Stable fibronectin domain compositions, methods and uses

Abstract

To provide protein scaffold molecules having enhanced thermal and chemical stability.SOLUTION: There is provided an isolated protein scaffold based on a fibronectin type III (FN3) domain comprising an amino acid sequence derived from a consensus sequence of the Tenascin FN3 domains, the protein scaffold being able to bind to human IgG.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to protein scaffolds having novel characteristics, including the ability to bind to intracellular targets. More specifically, the present invention relates to a fibronectin type III (FN3) repeat inhibitor. The objective of the present invention is to develop a protein scaffold based on the consensus sequence of [Background technology]

[0002] Monoclonal antibodies are most widely used when high affinity and specificity for a target molecule is desired. However, they are a class of therapeutic proteins that can be engineered to bind to such targets. Non-antibody proteins that can be synthesized using scaffolds are also of interest to the biopharmaceutical industry. The proteins are characterized by their low molecular weight, lack of disulfide bonds, high stability, and the ability to be isolated from prokaryotic cells. The possibility of expression in a cellular host may give them advantages over conventional antibodies. They are easily applied, they are conveniently conjugated with drugs / toxins, they penetrate efficiently into tissues, and It is easily formed into a multispecific binder (Skerra 2000 J Mol Re cognit 13(4):167~87;Binz and Pluckthun 2 005 Curr Opin Biotechnol 16(4):459~69).

[0003] One such alternative scaffold is the immunoglobulin (Ig) fold. This fold is found in the variable regions of antibodies, as well as thousands of non-antibody proteins. One such Ig protein, the 10th fibronectin of human fibronectin, The cutin type III (FN3) repeats are expressed in a manner that preserves the overall Ig fold structure. It has been shown that the ribozyme can tolerate multiple mutations in the surface-exposed loops. Therefore, these loops and specific binding sites selected against multiple different targets can be used to A library of amino acid variants has been constructed in a lab (Koide et al. l.1998 J Mol Biol 284(4):1141~51;Karatan et al.2004 Chem Biol 11(6):835~44). The engineered FN3 domain is capable of targeting with high affinity while retaining important biophysical properties. It has been found that the protein binds to the ribonucleic acid (Parker et al. 2005). in Eng Des Sel 18(9):435~44). Summary of the Invention [Problem to be solved by the invention]

[0004] Desirable physical properties of potential alternative scaffold molecules include high thermal stability and thermal The apparent reversibility of folding and unfolding of proteins and enzymes. To enhance the thermal stability of the , theoretical design was based on comparison with highly similar thermostable sequences. , design to stabilize disulfide bridges, mutations to increase α-helicality, salt bridge design, Various methods have been applied, including surface charge variation of proteins, directed evolution, and consensus sequence composition. (Lechmann and Wyss 2001 Curr Opin B iotechnol 12(4):371-5). The high thermal stability of the resulting recombinant transcription factor Increase protein yield, improve solubility of purified molecules, and enhance activity of intracellular scaffolds. Improves immunity, reduces immunogenicity, and minimizes the need for a cold chain in manufacturing This is a desirable property for such a scaffold. [Means for solving the problem]

[0005] The present invention relates to a protein based on the fibronectin type III (FN3) repeat protein. scaffolds, encoding or complementary nucleic acids, vectors, host cells, compositions, mixtures, processes In a preferred embodiment, methods, devices, and methods of making and using the same are provided. The protein scaffold is composed of multiple FNs of human tenascin-C (hereafter referred to as "tenascin"). In a further preferred embodiment, the protein sequence of the present invention is The caffold consists of the consensus sequences of 15 FN3 domains (SEQ ID NOs: 1 to 15) or variants thereof. In a particular embodiment of the invention, the protein scaffold of the invention is Substituted residues that cause selected proteins to exhibit enhanced ability to resist thermal and chemical denaturation The protein scaffolds of the present invention have a predetermined loop region within the scaffold. By inserting residues into the nuclease, a binding domain with high selectivity for the binding partner can be created. The binding partner may also be modified by methods known in the art. The polypeptide may be a polypeptide or a cell-associated molecule, such as an extracellular domain of a receptor protein.

[0006] In one embodiment, the compositions are selected for their inherent thermal and chemical stability as described herein. Consensus-based specific substitutions of sequence number 16 (Tencon) are Improved the thermal stability of the scaffold by up to 11°C, lowering the midpoint of GdmCl-induced denaturation to 3.4 M. to more than 5M. In one embodiment, the Specific substitutions may be single, such as N46V, E14P, and E86I, and alternative embodiments include In this embodiment, the substitutions are N46V and E86I, E14P and N46V and E86I, and and L17A, N46V, and E86I. Stability has been enhanced. Tencon-based polypeptides offer improved purification, ease of preparation, and long shelf life. The modified binding partners provide extended scaffolds with improved overall stability. They can be produced by introducing random peptides into the loops of a stabilized scaffold. do.

[0007] The protein scaffolds of the present invention may be used as monomer units or may be linked to form polymeric structures specific for the same or different binding partners. The Tencon protein scaffold-based molecules can be further modified to enhance biodistribution. and determining one or more in vivo properties associated with a therapeutic effect, such as bioretention, biopersistence, or association with a molecule. The molecule can enhance cell, particularly epithelial cell uptake, by a molecule that alters cell uptake, e.g. , the Fc region of an antibody, and molecules designed to bind to serum proteins, e.g. , albumin binding domain, etc. In a further embodiment, the protein scaffold of the invention is The fold may be bound to a nucleic acid molecule capable of encoding the protein scaffold.

[0008] The present invention also provides a method for the production of a medicament for the preparation of a medicament for the treatment of a cancer, comprising administering to a patient a therapeutically effective amount of the medicament, the method comprising administering to a patient a therapeutically effective amount of the medicament for the treatment of a cancer ... At least one method for expressing at least one protein scaffold comprising wherein at least one protein scaffold is detectable and / or retrievable. Also provided are methods comprising culturing a host cell as described herein under conditions in which the compound is expressed in an amount sufficient to produce a medicament for the treatment of atopic dermatitis. Provide.

[0009] The present invention also provides a method for producing a method for the preparation of a pharmaceutical composition comprising: (a) a plurality of FN3 domains and / or coding domains as described herein; (b) a protein scaffold based on a consensus sequence of a nucleic acid having a suitable and / or pharmaceutical and a commercially available carrier or diluent.

[0010] The present invention relates to a fibronectin type 3 (FN3) repeat protein, preferably a multi-repeat protein. and more preferably a consensus sequence of multiple FN3 domains of human tenascin. Library of protein scaffolds with enhanced thermal and chemical stability based on consensus sequences The present invention further includes methods for creating a loop by varying the amino acid composition of a single loop or by varying the amino acid composition of multiple loops. By simultaneously varying additional positions of the peptide or scaffold molecule, the library The loop in which the change occurs will be lengthened or shortened depending on the change. Such a library can be created and each position can be tested for possible amino acids. The library can contain all amino acids or a subset of the intended amino acids. The members can be displayed, for example by in vitro display (DNA, RNA, ribosomal Screening by yeast, bacteria, and phage display can be used.

[0011] The protein scaffolds of the present invention exhibit high osmolarity and solubility at high concentrations. The scaffold protein provides enhanced biophysical properties, such as stability. The domains are not disulfide bonded and therefore lack the enzymes required for disulfide bond formation. In vitro transcriptional systems (e.g., prokaryotic systems such as E. coli and in vitro transcriptional systems such as rabbit reticulocyte systems) This allows the region to be expressed and folded in a ribozyme / translation system.

[0012] In a further aspect, the present invention provides a method for the preparation of a scaffold of the present invention comprising the steps of: Panning the library to identify scaffold molecules that bind to specific targets In another related aspect, the present invention provides a method for making a compound having a desired activity, e.g., The affinity matured protein scaffold is capable of binding to a target protein with high affinity. Affinity maturation includes screening methods that can be used to prepare phage Repeated mutagenesis and selection can be carried out using systems such as spray or in vitro display. Mutagenesis during this process can be achieved by selectively activating specific scavenger receptors. Site-directed mutagenesis of selected residues, random mutagenesis by error-prone PCR, The present invention may be the result of NA shuffling, and / or a combination of these procedures. Further provided is any of the inventions described herein. [Brief description of the drawings]

[0013] [Figure 1] Figure 1 SDS-PAGE analysis of purified Tencon run on a NuPAGE 4–12% Bis-Tris gel (Invitrogen) and stained with Coomassie blue. N means as is, and R means reduced. [Diagram 2] Circular dichroism analysis of Tencon in PBS. [Diagram 3] Circular dichroism analysis of the third FN3 domain of tenascin and Tencon in PBS was performed, giving melting temperatures of 54° C. and 78° C., respectively. [Figure 4]The phagemid plasmid design of pTencon-pIX is shown. Expression is driven by the Lac promoter and secretion via the OmpA signal sequence. [Diagram 5] That myc-Tencon can be displayed on M13 phage is shown using ELISA, showing binding of phage to anti-Myc coated, CNTO95 coated, and uncoated wells. [Figure 6] Diagram depicting the loop structure of the third FN3 domain of human tenascin. [Figure 7] Screening of the IgG selection results by ELISA is shown, and each clone was tested for binding to biotinylated IgG or biotinylated HSA as a control. [Figure 8A] Graph showing GdmCl-induced unfolding of single mutants as measured by fluorescence excitation at 280 nm and emission at 360 nm. [Figure 8B] Graph showing GdmCl-induced unfolding of combinatorial mutants (B) as measured by fluorescence excitation at 280 nm and emission at 360 nm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Abbreviation ADCC=antibody-dependent cytotoxicity; CDC=complement-dependent cytotoxicity; DSC=differential chemotaxis Calorimetry; ΔG = Gibbs free energy; IgG = immunoglobulin G; Tm = melting temperature;

[0015] Definitions & Explanations The term "antibody" or "antibody portion" includes antibodies, fragments, specified portions, and variants thereof. It is contemplated that the present invention relates to a method for the preparation of a compound that mimics the structure and / or structure of an antibody, including, but not limited to, an antibody mimetic. It contains a portion of an antibody that mimics its function or a specific fragment or part thereof, and is a single chain antibody, a single domain antibody, Functional fragments include, but are not limited to, antibodies, bodies, and fragments thereof. For example, Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion and partial reduction), and F(ab')2 (e.g., , by pepsin digestion), facb (e.g., by plasmin digestion), pFc' (e.g. by pepsin or plasmin digestion), Fd (by pepsin digestion, partial reduction, and reassortment), Fv or scFv (e.g., by molecular biology techniques) fragments. The term includes, but is not limited to, antibody fragments capable of binding to a target antigen or a portion thereof. The term "antibody" includes, but is not limited to, human, mouse, rabbit, rat, and mouse antibodies. They may be derived from rodents, primates, camelids, caprines, or any combination thereof. and isolated human, primate, rodent, mammalian, chimeric, humanized, and / or CD4 R grafts, antibodies, immunoglobulins, cleavage products and other specified portions, and their variants. nothing.

