Increased yields and biological potency of knob into-hole-based soluble MHC class II molecules

Pau Serra, Nahir Garabatos, Santiswarup Singha, César Fandos, Josep Garnica, Patricia Solé,Daniel Parras , Jun Yamanouchi, Jesús Blanco, Meritxell Tort, Mireia Ortega, Yang Yang,Kristofor K. Ellestad2 & Pere Santamaria
Source: Nature
Publication Date: (2019)
Issue: 10: 1
Research Area:
Immunotherapy / Hematology
Regenerative medicine
Drug Discovery
Cells used in publication:
CHO-S [suspension]
Species: hamster
Tissue Origin: ovarian
Culture Media:


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-aß 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-aß
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.