Human Anti-FGF2 monoclonal antibody (MAB), pharmaceutical composition comprising same, use thereof and kit for the detection of cancer in a biological sample comprising said mab

Abstract

The present invention refers to a human anti-FGF2 monoclonal antibody (mAb) comprising the amino acid sequence of the light chain as defined by the SEQ ID NO: 1 or 3, and the amino acid sequence of the heavy chain as defined by the SEQ ID NO: 2 or 4. Additionally, the present invention refers to a pharmaceutical composition comprising said mAb and to the use thereof in the production of a medicament for treating cancer. Furthermore, the present invention relates to a kit for the detection of the FGF2 protein in a biological sample comprising said mAb or a biologically active fragment thereof bound to a detectable fraction, which can be used for prognosis or targeting cancer treatment. In addition to the therapeutic use associated with cancers, such as inhibition of angiogenesis, there are potential applications in the control of fibrotic diseases, neutralizing the excessive action of FGF, for example in intense tissue repair diseases (keloids), and idiopathic fibrosis or fibrosis secondary to chronic inflammatory diseases. In addition to their therapeutic potential, antibodies can be used for immunoscintigraphy, labeled with radioisotopes, for molecular imaging and prediction of fibrosing responses.

Description

[0001] “HUMAN ANTI-FGF2 MONOCLONAL ANTIBODY (MAB), PHARMACEUTICAL COMPOSITION COMPRISING THE SAME, USE THEREOF AND KIT FOR DETECTING CANCER IN A BIOLOGICAL SAMPLE COMPRISING SAID MAB” Field of Application:

[0001] The present invention falls within the field of preparations for medical purposes, more specifically, in the area of ​​medicinal preparations containing antigens or antibodies, since it relates to human anti-FGF2 monoclonal antibodies (mAbs). Background of the Invention and Prior Art:

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

[0003] Melanoma is the most aggressive and lethal type of cancer. In advanced stages, melanomas are difficult to treat because the tumors develop resistance to most therapies. Therefore, a poor prognosis persists for patients with metastatic melanoma, with a life expectancy of five years for 5 to 19% of cases and an average survival rate of 5.3 months, despite the most recent therapeutic attempts.

[0004] Given this technical challenge, it is necessary to develop new treatment options, especially those that are more effective or can be combined to combat this neoplasm. Inhibiting FGF2-mediated FGFR signaling may be an alternative treatment for patients with advanced or refractory melanoma, because FGF2 not only stimulates angiogenesis but also directly increases melanoma cell proliferation.

[0005] In order to solve the existing technical problem, 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 because they present the lowest risk of immunogenicity. Therefore, the strategy of the present invention for obtaining human anti-FGF2 antibodies was the cloning of the VL and VH regions of the functional human LC and Fd chains selected during the humanization process by guided selection (phage display) into vectors containing the constant region genes of the human immunoglobulin chains γ1HC, κLC, or λLC for expression of antibodies in the IgG1 format.

[0007] This combination of strategies allowed the selection of the human anti-FGF2 mAbs of the present invention, which bind to the FGF2 protein and, therefore, have potential for the treatment of different types of cancer with expected success, including melanoma.

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

[0009] Brazilian patent application No. PI 0912035-1 A2, published on May 23, 2009, in the name of GALAXY BIOTECH, LLC., entitled: “MONOCLONAL ANTIBODY, PHARMACEUTICAL COMPOSITION, USE OF A PHARMACEUTICAL COMPOSITION, MUSE MAB IN CHIMERIC OR HUMANIZED FORM, AND HUMANIZED ANTIBODY” refers to the combination of monoclonal antibody (mAb) and recombinant DNA technologies to develop new biologicals, more specifically, the production of mAbs that bind and neutralize FGF2, that is, anti-FGF2 mAbs, in which said mAbs are humanized and obtained through the guided selection technique. However, the document differs from the present invention, since the present invention proposes mAbs that 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.

