KR101255682B1 - Novel MD-2 binding polypeptide and the use thereof - Google Patents

Abstract

The present invention relates to novel polypeptides that specifically bind to Myeloid differentiation protein-2 (MD-2) proteins. More specifically, the polypeptide specifically binding to the MD-2 protein fused to the N-terminus of the internalin B protein, a modified repeat module of a variable lymphocyte receptor (VLR) protein and the C-terminus of the VLR protein, the poly Nucleic acid encoding a peptide, a vector comprising the nucleic acid, a host cell transformed with the vector, a method for producing the polypeptide by expressing the vector in the host cell and a composition for preventing or treating sepsis comprising the polypeptide It is about.

Description

Novel MD-2 binding polypeptide and the use thereof

The present invention relates to novel polypeptides that specifically bind to Myeloid differentiation protein-2 (MD-2) proteins. More specifically, the polypeptide specifically binding to the MD-2 protein fused to the N-terminus of the internalin B protein, a modified repeat module of a variable lymphocyte receptor (VLR) protein and the C-terminus of the VLR protein, the poly Nucleic acid encoding a peptide, a vector comprising the nucleic acid, a host cell transformed with the vector, a method for producing the polypeptide by expressing the vector in the host cell and a composition for preventing or treating sepsis comprising the polypeptide It is about.

Protein is a substance responsible for most of the in vivo regulation of living organisms, the technology to understand the nature of the protein itself or to study and regulate the interaction of the protein with other substances has become more important recently for the treatment of various diseases. In particular, a lot of efforts are being made to research and develop therapeutic agents to control signals transmitted into cells by enhancing or inhibiting protein interactions related to receptor proteins expressed on cell surfaces. Representative mechanisms through cell surface receptors are mostly mechanisms involving the action of hormones or immune substances, so immune-related protein therapeutics have been actively developed using them.

Sepsis is a disease in which various bacteria invade and multiply in the blood, causing poisoning by toxic substances produced or causing systemic infection. When the body is infected with Gram-negative bacteria, bacteria produce lipopolysaccharide (LPS), which binds to the body's Myeloid differentiation protein-2 (MD-2) protein. The conjugate again binds to TLR-4 (Toll-like receptor-4) on the surface of the immune cell and transmits the signal into the cell. The signal transmitted into the cell through the mechanism causes various immune responses through transcription factors such as NK-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) through various signaling pathways in the cell. This is a normal immune action, but when it is excessively instantaneously, death can result from systemic fever and organ damage in the body. Sepsis is known worldwide as a leading cause of morbidity and mortality in the ICU. In the United States, 750,000 sepsis patients occur each year. The mortality rate is as high as 40%, but there is only one FDA-approved treatment, and there are no effective treatments or substances yet. Efforts have been made to develop therapeutics that inhibit one of the various signaling systems through which bacterial LPS is delivered. Typically, LPS binds to MD-2 in a similar structure to LPS, but prevents subsequent signaling. Eritoran, an anti-septic drug, is being developed by Japanese pharmaceutical company Eisai.

To control protein interaction, there are two ways to mimic existing protein-protein bonds to competitively inhibit them, and to control them by binding to target proteins and changing their properties. As a substance used in the method, a protein therapeutic agent is more suitable than a small molecule compound in order to have fewer side effects and to specifically act on the protein. So far, most protein drugs have been concentrated on producing antibodies that bind to specific proteins to inhibit or enhance their function, accounting for about 50% of protein drugs, but the complexity of the antibody production process, limitation of binding sites, and patent entry Research into a new protein backbone to replace antibodies due to barriers, etc. is being expanded.

LRR (Leucine Rich Repeat) family protein is a term that encompasses proteins consisting of a combination of modules in which leucine is repeated at a certain position. Such LRR family proteins are N-terminus, various numbers of repeat modules, and C-terminus. This is a stable structure. LRR family proteins are designed as binding proteins for various protein targets by controlling the number of repeat modules, changing the shape of the C-terminus, increasing the binding area with other proteins, and creating new binding sites. It has the characteristic suitable for the following. Variable Lymphocyte Receptor (VLR) is a type of LRR family protein that has the ability to bind to a variety of proteins used in the immune function of the eel. However, the production method of the VLR has difficulty in securing proteins, such as extracting the VLR from the eel after injecting the antigenic material, or expressing it as an insoluble body in E. coli and using it after refolding.

Against this background, the present inventors have made intensive efforts to find a mass-producible protein that can be used for the treatment of sepsis. As a result, a novel protein that binds to MD-2 protein is prepared using an internalin-coupled VLR skeletal protein. , I confirmed the use that can alleviate sepsis to complete the present invention.

One object of the present invention is the Myeloid differentiation protein-2 (MD-2) protein, in which the N-terminus of the internalin B protein, the modified repeat module of the VLR (Variable Lymphocyte Receptor) protein, and the C-terminus of the VLR protein are fused. It is to provide a polypeptide that specifically binds to.

Still another object of the present invention is to provide a nucleic acid encoding the polypeptide, a vector comprising the nucleic acid, or a host cell transformed with the vector.

Still another object of the present invention is to provide a composition for preventing or treating sepsis comprising the polypeptide.

Still another object of the present invention is to provide a method for preventing or treating sepsis, which comprises administering the composition.

As one embodiment for achieving the above object, the present invention is an MD-2 (fused to the N-terminus of the internal B protein, a modified repeat module of VLR (Variable Lymphocyte Receptor) protein and the C-terminus of the VLR protein, Myeloid differentiation protein-2) relates to a polypeptide that specifically binds to a protein.

As used herein, the term “internaline B protein” is a type of LRR (Leucin-rich repeat) family protein expressed in Listeria strains and is different from LRR family proteins in which a hydrophobic core is uniformly distributed throughout the molecule. Because of its N-terminal structure, it is known to be stably expressed in microorganisms. The N-terminus of this internalin B protein is stably expressed in microorganisms because the N-terminus, which is most important for folding the repeat module, is not only derived from microorganisms but also has a more stable shape including an alpha helix. It is thought to be.

As used herein, the term "N-terminus of the Internal B protein" refers to the N-terminus of the Internalin B protein necessary for the water-soluble expression and folding of the protein, and the alpha helical capping motif and of the Internalin B protein. It means a repeat module. The N-terminus of the internalin B protein includes, without limitation, the N-terminus of the internalin B protein necessary for the soluble expression and folding of the protein, but may include, for example, the alpha helical capping motif "ETITVSTPIKQIFPDDAFAETIKANLKKKSVTDAVTQNE" and a repeat module. . Preferably, the repeating module pattern may include "LxxLxxLxLxxN". L in the repeating module pattern is alanine, glycine, phenylalanine, tyrosine, leucine, isoleucine, valine or tryptophan; N means asparagine, glutamine, serine, cysteine or threonine, x means hydrophilic amino acid. The N-terminus of the internalin B protein can be selected from the N-terminus having high structural similarity according to the type of variable lymphocyte receptor (VLR) .The most stable amino acid can be selected through calculation of binding energy and the like. Amino acid variations are possible.

