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WO2021245580A1 - Récepteur antigénique chimérique comprenant un variant du domaine 4 de pa63 en tant que domaine de liaison extracellulaire, et son utilisation - Google Patents

Récepteur antigénique chimérique comprenant un variant du domaine 4 de pa63 en tant que domaine de liaison extracellulaire, et son utilisation Download PDF

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Publication number
WO2021245580A1
WO2021245580A1 PCT/IB2021/054846 IB2021054846W WO2021245580A1 WO 2021245580 A1 WO2021245580 A1 WO 2021245580A1 IB 2021054846 W IB2021054846 W IB 2021054846W WO 2021245580 A1 WO2021245580 A1 WO 2021245580A1
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cancer
cells
domain
chimeric antigen
antigen receptor
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Korean (ko)
Inventor
이백승
이윤
이영관
전승현
이화진
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Mjcell Bio Co Ltd
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Mjcell Bio Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/54—Pancreas

Definitions

  • the present invention relates to an ANTXR (Anthrax toxin receptor) known to be overexpressed in various cancer cells; Anthrax toxin receptor) relates to a chimeric antigen receptor that optimizes safety for normal cells while maintaining anticancer efficacy by controlling the binding affinity of the chimeric antigen receptor that recognizes and binds antigen, ANTXRKanthrax toxin receptor 1) or ANTXR2 ( anthrax toxin receptor 2) relates to a ligand-based chimeric antigen receptor comprising a mutant of PA63 domain 4 that specifically binds to a cancer antigen, immune cells comprising the same, and uses thereof. background
  • Anthrax toxin receptor (ANTXR) antigens include ANTXR1 and ANTXR2, and anthrax toxin secreted by anthrax is known as a ligand for recognizing these antigens.
  • Anthrax toxin is secreted by Gram-positive bacteria, Bacillus anthracis 6 ) ) and Edema factor [EF, 89 kDa] (Morton, N. , New Engl. J. Med. 345: 1621-1626 [2001]).
  • PA protective antigen
  • LF lethal factor
  • ANTXR1 is an extracellular matrix protein, published in Brad St. It is known as TEM8 (Tumor endothelial marker 8) identified in colorectal cancer by Croix (Sciences 289:1197 [2000]). ANTXR1 is a type I transmembrane protein expressed in the endothelium of various tumors, and has been found to be a gene whose expression is increased during tumor angiogenesis (St. Croix et al., Sciences, 289).
  • ANTXR1 acts as a receptor for extracellular ligand (extracellular ligand), it has become a target antigen for the treatment of angiogenesis (Car son-Walter, EB et al., Cancer Res 61:6649 [2001]) ).
  • ANTXR2 Another name for ANTXR2 is CMG2 (capi 1 lary morphogenesis gene 2), and ANTXR2 is also known to be involved in angiogenesis. In addition, ANTXR2 is known to be involved in adhesion and migration of various types of cells, including epithelial cells and endothelial cells (Lin Ye et al., Int J Oncol 45: 1565-1573 [2014]).
  • CMG2 capi 1 lary morphogenesis gene 2
  • ANTXR2 is also known to be involved in angiogenesis.
  • ANTXR2 is known to be involved in adhesion and migration of various types of cells, including epithelial cells and endothelial cells (Lin Ye et al., Int J Oncol 45: 1565-1573 [2014]).
  • CAR-T immune cell therapy which has been booming in R&D by many global pharmaceutical companies, starting with FDA approval in 2017, is drawing attention from around the world as a genetically modified cell therapy.
  • a chimeric antigen receptor (CAR) gene having specificity as a surface antigen of cancer cells and anticancer activity (effectiveness) of cells is transduced into immune T cells or NK cells, and these chimeric antigen receptors are expressed
  • It is an anti-cancer immunotherapy (gene therapy) method in which T or NK cells are proliferated outside the body and administered to the patient for treatment.
  • This treatment has excellent anti-cancer effects compared to antibody drugs with a single administration and shows long-lasting results, so there is a lot of anticipation for anti-cancer efficacy in clinical practice.
  • the present inventors specifically recognize / bind to ANTXR, based on the CAR containing the PA63 domain 4, while maintaining the existing anti-cancer efficacy, differentiation / optimizing safety to minimize side effects on normal cells
  • the affinity was controlled by mutation of the amino acid of PA63 domain 4 (D4), which is a ligand site binding to the antigen, and the cancer treatment effect was off while maintaining
  • D4 the amino acid of PA63 domain 4
  • An object of the present invention is to provide a chimeric antigen receptor (CAR) with optimized safety while maintaining efficacy as an immune cell therapy agent by developing CARs having various binding affinities.
