CN114686436A

CN114686436A - Preparation and application of FSHR and FOLR1 targeting double targeting target point CAR T

Info

Publication number: CN114686436A
Application number: CN202011618643.4A
Authority: CN
Inventors: 周春燕; 张宇; 龚剑
Original assignee: Fosun Kaite Biotechnology Co ltd
Current assignee: Fosun Kaite Biotechnology Co ltd
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2022-07-01

Abstract

The invention provides a chimeric antigen receptor CAR-immune cell capable of targeting FOLR1 and FSHR double targets and application thereof. The application of the CAR-T cells of the invention can cover a wider range of ovarian cancer tumor cells. The CAR-T cell is structurally optimized on the basis of the traditional CAR-T cell, so that the CAR-T cell secretes BiTE, and double-target killing can be realized; meanwhile, the back-transfused T cells are mobilized to a greater extent, so that the tumor cells are killed more efficiently.

Description

Preparation and application of FSHR and FOLR1 targeting double targeting target point CAR T

Technical Field

The invention belongs to the technical field of biology, and particularly relates to preparation and application of a targeting FSHR and FOLR1 double targeting point CAR T.

Background

Globally, ovarian cancer is the seventh most common cancer in women, and is also the eighth most lethal cancer. Despite advances in surgical methods and chemotherapy, 5-year survival rates remain below 45%. More importantly, the number of cases diagnosed per year is also increasing, which poses a serious threat to female life.

Cellular immunotherapy is a novel precise targeted therapy, and has a tumor treatment mode with obvious curative effect, especially CAR-T. CAR-T cell therapy first collects cells from a patient and then modifies them by activation and genetic engineering so that the T cells surface express a Chimeric Antigen Receptor (CAR). The chimeric antigen receptor is composed of an extracellular antigen binding region, namely scFv for recognizing tumor-associated antigen, a transmembrane region, an intracellular signal domain and the like. These CAR-T cells are returned to the patient where they can recognize and destroy tumor cells expressing the indicated antigen.

Folate receptor 1(Folate receptor1, FOLR1), also known as Folate receptor alpha or Folate binding protein, is expressed less in normal tissues compared to tumor tissues. It is expressed on the plasma membrane of cells and can mediate the folic acid uptake of cells and influence the downstream pathway to promote the tumor growth. Studies have shown that FOLR1 is highly expressed in most ovarian cancers (76% of the expression in malignant ovarian cancer) as well as uterine, endometrial, pancreatic, renal, lung, and breast cancers, and mediates folate uptake or production of regulatory signals by cells to promote tumor cell growth. This expression pattern of FOLR1 makes it a good target for CAR-T cells to treat ovarian cancer.

Follicle Stimulating Hormone Receptor (FSHR) is expressed on blood vessels in tumor tissues of ovarian cancer, prostate cancer, urothelial cancer, and renal cell carcinoma, while no expression is detected in the surrounding healthy blood vessels. It is highly expressed in ovarian cancer, and is expressed in 50-70% of ovarian cancers. This surface antigen expression correlates with the prognosis of ovarian cancer, with patients with high FSHR expression having a significantly poorer prognosis than those with low FSHR expression. Normally, it is expressed in ovarian granulosa cells and endothelium, and not in gonadal healthy tissue, making it another suitable target for CAR-T cells to treat ovarian cancer. More importantly, FSHR is a receptor of FSH (follicle stimulating hormone), and FSHR can be targeted by using FSH instead of a single-chain antibody, so that CRS reaction can be effectively reduced.

However, due to tumor heterogeneity, ovarian cancer treatment is not well achieved using the single targets. In addition, because of the limitations of current genetic modification methods, it is difficult to achieve 100% modification of the reinfused T cells.

However, if the above FOLR1 is combined with FSHR two targets, the current method for killing the two targets simultaneously mainly comprises dual specificity CAR-T. However, there are still some drawbacks, and because of the limitations of the current genetic modification methods, it is difficult to achieve 100% modification of the returned T cells, and it is not possible to mobilize T cells in patients.

Therefore, there is an urgent need in the art to develop a more broad-spectrum bispecific CAR-T cell that can be efficiently modified, with a greater extent of mobilization back-transfusion.

Disclosure of Invention

The object of the present invention is to provide a more broad-spectrum bispecific CAR-T cell that can be efficiently modified, with a greater extent of mobilization back-transfusion.

In a first aspect of the invention, there is provided a chimeric antigen receptor CAR-immune cell, said CAR-immune cell comprising the following elements:

(a) a first CAR whose extracellular binding domain comprises a first binding element that targets a first target protein and whose extracellular binding domain comprises or does not comprise a second binding element that targets a second target protein;

(b1) optionally a second CAR whose extracellular binding domain comprises a second binding element that targets a second target protein;

(b2) optionally a nucleic acid sequence encoding a BiTE, wherein the BiTE targets a second target protein;

wherein when the extracellular binding domain of the first CAR does not contain a second binding element that targets a second target protein, the CAR-immune cell contains the (b1) and/or (b2) elements;

and, the first target protein is folate receptor 1(FOLR1) and the second target protein is Follicle Stimulating Hormone Receptor (FSHR); alternatively, the first target protein is Follicle Stimulating Hormone Receptor (FSHR), and the second target protein is folate receptor 1(FOLR 1).

