D-1462 – Method to express, purify, and biotinylate eukaryotic cell-derived major histocompatibility complexes.

Major histocompatibility complex (MHC) class I-specific reagents such as fluorescently-labeled multimers have greatly advanced the understanding of CD8 +T cells under normal and diseased states. While substantial advances have been made, recombinant MHC class I components are typically produced in bacterial culture followed by a lengthy purification protocol requiring additional non-covalent folding steps with exogenous peptide for complete molecular assembly. As such, there is a clear and unmet need for a more rapid and cost-effective approach to generating these important molecules.

This technology enables the large-scale and continuous batch production of soluble, fully folded and epitope-defined MHC class I molecules (at a purity of at least 95%) from an industry standard eukaryotic cell line.

Reference Number: D-1462

Market Applications:

  • Research tool enabling the detection of antigen-specific CD8+ T cells for use in basic, translational and clinical research Vaccine development;
  • Research tool for testing/evaluating immune response in vaccine development and during clinical trials.

Features, Benefits, and Advantages:

  • Combines the technical ease of CHO cell line cultivation and long-term expression of soluble, covalently-linked peptide/MHC molecules for multimerization based on end user’s needs for downstream assays;
  • Approach is based on Single Chain Trimer (SCT) methodology whereby MHC molecules presenting a range of class I peptides (e.g., low-to-high binding affinity) can be reliably generated;
  • Incorporates a SB transposon system providing for high insertion efficiency at close-to-random chromosomal sites, viral transduction to insert transgenes while eliminating the need to maintain genomic materials episomally (e.g., as with adeno-associated viruses) or near/in proto oncogenes (e.g., retroviral vectors);
  • Can be used for higher order reagents to better discriminate low frequency T cells (or bind “difficult” T cell receptors) such as fluorochrome conjugated dextramers since production process involves incubating biotinylated peptide/MHC with a dextran backbone containing streptavidin;
  • CHO cells enable instigation of post-translational modifications and can be easily grown at high density under serum-free conditions in suspension culture – protocol is amenable to freezing material at convenient stopping points;
  • Easily scaled and anticipated to reduce generation of folded protein/MHC molecule using traditional methods by a minimum of 50%.

Intellectual Property: A U.S. patent is currently pending and published as US20210380661.

Development Stage: Proof of concept has been achieved with a MHC complex generated with the popular ovalbumin epitope SIINFEKL. Purified and soluble peptide/MHC was successfully isolated from CHO cells and ligand binding specificity confirmed at the peptide/MHC class I interface. Stable cell lines have been produced and are readily available for licensing and technology transfer. Further validation to be conducted using high demand human peptide targets.

Contact Information

Name: Jennifer C Souter

Email: jesouter@ttu.edu

Phone: 806-834-7507