CN114853893A

CN114853893A - Target GPC3 chimeric antigen receptor T cell and application thereof

Info

Publication number: CN114853893A
Application number: CN202210168410.1A
Authority: CN
Inventors: 许元剑; 郭志刚
Original assignee: Nanjing Landun Biotechnology Co ltd
Current assignee: Nanjing Landun Biotechnology Co ltd
Priority date: 2022-02-23
Filing date: 2022-02-23
Publication date: 2022-08-05
Also published as: WO2023161846A1

Abstract

The invention provides a GPC3 targeted chimeric antigen receptor T cell (CAR-T) and application thereof. According to the invention, the scFv heavy chain and light chain targeting GPC3 are obtained by screening and used as CAR structures, and the scFv heavy chain and light chain have high affinity and high specificity, can well infiltrate into solid tumors, and can play an excellent tumor inhibition effect in animal bodies.

Description

Target GPC3 chimeric antigen receptor T cell and application thereof

Technical Field

The invention relates to the field of cellular immunotherapy. In particular to a GPC3 targeted chimeric antigen receptor T cell (CAR-T) and application thereof.

Background

The concept of CAR-T (Chimeric Antigen Receptor T cell) was first proposed by Zelig Ehhhar, Israel scientist in the PNAS article published in 1989, consisting of an Antigen binding region, a hinge region, a transmembrane region and an intracellular signal, undergoing several generations of structural development and optimization. Kymriah, a first global CAR-T product, was granted FDA approval in 2017 to be marketed for Acute Lymphocytic Leukemia (ALL) in children and young adults. Until now, a plurality of CAR-T products targeting CD19 or BCMA are marketed in a plurality of countries or regions, and achieve surprising treatment effects; however, these products are directed against hematological tumors, and no CAR-T product is currently approved for marketing against solid tumors, and most of the clinical outcomes of CAR-T for solid tumors are not as glaring as those of hematological tumors.

Glypican 3(GPC3) protein is a heparan sulfate glycoprotein on the surface of cell membranes, expressed in some tissues during infancy, particularly in the liver and kidney, and is an extracellular matrix protein associated with organ formation; the expression of GPC3 was hardly observed in adult tissues, but was highly expressed in various cancer tissues such as hepatocellular carcinoma, melanoma, clear ovarian cell carcinoma, and lung squamous cell carcinoma. Therefore, GPC3 is a good target for tumor therapy.

Therefore, there is a need in the art to develop a GPC 3-targeted chimeric antigen receptor T cell (CAR-T) product against solid tumors.

Disclosure of Invention

The invention aims to provide a GPC3 targeted chimeric antigen receptor T cell (CAR-T) and application thereof.

In a first aspect of the invention, there is provided a single chain variable fragment (scFv) targeting GPC3, the single chain variable fragment comprising a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region comprising the following three complementarity determining regions CDRs:

HCDR1 shown in SEQ ID NO.18,

HCDR2 shown in SEQ ID NO.19, and

HCDR3 shown in SEQ ID NO. 20; and/or

The light chain variable region comprises the following three complementarity determining regions CDRs:

LCDR1 shown in SEQ ID NO.21,

LCDR2 shown in SEQ ID NO.22, and

LCDR3 shown in SEQ ID NO. 23.

In another preferred embodiment, the single-chain variable fragment comprises SEQ ID NO: 3 and VH and SEQ ID NO: 7, VL of an amino acid sequence set forth in seq id no.

In another preferred embodiment, the scFv comprises a linker between VH and VL.

In another preferred embodiment, the scFv has the structure shown in formula a or b:

V _L -V _H (a)；

V _H -V _L (b)

wherein, V _H Is an antibody heavy chain variable region; v _L Is an antibody light chain variable region; "-" is a linker peptide or peptide bond.

In another preferred embodiment, the linker is a flexible linker, preferably the linker is (G4S) n, where n is 1-4, preferably n is 2-4, and more preferably n is 3.

In a second aspect of the invention, there is provided a Chimeric Antigen Receptor (CAR) fusion protein comprising, from N-terminus to C-terminus:

(i) the scFv according to the first aspect of the present invention,

(ii) (ii) a transmembrane domain which is capable of,

(iii) at least one co-stimulatory domain, and

(iv) an activation domain.

In another preferred embodiment, the chimeric antigen receptor fusion protein has the structure of formula I:

L-scFv-H-TM-C-CD3ζ(I)

in the formula (I), the compound is shown in the specification,

each "-" is independently a linker peptide or a peptide bond;

l is an optional signal peptide sequence;

the scFv is the scFv of the first aspect of the invention;

h is an optional hinge region;

TM is a transmembrane domain;

c is a costimulatory signal molecule;

CD3 ζ is the cytoplasmic signaling sequence derived from CD3 ζ.

In another preferred embodiment, L comprises the amino acid sequence shown in SEQ ID NO. 1.

In another preferred embodiment, said H is a hinge region selected from the group consisting of: CD8, CD28, CD137, or a combination thereof.

In another preferred embodiment, the H is a hinge region from which CD8 is derived.

In another preferred embodiment, the H comprises an amino acid sequence shown as SEQ ID NO. 9.

In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

In another preferred embodiment, the TM comprises a CD 8-derived transmembrane region.

In another preferred embodiment, the TM comprises the amino acid sequence shown in SEQ ID NO. 11.

In another preferred embodiment, C is a costimulatory signal molecule for a protein selected from the group consisting of: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

In another preferred embodiment, said C comprises a co-stimulatory signaling molecule from 4-1 BB.

In another preferred embodiment, said C comprises an amino acid sequence as shown in SEQ ID NO. 13.

In another preferred embodiment, the CAR fusion protein has the amino acid sequence of SEQ ID NO: 17.

