CN111040036A - anti-GPC 3 monoclonal antibody, immune effector cell modified by same and application thereof - Google Patents

Abstract

The invention relates to an anti-GPC 3 monoclonal antibody, an immune effector cell modified by the same and application thereof. Discloses an antibody for specifically recognizing tumor cell GPC3, which has good specificity, is particularly suitable for preparing immune effector cells based on CAR technology, and can also be applied to preparing various targeting antitumor drugs and drugs for diagnosing tumors.

Description

anti-GPC 3 monoclonal antibody, immune effector cell modified by same and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an anti-GPC 3 monoclonal antibody, an immune effector cell modified by the monoclonal antibody and application of the monoclonal antibody.

Background

Glypican 3(GPC3) is a cell surface membrane protein belonging to a member of the family of heparin proteoglycans, and its gene encodes a 70kD core protein which is cleaved by the proprotein convertase Furin to yield a 40kD amino-terminal subunit and a 30kD carboxy-terminal subunit with two Heparan Sulfate (HS) chains. The GPC3 protein is anchored to the cell membrane by a glycosylphosphatidylinositol anchor protein (GPI).

Chimeric antigen receptor T cell (CAR-T cell) immunotherapy is one of the more effective treatments for malignant tumors at present. The basic principle is to eliminate cancer cells by using the modified immune cells of a patient, and the method is different from the prior treatment method in that the method is a cell therapy instead of the traditional drug treatment.

The international cell therapy association (interna) in 2012 indicates that biological immune cell therapy has become a fourth means for treating tumors besides surgery, radiotherapy and chemotherapy, and will become a necessary means for treating tumors in the future. The immune cell therapy is to collect peripheral venous blood of a patient, separate peripheral blood mononuclear cells in a GMP laboratory, greatly expand immune effector cells with high-efficiency antitumor activity under the induction of various cytokines, and then return the cells into the body of the patient through intravenous injection, intradermal injection, intervention and the like so as to achieve the purposes of enhancing the immune function of the patient and killing tumor cells.

However, when a tumor is found clinically, the tumor is generally in the middle and late stages, at the moment, the tumor cells in the body of a patient are dominant, the immune function of the body is seriously damaged, the DC cell function is damaged under the microenvironment of the immune system, the T cell activation efficiency is low, the ability of attacking cancer cells is not enough, and the accuracy is not high enough; in addition, tumor cells escape from immune cell attack by escape mechanisms that either underexpress or do not express MHC molecules. There is a need to create a precisely guided, precisely targeted immune cell weapon that overcomes the MHC-mediated oncocidal mechanism. Thus, targeted anti-tumor cell CAR-T technology is in force.

In the cellular therapy of CAR-T, which is transfected into T cells of patients after splicing the gene sequence of the variable region of an antibody recognizing CD19 with the intracellular region sequence of a lymphocyte immunoreceptor, CD19 was used as the earliest therapeutic target for treating B-cell lymphoma, the formed CD19 CAR-T cells can specifically attack B cells with CD19 antigen, thereby promoting the body to eliminate tumor cells with CD19 antigen.

Currently, although CAR-T cell based therapy techniques have been the focus of research in the art, the difficulties of CAR-T cell therapy research are: first, it is not easy to find a suitable target; secondly, aiming at a specific target, an extracellular binding region which has ideal targeting property and is suitable for immune cell modification is not easy to obtain; third, the modified immune cells are less viable or proliferation is affected. In view of the above difficulties, although there are many tumor-specific expression markers identified in the art, there are still some CAR-T cell therapy technologies that can be successfully developed and used clinically and have clinically ideal effects, and further development, optimization and improvement are needed.

Disclosure of Invention

The invention aims to provide an anti-GPC 3 monoclonal antibody, an immune effector cell modified by the same and application thereof.

In a first aspect of the invention there is provided a monoclonal antibody specific against GPC3, the antibody having a light chain variable region selected from the group consisting of: 1-112 of SEQ ID NO. 1, 1-112 of SEQ ID NO. 3, 1-112 of SEQ ID NO. 4, 1-112 of SEQ ID NO. 7; and a heavy chain variable region selected from the group consisting of: positions 1-121 in SEQ ID NO. 2, positions 1-121 in SEQ ID NO. 5, positions 1-121 in SEQ ID NO. 6, and positions 1-121 in SEQ ID NO. 8.

In a preferred embodiment, the antibody is a murine antibody, a humanized antibody, a chimeric antibody, or a scFv antibody comprising the heavy chain variable region and the light chain variable region of said murine antibody, humanized antibody, chimeric antibody.

In another preferred embodiment, the antibody is a humanized antibody having a light chain variable region selected from the group consisting of: positions 1-112 in SEQ ID NO. 3 and positions 1-112 in SEQ ID NO. 4; and a heavy chain variable region selected from the group consisting of: positions 1-121 in SEQ ID NO. 5 and positions 1-121 in SEQ ID NO. 6; preferably, the antibody has the light chain variable region at positions 1-112 of SEQ ID NO. 3 and the heavy chain variable region at positions 1-121 of SEQ ID NO. 5.

In another preferred embodiment, the antibody is a murine antibody, which is produced from hybridoma cell line CCTCC No: c2018156.

In another preferred embodiment, the constant regions of the humanized antibody are human kappa chain and IgG1 constant region sequences.

In another aspect of the invention, nucleic acids encoding the antibodies are provided.

In another aspect of the invention, there is provided an expression vector comprising said nucleic acid.

In another aspect of the invention, there is provided a host cell comprising said expression vector or genome having said nucleic acid integrated therein.

In another aspect of the present invention, there is provided a use of the antibody of any one of the preceding claims for the preparation of a targeting agent, an antibody drug conjugate or a multifunctional (e.g. bifunctional) antibody for specifically targeting tumor cells expressing GPC 3; or for the preparation of an agent for the diagnosis of a tumour which expresses (overexpresses) GPC 3; or for preparing an immunohistochemical detection reagent; or for the preparation of chimeric antigen receptor-modified immune cells; preferably, the immune cell comprises: t lymphocytes, NK cells, or NKT lymphocytes.

In another aspect of the invention, there is provided a chimeric antigen receptor expressed on the surface of an immune cell, comprising an extracellular region comprising an antibody as described in any one of the preceding, a transmembrane region and an intracellular signaling region.

In a preferred embodiment, the transmembrane region comprises the transmembrane regions of CD8Hinge and CD 28; or said intracellular signaling region has an element selected from the group consisting of: 4-1BB, CD3Zeta, Fc ε RI γ, CD27, CD28, CD137, CD134, IL-12, or a combination thereof.

In another preferred embodiment, the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain and an intracellular signaling domain connected in this order as follows: scFv comprising the variable region of the light chain variable region or the heavy chain variable region of any of the antibodies described above, CD8Hinge, the transmembrane region of CD28, 4-1BB, CD3 Zeta; preferably, the kit also comprises GMCSF signal peptide and EGFR domains III-IV; more preferably, the GMCSF signal peptide and EGFR D III-D IV are linked to the intracellular signaling region via a 2A peptide (preferably selected from F2A, E2A, T2A, P2A).

In another preferred embodiment, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO. 9, or 1-492 of SEQ ID NO. 9.

In another aspect of the invention there is provided a nucleic acid encoding a chimeric antigen receptor as described in any one of the preceding.

In another aspect of the invention, there is provided an expression vector comprising said nucleic acid.

In another aspect of the invention, there is provided a virus comprising said vector or said nucleic acid encoding a chimeric antigen receptor.

In a preferred embodiment, the virus is a virus (genetically engineered virus) that is safe for organisms (particularly humans) or clinically safe.

In another preferred embodiment, the virus is a lentivirus or an adenovirus.

In another aspect of the present invention there is provided the use of a chimeric antigen receptor as described in any one of the preceding, or a nucleic acid as described, or an expression vector as described, or a virus as described, in the preparation of a genetically modified immune cell specifically targeting a tumour expressing GPC 3.

In another aspect of the invention, there is provided a genetically modified immune cell into which said nucleic acid, or said expression vector or said virus, has been introduced; or a surface thereof expresses said chimeric antigen receptor.

In a preferred embodiment, the immune cell comprises: t lymphocytes, NK cells or NKT cells.

In another aspect of the invention, the use of the genetically modified immune cell is provided for the preparation of a medicament for inhibiting a tumor, wherein the tumor is a tumor expressing (overexpressing) GPC 3.

In another aspect of the invention, there is provided an immune conjugate comprising: the antibody as described above; and a functional molecule attached thereto (including covalently linked, coupled, attached, adsorbed); the functional molecule is selected from: a molecule that targets a tumor surface marker, a molecule that inhibits a tumor, a molecule that targets a surface marker of an immune cell, or a detectable marker.

In a preferred embodiment, the molecule targeting the tumor surface marker is an antibody or ligand that binds to the tumor surface marker.

In another preferred embodiment, the tumor-inhibiting molecule is an anti-tumor cytokine or an anti-tumor toxin; preferably, the cytokines include (but are not limited to): IL-12, IL-15, IFN-beta, TNF-alpha.

