Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Chemical modification or the reaction product thereof – e.g.,...
Reexamination Certificate
1997-07-07
2001-01-09
Kulkosky, Peter F. (Department: 1615)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Chemical modification or the reaction product thereof, e.g.,...
C424S178100, C435S180000, C530S402000
Reexamination Certificate
active
06172202
ABSTRACT:
The present invention relates to a process for the preparation of a conjugate between a polymer and a bioactive substance.
In the last decade a steadily increasing number of proteins have entered routine clinical use as therapeutic or diagnostic agents (see, for example, Waldmann, T. A.,
Science,
252 : 1657-1662, 1991 and Jaffe, H. S. and Sherwin, S. A.,
Drugs of Today,
25 : 311-320, 1989 and Foon, K. A.,
Cancer Research,
49 : 1621-1639, 1989). The efficacy of many of these agents, however, is limited for two main reasons.
First, the in vivo half-life is often very short (see, for example, M{umlaut over (u)}hlradt, P. F. and Opitz, H. G.,
European Journal of Immunology,
12: 983-985, 1982 and Jacobs, C. A., Lyncl, D. H., Roux, E. R., Miller, R., Davis, B., Widmer, M. B., Wignall, J., VandenBos, T., Park, L. S. and Beeckmann, M. P.,
Blood,
77 : 2396-2403, 1991 and Blick, M., Sherwin, S.A., Rosenblum, M. and Gutterman, J.,
Cancer Research,
47 : 2986-2989, 1987).
Second, in case of heterologous proteins, another problem adds to the first. This is due to the proteins being recognized as foreign substances (antigens) by the immune system of the species treated, thereby leading to an immune response that abolishes, upon a second administration, the pharmacological activity of the heterologous protein (see, for example, Shawler, D. L., Bartholomew, R. M., Smith, L. M. and Dillman, R. O.,
Journal of Immunology,
125: 1530-1535, 1985 and Schroff, R. W., Foon, K. A., Beatty, S. M., Oldham, R. K. and Morgan, A. C.,
Cancer Research,
54 : 879-885, 1985 and Traub, U. C., De Jager, R. L., Primus, F. J., Losman, M. and Goldenberg, D. M.,
Cancer Research,
48 : 4002-4006, 1988).
For these reasons methods have been sought to overcome either one or both of the above mentioned problems, i.e. to prolong the in vivo half-life of proteins and to reduce their antigenicity in case of proteins heterologous with respect to the species to be treated. One particular approach that has been taken is to conjugate proteins to soluble synthetic polymers, in particular poly(ethylene glycol), poly(vinyl pyrrolidone), poly(vinyl alcohol), poly(amino acids), divinylether maleic anhydride, ethylene-maleic anhydride, N-(2-hydroxypropyl)methacrylamide and dextran (see, for example, Abuchowski, A., Van Es, T., Palczuk, N. C. and Davis, F. F.
Journal of Biological Chemistry
11, 3578-3581, 1977; Yasuda, Y., Fujita, T., Takakura, Y., Hashida, M., and Sezaki, H.
Chemistry and Pharmaceutical Bulletin
38, 2053-2056, 1990; Fagnani, R., Hagan, M. S. and Bartholomew,
Cancer Research
50, 3638-3645 (1990); Suck, J. M. and Wild, B. S. U.S. Pat. No. 3,679,653, 1972; Flanagan, P. A., Duncan, R., Rihova, B., Subr, V. and Kopecek, J.,
Journal of Bioactive and Compatible Polymers
5, 151-166, 1990.
More than 40 proteins have now been modified, mainly using polyethylene glycol), but it has been shown that a variety of other polymers can be substituted to provide reduced immunogenicity and protein stabilisation. In particular, the approach has been used to modify enzymes including arginase, asparaginase, adenosine deaminase galactosidase, lipase, pro-urokinase, streptokinase, superoxide dismutase, trypsin and uricase (see, for example, Nucci, M. L., Shorr, R. and Abuchowski, A.
Advanced Drug Delivery Reviews
6, 133-151, 1991; Veronese, F. M., Caliceti, P., Pastorino A., Schisvon, 0., and Sartore.
Journal of Controlled Release
10, 145-154, 1989); cytokines and growth factors such as interleukin 2 and human granulocyte colony-stimulating factor (see, for example, Katre, N. V.
