Epidermal growth factor receptor (EGFR) ligand

Abstract

The present invention relates to a superstable EGFR peptide ligand and variants thereof derived from plants, especially isolated from Pereskia belo, and also includes nucleic acids encoding them, host cells containing the nucleic acids, compositions containing the peptide ligands, and methods and therapeutic uses thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Singapore Patent Application No. 10201906403T filed on July 10, 2019, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] The present invention belongs to the field of peptide / protein technology, and in particular relates to an EGF receptor ligand with improved stability and a method and use thereof. Background Art

[0004] Chronic wounds and the attendant consequences of severe pain, amputation, and disability remain a significant health problem and add to the socioeconomic burden. At least 15% of diabetic patients will develop chronic ulcers on their feet. In the United States, the total medical costs of treating this disease are estimated to be $9-13 billion. In this regard, growth factors that play an important role in proliferation, migration, and differentiation hold promise as therapeutic interventions for wound management. Epidermal growth factor (EGF) was the first growth factor discovered in 1960 and has received clinical approval in several countries for the treatment of diabetic foot ulcers. However, EGF has poor proteolytic stability in the microenvironment of chronic wounds and requires frequent applications to achieve the desired therapeutic effect. Recent findings suggest that topical application of EGF in combination with protease inhibitors can improve wound healing compared to EGF alone.

[0005] The epidermal growth factor receptor (EGFR / ErbB1 / HER) is a major tyrosine kinase receptor (RTK) that initiates multiple cellular responses required for development and growth. Epidermal growth factor (EGF) was the first EGFR agonist discovered. Since then, EGFR agonists have been found in almost all forms of organisms, such as mammals, viruses, insects, and nematodes, but not in plants. They include seven related mammalian EGFR agonists, covering 53 to 132 amino acid residues, three of which are high-affinity agonists: EGF, transforming growth factor-α (TGF-α), and heparin-binding epidermal growth factor (hb-EGF). The remaining four, betacellulin, amphiregulin, epiregulin, and epigen, are low-affinity agonists. They all have a consistent EGF-like domain. To date, EGFR agonists have a typical EGF domain without exception. Due to the complex interactions of these agonists with different members of the Erb family of RTKs, they produce different biological responses. EGFR ligands can also be found in non-mammalian sources such as poxviruses (vaccinia growth factor, myxoma virus growth factor, and Shope fibroma virus growth factor), Caenorhabditis elegans (lin-3), and Drosophila (spitz).

[0006] EGFR ligands play an important role in cell proliferation, survival and differentiation through ligand-dependent activation of EGFR. All known EGFR peptide agonists have an evolutionarily conserved EGF-like domain consisting of a cysteine ​​motif, disulfide bonds (Cys I-III, Cys II-IV, Cys V-VI) and a three-ring structure with a double-stranded antiparallel β-fold. EGF has been introduced into the clinical setting for regenerative medicine, but its application is limited by its low in vivo stability.

[0007] To date, EGF is the main active ingredient in many drugs for treating diabetic foot ulcers and cosmetics used to help skin rejuvenation, with an estimated market size of $102 million in the skin care industry in 2016, with an average annual growth rate of 6.27%. Due to its nature as a protein, the disadvantage of EGF is that it is very susceptible to factors such as temperature and interaction with other agents, especially proteases, which affect its stability. Therefore, there is a need for an EGF substitute that exhibits improved stability (proteolytic and temperature) compared to EGF while maintaining high affinity binding to and activation of EGFR.

[0008] Plants are a rich source of bioactive compounds with great value for drug design and development. However, plant peptides and proteins are often underexplored due to their perceived instability.

[0009] The present invention addresses this need by providing ultrastable, plant-derived EGFR ligands that can be used in a variety of applications, including skin and wound applications (including wound healing, skin and tissue regeneration) and cell agriculture / food applications (including cultured meat) for cell expansion and improved cell survival. Summary of the invention

[0010] The present invention is based on the inventors' identification of novel cysteine-rich peptides (CRPs) derived from the plant Pereskia bleo, which have EGFR binding activity and high stability. Cysteine-rich peptides (CRPs) are generally small peptides characterized by a high cysteine ​​content and cross-linked by disulfide bridges. Their conformationally restricted structure and cysteine-rich core provide them with relatively high stability. The medicinal plant Pereskia bleo was found to be a rich source of cysteine-rich peptides, which are collectively referred to as bleogens. The prototype and cationic bleogen pB1 is a heparin-binding CRP with 36 residues, of which 5 are Lys / Arg and 6 are Cys, and the cysteine ​​motif is C(X)6C(X)7CC(X)3C(X) 10C (SEQ ID NO: 2). It differs structurally from EGFR ligands in that it has a more compact structure that is different from the typical EGF-like domain. Bleogen pB1 adopts a four-ring structure with disulfide bonds arranged in a cystine knot connection manner (Cys I-IV, Cys IIV and Cys III-VI). Bleogen pB1 is biosynthesized as a two-domain precursor, and the mature domain is released after signal peptidase cleavage. Bleogen pB1 has a cationic-polar-cationic motif that contributes to its heparin binding properties. Bleogen pB1 also has sequence homology with knottin-type antimicrobial peptides and exhibits anti-Candida properties. Therefore, Bleogen pB1 represents a first-class EGFR agonist 59 years after the discovery of EGF.

[0011] In a first aspect, the present invention therefore relates to an isolated peptide having EGFR binding activity, said peptide comprising or consisting of:

[0012] (i) the amino acid sequence set forth in SEQ ID NO: 1;

[0013] (ii) an amino acid sequence having at least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 over its entire length;

[0014] (iii) an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence homology to the amino acid sequence set forth in SEQ ID NO: 1 over its entire length; or

[0015] (iv) Any fragment of (i) to (iii).

[0016] The peptide consisting of SEQ ID NO: 1 is also referred to herein as "bleogen pB1" or "pB1".

[0017] In another aspect, the present invention also relates to nucleic acid molecules encoding the peptides described herein, and vectors containing such nucleic acids, in particular replication vectors or expression vectors.

[0018] In another aspect, the present invention also relates to a host cell, preferably a non-human host cell, comprising a nucleic acid as contemplated herein or a vector as contemplated herein. The host cell may be a bacterial cell, such as E. coli, or a plant cell.

[0019] Another aspect of the invention is a method for producing a peptide as described herein, comprising culturing a host cell as contemplated herein; isolating the peptide from the culture medium or the host cell. Another aspect relates to a method for producing a peptide as described herein by chemical synthesis (eg solid phase peptide synthesis).

[0020] In another aspect, the present invention relates to a composition, in particular a pharmaceutical, cosmetic or cosmeceutical composition, comprising the peptides described herein. The composition may further comprise a carrier and / or an excipient.

[0021] In another aspect, the present invention relates to the use of the peptides described herein or compositions containing them for activating EGFR, particularly in ex vivo applications such as cell culture.

[0022] In another aspect, the present invention relates to a method for preventing or treating an EGF or EGFR-related disease or condition in a subject in need thereof using one or more peptides of the present invention or a composition of the present invention. This aspect also encompasses the use of a peptide or cosmetic / pharmaceutical / cosmeceutical composition of the present invention in the preparation of a medicament for treating or preventing an EGF or EGFR-related disease or condition in a subject in need thereof, wherein the prevention or treatment may comprise administering a cosmetic, therapeutic or preventive effective amount of a peptide or composition of the present invention.

[0023] In another aspect, the invention relates to a method of treating or preventing an EGF or EGFR-related disease or disorder in a subject in need thereof, comprising administering to the subject a cosmetically, prophylactically or therapeutically effective amount of one or more compounds of the invention or compositions of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 .Sequence and structure comparison of bleogen pB1 and seven related mammalian EGFR agonists. (A) Leaves of Pereskia bleo; (B) Pro-structure, primary sequence and disulfide bond connections of bleogen pB1; (C) Sequence comparison of all seven related mammalian EGFR agonists (EGF, TGFα, hb-EGF, betacellulin, amphiregulin, epiregulin and epigen) displayed by WebLogo; YXGXR motif (X, any amino acid) is shaded in red. (D) Loop 4 of bleogen pB1 interacts with loop C of EGF (PDB entry: 1P9J), epiregulin (PDB entry: 1K36) and TGFα (PDB entry: 1YUF); (E) Interaction between bleogen pB1 and EGFR extracellular domain (PDB entry: 1IVO) was modeled using ClusPro version 2.0 server.

[0025] Figure 2.Chemical synthesis of bleogen pB1. (A) Synthesis scheme of bleogen pB1 and biotin-pB1 using stepwise solid phase Fmoc chemistry on Wang resin. Bleogen pB1 was synthesized using stepwise solid phase Fmoc chemistry on Wang resin to produce PG-pB1 (PG: protecting group). Biotin-pB1 was synthesized by coupling Fmoc-Lys(biotin) to the N-terminus of PG-pB1 on the resin. After TFA cleavage, the assembled linear precursor released from the resin support was immediately subjected to oxidative folding in 0.1 M ammonium bicarbonate and 10% dimethyl sulfoxide (DMSO) at pH 8.0 for 1 h, where the molar ratio of the redox reagent cysteamine / cystamine was 10:1; (B) Elution of native and synthetic bleogen pB1 using RP-HPLC; (C) HPLC chromatograms of native and synthetic bleogen pB1 using heparin affinity chromatography.

[0026] Figure 3.bleogen pB1 exerts EGF-like activity. (A,B) Natural and synthetic bleogen pB1 promoted the proliferation of HaCaT cells for 72 h in a dose-dependent manner (detected by crystal violet), with EGF as a positive control; all results are expressed as the mean ± SD of three independent experiments; (C) Bleogen pB1 promoted the proliferation of primary human keratinocytes for 72 h (detected by crystal violet), with EGF as a positive control; all results are expressed as the mean ± SD of three independent experiments; *p<0.05 compared with the control; (D) Bleogen pB1 promoted DNA synthesis in Ha-CaT cells in vitro (tested by EdU incorporation), with EGF as a positive control; all results are expressed as the mean ± SD of three independent experiments; *p<0.05 compared with the control. (E) Five consecutive days of subcutaneous injection of bleogen pB1 (3 mg / kg, 10 mice) accelerated the eruption of incisors in neonatal mice from 153 hours (saline control, 10 mice) to 125 hours by visual inspection, while the positive control EGF (3 mg / kg, 10 mice) accelerated the eruption of incisors to 100 hours; incisor eruption was defined as the time when a given tooth first pierced the oral epithelium; n = 10 mice per group; all results are expressed as mean ± SD; *p < 0.05 compared with the control group. (F) In a 5 mm full-thickness splinted excisional wound model using C57 mice (50 wounds in total; 25 mice), topical application of bleogen pB1 (10 nmol / wound; n = 10 wounds; 5 mice) and EGF (1 nmol / wound; n = 10 wounds; 5 mice) for three consecutive days after wounding accelerated wound healing compared with the saline vehicle control group (n = 30 wounds; 15 mice). Upper panel: Schematic representation of the treatment regimen. Lower panel: Percentage of wound healing over the course of 14 days post-wounding. All results are expressed as mean ± SD; *p < 0.05 for all treatment groups compared to saline control group.

[0027] Figure 4 .bleogen pB1 binds to EGFR. (A) Representative immunoblot images of biotin-pB1, biotin-aB1 (negative control), and biotin-EGF (positive control) pull-down experiments of EGFR in HaCaT cell lysates using anti-EGFR mAb; N = three independent experiments; (B) Anti-EGFR neutralizing mAb (clone LA1) blocked the proliferative effect of bleogen pB1 in HaCaT cells (detected by crystal violet). Mouse IgG1 was used as a control; all results are expressed as the mean ± SD of three independent experiments; *p < 0.05 compared with the pB1-treated group.