[0016] The term "epitope" refers to one or more loops of an antibody or scaffold base protein. By "specific binding to a protein determinant" is meant a protein determinant capable of specifically binding to a modified binding domain such as Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains. They are generally made up of molecules that have specific three-dimensional structural characteristics as well as specific charge characteristics. A synthetic epitope is one in which the binding to the former but not the latter is lost in the presence of denaturing solvents. Conformational epitopes are distinguished by their ability to bind amino acids from different parts of the linear sequence of the target molecule. Conformational folding of the target molecule that occurs when the amino acids come together in close proximity in three-dimensional space Such conformational epitopes are typically found on the extracellular side of the plasma membrane. is distributed above.

[0017] As used herein, "Fc," "Fc-containing protein," or "Fc-containing component" refers to a The term "antibody" refers to a monomeric antibody having at least an immunoglobulin CH2 and CH3 domain. The CH2 and CH3 domains are referred to as a monomeric, dimeric, or heterodimeric protein. capable of forming at least a portion of the dimeric region of a protein / molecule (e.g., an antibody) .

[0018] As used herein, the term "stability" refers to the ability of a compound to retain normal functional activity (e.g., cytokine activity). The antibody is designed to maintain at least one of the following binding properties: binding to a target molecule such as a cyclic amine or serum protein, Protein stability refers to the ability of a molecule to remain folded under physiological conditions. Measurements of protein integrity and protein instability can be viewed as the same or different aspects of protein integrity. Proteins can be destroyed by heat, ultraviolet light or ionizing radiation, and, when in solution, by the osmotic pressure of the surrounding environment. changes in ionization due to changes in ionization concentration and pH, filtration through small pore sizes, ultraviolet radiation, and gamma irradiation; Radiation, chemical or thermal dehydration, or any other substances that may cause the destruction of protein structure. susceptible to alterations caused by any other action or force, i.e., "incompatible" The stability of a molecule can be determined using standard methods. For example, The stability of can be determined by measuring the thermal melting ("TM") temperature. is the temperature (°C) at which half of the molecule is unfolded. Typically, the higher the TM, the The molecules are more stable. In addition to heat, the chemical environment also helps proteins to maintain their unique three-dimensional structure. This changes the ability to maintain

[0019] Chemical denaturation can also be measured by a variety of methods. Chemical denaturants include, for example, For example, hydrogen bonds, electrostatic bonds, van der Waals forces, hydrophobic interactions, or disulfides. Agents known to disrupt non-covalent interactions and covalent bonds within proteins, such as .beta.-binding Chemical denaturants include guanidine hydrochloride, guanidinium thiocyanate, and urea. , acetone, organic solvents (DMF, benzene, acetonitrile), salts (ammonium sulfate , lithium bromide, lithium chloride, sodium bromide, calcium chloride, sodium chloride), Reducing agents (e.g., dithiothreitol, β-mercaptoethanol, dinitrothiobenzene (dinitrothiobenzene), and hydrides, e.g. sodium borohydride), non-ionic and ionic detergents, acids (e.g., hydrochloric acid (HCl), acetic acid (CH3COOH), halogens acetic acid), hydrophobic molecules (e.g., phospho-fatty acids), and target modifiers (Jain RK a nd Hamilton AD,Angew.Chem.114(4),2002) Quantification of the extent of denaturation can include loss of functional properties, such as the ability to bind a target molecule, or aggregate, expose residues that were previously inaccessible to the solvent, or disulfide bonds. The disappearance may depend on physicochemical properties such as the tendency for bonds to be broken or formed.

[0020] With regard to loss of stability, i.e., the protein "denatures" or "denatures" the protein is defined as a state in which some or all of the three-dimensional conformations that confer the functional properties of a protein are Destruction during denaturation refers to the process by which a compound disappears with the concomitant loss of activity and / or solubility. The forces include electrostatic forces, hydrophobic forces, van der Waals forces, hydrogen bonds, and disulfide bonds. The denaturation of proteins includes, but is not limited to, intramolecular bonds such as tRNA and tRNA. Force applied to a protein or a solution containing a protein, e.g., mechanical force (e.g., compression force or shear stress), thermal stress, osmotic stress, pH, changes in electric or magnetic fields, ionizing radiation, ultraviolet radiation Dehydration and oxidation can be caused by a variety of factors, including oxidation and dehydration, and by chemical denaturants.

[0021] As used herein, a "therapeutically effective" treatment or amount refers to a treatment or amount effective to treat or prevent a disease or its symptoms. "Improve" refers to an amount sufficient to cause therapeutic relief or improvement. The subject of the present invention is preferably a human. However, any animal in need of treatment for an adverse condition, disorder, or disease may be treated with a It is envisioned that a patient may be treated with a scaffold-based protein.

[0022] Overview The present invention is based on the consensus sequence of the fibronectin type 3 (FN3) repeat protein. isolated recombinant and / or synthetic proteins, including but not limited to mammalian derived scaffolds; Protein scaffolds and compositions and compositions based on the consensus FN3 sequence and providing an encoding nucleic acid molecule comprising at least one polynucleotide encoding the code. The present invention can be used, for example, as a discovery platform and for the preparation of diagnostic compositions, therapeutic compositions, methods, and the like. Such nucleic acid and protein scaffolds have been produced for purposes of methods and devices. This includes, but is not limited to, methods using one or more of the above-mentioned methods.

[0023] The protein scaffolds of the present invention are small and compact, allowing for a broader range of immunoglobulins to be bound. This provides advantages to ribonucleic acid-based biological therapeutics. In particular, the size and shape of the biomolecule can be the ability to be administered topically, orally, or across the blood-brain barrier; Ability to be expressed; bispecific or binding to multiple targets or multiple epitopes of the same target Ability to be modified to specific molecules; suitability for binding, i.e., active substances, polymers, and compatibility with probes; the ability to be prepared in high concentrations; and the ability of such molecules to be isolated from diseased tissues and tumors. This can affect the ability of the drug to penetrate effectively into the bloodstream.

[0024] Additionally, protein scaffolds can be constructed in association with folds that mimic the variable regions of antibodies. This method allows the synthesis of DNA fragments similar to the complementarity determining regions (CDRs) of antibodies, which have many of the properties of antibodies. These should be able to bind to intracellular targets. This loop can then be altered, e.g., affinity matured, to improve specific binding or related properties. It can be improved.

[0025] Three of the six loops of the protein scaffold of the present invention are hypervariable in nature. The binding domains of antibodies are topologically related to the variable domain loops that are known to bind the According to Kabat, the complementarity determining regions (CDRs) of antibodies, i.e., the antigen-binding regions, are The remaining three loops are located in the hypervariable domain (HVL) at a defined position. R are exposed in a manner similar to that of R. These loops are shown in Table 3 below and in FIG. Thus, residues 13-16, 22-28, 38-43, 51-54, 60- 64, and 75-81 or thereabout. Preferably, residues 22-28, The loop regions 51-54 and 75-81 or thereabouts are used for determining binding specificity and affinity. One or more of these loop regions are altered to be different from other loop regions and / or strands. The strands are randomized to maintain their sequence and placed in a library. Selecting effective binders from libraries that have high affinity to protein targets Similar to the interaction of the antibody CDRs with the target protein, one or more loop regions can The agonist can interact with a target protein.

[0026] The scaffolds of the invention can be linked to other subunits, e.g., via covalent interactions. All or part of the antibody constant region can be attached to the scaffold. , antibody-like properties, particularly those associated with the Fc region, such as complement activation (ADCC), half-life, etc. Antibody-like properties can be imparted. For example, effector functions can be conferred, for example, C1q binding. and / or FcγR binding, thereby altering CDC activity and / or ADCC activity. An "effector function" can be provided and / or controlled by (e.g. Effector functions serve to activate or reduce biological activity (e.g., in a subject). Examples of functions include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; and antibody-dependent ADCC; phagocytosis; downregulation of receptors on the surface of cells (e.g. Examples of such antibodies include, but are not limited to, B cell receptors (BCRs). The vector function is associated with a binding domain (e.g., a protein scaffold loop) The Fc region may be required for various assays (e.g., Fc binding assays, ADCC Assays such as CDC assays can be used to assess the activity of the antibody.

[0027] The additional moiety may be attached to or associated with the scaffold base polypeptide, Toxin complexes, albumin or albumin binders, polyethylene glycols, etc., may be used for desired properties. Mutants such as polysaccharide (PEG) molecules may be attached to the scaffold molecule. may be fused in-line with the coding sequence of the scaffold, using standard techniques, e.g. , recombinant fusion coding vectors constructed using published coding nucleotide sequences Alternatively, the gene may be produced by expression of a fusion protein by a plasmid. and binding the moiety to a recombinantly produced scaffold base protein. good.

[0028] The scaffolds of the invention can be monospecific in monomeric form or bispecific or multimeric in multimeric form. or multispecific (against different target proteins or epitopes on the same target protein) The bonds between each scaffold unit can be covalent or non-covalent. For example, a dimeric bispecific scaffold may be one subunit having specificity for a protein or epitope, and a second target protein or a second subunit with specificity for the epitope. The subunits can be organized into various conformations that can increase the valency, or avidity, of the antigen. The structures may be connected in the same manner.

[0029] Scaffold protein production and engineering At least one scaffold protein of the present invention may optionally be prepared according to any of the methods described in the art. Produced by known cell lines, mixed cell lines, immortalized cells or clonal populations of immortalized cells in the For example, see Ausubel, et al., eds., Current Protocols in Molecular Biology,John Will ey & Sons, Inc.,NY,NY(1987~2001);Sambrook ,et al.,Molecular Cloning:A Laboratory M anual,2 nd Edition,Cold Spring Harbor,NY(1 989);Harlow and Lane, Antibodies, a Labora tory manual, Cold Spring Harbor, NY (1989); Colligan,etal.,eds.,Current Protocols in Immunology, John Wiley & Sons, Inc., NY (19 94~2001);Colligan et al.,Current Protoco ls in Protein Science,John Wiley & Sons, See NY, NY, (1997-2001).

[0030] As known in the art, amino acids may be modified from the scaffold protein. , addition, and / or deletion to reduce immunogenicity or to improve binding, affinity, association rate, dissociation speed, avidity, specificity, half-life, stability, solubility, or any other suitable property. It can be reduced, enhanced, or altered.