In another preferred embodiment, the immune cell comprises: t cells, NK cells, or a combination thereof.

In another preferred embodiment, the extracellular binding domain of the first CAR comprises, in order from N-terminus to C-terminus: optionally a signal peptide, a first binding element targeting a first target protein, optionally a linker sequence, and a second binding element targeting a second target protein; the extracellular binding domain of the first CAR sequentially comprises from N-terminus to C-terminus: an optional signal peptide, a second binding element targeting a second target protein, an optional linker sequence, and a first binding element targeting a first target protein.

In another preferred embodiment, the linker sequence is a flexible peptide.

In another preferred embodiment, the connection sequence is (G)₄S)_nWherein n is a positive integer (e.g., 1, 2, 3, 4, 5, or 6).

In another preferred embodiment, the CAR-immune cell contains the following elements:

(a) the extracellular binding domain of the first CAR contains a first binding element targeting a first target protein and does not contain a second binding element targeting a second target protein; and

(b1) a second CAR whose extracellular binding domain comprises a second binding element that targets a second target protein.

(a) the extracellular binding domain of the first CAR contains a first binding element that targets a first target protein and does not contain a second binding element that targets a second target protein; and

(b2) a nucleic acid sequence encoding a BiTE, wherein said BiTE targets a second target protein.

In another preferred embodiment, the first CAR or the second CAR has the structure shown in formula I below:

L-EB-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;

EB is the extracellular binding domain;

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, the sequence of "H-TM-C" in formula I can be the full-length sequence consisting of the extracellular domain of CD28, the transmembrane region of CD28, and the intracellular domain of CD28 (i.e., Extra _ CD28 TM _ ICD).

In another preferred example, the amino acid sequence of the Extra _ CD28 TM _ ICD is shown as SEQ ID NO. 1.

In another preferred embodiment, the signal peptide is a signal peptide of an immune cell surface molecule commonly used in the art, preferably a signal peptide of a protein selected from the group consisting of: CD8, CD28, GM-CSF, CD4, CD137, NKG2D, or a combination thereof.

In another preferred embodiment, the signal peptide is the leader sequence (signal peptide) of CD8, and the amino acid sequence is shown in SEQ ID NO: 2.

In another preferred embodiment, the hinge region may be a hinge region of a protein selected from the group consisting of: CD8, CD28, CD137, NKG2D, or a combination thereof.

In another preferred embodiment, the hinge region is that of CD8, and its amino acid sequence is shown in SEQ ID NO. 3.

In another preferred embodiment, the transmembrane domain may be a transmembrane region of an immune cell surface molecule commonly used in the art, preferably a transmembrane region of a protein selected from the group consisting of: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, NKG2D, DAP10, DAP12, or a combination thereof.

In another preferred embodiment, the transmembrane domain comprises a transmembrane region derived from CD8, and the amino acid sequence of the transmembrane domain is shown in SEQ ID NO. 4.

In another preferred embodiment, the costimulatory signal molecule 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, DAP10, DAP12, or a combination thereof.

In another preferred embodiment, the costimulatory signal molecule is a costimulatory signal molecule from 4-1BB source, and the amino acid sequence of the costimulatory signal molecule is shown in SEQ ID NO. 5.

In another preferred embodiment, the amino acid sequence of CD3 ζ is set forth as SEQ ID NO 6.

In another preferred embodiment, the first binding element or the second binding element targeting the first or second target protein may be an antigen binding domain ScFv of an antibody targeting said target protein, the ScFv having the structure of formula a or formula B:

V_H1-V_L1 (A)

V_L1-V_H1 (B)

in the formula, V_H1Is an antibody heavy chain variable region;

V_L1is an antibody light chain variable region;

"-" is a linker peptide or peptide bond.

In another preferred embodiment, the target protein is FOLR 1; wherein, V_H1Has the amino acid sequence shown as SEQ ID NO. 10, and V_L1The amino acid sequence of (A) is shown in SEQ ID NO. 11.

In another preferred embodiment, the amino acid sequence of the connecting peptide is shown in SEQ ID NO 12.

In another preferred embodiment, the first binding member or the second binding member targeting the first or second target protein may be a native sequence that specifically binds to the target protein.

In another preferred embodiment, said first or second target protein is FSHR and said first or second binding element is FSH or a fragment thereof.

In another preferred embodiment, the structure of said FSH or fragment thereof is represented by formula C or formula D below:

FSHβ-CGα (C)

CGα-FSHβ (D)

wherein FSH β is a subunit of FSH;

CG α is another subunit of FSH;

"-" is a linker peptide or peptide bond.

