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KR-20260064757-A - Extracellular vesicles comprising a stefin A protein variant specifically binding to target and uses thereof

KR20260064757AKR 20260064757 AKR20260064757 AKR 20260064757AKR-20260064757-A

Abstract

The present invention relates to a fusion protein for expressing a stepin A protein variant on a cell surface or in an extracellular vesicle that specifically binds to a target, a genetically modified cell or extracellular vesicle containing the same, and uses thereof. The fusion protein of the present invention can be efficiently expressed on a cell surface or in an extracellular vesicle, and the genetically modified cell and extracellular vesicle exhibit excellent target binding ability and selectively agonist or inhibitory activity against the target, making them useful as target-specific therapeutic agents or drug delivery vehicles.

Inventors

  • 안경훈
  • 최승현
  • 김기남
  • 김연주
  • 김혜림
  • 강현주
  • 강송화
  • 윤화인

Assignees

  • 주식회사 아피셀테라퓨틱스

Dates

Publication Date
20260508
Application Date
20241025

Claims (20)

  1. A fusion protein comprising a Stefin A protein variant that specifically binds to a target and a membrane-penetrating domain.
  2. The fusion protein according to claim 1, characterized in that the stepin A protein variant comprises the following amino acid sequence: MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVV-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF(sequence number 6); MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVD-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF (SEQ ID 7); or MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTGETYGKLEAVQYKTQVLA-(Xaa)n-GTNYYIKVRAGDNKYMHLKVFKSL-(Xaa)m-EDLVLTGYQVDKNKDDELTGF(Sequence No. 8) Here, Xaa are each independent amino acid residues, and n and m are each independent integers from 3 to 20.
  3. A fusion protein according to claim 1, wherein the membrane-penetrating domain is more preferably derived from a protein selected from the group consisting of Lamp2b, CD63, CD81, PDGFR, and PTGFRN.
  4. A fusion protein according to claim 1, further comprising a coiled coil domain.
  5. A fusion protein characterized in that, in paragraph 4, the coiled coil domain is a coiled coil domain derived from COMP.
  6. A fusion protein comprising a signal sequence in addition to the 1st paragraph.
  7. A fusion protein according to claim 1, characterized by comprising a Stepin A protein variant dimer that specifically binds to a target.
  8. In claim 7, the fusion protein is characterized in that the stepin A protein variant dimer that specifically binds to the target comprises two stepin A protein variants connected sequentially by a linker.
  9. In claim 7, a fusion protein comprising a structure selected from the following formula: Equation 1) [Affimer(dimer)]-[membrane-penetrating domain]; Equation 2) [Affimer(dimer)]-(L1)n-[membrane-penetrating domain]; Equation 3) [Affimer(dimer)]-(L1)n-[coiled coil domain]-(L2)m-[membrane penetrating domain]; Equation 4) [membrane-penetrating domain]-(L1)n-[Affimer(dimer)]; or Equation 5)[Through-film domain]-(L1)n-[Coiled-coil domain]-(L2)m-[Affimer(dimer)], Here, L1 and L2 are linkers selected independently, and The above n and m are each independently selected integers from 1 to 10.
  10. A fusion protein according to claim 8, characterized in that the linker included in the stepin A protein variant dimer that specifically binds to the target is a glycine-serine linker.
  11. A fusion protein according to claim 1, characterized by comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 389 to 456.
  12. A fusion protein according to claim 1, characterized in that the target is CD40L.
  13. Nucleic acid encoding the fusion protein of claim 1.
  14. A vector containing the nucleic acid of Clause 13.
  15. Genetically modified cells containing the nucleic acid of paragraph 13.
  16. In claim 15, the genetically modified cell is characterized by being selected from the group consisting of stem cells, immune cells, and somatic cells.
  17. In claim 15, the genetically modified cell is characterized in that the genetically modified cell is a mesenchymal stem cell.
  18. A method for producing extracellular vesicles, comprising the step of isolating and purifying extracellular vesicles from a culture medium of genetically modified cells of claim 15.
  19. Extracellular vesicle containing the fusion protein of claim 1.
  20. In paragraph 19, an extracellular vesicle additionally comprising a payload.

