PLoS One. 2015 Feb 20;10(2):e0117589. doi: 10.1371/journal.pone.0117589.

Novel receptor-derived cyclopeptides to treat heart failure caused by anti-β1-adrenoceptor antibodies in a human-analogous rat model. 
 

Boivin V1, Beyersdorf N2, Palm D3, Nikolaev VO4, Schlipp A5, Müller J6, Schmidt D2, Kocoski V2, Kerkau T2, Hünig T2, Ertl G7, Lohse MJ1, Jahns R1.
  • 1Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany; Rudolf-Virchow-Center/DFG-Research-Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany; Comprehensive Heart Failure Centre (CHFC), University Hospital of Würzburg, Würzburg, Germany.
  • 2Institute for Virology and Immunobiology, University of Würzburg, Würzburg, Germany.
  • 3Rudolf-Virchow-Center/DFG-Research-Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany.
  • 4Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany.
  • 5Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany; Rudolf-Virchow-Center/DFG-Research-Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany; Lehrstuhl Anatomie I, University of München (LMU), München, Germany.
  • 6Institute of Pathology, University of Würzburg, Würzburg, Germany.
  • 7Comprehensive Heart Failure Centre (CHFC), University Hospital of Würzburg, Würzburg, Germany.

 

Abstract:

Despite recent therapeutic advances the prognosis of heart failure remains poor. Recent research suggests that heart failure is a heterogeneous syndrome and that many patients have stimulating auto-antibodies directed against the second extracellular loop of the β1-adrenergic receptor (β1EC2). In a human-analogous rat model such antibodies cause myocyte damage and heart failure. Here we used this model to test a novel antibody-directed strategy aiming to prevent and/or treat antibody-induced cardiomyopathy. To generate heart failure, we immunised n=76/114 rats with a fusion protein containing the human β1EC2 (amino-acids 195-225) every 4 weeks; n=38/114 rats were control-injected with 0.9% NaCl. Intravenous application of a novel cyclic peptide mimicking β1EC2 (β1EC2-CP, 1.0 mg/kg every 4 weeks) or administration of the β1-blocker bisoprolol (15 mg/kg/day orally) was initiated either 6 weeks (cardiac function still normal, prevention-study, n=24 (16 treated vs. 8 untreated)) or 8.5 months after the 1st immunisation (onset of cardiomyopathy, therapy-study, n=52 (40 treated vs. 12 untreated)); n=8/52 rats from the therapy-study received β1EC2-CP/bisoprolol co-treatment. We found that β1EC2-CP prevented and (alone or as add-on drug) treated antibody-induced cardiac damage in the rat, and that its efficacy was superior to mono-treatment with bisoprolol, a standard drug in heart failure. While bisoprolol mono-therapy was able to stop disease-progression, β1EC2-CP mono-therapy -or as an add-on to bisoprolol- almost fully reversed antibody-induced cardiac damage. The cyclo­peptide acted both by scavenging free anti-β1EC2-antibodies and by targeting β1EC2-specific memory B-cells involved in antibody-production. Our model provides the basis for the clinical translation of a novel double-acting therapeutic strategy that scavenges harmful anti- β1EC2-antibodies and also selectively depletes memory B-cells involved in the production of such antibodies. Treatment with immuno-modulating cyclopeptides alone or as an add-on to β1-blockade represents a promising new therapeutic option in immune-mediated heart failure.

PMID: 25700031


Supplement:

When thinking about the huge size of the super-family of G protein-coupled receptors (GPCRs), it becomes evident that the substances having the potential to modulate such receptors are innumerable. Among them, we observe in the literature a constantly growing amount of reports of new noxious autoantibodies targeting GPCR, resulting in or exacerbating human pathologies. Substantial efforts have been made to understand their mechanisms of action that may lead to severe deregulations of cells targeted by such antibodies. This represents an opportunity but also a major challenge for the development of novel specific therapies. The aim of the present study was to develop a new strategy to specifically neutralise disease-inducing autoantibodies.