  • Another object of the present invention is a nucleic acid encoding the chimeric antigen receptor, 2021/245580 1» (: 1' year 2021/054846 To provide an expression vector containing a nucleic acid and a virus containing the expression vector.
  • Another object of the present invention is a composition for preventing or treating cancer comprising the immune cells, a method for preventing or treating cancer comprising administering the immune cells, the use of the immune cells for preventing or treating cancer, and cancer prevention or to provide the use of the immune cells for the manufacture of a therapeutic agent.
  • the present invention provides an extracellular binding domain containing a variant of PA63 ligand domain 4, a transmembrane domain, and an intracellular signaling domain comprising a It provides a chimeric antigen receptor (CAR).
  • the present invention also provides a nucleic acid encoding the chimeric antigen receptor, an expression vector containing the nucleic acid, a virus containing the expression vector, and immune cells expressing the chimeric antigen receptor.
  • the present invention also provides a composition for preventing or treating cancer comprising the immune cells, a method for preventing or treating cancer comprising administering the immune cells, the use of the immune cells for preventing or treating cancer, and preventing cancer or Provided is the use of said immune cells for the manufacture of a medicament for treatment.
  • 1 is melanoma (526-mel), pancreatic cancer (PANC-1, Mia_PaCa2), breast cancer (SK-BR3, 2021/245580 1»(:1'year2021/054846
  • FIG. 3 is a flow cytometry analysis of RFP fluorescent protein fused with each antigen using a flow cytometer in a chemochromatosis (526, el) cell line that simultaneously expresses ANTXRKTEM8) antigen protein or ANTXR2 (CMG2) antigen protein and RFP (Red Fluorescence Protein) fluorescent protein This is the result of confirming the expression.
  • Figure 4 is a picture expressing the position of the mutation of PA63 domain 4 (D4) wil type CAR and D4 specific to ANTXRKTEM8) or ANTXR2 (CMG2) according to the present invention and a representative plasmid DNA vector map.
  • the CAR of the present invention is an extracellular binding domain, a fragment containing D4 or D4 mutant essential for receptor binding of PA63 ligand, a transmembrane domain derived from CD8 a, a hinge region, a primary signaling domain of the CD3 chain, (3) 28 and CD137 costimulatory signaling domains.
  • 5 is a graph comparing cytotoxicity to ANTXR TEM8) and ANTXR2 (CMG2) overexpressing cell lines of D4 wild and D4 mutant #1 - #6 CAR-T cells prepared using PBMC.
  • Figure 6 shows the results of analyzing the anticancer activity against ANTXR TEM8) and ANTXR2 (CMG2) overexpressing cell lines of D4 wi ld and D4 mutant #1 - #6 CAR-T cells prepared using PBMC through IFN-gamma ELISA. This is the graph shown. 7 shows D4 wi ld and D4 prepared using PBMCs of 4 healthy donors. 2021/245580 1» (:1' year 2021/054846 mutant #4 and #6 This is a graph showing the results of analyzing the in vitro anticancer activity of CAR-T cells against ANTXR1 (TEM8) and ANTXR2 (CMG2).
  • a chimeric antigen receptor comprising PA63 ligand domain 4 specifically binding to ANTXRKTEM8) antigen and ANTXR2 (CMG2) antigen expressed in cancer tissue as an extracellular binding domain was developed.
  • anticancer activity efficiency
  • cancer cell specificity is increased and attack power against normal cells is increased
  • off-target toxicity was lowered, and it was confirmed that it can be maintained for a long time in the body by increasing the formation of memory T cells.
  • the present invention provides an extracellular binding domain containing a variant of PA63 ligand domain 4 (extracellular binding domain), a transmembrane domain and It relates to a chimeric antigen receptor (CAR) comprising an intracellular signaling domain.
  • extracellular binding domain containing a variant of PA63 ligand domain 4 (extracellular binding domain), a transmembrane domain and It relates to a chimeric antigen receptor (CAR) comprising an intracellular signaling domain.
  • CAR chimeric antigen receptor
  • CAR Chimeric antigen receptor
  • the first-generation CAR an extracellular domain including an antigen recognition site specifically expressed in cancer cells, a transmembrane domain, and an intracellular signaling domain were used, and only (3) 3 ⁇ 4 was used as the signaling domain, but the therapeutic effect on cancer was insignificant, and there was a problem that the duration was short.
  • This first-generation CAR is specifically described in US Patent No. 6,319,494, which is incorporated herein by reference.
  • a costimulatory domain (CD28 or 2021/245580 1»(:1' year2021/054846
  • a second-generation CAR combining (3) 137/4-1BB) and (3) 3 ⁇ 4 was prepared. Compared with the first-generation CAR, the number of CAR-containing immune cells remaining in the body significantly increased.
