Human anti-fgf2 monoclonal antibody (mAb), pharmaceutical composition comprising same, use thereof and kit for detecting cancer in biological sample comprising said mAb

Abstract

The present invention relates to a human anti-FGF2 monoclonal antibody (mAb) comprising an amino acid sequence of a light chain as shown in SEQ ID NO: 1 or 3 and an amino acid sequence of a heavy chain as shown in SEQ ID NO: 2 or 4, in addition, the present invention relates to a pharmaceutical composition comprising said mAb and use thereof in the production of a medicament for treating cancer, in addition, the present invention relates to a kit for detecting FGF2 protein in a biological sample, the kit comprises the mAb or a biologically active fragment thereof bound to a detectable moiety, and can be used for prognosis or targeting cancer therapy, in addition to cancer-associated therapeutic applications such as inhibition of angiogenesis, potential applications of controlling fibrotic diseases, neutralizing excessive effects of FGF, and in particular, cancer-associated therapeutic applications such as inhibition of angiogenesis. The antibodies can be used in addition to their therapeutic potential in immunoscintillation photography, radioisotope labeling, for molecular imaging and prediction of fibrosis responses, for example in diseases of high intensity tissue repair (keloids) and secondary fibrosis of idiopathic fibrosis or chronic inflammatory disease.

Description

TECHNICAL FIELD

[0001] The present invention belongs to the field of formulations for medical purposes, more specifically, to the field of pharmaceutical formulations containing antigens or antibodies, as they refer to human anti-FGF2 monoclonal antibodies (mAbs). BACKGROUND

[0002] Fibroblast growth factor 2 (FGF2) is considered a key protein in melanoma development and plays a key role in tumor growth, angiogenesis and metastasis.

[0003] Melanoma is the most aggressive and lethal type of cancer. In advanced stages, melanoma is difficult to treat, as the tumor is resistant to most therapies. Thus, despite recent therapeutic attempts, the prognosis for patients with metastatic melanoma remains poor, with 5% to 19% of cases having a life expectancy of 5 years and an average survival of 5.3 months.

[0004] In view of the technical problem, there is a need to develop new therapeutic options, particularly more effective therapeutic options, or combinable therapeutic options that can combat this tumor. Inhibition of FGF2-mediated FGFR signaling can be an alternative therapy for patients with advanced or refractory melanoma, as FGF2 not only stimulates angiogenesis, but also directly increases the proliferation of melanoma cells.

[0005] To solve the problem of the prior art, the present invention proposes human anti-FGF2 monoclonal antibodies (mAbs) capable of reducing FGF2-dependent cell proliferation and migration in human endothelial (HUVEC) and melanoma (SK-Mel-28) cells.

[0006] Fully human antibodies are currently considered the best option for therapy, as they present the lowest risk of immunogenicity. Thus, the strategy of the present invention to obtain human anti-FGF2 antibodies is to clone the functional VL and VH regions of the LC and Fd chains selected in the humanization process by directed selection (phage display) into vectors containing the genes of the constant region of the human immunoglobulin chain gamma 1 HC, kappa LC or lambda LC, for the expression of the antibody in the IgG1 form.

[0007] The combination of this strategy makes it possible to select human anti-FGF2 mAbs of the present invention that bind to the FGF2 protein, thus making it possible to expect successful treatment of various types of cancer, including melanoma.

[0008] Some prior art documents describe the development of human anti-FGF2 mAbs.

[0009] Brazilian patent application PI 0912035-1 A2, published by GALAXY BIOTECH, LLC on May 23, 2009, entitled: “MONOCLONAL ANTIBODY, PHARMACEUTICAL COMPOSITION, USE OF A PHARMACEUTICAL COMPOSITION, MOUSE MAB IN CHIMERIC OR HUMANIZED FORM, AND HUMANIZED ANTIBODY”, refers to the combination of monoclonal antibodies (mAbs) and recombinant DNA technology to develop new biologies, more specifically, to produce mAbs that bind and neutralize FGF2, i.e., anti-FGF2 mAbs, wherein the mAbs are humanized and obtained by directed selection techniques. However, since the mAbs presented by the present invention have only 55% and 46% identity with the light chain sequences of said document and only 35% and 65% identity with the heavy chain sequences of said document, said document is not relevant to the present invention.