Description

Extracellular vesicles comprising a stefin A protein variant specifically binding to a target and uses thereof The present invention relates to a fusion protein for expressing a stepin A protein variant on a cell surface or in an extracellular vesicle that specifically binds to a target, a genetically modified cell or extracellular vesicle containing the same, and uses thereof. This study was conducted with funding from the government (Ministry of Science and ICT, Ministry of Health and Welfare) and supported by the Korea Research Foundation for Regenerative Medicine (22A0201L1). The immune system is a collective term for a biological network composed of various cells and organs established to protect an organism from external invasions. The immune system operates based on the specific functions of the organs and cells that comprise it, as well as the signaling and interactions between these cells. The immune system maintains immunological homeostasis by achieving an appropriate balance between immune tolerance, which suppresses and regulates immunity, and immune response, which enhances immunity. Imbalances in immune tolerance and immune response can be induced by various causes, and such imbalances lead to the development of various diseases. For example, if the immune tolerance mechanism becomes stronger, it can facilitate the development of cancer or the invasion of external infectious agents, potentially causing cancer or infectious diseases; conversely, if the immune response mechanism becomes stronger, it can lead to inflammatory diseases such as autoimmune diseases and allergic diseases. Cytokines are very important targets in relation to the regulation of this immune system. A representative example is the CD40 ligand (also named CD40L or CD154), a protein that binds to CD40 on antigen-presenting cells and exhibits various effects depending on the target cell (The Journal of Experimental Medicine. 175 (4): 1091-101). CD40L typically has three binding partners: CD40, α5β1 integrin, and αIIbβ3. CD40L acts as a co-stimulatory molecule and is reported to be particularly important for a subset of T cells called T follicular helper cells (TFH cells) (Journal of Immunology. 149 (12): 3817-26). CD40L is primarily expressed in activated CD4+ T cells, but is also found in a soluble form and is reported to be expressed not only in T cells but also in non-hematopoietic cells such as platelets, mast cells, macrophages, basophils, NK cells, B cells, endothelial cells, and epithelial cells (Cellular and Molecular Life Sciences. 58 (1): 4-43). CD40L is classified as a tumor necrosis factor (TNF) superfamily, and as a co-stimulatory factor, CD40/CD40L signaling plays an important role in T cell activation and T cell-mediated differentiation and activation of B cells. Furthermore, stimulation of CD40/CD40L signaling plays an important role in the expression and regulation of the signaling mechanism of OX40/OX40L, which is the same co-stimulatory factor, thereby contributing to T cell survival and the development of memory T cells. In various immune homeostasis-related diseases, CD40L is reported to play a crucial role in the interaction between antigen-presenting cells (APCs) and T cells. In particular, the CD40L/CD40 interaction acts as a pathogenic factor in autoimmune or inflammatory diseases where the activation of T cells and B cells plays a major role in pathology. Specifically, it is a pathogenic factor in various diseases such as type 1 diabetes, thyroiditis, psoriasis, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and multiple sclerosis (MS). Various compounds or antibodies targeting CD40L are being developed for the treatment of these diseases (Semin Immunol. 2009;21(5):293-300; Advanced Drug Delivery Reviews Volume 141, 15 February 2019, Pages 92-103). Various studies have reported the importance of TNFR2 in the proliferation and function of Tregs. TNFR2-deficient mice showed a decrease in the number of thymic and peripheral Tregs (2013_Chen X, et al. PMID: 23277487), and TNFR2 -/- Tregs failed to control inflammatory responses in vivo (2008_Van Mierlo GJ, et al. PMID: 18292492). In humans, Tregs were shown to express higher levels of TNFR2 than T effector cells (2013_Okubo Y, et al. PMID: 24193319 & 2002_Annunzito F, et al. PMID: 12163566). TNFR2+ Tregs most potently inhibited the proliferation and cytokine production of co-cultured T-responder cells (2010_Chen X, et al. PMID: 20127680). In addition, TNFR2 is reported to play a major role in the initiation and growth of cancer by mediating the TNF response in immunosuppressive cells, thereby allowing immune evasion and tumor development (Sheng Y., et al. doi: 10.3389/fimmu.2018.01170). The inventors have developed a Stepin A protein variant that specifically binds to targeting such cytokines (e.g., CD40L, TNFR2, etc.) and genetically modified cells expressing the same, and have filed patent applications (Korean Patent Publication No. 10-