Humanized nano antibody targeting cadherin 17 and application thereof

Abstract

The invention relates to a humanized nano antibody targeting cadherin 17 and application of the humanized nano antibody, and relates to the technical field of immunology and molecular biology. The complementarity determining region of the humanized nano antibody comprises a CDR1 of which the amino acid sequence is shown as SEQ ID NO: 2, a CDR2 of which the amino acid sequence is shown as SEQ ID NO: 4 and a CDR3 of which the amino acid sequence is shown as SEQ ID NO: 6; a framework region of the humanized nano antibody comprises an FR1 of which the amino acid sequence is shown as any one of SEQ ID NO: 1 and SEQ ID NO: 8, an FR2 of which the amino acid sequence is shown as any one of SEQ ID NO: 3, SEQ ID NO: 9 and SEQ ID NO: 11, an FR3 of which the amino acid sequence is shown as any one of SEQ ID NO: 5 and SEQ ID NO: 10, and an FR4 of which the amino acid sequence is shown as SEQ ID NO: 7. According to the humanized nano-antibody, the immunogenicity of the nano-antibody is reduced, the targeted binding activity and killing activity are improved, and the humanized nano-antibody can be used for developing immunodetection reagents, CAR-T / NK cell drugs and antibody drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology and molecular biology, and in particular to a humanized nanobody targeting cadherin 17 and an application thereof. Background Art

[0002] 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. CDH17 was first discovered by Dietmar et al. in mouse hepatocytes in 1994. Since it is only expressed in the liver and small intestine in mice, it is named liver intestine-cadherin. Unlike classical cadherins, the extracellular domain of CDH17 has seven repeating structural units (EC1-EC7), and the intracellular domain of CDH17 contains only 24 amino acid residues, which lacks homology with the intracellular domain of classical cadherins consisting of 150-160 amino acids.

[0003] Nanobody (Nb) is a type of antibody that contains only the variable region (VHH) of heavy chain antibodies. In 1993, Belgian scientists first reported a special antibody found in the blood of camelids that naturally lacks light chains, namely heavy-chain antibodies (HCAbs). Although the molecular weight of nanobody is only 1 / 10 of that of complete traditional antibodies (about 15kDa), it retains the complete antigen recognition and binding ability of heavy chain antibodies. Compared with traditional antibodies, it has the advantages of strong specificity, strong affinity, high stability, strong targeting and tissue penetration, low immunogenicity, and easy humanization. The disadvantage of nanobody is that it has a short half-life, but it can be fused with anti-serum albumin or the Fc segment of the antibody to extend its half-life in the blood. Nanobody has been widely used in the study of biochemical mechanisms and structural biology, as well as in the development of diagnostic reagents and therapeutic drugs for diseases such as tumors.

[0004] Current studies have proven 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 immune cell drugs for diseases such as digestive system tumors. However, the currently available anti-CDH17 candidate antibody molecules are very limited, and they are mainly traditional antibodies. There is only one report of nano antibodies with outstanding advantages and good drugability, which comes from the research team of the University of Pennsylvania and is developed by Chimeric Therapeutics. The world's first CDH17 CAR-T has entered clinical phase II. Although nano antibodies have better properties than traditional antibodies, due to their animal origin, the potential immunogenicity risk of their clinical application cannot be ignored. This immunogenicity will reduce the durability and efficacy of targeted drugs and lead to safety risks. Therefore, nano antibodies need to be further humanized to reduce clinical risks. However, to date, there are no reports on the research of humanized anti-CDH17 nano antibodies. In order to promote the research and development of safer and more effective CDH17 targeted drugs, it is necessary to accelerate the development of more candidate anti-CDH17 nanoantibody molecules, especially humanized nanoantibodies. In view of this, the present invention provides humanized nanoantibodies targeting cadherin 17 and their applications. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a humanized nanobody targeting cadherin 17 and its application. The purpose is to obtain a humanized antibody by humanizing a camelid antibody.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] In the first aspect, a humanized nanobody targeting E-cadherin 17, the complementarity determining region of the humanized nanobody includes a CDR1 with an amino acid sequence as shown in SEQ ID NO: 2, a CDR2 with an amino acid sequence as shown in SEQ ID NO: 4, and a CDR3 with an amino acid sequence as shown in SEQ ID NO: 6; the framework region of the humanized nanobody includes a FR1 with an amino acid sequence as shown in any one of SEQ ID NO: 1 and SEQ ID NO: 8, an FR2 with an amino acid sequence as shown in any one of SEQ ID NO: 3, SEQ ID NO: 9 and SEQ ID NO: 11, an FR3 with an amino acid sequence as shown in any one of SEQ ID NO: 5 and SEQ ID NO: 10, and an FR4 with an amino acid sequence as shown in SEQ ID NO: 7.

[0008] Further, a humanized nanobody targeting E-cadherin 17, wherein the humanized nanobody includes any one of E046-VHH5, E046-VHH7, and E046-VHH10;

[0009] The framework region of the amino acid sequence of E046-VHH5 includes FR1 as shown in SEQ ID NO: 1, FR2 as shown in SEQ ID NO: 3, FR3 as shown in SEQ ID NO: 5, and FR4 as shown in SEQ ID NO: 7;

[0010] The framework region of the amino acid sequence of E046-VHH7 includes FR1 as shown in SEQ ID NO:8, FR2 as shown in SEQ ID NO:9, FR3 as shown in SEQ ID NO:10, and FR4 as shown in SEQ ID NO:7;

[0011] The framework region of the amino acid sequence of E046-VHH10 includes FR1 as shown in SEQ ID NO:8, FR2 as shown in SEQ ID NO:11, FR3 as shown in SEQ ID NO:5, and FR4 as shown in SEQ ID NO:7.

