Search

EP-4735483-A2 - IL-2 FUSION PROTEINS

EP4735483A2EP 4735483 A2EP4735483 A2EP 4735483A2EP-4735483-A2

Abstract

Provided herein are novel IL-2 fusion proteins that include an IL-2 moiety attached to an IL-2 masking moiety that includes an IL-13 mutein, an IL-13Ra2 binding mutein, an IL-13Ra2 antibody or antigen-binding fragment thereof, an extracellular domain of CD122, an extracellular domain of CD132, or an extracellular domain of CD25 by a protease sensitive linker (PSL). In some embodiments, the IL-2 fusion proteins include an IL-13 mutein or IL-13Ra2 antibody or antigen-binding fragment thereof that is capable of binding to IL- 13Ra2, and does not bind to IL-13Ro1, and the protease sensitive linker is cleavable by a protease in the tumor microenvironment. Such IL-2 fusion proteins are useful, for example, for the treatment of IL-13Ro2 expressing cancers.

Inventors

  • MERCHANT, FAHAR
  • LIU, QIAN

Assignees

  • Medicenna Therapeutics Inc.

Dates

Publication Date
20260506
Application Date
20240627

Claims (20)

  1. 1 . An IL-2 cytokine fusion protein comprising: a) an IL-2 moiety comprising an IL-2 or an IL-2 mutein, optionally an IL-2 mutein fusion; b) a protease sensitive linker (PSL); and c) an IL-2 masking moiety wherein the PSL attaches the IL-2 masking moiety to the IL-2 moiety.
  2. 2. The IL-2 cytokine fusion protein of claim 1, wherein the masking moiety comprises an IL-13, an IL-13 mutein, an IL-13Ra2 binding mutein, an IL-13Ra2 antibody, or an antigen-binding fragment thereof, wherein the IL-2 masking moiety is capable of binding to IL-13Ra2, and does not bind to IL-13Ra1 .
  3. 3. The IL-2 cytokine fusion protein of claim 1 or 2, wherein the masking moiety comprises an IL-13 mutein that has the amino acid sequence of any one of SEQ ID N0s:200-241 .
  4. 4. The IL-2 cytokine fusion protein of any one of claims 1 to 3, wherein the masking moiety comprises an IL-13 mutein that comprises the following amino acid substitutions compared to a wild-type human IL-13 (SEQ ID NQ:200): a) L10H, E15R, R86T, D87G, T88R, R108K, Q111 , b) L10H, E15R, R86T, D87G, T88R, R108K, R111 , c) L10H, R86T, D87G, T88R, and R108K, Q111, or d) L10H, R86T, D87G, T88R, and R108K, R111.
  5. 5. The IL-2 cytokine fusion protein of any one of claims 1 to 4, wherein the masking moiety comprises an IL-13 mutein that has the amino acid sequence of SEQ ID NO:216 or SEQ ID NO:228.
  6. 6. The IL-2 cytokine fusion protein of any one of claims 1 to 5, wherein the masking moiety further comprises an extracellular domain of CD122, an extracellular domain of CD132, or an extracellular domain of CD25.
  7. 7. The IL-2 cytokine fusion protein of any one of claims 1 to 6, wherein the IL-2 moiety comprises an IL-2 mutein having the amino acid sequence of any one of SEQ ID NOs:5-24 and 105.
  8. 8. The IL-2 cytokine fusion protein of claim 7, wherein the IL-2 mutein has the amino acid sequence of SEQ ID N0:5 or SEQ ID N0:9.
