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WO2024248490A1 - Chimeric antigen receptor targeting pd-l1 and t cell therapeutic agent expressing same - Google Patents

Chimeric antigen receptor targeting pd-l1 and t cell therapeutic agent expressing same Download PDF

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WO2024248490A1
WO2024248490A1 PCT/KR2024/007365 KR2024007365W WO2024248490A1 WO 2024248490 A1 WO2024248490 A1 WO 2024248490A1 KR 2024007365 W KR2024007365 W KR 2024007365W WO 2024248490 A1 WO2024248490 A1 WO 2024248490A1
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scfv
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Korean (ko)
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김미란
이상래
김주희
박춘호
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Ajou University Industry Academic Cooperation Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention relates to a composition for treating cancer using a chimeric antigen receptor (CAR) that binds to PD-L1 of tumor cells and a T cell transformed with an anti-PD-L1 CAR.
  • CAR chimeric antigen receptor
  • Chimeric antigen receptor (CAR)-T cells have revolutionized the field of cell therapy by demonstrating effective and sustained clinical treatment effects in hematological malignancies.
  • Chimeric antigen receptors are engineered synthetic receptors that activate the function of T lymphocytes to recognize and eliminate cancer cells expressing specific target antigens. The binding of CARs to target antigens expressed on the cell surface results in active T cell activation and potent anticancer effects.
  • CAR-T cell therapy still has many challenges to overcome, including life-threatening CAR-T cell-induced toxicity, limited efficacy against solid tumors compared to hematological malignancies, resistance to B-cell malignancies, and limited persistence. Therefore, several different approaches have been attempted recently, including combining CAR-T cell therapy with other anticancer therapies or using innovative CAR engineering strategies to improve anticancer efficacy, extend clinical efficacy duration, and attenuate toxicity.
  • PD-L1 (Programmed death-ligand 1) is a 40 kDa integral membrane protein.
  • cancer cells overexpress PD-L1 to evade attacks by tumor antigen-specific T cells.
  • PD-L1 overexpressed in cancer cells binds to PD-1, a receptor for PD-L1 expressed on tumor antigen-specific activated T cells, and inhibits the activation of tumor antigen-specific T cells through PD-1/PD-L1 signaling. Therefore, antibody therapeutics (immune checkpoint inhibitors) that can inhibit PD-1/PD-L1 signaling have been developed and are currently actively used as therapeutic agents that can more effectively eliminate solid cancers.
  • CAR-T cell therapy is being developed to overcome the tumor microenvironment of immunosuppressed solid cancers by expressing not only chimeric antigen receptors that bind to tumor cell antigens, but also various interleukin or chemokines with anticancer efficacy.
  • the inventors of the present invention took advantage of the fact that PD-L1 is overexpressed in various tumor cells, and produced CAR-T cells in which an interleukin gene that enhances the anticancer effect is additionally introduced into a chimeric antigen receptor using an antibody that binds to the PD-L1.
  • the interleukin gene is not constantly expressed, but is expressed at the time when T cells are activated, so that it is designed to enhance the killing ability of T cells while simultaneously activating other antitumor immune cells, such as natural killer cells and macrophages, while minimizing side effects such as cytokine release syndrome. It was confirmed that the CAR-T cells designed to have such enhanced function have cytotoxic ability specifically toward tumor cells expressing PD-L1.
  • the purpose of the present invention is to provide a T cell expressing a chimeric antigen receptor that specifically binds to PD-L1, which is overexpressed in various solid cancers.
  • the present invention provides a means for selectively expressing a cytokine that enhances anticancer efficacy in addition to a region encoding an anti-PD-L1 chimeric antigen receptor to the T cell.
  • the present invention provides a cell therapeutic agent composition for preventing or treating cancer, which comprises a T cell expressing an anti-PD-L1 chimeric antigen receptor produced according to the present invention.
  • the present invention provides a vector for producing T cells expressing a chimeric antigen receptor comprising an anti-PD-L1 scFv domain and an inducible cytokine domain.
  • the present invention provides a T cell expressing a chimeric antigen receptor including an anti-PD-L1 scFv transfected with the vector and selectively expressing a cytokine depending on whether the T cell is activated, and a cell therapeutic agent composition for preventing or treating cancer using the same.
  • an anti-PD-L1 chimeric antigen receptor comprising an anti-PD-L1 scFv domain is an antibody domain that specifically binds to PD-L1 specifically expressed in cancer cells, and T cells transformed with the anti-PD-L1 chimeric antigen receptor not only have improved specificity for tumor cells, but also have significantly improved cytotoxic effects since an inducible cytokine domain that selectively expresses cytokines depending on whether cells are activated is simultaneously transformed, thereby producing T cells expressing a chimeric antigen receptor with significantly improved cytotoxic effects.
  • Figure 1 is a schematic diagram showing the DNA region of each domain expressing the anti-PD-L1 chimeric antigen receptor according to the present invention.
  • FIG. 2 is a schematic diagram showing the DNA region of each domain additionally including an inducible cytokine domain in the anti-PD-L1 chimeric antigen receptor according to the present invention.
  • FIG. 3 is a schematic diagram of a lentiviral vector for expressing an anti-PD-L1 chimeric antigen receptor construct according to the present invention.
  • Figure 4 is a schematic diagram showing that an anti-PD-L1 chimeric antigen receptor according to the present invention is expressed on the surface of a T cell.
  • Figure 5 shows the results of measuring the cytotoxic effect of T cells expressing an anti-PD-L1 chimeric antigen receptor according to the present invention on MDA-MB-231 cells, a PD-L1 expressing breast cancer cell line.
  • the present invention provides a vector for producing T cells expressing a chimeric antigen receptor comprising an anti-PD-L1 scFv domain and an inducible cytokine domain.
  • the above chimeric antigen receptor comprises an anti-PD-L1 scFv region, a CD8 TM (transmembrane) region, a 4-1BB region and a CD3 ⁇ (zeta) region, and is produced as a vector comprising an antibody region binding to PD-L1, a cell membrane penetrating domain and a domain for T cell transduction and activation.
  • the above anti-PD-L1 scFv region may be composed of a light chain consisting of an amino acid sequence represented by SEQ ID NO: 1 and a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 2, or a light chain consisting of an amino acid sequence represented by SEQ ID NO: 3 and a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 4, or a light chain consisting of an amino acid sequence represented by SEQ ID NO: 5 and a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 6.
  • the above anti-PD-L1 scFv domain may sequentially include a gene encoding a light chain of anti-PD-L1 scFv represented by SEQ ID NO: 8 and a gene encoding a heavy chain of anti-PD-L1 scFv represented by SEQ ID NO: 9, or sequentially include a gene encoding a light chain of anti-PD-L1 scFv represented by SEQ ID NO: 10 and a gene encoding a heavy chain of anti-PD-L1 scFv represented by SEQ ID NO: 11, or sequentially include a gene encoding a light chain of anti-PD-L1 scFv represented by SEQ ID NO: 12 and a gene encoding a heavy chain of anti-PD-L1 scFv represented by SEQ ID NO: 13.
  • the above vector further comprises a CD8 TM (transmembrane) domain, a 4-1BB domain, and a CD3 ⁇ (zeta) domain sequentially after the anti-PD-L1 scFv domain.
  • the CD8 TM (transmembrane) domain consists of a base sequence represented by SEQ ID NO: 18, the 4-1BB domain consists of a base sequence represented by SEQ ID NO: 19, and the CD3 ⁇ (zeta) domain consists of a base sequence represented by SEQ ID NO: 20.
  • the inducible cytokine domain sequentially comprises a NFAT (Nuclear factor of activated T cells) binding motif and a gene encoding a cytokine.
  • the cytokine may be any one selected from IL-2, IL-12, IL-7, IL-15 or IL-18.
  • the inducible cytokine domain may be composed of a base sequence represented by SEQ ID NO: 14 that sequentially includes a NFAT (Nuclear factor of activated T cells) binding motif and a nucleotide encoding IL-2 so as to enable selective expression of IL-2.
  • SEQ ID NO: 14 that sequentially includes a NFAT (Nuclear factor of activated T cells) binding motif and a nucleotide encoding IL-2 so as to enable selective expression of IL-2.
  • the present invention provides a T cell that selectively expresses a cytokine depending on whether the T cell is activated while expressing a chimeric antigen receptor comprising an anti-PD-L1 scFv transfected with the vector.
  • T cells are CD4 + /CD8 + type T cells isolated from monocytes and stimulated with anti-CD3 and anti-hCD28.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising a transformed T cell expressing on its surface a chimeric antigen receptor comprising an anti-PD-L1 scFv manufactured according to the present invention and comprising an inducible cytokine domain.
  • the above cancers are 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, squa
  • the above pharmaceutical composition can be prepared in unit dose form by formulating it using a pharmaceutically acceptable carrier or can be prepared by placing it in a multi-dose container.
  • the pharmaceutically acceptable carriers mentioned above are those commonly used in the preparation of 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.
  • 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.
  • 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.
  • the vector is a tool that enables stable transfection of a cell, and the vector enables incorporation of the transgene(s) into the cell genome.
  • 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.
  • the vector is a plasmid vector, and more specifically, the vector is a viral vector, and can be arbitrarily replaced by a suitable vector by a person skilled in the art, and preferably, the vector may be a lentivirus vector, a retrovirus vector, a DNA vector, a plasmid, an RNA vector, an adenovirus vector, or an adenovirus-associated vector, but is not limited thereto.
  • protein 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 terms used herein refer to both chains, the shorter chains also commonly referred to as peptides, oligopeptides and oligomers in the art, and the longer chains commonly 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.
  • the polypeptide includes a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.
  • nucleotide is generally used in the same sense as polynucleotide, and includes DNA and RNA.
  • the polynucleotide is a nucleic acid, which is a polymer of nucleotides, and the polynucleotide can be hydrolyzed into monomeric nucleotides.
  • the polynucleotide includes, but is not limited to, any means available in the art, including cloning of nucleic acid sequences from a recombinant library or cell genome using a recombinant means, such as conventional cloning techniques and polymerase chain reaction (PCR), and all nucleic acid sequences obtained by synthetic means.
  • the anti-PD-L1 chimeric antigen receptor (hereinafter referred to as CAR) was produced by referencing the anti-CD-19 CAR configuration based on a genetic database as shown in Fig. 1 to produce a genetic construct encoding the anti-PD-L1 CAR.
  • the CAR it was designed to sequentially include a single chain variable fragment (scFv) region that binds to PD-L1, a CD8 TM (transmembrane) region, a 4-1BB region, and a CD3 ⁇ (zeta) region.
  • the scFv domain was designed to be exposed outside of the cell, and was produced in three types to have the configurations shown in Table 1 below.
  • the anti-PD-L1 CAR according to the present invention may be an A type consisting of a light chain consisting of an amino acid sequence represented by SEQ ID NO: 1 and a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 2 as an anti-PD-L1 scFv region, or a B type consisting of a light chain consisting of an amino acid sequence represented by SEQ ID NO: 3 and a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 4, or a C type consisting of a light chain consisting of an amino acid sequence represented by SEQ ID NO: 5 and a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 6.
  • Type Amino acid sequence SEQ. No.
  • the anti-PD-L1 CAR according to the present invention includes a CD8 TM (transmembrane) region as a cell membrane-penetrating region.
  • the CD8 TM (transmembrane) region includes an amino acid sequence represented by SEQ ID NO: 15.
  • the anti-PD-L1 CAR according to the present invention includes a 4-1BB region composed of an amino acid sequence represented by SEQ ID NO: 16 and a CD3 ⁇ (zeta) region composed of an amino acid sequence represented by SEQ ID NO: 17, wherein the 4-1BB region and the CD3 ⁇ (zeta) region are designed to be expressed inside a cell.
  • the anti-PD-L1 CAR gene construct according to the present invention was produced to sequentially include a gene encoding a light chain of anti-PD-L1 scFv, a linker sequence, a gene encoding a heavy chain of anti-PD-L1 scFv, a CD8 TM (transmembrane) domain, a 4-1BB domain, and a CD3 ⁇ (zeta) domain.
  • the above CAR gene construct type A includes a gene encoding a light chain of anti-PD-L1 scFv represented by SEQ ID NO: 8, and a gene encoding a heavy chain of anti-PD-L1 scFv represented by SEQ ID NO: 9.
  • the above CAR gene construct type B includes a gene encoding a light chain of anti-PD-L1 scFv represented by SEQ ID NO: 10, and a gene encoding a heavy chain of anti-PD-L1 scFv represented by SEQ ID NO: 11.
  • the above CAR gene construct type C includes a gene encoding a light chain of anti-PD-L1 scFv represented by SEQ ID NO: 12, and a gene encoding a heavy chain of anti-PD-L1 scFv represented by SEQ ID NO: 13.
  • the above CD8 TM (transmembrane) domain is composed of a base sequence represented by SEQ ID NO: 18.
  • the above 4-1BB domain is composed of a base sequence represented by SEQ ID NO: 19.
  • the above CD3 ⁇ (zeta) domain is composed of a base sequence represented by SEQ ID NO: 20.
  • the anti-PD-L1 CAR gene construct according to the present invention may additionally include an inducible cytokine domain.
  • the inducible cytokine domain is not designed to be constantly expressed, but is designed to be expressed according to the activity of T cells infected with the chimeric antigen receptor according to the present invention, and is designed to prevent toxicity and inflammation caused by excessive cytokines.
  • the 3' end of the anti-PD-L1 CAR configuration includes an inducible IL-2 domain sequentially including a NFAT (Nuclear factor of activated T cells) binding motif and a nucleotide encoding IL-2.
  • the gene encoding the inducible IL-2 domain is configured by the base sequence represented by SEQ ID NO: 14.
  • the gene encoding the inducible IL-2 domain can selectively induce the expression of IL-2 (interleukin-2) by including an NFAT (Nuclear factor of activated T cells) binding motif, and the IL-2 expressed through the inducible IL-2 domain is configured by the amino acid sequence represented by SEQ ID NO: 7.
  • a vector for expressing the anti-PD-L1 CAR according to the present invention was produced and a lentivirus was used as a means of delivering the same.
  • the third-generation lentiviral vector pSL-BBZ was used as the basic backbone.
  • the anti-PD-L1 CAR construct of the present invention was inserted into the vector.
  • a gene encoding the light chain of anti-PD-L1 scFv and a gene encoding the heavy chain of anti-PD-L1 scFv are located between the Bam H1 site and the Xho I site.
  • the CAR construct was designed to express an EGFP (Enhanced Green Fluorescent Protein) sequence at the end so that expression can be confirmed experimentally.
  • EGFP Enhanced Green Fluorescent Protein
  • the synthesized gene was subcloned into a lentiviral plasmid vector through polymerase chain reaction. Finally, the accuracy of the gene sequence was confirmed through base sequence sequencing using specific primers of the cloned plasmid.
  • the nucleotide sequences and regulatory regions of cytokine genes such as IL-2, IL-12, IL-7, IL-15, or IL-18 were confirmed in a gene database and synthesized, and subcloned into a lentiviral plasmid vector using the same method as above to finally produce an anti-PD-L1 CAR construct.
  • an anti-PD-L1 CAR construct was produced to sequentially include an inducible IL-2 domain sequentially including a NFAT (Nuclear factor of activated T cells) binding motif and nucleotides encoding IL-2 in the CAR gene construct type C.
  • NFAT Nuclear factor of activated T cells
  • HEK293T cells (5 Y 10 6 cells) were infected with 3 ⁇ g of lentiviral vector designed to include the above anti-PD-L1 CAR construct using Lipofectamine (Thermo Scientific #R0531). After culturing for 48 h, the supernatant was collected and only the virus particles were purified through a 0.45 ⁇ m filter. The purified virus particles were concentrated by ultracentrifugation at 28,000 rpm for 2 h.
  • T cells expressing the anti-PD-L1 CAR mononuclear cells were isolated from human peripheral blood.
  • CD8 + T cells were isolated from peripheral blood mononuclear cells by magnetic cell sorting (MACS), and CD4 - /CD8 + /CD3 + T cells were prepared by stimulating them with anti-hCD3 and anti-hCD28-coated immunobeads at a bead-to-cell ratio of 1:1 for 24 to 36 hours.
  • MCS magnetic cell sorting
  • activated T cells were cultured with concentrated anti-CAR lentiviral vectors in a 48-well plate flat plate coated with Novo Nectin (Novoprotein) at 32°C.
  • the immune beads were removed 6 to 7 days after transduction, and the T cells were adjusted to a cell density of 0.5 X 10 6 /mL to 2 ⁇ 10 6 /mL every 3 days.
  • the cancer cell killing effect on MDA-MB-231 cells was evaluated.
  • T cells expressing anti-CD-19 CAR were used as a control group.
  • the anti-CD-19 CAR used the anti-CD-19 CAR composition
  • the CAR expressing T cells were produced using lentivirus in the same manner as the anti-PD-L1 CAR.
  • MDA-MB-231 cells were placed in a 96-well plate at 2 X 10 4 cells/mL and cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing FBS for 24 hours.
  • DMEM Dulbecco's Modified Eagle's Medium
  • T cells transformed with each CAR were added to the wells at various ratios and then cultured at 37°C for 4 or 24 hours. After the culture was completed, the medium was removed from the wells, 0.2% MTT (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) was added at 50 ⁇ l/well, and incubated in an incubator at 37°C for 2 hours. After removing the supernatant, 100 ⁇ l/well of DMSO (dimethyl sulfoxide) was added, and the suspension was made for 5 minutes to dissolve the produced formazan, and the absorbance was measured at 570 nm using a microplate reader to determine the death rate of cancer cells.
  • MTT 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium
  • Every maximum numerical limitation given throughout this specification will include every lower numerical limitation, as if that lower numerical limitation were expressly written out.
  • Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if that higher numerical limitation were expressly written out.
  • Every numerical limitation given throughout this specification will include every better numerical range within that broader numerical range, as if that narrower numerical limitation were expressly written out.

