A single-domain antibody against tslp and uses thereof

Abstract

This invention belongs to the field of immunology and relates to a single-domain antibody against TSLP and its uses. The single-domain antibody is composed of heavy chains, including heavy chain CDR1 shown in any one of SEQ ID NO:42-SEQ ID NO:48 or SEQ ID NO:73, heavy chain CDR2 shown in any one of SEQ ID NO:49-SEQ ID NO:50, and heavy chain CDR3 shown in any one of SEQ ID NO:51-SEQ ID NO:61 or SEQ ID NO:74-SEQ ID NO:75. Compared with the prior art, the beneficial effects of this invention are: this invention uses bioengineering technology to screen for single-domain antibodies specifically targeting TSLP, and the antibody affinity is good.

Description

Technical Field

[0001] This invention relates to a single-domain antibody (hereinafter abbreviated as "TSLP single-domain antibody") capable of specifically binding to TSLP, a pharmaceutical composition containing the single-domain antibody as an active ingredient, and its use in pharmaceutical therapy. Background Technology

[0002] Thymic stromal lymphopoietin (TSLP) is primarily produced by non-hematopoietic cells such as fibroblasts, epithelial cells, and various types of stromal or stromal-like cells. It mainly affects bone marrow cells, induces monocytes to release T cell-capturing chemokines, and enhances the maturation of myeloid (CD11c+) dendritic cells. Studies have shown that TSLP can also activate the maturation of a specific dendritic cell subset located in the epidermis—Langerhans cells. Thymic TSLP activation of myeloid and plasmacytoid (CD123+) dendritic cells leads to the production of regulatory T cells. TSLP generates signals through a heterodimeric receptor complex composed of the thymic stromal lymphocyte receptor CRLF2 and the IL-7Rα chain. Binding to STAT5 induces its phosphorylation, leading to the expression of upstream transcription factors. Research indicates that TSLP plays a crucial regulatory role in lymphocyte development and differentiation, particularly in the activation and differentiation of dendritic cells.

[0003] The TSLP receptor complex is a heterodimer composed of the TSLP receptor (TSLPR) and the IL-7 receptor α (IL-7Ra), primarily expressed on dendritic cells, CD4+ T cells, eosinophils, basophils, mast cells, and type 2 innate lymphocytes (ILC2). Myeloid dendritic cells are the cells that express both TSLPR and IL-7Rα most highly. After TSLP binds to its receptor on the surface of myeloid dendritic cells, these cells secrete IL-8 and eotaxin-2 to recruit neutrophils and eosinophils, and secrete TARC and MDC to recruit Th2 cells. Furthermore, TSLP-activated dendritic cells induce CD4+ T cells to differentiate into Th2 cells. Th2 cells can produce IL-4, IL-5, IL-13, and TNF. These cytokines promote the production of IgE, eosinophils, and mucus, initiating allergic reactions and triggering diseases such as asthma and atopic dermatitis. Furthermore, TSLP induced fibroblast aggregation and collagen deposition in animals, confirming its role in promoting fibrosis.

[0004] The role of TSLP in the development and maintenance of allergic diseases has been confirmed in animal models. Mice with TSLP signaling deficiency resist the development of asthma, and antibody neutralization of TSLP or its receptor is effective in murine or primate models of asthma or rhinitis. For example, in a primate asthma model (cynomolgus monkeys naturally susceptible to Ascaris suum antigen), blocking TSLP with anti-TSLPPRmAb reduced eosinophilic airway resistance and IL13 levels.

[0005] Asthma is a common chronic disease, and symptoms can be controlled in many patients using bronchodilators, inhaled or oral corticosteroids. However, most patients with moderate to severe asthma still experience symptoms or poor control, impacting their quality of life and incurring a significant healthcare burden. In particular, many patients with severe asthma may be unresponsive or poorly responsive to high doses of steroids. TSLP is overexpressed in the epithelium and lamina propria of the lungs of asthmatic patients, even in patients using high doses of inhaled corticosteroids. Strong supporting data for the importance of TSLP in asthma come from the allergen challenge study of the anti-TSLP monoclonal antibody (AMG157 / MEDI9929) in patients with mild asthma: significant efficacy was observed in early and late responses, measured by changes in FEV1 and eosinophil counts and FeNO levels in blood and sputum, after 6 or 12 weeks of treatment with AMG157 (once monthly).

[0006] Therefore, the development of antibodies that can specifically recognize and bind to TSLP is of great significance in the diagnosis, treatment, and prognosis of diseases involving TSLP. Currently, there is a lack of single-domain anti-TSLP antibody products with strong affinity and pharmaceutical value in existing technologies. Summary of the Invention

[0007] The purpose of this invention is to provide a single-domain antibody that can specifically bind to TSLP and its uses.

[0008] A first aspect of the present invention provides a single-domain antibody against TSLP, said single-domain antibody being composed of heavy chains, the heavy chains including heavy chain CDR1 shown in any one of SEQ ID NO:42-SEQ ID NO:48 or SEQ ID NO:73, heavy chain CDR2 shown in any one of SEQ ID NO:49-SEQ ID NO:50, and heavy chain CDR3 shown in any one of SEQ ID NO:51-SEQ ID NO:61 or SEQ ID NO:74-SEQ ID NO:75. The single-domain antibody against TSLP is a single-domain antibody targeting TSLP.

[0009] Preferably, the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are one of the following (1)-(15):

[0010] (1) CDR1 shown in SEQ ID NO:42, CDR2 shown in SEQ ID NO:50, and CDR3 shown in SEQ ID NO:57;

[0011] (2) CDR1 shown in SEQ ID NO:43, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:60;

[0012] (3) CDR1 shown in SEQ ID NO:43, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:59;

[0013] (4) CDR1 shown in SEQ ID NO:43, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:61;

[0014] (5) CDR1 shown in SEQ ID NO:44, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:54;

[0015] (6) CDR1 shown in SEQ ID NO:45, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:56;

[0016] (7) CDR1 shown in SEQ ID NO:45, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:58;

[0017] (8) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:52;

[0018] (9) CDR1 shown in SEQ ID NO:47, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:53;

[0019] (10) CDR1 shown in SEQ ID NO:47, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:58;

[0020] (11) CDR1 shown in SEQ ID NO:48, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:55;

[0021] (12) CDR1 shown in SEQ ID NO:48, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:51.

[0022] (13) CDR1 shown in SEQ ID NO:43, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:74;

[0023] (14) CDR1 shown in SEQ ID NO:43, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:75;

[0024] (15) CDR1 shown in SEQ ID NO:73, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:59.

[0025] All of the above sequences can be replaced with sequences that have "at least 80% homology" or sequences that replace only one or a few amino acids; preferably "at least 85% homology", more preferably "at least 90% homology", even more preferably "at least 95% homology", and most preferably "at least 98% homology".

[0026] In one embodiment, in any one or more CDRs of the heavy chain CDR1, CDR2, and CDR3, one to five arbitrary amino acid residues may be substituted with their conserved amino acids. Specifically, in the heavy chain CDR1, one to five amino acid residues may be substituted with their conserved amino acids; in the heavy chain CDR2, one to five amino acid residues may be substituted with their conserved amino acids; and in the heavy chain CDR3, one to five amino acid residues may be substituted with their conserved amino acids.

[0027] As used herein, the term "sequence homology" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is typically expressed as a percentage. Preferably, homology is determined over the overall length of the sequences being compared. Thus, two copies having completely identical sequences have 100% homology.

[0028] In some embodiments, the inventive objective can also be achieved by substituting only one or a few amino acids compared to the aforementioned sequence, for example, by including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions. These variations include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, most preferably up to 5) at the C-terminus and / or N-terminus. In practice, when determining the degree of sequence homology between two amino acid sequences or when determining the combination of CDR1, CDR2, and CDR3 in a single-domain antibody, those skilled in the art can consider so-called “conserved” amino acid substitutions, in which case the substitution would preferably be a conserved amino acid substitution. A conserved amino acid can generally be described as an amino acid residue substituted by another amino acid residue having a similar chemical structure, and this substitution has little or no effect on the function, activity, or other biological properties of the polypeptide. The conserved amino acid substitutions are common in the art. For example, a conserved amino acid substitution is the substitution of one or a few amino acids in the following groups (a)-(d) by another or a few amino acids in the same group: (a) polar negatively charged residues and their non-charged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. The particularly preferred conserved amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg or Gln; Phe is replaced by Met, Leu, or Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe or Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr, or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser. Furthermore, those skilled in the art will understand that the frame region sequences FR1-4 are not immutable, and the sequences of FR1-4 can adopt conserved sequence variants of the sequences disclosed in this invention.

[0029] The term "anti-TSLP single-domain antibody" in this invention includes not only complete single-domain antibodies but also fragments, derivatives, and analogs of said anti-TSLP single-domain antibodies. As used herein, the terms "fragment," "derivative," and "analyte" have the same meaning and refer to polypeptides that substantially retain the same biological function or activity as the antibodies of this invention. The polypeptide fragments, derivatives, or analogs of this invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with an Fc tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0030] In a preferred embodiment, the antibody sequence further includes a framework region FR; the framework region FR includes the amino acid sequences of FR1, FR2, FR3, and FR4; the amino acid sequences of the framework region FR are as follows:

[0031] FR1 or a variant thereof shown in any one of SEQ ID NO:27, SEQ ID NO:29-32, wherein the variant thereof contains a substitution of up to 5 amino acids in the FR1;

[0032] The FR2 or a variant thereof shown in any one of SEQ ID NO:33-36, wherein the variant thereof contains a substitution of up to 5 amino acids in the FR2;

[0033] FR3 or a variant thereof shown in any one of SEQ ID NO:37-40, wherein the variant thereof contains a substitution of up to 5 amino acids in the FR3;

[0034] The FR4 or a variant thereof shown in SEQ ID NO:41, wherein the variant thereof contains substitutions of up to 5 amino acids.

