CN113831411B

CN113831411B - Single-domain antibody for L1CAM and derived protein and application thereof

Info

Publication number: CN113831411B
Application number: CN202111097189.7A
Authority: CN
Inventors: 苏志鹏; 孟巾果; 王乐飞; 张云
Original assignee: Nanjing Rongjiekang Biotechnology Co ltd
Current assignee: Nanjing Rongjiekang Biotechnology Co ltd
Priority date: 2021-09-18
Filing date: 2021-09-18
Publication date: 2023-05-02
Anticipated expiration: 2041-09-18
Also published as: CN113831411A

Abstract

The invention belongs to the field of immunology, and relates to a single-domain antibody aiming at L1CAM, a derivative protein thereof 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 CDR3; 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 the sequence with high homology. The invention uses biological gene engineering technology to screen out specific L1CAM single domain antibodies, the initial affinity of the antibodies is obvious, and the antibodies can block specific cells from releasing cytokines, and the antibodies have good binding activity through prokaryotic expression and certain drug property.

Description

Single-domain antibody for L1CAM and derived protein and application thereof

Technical Field

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

Background

The occurrence of tumor is a multi-factor and multi-stage evolution process, and in recent years, research discovers that L1CAM (L1 cell adhesion molecule, L1-cell adhesion molecule) has increased expression in various malignant tumors of human beings, is closely related to the progress, metastasis, invasion and prognosis of tumor, and is a potential target point of tumor treatment. Compared with three traditional treatment means of operation, radiotherapy and chemotherapy, the molecular targeting treatment has the effect of treating the root cause, has the characteristics of high energy efficiency and selectively killing tumor cells, and reduces the damage to normal tissues. Compared with other treatments, molecular targeting has fewer side effects and can treat tumors more pertinently.

L1CAM is a cell adhesion receptor, belongs to a neural cell adhesion molecule, and is a member of the immunoglobulin superfamily. L1CAM operates as a mature, non-mitotic cell adhesion receptor, and in addition to playing an important role in the development and physiology of the nervous system, L1CAM is also present in many tumor tissues and is involved in the proliferation, spread, metastasis and invasion of multiple types of tumors. In colon cancer studies, L1CAM exhibits high expression. L1CAM was detectable in 70% of colon cancers, whereas no expression was observed in normal colon tissues. When L1CAM is introduced into colon cancer cells lacking L1CAM, these cells become invasive and metastatic, and the liver in nude mice forms a general metastasis, which is promoted by cleavage of the extracellular domain of L1CAM. In breast cancer, expression of L1CAM allows cells to evade apoptosis, and L1CAM can promote cell movement by a mechanism that is not ERK dependent, but rather because the adhesive connection between cells is lost. In the breast cancer MCF7 cell line, overexpression of L1CAM leads to disruption of the adhesive linkage between cells, cell dispersion, and increased transcription activity of β -catenin-TCF. However, when siRNA gene inhibition was administered, endogenous L1CAM expression was significantly reduced, MCF7 cell motility was reduced, and cells were aggregated. In ovarian cancer, 79% expression of L1CAM in various tissue types is similar to colon cancer, and expression of L1CAM has a close relationship with poor prognosis and metastasis of ovarian cancer. The L1CAM may be cleaved by metalloproteases to form mL1CAM and sL1CAM. High expression levels of mL1CAM inhibited the activation of the oncogene p53 and was closely related to satisfactory ovarian cancer tumor cell debulking. The soluble L1CAM (sL 1 CAM) was detected by serum and ascites cells from patients with ovarian cancer. sL1CAM has been shown to be a diagnostic marker closely related to the type of clinical pathology and prognosis of patients, and its high expression predicts reduced sensitivity to chemotherapy and poor prognosis, and can promote cell migration, invasion and protect apoptosis in vitro. Overexpression of L1CAM increases IL-1β secretion and induces continued NF-kB activation, so tumor cell lysate levels can be determined indirectly by IL-1β levels. Of the renal cell carcinomas, 46% of clear cell renal cell carcinomas and 28% of papillary renal carcinomas express L1CAM, with normal kidneys not normally expressing L1CAM. In clear cell carcinoma, expression of L1CAM is associated with tumor metastasis mechanisms, and L1CAM is defined as an independent prognostic factor for tumorigenic metastasis by a multifactorial analysis combining the absence of cyclin d1 expression. Among the histological subtypes of various human skin malignant melanomas, the positive expression rate of L1CAM is 42%, and the positive expression proves that L1CAM is related to metastatic diseases, and becomes a positive predictor for evaluating prognosis of melanoma patients. In thyroid cancer, koonSoonKim et al detected the L1CAM expression level by immunohistochemical methods and found no expression in normal thyroid epithelial cells and thyroid differentiated cancer. There is significant overexpression in thyroid undifferentiated tumors (ATC).

