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. 2000 Mar 1;346(Pt 2):519–528.

Crystal structure of the anti-(carcinoembryonic antigen) single-chain Fv antibody MFE-23 and a model for antigen binding based on intermolecular contacts.

M K Boehm 1, A L Corper 1, T Wan 1, M K Sohi 1, B J Sutton 1, J D Thornton 1, P A Keep 1, K A Chester 1, R H Begent 1, S J Perkins 1
PMCID: PMC1220881  PMID: 10677374

Abstract

MFE-23 is the first single-chain Fv antibody molecule to be used in patients and is used to target colorectal cancer through its high affinity for carcinoembryonic antigen (CEA), a cell-surface member of the immunoglobulin superfamily. MFE-23 contains an N-terminal variable heavy-chain domain joined by a (Gly(4)Ser)(3) linker to a variable light-chain (V(L)) domain (kappa chain) with an 11-residue C-terminal Myc-tag. Its crystal structure was determined at 2.4 A resolution by molecular replacement with an R(cryst) of 19.0%. Five of the six antigen-binding loops, L1, L2, L3, H1 and H2, conformed to known canonical structures. The sixth loop, H3, displayed a unique structure, with a beta-hairpin loop and a bifurcated apex characterized by a buried Thr residue. In the crystal lattice, two MFE-23 molecules were associated back-to-back in a manner not seen before. The antigen-binding site displayed a large acidic region located mainly within the H2 loop and a large hydrophobic region within the H3 loop. Even though this structure is unliganded within the crystal, there is an unusually large region of contact between the H1, H2 and H3 loops and the beta-sheet of the V(L) domain of an adjacent molecule (strands DEBA) as a result of intermolecular packing. These interactions exhibited remarkably high surface and electrostatic complementarity. Of seven MFE-23 residues predicted to make contact with antigen, five participated in these lattice contacts, and this model for antigen binding is consistent with previously reported site-specific mutagenesis of MFE-23 and its effect on CEA binding.