[0035] In a preferred embodiment, the amino acid sequence of the frame region FR is as follows:

[0036] FR1 or a variant thereof shown in any one of SEQ ID NO:27 or SEQ ID NO:30, wherein the variant thereof contains substitutions of up to 5 amino acids in the FR1;

[0037] FR2 or a variant thereof shown in any one of SEQ ID NO:28 or SEQ ID NO:35, wherein the variant thereof contains substitutions of up to 5 amino acids in the FR2;

[0038] FR3 or a variant thereof shown in either SEQ ID NO:72 or SEQ ID NO:39, wherein the variant thereof contains substitutions of up to 5 amino acids in the FR3;

[0039] The FR4 or a variant thereof shown in SEQ ID NO:41, wherein the variant thereof contains substitutions of up to 5 amino acids.

[0040] A second aspect of the present invention is to provide an amino acid sequence of a single-domain antibody capable of binding TSLP, wherein the amino acid sequences of the single-domain antibody are as shown in SEQ ID NO. 1-13, or the single-domain antibody has at least 80% sequence homology with the amino acid sequences of SEQ ID NO. 1-13 and is capable of specifically binding TSLP protein.

[0041] Alternatively, the amino acid sequence of the single-domain antibody is shown in any one of SEQ ID NO. 62-66, or the single-domain antibody has at least 80% sequence homology with the amino acid sequences of SEQ ID NO. 62-66 and is able to specifically bind to TSLP protein.

[0042] In one embodiment, the anti-TSLP single-domain antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology with an amino acid sequence selected from SEQ ID NO: 1-13 or SEQ ID NO: 62-66, and is capable of specifically binding to the TSLP protein.

[0043] A third aspect of the present invention is to provide an Fc fusion antibody or humanized antibody of any of the aforementioned anti-TSLP single-domain antibodies.

[0044] A fourth aspect of the present invention is to provide nucleotide molecules encoding the aforementioned anti-TSLP single-domain antibody, the aforementioned Fc fusion antibody, or the aforementioned humanized antibody, wherein the nucleotide sequences are as shown in SEQ ID NO: 14-26, or have at least 95% sequence homology with any one of SEQ ID NO: 14-26; or wherein the nucleotide sequences are as shown in SEQ ID NO: 67-71, or have at least 95% sequence homology with any one of SEQ ID NO: 67-71.

[0045] In one embodiment, the nucleic acid molecule encoding the anti-TSLP single-domain antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology with nucleotide sequences selected from SEQ ID NO: 14-26 or SEQ ID NO: 67-71, and the anti-TSLP single-domain antibody encoded therein is capable of specifically binding to the TSLP protein.

[0046] A fifth aspect of the present invention is to provide an expression vector comprising a nucleotide molecule encoding a single-domain antibody, an Fc fusion antibody, or a humanized antibody against TSLP, the nucleotide sequences of which are shown in SEQ ID NO: 14-26 or SEQ ID NO: 67-71, respectively.

[0047] In a preferred embodiment, the expression vector used is RJK-V4-hFC1 (a nucleotide molecule encoding a single-domain antibody against TSLP or its Fc fusion antibody or humanized antibody is integrated into RJK-V4-hFC1 by genetic engineering). Other universal expression vectors may also be selected as needed.

[0048] A sixth aspect of the present invention is to provide a host cell capable of expressing the aforementioned anti-TSLP single-domain antibody, Fc fusion antibody, or humanized antibody, or an expression vector comprising the aforementioned. Preferably, the host cell is a bacterial cell, fungal cell, or mammalian cell.

[0049] In another preferred embodiment, the host cell includes prokaryotic or eukaryotic cells, including bacteria and fungi.

[0050] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0051] In another preferred embodiment, the prokaryotic cells are selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or combinations thereof.

[0052] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomiae, Trichoderma, or combinations thereof.

[0053] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of insect cells such as armyworms, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or combinations thereof.

[0054] In another preferred embodiment, the host cell is a suspension ExpiCHO-S cell.

[0055] In another preferred embodiment, the host cell is a suspension 293F cell.

[0056] A seventh aspect of the present invention is to provide a recombinant protein comprising the aforementioned anti-TSLP single-domain antibody. The recombinant protein may be a single-domain antibody shown in SEQ ID NO. 1-13 or SEQ ID NO. 62-66, or a single-domain antibody having at least 80% homology with SEQ ID NO. 1-13 or SEQ ID NO. 62-66, or a multi-epitope antibody, a multi-specific antibody, or a multivalent antibody; for example, the multi-epitope antibody may consist of more than one sequence from SEQ ID NO. 1-13 or SEQ ID NO. 62-66; the multivalent antibody may consist of one sequence from SEQ ID NO. 1-13 or SEQ ID NO. 62-66 repeated a certain number of times; the multi-specific antibody includes, but is not limited to, the aforementioned bispecific antibody and trispecific antibody; furthermore, the recombinant protein may be a fragment, derivative, or analog of the aforementioned antibody.

[0057] An eighth aspect of the invention is to provide a pharmaceutical composition comprising the aforementioned TSLP-binding single-domain antibody and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically determined based on the isoelectric point of the antibody (the pH of the aqueous carrier medium must deviate from the isoelectric point of the antibody by approximately 2).

[0058] The pharmaceutical compositions of the present invention can be directly used to bind TSLP protein molecules, and therefore can be used to treat allergic diseases such as asthma. Furthermore, they can be used in combination with other asthma treatments.

[0059] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the aforementioned single-domain antibody and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.

[0060] A ninth aspect of the present invention is to provide a medicament for treating asthma, comprising the aforementioned single-domain antibody for binding TSLP protein as an active ingredient.

[0061] A tenth aspect of the present invention is to provide a kit for detecting TSLP levels, comprising the aforementioned anti-TSLP single-domain antibody. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc.

[0062] In a preferred embodiment, the kit includes an antibody that recognizes the TSLP protein, a lysis medium for dissolving the sample, and universal reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0063] In a preferred embodiment, the kit further contains a second antibody and an enzyme or fluorescent or radiolabeled marker for detection, as well as a buffer solution.

[0064] In a preferred embodiment, the second antibody in the kit may be an antibody (as an anti-antibody) of the aforementioned anti-TSLP single-domain antibody, and may be a single-domain antibody, monoclonal antibody, polyclonal antibody, or any other form of antibody.

[0065] In an eleventh aspect of the present invention, a method for generating a single-domain antibody against TSLP is provided, comprising the steps of:

[0066] (a) Under conditions suitable for the production of single-domain antibodies, the host cells described in the sixth aspect of the present invention are cultured, thereby...

[0067] Obtain a culture containing the single-domain antibody against TSLP; and

[0068] (b) Isolating or recovering the anti-TSLP single-domain antibody from the culture; and

[0069] (c) Optionally, purify and / or modify the single-domain antibody of TSLP obtained in step (b).

[0070] The twelfth aspect of the present invention is to provide the use of the aforementioned anti-TSLP single-domain antibody or the aforementioned pharmaceutical composition in the preparation of a medicament for treating a disease.

[0071] In a preferred embodiment, the disease is asthma, allergic asthma, nasal polyps, sinusitis, chronic rhinosinusitis with nasal polyps, chronic urticaria, chronic spontaneous urticaria, chronic obstructive pulmonary disease, bronchial disease, hypersensitivity reaction, immediate-type hypersensitivity reaction, lung disease, obstructive pulmonary disease, respiratory allergy, eosinophilic esophagitis, or atopic dermatitis.

[0072] Beneficial effects

[0073] Compared with the prior art, the beneficial effects of the present invention are:

[0074] (1) The single-domain antibody of the present invention is specifically targeted at TSLP protein with the correct spatial structure.

[0075] (2) The single-domain antibody obtained by the present invention has a flexible expression system. It can be expressed in a prokaryotic system or in a eukaryotic system of yeast cells or mammalian cells. Moreover, its expression cost in the prokaryotic expression system is low, which can reduce the production cost in the later stage.

[0076] (3) The single-domain antibody obtained by the present invention has simple multi-combination form modification. It can be obtained by simple tandem through genetic engineering to obtain multivalent and multispecific antibodies. Moreover, its immune heterogeneity is very low and it will not produce a strong immune response without humanization modification.

[0077] (4) The present invention provides a single-domain antibody with a wider affinity range. Before affinity maturation, its affinity range can be from nM to pM, providing multiple options for antibodies for different purposes in the later stage. Attached Figure Description

[0078] To more clearly illustrate the technical solution of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0079] Figure 1 The enrichment status of the library for screening TSLP-targeting antibodies in Example 3;

[0080] Figure 2 , Figure 3 , Figure 4 , Figure 5 All are antibody-antigen binding dose-response curves from Example 12 (partial clones);

[0081] Figure 6 , Figure 7 , Figure 8 , Figure 9 All are antibody-antigen binding dose-response curve determination diagrams from Example 12 (another part of the clone, Tab, hIgG);

[0082] Figure 10 , Figure 11 , Figure 12 , Figure 13 , Figure 14 , Figure 15 , Figure 16 , Figure 17 All images show the results of antibody (eukaryotic sample) neutralization-induced proliferation of TSLPR-IL7R-BaF3 cells (first batch of samples, Tab, hIgG);

[0083] Figure 18 , Figure 19 , Figure 20 , Figure 21 , Figure 22 , Figure 23 , Figure 24 All images show the results of antibody (eukaryotic sample) neutralization-induced proliferation of TSLPR-IL7R-BaF3 cells (second batch of samples, Tab, hIgG). Detailed Implementation

[0084] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0085] As used herein, “sdAb” (also referred to as nanobodies or VHH by the developer Ablynx) is well known to those skilled in the art. A single-domain antibody is an antibody whose complementarity-determining region is part of a single-domain polypeptide. Therefore, a single-domain antibody contains a single complementarity-determining region (a single CDR1, a single CDR2, and a single CDR3). Examples of single-domain antibodies include antibodies containing only heavy chains (which naturally do not contain light chains), single-domain antibodies derived from conventional antibodies, and engineered antibodies.

[0086] Single-domain antibodies can be derived from any species, including mice, humans, camels, llamas, goats, rabbits, and cattle. For example, naturally occurring VHH molecules can be derived from antibodies provided by camelid species such as camels, dromedaries, llamas, and guanacos. Like complete antibodies, single-domain antibodies can selectively bind to specific antigens. Single-domain antibodies may contain only variable domains of the immunoglobulin chain, which have CDR1, CDR2, and CDR3, as well as a frame region.

