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EP-3600372-B1 - SYNTHEKINE COMPOSITIONS AND METHODS OF USE

EP3600372B1EP 3600372 B1EP3600372 B1EP 3600372B1EP-3600372-B1

Inventors

  • GONZALEZ, Ignacio Moraga
  • GARCIA, Kenan Christopher

Dates

Publication Date
20260513
Application Date
20180307

Claims (16)

  1. A synthetic ligand comprising: at least a first and a second binding domain, wherein each binding domain of the synthetic ligand specifically binds to the extracellular domain of a cytokine receptor polypeptide subunit expressed on the surface of a cell, wherein: the first binding domain specifically binds to the extracellular domain of a first cytokine receptor subunit, wherein the first cytokine receptor subunit has an intracellular domain comprising at least one JAK/STAT binding sequence; and the second binding domain specifically binds to the extracellular domain of a second cytokine receptor subunit, wherein the second cytokine receptor subunit has an intracellular domain comprising at least one JAK/STAT binding sequence, wherein the first and second cytokine receptor subunits do not naturally multimerize in response to contacting the cell with a naturally occurring cytokine ligand; wherein contacting the cell expressing the first and second cytokine receptor subunits with the synthetic ligand results in multimerization of the cytokine receptor subunits and activation of a JAK/STAT-mediated signal in the cell; and wherein each binding domain is an antibody derived binding protein.
  2. The synthetic ligand of claim 1, where the cytokine receptors are selected from interleukin 3 receptor alpha (IL-3Rα), beta interleukin 3 receptor (βIL-3R), granulocyte macrophage colony stimulating factor receptor (GM-CSFRα), interleukin 5 receptor alpha (IL-5Rα), ciliary neurotrophic receptor alpha (CNTFRα), cytokine receptor-like factor-1 (CRLF1), leukemia inhibitory factor receptor alpha (LIFRα), glycoprotein 130 (gp130), interleukin 6 receptor alpha (IL-6Rα), interleukin 11 receptor alpha (IL-11Rα), oncostatin M receptor beta subunit (OSMRβ), interleukin 2 receptor alpha (IL-2Rα), interleukin 2 receptor beta (IL-2Rβ), interleukin 2 receptor gamma (IL-2Rγ), interleukin 4 receptor alpha (IL-4Rα), interleukin 7 receptor alpha (IL-7Rα), interleukin 9 receptor alpha (IL-9Rα), interleukin 13 receptor alpha (IL-13Rα), interleukin 15 receptor alpha (IL-15Rα), interleukin 21 receptor alpha (IL-21Rα), interferon alpha receptor 2 (IFNAR2), interleukin 23 receptor (IL-23R), erythropoietin receptor (EpoR), interleukin 12 receptor beta (IL-12Rβ), interferon-alpha/beta receptor alpha chain (IFNAR1), interferon-alpha/beta receptor beta chain (IFNAR2), granulocyte colony stimulating factor receptor (G-CSFR), cluster of differentiation w210 (CDw210), interleukin 10 receptor beta (IL10Rβ), cluster of differentiation 212 (CD212), cluster of differentiation w119 (CDw119), and myeloproliferative leukemia protein receptor (c-MPLR).
  3. The synthetic ligand of claim 1 or claim 2 wherein an antibody derived binding protein comprises a scFv.
  4. The synthetic ligand of claim 3 wherein each antibody derived binding protein comprises a scFv.
  5. The synthetic ligand of claim 1 or claim 2 wherein an antibody derived binding protein comprises a VHH.
  6. The synthetic ligand of claim 5 wherein each antibody derived binding protein comprises a VHH.
  7. The synthetic ligand of any one of claims 1 to 6 wherein the binding affinity of each antibody derived binding protein of the synthetic ligand for the extracellular domain of each receptor subunit is at least 1 x 10 -7 M, optionally at least 1 x 10 -8 M, optionally at least 1 x 10 -9 M, optionally at least 1 x 10 -10 M.
  8. The synthetic ligand of any one of the preceding claims wherein the first and second binding domains are joined by a polypeptide linker of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length.
  9. The synthetic ligand of claim 1, wherein (a) the first binding domain specifically binds to the extracellular domain of IL-4Rα; (b) the second binding domain specifically binds to the extracellular domain of IFNAR2 or IL-2Rβ; (c) binding of the synthetic ligand to the first and the second receptor subunits results in multimerization of the receptor subunits and activation of JAK/STAT intracellular signaling.
  10. The synthetic ligand of any one of claims 1 to 7 wherein the first and the second binding domains are joined by a homo-bifunctional linker or a hetero-bifunctional linker.
  11. The synthetic ligand of claim 10 wherein the linker comprises a succinimidyl group which reacts with a primary amine, and a thiol-reactive maleimide which forms a covalent bond with the thiol of a cysteine residue.
  12. A synthetic ligand of any one of claims 1 to 11 for use in a method of treatment.
  13. A synthetic ligand of any one of claims 1 to 11 for use in a method of treating an inflammatory disease, an autoimmune disease, or cancer.
  14. A pharmaceutical formulation comprising a synthetic ligand of any one of claims 1 to 11.
  15. An in vitro method for selective activation of a non-native combination of cytokine receptor subunits in a cell, the method comprising contacting the cell with a synthetic ligand according to any one of claims 1 to 11, thereby causing multimerization of the cytokine receptor subunits and activation of signalling.
  16. The in vitro method of claim 15, wherein the cell is an immune effector cell.

