Available online at www.sciencedirect.com
ScienceDirect Neuromuscular Disorders 24 (2014) 195–200 www.elsevier.com/locate/nmd
Monoclonal antibodies for clinical trials of Duchenne muscular dystrophy therapy Le Thanh Lam a, Nguyen Thi Man a, Glenn E. Morris a,b,⇑ a
Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry SY10 7AG, UK b Institute for Science and Technology in Medicine, Keele University, Keele, UK Received 15 November 2013; accepted 27 November 2013
Abstract Most pathogenic mutations in Duchenne and Becker muscular dystrophies involve deletion of single or multiple exons from the dystrophin gene, so exon-specific monoclonal antibodies (mAbs) can be used to distinguish normal and mutant dystrophin proteins. In Duchenne therapy trials, mAbs can be used to identify or rule out dystrophin-positive “revertant” fibres, which have an internally-deleted dystrophin protein and which occur naturally in some Duchenne patients. Using phage-displayed peptide libraries, we now describe the new mapping of the binding sites of five dystrophin mAbs to a few amino-acids within single exons. The phage display method also confirmed previous mapping of MANEX1A (exon 1) and MANDRA1 (exon 77) by other methods. Of the 79 dystrophin exons, mAbs are now available against single exons 1, 6, 8, 12, 13, 14, 17, 21, 26, 28, 38, 41, 43, 44, 45, 46, 47, 50, 51, 58, 59, 62, 63, 75 and 77. Many have been used in clinical trials, as well as for diagnosis and studies of dystrophin isoforms. Ó 2013 Elsevier B.V. All rights reserved. Keywords: Duchenne muscular dystrophy; Dystrophin; Monoclonal antibody; Epitope mapping; Clinical trial; Exon-skipping; Therapy; Phage display
1. Introduction One of the long-term aims of the MDA Monoclonal Antibody Resource (www.glennmorris.org.uk/mabs.htm) has been to produce monoclonal antibodies (mAbs) against dystrophin that recognise specific amino-acid sequences spread throughout its 79 exons and 3684 amino-acids. We have paid particular attention to mAbs against the exons that are commonly deleted in Duchenne muscular dystrophy; these can be used to characterise mutant Becker dystrophins at the protein level in muscle biopsies [1]. The antibodies have also been used to characterise some of the short forms of dystrophin, including Dp71 [2–4], Dp140 [5] and Dp260 [6], and to identify ⇑ Corresponding author at: Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry SY10 7AG, UK. Tel.: +44 1691 404155; fax: +44 1691 404170. E-mail address: [email protected] (G.E. Morris).
0960-8966/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nmd.2013.11.016
nNOS-binding regions of dystrophin [7]. Exon-specific mAbs can also be used to distinguish tissue-specific isoforms of full-length dystrophin. Thus, our MANEX1 mAbs were mapped to the first three amino-acids of the muscle-specific isoform of dystrophin (first exon encodes LWWEEVEDCY) and so will not recognize either the brain isoform (first exon encodes ED) or the isoform in cardiac Purkinje cells (first exon encodes SEVSSD) [8]. They have also been used to investigate the nature and origin of revertant fibres in Duchenne patients [9] and mdx mice [10]. More recently, they have found a major application in monitoring the success of clinical trials of various experimental treatments for Duchenne MD. The dystrophin in revertant fibres always has missing exons, whereas dystrophin supplied in gene or cell therapy trials is usually full-length and will contain the exons deleted by the Duchenne mutation. Dystrophin-positive fibres in an early myoblast therapy trial were shown to be due to revertant fibres using exon-specific mAbs [11] and most subsequent trials have used this method to demonstrate
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successful dystrophin replacement [12,13]. In an early study, our exon-specific mAbs were used to distinguish revertant fibres in a Duchenne patient from dystrophin-positive fibres that might have arisen from stem cells in donor bone marrow transplants given several years earlier [14]. The exon-specific mAbs have also been used in trials of gene therapy [15], antisense oligo-based [16], morpholino-based [17,18] and stop codon readthrough [19,20] therapies. MANDYS106 has been particularly popular for “exon-skipping” approaches which aim to convert severe Duchenne patients into milder, Becker-like patients [16–18]. These applications require mAbs that work well for both immunolocalization and western blotting. Synthetic peptide immunogens do not work well for either globular regions or the triple-helical rod regions of dystrophin, although we had some success with peptides from the four non-helical linkers, or “hinges”, in the dystrophin rod [21]. For this reason, we have produced most dystrophin mAbs by using large recombinant fragments as immunogens and mapping the epitopes subsequently to single exons or groups of exons.
