Humanized nanoantibodies targeting E-cadherin 17 and their applications

Abstract

The present invention relates to humanized nanobodies targeting cadherin 17 and their applications, and to the technical fields of immunology and molecular biology. The complementary 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. The humanized nanobody of the present invention reduces the immunogenicity of the nanobody, improves the target binding activity and killing activity, and can be used to develop immune detection reagents, CAR-T / NK cell drugs, and antibody drugs.

Description

Technical Field

[0001] The present invention relates to the technical fields of immunology and molecular biology, and in particular to a humanized nanobody targeting E-cadherin 17 and applications 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 in mouse hepatocytes by Dietmar et al. in 1994. Because it is expressed exclusively in the liver and small intestine in mice, it was named liver intestine cadherin. Unlike classical cadherins, the extracellular domain of CDH17 has seven repeating structural units (EC1-EC7), while the intracellular domain of CDH17 consists of only 24 amino acid residues, lacking homology to the 150-160 amino acid domain of classical cadherins.

[0003] Nanobodies (Nb) are a class of antibodies that contain only the variable region (VHH) of heavy-chain antibodies. In 1993, Belgian scientists first reported the discovery of a special type of antibody naturally lacking light chains, namely heavy-chain antibodies (HCAbs), in the blood of camelids. Although the molecular weight of nanobodies is only 1 / 10 of that of intact traditional antibodies (approximately 15kDa), they retain the complete antigen recognition and binding capabilities of heavy-chain antibodies. Compared with traditional antibodies, they have advantages such as high specificity, strong affinity, high stability, strong targeting and tissue penetration, low immunogenicity, and ease of humanization. The disadvantage of nanobodies is their short half-life, but they can be fused with anti-serum albumin or the Fc region of antibodies to extend their half-life in the blood. Nanobodies have 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 research has demonstrated that CDH17 is an effective and safe target for targeted therapy of digestive system tumors. Anti-CDH17 antibody candidates can be used to develop diagnostic reagents, targeted antibody drugs, and CAR-T / NK cell therapy for digestive system tumors and other diseases. However, currently available anti-CDH17 antibody candidates are very limited and primarily traditional antibodies. Only one nanobody with significant advantages and good drugability has been reported. A team from the University of Pennsylvania, led by Chimeric Therapeutics, developed the world's first CDH17 CAR-T, which is currently in Phase II clinical trials. While nanobodies offer superior properties compared to traditional antibodies, their animal origin raises the potential for immunogenicity in clinical application. This immunogenicity could reduce the durability and efficacy of targeted drugs and pose safety risks. Therefore, further humanization of nanobodies is necessary to mitigate clinical risks. However, to date, no humanized anti-CDH17 nanobodies have been reported. 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 nanobody molecules, especially humanized nanobodies. In view of this, the present invention provides humanized nanobodies targeting E-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 E-cadherin 17 and its application. The purpose is to obtain a humanized antibody by humanizing a camel-derived 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] Furthermore, 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, wherein 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 described in the first aspect.

[0014] Furthermore, 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 signaling domain includes the CD28 intracellular co-stimulatory domain, the 4-1BB co-stimulatory domain and the CD3ζ signaling domain.

[0015] Furthermore, the amino acid sequence of the CD8α signal peptide 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 costimulatory 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 CD8α signal peptide 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 costimulatory 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] The 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, the nucleic acid, the lentiviral vector, or the 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 most potentially safe and effective target for the treatment of digestive system tumors: CDH17 is primarily expressed in the tight junctions between epithelial cells of 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 CDH17 CAR-T or bispecific antibodies do not damage 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 hematological 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 research has shown that CDH17 is a safe and effective therapeutic target for digestive system tumors, but the research on targeted drugs for CDH17 started late, and there are only a few drugs under development. The bottleneck is in the antibody discovery stage. There are only a few examples of CDH17 antibody molecules that can be developed as drugs, and there is only one example of a 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, screens out three humanized nanoantibodies that specifically bind to CDH17. 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 or comparable to that of the parent antibody. The humanized nanoantibodies of the present invention reduce the immunogenicity of nanoantibodies and 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 antibodies 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 ELISA test results of the binding activity of the full-length expressed proteins of the humanized Nanobodies of the present invention to the antigen CDH17-EC1-2-His;

