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  Ribosome display or mrna display method with selection for increased stability of the proteinUSPTO Application #: 20070298430Title: Ribosome display or mrna display method with selection for increased stability of the protein Abstract: A method of providing a subject polypeptide variant with improved stability compared with a parent subject polypeptide, employing translation and selection using an RNA expression system, wherein two or more stability selection pressures are applied simultaneously during translation, two or more stability selection pressures are applied simultaneously during selecting, or at least one stability selection pressure is applied during translation and continues to be applied during selecting, and at least one further stability selection pressure is applied during selecting. (end of abstract) Agent: Klarquist Sparkman, LLP - Portland, OR, US Inventors: Andrew Buchanan, Lutz Jermutus USPTO Applicaton #: 20070298430 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20070298430. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] The present invention relates to methods for selecting, obtaining or producing polypeptide variants with improved stability. It further relates to manufacture and use of the variants following selection, for example in therapy. [0002] The invention employs use of display technology incorporating in vitro translation and covalent or non-covalent linkage between genotype, such as RNA, and the encoded phenotype, such as a polypeptide of interest, to select for polypeptide variants that have improved stability compared with a parent polypeptide and that retain functional activity. [0003] Ribosome or polysome display and selection involves construction of nucleic acid libraries, screening for binding, and identification of binding entities of interest. The library is made by synthesising a DNA pool of diverse sequences that are then transcribed to produce a pool of mRNAs. In vitro translation is used to generate the encoded polypeptides or proteins displayed, and desirable binding interactions are selected using immobilised target antigen. mRNA encoding the binding entities can be used to make cDNA, which can then be amplified and the process may be repeated to enrich the population for genes encoding binders. The selected proteins may later be identified by cloning individual coding sequences and DNA sequencing. [0004] The technology has been reviewed extensively (Hanes et al., (2000) Meth. Enzymol. 328, 403-430; Pluckthun et al., (2000) Adv. Prot. Chem. 55, 367-403; Lipovsek and Pluckthun (2004) J. Immunological Methods 290, 51-67). [0005] The technology has been used for the display of antibody fragments, peptides and various proteins, including periplasmic and cytoplasmic proteins such as .beta.-lactamase and ankyrin-repeat proteins. [0006] Recovery of mRNA from polysome complexes was first reported in 1973 in a paper describing a protocol to capture mRNA coding for a mouse immunoglobulin L-chain using antibodies and immobilised oligothymidine (Schechter (1973) PNAS USA 70, 2256-2260). Improvements to the polysome immunoprecipitation protocols were made by Payvar and Schimke (Eur. J. Biochem. (1979) 101, 271-282) and cDNA clones for the heavy chain of HLA-DR antigens were obtained after immunoprecipitation of polysomes using a monoclonal antibody (PNAS USA (1982) 79, 1844-1848). Production of libraries of antibodies by ribosome display was proposed and patented by Kawasaki (U.S. Pat. No. 5,643,768 and U.S. Pat. No. 5,658,754, EP-B-0494955). [0007] There have been various examples of the use of ribosome display using either eukaryotic or prokaryotic translation systems. The first demonstration of selection of peptide ligands using an E. coli extract was by Mattheakis et al., (PNAS USA (1994) 91, 9022-9026 and Methods Enzymol (1996) 267, 195-207). This group demonstrated selection of peptide ligands that are similar to known peptides epitopes of a given antibody, using the antibody as a selection substrate. High-affinity peptide ligands which bind prostate-specific antigen have been identified using polysome selection from peptide libraries using a wheat germ extract translation system (Gersuk et al., (1997) Biotech and Biophys. Res. Com. 232, 578-582). The selection of functional antibody fragments was reported using an E. coli translation system designed for increased yield of ternary complexes and allowing disulphide bond formation (Hanes and Pluckthun, PNAS USA (1997) 94, 4937-4942). This experimental set up has subsequently been used to select antibodies from a murine library, and it was shown that affinity maturation occurs during the selection due to the combined effect of PCR errors and selection. A scFv fragment with a dissociation constant of about 10-.sup.11M was obtained (Hanes et al., PNAS USA (1998) 95, 14130-50). Enrichment for specific of antibodies from