Cd117 binding molecules and uses thereof

Abstract

This invention relates to CDH17 binding molecules and their applications. The 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, 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.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, specifically to anti-CDH17 binding molecules and their uses. Background Technology

[0002] Colorectal cancer (CRC) is a prevalent global health problem, 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. While early detection significantly improves prognosis, early-stage CRC often presents with nonspecific symptoms or remains asymptomatic, resulting in low early diagnosis rates. Surgical intervention remains the primary treatment for CRC.

[0003] cadherin-17 (CDH17), also known as hepato-gut cadherin, belongs to the calcium-dependent cell adhesion molecule cadherin family. CDH17 may play a role in the morphological organization of the liver and intestine and is involved in the transport of intestinal peptides. CDH17 has a total length of 832 amino acids, of which 23-787 are extracellular regions divided into seven domains. It is expressed in the gastrointestinal tract and pancreatic duct. It has not been detected in the kidney, lung, liver, brain, adrenal gland, and skin. Moreover, its expression level is not related to tumor phenotype. For example, CN102483414B discloses compositions and methods for diagnosing, treating, and / or preventing cancers characterized by CDH17 overexpression based on CDH17 detection or the use of CDH17 as a target for therapeutic or prophylactic interventions, achieving improvements in the early diagnosis of liver cancer, increasing diagnostic sensitivity and specificity, and significantly reducing liver tumor size in treatment.

[0004] CDH17 is a safe and promising target for TAAs in T-cell adjuvants. The CDH17 protein has a complex structure containing seven domains, and different epitopes may have a greater impact on TCE killing. However, no literature has systematically studied the effects of antibodies with different domains on TCE activity. Summary of the Invention

[0005] This invention utilizes CDH17 protein immunization of camels to obtain a high-quality single-domain antibody gene library. Then, phage display technology is used to screen the antibody gene library, thereby obtaining CDH17-specific single-domain antibody genes. These genes are then transformed into mammalian cells, resulting in antibody strains that can be efficiently expressed in mammalian cells and exhibit high specificity. High-affinity, high-specificity, and high-functional activity nanobodies are then identified using methods such as ELISA. The antibodies or their antigen-binding fragments possess good safety and targeting properties, specifically binding to the extracellular domain of human CDH17.

[0006] In this article, "CDH17 binding molecule" refers to a protein that specifically binds to CDH17, including but not limited to antibodies, heavy chain antibodies, nanobodies, or their antigen-binding fragments.

[0007] In this document, the term "antibody" includes monoclonal antibodies, antibody compositions with multi-epitope specificity, multi-specific antibodies (e.g., bispecific antibodies), biantibodies and single-chain molecules, and antibody fragments, especially antigen-binding fragments, such as VHH, ScFv, Fab, F(ab')2, and Fv. In this document, "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 and all 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 and is the smallest functional antigen-binding fragment, typically containing 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, 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.

[0010] Antibody variable region annotation schemes include: Chothia, Kabat, IMGT, and Contact. This article uses the IMGT annotation scheme as an example.

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

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

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

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

[0015] In some implementations, the CDH17 binding molecule is a T-cell adaptor.

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

[0017] In some embodiments, the nanobody is a camel family 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 the constant region of a camelid heavy chain antibody or a human antibody. In some embodiments, the heavy chain constant region comprises CH2 and CH3. In some embodiments, CH2 and CH3 are CH2 and CH3 of human IgG Fc, such as 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 described in any embodiment of the present invention is a chimeric antibody or a fully human antibody; preferably a fully human antibody.

[0020] In some embodiments, the binding molecule is an antibody containing 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 implementations, the antigen-binding fragment is selected from Fab, F(ab')2, Fv, and scFv.

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

[0024] (1) The coding sequence of the CDH17 binding molecule described in any of the embodiments herein;

[0025] (2)(1) complementary sequences.

[0026] The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. This invention also includes degenerate variants of polynucleotide sequences encoding fusion proteins, i.e., nucleotide sequences encoding the same amino acid sequence but with different nucleotide sequences. RNA can 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 molecules described herein.

[0028] In some implementations, the nucleic acid construct is a cloning vector, expression vector, or 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 phage.

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

[0032] (1) Expressing and / or secreting the CDH17-binding molecule as described in any of the embodiments herein;

[0033] (2) Contains the nucleic acid molecules described herein; and / or

[0034] (3) Includes the nucleic acid constructs described in this article.

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

[0036] The present invention also provides fusion proteins containing the anti-CDH17 binding molecule described herein and other peptides.

[0037] In some embodiments, other peptides are located at the N-terminus and / or C-terminus of the binding molecule.

