Anticancer composition comprising chimeric antigen receptor-expressing immune cell and traptavidin scaffold antibody-like protein, and use thereof

Abstract

The present invention relates to an anticancer cell therapeutic composition comprising immune cells expressing chimeric antigen receptors and traptavidin scaffold antibody-like proteins and a use thereof. The invention provides: an immune cell expressing a chimeric antigen receptor (CAR) that includes a biotin-tagged protein, and a co-stimulatory domain and an activation domain which are both capable of inducing an anticancer response; an antibody-like protein that contains a single-chain variable fragment (scFv) binding specifically to a tumor antigen, and traptavidin; wherein the antibody-like protein forms a tetramer through the self-binding of traptavidin and binds to a biotin-tagged protein expressed on the surface of the immune cell to allow the immune cell to have four single-chain variable fragments (scFvs), thereby providing an immune cell therapeutic agent having enhanced avidity to a tumor-specific antigen.

Description

Anticancer composition comprising an immune cell expressing a chimeric antigen receptor and a streptavidin scaffold antibody-like protein, and use thereof

[0001] The present invention relates to an anticancer cell therapy composition comprising an immune cell expressing a chimeric antigen receptor and a traptavidin scaffold antibody-like protein, and a use thereof.

[0002] When normal cells undergo genetic or acquired mutations and exhibit abnormal proliferation, they transform into cancer cells, and this proliferation leads to the development of cancer. Cancer cells express proteins and genes that differentiate them from normal cells. These are called tumor antigens, which distinguish cancer cells. Tumor antigens are recognized by the body's innate immune system, such as NK cells, T cells, and B cells, and are utilized as a means to kill cancer cells. They are also used as targets for various anticancer drugs. However, for certain cancers, there is a need to develop anticancer drugs with enhanced anticancer efficacy by evading the body's immune system and the various mechanisms by which anticancer drugs target cancer cells.

[0003] Among the anticancer drugs currently under development are cell therapies that utilize the immune system. Cell therapies are based on immune cells, which are modified to enhance cancer targeting ability or to have enhanced anticancer activity. Chimeric antigen receptors (CARs) are primarily used to modify the function of immune cells used in cell therapies. Currently developed chimeric antigen receptors consist of an antigen-binding domain, a transmembrane domain, and one or more intracellular signaling domains. The intracellular signaling domains in chimeric antigen receptors are involved in immune cell activation and can, for example, initiate the release of cytolytic molecules that induce tumor cell death.

[0004] Most currently used cell therapies utilize T cells. These T cells are easily proliferated and morphologically modified, and possess exceptional cancer-killing capabilities. Cell therapies utilizing T cells can be modified to target specific cancers and engineered to have tumor recognition sites that specifically bind to specific tumors. Chimeric antigen receptors (CARs) can be utilized as a method for modifying these T cells, and T cells expressing these CARs are called CAR-Ts.

[0005] [Prior Art Literature]

[0006] [Patent Document]

[0007] Korean Patent Publication No. 10-2018-0130534 (published on December 7, 2018)

[0008] The purpose of the present invention is to provide an immune cell therapeutic agent having an excellent anticancer effect by enhancing avidity for a tumor-specific antigen, and in the present invention, an immune cell expressing a chimeric antigen receptor including a biotin-tagged protein and a co-stimulatory region and a stimulatory region that induce an anticancer response is provided, and an antibody-like protein including a single chain variable fragment (scFv) that specifically binds to a tumor-specific antigen and Traptavidin is provided, and the antibody-like protein forms a tetramer due to self-association of Traptavidin and binds to a biotin-tagged protein expressed on the surface of the immune cell, thereby configuring the immune cell to have four single chain variable fragments (scFv), thereby providing an immune cell therapeutic agent having enhanced avidity for a tumor-specific antigen.

[0009] The present invention provides a chimeric antigen receptor comprising a tag protein, a hinge and transmembrane region (HTM region), a costimulatory region, and a stimulatory region.

[0010] Additionally, the present invention provides a polynucleotide encoding the chimeric antigen receptor.

[0011] Additionally, the present invention provides a vector for producing a chimeric antigen receptor comprising the polynucleotide.

[0012] In addition, the present invention provides an immune cell comprising a chimeric antigen receptor comprising a tag protein, a hinge and transmembrane region (HTM region), a costimulatory region, and a stimulatory region, and expressing the tag protein on the cell membrane surface.

[0013] In addition, the present invention provides a method for producing a CAR expressing immune cell, comprising the step of transducing an immune cell with a vector comprising a polynucleotide encoding a chimeric antigen receptor comprising a tag protein, a hinge and transmembrane region (HTM region), a costimulatory region, and a stimulatory region.

[0014] Additionally, the present invention provides an antibody-like protein comprising a single chain variable fragment (scFv) and Traptavidin.

[0015] In addition, the present invention provides a polynucleotide encoding the antibody-like protein.

[0016] In addition, the present invention provides a vector for producing an antibody-like protein comprising the polynucleotide.

[0017] In addition, the present invention provides an immune cell therapeutic composition for preventing or treating cancer, comprising: a chimeric antigen receptor comprising a tag protein, a hinge and transmembrane region (HTM region), a costimulatory region, and a stimulatory region; an immune cell expressing the tag protein on the cell membrane surface; and an antibody-like protein comprising a single chain variable fragment (scFv) and Traptavidin.

