396
New Technologies, Diagnostic Tools and Drugs
Epitope mapping via selection of anti-FVIII antibody-specific phagepresented peptide ligands that mimic the antibody binding sites Joerg Kahle1; Aleksander Orlowski1; Diana Stichel1; Karin Becker-Peters1; Ali Kabiri1; John F. Healey2; Kerstin Brettschneider1; Anja Naumann1; Anna Katharina Scherger1; Pete Lollar2; Dirk Schwabe1; Christoph Königs1,* 1Clinical
and Molecular Haemostasis and Immunodeficiency, Department of Paediatrics, Goethe University Hospital, Frankfurt am Main, Germany; 2Aflac Cancer & Blood Disorders Center, Department of Pediatrics, Children’s Healthcare of Atlanta and Emory University, Atlanta, Georgia; USA
Summary The most serious complication in today’s treatment of congenital haemophilia A is the development of neutralising antibodies (inhibitors) against factor VIII (FVIII). Although FVIII inhibitors can be eliminated by immune tolerance induction (ITI) based on repeated administration of high doses of FVIII, 20–30% of patients fail to become tolerant. Persistence of FVIII inhibitors is associated with increased morbidity and mortality. Data from recent studies provide evidence for a potential association between ITI outcome and epitope specificity of FVIII inhibitors. Nevertheless the determination of epitopes and their clinical relevance has not yet been established. In this study a general strategy for the identification of anti-FVIII antibody epitopes in haemophilia A patient plasma was to be demonstrated. Phage-displayed peptide libraries were screened against anti-FVIII antibodies to isolate specific peptides. Peptide specificity was confirmed by FVIII-sensitive
Correspondence to: Christoph Königs, MD PhD University Hospital Frankfurt Department of Paediatrics Clinical and Molecular Haemostasis Theodor-Stern-Kai 7 60596 Frankfurt am Main, Germany Tel.: +49 69 6301 83030, Fax: +49 69 6301 83991 E-mail: [email protected]
Introduction Current treatment of congenital haemophilia A is based on prophylactic or therapeutic infusion of recombinant or plasma derived factor VIII (FVIII) to restore haemostasis (1). Up to 30% of patients with severe haemophilia A develop inhibitory anti-FVIII antibodies (FVIII inhibitors) as a result of the protein replacement therapy (2). Inhibitor development renders FVIII replacement therapy ineffective and is the most serious complication in today’s treatment. FVIII inhibitors can be eliminated by repeated administration of high doses of FVIII (3). However, 20-30% of patients undergoing this so-called immune tolerance induction (ITI) fail to become tolerant, which results in increased morbidity and mortality (4, 5). Data of recent studies provide evidence for a potential association between outcome of ITI and domain (epitope) specificity of FVIII inhibitors (6, 7). The aim of this study was therefore to develop a general and highly specific strategy for the identification of FVIII-specific antibody epitopes in haemophilia A patients. FVIII inhibitors primarily consist of a polyclonal IgG population that recognise multiple epitopes (8, 9). Major FVIII in© Schattauer 2015
ELISA binding. Peptide residues essential for antibody binding were identified by mutational analysis and epitopes were predicted via FVIII homology search. The proposed mapping strategy was validated for the monoclonal murine antibody (mAb) 2–76. Binding studies with FVIII variants confirmed the location of the predicted epitope at the level of individual amino acids. In addition, anti-FVIII antibody-specific peptide ligands were selected for 10 haemophilia A patients with FVIII inhibitors. Detailed epitope mapping for three of them showed binding sites on the A2, A3 and C2 domains. Precise epitope mapping of anti-FVIII antibodies using antibody-specific peptide ligands can be a useful approach to identify antigenic sites on FVIII.
