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WO-2026093988-A1 - THE FGF-2 POLYPEPTIDES WITH IMPROVED THERMAL AND OXIDATIVE STABILITY, THE PROCESS FOR PREPARING THE FGF-2 POLYPEPTIDES AND USE THEREOF

WO2026093988A1WO 2026093988 A1WO2026093988 A1WO 2026093988A1WO-2026093988-A1

Abstract

The invention relates to a Fibroblast Growth Factor 2 (FGF-2) polypeptides with improved stability while maintaining biological activity compared to the wild-type of FGF-2. The invention provides the process for the preparation, stabilisation and the use thereof in biotechnological research and industrial applications, the pharmaceutical industry, cosmetics, clean meat industry, generation of organoids, 3D cell culture models, in combination with physical modalities and other related applications.

Inventors

  • SMOLA, Miroslav
  • DUCHON, Tomas
  • RASCHMANOVA, Hana
  • SIKORA, JAN

Assignees

  • BTL HEALTHCARE TECHNOLOGIES A.S.

Dates

Publication Date
20260507
Application Date
20251031
Priority Date
20241101

Claims (15)

  1. 1. An FGF-2 polypeptide having at least 90 % sequence identity to SEQ ID NO: 4.
  2. 2. An FGF-2 polypeptide having at least 90 % sequence identity to at least one of SEQ ID NO: 5 to 22.
  3. 3. An FGF-2 polypeptide derived from SEQ ID NO: 4, wherein the polypeptide comprises at least one amino acid substitution from the group of R31L, C34S, V52T, E54D, H59F, C78S, L92Y, S94I, C96N, C101A, C101V, S109E, and S121P.
  4. 4. An FGF-2 polypeptide derived from at least one of SEQ ID NO: 5 to 22, wherein the polypeptide comprises at least one amino acid substitution from the group of R31L, C34S, V52T, E54D, H59F, C78S, L92Y, S94I, C96N, C101A, C101V, S109E, and S121P.
  5. 5. An FGF-2 polypeptide derived from SEQ ID NO: 3, or 5, or 6, wherein the polypeptide comprises a deletion of up to 22 amino acids at the N-terminus.
  6. 6. An FGF-2 polypeptide derived from SEQ ID NO: 4,
  7. 7. wherein the polypeptide comprises a deletion of up to 22 amino acids at the N-terminus.
  8. 8. A composition comprising an FGF-2 polypeptide, wherein the composition comprises: (a) at least one of: solvents, stabilizers, emollients, humectants, preservatives, or colorants; and (b) at least one of: penetration enhancers, antioxidants, surfactants, rheological additives, or occlusives.
  9. 9. A method of administering an FGF-2 polypeptide, comprising: (a) applying an FGF-2 polypeptide to the tissue surface, to the surface of an applicator, or to both; and (b) inducing tissue irritation, tissue damage, or a both by at least one external stressor or procedure; wherein steps (a) and (b) are performed in any order.
  10. 10. A system for administering an FGF-2 polypeptide, comprising: (a) an FGF-2 polypeptide; and (b) a device that induce irritation of tissue, damage of tissue, or their combination by at least one external stressor or procedure, wherein the system is configured such that the application of the FGF-2 polypeptide and the induction of tissue irritation, tissue damage, or a combination thereof are performed in any order.
  11. 11. A method of administering an FGF-2 polypeptide, comprising: application of an FGF-2 polypeptide with one or more physical modalities consisting from the group of electromagnetic field (e.g. light energy, laser energy (ablative or non-ablative), radiofrequency (RF) energy, microwave energy, photobiomodulation) pulsed electric field (PEF) energy, ultrasound energy, acoustic shockwave energy, mechanical stimulation, mechanical microneedling or microperforation, magnetic field (including high power magnetic field of low power magnetic field or PEMF), electrical stimulation, vacuum-compression therapy, , thermal energy ( e.g. cryogenic therapy or heat therapy), plasma-based energy, oxygen-based therapy, or any combination thereof
  12. 12. A system for administering an FGF-2 polypeptide, comprising: (a) an FGF-2 polypeptide; and (b) a device configured to deliver at least one physical modality from the group of electromagnetic field (e.g. light energy, laser energy (ablative or non-ablative), radiofrequency (RF) energy, microwave energy, photobiomodulation) pulsed electric field (PEF) energy, ultrasound energy, acoustic shockwave energy, mechanical stimulation, mechanical microneedling or microperforation, magnetic field (including high power magnetic field of low power magnetic field or PEMF), electrical stimulation, vacuum-compression therapy, , thermal energy ( e.g. cryogenic therapy or heat therapy), plasma-based energy, oxygen-based therapy, or any combination thereof
  13. 13. A formulation comprising: an FGF-2 polypeptide and a sulfate stabilizer.
  14. 14. A method of storing of the FGF-2 polypeptide in a formulation comprising a sulfate stabilizer.
  15. 15. A composition comprising an FGF-2 polypeptide and at least one additional ingredient, wherein: (a) the FGF-2 polypeptide is present in an amount ranging from 500 ppm to 300000 ppm, and (b) the at least one additional ingredient is present in an amount ranging from 10000 ppm to 990,000 ppm relative to a total weight of the composition, and wherein the ratio of the concentration of the FGF-2 variant to the concentration of the additional ingredient is between 1:2 and 1:2,000.

