Nanobodies targeting cdh6 and uses thereof

Abstract

The present application relates to the field of immunology, in particular to a nanobody targeting CDH6 and its application. The present application obtains a human CDH6 nanobody sequence through natural phage library screening, verifies its affinity and designs a CAR structure, constructs it on a CAR-T, studies its anti-tumor function on ovarian cancer through in-vitro experiments, humanizes the sequence with better anti-tumor effect, further verifies its anti-tumor function in and out of animals, and finally obtains an optimized sequence, which can also be applied to the development of antibody drugs, ADC drugs and the like.

Description

Technical Field

[0001] This invention relates to the field of immunology, and in particular to the screening of nanobodies targeting CDH6 and their applications. Background Technology

[0002] Cadherin-6 (CDH6) is a member of the cadherin family, a single membrane glycoprotein composed of 790 amino acids. Belonging to type II cadherins, it consists of three distinct domains: an extracellular domain (ECD) containing five cadherin sequences, a transmembrane domain, and an intracellular tail. CDH6 is highly expressed in malignant tumors such as renal cell carcinoma, ovarian cancer, thyroid cancer, cholangiocarcinoma, and small cell lung cancer, especially in ovarian cancer (OVC) and renal cell carcinoma (RCC), where its expression is observed in approximately 65%–85% of OVC patients. Taking ovarian cancer as an example, approximately 70%–80% of patients with advanced ovarian cancer experience disease recurrence after receiving standard platinum-based chemotherapy. These patients still have unmet clinical needs for treatment and urgently require new therapies.

[0003] Chimeric antigen receptor modified T cell (CAR-T cell) therapy is one of the fastest-growing forms of adoptive cell immunotherapy. This therapy uses genetically engineered receptors to transduce the patient's T cells to target cancer cell surface antigens, thereby mediating an anti-tumor effect. CAR-T cell therapy primarily utilizes the patient's own immune system to attack cancer cells, offering advantages such as high targeting, long-lasting therapeutic effects, and fewer side effects.

[0004] The existing technology has the following disadvantages: (1) Antibody: Traditional single-chain antibodies have large steric hindrance and high immunogenicity; (2) Target: Currently, there is a lack of CAR-T products targeting CDH6 at home and abroad.

[0005] There is a significant unmet clinical need for treatment of refractory solid tumors such as ovarian cancer, renal cancer, and cholangiocarcinoma that highly express CDH6. Summary of the Invention

[0006] In view of this, this invention obtained the human CDH6 nanobody sequence through screening of a natural phage library, verified its affinity, and designed a CAR structure. The sequence was then constructed onto a CAR-T cell line, and its anti-tumor function against ovarian cancer and renal cancer was studied through in vitro experiments. Sequences with good anti-tumor effects were humanized to further verify their anti-tumor function in vivo and in vitro in animals, ultimately yielding an optimized sequence that can also be applied to the development of antibody drugs and ADCs. This invention uses alpaca-derived nanobodies, which have advantages over traditional single-chain antibodies, such as less steric hindrance and lower immunogenicity.

[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 sequence of CDR1 is SEQ ID NO: 4, the sequence of CDR2 is SEQ ID NO: 5, and the sequence of CDR3 is SEQ ID NO: 6.

[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 comprising 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 comprising the above-mentioned nucleic acid molecules.

[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 comprising: 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 application of the above-mentioned nanobodies, nucleic acid molecules, expression vectors or host cells in the preparation of chimeric antigen receptors or chimeric antigen receptor T cells.

[0024] The present invention also provides a chimeric antigen receptor, which is linked to the above-mentioned nanobody.

[0025] In some specific embodiments of the present invention, the chimeric antigen receptor described above has:

[0026] (4) An amino acid sequence as shown in SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 10; or

[0027] (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

[0028] (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).

[0029] The plurality of numbers can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0030] 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.

[0031] The present invention also provides a chimeric antigen receptor T cell having the above-mentioned chimeric antigen receptor.

[0032] The present invention also provides the application of the above-mentioned nanobody, the above-mentioned nucleic acid molecule, the above-mentioned expression vector, the above-mentioned host cell, the above-mentioned chimeric antigen receptor or the above-mentioned chimeric antigen receptor T cell in the preparation of drugs or detection reagents targeting CDH6.

[0033] 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, ovarian cancer, kidney cancer, and bile duct cancer.