[0010] International patent application PCT / US1999 / 11844, published on January 20, 2000, as WO 00 / 03245 (A1), published in the name of CHUGAI PHARMACEUTICAL COMPANY LIMITED, entitled: “PEPTIDE LIGANDS FOR THE HUMAN FIBROBLAST GROWTH FACTOR (FGF) RECEPTOR”, refers to clones isolated from a phage display library of probes that bind to the human fibroblast growth factor (FGF) receptor. DNA sequences encoding polypeptides of the clones obtained by directed selection techniques that bind to the FGF receptor, independent of the primary structure of the FGF, are disclosed. Differently, the present invention discloses human anti-FGF2 mAbs comprising novel heavy and light chain sequences and providing new, more effective therapeutic options.

[0011] United States patent application US 2020 / 0291120 (A1), published on September 17, 2020, under the name: “ANTI-CLEC2D ANTIBODIES AND METHODS OF USE THEREOF”, describes anti-CLEC2D antibodies and related compositions and methods of use thereof, wherein these antibodies are used as therapeutic agents and diagnostic and prognostic applications in different types of cancer and other diseases. Differently, the present invention presents human anti-FGF2 mAbs.

[0012] The article entitled "Blocking FGF2 with a new specific monoclonal antibody impairs angiogenesis and experimental metastatic melanoma, suggesting a potential role in adjuvant settings", published in Cancer Letters 371(2): 151-60, February 28, 2016, doi:10.1016 / j.canlet.2015.11.030, Aguiar RB et al., describes the therapeutic use of the anti-FGF2 monoclonal antibody (mAb) 3F12E7 using a B16-F10 melanoma model in vivo. In contrast, the present invention proposes a human anti-FGF2 mAb.

[0013] Thus, it is important to emphasize that the clinical efficiency of therapeutic antibodies depends on two types of functional properties: the specific binding to the antigen, mainly conferred by the complementarity determining regions (CDRs) within the VH and VL regions, and the effector functions mediated by the Fc.

[0014] Each variable region (VH and VL) has 3 CDRs that form the antigen-binding structure. The specificity differences of each antibody are mainly due to the differences in these regions.

[0015] Thus, the amino acid sequences of the CDRs of the mAb clones of the present invention were compared with the amino acid sequences of the CDRs of the murine clones in terms of identity and similarity. Moreover, according to the epitope prediction by molecular docking, the human anti-FGF2 mAbs of the present invention interact with FGF2 in a region of residues similar to the chimeric anti-FGF2 antibody, indicating that they recognize the same binding site (epitope) on the antigen.

[0016] The human anti-FGF2 mAb of the present invention interacts with FGF2 within the heparin-binding domain of FGF2 (heparan sulfate proteoglycan - HSPG) at residues Arg120 and Tyr124 and Lys119, Gln123 and Tyr124, respectively. This region is important in the function of FGF2 because, in order to have its function, FGF2 interacts with FGFR and HSPG in a ternary complex (FGF2-FGFR-HSPG) leading to the activation of signaling pathways mainly involved in inducing cell proliferation, migration and differentiation. Therefore, the binding of the human anti-FGF2 mAb to the heparin-binding domain (HSPG) can block the activation of the signaling cascade mediated by the FGF2-FGFR-HSPG complex. Moreover, the maintenance of the epitopes during the humanization process indicates that the specificity of the human anti-FGF2 mAb has been preserved and that the antibody of the present invention should bind the FGF2 antigen in the same region as the murine anti-FGF2 parental antibody that has been previously characterized in vitro and in vivo, showing promising results for use as an adjuvant in melanoma therapy.

[0017] Therefore, although there are anti-FGF2 mAbs developed by guided selection techniques in the state of the art, no literature discloses the mAb of the present invention and its therapeutic potential. SUMMARY As already mentioned, the present invention will provide a significant advantage for the development of new therapeutic options, particularly more effective therapeutic options, or therapeutic options combinable in the fight against melanoma.

