CN111690066B

CN111690066B - anti-IL-4R alpha single-domain antibody and application and medicament thereof

Info

Publication number: CN111690066B
Application number: CN202010576200.7A
Authority: CN
Inventors: 苏志鹏; 张云; 孟巾果; 王乐飞
Original assignee: Nanjing Rongjiekang Biotechnology Co ltd
Current assignee: Nanjing Rongjiekang Biotechnology Co ltd
Priority date: 2020-06-22
Filing date: 2020-06-22
Publication date: 2022-07-22
Anticipated expiration: 2040-06-22
Also published as: CN117327181A; CN114957472B; CN111690066A; CN114957472A

Abstract

The invention discloses an anti-IL-4R alpha single domain antibody, application and a medicament, and relates to the technical field of antibodies. The amino acid sequence of the CDR1 of the single domain antibody disclosed by the invention is shown in any one of SEQ ID NO.37-53, the amino acid sequence of the CDR2 is shown in any one of SEQ ID NO.54-68, and the amino acid sequence of the CDR3 is shown in any one of SEQ ID NO. 69-85. The single domain antibody can specifically bind to IL-4R alpha, and can be used for the prevention, diagnosis and/or treatment of IL-4R alpha related diseases such as autoimmune diseases.

Description

anti-IL-4R alpha single-domain antibody and application and medicament thereof

Technical Field

The invention relates to the technical field of antibodies, in particular to a single domain antibody for resisting IL-4R alpha, application and a medicament.

Background

IL-4 and IL-4R alpha are ideal drug therapy targets, and are mainly used for treating human diseases related to the abnormality of type II helper T (Th2) cells. The human IL-4R alpha subunit (Swiss Prot database accession number: P24394) is a type I membrane protein with a molecular weight of 140kDa and binds human IL-4 with high affinity. The IL-4/IL-4R α complex can dimerize IL-4R α with the CD132 or IL-13R α 1 subunit via a domain on the IL-4 molecule, thereby forming two distinct signaling molecule complexes corresponding to class I and class II receptors, respectively. In addition, the complex formed upon binding of IL-13 to IL-13R α 1 recruits the IL-4R α subunit, and a class II receptor complex may also be formed. IL-4R α can therefore modulate the biological activities associated with both IL-4 and IL-13 molecules (Gessner et al, immunology, 201:285,2000). In vitro studies have shown that the activation effects of IL-4 and IL-13 have an effect on a variety of cells, including: t lymphocytes, B lymphocytes, eosinophils, giant cells, basophils, airway smooth muscle cells, lung fibroblasts and endothelial cells.

IL4R α is not expressed at high levels on different cell types and typically expresses 100-5000 molecules per cell (Lowenthal et al, J Immunol,140:456,1988), such as peripheral blood T cells, monocytes, airway epithelial cells, B cells and lung fibroblasts. Type I receptors have an advantageous expression on the surface of hematopoietic cells, whereas type II receptors are expressed on the surface of both hematopoietic and non-hematopoietic cells.

The polymorphism of IL-4R α in human populations has been discussed (Gessner et al, immunology, 201:285,2000) and has been shown to correlate with IgE levels or clinical atopy in some populations, e.g., V75R576 IL-4R α is associated with allergic asthma and enhanced IL-4R α function (Risma et al, J.Immunol.169(3)1604-1610, 2002).

There is a lot of evidence that the IL-4/IL-13 pathway plays an important role in asthma pathology (for review see Chatila, Trends in Molecular Med,10:493,2004) as well as a number of other disorders listed elsewhere herein. It is presently believed that increased secretion of IL-4 and IL-13 initiates and sustains this disease process. IL-13 is thought to be an important ligand for triggering Airway Hyperresponsiveness (AHR), mucus hypersecretion, and airway remodeling, while IL-4 is thought to be a major inducer of Th2 polarization and IgE production (Wynn, Annu Rev Immunol,21:425,2003).

Evidence in animal models further supports the role of IL-4R α in asthma. IL-4R antagonists (IL-4 mutants; C118 deletion) inhibited the development of allergic airway eosinophilia and AHR in functional mice in vivo, caused by OVA sensitization, during allergen stimulation with ovalbumin (OVA, a model allergen) (Tomkinson et al, J.Immunol.166(9): 5792-. In addition, several in vivo studies have demonstrated the positive effects of blocking IL-13 or IL-14 in animal models of asthma. For example, in a chronic model of OVA-induced persistent airway inflammation, therapeutic doses of anti-IL-13 monoclonal antibodies inhibit AHR, halt subepithelial fibrosis and progression of inflammation, and restore mucus hypersecretion to basal levels (Yang et al, J.Pharmacol. Exp. Ther.313(1):8-15,2005). Inhibition of IL-4 with anti-IL-4 antibodies showed a significant reduction in eosinophil infiltration in the mouse OVA model, but when administered during immunization (Coyle et al, Am J Resp Cell Mol Biol, 13:54, 1995). In a similar model, IL-4 deficient mice had significantly fewer eosinophils in bronchoalveolar lavage fluid and much less peribronchial inflammation following OVA stimulation (Brussell et al, Clin Exp Allergy,24:73, 1994).

In humans, clinical phase IIa studies have shown that in asthmatic patients IL-4R α antagonists (so-called IL-4 muteins) reduce the allergen-induced delayed asthmatic response and attenuate the resting inflammatory state of the lungs of asthmatic patients (Wenzel et al, Lancet,370:1422,2007). These clinical data further strengthen the claim that IL-4 ra antagonists have clinical utility in asthma.

In addition to its role in asthma, IL-4R α is implicated in a number of other pathological conditions, such as COPD, including patient populations with various degrees of chronic bronchitis, small airway disease and emphysema. The underlying etiology of COPD is still unknown. The Dutch hypothesis states that COPD and asthma share common predisposition, and therefore, similar mechanisms may contribute to the pathogenesis of both conditions (Sluiser et al, Eur Respir J,4(4): p.479-89,1991). Zheng et al (J Clin Invest,106(9):1081-93,2000) demonstrated that overexpression of IL-13 in mouse lung causes emphysema, increases mucosal production and inflammation, which may reflect many of the features of human COPD. In addition, AHR has been shown to be predictive of lung function decline in smokers as an IL-13 dependent response in a mouse model of allergic inflammation (Tashkin et al, Am J Respir Crit Care Med,153(6Pt 1): 1802-. In addition, a correlation between IL-13 promoter polymorphisms and susceptibility to developing COPD has been established (Van Der Pouw Kraan et al, Genes Immun,3(7): 436-ion 9, 2002). Thus, it is the IL-4/IL-13 pathway, and IL-13 in particular, that plays an important role in the pathogenesis of COPD.

IL-13 may play a role in the pathogenesis of inflammatory bowel disease. Heller et al (Heller et al, Immunity,17(5):629-38,2002) reported that administration of soluble IL-13R α 2 to neutralize IL-13 improved colonic inflammation in a murine model of human ulcerative colitis. Correspondingly, the expression of IL-13 was higher in rectal biopsy samples from patients with ulcerative colitis compared to controls (Inoue et al, Am J Gastroenterol,94(9) JMl-6, I999).

In addition to asthma, the IL-4/IL-13 pathway has been associated with other fibrotic disorders, such as systemic scleroderma (Hasegawa et al, J Rheumatotol, 24(2):328-32,1997), pulmonary fibrosis (Hancock et al, Am J Respir Cell Mol Biol,18(1):60-5,1998), parasite-induced liver fibrosis (Fallon et al, J Immunol, 164 (5): 2585-91, 2000; Chiaramote et al, J Clin Invest,104(6):777-85, 1999; Chiaramote, Hepatology 34(2):273-82,2001), and bladder fibrosis (Hauber et al, J. Cyst fiber, 2189,2003).

IL-4 is crucial for B cell mediated activities such as B cell proliferation, immunoglobulin secretion, and Fc ε R expression. Clinical applications of IL-4 ra inhibitors include, for example, for suppressing IgE synthesis in allergy therapy (including, for example, atopic dermatitis and food allergy), for preventing graft rejection in transplant therapy, and for suppressing delayed-type hypersensitivity or contact hypersensitivity.

IL-4R antagonists may also be useful as adjuvants for allergy immunotherapy and as vaccine adjuvants. Antibodies against IL-4R α have been described. Two examples are neutralizing mouse anti-IL-4 ra monoclonal antibodies Μ Α β 230 (clone 25463) and 16146 (clone 25463.11), supplied by R & D company (R & D Systems) and Sigma company, respectively. These antibodies belong to the IgG2a subtype, were obtained by the mouse hybridoma method, and were developed from purified recombinant human IL-4R α (baculovirus-derived) immunized mice. Two additional neutralizing murine anti-IL-4 Ra antibodies, M57 and X2/45-12, were provided by BD Biosciences and eBioscience, respectively. These are IgGl antibodies, also obtained by mouse hybridoma technology from mice immunized with recombinant soluble IL-4R α.

There remains a need in the art for anti-IL-4 Ra antibodies, particularly anti-IL-4 Ra heavy chain single domain antibodies, that are capable of binding IL-4 Ra with high affinity and that are capable of blocking the binding of IL-4 to IL-4 Ra.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide an anti-IL-4R alpha single-domain antibody, application and a medicament. The single domain antibody can be specifically combined with IL-4R alpha, has higher affinity, easy preparation and low production cost, and has low immune heterogeneity, so that the single domain antibody can not generate stronger immune reaction when being used as a medicament and has wide application prospect.