2. Materials and methods 2.1. Epitope mapping Epitope mapping using phage-displayed random peptide libraries in filamentous phage was performed as previously described [22] using a modification of an earlier method [23]. Monoclonal antibody mixtures were diluted 1:50 with Tris-buffered saline (TBS) and immobilised onto sterile 35 mm Petri dishes coated directly with 1 ml of 1:200 dilution of rabbit-anti-[mouse Ig] in TBS (DAKOpatts, Denmark). Biopanning was performed using a 15-mer peptide library in phage f88–4, maintained in the K91Kan strain of Escherichia coli and generously supplied by Smith (University of Missouri). Any remaining binding sites on the dishes were blocked using 4% BSA in sterile TBS. A sample of the phage library (1013 virions) was pre-incubated in dishes coated with the rabbit anti-mouse antibodies alone to ensure any binding was specific for the target mAbs. Following the first round of biopanning, the bound phage were eluted and amplified by infection of K91Kan E. coli cells. Two rounds of biopanning were performed. Individual colonies of the phage-infected cells after the second round were grown on nitrocellulose membrane (BA85) and screened by western blotting to reveal positive clones. Positive clones were subjected to western blotting with individual mAbs from the mixture used for biopanning. After blocking non-specific sites with 5% skimmed milk protein in TBS, membranes were incubated with mAb supernatant (1/100 dilution in TBS). Antibody-reacting clones were visualized following development with biotinylated horse anti-mouse Ig in a
Fig. 1. Use of MANDYS126 to isolate phage expressing surface peptides which mimic its epitope. The four steps shown are described in the results section. (A) There are few mAb-positive clones after the first round of biopanning with a mixture of 13 mAbs. (B) After two rounds of biopanning, several E. coli. colonies reacted with the mAb mixture and ten of these (circled in B) were selected. (C) The ten colonies were streaked onto 13 horizontal strips and each strip was incubated with a single mAb from the mixture of 13. Only MANDYS126 gave a strong reaction. (D) Four of the ten colonies were cloned again and 10 clones were tested for mAb reaction – this was repeated until all 10 clones were positive to ensure that the phage contains a single sequence only. One colony from each of these clonings was amplified for isolation and sequencing of phage DNA.
L.T. Lam et al. / Neuromuscular Disorders 24 (2014) 195–200
197
Dystrophin mAbs: MANEX1216D: DWLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQ DSSPYLMSPLGLDFD Peptide1 SPDDPPLPDLLYRSG Peptide2 MANEX1216B: QQHKVLQEDLEQEQVRVNSLTHMVVVVDESSGDHATAA FAPDLTRFPSVVVST Peptide3 MANDYS19: ALKEKGQGPMFLDAD SSADGGQGPHLLVRY
Dystrophin
Dystrophin
Dystrophin Peptide4
MANDYS17: KDLSEMHEWMTQAEEEYLERDFEYKTPDELQKAVEEMKR EHFAPSSPDFLERHF Peptide5 NVSPDALEWLVGSKC Peptide6 MANDYS126: KASIPLKELEQFNSDIQKLLEPLEAEIQQGVNL DRRFFQSDILALFSP Peptide7 LPPPQQFHQDMMKLF Peptide8 RLPLDTFHSDLSRLT Peptide9
Dystrophin
Dystrophin Exon 38
MANEX1A: MLWWEEVEDCYEREDVQKKT DTADLWWNSGTFLPA Peptide10
Dystrophin Exon1
MANDRA1: EQLNNSFPSSRGRNTPGKPMREDTM THASYMSPSSAFTLQ Peptide11
Dystrophin Exon77
Fig. 2. Alignment of peptide sequences with human dystrophin sequence. For each of the seven mAbs, the full 15-aa sequence of the peptide from the phage library is aligned with a partial dystrophin that matches. The matching amino-acids are shown bold and underlined in both peptide and dystrophin sequences.
Table 1 Mapping of five monoclonal antibodies epitopes encoded by single dystrophin exons, using a phage-displayed peptide library. Name of mAb
Previous assignment
New mapping assignment
MANDYS1216D MANDYS1216B MANDYS19 MANDYS17 MANDYS126
Within Within Within Within Within
Exon Exon Exon Exon Exon
exons exons exons exons exons
12-16 12-16 20-21 26-27 38-39
Vectastain ABC kit (Vector Labs, Burlingame, CA) and diaminobenzidine substrate (Sigma; 0.4 mg/ml). Phage DNA was purified from positive clones by the phenol/ chloroform method and sequenced using primer: 50 -AGTAGCAGAAGCCTGAAGA-30 . 2.2. Immunofluorescence microscopy Culture supernatants containing monoclonal antibodies were diluted 1:10 in PBS and incubated on 5–7 micron frozen muscle sections without fixation for 1 h. Primary antibody was then removed by washing four times with
12: 13: 21: 26: 38:
aa478-486, PLGPDLEDL aa514-516, VVV aa901-904, GQGP aa1181-1188, LERDFEYK aa1787-1795, FNSDIQKL
PBS. Sections were then incubated with 5 lg/ml goat anti-mouse ALEXA 488 (Molecular Probes, Eugene, Oregon, USA) secondary antibody diluted in PBS containing 1% horse serum, 1% fetal bovine serum and 0.1% BSA, for 1 h. DAPI (diamidino phenylindole: 1 lg/ ml) was added for the final 5 min of incubation to counterstain nuclei before mounting in Hydromount (Merck). Images were obtained using a Leica SP5 confocal microscope with a 20 objective under identical capture conditions and laser power. Human muscle sections were obtained with the appropriate informed consent and ethical approval.
