Bispecific antibodies directed against alpha-folate receptor

Abstract

A bispecific antibody is described comprising a first antigen binding arm that binds FRa receptor and a second antigen-binding arm that binds CD3, wherein: a) the first antigen-binding arm (A) of the bispecific antibody comprises: three heavy chain complementarity determining regions (CDRs), A- VHCDR1, A-VHCDR2 and A- VHCDR3 comprising the aminoacid sequences of SEQ ID NO: 1, 2 and 3 respectively three light chain complementarity determining regions (CDRs), A- VLCDR1, A-VLCDR2 and A- VLCDR3 comprising the aminoacid sequences of SEQ ID NO: 4, 5 and 6 respectively b) the second antigen-binding arm (B) of the bispecific antibody comprises: three heavy chain complementarity determining regions (CDRs), B- VHCDR1, B-VHCDR2 and B- VHCDR3 comprising the aminoacid sequences of SEQ ID NO: 7, 8 and 9 respectively three light chain complementarity determining regions (CDRs), B- VLCDR1, B-VLCDR2 and B- VLCDR3 comprising the aminoacid sequences of SEQ ID NO: 10, 11 and 12 respectively. Furthermore, the pharmaceutical composition comprising said bispecific antibody and its pharmaceutical use are described.

Description

[0001] Title: Bispecific antibodies directed against alpha-folate receptor DESCRIPTION

[0002] Field of application

[0003] The invention relates to the field of antibodies for use in human therapy, in particular a bispecific antibody being directed to the a-folate receptor (FRa) and the co-receptor CD3, with utility in the treatment of tumors and other diseases related to the activity and expression of this receptor.

[0004] Prior art

[0005] Folate receptor a (FRa) came into focus as an anticancer target many decades after the successful development of drugs targeting intracellular folate metabolism, such as methotrexate and pemetrexed. Binding to FRa is one of several methods by which folate is taken up by cells; this receptor is an attractive anticancer drug target owing to its overexpression in a range of solid tumors, including ovarian, lung and breast cancers. Furthermore, using FRa to better localize effective anticancer therapies to their target tumors using platforms such as antibody-drug conjugates, small-molecule drug conjugates, radioimmunoconjugates and, more recently, chimeric antigen receptor T cells could further improve the outcomes of patients with FRa-overexpressing cancers1. FRa can also be harnessed for predictive biomarker research.

[0006] The FRa is a glycosylphosphatidyl-inositol (GPI)-linked protein and is overexpressed in more than 90% of epithelial ovarian carcinoma (EOC) and also in various tumors of mesodermic derivation, like pleural mesothelioma in which it is expressed in more than 50% of cases2. Although the function of FRa in cancers is not fully understood, its overexpression is associated with increased tumor aggressiveness3;4and might confer growth advantage by increasing folate availability to cancer cells5.

[0007] Folate receptor a (FRa) is a valid anticancer drug target for multiple reasons. These reasons include: the limited expression in non- malignant tissues coupled with overexpression in malignant tissues of certain cancers, making FRa a possible target using multiple platforms, for example monoclonal antibody or CAR-T6,7,8.

[0008] However, the monoclonal antibody recognizes and binds only one specific target, the antigen, but sometimes, to treat some tumors different targets are needed due to trigger different physiological or antitumor responses. Therefore, multi- treatment is the only way to treat tumors, but administering two different antibodies at the same time often leads to side effects that are too severe and not tolerated by the patient.

[0009] Furthermore, CAR T-cell therapy can be very effective against some types of hard-to-treat cancers, but it can also sometimes cause serious or even life-threatening side effects, for example cytokine release syndrome, nervous system problems and other possible serious side effects that can include allergic reactions during the infusion or a weakened immune system, with an increased risk of serious infections.

[0010] The technical problem underlying the present invention is to provide a new system that is highly specific against Folate receptor a expressed on tumor cells, thus overcoming the previously mentioned problems of the prior art.

