Platelet integrin α2 I-domain specific antibodies produced via domain specific DNA vaccination combined with variable gene phage display

Darren L. Hughes; Prachi Stafford; Samir W. Hamaia; Ian J. Harmer; Anne Schoolmeester; Hans Deckmyn; Richard W. Farndale; Willem H. Ouwehand; Nicholas A. Watkins

doi:10.1160/TH05-06-0410

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2005; 94(06): 1318-1326
DOI: 10.1160/TH05-06-0410

New Technologies and Diagnostic Tools

Schattauer GmbH

Platelet integrin α2 I-domain specific antibodies produced via domain specific DNA vaccination combined with variable gene phage display

Authors

Darren L. Hughes

¹Department of Haematology, University of Cambridge, UK
Prachi Stafford

¹Department of Haematology, University of Cambridge, UK
Samir W. Hamaia

¹Department of Haematology, University of Cambridge, UK
Ian J. Harmer^*

¹Department of Haematology, University of Cambridge, UK
Anne Schoolmeester

⁴Laboratory for Thrombosis Research, IRC, KU Leuven Campus Kortrijk, Belgium
Hans Deckmyn

⁴Laboratory for Thrombosis Research, IRC, KU Leuven Campus Kortrijk, Belgium
Richard W. Farndale

³Department of Biochemistry, University of Cambridge, UK
Willem H. Ouwehand

¹Department of Haematology, University of Cambridge, UK

²National Blood Service, Cambridge, UK
Nicholas A. Watkins

¹Department of Haematology, University of Cambridge, UK

Abstract

Summary

Antibodies are a powerful tool for structure/function studies of platelet proteins. However, classic immunisation frequently elicits antibody responses against domains of minor functional interest. Robust strategies to generate antibodies against defined domains would be of significant interest in post-genome research. In this study, we report a new strategy using a combination of DNA vaccination and V gene phage display that allows the rapid generation of domain specific single-chain Fv antibodies (scFvs).This system was validated using the I-domain of α2 integrin as a model. The α2β1 integrin, which is expressed on many cell types, is the dominant collagen attachment receptor on platelets, functioning in close interplay with the collagen signalling receptor glycoproteinVI. A novel set of I-domain specific antibodies was obtained by a DNA vaccination/V gene repertoire cloning approach. Mice were first immunized with a DNA vaccine in which the α2 I-domain is expressed as a fusion protein with fragment C of tetanus toxoid (FrC-TT).Then the heavy and kappa light chain variable gene repertoires were rescued from immune splenocytes using antibody phage display. A total of four α2 I-domain specific scFvs were isolated by selection on recombinant I-domain or native platelet α2β1 integrin. Characterisation of the scFvs indicated that they recognised distinct epitopes that had profound differences in accessibility between native and recombinant I-domain. Our data suggest DNA immunisation and phage display represent versatile alternatives to protein immunisation and hybridoma-fusion techniques for the isolation of recombinant antibody reagents. This approach will be particularly useful for the generation of domain or splicevariant specific antibodies that recognise native protein.

Keywords

Collagen - variable gene phage display - DNA immunisation - domain-specific - α2β1 integrin

