WO2024101942A1 - Cellules génétiquement modifiées et leur utilisation - Google Patents

Cellules génétiquement modifiées et leur utilisation Download PDF

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WO2024101942A1
WO2024101942A1 PCT/KR2023/018043 KR2023018043W WO2024101942A1 WO 2024101942 A1 WO2024101942 A1 WO 2024101942A1 KR 2023018043 W KR2023018043 W KR 2023018043W WO 2024101942 A1 WO2024101942 A1 WO 2024101942A1
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present
cells
peptide
amino acid
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정수영
서민구
안준엽
오민석
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주식회사 유씨아이테라퓨틱스
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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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 benefit from increased TGF- ⁇ expression and autocrine TGF- ⁇ signaling through inactivation of epithelial antiproliferative responses and 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.
  • 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 cells genetically engineered to express a peptide capable of inhibiting the transforming growth factor- ⁇ (TGF- ⁇ ) signaling pathway.
  • TGF- ⁇ transforming growth factor- ⁇
  • Another object of the present invention is a chimeric antigen receptor (CAR) that targets mesothelin as a cancer cell antigen along with a peptide capable of inhibiting the transforming growth factor beta (TGF- ⁇ ) signaling pathway. ) to provide cells that have been genetically engineered to express.
  • CAR chimeric antigen receptor
  • Another object of the present invention is to provide a method for producing genetically engineered cells as described above.
  • Another object of the present invention is to provide a cell therapeutic agent or pharmaceutical composition for various uses containing the genetically engineered cells.
  • the present invention provides a peptide capable of inhibiting the transforming growth factor- ⁇ (TGF- ⁇ ) signaling pathway, and in addition, a chimeric antigen receptor specific for a cancer cell antigen.
  • TGF- ⁇ transforming growth factor- ⁇
  • CAR chimeric antigen receptor
  • TGF- ⁇ transforming growth factor beta
  • “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.”
  • TGF- ⁇ Transforming growth factor beta
  • 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: 1, preferably consisting of the amino acid sequence represented by SEQ ID NO: 1.
  • sequence encoding the peptide may include the nucleotide sequence represented by SEQ ID NO: 2, preferably consisting of the nucleotide sequence represented by SEQ ID NO: 2.
  • 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 in addition to expressing a peptide or fragment thereof capable of inhibiting the transforming growth factor beta (TGF- ⁇ ) signaling pathway, additionally express a chimeric antigen receptor specific for a cancer cell antigen. It may be genetically engineered to express CAR).
  • TGF- ⁇ transforming growth factor beta
  • 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.
  • the chimeric antigen receptor described above in the present invention may be a chimeric antigen receptor targeting mesothelin (MSLN).
  • MSLN mesothelin
  • 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: 3, 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 signal transduction 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 high affinity.
  • 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 may be synthesized (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.
  • 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 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: 4; Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5; and a light chain variable region including a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6.
  • the mesothelin (MSLN) binding domain includes a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 7; Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 8; and a heavy chain variable region including a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 9.
  • 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 includes a variable region of at least about 120 to 130 amino acids, and the carboxy-terminal portion includes a constant region. do. 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 comprises a variable region of about 100 to about 110 or more amino acids, and the carboxy-terminal portion comprises 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 the light chain variable region (VL) of an antibody represented by the amino acid sequence of SEQ ID NO: 10, or the antibody encoded by the nucleotide sequence represented by SEQ ID NO: 11.
  • VL light chain variable region
  • the mesothelin (MSLN) binding domain may include the heavy chain variable region (VH) of the antibody represented by the amino acid sequence of SEQ ID NO: 12, or the antibody encoded by the nucleotide sequence represented by SEQ ID NO: 13.
  • VH heavy chain variable region
  • 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: 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 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: 12. 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: 13.
  • 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 (Gly4Ser)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: 14, or may be a linker encoded by the nucleotide sequence represented by SEQ ID NO: 15.
  • 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: 4, 5, and 6, respectively.