[0040] Figure 3 This is the FACS detection result of the binding activity 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 detection 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 to construct the CDH17 CAR lentiviral expression plasmid of the present invention;

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

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

[0046] Figure 9 Flow cytometric detection graphs of the CDH17 antigen positive rate and the green fluorescent protein (GFP) marker expression positive rate of the four target cells of the present invention;

[0047] Figure 10 These are the killing curves of the first batch of five CAR-T cells against two target cells, SNU5 (top) and ASPC1 (bottom);

[0048] Figure 11 These are the killing curves of the three CAR-T cells from the second batch of the present invention against two target cells, AGS (top) and COLO 205 (bottom);

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

[0050] Figure 13 The figures show the killing efficiency of the three CAR-T cells from 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. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. Where the manufacturers of the reagents or instruments used are 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 parental Nanobody E046-VHH (whose amino acid sequence is shown in SEQ ID NO: 15 and its 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] Humanized design was performed using the 10 most homologous allele sequences as a reference and the germline IGHV3-23 gene (its amino acid sequence is shown in SEQ ID NO: 17, and its encoding nucleotide sequence is shown in SEQ ID NO: 18) as a template. The E046-VHH framework region (FR) contains 16 camelid-derived sites. Based on these differentially located sites, sequences with varying degrees of humanization were designed. A total of 10 humanized sequences, E046-VHH1 to E046-VHH10, were designed. The amino acid sequences of E046-VHH5, E046-VHH7, and E046-VHH10 are shown in SEQ ID NOs: 19-21, respectively, and their encoding nucleotide sequences are shown in SEQ ID NOs: 22-24, 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 nanobody E046-VHH, E046-VHH5, E046-VHH7, and E046-VHH10 are shown in Table 1 .

[0057] Table 1 Amino acid sequences of FR and CDR regions 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 in sequence 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 parental nanoantibodies E046-VHH, E046-VHH5, E046-VHH7, and E046-VHH10 were fused to the coding nucleotide sequence of human IgG1-CS and fully synthesized. The nucleotide sequences were then constructed into the pcDNA3.4 vector (purchased from Invitrogen) to generate a full-length expression plasmid for the VHH-Fc fusion protein. Human IgG1-CS is the human IgG1 heavy chain constant region Fc, in which one amino acid C (cysteine) in the hinge region is mutated to S (serine) to reduce the presence of free C. Its amino acid sequence is shown in SEQ ID NO: 25, and its 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 for 7 days in a volume of 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 humanized antibody protein. 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: 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 40 mM and the non-reducing sample loading amount was 1 μg. The mixed sample was 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 destained before scanning with an EPSON V550 color scanner.

[0075] (4) Purity calculation: Use ImageJ 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.

[0076] System adaptability: The non-reduced molecular weight of the reference substance IPI (Ipilimumab) is approximately 150 kDa, and the purity is greater than 90%; the reduced heavy chain molecular weight is approximately 50 kDa, and the light chain molecular weight is approximately 25 kDa, and the purity of the heavy chain and 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 humanized antibody protein. The SEC identification method and steps 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: The system is considered to have passed if the purity of the reference product Herceptin (trastuzumab) monomer is greater than 95%, the resolution between BSA monomer and dimer is greater than 1.5, and the baseline is stable.

[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 comparable to that of the parent antibody, and both reach above 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 test the thermal stability of the full-length humanized antibody protein. The DSF test method and steps are as follows:

[0094] (1) Test sample preparation: In an eight-well tube strip 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 a final concentration of 5×, with a final volume of 20 μL. Gently tap the tube wall to mix, and centrifuge at 2000 rpm for 10 seconds; prepare three replicates for each sample.

[0095] (2) On-line PCR: Place the sample on an ABI7500 Fast Real-Time PCR instrument. Select the melting curve as the experimental type, adopt the continuous mode, scan the temperature from 25°C to 99°C, balance at 25°C for 5 minutes, and heat up at a rate of 1%. Use the reporter group ROX and the quencher group None.

