Humanized nanobody targeting cadherin 17 and use thereof

Abstract

Provided in the present invention are a humanized nanobody targeting cadherin 17 and the use thereof. The humanized nanobody has complementarity determining regions which comprise CDR1 having an amino acid sequence as shown in SEQ ID NO: 2, CDR2 having an amino acid sequence as shown in SEQ ID NO: 4, and CDR3 having an amino acid sequence as shown in SEQ ID NO: 6. The humanized nanobody has framework regions which comprise FR1 having an amino acid sequence as shown in any one of SEQ ID NO: 1 and SEQ ID NO: 8, FR2 having an amino acid sequence as shown in any one of SEQ ID NO: 3, SEQ ID NO: 9 and SEQ ID NO: 11, FR3 having an amino acid sequence as shown in any one of SEQ ID NO: 5 and SEQ ID NO: 10, and FR4 having an amino acid sequence as shown in SEQ ID NO: 7. The humanized nanobody of the present invention exhibits reduced nanobody immunogenicity and improved targeted binding activity and killing activity, and can be used in the development of immunoassay reagents, CAR-T / NK cell drugs and antibody drugs.

Description

Humanized nanobodies targeting cadherin 17 and uses thereof TECHNICAL FIELD

[0001] The present application relates to the field of immunology and molecular biology, in particular to humanized nanobodies targeting cadherin 17 and uses thereof. BACKGROUND

[0002] Cadherin-17 (CDH17), also known as liver intestine-cadherin (LI-cadherin) or human peptide transporter-1 (HPT-1), is a non-canonical member of the cadherin superfamily. CDH17 was first discovered by Dietmar et al. from mouse liver cells in 1994, and was named liver intestine-cadherin because it is only expressed in the liver and small intestine in mice. Unlike classic cadherins, the extracellular domain of CDH17 has seven repeat units (EC1-EC7), and the intracellular domain of CDH17 contains only 24 amino acid residues, which lacks homology with the intracellular domain of classic cadherins composed of 150-160 amino acids.

[0003] Nanobodies (Nb) are a type of antibodies that only contain the variable region (VHH) of heavy chain antibodies, and in 1993, Belgian scientists first reported a special antibody naturally lacking light chains found in camelid blood, i.e. heavy chain antibodies (HCAbs). Although the molecular weight of nanobodies is only 1 / 10 of that of complete traditional antibodies (about 15 kDa), it retains the complete antigen recognition and binding capacity of heavy chain antibodies, and compared with traditional antibodies, nanobodies have the advantages of strong specificity, strong affinity, high stability, strong targeting and tissue penetration, low immunogenicity, and easy humanization modification. The disadvantage of nanobodies is the short half-life, but they can be fused with anti-serum albumin or Fc segment of antibodies for expression to prolong their half-life in blood. Nanobodies have been widely used in biochemical mechanism and structural biology research, as well as for the development of diagnostic reagents and therapeutic drugs for diseases such as tumors.

[0004] The current study has proved that CDH17 is an effective and safe target for targeted therapy of digestive system tumors, and the candidate antibody molecules against CDH17 can be used for developing diagnostic reagents, targeted antibody drugs and CAR-T / NK immune cell drugs for diseases such as digestive system tumors, but the currently available candidate antibody molecules against CDH17 are very limited, and most of them are traditional antibodies, and only one example of nanobody with outstanding advantages and good drug properties has been reported, which is from the research team of the University of Pennsylvania, and the world's first CDH17 CAR-T developed by Chimeric Therapeutics company has entered clinical phase II. Although nanobody has more excellent properties than traditional antibody, the potential immunogenicity risk of its clinical application cannot be ignored, which will reduce the persistence and efficacy of the targeted drug and cause safety risks. Therefore, the nanobody needs to be further humanized to reduce the clinical risk. However, up to now, there is no report on the humanized nanobody against CDH17. In order to promote the development of safer and more effective CDH17 targeted drugs, more candidate nanobody molecules against CDH17, especially humanized nanobody, need to be developed. In view of this, the present application provides a humanized nanobody targeting cadherin 17 and application thereof. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a humanized nanobody targeting cadherin 17 and application thereof. The purpose is to obtain a humanized antibody by humanizing a camel-derived antibody.

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

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

[0008] Further, the humanized nanobody targeting cadherin 17 comprises any one of E046-VHH5, E046-VHH7 and E046-VHH10.

[0009] The framework region of the amino acid sequence of the 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 the 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 the 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] Further, the amino acid sequence of the humanized nanobody targeting cadherin 17 is shown in any one of SEQ ID NOs: 19-21.

[0013] In a second aspect, a CAR-T cell targeting cadherin 17, the CAR-T cell expressing 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 any one of the humanized nanobodies as claimed in claim 1 or 2.

[0014] Further, the signal peptide is a signal peptide of CD8a, the hinge region is a hinge region of IgG4 optimized by amino acid mutation, the transmembrane region is a transmembrane region of CD28, and the intracellular signaling domain includes a CD28 intracellular costimulatory domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain.