[0010] International patent application No. PCT / US1999 / 11844, published under No. WO 00 / 03245 (A1) on January 20, 2000, in the name of CHUGAI PHARMACEUTICAL COMPANY LIMITED, entitled “PEPTIDE LIGANDS FOR THE HUMAN FIBROBLAST GROWTH FACTOR (FGF) RECEPTOR” (Peptide ligands for the human fibroblast growth factor (FGF) receptor) relates to clones isolated from phage display libraries that bind to a human fibroblast growth factor (FGF) receptor probe. Disclosed are polypeptides unrelated to the primary structure of FGF and encoded by DNA sequences of the clones obtained by the guided selection technique that bind to the FGF receptor. In contrast, the present invention discloses human anti-FGF2 mAbs comprising novel heavy and light chain sequences and providing new, more effective treatment options.

[0011] U.S. Patent Application No. US 2020 / 0291120 (A1), published on September 17, 2020, in the name of ZUMUTOR BIOLOGIES INC., entitled: “ANTI-CLEC2D ANTIBODIES AND METHODS OF USE THEREOF” (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 therapeutics and in diagnostic and prognostic applications in various types of cancer and other diseases. In contrast, the present invention proposes 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, on February 28, 2016, under number doi: 10.1016 / j.canlet.2015.11.030, in the name of Aguiar RB et al. describes the therapeutic use of an anti-FGF2 monoclonal antibody (mAb), 3F12E7, using the B16-F10 melanoma model in vivo. In contrast, the present invention proposes human anti-FGF2 mAbs.

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

[0014] Each variable region (VH and VL) has three CDRs, which form an antigen-binding structure. The difference in specificity of each antibody is primarily due to 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 sequence of the CDRs of the murine clone with respect to identity and similarity. Furthermore, according to 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, suggesting that they recognize the same binding site on the antigen (epitope).

[0016] The human anti-FGF2 mAbs of the present invention interact with FGF2 at residues Arg120 and Tyr124, and Lys119, Gln123, and Tyr124, respectively, within the heparin-binding domain (hepan sulfate proteoglycans - HSPG) of FGF2. This region is important for FGF2 functionality, since to function, FGF2 interacts with FGFR and HSPG in a ternary complex (FGF2-FGFR-HSPG), resulting in the activation of signaling pathways mainly involved in the induction of cell proliferation, migration, and differentiation. Thus, the binding of human anti-FGF2 mAbs to the heparin-binding domain (HSPG) can block the activation of the signaling cascade mediated by the FGF2-FGFR-HSPG complex.Furthermore, the maintenance of the epitope in the humanization process indicates that the specificity of the human anti-FGF2 mAbs was preserved, and that the antibodies of the present invention should bind to the FGF2 antigen in the same region as the parental murine anti-FGF2 antibody that has already been previously characterized in vitro and in vivo showing promising results for use as an adjuvant in melanoma therapy.

[0017] Thus, although the development of anti-FGF2 mAbs through the guided selection technique already exists in the prior art, no document discloses the mAbs of the present invention and their therapeutic potential. Summary of the invention:

[0018] As already mentioned, the present invention will provide significant advantages for the development of new treatment options, especially more effective ones, or ones that can be combined 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) CDR1, CDR2 and CDR3 regions of the light chain having the amino acid sequences of positions 27-37, positions 55-57 and positions 94-102 of SEQ ID NO: 1, respectively; and CDR1, CDR2 and CDR3 regions of the heavy chain 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 invention relates to the use of said mAb for the production of a drug to treat cancer.

[0022] In a fourth aspect, the present invention relates to a kit for detecting the FGF2 protein in a biological sample comprising said mAb or a biologically active fragment thereof linked to a detectable fraction, which can be used for prognosis or targeting of cancer treatment.

[0023] In a fifth aspect, the present invention relates to the use of said mAb for the production of a drug for controlling fibrotic diseases, neutralizing the excessive action of FGF, for example in diseases of intense tissue repair (keloids), and idiopathic fibrosis or fibrosis secondary to chronic inflammatory diseases.

[0024] In a sixth aspect, the present invention relates to the use of said mAbs to be applied for immunoscintigraphy, labeled with radioisotopes, for molecular imaging and prediction of fibrosing responses. Brief description of the figures:

[0025] The present invention, together with its additional advantages, can be better understood by reference to the attached figures and the following description.

[0026] Figures 1 and 2 show the experimental flow for humanization of the anti-FGF2 monoclonal antibody by guided selection (phage display).

[0027] Figure 3 graphically shows the binding assay of human anti-FGF2 antibodies to rFGF2 antigen by SPR.

