Anti-EC peptide monoclonal antibody

Abstract

A monoclonal antibody, or an antigen binding fragment thereof, that specifically binds to Ec peptide and the use of the antibody, or an antigen binding fragment thereof, in the treatment, diagnosis, or prognosis of cancer.

Description

[0001] ANTI-EC PEPTIDE MONOCLONAL ANTIBODY

[0002] Technical field of the invention

[0003] The present invention relates to monoclonal antibodies and uses thereof. More particularly, the present invention relates to anti-Ec peptide (PEc) monoclonal antibodies and their use in the treatment, diagnosis, or prognosis of cancer.

[0004] Background of the invention

[0005] The current treatment options for cancer have shifted more towards targeted therapies rather than traditional chemotherapy. Immunotherapy, using engineered antibodies or antibody fragments, directly targeting tumour cells or inducing anti-tumour immune responses, is now considered a main component of cancer therapy.

[0006] The role of IGF-1 in cancer is well established. The lgf-1 gene is transcribed and translated in humans in the form of multiple precursor IGF-1 polypeptides, namely isoforms. These are the IGF-1 Ea predominant form, IGF-1 Eb and IGF-1 Ec, all of which undergo posttranslational modifications such as glycosylation and proteolytic cleavage, leading to the production of the mature IGF-1 (mlGF-1). Until recently, the role of the carboxy-terminal E peptides (PEa, PEb and PEc) deriving from the cleavage of the pro-IGF-1 isoforms, was not known. The Ec peptide, the part of the IGF-1 Ec isoform that is cleaved prior to the production of the mlGF-1 , has been associated with prostate cancer progression.

[0007] The IGF system is gaining interest and has become one of the most explored areas in targeted anticancer therapy over the last decade since it is involved in several aspects of cancer development such as tumour growth, metastasis and invasion in a broad range of malignancies. Current therapeutic targeting of the IGF-1 signalling pathways focuses on blocking the IGF-1 R or targeting the IGF-1 R major ligand (IGF-1). Targeting of the IGF-1 system, apart from the obvious toxicity defects associated to its natural function of the targeting of the IGF-1 system, seems to be problematic since none of these therapies were associated with significantly better overall survival in patients treated with chemotherapy and anti-IGF-1 R or anti-IGF-1 compared to patients treated with chemotherapy in various cancers. In the case of monoclonal antibodies against IGF-1 R, it has been suggested that they can act as a “biased” IGF-1 R agonist to promote IGF-1 R / b-arrestin1 association. This association results in ERK1 / 2 signalling pathway activation. Furthermore, these antibodies cannot neutralise the nuclear pool of the IGF-1 R that possesses pro-tumourigenic activities. In the case of monoclonal antibodies against IGF-1 there are several Phase II and Phase III clinical trials.

[0008] Unlike IGF-1 , the IGF-1 Ec isoform is not normally expressed in healthy tissues. Up regulation of IGF-1 Ec expression represents the molecular response at tissue damage and its function mediates mainly the mobilization of human Mesenchymal Stem Cells (HMSC) for tissue repair, locally. In cancer, IGF-1 Ec is only produced when cancer cells are challenged. In this case, there is the production of the IGF-1 Ea isoform (the main mature IGF-1 producing isoform) and the production of the IGF-1 Ec isoform that increases as tumour progresses. The IGF-1 Ec isoform is cleaved and it yields the mature IGF-1 and the PEc. Both molecules (IGF-1 and PEc) exert oncogenic actions through different receptors. Targeting of the IGF-1 or the IGF-1 R covers a broad range of molecules involved in the IGF-1 system, including the IGF-1 R and the heterohybrid receptors but it does not affect the action of PEc.

[0009] PEc secretion is central to tumour progression (proliferation, metastasis and repair). PEc overexpression in prostate cancer experimental models was associated with significant increase of cellular proliferation and induction of the epithelial to mesenchymal transition (EMT) process, which has a fundamental role in the mechanism of cancer cell invasion and metastasis. PEc effects are generated by activating the ERK 1 / 2 pathway, through an unknown receptor, other than IGF-1 R, IR, or any of the IGF-1 / IR hybrid receptors produced by the heterodimerization of the IR and IGF1 R respective monomers. The oncogenic role of PEc was also verified by the fact that the introduction of PEc overexpressing cells (normal and cancer cell lines) subcutaneously and orthotopically in SCID mice, leads to a significant elevation of both growth and metastatic rates of the resulting tumours compared to the tumours obtained from the wild type (wt) cell lines. Apart from its oncogenic effect, tumour secreted PEc was also found to be associated with host mesenchymal cells mobilization towards the tumour. The mobilisation of host MSC to the tumour has been associated with tumour repair.

[0010] Prostate cancer cell lines in in vitro conditions do not express the IGF-1 Ec isoform. However, the tumours developed by the introduction of unmodified prostate cancer cells into SCID mice, expressed increasing amounts of IGF-1 Ec as they progressed. Recent evidence suggests that the mechanism leading to tumour PEc secretion, is associated with the tumourhost immune cell-cell interaction. Immune cells secrete IL-6 upon prostate cancer cellular interaction. IL-6, through the JAK2 / STAT3 pathway, seems to be a key molecule regulating tumour igf-1Ec and H6 genes transcription. Tumour IL-6, acting in an autocrine / paracrine mode of action, further induces IGF-1 Ec expression in prostate cancer, consequently leading to the elevation of the PEc amount and to tumour progression. Consistent with the laboratory evidence is the fact that IGF1-Ec expression determined in human prostate cancer biopsies was found to be directly positively associated to tumour grade and stage.