In another preferred embodiment, the FSH sequence is an amino acid sequence consisting of the two subunits or fragments thereof.

In another preferred embodiment, the amino acid sequence of the beta subunit of FSH is shown in SEQ ID NO 7.

In another preferred embodiment, the amino acid sequence of the CG alpha subunit is shown in SEQ ID NO 8.

In another preferred embodiment, when said second target protein is FSHR, said BiTE is a CD3 ScFv.

In another preferred example, the BiTE may be an Fc region of an antibody and the immune cell is an NK cell.

In another preferred embodiment, in the CD3 ScFv, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 14.

In another preferred embodiment, when the CAR-immune cell comprises element (a) and element (b2), the element (a) and element (b2) may be derived from a peptide chain expressed from the same nucleic acid construct, the peptide chain having the structure of formula II from N-terminus to C-terminus:

SP1-B1-H-TM-C-CD3 zeta-CS-SP 2-B2-I-BiTE-M (formula II)

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

SP1 and SP2 are each independently an optional signal peptide sequence;

b1 and B2 are a first binding element targeting a first target protein, and a second binding element targeting a second target protein, respectively;

H. TM, C and CD3 ζ are as defined for formula I;

CS is a site sequence which can be specifically recognized and cleaved by protease;

i is a null or a connecting sequence;

BiTE is a sequence targeting a second target protein;

m is an optional tag sequence.

In another preferred embodiment, the amino acid sequence of the peptide chain with the structure of formula II is shown as SEQ ID NO. 15.

In another preferred embodiment, the encoding nucleotide sequence of the peptide chain with the structure of the formula II is shown as SEQ ID NO. 16.

In another preferred embodiment, the SP1 is a signal peptide derived from a membrane protein, which may be an immune cell surface molecule commonly used in the art; preferably, said membrane protein-derived signal peptide is a signal peptide of 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), GITR, TLR2, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD154, NKG2D, DAP10, 12, or a combination thereof.

In another preferred embodiment, the SP1 is a signal peptide sequence of CD8, and the amino acid sequence of the signal peptide sequence is shown as SEQ ID NO. 2.

In another preferred embodiment, the SP2 is a signal peptide sequence derived from a secretory protein, which may be a protein secreted by immune cells as is conventional in the art; preferably, the signal peptide of the secretory protein is a signal peptide of a protein selected from the group consisting of: immunoglobulin constitutive subunit, cytokine (IL-2, IL-12, IL-7, IFN-. gamma.) and the like.

In another preferred embodiment, SP2 is a signal peptide of Ig kappa chain, and its amino acid sequence is shown in SEQ ID NO. 9.

In another preferred embodiment, when element (a) and element (b2) are derived from the same peptide chain as expressed by the nucleic acid construct, the nucleic acid construct has two different promoters for promoter element (a) and promoter element (b 2).

In a second aspect of the invention there is provided a nucleic acid molecule encoding the elements (a), (b1) and/or (b2) in a CAR-immune cell according to the first aspect of the invention.

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

In another preferred embodiment, the vector comprises DNA and RNA.

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

In another preferred embodiment, the vector comprises a DNA virus or a retroviral vector.

In another preferred embodiment, the carrier is selected from the group consisting of: a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a combination thereof.

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

In a fourth aspect of the invention, there is provided a host cell comprising a vector or chromosome according to the third aspect of the invention and, integrated therein, an exogenous nucleic acid molecule according to the second 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 a fifth aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a CAR-immune cell 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.

In a sixth aspect of the invention, there is provided the use of a CAR-immune cell 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, for the manufacture of a medicament or formulation for the treatment of a tumour.

In another preferred embodiment, the tumor comprises an FSHR-positive tumor.

In another preferred embodiment, the tumor comprises a FOLR1 positive tumor.

In another preferred embodiment, the tumor comprises any tumor that is FSHR positive and FOLR1 positive.

In another preferred embodiment, the tumor comprises: ovarian cancer, breast cancer, gastric cancer, prostate cancer, colon cancer, lung cancer, or a combination thereof.

In another preferred embodiment, the tumor is ovarian cancer.

In a seventh aspect of the invention, there is provided a method of treating a disease comprising administering to a subject in need thereof an amount of a chimeric antigen receptor 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 cell according to the fourth aspect of the invention, or a pharmaceutical composition according to the fifth aspect of the invention.

In an eighth aspect of the invention there is provided a method of making a CAR-immune cell according to the first aspect of the invention, the method comprising the steps of: transducing a nucleic acid molecule according to the second aspect of the invention or a vector according to the third aspect of the invention into an immune cell, thereby obtaining the CAR-immune cell.