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3. Results The phage library consists of random 15-mer peptides expressed on a multiple-copy coat protein of the filamentous phage, fd-tet. Antibodies are used to enrich the phage for peptides recognised by the antibody by a method known as biopanning. Most mAbs against dystrophin appear to recognise epitopes with a conformational element so that only a small proportion can be mapped using peptides. For this reason, we always perform the biopanning with a mixture of randomly chosen mAbs, thirteen in the case of the example shown in Fig. 1. The enriched phage population was expanded by infection of E. coli, but since there were few mAb-positive colonies at this stage (Fig 1A), the biopanning step was performed a second time. The E. coli after the second biopanning were plated at cloning density and individual colonies were replicated onto a master plate and onto nitrocellulose for western blotting (Fig. 1B). Positive colonies were then replicated 13 times onto nitrocellulose so that they could be screened with individual mAbs and one mAb, MANDYS126, recognised most of the colonies. Four of these were then re-streaked from the master plate until all (10/10) sub-colonies were mAb-positive (Fig. 1D) and phage DNA for sequencing was isolated from one sub-colony of each group. The remaining 12 mAbs were re-used for biopanning without MANDYS126 to confirm that no phages were selected by them. The results for MANDYS126 are shown in Fig. 2, together with the data for all other positive mAbs. Only the 9 dystrophin mAbs in Fig. 2, out of 65 tested, were successful in recovering peptides from the library. Although, for MANDYS126, there were three different peptides, enabling confident identification of the epitope, it was common for most, if not all, of the four peptides sequenced to be identical. Even with a single peptide, four or more matching amino-acids in sequence, as with MANDYS19 in Fig. 2, is too unlikely to have occurred by chance (0.006%). Two peptides with only three sequential amino-acid matches in Fig. 2 agree well with previous data using different mapping techniques (LWW for MANEX1A was also obtained by peptide synthesis [8] and PSS for MANDRA1 is within the FPSSR identified using DNAseI fragment libraries [24]). The overall results and the refinements in epitope specificity achieved are shown in Table 1. Not all mAbs are suitable for immunolocalisation, since some work only on western blots; dystrophin mAbs suitable (“if”) and unsuitable (“xif”) for immunofluorescence microscopy, are identified in Ref. [26] and at http://www.glennmorris.org.uk/mabs/ Dystrophin.htm. Fig. 3 shows that MANDYS19 performs almost as well as the popular mAbs MANDYS1 (similar to MANDYS8; exon 31/32) and MANDYS106 (exon 43), giving only clear sarcolemmal
Fig. 3. Quality control of monoclonal antibodies for muscle biopsy sections. Each of four mAbs was used at 1:10 dilution for immunofluorescence microscopy on control muscle sections (left column) and sections from one Duchenne patient (right column). Images were captured under identical “exposure” conditions, as illustrated by the isolated dystrophin-positive “revertant” fibres present in each Duchenne section. Unexpectedly, the cross-reaction of MANDYS126 causing internal fibre staining is reduced in Duchenne, as well as the sarcolemmal dystrophin staining. The cross-reacting antigen is not known.
staining in control muscle and little or no sarcolemmal staining in Duchenne muscle, except in “revertant” fibres [9]. MANDYS126 is also usable for immunostaining, although it shows internal fibre staining due to an
L.T. Lam et al. / Neuromuscular Disorders 24 (2014) 195–200
unidentified cross-reaction (Fig. 3). Of the remaining mAbs mapped in Fig. 2, MANEX1A and MANDRA1 also work very well on frozen sections (data not shown), but the other three are recommended for western blotting only [26]. 4. Discussion Many different approaches have been used in the attempt to replace the missing dystrophin protein in Duchenne muscle. To assess whether any of these treatments has been effective in clinical trials, it is necessary to use antibodies against dystrophin to determine whether dystrophin has been produced during the therapy. Although improved muscle function is the ultimate aim, it is important to confirm that such improvement is due to new dystrophin production, or, if no improvement was observed, to determine whether new dystrophin is present, though ineffective. Where treatments are performed by local injection, it is usual to take a muscle biopsy near the site of injection and to determine its dystrophin status by immunocytochemistry. This technique can detect even a small number of dystrophin-positive fibres in a biopsy, whereas western blotting may only work well when the proportion of positive fibres is quite high. Because of the important requirement for good immunostaining of the sarcolemma, we have generally used large dystrophin fragments as immunogens and screened for mAbs that work well for immunocytochemistry of human muscle biopsies. This may explain why only 9 out of 65 dystrophin mAbs tested recognised 15-mer peptides in the phage library; the other 56 mAbs tested may require longer amino-acid sequences to mimic the dystrophin epitope that they recognise. These 56 mAbs include the popular MANDYS106 mAb, which has been mapped to exon 43 by a dystrophin fragmentation method [24]. Less likely is the possibility that the relevant peptide is missing from the library; sufficient phage particles (1013 which is >factorial 15) in the initial biopanning step should ensure that all possible random 15-mers are available for selection, but this assumes a truly random library (the libraries are generated by chemical synthesis of 45 bases using a mixture of all 4 bases at each synthetic step). We developed the method of biopanning with a mixture of 10–20 mAbs when it became clear that only a small proportion of mAbs raised against large recombinant immunogens can be mapped in this way (because many have a conformational element in their epitope which peptides cannot mimic). It is interesting that MANDYS17 has been mapped to amino-acids 1181–1188 for the first time here (Table 1), because MANDYS18 was previously mapped to 1181–1187 by a transposon mutagenesis method [25]. Using the transposon method, MANDYS18 did not bind to aa815–1181, but did bind to aa815–1187, whereas MANDYS17 bound neither of those two fragments but