First, we begged the question of whether peptides would be capable to act as epitope-specific antibody-scavengers in vivo. Second, we hypothesized that cyclisation of peptides would increase their stability in vivo and would better mimic the epitope-structure (Fig. 1). Furthermore, the use of a variety of epitope-specific peptides might, in the future, result in the development of more personalized therapeutic approaches.

 

 

fig1Fig. 1. Scheme of the human β1-adrenergic receptor highlighting the second extracellular loop β1-ECII and cyclopeptides (ECII-CP) mimicking its structure. The colored drawing shows the supposed mechanism of antibody-scavenging ECII-CP that prevent the binding of antibodies to the β1-adrenergic receptor (β1-AR).

 

In idiopathic dilated cardiomyopathy, which is characterized by a loss in cardiac contractility of unknown aetiology, an association of the disease with autoimmune reactions against various myocardial antigens has been reported. During the last two decades, we have focused our research on functionally active antibodies directed against the second loop of the human β1-adrenergic receptor (β1-ECII), which are supposed to play a paramount role in the pathogenesis of dilated cardiomyopathy.

In order to check the correctness of our assumptions, we induced autoimmune cardiomyopathy in a human-analogous rat model by monthly active immunization of the animals with β1-ECII fusion proteins as antigens. All immunized animals developed stimulatory anti-β1-antibodies and in the course of the study slowly progressive dilatation and pump failure of the left ventricle starting eight to nine months after the first immunization. The induced cardiomyopathic phenotype was monitored by echocardiography and was confirmed by invasive measurements and histological analysis of the excised hearts. The model appeared thus suitable for testing novel therapeutic approaches.

We first generated a β1-ECII-homolo­gous cyc­lic pep­tide (β1-ECII-CP) bearing the entire β1-ECII sequence composed of 25 amino acids (ECII-25CP). As peptide-length increases immunogenicity, but also increases the risk for unspecific cross-reactivity, we supposed that shortening the sequence would reduce the immunogenicity and improve specificity of the cyclopeptide. We then used our rat model of immune cardiomyopathy to assess the therapeutic effect of a long-term application (12 months, monthly injection) of the generated antibody-neutralizing ECII-25CP and, also, of a novel cysteine/serine ECII-CP mutant composed of 18 amino acids (ECII-18CPm). The obtained effects were compared with those of monthly injections of their non-cyclised linear counterparts (ECII-25LIN and ECII-18LINm).

In contrast to untreated immunized rats, in CP-treated animals the initial β1-ECII-antibody titres after four injections of ECII-25CP dropped to less than 20% (17.3%) of the starting-levels. Also, ECII-18CPm within 9 to 10 treatment-months achieved a significant decrease in β1-ECII-antibodies (35.6%) compared to starting-levels. By contrast, linear peptides were clearly less efficient and until study-end reduced β1-ECII-antibodies to only 56.9% (ECII-25LIN) or 53.7% (ECII-18LINm) of the initial antibody-concentrations (Fig. 2a). Functional parameters and histological morphometry of the hearts revealed a comparable efficacy regarding reversal of the cardiomyopathic phenotype in ECII-25CP and in ECII-18CPm treated rats, whereas their linear counterparts ECII-25LIN and ECII-18LINm had no obvious cardio-protective effects. In CP-treated animals left ventricular end-systolic (Fig. 2b) and end-diastolic diameters (not shown) were reverted to control levels; concordantly, cardiac function (i.e., cardiac index CI) improved significantly (not shown).

 

 

 

fig2

Fig. 2 . Course of anti-β1 ECII-antibodes and corresponding echocardiographic parameters in the therapy-study.

2a: Time course of anti–β1 ECII-titers in the therapy study measured before and 24h after CP-injections. Presented  are the remaining antibody-concentrations over 9 treatment-months in percent of the respective starting levels. For better readability error-bars are not shown in the graph.

