Anti-beta-catenin nano antibody as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an anti-beta-catenin nano antibody as well as a preparation method and application thereof. Specifically, the nano antibody with excellent affinity with beta-catenin is obtained through sample screening, and the nano antibody has the advantages and values of small molecular weight (about 15 thousand Daltons), small immunogenicity, better solubility and stability and longer CDR3 region, so that fundamental research and clinical experiments are facilitated, and the nano antibody has potential tumor diagnosis and treatment prospects. Therefore, the method has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to anti-β-catenin nano antibodies and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] The canonical Wnt signaling pathway plays an important role in the proliferation and differentiation of tumor cells. The core protein molecule β-catenin in this signaling pathway mediates the transmission of signals from the cytoplasm to the nucleus, and participates in the formation of a transcription complex in the nucleus to activate the transcription of downstream target genes. The entry of β-catenin into the nucleus is an important prerequisite for its activation of downstream transcription factors, and its entry and exit from the nucleus has not been fully resolved. The main ways for β-catenin to enter the nucleus are direct entry into the nucleus using key proteins of the canonical nuclear import pathway and the assistance of "molecular chaperones" to enter the nucleus, while the main way for β-catenin to exit the nucleus is the nuclear exit pathway that depends on chromosome maintenance region 1 (CRM1). Cadherin on the cell membrane, degradation complex-related proteins in the cytoplasm, and transcription complex-related proteins in the nucleus can all lead to the retention of β-catenin, thereby affecting its distribution inside and outside the nucleus.

[0004] At the same time, abnormal expression of β-Catenin protein in cells can be seen in a variety of human malignant tumors. It has been reported that abnormal subcellular localization and aggregation of β-Catenin protein are closely related to cell malignant transformation and proliferation, and similar conclusions have been obtained in breast cancer, colorectal cancer, hepatocellular carcinoma, prostate cancer, gastric cancer and lung cancer. Therefore, it is of great significance to design antibodies targeting β-Catenin protein to evaluate human health status, assess the progression of diseases, especially tumor-related diseases, and carry out targeted disease treatment.

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

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides anti-β-catenin nanobodies and preparation methods and applications. Specifically, the present invention successfully prepares anti-β-catenin nanobodies with high affinity, stability and specificity, which can be used for the detection and analysis of β-catenin and the diagnosis and treatment of diseases related to β-catenin protein, especially tumors with abnormally high expression of β-catenin. Based on the above research results, the present invention is completed.

[0007] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect of the present invention, there is provided an anti-β-catenin nanobody, wherein the heavy chain antibody heavy chain variable region (VHH) of the nanobody comprises a complementarity determining region (CDR), wherein the complementarity determining region has CDR1, CDR2 and CDR3 or has an amino acid sequence having a sequence identity of not less than 80% thereto;

[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 heavy chain antibody of the Nanobody has an amino acid sequence as shown in SEQ ID NO. 4 or 9 or an amino acid sequence having a sequence identity of not less than 80% thereto.

[0014] Furthermore, the anti-β-catenin nanobody can 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, which is not specifically limited here.

[0015] The second aspect of the present invention provides an isolated nucleic acid molecule, which can encode the above-mentioned anti-β-catenin nanobody.

[0016] The nucleic acid molecule has:

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

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

[0019] a3) a nucleotide sequence that encodes the same protein as the nucleotide sequence of a1) or a2) but is different from the nucleotide sequence of a1) or a2) due to the degeneracy of the genetic code;

[0020] a4) A nucleotide sequence obtained by replacing, deleting or adding one or two nucleotide sequences to the nucleotide sequence shown in any one of a1) to a3), and having the same or similar functions as the nucleotide sequence shown in any one of a1) to a3).

[0021] The third aspect of the present invention provides a recombinant expression vector comprising the above-mentioned nucleic acid molecule.

[0022] Furthermore, the recombinant expression vector is obtained by connecting the nucleic acid molecule with an expression vector; wherein the expression vector can be selected from: 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] The fourth aspect of the present invention provides a host cell, which comprises the above-mentioned recombinant expression vector, or comprises the above-mentioned nucleic acid molecule integrated into the host cell genome; or, the host cell expresses the above-mentioned anti-β-catenin nanobody.

