Camel nano antibody targeting CDH17, binding molecule and application thereof

Abstract

The invention relates to a CDH17-targeted camel nano antibody, a binding molecule and application of the CDH17-targeted camel nano antibody and the binding molecule. The invention provides a CDH17 binding molecule, the CDH17 binding molecule comprises an anti-CDH17 nano antibody or an antigen binding fragment thereof, a complementary determining region CDR of the anti-CDH17 nano antibody comprises CDR1, CDR2 and CDR3, the sequence of the CDR1 is shown as SEQ ID NO: 1, the sequence of the CDR2 is shown as SEQ ID NO: 2, and the sequence of the CDR3 is shown as SEQ ID NO: 3.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to a camel-like nanobody targeting CDH17, a binding molecule and uses thereof. Background Art

[0002] Colorectal cancer (CRC) is a widespread global health problem and the third most common cancer, accounting for approximately 8% of all cancer cases worldwide. It is also the second leading cause of cancer-related deaths. Although early detection significantly improves prognosis, early-stage CRC often presents with nonspecific symptoms or remains asymptomatic, resulting in a low rate of early diagnosis. Surgery remains the mainstay of treatment for CRC.

[0003] Cadherin-17 (CDH17), also known as liver-intestinal cadherin, belongs to the cadherin family of calcium-dependent cell adhesion molecules. CDH17 may play a role in the morphological organization of the liver and intestines and participate in the transport of intestinal peptides. The total length of CDH17 is 832 amino acids, of which 23-787 are the extracellular region, which is divided into seven domains. It is expressed in the gastrointestinal tract and pancreatic duct. It is not detected in the kidney, lung, liver, brain, adrenal gland and skin. And the expression level is not related to the tumor phenotype. For example, CN102483414B discloses a composition and method for diagnosing, treating and / or preventing cancer characterized by overexpression of CDH17 based on CDH17 detection or the use of CDH17 as a therapeutic intervention or preventive intervention target, which achieves improvements in the early diagnosis of liver cancer, improves the sensitivity and specificity of diagnosis, and significantly reduces the size of liver tumors in treatment.

[0004] CDH17 is a safe and promising TAA target for T cell engagers. The CDH17 protein has a complex structure, comprising seven domains. Different epitopes may have a greater impact on TCE killing, and no references have systematically studied the effects of antibodies with different domains on TCE activity. Summary of the Invention

[0005] The present invention utilizes CDH17 protein to immunize alpacas (alpaca) to obtain a high-quality single-domain antibody gene library. The antibody gene library is then screened using phage display technology to obtain CDH17-specific single-domain antibody genes. This gene is then transferred into mammalian cells to obtain an antibody strain that can be efficiently expressed in mammalian cells and has high specificity. Nanobodies with high affinity, high specificity, and high functional activity are then identified by methods such as ELISA. The antibodies or their antigen-binding fragments have good safety and targeting properties and can specifically bind to the extracellular domain of human CDH17.

[0006] Herein, "CDH17 binding molecules" are proteins that specifically bind to CDH17, including but not limited to antibodies, heavy chain antibodies, nanobodies, or antigen-binding fragments thereof.

[0007] As used herein, the term "antibody" includes monoclonal antibodies, antibody compositions with multiple epitope specificities, multispecific antibodies (e.g., bispecific antibodies), diabodies, and single-chain molecules, as well as antibody fragments, particularly antigen-binding fragments, e.g., VHH, ScFv, Fab, F(ab')2, and Fv. As used herein, "antibody" and "immunoglobulin" are used interchangeably.

[0008] As used herein, the terms "single-domain antibody," "anti-CDH17 single-domain antibody," "heavy chain variable region domain of a heavy chain antibody," and "VHH" are used interchangeably to refer to single-domain antibodies that specifically recognize and bind to CDH17. A single-domain antibody is the variable region of a heavy chain antibody, the smallest functional antigen-binding fragment, and typically contains three CDRs and four FRs.

[0009] The present invention provides a CDH17 binding molecule comprising an anti-CDH17 nanobody or an antigen-binding fragment thereof, wherein the complementarity determining region (CDR) of the anti-CDH17 nanobody comprises CDR1, CDR2 and CDR3, the sequence of CDR1 is shown in SEQ ID NO: 1, the sequence of CDR2 is shown in SEQ ID NO: 2, and the sequence of CDR3 is shown in SEQ ID NO: 3.

