenic protein is conjugated KLH; optionally wherein the portion of piAR ECL1, or modified version thereof (e.g., cyclic peptide) comprises at least 8 (e.g., 8, 9, 10, or 11) consecutive amino acids from WGRWEYGSFFC (SEQ ID NO: 1) or SEQ ID NOs: 31 and 32; and optionally wherein the portion of 01 AR ECL2 comprises at least 10 consecutive amino acids from HWWRAESDEARRCYNDPKCCDFVTNR (SEQ ID NO:3) or ECL2 portion of SEQ ID NOs: 33 or 35; v) a mixture of a third antigenic protein comprising at least a portion of human 01 AR ECL1, and a fourth antigenic protein comprising at least a portion of human 01 AR ECL2; vi) a vector comprising a nucleic acid sequence encoding: i) the anti-ECLl IgG3 01AR antibodies or antigen-binding portion thereof, ii) the anti-ECLl 01-Adrenergic Receptor monoclonal antibody or antigen-binding portion thereof, iii) the anti-ECLl 01- Adrenergic Receptor monoclonal nanobody or antigen-binding portion thereof, iv) the first and/or second antigenic protein, or v) the third and fourth antigen proteins; vii) a first mRNA sequence encoding at least an eight amino acid portion (e.g., at least 8, 9, 10, or 11) of ECL1 of human 01-Adrenergic Receptor, or modified version thereof (e.g., cyclic peptide), optionally wherein the first mRNA sequence comprises at least 24 (e.g., at least 24 . . . 40 . . . 55 ... 60 ... or more) consecutive nucleotides from the ECL1 mRNA sequences shown in any of SEQ ID NOs:2 and 8-12, 36 or 37, optionally wherein the first mRNA sequence comprises modified bases and is present in a liposome nanoparticle; and/or viii) the first mRNA sequence and a second mRNA sequence, wherein the second mRNA sequence encodes at least 10 amino acid portion of ECL2 of human p l -Adrenergic Receptor, and wherein the second mRNA sequence comprises at least 24 (e.g., 24 ... 50 ... 70 ... or 78) consecutive nucleotides from an ECL2 mRNA sequence shown in any of SEQ ID NOs:7 and 13-30, 38, and 40), and optionally wherein the first and second mRNA sequences comprise modified bases and are present in a liposome nanoparticle; and/or ix) a fifth antigenic protein comprising at least 8 or 10 consecutive amino acids from SEQ ID NOs. 4, 5, or 6, or a vector comprising a nucleic acid sequence encoding said at least 8 or 10 consecutive amino acids from SEQ ID NOs: 4, 5, or 6; and b) a P-blocker.

In some embodiments, provided herein are methods comprising: a) receiving results of, or conducting, an IgG3 31 -Adrenergic Receptor IgG3 P1AR antibodies (IgG3 P1AR antibodies) level analysis on a sample from a subject with cardiovascular disease (CVD) or Systemic Sclerosis (SS), and b) performing at least one of the following after identifying the sample as having higher levels of the IgG3 P1AR antibodies compared to control levels, i) treating the subject with any of the reagents recited above or herein, and/or ii) providing a report to the patient or medical personnel treating the patient, indicating the subject is suitable for, or should be, treated with any of the reagents recited above or herein. In particular embodiments, the control levels are from the general population or CVD patients not stratified by lgG3 31 AR antibody levels.

In some embodiments, provided herein are compositions comprising: a first engineered polynucleotide encoding at least a portion of human pi AR extra cellular loop 1 (ECL1), or modified version thereof (e.g., cyclic peptide) wherein the first engineered polynucleotide is at least partially optimized for enhanced expression, productive co- translational protein folding, increased stability, or a combination thereof. In certain embodiments, the first engineered polynucleotide comprises at least 24 consecutive nucleotides from the ECL1 mRNA (or modified version thereof) sequence shown in any of SEQ ID Nos:2, 8-12, 36, and 37. In other embodiments, the compositions further comprise: a second engineered polynucleotide encoding at least a portion of human pi AR extra cellular loop 2 (ECL2). In additional embodiments, the second engineered polynucleotide comprises at least 24 consecutive nucleotides from the ECL2 mRNA sequences shown in any of SEQ ID NOs:7, 13-30, 38, or 40.

In some embodiments, the polynucleotide comprises RNA, and optionally wherein the RNA is partially or fully human codon optimized. In other embodiments, the first (and/or second) engineered polynucleotide further comprises or encodes: a 5' untranslated region (UTR), a 5’ cap, a 3' UTR, a 3’ tailing sequence, or any combination thereof. In additional embodiments, the 5’ UTR, 3’ UTR, or both are heterologous to the polynucleotide encoding at least a portion of human P1AR ECL1.

In further embodiments, the 3’ tailing sequence comprises a polyA tail, a polyG quartet, a stem loop sequence, a triple helix forming sequence, a tRNA-like sequence, or any combination thereof. In other embodiments, the first engineered polynucleotide, and optionally the second engineered polynucleotide, comprises at least one chemically modified nucleotide. In certain embodiments, the at least one chemically modified nucleotide comprises a modified uracil. In other embodiments, at least 60% of the uracil in the first and/or second polynucleotide are chemically modified. In additional embodiments, the at least one chemically modified nucleotide comprises 5-methylcytosine or Nl- methylpseudouridine (ml ).

In particular embodiments, the first engineered polynucleotide comprises a nucleotide sequence having: i) at least 75% sequence identity to SEQ ID NO: 2, 36, or 37, or a complement or reverse complement thereof, wherein T may be replaced by U, and/or ii) at least 24 consecutive nucleotides from SEQ ID NOs: 2, 36, or 37, or a complement or reverse complement thereof, wherein T may be replaced by U. In other embodiments, the human P1AR ECL1 comprises: i) an amino acid sequence having at least 90% identity to SEQ ID NO:1, 4, 5, 6, 31, or 32 and/or ii) at least 12, at least 15, consecutive amino acids from SEQ ID NO: 1, 4, 5, 6, 31, or 32. In additional embodiments, the human 01AR ECL2 comprises: i) an amino acid sequence having at least 90% identity to any one of SEQ ID NO:3, 33, or 35, and/or ii) at least 12 or 15 consecutive amino acids from SEQ ID NO: 3, 33, or 35.

In some embodiments, provided herein are vaccines comprising: the antigenic compositions above or herein; and at least one adjuvant, a delivery vehicle, or a combination thereof. In further embodiments, the delivery vehicle comprises a lipid nanoparticle encapsulating the composition. In further embodiments, the lipid nanoparticle comprises a cationic lipid, a neutral and/or non-cationic lipid, a sterol, or any combination thereof. In some embodiments, the non-cationic lipid comprises a phospholipid. In certain embodiments, the sterol comprises cholesterol or a modification or ester thereof. In particular embodiments, the lipid nanoparticle comprises a polyethylene glycol (PEG)-lipid conjugate.

In some embodiments, provided herein are compositions comprising: a first immunogenic protein comprising at least a portion of human p l AR ECL1, wherein the first immunogenic protein is conjugated to a hapten, and/or wherein the composition further comprises an adjuvant and/or a delivery vehicle. In other embodiments, the first immunogenic protein comprises: i) an amino acid sequence having at least 90% identity to SEQ ID NO:1, 4, 5, 6, 31, or 32, and/or ii) at least 10 or at least 12 or at least 15 consecutive amino acids from SEQ ID NO:1 , 4, 5, 6, 31, or 32. In further embodiments, the hapten is selected from the group consisting of: fluorescein, biotin, digoxigenin, and dinitrophenol.

In additional embodiments, the compositions further comprise a second immunogenic protein comprising at least a portion of human [31 AR extra cellular loop 2 (ECL2). In certain embodiments, the second immunogenic protein comprises i) an amino acid sequence having at least 90% identity to SEQ ID NO:3, 33, or 35, and/or ii) at least 10 or at least 12 or at least 15 consecutive amino acids from SEQ ID NO:3, 33, or 35.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. ECL1 as AAb allosteric binding site for [31 AR signaling. Figure 1A shows structural modeling of IgGl and IgG3 bl AR AAbs illustrated hypothetical interacting sites between specific complementarity-determining regions (CDR) of the AAbs with epitopes on bl AR. Unique to IgG3, there is a potential CDR3 interface of AAb with ECL1 on bl AR Figure IB shows the predicted epitopes of bl AR, including: N-terminal (LVPASPPASLLPPASESPEPLSQQW, SEQ ID NO:4), ECL1 (WGRWEYGSFFCELWTSVDVL (SEQ ID NO: 1), ECL2 (WRAESDEARRCYNDPKCCFVTNR (SEQ ID NO:3), ECL3 (HRELVP, SEQ ID NO:5), and N-terminal (VLVLGASEPGNLSSAAPLPDGAAT, SEQ ID NO:6).

Figure 2. Hypothetical non-limiting model of Hypothesis: Human IgG3(+) [31 AR AAbs binding to the |31AR uniquely mediated signaling biases especially in the presence of beta-blocker therapy.

