Lipid NANO-particle having a ligand targeted to CD7 and chimeric antigen receptor and use thereof

Abstract

The present invention provides a lipid nanoparticle comprising the following (a) to (c), and having a ligand on its surface that targets an immunocyte: (a) a nucleic acid encoding a chimeric antigen receptor or an exogenous T cell receptor; (b) a cationic lipid; and (c) a non-cationic lipid, wherein the ligand is a polypeptide comprising a binding domain for CD7. The present invention also provides an in vivo therapeutic approach using the lipid nanoparticle for disease such as cancer and the like.

Description

LIPID NANO-PARTICLE HAVING A LIGAND TARGETED TO CD7 AND CHIMERIC ANTIGEN RECEPTOR AND USE THEREOF

[0001] The present invention relates to lipid nanoparticles having a ligand on its surface that targets an immunocyte, a method for expressing a chimeric antigen receptor or an exogenous T cell receptor in an immunocyte of interest by using the lipid nanoparticles, a pharmaceutical use thereof, and the like.

[0002] The research and development of cancer immunotherapy using CAR-T cells or TCR-T cells introduced with a gene of chimeric antigen receptor (CAR) or T-cell receptor (TCR) derived from cancer antigen-specific killer T cell is progressing rapidly. Current CAR-T cell therapy, such as Kymriah (trade name) and Yescarta (trade name), which were approved in the U.S., generally includes producing CAR-T cells by transfecting T cells collected from patients with CAR genes ex vivo using viral vectors such as lentiviral vector, and administering the CAR-T cells to the patients. However, this method has the problem that the production cost becomes high due to the cost of cell culture and preparation of viral vectors. To solve these problems, a technique using lipid nanoparticles has been reported as a technique for selectively introducing CAR or exogenous TCR into immune cells such as T cell and the like in vivo (Patent Literature 1).

[0003] Incidentally, many cell surface molecules highly expressed in cancer cells have been known as targets of cancer cells recognized by CAR. Among them, Glypican-3 (GPC3), CD19, and the like have been studied as some of the promising targets. CAR technology targeting GPC3 has been reported in, for example, Patent Literatures 2 to 4. In addition, CAR technology targeting CD19 has been described in, for example, Patent Literatures 5 and 6.

[0004] However, no reports showing sufficiently effective methods of treating cancer by introducing CAR or exogenous TCR binding to a tumor antigen such as GPC3 and CD19 into immune cells in vivo using lipid nanoparticles have been identified.

[0005] Patent Literature 1

[0006] WO 2019 / 131770Patent Literature 2

[0007] CN 116731196Patent Literature 3

[0008] WO 2014 / 180306Patent Literature 4

[0009] WO 2018 / 131586Patent Literature 5

[0010] WO 2021 / 092290Patent Literature 6

[0011] WO 2020 / 091869

[0012] The purpose of the present invention is to provide a novel in vivo cancer therapy based on a new concept.

[0013] The present inventors have conducted intensive studies in an attempt to achieve the above-mentioned purpose and found for the first time that lipid nanoparticles having the specific configuration of the present invention induce both CAR-T cell-mediated killing and CAR-NK cell-mediated killing in vivo. The present inventors have found that CAR-T cell-mediated killing and CAR-NK cell-mediated killing are also performed via not only direct cytotoxicity of CAR-target positive cancer cells but also cytokine / chemokine production to recruit immune infiltration. Furthermore, the present inventors have found that the lipid nanoparticles of the present invention induce endogenous T or NK cell activation / recruitment. To be specific, they have found that the lipid nanoparticles of the present invention induce NCR activation after initial CAR signaling and the activated NK cells can kill cancer cell in a target independent manner, and that the above-mentioned cytokine / chemokine production by CAR-T cells and CAR-NK cells activates endogenous T and NK cells and promote both MHC I dependent and independent killings. The present invention has been completed based on the above-mentioned findings.

[0014] Accordingly, the present invention provides the following.[1] A lipid nanoparticle having a ligand on its surface that targets an immunocyte, wherein the ligand is a polypeptide comprising a binding domain for CD7.[1A] The lipid nanoparticle according to [1], wherein the lipid nanoparticle comprising the following (a) to (c):(a) a nucleic acid encoding a chimeric antigen receptor or an exogenous T cell receptor;(b) a cationic lipid; and(c) a non-cationic lipid.[2] The lipid nanoparticle according to [1A], wherein the chimeric antigen receptor or exogenous T cell receptor is a polypeptide comprising a binding domain for glypican-3 (GPC3) or a polypeptide comprising a binding domain for CD19.[3] The lipid nanoparticle according to [1A] or [2], wherein the nucleic acid is encoding a chimeric antigen receptor, and the chimeric antigen receptor comprises the following (i) to (iv):(i) an extracellular antigen binding domain;(ii) a transmembrane domain;(iii) a co-stimulatory domain; and(iv) an intracellular signal transduction domain,wherein the extracellular antigen binding domain is a binding domain for GPC3 or a binding domain for CD19.[4] The lipid nanoparticle according to [3], wherein the extracellular antigen binding domain is a binding domain comprisingheavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1,heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2,heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3,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, andlight chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6,a binding domain comprisingheavy 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,heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 9,light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 10,light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 11, andlight chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 12, ora binding domain comprisingheavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13,heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14,heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15,light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16,light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17, andlight chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 18.[5] The lipid nanoparticle according to [3] or [4], wherein the extracellular antigen binding domain isa binding domain comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20,a binding domain comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22, ora binding domain comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 24.[6] The lipid nanoparticle according to any one of [3] to [5], wherein the extracellular antigen binding domain is scFv.[7] The lipid nanoparticle according to [6], wherein the scFv isa polypeptide comprising the amino acid sequence shown in SEQ ID NO: 25,a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 26, ora polypeptide comprising the amino acid sequence shown in SEQ ID NO: 27.[8] The lipid nanoparticle according to any one of [3] to [7], wherein the transmembrane domain is a T cell receptor (TCR) alpha chain, a TCR beta chain, a TCR zeta chain, CD28, a CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.[9] The lipid nanoparticle according to any one of [3] to [8], wherein the co-stimulatory domain is OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1(CD11a / CD18), ICOS(CD278), DAP10, DAP12, 4-1BB(CD137), or a combination thereof.

[0010] The lipid nanoparticle according to any one of [3] to [9], wherein the intracellular signal transduction domain is CD3 zeta, a CD3 zeta variant, 4-1BB, CD28, CD134, CD137, Lck, DAP10, ICOS, or a combination thereof.

[0011] The lipid nanoparticle according to any one of [3] to

[0010] , wherein the chimeric antigen receptor further comprises a hinge domain linking the extracellular antigen binding domain and the transmembrane domain.

[0012] The lipid nanoparticle according to any one of [3] to

[0011] , wherein the combination of the transmembrane domain, the co-stimulatory domain, and the intracellular signal transduction domain is CD28-CD28-CD3 zeta or a CD28-CD28-CD3 zeta variant.

[0013] The lipid nanoparticle according to any one of [1A] to

[0012] , wherein the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 28, the amino acid sequence shown in SEQ ID NO: 29, or the amino acid sequence shown in SEQ ID NO: 30.

[0014] The lipid nanoparticle according to any one of [1] to

[0013] , wherein the ligand comprises a binding domain comprisingheavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 34,heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 35,heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 36,light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 37,light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 38, andlight chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 39.

[0015] The lipid nanoparticle according to

[0014] , wherein the binding domain is a binding domain comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 40 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 41.

[0016] The lipid nanoparticle according to any one of [1] to

[0015] , wherein the ligand is Fab.

[0017] The lipid nanoparticle according to any one of [1] to

[0016] , wherein the ligand comprises the amino acid sequence shown in SEQ ID NO: 42 and 43.

[0018] The lipid nanoparticle according to any one of [1] to

[0017] , wherein the immunocyte comprises a T cell and / or an NK cell.

[0019] The lipid nanoparticle according to any one of [1] to

[0017] , wherein the immunocyte comprises a T cell and an NK cell.

[0020] A lipid nanoparticle comprising the following (a) to (c), for promoting MHC class I molecule-dependent and MHC class I molecule-independent cell killing by a T cell and / or an NK cell:(a) a nucleic acid encoding a chimeric antigen receptor or an exogenous T cell receptor;(b) a cationic lipid; and(c) a non-cationic lipid,wherein a surface of the lipid nanoparticle has a ligand that targets an immunocyte, wherein the ligand is a polypeptide comprising a binding domain for CD7.

[0021] The lipid nanoparticle according to

[0020] , wherein the chimeric antigen receptor is a polypeptide comprising a binding domain for GPC3, or a polypeptide comprising a binding domain for CD19.

[0022] A medicament comprising the lipid nanoparticle according to any one of [1A] to

[0021] .

[0023] The medicament according to

[0022] , wherein the medicament is a prophylactic or therapeutic drug for cancer.

[0024] The medicament according to

[0022] or

[0023] , wherein the medicament introduces a chimeric antigen receptor gene or an exogenous T cell receptor gene into an in vivo immunocyte to induce an expression thereof.

[0025] The medicament according to any one of

[0022] to

[0024] , wherein the medicament introduces a chimeric antigen receptor gene or an exogenous T cell receptor gene into an in vivo T cell and / or an NK cell to induce an expression thereof.

[0026] The medicament according to any one of

[0022] to

[0024] , wherein the medicament introduces a chimeric antigen receptor gene or an exogenous T cell receptor gene into an in vivo T cell and an NK cell to induce an expression thereof.

[0027] A method for introducing a chimeric antigen receptor or an exogenous T cell receptor into an in vivo immunocyte of a mammal to induce an expression thereof, comprising administering the lipid nanoparticle according to any one of [1A] to

[0021] to the mammal.

[0028] A method for introducing a chimeric antigen receptor or an exogenous T cell receptor into an in vivo T cell and / or an NK cell of a mammal to induce an expression thereof, comprising administering the lipid nanoparticle according to any one of [1A] to

[0021] to the mammal.

[0029] A method for introducing a chimeric antigen receptor or an exogenous T cell receptor into an in vivo T cell and an NK cell of a mammal to induce an expression thereof, comprising administering the lipid nanoparticle according to any one of [1A] to

[0021] to the mammal.

[0030] A method for preventing or treating cancer in a mammal, comprising administering the lipid nanoparticle according to any one of [1A] to

[0021] to the mammal.

[0031] The lipid nanoparticle according to any one of [1A] to

[0021] for use in the prophylaxis or treatment of cancer.

[0032] Use of the lipid nanoparticle according to any one of [1A] to

[0021] in the manufacture of an agent for the prophylaxis or treatment of cancer.

[0033] The lipid nanoparticle according to any one of [1A] to

[0021] for use in the induction of an expression of a chimeric antigen receptor or an exogenous T cell receptor.

[0015] According to the present invention, a novel in vivo cancer therapy based on a new concept can be provided.

[0016] Figure 1 shows the result of treatment effect in human T cell transferred tumor-bearing mouse model.Figure 2 shows the result of treatment effect in human T cell transferred tumor-bearing mouse model.Figure 3 shows the result of treatment effect in human NK cell transferred tumor-bearing mouse model (a, b). Also, Figure 3 shows the result of confirmation of CAR expression in human NK cell transferred tumor-bearing mouse model (c).Figure 4 shows the result of treatment effect in mouse tumor model.Figure 5 shows the result of confirmation of expression levels of CAR and interferon gamma in T cells and NK cells of LNP administered mice.Figure 6 shows the result of in vitro cytotoxicity of hCD19 CAR-CD7 LNP-treated T cells against NALM6-Luc cells.Figure 7 shows the result of in vivo anti-tumor efficacy of hCD19 CAR-CD7 LNP in the human T cell transferred NALM6-Luc xenograft model.Figure 8 shows the result of in vitro cytotoxicity of hCD19 CAR-CD7 LNP-treated human NK cells against NALM6 cells.Figure 9 shows the result of in vivo anti-tumor efficacy of hCD19 CAR-CD7 LNP in the human NK cell transferred NALM6-Luc xenograft model.Figure 10 shows the result of in vivo anti-tumor efficacy of hCD19 CAR-CD7 LNP in the human T / NK cell transferred NALM6-Luc xenograft model.Figure 11 shows the result of in vivo anti-tumor efficacy of CD19 CAR-CD7 LNP with IL-15 in the human T cell transferred NALM6-Luc xenograft model.

[0017] As used in this specification and the appended claims, singular articles such as “a,” “an,” and “the,” may refer to a single object or to a plurality of objects unless the context clearly indicates otherwise. Thus, for example, reference to a composition containing “a compound” may include a single compound or two or more compounds. The above description is intended to be illustrative and not restrictive. Many embodiments will be apparent to those of skill in the art upon reading the above description. Therefore, the scope of the present invention should be determined with reference to the appended claims and includes the full scope of equivalents to which such claims are entitled. The disclosures of all articles and references cited in the disclosure, including patents, patent applications and publications, are herein incorporated by reference in their entirety and for all purposes.

[0018] The term “comprise(s)” or “comprising” means inclusion of the element(s) following the word without limitations thereto. Accordingly, this suggests inclusion of the element(s) following the word, but does not suggest exclusion of any other element. The phrase “consist(s) of” or “consisting of” means inclusion of all the element(s) following the phrase and limitation thereto. Accordingly, the phrase “consist(s) of” or “consisting of” indicates that the enumerated element(s) is required or essential and substantially no other elements exist. The phrase “consist(s) essentially of” or “consisting essentially of” means inclusion of any element following the phrase and limitation of other elements to those that do not affect the activity or effect of the enumerated element(s) specified in the present disclosure. Accordingly, the phrase “consist(s) essentially of” or “consisting essentially of” indicates that the enumerated element(s) is required or essential, but other elements are optional and may exist or not exist depending on whether they affect the activity or effect of the enumerated element(s).

[0019] 1. Lipid nanoparticle (LNP) of the present inventionThe present invention provides a lipid nanoparticle comprising the following (a) to (c), and having a ligand on its surface that targets an immunocyte:(a) a nucleic acid encoding a chimeric antigen receptor or an exogenous T cell receptor;(b) a cationic lipid; and(c) a non-cationic lipid,wherein the ligand is a polypeptide comprising a binding domain for CD7 (hereinafter to be also referred to as “the lipid nanoparticle of the present invention”, “LNP of the present invention”).

[0020] In the present specification, the “lipid nanoparticle (LNP)” refers to particles with an average diameter of less than 1 mm and free of a small porous structure (e.g., mesoporous material) in a molecular assembly constituted of the above-mentioned (b) and (c).

[0021] The constituent elements (a) to (c) of the lipid nanoparticle of the present invention are explained below.

[0022] (a) Nucleic acid encoding chimeric antigen receptor (CAR) or exogenous T cell receptor (TCR).

[0023] The nucleic acid of the present invention may contain at least one type of modified nucleotide or nucleotide analogue. Such modified nucleotides and nucleotide analogues are not particularly limited, and examples thereof include 1-methyladenosine, 2-methyladenosine, N6-methyladenosine, 2’-0-methyladenosine, 2-methylthio-N6-methyladenosine, N6-isopentenyladenosine, 2-methylthio-N6-isopentenyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6-methyl-N6-threonylcarbamoyladenosine, N6-hydroxynorvalylcarbamoyladenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine, inosine, 3-methylcytidine, 2’-0-methylcytidine, 2-thiocytidine, N4-acetylcytidine, lysidine, 1-methylguanosine, 7-methylguanosine, 2’-0-methylguanosine, queuosine, epoxyqueuosine, 7-cyano-7-deazaguanosine, 7-aminomethyl-7-deazaguanosine, pseudouridine, N1-methylpseudouridine, dihydrouridine, 5-methyluridine, 2’-0-methyluridine, 2-thiouridine, 4-thiouridine, 5-methyl-2-thiouridine, 3-(3-amino-3-carboxypropyl)uridine, 5-hydroxyuridine, 5-methoxyuridine, uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, 5-aminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyl-2’-0-methyluridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thiouridine, 2-aminoadenosine, 5-(isopentenylaminomethyl)-2’-0-methyluridine, 2-thiothymidine, pyrrolopyrimidine, 3-methyladenosine, C5-propynyl-cytidine, C5-propynyl-uridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynylcytidine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, phosphorothioate, N3’-P5’-phosphoramidate, alkylphosphonate, nucleotides containing phosphorodithioate or alkylphosphonothioate internucleoside bonds, peptide nucleic acid, locked nucleic acid, and ethylene-bridged nucleic acid. The nucleic acid of the present invention may contain one type of modified nucleotide or nucleotide analogue, or two or more types thereof.

[0024] (a-1) Nucleic acid encoding CARCAR is an artificially constructed hybrid protein containing the antigen-binding domain (e.g., scFv) of an antibody coupled to a T cell signal transduction domain. CAR is characterized by the ability to utilize the antigen-binding property of the monoclonal antibody to redirect the specificity and responsiveness of immunocytes including T cells and NK cells to a selected target in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition confers on CAR-expressing T cells and CAR-expressing NK cells the ability to recognize antigens independently of antigen processing, thereby bypassing the major mechanism of tumor escape. Furthermore, when expressed in T cells, CAR advantageously does not dimerize with the endogenous TCR α chain and β chain.

[0025] The CAR used in the lipid nanoparticles of the present invention includes an extracellular antigen-binding domain of an antibody that can specifically recognize surface antigens (e.g., cancer antigen peptide, surface receptor showing promoted expression in cancer cells, etc.) that the target immunocyte (e.g., T cell, NK cell, NKT cell, monocyte, macrophage, dendritic cell, etc.), a transmembrane domain, a co-stimulatory domain and an intracellular signal transduction domain should recognize. The CAR used in the lipid nanoparticles of the present invention further comprises a hinge domain linking the extracellular antigen binding domain and the transmembrane domain. The lipid nanoparticles of the present invention may contain one or more CARs. In the case of two or more CARs, the CARs may be antigen-binding domains that recognize different surface antigens.In another embodiment, the CAR used in the lipid nanoparticles of the present invention can be CAR variants such as Chimeric Co-Stimulatory Receptor (CCR) which is a chimeric receptor that contains an extracellular antigen binding domain, a transmembrane domain, and an intracellular co-stimulatory signaling region comprising at least one co-stimulatory molecule but does not contain a T-cell signaling domain or a chimeric receptor that contains an extracellular antigen binding domain and a transmembrane domain, but does not contain a co-stimulatory signaling region or a T-cell signaling domain.

[0026] Examples of the surface antigens specifically recognized by antigen-binding domains include, but are not limited to, surface receptors showing promoted expression in various cancers (e.g., acute lymphocytic cancer, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anus, anal canal or anorectal cancer, cancer of the eye, cancer of the interhepatic bile duct, joint cancer, cervical, gallbladder or pleural cancer, nose, nasal cavity or middle ear cancer, oral cancer, vulvar cancer, chronic myelogenous cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia), liquid tumor, liver cancer, lung cancer (e.g., non-small cell lung cancer), lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B cell lymphoma, follicular lymphoma), malignant mesothelioma, mastocytoma, melanoma, multiple myeloma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer; peritoneal, omentum and mesenteric cancer; pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer and the like, for example, CD19, EGF receptor, BCMA, CD30, Her2, ROR1, MUC16, CD20, mesothelin, B-cell mutation antigen (BCMA), CD123, CD3, prostate specific membrane antigen (PSMA), CD33, MUC-1, CD138, CD22, GD2, PD-L1, CEA, chondroitin sulfate proteoglycan-4, IL-13 receptor α chain, IgG κ light chain, Claudin family (e.g., Claudin18.2, Claudin6, etc.), and cancer antigen peptides (e.g., peptides derived from WT1, GPC3, MART-1, gp100, NY-ESO-1, MAGE-A4, etc.). The surface antigens specifically recognized by antigen-binding domains are preferably GPC3 or CD19.

