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WO2022239986A1 - Récepteur antigénique chimérique comprenant le domaine extracellulaire de baff et son utilisation - Google Patents

Récepteur antigénique chimérique comprenant le domaine extracellulaire de baff et son utilisation Download PDF

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WO2022239986A1
WO2022239986A1 PCT/KR2022/005559 KR2022005559W WO2022239986A1 WO 2022239986 A1 WO2022239986 A1 WO 2022239986A1 KR 2022005559 W KR2022005559 W KR 2022005559W WO 2022239986 A1 WO2022239986 A1 WO 2022239986A1
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cells
baff
cell
chimeric antigen
receptor
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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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Definitions

  • the present invention relates to a chimeric antigen receptor (CAR) comprising a BAFF extracellular domain; a polynucleotide encoding the chimeric antigen receptor protein; a vector containing the polynucleotide; T cells or natural killer cells (NK cells) transformed with the vector; It relates to a cell therapy or a pharmaceutical composition for the treatment of cancer and systemic lupus erythematosus, comprising the cells as an active ingredient.
  • CAR chimeric antigen receptor
  • B-cell activating factor is a cytokine belonging to the tumor necrosis factor (TNF) ligand, BLyS (B-lymphocyte stimulator), TALL-1 (TNF- and ApoL related leukocyte-expressed ligand 1), THANK (TNF homologue that activates apoptosis, nuclear factor- ⁇ B and c-Jun NH2-terminal kinase), and is called TNFSF13B (TNFsuperfamily member 13B) or zTNF4 (Mackay and Browning, 2002).
  • TNFSF13B TNFsuperfamily member 13B
  • zTNF4 Mackay and Browning, 2002.
  • BAFF is a type 2 transmembrane protein originally composed of 285 amino acids.
  • BAFF is secreted from macrophages, monocytes, and dendritic cells (Mackay and Browning, 2002).
  • Receptors that bind to BAFF include the BAFF receptor, B-cell maturation antigen (BCMA), and Transmembrane activator and CAML interactor (TACI) (Mackay and Browning, 2002).
  • BAFF receptors are expressed on most human B cells, except for plasma cells in the bone marrow, and BCMA is mainly expressed on antibody-secreting human cells (Mackay and Schneider, 2009).
  • BCMA B-cell maturation antigen
  • TACI Transmembrane activator and CAML interactor
  • BAFF receptors are expressed on most human B cells, except for plasma cells in the bone marrow, and BCMA is mainly expressed on antibody-secreting human cells (Mackay and Schneider, 2009).
  • TACI is also expressed in most peripheral B cells (Mackay and Schneider, 2009). Therefore, signal transduction by the interaction between BAFF and three receptors that bind to BAFF, the BAFF
  • BCMA is a marker for multiple myeloma (Carpenter et al., 2013), breast cancer, T cell lymphoma, and B cell chronic lymphocytic leukemia (B -CLL) and non-small cell lung cancer (Haiat et al., 2006; Dou et al., 2016; Kampa et al., 2020).
  • the BAFF receptor is a B-cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, B cell It is expressed in various lymphomas such as B-cell non-Hodgkin lymphoma, and also in leukemias such as acute lymphoblastic leukemia (ALL) and chronic lymphoblastic leukemia (CLL).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphoblastic leukemia
  • BAFF receptors are overexpressed in B cells of autoimmune diseases such as systemic lupus erythematosus (SLE) (Haiat et al., 2006; Parameswaran et al., 2010; Takahata et al., 2010; Yang et al., 2010). al., 2014).
  • SLE systemic lupus erythematosus
  • TACI is found in multiple myeloma, breast cancer, thyroid carcinoma, and non-small cell lung cancer (Moreaux et al., 2009; Dou et al., 2016; Kampa et al., 2020).
  • cytotoxic T lymphocytes CTL
  • NK cells natural killer cells
  • a key obtained by grafting a single-chain variable fragment (scFv) of an antibody that recognizes an antigen to a domain grafted to CD3 zeta or the cytoplasmic signaling domain of another protein has been attempted as a method of delivering a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the chimeric antigen receptor is grafted onto T cells or natural killer cells, the anticancer activity of T cells or natural killer cells can be achieved only by recognizing the specific antigen of the single-chain Fv fragment, regardless of signal transduction by antigen presenting cells (APCs). It 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
  • CAR-T T cells recognizing BCMA or BAFF receptors as tumor antigens (US Patent Publication No. 2020-0283534) or water-soluble TACI immunoglobulin compositions and methods of use (US Patent Registration No. 08524232) have been disclosed.
