Abstract
Assembly of soluble peptide-major histocompatibility complex class II (pMHCII) monomers into multimeric structures enables the detection of antigen-specific CD4+ T cells in biological samples and, in some configurations, their reprogramming in vivo. Unfortunately, current MHCII-αβ chain heterodimerization strategies are typically associated with low production yields and require the use of foreign affinity tags for purification, precluding therapeutic applications in humans. Here, we show that fusion of peptide-tethered or empty MHCII-αβ chains to the IgG1-Fc mutated to form knob-into-hole structures results in the assembly of highly stable pMHCII monomers. This design enables the expression and rapid purification of challenging pMHCII types at high yields without the need for leucine zippers and purification affinity tags. Importantly, this design increases the antigen-receptor signaling potency of multimerized derivatives useful for therapeutic applications and facilitates the detection and amplification of low-avidity T cell specificities in biological samples using flow cytometry.
Highlights
Of soluble peptide-major histocompatibility complex class II monomers into multimeric structures enables the detection of antigen-specific CD4+ T cells in biological samples and, in some configurations, their reprogramming in vivo
We report a new molecular pMHC class II heterodimerization design based on the “knob-into-hole” (KIH) approach used for the generation of bispecific monoclonal antibodies[8,9]
The peptide-major histocompatibility complex class II (pMHCII) α and β chains were either transcribed from a single ORF as two chains separated by a P2A ribosomal skipping sequence (Fig. 1b), or from two different ORFs in different vectors (Fig. 1c)
Summary
Of soluble peptide-major histocompatibility complex class II (pMHCII) monomers into multimeric structures enables the detection of antigen-specific CD4+ T cells in biological samples and, in some configurations, their reprogramming in vivo. We show that fusion of peptide-tethered or empty MHCII-αβ chains to the IgG1-Fc mutated to form knob-into-hole structures results in the assembly of highly stable pMHCII monomers This design enables the expression and rapid purification of challenging pMHCII types at high yields without the need for leucine zippers and purification affinity tags. The rules that govern peptide binding to MHC class I molecules are well defined, including the identification of pockets capable of anchoring specific epitopes via complementary anchor residues that fix their binding register and stabilize the trimolecular complex These structural hallmarks facilitate the expression and purification of stable pMHC class I monomers from eukaryotic expression systems, as well as the refolding and assembly of the trimolecular complex in vitro, using peptide and heavy and light chains expressed separately in prokaryotic expression systems.
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