Anti-CDH17 antigen binding fragment, antibody and application thereof

Abstract

The invention provides an anti-CDH17 antigen binding fragment, an antibody and application thereof. CDR1 of a heavy chain variable region of the antigen binding fragment comprises amino acid sequences as shown in SEQ ID NO.1-3, CDR2 of the heavy chain variable region of the antigen binding fragment comprises amino acid sequences as shown in SEQ ID NO.4-6, and CDR3 of the heavy chain variable region of the antigen binding fragment comprises amino acid sequences as shown in SEQ ID NO.7-9; cDR1 of a light chain variable region of the antigen binding fragment comprises amino acid sequences as shown in SEQ ID NO.10 to 12, CDR2 of the light chain variable region of the antigen binding fragment comprises amino acid sequences as shown in SEQ ID NO.13 to 15, and CDR3 of the light chain variable region of the antigen binding fragment comprises amino acid sequences as shown in SEQ ID NO.16 to 18. The antigen binding fragment and the anti-CDH17 antibody provided by the invention have high specificity, and a chimeric antigen receptor and a CAR-T cell prepared from the antibody have obvious cytotoxicity on cells stably expressing CDH17 protein.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an anti-CDH17 antigen binding fragment, an antibody and applications thereof. Background Art

[0002] CDH17, the full name of transmembrane cadherin 17, is a transmembrane glycoprotein mainly expressed in gastrointestinal epithelial cells. It plays a vital role in maintaining intestinal barrier function and intercellular communication. CDH17 plays a key role in the normal physiological function of the intestine by participating in intercellular adhesion and signal transduction through its structural characteristics, such as transmembrane regions and extracellular domains. However, recent studies have found that the abnormal expression of CDH17 on the surface of a variety of tumor cells suggests its potential role in tumor occurrence and development. This abnormal expression may be related to malignant behaviors such as tumor cell proliferation, invasion and metastasis. Despite this, the current diagnostic and treatment methods for CDH17 are still limited, and existing antibody products often have problems such as low specificity and insufficient affinity, which seriously restricts its effectiveness and safety in clinical applications.

[0003] Therefore, providing an antibody that specifically binds to CDH17 has important therapeutic significance for cancers caused by tumor cells that express CDH17 positively. Summary of the invention

[0004] In view of the problems existing in the prior art, the purpose of the present invention is to provide an anti-CDH17 antigen-binding fragment, antibody and its application. The antigen-binding fragment and antibody can specifically bind to CDH17 protein, and are prepared into chimeric antigen receptors and CAR-T cells, which have obvious cytotoxicity to target cells expressing CDH17 protein.

[0005] To achieve this object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides an anti-CDH17 antigen-binding fragment, wherein the CDR1 of the heavy chain variable region of the antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO.1-SEQ ID NO.3, the CDR2 comprises the amino acid sequence shown in SEQ ID NO.4-SEQID NO.6, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO.7-SEQ ID NO.9;

[0007] The CDR1 of the light chain variable region of the antigen-binding fragment includes the amino acid sequence shown in SEQ ID NO.10-SEQ ID NO.12, the CDR2 includes the amino acid sequence shown in SEQ ID NO.13-SEQ ID NO.15, and the CDR3 includes the amino acid sequence shown in SEQ ID NO.16-SEQ ID NO.18.

[0008] The antigen-binding fragment and anti-CDH17 antibody provided by the present invention can specifically bind to CDH17 proteins from various sources and have high specificity; the chimeric antigen receptor and CAR-T cells prepared from the antibodies have obvious cytotoxicity to cells stably expressing CDH17 protein.

[0009] SEQ ID NO.1: DYYMY.

[0010] SEQ ID NO. 2: GYTMS.

[0011] SEQ ID NO.3: DYYIH.

[0012] SEQ ID NO.4: TISDGGNNTHYPGSVKG.

[0013] SEQ ID NO.5: TISSGSSFTYYPDSVKG.

[0014] SEQ ID NO. 6: WIYPGNVNTKFNEKFKG.

[0015] SEQ ID NO.7:RGLYGYAWFAY.

[0016] SEQ ID NO. 8: DFFGSTFDY.

[0017] SEQ ID NO.9: GAGTFAY.

[0018] SEQ ID NO. 10: KSSQNLLYSSNQKNYLA.

[0019] SEQ ID NO. 11: KSTKSLLNSDGFTYLD.

[0020] SEQ ID NO. 12: KAGQDVGTAVG.

[0021] SEQ ID NO.13: WASTRES.

[0022] SEQ ID NO. 14: LVSNRFS.

[0023] SEQ ID NO.15: WASTRHT.

[0024] SEQ ID NO. 16: QQYYSYPLT.

