Invented by Andreas Hohlbaum, Alexandra Baehre, Gabriele Matschiner, Stefan Trentmann, Klaus Kirchfeld, Hans-Juergen Christian, Pieris Pharmaceuticals GmbH, AstraZeneca AB
The Pieris Pharmaceuticals GmbH, AstraZeneca AB invention works as followsThe invention relates to novel muteins derived primarily from human tear lipocalin and which bind to IL 4 alpha. Certain combinations of amino acids make up the sequences of muteins. A mutated amino acids residue can be found at particular sequence positions, including 27, 28, 30, 31, 33 and 53. 57, 64, 65, 66, 66, 66, 66, 68, 66. 80, 83,104-106, and 108 of a linear polypeptide sequence for mature human tear lipocalin. An amino acid residue that has been mutated is also found at any two or more sequence positions 26, 32 and 34, 55 56, 58, 63, and 104 of the linear polypeptide of mature human tear lipocalin. The invention also contains a nucleic acids molecule that encodes such a mutation and a method of producing it.
Background for Tear lipocalin muteins binding IL-4 r Alpha
Proteins that selectively bind to targets via non-covalent interaction play an important role in biotechnology, medicine and bioanalytics, as well as in biological and life sciences in general.” Antibodies, i.e. Antibodies, i.e. immunoglobulins are a prominent example. Although there are many uses for these proteins, including recognition, binding, and/or separation targets/ligands, nearly all immunoglobulins are used at the moment. Other proteins that have defined ligand binding characteristics, such as the lectins or mRNA, are only used in special circumstances.
The members of the lipocalin proteinaceous binding family have additional functions that resemble antibodies. They have evolved to bind ligands. Lipocalins are found in many organisms including bacteria, insects, plants, and vertebrates. The lipocalin protein families (Pervaiz S. & Brew K. (1987 FASEB Journal. 1, 209-214) are small, secreted proteins that have one polypeptide chain. They are characterized by a range of different molecular-recognition properties: their ability to bind various, principally hydrophobic molecules (such as retinoids, fatty acids, cholesterols, prostaglandins, biliverdins, pheromones, tastants, and odorants), their binding to specific cell-surface receptors and their formation of macromolecular complexes. While they were originally classified as transport proteins in the past it is now apparent that lipocalins serve a wide range of physiological functions. These functions include retinol transport and olfaction as well as pheromone signaling and the synthesis prostaglandins. Lipocalins are also implicated in the regulation and mediation of cell homoeostasis. This has been reviewed in Flower, D. R. (1996) Biochem. J. J. (2000) Biochim, Biophys. Acta 1482, 9-24).
The sequence identity of lipocalins is often less than 20%, and they have an unusually low level of overall sequence conservation. Their overall folding pattern, however, is extremely conserved. The core of the lipocalin structure is a single, eight-stranded antiparallel??sheet that is folded back onto itself to form a continuous hydrogen-bonded?-barrel. This?-barrel creates a central cavity. The N-terminal peptide segment runs along the barrel’s bottom and three peptide loops connect the?-strands. One end of the barrel is sterically plugged. The solvent can be poured into the other end of the barrel. This is the target-binding area, which is covered by four flexible loops. This diversity of loops within the rigid lipocalin scaffold is what gives rise to many binding modes, each capable of accommodating targets with different sizes, shapes, and chemical characteristics (reviewed, for example, in Flower D. R. (1996); Flower D. R. et. al. Skerra, A. (2000) Biochim. Biophys. Acta 1482, 337-350).
International patent application WO99/16873 discloses four polypeptides belonging to the lipocalin familia with mutated amino acids positions in the region surrounding the binding pocket. These loops are located at the end of a cylindrical?-barrel structure that encompasses the binding pocket and correspond to the segments in the linear sequence of polypeptides that include the amino acid positions 28-45, 58-69, 86-99, 114-129 and the bilin-binding proteins of Pieris brassicae It has been reported that members of the lipocalin familia have been post-translationally modified (e.g. phosphorylation, glycosylation and synthesis of tear lipocalin (e.g. You, J., et al. (2010) Electrophoresis 31, 1853-1861. However, their molecular recognition properties are not affected by post-translational modifications.
