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CN-120888000-B - CD40L-IL-2 fusion protein and its preparation and use in preparing tumor treating medicine

CN120888000BCN 120888000 BCN120888000 BCN 120888000BCN-120888000-B

Abstract

The invention discloses a CD40L-IL-2 fusion protein, its preparation and application in preparing medicines for treating tumor. The fusion protein comprises a CD40L trimer or variant thereof and a cytokine IL-2 or variant thereof, wherein the CD40L trimer comprises three CD40L monomers connected. The fusion protein provided by the invention can synergistically activate APC-dependent antigen presentation and T cell-mediated immune killing to form a closed-loop anti-tumor immune response of antigen presentation-immune initiation-effect amplification, and provides an effective strategy for treating tumors.

Inventors

  • SHI HUI
  • GU HAO
  • LI YU
  • ZHOU ZUHAO
  • ZHANG CHANGSHENG

Assignees

  • 上海市胸科医院

Dates

Publication Date
20260508
Application Date
20250731

Claims (20)

  1. 1. A fusion protein is characterized in that the fusion protein is a CD40L trimer, an Fc domain and a cytokine IL-2 which are sequentially connected from N end to C end, wherein the CD40L trimer is three CD40L monomers connected through peptide joints; The CD40L trimer is connected with an Fc domain through a first connector, the Fc domain is connected with a cytokine IL-2 through a second connector, and the amino acid sequences of the peptide linker, the first connector and the second connector are respectively shown as (G 4 S) n, wherein n is an integer in 1-5; wherein the amino acid sequence of the CD40L trimer is shown as SEQ ID NO. 2.
  2. 2. The fusion protein of claim 1, wherein the fusion protein satisfies one or more of the following: 1) The amino acid sequence of the peptide linker is shown as SEQ ID NO. 5; 2) The amino acid sequence of the CD40L monomer is shown as SEQ ID NO. 3 or 4, or the CD40L monomer is a fragment of polypeptide with the amino acid sequence shown as SEQ ID NO. 3 or 4, and, 3) The Fc domain is an IgG Fc.
  3. 3. The fusion protein of claim 2, wherein the fusion protein satisfies one or more of the following: 1) The IgG Fc is derived from a mouse or a human, and/or the IgG Fc is an IgG1 Fc; 2) The amino acid sequence of the cytokine IL-2 is shown in SEQ ID NO. 8; 3) The amino acid sequence of the first linker is shown as SEQ ID NO. 6, and, 4) The amino acid sequence of the second linker is shown as SEQ ID NO. 7.
  4. 4. The fusion protein of claim 3, wherein the IgG1 Fc is human IgG1 Fc.
  5. 5. The fusion protein of claim 4, wherein the amino acid sequence of human IgG1 Fc is set forth in SEQ ID NO. 9.
  6. 6. The fusion protein of claim 5, wherein the fusion protein has an amino acid sequence as set forth in SEQ ID NO. 1.
  7. 7. An isolated nucleic acid encoding the fusion protein of any one of claims 1-6.
  8. 8. The isolated nucleic acid of claim 7, wherein the sequence of the isolated nucleic acid is set forth in SEQ ID No. 11.
  9. 9. A recombinant vector comprising the isolated nucleic acid of claim 7 or 8.
  10. 10. The recombinant vector of claim 9, wherein the recombinant vector is a recombinant expression vector or a recombinant cloning vector.
  11. 11. The recombinant vector of claim 10, wherein the recombinant expression vector is a prokaryotic expression vector or a eukaryotic expression vector.
  12. 12. The recombinant vector of claim 11, wherein the prokaryotic expression vector is selected from the group consisting of an E.coli expression vector, a B.subtilis expression vector and a Streptomyces expression vector, and wherein the eukaryotic expression vector is selected from the group consisting of a yeast expression vector, an insect expression vector and a mammalian expression vector.
  13. 13. The recombinant vector of claim 12, wherein the mammalian expression vector is pcdna3.4.
  14. 14. A transformant comprising the isolated nucleic acid of claim 7 or 8 or the recombinant vector of any one of claims 9-13, said transformant being of a non-animal variety and a non-plant variety.
  15. 15. The transformant of claim 14, wherein the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.
  16. 16. The transformant of claim 15, wherein the eukaryotic cell is a yeast cell or a mammalian cell.
  17. 17. The transformant of claim 16, wherein the mammalian cell is a CHO-S cell.
  18. 18. A method for producing a fusion protein, comprising the step of culturing the transformant according to any one of claims 14 to 17, and obtaining the fusion protein from the culture.
  19. 19. The method of claim 18, wherein the fusion protein is purified.
  20. 20. A pharmaceutical composition comprising the fusion protein of any one of claims 1-6 and a pharmaceutically acceptable carrier.