[0025] SEQ ID NO. 17: FQSNYPPLYT.

[0026] SEQ ID NO. 18: QQSSSYPWT.

[0027] Preferably, CDR1 of the heavy chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.1, CDR2 is the amino acid sequence shown in SEQ ID NO.4, and CDR3 is the amino acid sequence shown in SEQ ID NO.7; CDR1 of the light chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.10, CDR2 is the amino acid sequence shown in SEQ ID NO.13, and CDR3 is the amino acid sequence shown in SEQ ID NO.16.

[0028] Preferably, CDR1 of the heavy chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.2, CDR2 is the amino acid sequence shown in SEQ ID NO.5, and CDR3 is the amino acid sequence shown in SEQ ID NO.8; CDR1 of the light chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.11, CDR2 is the amino acid sequence shown in SEQ ID NO.14, and CDR3 is the amino acid sequence shown in SEQ ID NO.17.

[0029] Preferably, CDR1 of the heavy chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.3, CDR2 is the amino acid sequence shown in SEQ ID NO.6, and CDR3 is the amino acid sequence shown in SEQ ID NO.9; CDR1 of the light chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.12, CDR2 is the amino acid sequence shown in SEQ ID NO.15, and CDR3 is the amino acid sequence shown in SEQ ID NO.18.

[0030] In a second aspect, the present invention provides an anti-CDH17 antibody, wherein the anti-CDH17 antibody comprises the antigen-binding fragment as described in the first aspect.

[0031] Preferably, the amino acid sequence of the heavy chain variable region of the anti-CDH17 antibody is shown as SEQ ID NO.19-SEQ ID NO.22, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.23-SEQ ID NO.26.

[0032] 25E7-VH (SEQ ID NO. 19):

[0033] EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVATISDGGNN THYPGSVKGRFTISSRDNAKNSLYLQMTSLKSEDTAMYYCARRGLYGYAWFAYWGRGTLVT VSA.

[0034] 2D5-VH (SEQ ID NO. 20):

[0035] DVKLVESGGGLVKPGGSLKLSCAASGFTFSGYTMSWVRQTPEKRLEWVATISSGSSFT YYPDSVKGRFTISSRDNAKNTLHLQMSSLKSEDTAMYYCTRDFFGSTFDYWGQGTTLTVSS.

[0036] 9C11-VH (SEQ ID NO.21):

[0037] QVQLQQSGPELVKPGASVRISCKASGFTFTDYYIHWVKQRPGQGLEWIGWIYPGNVN TKFNEKFKGKATLTADKSSSTAYMQLSSSLTSEDSAVYFCGRGAGTFAYWGQGTLVTVSA.

[0038] H9C11-VH (SEQ ID NO.22):

[0039] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIHWVRQAPGQRLEWMGWIYPGN VNTKFNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARGAGTFAYWGQGTLVTV SA.

[0040] 25E7-VL (SEQ ID NO. 23):

[0041] DIVMTQSPSSLAVSVGEKISMSCKSSQNLLYSSNQKNYLAWYQQKPGQSPKLLIYWAS TRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK.

[0042] 2D5-VL (SEQ ID NO. 24):

[0043] DVVLTQTPLSLPVNIGDQASISCKSTKSLLNSDGFTYLDWYLQKPGQSPQLLIYLVSNR FSGVPDRFSGSGSGTDFTLMISRVEAEDLGVYYCFQSNYPPLYTFGGGTKLEIK.

[0044] In the present invention, the 9C11 antibody clone was humanized. Keeping the same CDR as the 9C11 antibody, the 9C11 antibody was successfully converted into the H9C11 antibody with high humanization by carefully selecting and transplanting the framework region FR of the human antibody. The modified H9C11 antibody is not only closer to the immunoglobulin in the human body in structure, but also functionally exhibits excellent binding ability with human and monkey CDH17 antigens. In order to further verify its specificity, advanced membrane protein array (MPA) technology was used to conduct a detailed analysis of the interaction between the H9C11 antibody and the target antigen. The results showed that the H9C11 antibody has a high specificity for the target antigen CDH17.

[0045] In the further development of the H9C11 antibody, a chimeric antigen receptor (CAR) was designed and a lentiviral expression vector was successfully constructed. CAR-T cells were prepared by introducing the CAR gene into T cells. In in vitro experiments, the specific killing ability of CAR-T cells on tumor target cells was evaluated. The experimental results showed that the modified CAR-T cells can effectively recognize and kill tumor cells expressing the CDH17 antigen, while having minimal toxic effects on normal cells. This discovery not only verifies the potential of the H9C11 antibody in tumor immunotherapy, but also provides an important experimental basis for future related clinical trials.

