Nanobodies targeting gcc and uses thereof

Abstract

The present application relates to the field of immunology, in particular to a nanobody targeting GCC and application thereof. The present application obtains an antibody sequence with high antigen affinity through screening and applies it to a CAR-T cell, and CDR1, CDR2 and CDR3 are represented by SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 in turn. The results show that the obtained CAR-T cell targeting GCC has a significant anti-tumor effect. The antibody sequence of the present application can be applied to the development of antibody drugs, cell therapy drugs, ADC drugs and the like.

Description

Technical Field

[0001] This invention relates to the field of immunology, and in particular to nanobodies targeting GCC and their applications. Background Technology

[0002] Guanylyl cyclase C (GUCY2C, GCC) is a transmembrane protein. The human GUCY2C gene is located on chromosome 12q12, with a molecular weight of approximately 120 kDa. It belongs to the receptor guanylyl cyclase family and is specifically expressed in intestinal epithelial cells, particularly highly along the entire medullary-caudate axis from the duodenum to the distal colon. GUCY2C, under the influence of its ligands, participates in signal transduction regulating water and electrolyte balance in the intestine. In colorectal cancer, the synthesis of ligand mRNA is inhibited, leading to the loss of ligands that activate GUCY2C, resulting in homeostasis imbalance in colorectal epithelial cells and promoting tumor development. GUCY2C is abnormally highly expressed in primary and metastatic colorectal cancer cells, at 2-10 times higher than in normal intestinal epithelial cells. It is not expressed in normal extraintestinal tissues. GUCY2C is also expressed in pancreatic cancer, gastric cancer, and esophageal cancer.

[0003] Colorectal cancer is a common malignant tumor of the gastrointestinal tract, occurring in the colon and rectum, most commonly the rectum and sigmoid colon. GUCY2C shows a high positive expression rate in the peripheral blood of colorectal cancer patients, suggesting that GUCY2C can serve as an early detection indicator for postoperative recurrence and metastasis in cases of colorectal cancer recurrence or metastasis. Numerous studies indicate that GUCY2C expression is often abnormal in the early stages of colon cancer. Research data shows that GUCY2C is specifically and highly expressed in metastatic colorectal cancer cells, demonstrating its great potential as a molecular marker for colorectal cancer.

[0004] CAR-T (Chimeric antigen receptor T cell) therapy refers to the use of gene modification technology to transfer genetic material containing specific antigen recognition domains and T cell activation signals into T cells. This allows the T cells to directly bind to specific antigens on the surface of tumor cells and be activated. By releasing perforin, granzyme B, and other substances, the T cells directly kill tumor cells. At the same time, the T cells also release cytokines to recruit endogenous immune cells to kill tumor cells, thereby achieving the goal of treating tumors. Furthermore, the T cells can be formed to form immune memory T cells, thus obtaining a specific and long-lasting anti-tumor mechanism.

[0005] However, existing technologies use traditional single-chain or double-chain antibodies. Compared to nanobodies, single-chain antibodies have a larger molecular weight, stronger immunogenicity, and are more difficult to identify hidden epitopes. They are also prone to heavy-light chain mismatch. When single-chain antibodies are applied to CAR-T, the heavy-light chain mismatch leads to CAR structure aggregation, which in turn causes spontaneous depletion of CAR-T. Summary of the Invention

[0006] In view of this, the present invention provides a novel anti-GCC antibody with high antigen affinity, targeting GUCY2C, with high target specificity and safety, while having the advantages of small molecular weight, strong specificity, high affinity, and weak immunogenicity to humans.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] The present invention provides a protein fragment having the sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0009] In some specific embodiments of the present invention, the protein fragments described above may be antibody fragments.

[0010] The present invention also provides nanobodies, the sequences of CDR1, CDR2, and CDR3 being shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

[0011] In some specific embodiments of the present invention, the above-mentioned nanobody has the following characteristics:

[0012] (1) The amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; or

[0013] (2) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (1), and whose function is the same as or similar to that of (1); or

[0014] (3) An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (1) or (2).

[0015] The plurality of numbers can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0016] The present invention also provides a nucleic acid molecule having a gene encoding the above-mentioned nanobody.

[0017] In some specific embodiments of the present invention, the above-mentioned nucleic acid molecules also include acceptable gene elements, including but not limited to promoters, signal peptides, hinge coding elements, transmembrane domain coding elements, co-stimulatory domain coding elements, and signal domain coding elements.

[0018] In some specific embodiments of the present invention, the morphology of the above-mentioned nucleic acid molecules can be linear or circular.

[0019] The present invention also provides an expression vector having the above-mentioned nucleic acid molecule.

[0020] In some specific embodiments of the present invention, the expression vectors described above include, but are not limited to, lentiviral vectors.

[0021] The present invention also provides a host cell having: the above-described nucleic acid molecule or the above-described expression vector.

[0022] In some specific embodiments of the present invention, the host cells mentioned above include, but are not limited to, immune cells, and the immune cells include, but are not limited to, T cells.

[0023] The present invention also provides the use of any of the following in the preparation of chimeric antigen receptors or chimeric antigen receptor T cells:

[0024] (a) the above-mentioned nanobody; (b) the above-mentioned nucleic acid molecule; (c) the above-mentioned expression vector; (d) the above-mentioned host cell.

