WO2024101962A1 - Genetically engineered cells and use thereof - Google Patents

Genetically engineered cells and use thereof Download PDF

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WO2024101962A1
WO2024101962A1 PCT/KR2023/018102 KR2023018102W WO2024101962A1 WO 2024101962 A1 WO2024101962 A1 WO 2024101962A1 KR 2023018102 W KR2023018102 W KR 2023018102W WO 2024101962 A1 WO2024101962 A1 WO 2024101962A1
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amino acid
acid sequence
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정수영
서민구
오민석
장미희
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주식회사 유씨아이테라퓨틱스
한국과학기술연구원
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C07K2319/00—Fusion polypeptide
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to genetically engineered immune effector cells and uses thereof to increase the therapeutic effect for diseases such as cancer by immunotherapy.
  • Cellular immunotherapy is a very promising treatment method for cancer treatment.
  • most immunotherapeutic approaches have the following limitations in their therapeutic effectiveness against most malignant tumors, including solid tumors: (1) Reduced expression of tumor antigens on the surface of tumor cells (which reduces detection of antigens by the immune system) Sikkim); (2) expression of ligands for inhibitory receptors such as PD1, NKG2A, and TIGIT; (3) upregulation of cellular checkpoints, such as CISH, leading to immune cell deactivation; and (4) induction of a microscopic environment that releases substances such as transforming growth factor- ⁇ (TGF- ⁇ ) and adenosine, which suppress immune responses and promote tumor cell proliferation and survival. Therefore, there is a need for an improved method of cellular immunotherapy that can solve at least one of the above-mentioned challenges.
  • TGF- ⁇ transforming growth factor- ⁇
  • TGF- ⁇ signaling plays an important role in cancer progression.
  • Most cancer cells inactivate epithelial antiproliferative responses and benefit from increased TGF- ⁇ expression and autocrine TGF- ⁇ signaling through effects on gene expression, release of immunosuppressive cytokines, and epithelial plasticity.
  • TGF- ⁇ plays a role in increasing invasion and metastasis of cancer cells, stem cell properties, and drug resistance.
  • TGF- ⁇ released from cancer cells, stromal fibroblasts and other cells in the tumor microenvironment further promotes cancer progression by shaping the structure of the tumor and suppressing the anti-tumor activity of immune cells, thus creating an immunosuppressive environment and anti-cancer. Prevents or attenuates the effectiveness of immunotherapy. Therefore, inhibition of TGF- ⁇ signaling is considered a prerequisite and key means to improve the efficacy of current and future immunotherapies, including for tumors containing cancer cells that are unresponsive to TGF- ⁇ .
  • MSLN Mesothelin
  • MSLN-targeted immunotherapies reported to date support a favorable safety profile.
  • MSLN includes at least oesophageal cancer, breast cancer, gastric cancer, cholangiocarcinoma, pancreatic cancer, colon cancer, lung cancer, and thymic carcinoma.
  • CAR targets in many common solid tumors, such as mesothelioma, ovarian cancer, and endometrial cancer [Morello, A. et al. (2016) Mesothelin-Targeted CARs: Driving T Cells to Solid Tumors. Cancer Disco. 6(2); 133-46].
  • One object of the present invention is to provide a peptide that can inhibit the transforming growth factor- ⁇ (TGF- ⁇ ) signaling pathway along with a chimeric antigen receptor (CAR) targeting mesothelin.
  • the aim is to provide cells that have been genetically engineered to express.
  • Another object of the present invention relates to a cell therapeutic agent comprising the genetically engineered cells.
  • Another object of the present invention relates to pharmaceutical compositions for various uses containing the genetically engineered cells.
  • a chimeric antigen receptor (CAR) targeting mesothelin MSLN
  • cells genetically engineered to express a peptide or fragment thereof capable of inhibiting the transforming growth factor- ⁇ (TGF- ⁇ ) signaling pathway a chimeric antigen receptor (CAR) targeting mesothelin (MSLN)
  • TGF- ⁇ transforming growth factor- ⁇
  • “recombinant” or “manipulated” in relation to a peptide means having an amino acid sequence that has been altered as a result of the application of genetic engineering techniques to the nucleic acid encoding the peptide and to the cell or organism expressing the peptide.
  • the terms “recombinant” or “engineered” mean having a nucleic acid sequence that has been altered as a result of the application of genetic engineering techniques. Genetic engineering technologies include PCR and DNA cloning technologies; Transfection, transduction, transformation and other gene transfer techniques; homologous recombination; site-specific mutation; and gene fusions.
  • the term “genetically engineered” or “genetically engineered” refers to a cell or organism, or an ancestor thereof, whose genomic DNA sequence has been intentionally modified by recombinant technology.
  • the “genetic manipulation” includes “gene transplantation.”
  • methods for genetically manipulating the expression of the peptide or its fragment in the cell include biological methods such as vectors, specific receptors, or cell fusion methods, microinjection methods, electroporation methods, gene guns, or ultrasonic genes.
  • biological methods such as vectors, specific receptors, or cell fusion methods, microinjection methods, electroporation methods, gene guns, or ultrasonic genes.
  • a method of introducing the gene encoding the peptide or its fragment through a physical method such as the introduction method, or a chemical method such as the calcium phosphate coprecipitation method, liposome method, lipofection method, DEAE dextran method, or alkali metal method. can do.
  • the chimeric antigen receptor; and peptides or fragments thereof; Foreign genes encoding the proteins can be introduced into the cells by transfecting the immune effector cells with vectors containing genes encoding each.
  • the “chimeric antigen receptor (CAR)” is defined as a cell-surface receptor comprising an extracellular target-binding domain, a transmembrane domain, and an intracellular signaling domain, all of which are formed on a single protein. They exist together in combinations that are not found naturally. This particularly includes receptors in which the extracellular and intracellular signaling domains are not naturally found together on a single receptor protein.
  • Mesothelin is a protein also called MSLN, and is a 40 kDa protein secreted by mesothelial cells.
  • the protein was first identified by reaction with the monoclonal antibody K1, and through continued research, the mesothelin gene was linked to a glycophosphatidylinositol linkage and a 31-kDa shed fragment, megakaryocyte-potentiating factor (MPF). ) is known to encode a precursor protein that is processed to produce mesothelin, which is attached to the cell membrane. It has been suggested that mesothelin may be involved in cell adhesion, etc., but its biological function has not yet been clearly identified.
  • the mesothelin may be composed of the amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto.
  • the chimeric antigen receptor includes a mesothelin binding domain, and may further include one or more selected from the group consisting of a hinge domain, a signal peptide domain, a transmembrane domain, and one or more signaling domains. .
  • the chimeric antigen receptor of the present invention includes a mesothelin (MSLN) binding domain.
  • MSLN mesothelin
  • the mesothelin (MSLN) binding domain provided in the present invention can bind to mesothelin with higher affinity than the conventionally known anti-MSLN binding domain.
  • the binding domain may be an antibody or a fragment thereof.
  • the “antibody” refers to an immunoglobulin molecule that specifically binds to an antigen.
  • the antibody may be a natural or recombinant intact immunoglobulin, or may be an immune-reactive portion of an intact immunoglobulin.
  • Antibodies are generally tetramers of immunoglobulin molecules.
  • the antibody is a tetrameric glycosylated protein composed of two light (L) chains of about 25 kDa each and two heavy (H) chains of about 50 kDa each. Two types of light chains, referred to as lambda and kappa, can be found in antibodies.
  • immunoglobulins can be classified into five main classes A, D, E, G and M, several of which are subclasses (isotypes), such as IgG1, IgG2 , can be further subdivided into IgG3, IgG4, IgA1 and IgA2.
  • Each light chain typically contains an N-terminal variable (V) domain (VL) and constant (C) domain (CL).
  • Each heavy chain typically contains an N-terminal V domain (VH), three or four C domains (CH1-3), and a hinge region. The CH domain located closest to VH is named CH1.
  • the VH and VL domains are composed of four regions of relatively conserved sequences, referred to as framework regions, which form the scaffold for three regions of hypervariable sequences (complementarity determining regions, CDRs) (FR1). , FR2, FR3 and FR4).
  • CDRs contain most of the residues responsible for the specific interaction of the antibody with the antigen.
  • the CDRs are referred to as CDR1, CDR2 and CDR3. That is, the CDR elements on the heavy chain are referred to as CDRH1, CDRH2, and CDRH3, while the CDR elements on the light chain are referred to as CDRL1, CDRL2, and CDRL3.
  • CDRs are typically described in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services (1991), eds. See Kabat CDR, as described in Kabat et al.
  • Another standard for specifying the antigen binding site is by reference to the hypervariable loop described by Chothia. For example, Chothia, D. et al. (1992) J. Mol. Biol. 227:799-817; and Tomlinson et al. (1995) EMBO J. 14:4628-4638.
  • Another standard is the AbM definition used in Oxford Molecular's AbM antibody modeling software. In general, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains.
  • the antibody may exist in various forms, including but not limited to polyclonal antibodies, monoclonal antibodies, Fv, Fab, F(ab)2, etc., and single chain antibodies and humanized antibodies (document.
  • antibodies against the antigen can be obtained from immunized transgenic mice using conventional hybridoma techniques.
  • Human immunoglobulin transgenes carried by transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Accordingly, using this technique, it is possible to prepare therapeutically useful IgG, IgA, IgM and IgE antibodies, such as, but not limited to, IgGl (gamma 1) and IgG3. Details of these techniques for preparing human antibodies and human monoclonal antibodies and the protocols for preparing such antibodies can be found, for example, in PCT Publication Nos.
  • “Humanized” antibodies retain similar antigenic specificity as the original antibody, i.e., the ability to bind, for example, MSLN in the present invention.
  • the “antibody fragment” or “antigen-binding fragment” is shorter than the full-length antibody, but includes at least a partial variable region (e.g., one or more CDRs and/or one or more antigen-binding sites) that binds to the antigen of the antibody. Therefore, it refers to any portion of a full-length antibody that retains the binding specificity and at least partial specific binding ability of the full-length antibody. Accordingly, antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds the same antigen as the antibody from which the antibody fragment was derived.
  • Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, and derivatives produced synthetically, such as those produced recombinantly. Antibodies include antibody fragments. Antibody fragments include, but are not limited to, single chain Fv (scFv), Fab, Fab', F(ab')2, Fv, dsFv, double-antibody, Fd and Fd' fragments, and other fragments, including modified fragments. It is not limited (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p3-25, Kipriyanov).
  • Fragments may comprise multiple chains linked together, for example, through disulfide bonds and/or through peptide linkers.
  • Antibody fragments generally contain at least or about 50 amino acids, and typically contain at least or about 200 amino acids.
  • the antigen-binding fragment is an antibody (e.g., through replacement of a corresponding region) to obtain an antibody that immunospecifically binds to the antigen (i.e., exhibits a Ka of at least or at least about 107-108 M-1).
  • a “functional fragment” or “anti-MSLN antibody analog” is a fragment or analog that can prevent or substantially reduce the ability of a receptor to bind a ligand or initiate signal transduction.
  • functional fragment generally has the same meaning as "antibody fragment” and, in the case of an antibody, is a fragment capable of preventing or substantially reducing the ability of a receptor to bind a ligand or initiate signal transduction, such as Fv. , Fab, and F(ab')2.
  • the “Fv” fragment consists of a dimer (VH-VL dimer) formed by the variable domains of one heavy chain and the variable domains of one light chain by non-covalent association.
  • VH-VL dimer dimer
  • the three CDRs of each variable domain interact to determine the target-binding site on the surface of the VH-VL dimer identical to the intact antibody.
  • the six CDRs together confer the target-binding specificity of the intact antibody.
  • a single variable domain or half of an Fv containing only three target-specific CDRs
  • the “single chain Fv (scFv)” is a single chain antibody fragment having the variable regions of the heavy and light chains of the antibody linked together.
  • scFv single chain Fv
  • the scFv is desirable because it can be genetically engineered to be expressed as part of a single chain together with other components that make up the chimeric antigen receptor.
  • the antigen binding domain is typically included as part of the extracellular portion of the chimeric antigen receptor and is capable of recognizing and binding the targeted antigen, here specifically mesothelin (MSLN).
  • the scFv can be prepared according to methods known in the art (e.g., Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad Sci USA 85:5879-5883).
  • ScFv molecules can be made by connecting the VH and VL regions using a flexible polypeptide linker.
  • the scFv molecule includes a linker with optimized length and/or amino acid composition (e.g., Ser-Gly linker). Linker length can greatly affect how the variable region of the scFv folds and interacts. In fact, if short polypeptide linkers (e.g. 5-10 amino acids) are used, intrachain folding is prevented.
  • interchain folding is required to bring the two variable regions into proximity to form a functional epitope binding site.
  • linker orientation and size see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCT Publication Nos. WO2006/020258 and WO2007/024715, the entire contents of each of which are incorporated herein by reference. Included.
  • the mesothelin (MSLN) binding domain may include an scFv capable of specifically recognizing mesothelin.
  • the mesothelin (MSLN) binding domain includes a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 2; Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 3; and a light chain variable region including a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 4.
  • the mesothelin (MSLN) binding domain includes a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 5; Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 6; and a heavy chain variable region including a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 7.
  • variable region In the present invention, the "variable region”, “variable domain”, “V region” or “V domain” is generally located at the amino-terminus of the light or heavy chain, about 120 to 130 amino acids in the heavy chain and about 100 amino acids in the light chain. Refers to the portion of the light or heavy chain of an antibody, ranging in length from 1 to 110 amino acids, and is used in the binding and specificity of each specific antibody for a specific antigen.
  • the variable region of the heavy chain may be referred to as “VH”.
  • VL variable region of the light chain
  • the term “variable” refers to the fact that certain segments of the variable region vary widely in sequence among antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody for a particular antigen.
  • variable region is a frame of about 15 to 30 amino acids separated by shorter regions of greater variability (e.g., extreme variability), referred to as “hypervariable regions,” which are each about 9 to 12 amino acids long. It consists of a less variable (i.e. relatively invariant) stretch called the work region (FR).
  • the variable regions of the heavy and light chains are composed of four FRs that predominantly adopt the ⁇ -sheet configuration, each connected by three hypervariable regions that form loops connecting, and in some cases form part of, the ⁇ -sheet structure. Includes.
  • the hypervariable regions within each chain are closely held by FRs, and hypervariable regions from other chains contribute to the formation of the antigen-binding site of the antibody (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)].
  • the constant region is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions, such as the antibody's participation in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • Variable regions vary widely in sequence between different antibodies.
  • the variable region is a human variable region.
  • the "heavy chain” refers to a polypeptide chain of about 50 to 70 kDa, wherein the amino-terminal portion comprises a variable region of at least about 120 to 130 amino acids, and the carboxy-terminal portion comprises a constant region.
  • Constant regions are of five distinct types, referred to as alpha ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ), and mu ( ⁇ ), based on the amino acid sequence of the heavy chain constant region, isotype).
  • the distinct heavy chains differ in size: ⁇ , ⁇ , and ⁇ contain approximately 450 amino acids, while ⁇ and ⁇ contain approximately 550 amino acids.
  • IgA immunoglobulin A
  • IgD immunoglobulin D
  • IgE immunoglobulin G
  • IgM immunoglobulin M
  • Subclasses include IgG1, IgG2, IgG3 and IgG4.
  • the “light chain” refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and the carboxy-terminal portion includes a constant region.
  • the approximate length of the light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two distinct types, referred to as kappa ( ⁇ ) or lambda ( ⁇ ).
  • CDR is used interchangeably with “hypervariable region,” “HVR,” and “complementarity determining region.”
  • CDR refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of an immunoglobulin (Ig or antibody) VH ⁇ -sheet framework or a non-framework region of the antibody VL ⁇ -sheet framework Refers to one of three hypervariable regions (L1, L2, or L3) within the hypervariable region.
  • CDR1, CDR2 and CDR3 within the VH domain are also referred to as HCDR1, HCDR2 and HCDR3, respectively.
  • CDR1, CDR2 and CDR3 within the VL domain are also referred to as LCDR1, LCDR2 and LCDR3, respectively. Accordingly, CDRs are variable region sequences interspersed within framework region sequences.
  • CDR regions are well known to those skilled in the art and have been defined by a well-known numbering system.
  • Kabat complementarity determining regions are based on sequence variability and are the most commonly used (e.g., Kabat et al., supra; Nick Deschacht et al., J Immunol 2010; 184 :5696-5704]).
  • Chothia instead refers to the position of a structural loop (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)).
  • the end of the Chotia CDR-H1 loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering system places insertions at H35A and H35B; 35A or If neither 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34.
  • the AbM hypervariable region represents a compromise between the Kabat CDRs and Chotia structural loops and is used by Oxford Molecular's AbM antibody modeling software. “Contact” hypervariable regions are based on analysis of available complex crystal structures.
  • IMGT ImMunoGeneTics
  • IMGT ImMunoGeneTics
  • IGs immunoglobulins
  • TCRs T-cell receptors
  • MHC major histocompatibility complexes
  • CDRs are referred to both in terms of amino acid sequence and position within the light or heavy chain. Because the "position" of the CDRs within the structure of immunoglobulin variable domains is conserved between species and exist in structures referred to as loops, a numbering system is used to align variable domain sequences according to structural features, CDRs and framework residues. is easily identified.
  • This information can be used to graft and replace CDR residues from one species of immunoglobulin into a recipient framework, typically from a human antibody.
  • An additional numbering system (AHon) is described in Honegger and Plckthun, J. Mol. Biol. 309: 657-70 (2001)].
  • the correspondence between numbering systems including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to those skilled in the art.
  • CDR complementarity determining region
  • a scheme is specified for the identification of a specific CDR or CDRs, such as CDRs defined by the IMGT, Kabat, Chotia or Contact methods.
  • the specific amino acid sequence of the CDR is provided.
  • the CDR region can also be defined by a combination of various numbering systems, for example a combination of the Kabat and Chotia numbering systems or a combination of the Kabat and IMGT numbering systems. Accordingly, terms such as “CDR1 as presented in a particular VH” include, but are not limited to, any CDR1 as defined by the exemplary CDR numbering system described above. Once a variable region (e.g., VH or VL) is given, one skilled in the art will understand that the CDRs within the region may be defined by different numbering systems or combinations thereof.
  • the "constant region” or “constant domain” refers to the carboxy-terminal portions of the light and heavy chains that are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions, such as interaction with Fc receptors. .
  • the term refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to other portions of the immunoglobulin, the variable region containing the antigen binding site.
  • the constant region may contain the CH1, CH2 and CH3 regions of the heavy chain and the CL region of the light chain.
  • the “framework” or “FR” refers to the variable region residues flanking the CDR.
  • FR residues are present in, for example, chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies.
  • FR residues are variable domain residues other than hypervariable region residues or CDR residues.
  • the mesothelin (MSLN) binding domain may include a light chain variable region (VL) of an antibody represented by SEQ ID NO: 8, or a light chain variable region of an antibody encoded by the nucleotide sequence represented by SEQ ID NO: 9. (VL) may be included.
  • the mesothelin (MSLN) binding domain may include the heavy chain variable region (VH) of the antibody represented by SEQ ID NO: 10, or the heavy chain variable region of the antibody encoded by the nucleotide sequence represented by SEQ ID NO: 11 (VH) may be included.
  • VH heavy chain variable region
  • VH the heavy chain variable region of the antibody represented by SEQ ID NO: 10
  • VH the heavy chain variable region of the antibody encoded by the nucleotide sequence represented by SEQ ID NO: 11
  • the light chain variable region includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 8. It can be done, and also includes those encoded by a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence shown in SEQ ID NO: 9.
  • the heavy chain variable region includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 10. It can be done, and also includes those encoded by sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the base sequence shown in SEQ ID NO: 11.
  • the scFv has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 amino acid residues between its VL region and VH region. , 18, 19, 20, 25, 30, 35, 40, 45, 50 or more linkers.
  • the linker sequence may include any naturally occurring amino acid.
  • the linker sequence may include the amino acids glycine and serine.
  • the linker sequence comprises a set of glycine and serine repeats, such as (Gly 4 Ser)n, where n may be a positive number of 1 or more, preferably 3 or 4.
  • the linker may include the amino acid sequence represented by SEQ ID NO: 12, or may be a linker encoded by the nucleotide sequence represented by SEQ ID NO: 13.
  • the mesothelin (MSLN) binding domain may include an scFv capable of specifically recognizing mesothelin, and the scFv is represented by the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively.
  • a light chain variable region (VL) comprising light chain CDR1, CDR2, and CDR3;
  • a linker comprising the amino acid sequence shown in SEQ ID NO: 12 or encoded by the nucleotide sequence shown in SEQ ID NO: 13;
  • a heavy chain variable region (VH) comprising heavy chain CDR1, CDR2, and CDR3 represented by the amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively, but is not limited thereto.
  • the mesothelin (MSLN) binding domain may include an scFv capable of specifically recognizing mesothelin, and the scFv may include the amino acid sequence shown in SEQ ID NO: 8, or Variable light chain region (VL) of the antibody encoded by the nucleotide sequence shown in SEQ ID NO: 9; A linker comprising the amino acid sequence shown in SEQ ID NO: 12 or encoded by the nucleotide sequence shown in SEQ ID NO: 13; and a variable heavy chain region (VH) of an antibody comprising the amino acid sequence represented by SEQ ID NO: 10 or encoded by the nucleotide sequence represented by SEQ ID NO: 11; but is not limited thereto.
  • the chimeric antigen receptor of the present invention can be designed with a signal peptide added to direct the translated chimeric protein to the membrane.
  • the signal peptide may be included in the amino-terminus (N-ter) of the chimeric antigen receptor.
  • this signal peptide can be selectively cleaved from the mesothelin binding domain (eg, scFv) while the chimeric antigen receptor is processed in the cell and localized to the cell membrane.
  • the signal peptide generally ranges from 15 to 30 amino acids.
  • Non-limiting examples of the signal peptide in the present invention include CD8 signal peptide (amino acids 21), CD33 signal peptide (amino acids 17), CD4 signal peptide (amino acids 25), and IL-2R (CD25) signal peptide (amino acids 21). dog), trypsinogen-2 signal peptide (15 amino acids), VEGFR1 signal peptide (26 amino acids), EGFR signal peptide (24 amino acids), GMCSFR signal peptide (22 amino acids), IgVL signal peptide, IgVK signal peptide Or there may be Ig VH signal peptide, etc.
  • the signal peptide may be a CD8 signal peptide, and preferably may include an amino acid sequence represented by SEQ ID NO: 14, or a signal peptide encoded by the nucleotide sequence represented by SEQ ID NO: 15. It can be.
  • the signal peptide may include an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence represented by SEQ ID NO: 14, , also includes those encoded by sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the base sequence shown in SEQ ID NO: 15.
  • the chimeric antigen receptor of the present invention may further include an extracellular spacer, that is, a hinge.
  • the "hinge” refers to a flexible polypeptide connector region (also referred to herein as a "hinge region”) that provides structural flexibility and spacing to the flanking polypeptide regions and is used in natural or synthetic polypeptides. It can be composed of:
  • the chimeric antigen receptor of the present invention can connect the mesothelin binding domain to the transmembrane domain described below through a hinge.
  • the hinge is sufficiently flexible to allow the antigen binding domain to be oriented in different directions to facilitate antigen binding.
  • the hinge may be a hinge region derived from IgG, and preferably may include the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 hinge region. Additionally, the hinge may contain one or more amino acid substitutions and/or insertions and/or deletions compared to the wild-type (naturally occurring) hinge region. For example, His229 of the human IgG1 hinge may contain a sequence substituted with Tyr.
  • the hinge includes all or part of the CD8, CD28, 4-1BB, OX40, CD3 zeta ( ⁇ ) chain, T cell receptor ⁇ or ⁇ chain, CD28, CD3 ⁇ , CD45 commonly used in the art.
  • CD8, 4-1BB, OX40, CD3 zeta ( ⁇ ) chain, T cell receptor ⁇ or ⁇ chain, CD28, CD3 ⁇ , CD45 commonly used in the art.
  • CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154 their functional derivatives, or combinations thereof.
  • CD4 CD5
  • CD8 CD9 CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS
  • the hinge may be a CD8-derived hinge region, and preferably may include an amino acid sequence represented by SEQ ID NO: 16, or a hinge encoded by the nucleotide sequence represented by SEQ ID NO: 17. You can.
  • the hinge may include an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence represented by SEQ ID NO: 16, and the sequence It also includes those encoded by sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the base sequence indicated by number 17.
  • the chimeric antigen receptor of the present invention may further include a transmembrane domain.
  • the transmembrane domain includes one or more additional amino acids adjacent to the transmembrane region, for example, one or more amino acids associated with the extracellular region of the protein from which the transmembrane domain is derived (e.g., amino acid 1 of the extracellular region, 2, 3, 4, 5, 6, 7, 8, 9, 10 and up to 15 amino acids) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g. It may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino acids in the region.
  • one or more amino acids associated with the extracellular region of the protein from which the transmembrane domain is derived e.g., amino acid 1 of the extracellular region, 2, 3, 4, 5, 6, 7, 8, 9, 10 and up to 15 amino acids
  • additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived e.g. It may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino acids in the region.
  • the transmembrane domain is selected or has amino acid substitutions to prevent binding of this domain to the transmembrane domain of the same or another surface membrane protein, for example to minimize interaction with other members of the receptor complex. It can be transformed by .
  • the transmembrane domain may be of natural origin or recombinant origin.
  • the domain may be derived from any membrane-bound or transmembrane protein.
  • the transmembrane domain can transmit a signal to the intracellular domain whenever the chimeric antigen receptor binds to the target antigen.
  • the transmembrane domain includes, for example, T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64. , CD80, CD86, CD134, CD137, and may include the transmembrane region of the alpha, beta or zeta chain of CD154, but are not limited thereto.
  • the transmembrane domain is a co-stimulatory signaling domain, such as MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein) ), activated NK cell receptor, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7 -H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta , IL2R gamma, IL7R alpha, ITGA4, V
  • the transmembrane domain when the transmembrane domain is a synthetic domain, it may include hydrophobic residues such as leucine and valine, or triplets consisting of phenylalanine, tryptophan and valine may be found at both ends of the synthetic transmembrane domain. .
  • the transmembrane domain may be a CD8 transmembrane domain, and preferably includes the amino acid sequence represented by SEQ ID NO: 18, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 19.
  • the transmembrane domain may include an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence represented by SEQ ID NO: 18. It also includes those encoded by a base sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the base sequence shown in SEQ ID NO: 19.
  • the chimeric antigen receptor of the present invention has a cytoplasmic domain and may further include an intracellular signaling domain.
  • the “intracellular signaling domain” generally induces activation of the normal effector function of the cell into which the chimeric antigen receptor has been introduced.
  • the “effector function” refers to a specialized function of a cell.
  • the effector function of a T cell may be, for example, cytolytic activity or helper activity, including secretion of cytokines.
  • the “intracellular signaling domain” refers to a portion of a protein that converts an effector function signal and instructs the cell to perform a specialized function. Typically the entire intracellular signaling domain can be used, but in many cases the entire domain is not required to be used.
  • intracellular signaling domain is thus meant to contain any truncated region of the intracellular signaling domain sufficient to transduce an effector function signal.
  • intracellular signaling domains in the present invention include cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act cooperatively to initiate signal transduction after binding to the antigen receptor, as well as any derivatives of these sequences. Or there may be variants, and any recombinant sequences with the same functional capacity, etc. It is known that signals generated through TCR fragments are insufficient to fully activate T cells, and secondary and/or costimulatory signals are also required.
  • TCR T cell receptor
  • the primary signaling domain controls the primary activation of the TCR complex in a stimulatory or inhibitory manner.
  • the primary intracellular cleavage domain that acts in a stimulatory manner may be a specific signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.
  • ITAMs containing primary intracellular signaling domains specifically used in the present invention include TCR zeta, FcR gamma, FcR beta, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278. (also known as “ICOS”), Fc.epsilon.RI, DAP10, DAP12, and ITAM of CD66d, but are not limited thereto.
  • the primary signaling domain may include a modified ITAM domain, for example, a mutated ITAM domain whose activity is altered (e.g., increased or decreased) compared to the original ITAM domain.
  • a modified ITAM domain for example, a mutated ITAM domain whose activity is altered (e.g., increased or decreased) compared to the original ITAM domain.
  • the intracellular signaling domain may comprise a primary signaling domain, e.g., the CD3 zeta signaling domain itself, or may be combined with any other preferred intracellular signaling domain available in the present invention. You can.
  • the intracellular signaling domain of a CAR may include one or more co-stimulatory signaling domains along with a primary signaling domain, such as a CD3 zeta chain region.
  • the “co-stimulatory signaling domain” includes the intracellular domain of a co-stimulatory molecule and is a cell surface molecule other than an antigen receptor or its ligand required for an effective response of lymphocytes to an antigen.
  • examples of such molecules in the present invention include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligands.
  • Receptor OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS ( CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA- 1, ITGB7,
  • the intracellular signaling domain may be designed to include one or more, or two or more, for example, 2, 3, 4, 5 or more co-stimulatory signaling domains.
  • neighboring molecules may be connected directly, but by linker molecules. They may also be placed spaced apart.
  • the linker molecule may be a glycine residue or an alanine residue, but is not limited thereto.
  • the intracellular signaling domain may be a CD3 zeta signaling domain, and preferably includes the amino acid sequence represented by SEQ ID NO: 20, or is encoded by the nucleotide sequence represented by SEQ ID NO: 21. You can.
  • the intracellular signaling domain may further include 4-1BB as a co-stimulatory signaling domain.
  • the 4-1BB preferably includes the amino acid sequence represented by SEQ ID NO: 22, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 23.
  • the intracellular signaling domain may include a CD3 zeta signaling domain as a primary signaling domain and 4-1BB as a co-stimulatory domain, preferably 4-1BB.
  • the CD3 zeta signaling domain may include the amino acid sequence represented by SEQ ID NO: 20, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 21, and the 4-1BB is the amino acid sequence represented by SEQ ID NO: 22. It may include or be encoded by the nucleotide sequence shown in SEQ ID NO: 23.
  • the intracellular signaling domain may be linked directly to the C-terminus of the transmembrane domain, but alternatively may be linked to a short oligopeptide or polypeptide linker, e.g., 2-10 amino acids in length. Can be connected by a linker.
  • the type of the linker is not limited, but a non-limiting example may be a glycine-serine doublet.
  • the chimeric antigen receptor may include the amino acid sequence represented by SEQ ID NO: 24, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 26, but is not limited thereto.
  • the chimeric antigen receptor may include the amino acid sequence represented by SEQ ID NO: 25, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 27, but is not limited thereto.
  • the cells may further include cells genetically engineered to express a peptide or fragment thereof capable of inhibiting the transforming growth factor- ⁇ (TGF- ⁇ ) signaling pathway.
  • TGF- ⁇ transforming growth factor- ⁇
  • the present invention includes cells genetically engineered to express both the chimeric antigen receptor and the peptide or fragment thereof that inhibit the transforming growth factor beta pathway, as well as cells genetically engineered to express the chimeric antigen receptor. It is also included in the purpose of the present invention to simultaneously include two types of cells and cells genetically engineered to express a peptide or fragment thereof that inhibits the transforming growth factor beta pathway.
  • the "transforming growth factor- ⁇ (TGF- ⁇ )” refers to a cytokine belonging to the TGF- ⁇ family.
  • TGF- ⁇ expressed in mammals include TGF- ⁇ 1 and TGF- ⁇ . Three are known: - ⁇ 2 and TGF- ⁇ 3.
  • Signal transduction by TGF- ⁇ plays a critical role in various biological processes and performs various functions such as cell growth inhibition, apoptosis, differentiation, and epithelial-mesenchymal transition (EMT).
  • EMT epithelial-mesenchymal transition
  • the TGF- ⁇ signaling system is tightly regulated and plays a critical role in development and organ formation as well as maintaining cellular homeostasis. Therefore, disruption of TGF- ⁇ signaling can cause life-threatening diseases such as cancer, fibrosis, and congenital malformations.
  • the peptide may include the amino acid sequence represented by SEQ ID NO: 28, preferably consisting of the amino acid sequence represented by SEQ ID NO: 28.
  • sequence encoding the peptide may include the nucleotide sequence represented by SEQ ID NO: 29, preferably consisting of the nucleotide sequence represented by SEQ ID NO: 29.
  • the peptide may bind to the TGF- ⁇ receptor (TGFBR1 and/or TGFBR2) to inhibit TGF- ⁇ signaling.
  • TGFBR1 and/or TGFBR2 TGF- ⁇ receptor
  • the peptide competes with TGF- ⁇ and binds to the TGF- ⁇ receptor. It may be that TGF- ⁇ signaling is inhibited through a mechanism that prevents TGF- ⁇ cytokines from binding to the TGF- ⁇ receptor.
  • the peptide may inhibit TGF- ⁇ signaling by suppressing the expression level of TGF- ⁇ in cells.
  • the peptide may inhibit TGF- ⁇ signaling in cells through an auto-inhibition pathway. It may be suppressing TGF- ⁇ signaling through a mechanism that reduces the expression level or reduces the extracellular emissions of TGF- ⁇ .
  • the cells may be immune effector cells.
  • the “immune effector cell” may be a lymphoid cell that participates in an immune response, such as promoting an immune effector response.
  • lymphocytes refers to cells that are commonly found in lymph and include natural killer cells (NK cells), T cells, and B cells. Those skilled in the art will understand that the immune cell types listed above can be further divided into subtypes.
  • the lymphocytes may be or include Natural Killer Cells (NK cells), but are not limited thereto.
  • NK cells Natural Killer Cells
  • the "Natural Killer Cells (NK cells)” are defined as large granular lymphocytes (LGL), which constitute three types of cells differentiated from common lymphoid progenitor cells-producing B and T lymphocytes.
  • NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus and enter the circulation.
  • the NK cells may include any type of NK cell without limitation, for example, cultured NK cells, such as primary NK cells, NK cells from a cultured NK cell line, or NK cells obtained from a mammal. It may be an NK cell, but is not limited thereto.
  • NK cells When NK cells are obtained from a mammal, the NK cells can be obtained from a number of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. NK cells may be concentrated or purified. The NK cells may preferably be human NK cells (eg, isolated from humans). NK cell lines are available, for example, from ATCC (American Type Culture Collection) and include, for example, NK-92 cells (ATCC CRL-2407), NK92MI cells (ATCC CRL-2408) or derivatives thereof, etc. .
  • ATCC American Type Culture Collection
  • a polynucleotide (or gene construct) encoding the above-mentioned chimeric antigen receptor and a polynucleotide (or gene) encoding a peptide or fragment thereof capable of inhibiting the transforming growth factor beta signaling pathway are included in one vector. construct), or a vector containing a polynucleotide (or gene construct) encoding the chimeric antigen receptor, and a peptide or fragment thereof capable of inhibiting the transforming growth factor beta signaling pathway.
  • Encoding may include both types of vectors containing polynucleotides (or gene constructs).
  • the "vector” refers to a recombinant vector that can be transfected into a suitable host cell to express a protein of interest, and refers to a genetic construct containing essential regulatory elements operably linked to express the gene insert.
  • operably linked means that the nucleic acid expression control sequence and the nucleic acid sequence encoding the protein of interest are functionally linked to perform a general function. Operational linkage with a recombinant vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cutting and ligation can be easily performed using enzymes generally known in the art. there is.
