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EP-4735473-A1 - IL-12 STABILITY VARIANTS

EP4735473A1EP 4735473 A1EP4735473 A1EP 4735473A1EP-4735473-A1

Abstract

The present disclosure provides IL-12 stability variants, nucleic acids encoding the variants, vectors comprising the nucleic acids, pharmaceutical compositions comprising the variants, therapeutic cells expressing the variants, and methods or uses of the same for the treatment or prevention of a disease or disorder.

Inventors

  • BOLDAJIPOUR, Bijan A.
  • BOYKEN, Scott Edward
  • DAVENPORT, Thaddeus M.
  • HUANG, SZU-HAN
  • LAJOIE, Marc Joseph
  • MOFFETT, Howell Franklin
  • SONG, YUN
  • WEITZNER, Brian D.
  • CASSEREAU, Luke

Assignees

  • Lyell Immunopharma, Inc.

Dates

Publication Date
20260506
Application Date
20240627

Claims (20)

  1. CLAIMS 1. An IL-12 variant comprising: a p40 subunit sequence with an amino acid sequence at least 90% identical to SEQ ID NO:1, and a p35 subunit sequence with an amino acid sequence at least 90% identical to SEQ ID NO:2, wherein the IL-12 variant lacks a native disulfide bond between the p40 subunit sequence and the p35 subunit sequence.
  2. 2. The IL-12 variant of claim 1, wherein: the p40 subunit sequence comprises a mutation removing a cysteine residue at a position corresponding to C177 of SEQ ID NO:1, optionally wherein the mutation is a C-to-S mutation; and/or the p35 subunit sequence comprises a mutation removing a cysteine residue at a position corresponding to C74 of SEQ ID NO:2, optionally wherein the mutation is a C-to-S mutation.
  3. 3. The IL-12 variant of claim 1 or 2, wherein the p35 subunit sequence comprises one or more of: a mutation at a position corresponding to R181 of SEQ ID NO:2, optionally a R-to-A mutation, a mutation at a position corresponding to R183 of SEQ ID NO:2, a mutation at a position corresponding to V185 of SEQ ID NO:2, optionally a V-to-A mutation, and a mutation at a position corresponding to R189 of SEQ ID NO:2, optionally a R-to-K mutation.
  4. 4. The IL-12 variant of claim 2 or 3, wherein the p40 subunit sequence comprises a substitution to a C at a position corresponding to A179 of SEQ ID NO:1, and the p35 subunit sequence comprises a substitution to a C at a position corresponding
  5. 5. The IL-12 variant of claim 1, wherein the p40 subunit sequence comprises SEQ ID NO:12, and the p35 subunit sequence comprises SEQ ID NO:13.
  6. 6. The IL-12 variant of claim 1, wherein the p40 subunit sequence comprises SEQ ID NO:12, and the p35 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:13 but for an R181A mutation.
  7. 7. The IL-12 variant of claim 1, wherein the p40 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:12 but for an A179C mutation, and the p35 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:13 but for an I52C mutation.
  8. 8. The IL-12 variant of claim 1, wherein the p40 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:12 but for an A179C mutation, and the p35 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:13 but for an I52C mutation and an R181A mutation.
  9. 9. The IL-12 variant of claim 1, wherein the p40 subunit sequence comprises SEQ ID NO:10, and the p35 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:11 but for a mutation at position C70, optionally a C70S mutation.
  10. 10. The IL-12 variant of claim 1, wherein the p40 subunit sequence comprises SEQ ID NO:10, and the p35 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:11 but for a mutation at position C70, optionally a C70S mutation and a mutation at position S177, optionally a S177R mutation.
  11. 11. The IL-12 variant of claim 1, wherein the p40 subunit sequence comprises SEQ ID NO:10, and the p35 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:11 but for a mutation at position N184, optionally a N184A mutation.
  12. 12. The IL-12 variant of any one of claims 1-11, wherein the IL-12 variant is a fusion protein in which the p40 subunit sequence and the p35 subunit sequence are linked by a cleavable peptide linker.
  13. 13. The IL-12 variant of claim 12, wherein the cleavable peptide linker comprises a furin- cleavage site, optionally wherein the cleavable linker comprises SEQ ID NO:5.
  14. 14. The IL-12 variant of claims 13, wherein the IL-12 variant comprises an amino acid sequence at least 90% sequence identical to amino acid residues 22-533 of SEQ ID NO:6.
  15. 15. An isolated nucleic acid molecule or isolated nucleic acid molecules encoding the IL- 12 variant of any one of claims 1-14, optionally wherein the nucleic acid molecule(s) are mRNA or DNA.
  16. 16. An expression vector or expression vectors comprising the isolated nucleic acid molecule(s) of claim 15.
  17. 17. The expression vector(s) of claim 16, wherein the expression vector(s) is/are selected from lentiviral vectors, adenoviral vectors, and adeno-associated viral (AAV) vectors.
  18. 18. The expression vector(s) of claim 16 or claim 17, further comprising a tissue-specific or inducible promoter.
  19. 19. The expression vector(s) of claim 18, wherein the inducible promoter comprises a combination of a nuclear factor of activated T cells (NFAT) domain and a human beta-globin (hBG) minimal promoter domain, optionally wherein the inducible promoter comprises SEQ ID NO:14.
  20. 20. A mammalian cell comprising the nucleic acid molecule(s) of claim 15 or the expression vector(s) of any one of claims 16-19.

