CN116410323A - anti-GPC 3 antibody and application thereof - Google Patents

Abstract

The invention relates to the technical field of biological medicine, in particular to an anti-GPC 3 antibody and application thereof, wherein the anti-GPC 3 antibody is denoted by B174, the anti-GPC 3 antibody comprises a heavy chain variable region, and the heavy chain variable region of the anti-GPC 3 antibody comprises a CDR-H1 with an amino acid sequence shown as SEQ ID No.1, a CDR-H2 shown as SEQ ID No.2 and a CDR-H3 shown as SEQ ID No. 3. The anti-GPC 3 antibody has high affinity and specificity, can effectively target tumor antigen GPC3, and can be used for preparing chimeric antigen receptor and further preparing chimeric antigen receptor cells, wherein the chimeric antigen receptor cells can kill tumor cells with high efficiency and have high specificity.

Description

anti-GPC 3 antibody and application thereof

Technical Field

The invention relates to the technical field of biological medicine, in particular to an anti-GPC 3 antibody and application thereof.

Background

Hepatocellular carcinoma (hepatocellular carcinoma, HCC) is the most common histological subtype in liver cancer (liver cancer), the sixth most common cancer, and the fourth leading cause of cancer-related death.

Currently, the clinical management of HCC presents considerable challenges. Only 15% -20% of HCC cases are diagnosed early and may be suitable for curative treatments such as surgical resection, liver transplantation, percutaneous ethanol injection local ablation, microwave ablation, radiofrequency ablation, etc. At the same time, most HCC patients have potential for chronic liver disease, and surgical excision therapy of such populations is fraught with the potential for various potential complications. In addition, there are many patients diagnosed with advanced HCC that cannot be surgically resected. HCC patients have a 5-year survival rate of only about 18%, and if cancer cells have spread to the surrounding tissue or distal parts of the body, the 5-year survival rate will drop to about 11% and 5%, respectively.

Glypican 3 (gpc 3) is a member of the heparan sulfate proteoglycan family and can play a role in controlling cell division and growth regulation. GPC3 is rarely expressed in healthy tissues, but is highly expressed in many solid tumors such as HCC. In HCC, GPC3 is often used as a biomarker for diagnosis and prognosis. The specific expression of GPC3 in tumor tissue, its accessible surface location and its role in various signaling pathways involved in tumor transformation make it an ideal target for immunotherapy of several solid tumors, including HCC.

Currently, HCC therapies targeting GPC3 in preclinical and clinical stages mainly include polypeptide vaccines, monoclonal antibodies, chimeric antigen receptor T cell immunotherapy, and the like.

Chimeric antigen receptor T cell (Chimeric antigen receptor T cells, CAR-T) therapy is a revolutionary cancer immune cell therapy in recent years. The T cells express chimeric antigen receptor by a gene transduction method to obtain CAR-T cells with tumor specificity, and the CAR-T cells specifically track, identify and kill tumor cells so as to treat tumors.

Therefore, the GPC3CAR-T prepared by introducing the chimeric antigen receptor gene against GPC3 into T cells by a gene transduction method can specifically identify and kill HCC cells expressing GPC3, thereby realizing the anti-tumor effect thereof and having a great clinical transformation value for the immunotherapy method targeting GPC 3.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide an anti-GPC 3 antibody and its use for solving the problems of the prior art.

To achieve the above and other related objects, the present invention provides an anti-GPC 3 antibody, which is denoted by B174 or GPC3-VHH-B174, the anti-GPC 3 antibody comprising a heavy chain variable region, the anti-GPC 3 antibody having one or more of the following technical features;

<1> the heavy chain variable region comprises CDR-H1 having an amino acid sequence as shown in SEQ ID No. 1;

<2> the heavy chain variable region comprises CDR-H2 having the amino acid sequence shown in SEQ ID No. 2;

<3> the heavy chain variable region includes CDR-H3 having an amino acid sequence shown in SEQ ID No. 3.

The invention also provides an isolated polypeptide comprising a transmembrane domain, an intracellular domain and an extracellular domain comprising the anti-GPC 3 antibody.

The invention also provides a chimeric antigen receptor immune cell that expresses the isolated polypeptide that membrane binds.

As described above, the anti-GPC 3 antibody and the use thereof of the present invention have the following advantageous effects:

(1) The anti-GPC 3 antibody only comprises a heavy chain variable region, has high affinity and specificity, can efficiently target GPC3 antigen, has a simple structure, and is easy to prepare;

(2) In the invention, the anti-GPC 3 antibody is utilized to construct a chimeric antigen receptor, and the chimeric antigen receptor can efficiently target GPC3;

(3) The chimeric antigen receptor cell of the invention can specifically identify GPC3 positive tumor cells, kill efficiently, and simultaneously release cytokines such as IFN-gamma factors and the like to play a cell killing role.

Drawings

FIG. 1 shows a graph of the affinity results of Biacore detection of anti-GPC 3 antibodies (GPC 3-VHH-B174);

FIG. 2 shows a graph of the results of FACS detection of anti-GPC 3 nanobodies recognizing GPC3 antigen;

FIG. 3 shows a plasmid map of a chimeric antigen receptor lentiviral vector targeting GPC3;

FIG. 4 is a schematic diagram showing the structure of a chimeric antigen receptor expressing anti-GPC 3 in example 4;

FIG. 5 is a graph showing the results of flow assay of the expression rate of chimeric antigen receptor (GPC 3-VHH-B174) of T lymphocytes;

FIG. 6A is a graph showing the killing effect of CAR-T cells of the present invention on 293T cells;

FIG. 6B is a graph showing the killing effect of CAR-T cells of the present invention on Huh7-GPC3 cells;

FIG. 6C is a graph showing the killing effect of CAR-T cells of the present invention on HepG2 cells;

FIG. 7 shows a graph of IFN-gamma cytokine secretion levels for CAR-T cells (GPC 3-VHH-B174).

Detailed Description

The invention provides an anti-GPC 3 antibody, code number B174 or GPC3-VHH-B174, the anti-GPC 3 antibody comprising a heavy chain variable region, the anti-GPC 3 antibody having one or more of the following technical characteristics;

<1> the heavy chain variable region comprises CDR-H1 having an amino acid sequence as shown in SEQ ID No. 1;

<2> the heavy chain variable region comprises CDR-H2 having the amino acid sequence shown in SEQ ID No. 2;

<3> the heavy chain variable region comprises CDR-H3 having the amino acid sequence shown in SEQ ID No. 3;

GFTLDYYA(SEQ ID No.1)

ISSTGHST(SEQ ID No.2)

AADIVRYYCSPVVYGDDYGV(SEQ ID No.3)。

CDRs (complementarity determining regions, complementarity determining region) generally refer to regions of an antibody that can be spatially complementary to an epitope. Variability in antibodies is typically not evenly distributed throughout the variable region of an antibody, the heavy chain variable region of a monoclonal antibody typically has 3 hypervariable regions (hypervariable region, HVR) which are typically complementary to antigenic determinants in spatial structure, so the hypervariable regions are also known as complementarity determining regions (complementarity determining region, CDRs), i.e., the heavy chain variable region typically comprises three complementarity determining regions, i.e., HCDR1, HCDR2 and HCDR3.

