Deimmunized therapeutic compositions and methods
This disclosure provides a bispecific ligand directed toxin (BLT) that includes a diphtheria toxin (DT) molecule that has been mutated to create a DT molecule that induces less of an immune response than native diphtheria toxin. The deimmunized DT molecule is fused with targeting ligands to create a fusion protein that can selectively deliver the deimmunized DT to target cells to kill the target cells.
1. A polypeptide comprising:
a diphtheria toxin (DT) domain comprising a variant of DT390, the variant of DT390 comprising:
the DT catalytic site;
the K125S, R173A and Q245S amino acid substitutions in the amino acid sequence of SEQ ID NO: 1; and
at least one targeting domain that selectively binds to a target cell.
2. The polypeptide of claim 1 , wherein the polypeptide exhibits measurable diphtheria toxin toxicity.
3. The polypeptide of claim 1 , wherein the target cell is a tumor cell and the targeting domain binds to a receptor on the tumor cell.
4. The polypeptide of claim 1 , wherein the targeting domain comprises an epidermal growth factor (EGF) polypeptide that selectively binds to epidermal growth factor receptor (EGFR).
5. The polypeptide of claim 1 , wherein the targeting domain comprises IL-13 polypeptide that selectively binds to IL-13 receptor.
6. The polypeptide of claim 1 , wherein the targeting domain comprises a polypeptide that selectively binds to CD22.
7. The polypeptide of claim 1 , wherein the targeting domain comprises a polypeptide that selectively binds to CD19.
8. The polypeptide of claim 1 , wherein the polypeptide comprises at least two targeting domains.
9. The polypeptide of claim 8 , wherein a first targeting domain comprises IL-13 polypeptide and a second targeting domain comprises an epidermal growth factor (EGF) polypeptide.
10. The polypeptide of claim 8 , wherein a first targeting domain comprises a polypeptide that selectively binds CD19 and a second targeting domain comprises a polypeptide that selectively binds CD22.
11. The polypeptide of claim 1 , wherein the variant of DT390 further comprises the K385G and E292S amino acid substitutions in the amino acid sequence of SEQ ID NO: 1.
12. The polypeptide of claim 11 , wherein the variant of DT390 further comprises the Q184S and K227S amino acid substitutions in the amino acid sequence of SEQ ID NO: 1.
13. A method of killing a target cell that expresses a receptor, the method comprising:
providing a polypeptide comprising:
a diphtheria toxin (DT) domain comprising a variant of DT390, the variant of DT390 comprising:
the DT catalytic site;
the K125S, R173A and Q245S amino acid substitutions in the amino acid sequence of SEQ ID NO: 1; and
at least one targeting domain that selectively binds to the receptor expressed on the target cell;
contacting the target cell with the polypeptide under conditions that allow the target cell to internalize the polypeptide; and
allowing the polypeptide to kill the target cell.
14. The method of claim 13 , wherein the target cell is present in vitro.
15. The method of claim 13 , wherein the target cell is an in vivo target cell.
16. The method of claim 13 , wherein the at least one targeting domain selectively binds to Her2, ROR1, CD19, CD22, CD133, CD20, CD33, CD52, EpCAM, CEA, UPA, or VEGFR.
17. The method of claim 13 , wherein the polypeptide is administered prior to, simultaneously with, or following administration of a chemotherapeutic drug.
18. A method of treating a subject having a tumor, the method comprising administering to the subject an amount of the polypeptide of claim 1 effective to ameliorate at least one symptom or clinical sign of the tumor, wherein the at least one targeting domain selectively binds to a target receptor expressed on cells of the tumor.
19. The method of claim 18 , wherein the target receptor is epidermal growth factor receptor (EGFR).
20. A method of producing a deimmunized diphtheria toxin polypeptide which comprises the diphtheria toxin catalytic site, the method comprising:
modifying a wild-type diphtheria toxin (DT) polypeptide by including the K125S, R173A and Q245S amino acid substitutions in the amino acid sequence of SEQ ID NO: 1 to produce a modified DT polypeptide;
screening the modified DT polypeptide for toxicity of the wild-type DT polypeptide;
screening the modified DT polypeptide for induction of anti-DT toxin antibodies; and
obtaining the modified DT polypeptide exhibiting measurable DT toxicity and reduced induction of anti-DT toxin antibodies compared to the wild-type DT to produce the deimmunized diphtheria toxin polypeptide.
21. The method of claim 20 , wherein the modifying further comprises including the K385G and E292S amino acid substitutions in the amino acid sequence of SEQ ID NO: 1.
22. The method of claim 21 , wherein the modifying further comprises including the Q184S and K227S amino acid substitutions in the amino acid sequence of SEQ ID NO: 1.