[0015] Further, the amino acid sequence of the signal peptide of CD8a is shown in SEQ ID NO: 31;

[0016] The amino acid sequence of the hinge region of IgG4 optimized by amino acid mutation 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 co-stimulatory domain sequence is shown as SEQ ID NO: 39;

[0020] The amino acid sequence of the CD3 zeta signaling domain is shown as SEQ ID NO: 41.

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

[0022] Further, the nucleotide sequence of the signal peptide of CD8a is shown as SEQ ID NO: 32;

[0023] The nucleotide sequence of the anti-CDH17 humanized nanobody is shown as SEQ ID NO: 22-24 or SEQ ID NO: 46-48;

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

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

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

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

[0028] The nucleotide sequence of the CD3 zeta signaling domain is shown as SEQ ID NO: 42.

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

[0030] In a fifth aspect, a pharmaceutical composition comprising the humanized nanobody targeting cadherin 17, or the CAR-T cell targeting cadherin 17, or the nucleic acid, or the lentiviral vector.

[0031] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0032] In a fifth aspect, the application provides use of the humanized nanobody targeting CDH17, the CAR-T cell targeting CDH17, the nucleic acid, the lentivirus vector, or the pharmaceutical composition in preparation of a detection reagent, an in vivo imaging probe, or a therapeutic product targeting CDH17.

[0033] The application has the following advantages:

[0034] (1) The CDH17 target selected by the application is the most safe and effective treatment target for digestive system tumors: CDH17 is mainly expressed in the tight junction between epithelial cells in the gastrointestinal system, and is highly expressed in digestive system tumors such as gastric cancer, cholangiocarcinoma, pancreatic cancer, esophageal cancer, neuroendocrine tumors, and colorectal cancer cells. Studies have shown that CDH17 CAR-T or double antibody drugs have no damage to normal tissues and can specifically kill CDH17-expressing tumor tissues.

[0035] (2) The application provides a humanized nanobody molecule targeting CDH17 for the first time: Compared with hematological tumors, the development of specific immunotherapy drugs for solid tumors is slow, and one of the constraints is the lack of effective and safe targets. Current studies have shown that CDH17 is a safe and effective treatment target for digestive system tumors, but the research on drugs targeting CDH17 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 have been developed, and only one nanobody is involved, and no humanization modification has been performed. The application provides a humanized nanobody molecule targeting CDH17 for the first time.

[0036] (3) The humanized nanobody of the present application has a wide application field: the present application reengineers the key amino acid sequences of the FR1-4 regions of the parent nanobody, and through antibody fusion construction and affinity detection, 3 humanized nanobodies that specifically bind to CDH17 are screened, which have binding activity to CDH17 antigen protein comparable to that of the parent nanobody, and good binding activity to gastric cancer cells SNU-16 and pancreatic cancer cells ASPC-1. Although the affinity level of the 3 humanized nanobodies to human CDH17 protein is significantly lower than that of the parent nanobody, the CAR-T cells expressing any of the humanized nanobodies have higher killing ability to tumor target cells positive for CDH17 antigen than the parent antibody or comparable to the parent antibody. The humanized nanobody of the present application reduces the immunogenicity of the nanobody and improves the targeted killing activity, and can be used for the development of immunodetection reagents, CAR-T / NK cell drugs and antibody drugs. Combining the characteristics of CDH17 target and the advantages of humanized nanobody, the candidate antibody of the present application will have better effect if used for immunodiagnostic reagents for cancer; if developed into cell or antibody immunotherapy drugs, it will have lower toxic side effects and better clinical efficacy, thereby providing more drug options for patients.

[0037] (4) It can be used for developing double-target drugs: research has found that Claudin 18.2 (Claudin 18.2) is also a safe and effective target for the treatment of digestive system tumors, and it is co-expressed with CDH17 in esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer and other digestive system tumors, and clinical or preclinical tests have proved that targeting these two targets can specifically kill tumor cells without harming normal tissues, so the humanized anti-CDH17 candidate nanobody of the present application can be used with CLDN18.2 antibody to develop anti-CLDN18.2 / CDH17 double-antibody drugs and double-target CAR-T drugs, which are expected to enhance the efficacy of monoclonal antibodies or single-target CAR-T / NK drugs. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is a comparison of the amino acid sequence analysis of the parent nanobody E046-VHH of the present application and human IGHV germline genes;

[0039] Figure 2 is the ELISA detection result of the binding activity of the full-length expressed protein of the humanized nanobody of the present application to antigen CDH17-EC1-2-His;

[0040] Figure 3 is the FACS detection result of the binding activity of the full-length expressed protein of the humanized nanobody of the present application on ASPC-1 cells;

[0041] Figure 4 is the FACS detection result of the binding activity of the full-length expressed protein of the humanized nanobody of the present application on SNU-16 cells;

[0042] Figure 5 is a schematic diagram of the universal CAR structure of the humanized nanobody of the present application and the parent control and positive control antibodies;

[0043] Figure 6 is a plasmid map of the third-generation lentiviral expression vector pCDH-EF1-Kan used for constructing the CDH17 CAR lentiviral expression plasmid of the present application;

