Nanobodies targeting cadherin 17 and uses thereof

Abstract

The present application relates to a nanobody targeting cadherin 17 and its application, and relates to the technical field of immunology and molecular biology, wherein the amino acid sequence of the complementarity determining region of the nanobody targeting cadherin 17 comprises CDR1 as shown in any one of SEQ ID NO: 21-32, CDR2 as shown in any one of SEQ ID NO: 34-45, and CDR3 with an amino acid sequence as shown in any one of SEQ ID NO: 47-58. The present application constructs an alpaca immune phage display library, screens a series of nanobody molecules targeting cadherin 17 (CDH17) from the library, and verifies the function and application of the nanobody at various levels such as CDH17 antigen protein, CDH17 target cells and CDH17 CAR-T cells.

Description

Technical Field

[0001] This invention relates to the fields of immunology and molecular biology, specifically to nanobodies targeting cadherin 17 and their applications. Background Technology

[0002] Cadherins are a superfamily of calcium-dependent cell adhesion molecules. They form connections with cadherin molecules in adjacent cells through their extracellular domains, thereby achieving cell adhesion and playing a crucial role in maintaining tissue structure and morphology. Cadherin-17 (CDH17), also known as liver intestine-cadherin (LI-cadherin) or human peptide transporter-1 (HPT-1), is a non-classical member of the cadherin superfamily. Unlike classic cadherins, CDH17's extracellular domain has seven repeating structural units (EC1-EC7) (e.g., ...). Figure 1 As shown, the intracellular domain of CDH17 contains only 24 amino acid residues, which is not homologous to the intracellular domain of classic cadherins, which consists of 150-160 amino acids.

[0003] Current research has demonstrated that CDH17 is an effective and safe target for targeted therapy of digestive system tumors. Anti-CDH17 candidate antibody molecules can be used to develop diagnostic reagents, targeted antibody drugs, and CAR-T / NK cell immunotherapy for diseases such as digestive system tumors. However, the number of available anti-CDH17 candidate antibody molecules is very limited, and they are mainly traditional antibodies. Only one nanobody with significant advantages and good druggability has been reported, from a research team at the University of Pennsylvania, and developed by Chimeric Therapeutics as the world's first CDH17 CAR-T, which is currently in Phase II clinical trials. To promote the development of CDH17-targeted diagnostic reagents and targeted drugs, it is necessary to accelerate the development of more anti-CDH17 candidate nanobody molecules. Therefore, this invention discloses a nanobody targeting cadherin 17 and its applications. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide nanobodies targeting cadherin 17 and their applications.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0006] In a first aspect, a nanobody targeting cadherin 17, wherein the amino acid sequence of the complementarity-determining region of the nanobody targeting cadherin 17 includes CDR1 as shown in any one of SEQ ID NO:21-32, CDR2 as shown in any one of SEQ ID NO:34-45, and CDR3 as shown in SEQ ID NO:47-58.

[0007] Furthermore, the nanobody targeting cadherin 17 includes any one of E001, E005, E042, E046, E101, E114, E116, E119, E121, E201, E242, and E243.

[0008] The complementarity-determining regions of the amino acid sequence of E001 include CDR1 as shown in SEQ ID NO:21, CDR2 as shown in SEQ ID NO:34, and CDR3 as shown in SEQ ID NO:47;

[0009] The complementarity-determining regions of the amino acid sequence of E005 include CDR1 as shown in SEQ ID NO:22, CDR2 as shown in SEQ ID NO:35, and CDR3 as shown in SEQ ID NO:48;

[0010] The complementarity-determining regions of the amino acid sequence of E042 include CDR1 as shown in SEQ ID NO:23, CDR2 as shown in SEQ ID NO:36, and CDR3 as shown in SEQ ID NO:49;

[0011] The complementarity-determining regions of the amino acid sequence of E046 include CDR1 as shown in SEQ ID NO:24, CDR2 as shown in SEQ ID NO:37, and CDR3 as shown in SEQ ID NO:50;

[0012] The complementarity-determining regions of the amino acid sequence of E101 include CDR1 as shown in SEQ ID NO:25, CDR2 as shown in SEQ ID NO:38, and CDR3 as shown in SEQ ID NO:51;

[0013] The complementarity-determining regions of the amino acid sequence of E114 include CDR1 as shown in SEQ ID NO:26, CDR2 as shown in SEQ ID NO:39, and CDR3 as shown in SEQ ID NO:52;

[0014] The complementarity-determining regions of the amino acid sequence of E116 include CDR1 as shown in SEQ ID NO:27, CDR2 as shown in SEQ ID NO:40, and CDR3 as shown in SEQ ID NO:53;

[0015] The complementarity-determining regions of the amino acid sequence of E119 include CDR1 as shown in SEQ ID NO:28, CDR2 as shown in SEQ ID NO:41, and CDR3 as shown in SEQ ID NO:54;

[0016] The complementarity-determining regions of the amino acid sequence of E121 include CDR1 as shown in SEQ ID NO:29, CDR2 as shown in SEQ ID NO:42, and CDR3 as shown in SEQ ID NO:55;

[0017] The complementarity-determining regions of the amino acid sequence of E201 include CDR1 as shown in SEQ ID NO:30, CDR2 as shown in SEQ ID NO:43, and CDR3 as shown in SEQ ID NO:56;

[0018] The complementarity-determining regions of the amino acid sequence of E242 include CDR1 as shown in SEQ ID NO:31, CDR2 as shown in SEQ ID NO:44, and CDR3 as shown in SEQ ID NO:57;

[0019] The complementarity-determining regions of the amino acid sequence of E243 include CDR1 as shown in SEQ ID NO:32, CDR2 as shown in SEQ ID NO:45, and CDR3 as shown in SEQ ID NO:58.

[0020] Furthermore, the amino acid sequence of the nanobody targeting cadherin 17 is shown in any one of SEQ ID NO: 9 to 20.

[0021] In a second aspect, a CAR-T cell targeting cadherin 17, said CAR-T cell expressing a chimeric antigen receptor (CAR) targeting cadherin 17; said chimeric antigen receptor targeting cadherin 17 includes a CDH17 antigen-binding domain, a hinge region, a transmembrane region, and an intracellular signal transduction domain; said CDH17 antigen-binding domain includes any one of a signal peptide and a nanobody as described in claim 1 or 2.

[0022] Furthermore, the signal peptide is a signal peptide derived from CD8α, the hinge region is the hinge region of IgG4 with amino acid mutation optimization, the transmembrane region is the transmembrane region of CD28, and the intracellular signal transduction domain includes the CD28 intracellular co-stimulatory domain, the 4-1BB co-stimulatory domain, and the CD3ζ signal transduction domain.

[0023] Furthermore, the amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO:60;

[0024] The amino acid sequence of the hinge region of the amino acid mutation-optimized IgG4 is shown in SEQ ID NO:62.

[0025] The amino acid sequence of the transmembrane region of CD28 is shown in SEQ ID NO:64;

[0026] The amino acid sequence of the intracellular co-stimulatory domain of CD28 is shown in SEQ ID NO:66;

[0027] The amino acid sequence of the 4-1BB co-stimulatory domain sequence is shown in SEQ ID NO:68;

[0028] The amino acid sequence of the CD3ζ signal transduction domain is shown in SEQ ID NO:70.