Journal of Biological Chemistry
144, 209-213, 1990; Tanaka, H., Satake-Ishikawa, R., Ishikawa, M., Natsuki, S. and Asano, K.
Cancer Research
51, 3710-3714, 1991) and antibodies (see, for example, Kitamura, K., Takahashi, T., Yamaguchi, T., Noguchi, A., Takashina, K., Tsurumi, H., Inagake, M., Toyokuni, T. and Hakomori, S.
Cancer Research
51, 4310-4315, 1991).
A number of methods have been described for linkage of polymers to bio-active protein molecules (see, for example, U.S. Pat. No. 4,179,337, U.S. Pat. No 4,732,863, Jackson C.-J., Charlton, J. L., Kuzminski, K., Lang, G. M. and Sehon A. H.
Analytical Biochemistry
165, 114-127, 1987; Veronese, F., M., Largajolli, R., Boccu, E., Benassi, C. A. and Schiavon, O.
Applied Biochemistry and Biotechnology
11, 141-152, 1985 ; WO93/15189). However, the methods currently available for conjugation have two major drawbacks. Usually, other than the Veronese approach in WO93/15189, first, the derivatisation procedures reported are inherently random thereby leading to the introduction of polymeric moieties into domains of the molecule that mediate the therapeutically or diagnostically desirable activity(ies). Consequently, the molecule may acquire a prolonged half-life in vivo and, in case of heterologous proteins, reduced immunogenicity, but at the expense of a significant or complete loss of the desired biological activity(ies) (see, for example, Kitamura, K., Takahashi,T.,Yamaguchi,T.,Noguchi,A.,Noguchi, A., Takashima, K.-i., Tsurumi, H., Inagake, M., Toyokuni, T. and Hakanori, S.-i.,
Cancer Research,
51: 4310-4315, 1991 and Maiti, P. K., Lang, G. M. and Sehon, A. H.,
International Journal of Cancer, Supplement
3: 17-22, 1988).
Loss of biological activity following polymer conjugation has been observed in the case of both antibodies and enzymes, particularly when access of the modified protein to a macromolecular substrate or receptor is essential to produce biological activity. However, it has been found that inactivation of enzymatic activity is not necessarily a result of polymer conjugation if the domain(s) mediating activity either do not contain functional groups suitable for polymer derivatisation, and/or the binding of polymer molecule(s) does not sterically hinder access of low molecular weight enzyme substrates.
In fact, enzymes like adenosine deaminase and L-asparaginase have been successfully conjugated with polyethylene glycol (see, for example, Hershfield, M. S., Buckley, R. H., Greenberg, M. L., Melton, A. L., Schiff, R., Hatem, C., Kurtzberg, J., Markert, M. L., Kobayashi, R. H., Kobayashi, A. L. and Abuchowski, A.,
The New England Journal of Medicine,
316 : 589-596, 1987 and Teske, E., Rutteman, G. R., van Heerde, P. and Misdorp, W.,
European Journal of Cancer,
26: 891-895, 1990). In one case (adznosine deaminase) the product thereby obtained has received approval for clinical use in humans. These examples, however, are the exception rather than the rule.
A second problem associated with synthesis of polymer-protein conjugates has been heterogeneity of the product formed. Polymers are by nature heterogeneous, displaying within any sample a range of molecular weights, ie. they are polydisperse, and in addition any preparation also displays a heterogeneity in the number of functional groups available for attachment to the protein to be modified. Thus, during the conjugation reaction there is opportunity to form a multitude of products. This problem has been previously been exacerbated by the need to control carefully the degree of protein modification to a minimum to ensure retention of substantial biological activity of the protein, whilst concurrently introducing a sufficient number of polymer molecules into the conjugate to facilitate the needed reduction in immunogenicity, and protein stabilisation.
According to the present invention, there is provided a process for the preparation of a conjugate between a polymer and a first substance having a biological activity mediated by a domain thereof, which process comprises:
(a) contacting the first substance with a second substance which specifically binds to the said domain of the first substance;
(b) conjugating a polymer to the first substance having the second substance bound thereto; and
(c) freeing the second substance from the first substance having the polymer conjugated thereto, and wherein, when the first substance is a proteolytic enzyme chosen from trypsin, urokinase, tissue plasminogen activator, plasmin, c
Gregory Ruth
Marcucci Fabrizio
Arent Fox Kintner Plotkin & Kahn
Kulkosky Peter F.
Pharmacia S.p.A.
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