[0010] Antibody variable region annotation schemes include: Chothia, Kabat, IMGT, and Contact. The IMGT annotation scheme is used as an example herein.

[0011] In some embodiments, the heavy chain variable region sequence of the anti-CDH17 Nanobody is as shown in SEQ ID NO:4.

[0012] In some embodiments, the CDH17 binding molecule comprises one, two or more anti-CDH17 nanobodies or antigen-binding fragments thereof; the CDH17 binding molecule is a monovalent, multivalent or multispecific nanobody or single domain antibody.

[0013] In one or more embodiments, the CDH17 binding molecule is a bispecific antibody or antigen-binding fragment thereof. In one or more embodiments, the CDH17 binding molecule is an antibody or antigen-binding fragment thereof having a first specificity for CDH17 and a second specificity. In one or more embodiments, the second antigen specificity is for a tumor-associated antigen or a pathogen antigen.

[0014] In one or more embodiments, the CDH17 binding molecule is an antibody or antigen-binding fragment thereof linked to one or more conjugates. In one or more embodiments, the conjugate is a chemotherapeutic agent, a toxin, a radioisotope, a luminescent label, or an enzyme substrate label.

[0015] In some embodiments, the CDH17 binding molecule is a T cell engager.

[0016] In some embodiments, the multivalent binding molecule or multispecific binding molecule is connected to one or more anti-CDH17 Nanobodies or antigen-binding fragments thereof via a linker. The linker consists of 1-15 amino acids selected from G and S.

[0017] In some embodiments, the Nanobody is a camelid heavy chain antibody or a cartilaginous fish heavy chain antibody.

[0018] In some embodiments, the Nanobody further comprises a heavy chain constant region, such as a constant region of a camelid heavy chain antibody or a constant region of a human antibody. In some embodiments, the heavy chain constant region comprises CH2 and CH3. In some embodiments, the CH2 and CH3 are the CH2 and CH3 of a human IgG Fc, such as the CH2 and CH3 of IgG1. Preferably, the heavy chain constant region is as shown in SEQ ID NO: 5. In some embodiments, the heavy chain constant region is the constant region of a cartilaginous fish heavy chain antibody, comprising CH1, CH2, CH3, CH4 and CH5.

[0019] In some embodiments, the CDH17 binding molecule according to any embodiment of the present invention is a chimeric antibody or a fully human antibody; preferably, it is a fully human antibody.

[0020] In some embodiments, the binding molecule is an antibody comprising the anti-CDH17 Nanobody as a heavy chain variable domain.

[0021] In some embodiments, the binding molecule further comprises a light chain variable domain, a heavy chain constant domain, and a light chain constant domain.

[0022] In some embodiments, the antigen binding fragment is selected from Fab, F(ab')2, Fv, scFv.

[0023] The present invention also provides a nucleic acid molecule having a sequence selected from any one of the following:

[0024] (1) the coding sequence of the CDH17 binding molecule described in any embodiment of the present invention;

[0025] (2) The complementary sequence of (1).

[0026] The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The present invention also encompasses degenerate variants of the polynucleotide sequence encoding the fusion protein, i.e., nucleotide sequences encoding the same amino acid sequence but differing in nucleotide sequence. RNA may be mRNA that expresses the binding molecule in vivo and / or in vitro.

[0027] The present invention also provides a nucleic acid construct comprising the nucleic acid molecule described herein.

[0028] In some embodiments, the nucleic acid construct is a cloning vector, an expression vector, or an integration vector.

[0029] The present invention also provides a phage comprising the CDH17 binding molecule described in any embodiment herein.

[0030] In some embodiments, the CDH17 binding molecule is displayed on the surface of the bacteriophage.

[0031] The present invention also provides a host cell selected from:

[0032] (1) expressing and / or secreting the CDH17 binding molecule described in any embodiment of the present invention;

[0033] (2) comprising a nucleic acid molecule as described herein; and / or

[0034] (3) comprising a nucleic acid construct as described herein.

[0035] The host cell suitable for introducing the nucleic acid construct described herein can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Examples of mammalian cells include immune cells, preferably immune effector cells. "Immune effector cells" are immune cells that can perform immune effector functions, including: T cells, NK cells, peripheral blood mononuclear cells (PBMCs), neutrophils, eosinophils, hematopoietic stem cells. T cells suitable for the present invention can be various types of T cells from various sources.