[0024] The host cells include cells of prokaryotic and eukaryotic origin. Among them, prokaryotic organisms are preferably Escherichia coli, and eukaryotic organisms include yeast and mammalian cells, wherein mammalian cells include CHO, NS0, HEK293, PERC6, etc., which are not specifically limited here.

[0025] A fifth aspect of the present invention provides a method for preparing an anti-β-catenin nanobody, comprising:

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

[0027] The sixth aspect of the present invention provides an immunoconjugate, which includes the anti-β-catenin nanobody described in the present invention, and a coupling part, wherein the coupling part is a detectable marker, a drug, a toxin, a cytokine, a viral coat protein or a virus-like particle, etc.

[0028] Wherein, the detectable marker includes a radioactive isotope, 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 folic acid antagonist, an antimetabolite drug, a chemosensitizer, a topoisomerase inhibitor, a vinca alkaloid, and the like.

[0030] The toxin can be chlortetracycline, maytansin, ricin, ethidium bromide, mitomycin, diphtheria toxin, abrin, gelonin, metotoxin, restrictocin, phenomycin, curcin, crotonin, calicheamicin, glucocorticoid, etc., and is not specifically limited here.

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

[0032] The seventh aspect of the present invention provides a detection kit, which includes the above-mentioned anti-β-catenin nanobody and / or the above-mentioned immunoconjugate; 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 to be tested. It is understood that the above detection kit can be used for tumor detection, monitoring, diagnosis and prognosis.

[0034] The eighth aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned anti-β-catenin nanobody or the above-mentioned immunoconjugate.

[0035] Furthermore, it also includes a pharmaceutically acceptable carrier and / or excipient. Moreover, according to the usual method, it can be made into a dosage form in the form of an oral preparation, an external preparation, a suppository, and a sterile injection solution such as a powder, a granule, a tablet, a capsule, a suspension, an emulsion, a syrup, a spray, etc.

[0036] The non-pharmaceutical active ingredients such as carriers and / or excipients that may be included are well known in the art, and those skilled in the art can determine whether they meet clinical standards.

[0037] Further, the pharmaceutical composition of the present invention can be applied to the body in a known manner. For example, it can be delivered to the tissue of interest by intravenous systemic delivery or local injection. It can be optionally applied via intravenous, percutaneous, intranasal, mucosal or other delivery methods. Such application can be carried out via single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be applied in the present invention can vary depending on various factors to a great extent, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0038] The subject to which the pharmaceutical composition is administered can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc. Among them, humans are the most preferred.

[0039] The ninth aspect of the present invention provides the use of the above-mentioned anti-β-catenin nanobody, immunoconjugate or pharmaceutical composition in any one or more of the following:

[0040] b1) Preparation of tumor detection, monitoring, diagnosis and / or prognosis products;

[0041] b2) Preparation of products for tumor prevention and / or treatment.

[0042] Tumor is used in the present invention as known to those skilled in the art. Benign tumors are defined as excessive proliferation of cells that are unable to form aggressive, metastatic tumors in the body. In contrast, malignant tumors are defined as cells with multiple cellular and biochemical abnormalities that are capable of forming systemic diseases (such as forming tumor metastases in distant organs).

[0043] Furthermore, the tumor is particularly a tumor with abnormally high expression of β-catenin, such as breast cancer, colorectal cancer, hepatocellular carcinoma, prostate cancer, gastric cancer and lung cancer, etc., which are not specifically limited here.

[0044] The tenth aspect of the present invention provides a method for treating a disease, the method comprising: administering a therapeutically effective dose of the above-mentioned anti-β-catenin nanobody, immunoconjugate or pharmaceutical composition to a subject.