  • the second-generation CAR used one co-stimulatory domain, whereas the third-generation CAR used two or more co-stimulatory domains.
  • a costimulatory domain can be combined with 4-1BB, CD28 or 0X40, and the like.
  • the second-generation CAR is specifically described in U.S. Patent Nos. 7,741,465, 7,446, 190 or 9,212,229, and the third-generation CAR is described in U.S. Patent No. 8,822,647 No., which is incorporated herein by reference.
  • the CAR-based immune protein of the cytokine can be further expressed, and the 5th generation CAR is an interleukin for strengthening immune cells It further comprises a receptor chain, for example, IL-2Rp.
  • 4th generation CAR is US Patent No. 10,316, 102,
  • extracellular binding domain of the present invention means a portion of the CAR having the ability to specifically bind to a target antigen of interest (antigen binding domain; antiigen binding domain) .
  • the extracellular binding domain is any protein that has the ability to specifically recognize and bind to a biological molecule (eg, a cell surface receptor or tumor protein, lipid, polysaccharide, or other cell surface target molecule, or a component thereof); Polypeptide, oligopeptide or 2021/245580 1» (: 1' year 2021/054846 peptide.
  • a binding domain includes any naturally occurring, synthetic, semisynthetic or recombinantly produced binding partner for a biological molecule of interest.
  • the term "specifically binds" refers to the binding of a molecule to another molecule with a greater binding affinity than background binding.
  • the extracellular binding domain binds to or associates with the target molecule with an affinity of about 10 _ 5 M or more or Ka (ie, the equilibrium dissociation constant of a specific binding interaction having a unit of 1/M). binds specifically to Alternatively, affinity may be defined as the equilibrium dissociation constant (Kd) of a specific binding interaction with units of (eg, 10 5 M to 10, or less).
  • the affinity of the extracellular binding domain and the CAR according to the present invention can be determined using conventional techniques, for example, competition ELISA (enzyme-linked immunosorbent assay) or labeled ligands, or using Biacore T100 (which is a piece of New Jersey). Combination using a surface-plasmon resonance device such as Cataway Biacore, Inc.) or optical biosensor technology such as EnSpire or the EPIC system available from Corning and Perkin Elmer, respectively. It can be readily determined by association or substitution analysis.
  • the PA63 ligand domain 4 may be characterized in that it recognizes ARTXRKanthrax toxin receptor 1) or ARTXR2 (anthrax toxin receptor 2).
  • the PA63 ligand domain 4 is the amino acid of SEQ ID NO: 1 2021/245580 1» (: 1' year 2021/054846 It can be characterized as represented by the sequence.
  • the variant of the PA63 ligand domain 4 may be characterized in that the affinity of the PA63 ligand domain 4 to the target is adjusted.
  • the affinity is a mutant regulated to recognize ANTXR expressed only in cancer cells, not in normal cells, but is not limited thereto.
  • the variant of the PA63 ligand domain 4 is one in which some amino acids are mutated in the PA63 ligand domain 4 represented by the amino acid sequence of SEQ ID NO: 1, preferably selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 7 It may be characterized as comprising one or more amino acid sequences, but is not limited thereto.
  • PA63 refers to a truncated 63kDa portion of protective anti-gen (PA, 83kDa), which is one of anthrax toxin proteins. Anthrax toxin is secreted by Bacillus anthracis 6 , a gram-positive bacterium.
  • the pathogenicity of anthrax is determined by its toxin-producing ability and capsular formation.
  • the proteins that affect the toxin production ability of anthrax are protective antigen (PA), edema factor (EF), and lethal factor (LF).
  • PA protective antigen
  • EF edema factor
  • LF lethal factor
  • PA is a receptor protein (Anthrax toxin) on the surface of infected cell lines such as macrophages.
  • furin family protease bound to 2021/245580 1 ⁇ (:1 ⁇ 2021/054846 receptor)
  • PA63 3 ⁇ 4 tamerized and is known to function as a toxin delivery channel that binds to LF or EF and transports them into cells.
  • the complex enters the cell through the endocytosis mechanism and when the pH of the endosome is lowered, a structural change occurs in the PA63 3 ⁇ 4 tamer and a pore is formed in the endosomal membrane.
  • Jiang J Atomic structure of anthrax protective antigen pore elucidates toxin translocation. Nature. 2015).
  • the term “ANTXRl (anthrax toxin receptor 1)” refers to an extracellular matrix protein, and another name is TEM8 (Tumor endothelial marker 8). Its specific nucleic acid sequence is known from the NCBI (NCBI Reference Sequence: NM-032208.2).
