CN115894712A

CN115894712A - Regulation-control fusion protein and application thereof

Info

Publication number: CN115894712A
Application number: CN202211140708.8A
Authority: CN
Inventors: 黄璟; 马丽雅; 谢海涛
Original assignee: Shenzhen Xiankangda Life Science Co ltd
Current assignee: Shenzhen Xiankangda Life Science Co ltd
Priority date: 2022-09-20
Filing date: 2022-09-20
Publication date: 2023-04-04

Abstract

The invention discloses a regulatory fusion protein and application thereof, wherein the fusion protein is PGLYRP2, and the amino acid sequence of the PGLYRP2 is shown as SEQ ID NO 1; the fusion protein is designed into immune cells and is expressed under control. After the PGLYRP2 fusion protein for regulated expression is designed into immune cells, the chimeric antigen receptor is expressed and can specifically recognize the targeted tumor cell surface antigen; the PGLYRP2 protein is expressed in the immune cells in a regulation mode, so that the infiltration capacity of the immune cells and the killing capacity to tumors can be enhanced.

Description

Regulation-control fusion protein and application thereof

Technical Field

The invention relates to the technical field of engineering immune cell modification, in particular to a regulatory fusion protein and application thereof.

Background

Tumor (tumor) refers to a new organism (neograwth) formed by local tissue cell proliferation of the body under the action of various tumorigenic factors, because the new organism is mostly in the form of space-occupying block-shaped protrusion, also called neoplasms (neoplasms). Among them, malignant tumors are easy to metastasize, and they are easy to recur after treatment and very difficult to cure in some special microenvironments.

Tumor Microenvironment (TME) consists of abnormal tumor vessels, extracellular matrix components, endothelial cells, pericytes, tumor-associated fibroblasts, smooth muscle cells, and immune cells. TME plays a crucial role in tumor development, growth and metastasis. Abnormal tumor vasculature, extracellular matrix components, and abundant stromal cells in the TME, affect the distribution and penetration of drugs in tumor tissues. Its immunosuppressive status is also one of the important causes of failure of various anti-tumors including immunotherapy. In recent years, much research has been devoted to improving therapeutic efficacy by targeting and remodeling TME.

Peptidoglycan recognition protein 2 (PGLYRP 2) is a bacterial peptidoglycan sensitive receptor that is expressed primarily at high levels in the liver and can also be induced to be expressed in keratinocytes and epithelial cells. PGLYRP2 has N-acetylmuramic acid-L-alanine amidase activity, acts on the connection between N-acetylmuramic acid and L-alanine residues, can hydrolyze PNG of bacteria and play a role in removing, and is also a key mechanism for the anti-inflammatory action of PGLYRP2 protein. Meanwhile, it has been shown that PGLYRP2 has a basic function in hepatocytes to inhibit tumor development by stimulating anti-tumor immune response, and PGLYRP2 binds to a gene untranslated region to regulate the expression of intracellular chemokine CCL5 and other proteins. The chemokine expression can recruit different immune cells such as NK cells, T cells and other immune cells to infiltrate into the tumor in the tumor microenvironment so as to overcome the inhibiting effect of the tumor microenvironment.

Pathogen antigens stimulate T cells by binding to the T Cell Receptor (TCR), activating a signaling cascade, promoting T cell proliferation and differentiation, and finally eliminating the pathogen. After TCR activation, tyrosine kinases are phosphorylated, thereby activating downstream signal transduction pathways. In response to activation, T cells reorganize their cytoskeleton, altering their metabolism and gene expression.

The three major pathways controlling gene expression (transcription) through the TCR are MAPK (mitogen-activated protein kinase), NF-kB (nuclear factor kB-B) and TCR-dependent MAPK pathways that activate Ras first, leading to activation of downstream Erk and formation of dimer activator protein-1 (ap-1). The AP-1 dimer, when bound to an AP-1response element (AP-1 response element, AP-1-RE), increases transcription and expression of T cell activation-associated genes. In addition, TCR can transmit signals through the LAT-SPL 76 complex, which in turn activates PKC0. Similarly, co-stimulatory factor CD28 may also signal PKC0 through PI3K and PDK 1. Activated PKC0 causes IKK activation. Further causing phosphorylation of IxB α, resulting in ubiquitination and degradation of IkB α, thereby translocating the NF-KB core, binding to NF-KB response elements (NF-KBRE), causing gene transcription of activated T cell Nuclear Factor (NFAT), a class of transcription factors associated with calcium signaling pathway, binding of activated NFAT to NFAT response elements (NFAT-RE), further regulating lymphocyte development, activation, and gene expression.

