Chimeric receptor with NKG2D specificity for use in cell therapy against cancer and infectious disease
The invention provides a chimeric receptor comprising NKG2D, DAP10 and CD3 zeta. Also disclosed is a composition comprising this chimeric receptor and methods for making and using it to enhance the cytotoxicity and antitumor capacity of NK cells. The invention also encompansses methods for use of NKG2D-DAP10-CD3 zeta polypeptides, vectors and cells in methods for treating cancer and other proliferative disorders, as well as infectious diseases.
1. A method of producing an expanded population of natural killer (NK) cells, said method comprising:
obtaining a peripheral blood sample,
isolating from said peripheral blood sample a mixed population of mononuclear cells comprising NK cells and T cells,
culturing said mixed population of cells under culture conditions resulting in the enhanced expansion of NK cells as compared to T cells,
depleting the mixed population of residual T cells, and
introducing into the NK cells nucleic acid encoding a chimeric receptor, the chimeric receptor comprising:
i) an extracellular receptor domain, wherein said extracellular receptor domain comprises a peptide that binds one or more native ligands of Natural Killer Group 2 member D (NKG2D) receptor, wherein the extracellular receptor domain is at least 95% identical to an extracellular domain of a native human NKG2D receptor, and
ii) one or more peptides that recruit downstream signaling effector molecules in response to binding of a native ligand by said extracellular receptor domain, wherein the one or more peptides comprise a CD3zeta cytoplasmic signaling domain, wherein the CD3zeta cytoplasmic signaling domain is at least 95% homologous to the amino acid sequence of SEQ ID NO: 10.
2. The method of claim 1 , wherein the culture conditions comprise culturing said mixed population of mononuclear cells with K562 myeloid leukemia cells, wherein said K562 myeloid leukemia cells are genetically modified to express membrane bound interleukin-15 and 4-1BB ligand, wherein the extracellular receptor domain is encoded by a first polypeptide that also encodes a CD3zeta signaling domain, and wherein the extracellular receptor domain binds the native ligand of NKG2D, and wherein the CD3zeta cytoplasmic signaling domain is at least 98% identical to the amino acid sequence of SEQ ID NO: 10.
3. The method of claim 1 , wherein said T cells are removed using beads coupled to an anti-CD3 antibody.
4. The method of claim 1 , wherein the nucleic acid is introduced into said NK cells by transduction.
5. The method of claim 1 , wherein the nucleic acid is introduced into said NK cells by electroporation.
6. The method of claim 1 , wherein the native ligand is one or more of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, or ULBP6.
7. The method of claim 1 , wherein the one or more peptides of the chimeric receptor further comprise a DNAX-activating protein 10 (DAP10) polypeptide.
8. The method of claim 7 , wherein the DNAX-activating protein 10 (DAP10) polypeptide is encoded by the amino acid sequence of SEQ ID NO: 14.
9. The method of claim 7 , wherein the NKG2D extracellular domain, the CD3 zeta cytoplasmic signaling domain and the DNAX-activating protein 10 (DAP10) polypeptide are in frame with one another.
10. The method of claim 1 , wherein the nucleic acid further comprises sequences encoding a viral vector.
11. The method of claim 1 , wherein the expanded population of NK cells has enhanced cytotoxicity as compared to NK cells stimulated with Interleukin-2.
12. A method of producing a population of natural killer (NK) cells, said method comprising:
obtaining a peripheral blood sample,
isolating from said peripheral blood sample a mixed population of mononuclear cells comprising NK cells and T cells,
culturing said mixed population of cells under culture conditions resulting in the enhanced expansion of NK cells as compared to T cells,
removing T cells from said cell culture, and
introducing into the NK cells a nucleic acid encoding a chimeric receptor comprising:
i) an extracellular receptor domain, wherein said extracellular receptor domain comprises a peptide that binds one or more native ligands of NKG2D, wherein the extracellular receptor domain is at least 90% homologous to a NKG2D extracellular domain, wherein the NKG2D extracellular receptor domain comprises amino acids 81-215 of SEQ ID NO: 12, and
ii) one or more peptides that recruit downstream signaling effector molecules in response to binding of a native ligand by said extracellular receptor domain, wherein the one or more peptides recruiting downstream signaling effector molecules comprises a CD3 zeta cytoplasmic signaling domain.
13. The method of claim 12 , wherein the extracellular receptor domain is at least 95% identical to the NKG2D extracellular domain.
14. The method of claim 13 , wherein the downstream signaling effector molecules trigger secretion of one or more of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon gamma (IFNγ).
15. The method of claim 12 , wherein said extracellular receptor domain comprises a peptide that binds one or more of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 or ULBP6.
16. A method of producing a population of natural killer (NK) cells, said method comprising:
isolating a mixed population of mononuclear cells comprising NK cells and T cells from a donor of a whole blood sample,
culturing the mixed population of cells under culture conditions resulting in the enhanced expansion of NK cells as compared to T cells,
removing T cells from said cell culture, and
introducing into the NK cells at least two polypeptides that together encode a chimeric receptor,
the first polypeptide encoding a extracellular receptor domain and a signaling domain, wherein said extracellular receptor domain comprises a peptide that binds one or more native ligands of NKG2D, wherein the first polypeptide encoding the extracellular receptor domain encodes an NKG2D extracellular domain,
the second polypeptide encoding a stimulatory molecule, wherein the stimulatory molecule comprises the cytoplasmic signaling domain of CD3 zeta, wherein upon binding of one or more native ligands of NKG2D by the extracellular receptor domain, the NK cells exhibit one or more of enhanced cytotoxicity and anti-tumor capacity against target cells, triggered signal transduction, triggered secretion of cytokines and chemokines, increased target cell apoptosis, or sustained cytotoxicity against target cells.
17. The method of claim 16 , wherein the NK cells are suitable for administration to the donor of the whole blood sample.
18. The method of claim 1 , wherein the native extracellular domain of the NKG2D receptor comprises amino acids 81-215 of SEQ ID NO: 12.