Knottin-drug conjugates and methods of using the same
Provided are knottin-drug conjugates. The conjugates include a knottin peptide that includes an engineered loop that binds to a target on a cancer cell surface, and a drug (e.g., a nucleoside drug) conjugated to the knottin peptide through a linker. Also provided are pharmaceutical compositions and kits that include the knottin-drug conjugates, as well as methods of using the knottin-drug conjugates, e.g., for therapeutic purposes.
1. A knottin-drug conjugate, comprising:
a knottin peptide comprising an engineered loop that binds to a receptor on a cancer cell surface; and
a chemotherapeutic nucleoside drug conjugated to the knottin peptide through an intracellularly cleavable linker.
2. The knottin-drug conjugate of claim 1 , wherein the knottin peptide is selected from the group consisting of: an EETI-II peptide, an AgRP peptide, a ω-conotoxin peptide, a Kalata B1 peptide, an MCoTI-II peptide, an agatoxin peptide, and a chlorotoxin peptide.
3. The knottin-drug conjugate of claim 1 , wherein the receptor is a cell adhesion receptor.
4. The knottin-drug conjugate of claim 3 , wherein the cell adhesion receptor is an integrin.
5. The knottin-drug conjugate of claim 4 , wherein the integrin is selected from the group consisting of: αvβ1 integrin, αvβ3 integrin, αvβ5 integrin, αvβ6 integrin, α5β1 integrin, and any combination thereof.
6. The knottin-drug conjugate of claim 1 , wherein the receptor is a chemokine receptor.
7. The knottin-drug conjugate of claim 6 , wherein the chemokine receptor is C-X-C chemokine receptor type 4 (CXCR4).
8. The knottin-drug conjugate of claim 1 , wherein the receptor is a growth factor receptor.
9. The knottin-drug conjugate of claim 1 , wherein the receptor is an immune cell receptor.
10. The knottin-drug conjugate of claim 9 , wherein the immune cell receptor is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).
11. The knottin-drug conjugate of claim 1 , wherein the receptor is neuropilin-1 (NRP1).
12. The knottin-drug conjugate of claim 1 , wherein the chemotherapeutic nucleoside drug comprises a nucleoside analogue.
13. The knottin-drug conjugate of claim 12 , wherein the nucleoside analogue is selected from the group consisting of: gemcitabine, cytarabine, troxacitabine, decitabine, cladribine, fludarabine, clofarabine, and 2′-C-cyano-2′-deoxy-1-β-D-arabino-pentofuranosylcytosine (CNDAC).
14. The knottin-drug conjugate of claim 13 , wherein the nucleoside analogue is gemcitabine.
15. The knottin-drug conjugate of claim 1 , wherein the intracellularly cleavable linker is a dipeptide-based intracellularly cleavable linker.
16. The knottin-drug conjugate of claim 15 , wherein the dipeptide-based intracellularly cleavable linker is a valyl-alanyl-para-aminobenzyloxy (Val-Ala-PAB)-based intracellularly cleavable linker.
17. The knottin-drug conjugate of claim 1 , wherein the knottin peptide comprises an unnatural amino acid to which the linker is attached.
18. The knottin-drug conjugate of claim 17 , wherein, prior to attachment, the unnatural amino acid comprises a functional group selected from the group consisting of: an azide, alkyne, alkene, amino-oxy, hydrazine, aldehyde, asaldehyde, nitrone, nitrile oxide, cyclopropene, norbornene, iso-cyanide, aryl halide, boronic acid, diazo, tetrazine, tetrazole, quadrocyclane, and iodobenzene.
19. The knottin-drug conjugate of claim 18 , wherein, prior to attachment, the unnatural amino acid comprises an azide functional group.
20. A pharmaceutical composition comprising:
the knottin-drug conjugate of claim 1 ; and
a pharmaceutically-acceptable excipient.
21. The pharmaceutical composition of claim 20 , wherein the composition is formulated for parenteral administration.
22. The pharmaceutical composition of claim 20 , wherein the composition is formulated for oral administration.
23. A kit comprising:
a therapeutically effective amount of the pharmaceutical composition of claim 20 ; and
instructions for administering the pharmaceutical composition to an individual in need thereof.
24. The kit of claim 23 , wherein the pharmaceutical composition is present in one or more unit dosages.
25. A method of treating an individual having cancer, comprising:
administering to an individual having cancer a therapeutically effective amount of a knottin-drug conjugate of claim 1 .
26. The method according to claim 25 , wherein the individual has a cancer selected from the group consisting of: brain cancer, breast cancer, ovarian cancer, and pancreatic cancer.
27. A method of making a knottin-drug conjugate, comprising:
conjugating a chemotherapeutic nucleoside drug to a knottin peptide comprising an engineered loop that binds to a receptor on a cancer cell surface, wherein the chemotherapeutic nucleoside drug is conjugated to the knottin peptide through an intracellularly cleavable linker.
28. The knottin-drug conjugate of claim 1 , wherein the intracellularly cleavable linker is an enzyme-labile linker.
29. The knottin-drug conjugate of claim 28 , wherein the intracellularly cleavable linker is cleavable by a lysosomal protease.
30. The knottin-drug conjugate of claim 29 , wherein the intracellularly cleavable linker is cleavable by cathepsin or plasmin.
31. The method according to claim 25 , wherein the intracellularly cleavable linker is an enzyme-labile linker.
32. The knottin-drug conjugate of claim 12 , wherein the nucleoside analogue is selected from the group consisting of: an adenoside/deoxyadenosine analogue, a guanosine/deoxyguanosine analogue, a thymidine/deoxythymidine analogue, and a deoxyuridine analogue.
33. The knottin-drug conjugate of claim 15 , wherein the dipeptide-based intracellularly cleavable linker is a maleimidocaproyl-valine-citruline-p-aminobenzyl (MC-vc-PAB)-based intracellularly cleavable linker.