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WO2024117781A1 - Protéine de fusion comprenant un domaine extracellulaire cd137, cellules immunitaires l'exprimant et utilisation associée - Google Patents

Protéine de fusion comprenant un domaine extracellulaire cd137, cellules immunitaires l'exprimant et utilisation associée Download PDF

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WO2024117781A1
WO2024117781A1 PCT/KR2023/019470 KR2023019470W WO2024117781A1 WO 2024117781 A1 WO2024117781 A1 WO 2024117781A1 KR 2023019470 W KR2023019470 W KR 2023019470W WO 2024117781 A1 WO2024117781 A1 WO 2024117781A1
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cells
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fusion protein
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cancer
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황도원
김신일
기영욱
박성진
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Therabest Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • 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
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a fusion protein containing the CD137 extracellular domain or a fragment thereof, immune cells expressing the fusion protein, and uses thereof.
  • T cells expressing CD3 are known as effector cells that can eliminate tumor cells.
  • Immune checkpoints are co-signaling molecules that play a central role in T cell activation and regulate the signal of the T cell receptor (TCR) to be suppressed or elevated (Immunological reviews, 2017, 276:52- 65).
  • NK cells natural killer cells
  • the activity of NK cells is regulated by a balance of various activating and inhibitory receptor signaling. It is known that the anticancer activity of NK cells is also achieved by distinguishing cancer cells through various immune receptors present on their surface.
  • NK cells can eliminate not only cancer cells but also cancer stem cells, so they are in the spotlight as a source of development for treatments that can not only suppress the occurrence, proliferation, and metastasis of cancer, but also reduce the recurrence of cancer after cure.
  • 4-1BB is one of the TNF-receptor superfamily (TNFRSF), a costimulatory molecule that is expressed upon activation of immune cells, both innate and adaptive immune cells, and regulates the activation of various immune cells.
  • TNFRSF TNF-receptor superfamily
  • the 4-1BB agonist enhances immune cell proliferation, survival, cytokine secretion, and CD8+ T cell lytic activity. This action has shown anticancer effects in several mouse tumor models and is attracting attention as a target for anticancer drugs. However, to this day, new methods that can enhance the anticancer activity of immune cells against cancer cells through 4-1BB activation are being studied.
  • the present inventors studied a new method that can enhance the anti-cancer activity of immune cells against cancer cells, and as a result, a fusion protein containing the extracellular domain and intracellular signaling domain of CD137 (or 4-1BB) was introduced into immune cells.
  • a fusion protein containing the extracellular domain and intracellular signaling domain of CD137 (or 4-1BB) was introduced into immune cells.
  • cytotoxicity against cancer cells increased compared to normal immune cells.
  • INF- ⁇ interferon gamma
  • one aspect of the present invention provides a fusion protein comprising the following (i) to (iii): (i) the extracellular domain of CD137 (4-1BB) or a fragment thereof; (ii) Transmembrane domain; and (iii) an intracellular signaling domain.
  • Another aspect of the present invention provides a polynucleotide encoding the fusion protein, a vector and a virus containing the polynucleotide.
  • Another aspect of the present invention provides immune cells expressing the fusion protein.
  • Another aspect of the present invention includes the steps of i) preparing the virus; and ii) processing the virus into immune cells.
  • Another aspect of the present invention is a pharmaceutical composition for preventing or treating cancer comprising the immune cells as an active ingredient; and pharmaceutical compositions and kits for cancer treatment comprising the immune cells and bispecific antibodies.
  • Another aspect of the invention provides CD137 extracellular domain variants or fragments thereof.
  • cBBR T cells overexpressing the 4-1BB/CD3 ⁇ fusion protein (cBBR) or a variant thereof specifically killed cancer cells overexpressing 4-1BBL In the case of T cells overexpressing cBBR containing the 4-1BB extracellular domain variant, it was confirmed that the binding affinity and cytotoxicity to 4-1BBL were reduced compared to cBBR T cells containing the native 4-1BB extracellular domain. did.
  • cBBR T cells overexpressing cBBR containing the above mutant were stimulated with Urelumab, their activity significantly increased compared to normal T cells.
  • anti-BCMA/anti-4-1BB bispecific antibodies it showed a significantly superior killing effect on BCMA-expressing cancer cells compared to normal T cells. Therefore, T cells overexpressing cBBR can be used as an anticancer agent.
  • Figure 1a is a schematic diagram of a nucleic acid cassette encoding a fusion protein combining 4-1BB and CD3 ⁇ intracellular domains.
  • SP represents the signal peptide
  • CRD represents the cysteine-rich domain
  • TM represents the transmembrane domain
  • ICD represents the intracellular domain of 4-1BB.
  • CD3 ⁇ represents the intracellular domain of CD3 ⁇ .
  • Figure 1b is a schematic diagram of a nucleic acid cassette encoding a fusion protein combining 4-1BB and CD3 ⁇ intracellular domains. Specifically, the domain structure of a fusion protein expressing only CRD1 and CDR1/CRD2 among CRD1, CRD2, CRD3, and CRD4 of the CD137 extracellular domain is schematized.
  • Figure 2 is a diagram showing that a fusion protein combining 4-1BB and CD3 ⁇ intracellular domains is stably expressed in transformed NK92MI cells.
  • Figure 3 shows the level of expression of 4-1BB after treating 1.0 x 10 6 NK92MI cells with Urelumab at different concentrations.
  • Figure 4 shows the expression level of 4-1BB quantified through FACS analysis after treatment with urelumab at different concentrations.
  • Figure 5 shows the measurement of the expression level of INF- ⁇ secreted by transformed NK92MI cells and normal NK cells cultured in the presence of urelumab. At this time, the group in which NK92MI cells and normal NK cells were cultured together with K562 cells was used as the control group.
  • Figure 6 shows a schedule for an experiment in which PBMC-derived NK cells were cultured, transfected with CD137 mRNA, and then co-cultured with Urelumab.
  • Figure 7 shows the expression level of 4-1BB over time using flow cytometry.
  • Figure 8 shows the amount of IFN- ⁇ secreted through ELISA.
  • Figure 9 shows that, despite the presence of only CRD1 and CRD1/CRD2 of the CD137 extracellular domain, it was confirmed through flow cytometry that Urelumab and the 4B4-1 antibody bind well.
  • FIG 10 is a diagram showing a schematic diagram of T (CER T, cBBR T) cells overexpressing chimeric engager receptor (cBBR) as an embodiment of the present invention.
  • Figure 11 is a graph showing the results of confirmation of cBBR (Chimeric 4-1BB receptor) or its variants (I64R, V71R) overexpressed in Jurkat T cells and 4-1BBL overexpressed in Nalm6 cells through flow cytometry.
  • cBBR Chimeric 4-1BB receptor
  • variants I64R, V71R
  • Figure 12 is a graph showing the results of confirming the expression of cBBR or its variants (I64R, V71R) overexpressed in blood-derived T cells through flow cytometry.
  • Figure 13 is a graph showing the results of confirming the binding rate of cBBR (Chimeric 4-1BB receptor) or its variants (I64R, V71R) overexpressed in Jurkat T cells and 4-1BBL overexpressed in Nalm6 cells through flow cytometry.
  • cBBR Chimeric 4-1BB receptor
  • variants I64R, V71R
  • Figure 14 shows the killing ability of T cells (cBBR T cells) overexpressing cBBR (Chimeric 4-1BB receptor) or its variants (I64R, V71R, I64R/V71R) against cancer cells overexpressing 4-1BBL by CFSE fluorescence staining and This is a graph showing the results measured by flow cytometry.