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did bind aa815–1205. The two mAbs recognise a sequence that lies on a turn between two helices of the triple-helical coiled-coil structure of the dystrophin rod domain [25], so it is possible that MANDYS17 prefers a larger epitope than MANDYS18 for efficient binding. As in all the data presented here, peptide mapping results should preferably supported by at least one [24], and preferably two [25], alternative mapping methods. All the dystrophin mAbs described in this study are freely available for non-profit research from the MDA Monoclonal Antibody Resource (www.glennmorris.org. uk/mabs.htm). Acknowledgements This study was supported by a Translational Infrastructure Research Grant from the Muscular Dystrophy Association (USA). We thank George P. Smith (University of Missouri) for the generous gift of phage libraries and Prof. Caroline Sewry (RJAH Orthopaedic Hospital, Oswestry, UK) for muscle biopsy sections and comments on the manuscript. References [1] Le TT, Nguyen thi Man, Hori S, Sewry CA, Dubowitz V, Morris GE. Characterization of genetic deletions in Becker muscular dystrophy using monoclonal antibodies against a deletion-prone region of dystrophin. Am J Med Genet 1995;58:177–86. [2] Lederfein D, Levy N, Augier N, et al. 71kD protein is a major product of the Duchenne muscular dystrophy gene in brain and other non-muscle tissues. Proc Natl Acad Sci USA 1992;89:5346–50. [3] Hugnot JP, Gilgenkrantz H, Vincent N, et al. Novel products of the dystrophin gene: a distal transcript initiated from a unique alternative first exon encoding a 75 kDa protein widely distributed in non-muscle tissues. Proc Natl Acad Sci USA 1992;89:7506–10. [4] Blake DJ, Love DR, Tinsley J, et al. Characterization of a 4.8kb transcript from the Duchenne muscular dystrophy locus expressed in Schwannoma cells. Hum Mol Genet 1992;1:103–9. [5] Morris GE, Simmons C, Nguyen thi Man. Apo-dystrophins (Dp140 and Dp71) and dystrophin splicing isoforms in developing brain. Biochem Biophys Res Commun 1995;215:361–7. [6] d’Souza VN, Nguyen thi Man, Morris GE, Karges W, Pillers DM, Ray PN. A novel dystrophin isoform is required for normal retinal electrophysiology. Hum Mol Genet 1995;4:837–42. [7] Lai Y, Zhao J, Yue Y, Duan D. a2 and a3 helices of dystrophin R16 and R17 frame a microdomain in the a1 helix of dystrophin R17 for neuronal NOS binding. Proc Natl Acad Sci USA 2013;110:525–30. [8] Le TT, Nguyen thi Man, Love DR, Helliwell TR, Davies KE, Morris GE. Monoclonal antibodies against the muscle-specific N-terminus of dystrophin: characterization of dystrophin in a muscular dystrophy patient with a frameshift deletion of exons 3–7. Am J Hum Genet 1993;53:131–9. [9] Le TT, Nguyen thi Man, Helliwell TR, Morris GE. Characterization of revertant muscle fibres in Duchenne muscular dystrophy using exon-specific monoclonal antibodies against dystrophin. Am J Hum Genet 1995;56:725–31. [10] Lu QL, Morris GE, Wilton SD, et al. Massive idiosyncratic exon skipping corrects the nonsense mutation in dystrophic mouse muscle and produces functional revertant fibres by clonal expansion. J Cell Biol 2000;148:985–96. [11] Partridge TA, Lu QL, Morris GE. Hoffmann EP Is myoblast transplantation effective? Nature Med 1998;4:1208–9.
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[12] Mendell JR, Kissel JT, Amato AA, et al. Myoblast transfer in the treatment of Duchenne’s muscular dystrophy. N Engl J Med 1993;333:832–8. [13] Skuk D, Roy B, Goulet M, et al. Dystrophin expression in myofibers of Duchenne muscular dystrophy patients following intramuscular injections of normal myogenic cells. Mol Ther 2004;9:475–82. [14] Gussoni E, Bennett RR, Gilgoff I, et al. Detection of donor nuclei in the muscles of a Duchenne muscular dystrophy patient 13 years following bone marrow transplantation. J Clin Invest 2002;110:807–14. [15] Romero NB, Braun S, Benveniste O, et al. Phase I study of dystrophin plasmid-based gene therapy in Duchenne/Becker muscular dystrophy. Hum Gene Therapy 2004;15:1065–76. [16] Goemans NM, Tulinius M, van den Akker JT, et al. Systemic administration of PRO051 in Duchenne’s muscular dystrophy. N Engl J Med 2011;364:1513–22. [17] Kinali M, Arechavala-Gomeza V, Feng L, et al. Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study. Lancet Neurol 2009;8:918–28. [18] Cirak S, Arechavala-Gomeza V, Guglieri M, et al. Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study. Lancet 2011;378:595–605.
[19] Welch EM, Barton ER, Zhuo J, et al. PTC124 targets genetic disorders caused by nonsense mutations. Nature (Lond) 2007;447:87–91. [20] Malik V, Rodino-Klapac LR, Viollet L, et al. Gentamicin-induced readthrough of stop codons in Duchenne muscular dystrophy. Ann Neurol 2010;67:771–80. [21] Ahmed N, Nguyen thi Man, Morris GE. Flexible hinges in dystrophin. Biochem Soc Trans 1998;26:S310. [22] Pereboev A, Morris GE. Reiterative screening of phage display peptide libraries for epitope mapping. In: Epitope Mapping Protocols Humana Press; (Methods in Mol. Biol. 66) 1996. pp. 195–206. [23] Scott JK, Smith GP. Searching for peptide ligands with an epitope library. Science 1990;249:386–90. [24] Nguyen thi Man, Morris GE. Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy. Am J Hum Genet 1993;52:1057–66. [25] Sedgwick SG, Nguyen thi Man, Ellis JM, Crowne H, Morris GE. Rapid mapping by transposon mutagenesis of epitopes on the muscular dystrophy protein, dystrophin. Nucleic Acids Res 1991;19:5889–94. [26] Morris GE, Nguyen M, Sewry CA. Monitoring Duchenne muscular dystrophy gene therapy with epitope-specific monoclonal antibodies. In: Dongsheng D, editor. Muscle Gene Therapy; 2011: 709, pp. 39– 62.
