Anti-glypican 3 antibody and use thereof

Abstract

The application relates to the technical field of biological medicine, in particular to an anti-Glypican 3 antibody and application thereof, the anti-Glypican 3 antibody is coded as B6 and B51, the anti-Glypican 3 antibody comprises a heavy chain variable region, the heavy chain variable region of the anti-Glypican 3 antibody comprises CDR-H1 with an amino acid sequence as shown in SEQ ID No. 1, CDR-H2 as shown in SEQ ID No. 2 and CDR-H3 as shown in SEQ ID No. 3. The anti-Glypican 3 antibody has high affinity and specificity, can effectively target tumor antigen Glypican 3, a chimeric antigen receptor is prepared by using the anti-Glypican 3 antibody, and a chimeric antigen receptor cell is further prepared, the chimeric antigen receptor cell can efficiently kill tumor cells and has high specificity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an anti-Glypican 3 antibody and its applications. Background Technology

[0002] Hepatocellular carcinoma (HCC) is the most common histological subtype of liver cancer, the sixth most common cancer, and the fourth leading cause of cancer-related deaths.

[0003] Currently, the clinical management of HCC remains a significant challenge. Only 15%-20% of HCC cases are diagnosed early enough to be suitable for curative treatments such as surgical resection, liver transplantation, percutaneous ethanol ablation, microwave ablation, and radiofrequency ablation. Meanwhile, most HCC patients have underlying chronic liver disease, and surgical resection in this population is fraught with potential complications. Furthermore, many patients are diagnosed with advanced HCC that is not surgically resectable. The 5-year survival rate for HCC patients is only about 18%, and if the cancer cells have spread to surrounding tissues or distant sites of the body, the 5-year survival rate drops to approximately 11% and 5%, respectively.

[0004] Glypican-3 (GPC3), a member of the heparan sulfate proteoglycan family, plays a role in controlling cell division and regulating growth. Glypican 3 is poorly expressed in healthy tissues but highly expressed in many solid tumors, such as hepatocellular carcinoma (HCC). In HCC, Glypican 3 is frequently used as a biomarker for diagnosis and prognosis. Its specific expression in tumor tissues, its accessible surface location, and its role in various signaling pathways involved in tumor transformation make it an ideal target for immunotherapy in several solid tumors, including HCC.

[0005] Currently, preclinical and clinical HCC therapies targeting Glypican 3 mainly include peptide vaccines, monoclonal antibodies, and chimeric antigen receptor T-cell immunotherapy.

[0006] Chimeric antigen receptor T cell (CAR-T) therapy is a revolutionary cancer immunotherapy that has emerged in recent years. By transducing T cells to express chimeric antigen receptors, tumor-specific CAR-T cells are obtained. These cells specifically target, recognize, and kill tumor cells, thus achieving the goal of treating tumors.

[0007] Therefore, Glypican 3 CAR-T cells prepared by introducing the chimeric antigen receptor gene against Glypican 3 into T cells via gene transduction can specifically recognize and kill HCC cells expressing Glypican 3, thereby achieving its anti-tumor effect. This has significant clinical translational value for immunotherapy targeting Glypican 3. Summary of the Invention

[0008] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an anti-Glypican 3 antibody and its application to solve the problems in the prior art.

[0009] To achieve the above and other related objectives, the present invention provides an anti-Glypican 3 antibody, designated B6 and B51, or Glypican 3-VHH-B6 and Glypican 3-VHH-B51, respectively, wherein the anti-Glypican 3 antibody includes a heavy chain variable region, and the anti-Glypican 3 antibody has one or more of the following technical features;

[0010] <1> The heavy chain variable region includes the amino acid sequence CDR-H1 as shown in SEQ ID No. 1;

[0011] <2> The heavy chain variable region includes the amino acid sequence CDR-H2 as shown in SEQ ID No. 2;

[0012] <3> The heavy chain variable region includes the amino acid sequence CDR-H3 as shown in SEQ ID No. 3.

[0013] The present invention also provides an isolated polypeptide comprising a transmembrane domain, an intracellular domain, and an extracellular domain, wherein the extracellular domain comprises the aforementioned anti-Glypican 3 antibody.

[0014] The present invention also provides a chimeric antigen receptor immune cell, wherein the chimeric antigen receptor immune cell expresses the isolated polypeptide that is bound to the membrane.

[0015] As described above, the anti-Glypican 3 antibody of the present invention and its application have the following beneficial effects:

[0016] (1) The anti-Glypican 3 antibody of the present invention only includes the heavy chain variable region, which has high affinity and specificity, can efficiently target Glypican 3 antigen, and has a simple structure and is easy to prepare;

[0017] (2) In this invention, a chimeric antigen receptor is constructed using the anti-Glypican 3 antibody, which can efficiently target Glypican 3;

[0018] (3) The chimeric antigen receptor cells of the present invention can specifically recognize Glypican 3 positive tumor cells and kill them efficiently. At the same time, they can also release cytokines such as IFN-γ to exert cell killing effects. Attached Figure Description

[0019] Figure 1A The image shows the affinity results of Biacore's detection of the anti-Glypican 3 antibody GPC3-VHH-B6;

[0020] Figure 1B The image shows the affinity results of Biacore's detection of the anti-Glypican 3 antibody GPC3-VHH-B51;

[0021] Figure 2 The image shows the FACS detection results of the anti-Glypican 3 nanobody recognizing the Glypican 3 antigen.

[0022] Figure 3 The image shows a lentiviral vector plasmid targeting the chimeric antigen receptor of Glypican 3.

[0023] Figure 4 The diagram shows the structure of the chimeric antigen receptor expressing anti-Glypican 3 in Example 4.

[0024] Figure 5 The graph shows the flow cytometry results of the expression rates of chimeric antigen receptors (GPC3-VHH-B6 and GPC3-VHH-B51) on T lymphocytes.

[0025] Figure 6A The image shown illustrates the killing effect of CAR-T cells on 293T cells according to the present invention.

[0026] Figure 6B The image shown illustrates the killing effect of CAR-T cells on Huh7-GPC3 cells according to the present invention.

[0027] Figure 6C The image shown illustrates the killing effect of CAR-T cells on HepG2 cells according to the present invention.

[0028] Figure 7 This graph shows the IFN-γ cytokine secretion levels of CAR-T cells (GPC3-VHH-B6 and GPC3-VHH-B51). Detailed Implementation

[0029] This invention provides an anti-Glypican 3 antibody, the anti-Glypican 3 antibody comprising a heavy chain variable region, and the anti-Glypican 3 antibody having one or more of the following technical features;

[0030] <1> The heavy chain variable region includes the amino acid sequence CDR-H1 as shown in SEQ ID No. 1;

[0031] <2> The heavy chain variable region includes the amino acid sequence CDR-H2 as shown in SEQ ID No. 2;

[0032] <3> The heavy chain variable region includes the amino acid sequence CDR-H3 as shown in SEQ ID No. 3;

[0033] DTLDYYA (SEQ ID No.1)

[0034] ISSTGAST (SEQ ID No. 2)

[0035] AADLHRYYCSAYVEERDFGS (SEQ ID No. 3).

[0036] A CDR (complementarity determining region) generally refers to a region in an antibody that is spatially complementary to the antigenic determinant. The variability in an antibody is usually not uniformly distributed throughout its variable region. The heavy chain variable region of a monoclonal antibody typically has three hypervariable regions (HVRs). These regions are often spatially complementary to the antigenic determinant, hence the term complementarity determining region (CDR). In other words, the heavy chain variable region typically includes three complementarity determining regions: HCDR1, HCDR2, and HCDR3.