In a third aspect of the invention, there is provided an antibody against GPC3, the antibody comprising a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region comprising the following three complementarity determining regions CDR:

HCDR1 shown in SEQ ID NO.18,

HCDR2 shown in SEQ ID NO.19, and

HCDR3 shown in SEQ ID NO. 20; and/or

LCDR1 shown in SEQ ID NO.21,

LCDR2 shown in SEQ ID NO.22, and

LCDR3 shown in SEQ ID NO. 23.

In another preferred embodiment, the antibody comprises SEQ ID NO: 3 and VH and SEQ ID NO: 7, VL of an amino acid sequence set forth in seq id no.

In another preferred embodiment, the antibody binds to human GPC3 protein.

In another preferred embodiment, the antibody is selected from the group consisting of: an antibody of animal origin, a chimeric antibody, a humanized antibody, or a combination thereof.

In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

In another preferred embodiment, the antibody is a monoclonal antibody.

In a fourth aspect of the present invention, there is provided a recombinant protein having:

(I) the scFv of the first aspect of the invention, the CAR fusion protein of the second aspect of the invention, or the antibody of the third aspect of the invention; and

(II) optionally a tag sequence to facilitate expression and/or purification.

In another preferred embodiment, the tag sequence comprises a 6His tag.

In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.

In a fifth aspect of the present invention, there is provided an antibody drug conjugate comprising:

(a) the scFv of the first aspect of the invention, the CAR fusion protein of the second aspect of the invention, or the antibody of the third aspect of the invention; and

(b) a coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

In another preferred embodiment, the antibody moiety is coupled to the coupling moiety via a chemical bond or a linker.

In a sixth aspect of the invention there is provided a nucleic acid molecule encoding a scFv according to the first aspect of the invention, a CAR fusion protein according to the second aspect of the invention or an antibody according to the third aspect of the invention.

In another preferred embodiment, the nucleic acid molecule encodes the scFv of the first aspect of the invention and has the nucleic acid sequence encoding VH shown in SEQ ID No.:4 and the nucleic acid sequence encoding VL shown in SEQ ID No.: 8.

In a seventh aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the sixth aspect of the invention.

In another preferred embodiment, the carrier is selected from the group consisting of: DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, transposons, or combinations thereof.

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

In an eighth aspect of the invention there is provided a host cell comprising a vector according to the seventh aspect of the invention or having integrated into the chromosome an exogenous nucleic acid molecule according to the sixth aspect of the invention or expressing an scFv according to the first aspect of the invention, a CAR fusion protein according to the second aspect of the invention or an antibody according to the third 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 cell is a mammalian cell.

In another preferred embodiment, the cell is a T cell.

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

In another preferred embodiment, the engineered immune cells comprise T cells or NK cells, preferably (i) chimeric antigen receptor T cells (CAR-T cells); or (ii) a chimeric antigen receptor NK cell (CAR-NK cell).

In a ninth aspect of the invention, there is provided a method of preparing an engineered immune cell expressing a CAR fusion protein according to the second aspect of the invention, comprising the steps of: transferring the nucleic acid molecule of the sixth aspect of the invention or the vector of the seventh aspect of the invention into a T cell or NK cell, thereby obtaining the engineered immune cell.

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

In a tenth aspect of the invention there is provided a formulation comprising a scFv of the first aspect of the invention, a CAR fusion protein of the second aspect of the invention, or a vector of the seventh aspect of the invention, or a host cell of the eighth aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

In an eleventh aspect of the invention, there is provided a use of the scFv of the first aspect of the invention, the CAR fusion protein of the second aspect of the invention, the antibody of the third aspect of the invention, the recombinant protein of the fourth aspect of the invention, or the antibody drug conjugate of the fifth aspect of the invention, or the cell of the eighth aspect of the invention, for the preparation of a medicament or formulation for the prevention and/or treatment of a GPC 3-related cancer or tumor.

In another preferred embodiment, the GPC 3-associated cancer or tumor is a solid tumor.

In another preferred embodiment, the GPC 3-related cancer or tumor is selected from the group consisting of: hepatocellular carcinoma, melanoma, ovarian cancer, lung squamous cell carcinoma, gastric cancer, breast cancer, or a combination thereof.

In a twelfth aspect of the invention, there is provided a kit for preparing a cell according to the eighth aspect of the invention, the kit comprising a container, and a nucleic acid molecule according to the sixth aspect of the invention, or a vector according to the seventh aspect of the invention, located in the container.

In a thirteenth aspect of the invention there is provided a use of a cell according to the eighth aspect of the invention, or a formulation according to the tenth aspect of the invention, for the prevention and/or treatment of a GPC 3-related cancer or tumour.

In a fourteenth aspect of the present invention there is provided a method of treating a disease comprising administering to a subject in need thereof an amount of a cell according to the eighth aspect of the present invention, or a formulation according to the tenth aspect of the present invention.

In another preferred embodiment, the disease is a cancer or tumor, preferably a GPC 3-related cancer or tumor.

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 schematic of GC76BB ζ structure.

FIG. 2 shows construction and detection of HuH7-Luc and HepG2-Luc cells.

Fig. 3 shows GC76BB ζ positive rate detection. Wherein, A represents T cells without transfected lentivirus and B represents CARR-T transduced lentivirus, and the same preparation process and CAR positive rate result of flow detection are adopted.

FIG. 4 shows GC76BB ζ kills HuH7-luc cells in vitro. Wherein, the A and B panels show the results of killing activity with E/T (effective target ratio) of 4 and E/T of 1, respectively. BM is the ScFv-constructed CAR-T of GPC3 antibody GC 33; UN-T is a T cell that is not transduced with lentivirus.

FIG. 5 shows GC76BB ζ kills HepG2-luc cells in vitro. Wherein, the A diagram and the B diagram respectively show the results of killing activity of E/T of 4 and E/T of 1.

Fig. 6 shows the in vivo tumor suppression effect of GC76BB ζ. Wherein, Panel A shows 1X10 dosing ⁶ CAR-T cell/mouse outcome; panel B shows 3x10 dosing ⁶ Results for CAR-T cells/mouse.

