Infliximab crystal structures reveal insights into self-association

Abstract

Aggregation and self-association in protein-based biotherapeutics are critical quality attributes that are tightly controlled by the manufacturing process. Aggregates have the potential to elicit immune reactions, including neutralizing anti-drug antibodies, which can diminish the drug's efficacy upon subsequent dosing. The structural basis of reversible self-association, a form of non-covalent aggregation in the native state, is only beginning to emerge for many biologics and is often unique to a given molecule. In the present study, crystal structures of the infliximab (Remicade) Fc and Fab domains were determined. The Fab domain structures are the first to be reported in the absence of the antigen (i.e., tumor necrosis factor), and are consistent with a mostly rigid complementarity-determining region loop structure and rotational flexibility between variable and constant regions. A potential self-association interface is conserved in two distinct crystal forms of the Fab domain, and solution studies further demonstrate that reversible self-association of infliximab is mediated by the Fab domain. The crystal structures and corresponding solution studies help rationalize the propensity for infliximab to self-associate and provide insights for the design of improved control strategies in biotherapeutics development.

Keywords: Aggregation; SEC; analytical ultracentrifugation; biophysical; biotherapeutic; crystallography; infliximab; remicade; self-association; structure.

Figure 1.

Crystal structures of infliximab Fc domain (A) and Fab domain (B). The Fc dimer is shown in (A), with one heavy chain shown in green and the symmetry-related chain shown in silver. N-linked glycans are shown in stick representation and the C_H2 and C_H3 sub-domains are labeled. The I2₁2₁2₁ form of the Fab domain is shown in (B), with the heavy chain in green and the light chain in silver. Variable (V_H and V_L) and constant (C_H1 and C_L) regions are labeled.

Figure 2.

Flexibility between the variable and constant regions of the infliximab Fab domain. Fab structures determined in C222₁ (blue) and I2₁2₁2₁ (green) crystal forms, aligned by their variable regions, show a ∼40° relative rotation of their constant regions, indicating intramolecular flexibility within the Fab domain.

Figure 3.

TNF binding does not affect the infliximab CDR loop structure. Fab structures in the unbound (blue; I2₁2₁2₁ form) and TNFα-bound (red) states are overlaid. CDR loop residues involved in TNF binding are shown as sticks in darker coloring and are labeled. The antigen binding interface structure is unchanged in the bound and free states, indicating that antigen binding does not affect the structure of infliximab.

Figure 4.

The Fab asymmetric unit contains a head-to-tail dimer, associated through light chain:light chain interactions in both crystal forms. Two Fab molecules (#1 and #2) and their variable and constant regions are labeled in the I2₁2₁2₁ form (left) and the C222₁ form (right). Heavy chains are shown in shades of green and light chains in shades of blue. In both crystal forms, Fab #1 is oriented with variable regions at the top, while Fab #2 variable regions are positioned at the bottom. The large contact area (> 1000 Ų) between light chains is larger than would be expected for typical crystal contacts, indicating a potential self-association interface in solution.

Figure 5.

Fab:Fab contact interface in the I2₁2₁2₁ crystal form. (A) A surface representation of the Fab asymmetric unit (dimer) is shown with heavy chains colored in shades of green and light chains in shades of blue. Interface residues are colored by residue type (green = polar, blue = basic, red = acidic, magenta = hydrophobic). (B) Each Fab domain is rotated outward 90° to expose the light chain:light chain interface residues, which are highlighted below in the light chain sequence. Residues common to both crystal form interfaces are underlined in the sequence. (C) Close-up view of Leu₃ from one light chain positioned into a pocket created by Thr₁₉₇, Lys₁₄₅ and Glu₁₄₃ in the adjacent light chain in the I2₁2₁2₁ crystal form.

Figure 6.

Fab:Fab contact interface in the C222₁ crystal form. (A) A surface representation of the Fab asymmetric unit (dimer) is shown with heavy chains colored in shades of green and light chains in shades of blue. Interface residues are colored by residue type (green = polar, blue = basic, red = acidic, magenta = hydrophobic). (B) Each Fab domain rotated outward 90° to expose the light chain:light chain interface residues, which are highlighted below in the light chain sequence. Residues common to both crystal form interfaces are underlined. (C) A close-up view of Leu₃ in the C222₁ interface, positioned within a pocket created by Val₁₉₁, Asp₁₅₁, Asn₁₅₂ and Lys₁₉₀ of the adjacent Fab. The surrounding hydrogen bond interactions are shown as dashed lines.

Figure 7.

Reversible self-association of full-length infliximab in solution. (A) SEC analysis of infliximab at increasing concentrations (0.5, 2.5, and 5 mg/mL) reveals a corresponding shift in the main peak to earlier retention times and a progressive asymmetry, indicative of self-association. (B) AUC-SV analysis of infliximab demonstrates that the sedimentation coefficient increases with concentration, and that two peaks become resolved at the highest concentration measured. Results from both methods are consistent with concentration-dependent, reversible self-association of infliximab.

Figure 8.

Measuring the affinity of infliximab self-association using AUC-SE. Experimental data (scatter plots) were fit to a monomer-dimer equilibrium model (C(cal), solid lines) to determine the equilibrium dissociation constant (K_D) of 21 μM. Infliximab was evaluated at 0.07 mg/mL (A), 0.2 mg/mL (B), and 0.6 mg/mL (C). Samples were run at rotor speeds of 7500 (red), 10000 (blue), 12500 (green) and 15000 rpm (purple).

Figure 9.

Infliximab self-association is localized to the Fab domain in solution. The infliximab Fc fragment does not show evidence of self-association by SEC (A) or AUC-SV (B). The SEC elution peak is symmetric and the retention time is consistent at all measured concentrations (A). Similarly, the sedimentation coefficient of the Fc fragment is consistent at all measured concentrations (B). In contrast, the Fab fragment shows evidence of reversible self-association by both SEC (C) and by AUC-SV (D), similar to the intact infliximab antibody (Fig. 7).