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KR-102962557-B1 - New IL-15 Prodrug and How to Use It

KR102962557B1KR 102962557 B1KR102962557 B1KR 102962557B1KR-102962557-B1

Abstract

The present invention provides an IL-15 cytokine prodrug and a method for manufacturing and using the same.

Inventors

  • 루, 유에펑
  • 유, 춘샤오
  • 루, 지안-펑

Assignees

  • 에스크진 파마, 아이엔씨.

Dates

Publication Date
20260508
Application Date
20200612
Priority Date
20190612

Claims (20)

  1. It includes an IL-15 cytokine moiety (A), a masking moiety (M), a carrier moiety (C), and a sushi domain (S), and The masking moiety binds to the IL-15 cytokine moiety and inhibits the biological activity of the IL-15 cytokine moiety, and the masking moiety comprises an anti-IL-15 antibody fragment, or an IL-2Rβ or IL-2Rγ extracellular domain (ECD), and The carrier moiety comprises an antibody Fc domain, or an antibody or its antigen-binding fragment, and The masking moiety and the receiving domain are fused to the carrier moiety, respectively, and IL-15 cytokine moiety fuses into the cyclic domain, Prodrug
  2. In paragraph 1, The masking moiety is fused to the carrier moiety through the first peptide linker, and The sucrose domain is fused to the carrier moiety through a second peptide linker, and An IL-15 cytokine moiety is fused to a subdomain via a third peptide linker, and at least one of the three peptide linkers is cleavable, Prodrug.
  3. In paragraph 2, A prodrug in which the third peptide linker is at least 15, 20, 25, or 30 amino acid lengths, and optionally the third peptide linker comprises SEQ ID NO. 139 or 140.
  4. It includes an IL-15 cytokine moiety (A), a masking moiety (M), a carrier moiety (C), and a receiving domain (S), and The masking moiety binds to the IL-15 cytokine moiety and inhibits the biological activity of the IL-15 cytokine moiety, and the masking moiety comprises an anti-IL-15 antibody fragment, or an IL-2Rβ or IL-2Rγ extracellular domain (ECD), and The carrier moiety comprises an antibody Fc domain, or an antibody or its antigen-binding fragment, and The IL-15 cytokine moiety and the transport domain are fused to the carrier moiety, respectively, and Masking moiety merging into the frequent domain, Prodrug.
  5. In paragraph 4, The IL-15 cytokine moiety is fused to the carrier moiety through the first peptide linker, and The sucrose domain is fused to the carrier moiety through a second peptide linker, and A masking moiety is fused to the subdomain via a third peptide linker, and optionally at least one of the three peptide linkers is cleavable, Prodrug.
  6. In paragraph 1, A prodrug containing the ECD of human IL-2Rβ, with a masking moiety.
  7. In paragraph 1, A prodrug containing the ECD of human IL-2Rγ, with a masking moiety.
  8. In Paragraph 7, A prodrug in which the ECD of human IL-2Rγ comprises SEQ ID NO. 6 or an amino acid sequence that is at least 90% identical thereto.
  9. In paragraph 6, A prodrug in which the ECD of human IL-2Rβ comprises SEQ ID NO. 3, 4, or 5, or an amino acid sequence that is at least 90% identical thereto.
  10. In paragraph 1, A prodrug comprising an antibody fragment in which a masking moiety binds to an IL-15 cytokine moiety.