[0044] Figure 7 is a flow cytometry detection diagram of the CAR positive rate of the first batch of 5 CAR-T cells of the present application;

[0045] Figure 8 is a flow cytometry detection diagram of the CAR positive rate of the second batch of 3 CAR-T cells of the present application;

[0046] Figure 9 is a flow cytometry detection diagram of the CDH17 antigen positive rate and the expression positive rate of the green fluorescent protein GFP carried by the four target cells of the present application;

[0047] Figure 10 is a killing curve of the first batch of 5 CAR-T cells of the present application against 2 target cells SNU5 (top) and ASPC1 (bottom), respectively;

[0048] Figure 11 is a killing curve of the second batch of 3 CAR-T cells of the present application against 2 target cells AGS (top) and COLO 205 (bottom), respectively;

[0049] Figure 12 is a killing efficiency of the first batch of 5 CAR-T cells of the present application against 2 target cells SNU5 (left) and ASPC1 (right), respectively;

[0050] Figure 13 is a killing efficiency of the second batch of 3 CAR-T cells of the present application against 2 target cells AGS (left) and COLO 205 (right), respectively. DETAILED DESCRIPTION

[0051] The principles and features of the present application are described below, and the examples are only used to explain the present application and are not intended to limit the scope of the present application. If the specific technology or condition is not specified in the examples, it is performed according to the technology or condition described in the literature in the art or according to the product manual. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be purchased through a regular channel.

[0052] EXAMPLE

[0053] 1. Humanization design of nanobody

[0054] The amino acid sequence of the parent nanobody E046-VHH (the amino acid sequence is shown as SEQ ID NO: 15, and the encoding nucleotide sequence is shown as SEQ ID NO: 16) was aligned with the IMGT database, and it was determined that it had the highest homology with 10 allelic sequences of germline genes IGHV3-23, IGHV3-23D, IGHV3-64, IGHV3-64D and IGHV3-NL1. The sequence alignment result is shown in Figure 1.

[0055] Taking the 10 allelic sequences with the highest homology as a reference, and taking the germline gene IGHV3-23 gene (the amino acid sequence is shown as SEQ ID NO: 17, and the encoding nucleotide sequence is shown as SEQ ID NO: 18) as a template, humanization design was performed. There are 16 camel-derived sites in the framework region (FR) of E046-VHH. According to the difference sites of the framework region, sequences with different degrees of humanization were designed, a total of 10 humanized sequences E046-VHH1-E046-VHH10 were designed, wherein the amino acid sequences of E046-VHH5, E046-VHH7 and E046-VHH10 are shown as SEQ ID NO: 19-21, and the encoding nucleotide sequences are shown as SEQ ID NO: 22-24, respectively.

[0056] The CDR region was defined by using the AbM scheme, and the FR region and CDR region amino acid sequences of the parent nanobody E046-VHH and E046-VHH5, E046-VHH7, E046-VHH10 are shown in Table 1.

[0057] Table 1 FR region and CDR region amino acid sequences of parent antibody and humanized antibody thereof

[0058] (2) Humanized antibody humanization degree calculation

[0059] The parent antibody E046-VHH and the designed humanized sequences VHH5, VHH7 and VHH10 were aligned with the human germline gene sequence, and the humanization degree of the nanobody was calculated, as shown in Table 2; it can be seen that the humanization degree is improved in turn, and all reaches more than 95%.

[0060] Table 2 Humanization degree information summary table

[0061] 2, Full-length expression and verification of humanized nanobody

[0062] 2.1 Construction of full-length antibody protein expression plasmid:

[0063] The coding nucleotide sequence of the maternal nanobody E046-VHH was fused with the coding nucleotide sequence of E046-VHH5, E046-VHH7, and E046-VHH10, respectively, and the coding nucleotide sequence of human IgG1-CS was fused and full gene synthesis was performed, and the VHH-Fc fusion protein full-length expression plasmid was constructed on pcDNA3.4 vector (purchased from Invitrogen). The human IgG1-CS is a human IgG1 heavy chain constant region Fc, and one amino acid C (cysteine) in the hinge region is mutated to S (serine) to reduce the presence of free C, and the amino acid sequence is shown as SEQ ID NO: 25, and the coding nucleotide sequence is shown as SEQ ID NO: 26.

[0064] 2.2 Expression and purification of full-length antibody protein

[0065] The constructed plasmid was transfected into Expi CHO cells (purchased from Gibco, A29133) for transient expression, and the expression time was 7 days, the expression volume was 10 mL, and protein purification and sub-packaging were performed after the expression was completed.

[0066] 2.3 SDS-PAGE identification of full-length antibody protein

[0067] Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) was used to identify the molecular weight and purity of the humanized antibody full-length expression protein. The identification method and steps of SDS-PAGE are as follows:

[0068] (1) Preparation of purified protein sample solution:

[0069] Preparation of non-reduced sample solution: The purified sample solution, 4x LDS loading buffer (Zeyebio, ZY6SL1197) and iodoacetamide (Zeyebio, ZY144) were mixed according to the proportion, and the final concentration of iodoacetamide was 40 mM, and the loading amount of non-reduced sample was 1 μg. The mixed sample was placed in a 75°C dry bath instrument for heating for 10 min.