  • the invention of using the BAFF receptor, BCMA, and TACI-expressing cancer cell-expressing extracellular domain (extracellular domain) of BAFF as an antigen-binding domain has not been described for use as an immunocancer therapeutic agent.
  • the present inventors have identified or induced the expression of BAFF receptors, BCMA, and TACI in various cancer cells, and the fact that the BAFF and BAFF receptors, BCMA, and TACI can bind to the extracellular domain of BAFF.
  • Cancer-specific chimeric antigen receptor-expressing T cells and natural killer cells expressing the BAFF receptor, BCMA, and TACI used are prepared, and the T cells or natural killer cells are specific for cells expressing the BAFF receptor, BCMA, and TACI It was confirmed through experiments that there is a cytotoxic ability. At this time, a fusion protein linking each of the extracellular domains of BAFF with the human immunoglobulin G 1 heavy chain constant region was created to amplify signal transduction strength.
  • chimeric antigen receptors have only one antigen-recognizing site by a single-chain Fv fragment, whereas the chimeric antigen receptor designed by the present inventors binds each of the extracellular domains of BAFF to human human immunoglobulin G 1 By linking with the heavy chain constant region (IgG 1 heavy chain constant region), two extracellular domains of BAFF per chimeric antigen receptor recognize antigens, thereby amplifying signal transduction strength.
  • the heavy chain constant region IgG 1 heavy chain constant region
  • Another object of the present invention is to provide a polynucleotide encoding the chimeric antigen receptor protein, a vector containing the polynucleotide, and T cells or natural killer cells transformed with the vector.
  • Another object of the present invention is to provide a cell therapy or a pharmaceutical composition for treating cancer and systemic lupus erythematosus expressing BAFF receptor, BCMA or TACI, including the transformed T cells or natural killer cells.
  • the present invention is an antigen binding site, an extracellular domain; transmembrane domain; And a chimeric antigen receptor (CAR) comprising an intracellular signaling domain, wherein the antigen binding domain comprises an extracellular domain of BAFF that specifically binds to a BAFF receptor, BCMA or TACI.
  • CAR chimeric antigen receptor
  • the present invention provides a polynucleotide encoding the chimeric antigen receptor protein, a vector containing the polynucleotide, and T cells or natural killer cells transformed with the vector.
  • the present invention provides a cell therapy or a pharmaceutical composition for treating cancer and systemic lupus erythematosus expressing BAFF receptor, BCMA or TACI, including the transformed T cells or natural killer cells.
  • a chimeric antigen receptor according to the present invention comprises an extracellular domain of BAFF that specifically binds to the BAFF receptor, BCMA or TACI. Therefore, in the case of cytotoxic T cells or natural killer cells transformed with a vector capable of overexpressing the chimeric antigen receptor, they have cytotoxicity specifically for carcinoma and autoimmune diseases expressing the BAFF receptor, BCMA or TACI. It can be usefully used as an immune cell therapy for the treatment of cancer and systemic lupus erythematosus.
  • FIG. 1 is a schematic diagram showing cDNA regions of each domain expressing a chimeric antigen receptor according to an embodiment of the present invention.
  • FIG. 2A is a schematic diagram of a lentiviral vector according to an embodiment of the present invention.
  • 2B is a schematic diagram of a retroviral vector according to an embodiment of the present invention.
  • FIG. 3 is a schematic diagram of a chimeric antigen receptor expressed on the surface of cytotoxic T cells or natural killer cells according to an embodiment of the present invention.
  • FIG. 4a is a schematic diagram of inserting BAFF receptor cDNA into a retroviral vector to express BAFF receptor, which is a target of the chimeric antigen receptor provided in the present invention, in Chinese hamster ovary (CHO) cells.
  • Figure 4b is a schematic diagram of inserting BCMA cDNA into a retroviral vector to express BCMA, which is the target of the chimeric antigen receptor provided in the present invention, in Chinese hamster ovary (CHO) cells.
  • FIG. 4c is a schematic diagram of inserting TACI cDNA into a retroviral vector to express TACI, which is the target of the chimeric antigen receptor provided in the present invention, in Chinese hamster ovary (CHO) cells.
  • Figure 5a is a diagram showing the results of measuring the expression level of BAFF receptor in Chinese hamster ovary (CHO) cells expressing BAFF receptor, which is a target of the chimeric antigen receptor provided in the present invention, using flow cytometry.
  • Figure 5b is a diagram showing the results of measuring the expression level of BCMA using flow cytometry in CHO cells expressing BCMA, which is the target of the chimeric antigen receptor provided in the present invention.