[0025] The present invention also provides a chimeric antigen receptor, which is linked to the above-mentioned nanobody.

[0026] In some specific embodiments of the present invention, the chimeric antigen receptor may further include at least one of a signal peptide, a hinge, a transmembrane domain, a co-stimulatory domain, and a signal domain.

[0027] In some specific embodiments of the present invention, the chimeric antigen receptor described above has:

[0028] (4) An amino acid sequence as shown in SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10; or

[0029] (5) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (4), and whose function is the same as or similar to that of (4); or

[0030] (6) An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (4) or (5).

[0031] The plurality of numbers can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0032] The present invention also provides a chimeric antigen receptor T cell having the above-mentioned chimeric antigen receptor.

[0033] This invention also provides the use of any of the following in the preparation of drugs or diagnostic reagents targeting GCC:

[0034] (a) the above-mentioned nanobody; (b) the above-mentioned nucleic acid molecule; (c) the above-mentioned expression vector; (d) the above-mentioned host cell; (e) the above-mentioned chimeric antigen receptor; (f) the above-mentioned chimeric antigen receptor T cell.

[0035] In some specific embodiments of the present invention, the drugs described above include anti-tumor drugs, and the tumors include, but are not limited to, colorectal cancer.

[0036] This invention also provides the use of any of the following in the treatment of colorectal cancer:

[0037] (a) the above-mentioned nanobody; (b) the above-mentioned nucleic acid molecule; (c) the above-mentioned expression vector; (d) the above-mentioned host cell; (e) the above-mentioned chimeric antigen receptor; (f) the above-mentioned chimeric antigen receptor T cell.

[0038] In some specific embodiments of the present invention, the colorectal cancer described above includes, but is not limited to, primary colorectal cancer and metastatic colorectal cancer.

[0039] The present invention also provides a product comprising any one of the following:

[0040] (a) the above-mentioned nanobody; (b) the above-mentioned nucleic acid molecule; (c) the above-mentioned expression vector; (d) the above-mentioned host cell; (e) the above-mentioned chimeric antigen receptor; (f) the above-mentioned chimeric antigen receptor T cell.

[0041] The present invention also provides a formulation for treating colorectal cancer, comprising acceptable excipients or adjuvants, and the aforementioned chimeric antigen receptor T cells.

[0042] In some specific embodiments of the present invention, the above-mentioned formulations include, but are not limited to, cell therapy formulations.

[0043] The present invention also provides treatment methods, including treatments based on any of the following:

[0044] (a) the above-mentioned nanobody; (b) the above-mentioned nucleic acid molecule; (c) the above-mentioned expression vector; (d) the above-mentioned host cell; (e) the above-mentioned chimeric antigen receptor; (f) the above-mentioned chimeric antigen receptor T cell.

[0045] In some specific embodiments of the present invention, the above-mentioned treatment method includes administering the chimeric antigen receptor T cells to a patient.

[0046] The present invention has the following beneficial effects:

[0047] (1) GUCY2C is stably expressed in primary colorectal cancer cells, while it is abnormally highly expressed in metastatic colorectal cancer cells, and is considered a specific marker molecule for metastatic colorectal cancer. It is also expressed in pancreatic cancer, gastric cancer, and esophageal cancer, suggesting that GUCY2C may serve as a potential target for these diseases.

[0048] (2) In this invention, 20 non-humanized nanobody sequences were designed for different epitopes of the GUCY2C protein, and the two sequences with the best function were obtained by screening through in vitro cell experiments;

[0049] (3) After humanizing the non-humanized nanobody sequences obtained by screening, two humanized nanobody sequences were obtained by screening with colorectal cancer target cells. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0051] Figure 1 This shows the antibody screening flowchart;

[0052] Figure 2 The results of titer calculation for GUCY2C CAR lentivirus concentrate are shown.

[0053] Figure 3 The image shows changes in CAR-T cells and the CD4 / CD8 ratio, where A shows changes in CAR and B shows the CD4 / CD8 ratio.

[0054] Figure 4 Construction of target cell lines for colon cancer;

[0055] Figure 5 The images show the killing effect of CAR-T cells on HCT116-GCC-LUC and HCT116-LUC target cells. In the images, A shows the killing effect of HCT116-GCC-LUC target cells, and B shows the killing effect of HCT116-LUC target cells.

[0056] Figure 6 The images show the release of cytokines after CAR-T cells are co-incubated with target cells. A shows the IFN-γ level after HCT116-GCC-LUC stimulation, B shows the IL-2 level after HCT116-LUC stimulation, and C shows the IFN-γ level after HCT116-LUC stimulation.

[0057] Figure 7 This demonstrates the release of CD107a from CAR-T cells after co-incubation with target cells;

[0058] Figure 8 Demonstrates the affinity of the humanized antibody for HCT116-GCC-LUC target cells;

[0059] Figure 9 The results of titer calculation for humanized GUCY2C CAR lentivirus concentrate are shown.

[0060] Figure 10 The image shows changes in CAR-T cells and the CD4 / CD8 ratio, where A shows changes in CAR and B shows the CD4 / CD8 ratio.

[0061] Figure 11 The results show the killing effect of CAR-T cells on HCT116-GCC-LUC and LS1034-LUC target cells. Among them, A shows the results of HCT116-GCC-LUC, B shows the results of LS1034-LUC, and C shows the results of HCT116-LUC.

