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WO2023113293A1 - Cellule présentatrice d'antigène professionnelle spécifique d'un antigène transformée contenant un récepteur antigénique chimérique (car) et son utilisation - Google Patents

Cellule présentatrice d'antigène professionnelle spécifique d'un antigène transformée contenant un récepteur antigénique chimérique (car) et son utilisation Download PDF

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WO2023113293A1
WO2023113293A1 PCT/KR2022/018876 KR2022018876W WO2023113293A1 WO 2023113293 A1 WO2023113293 A1 WO 2023113293A1 KR 2022018876 W KR2022018876 W KR 2022018876W WO 2023113293 A1 WO2023113293 A1 WO 2023113293A1
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cells
antigen
receptor
cell
cancer
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진화섭
전태훈
양준
이창희
이나영
박인병
강석진
박시원
이현정
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Immunological Designing Lab Inc
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a transformed antigen-specific professional antigen-expressing cell containing a chimeric antigen receptor (CAR), and specifically, the chimeric antigen receptor contains an antigen-binding domain that specifically binds to CD138. It relates to a pharmaceutical composition for the treatment of cell therapy or cancer comprising as an active ingredient.
  • CAR chimeric antigen receptor
  • Professional antigen presenting cells include macrophages, dendritic cells, and naive B cells (Makala et al., 2004).
  • macrophages and dendritic cells recognize antigens, they break the antigens into small pieces by phagocytosis, and the major histocompatibility complex (MHC) antigens expressed in macrophages and dendritic cells transfer antigenic determinants (epitopes) to T cells. (T cell) (Kambayashi and Laufer, 2014).
  • MHC major histocompatibility complex
  • naive B cells antigens are recognized by the B cell receptor (membrane bound IgM), and then major histocompatibility (major histocompatibility), which is expressed in naive B cells by crushing the antigen into small pieces Complex, MHC) antigen delivers epitopes to T cells (Kambayashi and Laufer, 2014).
  • MHC major histocompatibility complex
  • Professional antigen-expressing cells also convert na ⁇ ve T cells into effector T cells by delivery of antigenic determinants by major histocompatibility complex (MHC) antigens and transmission of co-stimulatory signals. effector T cells) (Kambayashi and Laufer, 2014).
  • professional antigen-expressing cells secrete various cytokines and chemokines to induce inflammatory responses or activate other immune cells (Kambayashi and Laufer, 2014). Therefore, an effective immune response is initiated by activation of professional antigen-expressing cells, and an effective immune response is not induced without activation of professional antigen-presenting cells (Makala et al., 2004).
  • tumor-specific antigens are treated with cancer patient-derived professional antigen-expressing cells, and then professional antigen-expressing cells activated by the tumor-specific antigens are introduced into the cancer patient's body (van Willigen et al., 2018).
  • Activated professional antigen-expressing cells transplanted into cancer patients induce the activation of cytotoxic T lymphocytes (CTL) and natural killer cells (NK cells) that directly target tumor cells, thereby killing tumor cells.
  • CTL cytotoxic T lymphocytes
  • NK cells natural killer cells
  • scFv single-chain variable fragment
  • a chimeric antigen receptor when grafted onto a T cell or natural killer cell, the anticancer action of the T cell or natural killer cell is achieved only by recognizing the specific antigen of the scFv, regardless of signal transduction by specialized antigen-expressing cells that recognize cancer-specific antigens. can be activated, and it is not limited to the HLA type, so it can be used as a more efficient treatment method that can be used universally by many people.
  • these chimeric antigen receptor-expressing T cells or natural killer cells show efficacy in various cancers (Holzinger et al., 2016; Pettitt et al., 2018; Wang et al., 2020).
  • CD138 also called syndecan-1
  • syndecan-1 is a type I transmembrane protein consisting of 288 amino acids and is known to be involved in the suppression of inflammatory responses by binding to ligands (Rops et al., 2007; Teng et al. , 2012).
  • CD138 appears to play an immunosuppressive role in certain immune diseases, although the exact mechanism is not yet known. known (Xu et al., 2005; Zhang et al., 2013).
  • CD138 is known to be expressed in various epithelial tissues and immune cells, and its expression is increased in some carcinomas, so it is used as a molecular marker or therapeutic target for carcinomas (Czarnowski, 2021).
  • Carcinomas expressing CD138 reported so far include multiple myeloma (Dhodapkar et al., 1998), breast cancer (Malek-Hosseini et al., 2017), and pancreatic cancer (Conejo et al.