In certain embodiments of the present invention, the complementarity determining regions of the heavy chain variable region of the anti-GPC 3 antibody comprise CDR-H1 of the amino acid sequence shown in SEQ ID No.1, CDR-H2 of the amino acid sequence shown in SEQ ID No.2 and CDR-H3 of the amino acid sequence shown in SEQ ID No. 3.

The anti-GPC 3 antibody is an antibody fragment, and/or a monoclonal antibody. Further, the monoclonal antibody is an IgG1 antibody.

An "antibody fragment" comprises a portion of an intact antibody, preferably comprising an antigen binding or variable region thereof. For example, antibody fragments include nanobodies (VHH), single chain antibodies (scFv), fab ', F (ab ') or F (ab ') 2.

In certain embodiments of the invention, the anti-GPC 3 antibody is a nanobody (Nb), i.e., a heavy chain single domain antibody VHH (variable domain of heavy chain of heavy-chain antibody). Nanobodies comprise only one heavy chain variable region (VHH) and CH2, CH3 regions, and nanobody light chains are naturally deleted compared to other antibodies. Nanobody crystals are about 2.5nm in diameter and about 4nm long, the smallest fragment that can bind to antigen in nature. The anti-GPC 3 antibody only comprises a heavy chain variable region, has high affinity and specificity, can efficiently target GPC3 antigen, has a simple structure, is easy to prepare, and has important application value in the preparation of medicines taking GPC3 as a target.

In certain embodiments of the invention, the anti-GPC 3 antibody is a single chain Fv (scFv). The single chain antibody may typically be V including antibodies _H (heavy chain variable region) and V _L A polypeptide chain (light chain variable region). In general, single chain antibodies may also include a linker peptide (linker), which is typically located at V _H And V _L To allow the scFv to form the desired structure for binding to the antigen. For example, the anti-GPC 3 antibody can include V _H And V _L ，V _H And V _L Can be provided with a connecting peptide, and the single-chain anti-GPC 3 antibody can sequentially comprise V from N end to C end _L Linker peptide and V _H The anti-GPC 3 single chain antibody may also include V from N-terminal to C-terminal in order _H Linker peptide and V _L . The linker peptide may be any of a variety of linker peptides suitable in the art for forming scFv, for example, the linker peptide may be a G4S3 linker, the selection or design of which may be found in references Michel Sadelain etc, science Translational Medicine,2013; carl h.june etc, science Translational Medicine,2015. The V is _L Can be obtained according to techniques conventional in the art.

In certain embodiments of the invention, the heavy chain variable region may further comprise a framework region, which may be located between or at either end of the complementarity determining regions. In some embodiments of the present invention, the sequence of the framework region is a human monoclonal antibody variable region, or a framework region sequence of a murine monoclonal antibody variable region is a framework region sequence obtained by substituting, deleting or adding one or more (specifically, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 1 to 3) amino acids, and the framework region sequence may have homology of 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more with the framework region sequence of the human monoclonal antibody variable region sequence.

In certain embodiments of the invention, the heavy chain variable region further comprises a framework region. The frame region includes frame regions FR 1-FR 4.

Preferably, the amino acid sequence of the FR1 is shown as SEQ ID No. 4: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID No. 4).

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

Preferably, the amino acid sequence of the FR3 is shown as SEQ ID No. 6: NYADSVKDRFTISRDNAKNTVYLQMNSLKPEDTAVYYC (SEQ ID No. 6).

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

Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No.8 as EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSTGHSTNYA DSVKDRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADIVRYYCSGYVVPDDYGVWGQG TQVTVSS (SEQ ID No. 8).

In certain embodiments of the invention, the anti-GPC 3 antibody is selected from a phage antibody library, and the heavy chain variable region nucleotide sequence is gaagtgcagctggtcgagtctggcggcggcctggtgcagccaggtgggagcctgagactgagctgcgcagcctccggctttacactggattactacgccatcggctggttcagacaggccccagggaaggagagggagggcgtgtcctgcatctcctccactggccactctaccaattatgccgacagcgtgaaagatagattcaccatctccagagataacgccaagaataccgtgtacctgcagatgaactccctgaaaccagaggacacagccgtgtactactgtgccgccgacatcgtgcgctattactgcagcggatacgtggtgcccgatgattacggggtgtgggggcagggcacccaggtgaccgtgtctagc (SEQ ID No. 9) as shown in SEQ ID No. 9.

In another aspect, the invention provides an isolated polynucleotide encoding the anti-GPC 3 antibody.

In certain embodiments of the invention, the polynucleotide has a sequence as set forth in SEQ ID No. 9.

In another aspect, the invention provides the use of the anti-GPC 3 antibody in the manufacture or screening of therapeutic agents, or in the manufacture of diagnostic agents.

The therapeutic agent may be an agent that targets GPC3 antigen, binds to or acts on the GPC3 antigen, thereby treating and/or preventing an indication.

In certain embodiments of the invention, the therapeutic agent may be a tumor therapeutic agent. The tumor is a tumor expressing GPC 3. The tumor therapeutic drug can be a drug which takes GPC3 antigen on the functional surface of the surface of tumor cells as a target and binds or acts on the GPC3 antigen so as to treat and/or prevent tumors. The tumor may be a tumor positive for GPC3 expression such as liver cancer, ovarian cancer, lung cancer, melanoma, gastric cancer, thyroid cancer, etc.

In certain embodiments of the invention, the therapeutic agent is a chimeric antigen receptor cell.

The chimeric antigen receptor cell therapeutic drug generally includes chimeric antigen receptor cells, which may be chimeric antigen receptor T cells, chimeric antigen receptor NK cells, or the like. The chimeric antigen receptor T cells typically comprise T lymphocytes, which also include chimeric antigen receptors. The chimeric antigen receptor NK cells generally 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 certain embodiments of the invention, the extracellular domain comprises the anti-GPC 3 antibody, i.e., the chimeric antigen receptor cell can express the anti-GPC 3 antibody on the cell surface, thereby directing the cell to act on cells expressing GPC3 antigen (e.g., tumor cells). The action on the cells expressing the GPC3 antigen may be killing of the cells expressing the GPC3 antigen, or the like.

The diagnostic drug specifically refers to a reagent for diagnosing an action target GPC3 antigen by taking the GPC3 antigen as a biomarker.

In another aspect, the invention provides an isolated polypeptide comprising a transmembrane domain, an intracellular domain, and an extracellular domain comprising the anti-GPC 3 antibody.

In certain embodiments of the invention, the polypeptide is a chimeric antigen receptor. In the present invention, the anti-GPC 3 antibody is used to construct a chimeric antigen receptor capable of efficiently targeting GPC3.

In certain embodiments of the invention, the transmembrane domain may be selected from any one or more of the transmembrane domains of the CD8 a transmembrane region, CD28 transmembrane region, DAP10 transmembrane region, and the like.