[0062] Figure 12 The images show the release of cytokines from CAR-T cells after co-incubation with target cells. A shows the IFN-γ level after HCT116-GCC-LUC stimulation, B shows the IL-2 level after HCT116-GCC-LUC stimulation, C shows the IFN-γ level after LS1034-LUC stimulation, D shows the IL-2 level after LS1034-LUC stimulation, E shows the IFN-γ level after HCT116-LUC stimulation, and F shows the IL-2 level after HCT116-LUC stimulation.

[0063] Figure 13 The images show the release of CD107a from CAR-T cells after co-incubation with target cells. In this image, A shows the results of HCT116-GCC-LUC, and B shows the results of LS1034-LUC.

[0064] Figure 14 This shows changes in tumor volume after GCC CAR-T cell administration;

[0065] Figure 15 The change in body weight in mice after administration of GCC CAR-T cells is shown. Detailed Implementation

[0066] This invention discloses nanobodies targeting GCC and their applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0067] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0068] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0069] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0070] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0071] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0072] This invention utilizes nanobody sequences. Compared to traditional antibodies, nanobodies have advantages such as small molecular weight, high specificity, high affinity, and weak immunogenicity in humans. GUCY2C is known to be stably expressed in primary colorectal cancer cells, while exhibiting abnormally high expression in metastatic colorectal cancer cells. It is not expressed in extraintestinal tumors or other tumors, demonstrating high target specificity and safety. Based on this, this invention screened and obtained both non-humanized and humanized nanobody sequences and provides their applications in colorectal cancer.

[0073] The amino acid sequence information of the nanobody is as follows:

[0074] The amino acid sequence of N3-B11 is as follows:

[0075] QVQLQESGGGSVQSGGSLRLSCGASGDTFDRFCMAWFRQAPGKEREEVARLTRAGLPTYADSVRGRFAISRDSAKSTLYLQMDRLRPDDTAMYYCAAEVTTLDWRCPGDYSYWGQGTQVTVSS (SEQ ID NO: 1);

[0076] The amino acid sequence of HU1206-VHH12 is as follows:

[0077] QVQLVESGGGVVQPGRSLRLSCAASGDTFDRFCMAWFRQAPGKEREEVARLTRAGLPTYADSVRGRFAISRDSSKNTLYLQMNRLRAEDTAVYYCAAEVTTLDWRCPGDYSYWGQGTLVTVSS (SEQ ID NO: 2);

[0078] The amino acid sequence of HU1206-VHH13 is as follows:

[0079] QVQLVESGGGVVQPGRSLRLSCAASGDTFDRFCMAWVRQAPGKEREEVARLTRAGLPTYADSVRGRFAISRDSSKNTLYLQMNRLRPEDTAVYYCAAEVTTLDWRCPGDYSYWGQGTLVTVSS (SEQ ID NO: 3);

[0080] The CDR regions of N3-B11, HU1206-VHH12, and HU1206-VHH13 are the same, and are as follows:

[0081] CDR1: GDTFDRFCMA (SEQ ID NO: 4);

[0082] CDR2:RLTRAGLPTYADSV (SEQ ID NO: 5);

[0083] CDR3: AAEVTTLDWRCPGDYSY (SEQ ID NO: 6).

[0084] Abbreviations or terms involved: GUCY2C refers to guanylate cyclase, also known as GCC; T refers to T lymphocytes; CAR-T refers to chimeric antigen receptor T cells; PBMC refers to peripheral blood mononuclear cells; VHH refers to heavy chain single-domain antibody; ELISA refers to enzyme-linked immunosorbent assay; Luc refers to luciferase; HCT116-LUC refers to HCT116 cells overexpressing luciferase; One-glo refers to luciferase substrate; Golgi Stop refers to Golgi apparatus terminator; UTD-T refers to untreated T cells; CD107a refers to lysosome-associated membrane protein 1, a marker of lymphocyte degranulation; IFN-γ refers to interferon-γ; IL-2 refers to interleukin-2; DPBS refers to phosphate-buffered saline.

[0085] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0086] The present invention will be further illustrated below with reference to the embodiments.

[0087] Example 1: VHH screening targeting GCC

[0088] 1. Experimental Methods

[0089] After co-incubating the natural library with the antigen (ACRO, GUC-H5257), multiple candidate monoclonal antibodies were obtained through two elution and amplification processes. The screening procedure is described in [link to screening process]. Figure 1 Select candidate antibodies with high affinity for functional validation.

[0090] 2. Experimental Results

[0091] Table 1 shows the affinity test data for two of the candidate antibodies.

[0092] Table 1: Affinity Test Data

[0093]

[0094] Example 2: Construction of GCC-CAR plasmid

[0095] 1. Experimental Methods

[0096] The coding gene sequences of multiple candidate nanobodies screened in Example 1 were homologously recombinated into a core plasmid vector to construct a GCC-CAR plasmid. The constructed plasmid was then transformed, and the transformed competent cells... E. coli The plasmid was spread onto a medium plate containing ampicillin and cultured. Positive clones were picked and sequenced. Correct sequencing indicated successful construction of the GCC-CAR plasmid. Table 2 shows the GCC-CAR structure on the core plasmid vector constructed based on two candidate nanobodies.