Detailed Description

The present inventors have extensively and intensively studied and, through a large number of screenings, obtained for the first time a chimeric antigen receptor T cell targeting GPC3, which targets the scFv of GPC3 as the antigen binding domain of CAR with high affinity and high specificity. The CAR-T cell disclosed by the invention has excellent target cell killing activity, can well infiltrate into a solid tumor, and can play an excellent tumor inhibition effect in an animal body. The present invention has been completed based on this finding.

Term(s) for

As used herein, a "CDR" is a complementarity determining region of an antibody VH or VL chain that is critical for binding to an antigen.

As used herein, "domain" refers to a region of a polypeptide that is independent of other regions and folds into a particular structure.

As used herein, "single chain variable fragment (scFv)" refers to a single chain polypeptide derived from an antibody that retains the ability to bind antigen. Examples of scfvs include antibody polypeptides formed by recombinant DNA techniques in which the Fv regions of immunoglobulin heavy (H chain) and light (L chain) chain fragments are linked via a spacer sequence. Various methods of engineering scFv are well known to those skilled in the art.

As used herein, "tumor antigen" refers to an antigenic biomolecule, the expression of which results in cancer.

GPC3

Chimeric Antigen Receptor (CAR)

As used herein, a "Chimeric Antigen Receptor (CAR)" is a fusion protein comprising an extracellular domain capable of binding an antigen, a transmembrane domain derived from a different polypeptide than the extracellular domain, and at least one intracellular domain. "Chimeric Antigen Receptors (CARs)" are also sometimes referred to as "chimeric receptors", "T-bodies" or "Chimeric Immunoreceptors (CIRs)". "extracellular domain capable of binding an antigen" refers to any oligopeptide or polypeptide capable of binding an antigen. "intracellular domain" refers to any oligopeptide or polypeptide known to function as a domain that transmits signals to activate or inhibit biological processes in a cell.

Specifically, the Chimeric Antigen Receptors (CARs) of the invention include an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding member (also referred to as an antigen-binding domain). The intracellular domain includes a costimulatory signaling region and a zeta chain moiety. The costimulatory signaling region refers to a portion of the intracellular domain that includes the costimulatory molecule. Costimulatory molecules are cell surface molecules required for efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands.

A linker may be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR.

As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to an extracellular domain or a cytoplasmic domain of a polypeptide chain. The linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids. Preferably, the linker is a flexible linker, e.g., the linker is (G4S) n, where n is 1-4.

The CARs of the invention, when expressed in T cells, are capable of antigen recognition based on antigen binding specificity. When it binds its associated antigen, it affects the tumor cells, causing the tumor cells to not grow, to be driven to death, or to otherwise be affected, and causing the patient's tumor burden to shrink or be eliminated. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and the zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of a combination of a CD28 signaling domain, and a CD3zeta signaling domain.

As used herein, "antigen binding domain" and "single chain antibody fragment" each refer to an Fab fragment, Fab 'fragment, F (ab') 2 fragment, or single Fv fragment having antigen binding activity. Fv antibodies contain the variable regions of the antibody heavy chain, the variable regions of the light chain, but no constant regions, and have the smallest antibody fragment of the entire antigen binding site. Generally, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding. The antigen binding domain is typically a scFv (single-chain variable fragment). The size of the scFv is typically 1/6 for a whole antibody. Single chain antibodies are preferably a sequence of amino acids encoded by a single nucleotide chain. In a preferred embodiment of the present invention, the scFv comprises an antibody, preferably a single-chain antibody, specifically recognizing the tumor highly expressed antigen GPC 3.

In a preferred embodiment of the invention, the antigen is GPC 3. In a preferred embodiment of the invention, the antigen binding domain of the CAR of the invention targets GPC 3.

Preferably, the antigen binding domain in the CAR of the invention comprises a single chain variable fragment (scFv) targeting GPC3, the structure of which is shown below (N-terminal to C-terminal):

V _L －V _H

wherein, V _H Is a heavy chain variable region; v _L Is a light chain variable region; "-" is a linker or a peptide bond.

In a preferred embodiment of the invention, the single chain variable fragment comprises a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region comprising the following three complementarity determining regions CDR:

HCDR1 shown in SEQ ID NO.18,

HCDR2 shown in SEQ ID NO.19, and

HCDR3 shown in SEQ ID NO. 20; and/or

LCDR1 shown in SEQ ID NO.21,

LCDR2 shown in SEQ ID NO.22, and

LCDR3 shown in SEQ ID NO. 23.

In the present invention, the scFv of the present invention also includes conservative variants thereof, which means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced with amino acids having similar or similar properties as compared with the amino acid sequence of the scFv of the present invention to form a polypeptide.

In the present invention, the number of amino acids to be added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20% of the total number of amino acids in the original amino acid sequence.

In the present invention, the number of the amino acids to be added, deleted, modified and/or substituted is usually 1, 2, 3, 4 or 5, preferably 1 to 3, more preferably 1 to 2, and most preferably 1.

For the hinge region and transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR.

In a preferred embodiment of the present invention, the amino acid sequence of the Chimeric Antigen Receptor (CAR) (GC76BB ζ) provided by the present invention is as follows:

MALPVTALLLPLALLLHAARPGSQVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLEWIGAIAPKTGNTAYNQKFKDKAILTADKSSSTAYMELRSLTSEDSAVYYCTRYYSYAYWGQGTLVTVSAGGGGSGGGGSGGGGSDVVMTQTPLSLSVSLGDQASISCRSSQSPVHSNGNTYLQWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEKKSGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO.17)

carrier

The invention also provides DNA constructs encoding the CAR sequences of the invention.

Nucleic acid sequences encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from vectors known to include the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically.

