Anti-CDH17 antibody, and preparation and use thereof

Abstract

The present invention relates to the technical field of biomedicine, and specifically relates to an anti-CDH17 antibody (such as a nanobody), and a biological material and product thereof. The provided anti-CDH17 antibody (such as a nanobody) can be used for preventing and / or treating cancer.

Description

An anti-CDH17 antibody, its preparation and uses

[0001] This application claims priority to Chinese Patent Application No. 202510064158.3, filed on January 15, 2025, entitled "An Anti-CDH17 Antibody and Its Preparation and Use Thereof", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of biomedical technology, specifically to an anti-CDH17 antibody and its biomaterials and products. Background Technology

[0003] CDH17 (Cadherin 17, also known as LI-cadherin or hepato-gut cadherin) is a non-classical member of the calcium-dependent protein CDH superfamily. CDH17 is silenced in healthy adult liver and stomach tissues but is aberrantly expressed in gastrointestinal malignancies (including hepatocellular carcinoma, pancreatic cancer, and colorectal cancer), and can serve as a potential diagnostic biomarker for digestive system malignancies. Global pharmaceutical companies have developed numerous new drugs targeting CDH17, including: CDH17-targeting monoclonal antibodies (such as ARB-101 and ARB-102), CDH17 / CD3 bispecific antibodies (such as ARB202), CDH17-targeting CAR-T therapies (such as CHM-2101), and CDH17-targeting ADCs (such as TORL-3-600). Research data on these drugs suggest that CDH17 can serve as an important therapeutic target for digestive system malignancies.

[0004] Nanobodies are a class of small, stable, single-domain antibodies derived from heavy-chain antibodies of camelids (such as camels and alpacas). Due to their small size, high affinity, stability, and ease of production, nanobodies have a variety of potential roles and applications in targeted therapy. Nanobodies exhibit high affinity and specificity for epitopes of target antigens, making them ideal candidates for targeted therapy; they can be used to develop novel inhibitors targeting immune checkpoints, enhancing the immune system's attack on tumors; they can be used to target biomarkers on the surface of tumor cells, achieving precise tumor treatment through conjugation with radionuclides or toxins; they can be used to target specific molecules in autoimmune diseases, modulating immune responses; their relatively low production cost helps reduce drug costs; and they possess better tissue penetration capabilities, reaching diseased tissues more effectively. Summary of the Invention

[0005] In view of this, this invention uses recombinant proteins from the CDH17EC1 region to immunize camels and alpacas, constructing a nanobody phage library. After biopanning, two CDH17-binding nanobodies were obtained. Both nanobodies exhibited high binding activity to human, monkey, and mouse CDH17 recombinant proteins, as well as to CDH17-positive cells.

[0006] This invention provides an anti-CDH17 antibody (e.g., a nanobody) and its biomaterials and products. The anti-CDH17 antibody specifically provided in this invention has at least one of the following advantages: small molecular weight, simpler structure, low immunogenicity, high tissue permeability, high stability, high solubility and low aggregation, and ease of cloning.

[0007] This invention uses recombinant CDH17 antigen to immunize camels and alpacas to construct a VHH phage library. After biopanning, two CDH17-binding nanobodies were obtained. Both nanobodies exhibited high binding activity to recombinant human CDH17 extracellular protein, CDH17-positive cells (Nalm-6 cells), or CDH17-overexpressing cells (293F-CDH17).

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] The present invention provides an anti-CDH17 antibody and its variants, or antigen-binding fragments thereof, wherein the anti-CDH17 antibody comprises three complementarity-determining regions CDR1, CDR2 and CDR3 of VHH (Table A) named HB01 and HCM17-105.

[0010] Table A: VHH sequences of HB01 and HCM17-105

[0011] The CDR and FR regions corresponding to the antibodies of this invention are exemplarily shown in Table B below:

[0012] Table B: CDR and FR regions in the VHH sequences of HB01 and HCM17-105

[0013] For example, the antibodies and their variants, or antigen-binding fragments thereof, described herein are humanized variants or identical variants (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%).

[0014] Furthermore, the antibodies and their variants, or their antigen-binding fragments, described herein are selected from camel Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, scFv, bis-scFv, (scFv)2, microantibodies, double-chain antibodies, triple-chain antibodies, tetra-chain antibodies, disulfide-stabilized Fv proteins, and single-domain antibodies (sdAb, nanobodies), bispecific antibodies, trispecific antibodies, or more specific antibodies.

