CLDN18.2-targeting nanobody, and preparation method therefor and use thereof

Abstract

Disclosed in the present invention are a CLDN18.2-targeting nanobody, and a preparation method therefor and the use thereof. Specifically disclosed are CLDN18.2-targeting nanobodies having amino acid sequences of SEQ ID NOs: 2, 4 and 20, respectively, and the uses thereof. The nanobody of the present invention has a high affinity and a good specificity, can bind to a CLDN18.2 antigen, has an anti-tumor activity, and cannot bind to other CLDN family members. The nanobody has few toxic and side effects, and thus can be prepared into a prophylactic and therapeutic drug for a Claudin 18.2 target-related disease, a diagnostic drug, a detection or in-vivo imaging product for a Claudin 18.2 protein, etc.

Description

Nanobodies targeting cldn18.2 and methods of making and using the same TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to nanobodies targeting CLDN18.2 and methods of making and using the same. BACKGROUND

[0002] Claudin (CLDN) is a tight junction molecule, an important protein in normal tissue tight junctions, with four transmembrane domains. Its function is mainly involved in the regulation of cell paracellular permeability and electrical conductivity, and the regulation of the permeability of the barrier structure. In human life activities, tight junctions play an important role. On the one hand, tight junctions can function as a "barrier" to select the size and charge of substances, thereby regulating the transport of substances through the paracellular pathway. For example, brain vascular endothelial cells can prevent blood from mixing with brain extracellular fluid through this barrier. On the other hand, tight junctions can also function as a "fence" to maintain cell polarity by regulating the free diffusion of lipids and proteins between the apical and basolateral membranes. Claudin protein is a four-transmembrane protein with a molecular weight of about 20-27 kDa, with the C and N terminals protruding into the cytoplasm. Studies have found that in epithelial or endothelial cells, Claudin proteins in tight junctions exist in the form of their subtypes, and the permeability of tight junctions is regulated by the interaction between different Claudin protein subtypes. The Claudin family contains at least 27 members, of which Claudin 18.2 (CLDN18.2) is a member of the Claudin protein family. It is a transmembrane protein with high tissue expression specificity, with very low expression levels in normal tissues, but highly selective expression in non-malignant gastric mucosa. It forms a tight junction complex with Claudin 18.2-molecules expressed on the surface of adjacent cells, forms a selectively permeable barrier, and realizes tissue-specific permeability. This tight junction protein is exposed on the surface of tumor cells during malignant transformation, making it a targetable target for cancer therapy. Existing studies have found that under normal physiological conditions, Claudin 18.2 is only expressed in differentiated epithelial cells in the gastric mucosa, but not in other healthy tissues. However, in gastrointestinal tumors such as gastric cancer, esophageal cancer, pancreatic cancer, colorectal adenocarcinoma, as well as lung cancer, liver cancer, kidney cancer, ovarian cancer, and other tumors, Claudin 18.2 is highly expressed. The disorder of cell polarity in tumor cells can lead to the exposure of Claudin 18.2 protein epitopes on the cell surface and abnormal activation. This normal tissue-limited expression and specific high expression in tumors make Claudin 18.2 an ideal target for solid tumor therapy. Currently, several CLDN18.2-targeted drugs have entered the clinical stage, and monoclonal antibodies, ADCs, bispecific antibodies, and CAR-T therapies have shown good safety and effectiveness in CLDN18.2-positive gastric and pancreatic cancer populations.Although Claudin 18.2 has potential in the development of solid tumor targeted therapy, research in this field is still in its infancy, and no drug targeting this site has been marketed in China.

[0003] The discovery and preparation of new CLDN18.2 antibodies with improved affinity and stronger anti-tumor activity can further improve efficacy, extend the benefits of CLDN18.2 targeted therapy to patients with lower target expression levels, and have wide clinical application value.

[0004] SUMMARY

[0005] One of the purposes of the present application is to provide a nanobody targeting CLDN18.2 and its use in tumor targeted therapy. The technical problems to be solved are not limited to the technical subject described, and other technical subjects not mentioned herein can be clearly understood by those skilled in the art through the following description.

[0006] To achieve the above-mentioned purposes, the present application first provides a nanobody targeting CLDN18.2, which can be any of the following:

[0007] A1) Nanobody A, comprising complementarity determining regions CDR1 of amino acid sequence 26-32 of SEQ ID NO: 2, CDR2 of amino acid sequence 50-59 of SEQ ID NO: 2, and CDR3 of amino acid sequence 98-111 of SEQ ID NO: 2;

[0008] A2) Nanobody B, comprising complementarity determining regions CDR1 of amino acid sequence 26-32 of SEQ ID NO: 4, CDR2 of amino acid sequence 50-59 of SEQ ID NO: 4, and CDR3 of amino acid sequence 98-111 of SEQ ID NO: 4;

[0009] A3) Nanobody C, comprising complementarity determining regions CDR1 of amino acid sequence 26-33 of SEQ ID NO: 20, CDR2 of amino acid sequence 51-57 of SEQ ID NO: 20, and CDR3 of amino acid sequence 95-109 of SEQ ID NO: 20.

[0010] The name of the nanobody A can be CLDN18.2-VHH-319. The name of the nanobody B can be CLDN18.2-VHH-357. The name of the nanobody C can be CLDN18.2-VHH-223.

[0011] The sequence of the complementarity determining region (CDR) is defined according to the Kabat numbering system.

[0012] The nanobody (also called single-domain antibody) only has a variable region (VHH) of a heavy chain antibody, which in turn consists of a framework region FR1, a complementarity determining region CDR1, a framework region FR2, a complementarity determining region CDR2, a framework region FR3, a complementarity determining region CDR3 and a framework region FR4.

[0013] The amino acid sequence of FR1 of the nanobody A can be as shown in SEQ ID NO: 2 at positions 1-25; the amino acid sequence of FR2 can be as shown in SEQ ID NO: 2 at positions 33-49; the amino acid sequence of FR3 can be as shown in SEQ ID NO: 2 at positions 60-97; and the amino acid sequence of FR4 can be as shown in SEQ ID NO: 2 at positions 112-122.

[0014] The amino acid sequence of FR1 of the nanobody B can be as shown in SEQ ID NO: 4 at positions 1-25; the amino acid sequence of FR2 can be as shown in SEQ ID NO: 4 at positions 33-49; the amino acid sequence of FR3 can be as shown in SEQ ID NO: 4 at positions 60-97; and the amino acid sequence of FR4 can be as shown in SEQ ID NO: 4 at positions 112-122.

[0015] The amino acid sequence of FR1 of the nanobody C can be as shown in SEQ ID NO: 20 at positions 1-25; the amino acid sequence of FR2 can be as shown in SEQ ID NO: 20 at positions 34-50; the amino acid sequence of FR3 can be as shown in SEQ ID NO: 20 at positions 58-94; and the amino acid sequence of FR4 can be as shown in SEQ ID NO: 20 at positions 110-120.

[0016] Further, the amino acid sequence of the nanobody A can be any one of the following:

[0017] B1) SEQ ID NO: 2, or an amino acid sequence having 80% or more identity compared to SEQ ID NO: 2, or an amino acid sequence having one or several amino acid substitutions, deletions or additions compared to SEQ ID NO: 2;

[0018] B2) an amino acid sequence obtained by connecting a tag or a signal peptide to the N-terminus and / or C-terminus of B1);

[0019] The amino acid sequence of the nanobody B can be any one of the following:

[0020] B3) SEQ ID NO: 4, or an amino acid sequence having 80% or more identity compared to SEQ ID NO: 4, or an amino acid sequence having one or several amino acid substitutions, deletions, or additions compared to SEQ ID NO: 4;

[0021] B4) an amino acid sequence obtained by connecting a tag or a signal peptide to the N-terminus and / or the C-terminus of B3);

[0022] The amino acid sequence of the Nanobody C can be any one of the following:

[0023] B5) SEQ ID NO: 20, or an amino acid sequence having 80% or more identity compared to SEQ ID NO: 20, or an amino acid sequence having one or several amino acid substitutions, deletions, or additions compared to SEQ ID NO: 20;

[0024] B6) an amino acid sequence obtained by connecting a tag or a signal peptide to the N-terminus and / or the C-terminus of B5).

[0025] Further, the substitution can be a conservative substitution.

[0026] Further, the discrepancy in the amino acid sequence can be in the framework region (FR), for example, having one or several amino acid substitutions, deletions, or additions in the framework region.

[0027] The framework region (FR) is known in the art, and refers to a region in the variable region other than the CDR, including FR1, FR2, FR3, and FR4.

[0028] The several can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0029] The connection in B2), B4), or B6) can be a direct connection through a peptide bond, or a connection through a linker.

[0030] The Nanobody shown in B2), B4), or B6) can be a Nanobody fused with a tag or a signal peptide and having the same function.

[0031] In order to facilitate the separation, purification, detection, and / or localization of the Nanobody shown in B1) or B3) or B5), a tag protein can be connected to the amino-terminal end or the carboxyl-terminal end of the Nanobody consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 20.

[0032] The tag includes, but is not limited to, a GST (glutathione S-transferase) tag protein, a Trx (thioredoxin) tag protein, a nitrogen utilization substrate A (NusA) tag protein, a His tag protein (His-tag), a MBP (maltose binding protein) tag protein, a Flag tag protein, a SUMO tag protein, a HA (influenza hemagglutinin) tag protein, a Myc tag protein, a LacZ tag protein, a CBD (cellulose binding domain) tag protein, a bacteriophage T7 protein kinase (T7PK) tag protein, a GFP (green fluorescent protein), a CFP (cyan fluorescent protein), a YFP (yellow-green fluorescent protein), a mCherry (monomeric red fluorescent protein), or an AviTag tag protein. Those skilled in the art know how to select a suitable tag protein according to the desired purpose. The use of the tag does not change the function of the target protein (nanobody), and the purpose is to isolate, purify, detect or track, so the tag protein suitable for the present application is not limited to a specific kind. The tag can be separated from the target protein (nanobody) by chemical cleavage or enzymatic cleavage known in the art, such as introducing a protease cleavage site to remove the tag using TEV protease.

[0033] The present application also provides a biological material, which can be any one of the following:

[0034] C1) a nucleic acid molecule encoding any one of the nanobodies described herein;

[0035] C2) an expression cassette containing the nucleic acid molecule of C1);

[0036] C3) a recombinant vector containing the nucleic acid molecule of C1);

[0037] C4) a recombinant microorganism containing the nucleic acid molecule of C1);

[0038] C5) a recombinant host cell containing the nucleic acid molecule of C1).

[0039] The biological material can express any one of the nanobodies (nanobody A, nanobody B or nanobody C) described herein.

[0040] Further, the recombinant vector can be a recombinant expression vector obtained by cloning a nucleic acid molecule encoding any one of the nanobodies described herein (such as a DNA molecule shown in SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 19) into an expression vector. Although the example provided by the present application utilizes a mammalian expression vector pCAG-Fc, the present application is not limited to this specific vector. Those skilled in the art can use other suitable vectors as long as the vector can clone or express a nucleic acid molecule encoding any one of the nanobodies described herein.

[0041] Further, the expression vector can be selected from the group consisting of prokaryotic expression vectors (including, but not limited to, E. coli (such as BL21, M15, Top10 and Origami i) expression vectors) and eukaryotic expression vectors (including, but not limited to, yeast (such as X33, GS115 and SMD1168) expression vectors, insect cell (such as Sf21, Sf-9 and Hi-5) expression vectors, and mammalian cell (such as HET293 and CHO) expression vectors).

[0042] In the above-mentioned biomaterials, the nucleic acid molecule can be any one of the following:

[0043] D1) the coding sequence or nucleotide sequence is a DNA molecule of SEQ ID NO: 1;

[0044] D2) the coding sequence or nucleotide sequence is a DNA molecule of SEQ ID NO: 3;

[0045] D3) the coding sequence or nucleotide sequence is a DNA molecule of SEQ ID NO: 19;

[0046] D4) the coding sequence or nucleotide sequence is a DNA molecule having 75% or more identity to the nucleotide sequence defined by SEQ ID NO: 1, and encoding a nanobody of the amino acid sequence of SEQ ID NO: 2;

[0047] D5) the coding sequence or nucleotide sequence is a DNA molecule having 75% or more identity to the nucleotide sequence defined by SEQ ID NO: 3, and encoding a nanobody of the amino acid sequence of SEQ ID NO: 4;

[0048] D6) the coding sequence or nucleotide sequence is a DNA molecule having 75% or more identity to the nucleotide sequence defined by SEQ ID NO: 19, and encoding a nanobody of the amino acid sequence of SEQ ID NO: 20.

[0049] The DNA molecule of SEQ ID NO: 1 encodes a nanobody CLDN18.2-VHH-319 (i.e., nanobody A) of the amino acid sequence of SEQ ID NO: 2.

[0050] The DNA molecule of SEQ ID NO: 3 encodes a nanobody CLDN18.2-VHH-357 (i.e., nanobody B) of the amino acid sequence of SEQ ID NO: 4.

[0051] The DNA molecule of SEQ ID NO: 19 encodes a nanobody CLDN18.2-VHH-223 (i.e., nanobody C) of the amino acid sequence of SEQ ID NO: 20.

[0052] The nucleotide sequences encoding any of the Nanobodies described herein can be readily subjected to mutagenesis by methods known to those of ordinary skill in the art, such as site-directed mutagenesis (including oligonucleotide primer-directed site- directed mutagenesis, PCR-mediated site-directed mutagenesis, and cassette mutagenesis), or directed evolution (including error-prone PCR, DNA shuffling, and in vitro random priming recombination). Those nucleotide sequences that are artificially constructed, which are more than 75% identical to the nucleotide sequences encoding any of the Nanobodies described herein (e.g., SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 19), and which encode any of the Nanobodies described herein and have the same function as any of the Nanobodies described herein, are derived from the nucleotide sequences of the present application and are equivalent to the nucleotide sequences of the present application.