Figure 3. Kaplan-Meier survival curves for the composite endpoint of all-cause death, cardiac transplantation, or hospitalization resulting from the exacerbation of heart failure. In our single-center cohort of 121 patients with heart failure, non-IgG3-01AR-AAb and IgG3-01AR-AAb were found to be positive in 20 (17%) and 26 patients (21%), respectively. During 2.2 ± 1.2 years of follow-up, the composite endpoint events were significantly more common in the patients without than in those with IgG3-01AR-AAb (P = 0.048, log-rank test). Conversely, patients with non-IgG3-01AR-AAb conferred worst clinical outcomes compared to those without 01AR-AAb or those with IgG3-|31 AR-AAb.

Figure 4. Schematic of experimental designs and plans for testing the cardiac effect of ECL1 immunization mice in a heart failure mouse model (a) and for testing the antibodies generated from ECL1, ECL2 or ECL1+ECL2 injected mice (b). Figure 5. Antibody titer after ECL1 peptide immunization in mice. ELISA analysis revealed the antibody response of mice to the ECL1 peptide across three experiments. The ELISA data indicates that mice produced higher antibody titers in the 2nd and 3rd experiments compared to the 1st experiment.

Figure 6. ECL1 peptide immunization reduces cardiac fibrosis in isoproterenol- induced heart failure. Mason's trichrome staining (A & C) and quantification (B & D) indicates that mice immunized with the ECL1 peptide exhibited reduced cardiac fibrosis in the isoproterenol -induced heart failure model in experiment 2 (A & B) and experiment 3 (C & D). Images of trichomes from two different mice were presented for each group in the experiment 3(C).

Figures 6E and 6F and 6G. Generating a murine model to recapitulate the anti- fibrotic effects induced by ECL1 signaling. Despite being highly unique to [31 AR, the ECL1 of pi AR is a relatively short segment that is highly hydrophobic, which may not be easy to stimulate AAb production in mice. To enhance the immune response in mice, the Betal AR ECL1 peptide was conjugated to the Keyhole Limpet Hemocyanin (KLH) carrier protein using a maleimide-activated KLH (mcKLH) kit (Thermo Scientific, 77666). We demonstrated that mice immunized by injecting subcutaneously the conjugated Betal AR ECL1 peptide can produce AAbs that can recognize the soluble ECL1 peptide, with antibody titers ranging from 1:500 to 1 : 125,000 (Fig 6E). We have shown the lack of adverse cardiac effects in mice immunized with the ECL1 of piAR using a 4-week isoproterenol (ISO)- induced HF mouse model designed to generate a hyperadrenergic state leading to cardiac hypertrophy and fibrosis (Fig 6F). Additionally, soluble Betal AR ECL1 peptide-immunized mice under ISO treatment exhibited less cardiac fibrosis than adjuvant treated mice (Fig 6). Our immunization strategy to generate Betal AR- AAbs did not show overt deleterious anti- ECL2 Betal AR Aab effects, (refs 1-3).

Figure 7. ECL1 as epitope for AAb against pi AR is associated with potential beneficial effects in human samples. In Figure 7 A we leveraged Phage Display Immunoprecipitation- sequencing (PhlP-Seq) on serum samples from healthy controls and patients with suspected autoimmune cardiomyopathy. We observed a patient with suspected autoimmune CM whose AAb binds to both ECL1 and ECL2 of (31 AR showed a reduced LVEF (47%), indicating a mild dysfunction of the heart. Conversely, the other two patients, whose AAbs bind solely to the ECL1 region, exhibited normal LVEF values (74% and 60%). This ECL1 specificity is also demonstrated in human-derived IgG3(+) samples. Figure 7B shows IgG3+ AAbs engage ECL1 and ECL2 to mediate unique allosteric modulation. Representative cAMP response to metoprolol (Meto) following competition with ECL1, ECL2 or both in presence of human IgG3(+). *p<0.005 vs. IgG3+ Ctrl; **p<0.01 vs. IgG3+ Meto Ctrl (n=3).

Figure 8. AAbs against ECL1 of piAR as epitope for allosteric modulation in mouse immunization model. Figure 8 A shows schematic of ECL1 as epitope for f> l AR AAb that mediates potential beneficial effects of either positive or negative allosteric modulation (PAM/NAM). In mouse immunized with ECL1 of piAR, pre-treatment of HEK 293 cells expressing human pi AR with pre-bleed (prior to immunization with ECL1 peptide) serum showed no appreciable increase in cAMP generation. However, post-bleed (serum from mice immunized with ECL1 peptide) showed significant increase in cAMP generation in response to metoprolol (Meto). Figure 8B shows cAMP response to Meto upon pre-treatment of cells with pre- and post-bleed following immunization with ECL1 in mice. *p<0.01 vs. pre-bleed (n=4).

Figure 9A shows exemplary ECL1 RNA sequences, including SEQ ID NOs:2 and 8- 12. Figure 9B shows exemplary ECL2 RNA sequences, including SEQ ID NOs:7 and 13-20. Figure 9C shows exemplary ECL1-ECL2 cyclic RNA sequences, including SEQ ID NOs: 36-40.

Figure 10 illustrates and provides sequence for inventor designer cyclic pi AR-ECL1 peptides (SEQ ID NO. 31 WGRWECYGSFFCE and SEQ ID NO. 32 WGRCWEYGSFFCE). These cyclic peptides aim to improve immunogenicity through the addition of an extra cysteine residue. Figure 10A, B, and C demonstrates molecular modeling utilized, design rationale, and sequence of an additional ECL1-ECL2 combined cyclic peptide. Modeling of ECL 1 (red) and ECL2 (blue) of piAR (A) allows design of cyclic ECL1/ECL2 peptide mimotope (SEQ ID NO. 33: RWEYGSFFAEHWWRAESDEARRAYNDPKCADFVTNR) as discontinuous epitopes, resulting in a unique allosteric effect beyond its interaction with pi AR ECL1 epitope; (B) with sequence almost parallel to that of original piAR ECL1/ECL2 domain (C) (original Bl AR sequence, SEQ ID NO. 34 WGRWEYGSFFCEHWWRAESDEARRCYNDPKCCDFVTNR and cyclic ECL1-ECL2 peptide, SEQ ID NO. 35 CGRWEYGSFFAEHWWRAESDEARRAYNDPKCADFVTNR.

DEFINITIONS

As used herein, the terms “cardiovascular disease” (CVD) or “cardiovascular disorder” are terms used to classify numerous conditions affecting the heart, heart valves, and vasculature (e.g., veins and arteries) of the body and encompasses diseases and conditions including, but not limited to arteriosclerosis, atherosclerosis, myocardial infarction, acute coronary syndrome, angina, dilated cardiomyopathy, congestive heart failure, aortic aneurysm, aortic dissection, iliac or femoral aneurysm, pulmonary embolism, primary hypertension, atrial fibrillation, stroke, transient ischemic attack, systolic dysfunction, diastolic dysfunction, myocarditis, atrial tachycardia, ventricular fibrillation, endocarditis, arteriopathy, vasculitis, atherosclerotic plaque, vulnerable plaque, acute coronary syndrome, acute ischemic attack, sudden cardiac death, peripheral vascular disease, coronary artery disease (CAD), peripheral artery disease (PAD), and cerebrovascular disease.

The phrase "dilated cardiomyopathy" refers to a condition in which the heart becomes enlarged and cannot pump blood effectively. Symptoms vary from none to feeling tired, leg swelling, and shortness of breath. It may also result in chest pain or fainting. Complications can include heart failure, heart valve disease, or an irregular heartbeat. Causes include genetics, alcohol, cocaine, certain toxins, complications of pregnancy, and certain infections. Coronary artery disease and high blood pressure may play a role, but are not the primary cause. In many cases the cause remains unclear.

As used herein “heart failure” refers to when the heart is unable to pump sufficiently to maintain blood flow to meet the body's needs. Signs and symptoms of heart failure commonly include shortness of breath, excessive tiredness, and leg swelling. The shortness of breath is usually worse with exercise, while lying down, and may wake the person at night. A limited ability to exercise is also a common feature. Common causes of heart failure include coronary artery disease including a previous or current myocardial infarction (heart attack), high blood pressure, atrial fibrillation, valvular heart disease, excess alcohol use, infection, and cardiomyopathy of an unknown cause.

The terms "individual," "host," "subject," and "patient" are used interchangeably herein, and generally refer to a mammal, including, but not limited to, primates, including simians and humans, equines (e.g., horses), canines (e.g., dogs), felines, various domesticated livestock (e.g., ungulates, such as swine, pigs, goats, sheep, and the like), as well as domesticated pets and animals maintained in zoos. In some embodiments, the subject is specifically a human subject (e.g., with dilated cardiomyopathy).

The term “monoclonal antibody,” as used herein, refers to an antibody produced by a single clone of B lymphocytes that is directed against a single epitope on an antigen. Monoclonal antibodies typically are produced using hybridoma technology, as first described in Kohler and Milstein, Eur. J. Immunol., 5: 511-519 (1976). Monoclonal antibodies may also be produced using recombinant DNA methods (see, e.g., U.S. Patent 4,816,567), isolated from phage display antibody libraries (see, e.g., Clackson et al. Nature, 352: 624-628 (1991)); and Marks et al., I. Mol. Biol., 222: 581-597 (1991)), or produced from transgenic mice carrying a fully human immunoglobulin system (see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181 : 69-97 (2008)). In contrast, “polyclonal” antibodies are antibodies that are secreted by different B cell lineages within an animal. Polyclonal antibodies are a collection of immunoglobulin molecules that recognize multiple epitopes on the same antigen.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to systems, kits, and methods for treating a subject that has cardiovascular disease or Systemic Sclerosis with at least one of the following: a) a first antigenic protein that elicits the production of anti-ECLl [31AR antibodies in said subject, b) a second antigenic protein comprising a portion of human [31 AR ECL1, or modified portion thereof (e.g., cyclic peptide) linked to a portion of human (31 AR ECL2; c) a mixture of an antigenic protein comprising at least a portion of human [31 AR ECL1, and an antigenic protein comprising at least a portion of human (31 AR ECL2; d) a vector comprising a nucleic acid sequence encoding such antigenic proteins, and g) a mRNA sequence encoding a portion of ECL 1 of human [31 -Adrenergic Receptor.

