Invented by Aaron K. Sato, Arthur C. Ley, Edward H. Cohen, Morphosys AG
The Morphosys AG invention works as follows
Compositions containing non-naturally occuring serum albumin binding molecules are described along with their methods of use, such as for detecting or isolating serum albumin in a solution, blood circulation imaging and linking therapeutics or another molecules to albumin. Preferred serum-albumin binding peptides with a high affinity for the human serum albumin, are disclosed.Background for Serum albumin binding moieties
The most abundant component of circulating blood in mammalian species, serum albumin is present at a normal concentration of 3 to 4.5 grams of whole blood per 100 milliters. Serum albumin, a blood-protein of about 70,000 daltons, plays several vital roles in the circulatory systems. It is a major transporter for fatty acids, bilirubin, and other organic molecules in the blood. Due to its abundance, it also acts as an osmotic controller of the circulating plasma. Human serum albumin is used in clinical practice as a plasma expansion agent for patients who have suffered blood loss due to surgery, trauma, burns or shock.
Commercial HSA is required to be purer than other therapeutic proteins, as patients receive large amounts of HSA during a single therapy. The protein should also be in the correct conformation, to avoid any antigenic reactions.
HSA can be obtained in large quantities by either purification of human serum from blood donors, or by expression from a recombinant system. Examples include transgenic murine (Shani et.al., Tranasgenic Res. 1: 195-208 (1992), Pichia Pastoris (Kobayashi and al. Ther. Apher, 2 : 257? 262 (1998), and transgenic leafy plants or tubers, such as potato and tobacco plants (Sijmons and al., Biotechnology, 8 : 217? 221 (1990). HSA derived from human serum is purified and tested to ensure that it does not contain any pathogens. Recombinant sources are an advantage because they do not have these transmissible pathogens.
Serum albumin is used in a wide range of protocols for research and testing. Serum albumin, for example, is used in tissue culture media to help grow mammalian and eukaryotic cells. Serum albumin can also be used in assay protocols such as enzyme-linked immunosorbent tests (ELISAs), Western immunoblots and other assays to block non-specific binding of other molecules. Serum albumin can also be used to bind antigens or to conjugate them to immunogenic compounds that elicit antibody response to an antigen. Its size makes serum albumin a useful standard molecular mass marker protein that can be used for estimating or calculating the size of proteins.
There is therefore a need to continue developing methods and means for producing HSA and serum albumins in a highly pure state with a higher yield, using fewer steps of production. There is also a need for methods and means to remove or trap more serum albumins in a solution including whole blood.
The invention provides a serum albumin-binding polypeptide described herein, as well as methods and means for producing or detecting highly purified serum albumins. These include human serum albums (HSA) or other mammalian albumins. The serum albumin-binding moieties are a serum binding polypeptide as described herein. They also include phages, phagemids or bacteria or host cells displaying the serum binding polypeptide.
The recombinant bacteria displaying small, non-natural cyclic polypeptides, which bind serum albumin in mammalian cells, were identified and isolated using phage display. These phage products, and the isolated polypeptides, have proven to be useful reagents in binding serum albumin specifically in different solutions including whole blood or fractions of it.
The invention includes serum albumin-binding polypeptides and albumin separation media. It also provides methods for detecting and isolating mammalian albumin in solutions such as whole blood, serum, other fractions of blood and other mixtures containing albumin (e.g. conditioned media). The invention includes recombinant bacteria (including phagemids), bacteriophage, mammalian cells, or other replicable genetic packs expressing exogenous DNA encoding serum-albumin binding polypeptides.
The invention provides an amino acid sequence polypeptide containing a serum albumin-binding moiety that is not naturally occurring.
The invention provides an amino acid sequence polypeptide containing a serum albumin-binding moiety that is not naturally occurring.
Xaa1-Xaa2-Xaa3-Cys-Xaa4-Xaa5-Xaa6-Xaa7-Cys-Xaa8-Xaa9-Xaa10 (SEQ ID NO:2),
wherein
In yet another embodiment of the invention, the invention provides an non-naturally occurring serum albumin binding moiety that comprises a polypeptide containing the amino acid sequence:
Ala-Glu-Gly-Thr-Gly-Ser-Xaa1-Xaa2-Xaa3-Cys-Xaa4-Xaa5-Xaa6-Xaa7-Cys-Xaa8-Xaa9-Xaa10-Ala-Pro-Glu (SEQ ID NO: 3),
wherein
The invention provides an amino acid sequence polypeptide containing a serum albumin-binding moiety that is not naturally occurring.
Cys-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Cys (SEQ ID NO: 130)
wherein
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