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Selected References

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  1. Al-Lazikani B., Lesk A. M., Chothia C. Standard conformations for the canonical structures of immunoglobulins. J Mol Biol. 1997 Nov 7;273(4):927–948. doi: 10.1006/jmbi.1997.1354. [DOI] [PubMed] [Google Scholar]
  2. Bajorath J., Harris L., Novotny J. Conformational similarity and systematic displacement of complementarity determining region loops in high resolution antibody x-ray structures. J Biol Chem. 1995 Sep 22;270(38):22081–22084. doi: 10.1074/jbc.270.38.22081. [DOI] [PubMed] [Google Scholar]
  3. Begent R. H., Verhaar M. J., Chester K. A., Casey J. L., Green A. J., Napier M. P., Hope-Stone L. D., Cushen N., Keep P. A., Johnson C. J. Clinical evidence of efficient tumor targeting based on single-chain Fv antibody selected from a combinatorial library. Nat Med. 1996 Sep;2(9):979–984. doi: 10.1038/nm0996-979. [DOI] [PubMed] [Google Scholar]
  4. Benchimol S., Fuks A., Jothy S., Beauchemin N., Shirota K., Stanners C. P. Carcinoembryonic antigen, a human tumor marker, functions as an intercellular adhesion molecule. Cell. 1989 Apr 21;57(2):327–334. doi: 10.1016/0092-8674(89)90970-7. [DOI] [PubMed] [Google Scholar]
  5. Boehm M. K., Mayans M. O., Thornton J. D., Begent R. H., Keep P. A., Perkins S. J. Extended glycoprotein structure of the seven domains in human carcinoembryonic antigen by X-ray and neutron solution scattering and an automated curve fitting procedure: implications for cellular adhesion. J Mol Biol. 1996 Jun 21;259(4):718–736. doi: 10.1006/jmbi.1996.0353. [DOI] [PubMed] [Google Scholar]
  6. Braden B. C., Goldman E. R., Mariuzza R. A., Poljak R. J. Anatomy of an antibody molecule: structure, kinetics, thermodynamics and mutational studies of the antilysozyme antibody D1.3. Immunol Rev. 1998 Jun;163:45–57. doi: 10.1111/j.1600-065x.1998.tb01187.x. [DOI] [PubMed] [Google Scholar]
  7. Braden B. C., Poljak R. J. Structural features of the reactions between antibodies and protein antigens. FASEB J. 1995 Jan;9(1):9–16. doi: 10.1096/fasebj.9.1.7821765. [DOI] [PubMed] [Google Scholar]
  8. Chester K. A., Begent R. H., Robson L., Keep P., Pedley R. B., Boden J. A., Boxer G., Green A., Winter G., Cochet O. Phage libraries for generation of clinically useful antibodies. Lancet. 1994 Feb 19;343(8895):455–456. doi: 10.1016/s0140-6736(94)92695-6. [DOI] [PubMed] [Google Scholar]
  9. Chothia C., Novotný J., Bruccoleri R., Karplus M. Domain association in immunoglobulin molecules. The packing of variable domains. J Mol Biol. 1985 Dec 5;186(3):651–663. doi: 10.1016/0022-2836(85)90137-8. [DOI] [PubMed] [Google Scholar]
  10. GOLD P., FREEDMAN S. O. DEMONSTRATION OF TUMOR-SPECIFIC ANTIGENS IN HUMAN COLONIC CARCINOMATA BY IMMUNOLOGICAL TOLERANCE AND ABSORPTION TECHNIQUES. J Exp Med. 1965 Mar 1;121:439–462. doi: 10.1084/jem.121.3.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hoedemaeker F. J., Signorelli T., Johns K., Kuntz D. A., Rose D. R. A single chain Fv fragment of P-glycoprotein-specific monoclonal antibody C219. Design, expression, and crystal structure at 2.4 A resolution. J Biol Chem. 1997 Nov 21;272(47):29784–29789. doi: 10.1074/jbc.272.47.29784. [DOI] [PubMed] [Google Scholar]
  12. Huston J. S., Levinson D., Mudgett-Hunter M., Tai M. S., Novotný J., Margolies M. N., Ridge R. J., Bruccoleri R. E., Haber E., Crea R. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5879–5883. doi: 10.1073/pnas.85.16.5879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
  14. Kabsch W., Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983 Dec;22(12):2577–2637. doi: 10.1002/bip.360221211. [DOI] [PubMed] [Google Scholar]
  15. Kortt A. A., Malby R. L., Caldwell J. B., Gruen L. C., Ivancic N., Lawrence M. C., Howlett G. J., Webster R. G., Hudson P. J., Colman P. M. Recombinant anti-sialidase single-chain variable fragment antibody. Characterization, formation of dimer and higher-molecular-mass multimers and the solution of the crystal structure of the single-chain variable fragment/sialidase complex. Eur J Biochem. 1994 Apr 1;221(1):151–157. doi: 10.1111/j.1432-1033.1994.tb18724.x. [DOI] [PubMed] [Google Scholar]
  16. Lee B., Richards F. M. The interpretation of protein structures: estimation of static accessibility. J Mol Biol. 1971 Feb 14;55(3):379–400. doi: 10.1016/0022-2836(71)90324-x. [DOI] [PubMed] [Google Scholar]
  17. Matthews B. W. Solvent content of protein crystals. J Mol Biol. 1968 Apr 28;33(2):491–497. doi: 10.1016/0022-2836(68)90205-2. [DOI] [PubMed] [Google Scholar]
  18. Mian I. S., Bradwell A. R., Olson A. J. Structure, function and properties of antibody binding sites. J Mol Biol. 1991 Jan 5;217(1):133–151. doi: 10.1016/0022-2836(91)90617-f. [DOI] [PubMed] [Google Scholar]