[0015] The present invention also provides a fusion protein comprising the antibody described herein and its variants, or an antigen-binding fragment thereof.

[0016] For example, the fusion protein described herein may further include a tag sequence (e.g., Poly-His, Hemagglutinin, c-Myc, GST, Flag-tag, etc.) or an IgG1-Fc protein sequence, or an additional epitope (e.g., an epitope targeting or different from CDH17) or an additional antibody or antibody active fragment (e.g., an antibody or antibody active fragment targeting or the same epitope targeting CDH17, or a ligand that can bind to CDH17, or an antibody targeting different from CDH17).

[0017] The present invention also provides an antibody-drug conjugate comprising the antibody described herein and its variants, or an antigen-binding fragment thereof.

[0018] For the antibody-drug conjugates described herein, the drug is selected from the following: radiolabeled substances, 32 P, 35 S, fluorescent dyes, electron-dense reagents, enzymes, biotin, streptavidin, digitalisin, haptens, immunogenic proteins, nucleic acid molecules having sequences complementary to the target, or any combination thereof; or immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, and antiparasitic agents, or any combination thereof.

[0019] The present invention also provides an isolated polynucleotide expressing an antibody or an antigen-binding fragment thereof, characterized in that the polynucleotide is capable of expressing the antibody and its variants described herein, or an antigen-binding fragment thereof; the polynucleotide is capable of expressing the fusion protein described herein; the polynucleotide is capable of expressing the antibody-drug conjugate described herein.

[0020] The present invention also provides a vector comprising the polynucleotides described herein, preferably a plasmid vector or a viral vector, etc.

[0021] The present invention also provides a host cell comprising a polynucleotide or a vector as described in this disclosure, or an antibody or antibody-drug conjugate expressing the present invention. Preferably, the host cell is a eukaryotic cell, such as a yeast cell or an immune cell.

[0022] For example, immune cells are a core component in maintaining the normal function of the body's immune system, and are mainly divided into innate immune cells and adaptive immune cells. Innate immune cells include macrophages, neutrophils, eosinophils, basophils, mast cells, natural killer cells (NK cells), and dendritic cells (DCs). They are responsible for rapidly responding to pathogen invasion, resisting infection through phagocytosis, secretion of cytokines, or direct killing. Adaptive immune cells mainly include T cells and B cells. T cells can be further subdivided into helper T cells (such as Th1, Th2, Th17, and Tfh cells), cytotoxic T cells (CD8+ T cells), regulatory T cells (Treg cells), and memory T cells, which are responsible for recognizing specific antigens and launching precise attacks. B cells differentiate into plasma cells to produce antibodies to neutralize pathogens. In addition, natural killer T cells (NKT cells), γδ T cells, and myeloid-derived suppressor cells (MDSCs) are also immune cells with special functions, playing an important role in connecting innate and adaptive immunity and regulating immune responses. Immune cells can also include engineered immune cells such as chimeric antigen receptor T cells (CAR-T cells) and CAR-NK cells, which are modified through genetic engineering technology and are widely used in the treatment of cancer and other diseases.

[0023] The present invention also provides a pharmaceutical composition comprising an antibody described herein and a variant thereof, or an antigen-binding fragment thereof, comprising a fusion protein described herein, comprising an antibody-drug conjugate or a host cell described herein, and optionally, comprising a pharmaceutically acceptable carrier.

[0024] The present invention also provides the use of the antibody described herein and its variants, or antigen-binding fragments thereof, the fusion protein described herein, and the antibody-drug conjugate described herein in the preparation of medicaments for the treatment and / or prevention of CDH17-related diseases.

[0025] The present invention also provides a method for treating and / or preventing CDH17-related diseases and related symptoms, comprising administering an effective amount of the antibody described herein and its variants, or antigen-binding fragments thereof, the fusion protein described herein, the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, or host cells (e.g., immune cells) to a subject.

[0026] The present invention also provides a method for detecting whether a sample contains T cells that highly express CDH17, comprising the steps of contacting the sample with the antibody described herein and its variants, or antigen-binding fragments thereof, or the fusion protein described herein, optionally, the detection may be for diagnostic purposes or for non-diagnostic purposes.