The invention is realized by the following steps:

in a first aspect, the invention provides a single domain antibody against IL-4R α comprising the following complementarity determining regions: CDR1, CDR2 and CDR 3;

wherein, the amino acid sequence of the CDR1 is shown as any one of SEQ ID NO.37-53, the amino acid sequence of the CDR2 is shown as any one of SEQ ID NO.54-68, and the amino acid sequence of the CDR3 is shown as any one of SEQ ID NO. 69-85.

The CDR1, CDR2 and CDR3 of the anti-IL-4 Ra single domain antibody provided by the invention can be respectively changed or selected in the sequence ranges of SEQ ID NO.37-53, SEQ ID NO.54-68 and SEQ ID NO.69-85, and the CDR1, CDR2 and CDR3 can be combined in any sequence, so that the obtained single domain antibody can be specifically combined with IL-4 Ra, has good affinity, can be used for preparing a reagent for treating IL-4 Ra mediated related diseases and detecting IL-4 Ra, and has wide and various applications.

In addition, the single-domain antibody provided by the invention has the advantages of simple structure, easiness in preparation and low production cost, and has low immune heterogeneity, so that when the single-domain antibody is used as a medicine for treating IL-4R alpha mediated related diseases, immune response is not easy to generate, and the single-domain antibody has a good application prospect.

It should be noted that, based on the above-mentioned structure of the complementarity determining regions disclosed in the present invention, those skilled in the art can easily conceive of performing substitution or deletion of one or more amino acids based on the above-mentioned structure to obtain single domain antibody mutants having substantially the same or improved binding activity or affinity for IL-4R α, which can be easily realized by those skilled in the art, and such single domain antibody mutants are also included in the scope of the present invention.

Alternatively, in some embodiments of the invention, the complementarity determining region of the single domain antibody is represented by any one of (1) to (18) below:

(1): CDR1 is shown as SEQ ID NO.53, CDR2 is shown as SEQ ID NO.61, CDR3 is shown as SEQ ID NO. 73;

(2): CDR1 is shown as SEQ ID NO.37, CDR2 is shown as SEQ ID NO.61, CDR3 is shown as SEQ ID NO. 72;

(3): CDR1 is shown as SEQ ID NO.39, CDR2 is shown as SEQ ID NO.58, and CDR3 is shown as SEQ ID NO. 78;

(4): CDR1 is shown as SEQ ID NO.53, CDR2 is shown as SEQ ID NO.63, and CDR3 is shown as SEQ ID NO. 72;

(5): CDR1 is shown as SEQ ID NO.42, CDR2 is shown as SEQ ID NO.64, and CDR3 is shown as SEQ ID NO. 74;

(6): CDR1 is shown as SEQ ID NO.46, CDR2 is shown as SEQ ID NO.66, CDR3 is shown as SEQ ID NO. 69;

(7): CDR1 is shown as SEQ ID NO.50, CDR2 is shown as SEQ ID NO.68, CDR3 is shown as SEQ ID NO. 85;

(8): CDR1 is shown as SEQ ID NO.48, CDR2 is shown as SEQ ID NO.55, CDR3 is shown as SEQ ID NO. 80;

(9): CDR1 is shown as SEQ ID NO.47, CDR2 is shown as SEQ ID NO.60, CDR3 is shown as SEQ ID NO. 83;

(10): CDR1 is shown as SEQ ID NO.49, CDR2 is shown as SEQ ID NO.67, and CDR3 is shown as SEQ ID NO. 81;

(11): CDR1 is shown as SEQ ID NO.38, CDR2 is shown as SEQ ID NO.58, and CDR3 is shown as SEQ ID NO. 77;

(12): CDR1 is shown as SEQ ID NO.45, CDR2 is shown as SEQ ID NO.54, and CDR3 is shown as SEQ ID NO. 70;

(13): CDR1 is shown as SEQ ID NO.41, CDR2 is shown as SEQ ID NO.59, and CDR3 is shown as SEQ ID NO. 84;

(14): CDR1 is shown as SEQ ID NO.43, CDR2 is shown as SEQ ID NO.57, and CDR3 is shown as SEQ ID NO. 82;

(15): CDR1 is shown as SEQ ID NO.40, CDR2 is shown as SEQ ID NO.56, and CDR3 is shown as SEQ ID NO. 76;

(16): CDR1 is shown as SEQ ID NO.44, CDR2 is shown as SEQ ID NO.58, and CDR3 is shown as SEQ ID NO. 75;

(17): CDR1 is shown as SEQ ID NO.52, CDR2 is shown as SEQ ID NO.62, CDR3 is shown as SEQ ID NO. 71;

(18): CDR1 is shown in SEQ ID NO.51, CDR2 is shown in SEQ ID NO.65, and CDR3 is shown in SEQ ID NO. 79.

The single domain antibody having the CDR structures shown in the above items (1) to (18) is capable of specifically binding to IL-4 ra and has a high affinity, particularly the CDR structures shown in the items (1), (3) and (6).

Alternatively, in some embodiments of the invention, the single domain antibody comprises the following framework regions: FR1, FR2, FR3 and FR 4;

wherein the amino acid sequence of FR1 is shown in any one of SEQ ID NO. 86-97; the amino acid sequence of FR2 is shown in any one of SEQ ID NO. 98-109; the amino acid sequence of FR3 is shown in any one of SEQ ID NO. 110-127; the amino acid sequence of FR4 is shown in SEQ ID NO. 128.

It should be noted that, the function of the single domain antibody to achieve specific binding with IL-4 ra mainly depends on the CDR regions, and based on the disclosure of the framework regions and CDR region sequences of the single domain antibody, one skilled in the art can easily think that, on the premise that the CDR region sequences are unchanged or slightly modified, the framework region sequences of the single domain antibody are similar to the framework region sequences disclosed in the present invention, such as sequences with homology of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more, so that the functions of the obtained single domain antibody have the same or similar functions as the aforementioned single domain antibody, which is also within the scope of the present invention.

Alternatively, in some embodiments of the invention, the framework regions FR1, FR2 and FR3 of the single domain antibody are as set forth in any one of (a) - (r) below:

(a) FR1 is shown as SEQ ID NO.88, FR2 is shown as SEQ ID NO.100, and FR3 is shown as SEQ ID NO. 115;

(b) FR1 is shown as SEQ ID NO.88, FR2 is shown as SEQ ID NO.100, and FR3 is shown as SEQ ID NO. 114;

(c) FR1 is shown as SEQ ID NO.95, FR2 is shown as SEQ ID NO.102, and FR3 is shown as SEQ ID NO. 123;

(d) FR1 is shown as SEQ ID NO.86, FR2 is shown as SEQ ID NO.100, FR3 is shown as SEQ ID NO. 117;

(e) FR1 is shown as SEQ ID NO.94, FR2 is shown as SEQ ID NO.105, FR3 is shown as SEQ ID NO. 113;

(f) FR1 is shown as SEQ ID NO.87, FR2 is shown as SEQ ID NO.99, FR3 is shown as SEQ ID NO. 110;

(g) FR1 is shown as SEQ ID NO.91, FR2 is shown as SEQ ID NO.109, FR3 is shown as SEQ ID NO. 111;

(h) FR1 is shown as SEQ ID NO.87, FR2 is shown as SEQ ID NO.100, and FR3 is shown as SEQ ID NO. 119;

(i) FR1 is shown as SEQ ID NO.89, FR2 is shown as SEQ ID NO.101, FR3 is shown as SEQ ID NO. 124;

(j) FR1 is shown as SEQ ID NO.93, FR2 is shown as SEQ ID NO.98, FR3 is shown as SEQ ID NO. 112;

(k) FR1 is shown as SEQ ID NO.94, FR2 is shown as SEQ ID NO.106, FR3 is shown as SEQ ID NO. 125;

(l) FR1 is shown as SEQ ID NO.92, FR2 is shown as SEQ ID NO.107, and FR3 is shown as SEQ ID NO. 127;

(m) FR1 is shown as SEQ ID NO.87, FR2 is shown as SEQ ID NO.103, and FR3 is shown as SEQ ID NO. 120;

(n) FR1 is shown as SEQ ID NO.87, FR2 is shown as SEQ ID NO.108, and FR3 is shown as SEQ ID NO. 126;

(o) FR1 is shown as SEQ ID NO.97, FR2 is shown as SEQ ID NO.105, and FR3 is shown as SEQ ID NO. 121;

(p) FR1 is shown as SEQ ID NO.90, FR2 is shown as SEQ ID NO.105, and FR3 is shown as SEQ ID NO. 122;

(q) FR1 is shown as SEQ ID NO.88, FR2 is shown as SEQ ID NO.100, FR3 is shown as SEQ ID NO. 116;

(r) FR1 is shown as SEQ ID NO.96, FR2 is shown as SEQ ID NO.104, and FR3 is shown as SEQ ID NO. 118.

Alternatively, in some embodiments of the invention, the amino acid sequence of the single domain antibody is as set forth in any one of SEQ ID nos. 1-18.

In a second aspect, the invention provides a fusion protein comprising a functional domain capable of specifically binding IL-4 Ra, said functional domain consisting of a single domain antibody against IL-4 Ra as described in the preceding paragraph.

The single domain antibody provided by the invention can be fused with any other protein or substance to achieve different purposes, such as combining with fluorescent protein, enzyme or radioactive element to achieve the purpose of easy detection, and further fusing with drug molecules for treating IL-4R alpha mediated related diseases to achieve better treatment purposes. The type of protein fused with the single domain antibody can be reasonably selected by those skilled in the art according to actual needs or purposes, and any type of fusion substance is included in the scope of the present invention.