  • various types of vectors such as nanoparticles, plasmids, viruses, and cosmids can be used as recombinant expression vectors for inserting the foreign genes.
  • the type of recombinant vector is not particularly limited as long as it functions to express the desired gene and produce the desired protein in various host cells of prokaryotic and eukaryotic cells, but specifically, it has a highly active promoter and strong expression ability while maintaining a natural state. Vectors that can produce large quantities of foreign proteins of a similar form can be used.
  • a variety of gene delivery vehicles are known in the art and include both viral and non-viral (e.g., naked DNA, plasmid) vectors.
  • Viral vectors suitable for gene transfer are known to those skilled in the art.
  • Non-limiting examples of the viral vectors include retroviral vectors (derived from Moloney murine leukemia virus vector (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., HIV-1, HIV-2) , derived from SIV, BIV, FIV, etc.), adenovirus (Ad) vectors, including replication-competent, replication-deficient and anergic forms thereof, adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors , bovine papilloma virus vector, Epstein Barr virus vector, herpes virus vector, chicken pox virus vector, Harvey rat sarcoma virus vector, rat mammary tumor virus vector, Rous s
  • Non-viral vectors for gene transfer include naked DNA, plasmids, transposons, and mRNA.
  • Non-limiting examples include pKK plasmid (Clonetech), pUC plasmid, pET plasmid (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmid (Invitrogen, San Diego, Calif.), pMAL plasmid (New England Biolabs, Beverly , Mass.).
  • vectors in the present invention can be introduced into many suitable host cells using methods disclosed or cited herein or otherwise known to those skilled in the art.
  • a suitable expression vector of the present invention may include a base sequence encoding a signal peptide for membrane targeting or secretion in addition to expression control elements such as a promoter, start codon, stop codon, polyadenylation signal, or enhancer.
  • the initiation codon and stop codon are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein and must be functional in the subject when the genetic construct is administered and must be in frame with the coding sequence.
  • the "promoter”, as used herein, refers to any sequence that regulates the expression of a coding sequence, such as a gene. Promoters can be, for example, constitutive, inducible, repressible, or tissue-specific.
  • a promoter is a control sequence, a region of polynucleotide sequence where the initiation and rate of transcription is controlled.
  • Non-limiting examples of the promoters in the present invention include Rous sarcoma virus (RSV) LTR promoter (optionally with RSV enhancer), cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase Promoter, ⁇ -actin promoter, phosphoglycerol kinase (PGK) promoter, U6 promoter, EF1alpha short form (EFS) promoter, human polypeptide chain elongation factor (EF1a) promoter, P5 promoter, Ubc promoter, CAG promoter, TRE promoter , UAS promoter, Ac5 promoter, polyhedrin promoter, CaMKIIa promoter, Gal1 promoter, TEF1 promoter, GDS promoter, ADH1 promoter, CaMV35S promoter, ubiquitin (Ubi) promoter
  • the promoter can be coupled to an enhancer to increase transcription efficiency.
  • the enhancer may include, but are not limited to, the RSV enhancer, the CMV enhancer, or the ⁇ -fetoprotein MERII enhancer.
  • the promoter may be the SSFV promoter, preferably the SSFV promoter represented by SEQ ID NO: 30, but is not limited thereto.
  • the polynucleotide (or gene construct) encoding the chimeric antigen receptor may include the base sequence represented by SEQ ID NO: 26, but is not limited thereto.
  • the polynucleotide encoding the chimeric antigen receptor may include the base sequence represented by SEQ ID NO: 27, but is not limited thereto.
  • the vector of the present invention may further include a sequence encoding a signal peptide upstream of the polynucleotide encoding a peptide or fragment thereof capable of inhibiting the transforming growth factor beta (TGF- ⁇ ) signaling pathway.
  • TGF- ⁇ transforming growth factor beta
  • the signal peptide may be an IL-2 signal peptide, and preferably includes the amino acid sequence represented by SEQ ID NO: 33, or is encoded by the nucleotide sequence represented by SEQ ID NO: 34. However, it is not limited to this.
  • the vector may include one or more additional polypeptides, for example, a polynucleotide encoding one or more markers and/or one or more effector molecules.
  • the one or more markers may include a transduction marker, a surrogate marker, and/or a selection marker.
  • the additional nucleic acid sequences introduced, encoding one or more additional polypeptides may be nucleic acid sequences that can improve the efficacy of the therapy, such as by promoting the viability and/or function of the transferred cells; Nucleic acid sequences that provide genetic markers for evaluation and/or selection of cells, such as to assess survival or localization in vivo; Lupton S. D. et al., Mol.
  • the marker may be a transduction marker or a surrogate marker.
  • the transduction marker or surrogate marker can be used to detect cells into which a polynucleotide (or gene construct) of the present invention, that is, a polynucleotide containing a sequence encoding the peptide of the present invention or a fragment thereof, has been introduced.
  • the transduction marker may indicate or confirm transformation of the cell
  • the surrogate marker may be a protein prepared to be co-expressed on the cell surface together with the peptide or fragment.
  • the surrogate marker may be a surface protein modified to have little or no activity.
  • the surrogate marker may be encoded on the same polynucleotide encoding the peptide or fragment.
  • the nucleic acid sequence encoding the peptide or fragment thereof may optionally be operably linked to a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, such as a 2A sequence.
  • the exemplary surrogate marker is a truncated form of a cell surface polypeptide, e.g., a non-functional, full-length form of the cell surface polypeptide that cannot or will not transmit signals or signals that would normally be transmitted and/or will be internalized. It may contain truncated forms that cannot or are not internalized.
  • Exemplary truncated cell surface polypeptides include truncated forms of growth factors or other receptors, such as truncated human epidermal growth factor receptor 2 (tHER2), truncated epidermal growth factor receptor (tEGFR), or It may include prostate-specific membrane antigen (PSMA) or a modified form thereof, such as truncated PSMA (tPSMA).
  • tEGFR may contain an epitope recognized by the antibody cetuximab (Erbitux) or other therapeutic anti-EGFR antibody or binding molecule, which identifies cells engineered with the tEGFR construct and the encoded exogenous protein. or to select and/or remove or isolate cells expressing the encoded exogenous protein.
  • cetuximab an epitope recognized by the antibody cetuximab (Erbitux) or other therapeutic anti-EGFR antibody or binding molecule, which identifies cells engineered with the tEGFR construct and the encoded exogenous protein. or to select and/or remove or isolate cells expressing the encoded exogenous protein.
  • the marker e.g., a surrogate marker
  • the marker includes all or part of CD34 (e.g., a truncated form), NGFR, CD19, or truncated CD19, e.g., truncated non-human CD19.
  • the marker is a detectable protein, such as a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), such as super-fold GFP. , sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue green fluorescent protein (BFP) ), enhanced blue fluorescent protein (EBFP) yellow fluorescent protein (YFP) and its variants, including species variants, monomer variants and codon-optimized, stabilized and/or enhanced variants of fluorescent proteins. It may be or contain a variant.
  • a fluorescent protein such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), such as super-fold GFP.
  • sfGFP red fluorescent protein
  • RFP red fluorescent protein
  • CFP blue green fluorescent protein
  • BFP blue green fluorescent protein
  • EBFP enhanced blue fluorescent protein
  • YFP yellow fluorescent protein
  • the marker is an enzyme such as luciferase, E. coli-derived lacZ gene, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyl transferase (CAT), and ⁇ -gal. It is or includes lactosidase or ⁇ -glucuronidase ( ⁇ -glucuronidase, GUS). Expression of the enzyme can be detected by addition of a substrate that can be detected upon expression and functional activity of the enzyme.
  • the marker may be green fluorescent protein (GFP), and preferably, the marker includes the amino acid sequence represented by SEQ ID NO: 31, or is encoded by the nucleotide sequence represented by SEQ ID NO: 32. It may be possible, but it is not limited to this.
  • GFP green fluorescent protein
  • the marker may be a selection marker.
  • the selection marker may be or include a polypeptide that confers resistance to an exogenous agent or drug.
  • the selection marker may be an antibiotic resistance gene, non-limiting examples include a puromycin resistance gene, hygromycin resistance gene, blasticidin resistance gene, neomycin resistance gene, geneticin resistance gene, or zeocin. It may be or contain a resistance gene or a modified form thereof.
  • the vector includes a polynucleotide (or gene construct) encoding the chimeric antigen receptor; A polynucleotide (or gene construct) encoding a peptide or fragment thereof capable of inhibiting the transforming growth factor beta signaling pathway; and a polynucleotide (or gene construct) encoding a marker, but is not limited thereto.
  • the vector includes a polynucleotide (or gene construct) encoding the chimeric antigen receptor in one vector; A polynucleotide (or gene construct) encoding a peptide or fragment thereof capable of inhibiting the transforming growth factor beta signaling pathway; and a polynucleotide (or gene construct) encoding a marker, but is not limited thereto.
  • the position order or combination order of each of these genes in the vector is not particularly limited.
  • the vector is a vector containing a polynucleotide (or gene construct) encoding the chimeric antigen receptor; And a vector containing a polynucleotide (or gene construct) encoding a peptide or fragment thereof capable of inhibiting the transforming growth factor beta signaling pathway; but is not limited thereto.
  • the vector is a vector containing a polynucleotide (or gene construct) encoding the chimeric antigen receptor; And a vector containing a polynucleotide (or gene construct) encoding a peptide or fragment thereof capable of inhibiting the transforming growth factor beta signaling pathway, wherein a marker is added to at least one of the two vectors.
  • Encoding polynucleotides (or gene constructs) may be additionally included, but are not limited thereto.
  • polynucleotide when two separate polynucleotides (or gene constructs) are included in one vector, for example, a polynucleotide (or gene construct) encoding the chimeric antigen receptor in one vector; A polynucleotide (or gene construct) encoding a peptide or fragment thereof capable of inhibiting the transforming growth factor beta signaling pathway; And when at least two of the polynucleotides (or gene constructs) encoding the marker are included simultaneously, they may be operably linked by one promoter, or may be operably linked by two or more promoters.
  • a self-cleavable peptide is provided so that each of them can be individually delivered or introduced into the cell for intracellular expression. It may further include a polynucleotide encoding.
  • the "self-cleavage peptide” refers to 10 to 50, 12 to 42, 14 to 34 peptides that can induce cleavage of proteins synthesized within cells. It refers to a peptide consisting of 16 to 26 or 18 to 22 amino acids.
  • the self-cleaving peptide may be derived from the 2A region of the viral gene.
  • the self-cleaving peptide may be derived from P2A, E2A, F2A or T2A.
  • the self-cleaving peptide may be derived from P2A.
  • a peptide that is cleaved by a degrading enzyme present in the cytoplasm can be used.
  • the self-cleavable peptide may be P2A or a peptide derived therefrom, and preferably includes the amino acid sequence represented by SEQ ID NO: 35, or is cleaved by the nucleotide sequence represented by SEQ ID NO: 36. It may be encrypted.
  • a cell therapeutic agent comprising the genetically engineered cells provided by the present invention as an active ingredient.
  • the “cell therapy” refers to a treatment using autologous, allogenic, or xenogenic cells to restore tissue function, and is used to suppress cancer. If the immune effector cells, for example, genetically modified natural killer cells, are included as active ingredients, they can be used as a cell therapy for the treatment and prevention of cancer.
  • the cell therapeutic agent may further include a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be, for example, saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, HSA (Human serum albumin), and a mixture of one or more of these ingredients.
  • Other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed.
  • the cell therapeutic agent may be used as a suspending agent, solubilizing agent, stabilizer, isotonic agent, preservative, anti-adsorption agent, surfactant, diluent, excipient, pH adjuster, analgesic agent, buffer, sulfur-containing agent, etc. ⁇ ) Reducing agents, antioxidants, etc. can be added appropriately.
  • the suspending agent include, but are not limited to, methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, traganmal, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate, etc. no.
  • the solution auxiliary agent includes polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinic acid amide, polyoxyethylene sorbitan monolaurate, mecrogol, castor oil fatty acid ethyl ester, etc.
  • Stabilizers include, but are not limited to, dextran 40, methylcellulose, gelatin, sodium sulfite, and sodium metasulfate.
  • the isotonic agent includes, for example, D-mannitol, sorbitol, etc., but is not limited thereto.
  • the preservative includes, for example, methyl paraoxybenzoate, ethyl paraoxybenzoate, sorbic acid, phenol, cresol, chlorocresol, etc., but is not limited thereto.
  • the anti-adsorption agent includes, for example, human serum albumin, lecithin, dextran, ethylene oxide propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hydrogenated castor oil, polyethylene glycol, etc. , but is not limited to this.
  • the sulfur-containing reducing agent includes, for example, N-acetylcysteine, N-acetylhomocysteine, thioctoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, Examples include those having a sulfuhydryl group such as glutathione and thioalkanoic acid having 1 to 7 carbon atoms, but are not limited thereto.
  • the antioxidants include, for example, erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, ⁇ -tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbic acid palmitate, Chelating agents such as L-ascorbate stearate, sodium bisulfite, sodium sulfite, triamyl gallate, propyl gallate or sodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate may be included, but are limited thereto. It doesn't work.
  • the cell therapeutic agent is used, for example, based on an adult patient weighing 70 kg, about 1,000 to 10,000 cells/time, 1,000 to 100,000 cells/time, 1,000 to 1,000,000 cells/time, 1,000 to 10,000,000. , 1,000 ⁇ 100,000,000 cells/time, 1,000 ⁇ 1,000,000,000 cells/time, 1,000 ⁇ 10,000,000,000 cells/circuit, can be administered in divided doses once or several times a day at regular time intervals, or can be administered multiple times at regular time intervals.
  • the injectable product according to the present invention can be manufactured in the form of a filled injection by taking the amount commonly known in the art depending on the patient's constitution and type of defect.
  • the present invention relates to a pharmaceutical composition for preventing, improving or treating cancer containing the genetically engineered cells provided by the present invention as an active ingredient.
  • cancer refers to or refers to a physiological condition typically characterized by uncontrolled cell growth in mammals.
  • the cancer subject to prevention, improvement, or treatment in the present invention may be a solid tumor consisting of a lump generated by abnormal cell growth in a solid organ, and preferably expresses mesothelin (MSLN).
  • MSLN mesothelin
  • Any cancer that has cancer can be included without limitation, and specific examples include stomach cancer, liver cancer, glioblastoma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, biliary tract cancer, It may be melanoma or lung cancer, but is not limited thereto.
  • prevention refers to all actions that inhibit cancer or delay its progression by administering the composition of the present invention.
  • treatment and “improvement” mean any action in which cancer symptoms are improved or beneficially changed by administration of the composition of the present invention.
  • the pharmaceutical composition of the present invention may be formulated to include one or more pharmaceutically acceptable carriers in addition to the active ingredients described above.
  • Pharmaceutically acceptable carriers may be saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and a mixture of one or more of these ingredients, and if necessary, antioxidants.
  • other common additives such as buffer solutions and bacteriostatic agents can be added.
  • diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets, and can act specifically on target organs.
  • Target organ-specific antibodies or other ligands can be used in combination with the carrier.
  • it can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA). there is.
  • the pharmaceutical composition of the present invention can be a solution, suspension, dispersion, emulsion, gel, injectable solution, or sustained-release preparation of the active compound, and is preferably an injection.
  • the pH is adjusted using a buffer solution such as an aqueous acid solution or phosphate that can be used as an injection to ensure product stability according to the distribution of the injection prescription, making the injection very physically and chemically stable. It can be manufactured with
  • the injection can be prepared by dissolving it in water for injection along with a stabilizer or solubilizing agent and then sterilizing it, especially by high-temperature reduced-pressure sterilization or aseptic filtration.
  • the water for injection may be distilled water for injection or a buffer solution for injection, for example, a phosphate buffer solution with a pH in the range of 3.5 to 7.5 or a sodium dihydrogen phosphate (NaH2PO4)-citric acid buffer solution.
  • the phosphate salt used may be in the form of a sodium salt or potassium salt, an anhydrous or hydrated form, and may be in the form of citric acid or an anhydrous or hydrated form.
  • the stabilizer used in the present invention includes sodium pyrosulfite, sodium bisulfite (NaHSO 3 ), sodium metabisulfite (Na 2 S 2 O 3 ), or ethylenediaminetetraacetic acid
  • Solubilizing agents include bases such as sodium hydroxide (NaOH), sodium bicarbonate (NaHCO 3 ), sodium carbonate (NaCO 3 ) or potassium hydroxide (KOH), or acids such as hydrochloric acid (HCl) or acetic acid (CH 3 COOH).
  • the injectable agent according to the present invention can be formulated to be bioabsorbable, biodegradable, and biocompatible.
  • bioabsorbable we mean that the injectable agent can disappear from the body upon initial application without decomposition or decomposition of the dispersed injectable agent.
  • Biodegradability means that the injectable agent can be broken down or decomposed in the body by hydrolysis or enzymatic degradation.
  • Biosynthesis means that all ingredients are non-toxic to the body.
  • the injection according to the present invention can be prepared using conventional fillers, weighting agents, binders, wetting agents, diluents such as surfactants, or excipients.
  • composition or active ingredient of the present invention may be administered by intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, subcutaneous, intrathecal, inhalational, topical, rectal, oral, intraocular or intradermal route depending on the purpose. It can be administered in a conventional manner, and preferably intravenously.
  • the composition or active ingredient of the present invention can be administered by injection or catheter.
  • the dosage of the active ingredient is 1 x 10 1 to 1 x 10 50 cells/kg, preferably 1 x 10 transformed host cells contained in the pharmaceutical composition, based on an adult weighing 60 kg. So that it can be administered within the range of 1 to 1 x 10 30 pieces/kg, more preferably 1 x 10 5 to 1 x 10 20 pieces/kg, and most preferably 1 x 10 7 to 1 x 10 9 pieces/kg. It can be adjusted.
  • the optimal dosage to be administered can be easily determined by a person skilled in the art, and can be determined based on the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of dosage form, and the patient's age, weight, and general health. It can be adjusted according to various factors, including condition, gender and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs.
  • the active ingredient may be contained in an amount of 0.001 to 50% by weight based on the total weight of the composition.
  • the content is not limited to this.
  • composition of the present invention may further include one or more anticancer agents.
  • the anticancer agents include nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, semasanib, bosutinib, axitinib, Cediranib, lestaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, bevacizumab, cisplatin, cetuximab, Viscum album, asparaginase, tretinoin, hydroxycarbamide , dasatinib, estramustine, gemtuzumab ozogamycin, ibritumab tusetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazin
  • tezolomide busulfan, ifosphamide, cyclophosphamide, melphalan, altretmin, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exemestane. , aminoglutethimide, anagrelide, navelvin, fadrazole, taciphen, toremifene, testolactone, anastrozole, letrozole, borozole, bicalutamide, lomustine and carmustine.
  • One or more types selected from the group may be used, but are not limited thereto.
  • a method for preventing, improving, or treating cancer which includes administering the cell therapy or pharmaceutical composition provided by the present invention to a subject.
  • the subject may include, without limitation, mammals, birds, reptiles, farmed fish, etc., including rats, livestock, humans, etc., that develop or are at risk of developing cancer due to a TGF- ⁇ -related disease.
  • the composition can be administered singly or multiple times in a pharmaceutically effective amount.
  • the composition can be formulated and administered in the form of a solution, powder, aerosol, injection, infusion solution (injection), capsule, pill, tablet, suppository, or patch.
  • the pharmaceutical composition for preventing or treating cancer may be administered through any general route as long as it can reach the target tissue.
  • the composition is not particularly limited thereto, but depending on the purpose, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, intrapulmonary administration, and intrarectal administration It can be administered through routes such as: However, when administered orally, it can be administered in an unformulated form, and since the active ingredients of the pharmaceutical composition may be denatured or decomposed by stomach acid, the oral composition must be coated with the active agent or protected from decomposition in the stomach. It can also be administered orally in formulated form or in the form of an oral patch. Additionally, the composition can be administered by any device that allows the active substance to move to target cells.
  • the genetically engineered immune effector cells can target only target tumor cells by a chimeric antigen receptor targeting mesothelin (MSLN) expressed in the cells,
  • MSLN chimeric antigen receptor targeting mesothelin
  • TGF- ⁇ transforming growth factor beta
  • TGF- ⁇ transforming growth factor beta
  • Figure 2 shows an NK cell line genetically engineered to express a chimeric antigen receptor according to the present invention and a peptide that inhibits the TGF- ⁇ signaling pathway in the HCC-1806 breast cancer cell line into which a luciferase reporter gene was introduced in Experimental Example 1 of the present invention. The results of comparing luciferase activity after processing are shown in a graph.
  • Figure 3 shows the MD-AMB-231 breast cancer cell line into which the luciferase reporter gene was introduced in Experimental Example 1 of the present invention, which was genetically engineered to express a chimeric antigen receptor according to the present invention and a peptide that inhibits the TGF- ⁇ signaling pathway.
  • the results of comparing luciferase activity after treating NK cell lines are shown graphically.
  • Figure 4 shows an NK cell line genetically engineered to express a peptide that inhibits the chimeric antigen receptor and TGF- ⁇ signaling pathway according to the present invention in the NCI-N87 gastric cancer cell line into which a luciferase reporter gene was introduced in Experimental Example 1 of the present invention. The results of comparing luciferase activity after processing are shown in a graph.
  • Figure 5 shows that in Experimental Example 1 of the present invention, the AGS gastric cancer cell line into which the luciferase reporter gene was introduced was treated with an NK cell line genetically engineered to express a peptide that inhibits the chimeric antigen receptor and the TGF- ⁇ signaling pathway according to the present invention. The results of comparing luciferase activity are shown in a graph.
  • Figure 6 shows a NK cell line genetically engineered to express a peptide that inhibits the chimeric antigen receptor and TGF- ⁇ signaling pathway according to the present invention in the NCI-H292 lung cancer cell line into which a luciferase reporter gene was introduced in Experimental Example 1 of the present invention. The results of comparing luciferase activity after processing are shown in a graph.
  • Figure 7 shows NK genetically engineered to express a chimeric antigen receptor according to the present invention and a peptide that inhibits the TGF- ⁇ signaling pathway in the SW-620 colon cancer cell line into which a luciferase reporter gene was introduced in Experimental Example 1 of the present invention.
  • the results of comparing luciferase activity after treating cell lines are shown graphically.
  • Figure 8 shows NK genetically engineered to express a chimeric antigen receptor according to the present invention and a peptide that inhibits the TGF- ⁇ signaling pathway in the SNU-1544 colon cancer cell line into which a luciferase reporter gene was introduced in Experimental Example 1 of the present invention. The results of comparing luciferase activity after treating cell lines are shown graphically.
  • Figure 9 shows the SK-HEP-1 liver cancer cell line into which the luciferase reporter gene was introduced in Experimental Example 1 of the present invention, which was genetically engineered to express a peptide that inhibits the chimeric antigen receptor and TGF- ⁇ signaling pathway according to the present invention.
  • the results of comparing luciferase activity after treating NK cell lines are shown graphically.
  • Figure 10 shows a NK cell line genetically engineered to express a peptide that inhibits the chimeric antigen receptor and TGF- ⁇ signaling pathway according to the present invention in the HEP-G2 liver cancer cell line into which the luciferase reporter gene was introduced in Experimental Example 1 of the present invention. The results of comparing luciferase activity after processing are shown in a graph.
  • Figure 11 shows an NK cell line genetically engineered to express a peptide that inhibits the chimeric antigen receptor and TGF- ⁇ signaling pathway according to the present invention in the AsPC-1 pancreatic cancer cell line into which the luciferase reporter gene was introduced in Experimental Example 1 of the present invention. The results of comparing luciferase activity after processing are shown in a graph.
  • Figure 12 shows a NK cell line genetically engineered to express a chimeric antigen receptor according to the present invention and a peptide that inhibits the TGF- ⁇ signaling pathway in the Capan-2 pancreatic cancer cell line into which a luciferase reporter gene was introduced in Experimental Example 1 of the present invention. The results of comparing luciferase activity after processing are shown in a graph.
  • Figure 13 shows the change in pSmad2/3 expression level in Aspc1, a pancreatic cancer cell line, according to treatment with the peptide of the present invention in Reference Example 1 of the present invention.
  • Figure 14 is a diagram showing the change in pSmad2/3 expression level in Bxpc3, a pancreatic cancer cell line, according to treatment with the peptide of the present invention in Reference Example 1 of the present invention.
  • Figure 15 is a diagram showing the change in pSmad2/3 expression level in Panc1, a pancreatic cancer cell line, according to treatment with the peptide of the present invention in Reference Example 1 of the present invention.
  • a chimeric antigen receptor (CAR) targeting mesothelin MSLN
  • cells genetically engineered to express a peptide or fragment thereof capable of inhibiting the transforming growth factor- ⁇ (TGF- ⁇ ) signaling pathway a chimeric antigen receptor (CAR) targeting mesothelin (MSLN)
  • TGF- ⁇ transforming growth factor- ⁇
  • the chimeric antigen receptor includes a mesothelin binding domain, and may further include one or more selected from the group consisting of a hinge domain, a signal peptide domain, a transmembrane domain, and one or more signaling domains. .
  • the mesothelin (MSLN) binding domain may include an scFv capable of specifically recognizing mesothelin, and the scFv is represented by the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively.
  • a light chain variable region (VL) comprising light chain CDR1, CDR2, and CDR3;
  • a linker comprising the amino acid sequence shown in SEQ ID NO: 12 or encoded by the nucleotide sequence shown in SEQ ID NO: 13;
  • a heavy chain variable region (VH) comprising heavy chain CDR1, CDR2, and CDR3 represented by the amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively, but is not limited thereto.
  • the mesothelin (MSLN) binding domain may include an scFv capable of specifically recognizing mesothelin, and the scFv may include the amino acid sequence shown in SEQ ID NO: 8, or Variable light chain region (VL) of the antibody encoded by the nucleotide sequence shown in SEQ ID NO: 9; A linker comprising the amino acid sequence shown in SEQ ID NO: 12 or encoded by the nucleotide sequence shown in SEQ ID NO: 13; and a variable heavy chain region (VH) of an antibody comprising the amino acid sequence represented by SEQ ID NO: 10 or encoded by the nucleotide sequence represented by SEQ ID NO: 11; but is not limited thereto.
  • the chimeric antigen receptor of the present invention can be designed with a signal peptide added to direct the translated chimeric protein to the membrane.
  • the signal peptide may be a CD8 signal peptide, and preferably may include an amino acid sequence represented by SEQ ID NO: 14, or a signal peptide encoded by the nucleotide sequence represented by SEQ ID NO: 15. It can be.
  • the chimeric antigen receptor of the present invention may further include an extracellular spacer, that is, a hinge.
  • the hinge may be a CD8-derived hinge region, and preferably may include an amino acid sequence represented by SEQ ID NO: 16, or a hinge encoded by the nucleotide sequence represented by SEQ ID NO: 17. You can.
  • the chimeric antigen receptor of the present invention may further include a transmembrane domain.
  • the transmembrane domain may be a CD8 transmembrane domain, and preferably includes the amino acid sequence represented by SEQ ID NO: 18, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 19.
  • the chimeric antigen receptor of the present invention has a cytoplasmic domain and may further include an intracellular signaling domain.
  • the intracellular signaling domain may be a CD3 zeta signaling domain, and preferably includes the amino acid sequence represented by SEQ ID NO: 20, or is encoded by the nucleotide sequence represented by SEQ ID NO: 21. You can.
  • the intracellular signaling domain may further include 4-1BB as a co-stimulatory signaling domain.
  • the 4-1BB preferably includes the amino acid sequence represented by SEQ ID NO: 22, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 23.
  • the intracellular signaling domain may include a CD3 zeta signaling domain as a primary signaling domain and 4-1BB as a co-stimulatory domain, preferably 4-1BB.
  • the CD3 zeta signaling domain may include the amino acid sequence represented by SEQ ID NO: 20, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 21, and the 4-1BB is the amino acid sequence represented by SEQ ID NO: 22. It may include or be encoded by the nucleotide sequence shown in SEQ ID NO: 23.
  • the cells may further include cells genetically engineered to express a peptide or fragment thereof capable of inhibiting the transforming growth factor- ⁇ (TGF- ⁇ ) signaling pathway.
  • TGF- ⁇ transforming growth factor- ⁇
  • the peptide may include the amino acid sequence represented by SEQ ID NO: 28, preferably consisting of the amino acid sequence represented by SEQ ID NO: 28.
  • sequence encoding the peptide may include the nucleotide sequence represented by SEQ ID NO: 29, preferably consisting of the nucleotide sequence represented by SEQ ID NO: 29.
  • the peptide may bind to the TGF- ⁇ receptor (TGFBR1 and/or TGFBR2) to inhibit TGF- ⁇ signaling.
  • TGFBR1 and/or TGFBR2 TGF- ⁇ receptor
  • the peptide competes with TGF- ⁇ and binds to the TGF- ⁇ receptor. It may be that TGF- ⁇ signaling is inhibited through a mechanism that prevents TGF- ⁇ cytokines from binding to the TGF- ⁇ receptor.
  • the cells may be immune effector cells.
  • the immune effector cells are lymphoid cells that participate in an immune response, such as promoting an immune effector response, and may be or include Natural Killer Cells (NK cells), but are not limited thereto.
  • NK cells Natural Killer Cells
  • a vector containing a gene encoding the peptide or its fragment can be transfected into the immune effector cell to introduce a foreign gene encoding the peptide or its fragment into the cell.
  • a polynucleotide (or gene construct) encoding the above-mentioned chimeric antigen receptor and a polynucleotide (or gene) encoding a peptide or fragment thereof capable of inhibiting the transforming growth factor beta signaling pathway are included in one vector. construct), or a vector containing a polynucleotide (or gene construct) encoding the chimeric antigen receptor, and a peptide or fragment thereof capable of inhibiting the transforming growth factor beta signaling pathway.
  • Encoding may include both types of vectors containing polynucleotides (or gene constructs).
  • a cell therapeutic agent comprising the genetically engineered cells provided by the present invention as an active ingredient.
  • the present invention relates to a pharmaceutical composition for preventing, improving or treating cancer containing the genetically engineered cells provided by the present invention as an active ingredient.
  • a peptide (P6) consisting of the amino acid sequence shown in SEQ ID NO: 28 targeting the TGF- ⁇ receptor was designed and produced.
  • pancreatic cancer cell lines Aspc1, Bxpc3, and Panc1 cell lines were seeded in a 6-well plate at a concentration of 1 (Control group: 15% ACN - candidate peptide solvent, DMSO - P144 peptide solvent, P144: TSLDASIWAMMQNA (SEQ ID NO: 37)) and the expression levels of pSmad2/3 and total Smad2/3 were analyzed by Western blotting method.
  • Example 1 Construction of a vector containing a chimeric antigen receptor and a TGF- ⁇ signaling pathway inhibitory gene
  • the chimeric antigen receptor expression cassette includes CD8 signal peptide, mesothelin binding domain (light chain variable region; linker; and heavy chain variable region), CD8 hinge, CD8 transmembrane domain, 4-1BB, and CD3 zeta signaling domain.
  • the recombinant gene was placed under the control of the SFFV single promoter.
  • the base sequence of the SFFV promoter used in the experiment and the base sequences of each other gene are shown in Table 1, and the amino acid sequences encoded by these base sequences are shown in Table 2 below.
  • Natural killer cells were used as immune effector cells to be genetically manipulated to express the chimeric antigen receptor and TGF- ⁇ signaling pathway inhibitory peptide.
  • the NK-92 cell line was purchased from ATCC.
  • the 293T cell line which is the cell line to be used to transduce the lentivirus, was also purchased from ATCC.
  • Both the NK-92 cell line and the lentivirus-transduced NK-92 cell line described below were treated with 100 ⁇ g/mL of streptomycin, 100 units/mL of penicillin, and 20% fetal bovine serum. serum; FBS), 10% MEM vitamin solution, 1 streptomycin), 100 units/mL of penicillin, and 10% fetal bovine serum (FBS). All cells were cultured at 37°C with 5% CO2 (95% CO2). Cultured in an environment maintained with air.
  • lentivirus In order to obtain a lentivirus of 5 , 5.5 ⁇ g of the lentiviral transfer vector (UCI-VD9 or UCI-VD35) prepared in Example 1, 3.7 ⁇ g of the packaging vector (UCI-VD6), and 1.8 ⁇ g of the envelope vector (UCI-VD11). Transfection was performed with lipofectamine 3000 transfection reagent. After transfection, the lentivirus produced in 293T cells was ejected out of the cell and existed in the cell culture medium in the form of virus particles. Accordingly, 48 or 72 hours after transfection, only the cell culture fluid was removed from the culture plate and concentrated 100 times with lenti-X concentration reagent (concentrator) to obtain lentivirus particles in the form of a pellet. Afterwards, 1 To select cell lines in which transduction was successful, cells expressing GFP were selected using a cell sorter (SH800S).
  • SH800S cell sorter
  • Pancreatic cancer cell lines (AsPC-1, Capan-2), breast cancer cell lines (HCC-1806, MDA-MB-231), stomach cancer cell lines (NCI-N87, AGS), lung cancer cell lines (NCI-H292), colon cancer cell lines (SNU- 1544, SW-620) and liver cancer cell lines (SK-HEP-1, Hep-G2) were purchased from the Korean Cell Line Bank (KCLB), and cell lines into which a luciferase reporter gene was introduced were used.
  • KCLB Korean Cell Line Bank
  • AsPC-1_luc_puro, Capan-2_luc_puro, HCC-1806_luc_puro, MDA-MB-231_luc_puro, NCI-N87_luc_puro, AGS_luc_puro, NCI-H292_luc_puro, SNU-1544_luc_puro and SW-620_luc_puro cell lines were cultured using RPMI-1640 medium.
  • EP- 1_luc_puro and Hep-G2_luc_puro were cultured using high-glucose Dulbecco's modified Eagle medium.
  • All media used for culturing cancer cell lines into which a luciferase reporter gene was introduced contained 100 ⁇ g/mL of streptomycin, 100 units/mL of penicillin, and 10% fetal bovine serum (FBS). ) and 4 ⁇ g/mL of puromycin were included. All cells were cultured at 37°C in an environment where CO 2 was maintained at 5% (95% air).
  • NK-92 cells MSLN CAR+p6 peptide
  • liver cancer cells were treated with an NK cell line genetically engineered to express a chimeric antigen receptor according to the present invention and a peptide that inhibits the TGF- ⁇ signaling pathway, the untreated group or the mock vector It was confirmed that the number of luciferase-expressing liver cancer cells was significantly reduced compared to the case of treating NK cells transfected with .
  • pancreatic cancer cells when pancreatic cancer cells are treated with NK cells transduced with a mock vector, the killing effect on gastric cancer cells is minimal compared to the untreated group, whereas the chimeric antigen receptor and TGF- according to the present invention
  • NK cell lines genetically engineered to express a peptide that inhibits the ⁇ signaling pathway were treated, it was confirmed that the death rate of pancreatic cancer cells was very high.
  • the NK cell line genetically engineered according to the present invention can target cancer cells expressing mesothelin due to the chimeric antigen receptor expressed in the cells, and can target cancer cells expressing mesothelin by the peptide additionally expressed in the cells. It can be seen that through inhibition of the TGF- ⁇ signaling pathway, the ability of cells to invade into microtumors is improved, and the killing effect on cancer cells is also significantly increased.
  • the present invention relates to immune effector cells genetically engineered to increase the therapeutic effect of diseases such as cancer by immunotherapy, and cell therapy products using the same.

