KR102390931B1 - Novel polypeptide binding with human complement component 3a and usese thereof - Google Patents

Abstract

The present invention relates to a novel polypeptide that selectively binds to the complement proteolysis product C3a, and more particularly, the present invention relates to a polypeptide capable of inhibiting its activity by binding to the human complement proteolysis product C3a, the polypeptide encoding the polypeptide It relates to a polynucleotide, a recombinant vector comprising the polynucleotide, a recombinant microorganism into which the recombinant vector is introduced, a method for producing the polypeptide using the recombinant microorganism, and a therapeutic pharmaceutical composition comprising the polypeptide. The novel polypeptide binding to the complement proteolysis product C3a of the present invention binds to the complement proteolysis product C3a with a higher affinity than the naturally occurring complement proteolysis product C3a receptor and inhibits its activity, thereby being associated with the complement proteolysis product C3a. It is useful because it can be widely used in the development of agents for the prevention or treatment of various diseases.

Description

Novel polypeptide binding with human complement component 3a and uses thereof

The present invention relates to a novel polypeptide that selectively binds to the complement proteolysis product C3a, and more particularly, the present invention relates to a polypeptide capable of inhibiting its activity by binding to the human complement proteolysis product C3a, the polypeptide encoding the polypeptide It relates to a polynucleotide, a recombinant vector comprising the polynucleotide, a recombinant microorganism into which the recombinant vector is introduced, a method for producing the polypeptide using the recombinant microorganism, and a therapeutic pharmaceutical composition comprising the polypeptide.

Because of the low side effects and high therapeutic efficacy compared to chemical agents, global pharmaceutical companies and biotechnology companies have invested heavily in the development of antibody therapeutics, and many antibody therapeutics are currently being used in clinical trials, and many therapeutic candidates are in clinical trials.

However, since antibody therapeutics are expensive to produce, difficult to break through the existing patent barriers, and difficult to penetrate into cells due to their large molecular weight, the therapeutic effect of actual patients is not as high as expected. is proceeding. Unlike antibody therapeutics, these artificial antibody framework proteins have greatly improved the efficiency of penetration into cancer tissues, and as a result, the advantages of improving the therapeutic effect have been revealed through many studies.

Under this background, the present inventors have successfully developed a repebody, a non-antibody protein scaffold, that can replace an existing antibody. Repibody is 1/5 the size of an antibody, and it is mass-produced in E. coli, and has little immunogenicity as a result of animal experiments. It has been demonstrated that the thermal and pH stability is very good, the binding force to the target can be very easily increased to the pico-mole level, and the specificity to the target is very excellent.

Complement proteolysis product C3a is an important mediator between the innate immune system and the adaptive immune system and is known to be a trigger of inflammatory response-based immune-related diseases such as asthma, rheumatoid arthritis, and lupus. In particular, it is well known as a target material for the development of a treatment for sepsis and asthma, which is caused by an excessive inflammatory reaction in the body as invaders such as bacteria infect and reproduce in the blood. Sepsis is known as a major cause of morbidity and mortality in intensive care units worldwide, and 750,000 sepsis occur every year in the United States, and although there are no accurate statistics in Korea, the incidence rate is very high. The fatality rate is also very high, about 40%, but as the treatment that received FDA approval in 2001 was withdrawn from the market in 2011, there is still a shortage of effective treatments or therapeutic substances. Therefore, research on the development of therapeutic agents targeting the complement protein degradation product C5a as a therapeutic agent for diseases that result from excessive inflammatory reactions such as sepsis is steadily progressing. has not been

The present inventors have developed a polypeptide called Repebody that can specifically bind to various proteins (Korean Patent Nos. 10-1356075, 10-1654890, 10-1587622, 10-1624702) , No. 10-1624703, No. 10-1713944, No. 10-1751501, No. 10-1818152 and No. 10-1875809), specific binding to various disease-related target proteins using the repibody backbone We have successfully manufactured a protein (specific protein binder), verified that it has a biological inhibitory effect with a cell-based method, and is actively conducting additional research.

Under this background, the present inventors made diligent efforts to successfully secure a protein that specifically binds to the complement protein degradation product C3a, which is known to be deeply related to various immune diseases, using the repeat body skeleton. As a result, structural characteristics of the repeat body Based on the random mutation library constructed through phosphorus modularity and overall structural analysis, novel proteins with specific binding affinity to the complement protein degradation product C3a are selected, and binding power is improved through the repeat module-based affinity enhancement method. The present invention was completed by confirming that the repeati body with improved binding strength can inhibit the activity of C3a.

An object of the present invention is a repeat body (Repebody) that specifically binds to the complement protein degradation product C3a; a polynucleotide encoding the repeat body; a recombinant vector comprising the polynucleotide; a recombinant microorganism into which the recombinant vector is introduced; And to provide a method for producing the repeat body using the recombinant microorganism.

Another object of the present invention is to provide a double-binding repebody (bispecific repebody) in which a repeati body specifically binding to vascular endothelial growth factor (VEGF) is fused to the repeati body.

Another object of the present invention comprises administering a composition for preventing or treating an immune disease containing the repeati body or a double-bonded repeat-body and a composition for preventing or treating an immune disease containing the repeat-body or a double-bonded repeat-body, To provide a method for preventing or treating an immune disease.

In order to achieve the above object, the present invention provides an N-terminus of Leucine rich repeat (LRR) family proteins having an alpha helical capping motif and a modified repeat module of VLR (Variable Lymphocyte Receptor) protein and C of VLR protein - a polypeptide that selectively binds to the fused end of the complement proteolytic product C3a, i) threonine at position 88 is substituted with methionine; ii) replacement of isoleucine at position 91 with leucine; iii) threonine at position 93 is replaced with methionine; iv) glycine at position 94 is replaced with proline; v) serine 99 is replaced with isoleucine; vi) valine 116 is replaced with tryptophan; There is provided a polypeptide that selectively binds to complement proteolysis product C3a, characterized in that it contains one or more amino acid mutations selected from the group consisting of vii) glutamic acid at position 117 is substituted with histidine.

The present invention also provides a polynucleotide encoding the polypeptide and a recombinant vector comprising the polynucleotide.

The present invention also provides a recombinant microorganism containing the recombinant vector.

The present invention also provides a method for producing a polypeptide comprising the steps of culturing the recombinant microorganism to express the polypeptide, and recovering the polypeptide from the cultured recombinant microorganism or culture.

The present invention also provides a bispecific repebody in which a polypeptide that specifically binds to the complement protein degradation product C3a and a repeatibody that specifically binds to vascular endothelial growth factor (VEGF) are fused. .

The present invention also provides a composition for preventing or treating immune diseases containing the polypeptide or double-bonded repeater body as an active ingredient.

The novel polypeptide binding to the complement proteolysis product C3a of the present invention binds to the complement proteolysis product C3a with a higher affinity than the naturally occurring complement proteolysis product C3a receptor and inhibits its activity, thereby being associated with the complement proteolysis product C3a. It is useful because it can be widely used in the development of agents for the prevention or treatment of various diseases.