[0018] According to the present invention, an immune cell expressing a chimeric antigen receptor that expresses a biotin tag on the membrane surface and includes a co-stimulatory region and a stimulus region that induce an anticancer response inside the cell is provided, and an antibody-like protein composed of a single chain variable fragment (scFv) and Traptavidin binds to the biotin tag expressed on the surface of the immune cell in the form of a tetramer due to self-association, so that the cell therapeutic agent manufactured according to the present invention has a single chain variable fragment (scFv) on the cell membrane surface of four cells, thereby enhancing the binding avidity to a tumor-specific antigen, and can be provided as an anticancer cell therapeutic agent with excellent anticancer effects.

[0019] Figure 1 is a schematic diagram of a plasmid for producing a chimeric antigen receptor according to the present invention.

[0020] Figure 2 shows a CD4 expressing chimeric antigen receptor (CAR) produced according to the present invention. + / CD8+ This is the result of analyzing the expression level of GFP (green fluorescent protein) using flow cytometry to confirm whether T cells were produced.

[0021] Figure 3 is a schematic diagram of producing a biotin-tagged CAR-T using biotin ligation of a T cell (CAR-T) expressing a chimeric antigen receptor (CAR) produced according to the present invention.

[0022] Figure 4 shows the results of observing the fluorescent area using a confocal microscope to confirm whether the biotin protein was exposed on the surface of a T cell (CAR-T) transduced with a biotin-tagged CAR protein produced according to the present invention.

[0023] Figure 5 is a schematic diagram of a vector for producing a Traptavidin scaffold antibody-like protein according to the present invention.

[0024] Figure 6 shows the results of detecting the Traptavidin scaffold antibody-like proteins manufactured according to the present invention using SDS-PAGE.

[0025] Figure 7 is a schematic diagram showing a tetramer form formed by self-assembly of a Traptavidin scaffold antibody-like protein manufactured according to the present invention.

[0026] Figure 8 is a Western blot result confirming the presence of a biotin binding site and FMC63 in a traptavidin scaffold antibody-like protein manufactured according to the present invention.

[0027] FIG. 9 is a schematic diagram of an immunotherapy comprising a tetramer-based universal CAR-T comprising a T cell (CAR-T) transduced with a biotin-tagged CAR protein and a Traptavidin scaffold antibody-like protein according to the present invention.

[0028] Figure 10 shows the results of evaluating the effect on cytokine levels to evaluate the anticancer activity of a tetramer-based universal CAR-T produced according to the present invention.

[0029] Figure 11 shows the results of evaluating cytotoxicity against cancer cells to evaluate the anticancer activity of a tetramer-based universal CAR-T produced according to the present invention.

[0030] The terms used in this specification have been selected from widely used, current terms, taking into account the functions of the present invention. However, these terms may vary depending on the intentions of those skilled in the art, precedents, the emergence of new technologies, etc. Furthermore, in certain cases, terms may be arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should not be defined simply as names, but rather based on their inherent meanings and the overall content of the present invention.

[0031] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0032] Numerical ranges are inclusive of the values ​​defined in the ranges above. Any maximum numerical limitation given throughout this specification includes any lower numerical limitation, as if that lower numerical limitation were explicitly stated. Any minimum numerical limitation given throughout this specification includes any higher numerical limitation, as if that higher numerical limitation were explicitly stated. Any numerical limitation given throughout this specification will include any better numerical range within the broader numerical range, as if that narrower numerical limitation were explicitly stated.

[0033] Hereinafter, the present invention will be described in more detail.

[0034] The present invention provides a chimeric antigen receptor comprising a tag protein, a hinge and transmembrane region (HTM region), a costimulatory region, and a stimulatory region.

[0035] The above tag protein is an avidin or biotin tag protein.

[0036] The above "HTM region (hinge and transmembrane region)" refers to a hydrophobic polypeptide that is naturally or artificially designed and spans from the cell membrane exposed to the outside of the cell to the cell membrane exposed to the inside of the cell or the cytoplasm, and is also called a transmembrane domain. The HTM region may be in the form of an alpha helix or a beta barrel, or a combination thereof, and may include a multi-component protein having multiple transmembrane segments, each alpha-helix, beta sheet, or a combination thereof.

[0037] In one embodiment of the present invention, the HTM region (hinge and transmembrane region) included in the chimeric antigen receptor may be, but is not limited to, the CD8 HTM represented by SEQ ID NO: 3, and the HTM region may be selected or modified by amino acid substitution.

[0038] The above "costimulatory region" is a polypeptide located inside a cell to stimulate or activate part or all of a signal peptide that transmits a signal for cellular activity such as an anti-cancer response or an immune response.

[0039] In one embodiment of the present invention, the costimulatory region may be one or more proteins selected from 4-1BB, CD2, CD7, CD27, CD28, CD30, CD40, CD83, CD258, NKG2C, NKG2D, B7-H3, OX40, ICAM-1, LFA-1, and ICOS, or an active region thereof, but is not limited thereto. Preferably, the costimulatory region of the present invention may be 4-1BB represented by SEQ ID NO: 5.

[0040] The above "stimulatory region" is a signal peptide located inside a cell that transmits signals for cell activity such as anticancer response or immune response.

[0041] In one embodiment of the present invention, the stimulatory region may be one or more proteins selected from CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRδ, TCRγ, CD79a, CD79b, DAP10, and DAP12, or an active region thereof, but is not limited thereto. Preferably, the stimulatory region of the present invention may be CD3ζ represented by SEQ ID NO: 7.