Autoimmune response to self-antigens results in autoantibodies (AAbs), wherein AAbs against extra cellular loop 2 (ECL2) of piAR is known to underlie dilated cardiomyopathy (DCM). Contrarily, recent studies show that patients with ( 1 AR AAbs belonging to the lgG3 subclass have beneficial outcomes. However, the signaling mechanisms that underlie the beneficial outcomes are not well understood. Work conducted during development of embodiments herein indicated that IgG3(+) (31AR AAbs facilitates uniquely biased [31AR signaling in response to (3-blockers. Since IgG3(+) piAR AAbs are associated with favorable patient outcomes, in silico studies were conducted to evaluate the interaction between the IgG3(+) antibody and the known structure of piAR. In silico epitope prediction suggested that the extracellular loop 1 (ECL1) could be an epitope, wherein binding by IgG3(+) AAbs at ECL1 could facilitate unique biased signaling in response to P- blockers. Peptides representing human piAR ECL 1 (e.g., WGRWEYGSFFC; SEQ ID NO: 1) was synthesized and antibodies targeting the human piAR ECL 1 was generated by immunizing the mice. HEK 293 cells expressing human pi AR were pre-treated with serum containing antibodies against ECL 1 of piAR followed by either piAR selective agonist dobutamine (DOB) or P-blocker metoprolol stimulation. Downstream i AR signaling was measured by cAMP generation. To test for specificity of ECL1 in the modulating iAR responses, the cells were incubated with ECL 1 peptides along with antibodies against ECL1 of human pi AR as competitors and cAMP generation assessed following metoprolol.

Treatment of human piAR expressing HEK 293 cells with serum from mouse immunized with ECL1 of human piAR showed no baseline changes in the cAMP generation. However, pretreatment of these cells with ECL1 antibodies followed by stimulation with DOB surprisingly showed marked reduction in cAMP generation in contrast to adjuvant controls which showed significant increase in cAMP. While, treatment of these ECL1 antibody pretreated cells with metoprolol unexpectedly resulted in cAMP generation which is blocked in the adjuvant controls. This shows that antibodies against the ECL1 of human i AR binds to the piAR and is able to allosterically modulate downstream signaling mediated by agonist (DOB) or P-blocker (metoprolol) as at baseline it does not alter pi AR function. To validate that antibodies against the ECL1 of human piAR allosterically modulates metoprolol signaling, the cells were also pre-treated with competing doses of ECL1 peptide followed by metoprolol treatment. Consistent with these results, incubation with ECL1 peptide remarkably blocked cAMP generation in response to metoprolol. This shows that antibody binding to the ECL1 of human piAR mediates this unique signaling in response to metoprolol wherein, a traditionally cAMP blocking P-blocker metoprolol now mediates cAMP generation. These observations indicate that the allosteric binding of piAR ECL1 by the antibodies mediates unexpected downstream signal that underlie the benefits observed in the patients harboring IgG3(+) AAbs.

The presence of circulating beta-1 adrenergic receptor (Pl AR) autoantibodies (AAbs), which target the second extracellular loop (ECL2) of pi AR, has been associated with worse outcomes than those without pi AR-AAbs. We have discovered that some HFrEF patients harboring the IgG3 subclass of piAR-Aabs unexpectedly experienced better rather than worse clinical outcomes and had greater improvement in cardiac structure and function with medical therapy than those without IgG3(+) or with no piAR-AAbs. Further, in vitro studies showed that IgG3(+) pi AR-AAbs targeting ECL2 augment the favorable response to the P- blocker metoprolol by promoting instead of inhibiting cAMP production and adenylyl cyclase activity, suggesting that some AAbs may interact with specific epitopes to promote cardio protection. When anti-ECLl pi AR-AAbs were generated and tested, they exhibited the same capability as patient-derived IgG3(+) pi AR-AAbs to modulate metoprolol-mediated PIAR signaling. In addition, this effect was reversed in the presence of soluble ECL1 and ECL2 peptides, indicating the relevance of both epitopes. Our structural modeling analysis revealed that one of the complementarity determining regions for IgG3(+) piARAAbs can interact with the adjacent first extracellular loop (ECL1) of pi AR that is in close proximity of ECL2 and accessible as discontinuous epitopes for AAbs.

Therefore, our data challenge the prevailing dogma that all pi AR-AAbs exert their P1AR agonistic effects via ECL2 binding. Instead, endogenous IgG3(+) pi AR-AAbs may exert beneficial effects by engaging both ECL1 (for allosteric modulation) and ECL2 (for pi AR epitope-binding specificity) to allosterically modulate piAR signaling. Pre-treatment of piAR cells with serum from mice immunized with a pi AR ECL1 peptide showed a unique immunoallosteric modulation (IAM) similar to that generated by human IgG3(+) pi AR- AAbs, thereby providing us with a mouse model to determine the modulation of downstream signaling pathway(s) of piAR. Meanwhile, binding to ECL2 of piAR by IgG3(+) pi AR- AAbs may still confer some potential advantages especially by competitively antagonizing otherwise pathogenic non-IgG3(+) pi AR-AAbs.

In certain embodiments, the present disclosure provides systems, kits, and methods for treating a subject that has cardiovascular disease (or systemic sclerosis) with at least one of the following: a) antibodies or nanobodies targeting the ECL1 portion of piAR (e.g., WGRWEYGSFFC), or antigen-binding portion thereof; b) an antigenic peptide (e.g., WGRWEYGSFFC) that elicits the production of anti-ECLl PAR antibodies in the subject; c) a vector comprising a nucleic acid sequence encoding the ECL1 antigenic peptide, or d) an mRNA encoding an antigenic peptide (e.g., WGRWEYGSFFC) that, for example, is in a lipid nanoparticle. In certain embodiments, the subject has dilated cardiomyopathy. In some embodiments, the subject is also administered a beta-blocker).

In silico work conducted during development of embodiments herein indicated that the extracellular loop 1 (ECL1) of pi AR may serve as a potential epitope, indicating a potential binding site for CDR3 of IgG3(+) AAbs that may facilitate unique biaser signaling with ECL1 of pi AR. Peptides representing human pi AR ECL1 were synthesized and AAbs targeting the human pi AR ECL1 can be generated by immunizing C57BL6 mice and detectable by ELISA. HEK 293 cells expressing human piAR were pre-treated with serum containing AAb raised against ECL1 of piAR followed by either pi AR selective agonist (dobutamine) or antagonist (P-blocker/metoprolol). We observed that AAb against pi AR ECL1 showed the same unique capability to biasing metoprolol signaling (affecting downstream pi AR signaling was measured by cAMP generation) like the IgG3(+) pi AR- AAbs targeting ECL2 in human studies. Certain in vivo data from pi AR ECL 1 -immunized mice with 2-week isoproterenol-induced HF model demonstrated less fibrosis and cardiac remodeling compared to vehicle-infused control. To test for specificity of ECL 1 and/or 2 in the modulating piAR biaser signaling responses, the cells were incubated with soluble ECL 1 or ECL2 peptides along with AAb against ECL1 of piAR as competitors and cAMP generation assessed following metoprolol. The biaser pi AR signaling pattern observed with anti-ECLl pi AR AAb were abolished in the presence of ECL1 and ECL2 peptides as soluble antagonists, suggesting both epitopes may be relevant. Identification of specific epitopes such as ECL1 piAR allow the generation of antibodies for allosteric modulation of favorable PIAR signaling patterns. By raising antibodies targeting against ECL1 of piAR as the presumed epitope leading to favorable biaser signaling pattern similar to the human IgG3(+) pi AR AAb effects, findings demonstrate specificity and in vivo effects on fibrosis and cardiac remodeling of anti-ECLl piAR antibodies.

Current pharmacologic therapy requires GPCR antagonists that require lifelong administration, and none has leveraged an immunotherapeutic approach. Identified specific epitope (ECL1 of piAR) demonstrated of our ability to general anti-ECLl pi AR antibodies in vivo. First-in-class immunotherapy that leverages unique biaser piAR signaling pattern that has been observed in patients with human IgG3(+) pi AR AAbs demonstrating a greater likelihood of myocardial recovery in DCM is provided.

Counter-regulatory processes have long been the target for clinical investigations to identify what nature can develop to counter disease mechanisms, and auto-antibody (AAb) generation is no exception. Endogenous self-reactive AAbs have long been associated with disease progression in cardiovascular diseases (CVD) as well as other autoimmune disorders and have been postulated to act as allosteric modulators of various G-protein-coupled receptor (GPCR) signaling. Contrary to AAbs mediating deleterious signals, we recently discovered that IgG3 subclass of piAR AAbs may facilitate beneficial outcomes. There synergistic effects with beta-blocker therapy may generate a unique “divergent allosterism” in pi AR signaling underlying its cardio- protective outcomes.