[0053] The nucleic acid molecules described herein can also include nucleic acid molecules that are codon-preferred versions of the nucleotide sequences set forth in SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 19. Codons can be chosen to optimize expression in a particular species, taking into account the degeneracy of the genetic code and the bias in the usage of certain codons in a particular species.

[0054] Variants having improved affinity and / or valency to the sequences of any of the Nanobodies described herein can be obtained by using methods known in the art and are included within the scope of the present application. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of the nucleotide sequence can also be used to improve the translation efficiency in the expression system used to produce the antibodies.

[0055] In certain embodiments, substitutions, insertions, or deletions can occur within one or more complementarity determining regions or framework regions of any of the Nanobodies of the present application, so long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions, well known to those skilled in the art, which do not change the properties and functions of the protein) can be made to the complementarity determining regions and / or framework regions that do not substantially reduce binding affinity. Such changes can be outside of antigen contacting residues in the complementarity determining regions, for example.

[0056] The present application also provides the use of any of the Nanobodies described herein or the biological material in any of the following:

[0057] E1) use in the manufacture of a product for the prevention or treatment of a tumor;

[0058] E2) use in the manufacture of a product for the prevention or treatment of a Claudin 18.2 target-related disease;

[0059] E3) use in the manufacture of a product for screening, diagnosis or aiding diagnosis of a Claudin 18.2 target-related disease;

[0060] E4) use in the manufacture of a product for detecting Claudin 18.2 protein or Claudin 18.2-expressing cells;

[0061] E5) use in the manufacture of a product for binding to Claudin 18.2 protein;

[0062] E6) use in the manufacture of a product for mediating specific recognition of tumors expressing Claudin 18.2 antigen by a drug;

[0063] E7) use in the manufacture of a product for in vivo imaging of Claudin 18.2 protein;

[0064] E8) use in the manufacture of a bispecific antibody, multispecific antibody, antibody drug conjugate or CAR cell targeting Claudin 18.2.

[0065] In the above uses, the tumor or Claudin 18.2 target-related disease can be a Claudin 18.2-positive tumor.

[0066] Further, the Claudin 18.2-positive tumor can be a Claudin 18.2-positive solid tumor. Further, the Claudin 18.2-positive solid tumor can be a Claudin 18.2-positive digestive system tumor.

[0067] Further, the Claudin 18.2-positive tumor can be a Claudin 18.2-positive cancer, or a Claudin 18.2-positive mesothelioma.

[0068] Further, the cancer can be selected from gastric cancer, esophageal cancer, pancreatic cancer, colorectal adenocarcinoma, lung cancer, liver cancer, kidney cancer, ovarian cancer, bronchial cancer, breast cancer, bladder cancer, head and neck cancer, gallbladder cancer, cholangiocarcinoma, and gastroesophageal junction (GEJ) adenocarcinoma.

[0069] Further, the cancer includes early-stage cancer, mid-stage cancer, mid-advanced stage cancer, advanced stage cancer, recurrent cancer, metastatic cancer, etc.

[0070] Claudin 18.2 is differentially expressed in normal and tumor tissues, involved in the occurrence and development of tumors, and located in the extracellular membrane, making it a cell lineage marker with high selectivity. E3) The use can include detecting Claudin 18.2 protein expression using the nanobodies of the application based on the principle of antigen-antibody specific reaction, and then used for screening and diagnosis of Claudin 18.2 target related diseases (including prognostic diagnosis, such as predicting the probability of patient death, evaluating disease risk stratification, etc.). For example, any of the nanobodies described herein can be prepared into an immunohistochemical kit for screening or diagnosing Claudin-18.2 positive tumor patients, and further guiding drug use and treatment.

[0071] E4) The detection of Claudin 18.2 protein or Claudin 18.2 expressing cells mentioned in the middle includes any in vivo or in vitro detection of Claudin 18.2 protein based on the principle of antigen-antibody specific reaction. The detection of Claudin 18.2 protein can be to detect whether the sample to be tested contains Claudin 18.2 protein and / or to detect the content of Claudin 18.2 protein in the sample to be tested. The Claudin 18.2 expressing cells can further be Claudin 18.2 expressing tumor cells.

[0072] E5) The product that binds to Claudin 18.2 protein mentioned in the middle can be a Claudin 18.2 protein inhibitor, or a product for isolating Claudin 18.2 protein, but is not limited to this. For example, any of the nanobodies of the application can be prepared into an immunoaffinity chromatography column, based on the principle that antigens can be captured by antibodies when passing through the chromatography column, and the antigens are dissociated from the column under environmental conditions such as changing pH value.

[0073] E6) The product that mediates the specific recognition of tumors expressing Claudin 18.2 antigen by drugs mentioned in the middle can be a drug-loaded system that specifically targets Claudin 18.2 protein, which can contain any of the nanobodies described herein or a combination of nanobody A and nanobody B. The drug-loaded system can be a liposome drug delivery system, a polymer micelle drug delivery system, a polymer disc drug delivery system, a nanoparticle drug delivery system, an emulsion drug delivery system, or a cell membrane nanovesicle drug delivery system. The drug-loaded system can be used for targeted transport and / or site-specific release of drugs.

[0074] E7) The product for in vivo imaging of Claudin 18.2 protein described in E7) can include imaging agents, contrast agents, tracers, etc., which can be used in immunological imaging technology. For example, the nanobody of any of the present application is coupled with a molecule having imaging function (including but not limited to radionuclide, near-infrared dye, luciferase, magnetic resonance imaging nanoparticle, magnetic resonance imaging quantum dot), after being injected into the patient, based on the targeting function of the nanobody, it can reach the corresponding tissue site by itself, and immunological imaging is carried out, that is, in vivo imaging of Claudin 18.2 protein is realized.

[0075] E8) The bispecific antibody, multispecific antibody, antibody drug conjugate or CAR cell targeting Claudin 18.2 described in E8) contains any of the nanobodies described herein.

[0076] A variety of different bispecific antibodies or multispecific antibodies structures are known in the art, and methods for constructing bispecific antibodies, multispecific antibodies based on nanobodies are known to those skilled in the art. For example, the nanobody of any of the present application (as a first antibody) is linked to one or more other antibodies (which can be other antibodies against different targets, or other antibodies against different epitopes of Claudin 18.2 target) using a hinge region or linker to construct a bispecific antibody or multispecific antibody. The other antibodies can be monoclonal antibodies, bispecific antibodies, multispecific antibodies, humanized antibodies (including chimeric antibodies, CDR grafting antibodies and SDR grafting antibodies), and fully human antibodies. The other antibodies not only include intact antibodies, but also antigen-binding fragments (such as Fab, Fab', F(ab')2, Fv, single-chain antibodies (ScFv), single-domain antibodies (sdAb, i.e. nanobodies) and minimal recognition units (MRU)) capable of specifically binding to target antigens.

[0077] The smallest bispecific antibody can be constructed by connecting two nanobody molecules head to tail using a linker (linker peptide), for example, using the nanobodies CLDN18.2-VHH-319 and CLDN18.2-VHH-357 of the present application to construct a bispecific nanobody targeting different epitopes of Claudin 18.2. Any of the nanobodies of the present application and a second nanobody can also be used to construct a bispecific nanobody targeting different targets.

[0078] The CAR cell described in E8) contains and / or expresses a chimeric antigen receptor (CAR) containing any of the nanobodies described herein. The CAR cell can include CAR-T cells, CAR-NK cells, CAR-NKT cells, CAR-γδT cells, CAR-macrophage (CAR-M cells), CAR-iPSC cells and CAR-PSC cells, etc. but is not limited thereto.

[0079] The present application also provides a bispecific antibody or a multispecific antibody comprising the Nanobody A or Nanobody B described herein.

[0080] Further, the bispecific antibody further contains a second antibody. The multispecific antibody further contains more than two other antibodies. The second antibody or other antibody can be an antibody against a different target (other targets different from the Claudin 18.2 target, such as CD3, 4-1BB, PD-L1, CD47, HER2, MUC1, CD16 and B7H3, etc.), or an antibody against a different epitope of the Claudin 18.2 target.

[0081] The second antibody or other antibody can be a monoclonal antibody, a bispecific antibody, a multispecific antibody, a humanized antibody (including chimeric antibodies, CDR-grafted antibodies and SDR-grafted antibodies), and a fully human antibody. The second antibody or other antibody not only includes intact antibodies, but also antigen-binding fragments (such as Fab, Fab', F(ab')2, Fv, single-chain antibodies (ScFv), single-domain antibodies (sdAb, i.e. nanobodies) and minimal recognition units (MRU) etc.) capable of specifically binding to target antigens. The second antibody or other antibody can be directly linked to the first antibody or linked through a linker.

[0082] Further, the linker can be a flexible peptide linker, such as a peptide linker comprising glycine, serine, proline and / or lysine residues. The peptide linker can consist of 1-40 amino acids. Further, the linker includes but is not limited to: (G)n, (S)n, (GxS)n, (SxG)n, (GSSGG)n(SEQ ID NO: 12), (GGSGG)n(SEQ ID NO: 13), (GSGGSG)n(SEQ ID NO: 14), (GSGSGS)n(SEQ ID NO: 15), (GGQGG)n(SEQ ID NO: 16), (EAAAK)n(SEQ ID NO: 17) and IEGRMD(SEQ ID NO: 18), and various combinations thereof. Wherein: n can be any integer between 1-10; x can be any integer between 1-6.

[0083] Further, the bispecific antibody can be a bispecific nanobody containing Nanobody CLDN18.2-VHH-319 and CLDN18.2-VHH-357.

[0084] Further, the present application also provides a preparation method of the bispecific antibody or the multispecific antibody, which comprises constructing a recombinant expression vector containing the gene of the bispecific antibody or the multispecific antibody; introducing the recombinant expression vector into a host cell to obtain a recombinant host cell; culturing the recombinant host cell, and obtaining the bispecific antibody or the multispecific antibody through separation and purification.

[0085] The present application also provides an antibody conjugate comprising an antibody moiety and a conjugate moiety, wherein the antibody moiety comprises the Nanobody A or the Nanobody B described herein.

[0086] Further, the antibody moiety and the conjugate moiety can be directly connected or covalently connected through a linker (such as a hydrazone bond, a disulfide bond, a thioether bond, a peptide bond).

[0087] Further, the conjugate moiety is selected from a chemical drug, a cytotoxin and a detectable label.

[0088] Further, the chemical drug can be a chemical drug for preventing or treating diseases, including a chemotherapeutic drug, an antitumor drug, an anti-inflammatory drug, etc., such as a cytokine, an apoptosis inducer (Bcl-xL inhibitor), a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, an antitumor antibiotic, an immunomodulator, an anti-angiogenic agent, an antimetabolite, a boron drug, an alkylating agent, a hormone and an anti-hormone drug, a corticosteroid, a photodynamic therapy drug, etc.

[0089] Further, the cytotoxin can be a substance that inhibits cell proliferation or induces cell apoptosis, including a microtubulin inhibitor (such as maytansine, auristatin (MMAE and MMAF)), a DNA damaging agent (such as a calicheamicin, a duocarmycin, a camptothecin, a duocarmycin), an RNA synthesis inhibitor (such as an amanitin, a tirapazamine and analogs thereof) or a protein synthesis inhibitor (such as a ricin).

[0090] The detectable label includes an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridinium ester compounds, acridinium sulfonamide compounds, luminol and its derivatives, ruthenium derivatives), a fluorescent dye (such as AMCA, FITC, PE, PI, PerCP-Cy5.5, PE-Cy7, APC, APC-H7, BV421, V500, Alexa 700, BV605), a near-infrared dye (such as cyanine dyes, BODIPY, rhodamine, squarine, porphyrin dyes), a radionuclide (such as124I,18F,11C,99mTc,123I), biotin, a magnetic resonance imaging nanoparticle, a magnetic resonance imaging quantum dot, a magnetic substance (such as magnetic beads, gadolinium-containing complex nanoparticles, superparamagnetic iron oxide nanoparticles) and colloidal gold, etc., but not limited thereto.

[0091] The present application also provides a pharmaceutical composition for preventing or treating a Claudin 18.2 target-related disease, the pharmaceutical composition comprising the nanobody, the biomaterial, the bispecific antibody or multispecific antibody, or the antibody conjugate according to any one of the present application, and one or more pharmaceutically acceptable carriers.

[0092] The pharmaceutically acceptable carrier is selected from the group consisting of diluents, excipients, fillers, binders, humectants, disintegrants, preservatives, stabilizers, absorption promoters, adsorption carriers, surfactants, lubricants, atomizing agents, suspending agents, plasticizers, and dispersants.

[0093] The dosage form of the pharmaceutical composition can include, but is not limited to, inhalation preparations, oral preparations, and injection preparations. The inhalation preparations can be inhalation aerosols, inhalation powder sprays, inhalation spray, inhalation liquid preparations, inhalation atomization preparations, inhalation lyophilized powder injections, or nasal sprays. The oral preparations can be tablets, powders, capsules, granules, pills, powders, pastes, solid beverages, or oral liquids. The injection preparations can be injection liquids or injection powders.

