EP2391637A1 - Novel peptide-homologues for inhibiting beta1-adrenoceptor antibodies - Google Patents

Novel peptide-homologues for inhibiting beta1-adrenoceptor antibodies

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Publication number
EP2391637A1
EP2391637A1 EP10706168A EP10706168A EP2391637A1 EP 2391637 A1 EP2391637 A1 EP 2391637A1 EP 10706168 A EP10706168 A EP 10706168A EP 10706168 A EP10706168 A EP 10706168A EP 2391637 A1 EP2391637 A1 EP 2391637A1
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EP
European Patent Office
Prior art keywords
cys
cyclic peptide
amino acid
peptide
arg
Prior art date
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EP10706168A
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German (de)
French (fr)
Inventor
Roland Jahns
Valérie JAHNS
Martin J. Lohse
Hans-Richard Rackwitz
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Julius Maximilians Universitaet Wuerzburg
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Julius Maximilians Universitaet Wuerzburg
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Publication of EP2391637A1 publication Critical patent/EP2391637A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor

Definitions

  • the present invention relates to novel ⁇ -AR homologous cyclopeptide-mutants comprising only two cysteine residues able to form an intramolecuiar linkage and comprising an ⁇ -aminobuty ⁇ c acid (ABu) residue, or an analogue thereof, which replaces the cysteine residue corresponding to cysteine 216 of the human ⁇ i-AR protein sequence.
  • the present invention further relates to linear peptides that can form these cyclopeptide-mutants and to nucleic acid molecules encoding peptides from which these cyclopeptide-mutants and linear peptides can be manufactured.
  • vectors comprising the nucleic acid molecules and recombinant host ceils comprising the nucleic acid molecules or vectors are provided. Further, a method for producing the disclosed cyclopeptide-mutants is provided. Further disclosed is a composition comprising the peptide(s), nucleic acid molecuie(s), vector( ⁇ ) or host celi(s) of the invention.
  • the present invention also relates to therapeutic and diagnostic means, methods and uses taking advantage of the peptide(s)s of the invention and to means, methods and uses for detecting anti- ⁇ -adrenergic receptor antibodies, in particular anti- ⁇ i-adrenergic receptor antibodies.
  • DCM dilated cardiomyopathy
  • Such injury most likely occurs upon acute infectious (myocarditis), toxic, or ischemic heart disease (myocardial infarction) resulting in myocyte apoptosis or necrosis (Caforio 2002, Rose 2001).
  • ischemic heart disease myocardial infarction
  • Myocyte apoptosis or necrosis apoptosis or necrosis
  • Presentation of myocardial self-antigens to the immune system may then induce an autoimmune response, which in the worst case results in perpetuation of immune-mediated myocyte damage involving either cellular (e.g., T- cell), or humoral (e.g., B-cell) immune responses, or co-activation of both the innate and the adaptive immune system (Eriksson 2003, Rose 2001).
  • anti-muscarinic antibodies ⁇ exhibiting an agonist-like action on the cardiac M2 acetylcholine-receptor have been mainly associated with negative chronotropic effects at the sinuatrial level (e.g., sinus node dysfunction, atrial fibrillation (Baba 2004, Wang. 1996))
  • agonistic anti- ⁇ i-AR antibodies have been associated with both the occurrence of severe arrhythmia at the ventricular level (Christ 2001 , iwata 2001a), and the development of (maladaptive) left ventricular hypertrophy, finally switching to left ventricular enlargement and progressive heart failure (Iwata 2001b, Jahns 1999b, Khoynezhad 2007). Both autoantibodies appear to be directed against the second extracellular loop of the respective receptors.
  • myocyte membrane proteins e.g., receptors
  • myocyte membrane proteins e.g., receptors
  • oligopeptides able to form a complex with a MHC or HLA class Il molecule of the host
  • Hoebeke 1996 the human ⁇ r AR computer-based analysis for potential immunogenic amino-acid ⁇ treches has shown, that the only portion of the receptor molecule containing B- and T-cell epitopes and being accessible to antibodies was in fact the predicted second extracellular receptor loop ( ⁇ rECu) (Hoebeke 1996). This might explain the successful use of second loop-peptides for the generation of ⁇ rspecific receptor antibodies in different animal-models (Iwata 2001b, Jahns. 2000, Jahns 1996).
  • DCM anti- ⁇ r AR induced dilated immune-cardiomyo-pathy
  • beta- blocking agents in order to attenuate or even abolish the autoantibody-mediated stimulatory effects, at least if ⁇ b!ockers can indeed prevent the antibody-induced activation of [J 1 -AR (Freedman 2004, Jahns 2000, Matsui 2001 , Jahns 2006).
  • New therapeutic approaches actually include elimination of stimulatory anti- ⁇ i-AR by nonselective or selective immunoadsorption (Hershko 2005, Wallukat 2002), or direct targeting of the anti- ⁇ rECn antibodies and/or the ant ⁇ - ⁇ rECn producing B-celis themselves (that is, induction of immune tolerance) (Anderton 2001).
  • Non-selective immunoadsorption because of an increased risk of infection after immunoglobulin depletion, requires the substitution of human IgG on the ground of safety (Felix 2000) with all possible side effects of substituted human proteins known in the art including severe anaphylactic reactions and death.
  • WO 01/21660 discloses certain peptides homologous to epitopes of the 1 st and the 2 n ⁇ loop of ⁇ i-AR, and proposes to apply these peptides for medical intervention of dilatative cardiomyopathy (DCM). Even if WO 01/21660 mentions marginally that peptides may be modified in order to protect them against serum proteases, for example by cyciization, corresponding examples and embodiments are not given and any in vitro or in vivo effect of the proposed peptides on the course of DCM or on the course of receptor-antibody titers is not shown. Moreover, in WO 01/21660 intends to rely on the above mentioned non-selective immunoadsorption approaches bearing the correspondingly mentioned risks.
  • DCM dilatative cardiomyopathy
  • ⁇ rECirhomologous cyclopeptides e.g. ⁇ rECn-CPs
  • ⁇ -i-ECn-CPs are cyclopeptides containing 3 cysteine (Cys) residues and hence, can form intramolecular bonds, whereby there is a potential option to form two different intramolecular bonds (besides the cyciization between the N- and C-terminus), individually.
  • ⁇ i-ECn-CP significantly reduced the amount of circulating anti- ⁇ rECn antibodies and effectively prevented development of cardiac dilatation and dysfunction (Boivin 2005).
  • the above-mentioned ⁇ -i-ECn-CPs were also disclosed in WO 2006/103101.
  • the technical problem underlying the present invention is the provision of improved and easily obtainable means and methods for the medical intervention of diseases related to anti- ⁇ -AR antibodies, in particular to anti- ⁇ -i-EC-H antibodies.
  • the technical problem is solved by provision of the embodiments characterized in the claims.
  • the present invention relates to ⁇ -AR homologous cyclopeptide-mutants (a!so termed herein as "cyclic peptides” or “cyclopeptides” and the like), particularly to Ji 1 -AR homologous cyclopeptide-mutants, namely ⁇ rEC-n homologous cyclopeptide-mutants ( ⁇ i-EC-n-CPs)
  • cyclic peptides or "cyclopeptides” and the like
  • Ji 1 -AR homologous cyclopeptide-mutants namely ⁇ rEC-n homologous cyclopeptide-mutants ( ⁇ i-EC-n-CPs)
  • cyclopeptide-mutants/cyciic peptides are structurally characterized in that they are able to form only one individual intramolecular disulphide bond and that they almost perfectly imitate/mimic the three-dimensional steric conformation of (the epitope(s) presented in) the native antibody-recognition and
  • the present invention relates to a cyclic peptide of formula I:
  • x is an amino acid other than Cys
  • b) h is any integer from 1 to 15
  • c) i is any integer from 0 to 14
  • d) one of x a , x b and x c is Pro
  • e) y is ⁇ -aminobutyric acid (ABu) or an ABu analogue
  • f) the cyclic peptide consists of at least 16 and of at most 25 amino acids.
  • cyclic peptide is, as discussed below, specific cyclic peptides as depicted in formulas VlI. IX, IX ' , Vl or VIII.
  • the present invention solves the above identified technical problem since, as documented herein below and in the appended examples, it was surprisingly found that in particular ⁇ i-EC ⁇ r CPs containing only two Cys residues, which can form one single defined, individual intramolecular disulfide bond, and having the Cys-residue corresponding to -Cys 216 of ⁇ -i-AR replaced by an ABu residue, are also able to inhibit anti- ⁇ -AR antibodies, and are particularly usefu! for the inhibition of stimulatory anti- ⁇ i-AR antibodies with high effectiveness.
  • ⁇ rEC )r CPs having the Cys residue corresponding to Cys 216 of ⁇ r AR replaced by an ABu residue have in fact a significantly higher antibody-blocking efficiency (at least in vivo) than ⁇ i-ECn-CPs having said Cys residue replaced by another Cys-like residue (for example by a serine (Ser) residue).
  • the Cys/ABu mutants as disclosed herein have a higher capacity to inhibit binding of anti-beta1-ECII antibodies to beta1-ECI!-peptides, i.e.
  • Non-invasive (Fig. 14 A-C) and invasive assessment (Fig. 14 D-F) of cardiac function made in the context of the appended examples also points towards a better cardioprotection in vivo achieved with cyclopeptides disclosed herein.
  • the cyc22AA mutants of this invention block (or scavenge) conformational antibodies even better than previously described ECIi-homologous larger cyclopeptides (e.g. cyc25AA peptides) or smaller cyclopeptides (e.g. cyc18AA peptides) known in the art (WO 2006/103101) with the 22AA Cys/Abu mutant being even more efficient than the 22AA Cys/Ser mutant (Fig.
  • inventive cyclic peptides comprising only two cysteines, which can form one single defined, individual intramolecular disulfide bond, can easily be obtained/manufactured, biochemically characterized and purified. This is particularly true when pure fractions of the same cyclopeptide isomers are required.
  • a mixture of cyclopeptide isomers, i.e. stereoisomers, comprising cyclopeptide isomers with different intramolecular disulphide bonds is avoided.
  • a specific and clean medical product fullfilling GLP/GMP standards
  • Cys-S-S-Cys disulfide bridge may either spontaneously occur, or be chemically installed by treatment with 3% DMSO (forced S-S bridge) in order to accomplish the stability criteria according to current GLP-/GMP-rules as a prerequisite for its use either as pharmaceutical substance or as diagnostic agent in human (heart) disease.
  • DMSO forced S-S bridge
  • a Cys ⁇ Ser exchange like that at position 18 of the herein exemplarily and preferably disclosed 25-meric cyclopeptide, at position 17 of the herein exemplar ⁇ y and preferably disclosed 22-meric cyclopeptide or at position 14 of the herein exempiarily and preferably disclosed 18-meric cyclopeptide, respectively, yields cyclic peptides (Cys-Ser cyclic peptides) with excellent antibody-neutralizing and pharmacological effects in vitro (Figs.
  • Cys ⁇ ABu exchange like that a position 17 of the herein exemplariiy and most preferably disciosed 22-meric cyclopeptide (formula IX') yields cyclic peptides (Cys-ABu cyclic peptides) with antibody-neutra ⁇ zing and pharmaceutical effects comparable (in vitro; see, e.g. Fig. 12) with respect to the corresponding 22-meric (and 18-meric) Cys-Ser cyclic peptides.
  • Cys ⁇ ABu exchange like that at position 18 of the herein exemplariiy and preferably disclosed 25-meric cyclopeptide (formulas VII/IX) or at position 14 of the herein exemplariiy and more preferably disclosed 18-meric cyclopeptide (formulas VI ⁇ /lll), respectively, yields further Cys-ABu cyclic peptides with comparably excellent or even more pronounced antibody-neutralizing and cardioprotective effects in vivo ⁇ Fig. 13B and Fig. 14A-F).
  • the cardioprotective and immunomoduiating activity of the cyclic peptides largely depends on their conformation. It was additionally found out in the context of this invention that an introduction of the smallest naturally occuring amino-acid glycine at the (predicted) ring closure site (or at the position corresponding thereto, Fig. 11) leads to an enhanced binding of anti- ⁇ rAR autoantibodies, i.e. apparently further enhances the similarity of, for example, the 22 AA cyclopeptide with the ECII-P 1 -AR domain.
  • the appended examples indicate that the 22AA cyclopeptides have a significantly higher antibody- blocking efficiency in vivo than other ECil-imitating cyclopeptides larger (i.e., 25AA cyclopeptides) or smaller (i.e., 18AA cyclopeptides).
  • Computer-aided modelling studies with said 22AA cyclopeptide confirmed an excellent imitation of the predicted second extracellular loop structure with a calculated difference in size of only 4.5 Angstrom (4.5 A) at the base of the cyclopeptide (opposed to the assumed antibody- binding site), when compared with the predicted native second extraceilufar loop backward helix (see also appended Fig. 11).
  • cyclic 22AA cyclopeptide Since repiacement of one of the three cysteines present in the cyclic 22AA peptide allows for the introduction of a reinforced disulfide bridge (as a second "internal" cycle, generated by double cyclization) between the two remaining cysteines, the resultant cyclic 22AA cyclopeptide also represents a biochemically unambiguously defined product (Fig. 16 (HPLC), Fig. 17 (MS/MS-spectra)).
  • cyclic peptides as disclosed herein show improved features, for example as compared to peptides comprising three Cys residues (for example the Cys-Cys cyciic peptides disclosed in WO 2006/103101 ).
  • improved features of the cyclic peptides of this invention are an extremely good capacity for blocking anti- ⁇ r AR antibodies both, in vitro (Fig.12) and in vivo (Fig. 13) and their advanced producibiiity according to GLP/GMP standards.
  • the in vitro findings were generally confirmed in in vivo tests (Figs. 5-10 and 13/14).
  • the established rat model of anti-beta 1 -adrenergic antibody-induced autoimmune- cardiomyopathy served to assess the efficacy of the generated beta1-ECII homologous cycSopeptide mutants in vivo.
  • the peptide-mutants with a cyclic structure are superior to their linear counterparts in terms of the recognition or scavenging of conformational anti- ⁇ -AR antibodies, their antibody-neutralizing (i.e. pharma-ceuticai) potential an their half-life in crude serum (for example: linear ⁇ 8h, cyclic >12h; in particular: linear 6 ⁇ 1h, cyclic 48 ⁇ 12h).
  • the antibody-blocking capacity of mutated cyclopeptides of this invention is advantageous high, as long as the peptide is not shorter than 18AA and not longer than 25AA. This was exempiar ⁇ y demonstrated by the reduction of the number of amino acids of the peptide from 25 to 18. However, within the range of 18 to 25 amino acids, cyclic peptides having 22 amino acids are most effective in accordance with this invention and, accordingly, are a particular preferred embodiment. An example of such a particular preferred 22-rner cyclic peptide is shown in formula IX ' .
  • cyclopeptide mutants of the present invention is - by mutating one particular cysteine (the Cys corresponding to Cys216 of the amino acid sequence of ⁇ rAR) to a ABu residue and by reinforcing formation of the unique possible intramolecular S-S bridge through a S-S specific cyclization procedure - that their conformational restraint is increased.
  • this increased restraint of the inventive peptides leads to a molecule that better mimics the epitope(s) presented in the native conformation of the second ⁇ i-ECn ioop on the celi surface.
  • cyclopeptide of the present invention was shown to block the activity of betai -receptor autoantibodies isolated from human DCM patients, as exemplariiy shown for IgG-fractions prepared from a 28 years-old female patient (see Fig.
  • Beta blockers such as bisoprolol, which are used in the art for the treatment of DCM and other diseases which are caused by stimulatory anti- ⁇ -i-AR antibodies, significantly reduce both heart rate and blood pressure.
  • an in wVo-application of the mutant cyclopeptides as disclosed in context of the present invention has no negative impact on lung function, heart rate or biood pressure (Figs. 10 and 14 D-F).
  • a number of important laboratory parameters to assess liver and kidney function were not influenced by repeated cyciopeptide injections (Fig. 14 G).
  • the cyclic peptides disclosed in context of the present invention are, inter alia, particularly suitable for the treatment of distinct patient groups which otherwise could not be treated by using a beta blocker, i.e. patients who, for example, already suffer from bradycardia or for whom the use of beta blockers is not possible because of contraindications (like those suffering from obstructive lung disease or hypotension).
  • the cyclic peptides of the present invention can easily be characterized and produced as pure fractions of the same isomer. This leads to a high reproducibility. Accordingly one particular advantage of the peptides of the present invention is that mixtures of isomers, which have to be separated and must be characterized in laborious testings, are avoided, and that at least one further production step (separation and/or biochemical characterization) can finaliy be omitted (see also Sewald 2002).
  • the present invention is, inter alia, based on the experiments described in the appended examples.
  • one of the cysteines either at position 17 or at position 18 of the ⁇ i -EC-ii 25AA-cyclopeptide or the cysteine at position 14 of the ⁇ rEC r 18AA-cyclopeptide was replaced by a serine residue ⁇ Cys 17 or i8 ⁇ Seri 7 or 1 8 mutation and Cysu ⁇ Ser ⁇ mutation, respectively), or the cysteine at position 17 of the ⁇ r EC ⁇ r 22AA-cyclopeptide was replaced by an ABu residue (Cys 17 ⁇ ABu 17 mutation), so that onSy one individual, single intramolecular disulfide bond (S-S) can be formed (Fig. 11 ,).
  • cyclic peptides of this invention can be obtained in contrast to the peptides of the prior art which form mixtures of isomers, by simple, robust and highly reproducible manufacturing processes. These can be scaled up efficiently. Furthermore these processes avoid separation of isomers mixtures and are suitable for GLP/GMP standards.
  • the appended examples provide for corresponding manufacturing/production methods.
  • the cyclization of the inventive peptides was, inter alia, obtained by the introduction of a "GIy” mutation, e.g. at the (ring) closure site of the cyclic peptide (Fig. 11 ).
  • the number of amino acids (AA) was reduced from 25AA to 22AA and further to 18AA in further sets of cyclopeptide-mutants of the present invention.
  • This measure provides the potential to minimize the potential immunologic side effects of the constructs and to screen for/identify the optional length of the cyctopepttde- mutants within the range of 18 to 25 amino acids.
  • the 18AA or 25AA cyclopeptide- mutants contained a cysteine- ⁇ ABu exchange at position 14 and 18, respectively, (18AA containing CyS 13 -ABu 14 mutant cyclopeptides and 25AA containing Cysi 8 ⁇
  • Ser 17 mutant-cyciopeptides optionally combined with a (further) glutamine-/D ⁇ glutamic acid-exchange, e.g. at the ring closure site of the cyc ⁇ c peptide (Gln ⁇ D-Glu mutation).
  • the preferred 22AA cyclopeptide-mutants contained a cysteine- ⁇ ABu exchange at position 17 (22AA containing Cys 16 -ABui 7 ), optionally combined with the introduction of a GIy residue at position 22 (a possible ring closure site of the cyclic peptide; Fig. 11).
  • the herein provided experimental in vitro data as weli as the in vivo data clearly demonstrate that the antibody-blocking capacity of the disclosed mutant cyciopeptides is not markedly affected by the reduction of the number of amino acids from a 25 ⁇ meric to a 22-meric or 18-meric cyclopeptide, for example, when using a dose ranging from 0.25 to 5.0 mg/kg body weight (Bw) or from 1.0 to 2.0 mg/kg Bw or, in particular, from 0.3 to 2.0 mg/kg Bwor.
  • a preferred example of such an "intermediate" cyclic peptide is a cyclic peptide comprising or consisting of the amino acid residues as shown in formula IX ' .
  • the exact nature of the exchange of one of the cysteine residues with a serine residue i.e., Cys/Ser (ABu) or Ser (ABu)/Cys-mutation) markedly determined the potency of the disclosed cyclic peptides in vitro and aiso in vivo (Figs. 2, 3, 6, 7, 13 and 14).
  • One preferred embodiment of the present invention is based on in vivo experiments addressing different modes of application/administration of the herein disclosed cyclic peptides, in particular of the aforementioned ⁇ r ECn epitope mimicking 22AA cyclopeptide mutants. These in vivo experiments are also described in the appended examples. In the context of these examples the cysteine at position 17 of the ⁇ rECIl- 22AA-cyclopeptide was replaced by an ABu residue (Cys17 ⁇ ABu17 mutation), so that only one individual, single intramolecular disulfide bond (S-S) can be formed (Fig. 11 ).
  • the different application/administration protocols as disclosed herein comprise either monthly intravenous applications of the herein disclosed cyclopeptide mutants (e.g.
  • cyc22AACys/Abu a triple injection (for example, one injection every week on 3 subsequent weeks) every three months.
  • Administration schemes fike these provide the potential to reduce the amount of injected cyciopeptides, to reduce the burden of (regular monthly) venipuncture and/or to increase the flexibility of application (for both, human patients and animals); whilst maintaining or increasing the biological efficiency of the injected constructs of the present invention.
  • ⁇ -adrenergic receptors ⁇ -ARs
  • ⁇ rARs particulary ⁇ i-adrenergic receptors
  • SEQ ID NO. 24 the nucleotide and amino acid sequence (SEQ ID NO. 24) of the human ⁇ rAR (also known as adrenergic ⁇ -1 -receptor (ADRB1 )) can be obtained from databank entry NM_000684 or NP_0O0675.
  • ⁇ -ARs are known to form two extracellular domains termed herein as ECi and ECn or p (1) -ECi and ⁇ ( i r ECn.
  • the cyclic peptides of the present invention share sequence similarity with ⁇ i-ECn, particularly with the amino acid stretch DEARRCYNDPKCCDFV (SEQ ID NO. 17) or RAESDEARRCYNDPKCCDFVTNR (SEQ ID NO. 18) of the human ⁇ r AR (amino acid positions 204 to 219 or 200 to 222, respectively) or, particularly, with the amino acid stretch DEARRCYNDPK (SEQ ID NO. 29) or ESDEARRCYNDPK (SEQ ID NO. 30) of the human ⁇ r AR.
  • DEARRCYNDPKCCDFV amino acid stretch DEARRCYNDPKCCDFV
  • RAESDEARRCYNDPKCCDFVTNR SEQ ID NO. 18
  • ⁇ -AR as used herein preferably refers to a ⁇ -adrenergic receptor (P 1 -AR) 1 more preferably to the human ⁇ i-AR as described above.
  • P 1 -AR ⁇ -adrenergic receptor
  • a cyclic peptide provided herein may have at least one of the features selected from the group consisting of: a) being capable of binding (auto-)antibodies against the ECu loop of ⁇ i- adrenergic receptor ( ⁇ -pAR); b) being capable of inhibiting the interaction between ⁇ r AR and (auto-)antibodies against the ECn ioop of ⁇ i-AR; c) mimicking at least one epitope presented in the native conformation of the ECn loop of ⁇ i-AR; and d) being capable of reducing an antibody-mediated activation of ⁇ i-AR.
  • the cyclic peptide of the present invention is defined by the genera! formula cycio(x-X h ⁇ Cys ⁇ x-x a -x b -x c -x-Cys-y-Xj-x) (formula I).
  • "y” is ABu ( ⁇ -aminobutyric acid) or an ABu analogue.
  • ABu is a non-naturaily occurring; i.e. non-genetically coded, amino acid weli known in the art. It is, for example, also known as alpha-aminobutyric acid, AABA 1 2-aminobutyhc acid, 2-aminobutanoic acid etc.
  • ABu is, for example, depicted by the chemical formulas CH 3 CH 2 CH(NH 3 )COOH or C 4 H 9 NO 2 . It is preferred in context of this invention that Abu is L-Abu (L- ⁇ -aminobutyric acid). However, Abu may also be D-Abu (D- ⁇ - aminobutyric acid).
  • Wlodaver (Science, 1989, 245 (4918): 616-21 ) describes the determination of the crystal structure of chemically synthesized HIV-1 protease based on the Rous sarcoma virus protease structure having the cysteines replaced by ABu.
  • “y” of formula I may be any ABu analogue, as long as this amino acid does not form an intramolecular linkage (e.g. a disulphide bond) with another amino acid of the cyclic peptide provided herein (in particular with another Cys of the cyclic peptide provided herein), “y” may be any ABu analogue similar to Cys (i.e. having a similar chemical structure and/or a similar "structural behavior" within a 3 dimentional peptide structure), with the exeption that it does not form an intramolecular linkage (e.g.
  • ABSu analogue in context of this invention means a residue, particularly an amino acid residue, having a structural character similar to that of ABu.
  • the term “ABu analogue” particularily refers to a(n) (amino acid) residue having a chemical structure and/or "behavior” within a 3 dimentional peptide structure which is more similar to that of ABu itself than to that of any other amino acid residue, like, for example, to that of Ser.
  • the meaning of the term “ABu analogue” as used throughout this application is clear to the skilled person.
  • Particular "ABu analogues” in accordance with the present invention may be those, the C-side chain of which is shortened or elongated as compared to ABu itself.
  • the C-residues of the C-chain may be up to 10, preferably up to 6.
  • such "ABu analogues" with a shortened or elongated C-side chain are 2-arninopropionic acid and, preferably, 2-aminopentanoic acid or 2-aminohexanoic acid.
  • ABS as referred to herein, in particular in the depicted formulas, refers to both, an ABu analogue or, which is preferred, ABu itself.
  • cyclic peptides of this invention comprise only two Cys able to form an intramolecular linkage.
  • Such cyclic peptides can, for example, be obtained by substituting a third Cys of a peptide homologous to the ⁇ i-ECn by a different amino acid.
  • the Cys to be substituted is the one corresponding to the 2 nd or, which is preferred, 3 rd Cys of the [VECn which lie in direct proximity to each other (amino acid position 215 and 216 of human ⁇ 1 -AR (see also NP__000675 and SEQ ID NO. 24).
  • Cys residues are referred to herein as "Cys-Cys”, “Cys/Cys”, “Cys2 15 -Cys2is” or “Cys2i5/Cys 2 i6” and the like).
  • mutant peptides or mutations or "Ser ⁇ Cys", “Ser/Cys”, “SePi 3 0 M 7 -CyS 14 0 Me” or "Ser 13 ⁇ r i7/Cysi 4 or ie” mutant peptides or mutations, depending on which of the Cys' is replaced and how many amino acids the mutant peptide comprises (18, 22 or 25).
  • the mutant peptides as disclosed herein are defined by referring to the particular amino acid exchanges at a certain position.
  • mutant peptides/ mutations are, for example, termed "CyS 14 , i 7 O r i ⁇ ABu- ⁇ 4 , 17 or is” mutant peptides/ mutations, Cysi 4 , i7 or i 8 ⁇ *Seri4, 17 or is” mutant peptides/mutations or “Cysi3 or i 7 ⁇ Seri 3 or i 7 " mutant peptides/mutations, depending on whether the Cys corresponding to Cys 2 ie or the Cys corresponding to the Cys 2 is, respectively, of ⁇ r AR is replaced by a different amino acid and how many amino acids the mutant peptide comprises.
  • indices "13, 16 or 17”, “14, 17 or 18", “13 or 17” or “14 or 18” relate to the position in the exemplified cyclic peptide of the invention, whereby position 1 corresponds to the first "x" as defined in formula I, i.e cycio ⁇ x-X h -Cys-x-x a -x b -x c -x-Cys- y-x,-x).
  • Cys 17 ⁇ ABur-i 7 respectively, mutant peptides/mutations and, in this particular example, refer to 22mer peptides disclosed herein.
  • Terms like “Cys 17 -Seri 8 " or “Cysi 7 /Ser 18 " mutant peptides/mutations are used in the same sense as “Cysi 8 ⁇ Ser 18 " mutant peptides/mutations and, in this particular example, refer to 25mer peptides disclosed herein.
  • the exemplar ⁇ y indices given above refer to the position of the indicated amino acid within the herein disclosed particular 18-mer, 22-mer or 25-mer peptide, respectively.
  • the starting point with respect to an indicated amino acid position given for a cyclic peptide disclosed herein is the N-terminal amino acid of the linearized backbone the cyclic peptide (like the first "x" in formula I, see above).
  • the starting point with respect to an indicated amino acid position given for a linear peptide disclosed herein is its N-terminai amino acid.
  • h can be any integer from 1 to 15, preferably from 5 to 9, and/or i can be any integer from 0 to 14, preferably from 1 to 14, more preferably from 0 to 6 and even more preferably from 1 to 6. Accordingly, h can be 1 , 2, 3, 4, 5, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 and/or i can be 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14.
  • h is 5, 8 or 9 and/or i is 3, 4 or 6. More preferably, h is 8 and/or i is 4.
  • x h stands for the particular amino acid stretches DEARR (SEQ ID NO. 19), AESDEARR (SEQ ID NO. 31 ) or RAESDEARR (SEQ ID NO. 20) and/or X 1 stands for the particular amino acid stretches DFV (SEQ ID NO. 21 ), DFVT (SEQ ID NO. 32) or DFVTNR (SEQ ID NO. 22).
  • x h stands for the particular amino acid stretch AESDEARR (SEQ ID NO. 31 ) and/or x, stands for the particular amino acid stretch DFVT (SEQ ID NO. 32).
  • the cyclic peptide of the present invention may consist of at least 18 amino acids and of at most 25 amino acids. Accordingly, the cyclic peptide of the present invention may consist of 18, 19, 20, 21 , 22, 23, 24 or 25 amino acids, whereby particularly 18 or 25 amino acids are preferred and particularly 22 amino acids are most preferred. In a less preferred embodiment, also smaller peptides, i.e. peptides comprising 16 or 17 amino acids are envisaged.
  • the number of amino acids and thus the length of the primary structure (i.e. the amino acid backbone) of cyclic peptides binding anti- ⁇ i-AR antibodies is crucial for their biologica! effects and/or successful/effective manufacture.
  • a peptide-length equal or above 26 amino acids may be stimulating directly (that is, without the use of carrier proteins) immunocompetent T- celts and thus may provoke an undesired paradoxal increase of anti- ⁇ 1 -receptor antibody production through T-cell mediated B-cell stimulation.
  • a peptide-length below 16 amino acids leads to undesired crystallization during the production process and problems in dissolving the synthesized products in an aqueous solution, e.g. for purposes of i.v. or s.c. injections
  • cyclic peptides falling under the above given definitions a) to f) of formula I but consisting of only 17 amino acids or, even less preferred, consisting of only 16 amino acids are provided.
  • a non-limiting example of such a less preferred cyclic peptide is the peptide cyclo(Ala-Arg-Arg-Cys- Tyr-Asn-Asp-Pro-Lys-Cys-ABu-Asp-Phe-Vaf-Tyr-Gln/DGIu) (formable by an amino acid backbone as depicted in SEQ ID NO. 23).
  • the disclosed cyclic peptide contains only one Pro. Accordingly, it is particularly preferred that the "x" of the formulas depicted herein, except of exactly one of x a , x b and x c , is not Pro. Within the amino acid stretch x a , x b and x c as depicted in formula I (or in other formulas), it is preferred that x c is Pro.
  • an acidic amino acid like Asp or GIu precedes the Pro contained in the cyclic peptide of the invention.
  • x b as depicted in formula I is an acidic amino acid, like GIu or, preferably, Asp.
  • x c is Pro
  • x b may be an acidic amino acid
  • x b is Pro
  • x a may be an acidic amino acid
  • the x of formula I (or of other formulas) lying between x a and the first Cys may be an acidic amino acid, wherein the acidic amino acid may be one as particularly described herein.
  • x a is Asn
  • x b is Asp
  • x c is Pro and/or x following x c is Lys.
  • the amino acid strech x a -x b -x c as depicted in the formulas provided herein is Asn-Asp-Pro (N-D-P).
  • the amino acid strech x a -x b -x c -x as depicted in the formulas provided herein is Asn-Asp-Pro- Lys (N-D-P-K).
  • cyclic peptide of the present invention may be defined by formula I ' or I " :
  • cyclic peptide of the present invention may be defined by formula Y “ or 1 " “ :
  • x ( and x M as depicted in formula I ' and I ' " (and in the other formulas depicted herein) may, as mentioned, any amino acid but Cys.
  • are such amino acids able to form a peptide bond, or the like, with each other under conditions of a "head to tail” cyclization.
  • Head to tail cyclizations are known in the art (e.g.
  • x N as referred to in formula I ' and Y" can be GIn or GIu, wherein GSu may also be DGIu (D-GIu; D-Glutamic acid).
  • GSu may also be DGIu (D-GIu; D-Glutamic acid).
  • D-GIu D-GIu; D-Glutamic acid
  • X ⁇ is GIn.
  • Xiii and X ⁇ v as depicted in formula I " and I " " may, as mentioned, also represent any amino acid but Cys.
  • V are such amino acids able to form a peptide bond, or the like, with each other under conditions of a "head to tail" cyclization.
  • a possible example of an amino acid that may be x m is Arg.
  • One possible, and most preferred, example of an amino acid that may be x ⁇ v is Giy or a GIy analogue.
  • Giy analogue in this context means a residue, particularly an amino acid residue, having a structural character similar than that of GIy.
  • GIy analogue refers to, for example, a(n) (amino acid) residue having the same (or even a smaller) size than a GIy residue. It was surprisingly found in context of this invention that particularly a small (amino acid) residue like GIy at the "Xiv" position leads to an improved mimicking of the ECU of ⁇ 1-AR by the corresponding cyclic peptides of the invention.
  • the cyclic peptides of this invention lack Trp and/or His. Accordingly, it is particularly envisaged in context of the invention, that neither an x nor y as depicted in any of Formula I to I " " is Trp or His. Furthermore, it is preferred that the provided cyclic peptides iack sites susceptible for hydrolysis or cleaving proteases, like, for example, serum proteases. The meanings of the terms “hydrolysis” and “(serum) proteases” are well known in the art.
  • a peptide as provided herein can also be described as a peptide consisting of or comprising an amino acid sequence homologous to SEQ ID NO. 17 (representing a wild type amino acid strech comprising epitopes of ⁇ i-EC-n), wherein (a) the amino acid corresponding to position 13 (or, less preferred, corresponding to position 12) of SEQ ID NO. 17 is not Cys and the amino acid corresponding to positions 6 and 12 (or, less preferred, corresponding to position 6 and 13) of SEQ ID NO, 17 is Cys, (b) wherein said amino acid sequence contains no further Cys able to form an intramolecular linkage within the peptide, i.e. within that part of the peptide being homologous to SEQ ID NO.
  • peptide can function as a cyclic peptide in accordance with this invention, e.g. is able to block anti- ⁇ -AR antibodies, or wherein the peptide can form such a cyclic peptide.
  • the further provisions given herein with respect to the structure of the disclosed linear and/or cyclic peptides apply here, mutatis mutandis.
  • the so defined peptide consists of a stretch of 16 amino acids being homologous to SEQ ID NO.
  • homologous means identical on amino acid level for at least 18.75%, at least 37.5%, at least 50%, at least 56.25%, at least 62.5%, at least 68.75%, at least 75%, at least 81.25%, at least 87.5% or 93.75%, wherein the higher values are preferred.
  • amino acid or “amino acid residue” is known in the art and is used herein accordingly. Thereby, it is of note that when an “amino acid” is a component of a peptide/protein the term “amino acid” is used herein in the same sense than "amino acid residue”.
  • an "amino acid” or “amino acid residue” as referred to herein, except the ABu or ABu analogue is preferably envisaged to be a naturally occurring amino acid, more preferably a naturally occurring L-amino acid (except the above mentioned DGSu).
  • an "amino acid” or “amino acid residue” in context of this invention (“x” referred to in formula ! and the other formulas given herein) may also be a non-naturally occurring (i.e. a synthetic) amino acid, like, for example, norleucine or ⁇ -aianine.
  • the term "acidic amino acid(s)” as used herein is intended to mean an amino acid selected from the group comprising Asp, Asn, GIu, and GIn, preferably Asp and GIu;
  • the term "basic amino acid(s)” as used herein is intended to mean an amino acid selected from the group comprising Arg, Lys and His, preferably Arg and Lys;
  • the term “aliphatic amino acid(s)” as used herein is intended to mean any amino acid selected from the group comprising GIy, AIa 1 Ser, Thr, VaI, Leu, lie, Asp, Asn, GIu, GIn, Arg s Lys, Cys and Met;
  • the term "polar amino acid(s)"as used herein is intended to mean any amino acid selected from the group comprising Cys, Met, Ser. Tyr, GIn 1 Asn and, less preferred, Trp.
  • the cyclic peptide as provided herein may be a cyclic peptide of formula II, III or 111 ' :
  • Xi is individually and independently selected from the group consisting of acidic amino acids; and/or b) X 2 is individually and independently selected from the group consisting of basic amino acids.
  • the cyclic peptide as provided herein may be a cyclic peptide of formula IV, V or V;
  • X 1 is individually and independently selected from the group consisting of acidic amino acids
  • X 2 is individually and independently selected from the group consisting of basic amino acids
  • X 3 is individually and independently selected from the group consisting of Leu. lie, VaI, Met, Trp, Tyr and Phe
  • X 4 is individually and independently selected from the group consisting of Ser, Thr, Ala and GIy
  • X 5 is individually and independently selected from the group consisting of GIn and Asn.
  • the cyclic peptides comprise an amino acid strech as defined by amino acid position 2-12 or 2-14 of formula Il or IV. an amino acid strech as defined by amino acid position 4-16 or 4-18 of formula Hl or V or an amino acid strech as defined by amino acid position 3-15 or 3-17 of formula or IH ' or V,
  • the cyclic peptide as provided herein may be a cyclic peptide of formula H, III or III ' .
  • the cyclic peptide as provided herein may comprise the amino acid stretch
  • aci-Glu-ASa-bas-bas-Cys-Tyr-neu-aci-neu-bas aci-neu-aci-Giu-Aia-bas-bas-Cys-Tyr-neu-aci-neu-bas
  • aci-Glu-Ala-bas-bas-Cys-Tyr-neu-aci-neu-bas-Cys-Ser aci-neu-aci-Glu-Aia-bas ⁇ bas-Cys-Tyr-neu-aci-neu-bas-Cys-Ser
  • amino acid residue of the above four amino acid stretches may also be defined independently as the corresponding amino acid residue of any one of formulas I, II, IH, III ' , IV, V 1 and V as provided herein.
  • the cyclic peptide as provided herein may comprise the amino acid stretch
  • XxX 1 is defined as V or '%
  • Xxx 3 is defined as “x” or “x 3”
  • Xxx 4 is defined as “x” or “x 4 " as mentioned in the above depicted formulas.
  • the above-mentioned amino acid stretch may be
  • the cyclic peptides of this invention comprise one or more epitopes born by ⁇ i-ECn, like, for example, epitopes born by any of the above mentioned amino acid stretches (or by parts of the disclosed cyclic peptides comprising these amino acid stretches).
  • epitope particularly refers to an amino acid stretch to which an (auto)anti- ⁇ i-AR antibody binds.
  • an epitope in context of this invention consists of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13 or at least 14 amino acids.
  • Non limiting examples of a cyciic peptide according to this invention are cyclic peptides selected from the group consisting of: a) cyclic peptides formable or formed by the amino acid sequence as depicted in any one of SEQ ID NO. 25, 27, 1 , 2 and 9 to 10; b) cyclic peptides formable by an amino acid sequence as encoded by a nucleotide sequence as depicted in any one of SEQ ID NO. 26, 28, 9, 10, 13 and 14; and c) cyclic peptides formabfe by an amino acid sequence as encoded by a nucleotide sequence which differs from the nucleotide sequence as depicted in any one of SEQ ID NO. 26, 28, 9, 10, 13 and 14 due to the degeneracy of the genetic code.
  • those cyclic peptides being Cys-Ser mutant peptides, i.e. having the Cys corresponding to the third Cys of the ⁇ r ECii (the Cys at position 216 of ⁇ r AR) exanged by ABu or an analogue thereof, are particularly preferred.
  • the above given examples refer to such particularly preferred cyclic peptides.
  • such cyclic peptides are particularly useful in inhibiting or diagnosing/detecting anti- ⁇ i-AR antibodies.
  • Non limiting exampfes of less preferred cyciic peptides according to this invention are cyclic peptides selected from the group consisting of: a) cyclic peptides formabte by the amino acid sequence as depicted in any one of SEQ iD NO. 5, 6 11 and 12; b) cyclic peptides formable by an amino acid sequence as encoded by a nucleotide sequence as depicted in any one of SEQ ID NO. 7, 8, 15 and 16; and c) cyclic peptides formable or formed by an amino acid sequence as encoded by a nucleotide sequence which differs from the nucleotide sequence as depicted in any one of SEQ ID NO. 7, 8, 15 and 16 due to the degeneracy of the genetic code.
  • cyclic peptides according to this invention are particularly those cyclic peptides, the pharmacological and/or diagnostic function of which has been demonstrated in the appended examples or the pharmacological and/or diagnostic function of which can at least be predicted on the basis of the appended examples (e.g. those characterized by any one of formula Vi to IX ' ).
  • the present invention also refers to cyclic peptides derived from the ECn of ⁇ 2 -AR, like those cyclic peptides corresponding to the ⁇ i-ECn-derived peptides of this invention but being structurally more closely related to EC U of ⁇ 2 -AR. These peptides may, for example, be used as reference/control peptides, when ⁇ r ECn-derived peptides are tested/assayed.
  • cyclic peptides derived from the ECn of ⁇ 2 -AR is the cyclic peptide as depicted by the following formula: cycio(Arg»Aia-Glu ⁇ Ser-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn- Glu-P ⁇ >Lys-Cys-Abu-Asp ⁇ Phe ⁇ Vai-Thr-Gly).
  • the skilled person is able to test whether a given variant of peptides of the present invention still has the desired function, for example the ability to specifically bind to ⁇ -AR antibodies, and therefore has the potential for a corresponding medical intervention, like the therapeutic and diagnostic applications described and provided herein.
  • Corresponding experimental guidance for such tests, i.e. respective assays, are exemplariiy provided and described herein, particularly in the appended examples.
  • variants of the herein disclosed and described peptides are still functionally active in accordance with this invention, i. e. functionally active as binding partners for anti- ⁇ -AR antibodies, particularly for anti- ⁇ rAR antibodies against the ⁇ rECn, more particularly functionally active as inhibitors of ⁇ rAR and even more preferably active in inhibiting the interaction between ⁇ r AR and anti- ⁇ i-AR antibodies against the ⁇ i-ECn, more preferably auto-anti- ⁇ rAR antibodies against the ⁇ i-EC-H; and, second, that these variants are not present in form of isomers mixtures or do not form isomers mixtures when cyclized in accordance with (the production method of) this invention.