[0028] Figure 5.bleogen pB1 activates EGFR and its downstream signaling pathways. (A) Representative immunoblot analysis of phosphorylated tyrosine (P-TYR) and EGFR in HaCaT cells using EGFR antibody (coupled to magnetic beads) in immunoprecipitated samples after incubation with 5 μM bleogen pB1; N = three independent experiments; (B) Representative immunoblot analysis of the expression of phosphorylated MEK1 / 2 (p-MEK1 / 2), total MEK (T-MEK1 / 2), phosphorylated ERK1 / 2 (p-ERK1 / 2), and total ERK1 / 2 (T-ERK1 / 2) in HaCaT cells after incubation with 5 μM bleogen pB1; N = three independent experiments; (C, D) Effects of AG1478 (an EGFR-specific tyrosine kinase inhibitor) and U0126 (a MEK-specific inhibitor) on bleogen pB1-induced proliferation of HaCaT cells in serum-free medium for 72 h. All results are expressed as the mean ± SD of three independent experiments; *p<0.05 compared with the pB1-treated group. (E) Bleogen pB1 culture for 6 hours increased luciferase activity in stably transfected SRE-luciferase reporter HaCaT cells. EGF was used as a positive control. All results are expressed as the mean ± SD of three independent experiments; *, #p<0.05 compared with the control group. (F) Bleogen pB1 treatment for 2 hours upregulated the gene expression of c-fos and c-Jun in HaCaT cells. All results are expressed as the mean ± SD of three independent experiments; *, #p<0.05 compared with the control group.

[0029] Figure 6 .Position scanning of the YAGQK region in bleogen pB1 was performed using D-amino acids.

[0030] (A) Biotin-EGF competitive displacement of EGFR based on TR-FRET was performed using different concentrations of bleogen pB1, D-analogs ([Y25y]pB1, [A26a]pB1, [Q28q]pB1, [K29k]pB1), aB1 (negative control), rT7 (negative control) or EGF (positive control). All results are expressed as the mean ± SD of three independent experiments; (B) Proliferative effect of 1 μM bleogen pB1 or D-analogs ([Y25y]pB1, [A26a]pB1, [Q28q]pB1, [K29k]pB1) on HaCaT cells for 72 hours (detected by crystal violet). All results are expressed as the mean ± SD of three independent experiments; *p<0.05 compared with the control group. #p<0.05 compared with the pB1 group. (C) Anti-EGFR neutralizing mAb (clone LA1) blocked the proliferative effect of [K29k]pB1 in HaCaT cells (detected by crystal violet). Mouse IgG1 was used as a control; all results are expressed as the mean ± SD of three independent experiments; #p < 0.05 compared with the [K29k]pB1 group. *p < 0.05 compared with the [K29k]pB1 and anti-EGFR mAb groups.

[0031] Figure 7 .bleogen pB1 and [K29k]pB1 accelerate wound healing in streptozotocin-induced diabetic mice. In a 5 mm full-thickness splinted excisional wound model using STZ-induced diabetic C57 mice (48 wounds in total; 24 mice), topical application of bleogen pB1 (1 nmol / wound; n=12 wounds; 6 mice), [K29k]pB1 (1 nmol / wound; n=12 wounds; 6 mice), and EGF (1 nmol / wound; n=12 wounds; 6 mice) for 5 consecutive days after injury accelerated wound healing compared with a saline vehicle control group (n=12 wounds; 6 mice). Upper panel: Schematic representation of the treatment regimen. Lower panel: Percentage of wound healing over the course of 14 days after injury. All results are expressed as mean ± SD; *p<0.05 for all treatment groups compared with saline control groups.

[0032] Figure 8.bleogen pB1 and [K29k] pB1 are ultrastable EGFR agonists. Stability of bleogen pB1, [K29k] pB1, EGF, and S-alkylated pB1 (iodoacetamide-) analyzed by RP-HPLC under (A) heat (100°C), (B) human serum, (C) pepsin, (D) trypsin, (E) pronase, and (F) neutrophil elastase treatment; all results are expressed as the mean ± SD of three independent experiments; ND: not detected.

[0033] Fig. 9 .Mass spectrum of MBP removal from MBP-pB1 fusion protein using enterokinase.

[0034] Fig.10 Co-elution of native and recombinant bleogen pB1 by reverse phase HPLC (RP-HPLC). DETAILED DESCRIPTION

[0035] The present invention is based on the inventors' identification of novel cysteine-rich peptides (CRPs) isolated from Pereskia bleo with EGFR binding activity. Specifically, the inventors successfully identified a novel ultrastable EGFR ligand named Bleogen pB1 from Pereskia bleo. The prototypical and cationic bleogen pB1 is a heparin-binding CRP with 36 residues, of which 5 are Lys / Arg and 6 are Cys, with a cysteine ​​motif of C(X)6C(X)7CC(X)3C(X) 10 C (SEQ ID NO: 2). Unlike previously known EGFR ligand structures, bleogen pB1 adopts a four-ring structure with disulfide bonds arranged in a cystine knot connection manner (Cys I-IV, Cys II-V and Cys III-VI). Bleogen pB1 is biosynthesized as a two-domain precursor, and the mature domain is released after signal peptidase cleavage. Bleogen pB1 has a cationic-polar-cationic motif that contributes to its heparin binding properties. Bleogen pB1 also has sequence homology with knottin-type antimicrobial peptides and exhibits anti-Candida properties.

[0036] The inventors also found that Bleogen pB1 does not contain an EGF-like domain and is therefore a first-in-class EGFR agonist 59 years after the discovery of EGF. The 36-residue bleogen pB1 is the smallest natural peptide-based EGFR agonist reported to date. It is 10 and 16 residues shorter than the two smallest EGFR agonists, namely 46-residue epiregulin and 52-residue TGF-α, respectively. EGF and TGF-α are classified as high-affinity EGFR agonists, while amphiregulin and epiregulin are classified as low-affinity agonists. Receptor displacement experiments have shown that bleogen pB1 is about 50-100 times less potent than EGF, making it a low-affinity EGFR agonist. Similar to EGF, bleogen pB1 promotes keratinocyte proliferation and keratinocyte migration. Loop 4 of bleogen pB1 has a high degree of sequence identity and structural similarity to loop C of TGF-α. The presence of a conserved YXGXK / R (SEQ ID NO: 3) motif suggests that they share a common EGFR interaction "hotspot".

[0037] Previous studies (US 5,182,261; Tam J & Ke X (1989) Systematic approach to study the structure-activity of transforming growth factor α. Peptides: chemistry and biology [Proceedings of the 11th American Peptide Symposium], (ESCOM, Leiden), pp75-77) have shown that mutations in the YXGXR loop C of TGF-α, particularly Y38 and R42, lead to a significant reduction in its EGFR affinity and EGF-like mitogenic potency in A431 cells. Similarly, in a separate study of EGF, its Y37 and R41 mutant analogs showed reduced EGF-like activity (Ogiso et al. (2002) Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains. Cell 110 (6): 775-78746, 62-65). Collectively, these findings suggest the importance of the YXGXR motif as a putative receptor contact site for EGFR agonists.

[0038] By point-replacing the YAGQK motif in bleogen pB1, it was demonstrated that all Ala-substituted pB1 analogs had reduced EGF-like bioactivity, which is consistent with observations for EGF and TGF-α. In contrast, two D-amino acid-substituted pB1 analogs, K29k pB1 and Y25y pB1, showed increased EGF-like bioactivity. By replacing the Lys at position 29 with D-Lys in K29k pB1, the low-affinity bleogen pB1 was converted to a high-affinity EGFR agonist. K29k pB1 was found to be as potent as EGF, with a 60-fold increase in potency compared to bleogen pB1. The in vitro results were supported by an in vivo STZ diabetic mouse model of wound healing, which showed that K29k pB1 and EGF acted comparably.

[0039] Sequence alignments show that K29 of bleogen pB1 corresponds to R41 of EGF and R42 of TGF-α. The Arg at this position is a key residue that is absolutely conserved in all known typical EGFR agonists (Ogiso, supra). Structural and mutational studies have also determined that the R41 residue of EGF is critical for forming a salt bridge with D355 of EGFR and is essential for receptor binding (Ogiso, supra). Consistent with previous mutational studies of TGF-α and EGF, these results indicate that K29 is an important molecular determinant of the EGF-like activity of bleogen pB1.

[0040] Compared with EGFR agonists, bleogen pB1 has similar functional characteristics, but its primary sequence, secondary and tertiary structure and biosynthesis are different. All known EGF family agonists, especially those of mammalian origin, contain EGF-like domains (Singh, Carpenter & Coffey (2016) EGF receptor ligands: recent advances. F1000 Research 5). In addition, mammalian EGFR agonists are biosynthesized as type 1 transmembrane precursors and released after proteolytic cleavage by dissociative proteins and metalloproteinases (ADAMs). In contrast, bleogen pB1 does not contain an EGF-like domain; instead, it contains a cysteine ​​motif of SEQ ID NO: 2, which is a typical feature of the 6-cysteine-hevein-like peptide family, and is biosynthesized as a two-domain precursor consisting of an ER signal peptide and a mature domain. This cysteine ​​motif is a cystine-knot disulfide bond connection that is more resistant to proteolytic degradation than EGF. Thus, bleogen pB1 is a prototypic member of a new class of hyperstable EGFR agonists with atypical primary sequence, secondary and tertiary structures, biosynthetic pathway, and high proteolytic stability.

[0041] Efforts have been made to extend the half-life of EGF by developing polymer-based systems, encapsulation and nanotechnology. It was found that bleogen pB1 and potent K29k pB1 exhibit EGF-like activity and share a structurally compact scaffold that is at least 100 times more stable to proteolytic degradation than EGF. Replacing Lys from its L-form to its D-form produced K29k pB1, which is as effective as EGF. By further developing the current method, it is feasible to scale up the production of modified peptides such as K29k pB1. The use of orthogonal tRNA / synthetase pairing can simplify the incorporation of unnatural amino acids for biosynthetic production in Escherichia coli (Liu & Schultz (2010) Adding new chemistries to the genetic code. Annu. Rev. Biochem. 79: 413-444; Liu et al. (2012) Genetic incorporation of d-lysine into diketoreductase in Escherichia coli cells. Amino Acids 43 (6): 2553-2559). In addition, highly time- and cost-effective chemical synthesis and fragment condensation methods can be used for industrial manufacturing. Commercial examples using these technologies include bivacrin (a thrombin inhibitor) and T-20 (an HIV fusion inhibitor). In summary, bleogen pB1 is an ultrastable, atypical, minimal plant-derived EGFR agonist, and its discovery is a promising start. The discovery that the improved pB1 analog, K29k pB1, is as effective as EGF could further advance the development of effective therapeutic analogs for use in wound healing, regenerative medicine, and skin care.

[0042] Based on the above findings, the present invention in a first aspect encompasses an isolated form of an active peptide, and more particularly relates to an isolated peptide comprising or essentially consisting of the amino acid sequence set forth in SEQ ID NO: 1 (Bleogen pB1).

[0043] The peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 is also referred to herein as "Bleogen pB1" or "pB1". As used herein, "isolated" refers to a form of the peptide that has been at least partially separated from other cellular components with which it may naturally occur or be associated. The peptide may be a recombinant peptide, i.e., a peptide produced in a genetically engineered organism that does not naturally produce the peptide.

[0044] The peptide according to the invention exhibits EGFR binding activity, i.e. it is able to recognize and bind to EGFR in a specific manner, i.e. preferentially to other receptor types, and generally binds with an affinity at least 10 or 100 times higher than that observed for non-specific binding. In addition, the peptide preferably also exhibits EGFR activating activity, i.e. acts as an EGFR agonist. In various preferred embodiments, EGFR binding is similar to that of EGF, i.e. the affinity is within ±50% of that of EGF.

[0045] As used herein, "peptide" refers to a polymer obtained from amino acids linked by peptide bonds. A peptide as defined herein may comprise 10 or more amino acids, preferably 20 or more, more preferably 25 or more amino acids, such as 25 to 50 amino acids, more preferably 30 to 40 or 32 to 36 amino acids. As used herein, "polypeptide" refers to a peptide comprising more than 100 amino acids.