[0070] Preparation of reduced sample solution: The purified sample solution, 4x LDS loading buffer and dithiothreitol (DTT) (Zeyebio, ZY3483) were mixed according to the proportion, and the final concentration of DTT was 5 mM, and the loading amount of reduced sample was 2 μg. The mixed sample was placed in a 100°C dry bath instrument for heating for 10 min.

[0071] (2) Electrophoresis: 140V, 75min.

[0072] (3) Staining, destaining and scanning: Coomassie brilliant blue staining, destaining and scanning by EPSON V550 color scanner.

[0073] (4) Purity calculation: The purity of reduced band or the sum of reduced heavy chain and light chain was calculated by ImageJ according to the peak area normalization method.

[0074] System suitability: The reference product IPI (Ipilimumab) had a non-reduced band molecular weight of about 150 kDa and a purity of more than 90%; the reduced heavy chain had a molecular weight of about 50 kDa, the light chain had a molecular weight of about 25 kDa, and the sum of the heavy chain and the light chain had a purity of more than 90%.

[0075] The SDS-PAGE quality inspection results are shown in Table 3: it can be seen that the purity of the humanized antibody expressed in full length is equivalent to that of the parent antibody, and is greater than 95.0%.

[0076] Table 3 SDS-PAGE detection results of humanized antibody full-length expressed protein

[0077] 2.4 SEC identification of full-length antibody protein

[0078] The purity of the humanized antibody full-length expressed protein was identified by size exclusion chromatography (SEC). The method and steps of SEC identification are as follows:

[0079] (1) Mobile phase preparation: 0.15M PB+NaCl, pH 6.0 was prepared.

[0080] (2) Sample treatment: The sample concentration was diluted to 0.5 mg / mL.

[0081] (3) Column conditions: XBridge BEH SEC 3.5μm, 7.8×300mm, column temperature was set to 20℃, and the detection baseline was stable.

[0082] (4) Parameter setting: flow rate was set to 0.8 mL / min; sample injection volume was set to 20μL; detection wavelength was 280nm, bandwidth was 4nm, reference wavelength was 360nm, bandwidth was 100nm, peak width (response time) was >0.1min (2s response time); slit was 4nm; negative absorbance baseline was 100mAU.

[0083] System suitability standard: the reference product Herceptin (Herceptin, Trastuzumab) had a monomer purity of more than 95%, the separation degree of BSA monomer and dimer was more than 1.5, and the baseline was stable, which was considered to pass the system suitability.

[0084] The SEC results of the humanized antibody full-length expression proteins are shown in Table 4. It can be seen that the purity of the humanized antibody full-length expression proteins is equivalent to that of the parent antibody, and is more than 98%.

[0085] Table 4. SEC detection results of humanized antibody full-length expression proteins Note: “--” represents not detected.

[0086] 2.5 DSF detection of full-length antibody proteins

[0087] The thermal stability of the humanized antibody full-length expression proteins was detected by differential scanning fluorimetry (DSF). The method and steps of DSF detection are as follows:

[0088] (1) Preparation of test samples: In an eight-tube or 96-well plate, add the sample diluted to 0.2 mg / mL with 1xPBS (pH 7.4), then add 100xSYPRO Orange working solution to make the final concentration 5x, the final volume 20 μL, mix by flicking the tube wall, centrifuge at 2000 rpm for 10 seconds; prepare 3 repeats for each sample.

[0089] (2) Machine: Place the sample on the ABI7500 Fast Real-Time PCR instrument, select the melting curve for the experiment type, take the continuous mode, scan the temperature from 25℃ to 99℃, 25℃ equilibrium for 5 min, the heating rate is 1%, the reporter group is ROX, and the quencher group is None.

[0090] (3) Result determination: The temperature corresponding to the first peak valley of the derivative function of the melting curve is determined as the denaturation temperature T m1 of the protein, the temperature corresponding to the second peak valley is determined as the denaturation temperature T m2 of the protein, and the temperature corresponding to the third peak valley is determined as the denaturation temperature T m3 of the protein.

[0091] System adaptability: The reference product Herceptin protein T m1 = 68.5℃ ± 1.0℃, T m2 = 81.0℃ ± 1.0℃.

[0092] The DSF detection results are shown in Table 5. It can be seen that the thermal stability of the humanized antibody full-length expression proteins is equivalent to that of the parent antibody.

[0093] Table 5. DSF detection results of humanized antibody full-length expression proteins

[0094] 2.6 ELISA detection of full-length antibody proteins

[0095] The binding activity of the full-length expressed humanized candidate antibody to the antigen CDH17 (EC1-2)-His was detected by ELISA. CDH17 (EC1-2) is the extracellular 1-2 structure region of human CDH17 antigen, the amino acid sequence of which is shown as SEQ ID NO: 27, and the nucleotide sequence encoding it is shown as SEQ ID NO: 28; His is a short peptide tag composed of 6 histidines.