  • Figure 5c is a diagram showing the results of measuring the expression level of TACI using flow cytometry in CHO cells expressing TACI, which is the target of the chimeric antigen receptor provided in the present invention.
  • Figure 6a is a diagram showing the result of comparing the expression ratio of GFP in cytotoxic T cells (BAFF CAR-T cells) transduced with an expression vector according to an embodiment of the present invention using a lentivirus system.
  • FIG. 6B is a diagram showing the results of comparing the expression ratio of BAFF in natural killer cells (BAFF CAR-NK cells) transduced with an expression vector according to an embodiment of the present invention using a retroviral system.
  • FIG. 7a is a diagram showing whether BAFF receptors are recognized by cytotoxic T cells (BAFF CAR-T cells) transduced with an expression vector according to an embodiment of the present invention using a lentivirus system, using flow cytometry.
  • Figure 7b is a diagram showing whether cytotoxic T cells (BAFF CAR-T cells) transduced with an expression vector according to an embodiment of the present invention using a lentivirus system recognize BCMA using flow cytometry.
  • Figure 7c is a diagram showing whether cytotoxic T cells (BAFF CAR-T cells) transduced with an expression vector according to an embodiment of the present invention using a lentivirus system recognize TACI using flow cytometry.
  • 8A is a diagram showing whether BAFF CAR-NK cells transduced with an expression vector according to an embodiment of the present invention using a retroviral system recognize BAFF receptors using flow cytometry.
  • 8B is a diagram showing whether BAFF CAR-NK cells transduced with an expression vector according to an embodiment of the present invention using a retroviral system recognize BCMA using flow cytometry.
  • 8C is a diagram showing whether TACI is recognized by natural killer cells (BAFF CAR-NK cells) transduced with an expression vector according to an embodiment of the present invention using a retroviral system, using flow cytometry.
  • BAFF CAR-NK cells natural killer cells
  • Figure 9a is a cytotoxic T cell (BAFF CAR-T cell) according to an embodiment of the present invention or a cytotoxic T cell (Mock T cell) transduced with an empty vector as an effector cell, BAFF receptor, It is a diagram showing the results of measuring cytotoxicity to CHO cells expressing BCMA and TACI, respectively.
  • FIG. 9B shows BAFF receptors, BCMA, It is a diagram showing the results of measuring cytotoxicity to CHO cells expressing TACI, respectively.
  • an antigen-binding domain transmembrane domain; And a chimeric antigen receptor (CAR) comprising an intracellular signaling domain, wherein the antigen binding domain comprises an extracellular domain of BAFF that specifically binds to a BAFF receptor, BCMA or TACI. do.
  • CAR chimeric antigen receptor
  • the "Chimeric antigen receptor (CAR)" naturally binds to a desired antigen without the mediation of an antigen-presenting cell (APC) or antibody necessary for the activation of T cells or natural killer cells, resulting in an antigen-antibody reaction. It refers to a fusion protein to be expressed in T cells or natural killer cells in order to induce activation of T cells and attack cells expressing the corresponding antigen. That is, it can be seen as a protein that binds to an antigen when expressed in T cells or natural killer cells and induces the activation of these cells. Through this, it may be a protein that recognizes an antigen specific to a cell to cause an immune response, and 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.
  • APC antigen-presenting cell
  • 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 that can specifically bind to the BAFF receptor, BCMA or TACI.
  • 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
  • B-cell activating factor is a cytokine belonging to the tumor necrosis factor (TNF) ligand, BLyS (B-lymphocyte stimulator), TALL-1 (TNF- and ApoL related leukocyte-expressed ligand 1), THANK (TNF homologue that activates apoptosis, nuclear factor- ⁇ B and c-Jun NH2-terminal kinase), is called TNFSF13B (TNFsuperfamily member 13B) or zTNF4.
  • TNFSF13B tumor necrosis factor-expressed ligand 1
  • THANK TNF homologue that activates apoptosis, nuclear factor- ⁇ B and c-Jun NH2-terminal kinase
  • TNFSF13B TNFsuperfamily member 13B
  • BAFF is a type 2 transmembrane protein originally composed of 285 amino acids. After being expressed on the cell surface as a homotrimer, the transmembrane portion is cut off and secreted out of the cell.
  • BAFF is secreted from macrophages, monocytes, and dendritic cells.
  • Receptors that bind to BAFF include the BAFF receptor, B-cell maturation antigen (BCMA), and Transmembrane activator and CAML interactor (TACI).
  • BCMA B-cell maturation antigen
  • TACI Transmembrane activator and CAML interactor
  • the extracellular domain of the BAFF may consist of SEQ ID NO: 1 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto.