  • These variants are envisaged to have only two certain Cys residues forming or being able to form only one individual intramolecular linkage (e.g. disulphide bond).
  • amino acids of said peptides are replaced by other one or more naturally occurring or synthetic amino acids.
  • this/these amino acid exchange(s) is/are (a)conservative amino acid exchange(s), i.e. that the replacement amino acid belongs to the same category of amino acids than the amino acid to be replaced.
  • an acidic amino acid may be replaced by another acidic amino acid
  • a basic amino acid may be replaced by another basic amino acid
  • an aliphatic amino acid may be replaced by another aliphatic amino acid
  • a polar amino acid may be replaced by another polar amino acid.
  • variants of the (cyclo) peptides of the present invention are variants wherein at least one of an acidic amino acid is replaced by a different amino acid selected from the group consisting of acidic amino acids, at least one of the basic amino acids is replaced by a different amino acid selected from the group consisting of basic amino acids, at least one of a polar amino acid is replaced by a different amino acid selected from the group consisting of polar amino acids and/or at least one of an aliphatic amino acid is replaced by a different amino acid selected from the group consisting of aliphatic amino acids (given that the above mentioned-requirements are fulfilled).
  • amino acid exchanges which lead to variants of the disclosed (cyclic) peptides are such, that the pattern of polarity and charge within the tertiary structure of the resulting variant still substantially mimics the three- dimensional structure of the corresponding EC M epitope(s) of ⁇ i-AR.
  • the herein defined Cys may also be replaced by other amino acids, as long as the replacement still leads to an individual intramolecular linkage, like that of a disuiphide bond, within the cyclopeptide, i.e. to the avoidance of isomers mixtures formation during cyclization and/or a correct mimicry of the ECn of ⁇ i-AR.
  • Such amino acid may, inter alia, be a non-naturally occurring amino acid, like a non-naturally occurring amino acid having an -SH group able to form a disuiphide bond.
  • the Cys given in formula I, above is indeed a naturally occurring amino acid, preferably Cys itself.
  • amino acids forming the (cyclic) peptide of the present invention may be modified.
  • any amino acid as used herein may also represent its modified form.
  • an alanine residue as used herein may comprise a modified alanine residue.
  • modifications may, among others, be a methyiation or acylation or the like.
  • modification(s) or modified amino acid(s) is (are) envisaged in the context the present invention as long as the thus modified amino acid and more particularly the peptide containing said thus modified amino acid is stil!
  • the invention also provides derivatives of the disclosed (cyclic) peptides such as salts with physiologic organic and anorganic acids like HCl, H 2 SO 4 , H 3 PO 4 , maiic acid, fumaric acid, citronic acid, tatratic acid, acetic acid.
  • physiologic organic and anorganic acids like HCl, H 2 SO 4 , H 3 PO 4 , maiic acid, fumaric acid, citronic acid, tatratic acid, acetic acid.
  • sequences of the peptides disclosed are indicated from the N- terminus to the C-terminus, whereby the N4erminus is at the left side and the C- terminus is at the right side of the respective depicted amino acid sequence.
  • the corresponding sequences are indicated from the side corresponding to the left side of formula f to the side corresponding to the right side of formula I.
  • a "cyclic peptide” or “cyclopeptide” and the like in accordance with the present invention is a peptide intramoiecularly forming a molecular ring structure within its amino acid backbone/primary amino acid sequence by at least one, preferably by at least two, more preferably by exactly two intramolecular linkages having covalent character.
  • the forming of this molecular ring structure is, in context of this invention, also termed "cyclization".
  • the cyclic peptide of this invention has two intramolecular linkages having covending character, wherein one of these linkages is an intramolecular linkage between the N- and C-termina! ends of a peptide being the amino acid backbone/primary amino acid sequence of the cyclic peptide disclosed and the other one is an intramolecular linkage between two non-terminal amino acids of this peptide.
  • these two non terminal amino acids may be two Cys.
  • the peptide bond as mentioned throughout this invention can be formed by the NH 2 group of an N-terminal amino acid and the COOH group of a C-terminal amino acid of a peptide forming the amino acid backbone/primary amino acid sequence of the cyclic peptide disclosed.
  • an intramolecular linkage between the N- and C-terrninal ends of a peptide forming the amino acid backbone/primary amino acid sequence of the cyclic peptide disclosed is a peptide bond and an intramolecular linkage between two nonterminal amino acids of this peptide is an S-S linkage (i, e. dlsulphide bond), in context of this invention, an intramolecular S-S linkage within the cyclic peptide provided can be formed between two Cys residues within the amino acid backbone/primary amino acid sequence of said cyclic peptide.
  • cyclic peptides of this invention not only the above mentioned two particular intramolecular covending linkage may be formed but also further intramolecular linkages may occur, with the proviso that the herein described functionality of the cyclic peptides is maintained and that the cyclic peptides can still easily be characterized biochemically, which, e.g., means that no isomers mixtures are formed during cyclization of the corresponding amino acid backbone/primary amino acid sequence.
  • Such further intramolecular linkages are additional bonds formed by a side chain of NH 2 groups and COOH groups of the constituent amino acids.
  • amino acid backbone or “primary amino acid sequence” as used throughout the present invention refer, on the one hand, to that structural component or part of a cyclic peptide which is formable or formed by its corresponding amino acid sequence. On the other hand, these terms refer to the linear peptides able to form the cyclic peptides of this invention by cyclization
  • a cyclic peptide which is obtainable by the method for producing a cyclic peptide as provided herein.
  • the definitions given herein-above also appiy with respect to this particularly provided cyclic peptide of the present invention
  • the disclosed cyclic peptides are formable by or are formed by.
  • these peptides are the linear peptides forming or being able to form the herein disclosed cyclic peptides, i e the amino acid backbone/primary amino acid sequence thereof
  • such a linear peptide can be any peptide, the covalent linkage of the N- and C-termtnus of which results in a cyclic peptide as disclosed in accordance with the present invention
  • such a linear peptide may be some kind of an intermediate compound in a procedure of producing the cyclic peptides of this invention, like the method for producing a cyclic peptide as disclosed herein
  • N- and C ⁇ terminal end of a linear peptide provided herein may be any amino acid pair lying in direct proximity to each other within the amino acid backbone of a cyclic peptide disclosed in context of this invention
  • cyciization (ring closure) of the cyclic peptide of this invention may generally occur between any of said amino acid pairs
  • the skilled person is readily in the position to find out such particular amino acid pairs which are effective/suitable to act as N- and C-terminal ends of a herein disclosed linear peptide, i e which are effective/suitable to act as an amino acid pair being involved in the ring closure/cyclization as defined in context of this invention
  • the cyclization (ring closure) of a linear peptide of this invention may occur between Ala and GIn or GIu, i.e the N-terminal amino acid of this linear peptide would be Ala and the C-terminal amino acid would be GIn or GIu.
  • Examples of such linear peptides able to form the cyclic peptide of the present invention are SEQ ID NO. 1 and 2 and, iess preferred SEQ ID NO. 3 and 4.
  • the cyciization (ring closure) of a linear peptide of this invention may occur between Lys and Pro, i.e. the N-termina!
  • linear peptides able to form the cyclic peptide of the present invention are SEQ !D NO. 9 and 10 and, less preferred, SEQ ID NO. 11 and 12.
  • the cyciization (ring closure) of a linear peptide of this invention may occur between Arg and GIy, i.e. the N-terminal amino acid of this linear peptide would be Arg and the C-terminal amino acid would be GIy.
  • SEQ ID NO. 25 An example of such a linear peptide able to form the cyclic peptide of the present invention is SEQ ID NO. 25.
  • the cyciization (ring closure) of a linear peptide of this invention may occur between Lys and Pro, i.e. the N-terminal amino acid of this linear peptide would be Lys and the C-terminal amino acid would be Pro.
  • An example of such a linear peptide able to form the cyclic peptide of the present invention is SEQ ID NO, 27.
  • the cyclic peptides of the invention may further comprise (e.g. have covalently bound) (a) further substituent(s). like labels, anchors (like proteinaceous membrane anchors), tags (like HIS tags) and the like. Appropriate substituents and methods for adding them to the cyclic peptide of this invention are known to those of ordinary skill in the art.
  • labels in this context include, inter alia, fiuorochromes (like fluorescein, rhodamine, Texas Red, etc), enzymes (like horse radish peroxidase, ⁇ - galactosidase, alkaline phosphatase), radioactive isotopes (like 32P, 33P, 35S, 1251 or 1231, 1351, 1241, 11 C, 15O) 5 biotin, digoxygenin, colloidal metais, chemi- or bioluminescent compounds (like dioxetanes, luminoi or acridiniums).
  • fiuorochromes like fluorescein, rhodamine, Texas Red, etc
  • enzymes like horse radish peroxidase, ⁇ - galactosidase, alkaline phosphatase
  • radioactive isotopes like 32P, 33P, 35S, 1251 or 1231, 1351, 1241, 11 C, 15O
  • One particularly envisaged label that may be bound to the peptide of this invention is a fiuorochrome belonging to a FRET pair of fiuorochromes, for example a GFP variant (e.g. GFP, eGFP, EYFP or ECFP).
  • GFP variant e.g. GFP, eGFP, EYFP or ECFP.
  • a variety of techniques are available for labeling biomolecules, are well known to the person skilled in the art and are considered to be within the scope of the present invention and comprise, inter alia, covended coupling of enzymes or biotinyl groups, phosphorylations, biotinylations, random priming, nick-translations, tailing (using terminal transferases).
  • Detection methods comprise, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, etc.
  • the substituent(s) can be bound (e.g. covalently) to the cyclic peptides of the invention directly or via linkers.
  • linkers The skilled person is readily in the position to find out appropriate linkers to be employed in this context.
  • the present invention relates to a nucieic acid molecule comprising a nucleotide sequence encoding an amino acid sequence which can form or which can be used to form or generate a cyclic peptide as disclosed in context of this invention, or an amino acid backbone/primary amino acid sequence thereof.
  • the present invention also relates to a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence which can form or which can be used to form or generate the linear peptides as provided and described herein.
  • such nucleic acid molecule may comprise a nucleotide sequence as depicted in any one of SEQ ID NO. 5, 6, 13, 14, 26 and 28 or, less preferred, SEQ ID NO. 7, 8, 15 and 16 or a nucleotide sequence which differs therefrom due to the degeneracy of the genetic code.
  • ABu or an ABu analogue is a non-genetical!y coded amino acid
  • the skilled person is readily in the position to provide nucleic acid molecules which encode amino acid sequences that can serve as a basic or intermediate product for the formation or generation of the cyclic peptides of the invention.
  • nucleic acid molecules are those coding for an amino acid stretch of ⁇ i-ECn corresponding to the amino acid backbone/primary amino acid sequence of the cyclic peptides of the invention having ABu (or the ABu analogue) deleted or replaced by any other genetically coded amino acid, preferably by an amino acid that can be replaced by ABu (or the ABu analogue) iater on.
  • nucleic acid moiecu!e(s) 7 "nucleic acid sequence(s)” and “nucleotide sequence(s)” and the like are well known in the art and are used accordingly in context of the present invention.
  • nucleotide sequences and/or nucleic acid sequences/molecules as wel! as to chemically synthesized nucleotide sequences and/or nucleic acid sequences/molecules.
  • nucleic acid analogues and nucieic acid derivatives such as e. g. locked DNA, PNA, oligonucleotide thiophosphates and substituted ribo-oligonucieotides.
  • these terms also refer to any molecule that comprises nucleotides or nucleotide analogues.
  • nucleic acid molecule(s) refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the "nucleic acid molecule(s)”, “nucleic acid sequence(s)” and “nucleotide sequence(s)” may be made by synthetic chemical methodology known to one of ordinary skill in the art, or by the use of recombinant technology, or may be isolated from natural sources, or by a combination thereof.
  • the nucleic acid molecules as provided herein are particularly useful for producing a cyclic peptide of the invention, for example by the corresponding method disclosed herein.
  • the present invention relates to a method for producing a cyclic peptide of the present invention, comprising the steps of a) (i) cultur ⁇ ng the recombinant host ceil of the present invention under conditions such that an amino acid sequence which can form or which can be used to form or to generate an amino acid backbone of the herein disclosed cyclic peptide (or the linear peptide of this invention) is expressed, recovering amino acid sequence and connecting said amino acid sequence into said amino acid backbone (or into said linear peptide of this invention); or
  • cyclization encompasses both, forming of the intramolecular bridge ⁇ disulphide bond) and the ring closure by covatently connecting the N- and C-termini of the backbones of the cyclic peptides to be produced.
  • the encoded amino acid sequence can be "converted” into the amino acid backbone of the cyclic peptides of the invention. For example, if the encoded amino acid sequence lacks the amino acid at the position corresponding to the position of ABu (or an ABu analogue) of the provided cyclic peptides, the encoded amino acid sequence can be "converted” in accordance with said method by introducing ABu (or an ABu analogue) at corresponding position, if the encoded amino acid sequence comprises an amino acid or amino acid stretch instead of ABu (or an ABu analogue), the amino acid sequence can be "converted” in accordance with said method by replacing said amino acid or amino acid stretch with ABu (or an ABu analogue).
  • the N-terminal amino acid of the amino acid backbone/linear peptide to be cyclized in order to produce a cyclic peptide of this invention is Ala, Arg or Lys and the corresponding C- terminal amino acid is Gin, GIy or Giu (also DGIu is possible) or Pro.
  • the N- and C-terminal amino acids are envisaged, i.e. also other cyciization (ring closure) sites can be employed in context of the disclosed method.
  • the cyclopeptide mutants were first synthesized in form of their linear peptides/amino acid backbones (for example by applying a chemical synthesis approach, like the Fmoc / tert butyl strategy (as described in WO 2006/103101 ; Chen W.C. and White P. D.: Fmoc Solid Phase Peptide Synthesis, Oxford University Press 2003)), and were then cyclized covalentiy on the backbone by condensation of the C-terminal carboxyi group with the amino group of the N-terminal amino acid ("head to tail" cyciization; Kates S. and Albericio F.: Solid phase synthesis, CRC-Press, 2000).
  • a disulphide bond is established between those two cysteine residues of the linear peptides which are able to form a disulphide bond (e.g. between Cys 7 and Cys- 13 of the 18mer peptide, between Cysi 0 and Cys 16 of the 22-mer peptide or between Cysn and Cysi 7 of the 25-mer peptide) by chemical interaction known in the art (e.g. Benoiton N. L.: Chemistry of Petide Synthesis. CRC-Press, 2005).
  • the ring closure of the linear backbone of the cyclic peptides to be produced may be performed before or after the formation of the S-S bridge.
  • the S-S bridge between the two Cys residues of the AA chain of the peptides may be the first step in the "cyciization" procedure of the described production process and the ring closure may be the second step, or vice versa.
  • the skilled person is able to find out which of these particular approaches is appropriate for a given setup of the production preconditions.
  • linear peptides/amino acid backbones of the cyclic peptides to be produced can also be produced by using recombinant engineering techniques. Such techniques are well known in the art (e. g. Sambrook, supra). As also mentioned above, by this kind of production of said linear peptides/amino acid backbones particular advantage can be taken of the herein disclosed and described nucleic acid molecules, vectors and/or host cells. The definitions correspondingly given above apply here, mutatis mutandis.
  • this invention also relates to a cycfic peptide obtainable or obtained by the above described method, but also to a corresponding linear peptide (amino acid backbone/primary sequence of the corresponding cyclic peptide) obtainable or obtained by the above described method as some kind of an intermediate product (particularly a product obtainable or obtained by step a) of the above described method).
  • the cyclic peptide according to the present invention may, inter alia, be used in medical intervention approaches.
  • Such approaches comprise the use as or in a diagnostic agent and for the manufacture of a medicament for the treatment of diseases or the use in or as a composition, preferably a pharmaceutical composition, a diagnostic composition or a diagnostic kit, preferably for the detection of anti- ⁇ -AR antibodies, more preferably for the detection of anti- ⁇ -i-AR antibodies.
  • the antibodies as defined or described herein are preferably autoantibodies.
  • Non-limiting uses and appiications of the compounds, particularly the cyclic peptide according to the present invention are described herein, for example further below.
  • the present invention also relates to a composition
  • a composition comprising a cyclic or a linear peptide, a nucleic acid molecule, a vector or a recombinant host cell as disclosed and provided in context of the present invention, and optionally a carrier.
  • said composition is a pharmaceutical composition and said carrier is a pharmaceutically acceptable carrier.
  • composition of this invention is particularly useful when employed in the treatment, amelioration or prevention as described and defined herein. Accordingly, the pharmaceutical composition of this invention may be used for the treatment, amelioration or prevention of a disease where the activity of a ⁇ -AR is enhanced or for the treatment of a patient having antibodies against a ⁇ -AR. Moreover, the pharmaceutical composition of this invention may be used for inducing immune tolerance of a patient, particularly immune tolerance of a patient with respect to immunogenic stretches of the endogenous ⁇ i-AR.
  • the (pharmaceutical) composition provided may either comprise two or a plurality (like at least 3 or at least 5) of cyclic peptides of the present invention.
  • the administration of said more than one of cyclic peptides may be simultaneously or successively.
  • the present invention relates to the pharmaceutical composition, the method or uses for medical intervention or the cyclic peptide or the pharmaceutical composition as disclosed herein, wherein said cyclic peptide is administered with or said pharmaceutical composition comprises at least one further pharmaceutically active agent.
  • Said at least one further pharmaceutically active agent may be a ⁇ -receptor blocker, preferably a selective ⁇ -AR blocker, like, for example, a ⁇ i-AR blocker selected from the group consisting of atenolol, metoproSol, nebivolol, bisoprolol and the like.
  • this kind of particular combination may provide for protection from antibody-induced, selective ⁇ i-AR downregulation by the herein provided cyclic peptides, since ⁇ i-AR is at the same time upreguiated by betablockers, like bisoprolol or metoproiol, and ultimately results in a synergistic effect of the cyclic peptides and the additional ⁇ -blocker(s).
  • the carrier optionally comprised in the (pharmaceutical) composition of the invention or to be administered together with the (pharmaceutical) composition or the cyclic peptide of the invention may particularly be a pharmaceutically acceptable carrier, excipient or diluent.
  • Such carriers are well known in the art. The skilled person is readily in the position to find out such carriers which are particularly suitable to be employed in accordance with the present invention.
  • compounds of the invention may be formulated in aqueous solution, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically saline buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • compositions of the present invention in particular those formulated as solutions, may be administered parenterally, such as by intravenous injection.
  • pharmaceutically acceptable carriers well known in the art into dosages suitable for subcutaneous or oral administration.
  • Such carriers enable the compounds according to the present invention to be formulated as tablets, pilis, capsules, dragees, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated.
  • Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered near the cell surface. Delivery systems involving liposomes are disclosed in U.S. Patent No. 4,880,635 to Janoff et al. The publications and patents provide useful descriptions of techniques for liposome drug delivery.
  • compositions comprising a compound according to the present invention for parenteral and/or subcutaneous administration include aqueous solutions of the active compound(s) in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil or castor oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injections suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions and to allow for a constantly slow release of the substance in the organism.
  • the disclosed pharmaceutical composition or cyclic peptide may be administered in a pharmaceuticals/therapeutically effective dose, which means that a pharmaceutically/therapeuticaliy effective amount of the compound administered is reached.
  • a pharmaceuticals/therapeutically effective dose refers to that amount of the compound administered (active ingredient) that produces amelioration of symptoms or a prolongation of survival of a subject which can be determined by the one skilled in the art doing routine testing,
  • the dosage regimen of the compounds to be administered in accordance with the present invention will be determined by the attending physician and clinical factors. As is well known in the medical arts, that dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A person skilled in the art is aware of and is able to test the relevant doses, the compounds to be medically applied in accordance with the present invention are to be administered in.
  • the effect of the cyclic peptides provided herein namely the blockage of anti- ⁇ t -AR antibodies, can be obtained in a dose dependent manner.
  • the efficiency of the Cys ⁇ Ser mutated cyclopeptides as disclosed herein depends on a threshold concentration (Figs. 6 and 7).
  • the Cys-ABu mutant as disclosed herein may act likewise.
  • the disclosed pharmaceutical composition or cyclic peptide may particularly be administered in a manner that it is present in a concentration, i.e. reaches a threshold concentration, of at least 0.05 mg per kg body weight, preferably in a concentration of at least 0.1 mg per kg body weight, more preferably in a range of 0.1 mg per kg body weight (100 ⁇ g/kg) to 100 mg per kg body weight, more preferably in a range of 1 mg per kg body weight to 100 mg per kg body weight and most preferably in a range of 1 mg per kg body weight to 10 mg per kg body weight.
  • the effective dose of the disclosed pharmaceutical composition or cyclic peptide may be at about 1 mg per kg body weight
  • higher concentrations of the disclosed pharmaceutical composition or cyclic peptide are generally envisaged to be reached by correspondingly applied administration schemes.
  • such higher concentrations may be at least 2, 3, 4 or 5 mg per kg body weight. Concentrations of at least 1 mg per kg body weight or at least 2 mg per kg body weight are preferred.
  • One particularly preferred, non-limiting administration scheme to be applied in context of this invention is an s.c. or i.v. application every two or four weeks.
  • a dose of 1 to 4 mg/kg i.v. every other month were sufficient to obtain therapeutic levels of the compounds according to the present invention, with the respective dosage for humans preferably being about 0.3-10 mg/kg i.v. or s.c, more preferably being about 1-10 mg/kg i.v. or s.c, even more preferably being about 1-5 mg/kg i.v. or s.c.
  • the respective dosage for humans are about 0.1-10 mg/kg i.v. or s.c, more preferably about 0.3-5 mg/kg i.v.
  • the administration of the disclosed cyclic peptides may initially trigger a transient opposite immune response, in particular when applied in higher doses. Such transient immune responses in the long run are compensated by the antibody-inactivating activity of the administered cyclic peptides. This may lead to a decelerated effect of the administered cyclic peptides, i.e. a decelerated elimination of anti- ⁇ rAR antibodies and hence a decelerated reduction of (aberrant) ⁇ i-AR activity.
  • the present invention also relates to a method for a) the treatment, amelioration or prevention of a disease where the activity of a ⁇ - AR, preferably ⁇ rAR, is enhanced; b) the treatment of a patient having antibodies against a ⁇ -AR, preferably against ⁇ r AR, or suffering from or being at risk to develop a disease as disclosed herein; or c) inducing immune tolerance, comprising the step of administering to a patient in need of such medical intervention a pharmaceutically active amount of a cyclic peptide and/or of a pharmaceutical composition as disclosed herein, and optionally a pharmaceutically acceptable carrier.
  • the present invention also relates to a cyc ⁇ c peptide or a pharmaceuticai composition as disclosed herein, and optionally a pharmaceutically acceptable carrier, for, or for use in, a) the treatment, ameiioration or prevention of a disease where the activity of a ⁇ - AR, preferably ⁇ i-AR, is enhanced; b) the treatment of a patient having antibodies against a ⁇ -AR, preferably against ⁇ rAR, or suffering from or being at risk to develop a disease as disclosed herein; or c) inducing immune tolerance.
  • the present invention further relates to a kit for use in a regimen for a) the treatment, amelioration or prevention of a disease where the activity of a ⁇ - adrenergic receptor ( ⁇ -AR) is enhanced; b) the treatment of a patient having antibodies against a ⁇ -AR; or c) inducing immune tolerance, said regimen comprising one or more cycles, preferably two or more cycles, each cycle consisting of a 2 to 70 day treatment/inducing period according to (a) to (c) followed by a 6 to 280 day rest period, wherein said kit comprises: (i) from 2 to 70 doses (e.g.
  • a cyclic peptide or a pharmaceutical composition according to the present invention daily, weekly or monthly doses of a cyclic peptide or a pharmaceutical composition according to the present invention; and, optionally, (ii) from 6 to 280 doses (e.g. daily, weekly or monthly doses) of a placebo or a nutrient supplement; and, optionally, (iti) a means for having the components arranged in a way as to facilitate compliance with the regimen, but not containing a cyclic peptide or pharmaceutical composition of the present invention.
  • the cyclic peptide or pharmaceutical composition of the present invention or the cyclic peptide or pharmaceutical composition to be administered in the context of the methods for treating, ameliorating, preventing or inducing of the present invention, are to be administered according to the above described regimen.
  • the rest period may be about 1 , 2, 3, 4 or 5 times the treatment/inducing period, wherein about 3 times the treatment/inducing period is preferred.
  • the treatment/inducing period may be about 2, 3, 4 or 5 weeks, wherein about 3 weeks are preferred.
  • the rest period may particularly be about 2 to 25 weeks.
  • the rest period is about 6, 9, 12 or 15 weeks, wherein about 9 weeks are preferred.
  • the cyclic peptide or pharmaceutical composition may, for example, be administered 1 to 7 times a week during the treatment/inducing period, wherein once per week during the treatment/inducing period is preferred.
  • the overall dose to be administered in the context of the regimen as defined herein can be determined by the attending physician and depends on clinical factors like patient's size, body surface area, age. This is described in more detail herein elsewhere.
  • the overali dose of the cyclic peptide or pharmaceutical composition to be administered in the context of the regimen defined herein corresponds to a monthly dose of 0.1-10 mg/kg, preferably of about 0.3-5 mg/kg, more preferably about 0.3-2 mg/kg and most preferably about 1 mg/kg.
  • the required overall dose is administered in weekly portions over a treatment/inducing period of 3 weeks followed by a rest period of 9 weeks.
  • Each of said weekly portions may particularly be one of the above described monthly doses, preferably the monthly dose of about 1.0 mg/kg.
  • the doses of a cyclic peptide or pharmaceutical composition as comprised in the kit of the invention may be provided in individual vials or several or ail of the doses may be combined in one or more common vial(s).
  • the amount of the cyclic peptide/pharmaceutical composition to be administered at a certain point in time (e.g. day) within the treatment/inducing period (e.g. weekly portion as described above) is combined in a single vial.
  • a monthly dose is envisaged to be provided in a single vial, wherein a corresponding kit comprises three of such vials.
  • said kit may further comprise an instruction manual/leaflet guiding the attending physician/patient to administer one of said 3 monthly doses once per week over a period of 3 weeks (the treatment/inducing period) foliowed by a period of 9 weeks (the rest period).
  • the cyclic peptide, pharmaceutical composition or kit of the present invention may be provided with/comprise an instruction manual/leaflet.
  • Said manual/leaflet may guide the attending physician/patient through the administration regimen of the cyclic peptide or pharmaceutical composition of the invention.
  • the instruction manual/leaflet may guide the attending physician/patient through the regimen as described herein-above.
  • Preferred cyciic peptides to be administered according to the above described regimen are the herein disclosed 22-mer cyclic peptides, in particular the 22-mer cyclic peptides formable or formed by the amino acid sequence as depicted in SEQ ID No. 25 or 27.
  • the diseases to be medically intervened (treated, ameliorated, prevented or diagnosed) in accordance with this invention or the diseases the patient as defined and described herein suffers from are preferably those, where the ⁇ rAR is activated in a non-physiological manner, more preferably is activated by antibodies, more preferably by auto-antibodies which are directed against the (VAR.
  • the diseases to be medically intervened in accordance with this invention or the diseases the patient as defined and described herein suffers from comprise, however, are not limited thereto, the group of heart diseases.
  • the heart diseases to be medically intervened in accordance with this invention or the heart diseases the patient as defined and described herein suffers from may comprise but are not limited to infectious and non-infectious heart disease, ischemic and non-ischemic heart disease, inflammatory heart disease and myocarditis, cardiac dilatation, idiopathic cardiomyopathy, (idiopathic) dilated cardiomyopathy (DCIvI) 5 immune-cardiomyopathy, heart failure, and any cardiac arrhythmia including ventricuiar and/or supraventricular premature capture beats as well as any atrial arrhythmia including atrial fibrillation and/or atrial flutter.
  • infectious and non-infectious heart disease ischemic and non-ischemic heart disease
  • inflammatory heart disease and myocarditis myocarditis
  • cardiac dilatation idiopathic cardiomyopathy
  • heart failure and any cardiac arrhythmia including ventricu
  • the heart disease as referred to in the descriptions and definitions given herein with respect to the methods or the cyclic peptide or the pharmaceutical composition of the invention may be heart diseases selected from the group comprising infectious and non-infectious heart disease, ischemic and non-ischemic heart disease, inflammatory heart disease and myocarditis, cardiac dilatation, idiopathic cardio-myopathy, (idiopathic) dilated cardiomyopathy (DCM), immune- card iomyopathy, heart failure, and any cardiac arrhythmia including ventricular and/or supraventricular premature capture beats as well as any atrial arrhythmia including atrial fibriilation and/or atrial flutter.
  • heart diseases selected from the group comprising infectious and non-infectious heart disease, ischemic and non-ischemic heart disease, inflammatory heart disease and myocarditis, cardiac dilatation, idiopathic cardio-myopathy, (idiopathic) dilated cardiomyopathy (DCM), immune- card iomyopathy, heart failure, and
  • DCM preferably idiopathic DCM.
  • a particular subgroup of the "patients" for the purpose of the present invention are those patients suffering from any of the diseases described herein, more particularly the group of heart diseases described herein and having at the same time antibodies directed against ⁇ -ARs, more preferably antibodies against the ⁇ -i-AR, whereby the antibodies are preferably auto-antibodies.
  • a disease to be medically intervened (treated, ameliorated, prevented or diagnosed) in accordance with this invention or a disease the patient as defined and described herein suffers from is intended to be induced by antibodies against a ⁇ -AR, preferably by antibodies against ⁇ i-AR. Preferably, these antibodies are autoantibodies.
  • the means and methods provided herein are particularly useful when provided in the prophylaxis/prevention of a disease as defined herein.
  • a patient may be treated with the cyclic peptide and/or pharmaceutical composition of the invention prior to the onset (of symptoms) of a disease as defined herein.
  • this preventive treatment may follow a preceding diagnostic application that, e.g., takes advantage of the diagnostic means and methods provided herein.
  • a preventive treatment taking advantage of the therapeutic means and methods of this invention is applied, when the risk to develop a disease as defined herein is diagnosed, e.g. when anti- ⁇ -AR (auto-)antibodies are detected.
  • a preferred "patient” is one bearing at risk to develop a disease as defined herein.
  • a patient is one having anti- ⁇ -AR (auto-)antibodies, preferably anti- ⁇ i-AR (auto-)antibodies, but not (yet) suffering from a disease as defined herein, or symptoms thereof.
  • the immune tolerance to be induced in context of this invention is envisaged to be particularly obtained by suppression of the production of antibodies against immunogenic stretches of the ⁇ -AR molecule, which, without being bound by theory, may be due to a blockade of the antigen-recognition sites of the antibody-producing early (mature) 8-celis and memory B-cells.
  • the provided pharmaceutical composition or cyclic peptide is particularly useful for the treatment, prevention and/or amelioration of any of the diseases and patient groups or patients as defined herein including the detection of anti- ⁇ -AR antibodies in these patients by using the aforementioned compounds.
  • a "patient” for the purposes of the present invention i. e. to whom a compound according to the present invention is to be administered or who suffers from the disease as defined and described herein or who is intended to be diagnosed in accordance with this invention, includes both humans and other animals and organisms.
  • the compounds and methods of this invention are applicable to or in connection with both human therapy and veterinary applications including diagnostic(s), diagnostic procedures and methods as wel! as staging procedures and methods.
  • the patient is a mammal, and in the most preferred embodiment the patient is human.
  • the mutant cyclic peptides according to the present invention may also be used for the preparation of a medicament for the treatment, prevention and/or amelioration of any of the diseases and patient groups/patients as defined herein.
  • What is said herein for the pharmaceutical composition applies also to the medicament for the manufacture of which the peptides of the present invention may be used.
  • the present invention is related to a diagnostic agent comprising or being a cyclic peptide or a composition according to this invention, and optionally at least one further biologicaiiy active compound.
  • the herein disclosed diagnostic agent consists of or comprises a mutant peptide of the present invention, whereby the mutant peptide comprises a label.
  • a label may be selected from the group comprising radioactive labels and fluorescent labels.
  • Respective labels are known to the ones skilled in the art. The definitions and descriptions of labels as given herein-above apply here, muatis mutandis.
  • the peptide is the part of the diagnostic agent conferring specific binding characteristics to the diagnostic agent, preferably binding to anti- ⁇ rAR antibodies, whereas the label confers the signalling characteristics to the diagnostic agent.
  • the diagnostic agent of this invention may comprise, apart from (a) labelled or unlabelled mutant peptide(s) of the present invention, a further biologically active compound.
  • a further biologically active compound may be a means to confer signalling characteristics to the diagnostic agent, particularly in case the mutant peptides of the present invention are unlabelled.
  • the further biologically active compound can be an antibody, preferably a monoclonal antibody, and more preferably a labelled antibody specificaSty binding to a mutant peptide of the present invention or to a complex consisting of a mutant peptide of the present invention and an anti- ⁇ -AR antibody, preferably an anti- ⁇ rAR antibody.
  • the present invention relates to a method for diagnosing a disease as defined and described herein comprising the steps of a) detecting antibodies against a ⁇ -AR (for example in a sample) using the cyclic peptide or the composition or the diagnostic agent of the present invention; and b) diagnosing for said disease, when the titer of said antibodies is increased.
  • the present invention is related to a method for diagnosing a patient which can be treated using the mutant peptides, pharmaceutical compositions and medicaments according to the present invention.
  • a step of detecting antibodies against a ⁇ -AR (for example in a sample) using the compounds of the present invention and/or a step of considering whether the outcome of said detection step indicates a disease as defined herein may be employed.
  • a disease as defined herein or the risk to develop a disease as defined herein is indicated, when the titer of said anti- ⁇ -AR antibodies is increased.
  • the present invention relates to a cyclic peptide, a composition or a diagnostic agent as provided and described herein for diagnosing (for example in a sample) a disease as defined herein.
  • a disease as defined herein or the risk to develop a disease as defined herein is indicated by an increased titer of anti- ⁇ -AR antibodies.
  • the term "increased titer of anti ⁇ AR antibodies” means that the titer of anti- ⁇ -AR antibodies (for example in a sample derived from a patient to be diagnosed in accordance with this invention) is higher than that of a healthy control patient, i.e. a patient not suffering from a disease as defined herein and/or a patient Sacking anti- ⁇ -AR antibodies.
  • an "increased titer of anti- ⁇ -AR antibodies" in accordance with the present invention preferably refers to any occurrence of anti- ⁇ - AR antibodies, i.e. any occurrence of a detectable amount of a ⁇ ti- ⁇ -AR antibodies.
  • sample in accordance with the present invention includes, but is not limited to, (a) biological or medical sampie(s), like, e.g. (a) sampie(s) comprising cell(s) or tissue(s).
  • sample(s) may comprise(s) biological material of biopsies.
  • biopsies comprise cell(s) or tissue(s) taken, e. g. by the attending physician, from a patient/subject as described herein.
  • the biological or medical sample to be analysed in context of the present invention is or is derived from blood, plasma, white blood cells, urine, semen, sputum, cerebrospinal fluid, lymph or lymphatic tissues or cells, muscle cells, heart cells, cells from veins or arteries, nerve cells, cells from spinal cord, brain cells, liver cells, kidney cells, cells from the intestinal tract, cells from the testis, ceils from the urogenital tract, colon cells, skin, bone, bone marrow, placenta, amniotic fluid, hair, hair and/or follicles, stem celis (embryonic, neuronal, and/or others) or primary or immortalized cell lines (lymphocytes, macrophages, or cell lines).
  • samples in accordance with the present invention are those derived from blood or plasma.
  • the biological or medical sample as defined herein may also be or be derived from biopsies, for example biopsies derived from heart tissue, veins or arteries.
  • the present invention relates to a diagnostic kit, for example a diagnostic kit for the detection of antibodies against a ⁇ -AR, comprising the cyclic peptide, composition or diagnostic agent of the invention.
  • the kit in accordance with the present invention comprises at least one of the compounds as disclosed according to the invention, like, for example a cyclic or linear peptide of the present invention, a nucleic acid molecule, vector or host cell of the invention or a composition or diagnostic agent according to the present invention.
  • the kit further comprises an instruction leaflet, and/or a buffer for use in the application of the kit, and/or at least one reaction vessel for carrying out the detection reaction for which the kit is or is to be used.
  • at least one, some or all of the reagents used in connection with the application of said kit are present as portions useful in carrying out the reaction(s) for which the kit is to be used.
  • One particular approach for using the compounds according to the present invention as a diagnostic and in a diagnostic method, respectively, is a three-step screening procedure.
  • this method comprises performing an ELISA with the cyclic peptides according to the present invention as well as determining immunofluorescence and determining cAMP responses in cells expressing native human ⁇ AR. It is to be acknowledged that each and any of the aforementioned steps can as such be preformed for the detection of said antibodies using the cyclic peptides according to the present invention.
  • a large number of patients, for example heart failure patients, may thus be screened for functionally active anti- ⁇ i-AR antibodies.
  • the definition of functionally active anti- ⁇ rAR antibodies is preferably based on their effects on receptor-mediated signalling, that is, their effects on cellular cAMP levels and on the activity of the cAMP-dependent protein kinase (PKA).
  • Cyclic AMP is a universal second messenger of many G protein-coupled receptors including the ⁇ - AR family. It exerts its effects via PKA, cAMP-gated ion channels, phosphodiesterases, and exchange proteins directly activated by cAMP, known as Epad and 2.
  • Epad and 2 exchange proteins directly activated by cAMP
  • single chain fluorescence indicators have been described in the art which are characterized by having an enhanced cyan (CFP) or yellow fluorescent protein (YFP) directly fused to the cAMP-binding domain of Epac-proteins, which allowed to achieve a higher sensitivity and better temporal resolution of the cAMP measurements.
  • CFP enhanced cyan
  • YFP yellow fluorescent protein
  • Such system is, among others described in WO 2005/052186.
  • Such system can be used in connection with any diagnostic procedure using the cyclic peptides or other corresponding compounds according to the present invention. Also such system can be used for, however is not limited thereto, analyzing the prevalence of functionally active anti- ⁇ i-AR antibodies.
  • Such diagnostic method is applied to a cohort of previously antibody-typed DCM patients or any individual to be assessed insofar or any individuai suspected of suffering from any of the diseases described herein or being at risk to suffer therefrom, in a further step of the diagnostic method and screening method, the abiiity of ⁇ -blockers to inhibit anti- ⁇ rAR antibodies-induced receptor activation may be assessed and determined, respectively.
  • the afore described assay which is a FRET-based method as described in WO 2005/052186 making use of the peptides according to the present invention is advantageous insofar as it is simpier, less time consuming, and at the same time discloses or identifies all DCM patients previously considered anti- ⁇ rECn antibody- positive.
  • This embodiment of a FRET based method of diagnosing making use of one or several of the peptides according to the present invention is based on detecting antibody-induced increases in cAMP.
  • the present invention is also related to the use of one or several of the peptides according to the present invention for use in an Epac-FRET assay. More preferably such Epac-FRET assay is used for diagnosis, even more preferably for the diagnosis of patients suffering from or suspected of suffering from any of the disease described herein.
  • the present invention relates to a method for detecting of antibodies against a ⁇ -AR (for example in a sample as defined herein) comprising the step of contacting the cyclic peptide of the invention with said antibodies to be detected.
  • the present invention relates to the cyclic peptide, composition or diagnostic agent as disclosed herein for detecting (for example in a sample as defined herein) antibodies against a ⁇ -AR.
  • ab antibody
  • Abs or abs antibodies
  • AR adrenergic receptor
  • EC extra cellular domain of a ⁇ -AR ECn extra cellular domain Il of a ⁇ -AR and AA amino acid.
  • Figure 1 is a diagram depicting the scheme of the mutated ⁇ i-ECn-25AA or 18AA- cycio-peptides (black rings with the original Cys-residues (white bails) or the Ser mutated Cysteines (biack balls; Cys/Ser or Ser/Cys, respectively), together with the amino-acids involved in forming the primary ring structure after head-to-tail closure (closure site either AIa-DGIu 5 or Pro-Lys).
  • Fmoc-Glu-ODmab or another Fmoc amino acid having a side chain protecting group which can be selectively cleaved off in an orthogonal manner is incorporated at the C-terminal end of the linear peptide.
  • the cleaving off of the cyclic peptide from the synthesis resin generates a peptide amide (in the case of D-G!u ⁇ Gln) and the removal of the protective groups of the side chain is done by treating the resin with triftuoro acetate acid/triisopropyisilane/ethandithiole/water for several hours.
  • Figure 2 is a diagram depicting the blocking capacity of ⁇ - ⁇ -ECn-18AA cyclopeptide mutants having a D-GSu ring closure on ⁇ r receptor-mediated signalling (functional cAMP-assay) using an approach by fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • rat number 4 with ⁇ i-ECn-18AA- cyclopeptide mutants (Cys/Ser mutation, dark blue (3); Ser/Cys mutation, tight blue (4)) was compared with the effect of a 3 Cys-containing 25AA Cys/Cys cyclopeptide (red (2)) or the result obtained with anti- ⁇ i-ECn IgG antibodies in the absence of blocking peptides (black (1)).
  • the y ⁇ axis represents the normalized YFP/CFP-ratio of the registered FRET emission signals, the x-axis corresponds to the registration time given in seconds (s).
  • Figure 3 is a diagram composed of two major (upper and lower) panels resuming the blocking effect of both 25AA- and 18AA-cyclopeptide mutants having a GIn closure site, as well as 18AA cyclopeptide mutants having a D-GIu closure site after preincubation (12h, 4°C, rotating incubation) with sera isolated from 69 different immunized antibody-positive rats in an ELISA-competition assay using the 3 Cys- containing Smear 25AA Cys/Cys-peptide as an antigen.
  • the first three columns on the left side within the two panels represent the results obtained with the (non mutated) 3 Cys-containing 25AA (Gln-)cyclopeptide (black columns) and the mutant ⁇ rE ⁇ Cn-25AA (Gln-)cyclopeptides (Cys/Ser mutation, white columns; Ser/Cys mutation, horizontally hatched columns).