Disclosure of Invention

Based on the above problems, the present invention provides a regulatory fusion protein and its application.

The technical scheme of the invention is as follows:

a PGLYRP2 regulation fusion protein contains the amino acid sequence shown in SEQ ID NO. 1.

The PGLYRP2 protein comprises an amino acid sequence shown as SEQ ID NO. 1, and the specific amino acid sequence is as follows:

MAQGVLWILLGLLLWSDPGTASLPLLMDSVIQALAELEQKVPAAKTRHTASAWLMSAPNSGPHNRLYHFLLGAWSLNATELDPCPLSPELLGLTKEVARHDVREGKEYGVVLAPDGSTVAVEPLLAGLEAGLQGRRVINLPLDSMAAPWETGDTFPDVVAIAPDVRATSSPGLRDGSPDVTTADIGANTPDATKGCPDVQASLPDAKAKSPPTMVDSLLAVTLAGNLGLTFLRGSQTQSHPDLGTEGCWDQLSAPRTFTLLDPKASLLTMAFLNGALDGVILGDYLSRTPEPRPSLSHLLSQYYGAGVARDPGFRSNFRRQNGAALTSASILAQQVWGTLVLLQRLEPVHLQLQCMSQEQLAQVAANATKEFTEAFLGCPAIHPRCRWGAAPYRGRPKLLQLPLGFLYVHHTYVPAPPCTDFTRCAANMRSMQRYHQDTQGWGDIGYSFVVGSDGYVYEGRGWHWVGAHTLGHNSRGFGVAIVGNYTAALPTEAALRTVRDTLPSCAVRAGLLRPDYALLGHRQLVRTDCPGDALFDLLRTWPHFTATVKPRPARSVSKRSRREPPPRTLPATDLQHHHHHH。

the fusion protein is designed into immune cells and is regulated and expressed, namely, the immune cells can regulate and express PGLYRP2 fusion protein under the regulation and control of a regulation promoter, and the immune cells simultaneously express chimeric antigen receptors.

The immune cells designed with PGLYRP2 have low expression of PGLYRP2 before and high expression of PGLYRP2 after approaching tumor antigen. In immune cells, the promoter of PGLYRP2 protein is a regulatory promoter, and PGLYRP2 protein can improve the infiltration effect of immune cells and the killing effect on tumors under the regulation and control of the regulatory promoter.

The fusion protein is designed into immune cells and is expressed under the regulation and control, and is realized by regulating and controlling a promoter, and the regulating and controlling sequence of the regulating and controlling promoter is one or more of NFAT, NF-KB and AP-1.

Wherein the NFAT regulatory sequence comprises a three-fold repeat of NFAT (i.e., 3 XNFAT); the NFAT comprises a nucleotide sequence shown as SEQ ID NO. 2, and the specific steps are as follows:

GGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGT。

any of the regulatory sequences NFAT, NF-KB, AP-1 can constitute the corresponding nucleic acid molecule.

The gene of the PGLYRP2 fusion protein and the chimeric antigen receptor is delivered by constructing expression cassettes (namely expression cassettes), wherein the number of the constructed expression cassettes is one or more. Further, the vector delivery means when constructing the expression cassette includes lentivirus, retrovirus, general plasmid, episome, nano delivery system, electrical transduction, or transposon.

The corresponding recombinant vector can contain an expression frame constructed by any one of nucleic acid molecules of regulatory sequences NFAT, NF-KB and AP-1 or the gene of the PGLYRP2 fusion protein and the chimeric antigen receptor.

In one embodiment, the chimeric antigen receptor is expressed as a chimeric antigen receptor that targets one target or multiple targets; wherein, the target of the chimeric antigen receptor comprises one or more of CLDN18.2, GPC3, HER2, TAA, GD2, MSLN, EGFR, NY-ESO-1, MUC1, PSMA and EBV.