Full Text

References

References
1 Kodadek T. Protein microarrays: prospects and problems. Chem Biol 2001; 8: 105-15.
2 Tang DC, DeVit M, Johnston SA. Genetic immunization is a simple method for eliciting an immune response. Nature 1992; 356: 152-4.
3 King CA, Spellerberg MB, Zhu D. et al DNA vaccines with single-chain Fv fused to fragment C of tetanus toxin induce protective immunity against lymphoma and myeloma [see comments]. Nat Med 1998; 4: 1281-6.
4 Winter G, Griffiths AD, Hawkins RE. et al Making antibodies by phage display technology. Annu Rev Immunol 1994; 12: 433-55.
5 Griffin HM, Ouwehand WH. A human monoclonal antibody specific for the leucine-33 (P1A1, HPA-1a) form of platelet glycoprotein IIIa from a V gene phage display library. Blood 1995; 86: 4430-6.
6 McCafferty J, Griffiths AD, Winter G. et al Phage antibodies: filamentous phage displaying antibody variable domains. Nature 1990; 348: 552-4.
7 Hoogenboom HR, Griffiths AD, Johnson KS. et al Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res 1991; 19: 4133-7.
8 Holt LJ, Herring C, Jespers LS. et al Domain antibodies: proteins for therapy. Trends Biotechnol 2003; 21: 484-90.
9 Smethurst PA, Joutsi-Korhonen L, O'Connor MN. et al Identification of the primary collagen-binding surface on human glycoprotein VI by site-directed mutagenesis and by a blocking phage antibody. Blood 2004; 103: 903-11.
10 Watkins NA, Dafforn TR, Kuijpers M. et al Molecular studies of anti-HLA-A2 using light-chain shuffling: a structural model for HLA antibody binding. Tissue Antigens 2004; 63: 345-54.
11 Joutsi-Korhonen L, Smethurst PA, Rankin A. et al The low frequency allele of the platelet collagen signalling receptor glycoprotein VI is associated with reduced functional responses and expression. Blood 2003; 101: 4372-9. .
12 Ames RS, Tornetta MA, Deen K. et al Conversion of murine Fabs isolated from a combinatorial phage display library to full length immunoglobulins. J Immunol Methods 1995; 184: 177-86.
13 Armour KL, Clark MR, Hadley AG. et al Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities. Eur J Immunol 1999; 29: 2613-24.
14 Holliger P, Prospero T, Winter G. „Diabodies“: small bivalent and bispecific antibody fragments. Proc Natl Acad Sci U S A 1993; 90: 6444-8.
15 Watkins NA, Armour KL, Smethurst PA. et al Rapid phenotyping of HPA-1a using either diabodybased hemagglutination or recombinant IgG1-based assays. Transfusion 1999; 39: 781-9.
16 Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 1992; 69: 11-25.
17 Santoro SA, Zutter MM. The alpha 2 beta 1 integrin: a collagen receptor on platelets and other cells. Thromb Haemost 1995; 74: 813-21.
18 Kamata T, Puzon W, Takada Y. Identification of putative ligand binding sites within I domain of integrin alpha 2 beta 1 (VLA-2, CD49b/CD29) [published erratum appears in J Biol Chem 1996 Aug 2; 271 (31): 19008]. J Biol Chem 1994; 269: 9659-63.
19 Depraetere H, Wille C, Gansemans Y. et al The integrin alpha 2 beta 1 (GPIa/IIa)-I-domain inhibits platelet-collagen interaction. Thromb Haemost 1997; 77: 981-5.
20 Emsley J, King SL, Bergelson JM. et al Crystal structure of the I domain from integrin alpha2beta1. J Biol Chem 1997; 272: 28512-7.
21 Emsley J, Knight CG, Farndale RW. et al Structural basis of collagen recognition by integrin alpha2beta1. Cell 2000; 101: 47-56.
22 Schoolmeester A, Vanhoorelbeke K, Katsutani S. et al Monoclonal antibody IAC-1 is specific for activated alpha2beta1 and binds to amino acids 199 to 201 of the integrin alpha2 I-domain. Blood 2004; 104: 390-6.
23 Tuckwell D, Calderwood DA, Green LJ. et al Integrin alpha 2 I-domain is a binding site for collagens. J Cell Sci 1995; 108 (Pt 4) 1629-37.
24 Tuckwell DS, Reid KB, Barnes MJ. et al The A-domain of integrin alpha 2 binds specifically to a range of collagens but is not a general receptor for the collagenous motif. Eur J Biochem 1996; 241: 732-9.
25 Aquilina A, Korda M, Bergelson JM. et al A novel gain-of-function mutation of the integrin alpha2 VWFA domain. Eur J Biochem 2002; 269: 1136-44.