[0011] Histopathology studies in cancer patients, using an anti-PEc polyclonal antibody, suggest that the presence of PEc is evident in various solid tumours including prostate, breast, endometrial, thyroid and colon cancer where it is significantly associated with advanced stages. Although anti-PEc polyclonal antibodies are known, there use in therapy is limited due to batch- to-batch variability and a high chance of cross-reactivity. There is a need for a targeted immunotherapy having high specificity, consistent and high binding affinity, and low crossreactivity. Summary of the invention

[0012] The present invention provides a monoclonal antibody, or an antigen binding fragment thereof, that binds specifically to Ec peptide (PEc).

[0013] A monoclonal antibody, or an antigen binding fragment thereof, according to the present invention comprises a heavy chain variable region comprising amino acid sequence SEQ ID NO: 2, or an amino acid sequence that is at least 90% identical to amino acid sequence SEQ ID NO: 2, and a light chain variable region comprising amino acid sequence SEQ ID NO: 3, or an amino acid sequence that is at least 90% identical to amino acid sequence SEQ ID NO: 3.

[0014] A monoclonal antibody, or an antigen binding fragment thereof, according to the present invention binds specifically to PEc and can be used in the diagnosis and / or prognosis and / or treatment of cancer.

[0015] The present invention provides also a pharmaceutical composition comprising a monoclonal antibody, or an antigen binding fragment thereof, as defined above and a pharmaceutically acceptable carrier.

[0016] The present invention provides also a monoclonal antibody, or an antigen binding fragment thereof, as defined above for use in the diagnosis and / or prognosis and / or treatment of cancer.

[0017] In addition, the present invention provides a method for the treatment of cancer comprising administering to a subject a monoclonal antibody, or an antigen binding fragment thereof, as defined above.

[0018] In addition, the present invention provides use of a monoclonal antibody, or an antigen binding fragment thereof, as defined above in the manufacture of a medicament for the treatment of cancer.

[0019] Furthermore, the present invention provides a method for the diagnosis and / or prognosis of cancer in a subject comprising contacting a sample obtained from the subject with a monoclonal antibody, or an antigen binding fragment thereof, as defined above.

[0020] Brief description of the drawings

[0021] Figure 1 shows the results of the binding of an anti-PEc monoclonal antibody (MAb) according to the present invention to PEc;

[0022] Figure 2 shows the effect of different MAb concentrations on the ERK1 / 2 phosphorylation (a key molecule for proliferation and metastases / invasion);

[0023] Figure 3 shows in vitro effects of an anti-PEc MAb according to the present invention in prostate cancer cells with respect to proliferation, migration, invasion;

[0024] Figure 4 shows in vivo effects of an anti-PEc MAb according to the present invention in established tumours size;

[0025] Figure 5 shows in vivo effects of an anti-PEc MAb according to the present invention in established tumours on their molecular profile and on their size over prolonged treatment;

[0026] Figure 6 shows the correlation of the expression of PEc with human prostate tumours; Figure 7 shows the correlation of the expression of PEc with human prostate tumours; and

[0027] Figure 8 shows a dot blot assay demonstrating that a monoclonal and a polyclonal antibody recognize different epitopes on PEc.

[0028] Detailed description of the invention

[0029] The present invention provides a monoclonal antibody, or an antigen binding fragment thereof, that binds specifically to Ec peptide (PEc).

[0030] The anti-PEc monoclonal antibody, or an antigen binding fragment thereof, according to the present invention comprises (a) a heavy chain variable region comprising amino acid sequence SEQ ID NO: 2, or an amino acid sequence that is at least 90% identical to amino acid sequence SEQ ID NO: 2, and (b) a light chain variable region comprising amino acid sequence SEQ ID NO: 3, or an amino acid sequence that is at least 90% identical to amino acid sequence SEQ ID NO: 3.

[0031] Preferably, the monoclonal antibody, or an antigen binding fragment thereof, according to the present invention comprises (a) a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 2, or an amino acid sequence that is at least 95% identical to amino acid sequence SEQ ID NO: 2, and (b) a light chain variable region comprising the amino acid sequence SEQ ID NO: 3, or an amino acid sequence that is at least 95% identical to amino acid sequence SEQ ID NO: 3.

[0032] More preferably, the monoclonal antibody, or an antigen binding fragment thereof, according to the present invention comprises (a) a heavy chain variable region comprising the amino acid sequence SEQ ID NO: 2, and (b) a light chain variable region comprising the amino acid sequence SEQ ID NO: 3.

[0033] The monoclonal antibody of the present invention, or an antigen binding fragment thereof, binds specifically to PEc and leads to a significant decrease in proliferation and metastatic rate of cancer cells that express PEc. Specifically, treatment of cancer cells that express PEc with the antibody of the present invention, or an antigen binding fragment thereof, leads to significant decrease of cellular proliferation and reversal of the cancer mesenchymal phenotype towards a more benign, epithelial phenotype, which explains the decrease in migration and invasion abilities of those cells. Therefore, the administration of the antibody of the present invention, or an antigen binding fragment thereof, to a subject inhibits tumour growth and metastasis.

[0034] The monoclonal antibody of the present invention, or an antigen binding fragment thereof, exhibits no toxicity towards non-cancerous cells.

[0035] An antibody according to the present invention includes an antibody of any class, such as IgG, IgA, or IgM.

[0036] Preferably, the monoclonal antibody of the present invention is an immunoglobulin G (IgG) antibody. Preferably, the monoclonal antibody of the present invention is a humanized antibody.

[0037] The antibody of the present invention can be produced by following methods well known in the art. For example, it can be produced by first cloning in an expression vector, for example a mammalian expression vector, the DNA that corresponds to the amino acid sequences of the heavy chain and the light chain. The vector is then introduced into an expression system, such as a Chinese hamster ovary expression system, which produces the MAb.