  • a light chain variable region (VL) comprising light chain CDR1, CDR2, and CDR3;
  • a linker comprising the amino acid sequence represented by SEQ ID NO: 14 or encoded by the nucleotide sequence represented by SEQ ID NO: 15;
  • a heavy chain variable region (VH) comprising heavy chain CDR1, CDR2, and CDR3 represented by the amino acid sequences of SEQ ID NOs: 7, 8, and 9, 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 represented by SEQ ID NO: 10, or Variable light chain region (VL) of an antibody encoded by the nucleotide sequence shown in SEQ ID NO: 11; A linker comprising the amino acid sequence represented by SEQ ID NO: 14 or encoded by the nucleotide sequence represented by SEQ ID NO: 15; and a variable heavy chain region (VH) of an antibody comprising the amino acid sequence represented by SEQ ID NO: 12 or encoded by the nucleotide sequence represented by SEQ ID NO: 13, 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” is also called a "leader sequence” and represents an amino acid sequence that can be linked to the amino terminus of the protein shown in the present invention.
  • a signal peptide/leader sequence typically directs protein positioning.
  • the signal peptide/leader sequence used in the present invention preferably promotes secretion of the protein from the cell in which it is produced.
  • the signal peptide/leader sequence is often cleaved from the remainder of the protein, often referred to as the mature protein, upon secretion from the cell.
  • a signal peptide/leader sequence is linked to the N terminus of the protein.
  • 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.
  • the signal peptide generally ranges from 15 to 30 amino acids.
  • Non-limiting examples of the signal peptide in the present invention include IL-2 signal peptide, CD8 signal peptide (21 amino acids), CD33 signal peptide (17 amino acids), CD4 signal peptide (25 amino acids), and IL-2R (CD25 signal peptide).
  • signal peptide 21 amino acids
  • 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 It may be a signal peptide, an IgVK signal peptide, or an Ig VH signal peptide.
  • the signal peptide may be a CD8 signal peptide, and preferably may include an amino acid sequence represented by SEQ ID NO: 16, or a signal peptide encoded by the nucleotide sequence represented by SEQ ID NO: 17. 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: 16, , 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: 17.
  • 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: 18, or a hinge encoded by the nucleotide sequence represented by SEQ ID NO: 19. 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: 18, 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 19.
  • 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: 20, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 21.
  • 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: 20. 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: 21.
  • 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: 22, or is encoded by the nucleotide sequence represented by SEQ ID NO: 23. 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: 24, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 25.
  • 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: 22, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 23, and the 4-1BB is the amino acid sequence represented by SEQ ID NO: 24. It may include or be encoded by the nucleotide sequence shown in SEQ ID NO: 25.
  • 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: 26, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 28, but is not limited thereto.
  • the chimeric antigen receptor may include the amino acid sequence represented by SEQ ID NO: 27, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 29, but is not limited thereto.
  • methods for genetically manipulating the cells, particularly immune effector cells, to express the above-described peptides or fragments thereof or additional chimeric antigen receptors include biological methods such as vectors, specific receptors, or cell fusion methods; Through physical methods such as microinjection, electroporation, gene gun or ultrasonic gene introduction, or chemical methods such as calcium phosphate co-precipitation, liposome method, lipofection, DEAE dextran method, or alkali metal method. This may be done by introducing a gene encoding a peptide or a fragment thereof.
  • a vector containing a gene encoding the peptide or a fragment thereof and a gene further encoding the chimeric antigen receptor is transfected into the immune effector cell to encode the peptide or a fragment thereof into the cell. foreign genes can be introduced.
  • 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 described above.
  • 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 vector may include a polynucleotide encoding a peptide or a fragment thereof capable of inhibiting the transforming growth factor beta (TGF- ⁇ ) signaling pathway, and preferably has the base sequence represented by SEQ ID NO: 2. It may contain polynucleotides containing.
  • TGF- ⁇ transforming growth factor beta
  • 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 further include a polynucleotide encoding the chimeric antigen receptor targeting mesothelin.