[0096] The method and steps of ELISA detection are as follows:

[0097] (1) Plate coating: dilute the antigen with 1xPBS to a concentration of 2 μg / mL, add 30 μL / well to the 96-well Elisa plate, and coat at 4°C overnight. (2) Blocking: wash the plate with PBST 3 times, add blocking solution (5% PBS-Milk), and block at room temperature for 2 h. (3) Incubation: wash the plate, add 30 μL / well of 1% Milk-diluted sample, and incubate at room temperature for 60 min. (4) Secondary antibody incubation: wash the plate with PBST 3 times, add the secondary antibody, and incubate at room temperature for 60 min. (5) Color development: wash the plate with PBST 3 times, add 30 μL TMB per well. (6) Termination: add 50 μl TMB color development termination solution (purchased from Biyun Tian, P0215) to terminate the reaction and detect OD 450 .

[0098] In the ELISA detection, the parent control antibody is E046-VHH, and the full-length expressed protein thereof is E046-P-hFc (P235705); NC is the negative control antibody, and BC is the blank control. In the present application, hFc and Fc have the same meaning.

[0099] The ELISA detection results are shown in FIG. 2 (the horizontal coordinate Antibody conc. is the antibody concentration) and 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.

[0100] Table 6 ELISA detection results of full-length expressed humanized antibody proteins

[0101] 2.7 FACS detection of full-length antibody proteins

[0102] The full-length expressed humanized candidate antibody was subjected to FACS detection with CDH17 positive target cells ASPC-1 (purchased from San Yu Biotechnology, C2305212) and SNU-16 (purchased from San Yu Biotechnology, C2312248) to identify the binding activity of the antibody to the target cells.

[0103] Method and steps:

[0104] (1) Cell plating: Transfer the cells in culture flask to centrifuge tube, centrifuge to remove supernatant, resuspend the cells in medium and count, then adjust the cell density to 1 x 10 6 (2) Addition of protein antibody: Dilute the antibody with FACS Buffer containing 2% FBS into 8 concentration gradients: 150.000, 37.500, 9.375, 2.3438, 0.5859, 0.1465, 0.0366 and 0.0092 nM, use 100 μL 12-channel pipette to add the antibody dilution to the 96-well cell plate, 100 μL per well. After mixing, incubate in 4°C refrigerator for 1 h.

[0105] In the above detection, the maternal control antibody is E046-VHH, and its full-length expression protein is E046-P-Fc (P235705); the isotype control antibody is IgG1 (P93950-1, purchased from San Yee), and the control labeled as "cell+sec" is only added with secondary antibody, and the blank cell control labeled as "cell only" is not added with any antibody.

[0106] The FACS detection results of the binding activity of the full-length expression protein of the humanized antibody to ASPC-1 cells are shown in FIG. 3, and the FACS detection results of the binding activity of the full-length expression protein of the humanized antibody to SNU-16 cells are shown in FIG. 4 (the horizontal coordinate Antibody conc. is the antibody concentration, and the vertical coordinate MFI is the average fluorescence intensity). It can be seen that the full-length expression 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 maternal antibody.

[0107] 2.8 Affinity kinetic detection of full-length antibody protein

[0108] The affinity kinetics analysis of the dissociation of the full-length expressed protein of the humanized antibody and the antigen CDH17 (EC1-2)-His was performed by using a high-throughput instrument Gator non-labeled biomolecular analyzer (purchased from Probe Life) based on the biofilm interference technology (BLI), and the method and steps were as follows:

[0109] (1) Select mode: turn on the Gator instrument and related software, and select the Kinetics experiment mode.

[0110] (2) On-machine: the analysis program is shown in Table 7:

[0111] Table 7 Affinity kinetics detection process of the full-length expressed protein of the humanized antibody

[0112] System adaptability: the Kd limit value detected by Gator is 1E-06, and the experimental results show that the correlation coefficient R of all antibodies in the Global fitting mode is greater than 0.95, meeting the system adaptability requirements, and the results are reliable. 2

[0113] The affinity kinetics analysis results of the dissociation of the full-length expressed protein of the humanized antibody and the antigen CDH17 (EC1-2)-His are shown in Table 8, and the results show that the affinity of the humanized antibody and the antigen is obviously lower than that of the parent antibody.

[0114] Table 8 Affinity kinetics detection results of the full-length expressed protein of the humanized antibody

[0115] 3, CAR-T construction and killing experiment

[0116] 3.1 Structure design of Anti-CDH17 CARs

[0117] ​Five kinds of chimeric antigen receptors targeting CDH17 (Anti-CDH17 CARs) were designed, and the schematic diagram of the general CAR structure is shown in Figure 5. Anti-CDH17 CARs include the signal peptide (SP) of CD8a, anti-CDH17 nanobody (anti-CDH17 VHH), hinge region of amino acid mutation optimized IgG4 (IgG4mH), transmembrane region of CD28 (CD28TM), CD28 intracellular costimulatory domain, 4-1BB costimulatory domain sequence and CD3 zeta signaling domain. The five Anti-CDH17 CARs are named E046 CAR, EV5 CAR, EV7 CAR, EV10 CAR and CDH17 CAR (positive control) respectively. The amino acid sequences of the expressed anti-CDH17 VHH are from the maternal nanobody clone E046-VHH, 3 humanized nanoclones E046-VHH5, E046-VHH7 and E046-VHH10, and anti-CDH17 VHH1 in the patent US20210253728A1 (the amino acid sequence and its encoding nucleotide sequence are shown in SEQ ID NO: 29-30, respectively).

