阅读说明：本技术 针对l1cam的单域抗体及其衍生蛋白和应用 (Single-domain antibody aiming at L1CAM and derivative protein and application thereof ) 是由 苏志鹏 孟巾果 王乐飞 张云 于 2021-09-18 设计创作，主要内容包括：本发明属于免疫学领域,涉及针对L1CAM的单域抗体及其衍生蛋白和应用。所述的单域抗体由重链构成,重链包括重链CDR1、重链CDR2和重链CDR3；所述的重链CDR1、重链CDR2和重链CDR3的氨基酸序列为(1)、(2)、(3)或者(4)的序列组合或与其同源性高的序列。本发明使用生物基因工程技术筛选出特异性针对L1CAM单域抗体,这些抗体初步亲和力明显,并且具有阻断特定细胞释放细胞因子,通过原核表达即具有良好的结合活性,具有一定的成药性。(The invention belongs to the field of immunology, and relates to a single domain antibody aiming at L1CAM, and a derivative protein and application thereof. The single domain antibody is composed of a heavy chain, wherein the heavy chain comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR 3; the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are the sequence combination of (1), (2), (3) or (4) or sequences with high homology with the sequence combination. The invention screens out specific L1CAM single-domain antibodies by using a biological genetic engineering technology, the antibodies have obvious initial affinity, block specific cells from releasing cytokines, have good binding activity through prokaryotic expression and have certain druggability.)

1. A single domain antibody directed to L1CAM, characterized by: the single domain antibody is composed of a heavy chain, wherein the heavy chain comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR 3;

the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are (1), (2), (3) or (4) as follows:

(1) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 12, CDR3 shown in SEQ ID NO. 17;

(2) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 13, CDR3 shown in SEQ ID NO. 17;

(3) CDR1 shown in SEQ ID NO. 11, CDR2 shown in SEQ ID NO. 14, CDR3 shown in SEQ ID NO. 16;

(4) CDR1 shown in SEQ ID NO. 9, CDR2 shown in SEQ ID NO. 15, and CDR3 shown in SEQ ID NO. 16.

2. The single domain antibody to L1CAM of claim 1, wherein: the sequence of the framework region FR of the single domain antibody is (a), (b), (c) or (d) below;

(a) FR1 shown by SEQ ID NO:20, FR2 shown by SEQ ID NO:21, FR3 shown by SEQ ID NO:24, FR4 shown by SEQ ID NO:28, or a variant thereof comprising substitutions of up to 3 amino acids in the FRs;

(b) FR1 shown by SEQ ID NO. 18, FR2 shown by SEQ ID NO. 21, FR3 shown by SEQ ID NO. 25, FR4 shown by SEQ ID NO. 28, or a variant thereof comprising substitutions of up to 3 amino acids in the FRs;

(c) FR1 shown by SEQ ID NO. 19, FR2 shown by SEQ ID NO. 23, FR3 shown by SEQ ID NO. 26, FR4 shown by SEQ ID NO. 28, or a variant thereof comprising substitutions of up to 3 amino acids in the FRs;

(d) FR1 shown in SEQ ID NO:19, FR2 shown in SEQ ID NO:22, FR3 shown in SEQ ID NO:27, FR4 shown in SEQ ID NO:28, or a variant thereof comprising substitutions of up to 3 amino acids in the FRs.

3. The single domain antibody to L1CAM of claim 1, wherein: the single domain antibody to L1CAM specifically binds to a sequence selected from SEQ ID NO:1-4 have at least 95% sequence homology and are capable of specifically binding to the L1CAM antigen.

4. A single domain antibody directed to L1CAM, characterized by: the single domain antibody is respectively shown as SEQ ID NO.1-4, or the single domain antibody has at least 95% sequence homology with the amino acid sequence of SEQ ID NO. 1-4.

5. The single domain antibody to L1CAM of claim 4, wherein: the coding sequence of the single-domain antibody is respectively shown in SEQ ID NO.5-8, or has at least 95% sequence homology with SEQ ID NO. 5-8.

6. An Fc fusion antibody of the single domain antibody against L1CAM of any one of claims 1-5.

7. A nucleotide molecule encoding the single domain antibody to L1CAM of any one of claims 1 to 5, wherein: the nucleotide sequences are respectively shown as SEQ ID NO: 5-8 or has at least 95% sequence homology with SEQ ID NO. 5-8.

8. An expression vector comprising a nucleotide molecule encoding the single domain antibody of any one of claims 1 to 5 or the Fc fusion antibody of claim 6 or the nucleotide molecule of claim 7.

9. A host cell capable of expressing a single domain antibody to L1CAM according to any one of claims 1 to 5 or comprising an expression vector according to claim 8.

10. Use of the single domain antibody against L1CAM of any one of claims 1-5 in the preparation of a medicament or anti-tumor medicament for inhibiting expression of L1CAM gene.

Technical Field

The invention belongs to the field of immunology, and relates to a single domain antibody aiming at L1CAM, and a derivative protein and application thereof.