  • the term “anthrax toxin receptor 2 (ANTXR2)” refers to a protein involved in angiogenesis, and another name is CMG2 (capi1lary morphogenesis gene 2). A specific nucleic acid sequence thereof is known from NCBI (NCBI). Reference Sequence: NM_058172.5).
  • the chimeric antigen receptor may be characterized in that it comprises a transmembrane domain.
  • the term “transmembrane domain” refers to extracellular binding 2021/245580 1»(:1' year2021/054846 min and intracellular signaling domains fused and transferred to the plasma membrane of immune effector cells
  • the transmembrane domain may be derived from a natural, synthetic, semi-synthetic or recombinant source.
  • the transmembrane domain is ⁇ alpha, beta or zeta chain of the cell receptor, 0028, 003 epsilon, 0045, 004, 005, 008, 009, 0) 16, 0022, 0033, 0037, 0064, 0080, It may be characterized in that it is selected from the group consisting of 0086, 0)134, 0)137 and )154, but is not limited thereto. can be attached.
  • the linker may be a short oligo_ or polypeptide linker of 2 to 10 amino acids in length, preferably a glycine 1 ⁇ 2) -serine) duplex ((101 ⁇ 1a), but is limited thereto no.
  • the binding domain of a shomyo is generally followed by one or more "hinge domains( 1 ⁇ 6 (1011 11) ").
  • the term "hinge domain” refers to an antigen-binding domain that plays an important role in positioning the antigen-binding domain away from the effector cell surface to enable proper cell/cell contact, antigen binding and activation. is a part It generally includes one or more hinge domains between the extracellular binding domain and the transmembrane domain.
  • the hinge domain may be derived from natural, synthetic, semi-synthetic or recombinant sources.
  • the hinge domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.
  • a “changed hinge region” is ) an amino acid change of up to 30% (e.g. up to 25%, 20%, 15%,
  • one or more cysteine residues in the naturally occurring immunoglobulin hinge region may be substituted with one or more other amino acid residues (eg, one or more serine residues).
  • the altered immunoglobulin hinge region may alternatively or additionally have a proline residue of the wild-type immunoglobulin hinge region substituted with another amino acid residue (eg, a serine residue).
  • the hinge domain is CD8a,
  • any transmembrane domain including the hinge domain can be used as long as it can connect the extracellular domain and the intracellular domain between the cell membrane.
  • it consists of a hinge domain and a transmembrane domain derived from (3) 8a, and may include the amino acid sequence represented by SEQ ID NO: 8, but is not limited thereto.
  • the chimeric antigen receptor may be characterized in that it comprises an intracellular signaling domain (intracellular signaling domain).
  • intracellular signaling domain is an effector cell function, for example, a CAR-bound target cell-to-cell Activation, including the release of toxic factors, cytokine production, proliferation and cytotoxic activity, or other cellular responses induced by antigen binding to the extracellular binding domain of the CAR into the interior of immune effector cells for the target antigen It refers to the portion of the CAR that is involved in the transmission of the message of effective CAR binding.
  • the agonistic function refers to a specific function of a cell, for example, the agonistic function of a T cell may be cytolytic activity, may support an activity including secretion of cytokines, or may be active. Accordingly, the intracellular signaling domain refers to a portion of a protein that transmits an operative function signal and directs the cell to perform a specific function.
  • T cell activation is mediated by two different classes of intracellular signaling domains.
  • T cell activation is mediated by a primary signaling domain that initiates antigen-dependent primary activation through a T cell receptor and a costimulatory signaling domain that acts in an antigen-independent manner to provide a secondary signal.
  • the intracellular signaling domain may be characterized as including a "primary signaling domain" and a "co-stimulatory signaling domain".
  • primary signaling domain of the present invention, T 2021/245580 1» (: 1' year 2021/054846 refers to a signaling domain that regulates the primary activation of the cell receptor complex in a stimulating or inhibitory manner.
  • a primary signaling domain that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITM.
  • the ITAM containing the primary signaling domain is TCRC FcRy,
  • the primary signaling domain is preferably SEQ ID NO:
  • co-stimulatory signaling domain refers to an intracellular signaling domain of a co-stimulatory molecule.
  • a costimulatory molecule is an antigen receptor or a cell surface molecule other than an Fc receptor that upon binding to an antigen provides a secondary signal required for efficient activation and function of T lymphocytes.
  • the costimulatory signaling domain is 0X40, (3) 2, CD27, CD28, (3)S, ICAM-1, LFA-1 (CDlla/CD18), ICOS (CD278) , CD7, CD30, CD40, PD-1 , LIGHT, NKG2C, B7-H3 , (3) 83 and 4-1BB ((3) 137) may be selected from the group consisting of, but is not limited thereto.