[0034] The present invention also provides the application of the above-mentioned nanobody, the above-mentioned nucleic acid molecule, the above-mentioned expression vector, the above-mentioned host cell, the above-mentioned chimeric antigen receptor or the above-mentioned chimeric antigen receptor T cell in the treatment of ovarian cancer, renal cancer or cholangiocarcinoma.

[0035] The present invention also provides products comprising the above-mentioned nanobody, the above-mentioned nucleic acid molecule, the above-mentioned expression vector, the above-mentioned host cell, the above-mentioned chimeric antigen receptor or the above-mentioned chimeric antigen receptor T cell.

[0036] The present invention also provides formulations for treating ovarian cancer, kidney cancer or bile duct cancer, comprising acceptable excipients or adjuvants, and the aforementioned chimeric antigen receptor T cells.

[0037] In some specific embodiments of the present invention, the above-mentioned formulations include, but are not limited to, cell therapy formulations.

[0038] The present invention also provides treatment methods, including treatment based on the above-described nanobodies, nucleic acid molecules, expression vectors, host cells, chimeric antigen receptors, or chimeric antigen receptor T cells.

[0039] In some specific embodiments of the present invention, the above-mentioned treatment method includes administering the chimeric antigen receptor T cells to a patient.

[0040] The present invention has the following beneficial effects:

[0041] Nanobodies have small molecular weight and good affinity: The molecular weight of nanobodies is only 10% of that of traditional antibodies, but they still retain a high antigen-binding capacity (HCAbs), which makes them more affinity and specific when targeting.

[0042] Simple structure, easy to prepare and express: Nanobodies are usually composed of VHH (monoclonal variants), and their heavy chain region has only one variable region, which makes the design and expression process simpler and also reduces production costs.

[0043] High specificity and strong binding force: Due to their optimized structure, nanobodies can bind to targets more efficiently and perform well in recognizing hidden sites of viruses such as HIV and SARS-CoV-2.

[0044] High stability: Compared with traditional antibodies, nanobodies have higher stability and are less likely to cause immune responses due to decomposition, thus prolonging their effective treatment time. Attached Figure Description

[0045] 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.

[0046] Figure 1 This shows the antibody screening flowchart;

[0047] Figure 2 Show the titers of SDT03-003 and SDT03-004 lentivirus concentrates;

[0048] Figure 3 This shows changes in CAR-T cells;

[0049] Figure 4 The results of flow cytometry analysis of OVCAR3-luc and SKOV3-AB003-luc-2 are shown.

[0050] Figure 5 This demonstrates the killing effect of CAR-T cells on target cells;

[0051] Figure 6 A diagram showing the gradient dilution steps;

[0052] Figure 7 This demonstrates the release of IFN-γ from CAR-T cells after co-incubation with target cells;

[0053] Figure 8 A diagram showing the gradient dilution steps;

[0054] Figure 9 This demonstrates IL-2 release after CAR-T cells are co-incubated with target cells;

[0055] Figure 10 This shows the release of CD107a from CAR-T cells after co-incubation with target cells;

[0056] Figure 11 This indicates the CD4 / CD8 ratio of CAR-T cells;

[0057] Figure 12 Showing the titer of the concentrated humanized lentivirus solution;

[0058] Figure 13 This shows changes in CAR-T cells;

[0059] Figure 14 This demonstrates the killing effect of CAR-T cells on OVCAR3-Luc.

[0060] Figure 15 The study showed that CAR-T cells killed SKOV3-AB003-LUC-2.

[0061] Figure 16This demonstrates the release of IFN-γ from CAR-T cells after co-incubation with target cells;

[0062] Figure 17 This demonstrates IL-2 release after CAR-T cells are co-incubated with target cells;

[0063] Figure 18 This indicates the proportion of CD4-positive CAR-T cells;

[0064] Figure 19 This indicates the proportion of CD8-positive CAR-T cells;

[0065] Figure 20 This indicates the release of CD107a from CAR-T cells;

[0066] Figure 21 This shows the changes in tumor volume after CAR-T cell administration;

[0067] Figure 22 This shows the change in body weight in mice after CAR-T cell administration. Detailed Implementation

[0068] This invention discloses the screening of CDH6-targeting nanobodies 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 is particularly important to note 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] CDH6 is significantly highly expressed in ovarian cancer, renal cancer, and cholangiocarcinoma, and is being studied as an emerging anti-tumor target. Currently, most drugs in development targeting this target are ADCs (antibody-adjuvant inhibitors), with two in clinical trials, but CAR-T therapy is lacking. This invention screens phage libraries to obtain multiple CDH6 sequences with high antigen affinity, then applies the discovered antibody sequences to CAR-T therapy. Through in vitro experiments, two sequences with significant anti-tumor effects are selected, humanized, and finally, in vivo animal efficacy studies show significant anti-tumor effects against ovarian cancer.