[0042] The above "chimeric antigen receptor" is a recombinant receptor protein having an antigen recognition site that specifically binds to cancer cells, and is composed of a site that recognizes surface molecules of cancer cells, a transmembrane region that penetrates the cell membrane, and an intracytoplasmic signal transduction site that induces cell activity. In one embodiment of the present invention, the chimeric antigen receptor includes an avidin or biotin tag protein as a tag protein, includes a CD8 HTM region as a transmembrane region, includes 4-1BB and CD3ζ as intracytoplasmic signal transduction sites, and may additionally include a fluorescent protein, GFP (green fluorescent protein). Specifically, the chimeric antigen receptor of the present invention is configured to sequentially include a tag protein, CD8 HTM, 4-1BB, and CD3ζ, and may include a fluorescent protein, GFP (green fluorescent protein), at the end of CD3ζ. More specifically, the chimeric antigen receptor of the present invention may be composed of a polypeptide represented by SEQ ID NO: 11.

[0043] The term "protein" as used herein is used interchangeably with "peptide" and "polypeptide," and refers to a compound having amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limitation on the maximum number of amino acids that can comprise the sequence of a protein or peptide. A polypeptide includes any peptide or protein having two or more amino acids linked to each other by peptide bonds. The term as used herein refers to both chains, with shorter chains also commonly referred to as peptides, oligopeptides, and oligomers in the art, and longer chains generally referred to as proteins in the art, of which there are many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0044] Additionally, the present invention provides a polynucleotide encoding the chimeric antigen receptor.

[0045] The above "polynucleotide" is a chain of nucleotides, including DNA and RNA. The polynucleotide is specifically a polymer of nucleotides as a nucleic acid, wherein the polynucleotide can be hydrolyzed into its monomeric nucleotides. The polynucleotide includes, but is not limited to, any nucleic acid sequence obtained by any means available in the art, including cloning of nucleic acid sequences from recombinant libraries or cellular genomes using conventional cloning techniques and polymerase chain reaction (PCR), and any nucleic acid sequence obtained by synthetic means.

[0046] Additionally, the present invention provides a vector for producing a chimeric antigen receptor comprising the polynucleotide.

[0047] The vector for producing the chimeric antigen receptor comprises an avidin gene encoding an avidin tag protein represented by SEQ ID NO: 2; a CD8 HTM gene encoding a CD8 HTM represented by SEQ ID NO: 4; a 4-1BB gene encoding a 4-1BB represented by SEQ ID NO: 6; and a CD3ζ gene encoding a CD3ζ represented by SEQ ID NO: 8. In addition, the vector comprises a first linker gene represented by the 46th to 51st base sequence of SEQ ID NO: 12 between the avidin gene and the CD8 HTM gene. Additionally, the vector for producing the chimeric antigen receptor may include a gene encoding GFP (green fluorescent protein) represented by SEQ ID NO: 10 at the 3' end of the CD3ζ gene, which is a stimulatory region, and may include a second linker gene represented by the 721st to 735th base sequence of SEQ ID NO: 12 between the CD3ζ gene and the GFP gene.

[0048] Specifically, the vector for producing the chimeric antigen receptor of the present invention may include a polynucleotide represented by SEQ ID NO: 12.

[0049] In the present invention, the "vector" is a tool that enables stable transfection of cells, and the vector enables incorporation of the transgene(s) into the cellular genome. Preferably, such a vector has a positive selection marker, and a suitable positive selection marker includes any gene that allows cells to grow under conditions that kill cells that do not express the gene. Non-limiting examples may include antibiotic resistance. Additionally, the vector is a plasmid vector, and more specifically, the vector is a viral vector, and can be replaced with any suitable vector by a person skilled in the art, and preferably, the vector may be a lentiviral vector, a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenovirus vector, or an adenovirus-associated vector, but is not limited thereto.

[0050] Below, repetitive features shared by each aspect of the invention are described without explanation.

[0051] In addition, the present invention provides an immune cell comprising a chimeric antigen receptor comprising a tag protein, a hinge and transmembrane region (HTM region), a costimulatory region, and a stimulatory region, and expressing the tag protein on the cell membrane surface.

[0052] The above immune cells may be, but are not limited to, T cells, NK cells, or B cells.

[0053] In addition, the present invention provides a method for producing a CAR expressing immune cell, comprising the step of transducing an immune cell with a vector comprising a polynucleotide encoding a chimeric antigen receptor comprising a tag protein, a hinge and transmembrane region (HTM region), a costimulatory region, and a stimulatory region.

[0054] If the tag protein is avidin, a biotin ligation step may be additionally included after the transduction step by reacting with biotin-AMP (biotin-adenosine monophosphate).

[0055] Additionally, the present invention provides an antibody-like protein comprising a single chain variable fragment (scFv) and Traptavidin.

[0056] The single chain variable fragment (scFv) binds to a tumor-specific antigen, and the tumor-specific antigen may be, but is not limited to, CD19, CD20, GD2, HER-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CSPG4, or B7-H4. Specifically, the single chain variable fragment (scFv) includes an FMC63 VL region (variable light chain region) which is a polypeptide represented by SEQ ID NO: 13, and an FMC63 VH region (variable heavy chain region) which is a polypeptide represented by SEQ ID NO: 15.

[0057] The above-mentioned Traptavidin is a biotin-binding protein with enhanced affinity, and is a protein in which the S52G and R53D mutations occur in streptavidin. The above-mentioned Traptavidin has a dissociation rate with respect to biotin that is 10 times slower than that of streptavidin, and has the characteristics of increased mechanical strength and improved thermal stability. In addition, the above-mentioned Traptavidin can form a tetramer through self-assembly without additional stimulation, and the above-mentioned Traptavidin monomer has a size of 42 kDa, and the above-mentioned Traptavidin tetramer has a size of 168 kDa. Specifically, the above-described Traptavidin of the present invention is a polypeptide represented by SEQ ID NO: 17.