[0087] As used in this article, the term "Fc fusion antibody" refers to a novel protein produced by fusing the Fc fragment of a target antibody with a biologically active functional protein molecule using genetic engineering techniques.

[0088] The term "humanized antibody" refers to an antibody obtained by fusing the heavy chain variable region of a target antibody (such as an animal antibody) with the constant region of a human antibody, or by transplanting the complementarity-determining region (CDR1-3 sequence) of a target antibody into the variable region of a human antibody, or by mutating the target antibody according to the characteristics of the human antibody backbone region (FR1-4). Humanized antibodies can be produced using synthetic methods or site-directed mutagenesis methods.

[0089] In this invention, sequences with high homology to the CDR1-3 sequences disclosed herein can also yield single-domain antibodies against TSLP. In some embodiments, sequences having "at least 80% homology," or "at least 85% homology," "at least 90% homology," "at least 95% homology," or "at least 98% homology" with the sequences in SEQ ID NO: 1-13 and SEQ ID NO: 62-66 can achieve the purpose of the invention.

[0090] The preferred host cell of the present invention is a bacterial cell, a fungal cell, or a mammalian cell.

[0091] This patent involves preparing a target protein and a truncated form of the target protein using genetic engineering technology. The obtained antigen protein is then used to immunize Bactrian camels in Alashan, Inner Mongolia. After multiple immunizations, peripheral blood lymphocytes or spleen cells of the camels are obtained. Through genetic engineering, the variable region coding sequence of the camel-derived antibody is recombined into a phage display vector. Specific antibodies against the antigen protein are screened using phage display technology, and their ability to bind to the antigen and their application in the treatment of allergic diseases are further tested.

[0092] The above technical solution will now be broken down and explained in detail, and described with specific embodiments:

[0093] Example 1: Preparation of human TSLP protein:

[0094] The human TSLP protein used in this patent was expressed and purified by the company itself. The specific design scheme of the human TSLP protein expression vector is as follows:

[0095] (1) The coding sequence of TSLP was obtained from NCBI, with accession number NM_033035.4 and accession number NP_149024.1 for the amino acid sequence encoded by this sequence. The full name of the target is Thymic stromal lymphopoietin, and the Uniprot accession number is Q969D9.

[0096] (2) The full-length nucleotide sequence encoding the TSLP protein was cloned into the vector pcDNA3.4 using gene synthesis.

[0097] (3) The constructed vector was subjected to Sanger sequencing. After comparing with the original sequence and confirming that there were no errors, the recombinant plasmid was extracted in batches to remove endotoxins. It was then transfected with suspension 293F to express and purify the target protein. The purified protein had a purity of up to 90%, which met the needs of animal immunity.

[0098] Example 2: Construction of a single-domain antibody library targeting the TSLP protein:

[0099] One mg of the purified human recombinant TSLP protein obtained in Example 1 was mixed with an equal volume of Freund's complete adjuvant and used to immunize a Bactrian camel from Alashan, Inner Mongolia. The camel was immunized once a week for a total of 7 weeks. Except for the first immunization, the remaining six immunizations were performed by mixing one mg of TSLP protein with an equal volume of Freund's incomplete adjuvant. This immunization process was intended to concentrate the stimulation of the camel to produce antibodies against TSLP protein.

[0100] After animal immunization, 150 mL of peripheral blood lymphocytes were collected from camels, and RNA was extracted from the cells. cDNA was synthesized using the extracted total RNA, and VHH (antibody heavy chain variable region) was amplified using nested PCR with the cDNA as a template.

[0101] Then, the pMECS vector and VHH fragment were digested with restriction endonucleases, and the digested fragments were ligated to the vector. The ligated fragments were electroporated into competent TG1 cells to construct a phage display library of the TSLP protein, and the library size was determined to be approximately 1 × 10⁻⁶. 9 Meanwhile, the correct insertion rate of the target fragment in the library was detected by colony PCR.

[0102] The results showed that after PCR amplification of 30 colonies randomly selected from the library, 27 clones were able to amplify bands of the predicted size, while 3 clones amplified bands incorrectly. Therefore, the correct insertion rate was 27 ÷ 30 × 100% ≈ 90%.

[0103] Example 3: Screening for single-domain antibodies against the TSLP protein:

[0104] Take 200 μL of the recombinant TG1 cells from Example 2 and culture them in 2×TY medium. During the culture, add 40 μL of helper phage VCSM13 to infect the TG1 cells and culture them overnight to amplify the phage. The next day, precipitate the phage with PEG / NaCl and collect the amplified phage by centrifugation.

[0105] 500 μg of TSLP protein diluted in 100 mM pH 8.3 NaHCO3 was coupled onto an ELISA plate and incubated overnight at 4°C. A negative control well (culture medium control) was also included. The next day, 200 μL of 3% skim milk was added, and the plate was blocked at room temperature for 2 hours. After blocking, 100 μL of the amplified phage library (approximately 2 × 10⁻⁶) was added. 11 (1 phage particle), incubate at room temperature for 1 hour; after 1 hour, wash 15 times with PBS + 0.05% Tween-20 to remove unbound phage.

[0106] Phages that specifically bind to TSLP protein were dissociated using trypsin at a final concentration of 25 mg / mL and then used to infect E. coli TG1 cells in the logarithmic growth phase. The cells were cultured at 37°C for 1 h to produce and collect phages for the next round of screening. The same screening process was repeated once to gradually enrich the cells.

[0107] When the enrichment factor reaches 10 times or more, the enrichment effect is as follows: Figure 1 As shown.

[0108] Figure 1 In this context, P / N = the number of monoclonal bacteria grown from phages eluted from positive wells in the biopanning process after infecting TG1 bacteria / the number of monoclonal bacteria grown from phages eluted from negative wells after infecting TG1 bacteria. This parameter gradually increases after enrichment occurs. I / E = the total number of phages added to positive wells in each round of the biopanning process / the total number of phages eluted from positive wells in each round of the biopanning process. This parameter gradually approaches 1 after enrichment occurs.

[0109] Example 4: Screening for TSLP-specific positive clones using phage enzyme-linked immunosorbent assay (ELISA):

[0110] According to the screening method in Example 3 above, three rounds of screening were performed on single-domain antibodies against TSLP protein. The phage enrichment factor against TSLP protein reached more than 10. After screening, 384 single colonies were selected from the positive clones and inoculated into 96-well plates of 2×TY medium containing 100 μg / mL ampicillin. A blank control was set up. After incubation at 37°C to the logarithmic phase, IPTG was added to a final concentration of 1 mM and incubated overnight at 28°C.

[0111] Crude antibody was obtained using the osmotic burst method. TSLP recombinant protein was released into 100 mM NaHCO3 (pH 8.3), and 100 μg of protein was coated overnight at 4°C in an ELISA plate. 100 μL of the obtained crude antibody extract was transferred to an ELISA plate containing the antigen and incubated at room temperature for 1 h. Unbound antibody was washed away with PBST, and 100 μL of Mouse Anti-HA tag Antibody (HRP) (Mouse anti-HA horseradish peroxidase labeled antibody, ThermoFisher) diluted 1:2000 was added. The plate was incubated at room temperature for 1 h. Unbound antibody was washed away with PBST, and horseradish peroxidase chromogenic solution was added. The reaction was carried out at 37°C for 15 min, and then stop solution was added. The absorbance was read at 450 nm using an ELISA reader.

[0112] When the OD value of the sample well is more than 5 times that of the control well, it is determined to be a positive clone well. The bacteria in the positive clone well are transferred to LB medium containing 100 μg / mL ampicillin for plasmid extraction and sequencing.

[0113] Gene sequences of each clone were analyzed using the sequence alignment software VectorNTI. Clones with identical CDR1, CDR2, and CDR3 sequences were considered the same clone, while clones with different sequences were considered different clones. Ultimately, single-domain antibodies specifically targeting the TSLP protein (including 1A6, 1A7, 1D1, 1F3, 1F9, 1H5, 2D10, 2H3, 4A1, 4B2, 4F3, 1B7, 2G5, 214D11, 21H6, and 212C) were obtained. 7, 27H4, 23G12, and antibody clones (sequences not shown) 1F2, 1A4, 1B10, 1C5, 1D8, 1E3, 1F7, 1F11, 2A9, 2B8, 2D1, 23G1, 26A7, 26C1, 27E2, 27F5, 29G1, 211C3, 212C10, 212D1, 21A12, 21B7, 21G3, 23C10, 211B5, 21H10, 213B6, 22B5).

[0114] The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, constituting the entire VHH. The obtained single-domain antibody recombinant plasmid can be expressed in a prokaryotic system to ultimately obtain the single-domain antibody protein.

[0115] The amino acid sequences of the first batch of single-domain antibodies 1A6, 1A7, 1B7, 1D1, 1F3, 1F9, 1H5, 2D10, 2G5, 2H3, 4A1, 4B2, and 4F3 are shown in SEQ ID NO: 1-13, and their nucleotide sequences are shown in SEQ ID NO: 14-26. The amino acid sequences of the second batch of single-domain antibodies 212C7, 214D11, 21H6, 23G12, and 27H4 are shown in SEQ ID NO: 62-66, and their nucleotide sequences are shown in SEQ ID NO: 67-71.

[0116] The CDR and FR sequences of 18 single-domain antibodies are shown in Table 1-7. Since the single-domain antibody samples were divided into first and second batches for the experiment, and the FR and CDR sequences of the first and second batches of antibodies were listed separately, the FR and CDR sequences of the first batch are partially the same as those of the second batch.

[0117] Table 1. CDR1 sequences of 18 single-domain antibodies

[0118]

[0119] Table 2. CDR2 sequences of 18 single-domain antibodies

[0120]

[0121] Table 3. CDR3 sequences of 18 single-domain antibodies

[0122]

[0123] Table 4. FR1 sequences of 18 single-domain antibodies

[0124]

[0125] Table 5. FR2 sequences of 18 single-domain antibodies

[0126]

[0127] Table 6. FR3 sequences of 18 single-domain antibodies

[0128]

[0129] Table 7. FR4 sequences of 18 single-domain antibodies

[0130]

[0131] Example 5: Purification and expression of a specific single-domain antibody against TSLP protein in the host bacterium *Escherichia coli*.