[0019] In a first aspect, the present invention relates to a human anti-FGF2 monoclonal antibody (mAb) comprising an antibody heavy chain and an antibody light chain, wherein said chains comprise complementarity determining regions (CDRs) selected from (i) - (ii): (i) light chain CDR1, CDR2 and CDR3 regions having the amino acid sequences of positions 27-37, positions 55-57 and positions 94-102 of SEQ ID NO: 1, respectively; and heavy chain CDR1, CDR2 and CDR3 regions having the amino acid sequences of positions 26-35, positions 53-59 and positions 98-114 of SEQ ID NO: 2, respectively; or (ii) light chain CDR1, CDR2 and CDR3 regions having the amino acid sequences of positions 26-33, positions 51-53 and positions 90-101 of SEQ ID NO: 3, respectively; and heavy chain CDR1, CDR2 and CDR3 regions having the amino acid sequences of positions 26-33, positions 51-58 and positions 97-110 of SEQ ID NO: 4, respectively.

[0020] In a second aspect, the present invention relates to a pharmaceutical composition comprising said mAb.

[0021] In a third aspect, the present application relates to the use of said mAbs for the production of a medicament for the treatment of cancer.

[0022] In a fourth aspect, the present application relates to a kit for the detection of FGF2 protein in a biological sample, said kit comprising said mAbs or biologically active fragments thereof bound to a detectable moiety and useful for the prognosis or targeted cancer treatment.

[0023] In a fifth aspect, the present application relates to the use of said mAbs for the production of a medicament for the control of fibrotic diseases by neutralizing the overaction of FGF, for example in diseases of high intensity tissue repair (keloids) and idiopathic fibrosis or fibrosis secondary to chronic inflammatory diseases.

[0024] In a sixth aspect, the present application relates to the use of these mAbs for immunoscintigraphy, labeled with a radioisotope, for molecular imaging and prediction of fibrotic response. SUMMARY The present application can be better understood with reference to the following drawings and description.

[0026] Figure 1 and 2 An experimental protocol for the humanization of anti-FGF2 monoclonal antibodies by guided selection (phage display) is given.

[0027] Figure 3 Binding assays of human anti-FGF2 antibodies to rFGF2 antigen are illustrated by SPR.

[0028] Figure 4A - B illustrates the evaluation of HUVEC (A) and SK-Mel-28 (B) cell proliferation in the presence of human anti-FGF2 antibodies.

[0029] Figure 5A - D illustrates the evaluation of the effect of human anti-FGF2 antibodies on HUVEC (A) and SK-Mel-28 (C) cell migration, where (B) is a representative image of the HUVEC cell migration assay and (D) is a representative image of the SK-Mel-28 cell migration assay.

[0030] Figure 6A- C illustrates the evaluation of the combined effect of human anti-FGF2 antibodies 62K98H and 85L117H on SK-Mel-28 cell proliferation and migration, where (A) is the number of viable SK-Mel-28 cells after 72 hours of treatment with 20 pg / mL of human anti-FGF2 antibodies 62K98H and 85L117H, an irrelevant human antibody, or PBS; (B) is the percentage migration of SK-Mel-28 cells after 48 hours of treatment with 20 pg / mL of human anti-FGF2 antibodies 62K98H and 85L117H, an irrelevant human antibody, or PBS; (C) is a representative image of the SK-Mel-28 cell migration assay.

[0031] Figure 7 A-C illustrate the prediction of human (and chimeric) anti-FGF2 mAb binding to FGF2 protein, where (A) is a chimeric antibody, (B) is human antibody 62K98H, (C) is human antibody 85L117H. DETAILED DESCRIPTION While the application can be susceptible to embodiments different from those described and illustrated herein, preferred embodiments are shown in the following detailed discussion, which should be considered in conjunction with the accompanying drawings, wherein like numbers represent like parts throughout the several views, and in which:

[0033] The present application relates to a human anti-FGF2 monoclonal antibody (mAb) comprising an antibody heavy chain and an antibody light chain, wherein said chains comprise complementarity determining regions (CDRs) selected from (i) to (ii): (i) light chain CDR1, CDR2 and CDR3 regions having the amino acid sequences of positions 27-37, 55-57 and 94-102, respectively, of SEQ ID NO: 1; and heavy chain CDR1, CDR2 and CDR3 regions having the amino acid sequences of positions 26-35, 53-59 and 98-114, respectively, of SEQ ID NO: 2; or (ii) light chain CDR1, CDR2 and CDR3 regions having the amino acid sequences of positions 26-33, 51-53 and 90-101, respectively, of SEQ ID NO: 3; and heavy chain CDR1, CDR2 and CDR3 regions having the amino acid sequences of positions 26-33, 51-58 and 97-110, respectively, of SEQ ID NO: 4.