Background

The occurrence of tumors is a multifactorial and multistage evolution process, and in recent years, L1CAM (L1 cell adhesion molecule, L1-cell adhesion molecule) is found to be highly expressed in various malignant tumors of human beings, closely related to the progression, metastasis, invasion and prognosis of the tumors and a potential target point for tumor treatment. Compared with three traditional treatment methods of operation and radiotherapy and chemotherapy, the molecular targeted therapy has the effect of treating the root cause, has the characteristics of high energy efficiency and selective killing of tumor cells, and reduces the damage to normal tissues. Compared with other treatments, the molecular targeting brings fewer side effects, and the tumor can be treated more specifically.

L1CAM is a cell adhesion receptor, belonging to the neural cell adhesion molecule, and is a member of the immunoglobulin superfamily. In addition to playing an important role in the development and physiological processes of the nervous system, L1CAM functions as a cell adhesion receptor in the mature and non-mitotic stages, and is also present in many tumor tissues and involved in the proliferation, spread, metastasis and invasion of various types of tumors. In the study of colon cancer, L1CAM exhibited high expression. L1CAM was detectable in 70% of colon cancers, but not in normal colon tissue. When L1CAM is introduced into colon cancer cells lacking L1CAM, these cells become invasive and metastatic, and the liver in nude mice forms a gross metastatic focus, and the molecular mechanism is that the extracellular domain of L1CAM is cleaved to promote the formation of metastatic mechanism. In breast cancer, expression of L1CAM allows cells to escape apoptosis, and L1CAM promotes cell movement by a mechanism that is not dependent on ERK but rather is due to the loss of adhesive connections between cells. In the breast cancer MCF7 cell line, the over-expression of L1CAM causes the adhesion connection among cells to be interrupted, the cells are scattered, and the transcription activity of beta-catenin-TCF is increased. However, when siRNA gene inhibition is given, the expression of endogenous L1CAM is obviously reduced, the movement of MCF7 cells is reduced, and the cells are aggregated. In ovarian cancer, 79% of all tissue types express L1CAM similar to colon cancer, and L1CAM expression is closely associated with poor prognosis and metastasis of ovarian cancer. L1CAM can be cleaved by metalloproteases to form mL1CAM and sL1 CAM. The high expression level of the mL1CAM can inhibit the activation of the cancer suppressor gene p53, and is closely related to whether satisfactory ovarian cancer tumor cell killing can be performed. Soluble L1CAM (sL1CAM) was detectable in sera and ascites cells from patients with ovarian cancer. The sL1CAM is proved to be a diagnostic marker closely related to clinical pathological types and patient prognosis, the high expression of the sL1CAM indicates that the sensitivity to chemotherapy is reduced and the prognosis is poor, and the sL1CAM can promote cell migration, invade and protect cell apoptosis in vitro. Overexpression of L1CAM increased IL-1 β secretion and induced a sustained activation of NF-kB, so tumor cell lysate levels could be measured indirectly by IL-1 β levels. Of the renal cell carcinomas, 46% of clear cell renal cell carcinomas and 28% of papillary renal carcinomas express L1CAM, and normal kidneys do not normally express L1 CAM. In clear cell carcinomas, expression of L1CAM is associated with a tumor metastasis mechanism, and L1CAM is defined as an independent prognostic factor for tumorigenic metastases by multifactorial analysis in combination with the absence of cyclin d1 expression. Among various histological subtypes of malignant melanoma on human skin, the positive expression rate of L1CAM is 42%, and the positive expression proves that L1CAM is related to metastatic diseases and becomes a positive prediction factor for evaluating prognosis of melanoma patients. In thyroid cancer, KoonsoKim et al detected the expression level of L1CAM by immunohistochemical method, and found that it was not expressed in normal thyroid epithelial cells and thyroid differentiated cancers. There was significant overexpression in thyroid undifferentiated tumors (ATC).

The mechanism of action of LI-CAM is related to the signal transduction pathway in addition to cell adhesion, as shown in FIG. 1. The binding of LI-CAM to the receptor initiates signaling within the cell and interfaces with the signaling pathways of other receptors. In cancer cells, L1CAM can bind to tyrosine kinase Receptors (RTKs) and integrins, leading to activation of extracellular signal-regulated kinases (ERKs), thereby promoting cell cycle progression, inducing angiogenesis and inducing apoptosis by activating the phosphoinositide 3 kinase/protein kinase B (PI3K/AKT) signaling pathway or BCL-2 pathway. New studies have shown that downstream signals of L1CAM, in addition to MAKPs/ERK, can also pass through two alternative signal pathways: the "forward" signal is through proteolysis in regulatory membranes and the "reverse" signal is through the Nuclear Factor (NF) -kB7 of transcription factors. There is currently no unified theory on the mechanism of action of L1CAM, but the Wnt/β -catenin signaling pathway is favored more. Abnormal activation of the Wnt signaling pathway plays a crucial role in the proliferation, invasion and metastasis processes of various tumors. The Wnt signal pathway in normal mature cells is in an inhibition state, and most of cytoplasmic beta-catenin is combined with E-cadherin to participate in cell adhesion, so that the stability of the cells is maintained. A small amount of free beta-catenin is phosphorylated by GSK-3p and degraded, so that cytoplasmic beta-catenin is kept at a very low concentration, Wnt/3-catenin signal channel signals enter a nucleus through excessive accumulation of free variant beta-catenin in cytoplasm, and are combined with TCF/Lef to activate the transcription process of corresponding target genes such as c-myc gene and cyclinD1, thereby regulating the proliferation and apoptosis of cells and participating in the occurrence of tumors. The level of the beta-catenin serving as a cell adhesion and signal transduction molecule of a Wnt signal path is influenced by beta-catenin gene mutation, and is also influenced by degradation complexes of the beta-catenin, such as L-CAM, APC, Axin, GSK-33, CK1, cell adhesion receptors CD44, uPAR, MMPs, extracellular short-matrix structures and other genes, so that the expression of target genes can be regulated, and late-stage tumor cells become more invasive and metastatic.