[0075] In one embodiment, the technical route of the present invention is as follows: (1) antibody discovery; (2) CAR gene synthesis and plasmid construction; (3) lentivirus packaging and CAR-T preparation; (4) target cell construction; (5) in vitro functional screening; (6) sequence humanization modification; (7) in vivo antitumor efficacy evaluation in animals.

[0076] The amino acid sequence of the CDH6 antibody used in CAR-T construction in this invention is as follows:

[0077] The amino acid sequence of 01-A1 is as follows:

[0078] EVQLVESGGGSVQAGGSLRLSCAASGYTYNRYYMGWFRQAPGKEREGVAVISAIAGRTYYADSVKGRFTISQANANNTLYLQMNSLQPEDTALYYCAADPRRYTPYDALSPRGYSFWGQGTQVTVSS (SEQ ID NO: 1);

[0079] The amino acid sequence of HU1207-VHH14 is as follows:

[0080] EVQLVESGGGLVQPGGSLRLSCAASGYTYNRYYMGWFRQAPGKEREGVAVISAIAGRTYYADSVKGRFTISRDANNSLYLQMNSLRPEDTAVYYCAADPRRYTPYDALSPRGYSFWGQGTMVTVSS (SEQ ID NO: 2);

[0081] The amino acid sequence of HU1207-VHH15 is as follows:

[0082] EVQLVESGGGLVQPGGSLRLSCAASGYTYNRYYMGWFRQAPGKEREGVAVISAIAGRTYYADSVKGRFTISRDANNSLYLQMNSLRAEDTAVYYCAADPRRYTPYDALSPRGYSFWGQGTMVTVSS (SEQ ID NO: 3);

[0083] The CDR regions of 01-A1, HU1207-VHH14, and HU1207-VHH15 mentioned above are the same, and are as follows:

[0084] CDR1: GYTYNRYYMG (SEQ ID NO: 4);

[0085] CDR2: VISAIAGRTYYADSVKG (SEQ ID NO: 5);

[0086] CDR3: AADPRRYTPYDALSPRGYSF (SEQ ID NO: 6).

[0087] Abbreviations or terms involved: NK refers to natural killer cells; CAR-NK refers to chimeric antigen receptor NK cells; T refers to T lymphocytes; UTD-T refers to untreated T cells; CAR-T refers to chimeric antigen receptor T cells; PBMC refers to peripheral blood mononuclear cells; CDH6 refers to K-cadherin, representing a subset of calcium-dependent cell adhesion proteins; PEI refers to polyethyleneimine, a water-soluble polymer widely used as a transfection reagent; ELISA refers to enzyme-linked immunosorbent assay; HSA refers to human serum albumin, an important protein and the most abundant protein in human plasma; FACS refers to flow cytometry; IFN-γ refers to interferon-γ, a water-soluble dimeric cytokine, the only member of type II interferon, formerly known as macrophage activating factor; IL-2 refers to interleukin-2, a type of interleukin among cytokines that plays an important role in the immune system; Luciferase refers to luciferase.

[0088] 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.

[0089] The present invention will be further illustrated below with reference to the embodiments.

[0090] Example 1: Antibody Discovery

[0091] 1. Experimental Methods

[0092] The phage library was coated with human CDH6 antigen protein (manufacturer: ACRO; catalog number: CA6-H5529; specification: 200 μg). After incubation with the immobilized antigen, the phage library was washed to remove non-bound phages, and two rounds of elution product amplification were performed. Plates used for the second round of titer determination were randomly selected for phage infection, the medium was changed, and the plates were incubated overnight at 30°C. Multiple candidate monoclonal antibodies were screened using a monoclonal phage ELISA method for functional validation. The screening procedure is described below. Figure 1 .

[0093] 2. Experimental Results

[0094] Table 1 shows the affinity test data for two of the candidate antibodies.