In the present invention, the term "VLR (Variable Lymphocyte Receptor) protein" is a kind of LRR family protein expressed in black eel and seven eel, and is a protein that performs immune function in black eel and seven eel as a skeleton capable of binding to various antigenic substances. It is usefully attracting attention. Polypeptides fused with the N-terminus and VLR protein of the internalin B protein are more useful for the preparation of novel protein therapeutics based on the increased water solubility and expression than the VLR proteins to which the internalin B protein is not fused. According to one embodiment of the present invention it was confirmed that the expression amount is significantly increased to 60 mg / L, 20 times more than when the expression amount of the protein is not fused with the N- terminal of the internalin B protein (3 mg / L) ( See Example 2-2). The improvement of expression amount suggests that the economic efficiency can be greatly improved by using the polypeptide of the present invention. As such, the expression amount of the fusion polypeptide fused with the internalin B protein and the VLR protein is increased. It was originally developed by the inventor.

In the present invention, the term "modified repeat module of the VLR protein" refers to a module in which leucine composed of 24 amino acids of the VLR protein is repeated at a predetermined position, and with MD-2 of TV3 proteins originally known to interact with MD-2. An important TV3 amino acid, known to bind, is implanted into a repeat module of the corresponding VLR protein. The repeat module has "LxxLxxLxLxxN" as a conserved pattern, where L is hydrophobic amino acids such as alanine, glycine, phenylalanine, tyrosine, leucine, isoleucine, valine and tryptophan, N is asparagine, glutamine, serine, Cysteine or threonine, x means any amino acid. The LRR family proteins of the present invention are not only known by their known sequences or newly discovered in vivo using mRNA or cDNA, but also have a sequence not known to nature through consensus design and the like. It may include all variants with skeletal structure of the module.

In a preferred embodiment, the polypeptide of the present invention has the amino acid sequence of the repeat module according to the definition of the LRR family, but the modified repeat module of the VLR protein is stable by combining a repeat module of a sequence existing in nature or through consensus design. It is a combination of the repeating module of the sequence increased. Preferably, the consensus design includes maintaining most of the structurally important amino acids and variously designing amino acids of the mutation region. For the purposes of the present invention, the polypeptide is characterized in that it specifically binds to the MD-2 protein, preferably a polypeptide having SEQ ID NO: 2, 3, 4 or 5 (Figs. 7 to 10). For the purpose of the present invention, a polypeptide that retains the activity of specifically binding to MD-2 protein is also seen in a polypeptide in which one or more amino acid residues are substituted, deleted or added in any one of the above SEQ ID NOs: 2 to 5. It is obvious to enter the scope of the invention.

According to an embodiment of the present invention, the polypeptide having SEQ ID NO: 2 has a binding force of MD-2 (K _D ) of 4.15 x 10 ^-8 M (FIG. 4), and the polypeptide having SEQ ID NO: 3 is 6.21 x 10 ^-8 M As shown in FIG. 3, there was no difference compared to the binding force of TV3 known to bind with MD-2, 7.6 × 10 ⁻⁸ M, and SEQ ID Nos. 4 and 5 also added one repeat module MD- The result of maintaining the binding force with 2 was confirmed (Fig. 5). These results demonstrate that the novel polypeptides prepared in the present invention specifically bind MD-2.

In the present invention, the term "MD-2 (Myeloid differentiation protein-2) protein" is a glycoprotein having a receptor capable of binding to LPS (Lipopolysaccharide), the bound LPS TLR-4 (Toll-) on the surface of immune cells like receptor-4) means a protein that transmits a signal into a cell.

According to the exemplary embodiment of the present invention, even when the protein is designed to bind the same amino acid MD-2, it was confirmed that the result of being expressed in water soluble when using the repeat module including the consensus design (FIG. 1).

Preferably, the repeating module of the sequence having increased stability through consensus design is the repeating module of the following Formula 1.

[SEQ ID NO: 1]

[LTNLxxLxLxxNQLQSLPxGVFDK]

Wherein x may comprise any amino acid. In the case of the VLR repeating module, the consensus portion is fixed and a portion where various amino acids can be represented by x, and is preferably an amino acid that is important for binding to the MD-2 protein.

In a preferred embodiment, the number of repeating modules of the polypeptide can be used in a variety of ways, but naturally having 2 to 9 modules, but is not limited thereto. According to one embodiment of the present invention, even when the repeat module was increased, it was confirmed that there is an effect of increasing the safety of itself, while maintaining the binding force with the MD-2 (Fig. 5).

Preferably, the C-terminal portion of the polypeptide may be variously substituted, and in particular, the loop structure frequently used for binding to the target material may be variously adjusted according to its length.

As another aspect, the present invention relates to a nucleic acid encoding the polypeptide. For the purposes of the present invention, the nucleic acid has a homology of 75%, preferably 85%, more preferably 90%, even more preferably 95% or more with a nucleic acid encoding SEQ ID NO: 2, 3, 4 or 5 It may be a nucleic acid molecule encoding a protein having a polypeptide activity specifically binding to -2.

As another aspect, the invention relates to a vector comprising a nucleic acid sequence encoding the fusion polypeptide.

The term "vector" of the present invention refers to a gene construct, which is an expression vector capable of expressing a protein of interest in a suitable host cell, comprising an essential regulatory element operably linked to express a gene insert. Operative linkage with recombinant vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation uses enzymes commonly known in the art and the like. Vectors of the present invention may include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals, enhancers and may be prepared in various ways depending on the purpose. have. The promoter of the vector may be constitutive or inducible. In addition, the expression vector includes a selectable marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, includes the origin of replication. The vector may be self-replicating or integrated into the host DNA.

Preferably, the vector may further include a tag sequence for protein purification, and the tag sequence for protein purification is not limited thereto. For example, the histidine-tag sequence (His-tag), an influenza hemagglutidine protein, may be used. It may be a HA-tag and a FLAG-tag corresponding to the epitope from which it is derived, preferably a histidine-tag sequence.

The vector used in the present invention is not particularly limited as long as it is replicable in a host cell, and any vector known in the art may be used. Such as plasmids, viral vectors, phage particles, or simply potential genomic inserts. The vector can be transformed into a suitable host and then replicated or integrated into the host genome independent of the host genome.

In a preferred embodiment, the nucleic acid sequence may be codon optimized for the host cell to be well expressed in the host cell to be introduced.