The mechanism of LI-CAM action is related to the signaling pathway in addition to cell adhesion, as shown in fig. 1. Binding of LI-CAM to a receptor initiates signal transmission intracellularly and interfaces with signaling pathways of other receptors. In cancer cells, L1 CAMs can bind to tyrosine kinase Receptors (RTKs) and integrins, resulting in activation of extracellular signal-regulated kinases (ERKs), thereby promoting cell cycle progression, induction of angiogenesis, and induction of apoptosis by activating phosphoinositide 3 kinase/protein kinase B (PI 3K/AKT) signaling pathways or BCL-2 pathway. New studies have shown that the downstream signal of L1CAM can be passed through two bypass signal pathways in addition to MAKPs/ERK: the "forward" signal passes through the Nuclear Factor (NF) -kB7 of the transcription factor by modulating intramembrane proteolysis and the "reverse" signal. There is currently no unified theorem about the mechanism of action of L1 CAMs, but there is a greater propensity for the Wnt/β -catenin signaling pathway. Aberrant activation of Wnt signaling pathways plays a critical role in the proliferation, invasion and metastasis processes of various tumors. The Wnt signal path in normal mature cells is in an inhibition state, and most cytoplasmic beta-catenin and E-cadherein are combined 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 to be degraded, so that the cytoplasmic beta-catenin is kept at a very low concentration, and Wnt/3-catenin signal channel signals are accumulated excessively through free variant beta-catenin in cytoplasm and enter the nucleus, and are combined with TCF/Lef to activate the transcription process of corresponding target genes such as c-myc genes and cyclinD1, thereby regulating and controlling proliferation and apoptosis of cells and participating in tumor generation. The beta-catenin is used as a cell adhesion and signal transduction molecule of a Wnt signal path, the level of the beta-catenin is influenced by beta-catenin gene mutation, and is also influenced by beta-catenin degradation complex, such as genes of L-CAM, APC, axin, GSK-33, CK1, cell adhesion receptors CD44, uPAR, MMPs, extracellular matrix structures and the like, and target gene expression can be regulated, so that late tumor cells become more invasive and metastatic.

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

In summary, L1CAM can be targeted for molecular targeted therapies by the use of monoclonal antibodies.

Disclosure of Invention

In order to overcome the defects, the invention aims to provide a single-domain antibody aiming at L1CAM, a derivative protein and application thereof, and biological genetic engineering technology is used for screening the single-domain antibody aiming at the L1CAM, wherein the single-domain antibody has obvious initial affinity, blocks specific cells from releasing cytokines, has good binding activity through prokaryotic expression and has certain drug forming property.

In a first aspect of the invention, there is provided a single domain antibody to an L1CAM, said single domain antibody consisting of a heavy chain comprising a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3;

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, CDR3 shown in SEQ ID NO. 16.

That is, the heavy chain includes complementarity determining region CDRs; the complementarity determining regions CDR include the amino acid sequences of heavy chain CDR1, CDR2 and CDR3. The CDR sequences (1) - (4) correspond in sequence to SEQ ID NO. 1-4. All of the above sequences may be replaced by sequences having "at least 80% homology" to the sequence or sequences with 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 regions FR include 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);

(a) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21, FR3 shown in SEQ ID NO. 24, FR4 shown in SEQ ID NO. 28 or a variant thereof comprising at most 3 amino acid substitutions in said FR;

(b) FR1 shown in SEQ ID NO. 18, FR2 shown in SEQ ID NO. 21, FR3 shown in SEQ ID NO. 25, FR4 shown in SEQ ID NO. 28 or a variant thereof comprising at most 3 amino acid substitutions in said FR;

(c) FR1 shown in SEQ ID NO. 19, FR2 shown in SEQ ID NO. 23, FR3 shown in SEQ ID NO. 26, FR4 shown in SEQ ID NO. 28 or a variant thereof comprising at most 3 amino acid substitutions in said FR;

(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 at most 3 amino acid substitutions in said FR.

In one embodiment, the single domain antibody to L1CAM hybridizes to a polypeptide selected from the group consisting of SEQ ID NOs: 1-4 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology and is capable of specifically binding to the L1CAM antigen.

In another preferred embodiment, the single domain antibody directed against L1CAM hybridizes to a polypeptide selected from the group consisting of SEQ ID NOs: 1-4 has at least 95% sequence homology and is capable of specifically binding to the L1CAM antigen.

In a second aspect of the invention there is provided a single domain antibody directed against L1CAM, said single domain antibody being shown as SEQ ID NO.1-4, respectively, or said single domain antibody having at least 95% sequence homology with the amino acid sequences of SEQ ID NO. 1-4.

In one embodiment, the nucleic acid molecule encoding the single domain antibody to L1CAM hybridizes to a nucleic acid molecule selected from the group consisting of SEQ ID NOs: 5-8 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology and encodes a polypeptide that specifically binds to the L1CAM antigen against the L1CAM single domain antibody.

Preferably, the coding sequences of the single domain antibodies are shown as SEQ ID NO.5-8, respectively, or have at least 95% sequence homology with SEQ ID NO. 5-8.

A third aspect of the invention is to provide the aforementioned Fc fusion antibody against a single domain antibody of L1CAM.