[0027] The present invention also provides a detection product comprising the antibodies described herein and their variants, or antigen-binding fragments thereof.

[0028] For example, the detection products described herein are selected from one or more of detection reagents, kits, chips, or test strips.

[0029] To achieve the above-mentioned objectives, the present invention further provides the following technical solutions:

[0030] A first aspect of the present invention provides an anti-CDH17 antibody (e.g., a nanobody), wherein the heavy chain variable region of the anti-CDH17 antibody (e.g., a nanobody) is composed of a framework region (FR) and a complementarity-determining region (CDR), the CDR comprising at least one of the following groups:

[0031] The CDR1, CDR2, and CDR3 shown in any of the VHHs named HB01, HCM17-105.

[0032] Nanobodies (Nb) are variable regions of heavy chain antibodies (IgG2 and IgG3) found in camel-dwelling animals, and are considered the smallest antigen-binding fragments found in nature. Compared to traditional full-length monoclonal antibodies (mAb, approximately 150 kDa), Nb offers advantages such as smaller molecular weight (12-15 kDa), simpler structure, lower immunogenicity, higher tissue permeability, higher stability, higher solubility, lower aggregation, and ease of cloning. Furthermore, compared to similar mAb products, Nb has significantly lower production costs, making it accessible to most cancer patients. In September 2018, the European Medicines Agency approved the first nanobody drug, caplacizumab (trade name Cablivi), primarily for the treatment of acquired thrombotic thrombocytopenic purpura (aTTP) in adults. Therefore, Nb holds promise for a wide range of applications in cancer treatment and diagnosis.

[0033] In this study, camels were immunized with recombinant CDH17 antigen to construct a VHH phage library. After biological screening, six functional anti-CDH17 antibodies were obtained.

[0034] In the embodiments provided by the present invention, the frame region FR includes at least one of the following groups:

[0035] FR1, FR2, FR3 or FR4 as shown in any of the VHHs in the names HB01, HCM17-105.

[0036] However, the frame region FR of this invention is not limited to the above sequence. Any sequence that can achieve its function is within the protection scope of this invention.

[0037] A second aspect of the present invention provides a polynucleotide that encodes the aforementioned anti-CDH17 antibody (e.g., nanobody).

[0038] A third aspect of the present invention provides a recombinant expression vector comprising the aforementioned polynucleotides.

[0039] In the embodiments provided by the present invention, the expression vector includes a prokaryotic expression vector or a eukaryotic expression vector.

[0040] A fourth aspect of the present invention provides a recombinant host cell comprising the above-mentioned polynucleotides or comprising the above-mentioned recombinant expression vector.

[0041] In the embodiments provided by the present invention, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0042] In specific embodiments provided by the present invention, the host cell is selected from Escherichia coli, yeast cells, or human cells, such as immune cells.

[0043] In specific embodiments provided by the present invention, the host cell is selected from human cells, yeast cells, and other immune cells.

[0044] The fifth aspect of this invention provides a method for preparing the above-mentioned anti-CDH17 antibody (e.g., nanobody), comprising the following steps:

[0045] The polynucleotide encoding the above-mentioned anti-CDH17 antibody (e.g., nanobody) is inserted into the expression vector to obtain the recombinant expression vector;

[0046] The recombinant expression vector was transferred into host cells to obtain recombinant host cells;

[0047] Recombinant host cells are cultured to obtain a culture.

[0048] The culture was purified to obtain anti-CDH17 antibodies (e.g., nanobodies).

[0049] In the embodiments provided by the present invention, purification is performed using Protein A agarose purification resin.

[0050] The sixth aspect of the present invention provides a bispecific antibody, a trispecific antibody, or a multispecific antibody, wherein the bispecific antibody, trispecific antibody, or multispecific antibody comprises the above-mentioned anti-CDH17 antibody (e.g., nanobody).

[0051] In the embodiments provided by the present invention, the second antibody includes, but is not limited to, 4-1BB nanobody, CD47 nanobody, VEGF nanobody, HER2 nanobody, EGFR nanobody, HER3 nanobody, B7H3 nanobody, TIGIT nanobody, OX-40 nanobody, CD40 nanobody or PD-L1 nanobody.