The present invention also provides a vector into which the expression cassette of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, since they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus, in that they can transduce non-proliferating cells such as hepatocytes. They also have the advantage of low immunogenicity.

In brief summary, an expression cassette or nucleic acid sequence of the invention is typically operably linked to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that may be used to regulate the expression of the desired nucleic acid sequence.

The expression constructs of the invention may also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Pat. nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the invention provides a gene therapy vector.

The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001, molecular cloning: A laboratory Manual, Cold spring harbor laboratory, N.Y.) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors comprise an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No.6,326,193).

Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

Additional promoter elements, such as enhancers, may regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that promoter function is maintained when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50bp apart, and activity begins to decline. Depending on the promoter, it appears that the individual elements may function cooperatively or independently to initiate transcription.

An example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1 α (EF-1 α). However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40(SV40) early promoter, the Mouse Mammary Tumor Virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr (Epstein-Barr) virus immediate early promoter, the rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch that is capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

To assess the expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cells can also comprise either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in a host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Typically, the reporter gene is the following: which is not present in or expressed by the recipient organism or tissue and which encodes a polypeptide whose expression is clearly indicated by some readily detectable property, such as enzymatic activity. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is assayed at an appropriate time. Suitable reporter genes may include genes encoding luciferase, β -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, the construct with the minimum of 5 flanking regions that showed the highest level of reporter gene expression was identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate the ability of an agent to modulate promoter-driven transcription.

Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vector may be readily introduced into a host cell by any method known in the art, e.g., mammalian, bacterial, yeast or insect cells. For example, the expression vector may be transferred into a host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing the polynucleotide into a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. patent nos. 5,350,674 and 5,585,362.

Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles).

In the case of non-viral delivery systems, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for use to introduce nucleic acids into host cells (ex vivo or in vivo). In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with the lipid may be encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution comprising the lipid, mixed with the lipid, associated with the lipid, contained as a suspension in the lipid, contained in or complexed with a micelle, or otherwise associated with the lipid. The lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution. For example, they may be present in bilayer structures, either as micelles or with a "collapsed" structure. They may also simply be dispersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include fatty droplets that occur naturally in the cytoplasm as well as such compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

The invention is illustratively accomplished using genome editing techniques, such as CRISPR-Cas9, ZFNs, or TALENs, where non-viral delivery systems are used.

In a preferred embodiment of the invention, the vector is a lentiviral vector.

The DNA construct further comprises a signal peptide coding sequence. Preferably, the signal peptide sequence is linked upstream of the nucleic acid sequence of the antigen binding domain.

Therapeutic applications

The invention includes cells transduced with a Lentiviral Vector (LV) encoding an expression cassette of the invention. The transduced T cells can induce CAR-mediated T cell responses.

Accordingly, the present invention also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue of a mammal comprising the steps of: administering to the mammal the CAR-T cells of the invention.

In one embodiment, the invention includes a class of cell therapies in which T cells are genetically modified to express a CAR of the invention, and the CAR-T cells are infused into a recipient in need thereof. The infused cells are capable of killing tumor cells in the recipient. Unlike antibody therapy, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

In one embodiment, the CAR-T cells of the invention can undergo robust in vivo T cell expansion and can last for an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the CAR-modified T cell induces an immune response specific to the antigen binding domain in the CAR.

Although the data disclosed herein specifically disclose lentiviral vectors comprising anti-GPC 3 scFv, hinge and transmembrane regions, and 4-1BB and CD3zeta signaling domains, the invention should be construed to include any number of variations on each of the construct components.

Diseases amenable to treatment include GPC 3-related cancers or tumors, such as GPC 3-positive tumors or cancers. GPC 3-associated cancers or tumors can include solid tumors, particularly hepatocellular carcinoma, melanoma, ovarian cancer, lung squamous cell carcinoma, gastric cancer, breast cancer, or combinations thereof.

The types of cancer treated with the CARs of the invention include, but are not limited to, carcinoma, blastoma and sarcoma, and certain benign and malignant tumors, such as sarcomas, carcinomas and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.

A solid tumor is an abnormal mass of tissue that generally does not contain cysts or fluid regions. Solid tumors can be benign or malignant. Different types of solid tumors are named for the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer.

The CAR-modified T cells of the invention may also be used as a type of vaccine for ex vivo immunization and/or in vivo therapy of mammals. Preferably, the mammal is a human.

For ex vivo immunization, at least one of the following occurs in vitro prior to administration of the cells into a mammal: i) expanding the cell, ii) introducing a nucleic acid encoding the CAR into the cell, and/or iii) cryopreserving the cell.

Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be a human, and the CAR-modified cells can be autologous with respect to the recipient. Alternatively, the cells may be allogeneic, syngeneic (syngeneic), or xenogeneic with respect to the recipient.

In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

In general, activated and expanded cells as described herein can be used to treat and prevent diseases that arise in immunocompromised individuals. In particular, the CAR-modified T cells of the invention are useful in the treatment of CCL. In certain embodiments, the cells of the invention are used to treat a patient at risk for CCL. Accordingly, the invention provides a method of treating or preventing CCL, comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the invention.

The CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, a pharmaceutical composition of the invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The compositions of the present invention are preferably formulated for intravenous administration.

The pharmaceutical compositions of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease-although the appropriate dosage may be determined by clinical trials.

When referring to an "immunologically effective amount", "an anti-tumor effective amount", "a tumor-inhibiting effective amount", or a "therapeutic amount", the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, extent of infection or metastasis, and individual differences in the condition of the patient (subject). It can be generally pointed out that: pharmaceutical compositions comprising T cells described herein can be in the range of 10 ⁴ To 10 ⁹ Dosage of individual cells/kg body weight, preferably 10 ⁵ To 10 ⁶ Doses of individual cells per kg body weight (including all integer values within those ranges) are administered. The T cell composition may also be administered multiple times at these doses. Cells can be administered by using infusion techniques well known in immunotherapy (see, e.g., Rosenberg et al, New Eng.J.of Med.319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the subject composition may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by i.v. injection. The composition of T cells can be injected directly into the tumor, lymph node or site of infection.