1 shows amino acid residues present in the LRRV2 module and the LRRV3 module, from which the first library was constructed to initially select a polypeptide for the complement proteolysis product C3a.
2 is a result of performing bio-panning of phage display on the complement protein degradation product C3a. The binding signal by enzyme immunoassay (ELISA) for the complement protein degradation product C3a compared to BSA was averaged (Normalization), and at this time, it was defined as a repeat body clone having a specific binding affinity to a clone in which the signal increased by 8 times or more.
3 is a result of measuring the dissociation constant using an isothermal titration calorimetry (ITC) for the clone H12 obtained in FIG. Confirmed.
FIG. 4 shows amino acid residues present in the LRRV4 module in which a second library was constructed based on H12, a clone that was primarily selected to increase binding affinity.
5 is a result of bio-panning of phage display using the second library. The binding signal by enzymatic immunoassay (ELISA) for the complement protein degradation product C3a compared to BSA was averaged (Normalization), and at this time, the clone with a 10-fold or more increase in signal was defined as a repeater body clone with increased binding affinity.
6 is a result of measuring the dissociation constant using an isothermal calorimeter for F6, a clone showing the highest binding affinity among the clones obtained in FIG. 5, showing that it has a binding affinity of 150 nM to the complement protein degradation product C3a Confirmed. This is about a 4-fold increase in binding force compared to the H12 clone before the binding force increase.
7 shows amino acid residues present in the LRR1 and LRRV1 modules in which a third library was constructed based on F5, a clone that was secondarily selected to increase binding affinity.
8 is a result of bio-panning of phage display using the third library. The binding signal by enzymatic immunoassay (ELISA) for BSA compared to the complement protein degradation product C3a was averaged (Normalization), and at this time, the clone with the signal increased by more than 12 times was defined as a repeater body clone with increased binding affinity.
9 is a result of measuring the dissociation constant using an isothermal calorimeter for clones having increased binding strength than F6 among the clones obtained in FIG. 8 . Clone E10 had the highest binding affinity to the complement proteolytic product C3a at about 0.6 nM, which was about 1,000-fold increased compared to the H12 clone before the binding strength was increased.
10 is a view showing the results of SDS-PAGE expression and purification of the final selected E10 having the highest binding force in E. coli.
11 shows whether the Lipibody E10 selected in the present invention specifically binds to the complement protein degradation product C3a, which was measured by enzyme immunoassay (ELISA) using phage.
12 is a diagram illustrating interleukin-6 and tumor necrosis factor-alpha, which are used as indicators of pro-inflammatory responses, when E10 selected in the present invention is treated with monocytes isolated from human peripheral blood mononuclear cells. It is the result of confirming the degree of generation of factor-α).
13 is a structural schematic diagram of a repeater body that is double-bonded to complement protein degradation product C3a and vascular endothelial growth factor by fusing E10 selected in the present invention and a repeater body that binds to vascular endothelial growth factor (VEGF) of the prior patent.
14 is a structural schematic diagram of a repeater body that is double-bonded to complement protein C3a and complement protein C5a by fusing E10 selected in the present invention with a repeater body that binds to complement protein C5a of the prior patent.
Figure 15 is a view showing the result of confirming the double bond of the C3a-VEGF double bond repeater body prepared in the present invention through enzyme immunoassay (ELISA).
16 is a view showing the result of confirming the double bond of the C3a-C5a double bond repeater body prepared in the present invention through enzyme immunoassay (ELISA).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.

In the present invention, a polypeptide that specifically binds to the complement protein degradation product C3a was selected, and the effect of inhibiting the activity of the polypeptide was confirmed.

In the present invention, the selection of polypeptides that specifically bind to the polypeptides included in the library for refibody development through biopanning using the C3a phagemid was performed, and the binding ability thereof was improved. As a result, a polypeptide specific for C3a was prepared.

That is, in one embodiment of the present invention, in order to develop a novel polypeptide capable of specifically binding to C3a, the N-terminus of the Internalin B protein, Variable Lymphocyte Receptor (VLR) A library was constructed that randomly includes a repeat module of a polypeptide to which a Leucine-rich repeat (LRR) protein portion is fused ( FIG. 1 ). The polypeptide contained in the library is encoded by the polynucleotide sequence of SEQ ID NO: 1 or is 75%, preferably 85%, more preferably 90%, more preferably 95% of the polynucleotide sequence of SEQ ID NO: 1 It may be encoded by a polynucleotide sequence having more than one homology.

In addition, the library may be in the form of a phagemid including the polynucleotide. As used herein, the term “phagemid” refers to a circular polynucleotide molecule derived from a phage, a virus with Escherichia coli as a host, and includes sequences of proteins and surface proteins necessary for propagation and proliferation. Recombinant phagemids may be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation may be performed using enzymes generally known in the art. The phagemid may include a signal sequence or leader sequence for secretion in addition to expression control elements such as a promoter, operator, start codon, stop codon, and enhancer, and mainly fuse a desired protein with a phage surface protein to label the phage surface method can be used for The promoter of the phagemid is primarily inducible and may contain selectable markers for selecting host cells. For the purpose of the present invention, the phagemid includes MalEss, DsbAss or PelBss, which is a signal sequence or leader sequence for expressing and secreting a polynucleotide encoding a polypeptide constituting the library, and the expression of the recombinant protein on the surface of the phage. The sequence described in the present inventor's prior patent (No. 10-2012-0019927) comprising a histidine-tag for identification and a polynucleotide encoding a gp3 domain, which is a kind of surface protein of M13 phage for expression on the phage surface It may be the polynucleotide of No. 2, but is not particularly limited thereto.

As used herein, the term “complement protein degradation product C3a (complement component C3a)” refers to a small protein fragment of about 9 kDa formed when complement protein C3 is cleaved during complement activation. Complement protein degradation product C3a is involved in various inflammatory reactions, such as increasing vascular permeability by releasing histamine by destroying mast cells and basophil granules, and promoting phagocytosis by attracting leukocytes. In particular, excessive and existing C3a in the body causes an excessive immune response to induce an abnormal inflammatory response, and this phenomenon has been found in various diseases such as asthma, rheumatoid arthritis, sepsis, and the like.

The present inventors used a phage display method using a library containing the phagemid to select a novel polypeptide (SEQ ID NO: 3) in the form of a repeat body having excellent binding affinity to the complement protein degradation product C3a (Fig. 1, Fig. 2) ). However, the selected polypeptide has a lower level of binding to the complement protein degradation product C3a than the complement component 5a recpetor existing in nature (FIG. 3), so that the mutation in the selected polypeptide In addition, it was attempted to produce a mutant polypeptide with improved binding to the complement protein degradation product C3a. To this end, a second library in which four amino acid residues located in the LRRV4 module were mutated was constructed with respect to the selected SEQ ID NO: 3 (FIG. 4), and the complement protein degradation product using the phage display method using the second library A novel polypeptide (SEQ ID NO: 4) with increased binding affinity to C3a was secondarily selected (FIG. 5). For each selected clone, binding affinity to the complement protein degradation product C3a was measured using isothermal titration calorimetry (FIG. 6), of which the polypeptide of SEQ ID NO: 4 had binding affinity to the complement protein degradation product C3a It was confirmed that this clone was the highest, and the binding force was confirmed to be 150 nM.

However, since the polypeptide binding to the selected complement proteolysis product C3a has a lower binding affinity than the complement proteolysis product C3a receptor existing in nature, the biological efficacy is expected to be low. Accordingly, the present inventors have developed a total of six amino acid residues present in the LRR1 and LRRV1 modules based on SEQ ID NO: 4 in order to secure a repeat body that can have biological efficacy while further improving the binding ability with the complement protein degradation product C3a. A mutated fourth library was constructed (FIG. 7).

Using the phage display method using the third library, two different polypeptides (SEQ ID NO: 5, SEQ ID NO: 6) with increased binding affinity to the complement proteolytic product C3a were selected, and among them, 0.6 nM of the A novel polypeptide (SEQ ID NO: 5) in the form of a repeater body having high binding affinity was finally selected.