[0095] Table 1: Affinity Test Data

[0096]

[0097] Example 2: CAR gene synthesis and plasmid construction

[0098] 1. Experimental Methods

[0099] The encoding gene sequences of several candidate CDH6 nanobodies screened in Example 1 were amplified by PCR and cloned into the core plasmid vector pCDH-EF1-eGFP via homologous recombination to construct a CDH6-CAR plasmid. The constructed plasmid was transformed, and the transformed competent E. coli cells were plated on a medium containing ampicillin. Positive clones were picked and sequenced to determine whether the CDH6-CAR was successfully constructed. Table 2 shows the CAR structures on the core plasmid vector constructed based on two of the candidate nanobodies.

[0100] Table 2: CAR structures on core plasmid vectors

[0101]

[0102] The amino acid sequence of the SDT03-003 CAR structure is as follows:

[0103] MALPVTALLLPLALLLHAARPEVQLVESGGGSVQAGGSLRLSCAASGYTYNRYYMGWFRQAPGKEREGVAVISAIAGRTYYADSVKGRFTISQANANNTLYLQMNSLQPEDTALYYCAADPRRYTPYDALSPRGYSFWGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQID NO: 7);

[0104] The amino acid sequence of the SDT03-004 CAR structure is as follows:

[0105] MALPVTALLLPLALLLHAARPQVQLVESGGGSVQAGGSLRLSCAASGYTYSRYYMGWFRQAPGKEREGVAVISGNAGRTLYADSVKGRFTISQDNAKNTLYLQMNSLQPEDTAMYYCAADPRRYTTYDALSPRGYSFWGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQID NO: 8).

[0106] 2. Experimental Results

[0107] Positive clones were selected for sequencing. The sequencing was correct, indicating that the CAR plasmid was successfully constructed.

[0108] Example 3: Lentiviral Packaging and CAR-T Preparation

[0109] 1. Experimental Methods

[0110] (1) Virus packaging

[0111] HEK293T is processed according to 6×10 6 Plasmids were plated in 15 cm dishes. After the plating density reached 70%~90%, plasmid transfection was performed. The transfection system is shown in Table 3. The medium was changed 6~8 h after plasmid transfection. The culture medium collected at 48 h and 72 h after transfection was used as the virus stock solution. The virus stock solution was centrifuged at 4000g and 4℃ for 10 min. The supernatant was collected and centrifuged at 4000g overnight. The supernatant was discarded and the precipitate was resuspended with 2% HSA and stored at -80℃.

[0112] Table 3: Lentiviral packaging system with three plasmids (15 cm culture dish)

[0113]

[0114] Virus titer detection: Centrifuge an appropriate amount of cells, collect the cell pellet and count them. Calculate the number of cells required for plating based on the results, using 5 × 10⁶ cells per well. 5 The virus concentrate was serially diluted with culture medium, and the diluted solution was then added to 24-well plates. The concentration was increased at 5 × 10⁻⁶. 5 Jurkat cells and polybrene were added to each well, and the cells were centrifuged at 800g for 1.5 h. After centrifugation, the cells were removed and placed in an incubator for culture. The supernatant was discarded the next day, and culture medium was added to continue culturing. Flow cytometry was performed 72 h after transduction.

[0115] Based on the flow cytometry results, infected wells with a positive rate of approximately 50% were selected for titer counting. The calculation formula is: Lentiviral titer (TU / mL) = (Positive rate / 100 × Cell count) / Infected volume × 10⁻¹⁰ 3 .

[0116] (2) Preparation of CAR-T cells

[0117] Prepare complete culture medium for T cells: KBM581 + 10% FBS + 200 IU / mL IL-2 + 10 ng / mL IL-7 + 5 ng / mL IL-15; 1% HSA; 1% human serum albumin and DPBS were used for the experiment.

[0118] On day 0 (D0), PBMC resuscitation was performed: 100 M PBMCs were resuscitated in a 37°C water bath, 20 mL of complete T cell culture medium was added, and the supernatant was discarded after centrifugation at 500g for 10 min. Cells were resuspended in 2 mL of 1% HSA and counted. The amount of magnetic beads used was calculated, and the magnetic beads were washed twice with 1% HSA. 5 × 10⁶ cells were added to the magnetic beads. 6Cells were sorted to a density of 103 / mL and incubated on a rotary oscillator for 30 min. After incubation on a magnetic rack for 2 min, the supernatant was collected as NK cells and counted; cells bound to the magnetic beads were identified as T cells. The magnetic beads were resuspended in culture medium and centrifuged at 500g for 10 min. The supernatant was discarded, and the pellet contained sorted T cells. The magnetic beads were resuspended in an appropriate amount of culture medium and counted. The cell density was adjusted to 1×103 using complete T cell culture medium. 6 After incubation at a density of 1 / mL.