  • Figure 15 is a graph showing the level of activation of cBBR T cells overexpressing cBBR variants (I64R, V71R, I64R/V71R) by urelumab, confirmed by flow cytometry after staining with anti-CD8a antibody and anti-CD107a antibody. am.
  • Figure 16 is a graph showing the results of confirming the degree of activation of cBBR T cells overexpressing cBBR variants (I64R, V71R, I64R/V71R) by urelumab by measuring the concentration of IFN- ⁇ .
  • Figure 17 shows cBBR T cells or Ramos cells overexpressing cBBR variants (I64R, V71R) treated with a bispecific antibody (anti-BCMA/anti-4-1BB), and then cBBR T cells or Ramos cells and the bispecific antibody.
  • BiTE anti-BCMA/anti-4-1BB bispecific antibody
  • 19F2 anti-BCMA antibody
  • 4B4-1 anti-4-1BB antibody
  • Figure 18 is a graph showing the results of confirming the cancer cell killing ability of cBBR T cells by co-culturing cBBR T cells overexpressing cBBR variants (I64R, V71R) with cancer cells expressing dual specific antibodies and BCMA.
  • Figure 19 confirms that tumor suppression occurred when transformed T cells and AB-101 antibody were administered.
  • One aspect of the present invention provides a fusion protein comprising (i) to (iii) below:
  • CD137 is a membrane protein belonging to the tumor necrosis factor receptor family.
  • CD137 is referred to as tumor necrosis factor receptor superfamily member 9 or 4-1BB.
  • CD137 is a tumor necrosis factor receptor and mainly acts as a co-stimulatory molecule in T cells.
  • the CD137 extracellular domain contains four cysteine-rich pseudo repeat (CRD) domains.
  • the CD137 protein may preferably be human CD137 protein, but is not limited thereto.
  • CD137 extracellular domain fragment refers to a fragment in which part of the N-terminus and/or C-terminus of the native CD137 extracellular domain protein is truncated, and is identical to or similar to the native CD137 extracellular domain 4- This may indicate 1BBL binding force.
  • wild type includes all proteins found in nature or nucleic acids encoding them and can be used interchangeably with wild type.
  • the CD137 extracellular domain or fragment thereof includes CRD1 comprising the amino acid sequence of SEQ ID NO: 2; CRD2 comprising the amino acid sequence of SEQ ID NO: 3; CRD3 comprising the amino acid sequence of SEQ ID NO: 6; CRD4 comprising the amino acid sequence of SEQ ID NO: 7; And it may include any one selected from the group consisting of combinations thereof.
  • the CD137 extracellular domain or fragment thereof may include CRD1. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD2. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD3. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD4. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD1 and CRD2. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD1 and CRD3. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD1 and CRD4. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD2 and CRD3. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD2 and CRD4.
  • the CD137 extracellular domain or fragment thereof may include CRD3 and CRD4. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD1, CRD2, and CRD3. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD1, CRD2, and CRD4. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD1, CRD3, and CRD4. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD2, CRD3, and CRD4. In one embodiment, the CD137 extracellular domain or fragment thereof may include CRD1, CRD2, CRD3, and CRD4.
  • the CD137 extracellular domain or fragment thereof may include or consist of the amino acid sequence of CRD1.
  • the CD137 extracellular domain or fragment thereof may include or consist of the amino acid sequences of CRD1 and CRD2.
  • the extracellular domain of CD137 or a fragment thereof may include or consist of the amino acid sequences of CRD1, CRD2, CRD3, and CRD4.
  • the CD137 extracellular domain or fragment thereof may include the amino acid sequence of SEQ ID NO: 15.
  • the protein consists of a sequence in which one or several amino acids are added, deleted, or substituted. It can be.
  • the CD137 extracellular domain has about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% amino acid identity with the amino acid sequence of SEQ ID NO: 15. It may contain or consist of sequences.
  • the CD137 extracellular domain or fragment thereof may further include mutations.
  • mutant refers to a form in which some amino acids of the above-described CD137 extracellular domain or fragment thereof are substituted. That is, the variant of the CD137 extracellular domain or fragment thereof may have a different sequence from the native CD137 extracellular domain or fragment thereof. In the present invention, the mutant may have reduced binding ability to 4-1BBL (CD137L) compared to native CD137.
  • 4-1BBL CD137L
  • the binding ability with 4-1BBL can be measured using methods known to those skilled in the art.
  • the mutation may be included in any one selected from the group consisting of CRD1, CRD2, CRD3, CRD4, and combinations thereof.
  • the mutation may be included in CRD1. In one embodiment, the mutation may be included in CRD2. In one embodiment, the mutation may be included in CRD3. In one embodiment, the mutation may be included in CRD4. In one embodiment, the mutation may be included in CRD1 and CRD2. In one embodiment, the mutation may be included in CRD1 and CRD3. In one embodiment, the mutation may be included in CRD1 and CRD4. In one embodiment, the mutation may be included in CRD2 and CRD3. In one embodiment, the mutation may be included in CRD2 and CRD4. In one embodiment, the mutation may be included in CRD3 and CRD4. In one embodiment, the mutation may be included in CRD1, CRD2, and CRD3.
  • the mutation may be included in CRD1, CRD2, and CRD4. In one embodiment, the mutation may be included in CRD1, CRD3, and CRD4. In one embodiment, the mutation may be included in CRD2, CRD3, and CRD4. In one embodiment, the mutation may be included in CRD1, CRD2, CRD3, and CRD4.
  • the mutation may be included in CRD2 and/or CRD3.
  • the mutation of CRD2 is any one amino acid selected from the group consisting of P3, S6, Q13, T15, C16, D17, I18, Q21, K23, V25, F26, and combinations thereof in the amino acid sequence of SEQ ID NO: 3. It may have been replaced. Additionally, the mutation of CRD3 may be a substitution of any one amino acid selected from the group consisting of S14, M15, C16, and combinations thereof in the amino acid sequence of SEQ ID NO: 6.
  • amino acids introduced by the substitution include glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, Methionine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, arginine, phenylalanine, tyrosine, tryptophan ( It may be any one selected from the group consisting of tryptophan, histidine, and proline.
  • the mutation of CRD2 may be a substitution of any one amino acid selected from the group consisting of I18R, V25R, and combinations thereof in the amino acid sequence of SEQ ID NO: 3.
  • the mutation in CRD2 may be one in which isoleucine (I), the 18th amino acid of SEQ ID NO: 3, is substituted with arginine (R) (I18R).
  • a mutation in CRD2 may be a substitution of valine (V), the 25th amino acid of SEQ ID NO: 3, with arginine (R) (V25R).
  • the mutation in CRD2 may be one in which isoleucine (I), the 18th amino acid of SEQ ID NO: 3, and valine (V), the 25th amino acid, are each substituted with arginine (R) (I18R, V25R).
  • CRD2 containing the mutation may include or be composed of any one amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 26.
  • the CD137 extracellular domain containing the above mutation may include any one amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 27.
  • the binding ability of the CD137 extracellular domain to 4-1BBL may be reduced compared to the native type.
  • transmembrane domain used in the present invention refers to a portion of the protein structure located in the cell membrane that connects the extracellular domain and the intracellular signaling domain and penetrates the cell membrane.