Accordingly, in one aspect, the present invention provides an N-terminal of a Leucine rich repeat (LRR) family protein having an alpha helical capping motif (capping mofit) and a modified repeat module of a VLR (Variable Lymphocyte Receptor) protein and a C- of the VLR protein A polypeptide that selectively binds to C3a, a product of complement proteolysis to which the terminus is fused, i) threonine at position 88 is substituted with methionine; ii) replacement of isoleucine at position 91 with leucine; iii) threonine at position 93 is replaced with methionine; iv) glycine at position 94 is replaced with proline; v) serine 99 is replaced with isoleucine; vi) valine 116 is replaced with tryptophan; It relates to a polypeptide that selectively binds to complement proteolysis product C3a, characterized in that it contains one or more amino acid mutations selected from the group consisting of vii) glutamic acid at position 117 is substituted with histidine.

In the present invention, the N-terminus of the LRR family protein having the alpha helical capping motif may be characterized as the N-terminus of the internalin protein, and the internalin protein is preferably internalin A , may be characterized as selected from the group consisting of internalin B, internalin C, internalin H, and internalin J, more preferably it may be characterized as internalin B protein.

As used herein, the term "internalin B protein" refers to a type of LRR family protein expressed in a Listeria strain, which is different from other LRR family proteins in which other hydrophobic cores are uniformly distributed throughout the molecule. It is known to be stably expressed in microorganisms because of the N-terminal structure. As for the N-terminus of these internalin proteins, the most important N-terminal site for the folding of the repeat module is derived from a microorganism and its shape includes a more stable structure including an alpha helix, so the LRR family proteins in the microorganism are It can be effectively used for stable expression.

As used herein, the term "N-terminus of an internalin protein" refers to the N-terminus of an internalin protein required for the soluble expression and folding of the protein, and includes an alpha helical capping motif and a repeating module of the internalin protein. it means. The N-terminus of the internalin protein includes, but is not limited to, the N-terminus of the internalin protein necessary for the soluble expression and folding of the protein, examples of which include an alpha helical capping motif "ETITVSTPIKQIFPDDAFAETIKANLKKKSVTDAVTQNE" (SEQ ID NO: 11) and a repeat module. can do. Preferably the repeating module pattern may comprise "LxxLxxLxLxxN" (SEQ ID NO: 12). In the repeating module pattern, L is alanine, glycine, phenylalanine, tyrosine, leucine, isoleucine, valine or tryptophan; N is asparagine, glutamine, serine, cysteine or threonine, and x is a hydrophilic amino acid. For the N-terminus of the internalin protein, the N-terminus with high structural similarity can be selected depending on the type of LRR family protein to which it can be fused, and the most stable amino acid is selected through calculation of binding energy, etc. Variation is possible

As used herein, the term "Variable Lymphocyte Receptor (VLR)" refers to a type of LRR family protein expressed in hagfish and lamprey eel. It can be usefully used as a backbone that can be bound. The polypeptide in which the N-terminus of the internalin B protein and the VLR protein are fused has higher water solubility and expression than the VLR protein to which the internalin B protein is not fused, so it can be used for the preparation of novel protein therapeutics based thereon. there is.

The term "LRR (Leucine rich repeat) protein" of the present invention refers to a protein consisting of a combination of modules in which leucine is repeated at a certain position, (i) has one or more LRR repeat modules, (ii) the LRR The repeat module consists of 20 to 30 amino acids, and (iii) the LRR repeat module has "LxxLxxLxLxxN" as a conserved pattern, where L is alanine, glycine, phenylalanine, tyrosine, leucine, isoleucine, valine and tryptophan. Hydrophobic amino acids, such as N, asparagine, glutamine, serine, cysteine or threonine, x means all kinds of 20 amino acids, (iv) LRR family proteins have a three-dimensional structure like a horseshoe protein that has The LRR family protein of the present invention is not only found using mRNA or cDNA whose sequence is already known or newly derived in vivo, but also has a sequence unknown in nature through design such as consensus design and repeats It can include all variants with a framework structure of the module.

As used herein, the term “repebody” refers to a polypeptide optimized by a consensus design by fusion based on the similarity of the structure of the VLR with the N-terminus of the internalin having an LRR structure. The repeat body protein can be structurally divided into a concave region and a convex region (Convex). Here, the concave region has high sequence diversity and is known to be an important site for protein interaction. Conversely, the convex region plays a role in stably maintaining the overall structure of the protein based on highly conserved sequences. The repeat body protein may include all fusion LRR family proteins with improved water-soluble expression and biophysical properties of proteins by the above method for all proteins belonging to the LRR family having a repeat module.

In the present invention, the polypeptide may be characterized in that it is represented by any one of the amino acid sequence of SEQ ID NOs: 3 to 5.

In the present invention, the polypeptide may be characterized in that it binds to the complement protein degradation product C3a and inhibits its activity.

In another aspect, the present invention relates to a polynucleotide encoding the polypeptide.

In the present invention, the polynucleotide is not particularly limited thereto, but may be a polynucleotide encoding an amino acid sequence of any one of SEQ ID NOs: 3 to 5, and 70% or more with the polynucleotide, more preferably 80 % or more, more preferably, it may be a polynucleotide having a nucleotide sequence having a homology of 90% or more, but is not particularly limited thereto.

In another aspect, the present invention relates to a recombinant vector comprising the polynucleotide.

As used herein, the term "vector" refers to a DNA preparation containing the nucleotide sequence of a polynucleotide encoding the target protein operably linked to a suitable regulatory sequence so that the target protein can be expressed in a suitable host. Such regulatory sequences include promoters capable of initiating transcription, optional operator sequences for regulating such transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences regulating the termination of transcription and translation. After transformation into an appropriate host, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself.

The vector used in the present invention is not particularly limited as long as it can replicate in a host, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, phagemids, cosmids, viruses and bacteriophages in a natural or recombinant state. For example, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pBR-based, pUC-based, and pBluescriptII-based plasmid vectors can be used. , pGEM-based, pTZ-based, pCL-based and pET-based and the like can be used. The vector usable in the present invention is not particularly limited, and a known expression vector may be used. Preferably, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pET-21a, pET-32a vectors and the like may be used. Most preferably, pET-21a or pET-32a vectors may be used.

In another aspect, the present invention relates to a recombinant microorganism into which the polynucleotide or a recombinant vector comprising the polynucleotide is introduced.

As used herein, the term "recombinant microorganism" refers to a cell transfected with a vector having a gene encoding one or more target proteins introduced into a host cell to express the target protein, and all cells such as eukaryotic cells and prokaryotic cells It may be, but is not particularly limited thereto, bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium; yeast cells; fungal cells such as Pichia pastoris; insect cells such as Drosophila and Spodoptera Sf9 cells; animal cells such as CHO, COS, NSO, 293, and Bow melanoma cells; or plant cells. The host cells usable in the present invention are not particularly limited, but preferably Escherichia coli can be used as the host cell. Most preferably, Escherichia coli BL21 (DE3) and OrigamiB (DE3) can be used as host cells.

As used herein, the term “transformation” refers to introducing a vector including a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. Transformed polynucleotides include all of them regardless of whether they are inserted into the chromosome of the host cell or located extrachromosomally, as long as they can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding a target protein. The polynucleotide may be introduced into a host cell and expressed in any form, as long as it can be expressed. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression. The expression cassette usually includes a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence necessary for expression in the host cell.

In another aspect, the present invention comprises the steps of (i) culturing the recombinant microorganism to express the polypeptide; And (ii) relates to a method for producing a polypeptide comprising the step of recovering the polypeptide from the cultured recombinant microorganism or culture.