[0052] The seventh aspect of the present invention provides the use of the above-mentioned anti-CDH17 antibody (e.g., nanobody) or bispecific antibody, trispecific antibody or multispecific antibody in the preparation of drugs for the prevention and / or treatment of cancer and in the detection of CDH17 protein.

[0053] In the embodiments provided by the present invention, cancers include, but are not limited to, lung cancer, stomach cancer, liver cancer, leukemia, kidney tumors, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumors, or bladder tumors.

[0054] The eighth aspect of the present invention provides a pharmaceutical composition comprising: the above-described anti-CDH17 antibody (e.g., nanobody) or bispecific antibody, trispecific antibody or multispecific antibody, or the host cell of the present invention; and pharmaceutically acceptable excipients.

[0055] In the embodiments provided by the present invention, the dosage form of the pharmaceutical composition includes, but is not limited to, injection, powder for injection, tablet or capsule.

[0056] The ninth aspect of the present invention provides a kit for detecting CDH17 protein, the kit comprising: the above-mentioned anti-CDH17 antibody (e.g., nanobody) or bispecific antibody, trispecific antibody or multispecific antibody; and a detection-acceptable reagent. Attached Figure Description

[0057] Figure 1 shows the SDS-PAGE electrophoresis images of the two nanobodies. Detailed Implementation

[0058] This invention discloses an anti-CDH17 antibody (e.g., a nanobody) and its biomaterials and products. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0059] Terminology Explanation:

[0060] Nanobodies (Nb) are naturally occurring antibodies lacking the light chain found in alpaca peripheral blood. These antibodies contain only a single heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. Unlike artificially engineered single-chain antibody fragments (scFv), they do not readily adhere to each other or aggregate. More importantly, the individually cloned and expressed VHH structure exhibits structural stability and antigen-binding activity comparable to the original heavy chain antibody, making it the smallest known unit capable of binding target antigens.

[0061] The framework region (FR) is the backbone region within the variable region of an antibody. Approximately 110 amino acid sequences near the N-terminus of the H and L chains of immunoglobulins exhibit significant variation, while the amino acid sequences of other parts remain relatively constant. Based on this, the light and heavy chains can be divided into variable (V) and constant (C) regions. The variable region contains the hypervariable region (HVR), also known as the complementarity-determining region (CDR), and the FR backbone region. The variability of the FR is less than that of the CDR. There are four FR molecules: FR1, FR2, FR3, and FR4. During antibody recognition, the four FR molecules coil, causing the CDR molecules to approach each other.

[0062] Complementarity determining region (CDR): The entire antibody molecule can be divided into two parts: constant region and variable region. Within the variable region, a small subset of amino acid residues exhibits particularly strong variations; these regions, where the composition and sequence of amino acid residues are more prone to variation, are called hypervariable regions. There are three hypervariable regions (HVRs) in the V region of the L and H chains. Because these regions can form precise complementarity with the antigenic determinants in their spatial structure, they are also called complementarity determining regions.

[0063] Bispecific antibodies are artificial antibodies containing two specific antigen-binding sites. They can bridge the gap between target cells and functional molecules (cells), stimulating a targeted immune response. They are a type of genetically engineered antibody and have become a hot topic in the field of antibody engineering, with broad application prospects in the immunotherapy of tumors.

[0064] The present invention will be further illustrated below with reference to the embodiments:

[0065] Example 1: Animal Immunization

[0066] The animal immunization program is as follows:

[0067] 1. Immunogen: Recombinant proteins from the human and monkey CDH17 EC1 region (with an hFc tag at the C-terminus) were used as immunoantigens.

[0068] 2. Laboratory animals and immunization sites:

[0069] Bactrian camel (1 animal): Multiple injections were administered subcutaneously and intramuscularly on the back of the neck.

[0070] Alpaca (1): Multiple injections were administered subcutaneously and intramuscularly on the back of the neck.

[0071] Follow up and observe the absorption of the subcutaneous injection mass after injection to confirm whether the immune process is normal.

[0072] 3. Immunization schedule and dosage:

[0073] Bactrian camel:

[0074] First immunization: 0.5 mg of antigen was emulsified with Freund's complete adjuvant at a 1:1 volume ratio and injected into each animal, with an injection volume of 1 mL.