  • the costimulatory signaling domain is preferably CD28 comprising the amino acid sequence shown in SEQ ID NO: 10 and ) 137 comprising the amino acid sequence shown in SEQ ID NO: 11, Therefore 2021/245580 1»(:1' year2021/054846 is not limited.
  • the chimeric antigen receptor according to the present invention may be characterized in that it includes two or more intracellular signaling domains, and when it includes two or more intracellular signaling domains, the intracellular signaling domains may be connected in series with each other have. Alternatively, it may be linked through an oligopeptide linker or a polypeptide linker consisting of 2 to 10 amino acids, and examples of such a linker sequence include a glycine-serine continuous sequence.
  • the CAR when designing the CAR according to the present invention, it may be characterized in that a nucleic acid encoding a signal peptide is inserted before the PA63 ligand domain 4 variant. Accordingly, the chimeric antigen receptor may be characterized in that it further comprises a signal peptide.
  • the present invention relates to a nucleic acid encoding the chimeric antigen receptor.
  • the nucleic acid (polynucleotide) encoding the chimeric antigen receptor according to the present invention may be modified by codon optimization, which is due to codon degeneracy, and many nucleotide sequences encoding polypeptides or variant fragments thereof Existence is well known to those of ordinary skill in the art.
  • nucleic acid a polynucleotide (nucleic acid) that is variable due to the difference in codon utilization, for example, a polynucleotide (nucleic acid) optimized for codon selection in humans, primates and/or mammals is preferable.
  • a nucleic acid encoding a mutant ( ⁇ 3 piece) of Sho63 domain 4 or 4) is introduced.
  • the nucleic acid encoding the 11 and 1111 may preferably include a nucleotide sequence selected from the group consisting of SEQ ID NO: 14 to SEQ ID NO: 19, but is not limited thereto.
  • the present invention relates to an expression vector comprising the nucleic acid and a virus comprising the expression vector.
  • the term "vector” refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule.
  • the transferred nucleic acid is generally linked to a vector nucleic acid molecule, for example, inserted into a vector nucleic acid molecule.
  • a vector may include sequences that direct autonomous replication in a cell, or it may include sequences sufficient to permit integration into a host cell.
  • the vector is It may be characterized in that it is selected from the group consisting of a plasmid, a lentiviral vector, an adenoviral vector and a retroviral vector, but is not limited thereto. 2021/245580 1» (: 1' year 2021/054846
  • virus means genetically modified to express the chimeric antigen receptor of the present invention for use in the treatment of cancer.
  • To be genetically modified is the addition of foreign genetic material in the form of DNA or RNA into the entire genetic material in a cell.
  • the nucleic acid or the vector is transfected or transfected with a virus.
  • the present invention relates to an immune cell expressing the chimeric antigen receptor on the surface.
  • the immune cells are capable of inducing the desired cancer treatment effect by inducing immunity, for example, T cells, NK cells, NKT cells, cytokine-induced killer cells (Cytokine Induced Killer cell, CIK) , It may be selected from the group consisting of macrophages and dendritic cells, but is not limited thereto. Preferably, it may be characterized as a T cell. Therefore, the immune cells expressing the chimeric antigen receptor according to the present invention are CAR-T cells (Chimeric Antigen Receptor T Cells), CAR-NK cells (Chimeric Antigen Receptor Natural Killer Cells) or CAR-NKT cells (Chimeric Antigen Receptor cells).
  • CAR-T cells Chimeric Antigen Receptor T Cells
  • CAR-NK cells Chimeric Antigen Receptor Natural Killer Cells
  • CAR-NKT cells Chimeric Antigen Receptor cells
  • the T cells are cytotoxic T lymphocytes; CTL); It may be characterized in that it is selected from the group consisting of T cells isolated from tumor infiltrating lymphocytes (TIL) and peripheral blood mononuclear cells (PBMC).
  • TIL tumor infiltrating lymphocytes
  • PBMC peripheral blood mononuclear cells
  • the present invention relates to a composition for preventing or treating cancer comprising immune cells (eg, T cells) expressing the chimeric antigen receptor.
  • immune cells eg, T cells
  • tumor infiltrating lymphocytes TIL
  • PBMC peripheral blood mononuclear cells
  • the term "prevention” refers to any action that inhibits or delays the progression of cancer by administering the composition of the present invention, and “treatment” refers to inhibition of cancer development, alleviation or elimination of symptoms.
  • the variant of PA63 ligand domain 4 according to the present invention can specifically bind to cancer cells or cancer tissues expressing ANTXR (anthrax toxin receptor), and PA63 ligand domain according to the present invention It is apparent to those skilled in the art that CAR-containing cells comprising a mutant of 4 have a cytotoxic effect on cancer expressing ANTXR.
