MHC class I molecules (Major Histocompatibility Complex Class I molecules) are transmembrane proteins of the cellular immune response which bind peptides from inside the cell, for example, from the cytosol or the lumen of the endocytic organelles, and present them to cytotoxic T-cells (CTL, cytotoxic T lymphocytes) at the cell surface. This is called antigen presentation.
The binding of a T-cell receptor of a CTL to a class I peptide complex of an antigen presenting cell (APC) leads (depending on the location of the reaction in the body, the type of APC (B-cell, dendritic cell, etc.) and the state of activation of the CTL) to the activation of the CTL and/or to the induction of cell death (apoptosis) of the APC by the CTL.
The immune response is effective because the CTL, which react with self-peptides (which are produced from the body's own proteins), are eliminated in the thymus. For this reason, the recognition of a peptide by the CTL implies that the APC is producing foreign proteins which stem from viruses or intracellular parasites (bacteria, protozoa); the overproduction of endogenous peptides in malignant degenerate tumor cells can also lead to recognition reactions. Almost all the proteins contained in the cell break down into peptides at the end of their lifecycle, which then bind to MHC class I molecules in the reticulum (an organelle inside the cell surrounded by a membrane); subsequently, the complex consisting of the peptide and the MHC class I molecule is transported to the surface of the cell and is available there for recognition by CTL. If, due to a tumorigenic malignant degeneration, novel or mutated proteins are produced, or if, due to a viral infection, viral proteins are produced from the genetic material of the virus, these “novel” proteins are also broken down into peptides, which are then presented at the surface of the cell in the complex with MHC class I molecules. These “novel” peptides are different from the cell's own peptides and trigger recognition by the CTL.
Presentation by MHC class I molecules also plays a role in allergic reactions, transplant rejection and a number of auto-immune diseases such a multiple sclerosis and rheumatoid arthritis.
Examining the immune responses that are mediated by MHC class I molecules often requires detecting CTL which react with at certain class I peptide complex (epitope). Reactions to a single immunodominant epitope often account for 10-20% of the entire T-cell population in an organism and observing the CTL frequency thus allows for a precise observation of the immune response (and, for example, of the success or failure of a therapy). For this reason, reagents that can identify CTL, which recognize a certain selected epitope, are indispensable.
In order to detect such epitope-specific CTL, recombinant MHC class I molecules, which are produced in bacteria and are available as insoluble inclusion bodies, have until now been used by first denaturing them in a solution of a chaotropic agent. The chaotrope is then removed (for example, by renaturation and refolding) in the presence of the desired peptide, and the peptide class I complex is separated from the unfolded protein by means of gel filtration chromatography. Since the low affinity of a single class I peptide complex with a single T-cell receptor does not lead to a strong bond, multimers of class I-peptide-complexes are used, which, due to the avidity effect, bind to the T-cell receptors of a T-cell strongly enough to allow for a durable bond. Such multimers are obtained, for example, by streptavidin-mediated tetramerization of biotinylated class I peptide complexes (class I tetramers) or by pentamerization by self-assembling coiled coil domain (class I pentamers).
Class I multimers are generally marked with fluorescent colorants which allow them to be detected by a microscope or by flow cytometry. Epitope-specific CTL can thus be directly colored.
Other uses of recombinant class I peptide complexes are:                In vitro—Selection and expansion of monospecific T-cells for reinfusion in cancer and viral diseases. (The selection can occur by means of cytofluorometry (flow cytometry) or, for increased throughput, in microarrays).        Ex vivo—Isolation and expansion of CTL for adoptive therapy after allogeneic stem cell transplantation.        Ex vivo—Removal of alloreactive T-cells after transplantation of peripheral stem cells. The removal of autoreactive T-cells, which cause type I diabetes, arthritis and other autoimmune diseases, is also interesting, as has already been described regarding MHC class II reagents. The use of isotope-marked multimers is described in US 2003/0228258 A1.        
The production of recombinant class I peptide complexes is complex and expensive. On the one hand, there are several thousand MHC class I allotypes, of which however, five alleles of HLA-A cover about 50% of the world population. Mainly, however, a new multimer must be produced for each peptide that is to be examined as an epitope so that new multimers, which must be specifically produced, are required for each patient or for each experiment.
It would be simpler to produce the multimers without the peptides and to subsequently add the respectively-required peptides as required, but this has not been possible so far because refolding the class I molecules without the peptide is extremely inefficient. A remedy has previously been described. A light-sensitive peptide was used which breaks down under UV radiation or periodate treatment, and can then be replaced by adding a peptide.
This method, however, remains expensive and does not function with all peptides or class I molecules.
USPTO Backgrounds
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