In the present invention, the step of culturing the recombinant microorganism is not particularly limited thereto, but is preferably performed by a known batch culture method, a continuous culture method, a fed-batch culture method, etc., and the culture conditions are not particularly limited thereto, A suitable pH (pH 5-9, preferably pH 6-8, most preferably pH 6.8) is adjusted using a basic compound (eg sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (eg phosphoric acid or sulfuric acid). aerobic conditions can be maintained by introducing oxygen or oxygen-containing gas mixture into the culture, and the culture temperature can be maintained at 20 to 45 °C, preferably 25 to 40 °C, and for about 10 to 160 hours Incubation is preferred. The polypeptide produced by the culturing may be secreted into the medium or may remain in the cell.

In addition, the culture medium used includes sugars and carbohydrates (eg, glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), oils and fats (eg, soybean oil, sunflower seed) as carbon sources. oil, peanut oil and coconut oil), fatty acids (such as palmitic acid, stearic acid and linoleic acid), alcohols (such as glycerol and ethanol), and organic acids (such as acetic acid) can be used individually or in combination; ; Nitrogen sources include nitrogen-containing organic compounds such as peptone, yeast extract, broth, malt extract, corn steep liquor, soy meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate) may be used individually or in combination; As the phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium-containing salts corresponding thereto, etc. may be used individually or in combination; Other metal salts (eg magnesium sulfate or iron sulfate), amino acids and vitamins may contain essential growth-promoting substances.

The method for recovering the polypeptide produced in the culturing step of the present invention is a culture method, for example, a desired polypeptide from a culture medium using a suitable method known in the art according to a batch, continuous or fed-batch culture method. can be collected

In another aspect, the present invention relates to a double-binding repebody in which a polypeptide that selectively binds to the complement protein degradation product C3a and a repeater body that specifically binds to vascular endothelial growth factor (VEGF) are fused to a bispecific repebody. it's about

In the present invention, the double bond repeater body may be characterized in that it is represented by the amino acid sequence of SEQ ID NO: 7 or 8.

In another aspect, the present invention relates to a composition for preventing or treating immune diseases containing the polypeptide or double bond repeater body as an active ingredient.

In the present invention, the prevention or treatment of the immune disease may be characterized in that the polypeptide binds to the complement protein degradation product C3a.

As used herein, the term “immune disease” refers to a disease caused by an abnormality in the immune system. The immune disease in the present invention is any autoimmune disease, but preferably rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, Behcet's syndrome, psoriasis, cirrhosis, ankylosing spondylitis, multiple sclerosis, Guillain-Barré syndrome, leukemia, Graves' disease, Crohn's disease, piliac disease, Wegener's granulomatosis, muscular dystrophy, uveitis, Tachiyasu's arteritis, temporal arteritis, recurrent polychondritis, multiple sclerosis, Chuck-Strauss syndrome, Henoch-Schoonlein purpura, Goodpasture syndrome , type 1 diabetes, aphthous stomatitis, pemphigus, and may be characterized as selected from the group consisting of pancreatic colitis, but is not limited thereto.

In the present invention, the term "treatment" refers to the administration of a composition to inhibit or alleviate an immune disease or one or more symptoms resulting therefrom, as well as treatment of cancer that reverses the symptoms of the disease or preventing the progression of cancer . In the present invention, the term "prevention" refers to any action that suppresses or delays the onset of an immune disease by administration of the composition. In the present invention, the prevention or treatment of immune diseases is achieved by binding the polypeptide obtained in the present invention to the complement protein degradation product C3a, and significantly inhibits its activity by binding to the complement protein degradation product C3a to prevent or treat immune diseases will do

The composition for preventing or treating immune diseases containing the polypeptide or double bond repeater body of the present invention as an active ingredient may further include a pharmaceutically acceptable carrier, and may be formulated together with the carrier.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound. Examples of acceptable pharmaceutical carriers for compositions formulated as liquid solutions include sterile and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.

The composition for preventing or treating immune diseases comprising the polypeptide of the present invention and a pharmaceutically acceptable carrier is applicable to any formulation containing the same as an active ingredient, and may be prepared as an oral or parenteral formulation. The pharmaceutical formulation of the present invention is oral, rectal, nasal, topical (including buccal and sublingual), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous). and those suitable for administration (including intravenous) or forms suitable for administration by inhalation or insufflation.

Formulations for oral administration containing the composition of the present invention as an active ingredient include, for example, tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. can do. For formulation into dosage forms such as tablets and capsules, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, an excipient such as dicalcium phosphate, a disintegrant such as corn starch or sweet potato starch, Masene stearate Lubricating oil such as calcium, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax may be included, and in the case of capsule formulation, a liquid carrier such as fatty oil may be further contained in addition to the above-mentioned substances.

Formulations for parenteral administration containing the composition of the present invention as an active ingredient include injections such as subcutaneous injection, intravenous injection or intramuscular injection, suppository injection method, or an aerosol for inhalation through the respiratory tract. can be formulated as In order to formulate an injectable formulation, the composition of the present invention may be mixed with a stabilizer or buffer in water to prepare a solution or suspension, which may be formulated for unit administration in ampoules or vials. For injection into a suppository, it may be formulated as a composition for rectal administration such as a suppository or enema containing a conventional suppository base such as cocoa butter or other glycerides. When formulated for spraying, such as an aerosol, a propellant or the like may be blended with an additive so that the water-dispersed concentrate or wet powder is dispersed.

In another aspect, the present invention relates to a method for preventing or treating an immune disease, comprising administering a composition for preventing or treating an immune disease containing the polypeptide as an active ingredient.

As used herein, the term "administration" means introducing the pharmaceutical composition of the present invention to a patient by any suitable method. The route of administration of the composition of the present invention may be administered through various routes, either oral or parenteral, 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, intranasal, inhalation, intraocular or intradermal routes.

The treatment method of the present invention includes administering the composition for preventing or treating immune diseases of the present invention in a pharmaceutically effective amount. It is apparent to those skilled in the art that a suitable total daily amount can be determined by a treating physician within the scope of sound medical judgment. A specific therapeutically effective amount for a particular patient will depend on the type and extent of the response to be achieved, the specific composition, including whether other agents are used, if necessary, the patient's age, weight, general health, sex and diet, time of administration; It is preferable to apply differently depending on various factors including the route of administration and secretion rate of the composition, the duration of treatment, the drug used together with or concurrently with the specific composition, and similar factors well known in the pharmaceutical field. Therefore, it is preferable to determine the effective amount of the composition for preventing or treating cancer suitable for the purpose of the present invention in consideration of the foregoing.

In addition, the treatment method of the present invention is applicable to any animal that can develop a disease due to excessive activity of the complement proteolysis product C3a, and the animal is not only humans and primates, but also cattle, pigs, sheep, horses, dogs and cats. including livestock such as

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Construction of a protein structure-based repeat body library

Like LRR proteins in nature, repeat units with a conserved leucine sequence are continuously connected to maintain the overall protein structure and modularity and the curvature of the shape of the entire structure. region) and a convex region. A hypervariable region, like a complementarity determining region (CDR) of an antibody, is located in the concave region to mediate protein-protein interaction. In addition, the convex region plays an important role in maintaining the overall structure of the LRR based on its well-conserved sequence. A random library was designed in the following manner by analyzing the protein structure of these repeat bodies.

Specifically, a random mutation library was constructed by selecting six amino acid residues 91, 93, 94, 115, 117, and 118 located in the concave regions of two consecutive modules (LRRV2, LRRV3 module) located in the direction of the amine group terminal. was done (FIG. 1).

Then, a mutagenic primer for library construction was synthesized so that the selected amino acid was substituted with an NNK degenerate codon. The primer sequence is as follows.

