JPWO2020023702A5

JPWO2020023702A5 -

Info

Publication number: JPWO2020023702A5
Application number: JP2021503843A
Authority: JP
Publication date: 2022-08-01

Claims

A prodrug comprising a human IL-21 agonist polypeptide, a masking moiety and a carrier moiety, wherein
said masking moiety binds to a human IL-21 agonist polypeptide and inhibits a biological activity of said human IL-21 agonist polypeptide;
the human IL-21 agonist polypeptide is fused to a carrier moiety, and the masking moiety is fused to the human IL-21 agonist polypeptide or carrier moiety via a cleavable peptide linker;
prodrug.

2. The prodrug of claim 1, wherein the human IL-21 agonist polypeptide comprises SEQ ID NO:1 or an amino acid sequence that is at least 90% identical to SEQ ID NO:1.

3. The prodrug of claim 2, wherein the human IL-21 agonist polypeptide comprises one or more mutations at positions selected from D18, Q19, E109 and K117 (numbering according to SEQ ID NO: 1).

2. The prodrug of claim 1, wherein the IL-21 agonist polypeptide has an amino acid sequence selected from SEQ ID NOS:2, 3, 4 and 5.

5. The prodrug of any of claims 1-4, wherein the masking moiety comprises the extracellular domain (ECD) of the human IL-21 receptor or a functional analogue thereof.

6. The prodrug of claim 5, wherein the IL-21 receptor is IL-21Rα or IL-21Rγ.

7. The prodrug of claim 6, wherein the IL-21 receptor ECD comprises SEQ ID NO:6 or 7 or an amino acid sequence that is at least 90% identical to SEQ ID NO:6 or 7.

8. The prodrug of any of claims 1-7, further comprising a second cytokine moiety.

the second cytokine part
(i) a human IL-2 agonist polypeptide comprising SEQ ID NO:8 or an amino acid sequence that is at least 90% identical to SEQ ID NO:8;
(ii) a human IL-7 agonist polypeptide;
(iii) a human IL-9 agonist polypeptide;
(iv) SEQ ID NO:9 or a human IL-15 agonist polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:9;
(v) a human IL-15 agonist polypeptide and a human IL-15 receptor alpha susi domain, or
9. The prodrug of claim 8, which is SEQ ID NO:27 or a human CCL19 polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:27.

further comprising a second masking moiety that binds to the second cytokine moiety and inhibits the biological activity of the second cytokine moiety, wherein the second masking moiety comprises a cleavable peptide linker to the second cytokine moiety or to the carrier moiety; 10. The prodrug of claim 8 or 9, fused via.

The second masking moiety is selected from an IL-2 receptor beta subunit ECD or functional analogue thereof, an IL-7 receptor ECD or functional analogue thereof and an IL-9 receptor ECD or functional analogue thereof 11. The prodrug of claim 10.

12. A prodrug according to any preceding claim, wherein the human IL-21 agonist polypeptide and/or the second cytokine moiety is fused to the carrier moiety via a non-cleavable peptide linker.

13. The prodrug of claim 12, wherein the non-cleavable peptide linker comprises an amino acid sequence selected from SEQ ID NOs:29-33.

14. The prodrug of any of claims 1-13, wherein the cleavable peptide linker comprises a substrate sequence for urokinase-type plasminogen activator (uPA), matrix metalloprotease (MMP) 2 or MMP9.

15. The prodrug of claim 14, wherein the cleavable peptide linker comprises (i) both uPA and MMP2, (ii) both uPA and MMP9, or (iii) substrate sequences of uPA, MMP2 and MMP9.

15. The prodrug of claim 14, wherein the cleavable peptide linker comprises an amino acid sequence selected from SEQ ID NOS: 11-26.

17. Any of claims 1-16, wherein the cleavable peptide linker is cleavable by one or more proteases located at the tumor site or its surrounding environment, and cleavage results in activation of the prodrug at the tumor site or surrounding environment. Prodrug as indicated.

18. A prodrug according to any preceding claim, wherein the carrier moiety is a PEG molecule, albumin, albumin fragment, antibody Fc domain or antibody or antigen binding fragment thereof.

19. The prodrug of claim 18, wherein the carrier moiety is an antibody Fc domain or antibody comprising L234A and L235A ("LALA") mutations (EU numbering).

an antibody Fc domain or antibody in which the carrier portion comprises a knob-into-hole mutation;
a human IL-21 agonist polypeptide and a masking portion thereof are fused to different polypeptide chains of an antibody Fc domain or different heavy chains of an antibody, and
the second cytokine portion and the second masking portion are fused to different polypeptide chains of an antibody Fc domain or different heavy chains of an antibody;
20. A prodrug according to claim 18 or 19.

21. The prodrug of claim 20, wherein the human IL-21 agonist polypeptide and masking portion thereof are fused to the C-termini of two different polypeptide chains of an Fc domain or to the C-termini of two different heavy chains of an antibody.