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Abstract

The present invention relates to a chimeric antigen receptor (CAR) binding to PD-L1 of tumor cells and a composition for treating cancer using T cells transformed with an anti-PD-L1 CAR and provides T cells expressing a chimeric antigen receptor binding specifically to PD-L1 overexpressed in various solid cancers. Moreover, in the present invention, T cells are provided with a means capable of selectively expressing a cytokine increasing anticancer efficacy together with a site encoding an anti-PD-L1 chimeric antigen receptor. In addition, the present invention provides a cell therapeutic composition for preventing or treating cancer, the composition comprising T cells expressing the anti-PD-L1 chimeric antigen receptor prepared according to the present invention.

Description

PD-L1을 표적으로 하는 키메라 항원 수용체 및 이를 발현하는 T 세포 치료제Chimeric antigen receptor targeting PD-L1 and T cell therapy expressing it

본 발명은 종양세포의 PD-L1과 결합하는 키메라 항원 수용체(Chimeric ntigen Receptor, CAR) 및 anti-PD-L1 CAR로 형질 전환된 T 세포를 이용한 암 치료용 조성물에 관한 것이다.The present invention relates to a composition for treating cancer using a chimeric antigen receptor (CAR) that binds to PD-L1 of tumor cells and a T cell transformed with an anti-PD-L1 CAR.