[0097] Table 2: GCC-CAR structure on the core plasmid vector

[0098]

[0099] The amino acid sequence of the SDT02-001 CAR structure is as follows:

[0100] MALPVTALLLPLALLLHAARPEVQLQESGGGSVQAGGSLRLSCVASGYDHCPVDMSWYRQAPGKEREFVSAITDDGSSTYAESVKGRFTISRDNNKNTVYLQMNSLKSEDTAMYYCKTQTSTVAYCWRRDRGGAWGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL DFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ IDNO: 7);

[0101] The amino acid sequence of the SDT02-017 CAR structure is as follows:

[0102] MALPVTALLLPLALLLHAARPQVQLQESGGGSVQSGGSLRLSCGASGDTFDRFCMAWFRQAPGKEREEVARLTRAGLPTYADSVRGRFAISRDSAKSTLYLQMDRLRPDDTAMYYCAAEVTTLDWRCPGDYSYWGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL DFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ IDNO: 8).

[0103] 2. Experimental Results

[0104] Positive clones were selected for sequencing, and the sequencing was correct, indicating that the GCC-CAR plasmid was successfully constructed.

[0105] Example 3: Lentiviral Preparation

[0106] 1. Experimental Methods

[0107] (1) 293T inoculation: at 15 cm 2 In the dish, add 2.0 × 10 mm per well. 7 Inoculation was performed, and virus packaging was carried out 18 h to 20 h after cell adhesion.

[0108] (2) Virus packaging: A three-plasmid virus packaging system was used. The core plasmid expressing GUCY2C CAR, the helper plasmid psPAX2 (Addgene#12260) expressing Gag-pol, and the envelope plasmid expressing VSV G (Addgene#12259) were added to DPBS in sequence. After thorough mixing, PEI at 3 times the amount of plasmid was added. After incubation for 15 min, the plasmid was added dropwise to 293T cells.

[0109] (3) Virus collection and concentration: The virus was collected at 48 h and 72 h and centrifuged at 2000 rpm for 10 min to remove cell debris. Then, it was concentrated by overnight centrifugation at 4000g, 4℃, and 9°C.

[0110] (4) Infect 5.0 × 10⁻⁶ virus concentrate with serially diluted virus solution. 5Jurkat cells were added, along with polybrene at a concentration of 8 μg / mL.

[0111] (5) Collect cells 72 h later to detect CAR+ and calculate viral titer.

[0112] 2. Experimental Results

[0113] See Figure 2 Two viruses with different GUCY2C sequences were successfully packaged, and the viral titers of both were higher than 1.0 × 10⁻⁶. 8 TU / mL.

[0114] Example 4: Preparation and Detection of GCC CAR-T Cells

[0115] 1. Experimental Methods

[0116] (1) PBMC recovery

[0117] ① Remove the purchased PBMC from liquid nitrogen and melt it quickly in a 37°C water bath;

[0118] ②Prepare 15 mL of KBM581 medium in a 50 mL centrifuge tube beforehand. Carefully transfer the cells from the cryopreservation tube to the 50 mL centrifuge tube and centrifuge at 500g (8 up, 6 down) for 10 min.

[0119] ③ Remove the supernatant, resuspend the cells in HPBS (1% HSA+DPBS) for cell counting and T cell sorting.

[0120] (2) T cell sorting and activation

[0121] ① Based on the PBMC resuscitation count results, sorting and activation magnetic beads (Gibco, 11132D) were added at a cell:magnetic bead ratio of 1:1.5.

[0122] ②Pipette the required volume of magnetic beads into a centrifuge tube containing HPBS, mix thoroughly, place the tube on a magnetic rack, let it stand for 2 minutes, discard the supernatant, remove the centrifuge tube from the magnetic rack, add 1 mL of HPBS, and mix well.

[0123] ③ T cell sorting: Add the mixed cell suspension to the washed magnetic bead cryovial, mix well, place in a four-dimensional mixer, and incubate at room temperature with shaking for 30 min.

[0124] ④ After incubation, insert the centrifuge tube into the magnetic rack, let it stand for 2 minutes, discard the supernatant, remove the tube, resuspend it in complete T cell culture medium, count the cells, and adjust the density to 1.0 × 10⁻⁶ cells / mL. 6 Continue culturing at a rate of [number] cells / mL, and perform virus transduction after 48 hours.

[0125] (3) Lentiviral transduction

[0126] ① After culturing the sorted T cells for 48 hours, the number of cells was counted.

[0127] ② The virus prepared in Example 3 was incubated with Novonectin, and transduction was performed at an MOI of 4 based on the virus titer and a transduction rate of 2.0 × 10⁻⁶. 6 T cells were added to 24-well plates with concentrated virus solution and 5 μg / mL Novonectin conversion aid, and incubated at 37°C for 30 min. Based on the T cell count, 2.0 × 10⁻⁶ cells were added. 6 T cells;

[0128] ③ Centrifugal transduction: Place the 24-well plate in a centrifuge and centrifuge at 800g, 5°C for 1.5 hours at 32°C; place the 24-well plate in a carbon dioxide incubator and centrifuge and change the medium after 24 hours.

[0129] (4) CAR+ detection

[0130] CAR-T cells cultured to day 7 and day 9 were collected, and CAR expression was detected after staining. Simultaneously, day 7 CAR-T cells were collected for flow cytometry analysis of CD4 and CD8 changes.