[0036] The present invention also provides fusion proteins comprising the anti-CDH17 binding molecules described herein and other polypeptides.

[0037] In some embodiments, the additional polypeptide is located at the N-terminus and / or C-terminus of the binding molecule.

[0038] In some embodiments, the additional polypeptides include polypeptides that localize the binding molecule to a different organelle, tags for purification or tags for immune response, transmembrane proteins or transmembrane regions thereof, chimeric antigen receptors or components thereof.

[0039] In some embodiments, the fusion protein is a chimeric antigen receptor, whose antigen binding domain comprises the CDH17 binding molecule. The chimeric antigen receptor comprises: an optional signal peptide sequence, a CDH17 binding molecule comprising a CDH17 single domain antibody, a hinge region, a transmembrane region, and an intracellular region.

[0040] The present invention also provides a method for producing a CDH17 binding molecule according to any embodiment of the present invention, comprising:

[0041] Culturing a host cell as described herein under conditions suitable for the production of a CDH17 binding molecule (e.g., a Nanobody or antigen-binding fragment thereof, a monovalent or multivalent Nanobody or single domain antibody, or a multispecific Nanobody or single domain antibody), and optionally purifying the CDH17 binding molecule from the culture, or

[0042] A nucleic acid molecule encoding a CDH17 binding molecule according to any embodiment of the present invention is incubated under conditions suitable for translation of DNA or RNA in a cell-free system (eg, solution).

[0043] The present invention also provides a pharmaceutical composition comprising the CDH17 binding molecule, nucleic acid molecule, nucleic acid construct, phage or host cell described in any embodiment herein, and a pharmaceutically acceptable excipient.

[0044] In some embodiments, the pharmaceutical composition is used to treat a disease or condition associated with CDH17 expression, such as cancer.

[0045] The present invention also provides use of the CDH17-binding molecule, nucleic acid molecule, nucleic acid construct, phage or host cell described in any embodiment herein in preparing immune cells (eg, T cells).

[0046] The present invention also provides use of the CDH17-binding molecule, nucleic acid molecule, nucleic acid construct, phage or host cell described in any embodiment herein in the preparation of a medicament for preventing or treating a disease or condition associated with CDH17 expression.

[0047] In some embodiments, the disease or condition is cancer, such as colorectal cancer (CRC), gastric cancer (GC), pancreatic cancer (PAC), esophageal cancer, or a neuroendocrine cancer.

[0048] The present invention also provides a method for treating or preventing a disease or condition associated with CDH17 expression, comprising administering to a patient in need thereof a therapeutically effective amount of the CDH17 binding molecule, nucleic acid molecule, nucleic acid construct or host cell according to any embodiment of the present invention, or the pharmaceutical composition according to any embodiment of the present invention.

[0049] The present invention also provides a kit for detecting CDH17, which is used, for example, to evaluate the effect of drug treatment or diagnose cancer. The kit comprises the CDH17 binding molecule, nucleic acid molecule, nucleic acid construct or host cell described in any embodiment of the present invention.

[0050] In some embodiments, the kit further comprises a reagent for detecting the binding of CDH17 to the CDH17 binding molecule, for example, a reagent for detecting the binding by enzyme-linked immunosorbent assay.

[0051] In some embodiments, the binding detection reagent is a detectable label that can be linked to the CDH17 binding molecule, such as biotin. The detectable label is linked to the CDH17 binding molecule or exists separately in the kit.

[0052] The present invention also provides a non-diagnostic method for detecting the presence of CDH17 in a sample, the method comprising: incubating the sample with a CDH17-binding molecule as described in any embodiment herein, and detecting binding of CDH17 to the CDH17-binding molecule, thereby determining the presence of CDH17 in the sample. The detection is performed by enzyme-linked immunosorbent assay (ELISA).

[0053] The present invention also provides use of the CDH17-binding molecule described in any embodiment herein in preparing a kit for detecting CDH17 in a sample, evaluating the effect of drug treatment, or diagnosing cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0055] Figure 1 :Detection of the titer of alpaca antiserum against CDH17 protein.

[0056] Figure 2 : ELISA binding curve of nanobody CT008-161 and human CDH17 protein.

[0057] Figure 3 : Binding detection of nanoantibody CT008-161 and COLO205 tumor cell line.