The term "codon optimization" in the present invention refers to changing the codons in the coding region or gene of the nucleic acid molecule to reflect the typical codon utilization of the host organism without changing the polypeptide encoded by the DNA. Codon optimization can be performed by known techniques.

As another aspect, the present invention relates to a host cell transformed with the vector comprising the fusion polypeptide.

The term "transformation" or "introduction" in the present invention means that the DNA is introduced into the host so that the DNA is replicable as an extrachromosomal factor or by chromosomal integration. The method of transforming the vector of the present invention includes any method of introducing a nucleic acid into a cell and may be carried out by selecting a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and Lithium acetate-DMSO method and the like, but is not limited thereto.

As the host cell, it is preferable to use a host having high DNA introduction efficiency and a high expression efficiency of the introduced DNA, but all microorganisms including prokaryotic and eukaryotic may be used, and preferably, E. coli is not limited thereto. E. coli can be selected according to the type of LRR protein to be fused. For example, when the C-terminus of the LRR protein has a disulfide bond, the strain has been modified in the related enzyme of the E. coli genome to help its expression. It is efficient to select.

In another aspect, the present invention provides a method for producing a composition comprising the steps of (a) preparing the vector; (b) introducing the prepared vector into a host cell; And (c) relates to a method for producing a polypeptide that specifically binds to MD-2, comprising the step of recovering the polypeptide from the host cell. In one preferred embodiment, the method may further include (d) purifying the fusion polypeptide using a column.

As another aspect, the present invention relates to a composition for preventing or treating sepsis comprising the polypeptide.

As used herein, the term "septicemia" refers to a condition in which a serious inflammatory response occurs in the whole body by infection with a microorganism. Fever symptoms that rise above 38 degrees or hypothermia below 36 degrees, respiratory rate increases above 24 times per minute (empty breathing), heart rate above 90 beats per minute (tachycardia), and increases or significant decreases in white blood cell counts on blood tests If you have more than two symptoms, this is called systemic inflammatory response syndrome (SIRS). This systemic inflammatory response syndrome is called sepsis when it is caused by infection of microorganisms. Sepsis usually occurs when the body is infected with Gram-negative bacteria, and a combination of LPS and bacteria's MD-2 protein, which binds to the body, binds to TLR-4 on the surface of the immune cell. It is a disease that occurs when you become excessively instantaneously.

The term "treatment" in the present invention means not only inhibiting or alleviating sepsis or one or more symptoms caused therefrom, but also preventing the progression of sepsis or the treatment of sepsis that reverses the symptoms of the disease. The term "prevention" in the present invention means any action that inhibits sepsis or delays the onset by administration of the composition.

Preferably, the prevention or treatment of sepsis is achieved by combining the polypeptide with MD-2, preventing LPS from binding with MD-2 in the human body, thereby inhibiting LPS signaling and treating sepsis.

According to one embodiment of the present invention, it was confirmed that IVLRc-MD2 of SEQ ID NO: 3 significantly reduces the secretion amount of TNF-α by LPS signal inhibition to 50% level (FIG. 2). This demonstrates that polypeptides can treat sepsis by effectively binding MD-2 and inhibiting signal transduction of LPS.

Sepsis prevention or treatment composition comprising the polypeptide of the present invention may further comprise a pharmaceutically acceptable carrier, it may be formulated with a carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate an organism and does not inhibit the biological activity and properties of the administered compound. Examples of the pharmaceutical carrier which is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water suitable for the living body such as saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

Sepsis prevention or treatment composition comprising the polypeptide of the present invention and a pharmaceutically acceptable carrier is applicable to any formulation containing it as an active ingredient, it can be prepared in oral or parenteral formulations. The pharmaceutical formulations of the present invention may be administered orally, rectally, nasal, topical (including under the ball and tongue), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous And intravenous), or forms suitable for administration by inhalation or insufflation.

Examples of formulations for oral administration comprising the composition of the present invention as an active ingredient include tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs can do. For formulation into tablets and capsules, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, stearic acid masne It may include a lubricating oil such as calcium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and in the case of a capsule, it may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.

Formulations for parenteral administration comprising the composition of the present invention as an active ingredient, for injection, such as subcutaneous injection, intravenous injection or intramuscular injection, a suppository injection method or aerosol for spraying by inhalation through the respiratory system It can be formulated as. To formulate injectable formulations, the compositions of the present invention may be mixed in water with stabilizers or buffers to prepare solutions or suspensions, which may be formulated for unit administration of ampoules or vials. For infusion into suppositories, it may be formulated in a rectal composition such as suppositories or enemas, including conventional suppository bases such as cocoa butter or other glycerides. When formulated for spraying such as aerosols, a propellant or the like may be combined with the additives to disperse the dispersed dispersion or wet powder.

As another aspect, the present invention relates to a method for treating sepsis, comprising administering a composition for preventing or treating sepsis comprising the polypeptide.

As used herein, the term "administration" means introducing a pharmaceutical composition of the present invention to a patient in any suitable manner. The route of administration of the composition of the present invention may be administered via various routes orally or parenterally as long as it can reach the target tissue, and specifically, oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, It may be administered in a conventional manner via transdermal, nasal, inhaled, intraocular or intradermal routes.

The treatment method of the present invention includes administering a composition for preventing or treating sepsis of the present invention in a pharmaceutically effective amount. It will be apparent to those skilled in the art that the appropriate total daily dose may be determined by the practitioner within the scope of sound medical judgment. The specific therapeutically effective amount for a particular patient will depend upon a variety of factors, including the type and extent of the response to be achieved, the specific composition, including whether or not other agents are used, the age, weight, general health status, sex and diet, The route of administration and the fraction of the composition, the duration of the treatment, the drugs used or co-used with the specific composition, and the like, well known in the medical arts. Therefore, the effective amount of the composition for preventing or treating sepsis suitable for the purpose of the present invention is preferably determined in consideration of the above-mentioned matters.

In addition, the method of treating sepsis of the present invention is applicable to any animal in which MD-2 and bacterial LPS are combined to cause an excessive immune response to cause sepsis due to systemic high fever and organ damage in the human body. Humans and primates, as well as domestic animals such as cattle, pigs, sheep, horses, dogs, and cats.

The novel polypeptide that binds to MD-2 of the present invention is prepared by a method for designing and preparing a sepsis therapeutic agent protein that binds to MD-2 using an internalin-fused VLR skeletal protein. By showing the possibility of treating sepsis in combination with -2, it is possible to secure proteins for the treatment of sepsis, while also demonstrating that it is a suitable framework for designing various binding proteins, which will contribute to further application research.

