Nanobodies against beta-catenin and methods of making and uses thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a nano antibody against beta-catenin and a preparation method and application thereof. Specifically, the nano antibody with excellent affinity to beta-catenin is obtained through sample screening, has the advantages of small molecular weight (about 15 kilodaltons), small immunogenicity, better solubility and stability, and a long CDR3 region, thereby being beneficial to basic research and clinical experiments, having potential tumor diagnosis and treatment prospects, and thus having good practical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a nano-antibody against beta-catenin and a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is for the purpose of increasing the understanding of the background of the present application and should not be taken as an acknowledgement or any form of suggestion that this information forms prior art with respect to any country.

[0003] The classical Wnt signaling pathway plays an important role in the proliferation and differentiation of tumor cells. The core protein molecule beta-catenin in this signaling pathway mediates the signal transmission from the cytoplasm to the nucleus and participates in the composition of the transcription complex in the nucleus to activate the transcription of the downstream target genes. The entry of beta-catenin into the nucleus is an important prerequisite for its activation of downstream transcription factors, and the mode of its entry and exit from the nucleus has not been fully analyzed. The main mode of beta-catenin into the nucleus is to directly enter the nucleus by using the key protein of the classical nuclear entry pathway and to enter the nucleus with the assistance of "molecular chaperone". The main mode of beta-catenin out of the nucleus is to rely on the nuclear export pathway of chromosomal maintenance region 1 (CRM1). The cadherin on the cell membrane, the degradation complex-related protein in the cytoplasm, and the transcription complex-related protein in the nucleus can all cause beta-catenin to be retained, thereby affecting its distribution in and out of the nucleus.

[0004] At the same time, abnormal expression of beta-Catenin protein can be found in many human malignant tumors. It has been reported that abnormal subcellular localization and aggregation of beta-Catenin protein are closely related to cell malignant transformation and proliferation, and similar conclusions have been drawn in breast cancer, colorectal cancer, hepatocellular carcinoma, prostate cancer, gastric cancer and lung cancer, etc. Therefore, it is of great significance to design antibodies targeting beta-Catenin protein for evaluating human health status, assessing the progression of diseases, especially tumor-related diseases, and carrying out targeted disease treatment.

[0005] However, the inventors have found that the current detection and research of beta-Catenin protein mainly apply traditional monoclonal antibodies and polyclonal antibodies. However, traditional antibody molecules have large molecular weight, low stability, high cost, and need to immunize a large number of experimental animals during production and preparation. Therefore, the application of existing detection and related disease research and treatment still cannot meet the requirements, and the specificity and affinity still need to be improved. Therefore, it is particularly necessary to prepare a new type of antibody with simple preparation process and good specificity and affinity. SUMMARY

[0006] To address the shortcomings of the existing technologies, this invention provides anti-β-catenin nanobodies, their preparation methods, and applications. Specifically, this invention successfully prepared anti-β-catenin nanobodies with high affinity, stability, and specificity, which can be used for the detection and analysis of β-catenin and for the diagnosis and treatment of diseases related to β-catenin protein, especially tumors with abnormally high β-catenin expression. This invention is based on the above research findings.

[0007] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides an anti-β-catenin nanobody, wherein the heavy chain antibody heavy chain variable region (VHH) of the nanobody includes a complementarity-determining region (CDR), the complementarity-determining region having CDR1, CDR2 and CDR3 or having an amino acid sequence having at least 80% sequence identity with it;

[0009] in,

[0010] The CDR1 has an amino acid sequence as shown in SEQ ID NO.1 or 6;

[0011] The CDR2 has an amino acid sequence as shown in SEQ ID NO.2 or 7;

[0012] The CDR3 has an amino acid sequence as shown in SEQ ID NO.3 or 8;

[0013] Furthermore, the heavy chain variable region of the nanobody has an amino acid sequence as shown in SEQ ID NO.4 or 9, or has an amino acid sequence that is at least 80% identical to it.

[0014] Furthermore, the anti-β-catenin nanobody may be a recombinant antibody, a fully human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antigen-binding fragment of these antibodies, without specific limitations.

[0015] In a second aspect, the present invention provides an isolated nucleic acid molecule capable of encoding the aforementioned anti-β-catenin nanobody.

[0016] The nucleic acid molecule has:

[0017] a1) A nucleotide sequence as shown in SEQ ID NO. 5 or 10;

[0018] a2) The complementary nucleotide sequence of the nucleotide sequence shown in SEQ ID NO.5 or 10;

[0019] a3) a nucleotide sequence encoding the same protein as the nucleotide sequence of a1) or a2), but being degenerate to the nucleotide sequence of a1) or a2) due to the degeneracy of the genetic code;

[0020] a4) a nucleotide sequence obtained by substitution, deletion, or addition of one or two nucleotides to the nucleotide sequence of any one of a1) to a3), and a nucleotide sequence having the same or similar function as the nucleotide sequence of any one of a1) to a3).