[0046] In various embodiments, the peptide comprises or consists of an amino acid sequence having at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.25%, or 99.5% sequence identity or homology to the amino acid sequence set forth in SEQ ID NO: 1 over its entire length. Alternatively, the identity or homology may be with respect to any one of SEQ ID Nos. 12-14. In some embodiments, it has an amino acid sequence that has at least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, 12, 13 or 14 over its entire length, or has an amino acid sequence that has at least 80%, preferably at least 90%, and more preferably at least 95% sequence homology with the amino acid sequence set forth in SEQ ID NO: 1, 12, 13 or 14 over its entire length.

[0047] In various embodiments, the peptide can be a precursor of a mature enzyme. In such embodiments, it may also include additional amino acid sequences in addition to those amino acid sequences listed in SEQ ID NO: 1, 12, 13 or 14. Such precursors typically may include an N-terminal signal peptide, which is typically 20-30 amino acids in length and may be cleaved during post-translational processing.

[0048] The identity of a nucleic acid sequence or an amino acid sequence is usually determined by sequence comparison. This sequence comparison is based on the BLAST algorithm established and commonly used in the prior art (see, for example, Altschul et al. (1990) "Basic local alignment search tool", J. Mol. Biol. 215: 403-410, and Altschul et al. (1997): "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs"; Nucleic Acids Res., 25, p. 3389-3402), which in principle associates the continuous sequences of similar nucleotides or amino acids in nucleic acid sequences and amino acid sequences. The tabular association of the relevant positions is referred to as "alignment". Usually, a computer program available and known to those skilled in the art is used to generate sequence comparisons (alignments), particularly multiple sequence comparisons.

[0049] This type of comparison also allows reporting the similarity between the compared sequences. This is usually expressed as a percentage identity, i.e., the ratio of identical nucleotides or amino acid residues in the same position or positions corresponding to each other in the alignment. In the context of amino acid sequences, the term "homology" is more broadly interpreted as also including consideration of conservative amino acid exchanges, i.e., amino acids with similar chemical activity, because they generally exhibit similar chemical activity within proteins. Conservative amino acid substitutions include, but are not limited to: conservative amino acid substitutions in the context of the present invention include, for example, G=A=S, I=V=L=M, D=E, N=Q, K=R, Y=F, S=T, A=I=V=L=M, Y=F=W. Therefore, the similarity of the comparison sequence can also be expressed as "percent homology" or "percent similarity". Indications of identity and / or homology can be encountered throughout the (poly)peptide or gene, or only in individual regions. Therefore, homologous regions and identical regions of various nucleic acid sequences or amino acid sequences are defined by matching in the sequence. These regions generally exhibit the same function. They can be very small and contain only a few nucleotides or amino acids. Such small regions often perform functions that are critical to the overall activity of the protein. Therefore, it may be useful to refer to only individual regions and optional small regions when matching sequences. However, unless otherwise indicated, indications of identity and homology herein refer to the full length of a nucleic acid sequence or amino acid sequence, respectively.

[0050] In various embodiments, the peptides described herein comprise an amino acid residue C at any one or more positions corresponding to positions 2, 9, 17, 18, 22 and 33 of SEQ ID NO: 1, preferably at least positions 2 and 18, 9 and 22 and / or 17 and 33, more preferably two groups of the aforementioned pairs, and most preferably all six positions.

[0051] In various embodiments, at least the cysteine-rich core motif C(X) is present n C(X) m CC(X) o C(X) p C, wherein X can be any amino acid except C, n is an integer from 4 to 8, m is an integer from 5 to 9, o is an integer from 1 to 5, and p is an integer from 8 to 12, preferably C(X)6C(X)7CC(X)3C(X) 10 C (SEQ ID NO: 2); and / or the amino acid sequence motif YXGXK / R (SEQ ID NO: 3), wherein X may be any amino acid except C, and preferably they are present in combination.

[0052] In various embodiments, the amino acid motif YXGXK / R (SEQ ID NO: 3) may be contained within the motif C(X) n C(X) m CC(X) o C(X) p C's (X) p (X) in the part or the motif of SEQ ID NO: 2 10 Thus, in various embodiments, the peptides of the invention comprise the amino acid sequence C(X)6C(X)7CC(X)3CXXYXGXK / RXXXC (SEQ ID NO:4), CK / RPXGXK / RCXXXXXPPCCXXXCXK / RYXGXK / RXGXCXXK / R (SEQ ID NO:5), or CKPXGXKCXEXXXPPCCXXXCXRYXGXKXGXCXXR (SEQ ID NO:6).

[0053] The motif of SEQ ID NO:3 may be YAGXK (SEQ ID NO:15), wherein X may be any amino acid except C, preferably D, E, N or Q, more preferably N or Q, most preferably Q. The motif of SEQ ID NO:3 may also be YXGQK (SEQ ID NO:16), wherein X may be any amino acid except C, preferably A, V, L, S, more preferably A, V or S, most preferably A. These embodiments also include peptides wherein the motif of SEQ ID NO:15 or 16 is included in a C-rich motif as defined above or in SEQ ID NO:2, 4, 5 or 6.

[0054] In various embodiments, the amino acids listed in SEQ ID Nos. 2-6 and 15-16 are retained in the backbone of SEQ ID NO: 1, while the remaining amino acids may vary within a given range of sequence identity and / or homology. In these embodiments, the substitution may include any other amino acid substitution for C.

[0055] In various embodiments, the peptide comprises the amino acids corresponding to residues 2-33 of SEQ ID NO: 1. In various embodiments, the fragments of the invention comprise an amino acid sequence corresponding to amino acids 2-33 of SEQ ID NO: 1. Thus, the length of the fragment is preferably at least 32 amino acids. Other fragments encompassed by the invention are fragments corresponding to N- and / or C-terminally truncated forms of SEQ ID NO: 1 or a corresponding sequence. N-terminally truncated fragments preferably lack only the first N-terminal amino acid (using position numbering of SEQ ID NO: 1), while C-terminally truncated fragments may lack 1-3 amino acids starting from the C-terminus (using position numbering of SEQ ID NO: 1). In various embodiments, the presence of the amino acid corresponding to position 36 of SEQ ID NO: 1 may be advantageous.

[0056] In various embodiments, the peptides of the invention differ from the sequences listed in SEQ ID NO: 1 in that they comprise one or more amino acid variations, selected from insertions, deletions and / or substitutions. In various embodiments, these variants of SEQ ID NO: 1 comprise any one or more of SEQ ID NOs. 2-6 and 15-16. In various embodiments, the peptides of the invention comprise one or more amino acid substitutions. These substitutions may include a naturally occurring L-amino acid being replaced by another L-amino acid, preferably selected as disclosed herein, in particular at the position designated as X in any of SEQ ID Nos. 2-6 and 15-16. In other embodiments, the substitution is a replacement of an L-amino acid by a D-amino acid, typically the corresponding D-amino acid. Thus, the peptide may comprise one or more D-amino acids. In various embodiments, these substitutions comprise Y25y and / or K29k, most preferably variants comprising a K29k substitution. The amino acid sequences of such variants are listed in SEQ ID Nos. 7-9. As defined below, capital letters in the single-letter code represent L-amino acids, while lowercase letters in the single-letter code represent D-amino acids. Thus, "K" is L-lysine and "k" is D-lysine. Thus "K29k" means that the L-lysine (K) at the position corresponding to position 29 in SEQ ID NO: 1 is replaced by D-lysine (k). If not otherwise indicated, all position numbers used herein refer to position numbers using SEQ ID NO: 1 as a reference.

[0057] In various embodiments, the peptide may have a positive net charge. This means that the peptide comprises amino acid residues such that the sum of all positive charges of the positively charged amino acids (H, K, R) is higher than the sum of all negative charges of the negatively charged amino acids (D, E). In other words, the sum of the amino acid residues of H, K, and R present in the peptide is higher than the sum of the amino acid residues D and E.

[0058] In various embodiments, the peptide comprises one, two or three disulfide bridges, preferably three, preferably selected from disulfide bridges between C2-C18, between C9-C22 and between C17-C33, using the position numbering of SEQ ID NO: 1.

[0059] In various embodiments, the stability of the peptides of the present invention to heat and / or proteases is at least 2 times, preferably at least 5 times, more preferably at least 10 times higher than that of wild-type EGF (whose amino acid sequence is set forth in SEQ ID NO: 10). These can be determined using routine assays known to those skilled in the art, wherein EGF and the peptides of the present invention are measured under the same conditions.

[0060] All amino acid residues are generally referred to herein by single-letter codes, and in some cases, their three-letter codes are used. This nomenclature is well known to those skilled in the art, and is used herein as understood in the art. Replacement refers to the starting amino acid, position number and target amino acid. For example, "K29A" means that the K at position 29 is replaced by A. In addition, all amino acids referred to herein with capital letters or without any further description are L-amino acids. Single-letter codes are used herein to represent D-amino acids with lowercase letters. Therefore, "K29k" means that the L-lysine at position 29 is replaced by D-lysine. In addition, in any sequence given herein, the amino acids separated by " / " mean that those can exist alternatively. Therefore, "K / R" means that K or R residues may exist at a given position.

[0061] In addition to the above modifications, the peptides according to the embodiments described herein may include amino acid modifications, particularly amino acid replacements, insertions or deletions. For example, such peptides are further developed by targeted genetic modification, i.e., by mutagenesis methods, and optimized for specific purposes or special properties (e.g., its activity, stability, etc.). If such additional modifications are introduced into the peptides of the present invention, they preferably do not affect, change or reverse the sequence motifs described in detail above, i.e., C-rich motifs (except variable residues X) and YXGXK / R loops. This means that the fixed positions defined above for these residues / motifs will not be changed by these additional mutations outside the above definitions.

[0062] In various embodiments, the peptides of the invention may be post-translationally modified, such as glycosylated. Such modifications may be performed recombinantly, i.e., directly in the host cell during production, or may be achieved chemically or enzymatically after polypeptide synthesis, such as in vitro.

[0063] The purpose of the modifications described herein can be to introduce targeted mutations (e.g., substitutions, insertions or deletions) into known molecules, for example, to increase binding specificity / affinity and / or improve activity. To this end, in particular, the surface charge and / or isoelectric point of the molecules can be changed, thereby changing their interaction with the target. Alternatively or in addition, the stability of the peptide can be enhanced by one or more corresponding mutations. The favorable properties of individual mutations (e.g., individual substitutions) can complement each other. Examples of such modifications have been described above, including replacing the selected amino acid with its D-amino acid counterpart, particularly the K29k variant.

[0064] In various embodiments, the peptide is characterized in that it can be obtained by using the above-mentioned polypeptide as the starting molecule of single or multiple conservative amino acid substitutions. The term "conservative amino acid substitution" refers to the exchange (substitution) of one amino acid residue for another amino acid residue, wherein this exchange does not cause a change in the polarity or charge at the position of the exchanged amino acid, such as replacing one non-polar amino acid residue with another non-polar amino acid residue. Conservative amino acid substitutions in the context of the present invention include, for example, substitutions disclosed above.

[0065] Alternatively or additionally, the peptide is characterized in that it can be obtained by fragmentation or deletion, insertion or substitution mutagenesis of the peptide herein as an initial molecule, and comprises an amino acid sequence matching the initial molecule listed in SEQ ID Nos. 1, 12-14 over the entire length of at least 32 consecutively linked amino acids thereof (corresponding to amino acids 2-33 of SEQ ID NO: 1). Preferably, in such embodiments, amino acids C2, C9, C17, C18, C22 and C33 and Y25, G27 and K29 ​​are still present in their natural L-conformation or artificially introduced D-form.

[0066] In various embodiments, the present invention therefore also relates to fragments of the peptides described herein, which retain the desired binding and activity. Preferably, they have at least 50%, more preferably at least 70%, most preferably at least 90% of the affinity and / or activity of the original molecule (preferably a peptide with the amino acid sequence of SEQ ID NO: 1). Preferred fragments have been defined above.