Keywords Haemophilia A, anti-FVIII antibodies, inhibitors, epitope mapping, phage display
Received: January 31, 2014 Accepted after major revision: August 15, 2014 Epub ahead of print: December 18, 2014 http://dx.doi.org/10.1160/TH14-01-0101 Thromb Haemost 2015; 113: 396–405
hibitor epitopes have been defined within the A2, A3-C1, and C2 domains by several methods including immunoprecipitation, western blotting, antibody neutralisation, and homolog scanning mutagenesis (10). Earlier studies also used the phage peptide display technique to isolate peptides that bind specifically to the monoclonal FVIII inhibitors ESH8 and BO2C11 (11, 12). The BO2C11-specific peptides were subsequently used to validate epitope prediction software (13, 14) by comparison with the structurally defined BO2C11 epitope (15). Selection of peptide epitope surrogates by phage display was successfully extended to polyclonal FVIII inhibitors from haemophilia A patients as a target (16, 17). The mapping strategy described in this study is based on these previous experiences and comprises the following three steps: (i) selection of antibody-specific peptides via phage display, (ii) identification of essential antibody binding peptide residues by mutational analysis, and (iii) alignments of essential peptide residues to FVIII and its verification. Firstly, this strategy was applied for the well-characterised monoclonal murine antibody (mAb) 2-76 and successfully estabThrombosis and Haemostasis 113.2/2015
Downloaded from www.thrombosis-online.com on 2015-06-09 | ID: 1000480351 | IP: 130.179.16.201 For personal or educational use only. No other uses without permission. All rights reserved.
397
Kahle et al. Epitope mapping of anti-FVIII antibodies
lished by identifying an epitope in the A2 region, which is known to contain its functional epitope (18). Eventually, evidence is presented that this strategy can also be used to map epitopes of antiFVIII antibodies in haemophilia A patients’ plasma. The potential use of this strategy for the identification of FVIII inhibitor epitopes in a larger cohort of haemophilia A patients is discussed.
logies and (ii) compared to the tertiary structure of B domain-deleted (BDD) FVIII (PDB 3CDZ) (19) with the program EpiSearch (http://curie.utmb.edu/episearch.html) using a patch size of 10 Å, area cut-off of 10 Å2, and accuracy cut-off of 2 (13). Amino acid residues of the predicted epitopes were visualised by PyMOL® software (DeLano Scientific, San Francisco, CA, USA).
Patients, materials and methods
Phagotope ELISA
Patients Ten haemophilia A patients with FVIII inhibitors (1.1 - 200.9 BU/ ml) were studied. Residual plasma from patients at a single time point was analysed. Institutional review board (IRB) approval was granted and patients provided informed consent.
Screening of phage displayed random peptide libraries (PDPLs) The affinity selection of phages (biopanning) was performed similarly to our previous studies (17). For positive selections, IgGs from FVIII inhibitor-positive plasma was immobilized on streptavidin-coated beads (MyOneTM Streptavidin DynaBeads®, Invitrogen, Darmstadt, Germany) via a biotin-conjugated goat antihuman IgG (Invitrogen, Darmstadt, Germany). For negative selections, IgGs from a plasma pool of healthy individuals negative for FVIII-specific antibodies were immobilised likewise. For selection of peptides that bind to the murine monoclonal anti-FVIII antibody (mAb) 2-76 (Green Mountain Antibodies, Burlington, MA, USA) immobilisation on streptavidin-coated beads was achieved via a biotin-conjugated rabbit anti-mouse IgG (Invitrogen). For negative selections, an appropriate IgG isotype (murine IgG2a) was used. Immobilised IgGs were screened with three different PDPLs (Ph.D™-12, -7 and C7C PDPL, New England Biolabs, Frankfurt, Germany). Binding peptides were eluted via pH-shift (using glycine–HCl, pH 2.2) or competition with rFVIII (Helixate NexGen®, CSL Behring or Kogenate™, Bayer, Leverkusen, Germany) in excess. Three positive and two negative selections were performed. The resulting phage pool was titred according to the manufacturer’s (New England Biolabs) instructions. Single phage clones (from each library) presenting individual peptides (phagotopes) were isolated and further characterised. DNA sequencing was performed using the -96 gIII sequencing primer (New England Biolabs, Frankfurt, Germany) and sequences were analyzed by DNAStar® software (DNASTAR Inc., Madison, WI, USA).
