CN110760005A - Chimeric antigen receptor modified T cell of targeted Glypican-3 antigen and application thereof - Google Patents

Abstract

The invention relates to a chimeric antigen receptor modified T cell of a targeted Glypican-3 antigen and application thereof, and particularly provides an anti-Glypican-3 chimeric antigen modified fusion protein and an immune cell expressing the fusion protein, wherein the fusion protein has a structure shown in an optimized formula I. The specific immune cell modified by the fusion protein can be used for targeted therapy of tumors specifically expressing the Glypican-3 antigen, and has the advantages of good curative effect, small side effect, low production cost and the like.

Description

Chimeric antigen receptor modified T cell of targeted Glypican-3 antigen and application thereof

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a chimeric antigen receptor modified T cell of a targeted Glypican-3 antigen and application thereof.

Background

With increasing morbidity and mortality, cancer is becoming the leading cause of death and an important public health problem in china and even globally. According to survey statistics, the most common 4 cancers in China are lung cancer, gastric cancer, liver cancer and esophageal cancer. The cancer accounts for 57% of Chinese cancer diagnosis, and the Chinese cancer accounts for 1/3 to 1/2 of the worldwide disease burden, and the survival rate of the cancer is very low. There are 78.2 ten thousand new cases of liver cancer and 74.5 ten thousand death cases in 2012 all over the world. Wherein, the number of new cases and death cases in China account for about 50 percent. The primary liver cancer is one of common malignant tumors in China and even the world, the incidence rate of adults is high, the disease is mostly in a late stage when being discovered, and the death rate is high, which is commonly called as 'king of cancer'. About 11 million people die of liver cancer in continental countries every year, accounting for 45% of the liver cancer deaths worldwide. The currently used methods for treating liver cancer are: the method comprises the following steps of surgical treatment, hepatic artery chemotherapy and embolization, absolute alcohol injection treatment, laser photodynamic treatment, microwave coagulation treatment under ultrasonic guidance, high-power focused ultrasound treatment, electrochemical therapy (direct current therapy), permanent implantation among radioactive particle tissues, radio frequency destruction, guide treatment, molecular drug targeted treatment, radiotherapy, liver transplantation and immunotherapy, and along with the gradual deepening of the research on a tumor immune response mechanism and an immune escape mechanism, the immunotherapy of the cancer is rapidly developed in the aspects of basic research and clinical application and even gradually becomes the most ideal weapon for thoroughly eliminating the cancer.

The tumor immunotherapy is to enhance the anti-tumor immune function of the body by mobilizing or exciting the immune function of the body in an active and passive mode, and further help a patient to inhibit and kill tumor cells. Three major fields of tumor immunotherapy currently include: adoptive cellular immunotherapy, immune checkpoint inhibitors and tumor vaccines.

Therefore, there is an urgent need in the art to develop an engineered immune cell that is effective, stable in therapeutic effect, and less in side effects, specifically against a new target.

Disclosure of Invention

The invention aims to provide an effective, stable-curative-effect and small-side-effect engineered immune cell specifically aiming at a new target.

In a first aspect, the present invention provides a fusion protein having the structure of formula I:

L-scFv-Z-TM-C-X (I)，

wherein,

l is a null or signal peptide sequence;

scFv is a variable region sequence of a targeting Glypican-3 antibody single chain;

z is a null or hinge region;

TM is a transmembrane domain;

c is a costimulatory signal molecule;

x is the cytoplasmic signaling sequence CD3 ζ;

each "-" represents a linking peptide or peptide bond linking the above-mentioned respective elements.

In another preferred embodiment, the scFv has the structure shown in formula A1 or A2:

V_L1-V_H1(A1) (ii) a Or

V_H1-V_L1(A2)；

Wherein, V_L1Is the light chain variable region of an anti-Glypican-3 antibody; v_H1Is the heavy chain variable region of an anti-Glypican-3 antibody; "- "is a linker peptide (or flexible linker) or a peptide bond.

In another preferred embodiment, V is_L1And V_H1Connected by a flexible joint.

In another preferred embodiment, the flexible linker is 1 to 5 (preferably, 2 to 4) consecutive sequences shown in SEQ ID NO:12 (GGGGS).

In another preferred embodiment, V_L1The amino acid sequence of (A) is shown in the 23 rd to 134 th positions of SEQ ID NO. 3, and V_H1The amino acid sequence of (1) is shown as position 150-263 of SEQ ID NO. 3.

In another preferred embodiment, the scFv has the structure shown in formula A3 or A4:

V_L2-V_H2(A3) (ii) a Or

V_H2-V_L2(A4)；

Wherein, V_L2Is the light chain variable region of an anti-Glypican-3 antibody; v_H2Is the heavy chain variable region of an anti-Glypican-3 antibody; "-" is a linker peptide (or flexible linker) or peptide bond.

In another preferred embodiment, V is_L2And V_H2Connected by a flexible joint.

In another preferred embodiment, the flexible linker is 1 to 5 (preferably, 2 to 4) consecutive sequences shown in SEQ ID NO:12 (GGGGS).

In another preferred embodiment, V_L2The amino acid sequence of (1) is shown in the 23 rd to 134 th positions of SEQ ID No. 5, and V_H2The amino acid sequence of (1) is shown in the position 150-264 of SEQ ID NO. 5.

In another preferred embodiment, the amino acid sequence of the scFv is shown in SEQ ID NO. 11 or 4.

In another preferred embodiment, the fusion protein has the structure shown in formula II or II':

L-V_L1-V_H1-Z-TM-C-CD3 ζ (II); or

L-V_H1-V_L1-Z-TM-C-CD3ζ(II’)

Wherein each element is as described above.

In another preferred embodiment, the fusion protein has the structure shown in formula III or III':

L-V_L2-V_H2-Z-TM-C-CD3 ζ (III); or

L-V_H2-V_L2-Z-TM-C-CD3ζ(III’)

Wherein each element is as described above.

In another preferred embodiment, L is a signal peptide of a protein selected from the group consisting of: CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

In another preferred embodiment, L is a signal peptide of a protein selected from the group consisting of: CD 8.

In another preferred embodiment, Z is a hinge region of a protein selected from the group consisting of: IgG4, CD8, IgGD, IgG1, or a combination thereof.

In another preferred embodiment, Z is a hinge region of a protein selected from the group consisting of:IgG 4.

In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD 28.

In another preferred embodiment, C is a costimulatory signal molecule for a protein selected from the group consisting of: CD28, OX40(CD134), ICOS, DAP10, 4-1BB (CD137), or a combination thereof.

In another preferred embodiment, C is a costimulatory signal molecule for a protein selected from the group consisting of: CD 28.

In another preferred embodiment, the fusion protein has an amino acid sequence as shown inSEQ ID NO 3 or 5.

In another preferred embodiment, the amino acid sequence of the fusion protein is shown inSEQ ID NO 3 or 5.

In another preferred embodiment, the fusion protein is a recombinant protein.

In a second aspect, the present invention provides a nucleic acid molecule encoding a fusion protein according to the first aspect of the invention.

In another preferred embodiment, the nucleic acid molecule is selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide as set forth in SEQ ID NO. 3 or 5;

(b) the polynucleotide with the sequence shown in SEQ ID NO. 2 or 6;

(c) a polynucleotide having a nucleotide sequence having a homology of 75% or more (preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more, still more preferably 99% or more) to the sequence of (b);

(d) a polynucleotide in which 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides are truncated or added to the 5 'end and/or the 3' end of the polynucleotide shown in (b);

(e) a polynucleotide complementary to any one of the polynucleotides of (a) - (d).

In another preferred embodiment, the nucleotide sequence of said nucleic acid molecule is as shown inSEQ ID NO 2 or 6.

In another preferred embodiment, the nucleic acid molecule is a polynucleotide.

In a third aspect, the invention provides a vector comprising a polynucleotide according to the second aspect of the invention.

In another preferred embodiment, the carrier is selected from the group consisting of: a plasmid, a lentiviral vector, an adenoviral vector, a retroviral vector, a transposon vector, or a combination thereof.

In another preferred embodiment, the vector is a lentiviral vector or a transposon vector.