SEQ ID NO: 13 M1: CTG CCG AAT GGC GTC TTT GAT AAA CTG ACG AAC CTG AAA GAA CTG NNK CTG NNK NNK AAT CAA CTG CAG TCT CTG CCG GAC

SEQ ID NO: 14 M2: CGT CAG TTT ATC AAA GAC GCC ATT CGG CAG GCT CTG CAG TTG GTT MNN MNN CAG MNN CAG ATA CGT CAG ATT GGT CAG TTC

Then, using the primers, an overlap polymerase chain reaction (overlap PCR) is performed on the two modules to obtain library DNA, and the phage of SEQ ID NO: 2 described in the present inventor's prior patent (Korea Patent No. 1,356,075) The final library phagemid was obtained by inserting it into the mid pBEL118M.

By introducing the obtained library into E. coli TG-1 by electroporation to obtain a transformant, a library having a diversity of 2.0x10 ⁸ level was constructed.

Example 2: Selection of polypeptides that specifically bind to C3a through a random phage library

Example 2-1: Selection of polypeptides binding to C3a through the panning process of the Lipibody library

A polypeptide capable of binding to the complement protein degradation product C3a was selected and purified using the library constructed in the prior patent. To select candidates capable of binding to the complement protein degradation product C3a, the complement protein degradation product C3a was added to an immuno-tube at a concentration of 50 μg/ml, and coated at 4° C. for 12 hours. The coated immune tube was washed three times with PBS, and blocked with a PBS solution (TPBSA) containing 1% BSA and 0.05% Tween 20 at 4° C. for 2 hours.

The purified phage was added to the coated immunotube at a concentration of 10 ¹² cfu/ml, and reacted at room temperature for 2 hours. After the reaction was completed, it was washed with PBS solution (TPBS) containing 0.05% Tween 20 5 times for a total of 2 minutes and twice with PBS. Finally, 1 ml 0.2 M Glycine-HCl (pH 2.2) was added to the immunotube and reacted at room temperature for 13 minutes to elute the phage expressing a candidate Lipibody capable of binding to the complement proteolytic product C3a on the surface. .

The eluate was neutralized by adding 60 μl of 1.0 M Tris-HCl (pH 9.0), and added to 9 ml E. coli XL1-Blue solution (OD 600 = 0.6) as a host cell, followed by biopanning (Bio-) plated on a 2xYT plate. panning) was repeated 4 times in the same manner. As a result, it was confirmed that phages specifically binding to the complement protein degradation product C3a were concentrated through each panning process. The above result was analyzed to mean that the library phage binding to the complement protein degradation product C3a specifically increased.

Example 2-2: Confirmation and sequence analysis of specific binding to complement protein degradation product C3a of the selected Repibody

The absorbance (OD 450) of the complement protein degradation product C3a compared to BSA was 8 by performing ELISA for the phage selected through the method of Example 2-1 using a 96-well plate coated with complement protein degradation product C3a and BSA. Candidates for repeat bodies more than twice as high were selected (FIG. 2), and after confirming their respective amino acid sequences, clones having the same amino acid sequence were excluded. As a result, a total of four types of repeat body sequences were identified in the selected phage, and as a result of measuring the dissociation constant using an isothermal calorimeter, clone H12 was found to bind to the complement protein degradation product C3a with a binding force of about 637 nM. was confirmed (FIG. 3). In H12, isoleucine at amino acid 91 is substituted with leucine, threonine at amino acid 93 is substituted with methionine, glycine at amino acid 94 is substituted with proline, valine is maintained at amino acid 114 with valine, and amino acid 116 is substituted for H12. It was confirmed that valine was substituted with tryptophan, and glutamic acid of amino acid 117 was substituted with histidine (SEQ ID NO: 3). Additionally, it was confirmed that threonine at position 88, which did not construct a library, was substituted with methionine, and serine at position 99 was substituted with isoleucine.

The above results were analyzed to mean that there is a residue that plays an important role in binding to the complement proteolytic product C3a.

Example 3: Performing an increase in binding force to C3a of the repeat body using a module-based method

The module-based affinity increase method described in the present invention was performed according to a prior patent (KR2012-0019927). This method is a technique that can be used universally for proteins having repeat modules, and has been successfully reproduced in this patent, enabling the design of proteins with a high level of affinity.

Example 3-1: Confirmation of additional library construction using a module and increase in binding force and confirmation of cross-linking to other proteins

The dissociation constant for the complement protein degradation product C3a of H12, a clone confirmed to specifically bind to the complement protein degradation product C3a from the results of Example 2-2, is 637 nM (FIG. 2), but a naturally occurring complement protein The dissociation constant of the degradation product C3a receptor for the complement protein degradation product C3a is at the level of 1 nM. Therefore, it was expected that the repeat body candidate of the present invention could not sufficiently inhibit the activity of the complement protein degradation product C3a.

In order to solve this problem, an additional library construction method was used to develop a mutant with improved binding ability.

Specifically, the first library for module-based affinity enhancement mutated a total of four amino acid residues of the LRRV4 module (FIG. 4), and through a total of five panning processes, F6 (SEQ ID NO: 4) with increased binding strength was obtained ( 5), as a result of measuring the dissociation constant using an isothermal calorimeter, it was confirmed that it had a binding force of 150 nM to the complement protein degradation product C3a (FIG. 6). In F6, it was confirmed that tyrosine at position 137 was substituted with arginine, asparagine at position 139 with lysine, alanine at position 141 with asparagine, and histidine at position 142 with glycine.

On the other hand, in order to develop a mutant exhibiting more improved binding affinity, a total of six residues of the LRRV1 and LRR1 modules were mutated using F6 as a basic polypeptide (FIG. 7), and then through the same panning process, E10 (SEQ ID NO: 5) and C9 (SEQ ID NO: 6) were selected. In E10, isoleucine at position 47 was substituted with tyrosine, asparagine at position 49 with threonine, asparagine at position 50 with serine, and alanine at position 69 and glycine at positions 71 and 72 were maintained as they were. Additionally, asparagine at position 139 was substituted with glutamine and glycine at position 142 with asparagine. In C9, isoleucine at position 47 was substituted with leucine, asparagine at position 49 with leucine, asparagine at position 50 remained as is, alanine at position 69 and glycine at position 71 with valine, and glycine at position 72 with serine. Additionally, asparagine at position 139 was substituted with glutamine and glycine at position 142 with asparagine. As a result of measuring the dissociation constant using an isothermal calorimeter, it was confirmed that E10 had the highest binding force of 0.6 nM to the complement protein degradation product C3a (FIG. 9). This is a result of about 1,000-fold improvement in binding force compared to the initially selected H12.

Through these results, the present inventors have successfully secured a repeater body having a higher binding affinity than the receptor of the naturally occurring complement protein degradation product C3a, and it was confirmed that this is a polypeptide having a specific binding affinity to the complement protein degradation product C3a.

Example 4: Confirmation of cross-linking to other proteins of the selected Repibody

The naturally occurring complement proteolysis product C3a has a high sequence similarity of 43.6% to the complement proteolysis product C5a. Therefore, in order to confirm that the selected E10 repeat body specifically exhibits strong binding affinity only to C3a, an experiment was performed to check whether the cross-linking with the complement protein degradation product C5a (RnD Systems, USA) and BSA.

After each protein was coated on a 96-well plate, phages displaying E10 repeat bodies were added and the degree of binding was confirmed by enzyme-linked immunoprecipitation (ELISA). As a result, the selected E10 repeat bodies specifically bind only to C3a. was confirmed (FIG. 11).

Through these results, the present inventors successfully secured a repeat body having high binding affinity to the complement protein degradation product C3a, and confirmed that it is a polypeptide having a specific binding ability to the target protein, the complement protein degradation product C3a.