[0038] In some embodiments, other peptides include peptides that target binding molecules to different organelles, tags for purification or for immune responses, transmembrane proteins or their transmembrane regions, chimeric antigen receptors or components thereof.

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

[0040] The present invention also provides a method for generating a CDH17-binding molecule according to any embodiment herein, comprising:

[0041] The host cells described herein are cultured under conditions suitable for generating CDH17-binding molecules (e.g., nanobodies or their antigen-binding fragments, monovalent or multivalent nanobodies or single-domain antibodies, or multispecific nanobodies or single-domain antibodies), and optionally, the CDH17-binding molecules are purified from the culture.

[0042] Nucleic acid molecules encoding CDH17-binding molecules of any embodiment described herein are incubated under conditions suitable for the translation of DNA or RNA in a non-cellular system (e.g., solution).

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

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

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

[0046] The present invention also provides the use of the CDH17 binding molecule, nucleic acid molecule, nucleic acid construct, bacteriophage or host cell described in any embodiment herein in the preparation of a medicament for the prevention or treatment of diseases or conditions associated with CDH17 expression.

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

[0048] The present invention also provides a method for treating or preventing diseases or conditions related to CDH17 expression, the method comprising administering to a patient in need a therapeutically effective amount of the CDH17 binding molecule, nucleic acid molecule, nucleic acid construct or host cell as described in any embodiment of the present invention, or a pharmaceutical composition as described in any embodiment of the present invention.

[0049] The present invention also provides a kit for detecting CDH17, for example, to evaluate the efficacy of drug treatment or to diagnose cancer, said kit comprising a CDH17 binding molecule, nucleic acid molecule, nucleic acid construct or host cell as described in any embodiment herein.

[0050] In some embodiments, the kit further includes reagents for detecting the binding of CDH17 to the CDH17-binding molecule. For example, reagents for detecting the binding by enzyme-linked immunosorbent assay (ELISA).

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

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

[0053] The present invention also provides the use of the CDH17 binding molecule described in any embodiment herein in the preparation of kits for detecting CDH17 in samples, evaluating the efficacy of drug treatments, or diagnosing cancer. Attached Figure Description

[0054] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0055] Figure 1 Detection of antiserum titer against CDH17 protein in camel.

[0056] Figure 2 ELISA binding curve of nanobody CT008-7101 to human CDH17 protein.

[0057] Figure 3 Detection of the binding of nanobody CT008-7101 to COLO205 tumor cell line.

[0058] Figure 4 Detection of the binding of nanobody CT008-7101 to SNU16 tumor cell line. Detailed Implementation

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

[0060] Example 1: Camel Immunization

[0061] 1.1 Immunogen Preparation:

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

[0063] 1.2 Camel Immunity:

[0064] The initial immunization dose was 400 μg of human CDH17 protein (His Tag), mixed with adjuvant (GERBU FAMA), and administered subcutaneously at four points on the camel's back, with each point receiving 1 mL. For the second to sixth immunizations, the dose of the immunization antigen was 200 μg, administered subcutaneously at four points on the camel's back, with each point receiving 1 mL. The interval between each immunization was two weeks.

[0065] 1.3 Detection of immune serum titer:

[0066] Human CDH17 Protein and His Tag antigen were coated overnight at 4℃. After blocking and washing, serially diluted serum was added to the ELISA plate for incubation, followed by incubation with anti-llama IgG HRP (Abcam) antibody. After washing, TMB chromogenic 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 as follows: Figure 1 As shown, the camel titer reached a high level (>256000) after 6 immunizations.

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

[0068] (1) After 7 immunizations, 100 mL of peripheral blood lymphocytes were extracted from camels and total RNA was extracted. RNA extraction was performed according to the instructions of the RNAiso reagent from TAKARA.

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

[0070] (3) The variable region encoding gene of the heavy chain antibody was obtained by nested PCR using PrimeSTAR high-fidelity DNA polymerase. The variable region fragment of the heavy chain antibody was amplified by nested PCR:

[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-guided peptide and antibody CH2 was amplified, annealed at 55°C for 30 cycles, and a DNA fragment of approximately 600 bp was recovered 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 fragment between the FR1 region of the heavy chain antibody and the long and short hinge regions (long and short fragments) was amplified, annealed at 55°C for 30 cycles, and the target fragment was recovered. The results showed that the size of the fragment was about 500 bp, that is, the nanobody gene electrophoresis band was about 500 bp.

[0079] (4) The phage particle pME207 and the PCR amplification product were 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 and ligated overnight at 16°C.

