Polynucleotides encoding interleukin-12 (IL12) and uses thereof

Patent metadata

Field	Value
Publication number	US-11000573-B2
Application number	US-202016842300-A
Country	US
Kind code	B2
Filing date	Apr 7, 2020
Priority date	May 18, 2016
Publication date	May 11, 2021
Grant date	May 11, 2021

Abstract

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.

First claim

What is claimed is:

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.

Assignees

• Modernatx Inc

Inventors

• Frederick Joshua
• Hewitt Susannah
• Bai Ailin
• Hoge Stephen
• Presnyak Vladimir
• Mcfadyen Iain
• Benenato Kerry
• Kumarasinghe Ellalahewage Sathyajith

Classifications

• C12N15/62

  DNA sequences coding for fusion proteins · CPC title

• C07K14/5434

  IL-12 · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• A61K48/005

  characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered · CPC title

• A61K48/0025

  wherein the non-active part clearly interacts with the delivered nucleic acid · CPC title