  • the fusion protein according to the present invention can be fixed to the cell membrane through the transmembrane domain.
  • the transmembrane domain includes T cell receptor (TCR), CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, It may be derived from any one molecule selected from the group consisting of CD137, CD152, and CD154. Specifically, the transmembrane domain may be derived from CD137.
  • the transmembrane domain may be derived from CD137. Specifically, the transmembrane domain may include the amino acid sequence of SEQ ID NO: 8.
  • the protein may be composed of a sequence in which one or several amino acids are added, deleted, or substituted. there is.
  • the transmembrane domain derived from CD137 may include or consist of an amino acid sequence having about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 8.
  • the (i) CD137 extracellular domain and (ii) transmembrane domain may be connected through a spacer.
  • the spacer refers to a linker and may be a protein or peptide. Additionally, it may be composed of about 1 to about 1,000 amino acids, and may include about 10 to about 300 amino acids, about 15 to about 100 amino acids, about 15 to about 60 amino acids, or about 15 to about 45 amino acids. Additionally, the linker may be a fragment of a protein in the human body, such as an Fc region.
  • the linker consists of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152 and CD154. It may be derived from any one molecule selected from the group.
  • the spacer may be derived from CD137. Specifically, the spacer may include or be composed of the amino acid sequence of SEQ ID NO: 11.
  • intracellular signaling domain refers to the case where an extracellular binding domain present on the cell surface binds to a biological target molecule (e.g., binding of CD137 extracellular domain and 4-1BBL). ), refers to a site that transmits signals into the cell to induce reactions such as cell activation, release of cytotoxic factors, cytokine production, and cell proliferation.
  • the intracellular signaling domain may include a co-stimulatory signaling domain and/or a primary signaling domain.
  • co-stimulatory domain refers to the intracellular signaling domain of a co-stimulatory molecule.
  • the “co-stimulatory molecule” is a cell surface molecule other than the Fc receptor that provides a secondary signal that induces efficient activation and function of immune cells when the extracellular binding site binds to the target molecule.
  • the costimulatory domain may be derived from any one molecule selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and CD137. .
  • the costimulatory domain may be derived from CD137.
  • the co-stimulatory domain may include or consist of the amino acid sequence of SEQ ID NO: 9.
  • primary signaling domain refers to a protein that constitutes the CD3 complex of T cells and refers to a signaling domain that regulates the primary activation of T cells in a stimulatory or inhibitory manner.
  • the primary signaling domain that stimulates activation of the T cell may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM (Immunoreceptor tyrosine-based activation motif).
  • ITAM containing the primary signaling domain may be derived from any one molecule selected from the group consisting of TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b and CD66d.
  • the primary signaling domain may be derived from CD3 ⁇ .
  • CD3 ⁇ used herein refers to a protein that constitutes the CD3 complex.
  • the CD3 complex is a protein complex and T cell co-receptor involved in activating both cytotoxic T cells and T helper cells.
  • the CD3 complex contains the CD3 ⁇ chain, CD3 ⁇ chain, CD3 ⁇ chain, CD3 ⁇ chain, CD3 ⁇ chain, and CD3 ⁇ .
  • the primary signaling domain may include or consist of the amino acid sequence of SEQ ID NO: 10.
  • CD28 is one of the proteins expressed on T cells that provide costimulatory signals necessary for T cell activation and survival. At this time, the intracellular domain of CD28 may have the amino acid sequence of SEQ ID NO: 34.
  • OX40 refers to tumor necrosis factor receptor superfamily, member 4, also known as CD134. At this time, the intracellular domain of OX40 may have the amino acid sequence of SEQ ID NO: 35.
  • each of the domains consists of a sequence in which one or several amino acids are added, deleted, or substituted. It can be.
  • the co-stimulatory domain may include or consist of an amino acid sequence having about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 9.
  • the primary signaling domain may include or consist of an amino acid sequence having about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 10. there is.
  • the intracellular signaling domain of the present invention may be an appropriate combination of the costimulatory domain and the primary signaling domain.
  • the intracellular signaling domain may include CD137 and CD3 ⁇ .
  • the co-stimulatory domain and the primary signaling domain may be connected through a linker.
  • the fusion protein of the present invention may contain the following structural formula (I).
  • N' is the N terminus of each polypeptide
  • the C' is the C terminus of each polypeptide
  • the TM is a transmembrane domain
  • B and C are intracellular signaling domains, each independently a co-stimulatory domain and a primary signaling domain;
  • L1, L2 and L3 are each peptide linkers
  • n, m and l are each independently 0 or 1.
  • extracellular domain or fragment thereof, transmembrane domain, and intracellular signaling domain of CD137 are the same as described above.
  • N'-CRD1 (SEQ ID NO: 2) - CRD2 (SEQ ID NO: 3) - CRD3 (SEQ ID NO: 6) - CRD4 (SEQ ID NO: 7) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular Domain (SEQ ID NO: 18)-CD3z (SEQ ID NO: 10)-C'
  • N'-CRD1 SEQ ID NO: 2
  • CRD4 SEQ ID NO: 7
  • Linker SEQ ID NO: 11
  • TM SEQ ID NO: 8
  • CD137 intracellular domain SEQ ID NO: 18
  • CD3z SEQ ID NO: 10
  • N'-CRD4 SEQ ID NO: 7
  • Linker SEQ ID NO: 11
  • T SEQ ID NO: 8
  • CD137 intracellular domain SEQ ID NO: 18
  • CD3z SEQ ID NO: 10
  • N'-CRD1 SEQ ID NO: 2
  • Linker SEQ ID NO: 11
  • T SEQ ID NO: 8
  • CD137 intracellular domain SEQ ID NO: 18
  • CD28 SEQ ID NO: 34
  • N'-CRD1 (SEQ ID NO: 2) - CRD2 (SEQ ID NO: 3) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular domain (SEQ ID NO: 18) - CD28 (SEQ ID NO: 34) - C'
  • N'-CRD1 (SEQ ID NO: 2) - CRD2 (SEQ ID NO: 3) - CRD3 (SEQ ID NO: 6) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular domain (SEQ ID NO: 18) - CD28 (SEQ ID NO: 34)-C'
  • N'-CRD1 (SEQ ID NO: 2) - CRD2 (SEQ ID NO: 3) - CRD3 (SEQ ID NO: 6) - CRD4 (SEQ ID NO: 7) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular Domain (SEQ ID NO: 18)-CD28 (SEQ ID NO: 34)-C'
  • N'-CRD1 (SEQ ID NO: 2) - CRD4 (SEQ ID NO: 7) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular domain (SEQ ID NO: 18) - CD28 (SEQ ID NO: 34) - C'
  • N'-CRD4 (SEQ ID NO: 7)-Linker (SEQ ID NO: 11)-TM (SEQ ID NO: 8)-CD137 intracellular domain (SEQ ID NO: 18)-CD28 (SEQ ID NO: 34)-C'
  • N'-CRD1 (SEQ ID NO: 2)-Linker (SEQ ID NO: 11)-TM (SEQ ID NO: 8)-CD137 intracellular domain (SEQ ID NO: 18)-OX40 (SEQ ID NO: 35)-C'
  • N'-CRD1 (SEQ ID NO: 2) - CRD2 (SEQ ID NO: 3) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular domain (SEQ ID NO: 18) - OX40 (SEQ ID NO: 35) - C'
  • N'-CRD1 (SEQ ID NO: 2) - CRD2 (SEQ ID NO: 3) - CRD3 (SEQ ID NO: 6) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular domain (SEQ ID NO: 18) - OX40 (SEQ ID NO: 35)-C'
  • N'-CRD1 (SEQ ID NO: 2) - CRD2 (SEQ ID NO: 3) - CRD3 (SEQ ID NO: 6) - CRD4 (SEQ ID NO: 7) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular Domain (SEQ ID NO: 18)-OX40 (SEQ ID NO: 35)-C'
  • N'-CRD1 (SEQ ID NO: 2) - CRD4 (SEQ ID NO: 7) - Linker (SEQ ID NO: 11) - TM (SEQ ID NO: 8) - CD137 intracellular domain (SEQ ID NO: 18) - OX40 (SEQ ID NO: 35) - C'
  • N'-CRD4 (SEQ ID NO: 7)-Linker (SEQ ID NO: 11)-TM (SEQ ID NO: 8)-CD137 intracellular domain (SEQ ID NO: 18)-OX40 (SEQ ID NO: 35)-C'
  • N' refers to the N terminus of the fusion protein
  • C' refers to the C terminus of the fusion protein
  • Another aspect of the present invention provides a polynucleotide encoding the fusion protein.