Sequence alignment and FVIII homology search To identify consensus motifs, linear peptide sequences derived from biopanning against a specific target were aligned with the program Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clusta lo/). Shading of alignment files was performed with the program Boxshade (http://www.ch.embnet.org/software/BOX_form.html). Peptide sequences containing the essential amino acid residues were (i) linearly aligned to the FVIII sequence to identify homo-
Phage binding to anti-FVIII antibodies (capture ELISA) was tested in microtitre plates coated overnight at 4°C with either (i) 0.05 µg/well (0.34 pmol/well) mAb 2-76 in 100 mM bicarbonate buffer, pH 9.6 or (ii) patient plasma 1:1,000 in PBS (Lonza, Walkersville, MD, USA). Plates were blocked for 2 hours (h) at room temperature with 5% milk powder, 0.05% Tween-20 in PBS (MPBST). Approximately 1x1010 plaque forming units (PFU) were added in MPBST for 2 h at room temperature or at 4°C overnight. In addition, phages were also purified according to the manufacturer’s instructions (New England Biolabs) prior to incubation (confirmatory ELISA). For competition of phage binding by FVIII (specificity ELISA) immobilised antibodies were pre-incubated for 30 minutes (min) with the indicated amount of FVIII in 50 μl MPBST followed by the addition of 1x109 PFU in 50 μl MPBST for 2 h at room temperature. Bound phages were detected by incubation with HRP-conjugated anti-M13 antibody (GE Healthcare Amersham, Munich, Germany) 1:5,000 in MPBST for 2 h at room temperature. Wells were developed with o-phenylenediamine (OPD) and absorption was measured at 492 nm and 620 nm (reference). Murine IgG2a of irrelevant specificity and pooled plasma of healthy individuals (negative for FVIII-specific antibodies) were used as negative controls.
Peptide ELISA Peptides selected via biopanning were purchased as N-terminally biotinylated peptides (BioTides™; JPT Peptide Technologies GmbH, Berlin, Germany) and analysed for binding to (i) mAb 2-76 or (ii) anti-FVIII antibodies from patient plasma. For that, biotinylated peptides (1 pmol/well for mAb 2-76 and 5-20 pmol/ well for patient plasma) were immobilised on streptavidin-coated microtitre plates (Microlon 600, Greiner BioOne, Solingen, Germany) and incubated with mAb 2-76 or diluted patient plasma after blocking. Specificity of antibody binding was tested with murine IgG2a of irrelevant specificity (isotype control) for mAb 2-76, pooled plasma of healthy individuals (for patient IgGs), and antibody binding in the presence of the indicated amount of FVIII. Bound murine mAb 2-76 antibodies were detected using an HRPconjugated rat anti-mouse IgG (H+L) antibody (Dianova, Hamburg, Germany) while IgGs from patient plasma were detected using an HRP-conjugated goat anti-human IgG (H+L) antibody (Invitrogen). Bound IgGs were detected with OPD and absorption was measured at 492 nm and 620 nm (reference). Streptavidincoated plates with biotin instead of biotinylated peptides were used as negative control.
Thrombosis and Haemostasis 113.2/2015 Downloaded from www.thrombosis-online.com on 2015-06-09 | ID: 1000480351 | IP: 130.179.16.201 For personal or educational use only. No other uses without permission. All rights reserved.