[0037] In some embodiments of the present invention, the complementarity-determining region of the heavy chain variable region of the anti-Glypican 3 antibody includes the amino acid sequence CDR-H1 as shown in SEQ ID No. 1, the amino acid sequence CDR-H2 as shown in SEQ ID No. 2, and the amino acid sequence CDR-H3 as shown in SEQ ID No. 3.

[0038] The anti-Glypican 3 antibody is an antibody fragment and / or a monoclonal antibody. Further, the monoclonal antibody is an IgG1 antibody.

[0039] An "antibody fragment" comprises a portion of a complete antibody, preferably including its antigen-binding region or variable region. For example, antibody fragments include nanobodies (VHH), single-chain antibodies (scFv), Fab, Fab', F(ab'), or F(ab')2.

[0040] In some embodiments of the present invention, the anti-Glypican 3 antibody is a nanobody (Nb), specifically a heavy chain single-domain antibody (VHH, variable domain of heavy chain). A nanobody contains only one heavy chain variable region (VHH) and CH2 and CH3 regions; unlike other antibodies, the light chain is naturally absent in nanobodies. The nanobody crystal has a diameter of approximately 2.5 nm and a length of approximately 4 nm, representing the smallest naturally occurring fragment capable of binding to an antigen. The anti-Glypican 3 antibody of the present invention comprises only the heavy chain variable region, possessing high affinity and specificity, enabling efficient targeting of the Glypican 3 antigen. It has a simple structure, is easy to prepare, and has significant application value in the field of preparing drugs targeting Glypican 3.

[0041] In some embodiments of the present invention, the anti-Glypican 3 antibody is a single-chain antibody (single-chain Fv, scFv). A single-chain antibody can typically be a V-type antibody comprising an antibody. H (heavy chain variable region) and V L A polypeptide chain (light chain variable region). Typically, single-chain antibodies may also include a linker, which is usually located in the V... H and V L Between these elements, the scFv is positioned to form a desired structure that allows it to bind to the antigen. For example, the anti-Glypican 3 antibody may include V... H and V L V H and V L Linking peptides may be present between the links, and the single-chain anti-Glypican 3 antibody may sequentially include V from the N-terminus to the C-terminus. L , linker peptides and V H The anti-Glypican 3 single-chain antibody may also sequentially include V from the N-terminus to the C-terminus. H , linker peptides and V L The linker peptide can be any linker peptide suitable for forming scFv in the art. For example, the linker peptide can be a G4S3 linker. The selection or design of the G4S3 linker can be found in the references Michel Sadelain et al., Science Translational Medicine, 2013; Carl H. June et al., Science Translational Medicine, 2015. The V L It can be obtained using conventional techniques in this field.

[0042] In some embodiments of the present invention, the heavy chain variable region may further include a framework region, which may be located between complementarity-determining regions or at both ends of the complementarity-determining regions. In some specific embodiments of the present invention, the framework region sequence is a human monoclonal antibody variable region or a mouse monoclonal antibody variable region framework region sequence obtained by substitution, deletion, or addition of one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, and the framework region sequence may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more homology with the framework region sequence of the human monoclonal antibody variable region sequence.

[0043] In some embodiments of the present invention, the heavy chain variable region further includes a framework region. The framework region includes framework regions FR1 to FR4. The amino acid sequences of the framework regions FR1 to FR4 are selected from any of the sequences shown in SEQ ID No. 4 to 7 or SEQ ID No. 23.

[0044] Preferably, the amino acid sequence of FR1 is as shown in SEQ ID No. 4 or SEQ ID No. 23: QLQLVESGGGLVQPGGSLRLSCAAS (SEQ ID No. 4)

[0045] QVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID No. 23).

[0046] Preferably, the amino acid sequence of FR2 is as shown in SEQ ID No. 5: IGWFRQAPGKEREGVSC (SEQ ID No. 5)

[0047] Preferably, the amino acid sequence of FR3 is as shown in SEQ ID No. 6: NYADSVKGRFTISKNNAKNTVYLQINSLKPEDTAVYFC (SEQ ID No. 6)

[0048] Preferably, the amino acid sequence of FR4 is as shown in SEQ ID No. 7: WGQGTQVTVSS (SEQ ID No. 7)

[0049] In some embodiments of the present invention, the anti-Glypican 3 antibody is designated as B6 or GPC3-VHH-B6, and the variable region of the heavy chain of the anti-Glypican 3 antibody includes amino acid sequences as shown in SEQ ID No. 1 (CDR-H1), amino acid sequences as shown in SEQ ID No. 2 (CDR-H2), amino acid sequences as shown in SEQ ID No. 3 (CDR-H3), and amino acid sequences of the frame regions FR1 to FR4 as shown in SEQ ID No. 4 to 7.

[0050] In some embodiments of the present invention, the anti-Glypican 3 antibody is designated as B51 or GPC3-VHH-B51, and the heavy chain variable region of the anti-Glypican 3 antibody includes amino acid sequences as shown in SEQ ID No. 1 (CDR-H1), amino acid sequences as shown in SEQ ID No. 2 (CDR-H2), amino acid sequences as shown in SEQ ID No. 3 (CDR-H3), and amino acid sequences of frame regions FR1-FR4 as shown in SEQ ID No. 23, 5-7.

[0051] Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 8 or SEQ ID No. 24.

[0052] QLQLVESGGGLVQPGGSLRLSCAASDTLDYYAIGWFRQAPGKEREGVSCISTGASTNYADSVKGRFTISKNNAKNTVYLQINSLKPEDTAVYFCAADLHRYYCSAYVEERDFGSWGQGTQVTVSS (SEQ ID No. 8)

[0053] QVQLVESGGGLVQPGGSLRLSCAASDTLDYYAIGWFRQAPGKEREGVSCISTGASTNYADSVKGRFTISKNNAKNTVYLQINSLKPEDTAVYFCAADLHRYYCSAYVEERDFGSWGQGTQVTVSS (SEQ ID No. 24).

[0054] In some embodiments of the present invention, the anti-Glypican 3 antibody is obtained from a phage antibody library through screening, and its heavy chain variable region nucleotide sequence is as shown in SEQ ID No. 9 or SEQ ID. No.25 shown cagctgcagctggtggagtccggcggcggactggtgcagcctggaggcagcctgaggctgtcttgcgcagcttccgacaccctggactac tatgccatcggctggttccggcaggccccaggcaaggagagggaggggagtgtcctgtatcagctccaccggcgccagcaccaattacgctgactccg tgaaaggcagatttacaatcagcaaaaacaatgccaagaacaccgtgtacctgcagattaactctctgaaaccagaggacaccgccgtgtacttttg cgccgccgacctgcacagatactactgctccgcctatgtggaagaacgggatttcggctcctggggtcagggaacccaggtgaccgtgagcagc (seq ID No.9)

[0055] caggtgcagctggtggagtccggcggcggactggtgcagcctggaggcagcctgaggctgtcttgcgcagcttccgacaccctggactactatgccatcggctggttccggcaggccccaggcaaggagagggagggagtgtcctgtatcagctccaccggcgccagcaccaattacgctgactccgtgaa aggcagatttacaatcagcaaaaacaatgccaagaacaccgtgtacctgcagattaactctctgaaaccagaggacaccgccgtgtacttttgcg ccgccgacctgcacagatactactgctccgcctatgtggaagaacgggatttcggctcctggggtcagggaacccaggtgaccgtgagcagc (seq ID No.25).

[0056] In another aspect, the present invention provides an isolated polynucleotide encoding the anti-Glypican 3 antibody.