[0021] In a third aspect of the present application, a recombinant expression vector is provided, which comprises the nucleic acid molecule.

[0022] Further, the recombinant expression vector is obtained by linking the nucleic acid molecule with an expression vector; wherein the expression vector can be selected from the group consisting of DNA, RNA, viral vector, plasmid, bacteriophage, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, transposon, other gene transfer system, or a combination thereof.

[0023] In a fourth aspect of the present application, a host cell is provided, which comprises the recombinant expression vector or the nucleic acid molecule integrated into the genome of the host cell; or the host cell expresses the anti-β-catenin nanobody.

[0024] The host cell includes cells of prokaryotic and eukaryotic origin. The prokaryotic is preferably Escherichia coli, and the eukaryotic includes yeast and mammalian cells, wherein the mammalian cells include CHO, NS0, HEK293, PERC6, etc., which are not specifically limited herein.

[0025] In a fifth aspect of the present application, a method for preparing the anti-β-catenin nanobody is provided, which comprises:

[0026] culturing the host cell to obtain a culture containing the anti-β-catenin nanobody; and isolating or recovering the anti-β-catenin nanobody from the culture.

[0027] In a sixth aspect of the present application, an immunoconjugate is provided, which comprises the anti-β-catenin nanobody and a conjugated moiety, wherein the conjugated moiety is a detectable label, a drug, a toxin, a cytokine, a viral coat protein or a virus-like particle, etc.

[0028] The detectable label includes a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or an enzyme.

[0029] The drug can be a cytotoxic drug, such as an anti-tubulin drug, a DNA minor groove binder, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folate antagonist, an antimetabolite, a chemosensitizer, a topoisomerase inhibitor, a vinca alkaloid, etc.

[0030] The toxin can be aureus, maytansinoid, ricin, ethidium bromide, mitomycin, diphtheria toxin, abrin, gelonin, maytansinol, restrictocin, phenomycin, curcin, calicheamicin, glucocorticoid, etc., which are not specifically limited herein.

[0031] The immunoconjugate contains a multivalent (e.g., bivalent) anti-β-catenin nanobody as described herein. The multivalent refers to the inclusion of multiple repeats of the anti-β-catenin nanobody as described herein in the amino acid sequence of the immunoconjugate.

[0032] In a seventh aspect of the present application, a detection kit is provided, which comprises the anti-β-catenin nanobody described above, and / or the immunoconjugate described above; the kit can be used to detect the presence or level of β-catenin in a sample.

[0033] The kit can be used to non-invasively detect the expression of β-catenin in a subject. It can be understood that the detection kit described above can be used for tumor detection, monitoring, diagnosis, and prognosis.

[0034] In an eighth aspect of the present application, a pharmaceutical composition is provided, which comprises the anti-β-catenin nanobody described above or the immunoconjugate described above.

[0035] Further, it further comprises a pharmaceutically acceptable carrier and / or excipient. Moreover, according to the usual method, it can be made into a dosage form of a powder, granule, tablet, capsule, suspension, emulsion, syrup, spray, etc. for oral administration, external use, suppository, and sterile injection solution.

[0036] The carrier and / or excipient, etc. non-pharmaceutically active ingredients that can be included are well known in the art, and a person of ordinary skill in the art can determine that they meet the clinical standards.

[0037] Further, the pharmaceutical composition of the present application can be administered into the body by known means. For example, by intravenous systemic delivery or local injection into the tissue of interest. Alternatively, administration can be via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration can be via single or multiple doses. The skilled person understands that the actual dose to be administered in the present application can vary to a large extent depending on a variety of factors, such as the target cell, the biological type or its tissue, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

[0038] The subject to which the pharmaceutical composition is administered can be a human and a non-human mammal, such as a mouse, a rat, a guinea pig, a rabbit, a dog, a monkey, a chimpanzee, and the like. Among them, a human is the most preferred.

[0039] In a ninth aspect of the present application, there is provided the use of the above anti-β-catenin Nanobody, immunoconjugate or pharmaceutical composition in any one or more of the following:

[0040] b1) the preparation of a tumor detection, monitoring, diagnosis and / or prognosis product;

[0041] b2) the preparation of a tumor prevention and / or treatment product.

[0042] Tumors are used in the present application as known to the skilled person. Benign tumors are defined as a hyperproliferation of cells that are not able to form aggressive, metastatic tumors in vivo. Conversely, malignant tumors are defined as cells with multiple cellular and biochemical abnormalities that are able to form systemic disease, such as tumor metastases in distant organs.