[0080] (5) After ethanol precipitation, the ligation product was dissolved in 100 μL of sterile water and electroporated ten times to transform *E. coli* TG1. 100 μL of the electroporated and cultured bacterial suspension was serially diluted and plated onto an ampicillin LB plate. The volume of the culture was calculated, and the remaining portion was plated onto an ampicillin 2×YT plate. The plates were incubated at 37°C inverted mode for 13–16 h. The bacterial growth on the plates was scraped off with 10 mL of 2×YT medium, and then 25% glycerol was added to the final concentration. The plates were aliquoted and stored at -80°C for later use. The volume of the culture was 2.89 × 10⁻⁶. 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 over 90%.

[0081] (6) Based on the calculated library capacity, live cells of 10 times the library capacity were inoculated into 200 mL of 2×YT (containing 2% glucose and 100 μg / mL ampicillin), cultured at 37°C and 200 r / min until the OD600 reached 0.5, helper phages were added at a multiplicity of infection of 20:1, and the mixture was incubated at 37°C for 30 min, then cultured at 37°C and 200 r / min for 30 min. Centrifuge the culture, resuspend the precipitate in 200 mL of 2×YT (containing 100 μg / mL ampicillin and 50 μg / mL kanamycin), incubate overnight at 37 °C and 250 rpm, centrifuge at 8000 rpm to collect the supernatant, add 5×PEG / NaCl solution, place on ice for 60 min, centrifuge at 8000 rpm for 30 min, resuspend the precipitate in 5 mL of PBS to obtain the anti-CDH17 single-domain heavy chain antibody (VHH) immunological library. Take 10 μL to determine the titer, and aliquot the remainder and store at -80 °C for later use.

[0082] (7) Using Human CDH17 Protein, His Tag Protein (CA7-H52H3), CDH17 was coated at 1 μg / mL, 100 μL per well, onto an ELISA plate and incubated overnight at 4°C. A negative control was also set up. The next day, 200 μL of 3% BSA was added to each of the five wells, and the plates were blocked at room temperature for 2 hours. After 2 hours, the plates were washed three times with PBST (PBS containing 0.05% Tween 20). After washing, 100 μL of phage pre-blocked with 5% skim milk (2-3 × 10⁻⁶) was added to each negative control well. 11 The TFU-immunized camel nanobody phage display gene library was used. After incubation at room temperature for 1.5 hours, the supernatant was transferred to the target antigen-coated wells and incubated at room temperature for another 1.5 hours. The cells were washed 12 times with PBST (containing 0.05% Tween 20) to remove unbound phages. Phages specifically bound to CDH17 were dissociated using Glycine (SIGMA). The eluted phages were neutralized with Tris (Invitrogen, 1M, pH 8.0) and then used to infect TG1 cells in the logarithmic growth phase. After amplification, the cells underwent another round of adsorption-elution. Finally, the eluted phages inoculated TG1 cells, and IPTG (Thermo) was used to induce TG1 nanobody expression. ELISA plates were coated with CDH17 protein, and the supernatant was used for ELISA detection. Clones with OD450 > 0.5 were selected for sequencing.

[0083] (8) After sequence analysis, the clone CT008-7101 that binds to CDH17 protein was obtained.

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

[0085] The nanobody CT008-7101 was constructed into the pCDNA3.4-IgG1 vector, and then subjected to ExpiCHO. TM The protein was expressed using a ThermoFisher expression system. After one week of expression, the supernatant was collected and purified using Protein A (GE) to obtain VHH protein linked to the IgG1 Fc structure. Protein concentration was then measured using Nanodrop, and protein purity was determined by HPLC. The obtained protein purity and yield met the requirements for subsequent experiments.

[0086] The amino acid sequence of CT008-7101 is shown below, with the CDR region underlined:

[0087] QMQLVESGGGLVQPGGSLRLSCAAS GFTLRDYP MNWVRQAPGKGLEWVSA I NSGEGIT FYADAVKGRFTISRDNAKNTLYLQMNSLKIEDTAMYYC ARGPIRDGI RG QGTQVTVSS (SEQ ID NO: 4).

[0088] Example 4, Protein-level affinity detection

[0089] The binding kinetics and affinity of heavy chain antibodies for human CDH17.His antigen were determined using surface plasmon resonance (SPR) technology. Purified antibodies were flowed through a sensor chip pre-immobilized with protein A, where they were captured. Five different concentrations of CDH17.His protein were used as the mobile phase, with binding and dissociation times of 30 min and 60 min, respectively. The binding rate (kon), dissociation rate (koff), and equilibrium constant (KD) were analyzed using Biacore Evaluation Software 2.0 (GE). BMK-2 (CDH17 nanobody from WO2019 / 210155A1) binding to CDH17 protein was selected as a positive control. The results are shown in Table 1.