26 Smith C, Estavillo D, Emsley J. et al Mapping the collagen-binding site in the I domain of the glycoprotein Ia/IIa (integrin alpha(2)beta(1)). J Biol Chem 2000; 275: 4205-9.
27 Hakes DJ, Dixon JE. New vectors for high level expression of recombinant proteins in bacteria. Anal Biochem 1992; 202: 293-8.
28 Panina-Bordignon P, Tan A, Termijtelen A. et al Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells. Eur J Immunol 1989; 19: 2237-42.
29 Valmori D, Pessi A, Bianchi E. et al Use of human universally antigenic tetanus toxin T cell epitopes as carriers for human vaccination. J Immunol 1992; 149: 717-21.
30 Kiefel V, Santoso S, Weisheit M. et al Monoclonal antibody–specific immobilization of platelet antigens (MAIPA): a new tool for the identification of plateletreactive antibodies. Blood 1987; 70: 1722-6.
31 Goodall AH. Platelet activation during preparation and storage of concentrates: detection by flow cytometry. Blood Coagul Fibrinolysis 1991; 2: 377-82.
32 Grisshammer R, Grunwald T, Sohal AK. Characterization of an antibody Fv fragment that binds to the human, but not to the rat neurotensin receptor NTS-1. Protein Expr Purif 2002; 24: 505-12.
33 Holt LJ, Bussow K, Walter G. et al By-passing selection: direct screening for antibody-antigen interactions using protein arrays. Nucleic Acids Res 2000; 28: E72.
34 Clackson T, Hoogenboom HR, Griffiths AD. et al Making antibody fragments using phage display libraries. Nature 1991; 352: 624-8.
35 Finnern R, Pedrollo E, Fisch I. et al Human autoimmune anti-proteinase 3 scFv from a phage display library. Clin Exp Immunol 1997; 107: 269-81.
36 Watkins NA, Smethurst PA, Allen D. et al Platelet alphaIIbbeta3 recombinant autoantibodies from the B-cell repertoire of a post-transfusion purpura patient. Br J Haematol 2002; 116: 677-85.
37 Marks JD, Hoogenboom HR, Bonnert TP. et al Bypassing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol 1991; 222: 581-97.
38 Watkins NA, Brown C, Hurd C. et al The isolation and characterisation of human monoclonal HLA-A2 antibodies from an immune V gene phage display library. Tissue Antigens 2000; 55: 219-28.
39 Whittaker CA, Hynes RO. Distribution and evolution of von Willebrand/integrin A domains: widely dispersed domains with roles in cell adhesion and elsewhere. Mol Biol Cell 2002; 13: 3369-87.
40 Chambers RS, Johnston SA. High-level generation of polyclonal antibodies by genetic immunization. Nat Biotechnol 2003; 21: 1088-92.
41 Tuckwell DS, Smith L, Korda M. et al Monoclonal antibodies identify residues 199–216 of the integrin alpha2 vWFA domain as a functionally important region within alpha2beta1. Biochem J 2000; 350 Pt 2 485-93.
42 Moroi M, Onitsuka I, Imaizumi T. et al Involvement of activated integrin alpha2beta1 in the firm adhesion of platelets onto a surface of immobilized collagen under flow conditions. Thromb Haemost 2000; 83: 769-76.
43 Cruz MA, Chen J, Whitelock JL. et al The platelet glycoprotein Ib-von Willebrand factor interaction activates the collagen receptor {alpha}2{beta}1 to bind collagen: activation-dependent conformational change of the {alpha}2-I domain. Blood 2005; 105: 1986-91.
44 Nykvist P, Tu H, Ivaska J. et al Distinct recognition of collagen subtypes by alpha(1)beta(1) and alpha(2)beta(1) integrins. Alpha(1)beta(1) mediates cell adhesion to type XIII collagen. J Biol Chem 2000; 275: 8255-61.
45 Calderwood DA, Tuckwell DS, Eble J. et al The integrin alpha1 A-domain is a ligand binding site for collagens and laminin. J Biol Chem 1997; 272: 12311-7.
46 Chowdhury PS, Viner JL, Beers R. et al Isolation of a high-affinity stable single-chain Fv specific for mesothelin from DNA-immunized mice by phage display and construction of a recombinant immunotoxin with anti-tumor activity. Proc Natl Acad Sci U S A 1998; 95: 669-74.
47 Nagata S, Salvatore G, Pastan I. DNA immunization followed by a single boost with cells: a proteinfree immunization protocol for production of monoclonal antibodies against the native form of membrane proteins. J Immunol Methods 2003; 280: 59-72.