[0058] In addition, the present invention provides a polynucleotide encoding the antibody-like protein.

[0059] In addition, the present invention provides a vector for producing an antibody-like protein comprising the polynucleotide.

[0060] The vector for producing the above antibody-like protein comprises a leader sequence represented by SEQ ID NO: 22; a gene encoding an FMC63 VL region (variable light chain region) represented by SEQ ID NO: 14; a gene encoding an FMC63 VH region (variable heavy chain region) represented by SEQ ID NO: 16; and a gene encoding streptavidin (Tav) represented by SEQ ID NO: 18.

[0061] In addition, the vector for producing the antibody-like protein includes a third linker gene represented by the 388th to 432nd base sequence of SEQ ID NO: 19 between the FMC63 VL gene and the FMC63 VH gene, an Sfil gene represented by SEQ ID NO: 21 between the FMC63 HL gene and the Tav gene and a fourth linker gene represented by the 808th to 861st base sequence of SEQ ID NO: 19, and a His tag gene represented by SEQ ID NO: 20 at the 3' end of the gene encoding streptavidin (Tav). In addition, one or two additional bases for frame shift may be included between the Sfil gene and the fourth linker gene, and specifically, adenosine (A) and guanine (G) may be sequentially included as the additional bases between the Sfil gene and the fourth linker gene.

[0062] Specifically, the vector for producing the antibody-like protein of the present invention may include a polynucleotide represented by SEQ ID NO: 19.

[0063] In addition, the present invention provides an immune cell therapeutic composition for preventing or treating cancer, comprising: a chimeric antigen receptor comprising a tag protein, a hinge and transmembrane region (HTM region), a costimulatory region, and a stimulatory region; an immune cell expressing the tag protein on the cell membrane surface; and an antibody-like protein comprising a single chain variable fragment (scFv) and Traptavidin.

[0064] The antibody-like protein forms a tetramer by self-assembly of the Traptavidin, and the Traptavidin forming the tetramer binds to the tag protein expressed on the surface of the immune cell, thereby exposing four single chain variable fragments (scFv) to the surface of the immune cell. Therefore, since the immune cell therapeutic agent of the present invention has four single chain variable fragments (scFv), its avidity for tumor-specific antigens is enhanced, and thus its anticancer effect is superior to that of conventional cell therapeutic agents.

[0065] The above cancers are leukemia, acute lymphoblastic leukemia, acute myeloblastic leukemia, chronic leukemia, chronic myeloblastic (granulocytic) leukemia, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary It may be one or more diseases selected from among adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, cholangiocarcinoma, seminoma, embryogenic carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0066] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains.

[0067] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0068] In the present invention, the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0069] The above pharmaceutical composition may be formulated in the form of one or more external preparations selected from the group consisting of injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, and cataplasmas.

[0070] The pharmaceutical composition of the present invention may further comprise pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients such as starches, sugars, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinyl pyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbates, cetyl alcohol, and glycerol. The pharmaceutically acceptable carriers and diluents may be biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents, and / or dispersion media.

[0071] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. In the case of oral administration, it may be formulated as tablets, troches, lozenges, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, or elixirs. In the case of parenteral administration, it may be formulated as injections, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for application, oils, creams, etc.

[0072] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition and weight, age, sex, health status, dietary constitution, nature of the formulation, severity of the disease, administration time of the composition, administration method, administration period or interval, excretion rate, and drug form, and may be appropriately selected by a person skilled in the art. For example, the dosage may range from about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day in divided doses.

[0073] The pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. The pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time, and / or administration route of the pharmaceutical composition, and a person skilled in the art can easily determine and prescribe an effective dosage for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or divided into several doses.

[0074] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0075]

[0076] Example 1. Production of immune cells expressing chimeric antigen receptors

[0077] 1-1. Production of CAR expression plasmid

[0078] The chimeric antigen receptor (hereinafter referred to as CAR) according to the present invention is sequentially composed of a tag protein, a hinge and transmembrane region (hereinafter referred to as HTM), a costimulatory region, and a stimulatory region, and in this embodiment, as one embodiment of the CAR, it is produced to sequentially include an avidin tag protein, CD8 HTM, 4-1BB, and CD3ζ.

[0079] In the above-described example, a plasmid containing an insert DNA encoding the CAR protein was constructed (Fig. 1). The insert DNA was designed to sequentially include an avidin gene encoding an avidin tag protein represented by SEQ ID NO: 2, a CD8 HTM gene encoding CD8 HTM represented by SEQ ID NO: 4, a 4-1BB gene encoding 4-1BB represented by SEQ ID NO: 6, and a CD3ζ gene encoding CD3ζ represented by SEQ ID NO: 8. The insert DNA was designed to have a first linker gene represented by the 46th to 51st base sequence of SEQ ID NO: 12 between the avidin gene and the CD8 HTM gene. In addition, in this embodiment, in order to easily confirm whether CAR is expressed or inserted using a fluorescent protein, a gene encoding GFP (green fluorescent protein) represented by SEQ ID NO: 10 is included at the 3' end of the CD3ζ gene, which is a stimulatory region, and a second linker gene represented by the 721st to 735th base sequence of SEQ ID NO: 12 is designed to exist between the CD3ζ gene and the GFP gene.