키메라 항원 수용체(Chimeric ntigen Receptor, CAR)-T 세포는 혈액암에서 효과적이고 지속적인 임상 치료효과를 나타내어 세포치료제 분야의 혁신을 가져왔다. 키메라 항원 수용체는 특정 표적 항원을 발현하는 암세포를 인식하고 제거하기 위해 T 림프구의 기능을 활성화시키기 위한 조작된 합성 수용체로서 세포 표면에 발현된 표적 항원에 대한 CAR의 결합은 활발한 T 세포 활성화 및 강력한 항암 효과를 나타낸다.Chimeric antigen receptor (CAR)-T cells have revolutionized the field of cell therapy by demonstrating effective and sustained clinical treatment effects in hematological malignancies. Chimeric antigen receptors are engineered synthetic receptors that activate the function of T lymphocytes to recognize and eliminate cancer cells expressing specific target antigens. The binding of CARs to target antigens expressed on the cell surface results in active T cell activation and potent anticancer effects.

B 세포 혈액암에 대한 Anti-CD19 CAR-T 세포의 획기적인 성공을 기반으로 미국식품의약국(FDA)은 2017년 키메라 항원 수용체 기반 세포치료제를 승인하기에 이르렀다. 하지만 CAR-T 세포치료제는 환자의 생명을 위협할 수 있는 CAR-T 세포유 발 독성의 문제, 혈액암에 비해 고형암에 대한 제한된 효능, B세포 악성종양에 대 한 내성, 제한된 지속성 등 여전히 해결해야 할 많은 과제를 안고 있다. 따라서 CAR-T 세포 요법을 다른 항암요법과 병행하거나 혁신적인 CAR 엔지니어링 전략을 사용하여 항암 효능을 개선하고 임상효과 지속성의 확장 및 독성을 약화시키는 것 을 포함하는 여러 다양한 접근법이 최근에 시도되고 있다. Based on the groundbreaking success of anti-CD19 CAR-T cells for B-cell hematological malignancies, the U.S. Food and Drug Administration (FDA) approved chimeric antigen receptor-based cell therapy in 2017. However, CAR-T cell therapy still has many challenges to overcome, including life-threatening CAR-T cell-induced toxicity, limited efficacy against solid tumors compared to hematological malignancies, resistance to B-cell malignancies, and limited persistence. Therefore, several different approaches have been attempted recently, including combining CAR-T cell therapy with other anticancer therapies or using innovative CAR engineering strategies to improve anticancer efficacy, extend clinical efficacy duration, and attenuate toxicity.

PD-L1(Programmed death-ligand 1)은 40 kDa의 내재성 막 단백질(integral membrane protein)로서 특히 암세포는 PD-L1을 과발현하여 종양 항원 특이적 T 세 포(tumor antigen-specific T cells)의 공격을 회피하게 된다. 암세포에서 과발현 된 PD-L1은 종양항원 특이적으로 활성화된 T 세포에서 발현되는 PD-L1의 수용체인 PD-1과 결합하여, PD-1/ PD-L1 신호전달을 통하여 종양 항원 특이적 T 세포의 활 성을 억제한다. 따라서 PD-1/ PD-L1 신호전달을 억제할 수 있는 항체치료제(면역관 문 억제제, immune checkpoint inhibitor)가 개발되어 고형암을 좀 더 효과적으로 제거 할 수 있는 치료제로 현재 활발히 사용되고 있다. 더불어서 CAR-T 세포의 고형암에 대한 효능을 높이기 위하여 종양세포 항원과 결합 하는 키메라 항원 수용체만 아니라 항암효능을 가지는 다양한 인터루킨(interleukin)이나 케모카인(chemokine)을 함께 발현하도록 하여 면역억제된 고 형암의 종양세포 미세환경(tumor microenvironment)을 극복하기 위한 새로운 개념 의 CART-T 세포치료제의 개발도 활발해지고 있다.PD-L1 (Programmed death-ligand 1) is a 40 kDa integral membrane protein. In particular, cancer cells overexpress PD-L1 to evade attacks by tumor antigen-specific T cells. PD-L1 overexpressed in cancer cells binds to PD-1, a receptor for PD-L1 expressed on tumor antigen-specific activated T cells, and inhibits the activation of tumor antigen-specific T cells through PD-1/PD-L1 signaling. Therefore, antibody therapeutics (immune checkpoint inhibitors) that can inhibit PD-1/PD-L1 signaling have been developed and are currently actively used as therapeutic agents that can more effectively eliminate solid cancers. In addition, in order to increase the efficacy of CAR-T cells against solid cancers, a new concept of CAR-T cell therapy is being developed to overcome the tumor microenvironment of immunosuppressed solid cancers by expressing not only chimeric antigen receptors that bind to tumor cell antigens, but also various interleukin or chemokines with anticancer efficacy.

이러한 배경하에서 본 발명자들은 다양한 종양세포에서 PD-L1이 과발현 된다 는 점을 이용하여 상기 PD-L1과 결합하는 항체를 이용한 키메라 항원 수용체에 항암 효과를 높여주는 인터루킨 유전자를 추가적으로 도입된 CAR-T세포를 제작하였다. 이 때 인터루킨 유전자는 항시적으로 발현되는 것이 아니라 T 세포가 활성화되는 시점에서 발현되어 T세포의 살상능력을 높이면서 다른 항종양 면역세포인 자연살해 세포나 대식세포를 함께 활성화시키면서도 사이토카인 방출 신드롬(cytokine release syndrome)등과 같은 부작용을 최소화시키도록 고안되었다. 이러한 향상된 기능을 가지도록 고안된 CAR-T 세포는 PD-L1을 발현하는 종양세포에 특이적으로 세 포 독성 능력이 있음이 확인되었다.Against this backdrop, the inventors of the present invention took advantage of the fact that PD-L1 is overexpressed in various tumor cells, and produced CAR-T cells in which an interleukin gene that enhances the anticancer effect is additionally introduced into a chimeric antigen receptor using an antibody that binds to the PD-L1. At this time, the interleukin gene is not constantly expressed, but is expressed at the time when T cells are activated, so that it is designed to enhance the killing ability of T cells while simultaneously activating other antitumor immune cells, such as natural killer cells and macrophages, while minimizing side effects such as cytokine release syndrome. It was confirmed that the CAR-T cells designed to have such enhanced function have cytotoxic ability specifically toward tumor cells expressing PD-L1.

[선행기술문헌][Prior art literature]

[특허문헌][Patent Document]

한국공개특허공보 제10-2019-0082241호 (2019. 07. 09. 공고)Korean Patent Publication No. 10-2019-0082241 (Published on July 9, 2019)

본 발명의 목적은 다양한 고형암에서 과발현되는 PD-L1에 특이적으로 결합하는 키메라 항원 수용체를 발현하는 T 세포를 제공하는 것이다. 또한, 본 발명에서는 anti-PD-L1 키메라 항원 수용체를 암호화하는 하는 부위와 더불어 항암 효능을 높이는 사이토카인을 선택적으로 발현할 수 있는 수단을 T 세포에게 제공하는 것이다. 또한, 본 발명은 본 발명에 따라 제작된 anti-PD-L1 키메라 항원 수용체를 발현하는 T 세포를 포함하는 암의 예방 또는 치료용 세포 치료제 조성물을 제공하는 것이다.The purpose of the present invention is to provide a T cell expressing a chimeric antigen receptor that specifically binds to PD-L1, which is overexpressed in various solid cancers. In addition, the present invention provides a means for selectively expressing a cytokine that enhances anticancer efficacy in addition to a region encoding an anti-PD-L1 chimeric antigen receptor to the T cell. In addition, the present invention provides a cell therapeutic agent composition for preventing or treating cancer, which comprises a T cell expressing an anti-PD-L1 chimeric antigen receptor produced according to the present invention.

본 발명은 anti-PD-L1 scFv 도메인 및 유도성 사이토카인(inducible cytokine) 도메인을 포함하는, 키메라 항원 수용체를 발현하는 T 세포의 제작을 위한 벡터를 제공한다.The present invention provides a vector for producing T cells expressing a chimeric antigen receptor comprising an anti-PD-L1 scFv domain and an inducible cytokine domain.

또한, 본 발명은 상기 벡터로 형질감염된 anti-PD-L1 scFv을 포함하는 키메라 항원 수용체를 발현하면서 T 세포의 활성화 여부에 따라 선택적으로 사이토카인을 발현하는 T 세포 및 이를 이용한 암의 예방 또는 치료를 위한 세포 치료제 조성물을 제공한다.In addition, the present invention provides a T cell expressing a chimeric antigen receptor including an anti-PD-L1 scFv transfected with the vector and selectively expressing a cytokine depending on whether the T cell is activated, and a cell therapeutic agent composition for preventing or treating cancer using the same.