© Schattauer 2015
398
Kahle et al. Epitope mapping of anti-FVIII antibodies
A
B
C
E
D Figure 1: Sequences, prevalence and consensus motif of phage peptides. Phage-displayed peptide libraries (PDPLs) were screened against mAb 2–76 (A) and haemophilia A patient IgGs (B-E). Two independent screenings were performed for patient 1 (B). The isolated phage-presented peptide sequences were aligned to each other by the use of the software Clustal Omega and subsequently shaded with the software Boxshade. Amino acids
shared by at least 45% (number of clones >10) or 50% (number of clones
New Technologies, Diagnostic Tools and Drugs
Epitope mapping via selection of anti-FVIII antibody-specific phagepresented peptide ligands that mimic the antibody binding sites Joerg Kahle1; Aleksander Orlowski1; Diana Stichel1; Karin Becker-Peters1; Ali Kabiri1; John F. Healey2; Kerstin Brettschneider1; Anja Naumann1; Anna Katharina Scherger1; Pete Lollar2; Dirk Schwabe1; Christoph Königs1,* 1Clinical
and Molecular Haemostasis and Immunodeficiency, Department of Paediatrics, Goethe University Hospital, Frankfurt am Main, Germany; 2Aflac Cancer & Blood Disorders Center, Department of Pediatrics, Children’s Healthcare of Atlanta and Emory University, Atlanta, Georgia; USA
Summary The most serious complication in today’s treatment of congenital haemophilia A is the development of neutralising antibodies (inhibitors) against factor VIII (FVIII). Although FVIII inhibitors can be eliminated by immune tolerance induction (ITI) based on repeated administration of high doses of FVIII, 20–30% of patients fail to become tolerant. Persistence of FVIII inhibitors is associated with increased morbidity and mortality. Data from recent studies provide evidence for a potential association between ITI outcome and epitope specificity of FVIII inhibitors. Nevertheless the determination of epitopes and their clinical relevance has not yet been established. In this study a general strategy for the identification of anti-FVIII antibody epitopes in haemophilia A patient plasma was to be demonstrated. Phage-displayed peptide libraries were screened against anti-FVIII antibodies to isolate specific peptides. Peptide specificity was confirmed by FVIII-sensitive
Correspondence to: Christoph Königs, MD PhD University Hospital Frankfurt Department of Paediatrics Clinical and Molecular Haemostasis Theodor-Stern-Kai 7 60596 Frankfurt am Main, Germany Tel.: +49 69 6301 83030, Fax: +49 69 6301 83991 E-mail: [email protected]
Introduction Current treatment of congenital haemophilia A is based on prophylactic or therapeutic infusion of recombinant or plasma derived factor VIII (FVIII) to restore haemostasis (1). Up to 30% of patients with severe haemophilia A develop inhibitory anti-FVIII antibodies (FVIII inhibitors) as a result of the protein replacement therapy (2). Inhibitor development renders FVIII replacement therapy ineffective and is the most serious complication in today’s treatment. FVIII inhibitors can be eliminated by repeated administration of high doses of FVIII (3). However, 20-30% of patients undergoing this so-called immune tolerance induction (ITI) fail to become tolerant, which results in increased morbidity and mortality (4, 5). Data of recent studies provide evidence for a potential association between outcome of ITI and domain (epitope) specificity of FVIII inhibitors (6, 7). The aim of this study was therefore to develop a general and highly specific strategy for the identification of FVIII-specific antibody epitopes in haemophilia A patients. FVIII inhibitors primarily consist of a polyclonal IgG population that recognise multiple epitopes (8, 9). Major FVIII in© Schattauer 2015
ELISA binding. Peptide residues essential for antibody binding were identified by mutational analysis and epitopes were predicted via FVIII homology search. The proposed mapping strategy was validated for the monoclonal murine antibody (mAb) 2–76. Binding studies with FVIII variants confirmed the location of the predicted epitope at the level of individual amino acids. In addition, anti-FVIII antibody-specific peptide ligands were selected for 10 haemophilia A patients with FVIII inhibitors. Detailed epitope mapping for three of them showed binding sites on the A2, A3 and C2 domains. Precise epitope mapping of anti-FVIII antibodies using antibody-specific peptide ligands can be a useful approach to identify antigenic sites on FVIII.
