Epidermal growth factor derivative and use thereof

Abstract

The present invention relates to an epidermal growth factor derivative, referred to as tEGFha, comprising or consisting of the amino acid sequence: NSDSECPLSHDGYCLHDGVCYYIENLDRYSCNCVVGYIGYRCQYR. The present invention relates to the epidermal growth factor derivative, referred to as tEGFha, for use in the prevention and / or treatment of a disease related to increased EGFR receptor activity, such as cancer.

Description

[0001] EPIDERMAL GROWTH FACTOR DERIVATIVE AND ITS USE

[0002] Field of invention

[0003] The present invention relates to the field of cancer treatment. In particular, the present invention relates to a protein that is an epidermal growth factor derivative with antitumor activity, designed using computational chemistry and developed biotechnologically.

[0004] Background

[0005] The epidermal growth factor receptor (EGFR) is a cell membrane receptor whose ligands are small extracellular proteins belonging to the epidermal growth factor (EGF) family. EGFR is a member of the ErbB tyrosine kinase receptor subfamily, which includes EGFR (ErbB-1, HER1 in humans), HER2 / c-neu (ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4). Mutations affecting EGFR expression or activity are known to cause cancer [1].

[0006] Currently, several treatments exist in clinical practice that act by blocking or inhibiting ErbB receptors [2], primarily used in colorectal, lung, head and neck, and breast cancers. EGFR-targeted therapies include the use of monoclonal antibodies that specifically recognize the receptor's extracellular domain, blocking ligand binding and inactivating its oncogenic action. They also include small molecules that inhibit EGFR kinase activity, which can easily diffuse into the tumor and tumor cells, blocking intracellular receptor signaling. In many cases, cancer cells activate resistance pathways in response to these treatments, leading to patient relapse.Therefore, efforts are currently being made to develop new agents targeting these receptors [1]. On the other hand, conventional chemotherapy with cytotoxic agents, which induce the death of tumor cells, has high efficacy, but greater side effects [3].

[0007] Therefore, there is a need to develop new products that overcome the aforementioned disadvantages. In response, the inventors have developed a protein, called tEGFha, which is a derivative of human epidermal growth factor (hEGF). The tEGFha protein mimics the natural ligand, so it is expected to easily diffuse to the tumor and bind to EGFR with high affinity, blocking it and preventing its activation by the natural ligand. In this way, tEGFha would inhibit tumor growth. Furthermore, tEGFha promotes the internalization of the receptor into the cell, so it could be used as a vehicle for targeted therapies against EGFR-overexpressing cancers by conjugating it to chemotherapeutic agents or radioisotopes. summary of the invention

[0008] In a first aspect, the present invention relates to a derivative of the epidermal growth factor called tEGFha comprising or consisting of the amino acid sequence (SEQ ID NO: 1):

[0009] NSDSECPLSHDGYCLHDGVCYYIENLDRYSCNCWGYIGYRCQYR. as well as a pharmaceutical composition comprising it.

[0010] In a second aspect, the present invention relates to the epidermal growth factor derivative called tEGFha or a composition comprising it according to the first aspect of the invention for use in the prevention and / or treatment of a disease related to increased EGFR receptor activity, which is cancer.

[0011] Brief description of the figures

[0012] Figure 1. Calculation of the absolute binding energies and amino acid breakdown values ​​of hEGF, truncated EGF (tEGF), and tEGFha in a dimeric system with EGFR (bottom of the graph) or a monomeric system with only the EGFR I ​​domain (top of the graph). The sequence changes corresponding to tEGFha are highlighted.

[0013] Figure 2. Purification and characterization of hEGF and tEGFha. A. Protein analysis of the different purification steps by silver nitrate-stained SDS-PAGE. Molecular weights (M); lane 1: E. coli culture supernatant before purification; lane 2: anion-exchange chromatography product; lane 3: purified product after gel filtration chromatography. B. Determination of the molecular weight of hEGF and tEGFha by mass spectrometry (MALDI-TOF). The analysis confirmed the corresponding molecular weight. C. Analysis of the folding state of purified hEGF and tEGF by RP-HPLC. The table shows the retention time of the peak corresponding to correctly folded EGF, and the percentage of this peak relative to the total protein loaded in the chromatography.

[0014] Figure 3. Determination of the active concentration of recombinant hEGF. BxPC3 cells were treated with 10 nM of commercial EGF or 30 nM of hEGF for 10 min. Left: Cell lysates were analyzed by Western blot with an antibody against total phosphotyrosine residues (only the EGFR band is shown) and against p-actin as a loading control. Right: The bands were quantified using ImageJ software, and the amount of phosphorylated EGFR normalized by the amount of p-actin is shown. C-, negative control in medium without growth factors.

[0015] Figure 4. Effect of tEGFha on EGFR phosphorylation. BxPC3 cells were treated with 10 nM commercial EGF or 30 nM hEGF or tEGFha for 10 min (left) or 30 min (right). Cell lysates were analyzed by Western blot using an antibody against total phosphotyrosine residues or specific phosphotyrosine residues (Y845, Y1045, Y1068, or Y1173). Antibodies against EGFR and p-actin were used as loading controls. C-, negative control in medium without growth factors. C+, positive control with commercial EGF.