In another preferred embodiment, the detectable label includes (but is not limited to): fluorescent markers, chromogenic markers.

In another preferred embodiment, the molecule targeting the surface marker of the immune cell is an antibody or ligand that binds to the surface marker of the immune cell; preferably, the immune cell surface markers include (but are not limited to): CD3, CD16, CD 28.

In another preferred embodiment, the molecule targeting the surface marker of the immune cell is an antibody that binds to a T cell surface marker, which forms a bifunctional antibody (BiTE) involved in T cell with the anti-GPC 3 monoclonal antibody of the present invention.

In another preferred embodiment, the antibody that binds to an immune cell surface marker is an anti-CD3 antibody.

In another preferred embodiment, the immune conjugate is a fusion polypeptide, and the anti-GPC 3 monoclonal antibody of the present invention and a functional molecule linked thereto further comprises a linking peptide.

In another preferred embodiment, the linker peptide has the sequence (GlyGlyGlyGlySer)_nWherein n is an integer of1 to 5; more preferably, n is 3.

In another aspect of the invention there is provided a nucleic acid encoding said immune conjugate.

In a further aspect of the invention there is provided the use of said immune conjugate for the preparation of an anti-tumour agent, or for the preparation of an agent for the diagnosis of a tumour which expresses GPC 3; or for the preparation of chimeric antigen receptor-modified immune cells; preferably, the immune cell comprises: t lymphocytes, NK cells, or NKT lymphocytes.

In another aspect of the present invention, there is provided a pharmaceutical composition comprising: an antibody as described in any of the preceding or a nucleic acid encoding the antibody; or a chimeric antigen receptor as described in any of the preceding or a nucleic acid encoding the chimeric antigen receptor; or said immune conjugate or a nucleic acid encoding said conjugate; or said genetically modified immune cell.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1 is a scanning image of a monoclonal antibody Western Blot detecting human GPC3 protein. Wherein, lanes 1-7 are protein Marker, liver cancer cell Huh7, LM3, HepG2, PLC, PVTT and SMMC-7721(7721) lysate respectively. Where "→" identifies endogenous GPC 3.

FIG. 2 is a tissue section diagram of monoclonal antibody GPC3 immunohistochemical detection of GPC3 protein in human liver cancer tissue. Wherein, A is liver cancer tissue (400 x) detected by monoclonal antibody immunohistochemistry; b is liver cancer tissue detected by monoclonal antibody immunohistochemistry (400X).

FIG. 3, competitive binding experiments of GPC3 antigen and humanized antibody.

FIG. 4 shows the restriction electrophoresis of the constructed plasmid.

FIG. 5 is a schematic diagram of the structure of a lentiviral expression vector for a GPC3 chimeric antigen receptor. Wherein VL is the light chain variable region and VH is the heavy chain variable region.

Figure 6, qPCR standard curve.

Figure 7, flow cytometry detection of T lymphocyte surface CAR protein expression levels.

Fig. 8, photograph of in vitro killing of HepG2 target cells by T lymphocytes.

FIG. 9, in vitro killing of HepG2 target cells by T lymphocytes; the experiment was repeated 2 times, and the results of 2 times were consistent.

FIG. 10, IL2 secretion levels after co-culture of T lymphocytes with target cells; the experiment was repeated 2 times, and the results of 2 times were consistent.

FIG. 11, IFN-. gamma.secretion levels after co-culture of T lymphocytes with target cells; the experiment was repeated 2 times, and the results of 2 times were consistent.

FIG. 12 cell proliferation assay 1-3 days after co-culture of T lymphocytes with target cells.

Detailed Description

The inventor obtains an antibody for specifically recognizing the tumor cell GPC3 through deep research screening and artificial modification, the antibody has good specificity, is particularly suitable for preparing immune effector cells based on CAR technology, and can be applied to preparing various targeting antitumor drugs and drugs for diagnosing tumors.

anti-GPC 3 antibody

The invention aims to optimize a specific antibody aiming at GPC3, obtains a monoclonal antibody with good performance through repeated screening, and on the basis, the invention further carries out amino acid mutation and humanization transformation to find an anti-GPC 3 monoclonal antibody with more excellent effect, and the monoclonal antibody has good selective binding capacity and is particularly suitable for preparing immune effector cells based on CAR technology.

The antibodies of the invention can be intact immunoglobulin molecules, as well as antigen-binding fragments, including but not limited to Fab fragments, Fd fragments, Fv fragments, F (ab')₂Fragments, Complementarity Determining Region (CDR) fragments, single chain antibodies (scFv), domain antibodies, bivalent single chain antibodies, single chain phage antibodies, bispecific double chain antibodies, triple chain antibodies, quadruple chain antibodies.

The antigen binding properties of an antibody can be described by 3 specific regions located in the variable regions of the heavy and light chains, called Complementarity Determining Regions (CDRs), which separate the variable regions into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction.

In a preferred embodiment of the present invention, the antibody is a humanized antibody, preferably having a light chain variable region selected from the group consisting of: positions 1-112 in SEQ ID NO. 3 and positions 1-112 in SEQ ID NO. 4; and a heavy chain variable region selected from the group consisting of: positions 1-121 in SEQ ID NO. 5 and positions 1-121 in SEQ ID NO. 6; more preferably, the antibody has the light chain variable region at positions 1-112 of SEQ ID NO. 3 and the heavy chain variable region at positions 1-121 of SEQ ID NO. 5. The mouse-derived monoclonal antibody can be used as a foreign protein to enter a human body, so that the human immune system can generate HAMA reaction (human anti-mouse antibody reaction), and certain risk exists, so that the antibody humanization can avoid the induction of the HAMA reaction, and the safety of the monoclonal antibody is improved.

Another aspect of the invention includes functional variants of the antibodies described herein. If the variant competes with the parent antibody for specific binding to SEQ ID NO. 11 and its ability to recognize tumor cell GPC3 is close to that of the specific antibody provided in the examples of the invention. The functional variants may have conservative sequence modifications, including nucleotide and amino acid substitutions, additions and deletions. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and can comprise natural as well as non-natural nucleotides and amino acids. Preferably, the sequence modification occurs in a region other than the CDR regions of the antibody.

Chimeric antigen receptor and genetically modified immune cell

The research of the inventor shows that the anti-GPC 3 monoclonal antibody has good selective binding capacity, is particularly suitable for preparing immune effector cells based on CAR technology, and the obtained immune effector cells have high killing efficiency on target cells; furthermore, it is more surprising that the immune effector cells obtained based on the humanized monoclonal antibody of the present invention are capable of producing large amounts of cytokines such as IL-2 and IFN- γ, at levels much higher than those of uninfected T cells and conventional CAR technology-based immune effector cells.

The present invention provides a chimeric antigen receptor expressed on the surface of an immune effector cell (immune cell), said chimeric antigen receptor comprising, linked in sequence: an extracellular binding region, a transmembrane region, and an intracellular signaling region, wherein the extracellular binding region comprises an antibody of the invention. The chimeric antigen receptor is expressed on the surface of immune effector cells, so that the immune effector cells have highly specific cytotoxicity on tumor cells expressing GPC 3.

As used herein, the term "immune cell" is used interchangeably with "immune effector cell" and includes: t lymphocytes, NK cells, NKT cells, or the like; preferably, it is a T lymphocyte.

In a preferred embodiment of the present invention, the chimeric antigen receptor comprises an antibody which is a single chain antibody linked to a transmembrane region of CD8 or CD28 (preferably, the transmembrane region of CD 28) via a CD8hinge region, followed by an intracellular signal region.

The invention also includes nucleic acids encoding the chimeric antigen receptors. The present invention also relates to variants of the above polynucleotides which encode polypeptides having the same amino acid sequence as the present invention or fragments, analogs and derivatives of the polypeptides.

The transmembrane region of the chimeric antigen receptor may be selected from the transmembrane regions of proteins such as CD8 or CD28 the human CD8 protein is a heterodimer consisting of either αβ or γ δ chains in a preferred embodiment of the invention, the transmembrane region is the transmembrane region of CD28 furthermore, the CD8 α hinge region (hinge) is a flexible region, and thus, CD8 or CD28 and the transmembrane region plus the hinge region are used to link the target scFv of the chimeric antigen receptor CAR to the intracellular signaling region.

The intracellular signaling region may be selected from 4-1BB, CD3Zeta, Fc ε RI γ, CD27, CD28, CD137, CD134, or a combination thereof. The CD3 molecule consists of five subunits, of which CD3Zeta subunit (also known as CD3 Zeta) contains 3 ITAM motifs, which are important signaling regions in the TCR-CD3 complex. CD3 δ Z is a truncated CD3 ζ sequence without an ITAM motif and is generally constructed as a negative control in the practice of the present invention. Fcsri γ is distributed primarily on the surface of mast cells and basophils, contains an ITAM motif and is similar in structure, distribution and function to CD3 ζ. In addition, as mentioned above, CD28, CD137, and CD134 are costimulatory signaling molecules, and the costimulation of their intracellular signaling segments upon binding to their respective ligands causes the sustained proliferation of immune effector cells (mainly T lymphocytes) and helps to increase the levels of cytokines such as IL-2 and IFN- γ secreted by immune effector cells, as well as to increase the survival cycle and anti-tumor effects of CAR immune effector cells in vivo.