[0012] Furthermore, the amino acid sequence of the humanized nanobody targeting E-cadherin 17 is shown in any one of SEQ ID NOs: 19 to 21.

[0013] In the second aspect, a CAR-T cell targeting E-cadherin 17, the CAR-T cell expresses a chimeric antigen receptor targeting E-cadherin 17; the chimeric antigen receptor targeting E-cadherin 17 includes a CDH17 antigen binding domain, a hinge region, a transmembrane region and an intracellular signaling domain; the CDH17 antigen binding domain includes a signal peptide and any one of the humanized nanoantibodies as described in claim 1 or 2.

[0014] Furthermore, the signal peptide is a signal peptide of CD8α, the hinge region is a hinge region of IgG4 with optimized amino acid mutations, the transmembrane region is a transmembrane region of CD28, and the intracellular signaling domain includes a CD28 intracellular co-stimulatory domain, a 4-1BB co-stimulatory domain and a CD3ζ signaling domain.

[0015] Furthermore, the amino acid sequence of the signal peptide of CD8α is shown in SEQ ID NO:31;

[0016] The amino acid sequence of the hinge region of the amino acid mutation-optimized IgG4 is shown in SEQ ID NO: 33;

[0017] The amino acid sequence of the transmembrane region of CD28 is shown in SEQ ID NO:35;

[0018] The amino acid sequence of the CD28 intracellular co-stimulatory domain is shown in SEQ ID NO: 37;

[0019] The amino acid sequence of the 4-1BB costimulatory domain sequence is shown in SEQ ID NO: 39;

[0020] The amino acid sequence of the CD3ζ signaling domain is shown in SEQ ID NO:41.

[0021] In the third aspect, a nucleic acid comprises a nucleic acid sequence encoding the humanized nanobody targeting E-cadherin 17 or its complementary sequence, or a nucleic acid sequence encoding the chimeric antigen receptor targeting E-cadherin 17 or its complementary sequence.

[0022] Furthermore, the nucleotide sequence of the signal peptide of CD8α is shown in SEQ ID NO:32;

[0023] The nucleotide sequence of the humanized anti-CDH17 nanobody is shown in SEQ ID NOs: 22 to 24 or SEQ ID NOs: 46 to 48;

[0024] The nucleotide sequence of the hinge region of the amino acid mutation-optimized IgG4 is shown in SEQ ID NO: 34;

[0025] The nucleotide sequence of the transmembrane region of CD28 is shown in SEQ ID NO:36;

[0026] The nucleotide sequence of the CD28 intracellular co-stimulatory domain is shown in SEQ ID NO: 38;

[0027] The nucleotide sequence of the 4-1BB costimulatory domain is shown in SEQ ID NO:40;

[0028] The nucleotide sequence of the CD3ζ signaling domain is shown in SEQ ID NO:42.

[0029] A fourth aspect provides a lentiviral vector, comprising the nucleic acid molecule.

[0030] A fifth aspect is a pharmaceutical composition, which includes the humanized nanoantibody targeting E-cadherin 17, or the CAR-T cell targeting E-cadherin 17, or the nucleic acid, or the lentiviral vector.

[0031] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0032] In the fifth aspect, a humanized nanobody targeting E-cadherin 17, or a CAR-T cell targeting E-cadherin 17, a nucleic acid, a lentiviral vector, or a pharmaceutical composition is used in the preparation of a detection reagent, an in vivo imaging probe or a therapeutic product targeting E-cadherin 17.

[0033] The beneficial effects of the present invention are:

[0034] (1) The CDH17 target selected by the present invention is the potential safest and most effective target for the treatment of digestive system tumors: CDH17 is mainly expressed in the tight junctions between epithelial tissue 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. Existing studies have shown that CDH17 CAR-T or dual-antibody drugs have no damage to normal expressing tissues and specifically kill CDH17-expressing tumor tissues.

[0035] (2) The present invention provides for the first time a humanized candidate nano-antibody molecule for CDH17 targeted therapy of digestive system tumors: Compared with blood tumors, the development of specific immune targeted therapy drugs for solid tumors has been slow, and one of the limiting factors is the lack of effective and safe targets. Current studies have shown that CDH17 is a safe and effective therapeutic target for digestive system tumors, but the research on targeted drugs for the CDH17 target started late, and there are only a few drugs under development. The bottleneck is in the antibody discovery stage. Only a few CDH17 antibody molecules that can be developed as drugs have been developed, and there is only one nano-antibody involved, and it has not been humanized. The present invention provides for the first time a humanized candidate nano-antibody molecule targeting CDH17.

[0036] (3) The humanized nanoantibodies of the present invention have a wide range of applications: The present invention modifies the key amino acid sequences of the FR1-4 region of the parent nanoantibody, and through fusion antibody construction and affinity testing, three humanized nanoantibodies that specifically bind to CDH17 are screened out, and their binding activity to the CDH17 antigen protein is comparable to that of the parent nanoantibody, and their binding activity to gastric cancer cell SNU-16 and pancreatic cancer cell ASPC-1 is good. Although the affinity levels of the three humanized nanoantibodies to human CDH17 protein are significantly lower than that of the parent nanoantibody, the killing ability of CAR-T cells expressing any humanized nanoantibody to CDH17 antigen-positive tumor target cells is higher than that of the parent antibody or comparable to that of the parent antibody. The humanized nanoantibodies of the present invention reduce the immunogenicity of nanoantibodies, improve the targeted killing activity, and can be used to develop immune detection reagents, CAR-T / NK cell drugs and antibody drugs. Combining the characteristics of the CDH17 target and the advantages of humanized nanobodies, the candidate antibodies of the present invention will have better effects if used as cancer immunodiagnostic reagents; if developed into cell or antibody immunotherapy drugs, they will have lower toxic side effects and better clinical efficacy, thereby providing patients with more drug options.