In a third aspect, the invention provides an anti-IL-4 Ra antibody, wherein said antibody is a conventional antibody or a functional fragment thereof, and wherein the heavy chain variable region of said antibody is comprised of a single domain anti-IL-4 Ra antibody as defined in any one of the above.

Traditional antibodies consist structurally of two identical heavy chains and two identical light chains, the light chains having a light chain variable region (VL) and a light chain constant region (CL); the heavy chain has a heavy chain variable region (VH) and a heavy chain constant region (CH1, CH2, CH3 and/or CH 4). On the premise that the present invention discloses a single domain antibody with a structure capable of specifically binding to IL-4R alpha, those skilled in the art can easily think of using the single domain antibody of the present invention to modify a conventional antibody, for example, applying the CDR region structure of the single domain antibody of the present invention to a conventional antibody to obtain a conventional antibody capable of specifically binding to IL-4R alpha, and such conventional antibodies also belong to the protection scope of the present invention; further, it is within the scope of the present invention that some of the conventional antibody structures, such as Fab, Fab ', (Fab') 2, Fv, scFv or sdFv structures, etc., also have IL-4R α binding specificity.

Alternatively, in some embodiments of the invention, the functional fragment is a Fab, Fab ', (Fab') 2, Fv, scFv or sdFv structure of the traditional antibody.

Use of an anti-IL-4 Ra single domain antibody as defined above, a fusion protein as defined above or an antibody as defined above in the manufacture of a medicament for targeting IL-4 Ra for the treatment of a disease.

Alternatively, in some embodiments of the invention, the disease is selected from asthma, atopic dermatitis, eczema, arthritis, herpes, chronic primary urticaria, scleroderma, hypertrophic scarring, Chronic Obstructive Pulmonary Disease (COPD), atopic dermatitis, idiopathic pulmonary fibrosis, kawasaki disease, sickle cell disease, graves ' disease, sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, barrett's esophagus, autoimmune uveitis, tuberculosis, and nephropathy.

The antibody of the invention can be used for treating IL-4R alpha as a target to neutralize IL-4R alpha so as to achieve the effect of treating diseases, including but not limited to the diseases, and the antibody also belongs to the protection scope of the invention.

In a fourth aspect, the present invention provides a medicament for the treatment of a disease, comprising an anti-IL-4 ra single domain antibody as defined in any one of the above, a fusion protein as defined above or an antibody as defined above, and a pharmaceutically acceptable excipient.

The pharmaceutically acceptable excipients refer to pharmaceutical excipients in the pharmaceutical field, such as: diluents, fillers, binders, wetting agents, absorption enhancers, surfactants, disintegrants, adsorption carriers, lubricants, and the like. In addition, other adjuvants such as flavoring agent and sweetener can also be added. The auxiliary materials are one or more than two of diluent, filler, adhesive, wetting agent, absorption enhancer, surfactant, disintegrant, adsorption carrier, lubricant, flavoring agent and sweetener.

The dosage form of the medicament provided by the invention is not strictly limited, and the medicament can be prepared into various dosage forms according to the existing method in the field of medicaments, and is applied to patients needing treatment in the modes of oral administration, nasal inhalation, rectum, parenteral administration or transdermal administration and the like.

In a fifth aspect, the invention provides an isolated nucleic acid molecule encoding a single domain antibody as described in any one of the above.

It should be noted that, based on the disclosure of the present invention, the polynucleotide molecules encoding the single domain antibodies and the fusion proteins described above can be easily obtained by those skilled in the art through the conventional techniques in the art, and based on the degeneracy of the codon, the polynucleotide molecules are varied, and there are many possibilities in the specific base sequences, and therefore, it is within the scope of the present invention that the polynucleotide molecules can be varied as long as they can encode the single domain antibodies or the fusion proteins of the present invention.

Alternatively, in some embodiments of the invention, the base sequence of the nucleic acid molecule is as set forth in any one of SEQ ID No. 19-36.

In a sixth aspect, the present invention provides a vector comprising a nucleic acid molecule as described above.

In a seventh aspect, the present invention provides a recombinant cell comprising a vector as described above.

In an eighth aspect, the present invention provides a method of preparing a single domain antibody as defined in any one of the above, comprising: culturing the recombinant cell as described above, and isolating and purifying the single domain antibody from the culture product.

It should be noted that the single domain antibody, fusion protein or antibody of the present invention can be prepared by chemical synthesis, genetic engineering techniques, or other methods, and any method for preparing the single domain antibody, fusion protein or antibody described above is within the scope of the present invention.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and those skilled in the art can also obtain other related drawings based on the drawings without inventive efforts.

FIG. 1 is an analysis diagram of the purification result of human recombinant protein IL-4R alpha.

FIG. 2 is a graph showing the measurement of the binding activity between a self-produced IL-4 Ra recombinant protein and a tool antibody, wherein the near-shore IL-4 Ra recombinant protein is designated by the reference number Novo, the self-produced IL-4 Ra recombinant protein is designated by the reference number RJK, the tool antibody is designated by the reference number Tab, and human IgG is a negative isotype control antibody.

Fig. 3 shows the enrichment of a single domain antibody library against the IL-4 ra recombinant protein after screening, where P/N is the number of monoclonal bacteria grown after TG1 bacteria infected with phage eluted from positive wells in biopanning/the number of monoclonal bacteria grown after TG1 bacteria infected with phage eluted from positive wells, which parameter increases gradually after enrichment has occurred; and I/E is the total amount of phages added into the positive hole in each round of biopanning/the total amount of phages eluted from the positive hole in each round of biopanning, the parameter gradually approaches to 1 after enrichment occurs, and the third round of enrichment is carried out, wherein P/N is 1000, and I/E is 2.

FIG. 4 shows SDS-PAGE analysis of single domain antibodies after one-step affinity chromatography on a nickel column.

FIG. 5 is an affinity dose-response curve of purified single domain antibody to IL-4R α.

FIG. 6 is a neutralization experiment dose-effect curve of the purified Fc fusion single-domain nano-antibody in the IL-4 induced TF-1 cell proliferation experiment.

FIG. 7 is a dose-effect curve of neutralization experiments of purified Fc fusion single-domain nanobodies in IL-13-induced TF-1 cell proliferation experiments.

Fig. 8 is the result of affinity kinetic determinations of single domain antibodies 1H1 and 4E9 curve and the fitting of the curve.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Expression vector for constructing human source recombinant IL-4R alpha protein

The method comprises the following steps:

(1) the coding sequence for IL-4R α was retrieved at NCBI and included as NM-000418.3, and the amino acid sequence generated by this sequence was assigned accession number NP-000409.1.

(2) The amino acid sequence corresponding to NP-000409.1 was analyzed for the transmembrane region and extracellular end of the protein by TMHMM and SMART websites, respectively.

(3) The analysis result shows that the extracellular end of the IL-4R alpha protein is amino acid 1-232, wherein, the 1-25 position is the signal peptide of the protein.

(4) The nucleotide sequence of 26-232 amino acids of the protein encoding IL-4R alpha is cloned into the vector pcDNA3.4 by means of gene synthesis.

(5) Sanger sequencing is carried out on the constructed vector, an original sequence is compared, after no error is confirmed, batch extraction is carried out on the recombinant plasmid, endotoxin is removed, transfection and suspension 293F are carried out to express and purify target protein, and the SDS-PAGE and western blot analysis results of the purified IL-4R alpha recombinant protein are shown in figure 1.

Example 2 determination of binding Activity of human-derived recombinant protein

The experimental method of the binding activity is carried out by referring to the method disclosed in the Chinese invention patent with the patent application number of 202010166444.8, namely the single domain antibody capable of specifically binding EpCAM and the application thereof, and the recombinant IL-4 Ra protein purchased from near shore is used as the positive control recombinant protein, and the human IgG is used as the negative isotype control of the tool antibody. The results are plotted in FIG. 2, and although the self-produced protein was less colored at high concentrations than the control, it was comparable to the tool antibody binding capacity (EC50), and neither the human IgG isotype control nor the IL-4R α recombinant protein purchased offshore or produced by the company.

Example 3

Construction of Single Domain antibody libraries directed against IL-4R α protein

The method comprises the following steps:

(1) 2mg of the IL-4 Ra recombinant protein obtained by purification in example 1 was mixed with an equal volume of Freund's complete adjuvant, and an inner Mongolian Alexandria was immunized once a week for 7 consecutive immunizations, and the animals were immunized by mixing 1mg of the IL-4 Ra recombinant protein with an equal volume of Freund's incomplete adjuvant for six consecutive immunizations except for the first immunization, and the immunization was performed in order to stimulate camels intensively to produce antibodies against IL-4 Ra.

(2) After animal immunization is finished, 100mL of camel peripheral blood lymphocytes are extracted, and RNA of the cells is extracted.

(3) cDNA was synthesized using the extracted total RNA, and VHH (heavy chain antibody variable region) was amplified by nested PCR reaction using the cDNA as a template.

(4) Respectively carrying out enzyme digestion on the pMECS vector and the VHH fragment by using restriction enzymes, and then connecting the enzyme-digested fragment with the vector.

(5) The ligated fragments were transformed into competent cells TG1, a phage display library of IL-4R α protein was constructed and the size of the library was determined to be about 1X 10⁹。

Example 4

Screening for Single Domain antibodies to IL-4R alpha protein

The method comprises the following steps:

(1) mu.L of the recombinant TG1 cells of example 2 were inoculated into 2 XTY medium and cultured, during which time 40. mu.L of helper phage VCSM13 was added to infect TG1 cells and cultured overnight to amplify phages, which were precipitated with PEG/NaCl the next day and collected by centrifugation.