  11. It comprises an IL-15 cytokine moiety (A), a masking moiety (M), a carrier moiety (C), and optionally a receiving domain (S), and The masking moiety comprises an antibody fragment that binds to the IL-15 cytokine moiety and inhibits the biological activity of the IL-15 cytokine moiety, and The carrier moiety comprises an antibody Fc domain, or an antibody or its antigen-binding fragment, and A masking moiety fused to a carrier moiety, IL-15 cytokine moiety, or a receiving domain via a peptide linker, Prodrug.
  12. In Paragraph 11, A prodrug comprising antibody fragments including heavy chain CDR (HCDR)1 containing SEQ ID NO 100, heavy chain CDR (HCDR)2 containing SEQ ID NO 101, heavy chain CDR (HCDR)3 containing SEQ ID NO 102 or 106, light chain CDR (LCDR)1 containing SEQ ID NO 103, light chain CDR (LCDR)2 containing SEQ ID NO 104, and light chain CDR (LCDR)3 containing SEQ ID NO 105.
  13. In Paragraph 11, A prodrug comprising an antibody fragment comprising (i) a heavy chain variable domain having an amino acid sequence identical to SEQ ID NO. 107 or at least 95% identical thereto, and a light chain variable domain having an amino acid sequence identical to SEQ ID NO. 108 or 123 or at least 95% identical thereto; (ii) SEQ ID NO. 109; (iii) SEQ ID NO. 110; or (iv) SEQ ID NO. 124.
  14. In Paragraph 12, Prodrug in which the Cys residue of heavy chain CDR3 is mutated to Ser, Thr, Met, Ala, Gly, Asn, or Gln.
  15. In paragraph 1, A prodrug in which the masking moiety does not interfere with or has minimal effect on the binding of the IL-15 cytokine moiety to IL-15Rα.
  16. In paragraph 1, A prodrug comprising a human IL-15 polypeptide having an IL-15 cytokine moiety of SEQ ID NO. 2.
  17. In paragraph 1, IL-15 cytokine moiety for SEQ ID NO. 2 N1A, N1D, N4A, N4D, I6T, S7A, D8A, D8T, D8E, D8N, K10A, K10D, K11A, K11D, E46, V49, L45, S51, L52, D61A, D61N, T62L, T62A, E64A, E64L, E64K, E64Q, N65A, N65L, N65D, L66D, L66E, I67D, I67E, I68S, I68E, L69S, L69E, N72A, N72D, V63E, V63D, L66E, L66D, I67E, I67D, Q108E, N112A, A prodrug comprising one or more mutations selected from N1D/D61N, N1D/E64Q, N4D/D61N, N4D/E64Q, D8N/D61N, D8N/E64Q, D61N/E64Q, E64Q/Q108E, N1D/N4D/D8N, D61N/E64Q/N65D, N1D/D61N/E64Q, N1D/Q108E, N1D/D61N/E64Q/Q108E, N4D/D61N/E64Q/Q108E and D30N/E64Q/N65D, which is a human IL-15 polypeptide.
  18. In paragraph 1, A prodrug in which the carrier moiety is a human antibody IgG 1 Fc domain containing mutants L234A and L235A ("LALA") (EU numbering) or a human IgG 1 antibody.
  19. In paragraph 1, A prodrug in which a carrier moiety is an antibody Fc domain containing a knob-into-hole mutation or an antibody, and an IL-15 cytokine moiety and a masking moiety are fused to different polypeptide chains of the antibody Fc domain or different heavy chains of the antibody.
  20. In Paragraph 19, The nop-into-hole mutation includes the T366Y "nop" mutation on the polypeptide chain of the Fc domain or the heavy chain of the antibody, and the Y407T "hole" mutation on another polypeptide of the Fc domain or another heavy chain of the antibody, or Nop-into-hole mutations including Y349C and/or T366W mutations in the CH3 domain of the "nop chain" and E356C, T366S, L368A and/or Y407V mutations (EU numbering) in the CH3 domain of the "hole chain", Prodrug.