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Abstract

The present invention relates to cells that have been genetically engineered to express a peptide or fragment thereof capable of inhibiting the transforming growth factor-β (TGF-β) signaling pathway, in conjunction with a chimeric antigen receptor (CAR) targeting mesothelin (MSLN).

Description

유전자 조작된 세포 및 이의 용도Genetically Engineered Cells and Their Uses
본 발명은 면역 요법에 의해 암 등의 질환에 대한 치료 효과가 증대되도록, 유전적으로 조작시킨 면역 이펙터 세포 및 이의 용도에 관한 것이다.The present invention relates to genetically engineered immune effector cells and uses thereof to increase the therapeutic effect for diseases such as cancer by immunotherapy.
세포 면역치료는 암 치료에 있어서 매우 장래성이 있는 치료법이다. 그러나, 대부분의 면역치료학적 접근법은 고형 종양을 비롯한 대부분의 악성 종양에 대한 치료 효과에 아래와 같은 제한점이 있다: (1) 종양 세포 표면 상의 종양 항원의 발현 감소 (이는 면역계에 의한 항원의 검출을 감소시킴); (2) PD1, NKG2A, TIGIT와 같은 억제성 수용체에 대한 리간드의 발현; (3) 면역 세포 비활성화를 유도하는 CISH와 같은 세포성 체크포인트의 상향조절; 및 (4) 면역 반응을 억제하여 종양 세포 증식과 생존률을 촉진하는 형질전환 성장 인자-β (TGF-β) 및 아데노신과 같은 물질들을 방출하는 미시적 환경의 유도 등이 있다. 따라서, 상기한 당면과제들 중 적어도 하나라도 해결할 수 있는 세포 면역치료의 개선된 방법이 필요한 실정이다.Cellular immunotherapy is a very promising treatment method for cancer treatment. However, most immunotherapeutic approaches have the following limitations in their therapeutic effectiveness against most malignant tumors, including solid tumors: (1) Reduced expression of tumor antigens on the surface of tumor cells (which reduces detection of antigens by the immune system) Sikkim); (2) expression of ligands for inhibitory receptors such as PD1, NKG2A, and TIGIT; (3) upregulation of cellular checkpoints, such as CISH, leading to immune cell deactivation; and (4) induction of a microscopic environment that releases substances such as transforming growth factor-β (TGF-β) and adenosine, which suppress immune responses and promote tumor cell proliferation and survival. Therefore, there is a need for an improved method of cellular immunotherapy that can solve at least one of the above-mentioned challenges.
한편, TGF-β 신호전달은 암 진행에 중요한 역할을 한다. 대부분의 암 세포는 상피 항증식 반응을 비활성화하고, 유전자 발현, 면역억제 사이토카인의 방출 및 상피 가소성에 대한 효과를 통해 증가된 TGF-β 발현 및 자가분비 TGF-β 신호로부터 이익을 얻고 있다. 그 결과, 암 세포에 있어서 TGF-β는 암 세포의 침윤 및 전이와, 줄기세포적 성질과 약제 내성을 증가시키는 역할을 한다. 종양 미세환경에서 암 세포, 기질 섬유아세포 및 기타 세포에서 방출되는 TGF-β는 종양의 구조를 형성하고 면역 세포의 항종양 활성을 억제함으로써 암 진행을 더욱 촉진하고, 따라서 면역억제 환경을 생성하여 항암 면역 요법의 효능을 예방하거나 약화시킨다. 따라서 TGF-β 신호전달의 억제는 TGF-β에 반응하지 않는 암 세포를 포함하는 종양을 포함하여 현재 및 향후 면역 요법의 효능을 향상시키기 위한 전제 조건 및 주요 수단으로 간주된다.Meanwhile, TGF-β signaling plays an important role in cancer progression. Most cancer cells inactivate epithelial antiproliferative responses and benefit from increased TGF-β expression and autocrine TGF-β signaling through effects on gene expression, release of immunosuppressive cytokines, and epithelial plasticity. As a result, in cancer cells, TGF-β plays a role in increasing invasion and metastasis of cancer cells, stem cell properties, and drug resistance. TGF-β released from cancer cells, stromal fibroblasts and other cells in the tumor microenvironment further promotes cancer progression by shaping the structure of the tumor and suppressing the anti-tumor activity of immune cells, thus creating an immunosuppressive environment and anti-cancer. Prevents or attenuates the effectiveness of immunotherapy. Therefore, inhibition of TGF-β signaling is considered a prerequisite and key means to improve the efficacy of current and future immunotherapies, including for tumors containing cancer cells that are unresponsive to TGF-β.
메소텔린(mesothelin; MSLN)은 정상 중피(mesothelial) 세포들에서 그것의 낮은 발현 및 광범위한 고형 종양들에서의 높은 발현을 고려할 때, 암 면역 요법(immunotherapy)의 매력적인 표적으로 알려졌다. 지금까지 보고된 MSLN-표적된 면역요법들은 유리한 안전성 프로파일을 뒷받침한다. MSLN은 적어도 식도암(oesophageal cancer), 유방암(breast cancer), 위암(gastric cancer), 담관암종(cholangiocarcinoma), 췌장암(pancreatic cancer), 결장암(colon cancer), 폐암(Lung cancer), 흉선 암종(Thymic carcinoma), 중피종(mesothelioma), 난소암(ovarian cancer), 및 자궁내막암(endometrial cancer)과 같은, 많은 흔한 고형 종양들에서 잠재적인 CAR 표적이 된다 [Morello, A. et al. (2016) Mesothelin-Targeted CARs: Driving T Cells to Solid Tumors. Cancer Discov. 6(2); 133-46].Mesothelin (MSLN) has been shown to be an attractive target for cancer immunotherapy, considering its low expression in normal mesothelial cells and high expression in a wide range of solid tumors. MSLN-targeted immunotherapies reported to date support a favorable safety profile. MSLN includes at least oesophageal cancer, breast cancer, gastric cancer, cholangiocarcinoma, pancreatic cancer, colon cancer, lung cancer, and thymic carcinoma. ), are potential CAR targets in many common solid tumors, such as mesothelioma, ovarian cancer, and endometrial cancer [Morello, A. et al. (2016) Mesothelin-Targeted CARs: Driving T Cells to Solid Tumors. Cancer Disco. 6(2); 133-46].
본 발명의 일 목적은 메소텔린(Mesothelin)을 표적화하는 키메라 항원 수용체(chimeric antigen receptor; CAR)와 함께 전환성장인자 베타(Transforming growth factor-β, TGF-β) 신호전달 경로를 억제할 수 있는 펩타이드를 발현하도록 유전적으로 조작된 세포를 제공하고자 한다. One object of the present invention is to provide a peptide that can inhibit the transforming growth factor-β (TGF-β) signaling pathway along with a chimeric antigen receptor (CAR) targeting mesothelin. The aim is to provide cells that have been genetically engineered to express.
본 발명의 다른 목적은 상기 유전적으로 조작된 세포를 포함하는 세포 치료제에 관한 것이다. Another object of the present invention relates to a cell therapeutic agent comprising the genetically engineered cells.
본 발명의 또 다른 목적은 상기 유전적으로 조작된 세포를 포함하는 다양한 용도의 약학 조성물에 관한 것이다. Another object of the present invention relates to pharmaceutical compositions for various uses containing the genetically engineered cells.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업계에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.
본 발명의 일 구현예에 따르면, 1) 메소텔린(Mesothelin; MSLN)을 표적화하는 키메라 항원 수용체(chimeric antigen receptor; CAR); 및 2) 전환성장인자 베타(Transforming growth factor-β, TGF-β) 신호전달 경로를 억제할 수 있는 펩타이드 또는 이의 단편을 발현하도록 유전적으로 조작된 세포에 관한 것이다. According to one embodiment of the present invention, 1) a chimeric antigen receptor (CAR) targeting mesothelin (MSLN); and 2) cells genetically engineered to express a peptide or fragment thereof capable of inhibiting the transforming growth factor-β (TGF-β) signaling pathway.
본 발명에서 펩타이드와 관련하여 본 명세서에 사용된 "재조합" 또는 "조작"은 펩타이드를 암호화하는 핵산 및 펩타이드를 발현하는 세포 또는 유기체에 대한 유전공학 기술의 적용 결과로 변경된 아미노산 서열을 갖는 것을 의미한다. 핵산과 관련하여, 용어 "재조합" 또는 "조작"은 유전공학 기술의 적용 결과로 변경된 핵산 서열을 갖는 것을 의미한다. 유전공학 기술은 PCR 및 DNA 클로닝 기술; 형질주입, 형질도입, 형질전환 및 다른 유전자 전달 기술; 상동 재조합; 부위-특이적 돌연변이; 및 유전자 융합을 포함하지만, 이로 제한되지 않는다. As used herein, “recombinant” or “manipulated” in relation to a peptide means having an amino acid sequence that has been altered as a result of the application of genetic engineering techniques to the nucleic acid encoding the peptide and to the cell or organism expressing the peptide. . With regard to nucleic acids, the terms “recombinant” or “engineered” mean having a nucleic acid sequence that has been altered as a result of the application of genetic engineering techniques. Genetic engineering technologies include PCR and DNA cloning technologies; Transfection, transduction, transformation and other gene transfer techniques; homologous recombination; site-specific mutation; and gene fusions.
본 발명에서 상기 "유전자 조작된 (engineered)" 또는 "유전적으로 조작된"이라는 용어는 게놈 DNA 서열이 재조합 기술에 의해 의도적으로 변형된 세포 또는 유기체, 또는 이의 조상을 의미한다. 본 발명에서 상기 "유전자 조작"은 "유전자 이식"을 포함한다.As used herein, the term “genetically engineered” or “genetically engineered” refers to a cell or organism, or an ancestor thereof, whose genomic DNA sequence has been intentionally modified by recombinant technology. In the present invention, the “genetic manipulation” includes “gene transplantation.”
본 발명에서 상기 세포에서 상기 펩타이드 또는 그 단편이 발현되도록 유전적으로 조작하는 방법으로는, 벡터, 특이적 수용체 또는 세포 융합법 등의 생물학적 방법, 마이크로 인젝션법, 일렉트로포레이션법, 유전자총 또는 초음파 유전자 도입법 등의 물리적 방법, 또는 인산칼슘 공침전법, 리포솜법, 리포펙션법, DEAE 덱스트런법, 또는 알칼리 금속법 등의 화학적 방법을 통하여 상기 펩타이드 또는 그 단편을 암호화하는 유전자를 도입하는 방법에 의할 수 있다. 상기 방법들이나 그 외에 다양한 기법이 당해 기술 분야에서 통상의 기술자에게 알려져 있으며, 예를 들면, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)에 개시되어 있다. 바람직하게는 본 발명에서는 상기 키메라 항원 수용체; 및 펩타이드 또는 그 단편; 각각을 암호화하는 유전자를 포함하는 벡터를 상기 면역 이펙터 세포에 형질 감염시켜 상기 세포에 상기 단백질들을 암호화하는 외래 유전자를 도입할 수 있다.In the present invention, methods for genetically manipulating the expression of the peptide or its fragment in the cell include biological methods such as vectors, specific receptors, or cell fusion methods, microinjection methods, electroporation methods, gene guns, or ultrasonic genes. By a method of introducing the gene encoding the peptide or its fragment through a physical method such as the introduction method, or a chemical method such as the calcium phosphate coprecipitation method, liposome method, lipofection method, DEAE dextran method, or alkali metal method. can do. These methods and various other techniques are known to those skilled in the art, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Preferably, in the present invention, the chimeric antigen receptor; and peptides or fragments thereof; Foreign genes encoding the proteins can be introduced into the cells by transfecting the immune effector cells with vectors containing genes encoding each.
1) 메소텔린(Mesothelin; MSLN)을 표적화하는 키메라 항원 수용체(chimeric antigen receptor; CAR) 1) Chimeric antigen receptor (CAR) targeting mesothelin (MSLN)
본 발명에서 상기 "키메라 항원 수용체(chimeric antigen receptor; CAR)"란 세포외 표적-결합 도메인, 막관통 도메인 및 세포내 신호전달 도메인을 포함하는 세포-표면 수용체로서 정의되며, 이들 모두는 단일 단백질 상에서 함께 천연적으로 발견되지 않는 조합으로 존재한다. 이는 특히, 세포외 도메인과 세포내 신호전달 도메인이 단일 수용체 단백질 상에서 함께 천연적으로 발견되지 않는 수용체를 포함한다. In the present invention, the “chimeric antigen receptor (CAR)” is defined as a cell-surface receptor comprising an extracellular target-binding domain, a transmembrane domain, and an intracellular signaling domain, all of which are formed on a single protein. They exist together in combinations that are not found naturally. This particularly includes receptors in which the extracellular and intracellular signaling domains are not naturally found together on a single receptor protein.
본 발명에서 상기 "메소텔린(Mesothelin)"은 MSLN이라고도 불리우는 단백질로서, 중피 세포(mesothelial cells)에서 분비되는 40 kDa 크기의 단백질이다. 상기 단백질은 단일클론항체 K1과의 반응에 의해 처음으로 규명되었으며, 이후 지속된 연구를 통해 메소텔린 유전자는 글리코포스파티딜이노시톨 연결과, 31-kDa 쉐드 단편인 거핵세포 강화 인자(megakaryocyte-potentiating factor; MPF)에 의해 세포막에 부착되는 메소텔린을 생성하도록 처리되는 전구체 단백질을 암호화하는 것으로 알려졌다. 메소텔린이 세포 부착 등에 관여할 수 있다고 제안되었으나, 아직까지는 그 생물학적 기능이 명확히 규명되지는 않았다. 본 발명에서 상기 메소텔린은 서열번호 1로 표시되는 아미노산 서열로 이루어지는 것일 수 있으나, 이에 제한되는 것은 아니다. In the present invention, “Mesothelin” is a protein also called MSLN, and is a 40 kDa protein secreted by mesothelial cells. The protein was first identified by reaction with the monoclonal antibody K1, and through continued research, the mesothelin gene was linked to a glycophosphatidylinositol linkage and a 31-kDa shed fragment, megakaryocyte-potentiating factor (MPF). ) is known to encode a precursor protein that is processed to produce mesothelin, which is attached to the cell membrane. It has been suggested that mesothelin may be involved in cell adhesion, etc., but its biological function has not yet been clearly identified. In the present invention, the mesothelin may be composed of the amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto.
본 발명에서 상기 키메라 항원 수용체는 메소텔린 결합 도메인을 포함하고, 그 외에, 힌지 도메인, 신호 펩티드 도메인, 막횡단 도메인 및 하나 이상의 신호전달 도메인으로 이루어진 군에서 선택된 1종 이상을 추가로 포함할 수 있다. In the present invention, the chimeric antigen receptor includes a mesothelin binding domain, and may further include one or more selected from the group consisting of a hinge domain, a signal peptide domain, a transmembrane domain, and one or more signaling domains. .
메소텔린 결합 도메인mesothelin binding domain
본 발명의 상기 키메라 항원 수용체는 메소텔린(MSLN) 결합 도메인을 포함한다. The chimeric antigen receptor of the present invention includes a mesothelin (MSLN) binding domain.
본 발명에서 제공되는 상기 메소텔린(MSLN) 결합 도메인은 종래에 알려진 항-MSLN 결합 도메인보다 더 높은 친화성으로 메소텔린에 결합할 수 있다.The mesothelin (MSLN) binding domain provided in the present invention can bind to mesothelin with higher affinity than the conventionally known anti-MSLN binding domain.
본 발명에서 상기 결합 도메인은 항체 또는 그 단편일 수 있다. In the present invention, the binding domain may be an antibody or a fragment thereof.
본 발명에서 상기 "항체"는 항원에 특이적으로 결합하는 면역글로불린 분자를 지칭한다. 항체는 천연 또는 재조합 무손상 면역글로불린일 수 있거나, 무손상 면역글로불린의 면역 반응 부분일 수 있다. 항체는 일반적으로 면역글로불린 분자의 사량체이다. 상세하게 상기 항체는 각각 약 25 kDa의 경(L)쇄 2개와 각각 약 50 kDa의 중(H)쇄 2개로 구성된, 테트라머 당화된 단백질이다. 람다 및 카파로 지칭되는 경쇄의 2가지 유형이 항체에서 발견할 수 있다. 중쇄 불변 도메인의 아미노산 서열에 따라, 면역글로불린은 5가지 주 클래스 A, D, E, G 및 M으로 분류할 수 있으며, 이들 중 몇 가지는 하위 클래스(이소형)로, 예를 들어, IgG1, IgG2, IgG3, IgG4, IgA1 및 IgA2로 더욱 세분될 수 있다. 각각의 경쇄는 전형적으로 N-말단 가변성 (V) 도메인 (VL)과 불변 (C) 도메인 (CL)을 함유한다. 각각의 중쇄는 전형적으로 N-말단 V 도메인 (VH), 3개 또는 4개의 C 도메인 (CH1-3) 및 힌지부를 함유한다. VH에 가장 가까이 위치한 CH 도메인은 CH1으로 명명된다. VH 도메인과 VL 도메인은, 과가변성 서열 (상보성 결정부, CDR)로 된 3개의 영역에 대한 스캐폴드를 형성하는, 프래임워크 영역으로 지칭되는 상대적으로 보존된 서열들로 된 4개의 영역들 (FR1, FR2, FR3 및 FR4)로 구성된다. CDR들은 항체와 항원의 특이적인 상호작용을 담당하는 잔기들 대부분을 함유한다. CDR들은 CDR1, CDR2 및 CDR3로 지칭된다. 즉, 중쇄 상의 CDR 구성 요소들은 CDRH1, CDRH2 및 CDRH3로 지칭되는 반면 경쇄 상의 CDR 구성 요소들은 CDRL1, CDRL2 및 CDRL3로 지칭된다. CDR들은 전형적으로 Sequences of Proteins of Immunological Interest, US Department of Health and Human Services (1991), eds. Kabat et al에 기술된 바와 같이, Kabat CDR을 참조한다. 항원 결합부를 특정하는 또 다른 표준은 Chothia에 의해 기술된 과가변성 루프를 참조하는 것이다. 예를 들어, Chothia, D. et al. (1992) J. Mol. Biol. 227:799-817; 및 Tomlinson et al. (1995) EMBO J. 14:4628-4638을 참조한다. 또 다른 표준은 Oxford Molecular's AbM antibody modeling software에서 사용되는 AbM 정의이다. 일반적으로, 예를 들어, Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S, and Kontermann, R., Springer-Verlag, Heidelberg)을 참조한다. Kabat CDR에 관하여 기술된 구현예들은 대안적으로 Chothia 과가변성 루프 또는 AbM-정의된 루프 또는 이들 방법들의 임의의 조합과 관련하여 유사하게 기술된 관계로 구현될 수 있다. 본 발명에서 상기 항체는 폴리클로날 항체, 모노클로날 항체, Fv, Fab, F(ab)2 등, 및 단일쇄 항체 및 인간화 항체를 포함하나 이에 제한되지는 않는 다양한 형태로 존재할 수 있다 (문헌 [Harlow, et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY]; [Harlow, et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York]; [Houston, et al., 1988, Proc. Natl. Acad. Sci., USA 85: 5879-5883]; 및 [Bird, et al., 1988, Science 242: 423-426]). In the present invention, the “antibody” refers to an immunoglobulin molecule that specifically binds to an antigen. The antibody may be a natural or recombinant intact immunoglobulin, or may be an immune-reactive portion of an intact immunoglobulin. Antibodies are generally tetramers of immunoglobulin molecules. In detail, the antibody is a tetrameric glycosylated protein composed of two light (L) chains of about 25 kDa each and two heavy (H) chains of about 50 kDa each. Two types of light chains, referred to as lambda and kappa, can be found in antibodies. Depending on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be classified into five main classes A, D, E, G and M, several of which are subclasses (isotypes), such as IgG1, IgG2 , can be further subdivided into IgG3, IgG4, IgA1 and IgA2. Each light chain typically contains an N-terminal variable (V) domain (VL) and constant (C) domain (CL). Each heavy chain typically contains an N-terminal V domain (VH), three or four C domains (CH1-3), and a hinge region. The CH domain located closest to VH is named CH1. The VH and VL domains are composed of four regions of relatively conserved sequences, referred to as framework regions, which form the scaffold for three regions of hypervariable sequences (complementarity determining regions, CDRs) (FR1). , FR2, FR3 and FR4). CDRs contain most of the residues responsible for the specific interaction of the antibody with the antigen. The CDRs are referred to as CDR1, CDR2 and CDR3. That is, the CDR elements on the heavy chain are referred to as CDRH1, CDRH2, and CDRH3, while the CDR elements on the light chain are referred to as CDRL1, CDRL2, and CDRL3. CDRs are typically described in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services (1991), eds. See Kabat CDR, as described in Kabat et al. Another standard for specifying the antigen binding site is by reference to the hypervariable loop described by Chothia. For example, Chothia, D. et al. (1992) J. Mol. Biol. 227:799-817; and Tomlinson et al. (1995) EMBO J. 14:4628-4638. Another standard is the AbM definition used in Oxford Molecular's AbM antibody modeling software. In general, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S, and Kontermann, R., Springer-Verlag, Heidelberg). Implementations described with respect to Kabat CDR can alternatively be implemented in relationships similarly described with respect to Chothia hypervariable loops or AbM-defined loops or any combination of these methods. In the present invention, the antibody may exist in various forms, including but not limited to polyclonal antibodies, monoclonal antibodies, Fv, Fab, F(ab)2, etc., and single chain antibodies and humanized antibodies (document. [Harlow, et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY]; [Harlow, et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York]; , et al., 1988, Proc. Acad. Sci., USA 85: 5879-5883; and [Bird, et al., 1988, Science 242: 423-426].
본 발명에서 상기 항원 (예를 들어, MSLN)에 대한 항체는 통상적인 하이브리도마 기법을 이용해 면역화된 형질전환 마우스로부터 수득할 수 있다. 형질전환 마우스가 보유한 인간 면역글로불린 전이유전자는 B 세포 분화 중에 재정렬하며, 이후 클래스 스위칭 및 체세포 돌연변이를 거치게 된다. 따라서, 이러한 기법을 이용해, 치료학적으로 유용한, 비-제한적으로, IgGl (gamma 1) 및 IgG3 등의, IgG, IgA, IgM 및 IgE 항체를 제조하는 것이 가능하다. 인간 항체 및 인간 단일클론 항체를 제조하기 위한 이러한 기술과 이러한 항체를 제조하는 프로토콜에 대한 상세한 내용은, 예를 들어, PCT 공개번호 WO2014/055771, WO 98/24893, WO 96/34096 및 WO 96/33735; 미국 특허 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 및 5,939,598을 참조하며, 이들 문헌들 각각은 그 전체가 원용에 의해 본 명세서에 포함된다. "인간화된" 항체는 오리지널 항체와 비슷한 항원 특이성을, 즉 본 발명에서 예를 들어 MSLN에 결합하는 능력을 유지한다.In the present invention, antibodies against the antigen (eg, MSLN) can be obtained from immunized transgenic mice using conventional hybridoma techniques. Human immunoglobulin transgenes carried by transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Accordingly, using this technique, it is possible to prepare therapeutically useful IgG, IgA, IgM and IgE antibodies, such as, but not limited to, IgGl (gamma 1) and IgG3. Details of these techniques for preparing human antibodies and human monoclonal antibodies and the protocols for preparing such antibodies can be found, for example, in PCT Publication Nos. WO2014/055771, WO 98/24893, WO 96/34096 and WO 96/ 33735; US Patent 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, each of which is incorporated herein by reference in its entirety. “Humanized” antibodies retain similar antigenic specificity as the original antibody, i.e., the ability to bind, for example, MSLN in the present invention.
본 발명에서 상기 "항체 단편" 또는 "항원 결합 단편"은 전장 항체보다 짧지만 적어도 항체의 항원에 결합하는 부분 가변 영역 (예를 들어, 하나 이상의 CDR 및/또는 하나 이상의 항원-결합 부위)을 포함하므로, 전장 항체의 결합 특이성 및 적어도 부분적 특이적 결합 능력을 보유하는 전장 항체의 임의의 부분을 지칭한다. 따라서, 항원-결합 단편은 항체 단편이 유래된 항체와 동일한 항원에 결합하는 항원-결합 부분을 포함하는 항체 단편을 지칭한다. 항체 단편은 전장 항체의 효소 처리에 의해 생산된 항체 유도체, 및 합성에 의해 생산된 유도체, 예컨대 재조합에 의해 생산된 유도체를 포함한다. 항체는 항체 단편을 포함한다. 항체 단편으로는 단일쇄 Fv (scFv), Fab, Fab', F(ab')2, Fv, dsFv, 이중-항체, Fd 및 Fd' 단편, 및 변형된 단편을 포함하는 기타 단편을 포함하나 이에 제한되지는 않는다 (예를 들어, 문헌 [Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p3-25, Kipriyanov] 참조). 단편은 예를 들어, 디술피드 결합을 통해 및/또는 펩티드 링커를 통해 함께 연결된 다중 쇄를 포함할 수 있다. 항체 단편은 일반적으로 적어도 또는 약 50개 아미노산을 포함하고, 전형적으로 적어도 또는 약 200개 아미노산을 포함한다. 항원-결합 단편은 항원에 면역특이적으로 결합하는 (즉, 적어도 또는 적어도 약 107-108 M-1의 Ka를 나타내는) 항체를 수득하기 위해 (예를 들어, 상응하는 영역의 대체를 통해) 항체 프레임워크에 삽입되는 임의의 항체 단편을 포함한다. "기능적 단편" 또는 "항-MSLN 항체 유사체"는 리간드에 결합하거나 신호 전달을 개시하는 수용체의 능력을 방지하거나 실질적으로 감소시킬 수 있는 단편 또는 유사체이다. 본원에서 사용된 바와 같은 기능적 단편은 일반적으로 "항체 단편"과 동일한 의미를 가지며, 항체의 경우, 리간드에 결합하거나 신호 전달을 개시하는 수용체의 능력을 방지하거나 실질적으로 감소시킬 수 있는 단편, 예컨대 Fv, Fab, 및 F(ab')2를 지칭할 수 있다. "Fv" 단편은 비공유 결합에 의해 하나의 중쇄의 가변 도메인 및 하나의 경쇄의 가변 도메인에 의해 형성된 이량체 (VH-VL 이량체)로 구성된다. 이 구성에서, 각 가변 도메인의 3개의 CDR이 상호작용하여 무손상 항체와 동일한 VH-VL 이량체의 표면 상의 표적-결합 부위를 결정한다. 6개의 CDR은 함께 무손상 항체의 표적-결합 특이성을 부여한다. 그러나, 심지어 단일 가변 도메인 (또는 3개의 표적-특이적 CDR만을 포함하는 Fv의 절반)도 여전히 표적을 인식하고 이에 결합하는 능력을 가질 수 있다.In the present invention, the “antibody fragment” or “antigen-binding fragment” is shorter than the full-length antibody, but includes at least a partial variable region (e.g., one or more CDRs and/or one or more antigen-binding sites) that binds to the antigen of the antibody. Therefore, it refers to any portion of a full-length antibody that retains the binding specificity and at least partial specific binding ability of the full-length antibody. Accordingly, antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds the same antigen as the antibody from which the antibody fragment was derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, and derivatives produced synthetically, such as those produced recombinantly. Antibodies include antibody fragments. Antibody fragments include, but are not limited to, single chain Fv (scFv), Fab, Fab', F(ab')2, Fv, dsFv, double-antibody, Fd and Fd' fragments, and other fragments, including modified fragments. It is not limited (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p3-25, Kipriyanov). Fragments may comprise multiple chains linked together, for example, through disulfide bonds and/or through peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically contain at least or about 200 amino acids. The antigen-binding fragment is an antibody (e.g., through replacement of a corresponding region) to obtain an antibody that immunospecifically binds to the antigen (i.e., exhibits a Ka of at least or at least about 107-108 M-1). Includes any antibody fragment inserted into the framework. A “functional fragment” or “anti-MSLN antibody analog” is a fragment or analog that can prevent or substantially reduce the ability of a receptor to bind a ligand or initiate signal transduction. As used herein, functional fragment generally has the same meaning as "antibody fragment" and, in the case of an antibody, is a fragment capable of preventing or substantially reducing the ability of a receptor to bind a ligand or initiate signal transduction, such as Fv. , Fab, and F(ab')2. The “Fv” fragment consists of a dimer (VH-VL dimer) formed by the variable domains of one heavy chain and the variable domains of one light chain by non-covalent association. In this configuration, the three CDRs of each variable domain interact to determine the target-binding site on the surface of the VH-VL dimer identical to the intact antibody. The six CDRs together confer the target-binding specificity of the intact antibody. However, even a single variable domain (or half of an Fv containing only three target-specific CDRs) may still have the ability to recognize and bind a target.
본 발명에서 상기 "단일쇄 Fv (scFv)"는 함께 연결되는 항체의 중쇄 및 경쇄의 가변 영역들을 가지는 단쇄 항체 단편이다. 미국 특허 제 7,741,465호, 및 6,319,494호 그리고 Eshhar et al., Cancer Immunol Immunotherapy (1997) 45: 131-136을 참고 가능하다. 표적 항원과 특이적으로 상호작용하는 모 항체의 능력을 유지한다. scFv는 키메라 항원 수용체를 이루는 다른 성분들과 함께 단쇄의 일부로서 발현되도록 유전적으로 조작될 수 있어 바람직하다. 항원 결합 도메인은 전형적으로 키메라 항원 수용체의 세포외 부분의 일부로 포함되어, 표적하는 항원, 여기서는 특히 메소텔린(MSLN)을 인식하여 결합할 수 있다. In the present invention, the “single chain Fv (scFv)” is a single chain antibody fragment having the variable regions of the heavy and light chains of the antibody linked together. Reference may be made to US Patent Nos. 7,741,465, and 6,319,494 and Eshhar et al., Cancer Immunol Immunotherapy (1997) 45: 131-136. Maintains the ability of the parent antibody to specifically interact with the target antigen. The scFv is desirable because it can be genetically engineered to be expressed as part of a single chain together with other components that make up the chimeric antigen receptor. The antigen binding domain is typically included as part of the extracellular portion of the chimeric antigen receptor and is capable of recognizing and binding the targeted antigen, here specifically mesothelin (MSLN).
본 발명에서 상기 scFv는 당해 기술 분야에 공지된 방법에 따라 제조할 수 있다 (예, Bird et al., (1988) Science 242:423-426 및 Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv 분자는 플렉서블 폴리펩타이드 링커를 사용해 VH와 VL 영역을 연결함으로써 만들 수 있다. scFv 분자는 최적화된 길이 및/또는 아미노산 조성을 가진 링커 (예를 들어, Ser-Gly 링커)를 포함한다. 링커 길이는 scFv의 가변부가 접히는 (folding) 방식과 상호작용하는 방식에 크게 영향을 미칠 수 있다. 실제, 짧은 폴리펩타이드 링커 (예, 아미노산 5-10개)가 사용된다면, 쇄내 접힘이 방지된다. 또한, 2개의 가변부를 근접시켜 기능성 에피토프 결합부를 형성하기 위해 쇄간 접힘이 필요하다. 링커 방향성 및 크기에 대한 예는, 예를 들어, Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, 미국 특허 출원 공개 번호 2005/0100543, 2005/0175606, 2007/0014794 및 PCT 공개번호 WO2006/020258 및 WO2007/024715를 참조하며, 이들 문헌 각각의 전체 내용이 원용에 의해 본 명세서에 포함된다.In the present invention, the scFv can be prepared according to methods known in the art (e.g., Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad Sci USA 85:5879-5883). ScFv molecules can be made by connecting the VH and VL regions using a flexible polypeptide linker. The scFv molecule includes a linker with optimized length and/or amino acid composition (e.g., Ser-Gly linker). Linker length can greatly affect how the variable region of the scFv folds and interacts. In fact, if short polypeptide linkers (e.g. 5-10 amino acids) are used, intrachain folding is prevented. Additionally, interchain folding is required to bring the two variable regions into proximity to form a functional epitope binding site. For examples of linker orientation and size, see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCT Publication Nos. WO2006/020258 and WO2007/024715, the entire contents of each of which are incorporated herein by reference. Included.
본 발명에서 상기 메소텔린(MSLN) 결합 도메인은 메소텔린을 특이적으로 인식할 수 있는 scFv를 포함할 수 있다. In the present invention, the mesothelin (MSLN) binding domain may include an scFv capable of specifically recognizing mesothelin.
본 발명에서 상기 메소텔린(MSLN) 결합 도메인은 서열번호 2로 표시되는 아미노산 서열로 이루어지는 경쇄 CDR1; 서열번호 3으로 표시되는 아미노산 서열로 이루어지는 경쇄 CDR2; 및 서열번호 4로 표시되는 아미노산 서열로 이루어지는 경쇄 CDR3를 포함하는 경쇄 가변 영역을 포함할 수 있다. In the present invention, the mesothelin (MSLN) binding domain includes a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 2; Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 3; and a light chain variable region including a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 4.
본 발명에서 상기 메소텔린(MSLN) 결합 도메인은 서열번호 5로 표시되는 아미노산 서열로 이루어지는 중쇄 CDR1; 서열번호 6으로 표시되는 아미노산 서열로 이루어지는 중쇄 CDR2; 및 서열번호 7로 표시되는 아미노산 서열로 이루어지는 중쇄 CDR3를 포함하는 중쇄 가변 영역을 포함할 수 있다. In the present invention, the mesothelin (MSLN) binding domain includes a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 5; Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 6; and a heavy chain variable region including a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 7.
본 발명에서 상기 "가변 영역", "가변 도메인", "V 영역" 또는 "V 도메인"은 일반적으로 경쇄 또는 중쇄의 아미노-말단에 위치하며, 중쇄에서 약 120 내지 130개 아미노산 및 경쇄에서 약 100 내지 110개 아미노산의 길이를 가지는, 항체의 경쇄 또는 중쇄의 부분을 지칭하며, 특정 항원에 대한 각각의 특정 항체의 결합 및 특이성에서 사용된다. 중쇄의 가변 영역은 "VH"로 지칭될 수 있다. 경쇄의 가변 영역은 "VL"로 지칭될 수 있다. 용어 "가변"은 가변 영역의 특정 세그먼트가 항체 중에서 서열이 광범위하게 달라진다는 사실을 나타낸다. V 영역은 항원 결합을 매개하고 특정 항원에 대한 특정 항체의 특이성을 정의한다. 그러나 가변성은 가변 영역의 110개 아미노산 범위에 걸쳐 고르게 분포되지 않는다. 대신, V 영역은 각각 약 9 내지 12개 아미노산 길이인 "초가변 영역"으로 지칭되는 더 큰 가변성(예를 들어, 극도의 가변성)의 더 짧은 영역에 의해 분리되는 약 15 내지 30개 아미노산의 프레임워크 영역(FR)으로 지칭되는 덜 가변성인(예컨대 상대적으로 불변성인) 스트레치로 이루어진다. 중쇄 및 경쇄의 가변 영역은 각각 β 시트 구조를 연결하는 루프를 형성하는, 일부 경우에 이의 일부를 형성하는 3개의 초가변 영역에 의해 연결된, 주로 β 시트 입체배치(configuration)를 채택하는 4개의 FR을 포함한다. 각각의 쇄 내의 초가변 영역은 FR에 의해 밀접하게 유지되고, 다른 사슬로부터의 초가변 영역은, 항체의 항원-결합 부위 형성에 기여한다(예를 들어, 문헌[Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)] 참조). 불변 영역은 항체가 항원에 결합하는 데 직접적으로 관여하지 않지만, 항체 의존성 세포성 세포독성(ADCC) 및 보체 의존성 세포독성(CDC)에서의 항체의 참여와 같은 다양한 이펙터 기능을 나타낸다. 가변 영역은 상이한 항체 사이에서 서열이 광범위하게 달라진다. 구체적인 실시형태에서, 가변 영역은 인간 가변 영역이다.In the present invention, the "variable region", "variable domain", "V region" or "V domain" is generally located at the amino-terminus of the light or heavy chain, about 120 to 130 amino acids in the heavy chain and about 100 amino acids in the light chain. Refers to the portion of the light or heavy chain of an antibody, ranging in length from 1 to 110 amino acids, and is used in the binding and specificity of each specific antibody for a specific antigen. The variable region of the heavy chain may be referred to as “VH”. The variable region of the light chain may be referred to as “VL”. The term “variable” refers to the fact that certain segments of the variable region vary widely in sequence among antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody for a particular antigen. However, the variability is not evenly distributed across the 110 amino acids of the variable region. Instead, the V region is a frame of about 15 to 30 amino acids separated by shorter regions of greater variability (e.g., extreme variability), referred to as “hypervariable regions,” which are each about 9 to 12 amino acids long. It consists of a less variable (i.e. relatively invariant) stretch called the work region (FR). The variable regions of the heavy and light chains are composed of four FRs that predominantly adopt the β-sheet configuration, each connected by three hypervariable regions that form loops connecting, and in some cases form part of, the β-sheet structure. Includes. The hypervariable regions within each chain are closely held by FRs, and hypervariable regions from other chains contribute to the formation of the antigen-binding site of the antibody (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)]. The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions, such as the antibody's participation in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Variable regions vary widely in sequence between different antibodies. In a specific embodiment, the variable region is a human variable region.