[0131] ① After transducing T cells with enveloped virus by centrifugation for 72 h, the cells were collected into 15 mL centrifuge tubes, placed on a magnetic rack for 2 min, and the supernatant was collected.

[0132] ② Centrifuge 500g of the collected supernatant for 10 min, resuspend in complete culture medium, and count 5×10⁻⁶ samples. 5 One cell;

[0133] ③ Wash once with FACS Buffer, then add 1 μL of CD3, CD4, D8 and anti-GUCY2C antibody, and stain at 4℃ for 20 min;

[0134] ④ After staining, wash again with FACS Buffer and then detect CAR+ by flow cytometry.

[0135] 2. Experimental Results

[0136] according to Figure 3 The CAR flow cytometry results showed that the two lentiviral sequences successfully infected T cells, with CAR expression levels exceeding 70% in both cases (see [link to data]). Figure 3 (A in the text); compared with UTD-T, CAR-T cells showed higher CD4 expression than CD8 (see A in the text). Figure 3 (B in the middle).

[0137] Example 5: Killing of target cells by GCC CAR-T cells in vitro

[0138] 1. Experimental Methods

[0139] (1) Construction of target cell lines for colon cancer

[0140] ① Cell plating: 293T cells were plated one day before transfection;

[0141] ② Transfection: Prepare the PEI / DNA complex. Take 2 mL of PBS (or opti-MEM) and transfer it to a 15 mL centrifuge tube. Add the core plasmid pCDH-CMV-GCC-EF1-Luc-T2A-Puro and pCDH-CMV-MCS-EF1-Luc-T2A-Puro (Aono Gene, catalog number HG-VMH1366), the helper plasmid psPAX2 (Gag-pol), and the envelope plasmid pMD2.G (VSV G) sequentially. Mix thoroughly by pipetting. Add PEI (YESEN, 40820ES10) at three times the weight of the plasmids and mix well. Incubate at room temperature for 15 min. Add the PEI / DNA complex dropwise to 293T cells and mix thoroughly. Place the culture dish in a 37℃, 5% CO2 incubator and incubate for 6-8 hours. Then replace the medium containing the transfection reagent with fresh complete medium (DMEM + 10% FBS) for further incubation. Viral supernatant was collected twice, at 48 h and 72 h post-transfection, and temporarily stored at 4°C. The virus was centrifuged to remove cell debris, and the supernatant was retained. The supernatant was concentrated by overnight centrifugation, and the supernatant was removed. The precipitate was resuspended in an appropriate amount of viral cryopreservation solution to obtain the concentrated virus solution, which was stored at -80°C for later use.

[0142] ③ Infect target cells: Add 1.5 × 10⁻⁶ cells to each well of a six-well plate. 6 Target cells with 10 μL of concentrated virus solution (activity titer approximately 1.0 × 10⁻⁶) 8 Add polybrene (TU / mL) to the working concentration, mix well, centrifuge at 32℃ and 800 g, and then incubate in an incubator; change the medium 6-8 h after infection, then subculture and add puromycin (puro) for selection, and observe cell death.

[0143] ④ Flow cytometry detection: Take 1×10 6 Tumor cells were stained with GUCY2C Recombinant Mouse Monoclonal Antibody (Thermo, MA5-49968) at 4°C for 20 min, washed once with FACS Buffer, stained with PE anti-mouse IgG2a Antibody (BioLegend, 407108) secondary antibody at 4°C for 20 min, washed once with FACS Buffer, and then analyzed.

[0144] (2) Detection of the killing effect of CAR-T cells on target cells

[0145] ① Add the prepared colon cancer target cell line to a 96-well white plate at a ratio of 10,000 cells / 100 μL;

[0146] ② CAR-T cells were prepared as effector cells according to the method in Example 4, and the effector cells were diluted with effector-target ratios of 2:1, 1:1, 0.5:1, 0.25:1, 0.125:1, and 0.625:1.

[0147] ③ After diluting the effector cells, transfer 100 μL to the target cells and co-culture in a CO2 incubator for 16-18 hours. Then, add ONE-Glo substrate (Promega, #E6120) and detect the cell death rate using a microplate reader. The formula for calculating the cell death rate is:

[0148] Cell killing % = [1 - (experimental group fluorescence reading - control group fluorescence reading) / control group fluorescence reading]%.

[0149] (3) Detection of cytokine release after co-culture of CAR-T cells and target cells

[0150] The detection was performed using an IFN-γ (ACRO, CRS-A017) and TNF-α (ACRO, CRS-A002) factor detection kit, and the specific method is as follows:

[0151] a. Reagent preparation

[0152] ① Before use, bring all reagents (see Table 3) to room temperature. If crystals are present, place the solution in a 37°C incubator for 10-15 minutes.

[0153] ② Preparation of Standards: Prepare a storage solution from the provided lyophilized product using ultrapure water. Dissolve the product at room temperature for 15 to 30 minutes, mixing gently by pipetting or aspirating, avoiding vigorous shaking or vortexing. The reconstituted storage solution should be stored at -70°C. It is recommended that the freeze-thaw cycle not exceed once, and the aliquot size should not be less than 10 μg.

[0154] Table 3: Reagent Information

[0155]

[0156] b. Preparation of working solution

[0157] ① Prepare 1×Washing Buffer: Take 50 mL of 10×Washing Buffer, dilute with ultrapure water / deionized water and bring the volume to 500 mL, then mix gently.