[0011] Summary of the invention

[0012] The above-mentioned technical problem is firstly solved by a bispecific antibody comprising a first antigen binding arm that binds FRa receptor and a second antigen-binding arm that binds CD3, wherein:

[0013] a) the first antigen-binding arm (A) of the bispecific antibody comprises: three heavy chain complementarity determining regions (CDRs), A- VHCDR1, A-VHCDR2 and A- VHCDR3 comprising the aminoacid sequences of SEQ ID NO: 1, 2 and 3 respectively

[0014] three light chain complementarity determining regions (CDRs), A- VLCDR1, A-VLCDR2 and A- VLCDR3 comprising the aminoacid sequences of SEQ ID NO: 4, 5 and 6 respectively

[0015] b) the second antigen-binding arm (B) of the bispecific antibody comprises:

[0016] three heavy chain complementarity determining regions (CDRs), B-VHCDR1, B-VHCDR2 and B- VHCDR3 comprising the aminoacid sequences of SEQ ID NO: 7, 8 and 9 respectively

[0017] three light chain complementarity determining regions (CDRs), B- VLCDR1, B-VLCDR2 and B- VLCDR3 comprising the aminoacid sequences of SEQ ID NO: 10, 11 and 12 respectively.

[0018] The bispecific antibodies containing the above mentioned CDRs show specificity to FRa and CD3.

[0019] The A- VH CDR1 is preferably located between bispecific antibody’s A-VH frameworks 1 and 2.

[0020] The A- VH CDR2 is preferably located between bispecific antibody’s A-VH frameworks 2 and 3.

[0021] The A- VH CDR3 is preferably located between bispecific antibody’s A-VH frameworks 3 and 4.

[0022] The A- VL CDR1 is preferably located between bispecific antibody’s A-VL frameworks 1 and 2.

[0023] The A- VL CDR2 is preferably located between bispecific antibody’s A-VL frameworks 2 and 3.

[0024] The A- VL CDR3 is preferably located between bispecific antibody’s A-VL frameworks 3 and 4.

[0025] The B- VH CDR1 is preferably located between bispecific antibody’s A-VH frameworks 1 and 2.

[0026] The B- VH CDR2 is preferably located between bispecific antibody’s A-VH frameworks 2 and 3.

[0027] The B- VH CDR3 is preferably located between bispecific antibody’s A-VH frameworks 3 and 4.

[0028] The B- VL CDR 1 is preferably located between bispecific antibody’s A-VL frameworks 1 and 2.

[0029] The B- VL CDR2 is preferably located between bispecific antibody’s A-VL frameworks 2 and 3.

[0030] The B- VL CDR3 is preferably located between bispecific antibody’s A-VL frameworks 3 and 4.

[0031] The above referred A-VH and B-VH frameworks 1, 2, 3 and A-VL and B-VL frameworks 1, 2, 3 are those defined according to the standard antibody structure of IMGT / Collier-de-Perles (Domain V): (Ruiz M, et al. Immunogenetics. 2002).

[0032] All said CDRs are constant in structure and simultaneously present in the antibodies of the invention.

[0033] According to the invention, the expression “bispecific antibody” or “bispecific monoclonal antibody” (BsMAb, BsAb) refers herein to an artificial protein that can simultaneously bind to two different types of antigen or two different epitopes on the same antigen. Naturally occurring antibodies typically only target one antigen. BsAbs can be manufactured in several structural formats. BsAbs can be designed to recruit and activate immune cells, in order to interfere with receptor signaling and inactivate signaling ligands, and to force association of protein complexes. Preferably, a sub-group of the first antigen-binding arm (A) of the bispecific antibody, containing particular aminoacids at certain positions of the VH and VL chains external to said CDRs have shown the additional unexpected property of a prolonged binding to the FRa. These first antigen-binding arm (A) of the bispecific antibody are characterized in that the VH comprises 120 aminoacids and meet at least ten of the conditions according to