  • the fusion protein is the same as described above.
  • the polynucleotide encoding the fusion protein may include or be composed of any one nucleotide selected from the group consisting of SEQ ID NO: 21 to SEQ ID NO: 23 and SEQ ID NO: 28.
  • one or more bases may be mutated by substitution, deletion, insertion, or a combination thereof.
  • a synthesis method well known in the art for example, a method described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), can be used. and triester, phosphite, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, and oligonucleotide synthesis methods on solid supports.
  • the polynucleotide is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, and at least about the base sequences of SEQ ID NO: 21 to SEQ ID NO: 23 and SEQ ID NO: 28, respectively. 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about It may include a nucleic acid sequence having an identity of 97%, at least about 98%, at least about 99%, or at least about 100%.
  • the polynucleotide may additionally include a nucleic acid encoding a signal sequence or leader sequence.
  • signal sequence used herein refers to a signal peptide that directs secretion of a target protein.
  • the signal peptide is cleaved after translation in the host cell.
  • the signal sequence is an amino acid sequence that initiates the movement of proteins through the ER (Endoplasmic reticulum) membrane.
  • the signal sequence has well-known characteristics in the art, and usually contains 16 to 30 amino acid residues, but may contain more or fewer amino acid residues.
  • a typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region.
  • the central hydrophobic region contains 4 to 12 hydrophobic residues that anchor the signal sequence throughout the membrane lipid bilayer while the immature polypeptide moves.
  • the signal sequence is cleaved within the lumen of the ER by a cellular enzyme commonly known as signal peptidase.
  • the signal sequence may be a secretion signal sequence of tPa (Tissue Plasminogen Activation), HSV gDs (Signal sequence of Herpes simplex virus glycoprotein D), or growth hormone.
  • tPa tissue Plasminogen Activation
  • HSV gDs Synignal sequence of Herpes simplex virus glycoprotein D
  • growth hormone a secretion signal sequence used in higher eukaryotic cells, including mammals.
  • the signal sequence can be used as a wild-type signal sequence, or by replacing it with a codon that is frequently expressed in host cells.
  • the signal sequence may be the signal sequence of CD137.
  • the signal sequence may include or consist of the amino acid sequence of SEQ ID NO: 1.
  • the vector may be plasmid DNA, phage DNA, etc., commercially developed plasmids (e.g., pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli-derived plasmids (e.g., pYG601BR322, pBR325, pUC118, pUC119, etc.) , Bacillus subtilis-derived plasmids (e.g., pUB110, pTP5, etc.), yeast-derived plasmids (e.g., YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, ⁇ gt10, ⁇ gt11, ⁇ ZAP, etc.), It may be an animal virus vector (e.g., retrovirus, adenovirus, vaccinia virus, etc.) or an insect virus vector (baculovirus, etc.
  • N' refers to the N terminus of the fusion protein
  • C' refers to the C terminus of the fusion protein.
  • CRD1, CRD2, CRD3, and CRD4 may include mutations. At this time, the mutation is as described above.
  • N' refers to the N terminus of the fusion protein
  • C' refers to the C terminus of the fusion protein
  • intracellular domain of CD137 the intracellular domain of CD3 ⁇ , the intracellular domain of CD28, and the intracellular domain of OX40 are the same as described above.
  • the immune cells may be natural killer cells, T cells, or macrophages.
  • the immune cell expressing the fusion protein is a cell transformed by introducing the polynucleotide, and can be transformed using a vector loaded with the polynucleotide or a virus containing the polynucleotide.
  • the polynucleotide, vector and virus are the same as described above.
  • NK cell naturally killer cell
  • cytotoxic lymphocyte that constitutes a major component of the innate immune system
  • LGL large granular lymphocyte
  • CLP lymphoid progenitor
  • T cell refers to a type of lymphocyte that hosts antigen-specific adaptive immunity, including immature T cells that have not come into contact with an antigen and effector T cells that have matured after contact with an antigen (helper T cells, cytotoxic T cells). cells, natural killer T cells) and memory T cells.
  • T cells Activation of T cells occurs when a na ⁇ ve T cell antigen receptor (TCR) binds to the MHC:antigen complex and activates the CD3 complexes of T cells: CD3 gamma ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), and zeta ( ⁇ ) is induced by phosphorylating the ITAM of the chain.
  • the ITAM phosphorylation induces phosphorylation of ZAP-70 protein, LAT protein, and SLP-76 protein, thereby inducing a signal response that activates transcription factors such as NF ⁇ B, AP-1, and NFAT.
  • Transcription factors such as NF ⁇ B, AP-1, and NFAT induce T cell division and differentiation by promoting the expression of interleukin-2 (IL-2).
  • IL-2 interleukin-2
  • macrophage is also referred to as a phagocytic cell and is a major cell responsible for innate immunity.
  • the macrophages may exist as settlers in tissues, such as Kupffer cells, Langerhans cells, and microglial cells, or may exist in the form of monocytes in the blood. At this time, the monocytes may differentiate into dendritic cells or macrophages. Macrophages control inflammatory responses through phagocytosis and secretion of cytokines, which absorb and digest foreign proteins such as cell tissue, foreign substances, microorganisms, and cancer cells. In addition, after removing the antigen, it acts as an antigen-presenting cell that delivers the antigen to lymphocytes, thereby inducing an immune response.
  • the immune cells can be obtained from an individual or from various tissues. Additionally, the immune cells include unmodified immune cells obtained from an individual and may include immature immune cells as well as mature immune cells.
  • the immune cells can be obtained by separating them from tissues, hematopoietic cells, hematopoietic stem cells, or hematopoietic progenitor cells.
  • the immune cells may include, but are not limited to, hematopoietic cells, hematopoietic stems or precursors from placental tissue, placental perfusate, umbilical cord blood, placental blood, peripheral blood, spleen, liver, etc.
  • the immune cells may be autologous or foreign cells.
  • the immune cells may be derived from mammals such as rats, mice, livestock, and humans.
  • the immune cells may be obtained from an individual seeking treatment.
  • the immune cells can be directly isolated from the blood of the individual and used, or they can be used by differentiating immature undifferentiated (or immature) immune cells or stem cells obtained from the individual.
