WO2023068584A1 - Récepteur antigénique chimérique se liant de manière spécifique à cd138 et ses utilisations - Google Patents

Récepteur antigénique chimérique se liant de manière spécifique à cd138 et ses utilisations Download PDF

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WO2023068584A1
WO2023068584A1 PCT/KR2022/014449 KR2022014449W WO2023068584A1 WO 2023068584 A1 WO2023068584 A1 WO 2023068584A1 KR 2022014449 W KR2022014449 W KR 2022014449W WO 2023068584 A1 WO2023068584 A1 WO 2023068584A1
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cells
cell
chimeric antigen
antigen receptor
cancer
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진화섭
전태훈
양준
이창희
이나영
박인병
강석진
박시원
이현정
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주식회사 이뮤노로지컬디자이닝랩
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • C12N2740/00—Reverse transcribing RNA viruses
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Definitions

  • the present invention relates to a chimeric antigen receptor (CAR) that specifically binds to CD138; a polynucleotide encoding the chimeric antigen receptor protein; a vector containing the polynucleotide; It relates to a T cell or natural killer cell (NK cell) transformed with the vector and a cell therapy or pharmaceutical composition for cancer treatment containing the cell as an active ingredient.
  • CAR chimeric antigen receptor
  • 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.
  • 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
  • death receptor 6 also called tumor necrosis factor (TNF) receptor superfamily 21 (TNFRSF21)
  • TNF tumor necrosis factor receptor superfamily 21
  • DR6 is a transmembrane protein composed of 392 amino acids. Upon receiving a signal, 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).
  • ADC antibody-drug conjugate
  • CAR chimeric antigen receptor
  • An invention using a chimeric antigen receptor comprising the DR6 extracellular domain as an antigen-binding domain as an immunocancer therapeutic agent has not been described.
  • the present inventors use the fact that CD138 expression is confirmed or induced in various cancer cells and that the CD138 and DR6 can bind with high affinity to develop a cancer-specific expression of CD138 using the extracellular domain of DR6.
  • Chimeric antigen receptor-expressing T cells and natural killer cells were prepared, and it was confirmed through experiments that the T cells or natural killer cells had cytotoxic ability specifically to cells expressing CD138.
  • a fusion protein linking each of the extracellular domains of DR6 to the human immunoglobulin G 1 heavy chain constant region (IgG 1 heavy chain constant region) was created to amplify signal transduction strength.
  • chimeric antigen receptors have only one antigen recognition site by a single-chain Fv fragment
  • the chimeric antigen receptor designed by the present inventors uses each of the extracellular domains of DR6 in human immunoglobulin G 1 heavy chain.
  • the constant region IgG 1 heavy chain constant region
  • two extracellular domains of DR6 per chimeric antigen receptor recognize the CD138 antigen, thereby amplifying signal transduction strength.
  • an object of the present invention is to provide a chimeric antigen receptor that specifically binds to CD138.
  • 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 agent or a pharmaceutical composition for treating cancer that kills cancer cells expressing CD138, 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 DR6 (Death receptor 6) that specifically binds to CD138.
  • 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 agent or a pharmaceutical composition for treating cancer that kills cancer cells expressing CD138, including the transformed T cells or natural killer cells.
  • the chimeric antigen receptor according to the present invention comprises an extracellular domain of DR6 that specifically binds to CD138. 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 to carcinoma expressing CD138, so they are useful as immune cell therapeutics for cancer treatment. can be used appropriately.
  • 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. 4 is a diagram showing the results of measuring the expression level of CD138 in cancer cells (RPMI-8226 cell line) expressing CD138, which is the target of the chimeric antigen receptor provided in the present invention, using flow cytometry.
  • FIG. 5 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. 6 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.
  • FIG. 7 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.
  • FIG. 8a 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 8b is a view showing the results 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
  • 8c is a diagram showing whether the prepared recombinant DR6 fusion protein recognizes the target cancer cells (RPMI-8226 cell line) expressing CD138 using flow cytometry.
  • 9a is a diagram showing the results of comparing the expression ratio of GFP in cytotoxic T cells (DR6 CAR-T cells) transduced with an expression vector according to an embodiment of the present invention using a lentivirus system.
  • 9B is a diagram showing the results of comparing the expression ratio of DR6 in natural killer cells (DR6 CAR-NK cells) transduced with an expression vector according to an embodiment of the present invention using a retroviral system.
  • Figure 10a shows CD138 expression using cytotoxic T cells (DR6 CAR-T cells) or cytotoxic T cells (Mock T cells) transduced with an empty vector according to an embodiment of the present invention as effector cells. It is a diagram showing the results of measuring cytotoxicity against cancer cells (RPMI-8226 cell line).
  • 10B shows cancer cells expressing CD138 using natural killer cells (DR6 CAR-NK cells) or mock NK cells transduced with an empty vector as effector cells according to an embodiment of the present invention.
  • RPMI-8226 cell line is a diagram showing the results of measuring cytotoxicity.
  • an antigen-binding domain comprising an intracellular signaling domain, wherein the antigen binding domain comprises an extracellular domain of DR6 (Death receptor 6) that specifically binds to CD138, providing a chimeric antigen receptor 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 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
  • DR6 (Death receptor 6), also called tumor necrosis factor (TNF) receptor superfamily 21 (TNFRSF21), is a transmembrane protein composed of 392 amino acids. It has a death domain that induces The function of DR6 known so far is known to be involved in the prognosis of several immune diseases by regulating T lymphocyte activity.