  • the five columns on the right side within the two panels represent the results obtained with the (non-mutated) 3 Cys-containing 18AA (Gln-)cyclopeptide (black columns) compared with the different 2 Cys-containing mutant 18AA cyclopeptides (18AA Cys/Ser mutant with a GIn closure site, white columns; 18AA Cys/Ser mutant with a D-GIu closure site, diagonally left hatched columns; 18AA Ser/Cys mutant with a GIn closure site, diagonally right hatched columns; 18AA Ser/Cys mutant with a D- Giu closure site, vertically hatched columns).
  • the error bars represent the standard error of the mean ( ⁇ SEM).
  • the y-axis represents the blocking efficiency of the various peptides used given in % of blocked versus non-blocked ELISA-reactivity of the sera.
  • Figure 5 is a diagram resuming the in vivo blocking capacity of in total five (prophylatic) applications of various linear and cyclic betal-ECII-peptides, started 3 months after the first immunization (and two subsequent beta1-ECH/GST-antigen- boosts, corresponding to a prevention protocol).
  • Serum-titers of the betai -receptor antibodies were determined before and 18-2Oh after each peptide injection (abscissa, time in months) and are given in % of the corresponding antibody-titers of immunized untreated rats (y-axis, ordinate).
  • the injected peptides were: 25AA Cys/Cys linear peptide (black squares), 25AA Cys/Cys cyclopeptide (white squares), 18AA Cys/Cys cyclopeptide (black diamond), 18AA Cys/Ser cyclopeptide mutant (white diamonds), and the 18AA Cys/Ser linear peptide mutant (vertically hatched diamonds). Also in vivo, the efficiency of the cyclic peptides was largely superior to their linear counterparts.
  • Figure 6 is a diagram resuming the tn-vivo blocking effect of both 25AA and 18AA cyclo-peptide mutants with a GIn closure site, determined after the first intravenous (i.v.) injection of 1.0 mg/kg body weight (Bw) into immunized antibody-positive rats. Sera were drawn 18-20 hours after i.v. injection of 1.0 or 0.25 mg/kg Bw of the indicated peptides and assayed for reactivity by ELISA using the 3 Cys-containing linear 25AA Cys/Cys-peptide as an antigen.
  • the bars and numbers (in boxes) of each row represent the mean values of the blocking capacity of the respectively indicated peptide given in % of the ELISA- immunoreactivity of the sera before and 18-20 hours after i.v. peptide injection (y- axis).
  • Figure 7 A is a diagram resuming the in vivo blocking effect of both 25AA and 18AA cyclopeptide mutants with a GIn closure site, determined after a total of ten intravenous (i.v.) injections of 1.0 mg/kg body weight (Bw) of the indicated peptides into immunized antibody-positive rats.
  • Sera were drawn before and 18-20 hours after i.v. injection of the various peptides every 4 weeks (abscissa: time in months of treatment) and assayed for reactivity by ELISA using the 3 Cys-containing linear 25AA Cys/Cys-peptide as an antigen.
  • the graph depicts the relative decrease (or increase) in specific anti-beta 1 -receptor antibody-titers in sera from antibody-positive immunized rats after injection of the indicated peptides and shows the respective mean value of the blocking capacity of the peptide given in % of the initial ELISA-immunoreactivity before starting treatment (y-axis, ordinate).
  • Figure TB is a diagram resuming the in vivo blocking effect of various concentrations of 18AA cyclopeptide mutants with a Gin closure site, determined after a total of ten intravenous (i.v.) injections of 0.25, 1.0, 2.0, and 4.0 mg/kg body weight (Bw) into immunized antibody-positive rats, irrespective of the cyclopeptide "responder-state" of individual animals.
  • Sera were drawn before and 18-20 hours after i.v, injection of the various peptides every 4 weeks (abscissa: time in months), and assayed for reactivity by ELISA using the 3 Cys-containing linear 25AA Cys/Cys-peptide as an antigen.
  • the graph depicts the relative decrease (or increase) in specific anti-beta1 -receptor antibody-titers in sera from antibody-positive immunized rats after injection of the indicated peptides and shows the respective mean values of the blocking capacity of the peptides given in % of the initial EUSA-immunoreactivity before starting treatment (y-axis, ordinate).
  • Figure 7C is a diagram resuming the in vivo blocking effect of various concentrations of 18AA cyclopeptide mutants with a GIn closure site, determined after a total of ten intravenous (i.v.) injections of 0.25, 1.0, 2.0, and 4.0 mg/kg body weight (Bw) into immunized antibody-positive rats, respecting only cycfopeptide-sensitive "responders", defined as animals having , after 7 cyclopeptide-injections, a maximum remaining receptor anti-body level equal or inferior to 80% of the respective titer at start of therapy (compare the curves between Fig. 7c and Fig. 7b, the latter representing the naturally occuring inhomogenous response of unselected animals).
  • Sera were drawn as described above and assayed for reactivity by ELlSA using the 3 Cys-containing linear 25AA Cys/Cys-peptide as an antigen.
  • the graph depicts the relative decrease in specific anti-beta 1 -receptor antibody-titers in sera from antibody-positive immunized responders after injection of the indicated peptides giving the blocking capacity in % of the initial ELISA-immunoreactivity (y- axis, ordinate).
  • Figure 8A is a diagram showing the time course (month 0 to 20) of the internal end- systolic and end-diastolic left ventricuSar diameters (LVES, LVED) of GST/ ⁇ i-ECn- immunized un-treated (black circles) versus GSTV ⁇ rECII-immunized animals treated with the indicated various cyciopeptides (see legend) as determined by echocardiography (echocardiographic system: Visual Sonics, Vevo 770 (version V2.2.3), equipped with a 15-17.5 MHz transducer), whereby LVES/LVED is left ventricular end-systolic diameter/left ventricular end ⁇ diastolic diameter.
  • echocardiography echocardiographic system: Visual Sonics, Vevo 770 (version V2.2.3), equipped with a 15-17.5 MHz transducer
  • Figure 8B is a similar diagram showing the time course (month 0 to 20) of the internal end-systolic and end-diastolic left ventricular diameters (LVES, LVED) of GST/ ⁇ i-ECii-immunized untreated (black circles) versus GST/ ⁇ rECII-imrnunized animals, treated with different concentrations of the 18AA Cys/Ser cyclopeptide mutant (see legend).
  • Figure 9A is a diagram depicting the time course ⁇ month 0 to 20) of the "Cardiac index" (Cl) in ml/min/g (body weight) as determined by echocardiography (echocardiography system see legend to figure 8a.).
  • Figure 9B is a similar diagram showing the time course (month 0 to 20) of the "Cardiac index" (Cl) in ml/min/g (body weight) as determined by echocardiography (echocardio-graphic system see legend to Figure 17a.)
  • FIGS 10A-C depict hemodynamic parameters obtained in the therapy study after
  • HF heart frequence
  • LV press. LV systolic blood pressure
  • Figure 10B on the left side depicts the contractility (+ dP/dt) in mmHg/s, and on the right side the relaxation (-dP/dt) in -mmHg/s;
  • FIG. 10C 1 shows the left ventricular end-diastolic pressure (LVEDP) as determined by cardiac catheterization in mmHg.
  • Figure 11 is a diagram depicting the scheme of mutated cysteine-containing betai- ECII-homologous cyclopeptides (amino-acids (AA)) are represented as white balls with the corresponding AA letter code written in each bail). Cysteine molecules and their substitutes are depicted as black balls. The assumed localization of the disulfide bridge is represented by a bold black line.
  • Left side scheme depicting the original sequence of the ECII-loop of the human betai adrenergic receptor; middle: cyclic 22AA ECII-homologous peptide with the glycine mutation at the assumed ring closure site (Position 222).
  • the right panels depict examples of a cyclic 22AA peptide-mutant containing only two cysteines (i.e., position 209 and 215).
  • the upper scheme shows the Cys/Ser mutant
  • the lower scheme depicts the Cys/ABu mutant of the cysteine at position 216 (Cyclic 22AA beta1-ECII peptide Cys 2 is ⁇ Ser 2 i6 and Cyclic 22AA beta1-ECll peptide Cys2i6 ⁇ ABu 2 i6, respectively).
  • Numbers given indicate the numbering of the amino-acids in the original primary sequence according to Frieile et a/. 1987, PNAS 84, pages 7920-7924.
  • 12B depicts the mean values ⁇ SEM of each of the treated groups of immunized beta1-ECII-antibody positive animals; error bars indicate ⁇ SEM.
  • the figure depicts the mean values ⁇ SEM of each of the treated groups of immunized beta 1 -ECI l-antibody positive cardiomyopathic rats (animal number per group is given in the legend).
  • the bar graph shows the mean effect of eight subsequent cyclopeptide-injections, determined 20-22 hours after application of the indicated constructs.
  • Figure 13B depicts the time course of antibody-titers after 11 (22cyc Cys/Abu) or 12 (22cyc Cys/Ser) subsequent cycio-peptide-injections, determined before and 20-22 hours after application of the indicated constructs. Values are given in per cent of increase or decrease in the respective antibody-titers after each cyciopeptide- injection compared with the antibody-titer determined at start of therapy (month 8), Error bars are not indicated in the graph.
  • Figure 14A is a diagram showing the time course (month 0 to 21) of the internal end- systolic and end-diastolic (eft ventricular diameters (LVES, LVED) of GST/beta 1 -EC 11- immunized untreated (black circles) versus GST/beta1-ECIi-immunized animals treated withe the indicated various cyclopeptides (see legend) as determined by 2D- and M-mode echocardiography (echocardiography system: Visual Sonics, Vevo 770 (version V2.2.3), equipped with a 15-17.5 MHz transducer), whereby LVES/LVED is left ventricular end-systolic diameter/left ventricular end-diastolic diameter.
  • Figure 14B is a diagram showing the time course (month 8 to 21) of the change in left ventricular fractional shortening, given in % of the values obtained at the initiation of therapy (LVED-LVES/LVED x 100) in GST/beta1-ECI!-immunized untreated rats (black circles) versus animals treated with the indicated different cyclopeptides (see legend) as determined by 2D- and M-mode echocardiography (echocardiographic system: Visual Sonics, Vevo 770 (version V2.2.3), equipped with a 17.5 MHz transducer), whereby LVES/LVED is left ventricular end-systolic diameter/left ventricular end-diastolic diameter.
  • 2D- and M-mode echocardiography echocardiographic system: Visual Sonics, Vevo 770 (version V2.2.3), equipped with a 17.5 MHz transducer
  • Figure 14C is a diagram depicting the time course (month 0 to 21) of the "Cardiac index" (Cl) in ml/min/g (body weight) as determined by 2D- and Doppler-echocardio- graphy (echocardiographic system see above).
  • Asterisk indicate level of significance as derived from repeated measures ANOVA and Bonferroni post hoc testing; * P ⁇ 0.05, ** P ⁇ 0.01 , ** *P ⁇ 0.005.
  • Figures 14D-F depict hemodynamic parameters of 0.9%NaCI-injected control animals (white columns) versus GST/beta 1 -EC I l-immunized untreated (black columns) versus GST/beta1-ECII-immunized animals injected with the indicated different cyclopeptides (see legend) after 12 months of treatment.
  • HF heart frequence
  • LV press. LV systolic blood pressure
  • Figure 14E on the left side depicts the contractility (+ dP/dt) in mmHg/s, and on the right side the relaxation (-dP/dt) in -mmHg/s;
  • Figure 14F shows the left ventricular end-diastolic pressure (LVEDP) as determined by cardiac catheterization in mmHg.
  • Figure 14G shows two panels (a and b) with different laboratory parameters determined in the serum of animals after 12 months of treatment.
  • the relative wet weights of the indicated organs are given in g/kg body weight.
  • Figure 15A is a scheme depicting the departing sequences of the 22AA Cys/Abu- cycio-pept ⁇ de mutant of the present invention.
  • the corresponding original 21 + 1AA of the second extracelluier loop of the human betai- (AA200 to AA221 ) and beta2-adre- nergic receptor (AA175 to AA 196) are depicted.
  • 4.5 angstrom units are the distance at the basis of the native ECIMoop between AA 200 and AA 220 (betai) or AA175 and AA 190 (beta2).
  • this gap was filled in by the smallest naturally occurring AA glycine.
  • sequences underneath depict the nine different cyclopeptides generated to identify further AA necessary for antibody-recognition and neutralization, derived from the beta2-ECII sequence (which was not able to block anti-beta1-ECIi antibodies) in subsequently performed blocking- and binding-competition assays using either 25AA beta1-ECII-peptides or (N- and C-termina!) biotinylated linear 16AA beta1-ECII-peptides, respectively (Ala-scan 22AA Cys/Abu-cyclopeptides).
  • Figure 15C is a diagram depicting the results of binding-competition assays using 22AA Cys/Ser versus 22AA Cys/Abu cyclopeptides in order to block binding of different rat anti-beta1-ECIi (e.g. monoclonal or polyclonal) to biotinylated linear 16AA beta1-ECII-peptides.
  • the respective EC 50 of the indicated cyclopeptides are given in the tables underneath each figure.
  • Panel 1 shows the (one site) binding competition curves obtained with a monoclonal rat anti-beta1-ECII antibody.
  • Panel 2 shows the (one site) binding competition curves obtained with a polyclonal rat anti-beta 1 -ECU antibody (K12R1), representative for 3 different rat sera tested.
  • Panel 3 shows the (one site versus two site) binding competition curves obtained with a polyclonal rabbit anti ⁇ beta1-ECil antibody, suggesting the presence of two different antibody-entities with different 18Cys/Ser cyclopeptide affinities in the probe.
  • Panel 4 shows the (one site) competition curves obtained for different site-specific ala-mutated 22AA-beta1 -cyclopeptides of the present invention, whereby 22AbuO represents the inventional product. The data shown in pane! 4 are representative for 3 different polyclonal rat sera tested.
  • Figure 16 shows the high pressure liquid chromatography (HPLC) elution profile of the two cysteine-containing mutant cyc22AA Cys/L-a!pha butyric acid (Abu) of the present invention, a cyclic peptide with a GIy closure site, HPLC was carried out in a Hewlett Packard Series 1050 analytical HPLC-system (Agilent Technologies Germany GmbH, B ⁇ blingen) equipped with a dual wavelength UV absorbance detector; absorbance was read at 216 nm.
  • HPLC high pressure liquid chromatography
  • Figure 17 shows the characterization of the cyclic ECH-22AA Cys/Abu mutant peptide (with a GIy closure site) by mass spectroscopy (MALDI-TOF).
  • the panel depicts MALDi-tracings of the cyc22AA Cys/Abu-mutant (2499.34 m/z).
  • the ordinate of each graph shows measured signal intensities ("a.u.” means arbitrary units), the abscissa indicates the molecular mass (given in m/z).
  • the MALDI-analysis was carried out using a reflex ll-mass spectroscope (Bruker Daltonic GmbH, Bremen), equipped with a Scout-26 sample carrier. In each case the simply protonated molecule was analyzed at 2200 m/z.
  • Figure 18 shows a panel demonstrating a high pressure liquid chromatography (HPLC) elution profile of the 3 cysteine-containing construct cyc22AA Cys/Cys.
  • HPLC high pressure liquid chromatography
  • Figure 19 is a diagram depicting the blocking capacity of different site-specific Ala- mutated 22AA Cys/Abu-cyclopeptides (Fig. 15A) on ⁇ -i-receptor-mediated signalling (functional cAMP-assay) using an approach by fluorescence resonance energy transfer (FRET) as described in Fig.2.
  • FRET fluorescence resonance energy transfer
  • the effect of preincubation (12h, 4°C, rotating incubator) of human anti- ⁇ i-ECI! IgG antibodies isolated from a representative 28 years-old female DCM patient with different 22Abu-mutants (as defined in Fig.15A) is shown. Numbers (n) indicated under columns correspond to the number of independent experiments performed under same conditions. Black column: patient IgG, unblocked: 18 ⁇ 3% pRET activity;
  • Diagonally hatched column patient IgG (inefficiently) blocked with the 22Abu8 cyclic peptide (17 ⁇ 2% FRET activity).
  • Figure 20 Is a diagram demonstrating the half-life of ECll-peptide-mutants in whole blood.
  • Betal -ECll-peptide-mutants cyc18AA Cys/Ser
  • a cyclic structure Le. the cyclic peptides as described herein
  • the data were obtained after incubation of linear versus cyclic peptides with human or rat whole biood in the presence of heparine (24 h, 4°C, rotating incubator) and monitoring the amount of intact peptide at the indicated time points.
  • the amount of remaining intact peptides was determined by competition ELISA with linear ⁇ i-ECl!-25AACys/Cys peptides at 2min, 10min, 30min, 1 , 2, 4, 8, 16 and 22h.
  • Black diamonds cyclic 18AACys/Ser peptides
  • White diamonds linear 18AACys/Ser peptides.
  • Figure 21A is a diagram resuming the in vivo blocking effect of 22AA Cys/ABu cyciopeptide mutants, determined after nine intravenous (i.v.) injections of 1.0 mg/kg body weight (Bw) of the cyclopeptides into immunized antibody-positive rats. Sera were drawn before and 18-20 hours after i.v. injection of the various peptides every 4 weeks (abscissa: time in months of treatment) and assayed for reactivity by ELISA using the 3 Cys-containing linear 25AA Cys/Cys-peptide as an antigen.
  • the graph depicts the relative decrease in specific anti- ⁇ i-ECIl antibody-titers in sera from antibody-positive immunized rats after injection of the indicated peptides and shows the respective mean value of the blocking capacity of the peptide given in % of the initial ELISA-immunoreactivity before starting treatment (y-axis, ordinate).
  • Figure 21 B is a diagram resuming the in vivo blocking effect of 22AA Cys/ABu cyciopeptide mutants (1.0 mg/kg body weight (Bw)), determined after nine intravenous (i.v.) (3/3/3) or eight triple injections (2/3/3) - one injection per week for three subsequent weeks - every 3 months into immunized antibody-positive rats, respectively.
  • Sera were drawn before and 18-20 hours after i.v. injection of the various peptides every 4 weeks (abscissa: time in months of treatment) and assayed for reactivity by ELlSA using the 3 Cys-containing linear 25AA Cys/Cys-peptide as an antigen.
  • the graph depicts the relative decrease in specific anti- ⁇ i-ECII antibody-titers in sera from antibody-positive immunized rats after injection of the indicated peptides at the indicated time points (white arrows) and shows the respective mean value of the blocking capacity of the respective peptides given in % of the initial ELISA- immunoreactivity before starting treatment (y-axis, ordinate).
  • Figure 22A is a diagram showing the time course (month 0 to 15) of the relative internal end-diastoiic left ventricular diameter (LVED) of GST/ ⁇ rECil-immunized untreated (black circles) versus GST/ ⁇ 1-EC!i-immunized animals treated with the indicated cyclopeptides/protocols (see legend) as determined by echocardiography (echocardiographic system: Visual Sonics, Vevo 770 (version V2.2.3), equipped with a 17.5 MHz transducer), whereby LVED is left ventricular end-diastolic diameter given in % of the respective values at initiation of treatment (month 9).
  • echocardiography echocardiographic system: Visual Sonics, Vevo 770 (version V2.2.3), equipped with a 17.5 MHz transducer
  • Figure 22B is a similar diagram showing the time course (month 0 to 15) of the relative internal end-systolic left ventricular diameter (LVED) of GST/ ⁇ 1 -ECII- immunized untreated (black circles) versus GST/ ⁇ 1-ECIl-immunized animals treated with the indicated cyciopeptides/protocols (see legend) as determined by echocardiography (echocardiographic system described above), whereby LVED is left ventricular end-diastolic diameter given in % of the respective values at initiation of treatment (month 9).
  • LVED left ventricular end-diastolic diameter
  • Figure 22C is a similar diagram showing the time course (month 0 to 15) of the "Cardiac index' 1 (Ci) in given in % of the respective values at initiation of treatment (month 9) of GST/ ⁇ 1-ECil-immunized untreated (black circles) versus GST/ ⁇ 1-ECII- immunized animals treated with the indicated cyclopeptides/protocols (see legend) as determined by echocardiography (echocardiographic system described above).
  • the symbols indicate the same therapy groups as in Fig.22A. Error bars are not indicated in the graph.
  • cyciopeptides which can form only one single individual disulfide bond are composed of 18, 22 or 25 amino acids (AA): EC ⁇ -18AA Cys/Ser mutant (Gln-)cyclopeptide, ECn-22AA Cys/ABu and Cys/Ser mutant (Gfy-)cyclopeptide and EC
  • the primary sequence is partially homologous to the human sequence of the P 1 -AR (amino acid positions 204 through 219. 200 through 220 and 200 through 222, respectively).
  • the 18AA, 22 or 25AA cyclopeptide mutant adopts a conformation which more closely mimics that of the epitope as presented on the surface of the native ⁇ - 1 -ECn protein loop.
  • cyclization has been employed as a tool to prolong the duration of action of peptide, since in general cyclic peptides are more stable to proteolysis than their linear counterparts.
  • Cyclo(K-18-P) Cyclic S-S, Cys/ABu or Cys/Ser mutant is: Cyclo-Ala-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-AButSerJ-Asp-Phe-
  • Cyclization can occur between Cys 7 and Cysi 3 (disulphide bond) and AIa 1 and Glnts ⁇ ring closure).
  • Cyclization can occur between Cysio and Cysie (disuiphide bond) and Argi and Gly 22 (ring closure).
  • Cyciization can occur between Cysn and Cys 17
  • the cyclopeptide mutants of the present invention are first synthesized as linear peptides, and are then cyclized covarrily on the backbone by condensation of the C-terminal carboxyl group with the amino group of the N-terminal amino acid. Subsequently, a disulphide bond between cysteine residues 7 and 13 (18mer cyclopeptide), cysteine residues 10 and 16 (22mer cyciopeptide) and cysteine residues 11 and 17 (25mer cyclopeptide) is established.
  • the linear peptide is assembled by stepwise solid phase peptide synthesis using an Fmoc / tert butyl strategy. Chlorotrityl is used as a starting resin.
  • the first amino acid (Fmoc-Pro-OH) is coupling with DIEA in DMF, the second with PYBOP/ HOBT/ DIEA in DMF and the following amino acids with diisopropylcarboimide, HOBT in DMF.
  • the peptide quality is monitored online by UV detection. Deprotection/coupling (two-fold excess) procedure is described below:
  • the fuily protected peptide with reactive N-terminal amino- and C-terminal carboxyi- groups is cleaved from the resin by treatment with hexaffuoroisopropanol/ dichloromethane.
  • Cyclization is carried out thereafter in solution according to the following protocol:
  • the "head to tai!-cyclization of the protected peptide is performed with PyBOP/ NaHCO 3 in high DMF dilution (10 mmol of linear peptide/1 L of DMF).
  • the cyclization is completed after 3 days.
  • the peptide is washed with 5% NaHCO 3 , H 2 O and pure H 2 O.
  • the reaction mixture is cooled down, and the peptide is deprotected excepting the cysteine groups. Afterwards, the partially protected peptide is isolated by precipitation with methyl t-butyl ether.
  • the crude peptide is pre-purified by liquid chromatography: Stationary phase: silica C18, 15 ⁇ m, 120 A Eluant: H 2 O acetonitrile + 0.1% TFA
  • the disulfide cyclization is performed in H 2 O (2 mg/mL) with the presence of dimethyl sulfoxyde (3%). The cyclization reaction is completed after 3 days.
  • the peptide is purified by HPLC, using the conditions described above. The fractions with purity greater than 95% are pooled. The peptide is exchanged on an ion exchange resin (Dowex 1X2) and the final solution lyophilized. The peptide content is determined by amino acid analysis (Edman sequencing).
  • 22mer cyclic peptide containing a cystein to Abu substitution can, for example, be synthesized as follows:
  • the peptide is first synthesized as a linear peptide and is then cyclized on resin covalentSy on the backbone by condensation of the C4erminal carboxy! group with the amino group of the N-terminai amino acid.
  • the linear peptide is assembled by stepwise solid phase peptide synthesis on a continouus flow peptide synthesizer using an Fmoc/tert butyl strategy.
  • Fmoc- Glu(Wang resin LL)-ODmab (Merck Biosciences, substitution 0,3 mmole/g) is used as a starting resin.
  • the Fmoc-L-amino acids except the cysteines are coupled with PyBOP/NMM in DMF in a double coupling procedure.
  • cysteines are incorporated also by double coupling as the preactivated Fmoc-L-Cys ⁇ Trt) ⁇ Opfp esters in DMF/HOBT (boid printed in the sequence below).
  • pseudoproline dipeptides are incorporated into the sequence at positions V-T and E-S (bold printed in the sequence below). It has been shown that those pseudoproline dipeptides are extremely useful tools for assisting the cyclization of peptides. (Schmiedeberg (2002) Org. lett. 4, 59 and a) Haack (1992) Tetrahedron lett. 33 » 1589; b) Mutter (1995) Pept. Res. 8, 145).
  • substitution of Ser or Thr by a pseudoproline residue has the effect of bringing the ends of the chain together, promoting cyclization and reducing oligomerization and cyclodimer formation.
  • the native sequence is regenerated on cleavage and deprotection.
  • the assembled linear peptide on resin thus has, for example, the following sequence:
  • the ODmab group is removed from the peptide resin by 2% Hydrazine-hydrate in DMF and the cyclization of the peptide is performed on resin with PyBOP/NMM in DMF by reacting 5 hours at 50 degree Celsius and overnight at room temperature. A Kaiser test performed after this time does not show residual amount of free amino groups.
  • the peptide is cleaved off the resin by 95% TFA, 4% triethylsiiane, and 1 % water.
  • Crude peptide is purified by HPLC liquid chromatography using a stationary phase of Silica C18 10 ⁇ m, 100 A with an eluant of acetonitrile (80%) in 0,1 % TFA-water and 0,1 % TFA in water (Fig. 16). The detection is done at 220 nm. Purified fractions are lyophilized and analysed by mass spectrometry (MALDI-TOF; Fig.17).
  • ⁇ - ⁇ -ECn-18AA cyciopeptide mutants (Cysi 3 ⁇ Ser 14 or Ser 13 - Cys t4 mutation having an additional D-Glu ⁇ Gln exchange, e.g. at the ring closure site) was compared with the 3 Cys-containing 25AA or 18AA Cys/Cys cyclopeptides after preincubation (12h, 4° C, rotating incubation over-night) of different numbers of sera from immunized antibody-positive rats in an ELISA-competition assay using the 3 Cys-containing linear 25AA Cys/Cys peptide as an antigen.
  • the normalized YFP/CFP-ratio of the registered FRET emission signals served to quantify the effect of the cyclo- peptide mutants in terms of blockade (in per cent) of antibody-induced cellular cAMP- production of transiently Epad-transfected stably P 1 -AR expressing human embryonic kidney ceils (HEK 293- ⁇ i cells).
  • the x-axis in Fig. 2 corresponds to the registration time given in seconds (s).
  • results of the tests performed herein demonstrate that the antibody-blocking capacity of mutated cyclopeptides was not affected by the reduction of the number of amino-actds from a 25-meric to a 18-meric peptide.
  • results also demonstrate an excellent comparability of 25AA Cys/Cys and 18AA Cys/Cys cyclopeptides with the cyclic 25AA or 18AA Cys/Ser mutants, but not with the cyclic 25AA or 18AA Ser/Cys mutants.
  • the animal model used in this example and any other example described herein, if not indicated to the contrary, is the human analogue rat model.
  • this human analogue rat model was treated as described herein-below using the various compounds of the present invention, more particularly compounds of formula Vl, VII, VIH and iX, and, as controls, a linear ECu-18AA Cys/Ser mutated (GIn 18 -)peptide (with the following amino-acid sequence: Aia-Asp- Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Ser-Asp-Phe-Val-Gln) and a linear non-mutated 3 Cys-containing ECn ⁇ 25AA Cys/Cys (GIn 2S ) peptide (with the following amino-acid sequence: Aia-Arg-Aia-Giu-Ser-Asp-Giu-
  • Sera were drawn 18-20 hours after i.v. injection and assayed for reactivity by ELISA using the 3 Cys-containing iinear 25AA Cys/Cys-peptide as an antigen.
  • the in vivo data confirmed a higher blocking capacity of the 18AA Cys/Ser mutated cyclo-peptides (1 mg/kg/Bw) compared with either 25AA Cys/Ser mutants or the clearly less effective 18AA Ser/Cys mutated cyciopeptides at a same concentration (Fig. 6).
  • the in vivo efficiency of the 18AA Cys/Ser cyclopeptide was also largely superior to that of the linear 18AA Cys/Ser peptide.
  • mutant cyclopeptides seems also not affected by a reduction in the number of amino acids from a 25-meric to a 22-meric cyciopeptide, which, however, seems to possess better antibody-blocking capacities than the 18-mehc cyciopeptide - regardless of the mutation at postion 216 of the ⁇ r ECI!-sequence.
  • the aforementioned ⁇ i-ECI! epitope-mimicking and 22AA cyciopeptide mutants of the present invention were employed for in vivo experiments in order to optimize their strategy of application/administration.
  • the different application protocols comprised either monthly intravenous applications of cyciopeptide mutants disclosed by the present invention (cyc22AACys/Abu), or a triple injection (one injection every week on 3 subsequent weeks) every three months.
  • Measures like these provide the potential to reduce the amount of injected cyclopeptides or to reduce the burden of (regular monthly) venipuncture and to increase the flexibility of application (for both, human patients and animais) whilst maintaining or increasing the biological efficiency of the injected constructs of the present invention.
  • Cys/Abu constructs triple injections (1 injection per week on 3 subsequent weeks) followed by a two-months intervention free time interval were at least as efficient as or even slightly superior to monthly applications in reducing the titer of circulating anti- P 1 -ECU antibodies (Fig. 21 A and B), and in reverting the cardiomypathic phenotype (Fig.22A-C).
  • each of the nine amino-acids diverging from the second loop sequence of the human beta2-adrenergic receptor, which had no beta 1 -antibody- blocking effect, has been mutated to alanine before cyclization and in wfro-testing for their respective antibody-biocking capacity.
  • ECU beta2-adrenergic receptor 175 RATHQEAlN CYANETCCD FFTG 196
  • ECU betai -adrenergic receptor 200
  • RAESPEARRCYNPPKCCD FVTG 221
  • Cvs-Cvs/Abu cvciopeptide RAESDEARRCYNDPKC Abu DFVTG
  • the by far most relevant AA to preserve the antibody-blocking capacity of the inventiona! cyclopeptide was the praline (P) at position 213 (overall 89% loss in blocking capacity compared with the inventional product), followed by aspartic acid (D) at position 212 (overall 57% loss in blocking capacity), asparagine (N) at position 211 (overall 41 % loss in blocking capacity), and the lysine (K) at postion 214 (overall 40% loss in blocking capacity; numbering according to Frielle et a/. 1987, PNAS 84, pages 7920-7924).
  • the "two-site" binding competition curve obtained with polyclonal rabbit anti-beta 1 -ECU antibodies indicated the presence of at least two different antibody-entities in the preparation with different beta1 ⁇ 18AA Cys/Ser cyciopeptde affinities (Fig. 15C, pane! 3).
  • the "one-site" competition curves obtained for selected out of the different site- specific alanine-mutated beta1 ⁇ 22AA Cys/Abu cyclopeptides of the present invention impressively confirmed the ELISA-competition results.
  • Example 6 In vitro blockade of activating human ⁇ i-receptor-autoantibodies
  • the blocking capacity of ⁇ i-EC ⁇ 22AA Cys/Abu cyclopeptide mutants on ⁇ -,-receptor- stimulation induced by activating human ⁇ -j-receptor autoantibodies was assayed using an approach by fluorescence resonance energy transfer (FRET) (Fig. 19)
  • FRET fluorescence resonance energy transfer
  • the normalized YFP/CFP-ratio of the registered FRET emission signals served to quantify the effect of the 22AA Cys/Abu cyciopeptide mutants in terms of blockade (in per cent) of antibody-induced cellular cAMP- production of transiently Epad-transfected stably ⁇ r AR expressing human embryonic kidney cells (HEK 293- ⁇ i cells).
  • results of the tests performed herein demonstrate that the antibody-blocking capacity of mutated ⁇ i-ECn-cyciopeptides was not affected by the reduction of the number of amino-acids from a 25-meric to a 22-meric (or a 18-meric) peptide.
  • the results also demonstrate a good comparability of 25AA Cys/Cys and 18AA Cys/Cys cyclopeptides with the cyclic 25AA, 22AA or 18AA Cys/Ser mutants, and also with the cyclic 22AA Cys/ABu mutants with respect to their capacities of blocking activating rat or rabbit anti ⁇ rECn ⁇ abs.
  • Aiso human anti- ⁇ rECn appear directed aginst an epitope within the second ⁇ -i-receptor loop which comprises either the key amino-acis Cys 2 o 9 /Pr ⁇ 2 i 3 , or the amino-acids Pro 2 - ⁇ 3 /Cys 2 i5 (numbering according to Frielle et al. 1987, PNAS 84, pages 7920-7924).
  • the present invention refers to the following nucleotide and amino acid sequences:
  • Amino acid sequence homologous to an ECn epitope of human ⁇ -i-AR (25AA;
  • Cysi 8 - ⁇ ABui 8 may occur between Alai and GIn 2 S
  • Amino acid sequence homologous to an ECn epitope of human ⁇ rAR 25AA;
  • Cys 17 ⁇ ABui 7 may occur between Alai and GIn 2 S
  • Nucleotide sequence encoding an amino acid sequence homologous to an ECn epitope of human ⁇ r AR 25AA; Cysis- ⁇ Xxx-is) gcncgggcggagagcgacgaggcgcgccgctgctacaacgaccccaagtgcXXgacttcgtcaccaaccggcar
  • Nucleotide sequence encoding an amino acid sequence homologous to an ECn epitope of human ⁇ r AR (18AA; Cys 13 ⁇ Xxxi3) gcngacgaggcgcgccgctgctacaacgaccccaagXXXtgcgacttcgtccar SEQ ID No. 8;
  • Nucleotide sequence encoding an amino acid sequence homologous to an ECn epitope of human ⁇ i-AR (25AA; Cys 17 ⁇ Xxxi 7 ) gcncgggcggagagcgacgaggcgcgccgctgctacaacgaccccaagXXXtgcgacttcgtcaccaaccggcar
  • Cys 3 - ⁇ ABu 3 Cyciization may occur between Lysi and Proi 8 Lys-Cys-ABu-Asp-Phe ⁇ /al-Gln-A ⁇ a-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro
  • Cys 3 ⁇ ABu 3 may occur between Lysi and Pro 2 5
  • Cys 2 ⁇ ABu 2 Cyciization may occur between Lys-i and Pro 18 Lys-ABu-Cys-Asp-Phe-Val-Gln-Ala-Asp-Glu-Aia-Arg-Arg-Cys-Tyr-Asn-Asp-Pro
  • Cys 2 ⁇ ABu 2 may occur between LyS 1 and Pro 2 5
  • SEQ ID No.14 Nucleotide sequence encoding an amino acid sequence homologous to an ECn epitope of human ⁇ r AR (25AA; Cys 3 ⁇ Xxx 3 ) aagtgcXXXgacttcgtcaccaaccggcargcncgggcggagagcgacgaggcgcgctgctacaacgacccc
  • Nucleotide sequence encoding an amino acid sequence homologous to an ECn epitope of human ⁇ i-AR 25AA; Cys2 ⁇ Xxx2 aagXXXtgcgacttcgtcaccaaccggcargcncgggcggagagcgacgaggcgcgctgctacaacgacccc
  • Amino acid sequence of an ECn epitope bearing portion of human ⁇ -i-AR (23AA; AA positions 200 to 222)
  • Nucleotide sequence encoding an amino acid sequence homologous to an ECn epitope of human ⁇ r AR (22AA; Cys 17 ⁇ Xxxi 7 ) cgggcggagagcgacgaggcgcgccgctgctacaacgaccccaagtgcXXgacttcgtcaccGLY
  • SEQ ID No.27 Amino acid sequence homologous to an ECn epitope of human ⁇ i-AR ⁇ 22AA;
  • CyS 3 - ⁇ ABu 3 Cyclization may occur between Lysi and Pr ⁇ 22
  • Nucleotide sequence encoding an amino acid sequence homologous to an ECn epitope of human Ji 1 -AR (22AA; Cys 3 ⁇ Xxx 3 ) aagtgcXXXgacttcgtcaccGLYcgggcggagagcgacgaggcgcgccgctgctacaacgacccc
  • xxx stands for any nucleotide triplet coding for an amino acid or amino acid stretch which can be replaced by ABu; Xxx may also mean that the nucleotide triplet is completely missing ,- "GLY” stands for any nucleotide triplet coding for GIy (Glycine), i.e. for ggn. n stands for any nucleotide, particularly a, c, g or t, y stands for t or e and r stands for a or g. in the amino acid sequence, "Xxx” stands for an amino acid or amino acid stretch which can be replaed by ABu; Xxx may also mean that the amino acid is completely missing.
  • sequences of the various peptides are indicated from the N- terminus to the C-terminus, whereby the N-terminus is at the left side and the C- terminus is at the right side of the respective depicted amino acid sequence.

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Abstract

The present invention relates to novel β-AR homologous cyclopeptide-mutants comprising only two cysteine residues able to form an intramolecular linkage and comprising an α-aminobutyric acid (ABu) residue, or an analogue thereof, which replaces the cysteine residue corresponding to cysteine 216 of the human β1-AR amino-acid sequence. The present invention further relates to linear peptides that can form these cyclopeptide-mutants and to nucleic acid molecules encoding peptides from which these cyclopeptide-mutants and linear peptides can be manufactured. Moreover, vectors comprising the nucleic acid molecules and recombinant host cells comprising the nucleic acid molecules or vectors are provided. Further, a method for producing the disclosed cyclopeptide-mutants is provided. Further disclosed is a composition comprising the peptide(s), nucleic acid molecuie(s), vector(s) or host cell(s) of the invention. The present invention also relates to therapeutic and diagnostic means, methods and uses taking advantage of the peptide(s) of the invention and to means, methods and uses for detecting anti-β- adrenergic receptor antibodies, in particular anti-β1 -adrenergic receptor antibodies.

Description

Novel peptide-homofogues for inhibiting (^-adrenoceptor antibodies
The present invention relates to novel β-AR homologous cyclopeptide-mutants comprising only two cysteine residues able to form an intramolecuiar linkage and comprising an α-aminobutyπc acid (ABu) residue, or an analogue thereof, which replaces the cysteine residue corresponding to cysteine 216 of the human βi-AR protein sequence. The present invention further relates to linear peptides that can form these cyclopeptide-mutants and to nucleic acid molecules encoding peptides from which these cyclopeptide-mutants and linear peptides can be manufactured. Moreover, vectors comprising the nucleic acid molecules and recombinant host ceils comprising the nucleic acid molecules or vectors are provided. Further, a method for producing the disclosed cyclopeptide-mutants is provided. Further disclosed is a composition comprising the peptide(s), nucleic acid molecuie(s), vector(ε) or host celi(s) of the invention. The present invention also relates to therapeutic and diagnostic means, methods and uses taking advantage of the peptide(s)s of the invention and to means, methods and uses for detecting anti-β-adrenergic receptor antibodies, in particular anti-βi-adrenergic receptor antibodies.
Progressive cardiac dilatation and pump failure of unknown etiology has been termed "idiopathic" dilated cardiomyopathy (DCM) (Richardson 1996 Circulation, 93, 841-842). DCM represents one of the main causes of severe heart failure with an annual incidence of up to 100 patients and a prevalence of 300-400 patients per million (AHA report 2007).
At present the large majority of DCM is thought to arise from an initial (mostly viral) infection leading to acute myocarditis which upon activation of the immune system may progress to (chronic) autoimmune myocarditis resulting in cardiac dilatation and severe congestive heart failure; the latter progression occurs particularly, when associated (a) with the development of autoantibodies against distinct myocyte sarcolemmai or membrane proteins which are essential for cardiac function (Freedman 2004, Jahns 2006), or (b) with chronic inflammation of the myocardium and virai persistence (Kϋhl 2005). These recent findings are further strengthened by the fact, that patients with DCM often have alterations in both cellular and humoral immunity (Jahns 2006, Limas 1997, Luppi 1998, Mahrholdt 2006). Under such conditions an initial acute inflammatory reaction may proceed into a kind of low-grade inflammation (MacLellan 2003) facilitating the development of abnormal or misled immune responses to the primary infectious trigger (Freedman 2004, Kϋhl 2005, MacLeilan and Lusis 2003, Maekawa 2007, Smuiski 2006). In the context of their humoral response a substantial number of DCM patients have been found to develop cross-reacting antibodies and/or autoantibodies to various cardiac antigens
Homologies between myocyte surface molecules such as membrane receptors and viral or bacterial proteins have been proposed as a mechanism for the elaboration of endogenous cardiac autoantibodies by antigen mimicry {Hoebeke 1996, Mobini 2004). Chagas' heart disease, a slowly evolving inflammatory cardiomyopathy, is one of the most prominent examples for this mechanism (Elies 1996, Smuiski 2006). Another -probably more relevant- mechanism leading to the production of endogenous cardiac autoantibodies would be primary cardiac injury followed by (sudden or chronic) liberation of a "critical amount" of antigenic determinants from the myocyte membrane or cytoplasm, previously hidden to the immune system. Such injury most likely occurs upon acute infectious (myocarditis), toxic, or ischemic heart disease (myocardial infarction) resulting in myocyte apoptosis or necrosis (Caforio 2002, Rose 2001). Presentation of myocardial self-antigens to the immune system may then induce an autoimmune response, which in the worst case results in perpetuation of immune-mediated myocyte damage involving either cellular (e.g., T- cell), or humoral (e.g., B-cell) immune responses, or co-activation of both the innate and the adaptive immune system (Eriksson 2003, Rose 2001). From a pathophysiological point of view, it seems reasonable to link the harmful (e.g., cardiomyopathy-inducing) potential of a heart-specific autoantibody to the accessibility and to the functional relevance of the corresponding target. Myocyte surface receptors are easily accessible to autoantibodies (Okazaki 2005). The two most promising candidates are the cardiac βrAR (representing the predominant adrenocepter subtype in the heart) and the M2-muscarinic acetylcholine receptor; against both receptors autoantibodies have been detected in DCM patients (Fu 1993, Jahns 1999b, Matsui 1995). Whereas anti-muscarinic antibodies {exhibiting an agonist-like action on the cardiac M2 acetylcholine-receptor) have been mainly associated with negative chronotropic effects at the sinuatrial level (e.g., sinus node dysfunction, atrial fibrillation (Baba 2004, Wang. 1996)), agonistic anti-βi-AR antibodies have been associated with both the occurrence of severe arrhythmia at the ventricular level (Christ 2001 , iwata 2001a), and the development of (maladaptive) left ventricular hypertrophy, finally switching to left ventricular enlargement and progressive heart failure (Iwata 2001b, Jahns 1999b, Khoynezhad 2007). Both autoantibodies appear to be directed against the second extracellular loop of the respective receptors. To generate an autoimmune response, myocyte membrane proteins (e.g., receptors) must be degraded to small oligopeptides able to form a complex with a MHC or HLA class Il molecule of the host (Hoebeke 1996). In case of the human βrAR computer-based analysis for potential immunogenic amino-acid εtreches has shown, that the only portion of the receptor molecule containing B- and T-cell epitopes and being accessible to antibodies was in fact the predicted second extracellular receptor loop (βrECu) (Hoebeke 1996). This might explain the successful use of second loop-peptides for the generation of βrspecific receptor antibodies in different animal-models (Iwata 2001b, Jahns. 2000, Jahns 1996). Moreover, in the last decade several groups have independently demonstrated that second loop antibodies preferentially recognize intact native βr AR in various immunological assays (whole ceil-ELISA, immunoprecipitation, immunofluorescence), indicating that they are "conformational" (Hoebeke 1996, Jahns 2006). Functional testing revealed that the same antibodies also affected receptor function, such as intracellular cAMP-production and/or cAMP-dependent protein kinase (PKA) activity, suggesting that they may act as allosteric regulators of βrAR activity (Jahns 2000, Jahns 2006), which is supported also by recent data on the detailed structure of the turkey βrAR protein by Warne et at. (2008 Nature, 454: 486-491).
