Method of producing bone-inducing agent

Details Method of producing bone-inducing agent Method of producing bone-inducing agent

1997-02-13

2001-11-27

Witz, Jean C. (Department: 1651)

Drug, bio-affecting and body treating compositions

Fermentate of unknown chemical structure

C514S002600, C514S021800, C424S573000, C435S366000

Reexamination Certificate

active

06322786

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of the biology of bone formation and protein chemistry. More specifically, the present invention relates to a novel method of producing bone-inducing agent by cultured human osteosarcoma cells.
2. Description of the Related Art
In recent years a number of purified bone-inducing proteins have been isolated, sequenced, and produced by recombinant technology using transfected animal tissue culture cells. These molecules are designated bone morphogenetic proteins (BMPs) 1 through 9, or, alternately, “osteogenin” which is identical to BMP-3 (27), and “osteoinductive protein-1” which is identical to BMP-7 (34). All the bone morphogenetic proteins (except BMP-1) have molecular structures similar to transforming growth factor-&bgr; (TGF-&bgr;) (47).
Several of the bone morphogenetic proteins are expressed at specific tissue sites and at specific stages of embryonic development (21). Early in embryogenesis, BMP-1 and BMP-4 are expressed in the central mesoderm and appear to play a role in dorso-ventral patterning (11,15,18,35,36). BMP-2, BMP-3, BMP-4, BMP-6, and BMP-7 mRNAs and proteins have been localized in the craniofacial mesenchyme, in developing limb buds and in tooth germs of mouse embryos (12,14,21,25,26,44,45) suggesting an important role for these bone morphogenetic proteins in cartilage and bone formation during embryogenesis. In fracture healing, BMP-4 mRNA is expressed by pre-osseous bone repair cells (29). Therefore, it is logical to predict that one or more of the bone morphogenetic proteins may be utilized in future therapy to promote normal craniofacial development or stimulate bone replacement or fracture repair.
Each of the bone morphogenetic proteins can induce bone formation in soft tissues when implanted subcutaneously in combination with extracted bone matrix, an incompletely-defined substance, which is believed to function as a slow release vehicle for bone morphogenetic protein. However, the possibility remains that trace amounts of one or more osteoinductive cofactors may persist in the extracted bone matrix and interact with exogenously added single bone morphogenetic proteins to induce bone (6). Semi-purified preparations of bone morphogenetic protein (19,20,22,24,33) and recombinant forms of BMP-2 or BMP-7 (10,16,41,49) mixed with bone matrix carrier have been reported to augment bone repair when implanted directly into bone defects.
Most of the bone morphogenetic proteins were isolated initially from large quantities of decalcified bovine bone (42). Once the primary structure of each bone morphogenetic protein was determined, production of recombinant bone morphogenetic proteins was achieved by transfecting Chinese hamster ovary (CHO) cells or other transformed animal cell lines with full length transcripts of human cDNA for bone morphogenetic proteins 1-7 (17,46) to initiate synthesis of bone morphogenetic proteins. Many investigators in the field would predict that a recombinant form of a single bone morphogenetic protein will ultimately be the best product for human clinical usage. However, there are drawbacks to this method of production, e.g., there may be residual, sensitizing hamster cell proteins contaminating the purified recombinant secretory product, or more than one pure bone morphogenetic protein may have to be combined (8) with other cofactors (such as may be present in bone matrix carrier) in order to maximally stimulate bone regeneration.
An alternative source of bone-inducing activity can be found in certain cultured cells. These include human amniotic cells (2), Hela cells (3), and, in recent years, osteoinductivity has been identified in murine (1,37) and human (5,6) osteosarcoma cells. However osteosarcoma cells that can induce bone are very unusual (5). Amitani, et al. (1) adapted the osteoinductive BFO strain of the Dunn murine osteosarcoma to cell culture, and Takaoka, et al. have shown that murine BFO cells express considerable quantities of BMP-4 (38). A bone-inducing agent is present in Saos-2 human osteosarcoma cells (5), but other osteosarcoma cell lines are non-osteoinductive (30,31) when compared to Saos-2 cells in a similar bioassay. Thus, Saos-2 cells are unique among cultured human bone cells in being able to elaborate a bone-inducing, morphogenetic agent.
The prior art is deficient in the lack of effective means of producing bone-inducing agents. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
The present invention isolates and purifies essential components required for bone-induction using extracts of Saos-2 cells or proteins released by Saos-2 cells into conditioned tissue culture medium. One object of the present invention is to purify biochemically the bone-inducing agent present within Saos-2 cells using sequential chromatography.
Another specific object of the present invention is to purify osteoinductive proteins that are secreted by Saos-2 cells and released thereby into serum-free conditioned culture medium. The present invention demonstrates that proteins secreted into the culture medium by Saos-2 cells are bone-inducing when implanted subcutaneously into Nu/Nu immunoincompetent mice.
In one embodiment of the present invention, there is provided a method of isolating a bone inducing agent in vitro, comprising the steps of: growing Saos-2 cells; harvesting said Saos-2 cells; pelleting said Saos-2 cells; washing said Saos-2 cells in acetone; drying said Saos-2 cells; dissolving said Saos-2 cells in GuHCl; centrifuging said dissolved Saos-2 cells at about 10,000 g to produce a pellet and a supernatant; running said supernatant through a Sephacryl 200 column in GuHCl to produce a first eluate; collecting fractions of said first eluate; pooling said fractions of said first eluate to produce a first pooled protein fraction, wherein said fractions pooled contain a highest bone inducing activity; dialyzing said first pooled protein fraction against about 6M urea to produce a first dialysate; running said first dialysate through an anion or cation exchange column to produce a second eluate; collecting fractions of said second eluate; pooling said fractions of said second eluate to produce a second pooled protein fraction, wherein said fractions pooled contain a highest bone inducing activity; running said a second pooled protein fraction through a Heparin/Sepharose column to produce a third eluate; collecting fractions of said third eluate; pooling said fractions of said third eluate to produce a third pooled protein fraction, wherein said fractions pooled contain a highest bone inducing activity; dialyzing said third pooled protein fraction to produce a second dialysate; applying said second dialysate to an HPLC column; eluting said second dialysate by an acetonitrile gradient; collecting fractions of said second dialysate; and pooling said fractions of said second dialysate to produce a fourth pooled protein fraction, wherein said fractions pooled contain isolated bone inducing agent.
Another embodiment of the present invention provides a method of isolating a bone inducing agent in vitro, comprising the steps of: growing Saos-2 cells to confluence in an appropriate cell culture medium with serum; removing said cell culture medium with serum; washing said Saos-2 cells; adding an appropriate cell culture medium without added serum; incubating said Saos-2 cells for about 48 hours; collecting said cell culture medium without added serum; and filtering said cell culture medium without added serum through a 0.45 micron pore filter to produce a filtrate and a retentate; wherein said retentate contains said isolated bone inducing agent. A particular embodiment of this method further includes the steps of: solublizing said retentate in about 6M urea and dialyzing said solublized retentate against water to produce dialyzed retentate.
Another object of the present invention provides a method of augmenting bone growth locally comprising implanting the bone inducing agen

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