[0075] Second to fourth immunizations: each time at 3-week intervals, use 0.25mg of antigen, emulsified with Freund's incomplete adjuvant at a 1:1 volume ratio, and inject 1mL per animal.

[0076] Alpaca:

[0077] First immunization: 0.5 mg of antigen was emulsified with Freund's complete adjuvant at a 1:1 volume ratio and injected into each animal, with an injection volume of 1 mL.

[0078] Second to fourth immunizations: each time at 2-week intervals, use 0.25mg of antigen, emulsified with Freund's incomplete adjuvant at a 1:1 volume ratio, and inject 1mL per animal.

[0079] 4. Immune response monitoring: After each immunization, observe the local reaction at the injection site, such as the formation and absorption of lumps, to ensure that there are no abnormalities in the immunization process.

[0080] 5. Serum Processing and Titer Detection: One week after the fourth immunization, 2 mL of peripheral blood was collected from camels and alpacas, and serum was separated. The recombinant protein (His tag) of the human and monkey CDH17EC1 region was coated onto 96-well ELISA plates, and the antibody titer in the serum was determined using ELISA. ELISA results showed that the ELISA serum titer of the fourth immunization serum from camels and alpacas binding to human and monkey CDH17EC1 was >1:32000, meeting the library construction criteria.

[0081] Example 2: Construction of a phage display immune antibody library

[0082] Since the serum titer of camels and alpacas was >1:32000 after the fourth immunization, it indicates the presence of high-affinity antibodies against human CDH17 in their serum. Therefore, we constructed a phage display antibody library according to the following steps:

[0083] 1. Collect 50 mL of peripheral blood from camels and camels after their fourth immunization, and isolate PBMCs (partial peripheral blood cells); take 2 × 10⁻⁶ cells. 7 Total RNA was extracted from the PBMCs using an RNA extraction kit; an appropriate amount of RNA (e.g., 3-5 μg) was taken and cDNA was obtained using an RT-PCR reverse transcription kit.

[0084] 2. The variable region sequences of the IgG2 and IgG3 heavy chains (the VHH region of the heavy chain of nanobodies) were obtained stepwise by nested PCR. The experimental steps are as follows: 1) Design a pair of specific nested outer primers and perform the first round of PCR amplification using cDNA as a template. The amplified region is the Leader-CH2 region of the camel and alpaca heavy chain antibody genes, and the product size is 700bp and 900bp. The 700bp PCR product was recovered by DNA gel electrophoresis. 2) Design nested inner primers (6 pairs for camels and 3 pairs for alpacas) and perform the second round of PCR amplification using the 700bp first round PCR product as a template. The amplified region is the VHH fragment of the camel and alpaca heavy chain antibody variable region, and the product size is 400bp. The second round PCR product was purified and recovered using a PCR product purification kit.

[0085] 3. The heavy chain variable region sequence was inserted into the enzyme-digested linearized phage vector VHH-libTemplate via homologous recombination or enzyme digestion ligation to obtain the recombinant vector. After purification and recovery, the vector was transformed into supercompetent SS320 cells (containing helper phage M13K07). The transformed bacterial culture was resuspended in SOC medium and activated for 1 hour. A small amount of the bacterial culture was serially diluted 10-fold to select an appropriate dilution titer and plated onto LB / tet10 and LB / Carb50 culture plates. The plates were incubated overnight at 37°C and used for library volume calculation the next day. The remaining bacterial culture was transferred to a large volume of 2YT / Carb50 / Kan25 liquid medium and cultured overnight at 37°C on a shaker. The supernatant was harvested the next day, and 1 / 4 volume of PEG / NaCl solution was added to precipitate the phages. The phages were then resuspended in an appropriate amount of PBT solution and diluted to the required concentration to obtain the phage display antibody library (stored at -80°C for later use).

[0086] 4. Count the number of clones on LB / Carb50 plates and calculate the library size: The library size of the camel antibody library Lib CDH17 Camel is 1.09 × 10⁻⁶. 10 The alpaca antibody library Lib CDH17 Alpaca has a library size of 0.52 × 10⁻⁶. 10Forty single clones were randomly selected from each plate for sequencing. The results showed that the VHH insertion efficiency of the camel antibody library Lib CDH17 Camel was >95%; the VHH insertion efficiency of the alpaca antibody library Lib CDH17 Alpaca was >95%.