[0031] The bioactive scaffold-based protein is characterized by its high affinity for antigens and other favorable properties. To this end, the scuffs can be designed with suitable biological properties. The folded protein is then compared to the parent sequence, optionally using three-dimensional models of the parent and modified sequences. The three-dimensional model can be prepared by a process of analysis of the sequence and various conceptual transformation products. Such sequences are generally available and are familiar to those skilled in the art. Computer programs are available that illustrate and display the three-dimensional conformation of potential The immunogenicity of a given antibody can be measured (e.g., Xencor, Inc. (Monrovi a, CA)'s Immunofilter program). By examining these displays , an analysis of the likely role of residues in the function of the candidate sequence, i.e. Allows analysis of residues that affect the ability of a scaffold protein to bind its antigen In this way, desired properties such as affinity for the target antigen can be achieved. Alternatively, residues can be selected and combined from the parent and reference sequences. Any other suitable modification method can be used as well as, or in addition to, the method.

[0032] screening Containing variable residues or domains that specifically bind to similar proteins or fragments A modified scaffold base protein or a light-binding protein comprising a scaffold base protein Screening of the library was performed using nucleotide (DNA or RNA display) or Peptide display libraries can be conveniently achieved using, for example, in vitro display. This method allows the creation of individual members with the desired function or structure. This involves screening a large collection of peptides. The length of the displayed peptides, with or without, is 3 to 5,000 or more nucleotides. A peptide or amino acid, often 5 to 100 amino acids, more often about 8 to 25 amino acids. In addition to direct chemical synthesis methods for generating peptide libraries, Several recombinant DNA methods have also been described. One type uses bacteriophages or Each bacteriophage or The cells contain a nucleotide sequence encoding a particular displayed peptide sequence. do.

[0033] The protein scaffolds of the present invention can be used to bind a wide range of human or other mammalian proteins. Affinity (K D In a preferred embodiment, at least one of the present invention The protein scaffolds of the present invention can be optionally provided with high affinity, e.g., as can be seen by those skilled in the art. As shown in Fig. 1, when measured by surface plasmon resonance or Kinexa, the K D About 10 - 7 M or less, for example, but not limited to, 0.1 to 9.9 (or any range therein) Range or value)X 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , 10 -15 or any range or value thereof that can bind to the target protein. do.

[0034] The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method. (See, for example, Berzofsky, et al., "Antibody -Antigen Interactions,” In Fundamental Im munology, Paul, WE, Ed., Raven Press: New Y ork,NY(1984);Kuby,Janis Immunology,WHF Reeman and Company: New York, NY (1992); and this book (See methods described herein.) The affinities measured may vary depending on the conditions (e.g., osmolarity, pH) Therefore, affinity and other antigen-binding parameters (e.g., K D , K on , K off The measurement of the solubility of the protein scaffold and the antigen is preferably carried out using a standardized dissolution method. The assay is carried out using standardized buffers such as those described herein.

[0035] Competing with the protein scaffold of the invention for binding to a target protein is, and / or or which proteins, antibodies, and other antagonists share the epitope region. To determine whether the protein scaffolds of the present invention are As is readily known to those of skill in the art, these assays can be used to detect Evaluate the competition between antagonists or ligands for limited binding sites. Immobilize, isolate, or capture proteins and / or antibodies, and also perform, for example, decanting ( (if the protein / antibody was preinsolubilized) or centrifugation (if the protein / antibody was precipitated after the competitive reaction) (if used) to separate the sample bound to the target protein from the unbound sample. In addition, the binding or non-binding of the protein scaffold to the target protein can be confirmed. For example, whether a protein scaffold molecule, e.g. For example, competitive binding may be determined by whether the enzyme activity of the label is inhibited or enhanced. ELISA and other functional assays, as are well known in the art, may be used. stomach.

[0036] nucleic acid molecule Nucleic acid molecules encoding the protein scaffolds of the present invention can be expressed as mRNA, hnRNA, in the form of RNA, such as tRNA or any other form, or obtained by cloning or synthetically produced cDNA and genomic DNA, but are not limited to these. The DNA may be triple-stranded, double-stranded, or any combination thereof. It may be double-stranded or single-stranded, or any combination thereof. DNA or RNA Any portion of at least one strand of A may be the coding strand, also known as the sense strand. Alternatively, it may be the non-coding strand, referred to as the antisense strand.

[0037] The isolated nucleic acid molecules of the present invention include those described herein and / or known in the art. As is known in the art, one or more introns (such as, but not limited to, at least At least one specific site of one protein scaffold) A nucleic acid molecule containing an open reading frame (ORF), a protein that binds to a target protein, Nucleic acid molecules comprising coding sequences for the matrix scaffold or loop regions, and substantially the same as those described above. They contain essentially different nucleotide sequences, but due to the degeneracy of the genetic code, they are often called protein scaffolds. Of course, the genetic code may include a nucleic acid molecule that still encodes the gene. Such code is well known in the art. Thus, those skilled in the art will appreciate that the present invention To generate these modified nucleic acid variants that encode specific protein scaffolds It would be routine to do so. See, e.g., Ausubel et al., supra. Such nucleic acid variants are encompassed by the present invention.

[0038] As described herein, the present invention provides a method for the preparation of a method for the preparation of a protein scaffold comprising the steps of: The nucleic acid molecules of the invention contain a nucleic acid sequence encoding, by itself, the amino acid sequence of a protein scaffold fragment. coding sequences for whole protein scaffolds or parts thereof, protein scaffolds Coding sequences for the fragments, fragments or portions thereof, as well as additional sequences, e.g. Role in transcription, mRNA processing, including isoforms and polyadenylation signals Transcribed and untranslated sequences that play important roles in the synthesis of the protein (e.g., ribosome binding and mRNA stability) Additional non-coding sequences, including but not limited to non-coding 5' and 3' sequences, may also be included. and / or with at least one additional coding sequence, such as at least one intron. coding sequence for at least one signal leader or fusion peptide, additional additional coding sequences encoding amino acids (e.g., those that provide additional functions); Thus, the protein scaffold may include, but is not limited to, The sequence to be synthesized may be a fusion containing a marker sequence, such as a protein scaffold fragment or portion. The protein scaffold may be fused to a sequence encoding a peptide that facilitates purification. stomach.

[0039] nucleic acid molecule The invention also relates to the nucleic acid sequences of the present invention, whether as isolated polynucleotides or in prokaryotic, eukaryotic, or filamentous form. Phage expression of, secretion of, and / or directed mutagenesis of, or The compositions of the invention can be expressed as part of an expression vector, including vectors compatible with display. The nucleic acid to be loaded is provided.

[0040] The isolated nucleic acid of the present invention comprises (a) recombinant methods; (b) synthetic methods; (c) purification methods; and / or (d) Combinations of these.

[0041] Polynucleotides useful in the practice of the present invention include those derived from the protein sequences described herein. The polynucleotides of the present invention encode functional portions of the protein of the present invention. Selective hybridization to a polynucleotide encoding a substrate scaffold. The present invention includes nucleic acid sequences available for the preparation of the polynucleotides disclosed herein. The present invention provides an isolated nucleic acid that hybridizes under selective hybridization conditions to Therefore, the polynucleotide of this embodiment provides such a polynucleotide. For example, the present invention can be used to isolate, detect, and / or quantify nucleic acids comprising the nucleic acid. The clear polynucleotides are used to identify partial or full-length clones in the compiled library. In some embodiments, the polynucleotides can be identified, isolated, or amplified. The cDNAs are isolated or otherwise derived from a human or mammalian nucleic acid library. A is a genomic or cDNA sequence complementary to A.

[0042] The nucleic acid may conveniently comprise sequences in addition to the polynucleotides of the invention. For example, a multiple cloning site containing one or more endonuclease restriction enzyme recognition sites, It can be inserted into a nucleic acid to aid in the isolation of a polynucleotide. Sequences can be inserted to aid in the isolation of translated polynucleotides of the invention. For example, a hexahistidine marker sequence provides a convenient means for purifying the proteins of the invention. The nucleic acid of the invention (excluding the coding sequence) can optionally be used as a polynucleotide of the invention. Vectors, adapters, or linkers for cloning and / or expression of nucleotides. do.

[0043] Additional sequences may be added to such cloned and / or expressed sequences to clone and / or Their function in expression can be optimized to aid in the isolation of polynucleotides. or to improve the introduction of polynucleotides into cells. The use of expression vectors, adapters, and linkers is well known in the art. is.

[0044] As described herein, the present invention provides a method for the preparation of a method for the preparation of a protein scaffold comprising the steps of: The nucleic acid molecules of the invention contain a nucleic acid sequence encoding, by itself, the amino acid sequence of a protein scaffold fragment. coding sequences for whole protein scaffolds or parts thereof, protein scaffolds Coding sequences for the fragments, fragments or portions thereof, as well as additional sequences, e.g. Role in transcription, mRNA processing, including isolating and polyadenylation signals Transcribed and untranslated sequences that play important roles in the synthesis of the protein (e.g., ribosome binding and mRNA stability) Additional non-coding sequences, including but not limited to non-coding 5' and 3' sequences, may also be included. and / or with at least one additional coding sequence, such as at least one intron. coding sequence for at least one signal leader or fusion peptide, additional additional coding sequences encoding amino acids (e.g., those that provide additional functions); Thus, the protein scaffold may include, but is not limited to, The sequence to be synthesized may be a fusion containing a marker sequence, such as a protein scaffold fragment or portion. The protein scaffold may be fused to a sequence encoding a peptide that facilitates purification. stomach.

[0045] For bacterial expression, such as in phage-infected bacteria, the preferred secretion signal is pelB or omp A secretion signal, but as described in U.S. Pat. No. 5,658,727, other secretion signals may be used. In phage display, downstream translational polypeptide domains may be used. Possible DNA sequences include those encoding filamentous phage outer membrane proteins, such as the pIII or pIX proteins. Preferred phage proteins are those encoded by filamentous phages M13, f1, fd, etc. , and equivalent filamentous phages. Thus, the downstream translatable DNA sequence is corresponding to the filamentous phage gene III or gene IX outer membrane polypeptide, and preferably The sequence of this outer membrane protein is known. These are available in public databases such as NCBI.