  • 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.
  • BAFF receptor In the present invention, "BAFF receptor”, “BCMA”, and “TACI” are observed in various malignant cells and autoimmune diseases, preferably systemic lupus erythematosus (SLE). So far, carcinomas expressing "BAFF receptor”, “BCMA”, and “TACI” are acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), and follicular lymphoma (follicular lymphoma).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphoblastic leukemia
  • follicular lymphoma follicular lymphoma
  • lymphoma mantle cell lymphoma
  • B-cell lymphoma diffuse large B-cell lymphoma (DLBCL)
  • B-cell non-Hodgkin lymphoma lymphoma B-cell non-Hodgkin lymphoma lymphoma
  • multiple myeloma T cell lymphoma, breast cancer, thyroid cancer, and non-small cell lung cancer. , but not limited thereto.
  • the "transmembrane domain” is a site that connects the extracellular domain of BAFF and the co-stimulatory and essential signaling domains between the cell membrane, and the intracellular signaling domain is an immune cell by binding of the antigen-binding domain. refers to the site that activates the immune response of
  • the transmembrane domain is selected from the group consisting of CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154 It may include one or more types, and preferably, the CD8 may be a transmembrane domain of CD8 ⁇ , which may consist of SEQ ID NO: 3 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto not.
  • intracellular signaling domain means a site that activates the immune response of immune cells by binding to an antigen binding domain.
  • the intracellular domain may be CD28, 4-1BB, CD3 zeta, or a combination thereof, but is not limited thereto.
  • the chimeric antigen receptor according to the present invention uses CD28, 4-1BB, and CD3 zeta as intracellular signaling domains, and has a high activity to kill cancer cells, especially cancer cells expressing BAFF receptors, BCMA, and TACI. can represent
  • the CD28 has SEQ ID NO: 4 or 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more sequence homology thereto, and the amino acid sequence represented by SEQ ID NO: 4 4-1BB (CD137) is SEQ ID NO: 5 or 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more.
  • sequence homology may consist of an amino acid sequence showing a function substantially equivalent to the amino acid sequence represented by SEQ ID NO: 5, CD3 zeta functions as an NK cell activation domain, and SEQ ID NO: 6 or 70 % or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more sequence homology, and an amino acid sequence that exhibits a function substantially equivalent to that of the amino acid sequence represented by SEQ ID NO: 6 It can be done.
  • the antigen-binding domain may include a CD8 ⁇ signal peptide, and the CD8 ⁇ signal peptide may consist of SEQ ID NO: 7 or an amino acid sequence showing 95% or more homology thereto, but is not limited thereto.
  • a polynucleotide encoding the chimeric antigen receptor protein is provided.
  • the polynucleotide encoding the antigen receptor of the present invention changes the amino acid sequence of the antigen receptor expressed from the coding region due to codon degeneracy or in consideration of codons preferred in organisms intended to express the antigen receptor.
  • Various modifications may be made to the coding region within a range not specified, and various modifications or modifications may be made to a part other than the coding region within a range that does not affect gene expression, and such modified genes are also within the scope of the present invention. It will be well understood by those skilled in the art.
  • nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included in the scope of the present invention.
  • a vector containing the polynucleotide and a cell transformed with the vector are provided.
  • Vectors 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 or a retro-virus vector can be used, and in the following embodiment of the present invention, the pCDH-CMV-MCS-EF1-copGFP vector (lenti-virus vector) and pLNCX2 (retro-viral vector) was used, but is not limited thereto.
  • cells can be transformed by introducing a chimeric antigen receptor that specifically binds to BAFF receptor, BCMA or TACI into cells through the vector.
  • the cells may be T cells, tumor-infiltrating lymphocytes, B cells, natural killer cells, or NKT cells, and preferably may be cytotoxic T cells or natural killer cells.
  • the cells may be obtained or prepared from bone marrow, peripheral blood, peripheral blood mononuclear cells or umbilical cord blood, and the cells may be human cells, but are not limited thereto.
  • Cells transformed by introducing the chimeric antigen receptor of the present invention as described above recognize BAFF receptor, BCMA or TACI as an antigen and bind strongly thereto.
  • chimeric antigen receptor T cell (hereinafter, simply abbreviated as 'CAR-T cell')" or “chimeric antigen receptor expressing NK cell (chimeric antigen receptor NK cell, hereinafter) Shortly abbreviated as 'CAR-NK cell')
  • 'CAR-NK cell' is a chimeric antigen receptor that specifically responds to cancer cells rather than the original T cell receptor or NK cell receptor by a method such as transduction of normal T cells or natural killer cells. refers to T cells or NK cells expressing T cells or NK cells having this receptor exhibit cytotoxicity by inducing apoptosis of target cells.