In another preferred example, the vector comprises a PiggyBac transposon vector, and/or a Sleeping bearty transposon vector.

In a fourth aspect, the invention provides a host cell comprising a vector or chromosome of the third aspect of the invention into which has been integrated an exogenous nucleic acid molecule of the second aspect of the invention or which expresses a fusion protein of the first aspect of the invention.

In another preferred embodiment, the cell is an isolated cell, and/or the cell is a genetically engineered cell.

In another preferred embodiment, the host cell is a prokaryotic cell or a eukaryotic cell.

In another preferred embodiment, the host cell is a mammalian cell.

In another preferred embodiment, the host cell is from a primate.

In another preferred embodiment, the host cell is a human cell.

In another preferred embodiment, the host cell is an NK cell, a T cell.

In another preferred embodiment, the host cell comprises an engineered immune cell.

In another preferred embodiment, the engineered immune cells comprise T cells or NK cells.

In another preferred embodiment, the engineered immune cell is selected from the group consisting of:

(i) a chimeric antigen receptor T cell (CAR-T cell);

(ii) chimeric antigen receptor NK cells (CAR-NK cells); or

(iii) Exogenous T Cell Receptor (TCR) T cells (TCR-T cells).

In another preferred embodiment, the immune cells are autologous.

In another preferred embodiment, the immune cells are non-autologous.

In another preferred embodiment, the immune cells target the Glypican-3 antigen.

In a fifth aspect, the invention provides a method of preparing an engineered immune cell expressing a fusion protein according to the first aspect of the invention, wherein the method comprises the steps of: transducing the nucleic acid molecule of the second aspect of the invention or the vector of the third aspect of the invention into an immune cell, thereby obtaining the engineered immune cell.

In another preferred embodiment, the introducing includes introducing simultaneously, sequentially or sequentially.

In another preferred embodiment, the immune cell is a T cell or NK cell.

In another preferred embodiment, the method further comprises the step of performing functional and validity detection on the obtained engineered immune cells.

In a sixth aspect, the present invention provides a pharmaceutical composition comprising: a fusion protein according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention or a host cell according to the fourth aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection.

In another preferred embodiment, the concentration of the cells in the pharmaceutical composition is 1 × 10⁵-1×10⁸One cell/ml, preferably 1X 10⁶-1×10⁷One cell/ml, more preferably 1X 10⁶-5×10⁶Individual cells/ml.

In another preferred embodiment, the pharmaceutical composition further comprises other drugs that selectively kill tumor cells (such as emerging antibody drugs, other CAR-T drugs, or chemotherapeutic drugs).

The seventh aspect of the present invention provides a use of the fusion protein according to the first aspect of the present invention, the nucleic acid molecule according to the second aspect of the present invention, the vector according to the third aspect of the present invention, or the host cell according to the fourth aspect of the present invention, for preparing a medicament or a preparation for selectively killing tumor cells.

In another preferred embodiment, the tumor cells are derived from a tumor that specifically expresses a Glypican-3 antigen.

In another preferred embodiment, the tumor specifically expressing the Glypican-3 antigen is selected from the group consisting of: liver cancer, lung cancer, gastric cancer, melanoma, liposarcoma, adrenal adenoma, cervical squamous cell carcinoma, or a combination thereof.

In an eighth aspect, the present invention provides a kit for selectively killing tumor cells, the kit comprising a container, and in the container, the fusion protein according to the first aspect of the present invention, the nucleic acid molecule according to the second aspect of the present invention, the vector according to the third aspect of the present invention, or the host cell according to the fourth aspect of the present invention.

In another preferred embodiment, the kit further comprises a label or instructions for use.

The ninth aspect of the present invention provides a method for selectively killing tumor cells, comprising:

administering to a subject in need thereof a safe and effective amount of a host cell according to the fourth aspect of the invention, or a pharmaceutical composition according to the sixth aspect of the invention.

In another preferred embodiment, the subject comprises a human or non-human mammal.

In another preferred embodiment, the non-human mammal includes a rodent (e.g., mouse, rat, rabbit), primate (e.g., monkey).

In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows a structural schematic of a chimeric antigen receptor targeting Glypican-3.

FIG. 2 shows the positive rates of flow-detecting GPC3CAR-T and GPC3CAR-T-0599 cellular CAR.

FIG. 3 shows the western blot detection of GPC3CAR-T and CAR gene expression in GPC3CAR-T-0599 cells.

FIG. 4 shows the results of RT-PCR detection of GPC3CAR-T and copy number of CAR gene expression in GPC3CAR-T-0599 cells.

FIG. 5 shows a comparison of killing of Mock-T, GPC3CAR-T and GPC3CAR-T-0599 cells on liver cancer cell lines, including Huh-7 and Hep 3B.

FIG. 6 shows a comparison of IL-2, IL-4, IL-6, IL-10, TNF- α and IFN- γ cytokine secretion levels by Mock-T, GPC3CAR-T and GPC3CAR-T-0599 cells, respectively, under stimulation with the GPC3 antigen.

FIG. 7 shows a comparison of Mock-T, GPC3CAR-T and GPC3CAR-T-0599 cells following stimulation with the GPC3 antigen for cell surface exhaustion phenotypes (including PD-1, TIM-3, and LAG-3), activation phenotypes (including CD107 α, CD69, and CD25), and subpopulations of memory T cells and effector T cells.

Detailed Description

The inventor unexpectedly obtains a fusion protein modified by anti-Glypican-3 chimeric antigen and an immune cell expressing the fusion protein through extensive and intensive research and a large amount of screening of targets, wherein the fusion protein has an optimized structure shown in formula I. Experimental results show that the specific immune cells modified by the fusion protein can be used for targeted therapy of tumors specifically expressing the Glypican-3 antigen, and have the advantages of good curative effect, small side effect, low production cost and the like. The present invention has been completed based on this finding.

Term(s) for

Glypican-3

Glypican 3 (GPC 3) is heparan sulfate glycoprotein (HSPG) on the surface of a cell membrane, is an oncofetal antigen of hepatocellular carcinoma, has high expression positive rate of up to 76.3 percent in liver cancer tissues, is hardly expressed in normal liver tissues, and is an ideal tumor treatment target. The GPC3 protein is anchored to the cell surface by a glycophospholipopeptide myoglobin (CPI), and binds to heparin-binding proteins such as growth factors, which are essential for the binding of growth factors such as IGF-2, BMP-7 and FGF-2 to their receptors, are extracellular matrix components that regulate cell growth, proliferation, differentiation/adhesion and migration, and also exert immunomodulatory effects, and may be involved in inhibiting or regulating the growth of most mesodermal tissues and organs. The inhibition of the function of GPC3 by down-regulation or competition has a profound negative effect on the proliferation of liver cancer cells. Unlike other tumor associated antigens known to date, GPC3 is a membrane-associated glycoprotein linked to phosphatidylinositol, with a large extracellular domain that facilitates antibody-mediated therapy.

The invention unexpectedly screens a fusion protein modified by anti-Glypican-3 chimeric antigen and an immune cell expressing the fusion protein through mass screening, and the fusion protein has obvious killing effect on tumor cells specifically expressing GPC3 or highly expressing GPC3, especially liver cancer cells.

Chimeric antigen receptor

The Chimeric Antigen Receptor (CAR) comprises an extracellular antigen binding region, a transmembrane region and an intracellular signal region, wherein the extracellular antigen binding region has a function of recognizing a specific tumor antigen, and the intracellular signal region mediates T intracellular signal conduction until T cells are activated and proliferated, so that the process of killing the tumor cells is finally completed. Recombinant plasmids were generated by in vitro genetic recombination of an antibody single chain variable region (scFv) that recognizes a Tumor Associated Antigen (TAA) and an intracellular signaling domain, "Immunoreceptor Tyrosine Activation Motif (ITAM)". The plasmid is then transferred into T cells, and the T cells which are genetically modified are called CAR-T cells. After the CAR-T cells are expanded in vitro in a large scale, the CAR-T cells are returned to a patient body, and tumor cells expressing specific antigens can be specifically recognized and killed in an MHC (major histocompatibility complex) non-limited form, so that the aim of killing the tumor cells is fulfilled.