Description

CD40L-IL-2 fusion protein and its preparation and use in preparing tumor treating medicine Technical Field The invention belongs to the technical field of biological medicines, and particularly relates to CD40L-IL-2 fusion protein and a preparation method and application thereof in preparing a tumor treatment medicine. Background Cytokine immunotherapy is a therapeutic strategy that activates or enhances anti-tumor immune responses by modulating key signaling molecules of the immune system (e.g., interleukins, interferons, etc.). The cytokine is used as a 'messenger' of an immune system, can regulate the functions of immune cells such as T cells, NK cells and the like, promote the presentation of tumor antigens and remodel an immunosuppressive microenvironment. Interlukin-2 (IL-2) is an important immunocytokine secreted by activated T cells and is effective in promoting proliferation, differentiation and survival of effector T cells, memory T cells and Natural Killer (NK) cells, thereby enhancing the cytotoxic immune response. IL-2 activates JAK-STAT and PI3K-AKT signaling pathway by binding to its receptor (IL-2R, composed of alpha, beta, gamma chains), driving immune cells to expand and secrete anti-tumor molecules such as interferon-gamma (IFN-gamma). In tumor immunotherapy, IL-2 is used to activate the host immune system (e.g., to treat metastatic melanoma and renal cancer), but its therapeutic effect is limited to some extent due to its short half-life and high dose toxicity (e.g., vascular leak syndrome). CD40 ligand (CD 40L) is a type II transmembrane protein member of the Tumor Necrosis Factor (TNF) superfamily, and CD40L activates downstream immune signaling pathways by binding to CD40 receptors on dendritic cells and other immune cell surfaces, promoting antigen presentation and enhancing T cell function. The effect of CD40L is not limited to the activation of T cells, but can also strengthen the tumor specific immune response and enhance the immune clearance of the organism to tumors. In recent years, the engineering fusion protein technology provides a new idea for integrating different immune regulation modules, such as optimizing the efficacy and safety through domain recombination or Fc fusion, and has demonstrated potential in the design of IL-15/IL-15Rα, PD-1/CTLA-4 diabodies and the like. However, when CD40L is fused to IL, the three-dimensional structures of the two may interfere with each other, resulting in steric hindrance or misfolding, affecting solubility and functional activity, receptor binding conflicts and signal crosstalk may impair the respective effects or induce unintended immunomodulatory effects, fusion proteins are inefficient and difficult to purify, linkers (Linker) may introduce immunogenicity, while differences in half-lives of the two may affect pharmacokinetics and targeting, and the risk of synergistically enhancing immunity or inducing cytokine storms is difficult to predict. Disclosure of Invention The technical problem to be solved by the invention is to overcome structural conflict, functional interference, expression difficulty and immunogenicity risk, and simultaneously ensure a synergistic effect and avoid fusion of IL-2 and CD40L under the condition of toxicity. The invention constructs CD40L-IL-2 fusion protein, breaks through the functional limitation of single cell factor, drives APC maturation and pro-inflammatory factor secretion by CD40L, promotes the activation of initial T cells, activates and expands IL-2 supporting effect T, NK cells, and cooperatively activates APC-dependent antigen presentation and T cell-mediated immune killing, thereby forming closed loop anti-tumor immune response of antigen presentation-immune initiation-effect expansion. In one aspect, the invention provides a fusion protein comprising a CD40L trimer or variant thereof and a cytokine IL-2 or variant thereof, wherein the CD40L trimer comprises three CD40L monomers linked. In some embodiments, three CD40L monomers in the CD40L trimer are linked by a peptide linker. In some embodiments, the peptide linker has an amino acid sequence as shown in (G 4S)n, where n is an integer from 1 to 5 (n is 1, 2, 3, 4, or 5), and n is preferably 2. In some embodiments, the peptide linker has an amino acid sequence as set forth in SEQ ID NO. 5. In some embodiments, the CD40L monomer has an amino acid sequence as shown in SEQ ID NO. 3 or 4, or the CD40L monomer is a fragment of a polypeptide having an amino acid sequence as shown in SEQ ID NO. 3 or 4. In some embodiments, the fusion protein further comprises an Fc domain, such as an IgG Fc, or a variant thereof. In some embodiments, the fusion protein further includes a linker, the amino acid sequence of which is, for example (shown in G 4S)n), wherein n is an integer from 1 to 5 (n is 1,2,3,4, or 5). In some embodiments, the fusion proteins provided herein include at least (i) IL-2 or variant thereof, (ii) Fc domain or variant thereof, and (ii