[0087] Example 3: Screening of antibody libraries

[0088] 1. Protein screening: Add 5 μg / mL of human CDH17 EC1 recombinant protein (his tag) to a 96-well plate (100 μL / well) and coat overnight at 4°C; streak NEB5αF' E. coli on 2YT / Tet10 plates and incubate overnight at 37°C; the next day, pick a single NEB5αF' colony from the overnight 2YT / Tet10 plate and add it to 3 mL of... In 2YT / Tet10 liquid medium, the bacteria were grown at 37°C until OD600 = 0.8. Simultaneously, the antigen supernatant of the 96-well plate was removed, and 200 μL of 1% BSA was added to each well for blocking. 200 μL of 1% BSA was added to each blank well as a negative control. The plates were then incubated at room temperature in a 3D rotary shaker for 2 hours. Afterward, the supernatant of the protein and control wells was removed, and the plates were washed with 200 μL of PT. 100 μL of phage antibody library was added to each well, and the plates were incubated at room temperature in a 3D rotary shaker for 2 hours. The supernatant of the protein and control wells was removed again, and the plates were washed with 200 μL of PT. 100 μL of 100 mM HCl was added to each well, and the plates were incubated at room temperature for 5 minutes. The supernatant was aspirated and transferred to a 1.5 mL centrifuge tube, and neutralized with 1M Tris-HCl. Add the above mixture to a centrifuge tube containing 1 mL of NEB5αF' bacteria, and incubate at 37°C for 1 hour. Take 20 μL of the culture medium from the centrifuge tube and dilute it appropriately, then plate it on an LB / Carb50 plate and incubate overnight at 37°C. The next day, use the diluted solution to calculate the titer and enrichment level. Add 1 μL of helper phage M13K07 (final concentration 10) to the remaining culture medium. 10 Incubate the culture medium (number of phages / mL) at 37°C on a shaker for 1 hour. Transfer the culture medium to 35 mL of 2YT / Carb50 / Kan25 medium, place on a shaker, and incubate overnight at 37°C. Collect the phages to form the antibody library for each round. Repeat the above steps 2-3 times until phage enrichment occurs. If the number of colonies in the antigen-binding wells on the LB / Carb50 plate is more than 10 times that of the negative control wells, enrichment is considered successful. In this experiment, after the second round of human CDH17 EC1 protein screening, the number of colonies in the antigen-binding wells of Lib CDH17 Camel was 100 times that of the negative control wells, indicating successful enrichment; after the second round of human CDH17 EC1 protein screening, the number of colonies in the antigen-binding wells of Lib CDH17 Alpaca was 10 times that of the negative control wells, indicating successful enrichment.

[0089] 2. Species Cross-Screening: The monkey CDH17 EC1 recombinant protein (his tag) was screened using the previous round of human CDH17 EC1 recombinant protein enrichment library, following the same procedure as above. After the second round of monkey CDH17 EC1 protein screening, the number of colonies in the antigen-binding wells of Lib CDH17Camel was 10 times that of the negative control wells, indicating successful enrichment; similarly, after the second round of monkey CDH17 EC1 protein screening, the number of colonies in the antigen-binding wells of Lib CDH17 Alpaca was 10 times that of the negative control wells, indicating successful enrichment.

[0090] 3. Cell screening: using 10 7 One CDH17-positive COLO-205 cell line was mixed with phages from the monkey CDH17 EC1 protein selection enrichment rounds of Lib CDH17 Camel and Lib CDH17 Alpaca, respectively, at 400 μL / well, and incubated at 4°C for 2 hours. Cells were washed 6-8 times with pre-chilled PT buffer at 4°C. The washed cells were then mixed with 1 mL of NEBalpha5F' cells and incubated at 37°C for 1 hour. 20 μL of the culture medium from the centrifuge tube was diluted appropriately and plated onto LB / Cab50 plates, incubated overnight at 37°C, and used the next day for titer and enrichment calculation. 1 μL of helper phage M13K07 (final concentration 10) was added to the remaining culture medium. 10 Incubate the culture medium (particles / mL) at 37°C on a shaker for 1 hour; then transfer the culture medium to 35 mL of 2YT / Carb50 / Kan25 medium, place on a shaker, and incubate overnight at 37°C. Collect the phages to form the antibody library for each round. Repeat the above steps twice.