In a fourth aspect, the present invention provides a nucleotide molecule encoding the aforementioned antibody directed against the L1CAM single domain, wherein the nucleotide sequences are shown in SEQ ID NO:5-8 or has at least 95% sequence homology with SEQ ID No. 5-8.

In a fifth aspect, the 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.

In a sixth aspect, the invention provides a host cell which can express a single domain antibody against L1CAM as described above, or which comprises an expression vector as described above.

The present invention also provides a method of producing a single domain antibody or Fc fusion antibody thereof to L1CAM, comprising the steps of: (a) Culturing the aforementioned host cell under conditions suitable for the production of the single domain antibody or Fc fusion antibody thereof, thereby obtaining a culture comprising said single domain antibody or Fc fusion antibody directed against L1 CAM; (b) Isolating or recovering the antibody directed against the L1CAM single domain or Fc fusion thereof from the culture; and (c) optionally purifying and/or modifying the antibody against the L1CAM single domain or Fc fusion thereof obtained in step (b).

A seventh aspect of the present invention provides a pharmaceutical composition comprising: (i) An Fc fusion antibody as described above for a single domain antibody of L1CAM, or a single domain antibody of L1 CAM; 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 drug for inhibiting the expression of the L1CAM gene or an anti-tumor drug. The drug inhibiting the expression of the L1CAM gene can be applied to any diseases with high expression of the L1CAM gene. Preferably, the neoplasm 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 single domain antibody against L1CAM or the Fc fusion antibody of the single domain antibody against L1CAM for preparing reagents, detection plates or kits; wherein the reagent, assay plate or kit is for: detecting the presence and/or amount of L1CAM protein in the sample.

The single domain antibody is a VHH, which comprises only the antibody heavy chain and does not comprise the antibody light chain.

The invention uses biological gene engineering technology to screen out the single domain antibody specific to L1CAM, the initial affinity of the antibody is obvious, and the antibody can block specific cells from releasing cell factor, and the single domain antibody has good frontal binding activity through prokaryotic expression and certain drug forming property, and the single domain antibody has the following advantages:

(1) The single domain antibody expression system is flexible to select, can be expressed in a prokaryotic system or a eukaryotic system of a yeast cell or a mammalian cell, has low expression cost in the prokaryotic expression system, and can reduce the post production cost.

(2) Because the single domain antibody is a single domain antibody, the multi-combination form of the antibody is simpler to reconstruct, multivalent and multi-specific antibodies can be obtained through simple serial connection in a genetic engineering mode, the immune heterogeneity is very low, and stronger immune response can not be generated under the condition of not carrying out humanized reconstruction.

(3) As reported in several documents, single domain antibodies have a broader range of affinities, ranging from nM to pM, before affinity maturation, providing multiple options for later antibodies for different uses.

Drawings

FIG. 1 SDS-PAGE analysis of human recombinant L1CAM protein;

FIG. 2 VHH sequence insertion analysis, where VHH1-30 is the PCR product of different clones randomly picked from the constructed library of antibodies against the L1CAM single domain;

FIG. 3 is a library enrichment profile targeting L1CAM panning;

FIG. 4L 1CAM target portion prokaryotic expression antibody SDS-PAGE;

FIG. 5L 1CAM target antibody antigen binding activity.

Detailed Description

The present invention is described in further detail below with reference to examples to enable those skilled in the art to practice the same by referring to the description.

Single domain antibodies (sdabs, also called 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 region is part of a single domain polypeptide. Thus, a single domain antibody comprises a single complementarity determining region (single CDR1, single CDR2, and single CDR 3). Examples of single domain antibodies are heavy chain-only antibodies (which naturally do not comprise light chains), single domain antibodies derived from conventional antibodies, and engineered antibodies.

The single domain antibodies may be derived from any species including mice, humans, camels, llamas, goats, rabbits, and cattle. For example, naturally occurring VHH molecules may be derived from antibodies provided by camelidae species (e.g. camels, dromedaries, llamas and dromedaries). Like whole antibodies, single domain antibodies are capable of selectively binding to a particular antigen. A single domain antibody may contain only the variable domains of an immunoglobulin chain, which domains have CDR1, CDR2 and CDR3, as well as 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 identical positions 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 with identical sequences have 100% homology.

