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. 2007 Oct;25(10):1171-6.
doi: 10.1038/nbt1336. Epub 2007 Sep 23.

Computational design of antibody-affinity improvement beyond in vivo maturation

Shaun M Lippow  1 , K Dane Wittrup, Bruce Tidor
Affiliations

Computational design of antibody-affinity improvement beyond in vivo maturation

Shaun M Lippow et al. Nat Biotechnol. 2007 Oct.

Abstract

Antibodies are used extensively in diagnostics and as therapeutic agents. Achieving high-affinity binding is important for expanding detection limits, extending dissociation half-times, decreasing drug dosages and increasing drug efficacy. However, antibody-affinity maturation in vivo often fails to produce antibody drugs of the targeted potency, making further affinity maturation in vitro by directed evolution or computational design necessary. Here we present an iterative computational design procedure that focuses on electrostatic binding contributions and single mutants. By combining multiple designed mutations, a tenfold affinity improvement to 52 pM was engineered into the anti-epidermal growth factor receptor drug cetuximab (Erbitux), and a 140-fold improvement in affinity to 30 pM was obtained for the anti-lysozyme model antibody D44.1. The generality of the methods was further demonstrated through identification of known affinity-enhancing mutations in the therapeutic antibody bevacizumab (Avastin) and the model anti-fluorescein antibody 4-4-20. These results demonstrate computational capabilities for enhancing and accelerating the development of protein reagents and therapeutics.