1 shows the amount of protein expression of a polypeptide comprising a VLR repeat module in nature and a polypeptide comprising a VLR repeat module comprising a consensus sequence. In the case of including 4 repeat modules in nature and 4 repeat modules including consensus sequences, the consensus-based results in the expression of a greater amount of protein. Lane 1 contains a standard marker, lane 2 contains a repeat module based on natural sequences (IVLRn-MD2, SEQ ID NO: 2), lane 3 contains a repeat module of consensus deception (IVLRc). -MD2, SEQ ID NO: 3).
Figure 2 shows that proteins made to bind MD-2 using IVLRc have the effect of preventing sepsis-related signaling from LPS and MD-2. It has proved to be similar to TV3, which is known to have a mitigating effect (the effect is that the protein must be mixed with MD-2 and reacted).
Figure 3 shows that the SLR method confirmed that the IVLRc-MD2 (SEQ ID NO: 3) made to respond to MD-2 in a repeat module using a consensus-based sequence has a binding capacity for MD-2.
Figure 4 shows that it was confirmed through the SPR method that IVLRn-MD2 (SEQ ID NO: 2) made to react to MD-2 as a repeat module using a sequence existing in nature is binding to MD2.
5 shows four repeat modules (IVLRn-MD2 and IVLRc-MD2, SEQ ID NO: 2 and SEQ ID NO: 3), and five (IVLRn5-MD2 and IVLRc5-MD2, SEQ ID NO: 4 and SEQ ID NO: 5) all MD- 2 and binding force. Both sequences present in nature and those designed from consensus sequences are shown to be binding. When mixed with MD-2, Lane 1: IVLRn-MD2, Lane 2: IVLRc-MD2, Lane 3: IVLRn5-MD2, Lane 4: IVLRc5-MD2 were confirmed to separate each protein into the form of MD-2. (Two bands). Lane 5: standard marker.
Figure 6 shows the IVLRn of SEQ ID NO: 1, wherein the repeat module derived from the N- terminal and VLR of the internalin B protein. Red is the internal part and black is the VLR repeat module. Blue represents the C-terminus.
Figure 7 shows the IVLRn-MD2 of SEQ ID NO: 2 to replace the amino acid of the repeating module to bind to MD-2 based on IVLRn. Red is the internal part and black is the VLR repeat module. Blue indicates the C-terminus and the underlined 12 amino acids indicate that the amino acid sequence has been substituted to bind MD-2.
Figure 8 shows the IVLRc-MD2 of SEQ ID NO: 3 to replace the amino acid of the repeat module to bind to MD-2 based on IVLRc. Red is the internal module and black is the consensus designed VLR's repeat module. Blue indicates the C-terminus and the underlined 12 amino acids indicate that the amino acid sequence has been substituted to bind MD-2.
Figure 9 shows the IVLRn5-MD2 of SEQ ID NO: 4 by adding the number of repeat modules based on IVLRn-MD2. Red is the internal part, purple is the VLR repeat module, and black is the VLR repeat module. Blue indicates the C-terminus and the underlined 24 amino acids indicate that the amino acid sequence has been substituted to bind MD-2.
Figure 10 shows the IVLRc5-MD2 of SEQ ID NO: 5 by adding the number of repeat module based on IVLRc-MD2. Red is the internal part, purple is the VLR repeat module, and black is the VLR repeat module. Blue indicates the C-terminus and the underlined 24 amino acids indicate that the amino acid sequence has been substituted to bind MD-2.
Figure 11 is a sequence that can be created when adding four repeat modules based on the consensus sequence, red is the internal portion, black is the repeat module, blue is the C-terminal part. The x portion of the repeat module can contain any amino acid sequence.

Hereinafter, the present invention will be described in more detail with reference to examples. These examples are merely for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples, and various applications and modifications are possible.

Example  One: Internal  Fused VLR  Using skeletal proteins MD Design of Amino Acids Binding to -2 Proteins and Measurement of Avidity

<1-1> Selection of an Internalin-fused VLR Skeletal Protein (IVLR) and Amino Acid Design to Simulate TV3-MD2 Interaction

The skeleton named IVLRn was used as a component. The skeleton is Listeria monocytogenes ) is a fusion of the N-terminal structure of the internalin B protein derived from the first repeating module and four repeating modules of the VLR protein in nature and the subsequent C-terminal structure having the configuration as shown in SEQ ID NO: ).

The TV3 protein is a fusion of a portion of the human TLR-4 receptor that originally interacts with MD-2 and a portion of the VLR protein introduced to enhance stability (Cell, 2007, 130, 906-917), which itself is a TV3 protein. And 7.6 x 10 ^-8 M are known to interact with each other. X-ray crystallography also reveals the three-dimensional structure. The amino acids that are important for the interaction were determined to be within 4.5 Å between the two proteins, and the corresponding TV3 amino acids were Q39, M41, E42, D60, S62, F63, D84, S86, R87, T110, V132. , V134, E135, N156, H159, S183, D209, S211, L212, W256, Q284 and the like.

The present inventors made the IVLRn-MD2 which can bind MD-2 by changing the amino acid type of TV3 which is important for the interaction determined previously to the corresponding amino acid position of IVLRn (SEQ ID NO: 2 and FIG. 7).

<1-2> Analysis of Expression of Design Protein and Adhesion with MD-2 in Escherichia Coli

DNA was synthesized based on the amino acid sequence (SEQ ID NO: 2) finally designed in Example <1-1>. The DNA sequence did not follow the DNA sequence of the existing Listeria or eel, and was newly synthesized according to the codon preference of E. coli. The synthesized DNA was cloned into a pET21a vector (Qiagen) that attaches six histidines to the C-terminal region of the protein for ease of expression and purification. The cloned vector was transformed into Origami B (Novagen) Escherichia coli to aid in the formation of disulfide bonds contained at the C-terminus of the VLR. E. coli transformed with the vector was grown to an absorbance (OD ₆₀₀ ) of 0.5, and then IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to a final concentration of 0.5 mM, and incubated at 18 ° C. for 20 hours for inter It was allowed to produce a naline-VLR fusion protein. After 20 hours, the cells were broken and compared with the amount of the internalin-VLR fusion protein produced in the cells. The small amount of original protein was purified through Ni-NTA resin using His-tag attached to the end of the protein, followed by concentration analysis.

MD-2 protein was produced and purified using a protein expression system using insect cells. The pAcGp67 vector was cloned together with MD-2 and Protein A gene for protein purification, and then expressed through an insect cell system using baculovirus. The protein expressed outside the Hi5 insect cell line was isolated and purified using lgG Sepharose affinity resin, and then obtained through the thrombin digestion to obtain MD-2 from which the protein A protein was cleaved. Further purification via gel filtration chromatography.