[0029] Thirdly, a nucleic acid comprising a nucleic acid sequence encoding the nanobody targeting cadherin 17 or a complementary sequence thereof, or a nucleic acid sequence encoding the chimeric antigen receptor targeting cadherin 17 or a complementary sequence thereof.

[0030] Furthermore, the nucleotide sequence encoding the signal peptide of CD8α is shown in SEQ ID NO:61;

[0031] The encoding nucleotide sequences of the anti-CDH17 nanobody are shown in SEQ ID NO:82-93 or SEQ ID NO:74-81;

[0032] The nucleotide sequence encoding the hinge region of the amino acid mutation-optimized IgG4 is shown in SEQ ID NO:63.

[0033] The nucleotide sequence encoding the transmembrane region of CD28 is shown in SEQ ID NO:65;

[0034] The nucleotide sequence encoding the intracellular co-stimulatory domain of CD28 is shown in SEQ ID NO:67;

[0035] The encoding nucleotide sequence of the 4-1BB co-stimulatory domain sequence is shown in SEQ ID NO:69;

[0036] The coding nucleotide sequence of the CD3ζ signal transduction domain is shown in SEQ ID NO:71.

[0037] Fourthly, a lentiviral vector comprising the aforementioned nucleic acid molecule.

[0038] Fifthly, a pharmaceutical composition comprising the aforementioned nanobody targeting cadherin 17, or the aforementioned CAR-T cells targeting cadherin 17, or the aforementioned nucleic acid, or the aforementioned lentiviral vector. Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0039] Sixthly, the application of the aforementioned nanobody targeting cadherin 17, or the aforementioned CAR-T cells targeting cadherin 17, or the aforementioned nucleic acid, the aforementioned lentiviral vector, or the aforementioned pharmaceutical composition in the preparation of detection reagents, in vivo imaging probes, and therapeutic products targeting cadherin 17.

[0040] The core innovation of this invention lies in providing a set of 12 nanobody molecules targeting CDH17. No antibodies with the same CDR1-3 sequence as this invention have been found in the prior art, thus solving the problem of the lack of candidate nanobody molecules targeting CDH17 in the prior art.

[0041] This invention constructs an alpaca immune phage display library, from which 12 nanobody molecules targeting cadherin 17 (CDH17) are screened. The function and application of these nanobodies are verified at multiple levels, including CDH17 antigen protein, CDH17 target cells, and CDH17 CAR-T cells. The fusion antibody (VHH-Fc) of the 12 nanobodies with the Fc fragment of human IgG1 exhibits good binding activity with human CDH17 antigen protein, as well as with gastric cancer cells SNU-16 and pancreatic cancer cells ASPC1 expressing human CDH17 antigen protein, and also shows cross-binding activity with mouse CDH17 antigen protein. Biomembrane interference (BLI) assay results show that the affinity (KD value) of the VHH-Fc fusion antibody of the 12 nanobodies for human CDH17 protein reaches the 100 pM to nM level. Among them, eight CAR-T cells expressing the amino acid sequences of nanobody clones E042, E046, E101, E116, E119, E121, E201, and E243, respectively, all exhibited significant killing ability against human CDH17 antigen-positive gastric and pancreatic cancer cells. The nanobodies of this invention can be used in the development of tumor diagnostic reagents, antibody drugs for tumor-targeted therapy, and immunocellular drugs such as CAR-T / NK cells.

[0042] The beneficial effects of this invention are:

[0043] (1) The CDH17 target selected in this invention is the safest and most effective therapeutic target for digestive system tumors: CDH17 is mainly expressed at the tight junctions between epithelial cells in the gastrointestinal system and is highly expressed in digestive system tumors such as gastric cancer, bile duct cancer, pancreatic cancer, esophageal cancer, neuroendocrine tumors and colorectal cancer cells; studies have shown that CDH17CAR-T or bispecific anti-tumor drugs do not damage normally expressed tissues and specifically kill CDH17-expressing tumor tissues.

[0044] (2) This invention provides candidate nanobody molecules for targeted therapy of digestive system tumors: Compared with hematologic malignancies, the development of specific immunotargeted therapy drugs for solid tumors has been slow, and one of the limiting factors is the lack of effective and safe targets. Current research has shown that CDH17 is a safe and effective therapeutic target for digestive system tumors, but research on targeted drugs targeting CDH17 started late, and there are only a few drugs under development at present. The bottleneck is in the antibody discovery stage. Only a few CDH17 candidate antibody molecules have been developed, and the nanobody involved is even rarer.

[0045] (3) The nanobodies of the present invention have a wide range of applications: (a) The series of candidate nanobodies against CDH17 obtained by screening from an alpaca immune library in this invention, when fused with the Fc fragment of human IgG1 at their C-terminus, showed better or at least comparable activity to the control antibody at both the protein and cellular levels. (b) CAR-T cells constructed using the series of candidate nanobodies of the present invention exhibit skillful killing activity against various tumor target cells of the digestive system. (c) Nanobodies have significant advantages over other antibodies, such as the ability to regulate their half-life through chemical modification or protein fusion, strong permeability, the ability to recognize hidden epitopes inaccessible to ordinary antibodies, resistance to pepsin, acid, and heat, ease of production, and ease of assembly with other types of antibodies to form bivalent and multivalent antibodies because they are single-chain. Therefore, the nanobodies of the present invention can be developed into immunoassay reagents, CAR-T / NK immune cell drugs, and antibody drugs. Based on the characteristics of the CDH17 target, the candidate antibodies of this invention will have better effects if used as immunodiagnostic reagents for cancer; if developed into cell or antibody immunotherapies, they will have lower toxicity and better clinical efficacy, thus providing patients with more drug options.

[0046] (4) It can be used to develop dual-target drugs: Studies have found that Claudin18.2, like CDH17, is a safe and effective therapeutic target for digestive system tumors. Both are co-expressed in digestive system tumors such as esophageal cancer, gastric cancer, pancreatic cancer, and colorectal cancer. Clinical or preclinical trials have shown that targeting these two targets can specifically kill tumor cells without harming normal tissues. Therefore, the anti-CDH17 candidate nanobody of this invention can be used in combination with CLDN18.2 antibody to develop anti-CLDN18.2 / CDH17 bispecific antibody drugs and dual-target CAR-T drugs, which are expected to enhance the efficacy of monoclonal antibodies or single-target CAR-T / NK and other immune cell drugs. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the CDH17 protein structure, where Extracellular represents the extracellular region and Cellular represents the cytoplasmic region.

[0048] Figure 2 The results of ELISA testing of alpaca serum titers after 3 and 4 immunizations are shown. Serum dilution fold is the serum dilution factor.

[0049] Figure 3 Flow cytometry was used to detect the MF1 value of alpaca quaternary immune serum binding activity to ASPC-1 and SNU-16 in CDH17 antigen-expressing cells.

[0050] Figure 4 Flowchart for constructing the CDH17 alpaca immune nanobody library;

[0051] Figure 5 The output set enriched at the nanobody level represents the ELISA detection results for cross-binding with three antigens: Human-CDH17-ECD-His, CDH17-EC1-2-His, and Mus-CDH17-ECD-His. The horizontal axis VHH Con. represents the nanobody concentration (μg / mL).