NK cells

Natural Killer (NK) cells are a major class of immune effector cells that protect the body from viral infection and tumor cell invasion through non-antigen specific pathways. In autoimmune diseases, NK cell imbalance (depletion) is an important mechanism leading to the pathogenesis of autoimmune diseases, and NK cell depletion leads to a decrease in its function of non-specifically inhibiting B cell secretion of antibodies. However, NK92 cells are the only cell line approved by FDA clinical test at present, the cytotoxic ability is strong, the survival time after killing tumor cells is short, the cells are easy to expand in vitro, and most of patients receiving treatment do not reject NK92-MI cells and have no risk of graft-versus-host reaction. In the present invention, NK cells are selected as effector cells.

NK92MI cell

Novel functions, including the ability to specifically recognize tumor antigens and having enhanced anti-tumor cytotoxic effects, are possible to be obtained by genetically modified NK92MI (CAR-NK92MI) cells. NK92MI cells have potent cytotoxic effects against a wide variety of tumors, such as leukemia, lymphoma, myeloma, and some solid tumors. Some clinical trials have shown that high doses of NK92MI cell transfusions are also of great safety.

CAR-NK92MI also has the following advantages compared to autologous CAR-T cells, for example: (1) directly kills tumor cells by releasing perforin and granzyme, but has no killing effect on normal cells of an organism; (2) they release very small amounts of cytokines and thus reduce the risk of cytokine storm; (3) is easy to be amplified in vitro and can be developed into ready-made products. In addition, similar to CAR-T cell therapy, CAR-NK92 or CAR-NK92MI did not elicit immune tolerance.

Antigen binding moieties

As used herein, the terms "antigen binding portion", "antigen binding domain" are used interchangeably.

In a preferred embodiment, the CAR of the invention comprises a target-specific binding member. The choice of moiety depends on the type and amount of ligand that defines the target cell surface. For example, the antigen binding domain can be selected to recognize ligands that function as cell surface markers on target cells associated with a particular disease state. Thus, examples of cell surface markers that may act as ligands for the antigen moiety domain in the CARs of the invention include those associated with viral, bacterial and parasitic infections, autoimmune diseases, and cancer cells.

In a preferred embodiment, the CARs of the invention can be engineered to target a tumor antigen of interest by engineering a desired antigen-binding moiety that specifically binds to the antigen on the tumor cell. In the present invention, "tumor antigen" refers to an antigen common to cancers.

The antigen binding domain of the CAR can target, for example, CD19, CD20, CD22, ROR1, mesothelin, CD33/IL3Ra, c-Met, CD37PSMA, glycolipid F77, HER2, EGFRvIII, GD-2, NY-ESO-1TCR, and MAGE A3TCR, or the antigen targeted by the antigen binding domain portion of the CAR includes, but is not limited to, FR α, CD24, CD44, CD133, CD166, epCAM, CA-125, HE4, ova, estrogen receptor, progesterone receptor, HER-2/neu, uPA, PAI-1, and the like.

The antigen binding domain may be any domain that binds an antigen, including but not limited to monoclonal antibodies, single chain antibodies (e.g., scFv), polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, and fragments thereof.

In a preferred embodiment, the antigen binding portion of the CAR of the invention targets the GPC3 antigen. In a preferred embodiment, the antigen binding portion of the CAR of the invention is anscFv targeting GPC 3.

In a preferred embodiment, the scFv has the structure of formula A1 or A2:

V_L1-V_H1(A1) (ii) a Or

V_H1-V_L1(A2)；

Wherein, V_L1Is the light chain variable region ofanti-GPC 3 antibody; v_H1Is the heavy chain variable region of the anti-GPC 3 antibody; "-" is a linker peptide or peptide bond.

In a preferred embodiment, V_L1Has the amino acid sequence shown in the 23 rd to 134 th positions of SEQ ID No. 3, and V_H1The amino acid sequence of (1) is shown in the 150-rd and 263-th positions of SEQ ID NO. 3.

In a preferred embodiment, the scFv has the structure of formula A3 or A4:

V_L2-V_H2(A3) (ii) a Or

V_H2-V_L2(A4)；

Wherein, V_L2Is the light chain variable region ofanti-GPC 3 antibody; v_H2Is the heavy chain variable region of the anti-GPC 3 antibody; "-" is a linker peptide or peptide bond.

In a preferred embodiment, V_L2The amino acid sequence of (1) is shown in the 23 rd to 134 th positions of SEQ ID No. 5, and V_H2The amino acid sequence of (1) is shown in the position 150-264 of SEQ ID NO. 5.

In a preferred embodiment, the scFV comprises a variant form, said variant having a homology of > 80%, > 85%, > 90%, > 95%, > 98% or > 99% to its wild type scFV sequence.

Fusion proteins

As used herein, the terms "fusion protein", "fusion protein of the invention", "active polypeptide" and "polypeptide of the invention" have the same meaning and all have the structure described in the first aspect of the invention. The fusion protein of the invention comprises: a leader sequence, a scFV capable of specific binding to GPC3, a hinge domain, a transmembrane domain TM (transmembrane region from CD28 molecule), a costimulatory domain (CD28), and a series of signal regions, CD3 ζ.

The fusion protein of the invention has the following characteristics:

a) after the fusion protein is expressed, the fusion protein can penetrate through a cell membrane and be positioned on the cell membrane to form a membrane protein which exposes an extracellular segment element to the outside. In addition, the fusion protein of the present invention also has a costimulatory molecule (or element) and CD3zeta, which are located intracellularly. In addition, the fusion protein of the present invention may also contain an optional signal peptide, a linker peptide element (linker), or other elements.

b) The invention constructs two CARs (preferably three-generation CARs, the structure of which comprises a single-chain antibody part capable of specifically recognizing GPC3 molecule, an Fc fragment maintaining spatial conformation, a cooperative co-stimulatory molecule CD28 and a CD3zeta molecule transmitting signals to the intracellular space) capable of specifically recognizing human GPC3 molecule, and transfects the constructed CARs onto NK92MI cells and human peripheral blood T cells to form CAR-NK and CAR-T cells by a form of lentivirus transfection, thereby carrying out a targeted killing test aiming at artificially constructed cell lines expressing GPC3 molecule and mononuclear cells of peripheral blood. The CAR-NK and CAR-T cells of the invention are capable of specifically killing tumors that specifically express a Glypican-3 antigen. In an in vitro killing test, the constructed CAR targeting GPC3 has better cytotoxicity and specificity, and provides a basis for clinical transformation research of CAR-NK and CAR-Ttherapies targeting GPC 3.

The term "fusion protein" as used herein also includes variants of the sequence ofSEQ ID NO 3 or 5 having the above-described activity. These variants include (but are not limited to): deletion, insertion and/or substitution of 1 to 3 (usually 1 to 2, more preferably 1) amino acids, and addition or deletion of one or several (usually up to 3, preferably up to 2, more preferably up to 1) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition or deletion of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the structure and function of the protein. In addition, the term also includes monomeric and multimeric forms of the polypeptides of the invention. The term also includes linear as well as non-linear polypeptides (e.g., cyclic peptides).

The invention also includes active fragments, derivatives and analogs of the above fusion proteins. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains the function or activity of a fusion protein of the invention. The polypeptide fragment, derivative or analogue of the present invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which an antigenic peptide is fused to another compound (such as a compound that increases the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) a polypeptide in which an additional amino acid sequence is fused to the polypeptide sequence (a fusion protein in which a tag sequence such as a leader sequence, a secretory sequence or 6His is fused). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

A preferred class of reactive derivatives refers to polypeptides formed by the replacement of up to 3, preferably up to 2, more preferably up to 1 amino acid with an amino acid of similar or analogous nature compared to the amino acid sequence of formula I. These conservative variants are preferably produced by amino acid substitutions according to Table A.

TABLE A

Initial residue(s)	Representative substitutions	Preferred substitutions
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

The analogs can differ from the polypeptides shown in SEQ ID NO. 3 or 5 by amino acid sequence differences, by modifications that do not affect the sequence, or by both, and also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., β, gamma-amino acids).

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.

In one embodiment of the invention, the amino acid sequence of the CAR is as set forth in SEQ ID No. 3 or 5.

