An antigen-binding fragment against CDH17, an antibody and its application

Abstract

This invention provides an antigen-binding fragment against CDH17, an antibody, and their applications. The heavy chain variable region of the antigen-binding fragment comprises the amino acid sequences shown in SEQ ID NO. 1-3 (CDR1), SEQ ID NO. 4-6 (CDR2), and SEQ ID NO. 7-9 (CDR3); the light chain variable region of the antigen-binding fragment comprises the amino acid sequences shown in SEQ ID NO. 10-12 (CDR1), SEQ ID NO. 13-15 (CDR2), and SEQ ID NO. 16-18 (CDR3). The antigen-binding fragment and anti-CDH17 antibody provided by this invention exhibit high specificity. The chimeric antigen receptor and CAR-T cells prepared from the antibody show significant cytotoxicity against cells stably expressing CDH17 protein.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to an antigen-binding fragment against CDH17, an antibody and application thereof. BACKGROUND

[0002] CDH17, full name transmembrane cadherin 17, is a transmembrane glycoprotein mainly expressed in gastrointestinal epithelial cells. It plays a crucial role in maintaining intestinal barrier function and intercellular communication. CDH17 participates in cell adhesion and signal transduction between cells through its structural characteristics such as transmembrane region and extracellular domain, thereby playing a key role in the normal physiological function of the intestine. However, recent research has found that CDH17 is abnormally expressed on the surface of various tumor cells, suggesting its potential role in tumor occurrence and development. This abnormal expression may be related to the malignant behaviors of tumor cells such as proliferation, invasion and metastasis. However, the current diagnostic and therapeutic means for CDH17 are still limited, and the existing antibody products often have problems such as low specificity and insufficient affinity, which seriously restricts the effectiveness and safety of its clinical application.

[0003] Therefore, providing an antibody that specifically binds to CDH17 has important therapeutic significance for cancer caused by CDH17-expressing tumor cells. SUMMARY

[0004] In view of the problems in the prior art, the purpose of the present application is to provide an antigen-binding fragment against CDH17, an antibody and application thereof. The antigen-binding fragment and the antibody can specifically bind to CDH17 protein, and the preparation of chimeric antigen receptor and CAR-T cells has obvious cytotoxicity to target cells expressing CDH17 protein.

[0005] To achieve this purpose, the present application adopts the following technical solutions:

[0006] In a first aspect, the present application provides an antigen-binding fragment against CDH17, 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-SEQ ID 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 comprises the amino acid sequence shown in SEQ ID NO. 10-SEQ ID NO. 12, the CDR2 comprises the amino acid sequence shown in SEQ ID NO. 13-SEQ ID NO. 15, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO. 16-SEQ ID NO. 18.

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

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

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

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

[0030] In a second aspect, the present application provides an anti-CDH17 antibody comprising the antigen binding fragment of the first aspect.

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

[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 this invention, the 9C11 antibody clone was humanized. Maintaining consistency with the 9C11 antibody's CDR, the FR region of the human antibody was carefully selected and transplanted, successfully converting the 9C11 antibody into a highly humanized H9C11 antibody. The modified H9C11 antibody not only structurally resembles human immunoglobulins but also exhibits excellent binding ability to the human and monkey CDH17 antigen. 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 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 against tumor target cells was evaluated. Experimental results showed that the modified CAR-T cells could effectively recognize and kill tumor cells expressing the CDH17 antigen, while having minimal toxicity to normal cells. This discovery not only validates the potential of the H9C11 antibody in tumor immunotherapy but also provides important experimental evidence 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 includes a constant region.

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

[0052] Meanwhile, the present invention also provides a method for preparing the anti-CDH17 antibody as described in the second aspect, specifically including the following steps:

[0053] (1) Hybridoma preparation was carried out by immunizing BALB / c mice with DNA. Five healthy female BALB / c mice aged 7-8 weeks were selected for immunization. After a certain immunization time, the serum titer of the immunized mice was detected. Spleen cells of mice whose serum titers met the requirements of the fusion experiment were selected and fused with myeloma cells SP2 / 0 in an appropriate ratio under the action of a fusion agent to prepare hybridoma monoclonal cells.