[0067] Nucleic acid molecules encoding the peptides described herein, as well as vectors, in particular replication vectors or expression vectors, containing such nucleic acids also form part of the present invention.

[0068] These can be DNA molecules or RNA molecules. They can exist as single strands, as single strands complementary to the single strands, or as double strands. In particular, for DNA molecules, the sequences of the two complementary strands in all three possible reading frames are considered in each case. It is also considered that different codons (i.e., base triplets) can encode the same amino acid, so a specific amino acid sequence can be encoded by a variety of different nucleic acids. Due to this degeneracy of the genetic code, all nucleic acid sequences that can encode one of the above-mentioned peptides are included in this subject of the present invention. The technician can clearly determine these nucleic acid sequences, because although the genetic code has degeneracy, the determined amino acid is associated with a single codon. Therefore, the technician can easily determine the nucleic acid encoding the amino acid sequence starting from the amino acid sequence. In addition, in the context of the nucleic acid according to the present invention, one or more codons can be replaced by synonymous codons. This aspect refers in particular to the heterologous expression of the peptides considered herein. For example, each organism (e.g., the host cell of the production strain) has a specific codon usage. "Codon usage" is understood as the translation of the genetic code into amino acids by the corresponding organism. If a codon located on a nucleic acid encounters a relatively small number of charged tRNA molecules in an organism, a bottleneck may occur in protein biosynthesis. It also encodes the same amino acid, so the codon is translated less efficiently in the organism than a synonymous codon encoding the same amino acid. Since a large number of tRNA molecules exist for the synonymous codon, the latter can be translated more efficiently in the organism.

[0069] By currently known methods, such as chemical synthesis or polymerase chain reaction (PCR) combined with standard methods of molecular biology or protein chemistry, the technician has the ability to produce corresponding nucleic acids or even complete genes based on known DNA sequences and / or amino acid sequences. Such methods are known, for example, from Sambrook, J., Fritsch, EF, and Maniatis, T, 2001, Molecular cloning: a lab manual, 3rd edition, Cold Spring Laboratory Press.

[0070] For the purposes of this article, "vector" is understood to be a composition consisting of nucleic acid, which contains the nucleic acid considered herein as a characteristic nucleic acid region. They enable the nucleic acid to be established as a stable genetic component in a species or cell line through multiple generations or cell division. Especially when used for bacteria, a vector is a special plasmid, i.e. a circular genetic component. In the context of this article, the nucleic acid as considered herein is cloned into a vector. For example, a vector includes a vector originating from a bacterial plasmid, a virus or a phage, or is mainly a synthetic vector or plasmid with a wide range of different derived components. Using other genetic components present in each case, a vector can establish itself as a stable unit in multiple generations of related host cells. They can be present outside the chromosome as a separate unit, or can be integrated into the chromosome into chromosomal DNA respectively.

[0071] The expression vector includes a nucleic acid sequence that can be replicated in a host cell, preferably a microorganism containing them, particularly preferably a bacterium, and expresses the nucleic acid contained therein. In various embodiments, the vector described herein therefore also includes a regulatory component for controlling the expression of the nucleic acid encoding the polypeptide of the present invention. Expression is particularly affected by one or more promoters that regulate transcription. In principle, expression can occur by a natural promoter that is initially located in front of the nucleic acid to be expressed, or by a host cell promoter provided on the expression vector, or by a modified, or completely different, promoter of another organism or another host cell. In the present invention, at least one promoter for expressing the nucleic acid considered herein is accessible and can be used for its expression. In addition, the expression vector can be regulated, for example, by changing the culture conditions or the host cells containing them to reach a specific cell density, or by adding specific substances, particularly gene expression activators. An example of such a substance is galactose derivative isopropyl-β-D-thiogalactopyranoside (IPTG), which is used as an activator of the bacterial lactose operon. In contrast to the expression vector, the nucleic acid contained is not expressed in the cloning vector.

[0072] On the other hand, the present invention also relates to a host cell, preferably a non-human host cell, comprising a nucleic acid as considered herein or a vector as considered herein. Preferably, the nucleic acid considered herein or a vector containing the nucleic acid is transformed into a microorganism, which represents a host cell according to an embodiment. Methods for transforming cells have been established in the prior art and are fully known to the technician. In principle, all cells are suitable as host cells, i.e., prokaryotic or eukaryotic cells. Preferably, a host cell that can be operated in a genetically advantageous manner (e.g., about the conversion using nucleic acids or vectors, and the stability of its construction) is used. In addition, preferred host cells are known for being easy to operate in microbiology and biotechnology. For example, this refers to easy cultivation, high growth rate, low demand for fermentation medium, and good production and secretion rate of exogenous proteins. Peptides can be modified by the cells that produce them after they are manufactured, for example, by adding sugar molecules, formylation, amination, etc. This type of post-translational modification can functionally affect the peptide.

[0073] The activity of the host cells of further embodiments can be regulated based on available genetic regulatory elements, for example on a vector, but can also be present innately in those cells. For example, their expression can be stimulated by controlling the addition of compounds used as activators, changing culture conditions or reaching a specific cell density. Thus, the proteins considered herein can be produced economically. As previously mentioned, an example of such a compound is IPTG.

[0074] Preferred host cells are prokaryotic or bacterial cells, such as E. coli cells. The notable feature of bacteria is that the generation time is short and the requirements for culture conditions are few. Therefore, economical culture methods and manufacturing methods can be established. In addition, technicians have extensive experience in bacteria in fermentation technology. In individual cases, according to various factors determined experimentally, such as nutrient sources, product formation rate, time requirements, etc., Gram-negative or Gram-positive bacteria may be suitable for specific production examples. In various embodiments, the host cell can be an E. coli cell.

[0075] The host cells considered herein may be modified according to their requirements for culture conditions, may contain other or additional selection markers, or may also express other or additional proteins / peptides. They may in particular be host cells transgenically expressing a variety of peptides.

[0076] However, the host cell can also be a eukaryotic cell, characterized in that it has a nucleus. Therefore, another embodiment is a host cell with a nucleus. Compared with prokaryotic cells, eukaryotic cells are able to post-translationally modify the formed protein / peptide. Examples are fungi such as actinomycetes, or yeasts such as Saccharomyces or Kluyveromyces or insect cells, such as Sf9 cells. This may be particularly advantageous, for example when proteins associated with their synthesis are subjected to specific modifications that may be achieved by such systems. The modifications that eukaryotic systems carry out in conjunction with protein synthesis, in particular, include, for example, combining low molecular weight compounds, such as membrane anchors or oligosaccharides. In various embodiments, the host cell is a eukaryotic cell.

[0077] The host cells considered herein are cultured and fermented in a conventional manner, for example in a discontinuous or continuous system. In the former case, the host cells are inoculated into a suitable nutrient medium and the product is harvested from the medium after a period of time determined experimentally. Continuous fermentation is known for achieving a flow equilibrium, in which over a relatively long period of time, the cells partially die but also partially renew, and the peptides formed can be removed from the medium at the same time.

[0078] The host cells contemplated herein are preferably used to produce the peptides described herein.

[0079] Therefore, another aspect of the invention is a method for manufacturing / producing a peptide as described herein, comprising culturing a host cell contemplated herein under conditions that allow expression of the peptide; isolating the peptide from the culture medium or the host cell. The culture conditions and culture medium can be selected by a person skilled in the art according to the host organism used, by employing common sense and techniques known in the art. For example, expression of the peptide can be carried out by using a fusion protein, in which the peptide of the invention is fused to another peptide / protein that facilitates expression / isolation / purification, for example by affinity chromatography. Such fusion constructs are usually processed by treatment with a site-specific protease, which cleaves the expression / affinity tag and thereby releases the target peptide.

[0080] In another aspect, the present invention relates to the use of the peptides disclosed herein as a medicament. The compounds of the present invention are therefore contemplated for use as medicaments.

[0081] On the other hand, the present invention relates to a method for preventing or treating EGF or EGFR-related diseases or conditions in a subject in need thereof using one or more peptides of the present invention. This aspect also encompasses the use of the peptides of the present invention in the manufacture of a drug for treating or preventing EGF or EGFR-related diseases or conditions in a subject in need thereof, wherein the prevention or treatment may include administering a therapeutic or preventive effective amount of the peptides of the present invention. "EGF or EGFR-related diseases or conditions" include not only diseases and conditions directly caused by abnormal activity of EGF / EGFR in a subject, but also diseases and conditions in which EGF and EGFR play a role in signaling pathways and benefit from EGF / EGFR activation. Such conditions include, but are not limited to, wound healing, ulcers, and neurodegenerative diseases, including, but not limited to, diabetic ulcers, gastric ulcers, esophageal ulcers, duodenal ulcers, burn wounds, surgical wounds, pressure wounds, wounds caused by chemotherapy, corneal wounds, Alzheimer's disease, and skin diseases. Skin-related applications include, but are not limited to, wrinkle improvement, skin hydration, pigmentation prevention, skin elasticity improvement, skin stem cell differentiation, etc. Therefore, the above-mentioned treatment includes promoting wound healing or treating any mentioned condition, such as ulcers and neurodegenerative diseases, in a subject.

[0082] In another aspect, the invention relates to a method of treating or preventing an EGF or EGFR-related disease or disorder in a subject in need thereof, comprising administering to the subject a prophylactically or therapeutically effective amount of one or more peptides of the invention.

[0083] Typically, the peptides of the present invention will be administered in a therapeutically / preventively effective amount alone or in combination with one or more other therapeutic agents by any common and acceptable means known in the art. The therapeutically / preventively effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the peptide used, and other factors.

[0084] The peptides of the present invention can be administered as pharmaceutical compositions by any conventional route, in particular topical administration, such as in the form of lotions, gels, eye drops, ointments or creams, and parenteral administration, such as in the form of injectable solutions or suspensions. Such applications also include dosage forms, such as band-aids, in which the composition can be provided on a carrier material, such as a fabric or fiber material, and a hydrogel.

[0085] Therefore, the present invention also relates to a pharmaceutical composition comprising one or more peptides of the present invention and a pharmaceutically acceptable excipient or carrier. The carrier may include a diluent and / or a solvent.

[0086] Pharmaceutical compositions comprising peptides of the present invention in free form or in the form of pharmaceutically acceptable salts and at least one pharmaceutically acceptable carrier or diluent can be manufactured in a conventional manner by mixing, granulating or coating methods. Compositions can be sterilized and / or contain adjuvants, such as preservatives, stabilizers, wetting agents or emulsifiers, solution promoters, salts and / or buffers for regulating osmotic pressure. In addition, they may also contain other substances with therapeutic value. Suitable formulations for topical applications such as skin and eyes are preferably aqueous solutions, ointments, creams or gels well known in the art. These may contain solubilizers, stabilizers, tension enhancers, buffers and preservatives.

[0087] The peptides of the present invention may be administered in therapeutically effective amounts in combination with one or more therapeutic agents (pharmaceutical combinations). Non-limiting examples of compounds that can be used in combination with the compounds of the present invention include: known protease inhibitors, EGF and EGF agonists.

[0088] When the peptides of the invention are administered in conjunction with other therapies, dosages of the co-administered compounds will of course vary depending on the type of co-drug employed, on the specific drug employed, on the condition being treated and so forth.

[0089] As used herein, the terms "co-administration" or "administered in combination" and the like are intended to include administration of selected therapeutic agents to a single patient and are intended to include treatment regimens in which the agents are not necessarily administered by the same route or at the same time.

[0090] As used herein, the term "pharmaceutical combination" refers to a product obtained by mixing or combining more than one active ingredient, and includes fixed and non-fixed combinations of active ingredients. The term "fixed combination" refers to active ingredients (e.g., peptides and adjuvants of the present invention) that are administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" refers to active ingredients (e.g., peptides and adjuvants of the present invention) that are administered to a patient simultaneously, simultaneously or sequentially as separate entities, without specific time limits, wherein such administration provides therapeutically effective levels of two compounds in the patient's body. The latter is also applicable to cocktail therapy, for example, administration of more than 3 active ingredients.