[0037] (4) Can be used to develop dual-target drugs: Studies have found that claudin 18.2, like CDH17, is a safe and effective target for the treatment of digestive system tumors. The two 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 humanized anti-CDH17 candidate nanoantibody of the present invention can be used in combination with CLDN18.2 antibody to develop anti-CLDN18.2 / CDH17 dual-antibody drugs and dual-target CAR-T drugs, which is expected to enhance the efficacy of monoclonal antibodies or single-target CAR-T / NK drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a comparison chart of the amino acid sequence analysis of the mother Nanobody E046-VHH of the present invention and the human IGHV germline gene;

[0039] Figure 2 The ELISA test results of the binding activity of the full-length expressed protein of the humanized Nanobody of the present invention with the antigen CDH17-EC1-2-His;

[0040] Figure 3 The binding activity FACS test results of the full-length expressed protein of the humanized Nanobody of the present invention on ASPC-1 cells;

[0041] Figure 4 This is the binding activity FACS test result of the full-length expressed protein of the humanized Nanobody of the present invention on SNU-16 cells;

[0042] Figure 5 Schematic diagram of the universal CAR structure of the humanized nanobody of the present invention and the parent control and positive control antibodies;

[0043] Figure 6 The plasmid map of the third-generation lentiviral expression vector pCDH-EF1-Kan used in constructing the CDH17 CAR lentiviral expression plasmid of the present invention;

[0044] Figure 7 This is a flow cytometry graph of the CAR positivity rate of the first batch of 5 CAR-T cells of the present invention;

[0045] Figure 8 This is a flow cytometry graph of the CAR positivity rate of the three CAR-T cells in the second batch of the present invention;

[0046] Fig. 9 It is a flow cytometry detection diagram of the CDH17 antigen positive rate of four target cells of the present invention and the expression positive rate of the green fluorescent protein GFP marker carried by them;

[0047] Fig.10 These are the killing curves of the first batch of five CAR-T cells of the present invention against two target cells, SNU5 (upper) and ASPC1 (lower);

[0048] Fig.11 These are the killing curves of the three CAR-T cells of the second batch of the present invention against two target cells, AGS (upper) and COLO 205 (lower);

[0049] Fig.12 The killing efficiency of the first batch of five CAR-T cells of the present invention on two target cells, SNU5 (left) and ASPC1 (right);

[0050] Fig.13 The killing efficiency of the three CAR-T cells in the second batch of the present invention against two target cells, AGS (left) and COLO 205 (right). DETAILED DESCRIPTION

[0051] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0052] Example

[0053] 1. Humanized design of nanobodies

[0054] The amino acid sequence of the parent nanobody E046-VHH (whose amino acid sequence is shown in SEQ ID NO: 15 and the encoding nucleotide sequence is shown in SEQ ID NO: 16) was compared with the IMGT database to determine that it had the highest homology with 10 allele sequences of germline genes such as IGHV3-23, IGHV3-23D, IGHV3-64, IGHV3-64D and IGHV3-NL1; the sequence alignment results are as follows Figure 1 shown.

[0055] The 10 most homologous allele sequences were used as references, and the germline gene IGHV3-23 gene (whose amino acid sequence is shown in SEQ ID NO: 17, and the encoding nucleotide sequence is shown in SEQ ID NO: 18) was used as a template for humanization design. There are 16 camel-derived sites in the E046-VHH framework region (FR). According to the differential sites in the framework region, humanized sequences of different degrees were designed. A total of 10 humanized sequences E046-VHH1 to E046-VHH10 were designed, among which the amino acid sequences of E046-VHH5, E046-VHH7 and E046-VHH10 are shown in SEQ ID NO: 19-21, respectively, and their encoding nucleotide sequences are shown in SEQ ID NO: 22 to 24, respectively.

[0056] The AbM scheme was used to define the CDR region. The amino acid sequences of the FR and CDR regions of the parent nanoantibodies E046-VHH and E046-VHH5, E046-VHH7, and E046-VHH10 are shown in Table 1 .

[0057] Table 1 FR and CDR amino acid sequences of the parent antibody and its humanized antibody

[0058]

[0059] (2) Calculation of the degree of humanization of humanized antibodies

[0060] The parent antibody E046-VHH and the designed humanized sequences VHH5, VHH7 and VHH10 were compared with the human germline gene sequences, and the degree of humanization of the nanobodies was calculated, as shown in Table 2; it can be seen that the degree of humanization increased successively and all reached more than 95%.

[0061] Table 2 Summary of humanization degree information

[0062] Sample name Number of camel-derived sites Degree of humanization E046-VHH 16 87.20％ E046-VHH5 6 95.20％ E046-VHH7 5 96.00％ E046-VHH10 2 98.40％

[0063] 2. Full-length expression and verification of humanized nanobodies

[0064] 2.1 Construction of expression plasmid for full-length antibody protein:

[0065] The coding nucleotide sequences of the parent nanoantibodies E046-VHH and E046-VHH5, E046-VHH7, and E046-VHH10 were fused to the coding nucleotide sequence of human IgG1-CS and subjected to full gene synthesis, and constructed into a pcDNA3.4 vector (purchased from Invitrogen) to obtain a full-length expression plasmid of the VHH-Fc fusion protein. Human IgG1-CS is the constant region Fc of the heavy chain of human IgG1, and an amino acid C (cysteine) in its hinge region is mutated to S (serine) to reduce the presence of free C, and its amino acid sequence is shown in SEQ ID NO: 25, and the coding nucleotide sequence is shown in SEQ ID NO: 26.