[0038] An antigen binding fragment according to the present invention incudes for example, Fab, Fab', F(ab')2, Fd, Fv, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.

[0039] The present invention provides also a pharmaceutical composition comprising a therapeutically effective amount of a monoclonal antibody, or an antigen binding fragment thereof, according to the present invention and a pharmaceutically acceptable excipient.

[0040] The composition may be formulated, for example, for oral, parenteral, intramuscular, intravenous, intraperitoneal, subcutaneous, or inhalation administration. The composition may have different forms, such as solid or liquid forms, for example it can have the form of solution, suspension, powder, emulsion, or capsule. Examples of pharmaceutically acceptable excipients, include solvents, buffering agents, emulsifying agents, preservatives, antioxidants, chelating agents, diluents, binders, dis integrants, or lubricating agents, which are well known in the art.

[0041] Examples of solvents, include water, saline, phosphate buffered saline, ethanol, isopropyl alcohol and mixtures thereof.

[0042] Examples of buffering agents include citric acid monohydrate, sodium citrate, sodium lactate, calcium lactate, acetic acid, sodium acetate, potassium acetate, dibasic potassium phosphate, monobasic potassium phosphate sodium hydrogen phosphate and sodium bicarbonate.

[0043] Examples of emulsifying agents include sodium lauryl sulfate, lecithin, polysorbate, sorbitan monooleate, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, poloxamer nonionic surfactants, acacia, agar, alginic acid and sodium alginate.

[0044] Examples of preservatives include sodium benzoate, benzyl alcohol, benzalkonium chloride, benzethonium chloride, bronopol, cetrimide, ethanol, and parahydroxy benzoic acids and their alkyl esters.

[0045] Examples of antioxidants include sodium metabisulfite, butylated hydroxyanisole, butylated hydroxytoluene and ascorbic acid.

[0046] Examples of chelating agents include ethylenediaminetetraacetic acid and salts thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, and the like), citric acid and salts thereof, fumaric acid and salts thereof, phosphoric acid and salts thereof, and tartaric acid and salts thereof.

[0047] Examples of diluents include lactose, sucrose, dextrose, mannitol sorbitol, inositol, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, microcrystalline cellulose, kaolin, sodium chloride, dry starch and cornstarch. Examples of binders include hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose, dextrose, xylitol, polyvinylpyrrolidone, polyethylene glycol, alginates and gelatin.

[0048] Examples of lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt and sodium lauryl sulphate.

[0049] The present invention provides also a monoclonal antibody, or an antigen binding fragment thereof, according to the present invention for use in the treatment of cancer in a subject.

[0050] The present invention provides also a method for the treatment of cancer in a subject comprising administering to the subject a monoclonal antibody, or an antigen binding fragment thereof, according to the present invention.

[0051] The present invention provides also use of a monoclonal antibody, or an antigen binding fragment thereof, according to the present invention in the manufacture of a medicament for the treatment of cancer in a subject.

[0052] Preferably, the subject is a human.

[0053] Preferably, the cancer is a solid cancer. More preferably, the cancer is selected from prostate cancer, breast cancer, colon cancer, bladder cancer, renal cancer, osteosarcoma, thyroid cancer, endometrial cancer, or neuroendocrine cancer. Even more preferably, the cancer is selected from prostate cancer, breast cancer, or colon cancer

[0054] The monoclonal antibody, or an antigen binding fragment thereof, is typically administered in the form of a pharmaceutical composition and it may be administered via a variety of routes including oral, intravenous, intramuscular, subcutaneous, transdermal, intraperitoneal, and topical route.

[0055] The monoclonal antibody, or an antigen binding fragment thereof, is generally administered daily, or more frequently, such as twice a day, or less frequently, such as every second day, or every week. The frequency of administration and the administered dosage depend on various factors, such as the type of cancer, the route of administration, the age of the subject, and the form of the administered composition, and can be determined by a person skilled in the art by using common general knowledge. For example, when the subject is a human, an effective dosage can be from 0.1 mg / kg to 80 mg / kg, preferably, from 1 mg / kg to 40 mg / kg, more preferably, from 6 mg / kg to 20 mg / kg

[0056] The present invention provides also an in vitro method for the diagnosis of cancer in a subject comprising contacting a sample obtained from the subject with a monoclonal antibody, or an antigen binding fragment thereof, according to the present invention. For example, a monoclonal antibody according to the present invention can be used in an ELISA assay for the detection of PEc in a sample, such as a blood sample, obtained from the subject. In another example, a monoclonal antibody according to the present invention can be used in an immunohistochemical assay for the detection of PEc in a sample of a tumour obtained from a subject. The present invention provides also an in vitro method for the prognosis of cancer in a subject comprising contacting a sample obtained from the subject with a monoclonal antibody, or an antigen binding fragment thereof, according to the present invention. For example, a monoclonal antibody according to the present invention can be used in an ELISA assay for the detection of the presence of PEc in the a sample, such as a blood sample obtained from the subject, wherein elevated PEc levels are associated with more aggressive cancer phenotypes, Furthermore, a monoclonal antibody according to the present invention can be used in an ELISA assay performed before and during cancer treatment to evaluate the efficiency of the treatment, as well as after treatment to evaluate the efficacy of the treatment, wherein an increase of PEc in a sample obtained from the subject is associated with a cancer relapse. In another example, a monoclonal antibody according to the present invention can be used in an immunofluorescence assay, for the detection of circulating tumour cells (CTCs). CTCs expressing PEc peptide are associated with more aggressive / metastatic cancer phenotypes.

[0057] Examples

[0058] Example 1

[0059] Anti-PEc monoclonal antibody (Mab) production.

