This application is a §371 of PCT/EP2009/066907filed Dec. 11, 2009, and claims priority from Italian Patent Application No. FI2008A000240 filed Dec. 12, 2008, both incorporated by reference in their entirety.
The present invention is referred to the field of diagnostics, in particular those that are capable of highly specific identifications through murine monoclonal antibodies and their conjugates.
Fibronectins (FNs) are adhesive and polymorphic glycoproteins present in biological fluids and extracellular matrices. These molecules are involved in several biological processes, such as cellular morphology, cellular migration, wound healing, angiogenic processes and neoplastic transformation. FN polymorphism is at least partially due to alternative splicing in three regions (IIICS, ED-A and FNIII-B) of the pre m-RNA coded for by a single gene. In all processes of tissue remodeling, both physiological (embryogenesis, wound healing, proliferative phase of endometrium) or pathological (for example, neoplasia), the pre-mRNA splicing pattern of FN is altered and generates an augmented expression of the isoforms containing the FNIII-B domain. In particular, the FN isoforms containing the FNIII-B domain (BFNs) are, with very rare exceptions, undetectable in normal adult tissues but show a considerable expression in fetal tissues, neoplastic tissues and tissues undergoing wound healing. Moreover, the accumulation of BFN around newly formed blood vessels makes it an important angiogenic marker (Carnemolla et al. 1989; Castellani et al. 1994; Castellani et al.2002).
Studies thus far have identified a single epitope localized within the type III repeat 7 (FNIII-7) that, while cryptic in FN molecules lacking the FNIII-B domain, is unmasked when FNIII-B is inserted into the molecule and can interact with specific ligands for BFN, i.e., antibodies, the prototype of which is the mouse monoclonal antibody (mab) BC-1.
The mab BC-1, specific for human BFN (Carnemolla et al. 1989), has been extensively used to study the distribution of BFN in angiogenic processes, in neoplasms and in different types of pathologies. This mab recognizes the epitope described above.
On the basis of the studies carried out with the murine mab BC-1, it was demonstrated that BFN may be a very good target for malignancies and for all angiogenesis-associated pathologies. In light of the above, the need is clear for mabs that can recognize new epitopes detectable only in the oncofetal BFN isoform, and that can be generated in substantial quantities and always on highly specific epitopes and thereafter utilized as an additional means for novel therapeutics or diagnostics.
The repeat FNIII-8 (
In particular, the invention refers to an epitope, hereinafter named C6, and to the murine monoclonal antibody (hereinafter named C6 mab), which is able to specifically recognize the above-mentioned epitope and hence the oncofetal BFN isoform. The invention also refers to all conjugates and derivatives formed by the said antibody and drugs or molecules able to act as diagnostic (e.g., radioactive isotopes, fluorescent substances, etc.) or therapeutic agents (e.g., radioactive isotopes, cytokines, chemotherapeutics, etc.) for neoplasms and other pathologies where BFN is expressed, the conjugation of the mab C6, subject of the invention, and the aforementioned agents being achieved following procedures normally used in this field for the preparation of similar products.
Experimental Description
Generation and characterization of the mab C6 and identification of a new epitope in the FNIII-8 repeat, cryptic in FN molecules lacking FNIII-B but available to the antibody when FNIII-B is inserted in the FN molecules.
FN was purified from human plasma and from the conditioned medium of WI38-VA13 cells according to Zardi et al. (1980).
FN recombinant fragments were produced and purified according to Carnemolla et al. (1992).
Murine hybridoma was generated by fusing splenocytes from immunized mice with murine myeloma SP2/0Ag14 cells, following a procedure described by Zardi et al. (1980).
ELISA was performed according to Carnemolla et al. (1996).
Immunohistochemistry on cryosections was performed according to Castellani et al. (2002).
SDS-PAGE and Western Blotting were carried out according to Zardi et al. (1987).
In order to sequence the variable regions of mab C6, total RNA was purified from C6 hybridoma cells and used as template to amplify the cDNA of the VH and VL by RT-PCR with the following primers: 5′ gatattgtgatgacccagtctccca 3′ (VL forward) (SEQ ID NO: 1); 5′ tggatacagttggtgcagc 3′ (VL, reverse) (SEQ ID NO: 2); 5′ aggtg(c)c(a)ctgcagg(c)agtct(a)gg 3′ (VH, forward) (SEQ ID NO: 3); 5′ ggccagtggatagac 3′ (VH, reverse) (SEQ ID NO: 4). The resulting amplification products were sequenced.
Female Balb/C mice, aged 5-6 weeks, were immunized following the protocol described by Zardi et al. (1980) using the FNIII-7B89 recombinant fragment (
The hybridoma clones obtained by fusing the splenocytes of the immunized mice and myeloma SP2/0Ag14 cells were tested by ELISA for reactivity with FNIII-7B89 and FNIII-789 fragments (
Clone C6 mab was chosen from among the clones that produced antibodies positive for FNIII-7B89 and negative for FNIII-789 and was then expanded for purification and characterization.
As reported in
Results of ELISA experiments using purified C6 mab (
In ELISA experiments, C6 mab showed no cross-reactivity with FNIII homology fragments of human TN-C and FN that were tested. The sequences of the cDNAs coding for the variable regions VH and VLk of the mab C6 are reported in
Immunohistochemical Analysis.
Immunohistochemical experiments on cryosections of human normal and neoplastic tissues were carried out using C6 mab; the results are reported in Table 1.
The staining pattern of C6 mab was compared with that of BC-1 mab and L19scFv (specific for FNIII-B) on serial sections of F9 murine teratocarcinoma , and SKMEL-28 human melanoma, both developed in SCID mice, and on sections of a patient derived glioblastoma specimen (
As demonstrated by the results reported above, C6 mab is a new tool for both diagnostic and therapeutic approaches to BFN-targeting, as well as for the continued study of the possible biological functions of FN regions unmasked by the insertion of FNIII-B.
Number | Date | Country | Kind |
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FI2008A000240 | Dec 2008 | IT | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/EP2009/066907 | 12/11/2009 | WO | 00 | 7/11/2011 |
Publishing Document | Publishing Date | Country | Kind |
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WO2010/066872 | 6/17/2010 | WO | A |
Entry |
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Schwarzbauer (Bioessays. Oct. 1991;13 (10): 527-33). |
Peters et al. (J. Lab. Clin. Med. Jun. 2003; 141 (6): 401-10). |
Campbell (Monoclonal Antibody Technology, Laboratory Techniques in Biochemistry and Molecular Biology, 1984, Elsevier Science Publishers B.V.: Amsterdam, The Netherlands, vol. 13, pp. 1-32). |
Number | Date | Country | |
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20110256060 A1 | Oct 2011 | US |