Neutralization of pathogenic beta1-receptor autoantibodiesby aptamers in vivo: the first successful proof of principlein spontaneously hypertensive rats

Annekathrin Haberland • Gerd Wallukat • Sabine Berg • Angela-Martina Schulz •

Ernst-Joachim Freyse • Roland Vetter • Eckhard Salzsieder • Johannes Muller •

Reinhold Kreutz • Ingolf Schimke

Received: 15 January 2014 / Accepted: 3 April 2014

� Springer Science+Business Media New York 2014

Abstract Autoantibodies (AABs) against the second

extracellular loop of the beta1-receptor (beta1(II)-AABs)

are found as a pathogenic driver in patients with idiopathic

dilated cardiomyopathy, Chagas cardiomyopathy, peripar-

tum cardiomyopathy, and myocarditis, and have been

increasingly seen as a treatment target. We recently iden-

tified an aptamer (single short DNA strand) that specifi-

cally binds and neutralizes beta1(II)-AABs. Via application

of this aptamer, a new treatment strategy for diseases

associated with the cardio-pathogenic beta1(II)-AABs

could be developed. Spontaneously hypertensive rats

(SHR) positive for beta1(II)-AABs were treated five times

at weekly intervals (bolus application of 2 mg/kg body

weight followed by an infusion of the same amount over

20 min). SHR responded to aptamer treatment with a

strong reduction in the cardio-pathogenic beta1(II)-AABs.

The AABs did not substantially return within the study

period. No signs for aptamer toxicity were observed by

visual examination of the heart, liver, and kidney, or by

measurement of plasma CK, ALT, and creatinine. The

aptamer’s potential for beta1(II)-AAB neutralization and

consequently for cardiomyopathy treatment has been

shown for the first time in vivo.

Keywords Aptamer � Autoantibody � Beta1-receptor �Cardiomyopathy � Heart failure

Introduction

Autoantibodies (AABs) directed to the second extracellular

loop of the beta1-adrenoceptor (beta1(II)-AABs) belonging

to the group of pathogenic AABs against G-protein-cou-

pled receptors (GPCRs) [1] are found in patients with

idiopathic dilated cardiomyopathy (DCM) and Chagas

cardiomyopathy [2, 3] and are well accepted to drive the

pathogenesis [4–6]. The AABs have been also found in

patients with peripartum cardiomyopathy and in those with

myocarditis. We recently selected a single short-strand

DNA aptamer for specific binding and neutralization of

human beta1(II)-AABs [7, 8].

Recently [9], we presented an apheresis column con-

taining our aptamer that had been successfully tested for

in vitro clearing of beta1(II)-AAB-positive serum from

DCM patients and for extracorporeal apheresis of sponta-

neously hypertensive rats (SHR) positive for beta1(II)-

AABs. However, the aptamer’s use for in vivo beta1(II)-

AAB neutralization—recently announced as ‘‘…hopeful

avenue for future drug development to treat DCM’’ [10]—

should be with respect to costs and logistics superior to the

apheresis treatment. We supply here the first evidence for

A. Haberland and G. Wallukat have contributed equally to this work.

A. Haberland � I. Schimke (&)

Abteilung Pathobiochemie und Medizinische Chemie,

Charite – Universitatsmedizin Berlin, Chariteplatz 1,

10117 Berlin, Germany

e-mail: ingolf.schimke@charite.de

G. Wallukat

Max-Delbruck-Centrum fur Molekulare Medizin, Berlin-Buch,

Germany

S. Berg � E.-J. Freyse � E. Salzsieder

Institut fur Diabetes ‘Gerhardt Katsch’, Karlsburg, Germany

A.-M. Schulz � R. Vetter � R. Kreutz

Institut fur Klinische Pharmakologie und Toxikologie,

Charite – Universitatsmedizin Berlin, Berlin, Germany

J. Muller

Berlin Heals UG, Berlin, Germany

123

Mol Cell Biochem

DOI 10.1007/s11010-014-2057-8

the aptamer’s potency for in vivo beta1(II)-AAB neutral-

ization, which we show by a strong reduction in the auto-

antibody titer in beta1(II)-AAB-positive SHR following the

aptamer treatment.