[0080] Finally, the insert DNA encoding the CAR manufactured in this example is composed of the base sequence represented by SEQ ID NO: 12, and the CAR protein manufactured finally was designed to have the amino acid sequence represented by SEQ ID NO: 11. Each gene was cloned into the pHR-SFFV vector (Addgene) by requesting a sequencing service, and the pHR-SFFV-CAR-GFP recombinant vector was manufactured, and the presence of the insert DNA was confirmed using agarose gel electrophoresis.

[0081]

[0082] 1-2. Production of lentivirus for CAR infection

[0083] Lentivirus capable of transfecting immune cells with the pHR-SFFV-CAR-GFP recombinant vector manufactured above was prepared. 6 × 10 cells were seeded in a 100π dish. 6 293T cells were prepared at a concentration of cell / mL and transfected with 8 μg each of the pHR-SFFV-CAR-GFP recombinant vector, pMD2G (Addgene, 12259), and psPAX2 (Addgene, 12260). The cells were cultured at 37°C and 5% CO2 for 3 to 5 days, and the culture medium was collected and centrifuged at 2,000 rpm for 10 minutes at 4°C to separate the supernatant. The separated supernatant was filtered through a 0.45 μm filter, and 1 / 4 of the filtered supernatant was added to the Lenti X concentrator (Takara, 631231) and reacted overnight at 4°C. The solution after the reaction was completed was centrifuged at 4°C and 1,500 xg for 45 minutes, and the obtained pellet was resuspended in PBS (phosphate buffer saline) and 1 × 10 13 Lentivirus for CAR infection was produced at a concentration of copies / mL.

[0084]

[0085] 1-3. Immune cell culture

[0086] EasySep from human peripheral blood mononuclear cells (PBMCs) TM CD4 using Human T cell Isolation kit (Stemcell, 17951) + / CD8 + T cells were isolated. The isolated CD4 + / CD8 + T cells were seeded at 1 × 10 in ImmunoCult XF T Cell Expansion Medium (Stemcell, 10981) containing 25 μL of Dynabeads human T activator (Gibco, 11131D) and 50 U / mL of IL-2 (Roche, 11011456001) in a 24-well plate. 6 cells / mL and cultured for 3 days. Cells were counted and 2 × 10 5 Cells were cultured by adding medium to obtain cells / mL.

[0087]

[0088] 1-4. CAR-T cell production using lentivirus

[0089] CD4 cultured above + / CD8 + T cells were seeded at 1 × 10 in a 24-well plate. 6 Dispense 1 mL at a concentration of cells / mL, and add 1 × 10 of the prepared lentivirus. 6 1 × 10 7copies / mL concentration, 8 μg of polybrene was added, and the mixture was centrifuged at 900 xg for 70 minutes at 32°C to induce binding of lentivirus to T cells. After incubation overnight at 37°C and 5% CO2, new medium was added. After incubation for 3 days by replacing the medium in the same manner as described above, the expression level of GFP was analyzed by flow cytometry to determine the CD4 + / CD8 + We confirmed whether CAR-T (Chimeric Antigen Receptor T cell) was produced by transducing CAR protein into T cells. The control group was wild-type CD4 not infected with lentivirus. + / CD8 + T cells (No-infection) were used.

[0090] As shown in Figure 2, wild-type CD4 + / CD8 + Unlike T cells (non-infected), GFP expression was detected in CAR-T (T cell-Avitag-CAR) transduced with CAR proteins. The above results demonstrate that the CAR protein produced according to the present invention was transduced into T cells.

[0091]

[0092] 1-5. Biotin ligation

[0093] According to the present invention, a biotin tag was ligated to the avidin tag protein of the CAR protein-transduced T cells (CAR-T) to produce biotin-tagged CAR-expressing immune cells. Biotin binding was performed using biotin-AMP (biotin-adenosine monophosphate) and BriA enzyme (Fig. 3). The CAR-T was added to 500 μL of PBS (phosphate buffer saline) at a density of 1 × 10 6 The cells were seeded at a cell concentration of 10 μM, AMP-biotin (Jena bioscience, NU-894-BIO-S) and 0.3 μM BirA (Abcam, ab135015) were added, and cultured at 37°C for 1 hour. After culture, the cells were stained with Cyanine5 Treptavidin, and the production of biotin-tagged CAR-T was confirmed by confocal microscopy.

[0094] As shown in Fig. 4, fluorescence of green fluorescent protein (GFP) bound to the terminal of the chimeric antigen receptor (CAR) was confirmed on the surface of the CAR protein-transduced T cell (CAR-T), and fluorescence of cyanine 5-labeled streptavidin (Cy5 labeled TAv) was detected at the same site. The above results demonstrate that streptavidin stably binds to the biotinylated protein exposed on the surface of the CAR protein-transduced T cell (CAR-T).

[0095]

[0096] Example 2. Production of a Traptavidin Scaffold Antibody-Like Protein

[0097] 2-1. Construction of the FMC63-Tav recombinant vector

[0098] The Traptavidin scaffold antibody-like protein according to the present invention is a protein (scFv-Tav protein) composed of a single chain variable fragment (hereinafter scFv) and Traptavidin (hereinafter Tav) that sequentially recognize cell surface molecules of specific cancer cells, and in this example, one embodiment of the scFv-Tav protein was produced to include the scFv of FMC63 and Traptavidin (Fig. 5).