[0118] The amino acid sequence and its encoding nucleotide sequence of the signal peptide (SP) are shown in SEQ ID NO: 31-32, respectively; the amino acid sequence and its encoding nucleotide sequence of IgG4mH are shown in SEQ ID NO: 33-34, respectively; the amino acid sequence and its encoding nucleotide sequence of CD28TM are shown in SEQ ID NO: 35-36, respectively; the amino acid sequence and its encoding nucleotide sequence of CD28 intracellular costimulatory domain are shown in SEQ ID NO: 37-38, respectively; the amino acid sequence and its encoding nucleotide sequence of 4-1BB costimulatory domain are shown in SEQ ID NO: 39-40, respectively; the amino acid sequence and its encoding nucleotide sequence of CD3 zeta signaling domain are shown in SEQ ID NO: 41-42, respectively; the full-length amino acid sequence and its encoding nucleotide sequence of CDH17 CAR as a positive control are shown in SEQ ID NO: 43-44, respectively.

[0119] 3.2 Construction of lentiviral expression plasmids of Anti-CDH17 CARs

[0120] (1) Codon optimization and synthesis of nucleotide encoding anti-CDH17 nanobody: The nucleotide encoding sequences of E046-VHH, E046-VHH5, E046-VHH7 and E046-VHH10 were first subjected to human codon optimization, and then DNA synthesis. The optimized nucleotide encoding sequences of the four nanobodies are shown in SEQ ID NO: 45-48, respectively.

[0121] (2) Vector enzyme digestion linearization: using the third generation lentiviral expression plasmid pCDH-EF1-Kan (purchased from Fuhui Biotechnology, the plasmid map is shown in Figure 6) as the vector backbone, double digestion at XbaI and EcoRI enzyme sites to linearize the vector.

[0122] (3) Seamless cloning: using conventional seamless cloning technology, all DNA fragments in the PCR amplified CAR structure are cloned into the linearized pCDH-EF1-Kan vector backbone to construct the Anti-CDH17 CAR expression plasmid of nanobody, called pCDH17 CAR.

[0123] 3.3 Lentivirus packaging and titer determination

[0124] The lentivirus packaging adopts the conventional four-plasmid system in the art, and the four plasmids are all kanamycin resistant. Adherent 293T cells (Thermo Fisher) are used as lentivirus packaging cells. The plasmid dosage ratio of co-transfected 293T cells with lentiviral expression plasmid pCDH17 CAR and three helper plasmids pMDLg / pRRE, pRSV-Rev and pMD2.G (all purchased from Fuhui Biotechnology) is 4:2:2:1; for a T75 cell culture flask, the total amount of plasmid is 20 ug, and the amount of the four plasmids is 8.8 ug, 4.4 ug, 4.4 ug and 2.2 ug, respectively. The amount of PEI transfection reagent is 3 times the total amount of the four plasmids; for a T75 culture flask, the amount of PEI is 60 ug (1 ug / ul, 60 ul). The above plasmids and PEI transfection reagent are mixed in serum-free medium, and then the mixture is incubated for 15 minutes. Then the mixture is added to the T75 culture flask with adherent 293T cells, mixed gently, and incubated at 37°C, 5% CO2 cell incubator for 6h. After 6h, replace the fresh medium containing 2% fetal bovine serum and continue to culture, collect the culture supernatant of lentivirus at 48h after transfection, centrifuge (2000 rpm, 15 min) to take the supernatant, filter through a 0.45 um filter, concentrate the supernatant by ultracentrifugation (25000 rpm, 3h), then resuspend the virus precipitate with the corresponding volume of medium according to the dilution ratio, aliquot and store at -80°C.

[0125] For titer determination of CDH17 CAR lentivirus, the lentivirus stock or concentrated solution is serially diluted and transfected into 293T cells, and the transfection efficiency is detected by flow cytometry 72h later. The active titer of lentivirus is calculated.

[0126] 3.4 CDH17 CAR-T preparation and culture

[0127] The frozen PBMC was resuscitated and cultured overnight, the suspended PBMC was centrifuged and resuspended with X-VIVO 15 medium (LONZA) containing 300 IU / mL of IL-2 and the cell density was adjusted to 1.5x10 6 / ml, and the activator MACS GMP T Cell TransAct (Miltenyi Biotec) was added at a ratio of 17.5:1 (PBMC volume: activator volume) for activation, and after 30 hours, the medium was replaced (X-VIVO 15 containing 300 IU / mL of IL-2), counted, and CAR lentivirus was added at a MOI of 2, mixed well, and cultured at 37°C, 5% CO2 in a cell incubator for 16h, centrifuged, and the supernatant was resuspended with twice the original volume of the same medium, and the cell density was maintained at 0.5-2x10 6 / ml by supplementing or replacing the medium every two days for continuous culture for 8-14 days.