[0096] (3) Result determination: The temperature corresponding to the first peak and valley of the melting curve derivative function 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 product Herceptin protein T m1 =68.5℃±1.0℃, T m2 =81.0℃±1.0℃.

[0098] The results of DSF testing are shown in Table 5 , which shows that the thermal stability of the fully 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] Serial number Protein name Molecular weight (kDa) <![CDATA[T m1 (℃)]]> <![CDATA[T m2 (℃)]]> / Herceptin 146.42 69.62 80.35 1 E046-P-FC 79.20 65.23 83.43 6 E046-VHH5-FC 79.34 66.43 81.20 8 E046-VHH7-FC 79.40 60.33 80.51 11 E046-VHH10-FC 79.22 61.23 81.20

[0101] 2.6 ELISA detection of full-length antibody protein

[0102] ELISA was performed to test the binding activity of the fully expressed humanized candidate antibodies to the antigen CDH17(EC1-2)-His. CDH17(EC1-2) represents the extracellular 1-2 domain of the human CDH17 antigen, the amino acid sequence of which is shown in SEQ ID NO:27, and the nucleotide sequence encoding it is shown in SEQ ID NO:28. His is a short peptide tag consisting of six histidine residues.

[0103] ELISA detection method and steps:

[0104] (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℃. (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 Biyuntian, P0215) to stop the reaction and detect OD at the same time 450 .

[0105] In the ELISA test, the parent control antibody is E046-VHH, and its full-length expressed protein is E046-P-hFc (P235705); NC is the negative control antibody, and BC is the blank control. In the present invention, hFc and Fc have the same meaning.

[0106] 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 fully expressed humanized antibody to the antigen CDH17(EC1-2)-His is comparable to that of the parent antibody.

[0107] Table 6 ELISA test results of humanized antibody full-length expression protein

[0108]

[0109] 2.7 FACS detection of full-length antibody protein

[0110] 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.

[0111] Methods and steps:

[0112] (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 using a 100μL pipette, adding 100μL to each well. Centrifuge at 300g / min for 5 minutes. Discard the supernatant. (2) Addition of protein antibodies: Dilute the antibody with FACS buffer containing 2% FBS to 8 concentration gradients: 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 solution to the 96-well cell plate, 100μL per well. Mix well and incubate in a 4℃ refrigerator for 1 hour. (3) Addition of secondary antibody: Dilute the secondary antibody PE-labeled anti-Human Fc (Jackson) 1:200 with FACS buffer containing 2% FBS. Centrifuge the cell culture plate to remove the supernatant. 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 incubate for 30 minutes. Centrifuge to remove the supernatant, wash the plate twice with FACS buffer, and resuspend the cells in each well with 120μL of FACS buffer. (4) Data acquisition: Turn on the flow cytometer (Beckman) and perform FACS detection after the instrument is cleaned.

[0113] In the above test, the parental 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 labeled "cell+sec", and the blank cell control without any antibody was labeled "cell only".

[0114] The results of FACS detection of the binding activity of the full-length expressed protein of the humanized antibody to ASPC-1 cells are as follows Figure 3 The results of FACS detection 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 fully 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.

[0115] 2.8 Affinity kinetics testing of full-length antibody proteins

[0116] A high-throughput instrument based on biofilm interferometry (BLI) technology, the Gator label-free biomolecule analyzer (purchased from Probe Life), was used to perform affinity kinetic analysis of the binding and dissociation of the full-length expressed humanized antibody protein with the antigen CDH17 (EC1-2)-His. The method and steps are as follows:

[0117] (1) Select the mode: Open the Gator instrument and related software, and select the Kinetics experimental mode.

[0118] (2) On-line analysis: The analysis procedure is shown in Table 7:

[0119] Table 7 Affinity kinetics detection process of full-length expressed humanized antibody

[0120]

[0121] 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, which meets the system adaptability requirements and the results are reliable.