[0046] Probes based on the sequences of the polynucleotides of the invention as disclosed herein The same can be used to screen a cDNA or genomic library. Or, to isolate different in vivo homologous genes, hybridization with genomic DNA or cDNA sequences is performed. A probe can be used to hybridize the nucleotides in the assay. Various degrees of hybridization stringency can be used in Either the hybridization or the washing medium can be stringent. As the conditions for hybridization become more stringent, Therefore, a greater degree of complementarity is required between the probe and the target for duplex formation to occur. The degree of stringency can be determined by a number of factors, including temperature, ionic strength, pH, and formamide. The presence of a selective denaturing solvent can be used to control the hybridization reaction. The stringency of the digestion can be determined by, for example, the concentration of formamide in the range of 0% to 50%. This can be conveniently altered by changing the polarity of the reaction solution through manipulation of the temperature. The degree of complementarity (sequence identity) required for binding depends on the hybridization medium and / or The degree of complementation varies depending on the stringency of the washing medium. or 70-100%, or any range or value therein. Slight sequence variations in the primers and primers may occur during hybridization and / or washing It should be understood that this can be compensated for by reducing the stringency of the

[0047] In one embodiment of the present invention, the polynucleotide is a polynucleotide obtained by repeating one or more specific residues. Randomization codes are used to diversify peptides or add residues at specific positions in the sequence. Random, anti-rational, and rational methods are used to Libraries of variegated polypeptide sequences can be generated using a variety of strategies, such as Rational and semi-rational methods can be used to more precisely characterize the effects of changes introduced into coding sequences. This strategy has the advantage over random strategies in that it allows for control. By focusing differently, every possible amino acid variant is identified at the selected position. It can be investigated.

[0048] Libraries constructed with the common NNK or NNS diversification schemes contain all positions and This library introduces 32 different codons for all 20 amino acids. In theory, every n residues increases by 32n. I is 10 9 ~10 10 The library is restricted to a sampling of variants of This means that if you are trying to include the complete sequence in a database, you can only target 6-7 residues. Therefore, it is important to identify key positions to be diversified and diversify along those positions. A semi-rational approach to generate libraries of scaffold variants by selecting a strategy A "codon set" is a set of sequences that represent the desired mutations. One of the various nucleotide triplet sequences used to code for amino acids The standard form of codon designation is the IUB code codon designation, which A "non-random codon set" refers to a set of codons that is selected based on known techniques in the art and described herein. The codon set that codes for a particular amino acid is called a nucleotide sequence. The synthesis of oligonucleotides having "degeneracy" is well known in the art, and is described, for example, in the TRIM The method is known (Knappek et al.; J. Mol. (1999), 29 6:57~86);Garrard & Henner, Gene (1993), 128 :103). Such sets of nucleotides having a particular codon set are commercially available. The nucleotide or nucleoside reagents and equipment used in the synthesis of nucleotides and nucleosides are do.

[0049] A codon set is a set of various nucleotide sequences used to code for a desired mutated amino acid. The codon set is shown below according to the IUB code: The symbols used to represent a particular nucleotide or equimolar mixture of nucleotides are: It can be done.

[0050] IUB Code G: Guanine A: Adenine T: Thymine C: Cytosine R (A or G) Y (C or T) M (A or C) K (G or T) S (C or G) W (A or T) H (A or C or T) B (C ​​or G or T) V (A or C or G) D (A or G or T) N(A or C or G or T)

[0051] For example, in the codon set DVK, D can be the nucleotide A or G or T, and V can be A, C, or G, and K can be G or T. This codon set contains 18 different The codons represent the amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, It can encode Ser, Arg, Asp, Glu, Gly, and Cys.

[0052] The focused (e.g., non-random) library uses the NNK codon, and can be created by focusing on the varigation of selected residues, Alternatively, variants with non-random substitutions may be used, for example, 11 amino acids (ACDEG It is constructed using the DVK codons that encode the nucleotide sequence KNRSYW and one stop codon. Alternatively, Kunkel-type mutagenesis can be used to modify desired residues or regions of a polypeptide. (Kunkel et al., Methods Enzyme mol.154:367~382,1987).

[0053] The library was cloned into a vector and expressed using standard cloning methods. The library can be expressed using known systems, for example, fusion proteins. The library can be expressed as a fusion protein. The fusion protein can be any suitable The antibody can be displayed on the surface of a bacteriophage. Methods for displaying fusion polypeptides containing fragments are well known (U.S. Pat. No. 6,966,333). No. 9,108 (Griffith), U.S. Pat. No. 6,172,197 (McCaffe rty), U.S. Patent No. 5,223,409 (Ladner), U.S. Patent No. 6,582, No. 915 (Griffiths), U.S. Pat. No. 6,472,147 (Janda). Libraries for the isolation of polypeptides can be displayed on pIX (WO2 009085462A1). The library can be, for example, a ribosome display ( nes and Pluckthun,Proc.Natl.Acad.Scie.Ac Ad.Scie.USA,94:4937,1997), mRNA display (Rob erts and Szostak,Proc.Natl.Acad.Sci.USA, 94:12297,1997), CIS display (Odegrip et.al., Proc. Natl. Acad. Sci. USA, 101:2806, 2004), or In vitro assays using other cell-free systems (US 5,643,768 to Kawasaki) It can also be translated in Roman.

[0054] The library with the diversified region encodes the Tencon sequence (SEQ ID NO: 16). The polynucleotide may be prepared using a vector containing the polynucleotide or a desired mutation thereof. The construct may have a promoter and a signal sequence for the polypeptide chain. To generate the library, the loop regions of the scaffold (A:B, B:C, C:D Mutagenesis using oligonucleotides encoding the sequences D:E, E:F, and F:G To ensure incorporation of all selected positions into the randomization scheme, In each region that is desired to be diversified, a stop codon (e.g., TAA) is inserted. Only clones in which the stop codon has been replaced are generated.

[0055] Modified Scaffold Polypeptides The modified protein scaffolds and fragments of the present invention can be directly or indirectly synthesized by other The polypeptide may include one or more moieties that are covalently attached to the protein.

[0056] When peptide residues are added or in-line fusion proteins are made, such residues The addition of the group can be accomplished by recombinant techniques of polynucleotide sequences as described herein. may be attached, bonded or conjugated to peptides, proteins, organic chemicals, inorganic chemicals, or atoms, or any combination thereof, of the scavenger of the protein of the invention. The attachment of the moiety to the bond or fragment is typically by other than a peptide bond. The protein scaffolds of the present invention are modified protein scaffolds or fragments. For example, the organic moiety can be prepared by reacting with an amine-reactive In a non-site-specific manner by utilizing a modifying agent, e.g., an NHS ester of PEG. The protein scaffolds of the present invention can be attached to the protein scaffolds. Modified protein scaffolds and fragments containing organic moieties attached to specific sites of the scaffold. Pieces can be prepared using a suitable method, such as reverse proteolysis (Fisch et al. l.,Bioconjugate Chem.,3:147~153(1992);We rlen et al.,Bioconjugate Chem.,5:411~417 (1994);Kumaran et al.,Protein Sci.6(10): 2233~2241(1997);Itoh et al.,Bioorg.Chem. ,24(1):59~68(1996);Capellas et al.,Biote chnol.Bioeng.,56(4):456~463(1997)),and t he methods described in Hermanson,GT,B ioconjugate Techniques,Academic Press:Sa n Diego, C. A. (1996).

[0057] When a polymer or chain is attached to the scaffold protein, the polymer or chain may be independently In particular, the group may be a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. As used, the term "fatty acid" includes mono- and di-carboxylic acids. As used herein, the term "hydrophilic polymeric group" refers to a polymer that is more hydrophilic than octane. For example, polylysine is more soluble in octane than in hexane. It is also highly soluble in water. Therefore, it is modified by covalently attaching polylysine. The protein scaffolds of the present invention are included in the present invention. Suitable hydrophilic polymers for modifying the can be linear or branched, for example, poly Alkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPE G), PPG, etc.), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides, etc. ), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartic acid, Polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide, etc.), Examples of the polyvinylpyrrolidone include polyvinylpyrrolidone and polyvinylpyrrolidone. The hydrophilic polymers that modify the protein scaffold range from about 800 to about 1000 individual compounds. It has a molecular weight of 50,000 daltons. For example, PEG 5000 and PEG 20,000 Use where the subscript is the average molecular weight of the polymer in Daltons. The alkyl group may be substituted with 1 to about 6 alkyl, fatty acid, or fatty acid ester groups. The hydrophilic polymer to be substituted with the fatty acid ester group can be prepared by adopting an appropriate method. For example, a polymer containing amine groups can be prepared by mixing the polymer with a fatty acid or fatty acid ester. Activated carboxylates on fatty acids or fatty acid esters can be coupled to carboxylates. Carboxylic acid salts (e.g., activated with N,N-carbonyldiimidazole) are added to the polymer The hydroxyl groups on the aryl group can be coupled to the aryl group on the aryl group.

[0058] Fatty acids and fatty acid esters suitable for modifying the protein scaffolds of the present invention are It may be saturated or may contain one or more units of unsaturation. Suitable fatty acids for modifying the scaffold include, for example, n-dodecanoic acid (C 12 , lauric acid), n-tetradecanoic acid (C 14 , myristic acid), n-octadecanoic acid (C 18 , stearic acid), n-eicosanoic acid (C 20 , arachidic acid), n-docosanoic acid (C 22 , behenic acid), n-triacontanoic acid (C 30 ), n-tetracontanoic acid (C 40 ), Sys- Δ9-octadecanoic acid (C 18 , oleic acid), all cis-Δ5,8,11,14-A Cosatetraenoic acid (C 20 , arachidonic acid), octanedioic acid, tetradecanedioic acid, Suitable fatty acid esters include: It includes monoesters of dicarboxylic acids containing linear or branched lower alkyl groups. A secondary alkyl group may contain from 1 to about 12, preferably from 1 to about 6, carbon atoms.

[0059] Fc-containing proteins are compared for functionality using several well-known in vitro assays. Specifically, the Fcγ receptors FcγRI, FcγRII, and FcγRIII can be These measurements are based on the affinity of the receptor to its family members. These can be made using either soluble or cell-bound forms of the receptor. The receptor responsible for the increased affinity and extended circulating half-life of IgG is recombinant soluble FcR. The ADCC assay can be performed using, for example, BIAcore. Cell-based functional assays, such as the ELISA and CDC assays, have been used to characterize the possible functions of specific mutant constructs. In one embodiment, the ADCC assay provides insight into the outcome of a primary It is constructed to have NK cells as effector cells, thereby FcγRII Reflects functional effects on IA receptors. Can measure cellular responses such as hyperoxia or the release of inflammatory mediators. To allow for the comparison of immune effector functions of different mutants, phagocytosis assays were used. In vivo models can also be used, for example, using mutant forms of anti-CD3 antibodies. It can be used to measure T cell activity in mice, and activity is measured by Fcγ receptor These depend on the Fc domain engaging a specific ligand, such as IgG1.