  • Death receptor 6 also called tumor necrosis factor (TNF) receptor superfamily 21 (TNFRSF21), is a transmembrane protein composed of 392 amino acids. When a signal is received, it enters the cytoplasmic tail. It has a death domain that induces apoptosis (Pan et al., 1998). The currently known function of DR6 is known to be involved in the prognosis of several immune diseases by regulating T lymphocyte activity (Liu et al., 2001; Schmidt et al., 2005).
  • the present inventors devised a new treatment method combining the functions of professional antigen-expressing cells and chimeric antigen receptors. That is, after recognizing a receptor capable of recognizing a tumor cell surface protein as a ligand and a ligand, a novel chimeric signaling domain capable of inducing the activity of professional antigen-expressing cells by the chimeric antigen receptor was devised, It was demonstrated that professional antigen-expressing cells activated by these chimeric signaling domains can have cytotoxic ability against cancer.
  • TLR-2 transmembrane domain of toll-like receptor 2
  • TLR-3 intracellular domain of TLR-4
  • IgE receptor gamma chain Fc ⁇ receptor I gamma chain, Fc ⁇ RI ⁇
  • IgG receptor 2A alpha chain Fc ⁇ receptor 2A alpha chain, Fc ⁇ R2A ⁇
  • IgE receptor beta chain Fc ⁇ receptor I beta chain, Fc ⁇ RI ⁇
  • ITAM immunoreceptor tyrosine-based activation motif
  • the extracellular domain of DR6 expressed in macrophages recognizes CD138 and, when combined with specific cancer cells expressing CD138, can transmit activation signals into these macrophages, and eventually these signals activate macrophages to release CD138. It can have cytotoxicity against expressing cancer. In fact, it was confirmed through in vitro experiments that the macrophages have a cytotoxic ability specifically to cells expressing CD138.
  • an object of the present invention is a transformed cell comprising a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an antigen binding domain that specifically binds to CD138, a transmembrane domain and an intracellular signaling domain.
  • the cell is an antigen-specific professional antigen-expressing cell having a targeted effector activity, and is to provide a transformed cell including macrophages, dendritic cells or naive B cells.
  • Another object of the present invention is to provide a cell therapy or a pharmaceutical composition for treating cancer that kills cancer cells expressing CD138 containing the transformed cells as an active ingredient.
  • the chimeric antigen receptor is an antigen-binding domain that specifically binds to CD138, a transmembrane domain and An intracellular signaling domain, wherein the cells are antigen-specific specialized antigen-expressing cells having targeted effector activity, including macrophages, dendritic cells, or naive B cells, transformed cells to provide.
  • the present invention provides a cell therapy or a pharmaceutical composition for treating cancer that kills cancer cells expressing CD138, including the transformed cells.
  • the antigen-binding domain of the chimeric antigen receptor may be an extracellular domain of death receptor 6 (DR6) that specifically binds to the CD138.
  • DR6 death receptor 6
  • the transmembrane domain may be TLR-2.
  • the intracellular signaling domain is TLR-3, TLR-4, IgE receptor gamma chain (Fc ⁇ receptor I gamma chain, Fc ⁇ RI ⁇ ), IgG receptor 2A alpha chain (Fc ⁇ receptor 2A alpha chain, Fc ⁇ R2A ⁇ ), immunoreceptor tyrosine-based activation motif (ITAM) of IgE receptor beta chain (Fc ⁇ receptor I beta chain, Fc ⁇ RI ⁇ ), or a combination thereof.
  • IgE receptor gamma chain Fc ⁇ receptor I gamma chain, Fc ⁇ RI ⁇
  • IgG receptor 2A alpha chain Fc ⁇ receptor 2A alpha chain, Fc ⁇ R2A ⁇
  • ITAM immunoreceptor tyrosine-based activation motif
  • Carcinomas expressing the CD138 include multiple myeloma, breast cancer, pancreatic cancer, lung cancer, hepatocellular carcinoma, bladder cancer, and ovarian cancer. , prostate cancer and colorectal cancer.
  • Transformed cells according to the present invention provide an enhanced chimeric signaling domain that induces the activity of professional antigen-presenting cells when they specifically bind to an antigen.
  • TLR-3, TLR-4 and IgE receptor gamma chain (Fc ⁇ receptor I gamma chain, Fc ⁇ RI ⁇ ) or IgG receptor 2A alpha chain which play an important role in signal transduction of the extracellular domain of DR6 and professional antigen-expressing cells (Fc ⁇ receptor 2A alpha chain, Fc ⁇ R2A ⁇ ) or immunoreceptor tyrosine-based activation motif (ITAM) of IgE receptor beta chain (Fc ⁇ receptor I beta chain, Fc ⁇ RI ⁇ ) as a subunit, and 3 ITAMs as a linker
  • ITAM immunoreceptor tyrosine-based activation motif
  • FIG. 1 is a schematic diagram showing the form of one of the cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.