[0027] The antigen-binding domain used in the present invention is not particularly limited as long as it is an antibody fragment that can specifically recognize the target antigen. Considering the ease of preparation of CAR, a single-chain antibody (scFv) in which a light chain variable region and a heavy chain variable region are linked via a linker peptide is desirable. The configuration of the light chain variable region and heavy chain variable region in single-chain antibody is not particularly limited as long as they can reconstitute a functional antigen-binding domain. They can generally be designed in the order of light chain variable region, linker peptide, and heavy chain variable region from the N-terminal side. As the linker peptide, a known linker peptide typically used for the production of single-chain antibodies can be used. For example, DNA encoding light chain variable region and heavy chain variable region can be prepared by cloning light chain gene and heavy chain gene respectively from antibody-producing cells and performing PCR using them as templates, or the like, or by chemically synthesizing them from the sequence information of existing antibodies. DNA encoding a single-chain antibody can be obtained by ligating each obtained DNA fragment with a DNA encoding linker peptide by an appropriate method. The N-terminal side of the antigen-binding domain is preferably further added with a reader sequence to present CAR to the surface of the immunocyte.

[0028] In one embodiment, the extracellular antigen binding domain is a binding domain comprisingheavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1,heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2,heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3,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, andlight chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6.

[0029] In one embodiment, the extracellular antigen binding domain is a binding domain comprisingheavy chain CDR1 consisting of an amino acid sequence shown in SEQ ID NO: 1, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids),heavy chain CDR2 consisting of an amino acid sequence shown in SEQ ID NO: 2, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids),heavy chain CDR3 consisting of an amino acid sequence shown in SEQ ID NO: 3, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids),light chain CDR1 consisting of an amino acid sequence shown in SEQ ID NO: 4, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids),light chain CDR2 consisting of an amino acid sequence shown in SEQ ID NO: 5, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids), andlight chain CDR3 consisting of an amino acid sequence shown in SEQ ID NO: 6, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids).

[0030]

[0031]

[0032] In another embodiment, the extracellular antigen binding domain is a binding domain comprisingheavy 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,heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 9,light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 10,light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 11, andlight chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 12.

[0033] In another embodiment, the extracellular antigen binding domain is a binding domain comprisingheavy chain CDR1 consisting of an amino acid sequence shown in SEQ ID NO: 7, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids),heavy chain CDR2 consisting of an amino acid sequence shown in SEQ ID NO: 8, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids),heavy chain CDR3 consisting of an amino acid sequence shown in SEQ ID NO: 9, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids),light chain CDR1 consisting of an amino acid sequence shown in SEQ ID NO: 10, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids),light chain CDR2 consisting of an amino acid sequence shown in SEQ ID NO: 11, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids), andlight chain CDR3 consisting of an amino acid sequence shown in SEQ ID NO: 12, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids).

[0034] In other embodiment, the extracellular antigen binding domain is a binding domain comprisingheavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13,heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14,heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15,light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16,light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17, andlight chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 18.

[0035] In other embodiment, the extracellular antigen binding domain is a binding domain comprisingheavy chain CDR1 consisting of an amino acid sequence shown in SEQ ID NO: 13, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids),heavy chain CDR2 consisting of an amino acid sequence shown in SEQ ID NO: 14, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids),heavy chain CDR3 consisting of an amino acid sequence shown in SEQ ID NO: 15, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids),light chain CDR1 consisting of an amino acid sequence shown in SEQ ID NO: 16, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids),light chain CDR2 consisting of an amino acid sequence shown in SEQ ID NO: 17, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids), andlight chain CDR3 consisting of an amino acid sequence shown in SEQ ID NO: 18, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids).

[0036] In one embodiment, the extracellular antigen binding domain is a binding domain comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20.

[0037] In one embodiment, the extracellular antigen binding domain is a binding domain comprising a heavy chain variable region comprising an amino acid sequence that is at least 80%, preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, identical to the amino acid sequence shown in SEQ ID NO: 19 and a light chain variable region comprising an amino acid sequence that is at least 80%, preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, identical to the amino acid sequence shown in SEQ ID NO: 20.

[0038] In another embodiment, the extracellular antigen binding domain is a binding domain comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22.

[0039] In another embodiment, the extracellular antigen binding domain is a binding domain comprising a heavy chain variable region comprising an amino acid sequence that is at least 80%, preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, identical to the amino acid sequence shown in SEQ ID NO: 21 and a light chain variable region comprising an amino acid sequence that is at least 80%, preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, identical to the amino acid sequence shown in SEQ ID NO: 22.

[0040] In other embodiment, the extracellular antigen binding domain is a binding domain comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 24.

[0041] In other embodiment, the extracellular antigen binding domain is a binding domain comprising a heavy chain variable region comprising an amino acid sequence that is at least 80%, preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, identical to the amino acid sequence shown in SEQ ID NO: 23 and a light chain variable region comprising an amino acid sequence that is at least 80%, preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, identical to the amino acid sequence shown in SEQ ID NO: 24.

[0042] In one embodiment, the scFv is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 25. In another embodiment, the scFv is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 26. In other embodiment, the scFv is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 27.

[0043] In one embodiment, the scFv is a polypeptide comprising an amino acid sequence that is at least 80%, preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, identical to the amino acid sequence shown in SEQ ID NO: 25. In another embodiment, the scFv is a polypeptide comprising an amino acid sequence that is at least 80%, preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, identical to the amino acid sequence shown in SEQ ID NO: 26. In other embodiment, the scFv is a polypeptide comprising an amino acid sequence that is at least 80%, preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, identical to the amino acid sequence shown in SEQ ID NO: 27.

[0044] As the transmembrane domain, T cell surface molecule-derived domains generally used in the relevant technical field can be used as appropriate. For example, they include, but are not limited to, domains derived from CD28, a CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0045] As the hinge domain, T cell surface molecule-derived domains generally used in the relevant technical field can be used as appropriate. For example, they include, but are not limited to, regions derived from CD8α and CD28.

[0046] Examples of the co-stimulatory domain include, but are not limited to, OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1(CD11a / CD18), ICOS(CD278), DAP10, DAP12, 4-1BB(CD137), and combinations thereof. Only one co-stimulatory domain may be present or two or more thereof may be present.

[0047] Examples of the intracellular signal transduction domain include, but are not limited to, domains derived from CD3 zeta, a CD3 zeta variant, 4-1BB(CD137), CD28, CD134, Lck, DAP10, ICOS, and a combination thereof. The CD3 zeta variant is not particularly limited. Examples of the CD3 zeta variant include those described in WO2019 / 133969, and CD3 zeta 1xx is preferred. In certain embodiments, CD3 zeta 1xx comprises a native ITAM1 having the amino acid sequence set forth in SEQ ID NO: 50, an ITAM2 variant having the amino acid sequence set forth in SEQ ID NO: 51 and an ITAM3 variant having the amino acid sequence set forth in SEQ ID NO: 52. Any domains normally used in the relevant technical field can be used in combination.

[0048]

[0049] The extracellular antigen binding domain, hinge domain, transmembrane domain, co-stimulatory domain, and intracellular signal transduction domain may be any combination of those described above. The combination of the transmembrane domain, the co-stimulatory domain, and the intracellular signal transduction domain is preferably CD28-CD28-CD3 zeta or a CD28-CD28-CD3 zeta variant.

[0050] In one embodiment, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 28, the amino acid sequence shown in SEQ ID NO: 29, or the amino acid sequence shown in SEQ ID NO: 30.

[0051] In one embodiment, the chimeric antigen receptor comprises an amino acid sequence that is at least 80%, preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, identical to the amino acid sequence shown in SEQ ID NO: 28, and has GPC3-binding activity.

[0052] In another embodiment, the chimeric antigen receptor comprises an amino acid sequence that is at least 80%, preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, identical to the amino acid sequence shown in SEQ ID NO: 29, and has GPC3-binding activity.

[0053] In other embodiment, the chimeric antigen receptor comprises an amino acid sequence that is at least 80%, preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, identical to the amino acid sequence shown in SEQ ID NO: 30, and has CD19-binding activity.

[0054] The binding activity of the chimeric antigen receptor to GPC3 or CD19 can be confirmed by any method known in the pertinent technical field.

[0055] Nucleic acid sequence information encoding extracellular antigen binding domain, hinge domain, transmembrane domain, co-stimulatory domain, and intracellular signal transduction domain is well known in the relevant technical field. Methods of producing CAR sequences are also well known in the relevant technical field. Those of ordinary skill in the art can easily obtain DNA fragments encoding each domain from T cells based on such information.DNA encoding CAR can be obtained by linking DNA fragments respectively encoding the thus-obtained extracellular antigen binding domain, hinge domain, transmembrane domain, co-stimulatory domain, and intracellular signal transduction domain, by a conventional method.

[0056] The obtained DNA encoding CAR can be inserted into an expression vector, preferably a plasmid vector, containing a functional promoter in T cells, either as is or after adding a suitable linker and / or nuclear translocation signal and the like. Examples of the functional promoter in T cells include, but are not limited to, constitutive SRα promoter in mammalian cells, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney mouse leukemia virus) LTR, HSV-TK (Herpes simplex virus thymidine kinase) promoter and the like. In addition, gene promoters such as CD3, CD4, and CD8, which are specifically expressed in T cells, can also be used. Furthermore, gene promoters such as CD34, CD117, CD56, CD94, and CD122, which are specifically expressed in NK cells, can also be used.

[0057] RNA encoding a CAR, preferably mRNA, can be prepared by transcription into mRNA in an in vitro transcription system known per se using an expression vector containing DNA encoding the above-mentioned CAR as a template.

[0058] (a-2) Nucleic acid encoding exogenous TCRIn the present specification, the “T cell receptor (TCR)” means a receptor that consists of dimers of the TCR chain (α-chain, β-chain) and recognizes an antigen or the antigen-HLA (human leukocyte type antigen) (MHC; major histocompatibility complex) complex and transduces a stimulatory signal to T cells. Each TCR chain consists of a variable region and a constant region, and the variable region contains three complementarity determining regions (CDR1, CDR2, CDR3). The TCR used in the present invention includes not only those in which the α and β chains of the TCR constitute a heterodimer but also those in which they constitute a homodimer. Furthermore, the TCR includes those with a part or all of the constant regions deleted, those with recombined amino acid sequence, and those with soluble TCR, and the like.

[0059] The “exogenous TCR” means being exogenous to T cell, which is the target cell of the lipid nanoparticle of the present invention. The amino acid sequence of the exogenous TCR may be the same as or different from that of the endogenous TCR expressed by T cell, which is the target cell of the lipid nanoparticle of the present invention.

[0060] The nucleic acid encoding TCR used in the lipid nanoparticle of the present invention is a nucleic acid encoding the α chain and β chain of TCR that can specifically recognize surface antigens (e.g., cancer antigen peptide etc.) to be recognized by the target T cell.

[0061] The nucleic acid can be prepared by a method known per se. When the amino acid sequence or nucleic acid sequence of the desired TCR is known, a DNA encoding the full-length or a part of the TCR of the present invention can be constructed based on the sequence by, for example, chemically synthesizing a DNA strand or an RNA strand, or connecting a synthesized, partially overlapping oligo-DNA short strand by the PCR method or the Gibson assembly method.

[0062] When the sequence of the desired TCR is not known, for example, T cell of interest is isolated from a population of cells containing the T cell expressing a TCR of interest, and a nucleic acid encoding the TCR can be obtained from the T cell. Specifically, a cell population (e.g., PBMC) containing T cells is collected from an organism (e.g., human), the cell population is cultured in the presence of epitopes of cell surface antigens recognized by the target TCR while stimulating the cell population, and T cell that specifically recognizes cells expressing the cell surface antigen can be selected from the cell population by a known method and using, as indices, specificity for cells expressing the cell surface antigen and cell surface antigens such as CD8 and CD4. The specificity for cells expressing the cell surface antigen of T cells can be measured, for example, by dextromer assay, ELISPOT assay, cytotoxic assay, or the like. The aforementioned cell population containing T cells is preferably collected from, for example, an organism having a large number of cells expressing a cell surface antigen recognized by the TCR of interest (e.g., patient with a disease such as cancer, or T cell-containing population contacted with an epitope of the antigen or dendritic cells pulsed with the epitope).

[0063] The nucleic acid of the present invention can be obtained by extracting DNA from the aforementioned isolated T cell by a conventional method, amplifying and cloning the TCR gene based on the nucleic acid sequence of the constant region of the TCR by using the DNA as a template. It can also be prepared by extracting RNA from a cell and synthesizing cDNA by a conventional method, and performing 5’-RACE (rapid amplification of cDNA ends) with the cDNA as templates using antisense primers complementary to the nucleic acids respectively encoding the constant regions of the TCR α chain and β chain. 5’-RACE may be performed by a known method and can be performed, for example, using a commercially available kit such as SMART PCR cDNA Synthesis Kit (manufactured by clontech). The DNA encoding the α chain and β chain of the obtained TCR can be inserted into an appropriate expression vector in the same way as the DNA encoding the above-mentioned CAR. The DNA encoding α chain and the DNA encoding β chain may be inserted into the same vector or separate vectors. When inserted into the same vector, the expression vector may express both strands in a polycistronic or monocistronic manner. In the former case, an intervening sequence that permits polycistronic expression, such as IRES or FMV 2A, is inserted between the DNA encoding both strands.

[0064] In addition, RNA encoding each strand of the TCR, preferably mRNA, can be prepared in the same way as the above-mentioned RNA encoding CAR, for example, by using the expression vector as a template.

[0065] In one embodiment, the CAR or exogenous TCR may be coexpressed with any cytokines in target cell. Such cytokines are not particularly limited and preferably IL-15 / IL-15Rα.

[0066] In the present invention, the “IL-15 / IL-15Rα” means a fusion protein containing IL-15 and IL-15Rα. In the IL-15 signal transduction system, IL-15Rα expressed on antigen-presenting cells generally binds to IL-15 and IL-15 is presented to IL-15 receptor consisting of a common γ chain (γc) with IL-15Rβ on CD8-positive and CD4-negative cell (trans-presentation), whereby the cytotoxic activity of CD8-positive CD4-negative cell is maintained. Therefore, when the CD3-positive cell expressing IL-15 / IL-15Rα is CD8-positive CD4-negative, the cell can transmit the IL-15 signal into its own cell via the IL-15 receptor. Alternatively, the CD3-positive cell expressing IL-15 / IL-15Rα can transmit the IL-15 signal into other CD8-positive CD4-negative cells via the IL-15 receptor. As described above, since IL-15 / IL-15Rα can maintain cytotoxic activity of CD8-positive CD4-negative cell, it is expected to show a continuous cytotoxic effect on cells targeted by CAR.

[0067] IL-15 / IL-15Rα may be a transmembrane type protein or a secretor protein. It is known that, in IL-15Rα, the IL-15 binding domain of 1-65 amino acids from the N-terminal of the mature protein is the region responsible for binding to IL-15 (Wei X. et al., J. Immunol., 167: 277-282, 2001). Therefore, the transmembrane type protein may be a protein that retains the IL-15 binding domain and retains the transmembrane domain of IL-15Rα. On the other hand, the secretor protein may be a protein that maintains the IL-15 binding domain and lacks the transmembrane domain of IL-15Rα.

[0068] A spacer may be incorporated between IL-15 and IL-15Rα of IL-15 / IL-15Rα. As the spacer, a peptide generally consisting of not more than 300 amino acids, preferably 10-100 amino acids, most preferably 20-50 amino acids, can be used. Specific examples thereof include, but are not limited to, GS linker and the like.

[0069] IL-15, IL-15Rα and IL-15 / IL-15Rα are not particularly limited.

[0070] In one embodiment, IL-15 comprises an amino acid sequence shown in SEQ ID NO: 46. In another embodiment, IL-15 comprises an amino acid sequence that is at least 80%, preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, identical to the amino acid sequence shown in SEQ ID NO: 46.

[0071] In one embodiment, IL-15Rα comprises an amino acid sequence shown in SEQ ID NO: 47. In another embodiment, IL-15Rα comprises an amino acid sequence that is at least 80%, preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, identical to the amino acid sequence shown in SEQ ID NO: 47.

[0072] In one embodiment, IL-15 / IL-15Rα comprises an amino acid sequence shown in SEQ ID NO: 48. In another embodiment, IL-15 / IL-15Rα comprises an amino acid sequence that is at least 80%, preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, identical to the amino acid sequence shown in SEQ ID NO: 48.

[0073]

[0074]

[0075] (b) Cationic lipidIn the present specification, the “cationic lipid” means a lipid that has a net positive charge at a selected pH, such as physiological pH. The cationic lipids used in the lipid nanoparticle of the present invention are not particularly limited. For example, cationic lipids and the like described in WO 2019 / 131770, WO 2015 / 011633, WO 2016 / 021683, WO 2019 / 131839, WO 2020 / 032184, WO 2011 / 153493, WO 2013 / 126803, WO 2010 / 054401, WO 2010 / 042877, WO 2016 / 104580, WO 2015 / 005253, WO 2014 / 007398, WO 2017 / 117528, WO 2017 / 075531, WO 2017 / 00414, WO 2015 / 199952, US 2015 / 0239834, WO2023 / 085299, and the like can be mentioned.

[0076] Preferred cationic lipids are represented by the following structural formulas and described in WO 2019 / 131770, WO 2015 / 011633.

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] and salts thereof.

[0085] Among the above-mentioned cationic lipids, cationic lipids represented by the following structural formulas are more preferred.

[0086] and salts thereof.

[0087] Preferred cationic lipid is represented by the following structural formula and described in WO 2016 / 021683. A compound represented by

[0088]

[0089] whereinW is the formula -NR1R2or the formula -N+R3R4R5(Z-),R1and R2are each independently a C1-4alkyl group or a hydrogen atom,R3, R4and R5are each independently a C1-4alkyl group,Z-is an anion,X is an optionally substituted C1-6alkylene group,YA, YBand YCare each independently an optionally substituted methine group,LA, LBand LCare each independently an optionally substituted methylene group or a bond, andRA1, RA2, RB1, RB2, RC1and RC2are each independently an optionally substituted C4-10alkyl group,or a salt thereof.

[0090] More preferably, cationic lipids represented by the following structural formulas can be mentioned.

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100] and salts thereof.

[0101] Among the above-mentioned cationic lipids, more preferred cationic lipids are represented by the following structural formulas.

[0102]

[0103]

[0104] and salts thereof.

[0105] In another preferable embodiment, a cationic lipid represented by the following formula (II) and described in WO2019 / 131839 (hereinafter to be also referred to as “compound (II)”) can be mentioned. A compound represented by

[0106]

[0107] whereinn is an integer of 2 to 5,R is a linear C1-5alkyl group, a linear C7-11alkenyl group or a linear C11alkadienyl group, andwavy lines are each independently shows a cis-type or trans-type bond,or a salt thereof.