  • the ANTXR includes ANTXR1 or ANTXR2.
  • the cancer or carcinoma that can be treated with the composition of the present invention is not particularly limited. 2021/245580 1» (:1' year 2021/054846, including both solid cancer and blood cancer.
  • the cancer may be a primary cancer or a metastatic cancer.
  • solid cancer means a tumor that is composed of blood vessels or connective tissue and has a certain hardness and shape, but is not limited thereto, for example, pancreatic cancer, stomach cancer, colon cancer, lung cancer, breast cancer, germ cell cancer , liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, etc.
  • the blood cancer include, but are not limited to, acute myeloid leukemia or multiple myeloma.
  • composition for preventing or treating cancer of the present invention may further include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound.
  • a pharmaceutically acceptable carrier as sterile and biocompatible, saline, sterile water, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol and among these components
  • One or more components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed.
  • diluents may be additionally added to form an injection formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.
  • the composition of the present invention may be in various oral or parenteral formulations.
  • commonly used thickening agents, extenders, binders, wetting agents, disintegrants, 2021/245580 1» (: 1' year 2021/054846 Prepared using a diluent or excipient such as a surfactant.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose or lactose. It is prepared by mixing (lactose), gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.
  • Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories.
  • Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • injectable esters such as ethyl oleate.
  • witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, etc. can be used as a base for the suppository.
  • Such pharmaceutically acceptable carriers and agents are Remington's
  • composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and rectal administration, etc. can be administered.
  • parenteral administration intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and rectal administration, etc.
  • the oral composition can be formulated to coat the active agent or to be protected from degradation in the stomach, and the composition of the present invention can be any device capable of moving the active substance to the target cell to be administered by 2021/245580 1» (:1' year 2021/054846 May.
  • a suitable dosage of the composition for preventing or treating cancer of the present invention depends on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. It varies, and an ordinary skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention.
  • the composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.
  • the present invention relates to a method for preventing or treating cancer comprising administering the immune cells.
  • the present invention relates to the use of the immune cells for the prevention or treatment of cancer.
  • the present invention relates to the use of the immune cells for the manufacture of a medicament for the prevention or treatment of cancer. Since the above-described "immune cells" are used for the prevention or treatment methods, uses, and uses of the present invention, redundant descriptions thereof will be omitted.
  • the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it is common knowledge in the art that the scope of the present invention is not to be construed as being limited by these examples. 2021/245580 1»(:1' year 20211/054846 It will be obvious to those who have it.
  • Example 1 Analysis of target antigen protein expression in cancer cell lines Melanoma cell line 526-mel, pancreatic cancer cell line (PANC1, Mia_PaCa2), breast cancer cell line (SK-BR3, MDA-MB-231, MCF7, ANTXRKTEM8 of ZR-517), As a result of analyzing ANTXR2 (CMG2) protein expression using western blot, ANTXR1 and ANTXR2 expression were increased in pancreatic cancer and breast cancer cell lines, melanoma cell line 526-mel and breast cancer cell line SK-BR3 did not express ANTXR1 and ANTXR2 was confirmed (FIG. 1).
  • Example 2 Preparation of target antigen protein overexpression cell line According to Example 1, TEM8-RFP, CMG2-RFP lentivirus was transducted into ANTXRKTEM8), ANTXR2 (CMG2) negative 526-mel cell line, and then western blot method was performed It was confirmed that the protein was normally expressed using the . After that, as a result of selection of positive cells using puromycin, a cell line with increased expression of 70% or more was prepared (FIG. 3) .
  • Example 3 ANTXR specific CAR design
  • the CAR specific for ANTXR1 or ANTXR2 was designed to include an extracellular binding domain, (3) a transmembrane domain derived from 8a, a hinge region, a primary signaling domain of the CD3 ⁇ 4 chain, (3) 28 and a CD137 costimulatory signaling domain.
  • extracellular binding 2021/245580 1» (: 1' year 2021/054846 Domains essential for receptor binding of PA63 ligands D4 wild-type (wild) or D4 mutants (mutant) #1 - #6 are designed, respectively, the introduced CAR, Figure 4
  • a vector was prepared by introducing it into the D4 site of the vector shown in .
  • the signal peptide is a short amino acid sequence and plays an important role in transporting the synthesized protein to the seedlings.
  • the signal peptide is removed, and the protein is transported to the Golgi complex through a carrier.
  • GM-CSF was used as a signal peptide for transporting the synthesized protein to its destination.