Although P-blockers which form the major line of therapy for heart disease, is thought to provide beneficial outcomes by impeding the pi -adrenergic receptor (pi AR) signals from the sympathetic overdrive, their role in up-regulation of pi AR may underlie the worsening outcomes of heart failure (HF) due to binding by piAR autoantibodies (piAR-AAb) that may prolong the signal. In contrast to these unfavorable outcomes observed in HF in presence of pi AR- A Ab targeting the second extracellular loop (ECL2), our prior studies have observed that patients characterized by the presence of IgG3(+) pi AR- A Ab responded better to P- blocker treatment compared to patients harboring IgG3(-) pi AR-AAb. Mechanistic data in vitro demonstrated that pretreatment of IgG3(+) pi AR-AAb targeting ECL2 promotes a significant increase in cAMP and adenylyl cyclase in response to metoprolol rather than inhibition. In silico modeling analysis showed that in addition to the IgG(+) piAR-AAb binding to ECL2 of pi AR, the IgG3(+) piAR-AAb may interact with the adjacent extracellular loop 1 (ECL1) of pi AR. Given this unexpected and novel observation, antibodies for the first open loop peptide (OLP) were generated, and treatment of cells expressing human pi ARs showed the same unique capability to biasing metoprolol signaling like the piAR-AAbs targeting ECL2. Such effects are reversed in the presence of soluble ECL1 and ECL2 OLP, suggesting both epitopes may be relevant. In contrast to the current paradigm that piAR- AAbs primarily targets ECL2 thereby rendering agonistic effects, our evidence shows that the previously observed beneficial effects of endogenous IgG3(+) piAR-AAbs may be in part due to their engagements in both ECL1 (for allosteric modulation) and ECL2 (for epitope binding specificity) to modulate pi AR signaling.

Autoimmune response to self-antigens results in autoantibodies (AAbs) which are known to play a determinant role in cardiovascular disease progression including dilated cardiomyopathy as up to -60% may harbor AAbs against pi -adrenergic receptor (pi AR). Occurrence of pi AR AAbs in patients with DCM reflects a causal role for these AAbs. Cellular and in vivo mouse studies show that pi AR AAbs are associated with deleterious outcomes. In contrast to these findings, recent studies show that presence of a subset of pi AR AAbs belonging to the IgG3 subclass is associated with beneficial outcomes ameliorating heart failure progression. However, the signaling mechanisms that underlie these beneficial outcomes are now well known, pi AR is a powerful regulator of cardiac function and is a seven trans-membrane G-protein coupled receptor (GPCR) with three extracellular loops (ECL 1, 2 & 3), which could act as self- antigens. Transfer of serum from mice immunized with ECL2 peptide of piAR led to DCM reflecting that the AAbs generated against ECL2 of piAR could underlie the deleterious outcomes. In contrast to these findings, recent studies showed that patients harboring IgG3(+) pi AR AAb responded better to P-blocker treatment compared to patients with non-IgG3(+) pi AR-AAbs. Pre-treatment of cells with IgG3(+) pi AR AAbs purified from these patients blunted piAR-selective agonist dobutamine (DOB)- mediated cAMP response. Moreover, IgG3(+) piAR AAbs pre-treatment resulted in significant increase in adenylyl cyclase activity and cAMP generation in response to P- blocker metoprolol. An unexpected finding in the context of current paradigm of GPCR signaling, wherein activation of pi AR with DOB leads to G-protein activation/cAMP generation. Use of antagonist metoprolol blocks G-protein coupling/cAMP generation but leads to biased P-arrestin recruitment. Thus, our surprising observation that presence of IgG3(+) pi AR AAbs promotes G-protein coupling/cAMP generation upon metoprolol reflects a yet to be determined role of IgG3(+) piAR AAbs in regulating pi AR responses to its agonist/blocker. This leads to our premise that allosteric engagement of IgG3(+) 01 AR AAb to the 1 AR uniquely biases downstream signals leading to unanticipated downstream signals, wherein presence of IgG3(+) 01 AR AAbs biases a) DOB signaling towards 0-arrestin recruitment impairing G-protein activation/cAMP generation, and b) metoprolol towards G- protein activation/cAMP generation. Interestingly, serum or purified antibodies from animals immunized with 01 AR ECL2 peptide showed similar biasing, wherein treatment of cells with 01 AR Abs resulted in significant cAMP generation with metoprolol. Since less is known about this unique signaling bias mediated by the TgG3(+) 01 AR AAb, in silico modeling was performed to attain insights on IgG3(+) 01 AR AAb binding to 01 AR. Modeling surprisingly showed that, in addition to binding the ECL2 on the 01AR, the IgG3(+) 01AR AAb seems to also engage ECL1 of the 01AR. Serum from animals immunized with ECL1 peptide of 01AR showed the same unique ability to bias metoprolol signaling like the 01 AR AAbs targeting ECL2. This is contrary to the current paradigm of 01 AR AAbs engaging ECL2 to mediate deleterious outcomes (“divergent allosterism”). This leads to our hypothesis that allosteric engagement of ECL1/2 by IgG3(+) 01 AR AAbs results in unique agonist-antagonist beneficial signaling that underlies favorable outcomes.

Various procedures known in the art may be used for the production of polyclonal antibodies directed against 01 AR ECL 1. For the production of antibody, various host animals can be immunized by injection with the peptide corresponding to an 01 AR ECL 1 epitope (e.g., WGRWEYGSFFC, SEQ ID NO:1 , or portion thereof) including hut not limited to rabbits, mice, rats, sheep, goats, etc. In certain embodiments, the peptide is conjugated to an immunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyhole limpet hemocyanin (KLH)). Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette Guerin) and Corynebacterium parvum)

Monoclonal antibodies against target 1 AR ECL 1 may be produced by a variety of techniques including conventional monoclonal antibody methodologies such as the somatic cell hybridization techniques of Kohler and Milstein, Nature, 256:495 (1975). Although in some embodiments, somatic cell hybridization procedures are preferred, other techniques for producing monoclonal antibodies are contemplated as well (e.g., viral or oncogenic transformation of B lymphocytes). In general, the preferred animal system for preparing hybridomas is the murine system. Hybridoma production in the mouse is a well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

Human monoclonal antibodies (mAbs) directed against [31AR ECL1 can be generated using transgenic mice carrying the complete human immune system rather than-the mouse system. Splenocytes from the transgenic mice are immunized with the antigen of interest, which are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein. (See e.g., Wood et al., WO 91/00906, Kucherlapati et al., WO 91/10741; Lonberg et al., WO 92/03918; Kay et al., WO 92/03917 [each of which is herein incorporated by reference in its entirety]; N. Lonberg et al., Nature, 368:856-859 [1994]; L.L. Green et al., Nature Genet., 7:13-21 [1994]; S.L. Morrison et al., Proc. Nat. Acad. Sci. USA, 81:6851-6855 [1994]; Bruggeman et al., Immunol., 7:33-40 [1993]; Tuaillon et al., Proc. Nat. Acad. Sci. USA, 90:3720-3724 [1993]; and Bruggeman et al. Eur. J. Immunol., 21 : 1323-1326 [1991]).

Monoclonal antibodies can also be generated by other methods known to those skilled in the art of recombinant DNA technology. An alternative method, referred to as the "combinatorial antibody display" method, has been developed to identify and isolate antibody fragments having a particular antigen specificity, and can be utilized to produce monoclonal antibodies. (See e.g., Sastry et al., Proc. Nat. Acad. Sci. USA, 86:5728 [1989]; Huse et al., Science, 246: 1275 [1989]; and Orlandi et al., Proc. Nat. Acad. Sci. USA, 86:3833 [1989]). After immunizing an animal with an immunogen (e.g., SEQ ID NO:1) as described above, the antibody repertoire of the resulting B-cell pool is cloned. Methods are generally known for obtaining the DNA sequence of the variable regions of a diverse population of immunoglobulin molecules by using a mixture of oligomer primers and the PCR. For instance, mixed oligonucleotide primers corresponding to the 5' leader (signal peptide) sequences and/or framework 1 (FR1) sequences, as well as primer to a conserved 3' constant region primer can be used for PCR amplification of the heavy and light chain variable regions from a number of murine antibodies. (See e.g., Larrick et al., Biotechniques, 1 1: 152-156 [1991]). A similar strategy can also been used to amplify human heavy and light chain variable regions from human antibodies (See e.g., Larrick et al., Methods: Companion to Methods in Enzymology, 2: 106-110 [1991]).

Chimeric mouse-human monoclonal antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted. (See e.g., Robinson et al., PCT/US86/02269; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; WO 86/01533; US 4,816,567; European Patent Application 125,023 [each of which is herein incorporated by reference in its entirety]; Better et al., Science, 240: 1041-1043 [1988]; Liu et al., Proc. Nat. Acad. Sci. USA, 84:3439-3443 [1987]; Liu et al., J. Immunol., 139:3521-3526 [1987]; Sun et al., Proc. Nat. Acad. Sci. USA, 84:214- 218 [ 1987]; Nishimura et al., Cane. Res., 47:999-1005 [1987]; Wood et al., Nature, 314:446- 449 [1985]; and Shaw et al., J. Natl. Cancer Inst., 80:1553-1559 [1988]).