[0028] Figures 4A-B graphically show the evaluation of the proliferation of HUVEC (A) and SK-Mel-28 (B) cells in the presence of human anti-FGF2 antibodies.

[0029] Figures 5A-D graphically show the evaluation of the action of human anti-FGF2 antibodies on the migration of HUVEC (A) and SK-Mel-28 (C) cells, in which (B) refer to representative images of the HUVEC cell migration assay, and (D) refer to representative images of the SK-Mel-28 cell migration assay.

[0030] Figures 6A-C graphically show the evaluation of the joint action of human anti-FGF2 antibodies 62K98H and 85L117H on the proliferation and migration of SK-Mel-28 cells, where (A) refers to the number of viable SK-Mel-28 cells 72 h after treatment with 20 μg / mL of human anti-FGF2 antibodies 62K98H and 85L117H, irrelevant human antibodies, or PBS; (B) refers to the percentage of migration of SK-Mel-28 cells after the 48 h period of treatment with 20 μg / mL of human anti-FGF2 antibodies 62K98H and 85L117H, irrelevant human antibodies, or PBS; and (C) are representative images of the SK-Mel-28 cell migration assay.

[0031] Figures 7A-C illustrate the prediction of binding of human (and chimeric) anti-FGF2 mAbs to FGF2 protein, wherein (A) refers to the chimeric antibody, (B) refers to the human antibody 62K98H; and (C) refers to the human antibody 85L117H. Detailed description of the invention:

[0032] Although the present invention may be susceptible to different embodiments, a preferred embodiment is shown in the following detailed discussion, with the understanding that the present embodiment should be considered an exemplification of the principles of the invention and is not intended to limit the present invention to what has been described in this report.

[0033] 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) to (ii): (i) CDR1, CDR2 and CDR3 regions of the light chain having the amino acid sequences of positions 27-37, positions 55-57 and positions 94-102 of SEQ ID NO: 1, respectively; and CDR1, CDR2 and CDR3 regions of the heavy chain 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.

[0034] In one embodiment, said anti-FGF2 human monoclonal antibody (mAb) comprises the light chain amino acid sequence as set forth by SEQ ID NO: 1 or 3, and the heavy chain amino acid sequence as set forth by SEQ ID NO: 2 or 4. Table 1 - CDR1, CDR2 and CDR3 regions of the light chain of the monoclonal antibody of the present invention Table 2 - CDR1, CDR2 and CDR3 regions of the heavy chain of the monoclonal antibody of the present invention

[0035] Although not claimed, the process of obtaining said mAbs is fundamental to obtain the essential characteristics of the monoclonal antibodies of the present invention.

[0036] Thus, the process of obtaining the referred mAbs comprises the steps of: a) Obtaining the cDNA of the 3F12E7 anti-FGF2 hybridoma; b) Amplifying the murine LC and Fd genes of the 3F12E7 anti-FGF2 hybridoma; c) Sequentially cloning the murine LC and Fd genes into a vector for the construction of the murine Fab combinatorial library; d) Enriching the library obtained in step "c" against the rFGF2 antigen (phage display, panning); e) Selecting the murine anti-FGF2 Fab clone (phage ELISA and ELISA); f) Clone the murine Fd gene from the selected murine Fab clone into a vector containing the human LC gene repertoire library for construction of the hybrid Fab combinatorial library; g) Enriching the human Fab combinatorial libraries against the rFGF2 antigen (phage display, panning); h) Select the human anti-FGF2 Fab clones (phage ELISA and ELISA); i) Clone the human light chain region of the selected hybrid clones into a vector;j) Clone the human heavy chain region of the selected hybrid clones into a vector; and k) Combine the light and heavy chain vectors to generate human anti-FGF2 antibodies. ;

[0037] Figures 1 and 2 show the experimental flow for humanization of the anti-FGF2 monoclonal antibody by phage display.

[0038] In one embodiment, said 3F12E7 anti-FGF2 hybridoma comprises the light chain amino acid sequence as set forth by SEQ ID NO: 5, and the heavy chain amino acid sequence as set forth by SEQ ID NO: 6. Table 3 - 3F12E7 anti-FGF2 hybridoma light chain CDR1, CDR2, and CDR3 regions Table 4 - CDR1, CDR2, and CDR3 regions of the heavy chain of the 3F12E7 anti-FGF2 hybridoma

[0039] In one embodiment, the vectors used in the process described above are AbVec vectors as described in the article entitled “Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning”, on behalf of Tiller T et al, published in the journal J Immunol Methods on January 1, 2008; 329(1-2):112-24, doi: 10.1016 / j.jim.2007.09.017, available at https: / / www.addgene.org / browse / article / 28189951 / .