In a preferred embodiment of the present invention, the chimeric antigen receptor may be linked in the following order: the variable region of the light chain of the antibody can be scFv of a variable region of a heavy chain, CD8Hinge, a transmembrane region of CD28, 4-1BB, CD3 Zeta; preferably, the kit also comprises GMCSF signal peptide and EGFR domains III-IV; more preferably, the GMCSF signal peptide and EGFRD III-D IV are linked to the intracellular signaling region via a 2A peptide (preferably selected from F2A, E2A, T2A, P2A).

In a preferred embodiment of the present invention, the lentiviral vector comprises an elongation factor-1 α (elongation factor-1 α -1 α) promoter regulating the expression of EGFR D III-D IV, and a segment comprising a target nucleic acid sequence encoding CAR is linked to the intracellular signaling region via a 2A sequence (preferably selected from F2A, E2A, T2A, and P2A), and the fragment of EGFR (epidermal growth factor receptor) DIII-IV receptor is expressed on the surface of T cells at the same time as GPC3 receptor is expressed, and serves as a positive selection marker and clearing antibody, thereby allowing clearance of CAR-T cells in vivo by ADCC (antibody-dependent cell-mediated cytotoxicity) of EGFR mAb, thus increasing the safety of use of the CAR-T.

The invention also provides genetically modified immune effector cells transduced with a nucleic acid of the invention or transduced with a recombinant plasmid of the invention comprising the nucleic acid, or a virus comprising the plasmid. Nucleic acid transduction methods conventional in the art, including non-viral and viral transduction methods, can be used in the present invention. Non-viral based transduction methods include electroporation and transposon methods. Recently, the Nucleofector nuclear transfectator developed by Amaxa corporation can directly introduce exogenous genes into cell nucleus to obtain high-efficiency transduction of target genes. In addition, based on the fact that transduction efficiency OF transposable subsystems such as Sleeping beauty transposons (Sleeping Beautysystems) or PiggyBac transposons and the like is greatly improved compared with that OF common electroporation, the combined application OF a nucleofector transfection instrument and the Sleeping beauty transposable subsystems has been reported [ Davies JK., ethyl. combining CD19redirection and allo-orientation generating mechanism-specific human hub T cells for the purpose OF exogenous cell thermal OF B-cell malignancies. cancer Res, 2010, 70(10): OF1-10 ]. In one embodiment of the invention, the method of transduction of immune effector cells to achieve genetic modification of a chimeric antigen receptor is a viral, e.g., retroviral or lentiviral, based transduction method. The method has the advantages of high transduction efficiency, stable expression of exogenous genes, shortened time for in vitro culture of immune effector cells to reach clinical level, etc. On the surface of the transgenic immune effector cell, the transduced nucleic acid is expressed on its surface by transcription and translation. In vitro cytotoxic experiments on various cultured tumor cells prove that the immune effector cells have a highly specific tumor cell killing effect (also called cytotoxicity). Thus, the nucleic acid encoding the chimeric antigen receptor protein, the plasmid containing the nucleic acid, the virus containing the plasmid and the transgenic immune effector cell transduced with the nucleic acid, the plasmid or the virus of the present invention can be effectively used for immunotherapy of tumors.

The invention also includes viruses comprising the vectors described above. The virus of the present invention includes a packaged virus having infectivity, and also includes a virus to be packaged which contains components necessary for packaging the virus having infectivity. Other viruses known in the art and their corresponding plasmid vectors that can be used to transfer foreign genes into immune effector cells can also be used in the present invention. When used for the purpose of disease treatment or for the preparation of a medicament for the treatment of disease, the art knows what viruses to select for safety.

The immune cell can further introduce an exogenous cytokine coding sequence; such cytokines include, but are not limited to: IL-12, IL-15 or IL-21, etc. These cytokines have immunomodulatory or anti-tumor activity, and can enhance the function of effector T cells and activated NK cells, or directly exert anti-tumor effects. Thus, it will be appreciated by those skilled in the art that the use of these cytokines helps the immune cells to function better.

The immune cells of the present invention may also express another chimeric antigen receptor in addition to the chimeric antigen receptor described above.

The immune cells of the invention may also express chemokine receptors; such chemokine receptors include, but are not limited to, CCR 2. It will be appreciated by those skilled in the art that the CCR2 chemokine receptor allows CCR2 in vivo to compete for binding, and is advantageous for blocking tumor metastasis.

The immune cells of the invention can also express siRNA capable of reducing PD-1 expression or protein capable of blocking PD-L1. It will be appreciated by those skilled in the art that competitive blockade of the interaction of PD-L1 with its receptor PD-1 is beneficial in restoring an anti-tumor T cell response, thereby inhibiting tumor growth.

The immune cells of the invention may also express a safety switch; preferably, the safety switch comprises: iCaspase-9 or RQR 8.

Immune conjugates

The invention also provides multifunctional immunoconjugates comprising the antibodies described herein and further comprising at least one other type of functional molecule. The functional molecule is selected from but not limited to: a molecule that targets a tumor surface marker, a molecule that inhibits a tumor, a molecule that targets a surface marker of an immune cell, or a detectable marker. The antibody and the functional molecule can form a conjugate through covalent connection, coupling, attachment, crosslinking and the like.

As a preferred mode, the immunoconjugate may comprise: the antibodies of the invention and at least one tumor surface marker targeting or tumor inhibiting molecule. The tumor-inhibiting molecule may be an anti-tumor cytokine or an anti-tumor toxin.

As a preferred mode, the immunoconjugate may comprise: antibodies of the invention and detectable labels. Such detectable labels include, but are not limited to: fluorescent markers, chromogenic markers; such as: enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and nonradioactive paramagnetic metal ions. More than one label may also be included. The label used to label the antibody for detection and/or analysis and/or diagnostic purposes depends on the particular detection/analysis/diagnostic technique and/or method used, e.g., immunohistochemical staining of (tissue) samples, flow cytometry, etc. Suitable labels are well known to those skilled in the art for detection/analysis/diagnostic techniques and/or methods known in the art.

As a preferred mode, the immunoconjugate may comprise: the antibodies of the invention and molecules targeting surface markers of immune cells. The molecules of the surface marker of the targeted immune cells can identify the immune cells, carry the antibody of the invention to reach the immune cells, and simultaneously, the antibody of the invention can target the immune cells to tumor cells, thereby triggering the immune cells to kill the tumor specifically.

As a means of chemically producing an immunoconjugate by conjugation, either directly or indirectly (e.g., through a linker), the immunoconjugate may be produced as a fusion protein comprising the antibody of the invention and a suitable other protein. Fusion proteins can be produced by methods known in the art, e.g., recombinantly by constructing a nucleic acid molecule comprising an in-frame nucleotide sequence encoding an antibody and a nucleotide sequence encoding a suitable marker, and then expressing the nucleic acid molecule.

In another aspect, the invention provides a nucleic acid molecule encoding at least one antibody, functional variant or immunoconjugate thereof of the invention. Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.

Pharmaceutical composition

The antibodies, immune conjugates comprising the antibodies, and genetically modified immune cells of the invention may be used to prepare pharmaceutical compositions or diagnostic reagents. The composition may further comprise a pharmaceutically acceptable carrier in addition to an effective amount of the antibody, immune conjugate or immune cell. The term "pharmaceutically acceptable" means that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human.

Specific examples of some substances that may serve as pharmaceutically acceptable carriers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; powdered gum tragacanth; 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; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, e.g.

Wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like.

The compositions of the present invention may be formulated into various dosage forms as desired, and may be administered by a physician in a dosage amount beneficial to the patient, depending on such factors as the type, age, weight and general condition of the patient, the mode of administration, and the like. Administration may be by injection or other therapeutic means, for example.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Example 1 preparation and characterization of murine anti-human GPC3 monoclonal antibody

1. Preparation of mouse anti-human GPC3 monoclonal antibody

Firstly, a mouse anti-human GPC3 monoclonal antibody is prepared by a hybridoma method, and the steps are as follows:

a. the GPC3 protein N-terminal fragment containing 1074 bases is amplified by PCR and inserted into a PH-CMV empty vector to construct PH-CMV-GPC3-NH₂' eukaryotic expression plasmid. The plasmid is transfected into eukaryotic cell strain, GPC3 molecule NH is expressed and purified₂' terminal protein.