[0066] 2.2 Expression and purification of full-length antibody protein

[0067] The constructed plasmid was transfected into Expi CHO cells (purchased from Gibco, A29133) for transient expression. The expression time was 7 days and the expression volume was 10 mL. After the expression, the protein was purified and packaged.

[0068] 2.3 SDS-PAGE identification of full-length antibody protein

[0069] Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) was used to identify the molecular weight and purity of the full-length expressed protein of the humanized antibody. The SDS-PAGE identification method and steps are as follows:

[0070] (1) Preparation of purified protein sample solution:

[0071] Preparation of non-reducing sample solution: Mix the purified sample solution, 4×LDS loading buffer (Zeye Biotechnology, ZY6SL1197) and iodoacetamide (Zeye Biotechnology, ZY144) in proportion to ensure that the final concentration of iodoacetamide is 40 mM and the non-reducing sample loading amount is 1 μg. The mixed sample is placed in a 75°C dry bath and heated for 10 min.

[0072] 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 5 mM and the reduced sample loading amount was 2 μg. The mixed sample was placed in a 100°C dry bath and heated for 10 min.

[0073] (2) Electrophoresis: 140 V, 75 min.

[0074] (3) Staining, destaining and scanning: The gel was stained with Coomassie Brilliant Blue and scanned using an EPSON V550 color scanner after destaining.

[0075] (4) Purity calculation: Use ImageJ to calculate the purity of the reduced band or the purity of the reduced heavy chain plus the light chain according to the peak area normalization method.

[0076] System adaptability: The non-reduced band molecular weight of the reference substance IPI (Ipilimumab) is about 150 kDa, and the purity is greater than 90%; the reduced heavy chain molecular weight is about 50 kDa, the light chain molecular weight is about 25 kDa, and the purity of the heavy chain plus the light chain is greater than 90%.

[0077] The SDS-PAGE quality inspection results are shown in Table 3: It can be seen that the purity of the full-length expressed humanized antibody is comparable to that of the parent antibody, and both are greater than 95.0%.

[0078] Table 3 SDS-PAGE detection results of humanized antibody full-length expression protein

[0079]

[0080]

[0081] 2.4 SEC characterization of full-length antibody protein

[0082] Size Exclusion Chromatography (SEC) was used to identify the purity of the full-length expressed protein of the humanized antibody. The method and steps of SEC identification are as follows:

[0083] (1) Mobile phase preparation: prepare 0.15 M PB + NaCl and adjust the pH to 6.0.

[0084] (2) Sample treatment: Dilute the sample concentration to 0.5 mg / mL.

[0085] (3) Column conditions: XBridge BEH SEC 3.5μm, 7.8×300mm, column temperature set at 20℃, detection baseline was stable.

[0086] (4) Parameter settings: flow rate set to 0.8 mL / min; sample injection volume set to 20 μL; detection wavelength 280 nm, bandwidth 4 nm, reference wavelength 360 nm, bandwidth 100 nm, peak width (response time) > 0.1 min (2 s response time); slit 4 nm; negative absorbance baseline 100 mAU.

[0087] System suitability criteria: If the purity of the reference product Herceptin (trastuzumab) monomer is greater than 95%, the separation between BSA monomer and dimer is greater than 1.5, and the baseline is stable, the system is considered to have passed the suitability test.

[0088] The SEC quality inspection results are summarized in Table 4: It can be seen that the purity of the full-length expressed humanized antibody is equivalent to that of the parent antibody, and both reach more than 98%.

[0089] Table 4 SEC test results of humanized antibody full-length expression protein

[0090]

[0091] Note: “--” means not detected.

[0092] 2.5 DSF detection of full-length antibody protein

[0093] Differential Scanning Fluorimetry (DSF) was used to detect the thermal stability of the full-length expressed protein of the humanized antibody. The method and steps of DSF detection are as follows:

[0094] (1) Test sample preparation: In an eight-well tube or 96-well plate, add a sample diluted to 0.2 mg / mL with 1× PBS (pH 7.4), then add 100× SYPRO Orange working solution to make the final concentration 5×, the final volume is 20 μL, tap the tube wall to mix, and centrifuge at 2000 rpm for 10 seconds; prepare 3 replicates for each sample.

[0095] (2) On-line: Place the sample on the ABI7500 Fast Real-Time PCR instrument, select the melting curve as the experiment type, adopt the continuous mode, scan the temperature from 25°C to 99°C, balance at 25°C for 5 min, the heating rate is 1%, the reporter group is ROX, and the quencher group is None.

[0096] (3) Result determination: The temperature corresponding to the first peak and valley of the derivative function of the melting curve is determined as the denaturation temperature T of the protein. m1 The temperature corresponding to the second peak valley is determined as the denaturation temperature T of the protein. m2 The temperature corresponding to the third peak valley is determined as the denaturation temperature T of the protein. m3 .

[0097] System suitability: Reference Herceptin Protein T m1 =68.5℃±1.0℃, T m2 =81.0℃±1.0℃.

[0098] The results of DSF detection are shown in Table 5 , and it can be seen that the thermal stability of the full-length expressed humanized antibody is comparable to that of the parent antibody.