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 the structural schematic diagram of FR1 scFV-FSH-BB, FR1scFV-BB and FSH-BB. Wherein Hinge TM represents the Hinge and transmembrane regions of CD 8; 4-1BB represents the intracellular signaling region of 4-1 BB.

FIG. 2 shows a comparison of cell killing by Tandem-CAR (FR1 scFV-FSH-BB) and single-target secondary CAR (FSH-BB and FR1 scFV-BB).

FIG. 3 shows the structural schematic diagram of FR1scFV-28- (FSH-BITE) and the second generation CAR (FSH-BB and FR1 scFV-BB).

FIG. 4 shows a comparison of BiTE partitioning-CAR FR1scFV-28- (FSH-BiTE) and single-target secondary CAR (FSH-BB and FR1scFV-BB) cell killing.

FIG. 5 shows the statistics of the number of target cells in the lower chamber of the Transwell.

FIG. 6 shows the detection of IFN-. gamma.in the Transwell supernatant.

FIG. 7 shows the results of detection of binding of BiTE-secreting-CAR supernatant (100-fold concentration) to Mock-T cells.

Figure 8 shows the results of the BiTE secreted-CAR supernatant mediated cell killing assay.

Detailed Description

The inventor develops a targeting FSHR and FOLR1 double targeting target CAR-T cell for the first time through extensive and intensive research and a large amount of screening. In the present invention, the inventors combined the FOLR1 antibody with FSH to construct bispecific CAR-T cells. The application of the CAR-T cells of the invention can cover a wider range of ovarian cancer tumor cells. The CAR-T cell is structurally optimized on the basis of the traditional CAR-T cell, so that the CAR-T cell secretes BiTE, and double-target killing can be realized; meanwhile, the back-transfused T cells are mobilized to a greater extent, so that the tumor cells are killed more efficiently. The present invention has been completed based on this finding.

Term(s) for

In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

The term "administering" refers to physically introducing the product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal cord or other parenteral routes of administration, e.g., by injection or infusion.

The term "antibody" (Ab) shall include, but is not limited to, an immunoglobulin that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains, or antigen-binding portions thereof, interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains CH1, CH2, and CH 3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises a constant domain CL. The VH and VL regions may be further subdivided into hypervariable regions, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

It should be understood that the amino acid names herein are given by the international single english letter designation, and the three english letters abbreviation corresponding to the amino acid names are: ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), I1e (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y) and Val (V).

Chimeric Antigen Receptor (CAR) -immune cells

As used herein, the terms "Chimeric Antigen Receptor (CAR) -immune cell", "CAR-immune cell", "immune cell of the invention" are used interchangeably and all refer to a CAR-immune cell with dual target specificity having elements (a), (b1) and/or (b2) according to the first aspect of the invention.

The CAR-immune cells of the invention express a first CAR and optionally a second CAR. Wherein the first CAR and the second CAR, except for the specific extracellular binding domain, each have the structure of a chimeric antigen receptor as is conventional in the art.

In one embodiment of the invention, the CAR-immune cell is capable of secreting a BiTE sequence that has the function of targeting a corresponding second CAR, which is capable of drawing the CAR-immune cell and unmodified immune cell into proximity with a target cell expressing the second CAR target protein, thereby further promoting killing of the target cell by the CAR-immune cell.

As used herein, the term "chimeric antigen receptor of the invention" refers to a first CAR and/or a second CAR in the invention.

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.

As used herein, "antigen binding domain" and "single chain antibody fragment" each refers to a Fab fragment, Fab 'fragment, F (ab')₂A fragment, or a single Fv fragment. 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 invention, the scFv comprises an antibody, preferably a single chain antibody, that specifically recognizes FOLR 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 one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domains may be selected, or modified by amino acid substitutions, to avoid binding such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex.

Carrier

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, New York) 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 cancer 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, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al, 2000FEBS Letters479: 79-82). 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 the 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 of introducing the polynucleotide into the 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.

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

Preparation

The invention provides a composition comprising a CAR-T cell according to the first aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injectionIt is used for injection. Preferably, the CAR-T cells are present in the formulation at a concentration of 1X 10³-1×10⁸Individual cells/ml, more preferably 1X 10⁴-1×10⁷Individual cells/ml.

In one embodiment, the formulation 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 formulations of the present invention are preferably formulated for intravenous administration.

Therapeutic applications

The invention includes therapeutic applications of cells (e.g., T cells) transduced with Lentiviral Vectors (LV) encoding expression cassettes of the invention. The transduced T cells can target markers FSHR and FOLR1 of tumor cells, can be used for autologous and allogeneic tumor treatment, can be prepared in a large scale, have uniform and stable quality, and can be used for any patient at any time.

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 injected into a recipient in need thereof. The injected cells are capable of killing tumor cells of 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. For example, anti-FSH CAR-T cells elicit a specific immune response against FSHR-expressing cells.