The N-terminal 1074 base sequences of GPC3 (SEQ ID NO:11) were as follows:

ATGGCCGGGACCGTGCGCACCGCGTGCTTGGTGGTGGCGATGCTGCTCAGCTTGGACTTCCCGGGACAGGCGCAGCCCCCGCCGCCGCCGCCGGACGCCACCTGTCACCAAGTCCGCTCCTTCTTCCAGAGACTGCAGCCCGGACTCAAGTGGGTGCCAGAAACTCCCGTGCCAGGATCAGATTTGCAAGTATGTCTCCCTAAGGGCCCAACATGCTGCTCAAGAAAGATGGAAGAAAAATACCAACTAACAGCACGATTGAACATGGAACAGCTGCTTCAGTCTGCAAGTATGGAGCTCAAGTTCTTAATTATTCAGAATGCTGCGGTTTTCCAAGAGGCCTTTGAAATTGTTGTTCGCCATGCCAAGAACTACACCAATGCCATGTTCAAGAACAACTACCCAAGCCTGACTCCACAAGCTTTTGAGTTTGTGGGTGAATTTTTCACAGATGTGTCTCTCTACATCTTGGGTTCTGACATCAATGTAGATGACATGGTCAATGAATTGTTTGACAGCCTGTTTCCAGTCATCTATACCCAGCTAATGAACCCAGGCCTGCCTGATTCAGCCTTGGACATCAATGAGTGCCTCCGAGGAGCAAGACGTGACCTGAAAGTATTTGGGAATTTCCCCAAGCTTATTATGACCCAGGTTTCCAAGTCACTGCAAGTCACTAGGATCTTCCTTCAGGCTCTGAATCTTGGAATTGAAGTGATCAACACAACTGATCACCTGAAGTTCAGTAAGGACTGTGGCCGAATGCTCACCAGAATGTGGTACTGCTCTTACTGCCAGGGACTGATGATGGTTAAACCCTGTGGCGGTTACTGCAATGTGGTCATGCAAGGCTGTATGGCAGGTGTGGTGGAGATTGACAAGTACTGGAGAGAATACATTCTGTCCCTTGAAGAACTTGTGAATGGCATGTACAGAATCTATGACATGGAGAACGTACTGCTTGGTCTCTTTTCAACAATCCATGATTCTATCCAGTATGTCCAGAAGAATGCAGGAAAGCTGACCACCACTATTGGCAAGTTATGTGCCCATTCTCAACAACGCCAATATAGA

b. and mixing the purified GPC 3-N-terminal recombinant antigen with Freund's adjuvant, and using the mixture as immunogen to immunize Balb/c mice. 5 female BALB/c mice, 5-6 weeks old, were injected subcutaneously around the shoulders and intramuscularly in the hind legs, approximately 1/8 immunogen was administered to each area and 1/2 of the remaining immunogen was intraperitoneally administered. After 1 week, 50 μ g of booster, complete Freund's adjuvant, was injected intraperitoneally. 2 weeks later, 50. mu.g of booster, Incomplete Freund's Adjuvant (IFA), was injected intraperitoneally. 3 weeks and later, 50 μ g of booster was directly injected intraperitoneally weekly. From week 5, tail vein blood was collected weekly, titer was measured by ELISA, coated with polypeptide KLH conjugate antigen 1.25 μ g/ml, blocked with 10% FCS, serum was diluted and added, goat anti-mouse IgG-labeled HRP was used as a secondary antibody, OPD was developed, and read at 492nm using a plate reader (Bio-Rad550 type). The OD values of ELISA results of the 4 th blood sampling are all larger than 0.75.

c. Mouse myeloma cells SP2/0 cells (purchased from ATCC) were prepared simultaneously with immunization of mice. After 3 days of the last boosting immunization, the spleen cells of the mice with the best Western Blot and immunohistochemical detection effects are taken, the ratio of the spleen cells to SP2/0 cells is 5:1, fusion reaction is carried out under the action of PEG1500, the spleen cells are implanted into a 96-well plate, and the spleen cells are cultured under the conditions of 37 ℃ and 5% CO 2. After 14 days of culture, the growing cloning pore is detected under a mirror to be a fusion positive pore, and the total fusion rate is calculated to be more than 95 percent. Supernatants from wells of monoclonal cells were selected as best for ELISA testing. ELISA positive clones were subcloned twice by Western Blot and immunohistochemical detection, with the best clones tested by Western Blot and immunohistochemical detection. Finally, cell clones positive to the GPC 3-N-terminal recombinant antigen reaction are screened out to be used as hybridoma cell strains of the anti-human GPC3 protein monoclonal antibody (3 clones are obtained by screening).

d.6-8 week-old female BALB/c mice were injected with paraffin oil in the abdominal cavity for 10 days, and hybridoma cells were selected at 2X 10⁶One cell/one intraperitoneal injection. After 7-14 days, ascites fluid rich in antibody was extracted from the abdominal cavity of the mouse for detection and purification. The purification was performed by ProteinG affinity chromatography. ProteinG affinity chromatography column was equilibrated with PBSAnd then ascites is taken to pass through a column, the column is washed by PBS until the OD value is close to zero, the elution is carried out by 50nmol/L glycine hydrochloric acid solution, the elution is collected, the OD value of each collecting pipe is measured, the elution in the peak area is reserved, and the monoclonal antibody of the antihuman GPC3 is obtained by dialysis and purification.

e. The hybridoma cell strain 4C11 obtained in the preparation process is sent to China center for type culture Collection for preservation, and the preservation number is CCTCC No: C2018156.

2. application of monoclonal antibody of anti-human GPC3 in identification and detection

Application of ① monoclonal antibody in Western Blot detection

Taking 30ug of denatured protein sample (liver cancer cell lysate) to perform SDS-PAGE protein electrophoresis; proteins were transferred to nitrocellulose membranes by an electrotransfer (Schleicher & Schell corporation); blocking with 5% BSA in TBST for 1h, washing with TBST once for 5 min; TBST containing 4% BSA diluted anti-GPC 3 monoclonal antibody primary antibody (1. mu.g/ml), incubated for 2h, TBST washed three times, each for 5 min; diluting the IR-800 labeled anti-mouse secondary antibody with TBST containing 4% BSA, incubating for 1h, washing with TBST four times, 5min each time; scanning with an infrared laser scanner Odyssey. The results are shown in FIG. 1. The results show that the monoclonal antibody can specifically identify human GPC3 molecules (Huh 7, LM3, HepG2 and SMMC-7721 cell strains express GPC3 at high level, and PLC and PVTT cell strains express GPC3 at low level).

② immunohistochemical detection of monoclonal antibodies

Placing the liver cancer tissue slices in a constant temperature box at 60 ℃ for baking for 20 minutes and dewaxing; 3% H₂O₂Endogenous peroxidase was inactivated (80% methanol) at room temperature for 10 min and washed 3 times with PBS for 5min each. High pressure repairing antigen with 0.01M sodium citrate buffer (pH6.0), washing with PBS for 5min for 3 times; sealing goat serum, standing at room temperature for 20 min, and removing excessive liquid; adding anti-GPC 3 monoclonal antibody primary antibody (10 μ g/ml) diluted by antibody diluent dropwise, incubating at room temperature for 2h, washing 3 times with PBS for 5 min; dripping 50 mu l of secondary antibody marked by HRP, incubating for 1h at room temperature, washing for 5 minutes by PBS for 3 times; DAB is developed for 5-10 minutes, the dyeing degree is controlled under a microscope, and PBS is washed for 3 times for 5 minutes respectively; counterstaining with hematoxylin for 2 min, differentiating with hydrochloric acid and alcohol, and washing with tap water for 10-15 min; dehydrating, transparent, sealingAnd (5) taking a picture and taking a picture. The results are shown in FIG. 2. The result shows that the monoclonal antibody can identify human GPC3 molecule more specifically.

Example 2 humanization optimization of monoclonal antibodies

③ monoclonal antibody amino acid sequence

Hybridoma CCTCC No: the amino acid sequence of the monoclonal antibody produced by C2018156 is shown in Table 1.

TABLE 1

2) Humanized design of monoclonal antibodies

The present inventors humanized the previously obtained GPC3 monoclonal antibody. In the humanized selection sequence, the sequence is selected,

the amino acid is the amino acid position which is different from the human mouse; italicized darkened amino acids were the amino acids after selection for mutations. The single underlined sequences are human kappa chain and IgG1 constant region sequences. As in table 2.

TABLE 2

The antibody variable region includes 4 framework FR regions and 3 highly variable CDR regions. The double underlined amino acids in the above table represent amino acids that are distinct from human and murine, and are located in the FR region. Theoretically, if complete humanization is to be achieved, all the different amino acids need to be replaced by human source, but the inventor finds that excessive amino acid changes may change the structure of the antibody and further influence the recognition of the antibody by the antibody variable region, so through repeated research and experiments, the humanized mutation of partial different amino acids is selected, and the italic black mark is marked.

Example 3 humanized antibody specific binding assay

GPC3 protein was diluted to 1ug/ml with coating buffer, 300ul was added to each well of 96-well plate, and incubated overnight at 4 deg.CThen blocked with PBST containing 2% (w/v) BSA at room temperature for 2 hours. After blocking, the cells were washed 3 times for 5 minutes with wash buffer (containing 0.05% Tween-20). Antigen PBS solution was prepared with a concentration gradient of 6nmol/L to 0.8umol/L and antibody was added to make the total volume 100 ul. After 1 hour incubation at room temperature, the cells were rinsed 3 times for 5 minutes each with wash buffer. Then, 100ul of HPR-labeled rabbit anti-human Fc antibody was added thereto at a dilution of 1:20000 per well, and the reaction was carried out at 37 ℃ for 1 hour. After 3 rinses with washing buffer TMB was added for 10 min and then 50ul of 2M H per well was used₂SO₄The color reaction was terminated. And finally, detecting by using an enzyme-labeling instrument at 450nm to obtain an absorbance value.