[0052] Figure 6 The results are ELISA results showing that all enriched output sets at the phage level cross-bind with the three antigens Human-CDH17-ECD-His, CDH17-EC1-2-His, and Mus-CDH17-ECD-His. The horizontal axis PhageTiter represents the phage titer (CFU).

[0053] Figure 7 A comparison diagram of the amino acid sequences of 12 candidate nanobody clones (VHH1 is the positive control);

[0054] Figure 8The results of ELISA assays show the binding activity of the full-length expressed proteins of the Yangshen antibody Anti-CDH17-VHH-Fc and 12 candidate nanobody clones (E001, E005, E042, E046, E101, E114, E116, E119, E121, E201, E242 and E243E) fused with the Fc fragment of human IgG1 to the antigens CDH17-EC1-2-His, Mus-CDH17-ECD-His and CDH17-EC1-2-His, respectively. The x-axis, Antibody conc., represents the concentration (μg / mL) of the full-length expressed antibody protein.

[0055] Figure 9 The binding activity of the full-length expressed proteins of the fusion of the Yangshen antibody Anti-CDH17-VHH-Fc and 12 candidate nanobody clones (E001, E005, E042, E046, E101, E114, E116, E119, E121, E201, E242 and E243E) with the Fc fragment of human IgG1 on ASPC-1 and SNU-16 cells was measured by FACS. The x-axis, Antibody conc., represents the concentration (nM) of the full-length expressed antibody protein.

[0056] Figure 10 This is a schematic diagram of the universal CAR structure of the positive control CDH17 CAR and eight CDH17 candidate nanobody clones (E042, E046, E101, E116, E119, E121, E201 and E243).

[0057] Figure 11 The plasmid map of the third-generation lentiviral expression vector pCDH-EF1-Kan used to construct CDH17 CAR lentiviral expression plasmids for eight candidate nanobody clones (E042, E046, E101, E116, E119, E121, E201 and E243) and control antibody.

[0058] Figure 12 Flow cytometry plots showing the CAR positivity rates of anti-CDH17 CAR-T and positive control CDH17 CAR-T for eight candidate nanobody clones (E042, E046, E101, E116, E119, E121, E201 and E243);

[0059] Figure 13 The results of flow cytometry analysis of the CDH17 antigen positivity rate and the GFP protein expression positivity rate after the introduction of the GFP fluorescent marker gene in three target cells (AGS, ASPC-1 and SNU5) that are CDH17 antigen positive.

[0060] Figure 14The graph shows the killing efficiency of anti-CDH17 CAR-T and positive control CDH17 CAR-T of eight candidate nanobody clones (E042, E046, E101, E116, E119, E121, E201 and E243) against three target cells (AGS, ASPC-1 and SNU5) labeled with CDH17 antigen. Detailed Implementation

[0061] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0062] Example

[0063] 1. Raw materials for alpaca immunization and immune bank screening: antigens, antibodies, and cells.

[0064] This invention utilizes CDH17 antigen protein and CDH17 antigen-highly expressing cells as immunogens to repeatedly immunize alpacas (Alpaca strain) to construct an alpaca immune library, from which antibody molecules specifically binding to the target antigen CDH17 are screened. All raw materials used in immunization and screening, including antigens, cell lines, and positive control antibodies, were prepared or provided by Sanyou Biotechnology Co., Ltd., as detailed in Table 1. CDH17-ECD refers to an antigen protein containing all seven extracellular domains (ECD1-7) of the CDH17 antigen, while CDH17-EC1-2 refers to an antigen protein containing only the first two extracellular domains (ECD1-2) of the CDH17 antigen.

[0065] Table 1 Raw materials for antigens, cell lines, and positive control antibodies used in immunization and screening.

[0066]

[0067]

[0068] The amino acid sequence of the Fc tag in the aforementioned antigen protein originates from hIgG1-CS, the constant region of the human IgG1 heavy chain, where an amino acid C (cysteine) in its hinge region is mutated to S (serine) to reduce the presence of free C. The amino acid sequence and encoding nucleotide sequence of the Fc are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The aforementioned amino acid and encoding nucleotide sequences, as shown in SEQ ID NO: 5 and SEQ ID NO: 6, represent the Anti-CDH17-VHH fused with the Fc, derived from the anti-CDH17 VHH1 (anti-CDH17 VHH1) in patent US20210253728A1.

[0069] 2. Alpaca Immunization

[0070] 2.1 Alpaca Immunization Methods

[0071] Alpaca were cross-immunized with Human-CDH17-ECD-Fc antigen and ASPC-1 cells for a total of 4 immunizations, once every two weeks, with each immunization using 0.5 mg of antigen or 1E+07 (scientific counting method, i.e., 1×10⁻⁷). 7 Alpaca immunization: 1 cell / alpaca. Initial immunization with Freund's complete adjuvant; subsequent immunizations with Freund's incomplete adjuvant; cellular immunizations without adjuvant (purchased from Sigma). Injection method: subcutaneous multiple-point injection. Alpaca immunization schedule is shown in Table 2.

[0072] Table 2 Alpaca Immunization Program

[0073]

[0074] 2.2 Detection of immune serum titer

[0075] Alpaca serum was collected after 3 and 4 immunizations, and antibody titers were detected by ELISA and FACS, respectively.

[0076] 2.2.1 Serum titer ELISA detection

[0077] The serum titer ELISA test includes the following steps: (1) Coating the plate with Human-CDH17-ECD-his antigen (2 μg / mL, 30 μL) and incubating overnight at 4°C; (2) Washing the plate three times with PBST and blocking it with 5% skim milk powder (purchased from Yili) prepared with PBS at room temperature for 2 h; (3) Washing the plate three times with PBST, adding 30 μL of serum serially diluted with PBS, and incubating at room temperature for 1 h; (4) Washing the plate three times with PBST, adding secondary antibody Goat anti-Llama IgG (H+L) Secondary Antibody, HRP (ThermoFisher, A16060), and incubating at room temperature for 50 min; (5) Adding 30 μl TMB (purchased from SurModics) for color development and incubating at room temperature for 15-30 min; (6) Adding 50 μl TMB color development stop solution (purchased from Beyotime, P0215) to stop the reaction and reading the results from the ELISA reader (Molecular). Devices) OD450 value.

[0078] The test samples and controls were triple-immunized serum (CDH17-3rd), quadruple-immunized serum (CDH17-4th), negative control antibody NC (NC-CDH17), and positive control antibody PC. Serum titers were determined based on 1.65 times the NC titer, with a titer greater than 16K considered acceptable. Results showed that after 3 and 4 immunizations, the titers of alpaca immune serum against Human-CDH17-ECD-his reached 256K, suitable for library construction. Serum titer ELISA test results are detailed in Table 3 and [Table data missing]. Figure 2 .