[0060] One hundred and twenty different monoclonal antibodies were produced using the hybridoma technique (Absea biotechnology ltd, Haidian district Beijing 100,085 China). Five mice were immunized with the human PEc or scrambled peptide (prior to the development of a valid isotype matching control antibody). Spleen cells were fused with immortal myeloma cells. The obtained monoclonal antibodies were tested for specificity and sensitivity against the antigen of interest (human PEc: YQPPSTNKNTKSQRRKGSTFEERK, SEQ ID NO:1) by an ELISA method. Different concentrations of each antibody ranged from 0 -1000 ng / ml were added in wells coated either with human Pec or with the scrambled peptide (1 pg / ml). Ten different monoclonal antibodies were selected for their specificity and sensitivity against human (and mouse) PEc and they were further tested by dot blot analysis. The scramble peptide (CSKNKTQSREGTKFSEKYRPNTRPQ, SEQ ID NO:4) was used as a negative control. The most potent anti-PEc monoclonal antibody was selected. This Mab comprises a heavy chain variable region having the amino acid sequence SEQ ID NO: 2 and a light chain variable region having the amino acid sequence SEQ ID NO: 3 (Table 1). Table 1

[0061] DNA coding for the amino acid sequence of this MAb was synthesized and cloned into the mammalian transient expression vector PETE V2 (Fusion Antibodies Pembroke Loop Road, Belfast Ireland). The MAb was expressed using CHO based transient expression system and the resulting antibody containing cell culture supernatants was clarified by centrifugation and filtration. MAb purification was obtained using AKTA affinity chromatography. Purified antibody was dialyzed into PBS. The purity of the MAb was determined to be >95% (reducing and denaturing SDS PAGE and size exclusion chromatography). MAb concentration was determined by measuring the absorbance at 280nm calculated using the standard extinction coefficient 205,000 M-1 cm-1 (or 1 .0mg / mL = A280 of 1 ,37, assuming a MW = 150,000 Da). This MAb was used in the Examples herein below.

[0062] Example 2 Binding analysis

[0063] Binding assays were performed by first capturing the IgG MAb of Example 1 by using an anti-mouse Fc Octet. The MAb capture biosensors were then submerged in wells containing 300nM of antigen. Steps were performed at 25°C at a constant flow rate of 1000 rpm. Dissociation rate constants (KD) were estimated using the Forte Bio data analysis software (Fusion Antibodies Pembroke Loop Road, Belfast Ireland). Due to the small size of the antigen (24aa) Octet binding analysis did not work. Therefore, binding analysis was carried out by a dot blot assay against different PEc fragments (aa: 1-12, 13-24, 1-6, 7-12, 13-19, 20-24. and against the whole PEc. Briefly, different concentrations of each fragment (10, 20, 40, 80 ng / mL) were applied onto a nitrocellulose strip, after drying the membrane was blocked by 5% BSA for 1 hour and then incubated with 2.5 pg / mL of the anti-PEc MAb for 1 hout at room temperature. After washing the membrane was incubated with an anti-mouse HRP antibody (1 :2,000 dilution); (Santa Cruz Biotechnology). Visualization took place by exposing the blots to x-ray film after incubation with freshly made ECL substrate for 3 min (SuperSignal, Pierce Biotechnology, Rockford, IL, USA). The results showed that the anti-PEc MAb is specific to the whole Ec peptide (24 amino acids) and to the last 12 amino acids and it does not attach efficiently to any of the smaller peptides.

[0064] Example 3

[0065] Competition assay

[0066] Wells of a microplate were coated with 1 pg / ml of PEc. After washing, the anti-PEc MAb of Example 1 was introduced in each well (5 pg / ml) together with PEc at different concentrations / well (0.2, 0.4, 0.6, 0.8, 1 , pg / ml). As a negative control human serum from 3 different individuals was used. Detection was carried out with a horseradish peroxidase conjugated goat anti-rabbit antibody at 1 :1 ,000 dilution (Thermo Scientific [Thermo Fisher Scientific Inc., Waltham, MA, USA]). As a substrate, 3,3',5,5'-Tetramethylbenzidine (TMB) was used. The reaction stopped by administrating 2 mol / L sulphuric acid solution. The absorption of the plate was measured within 20 min at 450 nm and 540 nm as reference using a microplate reader (VersaMax, Molecular Devices, Sunnyvale, CA, USA). The results (Fig. 1) show that the MAb of the present invention exhibits specificity and sensitivity towards PEc. Introduction of different concentrations of PEc resulted in the reduction of absorbance (MAb attached on the PEc on the walls of the wells).

[0067] Example 4

[0068] In vitro effects of anti-PEc MAb in prostate cancer cell lines

[0069] Prior to resemble this in-vivo characteristic of the prostate tumours, where PEc expression is gradually increased as tumour progresses, in in-vitro models, two prostate cancer cell lines (PC-3 androgen receptor (AR) positive and DU-145 AR negative) were modified to overexpress the PEc without affecting the mlGF-1 evels. Recent evidence suggests that prostate cancer cell lines that overexpress the PEc, present elevated cellular proliferation levels, and a shift of their epithelial phenotype towards a mesenchymal, metastatic phenotype by activating ERK1 / 2. Treatment of both modified cell lines with different concentrations of the anti-PEc MAb also leads to a gradual decrease of phosphorylated (activated) ERK 1 / 2 levels (Fig. 2). In prostate cancer, ERK1 / 2 activation apart from cellular proliferation, has been associated with the EMT and an increase in metastatic and invasive capacities.Treatment of both prostate cancer cell lines (PC-3PEc and DU145PEc) with the anti-PEc MAb of Example 1 lead to statistically significant decrease of the cellular proliferation (p<0.0005 in both cases) as measured at 24 and 48h (Fig.3a), in contrast to the unmodified prostate cancer cell lines, where treatment with anti- PEc did not significantly affect cellular proliferation. MAb treatment resulted in the significant reduction of migration (Fig. 3b) and invasion (Fig. 3c) capacities as was determined by trans-well migration and invasion assays (p<0.0005, in both cases). Verification of the anti-PEc MAb effect on the migration (metastatic) ability of the modified prostate cancer cells was obtained by a wound healing assay, were the MAb treated cell lines presented a significantly lower migration rate (p<0.0005 at 18 hours for both cell lines). Isotype control: The monoclonal antibody generated by the scrambled peptide (a peptide possessing the same amino acids as the PEc but placed in a random order).