In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) any number of relevant treatment modalities, including but not limited to treatment with: such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or efavirenz therapy for psoriasis patients or other therapy for PML patients. In further embodiments, the T cells of the invention may be used in combination with: chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In a further embodiment, the cell composition of the invention is administered to the patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) bone marrow transplantation with a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an injection of the expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered pre-or post-surgery.

The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The proportion of doses administered to a human can be effected in accordance with accepted practice in the art. Typically, 1X10 may be administered per treatment or per course of treatment ⁶ 1 to 10 ¹⁰ A modified T cell of the invention (e.g., a GC76BB ζ cell) is administered to a patient, for example, by intravenous infusion.

The main advantages of the invention include

(1) The scFV targeting GPC3 in the CAR structure of the CAR-T has high affinity and high specificity, can well infiltrate into solid tumors, and has an excellent tumor inhibition effect in animal bodies.

(2) The CAR-T has better safety and no or weak non-target cell killing.

The invention is further illustrated by the following 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. Experimental procedures without specifying the detailed conditions in the following examples, generally followed by conventional conditions such as Sambrook et al, molecular cloning: conditions described in a Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Example 1 Structure of GC76BB ζ

From about 60 clones, a scFv targeting GPC3 with excellent performance was obtained by screening through a specific antibody library screening, and the heavy chain variable region (SEQ ID NO: 3) and the light chain variable region (SEQ ID NO: 7) of the scFv were used as the antigen binding domain of the CAR.

The CAR structure in the CAR-T of the present invention comprises a signal peptide SP, an extracellular antigen-binding domain ScFv, a hinge region CD8, a transmembrane region CD8, an intracellular domain 4-1BB and a CD3Zeta, and is named as GC76BB ζ, and the specific structure is shown in FIG. 1.

Wherein, the specific sequence of each part is as follows:

GC76BB ζ sequence:

signal peptide SP amino acid (SEQ ID NO.1)

MALPVTALLLPLALLLHAARPGS

Signal peptide SP nucleic acid (SEQ ID NO.2)

ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCCCTGCTGCTCCACGCCGCTAGACCCGGAAGC

VH amino acid of heavy chain of scFv (SEQ ID NO.3)

QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLEWIGAIAPKTGNTAYNQKFKDKAILTADKSSSTAYMELRSLTSEDSAVYYCTRYYSYAYWGQGTLVTVSA

VH nucleic acid of heavy chain of scFv (SEQ ID NO.4)

CAGGTGCAGCTGCAGCAGTCCGGCGCCGAGCTGGTGAGACCCGGAGCTTCCGTGACCCTGTCCTGTAAGGCCAGCGGCTACACCTTCACCGATTACGAGATGCACTGGGTGAAGCAGACCCCTGTGCACGGCCTGGAGTGGATCGGCGCCATCGCCCCAAAGACCGGCAATACAGCCTACAATCAGAAGTTCAAGGATAAGGCCATCCTGACCGCCGATAAGTCCTCCAGCACCGCCTACATGGAGCTGAGGTCCCTGACATCCGAGGATAGCGCCGTGTACTACTGTACCAGATACTACAGCTACGCCTACTGGGGCCAGGGCACCCTGGTGACAGTGAGCGCC

Linker amino acid (SEQ ID NO. 5):

GGGGSGGGGSGGGGS

linker nucleic acid (SEQ ID NO. 6):

GGCGGAGGCGGAAGCGGAGGAGGAGGAAGCGGCGGAGGCGGTAGC

light chain VL amino acid of scFv (SEQ ID NO. 7):

DVVMTQTPLSLSVSLGDQASISCRSSQSPVHSNGNTYLQWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTKLEKK

light chain VL nucleic acid of scFv (SEQ ID NO. 8):

GATGTGGTGATGACACAGACCCCCCTGAGCCTGCCTGTGTCCCTGGGCGACCAGGCCTCCATCAGCTGTAGGAGCAGCCAGTCCCCTGTGCACTCCAATGGCAACACATACCTGCACTGGTACCTGCAGAAGCCCGGCCAGAGCCCTAAGCTGCTGATCTACAAGGTGAGCAACAGATTCTCCGGCGTGCCCGACAGATTCTCCGGAAGCGGCAGCGGCACAGACTTCACCCTGAAGATCTCCAGGGTGGAGGCCGAGGATCTGGGCGTGTACTTCTGCTCCCAGAGCACACACGTGCCTTACACCTTCGGCGGCGGCACAAAGCTGGAGATCAAG

hinge region Hinge amino acid (SEQ ID NO. 9):

SGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY

hinge region Hinge nucleic acid (SEQ ID NO. 10):

TCCGGCACAACCACCCCTGCCCCCAGACCCCCTACCCCAGCTCCTACAATCGCCAGCCAGCCCCTGAGCCTCAGGCCTGAGGCCTGCAGGCCCGCTGCTGGAGGAGCTGTGCACACCAGGGGCCTGGACTTCGCCTGTGACATCTAC

transmembrane region TM amino acid (SEQ ID No. 11):

IWAPLAGTCGVLLLSLVITLYC

transmembrane region TM nucleic acid (SEQ ID NO. 12):

ATCTGGGCCCCCCTGGCCGGCACCTGTGGAGTTCTGCTGCTGTCCCTGGTGATCACACTGTACTGC

costimulatory domain 4-1BB amino acid (SEQ ID No. 13):

RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

costimulatory domain 4-1BB nucleic acid (SEQ ID No. 14):

AGATTCTCCGTGGTGAAGAGAGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGGCCTGTGCAGACAACACAGGAGGAGGATGGCTGTTCCTGCAGATTCCCCGAGGAGGAGGAGGGCGGCTGTGAGCTG

intracellular signal CD 3-zeta amino acid (SEQ ID NO. 15):