Since L1CAM is present on the cell surface, according to this property, therapeutic intervention is performed from outside the cell, and by adding an antibody that interferes with the extracellular domain of L1CAM, tumor cells can be effectively inhibited in cell culture, and the tumor cells can be killed to a greater extent. Studies have shown that L-CAM is not expressed in normal mammary epithelial cells (nor is expression present in epithelial cells of most other normal tissues), and therefore the addition of anti-L1 CAM antibodies in culture (even in organisms) has no effect on cells and tissues, while antibodies against the extracellular region of L1CAM effectively inhibit proliferation of breast cancer cells. In the human colon cancer cell lines SW480 and HCT116, when the anti-L-CAM antibody was added to the medium, both the invasion and the metastatic ability of the cells decreased. Peritoneal dissemination of tumor cells was effectively inhibited by intraperitoneal injection of an anti-L1 CAM antibody into nude mice expressing L1 CAM-expressing ovarian cancer cells, and thus, treatment with an anti-L1 CAM antibody was shown to be effective in inhibiting tumor cell growth in nude mice. Other studies have demonstrated that the L1CAM antibody can also be directly applied in chemotherapy and radiotherapy of L1CAM expressing cancer cells. When the radioactive element copper-67 is combined with the monoclonal antibody of the anti-L1 CAM antibody and injected into the peritoneum of the nude mouse planted with the ovarian cancer cells of SKOV3ip, the accumulation amount of the radioactive element in the normal tissue is low, and the level of the radioactive element in the tumor tissue is high. The treatment result proves that SKOV3ip ovarian cancer cells are substantially inhibited in the nude mice, and the survival period of the nude mice is prolonged.

In conclusion, L1CAM can be used as a target for molecular targeted therapy through the application of monoclonal antibodies.

Disclosure of Invention

In order to overcome the defects, the invention aims to provide the single-domain antibody aiming at the L1CAM, the derived protein and the application thereof, the specific single-domain antibody aiming at the L1CAM is screened out by using a biological genetic engineering technology, the initial affinity of the antibodies is obvious, the antibodies have the functions of blocking specific cells from releasing cytokines, and have good binding activity and certain druggability through prokaryotic expression.

In a first aspect of the invention, there is provided a single domain antibody directed against L1CAM, said single domain antibody consisting of a heavy chain comprising heavy chain CDR1, heavy chain CDR2 and heavy chain CDR 3;

the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are (1), (2), (3) or (4) as follows:

(1) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 12, CDR3 shown in SEQ ID NO. 17;

(2) CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 13, CDR3 shown in SEQ ID NO. 17;

(3) CDR1 shown in SEQ ID NO. 11, CDR2 shown in SEQ ID NO. 14, CDR3 shown in SEQ ID NO. 16;

(4) CDR1 shown in SEQ ID NO. 9, CDR2 shown in SEQ ID NO. 15, and CDR3 shown in SEQ ID NO. 16.

That is, the heavy chain includes complementarity determining region CDRs; the complementarity determining region CDRs include the amino acid sequences of heavy chain CDR1, CDR2, and CDR 3. The above CDR sequences (1) - (4) correspond to SEQ ID NO.1-4 in sequence. All the above sequences may be replaced with a sequence having "at least 80% homology" with the sequence or a sequence having only one or a few amino acid substitutions; preferably "at least 85% homology", more preferably "at least 90% homology", more preferably "at least 95% homology", and most preferably "at least 98% homology".

