Human fibroblast growth factor 1 (FGF-1) muteins, dimers thereof and uses thereof

Abstract

The present invention relates to human fibroblast growth factor (FGF-1) muteins having reduced mitogenicity, dimers of human FGF-1 muteins, and to such human FGF-1 muteins and dimers of such muteins for use in lowering blood glucose levels, in particular for use in the treatment of diabetes.

Description

[Technical field]

[0001] The present invention relates to human fibroblast growth factor 1 (FGF-1) muteins having reduced mitogenicity, dimers of such FGF-1 muteins (both homodimers and heterodimers of such muteins) having reduced mitogenicity, which have the ability to lower glucose levels following administration, and their use in medicine, pharmacy and therapy, in particular in the treatment of diabetes, including type 2 diabetes (T2D). [Background technology]

[0002] Impairment of metabolic homeostasis manifests itself, among other things, as obesity and resistance of cells, especially peripheral cells, to the action of insulin (insulin resistance). This leads to weaker glucose absorption from the blood and, as a result, the development of type 2 diabetes. Diabetes is currently classified as one of the most serious civilization diseases worldwide. The number of subjects suffering from diabetes continues to increase, and the associated complications pose a threat to the health and life of patients. The main symptom of diabetes is high blood sugar levels (hyperglycemia). If such a condition continues for a long period of time, it causes damage to blood vessels and arteries. This leads to impaired blood supply and dysfunction of multiple organs and, as a result, chronic complications: diabetic retinopathy, diabetic nephropathy and diabetic neuropathy. Diabetes also accelerates the development of atherosclerosis in blood vessels, which leads to the development of ischemic heart disease and lower limbs, as well as stroke. Despite medical advances, the currently used therapies still have some undesirable side effects, such as the risk of developing excessively low blood sugar levels (hypoglycemia), fatty liver and bone loss. The continuous increase in the number of patients with T2D is observed, and thus new therapies aimed at removing insulin resistance are being sought. The most effective medicines currently used include thiazolidinediones (TZDs), which are antagonists of the nuclear PPARγ receptor. These receptors stimulate insulin mediators, which regulate lipogenesis, lipid metabolism, and mediate glucose uptake by peripheral tissues. Despite their high efficacy, the use of TZDs involves some side effects, such as weight gain, bone loss, or heart failure. This is why other mediators of the PPAR receptors are being sought that will continue to be characterized by a high insulin sensitizing capacity, but that will not necessarily entail the risk of side effects. Since the majority of patients do not respond to or are immediately refractory to a single drug, combination therapy, i.e. the simultaneous administration of several drugs, is also used in the treatment of T2D. Despite medical advances and the introduction of new drugs, it is still not possible to completely control the disease and its complications. One important aspect in the control of metabolic homeostasis is the ability of adipose tissue cells to remodel and alter basal metabolism in response to fluctuations in nutrient availability.In 2012, Jonker et al. presented a study aimed at identifying genes regulated by diet in different tissues involved in metabolism: muscle, liver, brown adipose tissue (BAT) and white adipose tissue (WAT). It has been demonstrated that FGF-1 is selectively induced in visceral (gonadal) white adipose tissue (gWAT) in response to a diet with high fat content (Jonker, JW, Suh, JM, Atkins, AR, Ahmadian, M., Li, P., Whyte, J., He, M., Juguilon, H., Yin, Y., Phillips, CT, Yu, RT, Olefsky, JM, Henry, RR, Downes, M., & Evans, RM (2012). A PPARγ-FGF1 axis is required for adaptive adipose remodelling and metabolic homeostasis. Nature, 485(7398), pp. 391-394. https: / / doi.org / 10.1038 / nature10998). Subsequent in vivo experiments demonstrated that deletion of the FGF-1 gene in mice resulted in systemic metabolic dysfunction and increased insulin resistance. Mice lacking the FGF-1 gene fed a high-fat diet developed a progressive diabetic phenotype resulting from restricted adipose tissue proliferation. In addition, the authors demonstrated that induction of FGF-1 in WAT is controlled by the nuclear PPARγ receptor (Jonker et al., 2012).

[0003] The family of fibroblast growth factors FGF in humans and rodents consists of 22 genes encoding structurally related polypeptides. These genes are located in different chromosomes, indicating that the FGF family was formed as a result of gene and chromosomal duplications and translocations. The FGF-1 protein is a paracrine protein, secreted by different types of cells (e.g., fibroblasts, adipocytes) and acts locally. Human FGF-1 (hFGF1) consists of 155 amino acids (aa), the first 14 aa of which constitute the propeptide and are removed during maturation in the course of cellular protein expression. There are known recombinant human (rh) forms of FGF-1 in the literature with three lengths: full length (155 aa) and two N-terminal deleted forms (containing amino acids 15-155 aa and 21-155 aa). According to current knowledge, all three forms have similar binding properties to FGFR and heparin, as well as similar mitogenic properties. FGF-1 is a known and potent mitogen (stimulates cell division and proliferation) for many cells. The FGF-1 ligand binds to all four cellular FGF receptors (FGFR1-4). The protein-receptor complex is stabilized by heparan, a polysaccharide that occurs naturally on cell surfaces. The literature shows that native (naturally occurring, wild-type) FGF-1 protein is characterized by low thermal stability (denaturation temperature is about 40° C.) and therefore a short biological half-life (half-life in DMEM is about 1.1-2 hours) (Zakrzewska, M., Krowarsch, D., Wiedlocha, A., & Otlewski, J. (2004). Design of fully active FGF-1 variants with increased stability. Protein Engineering Design and Selection, 17(8), pp. 603-611. https: / / pubmed.ncbi.nlm.nih.gov / 16126225 / ).

[0004] FGF-1 protein, produced endogenously by the organism, is secreted, inter alia, in adipose tissue and is necessary to maintain metabolic homeostasis of this tissue. Lack of the ability to synthesize FGF-1 leads to the development of T2D. Current literature shows studies aimed at determining the effect of exogenous administration of FGF-1 protein (more specifically, recombinant human FGF-1: rhFGF-1) to animals with type 2 diabetes induced by eating a high-fat diet. The aim of those studies was to determine what effect administration of FGF-1 ligand has on lowering blood glucose levels and restoring metabolic homeostasis in animals. It was demonstrated that rhFGF-1 lowers blood glucose concentrations. Moreover, it was demonstrated that rhFGF-1 lowers glucose concentrations without causing hypoglycemia, which is a significant advantage compared to the medicines currently used in the treatment of type 2 diabetes. However, due to its strong mitogenic properties and short half-life (rapid degradation), rhFGF-1 cannot be administered to patients with type 2 diabetes. The strong mitogenicity of FGF-1 may cause many negative effects, including neoplasia induction. Nevertheless, due to its advantageous mechanism of action of sensitizing cells to the action of endogenous insulin and the lack of risk of hypoglycemia, rhFGF-1 is an interesting candidate for further therapeutic development in the treatment of T2D.

[0005] In 2014, Suh et al. demonstrated the antidiabetic effects of subcutaneously administered FGF-1 in an animal model of diabetes induced by a high-fat diet (Suh, JM, Jonker, JW, Ahmadian, M., Goetz, R., Lackey, D., Osborn, O., Huang, Z., Liu, W., Yoshihara, E., van Dijk, TH, Havinga, R., Fan, W., Yin, Y.-Q., Yu, RT, Liddle, C., Atkins, AR, Olefsky, JM, Mohammadi, M., Downes, M., & Evans, RM (2014). Endocrinization of FGF1 produces a neomorphic and potent insulin sensitizer. Nature, 513(7518), pp. 436-439. https: / / doi.org / 10.1038 / nature13540). In their experiments, Suh et al. used the full-length (155 aa) rhFGF1 protein. Table 1 contains information related to the research model used by Suh et al.

[0006] [Table 1]

[0007] In all animal groups exhibiting a type 2 diabetic phenotype, a significant reduction in blood glucose concentration was observed after administration of rhFGF-1. As expected, in the case of the STZ model (mice characterized by the lack of insulin secretion by pancreatic β cells) exhibiting a type 1 diabetic phenotype, rhFGF-1 did not cause a reduction in blood glucose. This result allows us to conclude that this protein does not mimic the biological effect achieved by administration of insulin. In the case of healthy animals (with a physiological value of blood glucose concentration of about 135 mg / dl), the administration of the protein was neutral and did not reduce blood glucose concentration. The antidiabetic effect of a single administration of rhFGF-1 was maintained for at least the following 24 hours. The effect of rhFGF-1 proved to be dose-dependent; however, no hypoglycemic effect was observed for all doses used. In contrast to thiazolidinediones, rhFGF-1 did not cause weight gain or reduce bone mineral density, but it did contribute to a reduction in the level of hepatic steatosis (which TZDs increase), a fact observed during rhFGF-21 therapy.

[0008] The sequence of full-length human recombinant rhFGF-1 (155 aa) used by Suh et al. is presented below: MAEGEITTFTALTEKFNLPPGNYKKPKLLYCSNGGHFLRILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETGQYLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYISKKHAEKNWFVGLKKNGSCKRGPRTHYGQKAILFLPLPVSSD As shown above, native FGF-1 protein is characterized by high mitogenic potential, which leads to excessive cell proliferation and includes the risk of neoplasia. FGF-1 is known to exhibit strong affinity to its cellular receptor (FGFR), which results in strong intracellular signaling. FGF-1 ligand binding to the receptor initiates an intracellular signaling cascade that induces intensive cell division. Suh et al. tested whether it was possible to separate the mitogenic potential of FGF-1 from its antidiabetic activity. To this end, they developed an FGF-1 mutant lacking 24 N-terminal amino acids. They predicted that removal of these amino acids would reduce the strength of FGF-1 ligand binding to the FGFR receptor, which would result in a decrease in the mitogenic potential of the protein.

[0009] The recombinant human FGF-1 (FGF1 ΔNT ; K25 to D155) is as follows: MKPKLLYCSNGGHFLRILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETGQYLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYISKKHAEKNWFVGLKKNGSCKRGPRTHYGQKAILFLPLPVSSD As expected, the truncated ligands displayed relatively low affinity for FGFR, which resulted in a marked reduction in mitogenic activity; however, FGF1 ΔNT retained the antidiabetic potential observed for the wild-type protein.

[0010] To learn the mechanism of action of FGF-1 and the role of the basal receptor (FGFR) in binding to the FGF-1 ligand, Suh et al. ΔNT2 Such alterations blocked binding to FGFR1, depriving the protein of its antidiabetic properties.

[0011] The original sequence of rhFGF-1 used for modification was as follows:

number

[0012] [Table 2]

[0013] The work of Suh et al. was one of the first to explore the modification of mutant FGF-1 ligands for therapeutic use. To date, several strategies are known and have been described in the literature that aim to reduce mitogenic potential while preserving the positive biological effects in controlling metabolic homeostasis. These strategies include the purposeful introduction of changes in the amino acid sequence of the FGF-1 protein in various regions: 1. Deleting the first 24 aa (from the N-terminus) of the full-length hFGF-1 protein and shortening the chain from 155 aa to 131 aa (chain truncation). This method of separating the antidiabetic ability from the mitogenic ability was used by Suh et al. and described in their study (Suh et al., 2014).

[0014] 2. To generate partial FGFR agonists characterized by mutations in the heparan sulfate binding site. FGF-1 requires heparan sulfate for receptor binding, dimerization and activation of cellular pathways. Only stable FGF-1:FGFR complexes elicit mitogenic responses. Huang et al. developed a mutant FGF-1 protein with three local mutations in the heparan-binding site (Huang, Z., Tan, Y., Gu, J., Liu, Y., Song, L., Niu, J., Zhao, L., Srinivasan, L., Lin, Q., Deng, J., Li, Y., Conklin, DJ, Neubert, TA, Cai, L., Li, X., & Mohammadi, M. (2017). Uncoupling the Mitogenic and Metabolic Functions of FGF1 by Tuning FGF1-FGF Receptor Dimer Stability. Cell Reports, 20(7), pp. 1717-1728. https: / / doi.org / 10.1016 / j.celrep.2017.06.063). This resulted in a weakening of the effectiveness of FGF-1:FGFR:HS (heparan sulfate) complex formation, and the mitogenic response of cells was significantly reduced under the conditions studied. ΔHBS and has mutations at positions 127, 128, and 133 of the polypeptide chain (Huang et al., 2017).