본 발명에서 상기 "중쇄"는 약 50 내지 70 kDa의 폴리펩티드 쇄를 지칭하며, 여기서 아미노-말단 부분은 약 120 내지 130개 이상의 아미노산의 가변 영역을 포함하고, 카복시-말단 부분은 불변 영역을 포함한다. 불변 영역은 중쇄 불변 영역의 아미노산 서열에 기초하여, 알파(α), 델타(δ), 엡실론(ε), 감마(γ) 및 뮤(μ)로 지칭되는 5가지 별개의 유형(예를 들어, 아이소타입) 중 하나일 수 있다. 별개의 중쇄는 크기가 상이하다: α, δ 및 γ는 대략 450개 아미노산을 함유하는 반면, μ 및 ε은 대략 550개의 아미노산을 함유한다. 경쇄와 조합되는 경우, 이들 별개의 유형의 중쇄는 5개의 널리 공지되어 있는 부류(예를 들어, 아이소타입)의 항체, 각각 IgA, IgD, IgE, IgG 및 IgM를 생성하며, 이는 4개의 IgG의 하위부류, 즉 IgG1, IgG2, IgG3 및 IgG4를 포함한다.As used herein, the "heavy chain" refers to a polypeptide chain of about 50 to 70 kDa, wherein the amino-terminal portion comprises a variable region of at least about 120 to 130 amino acids, and the carboxy-terminal portion comprises a constant region. . Constant regions are of five distinct types, referred to as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant region, isotype). The distinct heavy chains differ in size: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with light chains, these distinct types of heavy chains produce five well-known classes (e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG, and IgM, respectively, which make up the four IgG classes. Subclasses include IgG1, IgG2, IgG3 and IgG4.
본 발명에서 상기 "경쇄"는 약 25 kDa의 폴리펩티드 사슬을 지칭하며, 여기서 아미노-말단 부분은 약 100 내지 약 110개 이상의 아미노산의 가변 영역을 포함하고, 카복시-말단 부분은 불변 영역을 포함한다. 경쇄의 대략적인 길이는 211 내지 217개 아미노산이다. 불변 도메인의 아미노산 서열에 기초하여, 카파(κ) 또는 람다(λ)로 지칭되는 2가지 별개의 유형이 존재한다.As used herein, the “light chain” refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids, and the carboxy-terminal portion includes a constant region. The approximate length of the light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two distinct types, referred to as kappa (κ) or lambda (λ).
본 발명에서 상기 "CDR"은 "초가변 영역", "HVR" 및 "상보성 결정 영역"과 상호교환 가능하게 사용된다. "CDR"은 면역글로불린(Ig 또는 항체) VH β 시트 프레임워크의 비-프레임워크 영역 내의 3개의 초가변 영역(H1, H2 또는 H3) 중 하나 또는 항체 VL β 시트 프레임워크의 비-프레임워크 영역 내의 3개의 초가변 영역(L1, L2 또는 L3) 중 하나를 지칭힌다. VH 도메인 내의 CDR1, CDR2 및 CDR3은 또한, 각각 HCDR1, HCDR2 및 HCDR3으로도 지칭된다. VL 도메인 내의 CDR1, CDR2 및 CDR3은 또한, 각각 LCDR1, LCDR2 및 LCDR3으로도 지칭된다. 따라서, CDR은 프레임워크 영역 서열 내에 산재된 가변 영역 서열이다.In the present invention, “CDR” is used interchangeably with “hypervariable region,” “HVR,” and “complementarity determining region.” “CDR” refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of an immunoglobulin (Ig or antibody) VH β-sheet framework or a non-framework region of the antibody VL β-sheet framework Refers to one of three hypervariable regions (L1, L2, or L3) within the hypervariable region. CDR1, CDR2 and CDR3 within the VH domain are also referred to as HCDR1, HCDR2 and HCDR3, respectively. CDR1, CDR2 and CDR3 within the VL domain are also referred to as LCDR1, LCDR2 and LCDR3, respectively. Accordingly, CDRs are variable region sequences interspersed within framework region sequences.
CDR 영역은 당업자에게 잘 알려져 있고 잘 알려진 넘버링 시스템에 의해 정의되었다. 예를 들어, 카바트 상보성 결정 영역(CDR)은 서열 가변성에 기초하며, 가장 흔히 사용된다(예를 들어, 상기 문헌[Kabat et al.]; 문헌[Nick Deschacht et al., J Immunol 2010; 184:5696-5704] 참조). 초티아는 대신 구조적 루프의 위치를 나타낸다(예를 들어, 문헌[Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)] 참조). 카바트 넘버링 규칙을 사용하여 넘버링될 때, 초티아 CDR-H1 루프의 끝은 루프의 길이에 따라 H32와 H34 사이에서 달라진다(이는 카바트 넘버링 체계가 H35A와 H35B에서 삽입을 배치하기 때문이며; 35A 또는 35B 중 어느 것도 존재하지 않는 경우, 루프는 32에서 끝나며; 35A만 존재하는 경우, 루프는 33에서 끝나고; 35A와 35B가 모두 존재하는 경우, 루프는 34에서 끝난다). AbM 초가변 영역은 카바트 CDR과 초티아 구조적 루프 간의 절충안을 나타내며, Oxford Molecular의 AbM 항체 모델링 소프트웨어에 의해 사용된다. "컨택트" 초가변 영역은 이용 가능한 복잡한 결정 구조의 분석에 기초한다. 개발되어 널리 채택된 또 다른 보편적인 넘버링 시스템은 ImMunoGeneTics(IMGT) Information System®이다. IMGT는 인간 및 기타 척추동물의 면역글로불린(IG), T-세포 수용체(TCR) 및 주요 조직적합성 복합체(MHC)에 특화된 통합 정보 시스템이다. 본원에서, CDR은 아미노산 서열 및 경쇄 또는 중쇄 내의 위치 둘 모두에 관하여 언급된다. 면역글로불린 가변 도메인의 구조 내의 CDR의 "위치"가 종 사이에 보존되고, 루프로 지칭되는 구조에 존재하기 때문에, 구조적 특징에 따라 가변 도메인 서열을 정렬하는 넘버링 시스템을 사용하여, CDR 및 프레임워크 잔기는 쉽게 식별된다. 이 정보는 하나의 종의 면역글로불린 유래의 CDR 잔기를 전형적으로 인간 항체 유래의, 수여자 프레임워크 내로 이식하고 대체하는 데 사용될 수 있다. 추가의 넘버링 시스템(AHon)은 문헌[Honegger and Plckthun, J. Mol. Biol. 309: 657-70 (2001)]에 의해 개발되었다. 예를 들어, 카바트 넘버링 및 IMGT 고유 넘버링 시스템을 포함하는 넘버링 시스템 간의 대응은 당업자에게 잘 알려져 있다.CDR regions are well known to those skilled in the art and have been defined by a well-known numbering system. For example, Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (e.g., Kabat et al., supra; Nick Deschacht et al., J Immunol 2010; 184 :5696-5704]). Chothia instead refers to the position of a structural loop (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)). When numbered using the Kabat numbering convention, the end of the Chotia CDR-H1 loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering system places insertions at H35A and H35B; 35A or If neither 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34. The AbM hypervariable region represents a compromise between the Kabat CDRs and Chotia structural loops and is used by Oxford Molecular's AbM antibody modeling software. “Contact” hypervariable regions are based on analysis of available complex crystal structures. Another common numbering system that has been developed and widely adopted is the ImMunoGeneTics (IMGT) Information System®. IMGT is an integrated information system specialized for immunoglobulins (IGs), T-cell receptors (TCRs), and major histocompatibility complexes (MHC) in humans and other vertebrates. Herein, CDRs are referred to both in terms of amino acid sequence and position within the light or heavy chain. Because the "position" of the CDRs within the structure of immunoglobulin variable domains is conserved between species and exist in structures referred to as loops, a numbering system is used to align variable domain sequences according to structural features, CDRs and framework residues. is easily identified. This information can be used to graft and replace CDR residues from one species of immunoglobulin into a recipient framework, typically from a human antibody. An additional numbering system (AHon) is described in Honegger and Plckthun, J. Mol. Biol. 309: 657-70 (2001)]. The correspondence between numbering systems, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to those skilled in the art.
주어진 CDR의 경계는 식별에 사용되는 체계에 따라 달라질 수 있다. 따라서, 달리 명시되지 않는 한, 주어진 항체 또는 이의 영역, 예컨대 가변 영역의 용어 "CDR" 및 "상보성 결정 영역", 뿐만 아니라 항체 또는 이의 영역의 개별 CDR(예를 들어, CDR-H1, CDR-H2)은 상기 본원에 기재된 임의의 공지된 체계에 의해 정의된 상보성 결정 영역을 포함하는 것으로 이해되어야 한다. 일부 경우에, IMGT, 카바트, 초티아 또는 컨택트 방법에 의해 정의된 CDR과 같은 특정 CDR 또는 CDR들의 식별을 위한 체계가 특정된다. 다른 경우에는 CDR의 특정 아미노산 서열이 제공된다. CDR 영역이 또한 다양한 넘버링 시스템의 조합, 예를 들어, 카바트 및 초티아 넘버링 시스템의 조합 또는 카바트 및 IMGT 넘버링 시스템의 조합에 의해 정의될 수 있는 것을 주의해야 한다. 따라서, "특정 VH에 제시된 바와 같은 CDR1"과 같은 용어는 상기 기재된 예시적인 CDR 넘버링 시스템에 의해 정의된 바와 같은 임의의 CDR1을 포함하지만, 이에 의해 제한되지 않는다. 일단 가변 영역(예를 들어, VH 또는 VL)이 주어지면, 당업자는 영역 내의 CDR이 상이한 넘버링 시스템 또는 이의 조합에 의해 정의될 수 있는 것을 이해할 것이다.The boundaries of a given CDR may vary depending on the scheme used for identification. Accordingly, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof, such as a variable region, as well as individual CDRs of an antibody or region thereof (e.g., CDR-H1, CDR-H2 ) should be understood to include complementarity determining regions defined by any known system described herein above. In some cases, a scheme is specified for the identification of a specific CDR or CDRs, such as CDRs defined by the IMGT, Kabat, Chotia or Contact methods. In other cases, the specific amino acid sequence of the CDR is provided. It should be noted that the CDR region can also be defined by a combination of various numbering systems, for example a combination of the Kabat and Chotia numbering systems or a combination of the Kabat and IMGT numbering systems. Accordingly, terms such as “CDR1 as presented in a particular VH” include, but are not limited to, any CDR1 as defined by the exemplary CDR numbering system described above. Once a variable region (e.g., VH or VL) is given, one skilled in the art will understand that the CDRs within the region may be defined by different numbering systems or combinations thereof.
본 발명에서 상기 "불변 영역" 또는 "불변 도메인"은 항원에 대한 항체의 결합에 직접 관여하지 않지만, Fc 수용체와의 상호작용과 같은 다양한 이펙터 기능을 나타내는, 경쇄 및 중쇄의 카복시 말단 부분을 지칭한다. 당해 용어는 항원 결합 부위를 함유하는 가변 영역인 면역글로불린의 다른 부분에 비해 더 보존된 아미노산 서열을 갖는 면역글로불린 분자의 부분을 지칭한다. 불변 영역은 중쇄의 CH1, CH2 및 CH3 영역과 경쇄의 CL 영역을 함유할 수 있다.In the present invention, the "constant region" or "constant domain" refers to the carboxy-terminal portions of the light and heavy chains that are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions, such as interaction with Fc receptors. . The term refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to other portions of the immunoglobulin, the variable region containing the antigen binding site. The constant region may contain the CH1, CH2 and CH3 regions of the heavy chain and the CL region of the light chain.
본 발명에서 상기 "프레임워크" 또는 "FR"은 CDR에 측접하는 가변 영역 잔기를 지칭한다. FR 잔기는, 예를 들어 키메라, 인간화, 인간, 도메인 항체, 디아바디, 선형 항체, 및 이중특이적 항체에 존재한다. FR 잔기는 초가변 영역 잔기 또는 CDR 잔기 이외의 가변 도메인 잔기이다.In the present invention, the “framework” or “FR” refers to the variable region residues flanking the CDR. FR residues are present in, for example, chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are variable domain residues other than hypervariable region residues or CDR residues.
본 발명에서 상기 메소텔린(MSLN) 결합 도메인은 서열번호 8로 표시되는 항체의 경쇄 가변 영역(VL)을 포함할 수 있고, 혹은 서열번호 9로 표시되는 염기 서열에 의해 암호화되는 항체의 경쇄 가변 영역(VL)을 포함할 수 있다. In the present invention, the mesothelin (MSLN) binding domain may include a light chain variable region (VL) of an antibody represented by SEQ ID NO: 8, or a light chain variable region of an antibody encoded by the nucleotide sequence represented by SEQ ID NO: 9. (VL) may be included.
본 발명에서 상기 메소텔린(MSLN) 결합 도메인은 서열번호 10으로 표시되는 항체의 중쇄 가변 영역(VH)을 포함할 수 있고, 혹은 서열번호 11로 표시되는 염기 서열에 의해 암호화되는 항체의 중쇄 가변 영역(VH)을 포함할 수 있다. In the present invention, the mesothelin (MSLN) binding domain may include the heavy chain variable region (VH) of the antibody represented by SEQ ID NO: 10, or the heavy chain variable region of the antibody encoded by the nucleotide sequence represented by SEQ ID NO: 11 (VH) may be included.
본 발명에서 상기 경쇄 가변 영역(VL)은 상기 서열번호 8로 표시되는 아미노산 서열과 적어도 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99% 동일한 아미노산 서열을 포함할 수 있고, 서열번호 9로 표시되는 염기 서열에 대하여 적어도 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99% 동일한 서열에 의해 암호화되는 것 또한 포함한다.In the present invention, the light chain variable region (VL) includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 8. It can be done, and also includes those encoded by a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence shown in SEQ ID NO: 9.
본 발명에서 상기 중쇄 가변 영역(VH)은 상기 서열번호 10으로 표시되는 아미노산 서열과 적어도 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99% 동일한 아미노산 서열을 포함할 수 있고, 서열번호 11로 표시되는 염기 서열에 대하여 적어도 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99% 동일한 서열에 의해 암호화되는 것 또한 포함한다.In the present invention, the heavy chain variable region (VH) includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 10. It can be done, and also includes those encoded by sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the base sequence shown in SEQ ID NO: 11.
본 발명에서 상기 scFv는 자체 VL 영역과 VH 영역 사이에 아미노산 잔기 적어도 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50개 또는 그 보다 많은 개수로 된 링커를 포함할 수 있다. 링커 서열은 임의의 자연 생성 아미노산을 포함할 수 있다. In the present invention, the scFv has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 amino acid residues between its VL region and VH region. , 18, 19, 20, 25, 30, 35, 40, 45, 50 or more linkers. The linker sequence may include any naturally occurring amino acid.
본 발명의 일 예시로, 상기 링커 서열은 아미노산 글리신과 세린을 포함할 수 있다. As an example of the present invention, the linker sequence may include the amino acids glycine and serine.
본 발명의 다른 예시로, 상기 링커 서열은 글리신과 세린 반복체 세트, 예를 들어 (Gly4Ser)n을 포함하며, 여기서 n은 1 이상의 양수일 수 있고, 바람직하게는 3 또는 4일 수 있다. In another example of the invention, the linker sequence comprises a set of glycine and serine repeats, such as (Gly 4 Ser)n, where n may be a positive number of 1 or more, preferably 3 or 4.
본 발명의 또 다른 예시로, 상기 링커는 서열번호 12로 표시되는 아미노산 서열을 포함할 수 있고, 혹은 서열번호 13으로 표시되는 염기 서열에 의해 암호화되는 링커일 수 있다. As another example of the present invention, the linker may include the amino acid sequence represented by SEQ ID NO: 12, or may be a linker encoded by the nucleotide sequence represented by SEQ ID NO: 13.
본 발명의 바람직한 일 예시로, 상기 메소텔린(MSLN) 결합 도메인은 메소텔린을 특이적으로 인식할 수 있는 scFv를 포함할 수 있고, 상기 scFv는 서열번호 2, 3 및 4 각각의 아미노산 서열로 표시되는 경쇄 CDR1, CDR2, CDR3를 포함하는 경쇄 가변 영역(VL); 서열번호 12로 표시되는 아미노산 서열을 포함하거나, 혹은 서열번호 13으로 표시되는 염기 서열에 의해 암호화되는 링커; 및 서열번호 5, 6 및 7 각각의 아미노산 서열로 표시되는 중쇄 CDR1, CDR2, CDR3를 포함하는 중쇄 가변 영역(VH);을 포함할 수 있으나, 이에 제한되는 것은 아니다.In a preferred example of the present invention, the mesothelin (MSLN) binding domain may include an scFv capable of specifically recognizing mesothelin, and the scFv is represented by the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively. a light chain variable region (VL) comprising light chain CDR1, CDR2, and CDR3; A linker comprising the amino acid sequence shown in SEQ ID NO: 12 or encoded by the nucleotide sequence shown in SEQ ID NO: 13; and a heavy chain variable region (VH) comprising heavy chain CDR1, CDR2, and CDR3 represented by the amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively, but is not limited thereto.
본 발명의 바람직한 다른 예시로, 상기 메소텔린(MSLN) 결합 도메인은 메소텔린을 특이적으로 인식할 수 있는 scFv를 포함할 수 있고, 상기 scFv는 서열번호 8로 표시되는 아미노산 서열을 포함하거나, 혹은 서열번호 9로 표시되는 염기 서열에 의해 암호화되는 항체의 가변 경쇄 영역(VL); 서열번호 12로 표시되는 아미노산 서열을 포함하거나, 혹은 서열번호 13으로 표시되는 염기 서열에 의해 암호화되는 링커; 및 서열번호 10으로 표시되는 아미노산 서열을 포함하거나, 혹은 서열번호 11로 표시되는 염기 서열에 의해 암호화되는 항체의 가변 중쇄 영역(VH);을 포함할 수 있으나, 이에 제한되는 것은 아니다.As another preferred example of the present invention, the mesothelin (MSLN) binding domain may include an scFv capable of specifically recognizing mesothelin, and the scFv may include the amino acid sequence shown in SEQ ID NO: 8, or Variable light chain region (VL) of the antibody encoded by the nucleotide sequence shown in SEQ ID NO: 9; A linker comprising the amino acid sequence shown in SEQ ID NO: 12 or encoded by the nucleotide sequence shown in SEQ ID NO: 13; and a variable heavy chain region (VH) of an antibody comprising the amino acid sequence represented by SEQ ID NO: 10 or encoded by the nucleotide sequence represented by SEQ ID NO: 11; but is not limited thereto.
신호 펩타이드signal peptide
본 발명의 키메라 항원 수용체는 번역된 키메라 단백질을 막으로 향하게 하기 위해 신호 펩타이드가 추가된 형태로 설계될 수 있다. The chimeric antigen receptor of the present invention can be designed with a signal peptide added to direct the translated chimeric protein to the membrane.
본 발명에서 상기 신호 펩타이드는 키메라 항원 수용체의 아미노-말단 (N-ter)에 포함될 수 있다. 다만, 이러한 신호 펩타이드는 키메라 항원 수용체가 세포에서 가공되어 세포 막으로 위치되는 동안에 메소텔린 결합 도메인 (예, scFv)로부터 선택적으로 절단될 수 있다. In the present invention, the signal peptide may be included in the amino-terminus (N-ter) of the chimeric antigen receptor. However, this signal peptide can be selectively cleaved from the mesothelin binding domain (eg, scFv) while the chimeric antigen receptor is processed in the cell and localized to the cell membrane.
본 발명에서 상기 신호 펩타이드는 일반적으로 아미노산 15 내지 30개 범위이다. In the present invention, the signal peptide generally ranges from 15 to 30 amino acids.
본 발명에서 상기 신호 펩타이드의 비제한적 예시로는 CD8 신호 펩타이드 (아미노산 21개), CD33 신호 펩타이드 (아미노산 17개), CD4 신호 펩타이드 (아미노산 25개), IL-2R (CD25) 신호 펩타이드 (아미노산 21개), 트립시노겐-2 신호 펩타이드 (아미노산 15개), VEGFR1 신호 펩타이드 (아미노산 26개), EGFR 신호 펩타이드 (아미노산 24개), GMCSFR 신호 펩타이드 (아미노산 22개), IgVL 신호 펩타이드, IgVK 신호 펩타이드 또는 Ig VH 신호 펩타이드 등이 있을 수 있다. Non-limiting examples of the signal peptide in the present invention include CD8 signal peptide (amino acids 21), CD33 signal peptide (amino acids 17), CD4 signal peptide (amino acids 25), and IL-2R (CD25) signal peptide (amino acids 21). dog), trypsinogen-2 signal peptide (15 amino acids), VEGFR1 signal peptide (26 amino acids), EGFR signal peptide (24 amino acids), GMCSFR signal peptide (22 amino acids), IgVL signal peptide, IgVK signal peptide Or there may be Ig VH signal peptide, etc.
본 발명의 일 예시로, 상기 신호 펩타이드는 CD8 신호 펩타이드일 수 있고, 바람직하게는 서열번호 14로 표시되는 아미노산 서열을 포함할 수 있으며, 혹은 서열번호 15로 표시되는 염기 서열에 의해 암호화되는 신호 펩타이드일 수 있다.As an example of the present invention, the signal peptide may be a CD8 signal peptide, and preferably may include an amino acid sequence represented by SEQ ID NO: 14, or a signal peptide encoded by the nucleotide sequence represented by SEQ ID NO: 15. It can be.
본 발명에서 상기 신호 펩타이드는 상기 서열번호 14로 표시되는 아미노산 서열에 대하여 적어도 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99% 동일한 아미노산 서열을 포함할 수 있고, 서열번호 15로 표시되는 염기 서열에 대하여 적어도 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99% 동일한 서열에 의해 암호화되는 것 또한 포함한다. In the present invention, the signal peptide may include an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence represented by SEQ ID NO: 14, , also includes those encoded by sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the base sequence shown in SEQ ID NO: 15.
힌지(hinge)hinge
본 발명의 키메라 항원 수용체는 세포외 스페이서, 즉 힌지(hinge)를 더 포함할 수 있다. The chimeric antigen receptor of the present invention may further include an extracellular spacer, that is, a hinge.
본 발명에서 상기 "힌지(hinge)"는 측면 폴리펩타이드 영역에 구조적 가요성 및 간격을 제공하는 가요성 폴리펩타이드 커넥터 영역(본원에서 "힌지 영역"으로도 지칭됨)을 지칭하고 천연 또는 합성 폴리펩타이드로 구성될 수 있다. 본 발명의 키메라 항원 수용체는 힌지를 통하여, 상기 메소텔린 결합 도메인을 이하 기재될 막관통 도메인에 연결시킬 수 있다. 본 발명에서 상기 힌지는 항원 결합 도메인이 항원 결합을 용이하게 하기 위해 상이한 방향으로 배향되도록 하기에 충분히 유연하다.In the present invention, the "hinge" refers to a flexible polypeptide connector region (also referred to herein as a "hinge region") that provides structural flexibility and spacing to the flanking polypeptide regions and is used in natural or synthetic polypeptides. It can be composed of: The chimeric antigen receptor of the present invention can connect the mesothelin binding domain to the transmembrane domain described below through a hinge. In the present invention, the hinge is sufficiently flexible to allow the antigen binding domain to be oriented in different directions to facilitate antigen binding.
본 발명에서 상기 힌지는 IgG 유래의 힌지 영역일 수 있고, 바람직하게는 인간 IgG1, IgG2, IgG3, 또는 IgG4 힌지 영역의 아미노산 서열을 포함할 수 있다. 또한, 상기 힌지는 야생형(자연 발생) 힌지 영역과 비교하여 하나 이상의 아미노산 치환 및/또는 삽입 및/또는 결실을 포함할 수 있다. 예를 들어, 인간 IgG1 힌지의 His229는 Tyr로 치환된 서열을 포함할 수 있다.In the present invention, the hinge may be a hinge region derived from IgG, and preferably may include the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 hinge region. Additionally, the hinge may contain one or more amino acid substitutions and/or insertions and/or deletions compared to the wild-type (naturally occurring) hinge region. For example, His229 of the human IgG1 hinge may contain a sequence substituted with Tyr.
또한, 본 발명에서 상기 힌지는 본 기술분야에서 통상적으로 사용하는 CD8, CD28, 4-1BB, OX40, CD3 제타(ζ) 사슬의 전부 또는 일부, T 세포 수용체 α 또는 β 사슬, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, 이들의 기능적 유도체 또는 이들의 조합을 포함한 인간 단백질의 힌지 영역을 포함할 수 있으나, 이에 제한되는 것은 아니다. In addition, in the present invention, the hinge includes all or part of the CD8, CD28, 4-1BB, OX40, CD3 zeta (ζ) chain, T cell receptor α or β chain, CD28, CD3ε, CD45 commonly used in the art. , CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, their functional derivatives, or combinations thereof. However, it is not limited to this.
본 발명의 일 예시로, 상기 힌지는 CD8 유래 힌지 영역일 수 있고, 바람직하게는 서열번호 16으로 표시되는 아미노산 서열을 포함할 수 있으며, 혹은 서열번호 17로 표시되는 염기 서열에 의해 암호화되는 힌지일 수 있다.As an example of the present invention, the hinge may be a CD8-derived hinge region, and preferably may include an amino acid sequence represented by SEQ ID NO: 16, or a hinge encoded by the nucleotide sequence represented by SEQ ID NO: 17. You can.
본 발명에서 상기 힌지는 상기 서열번호 16으로 표시되는 아미노산 서열과 적어도 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99% 동일한 아미노산 서열을 포함할 수 있고, 서열번호 17로 표시되는 염기 서열에 대하여 적어도 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99% 동일한 서열에 의해 암호화되는 것 또한 포함한다.In the present invention, the hinge may include an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence represented by SEQ ID NO: 16, and the sequence It also includes those encoded by sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the base sequence indicated by number 17.
막관통 도메인transmembrane domain
본 발명의 키메라 항원 수용체는 막관통 도메인을 더 포함할 수 있다. The chimeric antigen receptor of the present invention may further include a transmembrane domain.
본 발명에서 상기 막관통 도메인은 막관통 영역에 인접한 하나 이상의 부가적인 아미노산, 예를 들어 막관통 도메인이 유래된 단백질의 세포외 영역과 관련한 하나 이상의 아미노산 (예를 들어, 세포외 영역의 아미노산 1, 2, 3, 4, 5, 6, 7, 8, 9, 10개에서 최대 15개) 및/또는 막관통 단백질이 유래된 단백질의 세포내 영역과 관련한 하나 이상의 부가적인 아미노산 (예를 들어, 세포내 영역의 아미노산 1, 2, 3, 4, 5, 6, 7, 8, 9, 10개에서 최대 15개)을 함유할 수 있다. In the present invention, the transmembrane domain includes one or more additional amino acids adjacent to the transmembrane region, for example, one or more amino acids associated with the extracellular region of the protein from which the transmembrane domain is derived (e.g., amino acid 1 of the extracellular region, 2, 3, 4, 5, 6, 7, 8, 9, 10 and up to 15 amino acids) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g. It may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino acids in the region.
본 발명에서 상기 막관통 도메인은 동일한 또는 다른 표면 막 단백질의 막관통 도메인에 이 도메인이 결합하는 것을 방지하도록, 예를 들어 수용체 복합체의 다른 구성원과의 상호작용을 최소화하도록 선택되거나 또는 이를 위해 아미노산 치환에 의해 변형될 수 있다. In the present invention, the transmembrane domain is selected or has amino acid substitutions to prevent binding of this domain to the transmembrane domain of the same or another surface membrane protein, for example to minimize interaction with other members of the receptor complex. It can be transformed by .
본 발명에서 상기 막관통 도메인은 천연 유래 또는 재조합 유래일 수 있다. 천연 유래인 경우, 상기 도메인은 임의의 막-결합된 또는 막관통 단백질로부터 유래할 수 있다. 이때 상기 막관통 도메인은 키메라 항원 수용체가 표적 항원에 결합할 때마다 세포내 도메인에 신호를 전달할 수 있다. In the present invention, the transmembrane domain may be of natural origin or recombinant origin. When of natural origin, the domain may be derived from any membrane-bound or transmembrane protein. At this time, the transmembrane domain can transmit a signal to the intracellular domain whenever the chimeric antigen receptor binds to the target antigen.
본 발명에서 상기 막관통 도메인은 예를 들어 T 세포 수용체, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (예를 들어, CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154의 알파, 베타 또는 제타 쇄의 막관통 영역을 포함할 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the transmembrane domain includes, for example, T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64. , CD80, CD86, CD134, CD137, and may include the transmembrane region of the alpha, beta or zeta chain of CD154, but are not limited thereto.
또한, 본 발명에서 상기 막관통 도메인은 공동-자극성 신호전달 도메인, 예를 들어 MHC 클래스 I 분자, TNF 수용체 단백질, 면역글로불린-유사 단백질, 사이토카인 수용체, 인테그린, 신호전달성 림프구 활성화 분자 (SLAM 단백질), 활성화 NK 세포 수용체, BTLA, Toll 리간드 수용체, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a 및 CD83에 특이적으로 결합하는 리간드의 막관통 영역(들)을 함유할 수 있다. 막관통 도메인은 당해 기술 분야에 공지되거나 또는 본 발명에 언급된 임의의 방법을 이용해, 예를 들어 UniProt 데이터베이스를 이용함으로써 동정할 수 있다.In addition, in the present invention, the transmembrane domain is a co-stimulatory signaling domain, such as MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein) ), activated NK cell receptor, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7 -H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta , IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c , ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 ( CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT , GADS, SLP-76, PAG/Cbp, CD19a and CD83. Transmembrane domains can be identified using any method known in the art or mentioned herein, for example using the UniProt database.
또한, 본 발명에서 상기 막관통 도메인이 합성 도메인인 경우, 루신 및 발린과 같은 소수성 잔기를 포함할 수 있고, 혹은 페닐알라닌, 트립토판 및 발린으로 구성된 트리플렛이 합성 막관통 도메인의 양쪽 말단에서 발견될 수 있다. Additionally, in the present invention, when the transmembrane domain is a synthetic domain, it may include hydrophobic residues such as leucine and valine, or triplets consisting of phenylalanine, tryptophan and valine may be found at both ends of the synthetic transmembrane domain. .
본 발명에서 상기 막관통 도메인은 CD8 막관통 도메인일 수 있고, 바람직하게는 서열번호 18로 표시되는 아미노산 서열을 포함하거나, 서열번호 19로 표시되는 염기 서열에 의해 암호화되는 것일 수 있다. In the present invention, the transmembrane domain may be a CD8 transmembrane domain, and preferably includes the amino acid sequence represented by SEQ ID NO: 18, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 19.
본 발명에서 상기 막관통 도메인은 상기 서열번호 18로 표시되는 아미노산 서열에 대하여 적어도 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99% 동일한 아미노산 서열을 포함할 수 있고, 혹은 서열번호 19로 표시되는 염기 서열에 대하여 적어도 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99% 동일한 염기 서열에 의해 암호화되는 것 또한 포함한다. In the present invention, the transmembrane domain may include an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence represented by SEQ ID NO: 18. It also includes those encoded by a base sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the base sequence shown in SEQ ID NO: 19.
신호전달 도메인signaling domain
본 발명의 키메라 항원 수용체는 세포질 도메인으로, 세포내 신호전달 도메인을 더 포함할 수 있다. The chimeric antigen receptor of the present invention has a cytoplasmic domain and may further include an intracellular signaling domain.
본 발명에서 상기 "세포내 신호전달 도메인"은 일반적으로 키메라 항원 수용체가 도입된 세포의 정상적인 작동자 기능의 활성화를 유도한다. 여기서, 상기 "작동자 기능"이란 세포의 특화된 기능을 지칭하는 것으로, 예컨대 T 세포의 작동자 기능은 예를 들어 사이토카인의 분비를 비롯한 세포분해 활성 또는 헬퍼 활성일 수 있다. 따라서, 본 발명에서 상기 "세포 내 신호전달 도메인"은 작동자 기능 신호를 변환하여 세포가 특화된 기능을 수행하게 지시하는 단백질의 일부분을 지칭한다. 통상적으로 전체 세포내 신호전달 도메인이 사용될 수 있지만, 다수의 경우에 전체 도메인을 반드시 사용하여야 하는 것은 아니다. 세포내 신호전달 도메인의 잘린 영역이 사용되는 정도로, 이러한 잘린 영역이 작동자 기능 신호를 변환하는 한 온전한 도메인 대신 이용될 수 있다. 용어 세포내 신호전달 도메인은 따라서 작동자 기능 신호를 변환하기에 충분한 세포내 신호전달 도메인의 임의의 잘린 영역을 함유하는 것을 의미한다.In the present invention, the “intracellular signaling domain” generally induces activation of the normal effector function of the cell into which the chimeric antigen receptor has been introduced. Here, the “effector function” refers to a specialized function of a cell. For example, the effector function of a T cell may be, for example, cytolytic activity or helper activity, including secretion of cytokines. Accordingly, in the present invention, the “intracellular signaling domain” refers to a portion of a protein that converts an effector function signal and instructs the cell to perform a specialized function. Typically the entire intracellular signaling domain can be used, but in many cases the entire domain is not required to be used. To the extent that truncated regions of the intracellular signaling domain are used, these truncated regions can be used in place of the intact domain as long as they transduce an effector function signal. The term intracellular signaling domain is thus meant to contain any truncated region of the intracellular signaling domain sufficient to transduce an effector function signal.