Keywords Haemophilia A, anti-FVIII antibodies, inhibitors, epitope mapping, phage display
Received: January 31, 2014 Accepted after major revision: August 15, 2014 Epub ahead of print: December 18, 2014 http://dx.doi.org/10.1160/TH14-01-0101 Thromb Haemost 2015; 113: 396–405
hibitor epitopes have been defined within the A2, A3-C1, and C2 domains by several methods including immunoprecipitation, western blotting, antibody neutralisation, and homolog scanning mutagenesis (10). Earlier studies also used the phage peptide display technique to isolate peptides that bind specifically to the monoclonal FVIII inhibitors ESH8 and BO2C11 (11, 12). The BO2C11-specific peptides were subsequently used to validate epitope prediction software (13, 14) by comparison with the structurally defined BO2C11 epitope (15). Selection of peptide epitope surrogates by phage display was successfully extended to polyclonal FVIII inhibitors from haemophilia A patients as a target (16, 17). The mapping strategy described in this study is based on these previous experiences and comprises the following three steps: (i) selection of antibody-specific peptides via phage display, (ii) identification of essential antibody binding peptide residues by mutational analysis, and (iii) alignments of essential peptide residues to FVIII and its verification. Firstly, this strategy was applied for the well-characterised monoclonal murine antibody (mAb) 2-76 and successfully estabThrombosis and Haemostasis 113.2/2015
Downloaded from www.thrombosis-online.com on 2015-06-09 | ID: 1000480351 | IP: 130.179.16.201 For personal or educational use only. No other uses without permission. All rights reserved.
397
Kahle et al. Epitope mapping of anti-FVIII antibodies
lished by identifying an epitope in the A2 region, which is known to contain its functional epitope (18). Eventually, evidence is presented that this strategy can also be used to map epitopes of antiFVIII antibodies in haemophilia A patients’ plasma. The potential use of this strategy for the identification of FVIII inhibitor epitopes in a larger cohort of haemophilia A patients is discussed.
logies and (ii) compared to the tertiary structure of B domain-deleted (BDD) FVIII (PDB 3CDZ) (19) with the program EpiSearch (http://curie.utmb.edu/episearch.html) using a patch size of 10 Å, area cut-off of 10 Å2, and accuracy cut-off of 2 (13). Amino acid residues of the predicted epitopes were visualised by PyMOL® software (DeLano Scientific, San Francisco, CA, USA).
Patients, materials and methods
Phagotope ELISA
Patients Ten haemophilia A patients with FVIII inhibitors (1.1 - 200.9 BU/ ml) were studied. Residual plasma from patients at a single time point was analysed. Institutional review board (IRB) approval was granted and patients provided informed consent.
Screening of phage displayed random peptide libraries (PDPLs) The affinity selection of phages (biopanning) was performed similarly to our previous studies (17). For positive selections, IgGs from FVIII inhibitor-positive plasma was immobilized on streptavidin-coated beads (MyOneTM Streptavidin DynaBeads®, Invitrogen, Darmstadt, Germany) via a biotin-conjugated goat antihuman IgG (Invitrogen, Darmstadt, Germany). For negative selections, IgGs from a plasma pool of healthy individuals negative for FVIII-specific antibodies were immobilised likewise. For selection of peptides that bind to the murine monoclonal anti-FVIII antibody (mAb) 2-76 (Green Mountain Antibodies, Burlington, MA, USA) immobilisation on streptavidin-coated beads was achieved via a biotin-conjugated rabbit anti-mouse IgG (Invitrogen). For negative selections, an appropriate IgG isotype (murine IgG2a) was used. Immobilised IgGs were screened with three different PDPLs (Ph.D™-12, -7 and C7C PDPL, New England Biolabs, Frankfurt, Germany). Binding peptides were eluted via pH-shift (using glycine–HCl, pH 2.2) or competition with rFVIII (Helixate NexGen®, CSL Behring or Kogenate™, Bayer, Leverkusen, Germany) in excess. Three positive and two negative selections were performed. The resulting phage pool was titred according to the manufacturer’s (New England Biolabs) instructions. Single phage clones (from each library) presenting individual peptides (phagotopes) were isolated and further characterised. DNA sequencing was performed using the -96 gIII sequencing primer (New England Biolabs, Frankfurt, Germany) and sequences were analyzed by DNAStar® software (DNASTAR Inc., Madison, WI, USA).
