Use of transthyretin peptide/protein fusions to increase the serum half-life of pharmacologically active peptides/proteins

#Proteins, peptides and other drug molecules for therapeutic use are currently available in suitable forms in adequate quantities largely as a result of the advances in recombinant DNA technologies. The availability of such peptides and proteins has engendered advances in protein formulation and chemical modification. Chemical modification of biologically active peptides, proteins, oligonucleotides and other drugs for purposes of extending the serum half-life of such bioactive agents has been extensively studied. The ability to extend the serum half-life of such agents allows for the therapeutic potential of the agent to be realized without the need for high dosages and frequent administration.

#Chemical modification used to extend the half-lives of proteins in vivo includes the chemical conjugation of a water soluble polymer, such as polyethylene glycol (PEG), to the protein of interest. A variety of approaches have been used to attach the polyethylene glycol molecules to the protein (PEGylation). For example, Royer (U.S. Pat. No. 4,002,531) states that reductive alkylation was used for attachment of polyethylene glycol molecules to an enzyme. Davis et al. (U.S. Pat. No. 4,179,337) disclose PEG:protein conjugates involving, for example, enzymes and insulin. Shaw (U.S. Pat. No. 4,904,584) disclose the modification of the number of lysine residues in proteins for the attachment of polyethylene glycol molecules via reactive amine groups. Hakimi et al. (U.S. Pat. No. 5,834,594) disclose substantially non-immunogenic water soluble PEG:protein conjugates, involving for example, the proteins IL-2, interferon alpha, and IL-1ra. The methods of Hakimi et al. involve the utilization of unique linkers to connect the various free amino groups in the protein to PEG. Kinstler et al. (U.S. Pat. Nos. 5,824,784 and 5,985,265) teach methods allowing for selectively N-terminally chemically modified proteins and analogs thereof, including G-CSF and consensus interferon.

#Other approaches designed to extend the serum half-life of bioactive agents include: conjugation of the peptides to a large, stable protein which is too large to be filtered through the kidneys (e.g., serum albumin); G. D. Mao et al., Biomat., Art. Cells, Art. Org. 17:229-244 (1989); use of low- and high-density lipoproteins as transport vehicles and to increase serum half-life; P. Chris de Smidt et al., Nuc. Acids. Res., 19(17):4695-4700 (1991); the use of the Fc region of immunoglobulins to produce Fc-protein fusions; PCT WO 98/28427 (Mann et al, and references cited therein); and the use of the Fc domain to increase in vivo half-life of one or more biologically active peptides; PCT WO 00/24782 (Feige et al, and references cited therein).

#FIG. 1 is an SDS gel that depicts the purification of an E. coli expressed, recombinant human transthyretin (TTR) variant (C10A/G83C) with a Bradykinin peptide fused to the C-terminus of TTR. Lane 1 contains Novex Mark 12 molecular weight standards, and lanes 2-7 contain the following respectively: cell lysate, post-heating supernatant, pool from Q-sepharose chromatography step, pool from phenyl sepharose chromatography step, pool from hydroxyapatite chromatography step, and pool from source Q chromatography step.

#FIG. 2 demonstrates by size exclusion chromatography that fusion of peptides to the amino-terminus or carboxy-terminus of a TTR variant, TTR(C10A/G83C), does not alter its oligomeric structure. Solid line is TTR(C10A/G83C), dashed line is PTH fused to the amino-terminus of TTR(C10A/G83C), and the dotted line is Bradykinin fused to the carboxy-terminus of TTR(C10A/G83C).

#FIG. 3 demonstrates by size exclusion chromatography that fusion of proteins to the amino-terminus or carboxy-terminus of a TTR variant, TTR(C10A), does not alter its oligomeric structure. Solid line is TTR(C10A), dashed line is IL-1-ra fused to the carboxy-terminus of TTR(C10A), and the dotted line is IL-1-ra fused to the amino-terminus of TTR(C10A).

#For purposes of describing the present invention, the following terms are defined as set forth below.

#The term “biologically active agent” refers to any chemical material or compound useful for prophylactic, therapeutic or diagnostic application. The term “pharmacologically active compound” refers to a compound suitable for administration to a mammalian, preferably a human individual, which induces a desired local or systemic effect.

#The terms “peptide”, “polypeptide” and “protein” as used to describe biologically active agents herein are used interchangeably to refer to a naturally occurring, recombinantly produced or chemically synthesized polymer of amino acids. The terms are intended to include peptide molecules containing as few as 2 amino acids, chemically modified polypeptides, consensus molecules, analogs, derivatives or combinations thereof.