본 발명에서 상기 세포내 신호전달 도메인에 대한 예로는 T 세포 수용체 (TCR) 및 항원 수용체에 결합한 후 신호 전달을 개시하기 위해 협력하여 작용하는 공동-수용체의 세포질 서열뿐만 아니라 이들 서열에 대한 임의의 유도체 또는 변이체, 및 동일한 기능적 역량을 가진 임의의 재조합 서열 등이 있을 수 있다. TCR 단편을 통해 생성된 신호는 T 세포를 완전히 활성화하기에는 불충분하고, 2차적인 및/또는 공동 자극 신호가 또한 필요한 것으로 알려져 있다. Examples of such intracellular signaling domains in the present invention include cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act cooperatively to initiate signal transduction after binding to the antigen receptor, as well as any derivatives of these sequences. Or there may be variants, and any recombinant sequences with the same functional capacity, etc. It is known that signals generated through TCR fragments are insufficient to fully activate T cells, and secondary and/or costimulatory signals are also required.
본 발명에서 상기 1차 신호전달 도메인은 TCR 복합체의 1차 활성화를 자극 방식으로 또는 저해 방식으로 제어한다. 자극 방식으로 작동하는 1차 세포내 신호절단 도메인은 면역수용체 티로신-기반의 활성화 모티프 또는 ITAM으로 공지된 특정 신호전달 모티프일 수 있다. In the present invention, the primary signaling domain controls the primary activation of the TCR complex in a stimulatory or inhibitory manner. The primary intracellular cleavage domain that acts in a stimulatory manner may be a specific signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.
본 발명에서 특히 사용되는 1차 세포내 신호전달 도메인을 함유한 ITAM의 예로는 TCR zeta, FcR gamma, FcR beta, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 ("ICOS"라고도 함), Fc.epsilon.RI, DAP10, DAP12 및 CD66d의 ITAM 등이 있을 수 있으나, 이에 제한되는 것은 아니다. Examples of ITAMs containing primary intracellular signaling domains specifically used in the present invention include TCR zeta, FcR gamma, FcR beta, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278. (also known as “ICOS”), Fc.epsilon.RI, DAP10, DAP12, and ITAM of CD66d, but are not limited thereto.
또한, 본 발명에서 상기 1차 신호전달 도메인으로는 변형된 ITAM 도메인, 예를 들어, 본래의 ITAM 도메인과 비교해 활성이 변이된 (예, 증가 또는 감소된) 돌연변이된 ITAM 도메인을 포함할 수도 있다. Additionally, in the present invention, the primary signaling domain may include a modified ITAM domain, for example, a mutated ITAM domain whose activity is altered (e.g., increased or decreased) compared to the original ITAM domain.
본 발명에서 상기 세포내 신호전달 도메인은 1차 신호전달 도메인, 예를 들어, CD3 zeta 신호전달 도메인 자체를 포함할 수 있지만, 혹은 본 발명에서 이용가능한 임의의 다른 바람직한 세포내 신호전달 도메인과 조합될 수 있다. 예를 들어, CAR의 세포내 신호전달 도메인은 1차 신호전달 도메인, 예를 들어, CD3 zeta 쇄 영역과 함께 하나 이상의 공동-자극성 신호전달 도메인을 포함할 수 있다. In the present invention, the intracellular signaling domain may comprise a primary signaling domain, e.g., the CD3 zeta signaling domain itself, or may be combined with any other preferred intracellular signaling domain available in the present invention. You can. For example, the intracellular signaling domain of a CAR may include one or more co-stimulatory signaling domains along with a primary signaling domain, such as a CD3 zeta chain region.
본 발명에서 상기 "공동-자극성 신호전달 도메인"은 공동-자극성 분자의 세포내 도메인을 포함하는 것으로, 항원에 대한 림프구의 효과적인 반응을 위해 필요한 항원 수용체 또는 이의 리간드 이외의 다른 세포 표면 분자이다. 본 발명에서 이러한 분자에 대한 예로는 MHC 클래스 I 분자, TNF 수용체 단백질, 면역글로불린-유사 단백질, 사이토카인 수용체, 인테그린, 신호전달성 림프구 활성화 분자 (SLAM 단백질), 활성화 NK 세포 수용체, BTLA, Toll 리간드 수용체, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, 및 CD83에 특이적으로 결합하는 리간드 등이 있을 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the “co-stimulatory signaling domain” includes the intracellular domain of a co-stimulatory molecule and is a cell surface molecule other than an antigen receptor or its ligand required for an effective response of lymphocytes to an antigen. Examples of such molecules in the present invention include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligands. Receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS ( CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA- 1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, There may be, but are not limited to, ligands that specifically bind to CD19a and CD83.
본 발명에서 상기 세포내 신호전달 도메인은 1개 이상, 혹은 2개 이상, 예를 들어, 2, 3, 4, 5개 또는 그보다 많은 수의 공동-자극성 신호전달 도메인들을 포함하도록 설계될 수도 있다. 본 발명에서 공동-자극성 신호전달 도메인이 2개 이상, 예를 들어, 2, 3, 4, 5 또는 그 이상의 공동 자극성 신호전달 도메인이 포함되는 경우, 이웃하는 분자들이 직접 연결될 수 있지만 링커 분자에 의해 이격되어 배치될 수도 있다. 이때, 링커 분자는 글리신 잔기이거나 알라닌 잔기일 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the intracellular signaling domain may be designed to include one or more, or two or more, for example, 2, 3, 4, 5 or more co-stimulatory signaling domains. In the present invention, when two or more co-stimulatory signaling domains are included, for example, 2, 3, 4, 5 or more co-stimulatory signaling domains, neighboring molecules may be connected directly, but by linker molecules. They may also be placed spaced apart. At this time, the linker molecule may be a glycine residue or an alanine residue, but is not limited thereto.
본 발명의 일 예시로 상기 세포내 신호전달 도메인은 CD3 zeta 신호전달 도메인일 수 있고, 바람직하게는 서열번호 20으로 표시되는 아미노산 서열을 포함하거나, 서열번호 21로 표시되는 염기 서열에 의해 암호화되는 것일 수 있다. As an example of the present invention, the intracellular signaling domain may be a CD3 zeta signaling domain, and preferably includes the amino acid sequence represented by SEQ ID NO: 20, or is encoded by the nucleotide sequence represented by SEQ ID NO: 21. You can.
본 발명의 다른 예시로, 상기 세포내 신호전달 도메인은 공동-자극성 신호전달 도메인으로 4-1BB를 더 포함할 수 있다. 여기서, 상기 4-1BB는 바람직하게는 서열번호 22로 표시되는 아미노산 서열을 포함하거나, 서열번호 23으로 표시되는 염기 서열에 의해 암호화되는 것일 수 있다. As another example of the present invention, the intracellular signaling domain may further include 4-1BB as a co-stimulatory signaling domain. Here, the 4-1BB preferably includes the amino acid sequence represented by SEQ ID NO: 22, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 23.
본 발명의 또 다른 예시로, 상기 세포내 신호전달 도메인은 1차 신호전달 도메인으로 CD3 zeta 신호전달 도메인을 포함하고, 공동-자극성 도메인으로 4-1BB를 포함할 수 있고, 바람직하게는 4-1BB-CD3 zeta의 순으로 포함할 수 있다. 이때, 상기 CD3 zeta 신호전달 도메인은 서열번호 20으로 표시되는 아미노산 서열을 포함하거나, 서열번호 21로 표시되는 염기 서열에 의해 암호화되는 것일 수 있고, 상기 4-1BB는 서열번호 22로 표시되는 아미노산 서열을 포함하거나, 서열번호 23으로 표시되는 염기 서열에 의해 암호화되는 것일 수 있다.As another example of the present invention, the intracellular signaling domain may include a CD3 zeta signaling domain as a primary signaling domain and 4-1BB as a co-stimulatory domain, preferably 4-1BB. -Can be included in the order of CD3 zeta. At this time, the CD3 zeta signaling domain may include the amino acid sequence represented by SEQ ID NO: 20, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 21, and the 4-1BB is the amino acid sequence represented by SEQ ID NO: 22. It may include or be encoded by the nucleotide sequence shown in SEQ ID NO: 23.
본 발명에서 상기 1차 신호전달 도메인인 상기 CD3 zeta와 관련하여 기재된 서열이나 상기 공동-자극성 신호전달 도메인으로 상기 4-1BB와 관련하여 기재된 서열과 적어도 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99% 동일한 서열에 의해 정의된 것도 모두 포함한다. At least 80%, 85%, 90%, 95% of the sequence described in the present invention in relation to the CD3 zeta as the primary signaling domain or the sequence described in relation to the 4-1BB as the co-stimulatory signaling domain. Also includes those defined by sequences that are 96%, 97%, 98% or 99% identical.
본 발명의 키메라 항원 수용체에서 상기 세포내 신호전달 도메인은 상기 막관통 도메인의 C-말단에 직접적으로 연결될 수 있으나, 선택적으로, 짧은 올리고펩타이드 또는 폴리펩타이드 링커, 예를 들어, 아미노산 2-10개 길이의 링커에 의해 연결될 수 있다. 여기서, 상기 링커의 종류는 제한되지 않으나, 비제한적 예시로는 글리신-세린 더블렛일 수 있다. In the chimeric antigen receptor of the invention, the intracellular signaling domain may be linked directly to the C-terminus of the transmembrane domain, but alternatively may be linked to a short oligopeptide or polypeptide linker, e.g., 2-10 amino acids in length. Can be connected by a linker. Here, the type of the linker is not limited, but a non-limiting example may be a glycine-serine doublet.
본 발명의 바람직한 일 예시에서 상기 키메라 항원 수용체는 서열번호 24로 표시되는 아미노산 서열을 포함하는 것일 수 있고, 혹은 서열번호 26으로 표시되는 염기 서열에 의해 암호화되는 것일 수 있으나, 이에 제한되는 것은 아니다. In a preferred example of the present invention, the chimeric antigen receptor may include the amino acid sequence represented by SEQ ID NO: 24, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 26, but is not limited thereto.
본 발명의 바람직한 다른 예시에서 상기 키메라 항원 수용체는 서열번호 25로 표시되는 아미노산 서열을 포함하는 것일 수 있고, 혹은 서열번호 27로 표시되는 염기 서열에 의해 암호화되는 것일 수 있으나, 이에 제한되는 것은 아니다. In another preferred example of the present invention, the chimeric antigen receptor may include the amino acid sequence represented by SEQ ID NO: 25, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 27, but is not limited thereto.
2) TGF-β 신호전달 경로 억제 펩타이드 또는 그 단편 2) TGF-β signaling pathway inhibitory peptide or fragment thereof
본 발명에서 상기 세포는 전환성장인자 베타(Transforming growth factor-β, TGF-β) 신호전달 경로를 억제할 수 있는 펩타이드 또는 이의 단편을 발현하도록 유전적으로 조작된 세포를 더 포함할 수 있다. In the present invention, the cells may further include cells genetically engineered to express a peptide or fragment thereof capable of inhibiting the transforming growth factor-β (TGF-β) signaling pathway.
본 발명에서는 하나의 세포에 상기한 키메라 항원 수용체 및 상기 전환성장인자 베타 경로를 억제하는 펩타이드 또는 그 단편이 모두 발현되도록 유전적으로 조작된 것을 포함할 뿐만 아니라, 키메라 항원 수용체를 발현하도록 유전적으로 조작된 세포와, 전환성장인자 베타 경로를 억제하는 펩타이드 또는 그 단편을 발현하도록 유전적으로 조작된 세포의 2종을 동시에 포함하는 것 또한 본 발명의 목적에 포함된다. The present invention includes cells genetically engineered to express both the chimeric antigen receptor and the peptide or fragment thereof that inhibit the transforming growth factor beta pathway, as well as cells genetically engineered to express the chimeric antigen receptor. It is also included in the purpose of the present invention to simultaneously include two types of cells and cells genetically engineered to express a peptide or fragment thereof that inhibits the transforming growth factor beta pathway.
본 발명에서 상기 "전환성장인자 베타(Transforming growth factor-β, TGF-β)"란, TGF-β 초가계에 속하는 사이토카인으로서, 포유류에서 발현되는 TGF-β의 형태로는 TGF-β1, TGF-β2 및 TGF-β3의 3가지가 알려져 있다. 상기 TGF-β에 의한 신호 전달은 다양한 생물학적 과정에서 결정적인 역할을 하며 세포 성장 억제, 세포 사멸, 분화 및 상피-간충직 전이 (epithelial-mesenchymal transition; EMT)와 같은 다양한 기능을 수행한다. TGF-β 신호전달계는 엄격하게 조절되며 세포 항상성의 유지와 더불어 발생 및 기관 형성에 결정적인 역할을 한다. 따라서 TGF-β 신호전달의 교란은 암, 섬유증 및 선천성 기형과 같은 생명을 위협하는 질환을 유발할 수 있다. In the present invention, the "transforming growth factor-β (TGF-β)" refers to a cytokine belonging to the TGF-β family. The forms of TGF-β expressed in mammals include TGF-β1 and TGF-β. Three are known: -β2 and TGF-β3. Signal transduction by TGF-β plays a critical role in various biological processes and performs various functions such as cell growth inhibition, apoptosis, differentiation, and epithelial-mesenchymal transition (EMT). The TGF-β signaling system is tightly regulated and plays a critical role in development and organ formation as well as maintaining cellular homeostasis. Therefore, disruption of TGF-β signaling can cause life-threatening diseases such as cancer, fibrosis, and congenital malformations.
본 발명에서 상기 펩타이드는 서열번호 28로 표시되는 아미노산 서열을 포함하는 것, 바람직하게는 서열번호 28로 표시되는 아미노산 서열로 이루어지는 것일 수 있다.In the present invention, the peptide may include the amino acid sequence represented by SEQ ID NO: 28, preferably consisting of the amino acid sequence represented by SEQ ID NO: 28.
본 발명에서 상기 펩타이드를 암호화하는 서열은 서열번호 29로 표시되는 염기 서열을 포함하는 것, 바람직하게는 서열번호 29로 표시되는 염기 서열로 이루어진 것일 수 있다. In the present invention, the sequence encoding the peptide may include the nucleotide sequence represented by SEQ ID NO: 29, preferably consisting of the nucleotide sequence represented by SEQ ID NO: 29.
본 발명에서 상기 펩타이드는 TGF-β 수용체(TGFBR1 및/또는 TGFBR2)에 결합하여 TGF-β 신호전달을 억제하는 것일 수 있으며, 구체적으로 상기 펩타이드는 TGF-β와 경쟁하여 TGF-β 수용체에 결합함으로써 TGF-β 사이토카인이 TGF-β 수용체에 결합하는 것을 방해하는 기전을 통해 TGF-β 신호전달을 억제하는 것일 수 있다.In the present invention, the peptide may bind to the TGF-β receptor (TGFBR1 and/or TGFBR2) to inhibit TGF-β signaling. Specifically, the peptide competes with TGF-β and binds to the TGF-β receptor. It may be that TGF-β signaling is inhibited through a mechanism that prevents TGF-β cytokines from binding to the TGF-β receptor.
또한, 상기 펩타이드는 세포의 TGF-β 발현 수준 자체를 억제시켜 TGF-β 신호전달을 억제하는 것일 수 있으며, 구체적으로 상기 펩타이드는 자가-억제 경로(auto-inhibition pathway)를 통해 세포 내의 TGF-β 발현 수준을 감소시키거나, TGF-β의 세포외 배출량을 감소시키는 기전을 통해 TGF-β 신호전달을 억제하는 것일 수 있다.In addition, the peptide may inhibit TGF-β signaling by suppressing the expression level of TGF-β in cells. Specifically, the peptide may inhibit TGF-β signaling in cells through an auto-inhibition pathway. It may be suppressing TGF-β signaling through a mechanism that reduces the expression level or reduces the extracellular emissions of TGF-β.
3) 세포 3) cells
본 발명에서 상기 세포는 면역 이펙터 세포일 수 있다. 여기서, 상기 "면역 이펙터 세포"는 면역 이펙터 반응을 촉진하는 것과 같은 면역 반응에 참여하는 림프구 세포일 수 있다. In the present invention, the cells may be immune effector cells. Here, the “immune effector cell” may be a lymphoid cell that participates in an immune response, such as promoting an immune effector response.
본 발명에서 상기 "림프구(lymphocytes)"는 흔히 림프에서 발견되고, 그리고 자연살해 세포(NK 세포), T 세포, 및 B 세포를 포함하는 세포를 지칭한다. 당업계의 숙련자라면 상기에 나열된 면역세포 유형들이 아형으로 더 나눠질 수 있음을 이해할 것이다.As used herein, the term “lymphocytes” refers to cells that are commonly found in lymph and include natural killer cells (NK cells), T cells, and B cells. Those skilled in the art will understand that the immune cell types listed above can be further divided into subtypes.
본 발명의 일 예시에서 상기 림프구는 자연살해세포(Natural Killer Cells; NK cells)이거나 이를 포함할 수 있으나, 이에 제한되는 것은 아니다. In one example of the present invention, the lymphocytes may be or include Natural Killer Cells (NK cells), but are not limited thereto.
본 발명에서 상기 "자연살해세포(Natural Killer Cells; NK cells)"는 큰 과립 림프구 (LGL)로 정의되며, 일반적인 림프 전구세포-생성 B 및 T 림프구로부터 분화되는 3종류의 세포를 구성한다. NK 세포는 골수, 림프절, 비장, 편도선 및 흉선에서 분화되고 성숙하여 순환계로 진입하는 것으로 공지되어 있다. 본 발명에서 상기 NK 세포로는 어떠한 종류의 NK 세포라도 제한없이 포함될 수 있으며, 예컨대, 배양된 NK 세포, 예를 들면, 일차 NK 세포, 배양된 NK 세포주로부터의 NK 세포, 또는 포유동물로부터 수득된 NK 세포일 수 있으나, 이에 제한되는 것은 아니다. NK 세포가 포유동물로부터 수득되는 경우, NK 세포는 혈액, 골수, 림프절, 흉선, 또는 다른 조직 또는 체액을 포함하나 이들로 한정되지 않는 다수의 공급원들로부터 수득될 수 있다. NK 세포는 농축될 수도 있거나 정제될 수도 있다. NK 세포는 바람직하게는 인간 NK 세포일 수 있다(예를 들면, 인간으로부터 단리될 수 있다). NK 세포주는 예를 들면, ATCC (American Type Culture Collection)으로부터 입수가능하고, 예를 들면, NK-92 세포(ATCC CRL-2407), NK92MI 세포(ATCC CRL-2408) 또는 이들의 유도체 등을 포함한다.In the present invention, the "Natural Killer Cells (NK cells)" are defined as large granular lymphocytes (LGL), which constitute three types of cells differentiated from common lymphoid progenitor cells-producing B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus and enter the circulation. In the present invention, the NK cells may include any type of NK cell without limitation, for example, cultured NK cells, such as primary NK cells, NK cells from a cultured NK cell line, or NK cells obtained from a mammal. It may be an NK cell, but is not limited thereto. When NK cells are obtained from a mammal, the NK cells can be obtained from a number of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. NK cells may be concentrated or purified. The NK cells may preferably be human NK cells (eg, isolated from humans). NK cell lines are available, for example, from ATCC (American Type Culture Collection) and include, for example, NK-92 cells (ATCC CRL-2407), NK92MI cells (ATCC CRL-2408) or derivatives thereof, etc. .
본 발명의 다른 구현 예에 따르면, 상기 1) 메소텔린을 표적화하는 키메라 항원 수용체를 암호화하는 폴리뉴클레오티드; 및 2) TGF-β 신호전달 경로를 억제할 수 있는 펩타이드 또는 이의 단편을 암호화하는 폴리뉴클레오티드;를 포함하는 벡터에 관한 것이다. According to another embodiment of the present invention, 1) a polynucleotide encoding a chimeric antigen receptor targeting mesothelin; and 2) a polynucleotide encoding a peptide or fragment thereof capable of inhibiting the TGF-β signaling pathway.
본 발명에서는 하나의 벡터에 상기한 키메라 항원 수용체를 암호화하는 폴리뉴클레오티드(또는 유전자 컨스트럭트)와 상기 전환성장인자 베타 신호전달 경로를 억제할 수 있는 펩타이드 또는 그 단편을 암호화는 폴리뉴클레오티드(또는 유전자 컨스트럭트)가 모두 포함될 수 있고, 혹은 상기 키메라 항원 수용체를 암호화하는 폴리뉴클레오티드(또는 유전자 컨스트럭트)를 포함하는 벡터와, 상기 전환성장인자 베타 신호전달 경로를 억제할 수 있는 펩타이드 또는 그 단편을 암호화는 폴리뉴클레오티드(또는 유전자 컨스트럭트)를 포함하는 벡터 2종을 모두 포함할 수 있다. In the present invention, a polynucleotide (or gene construct) encoding the above-mentioned chimeric antigen receptor and a polynucleotide (or gene) encoding a peptide or fragment thereof capable of inhibiting the transforming growth factor beta signaling pathway are included in one vector. construct), or a vector containing a polynucleotide (or gene construct) encoding the chimeric antigen receptor, and a peptide or fragment thereof capable of inhibiting the transforming growth factor beta signaling pathway. Encoding may include both types of vectors containing polynucleotides (or gene constructs).
본 발명에서 상기 "벡터"란, 적당한 숙주 세포에 형질 주입되어 목적 단백질을 발현할 수 있는 재조합 벡터로서, 유전자 삽입물이 발현되도록 작동가능하게 연결된 필수적인 조절 요소를 포함하는 유전자 작제물을 말한다. 여기서, 상기 "작동가능하게 연결된(operably linked)"이란, 일반적 기능을 수행하도록 핵산 발현 조절 서열과 목적하는 단백질을 암호화하는 핵산 서열이 기능적으로 연결되어 있는 것을 의미한다. 재조합 벡터와의 작동적 연결은 당해 기술분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용하여 용이하게 할 수 있다.In the present invention, the "vector" refers to a recombinant vector that can be transfected into a suitable host cell to express a protein of interest, and refers to a genetic construct containing essential regulatory elements operably linked to express the gene insert. Here, the term “operably linked” means that the nucleic acid expression control sequence and the nucleic acid sequence encoding the protein of interest are functionally linked to perform a general function. Operational linkage with a recombinant vector can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cutting and ligation can be easily performed using enzymes generally known in the art. there is.
본 발명에서 상기 외래 유전자를 삽입하기 위한 재조합 발현 벡터로는 나노입자, 플라스미드, 바이러스, 코즈미드 등 다양한 형태의 벡터를 사용할 수 있다. 재조합 벡터의 종류는 원핵세포 및 진핵세포의 각종 숙주세포에서 원하는 유전자를 발현하고 원하는 단백질을 생산하는 기능을 하는 한 특별히 제한되지 않지만, 구체적으로 강력한 활성을 나타내는 프로모터와 강한 발현력을 보유하면서 자연 상태와 유사한 형태의 외래 단백질을 대량으로 생산할 수 있는 벡터가 이용될 수 있다. In the present invention, various types of vectors such as nanoparticles, plasmids, viruses, and cosmids can be used as recombinant expression vectors for inserting the foreign genes. The type of recombinant vector is not particularly limited as long as it functions to express the desired gene and produce the desired protein in various host cells of prokaryotic and eukaryotic cells, but specifically, it has a highly active promoter and strong expression ability while maintaining a natural state. Vectors that can produce large quantities of foreign proteins of a similar form can be used.
다양한 유전자 전달 비히클은 당 분야에 공지되어 있으며, 바이러스 및 비-바이러스(예를 들어, 네이키드 DNA, 플라스미드) 벡터 둘 모두를 포함한다. 유전자 전달에 적합한 바이러스 벡터는 당업자에게 공지되어 있다. 상기 바이러스 벡터의 비제한적 예시로는 레트로바이러스 벡터 (몰로니 쥐 백혈병 바이러스 벡터 (MoMLV), MSCV, SFFV, MPSV, SNV 등으로부터 유도), 렌티바이러스 벡터 (예를 들어, HIV-1, HIV-2, SIV, BIV, FIV 등으로부터 유도), 이의 복제 컴피턴트, 복제 결여 및 무기력한 형태를 포함하는 아데노바이러스 (Ad) 벡터, 아데노 관련 바이러스 (AAV) 벡터, 시미안 바이러스 40 (SV-40) 벡터, 소 유두종 바이러스 벡터, 엡스타인 바 바이러스 벡터, 헤르페스 바이러스 벡터, 수두 바이러스 벡터, 하비 쥐 육종 바이러스 벡터, 쥐 유방 종양 바이러스 벡터, 라우스 육종 바이러스 벡터, 파보바이러스 벡터, 소아 마비 바이러스 벡터, 수포성 구내염 바이러스 벡터, 마라바 바이러스 벡터 및 그룹 B 아데노바이러스 에나데노툭시레브 벡터 등을 포함할 수 있다.A variety of gene delivery vehicles are known in the art and include both viral and non-viral (e.g., naked DNA, plasmid) vectors. Viral vectors suitable for gene transfer are known to those skilled in the art. Non-limiting examples of the viral vectors include retroviral vectors (derived from Moloney murine leukemia virus vector (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., HIV-1, HIV-2) , derived from SIV, BIV, FIV, etc.), adenovirus (Ad) vectors, including replication-competent, replication-deficient and anergic forms thereof, adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors , bovine papilloma virus vector, Epstein Barr virus vector, herpes virus vector, chicken pox virus vector, Harvey rat sarcoma virus vector, rat mammary tumor virus vector, Rous sarcoma virus vector, parvovirus vector, polio virus vector, vesicular stomatitis virus vector. , Maraba virus vector, Group B adenovirus enadenotuxirev vector, etc.
유전자 전달을 위한 비-바이러스 벡터는 네이키드 DNA, 플라스미드, 트랜스포존 및 mRNA 등을 포함한다. 비제한적인 예는 pKK 플라스미드(Clonetech), pUC 플라스미드, pET 플라스미드(Novagen, Inc., Madison, Wis.), pRSET 또는 pREP 플라스미드(Invitrogen, San Diego, Calif.), pMAL 플라스미드(New England Biolabs, Beverly, Mass.)를 포함한다. Non-viral vectors for gene transfer include naked DNA, plasmids, transposons, and mRNA. Non-limiting examples include pKK plasmid (Clonetech), pUC plasmid, pET plasmid (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmid (Invitrogen, San Diego, Calif.), pMAL plasmid (New England Biolabs, Beverly , Mass.).
본 발명에서 상기 벡터는 본원에 개시되거나 인용되거나 관련 기술 분야의 당업자에게 달리 공지된 방법을 사용하여 많은 적절한 숙주 세포에 도입될 수 있다.The vectors in the present invention can be introduced into many suitable host cells using methods disclosed or cited herein or otherwise known to those skilled in the art.
본 발명의 적합한 발현 벡터는 프로모터, 개시코돈, 종결코돈, 폴리아데닐화 시그널 또는 인핸서 같은 발현 조절 엘리먼트 외에도 막 표적화 또는 분비를 위한 신호 펩타이드를 암호화하는 염기 서열을 포함할 수 있다. 개시 코돈 및 종결 코돈은 일반적으로 면역원성 표적 단백질을 암호화하는 뉴클레오타이드 서열의 일부로 간주되며, 유전자 작제물이 투여되었을 때 개체에서 반드시 작용을 나타내야 하며 암호화 서열과 인프레임(in frame)에 있어야 한다. 본 발명에서 상기 "프로모터"는, 본원에서 사용된 바와 같이, 유전자와 같은 암호화 서열의 발현을 조절하는 임의의 서열을 나타낸다. 프로모터는, 예를 들어, 구성적, 유도성, 억제성, 또는 조직-특이적일 수 있다. 프로모터는 전사의 시작 및 속도가 제어되는 폴리뉴클레오타이드 서열의 영역인 제어 서열이다. 본 발명에서 상기 프로모터의 비-제한적 예시로는 라우스 육종 바이러스 (Rous sarcoma virus: RSV) LTR 프로모터 (선택적으로 RSV 인핸서를 가짐), 시토메갈로바이러스 (CMV) 프로모터, SV40 프로모터, 디하이드로폴레이트 리덕타제 프로모터, β-액틴 프로모터, 포스포글리세롤 키나제 (PGK) 프로모터, U6 프로모터, EF1알파 짧은 형태 (EFS) 프로모터, 인간 폴리펩타이드 사슬 연장 인자 (EF1a) 프로모터, P5 프로모터, Ubc 프로모터, CAG 프로모터, TRE 프로모터, UAS 프로모터, Ac5 프로모터, 폴리헤드린 프로모터, CaMKIIa 프로모터, Gal1 프로모터, TEF1 프로모터, GDS 프로모터, ADH1 프로모터, CaMV35S 프로모터, 유비퀴틴 (Ubi) 프로모터, 예컨대 유비퀴틴 C (UbiC), H1 프로모터, U6 프로모터, 알파-1-안티트립신 프로모터, 비장 병소 형성 바이러스 (spleen focus-forming virus: SFFV) 프로모터 등을 포함할 수 있다. 또한, 본 발명에서 상기 프로모터는 전사 효율을 증가시키기 위해 인핸서에 커플링될 수 있다. 이때 상기 인핸서의 비-제한적 예시로는 RSV 인핸서, CMV 인핸서, 또는 α-태아 단백질 MERII 인핸서 등을 포함할 수 있으나, 이에 제한되는 것은 아니다. A suitable expression vector of the present invention may include a base sequence encoding a signal peptide for membrane targeting or secretion in addition to expression control elements such as a promoter, start codon, stop codon, polyadenylation signal, or enhancer. The initiation codon and stop codon are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein and must be functional in the subject when the genetic construct is administered and must be in frame with the coding sequence. In the present invention, the "promoter", as used herein, refers to any sequence that regulates the expression of a coding sequence, such as a gene. Promoters can be, for example, constitutive, inducible, repressible, or tissue-specific. A promoter is a control sequence, a region of polynucleotide sequence where the initiation and rate of transcription is controlled. Non-limiting examples of the promoters in the present invention include Rous sarcoma virus (RSV) LTR promoter (optionally with RSV enhancer), cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase Promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, U6 promoter, EF1alpha short form (EFS) promoter, human polypeptide chain elongation factor (EF1a) promoter, P5 promoter, Ubc promoter, CAG promoter, TRE promoter , UAS promoter, Ac5 promoter, polyhedrin promoter, CaMKIIa promoter, Gal1 promoter, TEF1 promoter, GDS promoter, ADH1 promoter, CaMV35S promoter, ubiquitin (Ubi) promoter such as ubiquitin C (UbiC), H1 promoter, U6 promoter, alpha- 1-Antitrypsin promoter, spleen focus-forming virus (SFFV) promoter, etc. may be included. Additionally, in the present invention, the promoter can be coupled to an enhancer to increase transcription efficiency. At this time, non-limiting examples of the enhancer may include, but are not limited to, the RSV enhancer, the CMV enhancer, or the α-fetoprotein MERII enhancer.
본 발명의 일 예시에서, 상기 프로모터는 SSFV 프로모터일 수 있고, 바람직하게는 서열번호 30으로 표시되는 SSFV 프로모터일 수 있으나, 이에 제한되는 것은 아니다. In one example of the present invention, the promoter may be the SSFV promoter, preferably the SSFV promoter represented by SEQ ID NO: 30, but is not limited thereto.
본 발명의 벡터에서 상기 키메라 항원 수용체의 구체적인 구성은 앞서 기재된 바와 중복되어 이하 자세한 기재를 생략한다. The specific configuration of the chimeric antigen receptor in the vector of the present invention overlaps with what was previously described, so detailed description is omitted below.
본 발명의 바람직한 일 예시에서 상기 키메라 항원 수용체를 암호화하는 폴리뉴클레오티드(또는 유전자 컨스트럭트)는 서열번호 26으로 표시되는 염기 서열을 포함하는 것일 수 있으나, 이에 제한되는 것은 아니다. In a preferred example of the present invention, the polynucleotide (or gene construct) encoding the chimeric antigen receptor may include the base sequence represented by SEQ ID NO: 26, but is not limited thereto.
본 발명의 바람직한 다른 예시에서 상기 키메라 항원 수용체를 암호화하는 폴리뉴클레오티드는 서열번호 27로 표시되는 염기 서열을 포함하는 것일 수 있으나, 이에 제한되는 것은 아니다. In another preferred example of the present invention, the polynucleotide encoding the chimeric antigen receptor may include the base sequence represented by SEQ ID NO: 27, but is not limited thereto.
본 발명의 상기 벡터에서 상기 전환성장인자 베타(TGF-β) 신호전달 경로를 억제할 수 있는 펩타이드 또는 이의 단편을 암호화하는 폴리뉴클레오티드의 상류에 신호 펩타이드를 암호화하는 서열을 더 포함할 수 있다. The vector of the present invention may further include a sequence encoding a signal peptide upstream of the polynucleotide encoding a peptide or fragment thereof capable of inhibiting the transforming growth factor beta (TGF-β) signaling pathway.
본 발명의 일 예시로, 상기 신호 펩타이드는 IL-2 신호 펩타이드일 수 있고, 바람직하게는 서열번호 33으로 표시되는 아미노산 서열을 포함하는 것이거나, 서열번호 34로 표시되는 염기 서열에 의해 암호화되는 것일 수 있으나, 이에 제한되는 것은 아니다. As an example of the present invention, the signal peptide may be an IL-2 signal peptide, and preferably includes the amino acid sequence represented by SEQ ID NO: 33, or is encoded by the nucleotide sequence represented by SEQ ID NO: 34. However, it is not limited to this.
또한, 본 발명에서 상기 벡터는 하나 이상의 추가 폴리펩타이드, 예를 들어 하나 이상의 마커 및/또는 하나 이상의 이펙터 분자를 암호화하는 폴리뉴클레오타이드를 포함할 수 있다. Additionally, in the present invention, the vector may include one or more additional polypeptides, for example, a polynucleotide encoding one or more markers and/or one or more effector molecules.
본 발명에서 상기 하나 이상의 마커는 형질 도입 마커, 대리 마커 및/또는 선택 마커를 포함할 수 있다. 예를 들어, 하나 이상의 추가 폴리펩타이드를 암호화하는 도입된 추가 핵산 서열 중에는, 예컨대 전달된 세포의 생존 능력 및/또는 기능을 촉진함으로써 요법의 효능을 개선할 수 있는 핵산 서열; 예컨대 생체 내에서 생존 또는 국소화를 평가하기 위해 세포의 평가 및/또는 선택을 위한 유전자 마커를 제공하는 핵산 서열; 문헌[Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); 및 Riddell et al., Human Gene Therapy 3:319-338 (1992)]에 기재된 바와 같이 예를 들어 생체 내에서 음성 선택에 세포를 예민하게 만들어 안정성을 개선하기 위한 핵산 서열이 포함되고; 또한 우성 양성 선택 가능 마커의 음성 선택 가능 마커와의 융합에서 유래된 2작용성 선택 가능 융합 유전자의 용도를 기술하는 문헌[WO 1992008796 및 WO 1994028143] 및 미국 특허 번호 제6,040,177호를 참조할 수 있다.In the present invention, the one or more markers may include a transduction marker, a surrogate marker, and/or a selection marker. For example, among the additional nucleic acid sequences introduced, encoding one or more additional polypeptides, may be nucleic acid sequences that can improve the efficacy of the therapy, such as by promoting the viability and/or function of the transferred cells; Nucleic acid sequences that provide genetic markers for evaluation and/or selection of cells, such as to assess survival or localization in vivo; Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); Reference may also be made to literature [WO 1992008796 and WO 1994028143] and US Patent No. 6,040,177, which describe the use of bifunctional selectable fusion genes derived from the fusion of a dominant positive selectable marker with a negative selectable marker.