[0132] The plasmids (pMECS-VHH) of different clones obtained from sequencing analysis in Example 4 were electroporated into Escherichia coli HB2151 and plated on LB+amp+glucose culture plates containing ampicillin and glucose, and incubated overnight at 37°C. Single colonies were selected and inoculated into 5 mL of LB culture medium containing ampicillin and incubated overnight on a shaker at 37°C.

[0133] Inoculate 1 mL of overnight cultured bacteria into 330 mL of TB culture medium and incubate at 37°C in a shaker. When the OD600nm value reaches 0.6-0.9, add 1 M IPTG and incubate overnight at 28°C in a shaker. Centrifuge to collect E. coli and obtain crude antibody extract using the osmotic rupture method.

[0134] The antibody was purified by nickel column affinity chromatography.

[0135] Example 6: Construction of a eukaryotic expression vector for an Fc fusion antibody against a single domain anti-TSLP antibody

[0136] (1) Subcloning the target sequence obtained in Example 4 into a eukaryotic expression vector: The antibody screened in Example 4 was obtained by Sanger sequencing to obtain its nucleotide sequence;

[0137] (2) The above nucleotide sequences (SEQ ID: 14-26, SEQ ID: 67-71 and other antibody nucleotide sequences not shown) were cloned into the vector RJK-V4-hFC1 designed and modified by our company by sequence synthesis to obtain a recombinant eukaryotic expression vector. The modification method of the vector is as described in Example 10.

[0138] (3) Transform the recombinant eukaryotic expression vector constructed in step (2) into DH5α Escherichia coli, culture it for plasmid extraction, and remove endotoxins;

[0139] (4) The extracted plasmids were then sequenced and identified.

[0140] (5) After confirming the recombinant vector, prepare it for subsequent eukaryotic cell transfection and expression. After expressing the Fc protein of VHH by the method of Example 7 or 8, purify the above antibody by the method of Example 9.

[0141] Example 7: Expression of single-domain antibody against TSLP protein in suspension ExpiCHO-S cells

[0142] (1) Three days before transfection, use 2.5×10 5 / mL cell passage and expansion culture ExpiCHO-S TM Cells, the calculated desired cell volume, were transferred to 120 mL (final volume) of fresh, preheated ExpiCHO. TM In a 500 mL shake flask of expression medium, the cell concentration was increased to approximately 4 × 10⁻⁶ cells / mL. 6 -6×10 6 live cells / mL;

[0143] (2) One day before transfection, ExpiCHO-S TM Cells were diluted to a concentration of 3.5 × 10⁻⁶. 6 Live cells / mL, incubate cells overnight;

[0144] (3) On the day of transfection, measure cell density and percentage of viable cells. Cell density should reach approximately 7 × 10⁻⁶ cells / day before transfection. 6 -10×10 6 live cells / mL;

[0145] (4) Use fresh ExpiCHO preheated to 37°C TM The expression medium was used to dilute the cells to 6 × 10⁶. 6 viable cells / mL. The calculated desired cell volume was transferred to 100 mL (final volume) of fresh, preheated ExpiCHO. TM In a 500 mL shake flask of expression medium;

[0146] (5) Gently invert to mix ExpiFectamine TM CHO reagent, using 3.7 mL OptiPRO TM Culture medium for diluting ExpiFectamine TM CHO reagent, vortex or mix well;

[0147] (6) Use 4 mL of refrigerated OptiPRO TM Dilute the plasmid DNA in the culture medium and vortex to mix.

[0148] (7) Incubate the ExpiFectamine CHO / plasmid DNA (the plasmid DNA is the Fc fusion antibody eukaryotic expression vector of the anti-TSLP single-domain antibody prepared in Example 6) complex at room temperature for 1-5 minutes, and then gently add it to the prepared cell suspension while gently shaking the flask during the addition process.

[0149] (8) The cells were cultured with shaking in humidified air at 37°C and 8% CO2.

[0150] (9) Add 600ul of ExpiFectamine on the first day after transfection (18-22 hours later). TM CHO Enhancer and 24mLExpiCHO feed.

[0151] (10) Collect the supernatant about 8 days after transfection (when cell viability is less than 70%).

[0152] Example 8: Expression of single-domain antibody against TSLP protein in suspension 293F cells

[0153] Recombinant single-domain antibody expression experimental procedure (taking a 500mL shake flask as an example):

[0154] (1) Three days before transfection, use 2.5×10 5 After passage and expansion of 293F cells at / mL, the calculated desired cell volume was transferred to a 500mL shake flask containing 120mL (final volume) of fresh, preheated OPM-293CD05 Medium to achieve a cell concentration of approximately 2×10⁻⁶. 6 -3×10 6 Live cells / mL.

[0155] (2) On the day of transfection, measure cell density and percentage of viable cells. Cell density should reach approximately 2 × 10⁻⁶ cells / day before transfection. 6 -3×10 6 Live cells / mL.

[0156] (3) Dilute the cells to 1×10⁻⁵ using preheated OPM-293CD05 Medium. 61 live cells / mL. Calculate the required cell volume and transfer it to a 500 mL shake flask containing 100 mL (final volume) of fresh, preheated culture medium.

[0157] (4) Dilute PEI (1 mg / mL) reagent with 4 mL Opti-MEM medium, and mix by swirling or pipetting; dilute plasmid DNA (the plasmid DNA is the eukaryotic expression vector of the Fc fusion antibody of the anti-TSLP single-domain antibody prepared in Example 6) with 4 mL Opt-MEM medium, mix by swirling, and filter with a 0.22 μm filter. Incubate at room temperature for 5 min.

[0158] (5) Add the diluted PEI reagent to the diluted DNA and mix by inverting. Incubate the PEI / plasmid DNA complex at room temperature for 15-20 minutes, then gently add it to the prepared cell suspension while gently shaking the flask during the addition process.

[0159] (6) Culture the cells at 37°C, 5% CO2, and 120 rpm with shaking.

[0160] (7) Add 5 mL of OPM-CHO PFF05 feed at 24 h and 72 h after transfection.

[0161] (8) Collect the supernatant about 7 days after transfection (when cell viability is less than 70%).

[0162] Example 9: Purification of single-domain antibody against TSLP protein

[0163] (1) The protein expression supernatant obtained in Example 7 or 8 was filtered with a 0.45 μm disposable filter to remove insoluble impurities;

[0164] (2) The above filtrate was purified by affinity chromatography using a protein purifier. The agarose packing material coupled with Protein A was used to purify the filtrate by utilizing the ability of human Fc to bind to Protein A.

[0165] (3) Pass the filtrate through a pre-packed Protein A column at a flow rate of 1 mL / min. In this step, the target protein in the filtrate will bind to the packing material.

[0166] (4) Wash the impurity proteins bound to the column with low-salt and high-salt buffers;

[0167] (5) Use a low pH buffer to systemically bind the target protein on the column;

[0168] (6) Quickly add the eluent to a Tris-HCl solution with a pH of 9.0 to neutralize it;

[0169] (7) After dialysis of the above-neutralized protein solution, perform SDS-PAGE analysis to confirm that the protein purity is above 95% and the concentration is above 0.5 mg / mL, and then store it at low temperature for later use.

[0170] Example 10: Construction of the single-domain antibody eukaryotic expression vector RJK-V4-hFC1

[0171] The aforementioned universal target vector for nanobodies, RJK-V4-hFC1, is a modified version of Invitrogen's commercial vector pCDNA3.4 (vector data link: https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / pcdna3_4_topo_ta_cloning_kit_man.pdf) by incorporating the Fc region of the human IgG1 heavy chain coding sequence. Specifically, this vector contains the CH2 and CH3 hinge regions of the IgG1 heavy chain. The specific modification scheme is as follows:

[0172] (1) Select the restriction enzyme sites XbaI and AgeI on pcDNA3.4;

[0173] (2) Multiple cloning sites (MCS) and 6×His tags were introduced at the 5' and 3' ends of the Fc fragment coding sequence, respectively, by overlapping PCR.

[0174] (3) The above fragment was amplified by PCR using a pair of primers with XbaI and AgeI restriction sites respectively;

[0175] (4) The recombinant DNA fragments in pcDNA3.4 and (3) were digested with restriction endonucleases XbaI and AgeI, respectively;

[0176] (5) The digested vector and the insert fragment were ligated with T4 ligase, and then the ligation product was transformed into E. coli, amplified, sequenced and verified to obtain the recombinant plasmid.

[0177] Example 11: Expression and purification of a tool antibody (Tab) targeting human TSLP

[0178] In this paper, Tab1 is tezepelumab, and the sequence is from IMGT; Tab2 (TSLP Fab1) was synthesized according to the method in US10000561B2.

[0179] The searched sequences were commissioned to General Biosystems (Anhui) Co., Ltd. for codon optimization in mammalian cell expression systems and cloned into the pcDNA3.1 vector. After antibiotic selection, plasmid-positive bacteria were selected for amplification, and plasmids were extracted using a plasmid extraction kit (Macherey Nagel, Cat#740412.50). 100 μg of plasmid (40 μg heavy chain + 60 μg light chain) was added per 100 mL of cells, and PEI was used to transiently express the plasmid in 293F cells (medium: FreeStyle 293Expression medium, Thermo, Cat#12338026+F-68, Thermo, Cat#24040032). 6–24 h after transfection, 5% volume of 10% Peptone (Sigma, Cat#P0521-100G) was added, and the cells were cultured in 8% CO2 at 130 rpm for approximately 7–8 days. When cell viability decreased to 50%, the expression supernatant was collected and purified using a gravity column with Protein A (GE, Cat#17-5438-02). After dialysis with PBS, the concentration was determined using Nanodrop, purity was identified by SEC, and binding capacity was verified by indirect ELISA.

[0180] The Tab obtained by this method has a concentration of not less than 2 mg / ml and a purity greater than 95%.