[0099] Table 5 DSF test results of humanized antibody full-length expression protein

[0100]

[0101]

[0102] 2.6 ELISA detection of full-length antibody protein

[0103] The binding activity of the fully expressed humanized candidate antibody to the antigen CDH17(EC1-2)-His was detected by ELISA. CDH17(EC1-2) is the extracellular 1-2 structural region of the human CDH17 antigen, the amino acids of which are shown in SEQ ID NO:27, and the encoding nucleotide sequence thereof is shown in SEQ ID NO:28; His is a short peptide tag consisting of 6 histidines.

[0104] ELISA test method and steps:

[0105] (1) Coating: Dilute the antigen with 1×PBS to a concentration of 2μg / mL, add 30μL / well to a 96-well Elisa plate, and coat overnight at 4°C. (2) Blocking: Wash the plate three times with PBST, add blocking solution (5% PBS-Milk) and block at room temperature for 2h. (3) Incubation: Wash the plate, add 1% Milk-diluted sample at 30μL / well, and incubate at room temperature for 60min. (4) Secondary antibody incubation: Wash the plate three times with PBST, add secondary antibody, and incubate at room temperature for 60min. (5) Color development: Wash the plate three times with PBST, add 30μL TMB to each well. (6) Stop: Add 50μl TMB color stop solution (purchased from Biotech, P0215) to stop the reaction and detect OD at the same time 450 .

[0106] In the ELISA test, the parent control antibody is E046-VHH, and its full-length expression protein is E046-P-hFc (P235705); NC is a negative control antibody, and BC is a blank control. In the present invention, hFc and Fc have the same meaning.

[0107] ELISA test results Figure 2 (The abscissa Antibody conc. is the antibody concentration) and are shown in Table 6. It can be seen that the binding activity of the full-length expressed humanized antibody to the antigen CDH17(EC1-2)-His is comparable to that of the parent antibody.

[0108] Table 6 ELISA test results of humanized antibody full-length expression protein

[0109]

[0110] 2.7 FACS detection of full-length antibody protein

[0111] FACS detection was performed on the full-length expressed humanized candidate antibodies and CDH17-positive target cells ASPC-1 (purchased from Sanyou Biotechnology, C2305212) and SNU-16 (purchased from Sanyou Biotechnology, C2312248) to identify the binding activity of the antibodies to the target cells.

[0112] Methods and steps:

[0113] (1) Cell plating: Transfer cells from the culture flask to a centrifuge tube, centrifuge to remove the supernatant, resuspend in culture medium, count, and adjust the cell density to 1×10 6 cells / mL. Take a 96-well round bottom plate and add the cells to the plate with a 100μL pipette, adding 100μL to each well. Centrifuge at 300g / min for 5min. Discard the supernatant. (2) Addition of protein antibodies: Dilute the antibody into 8 concentration gradients with FACS Buffer containing 2% FBS: 150.000, 37.500, 9.375, 2.3438, 0.5859, 0.1465, 0.0366 and 0.0092nM. Use a 100μL 12-channel pipette to add the antibody dilution to the 96-well cell plate, 100μL to each well. Mix well and incubate in a 4℃ refrigerator for 1h. (3) Addition of secondary antibody: dilute the secondary antibody PE labelled anti-Human Fc (Jackson) 1:200 with FACS Buffer containing 2% FBS, centrifuge the cell culture plate to remove the supernatant, and use a 100μL 12-channel pipette to add 100μL of the secondary antibody dilution to the cell culture plate. Place the cell culture plate in a 4°C refrigerator and culture for 30 minutes. Centrifuge to remove the supernatant, wash the plate twice with FACS Buffer, and resuspend the cells in each well with 120μL FACS Buffer. (4) Data acquisition: Turn on the flow cytometer (Beckman), and after the instrument is cleaned, perform FACS detection.

[0114] In the above test, the parent control antibody was E046-VHH, and its full-length expressed protein was E046-P-Fc (P235705); the isotype control antibody was IgG1 (P93950-1, purchased from Sanyou), the control with only secondary antibody added was marked as "cell+sec", and the blank cell control without any antibody was marked as "cell only".

[0115] The FACS test results of the binding activity of the full-length expressed protein of the humanized antibody to ASPC-1 cells are as follows Figure 3The FACS test results of the binding activity of the full-length expressed protein of the humanized antibody to SNU-16 cells are as follows Figure 4 (The horizontal axis Antibodyconc. is the antibody concentration, and the vertical axis MFI is the mean fluorescence intensity). It can be seen that the full-length expressed humanized antibody has good binding to the target cells ASPC-1 and SNU-16, but the binding activity is slightly lower than that of the parent antibody.

[0116] 2.8 Affinity kinetics testing of full-length antibody protein

[0117] The high-throughput instrument Gator non-labeled biomolecular analyzer (purchased from Probe Life) based on biomembrane interferometry (BLI) was used to perform affinity kinetic analysis of the binding and dissociation of the full-length expressed protein of the humanized antibody with the antigen CDH17 (EC1-2)-His. The method and steps are as follows:

[0118] (1) Select the mode: Open the Gator instrument and related software, and select the Kinetics experimental mode.

[0119] (2) On-line analysis: The analysis procedure is shown in Table 7:

[0120] Table 7 Affinity kinetics detection process of full-length expressed protein of humanized antibody

[0121]

[0122] System adaptability: The Kd limit detected by Gator is 1E-06. The experimental results show that the correlation coefficient R of all antibodies in the Global fitting mode is 2 They are all greater than 0.95, meeting the system adaptability requirements and the results are reliable.

[0123] The results of affinity kinetic analysis of the binding and dissociation of the full-length expressed protein of the humanized antibody with the antigen CDH17(EC1-2)-His are shown in Table 8, which shows that the affinity of the humanized antibody with the antigen is significantly lower than that of the parent antibody.

