Identification of functional and structural amino-acid residues by parsimonious mutagenesis

doi:10.1016/0378-1119(95)00821-7

Gene

Volume 169, Issue 2, 1996, Pages 147-155

https://doi.org/10.1016/0378-1119(95)00821-7 Get rights and content

Abstract

For in vitro evolution of protein function, we previously proposed using parsimonious mutagenesis (PM), a technique where mutagenic oligodeoxynucleotides (oligo) are designed to minimize coding sequence redundancy and limit the number of amino acid (aa) residues which do not retain parental structural features. For this work, PM was used to increase the affinity of C6.5, a human single-chain Fv (scFv) that binds the glycoprotein tumor antigen, c-erbB-2. A phage antibody library was created where 19 aa located in three of the heavy (H) and light (L) chain antigen-binding loops (L1, L3 and H2) were simultaneously mutated. After four rounds of selection, 50% of scFv had a lower dissociation rate constant (koff) than the parental scFv. The K_d of these scFv ranged from twofold (K_d=7.0 × 10⁹M) to sixfold (K_d=2.4 × 10⁹M) lower than the parental scFv (K_d = 1.6 × 10⁻⁸M). In higher affinity scFv, substitutions occurred at $1019$ of the positions, with $2128$ substitutions occurring at only four positions, two in H2, and one each in L1 and L3. Only the wild type (wt) aa was observed at $919$ aa. Based on a model of C6.5, seven of the nine conserved aa have a structural role in the variable domain, either in maintaining the main chain conformation of the loop, or in packing on the H-chain variable domain. Two of the conserved aa are solvent exposed, suggesting they may play a critical role in recognition. Thus, PM identified three types of aa: structural aa, functional aa which modulate affinity, and functional aa, which are critical for recognition. Since the sequence space was not completely sampled, higher affinity scFv could be produced by subjecting functional aa which modulate affinity to a higher rate of mutation. Furthermore, PM could prove useful for modifying function in other proteins that belong to structurally related families

References (38)

R.F. Balint et al.
Antibody engineering by parsimonious mutagenesis
Gene
(1993)
C. Chothia et al.
Canonical structures for the hypervariable regions of immunoglobulins
J. Mol. Biol.
(1987)
C. Chothia et al.
Structural repertoire of the human VH segments
J. Mol. Biol.
(1992)
R.E. Hawkins et al.
The contribution of contact and non-contact residues of antibody in the affinity of binding to antigen
R.E. Hawkins et al.
Selection of phage antibodies by binding affinity: mimicking affinity maturation
J. Mol. Biol.
(1992)
H.R. Hoogenboom et al.
Bypassing immunisation: human antibodies from synthetic repertoires of germ line VH-gene segments rearranged in vitro
J. Mol. Biol.
(1992)
B. Johnsson et al.
Immobilization of proteins to a carboxymethyldextran modified gold surface for BIAcore in surface plasmon resonance
Anal. Biochem.
(1991)
R. Karlsson et al.
Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system
J. Immunol. Methods
(1991)
H.B. Lowman et al.
Affinity maturation of human growth hormone by monovalent phage display
J. Mol. Biol.
(1993)
J.D. Marks et al.
By-passing immunization: human antibodies from V-gene libraries displayed on phage
J. Mol. Biol.
(1991)

M. Marquart et al.

Crystallographic refinement and atomic models of the intact immunoglobulin molecule KOL and its antigen binding fragment at 3.0Å and 1.9Å resolution

J. Mol. Biol.

(1980)

R. Schier et al.

In vitro and in vivo characterization of a human anti-c-erbB-2 single-chain Fv isolated from a filamentous phage antibody library

Immunotechnology

(1995)

R. Schier et al.

Isolation of high affinity monmeric human anti-c-erbB-2 single chain Fv using affinity driven selection

J. Mol. Biol.

(1996)

I.M. Tomlinson et al.

The repertoire of human germline V_H sequences reveals about fifty groups of V_H segments with different hypervariable loops

J. Mol. Biol.

(1992)

W.R. Tulip et al.

Refined crystal structure of the influenza virus neuraminidase-NC41 Fab complex

J. Mol Biol.

(1992)

I.A. Wilson et al.

Antibody-antigen interactions

Curr. Opin. Struct. Biol.

(1993)

G.P. Adams et al.

Highly specific in vivo tumor targeting by monovalent and divalent forms of 741F8 anti-c-erbB-2 single chain Fv

Cancer Res.

(1993)

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

S.D. Breitling et al.

A surface expression vector for antibody screening

Gene

(1991)

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Identification of functional and structural amino-acid residues by parsimonious mutagenesis

Abstract

Gene

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

Anal. Biochem.

J. Immunol. Methods

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

Immunotechnology

J. Mol. Biol.

J. Mol. Biol.

J. Mol Biol.

Curr. Opin. Struct. Biol.

Highly specific in vivo tumor targeting by monovalent and divalent forms of 741F8 anti-c-erbB-2 single chain Fv

Cancer Res.

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

A surface expression vector for antibody screening

Gene