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. 2014 May-Jun;6(3):637-48.
doi: 10.4161/mabs.28395. Epub 2014 Mar 3.

A combinatorial mutagenesis approach for functional epitope mapping on phage-displayed target antigen: application to antibodies against epidermal growth factor

Yanelys Cabrera Infante  1 , Amaury Pupo  1 , Gertrudis Rojas  1
Affiliations

A combinatorial mutagenesis approach for functional epitope mapping on phage-displayed target antigen: application to antibodies against epidermal growth factor

Yanelys Cabrera Infante et al. MAbs. 2014 May-Jun.

Abstract

Although multiple different procedures to characterize the epitopes recognized by antibodies have been developed, site-directed mutagenesis remains the method of choice to define the energetic contribution of antigen residues to binding. These studies are useful to identify critical residues and to delineate functional maps of the epitopes. However, they tend to underestimate the roles of residues that are not critical for binding on their own, but contribute to the formation of the target epitope in an additive, or even cooperative, way. Mapping antigenic determinants with a diffuse energetic landscape, which establish multiple individually weak interactions with the antibody paratope, resulting in high affinity and specificity recognition of the epitope as a whole, is thus technically challenging. The current work was aimed at developing a combinatorial strategy to overcome the limitations of site-directed mutagenesis, relying on comprehensive randomization of discrete antigenic regions within phage-displayed antigen libraries. Two model antibodies recognizing epidermal growth factor were used to validate the mapping platform. Abrogation of antibody recognition due to the introduction of simultaneous replacements was able to show the involvement of particular amino acid clusters in epitope formation. The abundance of some of the original residues (or functionally equivalent amino acids sharing their physicochemical properties) among the set of mutated antigen variants selected on a given antibody highlighted their contributions and allowed delineation of a detailed functional map of the corresponding epitope. The use of the combinatorial approach could be expanded to map the interactions between other antigens/antibodies.

Keywords: EGF; library; phage display; randomization; site-directed mutagenesis.

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Figures

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Figure 1. Conformation-sensitivity of the epitopes recognized by CB-EGF.1 and CB-EGF.2 mAbs. Microtiter plates coated with either recombinant EGF (A) or phage-displayed EGF fused to a c-myc tag (B) were sequentially treated with dithiothreitol (DTT) and iodoacetamide to disrupt disulfide bonds. CB-EGF.1 and CB-EGF.2 mAbs, as well as the control anti-c-myc tag 9E10 mAb, were added to the plates. Bound antibodies were detected with anti-mouse IgG polyclonal Abs conjugated to horseradish peroxidase. Relative antigenicity of treated EGF toward each mAb was calculated, using the signal on untreated control coated wells as the reference of maximal antigenicity (100%).
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Figure 2. Phage-displayed EGF fragments designed to identify the antigenic regions recognized by anti-EGF mAbs. The first set of fragments (pep1-pep5) covers the entire EGF sequence and keeps important structural elements. The fragments correspond to the three EGF loops (loop A, loop B and loop C) including the two Cys defining each loop, as well as to the extra-loop N-terminal and C-terminal segments. Additional unpaired Cys within each fragment were replaced by Ser (shaded in gray). The second set (pep6-pep10) includes protruding fragments not corresponding to any entire loop, where all the Cys were substituted by Ser (shaded in gray).
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Figure 3. Recognition of phage-displayed EGF fragments. Polyvinyl chloride microtiter plates were coated with anti-EGF mAbs (CB-EGF.1 and CB-EGF.2), the anti-c-myc tag 9E10 mAb or an unrelated antibody. Phages displaying the different EGF fragments (1011 viral particles/mL) were added to the plates. Phage-displayed EGF and phages displaying only the c-myc tag fused to M13 PVIII were used as controls. Bound phages were detected with an anti-M13 mAb conjugated to horseradish peroxidase.
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Figure 4. Sequence divergence between EGF mutated variants recognized by CB-EGF.1 and non-reactive variants. Residues in the segment P7-Y13 were substituted by a random aa mixture in a library. Sequence identity with the non-mutated EGF within the targeted segment was evaluated in two groups of EGF variants: non-selected phage-displayed molecules not recognized by CB-EGF.1 mAb (35 unique sequences) and positive variants selected on immobilized CB-EGF.1 (42 unique sequences). Composition differences between both groups of variants are shown in (A). A second library targeted the L15-V19 segment. Differences between negative (24 unique sequences) and positive variants (29 unique sequences) from this library are shown in (B). An additional analysis took into account not only strict aa conservation, but also conservative replacements, as indicators of sequence similarity to wt EGF. Results with P7-Y13 and L15-V19 libraries are shown in (C) and (D) respectively.
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Figure 5. Sequence divergence between EGF mutated variants recognized by CB-EGF.2 and non-reactive variants. Residues in the segment M21-L26 were substituted by a random aa mixture in a library. Sequence identity with the non-mutated EGF within the targeted segment was evaluated in two groups of EGF variants: non-selected phage-displayed molecules not recognized by CB-EGF.2 mAb (20 unique sequences) and positive variants selected on immobilized CB-EGF.2 (43 unique sequences). Composition differences between both groups of variants are shown in (A). A second library targeted the D27-N32 segment. Differences between negative (24 unique sequences) and positive variants (43 unique sequences) from this library are shown in (B). An additional analysis took into account not only strict aa conservation, but also conservative replacements, as indicators of sequence similarity to wt EGF. Results with M21-L26 and D27-N32 libraries are shown in (C) and (D) respectively.
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Figure 6. Locations of the epitopes recognized by CB-EGF.1 and CB-EGF.2 mAbs on EGF surface. EGF molecule (PDB code 1IVO_C) is represented as a cartoon with semi-transparent surface. CB-EGF.1 and CB-EGF.2 epitope residues are labeled and their side chains are shown as lines. EGF interface with the receptor (< 5Å) is colored yellow. CB-EGF.1 and CB-EGF.2 epitopes are highlighted in red and blue respectively. The resulting overlapping areas between each epitope and the EGF contact regions with receptor domains are shown in orange and green, and the rest of EGF is in gray. The figure was generated with Pymol.
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References

    1. Boyman O, Kovar M, Rubinstein MP, Surh CD, Sprent J. Selective stimulation of T cell subsets with antibody-cytokine immune complexes. Science. 2006;311:1924–7. doi: 10.1126/science.1122927. - DOI - PubMed
    1. Klein C, Lammens A, Schäfer W, Gorges S, Schwaiger M, Mössner E, Hopfner KP, Umaña P, Niederfellner G. Epitope interactions of monoclonal antibodies targeting CD20 and their relationships to functional properties. MAbs. 2013;5:1–12. - PMC - PubMed
    1. Rojas G, Pupo A, Leon K, Avellanet J, Carmenate T, Sidhu S. Deciphering the molecular bases of the biological effects of antibodies against Interleukin-2: a versatile platform for fine epitope mapping. Immunobiology. 2013;218:105–13. doi: 10.1016/j.imbio.2012.02.009. - DOI - PubMed
    1. Rojas G, Cabrera Infante Y, Pupo A, Carmenate T. Fine epitope specificity of antibodies against Interleukin-2 explains their paradoxical immunomodulatory effects. MAbs. 2013;6:273–85. - PMC - PubMed
    1. Greenspan NS, Di Cera E. Defining epitopes: It’s not as easy as it seems. Nat Biotechnol. 1999;17:936–7. doi: 10.1038/13590. - DOI - PubMed

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