  • the immune cells may be differentiated from iPSCs (Induced pluripotent stem cells).
  • Methods for introducing the vector or virus into immune cells can be methods known in the art, such as transient transfection, microinjection, transduction, cell fusion, and calcium phosphate precipitation.
  • Method, Liposome-mediated transfection, DEAE Dextran-mediated transfection, Polybrene-mediated transfection, electroporation can be introduced into cells by a gene gun or other known methods for introducing nucleic acids into cells (Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988). However, it is not limited to this.
  • Transduced or transfected immune cells can be propagated ex vivo following vector or viral infection.
  • the transfected immune cells are allowed to proliferate for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, It can be cultured for 13, 14, 15, 16, 17, 18, 19 or 20 days, preferably for 13 to 15 days.
  • Methods for confirming whether the vector has been successfully introduced into the immune cells include, for example, molecular biological assays (e.g., Southern and Northern blotting, RT-PCR and PCR) well known to those skilled in the art; It may include detection of the presence or absence of a particular peptide by biochemical assays (e.g., immunological methods (e.g., ELISAs, Western blots, flow cytometry)).
  • biochemical assays e.g., immunological methods (e.g., ELISAs, Western blots, flow cytometry)).
  • Another aspect of the present invention includes the steps of i) preparing a virus containing the polynucleotide; and ii) processing the virus into immune cells. It provides a method for producing immune cells expressing the fusion protein.
  • the polynucleotide, virus containing the polynucleotide, immune cell, and fusion protein are the same as described above.
  • Methods for producing the virus may use methods known in the art. Additionally, immune cells transfected with the virus can be proliferated by further culturing after infection.
  • culture used in the present invention refers to growing cells in appropriately controlled environmental conditions, and the culture process of the present invention can be carried out according to appropriate media and culture conditions known in the art. This culture process can be easily adjusted by a person skilled in the art depending on the cells selected.
  • the medium refers to a known medium used for culturing cells, and includes all known media for cell culture or modified media thereof.
  • immune cells transfected with the above virus persist for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 days after infection.
  • the production of immune cells expressing the fusion protein can be confirmed by confirming the expression of the fusion protein through methods such as molecular biological assays and biochemical assays well known to those skilled in the art.
  • Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising immune cells expressing the fusion protein as an active ingredient.
  • the pharmaceutical composition may further include an anti-CD137 antibody or a bispecific antibody.
  • the fusion protein and immune cells are the same as described above.
  • the above cancers include stomach cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, pancreas cancer, cervical cancer, thyroid cancer, laryngeal cancer, leukemia, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, and salivary gland cancer.
  • lymphoma, kidney cancer, melanoma multiple myeloma, brain cancer, osteosarcoma, glioblastoma, IgG-opsonized tumor, lymphoma, neuroma, mesothelioma, and esophageal cancer.
  • the term “antibody” refers to an immunoglobulin molecule that reacts immunologically with a specific antigen, and refers to a protein molecule that specifically recognizes the antigen.
  • the heavy and light chains of immunoglobulins may each include a constant region and a variable region.
  • the light and heavy chain variable regions of immunoglobulins include three variable regions called complementarity determining regions (CDRs) and four framework regions (FRs).
  • CDR complementarity determining regions
  • FRs framework regions
  • the antibody may refer to any one selected from IgG, IgE, IgM, IgD, and IgA, and may be a subclass of IgG, such as IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. Additionally, the antibody may be an agonistic antibody or an antagonistic antibody.
  • the antibodies include whole antibodies, monoclonal antibodies, polyclonal antibodies, single domain antibodies, single chain antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, intrabodies, scFvs, and Fab fragments. , F(ab') fragment, Fv(sdFv) linked by a disulfide bond, and any of the above epitope binding fragments, and is not limited thereto, as long as it specifically binds to the cancer antigen.
  • the term “dual specific antibody” refers to a substance that binds to at least one target or antigen.
  • the bispecific antibody includes a first domain that specifically binds to a tumor antigen; and a second domain that specifically binds to the extracellular domain of CD137.
  • the bispecific antibody may be a fragment of an anti-CD137 antibody, for example, scFv, bound to an antibody that specifically binds to an antigen.
  • the scFv may be bound to the C terminus of an antibody that specifically binds to an antigen, and at this time, the antibody and scFv may be bound through a peptide linker.
  • the peptide linker may be (G4S)n.
  • the linker of the peptide may be (G4S) 4 .
  • the bispecific antibody may be an engager.
  • the bispecific antibody may be in the form of BiTE (Bi-specific T-cell engager).
  • the first domain may be an antibody or scFv that specifically binds to a cancer antigen
  • the second domain may be an scFv that specifically binds to CD137.
  • the first domain and the second domain may be linked through a peptide linker.
  • the cancer antigen is alpha folate receptor, 5T4, ⁇ v ⁇ 6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a.
  • EGFR EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FR ⁇ , GD2, GD3, glypican -3(GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY- ESO-1, IL-11R ⁇ , IL-13R ⁇ 2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survi It may be any one selected from the group consisting of bin, TAG72, TEMs, VEGFR2, and W
  • the extracellular domain binding site of CD137 which is the second domain of the bispecific antibody, can serve as an agonistic antibody. Therefore, the bispecific antibody can activate CD137 by specifically binding to the extracellular domain of CD137.
  • the bispecific antibody specifically binds to the cancer antigen overexpressed on the tumor surface and specifically binds to the CD137 extracellular domain of the fusion protein of the present invention, thereby inhibiting tumor cells and the fusion protein of the present invention. It can be used as a cell engager to bind the expressed immune cells.
  • the bispecific antibody By using the bispecific antibody as a cell binder, it can target cancer and exhibit safer and superior anticancer activity by activating immune cells specifically for cancer cells.
  • T cells expressing the fusion protein of the present invention are treated in combination with anti-BCMA/anti-4-1BB bispecific antibodies, it is confirmed that cytotoxicity increases compared to wild type T cells. (Figure 17).
  • prevention refers to all actions that suppress or delay the onset of a disease by administering the pharmaceutical composition.
  • treatment refers to any action that improves or beneficially changes the symptoms of a disease by administering the pharmaceutical composition.
  • immune cells expressing the fusion protein may be included in any amount (effective amount) depending on the use, formulation, purpose of formulation, etc., as long as they can show activity.
  • the pharmaceutical composition of the present invention can be used to kill about 1 ⁇ 10 2 cells to about 1 ⁇ 10 16 cells, about 1 ⁇ 10 2 cells to about 1 ⁇ 10 15 cells, about 1 ⁇ 10 2 cells to about 1 ⁇ 10 14 cells, or about 1 cell. It may include immune cells expressing the fusion protein of ⁇ 10 2 cells to about 1 ⁇ 10 13 cells.
  • the pharmaceutical composition may include about 2 ⁇ 10 2 to about 2 ⁇ 10 11 cells of immune cells expressing the fusion protein.
  • effective amount refers to the amount of active ingredient that can induce an anti-aging effect. Such effective amounts can be determined experimentally within the scope of the ordinary ability of those skilled in the art.
  • Compositions of the present invention can be used unfrozen or frozen for later use. If freezing is required, use standard cryopreservation agents (e.g. DMSO, glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, id erythritol, D-ribitol, D-mannitol, D- Sorbitol, i-inositol, D-lactose, choline chloride, and Epilife ® cell freezing medium (Cascade biologics) can be added to the cells before freezing.