[0090] Table 1: Protein Affinity Detection

[0091] Antibody number <![CDATA[K on (1 / Ms)]]> <![CDATA[K off (1 / s)]]> KD(M) CT008-7101 3.46E+04 4.60E-03 1.33E-07 BMK2 1.31E+05 1.84E-04 1.40E-09

[0092] Example 5: ELISA detection of nanobody binding activity

[0093] Protein affinity assay: Human CDH17 Protein was coated at 1 μg / mL, 100 μL per well, onto an ELISA plate and incubated overnight at 4°C. Blocked with 3% BSA and incubated at 37°C for 2 hours. After 2 hours, washed three times with PBST (PBS containing 0.05% Tween 20). The nanobody was diluted in DPBS to 100 μL / well and incubated at room temperature for 1 hour. Secondary antibody Anti-Human IgG-Fc Fragment, HRP conjugated (Bethyl Laboratories, A80-104P), 100 μL / well, was added to DPBS for detection. Incubated at room temperature for 0.5 hours, washed, and then TMB chromogenic buffer was added for color development. The reaction was terminated with 2M HCl, and the absorbance at OD450 nM was measured using an ELISA reader. BMK-2 (CDH17 nanobody from WO2019 / 210155A1) and a blank were used as controls. Results are as follows. Figure 2 As shown.

[0094] Cell-level affinity assay: COLO205 cells or SNU16 cells were seeded in 96-well plates, 5 × 10⁶ cells per well. 5 Cells were then added with serially diluted antibodies, incubated for half an hour, and then the secondary antibody anti-human IgG PE (Jackson Immuno Research, Code: 109-117-008, Lot: 145501) was added and incubated again. The cells were then analyzed using a CytoFLEX flow cytometer. Results are as follows: Figure 3 and 4 As shown.

[0095] Therefore, it can be seen that the nanobody CT008-7101 has good binding activity with CDH17 protein or tumor cells expressing CDH17.

[0096] Part of the sequence in this article

Claims

1. A CDH17 binding molecule, which is an anti-CDH17 Nanobody, the complementarity determining regions (CDRs) of which comprise CDR1, CDR2 and CDR3, wherein the sequence of CDR1 is set forth in SEQ ID NO: 1, the sequence of CDR2 is set forth in SEQ ID NO: 2, and the sequence of CDR3 is set forth in SEQ ID NO:

3.

2. The CDH17 binding molecule of claim 1, wherein, The sequence of the variable region of the heavy chain of the anti-CDH17 Nanobody is set forth in SEQ ID NO:

4.

3. A CDH17 binding molecule, characterized in that, The CDH17 binding molecule is a heavy chain antibody, the complementarity determining regions (CDRs) of which comprise CDR1, CDR2 and CDR3, wherein the sequence of CDR1 is set forth in SEQ ID NO: 1, the sequence of CDR2 is set forth in SEQ ID NO: 2, and the sequence of CDR3 is set forth in SEQ ID NO: 3, the heavy chain antibody further comprising a heavy chain constant region.

4. A fusion protein, characterized in that, The CDH17 binding molecule of any one of claims 1-3, and a further polypeptide selected from a polypeptide that localizes the binding molecule to a different organelle or a tag for purification.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule of claim 5.

6. A nucleic acid construct, characterized in that, The nucleic acid molecule of claim 5.

7. The nucleic acid construct of claim 6, wherein The nucleic acid construct of claim 6 or 7.

8. A host cell, which: (1) expresses the CDH17 binding molecule of any one of claims 1-3 or the fusion protein of claim 4; (2) comprises the nucleic acid molecule of claim 5; and / or (3) comprises the nucleic acid construct of claim 6 or 7.

9. The host cell of claim 8, wherein The host cell is an immune effector cell.

10. The host cell of claim 8, wherein The host cell is a T cell.

11. A method of producing the CDH17 binding molecule of any one of claims 1-3, comprising: culturing the host cell of any one of claims 8-10 under conditions suitable for production of the CDH17 binding molecule, and optionally purifying the CDH17 binding molecule from the culture, or incubating the nucleic acid molecule of claim 5 under conditions suitable for DNA or RNA translation in a non-cellular system.

12. Use of the CDH17 binding molecule of any one of claims 1-3, the nucleic acid molecule of claim 5, the nucleic acid construct of claim 6 or 7, or the host cell of any one of claims 8-10, in the manufacture of an immune cell, which is a T cell.

13. A kit for detecting CDH17, characterized by, The kit comprises the CDH17 binding molecule of any one of claims 1-3, the nucleic acid molecule of claim 5, or the nucleic acid construct of claim 6 or 7.

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