Description

New IL-15 Prodrug and How to Use It Cross-reference regarding related applications This application claims priority to U.S. Provisional Application No. 62/860,635 filed June 12, 2019; No. 62/888,444 filed August 17, 2019; No. 62/891,190 filed August 23, 2019; No. 62/959,973 filed January 11, 2020; and No. 63/029,473 filed May 23, 2020. The disclosures of the aforementioned priority applications are incorporated herein by reference in their entirety. Sequence list The present application comprises a list of sequences submitted electronically in ASCII format, the entirety of which is incorporated herein by reference. The said ASCII copy created on June 8, 2020, is named 025471_WO004_SL.txt and has a size of 359,580 bytes. Interleukin-15 (IL-15) is a cytokine with a structure similar to IL-2. IL-15 is secreted by mononuclear phagocytes and other immune cells after viral infection. IL-15 induces the proliferation of natural killer (NK) cells and other cells of the immune system and is involved in the death of virus-infected cells and cancer cells. Like IL-2, IL-15 binds to the IL-2 receptor (IL-2R) β/γ complex, an intermediate affinity receptor, with a K D of approximately 1 nM (Giri et al., EMBO J. (1994) 13:2822-30). IL-15 binds to the IL-15 receptor (IL-15R) α with a higher affinity (K D = ~0.05 nM). IL-15Rα can form an IL-15-specific, functional high-affinity receptor (αβγ) by associating with the IL-2Rβ/γ complex (Minami et al., Annu Rev Immunol. (1993) 11:245-67; Giri et al., J Leukoc Biol. (1995) 5745:763-6; and Lehours et al., Eur Cytokine Netw. (2000) 11:207-15). The extracellular domain of IL-15Rα contains a Sushi domain, which is a common motif in protein-protein interactions. It has been shown that the N-terminal fragment of IL-15Rα with the first 65 amino acids is partially active, while the fragment with the first 85 amino acids is fully functional (Wei et al., J Immunol. (2001) 167(1):277-82). Mutations of IL-15 have been performed to study the interaction of IL-15 with its receptors. For example, D8 and Q108 have been shown to be involved in the binding of IL-15 to IL-2Rβ and γ subunits, respectively (Pettit et al., J Biol Chem. (1997) 272: 2312-18). Additional mutations of IL-15, including those at IL-15 residues L45, Q48, S51, L52, E64, N65, I68, and L69, have been disclosed (U.S. Patent No. 7,858,081), which are involved in the binding of IL-15 to IL-15Rα or IL-2Rβ. IL-15 mutant proteins with mutations E64K, N65K, N65D, L66D, L66E, I67D, I67E, or I68D were shown to have reduced biological activity in cell-based analyses (Zhu et al., J Immnol. (2009) 183(6):3598; and WO2005/085282A1). Mutations targeting IL-15 interactions with IL-15Rα have also been reported. For example, E46, V49, L45, S51, and L52 were shown to be involved in IL-15Rα binding (Bernard et al., J Biol Chem. (2004) 279:24313-22). E46 appears to be particularly decisive, as the substitution of its acidic side chain with a basic side chain (E46K) causes a complete loss of IL-15 binding to IL-15Rα and biological activity. Unfortunately, the adverse effects of recent IL-15 drug candidates are significant and limit the dosage of these drugs. Furthermore, the activation of T, NK, and other immune cells by these drug candidates is not site-specific. Additionally, despite a significantly reduced affinity for the IL-15/2 receptor, it has been shown to be a "PK sinker" for IL-15 mutant proteins. Moreover, there are many difficulties in the development of IL-15-based protein therapeutics. All of the above highlights the need for the development of improved IL-15-based therapeutics. The present invention provides a prodrug comprising an IL-15 cytokine moiety (A), a masking moiety (M), a carrier moiety (C), and a receiving domain (S), wherein the masking moiety binds to the IL-15 cytokine moiety and inhibits the biological activity of the IL-15 cytokine moiety, the masking moiety is fused to the carrier moiety, the receiving domain is fused to the carrier moiety, and the IL-15 cytokine moiety is fused to the receiving domain. In some embodiments, the masking moiety is fused to the carrier moiety via a first peptide linker, the receiving domain is fused to the carrier moiety via a second peptide linker, the IL-15 cytokine moiety is fused to the receiving domain via a third peptide linker, and at least one of the three peptide linkers (e.g., one, two, or three) is cleavable. In some embodiments, at least one of the three peptide linkers (e.g., one, two, or three) is non-cleavable. In some embodiments, all of the peptide linkers are non-cleavable. In a particular embodiment, the third peptide linker is at least 15, 20, 25, or 30 amino acid lengths (e.g., 15-50 or 15-100 amino acid lengths), and optionally, the third peptide linker comprises SEQ ID NO. 139 or 140. The present invention also provides a prodrug comprising an IL-15 cytokine moiety (A), a masking moiety (M), a carrier moiety (C), and a receiving domain (S), wherein the masking moie