본 발명에서 상기 마커는 형질 도입 마커 또는 대리 마커일 수 있다. 상기 형질 도입 마커 또는 대리 마커는 본 발명의 폴리뉴클레오타이드(또는 유전자 컨스트럭트), 즉 본 발명의 펩타이드 또는 그 단편을 암호화하는 서열을 포함하는 폴리뉴클레오타이드가 도입된 세포를 검출하는데 사용될 수 있다. 여기서, 상기 형질 도입 마커는 세포의 변형을 나타내거나 확인할 수 있고, 상기 대리 마커는 상기 펩타이드 또는 단편과 함께 세포 표면 상에 공발현되도록 제조된 단백질일 수 있다. 본 발명에서 상기 대리 마커는 거의 또는 전혀 활성을 갖지 않도록 변형된 표면 단백질일 수 있다. 본 발명에서, 상기 대리 마커는 상기 펩타이드 또는 단편을 암호화하는 동일한 폴리뉴클레오타이드 상에 암호화될 수 있다. 본 발명에서 상기 펩타이드 또는 그 단편을 암호화하는 핵산 서열은, 선택적으로 자가 절단 펩타이드 또는 리보솜 건너뛰기를 야기하는 펩타이드, 예컨대 2A 서열을 암호화하는 핵산에 작동 가능하게 연결될 수 있다. In the present invention, the marker may be a transduction marker or a surrogate marker. The transduction marker or surrogate marker can be used to detect cells into which a polynucleotide (or gene construct) of the present invention, that is, a polynucleotide containing a sequence encoding the peptide of the present invention or a fragment thereof, has been introduced. Here, the transduction marker may indicate or confirm transformation of the cell, and the surrogate marker may be a protein prepared to be co-expressed on the cell surface together with the peptide or fragment. In the present invention, the surrogate marker may be a surface protein modified to have little or no activity. In the present invention, the surrogate marker may be encoded on the same polynucleotide encoding the peptide or fragment. In the present invention, the nucleic acid sequence encoding the peptide or fragment thereof may optionally be operably linked to a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, such as a 2A sequence.
본 발명에서 상기 예시적인 대리 마커는 세포 표면 폴리펩타이드의 절단 형태, 예컨대 비기능적이며 전장 형태의 세포 표면 폴리펩타이드에 의한 신호 또는 이에 의해 일반적으로 전달되는 신호를 전달할 수 없거나 전달하지 않고/거나 내재화될 수 없거나 내재화되지 않는 절단 형태를 포함할 수 있다. 예시적인 절단형 세포 표면 폴리펩타이드는, 성장 인자 또는 다른 수용체의 절단 형태, 예컨대 절단형 인간 표피 성장 인자 수용체 2(truncated human epidermal growth factor receptor 2, tHER2), 절단형 표피 성장 인자 수용체(tEGFR) 또는 전립선 특이적 막 항원(prostate-specific membrane antigen, PSMA) 또는 이의 변형된 형태, 예컨대 절단형 PSMA(tPSMA)를 포함할 수 있다. 일부 측면에서, tEGFR은 항체 세툭시맙(Erbitux) 또는 다른 치료용 항-EGFR 항체 또는 결합 분자에 의해 인식되는 에피토프를 함유할 수 있으며, 이는 tEGFR 작제물 및 암호화된 외인성 단백질로 조작된 세포를 확인 또는 선택하기 위해 및/또는 암호화된 외인성 단백질을 발현하는 세포를 제거 또는 분리하기 위해 사용될 수 있다. 문헌[미국 특허 번호 8,802,374 및 Liu et al., Nature Biotech. 2016 April; 34(4): 430-434]을 참조한다. 일부 측면에서, 마커, 예를 들어 대리 마커는, CD34의 전부 또는 일부(예를 들어, 절단 형태), NGFR, CD19 또는 절단형 CD19, 예를 들어 절단형 비-인간 CD19를 포함한다. In the present invention, the exemplary surrogate marker is a truncated form of a cell surface polypeptide, e.g., a non-functional, full-length form of the cell surface polypeptide that cannot or will not transmit signals or signals that would normally be transmitted and/or will be internalized. It may contain truncated forms that cannot or are not internalized. Exemplary truncated cell surface polypeptides include truncated forms of growth factors or other receptors, such as truncated human epidermal growth factor receptor 2 (tHER2), truncated epidermal growth factor receptor (tEGFR), or It may include prostate-specific membrane antigen (PSMA) or a modified form thereof, such as truncated PSMA (tPSMA). In some aspects, tEGFR may contain an epitope recognized by the antibody cetuximab (Erbitux) or other therapeutic anti-EGFR antibody or binding molecule, which identifies cells engineered with the tEGFR construct and the encoded exogenous protein. or to select and/or remove or isolate cells expressing the encoded exogenous protein. See US Pat. No. 8,802,374 and Liu et al., Nature Biotech. April 2016; 34(4): 430-434]. In some aspects, the marker, e.g., a surrogate marker, includes all or part of CD34 (e.g., a truncated form), NGFR, CD19, or truncated CD19, e.g., truncated non-human CD19.
본 발명에서 상기 마커는 검출 가능한 단백질, 예컨대 형광 단백질, 예컨대 그린 형광 단백질(green fluorescent protein, GFP), 강화된 그린 형광 단백질(enhanced green fluorescent protein, EGFP), 예컨대 슈퍼-폴드 GFP(super-fold GFP, sfGFP), 레드 형광 단백질(red fluorescent protein, RFP), 예컨대 tdTomato, mCherry, mStrawberry, AsRed2, DsRed 또는 DsRed2, 시안 형광 단백질(cyan fluorescent protein, CFP), 블루 그린 형광 단백질(blue green fluorescent protein, BFP), 강화된 블루 형광 단백질(enhanced blue fluorescent protein, EBFP) 옐로우 형광 단백질(yellow fluorescent protein, YFP) 및 형광 단백질의 종 변이체, 단량체 변이체 및 코돈-최적화되고 안정화된 및/또는 강화된 변이체를 포함한 이의 변이체이거나 이를 포함할 수 있다. 또한, 본 발명에서 상기 마커는 효소, 예컨대 루시퍼라제, 대장균 유래 lacZ 유전자, 알칼리 포스파타제, 분비 배아 알칼리 포스파타제(secreted embryonic alkaline phosphatase, SEAP), 클로람페니콜 아세틸 트랜스퍼라제(chloramphenicol acetyl transferase, CAT), β-갈락토시다제 또는 β-글루쿠로니다제(β-glucuronidase, GUS)이거나 이를 포함한다. 상기 효소의 발현은, 효소의 발현 및 기능적 활성 시 검출될 수 있는 기질의 첨가로 검출될 수 있다.In the present invention, the marker is a detectable protein, such as a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), such as super-fold GFP. , sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue green fluorescent protein (BFP) ), enhanced blue fluorescent protein (EBFP) yellow fluorescent protein (YFP) and its variants, including species variants, monomer variants and codon-optimized, stabilized and/or enhanced variants of fluorescent proteins. It may be or contain a variant. In addition, in the present invention, the marker is an enzyme such as luciferase, E. coli-derived lacZ gene, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyl transferase (CAT), and β-gal. It is or includes lactosidase or β-glucuronidase (β-glucuronidase, GUS). Expression of the enzyme can be detected by addition of a substrate that can be detected upon expression and functional activity of the enzyme.
본 발명의 일 예시에서 상기 마커는 그린 형광 단백질(GFP)일 수 있고, 바람직하게는 상기 마커는 서열번호 31로 표시되는 아미노산 서열을 포함하는 것이거나, 서열번호 32로 표시되는 염기 서열에 의해 암호화되는 것일 수 있으나, 이에 제한되는 것은 아니다. In one example of the present invention, the marker may be green fluorescent protein (GFP), and preferably, the marker includes the amino acid sequence represented by SEQ ID NO: 31, or is encoded by the nucleotide sequence represented by SEQ ID NO: 32. It may be possible, but it is not limited to this.
본 발명에서 상기 마커는 선택 마커일 수 있다. 상기 선택 마커는 외인성 제제 또는 약물에 대한 내성을 부여하는 폴리펩타이드거나 이를 포함할 수 있다. 본 발명에서 상기 선택 마커는 항생제 내성 유전자일 수 있고, 비제한적 예시로는 퓨로마이신 내성 유전자, 히그로마이신 내성 유전자, 블라스티사이딘 내성 유전자, 네오마이신 내성 유전자, 게네티신 내성 유전자 또는 제오신 내성 유전자 또는 이의 변형된 형태이거나 이를 포함할 수 있다.In the present invention, the marker may be a selection marker. The selection marker may be or include a polypeptide that confers resistance to an exogenous agent or drug. In the present invention, the selection marker may be an antibiotic resistance gene, non-limiting examples include a puromycin resistance gene, hygromycin resistance gene, blasticidin resistance gene, neomycin resistance gene, geneticin resistance gene, or zeocin. It may be or contain a resistance gene or a modified form thereof.
본 발명의 바람직한 일 예시에서, 상기 벡터는 상기 키메라 항원 수용체를 암호화하는 폴리뉴클레오티드(또는 유전자 컨스트럭트); 상기 전환성장인자 베타 신호전달 경로를 억제할 수 있는 펩타이드 또는 그 단편을 암호화하는 폴리뉴클레오티드(또는 유전자 컨스트럭트); 및 마커를 암호화하는 폴리뉴클레오티드(또는 유전자 컨스트럭트)를 모두 포함할 수 있으나, 이에 제한되는 것은 아니다. In a preferred example of the present invention, the vector includes a polynucleotide (or gene construct) encoding the chimeric antigen receptor; A polynucleotide (or gene construct) encoding a peptide or fragment thereof capable of inhibiting the transforming growth factor beta signaling pathway; and a polynucleotide (or gene construct) encoding a marker, but is not limited thereto.
본 발명의 바람직한 다른 예시에서, 상기 벡터는 하나의 벡터에 상기 키메라 항원 수용체를 암호화하는 폴리뉴클레오티드(또는 유전자 컨스트럭트); 상기 전환성장인자 베타 신호전달 경로를 억제할 수 있는 펩타이드 또는 그 단편을 암호화하는 폴리뉴클레오티드(또는 유전자 컨스트럭트); 및 마커를 암호화하는 폴리뉴클레오티드(또는 유전자 컨스트럭트)를 모두 포함할 수 있으나, 이에 제한되는 것은 아니다. 이때 벡터 내 이들 각각 유전자의 위치 순서나 결합 순서는 특별히 제한하지 않는다. In another preferred example of the present invention, the vector includes a polynucleotide (or gene construct) encoding the chimeric antigen receptor in one vector; A polynucleotide (or gene construct) encoding a peptide or fragment thereof capable of inhibiting the transforming growth factor beta signaling pathway; and a polynucleotide (or gene construct) encoding a marker, but is not limited thereto. At this time, the position order or combination order of each of these genes in the vector is not particularly limited.
본 발명의 바람직한 또 다른 예시에서, 상기 벡터는 상기 키메라 항원 수용체를 암호화하는 폴리뉴클레오티드(또는 유전자 컨스트럭트)를 포함하는 벡터; 및 상기 전환성장인자 베타 신호전달 경로를 억제할 수 있는 펩타이드 또는 그 단편을 암호화하는 폴리뉴클레오티드(또는 유전자 컨스트럭트)를 포함하는 벡터;를 포함할 수 있으나, 이에 제한되는 것은 아니다. In another preferred example of the present invention, the vector is a vector containing a polynucleotide (or gene construct) encoding the chimeric antigen receptor; And a vector containing a polynucleotide (or gene construct) encoding a peptide or fragment thereof capable of inhibiting the transforming growth factor beta signaling pathway; but is not limited thereto.
본 발명의 바람직한 또 다른 예시에서, 상기 벡터는 상기 키메라 항원 수용체를 암호화하는 폴리뉴클레오티드(또는 유전자 컨스트럭트)를 포함하는 벡터; 및 상기 전환성장인자 베타 신호전달 경로를 억제할 수 있는 펩타이드 또는 그 단편을 암호화하는 폴리뉴클레오티드(또는 유전자 컨스트럭트)를 포함하는 벡터;를 포함하고, 이때 두 벡터 중 적어도 하나의 벡터에 마커를 암호화하는 폴리뉴클레오티드(또는 유전자 컨스트럭트)가 추가로 더 포함될 수 있으나, 이에 제한되는 것은 아니다.In another preferred example of the present invention, the vector is a vector containing a polynucleotide (or gene construct) encoding the chimeric antigen receptor; And a vector containing a polynucleotide (or gene construct) encoding a peptide or fragment thereof capable of inhibiting the transforming growth factor beta signaling pathway, wherein a marker is added to at least one of the two vectors. Encoding polynucleotides (or gene constructs) may be additionally included, but are not limited thereto.
본 발명에서 하나의 벡터에 2개의 별도의 폴리뉴클레오타이드(또는 유전자 컨스트럭트)가 포함되는 경우, 예컨대, 하나의 벡터에 상기 키메라 항원 수용체를 암호화하는 폴리뉴클레오티드(또는 유전자 컨스트럭트); 상기 전환성장인자 베타 신호전달 경로를 억제할 수 있는 펩타이드 또는 그 단편을 암호화하는 폴리뉴클레오티(또는 유전자 컨스트럭트)드; 및 마커를 암호화하는 폴리뉴클레오티드(또는 유전자 컨스트럭트) 중 적어도 2개가 동시에 포함되는 경우, 이들은 하나의 프로모터에 의해 작동 가능하게 연결될 수 있지만, 혹은 2개 이상의 프로모터에 의해 작동 가능하게 연결될 수 있다. In the present invention, when two separate polynucleotides (or gene constructs) are included in one vector, for example, a polynucleotide (or gene construct) encoding the chimeric antigen receptor in one vector; A polynucleotide (or gene construct) encoding a peptide or fragment thereof capable of inhibiting the transforming growth factor beta signaling pathway; And when at least two of the polynucleotides (or gene constructs) encoding the marker are included simultaneously, they may be operably linked by one promoter, or may be operably linked by two or more promoters.
본 발명에서 하나의 벡터에 2개 이상의 별도의 폴리뉴클레오티드(또는 유전자 컨스트럭트)가 포함되는 경우, 이들 각각이 세포 내 발현을 위해 세포로 개별적으로 전달되거나 도입될 수 있도록, 자가-절단성 펩타이드를 암호화하는 폴리뉴클레오티드를 추가로 추가로 더 포함할 수 있다. In the present invention, when two or more separate polynucleotides (or gene constructs) are included in one vector, a self-cleavable peptide is provided so that each of them can be individually delivered or introduced into the cell for intracellular expression. It may further include a polynucleotide encoding.
본 발명에서 상기 "자가-절단성 펩타이드(self-cleavage peptide)"란, 세포 내에서 합성되는 단백질의 절단을 유도할 수 있는 10개 내지 50개, 12개 내지 42개, 14개 내지 34개, 16개 내지 26개 또는 18개 내지 22개의 아미노산으로 구성된 펩타이드를 의미한다. 상기 자가-절단성 펩타이드는 바이러스 유전자의 2A 영역(region)에서 유래된 것일 수 있다. 상기 자가-절단성 펩타이드는 P2A, E2A, F2A 또는 T2A로부터 유래된 것일 수 있다. 구체적으로, 상기 자가-절단성 펩타이드는 P2A로부터 유래된 것일 수 있다. 또한, 상기 자가-절단성 펩타이드 대신 세포질 내 존재하는 분해효소에 의해 절단되는 펩타이드를 사용할 수 있다.In the present invention, the "self-cleavage peptide" refers to 10 to 50, 12 to 42, 14 to 34 peptides that can induce cleavage of proteins synthesized within cells. It refers to a peptide consisting of 16 to 26 or 18 to 22 amino acids. The self-cleaving peptide may be derived from the 2A region of the viral gene. The self-cleaving peptide may be derived from P2A, E2A, F2A or T2A. Specifically, the self-cleaving peptide may be derived from P2A. Additionally, instead of the self-cleaving peptide, a peptide that is cleaved by a degrading enzyme present in the cytoplasm can be used.
본 발명의 일 예시에서, 상기 자가-절단성 펩타이드는 P2A 또는 그 유래의 펩타이드일 수 있고, 바람직하게 서열번호 35로 표시되는 아미노산 서열을 포함하는 것이거나, 서열번호 36으로 표시되는 염기 서열에 의해 암호화되는 것일 수 있다. In one example of the present invention, the self-cleavable peptide may be P2A or a peptide derived therefrom, and preferably includes the amino acid sequence represented by SEQ ID NO: 35, or is cleaved by the nucleotide sequence represented by SEQ ID NO: 36. It may be encrypted.
본 발명의 다른 구현 예에 따르면, 본 발명에서 제공하는 유전적으로 조작된 세포를 유효 성분으로 포함하는, 세포 치료제에 관한 것이다. According to another embodiment of the present invention, it relates to a cell therapeutic agent comprising the genetically engineered cells provided by the present invention as an active ingredient.
본 발명에서 상기 "세포 치료제"는 조직의 기능을 복원하기 위하여 자가(autologous), 동종(allogenic), 이종(xenogenic) 세포를 이용한 치료제로, 암의 억제를 위해 사용되는 치료제를 의미한다. 상기 면역 이펙터 세포, 예를 들어 유전적으로 변형된 자연살해세포를 유효성분으로 포함하면 암의 치료 및 예방을 위한 세포 치료제로 활용할 수 있다.In the present invention, the “cell therapy” refers to a treatment using autologous, allogenic, or xenogenic cells to restore tissue function, and is used to suppress cancer. If the immune effector cells, for example, genetically modified natural killer cells, are included as active ingredients, they can be used as a cell therapy for the treatment and prevention of cancer.
본 발명에서 상기 세포 치료제는 약학적으로 허용가능한 담체를 더 포함할 수 있다. 상기 약학적으로 허용가능한 담체는 예컨대 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올, HSA(Human serum albumin) 및 이들 성분 중 1종 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액 및 정균제 등 다른 통상의 첨가제를 첨가할 수 있다.In the present invention, the cell therapeutic agent may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be, for example, saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, HSA (Human serum albumin), and a mixture of one or more of these ingredients. Other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed.
본 발명에서 상기 세포 치료제는 그 제형에 따라 필요한 경우, 현탁제, 용해보조제, 안정화제, 등장화제, 보존제, 흡착방지제, 계면활성화제, 희석제, 부형제, pH 조정제, 무통화제, 완충제, 함황(含硫)환원제, 산화방지제 등을 적절히 첨가할 수 있다. 상기 현탁제의 예로는, 메틸셀룰로오스, 폴리소르베이트 80, 히드록시에틸셀룰로오스, 아라비아고무, 트라간트말, 카르복시메틸셀룰로스나트륨, 폴리옥시에틸렌소르비탄모노라우레이트 등을 들 수 있으나, 이에 제한되는 것은 아니다.In the present invention, if necessary depending on the formulation, the cell therapeutic agent may be used as a suspending agent, solubilizing agent, stabilizer, isotonic agent, preservative, anti-adsorption agent, surfactant, diluent, excipient, pH adjuster, analgesic agent, buffer, sulfur-containing agent, etc.硫) Reducing agents, antioxidants, etc. can be added appropriately. Examples of the suspending agent include, but are not limited to, methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, traganmal, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate, etc. no.
본 발명에서 상기 용액 보조제로는, 폴리옥시에틸렌경화피마자유, 폴리소르베이트 80, 니코틴산아미드, 폴리옥시에틸렌소르비탄모노라우레이트, 메크로골, 피마자유지방산에틸에스테르 등을 들 수 있다. 안정화제로는, 덱스트란 40, 메틸셀룰로오스, 젤라틴, 아황산나트륨, 메타황산나트륨 등을 들 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the solution auxiliary agent includes polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinic acid amide, polyoxyethylene sorbitan monolaurate, mecrogol, castor oil fatty acid ethyl ester, etc. Stabilizers include, but are not limited to, dextran 40, methylcellulose, gelatin, sodium sulfite, and sodium metasulfate.
본 발명에서 상기 등장화제로는, 예를 들어 D-만니톨, 소르비톨 등을 들 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the isotonic agent includes, for example, D-mannitol, sorbitol, etc., but is not limited thereto.
본 발명에서 상기 보존제로는, 예를 들어 파라옥시벤조산메틸, 파라옥시벤조산에틸, 소르브산, 페놀, 크레졸, 클로로크레졸 등을 들 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the preservative includes, for example, methyl paraoxybenzoate, ethyl paraoxybenzoate, sorbic acid, phenol, cresol, chlorocresol, etc., but is not limited thereto.
본 발명에서 상기 흡착방지제로는, 예를 들어 인간혈청알부민, 레시틴, 덱스트란, 에틸렌옥사이드프로필렌옥사이드 공중합체, 히드록시프로필셀룰로오스, 메틸셀룰로오스, 폴리옥시에틸렌 경화피마자유, 폴리에틸렌글리콜 등을 들 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the anti-adsorption agent includes, for example, human serum albumin, lecithin, dextran, ethylene oxide propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hydrogenated castor oil, polyethylene glycol, etc. , but is not limited to this.
본 발명에서 상기 함황환원제로는, 예를 들어 N-아세틸시스테인, N-아세틸호모시스테인, 티옥토산, 티오디글리콜, 티오에탄올아민, 티오글리세롤, 티오소르비톨, 티오글리콜산 및 그 염, 티오황산나트륨, 글루타티온, 탄소원자수 1~7 의티오알칸산 등의 술푸히드릴기를 갖는 것 등을 들 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the sulfur-containing reducing agent includes, for example, N-acetylcysteine, N-acetylhomocysteine, thioctoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, Examples include those having a sulfuhydryl group such as glutathione and thioalkanoic acid having 1 to 7 carbon atoms, but are not limited thereto.
본 발명에서 상기 산화방지제로는, 예를 들어 에리소르브산, 디부틸히드록시톨루엔, 부틸히드록시아니솔, α-토코페롤, 아세트산토코페롤, L-아스코르브산 및 그 염, L-아스코르브산팔미테이트, L-아스코르브산스테아레이트, 아황산수소나트륨, 아황산나트륨, 갈릭산트리아밀, 갈릭산프로필 또는 에틸렌디아민테트라아세트산나트륨 (EDTA), 피로인산나트륨, 메타인산나트륨 등의 킬레이트제를 들 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the antioxidants include, for example, erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbic acid palmitate, Chelating agents such as L-ascorbate stearate, sodium bisulfite, sodium sulfite, triamyl gallate, propyl gallate or sodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate may be included, but are limited thereto. It doesn't work.
본 발명에서 상기 세포 치료제는 몸무게가 70 ㎏인 성인 환자를 기준으로 할 때, 예를 들어 약 1,000~10,000 세포/회, 1,000~100,000세포/회, 1,000~1000,000 세포/회, 1,000~10,000,000, 1,000~100,000,000 세포/회, 1,000~1,000,000,000 세포/회, 1,000~10,000,000,000 세포/회로, 일정시간 간격으로 1일 1회 내지 수회에 분할 투여할 수도 있고, 일정 시간 간격으로 여러 번 투여할 수 있다.In the present invention, the cell therapeutic agent is used, for example, based on an adult patient weighing 70 kg, about 1,000 to 10,000 cells/time, 1,000 to 100,000 cells/time, 1,000 to 1,000,000 cells/time, 1,000 to 10,000,000. , 1,000~100,000,000 cells/time, 1,000~1,000,000,000 cells/time, 1,000~10,000,000,000 cells/circuit, can be administered in divided doses once or several times a day at regular time intervals, or can be administered multiple times at regular time intervals.
본 발명에 따른 주사제품은 환자의 체질 및 결함의 종류에 따라 당 업계에 통상적으로 알려진 분량을 취하여 충전된 주사의 형태로 제조될 수 있다.The injectable product according to the present invention can be manufactured in the form of a filled injection by taking the amount commonly known in the art depending on the patient's constitution and type of defect.
본 발명의 또 다른 구현 예에 따르면, 본 발명에서 제공하는 유전적으로 조작된 세포를 유효 성분으로 포함하는 암의 예방, 개선 또는 치료용 약학 조성물에 관한 것이다. According to another embodiment of the present invention, the present invention relates to a pharmaceutical composition for preventing, improving or treating cancer containing the genetically engineered cells provided by the present invention as an active ingredient.
본 발명에서 상기 "암" 포유류에서 전형적으로 조절되지 않는 세포 성장으로 특징 지어진 생리적 상태를 나타내거나 가리킨다. 본 발명에서 예방, 개선 또는 치료의 대상이 되는 암은 고형 장기(solid organ)에서 비정상적으로 세포가 성장하여 발생한 덩어리로 이루어진 고형암(solid tumor)일 수 있고, 바람직하게는 메소텔린(MSLN)을 발현하는 암이라면 제한없이 포함될 수 있고, 구체적인 예를 들면, 위암, 간암, 교세포종, 난소암, 대장암, 두경부암, 방광암, 신장세포암, 유방암, 전이암, 전립선암, 췌장암, 담관계암, 흑색종 또는 폐암 등일 수 있으나, 이에 제한되는 것은 아니다.As used herein, "cancer" refers to or refers to a physiological condition typically characterized by uncontrolled cell growth in mammals. The cancer subject to prevention, improvement, or treatment in the present invention may be a solid tumor consisting of a lump generated by abnormal cell growth in a solid organ, and preferably expresses mesothelin (MSLN). Any cancer that has cancer can be included without limitation, and specific examples include stomach cancer, liver cancer, glioblastoma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, biliary tract cancer, It may be melanoma or lung cancer, but is not limited thereto.
본 발명에서 "예방"은 본 발명의 조성물의 투여로 암을 억제하거나 진행을 지연시키는 모든 행위를 의미한다. In the present invention, “prevention” refers to all actions that inhibit cancer or delay its progression by administering the composition of the present invention.
본 발명에서 "치료" 및 "개선"은 본 발명의 조성물의 투여로 암의 증상이 호전 또는 이롭게 변경되는 모든 행위를 의미한다.In the present invention, “treatment” and “improvement” mean any action in which cancer symptoms are improved or beneficially changed by administration of the composition of the present invention.
본 발명의 약학 조성물은 투여를 위해서 상기 기재한 유효성분 이외에 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 약학 조성물로 제제화할 수 있다. 약제학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올, 리포좀 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있으며, 표적 기관에 특이적으로 작용할 수 있도록 표적 기관 특이적 항체 또는 기타 리간드를 상기 담체와 결합시켜 사용할 수 있다. 더 나아가 당해 기술 분야의 적정한 방법으로 또는 레밍턴의 문헌(Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.For administration, the pharmaceutical composition of the present invention may be formulated to include one or more pharmaceutically acceptable carriers in addition to the active ingredients described above. Pharmaceutically acceptable carriers may be saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and a mixture of one or more of these ingredients, and if necessary, antioxidants. , other common additives such as buffer solutions and bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets, and can act specifically on target organs. Target organ-specific antibodies or other ligands can be used in combination with the carrier. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA). there is.
본 발명의 약학 조성물은 액제, 현탁제, 분산액, 유제, 겔제, 주사 가능한 액제 및 활성 화합물의 서방출형 제제 등이 될 수 있으며, 바람직하게는 주사제가 될 수 있다.The pharmaceutical composition of the present invention can be a solution, suspension, dispersion, emulsion, gel, injectable solution, or sustained-release preparation of the active compound, and is preferably an injection.
본 발명의 약학 조성물을 주사제로 제제화하는 경우, 주사제 처방의 유통에 따른 제품 안정성을 확보하기 위하여 주사제로 사용 가능한 산수용액 또는 인산염 등의 완충용액을 사용하여 pH를 조절함으로써 물리적으로나 화학적으로 매우 안정한 주사제로 제조될 수 있다.When the pharmaceutical composition of the present invention is formulated as an injection, the pH is adjusted using a buffer solution such as an aqueous acid solution or phosphate that can be used as an injection to ensure product stability according to the distribution of the injection prescription, making the injection very physically and chemically stable. It can be manufactured with
보다 구체적으로, 상기 주사제는 안정화제 또는 용해 보조제와 함께 주사용수에 용해시킨 후, 멸균처리, 특히 고온감압멸균법 또는 무균여과법에 의해 멸균처리하여 제조될 수 있다. 상기 주사용수로는 주사용 증류수 또는 주사용 완충용액, 예를 들어 pH 3.5 내지 7.5 범위의 인산염 완충용액 또는 인산이수소나트륨(NaH2PO4)-구연산 완충용액을 사용할 수 있다. 사용되는 인산염은 나트륨염 또는 칼륨염 형태이거나 무수물 또는 수화물 형태이어도 무방하고, 구연산 또는 무수물 또는 수화물 형태이어도 무방하다.More specifically, the injection can be prepared by dissolving it in water for injection along with a stabilizer or solubilizing agent and then sterilizing it, especially by high-temperature reduced-pressure sterilization or aseptic filtration. The water for injection may be distilled water for injection or a buffer solution for injection, for example, a phosphate buffer solution with a pH in the range of 3.5 to 7.5 or a sodium dihydrogen phosphate (NaH2PO4)-citric acid buffer solution. The phosphate salt used may be in the form of a sodium salt or potassium salt, an anhydrous or hydrated form, and may be in the form of citric acid or an anhydrous or hydrated form.
또한, 본 발명에서 사용되는 안정화제는 나트륨 피로설파이트(sodium pyrosulfite), 중아황산나트륨(NaHSO3), 메타중아황산나트륨(Na2S2O3) 또는 에틸렌디아민테트라아세트산(ethylenediaminetetraacetic acid)을 포함하고, 용해 보조제는 수산화나트륨(NaOH), 탄산수소나트륨(NaHCO3), 탄산나트륨(NaCO3) 또는 수산화칼륨(KOH)과 같은 염기, 또는 염산(HCl) 또는 아세트산(CH3COOH)과 같은 산을 포함한다.In addition, the stabilizer used in the present invention includes sodium pyrosulfite, sodium bisulfite (NaHSO 3 ), sodium metabisulfite (Na 2 S 2 O 3 ), or ethylenediaminetetraacetic acid, Solubilizing agents include bases such as sodium hydroxide (NaOH), sodium bicarbonate (NaHCO 3 ), sodium carbonate (NaCO 3 ) or potassium hydroxide (KOH), or acids such as hydrochloric acid (HCl) or acetic acid (CH 3 COOH). .
본 발명에 따른 주사제는 생체흡수성, 생체 분해성, 생체적합성으로 제형화될 수 있다. 생체흡수성이라 함은 주사제가 체내에서, 분산된 주사제의 분해 또는 분해 없이, 초기 적용에서 사라질 수 있음을 의미하는 것이다. 생체 분해성은 가수분해 또는 효소 분해에 의해 주사제가 체내에서 파쇄 또는 분해될 수 있음을 의미한다. 생체적 합성은 성분 모두가 체내에서 무독성임을 의미한다.The injectable agent according to the present invention can be formulated to be bioabsorbable, biodegradable, and biocompatible. By bioabsorbable we mean that the injectable agent can disappear from the body upon initial application without decomposition or decomposition of the dispersed injectable agent. Biodegradability means that the injectable agent can be broken down or decomposed in the body by hydrolysis or enzymatic degradation. Biosynthesis means that all ingredients are non-toxic to the body.
본 발명에 따른 주사제는 통상의 충진제, 중량제, 결합제, 습윤제, 계면활성제 등의 희석제, 또는 부형제 등을 사용하여 제조할 수 있다.The injection according to the present invention can be prepared using conventional fillers, weighting agents, binders, wetting agents, diluents such as surfactants, or excipients.
본 발명의 조성물 또는 유효성분은 목적에 따라 정맥내, 동맥내, 복강내, 근육내, 흉골내, 경피, 비측내, 피하, 자궁내 경막, 흡입, 국소, 직장, 경구, 안구내 또는 피내 경로 등을 통해 통상적인 방식으로 투여할 수 있으며, 바람직하게는 정맥내로 투여될 수 있다. 본 발명의 조성물 또는 유효성분은 주사 또는 카테터로 투여될 수 있다.The composition or active ingredient of the present invention may be administered by intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, subcutaneous, intrathecal, inhalational, topical, rectal, oral, intraocular or intradermal route depending on the purpose. It can be administered in a conventional manner, and preferably intravenously. The composition or active ingredient of the present invention can be administered by injection or catheter.
본 발명의 조성물에 있어서, 유효성분의 투여량은 체중 60 kg 성인기준으로 상기 약학 조성물 내에 포함되는 형질 전환된 숙주 세포가 1 x 101 ~ 1 x 1050개/kg, 바람직하게는 1 x 101 ~ 1 x 1030개/kg, 보다 바람직하게는 1 x 105 ~ 1 x 1020개/kg, 가장 바람직하게는 1 x 107 ~ 1 x 109개/kg의 범위 내로 투여될 수 있도록 조절할 수 있다. 다만, 투여될 최적의 투여량은 당업자에 의해 쉽게 결정될 수 있으며, 질환의 종류, 질환의 중증도, 조성물에 함유된 유효성분 및 다른 성분의 함량, 제형의 종류, 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다.In the composition of the present invention, the dosage of the active ingredient is 1 x 10 1 to 1 x 10 50 cells/kg, preferably 1 x 10 transformed host cells contained in the pharmaceutical composition, based on an adult weighing 60 kg. So that it can be administered within the range of 1 to 1 x 10 30 pieces/kg, more preferably 1 x 10 5 to 1 x 10 20 pieces/kg, and most preferably 1 x 10 7 to 1 x 10 9 pieces/kg. It can be adjusted. However, the optimal dosage to be administered can be easily determined by a person skilled in the art, and can be determined based on the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of dosage form, and the patient's age, weight, and general health. It can be adjusted according to various factors, including condition, gender and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs.
본 발명의 약학 조성물에 있어서 유효성분은, 조성물 총 중량에 대하여 0.001 내지 50 중량%로 함유될 수 있다. 그러나 함량은 이에 제한되지 않는다.In the pharmaceutical composition of the present invention, the active ingredient may be contained in an amount of 0.001 to 50% by weight based on the total weight of the composition. However, the content is not limited to this.
또한, 본 발명의 약학 조성물은 1종 이상의 항암제를 더 포함할 수 있다.Additionally, the pharmaceutical composition of the present invention may further include one or more anticancer agents.