[0128] The CAR expression positive rate of CDH17 CAR-T cells was detected by flow cytometry: 1E6 CAR-T cells were taken, centrifuged at 300g for 5min, the supernatant was discarded, and 50μL PBS was added for resuspension; 1-3ul CDH17 antigen CDH17 Protein-His Tag (Acro) was added, and the mixture was incubated at room temperature for 20min; 500μL PBS was added, mixed well, and centrifuged at 300g for 5min; the supernatant was discarded, and 50μL PBS was added for resuspension; CD3 antibody Anti-CD3-APC (Biolegend) and secondary antibody PE anti-His Tag (Biolegend) were added, mixed well, and incubated at room temperature for 20min; 500μL PBS was added, mixed well, and centrifuged at 300g for 5min; the supernatant was discarded, 200μL PBS was added to resuspend the cells, and the cells were detected on the machine. The proportion of CAR positive (PE positive) T cells in CD3 positive cell population (APC positive) was analyzed, which was the CAR positive rate of CAR-T cells.

[0129] Two batches of CAR-T preparation experiments were performed successively:

[0130] In the first batch, CAR-T cells of 5 kinds of CAR were prepared, and when the CAR positive rate was detected by flow cytometry, the amount of CDH17 antigen CDH17 Protein-His Tag was 1ul, which might be insufficient, resulting in that the CAR on the CAR-T cells could not be fully detected, and the CAR positive rate detection value was low, especially for EV5 CAR-T, EV7 CAR-T and EV10 CAR-T expressing humanized nanobodies, which might be more affected by insufficient antigen due to lower affinity of the antibody to the antigen than the parent antibody, resulting in particularly low CAR positive rate detection value.

[0131] The second batch prepared three kinds of CAR-T cells: E046 CAR-T, EV7 CAR-T and EV10 CAR-T. The CDH17 antigen CDH17 Protein-His Tag dosage was optimized to 3ul, and the positive rates of the three CAR-Ts were all improved to different degrees compared with the first batch.

[0132] The flow cytometry results of the CAR positive rates of the CAR-T cells in the above two batches are shown in Figures 7-8 and Table 9.

[0133] Table 9 CAR positive rate (%) of anti-CDH17 CAR-T cells

[0134] 3.5 CDH17 CAR-T in vitro killing function detection

[0135] Four kinds of target cells were used, which were CDH17 antigen naturally expressing cell lines with green fluorescent protein (GFP) marker gene 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 rates of CDH17 antigen and GFP marker protein of the four target cells are shown in Figure 9. In this invention, ASPC-1 and ASPC1, SNU-5 and SNU5 all represent the same kind of cells.

[0136] Killing experiment method: CAR-T cells and control T (MOCK-T) cells as effector cells were co-incubated with target cells, and the effector: target ratio was set to 2:1 (note: the total number of effector cells was 2E5 / ml). Each effector: target co-incubation was set up in 3 replicates, and a blank control with only target cells was set up. Live cell analyzer (purchased from SEDOLIS), set the photographing conditions of the instrument, and incubate at 37℃, 5% CO2. (2) The next 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 in the live cell analyzer (purchased from SEDOLIS) and continue to incubate at 37℃, 5% CO2 for 48-96h.

[0137] Result analysis: According to the green fluorescence pictures of target cells taken by IncuCyte in real time, the latest time point before the start of co-incubation of effector cells and target cells was taken as the normalization time point of target cell fluorescence area, and the picture point before the normalization time point was taken as the starting point (marked as 0h), and the end point of co-incubation was taken as the end point, and the growth curve of target cells was drawn, that is, the killing curve of effector cells on target cells. In the killing curve graph, the normalization time point of target cell fluorescence area is the second picture point

[0138] According to the green fluorescence value at the end point of co-incubation, the killing efficiency was calculated. The calculation formula is: killing efficiency = [(blank target cell end point fluorescence value-effector target co-incubation cell group end point fluorescence value) / blank target cell end point fluorescence value] x 100%. The killing efficiency graph was drawn according to the calculation results of killing efficiency.

[0139] The killing curves of the first batch of 5 kinds of CAR-T (E046 CAR-T, EV5 CAR-T, EV7 CAR-T and EV10 CAR-T and CDH17 CAR-T) EV10 CAR-T) and non-transduced MOCK T on target cells SNU-5 and ASPC-1 are shown in Figure 10 (upper and lower), and the killing efficiencies are shown in Figure 12 (left and right), respectively. The killing curves of the second batch of 3 kinds of CAR-T (E046 CAR-T, EV7 CAR-T and non-transduced MOCK T) on target cells AGS and COLO 205 are shown in Figure 11 (upper and lower), and the killing efficiencies are shown in Figure 13 (left and right), respectively.