For another example, the sequence of CD8 alpha can be referenced to NM-001145873, the sequence of CD28 can be referenced to NM-006139, and the sequence of DAP10 can be referenced to NM-014266.

In certain embodiments of the invention, the intracellular domain may comprise a costimulatory domain and/or a signaling domain. The signal transduction domain includes an immunoreceptor tyrosine activation motif. The immunoreceptor tyrosine activation motif may be selected from cd3ζ.

Preferably, the signal transduction domain further comprises a co-stimulatory molecule. For example, the costimulatory molecule may be selected from any of 4-1BB, CD28, OX40, ICOS, DAP10, etc., or a combination of at least two protein molecules. For another example, the amino acid sequence of 4-1BB may include the following:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID No.12)

For another example, the sequence of 4-1BB may refer to NM_001561, the sequence of CD28 may refer to NM_006139, the sequence of OX40 may refer to NM_003327, the sequence of ICOS may refer to NM_012092, the sequence of CD3 zeta may refer to NM_198053, the sequence of DAP 10 may refer to NM_014266.

In one embodiment of the present invention, the intracellular domain comprises 4-1BB and CD3 zeta in order from the N-terminus to the C-terminus.

In certain embodiments of the invention, the extracellular domain may include a signal peptide, an anti-GPC 3 antibody, a hinge region.

In certain embodiments of the invention, the signal peptide comprises a CD8 a signal peptide.

In certain embodiments of the invention, the hinge region is selected from the group consisting of a CD8 a hinge region.

In certain embodiments of the invention, the polypeptide comprises, in order from the N-terminus to the C-terminus, a CD8 a signal peptide, the anti-GPC 3 antibody, a transmembrane domain, an intracellular domain.

In some embodiments of the invention, the polypeptide comprises, in order from the N-terminus to the C-terminus, a CD8 a signal peptide, an anti-GPC 3 nanobody, a CD8 a hinge region, a CD8 a transmembrane region, a co-stimulatory molecule domain, a CD3 zeta signal domain.

In some embodiments of the invention, the polypeptide comprises, in order from the N-terminus to the C-terminus, a CD8 alpha signal peptide, an anti-GPC 3 nanobody, a CD8 alpha hinge region, a CD8 alpha transmembrane region, a 4-1BB co-stimulatory domain, and a CD3 zeta signal domain.

In a specific embodiment of the present invention, the polypeptide comprises, in order from the N-terminus to the C-terminus, a CD8 a signal peptide, the anti-GPC 3 nanobody, a CD8 a hinge region, a CD28 transmembrane region, a CD28 co-stimulatory domain, and a CD3 zeta signal domain.

In another embodiment of the present invention, the polypeptide comprises, in order from the N-terminus to the C-terminus, a CD8 a signal peptide, the anti-GPC 3 nanobody, a CD8 a hinge region, a CD8 a transmembrane region, an OX40 co-stimulatory domain, a CD3 zeta signal domain.

In another embodiment of the present invention, the polypeptide comprises, in order from the N-terminus to the C-terminus, a CD8 a signal peptide, the anti-GPC 3 nanobody, a CD8 a hinge region, a CD8 a transmembrane region, an ICOS co-stimulatory domain, a CD3 zeta signal domain.

In another embodiment of the present invention, the polypeptide comprises, in order from the N-terminus to the C-terminus, a CD 8. Alpha. Signal peptide, the anti-GPC 3 nanobody, a CD 8. Alpha. Hinge region, a CD 8. Alpha. Transmembrane region, a 4-1BB co-stimulatory domain, and a CD3 zeta.

In another embodiment of the present invention, the polypeptide comprises, in order from the N-terminus to the C-terminus, a CD8 a signal peptide, the anti-GPC 3 nanobody, a CD8 a hinge region, a CD28 transmembrane region, a CD28 co-stimulatory domain, an OX40 co-stimulatory domain, a CD3 zeta signal domain.

In certain embodiments of the invention, the polynucleotide sequence encoding the isolated polypeptide is set forth in SEQ ID No. 22:

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaagtgcagctggtcgagtctggcggcggc

ctggtgcagccaggtgggagcctgagactgagctgcgcagcctccggctttacactggattactacgccatcggctggttcagacaggcccca

gggaaggagagggagggcgtgtcctgcatctcctccactggccactctaccaattatgccgacagcgtgaaagatagattcaccatctccaga

gataacgccaagaataccgtgtacctgcagatgaactccctgaaaccagaggacacagccgtgtactactgtgccgccgacatcgtgcgctatt

actgcagcggatacgtggtgcccgatgattacggggtgtgggggcagggcacccaggtgaccgtgtctagcaccacgacgccagcgccgc

gaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcac

acgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttact

gcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccg

atttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaacca

gctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccg

cagagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcg

agcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctga(SEQ ID No.22)。

the invention also provides a nucleic acid construct comprising a polynucleotide encoding said isolated polypeptide.

The nucleic acid construct may be a lentiviral vector, a retroviral vector, or an adeno-associated viral vector. Taking lentiviral vectors as an example, lentiviral vectors include a vector backbone, i.e., an empty vector, and an expression frame. That is, the nucleic acid construct is a vector containing the gene encoding the chimeric antigen receptor.

The term "vector" refers to a nucleic acid fragment or polynucleotide fragment for introducing or transferring one or more nucleic acids or one or more polynucleotides into a target cell or tissue. Typically, the vector is used to introduce the exogenous DNA into another cell or tissue. The vector may comprise a bacterial resistance gene for growth in bacteria and a promoter for expression of the protein of interest in an organism. DNA may be generated in vitro by PCR or any other suitable technique or techniques known to those skilled in the art.

The term "expression cassette" refers to a sequence having the potential to encode a protein.

The invention also provides a lentivirus, which is formed by packaging the nucleic acid construct through viruses. The lentivirus contains the nucleic acid construct.

The invention also provides a lentiviral vector system, which is characterized in that the lentiviral vector system comprises the nucleic acid construct and a helper plasmid.

Still further, the helper plasmid encodes one or more nucleotide sequences for gag and pol proteins, as well as other necessary viral packaging component nucleotide sequences, and may include packaging plasmids and envelope plasmids. In one embodiment, the packaging plasmid is gag/pol, the envelope plasmid is VSVg, and both plasmids are commercially available, e.g., addgene accession numbers 14887 and 8454.

Further, the lentiviral vector system also includes a host cell, which may be a lentiviral producing cell, such as a mammalian cell, specifically 293T cell.

The lentivirus can be obtained after transfection of host cells with the nucleic acid construct and helper plasmid in the lentivirus vector system.

In another aspect, the invention provides a chimeric antigen receptor immune cell that expresses the isolated polypeptide membrane-bound.

Preferably, said chimeric antigen receptor immune cell comprises said nucleic acid construct and/or said lentivirus.

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

In another embodiment of the invention, the chimeric antigen receptor immune cell is a T lymphocyte.