  • TNF tumor necrosis factor
  • 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 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 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.
  • 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 for carcinomas or as a target for treatment.
  • Carcinomas expressing CD138 known so far include multiple myeloma, breast cancer, pancreatic cancer, lung cancer, hepatocellular carcinoma, bladder cancer, and ovarian cancer. cancer), prostate cancer, and colorectal cancer.
  • the "transmembrane domain” is a site that connects the extracellular domain of DR6 with the co-stimulatory and essential signaling domains between cell membranes, 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 the CD8 may be CD8 ⁇ or CD8 ⁇ , preferably a transmembrane domain of CD8 ⁇ , which may consist of SEQ ID NO: 3 or an amino acid sequence showing 95% or more homology thereto. However, it is not limited thereto.
  • 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, thereby exhibiting a high-activity killing effect on cancer cells, particularly cancer cells expressing CD138.
  • 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 may be transformed by introducing a chimeric antigen receptor that specifically binds to CD138 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 recognize CD138 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 can recognize and effectively destroy cancer cells expressing CD138.
  • 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 in which symptoms caused by cancer are improved or beneficially changed 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 treatment of CD138-expressing 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 treated by administering the pharmaceutical composition of the present invention to the subject.
  • Types of cancer expressing CD138 to be treated in the present invention include multiple myeloma, breast cancer, pancreatic cancer, lung cancer, liver cancer, bladder cancer, Ovarian cancer, prostate cancer, and colorectal cancer may be exemplified, but are not limited thereto.
  • an antigen recognition site in which a pair of extracellular domains of DR6 are linked to a human immunoglobulin G 1 heavy chain constant region, respectively, and a transmembrane domain of CD8 ⁇ ; Protein coding sequences of the intracellular domains of each of 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 .
  • the corresponding gene was used as a lentivirus-derived expression vector, pCDH-CMV -MCS-EF1-copGFP (System Biosciences) or retrovirus-derived expression vector pLNCX2 (Addgene) was cloned.
  • 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. Then, 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 DR6.CAR-T.pCDH-CMV-MCS-EF1- that recognizes CD138 and expresses a protein obtained by fusing a human immunoglobulin G 1 heavy chain constant region and a signaling protein domain (see FIG. 3).
  • copGFP and DR6.CAR-NK.pLNCX2 were completed (see Figs. 2a and 2b).
  • RPMI-8226 a cell line derived from multiple myeloma.
  • ATC a cell line derived from multiple myeloma.
  • flow cytometry 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. 5 .
  • 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.
  • a recombinant vector DR6-IgG.pLNCX2 expressing a protein obtained by fusing the extracellular domain of DR6 recognizing human CD138 with the human immunoglobulin G 1 heavy chain constant region was completed (see Fig. 6). .
  • 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. 8a).
  • 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 8b).
  • the purified soluble recombinant DR6 fusion protein was treated with a cell line (RPMI-8226 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 RPMI-8226 cell line
  • FIG. 8C The results are shown in FIG. 8C. .
  • RPMI-8226 cells expressing CD138 did not bind to control Ig (isotype control), but bound 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 the CD138-expressing RPMI-8226 cells, and recognized the prepared CD138 and produced human immunosuppressive proteins.
  • Cytotoxic T cells DR6.CAR-T cells
  • DR6.CAR-NK cells natural killer cells expressing a recombinant protein in which a globulin G 1 heavy chain constant region and a signaling protein domain are fused, CD138 protein-specific cells This means that it can be used for toxicity verification.
  • 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 8 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 DR6.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 the 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.
  • 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 to perform 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 to measure the amount of GFP expression inside the cells, and the transduced cytotoxic T cells (DR6 CAR-T cells) were compared with cytotoxic T cells (Mock CAR-T cells) transduced with a virus constructed with an empty vector. Transduction rates of T cells) were compared (see FIG. 9a).
  • 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.
  • NK92MI cells DR6 CAR-NK cells
  • DR6 CAR-NK cells DR6 CAR-NK cells
  • NK92MI cells NK92MI cells
  • pLNCX2 pLNCX2
  • RPMI-8226 cells CD138 positive cells
  • DR6 CAR-T cells Meric antigen receptor-expressing cytotoxic T cells
  • Mock CAR-T cells empty vector-transduced cytotoxic T cells
  • 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. After 6 hours of incubation, 50 ⁇ l of the supernatant from each well is removed and transferred to a transparent 96-well dish for measuring absorbance, and then the enzyme reaction is progressed and stopped by treatment with analysis 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 showed high toxicity of 36% or more to the CD138-expressing RPMI-8226 cell line, whereas the empty vector as a control In the case of cytotoxic T cells (Mock CAR-T cells) introduced with , showed 12% toxicity to the RPMI-8226 cell line.
  • RPMI-8226 cells CD138 positive cells
  • the target cells selected in Example 3 were used as chimeric antigens.
  • Receptor-expressing NK cells DR6 CAR-NK cells
  • empty vector-transduced NK cells Boost CAR-NK cells
  • a non-radioactive cytotoxicity assay was used to measure the degree of cytotoxicity of NK cells to target cells by 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 natural killer cells showed high toxicity of 33% or more to the CD138-expressing RPMI-8226 cell line, whereas the control empty vector In the case of the introduced natural killer cells (Mock CAR-NK cells), toxicity was less than 5% to the RPMI-8226 cell line.