[0108] More preferably, cationic lipids represented by the following structural formulas can be mentioned.

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119] and salts thereof.

[0120] Among the above-mentioned cationic lipids, more preferred cationic lipids are represented by the following structural formulas.

[0121]

[0122]

[0123] and salts thereof.

[0124] In another preferable embodiment, a cationic lipid represented by the following formula (III) and described in WO2020 / 032184 (hereinafter to be also referred to as “compound (III)”) can be mentioned. A compound represented by

[0125]

[0126] whereinn1 is an integer of 2 to 6,n2 is an integer of 0 to 2,n3 is an integer of 0 to 2,L is -C(O)O- or -NHC(O)O-,Ra is a linear C5-13alkyl group, a linear C13-17alkenyl group or a linear C17alkadienyl group,Rb is a linear C2-9alkyl group,Rc is a hydrogen atom or a linear C2-9alkyl group,Rd is a hydrogen atom or a linear C2-9alkyl group,Re is a linear C2-9alkyl group, andRf is a linear C2-9alkyl group,or a salt thereof.

[0127] More preferably, the following structural formula represented by cationic lipid can be mentioned.

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145] and salts thereof.

[0146] Among the above-mentioned cationic lipids, a cationic lipid represented by the following structural formula is more preferable.

[0147]

[0148] and salts thereof.

[0149] In other preferable embodiment, a cationic lipid represented by the following formula (IV) and described in WO2023 / 085299 (hereinafter to be also referred to as “compound (IV)”) can be mentioned. A compound represented by

[0150]

[0151] whereinW represents -NR1R2or -N+R11R12R13(Z-),R1and R2each independently represent H or an optionally substituted C1-5alkyl group,R11, R12and R13each independently represent an optionally substituted C1-5alkyl group,Z-represents an anion,X represents an optionally substituted C2-6alkylene group,RAand RBeach independently represent an optionally substituted C1-17alkyl group, an optionally substituted C3-17alkenyl group, an optionally substituted C15-17alkadienyl group, -R3-C(O)O-R4or -R3-OC(O)-R4,RCrepresents -R3-C(O)O-R4 or -R3-OC(O)-R4,R3represents an optionally substituted C1-16alkylene group, an optionally substituted C4-16alkenylene group or an optionally substituted C7-16alkadienylene group, andR4represents H, an optionally substituted C1-18alkyl group, an optionally substituted C3-18alkenyl group or an optionally substituted C15-18alkadienyl group,or a salt thereof.

[0152] More preferably, the following structural formula represented by cationic lipid can be mentioned.

[0153]

[0154]

[0155]

[0156] and salts thereof.

[0157] In another preferable embodiment, a cationic lipid represented by the following formula (V) (hereinafter to be also referred to as “compound (V)”) can be mentioned. A compound represented by the formula (V): …(V)in the formula (V),W is -NR1R2or -N+R11R12R13(Q-),R1and R2are each independently a hydrogen atom or an optionally substituted C1-5alkyl group,R11, R12and R13are each independently an optionally substituted C1-5alkyl group,Q-is an anion,X is an optionally substituted C2-6alkylene group,Y is -CO-OCH2- or -O-CO-,R3and R4are each an optionally substituted C3-18alkyl group, an optionally substituted C3-18alkenyl group or an optionally substituted C16-18alkadienyl group,Z is -CH2O-CO- or -COO-, andR5is an optionally substituted C3-20alkyl group, an optionally substituted C3-18alkenyl group or an optionally substituted C15-18alkadienyl group.

[0158] Compound (V) can be produced, for example, by the following production method. Compound (V) with a desired structure can be synthesized using an appropriate raw material according to the structure of the target compound (V), particularly during esterification. In addition, a salt of compound (V) can be obtained by appropriately mixing with an inorganic base, an organic base, an organic acid, or a basic or acidic amino acid.

[0159] The following scheme 1 shows one example of a method for producing compound (V). In scheme 1, P is a protecting group, L is a leaving group, and the other symbols are the same as in the formula (V) (the same applies to other schemes related to scheme 1).

[0160] [Corrected under Rule 26, 20.02.2026]

[0161] The following scheme 2 shows one example of a method for producing compound (V). In scheme 2, P is a protecting group, L is a leaving group, and the other symbols are the same as in the formula (V) (the same applies to other schemes related to scheme 2).

[0162] [Corrected under Rule 26, 20.02.2026]

[0163] The following scheme 3 shows a synthesis method of a compound (R5-COOH and R5-OH) used for esterification in schemes 1 and 2. In scheme 3, Ra, Rb, Rc, Rdand Reare each H, an optionally substituted alkyl group, an optionally substituted alkenyl group or an optionally substituted alkadienyl group, and constitute part of R5. The carbon numbers, substituents and structures of Ra, Rb, Rc, Rdand Reare appropriately adjusted according to the desired structure of R5.

[0164]

[0165] The following scheme 4 shows a synthesis method of a compound (R1R2N-X-COOH and R1R2N-X-OH) used for esterification in schemes 1 and 2. In scheme 4, Rfis an optionally substituted alkylene group. The carbon number, substituents and structure of Rfare appropriately adjusted according to the desired structure of X.

[0166]

[0167] In another preferable embodiment, a cationic lipid represented by the following formula (VI) (hereinafter to be also referred to as “compound (VI)”) can be mentioned. A compound represented by the formula (VI): …(VI)in the formula (VI),W is -NR1R2or -N+R11R12R13(Z-),R1and R2are each independently a hydrogen atom or an optionally substituted C1-5alkyl group,R11, R12and R13are each independently an optionally substituted C1-5alkyl group,Z-is an anion,X is an optionally substituted C2-6alkylene group,RAand RBare each independently an optionally substituted C1-17alkyl group, an optionally substituted C3-17alkenyl group, an optionally substituted C15-17alkadienyl group, or a structure represented by -R3-Y1-CH(R4)(Y2-R5),RCis a structure represented by -R3-Y1-CH(R4)(Y2-R5),Y1and Y2are each independently -CO-O- or -O-CO-,R3is an optionally substituted C1-3alkylene group,R4is a hydrogen atom, an optionally substituted C1-12alkyl group, an optionally substituted C3-18alkenyl group or an optionally substituted C15-18alkadienyl group,R5is an optionally substituted C1-12alkyl group, an optionally substituted C3-18alkenyl group or an optionally substituted C15-18alkadienyl group, andwhen RA, RBand RCare each a structure represented by -R3-Y1-CH(R4)(Y2-R5), R3, R4and R5in the structures of RA, RBand RCmay be the same or different.

[0168] Compound (VI) can be produced, for example, by the following production method. Compound (VI) with a desired structure can be synthesized using an appropriate raw material according to the structure of the target compound (VI), particularly during esterification. In addition, a salt of compound (VI) can be obtained by appropriately mixing with an inorganic base, an organic base, an organic acid, or a basic or acidic amino acid.

[0169] The following scheme 5 shows one example of a method for producing compound (VI). In scheme 5, P is a protecting group and the other symbols are the same as in the formula (VI) (the same applies to other schemes related to scheme 5). In scheme 5, L is a leaving group and RA, RBand RCmay be appropriately replaced with each other.

[0170]

[0171] The following scheme 6 shows a synthesis method of a compound (RAor RB-COOH) used for esterification when RAand / or RBin compound (VI) in scheme 5 is RA / Bgroup 1 (optionally substituted C1-17alkyl group, optionally substituted C3-17alkenyl group, optionally substituted C15-17alkadienyl group). In scheme 6, P2, P3, P4and P5are each a protecting group, and Ra, Rb, Rc, Rdand Rgare each H, an optionally substituted alkyl group, an optionally substituted alkenyl group or an optionally substituted alkadienyl group, and constitute part of RAand / or RB. The carbon numbers, substituents and structures of Ra, Rb, Rc, Rdand Rgare appropriately adjusted according to the desired structures of RAand / or RB.

[0172]

[0173] The following scheme 7 shows a synthesis method of a compound (RAor RB-COOH) used for esterification and a compound (RC-COOH) used for esterification of RCin compound (VI), when RAand / or RBin compound (VI) in scheme 5 is RA / Bgroup 2 (-R3-Y1-CH(R4)(Y2-R5)). In scheme 7, P6, P7and P8are each a protecting group.

[0174]

[0175] The following scheme 8 shows a synthesis method of a compound (R5-OH) used for esterification in scheme 7. In scheme 8, P4is a protecting group, and Ra, Rb, Rcand Rdare each H, an optionally substituted alkyl group, an optionally substituted alkenyl group or an optionally substituted alkadienyl group, and constitute part of R5. The carbon numbers, substituents and structures of Ra, Rb, Rcand Rdare appropriately adjusted according to the desired structure of R5.

[0176]

[0177] The following scheme 9 shows a synthesis method of a compound (R5-COOH) used for esterification in scheme 7. In scheme 9, P4and P5are each a protecting group, and Ra, Rb, Rcand Rdare each H, an optionally substituted alkyl group, an optionally substituted alkenyl group or an optionally substituted alkadienyl group, and constitute part of R5. The carbon numbers, substituents and structures of Ra, Rb, Rcand Rdare appropriately adjusted according to the desired structure of R5.

[0178]

[0179] The following scheme 10 shows a synthesis method of a compound (W-X-COOH) used for esterification in scheme 9. In scheme 10, Reis an optionally substituted alkylene group, L is a leaving group, and P9is a protecting group. The carbon number, substituents and structure of Reare appropriately adjusted according to the desired structure of X.

[0180]

[0181] The production method of the compounds (V) to (VI) of the present invention is described below.

[0182] When the compound obtained in each step is a free compound, it can be converted into the desired salt by a known method. Conversely, when the compound obtained in each step is a salt, it can be converted into a free form or other type of desired salt by a known method.

[0183] The compound obtained in each step can be used in the next reaction either as a reaction solution or as a crude product, or can be isolated and / or purified from the reaction mixture by a conventional separation method such as concentration, crystallization, recrystallization, distillation, solvent extraction, fractional distillation, chromatography, and the like.

[0184] When the raw materials and reagent compounds in each step are commercially available, the commercially available products can be used as they are.

[0185] In the reaction of each step, the reaction time may vary depending on the reagents and solvents to be used. Unless otherwise specified, it is generally 1 minute to 72 hours, preferably 10 minutes to 48 hours.

[0186] In the reaction of each step, the reaction temperature may vary depending on the reagents and solvents to be used. Unless otherwise specified, it is generally -78°C to 300°C, preferably -78°C to 150°C.

[0187] In the reaction of each step, the pressure may vary depending on the reagents and solvents to be used. Unless otherwise specified, it is generally 1 atm to 20 atm, preferably 1 atm to 3 atm.

[0188] In the reaction of each step, for example, a Microwave synthesis device such as Initiator manufactured by Biotage and the like may be used. The reaction temperature may vary depending on the reagents and solvents to be used. Unless otherwise specified, it is generally room temperature to 300°C, preferably room temperature to 250°C, more preferably 50°C to 250°C. The reaction time may vary depending on the reagents and solvents to be used. Unless otherwise specified, it is generally 1 minute to 48 hours, preferably 1 minute to 8 hours.

[0189] In the reaction of each step, unless otherwise specified, 0.5 to 20 equivalents, preferably 0.8 to 5 equivalents, of the reagent are used relative to the substrate. When a reagent is used as a catalyst, 0.001 to 1 equivalent, preferably 0.01 to 0.2 equivalents, of the reagent are used relative to the substrate. When the reagent also serves as a reaction solvent, the amount of the reagent to be used is the amount of the solvent.

[0190] In the reaction of each step, unless otherwise specified, these reactions are performed without a solvent, or by dissolving or suspending in an appropriate solvent. Specific examples of the solvent include the solvents described in Examples, or the following.

[0191] alcohols: methanol, ethanol, isopropanol, isobutanol, tert-butyl alcohol, 2-methoxyethanol, and the like;ethers: diethyl ether, diisopropyl ether, diphenyl ether, tetrahydrofuran, 1,2-dimethoxyethane, cyclopentyl methyl ether, and the like;aromatic hydrocarbons: chlorobenzene, toluene, xylene, and the like;saturated hydrocarbons: cyclohexane, hexane, heptane, and the like;amides: N,N-dimethylformamide, N-methylpyrrolidone, and the like;halogenated hydrocarbons: dichloromethane, carbon tetrachloride, and the like;nitriles: acetonitrile and the like;sulfoxides: dimethyl sulfoxide and the like;aromatic organic bases: pyridine and the like;acid anhydrides: acetic anhydride and the like;organic acids: formic acid, acetic acid, trifluoroacetic acid, and the like;inorganic acids: hydrochloric acid, sulfuric acid, and the like;esters: ethyl acetate, isopropyl acetate, and the like;ketones: acetone, methyl ethyl ketone, and the like;water.The above-mentioned solvents may be used in a mixture of two or more in an appropriate ratio.

[0192] When a base is used in the reaction of each step, for example, the bases shown below or the bases described in Examples are used.

[0193] inorganic bases: sodium hydroxide, potassium hydroxide, magnesium hydroxide, and the like;basic salts: sodium carbonate, calcium carbonate, sodium hydrogen carbonate, and the like;organic bases: triethylamine, diethylamine, N,N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine, N,N-dimethylaniline, 1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]-7-undecene, imidazole, piperidine, and the like;metal alkoxides: sodium ethoxide, potassium tert-butoxide, sodium tert-butoxide, and the like;alkali metal hydrides: sodium hydride and the like;metal amides: sodium amide, lithium diisopropylamide, lithium hexamethyldisilazide, and the like;organolithiums: n-butyllithium, sec-butyllithium, and the like.

[0194] When an acid or acid catalyst is used in the reaction of each step, for example, the acids or acid catalysts shown below or the acids or acid catalysts described in Examples are used.

[0195] inorganic acids: hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, phosphoric acid, and the like;organic acids: acetic acid, trifluoroacetic acid, citric acid, p-toluenesulfonic acid, 10-camphorsulfonic acid, and the like;Lewis acids: boron trifluoride diethyl ether complex, zinc iodide, anhydrous aluminum chloride, anhydrous zinc chloride, anhydrous iron chloride, and the like.

[0196] Unless otherwise specified, the reactions in each step are performed according to known methods, for example, the methods described in 5th Edition Jikken Kagaku Kouza, Vol. 13-19 (edited by The Chemical Society of Japan); New Jikken Kagaku Kouza, Vol. 14-15 (edited by The Chemical Society of Japan); Precision Organic Chemistry Revised 2nd Edition (L. F. Tietze, Th. Eicher, Nankodo); Revised Organic Named Reactions: Their Mechanisms and Key Points (Hideo Togo, Kodansha); ORGANIC SYNTHESES Collective Volume I - VII (John Wiley & SonsInc); Modern Organic Synthesis in the Laboratory A Collection of Standard Experimental Procedures (Jie Jack Li, OXFORD UNIVERSITY); Comprehensive Heterocyclic Chemistry III, Vol. 1-14 (Elsevier Japan Co., Ltd.); Organic Synthesis Strategies Learned from Named Reactions (translated and supervised by Kiyoshi Tomioka, published by Kagaku Dojin); Comprehensive Organic Transformations (VCH Publishers Inc.) 1989, and the like, or the methods described in Examples.

[0197] In each step, the protection or deprotection reaction of functional groups is performed according to known methods, for example, the methods described in “Protective Groups in Organic Synthesis, 4th Ed.” (Theodora W. Greene, Peter G. M. Wuts) published by Wiley-Interscience in 2007; “Protecting Groups 3rd Ed.” (P. J. Kocienski) published by Thieme in 2004, and the like, or the methods described in Examples.

[0198] Protective groups for hydroxyl groups of alcohols and phenolic hydroxyl groups include, for example, ether-type protective groups such as methoxymethyl ether, benzyl ether, p-methoxybenzyl ether, t-butyldimethylsilyl ether, t-butyldiphenylsilyl ether, and tetrahydropyranyl ether; carboxylate-type protective groups such as acetate; sulfonate-type protective groups such as methanesulfonate; and carbonate-type protective groups such as t-butyl carbonate.

[0199] Protective groups for carbonyl groups of aldehydes include, for example, acetal-type protective groups such as dimethylacetal; and cyclic acetal-type protective groups such as cyclic 1,3-dioxane.

[0200] Protective groups for the carbonyl group of ketones include, for example, ketal-type protective groups such as dimethyl ketal; cyclic ketal-type protective groups such as cyclic 1,3-dioxane; oxime-type protective groups such as O-methyloxime; and hydrazone-type protective groups such as N,N-dimethylhydrazone.

[0201] Protective groups for carboxyl groups include, for example, ester-type protective groups such as methyl ester and benzyl ester; and amide-type protective groups such as N,N-dimethylamide.

[0202] Protective groups for thiols include, for example, ether-type protective groups such as benzyl thioether; and ester-type protective groups such as thioacetate, thiocarbonate, and thiocarbamate.

[0203] Protective groups for amino groups and aromatic heterocycles such as imidazole, pyrrole, and indole include, for example, carbamate-type protective groups such as benzyl carbamate; amide-type protective groups such as acetamide; alkylamine-type protective groups such as N-triphenylmethylamine; and sulfonamide-type protective groups such as methanesulfonamide.

[0204] Protective groups can be removed (deprotected) by known methods, such as a method using an acid, a base, ultraviolet light, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate, or a trialkylsilyl halide (e.g., trimethylsilyl iodide, trimethylsilyl bromide), or a reduction method.

[0205] When a reduction reaction is performed in each step, examples of the reducing agent to be used include metal hydrides such as lithium aluminum hydride, sodium triacetoxyborohydride, sodium cyanoborohydride, diisobutylaluminum hydride (DIBAL-H), sodium borohydride, and tetramethylammonium triacetoxyborohydride; boranes such as borane tetrahydrofuran complex; Raney nickel; Raney cobalt; hydrogen; and formic acid. In addition, catalysts such as palladium-carbon, Raney nickel, and Raney cobalt can be used in the presence of hydrogen or formic acid.

[0206] When an oxidation reaction is performed in each step, examples of the oxidizing agent to be used include peracids such as m-chloroperbenzoic acid (MCPBA), hydrogen peroxide, and t-butyl hydroperoxide; perchlorates such as tetrabutylammonium perchlorate; chlorates such as sodium chlorate; chlorites such as sodium chlorite; periodates such as sodium periodate; high-valent iodine reagents such as iodosylbenzene; manganese-containing reagents such as manganese dioxide and potassium permanganate; lead compounds such as lead tetraacetate; chromium-containing reagents such as pyridinium chlorochromate (PCC), pyridinium dichromate (PDC), and Jones reagent; halogen compounds such as N-bromosuccinimide (NBS); oxygen; ozone; sulfur trioxide-pyridine complex; osmium tetroxide; zerene dioxide; and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ).

[0207] When a radical cyclization reaction is performed in each step, examples of the radical initiator to be used include azo compounds such as azobisisobutyronitrile (AIBN); water-soluble radical initiators such as 4-4’-azobis-4-cyanopentanoic acid (ACPA); triethylboron in the presence of air or oxygen; and benzoyl peroxide. Examples of the radical reaction reagent to be used include tributylstannane, tristrimethylsilylsilane, 1,1,2,2-tetraphenyldisilane, diphenylsilane, and samarium iodide.