The T lymphocytes may generally express the polypeptide, which may generally bind to a GPC3 antigen, more specifically may bind to a GPC3 antigen via an extracellular domain comprising the anti-GPC 3 antibody, and when the polypeptide binds to the GPC3 antigen, the T lymphocytes may generally be activated and/or stimulated to proliferate. In certain embodiments of the invention, the T lymphocytes, i.e., chimeric antigen receptor T cells, may express the anti-GPC 3 antibody on the surface of T lymphocytes, thereby directing the T lymphocytes to act on cells expressing GPC3 antigen (e.g., tumor cells), which may be killing cells expressing GPC3 antigen, etc.

In another embodiment of the invention, the chimeric antigen receptor immune cell is an NK cell.

The NK cells may generally express the polypeptide and may generally bind to the GPC3 antigen, more particularly may bind to the GPC3 antigen via an extracellular domain comprising the anti-GPC 3 antibody, and when the polypeptide binds to the antigen, the NK cells may generally be activated and/or stimulated to proliferate. In certain embodiments of the present invention, the NK cells, i.e., chimeric antigen receptor NK cells, can express the anti-GPC 3 antibody on the surface of NK cells, thereby allowing the NK cells to be directed to act on cells expressing GPC3 antigen (e.g., tumor cells), which may be killing cells expressing GPC3 antigen, etc.

In another aspect, the invention provides the use of the isolated polypeptide, isolated polynucleotide, nucleic acid construct, lentivirus, chimeric antigen receptor immune cell in the preparation or screening of a therapeutic agent, or in the preparation of a diagnostic agent.

The therapeutic or diagnostic agent may be an agent that targets the GPC3 antigen, binds to or acts on the GPC3 antigen, thereby treating and/or preventing an indication.

In certain embodiments of the invention, the therapeutic agent may be a tumor therapeutic agent. The tumor therapeutic agent may be an agent that binds to or acts on GPC3 antigen, which is functionally expressed on the surface of tumor cells, as a target, thereby treating and/or preventing tumors. The tumor can be tumor positive for GPC3 expression such as gastric cancer, lung cancer, pancreatic cancer, intestinal cancer, etc.

The present invention provides a pharmaceutical composition comprising said chimeric antigen receptor immune cells and/or said anti-GPC 3 antibody and/or said isolated polypeptide.

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or adjuvant.

By "pharmaceutically acceptable" is meant that the drug does not produce adverse, allergic or other untoward reactions when properly administered to an animal or human.

The "pharmaceutically acceptable carrier or adjuvant" should be compatible with the active ingredient, i.e. it can be blended therewith without substantially reducing the efficacy of the drug in the usual manner. Specific examples of some substances which may be pharmaceutically acceptable carriers or excipients are sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methyl cellulose, ethyl cellulose and methyl cellulose; tragacanth 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, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifying agents, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting and stabilizing agent; an antioxidant; a preservative; non-thermal raw water; isotonic saline solution; and phosphate buffer, etc. These substances are used as needed to aid stability of the formulation or to aid in enhancing the activity or its bioavailability or to produce an acceptable mouthfeel or odor in the case of oral administration.

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

Example 1

The present example constructs phage nanobody libraries and performs panning and ELISA primary screening.

1. Construction of phage nanobody library

(1) Immunization of Bactrian camels with extracellular domain expressing GPC3-Fc (purchased from Beijing Baizier organism) and ELISA validation titers were followed by 200mL peripheral blood withdrawal;

(2) Sorting lymphocytes to obtain peripheral blood mononuclear lymphocyte sediment, and extracting RNA;

(3) By using

III, reverse transcriptase synthesizes first strand cDNA by taking RNA as a template, and then utilizes nested PCR to amplify VHH genes;

(4) The VHH gene was inserted into a pMECS phage display vector (Du-Argania),after electrically transforming TG1 competent cells, taking bacterial liquid for library identification, uniformly coating all the rest cultures on an LB/AMP GLU plate, collecting lawn after bacteria grow out, adding 1/3 volume of 50% glycerol, uniformly mixing and packaging, and preserving at-80 ℃ to successfully construct a reservoir capacity of more than 10 ⁹ Is a phage display camelid VHH immune library.

2. Panning of phage nanobody libraries

Diluting purified GPC3-His recombinant protein to 4 mug/mL with PBS buffer solution, taking 96-well ELISA plate, selecting 3-well ELISA plate, adding 100 mug (400 ng/well) into each well, coating overnight at 4 ℃, and taking PBS as negative control; removing the coating liquid, adding 150 mu L of 2% degreasing inner powder into each hole, and sealing at 25 ℃ for 1h; washing with PBST for 4 times, collecting phage solution, diluting with 2% milk powder to 5×10 ¹¹ pfu/ml, adding an ELISA plate, 100 mu L/hole and incubating for 2h at 25 ℃; discarding phage samples, washing with PBST for 10 times, washing with PBS for 5 times, adding 100 μl of freshly prepared 0.1M triethylamine into each well, standing at 25deg.C for 10min, and rapidly neutralizing the eluate with equal volume of 1M Tris-HCl (pH 7.4); taking part of the eluent to determine phage titer; another 400. Mu.L of eluent was used to infect 4ml of fresh culture log phase TG1 bacterial liquid (OD 600 about 0.6), incubated at 37℃for 30min, 16mL of 2 XYT/AMP-GLU (ampicillin AMP and glucose GLU at 1. Mu.g/ml and 2% respectively) were added for cultivation, and 200r/min at 37℃until OD was reached ₆₀₀ Reaching 0.7. Taking 100 mu L of bacterial suspension, carrying out gradient dilution, and uniformly smearing on a 2 XYT/ampicillin/glucose agar plate so as to carry out library capacity and diversity measurement; inoculating 100 mu L of bacterial suspension, namely phage display vector library, into a 2 XYT/AMP-GLU culture medium, culturing to a logarithmic phase, adding auxiliary phage, carrying out library rescue, obtaining phage particles, measuring phage titer, concentrating and purifying to obtain phage particles for next round of screening; the residual bacterial liquid is subjected to centrifugation, then is resuspended by using a proper volume of 2 XYT culture solution, is smeared on a plate with screening resistance for overnight culture, is scraped from the plate by using a proper amount of liquid culture solution, is added with a 2 XYT culture solution containing 1/3 volume of 50% glycerol for resuspension and is sub-packaged, and all bacteria are stored at-80 ℃.

The above screening operation was repeated 3 times.

3 rounds of solid phase screening are carried out on the immune nanometer antibody library in vitro, so that phage clone with binding activity is effectively enriched. After prokaryotic induction expression of the monoclonal phage, phage clones that bind to the extracellular region of the antigen were further screened by ELISA.