[0057] In some embodiments of the present invention, the sequence of the polynucleotide is as shown in SEQ ID No. 9 or SEQ ID No. 25.

[0058] Another aspect of the present invention provides the use of the anti-Glypican 3 antibody in the preparation or screening of therapeutic drugs, or in the preparation of diagnostic drugs.

[0059] The therapeutic agent may be an agent that targets the Glypican 3 antigen, binds to or acts on the Glypican 3 antigen, and thereby treats and / or prevents the indication.

[0060] In some embodiments of the present invention, the therapeutic agent may be an antitumor drug. The tumor is a tumor expressing Glypican 3. The antitumor drug may be a drug that targets the Glypican 3 antigen on the functional surface of tumor cells, binding to or acting on the Glypican 3 antigen, thereby treating and / or preventing the tumor. The tumor may be a Glypican 3-positive tumor such as liver cancer, ovarian cancer, lung cancer, melanoma, gastric cancer, or thyroid cancer.

[0061] In some embodiments of the present invention, the therapeutic agent is a chimeric antigen receptor cell.

[0062] The chimeric antigen receptor cell therapy typically includes chimeric antigen receptor cells, which may be chimeric antigen receptor T cells, chimeric antigen receptor NK cells, etc. The chimeric antigen receptor T cells typically include T lymphocytes, which also include chimeric antigen receptors. The chimeric antigen receptor NK cells typically include NK cells, which also include chimeric antigen receptors. The chimeric antigen receptor includes a transmembrane domain, an intracellular domain, and an extracellular domain. In some embodiments of the present invention, the extracellular domain includes the anti-Glypican 3 antibody, meaning that the chimeric antigen receptor cells can express the anti-Glypican 3 antibody on their cell surface, thereby guiding the cells to act on cells expressing Glypican 3 antigen (e.g., tumor cells). The action on cells expressing Glypican 3 antigen may include killing cells expressing Glypican 3 antigen, etc.

[0063] The diagnostic drug specifically refers to a reagent that targets the Glypican 3 antigen and uses the Glypican 3 antigen as a biomarker for diagnosis.

[0064] Another aspect of the present invention provides an isolated polypeptide comprising a transmembrane domain, an intracellular domain, and an extracellular domain, wherein the extracellular domain comprises the anti-Glypican 3 antibody.

[0065] In some embodiments of the present invention, the polypeptide is a chimeric antigen receptor. In this invention, a chimeric antigen receptor is constructed using the anti-Glypican 3 antibody, which can efficiently target Glypican 3.

[0066] In some embodiments of the present invention, the transmembrane domain may be selected from any one or more transmembrane structural domains such as CD8α transmembrane region, CD28 transmembrane region, and DAP 10 transmembrane region.

[0067] For example, the sequence of CD8α can be found in NM_001145873, the sequence of CD28 in NM_006139, and the sequence of DAP10 in NM_014266.

[0068] In some embodiments of the present invention, the intracellular domain may include a co-stimulatory domain and / or a signal transduction domain. The signal transduction domain includes an immune receptor tyrosine activation motif. The immune receptor tyrosine activation motif may be selected from CD3ζ.

[0069] Preferably, the signal transduction domain further includes a co-stimulatory molecule. For example, the co-stimulatory molecule may be selected from any one or a combination of at least two protein molecules such as 4-1BB, CD28, OX40, ICOS, and DAP 10. As another example, the amino acid sequence of 4-1BB may include the following:

[0070] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID No.12)

[0071] For example, the sequence of 4-1BB can be found in NM_001561, the sequence of CD28 in NM_006139, the sequence of OX40 in NM_003327, the sequence of ICOS in NM_012092, the sequence of CD3 zeta in NM_198053, and the sequence of DAP10 in NM_014266.

[0072] In one specific embodiment of the present invention, the intracellular domain includes 4-1BB and CD3 zeta sequentially from the N-terminus to the C-terminus.

[0073] In some embodiments of the present invention, the extracellular domain may include a signal peptide, an anti-Glypican 3 antibody, or a hinge region.

[0074] In some embodiments of the present invention, the signal peptide includes the CD8α signal peptide.

[0075] In some embodiments of the present invention, the hinge region is selected from the CD8α hinge region.

[0076] In some embodiments of the present invention, the polypeptide comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, the anti-Glypican 3 antibody, a transmembrane domain, and an intracellular domain.

[0077] In some specific embodiments of the present invention, the polypeptide comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, an anti-Glypican 3 nanobody, a CD8α hinge region, a CD8α transmembrane region, a co-stimulatory molecular domain, and a CD3 zeta signal domain.

[0078] In some specific embodiments of the present invention, the polypeptide comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, an anti-Glypican 3 nanobody, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory domain, and a CD3 zeta signal domain.

[0079] In one specific embodiment of the present invention, the polypeptide comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, the anti-Glypican 3 nanobody, a CD8α hinge region, a CD28 transmembrane region, a CD28 co-stimulatory domain, and a CD3 zeta signal domain.

[0080] In another specific embodiment of the present invention, the polypeptide comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, the anti-Glypican 3 nanobody, a CD8α hinge region, a CD8α transmembrane region, an OX40 co-stimulatory domain, and a CD3 zeta signal domain.

[0081] In another specific embodiment of the present invention, the polypeptide comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, the anti-Glypican 3 nanobody, a CD8α hinge region, a CD8α transmembrane region, an ICOS co-stimulatory domain, and a CD3 zeta signal domain.

[0082] In another specific embodiment of the present invention, the polypeptide comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, the anti-Glypican 3 nanobody, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory domain, and a CD3 zeta.

[0083] In another specific embodiment of the present invention, the polypeptide comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, the anti-Glypican 3 nanobody, a CD8α hinge region, a CD28 transmembrane region, a CD28 co-stimulatory domain, an OX40 co-stimulatory domain, and a CD3 zeta signal domain.

[0084] In certain embodiments of the present invention, the polynucleotide sequence encoding the isolated polypeptide is shown in SEQ ID No. 22 or SEQ ID No. 26:

[0085]

[0086]

[0087] The present invention also provides a nucleic acid construct containing a polynucleotide encoding the isolated polypeptide.

[0088] The nucleic acid construct can be a lentiviral vector, a retroviral vector, or an adeno-associated virus vector. Taking a lentiviral vector as an example, a lentiviral vector includes a vector backbone, i.e., an empty vector, and an expression framework. That is, the nucleic acid construct is a vector containing the coding gene of the chimeric antigen receptor.

[0089] The term "vector" refers to a nucleic acid or polynucleotide fragment used to introduce or transfer one or more nucleic acids or polynucleotides into a target cell or tissue. Typically, a vector is used to introduce exogenous DNA into another cell or tissue. A vector may contain a bacterial resistance gene for growth in bacteria and a promoter for expression of a target protein in an organism. The DNA can be produced in vitro by PCR or one or more suitable techniques known to those skilled in the art.

[0090] The term "expression frame" refers to a sequence that has the potential to encode a protein.

[0091] The present invention also provides a lentivirus, which is formed by viral packaging of the nucleic acid construct. The lentivirus contains the nucleic acid construct.

[0092] The present invention also provides a lentiviral vector system, characterized in that the lentiviral vector system includes the aforementioned nucleic acid construct and auxiliary plasmid.

[0093] Furthermore, the helper plasmid encodes one or more nucleotide sequences of the gag and pol proteins, as well as other essential viral packaging component nucleotide sequences. The helper plasmid may include a packaging plasmid and an envelope plasmid. In one embodiment, the packaging plasmid is gag / pol, and the envelope plasmid is VSVg. Both plasmids are commercially available, for example, Addgene catalog numbers 14887 and 8454.