  • CAR-T cells or CAR-NK cells may be cells into which the chimeric antigen receptor of the present invention is introduced into cytotoxic T cells or NK cells.
  • the cells have the advantage of anticancer-specific targeted therapy, which is an existing advantage of CAR-T therapeutics.
  • the CAR-T cells or CAR-NK cells of the present invention recognize cancer cells expressing BAFF receptors, BCMA, and TACI to effectively can destroy
  • the present invention provides a cell therapeutic agent containing the cells or a pharmaceutical composition for treating cancer containing the same as an active ingredient.
  • cell therapy refers to cells and tissues prepared from a subject through isolation, culture, and special manipulation, and is a pharmaceutical product used for the purpose of treatment, and is a living autologous, allogeneic, or xenogeneic living agent to restore the function of a cell or tissue. It refers to drugs used for the purpose of treatment through a series of actions, such as proliferation and selection of cells in vitro or altering the biological characteristics of cells in other ways.
  • treatment refers to all activities that improve or beneficially change symptoms caused by cancer and autoimmune diseases 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. have.
  • 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 treatment of cancer expressing BAFF receptors, BCMA, and TACI. 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 humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, or animals that have or may develop cancer expressing BAFF receptors, BCMA, or TACI. All animals, including guinea pigs.
  • the type of subject is included without limitation as long as the disease can be effectively treated by administering the pharmaceutical composition of the present invention to the subject.
  • the autoimmune disease is preferably systemic lupus erythematosus (SLE), but is not limited thereto.
  • Types of the cancer include acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, B B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), B-cell non-Hodgkin lymphoma, multiple myeloma, T-cell lymphoma It may be selected from the group consisting of (T cell lymphoma), breast cancer, thyroid cancer, and non-small cell lung cancer, but is not limited thereto.
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphoblastic leukemia
  • follicular lymphoma mantle cell lymphoma
  • B B-cell lymphoma diffuse large B-cell lymphoma
  • B-cell non-Hodgkin lymphoma multiple myeloma
  • an antigen recognition site in which a pair of BAFF extracellular domains are linked to human immunoglobulin G 1 heavy chain constant region (IgG 1 heavy chain constant region) and transmembrane transmembrane of CD8 ⁇
  • the protein coding sequences of the intracellular domains of each of the domains CD28, 4-1BB, and CD3 zeta were identified in a gene database.
  • FIG. 1 A schematic diagram showing the cDNA region of each domain expressing the chimeric antigen receptor is shown in FIG. 1 .
  • lentivirus-derived expression of the corresponding gene It was cloned into a vector, pCDH-CMV-MCS-EF1-copGFP (System Biosciences) or a retrovirus-derived expression vector, pLNCX2 (Addgene).
  • a primer that creates a restriction enzyme XbaI cleavage site sequence is synthesized at the 5' end, a restriction enzyme cleavage site is created by polymerase chain reaction, and a restriction enzyme cleavage site 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 NotI and polymerase chain reaction. After that, the 5' end of the gene having a restriction enzyme cleavage site due to polymerase chain reaction was treated with XbaI, and the 3' end was treated with NotI. In addition, the multicloning site of the expression vector was treated with XbaI and NotI 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.
  • a primer that creates a restriction enzyme Bgl II cleavage site sequence is synthesized at the 5' end, a restriction enzyme cleavage site is made by polymerase chain reaction, and a restriction enzyme NotI cleavage site sequence is also added at the 3' end.
  • a primer was synthesized to make a restriction enzyme cleavage site by polymerase chain reaction. After that, the 5' end of the gene having a restriction enzyme cleavage site due to polymerase chain reaction was treated with BglII, and the 3' end was treated with NotI. In addition, the multicloning site of the expression vector was treated with BglII and NotI 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.
  • a recombinant vector BAFF receptor, BCMA, TACI.CAR- that recognizes BAFF receptor, BCMA, and TACI and expresses a protein obtained by fusing a human immunoglobulin G 1 heavy chain constant region and a signaling protein domain (see FIG. 3).
  • T.pCDH-CMV-MCS-EF1-copGFP and the BAFF receptor, BCMA, TACI.CAR-NK.pLNCX2 were completed (see Figures 2a and 2b).
  • each of the BAFF receptor, BCMA, and TACI genes had a restriction enzyme Bgl II cleavage site sequence at the 5' end and a restriction enzyme Hind III cleavage site at the 3' end. It was cloned into the pLNCX2 vector using the sequence (see FIGS. 4a, 4b and 4c).