Example 5: Confirmation of the pro-inflammatory response inhibitory ability of the selected Lipibody

Complement proteolysis product C3a is associated with inflammatory responses and is known to induce pro-inflammatory responses, particularly related to the secretion of cytokines such as interleukin-6 and tumor necrosis factor-α. there is. Accordingly, in order to confirm the activity of the selected Lipibody that specifically binds to the complement protein degradation product C3a can inhibit the pro-inflammatory response, interleukin-6 and tumor necrosis factor-alpha (Tumor) from monocytes. An experiment was performed to measure the degree of production and secretion of necrosis factor-α).

In order to confirm the inhibition, the selected Lipibody was treated on monocytes isolated from peripheral blood mononuclear cells, and the production level of Interleukin-6 and Tumor necrosis factor-α before and after treatment. was analyzed through enzyme immunoprecipitation (ELISA), it was confirmed that the production of interleukin-6 (Interleukin-6) and tumor necrosis factor-alpha (Tumor necrosis factor-α) decreased as the Lipibody was treated. (Fig. 12).

Example 6: Confirmation of binding potential as a double bond complex

Example 6-1: Preparation of double bond complex

The repeat body E10 and GGGGS linker that specifically binds to the complement protein degradation product C3a developed in the present invention with the repeat body that specifically binds to vascular endothelial growth factor (VEGF) described in Korean Patent No. 10-1713944-0000 A p ET21a expression vector (Novagen, USA) containing the sequence fused to the complex was prepared. Two linkers were fused between the two repeat bodies (X2) and four fused (X4) were prepared, and they were expressed in E. coli, and C2-X2-E10 and C2-X4-E10 complexes (SEQ ID NO: 7; SEQ ID NO: 8) was prepared (FIG. 13).

In addition, in the same strategy, the GGGGS linker sequence was fused with the repeat body E10 that specifically binds to the complement protein degradation product C3a developed in the present invention with the repeat body binding to complement protein C5a described in Korean Patent No. 10-1624702-0000. A p ET21a expression vector (Novagen, USA) containing one complex was prepared. Two linkers were fused between the two repeat bodies (X2) and four fused (X4) were prepared, which were expressed in E. coli, and the E8-X2-E10 and E8-X4-E10 complexes (SEQ ID NO: 9; SEQ ID NO: 10) was prepared (FIG. 14).

Example 6-2: Analysis of the presence of double bonds in the double bond complex

In order to confirm the double binding, the complex was simultaneously exposed to the complement protein degradation product C3a and vascular endothelial growth factor (VEGF), and as a result of confirming the double binding by enzyme immunoprecipitation (ELISA), the complex was the complement protein degradation product C3a and vascular endothelial growth factor (VEGF) were confirmed to bind simultaneously (FIG. 15).

In order to confirm the double bond, the complex was simultaneously exposed to the complement protein degradation product C3a and the complement protein C5a, and double binding was confirmed by enzyme immunoprecipitation (ELISA). As a result, the complex was both the complement protein degradation product C3a and the complement protein C5a. It was confirmed that it binds at the same time to (FIG. 16).

As the specific parts of the present invention have been described in detail above, for those of ordinary skill in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