본 발명에 따르면, anti-PD-L1 scFv 도메인을 포함하는 anti-PD-L1 키메라 항원 수용체는 암세포에서 특이적으로 발현하는 PD-L1에 특이적으로 결합하는 항체 도메인이며, 상기 anti-PD-L1 키메라 항원 수용체로 형질전환된 T 세포는 종양 세포에 대한 특이성이 향상될 뿐만 아니라, 세포의 활성화 여부에 따라 선택적으로 사이토카인을 발현하는 유도성 사이토카인(inducible cytokine) 도메인이 함께 형질전환됨에 따라 세포 독성 효과가 현저히 향상된 키메라 항원 수용체를 발현하는 T 세포를 제조할 수 있다.According to the present invention, an anti-PD-L1 chimeric antigen receptor comprising an anti-PD-L1 scFv domain is an antibody domain that specifically binds to PD-L1 specifically expressed in cancer cells, and T cells transformed with the anti-PD-L1 chimeric antigen receptor not only have improved specificity for tumor cells, but also have significantly improved cytotoxic effects since an inducible cytokine domain that selectively expresses cytokines depending on whether cells are activated is simultaneously transformed, thereby producing T cells expressing a chimeric antigen receptor with significantly improved cytotoxic effects.

도 1은 본 발명에 따른 anti-PD-L1 키메라 항원 수용체(Chimeric antigen receptor)를 발현시키는 각 도메인의 DNA 구역을 나타내는 모식도이다. Figure 1 is a schematic diagram showing the DNA region of each domain expressing the anti-PD-L1 chimeric antigen receptor according to the present invention.

도 2는 본 발명에 따른 anti-PD-L1 키메라 항원 수용체(Chimeric antigen receptor)에 유도성 사이토카인(inducible cytokine) 도메인을 추가적으로 포함하는 각 도메인의 DNA 구역을 나타내는 모식도이다.FIG. 2 is a schematic diagram showing the DNA region of each domain additionally including an inducible cytokine domain in the anti-PD-L1 chimeric antigen receptor according to the present invention.

도 3는 본 발명에 따른 anti-PD-L1 키메라 항원 수용체(Chimeric antigen receptor) 컨스트럭트(construct)를 발현하기 위한 렌티 바이러스 벡터의 모식도이다.FIG. 3 is a schematic diagram of a lentiviral vector for expressing an anti-PD-L1 chimeric antigen receptor construct according to the present invention.

도 4은 본 발명에 따른 anti-PD-L1 키메라 항원 수용체(Chimeric antigen receptor)가 T 세포의 표면에 발현되는 모식도이다.Figure 4 is a schematic diagram showing that an anti-PD-L1 chimeric antigen receptor according to the present invention is expressed on the surface of a T cell.

도 5는 본 발명에 따른 anti-PD-L1 키메라 항원 수용체(Chimeric antigen receptor)를 발현하는 T 세포가 PD-L1 발현 유방암 세포주인 MDA-MB-231 세포에 대한 세포 독성 효과를 측정한 결과이다.Figure 5 shows the results of measuring the cytotoxic effect of T cells expressing an anti-PD-L1 chimeric antigen receptor according to the present invention on MDA-MB-231 cells, a PD-L1 expressing breast cancer cell line.

본 명세서에서 사용되는 용어는 본 발명에서의 기능을 고려하면서 가능한 현재 널리 사용되는 일반적인 용어들을 선택하였으나, 이는 당 분야에 종사하는 기술자의 의도 또는 판례, 새로운 기술의 출현 등에 따라 달라질 수 있다. 또한, 특정한 경우는 출원인이 임의로 선정한 용어도 있으며, 이 경우 해당되는 발명의 설명 부분에서 상세히 그 의미를 기재할 것이다. 따라서 본 발명에서 사용되는 용어는 단순한 용어의 명칭이 아닌, 그 용어가 가지는 의미와 본 발명의 전반에 걸친 내용을 토대로 정의되어야 한다.The terms used in this specification are selected from the most widely used general terms possible while considering the functions of the present invention, but they may vary depending on the intention of engineers working in the field, precedents, the emergence of new technologies, etc. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meanings thereof will be described in detail in the description of the relevant invention. Therefore, the terms used in the present invention should be defined based on the meanings of the terms and the overall contents of the present invention, rather than simply the names of the terms.

다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.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 belongs. Terms defined in commonly used dictionaries, such as those defined in common usage, should be interpreted as having a meaning consistent with the meaning they have in the context of the relevant art, and will not be interpreted in an idealized or overly formal sense unless expressly defined in this application.

이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.

본 발명은 anti-PD-L1 scFv 도메인 및 유도성 사이토카인(inducible cytokine) 도메인을 포함하는, 키메라 항원 수용체를 발현하는 T 세포의 제작을 위한 벡터를 제공한다.The present invention provides a vector for producing T cells expressing a chimeric antigen receptor comprising an anti-PD-L1 scFv domain and an inducible cytokine domain.

상기 키메라 항원 수용체는 anti-PD-L1 scFv 영역, CD8 TM(transmembrane) 영역, 4-1BB 영역 및 CD3ζ(zeta) 영역을 포함하며, PD-L1과 결합하는 항체 부위, 세포막 관통 도메인 및 T 세포 신화전달 및 활성화를 위한 도메인을 포함하는 벡터로 제작된다.The above chimeric antigen receptor comprises an anti-PD-L1 scFv region, a CD8 TM (transmembrane) region, a 4-1BB region and a CD3ζ (zeta) region, and is produced as a vector comprising an antibody region binding to PD-L1, a cell membrane penetrating domain and a domain for T cell transduction and activation.

상기 anti-PD-L1 scFv 영역은, 서열번호 1로 표시되는 아미노산 서열로 구성된 경쇄(light chain) 및 서열번호 2로 표시되는 아미노산 서열로 구성된 중쇄(heavy chain)로 이루어지거나, 서열번호 3로 표시되는 아미노산 서열로 구성된 경쇄(light chain) 및 서열번호 4로 표시되는 아미노산 서열로 구성된 중쇄(heavy chain)로 이루어지거나, 서열번호 5로 표시되는 아미노산 서열로 구성된 경쇄(light chain) 및 서열번호 6로 표시되는 아미노산 서열로 구성된 중쇄(heavy chain)로 이루어진 것일 수 있다. The above anti-PD-L1 scFv region may be composed of a light chain consisting of an amino acid sequence represented by SEQ ID NO: 1 and a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 2, or a light chain consisting of an amino acid sequence represented by SEQ ID NO: 3 and a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 4, or a light chain consisting of an amino acid sequence represented by SEQ ID NO: 5 and a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 6.

상기 anti-PD-L1 scFv 도메인은, 서열번호 8로 표시되는 anti-PD-L1 scFv의 경쇄(light chain)를 암호화하는 유전자, 및 서열번호 9로 표시되는 anti-PD-L1 scFv의 중쇄(heavy chain)를 암호화하는 유전자를 순차적으로 포함하거나, 서열번호 10으로 표시되는 anti-PD-L1 scFv의 경쇄(light chain)를 암호화하는 유전자, 및 서열번호 11로 표시되는 anti-PD-L1 scFv의 중쇄(heavy chain)를 암호화하는 유전자를 순차적으로 포함하거나, 서열번호 12로 표시되는 anti-PD-L1 scFv의 경쇄(light chain)를 암호화하는 유전자, 및 서열번호 13으로 표시되는 anti-PD-L1 scFv의 중쇄(heavy chain)를 암호화하는 유전자를 순차적으로 포함할 수 있다. The above anti-PD-L1 scFv domain may sequentially include a gene encoding a light chain of anti-PD-L1 scFv represented by SEQ ID NO: 8 and a gene encoding a heavy chain of anti-PD-L1 scFv represented by SEQ ID NO: 9, or sequentially include a gene encoding a light chain of anti-PD-L1 scFv represented by SEQ ID NO: 10 and a gene encoding a heavy chain of anti-PD-L1 scFv represented by SEQ ID NO: 11, or sequentially include a gene encoding a light chain of anti-PD-L1 scFv represented by SEQ ID NO: 12 and a gene encoding a heavy chain of anti-PD-L1 scFv represented by SEQ ID NO: 13.

상기 벡터는 상기 anti-PD-L1 scFv 도메인 이후로 순차적으로 CD8 TM(transmembrane) 도메인, 4-1BB 도메인, 및 CD3ζ(zeta) 도메인을 추가로 포함한다. 상기 CD8 TM(transmembrane) 도메인은 서열번호 18로 표시되는 염기서열로 이루어지고, 상기 4-1BB 도메인은 서열번호 19로 표시되는 염기서열로 이루어지고, 상기 CD3ζ(zeta) 도메인은 서열번호 20으로 표시되는 염기서열로 이루어진다. The above vector further comprises a CD8 TM (transmembrane) domain, a 4-1BB domain, and a CD3ζ (zeta) domain sequentially after the anti-PD-L1 scFv domain. The CD8 TM (transmembrane) domain consists of a base sequence represented by SEQ ID NO: 18, the 4-1BB domain consists of a base sequence represented by SEQ ID NO: 19, and the CD3ζ (zeta) domain consists of a base sequence represented by SEQ ID NO: 20.

상기 유도성 사이토카인(inducible cytokine) 도메인은 NFAT(Nuclear factor of activated T cells) 결합 모티프 및 사이토카인을 암호화하는 유전자를 순차적으로 포함한다. 상기 사이토카인은 IL-2, IL-12, IL-7, IL-15 또는 IL-18 중 선택된 어느 하나일 수 있다. The inducible cytokine domain sequentially comprises a NFAT (Nuclear factor of activated T cells) binding motif and a gene encoding a cytokine. The cytokine may be any one selected from IL-2, IL-12, IL-7, IL-15 or IL-18.