  • a polynucleotide encoding the chimeric antigen receptor targeting mesothelin.
  • the vector may further include a polynucleotide encoding a mesothelin-targeting chimeric antigen receptor containing the base sequence represented by SEQ ID NO: 28 or 29, but is not limited thereto.
  • 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 the chimeric antigen receptor are added to one vector.
  • a vector containing a polynucleotide (or gene construct) encoding a peptide or fragment thereof capable of inhibiting the transforming growth factor beta signaling pathway, and the chimeric antigen It may contain both types of vectors containing polynucleotides (or gene constructs) encoding receptors.
  • the polynucleotide encoding the peptide or fragment thereof, or the polynucleotide encoding the chimeric antigen receptor may encode one or more additional polypeptides, such as one or more markers and/or one or more effector molecules. May contain polynucleotides.
  • 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, non-functional, full-length form of the cell surface polypeptide that cannot or will not transmit and/or will be internalized to transmit signals or signals normally transmitted by the cell surface polypeptide. 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. See US Pat. No.
  • 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 (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.
  • 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 (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), preferably the marker is green fluorescent protein (GFP) represented by SEQ ID NO: 31, or encoded by the nucleotide sequence represented by SEQ ID NO: 32. It may be green fluorescent protein (GFP), but is not limited thereto.
  • 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 polynucleotide (or gene construct) may include a base sequence encoding the peptide or a fragment thereof and a base sequence encoding one or more additional polypeptides.
  • the base sequence encoding the peptide or fragment thereof and the base sequence encoding one or more additional polypeptides, such as marker and/or effector molecules, are included in one polynucleotide (or gene construct). may be included.
  • the base sequence encoding the peptide or fragment thereof and the base sequence encoding one or more additional polypeptides, such as marker and/or effector molecules are each different polynucleotides (or gene constructs) ) can be included.
  • two separate polynucleotides (or gene constructs) are provided, and each can be individually delivered or introduced into a cell for intracellular expression.
  • one vector may contain a base sequence encoding the peptide or a fragment thereof, and another vector may contain a base sequence encoding one or more additional polypeptides.
  • the base sequence encoding the peptide or its fragment and the base sequence encoding one or more additional polypeptides may be included in one vector, but in this case, the nucleic acid encoding the peptide or its fragment is one Nucleic acids encoding one or more additional polypeptides may be operably linked to a promoter.
  • the nucleic acid encoding the peptide or fragment thereof may be upstream of the nucleic acid encoding one or more additional polypeptides, or the nucleic acid encoding the peptide fragment may be downstream of the nucleic acid encoding one or more additional polypeptides.
  • the base sequence encoding the peptide or its fragment and the base sequence encoding one or more additional polypeptides may be included in one vector, but in this case, the nucleic acid encoding the peptide or its fragment is one Nucleic acids encoding these additional polypeptides may be operably linked to two or more different promoters.
  • the nucleic acid encoding the peptide or fragment thereof may be upstream of the nucleic acid encoding one or more additional polypeptides, or the nucleic acid encoding the peptide fragment may be downstream of the nucleic acid encoding one or more additional polypeptides.
  • 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.
  • a polynucleotide (or gene construct) encoding the chimeric antigen receptor may all be included in one vector, 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.
  • one vector contains a polynucleotide (or gene construct) encoding the chimeric antigen receptor, and another vector is capable of inhibiting the transforming growth factor beta signaling pathway. It may include a polynucleotide (or gene construct) encoding a peptide or a fragment thereof, but is not limited thereto.
  • one vector contains a polynucleotide (or gene construct) encoding the chimeric antigen receptor, and another vector is capable of inhibiting the transforming growth factor beta signaling pathway. It includes a polynucleotide (or gene construct) encoding a peptide or a fragment thereof, and at least one of the two vectors may additionally contain a polynucleotide (or gene construct) encoding a marker. It is not limited.
  • 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.
  • 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 cell therapeutic agent comprising the genetically engineered immune effector 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.
