Polypeptides

#The present disclosure relates to a class of engineered polypeptides having a binding affinity for albumin. It also relates to new methods and uses that exploit binding by these and other compounds to albumin in different contexts, some of which have significance for the treatment of disease in mammals including humans.

#Serum Albumin

#Serum albumin is the most abundant protein in mammalian sera (40 g/I; approximately 0.7 mM in humans), and one of its functions is to bind molecules such as lipids and bilirubin (Peters, Advances in Protein Chemistry 37:161, 1985). Serum albumin is devoid of any enzymatic or immunological function. Furthermore, human serum albumin (HSA) is a natural carrier involved in the endogenous transport and delivery of numerous natural as well as therapeutic molecules (Sellers and Koch-Weser, Albumin Structure, Function and Uses, eds Rosenoer et al, Pergamon, Oxford, p 159, 1977). The half life of serum albumin is directly proportional to the size of the animal, where for example human serum albumin has a half life of 19 days and rabbit serum albumin has a half life of about 5 days (McCurdy et al, J Lab Clin Med 143:115, 2004). HSA is widely distributed throughout the body, in particular in the interstitial and blood compartments, where it is mainly involved in the maintenance of osmolarity. Structurally, albumins are single-chain proteins comprising three homologous domains and in total 584 or 585 amino acids (Dugaiczyk et al, Proc Natl Acad Sci USA 79:71, 1982). Albumins contain 17 disulfide bridges and a single reactive thiol, cysteine in position 34, but lack N-linked and O-linked carbohydrate moieties (Peters, 1985, supra; Nicholson et al, Br J Anaesth 85:599, 2000).

#FIG. 1A-1F is a listing of the amino acid sequences of examples of albumin binding polypeptides of the present disclosure (SEQ ID NO:1-152, SEQ ID NO:155-203), the GA3 domain from protein G of Streptococcus strain G148 extended by a N-terminal glycine residue (SEQ ID NO:153) and an albumin binding polypeptide derived from G148-GA3 as previously described by Jonsson et al (supra, SEQ ID NO:154).

#FIG. 2 shows the result of binding analysis performed in a Biacore instrument for investigating the binding of the albumin binding polypeptide PEP07912 (SEQ ID NO:157) to human serum albumin. Three different concentrations of purified protein (40 nM, fat gray line; 10 nM, black line; and 2.5 nM, gray line) were injected over a surface with 955 RU of immobilized human serum albumin.

#FIGS. 3A-C show the result of binding analysis performed by ELISA for investigating the binding of the albumin binding polypeptides PEP07913 (SEQ ID NO:153), PEP06923 (SEQ ID NO:154), PEP07271 (SEQ ID NO:155), PEP07912 (SEQ ID NO:157), PEP07554 (SEQ ID NO:156), PEP07914 (SEQ ID NO:158), PEP07968 (DOTA-conjugated PEP07911, SEQ ID NO:159) and PEP07844 (SEQ ID NO:161), to IgG molecules present in 126 individual normal human sera, where A) shows the average OD-value, B) shows the percentage of negative sera (defined as OD<0.15), and C) shows the percentage of positive sera (defined as OD>1.0).

#The invention will now be illustrated further through the non-limiting description of experiments conducted in accordance therewith. Unless otherwise specified, conventional chemistry and molecular biology methods were used throughout.

#In this example, ten different albumin binding polypeptides, PEP07913 (SEQ ID NO:153), PEP07912 (SEQ ID NO:157), PEP07914 (SEQ ID NO:158), PEP07968 (DOTA-conjugated PEP07911, SEQ ID NO:159), PEP06923 (SEQ ID NO:154), PEP07271 (SEQ ID NO:155), PEP07554 (SEQ ID NO:156), PEP07844 (SEQ ID NO:161), PEP07986 (SEQ ID NO:163) and PEP08296 (DOTA-conjugated PEP08185, SEQ ID NO:148), the amino acid sequences of which are set out in FIG. 1 and in the appended sequence listing, were cloned, purified and characterized.

#Material and Methods