[0181] Example 12: Determination of Antibody-Antigen Binding Dose-Reaction Curve

[0182] This embodiment uses the standard enzyme-linked immunosorbent assay (ELISA) procedure.

[0183] (1) Coat 50 μL of 1 μg / mL TSLP protein and incubate overnight at 4°C.

[0184] (2) Wash the plate; add 200 μL of 5% milk and seal at 37°C for 2 hours.

[0185] (3) VHH-hFc was diluted to 2 μg / mL, and then the antibody was serially diluted 5-fold to a total of 8 concentration gradients. Here, VHH-hFc refers to the Fc fusion antibody of the single-domain antibody against TSLP protein prepared in Example 8 (expressed in 293F cells) purified in Example 9. In addition, hIgG and Tab controls were set up respectively; Tab1 and Tab2 were prepared in Example 11;

[0186] (4) Wash the plate; add 50 μL of the single-domain antibody obtained by dilution in step (3), double replicates, and incubate at 37°C for 1 h.

[0187] (5) Wash the plate; add 50 μL of HRP-Goat anti hIgG secondary antibody and incubate at 37°C for 30 min.

[0188] (6) Wash the plate (wash several times); add 50 μL of TMB that has been brought to room temperature beforehand, and react at room temperature in the dark for 15 min.

[0189] (7) Add 50 μL of stop solution (1N HCl) and save the microplate reader reading.

[0190] (8) Plot the curve and calculate EC50, as follows: Figures 2-9 As shown, hIgG refers to the isotype control, an immunoglobulin molecule that does not bind to any target and is obtained through commercial purchase.

[0191] in, Figures 2-5 Results for the first batch of samples (including antibody strains with sequences not shown: 1A6, 1A7, 1D1, 1F3, 1F9, 1H5, 2D10, 2H3, 4A1, 4B2, 4F3, 1B7, 2G5, and others). Figures 6-9 Results for the second batch of samples (containing antibody clones with sequences not shown, including 214D11, 21H6, 212C7, 27H4, 23G12, and others).

[0192] It is evident that all 18 single-domain antibodies of this invention exhibit excellent binding efficacy and specificity to TSLP protein.

[0193] Example 13: Antibody (eukaryotic sample) neutralization induces TSLPR-IL7R-BaF3 cell proliferation

[0194] The following operations shall be performed in accordance with methods generally known to those skilled in the art:

[0195] The steps are as follows: After revival, passage TSLPR-IL7R-BaF3 3-4 times... ce11 cells were seeded at 10,000 cells per well in 96-well plates. A 10 μg / L solution of the Fc fusion protein (VHH-hFc) of the single-domain antibody provided in Example 8 (VHH-hFc refers to the Fc fusion antibody of the anti-TSLP protein single-domain antibody prepared in Example 8 (expressed in 293F cells) purified in Example 9) was prepared and serially diluted 5-fold. The serially diluted Tab, single-domain antibody, and TSLP protein at an EC80 concentration (0.5 ng / ml) obtained in the proliferation experiment were mixed at a 1:1 ratio to prepare a mixture. The mixture was added to the cell culture wells in an equal volume to the cell culture medium. After incubation for 72 h, cell viability was detected using a luminescent cell viability assay kit. Based on the detection results, the EC50 concentration of Tab and different single-domain antibodies in neutralizing TSLP-induced TSLPR-IL7R-BaF3cel1 proliferation was calculated. Since the first and second batches of samples were tested separately, all Tab samples were also tested twice. TSLP Ab(AF1398) is used to estimate whether Tab can be used normally in the system.

[0196] in, Figures 10-17 Results for the first batch of samples (including 1A6, 1A7, 1D1, 1F3, 1F9, 1H5, 2D10, 2H3, 4A1, 4B2, 4F3, 1B7, 2G5, and antibody clones, Tabs, and hIgG with sequences not shown). Figures 18-24 The results are for the second batch of samples (containing 214D11, 21H6, 212C7, 27H4, 23G12, and antibody clones, Tab, and hIgG whose sequences are not shown).