The chimeric antibody can be further humanized by replacing sequences of the Fv variable region that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General reviews of humanized chimeric antibodies are provided by S.L. Morrison, Science, 229: 1202-1207 (1985) and by Oi et al., Bio. Techniques, 4:214 (1986). Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from 7E3, an anti-GPIIbllla antibody producing hybridoma. The recombinant DNA encoding the chimeric antibody, or fragment thereof, can then be cloned into an appropriate expression vector.

Suitable humanized antibodies can alternatively be produced by CDR substitution (e.g., US 5,225,539 (incorporated herein by reference in its entirety); Jones et al., Nature, 321:552-525 [1986]; Verhoeyan et al., Science, 239:1534 [1988]; and Beidler et al., J. Immunol., 141 :4053 [1988]). All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to the Fc receptor.

An antibody can be humanized by any method that is capable of replacing at least a portion of a CDR of a human antibody with a CDR derived from a non-human antibody. The human CDRs may be replaced with non-human CDRs; using oligonucleotide site-directed mutagenesis.

Also within the scope of the invention are chimeric and humanized antibodies in which specific amino acids have been substituted, deleted or added. In particular, preferred humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, in a humanized antibody having mouse CDRs, amino acids located in the human framework region can be replaced with the amino acids located at the corresponding positions in the mouse antibody. Such substitutions are known to improve binding of humanized antibodies to the antigen in some instances.

In some embodiments, the monoclonal antibody is a murine antibody or a fragment thereof. In other embodiments, the monoclonal antibody is a bovine antibody or a fragment thereof. For example, the murine antibody can be produced by a hybridoma that includes a B cell obtained from a transgenic mouse having a genome comprising a heavy chain transgene and a light chain transgene fused to an immortalized cell. The antibodies can be full-length IgG3 antibody or can include only an antigen-binding portion (e.g., a Fab, F(ab')2, Fv or a single chain Fv fragment).

In preferred embodiments, the immunoglobulin is a recombinant antibody (e.g., a chimeric or a humanized antibody), a subunit, or an antigen binding fragment thereof (e.g., has a variable region, or at least a complementarity determining region (CDR)). In some embodiments, the immunoglobulin is monovalent (e.g., includes one pair of heavy and light chains, or antigen binding portions thereof). In other embodiments, the immunoglobulin is a divalent (e.g., includes two pairs of heavy and light chains, or antigen binding portions thereof).

In some embodiments, the present invention provides vaccine compositions comprising an antigenic protein that will generate anti-ECLl IgG3 p l AR antibodies in a subject. In certain embodiments, the antigenic protein is at least part of SEQ ID NO:1. The present invention is not limited by the particular formulation of a vaccine composition. Indeed, a vaccine composition of the present invention may comprise one or more different agents in addition to the fusion protein. These agents or cofactors include, but are not limited to, adjuvants, surfactants, additives, buffers, solubilizers, chelators, oils, salts, therapeutic agents, drugs, bioactive agents, antibacterials, and antimicrobial agents (e.g., antibiotics, antivirals, etc.). In some embodiments, a vaccine composition comprising an antigenic protein comprises an agent and/or co-factor that enhance the ability of the immunogen to induce an immune response (e.g., an adjuvant). In some preferred embodiments, the presence of one or more co-factors or agents reduces the amount of immunogen required for induction of an immune response (e.g., a protective immune response (e.g., protective immunization)). In some embodiments, the presence of one or more co-factors or agents can be used to skew the immune response towards a cellular (e.g., T cell mediated) or humoral (e.g., antibody mediated) immune response. The present invention is not limited by the type of co-factor or agent used in a therapeutic agent of the present invention.

Adjuvants are described in general in Vaccine Design-the Subunit and Adjuvant Approach, edited by Powell and Newman, Plenum Press, New York, 1995. The present invention is not limited by the type of adjuvant utilized (e.g., for use in a composition (e.g., pharmaceutical composition). For example, in some embodiments, suitable adjuvants include an aluminium salt such as aluminium hydroxide gel (alum) or aluminium phosphate. In some embodiments, an adjuvant may be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatised polysaccharides, or polyphosphazenes.

In some embodiments, the polynucleotides or mRNAs herein (e.g., UGGGGGCGGUGGGAAUACGGCUCCUUCUUCUGU (SEQ ID NO:2) for ECL1 , and CACUGGUGGAGGGCCGAGAGCGACGAAGCUCGCCGGUGUUAUAAUGACCCCAA AUGCUGUGACUUUGUAACGAAUAGA (SEQ ID NO:7) for ECL2 SEQ ID NO:7), as well as the sequences shown in SEQ ID Nos: 8-12 and 13-30, and those with at least 95% identity, comprise at least one chemical modification or chemically modified base, nucleoside, or nucleotide. The chemical modifications may comprise any modification which is not naturally present in said RNA or any naturally-occurring modification of adenosine (A), guanosine (G), uridine (U), or cytidine (C) ribonucleosides. For example, a single polynucleotide or mRNA may include both naturally-occurring and non-naturally-occurring modifications. Chemical modifications may be located in any portion of the polynucleotide or mRNA molecule and the polynucleotide or mRNA molecule may contain any percentage of modified nucleosides (1-100%, such as at least 20% ... at least 40% ... or at least 60%). In some embodiments, every particular base or nucleoside may be modified (e.g., every uridine is a modified uridine). In some embodiments, at least 20%, or 50%, or 80% of any single nucleotide (e.g., uracil) in the of the polynucleotide or mRNA is chemically modified. In some embodiments, a particular modification is used for every particular type of nucleoside or base (e.g., every uridine is modified to a 1-methyl-pseudouridine). Exemplary RNA modifications can be found in the RNA modification database (See, mods(dot)ma(dot)albany(dot)edu/home).

In some embodiments, the at least one chemical modification comprises a modified uridine residue. Exemplary modified uridine residues include, but are not limited to, pseudouridine, 1 -methylpseudouridine, 1 -ethylpseudouridine, 2-thiouridine, 4'- thiouridine, 5-methyluridine, 2-thio-l -methyl- 1 -deaza-pseudouridine, 2- thio-l-methyl-pseudouridine, 2- thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio- dihydrouridine, 2-thio- pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy- pseudouridine, 4-thio-l-methyl- pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-0-methyl uridine. In some embodiments, the at least one chemical modification comprises a modified cytosine residue. Exemplary nucleosides having a modified cytosine include 5 -aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5-methyl-cytidine, 5-halo-cytidine, 5-hydroxymethyl-cytidine, 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2- thio-cytidine, 2-thio-5- methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l- methyl-l-deaza-pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocytidine, zebularine, 5-aza- zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy- cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-l -methyl - pseudoisocytidine, lysidine, a-thio-cytidine, 2'-O-methyl-cytidine, 5,2'-O-dimethyl-cytidine, N4-acetyl-2'-O-methyl-cytidine, N4,2'-O-dimethyl-cytidine, 5-formyl-2'-O-methyl-cytidine, N4,N4,2'-O-trimethyl-cytidine, 1 -thio-cytidine, 2'-F-aracytidine, 2'-F-cytidine, and 2'-OH- aracytidine.

In some embodiments, the at least one chemical modification comprises a modified adenine residue. Exemplary nucleosides having a modified adenine include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, 2-amino-6-methyl-purine, 8-azido- adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza- 2- amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl- adenosine, 2-methyl-adenine, N6-methyl-adenosine, 2-methylthio-N6-methyl-adenosine, N6- isopentenyl-adenosine, 2-methylthio-N6-isopentenyl-adenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6- glycinylcarbamoyl-adenosine, N6-threonylcarbamoyl-adenosine, N6-methyl-N6- threonylcarbamoyl-adenosine, 2-methylthio-N6-threonylcarbamoyl-adenosine, N6,N6- dimethyl-adenosine, N6-hydroxynoryalylcarbamoyl-adenosine, 2-methylthio-N6- hydroxynoryalylcarbamoyl-adenosine, N6-acetyl-adenosine, 7-methyl-adenine, 2-methylthio- adenine, 2-methoxy-adenine, a-thio-adenosine, 2'-O-methyl-adenosine, N6,2'-O-dimethyl- adenosine, N6,N6,2'-O-trimethyl-adenosine, l,2'-O-dimethyl-adenosine, 2' 0- ribosyladenosine (phosphate), 2-amino-N6-methyl-purine, 1 -thio-adenosine, 8-azido- adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino- pentaoxanonadecyl)-adenosine.

