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EP-4739334-A1 - CELL SIGNALING COMPLEXES AND USES THEREOF

EP4739334A1EP 4739334 A1EP4739334 A1EP 4739334A1EP-4739334-A1

Abstract

Disclosed herein are cell signaling complexes such as chimeric cytokine complexes and uses thereof. In some embodiments, a chimeric cytokine complex comprises a protein cage polypeptide, a plurality of engineered Fc antibody domains bound to the protein cage polypeptide, and one or more cytokines linked to each of the plurality of engineered Fc antibody domains. Also disclosed are Fc-cytokine complexes and uses thereof. In some embodiments, an Fc-cytokine complex comprises an engineered Fc antibody domain and one or more cytokines linked to the engineered Fc antibody domain.

Inventors

  • HODGE, Curtis Daniel
  • IMAM, Zachary Ibrahim
  • HAINES, Luke Garland

Assignees

  • Nanotein Technologies, Inc.

Dates

Publication Date
20260513
Application Date
20240531

Claims (20)

  1. 1. A chimeric cytokine complex, comprising: a protein cage polypeptide; a plurality of engineered Fc antibody domains bound to the protein cage polypeptide; and one or more cytokines linked to each of the plurality of engineered Fc antibody domains.
  2. 2. The chimeric cytokine complex of claim 1, wherein the cytokines are interleukins.
  3. 3. The chimeric cytokine complex of claim 2, wherein the interleukins are at least one of interleukin-2s (IL-2s), IL-7s, and IL-15s.
  4. 4. The chimeric cytokine complex of claim 1 , wherein the plurality of engineered Fc antibody domains comprise between six and twelve engineered Fc antibody domains bound to the protein cage polypeptide.
  5. 5. The chimeric cytokine complex of claim 4, wherein between 12 and 24 cytokines are linked to the plurality of engineered Fc antibody domains in total.
  6. 6. The chimeric cytokine complex of claim 1 , wherein each of the one or more cytokines is linked to one of the engineered Fc antibody domains via an engineered metalloprotease-resistant linker sequence.
  7. 7. The chimeric cytokine complex of claim 6, wherein the engineered metalloprotease- resistant linker sequence is between 7 to 13 amino acid residues in length.
  8. 8. The chimeric cytokine complex of claim 1, wherein at least one of the engineered Fc antibody domains is C-terminally linked to an N-terminus of at least one of the cytokines.
  9. 9. The chimeric cytokine complex of claim 8, wherein one of the cytokines is interleukin- 2 (IL-2), wherein at least one of the engineered Fc antibody domains is linked to the IL- 2 via an engineered metalloprotease-resistant linker sequence comprising the amino acid sequence SLSPGKAPTS (SEQ ID NO: 20).
  10. 10. The chimeric cytokine complex of claim 8, wherein one of the cytokines is interleukin- 7 (IL-7), wherein at least one of the engineered Fc antibody domains is linked to the IL- 7 via an engineered metalloprotease-resistant linker sequence comprising the amino acid sequence SLSPGKDCDIEGK (SEQ ID NO:21).
  11. 1 1. The chimeric cytokine complex of claim 8, wherein one of the cytokines is interleukin- 15 (IL- 15), wherein at least one of the engineered Fc antibody domains is linked to the IL- 15 via an engineered metalloprotease-resistant linker sequence comprising the amino acid sequence SLSPGKN (SEQ ID NO:22).
  12. 12. The chimeric cytokine complex of claim 1, wherein at least one of the engineered Fc antibody domains is N-terminally linked to a C -terminus of at least one of the cytokines.
  13. 13. The chimeric cytokine complex of claim 12, wherein one of the cytokines is interleukin-2 (IL-2), wherein at least one of the engineered Fc antibody domains is linked to the IL-2 via an engineered metalloprotease-resistant linker sequence comprising the amino acid sequence TPKSCDKTHT (SEQ ID NO:23).
  14. 14. The chimeric cytokine complex of claim 12, wherein one of the cytokines is interleukin-7 (IL-7), wherein at least one of the engineered Fc antibody domains is linked to the IL-7 via an engineered metalloprotease-resistant linker sequence comprising the amino acid sequence HPKSCDKTHT (SEQ ID NO:24).
  15. 15. The chimeric cytokine complex of claim 12, wherein one of the cytokines is interleukin- 15 (IL- 15), wherein at least one of the engineered Fc antibody domains is linked to the IL- 15 via an engineered metalloprotease-resistant linker sequence comprising the amino acid sequence TSPKSCDKTHT (SEQ ID NO:25).
  16. 16. The chimeric cytokine complex of claim 1 , wherein the engineered Fc antibody domains are engineered human Fc antibody domains.
  17. 17. The chimeric cytokine complex of claim 16, wherein the engineered human Fc antibody domains are engineered human IgGl Fc antibody domains.
  18. 18. A method of activating and expanding immune cells, comprising: adding a chimeric cytokine complex to a population of immune cells, wherein the chimeric cytokine complex comprises: a protein cage polypeptide; a plurality of engineered Fc antibody domains bound to the protein cage polypeptide; and one or more cytokines linked to each of the plurality of Fc antibody domains.
  19. 19. An Fc-cytokine complex, comprising: an engineered Fc antibody domain; and one or more cytokines linked to the engineered Fc antibody domain.
  20. 20. A method of activating and expanding immune cells, comprising: adding an Fc-cytokine complex to a population of immune cells, wherein the Fc- cytokine complex comprises: an Fc antibody domain; and one or more cytokines linked to the Fc antibody domain.