[0208] When a Wittig reaction is performed in each step, examples of the Wittig reagent to be used include alkylidene phosphoranes. Alkylidene phosphoranes can be prepared by known methods, for example, by reacting a phosphonium salt with a strong base.

[0209] When a Horner-Emmons reaction is performed in each step, examples of the reagent to be used include phosphonoacetates such as methyl dimethylphosphonoacetate and ethyl diethylphosphonoacetate; and bases such as alkali metal hydrides and organolithiums.

[0210] When a Friedel-Crafts reaction is performed in each step, examples of the reagents to be used include a Lewis acid and an acid chloride or an alkylating agent (e.g., alkyl halides, alcohols, olefins, and the like). Alternatively, an organic acid or an inorganic acid can also be used instead of the Lewis acid, and an acid anhydride such as acetic anhydride can also be used instead of acid chloride.

[0211] When an aromatic nucleophilic substitution reaction is performed in each step, examples of the reagents to be used include a nucleophile (e.g., amines, imidazole, and the like) and a base (e.g., basic salts, organic bases, and the like).

[0212] When a nucleophilic addition reaction by a carbanion, a nucleophilic 1,4-addition reaction (Michael addition reaction) by a carbanion, or a nucleophilic substitution reaction by a carbanion is performed in each step, examples of the base to be used to generate the carbanion include organolithiums, metal alkoxides, inorganic bases, and organic bases.

[0213] When a Grignard reaction is performed in each step, examples of the Grignard reagent include arylmagnesium halides such as phenylmagnesium bromide; and alkylmagnesium halides such as methylmagnesium bromide and isopropylmagnesium bromide. Grignard reagents can be prepared by a known method, for example, by reacting an alkyl halide or aryl halide with metallic magnesium using ether or tetrahydrofuran as a solvent.

[0214] When a Knoevenagel condensation reaction is performed in each step, an active methylene compound sandwiched between two electron-withdrawing groups (e.g., malonic acid, diethyl malonate, malononitrile, etc.) and a base (e.g., organic bases, metal alkoxides, inorganic bases) are used as reagents.

[0215] When a Vilsmeier-Haack reaction is performed in each step, phosphoryl chloride and an amide derivative (e.g., N,N-dimethylformamide, etc.) are used as reagents.

[0216] When azidation reactions of alcohols, alkyl halides, and sulfonate esters are performed in each step, examples of the azidation agents to be used include diphenylphosphoryl azide (DPPA), trimethylsilyl azide, and sodium azide. For example, when azidation of alcohols is performed, there are methods using diphenylphosphoryl azide and 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) and methods using trimethylsilyl azide and Lewis acid.

[0217] When a reductive amination reaction is performed in each step, examples of the reducing agents to be used include sodium triacetoxyborohydride, sodium cyanoborohydride, hydrogen, and formic acid. When the substrate is an amine compound, examples of the carbonyl compounds to be used include paraformaldehyde, aldehydes such as acetaldehyde, and ketones such as cyclohexanone. When the substrate is a carbonyl compound, examples of the amines to be used include primary amines such as ammonia and methylamine; and secondary amines such as dimethylamine.

[0218] When a Mitsunobu reaction is performed in each step, azodicarboxylates (e.g., diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD)) and triphenylphosphine are used as reagents.

[0219] When an esterification reaction, an amidation reaction, or a urea reaction is performed in each step, examples of the reagents to be used include acyl halides such as esters, acid chlorides, and acid bromides; activated carboxylic acids such as acid anhydrides, active esters, and sulfates; and the like. Examples of the activators for carboxylic acids include carbodiimide-based condensing agents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N,N’-dicyclohexylcarbodiimide (DCC); triazine-based condensing agents such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride-n-hydrate (DMT-MM); carbonate ester condensing agents such as 1,1-carbonyldiimidazole (CDI); diphenylphosphoryl azide (DPPA); benzotriazol-1-yloxy-trisdimethylaminophosphonium salt (BOP reagent); 2-chloro-1-methyl-pyridinium iodide (Mukaiyama reagent); thionyl chloride; lower alkyl haloformates such as ethyl chloroformate; O-(7-aza benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU); sulfuric acid; and combinations of these. Additives such as 1-hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (HOSu), and dimethylaminopyridine (DMAP) may also be added to the reaction.

[0220] When a coupling reaction is performed in each step, examples of the metal catalyst to be used include palladium compounds such as palladium acetate (II), tetrakis(triphenylphosphine)palladium (0), dichlorobis(triphenylphosphine)palladium (II), dichlorobis(triethylphosphine)palladium (II), tris(dibenzylideneacetone)dipalladium (0), 1,1’-bis(diphenylphosphino)ferrocenepalladium (II) chloride, and palladium acetate (II); nickel compounds such as tetrakis(triphenylphosphine)nickel (0); rhodium compounds such as tris(triphenylphosphine)rhodium (III) chloride; cobalt compounds; copper compounds such as copper oxide and copper iodide (I); and platinum compounds. A base may also be added to the reaction, and examples of such bases include inorganic bases and basic salts.

[0221] When a thiocarbonylation reaction is performed in each step, diphosphorus pentasulfide is typically used as the thiocarbonylation agent. In addition to diphosphorus pentasulfide, a reagent having a 1,3,2,4-dithiadiphosphetane-2,4-disulfide structure such as 2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide (Lowesson reagent) may also be used.

[0222] When a Wohl-Ziegler reaction is performed in each step, examples of the halogenating agent to be used include N-iodosuccinimide, N-bromosuccinimide (NBS), N-chlorosuccinimide (NCS), bromine, and sulfuryl chloride. Furthermore, the reaction can be accelerated by adding a radical initiator such as heat, light, benzoyl peroxide, or azobisisobutyronitrile to the reaction.

[0223] When a halogenation reaction is performed in each step, examples of the halogenating agent to be used include halogens, hydrohalic acids, and acid halides of inorganic acids. Specifically, chlorine, hydrochloric acid, thionyl chloride, and phosphorus oxychloride are used for chlorination, and bromine, 48% hydrobromic acid, and combinations thereof are used for bromination. In addition, a method of obtaining an alkyl halide from an alcohol by the reaction of triphenylphosphine with carbon tetrachloride or carbon tetrabromide or the like may be used. Alternatively, a method of synthesizing an alkyl halide through a two-step reaction in which an alcohol is converted into a sulfonate ester and then reacted with lithium bromide, lithium chloride or sodium iodide may be used.

[0224] When the Arbuzov reaction is performed in each step, examples of the reagents to be used include alkyl halides such as ethyl bromoacetate; and phosphites such as triethyl phosphite and tri(isopropyl)phosphite.

[0225] When a sulfone esterification reaction is performed in each step, examples of the sulfonating agent to be used include methanesulfonyl chloride, p-toluenesulfonyl chloride, methanesulfonic anhydride, p-toluenesulfonic anhydride, trifluoromethanesulfonic anhydride, and the like.

[0226] When a hydrolysis reaction is performed in each step, an acid or a base is used as the reagent. When an acid hydrolysis reaction of t-butyl ester is performed, formic acid or triethylsilane may be added to reductively trap the by-produced t-butyl cation.

[0227] When a dehydration reaction is performed in each step, examples of the dehydrating agent to be used include sulfuric acid, diphosphorus pentoxide, phosphorus oxychloride, N,N’-dicyclohexylcarbodiimide, alumina, and polyphosphoric acid.

[0228] When a decarboxylation reaction is performed in each step, an acid may be used. Examples of acids include inorganic acids and organic acids.

[0229] When a nucleophilic substitution reaction is performed in each step, a base may be used. Examples of bases include metal alkoxides, inorganic bases, and organic bases.

[0230] In another embodiment, cationic lipids described in WO 2011 / 153493 can be mentioned.

[0231] Among the cationic lipids described in WO 2011 / 153493, cationic lipids represented by the following structural formulas are more preferable.

[0232] and salts thereof.

[0233] In another embodiment, cationic lipids described in WO 2013 / 126803 can be mentioned.

[0234] Among the cationic lipids described in WO 2013 / 126803, a cationic lipid represented by the following structural formula is more preferable.

[0235] and a salt thereof.

[0236] In another embodiment, cationic lipids K-E12, H-A12, Y-E12, G-O12, K-A12, R-A12, cKK-E12, cPK-E12, PK1K-E12, PK500-E12, cQK-E12, cKK-A12, KK-A12, PK-4K-E12, cWK-E12, PK500-O12, PK1K-O12, cYK-E12, cDK-E12, cSK-E12, cEK-E12, cMK-E12, cKK-O12, cIK-E12, cKK-E10, cKK-E14, and cKK-E16 synthesized by the following scheme described in Dong et al. (Proc Natl Acad Sci U S A. 2014 Apr 15; 111(15):5753) can be mentioned.

[0237] [Corrected under Rule 26, 20.02.2026]

[0238] Among the above-mentioned cationic lipids, cKK-E12, cKK-E14 are more preferable.

[0239] In another embodiment, cationic lipids C14-98, C18-96, C14-113, C14-120, C14-120, C14-110, C16-96, and C12-200 synthesized by the following scheme described in Love KT et al. (Proc Natl Acad Sci U S A. 2010 May 25; 107(21):9915) can be mentioned.

[0240]

[0241] Among the above-mentioned cationic lipids, C14-110, C16-96 and C12-200 are more preferable.

[0242] In a particularly preferable embodiment, a cationic lipid represented by the following formula (I) (hereinafter to be also referred to as “compound (I)”) can be mentioned.

[0243]

[0244] whereinL1is a C1-22alkylene group, a C2-22alkenylene group or a C3-22alkadienylene group,n is an integer of 0 or 1,R1is(1) a hydrogen atom,(2) a linear C1-22alkyl group optionally substituted by one or two substituents selected from a linear C1-22alkyl group and a linear C2-22alkenyl group,(3) a linear C2-22alkenyl group optionally substituted by one or two substituents selected from a linear C1-22alkyl group and a linear C2-22alkenyl group, or(4) a linear C3-22alkadienyl group optionally substituted by one or two substituents selected from a linear C1-22alkyl group and a linear C2-22alkenyl group,R2is -CH2-O-CO-R5, -CH2-CO-O-R5or -R5,R3is -CH2-O-CO-R6, -CH2-CO-O-R6or -R6,R4is a hydrogen atom, -CH2-O-CO-R7, -CH2-CO-O-R7or -R7,R5, R6and R7are each independently(1) a linear C1-22alkyl group optionally substituted by one or two substituents selected from a linear C1-22alkyl group and a linear C2-22alkenyl group,(2) a linear C2-22alkenyl group optionally substituted by one or two substituents selected from a linear C1-22alkyl group and a linear C2-22alkenyl group, or(3) a linear C3-22alkadienyl group optionally substituted by one or two substituents selected from a linear C1-22alkyl group and a linear C2-22alkenyl group, andR8and R9are each independently a C1-6alkyl group,or a salt thereof.

[0245] L1is a C1-22alkylene group, a C2-22alkenylene group or a C3-22alkadienylene group.L1is preferably a C1-22alkylene group.L1is more preferably a C1-12alkylene group.L1is further preferably a C1-6alkylene group.

[0246] n is an integer of 0 or 1.n is preferably an integer of 1.

[0247] R1is(1) a hydrogen atom,(2) a linear C1-22alkyl group optionally substituted by one or two substituents selected from a linear C1-22alkyl group and a linear C2-22alkenyl group,(3) a linear C2-22alkenyl group optionally substituted by one or two substituents selected from a linear C1-22alkyl group and a linear C2-22alkenyl group, or(4) a linear C3-22alkadienyl group optionally substituted by one or two substituents selected from a linear C1-22alkyl group and a linear C2-22alkenyl group.R1is preferably(1) a hydrogen atom,(2) a linear C1-22alkyl group (preferably linear C6-12alkyl group) optionally substituted by one or two linear C1-22alkyl groups (preferably linear C6-12alkyl groups), or(3) a linear C2-22alkenyl group (preferably linear C6-12alkenyl group) optionally substituted by one or two linear C2-22alkenyl groups (preferably linear C6-12alkenyl groups).R1is particularly preferably a hydrogen atom.

[0248] R2is -CH2-O-CO-R5, -CH2-CO-O-R5or -R5.R2is preferably -CH2-O-CO-R5or -R5.R2is more preferably -CH2-O-CO-R5.

[0249] R3is -CH2-O-CO-R6, -CH2-CO-O-R6or -R6.R3is preferably -CH2-O-CO-R6or -R6.R3is more preferably -CH2-O-CO-R6.

[0250] R4is a hydrogen atom, -CH2-O-CO-R7, -CH2-CO-O-R7or -R7.R4is preferably a hydrogen atom or -CH2-O-CO-R7.R4is more preferably -CH2-O-CO-R7.

[0251] R5, R6and R7are each independently(1) a linear C1-22alkyl group optionally substituted by one or two substituents selected from a linear C1-22alkyl group and a linear C2-22alkenyl group,(2) a linear C2-22alkenyl group optionally substituted by one or two substituents selected from a linear C1-22alkyl group and a linear C2-22alkenyl group, or(3) a linear C3-22alkadienyl group optionally substituted by one or two substituents selected from a linear C1-22alkyl group and a linear C2-22alkenyl group.

[0252] R5, R6and R7are each independently preferably(1) a linear C1-22alkyl group (preferably linear C4-18alkyl group) optionally substituted by one or two linear C1-22alkyl groups (preferably linear C1-10alkyl groups),(2) a linear C2-22alkenyl group (preferably linear C4-18alkenyl group), or(3) a linear C3-22alkadienyl group (preferably linear C4-18alkadienyl group).

[0253] R5, R6and R7are each independently more preferably(1) a linear C1-22alkyl group (preferably linear C4-18alkyl group) optionally substituted by one or two linear C1-22alkyl groups (preferably linear C1-10alkyl groups), or(2) a linear C2-22alkenyl group (preferably linear C4-18alkenyl group).

[0254] R8and R9are each independently a C1-6alkyl group.R8and R9are each independently a C1-3alkyl group (preferably methyl).

[0255] Preferably, compound (I) is a compound of the above-mentioned formula (I) whereinL1is a C1-22alkylene group (preferably C1-12alkylene group, more preferably C1-6alkylene group),n is an integer of 1,R1is(1) a hydrogen atom,(2) a linear C1-22alkyl group (preferably linear C6-12alkyl group) optionally substituted by one or two linear C1-22alkyl groups (preferably linear C6-12alkyl groups), or(3) a linear C2-22alkenyl group (preferably linear C6-12alkenyl group) optionally substituted by one or two linear C2-22alkenyl groups (preferably linear C6-12alkenyl groups),R2is -CH2-O-CO-R5or -R5,R3is -CH2-O-CO-R6or -R6,R4is a hydrogen atom or -CH2-O-CO-R7,R5, R6and R7are each independently(1) a linear C1-22alkyl group (preferably linear C4-18alkyl group) optionally substituted by one or two linear C1-22alkyl groups (preferably linear C1-10alkyl groups),(2) a linear C2-22alkenyl group (preferably linear C4-18alkenyl group), or(3) a linear C3-22alkadienyl group (preferably linear C4-18alkadienyl group), andR8and R9are each independently a C1-6alkyl group (preferably C1-3alkyl group, particularly preferably methyl).

[0256] More preferably, compound (I) is a compound of the above-mentioned formula (I) whereinL1is a C1-12alkylene group (preferably C1-6alkylene group),n is an integer of 1,R1is a hydrogen atom,R2is -CH2-O-CO-R5,R3is -CH2-O-CO-R6,R4is -CH2-O-CO-R7,R5, R6and R7are each independently(1) a linear C1-22alkyl group (preferably linear C4-18alkyl group) optionally substituted by one or two linear C1-22alkyl groups (preferably linear C1-10alkyl groups),(2) a linear C2-22alkenyl group (preferably linear C4-18alkenyl group), or(3) a linear C3-22alkadienyl group (preferably linear C4-18alkadienyl group), andR8and R9are each independently a C1-6alkyl group (preferably C1-3alkyl group, particularly preferably methyl).

[0257] More preferably, compound (I) is a compound of the above-mentioned formula (I) whereinL1is a C1-6alkylene group,n is an integer of 1,R1is a hydrogen atom,R2is -CH2-O-CO-R5,R3is -CH2-O-CO-R6,R4is -CH2-O-CO-R7,R5, R6and R7are each independently(1) a linear C1-22alkyl group (preferably linear C4-18alkyl group) optionally substituted by one or two linear C1-22alkyl groups (preferably linear C1-10alkyl groups), or(2) a linear C2-22alkenyl group (preferably linear C4-18alkenyl group), andR8and R9are each independently a C1-3alkyl group (preferably methyl).

[0258] A salt of the compound represented by the above-mentioned each structural formula is preferably a pharmacologically acceptable salt. Examples thereof include salts with inorganic bases (e.g., alkali metal salts such as sodium salt, potassium salt and the like; alkaline earth metal salts such as calcium salt, magnesium salt and the like; aluminum salt, ammonium salt), salts with organic bases (e.g., salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, tromethamine[tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, benzylamine, dicyclohexylamine, N,N-dibenzyl ethylenediamine), salts with inorganic acids (e.g., salts with hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydrogen iodide acid, nitric acid, sulfuric acid, phosphoric acid), salts with organic acids (salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid), salts with basic amino acids (salts with arginine, lysine, ornithine) or salts with acidic amino acids (salts with aspartic acid, glutamic acid).

[0259] The ratio (mol%) of the cationic lipid to the total lipids present in the lipid nanoparticle of the present invention is, for example, about 10% to about 80%, preferably about 20% to about 70%, more preferably about 40% to about 60%; however, the ratio is not limited to these.

[0260] Only one kind of the above-mentioned cationic lipid may also be used or two or more kinds thereof may be used in combination. When multiple cationic lipids are used, the ratio of the whole cationic lipid is preferably as mentioned above.

[0261] (c) Non-cationic lipidIn the present specification, the “non-cationic lipid” means a lipid other than the cationic lipid, and is a lipid that does not have a net positive electric charge at a selected pH such as physiological pH and the like. Examples of the non-cationic lipid used in the lipid nanoparticle of the present invention include phospholipid, steroids, PEG lipid and the like.

[0262] To enhance the delivery of nucleic acid encoding CAR or exogenous TCR into the target immunocyte, the phospholipid is not particularly limited as long as it stably maintains nucleic acid and does not inhibit fusion with cell membranes (plasma membrane and organelle membrane). For example, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidic acid, palmitoyloleoylphosphatidyl choline, lysophosphatidyl choline, lysophosphatidyl ethanolamine, dipalmitoylphosphatidyl choline, dioleoylphosphatidyl choline, distearoylphosphatidyl choline, dilinolenoylphosphatidyl choline and the like can be mentioned.

[0263] Preferred phospholipids include distearoylphosphatidyl choline (DSPC), dioleoylphosphatidyl choline (DOPC), dipalmitoylphosphatidyl choline (DPPC), dioleoylphosphatidyl glycerol (DOPG), palmitoyloleoylphosphatidyl glycerol (POPG), dipalmitoylphosphatidyl glycerol (DPPG), dioleoyl-phosphatidyl ethanolamine (DOPE), palmitoyloleoylphosphatidyl choline (POPC), palmitoyloleoyl-phosphatidyl ethanolamine (POPE), and dioleoylphosphatidyl ethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate (DOPE-mal), more preferably DOPC, DPPC, POPC, and DOPE.