Following Witebsky's postulates (Witebsky 1957) indirect evidence for the autoimmune etiology of a disease requires identification of the trigger (e.g., the responsible self-antigen), and induction of a self antigen-directed immune response in an experimentai animal, which then must develop a similar disease. Direct evidence, however, requires reproduction of the disease by transfer of homologous pathogenic antibodies or autoreactive T-celis from one to another animal of the same species (Rose 1993).
To analyze the pathogenetic potential of anti-βrAR antibodies, Jahns et a!, has choosen an experimental in vivo approach, which met the Witebsky criteria for direct evidence of autoimmune diseases. DCM was induced by immunizing inbred rats against βi-ECji (100% sequence homology between human and rat; indirect evidence); then the disease was reproduced in healthy animals by isogenic transfer of rat anti-βrAR "autoantibodies" (direct evidence) (Jahns 2004). The animals developed progressive left ventricular (LV)-dilatation and dysfunction, a relative decrease in LV wall-thickness, and selective downregulation of βi-AR, a feature that is also seen in human DCM (Lohse 2003).
These results, together with an agonist-like short-term effect of the antibodies in vivo (Jahns 2004), suggest that both the induced and the transferred cardiomyopathy phenotypes can be attributed mainly to the mild but sustained receptor activation achieved by stimulatory anti-βrAR antibodies (Jahns 2008). This hypothesis is supported by the large body of data available on the cardiotoxic effects of excessive and/or long term β^AR activation seen after genetic or pharmacological manipulation (Engelhardt 1999, Woodiwiss 2001). Therefore, anti- βrAR induced dilated immune-cardiomyo-pathy (DiCM) can now be regarded as a pathogenetic disease entity of its own, together with other established receptor-directed autoimmune diseases such as myasthenia gravis or Graves' disease (Freedman 2004, Hershko 2005, Jahns 2004, Jahns 2006).
The clinical importance of cardiac autoantibodies is difficult to assess, since low titers of such antibodies can also be detected in the healthy population as a part of the natural immunologic repertoire (Rose 2001 ). However, regarding functionally active anti-βrAR antibodies previous data from Jahns et al. has demonstrated that their prevalence is almost negligible in healthy individuals (<1 %) provided that a screening procedure based on cell-systems presenting the target (e.g., the βrAR) in its natural conformation is used (Jahns 1999b), By employing the latter screening method, occurrence of anti-βi-AR autoantibodies could also be excluded in patients with chronic valvular or hypertensive heart disease (Jahns 1999a). In contrast, the prevalence of stimulating anthβrAR was ~10% in ischemic (ICM) and -30% in dilated cardiomyopathy (DCM) (Jahns 1999b), which was significantly higher than in healthy controls, but in the lower range of previous reports on DCM collectives (33% to 95% prevalence) (Limas 1992; Magnusson 1994, Wallukat 1995). It seems conceivable that differences in screening methods aiming to detect functionally active anti-β-i-AR autoantibodies most iikely account for the wide range of prevalences reported in the past (Limas 1992). In fact, only a minor fraction of ELISA-defined human anti-β-AR autoantibodies was able to bind to cell surface located native β-AR. Only this fraction recognized (as determined by immunofluorescence) and activated (as determined by increases in cellular cAMP and/or PKA activity) human βi-AR expressed in the membrane of intact eukaryotic cells (Jahns 2000, Jahns 1999b). Therefore, eel! systems presenting the target in its natural conformation represent an essential tool in the screening for functionally relevant anti-β-AR autoantibodies (Nikolaev 2007, Jahns 2008).
Clinically, the presence of anti-βi-AR autoantibodies in DCM has been shown to be associated with a more severely depressed cardiac function (Jahns 1999b), the occurrence of more severe ventricular arrhythmia (Chiale 2001), and a higher incidence of sudden cardiac death (Iwata 2001a). Recent data comparing antibody- positive with antibody-negative DCM patients over a follow-up period of more than 10 years not only confirmed a higher prevalence of ventricular arrhythmia in the presence of activating anti-βrAR, but also revealed that antibody-positivity predicted an almost three-fo!d increased cardiovascular mortality-risk (Stork 2006). Taken together, the available clinical data underscore the pathophysiological relevance of functionally active anti-βi-AR antibodies in DCM (Jahns 2008).
One today generally accepted pharmacological strategy would be the use of beta- blocking agents in order to attenuate or even abolish the autoantibody-mediated stimulatory effects, at least if β~b!ockers can indeed prevent the antibody-induced activation of [J1 -AR (Freedman 2004, Jahns 2000, Matsui 2001 , Jahns 2006). New therapeutic approaches actually include elimination of stimulatory anti-βi-AR by nonselective or selective immunoadsorption (Hershko 2005, Wallukat 2002), or direct targeting of the anti-βrECn antibodies and/or the antϊ-βrECn producing B-celis themselves (that is, induction of immune tolerance) (Anderton 2001). Non-selective immunoadsorption, however, because of an increased risk of infection after immunoglobulin depletion, requires the substitution of human IgG on the ground of safety (Felix 2000) with all possible side effects of substituted human proteins known in the art including severe anaphylactic reactions and death.
WO 01/21660 discloses certain peptides homologous to epitopes of the 1st and the 2nό loop of βi-AR, and proposes to apply these peptides for medical intervention of dilatative cardiomyopathy (DCM). Even if WO 01/21660 mentions marginally that peptides may be modified in order to protect them against serum proteases, for example by cyciization, corresponding examples and embodiments are not given and any in vitro or in vivo effect of the proposed peptides on the course of DCM or on the course of receptor-antibody titers is not shown. Moreover, in WO 01/21660 intends to rely on the above mentioned non-selective immunoadsorption approaches bearing the correspondingly mentioned risks.
The newly developed βrECirhomologous cyclopeptides (e.g. βrECn-CPs) were employed six weeks after the active induction of stimulatory anti-βrECn antibodies. β-i-ECn-CPs are cyclopeptides containing 3 cysteine (Cys) residues and hence, can form intramolecular bonds, whereby there is a potential option to form two different intramolecular bonds (besides the cyciization between the N- and C-terminus), individually. βi-ECn-CP significantly reduced the amount of circulating anti-βrECn antibodies and effectively prevented development of cardiac dilatation and dysfunction (Boivin 2005). The above-mentioned β-i-ECn-CPs were also disclosed in WO 2006/103101.
In view of the present art, the technical problem underlying the present invention is the provision of improved and easily obtainable means and methods for the medical intervention of diseases related to anti-β-AR antibodies, in particular to anti-β-i-EC-H antibodies. The technical problem is solved by provision of the embodiments characterized in the claims.
Accordingly, in a first aspect, the present invention relates to β-AR homologous cyclopeptide-mutants (a!so termed herein as "cyclic peptides" or "cyclopeptides" and the like), particularly to Ji1-AR homologous cyclopeptide-mutants, namely βrEC-n homologous cyclopeptide-mutants (βi-EC-n-CPs) These cyclopeptide-mutants/cyciic peptides are structurally characterized in that they are able to form only one individual intramolecular disulphide bond and that they almost perfectly imitate/mimic the three-dimensional steric conformation of (the epitope(s) presented in) the native antibody-recognition and -binding regions of the βrEC-n.
Particularly, in the first aspect, the present invention relates to a cyclic peptide of formula I:
cyclo(x-Xh-Cys-x-xa-xb-xc-x~Cys-y-Xrx) (I),
wherein a) x is an amino acid other than Cys; b) h is any integer from 1 to 15; c) i is any integer from 0 to 14; d) one of xa, xb and xc is Pro; e) y is α-aminobutyric acid (ABu) or an ABu analogue; and f) the cyclic peptide consists of at least 16 and of at most 25 amino acids.
Particular preferred embodiments of this cyclic peptide are, as discussed below, specific cyclic peptides as depicted in formulas VlI. IX, IX', Vl or VIII.
The present invention solves the above identified technical problem since, as documented herein below and in the appended examples, it was surprisingly found that in particular βi-ECιrCPs containing only two Cys residues, which can form one single defined, individual intramolecular disulfide bond, and having the Cys-residue corresponding to -Cys 216 of β-i-AR replaced by an ABu residue, are also able to inhibit anti-β-AR antibodies, and are particularly usefu! for the inhibition of stimulatory anti-βi-AR antibodies with high effectiveness.
It was demonstrated in context of this invention that replacing the Cys residue corresponding to Cys 216 of the human βrAR amino-acid sequence particularly with an ABu residue results in βrECn-CPs with an enhanced efficacy in inhibiting anti-βi- AR antibodies. Evidences are provided herein that this enhanced inhibitory potential is due to an almost perfect steric imitation of the native antibody-recognition and binding regions of βrECn resulting from said Cys→ABu replacement.
In particular, in the context of this invention it was surprisingly found that βrEC)rCPs having the Cys residue corresponding to Cys 216 of βrAR replaced by an ABu residue have in fact a significantly higher antibody-blocking efficiency (at least in vivo) than βi-ECn-CPs having said Cys residue replaced by another Cys-like residue (for example by a serine (Ser) residue). In other words, the Cys/ABu mutants as disclosed herein have a higher capacity to inhibit binding of anti-beta1-ECII antibodies to beta1-ECI!-peptides, i.e. a higher affinity to antt-beta1-ECII antibodies, as compared to mutants having the corresponding Cys replaced by another amino acid (e.g. Cys/Ser mutants). This is, for example, indicated by smaller EC50 values for 22AA Cys/ABu mutants as compared to 22AA Cys/Ser mutants necessary to obtain comparable inhibitory effects on anti-beta1~ECII antibodies. These findings are, for example, evident from the binding competition assays performed in context of the appended examples (Fig. 15 C).
Non-invasive (Fig. 14 A-C) and invasive assessment (Fig. 14 D-F) of cardiac function made in the context of the appended examples also points towards a better cardioprotection in vivo achieved with cyclopeptides disclosed herein. In particular, the cyc22AA mutants of this invention block (or scavenge) conformational antibodies even better than previously described ECIi-homologous larger cyclopeptides (e.g. cyc25AA peptides) or smaller cyclopeptides (e.g. cyc18AA peptides) known in the art (WO 2006/103101) with the 22AA Cys/Abu mutant being even more efficient than the 22AA Cys/Ser mutant (Fig. 13 B and 14 B) without any adverse effects on kidney or liver function (Fig. 14 G and H). In addition, the inventive cyclic peptides comprising only two cysteines, which can form one single defined, individual intramolecular disulfide bond, can easily be obtained/manufactured, biochemically characterized and purified. This is particularly true when pure fractions of the same cyclopeptide isomers are required. In context of this invention, a mixture of cyclopeptide isomers, i.e. stereoisomers, comprising cyclopeptide isomers with different intramolecular disulphide bonds is avoided. As documented herein below, because of this avoidance a specific and clean medical product (fulfilling GLP/GMP standards) comprising isomers al! with the same intramolecular disulfide bond can be obtained. For example, only a sole Cys-S-S-Cys disulfide bridge may either spontaneously occur, or be chemically installed by treatment with 3% DMSO (forced S-S bridge) in order to accomplish the stability criteria according to current GLP-/GMP-rules as a prerequisite for its use either as pharmaceutical substance or as diagnostic agent in human (heart) disease.
A further surprising finding in context of the present invention was - as illustrated in the appended examples - that the exact nature of the exchange of one of the cysteine residues with an amino acid other than Cys. like, for example, a serine residue, markedly determined the antibody neutralizing potency of cyclic peptides derived from P1-ECn.
For example, a Cys→Ser exchange like that at position 18 of the herein exemplarily and preferably disclosed 25-meric cyclopeptide, at position 17 of the herein exemplarϋy and preferably disclosed 22-meric cyclopeptide or at position 14 of the herein exempiarily and preferably disclosed 18-meric cyclopeptide, respectively, yields cyclic peptides (Cys-Ser cyclic peptides) with excellent antibody-neutralizing and pharmacological effects in vitro (Figs. 2-4 and 12), whereas the Cys→Ser exchange at position 17, 16 or 13 of the herein exemplarily disclosed 25-meric, 22- meric or 18-meric cyclic peptide, respectively (Ser-Cys cyclic peptides), had, surprisingly, almost no inhibitory effect (Figs. 2/3). This inhibitory effect could neither be detected regarding their properties as antibody-scavengers nor in terms of their capability of inhibiting functional antibody-effects; as neutralization of receptor- stimulation in vitro as shown in, for example, Figs. 2-4. These findings demonstrate an comparability of 25AA, 22AA and 18AA cyclopeptides without any Cys mutation with the cyclic 25AA, 22AA or 18AA Cys18 17 or 14→ABu18 17 or 14 (Cys-ABu) mutants (or the respective Cys1g 17 oM4→Ser18 17 or 14 (Cys-Ser) mutants), but not with the cyclic 25AA or 18AA
Cys17 or 13→Ser17 or 13 (Ser-Cys) mutants.
Furthermore, it was demonstrated in context of this invention that a Cys→ABu exchange like that a position 17 of the herein exemplariiy and most preferably disciosed 22-meric cyclopeptide (formula IX') yields cyclic peptides (Cys-ABu cyclic peptides) with antibody-neutraϋzing and pharmaceutical effects comparable (in vitro; see, e.g. Fig. 12) with respect to the corresponding 22-meric (and 18-meric) Cys-Ser cyclic peptides. In view of this strong evidence is provided that also a Cys→ABu exchange like that at position 18 of the herein exemplariiy and preferably disclosed 25-meric cyclopeptide (formulas VII/IX) or at position 14 of the herein exemplariiy and more preferably disclosed 18-meric cyclopeptide (formulas VIΛ/lll), respectively, yields further Cys-ABu cyclic peptides with comparably excellent or even more pronounced antibody-neutralizing and cardioprotective effects in vivo {Fig. 13B and Fig. 14A-F).
It was a further finding in the context of the present invention that an almost perfect steric imitation of the ECll-βi-AR domain can be obtained by a second ioop- homoiogous Cys-ABu cyclized peptide particularly comprising 22 amino acids, for example 21 amino-acids of the published original primary sequence of the human βr AR, i.e. amino-acids 200 (R) to 221 (T) (numbering according to Frielle et ai. 1987, PNAS 84, pages 7920-7924), with an additional amino acid residue (for example glycine (G)) to close the synthetic cycle at position 222 of the human βrAR protein sequence to form a 22 AA cyciopeptide (Fig.11 ).
Without being bound by theory, the cardioprotective and immunomoduiating activity of the cyclic peptides largely depends on their conformation. It was additionally found out in the context of this invention that an introduction of the smallest naturally occuring amino-acid glycine at the (predicted) ring closure site (or at the position corresponding thereto, Fig. 11) leads to an enhanced binding of anti-βrAR autoantibodies, i.e. apparently further enhances the similarity of, for example, the 22 AA cyclopeptide with the ECII-P1-AR domain. Particularly, the appended examples, inter alia, indicate that the 22AA cyclopeptides have a significantly higher antibody- blocking efficiency in vivo than other ECil-imitating cyclopeptides larger (i.e., 25AA cyclopeptides) or smaller (i.e., 18AA cyclopeptides). Computer-aided modelling studies with said 22AA cyclopeptide confirmed an excellent imitation of the predicted second extracellular loop structure with a calculated difference in size of only 4.5 Angstrom (4.5 A) at the base of the cyclopeptide (opposed to the assumed antibody- binding site), when compared with the predicted native second extraceilufar loop backward helix (see also appended Fig. 11). Moreover, it was demonstrated herein and in the appended examples that particularly said 22 AA cyclopeptide reduces the titer of anti-βi-AR autoantibodies in vivo with an extraordinary high efficiency (Fig. 13A and B).
Since repiacement of one of the three cysteines present in the cyclic 22AA peptide allows for the introduction of a reinforced disulfide bridge (as a second "internal" cycle, generated by double cyclization) between the two remaining cysteines, the resultant cyclic 22AA cyclopeptide also represents a biochemically unambiguously defined product (Fig. 16 (HPLC), Fig. 17 (MS/MS-spectra)).
It was also surprisingly found that a Gln<→D-Glu exchange at position 25 (25-meric cyciopeptide-mutants) or 18 (18-meric cyclopeptide-mutants) did not significantly influence the blocking capacity of the cyclopeptides, regardless of their length; i.e., 25 versus 18 amino-acids as shown in Figs. 1 and 3).
The examples below also document that the cyclic peptides as disclosed herein show improved features, for example as compared to peptides comprising three Cys residues (for example the Cys-Cys cyciic peptides disclosed in WO 2006/103101 ). Examples of improved features of the cyclic peptides of this invention are an extremely good capacity for blocking anti-βrAR antibodies both, in vitro (Fig.12) and in vivo (Fig. 13) and their advanced producibiiity according to GLP/GMP standards. in context of the present invention, the in vitro findings were generally confirmed in in vivo tests (Figs. 5-10 and 13/14). interestingly, the difference in the blocking efficiency of the CyS18 17 or 14→Ser18 17 or 14 mutated cyclopeptides compared with that of the linear peptides was even more pronounced in vivo (Figs. 5-7). The same applies for the blocking efficacy of the Cysi8, i7 or i4→Ser18. i7 or i4 mutated cyclopeptides compared with said Cysi8, i7 or i4→Sert8, 17 or 14 mutated cyclopeptides (see, for example, Figs. 12, and 13).
The established rat model of anti-beta 1 -adrenergic antibody-induced autoimmune- cardiomyopathy (Jahns, 2004 and Jahns, 2008) served to assess the efficacy of the generated beta1-ECII homologous cycSopeptide mutants in vivo.
in addition, the in vivo experiments demonstrated that the antibody-blocking capacity of mutant cyciopeptides is seemingiy not affected by a reduction in the number of amino acids from a 25-meric to a 18-meric cyclopeptide; both in vitro and in vivo data demonstrate an excellent comparability of these two 2 cysteine-containing singie disulfide bond 25AA Cys/Ser or 18AA Cys/Ser cycfopeptide mutants (Figs. 7-10).
It was furthermore found in context of the present invention that the peptide-mutants with a cyclic structure (i.e. the cyclic peptides) as described and provided herein are superior to their linear counterparts in terms of the recognition or scavenging of conformational anti-β-AR antibodies, their antibody-neutralizing (i.e. pharma-ceuticai) potential an their half-life in crude serum (for example: linear < 8h, cyclic >12h; in particular: linear 6±1h, cyclic 48±12h). These results were obtained by the exemplariiy employment of ELiSA competition assays and functional (cAMP) FRET- assays, and incubation of linear versus cyclic peptides with human, rabbit or rat sera, respectively (Fig. 18 - 20).
Animals to which particularly the 18-meric, 22-meric or 25-meric cyclic peptide as disclosed herein -in particular the 22-meric Cys/Abu-mutant- was administered showed no signs of abnormalities (e.g. routine laboratory parameters of kidney and iiver function as well as organ weights Fig. 14 G and H), and only the desired effect of the administered peptide, nameiy the blockage of anti-βi-AR antibodies, was detected. Accordingly, the peptides as provided herein after 11 (eye 22AA Cys/Abu) or 12 applications (eye 22AA Cys/Ser ), corresponding to 12 months of treatment, display no undesired side effects or organ-specific toxicities at the applied dosage regimen (Fig. 14 G and H).
The antibody-blocking capacity of mutated cyclopeptides of this invention is advantageous high, as long as the peptide is not shorter than 18AA and not longer than 25AA. This was exempiarϋy demonstrated by the reduction of the number of amino acids of the peptide from 25 to 18. However, within the range of 18 to 25 amino acids, cyclic peptides having 22 amino acids are most effective in accordance with this invention and, accordingly, are a particular preferred embodiment. An example of such a particular preferred 22-rner cyclic peptide is shown in formula IX'.
One advantage of the cyclopeptide mutants of the present invention is - by mutating one particular cysteine (the Cys corresponding to Cys216 of the amino acid sequence of βrAR) to a ABu residue and by reinforcing formation of the unique possible intramolecular S-S bridge through a S-S specific cyclization procedure - that their conformational restraint is increased. In comparison to peptides known in the art, this increased restraint of the inventive peptides leads to a molecule that better mimics the epitope(s) presented in the native conformation of the second βi-ECn ioop on the celi surface.
Moreover, the cyclopeptide of the present invention was shown to block the activity of betai -receptor autoantibodies isolated from human DCM patients, as exemplariiy shown for IgG-fractions prepared from a 28 years-old female patient (see Fig. 19 and appended examples), in addition, by using cyclic Ala-mutated 22AA Cys/Abu peptides (according to Fig 15A) for these blocking experiments (pre-incubation of human IgG-preparations with 40-fold excess of the cyc22AA Cys/ABu Ala-mutants 0, 1 , and 8, respectively), the amino-acid proline (in βrECH loop-position 213; refered to herein aiso as xc) was identified as the most preferred amino-acid constituting the βr receptor epitope targeted by human receptor autoantibodies (Fig. 19). Beta blockers, such as bisoprolol, which are used in the art for the treatment of DCM and other diseases which are caused by stimulatory anti-β-i-AR antibodies, significantly reduce both heart rate and blood pressure. In contrast thereto, an in wVo-application of the mutant cyclopeptides as disclosed in context of the present invention has no negative impact on lung function, heart rate or biood pressure (Figs. 10 and 14 D-F). In addition, a number of important laboratory parameters to assess liver and kidney function were not influenced by repeated cyciopeptide injections (Fig. 14 G). Therefore, the cyclic peptides disclosed in context of the present invention are, inter alia, particularly suitable for the treatment of distinct patient groups which otherwise could not be treated by using a beta blocker, i.e. patients who, for example, already suffer from bradycardia or for whom the use of beta blockers is not possible because of contraindications (like those suffering from obstructive lung disease or hypotension).
As mentioned, a further advantage of the means and methods of the present invention, particularly over means and methods taking advantage of (cycHc)peptides derived from βrECn and stiil having 3 cysteines (as, for example disclosed in WO 2006/103101 ), is that the formation of mixtures of cycSopeptide isomers can be avoided.
The biochemical characterization of a mixture of different cyciopeptide isomers, formed during cyciization of peptides comprising three or more Cys residues, is laborious. Accordingly, the production of pure cyclic peptide fractions containing only one sort of a cyciopeptide isomer is time and cost intensive, when taking advantage of peptides comprising three or more Cys residues. This is particularly true, when the cyclic peptides are produced under GLP/GMP standards.
In contrast thereto, the cyclic peptides of the present invention can easily be characterized and produced as pure fractions of the same isomer. This leads to a high reproducibility. Accordingly one particular advantage of the peptides of the present invention is that mixtures of isomers, which have to be separated and must be characterized in laborious testings, are avoided, and that at least one further production step (separation and/or biochemical characterization) can finaliy be omitted (see also Sewald 2002). The present invention is, inter alia, based on the experiments described in the appended examples.
In context of these examples, one of the cysteines either at position 17 or at position 18 of the βi -EC-ii 25AA-cyclopeptide or the cysteine at position 14 of the βrECr 18AA-cyclopeptide was replaced by a serine residue {Cys17 or i8→Seri7 or 18 mutation and Cysu→Ser^ mutation, respectively), or the cysteine at position 17 of the βrECιr 22AA-cyclopeptide was replaced by an ABu residue (Cys17→ABu17 mutation), so that onSy one individual, single intramolecular disulfide bond (S-S) can be formed (Fig. 11 ,). Measures like this provide the potential to reduce side effects and to maintain or to increase the bioiogical efficacity of the constructs of the present invention. The cyclic peptides of this invention can be obtained in contrast to the peptides of the prior art which form mixtures of isomers, by simple, robust and highly reproducible manufacturing processes. These can be scaled up efficiently. Furthermore these processes avoid separation of isomers mixtures and are suitable for GLP/GMP standards. The appended examples provide for corresponding manufacturing/production methods.
In the appended examples, the cyclization of the inventive peptides was, inter alia, obtained by the introduction of a "GIy" mutation, e.g. at the (ring) closure site of the cyclic peptide (Fig. 11 ).
Furthermore, the number of amino acids (AA) was reduced from 25AA to 22AA and further to 18AA in further sets of cyclopeptide-mutants of the present invention. This measure provides the potential to minimize the potential immunologic side effects of the constructs and to screen for/identify the optional length of the cyctopepttde- mutants within the range of 18 to 25 amino acids. The 18AA or 25AA cyclopeptide- mutants contained a cysteine-→ABu exchange at position 14 and 18, respectively, (18AA containing CyS13-ABu14 mutant cyclopeptides and 25AA containing Cysi8~
Ser17 mutant-cyciopeptides) optionally combined with a (further) glutamine-/D~ glutamic acid-exchange, e.g. at the ring closure site of the cycϋc peptide (Gln÷→D-Glu mutation). The preferred 22AA cyclopeptide-mutants contained a cysteine-→ABu exchange at position 17 (22AA containing Cys16-ABui7), optionally combined with the introduction of a GIy residue at position 22 (a possible ring closure site of the cyclic peptide; Fig. 11). Taken together, the herein provided experimental in vitro data as weli as the in vivo data clearly demonstrate that the antibody-blocking capacity of the disclosed mutant cyciopeptides is not markedly affected by the reduction of the number of amino acids from a 25~meric to a 22-meric or 18-meric cyclopeptide, for example, when using a dose ranging from 0.25 to 5.0 mg/kg body weight (Bw) or from 1.0 to 2.0 mg/kg Bw or, in particular, from 0.3 to 2.0 mg/kg Bwor. In vitro and in vivo data indicate an excellent comparability of the two 2 cysteine-containg single disulfide bond 25AA Cys/ABu cyciopeptides (formulas VII/IX) or 18AA Cys/ABu cyciopeptides (formulas VIΛ/lil) cyclopeptide mutants at a dose of 1.0 mg/kg Bw. However, "intermediate" cyclic peptides of 19 to 24AA apparently exhibit an increased activity in accordance with this invention. Particularly, cyclic peptides of 22AA (22AA Cys/ABu cyciopeptides) exhibit an increased activity in accordance with this invention. A preferred example of such an "intermediate" cyclic peptide is a cyclic peptide comprising or consisting of the amino acid residues as shown in formula IX'. Moreover, the exact nature of the exchange of one of the cysteine residues with a serine residue (i.e., Cys/Ser (ABu) or Ser (ABu)/Cys-mutation) markedly determined the potency of the disclosed cyclic peptides in vitro and aiso in vivo (Figs. 2, 3, 6, 7, 13 and 14).
One preferred embodiment of the present invention is based on in vivo experiments addressing different modes of application/administration of the herein disclosed cyclic peptides, in particular of the aforementioned βrECn epitope mimicking 22AA cyclopeptide mutants. These in vivo experiments are also described in the appended examples. In the context of these examples the cysteine at position 17 of the βrECIl- 22AA-cyclopeptide was replaced by an ABu residue (Cys17→ABu17 mutation), so that only one individual, single intramolecular disulfide bond (S-S) can be formed (Fig. 11 ). The different application/administration protocols as disclosed herein comprise either monthly intravenous applications of the herein disclosed cyclopeptide mutants (e.g. cyc22AACys/Abu), or, preferably, a triple injection (for example, one injection every week on 3 subsequent weeks) every three months. Administration schemes fike these provide the potential to reduce the amount of injected cyciopeptides, to reduce the burden of (regular monthly) venipuncture and/or to increase the flexibility of application (for both, human patients and animals); whilst maintaining or increasing the biological efficiency of the injected constructs of the present invention.
With respect to the herein disclosed constructs (in particular cyc22AA Cys/Abu), triple injections (1 injection per week on 3 subsequent weeks) followed by a two-months intervention free time interval were shown to be of extraordinary efficient in reducing the titer of circulating anti-βi-ECu antibodies (Fig. 21 A and B), and in reverting the cardiomypathic phenotype (Fig.22A-C).
β-adrenergic receptors (β-ARs), particulary βi-adrenergic receptors (βrARs), are well known in the art. For example, the nucleotide and amino acid sequence (SEQ ID NO. 24) of the human βrAR (also known as adrenergic β-1 -receptor (ADRB1 )) can be obtained from databank entry NM_000684 or NP_0O0675. β-ARs are known to form two extracellular domains termed herein as ECi and ECn or p(1)-ECi and β(irECn. As mentioned above, the cyclic peptides of the present invention share sequence similarity with βi-ECn, particularly with the amino acid stretch DEARRCYNDPKCCDFV (SEQ ID NO. 17) or RAESDEARRCYNDPKCCDFVTNR (SEQ ID NO. 18) of the human βrAR (amino acid positions 204 to 219 or 200 to 222, respectively) or, particularly, with the amino acid stretch DEARRCYNDPK (SEQ ID NO. 29) or ESDEARRCYNDPK (SEQ ID NO. 30) of the human βrAR. The term "β-AR" as used herein preferably refers to a β^ -adrenergic receptor (P1-AR)1 more preferably to the human βi-AR as described above. The detailed structure of the turkey βrAR protein was, inter alia, analyzed by Warne et al. (2008 Nature, 454: 486-491 ).
A cyclic peptide provided herein may have at least one of the features selected from the group consisting of: a) being capable of binding (auto-)antibodies against the ECu loop of βi- adrenergic receptor (β-pAR); b) being capable of inhibiting the interaction between βrAR and (auto-)antibodies against the ECn ioop of βi-AR; c) mimicking at least one epitope presented in the native conformation of the ECn loop of βi-AR; and d) being capable of reducing an antibody-mediated activation of βi-AR.
As mentioned above, the cyclic peptide of the present invention is defined by the genera! formula cycio(x-Xh~Cys~x-xa-xb-xc-x-Cys-y-Xj-x) (formula I). In this formula, "y" is ABu (α-aminobutyric acid) or an ABu analogue. ABu is a non-naturaily occurring; i.e. non-genetically coded, amino acid weli known in the art. It is, for example, also known as alpha-aminobutyric acid, AABA1 2-aminobutyhc acid, 2-aminobutanoic acid etc. In the art, ABu is, for example, depicted by the chemical formulas CH3CH2CH(NH3)COOH or C4H9NO2. It is preferred in context of this invention that Abu is L-Abu (L-α-aminobutyric acid). However, Abu may also be D-Abu (D-α- aminobutyric acid).
For example, Wlodaver (Science, 1989, 245 (4918): 616-21 ) describes the determination of the crystal structure of chemically synthesized HIV-1 protease based on the Rous sarcoma virus protease structure having the cysteines replaced by ABu.
Besides ABu itself, "y" of formula I may be any ABu analogue, as long as this amino acid does not form an intramolecular linkage (e.g. a disulphide bond) with another amino acid of the cyclic peptide provided herein (in particular with another Cys of the cyclic peptide provided herein), "y" may be any ABu analogue similar to Cys (i.e. having a similar chemical structure and/or a similar "structural behavior" within a 3 dimentional peptide structure), with the exeption that it does not form an intramolecular linkage (e.g. a disulphide bond) with another amino acid of the cyclic peptide provided herein (in particular with another Cys of the cyclic peptide provided herein). "ABu analogue" in context of this invention means a residue, particularly an amino acid residue, having a structural character similar to that of ABu. The term "ABu analogue" particularily refers to a(n) (amino acid) residue having a chemical structure and/or "behavior" within a 3 dimentional peptide structure which is more similar to that of ABu itself than to that of any other amino acid residue, like, for example, to that of Ser. The meaning of the term "ABu analogue" as used throughout this application is clear to the skilled person. Hence, the skilled person is readily in the position to deduce which particular compounds fall under the meaning of this term, i.e. which compounds are, structurally and/or functionally, more clearly related to ABu itself, than to any other amino acid that can replace Cys in accordance with this invention, e.g. Ser or Selenocysteine.
Particular "ABu analogues" in accordance with the present invention may be those, the C-side chain of which is shortened or elongated as compared to ABu itself. The C-residues of the C-chain may be up to 10, preferably up to 6. For example, such "ABu analogues" with a shortened or elongated C-side chain are 2-arninopropionic acid and, preferably, 2-aminopentanoic acid or 2-aminohexanoic acid.
The term "ABu" as referred to herein, in particular in the depicted formulas, refers to both, an ABu analogue or, which is preferred, ABu itself.
As mentioned, one main feature of the cyclic peptides of this invention is that they comprise only two Cys able to form an intramolecular linkage. Such cyclic peptides can, for example, be obtained by substituting a third Cys of a peptide homologous to the βi-ECn by a different amino acid. Thereby, the Cys to be substituted is the one corresponding to the 2nd or, which is preferred, 3rd Cys of the [VECn which lie in direct proximity to each other (amino acid position 215 and 216 of human ^1-AR (see also NP__000675 and SEQ ID NO. 24). These two Cys residues, are referred to herein as "Cys-Cys", "Cys/Cys", "Cys215-Cys2is" or "Cys2i5/Cys2i6" and the like).
The resulting mutant peptides or mutations as disclosed herein are accordingly termed as "Cys-ABu", "Cys/ABu", Cysi3, i6 or i7-ABu14i 17 oπe" or "CySi31 16 Or IyZABu14 17 Or Is" mutant peptides or mutations, "Cys-Ser", "Cys/Ser", "Cysi3, 16 or 17-Ser14. 17 or i8" or "CyS13. 16 or i?/Seri4. 17 or W mutant peptides or mutations or "Ser~Cys", "Ser/Cys", "SePi3 0M7-CyS14 0Me" or "Ser13 θr i7/Cysi4 or ie" mutant peptides or mutations, depending on which of the Cys' is replaced and how many amino acids the mutant peptide comprises (18, 22 or 25). Alternatively, the mutant peptides as disclosed herein are defined by referring to the particular amino acid exchanges at a certain position. Then, the mutant peptides/ mutations are, for example, termed "CyS14, i7 Or iε→ABu-ι4, 17 or is" mutant peptides/ mutations, Cysi4, i7 or i8~*Seri4, 17 or is" mutant peptides/mutations or "Cysi3 or i7 →Seri3 or i7" mutant peptides/mutations, depending on whether the Cys corresponding to Cys2ie or the Cys corresponding to the Cys2is, respectively, of βr AR is replaced by a different amino acid and how many amino acids the mutant peptide comprises.
The indices "13, 16 or 17", "14, 17 or 18", "13 or 17" or "14 or 18" relate to the position in the exemplified cyclic peptide of the invention, whereby position 1 corresponds to the first "x" as defined in formula I, i.e cycio{x-Xh-Cys-x-xa-xb-xc-x-Cys- y-x,-x).
Accordingly, terms (ike "CyS13-SeH4" or "Cysi3/Ser14" mutant peptides/mutations are used in the same sense as "Cysi4→Seri4" mutant peptides/mutations and, in this particular example, refer to 18mer peptides disclosed herein. Terms like "Cys16-Ser17" or "Cysi6/Seri7" and "CyS16-ABu17" or 11CyS16ZABu17", respectively, mutant peptides/mutations are used in the same sense as "Cysi7→Ser17'! and "Cys17→ABur-i7", respectively, mutant peptides/mutations and, in this particular example, refer to 22mer peptides disclosed herein. Terms like "Cys17-Seri8" or "Cysi7/Ser18" mutant peptides/mutations are used in the same sense as "Cysi8→Ser18" mutant peptides/mutations and, in this particular example, refer to 25mer peptides disclosed herein.
Analogously, terms like "Ser13-Cysi4" or "SeH3ZCyS14" mutant peptides/mutations are used in the same sense as "Cysi3→Seri3" mutant peptides/mutations and, in this particular example, refer to 18mer peptides disclosed herein, and terms like "Sert7- Cys15" or "Ser17/Cys-ιS" mutant peptides/mutations are used in the same sense as "Cys17->Ser17'! mutant peptides/mutations and, in this particular example, refer to 25mer peptides disclosed herein.
The exemplarϋy indices given above refer to the position of the indicated amino acid within the herein disclosed particular 18-mer, 22-mer or 25-mer peptide, respectively. in context of this invention, the starting point with respect to an indicated amino acid position given for a cyclic peptide disclosed herein is the N-terminal amino acid of the linearized backbone the cyclic peptide (like the first "x" in formula I, see above). The starting point with respect to an indicated amino acid position given for a linear peptide disclosed herein is its N-terminai amino acid.
As also mentioned above, in the formulas of the cyclic peptide of the present invention, h can be any integer from 1 to 15, preferably from 5 to 9, and/or i can be any integer from 0 to 14, preferably from 1 to 14, more preferably from 0 to 6 and even more preferably from 1 to 6. Accordingly, h can be 1 , 2, 3, 4, 5, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 and/or i can be 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14. Preferably, h is 5, 8 or 9 and/or i is 3, 4 or 6. More preferably, h is 8 and/or i is 4. In particularly preferred embodiments of this invention, xh stands for the particular amino acid stretches DEARR (SEQ ID NO. 19), AESDEARR (SEQ ID NO. 31 ) or RAESDEARR (SEQ ID NO. 20) and/or X1 stands for the particular amino acid stretches DFV (SEQ ID NO. 21 ), DFVT (SEQ ID NO. 32) or DFVTNR (SEQ ID NO. 22). in more preferred particular embodiments of this invention, xh stands for the particular amino acid stretch AESDEARR (SEQ ID NO. 31 ) and/or x, stands for the particular amino acid stretch DFVT (SEQ ID NO. 32).
The cyclic peptide of the present invention (or the cyclic part thereof) may consist of at least 18 amino acids and of at most 25 amino acids. Accordingly, the cyclic peptide of the present invention may consist of 18, 19, 20, 21 , 22, 23, 24 or 25 amino acids, whereby particularly 18 or 25 amino acids are preferred and particularly 22 amino acids are most preferred. In a less preferred embodiment, also smaller peptides, i.e. peptides comprising 16 or 17 amino acids are envisaged.
A particularly preferred cyclic peptide in context of this invention is one of (21 +1 =)22AA length, having a defined maximum and minimum size of the cyclic molecule dependent on the respective amino-acid composition, constituted by 21 amino acids from the original primary sequence of the human βt-AR (i.e., amino acids 200 to 221 ; Frielle 1987, PNAS 84, 7920-7924) with an additional glycine as 22nd amino-acid at the assumed ring closure site (position 22). Without being bound by theory, the number of amino acids and thus the length of the primary structure (i.e. the amino acid backbone) of cyclic peptides binding anti-βi-AR antibodies is crucial for their biologica! effects and/or successful/effective manufacture.
A peptide-length equal or above 26 amino acids (primary structure) may be stimulating directly (that is, without the use of carrier proteins) immunocompetent T- celts and thus may provoke an undesired paradoxal increase of anti-β1 -receptor antibody production through T-cell mediated B-cell stimulation.
A peptide-length below 16 amino acids (primary structure) leads to undesired crystallization during the production process and problems in dissolving the synthesized products in an aqueous solution, e.g. for purposes of i.v. or s.c. injections
In a less preferred embodiment of this invention also cyclic peptides falling under the above given definitions a) to f) of formula I, but consisting of only 17 amino acids or, even less preferred, consisting of only 16 amino acids are provided. A non-limiting example of such a less preferred cyclic peptide is the peptide cyclo(Ala-Arg-Arg-Cys- Tyr-Asn-Asp-Pro-Lys-Cys-ABu-Asp-Phe-Vaf-Tyr-Gln/DGIu) (formable by an amino acid backbone as depicted in SEQ ID NO. 23).
It is particularly preferred herein that the disclosed cyclic peptide contains only one Pro. Accordingly, it is particularly preferred that the "x" of the formulas depicted herein, except of exactly one of xa, xb and xc, is not Pro. Within the amino acid stretch xa, xb and xc as depicted in formula I (or in other formulas), it is preferred that xc is Pro.
It is particularly envisaged herein that an acidic amino acid, like Asp or GIu, precedes the Pro contained in the cyclic peptide of the invention. Accordingly, it is preferred that xb as depicted in formula I (or other formulas) is an acidic amino acid, like GIu or, preferably, Asp. Particularly, when xc is Pro, xb may be an acidic amino acid, when xb is Pro, xa may be an acidic amino acid and when xa is Pro, the x of formula I (or of other formulas) lying between xa and the first Cys may be an acidic amino acid, wherein the acidic amino acid may be one as particularly described herein. Preferably, xa is Asn, xb is Asp, xc is Pro and/or x following xc is Lys. In a particularly preferred embodiment of the herein disclosed peptides, the amino acid strech xa-xb-xc as depicted in the formulas provided herein is Asn-Asp-Pro (N-D-P). In an even more preferred embodiment, the amino acid strech xa-xb-xc-x as depicted in the formulas provided herein is Asn-Asp-Pro- Lys (N-D-P-K).