[0015] 3. Reducing the affinity of CK2 kinase. After exogenous administration of FGF-1, FGF-1 binds to FGFR. Formation of the FGF-1:FGFR1 complex leads to receptor dimerization and, as a consequence, phosphorylation of the intracellular receptor domain. The complex is also internalized and FGF-1 is translocated to the nucleus where it stimulates DNA synthesis. Due to the pleiotropy of biological actions triggered by FGF-1 ligand, several cytoplasmic and nuclear proteins have been identified that are responsible for direct interaction with the ligand. One of them is the pleiotropic constitutively active CK2 kinase, which consists of two subunits α and two subunits β. Data from the literature indicate the fact that both subunits interact with FGF-1. A study by Skjerpen et al. demonstrates a correlation between the affinity of several different FGF-1 mutants for CK2 and their mitogenic potential (Skjerpen, C.S., Nilsen, T., Wesche, J., & Olsnes, S. (2002). Binding of FGF-1 variants to protein kinase CK2 correlates with mitogenicity. The EMBO Journal, 21(15), pp. 4058-4069. https: / / doi.org / 10.1093 / emboj / cdf402). Mutations in which the naturally occurring amino acid lysine (L) is replaced with neutral or negatively charged amino acids were found to result in reduced or absent protein binding to CK2. The mutants tested included proteins with lysine and serine (S) substitutions (K133R, K133A, K133E, S114A, S131E, S131E / K133E, substitutions counted relative to the full length chain 155 aa). Mutations S131A, S131A / K133A were characterized by a reduced mitogenic potential compared to the wild type. The K133E mutation did not show mitogenic potential (Skjerpen et al., 2002). These results are in agreement with data obtained from other studies (K133E mutation also downregulates FGF-1 ΔHBS It is also presented as one of three mutations in the mutant.

[0016] Another problem associated with the use of native human FGF-1 is its poor stability and short biological half-life (Culajay, JF, Blaber, SI, Khurana, A., & Blaber, M. (2000). Thermodynamic characterization of mutants of human fibroblast growth factor 1 with an increased physiological half-life. Biochemistry, 39(24), pp. 7153-7158. https: / / doi.org / 10.1021 / bi9927742). Poor stability is an essential limiting factor in the pharmacological application of proteins, as it interferes with the process of formulation or storage and use of biological medicines. (Wang, W. (1999). Instability, stabilization, and formulation of liquid protein pharmaceuticals. In International Journal of Pharmaceutics (Vol. 185, No. 2). https: / / doi.org / 10.1016 / S0378-5173(99)00152-0). In addition, proteins in living organisms are exposed to various factors such as non-optimal temperature, pH, the action of oxygen free radicals or proteases. These factors can lead to protein unfolding, aggregation or protein degradation. The denaturation temperature of FGF-1 is about 40°C, so at physiological pH and about 50% temperature, the protein unfolds. Therefore, increasing the thermal stability of a protein will have a direct impact on its biological activity and antidiabetic activity. Increasing the thermal stability of a protein can be achieved by introducing point mutations into the polypeptide chain of the protein. Culajay et al. demonstrated that a single H108G mutation in a truncated recombinant human rhFGF-1 protein increases its denaturation temperature by approximately 8° C., thus increasing its stability (Table 3 below) (Culajay et al., 2000).

[0017] Two other point mutations that increase the thermal stability of human FGF-1 have also been identified (Q55P and S62I). These mutations increased the denaturation temperature by 8.1°C and 9.3°C, respectively, compared to the native protein (Zakrzewska, Malgorzata, Krowarsch, D., Wiedlocha, A., Olsnes, S., & Otlewski, J. (2005). Highly Stable Mutants of Human Fibroblast Growth Factor-1 Exhibit Prolonged Biological Action. Journal of Molecular Biology, 352(4), pp. 860-875. https: / / doi.org / 10.1016 / j.jmb.2005.07.066). The same team created subsequent mutants containing multiple substitutions, including: Q55P / S62I and Q55P / S62I / H108G. The results obtained show that, of all the studied mutations, it was the triple substitution that proved to be the most stabilizing the protein, increasing the denaturation temperature and preventing proteolysis (Table 3 below). The triple mutation also extended the protein half-life by nearly 10 hours (for comparison, the half-life of the native FGF1 protein is about 1.5 hours). It should be pointed out that the data on the protein half-life concern preparations without added heparin (heparin protects the wild-type FGF-1 protein from undergoing thermal and proteolytic inactivation) (Malgorzata Zakrzewska et al., 2005). The analysis showed that digestion of the Q55P / S62I / H108G mutant with trypsin (protease) at 37°C for 1 hour did not cause protein fragmentation. In the case of the recombinant wild-type FGF1 protein, the mutant was almost completely degraded by the protease. The results obtained demonstrate a high proteolytic stability of the protein with three amino acid substitutions (Malgorzata Zakrzewska et al., 2005).

[0018] [Table 3]

[0019] In summary, the current literature identifies several different directions of modifying the primary structure of the FGF-1 protein in order to reduce its mitogenic potential, maintain or increase its biological activity for maintaining the homeostasis of cellular metabolism, or improve its physical and chemical properties, and consequently its pharmacological properties. However, the research carried out to date has not made it possible to develop muteins that exhibit the pharmacological properties and high efficacy expected from a therapeutic molecule. Thus, there is still an urgent need to develop FGF-1 muteins that would be optimal from the point of view of pharmacotherapeutics (i.e., would be characterized by high thermostability, resistance to proteolysis, and, consequently, a relatively long half-life in the organism), that do not exhibit mitogenic potential, but on the other hand, show high therapeutic efficacy (measured by effective reduction and stabilization of blood glucose levels), especially in the case of T2D, and at the same time do not pose the risk of hypoglycemia. Summary of the Invention [Problem to be solved by the invention]

[0020] The object of the present invention is to provide a human FGF-1 mutein and its constructs, such as dimers, that do not have the drawbacks known from the prior art. More specifically, the object of the present invention is to provide a human FGF-1 mutein that does not exhibit mitogenic potential. Furthermore, the object of the present invention is to provide a human FGF-1 mutein that is characterized by high thermostability, resistance to proteolysis and, consequently, a relatively long half-life after administration to an organism. The object of the present invention is also to provide a human FGF-1 mutein that has a hypoglycemic effect without the risk of causing hypoglycemia, characterized by physical, chemical and pharmacological properties that make it suitable for use in therapy, in particular in the treatment of type 2 diabetes.

[0021] These objects are provided by the present invention as defined in the appended claims. [Means for solving the problem]

[0022] BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENTS According to the present invention and disclosure, a mutein of human FGF-1 protein and its dimer have been developed, which comprises a point mutation that reduces the mitogenic ability of human FGF-1 protein. According to the present invention and disclosure, a mutein of human FGF-1 protein and its dimer have also been developed, which preferably comprises additional point mutations that increase the stability of human FGF-1 mutein by increasing its denaturation temperature, as well as mutations that result in a longer half-life in the bloodstream after administration to a subject due to increased resistance to proteolysis. Such properties allow efficient manufacturing processes on a large scale and therefore pharmacological development as a potential therapeutic agent, and prevent the protein from undergoing rapid denaturation in the organism, which ensures high therapeutic efficacy. The human FGF-1 mutein containing a combination of point mutations developed according to the present invention is unique, has not been known in the literature before, and provides an unobvious technical effect that allows its effective therapeutic action, especially for use in the treatment of diabetes, including type 2 diabetes.

[0023] The present invention provides a human FGF-1 mutein having reduced mitogenicity, characterized in that it contains two point mutations at amino acid position S114 and at amino acid position L150, the numbering of the amino acid positions being based on the full-length sequence of the wild-type FGF-1 protein as presented in SEQ ID NO:1 (SEK1).

[0024] Preferably, in an FGF-1 mutein according to the invention, the point mutation at position S114 is an S114A mutation (mutein having SEQ ID NO: 3 (SEK3)). Preferably, in an FGF-1 mutein according to the invention, the point mutation at position L150 is an L150D mutation (mutein having SEQ ID NO: 4 (SEK4)).

[0025] More preferably, an FGF-1 mutein according to the invention contains both the S114A and the L150D point mutations (the mutein having SEQ ID NO:5 (SEK5)).

[0026] Preferably, an FGF-1 mutein according to the invention further comprises at least one stabilizing mutation at an amino acid position selected from Q55, S62 and H108. More preferably, at least one stabilizing mutation at amino acid position Q55, S62 or H108 is a point mutation selected from Q55P, S62I and H108G, respectively.

[0027] Preferably, an FGF-1 mutein according to the invention contains three stabilizing mutations: Q55P, S62I and H108G. More preferably, the FGF-1 mutein according to the invention has the amino acid sequence set out in SEQ ID NO: 10 (SEK10).

[0028] Preferably, an FGF-1 mutein according to the present invention additionally comprises an N-terminal deletion of at least 19 consecutive amino acids of the full-length FGF-1 protein. More preferably, an FGF-1 mutein according to the present invention comprises an N-terminal deletion of amino acids E3 to G21 of the full-length FGF-1 protein.

[0029] Preferably, an FGF-1 mutein according to the present invention comprises an S114A point mutation, an L150D point mutation, and an N-terminal deletion of amino acids E3 to G21 of the full-length FGF-1 protein.

[0030] More preferably, the FGF-1 protein according to the invention has the amino acid sequence set out in SEQ ID NO: 18 (SEK18). The present invention further provides dimers of mutant FGF-1 proteins with reduced mitogenicity according to the invention as defined above.

[0031] Preferably, the dimer according to the invention is a homodimer. More preferably, the muteins forming a dimer according to the invention are linked by a linker, more preferably by an amino acid linker, most preferably such linker is the amino acid sequence GGGGSGGGGSGGGG.

[0032] Even more preferably, the dimer according to the invention has the amino acid sequence set out in SEQ ID NO: 21 or 22 (SEK21 or SEK22). The present invention further provides a human FGF-1 mutein with reduced mitogenicity according to the invention as defined above for use in lowering blood glucose levels.

[0033] The present invention further provides a human FGF-1 mutein with reduced mitogenicity according to the invention as defined above for use in the treatment of diabetes, in particular type 2 diabetes.

[0034] Preferably, an FGF-1 mutein for use according to the present invention has the amino acid sequence set out in SEQ ID NO: 10 (SEK10). Preferably, an FGF-1 mutein for use according to the present invention has the amino acid sequence set out in SEQ ID NO: 18 (SEK18).

[0035] The present invention also provides a dimer of a human FGF-1 mutein with reduced mitogenicity according to the invention as defined above for use in lowering blood glucose levels.

[0036] The present invention also provides a dimer of a human FGF-1 mutein with reduced mitogenicity according to the invention as defined above for use in the treatment of diabetes, in particular type 2 diabetes.

[0037] Preferably, a dimer for use according to the present invention has the amino acid sequence set out in SEQ ID NO: 21 (SEK21). Preferably, a dimer for use according to the present invention has the amino acid sequence set out in SEQ ID NO: 22 (SEK22).

[0038] Also disclosed herein is a mutein of human Fibroblast Growth Factor 1 (FGF-1) having reduced mitogenicity, characterized in that it contains a point mutation at amino acid position S153, where the numbering of the amino acid positions is based on the sequence of the full-length wild-type FGF-1 protein as presented in SEQ ID NO:1.

[0039] Such a point mutation at position S153 is an S153A mutation (mutein having SEQ ID NO: 32 (SEK32)) or an S153R mutation (mutein having SEQ ID NO: 31 (SEK31)).

[0040] Such human FGF-1 muteins according to the present disclosure may additionally comprise a S154 point mutation, in which the point mutation at position S154 is a S154D mutation and the point mutation at position S153 is a S153D point mutation (such a mutein is set forth in SEQ ID NO: 27).

[0041] Such human FGF-1 muteins according to the invention may further comprise at least one stabilizing mutation at an amino acid position selected from Q55, S62 and H108. Such at least one stabilizing mutation at amino acid position Q55, S62 or H108 is a point mutation selected from Q55P, S62I and H108G, respectively; such FGF-1 muteins according to the present disclosure in particular comprise three stabilizing mutations: Q55P, S62I and H108G; and in particular, such FGF-1 muteins according to the present invention have the amino acid sequence set out in SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:29.

[0042] Such FGF-1 muteins according to the present disclosure may additionally comprise an N-terminal deletion of at least 19 contiguous amino acids of the full-length FGF-1 protein, in particular an N-terminal deletion of amino acids E3 to G21 of the full-length FGF-1 protein.

[0043] Such FGF-1 muteins according to the present disclosure alternatively include an S153A point mutation, an S153R point mutation or a combination of S153D and S154D point mutations, as well as an N-terminal deletion of amino acids E3 to G21 of the full-length FGF-1 protein; in particular, such muteins according to the present disclosure have the amino acid sequence set out in SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:33 or SEQ ID NO:34.

[0044] Also disclosed herein are dimers of human fibroblast growth factor (FGF-1) muteins, including homodimers, having reduced mitogenicity as defined above and comprising a mutation at position S153.

[0045] Such dimer-forming muteins are linked by a linker, in particular an amino acid linker, in particular a linker with the amino acid sequence GGGGSGGGGSGGGG. Such dimers according to the present disclosure have an amino acid sequence selected from the sequences having numbers 35 to 38 (SEK35 to SEK38).

[0046] Also disclosed herein is a human fibroblast growth factor 1 (FGF-1) mutein having reduced mitogenicity, comprising a mutation at position S153, as defined above, and optionally at position S154, for use in lowering blood glucose levels.