(2) NaHCO diluted at 100mM pH 8.3₃The IL-4R alpha recombinant protein 500 mu g in the reagent kit is coupled on an enzyme label plate, is placed at 4 ℃ overnight, and is simultaneously provided with a negative control hole.

(3) The next day 200. mu.L of 3% skim milk was added and blocked for 2h at room temperature.

(4) After blocking, 100. mu.L of the amplified phage library (approx.2X 10) was added¹¹Individual phage particles) at room temperature for 1 h.

(5) After 1 hour of action, the unbound phage were washed off 5 times with PBS (containing 0.05% Tween-20).

(6) The phage specifically bound with the IL-4R alpha recombinant protein is dissociated by trypsin with the final concentration of 2.5mg/mL, escherichia coli TG1 cells in the logarithmic growth phase are infected, the cells are cultured at 37 ℃ for 1h, the phage are generated and collected for the next round of screening, the same screening process is repeated for 1 round, enrichment is gradually obtained, and when the enrichment multiple reaches more than 10 times, the enrichment effect is shown in figure 3.

Example 5

Phage enzyme-linked immunosorbent assay (ELISA) was used to screen specific positive clones for IL-4R α.

The method comprises the following steps:

(1) carrying out 3 rounds of screening on the IL-4R alpha recombinant protein according to the screening method of the single domain antibody in the embodiment 2, after the screening is finished, aiming at the phage enrichment factor of the IL-4R alpha recombinant protein to be more than 10, selecting 400 single colonies from positive clones obtained by screening, respectively inoculating the single colonies into a 96 deep-well plate of a TB culture medium containing 100 mu g/mL ampicillin, setting a blank control, culturing at 37 ℃ to a logarithmic phase, adding IPTG with the final concentration of 1mM, and culturing at 28 ℃ overnight;

(2) obtaining a crude antibody by using an osmotic bursting method; the IL-4R α recombinant proteins were released separately to 100mM NaHCO, pH 8.3₃In the enzyme labeling plate, 100 mug of IL-4R alpha recombinant protein is coated overnight at 4 ℃;

(3) transferring 100 mu L of the crude antibody extract obtained in the step to an ELISA plate added with an antigen, and incubating for 1h at room temperature;

(4) unbound antibody was washed away with PBST, 100. mu.l of Mouse anti-HA tag antibody (Mouse anti-HA antibody, Thermo Fisher) diluted at 1:2000 was added, and incubated at room temperature for 1 h;

(5) unbound antibody was washed off with PBST, 100ul of Anti-Rabbit HRP conjugate (goat Anti-Rabbit horseradish peroxidase labeled antibody, purchased from Thermo Fisher) diluted at 1:20000 was added, and incubated at room temperature for 1 h;

(6) washing away unbound antibodies by PBST, adding horseradish peroxidase developing solution, reacting at 37 ℃ for 15min, adding a stop solution, and reading an absorption value at a wavelength of 450nm on an enzyme-labeling instrument;

(7) when the OD value of the sample hole is more than 5 times of that of the control hole, judging the sample hole as a positive cloning hole;

(8) the positive colony well was transferred to LB medium containing 100. mu.g/. mu.L ampicillin to extract plasmids and sequence;

(9) analyzing the gene sequences of the clones according to Vector NTI (sequence alignment software), and regarding the strains with the same CDR1, CDR2 and CDR3 sequences as the same clones, and regarding the strains with different sequences as different clones to obtain the single-domain antibody specific to the IL-4R alpha protein. The amino acid sequence of the antibody is the structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the whole VHH is formed. The obtained single domain antibody recombinant plasmid can be expressed in a prokaryotic system to obtain the single domain antibody aiming at the IL-4R alpha protein.

The numbering of each single domain antibody and the results of its amino acid sequence analysis, i.e., the corresponding sequence identifier (SEQ ID No.) are given in table 1 below.

TABLE 1 sequence identifiers (SEQ ID NO.) corresponding to CDR and FR of each of the single domain antibodies of the examples

Example 5

And (3) purifying and expressing the specific single-domain antibody of the IL-4R alpha protein in host bacterium escherichia coli.

The method comprises the following steps:

(1) the plasmids (pMECS-VHH) of the different clones obtained by the above sequencing analysis were electrically transformed into E.coli HB2151, spread on LB + amp + glucose, i.e., a culture plate containing ampicillin and glucose, and cultured overnight at 37 ℃;

(2) selecting a single colony to be inoculated in 5mL LB culture solution containing shore penicillin, and carrying out shake culture at 37 ℃ overnight;

(3) inoculating 1mL of overnight cultured strain into 330mL of TB culture solution, performing shake culture at 37 ℃ until OD600nm value reaches 0.6-0.9, adding 1M IPTG, and performing shake culture at 28 ℃ overnight;

(4) centrifuging, collecting Escherichia coli, and obtaining crude antibody extractive solution by use of osmotic bursting method;

(5) the antibodies were purified by nickel column affinity chromatography, and the results of SDS-PAGE analysis of the purified single domain antibodies are shown in FIG. 4, in which VHH1-18 are assigned to the antibody numbers in Table 1, respectively.

Example 6

The Fc fusion antibody of the single-domain antibody is prepared by referring to the single-domain antibody with the patent application number of 202010166444.8 and the invention name of which is EpCAM specifically binding and the method disclosed in the Chinese invention patent application of the invention.

Example 7

And (3) determining the binding capacity and effect curve of the specific single-domain antibody of the IL-4R alpha protein.

The experimental method is carried out by referring to the method disclosed in the Chinese invention patent with the patent application number of 202010166444.8 and the invention name of the single-domain antibody capable of specifically binding EpCAM, drawing a curve and calculating EC₅₀The results are shown in fig. 5 and table 2 below.

TABLE 2 EC of Single Domain antibodies binding to IL-4R α recombinant proteins₅₀

Antibody numbering	1A9	1B1	1B2	1B4	1C4	1D9	1H1	2A7	2C10
										EC₅₀(nM)	2.724	0.850	5.816	0.404	2.686	0.103	1.132	3.060	4.150
Antibody numbering	2C9	2H2	3C11	3E10	3F9	3G10	4B3	4E9	4H11
										EC₅₀(nM)	26.29	2.168	1.379	1.838	1.602	5.207	1.364	2.135	16.290

It can be seen that in the binding detection of the 18 purified single-domain antibodies, the binding capacities of the antibodies except for 4H11 and 2C9 are above 10nM, the binding capacities of the other antibodies are below 10nM, and the binding capacities of the antibodies 1B4 and 1D9 are below 1nM, which proves that the single-domain antibody obtained by screening in this embodiment has good overall binding capacity.

Example 8

Expression and purification of a Tool antibody (Tool antibody, Tab) targeting human IL-4R alpha, Tab1(dupilumab) sequence from IMGT, Tab2(L1012H1031), Tab3(L1020H1031) sequence from patent No.: CN 107474134.

The method comprises the following steps:

(1) the Tab sequence was subjected to codon optimization of mammalian cell expression system by general biosystems (Anhui) Ltd and cloned into pcDNA3.1 vector.

(2) After resistance selection, plasmid positive bacteria were selected and amplified, and plasmids were extracted using a plasmid extraction kit (Macherey Nagel, Cat # 740412.50).

(3) Transient Expression in 293F cells (medium: FreeStyle 293Expression medium, Thermo, Cat #12338026+ F-68, Thermo, Cat #24040032) was performed using PEI as per 100. mu.g plasmid (50. mu.g heavy chain + 50. mu.g light chain) per 100mL of cells.

(4) After transfection for 6-24 h, 5% volume of 10% Peptone (Sigma, Cat # P0521-100G) and 8% CO were added₂And culturing at 130rpm for about 7-8 days.

(5) When the cell viability decreased to 50%, the expression supernatant was collected and purified using a ProteinA (GE, Cat #17-5438-02) gravity column.

(6) After PBS dialysis, concentration was determined using Nanodrop, purity was confirmed by SEC, and binding capacity was verified by indirect ELISA.

(7) Tab obtained by the method has the concentration of not less than 2mg/ml, the purity of more than 94 percent and the binding EC50 of IL-4R alpha (Novoprotein, Cat # CS33) and self-produced IL-4R alpha recombinant protein of 0.25 +/-0.05 nM.

Example 9

Human IL-4 induced proliferation of TF1 cells and tool antibody neutralization proliferation assay.

1 experimental method for TF1 cell proliferation induced by human IL-4:

(1) paving 10000 per hole TF-1 cells which are recovered and passaged for 3-4 times into a 96-hole plate;

(2) preparing a solution with the highest concentration of 500ng/mL of the human IL-4 protein, and performing 5-fold gradient dilution;

(3) adding the IL-4 solution which is diluted in a gradient manner into a cell culture hole according to the equal volume of the cell culture solution;

(4) after incubation for 72h, detecting the cell activity by using a luminescence method cell activity detection kit;

(5) and calculating the EC80 concentration of the IL-4 for inducing the TF-1 cells to proliferate according to the detection result.

2 tools antibody (Tab) neutralization of human IL-4 induced TF1 cell proliferation assay:

(1) paving the recovered TF-1 cells which are subjected to passage for 3-4 times into a 96-well plate according to 10000 cells per well;

(2) preparing different Tabs into 10 mu g/mL solution, and carrying out 5-time gradient dilution;

(3) mixing the Tab diluted in the gradient with IL-4 with EC80 concentration obtained in proliferation experiment at a ratio of 1:1 to obtain mixed solution;

(4) adding the mixed solution in the step into a cell culture hole according to the equal volume of the cell culture solution;

(5) after incubation for 72h, detecting the cell activity by using a luminescence method cell activity detection kit;

(6) the concentration of EC50 for neutralizing IL-4 by Tab to induce the proliferation of TF-1 cells was calculated based on the results of the assay, and the results are shown in Table 3.