구체적으로 상기 유도성 사이토카인(inducible cytokine) 도메인은 IL-2를 선태적으로 발현할 수 있도록 NFAT(Nuclear factor of activated T cells) 결합 모티프 및 IL-2을 암호화하는 뉴클레오타이드를 순차적으로 포함하는 서열번호 14로 표시되는 염기서열로 이루어질 수 있다.Specifically, the inducible cytokine domain may be composed of a base sequence represented by SEQ ID NO: 14 that sequentially includes a NFAT (Nuclear factor of activated T cells) binding motif and a nucleotide encoding IL-2 so as to enable selective expression of IL-2.

또한, 본 발명은 상기 벡터로 형질감염된 anti-PD-L1 scFv을 포함하는 키메라 항원 수용체를 발현하면서 T 세포의 활성화 여부에 따라 선택적으로 사이토카인을 발현하는 T 세포를 제공한다.Furthermore, the present invention provides a T cell that selectively expresses a cytokine depending on whether the T cell is activated while expressing a chimeric antigen receptor comprising an anti-PD-L1 scFv transfected with the vector.

상기 T 세포는 단핵 세포에서 분리된 CD4+/CD8+ 유형에서 anti-CD3 및 anti-hCD28 로 자극된 T 세포이다.The above T cells are CD4 + /CD8 + type T cells isolated from monocytes and stimulated with anti-CD3 and anti-hCD28.

또한, 본 발명은 본 발명에 따라 제조된 anti-PD-L1 scFv를 포함하는 키메라 항원 수용체를 표면에 발현하면서, 및 유도성 사이토카인(inducible cytokine) 도메인을 포함하는, 형질전환된 T 세포를 포함하는 암의 예방 또는 치료를 위한 약학적조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising a transformed T cell expressing on its surface a chimeric antigen receptor comprising an anti-PD-L1 scFv manufactured according to the present invention and comprising an inducible cytokine domain.

상기 암은 백혈병, 급성 림프구성 백혈병, 급성 골수구성 백혈병, 만성 백혈병, 만성 골수구성(과립구성) 백혈병, 만성 림프구성 백혈병, 진성 다혈구증, 림프종, 호지킨(Hodgkin) 병, 비-호지킨 병, 다발성 골수종, 발덴스트롬(Waldenstrom) 거대글로불린 혈증, 중쇄 질병, 섬유 육종, 점액 육종, 지방 육종, 연골 육종, 골육종, 척삭종, 혈관 육종, 내피 육종, 림프관 육종, 림프관 내피 육종(lymphangioendotheliosarcoma), 활막종, 중피종, 유윙(Ewing) 종양, 평활근 육종, 횡문근 육종, 결장 암종, 췌장암, 유방암, 난소암, 전립선암, 편평 세포 암종, 기저 세포 암종, 선암종, 한선 암종, 피지선 암종, 유두상 암종, 유두상 선암종, 낭선암종, 수질 암종, 기관지원성 암종, 신 세포 암종, 간종양, 담관 암종, 융모 암종, 고환종, 태생성 암종, 윌름(Wilm) 종양, 자궁경부암, 고환 종양, 폐 암종, 소세포 폐 암종, 방광 암종, 상피 암종, 신경교종, 성상세포종, 수모세포종, 두개인두종, 뇌실막종, 송과체종, 혈관모세포종, 청신경종, 희돌기교종, 수막종, 흑색종, 신경모세포종 및 망막모세포종 중에서 선택된 어느 하나 이상의 질병일 수 있다.The above cancers are 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 carcinoma, papillary carcinoma, papillary It may be any 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.

상기 약학적 조성물은 약제학적으로 허용되는 담체를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다.The above pharmaceutical composition can be prepared in unit dose form by formulating it using a pharmaceutically acceptable carrier or can be prepared by placing it in a multi-dose container.

상기 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸 히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.The pharmaceutically acceptable carriers mentioned above are those commonly used in the preparation of 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.

본 발명에 있어서, 상기 약학적 조성물에 포함되는 첨가제의 함량은 특별히 한정되는 것은 아니며 통상의 제제화에 사용되는 함량 범위 내에서 적절하게 조절될 수 있다.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.

본 발명에서 벡터는 세포의 안정적인 형질 감염을 가능하게 하는 도구로 상기 벡터는 전이 유전자(들)의 세포 게놈 내로의 편입을 가능하게 한다. 바람직하게는, 이러한 벡터는 양성 선택 마커를 갖고 적합한 양성 선택 마커는 유전자를 발현하지 않는 세포를 살해하는 조건 하에서 세포가 성장하도록 하는 임의의 유전자를 포함한다. 비-제한적인 예에는 항생제 저항성이 포함될 수 있다. 추가적으로 상기 벡터는 플라스미드 벡터이며, 보다 구체적으로 상기 벡터는 바이러스 벡터이고, 당업자에 의해 임의로 적합한 벡터로 대체하여 사용될 수 있으며, 바람직하게 상기 벡터는 렌티 바이러스 벡터, 레트로 바이러스 벡터, DNA 벡터, 플라스미드, RNA 벡터, 아데노 바이러스 벡터, 또는 아데노 바이러스 연관 벡터일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the vector is a tool that enables stable transfection of a cell, and the vector enables incorporation of the transgene(s) into the cell 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 arbitrarily replaced by a suitable vector by a person skilled in the art, and preferably, the vector may be a lentivirus vector, a retrovirus vector, a DNA vector, a plasmid, an RNA vector, an adenovirus vector, or an adenovirus-associated vector, but is not limited thereto.

본 발명에서 단백질”은 "펩타이드" 및 "폴리펩타이드"와 상호 교환적으로 사용되며, 펩타이드 결합에 의해 공유 연결된 아미노산 잔기를 갖는 화합물을 가리킨다. 단백질 또는 펩타이드는 적어도 2 개의 아미노산을 포함해야하며, 단백질 또는 펩타이드의 서열을 포함할 수 있는 아미노산의 최대 수에 대한 제한은 없다. 폴리펩타이드는 펩타이드 결합에 의해 서로 결합된 2 개 이상의 아미노산을 갖는 임의의 펩타이드 또는 단백질을 포함한다. 여기서 사용된 용어는 두가지 사슬을 모두 가리키며, 짧은 사슬은 또한 통상 당 업계에서의 펩타이드, 올리고펩타이드 및 올리고머를 예를 들어 지칭되며 더 긴 사슬에 대해서는 일반적으로 당 업계에서의 단백질로 지칭되는 데 많은 종류가 거기에 속한다. "폴리펩타이드"는 예를 들어 생물학적으로 활성 단편, 실질적으로 동종의 폴리 펩타이드, 올리고펩타이드, 동종이합체, 이질이합체, 폴리펩타이드의 변이체, 변형된 폴리펩타이드, 유도체, 유사체, 융합단백질, 등을 포함한다. 폴리펩타이드는 천연 펩타이드, 재조합 펩타이드, 합성 펩타이드, 또는 이들의 조합물을 포함한다.In the present invention, the term "protein" 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 terms used herein refer to both chains, the shorter chains also commonly referred to as peptides, oligopeptides and oligomers in the art, and the longer chains commonly 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. The polypeptide includes a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

본 발명에서 뉴클레오타이드는 통상적으로 폴리뉴클레오타이드와 동일한 의미로 사용되며, DNA 및 RNA를 포함한다. 상기 폴리뉴클레오타이드는 핵산으로 뉴클레오타이드의 중합체이며, 폴리뉴크레오타이드는 단량체인 뉴클레오타이드로 가수분해 될 수 있다. 상기 폴리뉴크레오타이드는 재조합 수단, 즉 통상적인 클로닝 기술 및 중합 효소 연쇄 반응 (PCR) 등을 사용한 재조합 라이브러리 또는 세포유전체로부터의 핵산 서열 클로닝을 포함하는 당 업계에서 이용 가능한 임의의 수단 그리고 합성 수단에 의해 획득된 모든 핵산 서열을 포함 하나, 이에 제한되는 것은 아니다.In the present invention, nucleotide is generally used in the same sense as polynucleotide, and includes DNA and RNA. The polynucleotide is a nucleic acid, which is a polymer of nucleotides, and the polynucleotide can be hydrolyzed into monomeric nucleotides. The polynucleotide includes, but is not limited to, any means available in the art, including cloning of nucleic acid sequences from a recombinant library or cell genome using a recombinant means, such as conventional cloning techniques and polymerase chain reaction (PCR), and all nucleic acid sequences obtained by synthetic means.

이하, 본 발명의 이해를 돕기 위하여 실험예 및 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실험예 및 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실험예 및 실시예에 한정되는 것은 아니다. 본 발명의 실험예 및 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, in order to help understand the present invention, experimental examples and examples will be given to explain in detail. However, the following experimental examples and examples are only intended to illustrate the content of the present invention, and the scope of the present invention is not limited to the following experimental examples and examples. The experimental examples and examples of the present invention are provided to more completely explain the present invention to a person having average knowledge in the art.