Description

Cross Reference This application claims benefit of U.S. Provisional Patent Application No. 62/479,993, filed March 31, 2017. BACKGROUND The manipulation of cells, particularly immune cells, to differentiate, develop specialized functions and expand in numbers is of great clinical interest. Many protein factors that affect these activities are known in the art, including in particular cytokines and chemokines. However, these signaling molecules also have pleiotropic effects on cells not targeted for manipulation, and thus methods of selectively activating signaling in a targeted cell population are desirable. Cytokines, chemokines, growth factor agonists and the like activate JAK/STAT; RTK linked; or death domain (TNF super family) receptors by multimerization, e.g. generating homo or hetero-dimers, or higher order oligomers to elicit signaling through intracellular trans-phosphorylation. The identity of the specific receptor chains within a multimer (e.g. dimer or trimer) determines the signaling and functional response. In the case of cytokines, they act as bi-specific ligands to specify which receptors are included in the dimers by forming specific contacts with each of the two receptor extracellular domains, thus acting to bridge or cross-link the dimeric signaling complex. Cytokine receptor dimerization leads to the activation of an intracellular JAK/STAT signaling pathway, comprised of four Janus Kinases (JAK1-3, TYK2) and seven signal transducer and activator of transcription (STAT1- 6) proteins. While the ligands are specific for the extracellular domains of their receptors, the JAK/TYK/STAT signaling modules are found in many combinations in endogenous receptor signaling complexes, and thus are capable of extensive cross-talk. Ligands for RTK receptors (such as EGF, VEGF, etc.) also compel signaling through receptor dimerization, although the molecular mechanisms can be quite distinct from cytokines. In both cases: JAK/STAT cytokines and RTK ligands, their role is to induce a positioning of their specific receptor subunits into dimers such that the intracellular kinases domains are in an orientation and proximity to enable trans-phosphorylation of both the kinases and the receptor intracellular domains. The sequence requirements (i.e. substrate specificity) of these tyrosine kinases can be rather degenerate, raising the possibility that these enzymes can be redirected by alternative receptor dimerizing ligands to phosphorylate receptor substrates other than those they are normally presented with in nature. Given that the ligands determine the composition of the receptor dimers, and the intracellular kinase degeneracy of JAK/TYK and RTK enzymes, the number of cytokine and growth factor receptor dimer pairings that occur in nature only represents a small proportion of the total number of signaling-competent receptor pairings theoretically allowed by the system. For example, the human genome encodes for approximately forty different JAK/STAT cytokine receptors. In principle, approximately 1600 unique homo- and hetero-dimeric cytokine receptor pairs could be generated with the potential to signal through different JAK/TYK/STAT combinations. However, the human genome encodes for less than fifty different cytokine ligands, limiting the scope of cytokine receptor dimers to those that can be assembled by the natural ligands. A similar argument can be made for the RTK family of receptors and ligands. Furthermore, given that is has been shown that Death receptors are capable of signaling as dimers or trimers, this concept can also be extended to this family. The ability to selectively activate signaling pathways of interest is of great interest. The present invention provides compositions and methods for this purpose. SUMMARY The invention is defined by the appended claims. In a first aspect, the invention provides a synthetic ligand comprising: at least a first and a second binding domain, wherein each binding domain of the synthetic ligand specifically binds to the extracellular domain of a cytokine receptor polypeptide subunit expressed on the surface of a cell, wherein: the first binding domain specifically binds to the extracellular domain of a first cytokine receptor subunit, wherein the first cytokine receptor subunit has an intracellular domain comprising at least one JAK/STAT binding sequence; and the second binding domain specifically binds to the extracellular domain of a second cytokine receptor subunit, wherein the second cytokine receptor subunit has an intracellular domain comprising at least one JAK/STAT binding sequence, wherein the first and second cytokine receptor subunits do not naturally multimerize in response to contacting the cell with a naturally occurring cytokine ligand; wherein contacting the cell expressing the first and second cytokine receptor subunits with the synthetic ligand results in multimerization of the cytokine receptor subunits and activation of a JAK/STAT-mediated si