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

intracellular signal CD 3-zeta nucleic acid (SEQ ID NO. 16):

AGAGTGAAGTTCAGCAGATCCGCCGATGCCCCCGCCTACCAGCAGGGACAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGAGAGGAGTACGATGTGCTGGATAAGAGGAGGGGCAGGGACCCTGAGATGGGCGGCAAGCCCAGGAGGAAGAACCCCCAGGAGGGCCTGTACAACGAACTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGAGAAGAAGAGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGTCCACAGCCACCAAGGACACATACGACGCCCTGCACATGCAGGCCCTGCCTCCCAGATGA

example 2 construction and detection of HuH7-Luc and HepG2-Luc cells

The luciferase gene (GenBank: ACF93193.1) sequence is searched, synthesized and constructed into a PLVX-Puro vector by CRO company, and the constructed vector is named as PLVX-Luc-Puro. A stable transfer cell line is constructed by a lentivirus transfection mode, namely pGpol, pVSVG packaging plasmid and a target plasmid PLVX-Luc-Puro are used, a company has a lentivirus packaging platform to produce high and low lentiviruses, and then target cells HuH7 and HepG2 are infected according to MOI (MOI-1) respectively. After several rounds of pressure screening of Purmycin, detecting, wherein the signal value of the qualified cell line luciferase is more than 100 times of that of the transfected lentivirus primary cells, and respectively naming the qualified cells as HuH7-Luc and HepG 2-Luc; the results of the detection are shown in FIG. 2.

The results show that the luciferase signal values (LUC values) of the HuH7-Luc and HepG2-Luc cells are more than 100 times of those of transfected lentivirus primary cells.

Example 3 Lentiviral preparation

1. One day before virus preparation, 15cm cell culture dishes were inoculated with 1.5 x10 ⁷ 293T cells, incubated at 37 ℃ with 5% CO ₂ And (5) an incubator for overnight culture.

2. The next day, the PEI, together with the lentiviral packaging plasmids (pGpol, pVSVG) and the plasmid of interest GC76BB ζ were removed from the refrigerator and thawed at room temperature

3.1 clean 15ml centrifuge tube is taken, 2ml opti-MEM culture medium is added, then 10 mug GC76BB zeta, 4 mug pGpol and 2 mug pVSVG are respectively added, after the pipette is blown up and down to be fully mixed, 18 mug PEI is added, the pipette is immediately blown up and down to be mixed, and the mixture is kept stand for 15 minutes at room temperature.

4. The DNA/PEI complex is added into a 15cm culture dish drop by drop, the culture dish is slightly shaken, and the mixture is fully mixed. Placing the culture dish at 37 ℃ and 5% CO ₂ And (3) after the culture in the incubator is carried out for 6-8 hours, removing the culture medium containing the transfection reagent, and replacing the culture medium with a fresh complete culture medium.

5. After 48 hours of continuous culture, the virus-containing culture supernatant in the petri dish was collected, filtered through a 0.45 μm filter, and then centrifuged at 20000g at 4 ℃ for 2 hours; after centrifugation, the liquid in the centrifuge tube was carefully aspirated, 400. mu.L of PBS buffer was added to resuspend the pellet, the virus was dispensed into 1.5ml centrifuge tubes, 100. mu.L each, and the virus was stored at-80 ℃.

Example 4 sorting and activation of T cells

1. Taking 1 frozen PBMC cell, and recovering in water bath at 37 ℃; adding the recovered PBMC into a 15ml centrifuge tube into which 10ml of pre-heated culture medium is added in advance; then centrifuged at 500g for 5 minutes

2. Cell counting was performed after cell reselection with sorting buffer

3. Centrifuge at 500g for 5 min and adjust cell density to 1.0 x10 ⁷ /ml

4. According to 1.0 x10 ⁷ The cells were added to a volume of 20. mu.l of the sorted magnetic beads, and the sorted magnetic beads were added

5. Incubation at 4 ℃ for 15 min

6. Adding the incubated cells into a sorting column to perform a sorting operation

7. Collecting effluent in the separation column, centrifuging at 500g for 5 min

8. Resuspending the cells in X-vivo 15 medium (containing 300U/mL IL-2), counting, and adjusting the cell density to 1.0X 10 ⁶ /ml

9. According to 1.0 x10 ⁶ Adding 10 μ l of activated magnetic beads into the cells, adding the activated magnetic beads, and placing the cells at 37 deg.C and 5% CO ₂ The incubator was incubated for 48 hours.

Example 5T cell infection

1. Activated T cells were collected, centrifuged at 500g for 5 min, resuspended in X-vivo 15 medium (containing 300U/mL IL-2), counted, and cell density adjusted to 1.0X 10 ⁶ /ml

2. The required amount of virus was calculated according to MOI of 10. The calculation formula is as follows: required amount of virus (mL) ═ cell number (MOI)/viral titer

3. Taking out the virus from a refrigerator at the temperature of-80 ℃, and then melting at room temperature; adding the desired virus to activated T cells

4. Sampling and counting every 2-3 days, and maintaining the cell density at 1.0 x10 ⁶ /ml, continuously culturing for 6-10 days

Example 6 CAR-T Positive Rate detection

1. 500g for 5 min and collecting the CAR-T cells 7 days after lentivirus infection

2. 500 u L PBS heavy suspension, 500g centrifugal 5 minutes washing cells once

3. Resuspend 1mL of PBS, take 20. mu.L of resuspended cells and count them on a cell counter

4. According to the cell counting result, 5 x10 is taken ⁵ Reaction cells for CAR-T Positive Rate detection

5. Adding detection antibody at concentration of 2 μ g/ml, reacting in volume of 100 μ L, and incubating at 4 deg.C for 30 min

6. After the incubation, the cells were centrifuged at 500g for 5 minutes

7. 500 u L PBS heavy suspension, 500g centrifugal 5 minutes washing cells once

8. Repeat step 7 operation

9. Resuspending with 200. mu.L PBS, and performing on-machine detection

As shown in fig. 3, the results indicated that GC76BB ζ positive rate was 52.13%, while T cells without lentivirus transfection were 0.31%.