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

[0046] One or more beneficial technical effects of the above technical solution:

[0047] The above technical scheme obtains nano-antibodies with excellent affinity and specificity for β-catenin through sample screening. It has the advantages of small molecular weight (~15 kilodaltons), low immunogenicity, better solubility and stability, and a longer CDR3 region, which is beneficial to basic research and clinical experiments, has potential prospects for tumor diagnosis and treatment, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

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

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

[0051] Figure 3 The size exclusion chromatography purification and SDS-PAGE images of β-catenin-NB1F3 (a) and β-catenin-NB1G9 (b) in the present invention.

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

[0053] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0054] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

[0055] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other 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 can be used interchangeably, referring to cloning the variable region of the antibody heavy chain to construct a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, antibodies that naturally lack light chains and heavy chain constant regions 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain 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 variable region (VHH) of the heavy chain antibody of camel. Nanobody (Nb) has excellent biological properties, with a molecular weight of 12-15kDa, which is one-tenth of the complete antibody. It has good tissue penetration, high specificity and good water solubility. Due to its special structural properties, it combines the advantages of traditional antibodies and small molecule drugs, and almost perfectly overcomes the defects of traditional antibodies such as long development cycle, low stability and harsh storage conditions. It has gradually become an emerging force in the new generation of antibody therapy, showing broad application prospects in immune diagnosis and treatment.

[0058] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conservative parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a b-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial beta folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region is not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0059] As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines (cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules combined with antibodies or fragments thereof of the present invention to form conjugates. The present invention also includes cell surface markers or antigens combined with the nano antibodies or fragments thereof against the new coronavirus.

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

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

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

[0063] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.

[0064] In the present invention, the terms "antibodies of the present invention", "proteins of the present invention", or "polypeptides of the present invention" are used interchangeably, and all refer to polypeptides that specifically bind to β-catenin, such as proteins or polypeptides having a heavy chain variable region. They may or may not contain an initial methionine.

[0065] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibodies of the present invention, preferably at least 95% homologous.

[0066] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four framework regions (FR). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

[0067] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because they are at least partially involved in binding to antigen. Therefore, the present invention includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.

[0068] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention 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 as the antibodies of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6xHis tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0070] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using the antiserum of the antibody of the present invention.

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

[0072] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the antibodies of the present invention.

[0073] Nanobodies targeting β-catenin

[0074] As used in the present invention, the terms "nanoantibody", "nanoantibody targeting β-catenin", and "β-catenin nanoantibody" are used interchangeably and all refer to nanoantibodies that specifically recognize and bind to β-catenin (including human β-catenin).

[0075] In an embodiment of the present invention, a nanobody targeting β-catenin is provided, wherein the complementarity determining region (CDR) of the VHH chain of the nanobody is one or more selected from the following group:

[0076] (1) CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, and CDR3 shown in SEQ ID NO.3 (corresponding to the CDR of Nanobody β-catenin-NB1F3);

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

[0078] (3) A sequence having β-catenin binding affinity by adding, deleting, modifying and / or replacing at least one (e.g., 1-5, 1-3, preferably 1-2, and more preferably 1) amino acid in any of the above amino acid sequences.

[0079] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or replacing at least one amino acid sequence is preferably an amino acid sequence with a homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

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

[0081] The antibody derivatives of the present invention can be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other known antibody derivatives in the field, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

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

[0083] In a preferred embodiment of the present invention, any one or several of the above SEQ ID NOs. 6, 7 and 8, or sequences thereof having binding affinity for the C-terminus of β-catenin after addition, deletion, modification and / or substitution of at least one amino acid, are located in the CDR region of the heavy chain variable region (VH).

[0084] In the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.

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

[0086] It should be understood that the antibodies of the present invention also include antibodies containing one or more mutations in the FR region and no mutation or only 1 or 2 conservative mutations in the CDR region, and still retaining specific binding and affinity to the N-terminus or C-terminus of β-catenin.

[0087] Antibody preparation

[0088] Any method suitable for producing antibodies can be used to produce the targeted β-catenin nanobodies of the present invention. For example, animals can be immunized with linked or naturally occurring β-catenin or fragments thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more approaches can be used.