Although the data disclosed herein specifically disclose lentiviral vectors comprising anti-FOLR 1-FSHRscFv, a human Fc hinge region, an ICOS transmembrane and intracellular region, and 4-1BB and CD3 zeta signaling domains, the invention should be construed to include any number of variations on each of the construct components.

Treatable cancers include tumors that are not vascularized or have not substantially vascularized, as well as vascularized tumors. The cancer may comprise a non-solid tumor (such as a hematological tumor, e.g., leukemia and lymphoma) or may comprise a solid tumor. The types of cancer treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas and sarcomas, and certain leukemias or lymphoid malignancies, 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-immune 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.

The invention provides a method of treating a tumor comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-immune cell of the invention.

The CAR-immune 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 is possible to generally point out: pharmaceutical compositions comprising T cells described herein can be in the range of 10⁴To 10⁹Dosage of individual cells/kg body weight, preferably 10⁵To 10⁶Doses of individual cells per kg body weight (including all integer values within those ranges) are administered. T cell compositions may also be used in these dosesAnd (4) performing secondary application. 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 may 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 bevacizumab, megestrol acetate dispersible tablet, paclitaxel injection, ifosfamide, treatment of ovarian cancer patients with ifosfamide for injection. In further embodiments, the CAR-immune cells of the invention can 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, 1X 10 may be used per treatment or per course of treatment ⁶1 to 10¹⁰The CAR-immune cells of the invention are administered to a patient, for example, by intravenous infusion.

The main advantages of the invention include:

1) because of tumor heterogeneity, ovarian cancer treatment may not be well achieved using a single target, in this patent we constructed CAR-T cells using FOLR1 antibody in combination with FSH. A wider range of ovarian cancer tumor cells can be covered.

2) Because of the limitations of current genetic modification methods, it is difficult to achieve 100% modification of the reinfused T cells. The invention optimizes the structure on the basis of CAR-T, so that the CAR-T secretes BiTE, can kill double targets, and meanwhile mobilizes the returned T cells to a greater extent to kill tumor cells.

The invention is further illustrated by the following examples: the following examples are illustrative and are intended to be purely exemplary of the invention and are not intended to limit the scope of the invention. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be included in the invention. The methods used in the present invention are, unless otherwise specified, conventional in the art; the reagents of the present invention are conventional in the art unless otherwise specified.

TABLE 1 sequences of the invention

Example 1: functional testing of Tandem-CAR

1.1 viral packaging

According to the structure shown in figure 1, the gene is synthesized and cloned to a virus transferred to a lentiviral vector pWPXld, and the vector can be used after being sequenced without errors. 293T cells (DMEM containing 10% FBS) cultured in 10cm dishes were controlled at a density of 70% -80%. Plasmids pWPXld, pMD2G, and psPAX2 were mixed with 2mL Opti-Mem at a ratio of 10ug:7.5ug:2.5ug to prepare a mixture of 3 transfection plasmids. This was then mixed with X-tremeGENE HP DNA Transfection Reagent. Generally, the ratio of DNA to Transfection Reagent is 1:3 (1000. mu.l per 10cm dish). Incubate at room temperature for 15 min.

The mixture was added dropwise to the 293 dish already prepared. After culturing in a cell culture box for 6h, the medium was replaced with 15ml of complete medium by washing and changing the medium. And after further culturing for 72h, collecting the virus.

The virus supernatant was centrifuged at about 500g for 5 minutes to remove cell debris, and the supernatant was collected. The supernatant recovered above was then filtered through a 0.45 μm filter. Then, the mixture was concentrated 15 times for use. If not used for a while, they should be dispensed in time and frozen in liquid nitrogen and transferred to a-80 ℃ freezer as soon as possible.

1.2 CAR-T cell preparation

Commercial PBMC cells were cultured with X-VIVO 15(LONZA, 04-418Q) containing 5 ‰ human serum albumin at an initial cell density of 1 × 10⁶Individual cells/mL.

CD3 antibody was added to a final concentration of 50ng/mL and IL-2 was added at 300IU/mL to activate T cell expansion. After 48 hours of cell activation, appropriate amounts of virus and polybrene were added to infect T cells. Centrifuging for 90min at 800g, and culturing in a cell culture box after centrifugation.

After 24h of lentivirus infection, the cell suspension was aspirated and expressed at 1X 10⁶The cells/mL concentration was supplemented with completely fresh X-VIVO 15 medium. Observing cell density every day, and timely supplementing T cell culture solution to maintain the density of T cells at 1 × 10⁶About one cell/mL. And continuing to expand for 5-10 days to complete the preparation of the CAR-T cells.