[0122] The results of affinity kinetic analysis of the binding and dissociation between the full-length expressed protein of the humanized antibody and the antigen CDH17(EC1-2)-His are shown in Table 8. The results show that the affinity of the humanized antibody to the antigen is significantly lower than that of the parent antibody.

[0123] Table 8 Affinity kinetics test results of full-length expressed proteins of humanized antibodies

[0124]

[0125] 3. CAR-T construction and killing experiments

[0126] 3.1 Structural Design of Anti-CDH17 CARs

[0127] Five CDH17-targeting chimeric antigen receptors (Anti-CDH17 CARs) were designed, and their general CAR structures are shown in the following figure. Figure 5Anti-CDH17 CARs include the CD8α signal peptide (SP), anti-CDH17 nanobody (anti-CDH17VHH), amino acid mutation-optimized IgG4 hinge region (IgG4mH), CD28 transmembrane region (CD28TM), CD28 intracellular costimulatory domain, 4-1BB costimulatory domain sequence and 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 VHHs were derived from the parental nanobody 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).

[0128] 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 costimulatory domain and its encoding nucleotide sequence are shown in SEQ ID NOs: 37 to 38, respectively; the amino acid sequence of the 4-1BB costimulatory 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 the CDH17 CAR used as a positive control and its encoding nucleotide sequence are shown in SEQ ID NOs: 43 to 4.

[0129] 3.2 Construction of Lentiviral Expression Plasmids for Anti-CDH17 CARs

[0130] (1) Codon Optimization and Synthesis of Anti-CDH17 Nanobody Encoding Nucleotides: The nucleotide sequences encoding the four Nanobodies, E046-VHH, E046-VHH5, E046-VHH7, and E046-VHH10, were first codon-optimized for human use and then subjected to DNA synthesis. The optimized nucleotide sequences encoding the four Nanobodies are shown in SEQ ID NOs: 45 to 48, respectively.

[0131] (2) Vector linearization by enzyme digestion: The third generation lentiviral expression plasmid pCDH-EF1-Kan (purchased from Fenghui Biotechnology, the plasmid map is as follows Figure 6 The vector backbone is shown in FIG, and the vector is linearized by performing double enzyme digestion at the XbaI and EcoRI restriction sites.

[0132] (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.

[0133] 3.3 Lentiviral packaging and titer determination

[0134] Lentiviral packaging uses a conventional four-plasmid system in the field, and all four plasmids are kanamycin-resistant. Adherent 293T cells (Thermo Fisher) were used as lentiviral packaging cells. The lentiviral expression plasmid pCDH17 CAR and three auxiliary plasmids pMDLg / pRRE, pRSV-Rev, and pMD2.G (all three plasmids were purchased from Fenghui Bio) were co-transfected into 293T cells at a plasmid dosage ratio of 4:2:2:1; for T75 cell culture flasks, the total amount of plasmid was 20ug, and the four plasmid dosages were 8.8ug, 4.4ug, 4.4ug, and 2.2ug, respectively. The amount of transfection reagent PEI was 3 times the total amount of the four plasmids; for T75 culture flasks, the PEI dosage was 60ug (1ug / ul, 60ul). The plasmid and PEI transfection reagent were added to serum-free culture medium and mixed. The mixture was then allowed to stand for 15 minutes. The mixture was then added to a T75 culture flask containing adherent 293T cells and gently mixed. The cells were cultured in a 37°C, 5% CO2 incubator for 6 hours. After 6 hours, fresh culture medium containing 2% fetal bovine serum was replaced and culture continued. 48 hours after transfection, the lentiviral culture supernatant was collected and centrifuged (2000 rpm, 15 minutes). The supernatant was filtered through a 0.45 μm filter and concentrated by ultracentrifugation (25000 rpm, 3 hours). The viral pellet was then resuspended in the appropriate volume of culture medium according to the dilution factor, aliquoted, and stored frozen at -80°C.

[0135] For the titer determination of CDH17 CAR lentivirus, the lentiviral stock solution or concentrate was serially diluted and then transfected into 293T cells. After 72 hours, the transfection efficiency was detected by flow cytometry, and the activity titer of the lentivirus was calculated.