  • Figure 2 is a schematic diagram showing the form of one of the cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.
  • Figure 3 is a schematic diagram showing the form of one of the cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.
  • Figure 4 is a schematic diagram showing the form of one of the cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.
  • FIG. 5 is a schematic diagram showing the shape of one of lentiviral vectors according to an embodiment of the present invention.
  • FIG. 6 is a schematic diagram showing the shape of one of lentiviral vectors according to an embodiment of the present invention.
  • FIG. 7 is a schematic diagram showing the shape of one of lentiviral vectors according to an embodiment of the present invention.
  • FIG. 8 is a schematic diagram showing the shape of one of lentiviral vectors according to an embodiment of the present invention.
  • FIG. 9 is a schematic diagram showing the form of one of the chimeric antigen receptors expressed on the surface of professional antigen-expressing cells according to an embodiment of the present invention.
  • FIG. 10 is a schematic diagram showing the form of one of the chimeric antigen receptors expressed on the surface of professional antigen-expressing cells according to an embodiment of the present invention.
  • FIG. 11 is a schematic diagram showing the form of one of the chimeric antigen receptors expressed on the surface of professional antigen-expressing cells according to an embodiment of the present invention.
  • FIG. 12 is a schematic diagram showing the form of one of the chimeric antigen receptors expressed on the surface of professional antigen-expressing cells according to an embodiment of the present invention.
  • FIG. 13 is a diagram showing the results of measuring the expression level of CD138 using flow cytometry in the AsPC-1 cell line, which is a cancer cell (CD138 positive cell) expressing CD138, which is a target of the chimeric antigen receptor provided in the present invention.
  • FIG. 14 is a schematic diagram showing cDNA regions of each domain expressing a recombinant DR6 fusion protein prepared to measure whether DR6 recognizes CD138 according to an embodiment of the present invention.
  • FIG. 15 is a schematic diagram of an expression vector (retroviral vector) expressing a recombinant DR6 fusion protein prepared to measure whether DR6 recognizes CD138 according to an embodiment of the present invention.
  • 16 is a schematic diagram of a recombinant DR6 fusion protein prepared to measure whether DR6 recognizes CD138 according to an embodiment of the present invention.
  • 17a is a diagram showing the result of expressing the prepared recombinant DR6 fusion protein in Chinese hamster ovary (CHO) cells and then purifying it by affinity chromatography using a protein A column.
  • Figure 17b is a diagram showing the result of Western blotting using an antibody (anti-human IgG heavy chain constant region antibody, anti-human IgG Fc region antibody) recognizing the human IgG heavy chain constant region of the prepared recombinant DR6 fusion protein.
  • an antibody anti-human IgG heavy chain constant region antibody, anti-human IgG Fc region antibody
  • 17c is a diagram showing whether the prepared recombinant DR6 fusion protein recognizes the target cancer cells (AsPC-1 cell line) expressing CD138 using flow cytometry.
  • FIG. 18 is a diagram showing the results of comparing the expression ratio of GFP in professional antigen-expressing cells transduced with 4 types of expression vectors according to an embodiment of the present invention using a lentivirus system.
  • Figure 19 shows the effector cells of professional antigen-expressing cells transduced with four types of chimeric antigen receptors or empty vectors according to an embodiment of the present invention, and cancer cells (AsPC-1 cell line) expressing CD138. It is a diagram showing the results of measuring the cytotoxicity for 24 hours after co-culture.
  • FIG. 20 shows the effector cells of professional antigen-expressing cells transduced with four types of chimeric antigen receptors or empty vectors according to an embodiment of the present invention, and cancer cells (AsPC-1 cell line) expressing CD138. It is a diagram showing the results of measuring the cytotoxicity for 48 hours after co-culture.
  • the present invention provides a transformed cell comprising a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an antigen-binding domain that specifically binds to CD138, a transmembrane domain, and an intracellular signaling domain wherein the cells are antigen-specific specialized antigen-expressing cells having targeted effector activity, and include macrophages, dendritic cells, or naive B cells, to provide transformed cells.
  • CAR chimeric antigen receptor
  • Chimeric antigen receptor proteins may include functional equivalents.