[0099] In the above-described example, a recombinant vector containing genes encoding each gene was constructed to produce the scFv-Tav protein, and was designed to sequentially include a leader sequence represented by SEQ ID NO: 22, a gene encoding the FMC63 VL region (variable light chain region) represented by SEQ ID NO: 14, a gene encoding the FMC63 VH region (variable heavy chain region) represented by SEQ ID NO: 16, and a gene encoding streptavidin (Tav) represented by SEQ ID NO: 18 between the EcoRI enzyme cleavage site (gaattc) and the Xhol enzyme cleavage site (ctcgag). The vector was designed to have a third linker gene represented by the 388th to 432nd base sequence of SEQ ID NO: 19 between the FMC63 VL gene and the FMC63 VH gene. The FMC63 HL gene and the Tav gene were designed to have an Sfil gene represented by SEQ ID NO: 21 and a fourth linker gene represented by base sequences 808 to 861 of SEQ ID NO: 19 between them. One or two additional bases for frame shift may exist between the Sfil gene and the fourth linker gene, and specifically, in this example, the Sfil gene and the fourth linker gene were designed to have adenosine (A) and guanine (G) as the additional bases. In addition, in this example, in order to easily separate the scFv-Tav protein, the 3'-end of the gene encoding streptavidin (Tav) was designed to have a His tag gene represented by SEQ ID NO: 20.

[0100] Finally, the gene encoding the scFv-Tav protein manufactured in this example is composed of a base sequence represented by SEQ ID NO: 19, and each gene was inserted into the pcDNA3.1 vector by requesting a sequencing service to produce a pcDNA3.1-FMC63-Tav recombinant vector, and the presence of the inserted DNA was confirmed using agarose gel electrophoresis.

[0101]

[0102] 2-2. Production of scFv-Tav scaffold antibody-like proteins

[0103] The FMC63-Tav protein was produced by transducing the recombinant vector produced above into expression cells. ExpiCHO cells (Gibco, A29133) with the vector expression system established were cultured in 25 mL of 6 × 10 6 Cells were prepared in a 125 mL flask at a concentration of 10 cells / mL. Transfection was performed with the pcDNA3.1-FMC63-Tav recombinant vector according to the manufacturer's instructions for ExpiCHO. After culturing for 7 to 9 days, the culture solution was harvested. The obtained culture solution was centrifuged at 2,900 × g for 10 min at 4°C to obtain the supernatant. The obtained supernatant was filtered through a 0.22 μm filter (Sartorius, 17573), and the FMC63-Tav protein was purified using fast protein liquid chromatography (FPLC). Protein purification was confirmed by SDS-PAGE. The purified FMC63-Tav protein was concentrated using Amicon (Merck, UFC805024), suspended in PBS, and stored frozen.

[0104] As shown in Figure 6, a 42 kDa band corresponding to a single FMC63-Tav (mono-FMC63-Traptavidin) protein was detected in the FPLC elution sample. In addition, a 168 kDa band corresponding to the t-FMC63-Tav protein (Figure 7), a tetramer formed by self-assembly of streptavidin, was detected.

[0105] To confirm the composition of the FMC63-Tav protein produced above, the biotin binding site in the FMC63-Tav protein was confirmed through Western blotting using biotin-HRP (Biotin Horseradish peroxidase). As a control, streptavidin protein was used. In addition, the presence of FMC63 was confirmed through Western blotting using an anti-FMC63 antibody.

[0106] As shown in Fig. 8, it was confirmed that biotin-HRP bound to the manufactured FMC63-Tav protein, and it was also shown that biotin-HRP bound to the streptavidin (Tav) protein. The above results demonstrate that the biotin binding site in the manufactured FMC63-Tav protein is the streptavidin (Tav) protein. In addition, the results of Western blotting using an anti-FMC63 antibody confirmed that the anti-FMC63 antibody bound to the manufactured FMC63-Tav protein, demonstrating that FMC63 is present in the manufactured FMC63-Tav protein.

[0107] Streptavidin (Tav) is a highly heat-resistant substance, not completely denaturing even at temperatures as high as 100°C. As shown in Figure 8, the FMC-Tav band retains its tetramer form even after 5 minutes at 120°C, demonstrating that denaturation begins at the linker connecting scFv and Tav. Tav is completely denatured in approximately 30 minutes of heating, demonstrating its potential as an antibody scaffold with extremely high heat stability.

[0108]

[0109] Example 3. Efficacy Evaluation of Tetramer Universal CAR Immunotherapy

[0110] An immune cell therapy comprising a tetramer-based universal CAR-T having four scFv regions was constructed by combining biotin-tagged CAR-expressing immune cells and scFv-Tav scaffold antibody-like proteins (Fig. 9). Specifically, the FMC63-Tav protein produced in Example 2 was combined with the biotin-tagged CAR-T produced in Example 1. After washing the biotin-tagged CAR-T three times with easySep buffer (Stemcell, 20144), 1 × 10 6Biotin-tagged CAR-T cells were constructed by adding 0.2 μg / μL of biotin-tagged CAR-T and 0.2 μg / μL of FMC63-Tav protein and incubating at 37°C for 1 hour to construct FMC63-Tav-Bio-CAR-T cells. To evaluate the anticancer activity of the constructed FMC63-Tav-Bio-CAR-T cells, changes in cytokine levels and cytotoxicity toward cancer cells were evaluated. To compare and evaluate the anticancer activity, Kymriah, a commercially used anticancer drug, and activated wild-type T cells without CAR trait (Untransduced) were used as positive controls.