[0034] In one embodiment, such human anti-FGF2 monoclonal antibody (mAb) comprises the amino acid sequence of the light chain set forth in SEQ ID NO: 1 or 3 and the amino acid sequence of the heavy chain set forth in SEQ ID NO: 2 or 4.

[0035] Table 1 - CDR1, CDR2 and CDR3 regions of the monoclonal antibody light chain of the present application

[0036] Table 2 - CDR1, CDR2 and CDR3 regions of the heavy chain of the monoclonal antibody of the application

[0037] Although not claimed, the method for obtaining said mAb is the basis for obtaining the essential features of the monoclonal antibody of the application.

[0038] Thus, the method for obtaining the aforementioned mAb comprises the following steps: a) obtaining cDNA from the anti-FGF2 hybridoma 3F12E7; b) amplifying the murine LC and Fd genes of the anti-FGF2 hybridoma 3F12E7; c) cloning the murine LC and Fd genes sequentially into vectors to construct a murine Fab combinatorial library; d) enriching the library obtained in step "c" against the rFGF2 antigen (phage display, panning); e) selecting murine anti-FGF2 Fab clones (phage ELISA and ELISA); f) cloning the murine Fd gene of the selected murine Fab clones into a vector containing a library of human LC gene sequences to construct a hybrid Fab combinatorial library; g) enriching the human Fab combinatorial library against the rFGF2 antigen (phage display, panning); h) selecting human anti-FGF2 Fab clones (phage ELISA and ELISA); i) cloning the human light chain region of the selected hybrid clones into a vector; j) cloning the human heavy chain region of the selected hybrid clones into a vector; and k) combining the light and heavy chain vectors to produce a human anti-FGF2 antibody.

[0039] Figure 1 and 2 An experimental protocol for humanizing the monoclonal antibody anti-FGF2 by phage display is presented.

[0040] In one embodiment, the anti-FGF2 hybridoma of 3F12E7 comprises the amino acid sequence of the light chain shown in SEQ ID NO: 5 and the amino acid sequence of the heavy chain shown in SEQ ID NO: 6.

[0041] Table 3 - CDR1, CDR2 and CDR3 regions of the light chain of the anti-FGF2 hybridoma 3F12E7

[0042] Table 4 - Heavy chain CDR1, CDR2 and CDR3 regions of anti-FGF2 hybridoma 3F12E7

[0043] In one embodiment, the vector used in the above method is the AbVec vector as described in the article by Tiller T et al. published on January 1, 2008 in J Immunol Methods; 329(1-2): 112-24, doi: 10.1016 / j.jim.2007.09.017, entitled “Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning”, available from https: / / www.addgene.org / browse / article / 28189951 / .

[0044] In one embodiment, to establish permanent stable lines, the variable region sequences of the light and heavy chains can be cloned into an expression vector for CHO cells, such as pCHO 3.0, or another vector for transfection of Expi-CHO cells (Thermofisher) for stable expression of the antibody.

[0045] Cloning to obtain a mixed stable population of monoclonal lines is performed by limiting dilution or robotic methods. Selection of the best clones is based on cell growth, productivity, stability, physicochemical characterization and in vitro test results (inhibition of cell proliferation and cell migration).

[0046] The selected clones are grown in increasing volumes in a culture medium suitable for the host cells to establish a fed-batch culture. The culture mode can be fed-batch, the culture temperature being optionally reduced.

[0047] After the culture, the clarified supernatant is purified to remove cells and debris, then purified in chromatography steps, such as capture on protein A and chromatography on ion exchange resin. There are two viral removal / inactivation steps by pH and membrane filtration.

[0048] The clarified supernatant can be purified using conventional monoclonal antibody purification methods, after affinity chromatography on protein A resin, retention at acidic pH (viral inactivation), cationic resin chromatography and anionic resin chromatography. These steps allow the purification of the monoclonal antibody to almost 100%, while removing residual DNA and residual proteins from the host cells.

[0049] Finally, the purity, structure, charge, and power of the obtained mAb are evaluated.

[0050] In addition, the present invention relates to pharmaceutical compositions comprising mAb, said mAb comprising the amino acid sequence of the light chain shown in SEQ ID NO:1 or 3 and the amino acid sequence of the heavy chain shown in SEQ ID NO:2 or 4.