  • a pharmaceutical composition for preventing, improving, or treating cancer comprising the genetically engineered immune effector 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, for example, stomach cancer, liver cancer, and 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, melanoma, or lung cancer.
  • 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 sodium salt or potassium salt, anhydrous or hydrated, and may be citric acid or in 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 through the like, and preferably intravenously.
  • the composition or active ingredient of the present invention may 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 provided by the present invention express the peptide or fragment thereof according to the present invention, and in addition to the basic anti-tumor activity of the immune effector cell, the peptide or fragment thereof expressed in the cell It has the advantage of excellent anti-cancer activity due to its improved ability to invade into microtumors and its cytotoxic effect on tumor cells due to the inhibitory effect of the transforming growth factor beta (TGF- ⁇ ) signaling pathway.
  • TGF- ⁇ transforming growth factor beta
  • the immune effector cells are genetically engineered to express, in addition to the peptide according to the invention, a chimeric antigen receptor targeting mesothelin (MSLN), thereby increasing the targeting ability against tumor cells and synergizing anti-tumor activity. Effect can be given.
  • MSLN chimeric antigen receptor targeting mesothelin
  • Figure 1 is a graph showing the results of treating HEK293_SBE_luc cells with the peptide of the present invention together with rhTGF- ⁇ in Experimental Example 1 of the present invention, and then confirming the effect of suppressing TGF- ⁇ signaling by confirming the change in the degree of luminescence through a luciferase assay. It is expressed as
  • Figure 2 shows a vector map containing the base sequence represented by SEQ ID NO: 2 encoding a peptide (P6S1) capable of inhibiting the TGF- ⁇ signaling pathway according to an example of the present invention.
  • Figure 3 shows a polynucleotide (SS CAR) encoding a chimeric antigen receptor targeting mesothelin and a peptide capable of inhibiting the transforming growth factor beta (TGF- ⁇ ) signaling pathway according to an example of the present invention. It shows a vector map containing all coding polynucleotides (P6S1).
  • Figure 4 shows that in Experimental Example 2 of the present invention, the HCC-1806 breast 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 TGF- ⁇ signaling pathway according to the present invention. The results of comparing luciferase activity are shown graphically.
  • Figure 5 shows the NCI-N87 gastric cancer cell line into which a luciferase reporter gene was introduced in Experimental Example 2 of the present invention, after treatment with an NK cell line genetically engineered to express a peptide that inhibits the TGF- ⁇ signaling pathway according to the present invention.
  • the results of comparing luciferase activity are shown graphically.
  • Figure 6 shows the NCI-H292 lung cancer cell line into which a luciferase reporter gene was introduced in Experimental Example 2 of the present invention, after treatment with an NK cell line genetically engineered to express a peptide that inhibits the TGF- ⁇ signaling pathway according to the present invention.
  • the results of comparing luciferase activity are shown graphically.
  • Figure 7 shows that in Experimental Example 2 of the present invention, the SW-620 colon cancer cell line into which a luciferase reporter gene was introduced was treated with an NK cell line genetically engineered to express a peptide that inhibits the TGF- ⁇ signaling pathway according to the present invention. The results of comparing luciferase activity are shown in a graph.
  • Figure 8 shows that in Experimental Example 2 of the present invention, the SK-HEP-1 liver 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 TGF- ⁇ signaling pathway according to the present invention. The results of comparing luciferase activity are shown in a graph.
  • Figure 9 shows the Capan-2 pancreatic cancer cell line into which a luciferase reporter gene was introduced in Experimental Example 2 of the present invention, after treatment with an NK cell line genetically engineered to express a peptide that inhibits the TGF- ⁇ signaling pathway according to the present invention.
  • the results of comparing luciferase activity are shown graphically.
  • Figure 10 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 HCC-1806 breast cancer cell line into which a luciferase reporter gene was introduced in Experimental Example 3 of the present invention. The results of comparing luciferase activity after processing are shown in a graph.
  • Figure 11 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-N87 gastric cancer cell line into which a luciferase reporter gene was introduced in Experimental Example 3 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 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 3 of the present invention. The results of comparing luciferase activity after processing are shown in a graph.