TABLE 3 detection of IL-4 induced TF-1 proliferation by neutralizing the tool antibody (Tab)

Name of antibody	Tab1	Tab2	Tab3
				EC₅₀(nM)	0.18	0.067	0.098

Example 10

Human IL-13 induced proliferation of TF1 cells and tool antibody neutralization proliferation assay.

1 human IL-13 induced proliferation of TF1 cells:

(3) adding the IL-4 solution which is diluted in a gradient manner into the cell culture hole according to the equal volume of the cell culture solution;

(5) and calculating the EC80 concentration of the IL-13 for inducing the TF-1 cells to proliferate according to the detection result.

2 tools antibody (Tab) neutralization of human IL-13 induced TF1 cell proliferation assay:

(3) mixing the Tab diluted in the gradient with IL-13 with EC80 concentration obtained in proliferation experiment at a ratio of 1:1 to obtain a mixed solution;

(6) and calculating the EC50 concentration of the Tab for neutralizing the IL-13 to induce the TF-1 cells to proliferate according to the detection result. The results are shown in Table 4.

TABLE 4 detection of IL-13 induced TF-1 proliferation by neutralization of tool antibody (Tab)

Name of antibody	Tab1	Tab2	Tab3
				EC₅₀(nM)	0.073	0.097	0.047

Example 11

Detection of TF1 cell proliferation induced by IL-4 neutralization of a single domain antibody specific for IL-4R α.

The method comprises the following steps:

(2) the different Tab, Fc fused single domain antibodies of example 8 (from example 6) were formulated as 10. mu.g/mL solutions and diluted 5-fold in gradient;

(3) respectively mixing the Tab and the single-domain antibody which are diluted in a gradient manner with IL-4 with EC80 concentration obtained in a proliferation experiment according to a ratio of 1:1 to prepare a mixed solution;

(4) adding the mixed solution in the previous step into a cell culture hole according to the equal volume of the cell culture solution;

(5) after incubation for 72h, detecting cell activity by using a luminescence method cell activity detection kit;

(6) the EC50 concentration for neutralizing IL-4 induced TF-1 cell proliferation by different single domain antibodies was calculated according to the results of the assay, and the results are shown in FIG. 6 and Table 5 below.

TABLE 5 detection results of experimental dose-response curve for neutralizing IL-4 induced TF-1 proliferation by single-domain antibody

Name of antibody	1A9	1B1	1B2	1B4	1C4	1D9	1H1	2A7	2C10
										EC₅₀(nM)	0.099	0.302	0.363	0.190	0.055	0.086	0.072	0.119	28.1
Name of antibody	2C9	2H2	3C11	3E10	3F9	3G10	4B3	4E9	4H11
										EC₅₀(nM)	2.375	0.149	0.083	0.175	0.133	0.507	0.231	0.071	16680000

It can be seen that, among the 18 antibodies, compared with other single domain antibodies in table 5, 4H11 has the worst neutralization effect, 2C10 has a general neutralization effect, although the neutralization effect of 2C9 is good, its EC50 is still above 1nM, and the other single domain antibodies have its EC50 at 1nM, which is better, wherein the neutralization effects of single domain antibodies 1C4, 4E9, and 1H1 are ranked first three, and EC50 of 1C4 reaches 55 pM.

Example 12

Detection of TF1 cell proliferation induced by neutralizing IL-13 with a single domain antibody specific for IL-4R α.

The method comprises the following steps:

(2) the different Tab, Fc fused single domain antibodies of example 8 (from example 6) were formulated as 10. mu.g/mL solutions and diluted 5-fold in a gradient;

(3) mixing the Tab and the single-domain antibody which are diluted in a gradient manner with IL-13 with EC80 concentration obtained in a proliferation experiment according to a ratio of 1:1 to prepare a mixed solution;

(6) the EC50 concentration for neutralizing IL-13 induced TF-1 cell proliferation by different single domain antibodies was calculated according to the results of the assay, and the results are shown in FIG. 7 and Table 6 below.

TABLE 6 detection results of experimental dose-response curve for neutralizing IL-13 induced TF-1 proliferation by single-domain antibody

It can be seen that, among the 18 antibodies, the neutralizing effects of 4H11 and 2C10 are inferior compared with those of other single domain antibodies in table 5, although the neutralizing effects of 2C9 and 1B2 are good, the EC50 is still above 1nM, and the EC50 of other single domain antibodies is below 1nM, wherein the neutralizing effects of the single domain antibodies 1H1, 3C11 and 4E9 are ranked three times, and the EC50 of 1H1 is 70 pM.

Example 13

Combining the experimental results of example 11 and example 12, the single domain antibodies 1H1 and 4E9 were the best overall neutralizing effect, and the two antibodies were selected for antigen-antibody affinity determination using the Octet RED96 instrument. The specific embodiment is as follows:

(1) preparing an SD buffer solution: appropriate amounts of bovine serum albumin and tween 20 were dissolved in 1 × PBS (ph7.4) so that the mass (or volume) fractions of bovine serum albumin and tween 20 were 0.1% and 0.02%, respectively.

(2) Preparing an antigen working solution: the antigen (IL-4R alpha recombinant protein) was prepared in SD buffer at 10. mu.g/mL.

(3) Preparing an antibody working solution: the antibody is prepared into 100nM by SD buffer solution, and then diluted according to 2 times of gradient, 5 concentration gradients are set, and a zero concentration is set.

(4) The system comprises: starting Octet 96 and Data Acquiston software in a computer matched with the Octet 96, cleaning the bottom surface and the side surface of the acquisition probe by taking a proper amount of 75% ethanol through a piece of lens wiping paper, and preheating the instrument for more than 15 min.

(5) Sensor prewetting: the Sensor is soaked in SD buffer solution for more than 10 minutes before the experiment is started for standby.

(6) The binding and dissociation times are shown in table 7:

TABLE 7 computer-on step

Experimental procedure	Time(s)	Sample (I)
			Baseline1	60	SD
Loading
		300	Antigens
Baseline2				60	SD
	Association
		300	Antibodies
	Dissociation
		300	SD

(7) And (5) after the analysis is finished, closing the instrument and the computer.

(8) And (3) data analysis: KD, Ka, Kd, Full R were calculated in Data Analysis software²The correlation constants were equalized, and the results are shown in FIG. 8 and Table 8

TABLE 8 summary of affinity kinetic data for clones 1H1 and 4E9

Clone name	KD(M)	Ka(1/Ms)	Kd(1/s)
				1H1	1.41E-11	5.32E+05	7.50E-06
4E9	2.39E-10	5.56E+05	1.33E-04

It was revealed from the prior art (patent CN107474134) that Tab2 and Tab3 have an affinity of 1.04X 10, respectively^-10M and 1.83X 10^-10M, the affinity of the single-domain antibody 1H1 obtained in the embodiment of the invention is obviously better than that of the control antibody and is 1.41 multiplied by 10^-11Whereas 4E9 was substantially equivalent to the control antibody.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Nanjing Rodgeikang Biotech Ltd

<120> anti-IL-4R alpha single domain antibody and application and medicine thereof

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Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser

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Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser

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Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser

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Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly Ser Leu Arg Val Ser

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Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser

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<400> 18

Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Glu Ser Ser Thr Ser Thr Ser Ser Ile Tyr Cys Met Ala Trp Phe