The binding force between the two proteins was measured by surface plasmon resonance (SPR) using the Biacore 3000 system. The produced IVLRn-MD2 was immobilized on a Biacore CM5 chip through an EDC / NHS reaction using sodium acetate buffer at pH4. The amount of the immobilized protein was used to have a value of 500 to 800 RU (resonance units). MD-2 at various concentrations was injected while flowing HBS-EP buffer to obtain sensorgram data (FIG. 4). The sensor data is then gram minus the value of the reference cell representing the non-specific binding, using Biacore Evaluation software affinity K _D Respectively.

As a result, it was confirmed that the binding force between IVLRn-MD2 and MD-2 was about 4.15 × 10 ⁻⁸ M, which was similar to the binding force between TV3 and MD-2 (FIG. 4).

Example 2 : Consensus  Added design Internal - VLR  Amino Acid Design and Adhesion Analysis of fusion Proteins with MD-2 as a Skeleton

<2-1> Internalin-VRL fusion protein with added consensus design and designed to bind MD-2

Consensus information can be obtained from amino acid frequency and substitution information of each sequence position from evolutionarily close sequences. Sequence positions that play an important role in the evolution of amino acids or interactions that are important for evolutionary structure and contribute to overall stability have high retention rates, while those that do not affect structural stability are relatively well substituted. have. In addition, the consensus information may help to limit the amino acid that can be substituted in the position based on the type and frequency of amino acids appearing at each position. Therefore, this evolutionary position-specific sequence information increases the possibility of designing various sequences while maintaining structural stability.

The advantage of VLR in this consensus sequence analysis is that the modularity of the repeat module is high because diversity is first obtained through recombination similar to antibodies. The phenylalanine spine portion of the VLR forms a hydrophobic core with four leucines around it, and this structure is expected to increase stability.

Selection of sequences for consensus design was done through BLAST for Uniprot DB, NCBI, and nr. In Uniprot DB, sequences were selected using the terms VLR and leucine, and the top 100 results obtained from BLAST using one protein sequence (PDB ID: 2O6Q) were used for NCBI and nr. For repeat module selection, a typical LRR motif was used to find a motif consisting of 24 amino acids (Typical LRR motif: LxxLxxLxLxxNxLxxLxxxxFxx).

Alignment scores were obtained using typical LRR motifs, and sequences with alignment scores greater than 39.0 were selected. Of the resulting sequences, 765 (Uniprot) and 500 (NCBI nr) were used. The logo plot was used to define the consensus, and the amino acid sequence was selected as the conservation part of the logo plot was used as it was, and the part showing the slight variation would help structural stability.

This result is shown below.

Consensus sequence

1) Uniprot DB

L TN L T x L x L xx NQL KSL P x G V FD N

2) NCBI nr

L TK L T x L x L xx NQL QS LP x G V FD K

Underlined: High Conservation Zone

Bold: Medium Conservative Area

x: transition region

For medium conserved regions (bolds), the following two selection criteria were applied to select the appropriate sequence; 1) frequency of amino acids appearing, 2) amino acids with no charge.

The reason for selecting an amino acid without charge is to prevent this because it uses a charged amino acid because the same charge will be in a similar position. Through this process, the sequence “ LTNLxxLxLxxNQLQSLPxGVFDK ” was confirmed as the sequence of the consensus module. Among these, the position of x is a position having evolutionarily diverse sequences, and the binding force and specificity with the target protein are thought to be controlled by the amino acid type of the position. In the consensus module of the 24 amino acids, x is any amino acid. As in Example 1, it was designed to have four VLR repeating modules, such as IVLRn, and was named IVLRc (SEQ ID NOs: 6 and 11).

In order to bind with MD-2, amino acids important for binding were designed as in the case of Example 1 by substituting the corresponding positions. The amino acid of the mutation region represented by x not involved in binding was selected as a natural VLR amino acid present at the position and named it IVLRc-MD2 (SEQ ID NOs: 3 and 8).

<2-2> Analysis of expression and binding ability of the designed protein

DNA was synthesized based on SEQ ID NO: 3 prepared in Example <2-1> and expressed in Escherichia coli in the same manner as in Example <1-2> to measure the binding force with MD-2 protein.

As a result, the binding force K _D value was 6.21 x 10 ^-8 M, which showed a similar degree of binding force as compared with TV3-MD2 binding and IVLRn-MD2. In addition, the expression level of the protein was 60 mg / L, which was significantly increased compared with the case of unfused with internalin (3 mg / L), and the stability (melting temperature (Tm) measured by circular dichroism) was increased at 70 ° C. IVLRc-MD2 was confirmed to increase to 83 ℃. These results suggest that the novel protein of the present invention can significantly increase the amount of expression compared to the existing protein, and at the same time, the consensus design can be applied to improve the properties of the protein even when used as a therapeutic agent in the future. .

Example 3 : IVLRc - MD2  On protein Intracellular LPS  Confirmation of Signaling Inhibition Effect and Confirmation of Sepsis Treatment Effect

<3-1> Production of IVLRc-MD2 Protein and Removal of Endotoxin

IVLRc-MD2 protein was basically produced and purified in the same manner as in Example <1-2>. To do this at the cellular or animal level, purely purified proteins are needed. However, unlike experiments on physicochemical properties, in biological cases, a large amount of endotoxins obtained during protein purification through Escherichia coli may cause problems. LPS, a kind of polysaccharide expressed on the surface of Gram-negative bacteria, is a substance that causes biological effects such as inflammatory reactions in very small amounts, and in order to prevent toxicity by removing endotoxins contained in E. coli in actual experiments. need. Inevitably, however, a large amount of LPS is obtained along with the protein at the time of protein purification. The LPS thus obtained causes an uninduced biological response, making it difficult to interpret meaningful experimental results. In order to overcome this advantage, the inventors have applied agarose resin (Sigma, Cat # p1441-10ML) to which a polymyxin-B is known to be effectively removed through specific binding with LPS in order to effectively remove LPS. After filling, the solution containing the LPS and the protein was passed through the column, and finally purified by eluting only the protein of the present inventors through PBS buffer. The initial protein purification was purified to a final 12.8 EU / ml (3.2 EU / mg protein) at an LPS content of> 30,000 EU / ml and used for subsequent experiments.

<3-2> Development of an Analysis System for MD-2 Inhibitory Activity at Cell Level and Confirmation of IVLRc-MD2 Activity

Experiments were performed to determine the effect of the InlB-VLR fusion protein of the present invention in combination with MD-2 to inhibit LPS signal in the form of InlB-VLR / MD-2 complex. The sTLR4 and MD-2 complexes, which consist of the soluble extracellular domain of the cell membrane receptor TLR4, have the ability to trap LPS, resulting in the inhibition of LPS signaling through the TLR4 receptor on the cell membrane. This is known to be a competitive inhibitory effect in which sTLR4 / MD-2 complexes capable of binding LPS compete with wtTLR4 / MD-2 complexes present in cell membranes (Mitsuzawa et al., J. Immunolo., 177: 8133-8139, 2006; Brandl et al., J. Endotoxin Res., 11: 197-206, 2005). Accordingly, the present inventors have the same mechanism as the sTLR4 / MD-2 complex that binds to LPS to inhibit signal transduction, and the following experiment was performed to confirm the effect of the InlB-VLR / MD-2 complex that competitively inhibits the LPS signal. Was performed.