[0047] Also disclosed herein is a human fibroblast growth factor 1 (FGF-1) mutein having reduced mitogenicity comprising a mutation at position S153, and optionally at position S154, as defined above, for use in the treatment of diabetes, in particular type 2 diabetes.

[0048] Such a human FGF-1 mutein has an amino acid sequence selected from SEQ ID NOs:23-30. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] In a first aspect, the present invention provides a mutant protein (mutein) of human fibroblast growth factor 1 (FGF-1) with reduced mitogenicity compared to wild-type FGF-1. Such mutant proteins of human FGF-1 are interchangeably referred to herein as human FGF-1 muteins or human FGF-1 mutants. Methods for introducing point mutations into protein sequences and testing for mitogenicity of protein drugs are known in the art, and preferred examples of such methods and tests suitable for carrying out the present invention are presented below. The human FGF-1 mutein according to the first aspect comprises at least two point mutations, namely a mutation of the naturally occurring amino acid S at position 114 of the full-length protein (155aa) and a mutation of the naturally occurring amino acid L at position 150 of the full-length FGF-1 protein as presented in sequence 1.

[0050] Point mutations at positions S114 and L150 of the full-length wild-type human FGF-1 protein result in reduced mitogenicity of the FGF-1 muteins. Preferably, the FGF-1 mutein according to the invention comprises the S114A mutation:

[0051] [ka]

[0052] The above point mutation notation is a standard way of designating a point mutation in a protein, in this case referring to a point mutation that replaces the amino acid serine naturally occurring in the polypeptide chain of human wild-type FGF-1 protein at position 114 with the alternative amino acid alanine (A).

[0053] The S114A mutation, as shown in the embodiments below and presented in the figures (e.g., FIG. 2), reduces the affinity of the mutant protein for CK2 kinase and results in reduced cell mitogenicity compared to the full-length wild-type human FGF-1 protein.

[0054] Preferably, the mutant of the full-length FGF-1 mutein according to the first aspect of the invention comprises a L150D mutation. The L150D mutation reduces the binding of the mutant FGF-1 protein according to the invention to the FGFR1 receptor. This mutation is located in the domain responsible for the affinity to the receptor, but is not critical for this binding. Figure 3 shows the reduced binding of a human FGF-1 mutein comprising a L150D mutation to the FGFR1 receptor. Such a protein is characterized by a reduced mitogenicity, as demonstrated below and presented in the figures (e.g., Figure 3).

[0055] More preferably, such an FGF-1 mutein according to the invention comprises both the S114A and L150D mutations. Such FGF-1 proteins according to the invention are characterized by an optimal reduction in mitogenicity, as shown below and presented in the figures (e.g., FIG. 4).

[0056] Guided by insights into the structure of the FGF1 protein, the inventors designed and obtained FGF1 muteins with reduced ability to bind to the FGFR1-4 receptors. The inventors achieved this with the substitution Leu150Asp (L150D), a well-known amino acid residue responsible for direct physical interaction with FGFR. The reduced binding affinity to FGFR leads to a reduced mitogenic potential of the protein, but also to a reduced thermal stability. A similar effect on protein stability was with the Ser114Ala (S114A) substitution. This point mutation is responsible for a reduced physical interaction with the proteins CK2 and p34, and is known to be a non-mitogenic FGF2. (S117A)It is a homologue of the mutant protein. CK2 and p34 are required for phosphorylation and nuclear translocation of exogenous FGF1, respectively, and as a consequence, the S114 position is the factor responsible for mitogenic activation. Lack of phosphorylation of FGF1 at the S114A position reduces the mitogenic properties, and thus the introduction of the S114A substitution increases the therapeutic potential of FGF1. Unfortunately, both substitutions S114A and L150D are required for phosphorylation and nuclear translocation of exogenous FGF1. (WT) Based on the above findings, we combined the mutation S114A / L150D with a very well-characterized stable triple mutation, also known as 3x: Gln55Pro / Ser62Ile / His108Gly (Q55P / S62I / H108G). Mutations at these residues radially increase the stability of the protein, but also its mitogenic potential. Introducing the quintuple 3x / S114A / L150D substitution, we created a novel FGF1 (3x / S114A / L150D) By a unique combination of five-fold substitutions, we obtained a thermostable protein that exhibits significantly increased glucose uptake both in vitro and in vivo and is not mitogenic.

[0057] Thus, preferably, the present invention provides a human FGF-1 mutein with reduced mitogenicity as described above, which additionally comprises at least one stabilizing point mutation at amino acid positions: Q55, S62 and H108 (according to the counting of amino acid positions of the full-length human wild-type protein-SEQ ID NO: 1); more preferably, the mutation is selected from Q55P, S62I and H108G. Even more preferably, the FGF-1 mutein according to the present invention comprises additional mutations at all three positions. Methods for determining the stability of FGF-1 proteins are known in the art, and exemplary methods for determining the stability are presented below and in Table 3.

[0058] Such FGF-1 muteins according to the present invention are characterized by both reduced mitogenicity and also increased stability, resistance to proteolysis, longer half-life after administration, and they effectively lower blood glucose levels without the concomitant induction of hypoglycemia; therefore, they exhibit optimal antidiabetic activity.

[0059] More preferably, such an FGF-1 mutein according to the first aspect of the invention further comprises a deletion of at least 19 amino acids from the N-terminus of the protein, in particular a deletion of amino acids E3 to G21 (from the N-terminus), which additionally reduces mitogenicity.

[0060] The amino acid sequence of the polypeptide chain of the FGF1 protein truncated at the N-terminus in the mutation-free version, in which the amino acid substitutions according to the invention have been made in the truncated version (protein / mutant described as "short" - truncated (shortened) sequence, i.e. with a deletion of at least 19 amino acids from the N-terminus) relative to the template, has a total chain length of 136 aa. SEQ ID NO: 2 (SEK2) is missing amino acids E3 to G21 (i.e. amino acids EGEITTFTALTEKFNLPPG) when compared to the full-length wild-type FGF-1 protein. According to the present invention and disclosure, the truncated version of the human FGF-1 protein, also referred to as the short variant of the human FGF-1 protein, preferably consists of 136 aa, whereas the full-length version of the human FGF-1 protein, also referred to as the long variant of the human FGF-1 protein, consists of 155 aa and does not include the deletion of amino acids E3 to G21.

[0061] Considering the pharmacologically advantageous parameters of such muteins of human FGF-1 apart from the reduced mitogenicity (i.e. higher thermal stability and longer half-life achieved in the presence of heparin), the triple stabilizing mutation was also tested in an in vivo test. As a result, such human FGF-1 muteins according to the present invention, including the five point mutations shown, also exhibit antidiabetic effects. Due to the above properties of the FGF1 muteins according to this embodiment of the present invention, they can be effectively used in medicine, especially in the treatment of diabetes, including type 2 diabetes, to lower blood glucose levels.

[0062] In a second aspect, the present invention provides a dimer of any of the above human FGF-1 muteins with reduced mitogenicity according to the invention. Methods for making protein dimers are known in the art, and a preferred method for obtaining a dimer of a mutein according to the invention is described below. Preferably, the dimer according to the invention is a homodimer of any of the above human FGF-1 muteins with reduced mitogenicity according to the invention, more preferably linked by a linker, in particular an amino acid linker. This makes it possible to obtain physical and chemical stabilization that promotes dimerization of the ligand. Ligand dimers more easily induce dimerization of cellular receptors. In addition, this makes it possible to increase the protein mass, since proteins with a mass of more than 30-40 kDa are not rapidly eliminated from the organism through the kidney and have a relatively long half-life. Even more preferably, such a linker is the amino acid sequence GGGGSGGGGSGGGG. Particularly advantageous variants of the dimer of the human FGF-1 mutein according to the invention have the amino acid sequences as presented in SEQ ID NOs: 21 and 22 (SEK21 and SEK22).

[0063] The properties of the above-mentioned human FGF-1 muteins according to the invention, as demonstrated below, allow their use in medicine, in particular for lowering blood glucose levels, in particular in the treatment of diabetes.

[0064] The human FGF-1 muteins and dimers thereof according to the present invention do not have the undesirable characteristics known from the prior art, which is why they can be used as medicines, in medicine, pharmacy and therapy.They are characterized by reduced mitogenicity and preferably increased thermostability and increased resistance to proteolysis, as well as favorable pharmacological properties.In addition, they are preferably characterized by hypoglycemic effect without risk of hypoglycemia, and therefore can be used to lower glucose levels, especially in the treatment of diabetes, especially type 2 diabetes.

[0065] Also disclosed herein are muteins of human fibroblast growth factor 1 (FGF-1) with reduced mitogenicity, comprising a point mutation at amino acid position S153, where the numbering of the amino acid positions is based on the full-length wild-type FGF-1 protein sequence as set forth in SEQ ID NO: 1. Such human FGF-1 muteins according to the herein described embodiments of the present disclosure comprise a point mutation selected from an S153A mutation (a mutein having SEQ ID NO: 32 (SEK32)) and an S153R mutation (a mutein having SEQ ID NO: 31 (SEK31)).

[0066] More preferably, such human FGF-1 muteins according to the present disclosure additionally comprise a point mutation at amino acid position S154. Even more preferably, such a mutation is provided by a S154D mutation, whereby the point mutation at position S153 is provided by a mutation S153D (such a mutein is shown in SEQ ID NO: 27 (SEK27)). The above mutations at positions S153 and S154 are located in the domain responsible for the affinity of the ligand to the receptor. The human FGF-1 muteins according to the embodiments described herein of the present disclosure are characterized by a reduced mitogenicity and an effective reduction in blood glucose levels, and therefore can be used to reduce blood glucose levels in the treatment of diabetes, especially type 2 diabetes.

[0067] Preferably, such human FGF-1 muteins according to the present disclosure further comprise at least one stabilizing mutation at an amino acid position selected from Q55P, S62I and H108G, in particular three such stabilizing mutations (Q55P, S62I and H108G). Most preferably, such FGF-1 muteins according to the herein described embodiments of the present disclosure have the amino acid sequence presented in SEQ ID NO: 23 (SEK23), SEQ ID NO: 25 (SEK25) or SEQ ID NO: 29 (SEK29). Such mutants are characterized by optimally reduced mitogenicity, optimal thermostability, and exhibit glucose level lowering capacity and, as a consequence, antidiabetic activity. Even more preferably, such muteins according to the present disclosure may also comprise an N-terminal deletion of at least 19 amino acids of the full-length human protein (short mutants), in particular amino acids E3 to G21 of the full-length human FGF-1 protein. Methods for determining the stability of FGF-1 proteins are known in the art, and exemplary methods for determining their stability are presented below.

[0068] Also disclosed herein are dimers of human fibroblast growth factor (FGF-1) muteins with reduced mitogenicity, comprising a mutation at position S153 as described above, and optionally at S154. Dimers of human FGF-1 muteins according to this embodiment of the disclosure are obtained in a similar manner as dimers of muteins according to the first embodiment of the invention. Preferably, such dimers are homodimers of any of the above human FGF-1 muteins with reduced mitogenicity according to the embodiment described herein of the disclosure, more preferably linked by a linker, in particular an amino acid linker, most preferably the amino acid sequence GGGGSGGGGSGGGG. This makes it possible to obtain physical and chemical stabilization that promotes ligand dimerization. Such dimers more easily induce dimerization of cellular receptors. In addition, the formation of a dimeric form makes it possible to increase the protein mass, which is desirable, since proteins with a mass of more than 30-40 kDa are not rapidly cleared from the organism through the kidney and have a relatively long half-life. Particularly advantageous variants of the dimer of the human FGF-1 mutein according to this aspect of the disclosure have an amino acid sequence selected from the sequences presented in SEQ ID NOs: 35-38.

[0069] The muteins according to the herein described aspects of the present disclosure have properties that make them suitable for use in lowering blood glucose levels, particularly in the treatment of diabetes, particularly type 2 diabetes.

[0070] The present invention will now be illustrated in the following figures and the following examples, which, however, are not intended to in any way limit the scope of the invention as defined in the claims. Unless otherwise indicated, all methods, reactants and parameters are the same as those commonly used in the art to which the invention pertains and recommended by the manufacturers.

[0071] Description of sequence listing All amino acid sequences claimed and included in this description are based on the cDNA FGF1_WT sequence, wild type variant, GenBank accession number NM001354952.2, encoding human Fibroblast Growth Factor 1 (FGF-1) with a total length of 155 amino acids.

[0072] SEQ ID NO:1 (SEK1) Based on the above sequence, a complete version of FGF1 (1-155aa) was generated, referred to herein as FGF1(155aa).