  • 'Functional equivalent' means at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably, the amino acid sequence of the chimeric antigen receptor protein as a result of addition, substitution, or deletion of amino acids. has a sequence homology of 95% or more, and refers to a protein that exhibits substantially the same physiological activity.
  • 'Substantially homogeneous physiological activity means having an activity capable of specifically binding to CD138.
  • the present invention also includes fragments, derivatives and analogs of chimeric antigen receptors.
  • fragments, derivatives and analogs of chimeric antigen receptors include (1) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted (the substituted amino acid residues are encoded by the genetic code).
  • polypeptide having substituent(s) on one or more amino acid residues (2) a polypeptide having substituent(s) on one or more amino acid residues, or (3) another compound (a compound capable of extending the half-life of a polypeptide, such as polyethylene glycol) a polypeptide derived from the combined mature polypeptide, or (4) an additional amino acid sequence (e.g., a leader sequence, a secreted sequence, a sequence used to purify the polypeptide, a proteinogen sequence, or a fusion protein) and It may be a polypeptide derived from the polypeptide to which it is linked.
  • additional amino acid sequence e.g., a leader sequence, a secreted sequence, a sequence used to purify the polypeptide, a proteinogen sequence, or a fusion protein
  • CD138 is found in various malignant cells. So far, carcinomas expressing CD138 include multiple myeloma, breast cancer, pancreatic cancer, lung cancer, hepatocellular carcinoma, bladder cancer, and ovarian cancer. ), prostate cancer or colorectal cancer, but is not limited thereto.
  • the "chimeric signaling domain” is expressed in professional antigen-expressing cells to induce activation of professional antigen-expressing cells through a ligand-receptor reaction by binding to a desired ligand and to attack cells expressing the corresponding ligand. It may mean a fusion protein for In other words, it can be seen as a protein that binds to a ligand when expressed in specialized antigen-expressing cells and induces the activation of these cells. Therefore, the novel chimeric signaling domain provided by the present invention is a novel chimeric signaling domain in a universal sense capable of inducing activation of professional antigen-expressing cells using all ligand-receptor reactions including all antigen-antibody reactions. to provide.
  • the chimeric antigen receptor can be regarded as a protein that, when expressed in specialized antigen-expressing cells, binds to an antigen and induces activation of these cells.
  • it may be a protein that recognizes an antigen specific to a cell to cause an immune response
  • the cell to cause an immune response may refer to a cell present in a specific tissue or forming a tissue that causes a lesion.
  • the antigen-binding domain of the chimeric antigen receptor may be an extracellular domain of death receptor 6 (DR6) that specifically binds to the CD138.
  • DR6 death receptor 6
  • the extracellular domain of DR6 may consist of SEQ ID NO: 1 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto.
  • the antigen-binding domain may be in the form of a dimer in which a pair of extracellular domains of DR6 are linked to a human immunoglobulin G 1 heavy chain constant region, respectively, to amplify signal transduction . It is not limited.
  • the human immunoglobulin G 1 heavy chain constant region may consist of SEQ ID NO: 2 or an amino acid sequence exhibiting 95% or more homology thereto, but is not limited thereto.
  • the intracellular signaling domain which is one component of the chimeric antigen receptor of the present invention, means a site that activates the immune response of immune cells by binding to the antigen binding domain.
  • the signaling domain may be TLR-3, TLR-4, tyrosine-based activation motif (ITAM), or a combination thereof.
  • ITAM tyrosine-based activation motif
  • the signaling domain may be in the form of connecting the ITAM as a subunit to the TLR-3-TLR-4 with a linker, and one or more ITAMs are linked. It may be, preferably three may be connected form, but is not limited thereto.
  • the ITAM is based on immunoreceptor tyrosine of IgE receptor gamma chain (Fc ⁇ receptor I gamma chain, Fc ⁇ RI ⁇ ), IgG receptor 2A alpha chain (Fc ⁇ receptor 2A alpha chain, Fc ⁇ R2A ⁇ ) or IgE receptor beta chain (Fc ⁇ receptor I beta chain, Fc ⁇ RI ⁇ ). It may be an activation motif, but is not limited thereto.
  • the TLR-3 is SEQ ID NO: 3 or an amino acid represented by SEQ ID NO: 3 having a sequence homology of at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% therewith. can consist of an amino acid sequence that exhibits a function substantially equivalent to that of the sequence;
  • the TLR-4 is SEQ ID NO: 4 or an amino acid represented by SEQ ID NO: 4 having a sequence homology of at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% therewith.