[0070] Example 5

[0071] In vivo effects of the anti-PEc MAb in Prostate cancer.

[0072] The in vivo effects of the anti-PEc MAb of Example 1 were also examined after subcutaneous (sc) inoculation of wild type (wt) unmodified prostate cancer cell lines (wtPC-3 and wtDU-145) into SCID mice (15). Both prostate cell lines used can generate tumours in 4 weeks after subcutaneous injection in SCID mice. In this study antibody treatment took place after the determination of palpable tumours in all the cases. Determination of the optimum MAb concentration for prostate cancer treatment was obtained by testing different concentrations of the anti-PEc MAb. The concentrations used were 5, 10 and 20 mg / kg, three times / week. Treatment of prostate tumours with different MAb concentrations resulted in significant tumour growth inhibition in 4 weeks, compared to the tumours treated with the isotype MAb (treatment with 20 mg / ml isotype control: tumour mass = 1 ,66g ± 0.3, with 0.5mg / ml anti-PEc MAb: tumour mass = 0.62g ± 0.1 , with 10mg / ml anti-PEc MAb: tumour mass = 0.18g ± 0.04, with 20 mg / ml anti-PEc MAb: tumour mass = 0.16g ± 0.04, with Docetaxel: tumour mass = 0.137g ± 0.05. n=5, p<0.0005, in every case) (Fig. 4). Tumours treated with 10 and 20 mg / kg did not present significant difference in respect to their size and weight, suggesting that the optimum MAb concentration was the 10 mg / kg. Tumours treated with 10 mg / kg MAb for 4 weeks were also compared with the tumours treated with docetaxel for the same time period, with no significant difference in respect to their size and weight.

[0073] Immunohistochemical examination of the tumours documented that treatment with the anti-PEc MAb was associated with significant increase of the epithelial marker E-Cadherin, the reduction of the mesenchymal marker Vimentin and a decrease of the cellular proliferation marker ki-67 (Fig. 5a). These results were also verified by qRT PCR where the results obtained suggested statistically significant differences in Vimentin, E-Cadherin and Ki-67 expression (p<0.0005 in all the treated vs untreated subjects) indicating the efficiency of anti-PEc MAb in ceasing tumour progression. In a different set of experiments, prostate tumours, resulting from subcutaneous (sc) inoculation of wtPC-3 and wtDU-145 cells into SCID mice, were treated with 10 mg / kg anti-PEc MAb three times I week, for a total of 8 weeks. Tumour volume was measured daily by calliper. Treatment for 5 weeks, led to significant tumour growth inhibition similar to that obtained by docetaxel (10 mg / kg, twice / week) and overall, to a statistically significant size difference when compared with the tumours treated with the isotype Mab control (for PC-3 cells: isotype control: 546 mm3± 57, anti-PEc MAb: 5mg / ml: 270mm3± 45, 10mg / ml: 20mm3± 4, 20mg / ml: 18mm3± 6, Docetaxel: 18mm3 ± 3, n=10 in every case, p<0.005 similar were the results for DU 145 cells), (Fig. 5b). The tumour size difference between isotype treated tumours and anti-PEc MAb treated tumours further increased at 60 days (for PC-3 cells: isotype control: 1320 mm3± 212, anti-PEc MAb: 5mg / ml: 532 mm3± 105, 10mg / ml: 31 mm3± 4, 20mg / ml: 26mm3± 6, Docetaxel: 28mm3± 3, n=10 in every case, p<0.0005 for every case for both cell lines). Tumours were also examined for the expression of P ERK1 / 2 (proliferation and EMT). Prostate tumours treated with the anti- PEc MAb presented a significant reduction of P ERK1 / 2 levels in comparison to the untreated tumours.

[0074] Example 6

[0075] Effects of the anti-PEc MAb of Example 1 in prostate cancer metastasis.

[0076] Epithelial to Mesenchymal Transition (EMT) is a central process to metastases, that normally occurs as tumours progresses as a response to harsh for the tumour conditions (space limitation, immune response of the host, hypoxia, NO2 species). The in vivo observation of the reversal of the cancer mesenchymal phenotype after the administration of the anti PEc MAb of Example 1 , was further examined in the context of metastasis. Sentinel nodes as well as different tissues (liver, lungs, kidneys) were collected from SCID mice that developed prostate cancer and they were treated with anti-PEc MAb (n=20). Similarly, the same tissues were collected from untreated controls (n=10). Collection took place at 50 days after tumour establishment. In every case, tissues were stained (H&E) and examined by an experienced pathologist till paraffin block exhaustion.

[0077] None of the 20 mice treated with the anti-PEc MAb presented evidence of a possible sentinel node infiltration or of distal metastases. Six out of the 10 untreated mice presented evidence of sentinel node infiltrations, as was determined by imunohistochemical analysis evaluation and / or by significant size increase. Furthermore 3 out of the 6 mice presented distal metastases in the liver.