[0046] 9C11-VL (SEQ ID NO.25):

[0047] DIVMTQSHKFMSTSVGDRVSITCKAGQDVGTAVGWYQQKPGQSPKLLIYWASTRHTG VPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQSSSYPWTFGGGTKLEIK.

[0048] H9C11-VL (SEQ ID NO.26):

[0049] DIQMTQSPSSVSASVGDRVTITCKAGQDVGTAVGWYQQKPGKAPKLLIYWASTRHTGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSYPWTFGGGTKLEIK.

[0050] Preferably, the anti-CDH17 antibody further comprises a constant region.

[0051] Wherein, the anti-CDH17 antibody can exist in the form of a monomer or a polymer. If it exists in the form of a polymer, one of its heavy chains forms an interchain disulfide bond with one of the light chains, the other heavy chain forms an interchain disulfide bond with the other light chain, and one of its heavy chains forms two interchain disulfide bonds with the other heavy chain.

[0052] At the same time, the present invention also provides a method for preparing the anti-CDH17 antibody as described in the second aspect, which specifically comprises the following steps:

[0053] (1) DNA-immunized BALB / c mice were used to prepare hybridomas. Five healthy female BALB / c mice aged 7 to 8 weeks were selected for immunization. After a certain immunization period, the serum titer of the immunized mice was tested. The spleen cells of mice whose serum titers met the requirements of the fusion experiment were selected and fused with myeloma cells SP2 / 0 at an appropriate ratio under the action of a fusion agent to prepare hybridoma monoclonal cells;

[0054] (2) Cultivating hybridoma cells using selective culture medium R1640-HAT, and after 7 to 10 days, switching to HT culture medium for 3 to 4 days, and detecting hybridoma supernatant samples using ELISA on the 10th to 14th day to obtain positive clones;

[0055] (3) Perform flow cytometry screening experiments, subclone the parent clones that bind to CDH17 but not to CDH17, and sequence to obtain clones with the correct sequence.

[0056] In a third aspect, the present invention provides a nucleic acid molecule encoding the antigen-binding fragment as described in the first aspect or the anti-CDH17 antibody as described in the second aspect.

[0057] In a fourth aspect, the present invention provides an expression vector, comprising the nucleic acid molecule as described in the third aspect.

[0058] In a fifth aspect, the present invention provides a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises the anti-CDH17 antibody as described in the second aspect.

[0059] In the present invention, the CAR-T cells containing the chimeric antigen receptor can highly express anti-CDH17 antibodies and have obvious cytotoxicity to cells that express CDH17 positively.

[0060] Preferably, the chimeric antigen receptor further includes a signal peptide (Leader), a hinge region (Hinge), a transmembrane domain (Transmembrane, TM), a co-stimulatory domain (ICD) and a signal transduction domain.

[0061] Preferably, the signal peptide comprises a CD8α signal peptide and / or an IgGκ light chain signal peptide, preferably an IgGκ light chain signal peptide.

[0062] Preferably, the hinge region is any one of CD8α, CD28, human IgG1, IgG2, IgG4 or IgA, preferably the CD8α hinge region.

[0063] Preferably, the transmembrane domain includes the CD8α transmembrane region and / or the CD28 transmembrane region, preferably the CD8α transmembrane region.

[0064] Preferably, the signaling domain comprises a CD3zeta signaling domain.

[0065] Preferably, the signaling domain further comprises a co-stimulatory domain;

[0066] Preferably, the co-stimulatory domain includes any one or a combination of at least two of 4-1BB, CD28 intracellular region, DAP10 or OX40.

[0067] In the present invention, the chimeric antigen receptor targeting CDH17 includes IgGκ light chain signal peptide, scFv, CD8α hinge region, CD8α transmembrane region, 4-1BB and CD3ζ.

[0068] In the present invention, the chimeric antigen receptor includes an IgGκ light chain signal peptide sequence, an antibody sequence scFv that specifically binds to the CDH17 antigen, a hinge region of CD8a, a transmembrane region sequence, a 4-1BB co-stimulatory domain sequence, and a CD3ζ signaling domain sequence.

[0069] Among them, the amino acid sequence of the IgGκ light chain signal peptide (SEQ ID NO.27) is:

[0070] MDMRVPAQLLGLLLLWLRGARC;

[0071] The amino acid sequence of the CD8α hinge region (hinge) (SEQ ID NO.28) is:

[0072] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD;

[0073] The amino acid sequence of the CD8α transmembrane region (TM) (SEQ ID NO.29) is:

[0074] IYIWAPLAGTCGVLLLSLVITLYC;

[0075] The amino acid sequence of the 4-1BB intracellular costimulatory domain (ICD) (SEQ ID NO.30) is:

[0076] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL;

[0077] The amino acid sequence of the CD3ζ signaling domain (SEQ ID NO.31) is:

[0078] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0079] Preferably, the amino acid sequence of the chimeric antigen receptor includes the sequence shown in SEQ ID NO.32-SEQ ID NO.35.