  • the ITAM is SEQ ID NO: 5; SEQ ID NO: 6; Alternatively, it may consist of an amino acid sequence that exhibits substantially equivalent functions to the amino acid sequence represented by SEQ ID NO: 7.
  • the linker may consist of an amino acid sequence that exhibits substantially the same function as the amino acid sequence represented by SEQ ID NO: 8.
  • the transmembrane domain of the present invention is a site that connects the extracellular domain of DR6 and the co-stimulatory and essential signaling domains between cell membranes, and the intracellular signaling domain is an immune response of immune cells by binding of the antigen-binding domain. indicates the site of activation.
  • the transmembrane domain which is one component of the chimeric antigen receptor of the present invention, is CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and a transmembrane domain of a protein selected from the group consisting of TLR-2.
  • the transmembrane domain may be TLR-2, which may consist of SEQ ID NO: 9 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto.
  • the antigen-binding domain may include a signal peptide, and the signal peptide may be a TLR-4 signal peptide, and may consist of SEQ ID NO: 10 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto no.
  • the vector used in the present invention may use various vectors known in the art, and depending on the type of host cell to produce the antigen receptor, a promoter, terminator, enhancer, etc.
  • Expression control sequences, sequences for membrane targeting or secretion, etc. may be appropriately selected and combined in various ways depending on the purpose.
  • Vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors and viral vectors.
  • Suitable vectors include expression control elements such as promoters, operators, initiation codons, stop codons, polyadenylation signals and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and may be prepared in various ways depending on the purpose.
  • a lenti-virus vector can be used, and in the following examples of the present invention, the pCDH-CMV-MCS-EF1-copGFP vector (a lenti-virus vector) was used (FIG. 5 to FIG. 8).
  • cells can be transformed by introducing the chimeric antigen receptor that specifically binds to CD138 of the present invention into cells through the vector.
  • the cells may be cytotoxic T cells and NK cells, tumor-infiltrating lymphocytes, B cells, NK cells, or NKT cells, preferably macrophages, dendritic cells, and unsensitized cells that are specialized antigen-expressing cells. It may be a naive B cell.
  • the cells may be obtained or prepared from bone marrow, peripheral blood, peripheral blood mononuclear cells, or umbilical cord blood.
  • the cell is a human cell.
  • professional antigen-expressing cells can be transformed with a chimeric antigen receptor that specifically binds to CD138 using the vector described above.
  • cells transformed by introducing the chimeric antigen receptor of the present invention recognize CD138 as an antigen and bind strongly thereto.
  • chimeric antigen receptor professional antigen presenting cell means a method such as transduction of normal professional antigen presenting cells It refers to specialized antigen-expressing cells that express chimeric antigen receptors that specifically react to cancer cells, rather than the original specialized antigen-expressing cell surface receptors. Professional antigen-expressing cells having this receptor exhibit cytotoxicity by inducing apoptosis of target cells.
  • CAR-pAPC cells may be cells into which the chimeric antigen receptor of the present invention has been introduced into professional antigen-expressing cells.
  • the cells have the advantage of anticancer-specific targeted therapy, which is an existing advantage of CAR-T therapeutics.
  • professional antigen-expressing cells equipped with the chimeric antigen receptor of the present invention can recognize and effectively destroy cancer cells expressing CD138. .
  • another aspect of the present invention is a cell therapy agent comprising the cells; A pharmaceutical composition for the treatment of cancer expressing CD138 comprising the same as an active ingredient; and administering the cells to a subject.
  • the cells may be cytotoxic T cells and NK cells, tumor infiltrating lymphocytes, B cells, NK cells, or NK-T cells, preferably, specialized antigen expression It may be a macrophage cell, a dendritic cell, or a naive B cell. or progenitor cells, for example, hematopoietic stem cells, mesenchymal stromal cells, stem cells, pluripotent stem cells, and embryonic stem cells, which can be used for cell therapy such as anticancer treatment.
  • the cells may come from a donor or may be cells obtained from a patient. Cells can be used for regeneration, eg replacing the function of diseased cells. Cells can also be modified to express heterologous genes so that biological agents can be delivered to specific microenvironments, such as, for example, diseased bone marrow or metastatic deposits.
  • cell therapy refers to cells and tissues prepared by isolation, culture, and special manipulation from a subject, and is a drug (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention, and restores the function of cells or tissues. It refers to medicines used for the purpose of treatment, diagnosis, and prevention through a series of actions, such as proliferating and selecting living autologous, allogeneic, or heterogeneous cells in vitro or changing the biological characteristics of cells in other ways.