  • a nucleic acid encoding copGFP was introduced into the vector of FIG. 4 in order to check the expression of CAR, but it may be omitted in order to use the CAR as an actual therapeutic agent. in the present invention
  • the amino acid sequences required for preparation are shown in Table 1.
  • Example 4 ANTXR-specific CAR construction
  • extracellular binding domain PA63 D4 wi ld and D4 mutant #1 - ⁇
  • extracellular binding domain PA63 D4 wi ld and D4 mutant #1 - ⁇
  • transmembrane domain CD8 transmembrane & hinge
  • intracellular signaling domain CD3, CD134, CD28
  • Gibson assembly was performed.
  • the virus was prepared using the lentiviral vector for CAR introduction prepared above, and the packaging cell 293FT cell (4X10 6 ) has an efficiency of about 50% or more.
  • 2021/245580 1» (:1' year 2021/054846 lentiviral vector was induced.
  • the lentivirus was prepared and the suspended cellmedia was recovered, which was concentrated 10 times for 30 minutes at 1,000 X g using a Centicon, and the concentrated lentivirus was transduced into 293FT, NIH3T3 cells and CAR It was possible to confirm the lentivirus titer into which the gene was introduced.
  • Each CAR-T cell was prepared by transduction of the prepared D4 wild or D4 mutant-containing CAR-introduced lentivirus into PBMCs isolated from healthy donors at a concentration of 3.5 M0I.
  • Example 5 Confirmation of cytotoxicity against ANTXR1, 2TEM8, CMG2) overexpressing cell lines of CAR-T cells prepared using T cells isolated from PBMC D4 wild-containing CAR introduced T cells and D4 mutant # The purpose of this study was to confirm the anticancer cytotoxicity of 1 - #6 containing CAR-introduced T cells. Cytotoxicity was confirmed against the 526-mel melanoma cell lines, which were both negative for ANTXR1 and 2 (TEM8, CMG2), and the 526-mel/TEM8jFP and 526-me1/CMG2_RFP cell lines overexpressing ANTXR1 and 2 (TEM8, CMG2), respectively. .
  • RFP Red Fluorescence Protein
  • Example 6 Confirmation of anticancer activity against ANTXR1, 2TEM8, CMG2) overexpressing cell lines of CAR-T cells prepared using T cells isolated from PBMC It was intended to confirm the anticancer activity of the CAR-introduced T cells. Anticancer activity was confirmed against 526-mel/TEM8jFP and 526-me1/CMG2_RFP cell lines overexpressing ANTXR1 and 2 (TEM8, CMG2), respectively, in 526-mel melanoma cell lines negative for both ANTXR1 and 2 (TEM8, CMG2). .
  • D4 mutant #1 - #6 CAR-T£1 IFN gamma secretion 2021/245580 1» (: 1' year, 2021/054846 decreased, which decreased as the binding force between the receptor and ligand for ANTXR1 and 2 (TEM8, CMG2) decreased, the anticancer activity of D4 mutant #1 #6 CAR-T decreased.
  • Fig. 6 Example 7: In vitro anticancer activity and safety analysis of CAR-T cells prepared by selection Using PBMCs from 4 healthy donors, 2 types of D4 mutant CAR vectors selected from 6 types of D4 mutants CAR- T cells were prepared, and the difference in in vitro anticancer activity according to each donor PBMC was investigated.
  • Example 6 In the same manner as in Example 6, NT (Non Transduced) T cell control and D4 (wild, mutant #4, #6) CAR-T cells were prepared and T cell activity was compared. As a result, it was possible to confirm the TEM8-specific activity of D4 mutants #4 and #6 in each of the CAR-T cells prepared from each of the four healthy donors, similar to the results of Example 6. In addition, it was reconfirmed that the CAR-T activity of D4 mutants maintained cytotoxicity compared to D4 wild, but the degree of IFNY secretion was decreased due to a decrease in affinity for antigen (Fig. 7 left) .
  • Example 8 Analysis of T cell characteristics after co-culture of TEM8 overexpressing cancer cell line and two selected D4 mutant CAR-T 2021/245580 1»(:1' year2021/054846
  • 526-me 1 /TEM8_RFP cells overexpressing ANTXR TEM8) in a 526-mel melanoma cell line negative for both ANTXR1 (TEM8) / ANTXR2 (CMG2) antigens were seeded in a 96-well plate with 4 lxlO cells each.