[0016] Figure 5. Proliferation assay. The ability of tEGFha to induce proliferation was tested in BxPC3 cells. tEGFha or hEGF proteins were used at a concentration of 30 nM in a medium containing 0.5% FBS and 1 pM insulin. The negative control (C-) consisted of a medium with 0.5% FBS and 1 pM insulin, while the positive control (C+) also contained 10 nM of commercial EGF. Cell proliferation was monitored for 4 days. Data are presented after normalization against the initial cell count (time = 0 h) and as a change in the variance factor relative to the negative control treatment. The mean and SD of three independent experiments are shown. Asterisks indicate the significance level of each treatment relative to baseline, t=Oh (* p<0.05, ** p<0.01). Figure 6. Competence capacity of tEGFha against hEGF for EGFR phosphorylation.BxPC3 cells were treated with 30 nM hEGF or tEGFha at different ratios (1:1, 1:2, and 1:5) for 30 min. Cell lysates were analyzed by Western blot using an antibody against total phosphotyrosine residues and against β-actin as a loading control (top). The change in phosphorylation relative to the positive control (level=1) is shown after normalization of the pTyr signal relative to actin levels (bottom). Asterisks indicate the level of significance with respect to hEGF treatment (* p<0.05, ** p<0.01, *** p<0.001). C-, negative control in medium without growth factors. Figure 7. Proliferation assay to determine the competitive ability of tEGFha against hEGF. BxPC3 cells were treated with 30 nM of hEGF or tEGFha at different ratios (1:1, 1:2 and 1:5), in a medium containing 0.5% FBS and 1 pM insulin.The negative control (C-) consisted of a medium containing 0.5% FBS and 1 pM insulin, while the positive control also contained 10 nM commercial EGF. Cell proliferation was determined at 4 days. Data are presented after normalization versus baseline cell count (time = 0 h) and as a change in the variance factor relative to the negative control treatment. Asterisks indicate the level of significance relative to the positive control (* p<0.05, ** p<0.01).

[0017] Figure 8. Dimerization assay. BxPC3 cells were treated with 10 nM commercial EGF or 30 nM hEGF or tEGF for 1 h. The samples were then treated with BS3, lysed, and analyzed by Western blot using an anti-EGFR antibody and a p-actin antibody as a loading control. The position of the EGFR monomers and dimers is indicated. C-, negative control in medium without growth factors. C+, positive control in medium with commercial EGF.

[0018] Figure 9. Effect of tEGFha on EGFR internalization compared with hEGF. BxPC3 cells were treated with 30 nM hEGF or tEGFha for 30 min at 4 °C to allow ligand binding to the receptor without internalization. The plates were then incubated for 15 or 30 min at 37 °C to allow receptor internalization. EGFR detection in the cell membrane was determined by flow cytometry without permeabilizing the cells. Each column of the graph represents the relative expression of EGFR in the cell membrane versus untreated control cells (C-) and represents the mean ± SD of three independent experiments (* p<0.05, ** p<0.01).

[0019] Figure 10. Effect of tEGFha treatment on EGFR degradation. BxPC3 cells were treated with cycloheximide and subsequently incubated with 30 nM hEGF or tEGFha for several hours. The cells were lysed, and the amount of EGFR was determined by Western blot. Actin levels were used as a load control. EGFR levels are plotted against the negative control (C-, cells in FBS medium, without cycloheximide), after normalization of the signal with respect to actin levels (mean and SD of three independent experiments). Asterisks indicate the level of significance with respect to the negative control (* p<0.05, ** p<0.01).

[0020] Detailed description of the invention

[0021] In a first aspect, the present invention relates to a derivative of the epidermal growth factor called tEGFha comprising the amino acid sequence (SEQ ID NO: 1):

[0022] NSDSECPLSHDGYCLHDGVCYYIENLDRYSCNCWGYIGYRCQYR.

[0023] The improved binding affinity of the tEGFha derivative to EGFR was achieved through molecular modeling, and the resulting optimized sequence features substitutions at four positions (M21, A25, K28, and A29) of natural EGF. The side chains of these residues target EGFR directly and participate in ligand binding to the receptor's domain I. Estimation of the relative binding energy of this derivative to EGFR domain I indicates a significantly enhanced interaction compared to the natural ligand. Therefore, the presence of this sequence in a protein would allow for its specific binding to EGFR and the blocking of natural ligand binding.Therefore, it can be stated that a person skilled in the art would consider any peptide comprising the sequence SEQ ID NO: 1 valid for the purposes of the present invention because the sequence of the invention is capable of achieving that EGFR binding enhancement and natural ligand binding blockade indicated above.

[0024] In another embodiment, the present invention also relates to an epidermal growth factor derivative called tEGFha consisting of the amino acid sequence (SEQ ID NO: 1):

[0025] NSDSECPLSHDGYCLHDGVCYYIENLDRYSCNCWGYIGYRCQYR.

[0026] In another embodiment, the present invention also relates to a pharmaceutical composition comprising the epidermal growth factor derivative tEGFha according to any of the above embodiments.

[0027] In the context of the present invention, the terms “comprises”, “comprising”, “containing” and “having” are open terms and can mean “includes”, “including” and the like; whereas the terms “consisting of” or “consisting of” refer to the elements mentioned after these terms and exclude others that are not mentioned.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary knowledge in the field to which the present invention pertains. The singular terms “a,” “an,” “the,” and “the” include plural referents unless the context clearly indicates otherwise. Similarly, the word “or” is intended to include “and,” unless the context clearly indicates otherwise.

[0029] It should be noted that the term “approximately” applied to the values ​​used earlier and later in this document includes a margin of error of ± 5%, such as, for example, ± 4%, ± 3%, ± 2%, ± 1%.

[0030] In a second aspect, the present invention relates to an epidermal growth factor derivative comprising or consisting of the amino acid sequence (SEQ ID NO: 1):

[0031] NSDSECPLSHDGYCLHDGVCYYIENLDRYSCNCWGYIGYRCQYR, or the pharmaceutical composition comprising it, for use in the prevention and / or treatment of a disease related to increased EGFR receptor activity that is cancer.