[0158] ② Prepare 1×Dilution Buffer: Take 50 mL of 2×Dilution Buffer, dilute with 1×Washing Buffer and bring the volume to 100 mL, then mix gently.

[0159] ③ Prepare Biotin-Anti-X Antibody working solution: Dilute the Biotin-Anti-IFN-γ Antibody reconstructed storage solution to 1.0 μg / mL with 1×Dilution Buffer; dilute the Biotin-Anti-TNF-α Antibody reconstructed storage solution to 0.5 μg / mL with 1×Dilution Buffer. This working solution should be prepared fresh before use.

[0160] ④ Prepare Streptavidin-HRP (Streptavidin-HRP peroxidase) working solution: Dilute Streptavidin-HRP 2000 times with 1×DilutionBuffer. This working solution should be stored away from light and prepared fresh before use.

[0161] c. Detection

[0162] ① Add the test sample and serially diluted standards to the reaction wells, 100 μL per well. Add 100 μL of Dilution Buffer to the blank control wells. Seal the plate with sealing film and incubate at room temperature for 1.0 h;

[0163] ② Carefully peel off the sealing film, discard the liquid in the wells, add 300 μL of 1×Washing Buffer to each well, soak for 10 seconds, and wash the plate 3 times in total. After each wash, pat the plate dry on absorbent paper;

[0164] ③ Add 100 μL of diluted Biotin-Anti-IFN-γAntibody (diluted to 1.0 μg / mL) / Biotin-Anti-TNF-α Antibody (diluted to 0.5 μg / mL) working solution to the corresponding well. Prepare the working solution fresh before use. Seal the plate with sealing film and incubate at room temperature for 1.0 h.

[0165] ④ Repeat step 2 to wash the plate;

[0166] ⑤ Add 100 μL of diluted Streptavidin-HRP (1:2000 dilution) working solution to the corresponding well. The working solution should be prepared fresh and stored in the dark. Seal the plate with sealing film and incubate at room temperature for 30 min.

[0167] ⑥ Repeat step 2 to wash the plate;

[0168] ⑦ Add 100 μL of Substrate Solution to each well. Seal the plate with a sealing film, protect from light, and incubate at room temperature for 20 min.

[0169] ⑧ Add 50 μL of Stop Solution to each well and gently shake the microplate until well mixed;

[0170] ⑨ Use an ELISA reader to measure the absorbance of each well at wavelengths of 450 nm and 630 nm, and take the readings within 10 minutes after termination.

[0171] (4) Detection of CD107a release after co-culture of CAR-T cells and target cells

[0172] ① Add 100 μL (1.0 × 10⁻⁶) of effector cells. 5 ), 100 μL of target cells (1.0 × 10⁻⁶) 5 Add a 96-well plate;

[0173] ② Add 2 μL of PE anti-human CD107a (LAMP-1) Antibody (BioLenged, 328608) and incubate at 37℃ for 1 h;

[0174] ③ Add 2 μL of GolgiStop TM Protein Transport Inhibitor (BD, 554724) was added to 3 mL of complete culture medium. 20 μL of the mixture was added to the cells and mixed well. After incubation for 2.5–3 h, 0.5 μL of APC anti-human CD56 (NCAM) Antibody (Biolegend, 362504) was added. The cells were incubated at 37 °C for 30 min. After washing once with FACS buffer, the expression of CD107a molecules was detected by instrumentation.

[0175] 2. Experimental Results

[0176] (1) Construction of target cell lines for colon cancer

[0177] according to Figure 4 The flow cytometry results showed that the selected colon cancer cells expressed GUCY2C at the required level, and the LUC overexpression cell line HCT116-GCC-LUC was successfully constructed.

[0178] (2) Detection of the killing effect of CAR-T cells on target cells

[0179] like Figure 5As shown, by analyzing the killing effect of HCT116-GCC-LUC target cells with high GCC expression, two types of CAR-T cells were found to have a strong killing effect on target cells, showing a significant difference compared with the control group UTD-T (e.g., Figure 5 (As shown in A); However, in the killing of GCC target cells with low HCT116-LUC expression, SDT02-001 and SDT02-017 were found to be insensitive to HCT116-LUC, and the killing effect was not different from that of the UTD-T control group (as shown in A). Figure 5 (As shown in B in the diagram).

[0180] (3) Detection of cytokine release after co-culture of CAR-T cells and target cells

[0181] Two types of CAR-T cells, after being stimulated by target cells with high GCC expression of HCT116-GCC-LUC, such as Figure 6 As shown, compared with the UTD-T control group, SDT02-001 and SDT02-017 released higher levels of IFN-γ and IL-2 cytokines (such as...). Figure 6 (As shown in A and B). After CAR-T cells were stimulated with HCT116-LUC target cells that weakly expressed GCC, the SDT02-001 and SDT02-017 groups of CAR-T cells released less IFN-γ, with the release amount being less than 50 pg / mL (as shown in A and B). Figure 6 (As shown in C). After stimulation of HCT116-LUC target cells, the level of IL-2 cytokine release was too low, and IL-2 release was not detected.

[0182] (4) Detection of CD107a release after co-culture of CAR-T cells and target cells

[0183] like Figure 7 As shown, after co-incubating HCT116-GCC-LUC, which is highly expressed in GCC, with CAR-T cells, the experimental groups released high levels of CD107a compared with the UTD-T control group cells.