[0043] Further, the tumor is in particular a tumor with an abnormally high expression of β-catenin, such as breast cancer, colorectal cancer, hepatocellular carcinoma, prostate cancer, gastric cancer and lung cancer, and the like, without being limited hereto.

[0044] In a tenth aspect of the present application, there is provided a method of treating a disease, the method comprising: administering to a subject a therapeutically effective dose of the above anti-β-catenin Nanobody, immunoconjugate or pharmaceutical composition.

[0045] Further, the disease can be a tumor, in particular a tumor with an abnormally high expression of β-catenin, such as breast cancer, colorectal cancer, hepatocellular carcinoma, prostate cancer, gastric cancer and lung cancer, and the like.

[0046] One or more of the above technical solutions have one or more of the following beneficial technical effects:

[0047] The above technical scheme obtains a nanobody with excellent affinity and specificity to β-catenin through sample screening, has the advantages of small molecular weight (about 15 kilodaltons), small immunogenicity, better solubility and stability, and a long CDR3 region, thereby being beneficial to basic research and clinical experiments, having potential tumor diagnosis and treatment prospects, and thus having good practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0048] The accompanying drawings, which form a part of this specification, are included to provide a further understanding of the application and are incorporated by reference herein. The drawings illustrate an exemplary embodiment of the present application and, together with the description, serve to explain the application.

[0049] Figure 1 Structural diagrams of the β-catenin-NB1F3 complex (a) and the β-catenin-NB1G9 complex (b) predicted by AlphaFold in the present application.

[0050] Figure 2 Affinity diagrams of the β-catenin-NB1F3 (a) and the β-catenin-NB1G9 (b) in the present application.

[0051] Figure 3 Size exclusion chromatography purification and SDS-PAGE diagrams of the β-catenin-NB1F3 (a) and the β-catenin-NB1G9 (b) in the present application.

[0052] Figure 4 HPLC chromatograms of the β-catenin-NB1F3 (a) and the β-catenin-NB1G9 (b) complex in the present application. DETAILED DESCRIPTION

[0053] It should be noted that the following detailed description is illustrative only and is not intended to limit the application in any way. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0054] It is to be understood that the terms used herein are for the purpose of describing specific embodiments and are not intended to limit the scope of the exemplary embodiments of the present application. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, devices, components, and / or combinations thereof, but do not preclude the presence or addition of one or more other features, steps, operations, devices, components, and / or combinations thereof.

[0055] In order that the disclosure can be more readily understood, certain terms are first defined. As used in this application, unless specifically stated otherwise, each of the following terms shall have the meaning given below. Additional definitions are set forth throughout the application.

[0056] The terms "single domain antibody," "VHH," "nanobody," "single domain antibody" (sdAb, or nanobody) have the same meaning and are used interchangeably to refer to the variable region of the heavy chain of a cloned antibody, which is constructed to consist of only one heavy chain variable region, and is the smallest antigen-binding fragment with full function. Usually, after obtaining an antibody naturally lacking a light chain and a heavy chain constant region 1 (CH1), the variable region of the heavy chain of the antibody is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

[0057] Nanobody / single domain antibody (Nanobody) is a new type of small molecule antibody fragment, which is cloned from the heavy chain variable region (VHH) of heavy chain antibody of camelids. Nanobody (Nb) has excellent biological properties, with a molecular weight of 12-15 kDa, which is one tenth of the complete antibody, has good tissue penetration, high specificity, and good water solubility. Due to its special structural properties, it has the advantages of traditional antibodies and small molecule drugs, and almost perfectly overcomes the defects of long development cycle, low stability, and harsh storage conditions of traditional antibodies, and gradually becomes a new force in the new generation of antibody therapy, showing broad application prospects in immunodiagnosis and treatment.

[0058] As used herein, the term "variable" refers to certain portions of the variable region of an antibody that differ in sequence among antibodies and are responsible for the binding and specificity of each particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the variable regions of an antibody. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the light chain and the heavy chain variable regions. The more highly conserved portions of the variable regions are called the framework regions (FRs). The variable regions of the heavy and light chains each comprise four FR regions, largely b- sheet in structure, connected by three CDRs, which form loops that connect, and in some cases pack against, the FRs of the opposite polypeptide chain. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, form the antigen binding site of antibodies (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647- 669 (1991)). The constant regions are not directly involved in binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0059] As known to those skilled in the art, immunoconjugates and fusion expression products include conjugates formed by the combination of a drug, a toxin, a cytokine, a radionuclide, an enzyme, and other diagnostic or therapeutic molecules with an antibody or a fragment thereof of the present application. The present application also includes cell surface markers or antigens conjugated with the nanobodies or fragments thereof against the novel coronavirus.