[0264] The ratio (mol%) of the phospholipid to the total lipids present in the lipid nanoparticle of the present invention may be, for example, about 0% to about 90%, preferably about 5% to about 30%, more preferably about 8% to about 15%.

[0265] Only one kind of the above-mentioned phospholipid may be used or two or more kinds thereof may be used in combination. When multiple phospholipids are used, the ratio of the whole phospholipid is preferably as mentioned above.

[0266] As the steroids, cholesterol, 5α-cholestanol, 5β-coprostanol, cholesteryl-(2’-hydroxy)-ethylether, cholesteryl-(4’-hydroxy)-butylether, 6-ketocholestanol, 5α-cholestane, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate can be mentioned, preferably cholesterol.

[0267] The ratio (mol%) of the steroid to the total lipids present in the lipid nanoparticle of the present invention when steroids are present may be, for example, about 10% to about 60%, preferably about 12% to about 58%, more preferably about 20% to about 55%.

[0268] Only one kind of the above-mentioned steroid may be used or two or more kinds thereof may be used in combination. When multiple steroids are used, the ratio of the whole steroid is preferably as mentioned above.

[0269] In the present specification, the “PEG lipid” means any complex of polyethylene glycol (PEG) and lipid. PEG lipid is not particularly limited as long as it has an effect of suppressing aggregation of the lipid nanoparticle of the present invention. For example, PEG conjugated with dialkyloxypropyl (PEG-DAA), PEG conjugated with diacylglycerol (PEG-DAG) (e.g., SUNBRIGHT GM-020 (NOF CORPORATION)), PEG conjugated with phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated with ceramide (PEG-Cer), PEG conjugated with cholesterol (PEG-cholesterol), or derivatives thereof, or mixtures thereof, mPEG2000-1,2-Di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG), 1-[8’-(1,2-dimyristoyl-3-propanoxy)-carboxamide-3’,6-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethylene glycol) (2KPEG-DMG) and the like can be mentioned. Preferred PEG lipid includes PEG-DAG, PEG-DAA, PEG-PE, PEG-Cer, and a mixture of these, more preferably, a PEG-DAA conjugate selected from the group consisting of a PEG-didecyl oxypropyl conjugate, a PEG-dilauryl oxypropyl conjugate, a PEG-dimyristyl oxypropyl conjugate, a PEG-dipalmityl oxypropyl conjugate, a PEG-distearyl oxypropyl conjugate, and mixtures thereof.

[0270] In addition to the methoxy group, maleimide group, thiol group, N-hydroxysuccinimidyl group, azide group, alkyne group such as DBCO (dibenzocyclooctyne) and the like for binding the T cell targeting ligand described later can be used as the free end of PEG. For example, SUNBRIGHT DSPE-020MA01, SUNBRIGHT DSPE-020GS (NOF) or DSPE-PEG(2000) Azide (AVANTI) can be used as a PEG lipid having a functional group for binding a T cell-targeting ligand (sometimes to be referred to as “terminal reactive PEG lipid” in the present specification).

[0271] The ratio (mol%) of the PEG lipid to the total lipids present in the lipid nanoparticle of the present invention may be, for example, about 0% to about 20%, preferably about 0.1% to about 5%, more preferably about 0.7% to about 2%.

[0272] Only one kind of the above-mentioned PEG lipid may be used or two or more kinds thereof may be used in combination. When multiple PEG lipids are used, the ratio of the whole PEG lipid is preferably as mentioned above.

[0273] The lipid nanoparticle of the present invention is used for gene transfer and expression of CAR or exogenous TCR in the immunocytes, particularly the immunocytes that express CD7, for example, T cells which are responsible for cellular immunity among acquired immunity and NK cells which are responsible for innate immunity. Therefore, the lipid nanoparticle of the present invention may further contain a ligand that may target the lipid nanoparticle to immunocytes, particularly T cells and / or NK cells, for efficient delivery to targeted immunocytes, particularly in vivo.

[0274] (d) Ligand capable of targeting lipid nanoparticle to an immunocyteThe ligand capable of targeting the lipid nanoparticle of the present invention to immune cells is not particularly limited as long as it is a polypeptide that can target immune cells, particularly cells expressing CD7. In other words, the ligand capable of targeting the lipid nanoparticle of the present invention to immune cells is a polypeptide comprising a binding domain for CD7. Here, the “binding domain” is synonymous with the binding domain that constitutes the above-mentioned CAR. However, since CAR needs to be prepared as a nucleic acid encoding same, restrictions occur and single-chain antibodies are generally used in many cases. Since the binding domain as an immunocyte targeting ligand is contained in a protein state in the lipid nanoparticle of the present invention, not only single-chain antibodies, but also any other antibody fragments, such as complete antibody molecules, Fab, F(ab’)2, Fab’, Fv, reduced antibody (rIgG), dsFv, sFv, diabody, triabody, and the like, can also be used preferably. Fab without an Fc moiety can be preferably used, especially for delivery to the target immunocyte in vivo. These antibody fragments can be prepared by treating the complete antibody (e.g., IgG) with a reducing agent (e.g., 2-mercaptoethanol, dithiothreitol) or peptidase (e.g., papain, pepsin, ficin), or by using a genetic recombination operation.

[0275]

[0276] In one embodiment, the ligand is a binding domain comprisingheavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 34,heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 35,heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 36,light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 37,light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 38, andlight chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 39.

[0277] In one embodiment, the ligand is a binding domain comprisingheavy chain CDR1 consisting of an amino acid sequence shown in SEQ ID NO: 34, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids),heavy chain CDR2 consisting of an amino acid sequence shown in SEQ ID NO: 35, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids),heavy chain CDR3 consisting of an amino acid sequence shown in SEQ ID NO: 36, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids),light chain CDR1 consisting of an amino acid sequence shown in SEQ ID NO: 37, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids),light chain CDR2 consisting of an amino acid sequence shown in SEQ ID NO: 38, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids), andlight chain CDR3 consisting of an amino acid sequence shown in SEQ ID NO: 39, which may be substituted, deleted, inserted, or added by one or several amino acids (e.g. one, two or three amino acids).

[0278] In another embodiment, the binding domain is a binding domain comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 40 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 41.

[0279] In another embodiment, the binding domain is a binding domain comprising a heavy chain variable region comprising an amino acid sequence that is at least 80%, preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, identical to the amino acid sequence shown in SEQ ID NO: 40 and a light chain variable region comprising an amino acid sequence that is at least 80%, preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, identical to the amino acid sequence shown in SEQ ID NO: 41.

[0280] In other embodiment, the immunocyte targeting ligand comprises the amino acid sequence shown in SEQ ID NO: 42 and 43.

[0281] In other embodiment, the immunocyte targeting ligand comprises an amino acid sequence that is at least 80%, preferably 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, identical to the amino acid sequence shown in SEQ ID NO: 42 or 43, and has CD7-binding activity. The binding activity of the immunocyte targeting ligand to CD7 can be confirmed by any method known in the pertinent technical field.

[0282] When the immunocyte targeting ligand is a complete antibody molecule, commercially available anti-CD7 antibody, etc. can be used, or the ligand can be isolated from the culture of the cells producing the antibody. On the other hand, when the ligand is any one of the aforementioned binding domain (antibody fragment), the nucleic acid encoding the binding domain, such as anti-CD7 antibody, is isolated in the same way as in the nucleic acid encoding the binding domain constituting the said CAR is obtained, and the binding domain can be recombinantly produced using the same.

[0283] In the lipid nanoparticle of the present invention, the immunocyte targeting ligand may bind to the outer shell in any manner as long as it is present on the surface of the lipid nanoparticle. For example, when a terminally reactive PEG lipid is contained as a non-cationic lipid, the ligand can be added to the terminal of PEG. For example, lipid nanoparticles labeled with a ligand (antibody) can be prepared by reacting a PEG lipid (e.g., SUNBRIGHT DSPE-0200MA) with a maleimide group introduced into the terminal with the thiol group of the above-mentioned reducing antibody (sometimes referred to as “antibody-LNP”).

[0284] 2. Production of lipid nanoparticle of the present inventionThe lipid nanoparticle of the present invention can be produced, for example, by the method described in US9,404,127. After preparation of the lipid nanoparticles, an immunocyte targeting ligand can be chemically bound to the lipid nanoparticles. As described in WO 2016 / 021683, for example, an organic solvent solution of the above-mentioned components (b) and (c) is prepared, the organic solvent solution is mixed with water or a buffer solution of (a) to prepare lipid nanoparticles, and then the immunocyte targeting ligand is chemically bound to produce same. Alternatively, for example, an organic solvent solution of the above-mentioned components (b) and (c) is prepared, the organic solvent solution is mixed with water or a buffer solution of (a) to prepare lipid nanoparticles, and then the targeting ligand-PEG-lipid, which is obtained from the reaction of terminal reactive PEG-lipid and targeting ligand are inserted into the lipid nanoparticles. The mixing ratio (molar ratio) of cationic lipid, phospholipid, cholesterol, and PEG lipid is, for example, 40 to 60:0 to 20:0 to 50:0 to 5, but the ratio is not limited thereto. When PEG lipid is blended as a non-cationic lipid and a immunocyte targeting ligand is added to the terminal of PEG, the mixing ratio (molar ratio) of the PEG lipid and the ligand may be, for example, 200:1 to 1:20. The above-mentioned PEG lipid may contain terminal reactive PEG at a ratio (mol%) of about 10% to about 100%. The above-mentioned mixing can be conducted using a pipette, a micro fluid mixing system (e.g. Asia microfluidic system (Syrris)) or Nanoassemblr (Precision Nanosystems)). The obtained lipid particles may be subject to purification by gel filtration, dialysis or sterile filtration.

[0285] The concentration of the total lipid component in the organic solvent solution is preferably 0.5 to 100 mg / mL.

[0286] As the organic solvent, for example, methanol, ethanol, 1-propanol, 2-propanol, 1- butanol, tert-butanol, acetone, acetonitrile, N,N-dimethylformamide, dimethylsulfoxide, or a mixture thereof can be recited. The organic solvent may contain 0 to 20% of water or a buffer solution. As the buffer solution, acidic buffer solutions (e.g. acetate buffer solution, citrate buffer solution) or neutral buffer solutions (e.g. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, (HEPE) buffer solution, tris(hydroxymethyl)aminomethane (Tris) buffer solution, a phosphate buffer solution, phosphate buffered saline (PBS)) can be recited.

[0287] In the case where a micro fluid mixing system is used for mixing, preference is given to mixing 1 part by volume of an organic solvent solution with 1 to 5 parts by volume of water or a buffer solution. In addition, in said system, the flow rate of the mixture (a mixture solution of an organic solvent solution and water or a buffer solution) is preferably 0.1 to 10 mL / min, and the temperature preferably is 4 to 45°C.

[0288] When a lipid particle dispersion is produced as described above, the dispersion containing components (a) to (d) can be produced by adding a nucleic acid encoding CAR or exogenous TCR to water or buffer solution. Addition of the nucleic acid in a manner to render the concentration thereof the active ingredient in water or a buffer solution 0.05 to 2.0 mg / mL is preferable.

[0289] In addition, the lipid nanoparticle of the present invention can also be produced by admixing a lipid particle dispersion with the nucleic acid by a method known per se.

[0290] In the lipid nanoparticle of the present invention, the content of the nucleic acid is preferably 1 - 20 wt%. The content can be measured using Quant-iTTMRibogreen (Registered) (Invitrogen). In the lipid nanoparticle of the present invention, the encapsulation ratio of the nucleic acid can be calculated based on the difference in fluorescence intensity in the presence or absence of the addition of a surfactant (e.g., Triton-X100).

[0291] A dispersion medium can be substituted with water or a buffer solution by dialysis. For the dialysis, ultrafiltration membrane of molecular weight cutoff 10 to 20K is used to carry out at 4°C to room temperature. The dialysis may repeatedly be carried out. For the dialysis, tangential flow filtration may be used.

[0292] The ratio (weight ratio) of the nucleic acid and the lipid in the lipid nanoparticle of the present invention obtained as mentioned above is about 0.01 to about 0.2.

[0293] The average particle size of the lipid nanoparticle of the present invention is preferably 10 to 200 nm. The average particle size of the lipid particles can be calculated using, for example, Zetasizer Nano ZS (Malvern Instruments) on cumulant analysis of an autocorrelation function.

[0294] 4. Medicament containing the lipid nanoparticle of the present inventionThe present invention provides a medicament containing the lipid nanoparticle of the present invention (hereinafter to be abbreviated as “the medicament of the present invention”).

[0295] The medicament of the present invention containing the lipid nanoparticle of the present invention is preferably prepared as a pharmaceutical composition by mixing the lipid nanoparticle with known pharmaceutically acceptable carriers (including excipient, diluent, bulking agent, binder, lubricant, flow aid, disintegrant, surfactant, and the like) and conventional additives, and the like. The excipients are well known to those of ordinary skill in the art and include, for example, phosphate-buffered saline (e.g., 0.01M phosphate, 0.138M NaCl, 0.0027M KCl, pH 7.4), aqueous solutions containing mineral acid salts such as hydrochloride, hydrobromate, phosphate, sulfate, and the like, saline solutions, solutions of glycol, ethanol, and the like, and salts of organic acids such as acetate, propionate, malonate, benzoate, and the like. In addition, adjuvants such as wetting agent or emulsifier, and pH buffering agents can also be used. In addition, preparation adjuvants such as suspension agent, preservative, stabilizer and dispersing agent may also be used. Alternatively, the above-mentioned pharmaceutical composition may be in a dry form which is reconstituted with a suitable sterile liquid prior to use. The pharmaceutical composition may be orally or parenterally administered systemically or topically, depending on the form in which it is prepared (oral agents such as tablet, pill, capsule, powder, granule, syrup, emulsion, suspension and the like; parenteral agents such as injection, drip transfusion, external preparation, suppository and the like). For parenteral administration, intravenous administration, intradermal administration, subcutaneous administration, rectal administration, transdermal administration and the like are available. When used in an injectable form, acceptable buffering agent, solubilizing agent, isotonic agent and the like can also be added.

[0296] The dosage of the medicament of the present invention containing the lipid nanoparticle of the present invention is, for example, in the range of 0.001 mg to 10 mg as the amount of a nucleic acid encoding CAR or exogenous TCR, per 1 kg body weight per dose. For example, when administered to a human patient, the dosage is in the range of 0.0001 to 50 mg for a patient weighing 60 kg. The above-mentioned dosage is an example, and the dosage can be appropriately selected according to the type of nucleic acid to be used, administration route, age, weight, symptoms, etc. of the subject of administration or patient.

[0297] By administration to a mammal (e.g., human or other mammal (e.g., mouse, rat, hamster, rabbit, cat, dog, bovine, sheep, monkey), preferably, human), the medicament of the present invention containing the lipid nanoparticle of the present invention can induce the expression of CAR or exogenous TCR in immunocytes, e.g., T cell, NK cell (to be also referred to as “in vivo immunocyte”, “in vivo T cell” or “in vivo NK cell” in the present specification) in the body of the animal. The in vivo immunocyte specifically recognizes cancer cells and the like expressing surface antigen targeted by CAR or exogenous TCR and kills the diseased cells, thereby demonstrating a prophylactic or therapeutic effect against the disease.

[0298] The medicament of the present invention may be a prophylactic or therapeutic drug for cancer. The cancer to be the application target of the medicament of the present invention is not particularly limited. Examples thereof include, but are not limited to, acute lymphocytic cancer, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anus, anal canal or anorectal cancer, cancer of the eye, cancer of the interhepatic bile duct, joint cancer, cervical, gallbladder or pleural cancer, nose, nasal cavity or middle ear cancer, oral cancer, vulvar cancer, chronic myelogenous cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia), liquid tumor, liver cancer, lung cancer (e.g., non-small cell lung cancer), lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B cell lymphoma, follicular lymphoma), malignant mesothelioma, mastocytoma, melanoma, multiple myeloma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer; peritoneal, omentum and mesenteric cancer; pharyngeal cancer, prostate cancer, colorectal cancer (e.g., rectal cancer), renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer and the like.

[0299] The present invention is explained in more detail in the following by referring to Examples which are mere exemplifications and do not limit the present invention.Example

[0300] [Example 1] Preparation of LNP(Production of hCD7Fab-DBCO)Fab-DBCO was produced by performing the following two steps. The DBCO / Antibody ratio (DAR) and protein concentration were quantified by LC-MS and BCA method, respectively. The physical property evaluation results are shown in Table 7.1. Production of Fab with LPETGG-His6 at the C-terminus: Plasmid pMG2.2 vector encoding the hCD7Fab (clone: Grisnilimab) gene was introduced into CHOZN cells by using an electroporation device (Maxcyte), and the cells were cultured for 6-8 days using EX-CELL Advanced CHO Feed 1 (with glucose). Thereafter, the cells were purified using a complete Ni column and a size exclusion column of Superdex 200 to produce LPETGG-His-tagged Fab.2. Production of Fab-DBCO: LPETGG-His-tagged Fab was mixed with CaCl2and 5-(glycylglycyl-beta-alanyl)-11,12-didehydro-5,6-dihydrodibenzo[b,f]azocine in pH 7.4 HEPES buffer, and then treated with Sortase A (Protein Science, 89, 15.3.1-15.3.19). After reacting for 4 hours at room temperature, the mixture was purified using a Ni column and dialysis step to produce Fab-DBCO.

[0301]

[0302] (Preparation of LNP1, LNP3 and LNP11)Preparation of Azide-LNPA lipid mixture (cationic lipid: DPPC:Cholesterol:SUNBRIGHT GM-020:DSPE-PEG (2000) Azide=60:10.6:27:1.4:1, mol%) was dissolved in 90% EtOH to obtain a 14 mg / ml lipid solution. As the cationic lipid, 2-(((4,5-dibutylnonanoyl)oxy)methyl)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propane-1,3-diyl didecanoate described in WO2020 / 032184 was used. The mRNA (SEQ ID NO: 31) encoding the GPC3-targeting CAR (GC33) (SEQ ID NO: 28) was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a 0.2 mg / ml nucleic acid solution. The obtained respective lipid solutions were mixed with the nucleic acid solution at room temperature using a NanoassemblrTMdevice (Precision Nanosystems) at a flow rate ratio of 3 ml / min:6 ml / min to obtain a dispersion containing the composition. The obtained dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialysis (20K fractional molecular weight, Thermo Fisher Scientific). The dispersion was then concentrated by ultrafiltration using Amicon Ultra (30K fractional molecular weight, Merck) and filtered using a 0.2 μm syringe filter. The final sucrose concentration was adjusted to 20% and stored at 4°C.

[0303] Binding reaction of hCD7Fab-DBCO and Azide-LNPThe hCD7Fab-DBCO solution was mixed with each LNP dispersion such that the molar concentration of Fab-DBCO to azide was 5 / 100, and the mixture was reacted at 25°C for 24 hours and stored at -80°C.