  • cryopreservation agents e.g. DMSO, glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, id erythritol, D-ribitol, D-mannitol, D- Sorbitol, i-inositol, D-
  • composition of the present invention may be formulated to further include a pharmaceutically acceptable carrier.
  • the term “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not significantly irritate living organisms and does not inhibit the biological activity and properties of the administered ingredient.
  • the pharmaceutical composition of the present invention can be applied as a cell therapy agent, and pharmaceutically acceptable carriers that can be included as a cell therapy agent include buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, bases, excipients, lubricants, etc. If it is known in the industry, it can be used without restrictions.
  • the pharmaceutical composition of the present invention can be prepared according to commonly used techniques in the form of various dosage forms.
  • the pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount, and can be administered through any route as long as it can induce movement to the diseased area.
  • administration means introducing a predetermined substance into an individual by an appropriate method.
  • loading the pharmaceutical composition of the present invention into a vehicle equipped with a means to direct it to the lesion may be considered.
  • it may be parenteral administration.
  • parenteral administration may include subcutaneous, intradermal, intramuscular, instillation, intravenous, intraarterial, intraarticular, and intracerebrospinal fluid administration.
  • the preferred administration method and formulation of the pharmaceutical composition of the present invention is injection.
  • Injections include aqueous solvents such as physiological saline solution, Ringer's solution, Hank's solution, or sterilized aqueous solution, vegetable oils such as olive oil, higher fatty acid esters such as ethyl oleic acid, and non-aqueous solvents such as ethanol, benzyl alcohol, propylene glycol, polyethylene glycol, or glycerin.
  • aqueous solvents such as physiological saline solution, Ringer's solution, Hank's solution, or sterilized aqueous solution
  • vegetable oils such as olive oil
  • higher fatty acid esters such as ethyl oleic acid
  • non-aqueous solvents such as ethanol, benzyl alcohol, propylene glycol, polyethylene glycol, or glycerin.
  • impermeable agents known in the art suitable for the barrier to pass through can be used, and as a stabilizer to prevent deterioration, ascorbic acid, sodium bisulfite, BHA, tocopherol, EDTA It may additionally contain pharmaceutical carriers such as emulsifiers, buffers for pH adjustment, and preservatives to inhibit microbial growth such as phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, and benzyl alcohol.
  • pharmaceutical carriers such as emulsifiers, buffers for pH adjustment, and preservatives to inhibit microbial growth such as phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, and benzyl alcohol.
  • the term “pharmaceutically effective amount” refers to an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is the patient's Factors including gender, age, weight, health status, type of disease, severity, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration and excretion rate, treatment period, drugs combined or used simultaneously, and other factors. It can be readily determined by a person skilled in the art according to factors well known in the medical field.
  • the preferred dosage of the pharmaceutical composition of the present invention is about 1 ⁇ 10 cells/kg to about 1 ⁇ 10 12 cells/kg per day, based on cells, depending on the patient's condition, weight, gender, age, patient's severity, and route of administration. It may be about 1 ⁇ 10 cells/kg to about 1 ⁇ 10 11 cells/kg or about 1 ⁇ 10 cells/kg to about 1 ⁇ 10 10 cells/kg.
  • the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and those skilled in the art will Taking these factors into consideration, the dosage can be adjusted appropriately.
  • the number of administrations can be once or twice or more within the range of clinically acceptable side effects.
  • the pharmaceutical composition of the present invention may be administered 1 to 3 times/day, 1 to 3 times/week, or 1 to 10 times/month.
  • the pharmaceutical composition of the present invention can be used 1 to 9 times/month, 1 to 8 times/month, 1 to 7 times/month, 1 to 6 times/month, 1 to 5 times/month, 1 It may be administered once to four times/month, once to three times/month, or once to twice per month.
  • the administration site it can be administered at one or two or more locations.
  • the dosage per kg or per individual is the same as that for humans, or, for example, the above-mentioned administration is based on the volume ratio (e.g., average value) of the organs (e.g., heart, etc.) between the target animal and human.
  • the converted dose can be administered.
  • the term "individual” used herein refers to a subject who has or may develop a disease to which the composition of the present invention can be applied (prescribed), and may be a mammal such as a rat, mouse, or livestock, including humans. . Preferably, it may be a human, but is not limited thereto.
  • the pharmaceutical composition of the present invention may additionally contain known substances that exhibit a preventive or therapeutic effect on the above diseases.
  • the pharmaceutical composition of the present invention may further include a substance that increases the activity of immune cells expressing the fusion protein.
  • the pharmaceutical composition of the present invention can be administered in combination with conventionally known substances for preventing or treating the above diseases.
  • the immune cells expressing the fusion protein, substances showing a preventive or therapeutic effect for the disease, and/or substances that increase the activity of the immune cells can be administered simultaneously or sequentially.
  • Another aspect of the present invention provides the use of immune cells expressing the fusion protein or a pharmaceutical composition containing the same for preventing or treating cancer.
  • Another aspect of the present invention provides a method for preventing or treating cancer, comprising administering to an individual an immune cell expressing the fusion protein or a pharmaceutical composition containing the same.
  • Immune cells expressing the fusion protein, pharmaceutical composition, cancer, prevention, treatment, subject, and administration are the same as described above.
  • Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an immune cell expressing the fusion protein and a dual-specific antibody as active ingredients.
  • kits for preventing or treating cancer including an immune cell expressing the fusion protein and a dual-specific antibody.
  • Immune cells and dual-specific antibodies expressing the fusion protein contained in the pharmaceutical composition and kit may be included in one formulation and may be administered in combination. At this time, the immune cells expressing the fusion protein and the dual-specific antibody may be administered simultaneously or sequentially.
  • Immune cells expressing the fusion protein, bispecific antibodies, cancer, prevention and treatment are the same as described above.
  • CD137 extracellular domain variants thereof or fragments thereof
  • CD137 extracellular domain variants or fragments thereof may include a mutation in any one selected from the group consisting of CRD1, CRD2, CRD3, CRD4, and combinations thereof.
  • the variant may include mutations in CRD2 and/or CRD3.
  • the mutations of CRD2 and CRD3 are the same as described above.
  • the CD137 extracellular domain variant or fragment thereof may include or consist of any one amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 27.
  • Antibody information used in the following experimental examples is shown in Table 1 below.
  • Fluorescently labeled antibodies of each FITC, PE, APC, BV421, BV605, and BV711 wavelength were used to stain NK cells. Specifically, 1.0 x 10 5 NK cells were harvested and washed once with Flow Cytometry Staining Buffer (eBioscience, 00-4222-26). Reaction was performed in a refrigerator at 4°C, shielded from light for 30 minutes, and analyzed using flow cytometry.
  • the transgene containing the 4-1BB and CD3 ⁇ domains was inserted into the pVAX1 vector using EcoRI and XhoI.
  • the vector was converted into a linear template strand using XbaI (NEB, R0145S) for mRNA production.
  • in vitro transcription was performed using the mMESSAGE mMACHINE TM T7 ULTRA Transcription Kit (Invitrogen, AM1345).
  • BBz Specific amino acid sequences and nucleic acid sequences are shown in Table 2.