[0089] Any suitable form of β-catenin can be used as an immunogen (antigen) to produce non-human antibodies specific for β-catenin and screen the biological activity of the antibodies. The stimulating 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: an adenovirus vector, an adeno-associated virus vector, a baculovirus vector, a plasmid and a non-viral vector.

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

[0091] Likewise, either type of light chain can be used in the compounds and methods herein. Specifically, kappa, lambda chains or variants thereof are useful in the compounds and methods of the invention.

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

[0093] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0094] In addition, artificial synthesis methods can also be used to synthesize the relevant sequence, especially when the fragment length is short. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0095] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[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, NS0, HEK293, PERC6 cells.

[0097] The step of transforming host cells with recombinant DNA described in the present invention can be carried out by techniques well known in the art. The obtained transformants can be cultured by conventional methods, and the transformants express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, conventional culture medium is used to cultivate under suitable conditions.

[0098] Typically, the transformed host cells are cultured under conditions suitable for the expression of the antibodies of the present invention, and then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc., conventional separation and purification means well known to those skilled in the art to obtain the antibodies of the present invention.

[0099] The resulting monoclonal antibodies can be characterized by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0100] Pharmaceutical composition

[0101] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, 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 5-8, preferably about 6-8, although the pH value may vary depending on the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

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

[0103] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned single-domain antibody (or its conjugate) of the present invention 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 match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. In addition, the antibody or its active fragment or its fusion protein of the present invention can also be used with other therapeutic agents.

[0104] When using a pharmaceutical composition, 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 micrograms / kg body weight, and in most cases does not exceed about 50 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight to about 10 milligrams / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

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

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

[0107] Step (1) Animal immunization: After mixing the β-catenin antigen and Freund's adjuvant in a ratio of 1:1, the mixture is immunized by multiple subcutaneous injections at the back of adult alpacas at a dose of 1 mg / time, for a total of 4 immunizations, with an immunization interval of 2 weeks.

[0108] Step (2) extracting total RNA: in step (1), 10 ml of peripheral blood from the immunized animal is taken, and the peripheral blood of the immunized animal is separated by density gradient centrifugation. 1 ml of peripheral blood from the immunized animal is added with 500 μl of nucleic acid extraction solution and 100 μl of chloroform, and the mixture is shaken vigorously for 15 seconds, and allowed to stand at room temperature for 5 minutes; centrifuged at 12,000 g, 4°C for 10 minutes, the supernatant is discarded, and 500 μl of 75% ethanol is added to wash the precipitate; centrifuged at 7500 g, 4°C for 5 minutes, the supernatant is discarded; dried at room temperature for 10 minutes, an appropriate amount of DEPC water is added, and the mixture is incubated at 55°C for 10 minutes. After the mixture is cut and recovered by agarose gel method, the concentration is adjusted to 1 μg / μl with RNase-free water.

[0109] Step (3) Obtaining antibody variable region genes: Use the reverse transcription kit instructions to reverse transcribe cDNA using the RNA obtained in step (2) as a template. Antibody variable region gene amplification: Use the cDNA obtained by reverse transcription as a template for polymerase chain reaction. Amplification is performed in 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 conditions and procedures were as follows: 95°C for 5 minutes; 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 30 seconds, 30 cycles; 72°C for 7 minutes; using an agarose gel recovery kit to recover a band of about 700 bp, and finally adjusting the nucleic acid concentration to 5 ng / μl with water.

[0113] The primer sequences for the second round of polymerase chain are as follows:

[0114] F: GATGTGCAGCTGCAGGAGTCTGGRGGAGG

[0115] R: CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT

[0116] The polymerase chain reaction conditions and procedures were as follows: 95°C for 5 minutes; 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, for 15 cycles; 72°C for 7 minutes, and the nanoantibody fragments were recovered by gel excision using agarose gel.

