Drug – bio-affecting and body treating compositions – Lymphokine
Reexamination Certificate
1995-06-07
2003-05-20
Russel, Jeffrey E. (Department: 1654)
Drug, bio-affecting and body treating compositions
Lymphokine
C424S085200, C424S085400, C424S094300, C424S178100, C424S425000, C424S486000, C424S487000, C435S180000, C435S182000, C514S002600, C514S003100, C514S006900, C514S008100, C514S012200, C514S021800, C525S054100, C530S345000, C530S410000, C530S815000, C530S817000
Reexamination Certificate
active
06565842
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for the modification of polypeptides. In a particular aspect, the present invention relates to modified polypeptides which can readily be crosslinked to produce a gel under extremely mild conditions. Such materials can be used, for example, for encapsulation of biologically active materials, including living cells.
BACKGROUND OF THE INVENTION
The crosslinking of proteins by various means has generated much interest in the fields of drug delivery, protein immobilization, enzyme and antibody immobilization, peptide-protein conjugation, vaccines, medical imaging, etc. The applications of such crosslinked protein systems are as diverse as the methods employed to achieve crosslinking.
The use of crosslinked proteins as scaffolds for drug delivery has been pursued by several investigators due to the intrinsic biodegradable nature of proteins in vivo. By far the most common method for protein crosslinking is the addition of external crosslinking agents.
Crosslinked protein compositions may take several forms. Microspheres comprising crosslinked proteins are typical in applications that involve drug delivery. Microspheres of proteins are typically prepared by emulsification of an aqueous protein solution with an organic phase and crosslinking by addition of multifunctional crosslinking agents such as glutaraldehyde (Langhein et al., 1987, J. Appl. Bacteriology 63: 443-448; Yan et al., 1988, Biotechnology and Applied Biochemistry 10: 13-20), or by heat denaturation (Law et al., 1991, Biomat. Art. Cells & Immob. Biotech. 19:613-629; Welz and Ofner, 1992, J. Pharmaceutical Sciences 81:85-90).
Immobilization of proteins on surfaces for enzymatic and chromatographic applications has also been reported in the literature. Proteins and peptides may be immobilized at surfaces by use of crosslinking agents such as glutaradehyde and carbodiimides (Benslimane et al., 1986, Biomaterials 7:268-72). Preparation of protein-protein or protein-peptide conjugates is commonly performed by use of glutaraldehyde as well as by use of heterobifunctional crosslinking agents such as N-succinimidyl bromoacetate (Bernatowitz and Matsueda, 1986, Anal. Biochem. 155: 95-102). Proteins have also been modified to introduce functional groups that may be polymerized upon exposure to free radicals resulting in the formation of crosslinked hydrogels (Park, 1988, Biomaterials 9:435-441).
Although most of the methods referred to above result in the formation of crosslinked proteins, the use of external agents (and the reaction conditions required for crosslinking) are too toxic for such processes to be carried out in the presence of living cells and tissues. Indeed none of the references noted above teach their respective art in the presence of living systems.
It is well known that agents of crosslinking such as those described above are in fact used as fixatives for cells and tissues. In a slightly different approach from the addition of external crosslinking agents, Park (1988), supra, describes the free radical polymerization of monomers such as acrylic acid and acrylamide along with derivatized proteins as multifunctional crosslinkers for the formation of polyacrylic acid and polyacrylamide gels. In this case the protein, derivatized with unsaturated groups capable of undergoing free radical polymerization, serves merely as the crosslinker, while the bulk of the resultant hydrogel is either polyacrylic acid or polyacrylamide. The formation of crosslinked hydrogels also necessitates the use of toxic free radical initiators, such as ammonium persulfate, and polymerization conditions that involve teperatures of 60° C. as well as polymerization times of an hour or more. No known living cells, except thermophilic organisms, are likely to survive such crosslinking conditions. Thus, in general, the encapsulation of living cells in a crosslinked protein gel has not been described in the art.
In general the encapsulation of cells requires conditions that are particularly fastidious with respect to mild temperatures,. absence of toxic chemicals, rigid maintainence of physiological conditions of pH and osmolarity, and processes that in general are fairly rapid so as to minimize the exposure of the living cells to adverse conditions. A good example of a nontoxic encapsulation process is the one using sodium alginate (a polysaccharide) that can be formulated in physiological saline (see, for example, Soon-Shiong et al., 1991, Transplantation Proceedings 23:758). Cells are simply suspended in a solution of polysaccharide, which is added dropwise into a solution of calcium chloride, resulting in the instantaneous formation of capsules of ionically crosslinked alginate containing entrapped cells.
Since proteins, in general, do not spontaneously form gels, external agents must be added to facilitate the formation of crosslinked hydrogels (an exception is gelatin, which can coagulate to form a gel below a certain temperature). The resulting protein hydrogels could potentially be utilized to entrap cells in a crosslinked protein matrix. Thus the solution of a protein may be stirred with an added external crosslinking agent to form a crosslinked protein mass or gel. Alternately the formation of a protein gel in the form of spheres or microspheres requires emulsification with a nonsolvent phase to form discrete droplets of the protein solution which can subsequently be crosslinked. However, as described above, common processes utilized to crosslink proteins suffer from the limitations of toxicity when contemplated for the encapsulation of living material.
There are several advantages attendant to the use of proteins as encapsulation materials for living cells and tissue. Proteins such as albumin, collagen, gelatin, and the like, being of natural origin, are well tolerated by living cells. For example, the use of albumin in culture media is well known and is in fact essential for the well being of cell cultures. Collagen is secreted by cells and forms the major component of the extracellular matrix. Gelatin is known to support cell adhesive behavior through its binding with fibronectin, another ubiquitous cell adhesion molecule. Thus a matrix of such proteins in the form of a microcapsule is favorable for the growth of the encapsulated cell. In fact commercially available gels such as Matrigel and Atrigel, both of which contain collagen, are known for their ability to support viable cells.
Albumin is considered to be an ‘inert’ protein since it does not bear epitopes that play a role in cell adhesion under normal physiological conditions. As a result, it does not support cell adhesion and is often utilized as a coating in applications that require a cell-free surface. Thus microcapsules or crosslinked gels of albumin are not expected to show a cell adhesive response when transplanted into a host organism. This effect in general is termed as ‘biocompatibilty’. Thus in applications such as cell therapy where foreign cells are encapsulated and transplanted to replace lost function in the host, such a ‘coating’ or encapsulation of the transplanted cell would prevent an inflammatory and fibrous reaction to the transplanted material. On the other hand, it is often required that transplanted tissue become vascularized or that the material of encapsulation become vascularized so that the encapsulated cells within the matrix of the crosslinked material are in reasonable proximity to a source of nutrients, and, more importantly, to a source of oxygen. In such a case, the use of crosslinked collagen or gelatin would be of great benefit in supporting the growth of vascularized tissue adjacent to the encapsulated cell.
Thus, it is essential to develop protein compositions and processes that can result in the formation of crosslinked protein gels in the presence of living cells in a manner that is innocuous to the well being of the cellular material. The essential requirements of such compositions and processes would be as follows:
the ability to crosslink in the presence of a suitable init
Desai Neil P.
Nagrani Shubhi
Sandford Paul A.
Sojomihardjo Soebianto A.
Soon-Shiong Patrick
American Bioscience, Inc.
Foley & Reiter
Reiter Stephen E.
Russel Jeffrey E.
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