Lessons from the Crystal Structure of the S. aureus Surface Protein Clumping Factor A in Complex With Tefibazumab, an Inhibiting Monoclonal Antibody

Abstract

The Staphylococcus aureus fibrinogen binding MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules), ClfA (clumping factor A) is an important virulence factor in staphylococcal infections and a component of several vaccines currently under clinical evaluation. The mouse monoclonal antibody aurexis (also called 12-9), and the humanized version tefibazumab are therapeutic monoclonal antibodies targeting ClfA that in combination with conventional antibiotics were effective in animal models but showed less impressive efficacy in a limited Phase II clinical trial. We here report the crystal structure and a biochemical characterization of the ClfA/tefibazumab (Fab) complex. The epitope for tefibazumab is located to the "top" of the N3 subdomain of ClfA and partially overlaps with a previously unidentified second binding site for fibrinogen. A high-affinity binding of ClfA to fibrinogen involves both an interaction at the N3 site and the previously identified docking of the C-terminal segment of the fibrinogen γ-chain in the N2N3 trench. Although tefibazumab binds ClfA with high affinity we observe a modest IC₅₀ value for the inhibition of fibrinogen binding to the MSCRAMM. This observation, paired with a common natural occurring variant of ClfA that is not effectively recognized by the mAb, may partly explain the modest effect tefibazumab showed in the initial clinic trail. This information will provide guidance for the design of the next generation of therapeutic anti-staphylococcal mAbs targeting ClfA.

Keywords: Aurexis; Clumping factor A; Fibrinogen; Staphylococcal infections; Tefibazumab; Therapeutic mAb.

Graphical abstract

Fig. 1

SPR analysis of the ligand interactions and inhibitions. (A) Two-fold serial dilution of ClfA_229–545 (from 32 to 1 nM) were injected to a tefibazumab surface (~ 150 RU captured by goat anti-human IgG (Fc) polyclonal F(ab’)₂). SPR sensorgrams shown in black were fitted to a 1:1 Langmuir binding model curves shown in red. The derived dissociation constant K_D (0.81 nM) was calculated from the rate constants (k_a = 5.94 × 10⁵ M^− 1 s^− 1 and k_d = 4.90 × 10^− 4 s^− 1). (B) ClfA_229–545 (from 1.28 to 0.04 μM) was injected to a Fg surface (10,000 RU on CM5 chip). The average responses at steady state (shown in red) were plotted as a function of the ClfA_229–545 concentration and fit to a one-site binding (hyperbola) model (inset). K_D of 0.66 μM was determined. (C & D) Dose response for the inhibition of different concentrations (from 0.125 to 4 μM) of tefibazumab (C) or Fg (D) on 1 μM of ClfA_229–545 binding to immobilized Fg. The maximum 100% response is ClfA_229–545 binding without any inhibitors and shown as dashed lines. (E) Inhibition data from C&D were plotted against inhibitor concentrations (the monomer Fg concentration was used) and fitted to a four-parameter logistic function. The control is for the binding response without any inhibitor. The tefibazumab IC₅₀ measured for inhibiting ClfA_229–545 binding to Fg is 0.39 μM and the fitted minimum is 27%, the Fg IC₅₀ is 0.48 μM and the fitted minimum is 0%. The binding of the inhibitors (at the highest concentration of 4 μM) to the Fg surface were very small (< 10 RU), compared to the ClfA response of ~ 550 RU, so they were not subtracted from the total responses.

Fig. 2

ClfA-tefibazumab interactions. (A) The structure of disulfide bond-closed ClfA_CC in complex with the Fab fragment of tefibazumab. ClfA_CC N2 and N3 domains are shown in green and yellow, respectively. The light and the heavy chains of the Fab fragment are shown in magenta and cyan, respectively. (B) Hydrogen bonding and key interactions between ClfA and the light chain of tefibazumab. Hydrogen bonds are shown as dashed lines. ClfA residues are shown in yellow and light chain residues are shown in magenta. (C) Hydrogen bonding and key interactions between ClfA and the heavy chain of tefibazumab. Hydrogen bonds are shown as dotted lines. ClfA residues are shown in yellow and heavy chain residues are shown in cyan.