[0040] In one embodiment, for the establishment of permanent stable lineages, the sequences of the variable regions of the light and heavy chains can be cloned into expression vectors for CHO cells, e.g., pCHO 3.0, or other used for transfection of Expi-CHO cells (Thermofisher) for stable expression of the antibody.

[0041] The stable mixed population cloned to obtain the monoclonal lineage is performed by limiting dilution or a robotic method. Selection of the best clone is based on cell growth, productivity, stability, physicochemical characterization, and in vitro assay results (inhibition of cell proliferation and cell migration).

[0042] The selected clone is grown in culture medium appropriate for the host cell, in increasing volumes, to establish fed-batch culture. The culture method can be fed-batch, with optional temperature reduction.

[0043] After cultivation, the supernatant is clarified to remove cells and debris and then purified through chromatographic steps, such as protein A capture and ion-exchange resin chromatography. There are two viral removal / inactivation steps: pH and membrane filtration.

[0044] The clarified supernatant can be purified using conventional monoclonal antibody purification methods, consisting of affinity chromatography on protein A resin, retention at acidic pH (viral inactivation), cation resin chromatography, and anion resin chromatography. These steps allow for nearly 100% purification of monoclonal antibodies, removing residual DNA and host cell proteins.

[0045] Finally, purity, structure, electrical charge and potency tests are applied to the obtained mAbs.

[0046] Additionally, the present invention relates to a pharmaceutical composition comprising a mAb comprising the light chain amino acid sequence as set forth by SEQ ID NO: 1 or 3, and the heavy chain amino acid sequence as set forth by SEQ ID NO: 2 or 4.

[0047] In one embodiment, the pharmaceutical composition comprises the mAb in a physiologically acceptable carrier, optionally with excipients or stabilizers, in the form of aqueous or lyophilized solutions.

[0048] Acceptable carriers, excipients, or stabilizers are nontoxic to the recipients at the dosages and concentrations used and optionally include buffers such as phosphate, citrate, or acetate at a pH typically of 5.0 to 8.0, more often 6.0 to 7.0; salts such as sodium chloride, potassium chloride, among others to manufacture isotonics; antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers such as polysorbate 80, amino acids, carbohydrates, chelating agents, sugars, and other standard ingredients known to one skilled in the art (Remington's Pharmaceutical Science, 16th edition, Osol, A. Ed. 1980). The mAb is typically present at a concentration of 10 to 100 mg / ml, e.g., 50 mg / ml.

[0049] Additionally, the present invention relates to the use of said mAb for the production of a drug proposed for the treatment of cancer.

[0050] Melanoma was the cancer type focused on in the preclinical model with the murine antibody. Furthermore, given that increased FGF2 expression is associated with several types of cancer, the therapeutic potential of the anti-FGF2 mAbs subject to the present invention could be extended.

[0051] Thus, in one embodiment of the invention, cancer is selected from the group consisting of melanoma, pancreatic cancer, non-small cell lung carcinoma, prostate tumors, hepatocellular carcinoma, Lewis lung carcinoma, among other cancers related to increased expression of FGF2. Preferably, the cancer is melanoma.

[0052] Additionally, in addition to the therapeutic use associated with cancers, such as inhibition of angiogenesis, the present invention relates to the use of said mAb for the production of a drug for controlling fibrotic diseases, neutralizing the excessive action of FGF, for example in diseases of intense tissue repair (keloids), and idiopathic fibrosis or fibrosis secondary to chronic inflammatory diseases.

[0053] In addition to the therapeutic expectation, the present invention refers to the use of said mAb for application in immunoscintigraphy, marked with radioisotopes, for molecular imaging and prediction of fibrosing responses.

[0054] Additionally, the present invention relates to a kit for detecting the FGF2 protein in a biological sample comprising said mAb or a biologically active fragment thereof linked to a detectable fraction, which can be used for prognosis or targeting of cancer treatment.