As a result, as shown in FIG. 3, all of the humanized antibodies (four combinations) in example 2 specifically bound to human GPC3, with HHa + HLa combination having the highest antibody binding activity.

Example 4 establishment of immune Effector cells modified by chimeric antigen receptors

1. Construction of Chimeric Antigen Receptor (CAR) lentivirus plasmid targeting GPC3

The full-length single chain antibody (scFv) was synthesized using humanized HHA + HLA Sequence and subcloned into lentiviral vector to form construct Lenti-EF1a-K011417S-EGFRT for expression of chimeric antigen receptor together with CD8Hinge, CD8 Transmembrane, 4-1BB, CD3Zeta, T2A Sequence, GMCSF Signalpeptide, EGFR D III-D IV as shown in FIG. 5.

The constructed lentivirus vector structure contains a section of truncated EGFR (Domain III & IV) sequence which is connected with a CAR sequence through a 2A polypeptide sequence. That is, the expression of the truncated EGFR and the expression of the CAR protein are driven by the same promoter. Therefore, by detecting the expression of the truncated EGFR, the expression of the CAR protein can be reflected.

The chimeric antigen receptor amino acid sequence is as follows (SEQ ID NO: 9):

the elements are as follows: signal peptide (1 to 21aa) -VL (22 to 133aa) -Linker (134 to 148aa) -VH (149 to 269aa) -CD 8Hinge (270 to 314aa) -CD8 Transmembrane (315 to 338aa) -4-1BB (339 to 380aa) -CD 3Zeta (381 to 492aa) -T2A Sequence (493 to 510aa) -GMCSF Signal peptide (511 to 527aa) -EGFR D III to D IV (528 to 862aa)

MALPVTALLLPLALLLHAARPDVVMTQSPLSLSVTLGQPASISCKSSQSLLDSFGQTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKLEIKGGGGSGGGGSGGGGSEVKLEESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWVAEIRLKSKNYATHYAESVKGRFTISRDDSKNSVYLQMNSLRAEDTGIYYCARGHYGTNYGDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMWLQSLLLLGTVACSISRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

The chimeric antigen receptor nucleotide sequence is as follows (SEQ ID NO: 10):

the nucleotide Sequence (code optimized) of the CAR cassette comprises sequentially linked EF1-alpha promoter-Signal peptide-VL-Linker-VH-CD8 Hinge-CD8 Transmembrane-4-1BB-CD3 Zeta-T2A Sequence-GMCSF Signal peptide-EGFR D III-D IV:

GAGTAATTCATACAAAAGGACTCGCCCCTGCCTTGGGGAATCCCAGGGACCGTCGTTAAACTCCCACTAACGTAGAACCCAGAGATCGCTGCGTTCCCGCCCCCTCACCCGCCCGCTCTCGTCATCACTGAGGTGGAGAAGAGCATGCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACGCCCCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCTTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGATTCGAATTCGCCGCCACCATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCGACGTGGTCATGACACAGAGCCCTCTGAGCCTGAGCGTGACACTGGGACAGCCTGCCAGCATCAGCTGCAAGTCTAGCCAGAGCCTGCTGGACAGCTTCGGCCAGACCTACCTGAATTGGCTGCTGCAGAGGCCTGGACAGAGCCCCAAGAGACTGATCTACCTGGTGTCCAAGCTGGACTCCGGCGTGCCCGATAGATTTTCTGGCAGCGGCAGCGGAACCGACTTCACCCTGAAGATCAGCAGAGTGGAAGCCGAGGACCTGGGCGTGTACTACTGTTGGCAGGGCACCCACTTTCCATACACCTTTGGCGGAGGCACAAAGCTGGAAATCAAAGGCGGCGGAGGAAGCGGAGGCGGAGGATCTGGTGGTGGTGGATCTGAAGTGAAGCTGGAAGAGTCTGGCGGCGGACTGGTTCAACCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAGCAACTACTGGATGAACTGGGTCCGACAGGCCCCTGGCAAAGGCCTTGAATGGGTTGCCGAGATCCGGCTGAAGTCCAAGAACTACGCCACACACTACGCCGAGAGCGTGAAGGGCAGATTCACCATCAGCCGGGACGACAGCAAGAACAGCGTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGATACCGGCATCTACTACTGTGCCAGAGGCCACTACGGCACCAACTACGGCGATTATTGGGGCCAGGGCACCAGCGTGACAGTGTCATCTACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGGGAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAACCCTGGCCCCATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTCGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGCCTCTTCATG

restriction enzymes EcoRI-SmaI and sequencing were used to determine whether the scFv was correctly inserted into the lentiviral vector. The restriction enzyme identification of the constructed lentiviral plasmid (FIG. 4) shows that scFv has been inserted into the lentiviral vector. The sequencing result of the plasmid shows that the sequence of the finally obtained plasmid is the same as the designed sequence.

2. Lentiviral packaging

a.15cm cell culture dish was charged with 25ml complete medium (DMEM + 10% fetal bovine serum + P/S) and inoculated with 5X 10⁶Cells, cultured overnight at 37 ℃ in a 5% carbon dioxide incubator, were ready for transfection.

b. Preparation of DNA/PEI complexes for transfection: after thawing 100uM PEI was mixed well with the viral packaging system (including the constructed CAR plasmid and viral packaging complex) at room temperature. 2ml PBS, 10ug Lenti-EF1a-Survivin-CAR plasmid and 11ul packaging complex were added to each well of a 6-well plate, followed by 26ul 100uM PEI and mixed well.

c. The DNA/PEI complex was slowly added to a 15cm cell culture dish and mixed well with gentle shaking. Placing the culture medium in a 5% carbon dioxide incubator at 37 ℃ for incubation for 6-8 hours, and then changing the culture medium into a fresh medium.

After d.48 hours, the virus particles in the culture broth were collected by filtration using a 0.45um filter and then centrifuged at 50000 Xg for 2 hours at 4 ℃. The supernatant from centrifugation was removed from the biosafety cabinet, the pellet from centrifugation was resuspended in 500ul PBS and stored in-80 ℃ freezer after packaging.

3. Recombinant lentivirus titer assay

a. HT1080 cells in the logarithmic growth phase are inoculated into a 24-well plate according to the density of 50000 cells per well and are placed in a 5% carbon dioxide incubator at 37 ℃ for overnight culture.

b.24 well cell culture plates 100ul of concentrated virus was added per well along with 6ug/ml polybrene and incubated at 37 ℃ in a 5% carbon dioxide incubator for 96 hours.

The cells were rinsed with pbs and then extracted with genomic DNA purification kit (Lifetech, CAT # K0512). The concentration of the extracted genomic DNA was measured using NanoDrop 2000.

d. pUC-WPRE and pUC-ALB plasmids were diluted 10-fold to prepare standard samples for fluorescent quantitative PCR.

e. The PCR reaction solution was prepared as shown in Table 3.

TABLE 3

The PCR primer information is shown in Table 4.

TABLE 4

Primer and method for producing the same	5’-3’	Fluorescent group
			LTR F	TGACAGCCGCCTAGCATTTC
LTR R	GCTCGATATCAGCAGTTCTTGAAG
			LTR Probe	CACGTGGCCCGAGAGCTGCATC	5’-FAM-BHQ1-3’
ALB F	GCTGTCATCTCTTGTGGGCTGT
			ALB R	ACTCATGGGAGCTGCTGGTTC
ALB Probe	CCTGTCATGCCCACACAAATCTCTCC	5’-FAM-BHQ1-3’

f.96 well plates 5ul of genomic DNA sample or standard and 15ul of PCR reaction solution were added to each well, sealed and centrifuged for 1 minute.

Pcr reaction conditions were set as in table 5.

TABLE 5

h. Drawing a qPCR standard curve.

i. The Ct values (Cycle threshold) of the virus samples are shown in Table 6.

TABLE 6

The qPCR standard curve is shown in figure 6.

j. The virus titer calculation formula is as follows:

k. the results of the virus titer calculations are shown in table 7.

TABLE 7

Sample	Lentivirus Titer
		Lenti-EF1a-K011417S-EGFRt	2.68*108TU/mL

The titer calculation result shows that the inventor successfully completes the virus packaging and obtains high packaging efficiency, and the virus titer reaches 10⁸The above.