[0073] [ka]

[0074] SEQ ID NO:2 (SEK2) Based on the above sequence, a truncated version of FGF1 (22-155aa) was generated, referred to herein as FGF1(Δ155aa). The protein was generated by deletion of the N'-terminal fragment. In the sequence, the bold font indicates the first two amino acids whose presence results from the method of cloning the truncated FGF1(aa22-155) sequence into the expression vector. Methionine (M) is encoded by the initiation codon AUG, from which the protein expression process is initiated, and its presence is required at the N'-terminus. Alanine (A) was added to the sequence as a result of the addition of two nucleotides to the sequence adjacent to the restriction site to preserve the proper reading frame in the transcription process. The addition of alanine does not cause any physical and chemical changes in the protein due to its non-polar and aliphatic nature.

[0075] [ka]

[0076] SEQ ID NO:3 (SEK3) Mutant full-length FGF1 (1-155aa) sequence with alanine (A) substituted for serine (S) at position 114. FGF1 (155aa; S114A) point mutation. Mutation serial number: M1.

[0077] [ka]

[0078] SEQ ID NO:4 (SEK4) Mutant full-length FGF1 (1-155aa) sequence with leucine (L) substituted by aspartic acid (D) at position 150. FGF1(155aa;L150D) point mutation. Mutation serial number: M2.

[0079] [ka]

[0080] SEQ ID NO:5 (SEK5) Mutant full-length FGF1 (1-155aa) sequence with alanine (A) substituted for serine (S) at position 114 (S114A) and aspartic acid (D) substituted for leucine (L) at position 150 (L150D). FGF1 (155aa; S114A / L150D) double mutation. Mutation serial number: M3.

[0081] [ka]

[0082] SEQ ID NO:6 (SEK6) Mutant full-length FGF1 (1-155aa) sequence with substitution of glutamine (Q) by proline (P) at position 55. FGF1(155aa;Q55P) point mutation. Mutation serial number: M4.

[0083] [ka]

[0084] SEQ ID NO:7 (SEK7) Mutant full-length FGF1 (1-155aa) sequence with a substitution of serine (S) by isoleucine (I) at position 62. FGF1(155aa;S62I) point mutation. Mutation serial number: M5.

[0085] [ka]

[0086] SEQ ID NO:8 (SEK8) Mutant full-length FGF1 (1-155aa) sequence with histidine (H) substituted by glycine (G) at position 108. FGF1(155aa;H108G) point mutation. Mutation serial number: M6.

[0087] [ka]

[0088] SEQ ID NO:9 (SEK9) Mutant full-length FGF1 (1-155aa) sequence with substitution of glutamine (Q) by proline (P) at position 55 (Q55P), serine (S) by isoleucine (I) at position 62 (S62I), and histidine (H) by glycine (G) at position 108 (H108G). FGF1(155aa;Q55P / S62I / H108G) triple mutation. Mutation serial number: M7.

[0089] [ka]

[0090] SEQ ID NO:10 (SEK10) A mutant full-length FGF1 (1-155aa) sequence in which glutamine (Q) is replaced by proline (P) at position 55 (Q55P), serine (S) is replaced by isoleucine (I) at position 62 (S62I), and histidine (H) is replaced by glycine (G) at position 108 (H108G); in addition, serine (S) is replaced by alanine (A) at position 114 (S114A), and leucine (L) is replaced by aspartic acid (D) at position 150 (L150D). FGF1 (155aa; Q55P / S62I / H108G / S114A / L150D) quintuple mutation. Mutation sequence number: M8.

[0091] [ka]

[0092] SEQ ID NO:11 (SEK11) Mutant truncated FGF1 (22-155aa) sequence with alanine (A) substituted for serine (S) at position 114. FGF1(Δ155aa;S114A) point mutation. Mutation serial number: M9.

[0093] [ka]

[0094] SEQ ID NO:12 (SEK12) Mutant truncated FGF1 (22-155aa) sequence with leucine (L) replaced by aspartic acid (D) at position 150. FGF1(Δ155aa;L150D) point mutation. Mutation serial number: M10.

[0095] [ka]

[0096] SEQ ID NO:13 (SEK13) Mutant truncated FGF1 (22-155aa) sequence with alanine (A) substituted for serine (S) at position 114 (S114A) and aspartic acid (D) substituted for leucine (L) at position 150 (L150D). FGF1(Δ155aa;S114A / L150D)d mutation. Mutation serial number: M11.

[0097] [ka]

[0098] SEQ ID NO:14 (SEK14) Mutant truncated FGF1 (22-155aa) sequence with substitution of glutamine (Q) by proline (P) at position 55. Point mutant FGF1 (Δ155aa; Q55P). Mutation serial number: M12.

[0099] [ka]

[0100] SEQ ID NO:15 (SEK15) Mutant truncated FGF1 (22-155aa) sequence with a substitution of serine (S) by isoleucine (I) at position 62. FGF1(Δ155aa;S62I) point mutation. Mutation serial number: M13.

[0101] [ka]

[0102] SEQ ID NO:16 (SEK16) Mutant truncated FGF1 (22-155aa) sequence with histidine (H) substituted by glycine (G) at position 108. FGF1(Δ155aa;H108G) point mutation. Mutation serial number: M14.

[0103] [ka]

[0104] SEQ ID NO:17 (SEK17) Mutant truncated FGF1 (22-155aa) sequence with substitution of glutamine (Q) by proline (P) at position 55 (Q55P), serine (S) by isoleucine (I) at position 62 (S62I), and histidine (H) by glycine (G) at position 108 (H108G). FGF1(Δ155aa;Q55P / S62I / H108G) triple mutation. Mutation serial number: M15.

[0105] [ka]

[0106] SEQ ID NO:18 (SEK18) A mutant truncated FGF1 (22-155aa) sequence in which glutamine (Q) is replaced by proline (P) at position 55 (Q55P), serine (S) is replaced by isoleucine (I) at position 62 (S62I), and histidine (H) is replaced by glycine (G) at position 108 (H108G); in addition, serine (S) is replaced by alanine (A) at position 114 (S114A), and leucine (L) is replaced by aspartic acid (D) at position 150 (L150D). FGF1(Δ155aa;Q55P / S62I / H108G / S114A / L150D) quintuple mutation. Mutation sequence number: M16.

[0107] [ka]

[0108] SEQ ID NO:19 (SEK19) A homodimer of full length FGF1 (1-155aa) in which two SEQ ID NO:1 are linked by a linker: GGGGSGGGGSGGGG: N'-terminus-(1-155-linker-2-155)-C'-terminus. The second FGF1 (155aa) monomer starts with an alanine (A) at position 170, and the methionine (M) was omitted. FGF1_DIMER(155aa).

[0109] [ka]

[0110] SEQ ID NO:20 (SEK20) Homodimer of truncated FGF1 (22-155aa) with two SEQ ID NO:2 linked by a linker: GGGGSGGGGSGGGG: N'-terminus-(22-155-linker-22-155)-C'-terminus. In the sequence, bold font indicates the first two amino acids of the first FGF1 (Δ155aa) monomer and the first amino acid of the second FGF1 (Δ155aa) monomer, whose presence results from the method of cloning of the truncated FGF1 (aa22-155) sequence into the expression vector. Methionine (M) is encoded by the initiation codon AUG, from which the protein expression process is initiated, and its presence is required at the N'-terminus; Alanine (A) was added to the sequence as a result of the addition of two nucleotides to the sequence adjacent to the restriction site to preserve the proper reading frame in the transcription process. The addition of alanine does not cause any physical and chemical changes in the protein due to its non-polar and aliphatic nature. The second FGF1(Δ155aa) monomer begins with an alanine (A) at position 70, and the methionine (M) was omitted: FGF1_DIMER(Δ155aa).

[0111] [ka]

[0112] SEQ ID NO:21 (SEK21) Homodimer of full length FGF1 (aa 1-155) mutant with serial number M8, in which two SEQ ID NO: 10 are linked by a linker: GGGGSGGGGSGGGG: N'-terminus-(1-155-linker-2-155)-C'-terminus. The second FGF1 (155aa; Q55P / S62I / H108G / S114A / L150D) monomer starts with an alanine (A) at position 170. The methionine (M) was omitted. FGF1_M8_DIMER(155aa).

[0113] [ka]

[0114] SEQ ID NO:22 (SEK22) Homodimer of truncated FGF1 (aa22-155) with two SEQ ID NO:18 linked by the linker: GGGGSGGGGSGGGG: N'-terminus-(22-155-linker-22-155)-C'-terminus. In the sequence, bold font indicates the first two amino acids of the first FGF1 (Δ155aa) monomer and the first amino acid of the second FGF1 (Δ155aa; Q55P / S62I / H108G / S114A / L150D) monomer, whose presence results from the method of cloning of the truncated FGF1 (aa22-155) sequence into the expression vector. A methionine (M) is encoded by the initiation codon AUG, from which the protein expression process is initiated, and its presence is required at the N'-terminus; an alanine (A) was added to the sequence as a result of the addition of two nucleotides to the sequence adjacent to the restriction site to preserve the proper reading frame in the transcription process. The addition of alanine does not cause any physical or chemical changes in the protein due to its non-polar and aliphatic nature. The second FGF1 (Δ155aa; Q55P / S62I / H108G / S114A / L150D) monomer begins with an alanine (A) at position 170. The methionine (M) was omitted. FGF1_M16_DIMER (Δ155aa).

[0115] [ka]

[0116] SEQ ID NO:23 (SEK23) Mutant full-length FGF1 (1-155aa) sequence with substitution of glutamine (Q) by proline (P) at position 55 (Q55P), serine (S) by isoleucine (I) at position 62 (S62I), histidine (H) by glycine (G) at position 108 (H108G); and alanine (A) by serine (S) at position 153 (S153A). FGF1 (155aa; Q55P / S62I / H108G / S153A) quadruple mutation. Mutation sequence number: M17.

[0117] [ka]

[0118] SEQ ID NO:24 (SEK24) A mutant truncated FGF1 (22-155aa) sequence in which glutamine (Q) is replaced by proline (P) at position 55 (Q55P), serine (S) is replaced by isoleucine (I) at position 62 (S62I), and histidine (H) is replaced by glycine (G) at position 108 (H108G); in addition, serine (S) is replaced by alanine (A) at position 153 (S153A). FGF1(Δ155aa; Q55P / S62I / H108G / S153A) quadruple mutation. Mutation sequence number: M18.

[0119] [ka]

[0120] SEQ ID NO:25 (SEK25) Mutant full-length FGF1 (1-155aa) sequence in which glutamine (Q) is replaced by proline (P) at position 55 (Q55P), serine (S) is replaced by isoleucine (I) at position 62 (S62I), histidine (H) is replaced by glycine (G) at position 108 (H108G); in addition, serine (S) is replaced by arginine (R) at position 153 (S153R). FGF1 (155aa; Q55P / S62I / H108G / S153R) quadruple mutation. Mutation sequence number: M19.

[0121] [ka]

[0122] SEQ ID NO:26 (SEK26) Mutant truncated FGF1 (22-155aa) sequence in which glutamine (Q) is replaced by proline (P) at position 55 (Q55P), serine (S) is replaced by isoleucine (I) at position 62 (S62I), and histidine (H) is replaced by glycine (G) at position 108 (H108G); in addition, serine (S) is replaced by arginine (R) at position 153 (S153R). Quadruple mutant FGF1 (Δ155aa; Q55P / S62I / H108G / S153R). Mutation sequence number: M20.

[0123] [ka]

[0124] SEQ ID NO:27 (SEK27) Mutant full-length FGF1 (1-155aa) sequence with a substitution of serine (S) by aspartic acid (D) at position 153 (S153D) and a substitution of serine (S) by aspartic acid (D) at position 154 (S154D). FGF1 (155aa; S153D / S154D) double mutation. Mutation serial number: M21.

[0125] [ka]

[0126] SEQ ID NO:28 (SEK28) Mutant truncated FGF1 (22-155aa) sequence with substitution of aspartic acid (D) by serine (S) at position 153 (S153D) and serine (S) by aspartic acid (D) at position 154 (S154D). FGF1(Δ155aa;S153D / S154D) double mutant. Mutation serial number: M22.

[0127] [ka]

[0128] SEQ ID NO:29 (SEK29) A mutant full-length FGF1 (1-155aa) sequence in which glutamine (Q) is replaced by proline (P) at position 55 (Q55P), serine (S) is replaced by isoleucine (I) at position 62 (S62I), and histidine (H) is replaced by glycine (G) at position 108 (H108G); in addition, serine (S) is replaced by aspartic acid (D) at position 153 (S153D), and serine (S) is replaced by aspartic acid (D) at position 154 (S154D). FGF1 (155aa; Q55P / S62I / H108G / S153D / S154D) quintuple mutation. Mutation sequence number: M23.

[0129] [ka]

[0130] SEQ ID NO:30 (SEK30) A mutant truncated FGF1 (22-155aa) sequence in which glutamine (Q) is replaced by proline (P) at position 55 (Q55P), serine (S) is replaced by isoleucine (I) at position 62 (S62I), histidine (H) is replaced by glycine (G) at position 108 (H108G); in addition, serine (S) is replaced by aspartic acid (D) at position 153 (S153D), and serine (S) is replaced by aspartic acid (D) at position 154 (S154D). FGF1(Δ155aa; Q55P / S62I / H108G / S153D / S154D) quintuple mutation. Mutation sequence number: M24.