[0094] To prepare the pharmaceutical composition into injection preparations such as solutions, emulsions, lyophilized powder injections, and suspensions, all diluents (carriers) commonly used in the art can be used, for example, water, saline, phosphate buffered saline, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid ester, and the like. In addition, to prepare isotonic injection liquids, an appropriate amount of sodium chloride, glucose, glycerol, or the like can be added to the injection preparation, and in addition, conventional cosolvents, buffers, pH adjustors, and the like can be added.

[0095] Further, the pharmaceutical composition can further include a drug delivery system. The drug delivery system can be used for targeted transport and / or site-specific release of drugs.

[0096] Further, the drug delivery system can be a liposome drug delivery system, a polymeric micelle drug delivery system, a polymeric disc drug delivery system, a nanoparticle drug delivery system, an emulsion drug delivery system, or a cell membrane nanovesicle drug delivery system.

[0097] Further, the drug delivery system includes liposomes, lipid nanoparticles (LNP), cationic lipids, ionizable cationic lipids, pegylated lipids, micellar nanoparticles, polymeric micelles, polymeric microspheres (also referred to as drug-loaded microspheres), nanoparticles (such as polylactic acid, chitosan, dendrimers, metal, carbon, mesoporous silica nanoparticles, etc.), emulsions, and cell membrane nanovesicles, but is not limited thereto.

[0098] The present application also provides a kit comprising the nanobody or the bispecific antibody or the multispecific antibody of any one of the present application.

[0099] The kit can have any one of the following uses:

[0100] (1) screening, diagnosing or aiding diagnosis of Claudin 18.2 target related diseases;

[0101] (2) detecting Claudin 18.2 protein or cells expressing Claudin 18.2;

[0102] (3) binding to Claudin 18.2 protein (such as isolation or purification);

[0103] (4) in vivo imaging of Claudin 18.2 protein.

[0104] Further, the kit can be used for detecting tumor cells expressing Claudin 18.2, or for detecting whether Claudin 18.2 positive cancer exists in a subject.

[0105] The sample to be detected by the kit can be a blood sample (such as whole blood, plasma, serum), a tissue sample, a cell sample, etc. but is not limited thereto.

[0106] The kit can be a chemiluminescence immunoassay kit, an enzyme-linked immunoassay kit, an immunoprecipitation assay kit, an immunoblotting assay kit, an immunochromatography assay kit, a flow cytometry assay kit, an immunohistochemistry assay kit, a colloidal gold immunoassay kit or a fluorescent immunoassay kit but is not limited thereto.

[0107] Further, the kit can further comprise reagents required for immunodetection, such as labeled antibodies or antigens, magnetic particles, blocking solution, dilution solution, washing solution, color developing solution, termination solution, etc. but is not limited thereto.

[0108] The various reagent components of the kit can be present in separate containers, or can be pre-combined into a reagent mixture, in whole or in part. The components of the kit can be provided in solution form, for example, in the form of an aqueous solution. In the case of being present in the form of an aqueous solution, the concentration or content of these components can be readily determined by those skilled in the art according to different needs. For example, for storage purposes, the components can be present in a higher concentration, and when in working condition or for use, the concentration can be reduced to the working concentration by diluting the above-mentioned solution of higher concentration.

[0109] The kit can also include, for example, a buffer, a preservative, or a protein stabilizer. The kit can also include components necessary for detecting a detectable label, such as an enzyme or a substrate. The kit can also contain a control sample or a series of control samples, which can be measured and compared with the test sample. The kit can include written material on or in the kit container. The written material describes how to use the reagents contained in the kit.

[0110] The present application also provides a method for detecting Claudin 18.2 protein, which comprises using any of the nanobodies, the bispecific antibodies or multispecific antibodies, or the kits described herein to detect Claudin 18.2 protein.

[0111] The detection can comprise detecting whether Claudin 18.2 protein exists in a sample, or the expression level or content of Claudin 18.2 protein in a sample. The sample can be a cell sample, a tissue sample, or a blood sample from a subject, etc., or can be a subject. The detection can comprise not only in vitro detection, but also in vivo detection.

[0112] Further, the method for detecting Claudin 18.2 protein comprises contacting any of the nanobodies, the bispecific antibodies or multispecific antibodies, or the kits described herein with a sample to be detected, and detecting whether Claudin 18.2 protein exists in the sample to be detected by antigen-antibody reaction.

[0113] Further, the method for detecting Claudin 18.2 protein comprises first labeling any of the nanobodies, or the bispecific antibodies or multispecific antibodies described herein with a detectable substance; and then contacting the labeled nanobodies with a sample to be detected, and detecting whether Claudin 18.2 protein exists in the sample to be detected, or the content of Claudin 18.2 protein in the sample to be detected, by detecting the detectable substance.

[0114] Further, the detectable substance can be selected from, but not limited to, an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridinium ester compounds, acridinium sulfonamide compounds, luminol and its derivatives, ruthenium derivatives), a fluorescent dye (such as AMCA, FITC, PE, PI, PerCP-Cy5.5, PE-Cy7, APC, APC-H7, BV421, V500, Alexa 700, BV605), a near-infrared dye (such as cyanine dyes, BODIPY, rhodamine, squarine, porphyrin dyes), a radionuclide (such as 124I, 18F, 11C, 99mTc, 123I), biotin, a magnetic resonance imaging nanoparticle, a magnetic resonance imaging quantum dot, a magnetic substance (such as magnetic beads, gadolinium-containing complex nanoparticles, superparamagnetic iron oxide nanoparticles), and colloidal gold, etc.

[0115] The method for detecting the Claudin 18.2 protein can be immunological detection, such as precipitation reaction, agglutination test, immunoblotting, enzyme immunoassay (such as ELISA), chemiluminescence immunoassay, fluorescence immunoassay, radioimmunoassay (RIA), colloidal gold immunological technology (GIC), colloidal gold immunochromatographic technology (GICA), immunohistochemistry, complement fixation reaction, multiplex immunoassay, and fluorescence immunoassay.

[0116] The method for detecting the Claudin 18.2 protein can be for disease diagnosis, disease prognosis, and / or disease treatment purposes, or for non-disease diagnosis, non-disease prognosis, and non-disease treatment purposes.

[0117] The present application also provides a method for preparing the nanobody as described herein, comprising expressing the nanobody as described herein in a host cell, and recovering or isolating the nanobody.

[0118] Further, the method for preparing the nanobody can comprise the following steps: constructing a recombinant expression vector containing a nucleic acid molecule encoding the nanobody as described herein; introducing the recombinant expression vector into a host cell to obtain a recombinant host cell expressing the nanobody; culturing the recombinant cell, and recovering or isolating the nanobody from the culture of the cultured recombinant host cell.

[0119] Further, the nucleic acid molecule encoding the nanobody as described herein can be any one of the following:

[0120] (1) the coding sequence or nucleotide sequence is a DNA molecule of SEQ ID NO: 1;

[0121] (2) the coding sequence or nucleotide sequence is a DNA molecule of SEQ ID NO: 3;

[0122] (3) the coding sequence or nucleotide sequence is a DNA molecule of SEQ ID NO: 19;

[0123] (4) the nucleotide sequence has more than 75% identity to the nucleotide sequence defined by SEQ ID NO: 1, and the amino acid sequence encoded is a Nanobody of SEQ ID NO: 2;

[0124] (5) the nucleotide sequence has more than 75% identity to the nucleotide sequence defined by SEQ ID NO: 3, and the amino acid sequence encoded is a Nanobody of SEQ ID NO: 4;

[0125] (6) the nucleotide sequence has more than 75% identity to the nucleotide sequence defined by SEQ ID NO: 19, and the amino acid sequence encoded is a Nanobody of SEQ ID NO: 20.

[0126] Further, the recovering or isolating can be by precipitation (e.g. salting-out, organic solvent precipitation, caprylic acid-saturated ammonium sulfate precipitation, isoelectric precipitation) or chromatography (e.g. ion exchange chromatography, gel filtration chromatography, affinity chromatography) from the culture (including all materials within the culture vessel).

[0127] In one embodiment of the present application, the host cell can be a HEK293 cell.

[0128] In one embodiment of the present application, the introducing can be cationic polymer method.

[0129] The present application also provides a method of preventing or treating a Claudin 18.2 target-related disease, comprising administering to a subject having a Claudin 18.2 target-related disease any of the Nanobodies, the biomaterials, the bispecific or multispecific antibodies, the antibody conjugates, or the pharmaceutical compositions described herein.

[0130] Further, the amount of administration can be a therapeutically effective amount. The therapeutically effective amount can refer to the amount of the drug that (i) treats or prevents a particular disease, condition, or disorder; (ii) attenuates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder; or (iii) prevents or delays onset of one or more symptoms of a particular disease, condition, or disorder described herein. A therapeutically effective amount can be determined by testing in known in vitro or in vivo (e.g. animal models) systems.

[0131] Further, the administration includes, but is not limited to, intramuscular injection, subcutaneous injection, intradermal injection, transdermal injection, intravenous injection, arterial injection, intraperitoneal injection, intraperitoneal injection, intrathecal injection, microneedle injection, intratumoral injection, mucosal administration, oral administration, oral and nasal cavity spray, aerosol inhalation, in vivo implantation and in vitro carrying device administration.

[0132] The preferred mode of administration (route of administration) is parenteral (e.g., intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intra-articular). The skilled person will understand that the route of administration will vary depending on the intended purpose. In certain embodiments, any of the nanobodies, bispecific antibodies or multispecific antibodies, isolated nucleic acid molecules, recombinant vectors, recombinant host cells, antibody conjugates or pharmaceutical compositions of the application can be administered by injection or continuous infusion (including, but not limited to, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intraocular and intraportal).

[0133] In some embodiments, the prophylactic or therapeutic methods provided by the present application can also be used in combination with other therapies, for example, treatment with a second therapeutic agent, which can be an antitumor drug, an anti-inflammatory drug, an immunosuppressant or other therapeutically active drug, including but not limited to gefitinib, erlotinib, icotinib hydrochloride, osimertinib, afatinib, dacomitinib, anlotinib hydrochloride, bevacizumab, imatinib, erlotinib, sorafenib, infliximab, adalimumab, rituximab, etanercept, filgrastim, pegfilgrastim, epirubicin, capecitabine, cisplatin, carboplatin, gemcitabine, pemetrexed, paclitaxel, oxaliplatin and fluorouracil.

[0134] The nanobodies, biomaterials, bispecific antibodies, multispecific antibodies, antibody conjugates or pharmaceutical compositions of the present application can be administered to the subject separately (by different routes at the same time or substantially at the same time), sequentially (at different times, the administration routes can be the same or different) or simultaneously (by the same route at the same time or substantially at the same time) with the second therapeutic agent.

[0135] The present application also provides a method for diagnosing or assisting in the diagnosis of a Claudin 18.2 target-related disease, which comprises: obtaining a sample from a subject, and then detecting the presence or absence of Claudin 18.2 protein or the content of Claudin 18.2 protein in the sample using any of the nanobodies, bispecific antibodies or multispecific antibodies, antibody conjugates or kits described herein, and diagnosing or assisting in the diagnosis of a Claudin 18.2 target-related disease according to the detection results.

[0136] Further, the method can further comprise a step of comparing the content of Claudin 18.2 protein in the detection result with a reference value (content of Claudin 18.2 protein in disease diagnosis standard). The reference value can be the level of Claudin 18.2 protein in a sample from a subject known not to have a disease related to Claudin 18.2 target (e.g. a healthy control) (also referred to as "negative reference value"). The result of comparison can indicate whether the subject has a disease related to Claudin 18.2 target or has a risk of having a disease related to Claudin 18.2 target, or indicate the progress of the disease and / or the recovery after treatment of the patient.

[0137] The present application also provides a method for screening a disease related to Claudin 18.2 target, which comprises: obtaining a sample from a subject, and then detecting whether Claudin 18.2 protein or the content of Claudin 18.2 protein in the sample using any of the nanobodies, the bispecific antibodies or multispecific antibodies, the antibody conjugates or the kits described herein, and screening a disease related to Claudin 18.2 target according to the detection result.

[0138] Further, the method can further comprise a step of comparing the content of Claudin 18.2 protein in the detection result with a reference value (content of Claudin 18.2 protein in disease screening standard). The reference value can be the level of Claudin 18.2 protein in a sample from a subject known not to have a disease related to Claudin 18.2 target (e.g. a healthy control) (also referred to as "negative reference value"). The result of comparison can indicate a wide range of population suitable for the therapeutic product of the present application.

[0139] Any of the nanobodies described herein for use as a medicament.

[0140] Any of the nanobodies described herein for use in preventing or treating a disease related to Claudin 18.2 target.

[0141] The disease related to Claudin 18.2 target described herein can be a Claudin 18.2 positive tumor.

[0142] The Claudin 18.2 positive tumor described herein can be a Claudin 18.2 positive solid tumor.

[0143] Further, the solid tumor can be a digestive system tumor (e.g. gastric cancer, esophageal cancer, pancreatic cancer, colorectal adenocarcinoma).

[0144] The Claudin 18.2-positive tumor described herein can be a Claudin 18.2-positive cancer or a Claudin 18.2-positive mesothelioma.

[0145] The cancer described herein can be selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, colorectal adenocarcinoma, lung cancer, liver cancer, kidney cancer, ovarian cancer, bronchial cancer, breast cancer, bladder cancer, head and neck cancer, gallbladder cancer, cholangiocarcinoma, and gastroesophageal junction adenocarcinoma.

[0146] The cancer described herein can include early-stage cancer, mid-stage cancer, mid-advanced stage cancer, advanced stage cancer, recurrent cancer, and metastatic cancer.