More specifically, the cyclic peptide of the present invention may be defined by formula I' or I":
cyclo(xrXh-Cys-x-xa-xb-xc-x-Cys-y-xrx) (!');
cyclo(xιιι-Xjy-Cys-x-xa-xb~xc-x-Cys-y-xrx) (I ").
Even more specifically, the cyclic peptide of the present invention may be defined by formula Y" or 1" ":
cyclo(xrxh-Cys-x-xa-xb-xc-x-Cys-y-x,-X[i) (I ' ") ;
cyc!o(xιιrXh-Cys-x-xa-xb-xc-x-Cys-y-xrX!v) (I" ").
fn general, x( and xM as depicted in formula I' and I' " (and in the other formulas depicted herein) may, as mentioned, any amino acid but Cys. However, particularly when the ring closure of the cyclic peptides of the invention occurs between xι and Xn, it is particularly envisaged that xι and xS| are such amino acids able to form a peptide bond, or the like, with each other under conditions of a "head to tail" cyclization. "Head to tail" cyclizations are known in the art (e.g. Kates and Albericio: Solid phase synthesis, CRC-Press, 2000; Williams, Chemical Approaches to the Synthesis of Peptides, CRC-Press 1997; Benoiton: Chemistry of Peptide Synthesis. CRC-Press, 2005) and examples thereof are given in the experimental part. Possible examples of amino acids that may be Xι are GIy, VaI, Thr, Ser and, preferably, Ala. Possible examples of amino acids that may be xfl are GIu and, preferably, GIn. Less preferred, Xi! may also be Asp or Asn. Most preferred, xs is Ala and Xn is Gin or GIu (preferrably DGIu). Accordingly, in the cyclic peptides of this invention xN as referred to in formula I' and Y" can be GIn or GIu, wherein GSu may also be DGIu (D-GIu; D-Glutamic acid). However, naturally amino acids are preferred herein. Therefore, it is more preferred that Xιι is GIn.
The skilled person is able chose amino acid residues appropriate to be Xι and/or Xn of formula I' and Y" in accordance with this invention on the basis of the teaching provided herein and his knowledge of the art (e.g. Williams, Chemical Approaches to the Synthesis of Peptides, CRC-Press 1997; Benoiton: Chemistry of Peptide Synthesis. CRC-Press, 2005).
Xiii and Xιv as depicted in formula I" and I" " (and in the other formulas depicted herein) may, as mentioned, also represent any amino acid but Cys. However, particularly when the ring closure of the cyclic peptides of the invention occurs between xm and X|V, it is particularly envisaged that Xm and X|V are such amino acids able to form a peptide bond, or the like, with each other under conditions of a "head to tail" cyclization. A possible example of an amino acid that may be xm is Arg. One possible, and most preferred, example of an amino acid that may be xιv is Giy or a GIy analogue. "Giy analogue" in this context means a residue, particularly an amino acid residue, having a structural character similar than that of GIy. Particularly, "GIy analogue" refers to, for example, a(n) (amino acid) residue having the same (or even a smaller) size than a GIy residue. It was surprisingly found in context of this invention that particularly a small (amino acid) residue like GIy at the "Xiv" position leads to an improved mimicking of the ECU of β1-AR by the corresponding cyclic peptides of the invention.
The skilled person is able chose amino acid residues appropriate to be Xm and/or X|V of formula I" and Y " in accordance with this invention on the basis of the teaching provided herein and his knowledge of the art (e.g. Williams, Chemical Approaches to the Synthesis of Peptides, CRC-Press 1997; Benoiton: Chemistry of Peptide Synthesis. CRC-Press, 2005).
It is further particularly preferred herein that the cyclic peptides of this invention lack Trp and/or His. Accordingly, it is particularly envisaged in context of the invention, that neither an x nor y as depicted in any of Formula I to I" " is Trp or His. Furthermore, it is preferred that the provided cyclic peptides iack sites susceptible for hydrolysis or cleaving proteases, like, for example, serum proteases. The meanings of the terms "hydrolysis" and "(serum) proteases" are well known in the art.
A peptide as provided herein can also be described as a peptide consisting of or comprising an amino acid sequence homologous to SEQ ID NO. 17 (representing a wild type amino acid strech comprising epitopes of βi-EC-n), wherein (a) the amino acid corresponding to position 13 (or, less preferred, corresponding to position 12) of SEQ ID NO. 17 is not Cys and the amino acid corresponding to positions 6 and 12 (or, less preferred, corresponding to position 6 and 13) of SEQ ID NO, 17 is Cys, (b) wherein said amino acid sequence contains no further Cys able to form an intramolecular linkage within the peptide, i.e. within that part of the peptide being homologous to SEQ ID NO. 17, or (c) wherein the peptide can function as a cyclic peptide in accordance with this invention, e.g. is able to block anti-β-AR antibodies, or wherein the peptide can form such a cyclic peptide. Optionally, the further provisions given herein with respect to the structure of the disclosed linear and/or cyclic peptides apply here, mutatis mutandis. The so defined peptide consists of a stretch of 16 amino acids being homologous to SEQ ID NO. 17 flanked at the N- and C-terminus by one or more amino acids, preferably naturally occurring amino acids, like the "X|Txιιι" at position 1 and the "x§ι"/"xιv" at the last position of formulas I ' to I " " given herein.
In context of the invention and in particular in context of the (wild type) SEQ ID NO. 17, "homologous" means identical on amino acid level for at least 18.75%, at least 37.5%, at least 50%, at least 56.25%, at least 62.5%, at least 68.75%, at least 75%, at least 81.25%, at least 87.5% or 93.75%, wherein the higher values are preferred.
In general, the meaning of the term "amino acid" or "amino acid residue" is known in the art and is used herein accordingly. Thereby, it is of note that when an "amino acid" is a component of a peptide/protein the term "amino acid" is used herein in the same sense than "amino acid residue".
Particularly, an "amino acid" or "amino acid residue" as referred to herein, except the ABu or ABu analogue, is preferably envisaged to be a naturally occurring amino acid, more preferably a naturally occurring L-amino acid (except the above mentioned DGSu). However, albeit less preferred, an "amino acid" or "amino acid residue" in context of this invention ("x" referred to in formula ! and the other formulas given herein) may also be a non-naturally occurring (i.e. a synthetic) amino acid, like, for example, norleucine or β-aianine.
Also known in the art is the meaning of the terms "acidic amino acid(s)", "basic amino acid(s)", "aliphatic amino acsd(s)" and "polar amino acid(s)" (for example, Stryer, Btochemie, Spectrum Akad. Verlag, 1991 , Item I. 2.). These terms are correspondingly used throughout this invention. Thereby, the particular provisos given herein with respect to the cyclic peptides of the invention also apply. Particularly, the term "acidic amino acid(s)" as used herein is intended to mean an amino acid selected from the group comprising Asp, Asn, GIu, and GIn, preferably Asp and GIu; the term "basic amino acid(s)" as used herein is intended to mean an amino acid selected from the group comprising Arg, Lys and His, preferably Arg and Lys; the term "aliphatic amino acid(s)" as used herein is intended to mean any amino acid selected from the group comprising GIy, AIa1 Ser, Thr, VaI, Leu, lie, Asp, Asn, GIu, GIn, Args Lys, Cys and Met; and the term "polar amino acid(s)"as used herein is intended to mean any amino acid selected from the group comprising Cys, Met, Ser. Tyr, GIn1 Asn and, less preferred, Trp.
in a further particular embodiment of the first aspect of this invention, the cyclic peptide as provided herein may be a cyclic peptide of formula II, III or 111':
cyc!o(xrXrXt~x~χ2~X2-Cys-x-xa-xb-xc-x-Cys-y-Xι-Xi!) (Ii); cyclo(xrX2-x-XrX-Xi-XrX-X2-X2-Cys-x-xa-xb-xc-x-Cys-y-xrXιι) (HI); cyclo(xui, 2-X-X1 -x-x-1 -Xi-x-X2-X2-Cys-x~xa~xb~xc-x-Cys-y-xrXιv) (IM '),
wherein a) Xi is individually and independently selected from the group consisting of acidic amino acids; and/or b) X2 is individually and independently selected from the group consisting of basic amino acids.
In a more specific embodiment of the first aspect of this invention, the cyclic peptide as provided herein may be a cyclic peptide of formula IV, V or V;
cycSo(X|-XrXrX4-X2-X2-Cys-x3-xa5--xb-xc~X2-Cys-y-x1"X3-X3-Xii) (IV); cyclo{X|-X2-X4'Xi-x4-XrXrX4-X2-X2-Cys-X3-xVxb-xc-X2-Cys-y-XrX3-x3-X4-x5-X2-Xii) (V); cyclo(xm, 2-x4-Xi-x4-XrXi-X4-X2-X2-Cys-X3-xa 5-xb-xc-x2~CyS"y~xrX3-X3--X4-Xtv) (V'),
wherein a) X1 is individually and independently selected from the group consisting of acidic amino acids; b) X2 is individually and independently selected from the group consisting of basic amino acids; c) X3 is individually and independently selected from the group consisting of Leu. lie, VaI, Met, Trp, Tyr and Phe; d) X4 is individually and independently selected from the group consisting of Ser, Thr, Ala and GIy; and/or e) X5 is individually and independently selected from the group consisting of GIn and Asn.
In a further embodiment of the first aspect of this invention, the cyclic peptides comprise an amino acid strech as defined by amino acid position 2-12 or 2-14 of formula Il or IV. an amino acid strech as defined by amino acid position 4-16 or 4-18 of formula Hl or V or an amino acid strech as defined by amino acid position 3-15 or 3-17 of formula or IH' or V, In a more general embodiment of the first aspect of this invention, the cyclic peptide as provided herein may be a cyclic peptide of formula H, III or III'. In a further particular embodiment of the first aspect of this invention, the cyclic peptide as provided herein may comprise the amino acid stretch
aci-Glu-ASa-bas-bas-Cys-Tyr-neu-aci-neu-bas; aci-neu-aci-Giu-Aia-bas-bas-Cys-Tyr-neu-aci-neu-bas; aci-Glu-Ala-bas-bas-Cys-Tyr-neu-aci-neu-bas-Cys-Ser; or aci-neu-aci-Glu-Aia-bas~bas-Cys-Tyr-neu-aci-neu-bas-Cys-Ser,
wherein "aci" stands for acidic amino acid, "neu" stands for neutral amino acid and "bas" stands for basic amino acid. Each amino acid residue of the above four amino acid stretches may also be defined independently as the corresponding amino acid residue of any one of formulas I, II, IH, III', IV, V1 and V as provided herein.
In a further particular embodiment of the first aspect of this invention, the cyclic peptide as provided herein may comprise the amino acid stretch
Asp-Xxxi-Xxx^Arg-Arg-Cys-Xxxs-Asn-Asp-Pro-lys (SEQ ID NO. 29) or Glu~Ser-Asp-Xxxi-Xxx4-Arg-Arg-Cys-Xxx3-Asn-Asp-Pro~Lys (SEQ ID NO. 30),
wherein XxX1 is defined as V or '%", Xxx3 is defined as "x" or "x3" and/or Xxx4 is defined as "x" or "x4" as mentioned in the above depicted formulas. For example, the above-mentioned amino acid stretch may be
Asp-G!u-Aia-Arg-Arg-Cys-Tyr-Asn-Asp-Pro~Lys (SEQ ID NO. 29) or Glu-Ser-Asp-G!u-Ala-Arg-Arg-Cys-Tyr-Asn~Asp-Pro-Lys (SEQ ID NO. 30).
It is particularly envisaged herein that the cyclic peptides of this invention comprise one or more epitopes born by βi-ECn, like, for example, epitopes born by any of the above mentioned amino acid stretches (or by parts of the disclosed cyclic peptides comprising these amino acid stretches). In this context, the term "epitope" particularly refers to an amino acid stretch to which an (auto)anti-βi-AR antibody binds. Particularly, an epitope in context of this invention consists of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13 or at least 14 amino acids. The person skilled in the art is in the position to deduce which particular amino acid residue(s) of βi-ECn contribute to (an) epitope(s) to which (auto)anti-βi-AR antibodies bind. Hence, he can deduce which particular amino acid residues have at least to be comprised in the cyclic peptides of this invention in order to ensure that these peptide bind (auto)anti-βi-AR antibodies. For this purpose, several means and methods known in the art can be employed (for example, means and methods for epitope mapping (like PepSpotε™, Biacore, Amino acid scans (iike alanine scans)).
Non limiting examples of a cyciic peptide according to this invention are cyclic peptides selected from the group consisting of: a) cyclic peptides formable or formed by the amino acid sequence as depicted in any one of SEQ ID NO. 25, 27, 1 , 2 and 9 to 10; b) cyclic peptides formable by an amino acid sequence as encoded by a nucleotide sequence as depicted in any one of SEQ ID NO. 26, 28, 9, 10, 13 and 14; and c) cyclic peptides formabfe by an amino acid sequence as encoded by a nucleotide sequence which differs from the nucleotide sequence as depicted in any one of SEQ ID NO. 26, 28, 9, 10, 13 and 14 due to the degeneracy of the genetic code.
Out of the cyclic peptides according to this invention, those cyclic peptides being Cys-Ser mutant peptides, i.e. having the Cys corresponding to the third Cys of the βr ECii (the Cys at position 216 of βrAR) exanged by ABu or an analogue thereof, are particularly preferred. The above given examples refer to such particularly preferred cyclic peptides. As demonstrated in the appended examples, such cyclic peptides are particularly useful in inhibiting or diagnosing/detecting anti-βi-AR antibodies.
The particular structure of the above exemplified particularly preferred cyclic peptides is given by any one of the following formulas Vl to IX': cyclotArg-Ala-Glu-Ser-Asp-Giu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-ABu-Asp-
PheΛ/al-Thr-GSy) (IX');
cyclotAla-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-ABu-Asp-Phe-Val-Gin)
(Vl);
cyclo(Ala~Arg-Ala-Glu-Ser-Asp-Glu-Ala-Arg-Arg-Cys-Tyr»Asn~Asp'Pro-LyS'Cys-ABu-
Asp-Phe-Va!~Thr-AsrvArg-GJn) (V!i);
cycio(Ala-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-ABu-Asp"Phe»Val-
DG!u) (ViII); and
cycfo(AIa-Arg-Ala-Glu-Ser-Asp-G!U"Ala»Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-ABu-
Asp-Phe-Val-Thr-Asn-Arg-DGIu) (IX).
Non limiting exampfes of less preferred cyciic peptides according to this invention are cyclic peptides selected from the group consisting of: a) cyclic peptides formabte by the amino acid sequence as depicted in any one of SEQ iD NO. 5, 6 11 and 12; b) cyclic peptides formable by an amino acid sequence as encoded by a nucleotide sequence as depicted in any one of SEQ ID NO. 7, 8, 15 and 16; and c) cyclic peptides formable or formed by an amino acid sequence as encoded by a nucleotide sequence which differs from the nucleotide sequence as depicted in any one of SEQ ID NO. 7, 8, 15 and 16 due to the degeneracy of the genetic code.
The particular structure of the above exemplified less preferred cycϋc peptides is given by any one of the following formulas X to XIII:
cyclo(Ala-Asp-Giu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-ABu-Cys~Asp-Phe'Val-Gln)
(X); cyclo(Ala-Arg-Ala-Glu-Ser-Asp-Glu-Ala-Arg-Arg"Cys-Tyr~Asn-Asp-Pro-Lys-ABu-Cys-
Asρ-Phe-Vaϊ-Thr-Asn-Arg-Gin) (XI);
cycIo(Ala-Asp-G!u-Aia-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-A8u-Cys-Asp-Phe-Va!-
DGiu) (XII);
cyciotAla-Arg-Ala-Glu-Ser-Asp-Glu-Aia-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-ABu-Cys-
Asp-Phe-Val-Thr-Asn-Arg-DGIu) (XIlI).
The above peptides are less preferred embodiments of this invention since peptides like the the corresponding "Cys-ABu" cyclic peptides (or "Cys-Ser" cyclic peptides) are, inter alia, in vivo more functional than the herein defined less preferred "ABu- Cys" cyclic mutant peptides ("Ser-Cys" cyclic peptides).
In this context it is of note that most preferred examples of the cyclic peptides according to this invention are particularly those cyclic peptides, the pharmacological and/or diagnostic function of which has been demonstrated in the appended examples or the pharmacological and/or diagnostic function of which can at least be predicted on the basis of the appended examples (e.g. those characterized by any one of formula Vi to IX').
The present invention also refers to cyclic peptides derived from the ECn of β2-AR, like those cyclic peptides corresponding to the βi-ECn-derived peptides of this invention but being structurally more closely related to ECU of β2-AR. These peptides may, for example, be used as reference/control peptides, when βr ECn-derived peptides are tested/assayed. One particular non-limiting example of such a cyclic peptides derived from the ECn of β2-AR is the cyclic peptide as depicted by the following formula: cycio(Arg»Aia-Glu~Ser-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn- Glu-Pπ>Lys-Cys-Abu-Asp~Phe~Vai-Thr-Gly).
It will be understood that for the various peptides of the present invention a certain flexibility and variability in the primary sequence, i. e. the amino acid sequence backbone, is possible as long as the overall secondary and tertiary structure of the respective peptides, which is defined by at least some fixed amino acid residues and by their spatial arrangement, is ensured (see, e.g., formuia I, supra). Based on the teaching provided herein, the skilled person is, one the one hand, readily in the position to find out corresponding variants of the peptides of the invention. One the other hand, the skilled person is able to test whether a given variant of peptides of the present invention still has the desired function, for example the ability to specifically bind to β-AR antibodies, and therefore has the potential for a corresponding medical intervention, like the therapeutic and diagnostic applications described and provided herein. Corresponding experimental guidance for such tests, i.e. respective assays, are exemplariiy provided and described herein, particularly in the appended examples.
Accordingly, also provided herein are variants of the herein disclosed and described peptides, given that, first, these variants are still functionally active in accordance with this invention, i. e. functionally active as binding partners for anti-β-AR antibodies, particularly for anti- βrAR antibodies against the βrECn, more particularly functionally active as inhibitors of βrAR and even more preferably active in inhibiting the interaction between βrAR and anti-βi-AR antibodies against the βi-ECn, more preferably auto-anti-βrAR antibodies against the βi-EC-H; and, second, that these variants are not present in form of isomers mixtures or do not form isomers mixtures when cyclized in accordance with (the production method of) this invention. These variants are envisaged to have only two certain Cys residues forming or being able to form only one individual intramolecular linkage (e.g. disulphide bond).
Within the variants of the peptides of the present invention it is, for example, envisaged that one or more amino acids of said peptides (V as referred to, for example, in formula I) are replaced by other one or more naturally occurring or synthetic amino acids. In this context, it is preferred that this/these amino acid exchange(s) is/are (a)conservative amino acid exchange(s), i.e. that the replacement amino acid belongs to the same category of amino acids than the amino acid to be replaced. For example, an acidic amino acid may be replaced by another acidic amino acid, a basic amino acid may be replaced by another basic amino acid, an aliphatic amino acid may be replaced by another aliphatic amino acid, and/or a polar amino acid may be replaced by another polar amino acid.
Accordingly, particularly preferred and provided variants of the (cyclo) peptides of the present invention are variants wherein at least one of an acidic amino acid is replaced by a different amino acid selected from the group consisting of acidic amino acids, at least one of the basic amino acids is replaced by a different amino acid selected from the group consisting of basic amino acids, at least one of a polar amino acid is replaced by a different amino acid selected from the group consisting of polar amino acids and/or at least one of an aliphatic amino acid is replaced by a different amino acid selected from the group consisting of aliphatic amino acids (given that the above mentioned-requirements are fulfilled).
It is particularly envisaged that the amino acid exchanges which lead to variants of the disclosed (cyclic) peptides are such, that the pattern of polarity and charge within the tertiary structure of the resulting variant still substantially mimics the three- dimensional structure of the corresponding ECM epitope(s) of βi-AR.
With respect to the "Variants" of the (cyclo) peptides of the present invention the herein defined Cys may also be replaced by other amino acids, as long as the replacement still leads to an individual intramolecular linkage, like that of a disuiphide bond, within the cyclopeptide, i.e. to the avoidance of isomers mixtures formation during cyclization and/or a correct mimicry of the ECn of βi-AR. Such amino acid may, inter alia, be a non-naturally occurring amino acid, like a non-naturally occurring amino acid having an -SH group able to form a disuiphide bond. However, it is preferred herein that the Cys given in formula I, above, is indeed a naturally occurring amino acid, preferably Cys itself.
It will also be acknowledged by the ones skilled in the art that one or several of the amino acids forming the (cyclic) peptide of the present invention may be modified. In accordance therewith any amino acid as used herein may also represent its modified form. For example, an alanine residue as used herein may comprise a modified alanine residue. Such modifications may, among others, be a methyiation or acylation or the like. Such modification(s) or modified amino acid(s) is (are) envisaged in the context the present invention as long as the thus modified amino acid and more particularly the peptide containing said thus modified amino acid is stil! functionally active as defined herein, like functionally active as an inhibitor of anti-βr AR antibodies, preferably active in inhibiting the interaction between βi-AR and antibodies, and more preferably auto-antibodies directed against βrAR. Corresponding assays for determining whether such a peptide, i. e. a peptide comprising one or several modified amino acids, fulfils this requirement are known to the one skilled in the art and, among others, are also described herein, particularly in the example part hereof.
The invention also provides derivatives of the disclosed (cyclic) peptides such as salts with physiologic organic and anorganic acids like HCl, H2SO4, H3PO4, maiic acid, fumaric acid, citronic acid, tatratic acid, acetic acid.
As used herein, the sequences of the peptides disclosed are indicated from the N- terminus to the C-terminus, whereby the N4erminus is at the left side and the C- terminus is at the right side of the respective depicted amino acid sequence. When referring to cyclic peptides, the corresponding sequences are indicated from the side corresponding to the left side of formula f to the side corresponding to the right side of formula I.
A "cyclic peptide" or "cyclopeptide" and the like in accordance with the present invention is a peptide intramoiecularly forming a molecular ring structure within its amino acid backbone/primary amino acid sequence by at least one, preferably by at least two, more preferably by exactly two intramolecular linkages having covalent character. The forming of this molecular ring structure is, in context of this invention, also termed "cyclization".
In one particularly preferred embodiment, the cyclic peptide of this invention has two intramolecular linkages having covaient character, wherein one of these linkages is an intramolecular linkage between the N- and C-termina! ends of a peptide being the amino acid backbone/primary amino acid sequence of the cyclic peptide disclosed and the other one is an intramolecular linkage between two non-terminal amino acids of this peptide. As mentioned, these two non terminal amino acids may be two Cys.
Generally, "cyclization" in accordance with this invention may occur by at least one linkage which is a covaient binding selected from the group consisting of S-S linkages, peptide bonds, carbon bonds such as C-C or C=C, ester bonds, ether bonds, azo bonds, C-S-C linkages, C-N-C linkages and C=N-C linkages. Particularly, the peptide bond as mentioned throughout this invention can be formed by the NH2 group of an N-terminal amino acid and the COOH group of a C-terminal amino acid of a peptide forming the amino acid backbone/primary amino acid sequence of the cyclic peptide disclosed.
Preferably, an intramolecular linkage between the N- and C-terrninal ends of a peptide forming the amino acid backbone/primary amino acid sequence of the cyclic peptide disclosed is a peptide bond and an intramolecular linkage between two nonterminal amino acids of this peptide is an S-S linkage (i, e. dlsulphide bond), in context of this invention, an intramolecular S-S linkage within the cyclic peptide provided can be formed between two Cys residues within the amino acid backbone/primary amino acid sequence of said cyclic peptide.
Within the cyclic peptides of this invention, not only the above mentioned two particular intramolecular covaient linkage may be formed but also further intramolecular linkages may occur, with the proviso that the herein described functionality of the cyclic peptides is maintained and that the cyclic peptides can still easily be characterized biochemically, which, e.g., means that no isomers mixtures are formed during cyclization of the corresponding amino acid backbone/primary amino acid sequence.
For example, such further intramolecular linkages are additional bonds formed by a side chain of NH2 groups and COOH groups of the constituent amino acids.
Terms like "amino acid backbone" or "primary amino acid sequence" as used throughout the present invention refer, on the one hand, to that structural component or part of a cyclic peptide which is formable or formed by its corresponding amino acid sequence. On the other hand, these terms refer to the linear peptides able to form the cyclic peptides of this invention by cyclization
In one particular embodiment of the first aspect of this invention, a cyclic peptide is provided which is obtainable by the method for producing a cyclic peptide as provided herein. The definitions given herein-above also appiy with respect to this particularly provided cyclic peptide of the present invention
In one embodiment of the first aspect of this invention also such peptides are provided, the disclosed cyclic peptides are formable by or are formed by. Particularly these peptides are the linear peptides forming or being able to form the herein disclosed cyclic peptides, i e the amino acid backbone/primary amino acid sequence thereof
In general, such a linear peptide can be any peptide, the covalent linkage of the N- and C-termtnus of which results in a cyclic peptide as disclosed in accordance with the present invention For example, such a linear peptide may be some kind of an intermediate compound in a procedure of producing the cyclic peptides of this invention, like the method for producing a cyclic peptide as disclosed herein
In general, the N- and C~terminal end of a linear peptide provided herein may be any amino acid pair lying in direct proximity to each other within the amino acid backbone of a cyclic peptide disclosed in context of this invention In other words, cyciization (ring closure) of the cyclic peptide of this invention may generally occur between any of said amino acid pairs The skilled person is readily in the position to find out such particular amino acid pairs which are effective/suitable to act as N- and C-terminal ends of a herein disclosed linear peptide, i e which are effective/suitable to act as an amino acid pair being involved in the ring closure/cyclization as defined in context of this invention
Sn one preferred but non-limiting example, the cyclization (ring closure) of a linear peptide of this invention may occur between Ala and GIn or GIu, i.e the N-terminal amino acid of this linear peptide would be Ala and the C-terminal amino acid would be GIn or GIu. Examples of such linear peptides able to form the cyclic peptide of the present invention are SEQ ID NO. 1 and 2 and, iess preferred SEQ ID NO. 3 and 4. In another preferred but non-limiting example, the cyciization (ring closure) of a linear peptide of this invention may occur between Lys and Pro, i.e. the N-termina! amino acid of this linear peptide would be Lys and the C-terminal amino acid would be Pro. Examples of such linear peptides able to form the cyclic peptide of the present invention are SEQ !D NO. 9 and 10 and, less preferred, SEQ ID NO. 11 and 12. In a more preferred but non-limiting example, particularly when a 22mer cyclic peptide is provided, the cyciization (ring closure) of a linear peptide of this invention may occur between Arg and GIy, i.e. the N-terminal amino acid of this linear peptide would be Arg and the C-terminal amino acid would be GIy. An example of such a linear peptide able to form the cyclic peptide of the present invention is SEQ ID NO. 25.
In another more preferred but non-limiting example, particularly when a 22mer cyclic peptide is provided, the cyciization (ring closure) of a linear peptide of this invention may occur between Lys and Pro, i.e. the N-terminal amino acid of this linear peptide would be Lys and the C-terminal amino acid would be Pro. An example of such a linear peptide able to form the cyclic peptide of the present invention is SEQ ID NO, 27.
Besides their amino acid backbone, the cyclic peptides of the invention may further comprise (e.g. have covalently bound) (a) further substituent(s). like labels, anchors (like proteinaceous membrane anchors), tags (like HIS tags) and the like. Appropriate substituents and methods for adding them to the cyclic peptide of this invention are known to those of ordinary skill in the art.
Examples of labels in this context include, inter alia, fiuorochromes (like fluorescein, rhodamine, Texas Red, etc), enzymes (like horse radish peroxidase, β- galactosidase, alkaline phosphatase), radioactive isotopes (like 32P, 33P, 35S, 1251 or 1231, 1351, 1241, 11 C, 15O)5 biotin, digoxygenin, colloidal metais, chemi- or bioluminescent compounds (like dioxetanes, luminoi or acridiniums). One particularly envisaged label that may be bound to the peptide of this invention is a fiuorochrome belonging to a FRET pair of fiuorochromes, for example a GFP variant (e.g. GFP, eGFP, EYFP or ECFP). A variety of techniques are available for labeling biomolecules, are well known to the person skilled in the art and are considered to be within the scope of the present invention and comprise, inter alia, covaient coupling of enzymes or biotinyl groups, phosphorylations, biotinylations, random priming, nick-translations, tailing (using terminal transferases). Such techniques are, e.g., described in Tijssen, "Practice and theory of enzyme immunoassays", Burden and von Knippenburg (Eds), Volume 15 (1985); "Basic methods in molecular biology", Davis LG, Dibmer MD, Battey Elsevier (1990); Mayer, (Eds) "Immunochemical methods in cell and molecular biology" Academic Press, London (1987); or in the series "Methods in Enzymology", Academic Press, Inc. Detection methods comprise, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, etc.
The substituent(s) can be bound (e.g. covalently) to the cyclic peptides of the invention directly or via linkers. The skilled person is readily in the position to find out appropriate linkers to be employed in this context.
in a further aspect, the present invention relates to a nucieic acid molecule comprising a nucleotide sequence encoding an amino acid sequence which can form or which can be used to form or generate a cyclic peptide as disclosed in context of this invention, or an amino acid backbone/primary amino acid sequence thereof. The present invention also relates to a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence which can form or which can be used to form or generate the linear peptides as provided and described herein. For example, such nucleic acid molecule may comprise a nucleotide sequence as depicted in any one of SEQ ID NO. 5, 6, 13, 14, 26 and 28 or, less preferred, SEQ ID NO. 7, 8, 15 and 16 or a nucleotide sequence which differs therefrom due to the degeneracy of the genetic code.
Since ABu or an ABu analogue is a non-genetical!y coded amino acid, there are no nucleic acid molecules existing which directly encode the cyclic peptide of the invention, its amino acid backbone/primary amino acid sequence or the corresponding linear peptide. However, the skilled person is readily in the position to provide nucleic acid molecules which encode amino acid sequences that can serve as a basic or intermediate product for the formation or generation of the cyclic peptides of the invention. Examples for such nucleic acid molecules are those coding for an amino acid stretch of βi-ECn corresponding to the amino acid backbone/primary amino acid sequence of the cyclic peptides of the invention having ABu (or the ABu analogue) deleted or replaced by any other genetically coded amino acid, preferably by an amino acid that can be replaced by ABu (or the ABu analogue) iater on.
The meanings of the terms "nucleic acid moiecu!e(s)"7 "nucleic acid sequence(s)" and "nucleotide sequence(s)" and the like are well known in the art and are used accordingly in context of the present invention.
For example, when used thoughout this invention, these terms refer to all forms of naturally occurring or recombinantly generated types of nucleotide sequences and/or nucleic acid sequences/molecules as wel! as to chemically synthesized nucleotide sequences and/or nucleic acid sequences/molecules. These terms aiso encompass nucleic acid analogues and nucieic acid derivatives such as e. g. locked DNA, PNA, oligonucleotide thiophosphates and substituted ribo-oligonucieotides. Furthermore, these terms also refer to any molecule that comprises nucleotides or nucleotide analogues.
Preferably, the terms "nucleic acid molecule(s)", "nucleic acid sequence(s)" and "nucleotide sequence(s)" and the like refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The "nucleic acid molecule(s)", "nucleic acid sequence(s)" and "nucleotide sequence(s)" may be made by synthetic chemical methodology known to one of ordinary skill in the art, or by the use of recombinant technology, or may be isolated from natural sources, or by a combination thereof. The nucleic acid molecules as provided herein are particularly useful for producing a cyclic peptide of the invention, for example by the corresponding method disclosed herein.
In yet another aspect, the present invention relates to a method for producing a cyclic peptide of the present invention, comprising the steps of a) (i) culturϊng the recombinant host ceil of the present invention under conditions such that an amino acid sequence which can form or which can be used to form or to generate an amino acid backbone of the herein disclosed cyclic peptide (or the linear peptide of this invention) is expressed, recovering amino acid sequence and connecting said amino acid sequence into said amino acid backbone (or into said linear peptide of this invention); or
(ii) chemically synthesizing the amino acid backbone of the herein disclosed cyclic peptide (or the linear peptide of this invention); and b) cyclization of said amino acid backbone (or said linear peptide) to form the herein disclosed cyclic peptide.
The definitions given herein-above with respect to the term "cyclization" apply here, mutatis mutandis. In the particular context of the above method, the meaning of the term "cyclization" encompasses both, forming of the intramolecular bridge {disulphide bond) and the ring closure by covatently connecting the N- and C-termini of the backbones of the cyclic peptides to be produced.
The definitions given herein with respect to the cyclic peptide, its amino acid backbone or the corresponding linear peptide according to this invention, as well as with respect to the host cell provided herein, apply here, mutatis mutandis.
In context of the above method for producing the cyclic peptides of this invention the skilled person knows how the encoded amino acid sequence can be "converted" into the amino acid backbone of the cyclic peptides of the invention. For example, if the encoded amino acid sequence lacks the amino acid at the position corresponding to the position of ABu (or an ABu analogue) of the provided cyclic peptides, the encoded amino acid sequence can be "converted" in accordance with said method by introducing ABu (or an ABu analogue) at corresponding position, if the encoded amino acid sequence comprises an amino acid or amino acid stretch instead of ABu (or an ABu analogue), the amino acid sequence can be "converted" in accordance with said method by replacing said amino acid or amino acid stretch with ABu (or an ABu analogue).
As already mentioned above with respect to the provided cyclic and linear peptides, in a preferred embodiment of this further aspect of the invention, the N-terminal amino acid of the amino acid backbone/linear peptide to be cyclized in order to produce a cyclic peptide of this invention is Ala, Arg or Lys and the corresponding C- terminal amino acid is Gin, GIy or Giu (also DGIu is possible) or Pro. However, as mentioned-above, also other N- and C-terminal amino acids are envisaged, i.e. also other cyciization (ring closure) sites can be employed in context of the disclosed method.
The person skilled in the art is readily able to put the herein disclosed method for producing a cyclic peptide into practice, based on his common general knowledge and the prior art like, for example, WO 2006/103101 , which discloses a general methology how to synthesize peptides and, particularly cyclic peptides. Also, the teachings of the invention, for example in the appended experimental part (example 1), provides for corresponding enabling technical guidance.
In the non-limiting appended Example 1 , the cyclopeptide mutants were first synthesized in form of their linear peptides/amino acid backbones (for example by applying a chemical synthesis approach, like the Fmoc / tert butyl strategy (as described in WO 2006/103101 ; Chen W.C. and White P. D.: Fmoc Solid Phase Peptide Synthesis, Oxford University Press 2003)), and were then cyclized covalentiy on the backbone by condensation of the C-terminal carboxyi group with the amino group of the N-terminal amino acid ("head to tail" cyciization; Kates S. and Albericio F.: Solid phase synthesis, CRC-Press, 2000).
Subsequently, a disulphide bond is established between those two cysteine residues of the linear peptides which are able to form a disulphide bond (e.g. between Cys7 and Cys-13 of the 18mer peptide, between Cysi0 and Cys16 of the 22-mer peptide or between Cysn and Cysi7 of the 25-mer peptide) by chemical interaction known in the art (e.g. Benoiton N. L.: Chemistry of Petide Synthesis. CRC-Press, 2005).
In general, in context of the "cyciization" step of the above described method, the ring closure of the linear backbone of the cyclic peptides to be produced may be performed before or after the formation of the S-S bridge. In other words, the S-S bridge between the two Cys residues of the AA chain of the peptides may be the first step in the "cyciization" procedure of the described production process and the ring closure may be the second step, or vice versa. The skilled person is able to find out which of these particular approaches is appropriate for a given setup of the production preconditions.
As mentioned above, the linear peptides/amino acid backbones of the cyclic peptides to be produced can also be produced by using recombinant engineering techniques. Such techniques are well known in the art (e. g. Sambrook, supra). As also mentioned above, by this kind of production of said linear peptides/amino acid backbones particular advantage can be taken of the herein disclosed and described nucleic acid molecules, vectors and/or host cells. The definitions correspondingly given above apply here, mutatis mutandis.
Several approaches of peptide synthesis, in particular synthesis approaches of cyclic peptides are known in the art (e.g. Williams, Chemical Approaches to the Synthesis of Peptides, CRC-Press 1997; Benoiton: Chemistry of Peptide Synthesis. CRC- Press, 2005). The skilled person is readily in the position to apply the prior art knowledge to the particular requirements of the production of the provided cyclic peptides, for example, of the disclosed method for producing the provided cyclic peptides, based of the herein provided teaching.
As already mentioned above, this invention also relates to a cycfic peptide obtainable or obtained by the above described method, but also to a corresponding linear peptide (amino acid backbone/primary sequence of the corresponding cyclic peptide) obtainable or obtained by the above described method as some kind of an intermediate product (particularly a product obtainable or obtained by step a) of the above described method).
In general, the cyclic peptide according to the present invention may, inter alia, be used in medical intervention approaches. Such approaches comprise the use as or in a diagnostic agent and for the manufacture of a medicament for the treatment of diseases or the use in or as a composition, preferably a pharmaceutical composition, a diagnostic composition or a diagnostic kit, preferably for the detection of anti-β-AR antibodies, more preferably for the detection of anti-β-i-AR antibodies. As mentioned, the antibodies as defined or described herein are preferably autoantibodies.
Non-limiting uses and appiications of the compounds, particularly the cyclic peptide according to the present invention are described herein, for example further below.
The present invention also relates to a composition comprising a cyclic or a linear peptide, a nucleic acid molecule, a vector or a recombinant host cell as disclosed and provided in context of the present invention, and optionally a carrier. In one particular embodiment of this aspect, said composition is a pharmaceutical composition and said carrier is a pharmaceutically acceptable carrier.
The composition of this invention, particularly pharmaceutical composition of this invention, is particularly useful when employed in the treatment, amelioration or prevention as described and defined herein. Accordingly, the pharmaceutical composition of this invention may be used for the treatment, amelioration or prevention of a disease where the activity of a β-AR is enhanced or for the treatment of a patient having antibodies against a β-AR. Moreover, the pharmaceutical composition of this invention may be used for inducing immune tolerance of a patient, particularly immune tolerance of a patient with respect to immunogenic stretches of the endogenous βi-AR.
Apart from containing at least one cyclic peptide of the present invention, the (pharmaceutical) composition provided may either comprise two or a plurality (like at least 3 or at least 5) of cyclic peptides of the present invention. Likewise, not only one, but also two or a plurality {like at least 3 or at least 5) of said cyclic peptides may be administered to a patient in need of medical intervention in accordance with the present invention. Thereby, the administration of said more than one of cyclic peptides may be simultaneously or successively.
Moreover, in on particular embodiment, the present invention relates to the pharmaceutical composition, the method or uses for medical intervention or the cyclic peptide or the pharmaceutical composition as disclosed herein, wherein said cyclic peptide is administered with or said pharmaceutical composition comprises at least one further pharmaceutically active agent.
Said at least one further pharmaceutically active agent may be a β-receptor blocker, preferably a selective β-AR blocker, like, for example, a βi-AR blocker selected from the group consisting of atenolol, metoproSol, nebivolol, bisoprolol and the like. Without being bound by theory, this kind of particular combination may provide for protection from antibody-induced, selective βi-AR downregulation by the herein provided cyclic peptides, since βi-AR is at the same time upreguiated by betablockers, like bisoprolol or metoproiol, and ultimately results in a synergistic effect of the cyclic peptides and the additional β-blocker(s).
The carrier optionally comprised in the (pharmaceutical) composition of the invention or to be administered together with the (pharmaceutical) composition or the cyclic peptide of the invention may particularly be a pharmaceutically acceptable carrier, excipient or diluent.
Such carriers are well known in the art. The skilled person is readily in the position to find out such carriers which are particularly suitable to be employed in accordance with the present invention.
In the following, several non-limiting administration schemes and the use of correspondingly suitable pharmaceutically acceptable carrier are described.
For an administration of the pharmaceutical composition and/or the cyclic peptides in accordance with this invention via subcutaneous (s.c.) or intravenous (i.v.) injection, compounds of the invention may be formulated in aqueous solution, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically saline buffer. For transmucosal and transpulmonal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
The use of pharmaceutical acceptable carriers to formulate the compounds according to the present invention into dosages or pharmaceutical compositions suitable for systemic, i.e. intravenous/intraarterial, or subcutaneous administration is within the scope of the present invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be readily formulated using pharmaceutically acceptable carriers well known in the art into dosages suitable for subcutaneous or oral administration. Such carriers enable the compounds according to the present invention to be formulated as tablets, pilis, capsules, dragees, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated.
Compounds according to the present invention, or medicaments comprising them, intended to be administered intracorporally/intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered near the cell surface. Delivery systems involving liposomes are disclosed in U.S. Patent No. 4,880,635 to Janoff et al. The publications and patents provide useful descriptions of techniques for liposome drug delivery.
Pharmaceutical compositions comprising a compound according to the present invention for parenteral and/or subcutaneous administration include aqueous solutions of the active compound(s) in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil or castor oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injections suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions and to allow for a constantly slow release of the substance in the organism. It is clear to the skilled person that, in accordance with the present invention, the disclosed pharmaceutical composition or cyclic peptide may be administered in a pharmaceuticals/therapeutically effective dose, which means that a pharmaceutically/therapeuticaliy effective amount of the compound administered is reached. Preferably, a pharmaceuticals/therapeutically effective dose refers to that amount of the compound administered (active ingredient) that produces amelioration of symptoms or a prolongation of survival of a subject which can be determined by the one skilled in the art doing routine testing,
It is of note that the dosage regimen of the compounds to be administered in accordance with the present invention will be determined by the attending physician and clinical factors. As is well known in the medical arts, that dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A person skilled in the art is aware of and is able to test the relevant doses, the compounds to be medically applied in accordance with the present invention are to be administered in.