[0058] Figure 4 : Binding detection of nanoantibody CT008-161 and SNU16 tumor cell line. DETAILED DESCRIPTION

[0059] The present invention will be described below by way of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and materials used in the examples are, unless otherwise stated, conventional materials and methods in the art.

[0060] Example 1, Alpaca immunization

[0061] 1.1 Immunogen Preparation:

[0062] According to the CDH17 protein sequence query on NCBI, Human CDH17 Protein, HisTag protein (CA7-H52H3) from ACRO was purchased as the immune protein. The protein was identified and the electrophoresis bands were correct with a purity of >90%.

[0063] 1.2 Alpaca Immunity:

[0064] For the first immunization, 400 μg of antigen (Human CDH17 Protein, His Tag) was mixed with an adjuvant (GERBU FAMA) and injected subcutaneously at four sites on the back of alpacas, with a volume of 1 mL per site. For the second to sixth immunizations, 200 μg of antigen was injected subcutaneously at four sites on the back of alpacas, with a volume of 1 mL per site. Each immunization was administered every two weeks.

[0065] 1.3 Immune serum titer detection:

[0066] Human CDH17 Protein, His Tag antigen was coated overnight at 4°C. After blocking and washing, the serum was added to the ELISA plate for incubation, and then anti-llama IgG HRP (Abcam) antibody was used for incubation. After washing, TMB colorimetric solution was added for color development, and the reaction was terminated with 2M HCl. The absorbance at OD450 nm was then measured using a microplate reader. The experimental results are shown in Figure 2. Figure 1 As shown, the titer of alpaca reached a high level (>128,000) after 6 immunizations.

[0067] Example 2: Construction and screening of nanoantibody immune libraries targeting antigens

[0068] (1) After the sixth immunization, 100 mL of peripheral blood lymphocytes were extracted from the alpaca and total RNA was extracted. RNA extraction was performed according to the instructions for the TAKARA RNAiso reagent.

[0069] (2) Using RNA as template and oligo dT as primer, synthesize the first strand of cDNA according to the instructions of TAKARA reverse transcriptase.

[0070] (3) Using PrimeSTAR high-fidelity DNA polymerase, nested PCR was performed to obtain the variable region encoding gene of the heavy chain antibody. Nested PCR was used to amplify the variable region fragment of the heavy chain antibody:

[0071] First round of PCR:

[0072] Upstream primer: GTCCTGGCTGCTCTTCTACAAGGC (SEQ ID NO: 6)

[0073] Downstream primer: GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO: 7)

[0074] The fragment between the heavy chain antibody guide peptide and antibody CH2 was amplified, annealed at 55°C for 30 cycles; a DNA fragment of approximately 600 bp was recovered and used as a template for the second round of PCR.

[0075] Second round of PCR:

[0076] Upstream primer: GATGTGCAGCTGCAGGAGTCTGGRGGAGG (SEQ ID NO: 8)

[0077] Downstream primer: GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT (SEQ ID NO: 9)

[0078] The fragments between the heavy chain antibody FR1 region and the long and short hinge regions (long fragments and short fragments) were amplified, annealed at 55°C for 30 cycles, and the target fragment was recovered. The results showed that the size of the fragment was approximately 500 bp, that is, the electrophoresis band of the nanobody gene was approximately 500 bp.

[0079] (4) Phagemid pME207 and the PCR amplification product were double-digested with Sfi I and Not I (NEB), respectively. After recovery and quantification, the two fragments were ligated with T4 DNA ligase (TaKaRa) at a molar ratio of 1:3 at 16°C overnight.

[0080] (5) After ethanol precipitation, the ligation product was dissolved in 100 μL sterile water and electroporated into Escherichia coli TG1 ten times. Take 100 μL of the bacterial solution after electroporation and culture, dilute it in multiple proportions, spread it on the ampicillin LB culture plate, calculate the storage capacity, and spread the rest on the ampicillin 2×YT culture plate, and culture it upside down at 37°C for 13 to 16 hours. Use 10 mL of 2×YT culture medium to scrape the bacterial moss on the culture plate, add glycerol to a final concentration of 25%, divide it into aliquots, and store it at -80°C for later use. The storage capacity is 2.89×10 10To detect the insertion rate of the library, 48 clones were randomly selected for colony PCR, and the results showed that the insertion rate had reached more than 90%.