  • Figure 13 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 3 of the present invention.
  • the results of comparing luciferase activity after treating cell lines are shown graphically.
  • Figure 14 shows a SK-HEP-1 liver cancer cell line into which a 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 15 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 Capan-2 pancreatic cancer cell line into which a luciferase reporter gene was introduced in Experimental Example 3 of the present invention. The results of comparing luciferase activity after processing are shown in a graph.
  • TGF- ⁇ transforming growth factor beta
  • the peptide may include the amino acid sequence represented by SEQ ID NO: 1, preferably consisting of the amino acid sequence represented by SEQ ID NO: 1.
  • sequence encoding the peptide may include the nucleotide sequence represented by SEQ ID NO: 2, preferably consisting of the nucleotide sequence represented by SEQ ID NO: 2.
  • the cells in addition to expressing a peptide or fragment thereof capable of inhibiting the transforming growth factor beta (TGF- ⁇ ) signaling pathway, additionally express a chimeric antigen receptor specific for a cancer cell antigen. It may be genetically engineered to express CAR).
  • TGF- ⁇ transforming growth factor beta
  • the chimeric antigen receptor described above may be a chimeric antigen receptor targeting mesothelin (MSLN).
  • MSLN mesothelin
  • 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 signal transduction domains. .
  • 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: 4; Light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5; and a light chain variable region including a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6.
  • the mesothelin (MSLN) binding domain includes a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 7; Heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 8; and a heavy chain variable region including a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 9.
  • the mesothelin (MSLN) binding domain may include the light chain variable region (VL) of an antibody represented by the amino acid sequence of SEQ ID NO: 10, or the antibody encoded by the nucleotide sequence represented by SEQ ID NO: 11.
  • VL light chain variable region
  • the mesothelin (MSLN) binding domain may include the heavy chain variable region (VH) of the antibody represented by the amino acid sequence of SEQ ID NO: 12, or the antibody encoded by the nucleotide sequence represented by SEQ ID NO: 13.
  • VH heavy chain variable region
  • the linker sequence may include the amino acids glycine and serine.
  • the linker sequence comprises a set of glycine and serine repeats, such as (Gly4Ser)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: 14, or may be a linker encoded by the nucleotide sequence represented by SEQ ID NO: 15.
  • 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: 4, 5, and 6, respectively.
  • a light chain variable region (VL) comprising light chain CDR1, CDR2, and CDR3;
  • a linker comprising the amino acid sequence represented by SEQ ID NO: 14 or encoded by the nucleotide sequence represented by SEQ ID NO: 15;
  • a heavy chain variable region (VH) comprising heavy chain CDR1, CDR2, and CDR3 represented by the amino acid sequences of SEQ ID NOs: 7, 8, and 9, 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 represented by SEQ ID NO: 10, or Variable light chain region (VL) of an antibody encoded by the nucleotide sequence shown in SEQ ID NO: 11; A linker comprising the amino acid sequence represented by SEQ ID NO: 14 or encoded by the nucleotide sequence represented by SEQ ID NO: 15; and a variable heavy chain region (VH) of an antibody comprising the amino acid sequence represented by SEQ ID NO: 12 or encoded by the nucleotide sequence represented by SEQ ID NO: 13, 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: 16, or a signal peptide encoded by the nucleotide sequence represented by SEQ ID NO: 17. It may be, but is not limited to this.
  • 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: 18, or a hinge encoded by the nucleotide sequence represented by SEQ ID NO: 19. However, it is not limited to this.
  • 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: 20, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 21. , but is not limited to this.
  • 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: 22, or is encoded by the nucleotide sequence represented by SEQ ID NO: 23.
  • SEQ ID NO: 22 the amino acid sequence represented by SEQ ID NO: 22
  • SEQ ID NO: 23 the nucleotide sequence represented by SEQ ID NO: 23.
  • 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: 24, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 25, but is not limited thereto.