[0147] The nanobody described in any of the present application can be prepared by various methods known in the art, for example, by obtaining a gene encoding the nanobody described in any of the present application through genetic engineering recombination technology. For example, the gene encoding the nanobody described in any of the present application is obtained by chemical synthesis or PCR amplification, inserted into an expression vector, then transfected into host cells, the transfected host cells are cultured under specific conditions, and the nanobody is expressed. The host cells, expression vectors, methods for introducing expression vectors into host cells, and methods for isolating and purifying antibodies can be selected as needed by those skilled in the art according to conventional methods.

[0148] The purposes of the uses (applications) and methods described in the present application can be disease diagnosis purposes, disease prognosis purposes, and / or disease treatment purposes, and their purposes can also be non-disease diagnosis purposes, non-disease prognosis purposes, and non-disease treatment purposes; their direct purposes can be to obtain information of intermediate results of disease diagnosis results, disease prognosis results, and / or disease treatment results, and their direct purposes can be non-disease diagnosis purposes, non-disease prognosis purposes, and / or non-disease treatment purposes.

[0149] The products described herein can include reagents, kits (such as therapeutic kits or detection kits), chips, test papers, detection cards, immunosensors, preparations, drugs, and pharmaceutical compositions, etc.

[0150] The samples described herein can be derived from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue associated cells (i.e., free cells), tissues (such as surgically resected tumor tissues, biopsy tissues, or fine needle aspiration tissues), etc.

[0151] The subject described herein can be a human or a non-human animal. In certain embodiments, the subject has a Claudin 18.2 target-related disease.

[0152] The non-human animal described herein can be a non-human mammal.

[0153] The non-human mammal described herein can be any one of, but not limited to, a mouse, a rat, a guinea pig, a hamster, a pig, a dog, a sheep, a monkey, a rabbit, a cat, a cow, a horse.

[0154] The nanobody targeting CLDN18.2 herein can also be referred to as an anti-CLDN18.2 nanobody.

[0155] The present application utilizes cell antigens to immunize two alpacas to construct a nanobody phage library, and obtains a high-affinity and high-specificity anti-Claudin18.2 nanobody from a nanobody expression library through phage display technology and affinity screening. After sequencing the antibody gene sequence, the nanobody (CLDN18.2-VHH-319, CLDN18.2-VHH-357 and CLDN18.2-VHH-223) is prepared by using a genetic engineering method. Further, the obtained nanobody is subjected to functional experiments such as affinity and specificity. The results show that the nanobody (CLDN18.2-VHH-319, CLDN18.2-VHH-357 and CLDN18.2-VHH-223) of the present application has high affinity and specificity. It can specifically bind to CLDN18.2 positive cells, but not to CLDN18.2 negative cells, especially not to human CLDN18.1 antigen which is extremely similar in structure (up to 92% homology), which shows that it has high specificity for the spatial structure of CLDN18.2 membrane protein. As a nanobody for treating tumors, it can not only specifically bind to the tumor surface antigen Claudin 18.2, trigger stronger complement-dependent cytotoxicity (CDC), and better induce tumor cell apoptosis and inhibit tumor cell proliferation, but also has stronger anti-tumor activity, can efficiently target and kill tumor cells expressing CLDN18.2, and can avoid the toxicity and side effects caused by non-specific binding of therapeutic antibodies to human CLDN18.1 and other proteins during targeted diagnosis and treatment. In addition, the nanobody of the present application can penetrate more easily into the core of the tumor than the complete traditional antibody molecule, more comprehensively detect the distribution of the target, and improve the detection rate of the tumor.

[0156] The nanobodies (CLDN18.2-VHH-319, CLDN18.2-VHH-357 and CLDN18.2-VHH-223) of the present application can be expressed and produced in prokaryotic cells, eukaryotic cells and any recombinant system, and can be made into prophylactic and therapeutic drugs for Claudin 18.2 target related diseases, diagnostic drugs, detection products of Claudin 18.2 protein or in vivo imaging products of Claudin 18.2 protein, etc. As small molecular weight nanobodies, they have low immunogenicity, small molecular weight, strong tissue penetration, stable structure, short half-life, can be quickly cleared in vivo, reduce side effects, and are easy to recombinantly express, low in production cost, can be used alone or as a drug carrying system to carry related drugs, and have very broad prospects and important significance in the fields of drug application and clinical diagnosis, etc.

[0157] Definitions of terms

[0158] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person skilled in the art. Meanwhile, in order to better understand the present application, the definitions and explanations of related terms are provided as follows.

[0159] The term "expression cassette" generally refers to a nucleic acid construct comprising nucleic acid elements sufficient to express a gene of interest. A typical expression cassette comprises a promoter, a MCS (multiple cloning site) and / or a terminator. The expression cassette can also include a gene of interest, a marker gene (such as a TK gene, a DHFR gene, a CAT gene and a NEO gene), a ribosome recognition and binding site (SD), a transcription factor binding site (TFBS), an enhancer, a silencer, a repressor, an intron, a poly(A) addition signal sequence and / or an mRNA splicing signal sequence, etc. The elements in the expression cassette can be directly connected or indirectly connected through a linker.

[0160] The term "vector" generally refers to a vehicle that is capable of carrying foreign DNA or a gene of interest into a host cell for amplification and / or expression. The vector can be a cloning vector or an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by the vector are amplified and / or expressed in the host cell. A person skilled in the art can select a suitable vector according to the purpose of genetic engineering and the nature of the recipient cell. The vector includes but is not limited to: plasmid, phage (such as lambda phage or M13 phage), cosmid (i.e. cos plasmid), phagemid, shuttle vector (such as yeast expression vector), Ti plasmid, artificial chromosome (such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), P1 artificial chromosome (PAC) or Ti plasmid artificial chromosome (TAC)), viral vector (such as baculovirus vector, retrovirus (including lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, papovavirus (such as SV40), herpesvirus (such as herpes simplex virus)). A vector can contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can also contain a replication initiation site.

[0161] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, spirochetes, algae, and the like. For example, the bacteria can be from Escherichia sp. (e.g., E. coli), Erwinia sp., Agrobacterium sp. (e.g., A. tumefaciens), Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., and Bacillus sp. (e.g., B. subtilis), and the like. The viruses can include rotavirus, baculovirus, retrovirus (e.g., lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, influenza virus, papovavirus (e.g., SV40), and herpesvirus (e.g., herpes simplex virus), and the like. The fungi can be from Saccharomyces sp. (e.g., S. cerevisiae, S. pombe, S. pichia), Fusarium sp., Rhizoctonia sp., Verticillium sp., Penicillium sp., Aspergillus sp., and Cephalosporium sp., and the like. The actinomycetes can be from Streptomyces sp. (e.g., S. coelicolor). The algae can be from Cyanophyta (e.g., cyanobacteria), Fucus sp., Achnanthes sp., Amphiprora sp., Amphora sp., Ankistrodesmus sp., Asteromonas sp., and Boekelovia sp., and the like.

[0162] The term "host cell" is also referred to as recipient cell, and generally refers to any type of cell that can be used for introducing a vector, such as plant cells and animal cells. The host cell can be understood to refer not only to the particular recipient cell, but also to the progeny of such a cell, which progeny will in most instances, but not all, contain the same genetic makeup as the parent cell, and which may, but not always, be capable of reproducing. Suitable host cells are known in the art, wherein: the plant cell can be, but is not limited to, Arabidopsis thaliana, Nicotiana tabacum, Zea mays, Oryza sativa, Triticum aestivum, etc.; the animal cell can be, but is not limited to, mammalian cells (e.g. Chinese hamster ovary cells (CHO cells), Chinese hamster ovary subline (CHO-K1 cells), African green monkey kidney cells (Vero cells), SV40-transformed African green monkey kidney cells (COS cells), baby hamster kidney cells (BHK cells), mouse mammary tumor cells (C127 cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells or NK cells, etc.), avian cells (e.g. chicken or duck cells), amphibian cells (e.g. Xenopus laevis cells or Andrias davidianus cells), fish cells (e.g. grass carp, common carp, rainbow trout or catfish cells), insect cells (e.g. Sf21 cells, Sf-9 cells or Hi-5), etc.

[0163] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed in vitro by ligating an exogenous gene of interest with a vector, which can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous gene of interest into a recipient cell, and provide the exogenous gene of interest with the ability of replication, integration, amplification and / or expression in the recipient cell.

[0164] The term "recombinant microorganism" generally refers to a recombinant microorganism obtained by manipulating and modifying the genes of a microorganism of interest, so that the function of the recombinant microorganism is changed. For example, an exogenous gene of interest or a recombinant vector is introduced into the microorganism of interest, or the endogenous genes of the microorganism of interest are directly genetically edited.

[0165] The term "recombinant host cell" generally refers to a recombinant host cell obtained by manipulating and modifying the genes of a host cell, so that the function of the recombinant host cell is changed. For example, an exogenous gene of interest or a recombinant vector is introduced into the host cell, or the endogenous genes of the host cell are directly genetically edited.

[0166] The term "linkage" generally refers to the association of two or more molecules. The linkage can be covalent or non-covalent. The linkage described herein can be directly linked by a peptide bond, or linked by a linker.

[0167] The term "linker" can also be referred to as a linking peptide, a peptide linker, or a linker, which is used to fuse, couple, link, or join two proteins or polypeptides, which can prevent steric hindrance. The linker is an amino acid chain that links two fusion proteins, which has a certain flexibility to allow the proteins on both sides to complete their respective independent functions. It should be understood that the presence of the linker is optional, and the length of the flexible linker can also be adjusted to allow the correct folding of the fusion protein or to achieve optimal biological activity. The characteristics of the linker and its suitability for a particular purpose are known in the art, and a person skilled in the art can independently select and optimize each peptide linker.

[0168] The term "signal peptide" generally refers to a short peptide chain (generally 5-30 amino acids in length) that directs the newly synthesized protein to the secretory pathway. The nanobodies described in the present application can further comprise a signal peptide, thereby facilitating their secretory expression in host cells.

[0169] The term "identity" generally refers to the extent to which two (nucleotide or amino acid) sequences have the same residues at a given position in an alignment, and is typically expressed as a percentage. Identity as described herein can refer to identity of an amino acid sequence or a nucleotide sequence. Two copies having exactly the same sequence have 100% identity. Those skilled in the art are aware that identity of an amino acid sequence or a nucleotide sequence can be determined using identity search sites on the internet, such as the BLAST page of the NCBI home page website. For example, the value of identity (%) can be obtained by searching using blastp as the program, setting Expect value to 10, setting all Filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, in High Score BLAST 2.1, and then calculating identity of an amino acid sequence. In addition, it can be determined using sequence analysis software (such as CLC Main Workbench and MegAlign™), for example, computer program BLAST, especially BLASTP or TBLASTN, using default parameters. Identity of 75% or more as described herein can be at least 75%, 80%, 85%, 90% or 95% or more. Identity of 80% or more as described herein can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more.

[0170] The term "conservative substitution" generally refers to the replacement of one amino acid residue by another possessing a similar physicochemical property. For example, conservative substitutions can be made between hydrophobic side chain amino acid residues (e.g., Met, Ala, Val, Leu, and He), between neutral hydrophilic side chain residues (e.g., Cys, Ser, Thr, Asn, and Gin), between acidic side chain residues (e.g., Asp, Glu), between basic side chain amino acids (e.g., His, Lys, and Arg), or between aromatic side chain residues (e.g., Trp, Tyr, and Phe). Conservative substitutions generally do not give rise to significant changes in the conformational structure of a protein, and thus are capable of preserving the biological activity of the protein, as is known in the art.

[0171] The term "antibody" generally refers to an immunoglobulin that is capable of specifically binding to a target antigen, including monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, humanized antibodies (including chimeric antibodies, CDR-grafted antibodies, and SDR-grafted antibodies), and fully human antibodies. When the term "antibody" is referred to, it includes not only intact antibodies, but also antigen-binding fragments of antibodies that are capable of specifically binding to a target antigen. Also, the antibody can be of different classes of antibodies, e.g., IgG, IgA, IgD, IgE, IgM, and subtypes thereof.

[0172] The term "nanobody" also referred to as single domain antibody (sdAb) generally refers to an antibody that consists of only one H chain V region, also referred to as VHH antibody. Nanobodies are extremely stable in their ability to bind to antigens and their stability is essentially identical to that of full antibodies. In this document, when the term "nanobody" is referred to, it includes not only intact nanobodies, but also antigen-binding fragments of nanobodies. An antigen-binding fragment refers to a polypeptide comprising a fragment of a nanobody that retains the ability to specifically bind to the same antigen to which the nanobody binds, and / or competes with the nanobody for specific binding to the antigen, which is also referred to as "antigen-binding portion". In some embodiments, the "antigen-binding fragment" of the nanobody can be truncated at the N- or C-terminus compared to the full-length nanobody to comprise only part of FR1 and / or FR4, or lack one or both of those framework regions, as long as it essentially retains antigen-binding and specificity.

[0173] The term "bispecific antibody (BsAb)" generally refers to an antibody that can bind to two targets with different structures at the same time, e.g., two different target antigens, two different epitopes on the same target antigen. There are various different bispecific antibody structures known in the art, such as IgG-like bispecific antibodies with Fc portion and non-IgG-like bispecific antibodies without Fc portion. Among the non-IgG-like bispecific antibodies without Fc portion, there are structures consisting of only two nanobodies, referred to as bispecific nanobodies (BsNb).

[0174] The term "multispecific antibody" refers herein to an antibody molecule that can bind to multiple (more than two) different antigen epitopes of the same antigen or multiple (more than two) different antigens.