[0119] Day 2 (D2) Transduction: After centrifuging and counting the T cells from D0, transduced according to the required number of T cells for each group. The virus and Novonectin were incubated at 37°C for 30 min, then the T cells were added and centrifuged at 800g. After completion, the cells were placed in an incubator for culture, and the medium was changed the next day.

[0120] Day 5 (D5): Remove magnetic beads: Transfer cells to centrifuge tubes, place on a magnetic rack, and remove magnetic beads. Aspirate supernatant and centrifuge. Resuspend cells in T-cell complete culture medium. Adjust the remaining cell density to 1×10⁻⁶ cells / mL. 6 Place the cells / mL in an incubator and incubate. Count and change the medium every two days, adjusting the density to 1×10⁻⁶. 6 Culturing per mL.

[0121] (3) CAR+ detection

[0122] On days 7 (D7), 9 (D9), and 12 (D12), 5 × 10⁵ samples were collected in 1.5 mL EP tubes. 5 Take about 100 cells, wash once with buffer, centrifuge to remove supernatant, add 0.5 μL of primary antibody to each tube, incubate in the dark for 20 min, wash once more, resuspend in FACS buffer, and then detect the proportion of CAR+ cells using an instrument.

[0123] 2. Experimental Results

[0124] Figure 2 The results showed that the viral titers of both SDT03-003 and SDT03-004 reached 1×10⁻⁶. 8 TU / mL or higher, and SDT03-004 is higher than SDT03-003.

[0125] Figure 3 The results showed that CAR+ was above 30% in both groups.

[0126] Example 4: Target Cell Construction

[0127] 1. Experimental Methods

[0128] Remove the necessary reagents, such as PEI MAX, plasmids, and culture media, from the refrigerator beforehand and allow them to reach room temperature. Transfer 2 mL of PBS (or opti-MEM) to a 15 mL centrifuge tube. Add the core plasmid, helper plasmid, and envelope plasmid in sequence, and mix thoroughly by pipetting. Add PEI MAX at three times the mass of the plasmid, mix well, and let stand at room temperature for 15 min. (If using opti-MEM, do not mix vigorously; gently pipette, vortex, and then centrifuge briefly.)

[0129] The DNA / PEI complex was added dropwise to HEK293T cells and mixed thoroughly. The culture dishes were placed in a 37°C, 5% CO2 incubator and incubated for 6–8 hours. Afterward, the culture medium containing the transfection reagent was removed and replaced with fresh complete culture medium (DMEM + 10% FBS). Viral supernatant was collected twice, at 48 h and 72 h post-transfection and temporarily stored at 4°C. Cell debris was removed by centrifugation, and the supernatant was retained. The supernatant was concentrated by overnight centrifugation, resuspended in an appropriate amount of viral cryopreservation buffer, and stored at -80°C for later use.

[0130] Add 1.5 × 10⁻⁶ ppm to each well of the six-well plate. 6 Each target cell was mixed with 10 μL of virus concentrate, and polybrene was added at the working concentration. The mixture was centrifuged at 32°C and 800g, and then incubated. The medium was changed 6–8 h post-infection, followed by passage culture and selection with POO. Cell death was observed. Target cells were digested with trypsin, and 5 × 10⁶ cells were collected. 5 The sample was transferred to a 1.5 mL EP tube, washed once with FACSbuffer, and 1 μL of CDH6 antibody (Biolegend) was added. After incubation at 4°C in the dark for 30 min, the sample was washed once and then tested.

[0131] 2. Experimental Results

[0132] like Figure 4 As shown in Table 4, OVCAR3-luc target cells showed high basal CDH6 expression, and only luciferase overexpression was constructed; SKOV3 target cells were constructed to simultaneously express both CDH6 and luciferase, and single-clonal cell lines were selected.