  • a retroviral system using 293GPG cells was used to introduce the pLNCX2 vector into Chinese hamster ovary (CHO) cells (ATCC) into which BAFF receptor, BCMA, and TACI genes had been inserted.
  • CHO Chinese hamster ovary
  • ATCC Chinese hamster ovary
  • 6 3 ⁇ 10 293GPG cells were dissolved in 10 ml of DMEM culture medium containing 10% calf serum, inoculated into a 100 ⁇ cell culture dish, and cultured for 24 hours.
  • Each of the previously prepared recombinant vectors (BAFF-R.pLNCX2 vector, BCMA.pLNCX2 vector, TACI.pLNCX2 vector) (20 ⁇ g) was crystallized using calcium phosphate and HEPES-buffered solution, and then added to the previously cultured 293GPG cell culture medium. has been added Thereafter, the culture medium was replaced at 24 hour intervals over 72 hours, and the culture supernatant of 293GPG cells containing the retrovirus was collected and stored.
  • the collected retrovirus-containing supernatant was centrifuged at 21,000 rpm for 2 hours using an ultra-high-speed centrifuge, and then reconstituted in DMEM (welgene) containing 10% calf serum to be concentrated 100 times compared to before concentration.
  • DMEM fetal calf serum
  • To transduce the concentrated retrovirus into CHO cells the CHO cells were inoculated in a 24-well cell culture dish at a density of 5 ⁇ 10 5 /ml. Then, a mixture obtained by adding 8 ⁇ g/ml of polybrene to the concentrated retrovirus was added to the culture medium of CHO cells, and cultured for 24 hours.
  • BAFF receptor After proliferation, the expression levels of BAFF receptor, BCMA, and TACI in CHO cells were measured by flow cytometry, respectively, and the recombinant vectors (BAFF-R.pLNCX2 vector, BCMA.pLNCX2 vector, TACI.pLNCX2 vector) were introduced into each CHO cell.
  • Anti-human BAFF receptor antibody Biolegend
  • anti-human BCMA receptor antibody Biolegend
  • anti-human TACI receptor antibody Biolegend
  • CHO cell lines that strongly express BAFF receptor, BCMA, and TACI were used as target cells, and mock CHO cell lines that do not express BAFF receptor, BCMA, and TACI were used as target cells. was used as a negative control.
  • cytotoxic T cells were first isolated from human peripheral blood mononuclear cells. After purchasing human peripheral blood mononuclear cells (Medi Lab Korea), magnetic-activated cell sorting was used to isolate cytotoxic T cells. Peripheral blood mononuclear cells are combined with an antibody (CD8 + T cell biotin-conjugated antibody cocktail, Miltenyi Biotec) capable of binding to other immune cells except for cytotoxic T cells, and then the antibodies are combined with magnetic microbeads (anti- biotin microbead) (Miltenyi Biotec).
  • an antibody CD8 + T cell biotin-conjugated antibody cocktail, Miltenyi Biotec
  • magnetic microbeads anti- biotin microbead
  • the microbeads, antibodies attached thereto, and cells were passed through a magnetic separation column (Miltenyi Biotec) to obtain cytotoxic T cells unlabeled with the antibodies.
  • flow cytometry was performed using CD8 and CD3 ⁇ , which are cell surface factors of cytotoxic T cells, and as a result, the purity was over 95%.
  • cytotoxic T cells Contains 1 ⁇ 10 6 /ml of human CD3 and CD28 antibody-coated magnetic beads (Thermo Fisher Scientific) and 10% calf serum supplemented with 100 U/ ⁇ l recombinant human IL-2 for activation of isolated cytotoxic T cells.
  • the cells were reconstituted in RPMI (Welgene) at a density of 1.5 ⁇ 10 6 cells/ml, inoculated into a 24-well cell culture dish, and cultured for 24 hours.
  • Example 5 Construction of chimeric antigen receptor-expressing cytotoxic T cells
  • Example 2 In order to introduce the recombinant vector prepared in Example 2 into cytotoxic T cells, a lentiviral system using 293FT cells (Thermo Fisher Scientific) 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 incubation, when the cells have grown to cover 60-70% of the dish, 20 ⁇ g of BAFF.CAR-T.pCDH-CMV-MCS-EF1-copGFP vector DNA was mixed with 10 ⁇ g of psPAX2 (Addgene) and 3 ⁇ g of pMD2. .G (Addgene) vector was crystallized using Calcium phosphate and Hepes-buffered solution and then introduced into 293FT cells.