ScienceDirect Neuromuscular Disorders 24 (2014) 195–200 www.elsevier.com/locate/nmd
Monoclonal antibodies for clinical trials of Duchenne muscular dystrophy therapy Le Thanh Lam a, Nguyen Thi Man a, Glenn E. Morris a,b,⇑ a
Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry SY10 7AG, UK b Institute for Science and Technology in Medicine, Keele University, Keele, UK Received 15 November 2013; accepted 27 November 2013
Abstract Most pathogenic mutations in Duchenne and Becker muscular dystrophies involve deletion of single or multiple exons from the dystrophin gene, so exon-specific monoclonal antibodies (mAbs) can be used to distinguish normal and mutant dystrophin proteins. In Duchenne therapy trials, mAbs can be used to identify or rule out dystrophin-positive “revertant” fibres, which have an internally-deleted dystrophin protein and which occur naturally in some Duchenne patients. Using phage-displayed peptide libraries, we now describe the new mapping of the binding sites of five dystrophin mAbs to a few amino-acids within single exons. The phage display method also confirmed previous mapping of MANEX1A (exon 1) and MANDRA1 (exon 77) by other methods. Of the 79 dystrophin exons, mAbs are now available against single exons 1, 6, 8, 12, 13, 14, 17, 21, 26, 28, 38, 41, 43, 44, 45, 46, 47, 50, 51, 58, 59, 62, 63, 75 and 77. Many have been used in clinical trials, as well as for diagnosis and studies of dystrophin isoforms. Ó 2013 Elsevier B.V. All rights reserved. Keywords: Duchenne muscular dystrophy; Dystrophin; Monoclonal antibody; Epitope mapping; Clinical trial; Exon-skipping; Therapy; Phage display
1. Introduction One of the long-term aims of the MDA Monoclonal Antibody Resource (www.glennmorris.org.uk/mabs.htm) has been to produce monoclonal antibodies (mAbs) against dystrophin that recognise specific amino-acid sequences spread throughout its 79 exons and 3684 amino-acids. We have paid particular attention to mAbs against the exons that are commonly deleted in Duchenne muscular dystrophy; these can be used to characterise mutant Becker dystrophins at the protein level in muscle biopsies [1]. The antibodies have also been used to characterise some of the short forms of dystrophin, including Dp71 [2–4], Dp140 [5] and Dp260 [6], and to identify ⇑ Corresponding author at: Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry SY10 7AG, UK. Tel.: +44 1691 404155; fax: +44 1691 404170. E-mail address: [email protected] (G.E. Morris).
0960-8966/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nmd.2013.11.016
nNOS-binding regions of dystrophin [7]. Exon-specific mAbs can also be used to distinguish tissue-specific isoforms of full-length dystrophin. Thus, our MANEX1 mAbs were mapped to the first three amino-acids of the muscle-specific isoform of dystrophin (first exon encodes LWWEEVEDCY) and so will not recognize either the brain isoform (first exon encodes ED) or the isoform in cardiac Purkinje cells (first exon encodes SEVSSD) [8]. They have also been used to investigate the nature and origin of revertant fibres in Duchenne patients [9] and mdx mice [10]. More recently, they have found a major application in monitoring the success of clinical trials of various experimental treatments for Duchenne MD. The dystrophin in revertant fibres always has missing exons, whereas dystrophin supplied in gene or cell therapy trials is usually full-length and will contain the exons deleted by the Duchenne mutation. Dystrophin-positive fibres in an early myoblast therapy trial were shown to be due to revertant fibres using exon-specific mAbs [11] and most subsequent trials have used this method to demonstrate
196
L.T. Lam et al. / Neuromuscular Disorders 24 (2014) 195–200
successful dystrophin replacement [12,13]. In an early study, our exon-specific mAbs were used to distinguish revertant fibres in a Duchenne patient from dystrophin-positive fibres that might have arisen from stem cells in donor bone marrow transplants given several years earlier [14]. The exon-specific mAbs have also been used in trials of gene therapy [15], antisense oligo-based [16], morpholino-based [17,18] and stop codon readthrough [19,20] therapies. MANDYS106 has been particularly popular for “exon-skipping” approaches which aim to convert severe Duchenne patients into milder, Becker-like patients [16–18]. These applications require mAbs that work well for both immunolocalization and western blotting. Synthetic peptide immunogens do not work well for either globular regions or the triple-helical rod regions of dystrophin, although we had some success with peptides from the four non-helical linkers, or “hinges”, in the dystrophin rod [21]. For this reason, we have produced most dystrophin mAbs by using large recombinant fragments as immunogens and mapping the epitopes subsequently to single exons or groups of exons.