[0055] Therefore, in order to elucidate the present invention, the following are presented experimental results and embodiments of the invention in order to demonstrate the inventive step of using the mAb of the present invention. Embodiments of the invention

[0056] The construction of human anti-FGF2 antibodies was performed using AbVec vectors (https: / / www.addgene.org / browse / article / 28189951 / ) containing the genes of the constant region of the human immunoglobulin chains γ1HC, κLC or λLC for expression of antibodies in HEK 293 or Expi-CHO mammalian cells (Thermofisher).

[0057] This system enabled the combination of each human LC with different human HC for the production of human anti-FGF2 antibodies. During the humanization process by guided selection, three human LC / murine HC hybrids and eight human HC / murine LC hybrids were obtained. All LC and HC variable regions were combined for transfection of the HC-LC pair, resulting in 24 possibilities.

[0058] Amplification of the human VH and VL regions was performed using specific primers according to the V and J family classification, and the amplified VL and VH regions were cloned into the respective vectors containing the constant region of the human immunoglobulin chains. The vectors obtained were used to transfect the LC and HC combinations into HEK 293 cells (Thermofisher, initially, and then ExpiCHO (Thermofisher)) for the production of human anti-FGF2 antibodies. CHO cells are the most widely used in the monoclonal antibody industry.

[0059] The antibodies were purified by affinity chromatography using a protein A-Sepharose resin column, and the integrity of the antibodies was analyzed by electrophoresis (SDS-PAGE) and WB. Of the 24 HC-LC combinations, not all were productive.

[0060] The two mAbs with significant results were evaluated in relation to the epitope recognized in the FGF2 molecule, and a recognition site similar to the site recognized by the original murine antibody was found, expressed in chimeric form to contain the same Fc region as human antibodies.

[0061] In a preferred embodiment of the invention, the two human anti-FGF2 antibodies, 62K98H and 85L117H, obtained can be expressed in expression vectors for CHO cells, such as the pCHO 3.0 vector, for synthesis in Expi-CHO cells. The culture method can be fed-batch, with an optional reduction in the culture temperature. The clarified supernatant can be purified by the conventional monoclonal antibody purification method, following affinity chromatography on protein A resin, retention at acidic pH (viral inactivation), chromatography on a cationic resin, and chromatography on an anionic resin. These steps allow for the purification of monoclonal antibodies to a level of nearly 100%, with removal of residual DNA and residual host cell proteins. Experimental Results - Binding assay of human anti-FGF2 antibodies to the rFGF2 antigen by SPR.

[0062] The antibodies, at a concentration of 25 µg / mL, were applied to the sensor immobilized with 1800 RU of rFGF2. The results show the RU values ​​at the point of binding stability (Figure 3).

[0063] The values ​​of k a , k d and K D of anti-FGF2 antibodies were determined by the kinetic affinity assay, as shown in Table 5 below: Table 5 - Values ​​of ka, kd and KD of anti-FGF2 antibodies determined by the kinetic affinity assay.

[0064] Thus, as can be seen in Figure 3, purified antibodies from 20 HC-LC combinations expressed as IgG were analyzed for antigen binding capacity, which showed a variety of results demonstrating that not all pairs function in the same way.

[0065] 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. - Evaluation of the proliferation of HUVEC and SK-Mel-28 cells in the presence of human anti-FGF2 antibodies:

[0066] The number of viable HUVEC (Figure 4A) and SK-Mel-28 (Figure 4B) cells after 72 h of treatment with 20 μg / mL of the human and chimeric anti-FGF2 antibodies, irrelevant human antibodies, or PBS. The assay was performed by trypan blue dye exclusion.

[0067] ANOVA followed by Bonferroni test was performed to verify the presence or absence of significant difference between the groups treated with anti-FGF2 antibodies and the control groups treated with irrelevant human antibodies (IgG control 1 and 2). ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

[0068] The human anti-FGF2 antibodies 62K98H and 85L117H, as well as the chimeric anti-FGF2 antibody, were able to significantly reduce the number of viable cells compared to groups treated with irrelevant human antibodies (IgG control 1 and 2) in proliferation assays with the two cell lines, HUVEC and SK-Mel-28. - Evaluation of the action of human anti-FGF2 antibodies on the migration of HUVEC and SK-Mel-28 cells:

[0069] Figure 5 shows the percentage of migration of HUVEC (Figure 5A) and SK-Mel-28 (Figure 5A C) cells after a 48-h treatment period with 20 μg / mL of human and chimeric anti-FGF2 antibodies, irrelevant human antibodies, or PBS.

