Opening opportunities for Kd determination and screening of MHC peptide complexes.

An essential element of adaptive immunity is selective binding of peptide antigens by major histocompatibility complex (MHC) class I proteins and their presentation to cytotoxic T lymphocytes. Using native mass spectrometry, we analyze the binding of peptides to an empty disulfide-stabilized HLA-A*02:01 molecule and, due to its unique stability, we determine binding affinities of complexes loaded with truncated or charge-reduced peptides. We find that the two anchor positions can be stabilized independently, and we further analyze the contribution of additional amino acid positions to the binding strength. As a complement to computational prediction tools, our method estimates binding strength of even low-affinity peptides to MHC class I complexes quickly and efficiently. It has huge potential to eliminate binding affinity biases and thus accelerate drug discovery in infectious diseases, autoimmunity, vaccine design, and cancer immunotherapy.

FoldAffinity: binding affinities from nDSF experiments.

Differential scanning fluorimetry (DSF) using the inherent fluorescence of proteins (nDSF) is a popular technique to evaluate thermal protein stability in different conditions (e.g. buffer, pH). In many cases, ligand binding increases thermal stability of a protein and often this can be detected as a clear shift in nDSF experiments. Here, we evaluate binding affinity quantification based on thermal shifts. We present four protein systems with different binding affinity ligands, ranging from nM to high µM. Our study suggests that binding affinities determined by isothermal analysis are in better agreement with those from established biophysical techniques (ITC and MST) compared to apparent Kds obtained from melting temperatures. In addition, we describe a method to optionally fit the heat capacity change upon unfolding ([Formula: see text]) during the isothermal analysis. This publication includes the release of a web server for easy and accessible application of isothermal analysis to nDSF data.

Structures of peptide-free and partially loaded MHC class I molecules reveal mechanisms of peptide selection.

Major Histocompatibility Complex (MHC) class I molecules selectively bind peptides for presentation to cytotoxic T cells. The peptide-free state of these molecules is not well understood. Here, we characterize a disulfide-stabilized version of the human class I molecule HLA-A*02:01 that is stable in the absence of peptide and can readily exchange cognate peptides. We present X-ray crystal structures of the peptide-free state of HLA-A*02:01, together with structures that have dipeptides bound in the A and F pockets. These structural snapshots reveal that the amino acid side chains lining the binding pockets switch in a coordinated fashion between a peptide-free unlocked state and a peptide-bound locked state. Molecular dynamics simulations suggest that the opening and closing of the F pocket affects peptide ligand conformations in adjacent binding pockets. We propose that peptide binding is co-determined by synergy between the binding pockets of the MHC molecule.

High-throughput peptide-MHC complex generation and kinetic screenings of TCRs with peptide-receptive HLA-A*02:01 molecules.

Major histocompatibility complex (MHC) class I molecules present short peptide ligands on the cell surface for interrogation by cytotoxic CD8+ T cells. MHC class I complexes presenting tumor-associated peptides such as neoantigens represent key targets of cancer immunotherapy approaches currently in development, making them important for efficacy and safety screenings. Without peptide ligand, MHC class I complexes are unstable and decay quickly, making the production of soluble monomers for analytical purposes labor intensive. We have developed a disulfide-stabilized HLA-A*02:01 molecule that is stable without peptide but can form peptide-MHC complexes (pMHCs) with ligands of choice in a one-step loading procedure. We illustrate the similarity between the engineered mutant and the wild-type molecule with respect to affinity of wild-type or affinity-matured T cell receptors (TCRs) and present a crystal structure corroborating the binding kinetics measurements. In addition, we demonstrate a high-throughput binding kinetics measurement platform to analyze the binding characteristics of bispecific TCR (bsTCR) molecules against diverse pMHC libraries produced with the disulfide-stabilized HLA-A*02:01 molecule. We show that bsTCR affinities for pMHCs are indicative of in vitro function and generate a bsTCR binding motif to identify potential off-target interactions in the human proteome. These findings showcase the potential of the platform and the engineered HLA-A*02:01 molecule in the emerging field of pMHC-targeting biologics.

Empty peptide-receptive MHC class I molecules for efficient detection of antigen-specific T cells.

The peptide-dependent stability of MHC class I molecules poses a substantial challenge for their use in peptide-MHC multimer-based approaches to comprehensively analyze T cell immunity. To overcome this challenge, we demonstrate the use of functionally empty MHC class I molecules stabilized by a disulfide bond to link the α1 and α2 helices close to the F pocket. Peptide-loaded disulfide-stabilized HLA-A*02:01 shows complete structural overlap with wild-type HLA-A*02:01. Peptide-MHC multimers prepared using disulfide-stabilized HLA-A*02:01, HLA-A*24:02, and H-2Kb can be used to identify antigen-specific T cells, and they provide a better staining index for antigen-specific T cell detection compared with multimers prepared with wild-type MHC class I molecules. Disulfide-stabilized MHC class I molecules can be loaded with peptide in the multimerized form without affecting their capacity to stain T cells. We demonstrate the value of empty-loadable tetramers that are converted to antigen-specific tetramers by a single-step peptide addition through their use to identify T cells specific for mutation-derived neoantigens and other cancer-associated antigens in human melanoma.