In a preferred embodiment, the sequence of the single domain antibody further comprises a framework region FR; the framework region FR comprises the amino acid sequences of FR1, FR2, FR3 and FR 4;

the sequence of the framework region FR of the single domain antibody is (a), (b), (c) or (d) below;

(a) FR1 shown by SEQ ID NO:20, FR2 shown by SEQ ID NO:21, FR3 shown by SEQ ID NO:24, FR4 shown by SEQ ID NO:28, or a variant thereof comprising substitutions of up to 3 amino acids in the FRs;

(b) FR1 shown by SEQ ID NO. 18, FR2 shown by SEQ ID NO. 21, FR3 shown by SEQ ID NO. 25, FR4 shown by SEQ ID NO. 28, or a variant thereof comprising substitutions of up to 3 amino acids in the FRs;

(c) FR1 shown by SEQ ID NO. 19, FR2 shown by SEQ ID NO. 23, FR3 shown by SEQ ID NO. 26, FR4 shown by SEQ ID NO. 28, or a variant thereof comprising substitutions of up to 3 amino acids in the FRs;

(d) FR1 shown in SEQ ID NO:19, FR2 shown in SEQ ID NO:22, FR3 shown in SEQ ID NO:27, FR4 shown in SEQ ID NO:28, or a variant thereof comprising substitutions of up to 3 amino acids in the FRs.

In one embodiment, the single domain antibody to L1CAM binds to a polypeptide selected from the group consisting of SEQ ID NO:1-4 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology and are capable of specifically binding to the L1CAM antigen.

In another preferred embodiment, the single domain antibody to L1CAM binds to a polypeptide selected from the group consisting of SEQ ID NO:1-4 have at least 95% sequence homology and are capable of specifically binding to the L1CAM antigen.

In a second aspect, the invention provides single domain antibodies to L1CAM as set forth in SEQ ID No.1-4, respectively, or having at least 95% sequence homology with the amino acid sequence of SEQ ID No. 1-4.

In one embodiment, the nucleic acid molecule encoding the single domain antibody to L1CAM is identical to a sequence selected from the group consisting of SEQ ID NO: 5-8 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology, and encodes a single domain antibody against L1CAM that is capable of specifically binding to the L1CAM antigen.

Preferably, the coding sequence of the single domain antibody is shown as SEQ ID NO.5-8, or has at least 95% sequence homology with SEQ ID NO.5-8, respectively.

A third aspect of the present invention is to provide the aforementioned Fc fusion antibody against a single domain antibody of L1 CAM.

In a fourth aspect of the present invention, there is provided a nucleotide molecule encoding the aforementioned single domain antibody against L1CAM, the nucleotide sequences of which are shown in SEQ ID NOs: 5-8 or has at least 95% sequence homology with SEQ ID NO. 5-8.

A fifth aspect of the present invention provides an expression vector comprising a nucleotide molecule encoding the aforementioned single domain antibody or the aforementioned Fc fusion antibody or the aforementioned nucleotide molecule.

A sixth aspect of the invention provides a host cell which can express the aforementioned single domain antibody against L1CAM, or which comprises the aforementioned expression vector.

The present invention also provides a method of producing a single domain antibody or Fc fusion antibody thereof against L1CAM, comprising the steps of: (a) culturing the aforementioned host cell under conditions suitable for the production of a single domain antibody or Fc fusion antibody thereof, thereby obtaining a culture comprising the single domain antibody to L1CAM or Fc fusion antibody thereof; (b) isolating or recovering said single domain antibody against L1CAM or Fc fusion antibody thereof from said culture; and (c) optionally, purifying and/or modifying the single domain antibody against L1CAM or Fc fusion antibody thereof obtained in step (b).

In a seventh aspect of the present invention, there is provided a pharmaceutical composition comprising: (i) a single domain antibody to L1CAM as described above, or an Fc fusion antibody to a single domain antibody to L1CAM as described above; and (ii) one or more pharmaceutically acceptable excipients.

The invention also provides application of the single domain antibody aiming at the L1CAM in preparing a medicament for inhibiting the expression of the L1CAM gene or an anti-tumor medicament. Drugs that inhibit the expression of the L1CAM gene may be applicable to any condition in which the L1CAM gene is highly expressed. Preferably, the tumor includes, but is not limited to, colon cancer, breast cancer, ovarian cancer, thyroid cancer, renal cell carcinoma, melanoma.

The invention also provides the use of the aforementioned single domain antibody against L1CAM, or the aforementioned Fc fusion antibody against a single domain antibody against L1CAM, for the preparation of a reagent, a detection plate or a kit; wherein the reagent, assay plate or kit is for: detecting the presence and/or amount of the L1CAM protein in the sample.

The single domain antibody is a VHH comprising only antibody heavy chains and no antibody light chains.

The invention screens out the single-domain antibodies specific to the L1CAM by using a biological genetic engineering technology, the antibodies have obvious initial affinity, block specific cells from releasing cytokines, have good additional binding activity through prokaryotic expression and certain druggability, and the single-domain antibodies have the following advantages:

(1) the expression systems of the single domain antibodies are flexible to select, can be expressed in prokaryotic systems and eukaryotic systems of yeast cells or mammalian cells, and have low expression cost in the prokaryotic expression systems, so that the later-stage production cost can be reduced.

(2) Because the single domain antibody is a single domain antibody, the multi-combination form of the antibody is simpler to modify, a multivalent and multi-specific antibody can be obtained by simply connecting in series in a genetic engineering mode, the immune heterogeneity is low, and stronger immune response can not be generated under the condition of not carrying out humanized modification.

