Journal of Molecular Biology
Volume 293, Issue 4, 5 November 1999, Pages 865-881
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Regular article
Selection and analysis of an optimized anti-VEGF antibody: crystal structure of an affinity-matured fab in complex with antigen1

https://doi.org/10.1006/jmbi.1999.3192Get rights and content

Abstract

The Fab portion of a humanized antibody (Fab-12; IgG form known as rhuMAb VEGF) to vascular endothelial growth factor (VEGF) has been affinity-matured through complementarity-determining region (CDR) mutation, followed by affinity selection using monovalent phage display. After stringent binding selections at 37°C, with dissociation (off-rate) selection periods of several days, high affinity variants were isolated from CDR-H1, H2, and H3 libraries. Mutations were combined to obtain cumulatively tighter-binding variants. The final variant identified here, Y0317, contained six mutations from the parental antibody. In vitro cell-based assays show that four mutations yielded an improvement of about 100-fold in potency for inhibition of VEGF-dependent cell proliferation by this variant, consistent with the equilibrium binding constant determined from kinetics experiments at 37°C. Using X-ray crystallography, we determined a high-resolution structure of the complex between VEGF and the affinity-matured Fab fragment. The overall features of the binding interface seen previously with wild-type are preserved, and many contact residues are maintained in precise alignment in the superimposed structures. However, locally, we see evidence for improved contacts between antibody and antigen, and two mutations result in increased van der Waals contact and improved hydrogen bonding. Site-directed mutants confirm that the most favorable improvements as judged by examination of the complex structure, in fact, have the greatest impact on free energy of binding. In general, the final antibody has improved affinity for several VEGF variants as compared with the parental antibody; however, some contact residues on VEGF differ in their contribution to the energetics of Fab binding. The results show that small changes even in a large protein-protein binding interface can have significant effects on the energetics of interaction.

Introduction

Angiogenic factors (Folkman & Klagsbrun, 1987), which stimulate endothelial cells leading to new vascularization, have roles in such disease states as cancer, rheumatoid arthritis, and macular degeneration (reviewed by Ferrara 1995, Folkman 1995, Iruela-Arispe and Dvorak 1997). Vascular endothelial growth factor (VEGF), a heparin-binding protein initially identified from pituitary cells (Ferrara & Henzel, 1989), is clearly a key angiogenic factor in development as well as in certain disease states, including the growth of solid tumors (reviewed by Ferrara, 1999). A murine monoclonal antibody, A.4.6.1, was found to block VEGF-dependent cell proliferation in vitro and to antagonize tumor growth in vivo(Kim et al., 1993). The murine mAb was previously humanized in Fab form to yield a variant known as Fab-12 (Presta et al., 1997). Both chimeric and humanized antibodies retained high affinity binding to VEGF, with an apparent equilibrium dissociation constant, Kd25°, of 0.9 to 3 nM Presta et al 1997, Baca et al 1997, Muller et al 1998a. The corresponding full-length IgG form of this antibody, rhumAb VEGF, is being developed as a possible therapeutic agent for the treatment of human solid tumors (Mordenti et al., 1999).

We became interested in obtaining higher affinity variants of Fab-12 in order to test whether affinity improvements of this antibody might improve its potency and efficacy. Phage display of randomized libraries of antibodies and other proteins has been extensively used to engineer proteins with improved affinity and specificity (Lowman et al., 1991; reviewed by Kay and Hoess 1996, Rader and Barbas 1997, Griffiths and Duncan 1998). In particular, a phage-based in vitro affinity maturation process has been successful in improving the binding affinity of antibodies previously identified from traditional monoclonal or naive-library sources (e.g. Hawkins et al 1992, Marks et al 1992, Barbas et al 1994, Yang et al 1995, Schier et al 1996, Thompson et al 1996).

In previous work, the humanized anti-VEGF antibody Fab-12 was adapted for improved monovalent phage display through selection of a CDR-L1 variant, designated Y0192 (Muller et al., 1998a). To select target residues for randomization and affinity optimization, we also previously screened all CDR residues, as defined by a combination of the hypervariable (Kabat et al., 1987) and structurally defined (Chothia & Lesk, 1987) CDR residues. Fab variants of Y0192 generated by alanine scanning were analyzed for side-chain contributions to antigen binding (Muller et al., 1998a). In addition, a crystal structure of Fab-12 in complex with the receptor-binding domain of VEGF, VEGF(109), was determined (Muller et al., 1998a). The results of these studies showed that the antigen binding site is almost entirely composed of residues from the heavy chain CDRs, CDR-H1, H2, and H3. Therefore, these CDRs appeared most likely to provide the opportunity for improved binding interactions with antigen.