[0184] Based on the responses of GCC CAR-T cells to target cells expressing different GCC values ​​(HCT116-GCC-LUC, 84.09% GCC expression) and HCT116-LUC, 5.2% GCC expression, it was found that both SDT02-001 and SDT02-017 CAR-T cells exhibited strong killing effects on HCT116-GCC-LUC and released related cytokine factors. However, after treatment of HCT116-LUC cells, compared with the control group UTD-T, there was no significant difference in killing effect and IFN-γ cytokine release between SDT02-001 and SDT02-017 CAR-T cells. Considering efficacy and safety, the VHH antibody sequence in the structures of SDT02-001 and SDT02-0017 was determined to be humanized.

[0185] Example 6: Sequence humanization modification and affinity detection

[0186] 1. Experimental Methods

[0187] (1) Humanized sequence design: The CDR region of the antibody was defined by predictive analysis using systems such as Kabat, IMGT, AbM, and Chothia. Human germline frames were selected as candidate templates, and the CDR grafting method was used to replace the camel-derived frame region (FR) with the selected human germline frame region (FR), retaining only the camel-derived CDR. In order to reduce the adverse effects of humanization on the spatial conformation, activity, and function of the antibody, it is also necessary to perform reverse mutations on some key amino acids in the human frame region, that is, to change them back to the amino acids at the corresponding sites in the camel-derived FR region.

[0188] (2) Humanized antibody expression: The designed humanized heavy chain antibody gene was linked to the human IgG1 Fc gene and cloned into expression plasmids to express it in the form of VHH-Fc. The plasmids were transfected into CHO cells, cultured for expression, and the culture supernatant was collected. After purification by Protein A affinity chromatography, the antibody affinity was detected.

[0189] (3) Humanized antibody affinity test: The kinetics and affinity properties of the antibody were tested using the Biacore 8K platform.

[0190] 2. Experimental Results

[0191] After humanization, the VHH antibodies 0102-B1 and N3-B11 in the structures of SDT02-001 and SDT02-0017 yielded 32 sequences. Based on the antibody affinity assay data for target cells, several candidate sequences with high affinity were screened for functional verification. Figure 8Table 4 shows the fluorescence signal levels and affinity data of two candidate sequences, HU1206-VHH12 and HU1206-VHH13.

[0192] Table 4 Affinity data for HU1206-VHH12 and HU1206-VHH13

[0193]

[0194] Example 7: Construction of humanized GCC-CAR plasmid and lentivirus packaging

[0195] 1. Experimental Methods

[0196] Humanized GCC CAR viruses were prepared according to Examples 2 and 3. Table 5 shows the GCC-CAR structure on the core plasmid vector constructed based on two candidate nanobodies.

[0197] Table 5: Humanized GCC-CAR structures on core plasmid vectors

[0198]

[0199] The amino acids in the CAR structure of SDT02-030 are as follows:

[0200] MALPVTALLLPLALLLHAARPQVQLVESGGGVVQPGRSLRLSCAASGDTFDRFCMAWFRQAPGKEREEVARLTRAGLPTYADSVRGRFAISRDSSKNTLYLQMNRLRAEDTAVYYCAAEVTTLDWRCPGDYSYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL DFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ IDNO: 9);

[0201] The amino acids in the CAR structure of SDT02-031 are as follows:

[0202] MALPVTALLLPLALLLHAARPQVQLVESGGGVVQPGRSLRLSCAASGDTFDRFCMAWVRQAPGKEREEVARLTRAGLPTYADSVRGRFAISRDSSKNTLYLQMNRLRPEDTAVYYCAAEVTTLDWRCPGDYSYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL DFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ IDNO: 10).

[0203] 2. Experimental Results:

[0204] like Figure 9 Table 6 shows that two candidate humanized GUCY2C-CAR viruses were successfully packaged, and the viral titers in all groups were higher than 3.0 × 10⁻⁶. 8 TU / mL can be used for subsequent sequence comparison and verification.

[0205] Table 6: Virus titer

[0206]

[0207] Example 8: Preparation and Detection of Humanized GCC-CAR-T Cells

[0208] 1. Experimental Methods

[0209] Humanized GCC-CAR-T cells were prepared according to the method in Example 4.

[0210] 2. Experimental Results

[0211] like Figure 10 As shown, the two humanized viral sequences successfully infected T cells, and the CAR content in all groups was above 60% (e.g., Figure 10 (As shown in A in the figure); compared with the control group UTD-T, the experimental group showed increased CD8 expression and decreased CD4 expression (as shown in A in the figure). Figure 10 (As shown in B in the diagram).

[0212] Example 9: Killing of target cells by humanized GCC-CAR-T cells in vitro

[0213] 1. Experimental Methods

[0214] The procedure was performed according to the method described in Example 5. LS1034-LUC was also prepared based on LS1034 cells according to the method described in Example 5.

[0215] 2. Experimental Results

[0216] (1) Cell killing

[0217] like Figure 11 As shown, after CAR-T cells killed target cells with high GCC expression, it was found that both groups of CAR-T cells significantly killed the target cells HCT116-GCC-LUC and LS1034-LUC with high GCC expression (as shown in Figures 1-2). Figure 11 As shown in A and B), it did not significantly kill target cells with low expression of HCT116-LUC in GCC (as shown in A and B). Figure 11 (as shown in C).