[0175] The term "Fab" or "Fab antibody" generally refers to a heterodimer comprising the heavy chain Fd and the entire light chain, with only one antigen-combining site. The coding genes of the heavy chain Fd and the entire light chain are ligated and fused with the bacterial protein signal peptide gene, and then the Fab antibody can be expressed and secreted in E. coli, with complete stereo-folding and intra-chain and inter-chain disulfide bonds. The heavy chain Fd refers to about 1 / 2 of the H chain portion in the Fab (including VH, CH1 and part of the hinge region).

[0176] The term "Fab'" or "Fab' antibody" or "Fab' fragment" generally refers to a portion comprising one light chain and one heavy chain comprising a VH domain and a CH1 domain and the region between CH1 and CH2 domains.

[0177] The term "F(ab')2" or "F(ab')2 antibody" or "F(ab')2 fragment" generally refers to a portion comprising two light chains and two heavy chains comprising the constant region between CH1 and CH2 domains, thereby forming inter-chain disulfide bonds between the two heavy chains. Thus, the F(ab')2 fragment is composed of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0178] The term "Fv" or "Fv antibody" generally refers to a vector containing VH and VL genes, respectively, co-transfecting cells to express, respectively, and then assembling a functional Fv antibody; or setting a stop codon between VH and VL in the vector, respectively, expressing two small molecular weight protein fragments, and then combining them through non-covalent bonds to form an Fv antibody.

[0179] The term "single-chain antibody (ScFv)" generally refers to connecting the light chain and heavy chain variable region genes with a suitable oligonucleotide linker to express a single polypeptide chain, called single-chain antibody (ScFv). The polypeptide chain can spontaneously fold into a natural conformation, maintaining the specificity and affinity of Fv.

[0180] The term "minimal recognition unit (MRU)" generally refers to a single CDR structure in the variable region, with a molecular weight of only about 1% of the complete antibody, which can bind to the corresponding antigen.

[0181] The term "complementarity determining region" or "CDR" generally refers to the amino acid residues in the variable region of an antibody responsible for antigen binding. The CDRs can be defined according to various numbering systems known in the art, such as according to the definition in the Kabat numbering system, the Chothia numbering system or the IMGT numbering system. For a given antibody, a person skilled in the art can easily identify the CDRs defined by each numbering system. And the correspondence between different numbering systems is well known to those skilled in the art.

[0182] The term "framework region (FR)" generally refers to those amino acid residues of an antibody variable region other than the CDR residues as defined above.

[0183] The term "epitope" generally refers to any antigenic determinant on an antigen that is bound by the paratope of an antibody. Both linear and conformational epitopes are included. Epitopes can be identified by any method well known in the art, such as conventional immunoassays, antibody competitive binding assays, or X-ray crystallography or related structural determination methods (e.g., nuclear magnetic resonance spectroscopy).

[0184] The term "antibody conjugate" herein not only includes antibody drug conjugates (ADC) formed by conjugation of an antibody (including nanobodies, monoclonal antibodies, bispecific antibodies, multispecific antibodies, etc.) with a drug (a substance for preventing, treating, or diagnosing a disease) via a linker, but also includes conjugates obtained by conjugation of an antibody (including nanobodies, monoclonal antibodies, bispecific antibodies, multispecific antibodies, etc.) with a detectable label.

[0185] The term "immunosensor" generally refers to a method of detecting an antigen or an antibody in a sample by immobilizing the antigen or the antibody on the surface of a solid support, combining a specific immune reaction with a highly sensitive sensing technology, and monitoring an antigen-antibody reaction. The immunosensor can achieve quantitative detection and real-time monitoring, and is a quantitative, automated immunodetection and diagnosis product.

[0186] The term "introducing" generally refers to the process of transferring an exogenous gene into a recipient cell such as a eukaryotic recipient cell or a prokaryotic recipient cell. The method of introduction is not particularly limited, and any known transformation method can be used as long as it can transfer the gene of interest (e.g., a gene encoding a nanobody of the present application) into a recipient cell. The method of introduction can include any one of the following: (1) introducing the gene of interest or a recombinant vector containing the gene of interest into a host bacterium by a chemical transformation method (e.g., Ca ion-induced transformation, polyethylene glycol-mediated transformation, or metal cation-mediated transformation) or a physical transformation method (e.g., electroporation transformation); (2) transducing the gene of interest into a host bacterium by a bacteriophage transduction method; (3) directly transferring the gene of interest into a plant recipient cell by a physical or chemical method, such as a chemical stimulation method, an electric shock method, a liposome-mediated method, a microinjection method, a gene gun method, a laser microbeam method, a pollen tube channel method, an ultrasonic wave method, an air gun method, and a vortex method; (4) transferring the gene of interest into a plant recipient cell using a vector as a medium, such as a Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector systems and binary vector systems) mediated method; and (5) introducing the gene of interest into an isolated animal cell (transfection) by a calcium phosphate coprecipitation method, a cationic polymer method, a liposome-mediated method, an electroporation method, microinjection, a gene gun method, or a viral vector method.

[0187] The term "Claudin 18.2 positive tumor" generally refers to a tumor expressing Claudin 18.2 protein. Methods for detecting Claudin 18.2 protein expression in a tumor are well known to those skilled in the art, for example, Claudin 18.2 protein expression can be determined by immunological detection methods based on antigen-antibody specific reaction (such as immunohistochemistry, FACS, etc.), Claudin 18.2 mRNA expression can also be determined by real-time fluorescent quantitative PCR, Northern blotting or RNA in situ hybridization methods.

[0188] The term "preventing" generally refers to a method implemented to stop or delay the occurrence of a disease or disorder or a symptom in a subject.

[0189] The term "treatment" generally refers to a method implemented to obtain a beneficial or desired clinical result. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, shrinkage of disease range, stabilization (i.e., not worsening) of the disease state, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Furthermore, treatment can also refer to prolonging survival as compared to expected survival of a subject if left untreated.

[0190] The term "diagnosis" generally refers to a method that can determine whether a subject is likely to have a given disease or disorder, which includes but is not limited to a disease or disorder characterized by an antigen or pathogen. A skilled person generally makes a diagnosis based on one or more diagnostic indicators, the presence, absence, amount or change in amount of which indicates the presence, severity or absence of a disease or disorder. Other diagnostic indicators can include patient history; physical symptoms, etc. The term "diagnosis" also includes the process of predicting and / or monitoring a disease or disorder, including monitoring the response of one or more patients to the administration of a drug or a candidate drug, for example, to determine its efficacy.

[0191] The term "reference value" generally refers to a reference level that can be obtained in advance from a subject, from another subject (including healthy subjects and patients), and the appropriate reference level can be measured and selected according to techniques known to those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0192] Figure 1 is the identification of CHOK1-CLDN18.2 stable expression cells.

[0193] Figure 2 is a total RNA electrophoresis map. In Figure 2, A is the total RNA of alpaca NB484-A; B is the total RNA of alpaca NB484-B. M: DNA marker; 1: alpaca A; 2: alpaca B.

[0194] Figure 3 is an electrophoretogram of the first round of PCR amplification products of NB484 VHH. Wherein: M-DNA marker, 1-First round of amplification of VHH fragments by nested PCR; 2-First round of amplification of VHH fragments by nested PCR; 3-First round of amplification of VHH fragments by nested PCR; 4-First round of amplification of VHH fragments by nested PCR; 5-First round of amplification of VHH fragments by nested PCR.

[0195] Figure 4 is an electrophoretogram of the second round of PCR amplification products of NB484 VHH. M: DNA marker, Line 1-2: VHH.

[0196] Figure 5 is the number of transformants of phage library. Wherein: Left side-After electroporation into E. coli, diluted to 10 -3 colony map after plating, right side-After electroporation into E. coli, diluted to 10 -4 colony map after plating.

[0197] Figure 6 is the identification and purity detection of recombinant antibodies (CLDN18.2-VHH-319 and CLDN18.2-VHH-357). Figure 6 A and B are SDS-PAGE electrophoresis and HPLC results of #319, #357 recombinant proteins, respectively.

[0198] Figure 7 is the flow cytometry identification results of recombinant antibodies (CLDN18.2-VHH-319 and CLDN18.2-VHH-357) on target cells.

[0199] Figure 8 is a standard curve of equilibrium dissociation constant (KD) of CLDN18.2-VHH-hFc (CLDN18.2-VHH-319 and CLDN18.2-VHH-357).

[0200] Figure 9 is the identification and purity detection of recombinant antibodies (CLDN18.2-VHH-223).

[0201] Figure 10 is a standard curve of equilibrium dissociation constant (KD) of CLDN18.2-VHH-hFc (CLDN18.2-VHH-223).

[0202] Figure 11 is the flow cytometry identification results of recombinant antibodies (CLDN18.2-VHH-223) on target cells. Embodiments of the present application

[0203] The present application will be further described in conjunction with the specific embodiments, and the examples given are only for the purpose of illustrating the present application, but not for limiting the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0204] The experimental methods in the following examples are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0205] The nucleotide sequence of the CLDN18.2 protein in the following examples is shown in SEQ ID NO: 8, and the amino acid sequence is shown in SEQ ID NO: 9. The nucleotide sequence of the CLDN18.1 protein is shown in SEQ ID NO: 10, and the amino acid sequence is shown in SEQ ID NO: 11.

[0206] The construction method of the CLDN18.2 overexpressing 293T cells in the following examples is as follows: the gene encoding human CLDN18.2 protein (SEQ ID NO: 8) is cloned into the eukaryotic expression vector pSecTag2A-BN (Invitrogen, catalog number: V90020) after removing the stop codon, to obtain a recombinant expression vector, which is transfected into 293T cells (Cooperative Cell Bank product, catalog number: 4201PAT-CCTCC01347), and the recombinant cells stably expressing human CLDN18.2 are obtained by screening, and are named 293T-CLDN18.2 cells.

[0207] The construction method of the CLDN18.1 overexpressing 293T cells in the following examples is as follows: the gene encoding human CLDN18.1 protein (SEQ ID NO: 10) is cloned into the eukaryotic expression vector pSecTag2A-BN after removing the stop codon, to obtain a recombinant expression vector, which is transfected into 293T cells, and the recombinant cells stably expressing human CLDN18.1 are obtained by screening, and are named 293T-CLDN18.1 cells.

[0208] Example 1, Screening and Preparation of Anti-CLDN18.2 Nanobody

[0209] 1, Animal Immunization and Detection of Serum Antibody Titer

[0210] 1.1, Animal Immunization

[0211] 1) The immunized animals are adult healthy llamas (2 llamas). The immunogen is CHOK1 cells stably overexpressing CLDN18.2 (CHOK1-CLDN18.2 cells).

[0212] The CHOK1-CLDN18.2 cells were constructed as follows: The gene encoding the human CLDN18.2 protein (GenBank Accession No. 51208 (CLDN18) (Update Date 13-May-2024), positions 137,998,816-138,033,649) was cloned into the eukaryotic expression vector pSecTag2A-BN to obtain a recombinant expression vector. This recombinant expression vector was transfected into CHOK1 cells (Kyoho Cell Bank product, catalog number 4201PAT-CCTCC01106). After screening, recombinant cells stably expressing human CLDN18.2 were obtained and named CHOK1-CLDN18.2 cells. The expression level of CLDN18.2 is shown in the flow cytometry detection and analysis results (Figure 1 and Table 1).

[0213] Table 1. Identification of cells stably expressing CHOK1-CLDN18.2

[0214] 2) CHOK1-CLDN18.2 cells were revived in advance and cultured in a carbon dioxide incubator at 37°C and 5% CO2.

[0215] 3) Each alpaca was immunized 5 times (see Table 2), with an injection volume of 2 × 10⁻⁶ cells per immunization. 7 Cells / animal, at 3-week intervals. For the first immunization, cells were emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously. For the second, third, fourth, and final immunizations, cells were emulsified with an equal volume of Freund's incomplete adjuvant and injected subcutaneously. For the final immunization, an additional 100 μg / animal of Human-CLDN18.2 full-length protein-VLP (purchased from Acrobiosystems, catalog number: CL2-H52P7) was added.

[0216] 4) One week after each immunization, 10ml of blood was drawn from the animal, the plasma was separated, and the titer of the previous immunization was tested. The final titer was tested one week after the injection.

[0217] 1.2 Serum antibody titer detection

[0218] Serum titers were determined by testing. Positive screening was performed using CLDN18.2-overexpressing 293T cells (293T-CLDN18.2 cells), and negative screening was performed using CLDN18.1-overexpressing 293T cells (293T-CLDN18.1 cells).

[0219] 1) Serum titer detection uses FACS detection, and the serum of alpaca after immunization is used as the sample to be tested. 10 ml of alpaca serum before the first immunization is taken as a control standard (0.02% Proclin 300 is stored), and the dilution ratio of the antibody to be tested is 1:100 and 1:1000, and the dilution ratio of the standard is the same as that of the sample to be tested.

[0220] 2) Collect 293T-CLDN18.2 cells and 293T-CLDN18.1 cells in logarithmic growth phase, centrifuge at 1500 rpm for 5 min, and resuspend;

[0221] 3) Adjust the cell density to 4x10 6 / mL with 1% horse serum or PBS;

[0222] 4) Take 50 μL of cell suspension into a 1.5 mL EP tube, add a certain volume of immune serum to be tested to the cells, and incubate at 4°C for 1 h;

[0223] 5) Add PBS washing solution, centrifuge at 1500 rpm for 5 min, and remove the supernatant;

[0224] 6) Resuspend the cells with 50 μL of diluted secondary antibody goat anti-alpaca IgG H&L conj-647 (NBbiolab, Cat#S001A647, Lot#20211120), and incubate at 4°C for 1 h;

[0225] 7) Wash the cells twice, resuspend them, and place them at 4°C for detection. The flow cytometry results are shown in Table 2.