[0032] Among the types of cancer that may be mentioned as targets of the present invention are, but are not limited to, cancer of the oral cavity and pharynx, cancer of other digestive organs, cancer of other respiratory organs, cancer of bone and articular cartilage, melanoma and other malignant neoplasms of the skin, cancer of mesothelial tissues and soft tissues, cancer of genital organs, cancer of the urinary tract, cancer of the eye, brain and other regions of the nervous system, cancer of the thyroid and other endocrine glands, malignant neuroendocrine tumors, cancer of lymphoid, hematopoietic and related tissues, carcinomas in situ, benign tumors, neoplasms of uncertain behavior, polycythemia vera and myelodysplastic syndromes, neoplasms of other locations, and neoplasms of unspecified behavior. Preferably, such cancer is selected from head and neck cancer, colon cancer, colorectal carcinoma, colorectal adenocarcinoma,prostate cancer, prostate adenocarcinoma, prostate carcinoma, breast cancer, breast carcinoma, breast adenocarcinoma, triple-negative breast cancer, brain cancer, brain adenocarcinoma, brain neuroblastoma, lung cancer, lung adenocarcinoma, lung carcinoma, small cell lung cancer, ovarian cancer, ovarian carcinoma, ovarian adenocarcinoma, uterine cancer, gastroesophageal cancer, renal cell carcinoma, clear cell renal cell carcinoma, endometrial cancer, endometrial carcinoma, endometrial stromal sarcoma, cervical carcinoma, thyroid carcinoma, metastatic papillary thyroid carcinoma, follicular thyroid carcinoma, bladder carcinoma, urinary bladder carcinoma, transitional cell carcinoma of the urinary bladder, liver cancer, metastatic liver cancer, pancreatic cancer, neuroendocrine cancers, squamous cell carcinoma, osteosarcoma, rhabdomyosarcomaEmbryonic cancers, glioma, neuroblastoma, medulloblastoma, retinoblastoma, nephroblastoma, hepatoblastoma, melanoma, hematologic neoplasms such as leukemias, lymphomas, and myelomas. More preferably, such cancer is selected from head and neck cancer, pancreatic cancer, biliary tract carcinoma, neuroblastoma, colon cancer, breast cancer, myeloma, gastric cancer, liver cancer, glioblastoma, ovarian cancer, colorectal cancer, non-Hodgkin lymphoma, lung cancer, prostate cancer, small cell lung cancer, large cell lung cancer, kidney cancer, esophageal cancer, stomach cancer, cervical cancer, or lymphoma tumors.

[0033] As previously stated, the epidermal growth factor derivative of the present invention, called tEGFha, has the ability to bind to and block the ErbB family epidermal growth factor receptor (EGFR) cell receptor, which is overexpressed in various types of tumors, and inhibits the ability of EGFR to promote cell proliferation and tumor growth.

[0034] In another preferred embodiment, the epidermal growth factor derivative, as defined above, is either alone, acting as an EGFR-blocking agent to stop cell proliferation, or conjugated with a chemotherapeutic agent—that is, known compounds used for the prevention and / or treatment of cancer that can potentially be chemically bound to the protein. In addition to chemotherapeutic agents, radioisotopes could also be conjugated for localized radiotherapy. Furthermore, photosensitizing agents could be conjugated for use in photodynamic therapy.

[0035] As used herein, the terms “treat” or “treating” or “treatment” and their equivalents refer to an improvement of at least one noticeable symptom of cancer. Likewise, the terms “treat” or “treating” or “treatment” refer to inhibiting the progression of at least one noticeable symptom of cancer, either physically (e.g., stabilization of a noticeable symptom), physiologically (e.g., stabilization of a physical parameter), or both. Additionally, the terms “treat” or “treating” or “treatment” refer to slowing the progression or reversing the progression of at least one noticeable symptom of cancer. In this document, the terms “prevent” or “prevention” and their equivalents refer to delaying the onset or reducing the risk of acquiring at least one noticeable symptom of cancer.

[0036] In this document, the term “prevent and / or treat” includes “prevent and treat” and “prevent or treat”.

[0037] In another embodiment, the epidermal growth factor derivative as defined above, or the pharmaceutical composition containing it, is administered orally (e.g., in the form of capsules, tablets, pills, powders, granules), rectally, nasally, ocularly, topically, vaginally, parenterally, transdermally, intraperitoneally, intrapulmonarily, intranasally, or by intravesical infusion. Preferably, administration is by parenteral route or by intravesical infusion. Depending on the route of administration, the epidermal growth factor derivative as defined above is mixed with one or more pharmaceutically acceptable excipients.

[0038] It should be noted that the present invention encompasses any combination of the above embodiments, cases, or examples that a person skilled in the art may consider feasible in the context of the present invention.

[0039] The different cases or particular examples indicated below are provided for illustrative purposes only and are not intended to limit in any way the scope of the present invention

[0040] EXAMPLES

[0041] 1. Methodology

[0042] 1.1. Computational design of the EGF derivative tEGFha

[0043] 1. 1. 1 Molecular dynamics (MD) simulations

[0044] MD simulations were performed using the crystal structure of the human EGF complex with the extracellular domains of the EGFR receptor (PDB 11VO), after modeling the first four amino acids of the EGF structure. The structure of the complex formed by tEGFha and the receptor was obtained using structure 1IVO after removing and modifying the EGF sequence with Pymol.

[0045] All simulations were performed using the AMBER 16 package [4] on an Nvidia GTX1080-based GPU cluster. Amino acid protonation states were predicted using the propkA program on the PDB2PQR server. The AMBER tleap module was used to create the parameter and topology files for the MD simulations, which were performed using the ff99SBildn force field [5]. Hydrogen atoms were added, and ionizable residues were adjusted to the predicted protonation states at pH 7.0. Na+ counterions were added to each system to achieve neutrality. TIP3P water molecules were added with a minimum separation of 10.0 Å from the box edges to the protein molecule. Energy minimization in each system was performed in a two-stage process; The first is to minimize only the positions of solvent molecules and ions, and the second is to minimize the position of all atoms in the simulation cell.The systems were then gently heated in six 50 ps steps, increasing the temperature by 50 K at each step (0–300 K) at constant volume, followed by an equilibration step of 2 ns with a time interval of 2 fs at a constant pressure of 1 atm (using the NPT set). Finally, conventional constant volume and temperature (NVT, 300 K) MD trajectories were collected. In total, three to four replicates of 500 ns–1000 ns MD simulations were performed for each system.

[0046] 1. 1.2 MM-P(G)BSA calculations

[0047] The binding energy between EGFR and tEGFha was calculated using the MM-PBSA / MM-GBSA (Molecular Mechanics-Poisson-Boltzmann or Generalized Born Surface Area) method in Amber16 [6]. For the EGF-EGFR dimehic system, the binding energy of each of the two ligands was evaluated. Since the energy obtained was the same, only the binding energy of one of the two ligands was evaluated for the tEGFha-EGFR dimehic system.