[0304] (Preparation of LNP2, LNP4, LNP5, LNP7, LNP10, LNP14 and LNP17)Preparation of Azide-LNPA lipid mixture (cationic lipid: DPPC:Cholesterol:SUNBRIGHT GM-020:DSPE-PEG (2000) Azide=60:10.6:27:1.4:1, mol%) was dissolved in 90% EtOH to obtain a 14 mg / ml lipid solution. As the cationic lipid, 2-(((4,5-dibutylnonanoyl)oxy)methyl)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propane-1,3-diyl didecanoate described in WO2020 / 032184 was used. The mRNA encoding the GFP was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a 0.2 mg / ml nucleic acid solution. The obtained respective lipid solutions were mixed with the nucleic acid solution at room temperature using a NanoassemblrTMdevice (Precision Nanosystems) at a flow rate ratio of 3 ml / min:6 ml / min to obtain a dispersion containing the composition. The obtained dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialysis (20K fractional molecular weight, Thermo Fisher Scientific). The dispersion was then concentrated by ultrafiltration using Amicon Ultra (30K fractional molecular weight, Merck) and filtered using a 0.2 μm syringe filter. The final sucrose concentration was adjusted to 20% and stored at 4°C.

[0305] Binding reaction of hCD7Fab-DBCO and Azide-LNPThe hCD7Fab-DBCO solution was mixed with each LNP dispersion such that the molar concentration of Fab-DBCO to azide was 5 / 100, and the mixture was reacted at 25°C for 24 hours and stored at -80°C.

[0306] (Preparation of LNP6 and LNP8)Preparation of Azide-LNPA lipid mixture (cationic lipid: DPPC:Cholesterol:SUNBRIGHT GM-020:DSPE-PEG (2000) Azide=60:10.6:27:1.4:1, mol%) was dissolved in 90% EtOH to obtain a 14 mg / ml lipid solution. As the cationic lipid, 2-(((4,5-dibutylnonanoyl)oxy)methyl)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propane-1,3-diyl didecanoate described in WO2020 / 032184 was used. The mRNA (SEQ ID NO: 45) encoding the mouse GPC3-targeting CAR (GC33) (SEQ ID NO: 44) was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a 0.2 mg / ml nucleic acid solution. The obtained respective lipid solutions were mixed with the nucleic acid solution at room temperature using a NanoassemblrTMdevice (Precision Nanosystems) at a flow rate ratio of 3 ml / min:6 ml / min to obtain a dispersion containing the composition. The obtained dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialysis (20K fractional molecular weight, Thermo Fisher Scientific). The dispersion was then concentrated by ultrafiltration using Amicon Ultra (30K fractional molecular weight, Merck) and filtered using a 0.2 μm syringe filter. The final sucrose concentration was adjusted to 20% and stored at 4°C.

[0307] Binding reaction of hCD7Fab-DBCO and Azide-LNPThe hCD7Fab-DBCO solution was mixed with each LNP dispersion such that the molar concentration of Fab-DBCO to azide was 5 / 100, and the mixture was reacted at 25°C for 24 hours and stored at -80°C.

[0308] (Preparation of LNP9)Preparation of Azide-LNPA lipid mixture (cationic lipid: DPPC:Cholesterol:SUNBRIGHT GM-020:DSPE-PEG (2000) Azide=60:10.6:27:1.4:1, mol%) was dissolved in 90% EtOH to obtain a 14 mg / ml lipid solution. As the cationic lipid, 2-(((4,5-dibutylnonanoyl)oxy)methyl)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propane-1,3-diyl didecanoate described in WO2020 / 032184 was used. The mRNA (SEQ ID NO: 32) encoding the mouse GPC3-targeting CAR (#5) (SEQ ID NO: 29) was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a 0.2 mg / ml nucleic acid solution. The obtained respective lipid solutions were mixed with the nucleic acid solution at room temperature using a NanoassemblrTMdevice (Precision Nanosystems) at a flow rate ratio of 3 ml / min:6 ml / min to obtain a dispersion containing the composition. The obtained dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialysis (20K fractional molecular weight, Thermo Fisher Scientific). The dispersion was then concentrated by ultrafiltration using Amicon Ultra (30K fractional molecular weight, Merck) and filtered using a 0.2 μm syringe filter. The final sucrose concentration was adjusted to 20% and stored at 4°C.

[0309] Binding reaction of hCD7Fab-DBCO and Azide-LNPThe hCD7Fab-DBCO solution was mixed with each LNP dispersion such that the molar concentration of Fab-DBCO to azide was 5 / 100, and the mixture was reacted at 25°C for 24 hours and stored at -80°C.

[0310] (Preparation of LNP12)Preparation of Null-LNPA lipid mixture (cationic lipid: DPPC:Cholesterol:SUNBRIGHT GM-020:DSPE-PEG (2000) Azide=60:10.6:27:1.4:1, mol%) was dissolved in 90% EtOH to obtain a 14 mg / ml lipid solution. As the cationic lipid, 2-(((4,5-dibutylnonanoyl)oxy)methyl)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propane-1,3-diyl didecanoate described in WO2020 / 032184 was used. The mRNA (SEQ ID NO: 33) encoding the CD19-targeting CAR (SEQ ID NO: 30) was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a 0.2 mg / ml nucleic acid solution. The obtained respective lipid solutions were mixed with the nucleic acid solution at room temperature using a NanoassemblrTMdevice (Precision Nanosystems) at a flow rate ratio of 3 ml / min:6 ml / min to obtain a dispersion containing the composition. The obtained dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialysis (20K fractional molecular weight, Thermo Fisher Scientific). The dispersion was then concentrated by ultrafiltration using Amicon Ultra (30K fractional molecular weight, Merck) and filtered using a 0.2 μm syringe filter. The final sucrose concentration was adjusted to 20% and stored at 4°C.

[0311] (Preparation of LNP13, LNP15, LNP16 and LNP18)Preparation of Azide-LNPA lipid mixture (cationic lipid: DPPC:Cholesterol:SUNBRIGHT GM-020:DSPE-PEG (2000) Azide=60:10.6:27:1.4:1, mol%) was dissolved in 90% EtOH to obtain a 14 mg / ml lipid solution. As the cationic lipid, 2-(((4,5-dibutylnonanoyl)oxy)methyl)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propane-1,3-diyl didecanoate described in WO2020 / 032184 was used. The mRNA (SEQ ID NO: 33) encoding the CD19-targeting CAR (SEQ ID NO: 30) was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a 0.2 mg / ml nucleic acid solution. The obtained respective lipid solutions were mixed with the nucleic acid solution at room temperature using a NanoassemblrTMdevice (Precision Nanosystems) at a flow rate ratio of 3 ml / min:6 ml / min to obtain a dispersion containing the composition. The obtained dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialysis (20K fractional molecular weight, Thermo Fisher Scientific). The dispersion was then concentrated by ultrafiltration using Amicon Ultra (30K fractional molecular weight, Merck) and filtered using a 0.2 μm syringe filter. The final sucrose concentration was adjusted to 20% and stored at 4°C.

[0312] Binding reaction of hCD7Fab-DBCO and Azide-LNPThe hCD7Fab-DBCO solution was mixed with each LNP dispersion such that the molar concentration of Fab-DBCO to azide was 5 / 100, and the mixture was reacted at 25°C for 24 hours and stored at -80°C.

[0313] (Preparation of LNP19)Preparation of Azide-LNPA lipid mixture (cationic lipid: DPPC:Cholesterol:SUNBRIGHT GM-020:DSPE-PEG (2000) Azide=60:10.6:27:1.4:1, mol%) was dissolved in 90% EtOH to obtain a 14 mg / ml lipid solution. As the cationic lipid, 2-(((4,5-dibutylnonanoyl)oxy)methyl)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propane-1,3-diyl didecanoate described in WO2020 / 032184 was used. Mixture (1:1 weight ratio) of mRNA (SEQ ID NO: 33) encoding CD19-targeting CAR (SEQ ID NO: 30) and mRNA (SEQ ID NO: 49) encoding the mbIL-15 (SEQ ID NO: 48) was dissolved in 10 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.5) to obtain a 0.2 mg / ml nucleic acid solution. The obtained respective lipid solutions were mixed with the nucleic acid solution at room temperature using a NanoassemblrTMdevice (Precision Nanosystems) at a flow rate ratio of 3 ml / min:6 ml / min to obtain a dispersion containing the composition. The obtained dispersion was dialyzed against water at room temperature for 1 hour and against PBS at 4°C for 24 hours using Slide-A-Lyzer Dialysis (20K fractional molecular weight, Thermo Fisher Scientific). The dispersion was then concentrated by ultrafiltration using Amicon Ultra (30K fractional molecular weight, Merck) and filtered using a 0.2 μm syringe filter. The final sucrose concentration was adjusted to 20% and stored at 4°C.Binding reaction of hCD7Fab-DBCO and Azide-LNPThe hCD7Fab-DBCO solution was mixed with each LNP dispersion such that the molar concentration of Fab-DBCO to azide was 5 / 100, and the mixture was reacted at 25°C for 24 hours and stored at -80°C.

[0314] (Particle size measurement)The particle size and polydispersity index (PDI) of LNP were measured by Zetasizer Nano ZS (MaIvern Panalytical).

[0315] (Nucleic acid concentration measurement)The mRNA encapsulation rate in LNP was measured using Quant-itTMRiboGreen RNA Assay Kit (Thermo Fisher Scientific). The mRNA concentration measured after dissolving LNP with 0.5% Triton X-100 was taken as the total mRNA concentration, and the mRNA concentration measured without adding Triton X-100 was taken as the mRNA concentration not encapsulated in LNP, and the mRNA encapsulation rate in LNP was calculated.

[0316] (LNP performance evaluation results)The performance evaluation results of LNP are shown in Table 8.

[0317]

[0318] [Experimental Example 1] In vivo anti-tumor efficacy in human T cell transferred tumor-bearing mouse modelIn vivo anti-tumor efficacy of GPC3 CAR (GC33, SEQ ID NO: 28)-CD7 LNP (LNP1) and GPC3 CAR (#5, SEQ ID NO: 29)-CD7 LNP (LNP9) produced in Example 1 against subcutaneously implanted human liver cancer was examined using a tumor-bearing model in which human T cells were transferred into immunodeficient mice.

[0319] NSG mice were subcutaneously inoculated with 5x106human liver cancer HepG2-RedFluc cells, in which the exogenous RedFluc gene was introduced into HepG2 cells that endogenously express human GPC3, and 7 days after inoculation, 10x106human T cells were administered into the tail vein. The human T cells used for transfer were those obtained by isolating and purifying from human PBMC and culturing for 4 days in X-VIVO15 medium containing 10 ng / mL human IL-2 and a specified amount of T cell TransAct. Three days after human T cell transfer was defined as day 0 of administration, and on days 0, 1, 3, 4, 7, and 8, GPC3 CAR (GC33)-CD7 LNP, GPC3 CAR(#5)-CD7 LNP, or GFP-CD7 LNP (LNP2) was each administered into the tail vein at a concentration of 0.8 mg / kg. As a control group, a non-treatment group in which 5.3% sucrose / PBS alone was administered after inoculation of HepG2-RedFluc cells, a T cell only transfer group in which 5.3% sucrose / PBS alone was administered after inoculation of HepG2-RedFluc cells and transfer of T cells, and an ex vivo GPC3 CAR-T cell treatment group in which CAR-T cells obtained by isolating T cells from human PBMC after inoculation of HepG2-RedFluc cells, retrovirally infecting the cells to express anti-GPC3CAR (GC33) or GPC3 CAR (#5) gene having the same amino acid sequence as the CAR mRNA introduced by LNP, and culturing the cells were administered at 2x106cells as CAR-positive cells into the tail vein on day 0 of administration were set up. The subcutaneous tumor volume of the mice was measured twice a week. In addition, the amount of residual tumor cells in the body was evaluated by measuring the chemiluminescence derived from the RedFluc gene after luciferin administration using a bioluminescence (BLI) analyzer on days 11, 14, and 17 after LNP administration.

[0320] In this test, blood, spleen, and lung were collected on day 2 (1 day after the second administration of LNP) from mice administered with LNP on days 0 and 1 after HepG2-RedFluc cell inoculation and T cell transfer, and from mice administered with ex vivo GPC3 CAR-T cells on day 0 after HepG2-RedFluc cell inoculation. The CAR-positive cell rate in human T cells was calculated by flow cytometry using a cell suspension prepared from each tissue. The mouse CD45-negative, human CD45-positive, and human CD3-positive cell fraction from which dead cells had been removed was defined as human T cell, and the CAR-positive rate in this fraction was defined according to the type of CAR binder as follows. For GPC3 CAR (GC33), the positive cell ratio to His-tagged recombinant human GPC3 and phycoerythrin-labeled anti-His antibody was defined as CAR-positive cell rate, and for GPC3 CAR (#5), the positive cell ratio to biotin-labeled protein L and Brilliant VioletTM421 (BV421)-conjugated streptavidin was defined as CAR-positive cell rate.

[0321] (Evaluation of GPC3 CAR(GC33)-CD7 LNP (LNP1))The results of changes in the tumor volume in the HepG2-RedFluc subcutaneous xenograft model are shown in Figure 1A. The horizontal axis is the number of days after the first administration of LNP or CAR-T cells to the mice, which is set as day 0, and the vertical axis is the tumor volume (tumor major axis x (tumor minor axis)2 / 2 (mm3)). The mean and standard deviation of tumor volume were calculated for each test group based on the measurement values of 5 mice per group. “Vehicle” is a group in which 5.3% sucrose / PBS was administered after tumor cell transplantation, “T+vehicle” is a group in which 5.3% sucrose / PBS was administered after tumor cell transplantation and T cell transfer, “T+GFP-CD7 LNP” is a group in which GFP-CD7 LNP (LNP2) was administered after tumor cell transplantation and T cell transfer, “T+ GPC3 CAR(GC33)-CD7 LNP” is a group in which GPC3 CAR-CD7 LNP (LNP1) was administered after tumor cell transplantation and T cell transfer, and “ex vivo GPC3 CAR(GC33)-T” is a group in which ex vivo hGPC3 CAR(GC33)-T cells were administered on day 0 and 5.3% sucrose / PBS on days 1, 3, 4, 7, and 8 after tumor cell transplantation.

[0322] As shown in Figure 1A, a decrease in the tumor volume was confirmed in the positive control, CAR-T cell treatment group (ex vivo GPC3 CAR(GC33)-T). When hGPC3 CAR(GC33)-CD7 LNP was administered, a remarkable decrease in tumor volume comparable to that of the ex vivo CAR-T cell treatment group was confirmed, compared with the administration of GFP-CD7 LNP, the T cell only transfer group (T+vehicle), and the non-treatment group (vehicle).

[0323] The results of BLI in this model are shown in Figure 1B. The chemiluminescence intensity measured in five mice per group on the number of days after the first administration of LNP or CAR-T cells is shown. As shown in Figure 1B, when hGPC3 CAR(GC33)-CD7 LNP was administered, an attenuating effect of tumor-derived chemiluminescence was confirmed compared to the GFP-CD7 LNP-administered group and the T cell only transfer group. Attenuation of chemiluminescence was also confirmed in the CAR-T cell treatment group (ex vivo GPC3 CAR(GC33)-T).

[0324] As shown in Figure 1C, a CAR positivity rate of 50% or more was detected in human T cells in the blood, spleen, and lungs of mice one day after the second administration of hGPC3 CAR(GC33)-CD7 LNP. A similar staining method confirmed a CAR positivity rate of about 80% for ex vivo GPC3 CAR(GC33)-T cells two days after administration.

[0325] Therefore, it was clarified, in a human T cell transferred tumor-bearing mouse model, that hGPC3 CAR(GC33)-CD7 LNP has the ability to induce the expression of functional CAR molecules in human T cells transferred into the mouse body, and has superior anti-tumor activity against subcutaneous human liver cancer xenograft.

[0326] (Evaluation of GPC3 CAR(#5)-CD7 LNP (LNP9))The results of the changes in the tumor volume in HepG2-RedFluc subcutaneous xenograft model are shown in Figure 2A. The horizontal axis is the number of days after the first administration of LNP or CAR-T cells to the mice, and the vertical axis is the tumor volume. The mean and standard deviation of tumor volume were calculated for each test group based on the measurement values of 5 mice per group. “Vehicle” is a group in which 5.3% sucrose / PBS was administered after tumor cell transplantation, “T+vehicle” is a group in which 5.3% sucrose / PBS was administered after tumor cell transplantation and T cell transfer, “T+GFP-CD7 LNP” is a group in which GFP-CD7 LNP (LNP2) was administered after tumor cell transplantation and T cell transfer, “T+ GPC3 CAR(#5)-CD7 LNP” is a group in which hGPC3 CAR(#5)-CD7 LNP (LNP9) was administered after tumor cell transplantation and T cell transfer, and “ex vivo GPC3 CAR(#5)-T” is a group in which ex vivo hGPC3 CAR(#5)-T cells were administered on day 0 and 5.3% sucrose / PBS on days 1, 3, 4, 7, and 8 after tumor cell transplantation.

[0327] As shown in Figure 2A, when GPC3 CAR(#5)-CD7 LNP was administered, a tumor growth suppression was confirmed, compared with the T cell only transfer group (T+vehicle), the non-treatment group (vehicle), and the GFP-CD7 LNP administration group. A decrease in tumor volume was also confirmed in the CAR-T cell treatment group which is a positive control (ex vivo GPC3 CAR(#5)-T). However, because there was a possibility that tumor pseudoprogression occurred due to accumulated T cells in the tumor tissue, chemiluminescence evaluation was performed, and the results are shown in Figure 2B. The chemiluminescence intensity measured in five mice per group on the number of days after the first administration of LNP or CAR-T cells is shown.

[0328] As shown in Figure 2B, also in this evaluation system, when GPC3 CAR(#5)-CD7 LNP was administered, an attenuating effect of tumor-derived chemiluminescence was confirmed compared to administration of GFP-CD7 LNP and T cell only transfer group (T+vehicle). Attenuation of chemiluminescence was also confirmed in the CAR-T cell treatment group (ex vivo GPC3 CAR(#5)-T).

[0329] As shown in Figure 2C, a CAR positivity rate of about 10% was detected in human T cells in the lungs of mice one day after the second administration of GPC3 CAR(#5)-CD7 LNP. GPC3 CAR(#5)-CD7 LNP was also confirmed to have the ability to induce expression of CAR molecules in human T cells transferred into the mouse body and to have anti-tumor activity against subcutaneous human liver cancer xenograft, in a human T cell transferred tumor-bearing mouse model.

[0330] These results clarified that the expression of GPC3 CAR in CD7-expressing human T cells using CD7-targeting LNPs shows superior anti-tumor activity against subcutaneous human liver cancer xenograft in a human T cell transferred tumor-bearing mouse model.

[0331] [Experimental Example 2] Treatment effect on human NK cell transferred tumor-bearing mouse modelThe treatment effect of GPC3 CAR(GC33)-CD7 LNP (LNP 3) against human lung cancer cells that were engrafted in mouse lungs by intravenous tail vein administration was examined using a tumor-bearing model in which human NK cells were transferred into immunodeficient mice.