(3) As reported in various documents, single domain antibodies have a broader affinity range, which can range from nM to pM before affinity maturation, providing multiple options for later use of the antibody.

Drawings

FIG. 1 is an SDS-PAGE analysis of the recombinant L1CAM protein from human;

FIG. 2 analysis of VHH sequence insertion rates, where VHH1-30 is the PCR product of different clones randomly picked from a constructed library of single domain antibodies against L1 CAM;

FIG. 3 library enrichment for targeted L1CAM panning;

FIG. 4 shows SDS-PAGE for prokaryotic expression of the L1CAM target portion;

FIG. 5L1CAM target antibody antigen binding activity.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.

Single domain antibodies (sdabs, also referred to as nanobodies or VHHs by the developer Ablynx) are well known to those skilled in the art. A single domain antibody is an antibody whose complementarity determining regions are part of a single domain polypeptide. Thus, single domain antibodies comprise a single complementarity determining region (single CDR1, single CDR2, and single CDR 3). Examples of single domain antibodies are heavy chain-only antibodies (which do not naturally contain a light chain), single domain antibodies derived from conventional antibodies, and engineered antibodies.

Single domain antibodies may be derived from any species, including mouse, human, camel, llama, goat, rabbit and cow. For example, naturally occurring VHH molecules may be derived from antibodies provided by species in the family camelidae (e.g. camel, dromedary, llama and guanaco). Like intact antibodies, single domain antibodies are capable of selectively binding to a particular antigen. Single domain antibodies may contain only the variable domains of immunoglobulin chains, with CDR1, CDR2 and CDR3, and the framework regions.

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 position in an alignment, and is typically expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compared. Thus, two copies of an identical sequence have 100% homology.

In the present invention, a nanobody against L1CAM can be obtained from a sequence having high sequence homology to CDR1-3 disclosed in the present invention. In some embodiments, sequences having "at least 80% homology" to the sequences in (1) - (4), or "at least 85% homology", "at least 90% homology", "at least 95% homology", "at least 98% homology" can all achieve the object of the invention (i.e., to derive proteins).

In some embodiments, sequences that replace only one or a few amino acids compared to the sequences in (1) - (4), e.g., comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, may also achieve the objects of the invention. Indeed, in determining the degree of sequence homology between two amino acid sequences or in determining the CDR1, CDR2, and CDR3 combination in a single domain antibody, the skilled person may consider so-called "conservative" amino acid substitutions, in which case the substitution will preferably be a conservative amino acid substitution, which may generally be described as an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and which has little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are common in the art, for example conservative amino acid substitutions are those in which one or a few amino acids within the following groups (a) - (d) are replaced by another or a few amino acids within the same group: (a) polar negatively charged residues and their uncharged amides: asp, Asn, Glu, Gln; (b) polar positively charged residues: his, Arg, Lys; (c) aromatic residue: phe, Trp, Tyr; (d) aliphatic nonpolar or weakly polar residues: ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val and Cys. Particularly preferred conservative amino acid substitutions are as follows: asp substituted by Glu; asn is replaced by Gln or His; glu is substituted with Asp; gln is substituted by Asn; his is substituted with Asn or Gln; arg is replaced by Lys; lys substituted by Arg, Gln; phe is replaced by Met, Leu, Tyr; trp is substituted by Tyr; tyr is substituted by Phe, Trp; ala substituted by Gly or Ser; ser substituted by Thr; thr is substituted by Ser; gly by Ala or Pro; met is substituted by Leu, Tyr or Ile; leu is substituted by Ile or Val; ile is substituted by Leu or Val; val is substituted by Ile or Leu; cys is substituted with Ser. In addition, the skilled person knows that the creativity of single domain antibodies is found in the CDR1-3 region, whereas the framework region sequence FR1-4 is not unalterable and the sequence of FR1-4 may take the form of conservative sequence variants of the sequences disclosed in the present invention.

Preferred host cells of the invention are bacterial cells, fungal cells or mammalian cells.

The method is characterized in that a target protein and a truncated form of the target protein are prepared by a genetic engineering technology, then the obtained antigen protein is used for immunizing inner Mongolian Alaan bactrian camel, after multiple immunizations, peripheral blood lymphocytes or spleen cells of the camel are obtained, a camel source antibody variable region coding sequence is recombined into a phage display carrier by the genetic engineering mode, specific antibodies aiming at L1CAM are respectively screened out by the phage display technology, and the binding capacity of the camel source antibody variable region coding sequence and the application of the camel source antibody variable region coding sequence in autoimmune disease treatment are further detected.

The above technical solution is now split and described in detail by way of specific examples:

example 1: preparation of human L1CAM recombinant extracellular domain protein:

the human recombinant extracellular domain protein used in the patent is obtained by expression and purification of a company, and the design scheme of the expression vector of the human recombinant L1CAM protein is as follows:

(1) the coding sequence for L1CAM was retrieved at NCBI and included as NM-000425.4, and the amino acid sequence generated by this sequence was accession number NP-000416.1.

(2) The amino acid sequence corresponding to NP-000416.1 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 L1CAM protein is 22-1120 amino acids.