1.3 verification of cell killing Effect

The target cell SKOV3-FSHR expressed by the Hibit + was constructed. For 96-well plate plating, 4E 3/well of target cells were added to CAR-T cells at different E: T ratios (0.625:1, 1.25:1), and the maximal release group (target cells only, but lysis treatment) and spontaneous group (target cells only) were set. 5% CO at 37 ℃₂The culture phase of (2) is cultured. When the preset incubation time is reached for 20h, the method is followed

The kit specification of the HiBiT excellular Detection System is used for detecting the killing activity of CAR-T cells, and the method refers to the kit specification. Cytotoxicity was calculated as follows:

cytotoxicity = (experimental-spontaneous)/(maximum release-spontaneous) × 100%

The results of the cell killing assay are detailed in FIG. 2. The results show that: the killing of Tandem-CAR FR1 scFV-FSH-BB modified CAR-T cells was superior to that of second generation CAR (FSH-BB and FSH-BB) modified CAR-T cells.

Example 2: BITE-secreting-CAR functional assays

2.1 viral packaging

According to the structure shown in FIG. 3, the gene was synthesized and cloned into a virus transferred to a lentiviral vector pwpxl, and the vector was used after sequencing without errors. 293T cells (DMEM containing 10% FBS) cultured in 10cm dishes were controlled at a density of 70% -80%. Plasmids pWPXld, pMD2G, psPAX2 were added to 2mL of Opti-Mem at a ratio of 10ug:7.5ug:2.5ug and mixed to prepare a mixture of 3 transfected plasmids. This was then mixed with X-tremeGENE HP DNA Transfection Reagent. Generally, the ratio of DNA to Transfection Reagent is 1:3 (1000. mu.l per 10cm dish). Incubate at room temperature for 15 min.

The mixture was added dropwise to the prepared 293 plate. After culturing in a cell culture box for 6h, the medium was replaced with 15ml of complete medium by washing and changing the medium. After further culturing for 72h, the virus was harvested.

2.2 CAR-T cell preparation

After 24h of lentivirus infection, the cell suspension was aspirated and expressed at 1X 10⁶The concentration of cells/mL was supplemented with completely fresh X-VIVO 15 medium. Observing cell density every day, and timely supplementing T cell culture solution to maintain the density of T cells at 1 × 10⁶About one cell/mL. And continuing to expand for 5-10 days to complete the preparation of the CAR-T cells.

2.3 verification of cell killing

Constructing target cells ES-2-Hibit-FR1-FSHR expressing Hibit +, FOLR1 and FSHR. In the case of plating with 96-well plates, 4E 3/well of target cells were added to CAR-T cells at different E: T ratios (1: 1, 3: 1), and the maximum release group (target cells only, but lysis treatment) and spontaneous group (target cells only, but lysis treatment) were set simultaneouslyTarget cells). The culture was carried out at 37 ℃ in an incubator containing 5% CO 2. When the preset incubation time is reached for 20h, the method is followed

cytotoxicity ═ 100% (experimental group-spontaneous)/(maximum release group-spontaneous) ×

The results are shown in FIG. 4. When the ratio of E to T is 3:1, the cell killing effect of FR1scFV-28- (FSH-BiTE) is better than that of FSH-28 and FR1 scFV-28.

2.4 BiTE secreted CAR functional validation

To further verify that BITE in FR1scFV-28- (FSH-BITE) plays a role, we performed a transwell experiment: ES-2 cells overexpressing FSHR and FR1 were constructed. FR1scFV-28- (FSH-BITE) CAR-T cell 1X 106 cell number was resuspended in 200. mu. L X-VIVO 15 medium, respectively, and then added to the upper chamber of 0.4- μm Transwell (hanging, 3413); unedited T cells and target cells (ES-2-FR1-FSHR) were resuspended in 500. mu.L of X-VIVO-15 medium at an E: T ratio (20:1 and 10:1) and added to the lower chamber of the Transwell, the number of target cells was 1.5E 5; carefully placing the upper chamber into the lower chamber, and culturing in an incubator for 48 hours; the plates were removed from the co-culture overnight and 500. mu.L of the lower chamber cells and supernatant were removed. Centrifugation at 300g for 5min, pipetting 150. mu.L of supernatant/well with a multi-channel pipette and transferring to a new 96-well cell culture plate for subsequent IFN-. gamma.ELISA detection, counting the cells in the pellet, according to CountBright^TMAbsolute Counting Beads, for flow cytometry (Thermo Fisher, C36950) instructions were used for Counting the operations. The counting results (see fig. 5) show: only the Mock-T and FR1scFV-28- (FSH-BITE) exist at the same time, the target cells can be killed, the number of the target cells is reduced, and the Mock-T or FR1scFV-28- (FSH-BITE) added alone has no obvious killing effect on the target cells.

2.5 cytokine Release assay

We performed cytokine assays on supernatants collected from the transwell experiments, and performed ELISA to detect the release of IFN-. gamma.cytokines upon killing of CAR-T cells according to the instructions of the IFN gamma Human Uncoated ELISA Kit (Invitrogen, # 88-7316-77). The results show (see fig. 6): the release amount of IFN-gamma in FR1scFV-28- (FSH-BiTE) group was higher than that in the control group.