  9. 9. The IL-2 cytokine fusion protein of claim 8, wherein the IL-2 mutein further comprises a T3A and a C125S amino acid substitution.
  10. 10. The IL-2 cytokine fusion protein of any one of claims 1 to 9, wherein the IL-2 moiety further comprises an albumin, an Fc domain, or an antibody attached to the IL-2 or IL-2 mutein.
  11. 11 . The IL-2 cytokine fusion protein of claim 10, wherein the albumin is a human albumin, optionally wherein the human albumin is recombinant human albumin.
  12. 12. The IL-2 cytokine fusion protein of claim 10, wherein the antibody is engineered as “knob-in-hole” (KiH) with mutations in constant region 3 (CH3) in its heavy chains.
  13. 13. The IL-2 cytokine fusion protein of claim 12, wherein the IL-2 moiety comprises an IL-2 x antibody (KiH) fusion protein comprising: a) a first polypeptide comprising an IL-2 or an IL-2 mutein, optionally an IL-2 fusion attached to the second and third constant regions (CH2 and CH3) of the “knob” heavy chain of the antibody (KiH); b) a second polypeptide comprising the “hole” heavy chain of the antibody (KiH); and c) a third polypeptide comprising the light chain of the antibody (KiH).
  14. 14. The IL-2 cytokine fusion protein of claim 12, wherein the IL-2 moiety comprises an IL-2 x antibody (KiH) fusion protein comprising: a) a first polypeptide comprising the “knob” heavy chain of the antibody (KiH) attached to an IL-2 or an IL-2 mutein, optionally an IL-2 fusion; b) a second polypeptide comprising the “hole” heavy chain of the antibody (KiH); c) a third polypeptide comprising the light chain of the antibody (KiH).
  15. 15. The IL-2 cytokine fusion protein of claim 12, wherein the IL-2 moiety comprises an IL-2 x antibody (KiH) fusion protein comprising: a) a first polypeptide comprising the “knob” heavy chain of the antibody (KiH); b) a second polypeptide comprising the “hole” heavy chain of the antibody (KiH) attached to an IL-2 or an IL-2 mutein or an IL-2 mutein fusion; c) a third polypeptide comprising the light chain of the antibody (KiH).
  16. 16 The IL-2 cytokine fusion protein of any one of claims 12 to 15, wherein the IL-2 masking moiety is attached to the IL-2 or the IL-2 mutein.
  17. 17 The IL-2 cytokine fusion protein of any one of claims 12 to 16, wherein the IL-2 masking moiety is attached to the antibody heavy chain that is not attached to the IL-2 or the IL-2 mutein.
  18. 18. The IL-2 cytokine fusion protein of any one of claims 12 to 17, wherein the antibody (KiH) is anti-PD1 (KiH).
  19. 19. The IL-2 cytokine fusion protein of any one of claims 12 to 18, wherein the IL-2 mutein has an amino acid sequence of any one of SEQ ID NOs: 5-24 and 105.
  20. 20. The IL-2 cytokine fusion protein of claim 19, wherein the IL-2 mutein further comprises a T3A and a C125S amino acid substitution.