[0094] Furthermore, the lentiviral vector system also includes a host cell, which can be a cell that produces lentiviruses, such as a mammalian cell, specifically a 293T cell.

[0095] The lentivirus can be obtained by transfecting host cells with the nucleic acid construct and helper plasmid in the lentiviral vector system.

[0096] Another aspect of the present invention provides a chimeric antigen receptor immune cell, wherein the chimeric antigen receptor immune cell expresses the isolated polypeptide that is bound to a membrane.

[0097] Preferably, the chimeric antigen receptor immune cell comprises the nucleic acid construct and / or the lentivirus.

[0098] The immune cells are selected from any one of T lymphocytes, B lymphocytes, NK cells, mast cells, or macrophages.

[0099] In another specific embodiment of the present invention, the chimeric antigen receptor immune cell is a T lymphocyte.

[0100] The T lymphocytes typically express the polypeptide, which can bind to the Glypican 3 antigen. More specifically, it can bind to the Glypican 3 antigen via an extracellular domain containing the anti-Glypican 3 antibody. When the polypeptide binds to the Glypican 3 antigen, the T lymphocytes can typically be activated and / or stimulated to proliferate. In some embodiments of the invention, the T lymphocytes, i.e., chimeric antigen receptor T cells, can express the anti-Glypican 3 antibody on their surface, thereby guiding the T lymphocytes to act on cells expressing the Glypican 3 antigen (e.g., tumor cells). This action can include killing cells expressing the Glypican 3 antigen, etc.

[0101] In another specific embodiment of the present invention, the chimeric antigen receptor immune cell is an NK cell.

[0102] The NK cells typically express the polypeptide and can bind to the Glypican 3 antigen, more specifically, through an extracellular domain containing the anti-Glypican 3 antibody. When the polypeptide binds to the antigen, the NK cells are typically activated and / or stimulated to proliferate. In some embodiments of the invention, the NK cells, i.e., chimeric antigen receptor NK cells, can express the anti-Glypican 3 antibody on their surface, thereby guiding the NK cells to act on cells expressing the Glypican 3 antigen (e.g., tumor cells), such action as killing cells expressing the Glypican 3 antigen.

[0103] Another aspect of the present invention provides the use of the isolated polypeptides, isolated polynucleotides, nucleic acid constructs, lentiviruses, and chimeric antigen receptor immune cells in the preparation or screening of therapeutic drugs, or in the preparation of diagnostic drugs.

[0104] The therapeutic or diagnostic drug may be a drug that targets the Glypican 3 antigen, binds to or acts on the Glypican 3 antigen, and thereby treats and / or prevents the indication.

[0105] In some embodiments of the present invention, the therapeutic drug may be an antitumor drug. The antitumor drug may be a drug that targets the functionally expressed Glypican 3 antigen on the surface of tumor cells, binding to or acting on the Glypican 3 antigen to treat and / or prevent tumors. The tumor may be a Glypican 3-positive tumor such as gastric cancer, lung cancer, pancreatic cancer, or colorectal cancer.

[0106] The present invention provides a pharmaceutical composition comprising the chimeric antigen receptor immune cells and / or the anti-Glypican 3 antibody and / or the isolated polypeptide.

[0107] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier or excipient.

[0108] "Pharmaceutical acceptable" means that when a drug is properly administered to animals or humans, it will not produce adverse, allergic, or other adverse reactions.

[0109] A "pharmaceutically acceptable carrier or excipient" should be compatible with the active ingredient, meaning it can be miscible with it without significantly reducing the drug's efficacy under normal circumstances. Specific examples of substances that can serve as pharmaceutically acceptable carriers or excipients include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0110] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0111] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0112] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0113] Example 1

[0114] In this embodiment, a phage nanobody library was constructed and subjected to panning and preliminary screening by ELISA.

[0115] 1. Construction of phage nanobody library

[0116] (1) Bactrian camels were immunized with GPC3-Fc (purchased from Beijing Baipusaisi Biotechnology) expressing the extracellular region. After verifying the titer by ELISA, 200 mL of peripheral blood was drawn.

[0117] (2) Sorting lymphocytes, obtaining peripheral blood mononuclear lymphocyte precipitates, and extracting RNA;

[0118] (3) First-strand cDNA was synthesized using SuperScript® III reverse transcriptase with RNA as a template, and then the VHH gene was amplified by nested PCR.

[0119] (4) The VHH gene was inserted into the pMECS phage display vector (Chengdu Apak Biotechnology). After electroporation of TG1 competent cells, the bacterial culture was collected for library identification. All remaining cultures were evenly spread on LB / AMP GLU plates. After bacterial growth, the bacterial colony was collected, 1 / 3 volume of 50% glycerol was added, mixed well, dispensed, and stored at -80°C. A library with a capacity greater than 10 was successfully constructed. 9 Phage display of camel VHH immune library.

[0120] 2. Selection of phage nanobody libraries

[0121] The purified GPC3-His recombinant protein was diluted to 4 µg / mL with PBS buffer. In a 96-well microplate, 100 μL (400 ng / well) was added to each well in triplet configurations and coated overnight at 4°C. PBS was used as a negative control. The coating solution was discarded, and 150 μL of 2% defatted phage powder was added to each well. The plate was blocked at 25°C for 1 h. The plate was washed four times with PBST. The prepared phage solution was then diluted to 5 × 10⁻⁶ with 2% milk powder. 11 Add pfu / ml to an ELISA plate, 100 μL / well, and incubate at 25°C for 2 h. Discard the phage sample, wash 10 times with PBST, then wash 5 times with PBS. Add 100 μL of freshly prepared 0.1 M triethylamine to each well, incubate at 25°C for 10 min, aspirate the eluent, and quickly neutralize with an equal volume of 1 M Tris-HCl (pH 7.4). Take a portion of the eluent to determine the phage titer. Take another 400 μL of the eluent and infect 4 ml of freshly cultured logarithmic-phase TG1 bacterial suspension (OD600 approximately 0.6), incubate at 37°C for 30 min, add 16 mL of 2×YT / AMP-GLU (ampicillin AMP and glucose GLU concentrations of 1 μg / ml and 2%, respectively), and incubate at 37°C and 200 r / min until OD600 reaches 0.6. 600 The target concentration was 0.7. 100 μL of bacterial suspension was serially diluted and evenly spread onto 2×YT / ampicillin / glucose agar plates for library volume and diversity determination. 100 μL of bacterial suspension (i.e., the phage display vector library) was inoculated into 2×YT / AMP-GLU medium and cultured to the logarithmic phase. Helper phages were added for library rescue. Phage particles were obtained, and their titers were measured. The phage particles were then concentrated and purified for the next round of screening. The remaining bacterial suspension was centrifuged and resuspended in an appropriate volume of 2×YT medium. The suspension was spread onto plates containing screening resistance and cultured overnight. Bacteria were scraped from the plates using an appropriate amount of liquid culture medium, resuspended in 2×YT medium containing 1 / 3 volume of 50% glycerol, and aliquoted. All bacteria were stored at -80℃.

[0122] Repeat the above filtering operation 3 times.

[0123] Three rounds of solid-phase screening were performed on the immune nanobody library in vitro, resulting in the effective enrichment of phage clones with binding activity. After prokaryotic induction and expression of monoclonal phages, phage clones capable of binding to the extracellular region of the antigen were further screened by ELISA.