[0060] Host Cell Selection or Host Cell Engineering As described herein, the scaffold base protein is selected for expression. The host cells in which the cells are grown are important contributors to the final composition, if any, e.g. The composition of oligosaccharide moieties that decorate proteins in the immunoglobulin CH2 domain Thus, one aspect of the present invention is to provide a method for the treatment of a desired Selection of a suitable host cell for use and / or development of a production cell expressing a protein of interest Includes.

[0061] Additionally, the host cells may be of mammalian origin, or may be COS-1, COS-7, HEK 293, BHK21, CHO, BSC-1, Hep G2,653, SP2 / 0, 293 , HeLa, myeloma, lymphoma, yeast, insect or plant cells, or any derivative thereof. The cell may be selected from a somatic, immortalized, or transformed cell.

[0062] Alternatively, the host cell may be incapable of glycosylation of polypeptides. Species or organisms that can be used to treat bacterial infections, such as prokaryotic cells or organisms, and natural or engineered E. coli spp., It may be selected from Buschera spp., or Pseudomonas spp.

[0063] Selection of binding domains The polypeptide or fusion protein or components and domains thereof are The invention provides a method for the preparation of a phage library of antibodies or components, for example, a phage library. Phage libraries may be used to isolate antibody domains from B cells of immunized animals or humans. A library of random oligonucleotides, such as a polypeptide containing a sequence of interest, can be created by inserting a library of oligonucleotides (Smith, G .P.1985.Science 228:1315~1317). Antibody phage live The library contains a pair of heavy chain (H) and light chain (L) variable regions in one phage, and produces a single-chain Fv fragment. Fab fragments (Hoogenboom, et al. 2000, Immunol.Today 21(8)371-8). Phagemid library The diversity can then be used to generate additional, desirable molecular properties and the polynucleotides that encode them. and to enhance and / or alter the specificity of the polypeptides in the library for subsequent identification. It can be operated in such a way that

[0064] Other libraries of target binding moieties, which may include other than antibody variable regions, have been developed using ribosome-dispersing display, CIS display, yeast display, bacterial display, and mammalian cell display Ribosome display is a method to display proteins while maintaining their attachment to RNA. It is a method of translating mRNAs into their cognate proteins. It can be recovered by PCR (Mattheakis, LC et al. 1994.P roc.Natl.Sci.USA 91,9022). The CIS display is live An alternative in vivo display method in which RAL is constructed as a fusion protein with RepA During in vitro translation, RepA binds in cis to the DNA on which it is made, It provides a direct link between genotype and phenotype (Odegrip et.al., Pro c. Natl. Acad. Sci. USA, 101:2806, 2004). Yeast Dis Prey constructs of fusion proteins of the membrane-bound α-agglutinin yeast adhesion receptor, aga1 and Based on aga2, it is part of the mating type system (Broder, et al. 1997 .Nature Biotechnology,15:553~7). Bacterial display are based on the fusion of targets with excreted bacterial proteins that are associated with the cell membrane or cell wall. (Chen and Georgiou 2002.Biotechnol Bioe ng, 79:496-503). Similarly, mammalian display systems have been used to generate randomized sequences. Formation of a fusion protein between a polypeptide containing a sequence and a secreted membrane-anchored protein Based on.

[0065] Applications of scaffold-based molecules Scaffold-based molecules as described herein and produced by any of the methods described above. The compositions of the present invention may be used to treat symptoms of a human disease or to treat cells, tissues, organs, body fluids, or generally the host. Diagnosing, monitoring, regulating, treating, mitigating, helping to prevent the occurrence of, or alleviating a particular condition The scaffold-based molecules can be modified for specific purposes. Uses include immune-mediated diseases or immune deficiencies, metabolic diseases, cardiovascular injuries or diseases, malignant diseases Neurological disorders or diseases; Infections such as bacterial, viral or parasitic infections; or swelling, pain, and other known or specific associated disorders such as tissue necrosis or fibrosis. It can be treated.

[0066] Such methods include those that are effective in regulating, treating, alleviating, preventing, or reducing such symptoms. or a cell, tissue, organ, animal or patient in need of the mechanism. The method may include administering an effective amount of a composition or pharmaceutical composition comprising the folded protein. The dosages can be determined using known methods, as described herein or as known in the relevant art. When determining whether or not to administer the drug, the dosage should be about 0.05 mg / kg / day per single (e.g., bolus), multiple, or continuous dose. 0.01 to 500 mg / kg or 0.01 to 5 mg / kg per single, multiple, or continuous dose 000 μg / mL, or any effective range or value therein. may include:

[0067] Compositions Comprising Scaffold Base Proteins Modified or unmodified, monovalent, bivalent or multivalent, single target, multi-target, Multitarget, target-binding scaffold proteins are useful for capture, immobilization, partitioning, or precipitation. The compounds are isolated using separation techniques well known in the art for the purposes of commercial application, and are then purified to the extent necessary for commercial application. It can be purified.

[0068] For therapeutic use, scaffold-based proteins can be administered parenterally, subcutaneously, intramuscularly, Intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intrasinus, intracavity, intracerebellar, intraventricular, intracellular In the intestine, in the cervical canal, in the stomach, in the liver, in the myocardium, in the bone, in the pelvis, in the pericardium, in the abdominal cavity, in the pleura, in the prostate , in the lungs, in the rectum, in the kidneys, in the retina, in the spinal cord, in the bursa, in the thorax, in the uterus, in the bladder, in the lesion , bolus, intravaginal, rectal, buccal, sublingual, intranasal, or transdermal means, among others. It can be prepared based on a suitable administration method, without limitation. The folded composition may be a tablet or capsule; a powder, a nasal spray or an aerosol; a gel, an ointment, It can be prepared for use in the form of a lotion, suspension, or the like, or can be used in the form of a liquid or liquid preparation, as described in the art. The present invention can be incorporated into a therapeutic dressing or "patch" delivery system as known in the art. The present invention provides a stable formulation of a scaffold-based protein. Preferably, aqueous phosphate buffered saline or mixed saline, as well as preserved solutions, are used. Liquids and preparations, as well as multi-purpose preserved preparations suitable for medicinal or veterinary use and the pharma- ceutical acceptable formulation comprises at least one scaffold base. Suitable media and preparations thereof (human proteins, e.g. (including human serum albumin) are described, for example, in Remington: The Science and Practice of Pharmacy, 21 stEdition,Tr oy,DBed.,Lipincott Williams and Wilkin s,Philadelphia,PA 2006,Part 5,Pharmaceut ical Manufacturing pp 691~1092,See espec ially pp.958-989.

[0069] The composition may be a single formulation or a combination of both that is effective in treating the indicated disease, condition, or disorder. It may be used with or incorporated into other active substances known to be Alternatively, the combination of the scaffold base protein with the novel composition and active agent It may be tested by preparing

[0070] Although the invention has been described in general terms, embodiments of the invention are set forth in the appended claims. This is further disclosed in the following examples, which should not be construed as limiting. EXAMPLES

[0071] Example 1. Construction of Fc glycosylation variants Tencon Design The third FN3 domain of human tenascin (SEQ ID NO: 3) was cloned into the complementarity determining region (C The surface-exposed loops are structurally similar to the DR (receptor-dependent ribosomal receptor) of the ribosomal receptor. This domain in its native form can be used as an alternative scaffold for The melting temperature of the peptide is 54°C in PBS. To prepare a scaffold molecule with improved thermal stability, 15 amino acids of human tenascin were synthesized. A consensus sequence was designed based on sequence comparison of the FN3 domains (SEQ ID NOs: 1 to 15).

[0072] The overlap sequence comparison analysis shown in Table 1 revealed that these 15 domains have 13-80% overlap. The results show that the two sequences share a similar sequence identity, with the average pairwise sequence identity being 29%. The consensus sequence (SEQ ID NO: 16) was compared to the most conserved amino acid sequence at each position in the sequence shown in Table 1. The design was based on incorporating amino acids with high frequency. The consensus sequence of the present invention, designated con (SEQ ID NO: 16), is the FN3 domain of tenascin. It is identical at 34 to 59% of positions, with an average sequence identity of 43%.

[0073] Protein expression and purification The amino acid sequence of Tencon (SEQ ID NO: 16) is reverse translated to D The resulting NA sequence was constructed by overlapping PCR and the modified p The vector was subcloned into the ET15 vector and transformed into BL21Star(DE3) E. coli (Invitrogen) ogen) and cultured on LB agar plates containing 75 μg / mL carbenicillin. Pick one colony and culture it in 2% glucose and 100 μg / mL carbenicillin at 37°C. The culture was cultured overnight in 50 mL of TB medium containing 100% ethanol. tra Yield flask (Thomson Instrument Company ) in autoinduction medium (Overnight Express Instant TB m The cells were seeded in 500 mL of 500 mM NaCl (Novagen). Growth and expression were monitored using ATR Mult Dual program in a itron shaker incubator (37°C, 300 rpm for 3 h, followed by 30°C , 250 rpm for 16 hours).

[0074] The culture medium was collected and centrifuged at 7000 rpm for 15 minutes in a JL8.1 rotor. The cells were then pelleted in 20 mM sodium phosphate (pH 7.5), 500 mM NaCl l, 10% glycerol, 20 mM imidazole, 0.37 mg / mL lysozyme; 1X Complete Protease inhibitor (EDTA-free, Roche), and Benzonase (Sigma-Aldrich, 0.25 μL / Resuspend in 30 mL of buffer containing Misonix XL2020 Ultra (final concentration: 1 mL). Lyse using a sonicator on ice for 5 minutes in pulse mode (5 seconds on, 30 seconds off). The insoluble material was removed by centrifugation at 17,000 rpm for 30 minutes in a JA-17 rotor. Removed.

[0075] Tencon proteins are purified from soluble lysates using a two-step chromatography method First, 2 mL of Ni-NTA agarose beads (Qiagen) was added to the lysate. , placed on a rocking platform at 4 °C for 1 h, and subjected to immobilized metal affinity chromatography. Proteins were captured using a Poly-Prep column (Bio-Rad). The resin was packed in 20 mM sodium phosphate (pH 7.5), 500 mM NaCl, Unbound material was removed by washing with 10% glycerol and 20 mM imidazole. 0 mM sodium phosphate (pH 7.5), 500 mM NaCl, 10% glycerol The protein was eluted from the resin with 500 mM imidazole. Immunology Consultants Laboratory Fractions were analyzed by SDS-PAGE, both Coomassie staining and Western blot. The desired fractions were pooled and dialyzed against PBS (pH 7.4). Then, the column was loaded onto a Superdex-75 HiLoad 16 / 60 column ( Proteins were loaded onto a gel plate (GE Healthcare). Fractions were analyzed by SDS-PAGE. The fractions containing Tencon were pooled and Centriprep UltraCel YM The mixture was concentrated using a -3 concentrator (Amicon).