  • culture supernatants containing lentivirus were collected at intervals of 24 hours 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 using an ultra-high speed centrifuge to concentrate the virus.
  • the concentrated virus was mixed with 4 ⁇ g/ml of polybrene and added to the culture medium of activated cytotoxic T cells, followed by centrifugation at 1,800 g for 75 minutes using a centrifuge for transduction.
  • the centrifuged cytotoxic T cells were further cultured for 4 hours and then replaced with RPMI culture medium containing 10% calf serum. After 48 hours, some of the transduced cytotoxic T cells were used to measure transduction efficiency.
  • the transduction efficiency was measured by flow cytometry for the amount of GFP expression inside the cell, and the transduced cytotoxic T cell (BAFF CAR-T cell) was transduced with a virus constructed with an empty vector. Cytotoxic T cell (Mock T cell) transduced ) The results compared with the transduction rate of ) are shown in Figure 6a.
  • the collected retrovirus-containing supernatant was centrifuged at 21,000 rpm for 2 hours using an ultra-high-speed centrifuge, and then reconstituted in Myelocult H5100 culture medium (STEMCELL) containing 10% calf serum to be concentrated 100 times compared to before concentration.
  • NK92MI cells American type culture collection, ATCC
  • NK92MI cells were inoculated in a 24-well cell culture dish at a density of 5 ⁇ 10 5 /ml. Then, a mixed solution in which 8ug/ml of polybrene was added to the concentrated retrovirus was added to the culture medium of NK92MI cells, and cultured for 24 hours.
  • NK92MI cells treated with retrovirus were cultured using Myelocult H5100 culture medium supplemented with neomycin antibiotic (600 ⁇ g/ml) 24 hours after the culture medium change for selection of cells into which the gene was introduced. After 14 days of neomycin selection, NK92MI cells were transferred to a fresh culture medium and grown for one week.
  • Example 7 chimeric antigen receptor and BAFF receptor, BCMA or TACI Affinity measurement of expressing human cell lines
  • the BAFF CAR-T cell and BAFF CAR-NK cell prepared in Examples 5 and 6 were To measure the binding to BAFF receptors, BCMA and TACI, soluble recombinant human BAFF receptor IgG (Acrobiosystems), soluble recombinant BCMA IgG (R&D systems), and soluble recombinant TACI IgG (R&D systems) were respectively tested in BAFF CAR-T cells and BAFF CAR-NK cells were treated and confirmed through flow cytometry.
  • soluble recombinant human BAFF receptor IgG, soluble recombinant BCMA IgG, and soluble recombinant TACI IgG do not bind to Mock T cells, but bind to BAFF CAR-T cells (see FIGS. 7a to 7c), and also to Mock NK cells. It did not bind, but bound to BAFF CAR-NK cells (see FIGS. 8a to 8c).
  • BAFF CAR-T cells and BAFF CAR-NK cells expressing chimeric antigen receptors prepared based on the above results were used as effector cells, and CHO cell lines expressing BAFF receptors, BCMA or TACI were used as target cells (target cells). ), BAFF receptor, BCMA, and TACI-specific cytotoxicity of BAFF CAR-T cells and BAFF CAR-NK cells were verified.
  • Example 8 CAR-T cells BAFF receptor, BCMA , TACI Verification of expression cell-specific cytotoxicity
  • BAFF receptor which is the target cell selected in Example 3, to determine whether the prepared chimeric antigen receptor-expressing cytotoxic T cell specifically recognizes cells expressing BAFF receptor, BCMA, or TACI and exhibits toxicity.
  • Expression CHO cells, BCMA-expressing CHO cells, and TACI-expressing CHO cells were co-cultured with BAFF.CAR-T cells or cytotoxic T cells (Mock T cells) into which an empty vector was introduced for 6 hours.
  • a non-radioactive cytotoxicity assay that measures the degree of cytotoxicity of cytotoxic T cells to target cells by the amount of lactate dehydrogenase present in the supernatant after co-culture used
  • cytotoxic T cells (1x10 5 cell) and target cells (1x10 4 cell) into wells of a 96-well cell culture dish, set the effector: target ratio to 10:1, and inoculate so that the volume per well is 100 ⁇ l. Centrifuge for 4 minutes under the condition of 250g using a centrifuge to close the gap between cells. Then, after culturing for 6 hours, 50 ⁇ l of the supernatant from each well was removed and transferred to a transparent 96-well dish for measuring absorbance, and then the enzyme reaction was progressed and stopped by treating the assay solution and 1M hydrochloric acid solution.