[0051] In one embodiment, the pharmaceutical composition comprises a mAb in a physiologically acceptable carrier, optionally containing excipients or stabilizers, in the form of an aqueous solution or a lyophilized solution.

[0052] Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used, and optionally include buffers such as phosphates, citrates, or acetates with a pH typically between 5.0 and 8.0, most often between 6.0 and 7.0; salts such as sodium chloride, potassium chloride, etc., to produce isotonicity; antioxidants, preservatives, low molecular weight peptides, proteins, hydrophilic polymers such as polysorbate 80, amino acids, carbohydrates, chelating agents, sugars, and other standard ingredients known to those skilled in the art (Remington's Pharmaceutical Science, 16th edition, Osol, A. Ed. 1980). mAb is typically present at a concentration of 10–100 mg / ml, for example, 50 mg / ml.

[0053] In addition, the present invention relates to the use of the mAb in producing the proposed medicament for cancer treatment.

[0054] Melanoma is a type of cancer studied in preclinical models using mouse antibodies. Furthermore, given that increased FGF2 expression is associated with different types of cancer, the therapeutic potential of the anti-FGF2 mAb objects of this invention can be broadened.

[0055] Therefore, in one embodiment of the invention, the cancer is selected from melanoma, pancreatic cancer, non-small cell lung cancer, prostate cancer, hepatocellular carcinoma, Lewis lung cancer, and other cancers associated with increased FGF2 expression. Preferably, the cancer is melanoma.

[0056] In addition to cancer-related therapeutic uses such as inhibiting angiogenesis, the present invention also relates to the use of the mAb in producing medicaments that control fibrotic diseases by neutralizing the excessive effects of FGF, such as in diseases of high-intensity tissue repair (keloids) and idiopathic fibrosis or fibrosis secondary to chronic inflammatory diseases.

[0057] In addition to therapeutic expectations, this invention relates to the application of the mAb in radioisotope-labeled immunoscintillation imaging for molecular imaging and prediction of fibrotic responses.

[0058] In addition, the present application relates to a kit for detecting FGF2 protein in a biological sample, said kit comprising said mAb or biologically active fragment thereof bound to a detectable moiety and useful for the prognosis or targeted cancer treatment.

[0059] Thus, in order to illustrate the present application, experimental results and embodiments of the present application are presented hereinafter to highlight the inventive step of using the mAb of the present application.

[0060] Particular embodiments The construction of human anti-FGF2 antibodies was performed using the AbVec vectors (https: / / www.addgene.org / browse / article / 28189951 / ) containing the genes of the constant region of the human immunoglobulin chains γΐ HC, κ LC or λ LC for antibody expression in HEK 293 or Expi-CHO mammalian cells (Thermofisher).

[0061] This system makes it possible to combine each human LC with different human HC to produce human anti-FGF2 antibodies. During the process of humanization by guided selection, three human LC / HC murine hybrids and eight human HC / LC murine hybrids were obtained. All LC and HC variable regions were combined with each other for transfection of HC-LC pairs, leading to 24 possibilities.

[0062] According to the classification of the V and J families, the human VH and VL regions were amplified using specific primers and the amplified VL and VH regions were cloned into the corresponding vectors containing the constant region of the human immunoglobulin chains. The obtained vectors were used to transfect the LC and HC combinations into HEK 293 cells (initially, Thermofisher, then ExpiCHO (Thermofisher)) for the production of human anti-FGF2 antibodies. CHO cells are the most widely used in the monoclonal antibody industry.

[0063] Antibodies were purified by affinity chromatography using A-sepharose protein resin columns and the integrity of the antibodies was analyzed by electrophoresis (SDS-PAGE) and WB. Of the 24 HC-LC combinations, not all were productive.

[0064] The two mAbs with significant results were evaluated with respect to the epitope recognized on the FGF2 molecule and it was found that similar recognition sites to those recognized by the original murine antibody were found, expressed in chimeric form to contain the same Fc region as the human antibody.