3. Phage packaging

100. Mu.L of frozen panning fungus solution from the previous round is added into 100mL of 2 XYT/AMP-GLU culture solution, and is cultured at 37deg.C with shaking (200 rpm) until logarithmic phase (OD) ₆₀₀ With a value of 0.6), 90. Mu.L of helper phage M13K07 (1.7X10) ¹³ PFU/mL), the mixture is firstly kept stand at 37 ℃ for 30min,2800 Xg is centrifuged for 10min to collect thalli, 200mL of 2 XYT/AMP-GLU culture medium is used for resuspension, shaking (200 rpm) is carried out at 37 ℃ for 12h,4 ℃ and 3800 Xg is centrifuged for 30min to remove thalli to collect supernatant, 1/5 volume of precooled PEG/NaCl is added for uniform mixing, phage is precipitated for 2h,4 ℃ and 3800 Xg is centrifuged for 30min to collect phage, 2mL of PBS solution with final volume is used for resuspension and transfer to a 15mL centrifuge tube, 4 ℃ and 12000 Xg is centrifuged for 15min to collect supernatant, 1/5 volume of precooled PEG/NaCl solution is added for uniform mixing up and down, and the mixture is kept stand on ice for 2h; centrifugation was performed at 4℃for 10min at 10000 Xg, the supernatant was discarded, phage pellet was resuspended in 1mL PBS and incubated overnight at 4℃with shaking bed to allow phage particles to dissolve well, phage solution was mixed with an equal volume of 60% glycerol and split into 1.5mL EP tubes and stored at-80 ℃.

In order to avoid losing sequence diversity, preliminary ELISA screening is carried out from round 2 and round 3 panning products by using GPC3 antigen to perform 3 rounds of panning on phage library, positive clones are randomly selected from panning products and induced to express, and the expression supernatant is crude extracted VHH antibody, and the VHH antibody sequence of the monoclonal strain is determined by sequencing.

Example 2

This example performed Fluorescence Activated Cell Sorting (FACS) candidate cloning.

Cell culture was performed according to standard cell culture protocols, GPC3 positive and negative cell suspensions were prepared using pancreatin digested cells, and after centrifugation (300 Xg, 5 min) to remove the culture broth, the cells were resuspended in Flow Buffer (PBS+2% FBS)Up to 2X 10 ⁶ cell/mL, 2X 10 wells per well in a V-bottom 96-well plate ⁵ Cell suspensions of individual cells were removed after centrifugation at 300 Xg for 5min, the cells were resuspended by adding VHH antibody crude extract and incubated at 4℃for 1h, the supernatant was removed after centrifugation at 300 Xg for 5min, the cells were resuspended by Flow Buffer, the APC anti-his antibodies were diluted to 2. Mu.g/mL with Flow Buffer, 100. Mu.L of cells were resuspended per well, incubated at 4℃for 1h, the cells were resuspended by 200. Mu.L of Flow Buffer after 3 washing with Flow Buffer and detected by Flow cytometry, ten candidate antibodies were selected, designated GPC3-VHH-B6, GPC3-VHH-B51, GPC3-VHH-B89, GPC3-VHH-B108, GPC3-VHH-B168, GPC3-VHH-B174 (V18), GPC 3-H-B174 (V32), 3-VHH-B174 (V33) and 3-VHH-B174 (V174) were only claimed as a single candidate antibody for the other application for VHH protection, since there was only a single problem in the application of GPC3-VHH protection.

Example 3

This example carries out VHH-mIgG2a Fc nanobody expression, purification and antibody affinity assay.

To further identify the antibodies screened in example 2, it was necessary to express the antibodies in mammalian cells, and therefore, a plasmid vector C-4pCP. Stuffer-mCg a-FC (purchased from Shanghai Baiying organism) with a mouse Fc tag expressing VHH was first constructed, comprising the steps of:

(1) The VHH fragments were amplified using PCR, the reaction system (reagents purchased from NEB) and PCR reaction conditions are shown in table 1 below;

TABLE 1

(2) The cleavage system and the reaction conditions are shown in Table 2, respectively, and the cleaved vector is used

Purifying by using a PCR purification kit, dissolving the air-dried DNA into 20 mu L of water, and detecting the concentration of the DNA;

TABLE 2

(3) The homologous recombination reaction system was 10. Mu.L (reagents were purchased from Norfluzan) as shown in Table 3;

TABLE 3 Table 3

(4) Adding all homologous recombination reaction systems into DH5 alpha competent cells, and transforming DH5 alpha competent cells under the transformation conditions shown in Table 4;

TABLE 4 Table 4

/>

(5) Transformation plate selection monoclonal PCR pre-identification, PCR identification system conditions are shown in Table 5 (reagents are purchased from Producer); sequencing and identification are carried out by a sequencing company, the sequencing result meets the expectations, and a plasmid vector with the mouse Fc tag for expressing VHH is successfully constructed.

TABLE 5

293E cells were passaged approximately 24h prior to plasmid transfection to a cell density of approximately 2.6X10 ⁶ cells/mL 293E cells were transfected with 0.15mg of VHH-mIgG2a (plasmid constructed as described above)/100 mL of 293E by PEI method and DNA: PEI=1:2. 37 ℃, 130rpm, 8% CO ₂ Shaking culture for 6 days at 3000rpm for 30min to collect cell culture supernatant, filtering the collected supernatant containing target antibody with Millex-GP Filter Unit 0.45 μm Sterile, and collecting the supernatant with MabSelect ^TM SuRe ^TM Centrifugal concentration, 1The column was washed with X PBS, the protein eluted with 0.1M (mol/L) Gly-HCl, neutralized with 1/10 volume of Tris-HCl at pH 8.5, dialyzed overnight at 4℃and quantified by the method of Nanodrop 2000 assay A280, and antibody purity was determined by SEC-HPLC.

Further, affinity of 10 purified GPC3 VHH antibodies (GPC 3-VHH-B6, GPC3-VHH-B51, GPC3-VHH-B89, GPC3-VHH-B108, GPC3-VHH-B168, GPC3-VHH-B174 (V18), GPC3-VHH-B174 (V32), GPC3-VHH-B174 (V33) and GPC3-VHH-B174 (V34)) was measured by Biacore. Biacore is a bioanalytical sensing technology developed based on surface plasmon resonance (surface Plasmon resonance, SPR), and can detect and track the whole change process of binding and dissociating molecules in a solution and molecules fixed on the surface of a chip, record the whole change process in the form of a sensor graph, provide kinetic and affinity data, solidify antibodies on the surface of the chip in the measuring process, enable a mobile phase to be a solution containing antigens, and the measuring result is shown in table 6 and fig. 1, and the 10 GPC3 VHH antibodies have high affinity as can be seen from the measuring result.

TABLE 6

VHH antibodies	ka(1/Ms)	kd(1/s)	KD(M)
				GPC3-VHH-B174	1.56E+05	0.006628	4.26E-08

Example 4

This example describes a flow assay against GPC3 nanobodies.

Huh7-GPC3 tumor cells were incubated with purified 10 recombinant anti-GPC3 VHH antibodies in an ice bath for 30min, no anti-GPC3 VHH antibody was added to the blank, and then incubated with APC-labeled goat anti-mouse IgG antibody or iF488-anti-VHHcocktail antibody for 30min, and detected by flow cytometry, as shown in FIG. 2, indicating that the anti-GPC3 antibodies prepared by the screening of the present invention were able to recognize GPC3 antigen on the cell surface.

Example 5

This example prepares lentiviral vectors expressing a chimeric antigen receptor targeting GPC3 (GPC 3 CAR).