[0124] Table 8 Affinity kinetic test results of full-length expressed proteins of humanized antibodies

[0125]

[0126] 3. CAR-T construction and killing experiment

[0127] 3.1 Structural design of Anti-CDH17 CARs

[0128] Five CDH17-targeting chimeric antigen receptors (Anti-CDH17 CARs) were designed, and their general CAR structure diagrams are shown in the figure. Figure 5As shown. Anti-CDH17 CARs include the signal peptide (SP) of CD8α, anti-CDH17 nanobody (anti-CDH17VHH), the hinge region of IgG4 optimized by amino acid mutation (IgG4mH), the transmembrane region of CD28 (CD28TM), the intracellular co-stimulatory domain of CD28, the sequence of 4-1BB co-stimulatory domain and the CD3ζ signaling domain. The five Anti-CDH17 CARs were named E046 CAR, EV5CAR, EV7CAR, EV10 CAR and CDH17 CAR (yang ginseng control), and the amino acid sequences of the expressed anti-CDH17 VHH were respectively from the parent nanoantibody clone E046-VHH, three humanized nanoclones E046-VHH5, E046-VHH7 and E046-VHH10, and the anti-CDH17 VHH1 in patent US20210253728A1 (the amino acid sequence and its encoding nucleotide sequence are shown in SEQ ID NO: 29-30, respectively).

[0129] The amino acid sequence of the signal peptide (SP) and its encoding nucleotide sequence are shown in SEQ ID NOs: 31 to 32, respectively; the amino acid sequence of IgG4mH and its encoding nucleotide sequence are shown in SEQ ID NOs: 33 to 34, respectively; the amino acid sequence of CD28TM and its encoding nucleotide sequence are shown in SEQ ID NOs: 35 to 36, respectively; the amino acid sequence of the CD28 intracellular co-stimulatory domain and its encoding nucleotide sequence are shown in SEQ ID NOs: 37 to 38, respectively; the amino acid sequence of the 4-1BB co-stimulatory domain and its encoding nucleotide sequence are shown in SEQ ID NOs: 39 to 40, respectively; the amino acid sequence of the CD3ζ signaling domain and its encoding nucleotide sequence are shown in SEQ ID NOs: 41 to 42, respectively; the full-length amino acid sequence of CDH17 CAR as a positive control and its encoding nucleotide sequence are shown in SEQ ID NOs: 43 to 4.

[0130] 3.2 Construction of lentiviral expression plasmid for Anti-CDH17 CARs

[0131] (1) Codon optimization and synthesis of anti-CDH17 nanobody encoding nucleotides: The encoding nucleotide sequences of four nanobodies, namely E046-VHH, E046-VHH5, E046-VHH7 and E046-VHH10, were firstly optimized for human codons and then subjected to DNA synthesis. The optimized encoding nucleotide sequences of the four nanobodies are shown in SEQ ID NOs: 45 to 48, respectively.

[0132] (2) Vector linearization by enzyme digestion: The third-generation lentiviral expression plasmid pCDH-EF1-Kan (purchased from Fenghui Biotechnology, the plasmid map is shown in Figure 6 ) is the vector backbone, and the vector is linearized by performing corresponding double enzyme digestion at the two restriction sites of XbaI and EcoRI.

[0133] (3) Seamless cloning: All DNA fragments in the CAR structure amplified by PCR were cloned into the linearized pCDH-EF1-Kan vector backbone using conventional seamless cloning technology to construct the Anti-CDH17 CAR expression plasmid of the nanobody, called pCDH17 CAR.

[0134] 3.3 Lentiviral packaging and titer determination

[0135] The conventional four-plasmid system in the field is used for lentiviral packaging, and all four plasmids are kanamycin-resistant. Adherent 293T cells (Thermo Fisher) are used as lentiviral packaging cells. The ratio of the plasmid dosage of lentiviral expression plasmid pCDH17 CAR and three auxiliary plasmids pMDLg / pRRE, pRSV-Rev and pMD2.G (all three plasmids were purchased from Fenghui Bio) to co-transfect 293T cells is 4:2:2:1; for T75 cell culture flasks, the total amount of plasmids is 20ug, and the dosage of the four plasmids is 8.8ug, 4.4ug, 4.4ug, and 2.2ug respectively. The dosage of transfection reagent PEI is 3 times the total amount of the four plasmids; for T75 culture flasks, the dosage of PEI is 60ug (1ug / ul, 60ul). The plasmid and PEI transfection reagent were added to serum-free medium and mixed. The mixture was placed for 15 minutes, and then added to the T75 culture flask with adherent 293T cells. The mixture was gently mixed and incubated at 37°C and 5% CO. 2 The cells were cultured in a cell culture incubator for 6 hours. After 6 hours, fresh culture medium containing 2% fetal bovine serum was replaced and culture was continued. The culture supernatant of the lentivirus was collected 48 hours after transfection, and the supernatant was taken after centrifugation (2000 rpm, 15 min). After filtering through a 0.45 um filter, the supernatant was concentrated by ultracentrifugation (25000 rpm, 3 h), and then the virus precipitate was resuspended with the corresponding volume of culture medium according to the dilution multiple, aliquoted, and stored at -80°C.

[0136] For the titer determination of CDH17 CAR lentivirus, the lentivirus stock solution or concentrate was serially diluted and transfected into 293T cells. After 72 hours, the transfection efficiency was detected by flow cytometry, and the active titer of the lentivirus was calculated.