[0081] (6) Based on the calculated reservoir capacity, 10 times the reservoir capacity of live cells was inoculated into 200 mL of 2×YT (containing 2% glucose and 100 μg / mL ampicillin) and cultured at 37°C, 200 rpm until the OD600 reached 0.5. Helper phage was added at a multiplicity of infection of 20:1. The cells were allowed to stand at 37°C for 30 min and then incubated at 37°C, 200 rpm for 30 min. The culture was centrifuged and the precipitate was resuspended in 200 mL of 2×YT (containing 100 μg / mL ampicillin and 50 μg / mL kanamycin). After overnight incubation at 37°C and 250 r / min, the supernatant was centrifuged at 8000 rpm, 5×PEG / NaCl solution was added, the mixture was placed on ice for 60 min, and centrifuged at 8000 rpm for 30 min. The precipitate was resuspended in 5 mL of PBS to obtain an anti-CDH17 single-domain heavy chain antibody (VHH) immune library. 10 μL was taken for titer determination, and the rest was aliquoted and stored at -80°C for later use.

[0082] (7) Using Human CDH17 Protein, His Tag protein (CA7-H52H3), CDH17 was coated on the ELISA plate at 1 μg / mL, 100 μL per well, and placed at 4°C overnight. A negative control was also set up. The next day, 200 μL of 3% BSA was added to each of the five wells and blocked at room temperature for 2 hours. After 2 hours, the plate was washed three times with PBST (PBS containing 0.05% Tween 20). After washing the plate, 100 μL of phage (2-3×10 11 The cells were then plated with a tfu-immunized alpaca nanobody phage display gene library) and allowed to react at room temperature for 1.5 hours. The supernatant after negative screening was then transferred to the target antigen-coated wells and allowed to react at room temperature for 1.5 hours. The cells were washed 12 times with PBST (PBS containing 0.05% Tween 20) to remove unbound phage. Phages specifically bound to CDH17 were dissociated with Glycine (SIGMA). The eluted phages were neutralized with Tris (Invitrogen, 1M, pH 8.0) and infected with TG1 in the logarithmic phase. After propagation and amplification, the next round of "adsorption-elution" was performed. Finally, the eluted phages were infected with TG1, and IPTG (Thermo) was used to induce TG1 to express nanobodies. ELISA plates were coated with CDH17 protein, and the supernatant was taken for ELISA detection. Clones with OD450>0.5 were selected for sequencing.

[0083] (8) After sequence analysis, clone CT008-161 that binds to CDH17 protein was obtained.

[0084] Example 3, Expression and purification of candidate antibodies

[0085] The nanobody CT008-161 was constructed into the pCDNA3.4-IgG1 vector and then TM The protein was expressed using the ThermoFisher expression system. After one week of expression, the supernatant was collected and purified using Protein A (GE) to obtain VHH linked to an IgG1 Fc construct. Protein concentration was then determined using Nanodrop and purity was determined using HPLC. The resulting protein purity and yield met the requirements of subsequent experiments.

[0086] The amino acid sequence of CT008-161 is shown below, with the CDR regions underlined:

[0087] QVQLVESGGGSVQAGGSLRLSCTAS GPTVSGAV MGWFRQAPGKERVLITA LS WSGART YYADSVKGRFTISRDNAKNTVHLQMSGLKSEDTAVYYC AAVSSWGRA ASPDGFHY WGQGTLVTVSS (SEQ ID NO: 4)

[0088] Example 4, protein level affinity detection

[0089] Surface plasmon resonance (SPR) was used to determine the binding kinetics and affinity of heavy chain antibodies to human CDH17.His antigen. The purified antibody was passed through a sensor chip pre-fixed with protein A, and the antibody was captured by protein A. Then, 5 different concentrations of CDH17.His protein were used as the mobile phase, with the association time and dissociation time being 30 min and 60 min, respectively. Biacore Evaluation Software 2.0 (GE) was used to analyze the association rate (kon), dissociation rate (koff), and equilibrium constant (KD). BMK-2 (CDH17 nanobody in WO2019 / 210155A1) that binds to CDH17 protein was selected as a positive control, and the results are shown in Table 1.