First, mononuclear THP-1 (TIB-202TM, ATCC) cell line was incubated at 37 ° C. and 5% CO ₂ in RPMI medium containing 10% FBS (Fetal Bovine Serum) and 50 μM 2-mercaptoethanol. When the cell density reached 90%, the cells were washed with DPBS buffer (pH 7.4), and then suspended in a medium containing 200 nM of PMA (phorbol 12-myristate-13-acetate) to increase sensitivity to LPS. The well plates were dispensed at 2.5 × 10 ⁴ / well and fully differentiated at 37 ° C., 5% CO ₂ conditions for 72 hours.

Prior to the experiment, RPMI culture medium with 10 μg / mL of InlB-VLR and MD-2 added simultaneously, 10 μg / mL of MD-2 and 10 μg / mL of IVLR, 10 μg / mL of TV3, and Cultures to which 10 μg / ml of TV-3 and MD-2 were simultaneously added were allowed to stand at 37 ° C. for 1 hour. Thereafter, THP-1 differentiated into macrophages by PMA was washed three times with DPBS buffer (pH 7.4), and RPMI cultures containing samples were treated with THP-1 with 20 ng / ml LPS at 37 ° C. Incubated for 6 hours at 5% CO ₂ . After 6 hours, a certain amount of culture was taken from each well, diluted 4-8 times with DPBS buffer (pH 7.4), and analyzed for secreted TNF-α by ELISA (enzyme-linked immunosorbent assays).

The results are shown in FIG. As shown in FIG. 2, the InlB-VLR / MD-2 complex and the TV3 / MD-2 complex significantly reduced the amount of TNF-α secreted by LPS signaling to 50%. On the other hand, the use of InlB-VLR and TV3 alone had no LPS inhibitory effect, and the use of MD-2 alone showed a weak inhibitory effect (Mitsuzawa et al., J. Immunolo., 177). : 8133-8139, 2006). On the other hand, the inhibitory effect of LPS signal by the InlB-VLR / MD-2 complex and TV3 / MD-2 complex was found to be almost the same level, which is the same as that of sTLR4 or TV3 in combination with MD-2 to inhibit LPS signal. It was determined to be effective (Mitsuzawa et al., J. Immunolo., 177: 8133-8139, 2006; Jung et al., PLoS ONE, 4: e7403, 2009). These results demonstrate that the InlB-VLR fusion protein of the present invention binds to MD-2 and exhibits the effect of inhibiting LPS signals in the form of InlB-VLR / MD-2 complexes, which is a novel method of the present invention. It is suggested that MD-2 binding polypeptide can treat sepsis.

Example 4 : IVLRc - MD2  Confirmation of module addition and binding effect of protein

Although it was confirmed that the proteins prepared in Examples <1-2> and <2-2> have a binding ability to MD-2, the inventors of the present invention utilize the characteristics of the IVLR backbone to improve the binding ability or the properties of the protein. Protein was designed. First, increase the number of repeating modules by one, add amino acids that can bind to the added module, and replace the new amino acids IVLRn5-MD2 and IVLRc5 by substituting amino acids that can affect the binding of the fused internalin module. -MD2 was designed (SEQ ID NOs: 4 and 5, FIGS. 9 and 10). The designed protein was expressed and purified in the same manner as in Example <1-2> to measure the binding force and stability.

As a result, as shown in FIG. 5, pull-down experiments using gel permeation chromatography (GPC) confirmed that the new IVLR proteins and MD-2 protein were combined at the same time. This shows that the binding force with the target protein MD-2 is maintained while controlling the number of modules of the IVLR protein, suggesting the possibility of various applications of the skeletal protein.

Melting temperature of IVLRc5-MD2 measured by CD (Circular dichroism) was 84 ℃, which was not significantly different from 83 ℃ of IVLRc-MD2.In case of IVLRn5-MD2, it increased by 5 ℃ than IVLRn-MD2. It was shown in ℃. This is an improvement of the properties of the protein itself without adversely affecting the binding force, suggesting that the design and manufacture of a protein that is more practical and industrially possible using IVLR skeletal protein.