[0070] ANOVA followed by the Bonferroni test was performed to verify the presence or absence of a significant difference between the groups treated with anti-FGF2 antibodies and the control groups treated with irrelevant human antibodies (IgG control 1 and 2). ** p ≤ 0.01; **** p ≤ 0.0001.

[0071] Representative images of the migration assay of HUVEC (Figure 5B) and SK-Mel-28 (Figure 5D) cells are also shown. The dashed lines indicate the cell-free area at time 0 h.

[0072] Here, we demonstrate the ability of human anti-FGF2 antibodies 62K98H and 85L117H to modulate cell migration in a cell-free area created in a confluent culture. It can be observed that in both cell lines, the two human anti-FGF2 antibodies tested, and the chimeric anti-FGF2 antibody, were able to significantly attenuate cell migration compared to groups treated with irrelevant human antibodies (control IgG 1 and 2).

[0073] Molecules capable of inhibiting cell proliferation and migration are of interest for the development of cancer therapies, since, in endothelial cells, these processes are necessary for tumor dissemination, invasion, and angiogenesis, and, in tumor cells, they are key steps in triggering metastasis and cancer progression. - Evaluation of the combined action of human anti-FGF2 antibodies 62K98H and 85L117H on the proliferation and migration of SK-Mel-28 cells:

[0074] Figure 6A shows the number of viable SK-Mel-28 cells 72 h after treatment with 20 μg / mL of the human anti-FGF2 antibodies 62K98H and 85L117H, irrelevant human antibodies, or PBS. The assay is performed by trypan blue dye exclusion.

[0075] Figure 6B shows the percentage of migration of SK-Mel-28 cells after a 48-h treatment period with 20 μg / mL of the human anti-FGF2 antibodies 62K98H and 85L117H, irrelevant human antibodies, or PBS.

[0076] ANOVA followed by the Bonferroni test was performed to verify the presence or absence of a significant difference between the groups treated with anti-FGF2 antibodies and the control groups treated with irrelevant human antibodies (IgG control 1 and 2). * p ≤ 0.05; **** p ≤ 0.0001.

[0077] Figure 6C shows representative images of the SK-Mel-28 cell migration assay. The dashed lines indicate the cell-free area at time 0 h.

[0078] This assay was performed to determine whether there is a synergistic potential between the human anti-FGF2 antibodies 262K98H 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 tested individually in a SK-Mel-28 cell proliferation and migration assay, as demonstrated above. - Prediction of binding of human (and chimeric) anti-FGF2 mAbs to FGF2 protein:

[0079] Figure 7 is a three-dimensional representation of the molecular complexes between the Fv region and FGF2 of the chimeric antibody (Figure 7A), and of the human antibodies 62K98H (Figure 7B) and 85L117H (Figure 7C).

[0080] Residues involved in the antigen-antibody interaction in FGF2 and antibodies (found in LigPlot+ / DIMPLOT analyses) are represented by magenta and orange, respectively. The dashed lines indicate hydrogen bonds. Figure 7D shows a schematic representation in yellow of the FGF2 residues predicted to interact with the human and chimeric mAbs. The FGFR receptor and the FGF2 heparin-binding site are represented in gray and black, respectively.

[0081] Therefore, the results presented here showed that, according to the molecular prediction, the chimeric anti-FGF2 mAb would contact FGF2 at residues Tyr111 and Tyr124 within the FGFR and heparin receptor binding domains, while the human anti-FGF2 mAbs 62K98H and 85L117H would contact FGF2 at residues Arg120, Tyr124, and Lys119, Gln123, Tyr124, respectively, within the heparin binding domains. Inhibition of these domains in the FGF2 molecule by human and chimeric anti-FGF2 mAbs may interfere with FGF2 function by compromising cell signaling and consequently reducing cell migration and proliferation, as observed in the results shown above. Furthermore, it was predicted that human anti-FGF2 mAbs 62K98H and 85L117H interact with FGF2 in a region of residues similar to the chimeric anti-FGF2 mAb.This result suggests that human anti-FGF2 mAbs recognize the same epitope as chimeric anti-FGF2, indicating success in humanization and generation of human antibodies corresponding to murine anti-FGF2 mAb 3F12E7.