  • prevention of the present invention refers to any activity that inhibits or delays the development of cancer expressing CD138 by administration of the composition.
  • treatment refers to all activities that improve or beneficially change symptoms caused by cancer expressing CD138 by administration of the composition.
  • composition may include a pharmaceutically acceptable carrier.
  • the "pharmaceutically acceptable carrier” may refer to a carrier or diluent that does not inhibit the biological activity and properties of the compound to be injected without irritating living organisms.
  • the type of the carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable can be used.
  • Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or in combination of two or more.
  • composition containing a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
  • solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose, and lactose in the compound. , can be prepared by mixing gelatin, etc.
  • lubricants such as magnesium stearate and talc may also be used.
  • Liquid preparations for oral use include suspensions, solutions for internal use, emulsions, and syrups.
  • various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is.
  • Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories.
  • Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.
  • Witepsol, Macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used as a base for the suppository.
  • composition can be administered in a pharmaceutically effective amount.
  • the "pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is dependent on the type and severity of the subject, age, sex, infected virus type, and drug activity, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including concomitantly used drugs and other factors well known in the medical arts.
  • the administration means introducing the composition of the present invention to a patient by any suitable method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue.
  • Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration may be administered, but is not limited thereto.
  • composition of the present invention may be administered daily or intermittently, and the number of administrations per day may be administered once or divided into 2 to 3 times.
  • the number of administrations when the two active ingredients are each a single agent may be the same or may be different.
  • the composition of the present invention can be used alone or in combination with other drug treatments for the prevention or treatment of blood cancer. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors, and can be easily determined by those skilled in the art.
  • the subject refers to all humans, including humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or guinea pigs that have or may develop cancer expressing CD138. means animals.
  • the type of subject is included without limitation as long as the disease can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject.
  • Carcinoma expressing CD138 to be treated in the present invention is multiple myeloma, breast cancer, pancreatic cancer, lung cancer, hepatocellular carcinoma, bladder cancer , ovarian cancer, prostate cancer, and colorectal cancer, but is not limited thereto.
  • chimeric antigen receptor of the present invention four types of antigen receptors having the sequences of the extracellular domain of DR6 (Death receptor 6) and the transmembrane domain of TLR-2 and encoding sequences for different intracellular signal transduction domains were prepared.
  • a chimeric antigen receptor protein coding sequence was synthesized.
  • the chimeric antigen receptor is an antigen recognition site in which a pair of extracellular domains of DR6 are linked to the human immunoglobulin G 1 heavy chain constant region, respectively, a transmembrane domain of TLR-2, and a TLR -4 signal peptide. Including in common.
  • Signal transduction domains were constructed in four different configurations. Table 1 below shows the configuration of the four types of signaling domains.
  • FIG. 1 to 4 show cDNA schematics of each domain expressing the constructed chimeric antigen receptor.
  • the protein coding sequence of each domain part was confirmed in a gene database, and the accuracy of gene synthesis was confirmed through sequencing after constructing a protein expression plasmid.
  • CD138 expression was confirmed in the human pancreatic adenocarcinoma (AsPC-1) (ATCC) cell line, which is a cell line derived from pancreatic cancer.
  • AsPC-1 as a cell line derived from pancreatic cancer.
  • flow cytometry analysis fluorescence-activated cell sorting
  • a soluble recombinant DR6 fusion protein was prepared.
  • a protein coding sequence in which each of the extracellular domains of DR6 is linked to the human immunoglobulin G1 heavy chain constant region was identified in a gene database.
  • gene synthesis was performed by connecting the remaining sequences except for the stop codon portion of the base sequence constituting the domains into one. The accuracy of gene synthesis was confirmed through sequencing after constructing a protein expression plasmid.
  • a schematic diagram showing the cDNA region of each domain expressing the recombinant protein is shown in FIG. 14 .
  • the recombinant DR6 fusion protein obtained by fusing the human CD138 recognition domain of Example 4 with the human immunoglobulin G 1 heavy chain constant region in Chinese hamster ovary (CHO) cells the corresponding gene was used in pLNCX2, a retrovirus-derived expression vector. (Addgene).
  • a primer that creates a restriction enzyme Xho I cleavage site sequence is synthesized at the 5' end, a restriction enzyme cleavage site is created by polymerase chain reaction (PCR), and restriction is also made at the 3' end.
  • a restriction enzyme cleavage site was prepared by synthesizing a primer that creates the cleavage site sequence of the enzyme Not I and polymerase chain reaction.
  • the 5' end of the gene having a restriction enzyme cleavage site due to polymerase chain reaction was treated with Xho I, and the 3' end was treated with Not I.
