Aug 2026· Journal of Immunology· Vol 215· 0 citations· 45 references
Medicine
TL;DR
A novel method is presented for the expression in eukaryotic cells of secreted peptide exchange–competent class I MHC proteins in their native conformation and it is demonstrated that tetramers produced in this way are equivalent to conventionally produced tetramers for T-cell staining.
Abstract
Abstract Class I MHC peptide (MHC-Ip) multimers are well-established reagents that detect antigen-specific T cells. The classical method for production of MHC-Ip multimers begins with the expression of MHC heavy chains (HCs) and β2-microglobulin (β2m) subunits as inclusion bodies in Escherichia coli and is followed by denaturant solubilization, in vitro folding in the presence of a defined peptide ligand, and purification by size exclusion chromatography. This protocol is labor intensive, difficult to scale, and represents a significant bottleneck in application of the technology. Herein, we present a novel method for the expression in eukaryotic cells of secreted peptide exchange–competent class I MHC proteins in their native conformation. In this method, expression constructs are engineered as bimolecular complexes composed of an MHC HC and a β2m molecule covalently linked at its amino terminus to an MHC-binding peptide through a flexible peptide linker containing a defined protease site. Upon proteolysis, the original peptide occupant of the MHC binding site dissociates and is easily replaced with a synthetic peptide. When leucine zippers are added to the carboxyl terminus of each subunit, protease cleavage of the linker results in a stable HC/β2m complex that can be isolated and stored for subsequent peptide loading. Using this method, we have produced homogeneous MHC-Ip complexes for 25 class I MHC alleles and demonstrated that tetramers produced in this way are equivalent to conventionally produced tetramers for T-cell staining.
An overview of the PLC as a major determinant of tumor immune escape and a potential therapy target is provided and existing or evolving therapeutic approaches to recover/reprogram PLC functions are explored.
O. Allela, Abdulkareem Shareef, Hayder Naji Sameer et al.· Current Medical Science· 0 citations
A glycan-dependent chaperone network is defined, finely tuned by a combination of low-micromolar interactions between the constituents, that ensures efficient MHC-I maturation and illustrates fundamental principles of ER protein quality control.
Tim J. Heinke, Amin Fahim, Niko Popovic et al.· EMBO Journal· 0 citations
Hydrophilic interaction chromatography is utilized to enrich glycopeptides from both peptides bound to HLA and digest of HLA proteins that obtained from immunoprecipitation of MHC complex from cell lysate that enables the characterization of glycosylation of MHC by database search.
Rui Chen· Methods in molecular biology· 0 citations
A unified structure-energy-dynamics model explaining how Post-translational modifications function as atomic-level chemical switches in antigen presentation is established, establishing a unified structure-energy-dynamics model explaining how PTMs function as atomic-level chemical switches in antigen presentation.
Adaptive immunity relies on T-cell receptor (TCR) recognition of peptides presented by the major histocompatibility complex (pMHC). Accurate prediction of TCR:pMHC binding pairs from sequence data remains a longstanding challenge in computational immunology, limiting the development of precision immunotherapies like cancer vaccines and adoptive cell therapies. Here, we present enFoldX (ensemble of Folded compleXes), a structure-based approach leveraging biophysical characterization of AlphaFold3-generated ensembles to classify TCR:pMHC sequence pairs as cognate versus non-cognate. Unlike previous methods reliant on only sequence data or a single, static predicted structure, enFoldX extracts features from an entire generated ensemble with a custom focus on the biophysical binding interface. Our model distinguishes T cell reactivity between peptides differing by a single amino acid substitution, the resolution required for cancer neoantigens, and generalizes to unseen peptides, MHCs, and TCRs, a major objective for artificial intelligence (AI) in immunology. Our performance on these crucial tasks demonstrates that diverse, structural sampling of biophysical interactions over an ensemble is fundamental for accurate AI-driven binding predictions and offers lessons for efficient future data generation to improve models. Our findings therefore offer a scalable framework to accelerate therapeutic binder design, and we provide access to a publicly available code repository.
O. Lyudovyk, JA Levine, M. Pathil et al.· bioRxiv· 1 citation
Nanobodies (Nbs) have been extensively utilized in medical diagnosis and therapies due to their advantages, such as ease of genetic manipulation and efficient soluble expression in prokaryotic systems. However, enhancing their binding affinity to antigens to match or even exceed that of polyclonal antibodies for high-sensitivity bioassays remains a challenge. Therefore, in the present study, a novel strategy to enhance the binding affinity of antibodies to antigens was developed by fusing an anti-C-reactive protein Nb (CRPNb) to the N-terminus of self-assembling peptides such as right-handed coiled coil [RHCC; derived from a right-handed coiled-coil peptide of an archaebacterium (Staphylothermus marinus)], verotoxin (VTB; the B-subunit of Escherichia coli verotoxin) and C4-binding protein (C4bp; derived from human plasma C4-binding protein α-chain). For functional detection, alkaline phosphatase (AP) was further fused to the C-terminus of RHCC, VTB and C4bp, respectively. This approach enabled the formation of CRPNb-RHCC-AP tetramers, CRPNb-VTB-AP pentamers and CRPNb-C4bp-AP heptamers. These were then expressed as soluble cytoplasmic proteins in the E. coli strain BL21 (DE3) and purified by imidazole elution. The protein expression was assessed by western blotting. Additionally, the stability of the multimeric constructs was evaluated using a direct ELISA, whilst their sensitivity was assessed using a competitive ELISA. The CRPNb-VTB-AP pentamers and CRPNb-RHCC-AP tetramers demonstrated antigen-specific recognition and enhanced affinity compared with the CRP-AP monomer based on a direct ELISA. Furthermore, they exhibited good thermal stability based on a direct ELISA. At 80˚C, CRPNb-VTB-AP, CRPNb-AP and CRPNb-RHCC-AP retained ~85, 75 and 60% activity, while CRPNb-H only retained ~40%. After incubation at 80˚C for 55 min, the two multimers still maintained ~40% activity. The results suggested them to be suitable for storage and transportation at ambient temperatures. In conclusion, a novel nanobody-fusion protein platform was established in the present study. The fusion proteins functioned as high-affinity binders for target antigens and heat-stable signal tracers for immunoassay detection and quantitative analysis, providing a novel type of thermostable immunoreagent.
Hong-Heng Li, Chen-Chen Zhou, Xiaomei Zhang et al.· Experimental and Therapeutic...· 0 citations
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