For years, recombinant major histocompatibility complexes (MHCs) have been expressed in bacterial systems with in vitro refolding. While this preserves the native MHC sequence, bacterial expression lacks post-translational modifications (PTMs) and risks misfolding. But what if we could bypass these challenges and produce fully functional MHCs directly in the form our cells naturally use?
What are MHC complexes and why are they important?
MHC complexes are composed of three chains: a peptide, an invariable light chain called β2-microglobulin (B2M), and a highly polymorphic heavy chain (HLA in humans) (Figure 1). MHC complexes present sets of 8 to 14 residue antigenic peptides on their surface. These peptides are presented to T cells and hence are critical in adaptive immunity. The interaction between MHCs and TCRs is the basis for immune surveillance to detect invading pathogens and also enlighten researchers of cancer immunotherapy development, such as adaptive T cell therapies and TCR-mimic antibody discovery. To facilitate development of these therapies, it is crucial to have biologically active and physiologically relevant MHC complexes as the study tools.
Figure 1. Structure of MHC-peptide complex.
Traditional Approach to MHC Production: E. coli expression and in vitro refolding
Traditionally, MHCs are expressed in E. coli and refolded in vitro, which is a complicated and labor-intensive process. This process involves expressing the heavy and light chains separately and refolding them in the presence of an excess of peptide. This process is laborious and carries the risk of misfolding MHCs. However, the biggest advantage of bacterial expression is that it maintains the original MHC sequence, something valued by researchers who want to ensure they’re accurately identifying epitope-specific antibodies or antigen-specific T cells.
Figure 2. Process of MHC expression in E. coli and in vitro refolding.
Modern Approach to MHC Production: Single-Chain Trimer MHC Expression from Mammalian Systems
A more novel approach to MHC production is the development of single-chain trimer (SCT) MHC complexes. The concept of SCT MHCs was first detailed in “Translational and basic applications of peptide-MHC single chain trimers” published in the Journal of Immunology (2), which laid the groundwork for their enhanced stability and functional advantages. These SCT MHCs combine the heavy chain, β2-microglobulin, and peptide into a single polypeptide chain, expressed in mammalian cells, effectively addressing several limitations of bacteria-refolded MHCs (Figure 3).
Figure 3. Process of single-chain trimer MHC expression in mammalian systems.
SCT expression simplifies the expression process and the number of steps to production. Additionally, it stabilizes MHC complexes and reduces the risk of peptide displacement by other peptides. This results in more accurate and reliable detection of antigen-specific T cells. Moreover, refolded proteins always pose a risk of structural uncertainty.
Building on this innovation, KACTUS recognized the inherent physiologic challenges and labor-intensive process of E. coli expression. Since our founding in 2018, we have made it our mission to engineer superior MHC complexes by leveraging the mammalian-expressed SCT design to eliminate the need for in vitro refolding. Our validation data has shown equivalent performance of mammalian-expressed SCT MHCs to E. coli refolded MHCs (Figure 4). Additionally, our mammalian expression systems mean the MHC complexes have PTMs including glycosylation.
Figure 4. Comparison of ELISA activity data for E. coli refolded MHCs versus mammalian expressed (Expi293) SCT MHCs.
Addressing Linker Concerns in SCT MHCs
Creating a single polypeptide chain between the peptide, heavy chain, and light chain, requires the addition of a linker. A common concern with SCTs is that the linker means a change in the original protein sequence. Researchers fear this change might interfere with TCR binding, affecting the physiological accuracy of antibodies or antigen-specific T cells identified using SCT MHCs. To address this concern, we performed extensive analysis of over 200 TCR-pMHC complex crystal structures to show that the linker in SCT MHCs does not interfere with the TCR-peptide-MHC interface. The linker is positioned away from the critical binding regions, ensuring that MHC-TCR interactions remain intact (Figure 5).
Figure 5. Structural analysis of TCR and peptide-MHC I/II interactions demonstrates the C-terminal linker does not interfere with the MHC-TCR binding interface.
Should you choose SCT MHCs for your research?
Selection of recombinant MHC complexes will always need to be considered in the context of experiment goals and applications. Researchers who are targeting biologically relevant MHCs that contain innate PTMs may be better off using MHCs derived from mammalian cells for immunization and antibody screening. In this scenario, E. coli-sourced MHCs could even be used to counterselect and remove antibodies that have an affinity towards MHCs without PTMs. Conversely, there might be situations where researchers want antibodies specifically targeting MHCs without PTMs or with low PTMs. Then, MHCs derived from E. coli would be suitable for immunization and screening.
That being said, advanced precision research in antibody drugs and T cell therapy increasingly requires the use of biologically relevant target proteins. The PTMs of mammalian-expressed SCT MHCs provide a distinct advantage of being more physiologically relevant when selecting for antibodies or T cells with high specificity and efficacy. E. coli-expressed MHCs may always have a place, but we believe SCT mammalian expression will soon become the gold standard for recombinant MHC complexes.
Our MHC Class I and Class II Products & Services
Owing to the development of TCR mimic antibodies and T cell therapy, we’ve developed a large catalog selection of mammalian-expressed MHC complexes. This includes MHC Class I and Class II monomers and tetramers as well as peptide-ready MHCs which are MHC monomers or tetramers that can be loaded with an antigenic peptide in-house. Browse our full suite of products here or request a custom MHC complex. Additionally, we offer soluble TCR expression services and SPR analysis services to address MHC/TCR binding interactions.
References
- Altman, J. D., H. Moss, P. A., R. Goulder, P. J., Barouch, D. H., McHeyzer-Williams, M. G., Bell, J. I., McMichael, A. J., & Davis, M. M. (1996). Phenotypic Analysis of Antigen-Specific T Lymphocytes. Science. https://doi.org/10.1126/science.274.5284.94
- Hansen, T. H., Connolly, J. M., Gould, K. G., & Fremont, D. H. (2010). Translational and basic applications of peptide-MHCI single chain trimers. Trends in Immunology, 31(10), 363. https://doi.org/10.1016/j.it.2010.07.003