[0133] Table 4: Flow Cytometry Results

[0134]

[0135] Example 5: In vitro functional verification

[0136] 1. External killing agent

[0137] (1) Experimental methods

[0138] After CAR-T cells were cultured for 48 h to remove IL-2 / 7 / 15, 1×10⁻⁶ ions were added to each well of a 96-well white plate. 4 One set of OVCAR3-luc target cells was prepared, with three replicates per group, and one control group was included. The CAR+ cell concentration was adjusted to 1×10⁻⁶. 6 Cells / mL (E:T=10:1), serially diluted and added to target cell wells, sealed with PBS, and co-cultured in an incubator for 20 h; 20 μL of one-glo working solution was added to each well, and the cells were detected using a microplate reader.

[0139] (2) Experimental results

[0140] like Figure 5 As shown, the cell killing results indicate that the two groups have similar in vitro killing efficiency against OVCAR3-luc target cells, and the latter is significantly stronger than the UTD-T group.

[0141] 2. IFN-γ release

[0142] (1) Experimental methods

[0143] After CAR-T cells were cultured for 48 h without IL-2 / IL-7 / IL-15, 1×10⁻⁶ cells were seeded per well in a 24-well plate. 5 1 target cell and 1×10 5 CAR-positive T cells were co-cultured for 20 h, and the supernatant was collected and detected using an IFN-γ ELISA kit. The kit steps are as follows:

[0144] according to Figure 6Prepare standard curves using standard plates Std.-1 to Std.-7 (1250 pg / mL ~ 19.5 pg / mL). Add 100 μL of the test sample and serially diluted standards to each well. Add 100 μL of Dilution Buffer to each blank control well. Seal the plate with sealing film and incubate at room temperature for 1.0 h. Carefully remove the sealing film, discard the liquid in the wells, and add 300 μL of 1×Washing Buffer to each well. Wash the plate three times, soaking for 10 s. After each wash, pat the plate dry on absorbent paper. Add 100 μL of diluted Biotin-Anti-IFN-γ Antibody (diluted to 1.0 μg / mL) working solution to the corresponding wells. Prepare the working solution fresh before use. Seal the plate with sealing film and incubate at room temperature for 1.0 h. Repeat the washing process. Add 100 μL of diluted Streptavidin-HRP (horseradish peroxidase) working solution (1:2000 dilution) to the corresponding well. Prepare the working solution fresh each time and store in the dark. Seal the plate with sealing film and incubate at room temperature for 30 min. Repeat the washing process. Add 100 μL of Substrate Solution to each well. Seal the plate with sealing film and incubate in the dark at room temperature for 20 min. Add 50 μL of Stop Solution to each well and gently vortex the plate until well mixed. Measure the absorbance of each well at 450 nm and 630 nm using a microplate reader, taking readings within 10 min after stop solution.

[0145] (2) Experimental results

[0146] like Figure 7 As shown, the ELISA results indicate that both groups of CAR-T cells co-cultured with OVCAR3-luc target cells released a large amount of IFN-γ, and the effect was significantly stronger than that of the UTD-T group.

[0147] 3. IL-2 release

[0148] (1) Experimental methods

[0149] After CAR-T cells were cultured for 48 h without IL-2 / IL-7 / IL-15, 1×10⁻⁶ cells were seeded per well in a 24-well plate. 5 1 target cell and 1×10 5 CAR-positive T cells were co-cultured for 20 h, and the supernatant was collected for detection using an IL-2 ELISA kit. The kit steps are as follows:

[0150] according to Figure 8Prepare standard curves using Std.-1 to Std.-8 (800 pg / mL~6.25 pg / mL). Add the test sample and serially diluted standards to the reaction wells, 50 μL per well. Simultaneously, add 50 μL of Biotin-Anti-IL-2 Antibody Solution to each well and incubate for 1 h. Carefully remove the sealing film, discard the liquid in the wells, add 300 μL of 1×Washing Buffer to each well, gently tap the plate for 1 minute, then discard the 1×Washing Buffer. After each wash, pat the plate dry on absorbent paper. Wash the plate a total of 5 times. Add 100 μL of Streptavidin-HRP Solution (streptavidin-horseradish peroxidase solution) to the corresponding wells. This working solution should be prepared fresh and stored away from light. Seal the plate with the sealing film and incubate at room temperature for 30 min. Repeat the washing process. Add 100 μL of Substrate Solution to each well. Seal the plate with sealing film, protect from light, and incubate at room temperature for 15 minutes. Add 50 μL of Stop Solution to each well and gently shake the plate until well mixed. Measure the absorbance of each well at 450 nm and 630 nm using a microplate reader, taking the readings within 10 minutes after termination.