In the present invention, a nanobody against L1CAM can be obtained from a sequence having high sequence homology with CDR1-3 disclosed in the present invention. In some embodiments, sequences having "at least 80% homology" or "at least 85% homology", "at least 90% homology", "at least 95% homology", "at least 98% homology" to the sequences of (1) - (4) may achieve the object of the invention (i.e., derived 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 object. In fact, in determining the degree of sequence homology between two amino acid sequences or in determining the CDR1, CDR2 and CDR3 combinations in a single domain antibody, the skilled person may consider so-called "conservative" amino acid substitutions, which in the case of substitution will preferably be conservative amino acid substitutions, which may generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and which substitution 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, e.g., conservative amino acid substitutions are those in which one or a few amino acids in the following groups (a) - (d) are substituted for another or a few amino acids in the same group: (a) a polar negatively charged residue and an uncharged amide thereof: asp, asn, glu, gln; (b) a polar positively charged residue: his, arg, lys; (c) aromatic residues: phe, trp, tyr; (d) aliphatic nonpolar or low polar residues: ala, ser, thr, gly, pro, met, leu, ile, val, cys. Particularly preferred conservative amino acid substitutions are as follows: asp is substituted with Glu; asn is substituted with Gln or His; glu is substituted with Asp; gln is substituted with Asn; his is substituted with Asn or Gln; arg is replaced by Lys; lys is substituted by Arg, gln; phe is replaced by Met, leu, tyr; trp is substituted with Tyr; tyr is substituted with Phe, trp; substitution of Ala with Gly or Ser; ser is substituted by Thr; thr is replaced by Ser; substitution of Gly with Ala or Pro; met is substituted with Leu, tyr or Ile; leu is substituted with Ile or Val; lie is substituted with Leu or Val; val is substituted with Ile or Leu; cys is replaced by Ser. In addition, those skilled in the art will recognize that the creativity of single domain antibodies is represented in the CDR1-3 regions, while the framework region sequences FR1-4 are not immutable, and that the sequences of FR1-4 may take the form of conservative sequence variants of the sequences disclosed herein.

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

The preparation method comprises the steps of preparing target protein and a truncated form of the target protein through a genetic engineering technology, immunizing an inner Mongolian alashan alpaca with the obtained antigen protein, obtaining peripheral blood lymphocytes or spleen cells of the alpaca after multiple immunization, recombining a camel source antibody variable region coding sequence into a phage display carrier through a genetic engineering mode, screening specific antibodies aiming at L1CAM through a phage display technology, and further detecting the binding capacity of the antibodies and the antigen and application of the antibodies in treatment of autoimmune diseases.

The above technical solutions will now be described in detail by way of specific embodiments:

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

the human recombinant extracellular domain protein used in the patent is obtained by self-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, designated NM-000425.4, was retrieved from NCBI and encoded to give the amino acid sequence accession NP-000416.1.

(2) The amino acid sequences corresponding to NP 000416.1 were analyzed for the transmembrane region and extracellular end of the protein via 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 coding L1CAM protein is cloned into a vector pcDNA3.4 by using a gene synthesis mode.

(5) And (3) carrying out Sanger sequencing on the constructed vector, comparing the original sequences, carrying out batch extraction on the recombinant plasmid after confirming no errors, removing endotoxin, carrying out expression and purification of target protein by transfecting suspension 293F, wherein the SDS-PAGE analysis result of the purified L1CAM recombinant protein is shown in figure 1, and the purity of the purified protein is as high as 90%, so that the animal immunity requirement is met.

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

(1) 1mg of the protein purified in example 1 was mixed with an equal volume of Freund's complete adjuvant, immunized one inner Mongolian Alexander camel once a week, and immunized 7 total times, with the exception of the first immunization, the remaining six times were animal immunized with 600. Mu. g L1CAM recombinant protein mixed with Freund's incomplete adjuvant equal volume, which was used to concentrate the camel to produce antibodies against L1CAM.

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

(3) Synthesizing cDNA using the extracted total RNA, and amplifying VHH (heavy chain antibody variable region) using the cDNA as a template by a nested PCR reaction;

(4) Respectively enzyme-cutting a pMECS vector and a VHH fragment by using restriction enzyme, and linking the enzyme-cut fragments with the vector;

(5) The ligated fragment spots were transformed into competent cells TG1, phage display library of L1CAM protein was constructed and the library capacity was determined, and the correct insertion rate of VHH fragments in the library was examined by PCR, as shown in FIG. 2, and the results showed that 28 clones amplified by PCR from 30 randomly selected colonies in the library had a band size of 600bp (predicted size) and 2 clones amplified incorrectly, so the correct insertion rate was 28.times.30.times.100%. Apprxeq.93.3%.

Example 3: single domain antibody screening against L1CAM protein:

(1) 200 mu L of recombinant TG1 cells are taken and cultured in a 2 XTY culture medium, 40 mu L of helper phage VCSM13 is added during the period to infect the TG1 cells, and the culture is carried out overnight to amplify phage, the phage is precipitated by PEG/NaCl on the next day, and the amplified phage is collected by centrifugation;

(2) NaHCO diluted at 100mM pH 8.3 ₃ 500 mug of the L1CAM protein is coupled on an ELISA plate, and the ELISA plate is placed at 4 ℃ overnight, and a negative control hole is formed;

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

(4) After blocking was completed, 100. Mu.L of amplified phage library (approximately 2X 10 ¹¹ Individual phage particles), 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 combined with L1CAM protein by trypsin with a final concentration of 2.5mg/mL, infecting E.coli TG1 cells in logarithmic growth phase, culturing at 37 ℃ for 1h, generating and collecting phage for the next round of screening, repeating the same screening process for 1 round to obtain enrichment gradually, when the enrichment multiple reaches more than 10 times, the enrichment effect is shown in figure 3, the number of monoclonal bacteria growing after phage infected with TG1 bacteria removed by P/N=biopanning positive Kong Xi/the number of monoclonal bacteria growing after phage infected with TG1 bacteria removed by positive Kong Xi, and the parameter gradually increases after enrichment occurs; I/E = total phage added to positive wells per round of biopanning/total phage removed from positive Kong Xi per round of biopanning, which parameter gradually approaches 1 after enrichment has occurred.

