Polynucleotides encoding tethered interleukin-12 (IL12) polypeptides and uses thereof
The present disclosure relates to polynucleotides encoding tethered interleukin-12 (IL-12) polypeptides comprising an IL-12 polypeptide and a membrane domain. The present disclosure also relates to vectors comprising the polynucleotides; host cells comprising the polynucleotides or vectors, polypeptides encoded by the polynucleotides; compositions comprising the polynucleotides, vectors, host cells, or polypeptides and a delivery agent; and uses thereof, including treatment of cancer.
1. A method of treating cancer in a subject in need thereof, comprising administering a lipid nanoparticle (LNP) comprising a messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a human interleukin-12 (IL-12) polypeptide operably linked to a membrane domain, optionally via a linker, wherein the human IL-12 polypeptide comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide, wherein the membrane domain comprises a transmembrane domain comprising a CD80 transmembrane domain, a PDGFR transmembrane domain, or a CD8 transmembrane domain, thereby reducing the size of the tumor or inhibiting growth of the tumor in the subject.
2. The method of claim 1 , wherein the membrane domain is operably linked to the IL-12A polypeptide by a peptide linker, or wherein the membrane domain is operably linked to the IL-12B polypeptide by a peptide linker.
3. The method of claim 1 , wherein the IL-12B polypeptide is operably linked to the IL-12A polypeptide by a peptide linker.
4. The method of claim 1 , wherein (i) the IL-12B polypeptide is located at the 5′ terminus of the IL-12A polypeptide, or the 5′ terminus of the peptide linker, or (ii) the IL-12A polypeptide is located at the 5′ terminus of the IL-12B polypeptide, or the 5′ terminus of the peptide linker.
5. The method of claim 1 , wherein the IL-12B polypeptide comprises amino acids 23 to 238 of SEQ ID NO: 48.
6. The method of claim 1 , wherein the IL-12A polypeptide comprises amino acids 336 to 532 of SEQ ID NO: 48.
7. The method of claim 1 , wherein the mRNA comprises a 3′ untranslated region (UTR) comprising a microRNA binding site.
8. The method of claim 7 , wherein the microRNA binding site is a miR-122 binding site.
9. The method of claim 8 , wherein the miR-122 binding site is a miR-122-3p or a miR-122-5p binding site.
10. The method of claim 1 , wherein the mRNA comprises at least one chemical modification.
11. The method of claim 10 , wherein the chemical modification is selected from the group consisting of pseudouridine or a psuedouridine analog.
12. The method of claim 10 , wherein the chemical modification is N1-methylpseudouridine.
13. The method of claim 10 , wherein the mRNA is fully modified with N1-methylpseudouridine.
14. The method of claim 1 , wherein the CD80 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 103, the PDGFR transmembrane domain comprises a PDGFR beta transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 102, and the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 101.
15. The method of claim 1 , wherein the membrane domain comprises an intracellular domain.
16. The method of claim 15 , wherein the intracellular domain is a CD80 intracellular domain or a PDGFR intracellular domain.
17. The method of claim 16 , wherein the CD80 intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 225 and the PDGFR intracellular domain comprises an amino acid sequence selected from SEQ ID NOs: 226-228.
18. The method of claim 1 , wherein the IL-12 polypeptide is operably linked to the membrane domain via a peptide linker.
19. The method of claim 1 , wherein the ORF encodes the amino acid sequence set forth in any one of SEQ ID NOs: 241, 243, 249, 253 and 255.
20. The method of claim 1 , comprising administering a second mRNA encoding a second polypeptide.
21. The method of claim 20 , wherein the LNP comprises the second mRNA.
22. The method of claim 1 , wherein treating comprising reducing the size of a tumor or inhibiting growth of a tumor in the subject.
23. The method of claim 1 , wherein the LNP is administered intratumorally.
24. The method of claim 1 , wherein the LNP comprises an ionizable amino lipid, a phospholipid, a sterol, and a PEG-modified lipid.
25. The method of claim 24 , wherein the LNP comprises a molar ratio of about 20-60% ionizable amino lipid: 5-25% phospholipid: 25-55% sterol: 0.5-15% PEG-modified lipid.
26. A method of treating cancer in a subject in need thereof, comprising administering a LNP comprising a mRNA comprising a 5′ untranslated region (UTR), an ORF, and a 3′ UTR, wherein the ORF comprises a nucleotide sequence encoding from 5′ to 3′:
5′-[IL-12B]-[L1]-[IL-12A]-[L2]-[MD]-3′, wherein
IL-12B is a human IL-12 p40 subunit polypeptide comprising amino acids 23 to 328 of SEQ ID NO: 48,
L1 is a first peptide linker,
IL-12A is a human IL-12 p35 subunit polypeptide comprising amino acids 336 to 532 of SEQ ID NO: 48,
L2 is a second peptide linker, and
MD is a membrane domain comprising a CD80 transmembrane domain and a CD80 intracellular domain.
27. The method of claim 26 , wherein the CD80 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 103 and the CD80 intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 225.
28. The method of claim 26 , wherein the 3′UTR comprises a microRNA binding site.
29. The method of claim 28 , wherein the microRNA binding site is a miR-122 binding site.
30. The method of claim 29 , wherein the miR-122 binding site is a miR-122-3p or a miR-122-5p binding site.
31. The method of claim 26 , wherein the mRNA comprises at least one chemical modification.
32. The method of claim 31 , wherein the chemical modification is selected from the group consisting of pseudouridine or a psuedouridine analog.
33. The method of claim 31 , wherein the chemical modification is N1-methylpseudouridine.
34. The method of claim 31 , wherein the mRNA is fully modified with N1-methylpseudouridine.
35. The method of claim 26 , wherein the ORF encodes a signal peptide.
36. The method of claim 35 , wherein the signal peptide is an IL-12B signal peptide comprising the amino acid sequence set forth in amino acids 1 to 22 of SEQ ID NO: 48.
37. The method of claim 26 , wherein [L1] and [L2] are each a Gly/Ser linker.
38. The method of claim 26 , wherein the ORF encodes the amino acid sequence set forth in SEQ ID NO: 249.
39. The method of claim 26 , comprising administering a second mRNA encoding a second polypeptide.
40. The method of claim 39 , wherein the LNP comprises the second mRNA.
41. The method of claim 26 , wherein treating comprising reducing the size of a tumor or inhibiting growth of a tumor in the subject.
42. The method of claim 26 , wherein the LNP is administered intratumorally.
43. The method of claim 26 , wherein the LNP comprises an ionizable amino lipid, a phospholipid, a sterol, and a PEG-modified lipid.
44. The method of claim 43 wherein the LNP comprises a molar ratio of about 20-60% ionizable amino lipid: 5-25% phospholipid: 25-55% sterol: 0.5-15% PEG-modified lipid.