  • the degree of cytotoxicity of the cytotoxic T cells in each well was quantified by measuring and converting the absorbance in the 490 nm wavelength band using a fluorescence/emission/absorption meter (multi-detection plate reader).
  • the prepared chimeric antigen receptor-expressing cytotoxic T cells were 27.99% in the BAFF receptor-expressing CHO cell line, 20% in the BCMA-expressing CHO cell line, and TACI-expressing CHO cell line. showed high toxicity of more than 25.42% to Mock CHO cell line, whereas it showed toxicity of 4.15% to Mock CHO cell line.
  • cytotoxic T cells (Mock T cells) introduced with the empty vector as a control group, they showed low toxicity of 3.02% to the BAFF receptor-expressing CHO cell line, 5.58% to the BCMA-expressing CHO cell line, and 4.85% to the TACI-expressing CHO cell line. , showed toxicity of 7.24% against Mock CHO cell line.
  • the chimeric antigen recognition receptor-expressing cytotoxic T cells containing the extracellular domain of BAFF showed cytotoxicity specific to the BAFF receptor, BCMA or TACI protein, and thus cancer-related cancers using the CAR-T cells It was confirmed that cell therapy is possible.
  • BAFF receptor-expressing CHO cells which are the target cells selected in Example 3, to determine whether the prepared chimeric antigen receptor-expressing NK cells specifically recognize BAFF receptor-, BCMA-, and TACI-expressing cells and exhibit toxicity.
  • BCMA-expressing CHO cells and TACI-expressing CHO cells were co-cultured with BAFF.CAR-NK cells or Mock NK cells into which an empty vector was introduced.
  • a non-radioactive cytotoxicity assay was used to measure the degree of cytotoxicity of NK cells to target cells with the amount of lactate dehydrogenase present in the supernatant. .
  • the degree of cytotoxicity of NK cells in each well was quantified by measuring and converting the absorbance in the 490 nm wavelength band using a fluorescence/luminescence/absorption meter (multi-detection plate reader).
  • the prepared chimeric antigen receptor-expressing cytotoxic T cells were 67.88% in the BAFF receptor-expressing CHO cell line, 57.06% in the BCMA-expressing CHO cell line, and 57.06% in the TACI-expressing CHO cell line. While it showed high toxicity of 60.84% or more, it showed toxicity of 31.68% against Mock CHO cell line.
  • mock NK cells introduced with the empty vector as a control group, they showed low toxicity of 22.72% to BAFF receptor-expressing CHO cell lines, 22.49% to BCMA-expressing CHO cell lines, and 27.00% to TACI-expressing CHO cell lines. , showed 26.18% toxicity against Mock CHO cell line.
  • chimeric antigen recognition receptor-expressing NK cells containing the extracellular domain of BAFF showed specific cytotoxicity to the BAFF receptor, BCMA or TACI protein, and thus related cancer cell treatment using the CAR-NK cells was able to confirm that it is possible.

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Abstract

La présente invention concerne : un récepteur antigénique chimérique se liant de manière spécifique à un récepteur BAFF, BCMA ou TACI et comprenant un domaine extracellulaire de BAFF ; un polynucléotide codant pour une protéine du récepteur antigénique chimérique ; un vecteur comprenant le polynucléotide ; une cellule transformée avec le vecteur ; et un produit de thérapie cellulaire ou une composition pharmaceutique pour le traitement du cancer ou du lupus érythémateux disséminé, comprenant la cellule en tant que principe actif. Le récepteur antigénique chimérique selon la présente invention comprend le domaine extracellulaire de BAFF qui se lie de manière spécifique au récepteur BAFF, BCMA ou TACI. Par conséquent, dans le cas de cellules T cytotoxiques ou de cellules tueuses naturelles qui sont transformées avec un vecteur qui peut surexprimer le récepteur antigénique chimérique, les cellules ont une cytotoxicité spécifique aux maladies auto-immunes et au carcinome qui expriment le récepteur BAFF, BCMA, ou TACI, et ainsi les cellules peuvent être utilisées de manière utile en tant qu'agents thérapeutiques de cellules immunitaires pour le traitement du cancer et du lupus érythémateux disséminé.
PCT/KR2022/005559 2021-05-11 2022-04-18 Récepteur antigénique chimérique comprenant le domaine extracellulaire de baff et son utilisation Ceased WO2022239986A1 (fr)

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US9790282B2 (en) * 2013-03-25 2017-10-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anti-CD276 polypeptides, proteins, and chimeric antigen receptors
WO2017222593A1 (fr) * 2016-06-24 2017-12-28 Icell Gene Therapeutics Llc Récepteurs d'antigènes chimériques (car), compositions et procédés associés
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