As shown herein, the effect of the cyclic peptides provided herein, namely the blockage of anti-βt-AR antibodies, can be obtained in a dose dependent manner. Thereby, the efficiency of the Cys→Ser mutated cyclopeptides as disclosed herein depends on a threshold concentration (Figs. 6 and 7). The Cys-ABu mutant as disclosed herein may act likewise.
Accordingly, the disclosed pharmaceutical composition or cyclic peptide may particularly be administered in a manner that it is present in a concentration, i.e. reaches a threshold concentration, of at least 0.05 mg per kg body weight, preferably in a concentration of at least 0.1 mg per kg body weight, more preferably in a range of 0.1 mg per kg body weight (100 μg/kg) to 100 mg per kg body weight, more preferably in a range of 1 mg per kg body weight to 100 mg per kg body weight and most preferably in a range of 1 mg per kg body weight to 10 mg per kg body weight. Particularly, the effective dose of the disclosed pharmaceutical composition or cyclic peptide may be at about 1 mg per kg body weight Also higher concentrations of the disclosed pharmaceutical composition or cyclic peptide are generally envisaged to be reached by correspondingly applied administration schemes. For example, such higher concentrations may be at least 2, 3, 4 or 5 mg per kg body weight. Concentrations of at least 1 mg per kg body weight or at least 2 mg per kg body weight are preferred.
One particularly preferred, non-limiting administration scheme to be applied in context of this invention is an s.c. or i.v. application every two or four weeks. In the rat model employed herein a dose of 1 to 4 mg/kg i.v. every other month were sufficient to obtain therapeutic levels of the compounds according to the present invention, with the respective dosage for humans preferably being about 0.3-10 mg/kg i.v. or s.c, more preferably being about 1-10 mg/kg i.v. or s.c, even more preferably being about 1-5 mg/kg i.v. or s.c. In a particular embodiment, the respective dosage for humans are about 0.1-10 mg/kg i.v. or s.c, more preferably about 0.3-5 mg/kg i.v. or s.c, even more preferably about 0.3-2 mg/kg i.v. or s.c. As demonstrated herein, the administration of the disclosed cyclic peptides may initially trigger a transient opposite immune response, in particular when applied in higher doses. Such transient immune responses in the long run are compensated by the antibody-inactivating activity of the administered cyclic peptides. This may lead to a decelerated effect of the administered cyclic peptides, i.e. a decelerated elimination of anti-βrAR antibodies and hence a decelerated reduction of (aberrant) βi-AR activity.
The present invention also relates to a method for a) the treatment, amelioration or prevention of a disease where the activity of a β- AR, preferably βrAR, is enhanced; b) the treatment of a patient having antibodies against a β-AR, preferably against βrAR, or suffering from or being at risk to develop a disease as disclosed herein; or c) inducing immune tolerance, comprising the step of administering to a patient in need of such medical intervention a pharmaceutically active amount of a cyclic peptide and/or of a pharmaceutical composition as disclosed herein, and optionally a pharmaceutically acceptable carrier. The present invention also relates to a cycϋc peptide or a pharmaceuticai composition as disclosed herein, and optionally a pharmaceutically acceptable carrier, for, or for use in, a) the treatment, ameiioration or prevention of a disease where the activity of a β- AR, preferably βi-AR, is enhanced; b) the treatment of a patient having antibodies against a β-AR, preferably against βrAR, or suffering from or being at risk to develop a disease as disclosed herein; or c) inducing immune tolerance.
The present invention further relates to a kit for use in a regimen for a) the treatment, amelioration or prevention of a disease where the activity of a β- adrenergic receptor (β-AR) is enhanced; b) the treatment of a patient having antibodies against a β-AR; or c) inducing immune tolerance, said regimen comprising one or more cycles, preferably two or more cycles, each cycle consisting of a 2 to 70 day treatment/inducing period according to (a) to (c) followed by a 6 to 280 day rest period, wherein said kit comprises: (i) from 2 to 70 doses (e.g. daily, weekly or monthly doses) of a cyclic peptide or a pharmaceutical composition according to the present invention; and, optionally, (ii) from 6 to 280 doses (e.g. daily, weekly or monthly doses) of a placebo or a nutrient supplement; and, optionally, (iti) a means for having the components arranged in a way as to facilitate compliance with the regimen, but not containing a cyclic peptide or pharmaceutical composition of the present invention.
in one particular embodiment, the cyclic peptide or pharmaceutical composition of the present invention, or the cyclic peptide or pharmaceutical composition to be administered in the context of the methods for treating, ameliorating, preventing or inducing of the present invention, are to be administered according to the above described regimen. In the described regimen, the rest period may be about 1 , 2, 3, 4 or 5 times the treatment/inducing period, wherein about 3 times the treatment/inducing period is preferred. The treatment/inducing period may be about 2, 3, 4 or 5 weeks, wherein about 3 weeks are preferred. Hence, the rest period may particularly be about 2 to 25 weeks. Preferably, the rest period is about 6, 9, 12 or 15 weeks, wherein about 9 weeks are preferred.
Further, in the described regimen, the cyclic peptide or pharmaceutical composition may, for example, be administered 1 to 7 times a week during the treatment/inducing period, wherein once per week during the treatment/inducing period is preferred. The overall dose to be administered in the context of the regimen as defined herein can be determined by the attending physician and depends on clinical factors like patient's size, body surface area, age. This is described in more detail herein elsewhere.
For example, the overali dose of the cyclic peptide or pharmaceutical composition to be administered in the context of the regimen defined herein corresponds to a monthly dose of 0.1-10 mg/kg, preferably of about 0.3-5 mg/kg, more preferably about 0.3-2 mg/kg and most preferably about 1 mg/kg.
In one particularly preferred example of the herein defined regimen, the required overall dose is administered in weekly portions over a treatment/inducing period of 3 weeks followed by a rest period of 9 weeks. Each of said weekly portions may particularly be one of the above described monthly doses, preferably the monthly dose of about 1.0 mg/kg.
The doses of a cyclic peptide or pharmaceutical composition as comprised in the kit of the invention may be provided in individual vials or several or ail of the doses may be combined in one or more common vial(s). Preferably, the amount of the cyclic peptide/pharmaceutical composition to be administered at a certain point in time (e.g. day) within the treatment/inducing period (e.g. weekly portion as described above) is combined in a single vial. With respect to the most preferred administration regimen, a monthly dose is envisaged to be provided in a single vial, wherein a corresponding kit comprises three of such vials. Optionally, said kit may further comprise an instruction manual/leaflet guiding the attending physician/patient to administer one of said 3 monthly doses once per week over a period of 3 weeks (the treatment/inducing period) foliowed by a period of 9 weeks (the rest period).
In general, the cyclic peptide, pharmaceutical composition or kit of the present invention may be provided with/comprise an instruction manual/leaflet. Said manual/leaflet may guide the attending physician/patient through the administration regimen of the cyclic peptide or pharmaceutical composition of the invention. In particular, the instruction manual/leaflet may guide the attending physician/patient through the regimen as described herein-above.
Preferred cyciic peptides to be administered according to the above described regimen are the herein disclosed 22-mer cyclic peptides, in particular the 22-mer cyclic peptides formable or formed by the amino acid sequence as depicted in SEQ ID No. 25 or 27.
The diseases to be medically intervened (treated, ameliorated, prevented or diagnosed) in accordance with this invention or the diseases the patient as defined and described herein suffers from are preferably those, where the βrAR is activated in a non-physiological manner, more preferably is activated by antibodies, more preferably by auto-antibodies which are directed against the (VAR. Exempiarily and preferably, the diseases to be medically intervened in accordance with this invention or the diseases the patient as defined and described herein suffers from comprise, however, are not limited thereto, the group of heart diseases.
Particularly, the heart diseases to be medically intervened in accordance with this invention or the heart diseases the patient as defined and described herein suffers from may comprise but are not limited to infectious and non-infectious heart disease, ischemic and non-ischemic heart disease, inflammatory heart disease and myocarditis, cardiac dilatation, idiopathic cardiomyopathy, (idiopathic) dilated cardiomyopathy (DCIvI)5 immune-cardiomyopathy, heart failure, and any cardiac arrhythmia including ventricuiar and/or supraventricular premature capture beats as well as any atrial arrhythmia including atrial fibrillation and/or atrial flutter. In other words, the heart disease as referred to in the descriptions and definitions given herein with respect to the methods or the cyclic peptide or the pharmaceutical composition of the invention may be heart diseases selected from the group comprising infectious and non-infectious heart disease, ischemic and non-ischemic heart disease, inflammatory heart disease and myocarditis, cardiac dilatation, idiopathic cardio-myopathy, (idiopathic) dilated cardiomyopathy (DCM), immune- card iomyopathy, heart failure, and any cardiac arrhythmia including ventricular and/or supraventricular premature capture beats as well as any atrial arrhythmia including atrial fibriilation and/or atrial flutter.
It is of note that the most preferred disease to be medically intervened (treated, ameliorated, prevented or diagnosed) in accordance with this invention or the most preferred disease the patient as defined and described herein suffers from is DCM, preferably idiopathic DCM.
A particular subgroup of the "patients" for the purpose of the present invention are those patients suffering from any of the diseases described herein, more particularly the group of heart diseases described herein and having at the same time antibodies directed against β-ARs, more preferably antibodies against the β-i-AR, whereby the antibodies are preferably auto-antibodies.
A disease to be medically intervened (treated, ameliorated, prevented or diagnosed) in accordance with this invention or a disease the patient as defined and described herein suffers from is intended to be induced by antibodies against a β-AR, preferably by antibodies against βi-AR. Preferably, these antibodies are autoantibodies.
The means and methods provided herein are particularly useful when provided in the prophylaxis/prevention of a disease as defined herein. This means that a patient may be treated with the cyclic peptide and/or pharmaceutical composition of the invention prior to the onset (of symptoms) of a disease as defined herein. For example, this preventive treatment may follow a preceding diagnostic application that, e.g., takes advantage of the diagnostic means and methods provided herein. Thereby, it is preferred that a preventive treatment taking advantage of the therapeutic means and methods of this invention is applied, when the risk to develop a disease as defined herein is diagnosed, e.g. when anti-β-AR (auto-)antibodies are detected. In this context, a preferred "patient" is one bearing at risk to develop a disease as defined herein. Particularly, such a patient is one having anti-β-AR (auto-)antibodies, preferably anti-βi-AR (auto-)antibodies, but not (yet) suffering from a disease as defined herein, or symptoms thereof.
The immune tolerance to be induced in context of this invention is envisaged to be particularly obtained by suppression of the production of antibodies against immunogenic stretches of the β-AR molecule, which, without being bound by theory, may be due to a blockade of the antigen-recognition sites of the antibody-producing early (mature) 8-celis and memory B-cells.
It is within the present invention that the provided pharmaceutical composition or cyclic peptide is particularly useful for the treatment, prevention and/or amelioration of any of the diseases and patient groups or patients as defined herein including the detection of anti-β-AR antibodies in these patients by using the aforementioned compounds.
A "patient" for the purposes of the present invention, i. e. to whom a compound according to the present invention is to be administered or who suffers from the disease as defined and described herein or who is intended to be diagnosed in accordance with this invention, includes both humans and other animals and organisms. Thus the compounds and methods of this invention are applicable to or in connection with both human therapy and veterinary applications including diagnostic(s), diagnostic procedures and methods as wel! as staging procedures and methods. In the preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient is human.
The mutant cyclic peptides according to the present invention may also be used for the preparation of a medicament for the treatment, prevention and/or amelioration of any of the diseases and patient groups/patients as defined herein. What is said herein for the pharmaceutical composition applies also to the medicament for the manufacture of which the peptides of the present invention may be used. In a still further aspect, the present invention is related to a diagnostic agent comprising or being a cyclic peptide or a composition according to this invention, and optionally at least one further biologicaiiy active compound.
Preferably the herein disclosed diagnostic agent consists of or comprises a mutant peptide of the present invention, whereby the mutant peptide comprises a label. Such label may be selected from the group comprising radioactive labels and fluorescent labels. Respective labels are known to the ones skilled in the art. The definitions and descriptions of labels as given herein-above apply here, muatis mutandis.Typically, the peptide is the part of the diagnostic agent conferring specific binding characteristics to the diagnostic agent, preferably binding to anti-βrAR antibodies, whereas the label confers the signalling characteristics to the diagnostic agent.
The diagnostic agent of this invention may comprise, apart from (a) labelled or unlabelled mutant peptide(s) of the present invention, a further biologically active compound. Such further biologically active compound may be a means to confer signalling characteristics to the diagnostic agent, particularly in case the mutant peptides of the present invention are unlabelled. For example, the further biologically active compound can be an antibody, preferably a monoclonal antibody, and more preferably a labelled antibody specificaSty binding to a mutant peptide of the present invention or to a complex consisting of a mutant peptide of the present invention and an anti-β-AR antibody, preferably an anti-βrAR antibody.
In a further aspect, the present invention relates to a method for diagnosing a disease as defined and described herein comprising the steps of a) detecting antibodies against a β-AR (for example in a sample) using the cyclic peptide or the composition or the diagnostic agent of the present invention; and b) diagnosing for said disease, when the titer of said antibodies is increased.
In a further aspect, the present invention is related to a method for diagnosing a patient which can be treated using the mutant peptides, pharmaceutical compositions and medicaments according to the present invention. In context of this particular method also a step of detecting antibodies against a β-AR (for example in a sample) using the compounds of the present invention and/or a step of considering whether the outcome of said detection step indicates a disease as defined herein, may be employed. As mentioned, a disease as defined herein or the risk to develop a disease as defined herein is indicated, when the titer of said anti-β-AR antibodies is increased.
In another aspect, the present invention relates to a cyclic peptide, a composition or a diagnostic agent as provided and described herein for diagnosing (for example in a sample) a disease as defined herein. Again, a disease as defined herein or the risk to develop a disease as defined herein is indicated by an increased titer of anti-β-AR antibodies.
In context of the present invention the term "increased titer of anti~β~AR antibodies" means that the titer of anti-β-AR antibodies (for example in a sample derived from a patient to be diagnosed in accordance with this invention) is higher than that of a healthy control patient, i.e. a patient not suffering from a disease as defined herein and/or a patient Sacking anti-β-AR antibodies.
As mentioned, in healthy patients, anti-β-AR antibodies are usually hardly or not at ail present or detectable. Accordingly, an "increased titer of anti-β-AR antibodies" in accordance with the present invention preferably refers to any occurrence of anti-β- AR antibodies, i.e. any occurrence of a detectable amount of aπti-β-AR antibodies.
A suitable "sample" in accordance with the present invention includes, but is not limited to, (a) biological or medical sampie(s), like, e.g. (a) sampie(s) comprising cell(s) or tissue(s). For example, such (a) sample(s) may comprise(s) biological material of biopsies. The meaning of "biopsies" is known in the art. For instance, biopsies comprise cell(s) or tissue(s) taken, e. g. by the attending physician, from a patient/subject as described herein. Exemplarify, but not limiting, the biological or medical sample to be analysed in context of the present invention is or is derived from blood, plasma, white blood cells, urine, semen, sputum, cerebrospinal fluid, lymph or lymphatic tissues or cells, muscle cells, heart cells, cells from veins or arteries, nerve cells, cells from spinal cord, brain cells, liver cells, kidney cells, cells from the intestinal tract, cells from the testis, ceils from the urogenital tract, colon cells, skin, bone, bone marrow, placenta, amniotic fluid, hair, hair and/or follicles, stem celis (embryonic, neuronal, and/or others) or primary or immortalized cell lines (lymphocytes, macrophages, or cell lines). Preferred "samples" in accordance with the present invention are those derived from blood or plasma. The biological or medical sample as defined herein may also be or be derived from biopsies, for example biopsies derived from heart tissue, veins or arteries.
In a further aspect, the present invention relates to a diagnostic kit, for example a diagnostic kit for the detection of antibodies against a β-AR, comprising the cyclic peptide, composition or diagnostic agent of the invention.
The kit in accordance with the present invention comprises at least one of the compounds as disclosed according to the invention, like, for example a cyclic or linear peptide of the present invention, a nucleic acid molecule, vector or host cell of the invention or a composition or diagnostic agent according to the present invention. In one particular embodiment the kit further comprises an instruction leaflet, and/or a buffer for use in the application of the kit, and/or at least one reaction vessel for carrying out the detection reaction for which the kit is or is to be used. In a further embodiment, at least one, some or all of the reagents used in connection with the application of said kit are present as portions useful in carrying out the reaction(s) for which the kit is to be used.
One particular approach for using the compounds according to the present invention as a diagnostic and in a diagnostic method, respectively, is a three-step screening procedure. For example, this method comprises performing an ELISA with the cyclic peptides according to the present invention as well as determining immunofluorescence and determining cAMP responses in cells expressing native human β~AR. It is to be acknowledged that each and any of the aforementioned steps can as such be preformed for the detection of said antibodies using the cyclic peptides according to the present invention. A large number of patients, for example heart failure patients, may thus be screened for functionally active anti-βi-AR antibodies. In connection with such (but also with other herein disclosed) diagnostic methods, the definition of functionally active anti-βrAR antibodies is preferably based on their effects on receptor-mediated signalling, that is, their effects on cellular cAMP levels and on the activity of the cAMP-dependent protein kinase (PKA). Cyclic AMP is a universal second messenger of many G protein-coupled receptors including the β- AR family. It exerts its effects via PKA, cAMP-gated ion channels, phosphodiesterases, and exchange proteins directly activated by cAMP, known as Epad and 2. The prior art describes several fluorescence methods for measuring cAMP in intact cells which can all be used in connection with the diagnostic method of the present invention. Fluorescence resonance energy transfer (pRj=f) between green fluorescent protein (GFP) variants fused to the regulatory and catalytic subunits of PKA has been described to study the spatio-temporal dynamics of cAMP in neurons (Hempel CM, Vincent P, Adams SR, Tsien RY, Selverston Al. Nature. 1996; 384:113- 114) or cardiac myocytes. (Zaccolo M, Pozzan T., Science. 2002; 295:1711-1715).
More recently, single chain fluorescence indicators have been described in the art which are characterized by having an enhanced cyan (CFP) or yellow fluorescent protein (YFP) directly fused to the cAMP-binding domain of Epac-proteins, which allowed to achieve a higher sensitivity and better temporal resolution of the cAMP measurements. Such system is, among others described in WO 2005/052186. Such system can be used in connection with any diagnostic procedure using the cyclic peptides or other corresponding compounds according to the present invention. Also such system can be used for, however is not limited thereto, analyzing the prevalence of functionally active anti-βi-AR antibodies. Preferably such diagnostic method is applied to a cohort of previously antibody-typed DCM patients or any individual to be assessed insofar or any individuai suspected of suffering from any of the diseases described herein or being at risk to suffer therefrom, in a further step of the diagnostic method and screening method, the abiiity of β-blockers to inhibit anti- βrAR antibodies-induced receptor activation may be assessed and determined, respectively.
The afore described assay which is a FRET-based method as described in WO 2005/052186 making use of the peptides according to the present invention is advantageous insofar as it is simpier, less time consuming, and at the same time discloses or identifies all DCM patients previously considered anti-βrECn antibody- positive. This embodiment of a FRET based method of diagnosing making use of one or several of the peptides according to the present invention is based on detecting antibody-induced increases in cAMP.
Taken together, screening by Epac-FRET, as, for example, described in WO 2005/052186, appears to represent a very sensitive single step approach, allowing to detect activating antibodies directed against the human βi-AR. Therefore, the present invention is also related to the use of one or several of the peptides according to the present invention for use in an Epac-FRET assay. More preferably such Epac-FRET assay is used for diagnosis, even more preferably for the diagnosis of patients suffering from or suspected of suffering from any of the disease described herein.
In view of the above, it is a particularly preferred use or apply the FRET technology, particularly a FRET-based detection system, in accordance with this invention.
In a further aspect, the present invention relates to a method for detecting of antibodies against a β-AR (for example in a sample as defined herein) comprising the step of contacting the cyclic peptide of the invention with said antibodies to be detected.
!n a further aspect, the present invention relates to the cyclic peptide, composition or diagnostic agent as disclosed herein for detecting (for example in a sample as defined herein) antibodies against a β-AR.
The above method for detecting of antibodies or cyclic peptide, composition or diagnostic agent is particularly useful to be employed in context of the diagnostic applications as described and provided in context of this invention.
Throughout the present application, the following abbreviations shall have the following meanings: Ab or ab: antibody, Abs or abs: antibodies, AR: adrenergic receptor, EC extra cellular domain of a β-AR, ECn extra cellular domain Il of a β-AR and AA amino acid.
The present invention is further described by reference to the following non-ϋmiting figures and examples. The Figures show:
Figure 1 is a diagram depicting the scheme of the mutated βi-ECn-25AA or 18AA- cycio-peptides (black rings with the original Cys-residues (white bails) or the Ser mutated Cysteines (biack balls; Cys/Ser or Ser/Cys, respectively), together with the amino-acids involved in forming the primary ring structure after head-to-tail closure (closure site either AIa-DGIu5 or Pro-Lys).
For the synthesis of cyclic βi-ECn-18AA (or βrECn-25AA) peptides on the solid phase, Fmoc-Glu-ODmab or another Fmoc amino acid having a side chain protecting group which can be selectively cleaved off in an orthogonal manner, is incorporated at the C-terminal end of the linear peptide. The cleaving off of the cyclic peptide from the synthesis resin generates a peptide amide (in the case of D-G!u→Gln) and the removal of the protective groups of the side chain is done by treating the resin with triftuoro acetate acid/triisopropyisilane/ethandithiole/water for several hours.
Figure 2 is a diagram depicting the blocking capacity of β-ι-ECn-18AA cyclopeptide mutants having a D-GSu ring closure on βr receptor-mediated signalling (functional cAMP-assay) using an approach by fluorescence resonance energy transfer (FRET). The effect of the preincubation (12h, 4°C, rotating incubator) of anti-β1-ECII IgG antibodies of a representative rat (serum) (Fig. 4, rat number 4) with βi-ECn-18AA- cyclopeptide mutants (Cys/Ser mutation, dark blue (3); Ser/Cys mutation, tight blue (4)) was compared with the effect of a 3 Cys-containing 25AA Cys/Cys cyclopeptide (red (2)) or the result obtained with anti-βi-ECn IgG antibodies in the absence of blocking peptides (black (1)). The y~axis represents the normalized YFP/CFP-ratio of the registered FRET emission signals, the x-axis corresponds to the registration time given in seconds (s).
Figure 3 is a diagram composed of two major (upper and lower) panels resuming the blocking effect of both 25AA- and 18AA-cyclopeptide mutants having a GIn closure site, as well as 18AA cyclopeptide mutants having a D-GIu closure site after preincubation (12h, 4°C, rotating incubation) with sera isolated from 69 different immunized antibody-positive rats in an ELISA-competition assay using the 3 Cys- containing Smear 25AA Cys/Cys-peptide as an antigen. The upper panel depicts rat sera preferentially reacting with the Cys/Ser mutated cyclopeptides (typei reaction, n=64), separated in cyc25AA(Gln)-peptides (left) and cyd 8AA(GIn and D-GIu)- peptides (right). The lower pane! depicts the rat sera reacting with both the Cys/Ser and the Ser/Cys mutated cyclopeptides (type2 reaction, n=5), again separated in the results obtained with cyc25AA(Gln)-peptides (left) and cyd 8AA(GIn and D-GIu)- peptides (right).
The first three columns on the left side within the two panels represent the results obtained with the (non mutated) 3 Cys-containing 25AA (Gln-)cyclopeptide (black columns) and the mutant βrEΞCn-25AA (Gln-)cyclopeptides (Cys/Ser mutation, white columns; Ser/Cys mutation, horizontally hatched columns).
The five columns on the right side within the two panels represent the results obtained with the (non-mutated) 3 Cys-containing 18AA (Gln-)cyclopeptide (black columns) compared with the different 2 Cys-containing mutant 18AA cyclopeptides (18AA Cys/Ser mutant with a GIn closure site, white columns; 18AA Cys/Ser mutant with a D-GIu closure site, diagonally left hatched columns; 18AA Ser/Cys mutant with a GIn closure site, diagonally right hatched columns; 18AA Ser/Cys mutant with a D- Giu closure site, vertically hatched columns).
The error bars represent the standard error of the mean (± SEM). The y-axis represents the blocking efficiency of the various peptides used given in % of blocked versus non-blocked ELISA-reactivity of the sera.
Figure 4 is a diagram resuming the dose-dependent (x-axis, abscissa: -fold molar excess of specific peptides) blocking capacity of various linear and cyclic betai -ECiI- peptides given in % of the unblocked antibody-titer (y-axis, ordinate), including 25AA Cys/Cys linear peptides (black squares), 25AA Cys/Ser cyclopeptide mutants (white squares), 18AA Cys/Cys cyclo-peptides (black diamonds), 18AA Cys/Ser cyclopeptide mutants (white diamonds), and 18AA Cys/Ser linear peptide mutants (vertically hatched diamonds) in an ELISA competition assay performed with sera from n=6 randomly choosen immunized antibody-positive rats. All sera were most efficiently blocked by the beta1-ECII-18AA Cys/Ser mutant cyclopeptide followed by the non-mutant 18AA Cys/Cys cylopeptide and the 25AA Cys/Ser mutant cyclopeptide. Al! cyclopeptides were largely superior to their linear counterparts (with or without mutation) in terms of their antibody blocking capacities (P<0.005).
Figure 5 is a diagram resuming the in vivo blocking capacity of in total five (prophylatic) applications of various linear and cyclic betal-ECII-peptides, started 3 months after the first immunization (and two subsequent beta1-ECH/GST-antigen- boosts, corresponding to a prevention protocol). Serum-titers of the betai -receptor antibodies were determined before and 18-2Oh after each peptide injection (abscissa, time in months) and are given in % of the corresponding antibody-titers of immunized untreated rats (y-axis, ordinate). The injected peptides were: 25AA Cys/Cys linear peptide (black squares), 25AA Cys/Cys cyclopeptide (white squares), 18AA Cys/Cys cyclopeptide (black diamond), 18AA Cys/Ser cyclopeptide mutant (white diamonds), and the 18AA Cys/Ser linear peptide mutant (vertically hatched diamonds). Also in vivo, the efficiency of the cyclic peptides was largely superior to their linear counterparts. The highest efficiency in terms of antibody-neutralization was achieved with 1.0 mg/kg body weight (Bw) of non mutant 25AA Cys/Cys or 18AA Cys/Cys- cyclo-peptides (87.7±2% or 89.9±3% decrease after 5 cyclopeptide injections, compared with untreated immunized animals; both P< 0.005), followed by the 18AA Cys/Ser mutant cyclopeptide (54.5+2% decrease after 5 cyclopeptide injections; P<0.05), whereas linear 25AA Cys/Cys peptides or linear 18AA Cys/Ser mutants at a same concentration had no significant blocking effects (25.8±3% or 4.5±11% decrease after 5 injections, P=O.16 or P=O.8, respectively). Black circles indicate antibody-titers of untreated regularly (every 4 weeks) immunized animals serving as reference, set at 100%.
Figure 6 is a diagram resuming the tn-vivo blocking effect of both 25AA and 18AA cyclo-peptide mutants with a GIn closure site, determined after the first intravenous (i.v.) injection of 1.0 mg/kg body weight (Bw) into immunized antibody-positive rats. Sera were drawn 18-20 hours after i.v. injection of 1.0 or 0.25 mg/kg Bw of the indicated peptides and assayed for reactivity by ELISA using the 3 Cys-containing linear 25AA Cys/Cys-peptide as an antigen.
The first row within the panel represents the group of rats (n=5) treated with the mutant 2 Cys-containing 25AA Cys/Ser (Gln)-cyclopeptide (schematically depicted on the top of the first row, hatched circles), the second and third row represent groups of rats treated with the mutant 18AA Cys/Ser (Gln-)cyclopeptide at two different concentrations {n=40.1 mg/kg Bw; n=9, 0.25 mg/kg Bw, scheme of the cyclic peptide depicted on the top of the second row, white circles), the fourth row represents n=4 animals treated with the mutant 18AA Ser/Cys (Gln-)cyclopeptide (1 mg/kg Bw, scheme of the cyclic peptide depicted on the top of the fourth row, black diamonds), and the fifth row represents the results obtained with i.v. injected (mutant) 2 Cys- containing linear 18AA Cys/Ser (Gln-)peptides (scheme of the linear peptide depicted on the top of the fifth row, black circles).
The bars and numbers (in boxes) of each row represent the mean values of the blocking capacity of the respectively indicated peptide given in % of the ELISA- immunoreactivity of the sera before and 18-20 hours after i.v. peptide injection (y- axis).
Figure 7 A is a diagram resuming the in vivo blocking effect of both 25AA and 18AA cyclopeptide mutants with a GIn closure site, determined after a total of ten intravenous (i.v.) injections of 1.0 mg/kg body weight (Bw) of the indicated peptides into immunized antibody-positive rats. Sera were drawn before and 18-20 hours after i.v. injection of the various peptides every 4 weeks (abscissa: time in months of treatment) and assayed for reactivity by ELISA using the 3 Cys-containing linear 25AA Cys/Cys-peptide as an antigen.
The graph depicts the relative decrease (or increase) in specific anti-beta 1 -receptor antibody-titers in sera from antibody-positive immunized rats after injection of the indicated peptides and shows the respective mean value of the blocking capacity of the peptide given in % of the initial ELISA-immunoreactivity before starting treatment (y-axis, ordinate). The symbols indicate: black circles, untreated regularly (every 4 weeks) immunized animals (n=9); white diamonds, 18AA Cys/Ser mutant (Gln-)cyclopeptide (1.0 mg/kg Bw, n=20); black squares, 25AA Cys/Ser mutant (Gln-)cyclopeptide (1.0 mg/kg Bw, n=5).
Figure TB is a diagram resuming the in vivo blocking effect of various concentrations of 18AA cyclopeptide mutants with a Gin closure site, determined after a total of ten intravenous (i.v.) injections of 0.25, 1.0, 2.0, and 4.0 mg/kg body weight (Bw) into immunized antibody-positive rats, irrespective of the cyclopeptide "responder-state" of individual animals. Sera were drawn before and 18-20 hours after i.v, injection of the various peptides every 4 weeks (abscissa: time in months), and assayed for reactivity by ELISA using the 3 Cys-containing linear 25AA Cys/Cys-peptide as an antigen.
The graph depicts the relative decrease (or increase) in specific anti-beta1 -receptor antibody-titers in sera from antibody-positive immunized rats after injection of the indicated peptides and shows the respective mean values of the blocking capacity of the peptides given in % of the initial EUSA-immunoreactivity before starting treatment (y-axis, ordinate). The symbols indicate: black circles, untreated regularly (every 4 weeks) immunized animals (n=9); white circles, 18AA Cys/Ser mutant (Gln-)cyclopeptide (0.25 mg/kg Bw, n=4); white diamonds, 18AA Cys/Ser mutant (Gln-)cyclopeptide (1.0 mg/kg Bw, n=20); vertically hatched diamonds, 18AA Cys/Ser mutant (Gln-)cyclopeptide (2.0 mg/kg Bw, n=5); black diamonds, 18AA Cys/Ser mutant (Gln-)cyclopeptide (4.0 mg/kg Bw, n=9).
Figure 7C is a diagram resuming the in vivo blocking effect of various concentrations of 18AA cyclopeptide mutants with a GIn closure site, determined after a total of ten intravenous (i.v.) injections of 0.25, 1.0, 2.0, and 4.0 mg/kg body weight (Bw) into immunized antibody-positive rats, respecting only cycfopeptide-sensitive "responders", defined as animals having , after 7 cyclopeptide-injections, a maximum remaining receptor anti-body level equal or inferior to 80% of the respective titer at start of therapy (compare the curves between Fig. 7c and Fig. 7b, the latter representing the naturally occuring inhomogenous response of unselected animals). Sera were drawn as described above and assayed for reactivity by ELlSA using the 3 Cys-containing linear 25AA Cys/Cys-peptide as an antigen.
The graph depicts the relative decrease in specific anti-beta 1 -receptor antibody-titers in sera from antibody-positive immunized responders after injection of the indicated peptides giving the blocking capacity in % of the initial ELISA-immunoreactivity (y- axis, ordinate). The symbols indicate (number of responders in bold): black circles, untreated regularly (every 4 weeks) immunized animals (n=9); white circles, 18AA Cys/Ser mutant (Gln-)cyclopeptide (0.25 mg/kg Bw, n=3/4); white diamonds, 18AA Cys/Ser mutant (Gln-)cyciopeptide (1.0 mg/kg Bw, n=16/20); vertically hatched diamonds, 18AA Cys/Ser mutant (Gln-)cyclopeptide (2.0 mg/kg Bw, n=2/5); black diamonds, 18AA Cys/Ser mutant (Gln-)cyciopeptide (4.0 mg/kg Bw, n=6/9).
Figure 8A is a diagram showing the time course (month 0 to 20) of the internal end- systolic and end-diastolic left ventricuSar diameters (LVES, LVED) of GST/βi-ECn- immunized un-treated (black circles) versus GSTVβrECII-immunized animals treated with the indicated various cyciopeptides (see legend) as determined by echocardiography (echocardiographic system: Visual Sonics, Vevo 770 (version V2.2.3), equipped with a 15-17.5 MHz transducer), whereby LVES/LVED is left ventricular end-systolic diameter/left ventricular end~diastolic diameter. The symbols indicate: white circles, untreated 0.9% NaCl-injected non immunized control animals (n=10); black circles, untreated regularly (every 4 weeks) immunized animals (n=9); white diamonds, 18AA Cys/Ser mutant (Gln-)cyclopeptide (1.0 mg/kg Bw, n=20); black squares, 25AA Cys/Ser mutant (Gin-)cyclopeptide (1.0 mg/kg Bw, n=5); white squares, 18AA Cys/Ser mutant linear (Gln-)peptide (1.0 mg/kg Bw, n=5).
Figure 8B is a similar diagram showing the time course (month 0 to 20) of the internal end-systolic and end-diastolic left ventricular diameters (LVES, LVED) of GST/βi-ECii-immunized untreated (black circles) versus GST/βrECII-imrnunized animals, treated with different concentrations of the 18AA Cys/Ser cyclopeptide mutant (see legend). The symbols indicate: white circles, untreated 0.9% NaCI-injected non immunized control animals (n=10); black circles, untreated regularly (every 4 weeks) immunized animals (n=9): white squares, 18AA Cys/Ser mutant (Gln-)cyclopeptide (0.25 mg/kg Bw, n=4); white diamonds, 18AA Cys/Ser mutant (Gln-)cyclopeptide (1.0 mg/kg Bw, n=20); vertically hatched diamonds, 18AA Cys/Ser mutant (Gln-)cyclopeptide (2.0 mg/kg Bw, n-5); black diamonds, 18AA Cys/Ser mutant (Gln-)cyciopeptide (4.0 mg/kg Bw, n=9). Figure 9A is a diagram depicting the time course {month 0 to 20) of the "Cardiac index" (Cl) in ml/min/g (body weight) as determined by echocardiography (echocardiography system see legend to figure 8a.). The symbols indicate: white circles, untreated 0.9% NaCI-injected non immunized control animals (n=10); black circles, untreated regularly (every 4 weeks) immunized animals (n=9); white diamonds, 18AA Cys/Ser mutant (Gln-)cyclopeptide (1.0 mg/kg Bw5 n=20); black squares, 25AA Cys/Ser mutant (Gln-)cyclopeptide (1.0 mg/kg Bw, n=5); white squares, 18AA Cys/Ser mutant linear (Gln-)peptide (1.0 mg/kg Bw, n=5).
Figure 9B is a similar diagram showing the time course (month 0 to 20) of the "Cardiac index" (Cl) in ml/min/g (body weight) as determined by echocardiography (echocardio-graphic system see legend to Figure 17a.)The symbols indicate: white circles, untreated 0.9% NaCI-injected non immunized control animals (n=10); black circles, untreated regularly (every 4 weeks) immunized animals (n=9); white squares, 18AA Cys/Ser mutant (Gln-)cyclopeptide (0.25 mg/kg Bw, n=4); white diamonds, 18AA Cys/Ser mutant (GJn-)cyciopeptide (1.0 mg/kg Bw, n-20); vertically hatched diamonds, 18AA Cys/Ser mutant (Gln-)cyclopeptide (2.0 mg/kg Bw, n=5).
Figures 10A-C, depict hemodynamic parameters obtained in the therapy study after
11 months of treatment.
Figure 10A on the left side depicts the heart frequence (HF) given in beats per minute (=bpm), and on the right side the LV systolic blood pressure (LV press.) given in mmHg;
Figure 10B on the left side depicts the contractility (+ dP/dt) in mmHg/s, and on the right side the relaxation (-dP/dt) in -mmHg/s;
Figure 10C1 shows the left ventricular end-diastolic pressure (LVEDP) as determined by cardiac catheterization in mmHg.
Left and right panels within each Figure separate data obtained with (left panels, constantly 1.0 mg/kg Bw of the different peptides) cyclic 18AA Cys/Ser (diagonally right hatched columns, n=20 animals), linear Cys/Ser mutants (horizontally hatched columns, n=5 animals), and cyclic 25AA Cys/Ser mutants (vertically hatched columns, n=5 animals). Columns in the right panels represent data obtained with various concentrations of the cyc18AA Cys/Ser mutant; with white filled, black dotted columns corresponding to 0.25 mg/kg Bw (n=4 animals), diagonally right hatched columns to 1.0 mg/kg Bw (n=10 animals), diagonally left hatched columns to 2.0 mg/kg Bw (n=5 animals), and black structured columns to 4.0 mg/kg/Bw (n=9 animals). Black and white columns in each panel serve as a reference and correspond to either untreated regularly (every 4 weeks) immunized animals (positive control, black, n=9), or to 0.9% NaCI-injected non immunized control animals (negative control, white, n=10).
In the legends "Betai untreated" means immunized antt-beta1 antibody positive cardiomyopathic not treated animals (n=9, black columns [No.2]), "Controls" means the 0.9% NaCI-injected control group (n=10, white columns [No.1]), "18cyc Cys/Ser." means immunized anti-beta 1 -positive cardiomyopathic animals therapeutically treated with the indicated linear 1SIIn Cys/Ser (n~5 [1.0 mg/kg Bw] [No.4]) or cyclic 18cyc Cys/Ser mutants (n=10 [1.0 mg/kg Bw] [No.3])), or cyclic 25AA Cys/Ser peptide mutants (n=4 [1.0 mg/kg Bw][No.5])) after 9 months of immunization. Panels on the right side depict the effects of different doses of intravenously injected beta1-ECII 18AA Cys/Ser cyclopeptide mutants (n=4 [0.25 mg/kg Bw]; n=20 [1.0 mg/kg Bw], n=5 [2.0 mg/kg Bw]1 and n=9 [4.0mg/kg Bw]1 [No.3-6] respectively). Differences between the groups were assessed by one way ANOVA; n.s. = not significant, *P< 0.05, **P< 0.005.
Figure 11 is a diagram depicting the scheme of mutated cysteine-containing betai- ECII-homologous cyclopeptides (amino-acids (AA)) are represented as white balls with the corresponding AA letter code written in each bail). Cysteine molecules and their substitutes are depicted as black balls. The assumed localization of the disulfide bridge is represented by a bold black line.
Left side: scheme depicting the original sequence of the ECII-loop of the human betai adrenergic receptor; middle: cyclic 22AA ECII-homologous peptide with the glycine mutation at the assumed ring closure site (Position 222). The right panels depict examples of a cyclic 22AA peptide-mutant containing only two cysteines (i.e., position 209 and 215). The upper scheme shows the Cys/Ser mutant, the lower scheme depicts the Cys/ABu mutant of the cysteine at position 216 (Cyclic 22AA beta1-ECII peptide Cys2is→Ser2i6 and Cyclic 22AA beta1-ECll peptide Cys2i6→ABu2i6, respectively). Numbers given indicate the numbering of the amino-acids in the original primary sequence according to Frieile et a/. 1987, PNAS 84, pages 7920-7924.
Figure 12A and B depict the in vitro blocking (-neutralizing) capacity of various cysteine-containing cyclopeptide variants of the second extracellular loop (ECU) of the human betai -adrenergic receptor, determined by testing n=6 individual sera (Fig. 12A) of immunized beta1-EC!l-antibody-positive rats after over-night incubation with the indicated cyclopeptides (12-14h, 4°C) by ELISA. Columns in Fig. 12A represent the receptor-antibody blocking efficiency of the indicated cycio-peptides in % of the antibody-(EL!SA-)signals obtained with unblocked antibody-positive rat sera. Fig. 12B depicts the mean values ± SEM of each of the treated groups of immunized beta1-ECII-antibody positive animals; error bars indicate ± SEM. White columns: cyc18AA Cys/Ser mutant (blocking-efficiency 60.0±8.3%, P=O.0014 when tested for significance against unblocked sera by two-sided Mest); Vertically hatched columns: cyc18AA Cys/Cys (blocking-efficiency 66.1 ±7.0%, P=O.00025). Black columns: cyc22AA Cys/Cys (blocking-efficiency 82.0±5.0%, P=O.000046); diagonally (right) hatched columns: cyc22AA Cys/Ser mutant (blocking-efficiency 74.9±5.0%, P=O.00026); diagonally (left) hatched columns: cyc22AA Cys/Abu mutant (blocking-efficiency 79.4±4.0%, P=O.00014); horizontally hatched columns: cyc25AA Cys/Cys (blocking-efficiency 73.4±5.0%, P=0.00011).
