Polynucleotides encoding interleukin-12 (IL12) and uses thereof
The present disclosure relates to polynucleotides comprising an open reading frame of linked nucleosides encoding human interleukin-12 (IL12), functional fragments thereof, and fusion proteins comprising IL12. In some embodiments, the open reading frame is sequence-optimized. In particular embodiments, the disclosure provides sequence-optimized polynucleotides comprising nucleotides encoding the polypeptide sequence of human IL12, or sequences having high sequence identity with those sequence optimized polynucleotides.
1. A method for treating cancer in a subject by inducing an anti-tumor immune response, comprising administering intratumorally to the subject a lipid nanoparticle (LNP) encapsulated messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a fusion protein comprising a human IL-12B polypeptide fused directly or by a linker to a human IL-12A polypeptide, wherein the IL-12B polypeptide comprises the amino acid sequence of SEQ ID NO: 1, wherein the IL-12A polypeptide comprises the amino acid sequence of SEQ ID NO: 3, wherein the LNP comprises an ionizable amino lipid; a phospholipid; a sterol; and a PEG-modified lipid, and wherein the mRNA encoded fusion protein induces an anti-tumor immune response thereby treating cancer.
2. The method of claim 1 , wherein the IL-12B polypeptide is fused directly to the IL-12A polypeptide without a linker.
3. The method of claim 1 , wherein the IL-12B polypeptide is fused directly to the IL-12A polypeptide with a peptide linker.
4. The method of claim 1 , wherein the fusion protein comprises a heterologous signal peptide.
5. The method of claim 1 , wherein the fusion protein comprises a human IL-12B signal peptide or a human IL-12A signal peptide.
6. The method of claim 5 , wherein the human IL-12B signal peptide comprises the amino acid sequence of SEQ ID NO: 45.
7. The method of claim 1 , wherein the ORF comprises from 5′ to 3′ a nucleotide sequence selected from the group consisting of:
(i) a nucleotide sequence encoding the IL-12B polypeptide, a nucleotide sequence encoding a peptide linker, and a nucleotide sequence encoding the IL-12A polypeptide;
(ii) a nucleotide sequence encoding the IL-12B polypeptide, and a nucleotide sequence encoding the IL-12A polypeptide;
(iii) a nucleotide sequence encoding the IL-12A polypeptide, a nucleotide sequence encoding a peptide linker, and a nucleotide sequence encoding the IL-12B polypeptide; and
(iv) a nucleotide sequence encoding the IL-12A polypeptide, and a nucleotide sequence encoding the IL-12B polypeptide.
8. The method of claim 1 , wherein the ORF comprises a nucleotide sequence encoding a signal peptide located at the 5′ terminus of the ORF.
9. The method of claim 1 , wherein the mRNA comprises (i) a 3′ UTR; (ii) a 5′ UTR and (iii) a polyA tail.
10. The method of claim 1 , wherein the mRNA comprises a chemically modified nucleotide.
11. A method for treating cancer in a subject by inducing an anti-tumor immune response, comprising administering intratumorally to the subject a LNP encapsulated mRNA comprising an ORF encoding a fusion protein, wherein the ORF comprises from 5′ to 3′:
(i) a nucleotide sequence encoding a signal peptide;
(ii) a nucleotide sequence encoding a human IL-12B polypeptide comprising the amino acid sequence of SEQ ID NO: 1;
(iii) a nucleotide sequence encoding a peptide linker;
(iv) a nucleotide sequence encoding a human IL-12A polypeptide comprising the amino acid sequence of SEQ ID NO: 3,
wherein the LNP comprises an ionizable amino lipid; a phospholipid; a sterol; and a PEG-modified lipid, and wherein the mRNA encoded fusion protein induces an anti-tumor immune response thereby treating cancer.
12. The method of claim 11 , wherein the signal peptide is selected from: (i) a heterologous signal peptide; (ii) a human IL-12A signal peptide; and (iii) a human IL-12B signal peptide.
13. The method of claim 12 , wherein the human IL-12B signal peptide comprises the amino acid sequence of SEQ ID NO: 45.
14. The method of claim 11 , wherein the mRNA comprises (i) a 3′ UTR; (ii) a 5′ UTR and (iii) a polyA tail.
15. The method of claim 11 , wherein the mRNA comprises a chemically modified nucleotide.
16. A method for treating cancer in a subject by inducing an anti-tumor immune response, comprising administering intratumorally to the subject a LNP encapsulated mRNA comprising an ORF encoding a fusion protein, wherein the ORF comprises from 5′ to 3′:
(i) a nucleotide sequence encoding a signal peptide;
(ii) a nucleotide sequence encoding a human IL-12B polypeptide comprising the amino acid sequence of SEQ ID NO: 1;
(iii) a nucleotide sequence encoding a human IL-12A polypeptide comprising the amino acid sequence of SEQ ID NO: 3,
wherein the LNP comprises an ionizable amino lipid; a phospholipid; a sterol; and a PEG-modified lipid, and wherein the mRNA encoded fusion protein induces an anti-tumor immune response thereby treating cancer.
17. The method of claim 16 , wherein the signal peptide is selected from: (i) a heterologous signal peptide; (ii) a human IL-12A signal peptide; and (iii) a human IL-12B signal peptide.
18. The method of claim 17 , wherein the human IL-12B signal peptide comprises the amino acid sequence of SEQ ID NO: 45.
19. The method of claim 16 , wherein the mRNA comprises (i) a 3′ UTR; (ii) a 5′ UTR and (iii) a polyA tail.
20. The method of claim 16 , wherein the mRNA comprises a chemically modified nucleotide.