First, a lentiviral vector HD-SIN03 GPC3 CAR carrying a GPC3 CAR chimeric antigen receptor was constructed, the vector profile was shown in FIG. 3, the chimeric antigen receptor structure was shown in FIG. 4, the nucleotide sequence of the CAR was shown in SEQ ID No.22, and the chimeric antigen receptor comprises a CD8 alpha signal peptide, an anti-GPC3 antibody (anti-GPC 3 VHH), a CD8 alpha hinge region, a transmembrane region and an immunoreceptor tyrosine activation motif (CD 3 zeta).

Wherein the amino acid sequence of the signal peptide is: MALPVTALLLPLALLLHAARP (SEQ ID No. 10).

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

The amino acid sequences of the CD8 a hinge region and the transmembrane region are:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC(SEQ ID No.11)。

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

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID No.12)。

the CD3 ζ amino acid sequence is:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ IDNo.13)。

the preparation method comprises the following steps:

(1) A PCR reaction system (reagents were purchased from TOYOBO) was prepared according to Table 8, and the signal peptide-GPC 3-VHH fragment was amplified using the primers shown in Table 7.

TABLE 7

TABLE 8

Reagent(s)	Volume (mu L)
		10x buffer	5
2mM dNTP	5
		25mM MgSO 4	3
10μM primer F	1
		10μM primer R	1
Template DNA (cDNAclone)	1
		PCR-grade pure water	33
KOD-Plus-Neo	1

The above reagents were derived from TOYOBO inc.

After completion of the preparation, the reaction was performed according to the PCR procedure shown in Table 9.

TABLE 9

After the reaction, the PCR product was subjected to 1% agarose gel electrophoresis, and a fragment of about 500bp was recovered and quantified by ultraviolet absorption. (2) The PCR reaction was carried out in accordance with Table 11, and the CD8a range-TM-41 BB-CD3Z fragment was amplified using the primers shown in Table 10.

Table 10

Primer(s)	Sequence(s)	Sequence number
			CD8H2-F	cgacgccagcgccgcgaccacc	SEQ ID No.16
Vector-R	tcgataagcttgatatcg	SEQ ID No.17

TABLE 11

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

(3) Mu.g of HD SIN03 CD19 41BBz (ka) plasmid was digested with BamHI and EcoRI, and reacted in a water bath at 37℃for 2 hours to recover a large fragment of the vector backbone.

(4) 2 fragments recovered in the steps 1 and 2 and the vector obtained in the step 3 are connected by using recombinase, a recombination reaction system is shown in a table 12, water bath reaction is carried out for 0.5h at 37 ℃ after preparation, the mixture is transformed into competent cells of escherichia coli stbl3 according to a conventional method, monoclonal is selected from a solid culture medium, overnight culture is carried out, PCR identification is carried out, a PCR reactant is prepared as shown in a table 13, a PCR program is shown in a table 14, positive clones are selected after PCR is finished, further sequencing identification is carried out, and a sequencing result accords with expectations.

Table 12

Reagent(s)	Dosage of
		HD SIN03 CD19 41BBz(ka)	150ng
CD8a signal GPC3 VHH	15ng
		CD8a hinge-TM-41BB-CD3Z	15ng
5x CE MultiS buffer (from Nuo Wei Zan)	2μL
		Exnase MultiS (from North Renzan)	1μL
PCR-grade pure water	Up to 10μL
		Total volume of	10μL

TABLE 13

TABLE 14

Example 6

The embodiment performs the packaging of lentiviruses, comprising the steps of:

(1) At 1.6X10 ⁷ Cell count 293T cells were seeded in 15cm dishes at 37℃with 5% CO ₂ Overnight incubation preparing packaging virus, medium DMEM, 10% fetal bovine serum (fetal bovine serum, FBS) added;

(2) 30. Mu.g of the lentiviral vector constructed in example 5 was dissolved in 2000. Mu.L of serum-free DMEM medium with 12.5. Mu.g of helper plasmid gag/pol and 10. Mu.g of envelope plasmid VSVg, respectively, and mixed well;

(3) 157.5. Mu.g PEI (1. Mu.g/. Mu.L) was dissolved in 2000. Mu.L serum-free DMEM medium and vortexed at 1000rpm for 5 seconds and incubated at 25℃for 5min;

(4) Formation of transfection complexes: adding PEI mixed solution into the DNA mixed solution, immediately vortex mixing or gently mixing after adding, and incubating for 20min at 25 ℃;

(5) 4mL of the transfection complex is dripped into a 15cm dish containing 25mL of DMEM culture medium, and after 4 hours, the fresh culture medium is replaced;

(6) After 48h, virus liquid supernatants were collected to obtain 10 lentiviruses expressing chimeric antigen receptors of different structures.

Example 7

This example was subjected to lentiviral concentration.

The virus supernatant prepared in example 6 was filtered with a 0.45 μm filter membrane and then collected in a 50mL centrifuge tube, 1/4 PEG-NaCl virus concentrate was added, mixed upside down, and left at 4℃overnight; centrifuging at 4 ℃ at 3500rpm for 30min; removing supernatant, adding RPMI 1640 medium (containing 10% FBS), and dissolving the resuspended viral pellet; the concentrated lentiviral suspension was split into 50. Mu.L portions each, stored in finished tubes and stored at-80 ℃.

Example 8

This example performs lentiviral titer detection.

mu.L of Jurkat cells (1X 10) ⁵ Individual cells) cells were seeded in 48-well plates; the lentivirus concentrated in example 7 was added to the cell suspension at 1. Mu.L, 0.2. Mu.L and 0.04. Mu.L, respectively, and polybrene was added to a final concentration of 5. Mu.g/mL; 37 ℃,5% CO ₂ After overnight incubation, fresh medium was changed; 72h after infection, 400 Xg was centrifuged for 5min, the supernatant was discarded to collect cells, 100. Mu.L PBS+2% FBS was added to resuspend the cells, 1. Mu.g of iF488-anti-VHH cocktail antibody (from gold Style) was added, and incubated on ice for 30min; after PBS+2% FBS is washed for 2 times, 300 mu L of PBS+2% FBS is added to resuspend cells, and a flow cytometer is used for detecting the infection efficiency; taking a sample of cells with a positive rate of 15%, and calculating the titer (TU/mL) =the number of cells (10) ⁵ ) X positiveRate/virus volume (mL).

Example 9

This example uses lentiviruses to transduce T lymphocytes.

Diluting the anti-human CD3 antibody and the anti-human CD28 antibody with PBS (phosphate buffered saline) to obtain final concentrations of 1 mug/mL and 0.5 mug/mL respectively, coating an orifice plate, and standing at 4 ℃ in a refrigerator overnight; discarding the antibody coating liquid in the pore plate, and washing twice with 1mL PBS; human PBMC were adjusted to a density of 1X 10 with T cell media (X-VIVO+10% FBS+IL-2 (300U/mL)) ⁶ /mL, then inoculated into CD3 and CD28 antibody coated well plates for activation for 48h; the activated T cells were collected and the cell density was adjusted to 1X 10 ⁶ Per mL, lentivirus prepared in example 7 was added at a multiplicity of infection (multiplicity of infection, MOI) =10, polybrene was added to a final concentration of 5 μg/mL; at 37℃with 5% CO ₂ After overnight incubation in the environment, fresh medium was changed and passaged every 2 days.