Example 7 killing of target cells by CAR-T cells

1. Collecting logarithmic growth phase target cells (HuH7-luc cells or HepG2-luc cells), and adjusting the target cell density to 2 x10 ⁵ A new 96-well plate was used for each ml, and the target cells were seeded at 50. mu.L/well. The unused wells around the 96-well plate were filled with 100. mu.L of medium per well to prevent evaporation of water from the middle experimental wells

2. Centrifuging and collecting the prepared CAR-T cells, and culturing with X-vivo 15 medium; then 50. mu.l of CAR-T cells were added according to the designed E/T ratio

3. Place the well plate in 5% CO ₂ Culturing at 37 deg.C for 14-18 hr

4. After the culture, the well plate was taken out from the incubator, 50. mu.l of One-Glo luciferase detection substrate 300g was added, the 96-well plate was centrifuged at room temperature for 3 minutes, the plate was gently taken out, and the reading was made on a microplate reader

5. Data calculation formula,% kill ═ 1- (sample read-Min)/(Max-Min)

The results of killing HuH7-luc cells and HepG2-luc cells of the human hepatoma cell line in vitro by GC76BB ζ are shown in FIGS. 4 and 5.

BM is the ScFv-constructed CAR-T of GPC3 antibody GC 33; UN-T is a T cell that is not transduced with lentivirus.

The results in fig. 4 show that GC76BB ζ in vitro killing activity against HuH7-luc cells at an E/T (effective target ratio) of 4, GC76BB ζ was able to kill nearly 90% of tumor cells, whereas BM yangshen CAR-T had only a 60% killing effect; even at an E/T (effective target ratio) of 1, GC76BB ζ had almost 60% killing ability, and BM positive control CAR-T had little or no killing effect.

The results in FIG. 5 show that GC76BB ζ in vitro killing activity against HepG2-luc cells was slightly better than BM parasol CAR-T at an E/T (effective to target ratio) of 4; at E/T (effective target ratio) of 1, GC76BB zeta has killing capacity of nearly 90%, and BM yangShen CAR-T has killing effect of nearly 48%.

Example 8 CAR T in vivo efficacy experiments

1. HuH7 cells in logarithmic growth phase were collected, washed twice with PBS, counted, and cell density was adjusted to 1X10 ⁸ /ml

2. Mice were inoculated subcutaneously with NCG mice, 100ul per mouse, at a density of 1 × 10 ⁸ /ml of HuH7 cells

3. The mice were further cultured and the tumor volume was measured weekly when the tumor volume of the mice had grown to 100mm ³ On the left and right, randomization grouping was performed according to tumor volume. Each group of 5 were given treatment on the day of the group, which was defined as D0. Each mouse was dosed with 1x10 ⁶ Or 3x10 ⁶ The CAR-T cell of (1).

4. Experimental observations and data collection, the effect of tumors on the normal behavior of animals was routinely monitored weekly after dosing. The specific contents include the activity of experimental animals, the condition of food intake and water drinking, the condition of weight increase or reduction, eyes, fur and other abnormal conditions. Tumor size and mouse body weight were measured and recorded. Tumor volume was calculated as: tumor volume (mm) ³ ) 0.5 × (tumor major diameter × tumor minor diameter) ² )。

The in vivo tumor suppression effect of GC76BB ζ is shown in fig. 6.

The results indicate that CAR-T cells are administered at lower doses (1x 10) ⁶ Cells/mouse), has excellent tumor-inhibiting effect.

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> Nanjing Landun Biotechnology Co., Ltd

<120> target GPC3 chimeric antigen receptor T cell and application thereof

<130> P2021-3264

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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 Gly Ser

20

<210> 2

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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atggccctgc ctgtgacagc cctgctgctg cctctggccc tgctgctcca cgccgctaga 60

cccggaagc 69

<210> 3

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Ala Ile Ala Pro Lys Thr Gly Asn Thr Ala Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ala

115

<210> 4

<211> 345

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<213> Artificial Sequence (Artificial Sequence)

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caggtgcagc tgcagcagtc cggcgccgag ctggtgagac ccggagcttc cgtgaccctg 60

tcctgtaagg ccagcggcta caccttcacc gattacgaga tgcactgggt gaagcagacc 120

cctgtgcacg gcctggagtg gatcggcgcc atcgccccaa agaccggcaa tacagcctac 180

aatcagaagt tcaaggataa ggccatcctg accgccgata agtcctccag caccgcctac 240

atggagctga ggtccctgac atccgaggat agcgccgtgt actactgtac cagatactac 300

agctacgcct actggggcca gggcaccctg gtgacagtga gcgcc 345

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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ggcggaggcg gaagcggagg aggaggaagc ggcggaggcg gtagc 45

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

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

20 25 30

Asn Gly 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

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

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

100 105 110

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<213> Artificial Sequence (Artificial Sequence)

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gatgtggtga tgacacagac ccccctgagc ctgcctgtgt ccctgggcga ccaggcctcc 60

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tacctgcaga agcccggcca gagccctaag ctgctgatct acaaggtgag caacagattc 180

tccggcgtgc ccgacagatt ctccggaagc ggcagcggca cagacttcac cctgaagatc 240

tccagggtgg aggccgagga tctgggcgtg tacttctgct cccagagcac acacgtgcct 300

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Ser Gly Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

1 5 10 15

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

20 25 30

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

35 40 45

Tyr

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<211> 147

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<213> Artificial Sequence (Artificial Sequence)