[0137] 3.4CDH17 CAR-T preparation and culture

[0138] The frozen PBMCs were revived and cultured overnight. The suspended PBMCs were 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 / ml, add the activator MACS GMP T Cell TransAct (Miltenyi Biotec) at 17.5:1 (PBMC volume: activator volume) for activation, centrifuge and replace the culture medium (X-VIVO 15 containing 300IU / mL IL-2) after 30 hours, count, add CAR lentivirus at MOI=2, pipette and mix, culture in a cell culture incubator at 37°C and 5% CO2 for 16 hours, centrifuge, take the supernatant, resuspend with twice the original volume of the same culture medium, and continue to culture. Count every two days and maintain the cell density at 0.5-2×10 by supplementing or replacing the culture medium. 6 / ml, and culture continuously for 8 to 14 days.

[0139] The CAR expression positive rate of CDH17 CAR-T cells was detected by flow cytometry: CAR-T cell solution containing 1E6 cells was taken, centrifuged at 300g for 5min, supernatant was discarded, and 50μL PBS was added for resuspending; 1-3ul CDH17 antigen CDH17 Protein-His Tag (Acro) was added, and incubated at room temperature and away from light for 20min; 500μL PBS was added, mixed, and centrifuged at 300g for 5min; supernatant was discarded, and 50μL PBS was added for resuspending; CD3 antibody Anti-CD3-APC (Biolegend) and secondary antibody PE anti-His Tag (Biolegend) were added, mixed, and incubated at room temperature and away from light for 20min; 500μL PBS was added, mixed, and centrifuged at 300g for 5min; supernatant was discarded, and cells were resuspended in 200μL PBS and tested on the machine. The proportion of CAR-positive (PE-positive) T cells in the CD3-positive cell population (APC-positive) was analyzed, which was the CAR positive rate of CAR-T cells.

[0140] Two batches of CAR-T preparation experiments were carried out:

[0141] The first batch of CAR-T cells with five types of CARs were prepared. When the CAR positivity rate was detected by flow cytometry, the amount of CDH17 antigen CDH17Protein-His Tag was 1ul, which may be insufficient, resulting in the failure to fully detect the CAR on the CAR-T cells and a low CAR positivity rate detection value. In particular, the three EV5 CAR-T, EV7 CAR-T and EV10CAR-T expressing humanized nanoantibodies may be more affected by the lack of antigen because the affinity between the antibody and the antigen is not as good as that of the parent antibody, resulting in a particularly low CAR positivity rate detection value.

[0142] The second batch prepared three types of CAR-T cells: E046 CAR-T, EV7 CAR-T and EV10 CAR-T. When the CAR positivity rate was detected by flow cytometry, the dosage of CDH17 antigen CDH17 Protein-His Tag was optimized and determined to be 3ul. The positivity rates of the three CAR-T cells were improved to varying degrees compared with the first batch.

[0143] The CAR positive rate flow cytometry results of the above two batches of CAR-T cells are as follows Figure 7-8 and as shown in Table 9.

[0144] Table 9 CAR positive rate of anti-CDH17 CAR-T cells (%)

[0145] E046 CAR-T EV5 CAR-T EV7 CAR-T EV10 CAR-T CDH17 CAR-T (Yang Shen) First batch 17.4 1.29 5.07 1.1 21.0 Second batch 27.8 / 28.0 20.0 /

[0146] 3.5 In vitro killing function detection of CDH17 CAR-T

[0147] There are four target cells used, which are CDH17 antigen natural expression cell lines into which green fluorescent protein (GFP) marker gene is introduced: human gastric adenocarcinoma cell AGS (purchased from Shanghai Academy of Sciences Cell Bank); human pancreatic cancer cell ASPC-1, human colon cancer cell COLO 205 and human gastric cancer cell SNU-5 (purchased from Chinese Academy of Sciences Cell Bank). The flow cytometry results of the expression positive rate of CDH17 antigen and GFP marker protein of the four target cells are shown in Figure 2. Fig. 9 In the present invention, ASPC-1 and ASPC1, SNU-5 and SNU5 all represent cells of the same type.

[0148] Killing experiment method: CAR-T cells and control T (MOCK-T) cells were co-incubated with target cells as effector cells, and the effector: target ratio was set to 2:1 (Note: effector cells refer to the total number of cells). Three replicate wells were set for each effector: target co-incubation, and a blank control with only target cells was set. (1) Target plating: In a 96-well plate, 100ul of target cells with a cell density of 2E5 / ml were added to each well. S3 live cell analyzer (purchased from Sartorius), set the instrument's photography conditions, and culture at 37°C, 5% CO2. (2) On the second day, take out the 96-well plate, add 100ul of effector cells with a cell density of 4E5 / ml to the corresponding wells, and add 100ul of culture medium to the blank control with only target cells. Put the 96-well plate back into the live cell sorting instrument (purchased from Sartorius) and culture at 37°C, 5% CO2. 2 Continue incubation for 48 to 96 hours.

[0149] Result analysis: According to the green fluorescence images of target cells taken by IncuCyte in real time, the target cell fluorescence area was normalized at the most recent shooting time before the addition of effector cells and the start of co-incubation with target cells. The target cell growth curve was drawn with the shooting point before the normalized time point as the starting point (marked as 0h) and the end point of co-incubation as the end point. That is, the killing curve of effector cells against target cells. On the killing curve, the time point of target cell fluorescence area normalization is the second shooting point.

[0150] The killing efficiency was calculated based on the green fluorescence value at the end of the co-incubation. The calculation formula was: killing efficiency = [(blank target cell end fluorescence value - effector target co-incubation cell group end fluorescence value) / blank target cell end fluorescence value] × 100%. The killing efficiency graph was drawn based on the killing efficiency calculation results.