[0076] A BioTek plate reader is used to measure the absorbance of the samples at 280 nm. Protein concentrations were determined by Coomassie staining (Figure 1) and Western blot analysis using anti-His antibody. Blot and a G3000SW-XL column (TOSOH Bios) equilibrated with PBS. The final preparation was analyzed by HPLC-SEC using SDS-P In the AGE analysis, Tencon predicted a mass of 10.7 for the monomeric protein. The protein migrates between 6 and 14 kDa, which corresponds to the molecular weight of the protein. The total yield of purified Tencon protein was >50 mg.

[0077] Biophysical characteristics The structure and stability of Tencon were characterized by circular dichroism spectroscopy and differential scanning calorimetry, respectively. The AVIV fraction was characterized at 0.2 mg / mL in PBS at 20°C. The CD measurements were performed using a photometer. The spectrum in Figure 8 shows a minimum at 218 nm, which is consistent with the design. This suggests a β-sheet structure predicted for proteins belonging to the FN3 family. Tenascin 3 FN3 domain or Tencon 0.5mg / mL in PBS solution, In the N-DSCII calorimeter (Applied Thermodynamics), DSC data was obtained by heating from 5°C to 95°C at a rate of 1°C / min. The curve for buffer alone was subtracted to obtain the profile shown in Figure 3. From this data, the third FN3 For each domain and Tencon, CpCalc (Applied Thermo Using the modynamics software, melting temperatures of 54°C and 78°C were calculated. The folding and denaturation of both domains is reversible at these temperatures.

[0078] Immunogenicity analysis Using a computer program to model the immunogenicity of amino acid sequences in humans , the third FN3 domain of human tenascin, Tencon, and various therapeutic antibodies (Table 2 The predicted immunogenicity of the amino acid sequences representing the ribozymes (shown in Tolerance limits were then applied to the chimeric mAb and the human mAb (adalimumab) (human live (removal of a 9-mer peptide that is 100% identical to the sequence encoded by the germline). No limits were applied to tenascin or Tencon. The tolerance limits were determined based on the human germline. Assuming broad T cell tolerance to the encoded mAb sequence, the sequence is primarily composed of CDRs and adjacent domains. We focus our analysis on novel sequences in the genome.

[0079] Based on the possibility that the 9-mer peptides derived from the analyzed sequences bind to one or more HLA molecules, Based on these analyses, both tenascin and Tencon have been shown to pose an immunogenic risk. The score is weighted according to the prevalence of each HLA allele. The scores for the model are summed for each sequence, and the overall score for each sequence is The results of this analysis are summarized in Table 2. Nascin was shown to have the lowest overall score (11.9). In addition, Tencon is developing agretopes that are inherently non-binders and have low predicted immunogenicity risk. (score=13.2). The sequences of tenascin and Tencon are It was scored as favorable compared to the therapeutic antibody.

[0080] Display of Tencon on M13 phage by pIX fusion Cloning the gene encoding the amino acid sequence of Tencon by PCR and restriction digestion The vector was then subcloned into the phagemid expression vector pPep9 by This system was developed by fusion of the C-terminus to the N-terminus of the M13 pIX protein. The N-terminal Myc-tagged Tencon is expressed (Figure 4). Expression levels are low in the absence of PTG and can be increased after addition of IPTG The OmpA signal sequence was added to the N-terminus of Tencon to facilitate efficient translocation to the periplasm. A short TSGGGGS linker (SEQ ID NO: 141) was inserted between Tencon and pIX. This structure was constructed to prevent steric interactions between these proteins.

[0081] To confirm the display on the M13 phage particle surface, XL was used with pTencon-pIX. Transform 1-Blue E. coli and use one colony to incubate in LB medium supplemented with ampicillin. The culture was grown at 37°C until it reached mid-log phase. As of 6 10 Add pfu of VCSM13 helper phage and incubate the culture at 37°C for 10 min. The plates were then incubated without shaking for 50 min, followed by 50 min with shaking. - The culture medium rescued by phage was cultured in 2YT medium supplemented with ampicillin and kanamycin for 50 min. Dilute to OD in mL 600 Grow at 37°C with shaking until the pH reaches 0.7. At this point, IPTG was added to a final concentration of 1 mM, and the temperature was lowered to 30°C. The cultures were centrifuged at 000 × g for 20 min, and the supernatants were collected and stored at 4°C for analysis.

[0082] The M13 phage was synthesized by binding to anti-Myc antibody (Invitrogen). Display of the Myc-Tencon construct on the phage surface was confirmed. Plates were coated overnight with anti-Myc or anti-αv antibodies (negative control) at a concentration of 2.5 μg / mL. The plate was then immersed in a SuperBlock T20 (Pierce) and blocked. The supernatant of the phagemid culture was serially diluted 2-fold in PBS and applied to the coated plates. After 1 hour, the plate was washed with TBST and anti-M13 HRP antibody was added to the wells. was added to each well, incubated for 1 hour, and then washed with TBST. Add che BD ELISA POD substrate and measure light emission with a plate reader (Tecan). Figure 5 shows that Myc-Tencon phage particles were cloned with anti-αv antibody on the plate. It did not bind to the coated wells or the uncoated control wells, but was highly concentrated in the anti-Myc wells. Specific display of Myc-Tencon on M13 phage particles through concentration-dependent binding This indicates that the following is confirmed:

[0083] Further phagemid vectors were constructed to express the nucleotide sequence as a fusion with the coding protein pIII. Tencon and library members (see Example 2) were then distributed on M13 phage. In this system, the pIX gene encodes a truncated pIII. In comparison with the system shown in Fig. 4, Further changes include replacement of the OmpA signal sequence with the DsbA signal sequence. This suggests that secretion using this sequence may lead to the display of stable alternative scaffold molecules. It has been shown to be useful (Steiner et al., 2006). .

[0084] Example 2: Creation of a Tencon library Tencon mutant libraries were quantified to the desired complexity and relative position of the mutants within the molecule. These can be generated in a variety of ways, depending on the location. DNA synthesis is the preferred method for creating variants. DNA containing mutations in different regions of the gene. Restriction enzyme cloning can also be used to recombine fragments. Saturation mutagenesis in small defined regions, such as on-loops, is Mutagenesis can be performed using oligonucleotides and oligonucleotide-directed mutagenesis (Kun kel et al., 1987).

[0085] Oligonucleotide-directed mutagenesis was used to modify the FG loop with seven random amino acids. We constructed a Tencon library (library FG7) designed to replace A degenerate sequence of 21 base pairs (bp) that is NNS at the position encoding the FG loop, and adjacent The gene also contains 20-27 bp of nucleotide sequences complementary to the Tencon coding sequence at two positions. To achieve this, an oligonucleotide (TconFG7-For-5'pho) was synthesized. In this design, all 20 amino acids can be represented in the FG loop. The calculated diversity is 1.3 × 10 9 It is.

[0086] TconFG7-For5'pho: (SEQ ID NO: 18) GAATACACCGTTTCTATCTACGGTGTTNNSNNSNNSNNS NNSNNSNNSCCGCTGTCTGCGGAATTCAC

[0087] pDsbA-Tencon-As, a template for oligonucleotide-directed mutagenesis c-loop-Myc-pIII, the sequence encoding the F:G loop of Tencon, This system was constructed by replacing the nucleotide sequence with a stem-loop sequence containing an AscI restriction site. The resulting DNA was digested with AscI prior to transformation to obtain the template DNA after mutagenesis. The background of A can be eliminated. Single-stranded DNA template in mutagenesis To purify the plasmid pDsbA-Tencon-Asc-loop-Myc-pIII, One colony of E. coli CJ236 was cultured with carbenicillin (50 μg / mL final concentration) and The cells were placed in 5 mL of 2YT growth medium containing chloramphenicol (10 μg / mL). Then, VCSM13 helper phage was added to a final concentration of 10 10 Add pfu / mL and shake. Incubate for 10 min without and with carbenicillin (10 μg / mL) and uridine (0 Transfer the mixture to 150 mL of 2YT containing 0.25 μg / mL of ethanol and shake at 200 rpm for 37 min. The cells were pelleted by centrifugation, the supernatant was collected, and the phages were isolated using the P EG NaCl precipitation. QIAprep Spin M13 kit (Qiagen Single-stranded DNA was purified from this precipitate using ELISA kit (http: / / www.dilution.net / ) according to the manufacturer's instructions.

[0088] To anneal the degenerate oligonucleotide to the template, 5 μg of template DNA was added to Tr Oligo-Tcon in is-HCl (50 mM, pH 7.5) and MgCl2 (10 mM) Mix with FG7-For-5-pho at a molar ratio of 10:1, and heat at 90℃ for 2 minutes and at 60℃ for 3 minutes. The mixture was incubated at 20°C for 5 min and at 20°C for 1 h. After the annealing reaction, ATP (10 mM) , dNTPs (25 mM of each), DTT (100 mM), T4 ligase (7 units), and T7 DNA polymerase (10 units) were added to the reaction mixture and incubated at 14°C for 6 hours. Afterwards, the mixture was incubated at 20°C for 12 hours. The resulting DNA was purified using a PCR purification kit (Qiage The library DNA was purified using 10 units of As The DNA was digested with cI for 4 hours and then purified again using the Qiagen PCR purification kit. The final library DNA was recovered in 50 μL of water. The resulting double-stranded DNA product was then The vector was transformed into E. coli MC1061F' by electroporation.

[0089] The transformants were harvested in 20 mL of SOC medium and allowed to recover for 1 hour at 37°C. Aliquots of the transformation solution were serially diluted and incubated in carbenicillin ( 100 μg / mL) plates and the total number of transformants was evaluated. The culture medium was used to inoculate 1 L of 2xYT medium containing carbenicillin and 1% glucose, and then incubated at 20°C for 1 h. D 600 The culture was grown until the affinity reached 0.6. 10 10 The cells were seeded at a concentration of 1 / mL, incubated at 37°C, and then centrifuged. The cell pellet was incubated with carbenicillin (100 μg / mL) and kanamycin (35 μg / mL). The cells were resuspended in 500 mL of fresh 2xYT medium containing 100% yeast extract, grown overnight at 30°C, and centrifuged. Phage particles were precipitated by adding PEG / NaCl and stored at -80°C.