In one embodiment, the binding region of the chimeric antigen receptor and the target can be scFv, fab, or a combination of scFv and Fab; the scFv region structure can be substituted by one or more of any single-chain antibody, single-chain variable fragment (scFv) and Fab fragment of any target point.

In one embodiment, the chimeric antigen receptor comprises a leader sequence, a scFv that recognizes a tumor-associated antigen, a hinge and transmembrane domain, an intracellular costimulatory domain, and an intracellular activation signal CD3Zeta; wherein the scFv is an scFv of an anti-idiotype antibody; the hinge region and transmembrane domain are CD28, or a CD8hinge region and transmembrane domain; the intracellular co-stimulatory domain is CD28, CD137 (4-1 BB), or an ICOS intracellular co-stimulatory domain.

In one embodiment, the binding region between the chimeric antigen receptor and the target can be a single target, a bispecific antibody that binds to two targets, or two or more chimeric antigen receptors that are formed across membranes and that recognize different targets.

In one embodiment, the chimeric antigen receptor comprises a structure comprising one or more of the signal peptide CD8SP, the transmembrane domain CD8Hinge, CD8TM, the intracellular activation element 4-1BB, and CD3 Zeta.

In one embodiment, the vector for the transfer of the gene of the immune cell into the chimeric antigen receptor comprises a lentivirus, a retrovirus, a general plasmid, an episome, a nano-delivery system, an electrical transduction, a transposon or other delivery system.

The immune cell may be one of a T cell, NK cell, NKT cell, macrophage, gamma-delta T cell, TIL cell, TCR-T cell or other tumor killing cell. The recombinant cell lines constructed by these immune cells include the above-described fusion proteins, nucleic acid molecules, expression cassettes, recombinant vectors, and the like.

The invention also provides a recombinant vector containing the nucleic acid molecule or the expression cassette.

The invention also provides a biological material, which comprises an expression cassette, a recombinant vector, a recombinant protein, a recombinant microorganism or a recombinant cell line which are constructed by the nucleic acid sequence or the amino acid sequence, and the nucleic acid sequence or the amino acid sequence is derived from the regulatory fusion protein. The biomaterial may also be formulated into a biological agent which is a pharmaceutically acceptable carrier, diluent or excipient. The biological agent or the biological material can be used in a medicament for treating and/or preventing cancer or tumor.

Compared with the prior art, the invention has the following beneficial effects:

after the PGLYRP2 fusion protein for regulated expression provided by the invention is designed into immune cells, the chimeric antigen receptor is expressed and the targeted tumor cell surface antigen can be specifically identified; the PGLYRP2 protein expressed in the immune cells in a regulated manner can enhance the infiltration capacity of the immune cells and the killing capacity of the tumor.

Drawings

FIG. 1 is a CAR-T cell nucleic acid molecule structure design;

FIG. 2 is a CAR-T amplification growth graph;

FIG. 3 is a flow chart of HGC-27-CLDN18.2 expression CLDN 18.2;

FIG. 4 shows the expression results of PGLYRP2 before and after stimulation with tumor antigen;

FIG. 5 is a result of in vitro recruitment of CAR-T cells;

FIG. 6 is a CAR-T animal experimental survival curve.

Detailed Description

The preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings.

The invention provides a PGLYRP2 regulatory protein with controllable expression, wherein the fusion protein is designed into immune cells and is subjected to controllable expression, namely the immune cells can controllably express the PGLYRP2 fusion protein under the control of a regulatory promoter, and the immune cells simultaneously express chimeric antigen receptors.

The immune cells designed with PGLYRP2 regulatory protein express PGLYRP2 in low level before approaching tumor antigen and express PGLYRP2 in high level after approaching tumor antigen; the promoter of PGLYRP2 protein in immune cells is a regulatory promoter, and the PGLYRP2 protein can improve the infiltration capacity of the immune cells and the killing effect on tumors under the regulation and control of the regulatory promoter. Wherein the regulation and control sequence for regulating and controlling the promoter is one or more of NFAT, NF-KB and AP-1.

In one embodiment, a nucleic acid molecule comprising any one or more of the regulatory sequences NFAT, NF-KB, AP-1 is also contemplated.

In one embodiment, the immune cell expresses a chimeric antigen receptor, e.g., a CAR cell. Chimeric antigen receptor expression may be a chimeric antigen receptor that targets one target or multiple targets; such as a CAR cell.