Materials and methods

Ten SHR (SHR/FubRkb, SHR, RGD:631696; age

*30–32 weeks, body mass 378 ± 18 g) bred at the

‘‘Forschungseinrichtung fur Experimentelle Medizin’’,

Charite – Universitatsmedizin Berlin, and positive for

beta1(II)-AABs were treated (experiment License Number:

7221.3-1,1-006/11) with the aptamer dissolved in 0.9 %

NaCl solution (n = 5). Control rats (n = 5) were treated

with only 0.9 % NaCl.

For this purpose, chronic catheters (PhysioCath, DSI, St.

Paul, MN, USA) were inserted under general anesthesia.

For details see [9]. Following a convalescence period of

*7 days, the catheters were functional for the following

treatment period of 4 weeks.

Due to the aptamers’ short half-life, which was derived

from data of structurally comparable aptamers [11], treat-

ment was performed in a biphasic mode by bolus appli-

cation of 2 mg/kg body weight followed by an infusion of

the same amount over 20 min. The treatment procedure

was repeated five times at weekly intervals (see also

Fig. 1).

The rat beta1(II)-AAB titer was estimated by measuring

the beta1-AABs’ chronotropic activity using the bioassay

first described in [12] and modified used in [3, 7–9]. For

this purpose, heparin-plasma was prepared from tail vein

blood sampled before catheter implantation for conforma-

tion of the rat beta1(II)-AAB positivity, then 2 days after

the second and 2 days after the fourth aptamer application

as well as 2, 6, and 13 weeks after the fifth aptamer

application. The last blood sampling was performed at the

rat age of 56–58 weeks (21 weeks after the fifth aptamer

application) before they were sacrificed. Plasma CK, ALT,

and creatinine were measured with commercially available

assays. Heart, liver, and kidney were removed for visual

examination of toxic signs. Thereafter, the hearts were

shock-frozen, and serial histological slides (7 lm sections)

were produced. After staining the slides with hematoxylin/

eosin, the thickness of the heart walls and septum was

measured at six different levels using a slide-scanning

system (ScanScope CS, Aperio, US) and Image Scope�

software.

Over the study time, the animal’s body weight was

monitored and their mobility and coat appearance visually

assessed.

Results

As the main result of this study (Fig. 1), treated rats versus

controls demonstrated a strong reduction in the blood

beta1(II)-AAB titer. This was already visible at the first

time of investigation (2 days after the second aptamer

application). The lowest beta1(II)-AAB titer presented

following the fifth aptamer application. Despite finishing

the treatment, thereafter, the beta1(II)-AABs did not sub-

stantially return within the study period.

No differences between treated SHR versus controls in

mobility, coat appearance, and body weight (407 ± 20 vs.

406 ± 14 g) were obvious. At study end, no signs for apt-

amer toxicity were observed by visual examination of the

heart, liver, and kidney. Plasma ALP, CK, and creatinine in

treated and control rats also did not differ (1.20 ± 0.38 vs.

1.09 ± 0.17 lkat/L; 1.00 ± 0.29 vs. 0.81 ± 0.12 lkat/L,

34.00 ± 1.71 vs. 33.65 ± 2.93 lmol/L, respectively).

Treated rats versus controls presented with lower means of

their thickness of left ventricle wall (3.49 ± 0.34 vs. 3.90 ±

0.44 mm) and septum (2.95 ± 0.45 vs. 3.28 ± 0.81 mm).

Discussion

We present here—using the model of beta1(II)-AAB-

positive SHR—for the first time evidence for reducing of

cardio-pathogenic beta1(II)-AABs due to in vivo treatment

with a recently selected aptamer [7, 8]. This aptamer has

been previously recognized, using cell [8] and apheresis [9]

experiments, as a highly specific binder and neutralizer of

Fig. 1 In vivo neutralization of cardio-pathogenic autoantibodies

against the second extracellular loop of the beta1-receptor (beta1(II)-

AABs) by aptamer treatment in spontaneously hypertensive rats.

Treatment was performed in a biphasic mode by bolus application of

2 mg/kg body weight dissolved in 0.9 % NaCl solution followed by

an infusion of the same amount over 20 min. The treatment procedure

was repeated five times at weekly intervals. Control rats were treated

with 0.9 % NaCl. Beta1(II)-AAB titers were estimated using the

bioassay as indicated under materials and methods. Titers represent

the chronotropic activity of beta1(II)-AABs expressed as Dbeats/min.