[0091] 4. Phage ELISA and Phage FACS: From the phages selected after cell selection, 96 clones were randomly selected and cultured in 96-well plates. After centrifugation, the supernatant was used for Phage ELISA and Phage FACS screening. In Phage ELISA screening, clones with an OD value >2 for binding to the recombinant protein (his tag) of the extracellular region of human CDH17 were defined as Phage ELISA positive clones, and their sequences were sequenced and aligned to obtain unique sequences. In Phage FACS screening, clones with a positive binding rate >2 for COLO-205 cells and 293F cells were defined as Phage FACS positive clones, and their sequences were sequenced and aligned to obtain unique sequences.

[0092] Example 4: Alignment and Statistical Analysis of Positive Sequences

[0093] The positive unique sequences from phage ELISA and phage FACS were compared to obtain two clones that were positive in both phage ELISA and phage FACS screenings. The results are shown in Table 1. It should be noted that the two positive clones obtained above were based on many (dozens) screening failures and the removal of many other positive clones.

[0094] Table 1. CDH17 nanobodies obtained from camel immunization

[0095] Example 5: Eukaryotic transient expression of nanobodies

[0096] For the two sequences in Table 1 of Example 4, eukaryotic expression was performed. The experimental steps were as follows: 1) The VHH fragments of these sequences were amplified by PCR, and the fragments were inserted into the eukaryotic expression vector pcDNA3 containing the hFc tag using homologous recombination or enzyme digestion ligation methods; the fragments were electroporated into E. coli trans5α host bacteria, and after bleomycin selection, single clones were sequenced to obtain the correct recombinant plasmids; then the host bacteria containing the recombinant plasmids were expanded and cultured, and sterile endotoxin-free plasmids were obtained using an endotoxin removal kit; 2) HEK293F or CHO cells were cultured in serum-free medium; the recombinant expression plasmids were transfected into HEK293F or CHO cells using transfection reagents for expression. On day 5, the supernatant was collected, and the antibodies were separated and purified using Protein A agarose resin and stored in PBS solution. The experimental results (Table 2, Figure 1) showed that the yield of the HB01 sequence expressed in 30 mL of CHO cells was 10.53 mg, and the yield of the HCM17-105 sequence expressed in 50 mL of HEK293F cells after transient transfection was 2.77 mg. SDS-PAGE electrophoresis confirmed that the protein bands of the two antibody sequences were of normal size and had a purity >95% under both reducing and non-reducing conditions.

[0097] Table 2.2 Results of eukaryotic transient expression of two nanobodies

[0098] Example 6: Affinity between nanobodies and recombinant antigen proteins ELISA EC50

[0099] The affinity of the VHH-hIgG1Fc recombinant antibodies of HB01 and HCM17-105 for recombinant CDH17 extracellular region protein (his tag), recombinant CDH16 extracellular region protein (his tag), and BSA was determined using ELISA. CDH16, CDH17 extracellular region protein (his tag), or BSA was added to 96-well ELISA plates at 200 ng / well and incubated overnight at 4°C. Nanobodies or control antibodies (nanobodies with clone number H17, derived from patent PCT / US2019 / 029333; our expression structure is VHH-hIgG1Fc) were diluted to different concentrations and reacted with the antigens using ELISA. HRP-labeled anti-hFc secondary antibody was used for color development, and the absorbance at 450 nm was measured using a microplate reader. The experimental results (Tables 3-1 and 3-2) show that the VHH-hIgG1Fc recombinant antibodies of HB01, HCM17-105 and control antibody H17 strongly bind to human, monkey and mouse CDH17 ECD protein, but do not bind to human CDH16 ECD protein and BSA.