<110> Korea Advanced Institute of Science and Technology          Korea Research Institute of Bioscience and Biotechnology <120> Novel MD-2 binding polypeptide and the use <130> PA100500 / KR <160> 6 <170> Kopatentin 1.71 <210> 1 <211> 266 <212> PRT <213> Artificial Sequence <220> <223> IVLRn <400> 1 Glu Thr Ile Thr Val Ser Thr Pro Ile Lys Gln Ile Phe Pro Asp Asp   1 5 10 15 Ala Phe Ala Glu Thr Ile Lys Ala Asn Leu Lys Lys Lys Ser Val Thr              20 25 30 Asp Ala Val Thr Gln Asn Glu Leu Asn Ser Ile Asp Gln Ile Ile Ala          35 40 45 Asn Asn Ser Asp Ile Lys Ser Val Gln Gly Ile Gln Tyr Leu Pro Asn      50 55 60 Val Lys Tyr Leu Arg Leu Gly Gly Asn Asn Leu Arg Asp Ile Ser Ala  65 70 75 80 Leu Glu Lys Leu Thr Ser Leu Thr Tyr Leu Asn Leu Ser Thr Asn Gln                  85 90 95 Leu Gln Ser Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Gln Leu Lys             100 105 110 Glu Leu Ala Leu Asn Thr Asn Gln Leu Gln Ser Leu Pro Asp Gly Val         115 120 125 Phe Asp Lys Leu Thr Lys Leu Thr Tyr Leu Ser Leu Gly Tyr Asn Glu     130 135 140 Leu Gln Ser Leu Pro Lys Gly Val Phe Asp Lys Leu Thr Ser Leu Lys 145 150 155 160 Glu Leu Arg Leu Tyr Asn Asn Gln Leu Lys Arg Val Pro Glu Gly Ala                 165 170 175 Phe Asp Lys Leu Thr Gln Leu Lys Asp Leu Arg Leu Tyr Gln Asn Gln             180 185 190 Leu Lys Ser Val Pro Asp Gly Val Phe Asp Arg Leu Thr Ser Leu Gln         195 200 205 Tyr Ile Trp Leu His Asp Asn Pro Trp Asp Cys Thr Cys Pro Gly Ile     210 215 220 Arg Tyr Leu Ser Glu Trp Ile Asn Lys His Ser Gly Val Val Arg Asn 225 230 235 240 Ser Ala Gly Ser Val Ala Pro Asp Ser Ala Lys Cys Ser Gly Ser Gly                 245 250 255 Lys Pro Val Arg Ser Ile Ile Cys Pro Thr             260 265 <210> 2 <211> 266 <212> PRT <213> Artificial Sequence <220> IV223n-MD2 <400> 2 Glu Thr Ile Thr Val Ser Thr Pro Ile Lys Gln Ile Phe Pro Asp Asp   1 5 10 15 Ala Phe Ala Glu Thr Ile Lys Ala Asn Leu Lys Lys Lys Ser Val Thr              20 25 30 Asp Ala Val Thr Gln Asn Glu Leu Asn Ser Ile Asp Gln Ile Ile Ala          35 40 45 Asn Asn Ser Asp Ile Lys Ser Val Gln Gly Ile Gln Tyr Leu Pro Asn      50 55 60 Val Lys Tyr Leu Arg Leu Gly Gly Asn Asn Leu Arg Asp Ile Ser Ala  65 70 75 80 Leu Glu Lys Leu Thr Ser Leu Thr Tyr Leu Ile Leu Thr Gly Asn Gln                  85 90 95 Leu Gln Ser Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Gln Leu Lys             100 105 110 Glu Leu Val Leu Val Glu Asn Gln Leu Gln Ser Leu Pro Asp Gly Val         115 120 125 Phe Asp Lys Leu Thr Lys Leu Thr Tyr Leu Asn Leu Ala His Asn Glu     130 135 140 Leu Gln Ser Leu Pro Lys Gly Val Phe Asp Lys Leu Thr Ser Leu Lys 145 150 155 160 Glu Leu Asp Leu Ser Tyr Asn Gln Leu Lys Arg Val Pro Glu Gly Ala                 165 170 175 Phe Asp Lys Leu Thr Gln Leu Lys Asp Leu Arg Leu Tyr Gln Asn Gln             180 185 190 Leu Lys Ser Val Pro Asp Gly Val Phe Asp Arg Leu Thr Ser Leu Gln         195 200 205 Tyr Ile Trp Leu His Asp Asn Pro Trp Asp Cys Thr Cys Pro Gly Ile     210 215 220 Arg Tyr Leu Ser Glu Trp Ile Asn Lys His Ser Gly Val Val Arg Asn 225 230 235 240 Ser Ala Gly Ser Val Ala Pro Asp Ser Ala Lys Cys Ser Gly Ser Gly                 245 250 255 Lys Pro Val Arg Ser Ile Ile Cys Pro Thr             260 265 <210> 3 <211> 266 <212> PRT <213> Artificial Sequence <220> IV223c-MD2 <400> 3 Glu Thr Ile Thr Val Ser Thr Pro Ile Lys Gln Ile Phe Pro Asp Asp   1 5 10 15 Ala Phe Ala Glu Thr Ile Lys Ala Asn Leu Lys Lys Lys Ser Val Thr              20 25 30 Asp Ala Val Thr Gln Asn Glu Leu Asn Ser Ile Asp Gln Ile Ile Ala          35 40 45 Asn Asn Ser Asp Ile Lys Ser Val Gln Gly Ile Gln Tyr Leu Pro Asn      50 55 60 Val Arg Tyr Leu Ala Leu Gly Gly Asn Lys Leu His Asp Ile Ser Ala  65 70 75 80 Leu Lys Glu Leu Thr Asn Leu Thr Tyr Leu Ile Leu Thr Gly Asn Gln                  85 90 95 Leu Gln Ser Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Asn Leu Lys             100 105 110 Glu Leu Val Leu Val Glu Asn Gln Leu Gln Ser Leu Pro Asp Gly Val         115 120 125 Phe Asp Lys Leu Thr Asn Leu Thr Tyr Leu Asn Leu Ala His Asn Gln     130 135 140 Leu Gln Ser Leu Pro Lys Gly Val Phe Asp Lys Leu Thr Asn Leu Thr 145 150 155 160 Glu Leu Asp Leu Ser Tyr Asn Gln Leu Gln Ser Leu Pro Glu Gly Val                 165 170 175 Phe Asp Lys Leu Thr Gln Leu Lys Asp Leu Arg Leu Tyr Gln Asn Gln             180 185 190 Leu Lys Ser Val Pro Asp Gly Val Phe Asp Arg Leu Thr Ser Leu Gln         195 200 205 Tyr Ile Trp Leu His Asp Asn Pro Trp Asp Cys Thr Cys Pro Gly Ile     210 215 220 Arg Tyr Leu Ser Glu Trp Ile Asn Lys His Ser Gly Val Val Arg Asn 225 230 235 240 Ser Ala Gly Ser Val Ala Pro Asp Ser Ala Lys Cys Ser Gly Ser Gly                 245 250 255 Lys Pro Val Arg Ser Ile Ile Cys Pro Thr             260 265 <210> 4 <211> 290 <212> PRT <213> Artificial Sequence <220> <223> IVLRn5-MD2 <400> 4 Glu Thr Ile Thr Val Ser Thr Pro Ile Lys Gln Ile Phe Pro Asp Asp   1 5 10 15 Ala Phe Ala Glu Thr Ile Lys Ala Asn Leu Lys Lys Lys Ser Val Thr              20 25 30 Asp Ala Val Thr Gln Asn Glu Leu Asn Ser Ile Asp Thr Ile Gln Ala          35 40 45 Met Glu Ser Asp Ile Lys Ser Val Gln Gly Ile Gln Tyr Leu Pro Asn      50 55 60 Val Lys Asn Leu Asp Leu Ser Phe Asn Asn Leu Arg Asp Ile Ser Ala  65 70 75 80 Leu Glu Lys Leu Thr