Wherein, the 1 st to the 22 nd positions in the SEQ ID NO. 3 are signal peptides; scFv at positions 23-263; 264 th-275 th-mentioned is a hinge region; position 492-519 is a transmembrane region (e.g., the transmembrane region of CD 28); position 520-560 is a costimulatory element (CD 28); the 561 st 672 bit is CD3 ζ.

Wherein, the 1 st to the 22 nd positions in the SEQ ID NO. 5 are signal peptides; scFv at positions 23-264; 265 th-276 is a hinge region; position 493-520 is a transmembrane region (e.g., the transmembrane region of CD 28); 521-561 is a co-stimulation element (CD 28); position 562-673 is CD3 ζ.

Coding sequence

The invention also relates to polynucleotides encoding the fusion proteins according to the invention.

The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence encoding the polypeptide set forth in SEQ ID No. 3 or 5 or may be a degenerate variant. As used herein, "degenerate variant" refers in the present invention to nucleic acid sequences which encode a polypeptide having the sequence shown in SEQ ID No. 3 or 5, but differ in the sequence of the corresponding coding region.

In a preferred embodiment of the invention, the sequence of the polynucleotide is as shown in SEQ ID No. 2 or 6.

The full-length nucleotide sequence or its fragment of the present invention can be obtained by PCR amplification, recombination, or artificial synthesis. At present, DNA sequences encoding the polypeptides of the present invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.

The invention also relates to vectors comprising the polynucleotides of the invention, and to genetically engineered host cells with the vector or polypeptide coding sequences of the invention. The polynucleotide, vector or host cell may be isolated.

As used herein, "isolated" refers to a substance that is separated from its original environment (which, if it is a natural substance, is the natural environment). If the polynucleotide or polypeptide in the natural state in the living cell is not isolated or purified, but the same polynucleotide or polypeptide is isolated or purified if it is separated from other substances coexisting in the natural state.

The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

The present invention also relates to variants of the above polynucleotides which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present invention. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polynucleotide encoding the fusion protein of the invention.

The full-length nucleotide sequence of the polypeptide of the present invention or a fragment thereof can be obtained by PCR amplification, recombination, or artificial synthesis. For the PCR amplification method, primers can be designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and the sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

In one embodiment of the invention, the polynucleotide sequence encoding the fusion protein is as shown in SEQ ID No. 2 or 6. 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.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

A method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

The invention also relates to a vector comprising the polynucleotide of the invention, as well as a genetically engineered host cell with the vector or protein coding sequence of the invention, and a method for expressing the fusion protein of the invention on the NK cells by recombinant techniques.

NK cells expressing the fusion protein of the present invention can be obtained by using the polynucleotide sequence of the present invention by a conventional recombinant DNA technique. Generally comprising the steps of: transferring the nucleic acid molecule according to the second aspect of the present invention or the vector according to the third aspect of the present invention into an NK cell, thereby obtaining the NK cell.

Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequences encoding the enzymes of the invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of 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. Representative examples are: bacterial cells of the genera escherichia coli, bacillus subtilis, streptomyces; fungal cells such as pichia, saccharomyces cerevisiae cells; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, NS0, COS7, or 293 cells. In a preferred embodiment of the invention, the NK cell is selected as a host cell. In another preferred embodiment of the invention, T cells are selected as host cells.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the protein encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The protein in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If desired, the proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

Preparation method

The fusion protein (polypeptide) of the present invention may be a recombinant polypeptide or a synthetic polypeptide. The polypeptides of the invention may be chemically synthesized, or recombinant. Accordingly, the polypeptides of the present invention can be artificially synthesized by a conventional method or can be produced by a recombinant method. The present invention uses conventional recombinant DNA techniques to express or produce the fusion proteins of the present invention using the polynucleotides of the present invention.

The present invention provides a method for preparing an engineered immune cell, the method comprising transfecting a polynucleotide or vector of the present invention into a T cell or NK cell, thereby obtaining the engineered immune cell.

Generally, the following steps are performed:

(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a fusion protein of the invention, or with a recombinant expression vector (especially a viral vector, such as a lentiviral vector) comprising the polynucleotide;

(2) a host cell (such as a T cell or NK cell) cultured in a suitable medium.

Pharmaceutical compositions and methods of administration

The engineered immune cell can specifically kill and kill tumors which specifically express the Glypican-3 antigen.

In another aspect, the invention provides a pharmaceutical (including vaccine) composition comprising (a) a safe and effective amount of an engineered immune cell of the invention; and (b) a pharmaceutically acceptable carrier or excipient.

The "active ingredient" in the pharmaceutical composition of the present invention refers to the engineered immune cells of the present invention.

The active ingredient and the pharmaceutical composition can be used for killing tumors specifically expressing the Glypican-3 antigen.

In a preferred embodiment, the pharmaceutical composition of the invention further comprises other drugs for treating cancer or tumor (such as emerging antibody drugs, CAR-T drugs, or chemotherapeutic drugs, etc.).

"safe and effective amount" means: the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects.

Typically, the pharmaceutical composition contains 1-2000mg of active ingredient per dose, more preferably, 10-200mg of active ingredient per dose. Preferably, the dose is a tablet or an injection.

"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity.

By "compatible" is meant herein that the components of the composition are capable of being combined with the active ingredients of the present invention and with each other without significantly diminishing the efficacy of the active ingredient.

Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), and the like

) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The mode of administration of the active ingredient or pharmaceutical composition of the present invention is not particularly limited, and representative modes of administration include, but are not limited to: oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and the like.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.

In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient or carrier, such as sodium citrate or dicalcium phosphate, or with one or more of the following:

(a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and/or (i) a lubricant, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof.

In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

The solid dosage forms may also be prepared using coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active ingredient in such compositions may be delayed in a certain portion of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, especially cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like. In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active ingredients, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these materials, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof. Generally, the therapeutic compositions can be prepared as injectables, e.g., as liquid solutions or suspensions; solid forms suitable for constitution with a solution or suspension, or liquid carrier, before injection, may also be prepared.

When the pharmaceutical composition of the present invention is used for practical treatment, it may be in various dosage forms depending on the use case, preferably injection or liquid formulation.

When the pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) to be treated, wherein the administration dose is a pharmaceutically-considered effective administration dose, and for a human body with a weight of 60kg, the daily administration dose is usually 1 to 2000mg, preferably 20 to 500 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The pharmaceutical compositions of the invention may be administered alone or in combination with (e.g., formulated in the same pharmaceutical composition as) other therapeutic agents.

The pharmaceutical compositions of the present invention may also be combined with other agents known to treat or ameliorate similar conditions. When the combination is administered, the mode and dosage of administration of the original drug is maintained, while the pharmaceutical composition of the present invention is administered simultaneously or subsequently. The combination also includes administering the pharmaceutical composition of the present invention in an overlapping time period with one or more other known drugs. When the pharmaceutical composition of the present invention is administered in combination with one or more other drugs, the dosage of the pharmaceutical composition of the present invention or known drugs may be lower than the dosage when they are administered alone.

The main advantages of the invention include:

(1) the engineered immune cell provided by the invention can specifically kill tumor specifically expressing a Glypican-3 antigen, so that the CAR-NK and CAR-T targeting GPC3 are expected to become a novel cellular immunotherapy strategy combining other antibody drugs and targets.

(2) The CAR designed by the invention has better targeting specificity and lower cytotoxicity.

(3) The engineered immune cells provided by the invention can be used together with other medicines for treating cancers or tumors, so that the cancers or tumors can be effectively treated.

(4) After the CAR-T cell interacts with an antigen, the CAR-T cell can efficiently secrete IFN-gamma cytokines and specifically kill GPC3 positive liver cancer cells, and a foundation is laid for further advancing GPC3CAR-T cell preclinical and clinical research.

(5) The CAR-T cell of the invention explores a new way for treating GPC3 positive liver cell liver cancer.

(6) The CAR-T cell lays an experimental foundation for further carrying out clinical transformation of the CAR-T cell targeting GPC3 for treating HCC.

