Cytokine-based bioactivatable drugs and methods of use thereof

Abstract

This disclosure provides a bioactivated drug construct, the “VitoKine” platform, designed to reduce toxicity based on systemic mechanisms and broaden the therapeutic use of proteins and cytokines in treating cancer, autoimmune diseases, inflammatory diseases, viral infections, transplantation, and various other disorders. The novel VitoKine construct of this invention comprises: 1) a tissue or disease site-targeting portion D1 domain (“D1”), 2) a bioactivated portion D2 domain (“D2”), and a masking portion D3 domain (“D3”). Because the “active portion” of the VitoKine construct remains inert until locally activated by upregulated proteases in diseased tissue, this prevents overactivation of the pathway and reduces undesirable “extra-tissue” “on-target” toxicity. Furthermore, the inertness of the VitoKine active portion prior to protease activation significantly reduces potential antigen or target uptake, thereby prolonging the in vivo half-life and resulting in improved biodistribution, bioavailability, and therapeutic efficacy.

Claims

1. A bioactivatable polypeptide drug construct D1-D2-D3, wherein the construct D1-D2-D3 comprises the following components in the N-terminus to C-terminus direction: 1) a dual-function domain D1, 2) a bioactive domain D2, and 3) a masking domain D3, wherein D1 functions to target the bioactive portion to an intended therapeutic site; wherein D3 maskes the functional activity of D2 until D2 is activated at the intended therapeutic site; and wherein the D2 domain is an IL-2 variant polypeptide with the amino acid sequence listed in SEQ ID NO: 240; wherein D1 is an antagonistic humanized PD-1 antibody with the following sequences: heavy and light chain amino acid sequences listed in SEQ ID NO: 195 and 196, heavy and light chain amino acid sequences listed in SEQ ID NO: 197 and 198, heavy and light chain amino acid sequences listed in SEQ ID NO: 275 and 276, or heavy and light chain amino acid sequences listed in SEQ ID NO: 277 and 278; and the amino acid sequence of the D3 domain is listed in SEQ ID NO:

268. D2 is attached to D1 via peptide linker L1, and D2 attaches to D3 via the peptide linker L2. L1 and L2 are respectively peptide linkers that can be cleaved by proteases or peptide linkers that cannot be cleaved.

2. The construct according to claim 1, wherein the protease-cleavable peptide linker is selected from the group consisting of sequences listed in SEQ ID NO:71-96 and 157-161.

3. The construct according to claim 1, wherein the cleavable peptide linker is selected from the group consisting of sequences listed in SEQ ID NO: 107-127.

4. The construct according to any one of claims 1 to 3, wherein both L1 and L2 are protease-cleavable peptide linkers.

5. The construct according to any one of claims 1 to 3, wherein both L1 and L2 are non-cleavable peptide linkers.

6. The construct according to any one of claims 1 to 3, wherein L1 is a protease-cleavable peptide linker and L2 is a non-cleavable peptide linker.

7. The construct according to any one of claims 1 to 3, wherein L1 is a non-cleavable peptide linker and L2 is a protease-cleavable peptide linker.

8. The construct according to any one of claims 1-3, wherein the D1, D2 and D3 domains of the construct are each in monomeric form, each in dimeric form, or a combination of dimer and monomeric form.

9. The construct according to claim 4, wherein the D1, D2 and D3 domains of the construct are each in monomeric form, each in dimeric form, or a combination of dimer and monomeric form.

10. The construct according to claim 5, wherein the D1, D2 and D3 domains of the construct are each in monomer form, each in dimer form, or together in a combination of dimer and monomer form.

11. The construct according to claim 6, wherein the D1, D2 and D3 domains of the construct are each in monomer form, each in dimer form, or together in a combination of dimer and monomer form.

12. The construct according to claim 7, wherein the D1, D2 and D3 domains of the construct are each in monomeric form, each in dimeric form, or a combination of dimer and monomeric form.

13. A pharmaceutical composition comprising a construct according to any one of claims 1 to 12, mixed with a pharmaceutically acceptable carrier.

14. Use of the construct according to any one of claims 1 to 12 in the preparation of a pharmaceutical composition for treating colon cancer.

15. The use according to claim 14, wherein the treatment further comprises a second therapeutic agent or therapy capable of treating the subject's cancer or cancer metastasis.

16. A nucleic acid molecule encoding a bioactive polypeptide drug construct according to any one of claims 1 to 12.

17. An expression vector comprising the nucleic acid molecule according to claim 16.

18. A host cell comprising the expression vector according to claim 17.

19. A method for producing a bioactive polypeptide drug construct according to any one of claims 1 to 12, the method comprising culturing a host cell according to claim 18 under conditions that promote the expression of the bioactive polypeptide drug construct, and recovering the bioactive polypeptide drug construct protein.

20. An isolated, bioactive polypeptide drug construct protein, said isolated, bioactive polypeptide drug construct protein being produced by the method according to claim 19.

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