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Figures

Figure 1
Figure 1
Designed high-affinity mutations in D44.1. (A) Experimental binding affinities, from left to right: 140-fold hex, 100-fold quad, L92 Asn-to-Ala, H58 Thr-to-Asp, H57 Ser-to-Val, H28 Thr-to-Asp, wild type. For each variant, different symbol shapes distinguish independent measurements. (B to D) Predicted structures for single mutations; green ribbon: lysozyme backbone; magenta ribbon: antibody backbone; atom color: design; grey: wild type. (B) L92 Asn-to-Ala. (C) H57 Ser-to-Val, top; H58 Thr-to-Asp, bottom. (D) H28 Thr-to-Asp. (E) Wild type in region of double mutation. The top-center arginine is from lysozyme and all other residues are from the antibody. (F) Designed double mutation: H99 Gly-to-Asp, H35 Glu-to-Ser. The Asp is predicted to displace a crystallographic water molecule.
Figure 1
Figure 1
Designed high-affinity mutations in D44.1. (A) Experimental binding affinities, from left to right: 140-fold hex, 100-fold quad, L92 Asn-to-Ala, H58 Thr-to-Asp, H57 Ser-to-Val, H28 Thr-to-Asp, wild type. For each variant, different symbol shapes distinguish independent measurements. (B to D) Predicted structures for single mutations; green ribbon: lysozyme backbone; magenta ribbon: antibody backbone; atom color: design; grey: wild type. (B) L92 Asn-to-Ala. (C) H57 Ser-to-Val, top; H58 Thr-to-Asp, bottom. (D) H28 Thr-to-Asp. (E) Wild type in region of double mutation. The top-center arginine is from lysozyme and all other residues are from the antibody. (F) Designed double mutation: H99 Gly-to-Asp, H35 Glu-to-Ser. The Asp is predicted to displace a crystallographic water molecule.
Figure 1
Figure 1
Designed high-affinity mutations in D44.1. (A) Experimental binding affinities, from left to right: 140-fold hex, 100-fold quad, L92 Asn-to-Ala, H58 Thr-to-Asp, H57 Ser-to-Val, H28 Thr-to-Asp, wild type. For each variant, different symbol shapes distinguish independent measurements. (B to D) Predicted structures for single mutations; green ribbon: lysozyme backbone; magenta ribbon: antibody backbone; atom color: design; grey: wild type. (B) L92 Asn-to-Ala. (C) H57 Ser-to-Val, top; H58 Thr-to-Asp, bottom. (D) H28 Thr-to-Asp. (E) Wild type in region of double mutation. The top-center arginine is from lysozyme and all other residues are from the antibody. (F) Designed double mutation: H99 Gly-to-Asp, H35 Glu-to-Ser. The Asp is predicted to displace a crystallographic water molecule.
Figure 1
Figure 1
Designed high-affinity mutations in D44.1. (A) Experimental binding affinities, from left to right: 140-fold hex, 100-fold quad, L92 Asn-to-Ala, H58 Thr-to-Asp, H57 Ser-to-Val, H28 Thr-to-Asp, wild type. For each variant, different symbol shapes distinguish independent measurements. (B to D) Predicted structures for single mutations; green ribbon: lysozyme backbone; magenta ribbon: antibody backbone; atom color: design; grey: wild type. (B) L92 Asn-to-Ala. (C) H57 Ser-to-Val, top; H58 Thr-to-Asp, bottom. (D) H28 Thr-to-Asp. (E) Wild type in region of double mutation. The top-center arginine is from lysozyme and all other residues are from the antibody. (F) Designed double mutation: H99 Gly-to-Asp, H35 Glu-to-Ser. The Asp is predicted to displace a crystallographic water molecule.
Figure 1
Figure 1
Designed high-affinity mutations in D44.1. (A) Experimental binding affinities, from left to right: 140-fold hex, 100-fold quad, L92 Asn-to-Ala, H58 Thr-to-Asp, H57 Ser-to-Val, H28 Thr-to-Asp, wild type. For each variant, different symbol shapes distinguish independent measurements. (B to D) Predicted structures for single mutations; green ribbon: lysozyme backbone; magenta ribbon: antibody backbone; atom color: design; grey: wild type. (B) L92 Asn-to-Ala. (C) H57 Ser-to-Val, top; H58 Thr-to-Asp, bottom. (D) H28 Thr-to-Asp. (E) Wild type in region of double mutation. The top-center arginine is from lysozyme and all other residues are from the antibody. (F) Designed double mutation: H99 Gly-to-Asp, H35 Glu-to-Ser. The Asp is predicted to displace a crystallographic water molecule.
Figure 1
Figure 1
Designed high-affinity mutations in D44.1. (A) Experimental binding affinities, from left to right: 140-fold hex, 100-fold quad, L92 Asn-to-Ala, H58 Thr-to-Asp, H57 Ser-to-Val, H28 Thr-to-Asp, wild type. For each variant, different symbol shapes distinguish independent measurements. (B to D) Predicted structures for single mutations; green ribbon: lysozyme backbone; magenta ribbon: antibody backbone; atom color: design; grey: wild type. (B) L92 Asn-to-Ala. (C) H57 Ser-to-Val, top; H58 Thr-to-Asp, bottom. (D) H28 Thr-to-Asp. (E) Wild type in region of double mutation. The top-center arginine is from lysozyme and all other residues are from the antibody. (F) Designed double mutation: H99 Gly-to-Asp, H35 Glu-to-Ser. The Asp is predicted to displace a crystallographic water molecule.
Figure 2
Figure 2
Designed high-affinity cetuximab mutant. Experimental binding affinity titrations as displayed for cetuximab (triangles) and 10-fold improved triple-mutant (squares).
Figure 3
Figure 3
Comparison of calculated and experimental binding free energies. Free energies are in kcal/mol relative to wild type, with the y=x line included to aid interpretation. diamonds: D1.3 mutants; filled triangles: D44.1 single mutants; open triangles: D44.1 combination mutants; filled squares: cetuximab single mutants; open squares: cetuximab combination mutants; asterisks: 4-4-20 mutants. (A) Calculated total free energy. (B) Calculated electrostatic free energy term.
Figure 3
Figure 3
Comparison of calculated and experimental binding free energies. Free energies are in kcal/mol relative to wild type, with the y=x line included to aid interpretation. diamonds: D1.3 mutants; filled triangles: D44.1 single mutants; open triangles: D44.1 combination mutants; filled squares: cetuximab single mutants; open squares: cetuximab combination mutants; asterisks: 4-4-20 mutants. (A) Calculated total free energy. (B) Calculated electrostatic free energy term.
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Comment in

  • No free energy lunch.
    Shoichet BK. Shoichet BK. Nat Biotechnol. 2007 Oct;25(10):1109-10. doi: 10.1038/nbt1007-1109. Nat Biotechnol. 2007. PMID: 17921992 No abstract available.

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