[0065] In a preferred embodiment of the application, the two human anti-FGF2 antibodies 62K98H and 85L117H obtained can be expressed in expression vectors of CHO cells, such as the pCHO 3.0 vector used for synthesis in Expi-CHO cells. The culture mode can be fed-batch, with an optional reduction of the culture temperature. The clarified supernatant can be purified using conventional methods for purification of monoclonal antibodies, a sequence of affinity chromatography on Protein A resin, retention at acidic pH (viral inactivation), chromatography on cationic resin and chromatography on anionic resin. These steps allow the purification of the monoclonal antibodies to almost 100%, while removing residual DNA and residual proteins from the host cells.

[0066] Results of the experiments Binding assay of human anti-FGF2 antibodies to rFGF2 antigen by SPR.

[0067] Antibodies at a concentration of 25 pg / mL were applied to sensors immobilized with 1800 RU of rFGF2. The results show the RU values at the point of binding stability ( Figure 3 ).

[0068] The k a , k d and K D values of the anti-FGF2 antibodies were determined by kinetic affinity assay, as shown in Table 5 below: Table 5 - K a , k d and K D values of the anti-FGF2 antibodies determined by kinetic affinity assay.

[0069] Thus, as can be seen in Figure 3 , the analysis of the ability of the antibodies purified from the 20 HC-LC combinations expressed as IgG to bind the antigen showed various results, demonstrating that not all pairs work in the same way.

[0070] As shown in Table 3, seven pairs of human HC-LC sequences with the highest antigen binding capacity were selected for functional experiments to inhibit the proliferation and migration of human endothelial (HUVEC) and melanoma (SK-Mel-28) cells.

[0071] Evaluation of HUVEC and SK-Mel-28 cell proliferation in the presence of human anti-FGF2 antibodies: Number of HUVEC ( Figure 4A ) and SK-Mel-28 ( Figure 4B ) cells that survived after 72 hours of treatment with 20 pg / mL of human and chimeric anti-FGF2 antibodies, an irrelevant human antibody or PBS. Determined by trypan blue dye exclusion.

[0072] ANOVA was performed, followed by a Bonferroni test to verify whether there was a significant difference between the groups treated with anti-FGF2 antibody and the control groups treated with irrelevant human antibodies (IgG controls 1 and 2). **p≤0.01; ***p≤0.001; ****p≤0.0001.

[0073] In proliferation assays using two cell lines, HUVEC and SK-Mel-28, human anti-FGF2 antibodies 62K98H and 85L117H, as well as chimeric anti-FGF2 antibodies, significantly reduced the number of viable cells compared to groups treated with unrelated human antibodies (IgG controls 1 and 2).

[0074] Evaluation of the effect of human anti-FGF2 antibodies on HUVEC and SK-Mel-28 cell migration: Figure 5 shows the HUVECs after 48 hours of treatment with 20 μg / mL human and chimeric anti-FGF2 antibody, irrelevant human antibody, or PBS. Figure 5A ) and SK-Mel-28 ( Figure 5A -C) Percentage of cell migration.

[0075] ANOVA was performed, followed by a Bonferroni test to verify whether there was a significant difference between the groups treated with anti-FGF2 antibody and the control groups treated with irrelevant human antibodies (IgG controls 1 and 2). p ≤ 0.01; **** p ≤ 0.0001.

[0076] HUVEC (also shown) Figure 5B ) and SK-Mel-28 ( Figure 5D Representative images from a cell migration assay. Dashed lines indicate cell-free regions at time 0h.

[0077] Here, we demonstrate the ability of human anti-FGF2 antibodies 62K98H and 85L117H to modulate cell migration in cell-free regions generated in confined cultures. It can be seen that in both cell lines, the two human anti-FGF2 antibodies and the chimeric anti-FGF2 antibody evaluated significantly attenuated cell migration compared to the groups treated with unrelated human antibodies (controls IgG1 and 2).

[0078] Molecules that can inhibit cell proliferation and migration events are a focus of attention in the development of cancer therapies because these processes are necessary for tumor spread, invasion and angiogenesis in endothelial cells, and are key steps in triggering metastasis and cancer progression in tumor cells.

[0079] Evaluation of the combined effect of human anti-FGF2 antibodies 62K98H and 85L117H on SK-Mel-28 cell proliferation and migration: Figure 6Ais the number of viable SK-Mel-28 cells after 72 hours of treatment with 20 pg / mL of human anti-FGF2 antibodies 62K98H and 85L117H, an irrelevant human antibody or PBS. Determined by trypan blue dye exclusion.