실시예 1. Anti-PD-L1 키메라 항원 수용체(Chimeric antigen receptor)의 제작 Example 1. Production of anti-PD-L1 chimeric antigen receptor

본 발명에 따른 anti-PD-L1 키메라 항원 수용체(Chimeric antigen receptor, 이하 CAR)는 유전자 데이터베이스를 토대로 도 1에 나타난 바와 같이, anti-CD-19 CAR 구성을 참고하여 anti-PD-L1 CAR 암호화하는 유전자 컨스트럭트(construct)를 제작하였다. 본 실시예에서는 상기 CAR의 하나의 양태로서 순차적으로 PD-L1과 결합하는 단일사슬 가변 단편(single chain variable fragment, scFv) 영역, CD8 TM(transmembrane) 영역, 4-1BB 영역, 및 CD3ζ(zeta) 영역을 포함하도록 설계되었다. 상기 scFv 도메인은 세포 밖으로 노출되도록 설계되었으며, 하기 표 1과 같은 구성을 갖도록 3 종류로 제작되었다. 구체적으로 본 발명에 따른 anti-PD-L1 CAR는 anti-PD-L1 scFv 영역으로 서열번호 1로 표시되는 아미노산 서열로 구성된 경쇄(light chain) 및 서열번호 2로 표시되는 아미노산 서열로 구성된 중쇄(heavy chain)로 이루어진 A 타입이거나, 서열번호 3로 표시되는 아미노산 서열로 구성된 경쇄(light chain) 및 서열번호 4로 표시되는 아미노산 서열로 구성된 중쇄(heavy chain)로 이루어진 B 타입이거나, 서열번호 5로 표시되는 아미노산 서열로 구성된 경쇄(light chain) 및 서열번호 6로 표시되는 아미노산 서열로 구성된 중쇄(heavy chain)로 이루어진 C 타입일 수 있다.According to the present invention, the anti-PD-L1 chimeric antigen receptor (hereinafter referred to as CAR) was produced by referencing the anti-CD-19 CAR configuration based on a genetic database as shown in Fig. 1 to produce a genetic construct encoding the anti-PD-L1 CAR. In this example, as one embodiment of the CAR, it was designed to sequentially include a single chain variable fragment (scFv) region that binds to PD-L1, a CD8 TM (transmembrane) region, a 4-1BB region, and a CD3ζ (zeta) region. The scFv domain was designed to be exposed outside of the cell, and was produced in three types to have the configurations shown in Table 1 below. Specifically, the anti-PD-L1 CAR according to the present invention may be an A type consisting of a light chain consisting of an amino acid sequence represented by SEQ ID NO: 1 and a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 2 as an anti-PD-L1 scFv region, or a B type consisting of a light chain consisting of an amino acid sequence represented by SEQ ID NO: 3 and a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 4, or a C type consisting of a light chain consisting of an amino acid sequence represented by SEQ ID NO: 5 and a heavy chain consisting of an amino acid sequence represented by SEQ ID NO: 6.

타입Type 아미노산 서열Amino acid sequence SEQ. No.SEQ. No. AA 경쇄
(light chain)Light chain
(light chain) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK 11 중쇄
(heavy chain)Double-sided
(heavy chain) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS 22 BB 경쇄
(light chain)Light chain
(light chain) EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIKEIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYCQQYGSLPWTFGQGTKVEIK 33 중쇄
(heavy chain)Double-sided
(heavy chain) EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSSEVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS 44 CC 경쇄
(light chain)Light chain
(light chain) QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL 55 중쇄
(heavy chain)Double-sided
(heavy chain) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSS 66

본 발명에 따른 anti-PD-L1 CAR는 세포막관통 영역으로 CD8 TM(transmembrane) 영역을 포함한다. CD8 TM(transmembrane) 영역은 서열번호 15로 표시되는 아미노산 서열을 포함한다. 본 발명에 따른 anti-PD-L1 CAR는 서열번호 16으로 표시되는 아미노산 서열로 구성된 4-1BB 영역 및 서열번호 17로 표시되는 아미노산 서열로 구성된 CD3ζ(zeta) 영역을 포함하며, 상기 4-1BB 영역 및 CD3ζ(zeta) 영역은 세포 내부에 발현되도록 설계되었다. The anti-PD-L1 CAR according to the present invention includes a CD8 TM (transmembrane) region as a cell membrane-penetrating region. The CD8 TM (transmembrane) region includes an amino acid sequence represented by SEQ ID NO: 15. The anti-PD-L1 CAR according to the present invention includes a 4-1BB region composed of an amino acid sequence represented by SEQ ID NO: 16 and a CD3ζ (zeta) region composed of an amino acid sequence represented by SEQ ID NO: 17, wherein the 4-1BB region and the CD3ζ (zeta) region are designed to be expressed inside a cell.

도 1에 나타난 바와 같이, 본 발명에 따른 anti-PD-L1 CAR 유전자 컨스트럭트는 순차적으로 anti-PD-L1 scFv의 경쇄(light chain)를 암호화하는 유전자, 링커 서열, anti-PD-L1 scFv의 중쇄(heavy chain)를 암호화하는 유전자, CD8 TM(transmembrane) 도메인, 4-1BB 도메인, 및 CD3ζ(zeta) 도메인을 포함되도록 제작되었다.As shown in FIG. 1, the anti-PD-L1 CAR gene construct according to the present invention was produced to sequentially include a gene encoding a light chain of anti-PD-L1 scFv, a linker sequence, a gene encoding a heavy chain of anti-PD-L1 scFv, a CD8 TM (transmembrane) domain, a 4-1BB domain, and a CD3ζ (zeta) domain.

상기 CAR 유전자 컨스트럭트 A 타입은 서열번호 8로 표시되는 anti-PD-L1 scFv의 경쇄(light chain)를 암호화하는 유전자, 및 서열번호 9로 표시되는 anti-PD-L1 scFv의 중쇄(heavy chain)를 암호화하는 유전자를 포함한다. The above CAR gene construct type A includes a gene encoding a light chain of anti-PD-L1 scFv represented by SEQ ID NO: 8, and a gene encoding a heavy chain of anti-PD-L1 scFv represented by SEQ ID NO: 9.

상기 CAR 유전자 컨스트럭트 B 타입은 서열번호 10으로 표시되는 anti-PD-L1 scFv의 경쇄(light chain)를 암호화하는 유전자, 및 서열번호 11로 표시되는 anti-PD-L1 scFv의 중쇄(heavy chain)를 암호화하는 유전자를 포함한다. The above CAR gene construct type B includes a gene encoding a light chain of anti-PD-L1 scFv represented by SEQ ID NO: 10, and a gene encoding a heavy chain of anti-PD-L1 scFv represented by SEQ ID NO: 11.

상기 CAR 유전자 컨스트럭트 C 타입은 서열번호 12로 표시되는 anti-PD-L1 scFv의 경쇄(light chain)를 암호화하는 유전자, 및 서열번호 13으로 표시되는 anti-PD-L1 scFv의 중쇄(heavy chain)를 암호화하는 유전자를 포함한다.The above CAR gene construct type C includes a gene encoding a light chain of anti-PD-L1 scFv represented by SEQ ID NO: 12, and a gene encoding a heavy chain of anti-PD-L1 scFv represented by SEQ ID NO: 13.

상기 CD8 TM(transmembrane) 도메인은 서열번호 18로 표시되는 염기서열로 구성된다. 상기 4-1BB 도메인은 서열번호 19로 표시되는 염기서열로 구성된다. 상기 CD3ζ(zeta) 도메인은 서열번호 20으로 표시되는 염기서열로 구성된다.The above CD8 TM (transmembrane) domain is composed of a base sequence represented by SEQ ID NO: 18. The above 4-1BB domain is composed of a base sequence represented by SEQ ID NO: 19. The above CD3ζ (zeta) domain is composed of a base sequence represented by SEQ ID NO: 20.

추가적으로 본 발명에 따른 anti-PD-L1 CAR 유전자 컨스트럭트는 유도성 사이토카인(inducible cytokine) 도메인을 추가적으로 포함할 수 있다. 상기 유도성 사이토카인 도메인은 항시 발현되는 것이 아니라 본 발명에 따른 키메라 항원 수용체에 감염된 T 세포의 활성에 따라 발현되도록 고안되었으며, 과도한 사이토카인으로 인한 독성 및 염증 발생을 방지할 수 있도록 설계되었다.Additionally, the anti-PD-L1 CAR gene construct according to the present invention may additionally include an inducible cytokine domain. The inducible cytokine domain is not designed to be constantly expressed, but is designed to be expressed according to the activity of T cells infected with the chimeric antigen receptor according to the present invention, and is designed to prevent toxicity and inflammation caused by excessive cytokines.

본 발명의 일 실시의 양태에서는 도 2에 나타난 바와 같이, anti-PD-L1 CAR 구성의 3` 말단으로 NFAT(Nuclear factor of activated T cells) 결합 모티프 및 IL-2을 암호화하는 뉴클레오타이드를 순차적으로 포함하는 유도성 IL-2 (inducible IL-2) 도메인을 포함한다. 상기 유도성 IL-2(inducible IL-2) 도메인을 암호화하는 유전자는 서열번호 14로 표시되는 염기서열로 구성된다. 상기 유도성 IL-2 (inducible IL-2) 도메인을 암호화하는 유전자는 NFAT(Nuclear factor of activated T cells) 결합 모티프를 포함함으로써, IL-2(interleukin-2)의 발현을 선택적으로 유도할 수 있으며, 상기 유도성 IL-2 (inducible IL-2) 도메인을 통해 발현된 IL-2는 서열번호 7로 표시되는 아미노산 서열로 구성된다.In one embodiment of the present invention, as shown in FIG. 2, the 3' end of the anti-PD-L1 CAR configuration includes an inducible IL-2 domain sequentially including a NFAT (Nuclear factor of activated T cells) binding motif and a nucleotide encoding IL-2. The gene encoding the inducible IL-2 domain is configured by the base sequence represented by SEQ ID NO: 14. The gene encoding the inducible IL-2 domain can selectively induce the expression of IL-2 (interleukin-2) by including an NFAT (Nuclear factor of activated T cells) binding motif, and the IL-2 expressed through the inducible IL-2 domain is configured by the amino acid sequence represented by SEQ ID NO: 7.