[0131] [ka]

[0132] SEQ ID NO:31 (SEK31) Mutant full-length FGF1 (1-155aa) sequence with a substitution of serine (S) by arginine (R) at position 153. FGF1(155aa;S153R) point mutation. Mutation serial number: M25.

[0133] [ka]

[0134] SEQ ID NO:32 (SEK32) Mutant full-length FGF1 (1-155aa) sequence with alanine (A) substituted for serine (S) at position 153. FGF1 (155aa; S153R) point mutation. Mutation serial number: M26.

[0135] [ka]

[0136] SEQ ID NO:33 (SEK33) Mutant truncated FGF1 (22-155aa) sequence with a substitution of serine (S) by arginine (R) at position 153. FGF1(Δ155aa;S153R) point mutation. Mutation serial number: M27.

[0137] [ka]

[0138] SEQ ID NO:34 (SEK34) Mutant truncated FGF1 (22-155aa) sequence with alanine (A) substituted for serine (S) at position 153. FGF1(Δ155aa;S153A) point mutation. Mutation serial number: M28.

[0139] [ka]

[0140] SEQ ID NO: 35 (SEK35) Homodimer of full length FGF1(1-155) mutant with sequence number M29, in which two SEQ ID NO:31 are linked by a linker: GGGGSGGGGSGGGG: N'-terminus-(1-155-linker-2-155)-C'-terminus. The second FGF1(155aa;S153R) monomer starts with an alanine (A) at position 170. The methionine (M) was omitted. FGF1_M29_DIMER(155aa).

[0141] [ka]

[0142] SEQ ID NO:36 (SEK36) A homodimer of full length FGF1(1-155) mutant with sequence number M30, in which two SEQ ID NO:32 are linked by a linker: GGGGSGGGGSGGGG: N'-terminus-(1-155-linker-2-155)-C'-terminus. The second FGF1(155aa;S153A) monomer starts with an alanine (A) at position 170. The methionine (M) was omitted. FGF1_M30_DIMER(155aa).

[0143] [ka]

[0144] SEQ ID NO:37 (SEK37) Homodimer of truncated FGF1(2-155) with two SEQ ID NO:33 linked by the linker: GGGGSGGGGSGGGG: N'-terminus-(22-155-linker-22-155)-C'-terminus. In the sequence, bold font indicates the first two amino acids of the first FGF1(Δ155aa;S153R) monomer and the first amino acid of the second FGF1(Δ155aa;S153R) monomer, whose presence results from the method of cloning of the truncated FGF1(aa22-155) sequence into the expression vector. A methionine (M) is encoded by the start codon AUG, from which the protein expression process is initiated, and its presence is required at the N'-terminus. An alanine (A) was added to the sequence as a result of the addition of two nucleotides to the sequence adjacent to the restriction site to preserve the proper reading frame in the transcription process. The addition of alanine does not cause any physical or chemical changes in the protein due to its non-polar and aliphatic nature. The second FGF1(Δ155aa;S153R) monomer begins with an alanine (A) at position 170. The methionine (M) was omitted. FGF1_M31_DIMER(Δ155aa).

[0145] [ka]

[0146] SEQ ID NO:38 (SEK38) Homodimer of truncated FGF1(2-155) with two SEQ ID NO:34 linked by the linker: GGGGSGGGGSGGGG: N'-terminus-(22-155-linker-22-155)-C'-terminus. In the sequence, bold font indicates the first two amino acids of the first FGF1(Δ155aa;S153A) monomer and the first amino acid of the second FGF1(Δ155aa;S153A) monomer, whose presence results from the method of cloning of the truncated FGF1(aa22-155) sequence into the expression vector. A methionine (M) is encoded by the start codon AUG, from which the protein expression process is initiated, and its presence is required at the N'-terminus. An alanine (A) was added to the sequence as a result of the addition of two nucleotides to the sequence adjacent to the restriction site to preserve the proper reading frame in the transcription process. The addition of alanine does not cause any physical or chemical changes in the protein due to its non-polar and aliphatic nature. The second FGF1 (Δ155aa; S15A) monomer begins with an alanine (A) at position 170. The methionine (M) was omitted. FGF1_M32_DIMER (Δ155aa).

[0147] [ka] [Brief description of the drawings]