[0048] MM-PBSA and MM-GBSA calculations were performed using 150 snapshots over 300 ns of simulations with 2 ns intervals. All energy values, including amino acid breakdown values, represent at least two independent MD simulation replicates for each ligand-receptor complex. All calculations were performed using the MMPBSA.py module in Amber 16 with an ionic strength of 0.1 M. For PBSA calculations, the PB equation was solved numerically using the PBSA program included in AmberTools. The hydrophobic contribution was approximated using the LCPO method implemented in the Sander simulator [7,8]. For MM-GBSA calculations, the GB(igb)8 model was used as the best configuration for protein analysis [6].

[0049] 1. 1.3 Design of the tEGFha with the Roseta software

[0050] The affinity of the tEGF protein for EGFR was enhanced using the Rosetta program. The tEGF-Doml complex was obtained using structure 1 IVO, after modeling residues 1 to 4 and retaining up to residue R45 of EGF to obtain tEGF, while retaining only residues E2 to S162 of EGFR (domain I). A 500 ns molecular dynamics simulation was performed, and three iterations of the Rosetta design were carried out using the four most populated clusters from the simulation after the structures were relaxed.

[0051] The polar M21 mutation was introduced to avoid the thioether group in preparation for future conjugation with metal-based drugs. Additionally, other mutations were limited to residues 21 to 32 of tEGF to improve EGFR binding characteristics. To this end, MM-PBSA and amino acid decomposition calculations were performed on 500 ns MD simulations of tEGFha complexed with EGFR domain I, following the procedure described above.

[0052] 1.2 tEGFha production

[0053] 1.2.1 Cloning

[0054] The tEGFha coding DNA sequence was synthesized by Integrated DNA Technologies Inc. This sequence includes the ompA sequence, along with the Xbal restriction site at the 5' end and the BamHI restriction site at the 3' end of the coding sequences. The DNA was then digested with the appropriate restriction enzymes and ligated to the plN-lll-ompA-2 expression vector, which contained the ampicillin resistance gene, using a T4 DNA ligase (Thermo Scientific). The resulting plasmid was transformed into E. coli DH5a cells. Positive clones were identified based on antibiotic resistance and subsequently sequenced to validate the accurate cloning of tEGFha (Macrogen). 1.2.2 Production and Purification

[0055] Colonies of the DH5a strain containing the correct plasmid were inoculated into 5 mL of Luria-Bertani (LB) medium supplemented with 50 mg / mL ampicillin and incubated overnight at 37°C with shaking at 250 rpm. The cultures were then centrifuged to separate the cells, and the plasmids were isolated and purified using the GeneJET Plasmid Miniprep Kit (Thermo Scientific). The pure plasmid was used to transform the Rosetta GamiB strain of E. coli. Once ampicillin-resistant colonies were obtained, they were cultured in 5 mL of LB medium supplemented with 50 mg / mL ampicillin and 100 mg / mL chloramphenicol. The cultures were incubated overnight at 37°C with shaking at 250 rpm. Subsequently, 2 mL of each overnight culture were inoculated into 200 mL of M9 minimal medium supplemented with 50 mg / mL of ampicillin and 100 mg / mL of chloramphenicol. The cultures were incubated at 37°C with shaking at 250 rpm for 3 h.Next, 100 pg / mL of IPTG (isopropyl pD-1-thiogalactopyranoside) was added to induce protein expression, and the cultures were incubated again for 3 h at 37°C with shaking at 250 rpm.

[0056] After the induction period, the culture medium was centrifuged at 4000 rpm and 4°C for 10 min. The supernatants were then filtered through 0.22 µm filters. The filtered samples were subsequently dialyzed against MilliQ water in preparation for fast-throughput liquid chromatography (FPLC) analysis, which was performed using an anion-exchange column (HiPrep DEAE FF 16 / 10) on an AKTAbasic system (GE Healthcare Bio-Sciences AB). Chromatography was carried out at a flow rate of 2 mL / min with elution buffers of 50 mM Tris-HCl, pH 7.5 (buffer A) and 50 mM Tris-HCl, 1 M NaCl, pH 7.5 (buffer B). Samples were eluted using a linear gradient of 0–10% buffer B for 4 minutes, 10–50% for 32 minutes, and 50–100% for 4 minutes. Recombinant proteins were collected approximately 20 minutes after the elution process, dialyzed against MilliQ water, and lyophilized.In the final purification step, the lyophilized samples were dissolved in 250 mL of elution buffer (50 mM Tris-HCl, 200 mM NaCl, pH 7.5) and subjected to atmospheric gel filtration chromatography. The proteins were eluted with approximately 25 mL of elution buffer. They were then dialyzed, lyophilized, and redissolved in sterile PBS. The proteins were quantified and their purity confirmed using the Pierce Silver Stain Kit (Thermo Fisher), revealing a single band of approximately 6 kDa.

[0057] 1.2.3 Product characterization by MALDI-TOF and reversed-phase chromatography At each stage of production, the presence of EGF or tEGFha was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 18%, followed by staining with the Pierce Silver Stain Kit (Thermo Fisher).

[0058] Subsequently, the molecular weight and purity of the proteins were evaluated by matrix-assisted laser desorption / time-of-flight mass spectrometry (MALDI-TOF MS) at the technical services of the University of Girona.

[0059] In addition, the folding states of EGF and tEGFha were analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) at the Laboratory of Innovation in Processes and Products of Organic Synthesis, also located at the University of Girona.

[0060] 1.3 Biological activity

[0061] 1.3. 1 Affinity

[0062] EGFR binding assays were performed on an Octet R2 (Sartorious) using streptavidin-coated biosensors (SA biosensors). The biosensors were equilibrated for at least 10 minutes in binding buffer (PBS, 0.05% Tween-20, 0.1% BSA). Experiments were designed and executed using Octet BLI Discovery 13.0 software. Biotinylated EGFR (Aero Biosystems) was immobilized on the biosensor by immersing the sensors in a 1 pg / mL solution for 500 s, followed by immersion of the sensor in fresh binding buffer for 200 s to establish a baseline. Titrations were performed at 25°C while rotating at 1000 rpm. In the association phase, the sensors were immersed in a solution containing EGF or tEGF at various concentrations in binding buffer for 150 s. After reaching equilibrium, the biosensors were immersed in a fresh binding solution to monitor dissociation kinetics for 400 s.Kinetic data were collected and processed using a 1:1 bonding model to obtain affinity constants using Octet Analysis Studio 13.0. The 1:1 bonding model was used to obtain the affinity constants.