20 25 30

Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val Ala Ser Ile Ser Pro

35 40 45

Ala Arg Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Ala

50 55 60

Ile Ser Gln Asp Asn Ala Lys Ser Val Leu Tyr Leu Gln Met Asn Ser

65 70 75 80

Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Arg Ser

85 90 95

Gly Thr Cys Asp Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser

100 105 110

Ser

<210> 19

<211> 333

<212> DNA

<213> Artificial sequence

<400> 19

gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccagcacc 60

tacacctaca ccccctactg catggcctgg ttcaggcagg cccccggcaa ggagagggag 120

gccgtggcca gcatcaggag cggcaggatc gcctactacg ccgacagcgt gaaggccagg 180

ttcaccatca gcctggacaa cgccaagaac accgtgtacc tggagatgaa cagcctgagg 240

cccgaggaca ccggcgtgta ctactgcgcc gccggcgacg tgggcagctg caacaactac 300

tggggccagg gcacccaggt gaccgtgagc agc 333

<210> 20

<211> 333

<212> DNA

<213> Artificial sequence

<400> 20

gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccagcagg 60

tacacctaca ccccctactg catggcctgg ttcaggcagg cccccggcaa ggagagggag 120

gccgtggcca gcatcaggag cggcaggatc gcctactacg ccgacagcgt gaaggccagg 180

ttcaccatca gccacgacaa cgccaagaac accgtgtacc tgcagatgaa cagcctgagg 240

cccgaggaca ccggcgtgta ctactgcgcc gccggcgacg tgggcagctg cgacaactac 300

tggggccagg gcacccaggt gaccgtgagc agc 333

<210> 21

<211> 339

<212> DNA

<213> Artificial sequence

<400> 21

gagagcggcg gcggcagcgt gcagaccggc ggcagcctga ggctgagctg cgccaccagc 60

gagtacacca gcaacctgta ctgcatggcc tggttcaggc aggcccccgg caaggagagg 120

gagggcatcg ccagcatcag ggccggcacc gacaggacct actacgccga cagcgtgaag 180

ggcaggttca ccatcagcag ggacaaggcc aagaacaccc tgtacctgca gatgaacagc 240

ctgaagcccg aggacaccgc catgtactac tgcgccgccg gccccagcgg cctgtgcacc 300

gactactggg gccagggcac ccaggtgacc gtgagcagc 339

<210> 22

<211> 333

<212> DNA

<213> Artificial sequence

<400> 22

gagagcggcg gcggcgccgt ggaggccggc ggcagcctga ggctgagctg cttcagcacc 60

tacacctaca ccccctactg catggcctgg ttcaggcagg cccccggcaa ggagagggag 120

gccgtggcca gcatcaggag cggcaggacc gcctactacg ccgacagcgt gaaggccagg 180

ttcaccatca gccaggacaa cgccaagaac accgtgtacc tgcagatgaa cagcctgagg 240

cccgaggaca ccggcgtgta ctactgcgcc gccggcgacg tgggcagctg cgacaactac 300

tggggccagg gcacccaggt gaccgtgagc agc 333

<210> 23

<211> 339

<212> DNA

<213> Artificial sequence

<400> 23

gagagcggcg gcggcagcgt gcagaccggc ggcagcctga ggctgagctg cgccgccagc 60

ggcgacaccg tgaacatgta ctgcatggcc tggttcaggc aggcccccgg caacgagagg 120

gaggccgtgg ccagcatcag gagcggcacc gacaggacct actacgccga cgccgtgaag 180

ggcaggttca ccatcagcag ggacaaggcc aagaacaccc tgtacctgca gatgaacagc 240

ctgaagcccg aggacaccgc catgtactac tgcgccgccg gccacagcgg cctgtgcacc 300

gactacaggg gccagggcac ccaggtgacc gtgagcagc 339

<210> 24

<211> 375

<212> DNA

<213> Artificial sequence

<400> 24

gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60

ggcagcaccg gcaggcacta cagcctgggc tggttcaggc aggcccccgg caaggagagg 120

gagggcgtgg ccatcatcta cagctgcggc agggacaccg actacgccga cagcgtgaag 180

ggcaggttca ccatcagcca ggacatcgcc gagagcaccc tgtacctgca gatgaacagc 240

ctgaagcccg aggacaccgg catgtactac tgcgccgccg ccctgggcct gagcaggctg 300

aggtgcgaca cccagagcat cagccccgac gagtacaacg tgtggggcca gggcacccag 360

gtgaccgtga gcagc 375

<210> 25

<211> 354

<212> DNA

<213> Artificial sequence

<400> 25

gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgaggtgagc 60

ggctacacca gctgcaggag gtacgacatg agctggtaca ggcaggcccc cggcaaggag 120

agggagttcg tgagcgtgat cgacggctac ggcggcatca actacgccga cagcgtgaag 180

ggcaggttca ccatcagcca ggacaacggc aagcacacca tgtacctgca gatggacaac 240

ctgcagcccg aggacaccgc catgtactac tgcaaggccg acggcaggag gtacaggggc 300

acctgcagca gcttcgccac ctggggccag ggcacccagg tgaccgtgag cagc 354

<210> 26

<211> 333

<212> DNA

<213> Artificial sequence

<400> 26

gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60

ggctacatca ccagctactg catggcctgg ttcaggcagg cccccggcaa ggagagggag 120

gccgtggcca gcatccacac caggggcacc acctactacg ccgacagcgt gaagggcagg 180

ttcaccatca gcaaggacat cgccaagaac accctgtacc tgcagatgaa catgctgaag 240

cccgaggaca ccgccatgta ctactgcgcc gccggcagca ccggctactg cagggactac 300

tggggccagg gcacccaggt gaccgtgagc agc 333

<210> 27

<211> 330

<212> DNA

<213> Artificial sequence

<400> 27

gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccaccagc 60

ggcgtgttct actgcatggc ctggttcagg caggcccccg gcaaggagag ggaggccgtg 120

gccaccatca ggagcggcgg caggaccgcc tactacgccg acagcgtgaa gggcaggttc 180

accatcagca gggacaacgc caaggacacc ctgtacctgc agatgaacag cctgaagccc 240

gaggacagcg ccatgtacta ctgcgccgtg ggcgtggacg gcgactgcag gaactactgg 300

ggccagggca cccaggtgac cgtgagcagc 330

<210> 28

<211> 378

<212> DNA

<213> Artificial sequence

<400> 28

gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg caccgtgagc 60

ggctacaact acaacagcgc ctacatcggc tggttcaggc aggcccccgg caaggagagg 120

gagggcgtgg ccgtgatcta cagcggcggc ggcagcaccg tgtacgccga cagcgtgaag 180

ggcaggttca ccatcagcct ggacatcgcc cagaacaccg tgtacctgca gatgaacagc 240

ctgaagcccg acgacaccgc catgtactac tgcgccgcca gcgtggccag gaggggcagc 300

accctgcccc agcccctgag gctgcagccc gcctactaca acaagtgggg ccagggcacc 360

caggtgaccg tgagcagc 378

<210> 29

<211> 333

<212> DNA

<213> Artificial sequence

<400> 29

gagagcggcg gcggcagcgt gcagaccggc ggcagcctga ggctgagctg cgccgccagc 60

gccgtggaca tgtactgcat ggcctggttc aggcagaccc ccggcaacga gagggagggc 120

gtggccagca tcagggccgg caccgacagg acctactacg ccgacagcgt gaagggcagg 180

ttcaccatca gcagggacag ggccaagaac accctgtacc tgcagatgaa cagcctgaag 240

cccgaggaca ccgccatgta ctactgcgcc gccggcccca gcggcgcctg caccgactac 300

tggggccagg gcacccaggt gaccgtgagc agc 333

<210> 30

<211> 366

<212> DNA

<213> Artificial sequence

<400> 30

gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg caccgccagc 60

ggcttcacct tcgacaacag cgacatgggc tggtacaggc aggcccccgg caacgagtgc 120

gagctggtga gcaagatcgg cagcaccagg accccctact acagcgacag cgtggccggc 180

aggttcacca tcagccagga caacgccaag aacaccgtgt acctgcagct gaacagcctg 240

aagcccgagg acaccgccgt gtactactgc gccgccgacg gccccttcta cgtgaggacc 300

agctgcaggg acgactacag gggccccagc tactggggcc agggcaccca ggtgaccgtg 360

agcagc 366

<210> 31

<211> 330

<212> DNA

<213> Artificial sequence

<400> 31

gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60

ggcgacttct actgcatggc ctggttcagg caggcccccg gcaaggagag ggagggcgtg 120

gccaccatca ggagcggcgg caggagcacc tactacgccg acagcgtgaa gggcaggttc 180

accatcagca aggacaacgc caaggacacc ctgtacctgc agatgaacag cctgaagccc 240

gaggacaccg ccatgtacta ctgcgccgtg ggcgtggacg gcaactgcag gaactactgg 300

ggccagggca cccaggtgac cgtgagcagc 330

<210> 32

<211> 336

<212> DNA

<213> Artificial sequence

<400> 32

gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60

ggcttcaagt tcggcaccag cgtgatgagc tgggtgaggc aggcccccgg caagggcctg 120

gagagggtga gcctgatcaa cagggacggc acctacacct actaccccga cagcgtgaag 180

ggcaggttca ccatcagcag ggacaacgcc aagaacatgc tgtacctgca gatgaacagc 240

ctgaagaccg acgacaccgc catgtactac tgcgccaagg actggacccc cggcacctgg 300

cccaggggcc agggcaccca ggtgaccgtg agcagc 336

<210> 33

<211> 339

<212> DNA

<213> Artificial sequence

<400> 33

gagagcggcg gcggcagcgt gcagaccggc ggcagcctga gggtgagctg cgccgccagc 60

gagtacacct acaacatgta ctgcatggcc tggttcaggc aggcccccgg caacgagagg 120

gaggccgtgg ccagcatcaa gaccgacacc gacaggacct actacgccga cagcgtgaag 180

