A general approach to antibody thermostabilization

Abstract

Antibody engineering to enhance thermostability may enable further application and ease of use of antibodies across a number of different areas. A modified human IgG framework has been developed through a combination of engineering approaches, which can be used to stabilize antibodies of diverse specificity. This is achieved through a combination of complementarity-determining region (CDR)-grafting onto the stable framework, mammalian cell display and in vitro somatic hypermutation (SHM). This approach allows both stabilization and maturation to affinities beyond those of the original antibody, as shown by the stabilization of an anti-HA33 antibody by approximately 10°C and affinity maturation of approximately 300-fold over the original antibody. Specificities of 10 antibodies of diverse origin were successfully transferred to the stable framework through CDR-grafting, with 8 of these successfully stabilized, including the therapeutic antibodies adalimumab, stabilized by 9.9°C, denosumab, stabilized by 7°C, cetuximab stabilized by 6.9°C and to a lesser extent trastuzumab stabilized by 0.8°C. This data suggests that this approach may be broadly useful for improving the biophysical characteristics of antibodies across a number of applications.

Keywords: CDR, complementarity-determining region; CH2, heavy chain constant domain 2; CH3, heavy chain constant domain 3; DSC, differential scanning calorimetry; HC, heavy chain; LC, light chain; NGF, β-nerve growth factor; SHM, somatic hypermutation; SPR, surface plasmon resonance; TNF, tumor necrosis factor; Tm, melting temperature; VH, heavy chain variable region; VL, light chain variable region; affinity maturation; monoclonal antibodies; protein engineering; solubility; somatic hypermutation; thermostability.

Figure 1.

Application of the stable IgG framework to improve antibody thermostability. (A) VH (H1, H2, H3) and VL (H1, H2, H3) CDR loops from the starting antibody (blue) are grafted (B) onto the IGHV3–23 framework (gold) containing stabilizing mutations (red) to generate a stable, full-length human IgG with the desired specificity. (C) Subsequent affinity maturation using SHM in vitro can be used to identify mutations (green) that further improve binding affinity. All stabilizing mutations have been made in the antibody framework and constant regions to minimize the effect on antigen binding affinity. IGHV3–23 stabilizing mutations (red) and mutations that improve affinity (green) for the grafted anti-HA33 antibody are labeled in panels (B) and (C), respectively, using Kabat numbering.

Figure 2.

Stability and affinity analysis of anti-HA33 antibody variants. (A) DSC analysis of the starting mouse anti-HA33 Fab (left), the chimeric full-length IgG (right, APE1148), and the initial CDR-grafted antibody (right, APE1146). Plots depict fitted peaks in red and original thermograms in black. Each of the full-length antibodies gave three melting transitions typical of an IgG, while the Fab unfolded in a single melting transition. (B) Sensorgrams show HA33 binding affinity of the starting Fab with a K_D equal to 6 nM (k_a = 8.1 x 10⁵ M⁻¹s⁻¹, k_d = 4.8 x 10⁻³ s⁻¹), the chimeric antibody, APE1148, with an identical K_D of 6 nM (k_a = 9.0 x 10⁵ M⁻¹s⁻¹, k_d = 5.1 x 10⁻³s⁻¹), the stable CDR-grafted antibody, APE1146, with a K_D of 9 nM (k_a = 7.0x10⁵ M⁻¹s⁻¹, k_d = 6.5 x 10⁻³s⁻¹), and the affinity matured stable CDR-grafted antibody, APE1553, with a K_D of 30 pM (k_a = 8.6x10⁵ M⁻¹s⁻¹, k_d = 2.5 x 10⁻⁵ s⁻¹). (C) Plot depicts percent activity of mature IgG APE1553 (y-axis) over a one hour thermal challenge at 70°C (x-axis), percent activity was normalized relative to an unheated control sample.

Figure 3.

Application of stable framework to improve antibody stability. (A) Thermofluor assay analysis comparing wild-type (dashed lines) and stable framework, CDR-grafted versions (solid lines) of four different antibodies of differing specificities. Unfolding temperatures are indicated, with arrows representing change in T_m resulting from grafting into the stable framework. Anti-βNGF, anti-C5, and anti-TNF antibodies demonstrated increased T_m, while the T_m of the anti-IL17-A antibody was decreased. (B) Summary of Thermofluor assay results comparing the T_m of original (blue) and stabilized (red) therapeutic antibodies. (C) Graph depicting change in T_m of five therapeutic antibodies upon CDR grafting into the stable framework.

Figure 4.

Thermal stability of therapeutic and stabilized antibodies. Adalimumab (right) and stabilized adalimumab (left) were heated at 70°C for up to 3 d with samples taken at indicated time points for TNF binding analysis by ELISA. Heated and unheated antibodies were evaluated by ELISA to determine activity. Plates were coated with 1 μg/mL TNF (R&D Systems) and incubated for 1 h with heated and unheated antibodies, each over a 1 μg/mL-100 pg/mL concentration range. Bound antibody was detected using a 1:10,000 dilution of goat anti-human IgG-HRP conjugate (Southern Biotech).