[0054] (2) Hybridoma cells were cultured in selective medium R1640-HAT for 7-10 days, and then cultured in HT medium for 3-4 days. On days 10-14, the hybridoma supernatant samples were detected by ELISA to obtain positive clones.

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

[0056] Thirdly, the present invention provides a nucleic acid molecule that encodes an antigen-binding fragment as described in the first aspect or an anti-CDH17 antibody as described in the second aspect.

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

[0058] Fifthly, the present invention provides a chimeric antigen receptor (CAR), said chimeric antigen receptor comprising the anti-CDH17 antibody as described in the second aspect.

[0059] In this invention, CAR-T cells containing the chimeric antigen receptor can highly express anti-CDH17 antibodies and exhibit significant cytotoxicity against CDH17-positive cells.

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

[0061] Preferably, the signal peptide includes CD8α signal peptide and / or IgGκ light chain signal peptide, and more preferably IgGκ light chain signal peptide.

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

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

[0064] Preferably, the signal conduction structure domain includes the CD3ζ signal conduction domain.

[0065] Preferably, the signal transduction structural domain further includes a co-stimulation 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 this invention, the target CDH17 chimeric antigen receptor includes IgGκ light chain signal peptide, scFv, CD8α hinge region, CD8α transmembrane region, 4-1BB, and CD3ζ.

[0068] In this 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 signal transduction domain sequence.

[0069] The amino acid sequence (SEQ ID NO.27) of the IgGκ light chain signal peptide is as follows:

[0070] MDMRVPAQLLGLLLLWLRGARC;

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

[0072] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD;

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

[0074] IYIWAPLAGTCGVLLLSLVITLYC;

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

[0076] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL;

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

[0078] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0079] Preferably, the amino acid sequence of the chimeric antigen receptor includes the sequences 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 (19C1 CAR amino acid sequence):

[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 a nucleic acid molecule as described in the third aspect, an expression vector as described in the fourth aspect, or a 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 includes an antitumor drug.

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

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

[0093] Eighthly, the present invention provides the use of the 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 in the preparation of cancer detection reagents and / or cancer treatment drugs.

[0094] Preferably, the cancer includes cancers that are positive for CDH17 expression.

[0095] Preferably, the cancer includes any one of stomach 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 this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all 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 CDH17 proteins from multiple species (including humans, mice and cynomolgus monkeys), providing more options for the selection of subsequent animal models; the membrane protein array experiment also shows that H9C11-scFv-hFc can specifically bind CDH17 without binding to other non-target proteins, indicating that the anti-CDH17 antibody has obvious specificity;

[0099] (2) This invention provides three chimeric antigen receptors (CARs) against CDH17 and one humanized chimeric antigen receptor against CDH17. After these chimeric antigen receptors are transfected into T cells via lentiviral vectors, CAR-T cells expressing anti-CDH17CAR are obtained. The CAR-T cells have significant cytotoxicity against cells that stably express CDH17 protein. Therefore, they have significant 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. Attached Figure Description

[0100] Figure 1 The graph shows the results of the antibody-cell line binding ability test.

[0101] Figure 2 This is a schematic diagram of the CDH17 CAR structure;

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

[0103] Figure 4A Image showing the results of humanized 19C1(H19C1)CAR combined with CDH17;

[0104] Figure 4B Image showing the results of humanized 19C1(H19C1)CAR combined with CDH17;

[0105] Figure 5 The graph shows the results of CDH17 CAR-T positivity rate detection.

[0106] Figure 6 The graph shows the killing rate of CDH17 CAR-T cells against CDH17-positive target cells.

[0107] Figure 7 The results of MPA detection of H9C11-scFv-hFc specifically binding to human CDH17 are shown in the figure. Detailed Implementation

[0108] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely 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 determined by the claims.

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

[0110] Example 1

[0111] In this embodiment, hybridoma monoclonal cells were prepared by DNA immunization of BALB / c mice, and the hybridoma cells were cultured in selective medium R1640-HAT, then replaced with HT medium, and the hybridoma supernatant samples were detected by ELISA, yielding 99 positive clones.