Figure 13A depicts the in vivo blocking (=neutralizing) capacity of two cysteine- containing 18AA or 22AA cyciopeptide-mutants of the second extracellular loop (ECU) of the human beta 1 -adrenergic receptor upon therapeutic injection of the different constructs into rats regularly immunized over 8 months (first = basic immunization followed by 7 antigen-boosts every 4 weeks). The effects of eight subsequent cyclopeptide-injections every 4 weeks are shown. The figure depicts the mean values ± SEM of each of the treated groups of immunized beta 1 -ECI l-antibody positive cardiomyopathic rats (animal number per group is given in the legend). The bar graph shows the mean effect of eight subsequent cyclopeptide-injections, determined 20-22 hours after application of the indicated constructs. The remaining receptor antibody-titers after each injection in % of the antibody-titers at initiation of therapy (month 8) are depicted (columns). Error bars indicate ± SEM. Numbers in columns indicate the number of (subsequent) monthly injection. Black columns: untreated antibody-positive animals (reference-titer after in total 8+ 8 (=16) antigen-boosts (compared to the titer at start of therapy) 87.7±10.7%; n=5, positive control). White columns: cyc18AA Cys/Ser mutant, n=5 animals (antibody-titer remaining after 8 injections in per cent of the titer at start of therapy: 50.3±14.6%, P=O.005 when tested for significance against the antibody-titer of untreated antibody-positive animals by two-sided f-test). Diagonally (right) hatched columns: eye 22AA Cys/Ser mutant, n=5 animals (antibody-titer remaining after 8 injections in per cent of the titer at start of therapy: 20.5±12.9%, P= 0.0001); diagonally (left) hatched columns: eye 22AA Cys/Abu mutant, n=5 animals (antibody- titer remaining after 8 injections in per cent of the titer at start of therapy: 9.8+4.3%. P= 2.0 x 10"8).
Figure 13B depicts the time course of antibody-titers after 11 (22cyc Cys/Abu) or 12 (22cyc Cys/Ser) subsequent cycio-peptide-injections, determined before and 20-22 hours after application of the indicated constructs. Values are given in per cent of increase or decrease in the respective antibody-titers after each cyciopeptide- injection compared with the antibody-titer determined at start of therapy (month 8), Error bars are not indicated in the graph.
Black circles: untreated antibody-positive animals (n= 5, positive control); white squares: cyc18AA Cys/Ser mutant, 12 injections, n= 5 animals; black diamonds' eye 22AA Cys/Ser mutant, 12 injections, n=5 animals; white diamonds: eye 22AA Cys/Abu mutant, 11 injections, n=5 animals.
Figure 14A is a diagram showing the time course (month 0 to 21) of the internal end- systolic and end-diastolic (eft ventricular diameters (LVES, LVED) of GST/beta 1 -EC 11- immunized untreated (black circles) versus GST/beta1-ECIi-immunized animals treated withe the indicated various cyclopeptides (see legend) as determined by 2D- and M-mode echocardiography (echocardiography system: Visual Sonics, Vevo 770 (version V2.2.3), equipped with a 15-17.5 MHz transducer), whereby LVES/LVED is left ventricular end-systolic diameter/left ventricular end-diastolic diameter. Error bars indicate mean ± SEIvI. White circles, untreated 0.9% NaCI-injected non immunized control animals (n=5); black circles, untreated regularly (every 4 weeks) immunized antibody-positive animals (n=5); white squares, cyc18AA Cys/Ser mutant (1.0 mg/kg Bw, n=5); black diamonds, eye 22AA Cys/Ser mutant (Gly-)peptide (1.0 mg/kg Bw, n=5); white diamonds, cyc22AA Cys/ABu mutant (Gly-)peptide (1.0mg/kg Bw, n=5). Asterisk indicate level of significance as derived from repeated measures ANOVA and Bonferroni post hoc testing; * = P<0.05, **= P<0.01 , ***= PO.005.
Figure 14B is a diagram showing the time course (month 8 to 21) of the change in left ventricular fractional shortening, given in % of the values obtained at the initiation of therapy (LVED-LVES/LVED x 100) in GST/beta1-ECI!-immunized untreated rats (black circles) versus animals treated with the indicated different cyclopeptides (see legend) as determined by 2D- and M-mode echocardiography (echocardiographic system: Visual Sonics, Vevo 770 (version V2.2.3), equipped with a 17.5 MHz transducer), whereby LVES/LVED is left ventricular end-systolic diameter/left ventricular end-diastolic diameter.
Differences between untreated rats and animals receiving cyc22AA beta1-ECII mutant cyclopeptides were highly significant, with 22AA Cys/Abu mutants being even more efficient than 22AA Cys/Ser mutants regarding reversal of cardiac dys-fu notion (after 4, 8 or 12 months of treatment; betai untreated versus 22Cys/Abu p<0.002 or p<0.0005; betai versus 22Cys/Ser p<0.004 or p<0.0007, respectiviely) .
Figure 14C is a diagram depicting the time course (month 0 to 21) of the "Cardiac index" (Cl) in ml/min/g (body weight) as determined by 2D- and Doppler-echocardio- graphy (echocardiographic system see above).
White circles, untreated 0.9% NaCi-injected non immunized control animals (n=5); black circles, untreated regularly (every 4 weeks) immunized antibody-positive animals (n=6); white squares, cyc18AA Cys/Ser mutant (1.0 mg/kg Bw1 n=5); black diamonds, cyc22AA Cys/Ser mutant (Gly-)peptide (1.0 mg/kg Bw, n=5); white diamonds, eye 22AA Cys/ABu mutant (Gly-)peptide (1.0 mg/kg Bw, n=5). Asterisk indicate level of significance as derived from repeated measures ANOVA and Bonferroni post hoc testing; *P< 0.05, **P< 0.01 , ***P< 0.005. Figures 14D-F, depict hemodynamic parameters of 0.9%NaCI-injected control animals (white columns) versus GST/beta 1 -EC I l-immunized untreated (black columns) versus GST/beta1-ECII-immunized animals injected with the indicated different cyclopeptides (see legend) after 12 months of treatment.
Figure 14D on the left side depicts the heart frequence (HF) given in beats per minute (=bpm), and on the right side the LV systolic blood pressure (LV press.) given in mmHg;
Figure 14E on the left side depicts the contractility (+ dP/dt) in mmHg/s, and on the right side the relaxation (-dP/dt) in -mmHg/s;
Figure 14F shows the left ventricular end-diastolic pressure (LVEDP) as determined by cardiac catheterization in mmHg.
Left and right panels in Figure 14D and 14E separate data obtained with (left panels, constantly 1.0 mg/kg Bw of the different peptides) cyclic 18AA Cys/Ser (4, diagonally right hatched columns, n=5 animals), cyclic 22AA Cys/Ser mutants (3, vertically hatched columns, n=5 animals), and cyclic 22AA Cys/ABu mutants (5, horizontally hatched columns, n=5 animals). Black and white columns in each panel serve as a reference and correspond to either untreated regularly (every 4 weeks) immunized animals (positive control, black, n=6), or to 0.9% NaCl-injected non immunized control animals (negative control, white, n=5). Differences between the groups were assessed by one way ANOVA; *P< 0.05, **P< 0.005.
Figure 14G shows two panels (a and b) with different laboratory parameters determined in the serum of animals after 12 months of treatment. "Betai untreated" and "Controls" in both panels means immunized anti-beta1 antibody positive cardio- myopathic not treated animals (n=6, black columns, positive control), and 0.9% NaCt- injected controls (n=5, white columns, negative control), respectively. In addition, the laboratory parameters of immunized anti-beta 1 -positive cardiomyopathic animals therapeutically treated with cyclic 18AA Cys/Ser (n=5 [1.0 mg/kg Bw], 4, diagonally hatched columns), or cyclic 22AA Cys/Ser mutants (n=5 [1.0 mg/kg Bw], 3, vertically hatched columns), or cyclic 22AA Cys/Abu mutants (n=5 [1.0 mg/kg Bw], 5, horizontally hatched columns) are given. In panel (a) "Crea" means creatinine, "GOT" means glutamic oxaloacetic transaminase, "GPT" means glutamic pyruvate transaminase, and "LDH" means lactate dehydrogenase. In panel (b) "gammaGT" means gamma glutamyl transpeptidase and "T-BiIi" means total bilirubin. Differences between the groups were assessed by one way ANOVA; P< 0.05, **P< 0.005.
Figure 14H depicts macro-anatomic parameters (organ wet weights) of immunized anti-beta 1 -positive cardiomyopathic rats therapeutically treated over 12 months with cyclic 18AA Cys/Ser mutants (n=5 [1.0 mg/kg Bw]1 4, diagonally hatched columns), or cyclic 22AA Cys/Ser mutants (n=5 [1.0 mg/kg Bw], 3, vertically hatched columns), or cyclic 22AA Cys/Abu mutants (n=5 [1.0 mg/kg Bw], 5, horizontally hatched columns). "Betai untreated" and "Controls" means immunized anti-beta1 antibody positive cardiomyopathic not treated animals (n=6, black columns, positive control), and 0.9% NaCI-injected controls (n=5, white columns, negative control), respectively. The relative wet weights of the indicated organs (from the left to the right: heart, spleen, right kidney, left kidney, lung, and liver (x10)) are given in g/kg body weight. Kidney R means right and Kidney L means left. Differences between the groups were assessed by one way ANOVA; n.s. = not significant, *P< 0.05, **P< 0.005.
Figure 15A is a scheme depicting the departing sequences of the 22AA Cys/Abu- cycio-peptϊde mutant of the present invention. The corresponding original 21 + 1AA of the second extracelluier loop of the human betai- (AA200 to AA221 ) and beta2-adre- nergic receptor (AA175 to AA 196) are depicted. 4.5 angstrom units are the distance at the basis of the native ECIMoop between AA 200 and AA 220 (betai) or AA175 and AA 190 (beta2). In case of the betai 22AA cyclopeptide this gap was filled in by the smallest naturally occurring AA glycine.
The sequences underneath depict the nine different cyclopeptides generated to identify further AA necessary for antibody-recognition and neutralization, derived from the beta2-ECII sequence (which was not able to block anti-beta1-ECIi antibodies) in subsequently performed blocking- and binding-competition assays using either 25AA beta1-ECII-peptides or (N- and C-termina!) biotinylated linear 16AA beta1-ECII-peptides, respectively (Ala-scan 22AA Cys/Abu-cyclopeptides).
Figure 15B is a diagram depicting the blocking-results of different betal-ECM-specific polyclonal rat (n=20; panel 1) or rabbit (n=1 ; panel 2) antibodies and previously generated monoclonal mouse (n=1; panel 3) or monoclonal rat (n=1 ; panel 4) anti- bodies with cyciopeptide mutants used for the alanine-scan of the 22AA Cys/Abu- cyclopeptide mutant of the present invention.
Nine different site-specific Ala-mutated cyclopeptides (Fig. 7a) were incubated overnight with sera from n=20 different anti-beta1-ECIl positive rats (panel 1 ) or a previously generated monoclonal rat anti-beta1-ECII (panel 2), or a polyclonal rabbit anti-beta1-ECII (panel 3), or a mouse monocional anti-beta 1 -ECIS (panel 4) to assess their respective antibody-recognition and neutralizing capacity by ELISA using the 3 Cys-containing linear 25AA beta1-ECIJ-peptide as an antigen. 22AbuO represents the inventtonal product. The sequences of the Ala-mutants 22Abu1 to 22Abu9 used for all experiments (panels 1-4) are listed underneath. In addition the sequence of the corresponding cyclic 22AA beta2~ECII peptide is indicated, which in all experiments exhibited no antibody-blocking effect, as exemplarily shown in panel 1.
ECI! beta 1 -22AA Cys-Cys/Abu cyciopeptide: 22AbuO RAESDEARRCYNDPKC Abu DFVTG
ECU beta1-22AA Cys-Cys/Abu cyclic alanine-substituted peptides:
22Abu 1 RAASDEARRCYN DPKC Abu DFVTG
22Abu2 RAEADEARRCYNDPKC Abu DFVTG
22Abu3. RAESAEARRCYNDPKC Abu DFVTG
22Abu4 RAESDEAARCYNDPKC Abu DFVTG
22Abu5 RAESDEARACYNDPKC Abu DFVTG
22Abu6 RAESDEARRCYADPKC Abu DFVTG
22Abu7 RAESDEARRCYNAPKC Abu DFVTG
22Abu8 RAESDEARRCYNDAKC Abu DFVTG
22Abu9 RAESDEARRCYNDPAC Abu DFVTG
ECU beta2-22AA Cys-Cys/Abu cyciopeptide: Beta-2 RATHQEA I N CYANETC Abu DFFTG
Figure 15C is a diagram depicting the results of binding-competition assays using 22AA Cys/Ser versus 22AA Cys/Abu cyclopeptides in order to block binding of different rat anti-beta1-ECIi (e.g. monoclonal or polyclonal) to biotinylated linear 16AA beta1-ECII-peptides. The respective EC 50 of the indicated cyclopeptides are given in the tables underneath each figure.
Panel 1 shows the (one site) binding competition curves obtained with a monoclonal rat anti-beta1-ECII antibody.
Panel 2 shows the (one site) binding competition curves obtained with a polyclonal rat anti-beta 1 -ECU antibody (K12R1), representative for 3 different rat sera tested. Panel 3 shows the (one site versus two site) binding competition curves obtained with a polyclonal rabbit anti~beta1-ECil antibody, suggesting the presence of two different antibody-entities with different 18Cys/Ser cyclopeptide affinities in the probe. Panel 4 shows the (one site) competition curves obtained for different site-specific ala-mutated 22AA-beta1 -cyclopeptides of the present invention, whereby 22AbuO represents the inventional product. The data shown in pane! 4 are representative for 3 different polyclonal rat sera tested.
Figure 16 shows the high pressure liquid chromatography (HPLC) elution profile of the two cysteine-containing mutant cyc22AA Cys/L-a!pha butyric acid (Abu) of the present invention, a cyclic peptide with a GIy closure site, HPLC was carried out in a Hewlett Packard Series 1050 analytical HPLC-system (Agilent Technologies Germany GmbH, Bδblingen) equipped with a dual wavelength UV absorbance detector; absorbance was read at 216 nm. After peptide-synthesis and cyciization, the samples were dissolved in H2CVO.1 % tri-fluoroacsd (TFA) and loaded on a analytic HPLC-column (Waters GmbH, Eschborn) XBridge BEH130, C18, 3,5 μm (column length 50 mm, lumen 4.6 mm) with a flow of 2 ml/min; then a separation-gradient from 0% to 75% acetonitril (ACN) in the presence of 0.1 % TFA was run over 5 minutes. The mutant cyclic 22AA Cys/Abu peptide containing only two cysteines (connected by a second reinforced disulfide bridge) gave sharp single elution peak appearing at 3.81 minutes.
Figure 17 shows the characterization of the cyclic ECH-22AA Cys/Abu mutant peptide (with a GIy closure site) by mass spectroscopy (MALDI-TOF). The panel depicts MALDi-tracings of the cyc22AA Cys/Abu-mutant (2499.34 m/z). The ordinate of each graph shows measured signal intensities ("a.u." means arbitrary units), the abscissa indicates the molecular mass (given in m/z). The MALDI-analysis was carried out using a reflex ll-mass spectroscope (Bruker Daltonic GmbH, Bremen), equipped with a Scout-26 sample carrier. In each case the simply protonated molecule was analyzed at 2200 m/z.
Figure 18 shows a panel demonstrating a high pressure liquid chromatography (HPLC) elution profile of the 3 cysteine-containing construct cyc22AA Cys/Cys. HPLC was carried out as described in context of Fig. 16, above. The fractions containing the cyclic beta1-ECH~22AA Cys/Cys peptides with three freely accessible cysteine molecules exhibit the typical mountain-like elution pattern indicating the presence of cystein-racemates (elution between 2.6 and 3.8 minutes).
Figure 19 is a diagram depicting the blocking capacity of different site-specific Ala- mutated 22AA Cys/Abu-cyclopeptides (Fig. 15A) on β-i-receptor-mediated signalling (functional cAMP-assay) using an approach by fluorescence resonance energy transfer (FRET) as described in Fig.2. The effect of preincubation (12h, 4°C, rotating incubator) of human anti- βi-ECI! IgG antibodies isolated from a representative 28 years-old female DCM patient with different 22Abu-mutants (as defined in Fig.15A) is shown. Numbers (n) indicated under columns correspond to the number of independent experiments performed under same conditions. Black column: patient IgG, unblocked: 18±3% pRET activity;
Diagonally hatched column: patient IgG (inefficiently) blocked with the 22Abu8 cyclic peptide (17±2% FRET activity).
Vertically hatched column: patient IgG (efficiently) blocked with the 22Abu1 cyclic peptide (8±5% FRET activity, PO.005 versus unblocked).
Horizontally hatched column: patient IgG (efficiently) blocked with the inventional product 22AbuO cyclic peptide (3+3% FRET activity, P<0.001 versus unblocked).
ECU beta1-22AA Cys-Cys/Abu cyclopeptide employed: 22AbuO RAESDEARRCYNDPKC Abu DFVTG
ECI! beta 1 -22AA Cys-Cys/Abu cyclic alanine-substituted peptides employed: 22Abu 1 RAASDEARRCYNDPKC Abu DFVTG 22Abu8 RAESDEARRCYNDAKC Abu DF\ATG.
Figure 20 Is a diagram demonstrating the half-life of ECll-peptide-mutants in whole blood. Betal -ECll-peptide-mutants (cyc18AA Cys/Ser) with a cyclic structure (Le. the cyclic peptides as described herein) have a highly significant longer half-life in whole biood drawn from both untreated (control) rats or healthy human (control) subjects than their linear counterparts (linear: 5.5h; cyclic: 64.5). The data were obtained after incubation of linear versus cyclic peptides with human or rat whole biood in the presence of heparine (24 h, 4°C, rotating incubator) and monitoring the amount of intact peptide at the indicated time points. The amount of remaining intact peptides was determined by competition ELISA with linear βi-ECl!-25AACys/Cys peptides at 2min, 10min, 30min, 1 , 2, 4, 8, 16 and 22h. Black diamonds: cyclic 18AACys/Ser peptides White diamonds: linear 18AACys/Ser peptides.
Figure 21A is a diagram resuming the in vivo blocking effect of 22AA Cys/ABu cyciopeptide mutants, determined after nine intravenous (i.v.) injections of 1.0 mg/kg body weight (Bw) of the cyclopeptides into immunized antibody-positive rats. Sera were drawn before and 18-20 hours after i.v. injection of the various peptides every 4 weeks (abscissa: time in months of treatment) and assayed for reactivity by ELISA using the 3 Cys-containing linear 25AA Cys/Cys-peptide as an antigen. The graph depicts the relative decrease in specific anti- βi-ECIl antibody-titers in sera from antibody-positive immunized rats after injection of the indicated peptides and shows the respective mean value of the blocking capacity of the peptide given in % of the initial ELISA-immunoreactivity before starting treatment (y-axis, ordinate). The symbols indicate: black circles, untreated regularly (every 4 weeks) immunized animals (n=5); white diamonds, 22AA Cys/ABu mutant (1.0 mg/kg Bw, n=5). Error bars are not indicated in the graph.
Figure 21 B is a diagram resuming the in vivo blocking effect of 22AA Cys/ABu cyciopeptide mutants (1.0 mg/kg body weight (Bw)), determined after nine intravenous (i.v.) (3/3/3) or eight triple injections (2/3/3) - one injection per week for three subsequent weeks - every 3 months into immunized antibody-positive rats, respectively. Sera were drawn before and 18-20 hours after i.v. injection of the various peptides every 4 weeks (abscissa: time in months of treatment) and assayed for reactivity by ELlSA using the 3 Cys-containing linear 25AA Cys/Cys-peptide as an antigen.
The graph depicts the relative decrease in specific anti- βi-ECII antibody-titers in sera from antibody-positive immunized rats after injection of the indicated peptides at the indicated time points (white arrows) and shows the respective mean value of the blocking capacity of the respective peptides given in % of the initial ELISA- immunoreactivity before starting treatment (y-axis, ordinate).
The symbols indicate: black circles, untreated regularly (every 4 weeks) immunized animals (n=5); black diamonds, 22AA Cys/ABu mutant (1.0 mg/kg Bw, n=5). Error bars are not indicated in the graph.
Figure 22A is a diagram showing the time course (month 0 to 15) of the relative internal end-diastoiic left ventricular diameter (LVED) of GST/βrECil-immunized untreated (black circles) versus GST/β1-EC!i-immunized animals treated with the indicated cyclopeptides/protocols (see legend) as determined by echocardiography (echocardiographic system: Visual Sonics, Vevo 770 (version V2.2.3), equipped with a 17.5 MHz transducer), whereby LVED is left ventricular end-diastolic diameter given in % of the respective values at initiation of treatment (month 9). The symbols indicate: black circles, untreated regularly (every 4 weeks) immunized animals (n=9); white diamonds, 22AA Cys/ABu mutant (monthly injections with 1.0 mg/kg Bw, n=5); black diamonds 22AA Cys/ABu mutant (3~monthly triple-injections of 1.0 mg/kg Bw, n=5). Error bars are not indicated in the graph.
Figure 22B is a similar diagram showing the time course (month 0 to 15) of the relative internal end-systolic left ventricular diameter (LVED) of GST/β1 -ECII- immunized untreated (black circles) versus GST/β1-ECIl-immunized animals treated with the indicated cyciopeptides/protocols (see legend) as determined by echocardiography (echocardiographic system described above), whereby LVED is left ventricular end-diastolic diameter given in % of the respective values at initiation of treatment (month 9).
The symbols indicate the same therapy groups as in Fig.22A. Error bars are not indicated in the graph.
Figure 22C is a similar diagram showing the time course (month 0 to 15) of the "Cardiac index'1 (Ci) in given in % of the respective values at initiation of treatment (month 9) of GST/β1-ECil-immunized untreated (black circles) versus GST/β1-ECII- immunized animals treated with the indicated cyclopeptides/protocols (see legend) as determined by echocardiography (echocardiographic system described above). The symbols indicate the same therapy groups as in Fig.22A. Error bars are not indicated in the graph.
The following, non-limiting examples illustrate the invention.
Example 1 : Synthesis of mutant cycfopeptides
Three particular examples of the herein disclosed cyciopeptides which can form only one single individual disulfide bond are composed of 18, 22 or 25 amino acids (AA): ECιι-18AA Cys/Ser mutant (Gln-)cyclopeptide, ECn-22AA Cys/ABu and Cys/Ser mutant (Gfy-)cyclopeptide and EC|}-25AA Cys/Ser mutant (Gln-)cyclopeptide, respectively. The primary sequence is partially homologous to the human sequence of the P1-AR (amino acid positions 204 through 219. 200 through 220 and 200 through 222, respectively). By restricting conformational flexibility through head-to-tail cyclization of the linear peptide followed by a second (single) disulfide-bond stabilizing cyclization procedure, the 18AA, 22 or 25AA cyclopeptide mutant adopts a conformation which more closely mimics that of the epitope as presented on the surface of the native β-1-ECn protein loop. Furthermore, cyclization has been employed as a tool to prolong the duration of action of peptide, since in general cyclic peptides are more stable to proteolysis than their linear counterparts. In detail, the peptide sequence of the Cyclo(K-18-P) Cyclic S-S, Cys/ABu or Cys/Ser mutant is: Cyclo-Ala-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-AButSerJ-Asp-Phe-
VaI-GIn; Cyclization can occur between Cys7 and Cysi3 (disulphide bond) and AIa1 and Glnts {ring closure).
In detail, the peptide sequence of the Cyclo(K-22-P) Cyclic S-S, Cys/Ser mutant is:
Cycϊo-Arg-Ala-Glu-Ser-Asp-Giu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Ser-Asp-
Phe-Val-Thr-Gly; Cyclization can occur between Cysio and Cysie (disuiphide bond) and Argi and Gly22 (ring closure).
In detail, the peptide sequence of the Cycio(K-25-P) Cyclic S-S, Cys/Ser mutant is:
Cyclo-Ala-Arg-Ala-Glu-Ser-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Ser-
Asp-Phe-Val-Thr-Asn-Arg-Gln; Cyciization can occur between Cysn and Cys17
(disulphide bond) and Aiai and GIn25 (ring closure).
The cyclopeptide mutants of the present invention are first synthesized as linear peptides, and are then cyclized covaiently on the backbone by condensation of the C-terminal carboxyl group with the amino group of the N-terminal amino acid. Subsequently, a disulphide bond between cysteine residues 7 and 13 (18mer cyclopeptide), cysteine residues 10 and 16 (22mer cyciopeptide) and cysteine residues 11 and 17 (25mer cyclopeptide) is established.
The linear peptide is assembled by stepwise solid phase peptide synthesis using an Fmoc / tert butyl strategy. Chlorotrityl is used as a starting resin. The first amino acid (Fmoc-Pro-OH) is coupling with DIEA in DMF, the second with PYBOP/ HOBT/ DIEA in DMF and the following amino acids with diisopropylcarboimide, HOBT in DMF. The peptide quality is monitored online by UV detection. Deprotection/coupling (two-fold excess) procedure is described below:
Table 1 : Deprotection / coupling (two-fold excess) procedure
coupling time is determined by Kaiser test For assembly, the following amino acids were used (exempiarily provided for the 18mer cyclic Cys/Ser peptide mutant):
Table 2: amino acids used for F-moc synthesis of the 18mer cyclic Cys/Ser peptide mutant
The fuily protected peptide with reactive N-terminal amino- and C-terminal carboxyi- groups is cleaved from the resin by treatment with hexaffuoroisopropanol/ dichloromethane.
Cyclization is carried out thereafter in solution according to the following protocol: The "head to tai!"-cyclization of the protected peptide is performed with PyBOP/ NaHCO3 in high DMF dilution (10 mmol of linear peptide/1 L of DMF). The cyclization is completed after 3 days. After DMF evaporation, the peptide is washed with 5% NaHCO3, H2O and pure H2O. The reaction mixture is cooled down, and the peptide is deprotected excepting the cysteine groups. Afterwards, the partially protected peptide is isolated by precipitation with methyl t-butyl ether.
The crude peptide is pre-purified by liquid chromatography: Stationary phase: silica C18, 15 μm, 120 A Eluant: H2O acetonitrile + 0.1% TFA
Detection: UV (210 nm)
The disulfide cyclization is performed in H2O (2 mg/mL) with the presence of dimethyl sulfoxyde (3%). The cyclization reaction is completed after 3 days. The peptide is purified by HPLC, using the conditions described above. The fractions with purity greater than 95% are pooled. The peptide is exchanged on an ion exchange resin (Dowex 1X2) and the final solution lyophilized. The peptide content is determined by amino acid analysis (Edman sequencing).
In particular the 22mer cyclic peptide containing a cystein to Abu substitution can, for example, be synthesized as follows:
The peptide is first synthesized as a linear peptide and is then cyclized on resin covalentSy on the backbone by condensation of the C4erminal carboxy! group with the amino group of the N-terminai amino acid.
The linear peptide is assembled by stepwise solid phase peptide synthesis on a continouus flow peptide synthesizer using an Fmoc/tert butyl strategy. Fmoc- Glu(Wang resin LL)-ODmab (Merck Biosciences, substitution 0,3 mmole/g) is used as a starting resin. The Fmoc-L-amino acids except the cysteines are coupled with PyBOP/NMM in DMF in a double coupling procedure. In order to prevent racemization durin cysteine couplings the corresponding cysteines are incorporated also by double coupling as the preactivated Fmoc-L-Cys{Trt)~Opfp esters in DMF/HOBT (boid printed in the sequence below).
To facilitate cyclisation of the linear peptide pseudoproline dipeptides are incorporated into the sequence at positions V-T and E-S (bold printed in the sequence below). It has been shown that those pseudoproline dipeptides are extremely useful tools for assisting the cyclization of peptides. (Schmiedeberg (2002) Org. lett. 4, 59 and a) Haack (1992) Tetrahedron lett. 33» 1589; b) Mutter (1995) Pept. Res. 8, 145). Due to the propensity of pseudoproline residues to adopt a cis- conformation, substitution of Ser or Thr by a pseudoproline residue has the effect of bringing the ends of the chain together, promoting cyclization and reducing oligomerization and cyclodimer formation. The native sequence is regenerated on cleavage and deprotection.
The assembled linear peptide on resin thus has, for example, the following sequence:
H»Ala-Arg(Pbf)-Arg(Pbf)-Cys{Trt)-Tyr{tBu)-Asn(Trt)-Asp(OtBu)-Pro-Lys(Boc)- Cys(Trt)-Abu-Asp(OtBu)-Phe-Vai-Thr(psϊWle,HΛepro)-Gly-Arg(Pbf)-Ala-GIu(OtBϋ}- Ser(psiMe,Mepro)-Asp(OtBu)-Glu(Wang resin LL)-ODmab. The ODmab group is removed from the peptide resin by 2% Hydrazine-hydrate in DMF and the cyclization of the peptide is performed on resin with PyBOP/NMM in DMF by reacting 5 hours at 50 degree Celsius and overnight at room temperature. A Kaiser test performed after this time does not show residual amount of free amino groups.
The peptide is cleaved off the resin by 95% TFA, 4% triethylsiiane, and 1 % water. Crude peptide is purified by HPLC liquid chromatography using a stationary phase of Silica C18 10μm, 100 A with an eluant of acetonitrile (80%) in 0,1 % TFA-water and 0,1 % TFA in water (Fig. 16). The detection is done at 220 nm. Purified fractions are lyophilized and analysed by mass spectrometry (MALDI-TOF; Fig.17).
All following in vitro and in vivo studies were carried out with these cyclopeptide mutants produced as described above.
Example 2: In vitro ELISA competition assay
The blocking capacity of β-ι-ECn-18AA cyciopeptide mutants (Cysi3~Ser14 or Ser13- Cyst4 mutation having an additional D-Glu→Gln exchange, e.g. at the ring closure site) was compared with the 3 Cys-containing 25AA or 18AA Cys/Cys cyclopeptides after preincubation (12h, 4° C, rotating incubation over-night) of different numbers of sera from immunized antibody-positive rats in an ELISA-competition assay using the 3 Cys-containing linear 25AA Cys/Cys peptide as an antigen. Fig. 3 shows the results from measurements performed with sera of n=69 immunized antibody-positive rats using different cyciopeptides of the present invention.
There were two patterns of reaction of the rat sera tested (type1/type2). A major fraction of the sera (93%, Fig. 3, upper panel, typei ) was very efficiently blocked by the 25AA or 18AA Cys/Ser mutant cyclopeptides (69±2% or 68±3% (Gin-closure)/ 69+2% (D-Glu-closure), respectively) which were even superior to the corresponding 3-Cys-containing 25AA or 18AA Cys/Cys cyclopeptides (65±2% or 59+2%, respectively), whereas the 25AA or 18AA Ser/Cys mutants had almost no inhibitory effect, irrespective of the amino-acid at the closure site (6±2%, 25AA Ser/Cys, P< 5x 10"30; 1±2%, 18AA Ser/Cys with GIn- (P< 3.7x10"33) or 1±2% with D-Glu-closure, (P< 6x10~55). A minor fraction of the sera (7%, Fig, 3, lower panel, type 2 and Fig.6) was blocked similarly by Cys/Ser or Ser/Cys mutated peptides - although to a lesser extent in terms of inhibitory capacity; in addition, for both 25AA and 18AA cyclopeptides the mutants were less effective than their 3 Cys-containing 25AA or 18AA Cys/Cys counterparts. Blocking capacity of the 25AA or 18AA Cys/Ser mutant cyclopeptides was 48±6% or 50±8% (Gln-ciosure)/47±5% (D~Glu-closure)t respectively, which was constantly inferior to that of the corresponding 3-Cys-containing 25AA or 18AA Cys/Cys cyclopeptides (72±5% or 67±δ%, respectively); however, in these animals the 25AA or 18AA Ser/Cys mutants revealed blocking capacities which were almost comparable to those of Cys/Ser-mutants (37±7% 25AA Ser/Cys, P=0.22 n.s.; 47±3% 18AA Ser/Cys with GIn- or 33±5% with D-Glu-ciosure, P=0.7 n.s. or P=O.08 n.s., respectively).
Subsequently, the dose-dependent blocking capacity of various linear and cyclic beta1-ECII-peptides in vitro was analyzed by using the same ELISA competition assay (Fig. 4):
Experiments including linear 25AA Cys/Cys peptides, cyclic 25AA Cys/Ser peptide mutants, cyclic 18AA Cys/Cys peptides, cyclic 18AA Cys/Ser peptide mutants and a linear 18AA Cys/Ser peptide mutant revealed, that all sera from n=6 randomly choosen immunized antibody-positive rats were best blocked in a dose-dependent manner by beta1-EC!l-18AA Cys/Ser mutant cyclopeptides, followed by non-mutant 18AA Cys/Cys cyclopeptides. and the 25AA Cys/Ser cyclopeptide mutant. All cyclopeptides were largely superior to their linear counterparts (with or without mutation) in terms of antibody neutralizing capacity (PO.005; Fig. 4), yielding a dose-dependent decrease in circulating free anti-beta 1 -ECU antibodies of 53% with an 8-fold excess, 66% with an 20-fold excess and about 85% with an 80-fold excess of 18AA Cys/Ser cyclopeptide mutants. The corresponding results for cyclic 18AA Cys/Cys- or cyclic 25AA Cys/Ser-peptides were: 46/30% [8-fold excess], 56/49% [20- fold excess], and 71/83% at an 80-fold excess. Linear peptides were clearly less efficient, yielding a dose-dependent decrease in receptor antibody-titers of only 24% [8-foid excess], 35% [20-foid excess], and about 50% at an 80-fold excess for both, linear 25AA Cys/Cys and linear 18AA Cys/Ser peptides. Additionally, the in vitro blocking (=neutraJization) capacity of the nove! cyciopeptide variants of the second extracellular loop (ECU) of the human beta 1 -adrenergic receptor including the Abu-variants described herein was tested with sera of immunized beta1-ECM antibody-positive rats after incubation for 12-14h at 4°C (Fig. 12). The in vitro blocking efficiency of the 22AA cyciopeptide cyc22AA Cys/Cys (blocking-efficiency 82.0±5.0% versus unblocked sera, P=O.000046) followed by the cyc22AA Cys/Abu mutant (blocking-efficiency 79.414.0%, P=0.00014) and the cyc22AA Cys/Ser mutant (blocking-efficiency 74.9+ 5.0%, P=0.00026), which underscores the relevance of optimally mimicking the second loop cyteines. The efficacy of these optimally beta-ECII (conformation) mimicking peptides was even higher than the blocking capacity of previously described 3 Cys-containig longer cyclopeptides, i.e., cyc25AA Cys/Cys (blocking-efficiency 73.4±5.0%, P=0.00011) or shorter cyd δAA Cys/Cys cyclopeptides (blocking-efficiency 66.1+ 7.0% versus unblocked sera, P=0.00025; see Figs. 12A and 12B).
The cyclic 22AA Cys/Abu peptide, although comparably efficient in vitro (antibody blocking efficacy cyc22AA Cys/Abu mutant 79.4±4.0% (P=O.00014) vs. untreated immunized rats; cyc22AA Cys/Ser 74.9±5.0% (P=0.00026) vs. untreated immunized rats; cyc22AA Cys/Abu versus cyc22AA Cys/Ser, P=O.5, Fig. 12B), had a clearly higher antibody-blocking efficiency than the cyc22AA Cys/Ser mutant in vivo, as evidenced by injection of the cyctopeptides in receptor-antibody positive immunized cardiomyopathic rats (Figs.13A and 13B).
Example 3; In vitro functional FRET-assay
The blocking capacity of βrECn 25AA or 18AA cyciopeptide mutants (having a D- Glu/Gin at the ring closure site or not) on β-, -receptor-mediated signalling (functional cAMP-assay) was assayed using an approach by fluorescence resonance energy transfer (FRET) (Fig. 2).
The effect of the pre-incubation (12h, 40C, rotating incubator) of anti-βt-EC-n IgG antibodies of a representative rat with β^ECn-i δAA cyctopeptide mutants (Cys/Ser or Ser/Cys mutations, respectively) was compared with the inhibitory effect of a 3 Cys- containing 25AA Cys/Cys cyciopeptide or with the effect of anti-βi-ECn igG antibodies not incubated with blocking peptides. The normalized YFP/CFP-ratio of the registered FRET emission signals served to quantify the effect of the cyclo- peptide mutants in terms of blockade (in per cent) of antibody-induced cellular cAMP- production of transiently Epad-transfected stably P1-AR expressing human embryonic kidney ceils (HEK 293- βi cells). The x-axis in Fig. 2 corresponds to the registration time given in seconds (s). Again, in terms of inhibiting measurable functional antibody-effects (blocking intracellular cAMP-increases) the cyclic prECtr 18AA Cys/Ser mutant was largely superior to its Ser/Cys counterpart, and even slightly more effective than a 3 Cys-containing 25AA Cys/Cys cyclopeptide (Fig. 2).
Taken together, the results of the tests performed herein demonstrate that the antibody-blocking capacity of mutated cyclopeptides was not affected by the reduction of the number of amino-actds from a 25-meric to a 18-meric peptide. The results also demonstrate an excellent comparability of 25AA Cys/Cys and 18AA Cys/Cys cyclopeptides with the cyclic 25AA or 18AA Cys/Ser mutants, but not with the cyclic 25AA or 18AA Ser/Cys mutants. Surprisingly, the exact nature of the exchange of one single cysteine residue with a serine residue markedly determines the neutralizing potency of the mutated peptides: the Cys→Ser exchange at position 18 (25-AA cyciopeptide) or at position 14 (18-AA cyclo-peptide), respectively, yielded cyclic peptides with excellent antibody-neutralizing and pharmacological effects in vitroin vitro (Figs. 2.3), whereas the CySi7- +Sen7 or Cys13→Seri3 mutants (25-AA or 18-AA peptide, respectively) had almost no inhibitory effect, neither regarding their properties as antibody-scavengers nor in terms of their capability of inhibiting functiona! antibody-effects (neutralization of receptor-stimulation in vitro; Figs. 2,3 and Example 3). The D-Giu/G!n exchange at position 25 (25AA cyciopeptide- mutants) or 18 (18AA cyclopeptide-mutants) did not significantly influence the blocking capacity of the cyclopeptides, regardless of their length (i.e., 25 versus 18 amino-acids; Figs. 3).
Example 4: Animal model, "in vivo" blockade of receptor antibodies
The animal model used in this example and any other example described herein, if not indicated to the contrary, is the human analogue rat model. Prior to evaluating and testing, respectively, this human analogue rat model was treated as described herein-below using the various compounds of the present invention, more particularly compounds of formula Vl, VII, VIH and iX, and, as controls, a linear ECu-18AA Cys/Ser mutated (GIn18-)peptide (with the following amino-acid sequence: Aia-Asp- Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-Ser-Asp-Phe-Val-Gln) and a linear non-mutated 3 Cys-containing ECn~25AA Cys/Cys (GIn2S) peptide (with the following amino-acid sequence: Aia-Arg-Aia-Giu-Ser-Asp-Giu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp- Pro-Lys-Cys-Cys-Asp-Phe-Val-Thr-Asn-Arg-Gin.
The in-vivo blocking effects of both 25AA and 18AA Cys/Ser mutant cyclopeptides (with a GIn closure site), the 18AA Ser/Cys mutant cyclopeptide, and a mutated linear 18AA Cys/Ser peptide were analyzed after intravenous (i.v.) injection of 1.0 mg/kg body weight (Bw) of each construct into freshly immunized antibody-positive rats (i.e., prophylactic use of cyclopeptides in a kind of "prevention" study), with a first cyclopeptide-application 3 months after the initial immunization (and two subsequent boost at months 2 and 3). In total, five prophylactic applications of the various constructs were given at 4-weekiy intervals, always two weeks after the monthly continued antigen boost. Sera were drawn 18-20 hours after i.v. injection and assayed for reactivity by ELISA using the 3 Cys-containing linear 25AA Cys/Cys- peptide as an antigen (Fig. 5).
This first (prophylactic) in vivo cyclopeptide-applications demonstrated, that the highest efficiency in terms of antibody-neutralization was achieved with 1.0 mg/kg body weight (Bw) of non mutant 25AA Cys/Cys or 18AA Cys/Cys-cylopeptides (87.7+2% or 89.9±3% decrease after five cyclopeptide injections, compared with untreated immunized animals; both P< 0.005), followed by the 18AA Cys/Ser mutant cyclopeptide (54.5±2% decrease after 5 cyciopeptide injections; P<0.05), whereas linear 25AA Cys/Cys peptides or linear 18AA Cys/Ser mutants at a same concentration had no significant blocking effects (25.8±3% or 4.5+11 % antibody-titer decrease after 5 injections. P=O.16 or P~0.8; Fig. 5).
Moreover, the in vivo blocking effects of therapeutically used 25AA and 18AA Cys/Ser cyclopeptide mutants, the 18AA Ser/Cys mutant (Gln-)cyclopeptide, and a mutated linear 18AA Cys/Ser peptide were assessed after a first intravenous (i.v.) dose (i.e., 1.0 mg/kg body weight (Bw)) of each construct injected into long term immunized anti-beta1-ECII antibody-positive rats, yet presenting a cardiomyopathic phenotype (after nine months of 1x monthly immunization with the betai -ECI I/GST antigen; Figs. 6-8). Sera were drawn 18-20 hours after the first i.v. injection of the various constructions and assayed for reactivity by ELlSA using the 3 Cys-containing ϋnear 25AA Cys/Cys-peptide as an antigen. "Therapeutic" application of various cyciopeptides in cardiomyopathic antibody-positive rats revealed a higher in vivo blocking capacity of 18AA Cys/Ser cyciopeptide mutants (1mg/kg/Bw) compared with either 25AA Cys/Ser cyciopeptide mutants or the clearly less efficient 18AA Ser/Cys cyciopeptide mutants at a same concentration (Figs. 6 and 7). Again, the in vivo efficiency of the 18AA Cys/Ser cyciopeptide mutant was largely superior to that of the linear 18AA Cys/Ser peptide mutant. However, when decreasing the applicated dose of cyclic 18AA Cys/Ser mutants to 0.25 mg/kg body weight (Bw), no relevant decrease in receptor-antibodies was achieved, suggesting a dose-and-effect relation for cyciopeptide mutants (Fig. 6).