[0117] Step (4) vector construction and clone selection: 10 μg of pMECS vector was double-digested with nuclease PstI and BstEII, 5 μl of 10x buffer was added to 50 μl, and digested at 37°C for 1 h; the antibody fragment in step (3) was double-digested with PstI and BstEII, 5 μl of 10x buffer was added to 50 μl, and digested at 37°C for 1 h. The vector and antibody fragment after digestion were recovered by agarose gel; 100 ng of antibody fragment recovered by digestion, 300 ng of vector recovered by digestion, 2 μl of T4 ligase, 2 μl of 10x buffer, and water was added to 20 μl, and ligated at 16°C overnight. 10 μl of the purified ligation product was taken and electroporated into 50 μl of E. coli TG1 competent cells, and diluted and plated after recovery culture, and clones were randomly selected for colony PCR identification. The library capacity was calculated based on the polymerase chain reaction positive rate (library capacity = number of clones × dilution factor × [positive rate] PCR identification × 10). The PCR primer sequences are as follows:

[0118] F:TTATGCTTCCGGCTCGTATG

[0119] R:CCACAGACAGCCCTCATAG

[0120] Step (5) Phage screening of nanoantibodies: Take an appropriate amount of the bacterial library in step (4) and inoculate it into 200 μl of culture medium containing tetracycline and ampicillin, culture it at 37°C and 250 rpm until the absorbance is 600 = 0.5, add 1 ml of helper phage M13KO7, and infect it at 37°C for 30 minutes. Add kanamycin at a final concentration of 50 μg / ml and 200 μm of isopropyl-β-D-thiogalactoside; thiogalactoside; isopropyl thio-β-D-galactoside; isopropyl thiogalactoside; galactose, culture overnight at 30°C and 250 rpm, precipitate with polyethylene glycol 8000 / sodium chloride, dissolve in phosphate buffer to obtain phage display library; coat β-catenin antigen at a concentration of 2 μg / ml, block with 3% bovine serum albumin, and wash with phosphate buffer; add 100 μl of phage, incubate at room temperature for 2h, wash 10 times with phosphate buffer, and the eluted phage enters the next round of screening after transfection with TG1. After 3 rounds of screening, the positive clones were verified by enzyme-linked immunosorbent assay and sequenced. Finally, two anti-β-catenin nanoantibodies were obtained, named NB1F3 and NB1G9.

[0121] Example 2 Expression and purification of β-catenin antibody

[0122] Step (1) Antibody expression: The nucleotide sequences of NB1F3 and NB1G9 were cloned into pMECS vectors with PelB signal peptide, C-terminal HA tag and His6 tag, and expressed in the periplasm of E. coli TOP10F' cells (WXR15-100S, Huayueyang Biotech, China).

[0123] Step (2): Antibody purification: Purification was performed using Ni-NTA, and the treated samples were subjected to column treatment. The specific operation was as follows: After induction, centrifugation was performed at 6000 rpm for 10 min, the bacteria were collected, 2 ml of bacterial lysis solution was added to every 100 mg of bacteria (wet weight), and the bacteria were lysed by ultrasound (300w, 5s working and 10s pause per cycle, a total of 10 min). Centrifugation was performed at 10,000 rpm and 4°C for 3 min, and the supernatant was collected. The bacterial lysate was diluted in equal times and loaded onto the column at a flow rate of 10 column volumes / hour, and the flow-through was collected. 15 column volumes of 20 mM HEPES (pH 7.4), 10 mM sodium chloride, 5% glycerol, and 30 mM imidazole were used to wash away impurities, and 5 column volumes of 20 mM HEPES (pH 7.4), 10 mM sodium chloride, 5% glycerol, and 300 mM imidazole were used for elution, and the eluate was collected.

[0124] Step (3) Antibody affinity determination: Immerse the StrepAvidin sensor in PBS buffer for 2 minutes to ensure a stable baseline signal. Load the biotinylated β-catenin onto the sensor and react at room temperature for 2 minutes until a stable binding response signal is reached. After loading is complete, rinse the sensor with cleaning 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 sequentially, and the binding response at each concentration was recorded for 1 minute, followed by dissociation. The bimolecular model (1:1) in the Octet software was used for curve fitting, and the binding and dissociation curves at each concentration were analyzed to calculate the binding rate constant, dissociation rate constant, and affinity constant. Results are shown in Figure 2 . This indicates that the above two nanoantibodies have good affinity for β-catenin protein.