[0112] Subsequently, flow cytometry screening was performed to obtain subclones, and sequencing yielded three correct sequence clones of anti-CDH17 antibodies: 25E7, 2D5, and 9C11. Sequencing identification confirmed the anti-CDH17 antibody sequences 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: The CDH17 antigen is coated onto a 96-well plate. The coating process must be carried out at room temperature to ensure the activity of the antigen.

[0128] 2. Antibody incubation: Different concentrations of anti-CDH17 antibody are incubated with the coated antigen at 37°C to simulate the environment inside the human body.

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

[0130] 4. Colorimetric reaction: Add TMB substrate and perform a colorimetric reaction based on the color change. The colorimetric reaction must be carried out under dark conditions to prevent substrate decomposition.

[0131] 5. Data Analysis: OD was measured using an ELISA reader. 450 The values ​​were determined, and 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 antibody affinity constant (KD value). The results are shown in Table 1.

[0133] Table 1

[0134]

[0135] Affinity assays were performed on the three antibodies (25E7, 2D5, and 9C11). The binding rate constants were comparable to those of the control antibody. All three antibodies (25E7, 2D5, and 9C11) showed high affinity. The KD value of the 25E7 antibody was 8.13E-09M, the KD value of the 2D5 antibody was 1.65E-09M, and the KD value of the 9C11 antibody was 9.11E-09M.

[0136] Example 3

[0137] The binding of the antibody of this invention to human CDH17 can be measured by flow cytometry. HEK293 cells were transiently transfected with a plasmid overexpressing CDH17 protein.

[0138] HEK293 cells (5×10) 5Cells were incubated in PBS + 5% BSA at 4°C for 30 min. Then, they were incubated together with experimental antibodies at concentrations from 1 μg / mL (serialized 10-fold to 0.01 μg / mL) + 5% BSA in PBS at 4°C for 60 min. After centrifugation and washing, the cells were incubated with secondary antibody (FITC-labeled, 1:200, Sigma, F9512) in PBS + 5% BSA on ice for 30 min (protected from light). Cells were washed three times and analyzed by flow cytometry. Results Figure 1 As shown: the EC50 values ​​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 embodiment, a chimeric antigen receptor against CDH17 and its expression vector were constructed.

[0141] (1) Sequence Design

[0142] This chimeric antigen receptor includes an IgGκ light chain signal peptide sequence (Leader), an antibody sequence that specifically binds to the CDH17 antigen (scFv), a CD8a hinge region and transmembrane region sequence, a 4-1BB co-stimulatory domain sequence, and a CD3ζ signal transduction domain sequence.

[0143] The specific structure is as follows: Figure 2 As shown in the figure; the amino acid sequence and nucleotide sequence of each part are shown in Table 2 below:

[0144] Table 2

[0145]

[0146] (2) Constructing a chimeric antigen receptor expression vector against CDH17

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

[0148] Then, the pCDH-EF1 vector was ligated to the CAR gene fragment, and the specific ligation system is shown in Table 3 below:

[0149] Table 3

[0150] Reagent Amount used pCDH-EFl vector 2 μL (50 ng) CAR gene 10 μL (150 ng) T4 DNA Ligase Buffer 2 μL T4 DNA Ligase (NEB) 1 μL ddH2O 5 μL Total 20 μL

[0151] Ligation was performed at 22℃ for 1 hour. The ligation product was directly transformed into Stbl3 Escherichia coli competent cells. 200 μL of the transformation product was spread on ampicillin-resistant LB plates and incubated upside down in an incubator at 37℃ overnight.

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

[0153] Example 5

[0154] Flow cytometry was used to detect the binding of CDH17 protein in 293T cells after transient transfection with CAR plasmid.

[0155] To further investigate the binding of CDH17 protein in 293T cells after transient transfection with CAR plasmids, we first prepared 293T cells and cultured them to an appropriate density. Then, we mixed the CAR plasmid DNA with the transfection reagent according to the manufacturer's instructions. The mixture was added to the culture medium containing 293T cells, and the culture dish was gently agitated to ensure uniform distribution. Next, the cells were placed in an incubator and cultured under appropriate conditions to promote transfection efficiency. Twenty-four hours after transfection, the cells were collected and washed with PBS. Subsequently, the cells were labeled with a solution containing CDH17 protein and incubated. Flow cytometry was then used to analyze the binding of CDH17 protein to the CAR plasmid-transfected 293T cells. Finally, the flow cytometry data were collected and analyzed to assess transfection efficiency and CAR protein expression levels. Results are as follows: Figure 4A and Figure 4B As shown, 25E7 can bind to CDH17 antigens from 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, at 19.08% and 39.48%, respectively. 9C11 can also only bind to human and monkey CDH17, at 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 embodiment, the lentiviral expression vector was packaged into lentiviruses, H9C11 CAR-T cells were constructed, and the infection efficiency of the lentivirus on T cells was detected.