Description

TITLE CELL SIGNALING COMPLEXES AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims priority to U.S. Patent Application No. 63/511,968 filed on July 05, 2023, the content of which is incorporated herein by reference in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING [0002] This application contains a Sequence Listing which has been submitted via Patent Center and is hereby incorporated by reference in its entirety. Said .xml copy, created on May 15, 2024 is named 04_NNTNZ00100WO_sequence_listing.xml and is 35,143 bytes in size. TECHNICAL FIELD [0003] This disclosure relates generally to the field of cell signaling complexes and, more specifically, to cell signaling complexes such as chimeric cytokine complexes and uses thereof. BACKGROUND [0004] A commonly used mechanism for cellular pathway activation from a cell surface is receptor oligomerization or receptor clustering. For example, receptor oligomerization/clustering is demonstrated through co-stimulatory receptor binding by ligands and agonistic antibodies [1] and by the binding of cell signaling molecules such as cytokines to certain cell receptors [2]. These receptors often include an extracellular domain on the surface of a cell, a transmembrane domain that passes through the cellular membrane, and an intracellular domain on the inside of the cell. In these receptor clustering paradigms, the extracellular domain of the receptors are engaged by cell signaling molecules, which can cause both conformational changes that translate through the membrane to contribute to activation/signaling, and/or oligomerization of multiple receptors that bring together intracellular signaling complexes that drive activation/signaling. [0005] As previously discussed, cytokines are a well-known class of cell signaling proteins. The well-established canonical mechanism for cytokines is the dimerization of extracellular cytokine receptors, often through engagement of two JAK (Janus kinase)- associated receptor subunits in a heterodimeric and sometimes homodimeric fashion [2, 3]. JAK dimerization leads to phosphorylation of STAT (signal transducer and activator of transcription) transcription factors to drive cellular activation through gene regulation [4]. [0006] Cytokines can further be classified into interleukins, interferons, chemokines, lymphokines, colony-stimulation factors (CSFs), and tumor necrosis factors (TGFs). One important family of cytokines is the common y-chain (yc) family of cytokines that play an important role in T cell expansion and survival. Some of the members of the yc family of cytokines include interleukin-2 (IL- 2), TL-7, and IL-15. [0007] The functional IL-2-engaged IL-2 receptor signaling complex can consist of sequential binding/complexing of IL-2Ra, IL-2R[3, and yc subunits as a high-affinity heterotrimer (Kd~10-11M) or an ~ 100-fold intermediate affinity dimeric complex of 1L- 2R(3, and c only (Kd~10"9M) [2, 5]. IL-2 has a low affinity to the IL-2Ra subunit (Kd~10" 8M), but this binding does not induce signaling. Naive T cells are thought to start out with low levels of IL-2Ra, but upon T cell receptor (TCR) activation, IL-2Ra expression is upregulated. IL-2R[3, and yc are constitutively expressed on lymphohematopoietic cells, which include low-density expression on naive T cells [5, 6]. In the activated T cell scenario, IL-2 will engage with the upregulated IL-2Ra subunit and the lowly expressed, intermediate affinity IL-2Rp, and yc heterodimer, leading to low activation in the later or inefficient recruitment of all receptor subunits to assemble the high-affinity signaling complex. Thus, approaches to increase IL-2 receptor signaling could take the form of: (i) increasing the affinity of IL-2 to the signaling-capable IL-2R[3/yc heterodimer to effectively increase the signaling stability, (ii) increasing the recruitment and assembly of all three IL- 2 receptor subunits to form the high-affinity signaling complex, or (iii) increasing the number of productive intermediate and high-affinity signaling complexes on the cell surface. [0008] While there have been successful cytokine engineering efforts aimed at modulating receptor signaling by focusing on modulating IL-2’s receptor binding affinity [2, 6], these efforts have focused on direct amino acid changes to the cytokine sequences to disrupt or enhance binding affinities to specific receptor subunits. Such efforts often require labor- intensive and time-intensive techniques such as directed evolution, X-ray crystallography, molecular dynamics simulations, and iterative cytokine-receptor interface engineering [2, 6], while only really taking advantage of the affinity approach. An example of these sequence-based engineering efforts is the development of interleukin-2 (IL-2) variants dubbed “superkines” or super-2s [6]. [0009] Therefore, a solution is needed that takes advantage of the various approaches for inducing cytokine receptor signaling de