[0078] Example 7

[0079] PEc expression in prostate cancer patient biopsies

[0080] The levels of expression of IGF-1 Ec were examined in 100 early stage (non-metastatic) prostate cancer patients (Fig. 6). Immunohistochemical expression of stained proteins was classified as either grade 1 (weak intensity), grade 2 (moderate intensity) or grade 3 (strong intensity). The H scoring system, incorporating both intensity and distribution of staining, was used for semi-quantitative analysis of protein immunoreactivity. More specifically, the percentage of positive cells was measured in every section and multiplied by 1 , 2 and 3, respectively (grade 1 score= percentage with grade 1 expression x 1 ; grade 2 score= percentage with grade 2 expression x 2; grade 3 score = percentage grade 3 expression X 3). A total score between 0 and 300 was obtained for each case (total score = grade 1 score + grade 2 score + grade 3 score). In all the cases elevated IGF-1 Ec was associated with, upregulation of vimentin and Ki- 67 and a downregulation of E cadherin and PTEN protein (Fig. 6).

[0081] The IGF-1 Ec h score was found significantly lower in prostate tumours in patients of stage T2N0 (AJCC) as compared to the tumours of patients of stage T3aN0 (p<0.0005). Patients of stage T3aN1 and T3bN0, presented significantly higher tumour IGF-1 Ec levels compared to that of T3N0 patients (p<0.0005) (Fig. 7). All of our patients were of Gleason Score 6, 7 and 9. IGF-1 Ec expression was significantly higher in the patients with gleason score 7 compared to those with Gleason score 6 (p<0.005). Patients with Gleason score 9 presented significantly higher levels of IGF-1 Ec compared to those with Gleason score 7 (p<0.0005) (Fig. 7). Similarly, patients that presented extraprostatic extension were significantly associated with elevated PEc expression (p<0.0005).

[0082] Tumour cribriform architecture seems to be another factor that is associated to the IGF- 1 Ec expression levels. Higher cribriform grade was associated with significantly elevated levels of IGF-1 Ec compared to tumours with lower cribriform (p<0.0005). Finally, elevated seminal vesicle infiltration presented significantly higher IGF-1 Ec levels than prostate tumours that were not associated with invasive potential (p<0.0005 in both cases).

[0083] Example 8

[0084] Effect of the anti-PEc MAb of Example 1 on human prostate tumour xenografts

[0085] Approximately 0.7 cm3of prostate tumours from 3 individuals with prostate cancer (all of which were of stage T3bN1 M0 and high gleason score 8-9) were donated after total prostatectomies and tumour characterization. Informed consent was obtained in every case.

[0086] Human xenografts from prostate cancers were generated after sc inoculation of about 0.5 cm3of tumour into SCID mice (n=3). The lump disappeared in 3 days and palpable tumours developed in 13 to 19 weeks (tumour 1 in 13 weeks, tumour 2 in 18 weeks, tumour 3 in 19 weeks). Tumours were allowed to grow up to 1 .5 cm3and they were then treated with 20 mg / ml anti PEc MAb of Example 1 intravenously for a period of 14 days during which mice were subjected to 4 injections. Starting point at time 0 was set as the point where tumour volume was about 1 .5 cm3, the second injection was obtained at 48 hrs, the 3rdat 100 hrs and the 4that 148 hrs. Treatment of SCID mice with anti PEc MAb resulted in a significant reduction in tumour volume in 76 hours (from 1.5 cm3±0.1 to 0.3 ± 0.02, p=0.0003). Tumour volume decrease continued over the whole period of monitoring but with lower rate. At day 14 (336 h) tumours were extracted and measured. Tumour volume in all the cases was about 0.1 cm3as measured by calliper.

[0087] Analysis and comparison of the epithelial marker E-cadherin, the mesenchymal marker Vimentin and of the proliferation marker Ki67 of the human tumours before inoculation into SCID mice and after treatment with anti PEc MAb indicated significant reduction in the levels of Vimentin (p <0.0005 combined cases), a significant increase of E-Cadherin levels (p<0.0005 combined cases) and a significant decrease of Ki67 levels (p<0.0005 combined cases). The results obtained indicate a reduction in cellular proliferation and a switch of the cancer mesenchymal cells which are associated to metastases and invasion, towards a more “benign” epithelial phenotype. Sentinel nodes as well as livers and lungs were examined for metastases (IHC). None of these tissues exhibited evidence of infiltration.

[0088] Example 9

[0089] Antibody toxicity and tissue distribution

[0090] -In vitro

[0091] The in vitro off target effects of the anti-PEc MAb of Example 1 were examined in two normal human cell lines, one prostate (HPrEc) and one fibroblast (HFL-1). In this case the normal cells were incubated with either, serum, peripheral blood mononuclear cells (PBMCs) or serum and PBMCs from 5 different individuals. After 24 hours the anti-PEc MAb was introduced for 12 hours and the non-cancerous cells were isolated from the PBMCs and analyzed with Anexin / PI, prior to determine the levels of apoptosis associated to the anti-PEc MAb. No significant differences were observed in respect to the induction of apoptosis after the administration of anti- PEc MAb in both normal cell lines used, nor when administration of the anti-PEc MAb was obtained in the presence of serum, PBMCs and serum +PBMCs.

[0092] In order to determine the effects of the anti-PEc MAb in normal cell survival over 48 h, non-cancerous cells were introduced with either, serum, PBMCs or serum and PBMCs from 5 healthy individuals for 24 hours followed by the introduction of the anti-PEc MAb. The non- cancerous prostate and fibroblast cells were isolated after 24 and 48 h and counted by trypan blue and MTT analysis. No statistically significant differences were observed in any of the comparisons between MAb treated and untreated cell lines, indicating that in in vitro conditions the anti-PEc MAb is not associated with any cytotoxic effects and this is evident due to the fact that non- cancerous cells produce almost negligible amounts of PEc even when challenged with factors of the immune system.