[0332] Human lung cancer A549 cells (hGPC3-A549-RedFluc cells) (3x105cells) that had been genetically modified to express human GPC3 and exogenous RedFluc genes were inoculated into the tail vein of NOG-hIL-15 transgenic mice, and 15x106human NK cells were administered into the tail vein three days after the administration. As the NK cells used for the transfer, human NK cells that had been isolated and purified from human PBMC, cultured for 5 days in NK MACS medium (Miltenyi Biotec) containing 500 U / mL human IL-2, and, after replacement with NK MACS medium containing 500 U / mL human IL-2 and 140 U / mL human IL-15, expansion cultured for another 9 days were harvested and used. Three days after human NK cell transfer was defined as day 0 of administration, and GPC3 CAR (GC33)-CD7 LNP (LNP3) or GFP-CD7 LNP (LNP4) was administered into the tail vein at a concentration of 0.8 mg / kg on days 0, 2, 4, 7, 9, 11, 14, 16, and 18. As control groups, a non-treatment group in which 5.3% sucrose / PBS was administered after inoculation of hGPC3-A549-RedFluc cells, an NK cell only transfer group in which 5.3% sucrose / PBS was administered after inoculation of hGPC3-A549-RedFluc cells and transfer of NK cells, and a CAR-NK treatment group in which CAR-NK cells (ex vivo GPC3 CAR(GC33)-NK) obtained by isolating NK cells from human PBMC, retrovirally infecting the cells to express anti-GPC3 CAR gene having the same amino acid sequence as the CAR mRNA introduced by LNP, and culturing the cells were administered at 12x106cells as CAR-positive cells into the tail vein on day 0 of administration were set up. To evaluate changes in tumor cell amount in vivo in mice, the chemiluminescence derived from the RedFluc gene after luciferin administration was measured twice a week using a BLI analyzer.

[0333] The results of changes in the tumor volume in hGPC3-A549-RedFluc lung tumor xenograft model are shown in Figures 3A and 3B. The horizontal axis of Figure 3A indicates the number of days after the first administration of LNP or CAR-NK cells, and the vertical axis indicates the chemiluminescence value (Total Flux (p / s)) in the mouse chest including the lungs after photographing the mouse with its abdomen facing up, as the amount of residual human tumor cells in the mouse lung tissue. The mean and standard deviation of chemiluminescent value were calculated based on the measurement values of 5 mice per group (3 mice in CAR-NK administration group). “Vehicle” is a group in which 5.3% sucrose / PBS was administered after tumor cell transplantation, “NK+vehicle” is a group in which 5.3% sucrose / PBS was administered after tumor cell transplantation and NK cell transfer, “NK+GFP-CD7 LNP” is a group in which GFP-CD7 LNP was administered after tumor cell transplantation and NK cell transfer, “NK+ GPC3 CAR(GC33)-CD7 LNP” is a group in which GPC3 CAR-CD7 LNP was administered after tumor cell transplantation and NK cell transfer, and “ex vivo GPC3 CAR(GC33)-NK” is a group in which CAR-NK cells were administered on day 0 and 5.3% sucrose / PBS on days 2, 4, 7, 9, 11, 14, 16 and 18 after tumor cell transplantation.

[0334] As shown in Figure 3A, a decrease in the tumor amount was confirmed in the positive control, CAR-NK cell treatment group (ex vivo GPC3 CAR(GC33)-NK). As compared with the non-treatment group (vehicle), a decrease in the tumor amount caused by the innate cell killing activity of transferred NK cells was confirmed in the NK cell only transfer group (NK+vehicle) and the GFP-CD7 LNP administration group. When GPC3 CAR(GC33)-CD7 LNP was administered, a greater decreasing effect on the tumor cell volume was observed.

[0335] In Figure 3B, in hGPC3-A549-RedFluc lung tumor engraftment model, the chemiluminescence intensity measured in 5 or 3 mice per group on the number of days after the initial administration of LNP or CAR-NK cells set as day 0 is visually shown as an abdominal-up image. Similar to Figure 3A, also in Figure 3B, an attenuating effect of tumor-derived chemiluminescence was confirmed by administration of GFP-CD7 LNP and in the NK cell only transfer group (NK+vehicle), and a greater attenuation of chemiluminescence was observed when GPC3 CAR (GC33)-CD7 LNP was administered.

[0336] Therefore, it was clarified that GPC3 CAR (GC33)-CD7 LNP has superior anti-tumor activity against human lung cancer xenografts in the human NK cell transferred tumor-bearing mouse model.

[0337] [Experimental Example 3] Confirmation of CAR expression in human NK cell transferred tumor-bearing mouse modelUsing a tumor-bearing model in which human NK cells were transferred into immunodeficient mice by using a method similar to that in Experimental Example 1, the ability of GPC3 CAR (GC33)-CD7 LNP (LNP 3) to induce expression of the CAR gene in human NK cells transferred into the mouse body was investigated. Human fetal kidney-derived HEK293 cells (HEK293-hGPC3) (5x106cells) that had been genetically modified to express human GPC3 gene were subcutaneously inoculated to NOG-hIL-15 transgenic mice, and 10x106human NK cells were administered into the tail vein 7 days after the inoculation. As the NK cells used for the transfer, human NK cells that had been isolated and purified from human PBMC, cultured for 5 days in NK MACS medium containing 500 U / mL human IL-2, and, after replacement with NK MACS medium containing 500 U / mL human IL-2 and 140 U / mL human IL-15, expansion cultured for another 9 days were harvested and used. Three days after human NK cell transfer was defined as day 0 of administration, and GPC3 CAR(GC33)-CD7 LNP or GFP-CD7 LNP (LNP 4) was administered into the tail vein at a concentration of 0.8 mg / kg on days 0 and 1. One day after the first administration of LNP, blood, spleen, and lungs were collected from the mice, and the expression rate of CAR in human NK cells in the tissue cell suspension was confirmed. The mouse CD45-negative, human CD45-positive, and human CD56-positive cell fraction from which dead cells had been removed was defined as human NK cell, and the ratio of cells positive for phycoerythrin-labeled anti-G4S linker antibody in that fraction was defined as the CAR-positive cell rate. As a control group, an NK cell only transfer group was set up in which 5.3% sucrose / PBS was administered on the same schedule after inoculation of hGPC3-HEK293 cells and transfer of NK cells.

[0338] The results are shown in Figure 3C. One day after the first administration of GPC3 CAR (GC33)-CD7 LNP, a CAR positive rate of 60% or more was confirmed in human NK cells in the spleen. A CAR expression rate of 90% or more was confirmed in human NK cells in the blood and lungs.

[0339] Therefore, it was confirmed that hGPC3 CAR (GC33)-CD7 LNP has the ability to induce the expression of CAR molecules in human NK cells transferred into the mouse body, in a human NK cell-transferred tumor-bearing mouse model.

[0340] [Experimental Example 4] Treatment effect in mouse tumor modelThe treatment effect of GPC3 mouse CAR (GC33) LNP (LNP6) in a mouse colorectal cancer tumor transplantation model was investigated using the following tumor-bearing mice.

[0341] Mouse colorectal cancer cell MC38 (hGPC3-MC38) (5x106cells) that had been genetically modified to express human GPC3 were subcutaneously inoculated to human CD7 knock-in C57BL / 6N mice genetically engineered to replace and express a gene expressing a chimeric protein of the extracellular domain of human CD7, and the transmembrane domain and intracellular domain of mouse CD7, at the mouse CD7 locus. Three days after inoculation, 0.5 mg / kg of GPC3 mouseCAR (GC33)-CD7 LNP (LNP6) or GFP-CD7 LNP (LNP 5) was administered into the tail vein at a concentration of 0.5 mg / kg. Three days after inoculation, 0.5 mg / kg of GPC3 mouseCAR (GC33)-CD7 LNP (LNP6) or GFP-CD7 LNP (LNP 5) was administered into the tail vein at a concentration of 0.5 mg / kg. As a control group, an LNP untreated group was set up that only received 3.3% sucrose / PBS administration. LNP was administered three times in total (3, 4, and 7 days after colon cancer cell inoculation). The tumor volume of the mice was measured twice a week. In this test, blood was collected from the mice administered with LNP or 3.3% sucrose / PBS 4 hours after the third administration of LNP, and the prepared cell suspension was used to calculate the CAR-positive cell rate in mouse NK cells and T cells by flow cytometry. Among the mouse CD45-positive cells from which dead cells had been removed, mouse CD90.2-positive cell fraction was defined as T cell, and mouse CD90.2-negative and mouse NKp46-positive cell fraction was defined as NK cell, and the ratio of cells positive for phycoerythrin-labeled anti-G4S linker antibody in that fraction was defined as the CAR-positive cell rate.

[0342] The results of the tumor volume of mouse colorectal cancer tumor transplantation model are shown in Figure 4A. The horizontal axis is the number of days after the first administration of LNP to the mice, which is set as day 0, and the vertical axis is the tumor volume (tumor major axis x (tumor minor axis)2 / 2 (mm3)). The mean and standard deviation of tumor volume of 8 mice per group were calculated for each test group.

[0343] As shown in Figure 4A, when GPC3 mouseCAR (GC33)-CD7 LNP) was administered, a remarkable tumor volume decreasing effect was confirmed compared to the GFP-CD7 LNP administration group and the LNP untreated group (vehicle). Figure 4B shows changes in the tumor volume for eight mice in each group. Furthermore, the expression of CAR in immune cells in the blood and spleen collected from the mice 4 hours after the third LNP administration was confirmed. As a result, the CAR positivity rate was 80% or more in NK cells in the blood, 50% or more in NK cells in the spleen, 20% in T cells in the blood, and 10% or more in T cells in the spleen (Figure 4C). Therefore, it was clarified that GPC3 mouseCAR (GC33)-CD7 LNP) induces the expression of functional CAR molecules in endogenous mouse T cells and NK cells in human CD7 knock-in mice and has superior anti-tumor activity in a mouse colorectal cancer tumor transplantation model.

[0344] [Experimental Example 5] Confirmation of expression levels of CAR and interferon gamma in T cells and NK cells of LNP administered mouseWith respect to a group in which hGPC3-MC38 cells were subcutaneously inoculated into human CD7 knock-in C57BL / 6N mice, and 3 days later, anti-NK1.1 antibody or isotype control antibody (anti-mouse IgG2a antibody) was administered intraperitoneally, and one day later, GPC3 mouseCAR(GC33)-CD7 LNP(LNP8), GFP-CD7 LNP(LNP7), or 3.3% sucrose / PBS (vehicle) was administered, the spleen was collected 4 hours after the first administration of LNP, and a cell suspension was prepared. The cell suspension was cultured for 4 hours in the presence of 50 ng / mL phorbol 12-myristate 13-acetate (PMA), 1 μg / mL ionomycin, and BD GolgiPlugTM(BD Biosciences). After cell fixation and cell membrane penetration treatment, antibody staining was performed using the method described below, and the expression level of anti-human GPC3 CAR on the cell surface and the interferon gamma production level were analyzed by flow cytometry.

[0345] Among mouse CD45 positive cells, mouse CD3 positive cell fraction was defined as T cell, and mouse CD3 negative and mouse NKp46 positive cell fraction was defined as NK cell. T cells were stained with CD8 antibody and CD4 antibody to define CD8 positive T cells (CD8 T cell) and CD4 positive T cells (CD4 T cell). Furthermore, cells positive for phycoerythrin-labeled anti-G4S linker antibody were defined as anti-GPC3 CAR (GC33) expressing cell fraction (CAR+), and negative cells were defined as CAR non-expressing cell fraction (CAR-). In addition, interferon gamma-producing cells were detected by staining with Brilliant VioletTM421(BV421)-labeled anti-interferon gamma (INFg) antibody.

[0346] The results are shown in Figure 5. An increase in the interferon gamma-positive cell rate was observed in CAR-expressing cells among spleen-derived NK cells (Figure 5A), CD3-positive T cells (Figure 5B), CD8-positive T cells (Figure 5C), and CD4-positive T cells (Figure 5D) of mice administered with GPC3 mouseCAR(GC33)-CD7 LNP, compared to the GFP-CD7 LNP administration group and the LNP-untreated group (vehicle). This supports the idea that NK cells and T cells in which CAR expression was induced by the administration of GPC3 mouseCAR(GC33)-CD7 LNP exhibit anti-tumor activity against target tumor cells in vivo. In addition, an increase in the interferon gamma positive cell rate was observed in the GPC3 mouseCAR (GC33)-CD7 LNP administration group also in the CAR non-expressing fraction of NK cells (Figure 5A) and CD3 positive T cells (Figure 5B), and a remarkable increase in the interferon gamma positive cell rate was observed in the CAR non-expressing cell fraction of CD8 positive T cells (Figure 5C) in the GPC3 mouseCAR (GC33)-CD7 LNP administration group. This result suggests that NK cells and T cells that do not express CAR were also induced to be in an activated state capable of inducing antitumor activity as a secondary action of the antitumor activity against target tumor cells by NK cells and T cells that were induced to express CAR by the administration of GPC3 mouseCAR (GC33)-CD7 LNP. To elucidate the contribution of NK cells to these actions, anti-NK1.1 antibody was further co-administered to reduce NK cells by 80-90% in mouse blood and tissues and then interferon gamma positive cells in CD8 T cells in the mouse spleen was detected. The results are shown in Figure 5E. The group co-administered with GPC3 mouseCAR (GC33)-CD7 LNP and anti-NK1.1 antibody (GPC3 CAR(GC33)-CD7LNP + anti-NK1.1) showed a decrease in the interferon gamma positive cell rate of non-CAR-expressing CD8 T cells, compared to the group administered with GPC3 mouseCAR (GC33)-CD7 LNP and an isotype control antibody that has no action on NK cells (GPC3 CAR(GC33)-CD7 LNP + isotype Ab). This suggests the involvement of NK cells in which functional CAR expression was induced by LNP administration, in the activation and anti-tumor activity of non-CAR-expressing CD8 T cells in the GPC3 mouseCAR (GC33)-CD7 LNP-administered group, as shown in Figure 5C, which is determined by the increased interferon gamma expression.

[0347] [Experimental Example 6] In vitro cytotoxicity of CD19 CAR-CD7 LNP-treated T cells against NALM6-Luc cellsInduction of CAR expression on human T cells using CD7 LNPT cells were isolated from frozen PBMC (Charles River Laboratories Cell Solutions) using Human T cell isolation kit (STEMCELL Technologies) following the instruction attached in the kit. T cells were cultured in the X-VIVOTM15 Serum-free Hematopoietic Cell Medium containing 10 ng / mL of IL-2 (Miltenyi Biotec) with vehicle (the same amount of “PBS-20% sucrose” as LNPs described below) or LNP12 (CD19 CAR-null LNP, 2 μg / mL as mRNA) or LNP14 (GFP-CD7 LNP, 2 μg / mL as mRNA) or LNP13 (CD19 CAR (SJ25C1, SEQ ID NO: 30)-CD7 LNP, 2 μg / mL as mRNA) for 3 days. After culture, cells were collected and CAR expression on the cell surface was evaluated using flow cytometry (Novocyte penteon, Agilent Technologies) by fluorescent staining of extracellular domain of CAR molecule using Phycoerythrin-conjugated human CD19 (20-291) protein (ACRO biosystems). To confirm the activation status of T cells after LNP treatment, CD69 expression was evaluated by fluorescent staining with anti-hCD69 antibody.

[0348] As shown in Figure 6A, CD19 CAR was expressed on over 70% of viable T cells and CAR-driven upregulation of CD69 expression was also observed by CD19 CAR-CD7 LNP treatment. CD19 CAR-null LNP failed to express CD19 CAR on T cells, suggesting the CD7 Fab-mediated potency of mRNA incorporation into T cells.

[0349] Co-culture of LNP-treated T cell with target tumor cellsA luciferase-expressing B cell precursor leukemia NALM6 cell line (hereinafter called to as NALM6-Luc), was engineered to express Red-Fluc gene with IVISbrite Red F-luc Lentiviral Particles (revvity) on parental NALM6 cells which endogenously express human CD19. NALM6-Luc cells were cultured using RPMI1640 Medium (Thermo Fisher Scientific) containing 10% of FBS (Biosera) and Penicillin-Streptomycin (Fujifilm Wako Pure Chemical Corporation). After collection, cells were counted and 3 x 104cells / 0.1 mL / well were seeded to 96-well plate. Serial dilutions of T cell suspensions treated with LNP for 3 days were added to the 96-well plate to make 6-point series of Effector (Total T cells): Target (Tumor cells) ratio (10:1, 3.3:1, 1.1:1, 0.37:1, 0.12:1, 0.04:1).

[0350] Measurement of in vitro tumor killing potencyCytotoxicity of human T cells treated with LNP13 (CD19 CAR-CD7 LNP) against NALM6-Luc cells were assessed using Bright-GloTMLuciferase Assay (Promega) according to the manufacture’s instruction. After 2 days co-culture of tumor cells and T cells, Bright-GloTMLuciferase Assay reagent was added into each well. After shaking plate for 10 min, luminescence of each well was measured using plate reader (EnSight, PerkinElmer). Relative tumor killing activity (%) was calculated by following formula: Relative killing tumor activity (%)=100- ((luminescence of each well / the luminescence value of tumor cell without co-culture) x 100).

[0351] In vitro cytotoxicity of CD19 CAR-CD7 LNP-treated human T cells against NALM6-Luc cellsResults of in vitro cytotoxicity of CD19 CAR-CD7 LNP-treated human T cells were shown in Figure 6B. Whereas T cells treated with neither of GFP-CD7 LNP and CD19 CAR-null LNP demonstrated cell cytotoxicity against NALM6-Luc cells, dose-dependent cell cytotoxicity was observed by T cells treated with CD19 CAR-CD7 LNP. In the CD19 CAR-CD7 LNP group, relative tumor killing activities at 0.37:1, 1.1:1, 3.3:1, and 10:1 E:T ratio were 76.7, 97.7, 99.4, and 99.6%, respectively.

[0352] In conclusion, human T cells treated with LNP13 (CD19 CAR-CD7 LNP) showed CAR expression and CAR-dependent target cell cytotoxicity on CD19-expressing NALM6-Luc cells.

[0353] [Experimental Example 7] In vivo anti-tumor efficacy of CD19 CAR-CD7 LNP in the human T cell transferred NALM6-Luc xenograft modelTransplantation of NALM6-Luc tumor cellsNALM6-Luc was cultured using RPMI 1640 Medium (Thermo Fisher Scientific) containing 10% of FBS (Biosera) and Penicillin-Streptomycin (Fujifilm Wako Pure Chemical Corporation). NALM6-Luc cell suspension was prepared using PBS. The tumor cell suspension was cooled on ice and mixed by pipetting before drawing into the syringe. The tumor cell suspension was drawn into a disposable syringe with a needle (Myjector, 27G [0.40 mm] × 1 / 2” [13 mm], Terumo Corporation) and injected into the tail vain of NSG mice. The transplantation volume was set at 100 μL / body (5×105cells / 0.1 mL).

[0354] Preparation of T cell for transferT cells were administered 3 days after tumor injection. T cells were isolated from frozen PBMC (Charles River Laboratories Cell Solutions) using Human T cell isolation kit (STEMCELL Technologies) following the instruction attached in the kit. T cells were cultured using X-VIVOTM15 Serum-free Hematopoietic Cell Medium containing 10 ng / mL of IL-2 (Miltenyi Biotec) and T Cell TransAct (Miltenyi Biotec) for 4 days. After culture, T cells were collected and cell suspension was prepared using PBS. The T cell suspension was cooled on ice and mixed by pipetting before drawing into a disposable syringe (Myjector, 27G [0.40mm] × 1 / 2” [13mm], Terumo Corporation) with a 27 G needle and injected into the tail vain of NSG mice inoculated with NALM-6-Luc cells. The transplantation volume was set at 200 μL / body (1×107cells / 0.2 mL).