[0148] [Figure 1]Figure 1 shows the proliferation assay measured by MTT method using the commercially available CellTiter96® non-radioactive cell proliferation assay kit (Promega). NIH3T3 cells were seeded at 2000 cells / well on 96-well plates using Dulbecco's modified Eagle medium (DMEM) supplemented with 10% calf serum (CS). The following day, cells were starved for 5 hours in DMEM without CS; subsequently, proteins were added at the specified concentrations and incubated for 48 hours. MTT assay was performed according to the manufacturer's protocol. The results of the experiment showed an increase in proliferation for both forms of FGF1, with no significant difference between truncated form FGF1 protein (Δ155aa) and full-length form FGF1 (155aa) protein. [Diagram 2] Figure 2 shows the results of proliferation assay measured by MTT method using commercially available CellTiter96® non-radioactive cell proliferation assay kit (Promega). NIH3T3 cells were seeded at 2000 cells / well on 96-well plates using DMEM supplemented with 10% CS. The following day, cells were starved for 5 hours in DMEM without CS; subsequently, proteins were added at the specified concentrations and incubated for 48 hours. MTT assay was performed according to the manufacturer's protocol. The results of the experiment showed a significant induction of proliferation by the full-length form of wild-type FGF1 protein (155aa), and an insignificant induction of proliferation by the S114A mutant (with reduced affinity for CK2) in both truncated form FGF1 (Δ155aa; S114A) and full-length form FGF1 (155aa; S114A). [Diagram 3]Figure 3 shows the results of proliferation assay measured by MTT method using commercial CellTiter96® non-radioactive cell proliferation assay kit (Promega). NIH3T3 cells were seeded at 2000 cells / well on 96-well plates using DMEM supplemented with 10% CS. The following day, cells were starved for 5 hours in DMEM without CS; subsequently, proteins were added in the amounts specified and incubated for 48 hours. MTT assay was performed according to the manufacturer's protocol. The results of the experiment showed a significant induction of proliferation by the full-length form of wild-type FGF1 protein (155aa), and an insignificant induction of proliferation by the L150D mutant (with reduced affinity for FGFR1 receptor) in both truncated form FGF1 (Δ155aa; L150D) and full-length form FGF1 (155aa; L150D). [Figure 4] Figure 4 shows the results of the proliferation assay measured by the MTT method using the commercially available CellTiter96® non-radioactive cell proliferation assay kit (Promega). NIH3T3 cells were seeded at 2000 cells / well on a 96-well plate using DMEM supplemented with 10% CS. The following day, cells were starved for 5 hours in DMEM without CS; subsequently, proteins were added at the concentrations specified and incubated for 48 hours. The MTT assay was performed according to the manufacturer's protocol. The results of the experiment showed a significant induction of proliferation by the full-length form of wild-type FGF1 protein (155aa), and an insignificant induction of proliferation by the S114A / L150D mutant (with reduced affinity for CK2 and FGFR1 receptors) in both the truncated form FGF1 (Δ155aa; S114A / L150D) and the full-length form FGF1 (155aa; S114A / L150D). [Diagram 5]Figure 5 shows the results of proliferation assay measured by MTT method using the commercially available CellTiter96® non-radioactive cell proliferation assay kit (Promega). NIH3T3 cells were seeded at 2000 cells / well on 96-well plates using DMEM supplemented with 10% CS. The following day, cells were starved for 5 hours in DMEM without CS; subsequently, proteins were added at the indicated concentrations and incubated for 48 hours. MTT assay was performed according to the manufacturer's protocol. The results of the experiment showed a significant induction of proliferation by the wild type form of FGF1 protein (155aa) and a less significant induction of proliferation by the Q55P / S62I / H108G / S114A / L150D mutant (a mutant with increased thermostability and reduced affinity for the CK2 and FGFR1 receptors) in both the truncated form FGF1 (Δ155aa; Q55P / S62I / H108G / S114A / L150D) and the full-length form FGF1 (155aa; Q55P / S62I / H108G / S114A / L150D). [Figure 6] Figure 6 shows the results of the proliferation assay measured by the MTT method using the commercially available CellTiter96® non-radioactive cell proliferation assay kit (Promega). NIH3T3 cells were seeded at 2000 cells / well on a 96-well plate using DMEM supplemented with 10% CS. The following day, cells were starved for 5 hours in DMEM without CS; subsequently, proteins were added at the concentrations specified and incubated for 48 hours. The MTT assay was performed according to the manufacturer's protocol. The protein used in the experiment is a mutant of the full-length FGF1 (155aa) protein. The results of the experiment showed a very high induction of proliferation by the mutant FGF1 (155aa; Q55P / S62I / H108G) with increased thermostability. Furthermore, experiments showed induction of proliferation by the wild-type form of the FGF1(155aa) protein as well as non-significant induction of proliferation by the mutants: FGF1(155aa;Q55P / S62I / H108G / S114A / L150D), FGF1(155aa;S114A), FGF1(155aa;L150D) and FGF1(155aa;S114A / L150D). [Figure 7] Figure 7 shows the results of proliferation assay measured by MTT method using commercial CellTiter96® non-radioactive cell proliferation assay kit (Promega). NIH3T3 cells were seeded at 2000 cells / well on 96-well plates using DMEM supplemented with 10% CS. The following day, cells were starved for 5 hours in DMEM without CS; subsequently, proteins were added at the concentrations specified and incubated for 48 hours. MTT test was performed according to the manufacturer's protocol. The protein used in the experiment is a mutant of the full-length form of FGF1 (155aa) protein. The results of the experiment showed a very high induction of proliferation by mutants with increased thermostability: FGF1(155aa;Q55P / S62I / H108G), FGF1(155aa;Q55P / S62I / H108G / S153A), FGF1(155aa;Q55P / S62I / H108G / S153R). Furthermore, the experiment showed induction of proliferation by the wild-type form of the FGF1(155aa) protein and mutants: FGF1(155aa;S153A), FGF1(155aa;S153R). The FGF1(155aa;Q55P / S62I / H108G / S114A / L150D) mutant showed a less pronounced induction of proliferation. [Figure 8]FIG. 8 shows the results of the proliferation assay measured by the MTT method using the commercially available CellTiter96® non-radioactive cell proliferation assay kit (Promega). NIH3T3 cells were seeded at 2000 cells / well on a 96-well plate with DMEM supplemented with 10% CS. The following day, cells were starved for 5 hours in DMEM without CS; subsequently, proteins were added at the concentrations specified and incubated for 48 hours. The MTT assay was performed according to the manufacturer's protocol. The protein used in the experiment is a mutant of the full-length form of the FGF1 (155aa) protein. The results of the experiment showed a very high induction of proliferation by the mutants: FGF1 (155aa; S153R) and FGF1 (155aa; Q55P / S62I / H108G / S114A / L150D). Furthermore, experiments demonstrated induction of proliferation by the wild-type form of the FGF1(155aa) protein and mutants: FGF1(155aa;S153A), FGF1(155aa;Q55P / S62I / H108G), and the FGF1(155aa;Q55P / S62I / H108G / S114A / L150D) mutant showed a less pronounced induction of proliferation. [Figure 9] Figure 9 shows the results of the proliferation assay measured by the MIT method using the commercially available CellTiter96® non-radioactive cell proliferation assay kit (Promega). NIH3T3 cells were seeded at 2000 cells / well on a 96-well plate using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% CS. The following day, cells were starved for 5 hours in DMEM without CS; subsequently, proteins were added at the concentrations specified and incubated for 48 hours. MTT assays were performed according to the manufacturer's protocol. The results of the experiment showed an increase in proliferation for both forms of FGF1, with no significant difference between the truncated form FGF1 (Δ155aa) protein and the truncated dimeric form FGF1_DIMER (Δ155aa) protein. [Figure 10]FIG. 10 shows the results of a proliferation assay measured by the MTT method using the commercially available CellTiter96® non-radioactive cell proliferation assay kit (Promega). NIH3T3 cells were seeded at 2000 cells / well on a 96-well plate with DMEM supplemented with 10% CS. The following day, cells were starved for 5 hours in DMEM without CS; subsequently, proteins were added at the concentrations specified (with or without the addition of heparin at 10 U / ml) and incubated for 48 hours. The MTT assay was performed according to the manufacturer's protocol. The results of the experiment showed a positive effect of heparin on increasing proliferation with respect to the wild-type form of the FGF1(Δ155aa) protein. The absence of heparin had an effect on the proliferation intensity but did not stop it. The results clearly show that heparin does not affect the efficacy of proliferation induction by FGF1(Δ155aa). Furthermore, experiments showed a non-significant induction of proliferation by the FGF1(Δ155aa;Q55P / S62I / H108G / S114A / L150D) mutant both with and without the addition of heparin. [Figure 11] FIG. 11 shows the results of a glucose uptake assay measured by chemiluminescence using a commercially available Glucose Uptake-Glo™ assay kit. In the test, adipocytes differentiated from mouse fibroblasts 3T3-L1 were used according to the protocol provided by the manufacturer Glucose Uptake-Glo (Promega). Differentiated adipocytes were seeded at 50,000 cells / well on 96-well poly-D-lysine coated plates using DMEM supplemented with 10% fetal bovine serum (FBS). The following day, proteins were added at the concentrations specified in DMEM without FBS and incubated for 16 hours. Glucose uptake assay was performed according to the manufacturer's protocol. The results of the experiment showed an increase in glucose uptake for both forms of FGF1, with no significant difference between truncated form FGF1 (Δ155aa) protein and full-length FGF1 (155aa) protein. [Figure 12]Figure 12 shows the results of a glucose uptake assay measured by chemiluminescence using a commercially available Glucose Uptake-Glo™ Assay Kit (Promega). In the test, adipocytes differentiated from mouse fibroblasts 3T3-L1 were used according to the protocol provided by the manufacturer Glucose Uptake-Glo (Promega). The differentiated adipocytes were seeded at 50,000 cells / well on a 96-well poly-D-lysine coated plate using DMEM supplemented with 10% FBS. The following day, proteins were added at the concentrations specified in DMEM without FBS and incubated for 16 hours. The glucose uptake assay was performed according to the manufacturer's protocol. The results of the experiment showed an increase in glucose uptake relative to the wild-type form of the FGF1(155aa) protein, as well as a significant increase in glucose uptake (greater than the effect of FGF1(155aa)) for the S114A mutant in both the truncated form FGF1(Δ155aa;S114A) and the full-length form FGF1(155aa;S114A). [Figure 13] Figure 13 shows the results of a glucose uptake assay measured by chemiluminescence using a commercially available Glucose Uptake-Glo™ Assay Kit (Promega). In the test, adipocytes differentiated from mouse fibroblasts 3T3-L1 were used according to the protocol provided by the manufacturer Glucose Uptake-Glo (Promega). The differentiated adipocytes were seeded at 50,000 cells / well on a 96-well poly-D-lysine coated plate using DMEM supplemented with 10% FBS. The following day, proteins were added at the concentrations specified in DMEM without FBS and incubated for 16 hours. The glucose uptake assay was performed according to the manufacturer's protocol. The results of the experiment showed an increase in glucose uptake for the wild-type form of the FGF1(155aa) protein, and a significant decrease in glucose uptake for the L150D mutant (compared to FGF1(155aa)) in both the truncated form FGF1(Δ155aa;L150D) and the full-length form FGF1(155aa;L150D). [Figure 14] Figure 14 shows the results of a glucose uptake assay measured by chemiluminescence using a commercially available Glucose Uptake-Glo™ Assay Kit (Promega). In the test, adipocytes differentiated from mouse fibroblasts 3T3-L1 were used according to the protocol provided by the manufacturer Glucose Uptake-Glo (Promega). The differentiated adipocytes were seeded at 50,000 cells / well on a 96-well poly-D-lysine coated plate using DMEM supplemented with 10% FBS. The following day, proteins were added at the concentrations specified in DMEM without FBS and incubated for 16 hours. The glucose uptake assay was performed according to the manufacturer's protocol. The results of the experiment showed an increase in glucose uptake for the wild type form of the FGF1(155aa) protein, and a significant decrease in glucose uptake for the S114A / L150D mutant (compared to FGF1(155aa)) in both the truncated form FGF1(Δ155aa;S114A / L150D) and the full length form FGF1(155aa;S114A / L150D). [Figure 15]Figure 15 shows the results of a glucose uptake assay measured by chemiluminescence using a commercially available Glucose Uptake-Glo™ Assay Kit (Promega). In the test, adipocytes differentiated from mouse fibroblasts 3T3-L1 were used according to the protocol provided by the manufacturer Glucose Uptake-Glo (Promega). The differentiated adipocytes were seeded at 50000 cells / well on a 96-well poly-D-lysine coated plate using DMEM supplemented with 10% FBS. The following day, proteins were added at the concentrations specified in DMEM without FBS and incubated for 16 hours. The glucose uptake assay was performed according to the manufacturer's protocol. The results of the experiment showed a significant increase in glucose uptake for the wild type form of FGF1 (155aa) protein, as well as an increase in glucose uptake for a 1000ng / ml dose of the Q55P / S62I / H108G / S114A / L150D mutant in both the truncated form FGF1 (Δ155aa; Q55P / S62I / H108G / S114A / L150D) and the full length form FGF1 (155aa; Q55P / S62I / H108G / S114A / L150D). [Figure 16]FIG. 16 shows the results of a glucose uptake assay measured by chemiluminescence using the commercially available Glucose Uptake-Glo™ Assay Kit (Promega). In the test, adipocytes differentiated from mouse fibroblasts 3T3-L1 were used according to the protocol provided by the manufacturer Glucose Uptake-Glo (Promega). The differentiated adipocytes were seeded at 50,000 cells / well on a 96-well poly-D-lysine coated plate using DMEM supplemented with 10% FBS. The following day, proteins were added at the concentrations specified in DMEM without FBS and incubated for 16 hours. The glucose uptake assay was performed according to the manufacturer's protocol. The protein used in the experiment is a mutant of the full-length FGF1 (155aa) protein. The results of the experiment showed a very high glucose uptake for the mutant FGF1 (155aa; Q55P / S62I / H108G) with increased thermal stability. Furthermore, increased glucose uptake was shown for FGF1(155aa), FGF1(155aa;Q55P / S62I / H108G / S114A / L150D) and FGF1(155aa;S114A). FGF1(155aa;L150D) and FGF1(155aa;S114A / L150D) showed a less significant or no increase in glucose uptake. [Figure 17]Figure 17 shows the results of a glucose uptake assay measured by chemiluminescence using the commercially available Glucose Uptake-Glo™ Assay Kit (Promega). In the test, adipocytes differentiated from mouse fibroblasts 3T3-L1 were used according to the protocol provided by the manufacturer Glucose Uptake-Glo (Promega). The differentiated adipocytes were seeded at 50000 cells / well on a 96-well poly-D-lysine coated plate using DMEM supplemented with 10% FBS. The following day, proteins were added at the concentrations specified in DMEM without FBS and incubated for 16 hours. The glucose uptake assay was performed according to the manufacturer's protocol. The protein used in the experiment is a mutant of the full-length FGF1 (155aa) protein. The results of the experiment showed a very high induction of glucose uptake for the mutants with increased thermostability: FGF1(155aa;Q55P / S62I / H108G), FGF1(155aa;Q55P / S62I / H108G / S153A), FGF1(155aa;Q55P / S62I / H108G / S153R). Furthermore, experiments showed increased glucose uptake for FGF1(155aa) and FGF1(155aa;Q55P / S62I / H108G / S114A / L150D), whereas FGF1(155aa;Q55P / S62I / H108G / S153A) and FGF1(155aa;Q55P / S62I / H108G / S153R) showed the highest induction of glucose uptake. [Figure 18]FIG. 18 shows the results of a glucose uptake assay measured by chemiluminescence using the commercially available Glucose Uptake-Glo™ Assay Kit (Promega). In the test, adipocytes were differentiated from mouse fibroblasts 3T3-L1 according to the protocol provided by the manufacturer Glucose Uptake-Glo (Promega). The differentiated adipocytes were seeded at 50,000 cells / well on a 96-well poly-D-lysine coated plate with DMEM supplemented with 10% FBS. The following day, proteins were added at the concentrations specified in DMEM without FBS and incubated for 16 hours. The glucose uptake assay was performed according to the manufacturer's protocol. The results of the experiment showed an increase in glucose uptake for both dimeric forms of FGF1, with no significant difference between the truncated form of FGF1_DIMER (Δ155aa) protein and the full-length form of FGF1_DIMER (155aa) protein. [Figure 19]FIG. 19 shows the results of the analysis of protein expression and phosphorylation of proteins of the FGF:FGFR-dependent pathway carried out by Western blot (WB) method by membrane scanning carried out by chemiluminescence method using a ChemiDock (Bio-Rad) device. In the test, adipocytes were differentiated from mouse fibroblasts 3T3-L1 according to the protocol provided by the manufacturer Glucose Uptake-Glo (Promega). The differentiated adipocytes were seeded at 50000 cells / well on 96-well poly-D-lysine coated plates with DMEM supplemented with 10% FBS. The following day, cells were starved for 5 hours in DMEM without FBS; subsequently, proteins were added at a concentration of 100 ng / mL and incubated for 10 minutes to study the phosphorylation of proteins of the FGF:FGFR-dependent pathway and for 16 hours to study the expression of protein Glut1. After incubation, proteins were washed out with cold PBS and cell lysis was carried out with RIPA buffer supplemented with protease and phosphatase inhibitors. Proteins were separated by SDS-PAGE electrophoresis and transferred onto nitrocellulose membranes. The blocked membranes were incubated with primary antibodies at 1:1000 dilution for 16 hours at 4°C. Anti-rabbit HRP secondary antibodies were incubated for 1 hour at room temperature. Clarity Max ECL buffer was used to acquire chemiluminescence signals. Protein concentration control was performed using Stain-Free signal analysis. The results of the experiment showed an increase in phosphorylation of FGFR1 receptor and FGFR1 receptor-dependent proteins, namely pErk1 / 2 and pFRS2a, for FGF1(155aa), FGF1(155aa;Q55P / S62I / H108G) and FGF1(155aa;S114A). FGF1(155aa;Q55P / S62I / H108G / S114A / L150D) activated the FGF1:FGFR pathway in a non-significant way, whereas FGF1(155aa;L150D) and FGF1(155aa;S114A / L150D) showed a lack of activation. [Figure 20]Figure 20 shows the results of an in vivo study of blood glucose concentration lowering effect in diabetic strain db / db mice (BKS.Cg-+Leprdb / +Leprdb / OlaHsd) after administration of 0.5 mg / kg mc wild type FGF1(Δ155aa) protein with or without the addition of 10 U / mL heparin. The control group was db / db mice administered with vehicle. The results clearly show the positive effect of FGF1(Δ155aa) on lowering glucose to normoglycemia and the lack of heparin effect on glucose lowering efficacy in db / db mice. [Figure 21] FIG. 21 shows the results of an in vivo study of blood glucose concentration lowering effect in diabetic strain db / db mice (BKS.Cg-+Leprdb / +Leprdb / OlaHsd) after administration of 1 mg / kg mc of wild type FGF1 protein (Δ155aa) and FGF1(Δ155aa;S114A) mutant. The control group was db / db mice administered with carrier. The results show the positive effect of FGF1(Δ155aa) and FGF1(Δ155aa;S114A) mutant on lowering glucose to normoglycemia in db / db mice. [Figure 22] FIG. 22 shows the results of an in vivo study of blood glucose concentration lowering effect in diabetic strain db / db mice (BKS.Cg-+Leprdb / +Leprdb / OlaHsd) after administration of 1 mg / kg mc of wild type FGF1 protein (Δ155aa) and FGF1(Δ155aa;L150D) mutant. The control group was db / db mice administered with carrier. The results show the positive effect of FGF1(Δ155aa) and the lack of effective effect of FGF1(Δ155aa;L150D) mutant on lowering glucose to normoglycemia in db / db mice. [Diagram 23]FIG. 23 shows the results of an in vivo study of blood glucose concentration lowering effect in diabetic strain db / db mice (BKS.Cg-+Leprdb / +Leprdb / OlaHsd) after administration of 1 mg / kg mc of FGF1 protein (Δ155aa) and FGF1 (Δ155aa;Q55P / S62I / H108G) mutant. The control group was db / db mice administered with carrier. The results show the positive effect of FGF1 (Δ155aa) and FGF1 (Δ155aa;Q55P / S62I / H108G) on lowering glucose to normoglycemia in db / db mice, with the efficacy of the action of FGF1 (Δ155aa;;Q55P / S62I / H108G) mutant being higher compared to FGF1 (Δ155aa). [Figure 24] Figure 24 shows the results of an in vivo study of blood glucose concentration lowering effect in diabetic strain db / db mice (BKS.Cg-+Leprdb / +Leprdb / OlaHsd) after administration of 1 mg / kg mc of FGF1(Δ155aa) protein and the Q55P / S62I / H108G / S114A / L150D mutant. The control group was db / db mice administered with the carrier. The results show a positive effect of FGF1(Δ155aa) and the Q55P / S62I / H108G / S114A / L150D mutant on lowering glucose to normoglycemia in db / db mice with no significant difference between the truncated form of FGF1(Δ155aa;Q55P / S62I / H108G / S114A / L150D) and the full-length form of FGF1(155aa;Q55P / S62I / H108G / S114A / L150D). [Diagram 25]Figure 25 shows the results of an in vivo study of blood glucose concentration lowering effect in diabetic strain db / db mice (BKS.Cg-+Leprdb / +Leprdb / OlaHsd) after administration of 1 mg / kg mc of FGF1 protein (Δ155aa), FGF1(155aa) and the truncated mutein dimeric form FGF1_DIMER(Δ155aa). The control group was db / db mice administered with the carrier. The results show the positive effect of FGF1(Δ155aa) and FGF1(155aa) on lowering glucose to normoglycemia in db / db mice and the lack of effective effect of the truncated dimeric form FGF1_DIMER(Δ155aa). [Figure 26] Figure 26 shows the results of an in vivo study of the blood glucose concentration lowering effect in diabetic mice strain db / db (BKS.Cg-+Leprdb / +Leprdb / OlaHsd) after administration of 5 mg / kg mc FGF1 protein (Δ155aa), mutant FGF1 (155aa; Q55P / S62I / H108G / S114A / L150D) and dimeric form FGF1_M8_DIMER (155aa). The control group was db / db mice administered with the carrier. The results show the positive effect of FGF1(Δ155aa) and FGF1(155aa;Q55P / S62I / H108G / S114A / L150D) on lowering glucose towards normoglycemia in db / db mice as well as the efficacy of the lower dimeric form FGF1_M8_DIMER(155aa) compared to the monomeric version. [Figure 27]Figure 27 shows the results of an in vivo study of blood glucose lowering activity in diabetic strain db / db mice (BKS.Cg-+Leprdb / +Leprdb / OlaHsd) after administration of 1 mg / kg mc of FGF1(Δ155aa) protein and three doses of FGF1(155aa;Q55P / S62I / H108G / S114A / L150D): 1, 2.5, 5 mg / kg mc. The control group was db / db mice administered with the vehicle. The results show a positive effect of FGF1(Δ155aa) and FGF1(155aa;Q55P / S62I / H108G / S114A / L150D) on lowering glucose to normoglycemia in db / db mice, with a clear dose-dependence for FGF1(155aa;Q55P / S62I / H108G / S114A / L150D). EXAMPLES