[0197] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. sequence list <110> Nanjing Rongjiekang Biotechnology Co., Ltd. <120> A single-domain antibody against TSLP and its uses <160> 75 <170> SIPOSequenceListing 1.0 <210> 1 <211> 120 <212> PRT <213> Artificial Sequence <400> 1 Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Thr Ser Arg Tyr Thr Tyr Gly Ser Ser Cys Met Ala Trp Phe 20 25 30 Arg Gln Ser Pro Gly Lys Glu Arg Glu Gln Val Ala Gly Ile Ala Met 35 40 45 Gly Val Val Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 50 55 60 Ser Gln Asp Asn Gly Lys Asn Thr Val Asp Leu Gln Met Asp Asn Leu 65 70 75 80 Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Pro Pro His 85 90 95 Tyr Lys Cys Tyr Ser Val Ser Gly Gly Arg Tyr Asn Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 2 <211> 120 <212> PRT <213> Artificial Sequence <400> 2 Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Thr Pro Arg Tyr Thr Tyr Gly Thr Ser Cys Met Gly Trp Phe 20 25 30 Arg Gln Ser Pro Gly Tyr Gln Arg Glu Gln Val Ala Gly Ile Ala Met 35 40 45 Gly Val Val Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 50 55 60 Ser Gln Asp Asn Gly Lys Asn Thr Val Asp Leu Gln Met Asp Asn Leu 65 70 75 80 Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Pro Ala His 85 90 95 Tyr Lys Cys Tyr Ser Ala Ser Gly Gly Arg Tyr Asn Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 3 <211> 121 <212> PRT <213> Artificial Sequence <400> 3 Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Thr Thr Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Ile Gly Trp Phe 20 25 30 Arg Gln Ala Pro Gly Lys Glu Arg Glu Glu Ile Ala Thr Ile Ser Arg 35 40 45 Val Gly Glu Ile Thr Phe Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 50 55 60 Ile Ser Gln Asp Asn Thr Glu Thr Thr Val Phe Leu Gln Met Asn Ser 65 70 75 80 Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Ser Pro Pro 85 90 95 Thr Tyr Gly Gly Cys Pro Thr Arg Ser Arg Asp Tyr Asp Lys Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 4 <211> 120 <212> PRT <213> Artificial Sequence <400> 4 Glu Ser Gly Gly Gly Ser Val Gln Val Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Thr Ser Arg Phe Thr Tyr Gly Ser Ala Cys Met Gly Trp Phe 20 25 30 Arg Gln Ser Pro Gly Lys Glu Arg Glu Gln Val Ala Gly Ile Ala Met 35 40 45 Gly Val Val Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 50 55 60 Ser Gln Asp Asn Gly Lys Asn Thr Val Asp Leu Gln Met Asp Asn Leu 65 70 75 80 Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Thr Gly Pro Ala His 85 90 95 Tyr Lys Cys Tyr Ser Ala Ser Gly Gly Arg Tyr Ser Asp Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 5 <211> 122 <212> PRT <213> Artificial Sequence <400> 5 Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Thr Ser Thr Ser Arg Phe Thr Tyr Gly Ser Ala Cys Met Gly 20 25 30 Trp Phe Arg Gln Ser Pro Gly Lys Glu Arg Glu Gln Val Ala Gly Ile 35 40 45 Ala Met Gly Val Val Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe 50 55 60 Thr Ile Ser Gln Asp Asn Gly Lys Asn Thr Val Asp Leu Gln Met Asp 65 70 75 80 Asn Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Pro 85 90 95 Ala His Tyr Asn Cys Tyr Ser Ala Ser Gly Gly Arg Tyr Asn Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 6 <211> 118 <212> PRT <213> Artificial Sequence <400> 6 Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Thr Ser Arg Tyr Thr Tyr Asn Cys Met Gly Trp Phe Arg Gln 20 25 30 Ser Pro Gly Lys Glu Arg Glu Gln Val Ala Gly Ile Ala Met Gly Val 35 40 45 Val Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln 50 55 60 Asp Asn Gly Lys Asn Thr Val Asp Leu Gln Met Asp Asn Leu Lys Pro 65 70 75 80 Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Pro Ala His Tyr Lys 85 90 95 Cys Tyr Ser Pro Ser Gly Gly Arg Tyr Asn Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 7 <211> 118 <212> PRT <213> Artificial Sequence <400> 7 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Thr Ser Arg Tyr Thr Tyr Asn Cys Met Gly Trp Phe Arg Gln 20 25 30 Ser Pro Gly Lys Glu Arg Glu Gln Val Ala Gly Ile Ala Met Gly Val 35 40 45 Val Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln 50 55 60 Asp Asn Gly Lys Asn Thr Val Asp Leu Gln Met Asp Asn Leu Lys Pro 65 70 75 80 Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Pro Ala His Tyr Lys 85 90 95 Cys Tyr Ser Pro Ser Gly Gly Arg Tyr Asn Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 8 <211> 120 <212> PRT <213> Artificial Sequence <400> 8 Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Thr Ser Arg Phe Thr Tyr Gly Ser Ala Cys Met Gly Trp Phe 20 25 30 Arg Gln Ser Pro Gly Lys Glu Arg Glu Gln Val Ala Gly Ile Ala Met 35 40 45 Gly Val Val Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 50 55 60 Ser Gln Asp Asn Gly Lys Asn Thr Val Asp Leu Gln Met Asp Asn Leu 65 70 75 80 Lys Pro Asp Asp Thr Ala Met Tyr Tyr Cys Ala Thr Gly Pro Ala His 85 90 95 Tyr Lys Cys Tyr Ser Ala Ser Gly Gly Arg Tyr Asn Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 9 <211> 120 <212> PRT <213> Artificial Sequence <400> 9 Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Thr Ser Arg Phe Thr Tyr Gly Ser Ala Cys Met Gly Trp Phe 20 25 30 Arg Gln Ser Pro Gly Lys Glu Arg Glu Gln Val Ala Gly Ile Ala Met 35 40 45 Gly Val Val Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 50 55 60 Ser Gln Asp Asn Gly Lys Asn Thr Val Asp Leu Gln Met Asp Asn Leu 65 70 75 80 Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Thr Gly Pro Ala His 85 90 95 Tyr Asn Cys Tyr Ser Ala Ser Gly Gly Arg Tyr Asn Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 10 <211> 122 <212> PRT <213> Artificial Sequence <400> 10 Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Thr Ser Thr Ser Arg Phe Thr Tyr Gly Ser Ala Cys Met Gly 20 25 30 Trp Phe Arg Gln Ser Pro Gly Lys Glu Arg Glu Gln Val Ala Gly Ile 35 40 45 Ala Met Gly Val Val Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe 50 55 60 Thr Ile Ser Gln Asp Asn Gly Lys Asn Thr Val Asp Leu Gln Met Asp 65 70 75 80 Asn Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Pro 85 90 95 Ala His Tyr Lys Cys Tyr Ser Ala Ser Gly Gly Arg Tyr Asn Ile Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 11 <211> 118 <212> PRT <213> Artificial Sequence <400> 11 Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Thr Ser Arg Tyr Thr Tyr Asn Cys Met Gly Trp Phe Arg Gln 20 25 30 Ser Pro Gly Lys Glu Arg Glu Gln Val Ala Gly Ile Ala Met Gly Val 35 40 45 Val Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln 50 55 60 Asp Asn Gly Lys Asn Thr Val Asp Leu Gln Met Asp Asn Leu Lys Pro 65 70 75 80 Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ser Gly Pro Pro His Tyr Lys 85 90 95 Cys Tyr Ser Ala Ser Gly Gly Arg Tyr Asn Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 12 <211> 120 <212> PRT <213> Artificial Sequence <400> 12 Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Thr Ser Arg Tyr Thr Tyr Gly Ser Ala Cys Met Gly Trp Phe 20 25 30 Arg Gln Ser Pro Gly Lys Glu Arg Glu Gln Val Ala Gly Ile Ala Met 35 40 45 Gly Val Val Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 50 55 60 Ser Gln Asp Asn Gly Lys Asn Thr Val Asp Leu Gln Met Asp Asn Leu 65 70 75 80 Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Pro Ala His 85, 90, 95 Tyr Leu Cys Tyr Ser Ala Ser Gly Gly Arg Tyr Thr Ser Trp Gly Gln 100, 105, 110 Gly Thr Gln Val Thr Val Ser Ser 115, 120 <210> 13 <211> 120 <212> PRT <213> Artificial Sequence <400> 13 Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser 1, 5, 10, 15 Cys Ala Thr Ser Arg Tyr Thr Tyr Gly Ser Ser Cys Met Gly Trp Phe 20, 25, 30 Arg Gln Ser Pro Gly Lys Glu Arg Glu Gln Val Ala Gly Ile Ala Met 35, 40, 45 Gly Val Val Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 50, 55, 60 Ser Gln Asp Asn Asn Lys Asn Thr Val Asp Leu Gln Met Asp Asn Leu 65, 70, 75, 80 Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ser Gly Pro Pro His 85, 90, 95 Tyr Lys Cys Tyr Ser Ala Ser Gly Gly Arg Tyr Asn Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 14 <211> 360 <212> DNA <213> Artificial Sequence <400> 14 gagtctgggg gaggctcggt gcaggctgga gggtctctga gactatcctg tgcaacctct 60 agatacacct acggtagcag ctgcatggcc tggttccgcc agtctccagg aaaagagcgc 120 gagcaggtcg caggtattgc tatgggtgtt gtcacatact atgccgactc cgtgaagggc 180 cggttcacca tctcccaaga caacggcaag aacacggtgg atctgcaaat ggacaacctg 240 aaacctgagg acactgccat gtactactgt gcggcggggc ccccgcacta taagtgctat 300 tcagtgtccg gagggcggta taactactgg ggccagggga cccaggtcac cgtctcctca 360 <210> 15 <211> 360 <212> DNA <213> Artificial Sequence <400> 15 gagtctggag gaggctcggt gcaggctgga gggtctctga gactatcctg tgcaacccca 60 agatacacct acggtaccag ctgcatgggc tggttccgtc agtctccagg ataccagcgc 120 gagcaggtcg caggtattgc tatgggtgtt gtcacatact atgccgactc cgtgaagggc 180 cggttcacca tctcccaaga caacggcaag aacacggtgg atctgcaaat ggacaacctg 240 aaacctgagg acactgccat gtactactgt gcggcggggc ccgcgcacta taagtgctat 300 tcagcgtccg gagggcggta taactactgg ggccagggga cccaggtcac cgtctcctca 360 <210> 16 <211> 363 <212> DNA <213> Artificial Sequence <400> 16 gagtctggag gaggctcggt gcaggctgga ggctctctga gactctcctg tacgacctct 60 ggatacacct acagtagtaa ttgtatcggc tggttccgcc aggctccagg gaaggagcgc 120 gaggagatcg caaccattag tcgtgtaggt gaaatcacat tctatgccga ctccgtgaag 180 ggccgattca ccatctccca agacaacacc gagaccacgg tgtttctgca aatgaacagc 240 ctgaaacctg aggacactgc cgtgtactac tgtgcggcaa gtcccccaac gtacggtgga 300 tgccccactc ggtctcgaga ctatgacaaa tggggccagg ggacccaggt caccgtctcc 360 tca 363 <210> 17 <211> 360 <212> DNA <213> Artificial Sequence <400> 17 gagtctggag gaggctcggt gcaggttgga gggtctctga gactatcctg tgcaacctct 60 agatttacct acggtagcgc ctgcatgggc tggttccgcc agtctccagg aaaggagcgc 120 gagcaggtcg caggtattgc tatgggtgtt gtcacatact atgccgactc cgtgaagggc 180 cggttcacca tctcccaaga caacggcaag aacacggtgg atctgcaaat ggacaacctg 240 aaacctgagg acactgccat gtactactgt gcgacggggc ccgcgcacta taagtgctat 300 tcagcgtccg gagggcggta tagcgactgg ggccagggga cccaggtcac cgtctcctca 360 <210> 18 <211> 366 <212> DNA <213> Artificial Sequence <400> 18 gagtctgggg gaggctcggt gcaggctgga gggtctctga gactatcctg tgcaacctct 60 acctctagat tcacctacgg tagcgcctgc atgggctggt tccgccagtc tccaggaaag 120 gagcgcgagc aggtcgcagg tattgctatg ggtgttgtca catactatgc cgactccgta 180 aagggccggt tcaccatctc ccaagacaac ggcaagaaca cggtggatct gcaaatggac 240 aacctgaaac ctgaggacac tgccatgtac tactgtgcgg cggggcccgc ccactataac 300 tgctattcag cgtccggagg gcggtataac tactggggcc aggggaccca ggtcaccgtc 360 tcctca 366 <210> 19 <211> 354 <212> DNA <213> Artificial Sequence <400> 19 gagtctgggg gaggctcggt acaggctgga gggtctctga gactatcctg tgcaacctct 60 agatacacct acaactgcat gggctggttc cgccagtctc caggaaagga gcgcgagcag 120 gtcgcaggta ttgctatggg tgttgtcaca tactatgccg actccgtgaa gggccggttc 180 accatctccc aagacaacgg caagaacacg gtggatctgc aaatggacaa cctgaaacct 240 gaggacactg ccatgtacta ctgtgcggcg gggcccgcac actataagtg ctattcaccg 300 tccggagggc ggtataacta ctggggccag gggacccagg tcaccgtctc ctca 354 <210> 20 <211> 354 <212> DNA <213> Artificial Sequence <400> 20 gagtctgggg gaggcttggt gcagcctggg gggtctctga gactatcctg tgcaacctct 60 agatacacct acaactgcat gggctggttc cgccagtctc caggaaagga gcgcgagcag 120 gtcgcaggta ttgctatggg tgttgtcaca tactatgccg actccgtgaa gggccggttc 180 accatctccc aagacaacgg caagaacacg gtggatctgc aaatggacaa cctgaaacct 240 gaggacactg ccatgtacta ctgtgcggcg gggcccgcac actataagtg ctattcaccg 300 tccggagggc ggtataacta ctggggccag gggacccagg tcaccgtctc ctca 354 <210> 21 <211> 360 <212> DNA <213> Artificial Sequence <400> 21 gagtctggag gaggctcggt gcaggctgga gggtctctga gactatcctg cgcaacctct 60 agatttacct acggtagcgc ctgcatgggc tggttccgcc agtctccagg aaaggagcgc 120 gagcaggtcg caggtattgc tatgggtgtt gtcacatact atgccgactc cgtgaagggc 180 cggttcacca tctcccaaga caacggcaag aacacggtgg atctgcaaat ggacaacctg 240 aaacctgacg acactgctat gtactactgt gcgacggggc ccgcgcacta taagtgctat 300 tcagcgtccg gagggcggta taactactgg ggccagggga cccaggtcac cgtctcctca 360 <210> 22 <211> 360 <212> DNA <213> Artificial Sequence <400> 22 gagtctggag gaggctcggt gcaggctgga gggtctctga gactatcctg tgcaacctct 60 agattcacct acggtagcgc ctgcatgggc tggttccgcc agtctccagg aaaggagcgc 120 gagcaggtcg caggtattgc tatgggtgtt gtcacatact atgccgactc cgtgaagggc 180 cggttcacca tctcccaaga caacggcaag aacacggtgg atctgcaaat ggacaacctg 240 aaacctgagg acactgccat gtactactgt gcgacggggc ccgcgcacta taattgctat 300 tcagcgtccg gagggcggta taactactgg ggccagggga cccaggtcac cgtctcctca 360 <210> 23 <211> 366 <212> DNA <213> Artificial Sequence <400> 23 gagtctggag gaggctcggt gcaggctgga gggtctctga gactatcctg tgcaacctct 60 acctctagat tcacctacgg tagcgcctgc atgggctggt tccgccagtc tccaggaaag 120 gagcgcgagc aggtcgcagg tattgctatg ggtgttgtca catactatgc cgactccgtg 180 aagggccggt tcaccatctc ccaagacaac ggcaagaaca