Here, we describe the selection of an affinity-improved anti-VEGF antibody by phage display and off-rate selection. We show that the affinity-matured antibody binds VEGF with at least 20-fold improved affinity and inhibits VEGF-induced cell proliferation with enhanced potency in a cell-based assay. We also report the crystal structure of an affinity-optimized antibody in complex with VEGF, to our knowledge, representing the first reported structure of an in vitro affinity-matured antibody:antigen complex. The structure, together with mutational analysis, shows that subtle changes in the antibody-antigen interface account for improved affinity.

Section snippets

Library design

We used the results of an alanine-scanning analysis, combined with a crystal structure of the wild-type Fab fragment in complex with VEGF (Muller et al., 1998a), to design targeted libraries within the antibody CDRs for random mutagenesis and affinity selection. This strategy enabled us to construct theoretically complete libraries with a small number of residues randomized in each CDR. Although sites remote from the antigen-combining region or buried within the protein could modulate antigen

Antibody binding selections and affinity improvement

Here we made use of results from alanine-scanning and the previous structure of a humanized antibody-antigen complex to design Fab-phage libraries that randomized the three heavy-chain CDRs as well as one framework region (FR-H3) for improving the binding affinity of an anti-VEGF antibody. Affinity-improved Fab variants were obtained, with the largest effects seen in variants from the CDR-H3 library, although significant improvement was also obtained from mutation of CDR-H1. We therefore

Construction of phage libraries and mutagenesis

A variant of the Fab-12 antibody (a humanized form of murine antibody A4.6.1) was previously identified from phage-displayed Fab libraries for improved expression on phage particles (Muller et al., 1998a). We made use of the plasmid pY0192, a phagemid construct with ampicillin (or carbenicillin) resistance, as the parental (“wild-type”) construct for libraries described here. To prevent contamination by wild-type sequence Lowman et al 1991, Lowman 1998, templates with the TAA stop codon at each

Acknowledgements

We thank Lyn Deguzman and Tom Zioncheck for providing 125[I]VEGF; Alan Padua and Bill Henzel for quantitative amino acid analysis; James Bourell for mass spectrometry; Vanessa Chisholm and Lynne Krummen for construction of cell lines; and Manuel Baca, Napoleone Ferrara, Yves Muller, Leonard Presta, and James Wells for many helpful discussions.

References (48)

  • B.K. Kay et al.

    Principles and applications of phage display

  • T.A. Kunkel et al.

    Efficient site-directed mutagenesis using uracil-containing DNA

    Methods Enzymol.

    (1991)
  • G. Küttner et al.

    A phage library-derived single-chain Fv fragment in complex with turkey egg-white lysozymecharacterization, crystallization and preliminary X-ray analysis

    Mol. Immunol.

    (1998)
  • H.B. Lowman et al.

    Affinity maturation of human growth hormone by monovalent phage display

    J. Mol. Biol.

    (1993)
  • Y.A. Muller et al.

    VEGF and the Fab fragment of a humanized neutralizing antibodycrystal structure of the complex at 2.4 Å resolution and mutational analysis of the interface

    Structure

    (1998)
  • P. Munson et al.

    Liganda versatile computerized approach for characterization of ligand-binding systems

    Anal. Biochem.

    (1980)
  • C. Rader et al.

    Phage display of combinatorial antibody libraries

    Curr. Opin. Biotechnol.

    (1997)
  • R. Schier et al.

    Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site

    J. Mol. Biol.

    (1996)
  • J. Thompson et al.

    Affinity maturation of a high-affinity human monoclonal antibody against the third hypervariable loop of human immunodeficiency virus: use of phage display to improve affinity and broaden strain reactivity

    J. Mol. Biol.

    (1996)
  • A. Tramontano et al.

    Framework residue 71 is a major determinant of the position and conformation of the second hypervariable region in the VH domains of immunoglobulins

    J. Mol. Biol.

    (1990)
  • J. Xiang et al.

    Framework residues 71 and 93 of the chimeric B72.3 antibody are major determinants of the conformation of heavy-chain hypervariable loops

    J. Mol. Biol.

    (1995)
  • W.-P. Yang et al.

    CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range

    J. Mol. Biol.

    (1995)
  • C.F. Barbas et al.

    In vitro evolution of a neutralizing human antibody to human immunodeficiency virus type 1 to enhance affinity and broaden strain cross-reactivity

    Proc. Natl Acad. Sci. USA

    (1994)
  • S. Bass et al.

    Hormone phagean enrichment method for variant proteins with altered binding properties

    Proteins: Struct. Funct. Genet.

    (1990)
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