[0149] All procedures, assays and experimental analyses described below were performed using commercially available test kits, reactants and equipment in accordance with the recommendations of the applicable kit, reagent and equipment manufacturers, unless otherwise expressly indicated herein; the inventors used standard, generally known methods applicable in the field to which the invention pertains.

[0150] Example 1 Creation of constructs encoding FGF1 protein variants according to the invention and disclosure, and their expression and purification A cDNA (GenBank accession number NM001354952.2, 468 bp) encoding wild-type mutant FGF1_WT-human fibroblast growth factor 1 (FGF-1) was optimized for expression in Escherichia coli (E. coli) cells and the gene was synthesized flanked by restriction sites for the enzymes: Ndel at the 5' end and XhoI at the 3' end (Gene Synthesis, Thermo Fisher Scientific). The synthetic gene was cloned into a modified expression vector pCPBT0010 made by Celon Pharma using the restriction enzymes identified above. The designed construct for FGF1 (155 aa) protein had no tag to facilitate purification and no additional amino acids at the N- or C-terminus.

[0151] All muteins constructed according to the invention, i.e. mutant sequences of proteins FGF1(Δ155aa) (short mutant) and FGF1(155aa) (full length mutant) containing 1, 2, 3 and 4 of the 5 point mutations, were obtained in standard fashion in PCR reactions following the methodology of site-directed mutagenesis and subsequent transformation into DH5 E. coli, unless otherwise indicated, according to the methodology described by Hanahan et al. (Hanahan, D., Jessee, J., & Bloom, FR (1991). Plasmid transformation of Escherichia coli and other bacteria. Methods in Enzymology, 204, 63-113. https: / / doi.org / 10.1016 / 0076-6879(91)04006-a).

[0152] Construction of muteins FGF1(155aa;L150D), FGF1(155aa;S114A)-single mutants were generated based on the FGF1 sequence (1-155aa, SEQ ID NO:1) in PCR reactions following the methodology of site-directed point mutagenesis described by Stratagene. All amplification reactions (50 μl) contained polymerase buffer, dNTPs, template DNA, primers complementary to the sense and antisense strands carrying the mutated codons and Q5 Hot Start High Fidelity Polymerase (New England Biolab). The reaction mixtures were transformed into DH5 E. coli cells according to the methodology of Hanahan (Hanahan et al., 1991).

[0153] Mutein: FGF1(155aa;S114A / L150D), Construction of FGF1(155aa;Q55P / S62I / H108G / S114A / L150D) - A mutein with two point mutations was generated by modifying the cDNA codons of the gene for the wild-type FGF1(aa1-155) protein. The DNA sequence was optimized for expression in E. coli cells, and the gene was synthesized and generated as described above.

[0154] Construction of the mutein: FGF1 (155aa; Q55P / S62I / H108G / S114A / L150D) - A complex mutein was generated by modifying the cDNA codons of the gene for the wild type FGF1 (aa1-155) protein (SEQ ID NO: 1). The DNA sequence was optimized for expression in E. coli cells and the gene was synthesized and generated as described above.

[0155] Construction of muteins: FGF1(155aa;S153A), FGF1(155aa;S153R) - Single mutants were generated based on the FGF1 sequence (1-155aa, SEQ ID NO:1) in PCR reactions following site-directed point mutagenesis as described by Stratagene. All amplification reactions (50 μl) contained polymerase buffer, dNTPs, template DNA, primers complementary to the sense and antisense strands carrying the mutated codons and Q5 Hot Start High Fidelity Polymerase (New England Biolab). Reaction mixtures were transformed into DH5 E. coli cells following the methodology of Hanahan (Hanahan et al., 1991).

[0156] Construction of muteins: FGF1(155aa;Q55P / S62I / H108G / S153A);FGF1(155aa;Q55P / S62I / H108G / S153R) - A composite mutein was generated by modifying the cDNA codons of the gene for wild type FGF1(aa1-155) protein (SEQ ID NO:1). The DNA sequence was optimized for expression in E. coli cells and the gene was synthesized and generated as described above.

[0157] Construction of the mutein: FGF1 (155aa; S153D / S154D) - Single mutants were generated based on the FGF1 sequence (1-155aa, SEQ ID NO: 1) in PCR reactions following the site-directed point mutagenesis methodology described by Stratagene. All amplification reactions (50 μl) contained polymerase buffer, dNTPs, template DNA, primers complementary to the sense and antisense strands carrying the mutated codons and Q5 Hot Start High Fidelity Polymerase (New England Biolab). The reaction mixture was transformed into DH5 E. coli cells according to the Hanahan methodology (Hanahan et al., 1991).

[0158] Construction of the mutein: FGF1 (155aa; Q55P / S62I / H108G / S153D / S154D) - A complex mutein was generated by modifying the cDNA codons of the gene for the wild type FGF1 (aa1-155) protein (SEQ ID NO: 1). The DNA sequence was optimized for expression in E. coli cells and the gene was synthesized and generated as described above.

[0159] Construction of muteins: FGF1(Δ155aa;Q55P / S62I / H108G / S153D / S154D); FGF1(Δ155aa;S114A); FGF1(Δ155aa;L150D); FGF1(Δ155aa;S114A / L150D); FGF1(Δ155aa;Q55P / S62I / H108G / S153A); FGF1(Δ155aa;Q55P / S62I / H108G / S153R); FGF1(Δ155aa;S153A), FGF1(Δ155aa;S153R) - the constructs for these mutant proteins contained cDNAs encoding the wild type form of the FGF1 protein truncated at the N-terminus and information for the truncated muteins. The DNA sequence was optimized for expression in E. coli cells and the synthetic gene was designed and constructed as described above. Expression and purification of FGF1 protein variants according to the present invention and disclosure FGF1 protein and its mutants in long FGF1 (155aa) and truncated FGF1 (Δ155aa) versions were expressed in E. coli cells in TB (Terrific Broth; Sigma) medium supplemented with antibiotics. Kanamycin-containing TB medium was inoculated with an overnight preculture. Protein expression was induced with IPTG and the culture was grown for 20 hours. The bacterial culture was centrifuged at 6,000×g for 15 min at 4° C. The resulting pellet was suspended in lysis buffer, incubated for 30 min, and subsequently subjected to sonication on ice for 5 min. The filtered soluble fraction (supernatant) obtained by centrifuging the sample at 20,000×g for 30 min at 4° C. was loaded onto a heparin column (Heparin 6 FastFlow, Cytiva). Resin-unbound proteins were washed with buffer A and FGF1 elution was performed with a gradient in buffer B. The eluted FGF1 protein was subjected to further process of gel filtration (HiLoad16 / 600 Superdex75 preparative grade column, Cytivia). The purified protein was subjected to quantitative analysis using extinction coefficient (A280) and qualitative analysis: purity using SDS / PAGE, capillary electrophoresis, thermal shift, SEC-HPLC, MS. The protein was divided into aliquots, frozen in liquid nitrogen and stored at -80°C.

[0160] Example 2 Confirmation of the efficacy of exogenous recombinant human FGF1 protein The purpose of the performed test was to confirm the antidiabetic activity of the obtained FGF1(Δ155aa) according to the invention. db / db mice were subcutaneously administered 0.5 mg / kg mc of FGF1 protein(Δ155aa) with the addition of heparin (unaltered FGF1 protein, which is why heparin was used to protect the protein from degradation and inactivation). The protein effectively reduces glucose concentrations up to 30 hours after injection. db / db mice administered only the carrier served as a control group (Figure 20).

[0161] Example 3 Verification of the efficacy of FGF1 (Δ155aa) and truncated mutant FGF1 (Δ155aa; Q55 / S62 / H108) without heparin addition. The study investigated the antidiabetic efficacy of FGF1 (Δ155aa) protein without the presence of heparin and the efficacy of truncated mutant FGF1 (Δ155aa; Q55 / S62 / H108). Finally, it was planned to administer the protein without the presence of heparin in the buffer, since the administration of heparin causes side effects, which is why it cannot be administered to human subjects.

[0162] Tests were performed using proteins FGF1(Δ155aa) and mutant FGF1(Δ155aa;Q55 / S62 / H108) at a concentration of 1 mg / kg mc. Both proteins showed antidiabetic activity, but the mutein FGF1(Δ155aa;Q55 / S62 / H108) with stabilizing mutations (stable mutant) maintained its action significantly longer (FIG. 23). Based on the results obtained, it was concluded that stabilizing FGF1 prolongs its antidiabetic activity.

[0163] By introducing known stabilizing mutations described in the literature, preferably all three of such stabilizing mutations as described above, into the mutant human FGF1 protein according to the present invention, a thermostable protein is obtained, which also has high antidiabetic ability.This also increases the pharmacological usefulness of this protein, since the stable protein can be efficiently produced on a large scale.

[0164] However, one of the main limitations of the clinical use of rhFGF1, its mitogenic effect, remains.

[0165] Example 4 Reduced mitogenic potential of human FGF1 protein The inventors investigated the mitogenic potential of a truncated human FGF1 protein containing an introduced S114A mutation in a heparin-free system in the cell line NIH3T3, using the method described by Skjerpen et al. (Skjerpen et al., 2002). In the experiments carried out in the cell line NIH3T3, the FGF1(Δ155aa;S114A) protein does not stimulate cell proliferation compared to the unaltered FGF1 (Figure 2). The results obtained are different from those obtained by Skjerpen et al. It is necessary to consider two variables that distinguish both experiments. First, in the study by Skjerpen et al., the experiments were carried out in a system that also included heparin, which stabilizes the protein. In the applicant's experiments, the effect was observed for a protein that was not additionally stabilized with heparin, since the protein was administered in vivo without heparin due to its toxic effect on the organism. Furthermore, the applicants demonstrated that the presence of heparin was not necessary to demonstrate the mitogenic potential of the wild-type protein (Figure 10). Second, while Skjerpen et al. worked with the full-length protein, the applicants performed experiments with both versions: truncated FGF1 (Δ155aa) and full-length FGF1 (155aa).

[0166] Example 5 Impairment of interaction with the FGFR1 receptor by introduction of mutations at sites other than the heparin-binding site. Activation and dimerization of the FGF1 receptor (FGFR1) are required to induce mitogenic responses. Impaired ligand binding to the receptor weakens the interaction, reduces signal transduction, and does not promote cell proliferation.

[0167] A mutant of FGF-1 was created that is characterized by impaired binding to the FGFR1 receptor. Mutations were developed within the domain responsible for affinity to the receptor, but the altered residues are not critical for this binding. The C-terminal leucine (L) residue was replaced by an aspartic acid (D) (L150D).

[0168] The applicant then verified whether the proteins obtained in this way, which are poorly bound to receptors, have a relatively weak mitogenic potential. For this purpose, experiments were carried out with NIH3T3 cells, exposing the cells to the action of the proteins studied for 48 hours. The MTT reaction was then added to the cells, and the level of absorption was measured. A reduced proliferation of cells treated with FGF1(Δ155aa;L150D), FGF1(Δ155aa;L150D) was observed compared to cells treated with FGF1(155aa) (Figure 3).