2.6 detection of binding of BiTE-sectioning-CAR-T culture supernatant to Mock-T

FR1scFV-28- (FSH-BITE) CAR-T and control Supernatant (SP) for day 12 were collected and concentrated by about 100X using an ultrafiltration tube. It was then incubated with Mock-T cells for about 30min, then stained with flow antibodies to APC-anti-Flag, and then detected with a flow meter using Flowjo for analysis.

The results are shown in FIG. 7. FR1scFV-28- (FSH-BITE) SP binds to Mock-T, i.e. FSH-BiTE protein is present in the supernatant, which binds to Mock-T.

2.7 BiTE-sectioning-CAR-T culture supernatant mediated killing assay

We collected FR1scFV-28- (FSH-BITE) CAR-T Supernatant (SP) for day 7 of day for killing experiments. Plating ES-2-hibit-FR1-FSHR cells, plating 4E3 cells in each well of a 96-well plate, removing supernatant after the cells are attached to the wall, and then mixing the cells according to the E: T ratio of 10:1 untransduced MOCK-T cells (20 uL/well) and finally 80uL of the supernatant collected above per well, while setting the maximum release group (target cells only, but lysis treatment) and the spontaneous group (target cells only). The culture was carried out at 37 ℃ in an incubator with 5% CO2 for about 20 h. Cell killing was tested as described above.

The results are shown in FIG. 8. FR1scFV-28- (FSH-BiTE) SP contains a BITE protein and the protein has activity, and can mediate Mock-T to kill tumor cells.

The comprehensive results show that: killing using dual-target CAR-T was stronger than single-target CARs. Namely, the CAR-T with double targets has better anti-tumor effect.

All documents mentioned in this application are incorporated by reference in 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

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

165 170 175

Cys Lys Ala Ser Gln Asp Ile Thr Asn Phe Ile Gly Trp Tyr Gln His

180 185 190

Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Ser Tyr Thr Ser Ile Leu

195 200 205

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

210 215 220

Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr

225 230 235 240

Tyr Cys Leu Gln Tyr Tyr Asn Leu Trp Thr Phe Gly Gly Gly Thr Lys

245 250 255

Val Glu Ile Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn

260 265 270

Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu

485 490 495

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

500 505 510

Leu Leu Trp Val Pro Gly Ser Thr Gly Asp Ala Pro Asp Val Gln Asp

515 520 525

Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro Phe Phe Ser Gln Pro Gly

530 535 540

Ala Pro Ile Leu Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro

545 550 555 560

Thr Pro Leu Arg Ser Lys Lys Thr Met Leu Val Gln Lys Asn Val Thr

565 570 575

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

580 585 590

Met Gly Gly Phe Lys Val Glu Asn His Thr Ala Cys His Cys Ser Thr

595 600 605

Cys Tyr Tyr His Lys Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly

610 615 620

Gly Ser Gly Gly Gly Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Ala

625 630 635 640

Ile Glu Lys Glu Glu Cys Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp

645 650 655

Cys Ala Gly Tyr Cys Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala

660 665 670

Arg Pro Lys Ile Gln Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu

675 680 685

Thr Val Arg Val Pro Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr

690 695 700

Tyr Pro Val Ala Thr Gln Cys His Cys Gly Lys Cys Asp Ser Asp Ser

705 710 715 720

Thr Asp Cys Thr Val Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Gly

725 730 735

Glu Met Lys Glu Gly Gly Gly Gly Ser Asp Ile Lys Leu Gln Gln Ser

740 745 750

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

755 760 765

Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln

770 775 780

Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg

785 790 795 800

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

805 810 815

Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr

820 825 830

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His

835 840 845

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

850 855 860

Val Glu Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly

865 870 875 880

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

885 890 895

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

900 905 910

Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp

915 920 925

Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser

930 935 940

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu

945 950 955 960

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro

965 970 975

Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

980 985

<210> 16

<211> 2967

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 16

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggaagtgc agctggtgga gagcggcggc ggcctggtgc agcctggcgg cagcctgaga 120

ctgagctgcg ccgtgagcgg cttcaccttc agcaattacg gcatgagctg ggtgagacag 180

gcccctggca agggcctgga gtgggtggcc acaatcagca gcggcggcag ctacacctac 240

taccctgact ccgtgaaggg cagattcacc atctccagag acaatagcaa gaataccctg 300

tacctgcaga tgaatagcct gagagccgag gacaccgccg tgtactactg cagcacccag 360

ggcagcagcg gctacgtggg ctactggggc cagggcaccc tggtgaccgt gagcagcggc 420

ggaggcggaa gtggaggcgg aggatctggc ggcggaggct ctgacatcca gatgacccag 480

agccctagca gcgtgagcgc cagcgtgggc gacagagtga ccatcacctg caaggccagc 540

caggacatca ccaatttcat cggctggtac cagcacaagc ctggcaaggc ccctaagctg 600

ctgatcagct acaccagcat cctggagagc ggcgtgccta gcagattctc cggcagcggc 660

tccggcaccg actacaccct gaccatcagc agcctgcagc ctgaggactt cgccacctac 720

tactgcctgc agtactacaa tctgtggacc ttcggcggcg gcaccaaggt ggagatcaag 780

attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc 840

catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagcccttt 900

tgggtgctgg tggtggttgg gggagtcctg gcttgctata gcttgctagt aacagtggcc 960

tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 1020

atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 1080

gacttcgcag cctatcgctc cagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 1140

cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 1200

gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac 1260

cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 1320

attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 1380

agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgcgga 1440

tcaggagcaa caaacttctc cttgcttaaa caagcaggag atgtggaaga gaatccggga 1500

cctatggaaa cagatacatt actcttatgg gtactactgt tatgggtacc gggttcaaca 1560

ggagatgctc ctgatgtgca ggattgccca gaatgcacgc tacaggaaaa cccattcttc 1620

tcccagccgg gtgccccaat acttcagtgc atgggctgct gcttctctag agcatatccc 1680

actccactaa ggtccaagaa gacgatgttg gtccaaaaga acgtcacctc agagtccact 1740

tgctgtgtag ctaaatcata taacagggtc acagtaatgg ggggtttcaa agtggagaac 1800

cacacggcgt gccactgcag tacttgttat tatcacaaat ctggtggtgg ttctggtggt 1860

ggatccggtg gtggttctgg tggtggtaat agctgtgagc tgaccaacat caccattgca 1920

atagagaaag aagaatgtcg tttctgcata agcatcaaca ccacttggtg tgctggctac 1980

tgctacacca gggatctggt gtataaggac ccagccaggc ccaaaatcca gaaaacatgt 2040

accttcaagg aactggtata cgaaacagtg agagtgcccg gctgtgctca ccatgcagat 2100

tccttgtata catacccagt ggccacccag tgtcactgtg gcaagtgtga cagcgacagc 2160

actgattgta ctgtgcgagg cctggggccc agctactgct cctttggtga aatgaaagaa 2220

ggaggaggag gatcagatat caaacttcaa caatcaggag cagaacttgc aagacctgga 2280

gcatcagtga agatgtcttg caagacgtcc ggatacacat ttacaagata cacaatgcac 2340

tgggtgaaac aaagacctgg acaaggactt gaatggatcg gatacatcaa cccttcaaga 2400

ggatacacaa actacaacca gaagttcaag gataaagcaa cacttacaac agataaatca 2460

tcatcaacag catacatgca actttcatca cttacatcag aagattcagc agtgtactac 2520

tgcgcaagat actacgatga tcactactgc cttgattact gggggcaggg cacaacactt 2580

acagtgtcat cagtggaagg tggctcgggt ggctccggag gaagcggagg gtcaggaggc 2640

gtcgacgata tccaacttac acaatcacct gcaatcatgt cagcatcacc tggagagaag 2700

gttactatga catgcagagc atcatcatca gtgtcataca tgaactggta ccaacagaag 2760

tccggaacgt cgcctaagcg gtggatctac gatacatcaa aggtagcctc aggagtgcct 2820

tacagattct ccggctcagg aagtggtact agctattcgc ttacaatctc atcaatggaa 2880

gcagaagatg cagcaacata ctactgccaa caatggtcat caaaccctct tacatttgga 2940

gcaggaacaa agttagagct taaataa 2967

Claims

1. A chimeric antigen receptor CAR-immune cell, wherein said CAR-immune cell comprises the following elements:

(a) a first CAR whose extracellular binding domain contains a first binding element that targets a first target protein and whose extracellular binding domain contains or does not contain a second binding element that targets a second target protein;

2. The CAR-immune cell of claim 1, wherein the first CAR or the second CAR has the structure shown in formula I below:

L-EB-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;

EB is the extracellular binding domain;

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.

3. The CAR-immune cell of claim 1, wherein when said second target protein is FSHR, said BiTE is a CD3 ScFv.

4. A nucleic acid molecule encoding element (a), (b1) and/or (b2) in the CAR-immune cell of claim 1.

5. A vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the vector or chromosome of claim 5 into which has been integrated an exogenous nucleic acid molecule of claim 4.

7. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the CAR-immune cell of claim 1, the nucleic acid molecule of claim 4, the vector of claim 5, or the host cell of claim 6.

8. Use of the CAR-immune cell of claim 1, the nucleic acid molecule of claim 4, the vector of claim 5, or the host cell of claim 6, for the preparation of a medicament or formulation for treating a tumor.

9. The use according to claim 8, wherein the tumour is ovarian cancer.

10. A method of making the CAR-immune cell of claim 1, comprising the steps of: transferring the nucleic acid molecule of claim 4 or the vector of claim 5 into an immune cell, thereby obtaining the CAR-immune cell.