[0034] Table 1:

[0035] Aminoacid at position

[0036] V 5

[0037] S 9

[0038] K 12

[0039] K 13

[0040] V 20

[0041] R 38

[0042] A 40

[0043] M 48

[0044] V 76

[0045] L 81

[0046] Q 82

[0047] I 83

[0048] s 84

[0049]

[0050] K 87 A 88

[0051] T 91

[0052] T 116

[0053]

[0054] S 120

[0055] and, at the same time, VL comprises 107 aminoacids and meets at least eight of the conditions according to:

[0056] Table 2

[0057] aminoacid at position

[0058] S 7

[0059] P 8

[0060] V 15

[0061] T 22

[0062] G 41

[0063] K 42

[0064] A 43

[0065] P 44

[0066] T 72

[0067] F 73

[0068] S 76

[0069]

[0070] S 77 P 80

[0071] T

[0072]

[0073] 85

[0074] More preferably, the first antigen-binding arm (A) of the bispecific antibody meets a higher number of conditions of Tables 1 and 2, in particular:

[0075] the VH meets at least fourteen of the conditions of Table 1 and the VL meets at least eleven of the conditions of Table 2, or

[0076] the VH meets at least fifteen of the conditions of Table 1 and the VL meets at least twelve of the conditions of Table 2, or

[0077] the VH meets at least sixteen of the conditions of Table 1 and the VL meets at least thirteen of the conditions of Table 2, or

[0078] the VH meets at least seventeen of the conditions of Table 1 and the VL meets at least fourteen of the conditions of Table 2, or

[0079] the VH meets all conditions of Table 1 and the VL meets all conditions of Table 2.

[0080] Preferably, the second antigen-binding arm (B) of the bispecific antibody is able to bind the co-receptor CD3 of lymphocyte. The lymphocytes bonded with the bispecific antibody are selected from neutrophils, macrophages, natural killer (NKs) and T Cells.

[0081] Preferably, the heavy chain of the first antigen-binding arm (A) comprising an aminoacid sequence having a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID NO: 13 (VH) and the light chain of the first antigen-binding arm (A) comprising an aminoacid sequence having a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID NO: 14 (VL).

[0082] Preferably, the heavy chain of the second antigen-binding arm (B) comprising an aminoacid sequence having a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID NO: 15 (VH) and the light chain of the second antigen-binding arm (B) comprising an aminoacid sequence having a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID NO: 16 (VL).

[0083] Preferably, the bispecific antibody comprising an aminoacid sequence having a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID NO: 17.

[0084] The VH chain of the both arm (A-VH and B-VH) and the VL chain of the both arm (A-VH and B-VL) could be linked through different linkers with aminoacid sequences SEQ ID NO: 18 and SEQ ID NO: 19 in order to have different configuration.

[0085] In particular the A-VH chain is linked with B-VL chain and B-VH chain is linked with A-VL chain through the linker with aminoacid sequence SEQ ID NO: 18. The B-VL chain is linked with B-VH chain through the linker with aminoacid sequence SEQ ID NO: 19.

[0086] Another preferred configuration is A-VH chain linked with A-VL chain and B-VH chain is linked with B-VL chain through the linker with aminoacid sequence SEQ ID NO: 19. The A-VL chain is linked with B-VH chain through the linker with aminoacid sequence SEQ ID NO: 18.

[0087] The above-mentioned technical problem is further solved by a bispecific antibody according to the invention for use in the treatment of tumour or other diseases characterized by an overexpression of FRa.

[0088] Preferably, the targeted tumors are ovarian carcinoma, more preferably epithelial ovarian carcinoma (EOC), and tumors of mesodermal derivation. More preferably, said tumors of mesodermal derivation is selected from pleural mesothelioma and peritoneal mesothelioma.