  • 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: 22, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 23, and the 4-1BB is the amino acid sequence represented by SEQ ID NO: 24. It may include or be encoded by the nucleotide sequence shown in SEQ ID NO: 25, but is not limited thereto.
  • the chimeric antigen receptor may include the amino acid sequence represented by SEQ ID NO: 26, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 28, but is not limited thereto.
  • the chimeric antigen receptor may include the amino acid sequence represented by SEQ ID NO: 27, or may be encoded by the nucleotide sequence represented by SEQ ID NO: 29, but is not limited thereto.
  • a method of genetically manipulating a peptide or fragment thereof capable of inhibiting the transforming growth factor beta (TGF- ⁇ ) signaling pathway described above in the cell or an additional chimeric antigen receptor is expressed in the cell. It can be transformed with a vector containing a polynucleotide encoding a peptide or a fragment thereof, and, if necessary, a polynucleotide further encoding the chimeric antigen receptor described above.
  • TGF- ⁇ transforming growth factor beta
  • 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 the chimeric antigen receptor are added to one vector.
  • a vector containing a polynucleotide (or gene construct) encoding a peptide or fragment thereof capable of inhibiting the transforming growth factor beta signaling pathway, and the chimeric antigen receptor may contain both types of vectors containing coding polynucleotides (or gene constructs).
  • the cells may be immune effector cells.
  • the immune effector cells may be natural killer cells (NK cells), T cells, or B cells, but are not limited thereto.
  • a cell therapeutic agent comprising the genetically engineered immune effector cells provided by the present invention as an active ingredient.
  • a pharmaceutical composition for preventing, improving, or treating cancer comprising the genetically engineered immune effector cells provided by the present invention as an active ingredient.
  • TGF- ⁇ 1 and TGF- ⁇ receptor complex To design a peptide that inhibits TGF- ⁇ signaling, the structure of the TGF- ⁇ 1 and TGF- ⁇ receptor complex (PDB ID: 3KFD) was searched on the PROTEIN DATA BANK site (https:/www.rcsb.org/). The structure of the TGF- ⁇ 1 and TGF- ⁇ receptor complex was observed using Pymol software to confirm the amino acid sequence involved in binding. In order to increase the solubility of the peptide while maintaining the inhibitory function of the peptide that binds to the TGF- ⁇ receptor instead of TGF- ⁇ 1, the peptide sequence was changed while maintaining the amino acid sequence involved in binding.
  • a basic amino acid sequence was added and changed to suppress non-specific binding between peptides due to the formation of disulfide bonds and TGF- ⁇ by changing the sequence from cysteine to serine.
  • Candidate amino acid sequences derived from TGF- ⁇ that target the receptor are listed in Table 1 below. However, in order to confirm the difference in the effect of inhibiting TGF- ⁇ signaling according to the isoelectric point of the peptide in the experiment below, the pI value was 3.12. A control sequence was produced.
  • Example 1 To confirm whether the candidate peptides produced in Example 1 could inhibit TGF- ⁇ signaling, the following experiment was performed.
  • HEK293_SBE_luc cells were inoculated into 100 ⁇ l of MEM- EBBS medium at a number of 5 24 hours after inoculation, the rhTGF- ⁇ solution was diluted to a final concentration of 100 pg/ml using MEM-EBBS medium and then added to each well, and the candidate peptide prepared in Example 1 was injected into MEM-EBBS medium. After diluting with EBBS medium to a final concentration of 10 ⁇ M or 50 ⁇ M, 50 ⁇ l was added to each well. The final volume for each well was 200 ⁇ l/well. After 24 hours, 100 ⁇ l of luciferase assay solution was added to each well, and the degree of luminescence was confirmed using a multiplate reader, and the results are shown in Figure 1.
  • the candidate peptide of P6S1 consisting of the amino acid sequence represented by SEQ ID NO: 1 according to the present invention binds to the TGF- ⁇ receptor and effectively inhibits TGF- ⁇ signaling, while the pI value is 3.12, which is acidic. It was confirmed that the phosphorus control sequence had a low binding affinity to the TGF- ⁇ receptor and had a minimal inhibitory effect on TGF- ⁇ signaling.