Description

IL-2 FUSION PROTEINS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority under 35 U.S.C. §119 to U.S. Patent Application Nos. 63/510,580, filed on June 27, 2023, 63/594,694, filed on October 31 , 2023, 63/595,044, filed on November 1 , 2023, 63/595,271 , filed on November 1, 2023, and 63/575,960, filed on April 8, 2024, all of which are expressly incorporated herein by reference in their entireties). BACKGROUND [0002] Interleukin 2 (IL-2) is a pluripotent cytokine produced primarily by activated CD4+ T cells, which plays a crucial role in producing a normal immune response. IL-2 promotes proliferation and expansion of activated T lymphocytes, potentiates B cell growth, and activates monocytes and natural killer cells. It was by virtue of these activities that IL-2 was tested and is used as an approved treatment of cancer (aldesleukin, Proleukin®). In eukaryotic cells, human IL-2 is synthesized as a precursor polypeptide of 153 amino acids, from which 20 amino acids are removed to generate mature secreted IL-2 (Taniguchi 1983). Recombinant human IL-2 has been produced in E. coli (Rosenberg 1984), in insect cells (Smith 1985) and in mammalian COS cells (Taniguchi 1983). [0003] lnterleukin-2 (IL-2) is a four a-helical bundle type I cytokine first identified as a T cell growth factor (Morgan et al., Science 193: 1007 (1976)) but subsequently shown to have broad actions. IL-2 promotes CD4+T helper differentiation (Zhu et al., Annual review of immunology 28: 445 (2010); Liao et al., Nat Immunol 9: 1288 (2008); and Liao et al , Nat Immunol 12: 551 (2011)) and the development of regulatory T (Treg) cells (Cheng et al., Immunol Rev 241 : 63 (2011)), induces natural killer and cytotoxic CD8+T cells (Liao et al., Immunity 38: 13 (2013)), and mediates activation-induced cell death (AICD) (Lenardo et al., Nature 353: 858 (1991)). [0004] IL-2 works by interacting with three different receptors: the interleukin 2 receptor alpha (I L-2Ra; CD25), the interleukin 2 receptor beta (IL-2RP; CD122), and the interleukin 2 receptor gamma (IL-2Ry;CD132; common gamma chain). The first receptor to be identified was the IL-2Ra, which is a 55 kD polypeptide (p55) that appears upon T cell activation and was originally called Tac (for T activation) antigen. The IL-2Ra binds IL-2 with a Kd of approximately 10'8 M and is also known as the “high affinity” IL-2 receptor. Binding of IL-2 to cells expressing only the IL-2Ra does not lead to any detectable biologic response. In most circumstances, IL-2 works through three different receptors: the IL-2Rct, the IL-2Rp, and the IL-2Ry. Most cells, such as resting T cells, are not responsive to IL-2 since they only express the IL-2RJ3, and the IL-2Ry, which have low affinity for IL-2. Upon stimulation, resting T cells express the relatively high affinity IL-2 receptor IL-2Ra. Binding of IL-2 to the IL-2Ra causes this receptor to sequentially engage the IL-2Rp, and the IL-2Ry, bringing about T cell activation. IL-2 “superkines” with augmented action due to enhanced binding affinity for IL-2R0 were previously developed (Levin et al., Nature 484: 529 (2012)) [0005] Despite the great potential of IL-2 for use in cancer therapies, its clinical application remains relatively restricted due in part to the severe toxicity associated with IL-2 administered at high doses. As IL-2 has a short serum half-life of several minutes, high doses of IL-2 are typically needed to achieve an optimal immunomodulatory effect. Such high doses, however, inevitably result in severe toxicities, including vascular leak syndrome (VLS), pulmonary edema, hypotension, and heart toxicities. Thus, there remains a need for novel effective IL-2 cancer therapies that minimize IL-2 associated toxicity. BRIEF SUMMARY [0006] Provided herein are novel IL-2 fusion proteins that include an IL-2 moiety attached to at least one IL-2 masking moiety comprising an IL-13 mutein, an IL-13Ra2 binding mutein or an IL-13Ra2 antibody or antigenbinding fragment thereof by a protease sensitive linker (PSL). In embodiments, the IL-2 fusion protein includes at least one IL-13 mutein, an IL-13Ra2 binding mutein or an IL-13Ra2 antibody or antigen-binding fragment thereof that is capable of binding to IL-13Ra2, and does not bind to IL-13Ro1 , and the protease sensitive linker is cleavable by a protease in the tumor microenvironment. When the IL-13 mutein or IL-13Ra2 antibody or antigen-binding fragment thereof is attached to the IL-2 by the PSL, the IL-13 mutein or IL-13Ra2 antibody or antigen-binding fragment thereof of the IL-2 fusion proteins described herein binds to and masks the activity of the IL-2 moiety. In addition, the IL-13 mutein or I L-13Ra2 antibody or antigen-binding fragment thereof allows the subject IL-2 fusion protein to bind to IL-13Ra2 expressing tumors. Once localized to the tumor microenvironment, the PSL of the IL-2 fusion protein undergoes proteolytic cleavage, thereby releasing the IL- 13 mutein or IL-1