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

(1) 3 rounds of screening are carried out on the L1CAM protein according to the single domain antibody screening method, after the screening is finished, phage enrichment factors aiming at the recombinant L1CAM protein reach more than 10, 400 single colonies are selected from positive clones obtained by screening and respectively inoculated into 96 deep well plates containing 100 mu g/mL ampicillin TB culture medium, blank control is arranged, after the culture is carried out at 37 ℃ to a logarithmic phase, IPTG with the final concentration of 1mM is added, and the culture is carried out at 28 ℃ for overnight;

(2) Obtaining a crude extract antibody by using a permeation swelling method; the L1CAM recombinant protein was released to 100mM NaHCO pH 8.3, respectively ₃ 100. Mu.g of protein was coated in an ELISA plate at 4℃overnight;

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

(4) Washing the unbound antibody with PBST, adding 100 μl of Mouse anti-HA tag antibody (Mouse anti-HA antibody, thermo Fisher) diluted 1:2000, and incubating for 1h at room temperature;

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

(6) Washing off unbound antibody with PBST, adding horseradish peroxidase chromogenic solution, reacting at 37deg.C for 15min, adding stop solution, and reading absorption value at 450nm wavelength on an enzyme-labeled instrument;

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

(8) The positive clone well was transferred and shaken in LB medium containing 100. Mu.g/. Mu.L ampicillin to extract plasmids and sequenced;

(9) The gene sequences of the individual clones were analyzed according to the sequence alignment software Vector NTI, the strains with the same CDR1, CDR2, CDR3 sequences were regarded as the same clone, and the strains with different sequences were regarded as different clones, and finally single domain antibodies specific for the L1CAM protein (SEQ ID nos. 1-4, and single domain antibodies 3G5, 3G6, 3G9, 3H6, 2C7, 2C8, 2C9, 2D1, 2G7, 2G8, 2H4, 3A5, 3B11, the sequences of which are not shown) were obtained. The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 structure, which forms the whole VHH. The obtained single-domain antibody recombinant plasmid can be expressed in a prokaryotic system, and finally the single-domain antibody protein is obtained.

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

TABLE 14 CDR and FR sequences of single domain antibodies

Table 24 amino acid sequences and nucleotide sequences of single domain antibodies

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

(1) Plasmids (pMECS-VHH) of the different clones obtained by the above sequencing analysis were electrotransformed into E.coli HB2151 and plated on LB+amp+glucose-containing culture plates, which were incubated overnight at 37 ℃;

(2) Selecting single colony to inoculate in 5mL LB culture solution containing shore penicillin, shaking culture at 37 ℃ overnight;

(3) Inoculating 1mL of overnight culture strain into 330mL of TB culture solution, shake culturing at 37deg.C, adding 1M IPTG when OD600nm value reaches 0.6-0.9, shake culturing at 28deg.C overnight;

(4) Centrifuging, collecting escherichia coli, and obtaining an antibody crude extract by using a osmotic bursting method;

(5) The antibodies are purified by nickel column affinity chromatography, the results of partial cloning and expression are shown in FIG. 4, VHH1-18 in FIG. 4 corresponds to 3G5, 3G6, 3G9, 3H1, 3H2, 3H6, 2C7, 2C8, 2C9, 2D1, 2D6, 2G7, 2G8, 2H4, 2H9, 3A5 and 3B11 respectively, wherein the sequences of 2D6, 3H1, 2H9 and 3H2 are shown as SEQ ID NO:1-4 respectively, and the sequences of the rest of the single domain antibodies are not shown (the single domain antibodies have insufficient technical effects or do not need 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) Subcloning the target sequence obtained in example 4 into a eukaryotic expression vector: the antibodies selected in example 4 were subjected to Sanger sequencing to obtain their nucleotide sequences;

(2) Synthesizing the nucleotide sequence (SEQ ID NO. 5-8) subjected to codon optimization into a vector RJK-V4-hFC designed and modified by the company in a sequence synthesis mode, wherein the modification method of the vector is as described in example 10;

(3) Transforming the recombinant eukaryotic expression vector constructed by the company into DH5 alpha escherichia coli, culturing to carry out plasmid large extraction, and removing endotoxin;

(4) Sequencing and identifying the sequence of the plasmid after large extraction;

(5) And preparing the recombinant vector after the determination of no error for subsequent eukaryotic cell transfection and expression.