2b: Echocardiographic follow-up in the therapy-study. Graphs show the time-course of left ventricular end-systolic diameters in mm over 20 study-months.

 

In conclusion, our pre-experiments demonstrated, that reversal of the β1-ECII antibody-induced DCM phenotype in a rat model of immune-cardiomyopathy appeared feasible and was indeed achieved. By scavenging harmful stimulatory β1-ECII-antibodies, both cyclopeptides (ECII-25CP and its mutant ECII-18CPm) were almost equipotent in reversing cardiac dilatation and in normalizing cardiac function. Moreover, concordant with our assumption in vivo only cyclic but not linear ECII-peptides were able to achieve relevant cardio-protective effects.

The described results encouraged us to further explore the neutralizing effects of cyclic peptides in our rat model and to develop therapeutic strategies as well as application schemes that could be suited also for human patients.

In previous studies, we were able to detect autoantibodies against recombinant human β1-receptors in about 30% of the patients suffering from heart failure due to idiopathic dilated cardiomyopathy; these patients had a significantly decreased cardiac function compared to β1-receptor antibody-negative patients (Jahns, 1999). In these former studies we have shown that the stimulatory effect of the antibodies could be at least partly abolished by the cardio-selective β1-receptor blocker bisoprolol. (Nikolaev, 2007). Although cardio-selective β1-blockers are standard therapeutics in heart failure, they may be not sufficient to cure dilated immune cardiomyopathy and, thus, alternative specific therapeutic approaches for the treatment of antibody-positive patients are needed.

The next logical step was to compare the therapeutic effects of our scavenger cyclopeptides alone or, in order to reproduce the condition of an already treated patient, in combination with bisoprolol.

It turned out that cyclopeptides mimicking the β1-ECII, in fact, prevented and treated antibody-induced heart failure in a larger study performed with our DCM rats, and that they were superior to the β1-receptor blocker bisoprolol alone. In overt disease, the cyclopeptide almost fully reversed the cardiomyopathic phenotype, whereas bisoprolol only stopped disease progression. The cyclopeptide not only scavenged anti-β1-ECII autoantibodies from the circulating blood but, unexpectedly, also blocked the expansion of anti β1-ECII-expressing memory B-cells in the spleen despite continued antigen-boosts.

The removal of potentially harmful β1-receptor antibodies from the circulation of patients by non-specific immunoadsorption or immunoapharesis has already been described. However, this both time and labour consuming procedure remains an expensive invasive intervention associated with hospitalization of the treated patients. In addition, due to the non-specific elimination of all immunoglobulins from the blood, numerous drawbacks arise such as an imbalance in the immune system resulting in altered immune responses. Nevertheless, it seems that antibody-positive DCM patients have at least a short-term hemodynamic benefit from such a procedure.

In this context, immunomodulating epitope-mimicking cyclopeptides might open new venues in the treatment of immune-mediated heart failure. Our study confirms the potential benefit of epitope-specific cyclic antibody-scavengers in a human-analogous rat model and thus, probably also in human autoimmune heart failure. More generally, it is tempting to broaden this concept by making the scavenger-principle which at the same time appears to address the cause of the disease, that is, the antibody-producing immune-cells themselves available also for the treatment of other autoimmune diseases.

 

References

  1. Jahns R, Boivin V, Siegmund C, Inselmann G, Lohse MJ, Boege F 1999 Autoantibodies activating human beta1-adrenergic receptors are associated with reduced cardiac function in chronic heart failure. Circulation 1999;99:649–654.
  2. Nikolaev VO, Boivin V, Störk S, Angermann CE, Ertl G, Lohse MJ, Jahns R 2007 A novel fluorescence method for the rapid detection of functional beta1-adrenergic receptor autoantibodies in heart failure. J Am Coll Cardiol 50:423–431.

 

 

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