[0090] A second library (BC6 / FG7) was designed and constructed using the Tencon B:C and F:G rules. To achieve this, we simultaneously introduced diversity into the genomic DNA. BC6-For-5'phos and POP149 were synthesized. The forward oligo was phosphorylated. It contains 18 bases of NNS codons at each position that encodes the B:C loop, while the reverse The directional oligos were biotinylated at the 5' end and had N at each position encoding the F:G loop. Both oligonucleotides contained 21 bases of the NS codon. The primers were flanked by two 18 bp nucleotide sequences identical to the following region (primer (See below for details.)

[0091] Tc-BC6-For-5'phos: (SEQ ID NO: 19) gactctctgcgtctgtcttggNNSNNSNNSNNSNNSNNS TTCGACTCTTTCCTGATCCAGTACC

[0092] POP 2149: (SEQ ID NO: 20) GTGAATTCCGCAGACAGCGGSNNSNNSNNNNSNNSNNS NNAACACCGTAGATAGAAACGGTG

[0093] To construct the library, oligos Tc-CB6-For5'phos and POP2 Tencon DNA template was amplified using 149 in sixteen 100 μL PCR reactions. In the process, NNS codons were simultaneously introduced into the B:C and F:G loops. Double-stranded PCR products were purified by magnetic streptavidin beads (Dynal) and B&W buffer. (10mM Tris-HCl (pH7.5), 1mM EDTA, 2M NaCl, 0 Mix in 1% Tween-20, incubate for 20 minutes, then drop on a magnet and The beads were washed twice with &W buffer. The forward strand was removed from the beads with 300 μL of 150 mM NaOH. This "megaprimer" (8 × 10 in theoretical diversity) 16 Longer than The single-stranded library template was annealed with a mixture of 1000 ribosomal inhibitors (a mixture of 1000 ribosomal inhibitors). Construction was performed as described above for the FG7 library.

[0094] Example 3: Selection of IgG binders To perform selection of Tencon library members that bind IgG, recombinant IgG (human IgG1 subtype) was immunolabeled with sulfo-NHS-LC-biotin (Pierce). The phage were biotinylated using 200 μL of 10 ... Block the ELISA library FG7 or BC6 / FG7 with 200 μL of chemiblocker. After the incubation, 500 nM (first round) or 100 nM (second and third rounds) of biotin were added. The bound phages were first detected by magnetic beads. (Seradyne), and the second and third runs were performed with streptavidin magnetic beads (Promega). Unbound phages were harvested with Tris-buffered saline containing tween (T Wash 5-10 times with 1 mL of Tris-buffered saline (TBS) followed by 1 mL of Tris-buffered saline (TBS). The beads were washed twice and then rinsed. E. coli MC1061F' in mid-logarithmic growth phase was added. Bound phages were eluted from the beads by adding carbenicillin and glucose. The infected cells were plated on LB agar plates supplemented with . The next day, the cells were scraped off the plates and The cells were grown to mid-log phase, then rescued with VCSM13 helper phage and incubated overnight. Phage particles isolated by PEG / NaCl precipitation were used in the next round of selection.

[0095] After testing the whole body against IgG three times, the Tencon gene was amplified by PCR. The result was cloned into a pET27 vector modified to contain a ligase-independent cloning site. The PCR product was annealed into the vector and transformed into BL21-G The plasmid was transformed into OLD(DE3) cells (Stratagene). Separate colonies were then cultured at 96 The cells were placed in 1 mL of culture medium in a deep-well plate (Corning) and incubated overnight at 37°C until saturated. The following day, 50 μL of the overnight culture was used to inoculate a new 1 mL culture. After the culture was grown at 37°C for 2 hours, IPTG was added to 1 mM and the temperature was increased to 30°C. After 16 hours of induction, the cells were harvested by centrifugation and then diluted with 100 μL of BugBus. The resulting lysate was cleared by centrifugation. This was used in an IgG binding test by ELISA.

[0096] Maxisorp plates (Nunc) were pre-coated with 0.1 μg of anti-His antibody (Qiagen). Coat overnight with 1 μg of Starting Block T20 (Thermo Scientific). The plates were diluted 1:4 in Arting Block and allowed to bind for 1 hour, after which the plates were Wash with BST. Add biotinylated IgG or biotinylated HSA at a concentration of 1 μg / mL. After incubation for 1 hour, the plate was washed with TBST. Jackson Immunoresearch) and POD chemiluminescence substrate Detection of bound IgG or HSA was achieved by ELISA. The ELISA results are shown in Figure 7. As judged by ELISA signal, biotinylated IgG was 10 times more effective than biotinylated HSA. After multiple selection rounds were completed, library FG 7, 60 unique binding sequences, and 10 unique sequences from library BC6FG7. Table 4 shows the extent to which the B:C and / or F:G loops differ from SEQ ID NO:16. Representative sequences of IgG binders are shown. Table 4 also lists other regions of the scaffold. It also shows many variations in the region.

[0097] The Tencon proteins designed, expressed and purified herein are alternative scavenger proteins. For the third FN3 domain of human tenascin, which is used as a fold molecule, It has improved thermal stability by 26°C. Based on this improved stability, the scaffold molecule It is believed that the ribozyme is more suitable for amino acid substitution and easier to manufacture. Decreasing mutations are considered to be more tolerated in the sense of a more stable scaffold. The resulting scaffolds, which therefore have improved stability, are The Ibraly is believed to result in a more functional, well-folded binder.

[0098] [Table 1]

[0099] [Table 2]

[0100] [Table 3]

[0101] [Table 4-1]

[0102] [Table 4-2]

[0103] [Table 4-3]

[0104] [Table 4-4]

[0105] array: SEQ ID NO:1: sppkdlvvtevteetvnlawdnemrvteylvvytptheg glemqfrvpgdqtstiiqelepgveyfirvfailenkksi pvsarvat

[0106] SEQ ID NO:2: tylpapeglkfksiketsvevewdpldiafetweiifrn mnkedegeitkslrrpetsyrqtglapgqeyeislhivkn ntrgpglkrvtttrld

[0107] SEQ ID NO:3: dapsqievkdvtdttalitwfkplaeidgieltygikdv pgdrttidltedenqysignlkpdteyevslisrrgdmss npaketftt

[0108] SEQ ID NO:4 tgldaprnlrrvsqtdnsitlewrngkaaidsyrikyap isggdhaevdvpksqqattkttltglrpgteygigvsavk edkesnpatinaateldtpkd

[0109] SEQ ID NO:5 dtpkdlqvsetaetsltllwktplakfdryrlnyslptg qwvgvqlprnttsyvlrglepgqeynvlltaekgrhkskp akskparvk

[0110] SEQ ID NO:6 qapelenltvtevgwdglrlnwtaadqayehfiiqvqea nkveaarnltvpgslravdipglkaatpytvsiygviqgy rtpvlsaeastge

[0111] SEQ ID NO:7 etpnlgevvvaevgwdalklnwtapegayeyffiqvqea dtveaaqnltvpgglrstdlpglkaathytitirgvtqdf sttplsvevlte

[0112] SEQ ID NO:8 evpdmgnltvtevswdalrlnwttpdgtydqftiqvqea dqveeahnltvpgslrsmeipglragtpytvtlhgevrgh strplavevvte

[0113] SEQ ID NO:9 dlpqlgdlavsevgwdglrlnwtaadnayehfviqvqev nkveaaqnltlpgslravdipgleaatpyrvsiygvirgy rtpvlsaeastakepe

[0114] SEQ ID NO:10 kepeignlnvsditpesfnlswmatdgifetftieiids nrlletveynisgaertahisglppstdfivylsglapsi rtktisatatte

[0115] SEQ ID NO:11 alpllenltisdinpygftvswmasenafdsflvtvvds gklldpqeftlsgtqrklelrglitgigyevmvsgftqgh qtkplraeivte

[0116] SEQ ID NO:12 aepevdnllvsdatpdgfrlswtadegvfdnfvlkirdt kkqsepleitllapertrdltglreateyeielygiskgr rsqtvsaiattam

[0117] SEQ ID NO:13 gspkevifsditensatvswraptaqvesfrityvpitg gtpsmvtvdgtktqtrlvklipgveylvsiiamkgfeese pvsg fttal

[0118] SEQ ID NO:14 dgpsglvtanitdsealarwqpaiatvdsyvisytgekv peitrtvsgntveyaltdlepateytlrifaekgpqksst itakfttdl

[0119] SEQ ID NO:15 dsprdltatevqsetalltwrpprasvtgyllvyesvdg tvkevivgpdttsysladlspsthytakiqalngplrsnm iqtifttigl

[0120] SEQ ID NO:16 LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESE KVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHR SNPLSAEFTT

[0121] SEQ ID NO:17 ctgccggcgccgaaaaacctggttgtttctgaagttacc gaagactctctgcgtctgtcttggaccgcgccggacgcgg cgttcgactctttcctgatccagtaccaggaatctgaaaa agttggtgaagcgatcaacctgaccgttccgggttctgaa cgttcttacgacctgaccggtctgaaaccgggtaccgaat acaccgtttctatctacggtgttaaaggtggtcaccgttc taacccgctgtctgcggaattcaccacc

[0122] TIFF0007643849000008.tif41128

[0123] Example 4: Stabilizing Mutations of Tencon The folding stability of the Tencon scaffold (SEQ ID NO: 16) described herein above was Mutants were designed to improve the sequence of each of SEQ ID NO:16 by generating point mutations. Residue substitutions, e.g., N46V (Tencon17-SEQ ID NO: 142), E14P (Te ncon18-SEQ ID NO: 143), E11N (Tencon19-SEQ ID NO: 144), E 37P (Tencon20-sequence number 145), and G73Y (Tencon21-sequence number No. 146), which are compiled into the program PoPMuSiC v2.0 (Dehou ck, Grosfils et al., 2009) is expected to improve stability. The mutant E86I (Tencon22-SEQ ID NO: 147) is a homologous protein, It has previously been found to stabilize the third FN3 domain of human tenascin (WO Finally, all loop residues of Tencon were independently activated. The L17A mutant significantly stabilized Tencon during alanine scanning experiments where it was substituted with lanine. (data not shown). After initial stability assays (see below) To further increase stability, the combinatorial mutant N46V / E86I (T encon 23-SEQ ID NO:148), E14P / N46V / E86I (Tencon2 4-SEQ ID NO: 149), and L17A / N46V / E86I (Tencon25-SEQ ID NO: No. 150).