본 발명에서 상기 항암제로는 나이트로젠 머스타드, 이마티닙, 옥살리플라틴, 리툭시맙, 엘로티닙, 네라티닙, 라파티닙, 제피티닙, 반데타닙, 니로티닙, 세마사닙, 보수티닙, 악시티닙, 세디라닙, 레스타우르티닙, 트라스투주맙, 게피티니브, 보르테조밉, 수니티닙, 카보플라틴, 베바시주맙, 시스플라틴, 세툭시맙, 비스쿰알붐, 아스파라기나제, 트레티노인, 하이드록시카바마이드, 다사티닙, 에스트라머스틴, 겜투주맵오조가마이신, 이브리투맙튜세탄, 헵타플라틴, 메칠아미노레불린산, 암사크린, 알렘투주맙, 프로카르바진, 알프로스타딜, 질산홀뮴 키토산, 젬시타빈, 독시플루리딘, 페메트렉세드, 테가푸르, 카페시타빈, 기메라신, 오테라실, 아자시티딘, 메토트렉세이트, 우라실, 시타라빈, 플루오로우라실, 플루다가빈, 에노시타빈, 플루타미드, 데시타빈, 머캅토푸린, 티오구아닌, 클라드리빈, 카르퍼, 랄티트렉세드, 도세탁셀, 파클리탁셀, 이리노테칸, 벨로테칸, 토포테칸, 비노렐빈, 에토포시드, 빈크리스틴, 빈블라스틴, 테니포시드, 독소루비신, 이다루비신, 에피루비신, 미톡산트론, 미토마이신, 블레로마이신, 다우노루비신, 닥티노마이신, 피라루비신, 아클라루비신, 페프로마이신, 템시롤리무스, 테졸로마이드, 부설판, 이포스파미드, 사이클로포스파미드, 멜파란, 알트레트민, 다카바진, 치오테파, 니무스틴, 클로람부실, 미토락톨, 레우코보린, 트레토닌, 엑스메스탄, 아미노글루테시미드, 아나그렐리드, 나벨빈, 파드라졸, 타시펜, 토레미펜, 테스토락톤, 아나스트로졸, 레트로졸, 보로졸, 비칼루타미드, 로무스틴 및 카르무스틴으로 이루어진 군에서 선택된 1종 이상을 사할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the anticancer agents include nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, semasanib, bosutinib, axitinib, Cediranib, lestaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, bevacizumab, cisplatin, cetuximab, Viscum album, asparaginase, tretinoin, hydroxycarbamide , dasatinib, estramustine, gemtuzumab ozogamycin, ibritumab tusetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, Gemcitabine, doxyfluridine, pemetrexed, tegafur, capecitabine, gimeracin, oteracil, azacitidine, methotrexate, uracil, cytarabine, fluorouracil, fludagabine, enocitabine, Flutamide, decitabine, mercaptopurine, thioguanine, cladribine, carper, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastine , teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleromycin, daunorubicin, dactinomycin, pyrarubicin, aclarubicin, pepromycin, temsirolimus. , tezolomide, busulfan, ifosphamide, cyclophosphamide, melphalan, altretmin, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exemestane. , aminoglutethimide, anagrelide, navelvin, fadrazole, taciphen, toremifene, testolactone, anastrozole, letrozole, borozole, bicalutamide, lomustine and carmustine. One or more types selected from the group may be used, but are not limited thereto.
본 발명의 또 다른 구현 예에 따르면, 개체에게 본 발명에서 제공하는 세포 치료제 또는 약학 조성물을 투여하는 단계를 포함하는 암의 예방, 개선 또는 치료 방법을 제공하는 것이다. According to another embodiment of the present invention, a method for preventing, improving, or treating cancer is provided, which includes administering the cell therapy or pharmaceutical composition provided by the present invention to a subject.
본 발명에서 상기 개체는 TGF-β 관련 질환으로 예컨대 암이 발병되거나 발병할 위험이 있는 쥐, 가축, 인간 등을 포함하는 포유동물, 조류, 파충류, 양식어류 등을 제한 없이 포함할 수 있다.In the present invention, the subject may include, without limitation, mammals, birds, reptiles, farmed fish, etc., including rats, livestock, humans, etc., that develop or are at risk of developing cancer due to a TGF-β-related disease.
본 발명에서 상기 조성물은 약학적으로 유효한 양으로 단일 또는 다중 투여될 수 있다. 이때, 조성물은 액제, 산제, 에어로졸, 주사제, 수액제(링겔), 캡슐제, 환제, 정제, 좌제 또는 패치의 형태로 제형화되어 투여할 수 있다. 상기 암 예방 또는 치료용 약학적 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여도 투여될 수 있다.In the present invention, the composition can be administered singly or multiple times in a pharmaceutically effective amount. At this time, the composition can be formulated and administered in the form of a solution, powder, aerosol, injection, infusion solution (injection), capsule, pill, tablet, suppository, or patch. The pharmaceutical composition for preventing or treating cancer may be administered through any general route as long as it can reach the target tissue.
본 발명에서 상기 조성물은 특별히 이에 제한되지 않으나, 목적하는 바에 따라 복강내 투여, 정맥내 투여, 근육내 투여, 피하 투여, 피내 투여, 경피패치투여, 경구 투여, 비내 투여, 폐내 투여, 직장내 투여 등의 경로를 통해 투여될 수 있다. 다만, 경구 투여 시에는 제형화되지 않은 형태로도 투여할 수 있고, 위산에 의하여 상기 약학적 조성물의 유효성분이 변성 또는 분해될 수 있기 때문에 경구용 조성물은 활성 약제를 코팅하거나 위에서의 분해로부터 보호되도록 제형화된 형태 또는 경구용 패치형태로 구강내에 투여할 수도 있다. 또한, 상기 조성물은 활성 물질이 표적세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다.In the present invention, the composition is not particularly limited thereto, but depending on the purpose, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, intrapulmonary administration, and intrarectal administration It can be administered through routes such as: However, when administered orally, it can be administered in an unformulated form, and since the active ingredients of the pharmaceutical composition may be denatured or decomposed by stomach acid, the oral composition must be coated with the active agent or protected from decomposition in the stomach. It can also be administered orally in formulated form or in the form of an oral patch. Additionally, the composition can be administered by any device that allows the active substance to move to target cells.
본 발명에서 제공하는, 유전적으로 조작된 면역 이펙터 세포, 특히 자연살해세포는, 상기 세포에서 발현되는 메소텔린(MSLN)을 표적화하는 키메라 항원 수용체에 의해 목표로 하는 종양 세포만을 표적화할 수 있고, 상기 세포의 기본적인 항-종양 활성에 더하여 상기 세포에서 추가로 발현되는 펩타이드 또는 이의 단편에 의해 전환성장인자 베타(TGF-β) 신호전달 경로가 억제되어 미세종양 내부로의 세포 침윤능이 향상되고, 종양 세포에 대한 세포 독성 효과 또한 현저히 향상되는 장점이 있다. The genetically engineered immune effector cells, especially natural killer cells, provided by the present invention can target only target tumor cells by a chimeric antigen receptor targeting mesothelin (MSLN) expressed in the cells, In addition to the basic anti-tumor activity of the cells, the transforming growth factor beta (TGF-β) signaling pathway is inhibited by the peptide or fragment thereof additionally expressed in the cells, thereby improving the cell invasion ability into the microtumor and tumor cells. There is also an advantage that the cytotoxic effect is significantly improved.
도 1은 본 발명의 일 예시에 따른 메소텔린(Mesothelin)을 표적화하는 키메라 항원 수용체를 암호화하는 폴리뉴클레오티드와 전환성장인자 베타(TGF-β) 신호전달 경로를 억제할 수 있는 펩타이드를 암호화하는 폴리뉴클레오티드(P6)를 모두 포함하는 벡터 맵을 도시한 것이다. 1 shows a polynucleotide encoding a chimeric antigen receptor targeting mesothelin and a polynucleotide encoding a peptide capable of inhibiting the transforming growth factor beta (TGF-β) signaling pathway according to an example of the present invention. A vector map containing all (P6) is shown.
도 2는 본 발명의 실험예 1에서 루시퍼라제 리포터 유전자가 도입된 HCC-1806 유방암 세포주에 본 발명에 따른 키메라 항원 수용체 및 TGF-β 신호전달 경로를 억제하는 펩타이드를 발현하도록 유전적으로 조작된 NK 세포주를 처리한 뒤 루시퍼라제 활성을 비교한 결과를 그래프로 나타낸 것이다. Figure 2 shows an NK cell line genetically engineered to express a chimeric antigen receptor according to the present invention and a peptide that inhibits the TGF-β signaling pathway in the HCC-1806 breast cancer cell line into which a luciferase reporter gene was introduced in Experimental Example 1 of the present invention. The results of comparing luciferase activity after processing are shown in a graph.
도 3은 본 발명의 실험예 1에서 루시퍼라제 리포터 유전자가 도입된 MD-AMB-231 유방암 세포주에 본 발명에 따른 키메라 항원 수용체 및 TGF-β 신호전달 경로를 억제하는 펩타이드를 발현하도록 유전적으로 조작된 NK 세포주를 처리한 뒤 루시퍼라제 활성을 비교한 결과를 그래프로 나타낸 것이다. Figure 3 shows the MD-AMB-231 breast cancer cell line into which the luciferase reporter gene was introduced in Experimental Example 1 of the present invention, which was genetically engineered to express a chimeric antigen receptor according to the present invention and a peptide that inhibits the TGF-β signaling pathway. The results of comparing luciferase activity after treating NK cell lines are shown graphically.
도 4는 본 발명의 실험예 1에서 루시퍼라제 리포터 유전자가 도입된 NCI-N87 위암 세포주에 본 발명에 따른 키메라 항원 수용체 및 TGF-β 신호전달 경로를 억제하는 펩타이드를 발현하도록 유전적으로 조작된 NK 세포주를 처리한 뒤 루시퍼라제 활성을 비교한 결과를 그래프로 나타낸 것이다.Figure 4 shows an NK cell line genetically engineered to express a peptide that inhibits the chimeric antigen receptor and TGF-β signaling pathway according to the present invention in the NCI-N87 gastric cancer cell line into which a luciferase reporter gene was introduced in Experimental Example 1 of the present invention. The results of comparing luciferase activity after processing are shown in a graph.
도 5는 본 발명의 실험예 1에서 루시퍼라제 리포터 유전자가 도입된 AGS 위암 세포주에 본 발명에 따른 키메라 항원 수용체 및 TGF-β 신호전달 경로를 억제하는 펩타이드를 발현하도록 유전적으로 조작된 NK 세포주를 처리한 뒤 루시퍼라제 활성을 비교한 결과를 그래프로 나타낸 것이다.Figure 5 shows that in Experimental Example 1 of the present invention, the AGS gastric cancer cell line into which the luciferase reporter gene was introduced was treated with an NK cell line genetically engineered to express a peptide that inhibits the chimeric antigen receptor and the TGF-β signaling pathway according to the present invention. The results of comparing luciferase activity are shown in a graph.
도 6은 본 발명의 실험예 1에서 루시퍼라제 리포터 유전자가 도입된 NCI-H292 폐암 세포주에 본 발명에 따른 키메라 항원 수용체 및 TGF-β 신호전달 경로를 억제하는 펩타이드를 발현하도록 유전적으로 조작된 NK 세포주를 처리한 뒤 루시퍼라제 활성을 비교한 결과를 그래프로 나타낸 것이다.Figure 6 shows a NK cell line genetically engineered to express a peptide that inhibits the chimeric antigen receptor and TGF-β signaling pathway according to the present invention in the NCI-H292 lung cancer cell line into which a luciferase reporter gene was introduced in Experimental Example 1 of the present invention. The results of comparing luciferase activity after processing are shown in a graph.
도 7은 본 발명의 실험예 1에서 루시퍼라제 리포터 유전자가 도입된 SW-620 대장암 세포주에 본 발명에 따른 키메라 항원 수용체 및 TGF-β 신호전달 경로를 억제하는 펩타이드를 발현하도록 유전적으로 조작된 NK 세포주를 처리한 뒤 루시퍼라제 활성을 비교한 결과를 그래프로 나타낸 것이다.Figure 7 shows NK genetically engineered to express a chimeric antigen receptor according to the present invention and a peptide that inhibits the TGF-β signaling pathway in the SW-620 colon cancer cell line into which a luciferase reporter gene was introduced in Experimental Example 1 of the present invention. The results of comparing luciferase activity after treating cell lines are shown graphically.
도 8은 본 발명의 실험예 1에서 루시퍼라제 리포터 유전자가 도입된 SNU-1544 대장암 세포주에 본 발명에 따른 키메라 항원 수용체 및 TGF-β 신호전달 경로를 억제하는 펩타이드를 발현하도록 유전적으로 조작된 NK 세포주를 처리한 뒤 루시퍼라제 활성을 비교한 결과를 그래프로 나타낸 것이다.Figure 8 shows NK genetically engineered to express a chimeric antigen receptor according to the present invention and a peptide that inhibits the TGF-β signaling pathway in the SNU-1544 colon cancer cell line into which a luciferase reporter gene was introduced in Experimental Example 1 of the present invention. The results of comparing luciferase activity after treating cell lines are shown graphically.
도 9는 본 발명의 실험예 1에서 루시퍼라제 리포터 유전자가 도입된 SK-HEP-1 간암 세포주에 본 발명에 따른 키메라 항원 수용체 및 TGF-β 신호전달 경로를 억제하는 펩타이드를 발현하도록 유전적으로 조작된 NK 세포주를 처리한 뒤 루시퍼라제 활성을 비교한 결과를 그래프로 나타낸 것이다.Figure 9 shows the SK-HEP-1 liver cancer cell line into which the luciferase reporter gene was introduced in Experimental Example 1 of the present invention, which was genetically engineered to express a peptide that inhibits the chimeric antigen receptor and TGF-β signaling pathway according to the present invention. The results of comparing luciferase activity after treating NK cell lines are shown graphically.
도 10은 본 발명의 실험예 1에서 루시퍼라제 리포터 유전자가 도입된 HEP-G2 간암 세포주에 본 발명에 따른 키메라 항원 수용체 및 TGF-β 신호전달 경로를 억제하는 펩타이드를 발현하도록 유전적으로 조작된 NK 세포주를 처리한 뒤 루시퍼라제 활성을 비교한 결과를 그래프로 나타낸 것이다.Figure 10 shows a NK cell line genetically engineered to express a peptide that inhibits the chimeric antigen receptor and TGF-β signaling pathway according to the present invention in the HEP-G2 liver cancer cell line into which the luciferase reporter gene was introduced in Experimental Example 1 of the present invention. The results of comparing luciferase activity after processing are shown in a graph.
도 11은 본 발명의 실험예 1에서 루시퍼라제 리포터 유전자가 도입된 AsPC-1 췌장암 세포주에 본 발명에 따른 키메라 항원 수용체 및 TGF-β 신호전달 경로를 억제하는 펩타이드를 발현하도록 유전적으로 조작된 NK 세포주를 처리한 뒤 루시퍼라제 활성을 비교한 결과를 그래프로 나타낸 것이다.Figure 11 shows an NK cell line genetically engineered to express a peptide that inhibits the chimeric antigen receptor and TGF-β signaling pathway according to the present invention in the AsPC-1 pancreatic cancer cell line into which the luciferase reporter gene was introduced in Experimental Example 1 of the present invention. The results of comparing luciferase activity after processing are shown in a graph.
도 12는 본 발명의 실험예 1에서 루시퍼라제 리포터 유전자가 도입된 Capan-2 췌장암 세포주에 본 발명에 따른 키메라 항원 수용체 및 TGF-β 신호전달 경로를 억제하는 펩타이드를 발현하도록 유전적으로 조작된 NK 세포주를 처리한 뒤 루시퍼라제 활성을 비교한 결과를 그래프로 나타낸 것이다.Figure 12 shows a NK cell line genetically engineered to express a chimeric antigen receptor according to the present invention and a peptide that inhibits the TGF-β signaling pathway in the Capan-2 pancreatic cancer cell line into which a luciferase reporter gene was introduced in Experimental Example 1 of the present invention. The results of comparing luciferase activity after processing are shown in a graph.
도 13은 본 발명의 참고예 1에서 췌장암 세포주인 Aspc1에서의 본 발명의 펩타이드 처리에 따른 pSmad2/3 발현 수준 변화를 나타낸 것이다.Figure 13 shows the change in pSmad2/3 expression level in Aspc1, a pancreatic cancer cell line, according to treatment with the peptide of the present invention in Reference Example 1 of the present invention.
도 14는 본 발명의 참고예 1에서 췌장암 세포주인 Bxpc3에서의 본 발명의 펩타이드 처리에 따른 pSmad2/3 발현 수준 변화를 나타낸 도면이다.Figure 14 is a diagram showing the change in pSmad2/3 expression level in Bxpc3, a pancreatic cancer cell line, according to treatment with the peptide of the present invention in Reference Example 1 of the present invention.
도 15는 본 발명의 참고예 1에서 췌장암 세포주인 Panc1에서의 본 발명의 펩타이드 처리에 따른 pSmad2/3 발현 수준 변화를 나타낸 도면이다.Figure 15 is a diagram showing the change in pSmad2/3 expression level in Panc1, a pancreatic cancer cell line, according to treatment with the peptide of the present invention in Reference Example 1 of the present invention.
본 발명의 일 구현예에 따르면, 1) 메소텔린(Mesothelin; MSLN)을 표적화하는 키메라 항원 수용체(chimeric antigen receptor; CAR); 및 2) 전환성장인자 베타(Transforming growth factor-β, TGF-β) 신호전달 경로를 억제할 수 있는 펩타이드 또는 이의 단편을 발현하도록 유전적으로 조작된 세포에 관한 것이다. According to one embodiment of the present invention, 1) a chimeric antigen receptor (CAR) targeting mesothelin (MSLN); and 2) cells genetically engineered to express a peptide or fragment thereof capable of inhibiting the transforming growth factor-β (TGF-β) signaling pathway.
본 발명에서 상기 키메라 항원 수용체는 메소텔린 결합 도메인을 포함하고, 그 외에, 힌지 도메인, 신호 펩티드 도메인, 막횡단 도메인 및 하나 이상의 신호전달 도메인으로 이루어진 군에서 선택된 1종 이상을 추가로 포함할 수 있다.In the present invention, the chimeric antigen receptor includes a mesothelin binding domain, and may further include one or more selected from the group consisting of a hinge domain, a signal peptide domain, a transmembrane domain, and one or more signaling domains. .
본 발명의 바람직한 일 예시로, 상기 메소텔린(MSLN) 결합 도메인은 메소텔린을 특이적으로 인식할 수 있는 scFv를 포함할 수 있고, 상기 scFv는 서열번호 2, 3 및 4 각각의 아미노산 서열로 표시되는 경쇄 CDR1, CDR2, CDR3를 포함하는 경쇄 가변 영역(VL); 서열번호 12로 표시되는 아미노산 서열을 포함하거나, 혹은 서열번호 13으로 표시되는 염기 서열에 의해 암호화되는 링커; 및 서열번호 5, 6 및 7 각각의 아미노산 서열로 표시되는 중쇄 CDR1, CDR2, CDR3를 포함하는 중쇄 가변 영역(VH);을 포함할 수 있으나, 이에 제한되는 것은 아니다.In a preferred example of the present invention, the mesothelin (MSLN) binding domain may include an scFv capable of specifically recognizing mesothelin, and the scFv is represented by the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively. a light chain variable region (VL) comprising light chain CDR1, CDR2, and CDR3; A linker comprising the amino acid sequence shown in SEQ ID NO: 12 or encoded by the nucleotide sequence shown in SEQ ID NO: 13; and a heavy chain variable region (VH) comprising heavy chain CDR1, CDR2, and CDR3 represented by the amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively, but is not limited thereto.
본 발명의 바람직한 다른 예시로, 상기 메소텔린(MSLN) 결합 도메인은 메소텔린을 특이적으로 인식할 수 있는 scFv를 포함할 수 있고, 상기 scFv는 서열번호 8로 표시되는 아미노산 서열을 포함하거나, 혹은 서열번호 9로 표시되는 염기 서열에 의해 암호화되는 항체의 가변 경쇄 영역(VL); 서열번호 12로 표시되는 아미노산 서열을 포함하거나, 혹은 서열번호 13으로 표시되는 염기 서열에 의해 암호화되는 링커; 및 서열번호 10으로 표시되는 아미노산 서열을 포함하거나, 혹은 서열번호 11로 표시되는 염기 서열에 의해 암호화되는 항체의 가변 중쇄 영역(VH);을 포함할 수 있으나, 이에 제한되는 것은 아니다.As another preferred example of the present invention, the mesothelin (MSLN) binding domain may include an scFv capable of specifically recognizing mesothelin, and the scFv may include the amino acid sequence shown in SEQ ID NO: 8, or Variable light chain region (VL) of the antibody encoded by the nucleotide sequence shown in SEQ ID NO: 9; A linker comprising the amino acid sequence shown in SEQ ID NO: 12 or encoded by the nucleotide sequence shown in SEQ ID NO: 13; and a variable heavy chain region (VH) of an antibody comprising the amino acid sequence represented by SEQ ID NO: 10 or encoded by the nucleotide sequence represented by SEQ ID NO: 11; but is not limited thereto.
본 발명의 키메라 항원 수용체는 번역된 키메라 단백질을 막으로 향하게 하기 위해 신호 펩타이드가 추가된 형태로 설계될 수 있다.The chimeric antigen receptor of the present invention can be designed with a signal peptide added to direct the translated chimeric protein to the membrane.
본 발명의 일 예시로, 상기 신호 펩타이드는 CD8 신호 펩타이드일 수 있고, 바람직하게는 서열번호 14로 표시되는 아미노산 서열을 포함할 수 있으며, 혹은 서열번호 15로 표시되는 염기 서열에 의해 암호화되는 신호 펩타이드일 수 있다.As an example of the present invention, the signal peptide may be a CD8 signal peptide, and preferably may include an amino acid sequence represented by SEQ ID NO: 14, or a signal peptide encoded by the nucleotide sequence represented by SEQ ID NO: 15. It can be.
본 발명의 키메라 항원 수용체는 세포외 스페이서, 즉 힌지(hinge)를 더 포함할 수 있다. 본 발명의 일 예시로, 상기 힌지는 CD8 유래 힌지 영역일 수 있고, 바람직하게는 서열번호 16으로 표시되는 아미노산 서열을 포함할 수 있으며, 혹은 서열번호 17로 표시되는 염기 서열에 의해 암호화되는 힌지일 수 있다.The chimeric antigen receptor of the present invention may further include an extracellular spacer, that is, a hinge. As an example of the present invention, the hinge may be a CD8-derived hinge region, and preferably may include an amino acid sequence represented by SEQ ID NO: 16, or a hinge encoded by the nucleotide sequence represented by SEQ ID NO: 17. You can.
본 발명의 키메라 항원 수용체는 막관통 도메인을 더 포함할 수 있다. 본 발명에서 상기 막관통 도메인은 CD8 막관통 도메인일 수 있고, 바람직하게는 서열번호 18로 표시되는 아미노산 서열을 포함하거나, 서열번호 19로 표시되는 염기 서열에 의해 암호화되는 것일 수 있다.The chimeric antigen receptor of the present invention may further include a transmembrane domain. In the present invention, the transmembrane domain may be a CD8 transmembrane domain, and preferably includes the amino acid sequence represented by SEQ ID NO: 18, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 19.
본 발명의 키메라 항원 수용체는 세포질 도메인으로, 세포내 신호전달 도메인을 더 포함할 수 있다. 본 발명의 일 예시로 상기 세포내 신호전달 도메인은 CD3 zeta 신호전달 도메인일 수 있고, 바람직하게는 서열번호 20으로 표시되는 아미노산 서열을 포함하거나, 서열번호 21로 표시되는 염기 서열에 의해 암호화되는 것일 수 있다. The chimeric antigen receptor of the present invention has a cytoplasmic domain and may further include an intracellular signaling domain. As an example of the present invention, the intracellular signaling domain may be a CD3 zeta signaling domain, and preferably includes the amino acid sequence represented by SEQ ID NO: 20, or is encoded by the nucleotide sequence represented by SEQ ID NO: 21. You can.
본 발명의 다른 예시로, 상기 세포내 신호전달 도메인은 공동-자극성 신호전달 도메인으로 4-1BB를 더 포함할 수 있다. 여기서, 상기 4-1BB는 바람직하게는 서열번호 22로 표시되는 아미노산 서열을 포함하거나, 서열번호 23으로 표시되는 염기 서열에 의해 암호화되는 것일 수 있다. As another example of the present invention, the intracellular signaling domain may further include 4-1BB as a co-stimulatory signaling domain. Here, the 4-1BB preferably includes the amino acid sequence represented by SEQ ID NO: 22, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 23.
본 발명의 또 다른 예시로, 상기 세포내 신호전달 도메인은 1차 신호전달 도메인으로 CD3 zeta 신호전달 도메인을 포함하고, 공동-자극성 도메인으로 4-1BB를 포함할 수 있고, 바람직하게는 4-1BB-CD3 zeta의 순으로 포함할 수 있다. 이때, 상기 CD3 zeta 신호전달 도메인은 서열번호 20으로 표시되는 아미노산 서열을 포함하거나, 서열번호 21로 표시되는 염기 서열에 의해 암호화되는 것일 수 있고, 상기 4-1BB는 서열번호 22로 표시되는 아미노산 서열을 포함하거나, 서열번호 23으로 표시되는 염기 서열에 의해 암호화되는 것일 수 있다.As another example of the present invention, the intracellular signaling domain may include a CD3 zeta signaling domain as a primary signaling domain and 4-1BB as a co-stimulatory domain, preferably 4-1BB. -Can be included in the order of CD3 zeta. At this time, the CD3 zeta signaling domain may include the amino acid sequence represented by SEQ ID NO: 20, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 21, and the 4-1BB is the amino acid sequence represented by SEQ ID NO: 22. It may include or be encoded by the nucleotide sequence shown in SEQ ID NO: 23.
본 발명에서 상기 세포는 전환성장인자 베타(Transforming growth factor-β, TGF-β) 신호전달 경로를 억제할 수 있는 펩타이드 또는 이의 단편을 발현하도록 유전적으로 조작된 세포를 더 포함할 수 있다.In the present invention, the cells may further include cells genetically engineered to express a peptide or fragment thereof capable of inhibiting the transforming growth factor-β (TGF-β) signaling pathway.
본 발명에서 상기 펩타이드는 서열번호 28로 표시되는 아미노산 서열을 포함하는 것, 바람직하게는 서열번호 28로 표시되는 아미노산 서열로 이루어지는 것일 수 있다.In the present invention, the peptide may include the amino acid sequence represented by SEQ ID NO: 28, preferably consisting of the amino acid sequence represented by SEQ ID NO: 28.
본 발명에서 상기 펩타이드를 암호화하는 서열은 서열번호 29로 표시되는 염기 서열을 포함하는 것, 바람직하게는 서열번호 29로 표시되는 염기 서열로 이루어진 것일 수 있다.In the present invention, the sequence encoding the peptide may include the nucleotide sequence represented by SEQ ID NO: 29, preferably consisting of the nucleotide sequence represented by SEQ ID NO: 29.
본 발명에서 상기 펩타이드는 TGF-β 수용체(TGFBR1 및/또는 TGFBR2)에 결합하여 TGF-β 신호전달을 억제하는 것일 수 있으며, 구체적으로 상기 펩타이드는 TGF-β와 경쟁하여 TGF-β 수용체에 결합함으로써 TGF-β 사이토카인이 TGF-β 수용체에 결합하는 것을 방해하는 기전을 통해 TGF-β 신호전달을 억제하는 것일 수 있다.In the present invention, the peptide may bind to the TGF-β receptor (TGFBR1 and/or TGFBR2) to inhibit TGF-β signaling. Specifically, the peptide competes with TGF-β and binds to the TGF-β receptor. It may be that TGF-β signaling is inhibited through a mechanism that prevents TGF-β cytokines from binding to the TGF-β receptor.
본 발명에서 상기 세포는 면역 이펙터 세포일 수 있다. 여기서, 상기 면역 이펙터 세포는 면역 이펙터 반응을 촉진하는 것과 같은 면역 반응에 참여하는 림프구 세포로, 자연살해세포(Natural Killer Cells; NK cells)이거나 이를 포함할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the cells may be immune effector cells. Here, the immune effector cells are lymphoid cells that participate in an immune response, such as promoting an immune effector response, and may be or include Natural Killer Cells (NK cells), but are not limited thereto.
본 발명에서는 상기 펩타이드 또는 그 단편을 암호화하는 유전자를 포함하는 벡터를 상기 면역 이펙터 세포에 형질 감염시켜 상기 세포에 상기 펩타이드 또는 그 단편을 암호화하는 외래 유전자를 도입할 수 있다.In the present invention, a vector containing a gene encoding the peptide or its fragment can be transfected into the immune effector cell to introduce a foreign gene encoding the peptide or its fragment into the cell.
본 발명의 다른 구현 예에 따르면, 상기 1) 메소텔린을 표적화하는 키메라 항원 수용체를 암호화하는 폴리뉴클레오티드; 및 2) TGF-β 신호전달 경로를 억제할 수 있는 펩타이드 또는 이의 단편을 암호화하는 폴리뉴클레오티드;를 포함하는 벡터에 관한 것이다. According to another embodiment of the present invention, 1) a polynucleotide encoding a chimeric antigen receptor targeting mesothelin; and 2) a polynucleotide encoding a peptide or fragment thereof capable of inhibiting the TGF-β signaling pathway.
본 발명에서는 하나의 벡터에 상기한 키메라 항원 수용체를 암호화하는 폴리뉴클레오티드(또는 유전자 컨스트럭트)와 상기 전환성장인자 베타 신호전달 경로를 억제할 수 있는 펩타이드 또는 그 단편을 암호화는 폴리뉴클레오티드(또는 유전자 컨스트럭트)가 모두 포함될 수 있고, 혹은 상기 키메라 항원 수용체를 암호화하는 폴리뉴클레오티드(또는 유전자 컨스트럭트)를 포함하는 벡터와, 상기 전환성장인자 베타 신호전달 경로를 억제할 수 있는 펩타이드 또는 그 단편을 암호화는 폴리뉴클레오티드(또는 유전자 컨스트럭트)를 포함하는 벡터 2종을 모두 포함할 수 있다.In the present invention, a polynucleotide (or gene construct) encoding the above-mentioned chimeric antigen receptor and a polynucleotide (or gene) encoding a peptide or fragment thereof capable of inhibiting the transforming growth factor beta signaling pathway are included in one vector. construct), or a vector containing a polynucleotide (or gene construct) encoding the chimeric antigen receptor, and a peptide or fragment thereof capable of inhibiting the transforming growth factor beta signaling pathway. Encoding may include both types of vectors containing polynucleotides (or gene constructs).
본 발명의 다른 구현 예에 따르면, 본 발명에서 제공하는 유전적으로 조작된 세포를 유효 성분으로 포함하는, 세포 치료제에 관한 것이다.According to another embodiment of the present invention, it relates to a cell therapeutic agent comprising the genetically engineered cells provided by the present invention as an active ingredient.
본 발명의 또 다른 구현 예에 따르면, 본 발명에서 제공하는 유전적으로 조작된 세포를 유효 성분으로 포함하는 암의 예방, 개선 또는 치료용 약학 조성물에 관한 것이다.According to another embodiment of the present invention, the present invention relates to a pharmaceutical composition for preventing, improving or treating cancer containing the genetically engineered cells provided by the present invention as an active ingredient.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
실시예Example
[참고예 1] 본 발명의 펩타이드의 TGF-β-신호전달 억제 효과 확인[Reference Example 1] Confirmation of the TGF-β-signaling inhibition effect of the peptide of the present invention
1. TGF-β-신호전달 억제 펩타이드 설계1. Design of TGF-β-signaling inhibitory peptide
TGF-β 신호전달을 억제하는 펩타이드를 개발하기 위해, TGF-β 수용체를 표적으로 하는 서열번호 28로 표시되는 아미노산 서열로 이루어지는 펩타이드(P6)를 설계하여 제작하였다. In order to develop a peptide that inhibits TGF-β signaling, a peptide (P6) consisting of the amino acid sequence shown in SEQ ID NO: 28 targeting the TGF-β receptor was designed and produced.
2. 펩타이드의 TGF-β-신호전달 억제효과 확인2. Confirmation of the TGF-β-signaling inhibition effect of the peptide
이렇게 제작한 펩타이드의 TGF-β 신호전달 억제효과를 확인하기 위하여, 하기와 같은 실험을 수행하였다. 이전의 실험에서 Bxpc3 및 Aspc1 세포주의 경우 TGF-β를 처리하고 12~24시간에 pSmad2/3 발현 수준이 높은 것을 확인하였는 바, 본 실험에서는 TGF-β를 처리하고 24시간 후의 pSmad2/3 발현 수준을 확인하여 TGF-β 신호전달 경로의 억제 여부를 확인하였다. 구체적으로, 췌장암 세포주인 Aspc1, Bxpc3 및 Panc1 세포주를 6웰 플레이트에 1X106/웰의 농도로 시딩하여 하룻동안 배양한 뒤, 다음날, TGF-β 또는 TGF-β+후보 펩타이드(P6)를 24시간동안 처리하고(대조군: 15% ACN - 후보 펩타이드 용매, DMSO - P144 펩타이드 용매, P144: TSLDASIWAMMQNA(서열번호 37)) pSmad2/3와 전체 Smad2/3의 발현 수준을 웨스턴 블랏팅 방법으로 분석하였다. In order to confirm the inhibitory effect of the peptide produced in this way on TGF-β signaling, the following experiment was performed. In previous experiments, it was confirmed that in the case of Bxpc3 and Aspc1 cell lines, the expression level of pSmad2/3 was high 12 to 24 hours after treatment with TGF-β. In this experiment, the expression level of pSmad2/3 24 hours after treatment with TGF-β It was confirmed whether the TGF-β signaling pathway was inhibited. Specifically, pancreatic cancer cell lines Aspc1, Bxpc3, and Panc1 cell lines were seeded in a 6-well plate at a concentration of 1 (Control group: 15% ACN - candidate peptide solvent, DMSO - P144 peptide solvent, P144: TSLDASIWAMMQNA (SEQ ID NO: 37)) and the expression levels of pSmad2/3 and total Smad2/3 were analyzed by Western blotting method.
그 결과, 도 13 내지 15에서 보는 바와 같이, Aspc1, Bxpc3 및 Panc1 세포주 모두에서 본 발명에 따르는 펩타이드가 pSmad2/3의 발현 수준을 현저히 억제하는 것을 확인할 수 있었다. 상기 결과를 토대로, 본 발명에서 제작한 TGF-β 신호전달 억제 펩타이드(P6)는 실제로 TGF-β 신호전달을 효과적으로 억제할 수 있음을 알 수 있다.As a result, as shown in Figures 13 to 15, it was confirmed that the peptide according to the present invention significantly suppressed the expression level of pSmad2/3 in all Aspc1, Bxpc3, and Panc1 cell lines. Based on the above results, it can be seen that the TGF-β signaling inhibitory peptide (P6) prepared in the present invention can actually effectively inhibit TGF-β signaling.
[실시예 1] 키메라 항원 수용체 및 TGF-β 신호전달 경로 억제 유전자가 포함된 벡터의 제작[Example 1] Construction of a vector containing a chimeric antigen receptor and a TGF-β signaling pathway inhibitory gene
도 1과 같이, 키메라 항원 수용체 발현 카세트로는 CD8 신호 펩타이드, 메소텔린 결합 도메인(경쇄 가변 영역; 링커; 및 중쇄 가변 영역), CD8 힌지, CD8 막관통 도메인, 4-1BB, CD3 zeta 신호전달 도메인을 암호화하는 폴리뉴클레오티드와, P2A, 그린 형광 단백질(GFP), P2A, IL-2 신호 펩타이드 및 TGF-β 신호전달 경로 억제 펩타이드(P6) 각각을 암호화하는 폴리뉴클레오티드를 렌티 바이러스 전달 벡터에 삽입하였다. 이때 상기 재조합 유전자는 SFFV 단일 프로모터의 제어 하에 배치하였다. 실험에 사용된 SFFV 프로모터의 염기 서열과, 그 외의 각 유전자의 염기 서열은 하기 표 1에 나타내었고, 이들 염기 서열에 의해 암호화되는 아미노산 서열은 하기 표 2에 나타내었다. As shown in Figure 1, the chimeric antigen receptor expression cassette includes CD8 signal peptide, mesothelin binding domain (light chain variable region; linker; and heavy chain variable region), CD8 hinge, CD8 transmembrane domain, 4-1BB, and CD3 zeta signaling domain. A polynucleotide encoding , and polynucleotides encoding P2A, green fluorescent protein (GFP), P2A, IL-2 signal peptide, and TGF-β signaling pathway inhibitory peptide (P6), respectively, were inserted into the lentiviral delivery vector. At this time, the recombinant gene was placed under the control of the SFFV single promoter. The base sequence of the SFFV promoter used in the experiment and the base sequences of each other gene are shown in Table 1, and the amino acid sequences encoded by these base sequences are shown in Table 2 below.