[0089] Preferably, further targeted tumors are non-mucinous adenocarcinomas of the ovary, uterus and cervix, testicular choriocarcinoma, trophoblastoma, ependymal brain tumors, laryngeal epithelial tumor, pituitary adenocarcinoma, colon, renal, breast, lung and kidney tumors. More preferably, the targeted tumor is ovary tumor.

[0090] The present invention also refers to a pharmaceutical composition comprising a bispecific antibody according to the invention.

[0091] A bispecific antibodies is now identified, having a high specificity to FRa, being useful in antitumor therapy, and for the treatment of other diseases involving an overexpression of FRa.

[0092] Said bispecific antibody shows the advantage of a time-extended binding to the FRa: this feature, which is different and additional to the CDR-driven receptor specificity (i.e. the capacity to distinguish the target cells from non-target cells) denotes an unexpectedly high binding strength; this improvement has been obtained by introducing specific aminoacids in the VH and VL chains, in areas laying outside the aforementioned CDRs, i.e. without modifying the CDRs.

[0093] Advantageously, said bispecific antibody binds not only FRa receptor, but also CD3 and can increase the number of intratumoral T cells and generate an inflammation in tumors.

[0094] Advantageously, the bispecific antibody has a conformation of the heavy and light chains that provides stability, thereby avoiding or reducing the formation of aggregates which can create immunogenicity problems. In fact, a major problem with protein-based therapeutics is their immunogenicity, namely their tendency to trigger an unwanted immune response against themselves. The stability and the purity of said bispecific antibody avoid said side effects, in particular an unwanted immune response.

[0095] Said bispecific antibody trigger the CD3 surface receptor on T cells, activating T-cell and subsequent killing tumor cell.

[0096] Features and advantages of the bispecific antibody according to the present invention will be disclosed with reference to the enclosed drawings relating to an indicative and a non-limiting implementation example.

[0097] Brief description of the drawings

[0098] Figure 1 shows a scheme of the bispecific antibody construct.

[0099] Figure 2 shows different analysis to prove the purity and integrity of bispecific antibody. In particular, figure 2a shows SDS-PAGE analysis (4- 12% gel). Figure 2b shows the chromatogram of bispecific antibody on SEC column (Size Exclusion Chromatography).

[0100] Figure 3 shows Biacore sensograms of binding analysis of bispecific antibody on FRa immobilized on sensor chip.

[0101] Figure 4 shows the immunofluorescence assay of bispecific reactivity on cell lines: IGROvl (positive for FRa), Jurkat (positive for CD3) and A431 (negative for both FRa and CD3). The light blue peak represents the binding of secondary antibody alone. The bispecific antibody of the present invention is compared with the specificity of the anti-FRa antibody Mov 18 for the anti-FR arm and the specificity of the TR66 (an anti CD3 antibody) for the anti-CD3 arm.

[0102] Figure 5 shows cytotoxic assay with CellTiter-Glo® Luminescent Cell Viability Assay on IGROvl, OV90 and A431 cell lines. Evaluation was performed from 24 to 96 hours after the addition of PBMC.

[0103] Figure 6 shows cytotoxic assay with CellTiter-Glo® Luminescent Cell Viability Assay on POCC3 and POCC4 cell line. Evaluation was performed from 24 to 120 hours after the addition of PBMC. Figure 7 shows cytotoxic assay with CellTiter-Glo® Luminescent Cell Viability Assay on peritoneal mesothelioma picked fresh from patient, In the graphic is represented the % of inhibition evaluated at 120 hours after the addition of PBMC.

[0104] Figure 8 shows different analysis. Figure 8a shows the cytotoxic assay with xCELLigence on a431tFR cell line. Evaluation in real time from 24 to 120 hours after the addition of PBMC, the cell index indicated the grow capacity of the FR positive cancer cells alone, in the presence of the bispecific, in the presence of lymphocytes or the combination of cancer cells plus bispecific plus lymphocyte. Figure 8b shows cytokine release in the medium after activation of lymphocytes for 24 hours with bispecific and / or PBMC. Figure 8c shows the FACS chromatograms activation of CD8 positive lymphocytes mediated by CD69 and CD25 markers after 24 hours of addition of bispecific and / or PBMC (said activation is proved by increased positivity for CD69 and CD25). Figure 8d shows the graphic of CD69 / CD25 positivity.

