Single chain trimers and uses therefor
Single chain trimer (SCT) molecules are disclosed, comprising an MHC antigen peptide sequence, a β 2 -microglobulin sequence and a full-length MHC class I heavy chain sequence, joined by linker sequences. Further described are nucleic acids encoding single chain trimers. Methods for expansion of antigen-specific T cell populations using single chain trimer molecules are also disclosed. In some configurations, these methods comprise co-culturing, in a first stage, CD8+ T cells from a donor with antigen presenting cells comprising an MHC antigen peptide, and co-culturing, in a second stage, the CD8+ T cells with cells comprising an SCT which has an MHC antigen peptide sequence identical to the sequence of the antigen peptide in the first stage. The methods can provide 10,000-100,000 fold expansion of antigen-specific CD8+ T cells within about 28 days after establishing culture, and can yield over 1 billion antigen-specific CD8+ T cells expanded from an individual donor.
1. A single chain trimer molecule comprising, in amino-to-carboxy terminal order, an antigen peptide sequence, a first flexible linker sequence, a β2-microglobulin sequence, a second flexible linker sequence and a human MHC class I heavy chain sequence wherein the MHC class I heavy chain sequence comprises a mutation of tyrosine-84 to a non-aromatic amino acid other than proline.
2. A single chain trimer molecule in accordance with claim 1 , wherein at least 80 percent of the amino acid residues comprising the first flexible linker sequence are selected from the group consisting of glycine, alanine and serine residues.
3. A single chain trimer molecule in accordance with claim 1 , wherein about 80 percent of the amino acid residues comprising the first flexible linker sequence are selected from the group consisting of glycine, alanine and serine residues.
4. A single chain trimer molecule in accordance with claim 1 , wherein at least 80 percent of the amino acid residues comprising the second flexible linker sequence are selected from the group consisting of glycine, alanine and serine residues.
5. A single chain trimer molecule in accordance with claim 1 , wherein about 80 percent of the amino acid residues comprising the second flexible linker sequence are selected from the group consisting of glycine, alanine and serine residues.
6. A single chain trimer molecule in accordance with claim 1 , wherein the non-aromatic amino acid other than proline is an alanine.