[0124] 3. Phage packaging

[0125] Add 100 μL of the previously frozen bacterial culture from the previous round to 100 mL of 2×YT / AMP-GLU culture medium and incubate at 37°C with shaking (200 rpm) until the logarithmic growth phase (OD50).600 With a value of 0.6, add 90 μL of helper phage M13K07 (1.7 × 10⁻⁶). 13 The mixture (PFU / mL) was first incubated at 37°C for 30 min, centrifuged at 2800×g for 10 min to collect bacterial cells, resuspended in 200 mL of 2×YT / AMP-GLU medium, and cultured at 37°C with shaking (200 rpm) for 12 h. After centrifugation at 4°C and 3800×g for 30 min to remove bacterial cells, the supernatant was collected and 1 / 5 volume of pre-chilled PEG / NaCl was added and mixed well. The phage was precipitated for 2 h, centrifuged at 4°C and 3800×g for 30 min to collect the phage, resuspended in a final volume of 2 mL of PBS solution, and transferred to a 15 mL centrifuge tube. After centrifugation at 4°C and 12000×g for 15 min, the supernatant was collected and 1 / 5 volume of pre-chilled PEG / NaCl solution was added, mixed by inverting, and incubated on ice for 2 h. After centrifugation at 4°C and 10000×g for 10 min, the supernatant was discarded and 1 mL of PBS solution was added. The phage pellet was resuspended in PBS and incubated overnight at 4°C on a shaker to allow the phage particles to fully dissolve. The phage solution was then mixed with an equal volume of 60% glycerol and dispensed into 1.5 mL EP tubes and stored at -80°C.

[0126] The phage library was panned three times using Glypican 3 antigen. To avoid losing sequence diversity, the initial ELISA screening was performed on the panning products from the second and third rounds. Positive clones were randomly selected from the panning products and induced to express. The expression supernatant was the crude VHH antibody. The VHH antibody sequence of the monoclonal strain was determined by sequencing.

[0127] Example 2

[0128] This embodiment describes the use of fluorescence activated cell sorting (FACS) candidate clones.

[0129] Cells were cultured according to standard cell culture protocols. Glypican 3 positive and negative cell suspensions were prepared by trypsin digestion. After centrifugation (300×g, 5 min) to remove the culture medium, the cells were resuspended in Flow Buffer (PBS + 2% FBS) to a final volume of 2×10⁶ cells / mL. 6 cell / mL, add 2×10⁻⁶ cells / mL to each well of a V-bottom 96-well plate. 5Cell suspensions of [number] cells were centrifuged at 300×g for 5 min, the supernatant was removed, and the cells were resuspended in crude VHH antibody extract. The cells were incubated at 4℃ for 1 h, centrifuged at 300×g for 5 min, the supernatant was removed, and the cells were resuspended in Flow Buffer. APC anti-his antibody was diluted to 2 μg / mL with Flow Buffer, and 100 μL of the solution was added to each well for resuspending. The cells were incubated at 4℃ for 1 h. After washing the cells three times with Flow Buffer, 200 μL of Flow Buffer was added to each well for further washing. Cells were resuspended in buffer and analyzed by flow cytometry, resulting in ten candidate antibodies, named GPC3-VHH-B6, GPC3-VHH-B51, GPC3-VHH-B89, GPC3-VHH-B108, GPC3-VHH-B168, GPC3-VHH-B174, GPC3-VHH-B174(V18), GPC3-VHH-B174(V32), GPC3-VHH-B174(V33), and GPC3-VHH-B174(V34). This application only claims protection for GPC3-VHH-B6 and GPC3-VHH-B51; the other candidate antibodies are claimed for protection in other patents.

[0130] Example 3

[0131] This embodiment describes the expression, purification, and antibody affinity determination of VHH-mIgG2a Fc nanobodies.

[0132] To further identify the antibodies obtained in Example 2, it is necessary to express them in mammalian cells. Therefore, a plasmid vector C-4 pCP.Stuffer-mCg2a-FC (purchased from Shanghai Baiying Biotechnology) expressing VHH with a mouse Fc tag was first constructed. The construction method includes the following steps:

[0133] (1) The VHH fragment was amplified by PCR. The reaction system (reagents were purchased from NEB) and PCR reaction conditions are shown in Table 1 below.

[0134] Table 1

[0135]

[0136] (2) The enzyme digestion system and reaction conditions are shown in Table 2. The digested vector was purified using the PureLink® PCR purification kit. The air-dried DNA was dissolved in 20 μL of water and the DNA concentration was detected.

[0137] Table 2

[0138]

[0139] (3) The homologous recombination reaction system was 10 μL (the reagent was purchased from Novizan), as shown in Table 3;

[0140] Table 3

[0141]

[0142] (4) Take the entire homologous recombination reaction system and add it to DH5α competent cells to transform DH5α competent cells. The transformation conditions are shown in Table 4.

[0143] Table 4

[0144] program Temperature (°C) Time (min) Ice bath 0 5 heat shock 42 1 Ice bath 0 3 Add 500 μL of LB medium and incubate with shaking at 220 RPM. 37 1.5 h Pipette 200 μL and spread it evenly on an LB / Amp plate. 37 Overnight (17 hours)

[0145] (5) Transformation plates were selected for single-clone PCR pre-identification. The PCR identification system conditions are shown in Table 5 (reagents were purchased from Sangon Biotech). The samples were sent to a sequencing company for sequencing identification. The sequencing results met expectations, and a plasmid vector expressing VHH with a mouse Fc tag was successfully constructed.

[0146] Table 5

[0147]

[0148] Approximately 24 hours before plasmid transfection, 293E cells were passaged to achieve a cell density of approximately 2.6 × 10⁻⁶. 6 0.15 mg VHH-mIgG2a (the constructed plasmid) / 100 mL of 293E cells were transfected into 293E cells using the PEI method, with a DNA:PEI ratio of 1:2. Cells were cultured at 37°C, 130 rpm, and 8% CO2 for 6 days. The cell culture supernatant was collected at 3000 rpm for 30 min. The collected supernatant containing the target antibody was filtered through a Millex-GP Filter Unit (0.45 μm Sterile), concentrated by MabSelect™ SuRe™ centrifugation, washed with 1×PBS, and eluted with 0.1 M (mol / L) Gly-HCl. The elution was then carried out with 1 / 10 volume of Tris-HCl at pH 8.5. After dialyzing at 4°C overnight, the A280 level was determined using a NanoDrop 2000 assay, and antibody purity was determined by SEC-HPLC.

[0149] In addition, the affinity of the purified GPC3 VHH antibodies (GPC3-VHH-B6 and GPC3-VHH-B51) was determined using Biacore. Biacore is a bioanalytical sensing technology based on surface plasmon resonance (SPR). It can detect and track the entire process of binding and dissociation between molecules in solution and molecules immobilized on the chip surface, recording the data in the form of a sensor map and providing kinetic and affinity data. During the assay, the antibody was immobilized on the chip surface, and the mobile phase was a solution containing the antigen. The assay results are shown in Table 6. Figure 1A and Figure 1B As shown in the results, these two GPC3 VHH antibodies have high affinity.

[0150] Table 6

[0151] VHH antibody ka (1 / Ms) kd (1 / s) KD (M) GPC3-VHH-B6 2.95E+05 0.01446 4.91E-08 GPC3-VHH-B51 4.32E+05 0.01574 3.65E-08

[0152] Example 4

[0153] In this embodiment, the anti-Glypican 3 nanobody was analyzed by flow cytometry.

[0154] Huh7-GPC3 tumor cells were incubated with two purified recombinant anti-GPC3 VHH antibodies on ice for 30 min. The blank control group received no anti-GPC3 VHH antibody. Cells were then incubated with either APC-labeled goat anti-mouse IgG antibody or iF488-anti-VHHcocktail antibody for 30 min. Flow cytometry analysis was performed, and the results are as follows: Figure 2 As shown, the anti-Glypican 3 antibody prepared by the present invention can recognize the Glypican 3 antigen on the cell surface.