[0111]

[0112] 3-1. Evaluation of changes in cytokine levels

[0113] The above-mentioned tetramer-based universal CAR-T (FMC63-Tav-Bio-CAR-T cell) was applied as an effector cell to the CD19 lymphoma cell line. + Changes in the levels of IFN-γ and IL-2, cytokines essential for the immune-oncogenic action against target cells, Raji cells, were measured. In a 96-well plate, CD19 target cells were measured per well. + 1 × 10 Raji cells 4 The cells were seeded at a concentration of 10 cells / well. FMC63-Tav-Bio-CAR-T cells, which are effector cells, were seeded into each well so that the effector cell to target cell ratio (E:T ratio) could be 0:1, 1:1, 2:1, 5:1, or 10:1, and co-cultured for 48 hours. The levels of IFN-γ and IL-2 in the cultures after culture were measured using ELISA (BD, 555142 and 555190).

[0114] As shown in Fig. 10, when FMC63-Tav-Bio-CAR-T cells were used as effector cells, the levels of cytokines IFN-γ and IL-2 were significantly higher than those of Kymriah and wild-type T cells (untransduced). The above results demonstrate that the tetramer-based universal CAR immunotherapy produced according to the present invention exhibits superior anticancer activity with superior immune anticancer responses compared to existing commercial anticancer agents and wild-type T cells.

[0115]

[0116] 3-2. Cytotoxicity evaluation

[0117] The above-mentioned FMC63-Tav-Bio-CAR-T cells were applied as effector cells to CD19 lymphoma cell line. + The cytotoxic effect on cancer cells was evaluated against target cells, Raji cells. Raji cells were transduced using lentivirus to express nanoLuc (nano luciferase). Then, target cells, CD19, were seeded in each well of a 96-well plate. + 1 × 10 Raji cells 4The cells were seeded at a concentration of 10 cells / well. FMC63-Tav-Bio-CAR-T cells, which are effector cells, were seeded in each well so that the effector cell to target cell ratio (E:T ratio) could be 0:1, 1:1, 2:1, 5:1, or 10:1, and co-cultured for 48 hours. After completing the culture, the cells were lysed using passive lysis buffer (Promega, E1941), and the luminescence level was measured using nanoluc substrate (Promega, N1150) to determine the CD19 + The reduction level of Raji cells was measured. CD19 was considered as 100% when no effector cells were added. + The presence of Raji cells was calculated as a relative %.

[0118] As shown in Figure 11, when FMC63-Tav-Bio-CAR-T cells were used as effector cells, compared to Kymriah and wild-type T cells (Untransduced), the effector cells were CD19 lymphoma cell lines. + Raji cells were found to be rapidly reduced. The above results demonstrate that the tetramer-based universal CAR immunotherapy produced according to the present invention exhibits superior anticancer activity, with superior immune anticancer responses compared to existing commercial anticancer agents and wild-type T cells.

[0119] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A chimeric antigen receptor comprising a tag protein, a hinge and transmembrane region (HTM region), a costimulatory region, and a stimulatory region.

2. A chimeric antigen receptor according to claim 1, characterized in that the tag protein is an avidin or biotin tag protein.

3. A chimeric antigen receptor according to claim 1, wherein the HTM region (hinge and transmembrane region) is a CD8 HTM represented by sequence number 3.

4. A chimeric antigen receptor according to claim 1, wherein the costimulatory region is at least one protein selected from among 4-1BB, CD2, CD7, CD27, CD28, CD30, CD40, CD83, CD258, NKG2C, NKG2D, B7-H3, OX40, ICAM-1, LFA-1, and ICOS, or an active region thereof.

5. A chimeric antigen receptor according to claim 1, characterized in that the costimulatory region is 4-1BB represented by sequence number 5.

6. A chimeric antigen receptor according to claim 1, wherein the stimulatory region is at least one protein selected from CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRδ, TCRγ, CD79a, CD79b, DAP10, and DAP12, or an active region thereof.

7. A chimeric antigen receptor according to claim 1, characterized in that the stimulatory region is CD3ζ represented by sequence number 7.

8. A chimeric antigen receptor according to claim 1, characterized in that the chimeric antigen receptor is a polypeptide represented by SEQ ID NO:

11.

9. A polynucleotide encoding a chimeric antigen receptor according to any one of claims 1 to 8.

10. A vector for producing a chimeric antigen receptor comprising the polynucleotide of claim 9.

11. In the 10th paragraph, the vector for producing the chimeric antigen receptor is, Avidin gene encoding the avidin tag protein represented by sequence number 2; CD8 HTM gene encoding CD8 HTM represented by sequence number 4; The 4-1BB gene encoding 4-1BB represented by sequence number 6; and A vector for producing a chimeric antigen receptor, characterized by comprising a CD3ζ gene encoding CD3ζ represented by sequence number 8.

12. In claim 11, the vector for producing the chimeric antigen receptor is characterized in that it includes a first linker gene represented by the 46th to 51st base sequence of SEQ ID NO: 12 between the avidin gene and the CD8 HTM gene.

13. A vector for producing a chimeric antigen receptor, characterized in that in claim 10, the vector for producing the chimeric antigen receptor comprises a polynucleotide represented by SEQ ID NO:

12.

14. A vector for producing a chimeric antigen receptor according to claim 10, characterized in that the vector for producing the chimeric antigen receptor is a lentivirus vector, a retrovirus vector, a DNA vector, a plasmid, an RNA vector, an adenovirus vector, or an adenovirus-associated vector.

15. An immune cell comprising a chimeric antigen receptor comprising a tag protein, a hinge and transmembrane region (HTM region), a costimulatory region, and a stimulatory region, and expressing the tag protein on the cell membrane surface.