International patent application WO 00/75308 discloses muteins of the bilin-binding protein, which specifically bind digoxigenin, whereas the international patent applications WO 03/029463 and WO 03/029471 relate to muteins of the human neutrophil gelatinase-associated lipocalin (hNGAL) and apolipoprotein D, respectively. A variety of approaches have been suggested to improve the ligand specificity, affinity, and folding stability of a variant of lipocalin (Skerra. (2001) Rev. Mol. Biotechnol. 74, 257-257; Schlehuher S. and Skerra A. (2002) Biophys. Chem. Chem. The PCT publication WO 2006/56464 discloses muteins of human neutrophile gelatinase-associated lipocalin with binding affinity for CTLA-4 in the low nanomolar range.
Human tear liquid lipocalin (TLPC, TIc), also known as lipocalin-1 or von Ebner protein, was initially described as a major component of tear fluid. It is also called lipocalin-1 or tear pre-albumin. However, it has been found in many other secretory tissues, including the prostate, adrenal glands, testis and nasal mucosa, as well as corticotrophs from the pituitary. The presence of homologous proteins has been confirmed in the rhesus monkey (chimpanzee), rat, mouse and pig as well as in cow, horse, hamster, pig, hamster and pig. Tear lipocalin, a unique lipocalin, is characterized by its unusually wide ligand specificity and high promiscuity for relative soluble lipids (see Redl., B. (2000) Biochim. Biophys. Acta 1482, 241-248). This characteristic of tear lipocalin can be attributed to the protein?s ability to inhibit bacterial and fungal growth at cornea. Endogenous ligands for this protein include a remarkable array of lipophilic compounds from different chemical classes, such as fatty acid, fatty alcohols or glycolipids. Contrary to other lipocalins, the length of the hydrocarbon tail for both alkylamides and fatty acid determines how strong the ligand (target), binding is. According to Glasgow, B. J., and others, tear lipocalin is most effective at binding the least soluble of lipids. (1995) Curr. Eye Res. 14, 363-372; Gasymov, O. K. et al. (1999) Biochim. Biophys. Acta 1433. 307-320). The 1.8-? The 1.8-???? crystal structure of tear lipocalin revealed an unusually big cavity within its [?-barrel] (Breustedt D. A. et.al. (2005) J. Biol. Chem. 280, 1, 484-493).
Despite all this progress, it would still be desirable to have a human tears lipocalin mutein with improved binding properties for IL-4 alpha, particularly of higher binding affinity, just for the purpose of further improving the suitability for muteins human tear lipocalin for diagnostic and therapeutic purposes.
Accordingly, the invention aims to provide a human tear lipocalin mutein that has high binding affinity for IL 4 alpha.
This object is achieved by a human tears lipocalin muein with the features described in the claims.
In a first embodiment, the invention provides a mutein for human tear lipocalin. The mutein binds with IL 4 receptor Alpha. Mutated amino acids can be found at any of the sequence positions 27, 30, 31, 33 and 53. The mutein also includes an amino acid residue that has been mutated at any of the sequence positions 26, 32 and 34, 55 56, 58, 63, and 62 of the linear polypeptide of mature human tear lipocalin. (1) Ser 26, Glu 34 and Lys 58; (2) Ser 26, Asn 35, Ala 55 & Lys 58; (3) Ser 26, Val 34 and (4) Pro 26 and Pro 56, respectively. (7) Leu 26 and Ala 55 are the mutiny sequences of human tear lipocalin. (8) Asn 26, Asp 36, Val 34 and (5) Pro 26 and Ala 55 are the mutiny sequences. (7) Leu 26, Trp34, Ala 55 is the 58.
The term ‘position’ is defined as: When used in accordance to the invention, the term?position? refers to either the position of an amino acid within an amino acid sequence depicted or the position a nucleotide inside a nucleic acids sequence depicted. The term “corresponding” is used herein. The term “corresponding” as used herein includes the fact that a position does not depend on the number of preceding nucleotides/amino acid. The position of an amino acid according to the invention that may be substituted can vary because of deletions or additions of amino acids in a wild-type or mutant lipocalin. The position of a nucleotide according to the invention that may be substituted can also vary because of deletions or additions elsewhere in a wild-type or mutein lipocalin 5′-untranslated area (UTR), including the promoter, and/or any other regulatory sequences and genes (including exons introns).