[0079] Table 3. Results of ELISA detection of serum titers

[0080] Sample dilution factor Triple immunity serum Four-immune serum NC PC 1:2K 2.67 1.98 0.05 2.34 1:4K 2.58 1.64 0.06 1.92 1:8K 1.92 1.15 0.05 2.44 1:16K 1.39 0.58 0.06 2.53 1:32K 0.97 0.37 0.05 2.35 1:64k 0.73 0.24 0.05 1.47 1:128k 0.38 0.15 0.05 0.56 1:256K 0.27 0.11 0.06 0.13

[0081] 2.2.2 FACS detection of the binding of immune serum to cells

[0082] Add 1E5 cells / well to each well, centrifuge, and wash cells with FACS buffer [1×PBS buffer containing 2% FBS (Gibco)]. Add 100 μL of serum diluent to the cells, incubate at 4°C for 60 min, centrifuge, and wash cells three times with FACS buffer. Add 100 μL of Goat anti-Llama IgG (H+L), Fluorescein (FITC) Conjugate (Thermo Fisher, A16061), and incubate at 4°C for 30 min. Resuspend the cells in FACS buffer; analyze by flow cytometry to determine the mean fluorescence intensity (MF1).

[0083] The test samples were four-immune serum (CDH17-4th), negative control non-immune alpaca serum (NC-CDH17), isotype control antibody IgG (purchased from Sanyou, P78791) and positive control antibody (Reference Ab, i.e. Anti-CDH17-VHH-Fc, P76670, purchased from Sanyou).

[0084] The results showed that the four-immune serum samples maintained good binding with SNU-16 and ASPC-1 cells at dilutions of 20, 80, 320, and 1280 times. The results are shown in Tables 4 and 5. Figure 3 .

[0085] Table 4. FACS Detection MF1 Values ​​of Alpaca Quadruple Immunological Serum Binding to ASPC-1

[0086] Dilution factor Four-immune serum NC-CDH17 IgG (P78791) Reference Ab 1:20 88324 1672 442 2792 1:80 90931 1169 405 2510 1:320 65455 590 419 1704 1:1280 29190 415 436 914

[0087] Table 5. FACS detection MF1 values ​​of alpaca tertiary immune serum binding to SNU-16.

[0088] Sample dilution factor Four-immune serum NC-CDH17 IgG (P78791) Reference Ab 1:20 164023 5965 567 45757 1:80 171792 4082 513 27913 1:320 129986 1750 526 8253 1:1280 63651 854 524 2539

[0089] 3. Construction of an alpaca immune bank

[0090] 3.1 Construction of the immune library

[0091] Blood (80 mL) was collected from one alpaca after four immunizations. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque density gradient separation medium (GE, 17144003S). RNA was extracted from the isolated PBMCs and reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, 6210A). Because alpaca antibodies differ from ordinary antibodies in molecular form (lacking a light chain and CH1 in the heavy chain), primers were designed at the VH germline gene terminus and CH2 to obtain two fragments of different sizes via PCR. The smaller target fragment was recovered by gel excision. Degenerate primers containing NcoI and NotI restriction sites were designed by comparing all sequences of the V and J genes of the VHH antibody. The recovered DNA fragment product was then used as a template to amplify all VHH genes. Finally, the target antibody gene fragment was inserted into a phage display vector via double enzyme digestion and ligation. The VHH gene was fused to the GIII gene at the C-terminus. The ligation product was recovered using a recovery kit (Omega, D6492-02) and then transformed into competent *E. coli* SS320 (Lucigen, MC1061F) cells using an electroporator (Bio-Rad, MicroPulser). The transformed cells were then plated onto ampicillin-conjugated 2-YT agar plates (prepared from 1.5% tryptone, 1% yeast extract, 0.5% NaCl, and 1.5% agar, at a mass / volume ratio of g / mL). The resulting alpaca immunophage display library was then used to obtain candidate antibodies through a series of screening processes. The above process is as follows: Figure 4 As shown.

[0092] 3.2 Determination of Single-Domain Antibody Library Size

[0093] The number of clones formed by electroporation, i.e., the library capacity, was calculated based on the number of clones formed on a plate after dilution with 1 μL of bacterial culture. The library capacity results are shown in Table 6, indicating that the single-domain antibody library meets the requirement of a capacity greater than or equal to 1.00 × 10⁻⁶. 8 CFU quality standards.

[0094] Table 6. Electroporation system and library capacity statistics for single-domain antibody libraries

[0095] Number of electronic transfers (pieces) Single-unit transfer of storage capacity (CFU) Total storage capacity (CFU) 2 <![CDATA[3.69×10 8 ]]> <![CDATA[7.38×10 8 ]]>

[0096] 3.3 Analysis of Sequencing Results of Single-Domain Antibody Library

[0097] The correct insertion rate of the antibody gene was verified by single-clone sequencing analysis. A total of 80 clones were selected for sequencing, and the successful sequencing results were further analyzed. Effective library capacity was calculated as: total library capacity × correct antibody expression rate × proportion of unique sequences = effective library capacity. The results are shown in Table 7.

[0098] Table 7. Statistical Table of Sequencing Information for Single-Domain Antibody Libraries

[0099]

[0100] The results showed that the antibody gene sequence insertion accuracy was greater than or equal to 80%, the CDR region alignment had high richness, the empty region ratio was less than or equal to 10%, and the enzyme restriction site accuracy was greater than or equal to 90%, meeting the quality standards for single-domain antibody library construction. The single-domain antibody library constructed in this invention is of qualified quality and can be used for subsequent screening.

[0101] 4. Alpaca immune bank screening

[0102] The antibody sequences of the constructed alpaca immune library are displayed on the surface of phages using phage display technology. Then, using specific proteins and cells as antigen materials, the phage-displayed antibodies that bind to the antigen are continuously enriched through multiple rounds of screening. Positive clones that specifically bind to the antigen are further screened by ELISA, and the antibody sequences of the positive clones are obtained by sequencing.

[0103] 4.1 Immune library screening methods

[0104] The immune library screening method includes the following steps: (1) Phage preparation: The alpaca immune library bacteria or the obtained output collection are inoculated, assisted phage infection, phage amplification, phage precipitation and resuspension, etc., to prepare enriched phages and put them into the next screening step. (2) Solid phase screening: The antigen is coated on the surface of the immune tube with high adsorption capacity, and then the prepared phages are added to the immune tube for incubation, washing and elution. After three rounds of screening, the specific monoclonal antibodies against the antigen are enriched. (3) Liquid phase screening: Biotin-labeled antigens are combined with streptavidin-coupled magnetic beads, and then incubated, washed and eluted with the prepared phages. After three rounds of screening, the specific monoclonal antibodies against the antigen are enriched. (4) Cell screening: The prepared negatively screened phages and antigen-overexpressing cells are incubated, washed and eluted. After three rounds of screening, the specific monoclonal antibodies against the antigen are enriched.

[0105] The preliminary selection methods and results are detailed in Table 8. The results show that enrichment of varying degrees occurred from the second round onwards, as detailed in Table 8. The methods were: a. solid phase, b. liquid phase, and c. cellular phase.

[0106] Table 8. Preliminary Selection Data Statistics

[0107]

[0108]

[0109]

[0110] Note: N / A indicates not detected or not applicable.

[0111] (5) Preliminary selection and testing

[0112] The enrichment effect was tested using an ELISA experiment on the initial selection output set.

[0113] ① The ELISA detection steps for the nanobody VHH level are as follows: Add 2 μg / mL antigen (Human-CDH17-ECD-His, CDH17-EC1-2-His or Mus-CDH17-ECD-His) to the wells of a 96-well plate and coat the plate → Block with 5% PBSM → Incubate with VHH Pool (VHH output collection) dilution buffer → Incubate with secondary antibody Anti-Flag-HRP → Incubate with TMB for color development → Incubate with TMB color development stop solution → Measure the OD450 value.