[0063] 1.3.2 EGFR Activation and its Signaling Pathway. BxPC3 cells were seeded in 6-well plates at a concentration of 1 million cells / plate. After 24 h of incubation, the cells were washed with PBS and FBS-free medium was added to avoid growth factors that could stimulate the EGF pathway. After 8 h, the cells were washed and hEGF or tEGFha solution was applied in FBS-free medium for the time required for each experiment. The cells were then washed with cold PBS and lysed using RIPA (20 mM sodium phosphate pH 7.4; 150 mM NaCl; 1% Triton X-100; a protease inhibitor cocktail – Cumplete, Roche Diagnostics – and 250 pg / ml sodium vanadate). The samples were homogenized using 0.9 mm syringes and centrifuged to recover the protein lysate. The protein concentration of the supernatant was quantified using the Pierce™ BCA Protein Assay Kit (Thermo Scientific).Next, SDS-PAGE (synthetic acrylamide gel electrophoresis) was performed using 15 pg of protein under denaturing conditions with 5% p-mercaptoethanol in loading buffer (120 mM Tris / HCl, 0.8% SDS, 40% glycerol, 0.05% bromophenol blue, pH 6.8) followed by heat treatment at 95 °C for 5 min. Electrophoresis gels were prepared using either 8% or 18% acrylamide, depending on the experiment. After adding the samples and the molecular weight marker (PageRuler™ Prestained Protein Ladder, Thermo Scientific), the SDS-PAGE gels were run at 100–120 V for 1–2 h.

[0064] The proteins were then transferred to Immobilon®-P polyvinylidene fluoride membranes (Millipore) for 3 h at 100 V and blocked for 1 h in 3% BSA blocking buffer (Merck) in TBS-T (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween-20) to prevent nonspecific antibody binding. The membranes were incubated overnight at 4 °C with the primary antibody diluted in blocking buffer. Overall and specific EGFR phosphorylation levels were analyzed by Western blot. The cells were treated with 30 nM hEGF or tEGFha for 10 min or 30 min. The primary antibodies used recognize pTyr, EGFR, p-EGFR (Y845), p-EGFR (Y1045), p-EGFR (Y1068), p-EGFR (Y1173), or p-actin. 10 nM of commercial EGF was used as a positive control, and untreated cells as a negative control.

[0065] The membranes were then washed with TBS-T and, if necessary, incubated with a secondary antibody (Goat anti-Rabbit peroxidase conjugate, GaR-POD) for 1 h at room temperature in blocking buffer. The results were visualized using the Immobilon® Western Chemiluminescent HRP Substrate (Millipore). Anti-actin detection was added as a control for protein content. Western blots were analyzed using Fiji / ImageJ software. 1.3.3 EGFR Dimerization

[0066] The ability of hEGF and tEGFha to induce receptor dimerization was determined using the protocol previously described by Turk et al. [9]. BxPC3 cells were seeded in 6-well plates and allowed to grow to 60% confluence. At this point, the cells were left unstimulated for 8 h in FBS-free medium and then washed three times with cold PBS. They were then treated with 30 nM hEGF or tEGFha in cold FBS-free medium and incubated for 1 h at 4 °C. This allows ligand-receptor interaction, producing dimerization, but does not allow internalization or subsequent recycling or degradation. Once the incubation with the treatment was completed, the cells were washed with cold PBS three times, and a cold dilution of 3 mM bis(sulfosuccinimidyl) suberate (BS3) (Thermo Scientific) was added to the wells for 20 min, to produce chemical crosslinking of nearby molecules.Following treatment with BS3, the reaction was stopped by washing with a cold 250 pM glycine solution for 5 min. The cells were then washed three times with cold PBS and lysed with RIPA buffer. The presence of dimers was detected by Western blot using anti-EGFR and anti-p-actin antibodies to detect the proteins of interest. The monomeric form of the receptor was detected at 170 kDa, and the dimeric form was detected at 340 kDa.

[0067] 1.3.4 Proliferation test

[0068] To evaluate the potential of tEGFha to arrest cell growth, BxPC3 cells were seeded in 96-well plates at a density of 2,000 cells per well. This seeding concentration was pre-optimized to ensure that the cells remained in the exponential growth phase throughout the experiment. Once seeded, the cells were grown for 72 h in culture medium and washed twice with PBS. They were then treated with either 30 nM hEGF or tEGFha in medium containing 0.5% FBS and 1 pM insulin. Cell proliferation was assessed every 24 h using the CyQuant cell proliferation assay (Thermo Scientific), according to the manufacturer's instructions. Next, the plates were incubated at 37 °C for 1 h and the fluorescence intensity of each well was measured using a microplate absorbance reader (ELx800; BioTek, Winooski, VT, USA) at a wavelength of 520 nm.Each treatment was performed in duplicate, with untreated cells (0.5% FBS and 1 pM insulin) as a negative control, and 10 nM commercial EGF as a positive control. The increase in cell proliferation was calculated by determining the increase in fluorescence at each time point relative to its initial starting point (0 h) and was normalized with respect to the control cells.

[0069] 1.3.5 Competency tests

[0070] Competition assays were performed to determine whether tEGFha was able to compete with hEGF for binding to EGFR. Competition was assessed using two different techniques:

[0071] 1) Western blot, analyzing EGFR phosphorylation levels in the simultaneous presence of hEGF and tEGFha. BxPC3 cells were seeded and cultured without stimulation as previously described for 8 h. The cells were then treated with hEGF at 30 nM and tEGFha at different ratios (1:1, 1:2, 1:5) for 10 min. The cells were then washed and lysed using RIPA buffer. Western blot was performed using anti-pTyr and anti-p-actin antibodies, as previously described.