[0151] The killing curves of the first batch of five CAR-Ts (E046 CAR-T, EV5 CAR-T, EV7 CAR-T, EV10 CAR-T and CDH17CAR-T) and untransduced MOCK T against target cells SNU-5 and ASPC-1 are shown in Figure 2. Fig.10 (above and below) show that the killing efficiency is Fig.12 (Left and right) The killing curves of the second batch of three CAR-Ts (E046 CAR-T, EV7CAR-T and untransduced MOCK T) against target cells AGS and COLO 205 are shown in Figure 2. Fig.11 (above and below) show that the killing efficiency is Fig.13 (Left and right) shown.

[0152] From the killing curve and killing efficiency graph, it can be seen that: (1) In the killing experiments of all effector-target co-incubation combinations of the two batches, the five CDH17 CAR-T cells tested all showed significant killing effects on the four CDH17 target cells tested, and the five CAR-T cells compared with the untransduced control MOCK T had a significant difference in killing efficiency against target cells: 3*(P<0.001) or 4*(P<0.0001). (2) The three humanized nanoantibody CAR-T cells (EV5CAR-T, EV7 CAR-T and EV10 CAR-T) of the present invention and the E046 CAR-T cells of their parent nanoantibodies had stronger killing abilities against the tested target cells SNU-5 and ASPC-1 than the positive control CDH17 CAR-T cells. (3) The killing abilities of the CAR-T cells of the three humanized nanoantibodies of the present invention against the four target cells are comparable to or higher than those of their parent E046 CAR-T cells.

[0153] In summary, the present invention transforms the key amino acid sequence of the FR1-4 region of the parent nanoantibody, and through fusion antibody construction and affinity detection, screens out 3 humanized nanoantibodies that specifically bind to CDH17, and its binding activity with the CDH17 antigen protein is comparable to that of the parent nanoantibody, and its binding activity with gastric cancer cell SNU-16 and pancreatic cancer cell ASPC-1 is good but slightly lower than that of the parent nanoantibody, although its affinity level with the antigen protein CDH17 is significantly lower than that of the parent nanoantibody, but the CAR-T cells expressing any humanized nanoantibody have the same killing ability as the parent antibody for CDH17 antigen-positive gastric cancer, pancreatic cancer and colon cancer target cells. The humanized nanoantibody of the present invention reduces the immunogenicity of the nanoantibody, maintains the targeted binding activity, and improves the killing activity, and can be used to develop immune detection reagents, CAR-T / NK cell drugs and antibody drugs.

[0154] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A humanized nanobody targeting cadherin 17, characterized in that: The complementarity determining region of the humanized Nanobody includes a CDR1 with an amino acid sequence as shown in SEQ ID NO: 2, a CDR2 with an amino acid sequence as shown in SEQ ID NO: 4, and a CDR3 with an amino acid sequence as shown in SEQ ID NO: 6; the framework region of the humanized Nanobody includes a FR1 with an amino acid sequence as shown in any one of SEQ ID NO: 1 and SEQ ID NO: 8, a FR2 with an amino acid sequence as shown in any one of SEQ ID NO: 3, SEQ ID NO: 9 and SEQ ID NO: 11, a FR3 with an amino acid sequence as shown in any one of SEQ ID NO: 5 and SEQ ID NO: 10, and a FR4 with an amino acid sequence as shown in SEQ ID NO:

7.

2. The humanized nanobody targeting E-cadherin 17 according to claim 1, characterized in that: The amino acid sequence of the humanized Nanobody is shown in any one of SEQ ID NOs: 19 to 21.

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

4. A CAR-T cell targeting E-cadherin 17 according to claim 3, characterized in that: The signal peptide is the signal peptide of CD8α, the hinge region is the hinge region of IgG4 with optimized amino acid mutations, 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. A CAR-T cell targeting E-cadherin 17 according to claim 4, characterized in that: The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO: 31; The amino acid sequence of the hinge region of the amino acid mutation-optimized IgG4 is shown in SEQ ID NO: 33; The amino acid sequence of the transmembrane region of CD28 is shown in SEQ ID NO:35; The amino acid sequence of the CD28 intracellular co-stimulatory domain is shown in SEQ ID NO: 37; The amino acid sequence of the 4-1BB costimulatory domain is shown in SEQ ID NO: 39; The amino acid sequence of the CD3ζ signaling domain is shown in SEQ ID NO:

41.

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

7. A nucleic acid according to claim 6, characterized in that The coding nucleotide sequence of the CD8α signal peptide is shown in SEQ ID NO: 32; The encoding nucleotide sequence of the humanized anti-CDH17 nanobody is shown in any one of SEQ ID NOs: 22 to 24 or SEQ ID NOs: 46 to 48; The nucleotide sequence encoding the hinge region of the amino acid mutation-optimized IgG4 is shown in SEQ ID NO: 34; The nucleotide sequence encoding the transmembrane region of CD28 is shown in SEQ ID NO:36; The coding nucleotide sequence of the CD28 intracellular co-stimulatory domain is shown in SEQ ID NO: 38; The encoding nucleotide sequence of the 4-1BB costimulatory domain sequence is shown in SEQ ID NO:40; The encoding nucleotide sequence of the CD3ζ signaling domain is shown in SEQ ID NO:

42.

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

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

10. Use of a humanized nanobody targeting E-cadherin 17 as described in any one of claims 1 to 2, or a CAR-T cell targeting E-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 any one of claims 8 to 9 in the preparation of a detection reagent, in vivo imaging probe or therapeutic product targeting E-cadherin 17.

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