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

(1) 3 days before transfection at 2.5X10 ⁵ ExpiCHO-S cell passage and expansion culture/mL ^TM The cells, calculated desired cell volume, were transferred to an ExpiCHO containing fresh pre-warmed 120mL (final volume) ^TM 500mL shake flask of expression medium; to achieve a cell concentration of about 4X 10 ⁶ -6×10 ⁶ Living cells/mL;

(2) One day prior to transfection, expiCHO-S was used ^TM Cell dilution concentration to 3.5X10 ⁶ Living cells/mL, allowing the cells to incubate overnight;

(3) The day of transfection, cell density and percent viable cells were determined. The cell density should reach about 7X 10 before transfection ⁶ -10×10 ⁶ Living cells/mL;

(4) Fresh ExpiCHO preheated to 37 ℃ ^TM Dilution of cells to 6X 10 in expression Medium ⁶ Each living cell/mL. The calculated desired cell volume was transferred to 100mL (final volume) of expcho filled with fresh pre-warmed ^TM 500mL shake flask of expression medium;

(5) Gently mixing the mixture with the mixture of the Expifectamine in a reverse manner ^TM CHO reagent with 3.7mL OptiPRO ^TM Dilution of Expifectamine in Medium ^TM CHO reagent, whipping or mixing;

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

(7) Incubating the ExpiFectamine CHO/plasmid DNA complex for 1-5 minutes at room temperature, then gently adding to the prepared cell suspension, gently agitating the shake flask during the addition;

(8) The cells were incubated at 37℃with 8% CO ₂ Shake culturing in humidified air;

(9) 600ul of Expiectamine was added on day 1 (18-22 hours post transfection) ^TM CHO enhancement and 24mL of expi CHO feed.

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

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

Recombinant single domain antibody expression experimental procedure (500 mL shake flask for example):

(1) 3 days before transfection at 2.5X10 ⁵ Cell passaging and expansion293F cells were grown in large cultures and the calculated desired cell volumes were transferred to 500mL shake flasks with fresh pre-warmed 120mL (final volume) OPM-293CD05 Medium. To achieve a cell concentration of about 2X 10 ⁶ -3×10 ⁶ Living cells/mL.

(2) The day of transfection, cell density and percent viable cells were determined. The cell density should reach about 2X 10 before transfection ⁶ -3×10 ⁶ Living cells/mL.

(3) Dilution of cells to 1X 10 with pre-warmed OPM-293CD05 Medium ⁶ Each living cell/mL. The calculated cell volume required was transferred to a 500mL shake flask containing fresh pre-warmed 100mL (final volume) of medium.

(4) Diluting PEI (1 mg/mL) reagent with 4mL of Opti-MEM culture medium, and stirring or blowing to mix uniformly; the plasmid DNA was diluted with 4mL of Opt-MEM medium, mixed back and forth, and filtered with a 0.22um filter. Incubate at room temperature for 5min.

(5) Diluted PEI reagent was added to the diluted DNA and mixed upside down. PEI/plasmid DNA complexes were incubated for 15-20 minutes at room temperature and then gently added to the prepared cell suspension, during which time the shake flask was gently swirled.

(6) The cells were incubated at 37℃with 5% CO ₂ Shake culturing at 120 rpm.

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

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

Example 9: purification of human Fc recombinant single domain antibodies

(1) The protein expression supernatant obtained in example 7 or 8 was filtered with a disposable filter head of 0.45 μm to remove insoluble impurities;

(2) Purifying the filtrate by using a Protein purifier to perform affinity chromatography, and purifying by using agarose filler coupled with Protein A by utilizing the binding capacity of human 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 combined with the packing;

(4) Washing the column-bound impurity proteins with a low-salt and high-salt buffer;

(5) The target protein combined on the column is subjected to a system by using a low pH buffer solution;

(6) Rapidly adding the eluent into Tris-HCl solution with pH of 9.0 for neutralization;

(7) And (3) dialyzing the neutralized protein solution, performing SDS-PAGE analysis to determine that the protein purity is above 95%, and preserving the protein at a low temperature for later use after the concentration is above 0.5 mg/mL.

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

The mentioned nanobody universal targeting vector RJK-V4-hFC is a commercial vector pCDNA3.4 (vector data link:

https:// assemes. Thermo-filter. Com/TFS-assems/LSG/manual/pcdna3_4_topo_ta_cloning_kit_man. Pdf) fused to the Fc region of the heavy chain coding sequence of human IgG (NCBI Accession No.: AB 776838.1), i.e., the vector contains the Hinge (Hinge) CH2 and CH3 regions of the IgG heavy chain. The concrete improvement scheme is as follows:

(1) Selecting restriction enzyme cutting sites XbaI and AgeI on pcDNA3.4;

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

(3) Amplifying the fragments by PCR using a pair of primers with XbaI and AgeI cleavage sites, respectively;

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

(5) And (3) connecting the digested vector and the inserted fragment under the action of T4 ligase, then converting the connection product into escherichia coli, amplifying, and checking by sequencing to obtain the recombinant plasmid.