[0091] The pharmaceutical compositions can be used in methods of preventing or treating an EGF or EGFR-related disease or disorder in a subject in need thereof.

[0092] In another aspect, the present invention relates to a method for treating or preventing an EGF or EGFR-related disease or disorder in a subject in need thereof, comprising administering to the subject a preventively or therapeutically effective amount of a pharmaceutical composition of the present invention.

[0093] All embodiments described above for pharmaceutical compositions are similarly applicable to cosmetic or cosmeceutical compositions that also constitute a part of the present invention. It should be understood that, in addition to the peptides of the present invention, all adjuncts present in these compositions are cosmetically or cosmeceutical acceptable. Since cosmetic / cosmeceutical compositions can generally be in the form of topical administration, the above disclosures about the topical forms of pharmaceutical compositions are similarly applicable to cosmetic / cosmeceutical compositions. As used herein, "cosmeceuticals" and "cosmeceuticals" refer to compositions having mixed therapeutic and beauty effects or compositions whose effects cannot be clearly distinguished. Such cosmetic applications include applications involving the skin, such as wrinkle improvement, skin hydration, pigmentation prevention, skin elasticity improvement, etc.

[0094] The peptides of the invention can also be used to activate EGFR in cells. Such uses may include ex vivo uses, such as in cell and tissue culture, including applications for agricultural or food purposes (e.g., cultured meat) and tissue engineering applications (e.g., tissue engineering of hard and soft tissues and osteochondral constructs).

[0095] All embodiments disclosed herein relating to peptides and nucleic acids are equally applicable to the uses and methods described herein, and vice versa.

[0096] The present invention is further illustrated by the following non-limiting examples and the appended claims.

[0097] Example

[0098] Materials and methods

[0099] Material

[0100] Unless otherwise stated, all chemicals and solvents were purchased from Sigma Aldrich and Fisher Scientific, USA. Bleogen pB1 referred to below has the amino acid sequence set forth in SEQ ID NO:1.

[0101] Extraction and purification of natural bleogen pB1

[0102] Fresh Pereskia bleo leaves were collected from the Nanyang Community Herb Garden of Nanyang Technological University, Singapore (provided by Mr. NgKim Chuan). Fresh leaves (1 kg) of Pereskia bleo were mixed with water for 15 minutes, then centrifuged at 9000 rpm for 10 minutes at 4 ° C (Beckman Coulter, USA), and the supernatant was filtered through a glass fiber filter paper with a pore size of 1 μm (Sartorius, Singapore). The filtrate was then loaded into a C18 fast column (Grace Davison, USA) and eluted with 60% ethanol. The eluted fraction was then loaded into an SP Sepharose resin column (GE Healthcare, UK), eluted with 1M NaCl (pH 3.0), and then ultrafiltered (ViVaflow 200, 2000MWCO hydrostatic). Further purification was performed by RP-HPLC and heparin affinity chromatography (Shimadzu, Japan). The presence of bleogen pB1 in the eluted fractions was identified using matrix-assisted laser desorption / ionization-time of flight mass spectrometry (MALDI-TOF MS).The eluted fractions were lyophilized for storage at room temperature.

[0103] Computer simulation

[0104] The interaction between the NMR structure of the bleogen pB1 and the crystal structure of human EGFR (PDB entry: 1VIO) was simulated using the protein-protein docking server ClusPro version 2.0. Docking involved global rigid docking using the fast Fourier transform correlation method. Two sets of 900 lowest energy structures were retained (using electrostatic energies, van der Waals attractions and van der Waals repulsions). The second step consisted of clustering the retained structures using pairwise RMSD. The energy of the complex was minimized and the 10 largest clusters were refined. The highest ranked clusters exhibited the most contacts with the protein.

[0105] Solid-phase peptide synthesis and oxidative folding of bleogen pB1

[0106] Synthetic bleogen pB1 (SEQ ID NO: 1), its Ala analogs ([Y25A]pB1, [G27A]pB1, [Q28A]pB1, [K29A]pB1) and D-analogs ([Y25y]pB1 (SEQ ID NO: 7), [A26a]pB1, [Q28q]pB1, [K29k]pB1 (SEQ ID NO: 8)) were artificially synthesized by Fmoc-based solid phase peptide synthesis on Wang resin at room temperature. The synthesized peptides were cleaved in cleavage mixture (92.5% TFA, 2.5% water; 2.5% 1,2-ethanedithiol; 2.5% triisopropylsilane) at room temperature for 1 hour. The crude cleavage product was then folded under the following folding conditions: 10% DMSO, 90% 0.1M NH4HCO3 aqueous solution with a pH of 8.0, cystamine (10 equivalents) and cysteamine (100 equivalents), and kept at room temperature for 1 hour. The folded pB1 and its Ala and D analogs were purified by preparative HPLC (250x 21mm, 5μm) (Phenomenex, USA) and identified using MALDI-TOF MS. A linear gradient of mobile phase A (0.1% TFA / H2O) and mobile phase B (0.1% TFA / acetonitrile (ACN)) was used. The folding yield was calculated to be 70% by HPLC analysis. 1 H NMR, RP-HPLC and heparin affinity chromatography were used to demonstrate the integrity of the synthetic bleogen pB1 compared to its native form.

[0107] N-terminal biotin labeling of pB1, [Y25y]pB1, and [K29k]pB1 was performed on the peptide resin using a mixture of Fmoc-lysin (biotin) -OH (4.0 eq.), N, N-diisopropylethylamine (DIPEA; 6.0 eq.), (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyBOP; 4.0 eq.) in 50% dimethylformamide (DMF) and 50% 1-methyl-2-pyrrolidone (NMP) at room temperature for 2 hours. After 2 hours of reaction, the biotin-labeled pB1 was cleaved and oxidatively folded under the conditions described above. The folded biotin-pB1 was purified by preparative HPLC and identified using MALDI-TOF MS.

[0108] Cell culture and transfection

[0109] HaCaT (human keratinocytes), HUVEC-CS (endothelial cells) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 100 U / mL penicillin and streptomycin. Normal human epidermal keratinocytes purchased from celltechnology The cells were cultured in DermaLife K medium. They were grown in a humidified incubator at 37°C with 5% CO2. HaCaT cells were stably transfected with pGL4.33[luc2P / SRE / Hygro] (Promega) and selected using 250 μg / mL hygromycin. Stable HaCaT cells expressing the SRE luciferase reporter gene were maintained with 250 μg / mL hygromycin.

[0110] Cell proliferation assay

[0111] Cell proliferation was determined using crystal violet staining. Briefly, 1.0 x 10 4 HaCaT cells were seeded in 96-well plates and bleogen pB1 or EGF (positive control) or 3417-Da peptide aB1 (negative control) were added in serum-free medium. 3417-Da-peptide aB1 is a CRP isolated from Achyranthes bidentata with an amino acid sequence of NCESGTSCIPGAQHNCCSGVCVPIVTIFYGVCY (SEQ ID NO:11). Crystal violet staining was performed as described previously. In brief, after the incubation period, the wells were fixed with 4% buffered paraformaldehyde for 20 minutes. The cells were then stained with 0.25% crystal violet in 20% methanol for 15 minutes. Excess crystal violet stain was rinsed 4-5 times with distilled water and air-dried. Glacial acetic acid (10%) in Milli-Q water was added to extract the crystal violet stain. The cells were then analyzed using a microplate reader (Tecan 200Pro, Switzerland) and the absorbance was measured at 595 nm.

[0112] EdU incorporation analysis

[0113] EdU incorporation assay was performed using the EdU proliferation kit (iFluor 488) according to the manufacturer's instructions. Briefly, 2.5 x 10 4 HaCaT cells were seeded in 96-well plates. After overnight culture, cells were incubated with EdU (10 μM) in serum-free medium with bleogen pB1 or EGF for 4 hours. Cells were then fixed, permeabilized and incubated in a reaction mixture containing iFluor 488 for 30 minutes. Cell nuclei were stained with DAPI. Plates were observed using a fluorescence microscope.

[0114] Cell migration assay

[0115] Cell migration was monitored using a scratch assay. Briefly, HaCaT cells were seeded into 2-well silicone inserts with a gap of 500 μm (ibidi, Germany). After overnight culture, the inserts were removed and cultured with bleogen pB1 or EGF in DMEM medium containing 0.1% FBS. After 8 hours of culture, the wells were photographed using an inverted phase contrast microscope. The images were analyzed for the percentage of cell-free area using the Wimasis image analysis platform.

[0116] Endothelial tube formation assay

[0117] Endothelial tube formation assay was performed using μ-slides (ibidi, Germany). Briefly, 10 μL of gel matrix (BD Matrigel TM Basement Membrane Matrix) was poured onto each μ-slide well and allowed to solidify. HUVEC-CS cells were inoculated with bleogen pB1 or EGF at a density of 2500 cells / well into each well. After 2 h of culture, the wells were photographed using an inverted phase contrast microscope. The branch point numbering of the images was analyzed using the Wimasis image analysis platform.

[0118] Pull-down test

[0119] Pull-down assays were performed using NeutrAvidin UltraLink resin (Thermo Fisher Scientific, USA). Briefly, the resin was washed three times with phosphate-buffered saline (PBS) and incubated with biotin-pB1 / biotin-[K29k]pB1 / biotin-[Y25y]pB1, or biotin (negative control) or biotin-aB1 (negative control) or biotin-EGF (positive control) at room temperature and rotated for 2 hours. 2% bovine serum albumin (BSA) in PBS was added to both tubes, incubated at room temperature and gently mixed by inversion for 2 hours. 600 μg of HaCaT cell lysate was added to each tube and incubated overnight at 4°C with rotation. After incubation, the resin was transferred to The column was centrifuged and washed 10 times with PBS. 6x loading dye containing β-mercaptoethanol was added to the resin and heated at 85°C for 10 minutes. The resulting mixture was centrifuged at 200g for 1 minute and then separated at 100V constant voltage for 120 minutes using 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The blot was transferred on polyvinylidene fluoride (PVDF) membrane (GE Healthcare, Sweden) at 250mA on ice for 120 minutes. The blot was blocked with 5% BSA tris buffered saline and Tween 20 (TBST), and then incubated overnight with anti-EGFR anti-rabbit antibody (1:1000 in 5% BSA TBST; Cell Signaling, USA) at 4°C. After overnight incubation, the membrane was washed 3 times with TBST at room temperature for 10 minutes each time. The blot was then incubated with secondary anti-rabbit horseradish peroxidase (HRP) (Cell Signaling, USA) (1:5000 in 5% BSA TBST) and incubated for another 1 hour at room temperature. The blot was washed 5 times with TBST at room temperature for 10 minutes each before adding chemiluminescent substrate (Advansta, USA) and exposing to X-ray film (Fujifilm, Japan).

[0120] Immunoprecipitation

[0121] Immunoprecipitation was performed using EGFR (D38B1) rabbit monoclonal antibody (Cell Signaling, USA) coupled to magnetic beads. In brief, the magnetic beads were washed 3 times with PBS, and then 50 μg of HaCaT cell lysate was added to each tube and cultured overnight with rotation at 4 ° C. After culture, the magnetic beads were washed 5 times with PBS. 6x loading dye containing β-mercaptoethanol was added to the resin and heated at 85 ° C for 10 minutes. The resulting mixture was centrifuged at 14000g for 1 minute and separated at a constant voltage of 100V for 120 minutes using 10% SDS-PAGE. Blot transfer was performed on PVDF membrane (GE Healthcare, Sweden) at 250mA on ice for 120 minutes. The blot was blocked with 5% BSA TBST and then cultured overnight at 4 ° C with anti-phosphotyrosine-HRP (R&D systems, USA) or anti-EGFR anti-rabbit antibody (1:1000 in 5% BSA TBST; Cell Signaling, USA). After incubation, the membrane was washed three times with TBST at room temperature for 10 minutes each time. The blot was then incubated with secondary anti-rabbit horseradish peroxidase (HRP) (1:5000 in 5% BSA TBST) and incubated for another hour at room temperature. The blot was washed five times with TBST at room temperature for 10 minutes each time before adding chemiluminescent substrate (Advansta, USA) and exposure on X-ray film (Fujifilm, Japan).