[0114] The control antibody in the ELISA assay is the nanobody VHH, which targets an unrelated target, as the negative control, i.e., NC(VHH). There are two positive control antibodies: Anti-CDH17-VHH-Fc (anti-human CDH17) is used as the positive control in the detection of binding to human antigens Human-CDH17-ECD-His and CDH17-EC1-2-His; and the VHH-Fc antibody against mouse CDH17, i.e., H_PR_A124 (purchased from Sanyou, P80612), is used as the positive control in the detection of binding to mouse antigen Mus-CDH17-ECD-His.

[0115] ② The ELISA detection procedure for phage levels is as follows: Add 2 μg / mL of antigen (Human-CDH17-ECD-His, CDH17-EC1-2-His, or Mus-CDH17-ECD-His) to the wells of a 96-well plate and coat the plate → Block with 5% PBSM → Incubate with Phage Pool dilution buffer → Incubate with secondary antibody Anti-M13-HRP → Incubate with TMB for color development → Incubate with TMB stop solution → Measure the OD450 value; Blank Phage is used as a negative control, and NC (Phage) is also included. Simultaneously, 1xPBS buffer is used as a blank control (BC) to replace the antigen, and parallel ELISA detection is performed.

[0116] The results of the preliminary selection and testing show that:

[0117] At the VHH level: all pools (output sets) showed cross-enrichment with Human-CDH17-ECD-His, CDH17-EC1-2-His, and Mus-CDH17-ECD-His antigens.

[0118] At the Phage level: all pools showed cross-enrichment with Human-CDH17-ECD-His, CDH17-EC1-2-His, and Mus-CDH17-ECD-His antigens; all pools did not bind nonspecifically to BC.

[0119] The ELISA test results of the preliminary selection group are shown below. Figure 5 (VHH level) and Figure 6 (Phage level)

[0120] (6) Initial screening of monoclonal antibodies

[0121] Single clones were initially screened from pools with enriched antigens. The process involved a series of steps, including target antigen coating, blocking, incubation, addition of secondary antibody, color development, termination, and OD value detection. Positive clones were identified based on a specific background value and then sequenced.

[0122] Screening was performed using separate plates for Human-CDH17-ECD-His, CDH17-EC1-2-His, and Mus-CDH17-ECD-His, resulting in 1656 clones selected from 19 monoclonal plates. Ultimately, 623 positive clones were obtained that cross-linked with Human-CDH17-ECD-His, CDH17-EC1-2-His, and Mus-CDH17-ECD-His. Sequence analysis yielded 36 unique single-domain antibody (also known as nanobody) molecules. The nucleotide sequences of 12 antibody clones (E001, E005, E042, E046, E101, E114, E116, E119, E121, E201, E242, and E243) are shown in SEQ ID NO:82–93, and their encoded amino acid sequences are shown in SEQ ID NO:9–20. Comparative analysis of their amino acid sequences yielded the following results: Figure 7 As shown in Table 9, the VHH amino acid sequences of the 12 nanobody clones were analyzed using the Kabat numbering and definition schemes, and their CDRs 1-3 were determined. VHH1 is the positive control antibody, derived from anti-CDH17VHH1 in patent US20210253728A1.

[0123] Table 9. Amino acid sequences of the three complementarity-determining regions (CDR1-3) of the twelve nanobodies.

[0124]

[0125]

[0126] 5. Full-length expression and physicochemical detection of candidate nanobody molecules

[0127] 5.1 Construction of expression plasmids for full-length antibody proteins:

[0128] The encoding nucleotide sequence of the nanobody was fused with the encoding nucleotide sequence of hIgG1-CS (Fc, whose amino acid sequence and encoding nucleotide sequence are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively) for whole-gene synthesis and constructed into the pcDNA3.4 vector (Invitrogen) to obtain the VHH-Fc fusion protein expression plasmid.

[0129] 5.2 Expression and purification of full-length antibody protein

[0130] The constructed antibody plasmid was transfected into Expi CHO cells (Gibco, A29133) for transient expression. The antibody expression time was 7 days, and the expression volume was 2 mL to 10 mL. After expression, the protein was purified and dispensed.

[0131] 5.3 SDS-PAGE Identification of Full-Length Antibody Proteins: Methods and Procedures for SDS-PAGE Identification of Full-Length Antibody Proteins:

[0132] (1) Preparation of purified protein sample solution:

[0133] Preparation of non-reducing sample solution: The purified sample solution, 4×LDS loading buffer (Zeye Biotechnology, ZY6SL1197) and iodoacetamide (Zeye Biotechnology, ZY144) were mixed in proportion to ensure that the final concentration of iodoacetamide was 40mM and the loading amount of non-reducing sample was 1μg. The mixed sample was heated in a 75℃ dry bath for 10min.

[0134] Preparation of reduced sample solution: The purified sample solution, 4×LDS loading buffer and dithiothreitol (DTT) (Zeye Biotechnology, ZY3483) were mixed in proportion to ensure that the final concentration of DTT was 5mM and the loading amount of reduced sample was 2μg. The mixed sample was heated in a 100℃ dry bath for 10min.

[0135] (2) Electrophoresis: 140V, 75min.

[0136] (3) Staining, destaining, and scanning: Gel Coomassie Brilliant Blue staining, followed by destaining and scanning with an EPSON V550 color scanner. (4) Purity calculation: ImageJ was used to calculate the purity of the reduced bands according to the peak area normalization method, or the purity of the reduced heavy chain plus the light chain.

[0137] System suitability: The reference IPI (Ipilimumab) has a non-reduced band molecular weight of approximately 150 kDa and a purity greater than 90%; the reduced heavy chain has a molecular weight of approximately 50 kDa, the light chain has a molecular weight of approximately 25 kDa, and the total purity of the heavy and light chains is greater than 90%.

[0138] 5.4 SEC Identification of Full-Length Antibody Protein

[0139] Methods and procedures for SEC identification of full-length antibody proteins: (1) Mobile phase preparation: Prepare 0.15M PB+NaCl and adjust the pH to 6.0. (2) Sample preparation: Dilute the sample concentration to 0.5 mg / mL. (3) Column conditions: Use XBridge BEH (Purchased from Waters), SEC 3.5μm, 7.8×300mm, column temperature set at 20℃, detection baseline stable. (4) Parameter settings: flow rate set at 0.8mL / min; sample injection volume set at 20μL; detection wavelength 280nm, bandwidth 4nm, reference wavelength 360nm, bandwidth 100nm, peak width (response time) > 0.1min (2s response time); slit 4nm; negative absorbance baseline 100mAU.

[0140] Systemic fitness criteria: The reference product Herceptin (trastuzumab) has a monomer purity greater than 95%, the separation degree between BSA monomer and dimer is greater than 1.5, and the baseline is stable, then the systemic fitness is considered to be passed.