[0082] In summary, the 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 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, 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; 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 of SEQ ID NO: 3, respectively; and CDR1, CDR2, and CDR3 regions of the heavy chain with the amino acid sequences of positions 26-33, positions 51-58, and positions 97-110 of SEQ ID NO: 4, respectively. 2.Human anti-FGF2 monoclonal antibody (mAb), according to item 1, in which it comprises the light chain amino acid sequence as established by SEQ ID NO: 1 or 3, and the heavy chain amino acid sequence as established by SEQ ID NO: 2 or 4. 3. Pharmaceutical composition comprising a mAb as defined in item 1 or 2. 4. Pharmaceutical composition, according to item 3, in which it comprises a physiologically acceptable carrier, optionally with excipients or stabilizers, in the form of aqueous or lyophilized solutions. 5. Pharmaceutical composition, according to items 2 or 3, in which the mAb is typically present in a concentration of 10 to 100 mg / ml, preferably 50 mg / ml. 6. Use of the mAb as defined in item 1 or 2 characterized by the fact that it is for the production of a medicament to treat cancer. 7.Use of the mAb as defined in item 1 or 2, in which it is for the production of a drug for the control of fibrotic diseases, neutralizing the excessive action of FGF, preferably in diseases of intense tissue repair (keloids), and idiopathic fibrosis or secondary to chronic inflammatory diseases. 8. Use of the mAb as defined in item 1 or 2, in which it is for application in immunoscintigraphy, labeled with radioisotopes, for molecular imaging and prediction of fibrosing responses. 9. Kit for the detection of cancer in a biological sample comprising the mAb as defined in item 1 or 2, or a biologically active fragment thereof bound to a detectable fraction. 10. Kit, according to item 9, in which it is applied for prognosis or direction of treatment for cancer.

[0083] Thus, the realizations presented in the present invention do not limit the totality of the possibilities, and it will be understood that various omissions, substitutions and alterations can be made by a person skilled in the art, without departing from the scope of the present invention.

[0084] It is expressly intended that all combinations of the elements that perform the same function in substantially the same manner to achieve the same results are within the scope of the invention. Substitutions of elements from one described embodiment to another are also fully intended and contemplated.

[0085] Technicians skilled in the art will appreciate the knowledge presented here and will be able to reproduce the invention in the embodiments presented and in other variants, covered by the scope of the claims.

Claims

CLAIMS 1. 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) CDR1, CDR2, and CDR3 regions of the light chain having the amino acid sequences of positions 27-37, positions 55-57, and positions 94-102 of SEQ ID NO: 1, respectively; and CDR1, CDR2, and CDR3 regions of the heavy chain 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. 2.Human anti-FGF2 monoclonal antibody (mAb) according to claim 1, characterized by the fact that it comprises the light chain amino acid sequence as established by SEQ ID NO: 1 or 3, and the heavy chain amino acid sequence as established by SEQ ID NO: 2 or 4.

3. Pharmaceutical composition characterized by the fact that it comprises a mAb as defined in claim 1 or 2.

4. Pharmaceutical composition according to claim 3, characterized by the fact that it comprises. a physiologically acceptable carrier, optionally with excipients or stabilizers, in the form of aqueous or lyophilized solutions.

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

6. Use of the monoclonal antibody (mAb) as defined in claim 1 or 2, characterized in that it is for the production of a medicament to treat cancer.

7. Use of the monoclonal antibody (mAb) as defined in claim 1 or 2, characterized in that it is for the production of a medicament to control fibrotic diseases, neutralizing the excessive action of FGF, preferably in diseases of intense tissue repair (keloids), and idiopathic fibrosis or fibrosis secondary to chronic inflammatory diseases. 8.Use of the monoclonal antibody (mAb) as defined in claim 1 or 2, characterized in that it is for application in immunoscintigraphy, labeled with radioisotopes, for molecular imaging and prediction of fibrosing responses.

9. Kit for detecting cancer in a biological sample comprising the monoclonal antibody (mAb) as defined in claim 1 or 2, or a biologically active fragment thereof, characterized in that said mAb or biologically active fragment thereof is linked to a detectable moiety.

10. Kit, according to claim 9, characterized by the fact that it is applied for prognosis or direction of cancer treatment.

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