[0060] As used herein, the term "heavy chain variable region" is used interchangeably with "VH".

[0061] As used herein, the term "variable region" is used interchangeably with "complementarity determining region" (CDR).

[0062] In a preferred embodiment of the present application, the heavy chain variable region of the antibody comprises three complementarity determining regions CDR1, CDR2, and CDR3.

[0063] In a preferred embodiment of the present application, the heavy chain of the antibody comprises the heavy chain variable region described above and a heavy chain constant region.

[0064] In the present application, the terms "antibody of the present application", "protein of the present application", or "polypeptide of the present application" are used interchangeably and refer to a polypeptide that specifically binds to β-catenin, such as a protein or polypeptide having a heavy chain variable region. They can or can not contain the initial methionine.

[0065] The present application also provides other proteins or fusion expression products having the antibodies of the present application. In particular, the present application includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugates and fusion expression products) having a heavy chain comprising a variable region, provided that the variable region is identical to or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of the antibodies of the present application.

[0066] Generally, the antigen binding properties of an antibody can be described by three specific regions of the variable region of the heavy chain, called the complementarity determining regions (CDRs), which are interspersed with four framework regions (FRs) whose amino acid sequences are relatively conserved and do not directly participate in binding interactions. The CDRs form loops or "hot spots" on the surface of the variable region, and are in close proximity to each other through the FRs that form a beta sheet. The CDRs on the heavy chain and the corresponding CDRs on the light chain make up the antigen binding site of the antibody. It is possible to determine which amino acids make up the FR or CDR regions by comparing the amino acid sequences of antibodies of the same class.

[0067] The variable regions of the heavy chains of the antibodies of the present application are of particular interest because at least some of them are involved in binding the antigen. Thus, the present application includes those molecules having the variable region of the heavy chain of the antibody with the CDRs, provided that the CDRs are at least 90% homologous, preferably at least 95% homologous, and most preferably at least 98% homologous to the CDRs identified herein.

[0068] The present application includes not only intact antibodies, but also fragments of the antibodies that are immunologically active or fusion proteins of the antibodies with other sequences. Thus, the present application also includes fragments, derivatives, and analogs of the antibodies.

[0069] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity of the antibodies of the present application. The polypeptide fragments, derivatives, or analogs of the present application can be (i) polypeptides having one or more conservative or non-conservative amino acid substitutions (preferably conservative amino acid substitutions) of the amino acid residues, where such substituted amino acid residues can or can not be encoded by the genetic code, or (ii) polypeptides having a substitution group at one or more amino acid residues, or (iii) polypeptides formed by fusing the mature polypeptide to another compound (such as a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) polypeptides formed by fusing additional amino acid sequences to the polypeptide sequence (such as a leader or secretion sequence, or a sequence or protein for purification of the polypeptide, or a proprotein sequence, or a fusion protein with a 6xHis tag). These fragments, derivatives, and analogs are within the purview of one of skill in the art in light of the teachings herein.

[0070] Variants of the polypeptide include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes to the DNA encoding the antibody of the present application under high or low stringency conditions, and polypeptides or proteins obtained using antisera to the antibody of the present application.

[0071] The present application also provides other polypeptides, such as fusion proteins comprising an antibody or fragment thereof. In addition to polypeptides that are nearly full-length, the present application also includes fragments of the antibody of the present application. Typically, the fragment has at least about 50 contiguous amino acids of the antibody of the present application, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

[0072] In the present application, "conservative variants of the antibody of the present application" refers to polypeptides in which up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced with amino acids of similar or analogous properties as compared to the amino acid sequence of the antibody of the present application.

[0073] Nanobodies targeting β-catenin

[0074] As used herein, the terms "nanobody," "nanobody targeting β-catenin," and "β-catenin nanobody" are used interchangeably and refer to a nanobody that specifically recognizes and binds to β-catenin, including human β-catenin.

[0075] In embodiments of the present application, a nanobody targeting β-catenin is provided, wherein the complementarity determining regions (CDRs) of the VHH chain of the nanobody are one or more selected from the group consisting of:

[0076] (1) CDR1 as set forth in SEQ ID NO. 1, CDR2 as set forth in SEQ ID NO. 2, and CDR3 as set forth in SEQ ID NO. 3 (corresponding to the CDRs of nanobody β-catenin-NB1F3);

[0077] (2) CDR1 as set forth in SEQ ID NO. 6, CDR2 as set forth in SEQ ID NO. 7, and CDR3 as set forth in SEQ ID NO. 8 (corresponding to the CDRs of nanobody β-catenin-NB1G9).

[0078] (3) a sequence having β-catenin binding affinity in which any one of the above-mentioned amino acid sequences is added, deleted, modified and / or substituted with at least one (e.g., 1-5, 1-3, preferably 1-2, more preferably 1) amino acid.