cggtggatct gcaaatggac 240 aacctgaaac ctgaggacac tgccatgtac tactgtgcgg cggggcccgc gcactataag 300 tgctattcag cgtccggagg gcggtataac atctggggcc aggggaccca ggtcaccgtc 360 tcctca 366 <210> 24 <211> 354 <212> DNA <213> Artificial Sequence <400> 24 gagtctggag gaggctcggt gcaggctgga gggtctctga gactatcctg tgcaacctct 60 agatacacct acaactgcat gggctggttc cgccagtctc caggaaagga gcgcgagcag 120 gtcgcaggta ttgctatggg tgttgtcaca tactatgccg actccgtgaa gggccggttc 180 accatctccc aagacaacgg caagaacacg gtggatctgc aaatggacaa cctgaaacct 240 gaggacactg ccatgtacta ctgtgcgtcg gggcccccgc attataagtg ctattcagcg 300 tccggagggc ggtataacta ctggggccag gggacccagg tcaccgtctc ctca 354 <210> 25 <211> 360 <212> DNA <213> Artificial Sequence <400> 25 gagtctggag gagggtcggt gcaggctgga gggtctctga gactatcctg tgcaacctct 60 agatacacct acggtagcgc gtgcatgggc tggttccgcc agtctccagg aaaggagcgc 120 gaacaggtcg caggtattgc tatgggtgtt gtcacatact atgccgactc cgtgaagggc 180 cggttcacca tctcccaaga caacggcaag aacacggtgg atctgcaaat ggacaacctg 240 aaacctgagg acactgccat gtactactgt gcggcgggac ccgcgcacta tttgtgctat 300 tcagcgtccg gagggcggta tacgtcctgg ggccagggga cccaggtcac cgtctcctca 360 <210> 26 <211> 360 <212> DNA <213> Artificial Sequence <400> 26 gagtctgggg gaggctcggt gcaggctgga gggtctctga gactatcctg tgcaacctct 60 agatacacct acggtagttc ctgcatgggc tggttccgcc agtctccagg aaaggagcgc 120 gagcaggtcg cgggtattgc tatgggggtt gtcacatact atgccgactc cgtgaagggc 180 cggttcacca tctcccaaga caacaacaag aacacggtgg atctgcaaat ggacaacctg 240 aaacctgagg acactgccat gtactactgt gcgtcggggc ccccgcatta taagtgctat 300 tcagcgtccg gagggcggta taactactgg ggccagggga cccaggtcac cgtctcctca 360 <210> 27 <211> 20 <212> PRT <213> Artificial Sequence <400> 27 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Thr Ser 20 <210> 28 <211> 17 <212> PRT <213> Artificial Sequence <400> 28 Met Gly Trp Phe Arg His Ala Pro Gly Lys Glu Arg Glu Gln Val Ala 1 5 10 15 Gly <210> 29 <211> 20 <212> PRT <213> Artificial Sequence <400> 29 Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Thr Pro 20 <210> 30 <211> 20 <212> PRT <213> Artificial Sequence <400> 30 Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Thr Ser 20 <210> 31 <211> 20 <212> PRT <213> Artificial Sequence <400> 31 Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Thr Thr Ser 20 <210> 32 <211> 20 <212> PRT <213> Artificial Sequence <400> 32 Glu Ser Gly Gly Gly Ser Val Gln Val Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Thr Ser 20 <210> 33 <211> 17 <212> PRT <213> Artificial Sequence <400> 33 Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Glu Ile Ala 1 5 10 15 Thr <210> 34 <211> 17 <212> PRT <213> Artificial Sequence <400> 34 Met Ala Trp Phe Arg Gln Ser Pro Gly Lys Glu Arg Glu Gln Val Ala 1 5 10 15 Gly <210> 35 <211> 17 <212> PRT <213> Artificial Sequence <400> 35 Met Gly Trp Phe Arg Gln Ser Pro Gly Lys Glu Arg Glu Gln Val Ala 1 5 10 15 Gly <210> 36 <211> 17 <212> PRT <213> Artificial Sequence <400> 36 Met Gly Trp Phe Arg Gln Ser Pro Gly Tyr Gln Arg Glu Gln Val Ala 1 5 10 15 Gly <210> 37 <211> 38 <212> PRT <213> Artificial Sequence <400> 37 Phe Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn 1 5 10 15 Thr Glu Thr Thr Val Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 38 <211> 38 <212> PRT <213> Artificial Sequence <\alpha> 38 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn 1 5 10 15 Gly Lys Asn Thr Val Asp Leu Gln Met Asp Asn Leu Lys Pro Asp Asp 20 25 30 Thr Ala Met Tyr Tyr Cys 35 <210> 39 <211> 38 <212> PRT <213> Artificial Sequence <400> 39 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn 1 5 10 15 Gly Lys Asn Thr Val Asp Leu Gln Met Asp Asn Leu Lys Pro Glu Asp 20 25 30 Thr Ala Met Tyr Tyr Cys 35 <210> 40 <211> 38 <212> PRT <213> Artificial Sequence <400> 40 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn 1 5 10 15 Asn Lys Asn Thr Val Asp Leu Gln Met Asp Asn Leu Lys Pro Glu Asp 20 25 30 Thr Ala Met Tyr Tyr Cys 35 <210> 41 <211> 11 <212> PRT <213> Artificial Sequence <400> 41 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 42 <211> 8 <212> PRT <213> Artificial Sequence <400> 42 Gly Tyr Thr Tyr Ser Ser Asn Cys 1 5 <210> 43 <211> 8 <212> PRT <213> Artificial Sequence <400> 43 Arg Phe Thr Tyr Gly Ser Ala Cys 1 5 <210> 44 <211> 8 <212> PRT <213> Artificial Sequence <400> 44 Arg Tyr Thr Tyr Gly Ser Ala Cys 1 5 <210> 45 <211> 8 <212> PRT <213> Artificial Sequence <400> 45 Arg Tyr Thr Tyr Gly Ser Ser Cys 1 5 <210> 46 <211> 8 <212> PRT <213> Artificial Sequence <400> 46 Arg Tyr Thr Tyr Gly Thr Ser Cys 1 5 <210> 47 <211> 6 <212> PRT <213> Artificial Sequence <400> 47 Arg Tyr Thr Tyr Asn Cys 1 5 <210> 48 <211> 10 <212> PRT <213> Artificial Sequence <400> 48 Thr Ser Arg Phe Thr Tyr Gly Ser Ala Cys 1 5 10 <210> 49 <211> 7 <212> PRT <213> Artificial Sequence <400> 49 Ile Ala Met Gly Val Val Thr 1 5 <210> 50 <211> 8 <212> PRT <213> Artificial Sequence <400> 50 Ile Ser Arg Val Gly Glu Ile Thr 1 5 <210> 51 <211> 19 <212> PRT <213> Artificial Sequence <400> 51 Ala Ala Gly Pro Ala His Tyr Lys Cys Tyr Ser Ala Ser Gly Gly Arg 1 5 10 15 Tyr Asn Ile <210> 52 <211> 19 <212> PRT <213> Artificial Sequence <400> 52 Ala Ala Gly Pro Ala His Tyr Lys Cys Tyr Ser Ala Ser Gly Gly Arg 1 5 10 15 Tyr Asn Tyr <210> 53 <211> 19 <212> PRT <213> Artificial Sequence <400> 53 Ala Ala Gly Pro Ala His Tyr Lys Cys Tyr Ser Pro Ser Gly Gly Arg 1 5 10 15 Tyr Asn Tyr <210> 54 <211> 19 <212> PRT <213> Artificial Sequence <400> 54 Ala Ala Gly Pro Ala His Tyr Leu Cys Tyr Ser Ala Ser Gly Gly Arg 1 5 10 15 Tyr Thr Ser <210> 55 <211> 19 <212> PRT <213> Artificial Sequence <400> 55 Ala Ala Gly Pro Ala His Tyr Asn Cys Tyr Ser Ala Ser Gly Gly Arg 1 5 10 15 Tyr Asn Tyr <210> 56 <211> 19 <212> PRT <213> Artificial Sequence <400> 56 Ala Ala Gly Pro Pro His Tyr Lys Cys Tyr Ser Val Ser Gly Gly Arg 1 5 10 15 Tyr Asn Tyr <210> 57 <211> 19 <212> PRT <213> Artificial Sequence <400> 57 Ala Ala Ser Pro Pro Thr Tyr Gly Gly Cys Pro Thr Arg Ser Arg Asp 1 5 10 15 Tyr Asp Lys <210> 58 <211> 19 <212> PRT <213> Artificial Sequence <400> 58 Ala Ser Gly Pro Pro His Tyr Lys Cys Tyr Ser Ala Ser Gly Gly Arg 1 5 10 15 Tyr Asn Tyr <210> 59 <211> 19 <212> PRT <213> Artificial Sequence <400> 59 Ala Thr Gly Pro Ala His Tyr Lys Cys Tyr Ser Ala Ser Gly Gly Arg 1 5 10 15 Tyr Asn Tyr <210> 60 <211> 19 <212> PRT <213> Artificial Sequence <400> 60 Ala Thr Gly Pro Ala His Tyr Lys Cys Tyr Ser Ala Ser Gly Gly Arg 1 5 10 15 Tyr Ser Asp <210> 61 <211> 19 <212> PRT <213> Artificial Sequence <400> 61 Ala Thr Gly Pro Ala His Tyr Asn Cys Tyr Ser Ala Ser Gly Gly Arg 1 5 10 15 Tyr Asn Tyr <210> 62 <211> 122 <212> PRT <213> Artificial Sequence <400> 62 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Thr Ser Thr Ser Arg Phe Thr Tyr Gly Ser Ala Cys Met Gly 20 25 30 Trp Phe Arg Gln Ser Pro Gly Lys Glu Arg Glu Gln Val Ala Gly Ile 35 40 45 Ala Met Gly Val Val Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe 50 55 60 Thr Ile Ser Gln Asp Asn Gly Lys Asn Thr Val Asp Leu Gln Met Asp 65 70 75 80 Asn Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Pro 85 90 95 Ala His Tyr Asn Cys Tyr Ser Ala Ser Gly Gly Arg Tyr Asn Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 63 <211> 120 <212> PRT <213> Artificial Sequence <400> 63 Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Thr Ser Arg Phe Thr Tyr Gly Ser Ala Cys Met Gly Trp Phe 20 25 30 Arg Gln Ser Pro Gly Lys Glu Arg Glu Gln Val Ala Gly Ile Ala Met 35 40 45 Gly Val Val Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 50 55 60 Ser Gln Asp Asn Gly Lys Asn Thr Val Asp Leu Gln Met Asp Asn Leu 65 70 75 80 Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Thr Gly Pro Ala His 85 90 95 Tyr Lys Cys Tyr Ser Ala Ser Gly Gly Arg Tyr Asp Ala Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 64 <211> 120 <212> PRT <213> Artificial Sequence <400> 64 Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Thr Ser Arg Trp Thr Tyr Gly Thr Ala Cys Met Gly Trp Phe 20 25 30 Arg Gln Ser Pro Gly Lys Glu Arg Glu Gln Val Ala Gly Ile Ala Met 35 40 45 Gly Val Val Thr Tyr Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr Ile 50 55 60 Ser Gln Asp Asn Glu Lys Asn Thr Val Asp Leu Gln Met Asp Asn Leu 65 70 75 80 Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Thr Gly Pro Ala His 85 90 95 Tyr Lys Cys Tyr Ser Ala Ser Gly Gly Arg Tyr Asn Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 65 <211> 120 <212> PRT <213> Artificial Sequence <400> 65 Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Thr Ser Arg Phe Thr Tyr Gly Ser Ala Cys Met Gly Trp Phe 20 25 30 Arg Gln Ser Pro Gly Lys Glu Arg Glu Gln Val Ala Gly Ile Ala Met 35 40 45 Gly Val Val Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 50 55 60 Ser Gln Asp Asn Gly Lys Asn Thr Val Asp Leu Gln Met Asp Asn Leu 65 70 75 80 Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Thr Gly Pro Ala His 85 90 95 Tyr Lys Cys Tyr Ser Ala Ser Gly Gly Arg Tyr Asn Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 66 <211> 120 <212> PRT <213> Artificial Sequence <400> 66 Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Thr Ser Arg Phe Thr Tyr Gly Ser Ala Cys Met Gly Trp Phe 20 25 30 Arg His Ala Pro Gly Lys Glu Arg Glu Gln Val Ala Gly Ile Ala Met 35 40 45 Gly Val Val Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 50 55 60 Ser Gln Asp Asn Gly Lys Asn Thr Val Asp Leu Gln Met Asp Asn Leu 65 70 75 80 Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Thr Gly Pro Ala His 85 90 95 Tyr Lys Cys Tyr Ser Pro Ser Gly Gly Arg Tyr Asn Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 67 <211> 366 <212> DNA <213> Artificial Sequence <400> 67 gagtctggag gaggcttggt gcagcctggg gggtctctga gactatcctg tgcgacctct 60 acctctagat tcacctacgg tagcgcctgc atgggctggt tccgccagtc tccaggaaag 120 gagcgcgagc aggtcgcagg tattgctatg ggtgttgtca catactatgc cgactccgta 180 aagggccggt tcaccatctc ccaagacaac ggcaagaaca cggtggatct gcaaatggac 240 aacctgaaac ctgaggacac tgccatgtac tactgtgcgg cggggcccgc ccactataac 300 tgctattcag cgtccggagg gcggtataac tactggggcc aggggaccca ggtcaccgtc 360 tcctca 366 <210> 68 <211> 360 <212> DNA <213> Artificial Sequence <400> 68 gagtctgggg gaggctcggt gcaggctgga gggtctctga gactatcctg tgcaacctct 60 agatttacct acggtagcgc ctgcatgggc tggttccgcc agtctccagg aaaggagcgc 120 gagcaggtcg caggtattgc tatgggtgtc gtcacatact atgccgactc cgtgaagggc 180 cggttcacca tctcccaaga caacggcaag aacacggtgg atctgcaaat ggacaacctg 240 aaacctgagg acactgccat gtactactgt gcgacggggc ccgcgcacta taagtgctat 300 tcagcgtccg gagggcggta tgacgcctgg ggccagggga cccaggtcac cgtctcctca 360 <210> 69 <211> 360 <212> DNA <213> Artificial Sequence <400> 69 gagtctgggg gaggctcggt gcaggctgga gggtctctga gactgtcctg tgcaacctcg 60 agatggacct acggtaccgc ctgcatgggc tggttccggc agtctccagg aaaggagcgc 120 gagcaggtcg caggtatcgc tatgggtgtt gtcacatact atgccgactc cgtggagggc 180 cggttcacca tctcccaaga caacgagaag aacacggtgg atctgcaaat ggacaacctg 240 aaacctgagg acactgccat gtactactgt gcgacggggc ccgcgcacta taagtgctat 300 tcagcgtccg gagggcggta taactactgg ggccagggga cccaggtcac cgtctcctca 360 <210> 70 <211> 360 <212> DNA <213> Artificial Sequence <400> 70 gagtctggag gaggctcggt gcaggctgga gggtctctga gactatcctg tgcaacctct 60 agattcacct acggtagcgc ctgcatgggc tggttccgcc agtctccagg aaaggagcgc 120 gagcaggtcg caggtattgc tatgggtgtt gtcacatact atgccgactc cgtgaagggc 180 cggttcacca tctcccaaga caacggcaag aacacggtgg atctgcaaat ggacaacctg 240 aaacctgagg acactgccat gtactactgt gcgacggggc ccgcgcacta taagtgctat 300 tcagcgtccg gcgggcggta taactactgg ggccagggga cccaggtcac cgtctcctca 360 <210> 71 <211> 360 <212> DNA <213> Artificial Sequence <400> 71 gagtctgggg gaggctcggt gcaggctgga gggtctctga gactatcctg tgcaacctct 60 cgatttacct acggtagcgc ctgcatgggc tggttccgcc acgctcccgg aaaggagcgc 120 gagcaagtcg caggtattgc tatgggtgtt gtcacatact atgccgactc cgtgaagggc 180 cggttcacca tctcccaaga caacggcaag aacacagtgg atctgcaaat ggacaacctg 240 aaacctgagg acactgccat gtactactgt gcgacggggc ccgcgcacta taagtgctat 300 tcaccgtccg gagggcggta taactactgg ggccagggga cccaggtcac cgtctcctca 360 <210> 72 <211> 38 <212> PRT <213> Artificial Sequence <400> 72 Tyr Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Gln Asp Asn 1 5 10 15 Glu Lys Asn Thr Val Asp Leu Gln Met Asp Asn Leu Lys Pro Glu Asp 20 25 30 Thr Ala Met Tyr Tyr Cys 35 <210> 73 <211> 8 <212> PRT <213> Artificial Sequence <400> 73 Arg Trp Thr Tyr Gly Thr Ala Cys 1 5 <210> 74 <211> 19 <212> PRT <213> Artificial Sequence <400> 74 Ala Thr Gly Pro Ala His Tyr Lys Cys Tyr Ser Ala Ser Gly Gly Arg 1 5 10 15 Tyr Asp Ala <210> 75 <211> 19 <212> PRT <213> Artificial Sequence <400> 75 Ala Thr Gly Pro Ala His Tyr Lys Cys Tyr Ser Pro Ser Gly Gly Arg 1 5 10 15 Tyr Asn Tyr