In one embodiment, the chimeric antigen receptor may also be targeted by one or more of the idiotypes CLDN18.2, GPC3, HER2, TAA, GD2, MSLN, EGFR, NY-ESO-1, MUC1, PSMA and EBV.

The binding region of the chimeric antigen receptor and the target can be scFv, fab or a combination of scFv and Fab; wherein, the scFv region structure can be replaced by one or more of any single-chain antibody, single-chain variable fragment (scFv), fab fragment and the like of any target point.

The chimeric antigen receptor comprises a leader sequence, scFv for recognizing a tumor-associated antigen, a hinge region and transmembrane domain, an intracellular costimulatory domain, and an intracellular activation signal CD3 ζ; wherein the scFv is an scFv of an anti-idiotype antibody; the hinge region and transmembrane domain are a CD28 or CD8hinge region and transmembrane domain; the intracellular co-stimulatory domain is CD28 or CD137 (4-1 BB) or an ICOS intracellular co-stimulatory domain.

The binding region of the chimeric antigen receptor and the target can be a binding region that binds to one target, or can be a bispecific antibody that binds to two targets, or can be a binding region in which two or more chimeric antigen receptors are formed across membranes and recognize different targets.

The PGLYRP2 fusion protein and the gene of the chimeric antigen receptor are delivered by constructing expression frames, namely expression cassettes, wherein the number of the constructed expression frames is one or more; when the expression frame is constructed, the delivery mode of the vector comprises lentivirus, retrovirus, common plasmid, episome, nano delivery system, electric transduction or transposon; that is, vectors for the transfer of genes of immune cells into chimeric antigen receptors include lentiviruses, retroviruses, common plasmids, episomes, nano-delivery systems, electrical transduction, transposons, or other delivery systems. The recombinant vector for constructing the expression frame can be a nucleic acid molecule containing a regulatory sequence NFAT, NF-KB or AP-1, or an expression frame constructed by gene fusion of PGLYRP2 fusion protein and a chimeric antigen receptor.

The immune cell of the invention may be one of a T cell, NK cell, NKT cell, macrophage, gamma-delta T cell, TIL cell, TCR-T cell or other tumor killing cell, preferably a T cell. The recombinant cell lines constructed by these immune cells include the above-described fusion proteins, nucleic acid molecules, expression cassettes, recombinant vectors, and the like.

The immune cells expressing PGLYRP2 protein in a regulated manner can be prepared into biological materials; the biological material comprises an expression cassette, a recombinant vector, a recombinant protein, a recombinant microorganism or a recombinant cell line which are constructed by a nucleic acid sequence or an amino acid sequence, wherein the nucleic acid sequence or the amino acid sequence is derived from the regulatory fusion protein.

The biological material can also be prepared into biological preparation which is a pharmaceutically acceptable carrier, diluent or excipient; and administration of the biological agent may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The biological agent can be applied to drugs for preventing and/or treating solid tumors.

The immune cell expressing PGLYRP2 in a regulated manner expresses a chimeric antigen receptor, and can specifically recognize a targeted tumor cell surface antigen; the immune cell regulated expresses PGLYRP2 protein, and can enhance the infiltration capacity of immune cells and the killing capacity to tumors.

In the following, CAR-T is described as a specific example, and any of NK cells, NKT cells, macrophages, gamma-delta T cells, TIL cells, and the like may be used as the immune cells.

EXAMPLE 1 preparation of Lentiviral vectors

1. Design of synthetic CAR nucleic acid molecule structures

1) Consisting of 3 × NFAT (nucleotide sequence shown in SEQ ID NO: 2), minP (weak promoter), PGLYRP2 (amino acid sequence is shown in SEQ ID NO: 1) and CAR molecules are connected in series to form the NFAT-P2-EF1 alpha-CAR molecule. The structure of the synthetic promoter-containing minP-containing CAR nucleic acid molecule is shown in part A of FIG. 1.

The amino acid sequence of PGLYRP2 is shown in SEQ ID NO: 1:

2) And 3 XNFAT nucleotide sequence is shown as SEQ ID NO:2, as shown in the figure:

3) And NF-KB-P2-EF1 alpha-CAR molecules formed by the tandem connection of 3 XNF-KB response elements, minP (weak promoter), PGLYRP2 and CAR molecules. The structure of the synthetic promoter-containing minP-containing CAR nucleic acid molecule is shown in part B of FIG. 1.