[0072] 2) Analyzing the proliferation capacity of BxPC3 cells in the simultaneous presence of hEGF and tEGFha. BxPC3 cells were seeded in 96-well plates at a density of 2,000 cells / well and incubated for 72 hours. The cells were then washed and treated with 30 nM hEGF plus tEGFha at different ratios (1:1, 1:2, 1:5) in media containing 0.5% FBS and 1 pM insulin. Cell proliferation was determined using the CyQuant cell proliferation assay, as previously described, at = 0, ht = 96 h. Each experimental treatment was performed in duplicate, with untreated cells cultured in medium containing 0.5% FBS and 1 pM insulin as a negative control, and 10 nM commercial EGF used as a positive control. To assess cell viability, the increase in cell proliferation was calculated, as described above.

[0073] 1.3.6 Internalization Trials

[0074] To explore whether tEGFha could be internalized via the EGFR receptor, BxPC3 cells were seeded in 12-well plates at a density of 100,000 cells / well and allowed to fix for 24 h. The cells were then deprived of growth factors for 8 h by washing with PBS and adding FBS-free medium. This was done to expose EGFR on the cell surface. The cells were then treated with 30 nM hEGF or tEGFha and incubated on ice for 30 min to allow binding to EGFR. The plates were then incubated for 15 or 30 min at 37 °C to allow receptor internalization. The cells were harvested by trypsinization and fixed with 1.5% formaldehyde in buffer (2% FBS, PBS) for 30 min at room temperature. Subsequently, the cells were incubated with anti-EGFR antibody (mAb225 Calbiochem) at a dilution of 1:100 and incubated for 30 min on ice.After washing the cells with buffer, a secondary antibody (Alexa Fluor 488 goat anti-mouse IgG) was added at a 1:200 dilution for 30 min on ice and protected from light. Finally, the cells were washed with buffer and fluorescence was measured using a Novocyte flow cytometer (Agilent Technologies) equipped with NovoExpress software. The median fluorescence intensity was established for 10,000 cells, and the increase compared to untreated control cells was determined.

[0075] 1.3.7 EGFR Degradation

[0076] For the analysis of EGFR degradation, BxPC3 cells were seeded in 6-well plates and incubated for binding. Subsequently, the cells were placed in FBS-free medium containing 10 pg / mL of the protein synthesis inhibitor cycloheximide (Sigma Aldrich), and hEGF or tEGFha at 30 nM was added for different times (30, 60, 180, and 300 min). After treatment, the cells were harvested and lysed, and the different samples were analyzed by Western blot using an anti-EGFR antibody and an anti-β-actin antibody, as previously described. Untreated cells were used as negative controls.

[0077] 2. Results

[0078] 2.1 Computational design of the tEGFha derivative

[0079] The design of tEGFha was based on a previous study of the interaction mechanism of tEGF with EGFR

[0010] . tEGF consists of a shortened version of hEGF, from which the last 8 residues of its C-terminus are cleaved. tEGF interacts with EGFR, but its truncated form prevents the receptor from forming dimers and becoming activated, thus blocking its activity and therefore cell proliferation. However, its low affinity for the receptor prevents it from accumulating in tumors in vivo

[0011] , which restricts its use as a therapeutic agent.

[0080] The main limitation of tEGF is the removal of the C-terminal end in its design. The C-terminal end of hEGF interacts with site 3 in domain III of EGFR with high affinity (Kd=400nM) [12,13]. The absence of this end in tEGF means that it primarily interacts with EGFR at site 1 in domain I, corresponding to a low-affinity interaction site (Kd=50-200pM)

[0012] . Consequently, tEGFha has been designed with increased affinity for EGFR, enhancing interactions with domain I of EGFR without losing its blocking properties. Furthermore, Met21 has been modified for potential application of the protein as a metallodrug carrier, since the thioether group of Met can compete with the complex for metal coordination

[0014] .

[0081] The design of tEGFha was performed using Rosetta software, and the absolute and relative binding energies were calculated considering both the interaction with domain I only and with the entire receptor, as well as the contribution of each amino acid in this interaction (fig 1).

[0082] After two rounds of refinement with Rosetta, a sequence with eight changes was obtained between residues 20 and 30 of EGF, corresponding to the segment that interacts with site 1 of EGFR domain I. Four of these positions (M21, A25, K28, and A29) are indeed oriented towards EGFR and participate in the interaction with site 1 in domain I. On the other hand, other positions suggested by Rosetta for modification are not oriented towards the receptor, and therefore, their modification did not appear to enhance its interaction; thus, they were discarded in the final design. Furthermore, a modification at position 40 was also included, since amino acid breakdown after the second round of Rosetta revealed a loss of stability at this residue, which plays a fundamental role in the interaction with domain III. Consequently, a mutation was introduced at this point to preserve stability (E40Y).

[0083] The estimation of relative binding energy indicates that tEGFha exhibits an improved interaction with EGFR domain I compared to its predecessor tEGF (table in Fig. 1), although this improvement is not seen in the analysis performed on the system with the complete receptor in dimeric form.

[0084] 2.2 tEGFha Production

[0085] The production, purification, and analysis of recombinant hEGF and tEGFha were carried out in parallel for comparative purposes. The coding sequences for hEGF and tEGFha were cloned into a periplasmic secretion system containing the OmpA signal sequence (which directs the protein to an outer membrane pore). This was done to obtain the proteins in the cell culture supernatant, as previously described

[0010] . To increase production yield, the expression system was transfected into the E. coli Rosetta-GammiB strain. The yield was 2–5 mg per liter of culture in a shaking flask.

[0086] Subsequently, the proteins were purified by anion-exchange chromatography on FPLC and atmospheric size-exclusion chromatography. Figure 2A shows the SDS-PAGE analysis of the samples obtained at each purification stage, confirming protein purity in the final step for all proteins. Protein identities were confirmed by MALDI-TOF analysis, which also demonstrated their high purity and expected molecular weight (Fig. 2B). Protein folding analysis was performed by reversed-phase HPLC, as previously described in

[0015] . The proportion of correctly folded protein was found to vary inconsistently among the samples. The hEGF sample exhibited the highest proportion of correctly folded protein, at almost 75%, while only two-thirds of the tEGF produced was correctly folded (66%).The fact that not all of the ligand is correctly folded has been taken into consideration to evaluate its effectiveness in the following biological tests.