  • polynucleotides encoding each of green fluorescent protein (GFP), P2A, IL-2 signal peptide, and TGF- ⁇ signaling pathway inhibitory peptide (P6S1) were inserted into the lentiviral delivery vector.
  • 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 2, and the amino acid sequences encoded by these base sequences are shown in Table 3 below.
  • Natural killer cells were used as immune effector cells to be genetically manipulated to express the 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 transduced with the lentivirus produced in Example 1, 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 , together with 5.5 ⁇ g of lentiviral transfer vector (UCI-VD9 or UCI-VD57) prepared in step 1 above, 3.7 ⁇ g of packaging vector (UCI-VD6), and 1.8 ⁇ g of 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
  • Capan-2_luc_puro, HCC-1806_luc_puro, NCI-N87_luc_puro, NCI-H292_luc_puro, and SW-620_luc_puro cell lines were cultured using RPMI-1640 medium, and SK-HEP-1_luc_puro was cultured using high-glucose Dulbecco's modified Eagle medium. did. All media used for culturing cancer cell lines into which a luciferase reporter gene has been introduced contain 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).
  • FBS fetal bovine serum
  • NK-92 cells P6S1-NK
  • luciferase activity was measured using a multiplate-reader (Spectra MAX iD3) using the Nano-Glo TM Luciferase Assay System, and the results are shown in Figures 4 to 9. shown in In addition, the change (%) in luciferase activity in each treated group compared to the untreated group was calculated, and the results are shown in Tables 4 to 9 below.
  • a control NK-92 cells transfected with a mock vector containing the GFP gene were used.
  • NCI-H292 division Luciferase activity change (%)
  • Control group (Mock-NK) 87.142 NK cell line expressing TGF- ⁇ signaling pathway inhibitory peptide (P6S1-NK) 70.769
  • Pancreatic cancer (Capan-2) division Luciferase activity change (%) Non-treated group 100 Control group (Mock-NK) 83.284 NK cell line expressing TGF- ⁇ signaling pathway inhibitory peptide (P6S1-NK) 68.372
  • breast cancer, stomach cancer, lung cancer, colon cancer, liver cancer, and pancreatic cancer cells are genetically engineered to express a peptide that inhibits the TGF- ⁇ signaling pathway according to the present invention.
  • NK cells P6S1-NK
  • the death rate of these cancer cells was very high, and when NK-92 cells (Mock-NK) transfected with a mock vector containing the GFP gene were treated, the death rate of cancer cells was much higher than when treated with NK-92 cells (Mock-NK) transfected with a mock vector containing the GFP gene. It was confirmed that the death rate was significantly high.
  • NK-92 cells Mock-NK
  • a mock vector containing the GFP gene luciferase activity was seen to increase.
  • the TGF- ⁇ signaling pathway was activated.
  • NK cells P6S1-NK
  • immune effector cells especially natural killer cells, genetically engineered according to the present invention, in addition to the basic anti-tumor activity of immune effector cells, transforming growth factor beta (TGF- ⁇ ) Due to the inhibitory effect of the signaling pathway, the ability to invade into microtumors is improved and the cytotoxic effect on tumor cells is also improved, which has the advantage of significantly increased anticancer activity compared to immune effector cells without genetic manipulation.
  • TGF- ⁇ transforming growth factor beta Due to the inhibitory effect of the signaling pathway, the ability to invade into microtumors is improved and the cytotoxic effect on tumor cells is also improved, which has the advantage of significantly increased anticancer activity compared to immune effector cells without genetic manipulation.
  • 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 10, and the amino acid sequences encoded by these base sequences are shown in Table 11 below.
  • NK-92 cells were prepared and cultured in the same manner as in Example 2, Section 2, and then a vector containing a mesothelin-targeting chimeric antigen receptor constructed in Section 1 above and a gene capable of suppressing the TGF- ⁇ signaling pathway. was introduced.