In some embodiments, the at least one chemical modification comprises a modified guanine residue. Exemplary nucleosides having a modified guanine include inosine, 1 - methyl-inosine, wyosine, methylwyosine, 4-demethyl-wyosine, isowyosine, wybutosine, peroxy wybutosine, hydroxywybutosine, undermodified hydroxywybutosine, 7-deaza- guanosine, queuosine, epoxy queuosine, galactosyl-queuosine, mannosyl-queuosine, 7-cyano- 7-deaza- guanosine, 7-aminomethyl-7-deaza- guanosine, archaeosine, 7-deaza-8-aza- guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7- methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1- methyl-guanosine, N2-methyl-guanosine, N2,N2-dimethyl-guanosine, N2,7-dimethyl- guanosine, N2,N2,7-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1- methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a- thio-guanosine, 2'-O-methyl-guanosine, N2-methyl-2'-O-methyl-guanosine, N2,N2-dimethyl- 2'-O-methyl-guanosine, 1 -methyl-2'-O-methyl-guanosine, N2,7-dimethyl-2'-O-methyl- guanosine, 2'-O-methyl-inosine, l,2'-O-dimethyl-inosine, and 2'-O-ribosylguanosine (phosphate).

The compositions herein (e.g., immunogenic compositions) may further comprise excipients or pharmaceutically acceptable carriers. The choice of excipients or pharmaceutically acceptable carriers will depend on factors including, but not limited to, the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

Excipients and carriers may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. Some examples of materials which can serve as excipients and/or carriers are sugars including, but not limited to, lactose, glucose and sucrose; starches including, but not limited to, com starch and potato starch; cellulose and its derivatives including, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients including, but not limited to, cocoa butter and suppository waxes; oils including, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols; including propylene glycol; esters including, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents including, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants including, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants. The compositions of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Techniques and formulations may be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). The compositions may be formulated for any particular mode of administration including for example, systemic administration, oral, rectal, nasal, sublingual, buccal, implants, or parenteral. The compositions or polynucleotides (e.g., mRNA) described herein may be used to prepare vaccines or another medicament (e.g., for treating or preventing cardiovascular disease) based on inducing an immune response against at least a portion of 01 AR ECL 1 (e.g., SEQ ID NO:1).

The vaccine and medicament may comprise an adjuvant or immunostimulant, or a polynucleotide encoding an adjuvant or immunostimulant (e.g., an adjuvantive polypeptide). Adjuvants and immunostimulants are compounds or compositions that either directly or indirectly stimulate the immune system’s response to a co-administered antigen. In some embodiments, the vaccines are not adjuvanted or are self-adjuvanting.

Suitable adjuvants are commercially available as, for example, Glucopyranosyl Lipid Adjuvant (GLA); Pam3CSK4; Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham); mineral salts (for example, aluminum, silica, kaolin, and carbon); aluminum salts such as aluminum hydroxide gel (alum), A1K(SO4)2, AlNa(SO4)2, A1NH4(SO4), and Al(0H)3; salts of calcium (e.g., Ca3(PO4)2), iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polynucleotides (for example, poly IC, poly AU acids, and CpG oligodeoxynucleotides (e.g., Class A or B)); polyphosphazenes; cyanoacrylates; polymerase- (DL-lactide-co- glycoside); bovine serum albumin; diphtheria toxoid; tetanus toxoid; edestin; keyhole-limpet hemocyanin; Pseudomonal Toxin A; choleragenoid; cholera toxin; pertussis toxin; viral proteins; Quil A; aminoalkyl glucosamine phosphate compounds. In addition, adjuvants such as cytokines (e.g., GM-CSF or interleukin-2, -7, or -12), interferons, or tumor necrosis factor, may also be used as adjuvants. Protein and polypeptide adjuvants may be obtained from natural or recombinant sources according to methods well known to those skilled in the art. When obtained from recombinant sources, the adjuvant may comprise a protein fragment comprising at least the immunostimulatory portion of the molecule.

Other known immunostimulatory macromolecules which can be used include, but are not limited to, polysaccharides, tRNA, non-metabolizable synthetic polymers such as polyvinylamine, polymethacrylic acid, polyvinylpyrrolidone, mixed polycondensates (with relatively high molecular weight) of 4',4-diaminodiphenylmethane-3,3'-dicarboxylic acid and 4-nitro-2- aminobenzoic acid (See, Sela, M., Science 166: 1365-1374 (1969)) or glycolipids, lipids or carbohydrates.

In some embodiments, the adjuvantive polypeptide comprises immune activator proteins, such as CD70, CD40 ligand, and constitutively active TLR4, or polycationic peptides (e.g., protamine). In some embodiments, the adjuvantive polypeptide is a flagellin polypeptide. Commercially available mRNA encoding adjuvantive polypeptides are available, for example, as TriMix (See Bonehill, A. et al. Mol. Ther. 16, 1170-1180 (2008), incorporated herein by reference). In some embodiments, the vaccine may comprise at least two separate polynucleotides, one encoding at least a portion of SCF1 and the other encoding an adjuvantive polypeptide (e.g., a flagellin polypeptide or immune activator protein).

The compositions may comprise a delivery vehicle. Exemplary delivery vehicles include, but are not limited to, microparticle compositions comprising poly(lactic acid) (PLA) and/or poly(lactic-co-glycolic acid) (PLGA), albumin nanoparticles, and liposomal compositions. The vaccines may further comprise a delivery vehicle. In some embodiments, the vaccine comprises a lipid nanoparticle encapsulating the disclosed compositions, polynucleotides, or mRNA.

Lipid nanoparticle compositions of the disclosure may include one or more cationic and/or ionizable lipids, phospholipids, neutral or non-cationic lipids, polyethylene glycol (PEG)-lipid conjugates, and/or sterols. In some embodiments, the lipid nanoparticle comprises a cationic lipid and/or ionizable lipid, a neutral or non-cationic lipid, and cholesterol. Cationic and/or ionizable lipids include, for example, amine-containing lipids that can be readily protonated and may have a positive or partial positive charge at physiological pH due to a pKa value between pH 5 and 8. The polar headgroup of the cationic lipids preferably comprises amine derivatives such as primary, secondary, and/or tertiary amines, quaternary ammonium, various combinations of amines, amidinium salts, or guanidine and/or imidazole groups as well as pyridinium, piperazine and amino acid headgroups such as lysine, arginine, ornithine and/or tryptophan. Cationic lipids include, but are not limited to, l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-di-O- octadeceny 1-3 -trimethylammonium propane (DOTMA) and/or l,2-dioleoyl-3- trimethylammonium propane (DOTAP), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium bromide (DMRIE), didodecyl(dimethyl)ammonium bromide (DDAB), l,2-dioleyloxypropyl-3 -dimethylhydroxyethyl ammonium bromide (DORIE), 3P-[N — (N\N'-dimethylamino- ethane)carbamoyl]cholesterol (DC-Chol) or dioleyl ether phosphatidylcholine (DOEPC). Ionizable lipids include, but are not limited to, l,2-dioleyloxy-3-dimethylamino-propane (DODMA).

In some embodiments, the lipid nanoparticle comprises a polyethylene glycol (PEG)- lipid conjugate. A PEG-lipid conjugate may include, but is not limited to, PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacyl glycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-DMG (1,2- dimyristoyl-rac-glycero-3-methoxypoly ethylene glycol), PEG-c-DOMG (R-3-[(co-methoxy poly(ethylene glycol)2000)carbamoyl)]-l,2-dimyristyloxlpropyl-3-amine), PEG-DMA (PEG- dimethacrylate), PEG-DLPE (l,2-didodecanoyl-sn-glycero-3-phosphoethanolamine-PEG), PEG-DMPE (PEG- l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), PEG-DPPC (PEG- dipalmitoyl phosphatidylcholine), PEG-N,N-di(tetradecyl)acetamide, or a PEG-DSPE (1, 2- distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)) lipid. In some embodiments, the lipid nanoparticle comprises PEG-DMG and/or PEG-N,N- di(tetradecyl)acetamide.

The sterol may comprise cholesterol, fecosterol, ergosterol, campesterol, sitosterol, stigmasterol, brassicasterol or a sterol ester, such as cholesteryl hemisuccinate, cholesteryl sulfate, or any other derivatives of cholesterol. A neutral or non-cationic lipid may include one or more phospholipids. Phospholipids include a phospholipid moiety and one or more fatty acid moieties. A phospholipid moiety may include, but is not limited to, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and sphingomyelin. A fatty acid moiety may include, but is not limited to, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

Phospholipids suitable for use in the compositions may include, but are not limited to, phosphatidylglycerol (PG) including dimyristoyl phosphatidylglycerol (DMPG) and 1 ,2- dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG); phosphatidylcholine (PC), including egg yolk phosphatidylcholine, dimyristoyl phosphatidylcholine (DMPC), 1,2- distearoyl-sn-glycero-3-phosphocholine (DS PC), l,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 ,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di- O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl-sn- glycero- 3 -phosphocholine (Cl 6 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-diarachidonoyl-sn-glycero-3- phosphocholine; phosphatidylethanolamine (PE) including l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- diarachidonoyl-sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine; phosphatidic acid (PA); phosphatidylinositol (PI); phosphatidylserine (PS); and sphingomyelin (SM).

The positively charged lipid structures described herein may also include other components typically used in the formation of vesicles (e.g., for stabilization). Examples of such other components includes, without being limited thereto, fatty alcohols, fatty acids, and/or any other pharmaceutically acceptable excipients which may affect the surface charge, the membrane fluidity and assist in the incorporation of the lipid into the lipid assembly.

The vaccine or medicament of the present disclosure may also contain other compounds, which may be biologically active or inactive. For example, one or more immunogenic and antigenic portions of polypeptides or polynucleotides encoding immunogenic polypeptides, or nucleic acid(s) encoding thereof, may be present within the vaccine. The vaccine or medicament may generally be used for prophylactic and therapeutic purposes.