  • the multicloning site of the expression vector was treated with xho I and Not I so that the gene could be inserted. After mixing the restriction enzyme-treated gene and the expression vector, they were ligated by treatment with a ligase.
  • Example 6 Affinity measurement of soluble recombinant DR6 fusion protein and human cell line expressing CD138
  • affinity chromatography was performed using a protein A column ( affinity chromatography) (GE healthcare) (see Fig. 17a).
  • the purified water-soluble chimeric antigen receptor was confirmed by Western blotting using an antibody (anti-human IgG heavy chain constant region antibody, anti-human IgG Fc region antibody, abcam) recognizing the human IgG heavy chain constant region (Fig. see 17b).
  • the purified soluble recombinant DR6 fusion protein was treated with a cell line (AsPC-1 cell line) expressing CD138, and it was confirmed through flow cytometry whether the soluble recombinant DR6 fusion protein could bind to the cell.
  • a cell line AsPC-1 cell line
  • FIG. 17C The results are shown in FIG. 17C. .
  • AsPC-1 cells expressing CD138 did not bind to control Ig (isotype control), but were confirmed to bind to soluble DR6 fusion protein (DR6-Ig).
  • the prepared soluble DR6 fusion protein had high affinity with the human cell line expressing CD138, which recognized AsPC-1 cells expressing CD138, and recognized the prepared CD138 as a human immunocompetent. This means that it can be used to verify CD138 protein-specific cytotoxicity in professional antigen-expressing cells (DR6.CAR-pAPC cells) expressing a recombinant protein in which a globulin G 1 heavy chain constant region and a signaling protein domain are fused.
  • Example 7 Production of specialized antigen-expressing cells expressing chimeric antigen receptors
  • Lentivirus using 293FT cells to introduce the four recombinant chimeric antigen receptor expression vectors prepared in Example 2 into THP-1 cells (human macrophage cell line), a type of professional antigen-expressing cells system was used.
  • 293FT cells were inoculated into 2.5 ⁇ 10 6 cells in a 100 ⁇ cell culture dish, and cultured in DMEM medium containing 10% calf serum. After 24 hours of culture, when the cells grow to cover about 60-70% of the dish, 20 ⁇ g of each of the 4 vector DNAs of Example 2 were dispensed, and 10 ⁇ g of psPAX2 (Addgene) and 3 ⁇ g of pMD2.G ( After crystallization of Calcium phosphate with Addgene) vector using Calcium phosphate and Hepes-buffered solution, it was introduced into 293FT cells. Thereafter, the culture supernatant containing the virus (lentivirus) was collected at 24 hour intervals after 48 hours of 293FT cells into which the expression vector was introduced.
  • the collected supernatant was centrifuged for 2 hours at 21,000 rpm in an ultra-high-speed centrifuge to concentrate the virus.
  • Transduction was performed by mixing the concentrated virus with 4 ⁇ g/ml of polybrene and adding it to the culture medium of THP-1 cells, a macrophage cell line.
  • the efficiency of transduction was increased by changing the culture medium of THP-1 cells to a culture medium into which the virus was concentrated twice at 24-hour intervals. 24 hours after the last change of the culture medium, some of the transduced THP-1 cells were used to measure the transduction efficiency.
  • the transduction efficiency was measured by flow cytometry to measure the amount of GFP expression inside the cell.
  • THP-1 cells transduced with each of the four recombinant chimeric antigen receptor expression vectors and THP-1 cells transduced with an empty vector (Mock) were analyzed for transduction efficiency. The result of comparison with the transduction rate of 1 cell is shown in FIG. 18 .
  • Example 8 Cells expressing CD138 (CD138 positive cells) co-culture Verification of CD138-specific cytotoxicity through
  • Example 3 the target cell selected in Example 3, to confirm whether the four types of chimeric antigen receptor-expressing THP-1 cells prepared above show toxicity by selectively recognizing CD138-expressing cells (CD138 positive cell, CD138 + cell).
  • One cell was co-cultured with 4 types of chimeric antigen receptor-expressing THP-1 cells or THP-1 cells (Mock) into which an empty vector was introduced.
  • 7-Aminoactinomycin D (7-AAD) was treated 24 or 48 hours later, and the fluorescence expression level of 7-AAD was compared by flow cytometry to measure the cytotoxicity of each chimeric antigen receptor-expressing THP-1 cell.
  • did 7-Aminoactinomycin D (7-AAD) binds to the DNA of apoptosis-induced cells and becomes fluorescent (Zembruski et al., 2012).