[0141] The physicochemical properties of the 12 candidate antibody Fc fusion proteins constructed and expressed in full length are shown in Table 10:

[0142] Table 10 Summary of physicochemical properties of Fc fusion proteins from twelve candidate nanobody clones

[0143]

[0144] Notes: 1) " / " indicates not detected. 2) SDS-PAGE test showed that the reference IPI band size was correct and the purity was greater than 95%. SEC test showed that the reference Herceptin monomer purity was greater than 95%, the separation between BSA monomer and dimer was greater than 1.5, and the baseline was stable, all of which met the system suitability requirements.

[0145] 5.5 Affinity kinetics of full-length antibody proteins

[0146] (1) Solution preparation

[0147] Q Buffer solution: Prepare a kinetic buffer solution with 1xPBS to a final concentration of 0.025% Tween 20 and 0.2% BSA (IgG-Free Protein-Free).

[0148] R Buffer solution: Prepare a solution containing 10 mM glycine and 150 mM sodium chloride using ultrapure water, and adjust the pH to 2.0 with hydrochloric acid.

[0149] (2) Affinity kinetics of full-length antibody protein

[0150] Methods and Procedures: ① Select Mode: Turn on the Gator Label-Free Biomolecular Analyzer (purchased from Probe Life) and related software, and select the Kinetics experimental mode. ② Analytical Procedure: The affinity assay process includes equilibration, capture, equilibration, binding, dissociation, and regeneration. First, after baseline equilibration, use the Protein A probe (purchased from Probe Life) to capture the antibody to be tested. After reequilibration, the antibody to be tested binds to and dissociates from serially diluted antigens. Finally, probe regeneration is performed. See Table 11 for details.

[0151] Table 11 Affinity kinetics detection process (Gator)

[0152]

[0153]

[0154] (2) Affinity kinetics test results

[0155] Table 12 shows the results of affinity tests between various antibody samples and the antigen using human CDH17-EC1-2-His (P67889) as the antigen.

[0156] K detected by the Gator label-free biomolecule analyzer D The limit is 1E-06M. Experimental results show that the correlation coefficient R of all antibodies in the Global fitting mode is... 2 All values ​​were greater than 0.95, meeting the system adaptability requirements and indicating reliable results. The affinity levels of the full-length protein samples of all 12 candidate antibodies to the antigen reached the nM or 100pM level, while the positive control antibody Anti-CDH17-VHH-Fc showed an affinity level of 100pM to the antigen.

[0157] Table 12 Affinity kinetics test results (Gator)

[0158]

[0159] Note: R 2 : Linear fitting constant; Rmax: Maximum response value of curve fitting.

[0160] The affinity test results for candidate antibody samples using the mouse antigen protein Mus-CDH17-ECD-His (P98752) as the antigen are shown in Table 13. The experimental results show that the correlation coefficient R for all antibodies in the Global Fitting Mode is... 2 All values ​​were greater than 0.95, meeting the system adaptability requirements and indicating reliable results. The results showed that test samples P210390 (E042) and P210434 (E243) could not fit the dissociation trend data with the antigen within a 180-second dissociation time. The affinity levels of the remaining 10 test samples with the mouse antigen reached the 10 nM-10 pM level. The positive control antibody Anti-CDH17-VHH-Fc showed an affinity level of 10 nM with the antigen.

[0161] Table 13 Affinity kinetics test results (Gator)

[0162]

[0163]

[0164] Note: 1) R 2 : Linear fitting constant; Rmax: Maximum response value of curve fitting.

[0165] 5.6 ELISA detection of full-length antibody protein

[0166] Methods and steps for ELISA detection of full-length antibody protein: (1) Plate coating: Dilute the antigen to a concentration of 2 μg / mL with 1×PBS, add 30 μL / well to a 96-well ELISA plate, and coat overnight at 4℃. (2) Blocking: Wash the plate 3 times with PBST, add blocking buffer (5% PBSM), and block at room temperature for 2 h. (3) Incubation: Wash the plate, add 30 μL / well of sample diluted with 1% PBSM, and incubate at room temperature for 60 min. (4) Secondary antibody incubation: Wash the plate 3 times with PBST, add secondary antibody, and incubate at room temperature for 50 min. (5) Color development: Wash the plate 3 times with PBST, add 30 μL TMB (purchased from SurModics) to each well. (6) Termination: Add 50 μL TMB colorimetric stop solution to terminate the reaction, and simultaneously detect OD. 450 .

[0167] In the above tests, the negative control antibody NC was IPI ( Ipilimumab The blank control BC was 1% PBSM.

[0168] The ELISA results of antibody samples and antigens using CDH17-EC1-2-His, Mus-CDH17-ECD-His, and CDH17-EC1-2-His as antigens are as follows: Figure 8 As shown in Table 14, all 12 full-length antibodies exhibited strong binding activity to CDH17-EC1-2-His and CDH17-EC1-2-His, and also showed varying degrees of binding activity to Mus-CDH17-ECD-His.

[0169] Table 14 Summary of ELISA detection EC50 (μg / mL) of full-length expressed proteins of purified candidate antibody clones

[0170]

[0171]

[0172] Note: / indicates not tested, N indicates no binding, W indicates weak binding, Y indicates binding, but EC cannot be calculated. 50 value.

[0173] 5.7 FACS detection of full-length antibody protein

[0174] The binding activity of 12 full-length antibody proteins to ASPC-1 and SNU-16 cells expressing two antigens was detected by FACS. The methods and procedures are as follows:

[0175] (1) Cell plating: Transfer cells from culture flasks to centrifuge tubes, centrifuge to remove supernatant, resuspend in culture medium, count, and adjust cell density to 1×10⁻⁶. 6 cells / mL. Take a 96-well round-bottom plate and add 100 μL of cells to each well using a 100 μL pipette. Centrifuge at 300 g / min for 5 min. Discard the supernatant.

[0176] (2) Addition of protein supernatant dilution buffer: The full-length antibody protein sample was diluted with FACS buffer (1×PBS buffer containing 2% FBS) to prepare eight concentration gradients: 150.000, 37.500, 9.375, 2.3438, 0.5859, 0.1465, 0.0366, and 0.0092 nM. Using a 100 μL 12-channel pipette, 100 μL of the antibody dilution buffer was added to each well of a 96-well cell plate. After mixing, the plate was incubated at 4°C for 1 h.

[0177] (3) Addition of secondary antibody: Dilute the secondary antibody PE labelled anti-Human Fc (purchased from Jackson) 1:200 with FACS buffer. Centrifuge the cell culture plate to remove the supernatant, and add 100 μL of the secondary antibody dilution buffer to the cell culture plate using a 100 μL 12-channel pipette. Incubate the cell culture plate at 4°C for 30 min. Centrifuge to remove the supernatant, wash the plate twice with FACS buffer, and resuspend the cells in 120 μL of FACS buffer in each well.

[0178] In the above tests, the positive control antibody was Anti-CDH17-VHH-Fc (P76673), the isotype control antibody was IgG1 (P93950-1, purchased from Sanyou), the control with only secondary antibody was labeled "cell sec", and the blank cell control without any antibody was labeled "cell only".

[0179] (4) Data acquisition: Turn on the flow cytometer (purchased from Beckman), and after the instrument is cleaned, perform FACS binding detection.

[0180] FACS detection results of antibody samples and antigen-expressing cells are as follows Figure 9 As shown in Table 15, the results indicate that all 12 full-length antibodies exhibit strong binding activity to ASPC-1 and SNU-16.