[0080] Figure 6B is the percentage of SK-Mel-28 cell migration after 48 hours of treatment with 20 pg / mL of human anti-FGF2 antibodies 62K98H and 85L117H, an irrelevant human antibody or PBS.

[0081] ANOVA was performed followed by Bonferroni test to verify if there was a significant difference between the groups treated with anti-FGF2 antibodies and the control groups treated with an irrelevant human antibody (control IgG 1 and 2). * p < 0.05; **** p < 0.0001.

[0082] Figure 6C Representative images of the SK-Mel-28 cell migration assay are shown. The dotted line indicates the cell-free area at time 0 h.

[0083] This test was performed to verify the presence or absence of synergistic potential between human anti-FGF2 antibodies 62K98H and 85L117H. The results show that no synergistic effect was observed between the two antibodies, with results similar to those found when the antibodies were evaluated individually in the SK-Mel-28 cell proliferation and migration assays, as shown above.

[0084] Prediction of human (and chimeric) anti-FGF2 mAbs binding to FGF2 protein: Figure 7 is the Fv region of the chimeric antibody (A), human antibody 62K98H (B) and 85L117H (C). Figure 7 A), human antibody 62K98H (B) and 85L117H (C). Figure 7 B) and 85L117H (C). Figure 7 C).

[0085] The residues that are part of the antigen-antibody interaction in FGF2 and the antibodies (found in the LigPlot+ / DIMPLOT analysis) are represented in magenta and orange, respectively. The dotted line represents a hydrogen bond. Figure 7 D shows a yellow schematic representation of the FGF2 residues predicted to interact with the human and chimeric mAbs. The FGFR receptor and heparin binding sites of FGF2 are represented in gray and black, respectively.

[0086] Accordingly, the results presented herein indicate that, according to molecular predictions, the chimeric anti-FGF2 mAbs will contact FGF2 at residues Tyr111 and Tyr124 within the FGFR and heparin receptor binding domains, while human anti-FGF2 mAbs 62K98H and 85L117H will contact FGF2 at residues Arg120, Tyr124 and Lys119, Gln123, Tyr124, respectively, within the heparin binding domain. Inhibition of these domains in the FGF2 molecule by the human and chimeric anti-FGF2 mAbs can interfere with the function of FGF2 by impairing cell signal transduction and thus reducing cell migration and proliferation, as observed in the results shown above. Furthermore, human anti-FGF2 mAbs 62K98H and 85L117H were predicted to repeat with FGF2 in a similar region of residues as the chimeric anti-FGF2 mAbs. This result indicates that the human anti-FGF2 mAbs recognize the same epitope as the chimeric anti-FGF2, indicating successful humanization and generation of human antibodies corresponding to the murine anti-FGF2 mAb 3F12E7.

[0087] Briefly, the present invention relates to the following aspects as defined in the following numbered items: 1. A human anti-FGF2 monoclonal antibody (mAb) comprising an antibody heavy chain and an antibody light chain, wherein the chains comprise complementarity determining regions (CDRs) selected from (i) to (ii): (i) light chain CDR1, CDR2 and CDR3 regions having the amino acid sequences of positions 27-37, positions 55-57 and positions 94-102, respectively, of SEQ ID NO: 1; and heavy chain CDR1, CDR2 and CDR3 regions having the amino acid sequences of positions 26-35, positions 53-59 and positions 98-114, respectively, of SEQ ID NO: 2; and (ii) light chain CDR1, CDR2 and CDR3 regions having the amino acid sequences of positions 26-33, positions 51-53 and positions 90-101, respectively, of SEQ ID NO: 3; and heavy chain CDR1, CDR2 and CDR3 regions having the amino acid sequences of positions 26-33, positions 51-58 and positions 97-110, respectively, of SEQ ID NO: 4.

[0088] 2. The human anti-FGF2 monoclonal antibody (mAb) according to item 1, comprising the amino acid sequence of the light chain set forth in SEQ ID NO: 1 or 3 and the amino acid sequence of the heavy chain set forth in SEQ ID NO: 2 or 4.

[0089] 3. A pharmaceutical composition comprising the mAb as set forth in item 1 or 2.

[0090] 4. Pharmaceutical composition according to item 3, comprising a physiologically acceptable carrier, optionally with excipients or stabilizers, in the form of an aqueous solution or a lyophilized solution.