[0080] SEQ ID NO.32 (25E7 CAR amino acid sequence):

[0081] MDMRVPAQLLGLLLLWLRGARCDIVMTQSPSSLAVSVGEKISMSCKSSQNLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVATISDGGNNTHYPGSVKGRFTISRDNAKNSLYLQMTSLKSEDTAMYYCARRGLYGYAWFAYWGRGTLVTVSATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

[0082] SEQ ID NO.33 (Amino acid sequence of 2D5 CAR):

[0083] MDMRVPAQLLGLLLLWLRGARCDVVLTQTPLSLPVNIGDQASISCKSTKSLLNSDGFTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFSGSGSGTDFTLMISRVEAEDLGVYYCFQSNYPPLYTFGGGTKLEIKGGGGSGGGGSGGGGSDVKLVESGGGLVKPGGSLKLSCAASGFTFSGYTMSWVRQTPEKRLEWVATISSGSSFTYYPDSVKGRFTISRDNAKNTLHLQMSSLKSEDTAMYYCTRDFFGSTFDYWGQGTTLTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

[0084] SEQ ID NO.34 (Amino acid sequence of 19C1 CAR):

[0085] MDMRVPAQLLGLLLLWLRGARCDIVMTQSHKFMSTSVGDRVSITCKAGQDVGTAVGWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQSSSYPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGPELVKPGASVRISCKASGFTFTDYYIHWVKQRPGQGLEWIGWIYPGNVNTKFNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCGRGAGTFAYWGQGTLVTVSATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

[0086] SEQ ID NO.35 (Humanized H19C1 CAR Amino Acid Sequence):

[0087] MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSVSASVGDRVTITCKAGQDVGTAVGWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSYPWTFG GGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIHWVRQAPGQRLEWMGWIYPGNVNTKFNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAR GAGTFAYWGQGTLVTVSATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0088] In a sixth aspect, the present invention provides a host cell, comprising the nucleic acid molecule as described in the third aspect, the expression vector as described in the fourth aspect, or the chimeric antigen receptor as described in the fifth aspect.

[0089] In a seventh aspect, the present invention provides a pharmaceutical composition, comprising the anti-CDH17 antibody described in the second aspect.

[0090] Preferably, the pharmaceutical composition further comprises an anti-tumor drug.

[0091] In the present invention, the pharmaceutical composition can also be used in combination with other anti-tumor drugs, including simultaneous administration, separate administration or sequential administration.

[0092] Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, diluent or excipient.

[0093] In an eighth aspect, the present invention provides an antigen-binding fragment as described in the first aspect, the anti-CDH17 antibody as described in the second aspect, the nucleic acid molecule as described in the third aspect, the expression vector as described in the fourth aspect, the chimeric antigen receptor as described in the fifth aspect, the host cell as described in the sixth aspect, or the pharmaceutical composition as described in the seventh aspect for use in the preparation of cancer detection reagents and / or cancer treatment drugs.

[0094] Preferably, the cancer comprises a cancer positive for CDH17 expression.

[0095] Preferably, the cancer includes any one of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer or gallbladder cancer.

[0096] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0097] Compared with the prior art, the present invention has at least the following beneficial effects:

[0098] (1) The antigen-binding fragment and anti-CDH17 antibody provided by the present invention can specifically bind to CDH17 proteins from multiple species (including humans, mice and cynomolgus monkeys), providing more options for the selection of subsequent animal models; membrane protein array experiments also show that H9C11-scFv-hFc can specifically bind to CDH17 but not to other non-target proteins, indicating that the anti-CDH17 antibody has obvious specificity;

[0099] (2) The present invention provides three anti-CDH17 chimeric antigen receptors (CARs) and one humanized anti-CDH17 chimeric antigen receptor. After these chimeric antigen receptors are transferred into T cells through lentiviral vectors, CAR-T cells expressing anti-CDH17 CAR are obtained. The CAR-T cells have obvious cytotoxicity to cells that stably express CDH17 protein. Therefore, they have obvious therapeutic value for cancers that are positive for CDH17 expression, such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 It is a curve chart of the test results of the binding ability between the antibody and the cell line;

[0101] Figure 2 Schematic diagram of the CDH17 CAR structure;

[0102] Figure 3 This is a map of the CDH17 CAR vector;

[0103] Figure 4A This is a diagram showing the results of humanized 19C1 (H19C1) CAR binding to CDH17;

[0104] Figure 4B This is a diagram showing the results of humanized 19C1 (H19C1) CAR binding to CDH17;

[0105] Figure 5 This is the CDH17 CAR-T positive rate test result diagram;

[0106] Figure 6 This is the result graph of the killing rate of CDH17 CAR-T on CDH17 positive target cells;

[0107] Figure 7 This is the result of MPA detection of H9C11-scFv-hFc specifically binding to human CDH17. DETAILED DESCRIPTION

[0108] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0109] In the following examples, unless otherwise specified, all reagents and consumables were purchased from conventional reagent manufacturers in the field; unless otherwise specified, the experimental methods and technical means used were conventional methods and means in the field.

