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

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.

Recognition of Multiple Hybrid Insulin Peptides by a Single Highly Diabetogenic T-Cell Receptor

“…To do this, we used plate-bound, knob-into-hole-based B2B-linker-IA g7 ab monomers secreted from transiently-transduced HEK-293T cells. As described previously, this design results in increased production yields and pMHCII structural stability than those using leucine zipper-heterodimerization domains (14,22). Although the peptidelinker-I-A g7 b library that was used to identify the B2B mimotope used four pre-defined anchor residues, it remained possible that the B2B sequence was preferentially displayed to the 4.1-TCR on alternative binding registers, such as those in which B2B uses the negatively charged Glu or Asp at P7 and P8, respectively, to anchor itself on I-A g7 's pocket 9.…”

“…b -Fc-hole/GFP and I-A d a -Fcknob/CFP were grown in a fed-batch culture (up to 1 L) in a shaking incubator (37°C and 8% CO 2 ) for 14 days with a temperature shift to 34°C when cell density reached 5 × 10 6 cells/ml. At day 14 or when cell viability dropped below 60%, cell culture supernatants were harvested and secreted pMHCII heterodimers purified on a protein G affinity column, as previously described (14). pMHCII monomers (50 mM) were biotinylated overnight with 10 mM biotin-protein ligase (Avidity) in 50 mM bicine buffer (pH 8.3) with 10 mM magnesium acetate, 10 mM ATP, and 5 mM dbiotin.…”

“…Although the peptidelinker-I-A g7 b library that was used to identify the B2B mimotope used four pre-defined anchor residues, it remained possible that the B2B sequence was preferentially displayed to the 4.1-TCR on alternative binding registers, such as those in which B2B uses the negatively charged Glu or Asp at P7 and P8, respectively, to anchor itself on I-A g7 's pocket 9. This was addressed by producing three different B2B-linker-I-A g7 pMHCIIs in which the B2B sequence was forced to bind to I-A g7 on three different registers via the strategic introduction of register-fixing Cysteine residues on the I-A g7 a chain and the C-terminus of B2B (cys-trap) (14,22). We then compared the luciferase activity in 4.1-JurMA cells challenged with platebound monomers.…”

Peptide-MHC-Based Nanomedicines for the Treatment of Autoimmunity: Engineering, Mechanisms, and Diseases

“…Low yields and the need to incorporate artificial affinity purification tags into the pMHC design represented significant obstacles for clinical translation. Expression in Chinese Hamster Ovary (CHO) cells and re-engineering of pMHC heterodimers as knob-into-hole-based Fc fusions addressed these limitations; KIH-based pMHC molecules are expressed at much higher yields than pMHCIIs heterodimerized using leucine zippers and can be purified to the desired levels of purity using protein A chromatography and additional polishing steps routinely used in the purification of biologics ( 15 ).…”

Nanotechnology's application in Type 1 diabetes

“…Moreover, this technique can be used not only on T1D but also for other autoimmune diseases such as EAE (Clemente‐Casares et al, 2016). In addition, we could significantly improve immune regulation ability and therapeutic effect of pMHC nanomaterials by optimizing the design variables besides the class of pMHC and the size and density of nanoparticles of different compounds (Clemente‐Casares, Tsai, Yang, & Santamaria, 2011; P. Serra et al, 2019; Singha et al, 2017). Therefore, pMHC‐based nano‐drug therapy is an auto‐immunotherapy with great potential.…”