  • Example 1.2 Isolation and proliferation of PB-NK cells
  • PBMC Peripheral Blood Mononuclear Cell
  • ficoll-Paque PLUS Cytiva, 17144002
  • Miltenyi Biotec's MACS Magnetically activated cell sorting
  • NK cells were electroporated using the Neon ® Transfection system (ThermoFisher) and MaxCyte's GTx.
  • NK cells were suspended at 5.0x10 6 cells/100 ⁇ L with Buffer T (Invitrogen, MPK10096). 2.5 ⁇ g of mRNA was added to the cell suspension, and electroporation was performed at 1850 V, 20 ms, 1 purse. GTx followed the manufacturer's instructions, and electroporation was performed with the code set to 'Expanded NK-5'.
  • a cell line expressing 4-1BB was isolated using anti-PE MicroBeads from Miltenyi Biotec (MiltenyiBiotec, 130-048-801).
  • Signal sequence (SEQ ID NO: 1), extracellular domain of CD137 (4-1BB) (SEQ ID NO: 15) or a variant thereof (SEQ ID NO: 16, 17 or 27), linker (SEQ ID NO: 11), transmembrane domain (SEQ ID NO: 8 ), a polynucleotide (SEQ ID NO: 12, 13, 14) encoding a fusion protein (SEQ ID NO: 18, 19, 20 or 31) containing a co-stimulatory domain (SEQ ID NO: 9) and a primary signaling domain (SEQ ID NO: 10) or 30) was synthesized and loaded into the pCDH-EF1-MCS-IRES-Puro vector (SBI, CD532A-2). The amino acid sequences of the fusion proteins are shown in Tables 3 to 6.
  • the fusion protein according to the present invention was named “cBBR (Chimeric 4-1BB receptor)”.
  • Fusion protein domain amino acid sequence sequence number cBBR (SEQ ID NO: 18) Signal peptide MGNSCYNIVATLLVLNFERTRS One CRD1 LQDPCSNCPAGTFCDNNRNQICS 2 CRD2 PCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAEC 3 CRD3 DCTPGFHCLGAGCSMCEQDCKQGQELTKKGCK 6 CRD4 DCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCG 7 linker PSPADLSPGASSVTPPAPAREPGHSPQ 11 Transmembrane (TM) IISFFLALTSTALLFLLFFLTLRFSVV 8 co-stimulatory domain KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 9 CD3z signal domain RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI
  • Fusion protein domain amino acid sequence sequence number cBBR_I64R (SEQ ID NO: 19) Signal peptide MGNSCYNIVATLLVLNFERTRS One CRD1 LQDPCSNCPAGTFCDNNRNQICS 2 CRD2 mutant PCPPNSFSSAGGQRTCD R CRQCKGVFRTRKECSSTSNAEC 4 CRD3 DCTPGFHCLGAGCSMCEQDCKQGQELTKKGCK 6 CRD4 DCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCG 7 linker PSPADLSPGASSVTPPAPAREPGHSPQ 11 Transmembrane (TM) IISFFLALTSTALLFLLFFLTLRFSVV 8 co-stimulatory domain KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 9 CD3z signal domain RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK
  • Fusion protein domain amino acid sequence sequence number cBBR_V71R (SEQ ID NO: 20) Signal peptide MGNSCYNIVATLLVLNFERTRS One CRD1 LQDPCSNCPAGTFCDNNRNQICS 2 CRD2 mutant PCPPNSFSSAGGQRTCDICRQCKG R FRTRKECSSTSNAEC 5 CRD3 DCTPGFHCLGAGCSMCEQDCKQGQELTKKGCK 6 CRD4 DCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCG 7 linker PSPADLSPGASSVTPPAPAREPGHSPQ 11 Transmembrane (TM) IISFFLALTSTALLFLLFFLTLRFSVV 8 co-stimulatory domain KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 9 CD3z signal domain RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK
  • Fusion protein domain amino acid sequence sequence number cBBR_ I64R/V71R (SEQ ID NO: 31) Signal peptide MGNSCYNIVATLLVLNFERTRS One CRD1 LQDPCSNCPAGTFCDNNRNQICS 2 CRD2 mutant PCPPNSFSSAGGQRTCD R CRQCKG R FRTRKECSSTSNAEC 26 CRD3 DCTPGFHCLGAGCSMCEQDCKQGQELTKKGCK 6 CRD4 DCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCG 7 linker PSPADLSPGASSVTPPAPAREPGHSPQ 11 Transmembrane (TM) IISFFLALTSTALLFLLFFLTLRFSVV 8 co-stimulatory domain KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 9 CD3z signal domain RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG
  • the 4-1BBL expression vector was constructed by synthesizing the polynucleotide of SEQ ID NO: 25 and loading it into the pCDH-EF1-MCS-IRES-Puro vector (SBI, CD532A-2).
  • pCDH-EF1-MCS-IRES-Puro vector loaded with polynucleotides encoding VSV-G, Rev, and cBBR or variants thereof (SEQ ID NO: 12, 13, 14 or 30) or a polynucleotide encoding 4-1BBL The pCDH-EF1-MCS-IRES-Puro vector loaded with SEQ ID NO: 25) was transduced into Lenti-X 293T cells (Clontech, 632180) using Lipofectamine 2000 (Thermo Fisher, 11668019).
  • each supernatant was harvested, mixed with Lenti-X Concentrator (Clontech, 631232) in a 1:3 ratio, and stored in the refrigerator for more than 1 hour.
  • Lenti-X Concentrator (Clontech, 631232) in a 1:3 ratio, and stored in the refrigerator for more than 1 hour.
  • Each of the above mixed solutions was centrifuged at 1,500g at 4°C, and the pellet was collected and used as virus particles capable of expressing cBBR or a variant thereof and virus particles expressing the 4-1BBL gene, respectively.
  • K562 cells and Nalm6 cells were purchased from the European collection of authenticated cell cultures (ECACC), and Jurkat T cells and Ramos cells were purchased from the Korean Cell Line Bank.
  • ECACC European collection of authenticated cell cultures
  • Various cBBR Jurkat T cells and 4-1BBL Nalm6 cells were transduced using the lentivirus prepared in Example 2.2 above. All cells were cultured using RPMI-1640 (Hyclone, SH30027.01) medium containing 10% FBS (Gibco, 10100147).
  • PBMC Peripheral Blood Mononuclear Cell
  • Ficoll Paque PLUS Cytiva, 17144002
  • T cells in PBMC were isolated using CD4+ T cell isolation kit, human (Miltenyi biotec, 130-096-533) and CD8+ T cell isolation kit, human (Miltenyi biotec, 130-096-495).
  • T cells isolated as above were activated by adding T Cell TransAct, human (Miltenyi biotec, 130-111-160).
  • Activated T cells were cultured using RPMI-1640 medium containing 10% FBS and 300 U/mL Proleukin IL-2 (NOVARTIS).
  • cBBR T cells The day after inducing activation, cBBR or its variants (I64R, V71R) were transduced into T cells using the lentivirus prepared in Example 2.2, and cultured for up to 14 days.
  • the cells prepared as above are hereinafter referred to as “cBBR T cells.”
  • Lymphoblast patient-derived NK cells NK92MI were supplemented with 12.5% FBS (fetal bovine serum, Gibco, 10100147), 12.5% horse serum (Gibco, 16050122), and 1% penicillin/streptomycin ( Gibco, 15140-122), alpha MEM (Cytiva, containing 0.2mM inositol (DAEJUNG, 5008-4105), 0.1mM 2-mercaptoethanol (Gibco, 21985023), and 0.02mM folic acid (Sigma Aldrich, F8758).