[0136] 3.4CDH17 CAR-T preparation and culture

[0137] 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, and activated by adding the activator MACS GMP T Cell TransAct (Miltenyi Biotec) at a ratio of 17.5:1 (PBMC volume: activator volume). After 30 hours, the cells were centrifuged and the culture medium (X-VIVO 15 containing 300 IU / mL IL-2) was replaced. The cells were counted and CAR lentivirus was added at an MOI of 2. The cells were mixed by pipetting and cultured in a cell culture incubator at 37°C and 5% CO2 for 16 hours. The cells were centrifuged, the supernatant was collected, and the cells were resuspended in twice the original volume of the same culture medium and cultured. The cells were counted every two days and the cell density was maintained at 0.5 to 2 × 10 by supplementing or replacing the culture medium. 6 / ml, and culture continuously for 8 to 14 days.

[0138] The CAR expression positivity rate of CDH17 CAR-T cells was determined by flow cytometry: 1E6 CAR-T cell suspension was centrifuged at 300g for 5 minutes, the supernatant discarded, and the suspension resuspended in 50μL PBS. 1-3μL CDH17 antigen CDH17 Protein-His Tag (Acro) was added and incubated at room temperature in the dark for 20 minutes. 500μL PBS was added, mixed, and centrifuged at 300g for 5 minutes. The suspension was discarded and the suspension was resuspended in 50μL PBS. CD3 antibody Anti-CD3-APC (Biolegend) and secondary antibody PE Anti-His Tag (Biolegend) were added, mixed, and incubated at room temperature in the dark for 20 minutes. 500μL PBS was added, mixed, and centrifuged at 300g for 5 minutes. The supernatant discarded, the suspension resuspended in 200μL PBS, and the cells were analyzed by flow cytometry. The CAR positivity rate of CAR-T cells was determined by analyzing the proportion of CAR-positive (PE-positive) T cells within the CD3-positive cell population (APC-positive).

[0139] Two batches of CAR-T preparation experiments were carried out:

[0140] The first batch of CAR-T cells with five types of CARs was prepared. When the CAR positivity rate was detected by flow cytometry, the amount of CDH17 antigen CDH17Protein-His Tag used 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. This was especially true for the three EV5 CAR-T, EV7 CAR-T, and EV10CAR-T cells expressing humanized nanobodies. This may be because the affinity between the antibody and the antigen is not as good as that of the parent antibody, and they are more affected by the insufficient antigen, resulting in a particularly low CAR positivity rate detection value.

[0141] 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 all improved to varying degrees compared with the first batch.

[0142] 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.

[0143] Table 9 CAR positive rate of anti-CDH17 CAR-T cells (%)

[0144] 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 /

[0145] 3.5 In vitro killing function detection of CDH17 CAR-T

[0146] Four target cells were used, including CDH17 antigen naturally expressing cell lines into which the green fluorescent protein (GFP) marker gene was introduced: human gastric adenocarcinoma cell line AGS (purchased from the Shanghai Academy of Sciences Cell Bank); human pancreatic cancer cell line ASPC-1, human colon cancer cell line COLO 205, and human gastric cancer cell line SNU-5 (purchased from the Chinese Academy of Sciences Cell Bank). The flow cytometry results of the positive expression rates of CDH17 antigen and GFP marker protein in the four target cells are shown in Figure 2. Figure 9 In the present invention, ASPC-1 and ASPC1, SNU-5 and SNU5 all refer to the same type of cells.

[0147] Killing experiment method: CAR-T cells and control T (MOCK-T) cells were co-incubated with target cells as effector cells, with the effector: target ratio set at 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 at the same time. (1) Target plating: 100ul of target cells with a cell density of 2E5 / ml were added to each well of a 96-well plate. The S3 live cell analyzer (purchased from Sartorius) was set to the instrument's photography conditions and incubated at 37°C, 5% CO2. (2) The next day, the 96-well plate was removed and 100 μl of effector cells at a cell density of 4E5 / ml were added to the corresponding wells. For the blank control containing only target cells, 100 μl of culture medium was added. The 96-well plate was placed back in place on the live cell sorting instrument (purchased from Sartorius) and incubated at 37°C, 5% CO2 for another 48 to 96 hours.