[0155] Example 5

[0156] This embodiment prepares a lentiviral vector expressing a chimeric antigen receptor (GPC3 CAR) targeting Glypican 3.

[0157] First, the lentiviral vector HD-SIN03 GPC3 CAR carrying the GPC3 CAR chimeric antigen receptor was constructed. The vector map is shown below. Figure 3 As shown, the structure of the chimeric antigen receptor is as follows: Figure 4 As shown, the nucleotide sequence of CAR, as indicated by SEQ ID No. 22 and 26, includes the CD8α signal peptide, anti-GPC3 antibody (anti-GPC3 VHH), CD8α hinge region, transmembrane region, and immune receptor tyrosine activation motif (CD3ζ).

[0158] The amino acid sequence of the signal peptide is: MALPVTALLLPLALLLHAARP (SEQ ID No. 10).

[0159] The amino acid sequence of anti-GPC3 VHH is shown in SEQ ID No. 8.

[0160] The amino acid sequences of the CD8α hinge region and transmembrane region are as follows:

[0161] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID No. 11).

[0162] The amino acid sequence of the intracellular region of 4-1BB is as follows:

[0163] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID No. 12).

[0164] The CD3ζ amino acid sequence is:

[0165] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID No. 13).

[0166] The specific preparation method is as follows:

[0167] (1) Prepare the PCR reaction system according to Table 8 (reagents purchased from TOYOBO), amplify the signal peptide-GPC3-VHH fragment, and use the primers shown in Table 7.

[0168] Table 7

[0169] Primers sequence Serial Number CD8a leader-F ctgcaggtcgactctagaggatcccgccaccatggccttaccagtga SEQ ID No. 14 CD8H-R gtcgcggcgctggcgtcgtggt SEQ ID No. 15

[0170] Table 8

[0171] reagents Volume (μL) 10x buffer 5 2 mM dNTP 5 <![CDATA[25 mM MgSO4]]> 3 10 µM primer F 1 10 µM primer R 1 Template DNA (cDNA clone) 1 PCR-grade pure water 33 KOD-Plus-Neo 1

[0172] The above reagents are from TOYOBO Inc.

[0173] After preparation, the PCR reaction was carried out according to the procedure shown in Table 9.

[0174] Table 9

[0175]

[0176] After the reaction, the PCR products were subjected to 1% agarose gel electrophoresis, and fragments of about 500 bp were recovered and quantified by ultraviolet absorption method.

[0177] (2) Prepare the PCR reaction system according to Table 11, amplify the CD8a hinge-TM-41BB-CD3Z fragment, and use the primers shown in Table 10.

[0178] Table 10

[0179] Primers sequence Serial Number CD8H2-F cgacgccagcgccgcgaccacc SEQ ID No. 16 Vector-R tcgataagcttgatatcg SEQ ID No. 17

[0180] Table 11

[0181] reagents Volume (μL) 10x buffer 5 2 mM dNTP 5 <![CDATA[25 mM MgSO4]]> 3 10 µM primer F 1 10 µM primer R 1 Template DNA (HD CD19 CAR) 1 PCR-grade pure water 33 KOD-Plus-Neo 1

[0182] After preparation, PCR reaction was performed according to the PCR procedure shown in Table 9. After the reaction, the PCR product was subjected to 1% agarose gel electrophoresis, and the fragment of about 700 bp was recovered and quantified by ultraviolet absorption method.

[0183] (3) 5 μg of HD SIN03 CD19 41BBz (ka) plasmid was digested with BamHI and EcoRI, and after reacting in a water bath at 37℃ for 2 h, the large fragment of the vector backbone was recovered.

[0184] (4) The two fragments recovered in steps 1 and 2 and the vector obtained in step 3 were ligated with recombinase. The recombinant reaction system is shown in Table 12. After preparation, the reaction was carried out in a water bath at 37°C for 0.5 h. The cells were transformed into Escherichia coli stbl3 competent cells according to conventional methods. Single clones were selected from solid culture medium, cultured overnight, and identified by PCR. The PCR reaction preparation is shown in Table 13, and the PCR program is shown in Table 14. After PCR, positive clones were selected for further sequencing identification. The sequencing results were in line with expectations.

[0185] Table 12

[0186] reagents Dosage HD SIN03 CD19 41BBz (ka) 150 ng CD8a signal GPC3 VHH 15 ng CD8a hinge-TM-41BB-CD3Z 15 ng 5 x CE MultiS buffer (purchased from Novizan) 2 μL Exnase MultiS (purchased from Novizan) 1μL PCR-grade pure water Up to 10 μL Total volume 10 μL

[0187] Table 13

[0188] reagents Volume (μL) Taq PCR Master Mix 10 10 µM LV-F2 1 10 µM LV-R 1 Template DNA bacterial culture 1 PCR-grade pure water 7 Total volume 20

[0189] Table 14

[0190]

[0191] Example 6

[0192] This embodiment describes the packaging of lentiviruses, including the following steps:

[0193] (1) With 1.6×107 293T cells were seeded in 15 cm culture dishes and cultured overnight at 37°C with 5% CO2 to prepare for virus packaging. The culture medium was DMEM with 10% fetal bovine serum (FBS) added.

[0194] (2) Dissolve 30 μg of the lentiviral vector constructed in Example 5, 12.5 μg of helper plasmid gag / pol and 10 μg of envelope plasmid VSVg in 2000 μL of serum-free DMEM culture medium and mix well;

[0195] (3) Dissolve 157.5 μg PEI (1 μg / μL) in 2000 μL of serum-free DMEM culture medium, vortex at 1000 rpm for 5 seconds, and incubate at 25℃ for 5 min;

[0196] (4) Formation of transfection complex: Add PEI mixture to DNA mixture, vortex mix or gently mix immediately after addition, and incubate at 25°C for 20 min;

[0197] (5) Add 4 mL of the transfection complex to a 15 cm culture dish containing 25 mL of DMEM medium. After 4 hours, replace with fresh medium.

[0198] (6) After 48 h, the viral supernatant was collected to obtain 10 lentiviruses expressing chimeric antigen receptors with different structures.

[0199] Example 7

[0200] This embodiment involves lentivirus concentration.

[0201] The viral supernatant prepared in Example 6 was filtered through a 0.45 μm filter membrane and collected into a 50 mL centrifuge tube. 1 / 4 of the PEG-NaCl viral concentrate was added, and the mixture was mixed by inverting the tube. The tube was then incubated overnight at 4°C. The tube was centrifuged at 3500 rpm for 30 min at 4°C. The supernatant was removed, and RPMI 1640 medium (containing 10% FBS) was added to dissolve and resuspend the viral precipitate. The concentrated lentivirus suspension was aliquoted into 50 μL portions and stored in finished tubes at -80°C.

[0202] Example 8

[0203] This embodiment performs lentivirus titer detection.

[0204] 500 μL of Jurkat cells (1×10⁶) 5Cells were seeded in 48-well plates. The concentrated lentivirus from Example 7 was added to the cell suspension at concentrations of 1 μL, 0.2 μL, and 0.04 μL, respectively, and polybrene was added to a final concentration of 5 μg / mL. Cells were cultured overnight at 37°C with 5% CO2, and then the medium was replaced with fresh medium. After 72 h of infection, cells were centrifuged at 400×g for 5 min, the supernatant was discarded, and the cells were resuspended in 100 μL of PBS + 2% FBS. 1 μg of iF488-anti-VHH cocktail antibody (purchased from GenScript) was added, and the cells were incubated on ice for 30 min. Cells were washed twice with PBS + 2% FBS, and then resuspended in 300 μL of PBS + 2% FBS. Infection efficiency was detected by flow cytometry. Cell samples with a positivity rate of 15% were preferred. The titer (TU / mL) was calculated as: cell number (102) / (102)2. 5 ) × Positive rate / Viral volume (mL).