[0169] Having obtained a weakened mitogenic effect, we verified whether the protein was still characterized by a high antidiabetic activity. In an in vitro assay, 3T3-L1 cells differentiated into adipocytes were treated with FGF1 protein or the mutants: FGF1(Δ155aa;L150D), FGF1(155aa;L150D). 16 hours after administration, a glucose uptake assay was performed according to the manufacturer (Glucose Uptake Glo assay, Promega). In an in vitro assay without the addition of heparin, the protein induced a slightly increased glucose uptake (Figure 13).

[0170] The in vitro results proved promising in both the mitogenic potential and antidiabetic efficacy evaluation of the novel mutant FGF1(Δ155aa;L150D). This is why the novel protein with the L150D mutation was tested for its ability to lower blood glucose levels in vivo in the db / db mouse animal model. A 1 mg / kg dose of the protein was administered subcutaneously to the mice. Blood glucose levels were then measured in the mice at several time points using a glucometer by puncturing the tip of the tail. Unfortunately, the FGF1(Δ155aa;L150D) mutant was unable to lower blood glucose in vivo (Figure 22).

[0171] The reason for the lack of effect probably consists in the physical properties of the protein; this mutant is characterized by a low stability, its denaturation temperature being about 28° C. Thus, the subcutaneously administered protein unfolds immediately and the heparan naturally present on the surface of the cells is not sufficient to stabilize the protein. At the physiological body temperature of the mouse (about 37° C.), the protein undergoes denaturation and loses its activity.

[0172] Example 6 Activity of FGF1 protein mutant with double mutation (FGF1(155aa;S114A / L150D)) In the experiment, the activity of mutants containing S114A and L150D mutations in NIH3T3 cells was tested by MTT assay. Such double mutants were found to show reduced mitogenic properties (Figure 4). Both S114A and L150D mutations and their combinations reduce proliferation compared to wild-type protein. Interestingly, the most reduced mitogenic activity was observed for the stabilized protein, i.e. FGF1(155aa;Q55P / S62I / H108G), which additionally contains three stabilizing mutations according to the present invention as shown below. In addition, the protein FGF1(155aa;Q55P / S62I / H108G / S114A / L150D) containing five mutations according to the present invention shows antidiabetic activity, unlike the L150D and S114A / L150D mutants that do not have stabilizing mutations.

[0173] In experiments examining the signaling cascade responsible for initiating proliferation pathways, FGF1(Δ155aa;L150D), an FGF1(Δ155aa;S114A / L150D) mutant, was less effective at activating the FGFR1 receptor, leading to weaker signaling and lower phosphorylation of Akt / PKB and ERK kinases (FIG. 19).

[0174] In the glucose uptake assay, which is the main assay in the context of the anti-diabetic activity of FGF-1 protein, the FGF1(Δ155aa;L150D), FGF1(Δ155aa;S114A / L150D) mutant according to the present invention, does not induce glucose uptake by mouse adipocytes even at the highest dose of 1000ng / ml (Figure 14). These mutants without stabilizing mutations do not show anti-diabetic efficacy; this is why a mutant containing multiple point mutations, in particular five point mutations, including both two mutations that reduce mitogenicity (S114A / L150D) and three stabilizing mutations (Q55P / S62I / H108G), was obtained, which shows anti-diabetic efficacy.

[0175] Example 7 Activity of FGF-1 muteins with multiple mutations FGF1(155aa;Q55P / S62I / H108G / S114A / L150D) and FGF1(Δ155aa;Q55P / S62I / H108G / S114A / L150D) mutants This protein combines all the desired properties: it is stable (denaturation temperature is above 60° C.) and has a reduced mitogenicity compared to the wild type. Even if it is not stabilized by the addition of heparin to the solution, the wild type FGF1 protein stimulates the proliferation of NIH3T3 cells. The FGF1 (155aa; Q55P / S62I / H108G / S114A / L150D) mutant at the concentrations studied does not show such an effect, regardless of the content or absence of heparin (FIG. 10). Moreover, the mutant shows antidiabetic activity both in vitro (in mouse 3T3-L1 cells differentiated into adipocytes) (FIGS. 15, 16, 17) and in vivo. The effect of lowering blood glucose in db / db mice was maintained for the following 30 hours after administration of the FGF1 (Δ155aa;Q55P / S62I / H108G / S114A / L150D) mutant and for 48 hours after administration of the wild-type protein. Although the mutant protein is effective, its activity is shorter than that of the wide-type protein (administered to animals at the same dose). This probably results from the mechanism of action of FGF1 and the role of the FGFR1 receptor in the blood glucose lowering effect (the mutant has impaired binding to FGFR1 due to the presence of the L150D mutation). Increasing the dose of the mutein to 5 mg / kg mc results in the achievement of an activity time equivalent to that of wild-type FGF1 administered at a dose of 1 mg / kg mc. The results of the antidiabetic activity assays performed are presented herein (Figure 24, Figure 26, Figure 27).

[0176] Example 8 Modification of full-length FGF1 (155aa; Q55P / S62I / H108G / S114A / L150D) mutant The full-length FGF1(155aa) sequence and the FGF1(155aa;Q55P / S62I / H108G / S114A / L150D) mutant, developed based on the full-length wild-type FGF1(155aa) mutant, were analyzed to clarify whether the 19aa fragment at the N-terminus of the protein has any effect on the biological activity of the protein and its muteins.

[0177] N-terminal deletion of amino acids E3 to G21 in the full-length sequence did not alter the glucose-lowering effect in vivo (FIG. 24), and the effect was similar to that of the analogous full-length mutein (FIG. 15).

[0178] Example 9 Dimeric proteins of truncated FGF1_DIMER(Δ155aa) mutant and full-length FGF1_DIMER(155aa) mutant Two molecules of FGF1 were linked by an amino acid linker in a standard manner. Dimerization was performed in both the short and full-length protein sequences. The activity of all of these forms was similar in in vitro conditions (Figure 18), but in vivo, the dimeric FGF1_DIMER(Δ155aa) version was relatively less active (Figure 25).

[0179] Example 10 Results showing activity of FGF1 protein mutants with single mutation: FGF1(155aa;S114A), FGF1(155aa;L150D) and double mutation: FGF1(155aa;S114A / L150D) FGF1(155aa;S114A) and FGF1(155aa;L150D) mutations limit cell mitogenic activity. Data are presented on NIH3T3 cells in MTT assay: both mutants FGF1(155aa;S114A) and FGF1(155aa;L150D) and their combination FGF1(155aa;S114A / L150D) reduce proliferation compared to wild-type protein, i.e., reduce the mitogenic potential of the mutant proteins (Fig. 2, Fig. 3, Fig. 4, Fig. 6). Interestingly, the most reduced mitogenic activity is observed for the additionally stabilized protein, i.e., FGF1(155aa;Q55P / S62I / H108G / S114A / L150D) that additionally contains the stabilizing mutations according to the invention (Fig. 6, Fig. 7, Fig. 8, Fig. 10). In addition, such stabilized FGF1 protein (155aa; Q55P / S62I / H108G / S114A / L150D) shows anti-diabetic activity, unlike mutants with single mutations: FGF1(155aa; S114A), FGF1(155aa; L150D) and double mutant FGF1(155aa; S114A / L150D) (Figure 21, Figure 22, Figure 24). In the experiment on the signal cascade responsible for the initiation of proliferation pathway, FGF1(155aa; S114A), FGF1(155aa; L150D) and FGF1(155aa; S114A / L150D) mutants are less effective in activating FGFR1 receptor, which leads to weaker signals and lower phosphorylation of Akt / PKB and ERK kinases (Figure 19).

[0180] In the glucose uptake assay, which is the main assay in the context of the antidiabetic activity of FGF1 protein, FGF1(155aa;S114A), FGF1(155aa;L150D) and FGF1(155aa;S114A / L150D) mutants according to the present invention do not induce glucose uptake by mouse adipocytes, even at the highest dose of 1000ng / mL (Figure 12, Figure 13, Figure 14, Figure 16). These mutants without stabilizing mutations do not show antidiabetic efficacy; this is why a mutant containing multiple point mutations, in particular five point mutations, including both two mutations that reduce mitogenicity (S114A / L150D) and three stabilizing mutations (Q55P / S62I / H108G), was obtained, which unexpectedly additionally shows antidiabetic activity.

[0181] Example 11 Activity of FGF1 muteins carrying a single mutation at position S153 of hFGF1 The inventors have studied the activity of the developed muteins containing point mutations located at position S153 in the domain responsible for FGF1 ligand affinity to the receptor, including the combination containing the stabilizing mutations according to the present disclosure (Q55P, S62I and H108G). The results of the carried out studies have shown that the developed muteins at position S153 are characterized by both reduced mitogenicity and reduced blood glucose activity (S153A and S153R: Figure 7, Figure 8, Figure 17). Furthermore, the combined mutants containing additional stabilizing mutations at three positions are characterized by high thermostability, resistance to proteolysis and, as a consequence, longer half-life in the organism.

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Claims

**Claim 1** A human fibroblast growth factor 1 (FGF-1) mutant having reduced mitogenicity, characterized by comprising two point mutations at amino acid position S114 and amino acid position L150, wherein the amino acid position numbering is based on the full-length wild-type FGF-1 protein sequence as shown in SEQ ID NO: 1, a human FGF-1 mutant. **Claim 2** The human FGF-1 mutant according to claim 1, wherein the point mutation at position S114 is an S114A mutation. **Claim 3** The human FGF-1 mutant according to claim 1 or 2, wherein the point mutation at position L150 is an L150D mutation. **Claim 4** The human FGF-1 mutant according to claim 1 or 2, characterized by comprising an S114A point mutation and an L150D point mutation. **Claim 5** The FGF-1 mutant according to claim 1 or 2, further characterized by comprising at least one stabilizing mutation at an amino acid position selected from Q55, S62 and H108. **Claim 6** The FGF-1 mutant according to claim 5, wherein at least one stabilizing mutation at amino acid position Q55, S62 or H108 is a point mutation selected from Q55P, S62I and H108G, respectively. **Claim 7** The FGF-1 mutant according to claim 1 or 2, characterized by comprising three stabilizing mutations: Q55P, S62I and H108G. **Claim 8** The FGF-1 mutant according to claim 7, characterized by having an amino acid sequence having SEQ ID NO:

10. **Claim 9** The FGF-1 mutant according to claim 1 or 2, further characterized by additionally comprising an N-terminal deletion of at least 19 consecutive amino acids of the full-length FGF-1 protein. **Claim 10** The FGF-1 mutant according to claim 9, characterized by comprising an N-terminal deletion of amino acids E3 - G21 of the full-length FGF-1 protein. **Claim 11** The FGF-1 mutant according to claim 1 or 2, characterized by comprising an S114A point mutation, an L150D point mutation and an N-terminal deletion of amino acids E3 - G21 of the full-length FGF-1 protein. **Claim 12** The FGF-1 mutant according to claim 11, characterized by having an amino acid sequence having SEQ ID NO:

18.

13. A dimer of a human fibroblast growth factor 1 (FGF-1) mutant having a reduced mitogenic promoting property as defined in claim 1 or 2.

14. The dimer according to claim 13, which is a homodimer.

15. The dimer according to claim 13, wherein the mutants forming the dimer are linked by a linker, preferably an amino acid linker.

16. The dimer according to claim 15, wherein the linker has the amino acid sequence GGGGSGGGGGSGGGGG.

17. The dimer according to claim 16, which has the amino acid sequence having SEQ ID NO:

21.

18. The dimer according to claim 16, which has the amino acid sequence having SEQ ID NO:

22.

19. A medicament for use in lowering blood glucose levels, comprising a human fibroblast growth factor 1 (FGF-1) mutant having a reduced mitogenic promoting property as defined in claim 1 or 2.

20. A medicament for use in the treatment of diabetes, particularly type 2 diabetes, comprising a human fibroblast growth factor 1 (FGF-1) mutant having a reduced mitogenic promoting property as defined in claim 1 or 2.

21. The medicament for use according to claim 19, wherein the FGF-1 mutant has the amino acid sequence having SEQ ID NO:

10.

22. The medicament for use according to claim 19, wherein the FGF-1 mutant has the amino acid sequence having SEQ ID NO:

18.

23. A medicament for use in lowering blood glucose levels, comprising a dimer of a mutant human fibroblast growth factor 1 (FGF-1) protein having a reduced mitogenic promoting property as defined in claim 13.

24. A medicament for use in the treatment of diabetes, particularly type 2 diabetes, comprising a dimer of a mutant human fibroblast growth factor (FGF-1) protein having a reduced mitogenic promoting property as defined in claim 13.

25. The medicament for use according to claim 23, wherein the dimer has the amino acid sequence having SEQ ID NO:

21.

26. The medicament for use according to claim 24, wherein the dimer has the amino acid sequence having SEQ ID NO: 22.