2. Materials and methods 2.1. Epitope mapping Epitope mapping using phage-displayed random peptide libraries in filamentous phage was performed as previously described [22] using a modification of an earlier method [23]. Monoclonal antibody mixtures were diluted 1:50 with Tris-buffered saline (TBS) and immobilised onto sterile 35 mm Petri dishes coated directly with 1 ml of 1:200 dilution of rabbit-anti-[mouse Ig] in TBS (DAKOpatts, Denmark). Biopanning was performed using a 15-mer peptide library in phage f88–4, maintained in the K91Kan strain of Escherichia coli and generously supplied by Smith (University of Missouri). Any remaining binding sites on the dishes were blocked using 4% BSA in sterile TBS. A sample of the phage library (1013 virions) was pre-incubated in dishes coated with the rabbit anti-mouse antibodies alone to ensure any binding was specific for the target mAbs. Following the first round of biopanning, the bound phage were eluted and amplified by infection of K91Kan E. coli cells. Two rounds of biopanning were performed. Individual colonies of the phage-infected cells after the second round were grown on nitrocellulose membrane (BA85) and screened by western blotting to reveal positive clones. Positive clones were subjected to western blotting with individual mAbs from the mixture used for biopanning. After blocking non-specific sites with 5% skimmed milk protein in TBS, membranes were incubated with mAb supernatant (1/100 dilution in TBS). Antibody-reacting clones were visualized following development with biotinylated horse anti-mouse Ig in a
Fig. 1. Use of MANDYS126 to isolate phage expressing surface peptides which mimic its epitope. The four steps shown are described in the results section. (A) There are few mAb-positive clones after the first round of biopanning with a mixture of 13 mAbs. (B) After two rounds of biopanning, several E. coli. colonies reacted with the mAb mixture and ten of these (circled in B) were selected. (C) The ten colonies were streaked onto 13 horizontal strips and each strip was incubated with a single mAb from the mixture of 13. Only MANDYS126 gave a strong reaction. (D) Four of the ten colonies were cloned again and 10 clones were tested for mAb reaction – this was repeated until all 10 clones were positive to ensure that the phage contains a single sequence only. One colony from each of these clonings was amplified for isolation and sequencing of phage DNA.
L.T. Lam et al. / Neuromuscular Disorders 24 (2014) 195–200
197
Dystrophin mAbs: MANEX1216D: DWLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQ DSSPYLMSPLGLDFD Peptide1 SPDDPPLPDLLYRSG Peptide2 MANEX1216B: QQHKVLQEDLEQEQVRVNSLTHMVVVVDESSGDHATAA FAPDLTRFPSVVVST Peptide3 MANDYS19: ALKEKGQGPMFLDAD SSADGGQGPHLLVRY
Dystrophin
Dystrophin
Dystrophin Peptide4
MANDYS17: KDLSEMHEWMTQAEEEYLERDFEYKTPDELQKAVEEMKR EHFAPSSPDFLERHF Peptide5 NVSPDALEWLVGSKC Peptide6 MANDYS126: KASIPLKELEQFNSDIQKLLEPLEAEIQQGVNL DRRFFQSDILALFSP Peptide7 LPPPQQFHQDMMKLF Peptide8 RLPLDTFHSDLSRLT Peptide9
Dystrophin
Dystrophin Exon 38
MANEX1A: MLWWEEVEDCYEREDVQKKT DTADLWWNSGTFLPA Peptide10
Dystrophin Exon1
MANDRA1: EQLNNSFPSSRGRNTPGKPMREDTM THASYMSPSSAFTLQ Peptide11
Dystrophin Exon77
Fig. 2. Alignment of peptide sequences with human dystrophin sequence. For each of the seven mAbs, the full 15-aa sequence of the peptide from the phage library is aligned with a partial dystrophin that matches. The matching amino-acids are shown bold and underlined in both peptide and dystrophin sequences.
Table 1 Mapping of five monoclonal antibodies epitopes encoded by single dystrophin exons, using a phage-displayed peptide library. Name of mAb
Previous assignment
New mapping assignment
MANDYS1216D MANDYS1216B MANDYS19 MANDYS17 MANDYS126
Within Within Within Within Within
Exon Exon Exon Exon Exon
exons exons exons exons exons
12-16 12-16 20-21 26-27 38-39
Vectastain ABC kit (Vector Labs, Burlingame, CA) and diaminobenzidine substrate (Sigma; 0.4 mg/ml). Phage DNA was purified from positive clones by the phenol/ chloroform method and sequenced using primer: 50 -AGTAGCAGAAGCCTGAAGA-30 . 2.2. Immunofluorescence microscopy Culture supernatants containing monoclonal antibodies were diluted 1:10 in PBS and incubated on 5–7 micron frozen muscle sections without fixation for 1 h. Primary antibody was then removed by washing four times with
12: 13: 21: 26: 38:
aa478-486, PLGPDLEDL aa514-516, VVV aa901-904, GQGP aa1181-1188, LERDFEYK aa1787-1795, FNSDIQKL
PBS. Sections were then incubated with 5 lg/ml goat anti-mouse ALEXA 488 (Molecular Probes, Eugene, Oregon, USA) secondary antibody diluted in PBS containing 1% horse serum, 1% fetal bovine serum and 0.1% BSA, for 1 h. DAPI (diamidino phenylindole: 1 lg/ ml) was added for the final 5 min of incubation to counterstain nuclei before mounting in Hydromount (Merck). Images were obtained using a Leica SP5 confocal microscope with a 20 objective under identical capture conditions and laser power. Human muscle sections were obtained with the appropriate informed consent and ethical approval.