Day of treatment and beta1(II)-AAB testing time are indicated

Mol Cell Biochem

123

not only of rat beta1(II)-AABs but also, and more impor-

tantly, of human beta1(II)-AABs. Consequently, in addi-

tion to the concept of DCM treatment with peptides—

derived from the beta1(II)-AAB-related epitope of the

second beta1-receptor loop—for beta1(II)-AAB neutral-

ization [13], an aptamer-based treatment of this important

human disease seems to be possible. Due to the chemical,

biochemical, and immunological properties of aptamers in

general [11, 14], such a treatment strategy may even be

superior to peptide treatment. The aptamer could also

potentially be used for treating patients suffering from

myocarditis, Chagas’ cardiomyopathy, and peripartum

cardiomyopathy whose pathogenic beta1(II)-AABs were

also neutralized by the aptamer [7]; an effect that must be

still shown for the peptides.

Most importantly, the reduced beta1(II)-AAB titer per-

sisted after finishing the treatment. For this lack of auto-

antibody re-expression despite treatment interruption, there

is presently no satisfactory explanation. However, ongoing

reduced beta1(II)-AAB levels have also been seen after

finishing beta1(II)-AAB immunoapheresis in humans [15].

The lower means for heart morphology measures in the

treated animals could be seen as the first indication for the

heart benefit of aptamer treatment for in vivo neutralization

of cardio-pathogenic beta1(II)-AABs. The lack of a statis-

tically significant difference in left ventricular wall thick-

ness between the treated and the untreated animals is in total

agreement with the observation in patients after immuno-

adsorption. In patients, it will take at least 3 months until

significant improvements in heart function will be observed.

Significant changes in the morphology of the heart will not

be seen before a follow-up period of 6 months [16].

In line with this, for statistical manifestation of the heart

benefit following the aptamer treatment, animal studies are

necessary in future with increasing group size, longer

observation time, and additional measurement of functional

parameters for heart benefit.

Study limitations

According to the prior aims, the study design (old SHR

positive for beta1-AABs as model, group size, observation

time) was adequate to evidence the in vivo beta1(II)-AAB

neutralization by the aptamer and to follow-up of the

autoantibody titer after the treatment. In contrast, the par-

allel analysis using this design of functional and structural

heart changes is limited. SHR present in this age already

with manifested functional heart pathology with only very

mild further progression. Consequently, extended group

size and methods with low variance for measurement of

function especially such as an implantable telemetry sys-

tem seem to be prerequisite to evidence any functional

heart benefit. To demonstrate significant treatment benefit

for heart structure, longer observation time is necessary.

Considering the economics and ethics as well as the prior

aims of the study, however, we believe that the chosen

study design is acceptable for the presented ‘‘proof of

concept study’’.

Conclusion

The reduction in cardio-pathogenic beta1(II)-AABs due to

aptamer treatment could open the door to a new treatment

concept suitable for patients with DCM, myocarditis,

Chagas cardiomyopathy, and peripartum cardiomyopathy.

However, a distinct number of these heart patients present

additionally with further cardio-pathogenic AABs against

GPCRs mainly such as AABs against the first extracellular

loop of the beta1-receptor (beta1(I)-AABs) and the mus-

carinergic 2-receptor (M2-AABs); for the last AABs, even

100 % of Chagas cardiomyopathy patients are positive.

Introducing an aptamer that is presently under-study [17]

and able to neutralize a numerous of the cardio-pathogenic

AABs against GPCRs including beta1(I)-, beta(II)-, and

M2-AABs could highlight the concept of aptamer-based

treatment of heart patients with cardiomyopathy and

myocarditis.

Acknowledgments We are grateful to Jens Peter Teifke (Friedrich-

Loeffler-Institut, Insel Riems, Germany) for providing the slide-

scanning system and his support in morphometric measurements as

well as to ‘‘European Regional Development Fund’’ (10141685),

Berlin, Germany and ‘‘Stiftung Pathobiochemie der Deutschen

Gesellschaft fur Klinische Chemie und Laboratoriumsmedizin’’ (105/

2011, 128/2013), Bonn, Germany for financial support.