  • Pancreatic cancer (Capan-2) division Luciferase activity change (%) Non-treated group 100
  • Control group (SS CAR-NK) 81.017 NK cell line expressing mesothelin CAR and TGF- ⁇ signaling pathway inhibitory peptide (SS CAR_P6S1-NK) 60.700
  • the mesothelin target chimeric antigen receptor and TGF- ⁇ signaling pathway according to the present invention are inhibited in all breast cancer, stomach cancer, lung cancer, colon cancer, liver cancer, and pancreatic cancer cells.
  • NK cells genetically engineered to express a peptide
  • SS CAR-NK mesothelin target chimeric antigen receptor
  • 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 the TGF- ⁇ signaling pathway by the peptide additionally expressed in the cells. It can be seen that through inhibition, 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 that have been genetically engineered to increase the therapeutic effect for diseases such as cancer by immunotherapy, and their application as a cell therapeutic agent.

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Abstract

La présente invention concerne des cellules génétiquement modifiées pour exprimer un peptide ou un fragment de celui-ci pouvant inhiber une voie de signalisation du facteur de croissance transformant-β (TGF-β).
PCT/KR2023/018043 2022-11-11 2023-11-10 Cellules génétiquement modifiées et leur utilisation WO2024101942A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060233708A1 (en) * 1997-06-19 2006-10-19 St. Louis University Peptide antagonists of TGF-beta family members and therapeutic uses thereof
KR100983182B1 (ko) * 2009-08-14 2010-09-20 (주)엔솔테크 신규 펩타이드 및 그 용도
KR20130012095A (ko) * 2011-06-13 2013-02-01 연세대학교 산학협력단 ＴＧＦ－β１ 발현을 억제하는 ｓｈＲＮＡ
US20150031624A1 (en) * 2012-03-23 2015-01-29 Office of Health and Human Services, NIH Anti-mesothelin chimeric antigen receptors
KR20210090503A (ko) * 2020-01-10 2021-07-20 주식회사 뉴클릭스바이오 TGF-β 수용체에 특이적으로 결합하는 항체 및 이의 이용방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060233708A1 (en) * 1997-06-19 2006-10-19 St. Louis University Peptide antagonists of TGF-beta family members and therapeutic uses thereof
KR100983182B1 (ko) * 2009-08-14 2010-09-20 (주)엔솔테크 신규 펩타이드 및 그 용도
KR20130012095A (ko) * 2011-06-13 2013-02-01 연세대학교 산학협력단 ＴＧＦ－β１ 발현을 억제하는 ｓｈＲＮＡ
US20150031624A1 (en) * 2012-03-23 2015-01-29 Office of Health and Human Services, NIH Anti-mesothelin chimeric antigen receptors
KR20210090503A (ko) * 2020-01-10 2021-07-20 주식회사 뉴클릭스바이오 TGF-β 수용체에 특이적으로 결합하는 항체 및 이의 이용방법

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CASTELLETTI LAURA, YEO DANNEL, VAN ZANDWIJK NICO, RASKO JOHN E. J.: "Anti-Mesothelin CAR T cell therapy for malignant mesothelioma", BIOMARKER RESEARCH, vol. 9, no. 1, 1 December 2021 (2021-12-01), XP093036960, DOI: 10.1186/s40364-021-00264-1 *
LI YUXIANG; XIAO FENGJUN; ZHANG AIMEI; ZHANG DAN; NIE WENBO; XU TIANXIN; HAN BING; SETH PREM; WANG HUA; YANG YUEFENG; WANG LISHENG: "Oncolytic adenovirus targeting TGF-β enhances anti-tumor responses of mesothelin-targeted chimeric antigen receptor T cell therapy against breast cancer", CELLULAR IMMUNOLOGY, ACADEMIC PRESS, SAN DIEGO, CA., US, vol. 348, 11 January 2020 (2020-01-11), US , XP085988481, ISSN: 0008-8749, DOI: 10.1016/j.cellimm.2020.104041 *

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