ggcaggttca ccatcagcca ggacaaggcc aagaacaccc tgtacctgca gatgaacagc 240

ctgaagcccg aggacaccgc catgtactac tgcgccgccg gcctgagcgg cagctgcacc 300

aactactggg gccagggcac ccaggtgacc gtgagcagc 339

<210> 34

<211> 339

<212> DNA

<213> Artificial sequence

<400> 34

gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgtgagc 60

ggcttcaccg acagcctgta ctgcatggcc tggttcaggc aggcccccgg caacgagagg 120

gaggccgtgg ccagcatcag ggccggcacc gacaggacct actacgccga cagcgtgaag 180

ggcaggttca ccatcagcca ggacaaggcc aagaacaccc tgtacctgca gatgaacagc 240

ctgaggcccg aggacaccgc catgtactac tgcgccgccg gccacagcgg cctgtgcacc 300

gactacagcg gccagggcac ccaggtgacc gtgagcagc 339

<210> 35

<211> 333

<212> DNA

<213> Artificial sequence

<400> 35

gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccagcacc 60

tacacctaca tcccctactg catggcctgg ttcaggcagg cccccggcaa ggagagggag 120

gccgtggcca gcatcaggag cggcaggatc acctactacg ccgacagcgt gaaggccagg 180

ttcaccatca gccaggacaa cgccaagaac accgtgtacc tgcagatgaa cagcctgaag 240

cccgaggaca ccgccgtgta ctactgcgcc gccggcgaca gcggcaactg caacaactac 300

tggggccagg gcacccaggt gaccgtgagc agc 333

<210> 36

<211> 339

<212> DNA

<213> Artificial sequence

<400> 36

gagagcggcg gcggcagcgt gcagaccggc ggcagcctga ggctgagctg cgagagcagc 60

accagcacca gcagcatcta ctgcatggcc tggttcaggc aggcccccgg caagggcagg 120

gagggcgtgg ccagcatcag ccccgccagg ggcaggacct actacgccga cagcgtgaag 180

ggcaggttcg ccatcagcca ggacaacgcc aagagcgtgc tgtacctgca gatgaacagc 240

ctgaagcccg aggacaccgc catgtactac tgcgccgccg gcaggagcgg cacctgcgac 300

gactactggg gccagggcac ccaggtgacc gtgagcagc 339

<210> 37

<211> 8

<212> PRT

<213> Artificial sequence

<400> 37

Arg Tyr Thr Tyr Thr Pro Tyr Cys

1 5

<210> 38

<211> 7

<212> PRT

<213> Artificial sequence

<400> 38

Ser Ala Val Asp Met Tyr Cys

1 5

<210> 39

<211> 9

<212> PRT

<213> Artificial sequence

<400> 39

Ser Glu Tyr Thr Ser Asn Leu Tyr Cys

1 5

<210> 40

<211> 9

<212> PRT

<213> Artificial sequence

<400> 40

Ser Glu Tyr Thr Tyr Asn Met Tyr Cys

1 5

<210> 41

<211> 6

<212> PRT

<213> Artificial sequence

<400> 41

Ser Gly Asp Phe Tyr Cys

1 5

<210> 42

<211> 9

<212> PRT

<213> Artificial sequence

<400> 42

Ser Gly Asp Thr Val Asn Met Tyr Cys

1 5

<210> 43

<211> 9

<212> PRT

<213> Artificial sequence

<400> 43

Ser Gly Phe Lys Phe Gly Thr Ser Val

1 5

<210> 44

<211> 9

<212> PRT

<213> Artificial sequence

<400> 44

Ser Gly Phe Thr Asp Ser Leu Tyr Cys

1 5

<210> 45

<211> 9

<212> PRT

<213> Artificial sequence

<400> 45

Ser Gly Phe Thr Phe Asp Asn Ser Asp

1 5

<210> 46

<211> 9

<212> PRT

<213> Artificial sequence

<400> 46

Ser Gly Ser Thr Gly Arg His Tyr Ser

1 5

<210> 47

<211> 6

<212> PRT

<213> Artificial sequence

<400> 47

Ser Gly Val Phe Tyr Cys

1 5

<210> 48

<211> 8

<212> PRT

<213> Artificial sequence

<400> 48

Ser Gly Tyr Ile Thr Ser Tyr Cys

1 5

<210> 49

<211> 9

<212> PRT

<213> Artificial sequence

<400> 49

Ser Gly Tyr Asn Tyr Asn Ser Ala Tyr

1 5

<210> 50

<211> 10

<212> PRT

<213> Artificial sequence

<400> 50

Ser Gly Tyr Thr Ser Cys Arg Arg Tyr Asp

1 5 10

<210> 51

<211> 9

<212> PRT

<213> Artificial sequence

<400> 51

Ser Thr Ser Thr Ser Ser Ile Tyr Cys

1 5

<210> 52

<211> 8

<212> PRT

<213> Artificial sequence

<400> 52

Thr Tyr Thr Tyr Ile Pro Tyr Cys

1 5

<210> 53

<211> 8

<212> PRT

<213> Artificial sequence

<400> 53

Thr Tyr Thr Tyr Thr Pro Tyr Cys

1 5

<210> 54

<211> 7

<212> PRT

<213> Artificial sequence

<400> 54

Ile Gly Ser Thr Arg Thr Pro

1 5

<210> 55

<211> 7

<212> PRT

<213> Artificial sequence

<400> 55

Ile His Thr Arg Gly Thr Thr

1 5

<210> 56

<211> 8

<212> PRT

<213> Artificial sequence

<400> 56

Ile Lys Thr Asp Thr Asp Arg Thr

1 5

<210> 57

<211> 8

<212> PRT

<213> Artificial sequence

<400> 57

Ile Asn Arg Asp Gly Thr Tyr Thr

1 5

<210> 58

<211> 8

<212> PRT

<213> Artificial sequence

<400> 58

Ile Arg Ala Gly Thr Asp Arg Thr

1 5

<210> 59

<211> 8

<212> PRT

<213> Artificial sequence

<400> 59

Ile Arg Ser Gly Gly Arg Ser Thr

1 5

<210> 60

<211> 8

<212> PRT

<213> Artificial sequence

<400> 60

Ile Arg Ser Gly Gly Arg Thr Ala

1 5

<210> 61

<211> 7

<212> PRT

<213> Artificial sequence

<400> 61

Ile Arg Ser Gly Arg Ile Ala

1 5

<210> 62

<211> 7

<212> PRT

<213> Artificial sequence

<400> 62

Ile Arg Ser Gly Arg Ile Thr

1 5

<210> 63

<211> 7

<212> PRT

<213> Artificial sequence

<400> 63

Ile Arg Ser Gly Arg Thr Ala

1 5

<210> 64

<211> 8

<212> PRT

<213> Artificial sequence

<400> 64

Ile Arg Ser Gly Thr Asp Arg Thr

1 5

<210> 65

<211> 8

<212> PRT

<213> Artificial sequence

<400> 65

Ile Ser Pro Ala Arg Gly Arg Thr

1 5

<210> 66

<211> 8

<212> PRT

<213> Artificial sequence

<400> 66

Ile Tyr Ser Cys Gly Arg Asp Thr

1 5

<210> 67

<211> 8

<212> PRT

<213> Artificial sequence

<400> 67

Ile Tyr Ser Gly Gly Gly Ser Thr

1 5

<210> 68

<211> 8

<212> PRT

<213> Artificial sequence

<400> 68

Val Ile Asp Gly Tyr Gly Gly Ile

1 5

<210> 69

<211> 24

<212> PRT

<213> Artificial sequence

<400> 69

Ala Ala Ala Leu Gly Leu Ser Arg Leu Arg Cys Asp Thr Gln Ser Ile

1 5 10 15

Ser Pro Asp Glu Tyr Asn Val Trp

20

<210> 70

<211> 22

<212> PRT

<213> Artificial sequence

<400> 70

Ala Ala Asp Gly Pro Phe Tyr Val Arg Thr Ser Cys Arg Asp Asp Tyr

1 5 10 15

Arg Gly Pro Ser Tyr Trp

20

<210> 71

<211> 12

<212> PRT

<213> Artificial sequence

<400> 71

Ala Ala Gly Asp Ser Gly Asn Cys Asn Asn Tyr Trp

1 5 10

<210> 72

<211> 0

<212> PRT

<213> Artificial sequence

<400> 72

<210> 73

<211> 12

<212> PRT

<213> Artificial sequence

<400> 73

Ala Ala Gly Asp Val Gly Ser Cys Asn Asn Tyr Trp

1 5 10

<210> 74

<211> 12

<212> PRT

<213> Artificial sequence

<400> 74

Ala Ala Gly His Ser Gly Leu Cys Thr Asp Tyr Arg

1 5 10

<210> 75

<211> 12

<212> PRT

<213> Artificial sequence

<400> 75

Ala Ala Gly His Ser Gly Leu Cys Thr Asp Tyr Ser

1 5 10

<210> 76

<211> 12

<212> PRT

<213> Artificial sequence

<400> 76

Ala Ala Gly Leu Ser Gly Ser Cys Thr Asn Tyr Trp

1 5 10

<210> 77

<211> 12

<212> PRT

<213> Artificial sequence

<400> 77

Ala Ala Gly Pro Ser Gly Ala Cys Thr Asp Tyr Trp

1 5 10

<210> 78

<211> 12

<212> PRT

<213> Artificial sequence

<400> 78

Ala Ala Gly Pro Ser Gly Leu Cys Thr Asp Tyr Trp

1 5 10

<210> 79

<211> 12

<212> PRT

<213> Artificial sequence

<400> 79

Ala Ala Gly Arg Ser Gly Thr Cys Asp Asp Tyr Trp

1 5 10

<210> 80

<211> 12

<212> PRT

<213> Artificial sequence

<400> 80

Ala Ala Gly Ser Thr Gly Tyr Cys Arg Asp Tyr Trp

1 5 10

<210> 81

<211> 25

<212> PRT

<213> Artificial sequence

<400> 81

Ala Ala Ser Val Ala Arg Arg Gly Ser Thr Leu Pro Gln Pro Leu Arg

1 5 10 15

Leu Gln Pro Ala Tyr Tyr Asn Lys Trp

20 25

<210> 82

<211> 11

<212> PRT

<213> Artificial sequence

<400> 82

Ala Lys Asp Trp Thr Pro Gly Thr Trp Pro Arg

1 5 10

<210> 83

<211> 12

<212> PRT

<213> Artificial sequence

<400> 83

Ala Val Gly Val Asp Gly Asp Cys Arg Asn Tyr Trp

1 5 10

<210> 84

<211> 12

<212> PRT

<213> Artificial sequence

<400> 84

Ala Val Gly Val Asp Gly Asn Cys Arg Asn Tyr Trp

1 5 10

<210> 85

<211> 17

<212> PRT

<213> Artificial sequence

<400> 85

Lys Ala Asp Gly Arg Arg Tyr Arg Gly Thr Cys Ser Ser Phe Ala Thr

1 5 10 15

Trp

<210> 86

<211> 19

<212> PRT

<213> Artificial sequence

<400> 86

Glu Ser Gly Gly Gly Ala Val Glu Ala Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Phe Ser