[0122] Immunoblot analysis

[0123] The blot was transferred to a PVDF membrane (GE Healthcare, Sweden) at 250 mA for 120 minutes on ice. The blot was blocked with 5% BSA TBST and then incubated at 4°C with anti-P-MEK1 / 2 rabbit antibody (1:2000 in 5% BSA TBST; CellSignaling, USA), anti-MEK1 / 2 rabbit antibody (1:2000 in 5% BSA TBST; Cell Signaling, USA), anti-P-ERK1 / 2 rabbit antibody (1:2000 in 5% BSA TBST; Cell Signaling, USA), anti-ERK1 / 2 rabbit antibody (1:2000 in 5% BSA TBST; Cell Signaling, USA) and anti-β-actin mouse antibody (1:10000 in 5% BSA TBST; Merck Millipore, USA). After incubation, the membrane was washed 3 times with TBST at room temperature for 10 minutes each time. The blots were then incubated with secondary anti-mouse or anti-rabbit horseradish peroxidase (HRP) (1:5000 in 5% BSA TBST; CellSignaling, USA) and incubated for another 1 hour at room temperature. The blots were washed five times with TBST at room temperature for 10 minutes each before adding chemiluminescent substrate (Advansta, USA) and exposure on X-ray film (Fujifilm, Japan).

[0124] TR-FRET EGFR ligand binding assay

[0125] Competitive displacement assays were performed using the EGF-EGFR LANCE Ultra TR-FRET Binding Kit (PerkinElmer, USA) according to the manufacturer's instructions. Streptavidin was conjugated to the LANCE Europium chelate, which was conjugated to biotin-EGF, while EGFR-Fc was conjugated to the LANCE Europium chelate. TMDye-labeled anti-human IgG interacts. In short, different concentrations of bleogen pB1 or its Ala- or D-amino acid analogs or EGF were mixed with the working solution and incubated at room temperature for 2 hours. The TR-FRET ratio was measured using a microplate reader in dual emission mode (excitation: 340nm, emission: 665nm and 615nm) (Cytation 1, USA). The results are expressed as the relative binding percentage of biotin-EGF. The aB1 with the amino acid sequence NCESGTSCIPGAQHNCCSGVCVPIVTIFYGVCY (SEQ ID NO: 11) or the Roseltide rT7 with the amino acid sequence CVSSGIVDACSECCEPDKCIIMLPTWPPRYVCSV (SEQ ID NO: 17) was used as a negative control. EGF (SEQ ID NO: 10) was used as a positive control.

[0126] Luciferase reporter gene assay

[0127] Stable HaCaT cells expressing the SRE luciferase reporter gene were tested for bioluminescent response to treatment with bleogen pB1 and EGF as positive controls. Briefly, cells were cultured in white 96-well plates to reach 90% confluence. Cells were washed with serum-free medium, starved overnight in serum-free DMEM medium, and treated with bleogen pB1 and EGF for 6 hours. Luciferase assays were performed using the ONE-Glo EX luciferase assay system (Promega, USA) according to the manufacturer's instructions. The luciferase was detected using a microplate reader (Tecan 200Pro, Switzerland) to measure luminescence.

[0128] Gene expression analysis

[0129] Using PureLink TM Total RNA was extracted from HaCaT and primary human keratinocytes using RNA mini kit (Thermo Fisher Scientific, USA). TMThe first-strand cDNA was synthesized from 600 ng of total RNA using 1 μg of SYBR Green reverse transcriptase and Oligo (dT) 12-18 (Thermo Fisher Scientific, USA). Quantitative PCR (qPCR) was performed for 40 cycles on a CFX96 Touch real-time PCR detection system (Bio-Rad, USA) using iTaq Universal SYBR Green Supermix (Bio-Rad, USA). PCR reaction (20 μL): 3 μL cDNA, 1 μL primer mixture (10 μM), 6 μL DEPC-treated water, and 10 μL reaction mixture (master mix). The pre-designed primer pairs (Origene, USA) used in the qPCR reactions were as follows: c-fos (NM_005252) was 5'-GCC TCT CTT ACT ACC ACT CACC-3' (forward; SEQ ID NO: 18) and 5'-AGA TGG CAG TGA CCG TGG GAA T-3' (reverse; SEQ ID NO: 19); c-Jun (NM_002228) was 5'-CCT TGA AAG CTC AGA ACT CGG AG-3' (forward; SEQ ID NO: 20) and 5'-TGC TGC GTT AGC ATG AGT TGG C-3' (reverse; SEQ ID NO: 21); the reference gene GAPDH (NM_001256799) was 5'-GTC TCC TCT GAC TTC AAC AGC G-3' (forward; SEQ ID NO: 22) and 5'-ACCACC CTG TTG CTG TAG CCA A-3' (reverse; SEQ ID NO: 23). GAPDH was used as a housekeeping gene for normalization. -ΔΔCT Methods The fold changes of c-fos and c-Jun gene expressions were calculated.

[0130] Full-thickness splinting of mouse wounds

[0131] C57BL / 6 mice were obtained from Vital Rital Laboratories (Beijing, China). Mice were housed in plastic cages at 23 ± 1 °C with a 12-h light / dark cycle and provided with free access to water and food. All experiments were approved and performed in accordance with the institutional guidelines of the Laboratory Animal Center of the Chinese Academy of Medical Sciences (Beijing, China).

[0132] Mice were subjected to full-thickness 5-mm splinted excisional wounds. Briefly, sodium pentobarbital (50 mg kg -1) was injected intraperitoneally to anesthetize mice. The hair on the back was shaved with an electric clipper and then a depilatory cream was applied. A symmetrical full-thickness excisional wound was made using a 5 mm diameter sterile biopsy punch. A splint was placed around the wound using glue and secured with four interrupted sutures. The wound and splint were covered with Tegaderm (3M, USA). The wound diameter was measured on designated days up to 14 days after the injury. The size of the wound was calculated as the average of the two diameter measurements along the x and y axes.

[0133] Management plan for full-thickness splinting wound healing in C57BL / 6 mice

[0134] C57BL / 6 mice were subjected to full-thickness 5 mm splinted excisional injuries as described above. After injury, wounds were treated with saline (vehicle control; n = 30 wounds; 15 mice), EGF (1 nmol / wound, positive control; n = 10 wounds; 5 mice), and bleogen pB1 (10 nmol / wound; n = 10 wounds; 5 mice) for three consecutive days.

[0135] Management plan for full-thickness splinting wound healing in streptozotocin-induced diabetic C57BL / 6 mice

[0136] Diabetes was induced in C57BL / 6 mice by two intraperitoneal injections of STZ (80 mg / kg) on ​​days 1 and 5. Blood glucose was monitored regularly for two weeks starting from day 8. Mice with blood glucose levels above 11.1 mmol / L were considered diabetic and underwent full-thickness 5 mm splinting as described above (a total of 24 mice, two wounds per mouse). After injury, saline (vehicle control; n = 12 wounds), bleogen pB1 (1 nmol / wound; n = 12 wounds), [K29k]pB1 (1 nmol / wound; n = 12 wounds) and EGF (1 nmol / wound; n = 12 wounds) were topically applied to the wound for five consecutive days. Blood glucose was also monitored on day 14 after injury to ensure that the mice were diabetic.

[0137] Neonatal mouse incisor eruption model

[0138] ICR mice were obtained from Vital Rital Laboratories (Beijing, China). The mice were kept in plastic cages at 23±1°C with a 12-hour light / dark cycle and provided with free access to drinking water and food. All experiments were approved and performed in accordance with the institutional guidelines of the Laboratory Animal Center of the Chinese Academy of Medical Sciences (Beijing, China). Subcutaneous injections were performed on the nape of the neck of newborn mice for five consecutive days starting from the day of birth (day 0). A total of 30 newborn mice were randomly divided into the following three test groups: PBS, EGF (3 mg / kg) and bleogen pB1 (3 mg / kg). Incisor eruption was recorded visually every day. Incisor eruption is defined as the time when a given tooth first pierces the oral epithelium.

[0139] Peptide stability assay

[0140] Thermal stability

[0141] 0.1 M purified bleogen pB1, [K29k]pB1, S-alkylated pB1 (iodoacetamide-), EGF, PBS (control) were incubated at 100° C. Samples were collected at different time points (0, 30, 60 and 120 min).

[0142] Acid stability

[0143] 0.1 M purified bleogen pB1, [K29k]pB1, S-alkylated pB1 (iodoacetamide-) and EGF were dissolved in 0.2 M HCl and incubated at 37° C. Samples were collected at different time points.

[0144] Pepsin stability

[0145] 0.1 M purified bleogen pB1, [K29k]pB1, S-alkylated pB1 (iodoacetamide-) and EGF were dissolved in 0.2 M HCl and incubated with pepsin (Roche Applied Science, USA) at a ratio of 50:1 (w / v) at 37° C. Samples were collected at different time points.

[0146] Pronase stability

[0147] 0.1 M purified bleogen pB1, [K29k]pB1, S-alkylated pB1 (iodoacetamide-), and EGF were dissolved in PBS and incubated with pronase (0.2 mg / mL; Roche Applied Science, USA) at 37° C. Samples were collected at different time points.

[0148] Neutrophil elastase stability

[0149] 0.1 M purified bleogen pB1, [K29k]pB1, S-alkylated pB1 (iodoacetamide-), and EGF were dissolved in PBS and incubated with human neutrophil elastase (0.05 mg / mL; Molecular Innovations, USA) at 37° C. Samples were collected at different time points.

[0150] Trypsin stability

[0151] 0.1 M purified bleogen pB1, [K29k]pB1, S-alkylated pB1 (iodoacetamide-), and EGF were dissolved in PBS and incubated with trypsin (0.2 mg / mL; Sigma Aldrich, USA) at 37° C. Samples were collected at different time points.

[0152] Human serum stability

[0153] 0.1 M purified bleogen pB1, [K29k] pB1, S-alkylated pB1 (iodoacetamide-) and EGF were prepared in 25% human serum in DMEM medium without phenol red. The test samples were incubated at 37°C. Samples were collected at different time points. The collected samples were protein precipitated with 100% ethanol and centrifuged at 180000g for 5 minutes at 4°C. The supernatant was collected for analysis.

[0154] Stability test analysis

[0155] All samples collected from various stability tests were analyzed by RP-HPLC using a linear gradient of mobile phase A (0.05% TFA / H2O) and mobile phase B (0.05% TFA / ACN) on an aeris peptide XB-C18 column (Phenomenex, USA). The resulting peaks were collected and identified by MALDI-TOF MS. The results were expressed as a percentage of the initial concentration using the peak area of ​​the HPLC curve.

[0156] Statistical analysis

[0157] Statistical comparisons were performed using GraphPad Version 8.2.1 (US). Data were analyzed using one-way analysis of variance (ANOVA) and Newman-Keuls post hoc test. Data are expressed as mean ± SD. p < 0.05 was considered statistically significant.

[0158] Example 1: Motif search and molecular docking of bleogen pB1 as an EGFR agonist to identify "hot spots" for structure-activity relationship studies

[0159] Seven related mammalian EGFR agonists, including EGF, TGF-α, Hb-EGF, betacellulin, amphiregulin, epiregulin, and epigen, share four conserved non-cysteine ​​residues, three of which are located in loop C (Cys V-VI), forming a specific YXGXR motif (X, any amino acid) ( Figure 1 C). Loop 4 of bleogen pB1 also contains a YXGXK motif that is similar in sequence and structure to loop C of the EGFR agonist ( Figure 1 D). Computer simulations of bleogen pB1 and EGFR (PDB entry 1IVO, chain A) using the protein-protein docking server ClusPro version 2.0 showed that bleogen pB1 loop 4 can bind to EGFR at the same site as EGF loop C, suggesting that this is a common "hotspot" for binding to EGFR ( Figure 1 E) (Ogiso, supra). Importantly, the finding that pB1 and EGFR agonists share the same "hotspot" provided the basis for our subsequent structure-activity relationship studies.