(4) The nucleotide sequence of 22-1120 amino acids of the encoded L1CAM protein is cloned into the pcDNA3.4 vector by a gene synthesis method.

(5) The constructed vector is subjected to Sanger sequencing, 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 used for expression and purification of target protein, the SDS-PAGE analysis result of the purified L1CAM recombinant protein is shown in figure 1, the purity of the purified protein is up to 90%, and the requirement of animal immunity is met.

Example 2: construction of a single domain antibody library against the L1CAM protein:

(1) 1mg of the protein purified from example 1 was mixed with an equal volume of Freund's complete adjuvant and immunized against a single Endomesha amata, once a week for 7 consecutive immunizations, except for the first immunization, animals were immunized against the 600. mu. g L1CAM recombinant protein mixed with an equal volume of Freund's incomplete adjuvant six times, in order to stimulate camels centrally to produce antibodies against L1 CAM.

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

(3) synthesizing cDNA (complementary deoxyribonucleic acid) by using the extracted total RNA, and amplifying VHH (heavy chain antibody variable region) by using the cDNA as a template through nested PCR (polymerase chain reaction);

(4) respectively carrying out enzyme digestion on a pMECS vector and a VHH fragment by using restriction enzymes, and then linking the enzyme-digested fragment with the vector;

(5) the ligated fragments were spotted into competent cells TG1, a phage display library of L1CAM protein was constructed and the library capacity was determined, correct insertion rate of VHH fragments in the library was detected by PCR, the detection results are shown in FIG. 2, which shows that after 30 colonies randomly selected from the library were PCR amplified, a band of 600bp (predicted size) was amplified by 28 clones, and that a band was not amplified by 2 clones, so correct insertion rate was 28 ÷ 30X 100% ≈ 93.3%.

Example 3: single domain antibody screening against L1CAM protein:

(1) culturing 200 μ L of recombinant TG1 cells in 2 × TY culture medium, adding 40 μ L of helper phage VCSM13 to infect TG1 cells, culturing overnight to amplify phage, precipitating phage with PEG/NaCl the next day, centrifuging, and collecting amplified phage;

(2) NaHCO diluted at 100mM pH 8.3₃500 mu g of the L1CAM protein in the kit is coupled on an enzyme label plate, is placed at 4 ℃ overnight, and is simultaneously provided with a negative control hole;

(3) adding 200 μ L of 3% skimmed milk the next day, sealing at room temperature for 2 hr;

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

(5) after 1 hour of action, wash 5 times with PBS + 0.05% Tween-20 to wash away unbound phage;

(6) dissociating phage specifically bound with L1CAM protein by trypsin with final concentration of 2.5mg/mL, infecting Escherichia coli TG1 cells in logarithmic growth phase, culturing at 37 ℃ for 1h, generating and collecting phage for next round of screening, repeating 1 round in the same screening process to gradually obtain enrichment, wherein when the enrichment multiple reaches more than 10 times, the enrichment effect is shown in figure 3, the number of monoclonal bacteria grown after phage eluted from a positive hole in biological elutriation infects TG1 bacteria/the number of monoclonal bacteria grown after phage eluted from the positive hole infects TG1 bacteria, and the parameter is gradually increased after enrichment; I/E ═ total number of phage added to positive wells per round of biopanning/total number of phage eluted from positive wells per round of biopanning, this parameter will gradually approach 1 after enrichment has occurred.

Example 4: screening of specific positive clones for L1CAM by phage enzyme-linked immunosorbent assay (ELISA):

(1) carrying out 3 rounds of screening on the L1CAM protein according to the single domain antibody screening method, after screening is finished, aiming at that the phage enrichment factor of the recombinant L1CAM protein reaches 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 ℃ until the logarithmic phase, adding IPTG with the final concentration of 1mM, and culturing at 28 ℃ overnight;

(2) obtaining a crude antibody by using a permeation cracking method; the L1CAM recombinant protein was released separately to 100mM NaHCO, pH 8.3₃Neutralizing and coating 100 ug protein in enzyme label plate at 4 deg.C overnight;

(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 away with PBST, 100ul of Anti-Rabbit HRP conjugate (goat Anti-Rabbit horseradish peroxidase labeled antibody, purchased from Thermo Fisher) diluted 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) the gene sequences of the respective clones were analyzed by Vector NTI using sequence alignment software, and strains having the same CDR1, CDR2, and CDR3 sequences were regarded as the same clones, while strains having different sequences were regarded as different clones, to finally obtain single domain antibodies specific for the L1CAM protein (SEQ ID nos. 1 to 4, and single domain antibodies 3G5, 3G6, 3G9, 3H6, 2C6, 2C7, 2C8, 2C9, 2D1, 2G7, 2G8, 2H4, 3a5, 3B11 having no shown sequences). The amino acid sequence of the antibody is in a 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, and finally single domain antibody protein is obtained.

The CDR and FR sequences of the 4 kinds of single domain antibodies are shown in Table 1, and the amino acid sequences and nucleotide sequences of the 4 kinds of single domain antibodies are shown in Table 2.