[0105] Detailed description

[0106] An example is described below explaining the steps for obtaining the bispecific antibody according to the present invention and the functionality of such bispecific antibody in terms of inhibition of the growth of tumor cells overexpressing FRa.

[0107] In general, the nomenclatures used in relation and the techniques of cell and tissue culture, molecular and chemical biology and hybridization of proteins and oligo- or polynucleotides described are those well-known and commonly used in the art.

[0108] EXPERIMENTALS

[0109] 1. Cell lines

[0110] Hereinafter, the derivation of cell lines is reported. IGROv1 (epithelial ovarian carcinomas) kindly provided from Dr. J. Bénard (Institute Gustave Roussy, Villejuif, France). OV90 (epithelial ovarian carcinomas), A431 (epidermoid epithelial carcinoma), Jurkat (immortalized line of human T lymphocytes) were purchased from American Type Culture Collection (Manassas, MD). A431tFR cells were obtained by transfection in house the Folate Receptor alpha into A431 cells.9

[0111] POCC3 and POCC4 are primary ovarian cancer cell line (POCC). The cell lines were established in house from the ascitic fluid obtained from two patients undergoing debulking surgery for ovarian cancers diagnosed as: high-grade serous carcinoma (POCC4) and low-grade serous carcinoma (POCC3).

[0112] Peritoneal Mesothelioma are fresh cells obtained from ascetic fluid of patient undergoing surgery for mesothelioma

[0113] 2. Bispecific Antibody soluble expression and characterization 2.1 Production

[0114] The soluble bispecific antibody of the present invention was produced in periplasmic fluid of E.coli (HB2151 competent cells). Periplasmic preparations were purified using L protein chromatography column (HiTrap Proteina L 5 ml, Cytiva) to obtain the bispecific antibody of the present invention (Figure 1).

[0115] 2.2 Biochemical analysis

[0116] The purified bispecific antibody was tested for integrity and purity by electrophoresis on 4-12% SDS-PAGE gel and stained with Comassie blue. Aggregation was tested by Size exclusion chromatography, on a SUPERDEX 75 5 / 15 column (Cytiva) on a HPLC (Perkin Elmer), flow 0.3 ml / min and buffer sodium phosphate 10 mM pH 7.4 + sodium chloride 150 mM. As shown in figure 2, the bispecific is pure and without aggregates.

[0117] 2.3 BIACORE binding studies Biacore is an instrument based on Surface Plasmon Resonance for the characterization of biomolecular interactions (Jason-Moller L et al Curr Protoc Protein Sci. 2006 Sep; Chapter 19: Unit 19.13), in this case antibodies and FRa. The bispecific antibody of said invention was tested for its binding to FRa. Kinetic analyses were performed with BIACORE T200 equipment. 400 RU of FRa was covalently bound to a CM 5 sensor chip using the amine coupling kit, using an antigen concentration of 5 μg / ml in 10 mM sodium Acetate (pH 4.5). Residual activated carboxyl groups were deactivated by 1.0 M ethanolamine pH 8.0. Different concentration, from 6.25 to 200 nM, of the bispecific antibody according to the present invention was analyzed at a flow rate of 30 μl / min and dissociation was allowed to proceed for at least 5 min. This experiment demonstrates the specificity of the molecule for the FRa and evaluates the kinetic of binding in real time (fig.3).

[0118] 2.4 FACS analysis

[0119] Fluorescence-activated cell sorting (FACS) is a subset of flow cytometry with the end goal of counting, sorting, and saving a subset population of cells from the initial sample.