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tccggcacaa ccacccctgc ccccagaccc cctaccccag ctcctacaat cgccagccag 60

cccctgagcc tcaggcctga ggcctgcagg cccgctgctg gaggagctgt gcacaccagg 120

ggcctggact tcgcctgtga catctac 147

<210> 11

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

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

1 5 10 15

Val Ile Thr Leu Tyr Cys

20

<210> 12

<211> 66

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<213> Artificial Sequence (Artificial Sequence)

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atctgggccc ccctggccgg cacctgtgga gttctgctgc tgtccctggt gatcacactg 60

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<210> 13

<211> 47

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

1 5 10 15

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

20 25 30

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40 45

<210> 14

<211> 141

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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agattctccg tggtgaagag aggcagaaag aagctgctgt acatcttcaa gcagcccttc 60

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gaggagggcg gctgtgagct g 141

<210> 15

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 16

<211> 339

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

agagtgaagt tcagcagatc cgccgatgcc cccgcctacc agcagggaca gaatcagctg 60

tacaacgagc tgaacctggg cagaagagag gagtacgatg tgctggataa gaggaggggc 120

agggaccctg agatgggcgg caagcccagg aggaagaacc cccaggaggg cctgtacaac 180

gaactgcaga aggacaagat ggccgaggcc tacagcgaga tcggcatgaa gggcgagaga 240

agaagaggca agggccacga cggcctgtac cagggcctgt ccacagccac caaggacaca 300

tacgacgccc tgcacatgca ggccctgcct cccagatga 339

<210> 17

<211> 495

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

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 Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala

20 25 30

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

35 40 45

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

50 55 60

Val His Gly Leu Glu Trp Ile Gly Ala Ile Ala Pro Lys Thr Gly Asn

65 70 75 80

Thr Ala Tyr Asn Gln Lys Phe Lys Asp Lys Ala Ile Leu Thr Ala Asp

85 90 95

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

100 105 110

Asp Ser Ala Val Tyr Tyr Cys Thr Arg Tyr Tyr Ser Tyr Ala Tyr Trp

115 120 125

Gly Gln Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Thr

145 150 155 160

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

165 170 175

Arg Ser Ser Gln Ser Pro Val His Ser Asn Gly Asn Thr Tyr Leu Gln

180 185 190

Trp Phe Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys

195 200 205

Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

290 295 300

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

305 310 315 320

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

325 330 335

Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

340 345 350

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

355 360 365

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

<210> 18

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Asp Tyr Glu Met His

1 5

<210> 19

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Ala Ile Ala Pro Lys Thr Gly Asn Thr Ala Tyr Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210> 20

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Tyr Tyr Ser Tyr Ala Tyr

1 5

<210> 21

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Arg Ser Ser Gln Ser Pro Val His Ser Asn Gly Asn Thr Tyr Leu Gln

1 5 10 15

<210> 22

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 23

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Ser Gln Ser Thr His Val Pro Tyr Thr

1 5

Claims

1. A single chain variable fragment (scFv) targeted to GPC3, comprising a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region comprising the following three complementarity determining regions CDRs:

HCDR1 shown in SEQ ID NO.18,

HCDR2 shown in SEQ ID NO.19, and

HCDR3 shown in SEQ ID NO. 20; and/or

LCDR1 shown in SEQ ID NO.21,

LCDR2 shown in SEQ ID NO.22, and

LCDR3 shown in SEQ ID NO. 23.

2. A Chimeric Antigen Receptor (CAR) fusion protein, comprising from N-terminus to C-terminus:

(i) the scFv of claim 1 wherein the scFv of the human,

(ii) (ii) a transmembrane domain which is capable of,

(iii) at least one co-stimulatory domain, and

(iv) an activation domain.

3. An antibody against GPC3, comprising a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region comprising the following three complementarity determining regions CDRs:

HCDR1 shown in SEQ ID NO.18,

HCDR2 shown in SEQ ID NO.19, and

HCDR3 shown in SEQ ID NO. 20; and/or

LCDR1 shown in SEQ ID NO.21,

LCDR2 shown in SEQ ID NO.22, and

LCDR3 shown in SEQ ID NO. 23.

4. A recombinant protein, said recombinant protein having:

(I) the scFv of claim 1, the CAR fusion protein of claim 2, or the antibody of claim 3; and

(II) optionally a tag sequence to facilitate expression and/or purification.

5. An antibody drug conjugate, comprising:

(a) the scFv of claim 1, the CAR fusion protein of claim 2, or the antibody of claim 3; and

6. A nucleic acid molecule encoding the scFv of claim 1, or the CAR fusion protein of claim 2, or the antibody of claim 3.

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

8. A host cell comprising the vector of claim 7, or having integrated into its chromosome the exogenous nucleic acid molecule of claim 6, or expressing the scFv of claim 1, or the CAR fusion protein of claim 2, or the antibody of claim 3.

9. A method of making an engineered immune cell that expresses the CAR fusion protein of claim 2, comprising the steps of: transforming the nucleic acid molecule of claim 6 or the vector of claim 7 into a T cell or NK cell, thereby obtaining the engineered immune cell.

10. A formulation comprising the scFv of claim 1, the CAR fusion protein of claim 2, the antibody of claim 3, or the vector of claim 7, or the cell of claim 8, and a pharmaceutically acceptable carrier, diluent, or excipient.

11. Use of the scFv of claim 1, the CAR fusion protein of claim 2, the antibody of claim 3, the recombinant protein of claim 4, or the antibody drug conjugate of claim 5, or the cell of claim 8, for the preparation of a medicament or formulation for the prevention and/or treatment of a GPC 3-related cancer or tumor.

12. A kit for preparing a cell according to claim 8, comprising a container and, located within the container, the nucleic acid molecule of claim 6, or the vector of claim 7.

13. Use of a cell according to claim 8, or a formulation according to claim 10, for the prevention and/or treatment of GPC 3-related cancers or tumors.