  • each of the four types of chimeric antigen receptor-expressing THP-1 cells was dispensed at 5 ⁇ 10 5 into wells of a 24-well cell culture dish, and target cells, CD138 + cells (AsPC-1 cells) were dispensed at 1 ⁇ 10 5 each, effector: target Coculture was performed at a ratio of 5:1.
  • the volume per well of the co-culture was 1 ml, and centrifugation was performed at 250 g for 4 minutes using a centrifuge to close the cell spacing.
  • the cells in each well were centrifuged for 3 minutes at 300 g using a centrifuge to remove the supernatant, and then the cells were stained with 7-AAD (Biolegend).
  • the degree of cytotoxicity of the four types of chimeric antigen receptor-expressing THP-1 cells prepared was quantified by measuring the ratio of cells showing fluorescence by 7-AAD using flow cytometry.
  • FIG. 19 As a result, as shown in FIG. 19, after 24 hours of co-culture of THP-1 cells expressing the chimeric antigen receptor with CD138 + cells (ASPC-1 cells), the THP that does not express the chimeric antigen receptor is shown in FIG. -1 cells (Mock) showed cytotoxicity of about 11.0%, whereas 3-4-3 THP-1 cells (TLR-3+TLR-4+Fc ⁇ RI ⁇ ITAM), which are THP-1 cells expressing four types of chimeric antigen receptors trimer), 3-4-2A THP-1 cell (TLR-3+TLR4+Fc ⁇ R2A ⁇ ITAM trimer), 3-4-1B THP-1 cell (TLR-3+TLR4+Fc ⁇ RI ⁇ ITAM trimer), 3-4-ABC In THP-1 cells (TLR-3 + TLR4 + Fc ⁇ R2A ⁇ ITAM monomer + Fc ⁇ RI ⁇ ITAM monomer + Fc ⁇ RI ⁇ ITAM monomer), it was confirmed that they showed very high cytotoxicity of 19.6%, 17.5%, 20
  • the transformed THP-1 cells containing the chimeric antigen receptor according to the present invention were CD138 By showing protein-specific cytotoxicity, it was confirmed that cancer cells can be treated using the transformed antigen-specific specialized antigen-expressing cells.

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Abstract

La présente invention concerne un agent thérapeutique cellulaire ou une composition pharmaceutique pour le traitement du cancer, comprenant, en tant que principe actif, une cellule présentatrice d'antigène professionnelle spécifique de l'antigène transformée comprenant un récepteur antigénique chimérique (CAR). En particulier, le récepteur antigénique chimérique comprend un domaine de liaison à l'antigène se liant spécifiquement à CD138. La cellule transformée selon la présente invention procure un domaine de signalisation chimérique amélioré qui induit l'activité de la cellule présentatrice d'antigène professionnelle lorsque cette dernière se lie de manière spécifique à un antigène. Par exemple, les cellules présentatrices d'antigène professionnelles transformées via un vecteur capable de surexprimer une protéine recombinée comprenant un domaine de signalisation dans une nouvelle cellule synthétique possèdent une cytotoxicité spécifique des carcinomes exprimant CD138, la protéine recombinée étant une protéine dans laquelle un dimère, dans lequel une paire de domaines extracellulaires de DR6 sont chacun liés à une région constante de chaîne lourde d'immunoglobuline humaine G1 (IgG1), et à un motif d'activation basé sur la tyrosine des immunorécepteurs (ITAM) d'un TLR-3, d'un TLR-4 et d'un récepteur IgE à chaîne gamma (récepteur Fcε I à chaîne gamma, FcεRIγ), d'un récepteur IgG 2A à chaîne alpha (récepteur Fcγ 2A à chaîne alpha, FcγR2Aα) ou un récepteur IgE à chaîne bêta (Fcε receptor I beta chain, FcεRIβ), ces derniers jouant un rôle important dans la transduction du signal des cellules présentatrices d'antigène professionnelles, sont des sous-unités, et trois ITAM sont liés par un lieur. Par conséquent, les cellules présentatrices d'antigène professionnelles surexprimant le récepteur antigénique chimérique selon la présente invention peuvent être utiles en tant que produit de thérapie cellulaire immunitaire pour traiter les carcinomes exprimant CD138.
PCT/KR2022/018876 2021-12-13 2022-11-25 Cellule présentatrice d'antigène professionnelle spécifique d'un antigène transformée contenant un récepteur antigénique chimérique (car) et son utilisation Ceased WO2023113293A1 (fr)

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