Engineered human antibody constant domains with increased stability

Abstract

The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising as scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. However, previous work has shown that an isolated murine CH2 domain is relatively unstable to thermally induced unfolding. To explore unfolding mechanisms of isolated human CH2 and increase its stability gamma1 CH2 was cloned and a panel of cysteine mutants was constructed. Human gamma1 CH2 unfolded at a higher temperature (T(m) = 54.1 degrees C, as measured by circular dichroism) than that previously reported for a mouse CH2 (41 degrees C). One mutant (m01) was remarkably stable (T(m) = 73.8 degrees C). Similar results were obtained by differential scanning calorimetry. This mutant was also significantly more stable than the wild-type CH2 against urea induced unfolding (50% unfolding at urea concentration of 6.8 m versus 4.2 m). The m01 was highly soluble and monomeric. The existence of the second disulfide bond in m01 and its correct position were demonstrated by mass spectrometry and nuclear magnetic resonance spectroscopy, respectively. The loops were on average more flexible than the framework in both CH2 and m01, and the overall secondary structure was not affected by the additional disulfide bond. These data suggest that a human CH2 domain is relatively stable to unfolding at physiological temperature, and that both CH2 and the highly stable mutant m01 are promising new scaffolds for the development of therapeutics against human diseases.

FIGURE 1.

A, amino acid sequence alignment of human CH2 (NCB Accession J00228) and mouse CH2 (NCB Accession J00453). Identical and similar residues were 67 and 92%, respectively. B, size exclusion chromatogram of human CH2. The inset is a standard curve. C, molecular size of CH2 compared with scFv, Fab, and IgG1. SDS-PAGE gel of purified CH2 (lane 1,10 μg; lane 2,5 μg), scFv m9 (lane 3), Fab X5 (lane 4), and IgG X5 (lane 5).

FIGURE 2.

Stability of human CH2 measured by CD and DSC. A, folding curve at 25 °C (—), unfolding at 90 °C (□□□), and refolding (– – –) at 25 °C measured by CD. B, fraction-folded of the protein (ff) was calculated as ff = ([θ] – [θ_M])/([θ_T] – [θ_M]). [θ_T] and [θ_M] were the mean residue ellipticities at 216 nm of folded state at 25 °C and unfolded state of 90 °C. The T_m value (54.1 ± 1.2 °C) from CD was determined by the first derivative [d(Fraction-folded)/dT] with respect to temperature (T). C, thermo-induced unfolding curve from DSC. T_m = 55.4 °C, which is similar to that from CD.

FIGURE 3.

Design of m01 and m02 based on the CH2 structure. The distance between two C_αs forming the native disulfide bond (indicated by black arrow) is 6.53 Å. An engineered disulfide bond was introduced between Leu-12 and Lys-104 (m01) or Val-10 and Lys-104 (m02), which were replaced by cysteines.

FIGURE 4.

High level of expression of m01 and m02. Soluble expression of m01 and m02 is compared with that of CH2 by SDS-PAGE. The expressed proteins are indicated by arrows.

FIGURE 5.

Increased stability of two mutants measured by CD (A–C), DSC (D), and spectrofluorimetry (E). Folding curve at 25 °C (—), unfolding at 90 °C (□□□), and refolding (– – –) at 25 °C of m01 (A) and m02 (B) are shown. C, fraction-folded and T_m of m01 and m02 were calculated by the same method as with CH2; T_m of m01 = 73.8 ± 1.7 °C, T_m of m02 = 65.3 ± 0.6 °C. D, thermo-induced unfolding curves of m01 (T_m = 73.4 °C) and m02 (T_m = 66.5 °C) were also recorded by DSC. E, urea-induced unfolding of CH2, m01, and m02 measure by spectrofluorimetry. Half-unfolding of CH2, m01, and m02 is at 4.2, 6.8, and 5.8 m, respectively.

FIGURE 6.

Size exclusion chromatograms of m01 and m02. The same standard curve as in Fig. 1B is used. CH2 are m01 monomeric, while m02 includes a small amount of dimers.

FIGURE 7.

C_α (A) and N (B) chemical shift differences between CH2 and m01. Asterisks at the bottom of graphs indicate the residues that were not assigned in one or both proteins. The p at the bottom of the graphs indicate proline residues.

FIGURE 8.

Ribbon structure presentations of CH2 domain structure. In A, the regions that show largest (red), medium (orange), and small (yellow) chemical shift changes are shown by the colors in parentheses, the regions that show insignificant changes in chemical shifts are in blue. In B, the rigid regions (¹H-¹⁵N NOE > 0.7) are shown in blue, and the mobile regions (¹H-¹⁵N NOE < 0.7) are shown in red. See details of the criteria under “Experimental Procedures.” Native cysteines and engineered cysteines are also shown by cyan sticks and pink sticks. In both A and B, the regions that were not assigned are shown in white.

FIGURE 9.

¹H-¹⁵N NOE values of CH2 (closed triangles) and m01 (open squares). Asterisks and at the bottom of graphs indicate the residues that were not assigned in one or both proteins. The p at the bottom of the graphs also indicate proline residues and cannot measure the ¹H-¹⁵N NOE values. The large errors indicated for the ¹H-¹⁵N NOE values of a few residues result from the weak peak intensity in the spectra, leading to large uncertainty.