실시예 2. Anti-PD-L1 CAR 발혈용 벡터 및 렌티바이러스의 제작Example 2. Production of anti-PD-L1 CAR hematopoietic vector and lentivirus

본 발명에 따른 anti-PD-L1 CAR를 발현시키기 위한 벡터를 제작하고 이를 전달하는 수단으로 렌티 바이러스를 사용하였다. 도 3에 나타난 바와 같이 3세대 렌티바이러스 벡터인 pSL-BBZ 를 기본 백본(backbone)으로 사용하였다. 벡터에 본 발명의 anti-PD-L1 CAR 컨스트럭트(construct)를 삽입하였다.A vector for expressing the anti-PD-L1 CAR according to the present invention was produced and a lentivirus was used as a means of delivering the same. As shown in Fig. 3, the third-generation lentiviral vector pSL-BBZ was used as the basic backbone. The anti-PD-L1 CAR construct of the present invention was inserted into the vector.

상기 anti-PD-L1 CAR 컨스트럭트(construct)에서 anti-PD-L1 scFv의 경쇄(light chain)를 암호화하는 유전자, 및 anti-PD-L1 scFv의 중쇄(heavy chain)를 암호화하는 유전자가 Bam H1 위치와 Xho I 위치 사이에 위치한다. 또한, 실험상 발현 여부를 확인할 수 있도록 상기 CAR 컨스트럭트(construct)의 말단으로 EGFP(Enhanced Green Fluorescent Protein) 서열이 발현되도록 설계되었다.In the above anti-PD-L1 CAR construct, a gene encoding the light chain of anti-PD-L1 scFv and a gene encoding the heavy chain of anti-PD-L1 scFv are located between the Bam H1 site and the Xho I site. In addition, the CAR construct was designed to express an EGFP (Enhanced Green Fluorescent Protein) sequence at the end so that expression can be confirmed experimentally.

합성한 유전자는 중합효소 연쇄반응(Polymerase Chain Reaction)을 통해 렌 티바이러스 플라스미드 벡터에 서브클로닝(subcloning) 하였다. 최종적으로 유전자 서열의 정확성은 클로닝된 플라스미드의 특이적 프라이머를 통한 염기서열 시퀀싱 을 통하여 확인하였다. 또한 IL-2, IL-12, IL-7, IL-15 또는 IL-18 등의 사이토카인 유전자들의 뉴클레오타이드 서열과 그 조절부위들도 유전자데이터베이스에서 확인하여 합성하였고 상기 와 같은 방법으로 렌티바이러스 플라스미드 벡터에 서브클로닝하여 최종적으로 anti-PD-L1 CAR 컨스트럭트(construct)로 제작하였다.The synthesized gene was subcloned into a lentiviral plasmid vector through polymerase chain reaction. Finally, the accuracy of the gene sequence was confirmed through base sequence sequencing using specific primers of the cloned plasmid. In addition, the nucleotide sequences and regulatory regions of cytokine genes such as IL-2, IL-12, IL-7, IL-15, or IL-18 were confirmed in a gene database and synthesized, and subcloned into a lentiviral plasmid vector using the same method as above to finally produce an anti-PD-L1 CAR construct.

본 실시예에서는 상기 CAR 유전자 컨스트럭트 C 타입에 NFAT(Nuclear factor of activated T cells) 결합 모티프 및 IL-2을 암호화하는 뉴클레오타이드를 순차적으로 포함하는 유도성 IL-2 (inducible IL-2) 도메인을 순차적으로 포함하도록 anti-PD-L1 CAR 컨스트럭트(construct)를 제작하였다.In this example, an anti-PD-L1 CAR construct was produced to sequentially include an inducible IL-2 domain sequentially including a NFAT (Nuclear factor of activated T cells) binding motif and nucleotides encoding IL-2 in the CAR gene construct type C.

상기 anti-PD-L1 CAR 컨스트럭트(construct)가 포함되도록 설계된 렌티 바이러스 벡터 3 μg을 Lipofectamine(Thermo Scientific #R0531)를 이용하여 HEK293T 세포(5 Y 106 cells)에 감염시켰다. 48 시간 동안 배양한 후 상등액을 회수하여 0.45μm 필터를 통해 바이러스 입자만을 정제하였다. 정제된 바이러스 입자는 28000 rpm에서 2시간 동안 초원심분리하여 농축하였다. HEK293T cells (5 Y 10 6 cells) were infected with 3 μg of lentiviral vector designed to include the above anti-PD-L1 CAR construct using Lipofectamine (Thermo Scientific #R0531). After culturing for 48 h, the supernatant was collected and only the virus particles were purified through a 0.45 μm filter. The purified virus particles were concentrated by ultracentrifugation at 28,000 rpm for 2 h.

실시예 3. 인간 T 세포 분리 및 활성화Example 3. Isolation and activation of human T cells

본 발명에 따른 상기 anti-PD-L1 CAR를 발현하는 T 세포를 제작하기 위해 사람의 말초 혈액으로부터 단핵 세포를 분리하였다. 말초혈액 단핵세포로부 터 자력이용 세포분리법(MACS)으로 CD8+ T 세포를 분리하였으며, 1:1의 비드 대 세포 비율로 anti-hCD3 및 anti-hCD28-코팅된 면역비드로 24 시간 내지 36 시간 동안 자극하여 분리된 CD4-/CD8+ /CD3+ T 세포로 준비하였다.To produce T cells expressing the anti-PD-L1 CAR according to the present invention, mononuclear cells were isolated from human peripheral blood. CD8 + T cells were isolated from peripheral blood mononuclear cells by magnetic cell sorting (MACS), and CD4 - /CD8 + /CD3 + T cells were prepared by stimulating them with anti-hCD3 and anti-hCD28-coated immunobeads at a bead-to-cell ratio of 1:1 for 24 to 36 hours.

실시예 4. Anti-PD-L1 CAR 발현 T 세포 제작 Example 4. Production of T cells expressing anti-PD-L1 CAR

상기 실시예 3에서 활성화된 T 세포를 32℃에서 Novo Nectin(Novoprotein) 코팅된 48well plate 플랫 플레이트에서 농축된 anti-CAR 렌티바이러스 벡터와 함께 배양하였다. 형질도입 후 6일 내지 7일 후에 면역 비드를 제거하고, T 세포를 세포 밀도를 0.5 X 106/ml 내지 2 Υ 106/mL로 3일 마다 조정하였다.In the above Example 3, activated T cells were cultured with concentrated anti-CAR lentiviral vectors in a 48-well plate flat plate coated with Novo Nectin (Novoprotein) at 32°C. The immune beads were removed 6 to 7 days after transduction, and the T cells were adjusted to a cell density of 0.5 X 10 6 /mL to 2 Υ 10 6 /mL every 3 days.

실시예 5. Anti-PD-L1 CAR T 세포의 PD-L1+ 암세포주에 대한 세포독성Example 5. Cytotoxicity of anti-PD-L1 CAR T cells against PD-L1+ cancer cell lines

본 발명에 따라 제조된 anti-PD-L1 CAR를 발현하는 T 세포의 암 세포에 대한 살상 효과를 확인하기 위해 유방암 세포주인 MDA-MB-231 세포에 대한 암세포 살상 효과를 평가하였다. 비교를 위해서 anti-CD-19 CAR를 발현하는 T 세포를 비교군을 사용하였다. 상기 anti-CD-19 CAR는 도 1에 나타난 바와 같이, anti-CD-19 CAR 구성을 사용하였으며, CAR 발현 T 세포는 anti-PD-L1 CAR와 동일하게 렌티 바이러스를 이용하여 제작하였다. MDA-MB-231 세포를 96 well 플레이트에 2 X 104 cell/mL로 넣고, FBS를 포 함하는 DMEM(Dulbecco's Modified Eagle's Medium) 배지에서 24시간 동안 배양하였다. 각각의 CAR로 형질전환된 T 세포를 다양한 비율로 well에 첨가한 후 37℃에서 4 시간 또는 24시간 동안 배양하였다. 배양이 완료된 웰에서 배지를 제거하고 다시 0.2% MTT(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium)를 50 ㎕/well씩 넣고, 2 시간 동안 37℃2 배양기에서 배양하였다. 상등액을 제거한 후 DMSO(dimethyl sulfoxide)을 100㎕/well씩 넣어준 후 5분간 현탁시켜 생성된 formazan 용해시키고 마이크로 플레이트 판독기(microplate reader)를 이용하여 570 nm에서 흡광도를 측정하 암세포의 사멸율 측정하였다.In order to confirm the cancer cell killing effect of T cells expressing the anti-PD-L1 CAR manufactured according to the present invention, the cancer cell killing effect on MDA-MB-231 cells, a breast cancer cell line, was evaluated. For comparison, T cells expressing anti-CD-19 CAR were used as a control group. As shown in Fig. 1, the anti-CD-19 CAR used the anti-CD-19 CAR composition, and the CAR expressing T cells were produced using lentivirus in the same manner as the anti-PD-L1 CAR. MDA-MB-231 cells were placed in a 96-well plate at 2 X 10 4 cells/mL and cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing FBS for 24 hours. T cells transformed with each CAR were added to the wells at various ratios and then cultured at 37°C for 4 or 24 hours. After the culture was completed, the medium was removed from the wells, 0.2% MTT (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) was added at 50 ㎕/well, and incubated in an incubator at 37℃ for 2 hours. After removing the supernatant, 100 ㎕/well of DMSO (dimethyl sulfoxide) was added, and the suspension was made for 5 minutes to dissolve the produced formazan, and the absorbance was measured at 570 nm using a microplate reader to determine the death rate of cancer cells.