[0226] 8) According to the final immunization results, select two alpacas to mix the library in a ratio of A:B of 3:1. Construct a phage library.

[0227] Table 2, five-immune serum titer detection results

[0228] 2, phage library construction and packaging

[0229] 2.1, phage library construction

[0230] 1) Collect 50 mL of peripheral blood from alpaca A and alpaca B after five immunizations, and separate PBMC according to the instructions for lymphocyte separation medium.

[0231] 2) Extract total RNA of alpaca A five immune and alpaca B five immune PBMC with RNAiso Plus reagent. Alpaca A total RNA: recovery volume 30 μL, 1445.2 ng / μL, A260 / A280 = 2.17, A260 / A230 = 2.27; alpaca B total RNA: recovery volume 30 μL, 1184.3 ng / μL, A260 / A280 = 2.13, A260 / A230 = 2.26.

[0232] 3) Take 1 μg RNA for electrophoresis, and gel electrophoresis is used to detect RNA purity. The results show that the RNA purity is good (Figure 2).

[0233] 4) According to the instructions of PrimeScript TM II 1st Strand cDNA Synthesis Kit (product of Takara Company, item number 6210A), perform reverse transcription, and a total of 5 μg RNA is transcribed. The specific process is as follows:

[0234] Take PrimeScript TM II 1st Strand cDNA Synthesis Kit components out of the -20℃ refrigerator, mix well, centrifuge briefly, and place on ice.

[0235] Add the components in Table 3 to a 0.2 μL centrifuge tube, and mix well.

[0236] Table 3, reverse transcription reaction system 1

[0237] Incubate at 65℃ for 5 min, and place on ice for more than 1 min.

[0238] Configure the cDNA synthesis system, add the components in Table 4, and mix well.

[0239] Table 4, reverse transcription reaction system 2

[0240] Gently mix, and perform the reaction according to the following Table 5.

[0241] Table 5, reverse transcription reaction program

[0242] After the reaction is completed, centrifuge the reaction product briefly, and store the cDNA at -20℃.

[0243] 5) First round of nested PCR

[0244] The cDNA stock solution obtained after immunizing alpaca A and alpaca B was mixed at a ratio of 3:1 and diluted 5 times for amplification. The reaction system and reaction conditions of the first round of nested PCR are shown in Table 6 and Table 7, and a total of 5 tubes were amplified. The electrophoresis result of the first round of nested PCR amplification product is shown in Figure 3.

[0245] Table 6, nested PCR reaction system

[0246] In Table 6, the nucleotide sequence of primer NPR-19001 is 5'-GTGGTCCTGGCTGCTCT-3' (SEQ ID NO: 5), the nucleotide sequence of primer NPR-19002 is 5'-CGCCATCAATRTACCAGTTGA-3' (SEQ ID NO: 6), and the concentration of primer NPR-19001 and primer NPR-19002 is both 10 nM.

[0247] Table 7, nested PCR reaction conditions

[0248] 6) Second round of nested PCR

[0249] After the first round of nested PCR amplification was completed, all PCR amplification products were collected, and the VHH fragments of about 750 bp were recovered by gel cutting. The second round of nested PCR was performed to amplify the target gene fragments, and 2% agarose electrophoresis was performed, as shown in Figure 4. The VHH fragments of about 500 bp were purified and recovered using a DNA product purification kit. The purified VHH fragments were stored at -20°C or below.

[0250] 7) The vector (Vector) pCANTAB5e (product of New England Biolabs, product number: V001082#) and the target gene fragment (VHH fragment) obtained in step 6) were respectively subjected to enzyme digestion with SfiI at 50°C overnight, and then the vector fragment and the target gene fragment were recovered using a universal DNA purification kit. After the enzyme digestion reaction was completed, T4 ligase was used for the ligation reaction at a molar ratio of Vector:VHH=1:3 at 4°C for 16h.

[0251] 8) Transform the ligation product into E. coli and perform 10 times of electroporation. Immediately after electroporation, add 1 mL of 2YT medium (preheated at 37°C) to the electroporation cup for recovery, aspirate the electroporation product and wash the electroporation cup with 2YT medium. A total of 100 mL of recovery product was obtained, which was recovered at 37°C, 180 rpm for 45 min, and 100 μL of gradient dilution was taken to 10 -3 and 10 -4Determine the number of library transformants, spread on 90 mm plates, centrifuge the rest, resuspend in 8 mL 2YT medium, spread on 8 200 mm plates (Figure 5). The next day determine the number of library transformants on the plates -4 There are 190 clones in total, so the number of bacterial library transformants is 1.90 x 10 9 cfu.

[0252] 2.2, Packaging of phage library

[0253] 1) Infect bacterial library into 300 mL 2YT+A+G (Amp: 100 ug / ml, Glu: 1%) medium to an initial OD600 of 0.1-0.2, incubate at 37°C, 230 rpm until OD600 = 0.8 or above.

[0254] 2) Add helper phage M13KO7 according to OD600 value (helper phage: bacteria = 20: 1), after adding M13KO7, mix well, and let stand at 37°C for 30 min.

[0255] 3) Incubate at 37°C, 180 rpm for 30 min. Centrifuge at 5000 rpm for 10 min, discard the supernatant, resuspend the pellet with an equal volume of 2YT+A+K (Amp: 100 ug / ml, Kan: 50 ug / ml) medium, and incubate at 30°C, 220 rpm overnight.

[0256] 4) Centrifuge the overnight culture at 10000 rpm for 20 min at 4°C, collect the supernatant and discard the pellet. Replace the centrifuge tube, centrifuge at 10000 rpm for 20 min at 4°C, collect the supernatant. Add PEG8000 / NaCl at 1 / 5 of the volume of the supernatant, mix well, and precipitate in an ice bath for 2 hours or more.

[0257] 5) Centrifuge at 10000 rpm for 20 min at 4°C, discard the supernatant, and remove the supernatant by emptying once. Resuspend the pellet with 1 mL of 1x PBS, and precipitate again with 1 / 5 volume of PEG8000 / NaCl for 1 h. Centrifuge at 12000 rpm for 10 min at 4°C, discard the supernatant, and remove the supernatant by emptying once. Resuspend the pellet with 1x PBS according to the amount of pellet.

[0258] 6) Add 100% glycerol to a final concentration of 50%, mix well, and aliquot into 1.5 mL EP tubes, and store at -80°C.

[0259] 7) Take 10 uL of library phage and dilute it in 2YT medium in a gradient, from 10 -8 and 10 -9 uL to 90 uL of TG1 bacterial solution, mix gently. Let stand at 37°C for 15 min, and spread on Amp-resistant plates, respectively, and incubate overnight.

[0260] 8) The next day, at 10 -9 There were 636 clones on the titer plate, and the phage library titer was 6.36 x 10 13 cfu / mL (636 x 10 9 x 100), ready for the next step of screening.

[0261] 3. Phage library screening

[0262] 3.1. Affinity panning

[0263] 1) Wash 293T-CLDN18.2 cells (5 x 10 6 cells): 500g centrifugation for 5 min, resuspend with 5% serum-PBS and wash the cell pellet twice; add 500uL 3% OVA blocking solution to the cell centrifuge tube, block at 4°C for 1h with slight shaking;

[0264] 2) Dilute the phage library with 5% serum-PBS and 3% OVA to an OVA concentration of 2%, 37°C block for 30 min for standby; centrifuge the cells after blocking, remove the supernatant, add the phage library dilution, 4°C, 1h with slight shaking; centrifuge, remove the unbound phage, and wash the cells with 5% serum-PBS 6 times;

[0265] 3) Add 100uL Gly-HCl elution solution, 37°C incubate for 8 min to elute the specifically bound phage; transfer the eluate to a 1.5mL sterile centrifuge tube, quickly neutralize with 10uL Tris-HCl neutralization buffer;

[0266] 4) Take 10uL for gradient dilution, determine the titer, calculate the panning recovery rate, mix the rest of the eluate for amplification and purification, and use it for the next round of affinity panning, change the panning conditions, and each round of panning conditions are shown in Table 8.

[0267] Table 8. Affinity panning conditions

[0268] 3.2. Amplification of the library after panning

[0269] 1) Mix the panning eluate with 20mL of E. coli TG1 culture in the early logarithmic growth phase, 37°C, stand for 30 min, add 1mL of 20% glucose, 220r / min shaking culture for 30 min, then add M13KO7 phage and 4uL Amp + at a ratio of cell: phage = 1:20, 37°C, stand for 30 min, then add 20mL of 2YT liquid medium, 220r / min shaking culture for 30 min;

[0270] 2) The culture was dispensed into centrifuge tubes, 4°C, 5000r / min, 10min, the cells were resuspended with 50mL 2xYT-AK liquid medium (2YT medium added Amp: 100μg / ml, Kan: 50μg / ml), 30°C, 250r / min shaking culture overnight;

[0271] 3) The overnight culture was centrifuged at 4°C, 10000r / min for 20min, the supernatant was transferred to a new centrifuge tube, 1 / 5 volume of PEG-NaCl was added, mixed and placed at 4°C for 2h or more;

[0272] 4) 4°C, 10000r / min, 20min, remove the supernatant, resuspend the precipitate in 1mL PBS, add 1 / 5 volume of PEG / NaCl, mix and place at 4°C for 1h or more;

[0273] 5) 4°C, 12000r / min, 2min, remove the supernatant, suspend the precipitate in 200μL PBS, which is the amplification product, determine the titer, for the next round of selection or analysis.

[0274] 3.3, Positive screening and identification of specific phage clones

[0275] 1) From the titer plate of the eluate of the selection, 480 clones were randomly selected from the second and third round titer determination plates with sterilized toothpicks, inoculated in 300uL 2xYT-A medium (2YT medium added Amp: 100μg / ml), 37°C, 230r / min shaking culture for 8h.

[0276] 2) Take 100μL of the above culture, add M13KO7 phage at a ratio of cell: phage = 1:20, 37°C, stand for 15min, 220r / min shaking culture for 45min. Add 300μL volume of 2xYT-AK, 30°C, shaking culture overnight. The next day, centrifuge at 12000rpm for 2min, take the supernatant, which is used for monoclonal ELISA identification.

[0277] 3) Prepare the identification of positive phage clones, culture 293T-CLDN18.2 cells and 293T-CLDN18.1 cells to cover the whole plate, wash with PBS twice, add 100uL 4% paraformaldehyde to fix the cells, 25°C for 20-30min;

[0278] 4) Wash with PBS twice, add 300uL 5% skim milk per well, 37°C for 1h; wash with PBST once, add 50μL phage culture supernatant and 50μL 5% skim milk per well, 37°C, incubate for 1h;

[0279] 5) PBST wash 5 times, add horseradish peroxidase-labeled anti-M13 antibody (diluted with PBS at 1:10000), 100 μL / well, 37°C for 1 h;

[0280] 6) Wash the plate with PBST 6 times. Add TMB color developing solution for color development, 100 μL / well, 37°C, 7 min, add stop solution to stop the reaction, 50 μL / well, measure optical density at 450 nm. A total of 142 positive clones were obtained.

[0281] 7) Sequence the positive clones, and the sequencing primer is: 5'-TCACTCATTAGGCACCCCAG-3' (SEQ ID NO: 7). After sequence determination of the 142 positive clones obtained, after excluding non-monoclonal and sequencing failure clones, data analysis after sequencing of the above positive clones, according to the different CDR3 sequences, 22 unique sequences were obtained.

[0282] In combination with the ELISA results and the data analysis results after sequencing of the positive clones, #319 and #357 positive clones were selected for subsequent identification.

[0283] The nucleotide sequence of the #319 VHH gene is shown as SEQ ID NO: 1, and the encoded amino acid sequence is shown as SEQ ID NO: 2.

[0284] The nucleotide sequence of the #357 VHH gene is shown as SEQ ID NO: 3, and the encoded amino acid sequence is shown as SEQ ID NO: 4.

[0285] In combination with the ELISA results and the data analysis results after sequencing of the positive clones, #223 positive clone was selected for subsequent identification.

[0286] The nucleotide sequence and amino acid sequence of the #223 VHH are as follows:

[0287] Nucleotide sequence of #223 VHH: 5'-CAGTTGCAGCTCGTGGAGTCAGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAACCTCTGCAAGCACCGTCAATATCTACTCCATGGGCTGGTACCGCCAGGGTCCAGGGAAGCAGCGCGAGTTGGTCGCAACAATGACTACCAGTGGTAGAATAAACTACGCGAACTCCGTGAAGGGCCGATTCACCATCTCCAGAGAGAACGCCAAGATGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATGTGCATCTTTTTAGTGCAGTGCCAGGTGGTGCGACAGATTACTGGGGCCAGGGGACCCAGGTCACCGTTTCCCCA-3' (SEQ ID NO: 19).

[0288] Amino acid sequence of #223 VHH: 5'-QLQLVESGGGLVQPGGSLRLSCATSASTVNIYSMGWYRQGPGKQRELVATMTTSGRINYANSVKGRFTISRENAKMVYLQMNSLKPEDTAVYYCNVHLFSAVPGGATDYWGQGTQVTVSP-3' (SEQ ID NO: 20).

[0289] 4. Recombinant antibody expression and purification

[0290] 4-1. Expression and purification of Nanobody A (CLDN18.2-VHH-319) and Nanobody B (CLDN18.2-VHH-357)

[0291] 1) The above two positive clones VHH genes (#319 VHH gene shown in SEQ ID NO: 1 and #357 VHH gene shown in SEQ ID NO: 3) were respectively cloned into a mammalian expression vector pCAG-Fc (purchased from Shanghai Jinpan Biotech Co., Ltd., item number: 165-27741) carrying a Human IgG Fc gene for expression. After the expression sequence was identified, the plasmid containing the target protein was transfected into E. coli competent cells for amplification to obtain sufficient amount of expression plasmid.

