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Structural consequences of humanizing an antibody

M A Holmes1, J Foote

  • 1Division of Molecular Medicine, Fred Hutchinson Cancer Research Center, Seattle, WA 98104, USA.

Journal of Immunology (Baltimore, Md. : 1950)
|March 1, 1997
PubMed
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We determined the crystal structure of a humanized antibody fragment (HuLys) against hen eggwhite lysozyme. Grafted antibody regions (CDRs) influenced framework structure, maintaining binding flexibility and showing humanization is viable for antibodies with induced fit mechanisms.

Area of Science:

  • Structural Biology
  • Immunology
  • Biochemistry

Background:

  • Humanized antibodies (HuLys) are crucial for therapeutic applications.
  • Understanding antibody structure-framework interactions is key to antibody engineering.
  • Previous studies on antibody humanization have focused on sequence rather than structural impact.

Purpose of the Study:

  • To determine the crystal structure of the Fv fragment of a humanized anti-hen eggwhite lysozyme antibody (HuLys).
  • To investigate the structural consequences of grafting complementarity-determining regions (CDRs) from a mouse antibody onto human antibody frameworks.
  • To assess the impact of humanization on antibody binding site conformation and flexibility.

Main Methods:

  • X-ray crystallography was used to determine the structure of the HuLys Fv fragment.

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  • Molecular replacement was employed for structure solution, using a composite model of parent antibody structures.
  • The crystal structure was resolved to 2.87 A resolution with an R factor of 22%.

    • Main Results:

    • The HuLys Fv fragment crystallized in space group P4(3)2(1)2 with two molecules in the asymmetric unit.
    • Framework regions of HuLys adopted conformations closer to the parent mouse antibody (D1.3) than to the human sequences.
    • Grafted CDRs maintained conformational equilibria, indicating humanization is feasible for antibodies utilizing induced fit or isomeric equilibrium binding mechanisms.
    • Systematic differences, resembling domain rotations, were observed between HuLys and D1.3, though the V(H)-V(L) interface remained unaffected.

    Conclusions:

    • Antibody complementarity-determining regions (CDRs) can significantly influence the conformation of antibody framework regions, even inducing a return to a more parent-like structure.
    • Humanization of antibodies, even those employing induced fit or isomeric equilibrium, is structurally feasible.
    • The observed domain rotations in HuLys are likely due to rearrangements distant from the variable heavy-variable light (V(H)-V(L)) interface.