[0151] (2) Experimental results

[0152] like Figure 9 As shown, the experimental results indicate that SDT03-003, SDT03-004 released less IL-2 when co-cultured with OVCAR3-luc.

[0153] 4. CD107a detection

[0154] (1) Experimental methods

[0155] Add 1×10 to each well of the 96-well plate 5 Each group of effector cells and target cells was prepared in triplicate. CD107a antibody was added and incubated at 37°C for 1 h. 0.5 μL of CD3 and CD8 antibodies were added and incubated at 37°C for 30 min. Diluted GolgiStop was added to each well, mixed, and incubated at 37°C for 2.5-3 h. Cells were washed once with FACS buffer and then analyzed.

[0156] (2) Experimental results

[0157] like Figure 10 As shown, the experimental results indicate that SDT03-003 and SDT03-004 can release a large amount of CD107a when co-cultured with OVCAR3-luc target cells, which is significantly stronger than that of UTD-T.

[0158] 5. CD8+ / CD4+T ratio detection

[0159] (1) Experimental methods

[0160] 5 × 10⁵ samples were collected on day 0 (D0) and day 7 (D7) of CAR-T culture, respectively. 5 About 100 cells were transferred to 1.5 mL EP tubes, washed once with buffer, centrifuged to remove supernatant, and 0.5 μL of CD3 / CD4 / CD8 antibody was added to each tube. The tubes were incubated in the dark for 20 min, washed once more, and resuspended in FACS buffer. The changes in the CD4 / CD8 ratio of each group were then detected by instrumentation.

[0161] (2) Experimental results

[0162] like Figure 11 As shown, comparing the CD4 / CD8 ratios from day 0 to day 7 (D0 to D7), it can be found that the CD8 ratio increases in both groups SDT03-003 and SDT03-004.

[0163] Comprehensive viral packaging (titer > 1×10) 8 Data on the CAR-T inhibitors, including CAR+, cell killing, IFN-γ, IL-2, and CD107a, were obtained. SDT03-003 and SDT03-004 showed high in vitro cell killing efficiency, IFN-γ release, and CD107a release. Considering efficacy and safety, the VHH antibody sequence in the structures of SDT03-003 and SDT03-004 were humanized.

[0164] Example 6: Humanization of Sequences

[0165] (1) Experimental methods

[0166] ① Humanized sequence design

[0167] The CDR regions of antibodies SDT03-003 and SDT03-004 were defined by predictive analysis using the Kabat system. Human germline frames were selected as candidate templates. 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.

[0168] ② Humanized antibody expression

[0169] The designed humanized heavy chain antibody gene was ligated into the human IgG1 Fc gene and cloned into expression plasmids, expressed as VHH-Fc. The plasmids were transfected into CHO cells, cultured for expression, and the culture supernatant was collected. After purification using a Protein A affinity chromatography column, antibody affinity was detected.

[0170] (2) Experimental results

[0171] Humanized antibodies were designed by modifying the VHH antibody sequences 01-A1 and N60-D12 in the parent sequences SDT03-003 and SDT03-004. Based on antibody expression and affinity detection data, several candidate sequences with high affinity were selected for subsequent functional validation. Table 5 shows the affinity data of two candidate sequences, HU1207-VHH14 and HU1207VHH15.

[0172] Table 5: Affinity data of humanized antibodies

[0173]

[0174] Example 7: Humanized CAR gene synthesis and plasmid construction

[0175] 1. Experimental Methods

[0176] The core plasmid vector was constructed according to the method in Example 2, and the CAR structure on the core vector is shown in Table 6.

[0177] Table 6: Humanized CDH6-CAR structures on plasmid vectors

[0178]

[0179] The amino acids in the CAR structure of SDT03-025 are as follows:

[0180] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGYTYNRYYMGWFRQAPGKEREGVAVISAIAGRTYYADSVKGRFTISRDANNSLYLQMNSLRPEDTAVYYCAADPRRYTPYDALSPRGYSFWGQGTMVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQID NO: 9);

[0181] The amino acids in the CAR structure of SDT03-026 are as follows:

[0182] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGYTYNRYYMGWFRQAPGKEREGVAVISAIAGRTYYADSVKGRFTISRDANNSLYLQMNSLRAEDTAVYYCAADPRRYTPYDALSPRGYSFWGQGTMVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQID NO: 10).