[0087] 2.3 Biological characterization of tEGFha. Blocking and antipro-iterative properties

[0088] To characterize the biological activity of tEGFha, the BxPC3 pancreatic cancer cell line has been used, since it is a cell model in which the MAPK pathway is not mutated, and therefore is not permanently activated, and in which EGFR is overexpressed.

[0089] In the different experiments, a commercial EGF was used as a control and recombinantly produced hEGF in the laboratory as a reference to compare with tEGFha. Initially, it was determined by WB that a concentration of 30 nM of recombinant hEGF had the same activity as 10 nM of commercial EGF, so this concentration was also used to test the activity of tEGFha (Fig. 3).

[0090] Initially, the dissociation constant (Kd) of each recombinant protein and commercial EGF was determined (Table 1). The Kd of recombinant hEGF is approximately three times higher than that of commercial EGF, which is consistent considering that we need three times more recombinant hEGF to activate EGFR to the same level as commercial EGF. It should be noted that approximately 25% of all hEGF produced is misfolded.

[0091] The tEGFha ligand, designed to have a higher affinity for EGFR than tEGF, has a Kd 1.2 times greater than that of hEGF. It should be noted that in this case, we obtained a lower percentage of well-folded protein compared to hEGF production, which could partially explain why we obtained a mixture with less affinity for EGFR. The affinity of tEGFha for EGFR is likely similar to that of recombinant hEGF.

[0092] Table 1. Kd values ​​of commercial EGF, recombinant hEGF and tEGFha, calculated by biolayer interferometry.

[0093] The ability to activate EGFR was analyzed by Western blot (WB), determining the level of EGFR receptor phosphorylation after stimulating BxPC3 cells with hEGF or tEGFha. The assay was performed by incubating the cells for 10 minutes and 30 minutes with 30 nM of hEGF or tEGFha, using 10 nM of commercial EGFR as a positive control (Figure 4).

[0094] tEGFha is unable to activate the receptor in either 10 or 30 minutes, using concentrations equivalent to those of hEGF or commercial EGF. Its ability to promote the phosphorylation of certain specific tyrosine residues has also been determined: Y845, related to the stabilization of the receptor activation loop; Y1045, related to the ubiquinone and degradation process; Y1068, related to the activation of the MAPK proliferation pathway; and Y1173, related to receptor dephosphorylation and deactivation

[0016] . Stimulation of cells with tEGFha triggers some activation of Y1045, much less than that caused by hEGF. To determine whether tEGFha induces cell growth, MTT proliferation assays were performed with tEGFha and recombinant hEGF. A minimal medium containing only 0.5% FBS and insulin was used as a negative control.Insulin is necessary to facilitate cell cycle progression upon the addition of EGF [17,18]. Minimal medium with 10 nM of commercial EGF was used as a positive control. Treatments with hEGF and tEGFha were performed at a concentration of 30 nM in minimal medium. hEGF induces growth equivalent to that obtained in the positive control, consistent with previous results (Fig. 5). tEGFha does not induce cell proliferation, as the proliferation measured at each time point is not significantly higher than that at time point Oh.

[0095] Biofilm interferometry has shown that tEGFha can bind to EGFR in vitro, but its inability to activate the receptor, as determined by phosphorylation or to induce proliferation in an EGFR-overexpressing cell line, could be due to tEGFha's inability to bind correctly to EGFR under cell culture conditions. Therefore, the ability of tEGFha to compete with hEGF and displace it from its EGFR binding site when co-incubated was determined. The competition assay was performed by co-incubating 30 nM hEGF with tEGFha in 1:1, 1:2, and 1:5 ratios for 30 minutes, and determining EGFR activation capacity by Western blot (Fig. 6). The results show that, in the presence of tEGFha, EGFR activation progressively decreases with increasing tEGFha concentrations.

[0096] tEGFha has a strong capacity to compete with hEGF, inhibiting EGFR activation by approximately 50% at a 1:1 ratio. This is consistent with the fact that this ligand has a similar affinity for the receptor to hEGF, meaning that EGFR appears to bind to either ligand indiscriminately. At concentrations five times higher than that of hEGF, tEGFha almost completely displaces it and blocks EGFR.

[0097] This competitive ability has also been demonstrated by a proliferation assay. BxPC3 cells were treated with 1:1, 1:2, or 1:5 ratios of hEGF and tEGFha for 4 days (Fig. 7). Proliferation of the culture was progressively inhibited with increasing tEGFha ratios, reaching complete inhibition at a tEGFha concentration 5 times higher than that of hEGF. The EGFR activation process involves a conformational change of the receptor followed by dimerization, which activates the intracellular domain. Having demonstrated the ability of tEGFha to bind to EGFR present in cultured cells, and to determine whether this binding allowed receptor dimerization, a cross-linking assay was performed following the protocol of Turk et al. [9]. For this, BxPC3 cells were deprived of growth factors overnight and incubated with hEGF or tEGFha for 1 h.Subsequently, the samples were treated with BS3, a crosslinking reagent that reacts with primary amines to form stable amide bonds and, therefore, covalent bonds between nearby molecules. Thus, if a receptor dimer has formed, the dimers remain linked and can be identified by electrophoretic separation (Fig. 8). As can be seen, treatment with tEGFha allows the formation of EGFR dimers, indicating that the lack of activation of the receptor's Tyr-kinase activity is not due to inhibition of the dimerization process. Consequently, a dimer with an inactive conformation is likely formed.

[0098] Since the receptor can form dimers when exposed to tEGFha, it is interesting to see if it can also be internalized within the cell, so that this ligand could act as a drug delivery system. To this end, cells were treated with 30 nM of hEGF and tEGFha, and the amount of receptor present on the cell surface was determined initially and after 15 and 30 minutes of incubation, by flow cytometry (Fig. 9).