도 5에 나타난 바와 같이, 각각의 CAR로 형질전환된 T 세포를 암세포와 반응한 경우 4시간에서는 효과기(Effector; E) T 세포와 타겟(Target; T) 암세포간의 비율(E:T ratio)의 변화에도 세포독성이 크게 변화되지 않는 것으로 확인되었다. 24 시간 반응한 경우에는 E:T 비율이 10:1 에서 1:4로 변화하는 경우 anti-CD-19 CAR T 세포와 반응한 암세포의 사멸율이 급격히 낮아지는 것으로 나타났다. 반면, anti-PD-L1 CAR T 세포와 반응한 암세포의 사멸율은 E:T 비율이 10:1 에서 1:4로 변화하여도 암세포의 사멸율이 현저히 우수하게 나타났다.As shown in Fig. 5, when T cells transformed with each CAR were reacted with cancer cells, it was confirmed that the cytotoxicity did not change significantly even when the ratio (E:T ratio) between effector (E) T cells and target (T) cancer cells was changed after 4 hours. When reacted for 24 hours, the death rate of cancer cells reacted with anti-CD-19 CAR T cells was found to decrease rapidly when the E:T ratio changed from 10:1 to 1:4. On the other hand, the death rate of cancer cells reacted with anti-PD-L1 CAR T cells was found to be significantly superior even when the E:T ratio changed from 10:1 to 1:4.

이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 즉, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다.While the specific parts of the present invention have been described in detail above, it is obvious to those skilled in the art that such specific description is merely a preferred embodiment and that the scope of the present invention is not limited thereby. In other words, the actual scope of the present invention is defined by the appended claims and their equivalents.

수치 범위는 상기 범위에 정의된 수치를 포함한다. 본 명세서에 걸쳐 주어진 모든 최대의 수치 제한은 낮은 수치 제한이 명확히 쓰여 있는 것처럼 모든 더 낮은 수치 제한을 포함한다. 본 명세서에 걸쳐 주어진 모든 최소의 수치 제한은 더 높은 수치 제한이 명확히 쓰여 있는 것처럼 모든 더 높은 수치 제한을 포함한다. 본 명세서에 걸쳐 주어진 모든 수치 제한은 더 좁은 수치 제한이 명확히 쓰여 있는 것처럼, 더 넓은 수치 범위 내의 더 좋은 모든 수치 범위를 포함할 것이다.The numerical ranges are inclusive of the numbers defined in the above ranges. Every maximum numerical limitation given throughout this specification will include every lower numerical limitation, as if that lower numerical limitation were expressly written out. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if that higher numerical limitation were expressly written out. Every numerical limitation given throughout this specification will include every better numerical range within that broader numerical range, as if that narrower numerical limitation were expressly written out.

Claims (13)

anti-PD-L1 scFv 도메인 및 유도성 사이토카인(inducible cytokine) 도메인을 포함하는, 키메라 항원 수용체를 발현하는 T 세포의 제작을 위한 벡터.A vector for producing T cells expressing a chimeric antigen receptor comprising an anti-PD-L1 scFv domain and an inducible cytokine domain. 제1항에 있어서, 상기 키메라 항원 수용체는 anti-PD-L1 scFv 영역, CD8 TM(transmembrane) 영역, 4-1BB 영역 및 CD3ζ(zeta) 영역을 포함하는 것을 특징으로 하는 벡터.A vector according to claim 1, characterized in that the chimeric antigen receptor comprises an anti-PD-L1 scFv region, a CD8 TM (transmembrane) region, a 4-1BB region, and a CD3ζ (zeta) region. 제2항에 있어서, 상기 anti-PD-L1 scFv 영역은,In the second paragraph, the anti-PD-L1 scFv region is 서열번호 1로 표시되는 아미노산 서열로 구성된 경쇄(light chain) 및 서열번호 2로 표시되는 아미노산 서열로 구성된 중쇄(heavy chain)로 이루어지거나,It consists of a light chain consisting of an amino acid sequence represented by sequence number 1 and a heavy chain consisting of an amino acid sequence represented by sequence number 2, or 서열번호 3로 표시되는 아미노산 서열로 구성된 경쇄(light chain) 및 서열번호 4로 표시되는 아미노산 서열로 구성된 중쇄(heavy chain)로 이루어지거나, Consisting of a light chain consisting of an amino acid sequence represented by sequence number 3 and a heavy chain consisting of an amino acid sequence represented by sequence number 4, or 서열번호 5로 표시되는 아미노산 서열로 구성된 경쇄(light chain) 및 서열번호 6로 표시되는 아미노산 서열로 구성된 중쇄(heavy chain)로 이루어진 것을 특징으로 하는 벡터.A vector characterized by comprising a light chain consisting of an amino acid sequence represented by sequence number 5 and a heavy chain consisting of an amino acid sequence represented by sequence number 6. 제1항에 있어서, 상기 anti-PD-L1 scFv 도메인은, In the first paragraph, the anti-PD-L1 scFv domain, 서열번호 8로 표시되는 anti-PD-L1 scFv의 경쇄(light chain)를 암호화하는 유전자, 및 서열번호 9로 표시되는 anti-PD-L1 scFv의 중쇄(heavy chain)를 암호화하는 유전자를 순차적으로 포함하거나, Sequentially comprising a gene encoding a light chain of anti-PD-L1 scFv represented by SEQ ID NO: 8, and a gene encoding a heavy chain of anti-PD-L1 scFv represented by SEQ ID NO: 9, or 서열번호 10으로 표시되는 anti-PD-L1 scFv의 경쇄(light chain)를 암호화하는 유전자, 및 서열번호 11로 표시되는 anti-PD-L1 scFv의 중쇄(heavy chain)를 암호화하는 유전자를 순차적으로 포함하거나,Sequentially comprising a gene encoding a light chain of anti-PD-L1 scFv represented by SEQ ID NO: 10, and a gene encoding a heavy chain of anti-PD-L1 scFv represented by SEQ ID NO: 11, or 서열번호 12로 표시되는 anti-PD-L1 scFv의 경쇄(light chain)를 암호화하는 유전자, 및 서열번호 13으로 표시되는 anti-PD-L1 scFv의 중쇄(heavy chain)를 암호화하는 유전자를 순차적으로 포함하는 것을 특징으로 하는 벡터.A vector characterized by sequentially including a gene encoding a light chain of anti-PD-L1 scFv represented by SEQ ID NO: 12, and a gene encoding a heavy chain of anti-PD-L1 scFv represented by SEQ ID NO: 13. 제1항에 있어서, 상기 벡터는 상기 anti-PD-L1 scFv 도메인 이후로 순차적으로 CD8 TM(transmembrane) 도메인, 4-1BB 도메인, 및 CD3ζ(zeta) 도메인을 추가로 포함하는 것을 특징으로 하는 벡터.In the first paragraph, the vector is characterized in that it additionally sequentially comprises a CD8 TM (transmembrane) domain, a 4-1BB domain, and a CD3ζ (zeta) domain after the anti-PD-L1 scFv domain. 제5항에 있어서,In paragraph 5, 상기 CD8 TM(transmembrane) 도메인은 서열번호 18로 표시되는 염기서열로 이루어지고,The above CD8 TM (transmembrane) domain is composed of a base sequence represented by sequence number 18, 상기 4-1BB 도메인은 서열번호 19로 표시되는 염기서열로 이루어지고,The above 4-1BB domain is composed of a base sequence represented by sequence number 19, 상기 CD3ζ(zeta) 도메인은 서열번호 20으로 표시되는 염기서열로 이루어진 것을 특징으로 하는 벡터.A vector characterized in that the CD3ζ(zeta) domain is composed of a base sequence represented by sequence number 20. 제1항에 있어서, 상기 유도성 사이토카인(inducible cytokine) 도메인은 NFAT(Nuclear factor of activated T cells) 결합 모티프 및 사이토카인을 암호화하는 유전자를 순차적으로 포함하는 것을 특징으로 하는 벡터.A vector in claim 1, characterized in that the inducible cytokine domain sequentially includes a NFAT (Nuclear factor of activated T cells) binding motif and a gene encoding a cytokine. 제7항에 있어서, 상기 사이토카인은 IL-2, IL-12, IL-7, IL-15 또는 IL-18 중 선택된 어느 하나인 것을 특징으로 하는 벡터.A vector, characterized in that in claim 7, the cytokine is any one selected from IL-2, IL-12, IL-7, IL-15, or IL-18. 제1항에 있어서, 상기 유도성 사이토카인(inducible cytokine) 도메인은 서열번호 14로 표시되는 염기서열로 이루어진 것을 특징으로 하는 벡터.A vector characterized in that in claim 1, the inducible cytokine domain consists of a base sequence represented by SEQ ID NO: 14. 제1항에 있어서, 상기 벡터는 렌티 바이러스 벡터인 것을 특징으로 하는 벡터.A vector according to claim 1, characterized in that the vector is a lentiviral vector. 제1항 내지 제10항 중 어느 하나의 벡터로 형질감염된 T 세포.A T cell transfected with any one of the vectors of claims 1 to 10. 제11항에 있어서, 상기 T 세포는 단핵 세포에서 분리된 CD4+/CD8+ 유형에서 anti-CD3 및 anti-hCD28 로 자극된 T 세포인 것을 특징으로 하는 T 세포.In claim 11, the T cell is a T cell characterized in that it is a T cell stimulated with anti-CD3 and anti-hCD28 of the CD4 + /CD8 + type isolated from mononuclear cells. 제11항의 T 세포를 포함하는 암의 예방 또는 치료를 위한 약학적 조성물.A pharmaceutical composition for preventing or treating cancer comprising the T cell of claim 11.
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