[0091] 5. Pharmaceutical composition according to item 2 or 3, wherein the mAb is present in a concentration of typically 10-100 mg / ml, preferably 50 mg / ml.

[0092] 6. Use of the mAb according to item 1 or 2, characterized in that it is for the production of a medicament for the treatment of cancer.

[0093] 7. Use of the mAb according to item 1 or 2, wherein it is for the production of a medicament for the control of fibrotic diseases by neutralizing the overaction of FGF, preferably in diseases of high intensity tissue repair (keloids) and idiopathic fibrosis or fibrosis secondary to chronic inflammatory diseases.

[0094] 8. Use of the mAb according to item 1 or 2, wherein it is applied to immunoscintigraphy, labeled with a radioisotope, for molecular imaging and prediction of fibrotic response.

[0095] 9. Kit for the detection of cancer in a biological sample, comprising the mAb or biologically active fragment thereof according to item 1 or 2, bound to a detectable moiety.

[0096] 10. Kit according to item 9, wherein it is applied to the prognosis or therapeutic targeting of cancer.

[0097] Therefore, the embodiments presented in the present application do not limit the totality of possibilities and it should be understood that various omissions, substitutions and changes can be made by those skilled in the art without departing from the scope of the present application.

[0098] It is expressly provided that all combinations of elements performing the same function in substantially the same way to achieve the same results are within the scope of the present application. The substitution of elements from one described embodiment to another is also fully intended and contemplated.

[0099] The person skilled in the art will understand the knowledge presented herein and will be able to reproduce the present application in the presented embodiments and in other variants covered by the scope of the claims.

Claims

1. A human anti-FGF2 monoclonal antibody (mAb) characterized in that comprising an antibody heavy chain and an antibody light chain, wherein said chains comprise complementarity determining regions (CDRs) selected from (i) to (ii): (i) light chain CDR1, CDR2 and CDR3 regions having the amino acid sequences of positions 27-37, 55-57 and 94-102 of SEQ ID NO: 1, respectively; and heavy chain CDR1, CDR2 and CDR3 regions having the amino acid sequences of positions 26-35, 53-59 and 98-114 of SEQ ID NO: 2, respectively; or (ii) light chain CDR1, CDR2 and CDR3 regions having the amino acid sequences of positions 26-33, 51-53 and 90-101 of SEQ ID NO: 3, respectively; and heavy chain CDR1, CDR2 and CDR3 regions having the amino acid sequences of positions 26-33, 51-58 and 97-110 of SEQ ID NO: 4, respectively.

2. The human anti-FGF2 monoclonal antibody (mAb) according to claim 1, characterized by comprising the amino acid sequence of the light chain as set forth in SEQ ID NO: 1 or 3 and the amino acid sequence of the heavy chain as set forth in SEQ ID NO: 2 or 4.

3. A pharmaceutical composition, characterized by comprising the mAb of claim 1 or 2.

4. The pharmaceutical composition of claim 3, wherein comprising a physiologically acceptable carrier, optionally excipients or stabilizers, in the form of an aqueous or lyophilized solution.

5. The pharmaceutical composition according to claim 3 or 4, characterized in that The mAb is typically present in a concentration of 10-100 mg / ml, preferably 50 mg / ml.

6. Use of a monoclonal antibody (mAb) according to claim 1 or 2, characterized in that for use in the manufacture of a medicament for the treatment of cancer.

7. Use of a monoclonal antibody (mAb) according to claim 1 or 2, characterized in that for use in the manufacture of a medicament for the control of fibrotic diseases, neutralization of the overaction of FGF, preferably for high intensity tissue repair (keloids) and idiopathic fibrosis or diseases secondary to chronic inflammatory diseases.

8. Use of a monoclonal antibody (mAb) according to claim 1 or 2, characterized in that for use in immunoscintigraphy, labeled with a radioisotope, for molecular imaging and prediction of fibrotic response.

9. A kit for detecting cancer in a biological sample, said kit comprising a monoclonal antibody (mAb) or biologically active fragment thereof according to claim 1 or 2, characterized in that said mAb or biologically active fragment thereof is conjugated to a detectable moiety.

10. The kit of claim 9, wherein for use in the prognosis or treatment of targeted cancer. for use in the prognosis or treatment of targeted cancer.

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