Fig. 3

Analysis of ClfA N3 site interactions with tefibazumab and fibrinogen. (A) Ribbon diagram of ClfA N3 domain showing alanine substitutions at the positions of P467, Y512 and W518 in blue. The Fg γ-peptide is shown as a red ribbon. (B) Biacore sensorgrams showing 0.1 μM of ClfA_221–559 wt or variants run over immobilized tefibazumab and fibrinogen. The variant proteins used are indicated in panel A. (C) ClfA_PWY and Fg-γ P16 synthetic peptide interaction measured by ITC. Thermodynamic parameters values (K_D = 5.8 μM, ΔH = − 6.79 kcal/mol, ΔS = 1.6 cal/mol/K) are derived from this experiments. (D) Overlay of crystal structures of ClfA_CC/Fg γ-peptide and the ClfA_CC/tefibazumab Fab complex. The N2 and the N3 subdomains of ClfA and Fab complex are shown in green and yellow respectively and ClfA from the Fg γ-peptide complex (PDB: 2VR3) is shown in grey. Tefibazumab light and heavy chains are shown in magenta and cyan, respectively, and the Fg γ-peptide bound in the N2N3 trench is shown as a red ribbon. Note that the N2N3 site and the mAb epitope do not overlap. (E) Molecular surface representation of ClfA shown in green (N2 domain), yellow (N3 domain). The surface that contacts Fg γ-peptide and the light chain and heavy chains of tefibazumab are shown in red, gold and grey, respectively. (F) Molecular model of ClfA/Fg D-fragment interaction. ClfA N2 and N3 domains are colored green and yellow, respectively. The α, β, and γ chains of Fg D-fragment are shown in orange, light blue and red, respectively.

Fig. 4

Comparison of ClfA-Fg interactions. (A & B) Analysis of the interaction using ITC. Fg was placed in the cell and ClfA_CC was titrated in A, whereas ClfA_CC was placed in the cell and P16 was titrated in B. (C) SPR measurement of the interactions between ClfA_229–545 and Fg molecules. Two-fold dilution of ClfA_229–545 (1.28 to 0.04 μM, 8.0 to 0.125 μM, and 32 to 1 μM) were injected to Fg (600 RU), D-fragment (700 RU) and GST-γ (1500 RU) respectively. (D) SPR measurement of the interactions between ClfA N3 and Fg molecules. Two-fold dilution of ClfA N3 (12.8 to 0.1 or 0.4 μM) were injected to Fg (1000 RU), D-fragment (1000 RU) and GST-γ (2500 RU) respectively. SPR sensorgrams for binding at different concentration of proteins are shown as black lines. The average responses at steady state (shown in red) were used to generate a binding curve (inset) and the equilibrium dissociation constant K_D was determined.

Fig. 5

Effect of tefibazumab on ClfA binding to immobilized GST-γ. (A) SPR experiments for 5 μM ClfA_229–545 binding to GST-γ surface (700 RU, captured by anti-GST pAb), in the presence (dashed line, with the tefibazumab background response subtracted) or absence (solid line) of 2 μM tefibazumab. (B) Taking advantage of the slow off-rate of ClfA_CC (5 μM) binding to GST-γ (black line), strong binding of 2 μM of tefibazumab to the GST-γ bound ClfA_CC was shown (in green), compared to non-specific binding of tefibazumab (2 μM, in blue) to the GST-γ.

Fig. 6

ClfA_229–545 and some natural variants binding to tefibazumab and Fg. (A) Sensorgrams generated by binding of each ClfA protein at 32 nM concentration to the surface of tefibazumab (~ 200 RU captured through goat anti-human IgG (Fc) polyclonal F(ab’)₂). Dissociation constants K_D for each ClfA/tefibazumab interaction are listed, and sensorgrams with fitting are shown in Supplementary Figure S3. (B) Binding of 320 nM of each protein to the immobilized Fg (about 600 RU) are overlaid and the K_D for each of the ClfA/Fg interactions is listed. Standard errors for the K_D measurements from different experiments (n ≥ 2).

Fig. 7

Schematic representation of the binding and inhibition mechanism of Fg and tefibazumab respectively to ClfA. (A) Domain organization of fibrinogen and ClfA. The inter chain disulfide bonds linking the individual α, β, and γ chains and the dimeric Fg molecule are shown as black lines. The α, β, and γ chain regions corresponding to D-fragment are shown in orange, light blue and red respectively. The N2 and the N3 subdomains in ClfA are colored green and yellow respectively. (B) A schematic model showing the Fg binding regions on ClfA and the tefibazumab epitope. Tefibazumab can only partially inhibit Fg binding to ClfA by competing for the second Fg binding site on the MSCRAMM.