4. Initial T cell isolation

a. A lymphocyte isolate was prepared and 15ml of the isolate was added to a 50ml centrifuge tube.

b. The blood sample was diluted 1: 1 by volume with PBS buffer and the diluted blood sample was then slowly added to the separation medium, taking care to keep the interface clear.

c. Centrifuge at 800Xg for 30 min at 20 ℃.

d. After centrifugation, the mononuclear cell layer (PBMC) between the upper yellow serum layer and the lower colorless fraction was carefully collected, and the collected peripheral blood mononuclear cells were washed once with PBS, and finally the cell concentration was adjusted to 1X 10 per ml⁶And (4) cells.

e. Washed CD3/CD28 magnetic beads were added to the cell suspension (3: 1 bead to cell ratio) and incubated at room temperature for 30 minutes.

f. The supernatant was removed using a magnet, and the cells and magnetic beads were resuspended in 3mL of X-VIVO 15 medium, and 200IU/mL IL-2, 10ng/mL IL-7, 5ng/mL IL-15, and 5ng/mL IL-21 were added. Finally, adjusting the cell concentration to 0.5-1 × 10/ml⁶And (4) cells.

g. The cells were cultured for an additional 48 hours and resuspended with a pipette and magnetic beads once a day starting on day 3. When the cell concentration reaches 1X 10 per ml⁶Fresh medium was added at the time of individual cells to adjust the cell concentration to 0.5X 10 per ml⁶And (4) cells.

5. Lentiviral infection of human T cells

a. Adjusting the multiplicity of infection (MOI) to 10-20 for virus infection, and calculating the virus volume for infection by the following formula:

virus volume (mL) ═ MOI × cell number)/virus titer

b. 6ug/mL polybrene was mixed well with the virus, added to the cell culture plate and centrifuged at 800Xg for 1 hour. Then, the cells were incubated at 37 ℃ in a 5% carbon dioxide incubator for 24 hours.

h. After incubation, centrifugation at 250 Xg for 10 min was carried out, the supernatant was removed, and the cell mass was resuspended in fresh medium and cultured for 5 days. Resuspend the beads/cell pellet daily with a pipette at a cell concentration greater than 1X 10 cells per ml⁶Fresh medium was added at the time of individual cells to adjust the cell concentration to 0.5X 10 per ml⁶And (4) cells.

i. A portion of the cells was taken for identification of CAR expression.

6. Flow cytometry for detecting expression of CAR protein on surface of T lymphocyte after infection

Expression of the CAR protein was obtained by detecting the expression of this truncated EGFR. The method comprises the following specific steps:

a. collecting 2X 10⁶Individual CAR-T cells, divided into two tubes, 1X 10 per tube⁶And (4) cells. Resuspend cells with 100. mu.L PBS.

b. mu.L of anti-EGFR antibody (Cetuximab) was added to each tube and incubated for 30 minutes at room temperature.

c.800 Xg speed centrifugal 5 minutes, remove the supernatant after 1mL PBS rinse cell three times.

d. mu.L of APC-Anti-human IgG was added to each tube and incubated for 30 minutes at room temperature in the absence of light.

e.800 Xg speed of centrifugation for 5 minutes, remove the supernatant after 1mL PBS rinse cell three times.

f. Cells were resuspended in 500 μ L PBS and then the expression of CAR protein on the surface of T lymphocytes was detected using flow cytometry.

The results of FACS measurements are shown in FIG. 7, which shows 32.4% of the T lymphocytes expressing the CAR protein.

Example 5 functional study of immune Effector cells

1. CAR-T in vitro toxicity effect experiment

The inventors selected HepG2 cells as target cells. HepG2 cells were co-cultured with T lymphocytes according to different effective target ratios (E/T). Culture supernatants were then collected for LDH levels and pictures of cell killing were taken by microscope. LDH (lactate dehydrogenase) is an enzyme present in the cytoplasm, and is released into the medium when the cell membrane is damaged. Due to the stability of the released LDH, the detection of the LDH content in the culture medium can be used as an index for determining the number of dead cells and damaged cells.

a. Target cells were passaged twice before the experiment to reach logarithmic growth phase.

b. After the target cells were collected by digestion, the cell density was adjusted to 5X 10/ml⁵The number of the individual cells is one,after inoculating 100. mu.L of cell suspension per well of the 96-well plate, the plate was incubated overnight at 37 ℃ in a 5% carbon dioxide incubator.

c. The previously prepared CAR-T cells were harvested by centrifugation and then resuspended in serum-free RPMI1640 medium. The 96-well plates inoculated with the target cells were removed from the incubator, the cells were washed with sterile PBS after complete removal of the medium, CAR-T cells were added to each well according to the E/T ratio, while 4 wells were kept without CAR-T cells as positive and negative controls, respectively. The cell culture plate was incubated at 37 ℃ in a 5% carbon dioxide incubator for 6 hours.

d. After the incubation was finished, the photographs were observed by a microscope. Then adding cell lysate into the cell culture plate, centrifuging for 5 minutes at the rotating speed of 1200Xg, taking 50 mu L of supernatant, transferring the supernatant into another plate, adding LDH detection reagent, and finally detecting the OD value by an enzyme labeling instrument.

e. The percentage of target cell lysis is calculated as follows:

as shown in fig. 8, 10:1 and 5:1, a large number of CAR-T cells aggregated on the surface of HepG2 cells, and the target cells were morphologically rounded; the target cell morphology of the control T cell group did not change much.

The LDH detection result is shown in figure 9, and shows that the CAR-T cell has a remarkable killing effect on target cells in vitro, and the killing effect of the CAR-T cell has a dose-dependent relationship with the CAR-T cell. When the effective target ratio is 10:1, the killing efficiency on target cells can reach about 90%.

2. ELISA detection of the Effect of CAR-T on the expression level of target cytokines

The inventors selected HepG2 cells as target cells. HepG2 cells were co-cultured with T lymphocytes according to different effective target ratios (E/T). Culture supernatants were then collected and ELISA assays were performed to detect the expression levels of cytokines IL-2 and IFN- γ.

a. Target cells were passaged twice before the experiment to reach logarithmic growth phase.

b. After the target cells were collected by digestion, the cell density was adjusted to 5X 10/ml⁵Is smallCells, 96-well plates were inoculated with 100. mu.L of cell suspension per well and incubated overnight at 37 ℃ in a 5% carbon dioxide incubator.

c. The previously prepared CAR-T cells were harvested by centrifugation and then resuspended in serum-free RPMI1640 medium. The 96-well plates inoculated with the target cells were removed from the incubator, the cells were washed with sterile PBS after complete removal of the medium, and CAR-T cells were added to each well according to the E/T ratio. The cell culture plate was incubated at 37 ℃ in a 5% carbon dioxide incubator for 6 hours.

d. After the incubation, the cells were centrifuged at 1200Xg for 5 minutes at room temperature. 50 μ L of supernatant was taken from each well and IL-2 and IFN-. gamma.cytokine levels were measured using an ELISA kit.

e. Data were analyzed using GraphPad Prism software

The cytokine detection results are shown in FIGS. 10-11, and after the cytokine detection results are incubated with target cells, the CAR-T cells can produce more IL-2 and IFN-gamma cytokines, and the expression level of the cytokine is far higher than that of uninfected T cells and is improved by about 3 times or more.

3. CART cell proliferation assay

CFSE is a cell membrane permeable fluorescent dye that binds to intracellular cytoskeletal proteins after entering the cell to form a fluorescent protein adduct. Therefore, when the cell undergoes division and proliferation, cytoplasmic proteins with fluorescence are evenly distributed to the second generation cells, so that the fluorescence intensity is reduced by half compared with the first generation cells; by analogy, the fluorescence intensity of the third generation of cells obtained by division is reduced again compared with that of the second generation. The present inventors labeled CAR-T and control T cells with CFSE dye and then detected using flow cytometry for further analysis to determine cell division proliferation.

a. Target cells were passaged twice before the experiment to reach logarithmic growth phase.

b.24 plates were seeded with 100. mu.L of cell suspension per well and cell concentrations were adjusted to 5X 10 per well⁴Individual cell

c. The CAR-T cells, which were prepared before collection, were centrifuged and the X-VIVO medium was resuspended. CFSE was added for cell staining. CAR-T cells were then added to 24-well plates at an E/T ratio of 10:1, and each well was made up to 500. mu.L. The cell culture plate was placed in a 5% carbon dioxide incubator at 37 ℃.

d. Cells were collected and labeled with APC-conjugated anti-CD3 antibody for final detection using flow cytometry.

Cell proliferation assay results as shown in fig. 12, after incubation and culture with target cells, both CAR-T cells and uninfected control T cells were still able to proliferate, and the fluorescence values of the assays decreased with the number of days in culture. Also, as can be seen in figure 12, the proliferation rate of CAR-T cells was higher than control T cells.

IL-2 can regulate the proliferation speed of T cells and accelerate the proliferation of the T cells, but the IL-2 can cause over-differentiation and apoptosis of the T cells after reaching a certain high level. From the above results of the present invention, it can be seen that the level of IL2 expressed by CART cells is just achieving promotion of T cell proliferation without causing apoptosis of T cells.