Example 11: binding dose-response curve assay for specific single domain antibodies to L1CAM proteins

(1) 50. Mu.L of 1. Mu.g/mL L1CAM was coated overnight at 4 ℃.

(2) Washing the plate; 200. Mu.L of 5% milk was added and blocked at 37℃for 1h.

(3) The VHH, i.e., the specific single domain antibody of the L1CAM protein prepared by prokaryotic expression in example 5, was diluted to 2. Mu.g/mL and then 5-fold gradient diluted for a total of 8 concentration gradients.

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

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

(6) Washing the plate (washing several times); 50. Mu.L of TMB which had previously recovered the room temperature was added thereto, and the reaction was continued at the normal temperature in the dark for 15 minutes.

(7) Add 50. Mu.L of stop solution (1N HCl) and store the microplate reader reading.

(8) The EC50 was calculated by plotting the curves, and as shown in fig. 5 and table 3, it was seen that the single domain antibodies 2D6, 3H1, 2H9, 3H2 against L1CAM of the present invention were excellent in binding potency and specificity to L1CAM proteins.

3G5

3G6

3G9

3H1

3H2

3H6

EC50

～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

Although embodiments of the present invention have been disclosed above, it is not limited to the use of the description and embodiments, it is well suited to various fields of use for the invention, and further modifications may be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the particular details without departing from the general concepts defined in the claims and the equivalents thereof.

Sequence listing

<110> Nanjing Rongjiekang biotechnology Co., ltd

<120> Single-domain antibodies against L1CAM and derived proteins and uses thereof

<130> 2021091801

<141> 2021-09-18

<160> 28

<170> SIPOSequenceListing 1.0

<210> 1

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<212> PRT

<213> Artificial sequence (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 Val Val Ser Gly Tyr Thr Ser Ser Ile Trp Tyr Met Gly Trp Phe

20 25 30

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

35 40 45

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

50 55 60

Ile Ser Gln Asp Lys Thr Lys Asn Thr Val Tyr Leu Gln Met Asn Gly

65 70 75 80

Leu Thr Leu Glu Asp Ser Ala Met Tyr Phe Cys Ala Ala Thr Arg Arg

85 90 95

Ser Trp Phe Pro Gly Thr Leu Pro Ser Glu Glu Val Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 2

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

1 5 10 15

Cys Val Val Ser Gly Tyr Thr Ser Ser Ile Trp Tyr Met Gly Trp Phe

20 25 30

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

35 40 45

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

50 55 60

Ile Ser Gln Asp Lys Thr Lys Asn Thr Val Tyr Leu Gln Met Asn Gly

65 70 75 80

Leu Thr Pro Glu Asp Ser Ala Met Tyr Phe Cys Ala Ala Thr Arg Arg

85 90 95

Ser Trp Phe Pro Gly Thr Leu Pro Ser Glu Glu Val Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 3

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

1 5 10 15

Cys Ala Ala Ser Gly Tyr Val Ile Gly Ser Asp Trp Met Gly Trp Phe

20 25 30

Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Thr Ile Tyr Thr

35 40 45

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

50 55 60

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

65 70 75 80

Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala Arg Pro Arg

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Pro Met Tyr Gly Ser Glu Trp Leu Ser Pro Asn Glu Tyr Asn Tyr Trp

100 105 110

Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

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<213> Artificial sequence (Artificial Sequence)

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

1 5 10 15

Cys Ala Ala Ser Ala Tyr Thr Tyr Thr Asn Ala Phe Met Gly Trp Phe

20 25 30

Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Tyr Thr

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Glu Thr Trp Asn Gly Val Leu Ala Ala Gly Ser Tyr Asn Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