  19. Morea V., Tramontano A., Rustici M., Chothia C., Lesk A. M. Conformations of the third hypervariable region in the VH domain of immunoglobulins. J Mol Biol. 1998 Jan 16;275(2):269–294. doi: 10.1006/jmbi.1997.1442. [DOI] [PubMed] [Google Scholar]
  20. Oliva B., Bates P. A., Querol E., Avilés F. X., Sternberg M. J. Automated classification of antibody complementarity determining region 3 of the heavy chain (H3) loops into canonical forms and its application to protein structure prediction. J Mol Biol. 1998 Jun 26;279(5):1193–1210. doi: 10.1006/jmbi.1998.1847. [DOI] [PubMed] [Google Scholar]
  21. Padlan E. A., Abergel C., Tipper J. P. Identification of specificity-determining residues in antibodies. FASEB J. 1995 Jan;9(1):133–139. doi: 10.1096/fasebj.9.1.7821752. [DOI] [PubMed] [Google Scholar]
  22. Padlan E. A. Anatomy of the antibody molecule. Mol Immunol. 1994 Feb;31(3):169–217. doi: 10.1016/0161-5890(94)90001-9. [DOI] [PubMed] [Google Scholar]
  23. Padlan E. A. On the nature of antibody combining sites: unusual structural features that may confer on these sites an enhanced capacity for binding ligands. Proteins. 1990;7(2):112–124. doi: 10.1002/prot.340070203. [DOI] [PubMed] [Google Scholar]
  24. Perisic O., Webb P. A., Holliger P., Winter G., Williams R. L. Crystal structure of a diabody, a bivalent antibody fragment. Structure. 1994 Dec 15;2(12):1217–1226. doi: 10.1016/s0969-2126(94)00123-5. [DOI] [PubMed] [Google Scholar]
  25. Perkins S. J. Protein volumes and hydration effects. The calculations of partial specific volumes, neutron scattering matchpoints and 280-nm absorption coefficients for proteins and glycoproteins from amino acid sequences. Eur J Biochem. 1986 May 15;157(1):169–180. doi: 10.1111/j.1432-1033.1986.tb09653.x. [DOI] [PubMed] [Google Scholar]
  26. Raag R., Whitlow M. Single-chain Fvs. FASEB J. 1995 Jan;9(1):73–80. doi: 10.1096/fasebj.9.1.7821762. [DOI] [PubMed] [Google Scholar]
  27. Ramachandran G. N., Sasisekharan V. Conformation of polypeptides and proteins. Adv Protein Chem. 1968;23:283–438. doi: 10.1016/s0065-3233(08)60402-7. [DOI] [PubMed] [Google Scholar]
  28. Sali A., Blundell T. L. Definition of general topological equivalence in protein structures. A procedure involving comparison of properties and relationships through simulated annealing and dynamic programming. J Mol Biol. 1990 Mar 20;212(2):403–428. doi: 10.1016/0022-2836(90)90134-8. [DOI] [PubMed] [Google Scholar]
  29. Saul F. A., Poljak R. J. Structural patterns at residue positions 9, 18, 67 and 82 in the VH framework regions of human and murine immunoglobulins. J Mol Biol. 1993 Mar 5;230(1):15–20. doi: 10.1006/jmbi.1993.1121. [DOI] [PubMed] [Google Scholar]
  30. Schlom J., Eggensperger D., Colcher D., Molinolo A., Houchens D., Miller L. S., Hinkle G., Siler K. Therapeutic advantage of high-affinity anticarcinoma radioimmunoconjugates. Cancer Res. 1992 Mar 1;52(5):1067–1072. [PubMed] [Google Scholar]
  31. Shirai H., Kidera A., Nakamura H. Structural classification of CDR-H3 in antibodies. FEBS Lett. 1996 Dec 9;399(1-2):1–8. doi: 10.1016/s0014-5793(96)01252-5. [DOI] [PubMed] [Google Scholar]
  32. Verhaar M. J., Chester K. A., Keep P. A., Robson L., Pedley R. B., Boden J. A., Hawkins R. E., Begent R. H. A single chain Fv derived from a filamentous phage library has distinct tumor targeting advantages over one derived from a hybridoma. Int J Cancer. 1995 May 16;61(4):497–501. doi: 10.1002/ijc.2910610412. [DOI] [PubMed] [Google Scholar]
  33. Waldmann T. A. Monoclonal antibodies in diagnosis and therapy. Science. 1991 Jun 21;252(5013):1657–1662. doi: 10.1126/science.2047874. [DOI] [PubMed] [Google Scholar]
  34. Wilson I. A., Stanfield R. L. Antibody-antigen interactions: new structures and new conformational changes. Curr Opin Struct Biol. 1994 Dec;4(6):857–867. doi: 10.1016/0959-440x(94)90267-4. [DOI] [PubMed] [Google Scholar]
  35. Wu T. T., Kabat E. A. An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity. J Exp Med. 1970 Aug 1;132(2):211–250. doi: 10.1084/jem.132.2.211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yokota T., Milenic D. E., Whitlow M., Schlom J. Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms. Cancer Res. 1992 Jun 15;52(12):3402–3408. [PubMed] [Google Scholar]
  37. Zdanov A., Li Y., Bundle D. R., Deng S. J., MacKenzie C. R., Narang S. A., Young N. M., Cygler M. Structure of a single-chain antibody variable domain (Fv) fragment complexed with a carbohydrate antigen at 1.7-A resolution. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6423–6427. doi: 10.1073/pnas.91.14.6423. [DOI] [PMC free article] [PubMed] [Google Scholar]

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