[0158] (1) Lentiviral packaging using a four-plasmid system

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

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

[0161] After 6 hours, the culture medium was replaced with fresh medium and cultured again. 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 peripheral blood with physiological saline at a ratio of 1:1, add Ficoll to a centrifuge tube, slowly add the diluted peripheral blood, centrifuge at 1500 rpm for 30 min, and gently aspirate the PBMC layer into another centrifuge tube.

[0164] The PBMCs were washed multiple times with physiological saline and then transferred to CAR-T cell culture medium (containing 50 ng / mL OKT3 and 300 IU / mL IL-2) for culture.

[0165] After PBMC isolation, it needs to be activated with CAR-T cell culture medium containing 50 ng / mL OKT3 and 300 IU / mL IL-2;

[0166] Two days later, the culture medium was replaced with CAR-T cell culture medium containing 300 IU / mL for scale-up culture.

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

[0168] (3) Lentiviral infection of T cells

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

[0170] RetroNectin was aspirated and the coated 6-well plates were blocked with Hank's solution containing 2.5% BSA and placed in a cell culture incubator at 37°C for 0.5 h.

[0171] Aspirate the blocking solution, wash the 6-well plate with Hank's solution containing 2% Hepes, add X-VIVO medium, add an appropriate amount of lentivirus solution, centrifuge at 2000g for 2h;

[0172] Discard the supernatant and add 1×10 6 T cells were centrifuged at 1000g for 10 min and cultured in a 37℃, 5% CO2 cell culture incubator. The above process was repeated on the second day. The expression of H9C11 CAR was measured 5 days after infection. CAR expression was detected by flow cytometry by using FITC-Protein L to bind to H9C11 CAR.

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

[0174] Example 6

[0175] In this embodiment, tumor cells overexpressing the luciferase gene were used as target cells for a killing assay, and blank control NC (untransfected T cells) were used as a control.

[0176] The cytotoxicity 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 was detected using the luciferase assay.

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

[0178] Example 7

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

[0180] First, the expressed antibody fusion protein H9C11-scFv-hFc is a single-chain antibody sequence of H9C11 fused with 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 approximately 6,000 membrane protein clones (representing more than 94% of the human membrane proteome) were transfected into HEK-293T cells (ATCC, CRL-3216); or QT6 cells (ATCC, CRL-1708); or into 384-well cell culture plates (Corning, 3764), at a density of 18,000 cells / well.

[0182] After 36 hours of incubation, the experimental antibody was added to a membrane proteome array matrix plate at a predetermined concentration, and the binding of the antibody H9C11-scFv-hFc to cells expressing approximately 6000 membrane proteins was directly detected using flow cytometry. Therefore, all target proteins possessed native conformations and appropriate post-translational modifications.

[0183] Membrane protein array results as follows Figure 7 As shown, H9C11-scFv-hFc can specifically bind to human CDH17 without binding to other non-target proteins.

[0184] In summary, the anti-CDH17 antibody provided by this invention can specifically bind to CDH17 proteins from multiple sources, including human, mouse, and monkey sources, and has virtually no binding ability to other proteins, exhibiting high specificity. Furthermore, the CAR provided by this invention and T cells containing this CAR exhibit significant cytotoxicity against cells expressing CDH17 protein. Therefore, the anti-CDH17 antibody provided by this invention has a specific therapeutic effect on diseases targeting the CDH17 protein.