[0183] 2. Experimental Results

[0184] Positive clones were selected for sequencing. The sequencing was correct, indicating that the CAR plasmid was successfully constructed.

[0185] Example 8: Lentiviral Packaging and Preparation of Humanized CDH6-CART Cells

[0186] 1. Experimental Methods

[0187] The humanized CAR-T lentivirus packaging was prepared according to the method in Example 3.

[0188] 2. Experimental Results

[0189] Figure 12 Table 7 shows the successful packaging of viruses with the method structures of two alternative reference examples 3, and the viral titers in all groups are higher than 2 × 10⁻⁶. 8 TU / mL can be used for subsequent sequence comparison and verification.

[0190] Table 7: Virus titer

[0191]

[0192] Figure 13 The results showed that the CAR+ groups SDT03-025 and SDT03-026 had a positivity rate of around 80% on day 7 (D7), day 9 (D9), and day 11 (D11).

[0193] Example 9: In vitro functional verification of humanized CDH6-CART cells

[0194] 1. Experimental Methods

[0195] The method described in Example 4 shall be followed.

[0196] 2. Experimental Results

[0197] Figure 14 , Figure 15 The results showed that SDT03-025 and SDT03-026 had significantly stronger killing ability against OVCAR3-Luc and SKOV3-AB003-Luc cells that highly expressed CDH6 target than the UTD group.

[0198] Figure 16 The results showed that the IFN-γ release of CAR-T cells in the SDT03-025 and SDT03-026 groups was significantly stronger than that in the UTD group.

[0199] Figure 17 The results showed that the IL-2 release of CAR-T cells in the SDT03-025 and SDT03-026 groups was significantly stronger than that in the UTD group.

[0200] Figure 18 , Figure 19 The results showed that the percentage of CD4 positive cells in the SDT-CT025 and SDT-CT026 groups was above 40% on day 0 (D0), day 9 (D9), and day 11 (D11), and the proportion of CD8 cells was also increased.

[0201] Figure 20 The results showed that SDT03-025 and SDT03-026 were significantly different from the UTD group, with SDT03-025 showing stronger CD107a release.

[0202] Example 10: In vivo efficacy evaluation in animals

[0203] 1. Experimental Methods

[0204] For tumor cell preparation and mouse tumor-bearing information, please refer to Table 8. NOG mice (severely immunodeficient mice) were selected, and a total of 30 mice were required.

[0205] Table 8: Tumor Bearing Information Table

[0206]

[0207] The experimental groups are shown in Table 9. 47 days after tumor implantation (tumor volume reaches 60 mm) 3 ~100 mm 3CAR-T cells were injected between [times]. Mouse weight and tumor volume were measured regularly to observe the mice's condition; the tumor volume reached 2000 mm. 3 euthanize mice.

[0208] Table 9: Experimental Groups

[0209]

[0210] (2) Experimental results

[0211] Figure 21 , Figure 22 The results in Tables 10 and 11 show that both SDT03-025 and SDT03-026 exhibited significant tumor-suppressing effects after CAR-T cell administration, with SDT03-026 showing the best tumor-suppressing effect; almost no tumor was observed 30 days after administration. The mice in all groups maintained normal body weight, indicating that the CAR-T therapy in each group was safe.

[0212] Table 10: Statistical analysis of significant changes in tumor volume

[0213]

[0214] Table 11: Statistical analysis of significant changes in mouse body weight

[0215]

[0216] 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 CDH6, characterized in that, It includes CDR1, CDR2 and CDR3, wherein the sequence of CDR1 is SEQ ID NO: 4, the sequence of CDR2 is SEQ ID NO: 5 and the sequence of CDR3 is SEQ ID NO:

6.

2. The nanobody as described in claim 1, characterized in that, The sequence is SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:

3.

3. A nucleic acid molecule, characterized in that, It contains a gene encoding the nanobody as described in claim 1 or 2.

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

5. A host cell, characterized in that, Include: (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 CDH6 or chimeric antigen receptor T cells targeting CDH6: (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 CDH6, 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 CDH6, 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 targeting CDH6 or assays targeting CDH6: (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; (e) The chimeric antigen receptor as claimed in claim 7; (f) The chimeric antigen receptor T cell as described in claim 8; The drug in question is an anti-CDH6-overexpressing ovarian cancer drug.

10. The product, characterized in that, Includes acceptable excipients or adjuvants, and the chimeric antigen receptor T cells as described in claim 8; The products mentioned do not include food.

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