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<210> 5

<211> 366

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgtggtgagc 60

ggctacacca gcagcatctg gtacatgggc tggttcaggc aggcccccgg caaggagagg 120

gaggccgtgg ccaggatcgc caccgccggc ggcgccaccg cctacagcga caccgtgaag 180

ggcaggttca ccatcagcca ggacaagacc aagaacaccg tgtacctgca gatgaacggc 240

ctgaccctgg aggacagcgc catgtacttc tgcgccgcca ccaggaggag ctggttcccc 300

ggcaccctgc ccagcgagga ggtgatggac tactggggcc agggcaccca ggtgaccgtg 360

agcagc 366

<210> 6

<211> 366

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

gagagcggcg gcggcagcgt gcaggccggc gagaccctga ggctgagctg cgtggtgagc 60

ggctacacca gcagcatctg gtacatgggc tggttcaggc aggcccccgg caaggagagg 120

gaggccgtgg ccaggatcgc caccagcggc ggcgccaccg cctacagcga caccgtgaag 180

ggcaggttca ccatcagcca ggacaagacc aagaacaccg tgtacctgca gatgaacggc 240

ctgacccccg aggacagcgc catgtacttc tgcgccgcca ccaggaggag ctggttcccc 300

ggcaccctgc ccagcgagga ggtgatggac tactggggcc agggcaccca ggtgaccgtg 360

agcagc 366

<210> 7

<211> 366

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60

ggctacgtga tcggcagcga ctggatgggc tggttcaggc aggcccccgg caaggagagg 120

gagggcgtgg ccaccatcta caccgagtac agcatcacct actacgccga cagcgtgaag 180

ggcaggttca ccatcagcca ggacaacgcc aagaacaccg tgtacctgga gatgaacagc 240

ctgaagcccg aggacaccgc catctactac tgcgccgcca ggcccaggcc catgtacggc 300

agcgagtggc tgagccccaa cgagtacaac tactggggcc agggcaccca ggtgaccgtg 360

agcagc 366

<210> 8

<211> 363

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60

gcctacacct acaccaacgc cttcatgggc tggttcaggc aggcccccgg caaggagagg 120

gagggcgtgg ccgccatcta caccggcgcc gacgccaagt actacgccga cagcgtgaag 180

ggcaggttca ccatcagcca ggacaacgcc aagaacaccg tgtacctgca gatgaacgac 240

ctgaagcccg aggacaccgc catgtactac tgcgccacca gggccggcga gacctggaac 300

ggcgtgctgg ccgccggcag ctacaactac tggggccagg gcacccaggt gaccgtgagc 360

agc 363

<210> 9

<211> 8

<212> PRT

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Ala Tyr Thr Tyr Thr Asn Ala Phe

1 5

<210> 10

<211> 8

<212> PRT

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<400> 10

Gly Tyr Thr Ser Ser Ile Trp Tyr

1 5

<210> 11

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

Gly Tyr Val Ile Gly Ser Asp Trp

1 5

<210> 12

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

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Ile Ala Thr Ala Gly Gly Ala Thr

1 5

<210> 13

<211> 8

<212> PRT

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<400> 13

Ile Ala Thr Ser Gly Gly Ala Thr

1 5

<210> 14

<211> 8

<212> PRT

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Ile Tyr Thr Glu Tyr Ser Ile Thr

1 5

<210> 15

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

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Ile Tyr Thr Gly Ala Asp Ala Lys

1 5

<210> 16

<211> 20

<212> PRT

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Ala Ala Arg Pro Arg Pro Met Tyr Gly Ser Glu Trp Leu Ser Pro Asn

1 5 10 15

Glu Tyr Asn Tyr

20

<210> 17

<211> 20

<212> PRT

<213> Artificial sequence (Artificial Sequence)

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Ala Ala Thr Arg Arg Ser Trp Phe Pro Gly Thr Leu Pro Ser Glu Glu

1 5 10 15

Val Met Asp Tyr

20

<210> 18

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<212> PRT

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

1 5 10 15

Cys Val Val Ser

20

<210> 19

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<212> PRT

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

1 5 10 15

Cys Ala Ala Ser

20

<210> 20

<211> 20

<212> PRT

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

1 5 10 15

Cys Val Val Ser

20

<210> 21

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

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

1 5 10 15

Arg

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<211> 17

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<213> Artificial sequence (Artificial Sequence)

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

1 5 10 15

Ala

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<211> 17

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<213> Artificial sequence (Artificial Sequence)

<400> 23

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

1 5 10 15

Thr

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<211> 38

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 24

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

1 5 10 15

Thr Lys Asn Thr Val Tyr Leu Gln Met Asn Gly Leu Thr Leu Glu Asp

20 25 30

Ser Ala Met Tyr Phe Cys

35

<210> 25

<211> 38

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 25

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

1 5 10 15

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

20 25 30

Ser Ala Met Tyr Phe Cys

35

<210> 26

<211> 38

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 26

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

1 5 10 15

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

20 25 30

Thr Ala Ile Tyr Tyr Cys

35

<210> 27

<211> 38

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 27

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

1 5 10 15

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

20 25 30

Thr Ala Met Tyr Tyr Cys

35

<210> 28

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 28

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

Claims

1. A single domain antibody to L1CAM, characterized in that: 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 CDR3;

the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are as follows:

CDR1 shown in SEQ ID NO. 10, CDR2 shown in SEQ ID NO. 12, CDR3 shown in SEQ ID NO. 17.

2. The single domain antibody against L1CAM of claim 1, wherein: the sequence of the framework region FR of the single domain antibody is as follows:

FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21, FR3 shown in SEQ ID NO. 24 and FR4 shown in SEQ ID NO. 28.

3. A single domain antibody to L1CAM, characterized in that: the amino acid sequence of the single domain antibody is shown as SEQ ID NO: 1.

4. The Fc fusion antibody of any one of claims 1-3 to a single domain antibody of L1CAM.

5. A nucleic acid molecule encoding the antibody of any one of claims 1-3 to L1CAM single domain antibody, wherein: the nucleotide sequence is shown as SEQ ID NO: shown at 5.

6. An expression vector comprising a nucleic acid molecule encoding the single domain antibody of any one of claims 1-3 or the Fc fusion antibody of claim 4 or the nucleic acid molecule of claim 5.

7. A host cell capable of expressing a single domain antibody against L1CAM according to any one of claims 1-3, or comprising an expression vector according to claim 6.