Multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange

Protein Eng. 2001 Dec;14(12):1025-33. doi: 10.1093/protein/14.12.1025.


A series of single-chain anti-CD20 antibodies was produced by fusing single-chain Fv (scFv) with human IgG1 hinge and Fc regions, designated scFv-Fc. The initial scFv-Fc construct was assembled using an 18 amino acid (aa) linker between the antibody light- and heavy-chain variable regions, with the Cys residue in the upper hinge region (Kabat 233) mutagenized to Ser. Anti-CD20 scFv-Fc retained specific binding to CD20-positive cells and was active in mediating complement-dependent cytolysis. Size-exclusion HPLC analysis revealed that the purified scFv-Fc included multimeric as well as monomeric components. Variant scFv-Fcs were constructed incorporating four different hinges between the scFv and Fc regions, or three different linkers in the scFv domain. All formed multimers, with the highest level of multimerization found in the scFv-Fc with the shortest linker (8 aa). Elimination of an unusual salt bridge between residues L38 and H89 in the V(L)-V(H) domain interface failed to reduce the formation of higher order forms. Structural analysis of the scFv-Fc constructed with 18 or 8 aa linkers by pepsin or papain cleavage suggested the proteins contained a form in which scFv units had cross-paired to form a 'diabody'. Thus, domain exchange or cross-pairing appears to be the basis of the observed multimerization.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies / chemistry
  • Antibodies / genetics
  • Antibodies / immunology*
  • Antibodies / isolation & purification
  • Antigens, CD20 / chemistry
  • Antigens, CD20 / immunology*
  • Cloning, Molecular
  • Dimerization
  • Immunoglobulin Fragments / genetics
  • Immunoglobulin Fragments / immunology
  • Mice
  • Molecular Sequence Data
  • Papain
  • Pepsin A
  • Protein Conformation
  • Protein Isoforms
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / chemistry
  • Tumor Cells, Cultured


  • Antibodies
  • Antigens, CD20
  • Immunoglobulin Fragments
  • Protein Isoforms
  • Recombinant Fusion Proteins
  • immunoglobulin Fv
  • Papain
  • Pepsin A