The vaccines or medicaments may be formulated for any appropriate manner of administration, and thus administered, including for example, topical, oral, nasal, intravenous, intravaginal, epicutaneous, sublingual, intracranial, intradermal, intraperitoneal, subcutaneous, intramuscular administration, or via inhalation.

The vaccines or medicaments may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, solutes that render the formulation isotonic, hypotonic, or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives. Alternatively, vaccines or medicaments may be formulated as a lyophilisate. The compositions and vaccines may be prepared, packaged, or sold in a form suitable for bolus administration or sold in unit dosage forms, such as in ampules or multidose containers. In some embodiments, the compositions and vaccines contain a preservative.

EXAMPLES

EXAMLPE 1

Immunization mice with the betal adrenergic receptor protein Five-to-six-week-old mice were injected with 50ug of the 1st extracellular loop (ECL1) of the betal adrenergic receptor protein peptide (Genescript) (with sequence WGRWEYGSFFC SEQ ID NO: 1), emulsified in 200uL of Incomplete Freund’s Adjuvant (Sigma- Aldrich). A general schematic of the protocols for this example is shown in Figure 4A. Before the first injection, cardiac function was assessed via echocardiography, and blood was collected from the submandibular vein. The peptide was injected every 2 weeks for a total of 3 doses. One week after the final dose, blood was drawn from each mouse for an ELISA to determine the antibody response titer and assess if additional doses were needed. The final boost was administered three weeks after the ELISA confirmed an antibody response.

Osmotic pumps (Alzet, 2002) infused with either saline or isoproterenol (ISO) were implanted subcutaneously in the mice for 2 weeks. Cardiac function was assessed both before and after the osmotic pump implantation on a weekly basis. Tissues (heart, plasma, and spleen cells) were harvested two weeks post-implantation. Plasma samples were utilized for in vitro testing and ELISA. One set of heart tissues was allocated for molecular or biochemical studies, while another set was reserved for histology.

For the ECL1 and ECL2 injection experiments, plasma samples were collected at prebleed, one week post-final dose, and at the endpoint (see Figure 4B). The plasma from the ECL1 and ECL2 injections was used for in vitro experiments. It is noted that mice could be injected with both ECL1 peptide (e.g., WGRWEYGSFFC SEQ ID NO: 1, or 8-12 consecutive amino acids therefrom) and ECL2 peptide (e.g., HWWRAESDEARRCYNDPKCCDFVTNR, SEQ ID NO:3, or 10-15 consecutive amino acids therefrom).

We have done three experiments. There were four groups in each experiment. Group 1 : ECL1 immunized mice with ISO pumps. Group 2: non-immunized mice with ISO pumps. Group 3: control mice with saline pumps. Group 4 ECL1 immunized mice with saline pumps Experiment 1: n= 5 for each group. However, we did not conjugate the ECL1 peptide with KLH, which should help with the enhancement of the immune response. The ECL1 antibody reaction was not ideal. All samples were fixed for histology assays. The results of antibody titer tested using ELSIA are shown in Figure 5.

Experiment 2: n=3-6. ECL1 peptide was conjugated with KLH. ECL1 antibody titer for each mouse was between 1:500-1 :2500, some with a better reaction around 1 :2500- 1 : 12500. Three mice per group for biochemistry study and two for histology analysis. The antibody titer is better than experiment 1. Antibody titer comparison among three experiments are shown in Figure 5. Histology results are shown in Figure 6A-B. Experiment 3: n=6 for groups 1, 2, 4. n=4 for the Saline control group. Three for histology and three for molecular and biochemistry study. Spleen cells were collected from 12 ECL-injected mice. Antibody titer comparison among three experiments are shown in Figure 5. Histology results are shown in Figures 6B and 6C-D.

Discussion of results: The immunized mice conjugated with ECL1 were able to produce an antibody response with a titer ranging from 1 :500 to 1:125,000. ECL1 peptide- immunized mice showed reduced cardiac fibrosis induced by isoproterenol, although the reduction was not statistically significant when compared to the control+ISO group.

REFERENCES

1. Jahns R, Boivin V, Schwarzbach V, Ertl G, Lohse MJ. Pathological autoantibodies in cardiomyopathy. Autoimmunity. 2008;41(6):454-61. Epub 2008/09/11.

2. Stork S, Boivin V, Horf R, Hein L, Lohse MJ, Angermann CE, Jahns R. Stimulating autoantibodies directed against the cardiac beta 1 -adrenergic receptor predict increased mortality in idiopathic cardiomyopathy. Am Heart J. 2006;152(4):697-704. Epub 2006/09/26.

3. Jahns R, Boivin V, Hein L, Triebel S, Angermann CE, Ertl G, Lohse MJ. Direct evidence for a beta 1 -adrenergic receptor-directed autoimmune attack as a cause of idiopathic dilated cardiomyopathy. J Clin Invest. 2004; 113(10) : 1419-29. Epub 2004/05/18.

4. Nagatomo et al.,. Autoantibodies Specifically Against pi Adrenergic Receptors and Adverse Clinical Outcome in Patients With Chronic Systolic Heart Failure in the P-Blocker Era: The Importance of Immunoglobulin G3 Subclass. J Card Fail. 2016 Jun;22(6):417-22.

5. Nagatomo et al., IMAC-2 Investigators. Myocardial Recovery in Patients With Systolic Heart Failure and Autoantibodies Against pi -Adrenergic Receptors. J Am Coll Cardiol. 2017 Feb 28;69(8):968-977.

6. Mohan et al., The IgG3 subclass of pi -adrenergic receptor autoantibodies is an endogenous biaser of piAR signaling. Mol Biol Cell. 2021 Apr l;32(7):622-633.

7. Tang WHW, Naga Prasad SV. Autoantibodies and Cardiomyopathy: Focus on Beta-1 Adrenergic Receptor Autoantibodies. J Cardiovasc Pharmacol. 2022 Sep 1 ;80(3) :354-363.

Although only a few exemplary embodiments have been described in detail, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this disclosure. Accordingly, all such modifications and alternative are intended to be included within the scope of the invention as defined in the following claims. Those skilled in the art should also realize that such modifications and equivalent constructions or methods do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.

Claims

CLAIMS What is claimed is:

1. A method of treating a subject that has cardiovascular disease (CVD), or Systemic

Sclerosis (SS), comprising: treating a subject that has CVD or SS with at least one of the following: a) a first antigenic protein that elicits the production of anti-ECLl pi AR antibodies in said subject, optionally wherein said anti-ECLl (31AR antibodies are IgG3 antibodies; optionally wherein said first antigenic protein comprises a least eight consecutive amino acids from SEQ ID NOs: l, 31, or 32, optionally wherein said first antigenic protein is conjugated to a carrier protein which is optionally KLH; b) a second antigenic protein comprising a portion of human (31 AR ECL1, or modified portion thereof, linked to a portion of human [31 AR ECL2, optionally wherein said second antigenic protein is conjugated to a carrier protein which is optionally KLH; optionally wherein said portion of [ 1 AR ECL1 comprises at least 8 consecutive amino acids from WGRWEYGSFFC (SEQ ID NO:1); and optionally wherein said portion of [ 1 AR ECL2 comprises at least 10 consecutive amino acids from HWWRAESDEARRCYNDPKCCDFVTNR (SEQ ID NO:3), and optionally wherein said antigenic protein comprises at least 8 consecutive amino acids from SEQ ID NOs: 33 and 35; c) a mixture of a third antigenic protein comprising at least a portion of human 31 AR ECL1 or modified portion thereof, and a fourth antigenic protein comprising at least a portion of human piAR ECL2; d) a vector comprising a nucleic acid sequence encoding: i) said first and/or second antigenic protein, or ii) said third and fourth antigen proteins; e) a first mRNA sequence encoding at least an eight amino acid portion of ECL1 of human pi -Adrenergic Receptor, or modified portion thereof, optionally wherein said first mRNA sequence comprises at least 24 consecutive nucleotides from the ECL1 mRNA sequence in SEQ ID NO:2, 8-12, 36, or 37 optionally wherein said first mRNA sequence comprises modified bases and is present in a liposome nanoparticle; f) said first mRNA sequence and a second mRNA sequence, wherein said second mRNA sequence encodes at least 10 amino acid portion of ECL2 of human pi -Adrenergic Receptor or modified portion thereof, and wherein said second mRNA sequence comprises at least 24 consecutive nucleotides from the ECL2 mRNA nucleic acid sequence in SEQ ID N0:7, 13-30, 38, or 40, and optionally wherein said first and second mRNA sequences comprise modified bases and are present in a liposome nanoparticle; and/or g) a fifth antigenic protein comprising at least 8 or 10 consecutive amino acids from SEQ ID NOs. 4, 5, or 6, or a vector comprising a nucleic acid sequence encoding said at least 8 or 10 consecutive amino acids from SEQ ID NOs: 4, 5, or 6.

2. The method of Claim 1, wherein said subject is negative, or lower than a control, for IgG3 pi AR antibodies prior to said treatment.

3. The method of Claim 1, wherein said CVD comprises dilated cardiomyopathy.

4. The method of Claim 1, further comprising: testing a blood, serum, or plasma sample from said subject for the presence of IgG3 pi AR antibodies prior to said treatment.