[0110] Example 1

[0111] In this example, hybridoma monoclonal cells were prepared by DNA immunization of BALB / c mice, and the hybridoma cells were cultured using the selective culture medium R1640-HAT, and then replaced with HT culture medium for culture, and the hybridoma supernatant samples were tested by ELISA method, and 99 positive clones were obtained;

[0112] Subsequently, flow cytometry screening experiments were performed to obtain subclones, and three anti-CDH17 antibody correct sequence clones 25E7, 2D5, and 9C11 were obtained by sequencing. After sequencing and identification, it was found that the anti-CDH17 antibody sequence was as follows:

[0113] 25E7-VH (SEQ ID NO. 19):

[0114] EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVATISDGGNN THYPGSVKGRFTISSRDNAKNSLYLQMTSLKSEDTAMYYCARRGLYGYAWFAYWGRGTLVT VSA.

[0115] 2D5-VH (SEQ ID NO. 20):

[0116] DVKLVESGGGLVKPGGSLKLSCAASGFTFSGYTMSWVRQTPEKRLEWVATISSGSSFT YYPDSVKGRFTISRDNAKNTLHLQMSSLKSEDTAMYYCTRDFFGSTFDYWGQGTTLTVSS。

[0117] 9C11-VH (SEQ ID NO.21):

[0118] QVQLQQSGPELVKPGASVRISCKASGFTFTDYYIHWVKQRPGQGLEWIGWIYPGNVN TKFNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCGRGAGTFAYWGQGTLVTVSA。

[0119] 25E7-VL (SEQ ID NO.23):

[0120] DIVMTQSPSSLAVSVGEKISMSCKSSQNLLYSSNQKNYLAWYQQKPGQSPKLLIYWAS TRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLELK。

[0121] 2D5-VL (SEQ ID NO.24):

[0122] DVVLTQTPLSLPVNIGDQASISCKSTKSLLNSDGFTYLDWYLQKPGQSPQLLIYLVSNR FSGVPDRFSGSGSGTDFTLMISRVEAEDLGVYYCFQSNYPPLYTFGGGTKLEIK。

[0123] 9C11-VL (SEQ ID NO.25):

[0124] DIVMTQSHKFMSTSVGDRVSITCKAGQDVGTAVGWYQQKPGQSPKLLIYWASTRHTG VPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQSSSYPWTFGGGTKLEIK。

[0125] Example 2

[0126] Antibody affinity detection.

[0127] 1. Antigen coating: Coat the CDH17 antigen on a 96-well plate. The coating process must be performed at room temperature to ensure the activity of the antigen.

[0128] 2. Antibody incubation: Incubate the coated antigen with different concentrations of anti-CDH17 antibodies at 37°C to simulate the environment in the human body.

[0129] 3. Binding detection: Add biotin-labeled CDH17 antigen, followed by streptavidin-labeled HRP. The binding detection process must be performed at room temperature to ensure enzyme activity.

[0130] 4. Color development reaction: Add TMB substrate and perform color development reaction according to the color change. The color development reaction must be carried out under light-proof conditions to prevent substrate decomposition.

[0131] 5. Data Analysis: OD Measurement by Microplate Reader 450 The affinity constant of the antibody was calculated using the Scatchard analysis method.

[0132] The binding and dissociation data were processed using ForteBIO analysis software to calculate the affinity constant (KD value) of the antibody. The results are shown in Table 1.

[0133] Table 1

[0134]

[0135] Affinity detection of 3 antibodies (25E7, 2D5, 9C11) showed that the binding rate constants were comparable to those of the control antibody. All 3 antibodies (25E7, 2D5, 9C11) showed high affinity, with the KD value of 25E7 antibody being 8.13E-09M, the KD value of 2D5 antibody being 1.65E-09M, and the KD value of 9C11 antibody being 9.11E-09M.