[0079] In another preferred embodiment, the sequence formed by the addition, deletion, modification and / or substitution of at least one amino acid sequence is preferably an amino acid sequence having a homology of at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%.

[0080] The antibody of the present application can be a double-chain or single-chain antibody, and can be selected from an animal-derived antibody (e.g., a camel-derived antibody), a chimeric antibody, a humanized antibody, more preferably a humanized antibody, a human-animal chimeric antibody, more preferably a fully humanized antibody.

[0081] The antibody derivative of the present application can be a single-chain antibody, and / or an antibody fragment such as Fab, Fab', (Fab')2, or other known antibody derivatives in the art, and any one or several of IgA, IgD, IgE, IgG, and IgM antibodies or other subtypes of antibodies.

[0082] In a preferred embodiment of the present application, the sequence of any one or several of SEQ ID NO. 1, 2 and 3, or a sequence having a binding affinity to the N-terminus of β-catenin formed by the addition, deletion, modification and / or substitution of at least one amino acid thereof, is located in the CDR region of the heavy chain variable region (VH).

[0083] In a preferred embodiment of the present application, the sequence of any one or several of SEQ ID NO. 6, 7 and 8, or a sequence having a binding affinity to the C-terminus of β-catenin formed by the addition, deletion, modification and / or substitution of at least one amino acid thereof, is located in the CDR region of the heavy chain variable region (VH).

[0084] In the present application, the number of added, deleted, modified and / or substituted amino acids is preferably not more than 40% of the total number of amino acids of the original amino acid sequence, more preferably not more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.

[0085] In the present application, the number of added, deleted, modified and / or substituted amino acids is typically 1, 2, 3, 4 or 5, more preferably 1-3, more preferably 1-2, most preferably 1.

[0086] It should be understood that the antibody of the present application also includes an antibody having one or more mutations in the FR region without mutations or only 1 or 2 conservative mutations in the CDR region, and still retaining the specific binding and affinity to the N-terminus or C-terminus of β-catenin.

[0087] Preparation of antibody

[0088] Any method suitable for generating antibodies can be used to generate the β-catenin-targeting nanobodies of the application. For example, an animal can be immunized with a linked or naturally occurring β-catenin or fragment thereof. Suitable immunization protocols can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes of administration can be used.

[0089] Any suitable form of β-catenin can be used as an immunogen (antigen) for generating non-human antibodies specific for β-catenin, and for screening the antibodies for biological activity. The immunogen can be recombinant β-catenin or a fragment thereof. The immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art. The immunogen can be purified from a natural source or produced in a genetically modified cell. The DNA encoding the immunogen can be genomic or non-genomic (e.g., cDNA) in origin. The DNA encoding the immunogen can be expressed using a suitable genetic vector, including but not limited to: adenoviral vectors, adeno-associated viral vectors, baculoviral vectors, plasmids, and non-viral vectors.

[0090] The antibodies of the application can be selected from any class of immunoglobulin, including IgG and IgE, of any species. Preferred antibodies are IgG antibodies, such as the IgG1 subtype. Optimization of the necessary constant domain sequences to produce the desired biological activity is readily achieved by screening antibodies using the biological assays described in the Examples below.

[0091] Likewise, any class of light chain can be used in the compounds and methods herein. In particular, kappa, lambda chains, or variants thereof are useful in the compounds and methods of the application.

[0092] The sequence of the DNA molecule of the antibody or fragment thereof of the application can be obtained using conventional techniques, such as by PCR amplification or screening of genomic libraries.

[0093] Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods. This is typically done by cloning it into a vector, which is then introduced into a cell, and then the relevant sequence is isolated from the propagated host cell using conventional methods.

[0094] In addition, the relevant sequence can be synthesized using artificial synthesis methods, particularly for shorter fragments. Typically, longer fragments are obtained by first synthesizing a number of smaller fragments, which are then ligated. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art.

[0095] The application also relates to vectors comprising the appropriate DNA sequence described above, as well as an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells, so that they are able to express the protein.

[0096] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include CHO, NSO, HEK293, PERC6 cells.

[0097] The step of transforming a host cell with recombinant DNA described in the present application can be performed using techniques well known in the art. The transformants obtained can be cultured using conventional methods, and the transformants express the polypeptides encoded by the genes of the present application. The culture is performed under suitable conditions using a conventional medium, depending on the host cell used.

[0098] Generally, the host cell obtained by transformation is cultured under conditions suitable for the expression of the antibody of the present application. The antibody of the present application is then purified using conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, size exclusion chromatography, or affinity chromatography, as well as other conventional separation and purification methods known to those skilled in the art.