[0160] Example 2: Synthesis and characterization of bleogen pB1

[0161] For in vitro and in vivo assays of EGF-like activity, natural bleogen pB1 isolated from Pereskia plants was used. Natural bleogen pB1 was isolated from leaf extracts of Pereskia bleo using C-18 reverse phase high performance liquid chromatography (RP-HPLC) (Loo et al. (2017) Bleogens: cactus-derived anti-candida cysteine-rich peptides with three different precursor arrangements. Front. Plant Sci. 8:2162). For the preparation of synthetic bleogen pB1, a stepwise solid phase method and Fmoc chemistry were used ( Figure 2 A). After removal of protecting groups and cleavage of the unprotected peptide from the resin support by trifluoroacetic acid (TFA), the crude pB1 product was oxidatively refolded using a redox reagent combination consisting of cysteamine and cystamine in a 10:1 molar ratio in 0.1 M ammonium bicarbonate, pH 8 for 1 hour to yield 70% bleogen pB1. Purified synthetic and natural bleogen pB1 were indistinguishable as demonstrated by RP-HPLC, heparin affinity chromatography, and two-dimensional nuclear magnetic resonance (NMR) Figure 2 B, C).

[0162] In order to equip bleogen pB1 with a chemical affinity probe for target recognition ( Figure 2 A), again fully synthesized by coupling Fmoc-Lys(biotin) to the N-terminus of the protected pB1 peptide still attached to the resin support to provide biotinylated-pB1 (biotin-pB1). These total syntheses provided clear site-specific labeling of the bleogen pB1 at its N-terminus, maintaining the integrity of its side chain functional groups.

[0163] In addition, bleogen pB1 was recombinantly produced in E. coli as an MBP (maltose binding protein) fusion protein using LB medium with IPTG induction expression (0.4 mM, 4 hours at 37°C; 0.1 mM, 20 hours at 15°C; data not shown). The expressed fusion protein was isolated by affinity chromatography using maltose resin and eluted with 10 mM maltose in PBS for purification. The purified yield of MBP-pB1 was approximately 10-15 mg / L LB broth. As shown by mass spectrometry analysis, the MBP tag was removed by treatment with enterokinase ( Fig. 9 ). Fig.10 It is shown that native and recombinant bleogen pB1 co-elute at the same retention time using RP-HPLC.

[0164] Example 3: Bleogen pB1 exhibits EGF-like biological activity

[0165] To determine whether bleogen pB1 is an EGF-like mitogen, its biological effects on the proliferation of HaCaT keratinocytes were examined using EGF as a positive control. The results showed that bleogen pB1 and EGF promoted the proliferation of HaCaT cells with EC50 values ​​of 130 nM and 1.2 nM, respectively ( Figure 3 A, B). The 100-fold difference in mitogenic potency between EGF and pB1 suggests that bleogen pB1 belongs to a family of low-affinity EGFR agonists, such as epigen and amphiregulin. Natural and synthetic bleogen pB1 showed equivalent proliferative activity against HaCaT cells, confirming that the observed proliferative effect was not due to contaminants from plant extracts ( Figure 3 B). As a negative control, the 3417-Da peptide aB1 isolated from Achyranthes bidentata, a 6CHLP belonging to the same CRP family as bleogen pB1, with similar cysteine ​​motifs and disulfide bonds, was active in this assay up to 10 μM. The results also showed that bleogen pB1 promoted the proliferation of primary human keratinocytes ( Figure 3C), and enhanced DNA synthesis in HaCaT cells by 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay ( Figure 3 D). In addition, both EGF and bleogen pB1 accelerated HaCaT cell migration and endothelial cell tube formation (data not shown). This finding was revealed by a landmark EGF assay, the eruption of incisors in newborn mice. The results showed that subcutaneous injection of bleogen pB1 (3 mg / kg) or EGF (3 mg / kg) for 5 consecutive days accelerated the eruption of incisors in newborn mice from 153 hours (saline control) to 125 hours and 100 hours ( Figure 3 E). The results also showed that bleogen pB1 showed in vivo wound healing. Using a full-thickness excisional wound model in C57 mice, three consecutive days of treatment with bleogen pB1 (10 nmol / wound) or EGF (1 nmol / wound) accelerated wound healing from day 3 to day 11 ( Figure 3 F) (Wang et al. (2013), Nat. Protoc. 8(2):302). In summary, these six different in vitro and in vivo experiments strongly support that bleogenpB1 is a mitogen and exerts EGF-like biological activities.

[0166] Example 4: Interaction of Bleogen pB1 with EGFR

[0167] To show that the mitogenic activity of bleogen pB1 is a result of its interaction with EGFR, pull-down and neutralizing antibody assays were used to examine their interaction. Biotin-pB1 was able to pull down EGFR, indicating the presence of a putative ligand-receptor interaction ( Figure 4 A). Co-culture with EGFR neutralizing antibody (clone LA1) blocked the bleogen pB1-induced proliferation of HaCaT cells ( Figure 4 B) These results strongly support that the interaction between the bleogen pB1 and EGFR is specific and that the proliferative effect is EGFR-dependent.

[0168] Example 5: The proliferation effect of bleogen pB1 is associated with the EGFR / MEK / ERK signaling pathway

[0169] EGF activates the EGFR / MEK / ERK signaling pathway, which plays an important role in regulating cell proliferation. We investigated whether bleogen pB1 would undergo a similar EGF signaling pathway to induce keratinocyte proliferation. We found that bleogen pB1 induced phosphorylation of EGFR, MEK1 / 2, and ERK1 / 2 ( Figure 5A, B). In addition, co-culture with a small molecule EGFR tyrosine kinase inhibitor (AG1478) or a MEK inhibitor (U0126) significantly inhibited the proliferation effect of bleogen pB1 in HaCaT cells ( Figure 5 C, D). Collectively, these results indicate that bleogen pB1 binds to EGFR and activates the EGFR / MEK / ERK signaling pathway to trigger HaCaT cell proliferation.

[0170] EGF is known to activate transcription factors that bind to serum response elements (SREs) and initiate transcription of immediate early genes involved in EGFR-mediated cell proliferation regulation, such as c-fos and c-Jun (Lee et al. (2018) Sci. Rep. 8(1):162). It could be demonstrated that bleogen pB1 increased luciferase activity in a dose-dependent manner, similar to EGF in the stably expressed SRE-luciferase reporter HaCaT cell line ( Figure 5 E), indicating that bleogen pB1 stimulates SRE-mediated gene transcription. The effects of EGF- and bleogen pB1-induced transcriptional responses on the expression of EGF-related immediate early genes were also examined using qPCR (Brankatschk et al. (2012), Sci. Signal. 5(215): ra21-ra21). It has been demonstrated that both bleogen pB1 and EGF significantly upregulated the mRNA expression of c-fos and c-Jun in HaCaT cells ( Figure 5 F) In summary, this series of experiments supports the similarity of signaling pathways and transcriptional responses induced by EGF and pB1.

[0171] Example 6: Positional scanning of loop 4 containing the YAGQK motif in bleogen pB1 using Ala- and D-amino acids

[0172] It was hypothesized that the YXGXK / R motif (X, any amino acid; SEQ ID NO: 3) in loop 4 of bleogen pB1 and loop C of typical EGFR agonists may be a common "hotspot" for EGFR interaction. It has been previously demonstrated that mutations of Y38 and R42 in loop C of the TGF-α YXGXR motif using Ala or D-amino acid scanning resulted in a significant reduction in EGFR affinity and EGF-like mitogenic potential (Tam and Tam et al., supra). Therefore, a focused Ala and D-amino acid library of the corresponding "hotspot" of bleogen pB1, YAGQK loop 4 was chemically synthesized. This included an Ala substitution series of [Y25A]pB1, [G27A]pB1, [Q28A]pB1, and [K29A]pB1 and a D-amino acid substitution series of [Y25y]pB1, [A26a]pB1, [Q28q]pB1, and [K29k]pB1. Each peptide was compared to bleogen pB1 in two different studies to test its affinity for EGFR using competitive displacement based on time-resolved fluorescence energy transfer (TR-FRET) and its mitogenic potential using a HaCaT cell proliferation assay. The results showed that bleogen pB1 displaced biotin-EGF with an IC50 of 1720±0.075nM, while the IC50 of EGF was 31±0.016nM ( Figure 6 A). All Ala analogs showed reduced EGF-like bioactivity by EGFR replacement and cell proliferation assays (data not shown). In contrast, the two D analogs [K29k]pB1 and [Y25y]pB1 showed higher affinity than bleogen pB1, with IC50 of 27±0.050nM and 580±0.089nM, respectively. Compared with bleogen pB1, [K29k]pB1 was approximately 60-fold more potent ( Figure 6 B). The results showed that biotin-[K29k]pB1 and biotin-[Y25y]pB1, which were chemically synthesized as previously described for biotin-pB1, interacted with EGFR using a pull-down assay (data not shown). This data indicates that [K29k]pB1 and [Y25y]pB1 are more mitogenic than bleogen pB1, and that their mitogenic effects can be inhibited by an EGFR neutralizing antibody (clone LA1), indicating that their activity is EGFR-dependent ( Figure 6 C).

[0173] Example 7: Bleogen pB1 accelerates wound healing in an excisional wound model using streptozotocin-induced diabetic mice

[0174] We investigated whether the enhanced mitogenic effect of [K29k]pB1 observed in vitro would also accelerate wound healing in streptozotocin (STZ)-induced diabetic mice. Diabetes was induced in C57 mice by two intraperitoneal injections of STZ (80 mg / kg) on ​​days 1 and 5. Blood glucose was monitored regularly for two weeks starting on day 8. Mice with blood glucose levels above 11.1 mmol / L were considered diabetic and subjected to a splinting wound healing model (Huang et al. (2018), FASEB J. 33(1): 953-964). The results showed that wound healing in STZ-induced diabetic mice was significantly delayed compared with that in non-diabetic mice. The results also showed that wounds topically treated with EGF (1 nmol / wound), bleogen pB1 (1 nmol / wound) or [K29k]pB1 (1 nmol / wound) for five consecutive days healed at a significantly faster rate starting on day 4, with macroscopic changes compared with vehicle controls ( Figure 7 ). Notably, [K29k]pB1 promoted wound healing at a similar rate to EGF and much faster than the bleogen pB1. These in vivo results are consistent with the in vitro results of EGFR affinity and mitogenicity, indicating that [K29k]pB1 is as potent as EGF.