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3. Results The phage library consists of random 15-mer peptides expressed on a multiple-copy coat protein of the filamentous phage, fd-tet. Antibodies are used to enrich the phage for peptides recognised by the antibody by a method known as biopanning. Most mAbs against dystrophin appear to recognise epitopes with a conformational element so that only a small proportion can be mapped using peptides. For this reason, we always perform the biopanning with a mixture of randomly chosen mAbs, thirteen in the case of the example shown in Fig. 1. The enriched phage population was expanded by infection of E. coli, but since there were few mAb-positive colonies at this stage (Fig 1A), the biopanning step was performed a second time. The E. coli after the second biopanning were plated at cloning density and individual colonies were replicated onto a master plate and onto nitrocellulose for western blotting (Fig. 1B). Positive colonies were then replicated 13 times onto nitrocellulose so that they could be screened with individual mAbs and one mAb, MANDYS126, recognised most of the colonies. Four of these were then re-streaked from the master plate until all (10/10) sub-colonies were mAb-positive (Fig. 1D) and phage DNA for sequencing was isolated from one sub-colony of each group. The remaining 12 mAbs were re-used for biopanning without MANDYS126 to confirm that no phages were selected by them. The results for MANDYS126 are shown in Fig. 2, together with the data for all other positive mAbs. Only the 9 dystrophin mAbs in Fig. 2, out of 65 tested, were successful in recovering peptides from the library. Although, for MANDYS126, there were three different peptides, enabling confident identification of the epitope, it was common for most, if not all, of the four peptides sequenced to be identical. Even with a single peptide, four or more matching amino-acids in sequence, as with MANDYS19 in Fig. 2, is too unlikely to have occurred by chance (0.006%). Two peptides with only three sequential amino-acid matches in Fig. 2 agree well with previous data using different mapping techniques (LWW for MANEX1A was also obtained by peptide synthesis [8] and PSS for MANDRA1 is within the FPSSR identified using DNAseI fragment libraries [24]). The overall results and the refinements in epitope specificity achieved are shown in Table 1. Not all mAbs are suitable for immunolocalisation, since some work only on western blots; dystrophin mAbs suitable (“if”) and unsuitable (“xif”) for immunofluorescence microscopy, are identified in Ref. [26] and at http://www.glennmorris.org.uk/mabs/ Dystrophin.htm. Fig. 3 shows that MANDYS19 performs almost as well as the popular mAbs MANDYS1 (similar to MANDYS8; exon 31/32) and MANDYS106 (exon 43), giving only clear sarcolemmal
Fig. 3. Quality control of monoclonal antibodies for muscle biopsy sections. Each of four mAbs was used at 1:10 dilution for immunofluorescence microscopy on control muscle sections (left column) and sections from one Duchenne patient (right column). Images were captured under identical “exposure” conditions, as illustrated by the isolated dystrophin-positive “revertant” fibres present in each Duchenne section. Unexpectedly, the cross-reaction of MANDYS126 causing internal fibre staining is reduced in Duchenne, as well as the sarcolemmal dystrophin staining. The cross-reacting antigen is not known.
staining in control muscle and little or no sarcolemmal staining in Duchenne muscle, except in “revertant” fibres [9]. MANDYS126 is also usable for immunostaining, although it shows internal fibre staining due to an
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unidentified cross-reaction (Fig. 3). Of the remaining mAbs mapped in Fig. 2, MANEX1A and MANDRA1 also work very well on frozen sections (data not shown), but the other three are recommended for western blotting only [26]. 4. Discussion Many different approaches have been used in the attempt to replace the missing dystrophin protein in Duchenne muscle. To assess whether any of these treatments has been effective in clinical trials, it is necessary to use antibodies against dystrophin to determine whether dystrophin has been produced during the therapy. Although improved muscle function is the ultimate aim, it is important to confirm that such improvement is due to new dystrophin production, or, if no improvement was observed, to determine whether new dystrophin is present, though ineffective. Where treatments are performed by local injection, it is usual to take a muscle biopsy near the site of injection and to determine its dystrophin status by immunocytochemistry. This technique can detect even a small number of dystrophin-positive fibres in a biopsy, whereas western blotting may only work well when the proportion of positive fibres is quite high. Because of the important requirement for good immunostaining of the sarcolemma, we have generally used large dystrophin fragments as immunogens and screened for mAbs that work well for immunocytochemistry of human muscle biopsies. This may explain why only 9 out of 65 dystrophin mAbs tested recognised 15-mer peptides in the phage library; the other 56 mAbs tested may require longer amino-acid sequences to mimic the dystrophin epitope that they recognise. These 56 mAbs include the popular MANDYS106 mAb, which has been mapped to exon 43 by a dystrophin fragmentation method [24]. Less likely is the possibility that the relevant peptide is missing from the library; sufficient phage particles (1013 which is >factorial 15) in the initial biopanning step should ensure that all possible random 15-mers are available for selection, but this assumes a truly random library (the libraries are generated by chemical synthesis of 45 bases using a mixture of all 4 bases at each synthetic step). We developed the method of biopanning with a mixture of 10–20 mAbs when it became clear that only a small proportion of mAbs raised against large recombinant immunogens can be mapped in this way (because many have a conformational element in their epitope which peptides cannot mimic). It is interesting that MANDYS17 has been mapped to amino-acids 1181–1188 for the first time here (Table 1), because MANDYS18 was previously mapped to 1181–1187 by a transposon mutagenesis method [25]. Using the transposon method, MANDYS18 did not bind to aa815–1181, but did bind to aa815–1187, whereas MANDYS17 bound neither of those two fragments but