[0218] (2) Cytokine release

[0219] The release of IFN-γ and IL-2 was detected after CAR-T cells were co-incubated with three types of target cells. Figure 12 As shown in the figure, HCT116-GCC-LUC and LS1034-LUC target cells, which highly express GCC, released higher levels of IFN-γ (as shown in A and C in the figure) and IL-2 (as shown in B and D in the figure) after stimulating CAR-T cells, and the release of IFN-γ was higher than that of IL-2; after stimulation with HCT116-LUC (weakly expressing GCC) cells, CAR-T cells released trace amounts of IFN-γ and IL-2, both of which were less than 100 pg / mL (as shown in E and F in the figure).

[0220] (3) CD107a release

[0221] like Figure 13 As shown, after stimulation with HCT116-GCC-LUC (Figure A) and LS1034-LUC (Figure B), SDT02-030 CAR-T and SDT02-031 CAR-T cells released large amounts of CD107a.

[0222] Experiments involving CAR-T cell characterization, cell killing, and cytokine release revealed that SDT02-030 and SDT02-031 CAR-T cells significantly killed target cells with high GCC expression, releasing large amounts of cytotoxic factors such as IFN-γ, IL-2, and CD107a. However, they did not significantly kill target cells with low GCC expression or release cytokines. Based on these findings, the SDT02-030 and SDT02-031 nanobody sequences were selected for in vivo animal validation.

[0223] Example 10: In vivo animal validation of GCC CAR-T

[0224] 1. Experimental Methods

[0225] The tumor animal model was established using 5-6 week old female NCG mice purchased from Chengdu Yaokang Biotechnology Co., Ltd. Tumor cells were selected from LS1034-LUC cells constructed according to the method in Example 5, at a concentration of 5.0 × 10⁻⁶. 6 Subcutaneous transplantation of tumor-bearing cells was performed at a dose per tumor-bearing cell. GCC CAR-T cells were prepared according to the method in Example 4 and at a dose of 5.0 × 10⁻⁶ cells / cell. 6 The mice were administered the drug at the prescribed doses. A blank control group (DPBS, phosphate-buffered saline) and a negative control group (UTD-T cells, untreated T cells) were included, with four mice in each group. The antitumor effect of GCC CAR-T cells in mice was observed by periodically monitoring changes in tumor volume and mouse body weight.

[0226] 2. Experimental Results

[0227] Mice were inoculated with experimental samples on the second day after tumor implantation, and tumor volume changes were measured every 3 days. The results are as follows: Figure 14 , Figure 15 As shown in Tables 7 and 8, after CAR-T cell administration, the body weight of mice in the SDT02-030 and SDT02-031 experimental groups remained stable, indicating that 5 × 10⁻⁶ cells were administered. 6 At the prescribed dosage, CAR-T cells showed no toxicity in mice. Observations of tumor growth in mice revealed that after 28 days of administration, the SDT02-030 sample exhibited a tumor inhibition rate of 62.9%, while the SDT02-031 sample showed an inhibition rate of 25.7%. This indicates that both SDT02-030 and SDT02-031 samples significantly inhibited tumor growth in mice, with the SDT02-030 sample demonstrating a more significant effect.

[0228] Table 7: Statistical analysis of tumor volume changes after GCC CAR-T cell administration

[0229]

[0230] Table 8: Statistical analysis of body weight changes in mice after GCC CAR-T cell administration

[0231]

[0232] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nanobody targeting GCC, characterized in that, The nanobody comprises CDR1, CDR2 and CDR3, and the sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

2. The nanobody as described in claim 1, characterized in that, The sequences are shown as SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:

3.

3. A nucleic acid molecule, characterized in that, Genes encoding the nanobody as described in claim 1 or 2.

4. An expression carrier, characterized in that, It has the nucleic acid molecule as described in claim 3.

5. A host cell, characterized in that, have: (i) the nucleic acid molecule of claim 3; or (ii) The expression vector as described in claim 4.

6. Any of the following applications in the preparation of chimeric antigen receptors targeting GCC or chimeric antigen receptor T cells targeting GCC: (a) The nanobody according to claim 1 or 2; (b) The nucleic acid molecule of claim 3; (c) The expression vector as claimed in claim 4; (d) The host cell as described in claim 5.

7. A chimeric antigen receptor targeting GCC, characterized in that, It includes a signal peptide, a hinge, a transmembrane domain, a co-stimulatory domain, and a signal domain, and is connected with the nanobody as described in claim 1 or 2 as an antigen recognition domain. The co-stimulatory domain is 4-1BB; The signal structure domain is CD3ζ.

8. Chimeric antigen receptor T cells targeting GCC, characterized in that, It has the chimeric antigen receptor as described in claim 7.

9. Any of the following applications in the preparation of drugs or diagnostic reagents targeting GCC: (a) The nanobody according to claim 1 or 2; (b) The nucleic acid molecule of claim 3; (c) The expression vector according to claim 4; (d) The host cell as described in claim 5; (e) The chimeric antigen receptor as described in claim 7; (f) The chimeric antigen receptor T cell as described in claim 8; The drug is used to treat colon cancer with high GCC expression.

10. The product, characterized in that, Includes acceptable excipients or adjuvants, and the chimeric antigen receptor T cells as described in claim 8.

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