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, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Example 1: construction of the recombinant plasmid pS338B empty vector, pNB338B-M3C11 and pNB338B-0599

The Shanghai Jervy organism company was entrusted with the synthesis of the sequence sDTS (SEQ ID NO:1), and XbaI cleavage sites were introduced upstream of the sequence and PvuI cleavage sites were inserted downstream thereof, and EF1 in the XbaI + PvuI double-digested pNB328 vector (the structure and sequence of the pNB328 vector are referred to CN201510638974.7, which is herein incorporated by reference in its entirety and contains the EF1 α promoter) was incorporated as a recombinant plasmid designated pNB 338B.

Shanghai Czeri Bio Inc. was entrusted with the synthesis of GPC3-CAR gene, the structural pattern of which is shown in FIG. 1. The GPC3-CAR gene was packaged into pNB338B vector double-digested with EcoR1+ SalI, and named pNB338B-M3C 11. In the structural mode diagram, the nucleotide sequence of a GPC3-CAR gene is shown as SEQ ID NO. 2, the amino acid sequence of a GPC3-CAR is shown as SEQ ID NO. 3, and the GPC3-CAR gene sequentially comprises a CD8 signal peptide, a single-chain antibody M3C11 (the amino acid sequence is shown as SEQ ID NO. 11) targeting GPC3, an IgG4 hinge region, a CD28 transmembrane region, a CD28 intracellular costimulatory region and a CD3zeta signaling domain.

The invention also contemplates a CAR, wherein the V of the scFv in the CAR_LThe sequence is 23-134, V of SEQ ID No. 5_HThe sequence is 150-264 of SEQ ID NO. 5, and the linker connecting VL and VH is (GGGGS)₃The amino acid sequence of the scFv sequence is shown as SEQ ID NO. 4. The amino acid residues 23-263 of SEQ ID NO. 3 were substituted with SEQ ID NO. 4 to form GPC3CAR-0599, the sequence of GPC3CAR-0599 is shown in SEQ ID NO. 5, and the nucleotide sequence encoding GPC3CAR-0059 is shown in SEQ ID NO. 6. As described above, the vector of pNB338B, which was double digested with EcoR1+ SalI and designated as pNB338B-0599, was loaded withSEQ ID NO 6.

Example 2: positive rate of CAR-T cells detected by parallel flow of electrotransformation PBMC

1. Control Mock-T cells and CAR-T cells were obtained by electroporating PBMC (PBMC purchased from AllCells, lot: A0043) as follows:

(1) collecting the suspension cells into a 50ml centrifuge tube, centrifuging for 3min at 1200 rmp;

(2) discarding the supernatant, adding physiological saline for resuspension, 1200rmp, centrifuging for 3min, discarding the physiological saline, repeating the steps, and counting the cells;

(3) two 1.5ml centrifuge tubes were added to each tube at5X 10⁶Individual cells, No. A, B, C, 1200rmp, centrifuged for 3 min;

(4) discarding the supernatant, taking an electric transfer kit (purchased from Lonza company), adding 18 μ l of solubilization I reagent and 82 μ l of solubilization II reagent, adding 6 μ g of pNB338B-MCS no-load plasmid into tube A for comparison, adding 6 μ g of pNB338B-M3C11 plasmid into tube B, and adding 6 μ g of pNB338B-0599 into tube C;

(5) transferring the cells in the centrifuge tube into an electric rotor, putting the cells into an electric rotor, selecting a required program, and performing electric shock;

(6) transferring the cell suspension into a twelve-well plate (AIM-V culture solution containing 2% FBS) containing AIM-V culture solution by using a micropipette in the kit, mixing uniformly, and culturing at 37 deg.C in a 5% CO2 incubator;

(7) transferring the cells into a six-hole plate coated with antigen in advance after six hours, adding a stimulating factor IL-2, supplementing culture solution until 3ml, culturing for 4-5 days, observing the growth condition of T cells, and respectively obtaining GPC3CAR-T cells and GPC3CAR-T-0599 cells from the cells transferred with pNB338B-M3C11 and pNB338B-0599 plasmids; the cells transfected with pNB338B-MCS in the unloaded state were Mock-T cells as controls.

2. Flow detection of CAR-T cell positivity

(1) Mock-T, GPC3CAR-T and GPC3CAR-T-0599 cells were collected separately, each at1X 10⁶Centrifuging the cells at 1000rpm for 3 min;

(2) discarding the supernatant, adding physiological saline to resuspend the cells respectively, centrifuging at 1000rpm for 3 min;

(3) the supernatant was discarded, 100. mu.l of physiological saline was added to each tube to resuspend the cells, 1. mu.l of Biotin-labeled GPC3 antigen (Biotin labeling was assigned to King.) was added to each tube, and the mixture was incubated at 4 ℃ for 30 minutes;

(4) adding proper amount of normal saline respectively, centrifuging at 1000rpm for 3min, washing twice, and discarding the supernatant;

(5) adding 100 μ l physiological saline, adding 1ul PE-labeled streptavidin, mixing, and incubating at 4 deg.C for 30 min;

(6) adding proper amount of normal saline respectively, centrifuging at 1000rpm for 3min, washing twice, and discarding the supernatant;

(7) resuspend with 400. mu.l physiological saline and test on a machine.

The results are shown in FIG. 2. FIG. 2 shows that the Mock-T cell positivity rate is substantially close to 0, and the GPC3CAR-T cell positivity rate is close to 60%, which is significantly higher than 41.54% of the GPC3CAR-T-0599 cell positivity rate.

Example 3: detection of CAR gene expression in CAR-T cells by Western Blotting method

1. Protein extraction

(1) Collecting cells to 1.5ml EP tube, centrifuging at 1000rpm for 3min, discarding supernatant, adding physiological saline or PBS to wash cells, continuing to rotate at 1000rpm, centrifuging for 3min, discarding supernatant, and leaving cell precipitate;

(2) adding lysis solution, blowing uniformly to break up cells, lysing on crushed ice for 10min, centrifuging at 4 deg.C and 12000rpm for 10min, and collecting supernatant;

(3) adding a proper amount of 5x sample buffer into the collected supernatant, uniformly mixing in a water bath at 56 ℃, and boiling for 10min at 98 ℃;

(4) placing on ice for 2min after boiling, storing at-20 deg.C for a short period, and storing at-80 deg.C for a long period.

SDS-PAGE electrophoresis

(1) Assembling the glass plate and the rubber frame according to the program of the device, and preparing separation rubber and concentrated rubber according to the kit specification;

(2) filling separation glue between grooves of a glass plate, wherein the separation glue approximately occupies the volume of thegrooves 2/3, pressing and standing for 15mins by using ethanol, removing ethanol, adding concentrated glue, inserting a comb, and standing for 15min when an obvious boundary line is formed between the separation glue and the ethanol;

(3) assembling the SDS-PAGE electrophoresis device according to the program of the device, filling the assembled SDS-PAGE electrophoresis device into a glass plate for glue making, fixing the assembled SDS-PAGE electrophoresis device, adding electrophoresis buffer solution (if only one rubber plate is used, and the balance plate is needed on the other side), wherein the liquid does not exceed the volume between the two rubber plates in the electrophoresis tank, and the buffer solution outside the electrophoresis tank only needs to be added to the scales on the tank;

(4) carefully pulling out the comb for preparing the gel, carefully keeping the integrity of the sample hole, adjusting the sample loading amount (10-20ul) according to the size of the sample hole and the concentration of the sample, and correspondingly reducing the Marker amount;

(5) setting electrophoresis program, constant voltage/constant current (currently, the common electrophoresis condition: 80V 30mins 120V60mins (voltage is adjustable, maximum is not more than 200V)

3. Rotary film

(1) After electrophoresis is finished, slightly disassembling the glass plate, cutting off redundant glue, and putting the cut glue blocks into a membrane transferring buffer solution to keep a wet state; cutting a PVDF membrane with a proper size, and placing the PVDF membrane in methanol for activation for 30 s; 6 pieces of filter paper slightly larger than the PVDF film (0.3-0.5 cm in length and width) are cut

(2) Wet rotation: placing the sponge cushion, the filter paper, the PVDF membrane and the glue into a membrane transferring buffer solution for balancing for 10min, assembling membrane transferring equipment (white splint-sponge cushion-three layers of filter paper-PVDF membrane-glue-three layers of filter paper-sponge cushion-black splint) in the following sequence, filling the membrane transferring solution into an electrophoresis tank, placing an ice bag, and starting to transfer the membrane under the condition of the membrane transferring being good. At present, the laboratory is usually used under the conditions of 150-.