[0205] Example 9

[0206] This embodiment utilizes lentivirus transduction of T lymphocytes.

[0207] Anti-human CD3 antibody and anti-human CD28 antibody were diluted with PBS to final concentrations of 1 μg / mL and 0.5 μg / mL, respectively, and coated into well plates. The plates were then incubated overnight at 4°C. The antibody coating solution was discarded, and the plates were washed twice with 1 mL of PBS. Human PBMCs were adjusted to a density of 1 × 10⁻⁶ cells / well using T cell culture medium (X-VIVO + 10% FBS + IL-2 (300 U / mL)). 6 / mL, and then seeded into CD3 and CD28 antibody-coated well plates for activation for 48 h; the activated T cells were collected and the cell density was adjusted to 1×10⁶. 6 / mL, add the lentivirus prepared in Example 7 according to the multiplicity of infection (MOI) = 10, add polybrene to the final concentration of 5 μg / mL; incubate overnight at 37°C and 5% CO2, then replace with fresh medium, and passage every 2 days.

[0208] Example 10

[0209] This embodiment describes the construction of GPC3 overexpression cell lines (Huh7-GPC3 and Sk-hep1-GPC3), including the following steps:

[0210] 1. Construction of GPC3 overexpression plasmid:

[0211] Prepare the PCR reaction system according to Table 16, and amplify the GPC3 CDS fragment and the P2A-PuroR fragment respectively. The primers used are shown in Table 15.

[0212] Table 15

[0213] Primers sequence Serial Number GPC3-F caggtcgactctagaggatcccgccaccatggccgggaccgtgc SEQ ID No. 18 GPC3-R tcaaagtctgtttcacgtgcaccaggaagaag SEQ ID No. 19 F2A-F gtgaaacagactttgaattt SEQ ID No. 20 LV-EcoRI-R ggttgattatcgataagcttgatatcgaattctcaggcaccgggcttgcgggtcat SEQ ID No. 21

[0214] Table 16

[0215] reagents Volume (μL) 10x buffer 5 2 mM dNTP 5 <![CDATA[25 mM MgSO4]]> 3 10 µM primer F 1 10 µM primer R 1 Template DNA (cDNA clone) 1 PCR-grade pure water 32 KOD-Plus-Neo 2

[0216] The above reagents are from TOYOBO Inc.

[0217] After preparation, the PCR reaction was carried out according to the procedure shown in Table 17.

[0218] Table 17

[0219]

[0220] After the reaction, the PCR products were subjected to 1% agarose gel electrophoresis. The GPC3 CDS fragment of about 1800 bp and the P2A-PuroR fragment of about 665 bp were recovered and quantified by UV absorption.

[0221] (2) 4 μg of pSIN CEA 1A6-41BBz (kanaR) plasmid was digested with BamHI and EcoRI, and after reacting in a water bath at 37℃ for 2 h, the large fragment of the vector backbone was recovered.

[0222] (3) The two fragments recovered in step 1 and the vector obtained in step 2 were ligated with recombinase. The recombinant reaction system is shown in Table 18 (the reagents were purchased from Novizan). After preparation, the reaction was carried out in a water bath at 37°C for 0.5 h. The cells were transformed into Escherichia coli stbl3 competent cells according to conventional methods. Single clones were selected from the solid culture medium, cultured overnight, and identified by PCR. The PCR reaction preparation is shown in Table 13, and the PCR program is shown in Table 14. After PCR, positive clones were selected for further sequencing identification. The sequencing results were in line with expectations.

[0223] Table 18

[0224] reagents Dosage pSIN CEA1A6-41BBz (kanaR) 170 ng GPC3 CDS 72 ng P2A-PuroR 27 ng 5 x CE MultiS buffer 4 μL Exnase MultiS 2 μL PCR-grade pure water Up to 20 μL Total volume 20 μL

[0225] 2. Packaging of lentiviruses overexpressing Glypican 3:

[0226] The packaging process for Glypican 3 overexpression lentivirus is the same as in Example 6.

[0227] 3. Construction of Glypican 3 overexpression cell lines:

[0228] (1) Huh7 and Sk-hep1 cells (both purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were digested and counted, then resuspended in DMEM + 10% FBS medium. 1×10⁶ cells were then collected.5 Cells were seeded in 6-well plates, and 1 mL of Glypican 3 overexpressing lentivirus was added to each well. The medium was then added to 2 mL, and polybrene was added to a final concentration of 5 μg / mL. The plates were incubated overnight at 37°C with 5% CO2.

[0229] (2) Replace the virus-infected Huh7 cells and Sk-hep1 cells with 2 mL of fresh DMEM + 10% FBS medium and continue to culture at 37°C and 5% CO2.

[0230] (3) After culturing for 3 days, puromycin was added at final concentrations of 0.5 μg / mL and 1 μg / mL to screen for positive cells;

[0231] (4) After 3 days of culture, the virus-infected Huh7 cells and Sk-hep1 cells were passaged and puromycin was added to the cells for further selection at a final concentration of 1 μg / mL.

[0232] (5) Continue passage and puromycin screening of virus-infected Huh7 and Sk-hep1 cells in a cycle of 3-4 days. After a total screening time of about 2 weeks, detect the expression of Glypican 3 and freeze the cells.

[0233] Example 11

[0234] This embodiment demonstrates T-lymphocyte chimeric antigen receptor expression, including the following steps:

[0235] 1. Five days after infection, collect 3×10 5 T cells were centrifuged at 400×g for 5 min at 4℃, the supernatant was discarded, and the cells were washed once with PBS + 2% FBS.

[0236] 2. Resuspend cells in 100 μL PBS + 2% FBS, add 1 μg of iF488-anti-VHH cocktail antibody, and incubate on ice for 30 min; wash twice with PBS + 2% FBS, then resuspend cells in 300 μL PBS + 2% FBS. Use uninfected T cells as a control. Flow cytometry was used to detect infection efficiency. Results are shown below. Figure 5 As shown, the infected CAR-T cells showed a significant positive cell population, indicating that the present invention successfully constructed two types of CAR-T cells expressing chimeric antigen receptors with different structures, labeled as GPC3-VHH-B6 and GPC3-VHH-B51, respectively.

[0237] Example 12

[0238] In this embodiment, an in vitro toxicity experiment was conducted on CAR-T cells.

[0239] 1. Target cell seeding:

[0240] 293T (GPC3-, purchased from ATCC), Huh7-GPC3 (GPC3+), and HepG2 (GPC3+), purchased from Shanghai Enzyme Research Institute Biotechnology, were used as target cells, and the target cell concentration was adjusted to 1×10⁻⁶. 5 / mL, take 100 µL and inoculate it into a 96-well plate;

[0241] 2. Effector cell seeding:

[0242] GPC3 CAR-T cells and control T cells were used as effector cells. CAR-T cells and control T cells were added to 96-well plates at effector-to-target ratios of 0.3:1, 1:1 and 3:1.

[0243] 3. Each group had 3 replicates, and the average value of the 3 replicates was taken. The experimental groups and control groups are as follows:

[0244] Experimental group: each target cell + CAR-T;

[0245] Control group 1: Target cells release maximum LDH;

[0246] Control group 2: Target cells spontaneously release LDH;

[0247] Control group 3: Effector cells spontaneously release LDH;

[0248] 4. Detection method:

[0249] After effector cells and target cells were co-cultured for 18 h, the CytoTox 96 non-radioactive cytotoxicity assay kit (Promega) was used.

