Immune Netw.  2012 Aug;12(4):155-164.

Affinity Maturation of an Epidermal Growth Factor Receptor Targeting Human Monoclonal Antibody ER414 by CDR Mutation

• Chang KH
• Kim MS
• Hong GW
• Seo MS
• Shin YN
• Kim SH

Abstract

It is well established that blocking the interaction of EGFR with growth factors leads to the arrest of tumor growth, resulting in tumor cell death. ER414 is a human monoclonal antibody (mAb) derived by guided selection of the mouse mAb A13. The ER414 exhibited a ~17-fold lower affinity and, as a result, lower efficacy of inhibition of the EGF-mediated tyrosine phosphorylation of EGFR when compared with mAb A13 and cetuximab. We performed a stepwise in vitro affinity maturation to improve the affinity of ER414. We obtained a 3D model of ER414 to identify the amino acids in the CDRs that needed to be mutated. Clones were selected from the phage library with randomized amino acids in the CDRs and substitution of amino acids in the HCDR3 and LCDR1 of ER414 led to improved affinity. A clone, H3-14, with a ~20-fold increased affinity, was selected from the HCDR3 randomized library. Then three clones, ER2, ER78 and ER79, were selected from the LCDR1 randomized library based on the H3-14 but did not show further increased affinities compared to that of H3-14. Of the three, ER2 was chosen for further characterization due to its better expression than others. We successfully performed affinity maturation of ER414 and obtained antibodies with a similar affinity as cetuximab. And antibody from an affinity maturation inhibits the EGF-mediated tyrosine phosphorylation of EGFR in a manner similar to cetuximab.

Keyword

Affinity maturation; EGFR; Epitope; Phage-display; CDR randomization; Tyrosine phosphorylation

MeSH Terms

Amino Acids
Animals
Antibodies
Antibodies, Monoclonal, Humanized
Bacteriophages
Cell Death
Cetuximab
Clone Cells
Deoxycytidine
Epidermal Growth Factor
Humans
Intercellular Signaling Peptides and Proteins
Mice
Phosphorylation
Receptor, Epidermal Growth Factor
Tyrosine
Amino Acids
Antibodies
Antibodies, Monoclonal, Humanized
Deoxycytidine
Epidermal Growth Factor
Intercellular Signaling Peptides and Proteins
Receptor, Epidermal Growth Factor
Tyrosine

Figure

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  Figure 1 3D model of ER414 using the program Web Antibody Modeling (WAM; http://antibody.bath.ac.uk). Amino acids are numbered sequentially. VH is in brown, and VL is in cyan; HCDR3 (green) and LCDR1 (red) are highlighted.

• 

  Figure 2 The arrangement of genes in the pSC73 vector. LacZ denotes the lac promoter. PelB denotes the leader peptide of pectate lyase B of Erwinia carotovora, and GeneIII denotes gene3, which encodes the minor coat protein of the filamentous phage M13. His denotes a tag of 6 histidine repeats. Restriction enzyme sites used for cloning of scFv are shown.

• 

  Figure 3 The arrangement of genes in the expression vectors for the heavy (pRC12) and light (pKC12) chains. PCMV denotes cytomegalovirus promoter, and Sig denotes signal peptide of human immunoglobulin. Restriction enzyme sites used for cloning are shown. The light chain expression vector contains the dihydrofolate reductase (dhfr) gene (not shown) used for amplification of integrated genes by adjusting the cells in increasing concentrations of methotrexate (MTX).

• 

  Figure 4 Competition ELISA for measuring relative affinities of anti-EGFR IgGs from random mutations in the HCDR3 of ER414. The inhibition of anti-EGFR IgG binding to EGFR was analyzed on EGFR-coated plates with different concentrations of free EGFR, and the curves were fitted to a 4-parameter model using SoftMaxPro software. The antibodies are H3-14 (▴), H3-15 (♦) and ER414 (•).

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  Figure 5 Competition ELISA for measuring the relative affinities of anti-EGFR IgGs from random mutations in the LCDR1 of H3-14. The inhibition of anti-EGFR IgG binding to EGFR was analyzed on EGFR-coated plates with different concentrations of free EGFR, and the curves were fitted to a 4-parameter model using the SoftMaxPro software. The antibodies are ER2 (▪), ER78 (♦), ER79 (▴), ER414 (•) and cetuximab (○).

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  Figure 6 Binding of anti-EGFR mAbs to cell surface-expressed EGFR. Differential binding of mAbs to EGFR-positive A431 tumor cells was determined by flow cytometry. Histograms in different colors represent the reactions of labeled secondary Ab with A431 cells (black), ER414 with A431 cells (green), ER2 with A431 cells (blue), and mAb A13 with A431 cells (red).

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  Figure 7 Blocking of the EGF-induced tyrosine phosphorylation of EGFR by A13 mAb, ER414, ER2 and cetuximab in MDA-MB-231 cells. The serum-starved cells were untreated, treated with EGF (16 nM) only, or treated with EGF (16 nM) and 33.3, 166.7, 333.3 or 666.7 pM of mAbs for 30 min, as indicated in the panels, before the Western blotting analyses. The arrows indicate the expected size (~170 kDa) of the EGFR.

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  Figure 8 Domain-level epitope mapping of ER2 using the yeast surface-expressed EGFR fragments. Representative flow cytometry histograms depict anti-c-myc 9e10 labeling for the expression of EGFR fragments (black) and ER2 binding (blue) to the yeast surface-expressed EGFR fragments, as indicated in each panel. For the whole EGFR (1~621), the 404SG mutant was used (described in detail in the text).

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  Figure 9 Comparison of the ER2 and cetuximab binding sites on EGFR by competitive SPR assay. The first part of the curve shows the response for the binding of cetuximab (500 nM) to the surface-immobilized sEGFR, and the second part of the curve shows the binding upon injection of ER2 (500 nM) before the dissociation phase.

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