SEQUENCE LISTING <110> Korea Advanced Institute of Science and Technology <120> Novel polypeptide binding with human complement component 3a and use thereof <130> P19-B341 <160> 14 <170> PatentIn version 3.2 <210> 1 <211> 798 <212> DNA <213> Repebody-Library <220> <221> misc_feature <222> (139)..(140) <223> n is a, c, g, or t <220> <221> misc_feature <222 > (145)..(146) <223> n is a, c, g, or t <220> <221> misc_feature <222> (148)..(149) <223> n is a, c, g , or t <220> <221> misc_feature <222> (205)..(206) <223> n is a, c, g, or t <220> <221> misc_feature <222> (211)..( 212) <223> n is a, c, g, or t <220> <221> misc_feature <222> (214)..(215) <223> n is a, c, g, or t <400> 1 gaaaccatta ccgtgagcac cccgatcaaa cagatttttc cggatgacgc gttcgccgaa 60 acgatcaaag caaacctgaa gaaaaagagc gttaccgatg ctgtcacgca aaatgaactg 120 aacagtattg accagatcnn kgcgnnknnk tccgatatca aatcagtgca aggcattcag 180 tatctgccga atgttcgtta cctgnnkctg nnknnkaaca aactgcatga catctcggca 240 ctgaaagaac tgaccaatct gacgtatctg attctgaccg gtaaccaact gcagagcctg 300 ccgaatggcg tctttgataa actgacgaac ctgaaagaac tggtgctggt tgaaaatcaa 360 ctgcagtctc tgccggacgg tgtcttcgat aaactgacca acctgacgta cctgaatctg 420 gctcacaacc aactgcagag tctgccgaaa ggcgtgtttg acaaactgac caatctgacg 480 gaactggatc tgtcctataa ccaactgcag tcactgccgg aaggtgtttt cgacaaactg 540 acccagctga aagatctgcg cctgtaccag aatcagctga aatcggtccc ggacggcgtg 600 tttgatcgtc tgaccagcct gcagtatatc tggctgcatg ataacccgtg ggattgcacc 660 tgtccgggta ttcgctacct gtctgaatgg atcaataaac acagtggcgt tgtccgtaac 720 tccgcgggtt cagttgcccc ggattcggcg aaatgctccg gcagcggtaa accggtgcgt 780 agcattattt gcccgacc 798 <210> 2 <211> 266 ValThr PRT <213> Ser Thr Glu Phe Ip Lyp As 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> 3 <211> 266 <212> PRT <213> Repebody-H12 <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 Met Tyr Leu Leu Leu Met Pro Asn Gln 85 90 95 Leu Gln Ile Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Asn Leu Lys 100 105 110 Glu Leu Val Leu Trp His 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> 266 <212> PRT <213> Repebody-F6 <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 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 Met Tyr Leu Leu Leu Met Pro Asn Gln 85 90 95 Leu Gln Ile Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Asn Leu Lys 100 105 110 Glu Leu Val Leu Trp His Asn Gln Leu Gln Ser Leu Pro Asp Gly Val 115 120 125 Phe Asp Lys Leu Thr Asn Leu Thr Arg Leu Lys Leu Asn Gly 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> 5 <211> 266 <212> PRT <213> Repebody-E10 <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 Gln Ile Tyr Ala 35 40 45 Thr Ser 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 Met Tyr Leu Leu Leu Met Pro Asn Gln 85 90 95 Leu Gln Ile Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Asn Leu Lys 100 105 110 Glu Leu Val Leu Trp His Asn Gln Leu Gln Ser Leu Pro Asp Gly Val 115 120 125 Phe Asp Lys Leu Thr Asn Leu Thr Arg Leu Gln Leu Asn Asn 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> 6 <211> 266 <212> PRT <213> Repebody-C9 <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 Leu Ala 35 40 45 Leu Asn Ser Asp Ile Lys Ser Val Gln Gly Ile Gln Tyr Leu Pro Asn 50 55 60 Val Arg Tyr Leu Val Leu Val Ser Asn Lys Leu His Asp Ile Ser Ala 65 70 75 80 Leu Lys Glu Leu Thr Asn Leu Met Tyr Leu Leu Leu Met Pro Asn Gln 85 90 95 Leu Gln Ile Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Asn Leu Lys 100 105 110 Glu Leu Val Leu Trp His Asn Gln Leu Gln Ser Leu Pro Asp Gly Val 115 120 125 Phe Asp Lys Leu Thr Asn Leu Thr Arg Leu Gln Leu Asn Asn 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> 7 <211> 542 <212> PRT <213> Repebody-C2-X2-E10 <400> 7 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 Tyr Gln Ser Asp Ile Lys Ser Val Gln Gly Ile Gln Tyr Leu Pro Asn 50 55 60 Val Arg Tyr Leu Ile Leu Leu Gln 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 Gly Gly Gly Gly Ser Gly 260 265 270 Gly Gly Gly Ser Glu Thr Ile Thr Val Ser Thr Pro Ile Lys Gln Ile 275 280 285 Phe Pro Asp Asp Ala Phe Ala Glu Thr Ile Lys Ala Asn Leu Lys Lys 290 295 300 Lys Ser Val Thr Asp Ala Val Thr Gln Asn Glu Leu Asn Ser Ile Asp 305 310 315 320 Gln Ile Tyr Ala Thr Ser Ser Asp Ile Lys Ser Val Gln Gly Ile Gln 325 330 335 Tyr Leu Pro Asn Val Arg Tyr Leu Ala Leu Gly Gly Asn Lys Leu His 340 345 350 Asp Ile Ser Ala Leu Lys Glu Leu Thr Asn Leu Met Tyr Leu Leu Leu 355 360 365 Met Pro Asn Gln Leu Gln Ile Leu Pro Asn Gly Val Phe Asp Lys Leu 370 375 380 Thr Asn Leu Lys Glu Leu Val Leu Trp His Asn Gln Leu Gln Ser Leu 385 390 395 400 Pro Asp Gly Val Phe Asp Lys Leu Thr Asn Leu Thr Arg Leu Gln Leu 405 410 415 Asn Asn Asn Gln Leu Gln Ser Leu Pro Lys Gly Val Phe Asp Lys Leu 420 425 430 Thr Asn Leu Thr Glu Leu Asp Leu Ser Tyr Asn Gln Leu Gln Ser Leu 435 440 445 Pro Glu Gly Val Phe Asp Lys Leu Thr Gln Leu Lys Asp Leu Arg Leu 450 455 460 Tyr Gln Asn Gln Leu Lys Ser Val Pro Asp Gly Val Phe Asp Arg Leu 465 470 475 480 Thr Ser Leu Gln Tyr Ile Trp Leu His Asp Asn Pro Trp Asp Cys Thr 485 490 495 Cys Pro Gly Ile Arg Tyr Leu Ser Glu Trp Ile Asn Lys His Ser Gly 500 505 510 Val Val Arg Asn Ser Ala Gly Ser Val Ala Pro Asp Ser Ala Lys Cys 515 520 525 Ser Gly Ser Gly Lys Pro Val Arg Ser Ile Ile Cys Pro Thr 530 535 540 <210> 8 <211> 552 <212> PRT <213> Repebody- C2-X4-E10 <400> 8 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 Tyr Gln Ser Asp Ile Lys Ser Val Gln Gly Ile Gln Tyr Leu Pro Asn 50 55 60 Val Arg Tyr Leu Ile Leu Leu Gln 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 Gly Gly Gly Gly Ser Gly 260 265 270 Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Thr 275 280 285 Ile Thr Val Ser Thr Pro Ile Lys Gln Ile Phe Pro Asp Asp Ala Phe 290 295 300 Ala Glu Thr Ile Lys Ala Asn Leu Lys Lys Lys Ser Val Thr Asp Ala 305 310 315 320 Val Thr Gln Asn Glu Leu Asn Ser Ile Asp Gln Ile Tyr Ala Thr Ser 325 330 335 Ser Asp Ile Lys Ser Val Gln Gly Ile Gln Tyr Leu Pro Asn Val Arg 340 345 350 Tyr Leu Ala Leu Gly Gly Asn Lys Leu His Asp Ile Ser Ala Leu Lys 355 360 365 Glu Leu Thr Asn Leu Met Tyr Leu Leu Leu Met Pro Asn Gln Leu Gln 370 375 380 Ile Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Asn Leu Lys Glu Leu 385 390 395 400 Val Leu Trp His Asn Gln Leu Gln Ser Leu Pro Asp Gly Val Phe Asp 405 410 415 Lys Leu Thr Asn Leu Thr Arg Leu Gln Leu Asn Asn Asn Gln Leu Gln 420 425 430 Ser Leu Pro Lys Gly Val Phe Asp Lys Leu Thr Asn Leu Thr Glu Leu 435 440 445 Asp Leu Ser Tyr Asn Gln Leu Gln Ser Leu Pro Glu Gly Val Phe Asp 450 455 460 Lys Leu Thr Gln Leu Lys Asp Leu Arg Leu Tyr Gln Asn Gln Leu Lys 465 470 475 480 Ser Val Pro Asp Gly Val Phe Asp Arg Leu Thr Ser Leu Gln Tyr Ile 485 490 495 Trp Leu His Asp Asn Pro Trp Asp Cys Thr Cys Pro Gly Ile Arg Tyr 500 505 510 Leu Ser Glu Trp Ile Asn Lys His Ser Gly Val Val Arg Asn Ser Ala 515 520 525 Gly Ser Val Ala Pro Asp Ser Ala Lys Cys Ser Gly Ser Gly Lys Pro 530 535 540 Val Arg Ser Ile Ile Cys Pro Thr 545 550 <210> 9 <211> 537 <212> PRT <213> Repebody-E8-X2 -E10 <400> 9 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 Phe Leu Asp