Directed evolution of gene-shuffled IFN-alpha molecules with activity profiles tailored for treatment of chronic viral diseases

Abstract

Type I IFNs are unusually pleiotropic cytokines that bind to a single heterodimeric receptor and have potent antiviral, antiproliferative, and immune modulatory activities. The diverse effects of the type I IFNs are of differential therapeutic importance; in cancer therapy, an enhanced antiproliferative effect may be beneficial, whereas in the therapy of viral infections (such as hepatitis B and hepatitis C), the antiproliferative effects lead to dose limiting bone marrow suppression. Studies have shown that various members of the natural IFN-alpha family and engineered variants, such as IFN-con1, vary in the ratios between various IFN-mediated cellular activities. We used DNA shuffling to explore and confirm the hypothesis that one could simultaneously increase the antiviral and Th1-inducing activity and decrease the antiproliferative activity. We report IFN-alpha hybrids wherein the ratio of antiviral:antiproliferative and Th1-inducing: antiproliferative potencies are markedly increased with respsect to IFN-con1 (75- and 80-fold, respectively). A four-residue motif that overlaps with the IFNAR1 binding site and is derived by cross breeding with a pseudogene contributes significantly to this phenotype. These IFN-alphas have an activity profile that may result in an improved therapeutic index and, consequently, better clinical efficacy for the treatment of chronic viral diseases such as hepatitis B virus, human papilloma virus, HIV, or chronic hepatitis C.

Fig. 1.

Genealogies of shuffled IFN B9.1.1 and B9.1.2. (A) Graphic representation of the genealogies of evolved IFNs B9.1.1 and B9.1.2 and the wild type IFNs from which they are derived. The gene segments are colored according to the parental gene from which they were derived. The vertical red bars in B9.1.1 represent the location of the pseudogene-derived amino acids. (B) Sequence alignment of B9.1.1 and B9.1.2 with IFN-α2a and IFN-con1.

Fig. 2.

Antiviral activity of shuffled IFN-α compounds compared with IFN-con1, IFN-α2a, and IFN-α2b. (A) Graphic representation of the fold improvement of the 15 shuffled IFN-αs over IFN-con1 in the Huh7, WISH, and HeLa EMCV antiviral assays. The samples are listed in order of increasing potency in the Huh7 EMCV antiviral assay. Fold improvements were calculated as a ratio of mean EC₅₀ (ng/ml) values relative to IFN-con1 (SI Tables 3–5). (B) Exemplary dose-response curve for B9X14 and B9X25 compared with IFN-con1 and IFN- α2a in the HeLa EMCV antiviral assay. The calculated EC₅₀ value in this experiment for B9X25 is 0.00164 ng/ml, for B9X14 is 0.00314 ng/ml, for IFN-con1 is 0.133 ng/ml and for IFN-α2a is 0.415 ng/ml (mean ± SEM, n = 3).

Fig. 3.

Th1-inducing activity of shuffled IFN-α compounds compared with IFN-con1 and IFN-α2a. Exemplary dose-response curves are shown as follows: (A) B9X14 and IFN- α2a, EC₅₀ = 0.096 and 6.95 ng/ml, respectively; (B) B9X14 and IFN-con1, EC₅₀ = 0.10 and 0.82 ng/ml, respectively; (C) B9X25 and IFN-α2a, EC₅₀ = 0.053 and 3.94 ng/ml, respectively; (D) B9X25 and IFN-con1, EC₅₀ = 0.033 and 0.305 ng/ml, respectively. The pair of curves in each panel is from triplicate samples of each IFN run in a single plate to minimize between-plate variance (mean ± SEM, n = 4). The EC50s were calculated as described in SI Table 6. We tested these four variants in 8–12 independent donors, and in all cases, both B9X14 and B9X25 were significantly more potent than both IFN-α2a and IFN-con1 (SI Table 6).

Fig. 4.

The presence of pseudogene motif 2 is highly correlated with high ratio of antiproliferative:antiviral and antiproliferative:Th1 inducing activity. (A) The ratios of calculated antiviral:antiproliferative and Th1:antiproliferative potencies (SI Table 7) are plotted. Red squares correspond to variants containing pseudogene motif 2 (FLFY), and blue diamonds correspond to variants containing motif 2 from B9.1.2. (B) Summary of the fold potency of B9X14, B9X25, B9.1.2, IFN-α2a, and IFN-α2b versus IFN-con1 in the HeLa EMCV antiviral assay (striped bars), the Th1 differentiation assay (black bars), the Daudi binding assay (stippled bars) and the Daudi antiproliferation assay (open bars). (C). IFNAR1 (red) and IFNAR2 (green) binding sites are displayed on the coordinates of IFN-α2 (Protein Data Bank accession no. 1997-12-03). Residues corresponding to the pseudogene derived motif 2 (Phe-Leu-Phe; residues 54–56) are shown in blue, and residue Y57, which arose by a mutation C>T in the H57 codon of IFN-α4, is shown in mauve.