Biological material preservation

The hybridoma cell strain 4C11 is preserved in China center for type culture Collection (CCTCC, Wuhan, China), the preservation number is CCTCC NO: C2018156, and the preservation date is 2018, 8 and 30 days.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Katiu (Shanghai) Biotechnology Ltd

<120> anti-GPC 3 monoclonal antibody, immune effector cell modified by the same, and use thereof

<130>178367

<160>11

<170>SIPOSequenceListing 1.0

<210>1

<211>219

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<213> mouse (Mus musculus)

<400>1

Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Phe Gly Gln Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 5560

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

115 120 125

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

130 135 140

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

145 150 155 160

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

180 185 190

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

195 200 205

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

210 215

<210>2

<211>441

<212>PRT

<213> mouse (Mus musculus)

<400>2

Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Leu Lys Ser Lys Asn Tyr Ala Thr His Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Ile Gly Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr

85 90 95

Tyr Cys Ala Arg Gly His Tyr Gly Thr Asn Tyr Gly Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser

115 120 125

Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val

130135 140

Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro

180 185 190

Ser Ser Pro Arg Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro

195 200 205

Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly

210 215 220

Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Leu Ile Thr Val Thr Pro Lys

245 250 255

Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln

260 265 270

Phe Ser Trp Phe Val Asp Asn Val Glu Val His Thr Ala Gln Thr Gln

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Ala Leu

290295 300

Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg

305 310 315 320

Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Thr Lys Gly Lys Pro Arg Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro

340 345 350

Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr

355 360 365

Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Ser Asp Gly Gln

370 375 380

Ala Pro Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly

385 390 395 400

Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu

405 410 415

Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn

420 425 430

His His Thr Glu Lys Ser Leu Ser His

435 440

<210>3

<211>219

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>PEPTIDE

<222>(1)..(219)

<223> humanized antibody light chain

<400>3

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Ser Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Phe Gly Gln Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210>4

<211>219

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>PEPTIDE

<222>(1)..(219)

<223> humanized antibody light chain

<400>4

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Ser Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Phe Gly Gln Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210>5

<211>451

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>PEPTIDE

<222>(1)..(451)

<223> humanized antibody heavy chain

<400>5

Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Leu Lys Ser Lys Asn Tyr Ala Thr His Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Ile Tyr

85 90 95

Tyr Cys Ala Arg Gly His Tyr Gly Thr Asn Tyr Gly Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210>6

<211>451

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>PEPTIDE

<222>(1)..(451)

<223> humanized antibody heavy chain

<400>6

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Leu Lys Ser Lys Asn Tyr Ala Thr His Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Gly Val Tyr

85 90 95

Tyr Cys Ala Arg Gly His Tyr Gly Thr Asn Tyr Gly Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

AspLys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu SerAsn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210>7

<211>219

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>PEPTIDE

<222>(1)..(219)

<223> chimeric antibody light chain

<400>7

Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Phe Gly Gln Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 4045

Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210>8

<211>451

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>PEPTIDE

<222>(1)..(451)

<223> chimeric antibody heavy chain

<400>8

Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Leu Lys Ser Lys Asn Tyr Ala Thr His Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Ile Gly Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr

85 90 95

Tyr Cys Ala Arg Gly His Tyr Gly Thr Asn Tyr Gly Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210>9

<211>862

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>PEPTIDE

<222>(1)..(862)

<223> chimeric antigen receptor amino acid sequence

<400>9

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu

20 25 30

Ser Val Thr Leu Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln

35 40 45

Ser Leu Leu Asp Ser Phe Gly Gln Thr Tyr Leu Asn Trp Leu Leu Gln

50 55 60

Arg Pro Gly Gln Ser Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu

65 70 75 80

Asp Ser GlyVal Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

85 90 95

Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr

100 105 110

Tyr Cys Trp Gln Gly Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr

115 120 125

Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val

145 150 155 160

Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

165 170 175

Phe Ser Asn Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly

180 185 190

Leu Glu Trp Val Ala Glu Ile Arg Leu Lys Ser Lys Asn Tyr Ala Thr

195 200 205

His Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp

210 215 220

Ser Lys Asn Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

225 230 235 240

Thr Gly Ile Tyr Tyr Cys Ala Arg Gly His Tyr Gly Thr Asn Tyr Gly

245 250 255

Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Thr Thr Thr

260 265 270

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

275 280 285

Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val

290 295 300

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

305 310 315 320

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

325 330 335

Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro

340 345 350

Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

355 360 365

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

370 375 380

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu

385 390 395 400

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

405 410 415

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

420 425 430

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

435 440 445

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

450 455 460

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

465 470 475 480

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Glu Gly Arg Gly

485 490 495

Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Trp

500 505 510

Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile Ser Arg

515 520 525

Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser

530 535 540

Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser

545 550 555 560

Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr

565 570 575

His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val

580 585 590

Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg

595 600 605

Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr

610 615 620

Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr

625 630 635 640

Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile

645 650 655

Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys

660 665 670

Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg

675 680 685

Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys

690 695 700

Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys

705 710 715 720

Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu

725 730 735

Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys

740 745 750

His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg

755 760 765

Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His

770 775 780

Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu

785 790 795 800

Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro

805 810 815

Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr

820 825 830

Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu

835 840 845

Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met

850 855 860

<210>10

<211>3870

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>misc_feature

<222>(1)..(3870)