[0181] Table 15. FACS detection of full-length expressed proteins of purified candidate antibody clones (EC). 50 Summary of Results (nM)

[0182]

[0183]

[0184] 6. Structural Design of Anti-CDH17 CARs

[0185] Nine chimeric antigen receptors (Anti-CDH17 CARs) targeting CDH17 were designed, and their general CAR structure diagram is shown below. Figure 10As shown. Anti-CDH17 CARs include a CD8α signal peptide (SP), an anti-CDH17 nanobody (anti-CDH17VHH), an amino acid mutation-optimized hinge region of IgG4 (IgG4mH), a transmembrane region of CD28 (CD28TM), an intracellular co-stimulatory domain of CD28, a 4-1BB co-stimulatory domain sequence, and a CD3ζ signaling domain. The amino acid sequences of the VHH nanobody expressed by the eight CARs are derived from eight nanobody clones of this invention: E042, E046, E101, E116, E119, E121, E201, and E243, respectively, and their corresponding CAR names are abbreviated as E042 CAR, E046 CAR, E101 CAR, E116 CAR, E119 CAR, E121 CAR, E201 CAR, and E243 CAR. The amino acid and encoding nucleotide sequences of the nanobody expressed by the positive control CAR (abbreviated as CDH17 CAR) are the anti-CDH17 VHH1 sequence in patent US20210253728A1.

[0186] The amino acid and encoding nucleotide sequences of SP are shown in SEQ ID NO:60-61, respectively; the amino acid and encoding nucleotide sequences of IgG4mH are shown in SEQ ID NO:62-63, respectively; the amino acid and encoding nucleotide sequences of CD28TM are shown in SEQ ID NO:64-65, respectively; the amino acid and encoding nucleotide sequences of the intracellular co-stimulatory domain of CD28 are shown in SEQ ID NO:66-67, respectively; the amino acid and encoding nucleotide sequences of the 4-1BB co-stimulatory domain are shown in SEQ ID NO:68-69, respectively; the amino acid and encoding nucleotide sequences of the CD3ζ signal transduction domain are shown in SEQ ID NO:70-71, respectively; and the full-length amino acid and encoding nucleotide sequences of CDH17 CAR as a positive control are shown in SEQ ID NO:72-73, respectively.

[0187] 7. Construction of lentiviral expression plasmids for Anti-CDH17 CARs

[0188] (1) Codon optimization and synthesis of the encoding nucleotides of Anti-CDH17 VHH: The VHH encoding nucleotide sequences of eight nanobody clones were first optimized using human codons, and then DNA was synthesized. The encoding nucleotide sequences of the eight optimized candidate antibody clones E042, E046, E101, E116, E119, E121, E201 and E243 are shown in SEQ ID NO:74~81, respectively.

[0189] (2) Vector linearization by enzyme digestion: The expression plasmid pCDH-EF1-Kan of the third-generation lentivirus (purchased from Fenghui Biotechnology, its plasmid map is shown below) was used. Figure 11 The vector backbone is shown. The vector is linearized by double digestion at the XbaI and EcoRI restriction sites.

[0190] (3) Seamless cloning: Using conventional seamless cloning technology, all DNA fragments in the PCR-amplified CAR structure are cloned into the linearized pCDH-EF1-Kan vector backbone to construct an Anti-CDH17CAR expression plasmid with 8 nanobodies and 1 positive control, called pCDH17 CAR.

[0191] 8. Lentiviral packaging and titer determination

[0192] Lentiviral packaging employed a standard four-plasmid system, with all four plasmids being kanamycin resistant. 293T cells (ThermoFisher) were used as the lentiviral packaging cells. The lentiviral expression plasmid pCDH17 CAR was co-transfected with three helper plasmids (pMDLg / pRRE, pRSV-Rev, and pMD2.G, all purchased from Fenghui Biotechnology) in a plasmid ratio of 4:2:2:1. For T75 cell culture flasks, the total plasmid volume was 20 μg, resulting in the use of 8.8 μg, 4.4 μg, 4.4 μg, and 2.2 μg of the four plasmids, respectively. The transfection reagent PEI was used at a volume three times the total plasmid volume; for T75 culture flasks, the PEI volume was 60 μg (1 μg / μl, 60 μl). The plasmid and PEI transfection reagent were added separately to serum-free culture medium and mixed thoroughly. After mixing, the mixture was incubated for 15 minutes. Then, the mixture was added to a T75 culture flask containing 293T cells, gently mixed, and incubated at 37°C in a 5% CO2 cell culture incubator for 6 hours. After 6 hours, the medium was replaced with fresh medium containing 2% FBS, and the culture was continued. 48 hours after transfection, the lentivirus culture supernatant was collected, centrifuged (2000 rpm, 15 min), and the supernatant was filtered through a 0.45 μm filter. The supernatant was then concentrated by ultracentrifugation (25000 rpm, 3 h). The viral pellet was then resuspended in the appropriate volume of culture medium according to the dilution factor, aliquoted, and stored at -80°C.

[0193] For the titer determination of CDH17 CAR lentivirus, the lentivirus stock solution or concentrate was serially diluted and transfected into 293T cells. The transfection efficiency was detected by flow cytometry after 72 hours, and the active titer of the lentivirus was calculated.

[0194] 9. Preparation and culture of CDH17 CAR-T

[0195] Frozen PBMCs were thawed and cultured overnight. The suspended PBMCs were then centrifuged and resuspended in X-VIVO15 medium (LONZA) containing 300 IU / mL IL-2, and the cell density was adjusted to 1.5 × 10⁻⁶ cells / mL. 6 At a concentration of 17.5:1 (PBMC volume: activator volume), MACS GMP T Cell TransAct (Milteny Biotec) was added to activate the cells. After 30 hours, the cells were centrifuged and the medium (containing 300 IU / mL IL-2 in X-VIVO 15) was replaced. Cell counts were performed, and CDH17 CAR lentivirus was added at an MOI of 5. The mixture was aspirated and incubated at 37°C and 5% CO2 for 16 hours. An equal volume of medium was added, and the cells were cultured for an additional 16 hours. Cell counts were then performed every two days, and the cell density was maintained at 0.5–2 × 10⁶ cells / ml by adding or replacing the medium. 6 / ml, and continue culturing for 8-14 days (starting from cell activation). During this period, the CAR expression positivity rate of CDH17 CAR-T cells was detected by flow cytometry, as follows:

[0196] Take CAR-T cell suspension containing 1E6 cells, centrifuge at 300g for 5 min, discard the supernatant, and resuspend in 50 μL PBS; add 1-3 μL CDH17 antigen CDH17 Protein-His Tag (Acro), and incubate at room temperature in the dark for 20 min; add 500 μL PBS, mix well, and centrifuge at 300g for 5 min; discard the supernatant, and resuspend in 50 μL PBS; add CD3 antibody Anti-CD3-APC (Biolegend) and secondary antibody PE anti-His Tag (Biolegend), mix well, and incubate at room temperature in the dark for 20 min; add 500 μL PBS, mix well, and centrifuge at 300g for 5 min; discard the supernatant, resuspend the cells in 200 μL PBS, and analyze. Analyze the proportion of CAR-positive T cells in the CD3-positive cell population, which is the CAR positivity rate of CAR-T cells. In one specific embodiment, the flow cytometry results of the CAR positivity rate of nine types of CAR-T cells 9 days after activation (D9) are as follows: Figure 12 As shown in Table 16.

