Therapeutic composition for use in the prevention and treatment of bone metastases

#The invention relates to medicaments for use in the prevention and treatment of bone metastases which are based on antibodies against non-cellular bone matrix proteins.

#A primary cancer is often followed by secondary bone tumors. Despite all medical advances, bone tumors cannot be healed as they generally occur spread-out and can hardly be treated surgically. The probability of bone metastases is larger than 96% in the case of multiple myeloma, between 65% and 75% in the case of cancers of the breast and prostate, and between 30% and 50% in the case of cancers of the lung, kidney, cervix and bladder. Systemic chemotherapies have hardly any effect. Initial successes have been achieved using therapeutic antibodies. However, when therapeutic antibodies are used, there is the problem that they are mostly directed against targets which are only present on proliferating tumor cells. Generally not detectable are non-proliferating tumor cells in the blood circulation and latent metastases.

#There is a close relationship between the occurrence of the bone matrix proteins osteopontin (OPN), osteonectin (ON) and bone-sialoprotein II (BSP) in the primary tumor on one hand, and the later occurrence of secondary blastomas of the bones on the other hand. The cells of an osteotropic metastasizing primary tumor nearly always express significant amounts of BSP or OPN (I. J. Diel et al., Clinic Cancer Res, 1999, 5:3914; A. B. Tuck et al., J Mammary Gland Biol Neplasia, 2001, 6:419; P. S. Rudland et al., Cancer Res 2002, 62:3417; D. Agrawal et al., J Natl Cancer Inst, 2002, 94:513; D. Waltregny et al., J Bone Miner Res, 2000, 15(5):834; A. Bellahcène et al., J Bone Miner Res, 1996, 11:665; D. Waltregny et al., J Nat Cancer Inst, 1998, 90:1000; A. Bellahcène et al., Int. J. Cancer, 1996, 69:350; WO 02/100901 (Immundiagnostik AG), WO 02/25285 (Smith et al.)).

#FIG. 1 shows a block diagram of the process of the invention for the production of bacteria having antigen determinants from BSP, which elicit an immune reaction against osteotropic tumor cells and the settling of tumor cells in bones;

#FIG. 2 shows a representation of generated BSP fragments for recombinant expression of BSP antigen segments on carrier bacteria; RGD: cell-binding motif; YXY: tyrosine-rich region; E8: glutamic acid-rich region; T-Epitop: BSP tumor epitope (BSP-position 125-130);

#FIG. 3A shows a representation of the proportion of positive bacteria which after FACS analysis have on their surface functionally anchored BSP fragments (vBSP-1, -2, -3, -4 or eBSP), as evidenced by a tumor-epitope specific polyclonal antiserum (rabbit)—carrier bacteria with no BSP on the surface were used as negative control;

#Osteotropic tumors of the prostate, breast, lung, kidney and thyroid differ from less malign tumors inter alia by the fact that they contain BSP expressing cells and can disseminate into the bone tissue. Even though the process of metastasis into bones is complex and not understood, the presence of tumorgenic BSP in serum allows for a safe prognosis of bone metastases. The presence of tumorgenic BSP in the bone matrix may further be used as a rating of the bone remodeling by bone metastases. BSP from tumor cells possesses other posttranslational modifications compared to endogenous BSP of normal osteoblasts. Thus, IgY-antibodies could be produced in chicken which specifically recognize a BSP secreted from cells of osteotropic tumors, called herein tumor-eBSP. In animal experiments, these antibodies are strikingly active against induced bone metastases of human cancer cells (WO 02/100899). The therapeutic composition of the invention for treatment and prevention of bone tumors elicits in the patient endogenous antibodies against BSP and in particular against tumor specific eBSP. Presumably, the so induced autoantibodies against tumorgenic eBSP interfere with the docking mechanism of the osteotropic cells from the primary tumor. Since BSP is bound and masked by factor H of the complement system, many eBSP expressing secondary cells of the primary tumor can evade from being attacked by the immune system and necrosis by the complement system. Also the bringing about of apoptosis would be very important. The death of autoreactive lymphocytes in the thyroid gland usually ensures that the immune system does not attack endogenous antigens. The therapeutic composition of the invention overcomes these false self-protection mechanisms, because, inter alia, an immune reaction against tumorgenic BSP from tumor cells is specifically induced, and the antibodies bind to an epitope of BSP, which remains free and accessible even when enshrouded by complement factor H from serum. The generated cell-mediated cytotoxicity causes a necrotic or apoptotic death of the target cells. Contrary to conventional therapeutic approaches, no tumor BSP specific antibodies are administered but a cell-mediated immunity produced in the patient by a combined xeno- and idio-immunization against a protein specific for tumors metastasizing into bones and presumably required for their dissemination into bones.

#The therapeutic composition may contain dead or weakly pathogenic microorganisms, which contain a DNA sequence encoding BSP or a fragment thereof integrated in an episomal vector. Consequently, the therapeutic composition corresponds to a vaccine, or a live vaccine, in which a foreign BSP-encoding DNA is translated and expressed as BSP antigens and tumor-eBSP antigens, respectively, so that the patient develops antibodies against presented foreign BSP and tumor eBSP antigens. The so induced auto-immunity against tumor BSP presumably protects against the docking of metastasizing osteotropic cells of the primary tumor to the bone tissue and leads to a cell-mediated cytotoxicity against tumor BSP producing tumor cells. Both mechanisms evolve into a prophylactic and a therapeutic activity against bone metastases.

#Further, the activity of the therapeutic composition may be enhanced and modulated by the addition of antibodies, especially anti-tumor BSP antibodies, ligands, especially RGD binding ligands, inhibitors which interact with adhesion molecules, membrane associated proteases or receptors mediating chemotaxis, for example chemokine receptors, as well as apoptosis inducing substances such as antibodies or proteins and peptides obtained from natural and artificial peptide libraries. Peptides from BSP and eBSP, which were made immunogenic by coupling with beta-alanine, appear to be especially promising.