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Abstract

La présente invention concerne un récepteur antigénique chimérique (CAR) qui se lie de manière spécifique à CD138 ; un polynucléotide codant pour la protéine du récepteur antigénique chimérique ; un vecteur comprenant le polynucléotide ; et un lymphocyte T ou une cellule tueuse naturelle (cellule NK) transformé au moyen du vecteur, et un agent thérapeutique cellulaire ou une composition pharmaceutique, destiné au traitement du cancer comprenant la cellule en tant que principe actif. Le récepteur antigénique chimérique selon la présente invention comprend un domaine extracellulaire de DR6 qui se lie de manière spécifique à CD138. En conséquence, les lymphocytes T cytotoxiques ou les cellules tueuses naturelles, transformés au moyen d'un vecteur capable de surexprimer le récepteur antigénique chimérique, présentent une cytotoxicité spécifique à l'égard du carcinome exprimant CD138, lesdits lymphocytes T cytotoxiques ou lesdites cellules tueuses naturelles peuvent ainsi être utilisés de manière efficace en tant qu'agent thérapeutique de cellules immunitaires pour le traitement du cancer.
PCT/KR2022/014449 2021-10-22 2022-09-27 Récepteur antigénique chimérique se liant de manière spécifique à cd138 et ses utilisations WO2023068584A1 (fr)

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