Sequence alignment and FVIII homology search To identify consensus motifs, linear peptide sequences derived from biopanning against a specific target were aligned with the program Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clusta lo/). Shading of alignment files was performed with the program Boxshade (http://www.ch.embnet.org/software/BOX_form.html). Peptide sequences containing the essential amino acid residues were (i) linearly aligned to the FVIII sequence to identify homo-
Phage binding to anti-FVIII antibodies (capture ELISA) was tested in microtitre plates coated overnight at 4°C with either (i) 0.05 µg/well (0.34 pmol/well) mAb 2-76 in 100 mM bicarbonate buffer, pH 9.6 or (ii) patient plasma 1:1,000 in PBS (Lonza, Walkersville, MD, USA). Plates were blocked for 2 hours (h) at room temperature with 5% milk powder, 0.05% Tween-20 in PBS (MPBST). Approximately 1x1010 plaque forming units (PFU) were added in MPBST for 2 h at room temperature or at 4°C overnight. In addition, phages were also purified according to the manufacturer’s instructions (New England Biolabs) prior to incubation (confirmatory ELISA). For competition of phage binding by FVIII (specificity ELISA) immobilised antibodies were pre-incubated for 30 minutes (min) with the indicated amount of FVIII in 50 μl MPBST followed by the addition of 1x109 PFU in 50 μl MPBST for 2 h at room temperature. Bound phages were detected by incubation with HRP-conjugated anti-M13 antibody (GE Healthcare Amersham, Munich, Germany) 1:5,000 in MPBST for 2 h at room temperature. Wells were developed with o-phenylenediamine (OPD) and absorption was measured at 492 nm and 620 nm (reference). Murine IgG2a of irrelevant specificity and pooled plasma of healthy individuals (negative for FVIII-specific antibodies) were used as negative controls.
Peptide ELISA Peptides selected via biopanning were purchased as N-terminally biotinylated peptides (BioTides™; JPT Peptide Technologies GmbH, Berlin, Germany) and analysed for binding to (i) mAb 2-76 or (ii) anti-FVIII antibodies from patient plasma. For that, biotinylated peptides (1 pmol/well for mAb 2-76 and 5-20 pmol/ well for patient plasma) were immobilised on streptavidin-coated microtitre plates (Microlon 600, Greiner BioOne, Solingen, Germany) and incubated with mAb 2-76 or diluted patient plasma after blocking. Specificity of antibody binding was tested with murine IgG2a of irrelevant specificity (isotype control) for mAb 2-76, pooled plasma of healthy individuals (for patient IgGs), and antibody binding in the presence of the indicated amount of FVIII. Bound murine mAb 2-76 antibodies were detected using an HRPconjugated rat anti-mouse IgG (H+L) antibody (Dianova, Hamburg, Germany) while IgGs from patient plasma were detected using an HRP-conjugated goat anti-human IgG (H+L) antibody (Invitrogen). Bound IgGs were detected with OPD and absorption was measured at 492 nm and 620 nm (reference). Streptavidincoated plates with biotin instead of biotinylated peptides were used as negative control.
Thrombosis and Haemostasis 113.2/2015 Downloaded from www.thrombosis-online.com on 2015-06-09 | ID: 1000480351 | IP: 130.179.16.201 For personal or educational use only. No other uses without permission. All rights reserved.
© Schattauer 2015
398
Kahle et al. Epitope mapping of anti-FVIII antibodies
A
B
C
E
D Figure 1: Sequences, prevalence and consensus motif of phage peptides. Phage-displayed peptide libraries (PDPLs) were screened against mAb 2–76 (A) and haemophilia A patient IgGs (B-E). Two independent screenings were performed for patient 1 (B). The isolated phage-presented peptide sequences were aligned to each other by the use of the software Clustal Omega and subsequently shaded with the software Boxshade. Amino acids
shared by at least 45% (number of clones >10) or 50% (number of clones