[0136] Example 3

[0137] The binding of the antibodies of the present invention to human CDH17 can be measured in a flow cytometry assay. After HEK293 cells are transiently transformed with a plasmid overexpressing CDH17 protein,

[0138] HEK293 cells (5×10 5) in PBS + 5% BSA at 4°C for 30 min. Incubate with the experimental antibody + 5% BSAPBS at a minimum concentration of 1 μg / mL (in 10-fold gradient dilution) to 0.01 μg / mL for 60 min at 4°C, wash by centrifugation, and incubate with the secondary antibody (FITC-labeled, 1:200, Sigma, F9512) in PBS + 5% BSA for 30 min on ice (protected from light). Wash the cells 3 times and analyze them by flow cytometry. Results Figure 1 As shown: The EC50 of the three chimeric antibodies are 2D5 (0.5662 μg / mL), 9C11 (0.5671 μg / mL), and 25E7 (0.634 μg / mL).

[0139] Example 4

[0140] In this example, a chimeric antigen receptor against CDH17 and its expression vector were constructed.

[0141] (1) Sequence design

[0142] The chimeric antigen receptor includes an IgGκ light chain signal peptide sequence (Leader), an antibody sequence (scFv) that specifically binds to the CDH17 antigen, a hinge region (Hinge) and a transmembrane region sequence (Transmembrane) of CD8a, a 4-1BB costimulatory domain sequence, and a CD3ζ signaling domain sequence;

[0143] Specific structure such as Figure 2 The amino acid sequence and nucleotide sequence of each part are shown in Table 2 below:

[0144] Table 2

[0145]

[0146] (2) Construction of anti-CDH17 chimeric antigen receptor expression vector

[0147] First, the CAR sequence was fully synthesized, and the fully synthesized CAR and empty vector were double-digested with EcoRI and BamHI. After digestion in a 37°C water bath for 30 minutes, DNA was electrophoresed using 1.5% agarose gel, and then purified and recovered using Tiangen's agarose gel kit;

[0148] Then, the pCDH-EF1 vector and the CAR gene fragment were connected. The specific connection system is shown in Table 3 below:

[0149] Table 3

[0150] Reagents Usage pCDH-EF1 vector 2μL(50ng) CAR gene 10μL (150ng) T4DNALigaseBuffer 2μL T4 DNA Ligase (NEB) 1μL <![CDATA[ddH2O]]> 5μL Total 20μL

[0151] The cells were connected at 22°C for 1 hour, and the ligation products were directly transformed into Stbl3 E. coli competent cells. 200 μL of the transformation products were coated on an ampicillin-resistant LB plate, and the LB plate was inverted and cultured in a 37°C incubator overnight.

[0152] The next morning, three single clones were randomly selected for colony PCR identification, and the positive clones were sent for sequencing to obtain the sequence of the anti-CDH17 chimeric antigen receptor lentiviral expression plasmid chimeric antigen receptor. Among them, the humanized H9C11 CAR vector was as follows Figure 3 shown.

[0153] Example 5

[0154] Flow cytometry was used to detect the binding of CDH17 protein to 293T cells transiently transfected with CAR plasmid.

[0155] The binding of 293T cells to CDH17 protein after transient transfection of CAR plasmid was further studied. First, 293T cells were prepared and cultured to an appropriate density. Then, the CAR plasmid DNA was mixed with the transfection reagent and operated according to the manufacturer's instructions. The mixture was added to the culture medium containing 293T cells, and the culture dish was gently shaken to ensure even distribution. Next, the cells were placed in an incubator and continued to be cultured, giving appropriate conditions to promote transfection efficiency. 24h after transfection, the cells were collected and washed with PBS. Subsequently, the cells were labeled with a solution containing CDH17 protein and incubated. After that, the labeled cells were detected using a flow cytometer to analyze the binding of CAR plasmid-transfected 293T cells to CDH17 protein. Finally, the flow cytometry data were collected and analyzed to evaluate the transfection efficiency and the expression level of CAR protein. The results are shown in Figure 4A and Figure 4B As shown, 25E7 can bind to CDH17 antigens of three species, with a positive rate of 20.31% for human CDH17, 25.99% for mouse CDH17, and 55.81% for monkey CDH17. 2D5 can only bind to human and monkey CDH17 antigens, 19.08% and 39.48% respectively. 9C11 can also only bind to human and monkey CDH17, 24.2% and 52.09% respectively. Humanized 9C11 (H9C11) binds to human and monkey CDH17 at 20.5% and 72.25% respectively.

[0156] Example 5

[0157] In this example, the lentiviral expression vector was packaged with lentivirus, H9C11 CAR-T cells were constructed, and the infection efficiency of the lentivirus on T cells was detected.