[0292] 2) Before the small-scale antibody production expression of 40 mL / clone, the HEK293 cells were cultured in advance using serum-free CD medium (Yiqiao God, item number: SMM 293-TI), and continuously passaged for 3 times. On the day of transfection, the HEK293 cells were adjusted to a cell density of 2x10 6 / mL using serum-free CD medium.

[0293] 3) The culture system was 40 mL of cells. The target protein expression plasmid was mixed with transfection reagent TF1 (Yiqiao God, item number STF02) at 1:10 (w / v) and then added to 40 mL of cells to start transfection, which was recorded as day 0. The transfection reagent was used according to the instructions. On the 1st, 3rd and 5th day after the start of transfection, 293 serum-free feeding liquid (Yiqiao God, item number: M293-SUPI-100) was added, and the feeding liquid was used according to the instructions.

[0294] 4) The cells were cultured for 5-7 days. The whole process of fine culture was carried out in a shaker containing CO2 (5%-8%). The shaker speed was maintained at 150-175 rpm.

[0295] 5) The supernatant was centrifuged and prepared for the next step of antibody purification. The supernatant was filtered with a filter to remove the remaining insoluble substances.

[0296] 6) The antibody was purified by Protein A affinity chromatography column. The chromatography column was equilibrated with 20 mL of equilibration buffer (1xPBS) at a flow rate of 1 ml / min. The centrifuged and filtered supernatant was loaded at a flow rate of 1 ml / min, and then 20 mL of equilibration buffer was used to wash away part of the impure proteins at a flow rate of 1 ml / min. After the impurities were removed, elution was performed. 1M sodium acetate buffer (pH 3.4) was added to elute the target protein at a speed of 1 ml / min. The elution used 20 mL of sodium acetate buffer. The obtained sample was collected in separate tubes, and then 500 uL of 1M NaHCO3 (pH 9.0) solution was added to neutralize the eluted antibody. A total of 10 tubes were collected. After collecting the elution components, the high-concentration protein was absorbed into a dialysis bag and placed in a beaker containing 1xPBS for dialysis. Two VHH-hIgG1Fc fusion recombinant proteins (#319, #357) were obtained by purification, i.e. VHH antibodies fused with hIgG1Fc tags.

[0297] 7) The recombinant protein concentration was determined by BCA protein quantification kit. The recombinant antibody was stored at -80°C after aliquot.

[0298] The SDS-PAGE electrophoresis and HPLC results of the purified recombinant antibody are shown in Figure 6. The protein purity was greater than 95%. Antibodies of 4.34 mg and 2.66 mg were obtained, respectively.

[0299] The obtained VHH antibodies (also known as nanobodies or single-domain antibodies) are named CLDN18.2-VHH-319 and CLDN18.2-VHH-357, respectively.

[0300] The amino acid sequence of the anti-CLDN18.2 nanobody CLDN18.2-VHH-319 is shown as follows:

[0301] 5'-QVQLVESGGGLVQAGGSLRLSCAASERFSIYNMAWFRQAPGKEREFVARFEWVWNGINTYYADSVKGRFTISGENAKSTLYLQMNSLKPEDTAVYYCAGKPIGSPRNEYDYWGQGTQVTVSS-3' (SEQ ID NO: 2);

[0302] The amino acid sequence of the anti-CLDN18.2 nanobody CLDN18.2-VHH-357 is shown as follows:

[0303] 5'-QVQLVESGGGLVQAGGSLRLSCAASRRFSIYNMAWFRQAPGKEREFVARFEWVWNGINTYYADSVKGRFTISGENAKSTLYLQMNSLKPEDTAVYYCAGKPIQSPRNEYDYWGQGTQVTVSS-3' (SEQ ID NO: 4).

[0304] The three underlined parts are CDR1, CDR2 and CDR3, respectively.

[0305] 4-2, Expression and purification of nanobody C (CLDN18.2-VHH-223)

[0306] (1) The VHH gene of the positive clone (DNA molecule shown in SEQ ID NO: 19) was cloned into the mammalian expression vector pCAG-Fc (purchased from Shanghai Jinpan Biotech Co., Ltd., item number: 165-27741) carrying the Human IgG Fc gene for expression. After the expression sequence was identified, the plasmid containing the target protein was transfected into E. coli competent cells for amplification to obtain sufficient amount of expression plasmid.

[0307] (2) Before small-scale antibody production expression of 40 mL / clone, the HEK293 cells were cultured in advance using serum-free CD medium (Yiqiao God, item number: SMM 293-TI) and subcultured for 3 times. On the day of transfection, the HEK293 cells were adjusted to a cell density of 2 x 10 6 / mL using serum-free CD medium.

[0308] (3) The culture system is 40 mL of cells. The target protein expression plasmid is mixed with transfection reagent TF1 (Yiqiao God, item number STF02) at a ratio of 1:10 (w / v) and then added to 40 mL of cells to start transfection, which is recorded as day 0. The transfection reagent is used according to the instructions. On the 1st, 3rd and 5th day after the start of transfection, 293 serum-free feed solution (Yiqiao God, item number: M293-SUPI-100) is added, and the feed solution is used according to the instructions.

[0309] (4) The cells are cultured for 5-7 days. The whole process of cell culture is carried out in a shaker containing CO2 (5%-8%), and the shaker speed is maintained at 150-175 rpm.

[0310] (5) The supernatant is centrifuged and prepared for the next step of antibody purification. The supernatant is filtered with a filter to remove residual insoluble substances.

[0311] (6) The antibody is purified by Protein A affinity chromatography column. The chromatography column is equilibrated with 20 mL of equilibration buffer (1x PBS) at a flow rate of 1 ml / min. The centrifuged and filtered supernatant is loaded at a flow rate of 1 ml / min, and then 20 mL of equilibration buffer is used to wash away part of the impure proteins at a flow rate of 1 ml / min. After the impurities are removed, elution is performed. 1M sodium acetate buffer (pH 3.4) is added, and the target protein is eluted at a speed of 1 ml / min. The elution uses 20 mL of sodium acetate buffer, and the obtained sample is collected in separate tubes. Then, 500 uL of 1M NaHCO3 (pH 9.0) solution is added to neutralize the eluted antibody. A total of 10 tubes are collected. After collecting the elution components, the high-concentration protein is absorbed into a dialysis bag and placed in a beaker containing 1x PBS for dialysis. The purified VHH-hIgG1Fc fusion recombinant protein, which is a VHH antibody fused with hIgG1Fc tag, is obtained.

[0312] (7) The recombinant protein purity is identified and determined by SDS-PAGE and SEC-HPLC methods. The recombinant protein concentration is determined by BCA protein quantification kit. The recombinant antibody is aliquoted and stored at -80°C.

[0313] The SDS-PAGE electrophoresis and HPLC results of the purified recombinant antibody are shown in Figure 9. The harvested antibody is 4.36 mg, and the protein purity is greater than 95%.

[0314] The obtained VHH antibody (also known as nanobody) is named: CLDN18.2-VHH-223. The amino acid sequence of the anti-CLDN18.2 nanobody CLDN18.2-VHH-223 is as follows:

[0315] 5'-QLQLVESGGGLVQPGGSLRLSCATSASTVNIYSMGWYRQGPGKQRELVATMTTSGRINYANSVKGRFTISRENAKMVYLQMNSLKPEDTAVYYCNVHLFSAVPGGATDYWGQGTQVTVSP-3' (SEQ ID NO: 20).

[0316] The three underlined parts are CDR1, CDR2 and CDR3, respectively.

[0317] Example 2, Specific detection of anti-CLDN18.2 Nanobodies (Nanobody A and Nanobody B)

[0318] This example relates to the specific detection of Nanobody A (CLDN18.2-VHH-319) and Nanobody B (CLDN18.2-VHH-357).

[0319] Using the above 2 VHH-hIgG1 Fc fusion recombinant proteins (#319, #357), respectively, Pierce TM NHS-Fluorescein Antibody Labeling Kit (Thermo Scientific product, item number: 53029) was labeled to obtain FITC directly labeled antibody FITC-CLDN18.2-VHH-hFc, which was used for flow detection of CLDN18.2 protein expression in different cell lines.

[0320] 1) CLDN18.2 negative cell lines: 293T cells and 293T-CLDN18.1 cells; CLDN18.2 positive cell lines: 293T-CLDN18.2 cells. The above cells were respectively resuscitated at 37°C and added to cell culture bottles, cultured at 37°C, 5% CO2, and subcultured twice for detection of anti-CLDN18.2 nanobodies (FITC-CLDN18.2-VHH-hFc).

[0321] 2) Adjust the concentration of each cell to 2x10 6 6x105 / mL, 200μL / well into a 96-well plate, centrifuge at 1500rpm for 5min;

[0322] 3) Wash with FACS buffer and centrifuge;

[0323] 4) Add 20μL of diluted FITC-CLDN18.2-VHH-hFc antibody (1:400) and mix, incubate at room temperature for 10min;

[0324] 5) Add FACS buffer, centrifugal to wash away the antibody, then add buffer to resuspend, and detect by flow cytometry.

[0325] Flow cytometry results showed that the FITC-labeled CLDN18.2-VHH-hFc recombinant antibody could specifically bind to the CLDN18.2 molecules expressed on the cells (Figure 7), and had no reaction with the human CLDN18.1 antigen which was extremely similar in structure.

[0326] Example 3, Affinity determination of anti-CLDN18.2 nanobodies (nanobody A and nanobody B)

[0327] This example relates to the affinity determination of nanobody A (CLDN18.2-VHH-319) and nanobody B (CLDN18.2-VHH-357).

[0328] Gradient-diluted FITC-labeled CLDN18.2-VHH-hFc antibodies were mixed with the same number of CLDN18.2-positive cells 293T-CLDN18.2 cells (target cells), respectively, incubated at room temperature for 10 min, then centrifuged to wash, resuspended the cells in FACS buffer for flow cytometry reading, and the average fluorescence intensity (MFI) obtained by flow cytometry analysis was used to calculate the equilibrium dissociation constant (KD) of CLDN18.2-VHH-hFc antibody binding to target cells to determine the strength of affinity. The smaller the KD value, the stronger the affinity.

[0329] Specific experimental steps:

[0330] 1) Resuspend the cells to a density of 2 x 10 6 cells / mL, take 100 μL and place in a 96-well V-bottom plate, centrifuge to discard the supernatant and collect the cells.

[0331] 2) Incubate 40 μL of different gradient-diluted FITC-CLDN18.2-VHH-hFc antibodies with the above cells at room temperature in the dark for 10 min.

[0332] 3) After incubation, wash twice with 200 μL FACS buffer, resuspend with 200 μL FACS buffer, and immediately detect by flow cytometry.

[0333] 4) Calculate the affinity

[0334] Calculation formula: 1 / (MFI-Con) = 1 / Fmax + (KD / Fmax)(1 / [CLDN18.2-scfv IgG Fc])

[0335] MFI-Con represents the relative average fluorescence intensity, which is the value of the average fluorescence intensity of the experimental group minus the background average fluorescence intensity; CLDN18.2-scfv IgG Fc represents the amount of antibody used (unit: ng).

[0336] According to the formula calculation method: taking the reciprocal of the amount of antibody used as the abscissa, and the reciprocal of the relative average fluorescence intensity as the ordinate, a standard curve is drawn, and a linear regression equation is obtained. According to the linear regression equation calculation, the intersection of the standard curve and the ordinate is 1 / Fmax, and KD is the slope of the straight line multiplied by Fmax.

[0337] According to the above calculation method, the affinity constant (KD) of recombinant CLDN18.2 VHH#319 antibody (i.e. nanobody CLDN18.2-VHH-319) is 42.2 nM, and the affinity constant (KD) of recombinant CLDN18.2 VHH#357 antibody (i.e. nanobody CLDN18.2-VHH-357) is 25.4 nM. The standard curve is shown in Figure 8, and the affinity constant (KD) is shown in Table 9.

[0338] Table 9, affinity constant (KD) of CLDN18.2-VHH-hFc to 293T-CLDN18.2 cells

[0339] Example 4, affinity detection of anti-CLDN18.2 nanobody (nanobody C)

[0340] This example relates to the affinity determination of nanobody C (CLDN18.2-VHH-223).

[0341] First, the above VHH-hIgG1Fc fusion recombinant protein (#223) was labeled using Pierce TM NHS-Fluorescein Antibody Labeling Kit (Thermo Scientific product, item number: 53029) to obtain FITC straight-labeled antibody FITC-CLDN18.2-VHH-hFc.

[0342] Gradient dilution of FITC-labeled CLDN18.2-VHH-hFc antibody (FITC-CLDN18.2-VHH-hFc) was mixed with the same number of CLDN18.2 positive cells 293T-CLDN18.2 cells (target cells) respectively, incubated at room temperature for 10 min, then centrifuged and resuspended in FACS buffer for flow cytometry analysis. The average fluorescence intensity (MFI) was obtained by flow cytometry analysis to calculate the equilibrium dissociation constant (KD) of CLDN18.2-VHH-hFc antibody binding to target cells to determine the strength of affinity. The smaller the KD value, the stronger the affinity. The specific experimental steps are as follows:

[0343] (1) Resuspend the cells to a density of 2 x 10 6 cells / mL, take 100 μL and centrifuge to discard the supernatant and collect the cells.