4) An AP-1-P2-EF1 alpha-CAR molecule formed by tandem connection of a 3 × AP-1 reactive element, a minP, a PGLYRP2, and a CAR molecule. The structure of the synthetic promoter-containing minP-containing CAR nucleic acid molecule is shown in part C of FIG. 1.

5) EF1 α -CAR molecules formed by the tandem connection of the nucleic acid sequences of EF1 α promoter, CD8 α signal peptide, anti-CLDN 18 single chain antibody, CD8 α transmembrane domain, 4-1BB and CD3 ζ, and the structure of the CAR nucleic acid molecule designed to synthesize a promoter-free minP is shown in part D of fig. 1.

Constructing the CAR molecule synthesized by the part A in the figure 1, a regulatory promoter and a PGLYRP2 molecule into a lentiviral expression vector P161 to form a pNFAT-P2-EF1 alpha-CAR plasmid; the partially D synthetic CAR molecule of figure 1 was constructed into lentiviral expression vector p161, forming the pEF1 α -CAR plasmid.

2. Construction of CLDN18.2 molecular nucleic acid sequences

The synthetic CLDN18.2 molecular nucleic acid sequence was constructed into p161 to form a p CLDN18.2 plasmid as shown in section E of fig. 1.

Example 2 packaging of recombinant lentiviruses

The pEF 1. Alpha. -CAR prepared in example 1 was transferred into 293T cells with three plasmids, lentiviral envelope Plasmid pMD2.G (Addgene, plasmid # 12259) and lentiviral packaging Plasmid psPAX2 (Addgene Plasmid # 12260), using Lipofectamine3000, to prepare a lentiviral complete expression vector LV-EF 1. Alpha. -CAR.

pNFAT-P2-EF 1. Alpha. -CAR was transformed with the lentiviral envelope Plasmid pMD2.G (Addgene, plasmid # 12259) and the lentiviral packaging Plasmid psPAX2 (Addgene Plasmid # 12260) using Lipofectamine3000 into 293T cells to prepare the lentiviral complete expression vector LV-NFAT-P2-EF 1. Alpha. -CAR.

The entire lentiviral expression vector LV-CLDN18.2 was prepared by transferring p CLDN18.2 with the three lentiviral envelope Plasmid pMD2.G (Addge, plasmid # 12259) and the lentiviral packaging Plasmid psPAX2 (Addge Plasmid # 12260) into 293T cells using Lipofectamine 3000.

The three virus supernatants were collected at 48h and 72h, respectively, and ultracentrifugation concentration (Merck Millipore) was performed on the collected virus supernatants, respectively, to yield three concentrated viruses, i.e., LV-EF1 α -CAR, LV-NFAT-P2-EF1 α -CAR, LV-CLDN18.2, respectively.

Example 3 preparation of PGLYRP 2-expressing regulatory CAR-T cells

1. T cell harvesting

Separating mononuclear cells from peripheral blood of a donor, performing density gradient centrifugation using a ficoll method, and enriching T cells using a T cell sorting kit, such as CD3 MicroBeads, human-lysophilized, or 130-097-043, and activating cultured and expanded T cells using anti-CD3/anti-CD28 coupled magnetic beads;

2. expansion of T cells

For T cell culture, texMACS GMP Medium (Miltenyi Biotec, 170-076-309) Medium containing 10% of FBS, 2mM L-glutamine and 100IU/ml rhIL2 was used, and the cells were cultured at 37 ℃ and 5% CO ₂ Culturing in a constant-temperature incubator;

infecting the concentrated recombinant lentiviruses LV-EF1 alpha-CAR and LV-NFAT-P2-EF1 alpha-CAR with activated T cells respectively to obtain CAR-T cells (CAR-T-CLDN 18.2) only expressing CAR and CAR-T cells (CAR-T-CLDN 18.2-P2) expressing CAR and simultaneously expressing PGLYRP2 in a regulated manner. The two CAR-T proliferation profiles obtained are shown in figure 2; the proliferation expansion fold of CAR-T-CLDN18.2 and CAR-T-CLDN18.2-P2 cells was 1958 fold and 2221 fold, respectively, by day 12 of culture.