[0175] Example 8: Bleogen pB1 and [K29k]pB1 are ultrastable against proteolytic degradation

[0176] It was determined whether bleogen pB1 and [K29k] pB1, which are more compact in structure than EGF, are more proteolytically stable and resistant to heat and acid treatment. All three tested peptides, bleogen pB1, [K29k] pB1, and EGF were relatively stable to acid with calculated t1 / 2 values ​​> 500 minutes (data not shown). However, it was observed that bleogen pB1 and [K29k] pB1 were significantly more stable than EGF in heat treatment with calculated t1 / 2 values ​​> 500 minutes ( Figure 8 A). More importantly, they also tested its proteolytic stability with a panel of different proteases. These included pepsin, human serum, trypsin, pronase, and human neutrophil elastase ( Figure 8BF). Bleogen pB1 and [K29k]pB1 showed a stark contrast in their sensitivity to our panel of proteases compared to EGF. The calculated t1 / 2 values ​​for bleogen pB1 and [K29k]pB1 were >500 min in the panel of proteases and >800 h in human serum. In comparison, the t1 / 2 of EGF ranged from 0.9 to 23.5 min in the presence of this panel of proteases and was 3.1 h in human serum. EGF was at least 100-fold less stable under proteolytic conditions than bleogen pB1 and [K29k]pB1. The control peptide S-alkylated bleogen pB1, in which all disulfide bonds were reduced and S-alkylated by iodoacetamide, was degraded by proteases within minutes, suggesting that the structural integrity of bleogen pB1 and [K29k]pB1 contributes to their proteolytic stability. SEQUENCE LISTING <110> Nanyang Technological University <120> Epidermal growth factor receptor (EGFR) ligand <130> P118398 <150> SG10201906403T <151> 2019-07-10 <160> twenty three <170> PatentIn version 3.5 <210> 1 <211> 36 <212> PRT <213> Pereskia bleo <400> 1 Gln Cys Lys Pro Asn Gly Ala Lys Cys Thr Glu Ile Ser Ile Pro Pro 1 5 10 15 Cys Cys Ser Asn Phe Cys Leu Arg Tyr Ala Gly Gln Lys Ser Gly Thr 20 25 30 Cys Ala Asn Arg 35 <210> 2 <211> 32 <212> PRT <213> Artificial <220> <223> C-rich motif <220> <221> MISC_FEATURES <222> (2)…(7) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURES <222> (9)…(15) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURES <222> (18)..(20) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURES <222> (22)..(31) <223> X can be any amino acid with the exception of C <400> 2 Cys Free Free Free Cys Free Free Cys 1 5 10 15 Cys Free Free Cys Free Free Free Free Cys 20 25 30 <210> 3 <211> 5 <212> PRT <213> Artificial <220> <223> Loop 4 patterns <220> <221> MISC_FEATURE <222> (2)..(2) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (4)..(4) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (5)..(5) <223> X is R or K <400> 3 Tyr Xaa Gly Xaa Xaa 1 5 <210> 4 <211> 32 <212> PRT <213> Artificial <220> <223> C-rich peptide motif <220> <221> MISC_FEATURE <222> (2)..(7) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (9)..(15) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (18)..(20) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (22)..(23) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (25)..(25) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (27)..(27) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (28)..(28) <223> X is K or R <220> <221> MISC_FEATURE <222> (29)..(31) <223> X can be any amino acid with the exception of C <400> 4 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 15 Cys Xaa Xaa Xaa Cys Xaa Xaa Tyr Xaa Gly Xaa Xaa Xaa Xaa Xaa Cys 20 25 30 <210> 5 <211> 35 <212> PRT <213> Artificial <220> <223> C-rich peptide motif <220> <221> MISC_FEATURE <222> (2)..(2) <223> X is K or R <220> <221> MISC_FEATURE <222> (4)..(4) <223> X is any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (6)..(6) <223> X is any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (7)..(7) <223> X is K or R <220> <221> MISC_FEATURE <222> (9)..(13) <223> X is any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (18)..(20) <223> X is any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (22)..(22) <223> X is any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (23)..(23) <223> X is K or R <220> <221> MISC_FEATURE <222> (25)..(25) <223> X is any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (27)..(27) <223> X is any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (28)..(28) <223> X is K or R <220> <221> MISC_FEATURE <222> (29)..(29) <223> X is any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (31)..(31) <223> X is any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (33)..(34) <223> X is any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (35)..(35) <223> X is K or R <400> 5 Cys Xaa Pro Xaa Gly Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Pro Pro Cys 1 5 10 15 Cys Xaa Xaa Xaa Cys Xaa Xaa Tyr Xaa Gly Xaa Xaa Xaa Gly Xaa Cys 20 25 30 Xaa Xaa Xaa 35 <210> 6 <211> 35 <212> PRT <213> Artificial <220> <223> C-rich peptide motif <220> <221> MISC_FEATURE <222> (4)..(4) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (6)..(6) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (9)..(9) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (11)..(13) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (18)..(20) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (22)..(22) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (25)..(25) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (27)..(27) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (29)..(29) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (31)..(31) <223> X can be any amino acid with the exception of C <220> <221> MISC_FEATURE <222> (33)..(34) <223> X can be any amino acid with the exception of C <400> 6 Cys Lys Pro No Gly No Lys Cys No Glu No No Pro Pro Cys 1 5 10 15 Cys cells, Cys cells, Arg cells, Tyr cells, Gly cells, Lys cells, Gly cells, Cys cells 20 25 30 Dear Dear Arg 35 <210> 7 <211> 36 <212> PRT <213> Artificial <220> <223> Variant Bleogen pB1 <220> <221> MISC_FEATURES <222> (25)..(25) <223> X is D-tyrosine. <400> 7 Gln Cys Lys Pro Asn Gly Ala Lys Cys Thr Glu Ile Ser Ile Pro Pro 1 5 10 15 How To Make Gly Gln Lys Make Gly Thr 20 25 30 Cys Only Asn Arg 35 <210> 8 <211> 36 <212> PRT <213> Artificial <220> <223> Variant Bleogen pB1 <220> <221> MISC_FEATURE <222> (29)..(29) <223> X is D-lysine <400> 8 Gln Cys Lys Pro Asn Gly Ala Lys Cys Thr Glu Ile Ser Ile Pro Pro 1 5 10 15 Cys Cys Ser Asn Phe Cys Leu Arg Tyr Ala Gly Gln Xaa Ser Gly Thr 20 25 30 Cys Ala Asn Arg 35 <210> 9 <211> 36 <212> PRT <213> Artificial <220> <223> Bleogen pB1 variant <220> <221> MISC_FEATURE <222> (25)..(25) <223> X is D-tyrosine <220> <221> MISC_FEATURE <222> (29)..(29) <223> X is D-lysine <400> 9 Gln Cys Lys Pro Asn Gly Ala Lys Cys Thr Glu Ile Ser Ile Pro Pro 1 5 10 15 Cys Cys Ser Asn Phe Cys Leu Arg Xaa Ala Gly Gln Xaa Ser Gly Thr 20 25 30 Cys Ala Asn Arg 35 <210> 10 <211> 53 <212> PRT <213> Homo sapiens <400> 10 Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His 1 5 10 15 Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30 Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45 Trp Trp Glu Leu Arg 50 <210> 11 <211> 33 <212> PRT <213> Achyranthes bidentata <400> 11 Asn Cys Glu Ser Gly Thr Ser Cys Ile Pro Gly Ala Gln His Asn Cys 1 5 10 15 Cys Ser Gly Val Cys Val Pro Ile Val Thr Ile Phe Tyr Gly Val Cys 20 25 30 Tyr <210> 12 <211> 36 <212> PRT <213> Salix viminalis <400> 12 Gln Cys Lys Pro Asn Gly Ala Arg Cys Thr Glu Ser Ser Ile Pro Pro 1 5 10 15 Cys Cys Ser Arg Phe Cys Leu Arg Tyr Pro Gly Gln Arg Trp Gly Arg 20 25 30 Cys Ala Asn Arg 35 <210> 13 <211> 36 <212> PRT <213> Salix viminalis <400> 13 Gln Cys Lys Pro Asn Gly Ala Lys Cys Thr Glu Ile Ser Ile Pro Pro 1 5 10 15 Cys Cys Ser Gly Tyr Cys Leu Arg Tyr Ala Gly Gln Lys Ser Gly Thr 20 25 30 Cys Thr Asn Arg 35 <210> 14 <211> 33 <212> PRT <213> Cimicifuga racemose <400> 14 Gln Cys Lys Pro Asn Gly Ala Lys Cys Thr Glu Ile Ser Ile Pro Pro 1 5 10 15 Cys Cys Ser Gly Tyr Cys Leu Arg Tyr Ala Gly Gln Lys Ser Gly Thr 20 25 30 Cys <210> 15 <211> 5 <212> PRT <213> Artificial <220> <223> Peptide motif <220> <221> MISC_FEATURE <222> (4)..(4) <223> X may be any amino acid with the exception of C <400> 15 Tyr Ala Gly Xaa Lys 1 5 <210> 16 <211> 5 <212> PRT <213> Artificial <220> <223> Peptide motif <220> <221> MISC_FEATURE <222> (2)..(2) <223> X may be any amino acid with the exception of C <400> 16 Tyr Xaa Gly Gln Lys 1 5 <210> 17 <211> 34 <212> PRT <213> Hibiscus sabdariffa <400> 17 Cys Val Ser Ser Gly Ile Val Asp Ala Cys Ser Glu Cys Cys Glu Pro 1 5 10 15 Asp Lys Cys Ile Ile Met Leu Pro Thr Trp Pro Pro Arg Tyr Val Cys 20 25 30 Ser Val <210> 18 <211> 22 <212> DNA <213> Artificial <220> <223> Primer <400> 18 gcctctctta ctaccactca cc 22 <210> 19 <211> 22 <212> DNA <213> Artificial <220> <223> Primer <400> 19 agatggcagt gaccgtggga at 22 <210> 20 <211> 23 <212> DNA <213> Artificial <220> <223> Primer <400> 20 ccttgaaagc tcagaactcg gag 23 <210> 21 <211> 22 <212> DNA <213> Artificial <220> <223> Primer <400> 21 tgctgcgtta gcatgagttg gc 22 <210> 22 <211> 22 <212> DNA <213> Artificial <220> <223> Primer <400> 22 gtctcctctg acttcaacag cg 22 <210> 23 <211> 22 <212> DNA <213> Artificial <220> <223> Primer <400> 23 accaccctgt tgctgtagcc aa 22

Claims

1. An isolated peptide having EGFR binding activity, the peptide consisting of: (i) the amino acid sequence set forth in SEQ ID NO: 1; or (ii) a fragment of (i), wherein the fragment is at least 32 amino acids in length; wherein the isolated peptide comprises three disulfide bridges, between C2 and C18, between C9 and C22, and between C17 and C33, using the position numbering of SEQ ID NO: 1; and wherein the isolated peptide comprises: (a) D-tyrosine at the position corresponding to position 25 of SEQ ID NO: 1; or (b) D-lysine at the position corresponding to position 29 of SEQ ID NO:

1.

2. The isolated peptide of claim 1, wherein the peptide consists of the amino acid sequence listed in SEQ ID NO:

7.

3. The isolated peptide of claim 1, wherein the peptide consists of the amino acid sequence listed in SEQ ID NO:

8.

4. The isolated peptide of claim 1, wherein the fragment (ii) comprises: amino acids corresponding to amino acid residues 2-33 of SEQ ID NO:

1.

5. The isolated peptide of claim 1, wherein the peptide has a net positive charge.

6. The isolated peptide of claim 1, wherein the peptide is at least 2 times more stable to heat and / or proteases than human EGF having the amino acid sequence set forth in SEQ ID NO:

10.

7. A nucleic acid molecule encoding the peptide according to any one of claims 1 to 6.

8. A vector comprising the nucleic acid molecule according to claim 7.

9. The vector of claim 8, wherein the vector further comprises a regulatory element for controlling the expression of the nucleic acid molecule.

10. A host cell comprising the nucleic acid molecule of claim 7 or the vector of claim 8 or 9.

11. A method for producing a peptide according to any one of claims 1 to 6, comprising culturing the host cell according to claim 10 under conditions allowing expression of the peptide, and isolating the peptide from the host cell or a culture medium.

12. A composition comprising a peptide as claimed in any one of claims 1 to 6 and a carrier and / or an excipient.

13. The composition of claim 12, wherein the composition is a cosmetic, pharmaceutical or cosmeceutical composition.

14. An in vitro method for activating EGFR in a cell, the method comprising contacting the cell with the peptide of claim 1.

15. The method of claim 14, wherein the method is an in vitro method and the cells are in cell or tissue culture.

16. The method of claim 15, wherein the method is used in tissue engineering applications and / or cell agriculture and food applications.

17. Use of the peptide of claim 1 in the manufacture of a medicament for wound healing in a subject in need thereof.

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