CDR and FR sequences of Table 14 single-domain antibodies

Amino acid sequence and nucleotide sequence of 24 kinds of single domain antibody in table

Example 5: purification and expression of specific single-domain antibody of L1CAM protein in host bacterium escherichia coli

(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 antibody is purified by a nickel column affinity chromatography method, the partial cloning expression result of the purified single-domain antibody is shown in figure 4, VHH1-18 in figure 4 respectively corresponds to 3G5, 3G6, 3G9, 3H1, 3H2, 3H6, 2C6, 2C7, 2C8, 2C9, 2D1, 2D6, 2G7, 2G8, 2H4, 2H9, 3a5 and 3B11, wherein the sequences of 2D6, 3H1, 2H9 and 3H2 are respectively shown in SEQ ID NOs: 1-4, and the sequences of the rest single-domain antibodies are not shown (the single-domain antibodies which are not good in technical effect or need not to be protected in the present application).

Example 6: construction of Fc fusion antibody eukaryotic expression vector of specific single domain antibody of L1CAM protein

(1) The target sequence obtained in example 4 was subcloned into eukaryotic expression vectors: the antibody screened out in the example 4 is subjected to Sanger sequencing to obtain a nucleotide sequence;

(2) the above-mentioned nucleotide sequence (SEQ ID NO.5-8) after codon optimization was synthesized into the carrier RJK-V4-hFC designed and modified by this company by means of sequence synthesis, and the modification method of the carrier was as described in example 10;

(3) transforming a recombinant eukaryotic expression vector constructed by a company into DH5 alpha escherichia coli, culturing, carrying out plasmid macro-extraction, and removing endotoxin;

(4) carrying out sequencing identification on the greatly extracted plasmid;

(5) and preparing the recombinant vector which is determined to be error-free for subsequent eukaryotic cell transfection expression.

Example 7: fc fusion antibody of specific single domain antibody of L1CAM protein is expressed in suspension ExpicHO-S cells

(1) 3 days before transfection at 2.5X 10⁵/mL cell passage and expanded culture ExpCHO-S^TMCells, calculated required cell volume transferred to ExpCHO filled with fresh preheated 120mL (final volume)^TMIn a 500mL shake flask of expression medium; to achieve a cell concentration of about 4X 10⁶-6×10⁶Viable cells/mL;

(2) one day before transfection, ExpicHO-S^TMCell dilution to 3.5X 10⁶Viable cells/mL, cells were cultured overnight;

(3) on the day of transfection, cell density and percentage of viable cells were determined. The cell density before transfection should reach about 7X 10⁶-10×10⁶Viable cells/mL;

(4) with fresh ExpiCHO preheated to 37 ℃^TMExpression media cells were diluted to 6X 10⁶Viable cells/mL. The calculated required cell volume was transferred to ExpicHO containing fresh preheated 100mL (final volume)^TMIn a 500mL shake flask of expression medium;

(5) expifeacmine was mixed by gentle inversion^TMCHO reagent, 3.7mL OptiPRO^TMDilution of Expifeacylamine in culture Medium^TMCHO reagent, swirling or mixing;

(6) by refrigerated 4mL OptiPRO^TMDiluting plasmid DNA with a culture medium, and mixing uniformly; the plasmid DNA is the Fc fusion antibody eukaryotic expression vector of the specific single domain antibody of the H1CAM protein prepared in example 6;

(7) incubating the Expifactamine CHO/plasmid DNA complex for 1-5 minutes at room temperature, then gently adding the Expifactamine CHO/plasmid DNA complex into the prepared cell suspension, and gently swirling the shake flask in the adding process;

(8) cells were incubated at 37 ℃ with 8% CO₂Carrying out shake culture in humidified air;

(9) day 1 post transfection (18-22 hours later) 600ul Expifeacylamine was added^TMCHO Enhancer and 24mL ExpicHO feed.

(10) Supernatants were collected approximately 8 days after transfection (cell viability below 70%).

Example 8: expression of Fc fusion antibodies of specific single domain antibodies of the L1CAM protein in 293F cells in suspension

Recombinant single domain antibody expression experimental protocol (taking 500mL shake flask as an example):

(1) 3 days before transfection at 2.5X 10⁵The 293F cells were passaged and expanded and the calculated required cell volume was transferred to 500mL shake flasks containing fresh pre-warmed 120mL OPM-293 CD05 Medium (final volume). The cell concentration is about 2X 10⁶-3×10⁶Viable cells/mL.

(2) On the day of transfection, cell density and percentage of viable cells were determined. The cell density before transfection should reach about 2X 10⁶-3×10⁶Viable cells/mL.

(3) Cells were diluted to 1X 10 with pre-warmed OPM-293 CD05 Medium⁶Viable cells/mL. The required cell volume was calculated and transferred to a 500mL shake flask containing fresh pre-warmed 100mL (final volume) of medium.

