Abstract
We have generated transgenic mice that harbor humanized type I interferon receptors (IFNARs) enabling the study of type I human interferons (Hu-IFN-Is) in mice. These “HyBNAR” (Hybrid IFNAR) mice encode transgenic variants of IFNAR1 and IFNAR2 with the human extracellular domains being fused to transmembrane and cytoplasmic segments of mouse sequence. B16F1 mouse melanoma cells harboring the HyBNAR construct specifically bound Hu-IFN-Is and were rendered sensitive to Hu-IFN-I stimulated anti-proliferation, STAT1 activation and activation of a prototypical IFN-I response gene (MX2). HyBNAR mice were crossed with a transgenic strain expressing the luciferase reporter gene under the control of the IFN-responsive MX2 promoter (MX2-Luciferase). Both the HyBNAR and HyBNAR/MX2-Luciferase mice were responsive to all Hu-IFN-Is tested, inclusive of IFNα2A, IFNβ, and a human superagonist termed YNSα8. The mice displayed dose-dependent pharmacodynamic responses to Hu-IFN-I injection, as assessed by measuring the expression of IFN-responsive genes. Our studies also demonstrated a weak activation of endogenous mouse interferon response, especially after high dose administration of Hu-IFNs. In sharp contrast to data published for humans, our pharmacodynamic readouts demonstrate a very short-lived IFN-I response in mice, which is not enhanced by sub-cutaneous (SC) injections in comparison to other administration routes. With algometric differences between humans and mice taken into account, the HyBNAR mice provides a convenient non-primate pre-clinical model to advance the study of human IFN-Is.
Highlights
Type I Interferons (IFN-Is) comprise a family of sixteen human cytokines, which collectively feed into a signaling network through binding to two-receptor components, IFNAR1 and IFNAR2
The HyBNAR construct constitutes the human-encoded extracellular domains of both IFNAR1 and IFNAR2, whereas their transmembrane and intracellular domains are encoded by mouse sequence (Fig. 1A)
Cell surface expression of the HyBNAR receptors for B16F1-HyBNAR cells was detected by live FACS staining using antibodies directed against the extracellular domains of human IFNAR1 or IFNAR2 (Fig. 2A)
Summary
Type I Interferons (IFN-Is) comprise a family of sixteen human cytokines, which collectively feed into a signaling network through binding to two-receptor components, IFNAR1 and IFNAR2. This is supported by the finding that a variant of human IFNa2 genetically engineered to bind tightly to IFNAR1 converts this ‘‘IFNa’’ into a superagonist with anti-proliferative potency surpassing that of IFNb [17,18] This tightly-binding superagonist was generated by exchanging three amino acids receptor-binding interface (HEQRYNS). Our variant YNS and YNSa8 mutants bind the human IFNARs with higher affinity than any known IFN-I found in nature, and to our knowledge better than any other IFN-I engineered mutant Their potency in inducing anti-proliferative activity relates directly to their increase in binding affinity. These ‘‘HyBNAR’’ (Hybrid IFNAR) mice encode transgenic interferon receptors, whose extracellular and cytoplasmic segments are encoded by human and mouse sequences respectively This allows the binding of human IFN-Is to the recombinant receptors but once activated, transduces their signal effectively in a mouse cellular environment. We describe the generation of the HyBNAR mice and a mouse cell line harboring the HyBNAR transgenes, and demonstrate to proof-of-concept their ability to induce a sensitive response to Hu-IFN-Is, and provide some insight into suggested dosing regiments, this which differs significantly to the human scenario
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