  • SH30265.01 was cultured in a CO 2 incubator using medium.
  • cBBR NK92MI cells 4-1BB expressing NK92MI cells.
  • a cell line was developed to determine whether NK92MI cells expressing 4-1BB are activated by the monoclonal antibody urelumab (Figure 2). Titration evaluation was performed to confirm the appropriate concentration of urelumab ( Figures 3 and 4). Then, in order to confirm the IFN- ⁇ produced by NK cells, the concentration of IFN- ⁇ was measured in the NK cell culture medium using the Human IFN-gamma Quantikine ELISA kit (R&D systems, DIF50C).
  • each cBBR Jurkat T cell, 4-1BBL Nalm6 cell, and cBBR T cell (1.0 ⁇ 10 5 cells) were washed once with Flow Cytometry Staining Buffer (eBioscience, 00-4222-26), and then incubated with each antibody. was treated and reacted at 4°C, shielded from light for 30 minutes. After the reaction was completed, the stained cells were analyzed using flow cytometry. At this time, the antibodies used are shown in Table 7.
  • cBBR Jurkat T cells were stained with 1 ⁇ M CellTracker Deep Red Dye (Invitrogen, C34565), and 4-1BBL Nalm6 cell line was stained with 5 ⁇ M CellTrace CFSE Cell Proliferation Kit (Invitrogen, C34554).
  • the two types of stained cells were co-cultured for 30 minutes and then fixed by adding 4% paraformaldehyde solution (Biosesang, PC2031-100-00).
  • the fixed cells were analyzed using flow cytometry (% of CFSE+ 7-AAD+ cells).
  • cBBR T cells and 4-1BBL Nalm6 cells were co-cultured to measure the cytotoxicity of cBBR T cells against cancer cells.
  • cBBR T cells were toxicity of cBBR T cells using the Cytotoxicity Assay Kit (Abcam, ab270780). 4-1BBL Nalm6 cells were stained with the CFSE reagent included in the kit, mixed with cBBR T cells at a ratio of 0.5:1, 1:1, and 2:1, respectively, and co-cultured for 24 hours. At this time, Nalm6, in which 4-1BBL was not transduced, was used as a control. After co-culture, the cells were stained with 7-AAD and dead cells were confirmed through flow cytometry (% of CFSE+ 7-AAD+ cells).
  • cBBR T cells (including mutants) exhibited cytotoxicity against 4-1BBL Nalm6 cells due to the binding of the 4-1BB extracellular domain and 4-1BBL.
  • cytotoxicity was reduced in cBBR T cells (I64R, V71R, I64R/V71R) in which some amino acids of cBBR were substituted (mutants). The above results are believed to be due to a decrease in binding force between the CD137 (4-1BB) extracellular domain of the cBBR variant and 4-1BBL.
  • a 96-well plate was coated with urelumab (MedChemExpress, HY-P99055) (1 ⁇ g/mL, 10 ⁇ g/mL), and then cBBR T cells (I64R, V71R, I64R/V71R) prepared by the method of Example 2 above were added. were inoculated at a concentration of 5 ⁇ 10 5 cell/mL and reacted for 24 hours. After the reaction was completed, the cells were stained with anti-CD8a antibody (APC) and anti-CD107a antibody (PE), respectively, and analyzed using flow cytometry. As a result, as shown in Figure 15, the binding affinity of cBBR T cells with amino acid substitutions (mutants) to 4-1BBL decreased. However, it was confirmed that the activity caused by Urelumab, an anti-CD137 antibody (agonist), was not reduced.
  • urelumab MedChemExpress, HY-P99055
  • cBBR T cells (I64R, V71R, I64R/V71R) produced by the method of Example 2 were treated with urelumab at a concentration of 1 ⁇ g/mL or 10 ⁇ g/mL, and then incubated for 24 hours to induce activation. After the reaction, the concentration of IFN- ⁇ secreted from activated cBBR T cells into the culture medium was measured using the Human IFN-gamma Quantikine ELISA Kit (R&D systems, SIF50C). As a result, as shown in Figure 16, the binding affinity of cBBR T cells with amino acid substitutions (mutants) to 4-1BBL decreased. However, it was confirmed that the activity caused by Urelumab, an anti-CD137 antibody (agonist), was not reduced.
  • the bispecific antibody used in the present invention was produced by Genescript Probio.
  • the amino acid sequence of the bispecific antibody is shown in Table 8 below.
  • the bispecific antibody is an anti-BCMA antibody, and an scFv that binds to 4-1BB is bound to the C terminus.
  • each cBBR T cell Control, I64R, V71R
  • Ramos cell 1.0 ⁇ 10 5 cells
  • Flow cytometry staining buffer eBioscience, 00-4222-26
  • the bispecific antibody prepared by the method was treated and reacted at 4°C for 30 minutes, shielded from light. After the reaction was completed, the stained cells were analyzed using flow cytometry.
  • cBBR T cells In order to confirm the killing ability of cBBR T cells whose activation was induced by a bispecific antibody (BiTE) against cancer cells, cBBR T cells (I64R, V71R) were treated with a bispecific antibody, and then cancer cells were killed using the method of Experimental Example 3. Cytotoxicity was measured. At this time, the bispecific antibody was treated at a concentration of 1 or 10 ⁇ g/mL for 24 hours.

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Abstract

Des lymphocytes T cBBR surexprimant une protéine de fusion 4-1BB/CD3ζ (cBBR) ou un variant de celle-ci détruisent de manière spécifique les cellules cancéreuses surexprimant 4-1BBL. Par comparaison avec des lymphocytes T cBBR comprenant un domaine extracellulaire de 4-1BB de forme native, les lymphocytes T surexprimant cBBR comprenant un variant de domaine extracellulaire de 4-1BB ont une liaison inférieure à 4-1BBL et une cytotoxicité. Les lymphocytes T cBBR surexprimant cBBR stimulés par Urelumab comprenant le variant ont une activité remarquablement meilleure que les lymphocytes T normaux. En outre, les lymphocytes T qui sont mis à réagir avec un anticorps bispécifique anti-BCMA/anti-4-1BB, ont une activité destructrice remarquablement meilleure, sur des cellules cancéreuses surexprimant BCMA, que les lymphocytes T normaux. Par conséquent, des lymphocytes T surexprimant la protéine de fusion cBBR peuvent être utilisés en tant qu'agent anticancéreux. De plus, il a été identifié que des cellules NK surexprimant une protéine de fusion cBBR sont activées de manière efficace par un anticorps anti-4-1BB, ces cellules peuvent ainsi être utilisées en tant qu'agent anticancéreux.
PCT/KR2023/019470 2022-11-29 2023-11-29 Protéine de fusion comprenant un domaine extracellulaire cd137, cellules immunitaires l'exprimant et utilisation associée Ceased WO2024117781A1 (fr)

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EP3636320A1 (fr) * 2018-10-09 2020-04-15 Numab Therapeutics AG Anticorps dirigés contre cd137 et leurs procédés d'utilisation
KR102426765B1 (ko) * 2016-04-22 2022-07-29 엘리게이터 바이오사이언스 에이비 Cd137에 대한 신규한 이중특이성 폴리펩타이드

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US20200009190A1 (en) * 2017-03-17 2020-01-09 Fred Hutchinson Cancer Research Center Immunomodulatory fusion proteins and uses thereof
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