Phe Asn Gln 85 90 95 Leu Gln Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Asn Leu Lys Glu 100 105 110 Leu Tyr Leu Ser Pro Asn Gln Leu Gln Ser Leu Pro Asp Gly Val Phe 115 120 125 Asp Lys Leu Thr Asn Leu Thr Ile Leu Gly Leu Asp Met Asn Gln Leu 130 135 140 Gln Ser Leu Pro Lys Gly Val Phe Asp Lys Leu Thr Asn Leu Thr Glu 145 150 155 160 Leu Asp Leu Ser Tyr Asn Gln Leu Gln Ser Leu Pro Lys Gly Val Phe 165 170 175 Asp Lys Leu Thr Gln Leu Lys Asp Leu Ser Leu Ser Tyr Asn Gln Leu 180 185 190 Lys Ser Val Pro Asp Gly Val Phe Asp Arg Leu Thr Ser Leu Gln Tyr 195 200 205 Ile Trp Leu His Asp Asn Pro Trp Asp Cys Thr Cys Pro Gly Ile Arg 210 215 220 Tyr Leu Ser Glu Trp Ile Asn Lys His Ser Gly Val Val Gly Gly Pro 225 230 235 240 Asp Ser Ala Lys Cys Ser Gly Ser Gly Lys Pro Val Arg Ser Ile Ile 245 250 255 Cys Pro Thr Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Thr Ile 260 265 270 Thr Val Ser Thr Pro Ile Lys Gln Ile Phe Pro Asp Asp Ala Phe Ala 275 280 285 Glu Thr Ile Lys Ala Asn Leu Lys Lys Lys Ser Val Thr Asp Ala Val 290 295 300 Thr Gln Asn Glu Leu Asn Ser Ile Asp Gln Ile Tyr Ala Thr Ser Ser 305 310 315 320 Asp Ile Lys Ser Val Gln Gly Ile Gln Tyr Leu Pro Asn Val Arg Tyr 325 330 335 Leu Ala Leu Gly Gly Asn Lys Leu His Asp Ile Ser Ala Leu Lys Glu 340 345 350 Leu Thr Asn Leu Met Tyr Leu Leu Leu Leu Met Pro Asn Gln Leu Gln Ile 355 360 365 Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Asn Leu Lys Glu Leu Val 370 375 380 Leu Trp His Asn Gln Leu Gln Ser Leu Pro Asp Gly Val Phe Asp Lys 385 390 395 400 Leu Thr Asn Leu Thr Arg Leu Gln Leu Asn Asn Asn Gln Leu Gln Ser 405 410 415 Leu Pro Lys Gly Val Phe Asp Lys Leu Thr Asn Leu Thr Glu Leu Asp 420 425 430 Leu Ser Tyr Asn Gln Leu Gln Ser Leu Pro Glu Gly Val Phe Asp Lys 435 440 445 Leu Thr Gln Leu Lys Asp Leu Arg Leu Tyr Gln Asn Gln Leu Lys Ser 450 455 460 Val Pro Asp Gly Val Phe Asp Arg Leu Thr Ser Leu Gln Tyr Ile Trp 465 470 475 480 Leu His Asp Asn Pro Trp Asp Cys Thr Cys Pro Gly Ile Arg Tyr Leu 485 490 495 Ser Glu Trp Ile Asn Lys His Ser Gly Val Val Arg Asn Ser Ala Gly 500 505 510 Ser Val Ala Pro Asp Ser Ala Lys Cys Ser Gly Ser Gly Lys Pro Val 515 520 525 Arg Ser Ile Ile Cys Pro Thr Leu Glu 530 535 <210> 10 <211> 547 <212> PRT <213> Repebody-E8-X4-E10 < 400> 10 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 Phe Leu Asp Phe Asn Gln 85 90 95 Leu Gln Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Asn Leu Lys Glu 100 105 110 Leu Tyr Leu Ser Pro Asn Gln Leu Gln Ser Leu Pro Asp Gly Val Phe 115 120 125 Asp Lys Leu Thr Asn Leu Thr Ile Leu Gly Leu Asp Met Asn Gln Leu 130 135 140 Gln Ser Leu Pro Lys Gly Val Phe Asp Lys Leu Thr Asn Leu Thr Glu 145 150 155 160 Leu Asp Leu Ser Tyr Asn Gln Leu Gln Ser Leu Pro Lys Gly Val Phe 165 170 175 Asp Lys Leu Thr Gln Leu Lys Asp Leu Ser Leu Ser Tyr Asn Gln Leu 180 185 190 Lys Ser Val Pro Asp Gly Val Phe Asp Arg Leu Thr Ser Leu Gln Tyr 195 200 205 Ile Trp Leu His Asp Asn Pro Trp Asp Cys Thr Cys Pro Gly Ile Arg 210 215 220 Tyr Leu Ser Glu Trp Ile Asn Lys His Ser Gly Val Val Gly Gly Pro 225 230 235 240 Asp Ser Ala Lys Cys Ser Gly Ser Gly Lys Pro Val Arg Ser Ile Ile 245 250 255 Cys Pro Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 260 265 270 Gly Ser Gly Gly Gly Gly Gly Ser Glu Thr Ile Thr Val Ser Thr Pro Ile 275 280 285 Lys Gln Ile Phe Pro Asp Asp Ala Phe Ala Glu Thr Ile Lys Ala Asn 290 295 300 Leu Lys Lys Lys Lys Ser Val Thr Asp Ala Val Thr Gln Asn Glu Leu Asn 305 310 315 320 Ser Ile Asp Gln Ile Tyr Ala Thr Ser Ser Asp Ile Lys Ser Val Gln 325 330 335 Gly Ile Gln Tyr Leu Pro Asn Val Arg Tyr Leu Ala Leu Gly Gly Asn 340 345 350 Lys Leu His Asp Ile Ser Ala Leu Lys Glu Leu Thr Asn Leu Met Tyr 355 360 365 Leu Leu Leu Met Pro Asn Gln Leu Gln Ile Leu Pro Asn Gly Val Phe 370 375 380 Asp Lys Leu Thr Asn Leu Lys Glu Leu Val Leu Trp His Asn Gln Leu 385 390 395 400 Gln Ser Leu Pro Asp Gly Val Phe Asp Lys Leu Thr Asn Leu Thr Arg 405 410 415 Leu Gln Leu Asn Asn Asn Gln Leu Gln Ser Leu Pro Lys Gly Val Phe 420 425 430 Asp Lys Leu Thr Asn Leu Thr Glu Leu Asp Leu Ser Tyr Asn Gln Leu 435 440 445 Gln Ser Leu Pro Glu Gly Val Phe Asp Lys Leu Thr Gln Leu Lys Asp 450 455 460 Leu Arg Leu Tyr Gln Asn Gln Leu Lys Ser Val Pro Asp Gly Val Phe 465 470 475 480 Asp Arg Leu Thr Ser Leu Gln Tyr Ile Trp Leu His Asp Asn Pro Trp 485 490 495 Asp Cys Thr Cys Pro Gly Ile Arg Tyr Leu Ser Glu Trp Ile Asn Lys 500 505 510 His Ser Gly Val Val Arg Asn Ser Ala Gly Ser Val Ala Pro Asp Ser 515 520 525 Ala Lys Cys Ser Gly Ser Gly Lys Pro Val Arg Ser Ile Ile Cys Pro 530 535 540 Thr Leu Glu 545 <210> 11 <211> 39 <212> PRT <213> capping motif <400> 11 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 35 <210> 12 <211> 12 <212> PRT <213> repeat module <220 > <221> misc_feature <222> (2)..(3) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (5)..(6) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (8)..(8) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (10).. (11) <223> Xaa can be any naturally occurring amino acid <400> 12 Leu Xaa Xaa Leu Xaa Xaa Leu Xaa Leu Xaa Xaa Asn 1 5 10 <210> 13 <211> 81 <212> DNA <213> M1 < 220> <221> misc_feature <222> (46)..(47) <223> n is a, c, g, or t <220> <221> misc_feature <222> (52)..(53) <223 > n is a, c, g, or t <220> <221> misc_feature <222> (55)..(56) <223> n is a, c, g, or t <400> 13 ctgccgaatg gcgtctttga taaactgacg aacctgaaag aactgnnkct gnnknnkaat 60 caactgcagt ctctgccgga c 81 <210> 14 <211> 81 <212> DNA <2 13> M2 <220> <221> misc_feature <222> (47)..(48) <223> n is a, c, g, or t <220> <221> misc_feature <222> (50)..( 51) <223> n is a, c, g, or t <220> <221> misc_feature <222> (56)..(57) <223> n is a, c, g, or t <400> 14 cgtcagttta tcaaagacgc cattcggcag gctctgcagt tggttmnnmn ncagmnncag 60atacgtcaga ttggtcagtt c 81

Claims (17)

A polypeptide represented by the amino acid sequence of SEQ ID NO: 5 and selectively binding to complement proteolytic product C3a.
delete delete delete delete delete The polypeptide according to claim 1, wherein the polypeptide binds to complement proteolysis product C3a and inhibits its activity.
A polynucleotide encoding the polypeptide of claim 1 .
A recombinant vector comprising the polynucleotide of claim 8 .
A recombinant microorganism into which the recombinant vector of claim 9 is introduced.
A method for producing a polypeptide that specifically binds to complement proteolytic product C3a, comprising the steps of:
(i) culturing the recombinant microorganism of claim 10 to express the polypeptide of claim 1; and
(ii) recovering the polypeptide of claim 1 from the cultured recombinant microorganism or culture.
Represented by the amino acid sequence of SEQ ID NO: 7 or 8, a double-binding repebody that specifically binds to complement protein degradation product C3a and vascular endothelial growth factor (VEGF).
delete A double bond repebody represented by the amino acid sequence of SEQ ID NO: 9 or 10 and specifically binding to complement proteolysis product C3a and complement protein C5a. delete delete delete

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