[0124] Expression and purification Using the QuikChange mutagenesis kit (Stratagene), A mutant of the on coding sequence was generated. The resulting plasmid was transformed into BL21-GOLD(D E3) Transform into E. coli (Stratagene) and express. Pick a single colony. The bacteria were cultured overnight at 37° C. in 2 mL of TB medium containing 100 μg / mL ampicillin. This culture medium was used to grow autoinduction medium (Overnig medium) in a 500 mL baffled flask. ht Express Instant TB media,Novagen)100m L and cultured at 37°C for 16 hours.

[0125] The culture medium was harvested by centrifugation at 4000 x g for 20 minutes, and the pelleted cells were The cells were cultured in 5 mL of BugBuster HT (Nov) per gram of wet cell pellet. After incubation at room temperature for 30 minutes, the cells were centrifuged at 30,000 × g. The lysate was clarified by centrifugation at 4°C for 20 min and added to 3 mL of Ni-NTA super The column was loaded by gravity using 50 mM sodium phosphate buffer. (pH 7.4), 500 mM NaCl, and 10 mM imidazole Each column was washed with 15 mL of the buffer containing 50 mM sodium phosphate (pH 7.4) 10 mL containing 500 mM NaCl and 250 mM imidazole The bound protein was eluted from the column using a buffer of 0.1 ml. The purity of the protein was confirmed by SDS-PAGE. Prior to biophysical analysis, each mutant was incubated in PBS (pH 7.4) were thoroughly dialyzed into the 100 mL culture medium. mg of purified protein was obtained.

[0126] Characterization of thermal stability The thermal stability of Tencon and each mutant of the present invention was examined by capillary differential scanning calorimetry (DSC). Each sample was dialyzed against PBS (pH 7.4) and diluted to 2–3 mg / mL. The VP with an autosampler (MicroCal, LLC) was used to dilute the solution to a concentration of 100 mL. -The melting temperatures of these samples were measured using a DSC instrument. The samples were heated from 10°C to The temperature was increased to 95°C or 100°C at a rate of 1°C per minute. To obtain a good understanding of the buffer-only properties, a buffer-only scan was performed between each sample scan. The signal was fitted to a two-state denaturation model after subtraction. The reversibility of thermal denaturation was determined by repeating the scan without activating the denaturation. It is calculated by comparing the area under the first scan curve with the area under the second scan curve. The results of the DSC experiments are shown in Table 5 as values ​​derived from the complete melting curves. The mutants Tencon17, Tencon18, Tencon19, and Tencon on22 has improved thermostability compared to the parent Tencon sequence. Combinatorial mutant samples Tencon23, Tenc on24, and Tencon25 all showed significantly greater stability improvements, with the designed mutations was shown to act additively on improving thermal stability.

[0127] Denaturation with Guanidine Hydrochloride Treatment with high concentrations of guanidine hydrochloride (GdmCl) as measured by tryptophan fluorescence The ability of Tencon and each mutant to maintain folding when Tencon contains only one tryptophan residue. The amino acid residues are embedded in the hydrophobic core, and therefore the fluorescence emission at 360 nm is This provides a sensitive measure of protein folding. For the 17-point titration, 50 mM phosphate was used. Sodium (pH 7.0), 150 mM NaCl, and variable pH from 0.48 to 6.63 M Add 200 µL of a solution containing 100 µL of GdmCl to a black, non-binding 96-well plate. The Tencon mutants were then pipetted into a 10 Add 1 μL of the solution to each well across the plate for a final protein concentration of 23 μM. The solution was added and mixed by gently pipetting up and down. The solution was incubated at room temperature for 24 hours. After incubation, the plates were read using a SpectraMax M5 plate reader (Molecule The fluorescent fluorescein was generated by Fluorescence Imaging (FIM) (Far Devices) with excitation at 280 nm and emission at 360 nm. The light was read. The data generated from such a curve is shown in FIG. 8. The following formula (Pa ce 1986 Methods Enzymol 131:266-80) The light signal was converted into percent unfolding.

[0128]

number

[0129] The midpoint of the transition to unfolding and the transition slope are calculated using the following formula (Clarke, Hamill et al., 1997).

[0130]

number

[0131]

number

[0132] It is often difficult to accurately measure the transition slope m of such a curve. The mutations described are not expected to alter the folding mechanism of Tencon. Therefore, we measured the m value for each mutant and averaged these values ​​(see Pace 198 above). 6), and m = 14.83 kJ / mol / M (354 The results of these calculations are shown in Table 5. The results of Cl-denaturation experiments show that the same mutants that stabilize Tencon with respect to thermal stability It has also been shown to stabilize proteins against GdmCl-induced denaturation.

[0133] Size Exclusion Chromatography Size exclusion chromatography (SEC) was used to identify WT Tencon and each mutant. The aggregation state of the variants was evaluated. 5 μL of each sample was diluted with PBS mobile phase and diluted with 0.3 mL / Superdex 75 5 / 150 column (GE Healthcare) at a flow rate of 1 min The elution from the column was monitored by absorbance at 280 nm. The aggregation state was evaluated. To determine the molecular weight of the globular samples, globular molecular weight standards (Sigma) were used. All samples tested except Tencon21 were pre-calibrated with -Eluted in a single peak with an elution volume that matched the elution volume of the sample. Tencon21 was eluted in two peaks, indicating the presence of aggregation.

[0134] [Table 5]

[0135] It will be apparent that the invention can be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings. Accordingly, they are intended to fall within the scope of the appended claims. The present invention includes the following embodiments. [1] A protein scaffold having an amino acid sequence based on the consensus amino acid sequence of SEQ ID NO: 16, wherein one or more specific residues of SEQ ID NO: 16 are substituted, such that the thermal and chemical stability of the protein scaffold is enhanced compared to the thermal and chemical stability of a protein scaffold having the amino acid sequence of SEQ ID NO: 16, and the substitutions are selected from the group consisting of N46V, E14P, L17A, E86I, a combination of N46V and E86I, a combination of E14P, N46V and E86I, and a combination of L17A, N46V and E86I. [2] The protein scaffold according to the above-mentioned [1], wherein the thermal stability is measured as melting temperature (Tm) by differential scanning calorimetry, and the chemical stability is measured by resistance to guanidinium chloride denaturation, and the Tm is increased by about 1 Kcal to about 12 Kcal compared to a protein scaffold having the amino acid sequence of SEQ ID NO: 16. [3] The protein scaffold according to the above-mentioned [1], comprising a loop region that contacts a target protein, the loop region being modified to include a mutation at one or more positions selected from the group consisting of residues 13, 15 and 16, 22 to 28, 38 to 43, 51 to 54, 60 to 64, and 75 to 81 of an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 142, 143, and 147 to 151. [4] The protein scaffold has a molecular weight of at least 10 as measured by surface plasmon resonance or Kinexa. -9 M or less, at least 10 -10 M or less, at least 10 -11 M or less and at least 10 -12 M or less, at least 10 -13 M or less, at least 10 -14 M or less and at least 10 -15 K who is M D The protein scaffold according to [3] above, which binds to a target protein with at least one affinity selected from the following: [5] A protein scaffold comprising an amino acid sequence selected from the group consisting of amino acid sequences selected from the group consisting of SEQ ID NOs: 142-144 and 147-151.

Claims

1. 1. A composition comprising a protein scaffold comprising an amino acid sequence based on the consensus amino acid sequence of SEQ ID NO: 16, said protein scaffold binding to human IgG1, wherein one or more specific residues in the amino acid sequence of SEQ ID NO: 16 are substituted, said substitutions in the amino acid sequence of SEQ ID NO: 16 being selected from the group consisting of N46V, E14P, L17A, E86I, a combination of N46V and E86I, a combination of E14P, N46V and E86I, and a combination of L17A, N46V and E86I, and wherein the B:C loop and F:G loop of said protein scaffold corresponding to the B:C loop at amino acids 22-28 and the F:G loop at amino acids 75-81 in the amino acid sequence of SEQ ID NO: 16 are, respectively: (a) the B:C loop of SEQ ID NO:21 and the F:G loop of SEQ ID NO:46 or 52; (b) the B:C loop of SEQ ID NO:22 and the F:G loop of SEQ ID NO:46; (c) the B:C loop of SEQ ID NO:23 and the F:G loop of SEQ ID NO:46; (d) the B:C loop of SEQ ID NO:24 and the F:G loop of SEQ ID NO:47; (e) the B:C loop of SEQ ID NO:25 and the F:G loop of SEQ ID NO:48; (f) the B:C loop of SEQ ID NO:26 and the F:G loop of SEQ ID NO:49; (g) the B:C loop of SEQ ID NO:27 and the F:G loop of SEQ ID NO:50; (h) the B:C loop of SEQ ID NO:28 and the F:G loop of SEQ ID NO:51; (i) the B:C loop of SEQ ID NO:29 and the F:G loop of SEQ ID NO:52; (j) the B:C loop of SEQ ID NO:30 and the F:G loop of SEQ ID NO:53; (k) the B:C loop of SEQ ID NO:31 and the F:G loop of SEQ ID NO:54; (l) the B:C loop of SEQ ID NO:32 and the F:G loop of SEQ ID NO:55; (m) the B:C loop of SEQ ID NO:33 and the F:G loop of SEQ ID NO:56; (n) the B:C loop of SEQ ID NO:34 and the F:G loop of SEQ ID NO:57; (o) a B:C loop of SEQ ID NO:35 and an F:G loop of SEQ ID NO:58; (p) a B:C loop of amino acids 22 to 28 in the amino acid sequence of SEQ ID NO:16 and an F:G loop selected from the group consisting of SEQ ID NOs:46 and 59 to 133; (q) the B:C loop of SEQ ID NO:36 and the F:G loop of SEQ ID NO:134; (r) the B:C loop of SEQ ID NO:37 and the F:G loop of SEQ ID NO:62; (s) a B:C loop of SEQ ID NO:38 and an F:G loop of SEQ ID NO:135; (t) the B:C loop of SEQ ID NO:39 and the F:G loop of SEQ ID NO:136; (u) the B:C loop of SEQ ID NO:40 and the F:G loop of SEQ ID NO:137; (v) the B:C loop of SEQ ID NO:41 and the F:G loop of SEQ ID NO:138; (w) the B:C loop of SEQ ID NO:42 and the F:G loop of SEQ ID NO:62 or 63; (x) the B:C loop of SEQ ID NO:43 and the F:G loop of SEQ ID NO:139; (y) the B:C loop of SEQ ID NO:44 and the F:G loop of SEQ ID NO:62; or (z) the B:C loop of SEQ ID NO:45 and the F:G loop of SEQ ID NO:140; The composition,

2. 10. The composition of claim 1, administered by a method of administration selected from parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intrachondral, intrasinus, intracavity, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, bolus, intravaginal, rectal, buccal, sublingual, intranasal, and transdermal.

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