5. The method of Claim 1, wherein said subject is a human.

6. The method of Claim 1, wherein said portion of iAR ECL1 comprises at least 8 consecutive amino acids from WGRWEYGSFFC (SEQ ID NO:1)

7. The method of Claim 1 , wherein said treating comprises intravenous treatment.

8. The method of Claim 1, wherein said vectors comprises a plasmid, adeno-associated virus, or adeno-associated virus.

9. The method of Claim 1, wherein said subject is on a P-blocker prior to said treatment.

10. The method of Claim 1, further comprising: administering a P-blocker to said subject prior to, during, or after said treatment.

11. The method of Claim 10, wherein said P-blocker is selected from the group consisting of: Acebutolol, Atenolol, Bisoprolol, Metoprolol, Nadolol, Nebivolol, and Propranolol.

12. The method of Claim 1, wherein said portion of piAR ECL2 comprises at least 10 consecutive amino acids from HWWRAESDEARRCYNDPKCCDFVTNR (SEQ ID NOG);

13. A kits, system, or composition comprising: a) at least one of the following: i) a first antigenic protein that elicits the production of anti-ECLl [51 AR antibodies in said subject, optionally wherein said anti-ECLl (31 AR antibodies are IgG3 antibodies; optionally wherein said first antigenic protein comprises a least eight consecutive amino acids from WGRWEYGSFFC (SEQ ID NO: 1) or SEQ ID NOs:31 or 32, optionally wherein said first antigenic protein is conjugated to a carrier protein, which is optionally KLH; ii) a second antigenic protein comprising a portion of human (31 AR ECL1 , or modified version thereof, linked to a portion of human [31 AR ECL2, optionally wherein said second antigenic protein is conjugated to a carrier protein, which is optionally KLH; optionally wherein said portion of [31AR ECL1 comprises at least 8 consecutive amino acids from WGRWEYGSFFC (SEQ ID NO: 1) or SEQ ID NOs: 31 or 33; and optionally wherein said portion of [31 AR ECL2 comprises at least 10 consecutive amino acids from HWWRAESDEARRCYNDPKCCDFVTNR (SEQ ID NO:3) or SEQ ID NOs:33 or 35; iii) a mixture of a third antigenic protein comprising at least a portion of human (31 AR ECL1, and a fourth antigenic protein comprising at least a portion of human [31AR ECL2; iv) a vector comprising a nucleic acid sequence encoding: i) said first and/or second antigenic protein, or ii) said third and fourth antigen proteins; vii) a first mR A sequence encoding at least an eight amino acid portion of ECL1 of human [31 -Adrenergic Receptor, optionally wherein said first mRNA sequence comprises at least 24 consecutive nucleotides from the ECL1 mRNA, or modified version thereof, sequence shown in SEQ ID NO:2, 8-12, 36, or 37, optionally wherein said first mRNA sequence comprises modified bases and is present in a liposome nanoparticle; and/or viii) said first mRNA sequence and a second mRNA sequence, wherein said second mRNA sequence encodes at least 10 amino acid portion of ECL2 of human [31- Adrenergic Receptor, and wherein said second mRNA sequence comprises at least 24 consecutive nucleotides from ECL2 mRNA sequence shown in SEQ ID NO:7, 13-30, 38, or 40, and optionally wherein said first and second mRNA sequences comprise modified bases and are present in a liposome nanoparticle; and/or viii) a fifth antigenic protein comprising at least 8 or 10 consecutive amino acids from SEQ ID NOs. 4, 5, or 6, or a vector comprising a nucleic acid sequence encoding said at least 8 or 10 consecutive amino acids from SEQ ID NOs: 4, 5, or 6, and b) a P-blocker.

14. A method comprising: a) receiving results of, or conducting, an IgG3 p I -Adrenergic Receptor IgG3 P1AR antibodies (IgG3 [31AR antibodies) level analysis on a sample from a subject with cardiovascular disease (CVD) or Systemic Sclerosis (SS), and b) performing at least one of the following after identifying said sample as having higher levels of said IgG3 (31 AR antibodies compared to control levels, i) treating said subject with any of the reagents recited in claim 1 , and/or ii) providing a report to said patient or medical personnel treating said patient, indicating said subject is suitable for, or should be, treated with any of the reagents recited in claim 1.

15. The method of Claim 14, wherein said control levels are from the general population or CVD patients not stratified by IgG3 p I AR antibody levels.

16. A composition comprising: a first engineered polynucleotide encoding at least a portion of human P1AR extra cellular loop 1 (ECL1) or modified version thereof, wherein the first engineered polynucleotide is at least partially optimized for enhanced expression, productive co-translational protein folding, increased stability, or a combination thereof.

17. The composition of claim 16, wherein said first engineered polynucleotide comprises at least 24 consecutive nucleotides from any of SEQ ID NOs:2 or 8-12 or 36-37.

18. The composition of claim 16, further comprising a second engineered polynucleotide encoding at least a portion of human piAR extra cellular loop 2 (ECL2).

19. The composition of claim 18, wherein said second engineered polynucleotide comprises at least 24 consecutive nucleotides from any of SEQ ID NOs:7, 13-30, 38, or 40.

20. The composition of claim 16, wherein the polynucleotide comprises RNA, and optionally wherein said RNA is partially or fully human codon optimized.

21. The composition of claims 16-20, wherein the first engineered polynucleotide further comprises or encodes: a 5' untranslated region (UTR), a 5’ cap, a 3' UTR, a 3’ tailing sequence, or any combination thereof.

22. The composition of claim 21, wherein the 5’ UTR, 3’ UTR, or both are heterologous to the polynucleotide encoding at least a portion of human [31 AR ECL1.

23. The composition of claim 21, wherein the 3’ tailing sequence comprises a poly A tail, a polyG quartet, a stem loop sequence, a triple helix forming sequence, a tRNA-like sequence, or any combination thereof.

24. The composition of any of claims 16-23, wherein the first engineered polynucleotide, and optionally said second engineered polynucleotide, comprises at least one chemically modified nucleotide.

25. The composition of claim 24, wherein the at least one chemically modified nucleotide comprises a modified uracil.

26. The composition of claim 25, wherein at least 60% of the uracil in the first and/or second polynucleotide are chemically modified.

27. The composition of any of claims 24-26, wherein the at least one chemically modified nucleotide comprises 5-methylcytosine or N 1 -methylpseudouridine (ml ).

28. The composition of any of claims 16-27, wherein said first engineered polynucleotide comprises a nucleotide sequence having: i) at least 75% sequence identity to SEQ ID NOs: 2, 8-12, 36, or 37, or a complement or reverse complement thereof, wherein T may be replaced by U, and/or ii) at least 24 consecutive nucleotides from SEQ ID NOs: 2, 8-12, 36, or 37, or a complement or reverse complement thereof, wherein T may be replaced by U.

29. The composition of any of claims 16-28, wherein the human [31 AR ECL1 comprises: i) an amino acid sequence having at least 90% identity to SEQ ID NO:1, 31, or 32 and/or ii) at least 12, at least 15, consecutive amino acids from SEQ ID NO:1, 31, or 32.

30. The composition of any of claims 18, wherein said human piAR ECL2 comprises: i) an amino acid sequence having at least 90% identity to any one of SEQ ID NO:3, 33, or 35, and/or ii) at least 12 or 15 consecutive amino acids from SEQ ID NO: 3, 33, or 35.

31. A vaccine comprising: the composition of any of claims 16-30; and at least one adjuvant, a delivery vehicle, or a combination thereof.

32. The vaccine of claim 31, wherein the delivery vehicle comprises a lipid nanoparticle encapsulating the composition.

33. The vaccine of claim 32, wherein the lipid nanoparticle comprises a cationic lipid, a neutral and/or non-cationic lipid, a sterol, or any combination thereof.

34. The vaccine of claim 33, wherein the non-cationic lipid comprises a phospholipid.

35. The vaccine of claim 33, wherein the sterol comprises cholesterol or a modification or ester thereof.

36. The vaccine of any of claims 32-35, wherein the lipid nanoparticle comprises a polyethylene glycol (PEG)-lipid conjugate.

37. A composition comprising: a first immunogenic protein comprising at least a portion of human 01AR ECL1, wherein said first immunogenic protein is conjugated to a hapten, and/or wherein said composition further comprises an adjuvant and/or a delivery vehicle.

38. The composition of claim 37, wherein said first immunogenic protein comprises: i) an amino acid sequence having at least 90% identity to SEQ ID NO: 1, 4, 5, 6, 31, or 32, and/or ii) at least 10 or at least 12 or at least 15 consecutive amino acids from SEQ ID NO:1, 4, 5, 6, 31, or 32.

39. The composition of claim of any of claims 37-38, wherein said hapten is selected from the group consisting of: fluorescein, biotin, digoxigenin, and dinitrophenol.

40. The composition of claim 37, further comprising a second immunogenic protein comprising at least a portion of human [31 AR extra cellular loop 2 (ECL2).

41. The composition of claim 40, wherein said second immunogenic protein comprises i) an amino acid sequence having at least 90% identity to SEQ ID NO:3, 33, or 35, and/or ii) at least 10 or at least 12 or at least 15 consecutive amino acids from SEQ ID NO:3, 33, or 35.