Methods related to engineered erythoid cells comprising 4-1BBL
The invention includes compositions and methods related to multimodal therapies, e.g., for treating a cancer. A multimodal therapy described herein provides and/or administers a plurality of agents that function in a coordinated manner to provide a therapeutic benefit to a subject in need thereof, e.g., a subject having a cancer.
1. A method of making a genetically engineered enucleated erythroid cell comprising a first exogenous polypeptide comprising a 4-1BB-binding fragment of 4-1BBL at the surface of the genetically engineered enucleated erythroid cell, comprising:
(i) providing a nucleated erythroid cell, or a precursor thereof, comprising a first exogenous nucleic acid encoding the first exogenous polypeptide; and
(ii) culturing the nucleated erythroid cell under conditions suitable for enucleation of the nucleated erythroid cell and for production of the first exogenous polypeptide.
2. The method of claim 1 , wherein the first exogenous polypeptide further comprises a transmembrane domain.
3. The method of claim 2 , wherein the transmembrane domain comprises a polypeptide having at least 80% identity to the amino acid sequence of SEQ ID NO: 9.
4. The method of claim 2 , wherein the transmembrane domain comprises a type I red blood cell transmembrane domain.
5. The method of claim 2 , wherein the transmembrane domain comprises a glycophorin A (GPA) transmembrane domain.
6. The method of claim 1 , wherein the first exogenous polypeptide comprises a 4-1BB-binding fragment of an amino acid sequence of SEQ ID NO: 21.
7. The method of claim 1 , wherein the first exogenous polypeptide lacks a sortase transfer signature.
8. The method of claim 1 , wherein the genetically engineered enucleated erythroid cell comprises a plurality of exogenous fusion proteins on the surface of the genetically engineered enucleated erythroid cell, wherein at least 50% of the exogenous fusion proteins comprised by the cell have an identical sequence.
9. The method of claim 1 , wherein the first exogenous nucleic acid comprises DNA.
10. The method of claim 1 , wherein the first exogenous nucleic acid comprises RNA.
11. The method of claim 1 , wherein the genetically engineered enucleated erythroid cell further comprises a second exogenous polypeptide.
12. The method of claim 11 , wherein the first exogenous nucleic acid encodes the second exogenous polypeptide.
13. The method of claim 11 , wherein the second exogenous polypeptide is encoded by a second exogenous nucleic acid.
14. The method of claim 11 , wherein the second exogenous polypeptide comprises IL-15 or an IL15Rα-binding fragment thereof.
15. The method of claim 11 , wherein the second exogenous polypeptide comprises IL-12 or a receptor-binding fragment thereof.
16. The method of claim 1 , further comprising introducing the first exogenous nucleic acid into the nucleated erythroid cell, or the precursor thereof.
17. The method of claim 16 , wherein the introducing comprises transduction.
18. The method of claim 17 , wherein transduction comprises use of a lentivirus vector.
19. The method of claim 16 , wherein the introducing comprises transfection or electroporation.
20. The method of claim 1 , wherein culturing comprises expanding the nucleated erythroid cell, or the precursor thereof, by at least 1,000-fold in culture.
21. The method of claim 1 , which produces a plurality of genetically engineered erythroid cells wherein at least 60% of cells in the plurality are genetically engineered enucleated erythroid cells.
22. The method of claim 1 , which produces a plurality of genetically engineered erythroid cells wherein at least 90% of cells in the plurality are genetically engineered enucleated erythroid cells.
23. The method of claim 1 , wherein the genetically engineered enucleated erythroid cell is a reticulocyte.
24. The method of claim 1 , wherein the genetically engineered enucleated erythroid cell is a mature red blood cell.
25. The method of claim 1 , wherein the precursor is a CD34+ cell.
26. The method of claim 1 , wherein the genetically engineered enucleated erythroid cell exhibits substantially the same osmotic membrane fragility as an isolated, unmodified, uncultured enucleated erythroid cell.
27. The method of claim 1 , wherein the genetically engineered enucleated erythroid cell is capable of promoting T cell proliferation.
28. The method of claim 1 , wherein the nucleated erythroid cell, or the precursor thereof, is a human cell.