<223> chimeric antigen receptor nucleotide sequence

<400>10

gagtaattca tacaaaagga ctcgcccctg ccttggggaa tcccagggac cgtcgttaaa 60

ctcccactaa cgtagaaccc agagatcgct gcgttcccgc cccctcaccc gcccgctctc 120

gtcatcactg aggtggagaa gagcatgcgt gaggctccgg tgcccgtcag tgggcagagc 180

gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct 240

agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc 300

ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca 360

acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct 420

ttacgggtta tggcccttgc gtgccttgaa ttacttccac gcccctggct gcagtacgtg 480

attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa 540

ggagcccctt cgcctcgtgc ttgagttgag gcctggcttg ggcgctgggg ccgccgcgtg 600

cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa 660

aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc 720

caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg 780

tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg 840

gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc 900

ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg 960

cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg 1020

ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac 1080

tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg 1140

tcgtctttag gttgggggga ggggttttat gcgttggaat ttgccctttt tgagtttgga 1200

tcttggttca ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg 1260

tcgtgattcg aattcgccgc caccatggcc ctccctgtca ccgccctgct gcttccgctg 1320

gctcttctgc tccacgccgc tcggcccgac gtggtcatga cacagagccc tctgagcctg 1380

agcgtgacac tgggacagcc tgccagcatc agctgcaagt ctagccagag cctgctggac 1440

agcttcggcc agacctacct gaattggctg ctgcagaggc ctggacagag ccccaagaga 1500

ctgatctacc tggtgtccaa gctggactcc ggcgtgcccg atagattttc tggcagcggc 1560

agcggaaccg acttcaccct gaagatcagc agagtggaag ccgaggacct gggcgtgtac 1620

tactgttggc agggcaccca ctttccatac acctttggcg gaggcacaaa gctggaaatc 1680

aaaggcggcg gaggaagcgg aggcggagga tctggtggtg gtggatctga agtgaagctg 1740

gaagagtctg gcggcggact ggttcaacct ggcggatctc tgagactgtc ttgtgccgcc 1800

agcggcttca ccttcagcaa ctactggatg aactgggtcc gacaggcccc tggcaaaggc 1860

cttgaatggg ttgccgagat ccggctgaag tccaagaact acgccacaca ctacgccgag 1920

agcgtgaagg gcagattcac catcagccgg gacgacagca agaacagcgt gtacctgcag 1980

atgaacagcc tgagagccga ggataccggc atctactact gtgccagagg ccactacggc 2040

accaactacg gcgattattg gggccagggc accagcgtga cagtgtcatc taccactacc 2100

ccagcaccga ggccacccac cccggctcct accatcgcct cccagcctct gtccctgcgt 2160

ccggaggcat gtagacccgc agctggtggg gccgtgcata cccggggtct tgacttcgcc 2220

tgcgatatct acatttgggc ccctctggct ggtacttgcg gggtcctgct gctttcactc 2280

gtgatcactc tttactgtaa gcgcggtcgg aagaagctgc tgtacatctt taagcaaccc 2340

ttcatgaggc ctgtgcagac tactcaagag gaggacggct gttcatgccg gttcccagag 2400

gaggaggaag gcggctgcga actgcgcgtg aaattcagcc gcagcgcaga tgctccagcc 2460

tacaagcagg ggcagaacca gctctacaac gaactcaatc ttggtcggag agaggagtac 2520

gacgtgctgg acaagcggag aggacgggac ccagaaatgg gcgggaagcc gcgcagaaag 2580

aatccccaag agggcctgta caacgagctc caaaaggata agatggcaga agcctatagc 2640

gagattggta tgaaagggga acgcagaaga ggcaaaggcc acgacggact gtaccaggga 2700

ctcagcaccg ccaccaagga cacctatgac gctcttcaca tgcaggccct gccgcctcgg 2760

gagggcagag gcagcctgct gacatgtggc gacgtggaag agaaccctgg ccccatgtgg 2820

ctgcagagcc tgctgctctt gggcactgtg gcctgcagca tctctcgcaa agtgtgtaac 2880

ggaataggta ttggtgaatt taaagactca ctctccataa atgctacgaa tattaaacac 2940

ttcaaaaact gcacctccat cagtggcgat ctccacatcc tgccggtggc atttaggggt 3000

gactccttca cacatactcc tcctctggat ccacaggaac tggatattct gaaaaccgta 3060

aaggaaatca cagggttttt gctgattcag gcttggcctg aaaacaggac ggacctccat 3120

gcctttgaga acctagaaat catacgcggc aggaccaagc aacatggtca gttttctctt 3180

gcagtcgtca gcctgaacat aacatccttg ggattacgct ccctcaagga gataagtgat 3240

ggagatgtga taatttcagg aaacaaaaat ttgtgctatg caaatacaat aaactggaaa 3300

aaactgtttg ggacctccgg tcagaaaacc aaaattataa gcaacagagg tgaaaacagc 3360

tgcaaggcca caggccaggt ctgccatgcc ttgtgctccc ccgagggctg ctggggcccg 3420

gagcccaggg actgcgtctc ttgccggaat gtcagccgag gcagggaatg cgtggacaag 3480

tgcaaccttc tggagggtga gccaagggag tttgtggaga actctgagtg catacagtgc 3540

cacccagagt gcctgcctca ggccatgaac atcacctgca caggacgggg accagacaac 3600

tgtatccagt gtgcccacta cattgacggc ccccactgcg tcaagacctg cccggcagga 3660

gtcatgggag aaaacaacac cctggtctgg aagtacgcag acgccggcca tgtgtgccac 3720

ctgtgccatc caaactgcac ctacggatgc actgggccag gtcttgaagg ctgtccaacg 3780

aatgggccta agatcccgtc catcgccact gggatggtgg gggccctcct cttgctgctg 3840

gtggtggccc tggggatcgg cctcttcatg 3870

<210>11

<211>1074

<212>DNA

<213> Intelligent (Homo sapiens)

<400>11

atggccggga ccgtgcgcac cgcgtgcttg gtggtggcga tgctgctcag cttggacttc 60

ccgggacagg cgcagccccc gccgccgccg ccggacgcca cctgtcacca agtccgctcc 120

ttcttccaga gactgcagcc cggactcaag tgggtgccag aaactcccgt gccaggatca 180

gatttgcaag tatgtctccc taagggccca acatgctgct caagaaagat ggaagaaaaa 240

taccaactaa cagcacgatt gaacatggaa cagctgcttc agtctgcaag tatggagctc 300

aagttcttaa ttattcagaa tgctgcggtt ttccaagagg cctttgaaat tgttgttcgc 360

catgccaaga actacaccaa tgccatgttc aagaacaact acccaagcct gactccacaa 420

gcttttgagt ttgtgggtga atttttcaca gatgtgtctc tctacatctt gggttctgac 480

atcaatgtag atgacatggt caatgaattg tttgacagcc tgtttccagt catctatacc 540

cagctaatga acccaggcct gcctgattca gccttggaca tcaatgagtg cctccgagga 600

gcaagacgtg acctgaaagt atttgggaat ttccccaagc ttattatgac ccaggtttcc 660

aagtcactgc aagtcactag gatcttcctt caggctctga atcttggaat tgaagtgatc 720

aacacaactg atcacctgaa gttcagtaag gactgtggcc gaatgctcac cagaatgtgg 780

tactgctctt actgccaggg actgatgatg gttaaaccct gtggcggtta ctgcaatgtg 840

gtcatgcaag gctgtatggc aggtgtggtg gagattgaca agtactggag agaatacatt 900

ctgtcccttg aagaacttgt gaatggcatg tacagaatct atgacatgga gaacgtactg 960

cttggtctct tttcaacaat ccatgattct atccagtatg tccagaagaa tgcaggaaag 1020

ctgaccacca ctattggcaa gttatgtgcc cattctcaac aacgccaata taga 1074

Claims (22)

1. a specific anti-GPC3 monoclonal antibody, characterized in that the antibody has a light chain variable region selected from the group consisting of: positions 1-112 in SEQ ID NO: 1, positions 1-112 in SEQ ID NO: 3 position, position 1-112 in SEQ ID NO:4, position 1-112 in SEQ ID NO:7; and A heavy chain variable region selected from the group consisting of positions 1-121 in SEQ ID NO:2, positions 1-121 in SEQ ID NO:5, positions 1-121 in SEQ ID NO:6, SEQ ID NO: 1-121 of 8. 2. The monoclonal antibody of claim 1, wherein the antibody is a murine antibody, a humanized antibody, a chimeric antibody, or an antibody comprising the murine antibody, humanized antibody, or chimeric antibody Heavy chain variable region and light chain variable region scFv antibodies. 3. The monoclonal antibody of claim 1, wherein the antibody is a humanized antibody and has a light chain variable region selected from the group consisting of positions 1-112 in SEQ ID NO: 3, SEQ ID Positions 1-112 in NO:4; and a heavy chain variable region selected from the group consisting of positions 1-121 in SEQ ID NO:5, positions 1-121 in SEQ ID NO:6; preferably, The antibody has the light chain variable region at positions 1-112 in SEQ ID NO:3, and the heavy chain variable region at positions 1-121 in SEQ ID NO:5. 4. The monoclonal antibody of claim 1, wherein the antibody is a murine antibody produced by a hybridoma cell line CCTCC No: C2018156. 5. A nucleic acid encoding the antibody of any one of claims 1 to 4. 6. An expression vector comprising the nucleic acid of claim 5. 7. A host cell comprising the expression vector of claim 6 or the nucleic acid of claim 5 integrated into the genome. 8. Use of the antibody according to any one of claims 1 to 4, for preparing a targeted drug, antibody-drug conjugate or multifunctional antibody that specifically targets tumor cells expressing GPC3; or For preparing a reagent for diagnosing tumors, the tumor expresses GPC3; or for preparing immunohistochemical detection reagents; or for preparing chimeric antigen receptor-modified immune cells; preferably, the immune cells include: T lymphocytes , NK cells or NKT lymphocytes. 9. A chimeric antigen receptor, expressed on the surface of immune cells, comprising an extracellular region, a transmembrane region and an intracellular signal region, the extracellular region comprising the antibody according to any one of claims 1 to 4, the transmembrane region and intracellular signaling regions. 10. The chimeric antigen receptor of claim 9, wherein the transmembrane region comprises the transmembrane region of CD8 Hinge and CD28; or The intracellular signal region has elements selected from the group consisting of 4-1BB, CD3 Zeta, FcεRIγ, CD27, CD28, CD137, CD134, IL-12 or a combination thereof. 11. The chimeric antigen receptor of claim 10, wherein the chimeric antigen receptor comprises the following sequence-connected extracellular region, transmembrane region and intracellular signal region: containing claim 1 ～4 The light chain variable region of any one of the antibodies can be the scFv of the heavy chain variable region, CD8 Hinge, the transmembrane region of CD28, 4-1BB, CD3 Zeta; preferably, GMCSF signal peptide and EGFR are also included Domain III to IV; more preferably, the GMCSF signal peptide and EGFR D III to D IV are linked to the intracellular signal region through the 2A peptide. 12. The chimeric antigen receptor according to claim 11, wherein the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO: 9, or as shown in SEQ ID NO: 9, 1- 492 bits shown. 13. A nucleic acid encoding the chimeric antigen receptor of any of claims 9-12. 14. An expression vector, characterized in that it comprises the nucleic acid of claim 13. 15. A virus, characterized in that the virus comprises the vector of claim 14 or the nucleic acid encoding the chimeric antigen receptor of claim 13. 16. Use of the chimeric antigen receptor according to any one of claims 9 to 12, or the nucleic acid according to claim 13, or the expression vector according to claim 14, or the virus according to claim 15, for use in Generation of genetically modified immune cells that specifically target GPC3-expressing tumors. 17. A genetically modified immune cell, wherein the nucleic acid of claim 13, the expression vector of claim 14 or the virus of claim 15 are introduced into it; or The chimeric antigen receptor according to any one of claims 9 to 12 is expressed on its surface. 18. The genetically modified immune cell of claim 17, wherein the immune cell comprises: T lymphocytes, NK cells or NKT cells. 19. Use of the gene-modified immune cell according to claim 17 or 18, characterized in that it is used to prepare a drug for inhibiting tumor, and the tumor is a tumor expressing GPC3. 20. An immunoconjugate, wherein the immunoconjugate comprises: The antibody of any one of claims 1 to 4; and A functional molecule connected to it; the functional molecule is selected from: a molecule targeting tumor surface markers, a tumor-inhibiting molecule, a molecule targeting immune cell surface markers or a detectable marker. 21. A pharmaceutical composition, characterized in that it comprises: The antibody of any one of claims 1 to 4 or a nucleic acid encoding the antibody; or The chimeric antigen receptor of any one of claims 9 to 12 or the nucleic acid encoding the chimeric antigen receptor; or The immunoconjugate of claim 20 or a nucleic acid encoding the conjugate; or The genetically modified immune cell of claim 17 or 18. 22. A hybridoma cell line, the deposit number of which is CCTCC NO: C2018156 in the China Center for Type Culture Collection.

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