[0120] 10 μg / ml bispecific antibody was added to tumor cells (5x105) in 100 μl PBS+ 0.03% BSA, and the mixture was incubated for 30 min on ice. The binding was detected by secondary antibody anti HIS Tag (produced in mouse) for 30 min on ice. All antibodies binding was revealed with an Alexa488 conjugated anti mouse antibody for 30 min on ice. MOvl8 antibody and TR66 are the positive control antibodies for FRa and CD3 respectively. For each sample 5000 cells were analyzed with FACS Canto using DIVA software. The results are in Figure 4: it can be seen that the bispecific antibody shows specificity for FRa receptor on IGROvl cells and for CD3 on Jurkat cells whereas there is no binding on A431 cells, which do not express the receptor target of this bispecific antibody (negative control cells). Said analysis indicates that the bispecific antibody is specific for the two targets. 3. Cytotoxic assay

[0121] 3.1 Isolation of PBMC.

[0122] Peripheral blood mononuclear cells (PBMC) were isolated from healthy donors’ huffy coats provided by the Immuno-Hematology and Transfusion Medicine Unit of our Institute, after signing informed consent, and stored at -80°C. Before each experiment PBMC was thawed and grown up in TexMACS TM culture medium (Miltenyi Biotec) for 48hours (no interleukins added).

[0123] 3.2 Cytotoxicity assay

[0124] For the evaluation of cytotoxicity of bispecific antibody, different cell lines positive for FRα (IGROV1, OV90, A431tFR, POCC3, POCC4, mesothelioma cell fresh picked from patient and A431 negative for the target as a control were used. Cells were seeded on 96- well flat bottom plates (Corning Inc., Corning, NY) and after 24 hours of adhesion they were treated with bispecific antibodies alone at 1 pg / ml or with bispecific plus PBMCs. In particular the ratios between the lymphocyte (E) and the tumor cell (T) are E: T, 2:1, 1:1 and 0.5:1. Cells alone and cells with PBMCs alone were used as controls. After a time varying from 24 to 120 hours, the remaining live cells were counted with CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Southampton, UK) and the % of growth inhibition was calculated with the following formula:

[0125] % of inhibition = (1- chemiluminescence reading of treated cells / chemiluminescence reading of untreated cells) ×100.

[0126] For all cell lines treated with bispecific antibody, the inhibition is more than 50% (Figure 5 and 6) with all the ratio provided. In particular, for POCC3 and POCC4 cell line of the ovarian cancer, the inhibition is more than 80%. The peritoneal mesothelioma and serous carcinoma (from which POCC cells derive) share similarities in histogenesis because they both originate from mesothelial or mesothelium-derived cells and, as shown in figure 7, they behave in the same way in the presence of the bispecific reaching more than 80% of inhibition at an E:T of 2:1

[0127] 3.3 Cytotoxicity assay in real time (xCELLigence, Agilent Technologies)

[0128] The A431tFR cells were seeded in particular chambers (E-plate) with electrodes on the bottom and are inserted into the instrument. After 24 hours of adhesion, they were treated with bispecific antibody alone at 1 pg / ml or with bispecific plus PBMCs (E: T, 5: 1). Cells alone and cells with PBMCs alone were used as controls. The instrument records the impedance (Cell index) that decreases when the cells are died.

[0129] The % of growth inhibition was calculated with the following formula: % of inhibition = (1- cell index reading of treated cells / cell index reading of untreated cells) ×100.

[0130] Also, the cytotoxicity assay in real time demonstrated that the bispecific antibody according to the invention has a high cytotoxicity against ovarian tumor (figure 8a).