[0197] Table 16 CAR positivity rate of anti-CDH17 CAR-T cells (D9)

[0198]

[0199] 10. CDH17 CAR-T in vitro killing function test

[0200] Three target cell lines were used: gastric adenocarcinoma AGS (purchased from the Shanghai Academy of Sciences Cell Bank), pancreatic cancer ASPC-1 (purchased from the Chinese Academy of Sciences Cell Bank), and gastric cancer SNU-5 (purchased from the Chinese Academy of Sciences Cell Bank), all of which were natively expressing the CDH17 antigen and infused with the green fluorescent protein (GFP) marker gene. Flow cytometry results of the positive expression rates of CDH17 antigen and GFP marker protein in the three target cell lines are shown below. Figure 13 express.

[0201] Killing experiment setup: Nine types of CAR-T cells and control T cells (MOCK-T cells) were used as effector cells and co-incubated with three types of target cells. The effector-to-target ratio was set at 2:1 (Note: effector cells are the total number of cells). Each effector-target cell was co-incubated in three replicates. A blank control containing only target cells was also set up. (1) Target plating: In a 96-well plate, 100 μL of target cells with a cell density of 2E5 / ml were added to each well. S3 real-time live cell imaging instrument (purchased from Sartorius), set the target cell green fluorescence imaging parameters, and culture overnight at 37℃ and 5% CO2; (2) the next day, add 100ul of effector cells with a cell density of 4E5 / ml to the corresponding well, and add 100ul of culture medium to the blank control with only target cells. Place the 96-well plate into The S3 real-time live cell imaging system (purchased from Sartorius) was incubated for another 48 hours at 37°C and 5% CO2.

[0202] Results Analysis: Analysis was performed based on real-time green fluorescence images of target cells captured by IncuCyte. The most recent image capture point before the start of co-incubation with effector cells was used as the time point for normalizing the target cell fluorescence area. The endpoint of co-incubation was used as the endpoint to plot the target cell growth curve, i.e., the effector cell killing curve. The killing efficiency of effector cells was calculated based on the target cell green fluorescence value corresponding to the endpoint of co-incubation on the killing curve. The calculation formula is: Killing efficiency = [(Endpoint fluorescence value of blank target cells - Endpoint fluorescence value of effector-target co-incubated cell group) / Endpoint fluorescence value of blank target cells] × 100%. A bar chart of killing efficiency was plotted based on the calculated killing efficiency results (see...). Figure 4 As can be seen, all nine CAR-T cells exhibited significant killing effects against the three CDH17 target cell types, with statistically significant differences in killing efficacy between 1* (P<0.05) and 4* (P<0.0001) compared to MOCK-T. Among them, the killing efficiency of E119CAR-T and E121CAR-T was relatively low, which is related to their lower CAR-positive rates. Except for E119CAR-T and E121CAR-T, the CAR-T killing efficiency of the other six candidate antibodies was comparable to or higher than that of the control CAR-T. Furthermore, the nine CAR-T cells showed better killing effects against AGS and SNU-5 gastric cancer cells than against ASPC-1 pancreatic cancer cells.

[0203] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A nanobody targeting cadherin 17, characterized in that, The complementarity-determining regions of the nanobody targeting cadherin 17 include CDR1 (amino acid sequence as shown in SEQ ID NO:24), CDR2 (amino acid sequence as shown in SEQ ID NO:37), and CDR3 (amino acid sequence as shown in SEQ ID NO:50).

2. The nanobody targeting cadherin 17 according to claim 1, characterized in that, The amino acid sequence of the nanobody targeting cadherin 17 is shown in SEQ ID NO:

12.

3. A CAR-T cell targeting cadherin 17, characterized in that, The CAR-T cells express a chimeric antigen receptor targeting cadherin 17; the chimeric antigen receptor targeting cadherin 17 includes a CDH17 antigen-binding domain, a hinge region, a transmembrane region, and an intracellular signal transduction domain; the CDH17 antigen-binding domain includes a signal peptide and a nanobody as described in claim 1 or 2.

4. The CAR-T cell targeting cadherin 17 according to claim 3, characterized in that, The signal peptide is the CD8α signal peptide, the hinge region is the hinge region of IgG4 with amino acid mutation optimization, the transmembrane region is the transmembrane region of CD28, and the intracellular signal transduction domain includes the CD28 intracellular co-stimulatory domain, the 4-1BB co-stimulatory domain, and the CD3ζ signal transduction domain.

5. The CAR-T cell targeting cadherin 17 according to claim 4, characterized in that, The amino acid sequence of the signal peptide of CD8α is shown in SEQ ID NO:60; The amino acid sequence of the hinge region of the amino acid mutation-optimized IgG4 is shown in SEQ ID NO:

62. The amino acid sequence of the transmembrane region of CD28 is shown in SEQ ID NO:64; The amino acid sequence of the intracellular co-stimulatory domain of CD28 is shown in SEQ ID NO:66; The amino acid sequence of the 4-1BB co-stimulatory domain is shown in SEQ ID NO:68; The amino acid sequence of the CD3ζ signal transduction domain is shown in SEQ ID NO:

70.

6. A nucleic acid, characterized in that, It includes a nucleic acid sequence encoding a nanobody targeting cadherin 17 as described in any one of claims 1 to 2, or a nucleic acid sequence encoding a chimeric antigen receptor targeting cadherin 17 as described in any one of claims 3 to 5.

7. A nucleic acid according to claim 6, characterized in that, The nucleotide sequence encoding the signal peptide of CD8α is shown in SEQ ID NO:61; The encoding nucleotide sequence of the anti-CDH17 nanobody is shown in SEQ ID NO:75; The nucleotide sequence encoding the hinge region of the amino acid mutation-optimized IgG4 is shown in SEQ ID NO:

63. The nucleotide sequence encoding the transmembrane region of CD28 is shown in SEQ ID NO:65; The nucleotide sequence encoding the intracellular co-stimulatory domain of CD28 is shown in SEQ ID NO:67; The nucleotide sequence encoding the 4-1BB co-stimulatory domain is shown in SEQ ID NO:69; The coding nucleotide sequence of the CD3ζ signal transduction domain is shown in SEQ ID NO:

71.

8. A lentiviral vector, characterized in that, The lentiviral vector comprises the nucleic acid as described in claim 6 or 7.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a nanobody targeting cadherin 17 as described in any one of claims 1 to 2, or a CAR-T cell targeting cadherin 17 as described in any one of claims 3 to 5, or a nucleic acid as described in any one of claims 6 to 7, or a lentiviral vector as described in claim 8.

10. The use of a nanobody targeting cadherin 17 as described in any one of claims 1 to 2, or a CAR-T cell targeting cadherin 17 as described in any one of claims 3 to 5, or a nucleic acid as described in any one of claims 6 to 7, or a lentiviral vector as described in claim 8, or a pharmaceutical composition as described in claim 9, in the preparation of a detection reagent or in vivo imaging probe targeting cadherin 17.

11. The use of the CAR-T cells targeting cadherin 17 as described in any one of claims 3 to 5 in the preparation of products for treating any tumor of pancreatic cancer or gastric cancer.

Images