[0140] From the killing curve and killing efficiency graph, it can be seen that: (1) In all effector target co-incubation combinations of the two batches, the five kinds of CDH17 CAR-T cells tested all showed significant killing effect on the four kinds of CDH17 target cells tested, and the difference in killing efficiency of the five kinds of CAR-T cells compared with the non-transduced control MOCK T was significantly different, reaching extremely significant and above level: 3*(P<0.001) or 4*(P<0.0001).(2) The CAR-T cells of the three kinds of humanized nanobodies (EV5 CAR-T, EV7 CAR-T and EV10 CAR-T) and the E046 CAR-T cells of their parent nanobodies in the application have stronger killing ability on the tested target cells SNU-5 and ASPC-1 than the positive control CDH17 CAR-T cells.(3) The CAR-T cells of the three kinds of humanized nanobodies in the application have the same or higher killing ability on the four kinds of target cells than their parent E046 CAR-T cells.

[0141] From the above, the application screens 3 humanized nanobodies that specifically bind to CDH17 by modifying the key amino acid sequences of the FR1-4 regions of the parent nanobody, constructing and detecting the affinity of the fusion antibody, the binding activity of the 3 humanized nanobodies to the CDH17 antigen protein is equivalent to that of the parent nanobody, the binding activity to the gastric cancer cell SNU-16 and the pancreatic cancer cell ASPC-1 is good but slightly lower than that of the parent nanobody, although the affinity level of the 3 humanized nanobodies to the antigen protein CDH17 is significantly lower than that of the parent nanobody, the killing ability of the CAR-T cells expressing any of the 3 humanized nanobodies to the gastric cancer, pancreatic cancer and colon cancer target cells that are positive for the CDH17 antigen is equivalent to or even higher than that of the parent nanobody. The humanized nanobody of the application reduces the immunogenicity of the nanobody, maintains the targeted binding activity, and improves the killing activity, and can be used for developing immunoassay reagents, CAR-T / NK cell drugs and antibody drugs.

[0142] Although the embodiments of the application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the application.

Claims

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

7.

2. The humanized nanobody targeting cadherin 17 according to claim 1, characterized in that, The amino acid sequence of the humanized nanobody is as set forth in any one of SEQ ID NO: 19-21.

3. A CAR-T cell targeting Cadherin 17, characterized in that, The CAR-T cell expresses a chimeric antigen receptor targeting cadherin 17; the chimeric antigen receptor targeting cadherin 17 comprises a CDH17 antigen binding domain, a hinge region, a transmembrane region and an intracellular signaling domain; the CDH17 antigen binding domain comprises a signal peptide and any one of the humanized nanobodies according to claim 1 or 2.

4. The CAR-T cell targeting Cadherin 17 according to claim 3, characterized in that, The signal peptide is a signal peptide of CD8a, the hinge region is a hinge region of IgG4 optimized by amino acid mutation, the transmembrane region is a transmembrane region of CD28, and the intracellular signaling domain comprises a CD28 intracellular costimulatory domain, a 4-1BB costimulatory domain and a CD3 zeta signaling domain.

5. The CAR-T cell targeting Cadherin 17 according to claim 4, characterized in that, The amino acid sequence of the signal peptide of CD8a is as set forth in SEQ ID NO: 31; The amino acid sequence of the hinge region of IgG4 optimized by amino acid mutation is as set forth in SEQ ID NO: 33; The amino acid sequence of the transmembrane region of CD28 is as set forth in SEQ ID NO: 35; The amino acid sequence of the CD28 intracellular costimulatory domain is as set forth in SEQ ID NO: 37; The amino acid sequence of the 4-1BB costimulatory domain is as set forth in SEQ ID NO: 39; The amino acid sequence of the CD3 zeta signaling domain is as set forth in SEQ ID NO:

41.

6. A nucleic acid, characterized in that, The nucleotide sequence encoding the humanized nanobody targeting cadherin 17 according to any one of claims 1-2, or the nucleotide sequence encoding the chimeric antigen receptor targeting cadherin 17 according to any one of claims 3-5.

7. The nucleic acid of claim 6, wherein The nucleotide sequence encoding the signal peptide of CD8a is as set forth in SEQ ID NO: 32; The nucleotide sequence encoding the humanized nanobody against CDH17 is as set forth in any one of SEQ ID NO: 22-24 or SEQ ID NO: 46-48; The nucleotide sequence encoding the hinge region of IgG4 optimized by amino acid mutation is as set forth in SEQ ID NO: 34; The nucleotide sequence encoding the transmembrane region of CD28 is as set forth in SEQ ID NO: 36; The nucleotide sequence encoding the CD28 intracellular co-stimulatory domain is shown as SEQ ID NO: 38; The nucleotide sequence encoding the 4-1BB co-stimulatory domain sequence is shown as SEQ ID NO: 40; The nucleotide sequence encoding the CD3 zeta signaling domain is shown as 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 by, The pharmaceutical composition comprises the humanized nanobody targeting cadherin 17 of any one of claims 1 to 2, or the CAR-T cell targeting cadherin 17 of any one of claims 3 to 5, or the nucleic acid of any one of claims 6 to 7, or the lentiviral vector of claim 8.

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

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