[0158] (1) Using the four-plasmid system for lentiviral packaging

[0159] The four-plasmid system respectively expressed gag / pol, Rev, VSV-G required for lentiviral vector packaging and an artificial chimeric antigen receptor composed of an engineered stable single-chain antibody. The four plasmids were transiently transfected into 293T cells with a total mass of 10 μg;

[0160] The plasmids were added to serum-free DMEM, mixed and placed for 15 min, and the mixture was added to a T75 culture flask containing 293T cells, mixed gently, and cultured in a cell culture incubator at 37°C and 5% CO2 for 6 h.

[0161] After 6 hours, fresh culture medium was replaced, culture was continued, and 10 mM sodium butyrate solution was added. After 72 hours, the culture supernatant of the lentivirus was collected for purification and detection.

[0162] (2) Expansion of CAR-T cells

[0163] Collect 30 mL of whole blood, dilute the peripheral blood with saline in a ratio of 1:1, add Ficoll to a centrifuge tube, slowly add the diluted peripheral blood, centrifuge at 1500 rpm for 30 min, gently aspirate the PBMC layer and transfer it to another centrifuge tube;

[0164] PBMCs were washed several times with saline and transferred into CAR-T cell culture medium (containing 50 ng / mL OKT3 and 300 IU / mL IL-2) for culture;

[0165] After PBMCs are isolated, they need to be activated with CAR-T cell culture medium containing 50 ng / mL OKT3 and 300 IU / mL IL-2;

[0166] After 2 days, the culture medium was replaced with CAR-T cell culture medium containing 300 IU / mL for expanded culture;

[0167] Then, the cells were counted and the CAR-T cell culture medium containing 300 IU / mL was replaced every two days, and the cell concentration was maintained at 0.5×10 6 ~1×10 6 / mL, observed for 10 consecutive days;

[0168] (3) Lentivirus infection of T cells

[0169] RetroNectin was used to improve the infection efficiency of lentivirus on T cells. 30 μg of RetroNectin was coated in a 6-well plate and placed in a 37°C cell culture incubator for 2 h;

[0170] Pipette RetroNectin, block the coated 6-well plate with Hank's solution containing 2.5% BSA, and place in a 37°C cell culture incubator for 0.5 h;

[0171] The blocking solution was aspirated, and the 6-well plate was washed with Hank's solution containing 2% Hepes, and X-VIVO medium was added, and an appropriate amount of lentivirus solution was added, and centrifuged at 2000 g for 2 h;

[0172] Discard the supernatant and add 1×10 6 T cells were centrifuged at 1000 g for 10 min and cultured in a cell culture incubator at 37° C. and 5% CO 2 . The above process was repeated on the second day. The expression of H9C11 CAR was determined 5 days after infection. FITC-Protien L was combined with H9C11 CAR and the expression of CAR was detected by flow cytometry.

[0173] The results are as follows Figure 5 As shown, the positive expression rate of H9C11 CAR was 51.3%.

[0174] Example 6

[0175] In this example, tumor cells overexpressing the luciferase gene were used as target cells to perform a killing test, and a blank control NC (untransfected T cells) was used as a control.

[0176] The luciferase method was used to detect the toxicity of CAR-T cells to SW480-CDH17-luc (SW480 cells stably expressing human CDH17), 293T-CDH17 (293T cells stably expressing human CDH17) and Raji cells.

[0177] The results are as follows Figure 6 As shown, anti-CDH17 CAR-T cells can specifically kill SW480 cells stably expressing human CDH17 (SW480-CDH17) and 293T cells stably expressing human CDH17 (293T-CDH17), but have almost no killing ability against target cells that do not express CDH17 (SW480-luc, 293T-luc, Raji-luc).

[0178] Example 7

[0179] In this example, a membrane protein array was used to verify the non-target binding interactions of the antibody.

[0180] First, the antibody fusion protein H9C11-scFv-hFc was expressed, which is the single-chain antibody sequence of H9C11 fused to the Fc segment of human IgG1; the membrane proteome array (MPA) is a platform for analyzing specific antibodies and other ligands targeting human membrane proteins, which can be used to determine the specificity of antibody targets;

[0181] Plasmids containing about 6,000 membrane protein clones (accounting for more than 94% of the human membrane protein group) were transfected into HEK-293T cells (ATCC, CRL-3216); or QT6 cells (ATCC, CRL-1708); 384-well cell culture plates (Corning, 3764), 18,000 cells / well;

[0182] After 36 hours of incubation, the test antibodies were added to the membrane proteome array matrix plate at a predetermined concentration, and the binding of antibody H9C11-scFv-hFc to cells expressing approximately 6,000 membrane proteins was directly detected using flow cytometry. Therefore, all target proteins have native conformations and appropriate post-translational modifications.

[0183] Membrane protein array results Figure 7 As shown: H9C11-scFv-hFc can specifically bind to human CDH17 but not to other non-target proteins.