Description

IL-12 STABILITY VARIANTS CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims priority from U.S. Provisional Application 63/510,579, filed June 27, 2023, the content of which is incorporated by reference herein in its entirety. SEQUENCE LISTING [0002] The instant application contains a Sequence Listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 27, 2024, is named 026225_WO032_SL.xml and is 36,143 bytes in size. BACKGROUND OF THE INVENTION [0003] Interleukin-12 (IL-12) is a pleiotropic proinflammatory cytokine that can stimulate the proliferation of natural killer (NK) and T cells and drive secretion of IFN-γ and TNF-α (Tugues et al., Cell Death Differ. (2015) 22(2):237-46). IL-12 is a heterodimer composed of two subunits, p35 (a.k.a. IL-12A) and p40 (aka. IL-12B). The heterodimer is also called p70. IL-12 binds to IL-12 receptor (IL-12R), which in turn is composed of two subunits, IL- 12Rβ1 and IL-12Rβ2. IL-12Rβ1 primarily binds the IL-12 p40 subunit, while IL-12Rβ2 primarily binds the IL-12 p35 subunit (Presky et al., J Immunol. (1998) 160(5):2174-9). There is no complete co-crystal structure of IL-12 bound to IL-12R. Simultaneous binding of IL-12 to both IL-12Rβ1 and IL-12Rβ2 may be required to drive intracellular signaling (Presky et al., PNAS (1996) 93(24):14002-7; Presky et al., Ann NY Acad Sci. (1996) 795:390- 3; Robinson, Cytokine (2015) 71(2):348-59). Recent cryogenic electron microscopy studies produced a structure of IL-12 in complex with IL-12R, but they were unable to solve the detailed interactions between p35 and IL-12Rβ2 (Glassman et al., Cell (2021) 184:983-99). [0004] IL-12 is a key cytokine in the initiation of a Th1 response and has been explored as a potential therapy to treat cancer (Lasek et al., Cancer Immunol Immunother. (2014) 63(5):419-35). But due to significant systemic toxicity, the approaches to IL-12-based immunotherapy have been focused on direct injections of IL-12 to tumor sites and on fusions of IL-12 to tumor-targeting moieties. Some researchers have attempted to use a cell-based approach to deliver IL-12 in which cells engineered to express IL-12 are administered in vivo (Wei et al., J Cell Mol Med. (2013) 17(11):1465-74; Zhang et al., Clin Cancer Res. (2015) 21(10):2278-88). However, this approach is also challenging because IL-12 activates proinflammatory signaling even at very low concentrations in circulation. Furthermore, efficacy is still a critical consideration as the immunosuppressive TME can restrain cell activity even in the presence of IL-12 (Lasek, supra). [0005] Thus, there is a long-felt and unmet need for a safe and effective form of IL-12 for stimulation of the immune system, either as a monotherapy or as a combination therapy. SUMMARY OF THE INVENTION [0006] The present disclosure provides an IL-12 variant comprising: a p40 subunit sequence with an amino acid sequence at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO:1, and a p35 subunit sequence with an amino acid sequence at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to SEQ ID NO:2, wherein the IL-12 variant lacks a native disulfide bond between the p40 subunit sequence and the p35 subunit sequence. [0007] In some embodiments, the IL-12 p40 subunit sequence comprises a mutation removing a cysteine residue at a position corresponding to C177 of SEQ ID NO:1, optionally wherein the mutation is a C-to-S mutation; and/or the p35 subunit sequence comprises a mutation removing a cysteine residue at a position corresponding to C74 of SEQ ID NO:2, optionally wherein the mutation is a C-to-S mutation. In some embodiments, the IL-12 variant comprises mutations at both positions (e.g., both to a serine). [0008] In some embodiments, the IL-12 variant p35 subunit sequence comprises one or more of: a mutation at a position corresponding to R181 of SEQ ID NO:2 (e.g., a R-to-A mutation); a mutation at a position corresponding to R183 of SEQ ID NO:2; a mutation at a position corresponding to V185 of SEQ ID NO:2 (e.g., a V-to-A mutation); and a mutation at a position corresponding to R189 of SEQ ID NO:2 (e.g., a R-to-K mutation). [0009] In some embodiments, the IL-12 variant p40 subunit sequence further comprises a substitution to a C at a position corresponding to A179 of SEQ ID NO:1, and the p35 subunit sequence further comprises a substitution to a C at a position corresponding to I52 of SEQ ID NO:2. [0010] In some embodiments, the IL-12 variant p40 subunit sequence comprises SEQ ID NO:12, and the p35 subunit sequence comprises SEQ ID NO:13. [0011] In some embodiments, the IL-12 variant p40 subunit sequence comprises SEQ ID NO:12, and the p35 subunit sequence comprises an amino acid sequence identical to SEQ ID NO:13 but for an R181A mutation. [0012] In some embodiments, the IL-12 variant p40 subunit sequence comprises an amin