4. Sealing of

The PVDF membrane was taken out from the electrotransformation apparatus, placed in a special small box, soaked in 5% BSA or 5% skim milk, and blocked on a shaker at room temperature for 2 h.

5. Incubation primary antibody

(1) Taking out the PVDF membrane from the sealing liquid, washing for 2-3 times and 5 mins/time by TBST;

(2) the primary anti-Mouse anti-human CD247 was diluted according to the antibody instructions, and the diluted primary antibody was added to the antibody incubation cassette, overnight at 4 ℃, and after completion washed 5 times 5 mins/time on a shaker with TBST.

6. Incubation secondary antibody

Diluting the secondary antibody HRP Goat anti mouse IgG according to the secondary antibody specification, adding the diluted secondary antibody into an antibody incubation box, incubating at normal temperature for 40-60mins, and washing with TBST on a shaking table for 5 times and 5 mins/time

7. Development

Determining the dosage of the luminous liquid according to the size of the PVDF film, taking the liquid A and the liquid B with equal volumes, uniformly mixing and storing for later use, taking out the film by using forceps, spreading the film on a layer of preservative film, adding the mixed solution in the center of the film, and placing the film in an AI600 chemiluminescence imager for photographing.

The results are shown in figure 3 and indicate that GPC3CAR-T cells electroporated with plasmids pNB338B-M3C11 and pNB338B-0599 can express exogenous CAR protein, wherein GPC3CAR-T and GPC3CAR-T-0599 both express exogenous CD3 ζ molecular weight of about 74KD, whereas Mock-T does not express exogenous CD3 ζ; in addition both Mock-T, GPC3CAR-T and GPC3CAR-T-0599 cells expressed endogenous CD3 ζ with a molecular weight of 17 kD.

Example 4: RT-PCR detection of copy number of GPC3CAR-T cell expression

Mock-T cells and GPC3CAR-T cells from day 13 after electroporation in example 2 were treated at1X 10⁶Cell number cell pellets were collected, washed 1 time with PBS, and cell genomic dna (gDNA) was extracted using gDNA extraction kit (9765, Takara). Experimental procedures the copy number of the CAR gene (for CD28 detection) inserted in the genomic DNA was determined using real-time fluorescent quantitative PCR (qRT-PCR) with reference to the instructions attached to the kit. The reaction procedure is as follows: 50 ℃, 2min → 95 ℃,10min → 95 ℃,15s → 60 ℃,1min, 40 cycles. The primer sequences used for the fluorescent quantitative PCR were as follows:

CD28-F 5’CTCCTGCACAGTGACTACATG(SEQ ID NO:7)

CD28-R 5’GAACTTCACTCTGGAGCGATAG(SEQ ID NO:8)

CD28LNA probe 5’ccgCaaGcaTtaCcagcc(SEQ ID NO:9)

wherein the capital letters in SEQ ID NO 9 are nucleotides containing a locked nucleic acid structure.

And calculating the absolute copy number content according to the obtained CT value of the GPC3-CAR genome and the CT value of Actin according to corresponding formulas.

The results are shown in FIG. 4. No copies of CAR were detected in the Mock-T cell assay, the copy number of CAR detected in the GPC3-CAR-T cells was about 4.4 per cell, as large as 2.7 per cell in the GPC3-CAR-T-0599 cells, and slightly higher than that of GPC3-CAR-T-0599 cells, but at essentially the same level in the GPC3-CAR-T-0599 cells.

Example 5: testing of GPC3CAR-T and GPC3CAR-T-0599 cell killing function

The in vitro killing activity of the GPC3CAR-T cells and GPC3CAR-T-0599 obtained in example 2 was examined using a real-time unlabelled cell function Analyzer (RTCA) from the company Aisen, as follows:

(1) zero setting: adding 50 mul of DMEM culture solution into each hole, putting the DMEM culture solution into an instrument, selecting thestep 1, and adjusting zero;

(2) target cell plating: human hepatoma cell Huh-7 and human hepatoma cell Hep3B (purchased from American type culture Collection ATCC) 10 per well⁴Laying 50 mu l of each cell in a plate containing a detection electrode, standing for several minutes, placing the cell in an instrument after the cell is stabilized, starting thestep 2, and culturing the cell;

(3) adding effector cells: after the target cells are cultured for 24 hours, suspending thestep 2, respectively adding effector cells Mock-T, GPC3CAR-T and GPC3CAR-T-0599 with the effective-target ratio of 8:1 in 50 ul per well, starting thestep 3, and observing a cell proliferation curve after co-culturing for 24 hours;

the results are shown in FIG. 5. FIG. 5 shows a comparison of the killing effect of GPC3CAR-T and GPC3CAR-T-0599 cells prepared in example 2 on Huh-7 cells and Hep3B cells. The results show that the killing effect of GPC3CAR-T and GPC3CAR-T-0599 cells on various tumor cells is obviously stronger than that of MOCK-T cells, and the killing effect of GPC3CAR-T on target cells is stronger than that of GPC3 CAR-T-0599.

Example 6: contrast of cytokine release by MOCK-T, GPC3CAR-T and GPC3CAR-T-0599 cells under specific stimulation by GPC3 antigen

The sequence of the artificially synthesized and coded GPC3 protein is a DNA sequence shown in SEQ ID NO. 10, and the 5 'end and the 3' end are respectively connected with restriction enzyme site linkers of EcoRI and XbaI and then connected to a pCDNA3.4 vector to construct an expression vector for over-expressing the GPC3 protein. According to ExpicHO^TMInstructions for expression System, Using ExpicHO^TMAfter the above fusion protein was overexpressed by the expression system, the expression product was purified by using MabSelect affinity chromatography resin from GE Healthcare according to the procedures described in the specification, to obtain purified GPC3 antigen.

Coating 24-well plate with 5 μ g/ml GPC3 antigen, coating overnight at 4 deg.C, washing 3 times with PBS, and adding 1 × 10⁶Each (1ml volume) of the Mock-T, GPC3CAR-T, GPC3CAR-T-0599 cells prepared in example 2 was cultured for 24h and the cell supernatant was collected. Using BD^TMThe CBA Human Th1/Th2Cytokine Kit II detects the secretion of the cytokines after the two T cells are stimulated by GPC3 antigen, and the specific steps are as follows:

(1) mixing human IL-2, IL-4, IL-6, IL-10, TNF and IFN-gamma capture magnetic beads, vortexing, vibrating, mixing the capture magnetic beads uniformly, and adding 50 mu l of the mixed capture magnetic beads into each tube;

(2) mu.l of human Th1/Th2cytokine standard (diluted 2-fold in diluent) and 50. mu.l of test sample (5000 pg/ml, 2500pg/ml, 1250pg/ml, 625pg/ml, 312.5pg/ml, 156pg/ml, 80pg/ml, 40pg/ml, 20pg/ml, 0pg/ml) were added.

(3) Add 50. mu.l of human Th1/Th2-II-PE detection antibody to each tube;

(4) incubating for 3h at room temperature in a dark place;

(5) adding 1ml of washing buffer solution into each tube, centrifuging for 5min at 200 ℃, and removing supernatant;

(6) add 300. mu.l of wash buffer to each tube, resuspend the cells, and transfer to a flow tube and detect fluorescence using a flow cytometer.

Results As shown in FIG. 6, FIG. 6 shows the secretion levels of IL-2, IL-4, IL-6, IL-10, TNF- α and IFN- γ after stimulation with GPC3 antigen both GPC3CAR-T and GPC3CAR-T-0599 cells secrete significantly higher amounts of IL-2, IL-4, IL-6, IL-10, TNF- α and IFN- γ than MOCK-T cells, and GPC3CAR-T cells secrete higher levels of various cytokines after antigen stimulation than GPC3CAR-T-0599 cells.