<210> 87

<211> 19

<212> PRT

<213> Artificial sequence

<400> 87

Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Ala Ala

<210> 88

<211> 19

<212> PRT

<213> Artificial sequence

<400> 88

Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Ala Ser

<210> 89

<211> 19

<212> PRT

<213> Artificial sequence

<400> 89

Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Ala Thr

<210> 90

<211> 19

<212> PRT

<213> Artificial sequence

<400> 90

Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Ala Val

<210> 91

<211> 19

<212> PRT

<213> Artificial sequence

<400> 91

Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Glu Val

<210> 92

<211> 19

<212> PRT

<213> Artificial sequence

<400> 92

Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Thr Ala

<210> 93

<211> 19

<212> PRT

<213> Artificial sequence

<400> 93

Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Thr Val

<210> 94

<211> 19

<212> PRT

<213> Artificial sequence

<400> 94

Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Ala Ala

<210> 95

<211> 19

<212> PRT

<213> Artificial sequence

<400> 95

Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Ala Thr

<210> 96

<211> 19

<212> PRT

<213> Artificial sequence

<400> 96

Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Glu Ser

<210> 97

<211> 19

<212> PRT

<213> Artificial sequence

<400> 97

Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly Ser Leu Arg Val Ser

1 5 10 15

Cys Ala Ala

<210> 98

<211> 17

<212> PRT

<213> Artificial sequence

<400> 98

Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala

1 5 10 15

Val

<210> 99

<211> 17

<212> PRT

<213> Artificial sequence

<400> 99

Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala

1 5 10 15

Ile

<210> 100

<211> 17

<212> PRT

<213> Artificial sequence

<400> 100

Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val Ala

1 5 10 15

Ser

<210> 101

<211> 17

<212> PRT

<213> Artificial sequence

<400> 101

Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val Ala

1 5 10 15

Thr

<210> 102

<211> 17

<212> PRT

<213> Artificial sequence

<400> 102

Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Ile Ala

1 5 10 15

Ser

<210> 103

<211> 17

<212> PRT

<213> Artificial sequence

<400> 103

Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala

1 5 10 15

Thr

<210> 104

<211> 17

<212> PRT

<213> Artificial sequence

<400> 104

Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val Ala

1 5 10 15

Ser

<210> 105

<211> 17

<212> PRT

<213> Artificial sequence

<400> 105

Met Ala Trp Phe Arg Gln Ala Pro Gly Asn Glu Arg Glu Ala Val Ala

1 5 10 15

Ser

<210> 106

<211> 17

<212> PRT

<213> Artificial sequence

<400> 106

Met Ala Trp Phe Arg Gln Thr Pro Gly Asn Glu Arg Glu Gly Val Ala

1 5 10 15

Ser

<210> 107

<211> 17

<212> PRT

<213> Artificial sequence

<400> 107

Met Gly Trp Tyr Arg Gln Ala Pro Gly Asn Glu Cys Glu Leu Val Ser

1 5 10 15

Lys

<210> 108

<211> 17

<212> PRT

<213> Artificial sequence

<400> 108

Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Arg Val Ser

1 5 10 15

Leu

<210> 109

<211> 16

<212> PRT

<213> Artificial sequence

<400> 109

Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser

1 5 10 15

<210> 110

<211> 38

<212> PRT

<213> Artificial sequence

<400> 110

Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Ile

1 5 10 15

Ala Glu Ser Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp

20 25 30

Thr Gly Met Tyr Tyr Cys

35

<210> 111

<211> 38

<212> PRT

<213> Artificial sequence

<400> 111

Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn

1 5 10 15

Gly Lys His Thr Met Tyr Leu Gln Met Asp Asn Leu Gln Pro Glu Asp

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210> 112

<211> 38

<212> PRT

<213> Artificial sequence

<400> 112

Val Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Leu Asp Ile

1 5 10 15

Ala Gln Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Asp Asp

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210> 113

<211> 38

<212> PRT

<213> Artificial sequence

<400> 113

Tyr Tyr Ala Asp Ala Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys

1 5 10 15

Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210> 114

<211> 38

<212> PRT

<213> Artificial sequence

<400> 114

Tyr Tyr Ala Asp Ser Val Lys Ala Arg Phe Thr Ile Ser His Asp Asn

1 5 10 15

Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

20 25 30

Thr Gly Val Tyr Tyr Cys

35

<210> 115

<211> 38

<212> PRT

<213> Artificial sequence

<400> 115

Tyr Tyr Ala Asp Ser Val Lys Ala Arg Phe Thr Ile Ser Leu Asp Asn

1 5 10 15

Ala Lys Asn Thr Val Tyr Leu Glu Met Asn Ser Leu Arg Pro Glu Asp

20 25 30

Thr Gly Val Tyr Tyr Cys

35

<210> 116

<211> 38

<212> PRT

<213> Artificial sequence

<400> 116

Tyr Tyr Ala Asp Ser Val Lys Ala Arg Phe Thr Ile Ser Gln Asp Asn

1 5 10 15

Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 117

<211> 38

<212> PRT

<213> Artificial sequence

<400> 117

Tyr Tyr Ala Asp Ser Val Lys Ala Arg Phe Thr Ile Ser Gln Asp Asn

1 5 10 15

Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

20 25 30

Thr Gly Val Tyr Tyr Cys

35

<210> 118

<211> 38

<212> PRT

<213> Artificial sequence

<400> 118

Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Gln Asp Asn

1 5 10 15

Ala Lys Ser Val Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210> 119

<211> 38

<212> PRT

<213> Artificial sequence

<400> 119

Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Ile

1 5 10 15

Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Met Leu Lys Pro Glu Asp

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210> 120

<211> 38

<212> PRT

<213> Artificial sequence

<400> 120

Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn

1 5 10 15

Ala Lys Asp Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210> 121

<211> 38

<212> PRT

<213> Artificial sequence

<400> 121

Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Lys

1 5 10 15

Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210> 122

<211> 38

<212> PRT

<213> Artificial sequence

<400> 122

Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Lys

1 5 10 15

Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210> 123

<211> 38

<212> PRT

<213> Artificial sequence

<400> 123

Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys

1 5 10 15

Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210> 124

<211> 38

<212> PRT

<213> Artificial sequence

<400> 124

Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Ala Lys Asp Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp

20 25 30

Ser Ala Met Tyr Tyr Cys

35

<210> 125

<211> 38

<212> PRT

<213> Artificial sequence

<400> 125

Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Arg

1 5 10 15

Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210> 126

<211> 38

<212> PRT

<213> Artificial sequence

<400> 126

Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Ala Lys Asn Met Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Asp Asp

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210> 127

<211> 38

<212> PRT

<213> Artificial sequence

<400> 127

Tyr Tyr Ser Asp Ser Val Ala Gly Arg Phe Thr Ile Ser Gln Asp Asn

1 5 10 15

Ala Lys Asn Thr Val Tyr Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 128

<211> 10

<212> PRT

<213> Artificial sequence

<400> 128

Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

Claims

1. An anti-IL-4 ra nanobody, characterized in that it comprises the following heavy chain complementarity determining regions: CDR1, CDR2 and CDR 3;

the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID Nos. 48, 55 and 80, or SEQ ID Nos. 41, 59 and 84 in sequence.

2. The nanobody against IL-4 ra according to claim 1, characterized in that it comprises the following framework regions: FR1, FR2, FR3 and FR 4.

3. The nanobody against IL-4 ra according to claim 2, characterized in that the skeleton regions FR1, FR2 and FR3 of said nanobody are represented by any one of the following (a) to (R):

(b) FR1 is shown as SEQ ID NO.88, FR2 is shown as SEQ ID NO.100, FR3 is shown as SEQ ID NO. 114;

(g) FR1 is shown as SEQ ID NO.91, FR2 is shown as SEQ ID NO.109, and FR3 is shown as SEQ ID NO. 111;

(k) FR1 is shown as SEQ ID NO.94, FR2 is shown as SEQ ID NO.106, and FR3 is shown as SEQ ID NO. 125;

(l) FR1 is shown as SEQ ID NO.92, FR2 is shown as SEQ ID NO.107, FR3 is shown as SEQ ID NO. 127;

(p) FR1 is shown as SEQ ID NO.90, FR2 is shown as SEQ ID NO.105, FR3 is shown as SEQ ID NO. 122;

(q) FR1 is shown as SEQ ID NO.88, FR2 is shown as SEQ ID NO.100, and FR3 is shown as SEQ ID NO. 116;

4. The nanobody against IL-4 ra according to claim 3, characterized in that it has the amino acid sequence shown in SEQ ID No.8 or 13.

5. Use of a nanobody against IL-4 ra according to any of claims 1 to 4 for the preparation of a medicament targeting IL-4 ra for the treatment of a disease selected from at least one of asthma, chronic obstructive pulmonary disease, atopic dermatitis and chronic primary urticaria.

6. A medicament, comprising the nanobody against IL-4 ra of any one of claims 1 to 4 and a pharmaceutically acceptable excipient.

7. An isolated nucleic acid molecule encoding the nanobody of any one of claims 1 to 4.

8. The isolated nucleic acid molecule of claim 7, wherein the base sequence of said nucleic acid molecule is set forth in SEQ ID No.26 or 31.

9. A vector comprising the nucleic acid molecule of claim 7 or 8.

10. A recombinant cell comprising the vector of claim 9.

11. A method of preparing the nanobody of any one of claims 1 to 4, which comprises: culturing the recombinant cell of claim 10, and separating and purifying the nanobody from the culture product.