Thus, in a?corresponding situation? It is preferred to understand that nucleotides/amino acid may differ in the indicated numbers but still have similar neighboring nucleotides/amino acid. The term “corresponding position” refers to the nucleotides/amino acid that can be deleted, added, or exchanged.
Specifically, to determine if a nucleotide or amino acid residue in the amino acids sequence of a lipocalin mutein different from TIc lipocalin mutein, the invention corresponds with a position in the nucleotide sequencing or the amino sequence of TIc lipocalin mutein (SEQ ID No: 20), a skilled artisan may use means and methods well-known to the art, e.g. alignments. A lipocalin-mutein can be any of the SEQ ID Nos. 2-11, or one or more amino acids substitutions at position 27, 30, 31, 53, 56, 61 and 64, 66. 66. 66. 66. 66. 66. 66. 66. 66. 66. 66, 67, 68, 69, 66. 66. 66. 66. 68. 63. 66. 66. 68.
In a second aspect, the present invention provides a method for generating a human tear lipocalin mutein. The mutein binds with IL-4 receptor alpha. A nucleic acid molecule that encodes a human tear lipid is subjected to mutation at any of the following amino acid sequence positions: 27, 2.8, 31, 33, 53 and 57, 61, 64 and 66, 66, 66, 68, 66, 66, 67, 66, 66, 69, 64, 65, 66, 76, 86, 80,83,104-106, 108 of the linear polypeptide of mature human tear lipocalin. The method also includes subjecting the nucleic acids molecule encoding human tear lipocalin for mutagenesis at any of the amino acid sequence position 26, 32, 34 and 55, 56. 58. 63 of the linear protein sequence of mature human tear lipocalin. One or more of the following amino acid sequence positions 26-34, 55, 56. 58, 63, and 63 are required for the linear polypeptide sequence in mature human tear lipocalin. One or more nucleic acid sequences encoding a human tear lipocalin mutein are obtained. The encoded mutein amino acid sequence includes the following combinations: (1) Ser 26, Glu 34 and Leu 55, Lys 58; (2) Ser 26, Asn 34 and Ala 55. (4) Pro 26, Ser 34 and (5) Pro 26, Ala 55. (7) Leu 26, Trp 35, Ala 55. (8) Asn 26. (7) Leu 34. (8) Leu 26, Trp 36, Ala 56. (7) Leu 26, Trp 44, Ile 58. (8) Asn 26, Arg 34, Ala 58. (13) Lys 26, Arg 35, Asn 58 and (14) Glu 26 Gly 34 Ala 55. (15) Glu 36, Gly 34 Ala 58. This method includes the expression of one or more mutein-encoding nucleic acids molecules that have been obtained in an expression system. This method allows you to obtain one or more muteins. The method also includes enriching one or more muteins that bind to IL 4 receptor Alpha through isolation and/or selection.
The present invention provides a nucleic acids molecule in a third aspect.” A nucleotide sequence encodes mutein according the first aspect of the nucleic acid molecule.
In a fourth aspect, the present invention provides host cells. According to the third aspect, the host cell contains a nucleic acids molecule.
The present invention also provides a pharmaceutical composition in a fifth aspect. According to the first aspect, the pharmaceutical composition contains a mutein human tear lipocalin. A pharmaceutically acceptable excipient is also included in the pharmaceutical composition.
The detailed description of the invention will help you understand it better when it is viewed in conjunction with the accompanying drawings and non-limiting examples.
The present invention provides muteins for human tear lipocalin with a high affinity to IL-4R alpha. The IL-4 receptor beta is a target for a mutein according to the present invention. It can be a mammalian protein such as a human protein. In vivo, IL-4 receptor beta can bind interleukin 4 or 13 to regulate IgE antibody formation in B cells.Click here to view the patent on Google Patents.