  • the TEM8 antigen-overexpressing human pancreatic cancer cell line (Mia-PaCa2/TEM8) was implanted by subcutaneous injection at a dose of 5 x 10 6 cels/head. 21 days after transplantation (tumor size: about ⁇ 100mm 3 ) D4 2021/245580 1» (: 1' year 2021/054846 wi ld and D4 mutant #6 CAR-T cells finally selected from the above results 1 x 10 7 cel ls/head(total cel l: 2 x 10 7 cel ls/head) was administered to tumor-implanted mice by tail vein injection, and the tumor growth inhibitory effect was compared and investigated (FIG. 9A).
  • the tumor growth was inhibited by about 61% (FIG. 9B).
  • the D4 mutant-containing CAR-introduced T cells had excellent efficacy in an animal model using solid carcinoma expressing ANTXR1 (TEM8).
  • TEM8 solid carcinoma expressing ANTXR1
  • Industrial Applicability Most of the target antigens of solid cancer are cancer-associated antigens, not cancer-specific antigens, and the target antigen ANTXR of the present invention is also a cancer-associated antigen. It is important to secure safety while maintaining CAR-T anticancer efficacy.
  • a chimeric antigen receptor comprising a mutant of PA63 ligand domain 4 as an extracellular binding domain, it shows an excellent anticancer effect against cancer expressing an ARTXR antigen, and controls the binding affinity to the ARTXR antigen to control normal cells Since safety can be optimized so that there is no effect on cancer, it is possible to treat cancer with very effective anticancer treatment and minimizing side effects of existing CAR-T immune cell therapies.
  • a specific part of the content of the present invention has been described in detail, and for those of ordinary skill in the art, this specific description is only a preferred implementation.

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Abstract

La présente invention concerne un récepteur antigénique chimérique permettant d'optimiser la sécurité pour des cellules normales tout en maintenant l'efficacité anticancéreuse par contrôle de l'affinité de liaison d'un récepteur antigénique chimérique reconnaissant un antigène du récepteur de la toxine de l'anthrax (ANTXR) et se liant à celui-ci. La présente invention concerne également : un récepteur antigénique chimérique à base de ligand comprenant un variant du domaine 4 de PA63 se liant de manière spécifique à un antigène du cancer ANTXR1 ou ANTXR2; des cellules immunitaires le comprenant; et une utilisation associée. Selon la présente invention, le récepteur antigénique chimérique comprenant un variant du domaine 4 du ligand PA63 en tant que domaine de liaison extracellulaire peut être utilisé pour présenter un excellent effet anticancéreux pour le cancer exprimant l'antigène ARTXR et pour optimiser la sécurité par contrôle de l'affinité de liaison pour l'antigène ARTXR de sorte que les cellules normales ne soient pas affectées, et, par conséquent, le récepteur antigénique chimérique est capable de traiter le cancer qui est très efficace tout en minimisant les effets secondaires d'un agent de traitement de cellules immunitaires CAR-T existant.
PCT/IB2021/054846 2020-06-04 2021-06-03 Récepteur antigénique chimérique comprenant un variant du domaine 4 de pa63 en tant que domaine de liaison extracellulaire, et son utilisation Ceased WO2021245580A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20080052566A (ko) * 2005-08-02 2008-06-11 플래닛 바이오테크놀로지, 인코포레이티드 개선된 키메라 독소 수용체 단백질 및 탄저병의 예방 및치료를 위한 키메라 독소 수용체 단백질
US20160015750A1 (en) * 2013-03-09 2016-01-21 Baylor College Of Medicine Vascular-targeted t-cell therapy
US20160303230A1 (en) * 2012-04-20 2016-10-20 Baylor College Of Medicine Chimeric antigen receptor for bispecific activation and targeting of t lymphocytes
US20170114133A1 (en) * 2014-06-09 2017-04-27 Biomed Valley Discoveries, Inc. Tem8 antibodies and methods of use
KR20190013612A (ko) * 2017-07-27 2019-02-11 고려대학교 산학협력단 인간 항-antxr 키메라 항원 수용체 및 이의 용도

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20080052566A (ko) * 2005-08-02 2008-06-11 플래닛 바이오테크놀로지, 인코포레이티드 개선된 키메라 독소 수용체 단백질 및 탄저병의 예방 및치료를 위한 키메라 독소 수용체 단백질
US20160303230A1 (en) * 2012-04-20 2016-10-20 Baylor College Of Medicine Chimeric antigen receptor for bispecific activation and targeting of t lymphocytes
US20160015750A1 (en) * 2013-03-09 2016-01-21 Baylor College Of Medicine Vascular-targeted t-cell therapy
US20170114133A1 (en) * 2014-06-09 2017-04-27 Biomed Valley Discoveries, Inc. Tem8 antibodies and methods of use
KR20190013612A (ko) * 2017-07-27 2019-02-11 고려대학교 산학협력단 인간 항-antxr 키메라 항원 수용체 및 이의 용도

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