Example 10

In this example, GPC3 overexpressing cell lines (Huh 7-GPC3 and Sk-hep1-GPC 3) were constructed, comprising the steps of:

1. construction of GPC3 overexpression plasmid:

the PCR reaction system was prepared in accordance with Table 16, and GPC3 CDS fragment and P2A-PuroR fragment were amplified, respectively, using the primers shown in Table 15.

TABLE 15

Table 16

The above reagents were derived from TOYOBO inc.

After completion of the preparation, the reaction was performed according to the PCR procedure shown in Table 17.

TABLE 17

After the reaction, the PCR product was subjected to 1% agarose gel electrophoresis, and a GPC3 CDS fragment of about 1800bp and a P2A-PuroR fragment of about 665bp were recovered and quantified by an ultraviolet absorption method.

(2) After 4. Mu.g of pSIN CEA 1A6-41BBz (kanaR) plasmid was digested with BamHI and EcoRI, and reacted in a water bath at 37℃for 2 hours, the large fragment of the vector backbone was recovered.

(3) The 2 fragments recovered in the step 1 and the vector obtained in the step 2 are connected by using recombinase, a recombination reaction system is shown in a table 18 (the reagent is purchased from the Norfluzan), the mixture is subjected to water bath reaction at 37 ℃ for 0.5h after preparation, the mixture is transformed into competent cells of escherichia coli stbl3 according to a conventional method, single clones are selected from a solid culture medium, the single clones are cultured overnight, PCR identification is carried out, the preparation of PCR reactants is shown in a table 13, the PCR procedure is shown in a table 14, positive clones are selected after the PCR is finished, further sequencing identification is carried out, and the sequencing result meets the expectations.

TABLE 18

2. Packaging of GPC3 over-expressed lentiviruses:

GPC3 overexpression packaging procedure for lentivirus virus was the same as in example 6.

3. Construction of GPC3 overexpressing cell lines:

(1) Huh7 cells and Sk-hep1 cells (both purchased from Shanghai cell Bank of the national academy of sciences) were digested and counted, resuspended in DMEM+10% FBS medium, and 1X 10 was taken ⁵ Cells were plated in 6-well plates, 1mL of GPC3 overexpressing lentivirus was added, medium was supplemented to 2mL, polybrene was added to final concentrationThe temperature was 5. Mu.g/mL, 37℃and 5% CO ₂ Culturing overnight;

(2) The virus-infected Huh7 cells and Sk-hep1 cells were replaced with fresh DMEM+10% FBS medium 2mL, and the temperature was continued at 37℃with 5% CO ₂ Culturing;

(3) After 3 days of culture, positive cells were selected by adding puromycin at a final concentration of 0.5. Mu.g/mL and 1. Mu.g/mL, respectively;

(4) After 3 days of culture, the virus-infected Huh7 cells and Sk-hep1 cells were passaged, and selection was continued by adding puromycin at a final concentration of 1. Mu.g/mL;

(5) The passage of virus-infected Huh7 cells and Sk-hep1 cells and the puromycin selection were continued for a period of 3-4 days, and after a total screening time of about 2 weeks, GPC3 expression was detected and cell cryopreservation was performed.

Example 11

This example performs T lymphocyte chimeric antigen receptor expression comprising the steps of:

1. 5 days after infection, 3X 10 is taken ⁵ Centrifuging at 4 ℃ for 5min at 400 Xg, discarding the supernatant, and washing once with PBS+2% FBS;

2. cells were resuspended in 100. Mu.L PBS+2% FBS, 1. Mu.g of iF488-anti-VHH cocktail antibody was added and incubated on ice for 30min; after PBS+2% FBS was washed 2 times, 300. Mu.L of PBS+2% FBS was added to resuspend cells, and the infection efficiency was examined by a flow cytometer using uninfected T cells as a control, and the results are shown in FIG. 5, in which the infected CAR-T cells have a significant positive cell population, indicating that 10 kinds of CAR-T cells expressing chimeric antigen receptors of different structures, labeled GPC3-VHH-B6, GPC3-VHH-B51, GPC3-VHH-B89, GPC3-VHH-B108, GPC3-VHH-B168, GPC3-VHH-B174 (V18), GPC3-VHH-B174 (V32), GPC3-VHH-B174 (V33) and GPC3-VHH-B174 (V34), respectively, were successfully constructed.

Example 12

In vitro toxicity experiments were performed on CAR-T cells in this example.

1. Target cell inoculation:

293T (GPC 3-, available from ATCC), huh7-GPC3 (GPC 3+), hepG2 (GPC 3+), available from Shanghai Engineers) was used as target cells, and the target cells were adjustedThe concentration is 1 multiplied by 10 ⁵ 100. Mu.L of the solution is inoculated into a 96-well plate;

2. effector cell inoculation:

GPC3 CAR-T and control T cells are effector cells, and CAR-T cells and control T cells are added to a 96-well plate according to the effective target ratio of 0.3:1, 1:1 and 3:1;

3. each group is provided with 3 compound holes, and the average value of the 3 compound holes is taken. Wherein each experimental group and each control group were as follows:

experimental group: each target cell + CAR-T;

control group 1: target cells release LDH maximally;

control group 2: target cells spontaneously release LDH;

control group 3: effector cells spontaneously release LDH;

4. the detection method comprises the following steps:

after 18h of co-culture of effector cells with target cells, cytoTox 96 non-radioactive cytotoxicity detection kit (Promega corporation) was used.

The method is a colorimetric-based detection method, and reflects the cell lysis degree by detecting the content of Lactate Dehydrogenase (LDH). LDH is a stable cytoplasmic enzyme that is released upon cell lysis in a manner substantially similar to that of 51Cr in radioassays. The released LDH medium supernatant can be detected by a coupled enzymatic reaction in which LDH converts a tetrazolium salt (INT) to red formazan. The amount of red product produced is proportional to the number of cells lysed.

Reference is made in particular to the instructions for the CytoTox 96 nonradioactive cytotoxicity test kit.

5. The cytotoxicity calculation formula is:

the results are shown in fig. 6A-6C, the constructed CAR-T cells have no obvious killing effect on GPC3 negative cells, and have stronger killing activity on GPC3 positive tumor cells.

Example 13

This example detects CAR-T cytokine secretion.

1. Cell culture supernatant

The cell culture of example 12, with an effective target ratio of 1:1, was centrifuged at 400 Xg for 10min to remove the precipitate, and the supernatant was stored at-80℃for examination.

2. Reagent preparation

Detection was performed using a Union ELISA kit (product number: human gamma interferon ELISA kit: EK 180-96), all reagents and samples were returned to 25℃before detection, and 1 Xwash solution, 1 Xdetection buffer, and detection antibody were prepared according to the instructions of use.

3. Standard substance and sample preparation

Standard substance: standard stock was 2-fold diluted with 5%1640 medium for a total of 8 dilution gradients, including zero concentration. Sample: samples were diluted in ratio using 5%1640 medium.