[0148] Result analysis: Based on the real-time green fluorescence images of target cells taken by IncuCyte, the target cell fluorescence area was normalized at the most recent time point before the addition of effector cells and the start of co-incubation with target cells. The target cell growth curve, i.e., the killing curve of effector cells against target cells, was drawn with the photographic point before the normalized time point as the starting point (marked as 0h) and the end point of co-incubation as the end point. On the killing curve, the time point for the normalization of the target cell fluorescence area is the second photographic point.

[0149] Calculate the killing efficiency based on the green fluorescence value at the end of the co-incubation. The calculation formula is: Killing efficiency = [(blank target cell endpoint fluorescence value - effector target co-incubation cell endpoint fluorescence value) / blank target cell endpoint fluorescence value] × 100%. Plot a killing efficiency graph based on the killing efficiency calculation results.

[0150] 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. Figure 10 (above and below) show that the killing efficiency is as follows: Figure 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 as follows: Figure 11 (above and below) show that the killing efficiency is as follows: Figure 13 (Left and right) shown.

[0151] 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 differences in the killing efficiency of the five CAR-T cells against the target cells compared with the untransduced control MOCK T were all extremely significant or above: 3*(P<0.001) or 4*(P<0.0001). (2) The CAR-T cells of the three humanized nanoantibodies of the present invention (EV5CAR-T, EV7CAR-T and EV10CAR-T) and their parent nanoantibody E046 CAR-T cells had stronger killing abilities against the 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.

[0152] In summary, the present invention modified the key amino acid sequence of the FR1-4 region of the parent nanoantibody and screened out three humanized nanoantibodies that specifically bind to CDH17 through fusion antibody construction and affinity testing. The binding activity of the nanoantibody to the CDH17 antigen protein was comparable to that of the parent nanoantibody, and the binding activity to gastric cancer cell SNU-16 and pancreatic cancer cell ASPC-1 was good but slightly lower than that of the parent nanoantibody. Although the affinity level of the nanoantibody to the antigen protein CDH17 was significantly lower than that of the parent nanoantibody, the killing ability of CAR-T cells expressing any humanized nanoantibody to CDH17 antigen-positive gastric cancer, pancreatic cancer and colon cancer target cells was comparable to or even higher than that of the parent antibody. The humanized nanoantibody of the present invention reduces the immunogenicity of the nanoantibody, maintains the target binding activity, and improves the killing activity, and can be used to develop immune detection reagents, CAR-T / NK cell drugs and antibody drugs.

[0153] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A humanized nanobody targeting E-cadherin 17, characterized in that: The complementarity determining regions of the humanized Nanobody include 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 regions of the humanized Nanobody include 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 cadherin 17; the chimeric antigen receptor targeting 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 a humanized nanobody 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 signaling domain includes the CD28 intracellular co-stimulatory domain, the 4-1BB co-stimulatory domain and the CD3ζ signaling domain.

5. The 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 costimulatory 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 E-cadherin 17 as described in any one of claims 1 to 2, or a nucleic acid sequence encoding a chimeric antigen receptor targeting E-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 CD8α signal peptide is shown in SEQ ID NO: 32; The encoding nucleotide sequence of the humanized nanobody targeting E-cadherin 17 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 nucleotide sequence encoding the CD28 intracellular costimulatory domain is shown in SEQ ID NO: 38; The nucleotide sequence encoding the 4-1BB costimulatory domain sequence is shown in SEQ ID NO: 40; The nucleotide sequence encoding 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 of claim 6 or 7.

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

10. Use of a humanized nanobody targeting E-cadherin 17 according to any one of claims 1 to 2, or a CAR-T cell targeting E-cadherin 17 according to any one of claims 3 to 5, or a nucleic acid according to any one of claims 6 to 7, or a lentiviral vector according to claim 8, or a pharmaceutical composition according to any one of claims 8 to 9 in the preparation of a detection reagent targeting E-cadherin 17, an in vivo imaging probe, or a product for treating gastric cancer, pancreatic cancer, or colon cancer.

Images