Claims

1. An anti-TSLP single-domain antibody, characterized in that: The single-domain antibody is composed of heavy chains, including heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3; The amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are as follows (1) or (2): (1) CDR1 shown in SEQ ID NO:48, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:51; (2) CDR1 shown in SEQ ID NO:73, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:

59.

2. The anti-TSLP single-domain antibody according to claim 1, characterized in that: The single-domain antibody further includes a frame region FR; the frame region FR includes the amino acid sequences of FR1, FR2, FR3, and FR4; the amino acid sequences of the frame region FR are as follows: The FR1 or a variant thereof shown in SEQ ID NO:30, wherein the variant thereof contains up to 5 amino acid substitutions in the FR1; The FR2 or a variant thereof shown in SEQ ID NO:35, wherein the variant thereof contains substitutions of up to 5 amino acids; The FR3 or a variant thereof shown in SEQ ID NO:39 or SEQ ID NO:72, wherein the variant thereof contains substitutions of up to 5 amino acids in the FR3; The FR4 or a variant thereof shown in SEQ ID NO:41, wherein the variant thereof contains substitutions of up to 5 amino acids.

3. An anti-TSLP single domain antibody, characterized in that: The amino acid sequences of the single-domain antibodies are shown in SEQ ID NO. 10 or 64, respectively.

4. The Fc fusion antibody or humanized antibody of the anti-TSLP single-domain antibody according to any one of claims 1 to 3.

5. A polynucleotide molecule encoding the anti-TSLP single-domain antibody of any one of claims 1 to 3, characterized in that: Their nucleotide sequences are shown in SEQ ID NO: 23 or 69, respectively.

6. An expression vector, characterized by: It comprises a single-domain antibody encoding the anti-TSLP of any one of claims 1 to 3, or a multinucleotide molecule of the Fc fusion antibody or humanized antibody of claim 4, or a multinucleotide molecule of claim 5.

7. A host cell, characterized in that: It can express the anti-TSLP single-domain antibody as described in any one of claims 1 to 3, or the Fc fusion antibody or humanized antibody as described in claim 4, or it may contain the expression vector as described in claim 6.

8. A pharmaceutical composition, characterized by: The pharmaceutical composition comprises an anti-TSLP single-domain antibody selected from any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

9. A medicament for the treatment of asthma, characterized in that: It comprises an anti-TSLP single-domain antibody as the active ingredient of any one of claims 1 to 3.

10. A kit for detecting TSLP levels, characterized by: The kit contains the anti-TSLP single-domain antibody as described in any one of claims 1 to 3.

11. Use of the anti-TSLP single-domain antibody of any one of claims 1 to 3 or the pharmaceutical composition of claim 8 in the manufacture of a medicament for the treatment of a disease, characterized in that, The diseases mentioned are asthma, chronic rhinosinusitis with nasal polyps, chronic obstructive pulmonary disease, or hypersensitivity reactions.

12. Use according to claim 11, characterized in that: The hypersensitivity reaction is atopic dermatitis, chronic urticaria, or eosinophilic esophagitis.

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