Asn Leu Thr Val Leu Asp Leu Ser Arg Asn Gln                  85 90 95 Leu Gln Ser Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Ser Leu Thr             100 105 110 Tyr Leu Ile Leu Thr Gly Asn Gln Leu Gln Ser Leu Pro Asn Gly Val         115 120 125 Phe Asp Lys Leu Thr Gln Leu Lys Glu Leu Val Leu Val Glu Asn Gln     130 135 140 Leu Gln Ser Leu Pro Asp Gly Val Phe Asp Lys Leu Thr Lys Leu Thr 145 150 155 160 Tyr Leu Asn Leu Ala His Asn Glu Leu Gln Ser Leu Pro Lys Gly Val                 165 170 175 Phe Asp Lys Leu Thr Ser Leu Lys Glu Leu Asp Leu Ser Tyr Asn Gln             180 185 190 Leu Lys Arg Val Pro Glu Gly Ala Phe Asp Lys Leu Thr Gln Leu Lys         195 200 205 Asp Leu Arg Leu Tyr Gln Asn Gln Leu Lys Ser Val Pro Asp Gly Val     210 215 220 Phe Asp Arg Leu Thr Ser Leu Gln Tyr Ile Trp Leu His Asp Asn Pro 225 230 235 240 Trp Asp Cys Thr Cys Pro Gly Ile Arg Tyr Leu Ser Glu Trp Ile Asn                 245 250 255 Lys His Ser Gly Val Val Arg Asn Ser Ala Gly Ser Val Ala Pro Asp             260 265 270 Ser Ala Lys Cys Ser Gly Ser Gly Lys Pro Val Arg Ser Ile Ile Cys         275 280 285 Pro Thr     290 <210> 5 <211> 290 <212> PRT <213> Artificial Sequence <220> <223> IVLRc5-MD2 <400> 5 Glu Thr Ile Thr Val Ser Thr Pro Ile Lys Gln Ile Phe Pro Asp Asp   1 5 10 15 Ala Phe Ala Glu Thr Ile Lys Ala Asn Leu Lys Lys Lys Ser Val Thr              20 25 30 Asp Ala Val Thr Gln Asn Glu Leu Asn Ser Ile Asp Thr Ile Gln Ala          35 40 45 Met Glu Ser Asp Ile Lys Ser Val Gln Gly Ile Gln Tyr Leu Pro Asn      50 55 60 Val Arg Asn Leu Asp Leu Ser Phe Asn Lys Leu His Asp Ile Ser Ala  65 70 75 80 Leu Lys Glu Leu Thr Asn Leu Thr Val Leu Asp Leu Ser Arg Asn Gln                  85 90 95 Leu Gln Ser Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Asn Leu Thr             100 105 110 Tyr Leu Ile Leu Thr Gly Asn Gln Leu Gln Ser Leu Pro Asn Gly Val         115 120 125 Phe Asp Lys Leu Thr Asn Leu Lys Glu Leu Val Leu Val Glu Asn Gln     130 135 140 Leu Gln Ser Leu Pro Asp Gly Val Phe Asp Lys Leu Thr Asn Leu Thr 145 150 155 160 Tyr Leu Asn Leu Ala His Asn Gln Leu Gln Ser Leu Pro Lys Gly Val                 165 170 175 Phe Asp Lys Leu Thr Asn Leu Thr Glu Leu Asp Leu Ser Tyr Asn Gln             180 185 190 Leu Gln Ser Leu Pro Glu Gly Val Phe Asp Lys Leu Thr Gln Leu Lys         195 200 205 Asp Leu Arg Leu Tyr Gln Asn Gln Leu Lys Ser Val Pro Asp Gly Val     210 215 220 Phe Asp Arg Leu Thr Ser Leu Gln Tyr Ile Trp Leu His Asp Asn Pro 225 230 235 240 Trp Asp Cys Thr Cys Pro Gly Ile Arg Tyr Leu Ser Glu Trp Ile Asn                 245 250 255 Lys His Ser Gly Val Val Arg Asn Ser Ala Gly Ser Val Ala Pro Asp             260 265 270 Ser Ala Lys Cys Ser Gly Ser Gly Lys Pro Val Arg Ser Ile Ile Cys         275 280 285 Pro Thr     290 <210> 6 <211> 266 <212> PRT <213> Artificial Sequence <220> <223> IVLRc <220> <221> VARIANT (222) (88) .. (166) <223> Xaa is any amino acid <400> 6 Glu Thr Ile Thr Val Ser Thr Pro Ile Lys Gln Ile Phe Pro Asp Asp   1 5 10 15 Ala Phe Ala Glu Thr Ile Lys Ala Asn Leu Lys Lys Lys Ser Val Thr              20 25 30 Asp Ala Val Thr Gln Asn Glu Leu Asn Ser Ile Asp Gln Ile Ile Ala          35 40 45 Asn Asn Ser Asp Ile Lys Ser Val Gln Gly Ile Gln Tyr Leu Pro Asn      50 55 60 Val Arg Tyr Leu Ala Leu Gly Gly Asn Lys Leu His Asp Ile Ser Ala  65 70 75 80 Leu Lys Glu Leu Thr Asn Leu Xaa Xaa Leu Xaa Leu Xaa Xaa Asn Gln                  85 90 95 Leu Gln Ser Leu Pro Xaa Gly Val Phe Asp Lys Leu Thr Asn Leu Xaa             100 105 110 Xaa Leu Xaa Leu Xaa Xaa Asn Gln Leu Gln Ser Leu Pro Xaa Gly Val         115 120 125 Phe Asp Lys Leu Thr Asn Leu Xaa Xaa Leu Xaa Leu Xaa Xaa Asn Gln     130 135 140 Leu Gln Ser Leu Pro Xaa Gly Val Phe Asp Lys Leu Thr Asn Leu Xaa 145 150 155 160 Xaa Leu Xaa Leu Xaa Xaa Asn Gln Leu Gln Ser Leu Pro Xaa Gly Val                 165 170 175 Phe Asp Lys Leu Thr Gln Leu Lys Asp Leu Arg Leu Tyr Gln Asn Gln             180 185 190 Leu Lys Ser Val Pro Asp Gly Val Phe Asp Arg Leu Thr Ser Leu Gln         195 200 205 Tyr Ile Trp Leu His Asp Asn Pro Trp Asp Cys Thr Cys Pro Gly Ile     210 215 220 Arg Tyr Leu Ser Glu Trp Ile Asn Lys His Ser Gly Val Val Arg Asn 225 230 235 240 Ser Ala Gly Ser Val Ala Pro Asp Ser Ala Lys Cys Ser Gly Ser Gly                 245 250 255 Lys Pro Val Arg Ser Ile Ile Cys Pro Thr             260 265

Claims (12)

delete A polypeptide that specifically binds to a Myeloid differentiation protein-2 (MD-2) protein, defined by the amino acid sequence set forth in SEQ ID NO: 2, 3, 4 or 5.
delete delete delete A nucleic acid encoding the polypeptide of claim 2.
A vector comprising the nucleic acid of claim 6.
A host cell transformed with the vector of claim 7.
(a) preparing the vector of claim 7;
(b) introducing the prepared vector into a host cell; And
(c) a method for producing a polypeptide that specifically binds to MD-2, comprising recovering the polypeptide from the host cell.
Sepsis prevention or treatment composition comprising the polypeptide of claim 2.
The composition of claim 10, wherein the prevention or treatment of sepsis is by combining the polypeptide with MD-2.
The composition of claim 10, wherein the composition further comprises a pharmaceutically acceptable carrier.

Images