Repeated therapeutic injections of mutant single S-S cyciopeptides every 4 weeks into long-term immunized rats with antibody-induced immune-cardiomyopathy confirmed a kind of "critical minimal dose"-and-effect relation for single S-S cycSopeptide mutants: a dose of 0.25 mg/kg Bw of the 18AA Cys/Ser cyciopeptide - albeit capable of scavenging receptor-anti-bodies to some extent - was clearly less efficient in terms of both, (1) the achieved decrease in circuiating receptor-antibodies (even when respecting only cyclopeptide-sensitive "responders", defined as animals having, after 7 cyclopeptide-injections, a maximum remaining antibody-level equal or inferior to 80% of the titer at start of therapy (Figs. 7B and C), and (2) in the achieved cardioprotective effect (Figs. 7B, 8B, 9B, and 10) compared with either a dose of 1.0 or 2.0 mg/kg body weight 18AA Cys/Ser cyciopeptide. The latter doses were almost equally efficient in terms of both, neutralization of circuiating receptor antibodies (Figs. 7B and C), and reversal of antibody-induced cardiomyopathic features (Figs. 8B, 9B, and 10). A further increase in the applicated dose to 4.0 mg/kg body weight, however, did not result in higher efficiency - neither regarding antibody scavenging capacity (Figs. 7B and C), nor regarding cardioprotective effects (Figs. 8B, 9B, 10). Upon injection of the peptides no serious local or systemic side effects were observed. In addition, after injection of the various mutant cyciopeptides, both the heart rate and the blood pressure of the animals were not affected (Fig. 10A). As mentioned, the in-vivo blocking effects of both 25AA and 18AA Cys/Ser mutated cyciopeptides (with a GIn closure site), the 18AA Ser/Cys mutant (Gln-)cyclopeptide, and a mutated linear 18AA Cys/Ser peptide were analyzed after a first intravenous (i.v.) injection of 1 mg/kg body weight (Bw) of each construct into immunized antibody-positive rats. Sera were drawn 18-20 hours after i.v. injection and assayed for reactivity by ELISA using the 3 Cys-containing iinear 25AA Cys/Cys-peptide as an antigen. The in vivo data confirmed a higher blocking capacity of the 18AA Cys/Ser mutated cyclo-peptides (1 mg/kg/Bw) compared with either 25AA Cys/Ser mutants or the clearly less effective 18AA Ser/Cys mutated cyciopeptides at a same concentration (Fig. 6). The in vivo efficiency of the 18AA Cys/Ser cyclopeptide was also largely superior to that of the linear 18AA Cys/Ser peptide. Interestingly, the difference in the blocking efficiency of the Cys/Ser mutated cyciopeptides compared with that of the linear peptides was even more pronounced in vivo (Figs. 6, 7). This finding was supported by comparative kinetic measurements demonstrating that the half-life of ECII-peptide-mutants in whole blood (β-i-ECII-peptides, e.g. eye 25AA- Cys/Cys or eye 18AA- or 25AA-Cys/Ser mutants with a cyclic structure as the cyclic peptides described herein) is more than 10fold than the half-life of their linear counterparts (linear: ca. 5.5h; cyclic: ca. 64.5h). The data were obtained after incubation of linear or cyclic peptides with whole blood drawn from both untreated (control) rats or healthy human (control) subjects in the presence of heparine (24 h, 4°C, rotating incubator), respectively. The amount of remaining intact peptides was determined by competition ELISA with linear βrECil-25AACys/Cys peptides at 2min, 10min, 30min, 1 , 2, 4, 8, 16 and 22h. (Fig. 20).
Upon injection of the peptides no serious local or systemic side effects were observed. In addition, after injection of the various mutant cyciopeptides, both the heart rate and the blood pressure of the animals were not affected. However, the in vivo data also indicate, that the efficiency of the 18AA Cys/Ser mutated cyclopeptide might equally depend on the applied dose (Figs. 6, 7). The obtained results are compatibie with a (minimal) dose-and -effect relation for single S- S cyclopeptide mutants: a dose of 0.25 mg/kg of the 18AACys/Ser cyciopeptide mutant was largely less efficient in terms of both, the achieved decrease in circulating receptor-antibodies and in the achieved cardioprotective effect compared with either a dose of 1.0 or 2.0 mg/kg body weight (Bw) (Figs. 6, 7). These doses were almost equally efficient in terms of both, neutralization of circulating receptor antibodies and reversal of antibody-induced cardiomyopathic features (Figs, 7, 8, 9 and 10). A further increase in the appiicated dose to 4.0 mg/kg Bw, however, did not result in higher efficiency - neither regarding antibody scavenging (Fig.7) capacity nor regarding cardioprotective effects in vivo (Figs. 8-10). A high dose (=4.0 mg/kg Bw) of cyc18AA Cys/Ser mutants did not increase the efficiency; instead, it led to an transient increase in antibody-titers, allowing for significant reductions in receptor- antibody titers only after the third or fourth cyclopeptide-injection. Most notably, the effect on the antibody-neutralizing capacity of the different injected concentrations of cyc18AA Cys/Ser mutant cyclopeptides was also confirmed in terms of reversal of antibody-induced cardiomyopathic features in the course of the study with the best cardioprotection achieved by 1.0 or 2.0 mg/kg Bw 18AA Cys/Ser cyciopeptide mutants (Figs. SB, 9B, and 10,). in addition, the in vivo experiments demonstrated that the antibody-blocking capacity of mutant cyclopeptides is seemingly not affected by a reduction in the number of amino acids from a 25-meric to a 18-meric cyciopeptide; both in vitro and in vivo data demonstrate an excellent comparability of the two 2 cysteine-containing single disulfide bond 25AA Cys/Ser or 18AA Cys/Ser cyciopeptide mutants (Fig. 8A). It should be noted, however, that both 1.0 mg/kg 25AA-meric Cys/Ser as well as high dose (i.e., 4.0 mg/kg Bw) 18AA-meric Cys/Ser mutants led to an initial transient increase in antibody-titers (Fig. 7A and B), and thus postponed a significant reduction in receptor antibody titers to the third or fourth cyclo-peptide-application (third or fourth month of therapy). This phenomenon did not occur with either 1.0 or 2.0 mg/kg Bw doses of 18AA Cys/Ser cyciopeptide mutants.
Further, the antibody-blocking capacity of mutant cyclopeptides seems also not affected by a reduction in the number of amino acids from a 25-meric to a 22-meric cyciopeptide, which, however, seems to possess better antibody-blocking capacities than the 18-mehc cyciopeptide - regardless of the mutation at postion 216 of the βr ECI!-sequence. The in vivo blocking (=neutralizing) capacity of the novel cysteine- containing cyciopeptide mutants (cyc22AA Cys/Ser and cyc22 Cys/Abu mutants) of the second extracellular loop (ECU) of the human betai -adrenergic receptor described herein were tested by therapeutic injection into rats which had been regularly immunized over 8 months (basic immunization and seven subsequent antigen-boosts every 4 weeks (Figs. 13 and 14), and compared with the effects of cyclic beta1-ECI! 18AA Cys/Ser-mutants. After 12 (cyc18 Cys/Ser) regular cyclo- . peptide injections every 4 weeks, the titers in untreated antibody-positive animals (significantly) decreased to 47.9±6.0% of the values at start of therapy (n=5 positive controls). In contrast, 12 injections of cyc22AA Cys/Ser mutants (n=5 animals) significantly decreased the antibody titers to 15.0±10.6% (P=O.0001), or 11 injections of the inventionai cyc22AA Cys/Abu mutants (n=5 animals) even more significantly decreased the titers to 7.4 ± 4.0% of the antibody-titers at start of therapy (P= 2.0 x 10-8) (Figs. 13A and B).
The in vivo-efficiency of both novel cyc22AA-mutants was thus largely superior to the previously described 18AACys/Ser cyclopeptide-mutant (n= 5 animals), which after 12 injections decreased the antibody-titers to roughly 50% of the titers at start of therapy (P= 0.005 versus untreated antibody-positive animals; see Fig. 13B). The mutant 22AA Cys216→Abu216 cyclopeptide, although similarly efficient in vitro (t-test: P- 0.5 versus cyc22AA Cys216-→Ser216), had in fact a trend towards a higher antibody-bfocking efficacy in vivo in receptor-antibody positive immunized rats than the cyc22AA Cys216/Ser mutant, as evidenced in receptor-antibody positive long term immunized rats (Fig. 13B).
In addition, assessment of the biological cardioprotective efficacy of the cyciopeptide mutants by echocardiographic follow-up during 12 months of treatment also suggests a superiority of the cyc22AA Cys/Abu mutant over the cyc22AA Cys/Ser mutant. Both 22AA cyclopeptides appeared more efficient than a eyelid 8AA Cys/Ser betai- ECII peptide mutant regarding their cardioprotective effects in vivo, as evidenced by the (non~invasiveiy determined) decreases in left ventricular end-diastolic (LVED) and end-systolic (LVES) diameters (Figs. 14A and B), and the increase in "Cardiac Index" (Cl, given in ml/min/g body weight; Fig. 14C; derived from two dimensional- and Doppler-echocardiography using a Visual Sonics echocardiographic system (Vevo 770, version V2.2.3), equipped with a 17.5 MHz transducer). Similarly, invasive assessment of cardiac functional parameters suggest a (non significant) trend for a higher efficacy of the cyc22AA Cys/Abu mutant compared with the cyc22AA Cys/Ser mutant regarding recovery of left ventricular contractility (Fig. 14 E) and reversal of LV end-diastolic pressure (Fig. 14F).
in the next step, the aforementioned βi-ECI! epitope-mimicking and 22AA cyciopeptide mutants of the present invention were employed for in vivo experiments in order to optimize their strategy of application/administration. The different application protocols comprised either monthly intravenous applications of cyciopeptide mutants disclosed by the present invention (cyc22AACys/Abu), or a triple injection (one injection every week on 3 subsequent weeks) every three months. Measures like these provide the potential to reduce the amount of injected cyclopeptides or to reduce the burden of (regular monthly) venipuncture and to increase the flexibility of application (for both, human patients and animais) whilst maintaining or increasing the biological efficiency of the injected constructs of the present invention.
With respect to the herein disclosed cyc22AA Cys/Abu constructs triple injections (1 injection per week on 3 subsequent weeks) followed by a two-months intervention free time interval were at least as efficient as or even slightly superior to monthly applications in reducing the titer of circulating anti- P1-ECU antibodies (Fig. 21 A and B), and in reverting the cardiomypathic phenotype (Fig.22A-C).
Taken together, because the cardioprotective and immunomodulating activity of the ECIl-homoIogous cyclic peptides appears to depend largely on their conformation, an sntramolecularly localized disulfide bridge is essential to stabilize and maintain the three-dimensional structure of the ECM-loop mimicking construction. In the cyclic 21 +1 (=22)AA peptide, the remaining cysteines (i.e. in position 209 and 215, in case 216 has been mutated to the non-naturally occuring amino-acid L-alpha amino- butyric acid (Abu)) maintain a defined (intramolecular) distance, further strengthened by introduction of the smallest naturally occuring amino-acid glycine at the (predicted) ring closure site, in order to allow for the formation of a structure-defining intramolecular disulfide bridge. Example 5: Aianine-scan of beta1-ECH
To determine further amino-acids essential for the specific beta1-ECi!-mimicking conformation and thus antibody-blocking efficacy of the novel (21+1=) 22AA Cys/Abu cyclopeptide, an alanine-scan was performed with the here presented investigational product (Fig. 15A).
For the Aia-scan, each of the nine amino-acids diverging from the second loop sequence of the human beta2-adrenergic receptor, which had no beta 1 -antibody- blocking effect, has been mutated to alanine before cyclization and in wfro-testing for their respective antibody-biocking capacity.
Domain (first) AA-sequence (last)
ECU beta2-adrenergic receptor: 175 RATHQEAlN CYANETCCD FFTG 196 ECU betai -adrenergic receptor: 200 RAESPEARRCYNPPKCCD FVTG 221 ECIi petal -22AA Cvs-Cvs/Abu cvciopeptide: RAESDEARRCYNDPKC Abu DFVTG
ECU beta1-22AA Cvs-Cvs/Abu cvciic aia-substituted peptides 1. RAASDEARRCYNDPKC Abu DFVTG
2. RAEADEARRCYNDPKC Abu DFVTG
3. RAESAEARRCYNDPKC Abu DFVTG
4. RAESDEAARCYNDPKC Abu DFVTG
5. RAESDEARACYNDPKC Abu DFVTG
6. RAESDEARRCYADPKC Abu DFVTG
7. RAESDEARRCYNAPKC Abu DFVTG
8. RAESDEARRCYNDAKC Abu DFVTG
9. RAESDEARRCYNDPAC Abu DFVTG
Analysis of their respective antibody-blocking capacities surprisingly revealed, that mutation of AA situated in the second half of the beta1-22AA cyclopepide {e.g., amino-acids 211-214: NDPK) dramatically decreased blocking-efficiency (Fig. 15B),
This finding was the same for both, conformational polyclonal and monoclonal rat anti-beta1-ECII antibodies (Fig. 15B, panels 1 and 2), and also for purified conforma- tionai polyclonal rabbit (Fig. 15B, pane) 3) or monoclonal mouse anti-beta1-ECII antibodies (Fig. 15B, panel 4).
Consistently and irrespective of the species in which the anti-beta1-EC!l was generated, the by far most relevant AA to preserve the antibody-blocking capacity of the inventiona! cyclopeptide was the praline (P) at position 213 (overall 89% loss in blocking capacity compared with the inventional product), followed by aspartic acid (D) at position 212 (overall 57% loss in blocking capacity), asparagine (N) at position 211 (overall 41 % loss in blocking capacity), and the lysine (K) at postion 214 (overall 40% loss in blocking capacity; numbering according to Frielle et a/. 1987, PNAS 84, pages 7920-7924).
In addition, in case of the mouse monoclonal anti-beta1 -ECIl5 also an exchange of each of the two arginines at position 207 (R; 81% loss in blocking capacity), and 208 (R; 96% loss in blocking capacity), and also, although to a lesser extent, an exchange of the aspartic acid at position 204 (D; 33% loss in biocking capacity) significantly decreased the blocking capacity of the inventional beta1-22AA Cys/Abu cyclopeptide.
In contrast, for most anti-beta1-ECil antibodies (rat/rabbit) a substitution of amino- acids within the first half of the cyc22AA Cys/Abu beta1-ECII homologous cyclopeptide (e.g., amino-acids 202-208: ESDEARR) did not significantly affect the blocking efficacy of the inventionai cyclopeptide. Interestingly, a substitution of serine in position 203 by alanin for distinct anti-beta1-ECI! antibodies even slightly increased its blocking capacity (e.g., the rat monoclonal, the rabbit polyclonal, and also a few of the rat polyclonal antibodies, see also Fig. 15B).
By the above-described Ala-scan essential amino-acids for antibody-recognition of the disclosed cyclopeptide(s) were identified. Most notably, amino-acids at position 12, 13, 14, and 15 of the cyclic peptide of the present invention (as depicted in Fig. 31A) are required to preserve the antibody-blocking capacity of the cyclopeptide, with (apart from cysteines) proline at position 14 being one of the the most essential AA in the 22AA cyclopeptide (Figs. 15A and B).
Subsequent binding-competition assays (Fig. 15C) to compare the dose-response of 22AA Cys/Ser compared to inventional 22AA Cys/Abu cyclopeptides regarding their capacity to inhibit binding of different rat or rabbit anti-beta 1-ECIi to biotinyiated linear 16AA beta1-ECil-peptides (i.e., to compare peptide/antibody-affinities) confirmed the about 2-fold lower EC50 of 22AA Cys/Abu versus 22AA Cys/Ser-CP for monoclonal (Fig. 15C, panel 1) and polyclonal rat antibodies, but equally for polyclonal rabbit anti-beta 1 -ECl! (Fig. 15C, panel 2 and 3).
Surprisingly, in case of the eyelid 8AA Cys/Ser peptide mutant, the "two-site" binding competition curve obtained with polyclonal rabbit anti-beta 1 -ECU antibodies indicated the presence of at least two different antibody-entities in the preparation with different beta1~18AA Cys/Ser cyciopeptde affinities (Fig. 15C, pane! 3). The "one-site" competition curves obtained for selected out of the different site- specific alanine-mutated beta1~22AA Cys/Abu cyclopeptides of the present invention impressively confirmed the ELISA-competition results.
Whereas the inventional 22AA Cys/Abu cyclopeptide (22Abu0) or a serine/alanine exchange (22Abu2) efficiently blocked antibody-binding to biotinyiated linear 16AA beta1-ECIl-peptides to about the same extent, a proline/alanine exchange (22Abu8) decreased antibody-binding efficacy (=antibody-affinity to the 22Abu8-mutant) by about 400-fold (EC50: 5,54x10"5 (22Abu8) versus 1 ,67x10"7 (22AbuO); Fig. 15C, panel 4).
Example 6; In vitro blockade of activating human βi-receptor-autoantibodies
The blocking capacity of βi-ECϋ 22AA Cys/Abu cyclopeptide mutants on β-,-receptor- stimulation induced by activating human β-j-receptor autoantibodies (functional cAMP-assay) was assayed using an approach by fluorescence resonance energy transfer (FRET) (Fig. 19) The normalized YFP/CFP-ratio of the registered FRET emission signals served to quantify the effect of the 22AA Cys/Abu cyciopeptide mutants in terms of blockade (in per cent) of antibody-induced cellular cAMP- production of transiently Epad-transfected stably βrAR expressing human embryonic kidney cells (HEK 293-βi cells).
For this purpose, human anti~βrECn IgG antibodies isolated from a (representative) 28 years-old female DCM patient were preincubated (12h, 40C1 rotating incubator) with different cyc22AA Cys/Abu-Ala exchange-mutants (as given in Fig.15A). Unblocked (human) patient IgG yielded 18±3% FRET activity. The 22Abu8 cyclic peptide (eye RAESDEARRCYNDAKC Abu DFVTG) with an Pro2i6>A!a exchange was not able to block the patient's SgG (17±2% residual FRET activity), indicating that also in humans the proline in position 216 of the βrECιrsequence represents an essential amino-acid within the receptor epitope recognized and bound by human βr receptor autoantibodies. In contrast, patient IgG was very efficiently biocked with the unchanged cyc22AACys/Abu mutant βrECn-peptide referred to as 22AbuO (= eye RAESDEARRCYNDPKC Abu DFVTG) showing only negligible residual FRET activity after preincubation (3±3%, P<0.001 versus unblocked). Similarly, the 22Abu1 cyclic peptide had a strong blocking effect (8±5% FRET activity, P<0.005 versus unblocked patient IgG), indicating that Glu2o2 is not an important epitope-constituting amino-acid and not a relevant target for human βi -receptor autoantibodies.
Taken together, the results of the tests performed herein demonstrate that the antibody-blocking capacity of mutated βi-ECn-cyciopeptides was not affected by the reduction of the number of amino-acids from a 25-meric to a 22-meric (or a 18-meric) peptide. The results also demonstrate a good comparability of 25AA Cys/Cys and 18AA Cys/Cys cyclopeptides with the cyclic 25AA, 22AA or 18AA Cys/Ser mutants, and also with the cyclic 22AA Cys/ABu mutants with respect to their capacities of blocking activating rat or rabbit antiβrECn~abs. This was also the case for activating human β-j-receptor autoantibodies (aabs). Aiso human anti-βrECn appear directed aginst an epitope within the second β-i-receptor loop which comprises either the key amino-acis Cys2o9/Prθ2i3, or the amino-acids Pro2-ι3/Cys2i5 (numbering according to Frielle et al. 1987, PNAS 84, pages 7920-7924).
The present invention refers to the following nucleotide and amino acid sequences:
SEQ ID No. 1 :
Amino acid sequence homologous to an ECN epitope of human βt-AR (18AA;
Cys14→ABui4); Cycϋzation may occur between Alai and GIn-I8
Ala-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-ABu-Asp-Phe-Val-GIn SEQ ID No. 2:
Amino acid sequence homologous to an ECn epitope of human β-i-AR (25AA;
Cysi8-→ABui8); Cyclization may occur between Alai and GIn2S
Ala-Arg-Ala-Glu-Ser-Asp-Glu-Aia-Arg-Arg-Cyε-Tyr-Asn-Asp-Pro-Lys-Cys-ABu-Asp-Phe-Val-Thr-Asn- Arg-GSn
SEQ ID No. 3:
Amino acid sequence homologous to an ECn epitope of human βrAR (18AA;
Cysi3→ABui3); Cyclization may occur between Alai and GIn18
Ala-Asp-Glu-Ala-Arg-ArgXyε-Tyr-Asn-Asp-Pro-Lyε-ABu-Cys-Asp-Phe-Vat-GIn
SEQ ID No. 4:
Amino acid sequence homologous to an ECn epitope of human βrAR (25AA;
Cys17→ABui7); Cyclization may occur between Alai and GIn2S
Ala-Arg-Ala-Glu-Ser-Asp-Gtu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-ABu-Cys-Aεp-Phe-Val-Thr-Asn- Arg-Gln
SEQ ID No. 5:
Nucleotide sequence encoding an amino acid sequence homologous to an ECn epitope of human βrAR (18AA; Cysu→Xxxu) gcngacgaggcgcgccgctgctacaacgaccccaagtgcXXXgacttcgtccar
SEQ ID No. 6:
Nucleotide sequence encoding an amino acid sequence homologous to an ECn epitope of human βrAR (25AA; Cysis-→Xxx-is) gcncgggcggagagcgacgaggcgcgccgctgctacaacgaccccaagtgcXXXgacttcgtcaccaaccggcar
SEQ ID No. 7:
Nucleotide sequence encoding an amino acid sequence homologous to an ECn epitope of human βrAR (18AA; Cys13→Xxxi3) gcngacgaggcgcgccgctgctacaacgaccccaagXXXtgcgacttcgtccar SEQ ID No. 8;
Nucleotide sequence encoding an amino acid sequence homologous to an ECn epitope of human βi-AR (25AA; Cys17→Xxxi7) gcncgggcggagagcgacgaggcgcgccgctgctacaacgaccccaagXXXtgcgacttcgtcaccaaccggcar
SEQ ID No. 9:
Amino acid sequence homologous to an ECn epitope of human P1-AR (18AA;
Cys3-→ABu3); Cyciization may occur between Lysi and Proi8 Lys-Cys-ABu-Asp-PheΛ/al-Gln-Aϊa-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro
SEQ iD No. 10:
Amino acid sequence homologous to an ECn epitope of human P1-AR (25AA;
Cys3→ABu3); Cyciization may occur between Lysi and Pro25
Lys-Cys-ABu-Asp-Phe-Val-Thr-Asn-Arg-Gln-Ala-Arg-Ala-Glu-Ser-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn- Asp-Pro
SEQ ID No. 11 :
Amino acid sequence homologous to an ECn epitope of human P1-AR (18AA;
Cys2→ABu2): Cyciization may occur between Lys-i and Pro18 Lys-ABu-Cys-Asp-Phe-Val-Gln-Ala-Asp-Glu-Aia-Arg-Arg-Cys-Tyr-Asn-Asp-Pro
SEQ ID No. 12:
Amino acid sequence homologous to an ECn epitope of human βrAR (25AA:
Cys2→ABu2); Cyciization may occur between LyS1 and Pro25
Lys-ABu-Cys-Asp-Phe-Val-Thr-Asn-Arg-Gln-Ala-Arg-Aia-Glu-Ser-Asp-Giu-Ala-Arg-Arg-Cys-Tyr-Asn-
Asp-Pro
SEQ lD No. 13:
Nucleotide sequence encoding an amino acid sequence homologous to an ECn epitope of human βi-AR (18AA; Cys3→Xxx3) aagtgcXXXgacttcgtccargcngacgaggcgcgccgctgctacaacgacccc
SEQ ID No.14: Nucleotide sequence encoding an amino acid sequence homologous to an ECn epitope of human βrAR (25AA; Cys3→Xxx3) aagtgcXXXgacttcgtcaccaaccggcargcncgggcggagagcgacgaggcgcgccgctgctacaacgacccc
SEQ lD No. 15:
Nucleotide sequence encoding an amino acid sequence homologous to an ECn epitope of human P1-AR (18AA; Cys2-→Xxx2) aagXXXtgcgacttcgtccargcngacgaggcgcgccgctgctacaacgacccc
SEQ ID No. 16:
Nucleotide sequence encoding an amino acid sequence homologous to an ECn epitope of human βi-AR (25AA; Cys2→Xxx2) aagXXXtgcgacttcgtcaccaaccggcargcncgggcggagagcgacgaggcgcgccgctgctacaacgacccc
SEQ ID No. 17:
Amino acid sequence of an ECn epitope bearing portion of human βrAR (16AA; AA positions 204 to 219)
DEARRCYNDPKCCDFV
SEQ ID No. 18:
Amino acid sequence of an ECn epitope bearing portion of human β-i-AR (23AA; AA positions 200 to 222)
RAESDEARRCYNDPKCCDFVTNR
SEQ ID No. 19:
Amino acid sequence of an ECn epitope of human βi-AR
DEARR
SEQ ID No. 20:
Amino acid sequence of an ECn epitope (bearing portion) of human β-i-AR
RAESDEARR
SEQ ID No. 21 :
Amino acid sequence of an ECn epitope of human βf-AR DFV
SEQ ID No. 22:
Amino acid sequence of an ECu epitope of human βi-AR
DFVTNR
SEQ ID No. 23:
Amino acid sequence homologous to an ECn epitope of human P1-AR (16AA;
Cys-n→ABun; N-terminal AA: GIn16 or DGIu16); Cyclization may occur between Alai
Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-ABu-Asp-Phe-Val-Tyr-Gln
SEQ ID No. 24:
Amino acid sequence of the human βrAR
1 mgagvlvlga sepgnlssaa plpdgaataa rllvpaεppa sllppasesp eplsqqwtag 61 mgllmalivl livagnvlvi vaiaktprlq tltnlfimsl aεadlvmgll vvpfgativv 121 wgrweygsff celwtsvdvl cvtasietlc vialdrylai tspfryqsll trararglvc 181 tvwaisalvs flpilmhwwr aesdearrcy ndpkccdfvt nrayaiassv vsfyvplcim 241 afvylrvfre aqkqvkkids cerrflggpa rppspspspv papapppgpp rpaaaaatap 301 langragkrr psrlvalreq kalktlgiim gvftlcwlpf flanvvkafh relvpdrlfv 361 ffnwlgyans afnpiiycrs pdfrkafqgi lccarraarr rhathgdrpr asgclarpgp 421 ppspgaasdd ddddwgatp parilepwag cnggaaadsd ssldepcrpg faseskv
SEQ ID No. 25:
Amino acid sequence homologous to an ECn epitope of human βrAR (22AA;
Cysi7→ABu17); Cyclization may occur between Argi and Gly22
Arg-Ala-Glu-Ser-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-ABu-Asp-Phe-Val-Thr-Gly
SEQ ID No. 26:
Nucleotide sequence encoding an amino acid sequence homologous to an ECn epitope of human βrAR (22AA; Cys17→Xxxi7) cgggcggagagcgacgaggcgcgccgctgctacaacgaccccaagtgcXXXgacttcgtcaccGLY
SEQ ID No.27: Amino acid sequence homologous to an ECn epitope of human βi-AR {22AA;
CyS3-^ABu3); Cyclization may occur between Lysi and Prθ22
Lys-Cys-ABu-Asp-Phe-Val-Thr-Gly-Arg-ASa-Glu-Ser-Asp-Giu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro
SEQ ID No. 28:
Nucleotide sequence encoding an amino acid sequence homologous to an ECn epitope of human Ji1-AR (22AA; Cys3→Xxx3) aagtgcXXXgacttcgtcaccGLYcgggcggagagcgacgaggcgcgccgctgctacaacgacccc
SEQ ID No. 29:
Amino acid sequence of an ECn epitope (bearing portion) of human βi-AR
DEARRCYNDPK
SEQ ID No. 30:
Amino acid sequence of an ECn epitope (bearing portion) of human βi-AR
ESDEARRCYNDPK
SEQ ID No. 31 :
Amino acid sequence of an ECn epitope of human βrAR
AESDEARR
SEQ ID No. 32:
Amino acid sequence of an ECn epitope of human βrAR DFVT
In the nucleotide sequences, "xxx" stands for any nucleotide triplet coding for an amino acid or amino acid stretch which can be replaced by ABu; Xxx may also mean that the nucleotide triplet is completely missing ,- "GLY" stands for any nucleotide triplet coding for GIy (Glycine), i.e. for ggn. n stands for any nucleotide, particularly a, c, g or t, y stands for t or e and r stands for a or g. in the amino acid sequence, "Xxx" stands for an amino acid or amino acid stretch which can be replaed by ABu; Xxx may also mean that the amino acid is completely missing.
As used herein, the sequences of the various peptides are indicated from the N- terminus to the C-terminus, whereby the N-terminus is at the left side and the C- terminus is at the right side of the respective depicted amino acid sequence.
The following additional abbreviations are used herein:
amino actd: 3-letter code: 1 -letter code:
Alanine Ala A α-aminobutyric acid ABu
Arginine Arg R
Asparagine Asn N
Aspartic acid Asp D
Cysteine Cys C
Glutamic acid GIu E
Giutamine GIn Q
Glycine GIy G
Histidine Hts H
Isoleucine He I
Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr Y
Valine VaI V
Asparagine or aspartic acid Asx B
Giutamine or glutamic acid GIx Z
Leucine or isoleucine XIe J
Unspecified or unknown amino acid Xaa X Additional references cited
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Claims

1. A cyclic peptide of formula I:
cycio(x-Xh-Cys-x-xa-xb-xc-x-Cys-y-Xi-x) (I),
wherein a) x is an amino acid other than Cys; b) h is any integer from 1 to 15; c) i is any integer from 0 to 14; d) one of xa, xb and xc is Pro; e) y is α-aminobutyric acid (ABu) or an ABu analogue; and f) the cyclic peptide consists of at least 16 and of at most 25 amino acids, and wherein said peptide i) is capable of binding (auto-)antibodies against the EC» loop of βr adrenergic receptor (βi-AR); ii) is capable of inhibiting the interaction between βi-AR and
(auto-)antibodies against the ECu !oop of βi-AR; iii) mimics the epitope(s) presented in the native conformation of the ECn iv) is capable of reducing an antibody-mediated activation of βt-AR.
2. The cyclic peptide of claim 1 , wherein h is 5, 8 or 9.
3. The cyclic peptide of claim 1 or 2, wherein i is 3, 4 or 6.
4. The cyclic peptide of any one of claims 1 to 3, being a cyclic peptide of formula I' or I":
cyclo(xrXb-Cys-x-xa-xb-xc~x~Cys~y~XrX) (!');
cycio(xuι-Xh-Cys-x~xa-xb-xc-x-Cys-y-xrx) (I "), wherein xt is Ala, GIy, VaI, Thr or Ser and Xm is Arg.
5. The cyclic peptide of any one of claims 1 to 4, being a cyclic peptide of formula Y" or I" ":
cyclo(x|-xh-Cys-x-xa-xb-xc-x~Cys-y-xrXii) (I' ");
cyc(o(Xιιι-Xh~Cys-x-xa-xb-xc-x-Cys-y-Xi-Xιv) (I " ") ,
wherein xn is GIn, GIu, Asp or Asn and x)V is GIy or a GIy analogue.
6. The cyclic peptide of claim 4 or 5, wherein xi is Ala.
7. The cyclic peptide of any one of claims 4 to 6, wherein xS| is GIn or GIu.
8. The cyclic peptide of any one of claims 4 to 7, wherein xn is DGIu.
9. The cyclic peptide of any one of claims 1 to 8, wherein xc is Pro
10. The cyclic peptide of any one of claims 1 to 9, wherein xb is an acidic amino acid.
11. The cyclic peptide of any one of claims 1 to 10, wherein x except xa, xb or xc is not Pro.
12. The cyclic peptide of any one of claims 1 to 11 being a cyclic peptide of formula II, III or III':
cyc!o(X|-XrXrX-X2-X2~Cys~x~xa-xb~xc-x-Cys-y-XrXii) (II); cyclo(xrX2~x-Xi-x-Xi-XrX-X2-X2-Cys-x-xa-xb-x°-x-Cys-y-Xi-X|f) (til); cyc!o(X|[i, 2-X-X1-X-X1 -Xi -x-X2-X2-Cys-x-xa-xb-xc-x-Cys-y-Xi-Xιv) (IN'), wherein a) x-i is individually and independently selected from the group consisting of acidic amino acids; and/or b) X2 is individually and independently selected from the group consisting of basic amino acids.
13, The cyclic peptide of any one of claims 1 to 12 being a cyclic peptide of formula IV, V or V:
cyclo(xi-Xi-Xt-x4-X2-X2-Cys-x3-xa5-xb--xc-X2-Cys-y-XrX3-X3'Xιι) (IV); cyclo(xrX2-X4-Xi-X4"XrXi-X4"X2-X2-Cys-X3-xa5-xb-xc-X2-Cys-y-XrX3-X3-X4-X5-X2-Xιι)
(V);
cyclo(xιn. 2-x4-Xi-x4-Xi-xrX4-X2-X2-CyS"X3-xVxb'Xc-X2-Cys-y-XrX3-X3-X4-Xιv) (V'),
wherein a) Xi is individually and independently selected from the group consisting of acidic amino acids; b) X2 is individually and independently selected from the group consisting of basic amino acids; c) X3 is individually and independently selected from the group consisting of Leu, lie, VaI, Met, Trp, Tyr and Phe; d) X4 is individually and independently selected from the group consisting of Ser, Thr, Ala and GIy; and/or e) X5 is individually and independently selected from the group consisting of GIn and Asn.
14. The cyclic peptide of any one of claims 1 to 13, comprising the amino acid stretch Asp-Xxxi -XxX4- Arg-Arg-Cys-Xxx3-Asn-Asp-Pro-Lys; or
Glu-Ser-Asp~Xxxi-Xxx4-Arg-Arg-Cys-Xxx3-Asn-Asp-Pro-Lys,
wherein Xxxi is defined as in any one of claims 1a), 12a) and 13a), Xxx3 is defined as in claim 1a) or 13c) and/or Xxx4 is defined as in claim 1a) or 13d).
15. The cyclic peptide of any one of claims 1 to 14, comprising the amino acid stretch
Asp-G!u-Ala~Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys; or
Glu-Ser-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys.
16. The cyclic peptide of any one of claims 1 to 15 being a cyclic peptide selected from the group consisting of: a) a cyclic peptide formable or formed by the amino acid sequence as depicted in any one of SEQ ID NO. 25, 27, 1 , 2, 9 and 10; b) a cyclic peptide formable by an amino acid sequence as encoded by a nucleotide sequence as depicted in any one of SEQ ID NO. 26, 28, 5, 6, 13 and 14; c) a cyclic peptide formable by an amino acid sequence as encoded by a nucleotide sequence which differs from the nucleotide sequence as depicted in any one of SEQ ID NO. 26, 28, 5, 6, 13 and 14 due to the degeneracy of the genetic code; and d) a cyclic peptide of any one of formula Vl to IX':
cyclo(Arg-Ala-Glu-Ser-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys- Cys-ABu-Asp-Phe-Val-Thr-Gly) (IX')
cyclo(Ala-Asp~Glu-AIa-Arg-Arg~Cys-Tyr-Asn-Asp-Pro-Lys-Cys-ABu-
Asp-Phe-Val-Gln) (VI); cyciotAia-Arg-Ala-Giu-Ser-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro- Lys-Cys-ABu-Asp-Phe-Vai-Thr-Asn-Arg-GJn) (ViI);
cyclo(Aia-Asp~Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro-Lys-Cys-ABu-
Asp-Phe-Val-DGIu) (VIII);
cyclo(Ala-Arg-Ala-Giu--Ser-Asp-Glu-Ala-Arg-Arg-Cys-Tyr-Asn-Asp-Pro- Lys-Cys-ABu-Asp-Phe-Vai-Thr-Asn-Arg-DGlu) (IX).
17. The cyclic peptide of claim 16, wherein at least one of acidic amino acids is replaced by a different amino acid selected from the group consisting of acidic amino acids.
18. The cyclic peptide of claim 16 or 17, wherein at least one of the basic amino acids is replaced by a different amino acid selected from the group consisting of basic amino acids.
19. The cyclic peptide of any one of claims 16 to 18, wherein at least one of the aliphatic amino acid residues is replaced by a different amino acid selected from the group consisting of aSiphatic amino acids.
20 The cyclic peptide of any one of claims 1 to 19, wherein cycϋzation occurs by at least one linkage which is a covalent binding selected from the group consisting of S-S linkages, peptide bonds, carbon bonds such as C-C or C=C, ester bonds, ether bonds, azo bonds, C-S-C linkages, C-N-C linkages and C=N-C linkages.
21. The cyclic peptide of any one of claims 1 to 20, wherein cyclization occurs by at least two linkages which are an S-S linkage and a peptide bond.
22. The cyclic peptide of claim 20 or 21 , wherein said S-S linkage is formed by two Cys residues of the peptide.
23. The cyclic peptide of any one of claims 20 to 22, wherein said peptide bond is formed by the NH2 group of an N-terminal amino acid and the COOH group of an C4erminal amino acid.
24. The cyclic peptide of any one of claims 20 to 23, wherein additional bonds are formed by a side chain of NH2 groups and COOH groups of the constituent amino acids.
25. A nucleic acid molecule encoding an amino acid sequence which can (be modified to) form or can be used to form or to generate the amino acid backbone of the cyclic peptide of any one of claims 1 to 24.
26. The nucleic acid molecule of claim 25, comprising a nucleotide sequence as depicted in any one of SEQ ID NO. 5, 6, 13, 14, 26 and 28 or a nucleotide sequence which differs therefrom due to the degeneracy of the genetic code.
27. A vector comprising the nucleic acid molecule of claim 25 or 26.
28. A recombinant host cell comprising the nucleic acid molecule of claim 25 or 26 or the vector of claim 27.
29 A method for producing a cyclic peptide of any one of claims 1 to 24. comprising the steps of a) (i) culturing the recombinant host cell of claim 28 under conditions such that an amino acid sequence which can (be modified to) form or can be used to form or to generate the amino acid backbone of the polypeptide of any one of claims 1 to 24 is expressed, recovering said amino acid sequence and converting said amino acid sequence into said amino acid backbone; or
(ii) chemically synthesizing the amino acid backbone of the polypeptide of any one of claims 1 to 24; and b) cyclization of said amino acid backbone to form the cyclic peptide of any one of claims 1 to 24.
30. The method of claim 29, wherein said cyclization is defined as in any one of claims 20 to 24.
31. The method of claim 30, wherein said N-terminal amino acid is Ala or Arg and said C-terminal amino acid is GIn or GIu or GIy, respectively, or said N- terminal amino acid is Lys and said C~terminaS amino acid is Pro.
32. The method of claim 31 , wherein GIu is DGIu.
33. A cyclic peptide obtainable by the method of any one of claims 29 to 32.
34. A composition comprising a cyclic peptide of any one of claims 1 to 24 and 33, a nucleic acid molecule of claim 25 or 26, the vector of claim 27 or the recombinant host ceil of claim 28, and optionally a carrier.
35. The composition of ciaim 34, wherein said composition is a pharmaceutical composition and said carrier is a pharmaceutically acceptable carrier.
36. A method for a) the treatment, amelioration or prevention of a disease where the activity of a β-adrenergic receptor (β-AR) is enhanced; b) the treatment of a patient having antibodies against a β-AR; or c) inducing immune tolerance, comprising the step of administering to a patient in need of such medical intervention a pharmaceutically active amount of a cyclic peptide of any one of claims 1 to 24 and 33 and/or of the pharmaceutical composition of claim 35, and optionally a pharmaceutically acceptable carrier.
37. A cyclic peptide of any one of claims 1 to 24 and 33 or a pharmaceutical composition of claim 35, and optionally a pharmaceutically acceptable carrier, for a) the treatment, amelioration or prevention of a disease where the activity of a β-AR is enhanced; b) the treatment of a patient having antibodies against a β-AR; or c) inducing immune toierance.
38. The pharmaceutical composition, the method or the cyclic peptide of any one of claims 35 to 37, wherein said cyclic peptide is administered with or said pharmaceutical composition comprises at least one further pharmaceutically active agent.
39. The pharmaceutical composition, the method or the cyclic peptide of claim 38, wherein said at least one further pharmaceutically active agent is a β-receptor blocker.
40. The pharmaceutical composition, the method or the cyclic peptide of claim 39, wherein said β-receptor blocker is a selective β-AR blocker.
41. The pharmaceutical composition, the method or the cyclic peptide of claim 40, wherein said selective β-AR blocker is atenolol, metoproiol, nebivolol, and bisoproloS.
42. The method, the cyclic peptide or the pharmaceutical composition of any one of claims 36 to 41 , wherein said disease where the activity of a β-adrenergic receptor is enhanced is a heart disease or wherein said patient suffers from a heart disease.
43. The method or the cyclic peptide or the pharmaceutical composition of claim 42, wherein said heart disease is selected from the group consisting of infectious and non-infectious heart disease, ischemic and non-ischemic heart disease, inflammatory heart disease and myocarditis, cardiac dilatation, idiopathic cardio-myopathy, (idiopathic) dilated cardiomyopathy (DCM), immune-card iomyopathy, heart failure, and any cardiac arrhythmia including ventricular and/or supraventricular premature capture beats as well as any atrial arrhythmia including atrial fibriilation and/or atrial flutter.
44. The method or the cycϋc peptide or the pharmaceutical composition of claim 42 or 43, wherein said heart disease is (idiopathic) DCIvI.
45. The method, the cyclic peptide or the pharmaceutical composition of any one of claims 36 to 44, wherein said disease is induced by antibodies against a β- AR.
46. The method, the cyclic peptide or the pharmaceutical composition of any one of claims 36 to 41 , wherein said induction of immune tolerance is obtained by suppression of the production of antibodies against a β-AR.
47. The method, the cyclic peptide or the pharmaceutical composition of claim 46, wherein said induction of immune tolerance is obtained by suppression of the production of antibodies against a β-AR through blockade of the antigen- recognition sites of the antibody-producing early B-cetls and memory B-cells.
48. The method, the cyclic peptide or the pharmaceutical composition of any one of claims 36 to Al, wherein said cyclic peptide or said pharmaceutical composition is administered in that at least 0,05 mg of said cyclic peptide per kg body weight is reached.
49. Method for detecting antibodies against a β-AR (in a sample) comprising the step of contacting the cyclic peptide of any one of claims 1 to 24 and 33 with said antibodies to be detected.
50. The method, the cyclic peptide or the pharmaceutical composition of any one of claims 36 to 49, wherein said β-AR is β-i-AR.
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