[0131] 3.4 CD69 and CD25 activation markers evaluation

[0132] A431tFR cell line were used as target cells and were grown in RPMI 1640 into 48 well plates (Corning) at a density of 3.5 × 104cells for well. After 24 hours they were treated with bispecific antibody alone at 1 pg / ml or with bispecific plus PBMCs (E: T, 5:1) After incubation for 24 h at 37 °C in 5% CO2, PBMCs contained in the supernatant were recovered, washed with PBS and stained with: a BB515 conjugated mouse anti-human CD4; an APC conjugated mouse anti-human CD8; a PerCP Cy5.5 conjugated mouse anti-human CD69 and a PE conjugated mouse anti-human CD25 for 30 min on ice. After washing three times with PBS, the cells were analyzed by flow cytometry at FACS Canto. As can be observed in figure 8c and 8d, in the A431tFR cell line the bispecific antibody shows a higher frequency of the CD69 / CD25 markers in the CD8 positive population compared to control indicating the ability of the bispecific to activate lymphocytes.

[0133] 3.5 Determination of cytokines release (Bioplex)

[0134] To determine amounts of secreted GM-CSF, IFN-y, IL-2, IL-8, IL- 10 and TNF-a after activation of PBMCs, supernatants were collected 24 hours after the start of the treatment of tumor cells with bispecific plus PBMCs. Supernatants were analyzed for cytokine secretion using Bio-plex ProTM Human Cytokine standard 27-Plex, Group I (BIORAD), according to the manufacturer’s protocol. As shown in Figure 8b, the bispecific antibody according to the invention induces significant cytokine release from immune cells in vivo explaining the cytotoxic results. SEQUENCE LISTINGS SEQ.ID.NO:1

[0135] A-VH CDR1 DYIFTNYD SEQ. ID. NO:2

[0136] A-VH CDR2 IDPRSGKS SEQ. ID. NO:3

[0137] A-VH CDR3 ATMYYYGSSPPMDY SEQ. ID. NO:4

[0138] A-VL CDR1

[0139] QDINNF SEQ. ID. NO:5

[0140] A-VL CDR2

[0141] YTS SEQ.ID.NO:6

[0142] A-VL CDR3

[0143] QQSSTIPRT SEQ. ID. NO:7

[0144] B-VH CDR1 GYSFTGYT

[0145] SEQ. ID. NO:8

[0146] B-VH CDR2 INPYKGVS

[0147] SEQ. ID. NO:9

[0148] B-VH CDR3 ARSGYYGDSDWYFDV

[0149] SEQ.ID.NO:10

[0150] B-VL CDR1 QDIRNY

[0151] SEQ. ID. NO: 11

[0152] B-VL CDR2

[0153] YTS

[0154] SEQ.ID.NO:12

[0155] B-VL CDR3 QQGNTLPWT SEQ. ID. NO: 13

[0156] VH FRAbl: QVQLVQSGSELKKPGASVKVSCKASDYIFTNYDITWVRQAPGQGLEWMGE IDPRSGKSYYNEKFKGKSTLTADKSVSTAYLQISSLKAEDTAVYFCATMYYY GSSPPMDYWGQGTTVTVSS

[0157] SEQ. ID. NO:14

[0158] VL FRAbl:

[0159] DIQMTQSPSSLSASVGDRVTITCRASQDINNFLNWYQQKPGKAPKLLIYYTS RLHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYFCQQSSTIPRTFGQGTKL EIK

[0160] SEQ. ID. NO:15

[0161] VH anti-CD3:

[0162] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVA LINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSG YYGDSDWYFDVWGQGTLVTVSS

[0163] SEQ. ID. NO:16

[0164] VL anti-CD3:

[0165] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTS RLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTK VEIKR

[0166] SEQ. ID. NO:17

[0167] VH FRAb1-VL anti-CD3---VH anti-CD3-VL FRAb1

[0168] QVQLVQSGSELKKPGASVKVSCKASDYIFTNYDITWVRQAPGQGLEWMGE IDPRSGKSYYNEKFKGKSTLTADKSVSTAYLQISSLKAEDTAVYFCATMYYY GSSPPMDYWGQGTTVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS QDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSL QPEDFATYYCQQGNTLPWTFGQGTKVEIKRGGGGSGGGGSGGGGSEVQ LVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINP YKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYG DSDWYFDVWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRAS QDINNFLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTFTISSL QPEDIATYFCQQSSTIPRTFGQGTKLEIK

