Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Conjugate or complex
Reexamination Certificate
1993-12-08
2001-07-31
Spector, Lorraine (Department: 1647)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Conjugate or complex
C424S192100, C424S193100, C424S195110, C435S069700, C530S350000, C536S023400, C536S023500, C536S023700
Reexamination Certificate
active
06267964
ABSTRACT:
The present invention relates to the use of biologically active proteins or peptides in the form of products showing improved stability in vivo, i.e. extended half-life, and the invention relates inter alia to the use of recombinant vectors capable of replication in a host cell to produce such useful products. More specifically, the invention relates to a protein or peptide conjugate capable of selective binding to a host protein or macromolecule, thus stabilizing the biologically active protein or peptide in this host. The invention also extends to a process for extending the half life in vivo of a biologically active protein or peptide and to the use of a protein or peptide conjugate resulting from such process for the manufacture of a medicament.
Although the present invention in the following will be mainly illustrated through protein or peptide conjugates produced by recombinant DNA technology the invention is not limited to such production system but is equally useful when such protein or peptide conjugate is prepared by chemical covalent coupling of its constituents.
Gene fusion is a procedure wherein the coding sequence of two or more genes are spliced together to form a combined gene which on expression in a suitable host organism will produce a fusion product wherein the separate proteins or polypeptides coded for by the respective genes are fused together into a single molecule.
The rapid depletion of bioactive proteins in vivo is, in some cases, a limiting factor for the efficiency of therapeutic compounds. Examples of products of potential clinical interest with short half-lives in vivo are components such as soluble human CD4, having an interest in the treatment of AIDS, which have a half-life in rabbit of 15 minutes (Watanabe et al., Nature, 337, 1984, 267-270) and human t-PA used in the treatment of blood clots with a halflife of only 2-3 minutes in humans (Hollander, Critical Reviews in Biotechn. 6, 1987, 253-271). Such short half-lives of therapeutically interesting proteins might make it necessary to distribute the compound to the patient either with a high initial dose or with many repeated distributions in order to keep the level of the compound at a clinically relevant level. This reduces the cost effectiveness of the drug and might cause negative side-effects due to the high doses necessary.
To overcome these problems several systems for slow release of drugs have been designed, in which the therapeutic agent is encapsulated by physical means to release the drug in a delayed manner (i.e. entero or depot tablets) or is delivered as a pro drug, inactive until chemically modified within the patient. In this way, it is in some cases possible to prolong the action of the therapeutic agent, although the actual in vivo half-life of the compound in circulation has not increased.
Recently, an alternative strategy has been described using fusions between a recombinant protein and a host protein such as IgG (Capon et al., Nature 337, 1989, 525-531) or IgM (Karjalainen et al., Nature 339 (1989) 68-70). In this way, the half-life of recombinant soluble CD4 in vivo was shown to be substantially prolonged. However, this strategy of distributing therapeutically interesting compounds have the disadvantage that unwanted immunological reactions are possible and that the half-life of the thus produced recombinant fusion protein might not be substantially prolonged.
Also for the development of vaccines and other immuno-stimulatory preparations, a rapid depletion of the antigen from the circulation might decrease the immune response. In order to present the antigen to the immune system in an efficient manner different vehicles have been developed, thereby increasing the immune response. (Allison et al. Journ. of Imm. Methods, 95 (1986), 157-168).
This strategy is often accompanied by simultaneous injection with weakened or killed pathogens such as in Freund's complete adjuvant (FCA). However, these formula have the potential risk of being toxic to the recipient and might lead to denaturation of the protein, thus limiting the use of this strategy for the distribution of therapeutic proteins.
The present invention provides new improved means to facilitate the stabilization of proteins and polypeptide products in vivo. According to the present invention this is achieved by coupling, such as by fusion of the desired biologically active protein or polypeptide to a binding-protein capable of selective binding to a host protein or macromolecule thus stabilizing the desired protein in said host. By selective binding to a patient protein with a relatively long half-life, the depletion of the fusion protein is retarded. By the term “patient” used in the present disclosure is intended a living animal, especially a mammal including man.
In accordance with a preferred aspect of the present invention, gene fusion is used to combine a first DNA-sequence coding for a binding protein with a second DNA-sequence coding for a desired protein or polypeptide into a functional gene capable of expressing the fusion product of said desired binding protein part.
Due to the binding ability the produced protein or polypeptide is stabilized in vivo in the receptor host.
Accordingly, the present invention is based on the surprising finding that the half-life in vivo of a biologically active protein or peptide can be substantially prolonged by covalently coupling such protein or peptide to a polypeptide fragment capable of binding to a serum protein. This finding was totally unexpected and could not be predicted from available scientific knowledge.
Thus, according to one aspect of the invention, there is provided a process for extending the half-life in vivo of a biologically active protein or peptide, such process comprising the steps of covalently coupling the protein or peptide to a polypeptide fragment which is capable of binding to a serum protein. When administering the protein or peptide conjugate resulting from such process the binding thereof to the serum protein results in substantially extended biological activity due to increased half-life thereof.
According to a preferred embodiment of this aspect of the invention said polypeptide fragment is capable of binding to serum albumin, such as a serum albumin of mammal origin, for example human serum albumin.
The binding polypeptide fragment of the conjugate preferably originates from streptococcal protein G.
Another aspect of the invention is constituted by the use of the protein or peptide conjugate as defined above for the manufacture of a dug or medicament which, when administered to a mammal including man, shows extended half life in vivo thus prolonging the biological activity of the conjugate.
A preferred aspect of the present invention is thus the provision of a recombinant DNA cloning vehicle or vector comprising a DNA sequence coding for a desired protein or polypeptide operatively linked to a DNA sequence coding for a binding mediating part, such that said DNA-sequences together code for a fusion protein of said desired protein or polypeptide, said binding mediating part being capable of selectively binding to a protein or macromolecule present in the patient to be treated.
By transforming a compatible host organism with said vector to permit expression of the above combined DNA sequence and culturing the host organism in a nutrient medium the corresponding binding mediating fusion protein or polypeptide will be produced. Host cells producing functional fusion proteins should be used, which could be bacterial cells, such as Escherichia or eukaryotic cells, such as fungi, insect cells, plant or mammalian cell cultures. The transformation of the hosts may be effected with well-known methods.
Said fusion protein of said desired protein or polypeptide and said binding mediating protein produced by the cultured host organism can be efficiently isolated from the cell culture by means of standard protein purification methods such as size exclusion chromatography, ion exchange chromatography or affinity purification using a suitable ligand i
Nygren Per Åke
Uhlen Mathias
Wigzell Hans
Affibody Technology Sweden AB
Burns Doane Swecker & Mathis L.L.P.
Spector Lorraine
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