[0100] Table 3-1. ELISA binding affinity of antibodies to human CDH17 ECD (his tag)

[0101] Table 3-2. ELISA binding affinity of antibodies to human CDH16 ECD (his tag) and BSA

[0102] Example 7: Binding of nanobodies to human CDH17 positive cells or overexpressing cells

[0103] The affinity of HB01, HCM17-105, and H17 antibodies for CDH17-positive cells (COLO-205 cells) or HEK293F cells was detected using flow cytometry. FACS detection method: 1) COLO-205 or HEK293F cells were incubated with 0.1 or 1 μg / mL antibodies for 1 hour; 2) After washing the cells three times with PBS, the cells were resuspended in 100 μL PBS, and 0.2 μg / mL of PE-labeled Anti-hFc antibody was added, followed by incubation for 1 hour; 3) After washing the cells three times with PBS, the cells were resuspended in 300 μL PBS, and fluorescence was detected using flow cytometry. The results are shown in Table 4. The experimental results showed that the VHH-hIgG1Fc recombinant antibodies of HB01, HCM17-105, and control antibody H17 had strong binding activity with COLO-205 cells, but no binding activity with HEK293F cells.

[0104] Table 4. Binding affinity of CDH17 antibody to FACS cells

[0105] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An anti-CDH17 antibody and a variant thereof, or an antigen-binding fragment thereof, said anti-CDH17 antibody comprising three complementarity-determining regions CDR1, CDR2, and CDR3 of VHH named HB01 and HCM17-105, Exemplarily, for the antibodies and their variants, or antigen-binding fragments thereof, the variants are humanized variants or identical variants (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) variants; Alternatively, by way of example, the antibody and its variants, or the antigen-binding fragment thereof, are selected from camel Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, scFv, bis-scFv, (scFv)2, microantibodies, double-chain antibodies, triple-chain antibodies, tetra-chain antibodies, disulfide-stabilized Fv proteins, and single-domain antibodies (sdAb, nanobodies), camel antibodies, bispecific antibodies, or trispecific antibodies. Further, the antibody is an antibody named HB01 or HCM17-105. For example, the CDR sequences of the antibodies HB01 and HCM17-105 are shown in the table below: CDR and FR regions in the VHH sequences of HB01 and HCM17-105 2. A fusion protein comprising the antibody of claim 1 or a variant thereof, or an antigen-binding fragment thereof. For example, the fusion protein may further include a tag sequence (e.g., Poly-His, Hemagglutinin, c-Myc, GST, Flag-tag, etc.) or an IgG1-Fc protein sequence, or an additional epitope (e.g., an epitope targeting or different from CDH17) or an additional antibody active fragment (e.g., an antibody or antibody active fragment targeting or targeting the same epitope as CDH17, or a ligand capable of binding to CDH17).

3. An antibody-drug conjugate comprising the antibody of claim 1 and its variants, or an antigen-binding fragment thereof.

4. A polynucleotide expressing an antibody or its antigen-binding fragment, characterized in that, The polynucleotide is capable of expressing the antibody and its variants as claimed, or its antigen-binding fragment; the polynucleotide is capable of expressing the fusion protein described herein; or the polynucleotide is capable of expressing the antibody-drug conjugate described herein.

5. A vector comprising the polynucleotide of claim 4, preferably a plasmid vector.

6. A host cell comprising the polynucleotide of claim 4 or the vector of claim 5, preferably, the host cell being a eukaryotic cell.

7. A pharmaceutical composition comprising the antibody of claim 1 and a variant thereof, or an antigen-binding fragment thereof, comprising the fusion protein of claim 2, comprising the antibody-drug conjugate of claim 3, and optionally, comprising a pharmaceutically acceptable carrier.

8. Use of the antibody of claim 1 and its variants, or antigen-binding fragments thereof, the fusion protein of claim 2, and the antibody-drug conjugate of claim 3 in the preparation of medicaments for the treatment and / or prevention of CDH17-related diseases.

9. A method for treating and / or preventing CDH17-related diseases and related symptoms, comprising administering an effective amount of the fusion protein of claim 2, the antibody-drug conjugate of claim 3, or the pharmaceutical composition of claim 7 to a subject.

10. An immune cell capable of expressing CDH17 antibody, characterized in that, The immune cells are genetically modified or treated in vitro to express or bind antibodies that specifically recognize CDH17 molecules and are used to kill target cells expressing CDH17, wherein the antibody is the anti-CDH17 antibody of claim 1.

11. The immune cells as described in claim 10, characterized in that, The immune cells are one or more of T cells, natural killer cells (NK cells), natural killer T cells (NKT cells), or γδT cells.

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