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

[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] QVQLQESGGGSVQAGGSLRLSCAASGYAYCMGWFRQAPGKEREGVAAIDSDGITSYADSVKGRFTISKDNAKNTLYLQMNSLKPEDTAMYYCTADEDVCVNYGLGTGQGIHPADFGYWGQGTQVTVSS(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-NB1G9 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] CCGCCAGGCTCCAGGGAAGGAGCGCGAGGGGGTAGCAGATATTGATAGTGATGGT

[0150] AGCACAAGCTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAAAGACAAC

[0151] GCCAAGAACACTCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACTGCCA

[0152] GATACTACTGTGCGGCAGCTCTGGGGTGCGACTATGACATATGGGAGTATAACTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA(SEQ ID NO.10)

[0153] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. An anti-β-catenin nanobody, characterized in that: The heavy chain variable region of the heavy chain antibody of the Nanobody comprises a complementarity determining region, and the complementarity determining region has CDR1, CDR2 and CDR3 or has an amino acid sequence with no less than 80% sequence identity thereto; in, The CDR1 has an amino acid sequence as shown in SEQ ID NO.1 or 6; The CDR2 has an amino acid sequence as shown in SEQ ID NO. 2 or 7; The CDR3 has an amino acid sequence as shown in SEQ ID NO. 3 or 8.

2. The anti-β-catenin nanobody according to claim 1, characterized in that The heavy chain variable region of the heavy chain antibody of the Nanobody has an amino acid sequence as shown in SEQ ID NO. 4 or 9 or an amino acid sequence having a sequence identity of not less than 80% thereto. Furthermore, the anti-β-catenin nanobody is a recombinant antibody, a fully human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody or an antigen-binding fragment of these antibodies.

3. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule is capable of encoding the anti-β-catenin nanobody according to claim 1 or 2; Furthermore, the nucleic acid molecule has: a1) the nucleotide sequence shown in SEQ ID NO.5 or 10; a2) a complementary nucleotide sequence to the nucleotide sequence shown in SEQ ID NO. 5 or 10; a3) a nucleotide sequence that encodes the same protein as the nucleotide sequence of a1) or a2) but is different from the nucleotide sequence of a1) or a2) due to the degeneracy of the genetic code; a4) A nucleotide sequence obtained by replacing, deleting or adding one or two nucleotide sequences to the nucleotide sequence shown in any one of a1) to a3), and having the same or similar functions as the nucleotide sequence shown in any one of a1) to a3).

4. A recombinant expression vector, characterized in that: It comprises the nucleic acid molecule according to claim 3.

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

6. A method for preparing the anti-β-catenin nanobody according to claim 1 or 2, characterized in that: include: Cultivating the host cell according to claim 5 to obtain a culture containing the anti-β-catenin nanobody; and separating or recovering the anti-β-catenin nanobody from the culture.

7. An immunoconjugate, characterized in that: It comprises the anti-β-catenin nanobody according to claim 1 or 2, and a coupling portion, wherein the coupling portion is a detectable marker, a drug, a toxin, a cytokine, a viral coat protein or a virus-like particle.

8. A detection kit, characterized in that: It comprises the anti-β-catenin nanobody according to claim 1 or 2, and / or the immunoconjugate according to claim 7; the kit can be used to detect the presence or level of β-catenin in a sample.

9. A pharmaceutical composition, characterized in that It comprises the β-catenin according to claim 1 or 2 or the immunoconjugate according to claim 7.

10. Use of the anti-β-catenin nanobody according to claim 1 or 2, the immunoconjugate according to claim 7, or the pharmaceutical composition according to claim 9 in any one or more of the following: b1) Preparation of tumor detection, monitoring, diagnosis and / or prognosis products; b2) Preparation of products for tumor prevention and / or treatment; The tumor is a tumor with abnormally high expression of β-catenin, including breast cancer, colorectal cancer, hepatocellular carcinoma, prostate cancer, gastric cancer and lung cancer.

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