Methods of delivery of exogenous proteins to the cytosol and uses thereof
View Patent ↗The present invention is directed to a method for delivering exogenous proteins to the cytosol, by binding a target antigen (such as a protein) to a transport factor that contains a fragment of a bipartite protein exotoxin, but not the corresponding protective antigen. Preferably, the target antigen is fused to the transport factor. Preferred transport factors include the protective antigen binding domain of lethal factor (LFn) from B. anthracis , consisting of amino acids 1-255, preferably a fragment of at least 80 amino acids that shows at least 80% homology to LFn, and a fragment of about 105 amino acids from the carboxy portion that does not bind PA. The target antigen can include any molecule for which it would be desirable to elicit a CMI response, including viral antigens and tumor antigens.
1. A method for measuring a cell mediated immune response, comprising contacting a cell with an isolated polypeptide comprising a target antigen and a transport factor, wherein the transport factor contains a fragment of at least 104 C-terminal amino acids of a bipartate protein exotoxin which is not toxic to the cell and functions to deliver the target antigen to the cytosol of the cell, and wherein a protective antigen (PA) of a bipartate exotoxin is not present, and measuring the increase in at least one cytokine or the increase in a cell surface antigen expressed on the cell in response to delivering the target antigen to the cytosol of the cell, wherein the increase of at least one cytokine or expression of a cell surface antigen by the cell, as compared to the corresponding at least one cytokine or expression of a cell surface antigen in the absence of the target antigen and transport factor, is indicative of a cell mediated immune response.
2. A method for measuring a cell mediated immune response in vitro, comprising contacting a cell with a polypeptide comprising a target antigen linked to transport factor from B. anthracia , wherein the transport factor is a bipartate protein exotoxin that is not toxic to the cell, and wherein the protective antigen (PA) of the bipartate exotoxin is not present, and measuring the increase in at least one cytokine or the increase in a cell surface antigen expressed on the cell in response to delivering the target antigen to the cytosol of the cell, wherein the increase in at least one cytokine or expression of a cell surface antigen by the cell, as compared to the corresponding at least one cytokine or expression of a cell surface antigen in the absence of the target antigen linked to the transport factor, is indicative of a cell mediated immune response.
3. The method of claim 1 or 2 , wherein the transport factor comprises at least residues 185-288 of SEQ ID NO: 2.
4. The method of claim 1 or 2 , wherein the transport factor comprises at least residues 35-288 of SEQ ID NO: 2.
5. The method of claim 2 , wherein the polypeptide is a bacterial target antigen.
6. The method of claim 5 , wherein the bacterial target antigen is a polypeptide from S. typhi or Mycobacteria.
7. The method of claim 6 , wherein the Mycobacteria is M. tuberculosis .
8. A kit for measuring a cell mediated immune response in vitro comprising a cocktail of different polypeptides, wherein each polypeptide is a target antigen which is bound to a transport factor, wherein the transport factor comprises at least residues 185-288 of SEQ ID NO: 2 and wherein the protective antigen (PA) of B. anthracis bipartate exotoxin is not present.
9. The kit of claim 8 , wherein the transport factor comprises at least residues 34-288 of SEQ ID NO: 2.
10. The kit of claim 8 , wherein at least one polypeptide comprises a target antigen which is a bacterial target antigen.
11. The kit of claim 10 , wherein the bacterial target antigen is a polypeptide from S. typhi or Mycobacteria .
12. The kit of claim 11 , wherein the Mycobacteria is M. tuberculosis .
13. The method of claim 1 or 2 , wherein the cytokine is selected from the group consisting of: interferon, IL-2, IL-12, IL-4, GM-CSF, CpG.
14. The method of claim 13 , wherein the interferon is INF-γ.
15. The method of claim 1 or 2 , wherein the cell-surface marker is expressed by a T-cell.
16. The method of claim 15 , wherein the cell-surface marker expressed by a T-cell is selected from the group consisting of: CD96, CD8, CD3.
17. The method of claim 1 or 2 , wherein the cytokine is measured by any one of: ELISA assay, ElisSPOT assay or flow-based intracellular cytokine assay.
18. The method of claim 1 or 2 , wherein the cell is an immune cell.
19. The method of claim 18 , wherein the immune cell is selected from PBMCs, T-cell.