mixed populations using rabbit reticulyocyte lysate extracts has also been demonstrated (He and Taussig (1997) NAR, 5132-5234). [0008] mRNA display, like ribosome display, uses a complex between mRNA and the encoded polypeptide as the basic selection unit. What distinguishes mRNA display from ribosome display is the covalent nature of the linkage between the mRNA and the protein. The linkage is achieved through a small adaptor molecule, typically puromycin (Nemoto et al., (1997) FEBS Lett 414:405; Roberts and Szostak, (1997) Proc. Natl. Acad. Sci. USA 94: 12297; Takahashi et al., (2003) Trends Biochem. Sci. 28:159). mRNA display is not limited to 4.degree. C., which is the usual temperature at which ribosome display is carried out. Typically the temperature at which selections are performed is limited by the stability of the protein target. mRNA display has been used for affinity selections of peptides and antibodies (Reviewed in Lipovsek and Pluckthun, (2004) J Immunological Methods 290:51-97). [0009] The present invention provides methodology that is applicable to display technology incorporating in vitro translation and covalent or non-covalent linkage between genotype, such as RNA, and the encoded phenotype, such as a polypeptide of interest, for selection of subject polypeptide variants with improved stability and retained functionality compared with a parent polypeptide. Methods of the invention incorporate a number of features not previously used together in ribosome or mRNA display. [0010] In embodiments of the present invention, two or more than two stability selection pressures are employed, simultaneously, especially two or more than two stability selection pressures, allowing for selection of stable polypeptides. [0011] A stability selection pressure may be any factor that can be used in display technology incorporating in vitro translation and covalent or non-covalent linkage between genotype, such as RNA, and the encoded phenotype, such as a polypeptide of interest, that allows for selection of a variant polypeptide on the basis of its stability. Stability can generally be defined as the propensity of a molecule to exist in its folded and active state. A stability selection pressure may disrupt or prevent a polypeptide folding correctly such that it does not attain an active or fully active state. A stability selection pressure may affect the ability of a polypeptide to remain in its folded and active state. A stability selection pressure may differentiate in some way between polypeptides that are in a folded and active state and those that are not. [0012] For example, a stability selection pressure may be a chemical denaturant, such as urea, guanidine HCl (GuHCl) or thiocyanate, for example, sodium thiocyanate. A stability selection pressure may be a reducing agent, such as dithiothreitol (DTT), Tris[2-carboxyethyl] phosphine hydrochloride (TCEP), mercaptoethanol or glutathione. A stability selection pressure may be a physical denaturant, such as pH or temperature, in particular increased temperature. A selection pressure may be a protease or enzyme capable of degrading protein. A selection pressure may be depletion of chaperons or small molecule protein folding inhibitors. [0013] A stability selection pressure may be the use of hydrophobic interaction chromatography (HIC). [0014] Hydrophobic interaction chromatography (HIC) is a technique for the separation of biomolecules based on differences in their surface hydrophobicity. HIC techniques have been used as a part of protein purification strategies as well as an analytical tool for the detection of protein conformational changes (reviewed in Queiroz et al., (2001) J. Biotech. 87, 143-159; Fulton and Vanderburgh (1996) Biomolecule Chromatography PerSeptive Biosystems). HIC is based on hydrophobic attraction between the HIC matrix and the protein molecules. The HIC matrix consists of small non-polar groups (butyl, octyl or phenyl) attached to a hydrophilic polymer backbone (e.g. cross-linked dextran or agarose). Many proteins, generally considered to be hydrophilic, also have sufficient numbers of hydrophobic groups allowing interaction with the HIC matrix. HIC is sensitive enough to interact with non-polar groups normally buried within the tertiary structure of the protein but exposed due to incorrect folding. The strength of the interaction is dependent upon the type of matrix, type and concentration of salt, pH, additives and temperature. [0015] The present inventors' work described herein demonstrates for the first time use of at least two selection pressures, such as DTT, HIC and increased temperature, simultaneously to improve the pharmacological properties of therapeutic proteins using display technology such as ribosome or mRNA display, in particular to improve shelf-life stability. [0016] Among the advantages of using at least two selection pressures, especially simultaneously, compared with single or