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 method of expanding a population of antigen-specific CD8+ T cells ex vivo, the method comprising:
establishing a first culture comprising a population of antigen-specific CD8+ T cells, a first population of antigen presenting cells comprising an MHC antigen peptide and a culture medium comprising IL-7;
co-culturing the first culture for no greater than 14 days, wherein IL-2 is added to the first culture every 2-4 days; and
subsequently establishing a second culture comprising the population of antigen-specific CD8+ T cells, a population of cells comprising a single chain trimer and a culture medium comprising IL-2;
co-culturing the second culture for about 14 days, wherein the single chain trimer comprises, in amino-to-carboxy terminal order, a segment comprising the sequence of the MHC antigen peptide, a segment comprising a human β2-microglobulin sequence, and a segment comprising a human MHC class I heavy chain sequence.
2. A method in accordance with claim 1 , wherein the first population of antigen presenting cells is selected from the group consisting of dendritic cells, B cells, macrophages and a combination thereof.
3. A method in accordance with claim 1 , wherein the population of cells comprising the single chain trimer is a population of antigen presenting cells expressing the single chain trimer.
4. A method in accordance with claim 3 , wherein the population of antigen presenting cells expressing the single chain trimer is a population of human hematopoietic cells expressing the single chain trimer.
5. A method in accordance with claim 1 , wherein the MHC antigen peptide comprises a sequence selected from the group consisting of NLVPMVATV (SEQ ID NO: 1), GLCTLVAML (SEQ ID NO: 2), GILGFVFTL (SEQ ID NO: 3), IMDQVPFSV (SEQ ID NO: 4) and YLEPGPVTV (SEQ ID NO: 5).
6. A method in accordance with claim 1 , wherein expanding the population of antigen-specific CD8+ T cells comprises obtaining about 1×10 9 of the antigen-specific CD8+ T cells.
7. A method in accordance with claim 1 , wherein expanding the population of antigen-specific CD8+ T cells comprises expanding the population at least about 10,000 fold.
8. A method in accordance with claim 1 , wherein expanding the population of antigen-specific CD8+ T cells comprises expanding the population at least about 100,000 fold.
9. A method in accordance with claim 1 , wherein the population of antigen-specific CD8+ T cells following the expanding is at least about 55% pure.
10. A method in accordance with claim 1 , wherein the population of antigen-specific CD8+ T cells following the expanding is at least about 95% pure.
11. A method in accordance with claim 1 , wherein the population of antigen-specific CD8+ T cells following the expanding comprises at least about 1.1×10 5 fold increase in tetramer-positive cells.
12. A method in accordance with claim 1 , wherein the population of antigen-specific CD8+ T cells following the expanding comprises at least about 4.1×10 6 fold increase in tetramer-positive cells.