The Dos and Don’ts of designing peptide HLA-I single-chain trimers

From the Strong Lab, Basic Sciences and Vaccine and Infectious Disease Divisions

The human leukocyte antigens (HLA-I) are multi-subunit complexes that localize to the cell surface and present bound peptides to nearby immune cells. The study of these HLA peptide-presenting complexes and features of the bound peptides have provided insights into the tuning of adaptive immune responses and facilitated advancements in diagnostic and therapeutic applications. In order to study specific HLA-I complexes and peptide pairs, synthetic single-chain trimer (SCT) molecules were constructed to allow for co-expression of each component (α-chain, β2-chain, and peptide) in a single polypeptide via conjoining linker sequences. The Strong lab in the Basic Sciences division at Fred Hutchinson Cancer Center wanted to better understand the effect of stabilizing mutations in the design of these SCTs and identify caveats of this tool to improve its utility and data interpretation. Their findings were published recently in Frontiers in Immunology.

(Left) Diagram of a native peptide/HLA-I complex (pHLA) and an engineered single-chain trimer (SCT). (Right) Different length peptides are color coded, and exhibit altered peptide structure and orientation in the peptide-binding pocket of HLA α1/α2 platform.
(Left) Diagram of a native peptide/HLA-I complex (pHLA) and an engineered single-chain trimer (SCT). (Right) Different length peptides are color coded, and exhibit altered peptide structure and orientation in the peptide-binding pocket of HLA α1/α2 platform. Image provided by Dr. Finton

“The engineered SCT format has mixed advantages and limitations as a surrogate for native [peptide/HLA-I complex] pHLAs in biochemical studies,” stated the researchers. “The key advantage is permitting expression in eukaryotic systems, where the key caveats are destabilization, which can foil expression of weakly binding peptides, or over-stabilization by point mutations and engineered disulfide linkages, leading to decoupling of linked peptide binding from native peptide presentation. Another practical SCT disadvantage is reducing crystallizability.” The Strong lab conducted biochemical and structural studies on engineered classical and non-classical HLA-I complexes in combination with several stabilizing mutations that included substitutions enabling disulfide bond formation proximal to the HLA-I peptide binding groove. In addition to these mutations in the HLA-I complex and linker sequences, the researchers generated versions with one of 44 different peptides that ranged in length from eight to 14 amino acids. Due to variation in the stability of the engineered SCTs, the researchers encountered significant challenges in crystalizing these complexes for structural analyses. To overcome this challenge, the Strong lab employed a crystallization chaperone to stabilize the SCTs. This chaperone was discovered by screening a library of antibodies produced by a llama immunized with SCT groove-open mutant (SCTY84A). Llamas, unlike most mammals, produce antibodies composed of a single polypeptide chain, unlike the more common heavy- and light-chain antibodies from mice, rabbits, and humans. The single chain antibodies are easier to express and often more stable than their two-chain counterparts. The researchers were now able to successfully crystalize the challenging SCT complexes. Their findings revealed that the tested stabilizing mutations did not significantly alter the HLA-I complex structure, and that SCTs with 9-mer peptides produced structures similar to the native complexes. However, differences between predicted and experimentally validated structures were observed for longer peptide-containing SCTs, and mutations that altered the stability of the complex changed polypeptide yields and sensitivities to temperature.

Based on their findings, the researchers suggest a few general guidelines for choosing appropriate SCT designs: “SCT Y84C/A139C should be avoided due to over-stabilization resulting in expression of any SCT, regardless of whether peptide is bound. The Y84C mutation should be avoided for cysteine-containing peptides due to unintended disulfide formation between the peptide and heavy chain resulting in off register binding; use Y84A instead. Verification of binding register is needed when designing SCTs with peptides longer than 10 residues, especially when peptides contain alternate anchor positions. SCT H74L/Y84C should be used when incorporating low affinity peptides to ensure expression; H74L alone can rescue expression of some SCTs with low affinity peptides.” Together, several caveats are now better appreciated from these findings and when included in the design strategy of SCTs, may ensure that these tools are representative of native peptide/HLA complexes.

Principal investigator Dr. Roland Strong commented on one key takeaway from this project: “Unappreciated complexities can pop up in all sorts of ways with even widely used methodologies - but can lead to new scientific insights, as in our study of commonly used approaches for MHC class I peptide presentation in adaptive immunity.” Their findings now provide additional guidance on HLA-I SCT design that can be applied to the use of these constructs in basic, translational, and clinical research.

The spotlighted research was funded by the National Institutes of Allergy and Infectious Diseases of the National Institutes of Health, the National Cancer Institute, and Fred Hutchinson Cancer Center Joint Translational Data Science/Immunotherapy award.

Fred Hutch/University of Washington/Seattle Children's Cancer Consortium member Roland Strong contributed to this work.

Finton KAK, Rupert PB, Friend DJ, Dinca A, Lovelace ES, Buerger M, Rusnac DV, Foote-McNabb U, Chour W, Heath JR, Campbell JS, Pierce RH, Strong RK. 2023. Effects of HLA single chain trimer design on peptide presentation and stability. Front Immunol. 14:1170462.