Example 4 construction of cell line expressing CLDN18.2

The virus LV-CLDN18.2 concentrated in example 2 was used to infect HGC-27 cells, and a HGC-27 cell line overexpressing CLDN18.2 was finally obtained and designated HGC-27-CLDN18.2. The expression results for CLDN18.2 are shown in fig. 3.

Example 5 expression of CAR-T cell PGLYRP2

Taking two CAR-T cells prepared in example 3, resuscitating and culturing the cells by using a fresh T cell culture medium for 24 hours, and collecting the resuscitated CAR-T cells; co-culturing the HGC-27-CLDN18.2 overexpression cell line constructed in the embodiment 4 and the recovered CAR-T cells for 24h, and collecting cell supernatant; the expression of PGLYRP2 of each cell was measured by ELISA, and the results are shown in FIG. 4; the CAR-T-CLDN18.2 cells do not secrete PGLYRP2 fusion protein (i.e., PGLYRP2 fusion protein is secreted in an amount of 0), while CAR-T-CLDN18.2-P2 cells secrete 300pg/ml PGLYRP2 fusion protein.

Example 6 CAR-T cell in vivo functional assessment

Selecting 6-8 weeks old NSG mice (weight 18-22 g) 24 mice, adaptively feeding for one week, subcutaneously inoculating HGC-27-CLDN18.2 positive tumor cell strain, and inoculating 1 x 10 for each mouse ⁷ Closely observing the state of the animal, measuring the tumor volume of the mouse every three days by using a vernier caliper, and when the tumor volume reaches 100mm ³ CAR-T cells or control T cells were infused via tail vein after randomized grouping according to mouse body weight and tumor size. The detailed administration method, administration dose and administration route are shown in table 1.

TABLE 1 animal protocol

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As shown in the results of FIG. 5, the PGLYRP 2-expressing CAR-T can greatly prolong the survival time of mice. As shown in FIG. 6, regulated PGLYRP 2-expressing CAR-T produced a significant increase in tumor-infiltrating CAR-T cells (CAR-T-CLDN 18.2:3.5%; CAR-T-CLDN18.2-P2: 6.2%).

The above examples demonstrate that: the regulated CAR-T cells expressing PGLYRP2 have stronger proliferation capacity and in-vitro tumor killing activity on tumors compared with CAR-T cells not expressing other proteins.

It should be understood that the above description is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting the scope of the invention, which is defined by the appended claims.

Claims

1. A regulatory fusion protein is characterized in that the fusion protein is PGLYRP2, and the amino acid sequence of the PGLYRP2 is shown as SEQ ID NO 1; the fusion protein is designed into immune cells and is expressed under control.

2. The regulatory fusion protein of claim 1, wherein the regulated expression of the fusion protein is achieved by a regulatory promoter, and the regulatory sequence of the regulatory promoter is one or more of NFAT, NF-KB, and AP-1.

3. The modulatory fusion protein of claim 2, wherein said NFAT regulatory sequence comprises a three-way repeat of NFAT; the NFAT with the three repeated segments comprises a nucleotide sequence shown as SEQ ID NO. 2.

4. A nucleic acid molecule comprising the NFAT, NF-KB or AP-1 regulatory sequences of claim 2.

5. An expression cassette comprising the fusion protein of claim 1 or the nucleic acid molecule of claim 4.

6. A recombinant vector comprising the fusion protein of claim 1, or comprising the nucleic acid molecule of claim 4, or comprising the expression cassette of claim 5.

7. A recombinant cell line comprising the fusion protein of claim 1, or comprising the nucleic acid molecule of claim 4, or comprising the expression cassette of claim 5, or comprising the recombinant vector of claim 6.

8. A chimeric antigen receptor comprising the modulatory fusion protein of any one of claims 1-3.

9. A biological agent comprising the fusion protein of claim 1, or comprising the nucleic acid molecule of claim 4, or comprising the expression cassette of claim 5, or comprising the recombinant vector of claim 6, or comprising the recombinant cell line of claim 7.

10. Use of the biological agent according to claim 9 in a medicament for the treatment and/or prevention of cancer or a tumor.