16. An immune cell according to claim 15, characterized in that the tag protein is an avidin or biotin tag protein.

17. An immune cell according to claim 15, wherein the HTM region (hinge and transmembrane region) is CD8 HTM represented by SEQ ID NO: 3, the costimulatory region is 4-1BB represented by SEQ ID NO: 5, and the stimulatory region is CD3ζ represented by SEQ ID NO:

7.

18. An immune cell according to claim 15, characterized in that the immune cell is a T cell, an NK cell, or a B cell.

19. A method for producing a CAR expressing immune cell, comprising the step of transducing an immune cell with a vector comprising a polynucleotide encoding a chimeric antigen receptor comprising a tag protein, a hinge and transmembrane region (HTM region), a costimulatory region, and a stimulatory region.

20. A method for producing a CAR expressing immune cell, characterized in that in claim 19, if the tag protein is avidin, a biotin ligation step is additionally included after the transduction step by reacting with biotin AMP (biotin-adenosine mono phosphate).

21. An antibody-like protein comprising a single chain variable fragment (scFv) and Traptavidin.

22. An antibody-like protein according to claim 21, wherein the single chain variable fragment (scFv) binds to a tumor-specific antigen.

23. An antibody-like protein according to claim 22, wherein the tumor-specific antigen is CD19, CD20, GD2, HER-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CSPG4, or B7-H4.

24. An antibody-like protein according to claim 21, wherein the single chain variable fragment (scFv) comprises a FMC63 VL region (variable light chain region) which is a polypeptide represented by SEQ ID NO: 13 and a FMC63 VH region (variable heavy chain region) which is a polypeptide represented by SEQ ID NO:

15.

25. An antibody-like protein according to claim 21, characterized in that the Traptavidin is a polypeptide represented by SEQ ID NO:

17.

26. A polynucleotide encoding an antibody-like protein according to any one of claims 21 to 25.

27. A vector for producing an antibody-like protein comprising the polynucleotide of claim 26.

28. In paragraph 27, the vector for producing the antibody-like protein is, The leader sequence represented by sequence number 22; A gene encoding the FMC63 variable light chain region (VL region) represented by sequence number 14; A gene encoding the FMC63 VH region (variable heavy chain region) represented by sequence number 16; and A vector for producing an antibody-like protein, characterized by comprising a gene encoding streptavidin (Tav) represented by sequence number 18.

29. In paragraph 28, the vector for producing the antibody-like protein is A third linker gene is included between the FMC63 VL gene and the FMC63 VH gene, represented by the 388th to 432nd base sequence of SEQ ID NO: 19, Between the FMC63 HL gene and the Tav gene, there is included a Sfil gene represented by SEQ ID NO: 21 and a fourth linker gene represented by the 808th to 861st base sequence of SEQ ID NO: 19, A vector for producing an antibody-like protein, characterized in that the 3' end of a gene encoding streptavidin (Tav) includes a His tag gene represented by SEQ ID NO:

20.

30. A vector for producing an antibody-like protein, characterized in that in claim 29, one or two additional bases for frame shift are included between the Sfil gene and the fourth linker gene.

31. In claim 27, a vector for producing an antibody-like protein, characterized in that the vector for producing the antibody-like protein comprises a polynucleotide represented by SEQ ID NO:

19.

32. An immune cell comprising a chimeric antigen receptor comprising a tag protein, a hinge and transmembrane region, a costimulatory region, and a stimulatory region, and expressing the tag protein on the cell membrane surface; and An immune cell therapeutic composition for the prevention or treatment of cancer, comprising an antibody-like protein comprising a single chain variable fragment (scFv) and Traptavidin.

33. In claim 32, an immune cell therapeutic composition for preventing or treating cancer, characterized in that the antibody-like protein forms a tetramer by self-assembly of the Traptavidin, and the Traptavidin formed into the tetramer binds to the tag protein expressed on the surface of the immune cell.

34. An immune cell therapeutic composition for the prevention or treatment of cancer, characterized in that in claim 32, the tag protein is a biotin tag protein, the HTM region (hinge and transmembrane region) is CD8 HTM represented by SEQ ID NO: 3, the costimulatory region is 4-1BB represented by SEQ ID NO: 5, and the stimulatory region is CD3ζ represented by SEQ ID NO:

7.

35. An immune cell therapeutic composition for preventing or treating cancer, characterized in that the immune cell in claim 32 is a T cell, an NK cell, or a B cell.

36. An immune cell therapeutic composition for the prevention or treatment of cancer, characterized in that the single chain variable fragment (scFv) of claim 32 binds to a tumor-specific antigen.

37. An immune cell therapeutic composition for the prevention or treatment of cancer, characterized in that in claim 36, the tumor-specific antigen is CD19, CD20, GD2, HER-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CSPG4, or B7-H4.

38. An immune cell therapeutic composition for the prevention or treatment of cancer, characterized in that in claim 32, the single chain variable fragment (scFv) comprises an FMC63 variable light chain region (VL) represented by SEQ ID NO: 13 and an FMC63 variable heavy chain region (VH) represented by SEQ ID NO:

15.

39. In the 32nd paragraph, the cancer is leukemia, acute lymphoblastic leukemia, acute myeloblastic leukemia, chronic leukemia, chronic myeloblastic (granulocytic) leukemia, chronic lymphocytic leukemia, true polycythemia, lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland An immune cell therapeutic composition for the prevention or treatment of cancer, characterized in that the cancer is at least one disease selected from among carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, cholangiocarcinoma, seminoma, embryogenic carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

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