[0099] It is observed that, after treatment with tEGFha, the receptor is internalized and can be detected to a lesser extent on the cell surface. When cells are treated with the natural ligand hEGF, the receptor decreases drastically from the membrane as early as 15 minutes, while at 30 minutes there is a proportion, although still lower, higher than at the previous incubation time. This is due to receptor recycling. The receptor is internalized, binds to lysosomal vesicles where the ligand is cleaved, and part of the receptor is recycled back to the membrane

[0019] , although another part is degraded and synthesized again. When cells are treated with tEGFha, this process is slower, and the proportion of EGFR still present on the membrane at 15 minutes is higher than at 30 minutes, a time when the receptor is still internalizing and no recycling back to the membrane has occurred (or has occurred to a lesser extent).The fact that the receptor internalizes into the cell indicates that tEGFha has interesting characteristics for use as a carrier protein for antitumor drugs, directing them directly to tumor cells with EGFR overexpression. The cell's ability to regenerate the EGFR recycled from the membrane affects the effectiveness of the blocking agent. Therefore, it was determined whether EGFR was being reused after internalization or if it was being degraded. To analyze this degradation, cells were treated for 8 h with cycloheximide, a compound that inhibits protein synthesis in cells, and subsequently hEGF or tEGFha was added at different time points. Finally, the amount of EGFR in the cells was evaluated by Western blot (Fig. 10).

[0100] Although EGFR degradation was detected after 3 hours following hEGF treatment, total EGFR levels in the cell were maintained for a longer period when cells were treated with tEGFha, and a significant decrease in total EGFR levels was not observed until 5 hours. Therefore, tEGFha is able to block EGFR and prevent its binding to the natural ligand, and it also promotes less recycling of this receptor because internalization is slower, as is the degradation of the receptor once internalized.

[0101] 3. Conclusion

[0102] In conclusion, we have demonstrated that the tEGFha protein binds to EGFR, and although it induces receptor dimehasis, the receptor is not active. Therefore, EGFR cannot be transphosphorylated at the tyrosine residues involved in stabilizing the activation loop, in proliferation signaling, or in receptor recycling. Furthermore, tEGFha can compete with the natural ligand with high affinity, displacing it at equivalent concentrations. Once bound to the receptor, tEGFha does promote its internalization, which would allow its use as a drug delivery agent into tumor cells. Receptor degradation is slower than with the natural ligand, so EGFR remains blocked for a longer period, and therefore, the inhibition would have a persistent effect.

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Claims

CLAIMS 1. Derived from the epidermal growth factor called tEGFha comprising the amino acid sequence SEQ ID NO:

1.

2. Derivative of the epidermal growth factor called tEGFha, according to claim 1, consisting of the amino acid sequence SEQ ID NO:

1.

3. Pharmaceutical composition comprising the epidermal growth factor derivative tEGFha according to claim 1 or 2.

4. Epidermal growth factor derivative, according to claim 1 or 2, or composition, according to claim 3, for use in the prevention and / or treatment of a disease related to increased EGFR receptor activity, which is cancer.

5. Epidermal growth factor derivative or pharmaceutical composition for use according to claim 4, wherein said cancer is selected from cancer of the oral cavity and pharynx, cancer of other digestive organs, cancer of other respiratory organs, cancer of bone and articular cartilage, melanoma and other malignant neoplasms of the skin, cancer of mesothelial tissues and soft tissues, cancer of genital organs, cancer of the urinary tract, cancer of the eye, brain and other regions of the nervous system, cancer of the thyroid and other endocrine glands, malignant neuroendocrine tumors, cancer of lymphoid, hematopoietic and related tissues, carcinomas in situ, benign tumors, neoplasms of uncertain behavior, polycythemia vera and myelodysplastic syndromes, neoplasms of other locations and neoplasm of unspecified behavior.

6. An epidermal growth factor derivative or pharmaceutical composition for use according to claim 5, wherein said cancer is selected from head and neck cancer, colon cancer, colorectal carcinoma, colorectal adenocarcinoma, prostate cancer, prostate adenocarcinoma, prostate carcinoma, breast cancer, breast carcinoma, breast adenocarcinoma, triple-negative breast cancer, brain cancer, brain adenocarcinoma, brain neuroblastoma, lung cancer, lung adenocarcinoma, lung carcinoma, small cell lung cancer, ovarian cancer, ovarian carcinoma, ovarian adenocarcinoma, uterine cancer, gastroesophageal cancer, renal cell carcinoma, carcinoma of clear cell renal cell carcinoma, endometrial cancer, endometrial carcinoma, endometrial stromal sarcoma, cervical carcinoma, thyroid carcinoma, metastatic papillary thyroid carcinoma, follicular thyroid carcinoma, bladder carcinoma, urinary bladder carcinoma, transitional cell carcinoma of the urinary bladder, liver cancer, metastatic liver cancer, pancreatic cancer, neuroendocrine cancers, squamous cell carcinoma, osteosarcoma, rhabdomyosarcoma, embryonal cancers, glioma, neuroblastoma, medulloblastoma, retinoblastoma, nephroblastoma, hepatoblastoma, melanoma, hematologic neoplasms such as leukemias, lymphomas and myelomas.

7. Epidermal growth factor derivative or pharmaceutical composition for use according to claim 6, wherein said cancer is selected from head and neck cancer, pancreatic cancer, biliary tract carcinoma, neuroblastoma, colon cancer, breast cancer, myeloma, gastric cancer, liver cancer, glioblastoma, ovarian cancer, colorectal cancer, non-Hodgkin lymphoma, lung cancer, prostate cancer, small cell lung cancer, large cell lung cancer, kidney cancer, esophageal cancer, stomach cancer, cervical cancer, or lymphoma tumors.

8. Epidermal growth factor derivative or pharmaceutical composition for use therewith, according to any of claims 4 to 7, wherein said derivative is alone or conjugated with a chemotherapeutic agent or a radioisotope or a photosensitizing agent.

9. Epidermal growth factor derivative or pharmaceutical composition for use according to any of claims 4 to 8, wherein said derivative or pharmaceutical composition is administered parenterally or by intravesical infusion.

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