(4) Diluting PEI (1mg/mL) reagent with 4mL of Opti-MEM medium, and swirling or blowing to mix evenly; the plasmid DNA was diluted with 4mL Opt-MEM medium, vortexed, mixed well, and filtered through a 0.22um filter tip. Incubate at room temperature for 5 min.

(5) Diluted PEI reagent was added to the diluted DNA and mixed by inversion. The PEI/plasmid DNA complex was incubated for 15-20 minutes at room temperature and then gently added to the prepared cell suspension, with gentle swirling of the flask during the addition.

(6) Cells were incubated at 37 ℃ with 5% CO₂And shake culturing at 120 rpm.

(7) 5mL OPM-CHO PFF05 feed was added at 24h, 72h post transfection.

(8) Supernatants were collected approximately 7 days after transfection (cell viability below 70%).

Example 9: purification of human Fc recombinant Single Domain antibodies

(1) Filtering the protein expression supernatant obtained in example 7 or 8 with a 0.45 μm disposable filter to remove insoluble impurities;

(2) performing affinity chromatography purification on the filtrate by using a Protein purifier, and purifying by using agarose filler coupled with Protein A by utilizing the binding capacity of human-derived Fc and Protein A;

(3) passing the filtrate through a Protein A pre-packed column at a flow rate of 1 mL/min, wherein the target Protein in the filtrate is bound to the packing;

(4) washing the impurity protein bound on the column by low-salt and high-salt buffer solutions;

(5) performing a system of target proteins bound to the column with a low pH buffer;

(6) adding the eluent into Tris-HCl solution with pH9.0 rapidly for neutralization;

(7) dialyzing the neutralized protein solution, performing SDS-PAGE analysis to determine that the protein has a purity of 95% or more and a concentration of 0.5mg/mL or more, and storing at low temperature for later use.

Example 10: construction of nano antibody eukaryotic expression vector RJK-V4-hFc

The target vector RJK-V4-hFC for the general use of the nano-antibody is the Invitrogen commercial vector pCDNA3.4 (vector data link:

https:// associations. thermofisher. com/TFS-Assets/LSG/manuals/pcdna 3-4 _ topo _ ta _ cloning _ kit _ man. pdf) is fused with the Fc segment in the heavy chain coding sequence of human IgG (NCBI Accession No.: AB776838.1), i.e. the vector comprises the Hinge region (Hinge) CH2 and CH3 regions of IgG heavy chain. The specific modification scheme is as follows:

(1) selecting restriction sites XbaI and AgeI on pcDNA3.4;

(2) introducing a Multiple Cloning Site (MCS) and a 6 XHis tag at the 5 'end and the 3' end of the Fc fragment coding sequence respectively by means of overlapping PCR;

(3) amplifying the fragment by using a pair of primers with XbaI and AgeI enzyme cutting sites respectively in a PCR mode;

(4) the recombinant DNA fragments in pcDNA3.4 and (3) are digested with restriction enzymes XbaI and AgeI respectively;

(5) and (3) connecting the vector and the insert after enzyme digestion under the action of T4 ligase, then transforming the connection product into escherichia coli, amplifying, sequencing and verifying to obtain the recombinant plasmid.

Example 11: binding capacity-response curve determination of specific single-domain antibodies of L1CAM protein

(1) Coating 50. mu.L of 1. mu.g/mL L1CAM, overnight at 4 ℃.

(2) Washing the plate; add 200. mu.L of 5% milk and block for 1h at 37 ℃.

(3) VHH, a single domain antibody specific for the L1CAM protein produced by prokaryotic expression in example 5, was diluted to 2. mu.g/mL and then the antibody was diluted in 5-fold gradients for 8 concentration gradients.

(4) Washing the plate; add 50. mu.L of the single domain antibody diluted in step (3), duplicate wells, and incubate for 1h at 37 ℃.

(5) Washing the plate; mu.L of a murine anti-HA-labeled secondary HRP antibody was added and incubated at 37 ℃ for 30 min.

(6) Washing the plate (washing for several times); adding 50 μ L of TMB recovered to normal temperature in advance, and reacting for 15min at normal temperature in the dark.

(7) Add 50. mu.L of stop buffer (1N HCl) and read by microplate reader.

(8) Curves were drawn, and EC50 was calculated, as shown in fig. 5 and table 3, and it was found that the single domain antibodies 2D6, 3H1, 2H9, 3H2 against L1CAM of the present invention were excellent in binding potency and specificity to L1CAM protein.

3G5

3G6

3G9

3H1

3H2

3H6

EC5O

～0.03140

37.42

～27.91

2.829

8.087

～0.000

2C6

2C7

2C8

2C9

2D1

2D6

EC50

9.045

～69.21

～31.49

102.0

～31.40

1.331

2G7

2G8

2H4

2H9

3A5

3B11

EC50

165.7

258.6

～26.60

4.782

～97098

19.57

TABLE 3 EC50 values for each single domain antibody

While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.

Sequence listing

<110> Nanjing Congjiekang Biotech Co., Ltd

<120> single domain antibody against L1CAM and derived protein and application thereof

<130> 2021091801

<141> 2021-09-18

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Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

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