[0093] -In vivo

[0094] The anti PEc MAb of Example 1 was examined for toxic side effects as well as for its distribution in mouse tissues. For the toxicity determination, 10 wt mice (n=10) were injected 4 times I week with 30 mg / kg anti PEc MAb for 60 days. Mice were assessed daily in respect to their appearance (body weight and coat condition), body function (dyspnoea and / or tachypnoea, food intake), environment (loose stools or diarrhoea, blood in diarrhoea) and behaviour (handling, aggression, abnormal gait, abnormal posture, reluctance to move), according to the Severity Assessment Framework of the European Commission.

[0095] None of the characteristics examined presented significant differences between treated and untreated mice. At the end of 60 days mice were sacrificed and tissues were collected stained and analysed in respect to tissue and cellular integrity. The tissues examined were lungs, liver, kidneys, heart and muscle. None of the tissues examined presented evidence of deterioration or structural alterations as assessed by an experienced pathologist.

[0096] Tissue distribution

[0097] Tissue distribution of the MAb examined in this study was carried out using the biotinylated version of the anti-PEc Mab of Example 1. Five SCID mice (n=5) with already established tumours were injected with 20 mg / kg biotynilated MAb. After 24 h, mice were sacrificed and tissues were collected and analysed with an anti-biotin MAb. Tissues and tumours were then collected and examined for the presence of the anti-PEc MAb with an anti-biotin antibody (Abeam). The tissues examined were lung, liver, muscle, and kidney. It was determined that anti-PEc MAb was only present in the tumour and it was undetectable in all the healthy tissues examined.

[0098] Example 10

[0099] Alternative means of targeting the pathway

[0100] The IGF axis plays important roles in cancer progression and metastasis (15). The PEc arises from the proteolytic cleavage of the IGF-1 Ec isoform. Recent evidence suggests that the PEc exerts its actions similarly to the IGF-1 through ERK1 / 2. anti-IGF-1 R MAb and anti-IGF- 1 MAbs are in clinical trials for a number of different solid neoplasms. We therefore compared the effects of anti IGF-1 R (10) and anti-IGF-1 (clone 7973) MAbs to those exerted by the anti-PEc MAb in prostate tumours. Subcutaneous prostate tumours were obtained by the introduction of PC-3 or DU145 cells in SCID mice. Tumours were treated for 4 weeks with 10 mg / kg of anti-IGF- 1 MAb, anti-IGF-1 R MAb and anti-PEc MAb. All the tumours extracted presented a significant size reduction compared to the tumours treated with the isotype control (Isotype treatment: 1 ,73g ± 0,19, anti-IGF-1 R: 1.148g ± 0.082, anti-IGF-1 : 1 .07g ± 0.082, anti-PEc MAb: 0.062g ± 0.012, n=10 in every case, p<0.005). They also presented a significant increase in E cadherin expression (p<0.005) and a decrease in Vimentin and Ki-67expression (p<0.005 in both cases) as determined by qRT-PCR. Comparison of the tumours treated with the anti-IGF-1 and anti IGF- 1 R antibody (both from R&D Systems, Inc., MN, USA.) did not present significant difference in the aforementioned markers. Comparison of the tumours treated either with the anti IGF-1 Ab or the anti-IGF-1 R antibody with the anti-PEc MAb indicated a significant difference in tumour size and in the expression of E cadherin and vimentin (p<0.0005 in every case). Sentinel nodes as well as livers and lungs were examined for metastases (IHC). None of these tissues exhibited evidence of infiltration. Toxicity: IGF-1 R and IGF-1 MAb treated mice appeared to be lethargic and in 2 and 3 out of 10 respectively presented diarrhoea.

[0101] Example 11

[0102] The effect of anti-PEc MAb of Example 1 in breast and colon cancer

[0103] The effect of the anti-PEc MAb of Example 1 was examined in SCID mice that developed breast cancer after orthotopic inoculation of MCF-7 cells (n=20) and colon cancer after subcutaneous injection of DLD-1 cells (n=10), with similar results. In both cases the protocol for the antibody administration was kept the same as in prostate cancer. Therefore, after tumour establishment, 10 mg / kg were administrated into the SCID mice.

[0104] In breast cancer 2 out of 20 mice treated with the anti-PEc MAb retained palpable tumours compared to 10 out of 10 obtained in the untreated mice, 4 weeks aftertumourdetection. Due to the small size of the tumours obtained by the MAb treated mice we were not able to proceed with IHC. Therefore, we proceeded with qRT-PCR to examine the levels of E-Cadherin, Vimentin and Ki-67. Similarly, to the results observed in prostate cancer, breast tumours presented evidence of EMT reversal associated with a significant decrease of Vimentin and Ki- 67 (p<0.0005 for both markers) and an increase of E-Cadherin compared to the untreated tumours (p<0.0001).

[0105] SCID mice with subcutaneous colon cancer tumours have been developed using wt DLD-1 cells. Treatment of palpable tumours with the anti-PEc MAb of Example 1 (4 weeks, 3 times / week, 10 mg / kg) resulted in the development of significantly smaller tumours compared to the untreated tumours (Isotype MAb treatment: 2.36g ± 0.29, anti-PEc MAb treatment: 1 ,23g ± 0.16, Docetaxel treatment: 0.38g ± 0.06, n=10, p<0.0005). Treated tumours presented significant lower levels of Vimentin and Ki-67 expression and elevated levels of E cadherin as was observed by IHC and qRTPCR (p<0.0005 in every case). Western blot analysis also presented evidence of pERK 1 / 2 reduction compared to the untreated tumours. Tumours that are given raise by DLD-1 cells are very aggressive and they have high infiltrating capacity repair. What was observed in respect to tumour infiltration is the fact that DLD-1 tumours treated with the anti-PEc MAb did not infiltrate into the mouse body, compared to the untreated tumours where in all cases tumours infiltrated the ribcage and other tissues.