4. Detection step

(1) Soaking the ELISA plate: adding 300 mu L of 1 Xwashing liquid, standing and soaking for 30s, removing the washing liquid, and then beating the micro-porous plate on water-absorbing paper;

(2) Adding a standard substance: standard wells were filled with 100 μl of 2-fold diluted standard and blank wells were filled with 100 μl of 5%1640 medium;

(3) Adding a sample: sample wells were added with 100 μl of cell culture supernatant;

(4) Adding a detection antibody: 50. Mu.L of diluted detection antibody (1:100 dilution) was added to each well;

(5) Incubation: sealing plates by using sealing plates, vibrating at 300rpm, and incubating at 25 ℃ for 2 hours;

(6) Washing: liquid was discarded, and 300. Mu.L of wash solution was added to wash the plate 6 times per well;

(7) And (3) enzyme adding and incubation: mu.L of diluted horseradish peroxidase-labeled streptavidin (1:100 dilution) was added to each well;

(8) Incubation: using a new sealing plate membrane sealing plate, oscillating at 300rpm, and incubating at 25 ℃ for 45min;

(9) Washing: repeating step (6);

(10) And (3) color development of the substrate: 100 mu L of chromogenic substrate TMB is added into each hole, and incubated for 15min at 25 ℃ in the dark;

(11) Adding a stop solution: adding 100 mu L of stop solution into each hole, and fully and uniformly mixing;

(12) Detecting and reading: the dual wavelength detection was performed using an enzyme-labeled instrument, and the OD at the 450nm maximum absorption wavelength and the 630nm reference wavelength was measured, and the OD after calibration was measured at 450nm minus the measured at 630 nm.

The IFN-gamma factor secretion results are shown in figure 7, wherein the results are shown in the figure 7, the results are that the results are a CAR-T cell independent culture, a trace amount of IFN-gamma factor is detected in the results of the spontaneous culture and the CAR-T culture, and the results are that the Huh7-GPC3, sk-hep1-GPC3 and HepG2 respectively detect the IFN-gamma factor with higher content in the CAR-T culture, so that the CAR-T cell constructed by the invention can also release cytokines to tumor cells positive for GPC3 to play a killing function, has high specificity and has no obvious cytokine secretion to GPC3 negative cells.

In summary, the anti-GPC 3 antibody is screened and prepared, the anti-GPC 3 antibody has high affinity and specificity, can effectively target tumor antigen GPC3, and can be used for preparing chimeric antigen receptor cells by utilizing the anti-GPC 3 anti-chimeric antigen receptor, wherein the chimeric antigen receptor cells can effectively kill tumor cells, can secrete various cytokines to exert killing functions, and have high specificity.

The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. Further, various modifications of the methods set forth herein, as well as variations of the methods of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.

Claims (18)

1. An anti-GPC 3 antibody, wherein the anti-GPC 3 antibody comprises a heavy chain variable region, the anti-GPC 3 antibody having one or more of the following technical features;

<1> the heavy chain variable region comprises CDR-H1 having an amino acid sequence as shown in SEQ ID No. 1;

<2> the heavy chain variable region comprises CDR-H2 having the amino acid sequence shown in SEQ ID No. 2;

<3> the heavy chain variable region includes CDR-H3 having an amino acid sequence shown in SEQ ID No. 3.

2. The anti-GPC 3 antibody of claim 1, wherein the heavy chain variable region further comprises a framework region comprising framework regions FR1 to FR4, the amino acid sequences of framework regions FR1 to FR4 being as set forth in SEQ ID nos. 4 to 7.

3. The anti-GPC 3 antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region is as set forth in seq id No. 8.

4. The anti-GPC 3 antibody of claim 1, wherein the anti-GPC 3 antibody is a nanobody or a single chain antibody.

5. Use of an anti-GPC 3 antibody according to any one of claims 1 to 4 in the manufacture or screening of a medicament for the treatment of a tumour.

6. The use according to claim 5, wherein the tumor is a GPC 3-expressing tumor; preferably, the tumor is selected from liver cancer, ovarian cancer, lung cancer, melanoma, gastric cancer or thyroid cancer; preferably, the tumor therapeutic agent is a chimeric antigen receptor cell.

7. An isolated polypeptide comprising a transmembrane domain, an intracellular domain and an extracellular domain comprising the anti-GPC 3 antibody of any one of claims 1 to 4.

8. The polypeptide of claim 7, further comprising any one or more of the following features:

1) The polypeptide is a chimeric antigen receptor;

2) The transmembrane domain is selected from any one or more of a CD8 alpha transmembrane region, a CD28 transmembrane region and a DAP 10 transmembrane region;

3) The intracellular domain comprises a costimulatory molecule domain and/or a signaling domain; preferably, the signal transduction domain comprises an immunoreceptor tyrosine activation motif; more preferably, the immunoreceptor tyrosine activation motif is selected from the group consisting of cd3ζ; preferably, the co-stimulatory molecule is selected from any one or a combination of at least two of 4-1BB, CD28, OX40, ICOS, DAP 10;

4) The extracellular domain comprises a signal peptide, an anti-GPC 3 antibody, and a hinge region; preferably, the signal peptide comprises a CD8 a signal peptide; preferably, the hinge region is selected from the group consisting of a CD8 a hinge region;

5) The polypeptide sequentially comprises a CD8 alpha signal peptide, an anti-GPC 3 nanometer antibody, a CD8 alpha hinge region, a CD8 alpha transmembrane region, a co-stimulatory molecule domain and a CD3 zeta signal domain from the N end to the C end.

9. An isolated polynucleotide encoding the anti-GPC 3 antibody of any one of claims 1 to 4 or the polypeptide of any one of claims 7 to 8.

10. A nucleic acid construct comprising a polynucleotide encoding the isolated polypeptide of any one of claims 7-8.

11. The nucleic acid construct of claim 10, wherein the nucleic acid construct is any one of a lentiviral vector, a retroviral vector, or an adeno-associated viral vector.

12. A lentivirus, wherein the lentivirus is packaged by a nucleic acid construct according to claim 10 or 11.

13. A lentiviral vector system comprising the nucleic acid construct of claim 10 or 11 and a helper plasmid or host cell.

14. A chimeric antigen receptor immune cell expressing the isolated polypeptide of any one of claims 7-8 in membrane-bound form.

15. The chimeric antigen receptor immune cell according to claim 14, wherein the immune cell is selected from any one of T lymphocytes, B lymphocytes, NK cells, mast cells or macrophages.

16. Use of an isolated polypeptide according to any one of claims 7 to 8, a chimeric antigen receptor immune cell according to any one of claims 14 to 15, in the preparation or screening of a medicament for the treatment of a tumor.

17. The use according to claim 16, wherein the tumor is a GPC 3-expressing tumor; preferably, the tumor is selected from liver cancer, ovarian cancer, lung cancer, melanoma, gastric cancer or thyroid cancer.

18. A pharmaceutical composition comprising the chimeric antigen receptor immune cell of any one of claims 14 to 15 and a pharmaceutically acceptable carrier or adjuvant.

Images