유전자 이름gene name 염기 서열base sequence 서열번호 sequence number
SFFV 프로모터SFFV promoter GTAACGCCATTTTGCAAGGCATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACATGAAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGGCCAAGAACAGATGGTCACCGCAGTTTCGGCCCCGGCCCGAGGCCAAGAACAGATGGTCCCCAGATATGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCTGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATAAAAGAGCTCACAACCCCTCACTCGGCGCGCCAGTCCTCCGACAGACTGAGTCGCCCGGGGTAACGCCATTTTGCAAGGCATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACATGAAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGGCCAAGAACAGATGGTCACCGCAGTTTCGGCCCCGGCCCGAGGCCAAGAACAGATGGTCCCCAGATATGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCCA AGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCTGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATAAAAGAGCTCACAACCCCTCACTCGGCGCGCCAGTCCTCCGACAGACTGAGTCGCCCGGG 3030
CD8 signal peptideCD8 signal peptide ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCG CTGGCCTTGCTGCTCCACGCCGCCAGGCCGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG 1515
VLV.L. GATATCGAACTCACTCAGTCCCCAGCAATCATGTCCGCTTCACCGGGAGAAAAGGTGACCATGACTTGCTCGGCCTCCTCGTCCGTGTCATACATGCACTGGTACCAACAAAAATCGGGGACCTCCCCTAAGAGATGGATCTACGATACCAGCAAACTGGCTTCAGGCGTGCCGGGACGCTTCTCGGGTTCGGGGAGCGGAAATTCGTATTCGTTGACCATTTCGTCCGTGGAAGCCGAGGACGACGCAACTTATTACTGCCAACAGTGGTCAGGCTACCCGCTCACTTTCGGAGCCGGCACTAAGCTGGAGATC GATATCGAACTCACTCAGTCCCCAGCAATCATGTCCGCTTCACCGGGAGAAAAGGTGACCATGACTTGCTCGGCCTCCTCGTCCGTGTCATACATGCACTGGTACCAACAAAAATCGGGGACCTCCCCTAAGAGATGGATCTACGATACCAGCAAACTGGCTTCAGGCGTGCCGGGACGCTTCTCGGGTTCGGGGAGCGGAAATTCGTATTCGTTGACCATTTCGTCCGTGGAAGCCGAGGACGACGCAACTTATT ACTGCCAACAGTGGTCAGGCTACCCGCTCACTTTCGGAGCCGGCACTAAGCTGGAGATC 99
LinkerLinker GGAGGCGGAGGGAGCGGAGGAGGAGGCAGCGGAGGTGGAGGGTCGGGAGGGCGGAGGGAGCGGAGGAGGAGGCAGCGGAGGTGGAGGGTCG 1313
VH VH CAAGTCCAGCTCCAGCAGTCGGGCCCAGAGTTGGAGAAGCCTGGGGCGAGCGTGAAGATCTCATGCAAAGCCTCAGGCTACTCCTTTACTGGATACACGATGAATTGGGTGAAACAGTCGCATGGAAAGTCACTGGAATGGATCGGTCTGATTACGCCCTACAACGGCGCCTCCAGCTACAACCAGAAGTTCAGGGGAAAGGCGACCCTTACTGTCGACAAGTCGTCAAGCACCGCCTACATGGACCTCCTGTCCCTGACCTCCGAAGATAGCGCGGTCTACTTTTGTGCACGCGGAGGTTACGATGGACGGGGATTCGACTACTGGG GCCAGGGAACCACTGTCACCGTGTCGAGCCAAGTCCAGCTCCAGCAGTCGGGCCCAGAGTTGGAGAAGCCTGGGGCGAGCGTGAAGATCTCATGCAAAGCCTCAGGCTACTCCTTTACTGGATACACGATGAATTGGGTGAAACAGTCGCATGGAAAGTCACTGGAATGGATCGGTCTGATTACGCCCTACAACGGCGCCTCCAGCTACAACCAGAAGTTCAGGGGAAAGGCGACCCTTACTGTCGACAAGTCGTCAAGCACCGCCTACATGGACCTCCTGTCCCTGAC CTCCGAAGATAGCGCGGTCTACTTTTGTGCACGCGGAGGTTACGAATGGACGGGGATTCGACTACTGGGGCCAGGGAACCACTGTCACGTGTCGAGC 1111
CD8 hingeCD8 hinge ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT 1717
CD8 transmembraneCD8 transmembrane ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC 1919
4-1BB4-1BB AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAAGAAGGAGGATGTGAACTG 2323
CD3 zetaCD3 zeta AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGG CAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC 2121
P2AP2A GCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT 3636
GFPGFP ATGCCCGCCATGGAGATCGAGTGCCGCATCACCGGCACCCTGAACGGCGTGGAGTTCGAGCTGGTGGGCGGCGGAGAGGGCACCCCCAAGCAGGGCCGCATGACCAACAAGATGAAGAGCACCAAAGGCGCCCTGACCTTCAGCCCCTACCTGCTGAGCCACGTGATGGGCTACGGCTTCTACCACTTCGGCACCTACCCCAGCGGCTACGAGAACCCCTTCCTGCACGCCATCAACAACGGCGGCTACACCAACACCCGCATCGAGAAGTACGAGGACGGCGGCGTGCTGCACGTGAGCTTCAGCTACCGCTACGAGGCCGGCCGCGTGATCGGCGACTTCAAGGTGGTGGGCACCGGCTTCCCCGAGGACAGCGTGATCTTCACCGACAAGATCATCCGCAGCAACGCCACCGTGGAGCACCTGCACCCCATGGGCGATAACGTGCTGGTGGGCAGCTTCGCCCGCACCTTCAGCCTGCGCGACGGCGGCTACTACAGCTTCGTGGTGGACAGCCACATGCACTTCAAGAGCGCCATCCACCCCAGCATCCTGCAGAACGGGGGCCCCATGTTCGCCTTCCGCCGCGTGGAGGAGCTGCACAGCAACACCGAGCTGGGCATCGTGGAGTACCAGCACGCCTTCAAGACCCCCATTGCCTTCGCCATGCCCGCCATGGAGATCGAGTGCCGCATCACCGGCACCCTGAACGGCGTGGAGTTCGAGCTGGTGGGCGGCGGAGAGGGCACCCCCAAGCAGGGCCGCATGACCAACAAGATGAAGAGCACCAAAGGCGCCCTGACCTTCAGCCCCTACCTGCTGAGCCACGTGATGGGCTACGGCTTCTACCACTTCGGCACCTACCCCAGCGGCTACGAGAACCCCTTCCTGCACGCCATCAACAACGGCGGCTACACCAA CACCCGCATCGAGAAGTACGAGGACGGCGGCGTGCTGCACGTGAGCTTCAGCTACCGCTACGAGGCCGGCCGCGTGATCGGCGACTTCAAGGTGTGTGGGCACCGGCTTCCCCGAGGACAGCGTGATCTTCACCGACAAGATCATCCGCAGCAACGCCACCGTGGAGCACCTGCACCCCATGGGCGATAACGTGCTGGTGGGCAGCTTCGCCCGCACCTTCAGCCTGCGCGACGGCGGCTACTACAGCT TCGTGGTGGACAGCCACATGCACTTCAAGAGCGCCATCCACCCCAGCATCCTGCAGAACGGGGGCCCCATGTTCGCCTTCCGCCGCGTGGAGGAGCTGCACAGCAACACCGAGCTGGGCATCGTGGAGTACCAGCACGCCTTCAAGACCCCCATTGCCTTCGCC 3232
P2AP2A GCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT 3636
IL-2 신호 서열IL-2 signal sequence ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGTATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAAGTCTTGCACTTGTCACAAACAGT 3434
TGF-β 신호전달 경로 억제 유전자TGF-β signaling pathway inhibition gene TTCTGCCTGGGCCCCTGCCCCTACATCTGGAGCCTGGACACCGTGCTGAGCCTGTTCTGCCTGGGCCCCTGCCCCTACATCTGGAGCCTGGACACCGTGCTGAGCCTG 2929
펩타이드 이름peptide name 아미노산 서열amino acid sequence 서열번호 sequence number
CD8 signal peptideCD8 signal peptide MALPVTALLLPLALLLHAARPMALPVTALLLPLALLLHAARP 1414
VLV.L. DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSGYPLTFGAGTKLEIDIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSGYPLTFGAGTKLEI 88
LinkerLinker GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 1212
VHVH QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSQVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATTLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSS 1010
CD8 hingeCD8 hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD 1616
CD8 transmembraneCD8 transmembrane IYIWAPLAGTCGVLLLSLVITLYCIYIWAPLAGTCGVLLLSLVITLYC 1818
4-1BB4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 2222
CD3 zetaCD3 zeta RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 2020
P2A P2A ATNFSLLKQAGDVEENPGPATNFSLLKQAGDVEENPGP 3535
GFPGFP MPAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNKMKSTKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYRYEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDGGYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFAMPAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNKMKSTKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYRYEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDGGYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVE YQHAFKTPIAFA 3131
P2A P2A ATNFSLLKQAGDVEENPGPATNFSLLKQAGDVEENPGP 3535
IL-2 신호 서열IL-2 signal sequence MYRMQLLSCIALSLALVTNSMYRMQLLSCIALSLALVTNS 3333
TGF-β 신호전달 경로 억제 유전자TGF-β signaling pathway inhibition gene FCLGPCPYIWSLDTVLSLFCLGPCPYIWSLDTVLSL 2828
[실시예 2] 렌티바이러스의 NK 세포로의 형질 도입[Example 2] Transduction of lentivirus into NK cells
1. 세포의 준비 및 배양1. Preparation and culture of cells
상기 키메라 항원 수용체 및 TGF-β 신호전달 경로 억제 펩타이드가 발현되도록 유전적으로 조작할 면역 이펙터 세포로는 자연살해 세포를 이용하였다. 이러한 자연살해 세포주 (natural killer cell)로는 NK-92 세포주를 ATCC로부터 구매하였다. 또한, 렌티바이러스를 형질 도입할 세포주인 293T 세포주 역시 ATCC로부터 구매하였다. NK-92 세포주와 이하 기재할 렌티바이러스가 형질 도입된 NK-92 세포주는 모두 100 μg/mL의 스트렙토마이신(streptomycin), 100 units/mL의 페니실린(penicillin), 20%의 소 태아 혈청(fetal bovine serum; FBS), 10%의 MEM 비타민 용액, 1X의 2-머캅토에탄올(2-mercaptoethanol) 및 400 IU의 rhIL-2가 포함된 배지에서 배양하였고, 293T 세포주는 100 μg/mL의 스트렙토마이신(streptomycin), 100 units/mL의 페니실린(penicillin), 10%의 소 태아 혈청(fetal bovine serum; FBS)이 포함된 배지에서 배양하였으며, 모든 세포의 배양은 37 ℃에서 CO2가 5% (95% 공기)로 유지되는 환경에서 배양하였다.Natural killer cells were used as immune effector cells to be genetically manipulated to express the chimeric antigen receptor and TGF-β signaling pathway inhibitory peptide. As a natural killer cell line, the NK-92 cell line was purchased from ATCC. In addition, the 293T cell line, which is the cell line to be used to transduce the lentivirus, was also purchased from ATCC. Both the NK-92 cell line and the lentivirus-transduced NK-92 cell line described below were treated with 100 μg/mL of streptomycin, 100 units/mL of penicillin, and 20% fetal bovine serum. serum; FBS), 10% MEM vitamin solution, 1 streptomycin), 100 units/mL of penicillin, and 10% fetal bovine serum (FBS). All cells were cultured at 37°C with 5% CO2 (95% CO2). Cultured in an environment maintained with air.
2. 렌티바이러스의 형질 도입2. Lentiviral transduction
바이러스의 물리적 역가(Physical titer) (One-Wash Lentivirus Titer Kit이용) 기준 5 x 108 TU/mL 이상의 렌티바이러스를 얻기 위하여, 1.2 x 106개의 293T 세포를 60 mm 배양 디쉬에 24시간 배양한 뒤, 상기 실시예 1에서 제조한 렌티바이러스 전달 벡터 (UCI-VD9 or UCI-VD35) 5.5 μg, 패키징 벡터(UCI-VD6) 3.7 μg, 및 외피 벡터(envelope vector)(UCI-VD11) 1.8 μg와 함께 리포펙타민(lipofectamine) 3000 형질 감염 시약으로 형질 감염시켰다. 형질 감염 후 293T 세포에서 제작된 렌티바이러스는 세포 밖으로 분출되어 나와 바이러스 입자(virus particle) 형태로 세포 배양액에 존재하였다. 이에 형질주입 48 또는 72시간 뒤, 배양 플레이트에서 세포 배양액만을 걷어내 lenti-X 농축 시약(concentrator)으로 100배 농축해 렌티바이러스 입자를 펠렛 형태로 얻었다. 이후, 24-웰 플레이트에 1 x 106 개의 NK-92 세포주와 농축된 바이러스 상층액을 MOI 100이 되도록 넣어준 후 형질 도입 유도를 위해 8 μg/mL의 폴리브렌(polybrene)을 함께 넣어주었다. 형질 도입이 성공적으로 일어난 세포주의 선정을 위하여 세포 선별기(cell sorter) (SH800S)를 이용하여 GFP 가 발현되는 세포들을 선별하였다. In order to obtain a lentivirus of 5 , 5.5 μg of the lentiviral transfer vector (UCI-VD9 or UCI-VD35) prepared in Example 1, 3.7 μg of the packaging vector (UCI-VD6), and 1.8 μg of the envelope vector (UCI-VD11). Transfection was performed with lipofectamine 3000 transfection reagent. After transfection, the lentivirus produced in 293T cells was ejected out of the cell and existed in the cell culture medium in the form of virus particles. Accordingly, 48 or 72 hours after transfection, only the cell culture fluid was removed from the culture plate and concentrated 100 times with lenti-X concentration reagent (concentrator) to obtain lentivirus particles in the form of a pellet. Afterwards, 1 To select cell lines in which transduction was successful, cells expressing GFP were selected using a cell sorter (SH800S).
[실험예 1] 항암 활성 평가[Experimental Example 1] Evaluation of anticancer activity
1. 암 세포의 준비 및 배양1. Preparation and culture of cancer cells
췌장암 세포주 (AsPC-1, Capan-2), 유방암 세포주 (HCC-1806, MDA-MB-231), 위암 세포주 (NCI-N87, AGS), 폐암 세포주 (NCI-H292), 대장암 세포주 (SNU-1544, SW-620) 그리고 간암 세포주 (SK-HEP-1, Hep-G2)를 한국 세포주 은행 (KCLB)로부터 구매 후 루시퍼라제 리포터(luciferase reporter) 유전자가 도입된 세포주를 사용하였다. AsPC-1_luc_puro, Capan-2_luc_puro, HCC-1806_luc_puro, MDA-MB-231_luc_puro, NCI-N87_luc_puro, AGS_luc_puro, NCI-H292_luc_puro, SNU-1544_luc_puro 그리고 SW-620_luc_puro 세포주는 RPMI-1640 배지를 이용하여 배양하였고 SK-HEP-1_luc_puro, Hep-G2_luc_puro는 high-glucose Dulbecco's modified Eagle medium 배지를 이용하여 배양하였다. 루시퍼라제 리포터 유전자가 도입된 암 세포주의 배양에 사용되는 모든 배지는 100 μg/mL의 스트렙토마이신(streptomycin), 100 units/mL의 페니실린(penicillin), 10%의 소 태아 혈청(fetal bovine serum; FBS) 및 4 μg/mL의 퓨로마이신(puromycin)이 포함되었다. 모든 세포는 37 ℃에서 CO2가 5% (95% 공기)로 유지되는 환경에서 배양하였다.Pancreatic cancer cell lines (AsPC-1, Capan-2), breast cancer cell lines (HCC-1806, MDA-MB-231), stomach cancer cell lines (NCI-N87, AGS), lung cancer cell lines (NCI-H292), colon cancer cell lines (SNU- 1544, SW-620) and liver cancer cell lines (SK-HEP-1, Hep-G2) were purchased from the Korean Cell Line Bank (KCLB), and cell lines into which a luciferase reporter gene was introduced were used. AsPC-1_luc_puro, Capan-2_luc_puro, HCC-1806_luc_puro, MDA-MB-231_luc_puro, NCI-N87_luc_puro, AGS_luc_puro, NCI-H292_luc_puro, SNU-1544_luc_puro and SW-620_luc_puro cell lines were cultured using RPMI-1640 medium. EP- 1_luc_puro and Hep-G2_luc_puro were cultured using high-glucose Dulbecco's modified Eagle medium. All media used for culturing cancer cell lines into which a luciferase reporter gene was introduced contained 100 μg/mL of streptomycin, 100 units/mL of penicillin, and 10% fetal bovine serum (FBS). ) and 4 μg/mL of puromycin were included. All cells were cultured at 37°C in an environment where CO 2 was maintained at 5% (95% air).
2. 항암 활성 평가2. Evaluation of anticancer activity
루시퍼라제 세포주 기반 인-비트로(in vitro) 세포사멸 시험을 수행하기 위하여, 5 x 103개의 각 암 세포주를 100 μL의 암 세포 배양배지와 섞어 화이트(white) 96-웰 플레이트에 분주 후 37 ℃, 5% CO2 인큐베이터(incubator)에서 48시간 동안 배양하였다. 48 시간 배양 뒤, 각 웰의 사용 배지를 제거한 후 상기 실시예 2에서 구축한 유전적 조작된 NK-92 세포(MSLN CAR+p6 peptide) 1 x 104개를 200 μL의 NK-92 세포 배양 배지와 섞어 암 세포가 배양되고 있는 웰 플레이트에 분주하고, 37 ℃, 5% CO2 인큐베이터에서 추가 배양하였다. 추가 배양 8시간 또는 24시간 뒤, 모든 웰을 DPBS로 세척후 Nano-GloTM Luciferase Assay System을 이용하여 multiplate-reader (Spectra MAX iD3) 장비로 루시퍼라제 활성(luciferase activity)을 측정하였다. 실험 결과는 도 2 내지 12에 나타내었다. 단, 본 실험에서 대조군으로는 GFP 발현 mock 벡터가 형질 감염된 NK-92 세포를 이용하였다. To perform a luciferase cell line-based in vitro apoptosis test, 5 , cultured in a 5% CO 2 incubator for 48 hours. After 48 hours of incubation, the used medium from each well was removed, and then 1 x 10 4 of the genetically engineered NK-92 cells (MSLN CAR+p6 peptide) constructed in Example 2 were added to 200 μL of NK-92 cell culture medium. It was mixed and dispensed into a well plate where cancer cells were cultured, and further cultured in an incubator at 37°C and 5% CO 2 . After 8 or 24 hours of additional culture, all wells were washed with DPBS and luciferase activity was measured using a multiplate-reader (Spectra MAX iD3) using the Nano-Glo TM Luciferase Assay System. The experimental results are shown in Figures 2 to 12. However, in this experiment, NK-92 cells transfected with a GFP expression mock vector were used as a control group.
도 2 및 3에서 보는 바와 같이, 유방암 세포에 본 발명에 따른 키메라 항원 수용체 및 TGF-β 신호전달 경로를 억제하는 펩타이드를 발현하도록 유전적으로 조작된 NK 세포를 처리한 경우, 무처리군이나 mock 벡터로 형질 주입된 NK 세포를 처리한 경우에 비하여 유방암 세포의 사멸율이 매우 높은 것을 확인할 수 있었다. As shown in Figures 2 and 3, when breast cancer cells are treated with NK cells genetically engineered to express a chimeric antigen receptor according to the present invention and a peptide that inhibits the TGF-β signaling pathway, the untreated group or the mock vector It was confirmed that the death rate of breast cancer cells was very high compared to the case of treatment with NK cells transfected with .
도 4 및 5에서 보는 바와 같이, 위암 세포에 mock 벡터가 형질 도입된 NK 세포를 처리한 경우는 무처리 군과 비교하여 위암 세포의 사멸 효과가 미미한 반면, 본 발명에 따른 키메라 항원 수용체 및 TGF-β 신호전달 경로를 억제하는 펩타이드를 발현하도록 유전적으로 조작된 NK 세포주를 처리한 경우, 루시퍼라제 발현 위암 세포의 사멸 효과가 현저히 뛰어난 것을 확인할 수 있었다. As shown in Figures 4 and 5, when gastric cancer cells are treated with NK cells transduced with a mock vector, the killing effect on gastric cancer cells is minimal compared to the untreated group, whereas the chimeric antigen receptor and TGF- according to the present invention When NK cell lines genetically engineered to express a peptide that inhibits the β signaling pathway were treated, it was confirmed that the killing effect on luciferase-expressing gastric cancer cells was significantly superior.
도 6에서 보는 바와 같이, 폐암 세포에 본 발명에 따른 키메라 항원 수용체 및 TGF-β 신호전달 경로를 억제하는 펩타이드를 발현하도록 유전적으로 조작된 NK 세포주를 처리한 경우, 폐암 세포의 사멸율이 높게 관찰되었다. As shown in Figure 6, when lung cancer cells were treated with a NK cell line genetically engineered to express a chimeric antigen receptor according to the present invention and a peptide that inhibits the TGF-β signaling pathway, a high death rate of lung cancer cells was observed. It has been done.
도 7 및 8에서 보는 바와 같이, 대장암 세포에서 본 발명에 따른 키메라 항원 수용체 및 TGF-β 신호전달 경로를 억제하는 펩타이드를 발현하도록 유전적으로 조작된 NK 세포주를 처리한 경우, 루시퍼라제 발현 대장암 세포의 수가 현저히 감소한 것을 확인할 수 있었다. As shown in Figures 7 and 8, when colon cancer cells are treated with NK cell lines genetically engineered to express the chimeric antigen receptor according to the present invention and a peptide that inhibits the TGF-β signaling pathway, luciferase-expressing colon cancer It was confirmed that the number of cells was significantly reduced.
도 9 및 10에서 보는 바와 같이, 간암 세포에 본 발명에 따른 키메라 항원 수용체 및 TGF-β 신호전달 경로를 억제하는 펩타이드를 발현하도록 유전적으로 조작된 NK 세포주를 처리한 경우, 무처리군이나 mock 벡터로 형질 주입된 NK 세포를 처리한 경우에 비하여 루시퍼라제 발현 간암 세포의 수가 현저히 감소한 것을 확인할 수 있었다.As shown in Figures 9 and 10, when liver cancer cells were treated with an NK cell line genetically engineered to express a chimeric antigen receptor according to the present invention and a peptide that inhibits the TGF-β signaling pathway, the untreated group or the mock vector It was confirmed that the number of luciferase-expressing liver cancer cells was significantly reduced compared to the case of treating NK cells transfected with .
도 11 및 12에서 보는 바와 같이, 췌장암 세포에 mock 벡터가 형질 도입된 NK 세포를 처리한 경우는 무처리 군과 비교하여 위암 세포의 사멸 효과가 미미한 반면, 본 발명에 따른 키메라 항원 수용체 및 TGF-β 신호전달 경로를 억제하는 펩타이드를 발현하도록 유전적으로 조작된 NK 세포주를 처리한 경우, 췌장암 세포의 사멸율이 매우 높은 것을 확인할 수 있었다.As shown in Figures 11 and 12, when pancreatic cancer cells are treated with NK cells transduced with a mock vector, the killing effect on gastric cancer cells is minimal compared to the untreated group, whereas the chimeric antigen receptor and TGF- according to the present invention When NK cell lines genetically engineered to express a peptide that inhibits the β signaling pathway were treated, it was confirmed that the death rate of pancreatic cancer cells was very high.
상기 결과를 종합할 때, 본 발명에 따라 유전적으로 조작된 NK 세포주는 상기 세포에서 발현되는 키메라 항원 수용체로 인하여 메소텔린 발현하는 암 세포를 표적화할 수 있고, 상기 세포에서 추가로 발현되는 펩타이드에 의하여 TGF-β 신호전달 경로 억제를 통해 세포의 미세종양 내부로의 침윤능이 향상되고, 암 세포에 대한 사멸 효과 역시 현저히 증가된 것을 알 수 있다. Considering the above results, the NK cell line genetically engineered according to the present invention can target cancer cells expressing mesothelin due to the chimeric antigen receptor expressed in the cells, and can target cancer cells expressing mesothelin by the peptide additionally expressed in the cells. It can be seen that through inhibition of the TGF-β signaling pathway, the ability of cells to invade into microtumors is improved, and the killing effect on cancer cells is also significantly increased.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.
본 발명은 면역 요법에 의해 암 등의 질환에 대한 치료 효과가 증대되도록, 유전적으로 조작시킨 면역 이펙터 세포와, 이를 이용한 세포치료제에 관한 것이다.The present invention relates to immune effector cells genetically engineered to increase the therapeutic effect of diseases such as cancer by immunotherapy, and cell therapy products using the same.

Claims (24)

1) 메소텔린(Mesothelin; MSLN)을 표적화하는 키메라 항원 수용체(chimeric antigen receptor; CAR); 및 2) 전환성장인자 베타(Transforming growth factor-β, TGF-β) 신호전달 경로를 억제할 수 있는 펩타이드 또는 이의 단편을 발현하도록 유전적으로 조작된 세포.1) Chimeric antigen receptor (CAR) targeting mesothelin (MSLN); and 2) cells genetically engineered to express a peptide or fragment thereof capable of inhibiting the transforming growth factor-β (TGF-β) signaling pathway.
제1항에 있어서,According to paragraph 1,
상기 키메라 항원 수용체는 메소텔린 결합 도메인을 포함하는, 세포.The chimeric antigen receptor comprises a mesothelin binding domain.
제2항에 있어서,According to paragraph 2,
상기 메소텔린 결합 도메인은 서열번호 2로 표시되는 아미노산 서열로 이루어지는 경쇄 CDR1; 서열번호 3으로 표시되는 아미노산 서열로 이루어지는 경쇄 CDR2; 및 서열번호 4로 표시되는 아미노산 서열로 이루어지는 경쇄 CDR3를 포함하는 경쇄 가변 영역; 및The mesothelin binding domain includes a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 2; Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 4; and
서열번호 5로 표시되는 아미노산 서열로 이루어지는 중쇄 CDR1; 서열번호 6으로 표시되는 아미노산 서열로 이루어지는 중쇄 CDR2; 및 서열번호 7로 표시되는 아미노산 서열로 이루어지는 중쇄 CDR3를 포함하는 중쇄 가변 영역;을 포함하는, 세포. Heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 5; Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 6; and a heavy chain variable region comprising a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 7. A cell comprising a.
제2항에 있어서,According to paragraph 2,
상기 메소텔린 결합 도메인은 서열번호 8로 표시되는 항체의 경쇄 가변 영역(VL); 및 서열번호 10으로 표시되는 항체의 중쇄 가변 영역(VH);을 포함하는, 세포. The mesothelin binding domain includes the light chain variable region (VL) of the antibody represented by SEQ ID NO: 8; And a heavy chain variable region (VH) of the antibody represented by SEQ ID NO: 10. A cell comprising a.
제4항에 있어서,According to clause 4,
상기 메소텔린 결합 도메인은 서열번호 12로 표시되는 아미노산 서열을 포함하는 링커를 더 포함하는, 세포. The cell wherein the mesothelin binding domain further includes a linker containing the amino acid sequence represented by SEQ ID NO: 12.
제2항에 있어서,According to paragraph 2,
상기 키메라 항원 수용체는 힌지; 막관통 도메인; 및 신호전달 도메인을 포함하는, 세포. The chimeric antigen receptor includes a hinge; transmembrane domain; and a cell comprising a signaling domain.
제6항에 있어서,According to clause 6,
상기 힌지는 CD8, CD28, 4-1BB, OX40, CD3 제타(ζ) 사슬의 전부 또는 일부, T 세포 수용체 α 또는 β 사슬, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154 또는 이들의 조합을 포함하는, 세포. The hinge includes CD8, CD28, 4-1BB, OX40, all or part of the CD3 zeta (ζ) chain, T cell receptor α or β chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, A cell comprising CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or a combination thereof.
제6항에 있어서,According to clause 6,
상기 힌지는 서열번호 16으로 표시되는 아미노산 서열을 포함하는, 세포. A cell wherein the hinge includes the amino acid sequence represented by SEQ ID NO: 16.
제6항에 있어서,According to clause 6,
상기 막관통 도메인은 T 세포 수용체, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 또는 CD154의 막관통 도메인인, 세포. The transmembrane domain is a transmembrane domain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154. .
제6항에 있어서,According to clause 6,
상기 막관통 도메인은 서열번호 18로 표시되는 아미노산 서열을 포함하는, 세포. A cell, wherein the transmembrane domain includes the amino acid sequence represented by SEQ ID NO: 18.
제6항에 있어서,According to clause 6,
상기 신호전달 도메인은 TCR zeta, FcR gamma, FcR beta, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278, Fc.epsilon.RI, DAP10, DAP12 또는 CD66d의 ITAM의 1차 신호전달 도메인을 포함하는, 세포. The signaling domain is the ITAM of TCR zeta, FcR gamma, FcR beta, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278, Fc.epsilon.RI, DAP10, DAP12 or CD66d. A cell comprising a primary signaling domain.
제11항에 있어서,According to clause 11,
상기 신호전달 도메인은 서열번호 20으로 표시되는 아미노산 서열을 포함하는 CD3 zeta를 포함하는 것인, 세포. The cell wherein the signaling domain includes CD3 zeta comprising the amino acid sequence represented by SEQ ID NO: 20.
제12항에 있어서,According to clause 12,
상기 신호전달 도메인은 MHC 클래스 I 분자, TNF 수용체 단백질, 면역글로불린-유사 단백질, 사이토카인 수용체, 인테그린, 신호전달성 림프구 활성화 분자 (SLAM 단백질), 활성화 NK 세포 수용체, BTLA, Toll 리간드 수용체, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a 및 CD83에 특이적으로 결합하는 리간드로 이루어진 군에서 선택되는 하나 이상의 공동-자극성 신호전달 도메인을 더 포함하는, 세포. The signaling domain includes MHC class I molecules, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR , BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a and CD83 A cell further comprising one or more co-stimulatory signaling domains selected from the group consisting of a specifically binding ligand.
제13항에 있어서,According to clause 13,
상기 신호전달 도메인은 서열번호 22로 표시되는 아미노산 서열을 포함하는 공동-자극성 신호전달 도메인을 더 포함하는, 세포. The cell, wherein the signaling domain further comprises a co-stimulatory signaling domain comprising the amino acid sequence represented by SEQ ID NO: 22.
제2항에 있어서,According to paragraph 2,
키메라 항원 수용체는 신호 펩타이드를 더 포함하는, 세포. Chimeric antigen receptors further contain a signal peptide.
제1항에 있어서,According to paragraph 1,
상기 키메라 항원 수용체는 서열번호 24 또는 25로 표시되는 아미노산 서열을 포함하는, 세포. The chimeric antigen receptor is a cell comprising the amino acid sequence represented by SEQ ID NO: 24 or 25.
제1항에 있어서,According to paragraph 1,
상기 전환성장인자 베타 신호전달 경로를 억제할 수 있는 펩타이드는 서열번호 28로 표시되는 아미노산 서열을 포함하는, 세포. The peptide capable of inhibiting the transforming growth factor beta signaling pathway includes the amino acid sequence represented by SEQ ID NO: 28.
제1항에 있어서,According to paragraph 1,
상기 세포는 면역 이펙터 세포인, 세포. A cell, wherein the cell is an immune effector cell.
1) 메소텔린을 표적화하는 키메라 항원 수용체를 암호화하는 폴리뉴클레오티드; 및 1) a polynucleotide encoding a chimeric antigen receptor targeting mesothelin; and
2) 전환성장인자 베타(Transforming growth factor-β, TGF-β) 신호전달 경로를 억제할 수 있는 펩타이드 또는 이의 단편을 암호화하는 폴리뉴클레오티드;를 포함하는 벡터.2) A vector containing a polynucleotide encoding a peptide or fragment thereof capable of inhibiting the transforming growth factor-β (TGF-β) signaling pathway.
제19항에 있어서,According to clause 19,
상기 키메라 항원 수용체는 서열번호 8로 표시되는 항체의 경쇄 가변 영역(VL), 서열번호 12로 표시되는 아미노산 서열을 포함하는 링커 및 서열번호 10으로 표시되는 항체의 중쇄 가변 영역(VH)을 포함하는 메소텔린 결합 도메인; 서열번호 16으로 표시되는 아미노산 서열을 포함하는 힌지; 서열번호 18로 표시되는 아미노산 서열을 포함하는 막관통 도메인; 서열번호 22로 표시되는 아미노산 서열을 포함하는 공동-자극성 신호전달 도메인; 및 서열번호 20으로 표시되는 아미노산 서열을 포함하는 신호전달 도메인;을 포함하는, 벡터.The chimeric antigen receptor includes a light chain variable region (VL) of the antibody shown in SEQ ID NO: 8, a linker containing the amino acid sequence shown in SEQ ID NO: 12, and a heavy chain variable region (VH) of the antibody shown in SEQ ID NO: 10. mesothelin binding domain; A hinge comprising the amino acid sequence represented by SEQ ID NO: 16; A transmembrane domain comprising the amino acid sequence represented by SEQ ID NO: 18; A co-stimulatory signaling domain comprising the amino acid sequence represented by SEQ ID NO: 22; And a signaling domain comprising the amino acid sequence represented by SEQ ID NO: 20. A vector comprising a.
제19항에 있어서,According to clause 19,
상기 키메라 항원 수용체를 암호화하는 폴리뉴클레오티드는 서열번호 26 또는 27로 표시되는 염기 서열을 포함하는, 벡터. A vector wherein the polynucleotide encoding the chimeric antigen receptor includes the base sequence represented by SEQ ID NO: 26 or 27.
제1항 내지 제18항 중 어느 한 항의 유전적으로 조작된 세포를 유효 성분으로 포함하는 세포 치료제. A cell therapeutic agent comprising the genetically engineered cell of any one of claims 1 to 18 as an active ingredient.
제1항 내지 제18항 중 어느 한 항의 유전적으로 조작된 세포를 유효 성분으로 포함하는 암의 예방 또는 치료용 약학 조성물. A pharmaceutical composition for preventing or treating cancer comprising the genetically engineered cells of any one of claims 1 to 18 as an active ingredient.
개체에게 제23항의 약학 조성물을 투여하는 단계를 포함하는, 암의 예방 또는 치료 방법. A method for preventing or treating cancer, comprising administering the pharmaceutical composition of claim 23 to an individual.
PCT/KR2023/018102 2022-11-11 2023-11-10 Genetically engineered cells and use thereof WO2024101962A1 (en)

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KR20210108290A (en) * 2020-02-25 2021-09-02 (주)녹십자셀 Mesothelin-specific chimeric antigen receptor and Uses Thereof
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