Example 7: cellular phenotyping analysis of GPC3CAR-T cells

MOCK-T, GPC3CAR-T and GPC3CAR-T-0599 cells obtained in example 2 were collected, counted at1X 10⁶Adding each cell/tube into 1.5ml of EP tube, washing with PBS twice, centrifuging at 1200rpm for 5min, discarding supernatant, adding flow antibodies anti-PD-1-PE, anti-TIM-3-PE and anti-LAG-3-APC for detecting T cell exhaustion phenotype, flow antibodies anti-CD107 α -PECY5, anti-CD69-PECY5 and anti-CD25-PECY5 for activating phenotype, adding flow antibodies anti-CD45RO-PECY5+ anti-CD197-FITC + anti-CD62L-PE for detecting memory T cell phenotype into 1 tube, mixing the mixture by flicking, incubating at room temperature for 30min, washing, centrifuging at 1200rpm for 5min, discarding supernatant, adding 400 μ l of physiological saline, transferring the cells into flow tubes, and detecting on machine.

Experimental results as shown in figure 7, flow-assay GPC3CAR-T and GPC3CAR-T-0599 cells showed significantly higher expression of exhaustion phenotype PD1 than MOCK-T cells, again, due to a corresponding increase in expression of exhaustion markers on the cell surface after activation of GPC3CAR-T cells, and GPC3CAR-T cells showed higher PD-1 expression levels than GPC3 CAR-T-0599. likewise,exhaustion phenotype markers 3 and LAG3 were also higher than MOCK-T cells, and also expression ofLAG 7 of GPC3CAR-T cells was significantly higher than both expression levels of GPC3CAR-T-0599, 3 were substantially equivalent, expression of activation phenotype CD107, CD69 and CD25 of GPC3CAR-T-0599 cells were all significantly higher than MOCK-T cells, indicating that GPC3CAR-T cells were fully activated after antigen stimulation and that CAR-T-052 cells were stimulated by GPC3CAR-T cells, CD 369 and CD 3699 cells were significantly more responsive to GPC3CAR-T-0599 cells than central CD-T cells, CD 5634 and CD 5634-T-0599 cells showed that the expression of GPC3CAR-T cells were significantly higher than CD-T-0599 cells, CD-T cells were stimulated by GPC3 cells, and CD-T-CD-T-c-T-c.

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> Shanghai cell therapy engineering research center group Co., Ltd

Shanghai cell therapy research institute

<120> chimeric antigen receptor modified T cell of targeted Glypican-3 antigen and application thereof

<130>P2018-0956

<160>12

<170>PatentIn version 3.5

<210>1

<211>144

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>1

ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 60

agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 120

tgcatctcaa ttagtcagca acca 144

<210>2

<211>2019

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>2

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgagcgatg ttgtgatgac ccagactcca ctcactttgt cggttaccat tggacaacca 120

gcctccatct cttgcaagtc aagtcagagc ctcttagata gtgatggaaa gacatatttg 180

aattggttgt tacagaggcc aggccagtct ccaaagcgcc taatctatct ggtgtctaaa 240

ttggactctg gagcccctga caggttcact ggcagtggat cagggacaga tttcacactg 300

aaaatcagta gagtggaggc tgaggatttg ggaatttatt attgctggca aggtacacat 360

tttccgctca cgttcggtgc tgggaccaag ctggagctga aaggtggagg cggttcaggc 420

ggaggtggca gcggcggtgg cgggtcggag gtgcaactgg tggagtctgg gggaggctta 480

gtgaagcctg gaggatccct gaaactctcc tgtgcagcct ctggattcac tttcagtcgc 540

tatgccatgt cttgggttcg ccagattcca gagaagatac tggagtgggt cgcagccatt 600

gatagtagtg gtggtgacac ctactattta gacactgtga aggaccgatt caccatctcc 660

agagacaatg ccaataatac cctgcacctg caaatgcgca gtctgaggtc tgaggacaca 720

gccttgtatt actgtgtaag acaggggggg gcttactggg gccaagggac tctggtcact 780

gtctctgcag agtccaaata tggtccccca tgcccaccat gcccagcacc tcccgtggcc 840

ggaccatcag tcttcctgtt ccccccaaaa cccaaggaca ctctcatgat ctcccggacc 900

cctgaggtca cgtgcgtggt ggtggacgtg agccaggaag accccgaggt ccagttcaac 960

tggtacgtgg atggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagttc 1020

cagagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaacggc 1080

aaggagtaca agtgcaaggt ctccaacaaa ggcctcccgt cctccatcga gaaaaccatc 1140

tccaaagcca aagggcagcc ccgagagcca caggtgtaca ccctgccccc atcccaggag 1200

gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta ccccagcgac 1260

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1320

gtgctggact ccgacggctc cttcttcctc tacagcaggc taaccgtgga caagagcagg 1380

tggcaggagg ggaatgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1440

acacagaaga gcctctccct gtctctgggt aaaccctttt gggtgctggt ggtggttggt 1500

ggagtcctgg cttgctatag cttgctagta acagtggcct ttattatttt ctgggtgagg 1560

agtaagagga gcaggctcct gcacagtgac tacatgaaca tgactccccg ccgccccggg 1620

cccacccgca agcattacca gccctatgcc ccaccacgcg acttcgcagc ctatcgctcc 1680

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 1740

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 1800

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 1860

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 1920

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 1980

tacgacgccc ttcacatgca ggccctgccc cctcgctga 2019

<210>3

<211>672

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>3

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 Ser Asp Val Val Met Thr Gln Thr Pro Leu Thr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Leu Asp Ser Gly Ala Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr

85 90 95

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

100 105 110

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

115 120 125

Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

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

145 150 155 160

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

165 170 175

Thr Phe Ser Arg Tyr Ala Met Ser Trp Val Arg Gln Ile Pro Glu Lys

180 185 190

Ile Leu Glu Trp Val Ala Ala Ile Asp Ser Ser Gly Gly Asp Thr Tyr

195 200 205

Tyr Leu Asp Thr Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala

210 215 220

Asn Asn Thr Leu His Leu Gln Met Arg Ser Leu Arg Ser Glu Asp Thr

225 230 235 240

Ala Leu Tyr Tyr Cys Val Arg Gln Gly Gly Ala Tyr Trp Gly Gln Gly

245 250 255

Thr Leu Val Thr Val Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro

260 265 270

Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

275 280 285

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

290 295 300

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

305 310 315 320

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

325 330 335

Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

340 345 350

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

355 360 365

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

370 375 380

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

385 390 395 400

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

405 410 415

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

420 425 430

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

435 440 445

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

450 455 460

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

465 470 475 480

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Pro Phe Trp Val Leu

485 490 495

Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val

500 505 510

Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His

515 520 525

Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys

530 535 540

His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser

545 550 555 560

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

565 570 575

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

580 585 590

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

595 600 605

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

610 615 620

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

625 630 635 640

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

645 650 655

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

660 665 670

<210>4

<211>242

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>4

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

AsnGly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe 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 Phe Cys Ser Gln Asn

85 90 95

Thr His Val Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

115 120 125

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

130 135 140

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

145 150 155 160

Met His Trp Val Lys Gln Thr Pro Val His Gly Leu Lys Trp Ile Gly

165 170 175

Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys

180 185 190

Gly Lys Ala ThrLeu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met

195 200 205

Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr

210 215 220

Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ala

<210>5

<211>673

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>5

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 Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser

