Structural basis of selectivity and neutralizing activity of a TGFα/epiregulin specific antibody

Abstract

Recent studies have implicated a role of the epidermal growth factor receptor (EGFR) pathway in kidney disease. Skin toxicity associated with therapeutics which completely block the EGFR pathway precludes their use in chronic dosing. Therefore, we developed antibodies which specifically neutralize the EGFR ligands TGFα (transforming growth factor-alpha) and epiregulin but not EGF (epidermal growth factor), amphiregulin, betacellulin, HB-EGF (heparin-binding epidermal growth factor), or epigen. The epitope of one such neutralizing antibody, LY3016859, was characterized in detail to elucidate the structural basis for ligand specificity. Here we report a crystal structure of the LY3016859 Fab fragment in complex with soluble human TGFα. Our data demonstrate a conformational epitope located primarily within the C-terminal subdomain of the ligand. In addition, point mutagenesis experiments were used to highlight specific amino acids which are critical for both antigen binding and neutralization, most notably Ala⁴¹ , Glu⁴⁴ , and His⁴⁵ . These results illustrate the structural basis for the ligand specificity/selectivity of LY3016859 and could also provide insight into further engineering to alter specificity and/or affinity of LY3016859.

Keywords: X-ray crystallography; epiregulin; epitope mapping; kidney disease; transforming growth factor alpha.

Figure 1

Alignment of the sequences of the seven human EGFR ligands as well as mouse TGFα, epiregulin, and epigen. Measured affinities (K _D) of LY3016859 to these ligands are shown on the right side. Based on sequence homology of the binding/non‐binding ligands, the C‐terminal sequence indicated by the box was initially proposed as the epitope region. Further inspection of the alignment showed the conserved (A/V)RCEH sequence for the ligands with sub nM affinity: human TGFα, mouse TGFα, and human epiregulin.

Figure 2

Overview of the LY3016859 Fab/human TGFα complex showing each complex in the asymmetric unit. The N‐ and C‐termini of TGFα are indicated. The coordinated Zn²⁺ metal ion is represented as a gray sphere. Heavy chain variable domain (V _H), heavy chain constant domain 1 (C _H1), light chain variable domain (V _L), and light chain kappa constant domain (C _L) are indicated.

Figure 3

The primary TGFα binding surface of LY3016859 is shown. Only residues 39–49 of TGFα (in magenta) are depicted. The LY3016859 surface is colored by electrostatic potential using Pymol, and several residues which make up the binding interface are annotated with HC amino acids in green and LC amino acids in cyan. Glu⁴⁴ and His⁴⁵ sit in a binding pocket, and a coordinated water molecule (red sphere) is observed in this pocket in both complexes in the asymmetric unit. Pockets which accommodate Ala⁴¹ and Leu⁴⁸ are also observed while Val³⁹, Arg⁴², Ala⁴⁶, Asp⁴⁷, and Leu⁴⁹ all sit on the surface of the Fab.

Figure 4

Comparison of the EGFR (1MOX) and LY3016859 binding sites. Structures are aligned by TGFα. TGFα from 1MOX is shown in yellow, and LY3016859 bound TGFα is shown in magenta. EGFR is shown in grey while the LY3016859 Fab is colored cyan (LC) and green (HC). A Cd²⁺ ion is observed in the 1MOX structure (yellow sphere) and is bound by TGFα similarly to the Zn²⁺ ion in the LY3016859 bound structure. It is clear from the superposition of the complexes that EGFR and LY3016859 cannot simultaneously bind TGFα, and this provides a structural basis for the neutralization of TGFα by LY3016859.

Figure 5

Interactions and key amino acid sidechains in the Glu⁴⁴/His⁴⁵ binding pocket are shown. Hydrogen bonds between TGFα (magenta)/Fab (LC in cyan, HC in green) and the bound water molecule (red sphere) are indicated by dashed lines. The water in the binding pocket forms a network of hydrogen bonds with the Glu⁴⁴ and His⁴⁵ sidechains, likely stabilizing the epitope conformation. Tyr¹⁰¹ of the LC and Tyr³³ of the heavy chain are critical for binding as they form numerous hydrogen bonds to both TGFα and the bound water molecule. The Ala⁴¹/HC Trp⁵² interaction which is important for selectivity is also shown. Substitution with bulkier amino acids such as glutamic acid at this position results in significant loss in affinity.

Figure 6

Biacore sensorgrams of LY3016859 binding to wt TGFα and three point mutants within the LY3016859 epitope. Binding kinetics are reported in Table II. The E44A mutation has the most dramatic effect with a 2,500‐fold loss in affinity resulting from an 8‐fold reduction in on‐rate and a 280‐fold increase in off‐rate. While the A41E and H45A mutants have affinity losses of ∼80‐fold, the decrease in H45A affinity is nearly entirely off‐rate driven while A41E suffers from both a decreased on‐rate and an increased off‐rate.