[0344] (2) Incubate 40 μL of different gradient dilutions of FITC-CLDN18.2-VHH-hFc antibody with the above cells at room temperature for 10 min.

[0345] (3) After incubation, wash twice with 200 μL FACS buffer, resuspend with 200 μL FACS buffer and immediately flow cytometry detection. Analyze the average fluorescence intensity (MFI).

[0346] (4) Calculate affinity

[0347] Calculation formula: 1 / (MFI-Con) = 1 / Fmax + (KD / Fmax)(1 / [CLDN18.2-scfv IgG Fc])

[0348] MFI-Con represents the relative average fluorescence intensity, which is the value of the average fluorescence intensity of the experimental group minus the background average fluorescence intensity; CLDN18.2-scfv IgG Fc represents the amount of antibody used (unit: ng).

[0349] According to the formula calculation method: take the reciprocal of the amount of antibody used as the abscissa, and the reciprocal of the relative average fluorescence intensity as the ordinate, draw a standard curve, and obtain a linear regression equation. According to the linear regression equation, the intersection of the standard curve and the ordinate is 1 / Fmax, and KD is the slope multiplied by Fmax.

[0350] According to the above calculation method, the affinity constant (KD) of recombinant CLDN18.2 VHH antibody (i.e. nanobody CLDN18.2-VHH-223) is 13.6 nM, the standard curve is shown in Figure 10, and the affinity constant (KD) is shown in Table 10.

[0351] Table 10, Affinity constant (KD) of CLDN18.2 VHH-hFc to 293T-CLDN18.2 cells

[0352] Example 5, Specific detection of anti-CLDN18.2 Nanobody (Nanobody C)

[0353] This example relates to the specific detection of Nanobody C (CLDN18.2-VHH-223).

[0354] The above VHH-hlgG1 Fc fusion recombinant protein (#223) was labeled using Pierce TM NHS-Fluorescein Antibody Labeling Kit to obtain FITC-labeled antibody FITC-CLDN18.2-VHH-hFc for flow detection of CLDN18.2 protein expression in different cell lines.

[0355] (1) CLDN18.2 negative cell lines: 293T cells and 293T-CLDN18.1 cells; CLDN18.2 positive cell lines: 293T-CLDN18.2 cells. The above cells were respectively resuscitated at 37°C and added to cell culture bottles, cultured at 37°C, 5% CO2, and subcultured twice for detection of anti-CLDN18.2 Nanobody (FITC-CLDN18.2-VHH-hFc).

[0356] (2) Adjust the concentration of each cell to 2x10 6 / mL, 200 μL / well into a 96-well plate, centrifuge at 1500 rpm for 5 min.

[0357] (3) Wash with FACS buffer and centrifuge.

[0358] (4) Add 20 μL of diluted FITC-CLDN18.2-VHH-hFc antibody (1:400) and mix, incubate at room temperature for 10 min.

[0359] (5) Add FACS buffer and centrifuge to remove the antibody, then add buffer to resuspend, and detect using a flow cytometer.

[0360] The flow results show that the FITC-labeled CLDN18.2-VHH-hFc recombinant antibody can specifically bind to the CLDN18.2 molecules expressed on the cells (Figure 11), and has no reaction with the human CLDN18.1 antigen which is extremely similar in structure.

[0361] The application has been described in detail above. For those skilled in the art, the application can be implemented within a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the application and without unnecessary experiments. Although specific examples are given in the application, it should be understood that further improvements can be made to the application. In summary, according to the principles of the application, the application is intended to include any changes, uses or improvements to the application, including changes made by conventional techniques known in the art, which depart from the scope disclosed in the application. Industrial applicability

[0362] The nanobodies (CLDN18.2-VHH-319, CLDN18.2-VHH-357 and CLDN18.2-VHH-223) of the application have high affinity and specificity. Not only have stronger antitumor activity, but also can efficiently target and kill tumor cells expressing CLDN18.2, and can avoid the toxicity and side effects caused by non-specific binding of therapeutic antibodies to human CLDN18.1 and other proteins during targeted diagnosis and treatment. The nanobodies of the application can be expressed and produced in prokaryotic cells, eukaryotic cells and any recombinant system, and can be made into prophylactic and therapeutic drugs for Claudin 18.2 target-related diseases, diagnostic drugs, detection products of Claudin 18.2 protein or in vivo imaging products of Claudin 18.2 protein, etc. As small molecular weight nanobodies, they have low immunogenicity, small molecular weight, strong tissue penetration, stable structure, short half-life, can be quickly cleared in vivo, reduce side effects, and are easy to recombinantly express, low in production cost, can be used alone or as a drug delivery system to carry related drugs, and have very broad prospects and important significance in the fields of drug application and clinical diagnosis, etc.

[0363] Cross-reference to related applications

[0364] The present application claims priority to the Chinese patent application No. 202410766891.5, filed on June 14, 2024, and entitled “Anti-CLDN18.2 Nanobodies and Uses Thereof”, the entire content of which is incorporated herein by reference.

[0365] The present application claims priority to the Chinese patent application No. 202410766888.3, filed on June 14, 2024, and entitled “Nanobodies Targeting CLDN18.2 and Methods of Making and Using Same”, the entire content of which is incorporated herein by reference.

Claims

1. A nanobody targeting CLDN18.2, characterized in that, The nanobody is any one of the following: A1) a nanobody A comprising a complementarity determining region CDR1 of amino acid sequence 26-32 of SEQ ID NO: 2, a complementarity determining region CDR2 of amino acid sequence 50-59 of SEQ ID NO: 2 and a complementarity determining region CDR3 of amino acid sequence 98-111 of SEQ ID NO: 2; A2) a nanobody B comprising a complementarity determining region CDR1 of amino acid sequence 26-32 of SEQ ID NO: 4, a complementarity determining region CDR2 of amino acid sequence 50-59 of SEQ ID NO: 4 and a complementarity determining region CDR3 of amino acid sequence 98-111 of SEQ ID NO: 4; A3) a nanobody C comprising a complementarity determining region CDR1 of amino acid sequence 26-33 of SEQ ID NO: 20, a complementarity determining region CDR2 of amino acid sequence 51-57 of SEQ ID NO: 20 and a complementarity determining region CDR3 of amino acid sequence 95-109 of SEQ ID NO:

20.

2. The Nanobody according to claim 1, characterized in that, The amino acid sequence of the nanobody A is any one of the following: B1) SEQ ID NO: 2, or an amino acid sequence having more than 80% identity to SEQ ID NO: 2, or an amino acid sequence having one or several amino acid substitution(s), deletion(s) or addition(s) compared to SEQ ID NO: 2; B2) an amino acid sequence resulting from the N-terminal and / or C-terminal linkage of a tag or a signal peptide to B1); The amino acid sequence of the nanobody B is any one of the following: B3) SEQ ID NO: 4, or an amino acid sequence having more than 80% identity to SEQ ID NO: 4, or an amino acid sequence having one or several amino acid substitution(s), deletion(s) or addition(s) compared to SEQ ID NO: 4; B4) an amino acid sequence resulting from the N-terminal and / or C-terminal linkage of a tag or a signal peptide to B3); The amino acid sequence of the nanobody C is any one of the following: B5) SEQ ID NO: 20, or an amino acid sequence having more than 80% identity to SEQ ID NO: 20, or an amino acid sequence having one or several amino acid substitution(s), deletion(s) or addition(s) compared to SEQ ID NO: 20; B6) an amino acid sequence resulting from the N-terminal and / or C-terminal linkage of a tag or a signal peptide to B5).

3. A biomaterial, characterized in that, The biomaterial is any one of the following: C1) a nucleic acid molecule encoding a nanobody according to claim 1 or 2; C2) an expression cassette comprising the nucleic acid molecule of C1); C3) a recombinant vector comprising the nucleic acid molecule of C1); C4) a recombinant microorganism comprising the nucleic acid molecule of C1); C5) a recombinant host cell comprising the nucleic acid molecule of C1).

4. The biomaterial of claim 3, wherein, The nucleic acid molecule is any one of the following: D1) a DNA molecule having a coding sequence or a nucleotide sequence of SEQ ID NO: 1; D2) a DNA molecule having a coding sequence or a nucleotide sequence of SEQ ID NO: 3; D3) a DNA molecule encoding a sequence or nucleotide sequence is SEQ ID NO: 19; D4) a DNA molecule having 75% or more identity to the nucleotide sequence defined by SEQ ID NO: 1, and encoding an amino acid sequence is the Nanobody of SEQ ID NO: 2; D5) a DNA molecule having 75% or more identity to the nucleotide sequence defined by SEQ ID NO: 3, and encoding an amino acid sequence is the Nanobody of SEQ ID NO: 4; D6) a DNA molecule having 75% or more identity to the nucleotide sequence defined by SEQ ID NO: 19, and encoding an amino acid sequence is the Nanobody of SEQ ID NO:

20.

5. Use of the Nanobody of claim 1 or 2 or the biological material of claim 3 or 4 in any of the following: E1) use in the manufacture of a product for preventing or treating a tumor; E2) use in the manufacture of a product for preventing or treating a Claudin 18.2 target related disease; E3) use in the manufacture of a product for screening, diagnosing or aiding diagnosis of a Claudin 18.2 target related disease; E4) use in the manufacture of a product for detecting a Claudin 18.2 protein or a cell expressing Claudin 18.2; E5) use in the manufacture of a product for binding to a Claudin 18.2 protein; E6) use in the manufacture of a product for mediating specific recognition of a tumor expressing a Claudin 18.2 antigen by a drug; E7) use in the manufacture of a product for in vivo imaging of a Claudin 18.2 protein; E8) use in the manufacture of a bispecific antibody, a multispecific antibody, an antibody drug conjugate or a CAR cell targeting Claudin 18.

2.

6. Use according to claim 5, characterized in that, The tumor or the Claudin 18.2 target related disease is a Claudin 18.2 positive tumor.

7. A bispecific antibody or a multispecific antibody, characterized in that The bispecific antibody or the multispecific antibody comprises the Nanobody A or the Nanobody B as described in claim 1 or 2.

8. An antibody conjugate comprising an antibody moiety and a conjugate moiety, characterized in that, The antibody moiety comprises the Nanobody A or the Nanobody B as described in claim 1 or 2.

9. A pharmaceutical composition for preventing or treating a disease associated with Claudin 18.2 target, characterized in that, The pharmaceutical composition comprises the Nanobody of claim 1 or 2, the biological material of claim 3 or 4, the bispecific antibody or the multispecific antibody of claim 7 or the antibody conjugate of claim 8, and one or more pharmaceutically acceptable carriers.

10. A kit characterized in that, The kit contains the Nanobody of claim 1 or 2 or the bispecific antibody or the multispecific antibody of claim 7.

11. A method of detecting Claudin 18.2 protein characterized in that, The method comprises detecting a Claudin 18.2 protein using the Nanobody of claim 1 or 2, the bispecific antibody or the multispecific antibody of claim 7 or the kit of claim 10.

12. A method of producing a Nanobody according to claim 1 or 2, characterized in that, The method of preparation comprises expressing the Nanobody of claim 1 or 2 in a host cell, and recovering or isolating the Nanobody.

13. A method for preventing or treating a Claudin 18.2 target-related disease, characterized by, The method comprises administering the nanobody of claim 1 or 2, the biomaterial of claim 3 or 4, the bispecific antibody or multispecific antibody of claim 7, the antibody conjugate of claim 8, or the pharmaceutical composition of claim 9 to a subject suffering from a Claudin 18.2 target-related disease.

14. A method for diagnosing or aiding in the diagnosis of a disease related to Claudin 18.2 target, characterized in that, The method comprises: obtaining a sample from a subject, and then detecting the presence or content of Claudin 18.2 protein in the sample using the nanobody of claim 1 or 2, the bispecific antibody or multispecific antibody of claim 7, the antibody conjugate of claim 8, or the kit of claim 10, and making a diagnosis or auxiliary diagnosis of a Claudin 18.2 target-related disease based on the detection result.

15. A method of screening for a disease related to Claudin 18.2 target, characterized in that, The method comprises: obtaining a sample from a subject, and then detecting the presence or content of Claudin 18.2 protein in the sample using the nanobody of claim 1 or 2, the bispecific antibody or multispecific antibody of claim 7, the antibody conjugate of claim 8, or the kit of claim 10, and making a diagnosis or auxiliary diagnosis of a Claudin 18.2 target-related disease based on the detection result.

16. The method according to any one of claims 13-15, characterized by, The Claudin 18.2 target-related disease is a Claudin 18.2-positive tumor.

17. Use according to claim 5 or 6 or method according to claim 16, characterized in that, The Claudin 18.2-positive tumor is a Claudin 18.2-positive solid tumor.

18. Use according to claim 5 or 6 or method according to claim 16, characterized in that, The Claudin 18.2-positive tumor is a Claudin 18.2-positive cancer or a Claudin 18.2-positive mesothelioma.

19. The method of claim 18, wherein, The cancer is selected from gastric cancer, esophageal cancer, pancreatic cancer, colorectal adenocarcinoma, lung cancer, liver cancer, kidney cancer, ovarian cancer, bronchial cancer, breast cancer, bladder cancer, head and neck cancer, gallbladder cancer, bile duct cancer, and gastroesophageal junction adenocarcinoma.

20. The method of claim 18 or 19, wherein, The cancer includes early-stage cancer, mid-stage cancer, mid-advanced-stage cancer, advanced-stage cancer, recurrent cancer, and metastatic cancer. The cancer includes early-stage cancer, mid-stage cancer, mid-advanced-stage cancer, advanced-stage cancer, recurrent cancer, and metastatic cancer.

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