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did bind aa815–1205. The two mAbs recognise a sequence that lies on a turn between two helices of the triple-helical coiled-coil structure of the dystrophin rod domain [25], so it is possible that MANDYS17 prefers a larger epitope than MANDYS18 for efficient binding. As in all the data presented here, peptide mapping results should preferably supported by at least one [24], and preferably two [25], alternative mapping methods. All the dystrophin mAbs described in this study are freely available for non-profit research from the MDA Monoclonal Antibody Resource (www.glennmorris.org. uk/mabs.htm). Acknowledgements This study was supported by a Translational Infrastructure Research Grant from the Muscular Dystrophy Association (USA). We thank George P. Smith (University of Missouri) for the generous gift of phage libraries and Prof. Caroline Sewry (RJAH Orthopaedic Hospital, Oswestry, UK) for muscle biopsy sections and comments on the manuscript. References [1] Le TT, Nguyen thi Man, Hori S, Sewry CA, Dubowitz V, Morris GE. Characterization of genetic deletions in Becker muscular dystrophy using monoclonal antibodies against a deletion-prone region of dystrophin. Am J Med Genet 1995;58:177–86. [2] Lederfein D, Levy N, Augier N, et al. 71kD protein is a major product of the Duchenne muscular dystrophy gene in brain and other non-muscle tissues. Proc Natl Acad Sci USA 1992;89:5346–50. [3] Hugnot JP, Gilgenkrantz H, Vincent N, et al. Novel products of the dystrophin gene: a distal transcript initiated from a unique alternative first exon encoding a 75 kDa protein widely distributed in non-muscle tissues. Proc Natl Acad Sci USA 1992;89:7506–10. [4] Blake DJ, Love DR, Tinsley J, et al. Characterization of a 4.8kb transcript from the Duchenne muscular dystrophy locus expressed in Schwannoma cells. Hum Mol Genet 1992;1:103–9. [5] Morris GE, Simmons C, Nguyen thi Man. Apo-dystrophins (Dp140 and Dp71) and dystrophin splicing isoforms in developing brain. Biochem Biophys Res Commun 1995;215:361–7. [6] d’Souza VN, Nguyen thi Man, Morris GE, Karges W, Pillers DM, Ray PN. A novel dystrophin isoform is required for normal retinal electrophysiology. Hum Mol Genet 1995;4:837–42. [7] Lai Y, Zhao J, Yue Y, Duan D. a2 and a3 helices of dystrophin R16 and R17 frame a microdomain in the a1 helix of dystrophin R17 for neuronal NOS binding. Proc Natl Acad Sci USA 2013;110:525–30. [8] Le TT, Nguyen thi Man, Love DR, Helliwell TR, Davies KE, Morris GE. Monoclonal antibodies against the muscle-specific N-terminus of dystrophin: characterization of dystrophin in a muscular dystrophy patient with a frameshift deletion of exons 3–7. Am J Hum Genet 1993;53:131–9. [9] Le TT, Nguyen thi Man, Helliwell TR, Morris GE. Characterization of revertant muscle fibres in Duchenne muscular dystrophy using exon-specific monoclonal antibodies against dystrophin. Am J Hum Genet 1995;56:725–31. [10] Lu QL, Morris GE, Wilton SD, et al. Massive idiosyncratic exon skipping corrects the nonsense mutation in dystrophic mouse muscle and produces functional revertant fibres by clonal expansion. J Cell Biol 2000;148:985–96. [11] Partridge TA, Lu QL, Morris GE. Hoffmann EP Is myoblast transplantation effective? Nature Med 1998;4:1208–9.
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[12] Mendell JR, Kissel JT, Amato AA, et al. Myoblast transfer in the treatment of Duchenne’s muscular dystrophy. N Engl J Med 1993;333:832–8. [13] Skuk D, Roy B, Goulet M, et al. Dystrophin expression in myofibers of Duchenne muscular dystrophy patients following intramuscular injections of normal myogenic cells. Mol Ther 2004;9:475–82. [14] Gussoni E, Bennett RR, Gilgoff I, et al. Detection of donor nuclei in the muscles of a Duchenne muscular dystrophy patient 13 years following bone marrow transplantation. J Clin Invest 2002;110:807–14. [15] Romero NB, Braun S, Benveniste O, et al. Phase I study of dystrophin plasmid-based gene therapy in Duchenne/Becker muscular dystrophy. Hum Gene Therapy 2004;15:1065–76. [16] Goemans NM, Tulinius M, van den Akker JT, et al. Systemic administration of PRO051 in Duchenne’s muscular dystrophy. N Engl J Med 2011;364:1513–22. [17] Kinali M, Arechavala-Gomeza V, Feng L, et al. Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study. Lancet Neurol 2009;8:918–28. [18] Cirak S, Arechavala-Gomeza V, Guglieri M, et al. Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study. Lancet 2011;378:595–605.
[19] Welch EM, Barton ER, Zhuo J, et al. PTC124 targets genetic disorders caused by nonsense mutations. Nature (Lond) 2007;447:87–91. [20] Malik V, Rodino-Klapac LR, Viollet L, et al. Gentamicin-induced readthrough of stop codons in Duchenne muscular dystrophy. Ann Neurol 2010;67:771–80. [21] Ahmed N, Nguyen thi Man, Morris GE. Flexible hinges in dystrophin. Biochem Soc Trans 1998;26:S310. [22] Pereboev A, Morris GE. Reiterative screening of phage display peptide libraries for epitope mapping. In: Epitope Mapping Protocols Humana Press; (Methods in Mol. Biol. 66) 1996. pp. 195–206. [23] Scott JK, Smith GP. Searching for peptide ligands with an epitope library. Science 1990;249:386–90. [24] Nguyen thi Man, Morris GE. Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy. Am J Hum Genet 1993;52:1057–66. [25] Sedgwick SG, Nguyen thi Man, Ellis JM, Crowne H, Morris GE. Rapid mapping by transposon mutagenesis of epitopes on the muscular dystrophy protein, dystrophin. Nucleic Acids Res 1991;19:5889–94. [26] Morris GE, Nguyen M, Sewry CA. Monitoring Duchenne muscular dystrophy gene therapy with epitope-specific monoclonal antibodies. In: Dongsheng D, editor. Muscle Gene Therapy; 2011: 709, pp. 39– 62.