[0169] SEQ.ID.NO:18

[0170] Linker

[0171] GGGGS

[0172] SEQ.ID.NO:19

[0173] Linker

[0174] GGGGSGGGGSGGGGS

[0175]

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Claims

CLAIMS1. A bispecific antibody comprising a first antigen binding arm that binds FRa receptor and a second antigen-binding arm that binds CD3, wherein:a) the first antigen-binding arm (A) of the bispecific antibody comprises: three heavy chain complementarity determining regions (CDRs), A- VHCDR1, A-VHCDR2 and A- VHCDR3 comprising the aminoacid sequences of SEQ ID NO: 1, 2 and 3 respectivelythree light chain complementarity determining regions (CDRs), A-VLCDR1, A-VLCDR2 and A- VLCDR3 comprising the aminoacid sequences of SEQ ID NO: 4, 5 and 6 respectivelyb) the second antigen-binding arm (B) of the bispecific antibody comprises:three heavy chain complementarity determining regions (CDRs), B-VHCDR1, B-VHCDR2 and B- VHCDR3 comprising the aminoacid sequences of SEQ ID NO: 7, 8 and 9 respectivelythree light chain complementarity determining regions (CDRs), B- VLCDR1, B-VLCDR2 and B- VLCDR3 comprising the aminoacid sequences of SEQ ID NO: 10, 11 and 12 respectively.

2. The bispecific antibody according to claim 1, wherein first antigenbinding arm (A) of the bispecific antibody are characterized in that the VH comprises 120 aminoacids and meet at least ten of the conditions according toTable 1:Aminoacid at positionV 5s 9K 12K 13V 20R 38A 40M 48V 76L 81Q 82I 83s 84K 87A 88T 91T 116S 120and, at the same time, VL comprises 107 aminoacids and meets at least eight of the conditions according to:Table 2Aminoacid at positionS 7P 8V 15T 22G 41K 42A 43P 44T 72F 73S 76S 77Q 79P 80T 853. The bispecific antibody according to claim 2, wherein the heavy chain of the first antigen-binding arm (A) comprising an aminoacid sequence having a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID NO: 13 (VH) and the light chain of the first antigen-binding arm (A) comprising an aminoacid sequencehaving a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID NO: 14 (VL).

4. The bispecific antibody according to claim 1, wherein the heavy chain of the second antigen-binding arm (B) comprising an aminoacid sequence having a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID NO: 15 (VH) and the light chain of the second antigen-binding arm (B) comprising an aminoacid sequence having a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID NO: 16 (VL).

5. The bispecific antibody according to any one of claims 1-4, wherein the bispecific antibody comprising an aminoacid sequence having a sequence identity between 80% and 100%, preferably between 85% and 95%, with respect to SEQ ID NO: 17.

6. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1-5.

7. A bispecific antibody according to any one of claims 1-5 for use in therapy in the treatment of tumor or other diseases characterized by an overexpression of FRa.

8. The bispecific antibody for the use according to claim 7, wherein the targeted tumors are ovarian carcinoma, preferably epithelial ovarian carcinoma (EOC), and tumors of mesodermal derivation, preferably, said tumors of mesodermal derivation is selected from pleural mesothelioma and peritoneal mesothelioma.

9. The bispecific antibody for the use according to claim 7 or 8, wherein the further targeted tumors are non-mucinous adenocarcinomas of the ovaiy, uterus and cervix, testicular choriocarcinoma, trophoblastoma, ependymal brain tumors, laryngeal epithelial tumor, pituitary adenocarcinoma, colon, renal, breast, lung and kidney tumors.

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