References

1. Xia Y, Kellems RE (2011) Receptor-activating autoantibodies

and disease: preeclampsia and beyond. Expert Rev Clin Immunol

7:659–674

2. Wallukat G, Nissen E, Muller J, Brinkmann R, Schimke I, Kunze

R (2003) The pathophysiological role of autoantibodies directed

to G-protein coupled receptors and therapeutic strategies of

antibody removal. In: Kunze R, Brinkmann R (eds) G-protein

coupled receptors and autoantibodies. Pabst Science Publishers,

Lengerich, pp 7–47

3. Wallukat G, Munoz Saravia SG, Haberland A, Bartel S, Araujo

R, Valda G, Duchen D, Diaz Ramirez I, Borges AC, Schimke I

(2010) Distinct patterns of autoantibodies against G-protein-

coupled receptors in Chagas’ cardiomyopathy and megacolon:

their potential impact for early risk assessment in asymptomatic

Chagas’ patients. J Am Coll Cardiol 55:463–468

4. Matsui S, Fu ML, Hayase M, Katsuda S, Yamaguchi N, Teraoka

K, Kurihara T, Takekoshi N (1999) Active immunization of

combined beta1-adrenoceptor and M2-muscarinic receptor pep-

tides induces cardiac hypertrophy in rabbits. J Card Fail

5:246–254

Mol Cell Biochem

123

5. Jahns R, Boivin V, Hein L, Triebel S, Angermann CE, Ertl G,

Lohse MJ (2004) Direct evidence for a beta 1-adrenergic recep-

tor-directed autoimmune attack as a cause of idiopathic dilated

cardiomyopathy. J Clin Invest 113:1419–1429

6. Zuo L, Bao H, Tian J, Wang X, Zhang S, He Z, Yan L, Zhao R,

Ma XL, Liu H (2011) Long-term active immunization with a

synthetic peptide corresponding to the second extracellular loop

of b1-adrenoceptor induces both morphological and functional

cardiomyopathic changes in rats. Int J Cardiol 149:89–94

7. Schimke I, Haberland A, Kage A, Wallukat G, Dahmen C (2012)

Aptamers that inhibit interaction between antibody and 2nd

extracellular loop of human beta-1-adrenergic receptor. WO

2012000889, 05 Jan 2012

8. Haberland A, Wallukat G, Dahmen C, Kage A, Schimke I (2011)

Aptamer neutralization of beta1-adrenoceptor autoantibodies iso-

lated from patients with cardiomyopathies. Circ Res 109:986–992

9. Wallukat G, Haberland A, Berg S, Schulz A, Freyse EJ, Dahmen C,

Kage A, Dandel M, Vetter R, Salzsieder E, Kreutz R, Schimke I

(2012) The first aptamer-apheresis column specifically for clearing

blood of b1-receptor autoantibodies. Circ J 76:2449–2455

10. Patel PA, Hernandez AF (2013) Targeting anti-beta-1-adrenergic

receptor antibodies for dilated cardiomyopathy. Eur J Heart Fail

15:724–729

11. Keefe AD, Pai S, Ellington A (2010) Aptamers as therapeutics.

Nat Rev Drug Discov 9:537–550

12. Wallukat G, Wollenberger A (1987) Effects of the serum gamma

globulin fraction of patients with allergic asthma and dilated

cardiomyopathy on chronotropic beta adrenoceptor function in

cultured neonatal rat heart myocytes. Biomed Biochim Acta

46:S634–S639

13. Jahns R, Jahns V, Lohse M, Palm D (2006) Means for the inhibition

of anti-beta1-adrenergic receptor antibodies. WO 2006103101 A2,

31 Mar 2006

14. Marquis JK, Grindel JM (2000) Toxicological evaluation of oli-

gonucleotide therapeutics. Curr Opin Mol Ther 2:258–263

15. Dandel M, Wallukat G, Englert A, Lehmkuhl HB, Knosalla C,

Hetzer R (2012) Long-term benefits of immunoadsorption in

b(1)-adrenoceptor autoantibody-positive transplant candidates

with dilated cardiomyopathy. Eur J Heart Fail 14:1374–1388

16. Mueller J, Wallukat G, Dandel M, Bieda H, Brandes K, Spiegels-

berger S, Nissen E, Kunze R, Hetzer R (2000) Immunoglobulin

adsorption in patients with idiopathic dilated cardiomyopathy.

Circulation 101:385–391

17. Schimke I, Haberland A, Wallukat G (2012) Use of aptamers in

therapy and/or diagnosis of autoimmune diseases. WO 201211

9938, 13 Sept 2012

Mol Cell Biochem

123