Description

THE FGF-2 POEYPEPTIDES WITH IMPROVED THERMAL AND OXIDATIVE STABILITY, THE PROCESS FOR PREPARING THE FGF-2 POLYPEPTIDES AND USE THEREOF Field of the invention [1] The invention relates to a Fibroblast Growth Factor 2 (FGF-2) polypeptide with improved stability, especially thermal and oxidative stability, compared to the wild-type FGF-2 and the use thereof in research and industrial applications, such as biotechnological research, medicine, pharmaceutical industry, cosmetics, clean meat industry, generation of organoids and 3D cell culture models and other related applications. Background of the invention [2 ] Fibroblast growth factors (FGFs) are a family of cell signaling proteins. They are involved in a variety of processes, especially as key elements for normal development of animal cells. These growth factors bind to heparin and heparan sulphate and typically activate cell surface receptors. [3] Fibroblast growth factors that signal through FGF receptors (FGFRs) regulate fundamental developmental pathways, including the regulation of angiogenesis and wound repair. FGFRs are expressed on many different cell types and regulate key cell processes, such as proliferation, differentiation and survival, which make FGF signaling susceptible to subversion by cancer cells. [4] FGFs are secreted glycoproteins that are generally readily sequestered to the extracellular matrix, as well as the cell surface, by heparan sulphate proteoglycans (HPSGs). For cellular signaling, FGFs are released from the extracellular matrix by heparinases, proteases or specific FGF-binding proteins, and the liberated FGFs subsequently bind to cell surface HPSGs. Cell surface HPSGs also stabilize the FGF ligand-receptor interaction, forming a ternary complex with FGFR. [5] FGF receptors signal as dimers, and ligand-dependent dimerization leads to a conformational shift in receptor structure that activates the intracellular kinase domain, resulting in intermolecular transphosphorylation of the tyrosine kinase domains and intracellular tail. Phosphorylated tyrosine residues on the receptor function as docking sites for adaptor proteins, which themselves may also be directly phosphorylated by FGFR, leading to the activation of multiple signal transduction pathways. [6 ] The human FGF-2 gene encodes not one protein, but a complex set of isoforms. The secreted isoform is a single-chain, non-glycosylated polypeptide with 154 amino acids. The amino acid sequence of human FGF-2 is 99% homologous to that of bovine FGF-2 and has high homology with bovine and rodent FGF- 2, suggesting strong sequence conservation for structure and function. [7] The stability of FGF-2 is widely accepted to be a major concern in the development of useful medicinal products or serum replacement in biotechnological research. The manufacturers usually state that solutions of reconstituted FGF-2 are stable for up to 12 months only when stored at -20 °C or lower. The reconstituted FGFs solutions are stable for only about a week at 4 °C and are recommended to be used within 24 h when at ambient temperatures (around 25 °C). Nevertheless, a 50% loss of functionality of FGF-2 solutions at a concentration of 72 pg/mL was observed after just 4 min at 25 °C. The functional half-life is decreased to 37, 33 and 10 min, as the storage temperature is increased to 37 °C, 42 °C and 50 °C, respectively. [8] One of the challenges is to preserve the biological activity of FGF-2. Most efforts are directed at sustaining FGF-2 activity for cell culture research, or to develop sustained-release formulations of FGF- 2 for tissue engineering applications. For example, approaches to maintain the stability and biological activity of FGF-2 include modulating ionic interactions in solution and chemical modifications of FGF- 2. [9] The addition of excipients to aqueous solutions of FGF-2 is among the simplest methods for FGF-2 stabilization by way of modulating ionic interactions in solution. Common strategies include the complexation of FGF-2 with its endogenous stabilizer, heparin or heparin-like polymers, or polycations. Ionic interactions between FGF-2 and the additives reduce the structural energy at the heparin-binding site, stabilize the FGF-2 native conformation and prolong its bioactivity in aqueous media. This method, unfortunately, has some disadvantages. For example, there are safety concerns because the pharmaceutical-grade heparins are isolated from porcine intestines and bovine lung tissues and the heparins are susceptible to batch-to-batch variability. This variability can induce immune responses or be contaminated or adulterated by natural and synthetic heparinoids that may lead to anaphylactoid type responses and death. Additionally, natural heparin is susceptible to degradation and desulphation by heparinases, which may adversely affect its effectiveness at stabilizing FGF-2. [10] Methods of prolonging the bioactivity of FGF-2 in aqueous media include point mutations of the prote