[0099] The monoclonal antibodies obtained can be identified using conventional means. For example, the binding specificity of the monoclonal antibodies can be determined using immunoprecipitation or an in vitro binding assay, such as a radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA).

[0100] Pharmaceutical composition

[0101] The present application also provides a composition. Preferably, the composition is a pharmaceutical composition, which comprises the antibody or active fragment thereof or fusion protein thereof described above, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5 to 8, preferably about 6 to 8, although the pH value can vary depending on the nature of the substance to be formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by a conventional route, including (but not limited to) intratumoral, intraperitoneal, intravenous, or local administration.

[0102] The pharmaceutical composition of the present application can be used directly to bind to the β-catenin protein molecule, and thus can be used to treat tumors or cancer. In addition, other therapeutic agents can also be used simultaneously.

[0103] The pharmaceutical composition of the present application contains a safe and effective amount (e.g. 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the single-domain antibody (or conjugate thereof) described above and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched with the administration mode. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition such as a needle, a solution is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, about 10 μg / kg body weight to about 50 mg / kg body weight per day. In addition, the antibody or active fragment thereof or fusion protein thereof of the present application can also be used with other therapeutic agents.

[0104] When the pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight, preferably the dose is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dose should also take into account the administration route, patient health status, etc., which are within the skill of the skilled physician.

[0105] The present application is further explained by the following examples, which do not limit the present application. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application.

[0106] Example 1 Construction and screening of nanobody phage display library

[0107] Step (1) Animal immunization: The β-catenin antigen was mixed with Freund's adjuvant at a ratio of 1:1, and the adult alpaca was immunized by subcutaneous injection of multiple points on the back at a dose of 1 mg / time. The immunization was performed for 4 times with an interval of 2 weeks.

[0108] Step (2) Extraction of total RNA: 10 ml of peripheral blood of the immunized animal was taken, and the peripheral blood of the immunized animal was separated by density gradient centrifugation. 1 ml of peripheral blood of the immunized animal was taken, 500 μl of nucleic acid extraction solution and 100 μl of chloroform were added, and the mixture was shaken vigorously for 15 s at room temperature. After standing for 5 min, centrifugation was performed at 12,000 g and 4°C for 10 min, and the supernatant was discarded. 500 μl of 75% ethanol was added to wash the precipitate. Centrifugation was performed at 7500 g and 4°C for 5 min, and the supernatant was discarded. The precipitate was dried at room temperature for 10 min, and then an appropriate amount of DEPC water was added. Incubation was performed at 55°C for 10 min. After the gel was recovered by agarose gel method, the concentration was adjusted to 1 μg / μl with RNase-free water.

[0109] Step (3) Obtain antibody variable region gene: Use reverse transcription kit instruction to carry out reverse transcription cDNA with RNA obtained in step (2) as template. Amplify antibody variable region gene: use the cDNA obtained by reverse transcription as template to carry out polymerase chain reaction. Amplify for two rounds, the primer sequence of the first round of polymerase chain is as follows:

[0110] F: GTCCTGGCTGCTCTTCTACAAGG

[0111] R: GGTACGTGCTGTTGAACTGTTCC

[0112] The polymerase chain reaction condition and procedure are: 95℃ for 5 minutes; 95℃ for 30 seconds, 57℃ for 30 seconds, 72℃ for 30 seconds, 30 cycles; 72℃ for 7 minutes; use agarose gel recovery kit to recover the band of about 700bp, finally adjust the nucleic acid concentration to 5 nanograms / microliter with water.

[0113] The primer sequence of the second round of polymerase chain is as follows:

[0114] F: GATGTGCAGCTGCAGGAGTCTGGRGGAGG

[0115] R: CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT

[0116] The polymerase chain reaction condition and procedure are: 95℃ for 5 minutes; 95℃ for 30 seconds, 55℃ for 30 seconds, 72℃ for 30 seconds, 15 cycles; 72℃ for 7 minutes, use agarose gel to cut and recover nanobody fragment.

[0117] Step (4) Vector construction and clone selection: use endonuclease PstI, BstEII to double enzyme cut 10 micrograms of pMECS vector, 5 microliters of 10x buffer, add water to 50 microliters, 37℃ enzyme cut for 1h; use PstI, BstEII to double enzyme cut the antibody fragment in step (3), 5 microliters of 10x buffer, add water to 50 microliters, 37℃ enzyme cut for 1h. Recover the enzyme cut vector and antibody fragment with agarose gel; take 100ng of enzyme cut and recovered antibody fragment, 300ng of enzyme cut and recovered vector, 2 microliters of T4 ligase, 2 microliters of 10x buffer, add water to 20 microliters, 16℃ connect overnight. Take 10 microliters of purified connection product, electrotransfer to 50 microliters of E. coli TG1 competent cells, recover and culture, then dilute and plate, randomly select clones, and carry out colony PCR identification. According to the positive rate of polymerase chain reaction, calculate the library capacity (library capacity = number of clones x dilution factor x [positive rate] PCR identification x 10). The PCR primer sequence is as follows:

[0118] F: TTATGCTTCCGGCTCGTATG

[0119] R: CCACAGACAGCCCTCATAG

[0120] Step (5) Phage screening of nanobodies: inoculate the bacterial library of step (4) into 200 microliter culture medium containing tetracycline and ampicillin, incubate at 37°C, 250 rpm until the absorbance at 600 nm is 0.5, add 1 microliter helper phage M13KO7, incubate at 37°C for 30 minutes. Add kanamycin at a final concentration of 50 micrograms per milliliter and 200 micromolar isopropyl-beta-D-thiogalactopyranoside; thiogalactopyranoside; isopropyl thiio-beta-D-galactoside; isopropyl thiogalactoside; galactose, incubate at 30°C, 250 rpm overnight, precipitate by polyethylene glycol 8000 / sodium chloride, dissolve in phosphate buffer to obtain the phage display library; coat the beta-catenin antigen at a concentration of 2 micrograms per milliliter, block with 3% bovine serum albumin, wash with phosphate buffer; add 100 microliters of phage, incubate at room temperature for 2 hours, wash 10 times with phosphate buffer, and the eluted phage is used to transfect TG1 for the next round of screening. After 3 rounds of screening, positive clones are verified by enzyme-linked immunoassay and sequenced. Finally, two anti-beta-catenin nanobodies are obtained, designated as NB1F3 and NB1G9.

[0121] Example 2 Expression and purification of beta-catenin antibodies

[0122] Step (1) Antibody expression: the nucleotides of NB1F3 and NB1G9 are cloned into the pMECS vector with a PelB signal peptide and a C-terminal HA tag and His6 tag, respectively. Expression is carried out in the periplasm of Escherichia coli TOP10F' cells (WXR15-100S, Huayueyang Biotech, China).

[0123] Step (2) Antibody purification: purification is carried out using Ni-NTA, and the treated sample is subjected to column treatment. Specifically, after induction, centrifuge at 6000 rpm for 10 min, collect the bacterial cells, add 2 ml bacterial lysis solution per 100 mg of bacterial cells (wet weight), and lyse the bacterial cells by ultrasonication (300 w, 5 s on and 10 s off per cycle, for a total of 10 min). Centrifuge at 10,000 rpm, 4°C for 3 min, and collect the supernatant. Load the diluted bacterial lysis solution onto the column at a flow rate of 10 column volumes per hour, and collect the flow-through. Wash away the impurities using 15 column volumes of 20 mM HEPES (pH 7.4), 10 mM sodium chloride, 5% glycerol, and 30 mM imidazole, and elute using 5 column volumes of 20 mM HEPES (pH 7.4), 10 mM sodium chloride, 5% glycerol, and 300 mM imidazole, and collect the eluate.

[0124] Step (3) Antibody affinity assay: StrepAvidin sensor was immersed in PBS buffer for 2 minutes to ensure stable baseline signal. Biotinylated β-catenin was loaded onto the sensor, reacted at room temperature for 2 minutes until a stable binding response signal was reached. After loading was completed, the sensor was rinsed with wash buffer to remove unbound β-catenin molecules. Different concentrations of NB1F3 antibody and NB1G9 antibody (3nM to 100nM, diluted in the same reaction buffer) were added in turn, and the binding response at each concentration was recorded for 1 minute, followed by dissociation. The binding and dissociation curves at each concentration were analyzed using the bimolecular model (1:1) in the Octet software to calculate the association rate constant, dissociation rate constant and affinity constant. The results are shown in Figure 2 . It is shown that the above two nanobodies have good affinity for β-catenin protein.

[0125] Amino acid and nucleotide sequence information used in the present application:

[0126] β-catenin-NB1F3 nanobody

[0127] CDR1: AASGYAYC (SEQ ID NO. 1)

[0128] CDR2: IDSDGIT (SEQ ID NO. 2)

[0129] CDR3: TADEDVCVNYGLGTGQGIHPADFGY (SEQ ID NO. 3)

[0130] VHH amino acid sequence:

[0131] QVQLQESGGGSVQAGGSLRLSCAASGYAYCMGWFRQAPGKEREGVAAIDSDGITSYADSVKGRFTISKDNAKNTLYLQMNSLKPEDTAMYYCTADE DVCVNYGLGTGQGIHPADFGYWGQGTQVTVSS (SEQ ID NO. 4)

[0132] VHH nucleotide sequence:

[0133] CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTG

[0134] AGACTCTCCTGTGCAGCCTCTGGATACGCCTACTGCATGGGCTGGTTCCGCCAGGC

[0135] TCCAGGAAAGGAGCGCGAGGGGGTCGCGGCTATTGATAGTGATGGTATCACAAGC

[0136] TACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAAAGACAACGCCAAGAAC

[0137] ACTCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACTGCCATGTACTACTG

[0138] TACGGCAGATGAGGATGTGTGTGTGAACTATGGGTTGGGTACGGGGCAGGGAATC

[0139] CACCCCGCTGACTTTGGTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA(SEQ ID NO.5)

[0140] β-catenin-NB1 G9 Nanobody

[0141] CDR1: GYTYSSYC (SEQ ID NO. 6)

[0142] CDR2: IDSDGST (SEQ ID NO. 7)

[0143] CDR3: AAALGCDYDIWEYNS (SEQ ID NO. 8)

[0144] VHH amino acid sequence:

[0145] QVQLQESGGGSVQAGGSLRLSCAASGYTYSSYCMGWFRQAPGKEREGVADIDSDGSTSYADSVKGRFTISKDNAKNTLYLQMNSLKPEDTARYYCAAALGCDYDIWEYNSWGQGTQVTVSS (SEQ ID NO. 9)

[0146] VHH nucleotide sequence:

[0147] CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTG

[0148] AGACTCTCCTGTGCAGCCTCTGGATACACCTACAGTAGCTACTGCATGGGCTGGTT

[0149] CCGCCAGGCT CCAGGGAAGG AGCGCGAGGG GGTA GCAGATATT GATAGTGATG GT

[0150] AGCACAAGCT ACGCAGACTC CGTGAAGGGC CGATTCACCA TCTCCAAAGA CAAC

[0151] GCCAAGAACACTCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACTGCCA

[0152] GATACTACTGTGCGGCAGCTCTGGGGTGCGACTATGACATATGGGAGTATAACTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA (SEQ ID NO. 10)

[0153] It should be noted that the above examples are only used to illustrate the technical solutions of the present application but not to limit it. Although the present application is explained in detail with reference to the examples given, the ordinary skilled in the art can modify or equivalently replace the technical solutions of the present application according to the needs without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. A nanobody against β-catenin, characterized in that, The heavy chain variable region of the nanobody comprises complementarity determining regions CDR1, CDR2 and CDR3; the CDRs are selected from: (1) CDR1 as shown in SEQ ID NO. 1, CDR2 as shown in SEQ ID NO. 2 and CDR3 as shown in SEQ ID NO. 3; (2) CDR1 as shown in SEQ ID NO. 6, CDR2 as shown in SEQ ID NO. 7 and CDR3 as shown in SEQ ID NO.

8.

2. The anti-β-catenin Nanobody of claim 1, wherein, The heavy chain variable region of the nanobody has an amino acid sequence as shown in SEQ ID NO. 4 or 9 or an amino acid sequence having no less than 80% sequence identity thereto.

3. An isolated nucleic acid molecule, comprising, The nucleic acid molecule is capable of encoding the anti-β-catenin nanobody of claim 1 or 2.

4. The nucleic acid molecule of claim 3, wherein, The nucleic acid molecule has: a1) a nucleotide sequence as shown in SEQ ID NO. 5 or 10; a2) a complementary nucleotide sequence of the nucleotide sequence as shown in SEQ ID NO. 5 or 10; a3) a nucleotide sequence encoding the same protein as the nucleotide sequence of a1) or a2), but different from the nucleotide sequence of a1) or a2) due to the degeneracy of the genetic code.

5. A recombinant expression vector, characterized in that, It comprises the nucleic acid molecule of claim 3 or 4.

6. A host cell, characterized in that, It comprises the recombinant expression vector of claim 5, or the nucleic acid molecule of claim 3 or 4 integrated into the genome of a host cell; or the host cell expresses the anti-β-catenin nanobody of claim 1 or 2.

7. A method of preparing the anti-β-catenin Nanobody of claim 1 or 2, characterized in that, It comprises: culturing the host cell of claim 6, thereby obtaining a culture comprising the anti-β-catenin nanobody; and isolating or recovering the anti-β-catenin nanobody from the culture.

8. An immunoconjugate, comprising, It comprises the anti-β-catenin nanobody of claim 1 or 2, and a conjugated moiety, wherein the conjugated moiety is a detectable label.

9. A test kit comprising, It comprises the anti-β-catenin nanobody of claim 1 or 2, and / or the immunoconjugate of claim 8; the kit can be used for detecting the presence of β-catenin in a sample.

10. Use of the anti-β-catenin nanobody of claim 1 or 2 or the immunoconjugate of claim 8 in the manufacture of a product for detecting β-catenin.

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