sequential selection pressures, is that the strategy is more generic and robust. From the inventors' experience some proteins are not susceptible to one selection pressure alone, i.e. are not significantly destabilised by one pressure alone, but are susceptible to two pressures, especially when applied simultaneously. For example, the inventors have seen that DTT may be insufficient on its own and is useful only for certain types of protein, for example, those with disulphide linkages. Thus, the present invention using selection allows for a more stringent selection method and the selection of any protein on the basis of stability. [0017] In methods of the invention, a library of genes encoding polypeptide variants may be constructed. Polypeptide variants may be subject to a stability selection pressure as they are produced, for example by translating them in the presence of DTT. A stability selection pressure may act during translation of a polypeptide, during folding of a nascent polypeptide and/or following folding of a polypeptide. [0018] A stability selection pressure that may be used while a polypeptide is produced, that is during translation and folding of the nascent polypeptide, includes DTT, glutathione, Tris[2-carboxyethyl] phosphine hydrochloride (TCEP), mercaptoethanol, urea, guanidine HCl (GuHCl), depletion of chaperons, pH, and small molecule protein folding inhibitors. Chaperons act to aid folding of a polypeptide. Their depletion thus affects the ability of a polypeptide to fold correctly. Chaperones can be depleted from cell-free extracts by using immunoprecipitation (IP). Antibodies against all major chaperons such as GroEL/GroES, DnaJ, DnaK are available commercially and could be used for IP-based removal of specific components of a ribosome display system. The same approach has been demonstrated for the removal of phosphatases from cell-free abstracts which improved the abundance of ATP and in consequence expression yield (Shen et al. (1998) Biochem. Eng. J. 2:23-28). Also, folding may be inhibited by specifically designed small molecules (Gestwicki et al. (2004) Science 306:865-869). A small molecule protein folding inhibitor may be any small molecule that is capable of disrupting or preventing correct folding of a protein. [0019] Two selection pressures are applied simultaneously within the overall process of producing the variants (e.g. during translation) and selection (e.g. by binding or biological activity). Preferably one or more than one selection pressure is applied during the selection part of the process, more preferably two selection pressures in the selection part or more than two. There may be one or more than one selection pressure in the translation part of the process and one or more than one selection pressure in the selection part of the process. [0020] Selection is made for variants that are more stable and still bind their cognate ligand, receptor or specific binding pair member. Variants are incubated with two or more than two stability selection pressures simultaneously. In methods of the invention, the stability selection pressures used, preferably used simultaneously, may be for example, HIC and increased temperature. Improved, active variants are captured via their cognate ligand, receptor or specific binding pair member. The selection pressures may be present during capture of active variants or may be removed prior to incubation of variants with their cognate ligand, receptor or specific binding pair member. [0021] Stability selection pressures that may be used include DTT, glutathione, TCEP, mercaptoethanol, urea, GuHCl, sodium thiocyanate, proteases, HIC and increased temperature. [0022] HIC may use a hydrophobic interaction chromatography matrix or any other suitable hydrophobic matrix, for example, a matrix that is used for reverse phase chromatography. For example, a suitable matrix for HIC is butyl-Sepaharose.TM. (Amersham). [0023] By "increased temperature" is meant a temperature that is higher than the temperature at which the display technique is usually carried out and which is at least partly destabilising for proteins [0024] For example, as described herein, ribosome display is usually carried out at 4.degree. C. to ensure the stability of the ribosome/mRNA/polypeptide complex. Thus, increased temperature in a method using ribosome display is a temperature above 4.degree. C. For example, increased temperature may be above 15.degree. C., or at or above room temperature, by which is meant any temperature between about 20.degree. C. and about 30.degree. C. inclusive. An increased temperature may be or be about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.degree. C. Preferably, an increased temperature is or is about 25.degree. C. Preferably, a method of the invention uses HIC carried out at room temperature, preferably at about 25.degree. C. Continue reading... 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