[0106] Example 12

[0107] The epitope-binding effect of an anti-PEc MAb

[0108] Prior work by the inventors provided an anti-PEc polyclonal antibody (Papageorgiou E, Philippou A, Armakolas A, Christopoulos PF, Dimakakos A, Koutsilieris M. The human Ec peptide: the active core of a progression growth factor with species-specific mode of action. Hormones (Athens). 2016 Jul; 15(3):423-434. doi: 10.14310 / horm.2002.1699. PMID: 27838607). However, certain drawbacks were associated with the anti-PEc polyclonal antibody for use in therapy. To assess specificity, potential binding affinity, and likelihood of cross-reactivity, the anti- PEc polyclonal antibody was compares to an anti-PEc Mab according to the invention.

[0109] The monoclonal and the polyclonal antibodies recognize different epitopes on Ec peptide as determined by performing a dot blot assay (Fig. 8). In this case, both antibodies were compared side by side for their ability to recognize different fragments of the Ec peptide (aa: 1- 12, 13-24, 17-24) and against the whole PEc. Briefly, different concentrations of each fragment (50 ng / mL) were applied onto a nitrocellulose strip and, after drying, the membrane was blocked by 5% BSA for 1 hour and then incubated with 2.5 pg / mL of the anti-PEc MAb for 1 h at RT. After washing the membrane was incubated with an anti-mouse and an anti-rabbit HRP antibody (1 :2,000 dilution); (Santa Cruz Biotechnology). Visualization took place by exposing the blots using the iBright 1500 imaging system (Invitrogen) after incubation with freshly made ECL substrate for 3 min (SuperSignal, Pierce Biotechnology, Rockford, IL, USA). It was observed that both antibodies recognize the whole Ec peptide. In contrast to the polyclonal anti-PEc antibody, the anti PEc monoclonal antibody recognizes the second half of the EC peptide where the PEc active core is located. This results in target recognition consistency. Since the polyclonal antibody was produced after injecting an animal with a certain antigen, as part of the immune response of the animal, there are several antibodies produced against the injected antigen, such that these antibodies have been raised against different epitopes of the antigen, whereby the polyclonal antibody pool may differ from injection to injection in respect to the recognition epitopes.

Claims

Claims1 . A monoclonal antibody, or an antigen binding fragment thereof, that binds specifically to peptide Ec, wherein the antibody, or an antigen binding fragment thereof, comprises(a) a heavy chain variable region comprising amino acid sequence SEQ ID NO: 2, or an amino acid sequence that is at least 90% identical to amino acid sequence SEQ ID NO:2, and(b) a light chain variable region comprising amino acid sequence SEQ ID NO: 3, or an amino acid sequence that is at least 90% identical to amino acid sequence SEQ ID NO:3.

2. The monoclonal antibody, or an antigen binding fragment thereof, according to claim 1 , wherein the antibody, or an antigen binding fragment thereof, comprises(a) a heavy chain variable region comprising amino acid sequence SEQ ID NO: 2, or an amino acid sequence that is at least 95% identical to amino acid sequence SEQ ID NO:2, and(b) a light chain variable region comprising amino acid sequence SEQ ID NO: 3, or an amino acid sequence that is at least 95% identical to amino acid sequence SEQ ID NO:3.

3. The monoclonal antibody, or an antigen binding fragment thereof, according to claim 1 or 2, wherein the antibody, or an antigen binding fragment thereof, comprises(a) a heavy chain variable region comprising amino acid sequence SEQ ID NO: 2, and(b) a light chain variable region comprising amino acid sequence SEQ ID NO: 3.

4. The monoclonal antibody, or an antigen binding fragment thereof, according to any one of the preceding claims, wherein the antibody is a humanized antibody.

5. The monoclonal antibody, or an antigen binding fragment thereof, according to any one of the preceding claims, wherein the antibody is an IgG antibody.

6. The monoclonal antibody, or an antigen binding fragment thereof, according to any one of the preceding claims for use in the treatment of cancer in a subject.

7. The monoclonal antibody, or an antigen binding fragment thereof, for use according to claim 6, wherein the subject is a human.

8. The monoclonal antibody, or an antigen binding fragment thereof, for use according to claim 6 or 7, wherein the cancer is a solid cancer.

9. The monoclonal antibody, or an antigen binding fragment thereof, for use according to any one of claims 6 to 8, wherein the cancer is selected from prostate cancer, breast cancer, colon cancer, bladder cancer, renal cancer, osteosarcoma, thyroid cancer, endometrial cancer, or neuroendocrine cancer.

10. The monoclonal antibody, or an antigen binding fragment thereof, for use according to any one of claims 6 to 9, wherein the cancer is selected from prostate cancer, breast cancer, or colon cancer.11 . A pharmaceutical composition comprising a therapeutically effective amount of a monoclonal antibody, or an antigen binding fragment thereof, according to any one of claims 1 to 5 and a pharmaceutically acceptable excipient.

12. The pharmaceutical composition according to claim 11 , wherein the composition is suitable for oral, parenteral, intramuscular, intravenous, intraperitoneal, subcutaneous, or inhalation administration.

13. A method for the diagnosis of cancer in a subject comprising contacting a sample obtained from a subject with the monoclonal antibody, or an antigen binding fragment thereof, according to any one of claims 1 to 5.

14. A method for the prognosis of cancer in a patient comprising contacting a sample obtained from a subject with the monoclonal antibody, or an antigen binding fragment thereof, according to any one of claims 1 to 5.

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