[0184] In summary, the anti-CDH17 antibody provided by the present invention can specifically bind to CDH17 proteins from various sources, including human, mouse and monkey, and has basically no binding ability to other proteins, and is highly specific. At the same time, the CAR and T cells containing the CAR provided in the present invention have obvious cytotoxicity to cells expressing CDH17 protein; therefore, the anti-CDH17 antibody provided by the present invention has a specific therapeutic effect on diseases targeting CDH17 protein.

[0185] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. An anti-CDH17 antigen-binding fragment, characterized in that: The CDR1 of the heavy chain variable region of the antigen-binding fragment includes the amino acid sequence shown in SEQ ID NO.1-SEQ ID NO.3, the CDR2 includes the amino acid sequence shown in SEQ ID NO.4-SEQ ID NO.6, and the CDR3 includes the amino acid sequence shown in SEQ ID NO.7-SEQ ID NO.9; The CDR1 of the light chain variable region of the antigen-binding fragment includes the amino acid sequence shown in SEQ ID NO.10-SEQ ID NO.12, the CDR2 includes the amino acid sequence shown in SEQ ID NO.13-SEQ ID NO.15, and the CDR3 includes the amino acid sequence shown in SEQ ID NO.16-SEQ ID NO.

18.

2. The antigen-binding fragment according to claim 1, characterized in that The CDR1 of the heavy chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.1, CDR2 is the amino acid sequence shown in SEQ ID NO.4, and CDR3 is the amino acid sequence shown in SEQ ID NO.7; the CDR1 of the light chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.10, CDR2 is the amino acid sequence shown in SEQ ID NO.13, and CDR3 is the amino acid sequence shown in SEQ ID NO.16; Preferably, the CDR1 of the heavy chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.2, CDR2 is the amino acid sequence shown in SEQ ID NO.5, and CDR3 is the amino acid sequence shown in SEQ ID NO.8; the CDR1 of the light chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.11, CDR2 is the amino acid sequence shown in SEQ ID NO.14, and CDR3 is the amino acid sequence shown in SEQ ID NO.17; Preferably, CDR1 of the heavy chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.3, CDR2 is the amino acid sequence shown in SEQ ID NO.6, and CDR3 is the amino acid sequence shown in SEQ ID NO.9; CDR1 of the light chain variable region of the antigen-binding fragment is the amino acid sequence shown in SEQ ID NO.12, CDR2 is the amino acid sequence shown in SEQ ID NO.15, and CDR3 is the amino acid sequence shown in SEQ ID NO.

18.

3. An anti-CDH17 antibody, characterized in that The anti-CDH17 antibody comprises the antigen-binding fragment of claim 1 or 2; Preferably, the amino acid sequence of the heavy chain variable region of the anti-CDH17 antibody is as shown in SEQ ID NO.19-SEQ ID NO.22, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.23-SEQ ID NO.26; Preferably, the anti-CDH17 antibody further comprises a constant region.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleic acid encoding the anti-CDH17 antigen-binding fragment of claim 1 or 2 or the anti-CDH17 antibody of claim 3.

5. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule of claim 4.

6. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises the anti-CDH17 antibody of claim 3; Preferably, the chimeric antigen receptor further comprises a signal peptide, a hinge region, a transmembrane domain and a signaling domain; Preferably, the signal peptide comprises a CD8α signal peptide and / or an IgGκ light chain signal peptide, preferably an IgGκ light chain signal peptide; Preferably, the hinge region comprises any one of CD8α, CD28, human IgG1, IgG2, IgG4 or IgA, preferably the CD8α hinge region; Preferably, the transmembrane domain includes a CD8α transmembrane region and / or a CD28 transmembrane region, preferably a CD8α transmembrane region; Preferably, the signaling domain comprises a CD3ζ signaling domain; Preferably, the signaling domain further comprises a co-stimulatory domain; Preferably, the co-stimulatory domain includes any one or a combination of at least two of 4-1BB, CD28 intracellular region, DAP10 or OX40.

7. A host cell, characterized in that The host cell comprises the nucleic acid molecule of claim 4, the expression vector of claim 5 or the chimeric antigen receptor of claim 6.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the anti-CDH17 antibody of claim 3; Preferably, the pharmaceutical composition further comprises an anti-tumor drug; Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, diluent or excipient.

9. Use of the antigen-binding fragment according to claim 1 or 2, the anti-CDH17 antibody according to claim 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the chimeric antigen receptor according to claim 6, the host cell according to claim 7 or the pharmaceutical composition according to claim 8 in the preparation of cancer detection reagents and / or cancer therapeutic drugs.

10. The use according to claim 9, characterized in that: The cancer includes cancer that is positive for CDH17 expression; Preferably, the cancer includes any one of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer or gallbladder cancer.

Images