21. The prodrug of claim 20, wherein the human IL-21 agonist polypeptide and masking portion thereof are fused to the N-termini of two different polypeptide chains of an Fc domain or to the N-termini of two different heavy chains of an antibody.

A second cytokine portion and a second masking portion are fused to opposite ends of two different polypeptide chains of an Fc domain from a human IL-21 agonist polypeptide and a masking portion thereof or opposite ends of two different heavy chains of an antibody 23. The prodrug of claim 21 or 22, which is

Knob-into-hole mutations introduce a T366Y "knob" mutation (EU numbering) in a polypeptide chain of the Fc domain or heavy chain of the antibody and a Y407T "hole" mutation (EU numbering) in another polypeptide chain of the Fc domain or other heavy chain of the antibody. A prodrug according to any of claims 20-23, comprising

20. The knob-into-hole mutations comprise Y349C and/or T366W mutations in the CH3 domain of the "knob chain" and E356C, T366S, L368A and/or Y407V mutations (EU numbering) in the CH3 domain of the "whole chain". 24. A prodrug according to any one of 1 to 23.

The carrier moiety is guanylate cyclase C (GCC), carbohydrate antigen 19-9 (CA19-9), glycoprotein A33 (gpA33), mucin 1 (MUC1), carcinoembryonic antigen (CEA), insulin-like growth factor 1 receptor body (IGF1-R), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), delta-like protein 3 (DLL3), delta-like protein 4 (DLL4), epithelial cell proliferation Factor Receptor (EGFR), Glypican-3 (GPC3), c-MET, Vascular Endothelial Growth Factor Receptor 1 (VEGFR1), Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), Nectin-4, Liv-1, Glycoprotein NMB (GPNMB), Prostate Specific Membrane Antigen (PSMA), Trop-2, Carbonic Anhydrase IX (CA9), Endothelin B Receptor (ETBR), Prostate 6 Transmembrane Epithelial Antigen 1 (STEAP1), Folic Acid receptor alpha (FR-α), SLIT and NTRK-like protein 6 (SLITRK6), carbonic anhydrase VI (CA6), ectonucleotide pyrophosphatase/phosphodiesterase family member 3 (ENPP3), mesothelin, trophoblast glycoprotein (TPBG) , CD19, CD20, CD22, CD33, CD40, CD56, CD66e, CD70, CD74, CD79b, CD98, CD123, CD138, CD352, CD47, signal regulatory protein alpha (SIRPα), PD1, claudin 18.2, claudin 6 , 5T4, BCMA, PD-L1, PD-1, fibroblast activation protein alpha (FAP alpha), melanoma-associated chondroitin sulfate proteoglycan (MCSP) and EPCAM. 26. The prodrug of any of claims 18-25, which is an antibody or antigen-binding fragment thereof.

The prodrugs have amino acid sequences of SEQ ID NOS: 36 and 38, respectively;
SEQ ID NOS: 37 and 38,
SEQ ID NOS: 39 and 41,
SEQ ID NOS: 40 and 41,
SEQ ID NOS: 42 and 44,
SEQ ID NOS: 43 and 44,
SEQ ID NOs:45 and 47 or SEQ ID NOs:46 and 47
21. The prodrug of claim 20, comprising two polypeptide chains comprising:

19. The prodrug of claim 18, wherein the carrier moiety comprises two heavy chain polypeptides whose amino acid sequences comprise SEQ ID NOs:48 and 49 and two light chain polypeptides whose amino acid sequences comprise SEQ ID NOs:50 or 51, respectively.

4. Claims wherein the carrier moiety is an antibody or antigen-binding fragment thereof that binds FAPα or 5T4 and further comprises two or three ectodomains of a tumor necrosis factor (TNF) ligand family member or a 4-1BB ligand or fragment thereof. 18. A prodrug according to 18.

A pharmaceutical composition comprising the prodrug according to any one of claims 1 to 29 and a pharmaceutically acceptable excipient.

A polynucleotide encoding a prodrug according to any one of claims 1-29.

32. One or more expression vectors comprising the polynucleotide of claim 31.

A host cell comprising the vector of claim 32.

34. The host cell of claim 33, wherein the genes encoding uPA, MMP2 and/or MMP9 have been knocked out in the host cell.

A method for producing a prodrug according to any one of claims 1 to 29,
culturing the host cell of claim 33 or 34, wherein the host cell is a mammalian cell, under conditions that allow expression of the prodrug, and isolating the prodrug,
Method.

A prodrug according to any one of claims 1 to 29 for use in treating cancer or infectious disease or stimulating the immune system in a patient in need thereof.

Use of a prodrug according to any of claims 1-29 in the manufacture of a medicament for the treatment of cancer or infectious diseases or stimulation of the immune system in a patient in need of such treatment.

the patient is HIV, HBV, HCV or HPV infected; or is selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colorectal cancer and gastric cancer; 29. The prodrug of claim 28 , wherein the prodrug has cancer.

the patient is HIV, HBV, HCV or HPV infected; or is selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colorectal cancer and gastric cancer; 38. Use according to claim 37 , wherein the cancer is