[0250] This method is a colorimetric detection approach that reflects the degree of cell lysis by detecting the content of lactate dehydrogenase (LDH). LDH is a stable cytoplasmic enzyme released during cell lysis, and its release mechanism is essentially the same as that of 51Cr in radiometric analysis. The released LDH in the culture supernatant can be detected by a coupled enzyme reaction, in which LDH converts a tetrazolium salt (INT) into red formazan. The amount of red product generated is directly proportional to the number of lysed cells.

[0251] For details, please refer to the CytoTox 96 non-radioactive cytotoxicity assay kit instructions.

[0252] 5. The formula for calculating cytotoxicity is:

[0253]

[0254] The results are as follows Figures 6A-6C As shown, the constructed CAR-T cells had no significant killing effect on Glypican 3 negative cells, but exhibited strong killing activity against Glypican 3 positive tumor cells.

[0255] Example 13

[0256] This embodiment detects the secretion of CAR-T cytokines.

[0257] 1. Cell culture supernatant

[0258] The cell culture with an effect-to-target ratio of 1:1 in Example 12 was centrifuged at 400×g for 10 min to remove the precipitate, and the supernatant was stored at -80℃ for testing.

[0259] 2. Reagent Preparation

[0260] The detection was performed using the Linko Bio ELISA kit (catalog number: Human Gamma Interferon ELISA Kit: EK180-96). Before the detection, all reagents and samples were brought to 25°C. 1× wash buffer and 1× detection buffer were prepared according to the instructions for use, and the antibody was detected.

[0261] 3. Preparation of Standards and Samples

[0262] Standards: The stock solution of the standard was diluted twice using 5% 1640 culture medium, with a total of 8 dilution gradients, including zero concentration.

[0263] Samples: Dilute the samples using 5% 1640 medium as directed.

[0264] 4. Testing Procedures

[0265] (1) Soaking the microplate: Add 300 μL of 1× washing buffer and let it stand for 30 seconds. After discarding the washing buffer, pat the microplate dry on absorbent paper.

[0266] (2) Add standard: Add 100 μL of 2-fold serially diluted standard to the standard wells, and add 100 μL of 5% 1640 culture medium to the blank wells;

[0267] (3) Add sample: Add 100 μL of cell culture supernatant to the sample well;

[0268] (4) Add detection antibody: Add 50 μL of diluted detection antibody (1:100 dilution) to each well.

[0269] (5) Incubation: Seal the plate with sealing film, shake at 300 rpm, and incubate at 25°C for 2 h;

[0270] (6) Washing: Discard the liquid, add 300 μL of washing solution to each well and wash the plate 6 times;

[0271] (7) Enzyme incubation: Add 100 μL of horseradish peroxidase-labeled streptavidin (1:100 dilution) to each well.

[0272] (8) Incubation: Seal the plate with a new sealing film, shake at 300 rpm, and incubate at 25°C for 45 min;

[0273] (9) Washing: Repeat step (6);

[0274] (10) Adding substrate for color development: Add 100 μL of TMB substrate to each well, incubate in the dark at 25°C for 15 min;

[0275] (11) Add stop solution: Add 100 μL of stop solution to each well and mix thoroughly;

[0276] (12) Detection reading: The OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm was measured using an ELISA reader. The calibrated OD value is the measured value at 450 nm minus the measured value at 630 nm.

[0277] IFN-γ factor secretion results as follows Figure 7 As shown, spontaneous CAR-T cell culture alone was detected. Trace amounts of IFN-γ were detected in spontaneous, 293T, and CAR-T cultures. High levels of IFN-γ were detected in Huh7-GPC3, Sk-hep1-GPC3, and HepG2 cultures, respectively, indicating that the CAR-T cells constructed in this invention can also release cytokines to kill Glypican 3-positive tumor cells and have high specificity. No significant cytokine secretion was observed in Glypican 3-negative cells.

[0278] In summary, this invention screens and prepares anti-Glypican 3 antibodies, which possess high affinity and specificity, effectively targeting the tumor antigen Glypican 3. The anti-Glypican 3 antibodies are then used to target chimeric antigen receptors, and chimeric antigen receptor cells are further prepared. These chimeric antigen receptor cells can efficiently kill tumor cells and secrete various cytokines to exert their killing function, exhibiting high specificity.

[0279] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. An anti-Glypican 3 antibody, characterized in that, The anti-Glypican 3 antibody is a nanobody, and the anti-Glypican 3 antibody includes a heavy chain variable region. The anti-Glypican 3 antibody has the following technical features: the heavy chain variable region includes amino acid sequences as shown in SEQ ID No. 1 (CDR-H1), amino acid sequences as shown in SEQ ID No. 2 (CDR-H2), and amino acid sequences as shown in SEQ ID No. 3 (CDR-H3).

2. The anti-Glypican 3 antibody according to claim 1, characterized in that, The heavy chain variable region further includes a framework region, which includes framework regions FR1 to FR4, and the amino acid sequences of framework regions FR1 to FR4 are shown in SEQ ID No. 4 to 7, respectively; or, the amino acid sequences of framework regions FR1 to FR4 are shown in SEQ ID No. 23, SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7, respectively.

3. The anti-Glypican 3 antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 8 or SEQ ID No.

24.

4. The use of the anti-Glypican 3 antibody according to any one of claims 1 to 3 in the preparation or screening of tumor therapeutic drugs, characterized in that, The tumor is a tumor expressing Glypican 3; the tumor is selected from liver cancer.

5. The use according to claim 4, characterized in that, The tumor treatment drug is a chimeric antigen receptor cell.

6. An isolated polypeptide, characterized in that, The polypeptide is a chimeric antigen receptor; the polypeptide comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, an anti-Glypican 3 antibody as described in any one of claims 1 to 3, a CD8α hinge region, a CD8α transmembrane region, a co-stimulatory molecular domain, and a CD3 zeta signal domain.

7. An isolated polynucleotide, characterized in that, Encoding the anti-Glypican 3 antibody as described in any one of claims 1 to 3 or the polypeptide as described in claim 6.

8. A nucleic acid construct, characterized in that, The nucleic acid construct contains a polynucleotide encoding the isolated polypeptide of claim 6; the nucleic acid construct is a vector.

9. The nucleic acid construct according to claim 8, characterized in that, The nucleic acid construct is any one of a lentiviral vector, a retroviral vector, or an adeno-associated virus vector.

10. A lentivirus, characterized in that, The lentivirus is formed by viral packaging of the nucleic acid construct as described in claim 8 or 9.

11. A lentiviral vector system, characterized in that, The lentiviral vector system includes the nucleic acid construct as described in claim 8 or 9, as well as an auxiliary plasmid or host cell.

12. A chimeric antigen receptor immune cell, characterized in that, The chimeric antigen receptor immune cell expresses the isolated polypeptide of claim 6, which is membrane-bound.

13. The chimeric antigen receptor immune cell according to claim 12, characterized in that, The immune cells are selected from any one of T lymphocytes, B lymphocytes, NK cells, mast cells, or macrophages.

14. Use of the isolated polypeptide of claim 6 and the chimeric antigen receptor immune cell of any one of claims 12-13 in the preparation of a tumor therapeutic drug, wherein the tumor is a tumor expressing Glypican 3; the tumor is selected from liver cancer.

15. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the chimeric antigen receptor immune cells as described in any one of claims 12-13 and a pharmaceutically acceptable carrier or excipient.

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