[0185] The applicant declares that the above description is only a specific embodiment 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 conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. An antigen binding fragment against CDH17, characterized in that, The antigen binding fragment is selected from one of the following three groups: (1) the CDR1 of the heavy chain variable region of the antigen binding fragment is the amino acid sequence of SEQ ID NO. 1, the CDR2 is the amino acid sequence of SEQ ID NO. 4, and the CDR3 is the amino acid sequence of SEQ ID NO. 7; the CDR1 of the light chain variable region of the antigen binding fragment is the amino acid sequence of SEQ ID NO. 10, the CDR2 is the amino acid sequence of SEQ ID NO. 13, and the CDR3 is the amino acid sequence of SEQ ID NO. 16; (2) the CDR1 of the heavy chain variable region of the antigen binding fragment is the amino acid sequence of SEQ ID NO. 2, the CDR2 is the amino acid sequence of SEQ ID NO. 5, and the CDR3 is the amino acid sequence of SEQ ID NO. 8; the CDR1 of the light chain variable region of the antigen binding fragment is the amino acid sequence of SEQ ID NO. 11, the CDR2 is the amino acid sequence of SEQ ID NO. 14, and the CDR3 is the amino acid sequence of SEQ ID NO. 17; (3) the CDR1 of the heavy chain variable region of the antigen binding fragment is the amino acid sequence of SEQ ID NO. 3, the CDR2 is the amino acid sequence of SEQ ID NO. 6, and the CDR3 is the amino acid sequence of SEQ ID NO. 9; the CDR1 of the light chain variable region of the antigen binding fragment is the amino acid sequence of SEQ ID NO. 12, the CDR2 is the amino acid sequence of SEQ ID NO. 15, and the CDR3 is the amino acid sequence of SEQ ID NO.

18.

2. An anti-CDH17 antibody, characterized in that, The anti-CDH17 antibody comprises the antigen binding fragment of claim 1.

3. The anti-CDH17 antibody of claim 2, wherein, The anti-CDH17 antibody is selected from one of the following three groups: (1) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 19, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 23; (2) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 20, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 24; (3) the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 21, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.

25.

4. The anti-CDH17 antibody of claim 2, wherein The anti-CDH17 antibody further comprises a constant region.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises the anti-CDH17 antigen binding fragment of claim 1 or the anti-CDH17 antibody of any one of claims 2-4.

6. An expression vector, characterized by, The expression vector comprises the nucleic acid molecule of claim 5.

7. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises the anti-CDH17 antibody of any one of claims 2-4.

8. The chimeric antigen receptor of claim 7, wherein, The chimeric antigen receptor further comprises a signal peptide, a hinge region, a transmembrane domain, and a signaling domain.

9. The chimeric antigen receptor of claim 8, wherein, The signal peptide comprises a CD8a signal peptide and / or an IgG kappa light chain signal peptide.

10. The chimeric antigen receptor of claim 9, wherein, The signal peptide is an IgG kappa light chain signal peptide.

11. The chimeric antigen receptor of claim 8, wherein, The hinge region comprises any one of CD8a, CD28, human IgGl, IgG2, IgG4, or IgA.

12. The chimeric antigen receptor of claim 11, wherein, The hinge region is a CD8a hinge region.

13. The chimeric antigen receptor of claim 8, wherein, The transmembrane domain comprises a CD8a transmembrane region and / or a CD28 transmembrane region.

14. The chimeric antigen receptor of claim 13, wherein, The transmembrane domain is a CD8a transmembrane region.

15. The chimeric antigen receptor of claim 8, wherein, The signaling domain comprises a CD3 zeta signaling domain.

16. The chimeric antigen receptor of claim 8, wherein, The signaling domain further comprises a costimulatory domain.

17. The chimeric antigen receptor of claim 16, wherein, The costimulatory domain comprises any one or a combination of at least two of 4-1BB, CD28 intracellular region, DAP10, or OX40.

18. A host cell, characterized in that, The host cell comprises the nucleic acid molecule of claim 5, the expression vector of claim 6, or the chimeric antigen receptor of any one of claims 7-17.

19. A pharmaceutical composition comprising, The pharmaceutical composition comprises the anti-CDH17 antibody of any one of claims 2-4.

20. The pharmaceutical composition of claim 19, wherein, The pharmaceutical composition further comprises an anti-tumor drug.

21. The pharmaceutical composition of claim 19, wherein, The pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, diluent, or excipient.

22. Use of the host cell of claim 18 in the preparation of a medicament for treating colon cancer positive for CDH17 expression.

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