[0090] Table 1: Protein level affinity detection

[0091] Antibody No. <![CDATA[K on (1 / Ms)]]> <![CDATA[K off (1 / s)]]> KD(M) CT008-161 3.27E+04 2.29E-03 7.00E-08 BMK2 1.31E+05 1.84E-04 1.40E-09

[0092] Example 4, ELISA detection of nanobody binding activity

[0093] Protein level affinity detection: Human CDH17 Protein was coated on an enzyme-labeled plate at 1 μg / mL, 100 μL per well, and placed at 4°C overnight. Blocked with 3% BSA, incubated at 37°C for 2 hours, and washed 3 times with PBST (PBS containing 0.05% Tween 20) after 2 hours. Nanobodies were diluted in DPBS, added at a volume of 100 μL / well, and incubated at room temperature for 1 hour. Secondary antibody Anti-Human IgG-Fc Fragment, HRP conjugated (Bethyl Laboratories, A80-104P) was detected in DPBS at 100 μL / well. Incubate at room temperature for 0.5 hours, wash, add TMB colorimetric solution for color development, terminate the reaction with 2M HCl, and then use an enzyme-labeled instrument to detect the absorbance at OD450nM. BMK-2 (CDH17 nanobody in WO2019 / 210155A1) and blank were used as controls, and the results are as follows. Figure 2 shown.

[0094] Cell-level affinity assay: COLO205 cells or SNU16 cells were plated in 96-well plates, with 5×10 cells per well. 5 Cells were then added with gradiently diluted antibodies to the cells. After incubation for half an hour, the secondary antibody anti-human IgG PE (Jackson Immuno Research, Code: 109-117-008, Lot: 145501) was added and incubated. The cells were then detected using a CytoFLEX flow cytometer. The results are shown in Figure 2. Figure 3 and 4 shown.

[0095] It can be seen that the nanobody CT008-161 has good binding activity with CDH17 protein or tumor cells expressing CDH17.

[0096] Part of this article

[0097]

Claims

1. A CDH17 binding molecule comprising an anti-CDH17 nanobody or an antigen-binding fragment thereof, wherein the complementarity determining region (CDR) of the anti-CDH17 nanobody comprises CDR1, CDR2 and CDR3, wherein the sequence of CDR1 is shown in SEQ ID NO: 1, the sequence of CDR2 is shown in SEQ ID NO: 2, and the sequence of CDR3 is shown in SEQ ID NO:

3.

2. The CDH17 binding molecule according to claim 1, wherein The heavy chain variable region sequence of the anti-CDH17 nanobody is shown in SEQ ID NO: 4, and / or the CDH17 binding molecule comprises one, two or more anti-CDH17 nanobodies or antigen-binding fragments thereof, and the CDH17 binding molecule is a monovalent, multivalent or multispecific nanobody or single domain antibody.

3. The CDH17 binding molecule according to claim 1 or 2, wherein Said Nanobody further comprises a heavy chain constant region.

4. A fusion protein, characterized in that Comprising the CDH17 binding molecule according to any one of claims 1 to 3 and other polypeptides; preferably, the fusion protein is a chimeric antigen receptor.

5. A nucleic acid molecule, characterized in that A coding sequence of the CDH17 binding molecule according to any one of claims 1 to 3 or the fusion protein according to claim 4.

6. A nucleic acid construct, characterized in that Comprising the nucleic acid molecule according to claim 5, preferably, the nucleic acid construct is a cloning vector, an expression vector or an integration vector.

7. A host cell selected from: (1) expressing the CDH17 binding molecule according to any one of claims 1 to 3 or the fusion protein according to claim 4; (2) comprising the nucleic acid molecule according to claim 5; and / or (3) comprising the nucleic acid construct according to claim 6, Preferably, the host cell is an immune effector cell, more preferably a T cell.

8. A method for producing the CDH17-binding molecule of any one of claims 1 to 3, comprising: The host cell of claim 7 is cultured under conditions suitable for producing the CDH17 binding molecule, and optionally the CDH17 binding molecule is purified from the culture, or the nucleic acid molecule encoding the nucleic acid of claim 5 is incubated under conditions suitable for translation of DNA or RNA in a cell-free system.

9. Use of the CDH17 binding molecule according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the nucleic acid construct according to claim 5, or the host cell according to claim 6 in preparing an immune cell, preferably, the immune cell is a T cell.

10. A kit for detecting CDH17, characterized in that: The kit comprises the CDH17 binding molecule according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, or the nucleic acid construct according to claim 5.

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