20 25 30

Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser

35 40 45

Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu

50 55 60

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

65 70 75 80

Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

85 90 95

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

100 105 110

Tyr Phe Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Ser Gly

115 120 125

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

130 135 140

Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu

145 150 155 160

Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr

165 170 175

Thr Phe Thr Asp Tyr Glu Met His Trp Val Lys Gln Thr Pro Val His

180 185 190

Gly Leu Lys Trp Ile Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala

195 200 205

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

210 215 220

Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser

225 230 235 240

Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln

245 250 255

Gly Thr Leu Val Thr Val Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys

260 265 270

Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe

275 280 285

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

290 295 300

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

305 310 315 320

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

325 330 335

Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr

340 345 350

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

355 360 365

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

370 375 380

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

385 390 395 400

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

405 410 415

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

420 425 430

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

435 440 445

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

450 455 460

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

465 470 475 480

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Pro Phe Trp Val

485 490 495

Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr

500 505 510

Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu

515 520 525

His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg

530 535 540

Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg

545 550 555 560

Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

565 570 575

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

580 585 590

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

595 600 605

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

610 615 620

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

625 630 635 640

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

645 650 655

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

660 665 670

Arg

<210>6

<211>2022

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>6

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgagcgatg ttgtgatgac ccaaactcca ctctccctgc ctgtcagtct tggagatcaa 120

gcctccatct cttgcagatc tagtcagagc cttgtacaca gtaatggaaa cacctattta 180

cattggtacc tgcagaagcc aggccagtct ccaaagctcc tgatctacaa agtttccaac 240

cgattttctg gggtcccaga caggttcagt ggcagtggat cagggacaga tttcacactc 300

aagatcagca gagtggaggc tgaggatctg ggagtttatt tctgctctca aaatacacat 360

gttcctccta cgttcggatc ggggaccaag ctggaaataa aaggtggagg cggttcaggc 420

ggaggtggca gcggcggtgg cgggtcgcag gttcaactgc agcagtctgg ggctgagctg 480

gtgaggcctg gggcttcagt gaagctgtcc tgcaaggctt cgggctacac atttactgac 540

tatgaaatgc actgggtgaa gcagacacct gtgcatggcc taaaatggat tggagctctt 600

gatcctaaaa ctggtgatac tgcctacagt cagaagttca agggcaaggc cacactgact 660

gcagacaaat cctccagcac agcctacatg gagctccgca gcctgacatc tgaggactct 720

gccgtctatt actgtacaag attctactcc tatacttact ggggccaagg gactctggtc 780

actgtctctg cagagtccaa atatggtccc ccatgcccac catgcccagc acctcccgtg 840

gccggaccat cagtcttcct gttcccccca aaacccaagg acactctcat gatctcccgg 900

acccctgagg tcacgtgcgt ggtggtggac gtgagccagg aagaccccga ggtccagttc 960

aactggtacg tggatggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 1020

ttccagagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaac 1080

ggcaaggagt acaagtgcaa ggtctccaac aaaggcctcc cgtcctccat cgagaaaacc 1140

atctccaaag ccaaagggca gccccgagag ccacaggtgt acaccctgcc cccatcccag 1200

gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctaccccagc 1260

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1320

cccgtgctgg actccgacgg ctccttcttc ctctacagca ggctaaccgt ggacaagagc 1380

aggtggcagg aggggaatgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1440

tacacacaga agagcctctc cctgtctctg ggtaaaccct tttgggtgct ggtggtggtt 1500

ggtggagtcc tggcttgcta tagcttgcta gtaacagtgg cctttattat tttctgggtg 1560

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 1620

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 1680

tccagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 1740

ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 1800

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1860

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1920

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1980

acctacgacg cccttcacat gcaggccctg ccccctcgct ga 2022

<210>7

<211>21

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>7

ctcctgcaca gtgactacat g 21

<210>8

<211>22

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>8

gaacttcact ctggagcgat ag 22

<210>9

<211>18

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>9

ccgcaagcat taccagcc 18

<210>10

<211>1302

<212>DNA

<213> Artificial sequence (artificial sequence)

<400>10

atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60

gctcatgtag aacatgaaga aaccttatcc agccgaagaa gggaactaat tcagaagttg 120

aagtctttca tcagcttcta tagtgctttg cctggctaca tctgcagcca tagccctgtg 180

gcggaaaacg acaccctttg ctggaatgga caagaactcg tggagagata cagccaaaag 240

gcagcaagga atggaatgaa aaaccagttc aatctccatg agctgaaaat gaagggccct 300

gagccagtgg tcagtcaaat tattgacaaa ctgaagcaca ttaaccagct cctgagaacc 360

atgtctatgc ccaaaggtag agttctggat aaaaacctgg atgaggaagg gtttgaaagt 420

ggagactgcg gtgatgatga agatgagtgc attggaggct ctggtgatgg aatgataaaa 480

gtgaagaatc agctccgctt ccttgcagaa ctggcctatg atctggatgt ggatgatgcg 540

cctggaaaca gtcagcaggc aactccgaag gacaacgaga taagcacctt tcacaacctc 600

gggaacgttc atgagtccaa atatggtccc ccatgcccac catgcccagc acctgagttc 660

ctggggggac catcagtctt cctgttcccc ccaaaaccca aggacactct catgatctcc 720

cggacccctg aggtcacgtg cgtggtggtg gacgtgagcc aggaagaccc cgaggtccag 780

ttcaactggt acgtggatgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 840

cagttcaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 900

aacggcaagg agtacaagtg caaggtctcc aacaaaggcc tcccgtcctc catcgagaaa 960

accatctcca aagccaaagg gcagccccga gagccacagg tgtacaccct gcccccatcc 1020

caggaggaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctacccc 1080

agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1140

cctcccgtgc tggactccga cggctccttc ttcctctaca gcaggctaac cgtggacaag 1200

agcaggtggc aggaggggaa tgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1260

cactacacac agaagagcct ctccctgtct ctgggtaaat ga 1302

<210>11

<211>241

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>11

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

Asp Gly Lys 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 Ala 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 Ile Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

115 120 125

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

130 135 140

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

145 150 155 160

Met Ser Trp Val Arg Gln Ile Pro Glu Lys Ile Leu Glu Trp Val Ala

165 170 175

Ala Ile Asp Ser Ser Gly Gly Asp Thr Tyr Tyr Leu Asp Thr Val Lys

180 185 190

Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Thr Leu His Leu

195 200 205

Gln Met Arg Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys Val

210 215 220

Arg Gln Gly Gly Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

225 230 235 240

Ala

<210>12

<211>5

<212>PRT

<213> Artificial sequence (artificial sequence)

<400>12

Gly Gly Gly Gly Ser

1 5

Claims (11)

1. A fusion protein having the structure of formula I:

L-scFv-Z-TM-C-X (I)，

wherein,

l is a null or signal peptide sequence;

scFv is a variable region sequence of a targeting Glypican-3 antibody single chain;

z is a null or hinge region;

TM is a transmembrane domain;

c is a costimulatory signal molecule;

x is the cytoplasmic signaling sequence CD3 ζ;

each "-" represents a linking peptide or peptide bond linking the above-mentioned respective elements.

2. The fusion protein of claim 1, wherein the scFv has the structure of formula a1 or a 2:

V_L1-V_H1(A1) (ii) a Or

V_H1-V_L1(A2)；

Wherein, V_L1Is the light chain variable region of an anti-Glypican-3 antibody; v_H1Is the heavy chain variable region of an anti-Glypican-3 antibody; "-" is a linker peptide (or flexible linker) or peptide bond.

3. The fusion protein of claim 1, wherein the scFv has the structure of formula A3 or a 4:

V_L2-V_H2(A3) (ii) a Or

V_H2-V_L2(A4)；

Wherein, V_L2Is the light chain variable region of an anti-Glypican-3 antibody; v_H2Is the heavy chain variable region of an anti-Glypican-3 antibody; "-" is a linker peptide (or flexible linker) or peptide bond.

4. The fusion protein of claim 1, wherein the fusion protein has an amino acid sequence as set forth in SEQ ID NO 3 or 5.

5. A nucleic acid molecule encoding the fusion protein of claim 1.

6. A vector comprising the nucleic acid molecule of claim 5.

7. A host cell comprising the vector or chromosome of claim 6 having integrated therein an exogenous nucleic acid molecule of claim 5 or expressing the fusion protein of claim 1.

8. A method of preparing an engineered immune cell expressing the fusion protein of claim 1, wherein the method comprises the steps of: transferring the nucleic acid molecule of claim 5 or the vector of claim 6 into an immune cell, thereby obtaining the engineered immune cell.

9. A pharmaceutical composition, wherein the composition comprises: the fusion protein of claim 1, the nucleic acid molecule of claim 5, the vector of claim 6, or the host cell of claim 7, and a pharmaceutically acceptable carrier, diluent, or excipient.

10. Use of the fusion protein of claim 1, the nucleic acid molecule of claim 5, the vector of claim 6, or the host cell of claim 7 for the preparation of a medicament or formulation for selective killing of tumor cells.

11. A kit for selectively killing tumor cells, comprising a container, and the fusion protein of claim 1, the nucleic acid molecule of claim 5, the vector of claim 6, or the host cell of claim 7 disposed within the container.

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