[0355] LNP dosingFrozen stock of LNP14 (GFP-CD7 LNP) and LNP13 (CD19 CAR-CD7 LNP) were thawed and diluted to target concentration using PBS, PBS-20% sucrose and water for injection. The first dose of LNP was administered 3 days after T cell transfer, with the first dosing day designated as day 0, followed by additional doses of LNP on days 3, 7 and 10. The dose of LNP14 was 0.8 mg / kg and the doses of LNP13 were 0.125, 0.375 and 0.8 mg / kg.

[0356] Ex vivo CAR-T administrationTo demonstrate that the CD19 CAR mRNA introduced by LNP was functional, a control group was set in which CAR molecule consisting of the same amino acid sequence was expressed on human T cells using a retrovirus and T cells including 2x106cells of CAR-positive CAR-T cells were administered via tail vein on the same day as the first dose of LNP (day 0).

[0357] Tumor measurementChanges in the amount of inoculated tumor cells in mice after LNP administration were evaluated twice weekly by measuring the chemiluminescence values derived from the luciferase gene in tumor cells. The IVIS imaging reagent (working solution) was injected intraperitoneally (15 mg / mL, 150 mg / kg, 10 mL / kg) to animals anesthetized by inhalation of isoflurane (2.0 to 4.0%; Isoflurane Inhalation Solution [VTRS] (VIATRIS Inc)[Pfizer], Mylan N.V.). After 10 min after injection, whole body (supine or prone) images of animals were captured using IVIS Lumina III (Perkin Elmer Inc.). Whole body image data (excluding the tail) were analyzed using Living Image Software, Version 4.4 (Perkin Elmer, Inc.), and numerical data (total flux, p / s) were obtained.

[0358] Anti-tumor effect of CD19 CAR LNP on the T cell transferred NALM6-Luc xenograft modelAs shown in Figure 7B, compared to the non-treated group (vehicle), the T cell alone transfer group (T+vehicle) and the group treated with GFP-CD7 LNPs after T cell transfer showed a similar level of tumor cell amount reduction, while the groups treated with the tested three doses of CD19 CAR-CD7 LNPs showed a significant tumor volume reduction, which were comparable to the ex vivo CD19 CAR-T cell treatment group (ex vivo hCD19 CAR-T). Figure 7A shows the chemiluminescence intensity from day 3 to day 14 in the groups excluding the non-treated group. CD19 CAR-CD7 LNP-treated group showed a remarkable drug efficacy at an early stage compared to the ex vivo CD19 CAR-T treated group.

[0359] In vivo CAR expression after LNP administrationIn this study, non-tumor bearing mice administered with CD19 CAR-CD7 LNP or GFP-CD7 LNP had their spleen and lung tissues collected one day after the second LNP injection. Using cell suspensions prepared from each tissue, the percentage of CAR-positive cells in human T cells was calculated by flow cytometry. Dead cells were excluded, and mouse CD45-negative, human CD45-positive, and human CD3-positive cell fraction was defined as human T cells. The CAR-positive cell rate in this fraction was defined as the proportion of cells detected using Phycoerythrin-conjugated human CD19 protein (20-291).

[0360] As shown in Figure 7C, in human T cells in the spleen and lung, CD19 CAR-CD7 LNP-administered group showed more than 40% CAR-positive rate. Additionally, as shown in Figure 7D, an increase in the percentage of CD69-positive cells was observed in the same group, which was due to T cell activation by CAR construct.

[0361] These results indicate that CD19 CAR-LNP induces the expression of functional CAR gene in human T cells engrafted into mice, demonstrating anti-tumor activity against tumors expressing the target molecule.

[0362] [Experimental Example 8] In vitro cytotoxicity of hCD19 CAR-CD7 LNP-treated human NK cells against NALM6 cells (Figure 8)Induction of CAR expression on human NK cells using CD7 LNPNK cells were isolated from frozen PBMC (Charles River Laboratories Cell Solutions) using Human NK cell isolation kit (STEMCELL Technologies) following the instruction attached in the kit. NK cells were cultured using NK MACS (Registered) Medium, human (Miltenyi Biotec) containing 500 U / mL of IL-2 (Miltenyi Biotec) for 4 days. After culture, culture medium was replaced with NK MACS medium containing 500 U / mL of IL-2 and 140 U / mL of IL-15 (Miltenyi Biotec), then NK cells were cultured for another 9 days. One million cells of NK cells were treated with LNP15 (CD19 CAR-CD7 LNP, 3.0 μg / mL as mRNA) for 3 days. Cells were collected and CAR expression on the cell surface was evaluated using flow cytometry (Novocyte 3005, Agilent Technologies) by fluorescent staining of extracellular domain of CAR molecule using Phycoerythrin-conjugated human CD19 (20-291) protein.

[0363] As shown in Figure 8A, CD19 CAR was expressed on over 60% of viable NK cells by CD19 CAR-CD7 LNP treatment.

[0364] Co-culture of LNP-treated NK cell with target tumor cellsNALM6 cell line was cultured using RPMI1640 Medium containing 10% of FBS and Penicillin-Streptomycin. After collection, cells were counted and 3 x 104cells / 0.1 mL / well were seeded to 96-well plate. NK cells treated with LNP for 3 days were labeled with CellTrace Violet (ThermoFisher scientific), then serial dilutions of labeled NK cell suspensions were added to the 96-well plate to make 3-point series of Effector (Total NK cells): Target (Tumor cells) ratio (5:1, 1:1, and 0.2:1). To demonstrate that the CD19 CAR mRNA introduced by LNP was functional, a control group was set in which CAR molecule consisting of the same amino acid sequence was expressed on human NK cells using a retrovirus.

[0365] Measurement of in vitro tumor killing potencyCytotoxicity of human NK cells treated with LNP15 (CD19 CAR-CD7 LNP) against NALM6 cells were assessed by flow cytometry. After 4 hours co-culture, total cell suspensions were stained with 7AAD, then relative tumor killing activity (%) was calculated by following formula: Relative tumor killing activity (%)=7-AAD positive cell ratio in Cell Trace Violet positive cells.

[0366] In vitro cytotoxicity of CD19 CAR-CD7 LNP-treated human NK cells against NALM6 cellsThe results of in vitro cytotoxicity of CD19 CAR-CD7 LNP-treated human NK cells are shown in Figure 8B. Even NK cells without gene introduction (UTD-NK groups) exhibit concentration-dependent tumor cell cytotoxicity. However, NK cells treated with CD19 CAR-CD7 LNP showed concentration-dependent cytotoxicity and exhibited stronger activity.

[0367] In conclusion, human NK cells treated with LNP15 (CD19 CAR-CD7 LNP) showed CAR expression and enhancement of target cell cytotoxicity on CD19-expressing NALM6 cells.

[0368] [Experimental Example 9]In vivo anti-tumor efficacy of CD19 CAR-CD7 LNP in the human NK cell transferred NALM6-Luc xenograft model (Figure 9)Transplantation of NALM6-Luc tumor cellsNALM6-Luc cell suspension was prepared using PBS. The tumor cell suspension using cold PBS was injected into the tail vain of human IL15 transgenic NOG mice using a disposable syringe with a needle. The transplantation volume was set at 100 μL / body (5×105cells / 0.1 mL).

[0369] Preparation of NK cell for transferNK cells were administered 2 days after tumor injection. Isolation of NK cells from human PBMC and culture method was following the method used in Experimental Example 8. After culture, NK cells were collected and cell suspension was prepared using PBS. The NK cell suspension was cooled on ice and mixed by pipetting before drawing into a disposable syringe (Myjector, 27G [0.40mm] × 1 / 2” [13mm], Terumo Corporation) with a 27 G needle and injected into the tail vain of NSG mice inoculated with NALM6-Luc cells. The transplantation volume was set at 500 μL / body (9.5×106cells / 0.5 mL).

[0370] LNP dosingFrozen stock of LNP17 (GFP-CD7 LNP) and LNP16 (CD19 CAR-CD7 LNP) were thawed and diluted to target concentration using PBS, PBS-20% sucrose and water for injection. The first dose of LNP was administered 3 days after NK cell transfer, with the first dosing day designated as day 0, followed by additional doses of LNP on days 3, 7 and 10. The dose of LNP16 and LNP17 was 0.8 mg / kg.

[0371] Ex vivo CAR-NK administrationTo demonstrate that the CD19 CAR mRNA introduced by LNP was functional in human NK cells, a control group was set in which CAR molecule consisting of the same amino acid sequence was expressed on human NK cells using a retrovirus. CAR-NK cells including 2x106cells of CAR-positive cells were administered via tail vein on the same day as the first dose of LNP (day 0).

[0372] Tumor measurementChanges in the amount of inoculated tumor cells in mice after LNP administration were evaluated twice weekly by measuring the chemiluminescence values derived from the luciferase gene in tumor cells. The IVIS imaging reagent was injected intraperitoneally (15 mg / mL, 150 mg / kg, 10 mL / kg) to animals anesthetized by inhalation of isoflurane. After 10 min after injection, whole body (supine or prone) images of animals were captured using IVIS Lumina III. Whole body image data (excluding the tail) were analyzed using Living Image Software, Version 4.4, and numerical data (total flux, p / s) were obtained.

[0373] Anti-tumor effect of CD19 CAR LNP on the NK cell transferred NALM6-Luc xenograft modelAs shown in Figures 9A and 9B, compared to the non-treated group (vehicle), administration of ex vivo CD19 CAR-NK cells demonstrated tumor cell amount reduction. As shown in Figures 9C and 9D, whereas significant change was not observed in the tumor growth between NK alone transfer group and the group treated with GFP-CD7 LNP, the group treated with CD19 CAR-CD7 LNP showed a significant tumor volume reduction.

[0374] In summary, CD19 CAR-CD7 LNP demonstrated in vivo anti-tumor efficacy against CD19-expressing NALM6-Luc tumors by in vivo CAR expression on transferred human NK cells.

[0375] [Experimental Example 10] In vivo anti-tumor efficacy of CD19 CAR-CD7 LNP in the human T / NK cell transferred NALM6-Luc xenograft model (Figure 10)Transplantation of NALM6-Luc tumor cellsNALM6-Luc cell suspension was prepared using PBS. The tumor cell suspension using cold PBS was injected into the tail vain of human IL15 transgenic NOG mice using a disposable syringe with a needle. The transplantation volume was set at 100 μL / body (5×105cells / 0.1 mL).

[0376] Preparation of T cells and NK cells for transferHuman T cells and NK cells were administered 2 days after tumor injection. Isolation of NK cells from human PBMC and culture method was following the method used in Experimental Example 8. T cells were isolated from the same lot of PBMCs used for NK cell isolation following the method described in Experimental Example 7. After culture, T cells and NK cells were collected and cell suspension was prepared using PBS. The T / NK cell suspension was cooled on ice and mixed by pipetting before drawing into a disposable syringe (Myjector, 27G [0.40mm] × 1 / 2” [13mm], Terumo Corporation) with a 27 G needle and injected into the tail vain of human IL15 transgenic NOG mice inoculated with NALM6-Luc cells. The transplantation volume was set at 200 μL / body as a T and NK cell mixed suspension (1×106cells of T cells and 1x107cells of NK cells / 0.2 mL).

[0377] LNP dosingFrozen stock of LNP14 (GFP-CD7 LNP) and LNP13 (CD19 CAR-CD7 LNP) were thawed and diluted to target concentration using PBS, PBS-20% sucrose and water for injection. The first dose of LNP was administered 3 days after T / NK cell transfer, with the first dosing day designated as day 0, followed by additional doses of LNP on days 4, 7 and 10. The dose of LNP13 and LNP14 was 0.8 mg / kg.

[0378] Administration of ex vivo CAR-T and ex vivo CAR-NKTo demonstrate that the CD19 CAR mRNA introduced by LNP was functional, a control group was set in which CAR molecule consisting of the same amino acid sequence was expressed on human T cells or human NK cells using a retrovirus and CAR-T cells including 1x106cells of CAR-positive T cells or CAR-NK cells including 5x106cells of CAR-positive NK cells were administered via tail vein on the same day as the first dose of LNP (day 0).

[0379] Tumor measurementChanges in the amount of inoculated tumor cells in mice after LNP administration were evaluated twice weekly by measuring the chemiluminescence values derived from the luciferase gene in tumor cells. The IVIS imaging reagent was injected intraperitoneally (15 mg / mL, 150 mg / kg, 10 mL / kg) to animals anesthetized by inhalation of isoflurane. After 10 min after injection, whole body (supine or prone) images of animals were captured using IVIS Lumina III. Whole body image data (excluding the tail) were analyzed using Living Image Software, Version 4.4, and numerical data (total flux, p / s) were obtained.

[0380] Anti-tumor effect of CD19 CAR LNP on the T / NK cell transferred NALM6-Luc xenograft modelAs shown in Figures 10A and 10B, compared to the non-treated group (vehicle), administration of either of ex vivo CD19 CAR-T cells or CD19 CAR-NK cells demonstrated tumor cell amount reduction. The group treated with CD19 CAR-CD7 LNP after T / NK injection demonstrated a significant tumor amount reduction compared to the T / NK transfer group or GFP-CD7 LNP-treated group.

[0381] In vivo CAR expression after LNP administrationIn this study, non-tumor bearing mice administered with CD19 CAR-CD7 LNP had their lung tissues collected one day after the first LNP injection. Using cell suspensions prepared from the tissue, the percentage of CAR-positive cells in human T cells and NK cells were calculated by flow cytometry. Dead cells were excluded, and mouse CD45-negative, human CD45-positive was gated. In this gate, human CD3-positive cell fraction was defined as human T cells, and human CD56-positive cell fraction was defined as human NK cells. The CAR-positive cell rate in these fractions was defined as the proportion of cells detected using Phycoerythrin-conjugated recombinant human CD19 protein.

[0382] As shown in Figure 10C, in both of human T cells and NK cells in lung, CD19 CAR-CD7 LNP-administered group showed more than 70% CAR-positive rate.

[0383] These results indicate that CD19 CAR-LNP induces the expression of functional CAR gene in both of human T cells and NK cells engrafted into mice, demonstrating potent anti-tumor activity against tumors expressing the target molecule.

[0384] [Experimental Example 11] In vivo anti-tumor efficacy of CD19 CAR-CD7 LNP with IL-15 in the human T cell transferred NALM6-Luc xenograft modelTransplantation of NALM6-Luc tumor cellsNALM6-Luc was cultured using RPMI 1640 Medium containing 10% of FBS and Penicillin-Streptomycin. NALM6-Luc cell suspension was prepared using PBS. The tumor cell suspension was cooled on ice and mixed by pipetting before drawing into the syringe. The tumor cell suspension was drawn into a disposable syringe with a needle (Myjector, 27G [0.40 mm] × 1 / 2” [13 mm], Terumo Corporation) and injected into the tail vain of NSG mice. The transplantation volume was set at 100 μL / body (5×105 cells / 0.1 mL).

[0385] Preparation of T cell for transferT cells were administered 2 days after tumor injection. T cells were isolated from frozen PBMC using Human T cell isolation kit following the instruction attached in the kit. T cells were cultured using X-VIVOTM15 Serum-free Hematopoietic Cell Medium containing 10 ng / mL of IL-2 and T Cell TransAct for 4 days. After culture, T cells were collected and cell suspension was prepared using PBS. The T cell suspension was cooled on ice and mixed by pipetting before drawing into a disposable syringe (Myjector, 27G [0.40mm] × 1 / 2” [13mm], Terumo Corporation) with a 27 G needle and injected into the tail vain of NSG mice inoculated with NALM-6-Luc cells. The transplantation volume was set at 200 μL / body (1×106or 3×106cells / 0.2 mL).

[0386] LNP dosingFrozen stock of LNP18 (CD19 CAR-CD7 LNP) and LNP19 (CD19 CAR / mbIL15-CD7 LNP) were thawed and diluted to target concentration using PBS, PBS-20% sucrose and water for injection. The first dose of LNP was administered 3 days after T cell transfer, with the first dosing day designated as day 0, followed by additional doses of LNP on days 1, 4, 7 and 8. The dose of LNP18 was 0.375 mg / kg and the doses of LNP19 were 0.75 mg / kg.

[0387] Ex vivo CAR-T administrationTo demonstrate that the CD19 CAR mRNA introduced by LNP was functional, a control group was set in which CAR molecule and membrane-bound IL-15 (mbIL-15) consisting of the same amino acid sequence was expressed on human T cells using a retrovirus and T cells including 2.5x105cells of CAR-positive CAR-T cells were administered via tail vein on the same day as the first dose of LNP (day 0).

[0388] Tumor measurementChanges in the amount of inoculated tumor cells in mice after LNP administration were evaluated twice weekly by measuring the chemiluminescence values derived from the luciferase gene in tumor cells. The IVIS imaging reagent (working solution) was injected intraperitoneally (15 mg / mL, 150 mg / kg, 10 mL / kg) to animals anesthetized by inhalation of isoflurane (2.0 to 4.0%; Isoflurane Inhalation Solution [VTRS]. After 10 min after injection, whole body (supine or prone) images of animals were captured using IVIS Lumina III. Whole body image data (excluding the tail) were analyzed using Living Image Software, Version 4.4, and numerical data (total flux, p / s) were obtained.

[0389] Anti-tumor effect of CD19 CAR LNP on the T cell transferred NALM6-Luc xenograft modelAs shown in Figure 11A, compared to the non-treated group (vehicle) or the T cell alone transfer group (1M T+vehicle, or 3M T+vehicle), the group treated with CD19 CAR-CD7 LNPs after T cell transfer showed tumor cell amount reduction in a T cell dose-dependent manner. The group treated with CD19 CAR / mbIL-15-CD7 LNP demonstrated more potent suppressive effect on tumor growth compared to the group treated with CD19 CAR-CD7 LNP. Ex vivo mbIL-15-armored CD19 CAR-T cell treatment group (ex vivo mbIL-15-armored hCD19 CAR-T) well controlled the tumor growth in this study. Figure 11B shows the chemiluminescence intensity from day 3 to day 21 in the groups. CD19 CAR / mbIL-15-CD7 LNP-treated group showed a remarkable drug efficacy in a later stage compared to the CD19 CAR-CD7 LNP treated group.

[0390] In vivo CAR expression after LNP administrationIn this study, mice administered with CD19 CAR-CD7 LNP or CD19 CAR / mbIL-15-CD7 LNP 3 days after tumor inoculation had their lung tissues collected one day after the 2nd LNP injection. Using cell suspensions prepared from each tissue, the percentage of CAR-positive cells in human T cells was calculated by flow cytometry. Dead cells were excluded, and human CD3-positive cell fraction was defined as human T cells. The CAR-positive cell rate in this fraction was defined as the proportion of cells detected using Phycoerythrin-conjugated human CD19 protein (20-291) and mbIL-15-positive cell rate in this fraction was also defined as the proportion of the cells detected using fluorescence-tagged anti-IL-15Rα antibody.

[0391] As shown in Figure 11C, in human T cells in the spleen and lung, Both of the CD19 CAR-CD7 LNP-administered group and CD19 CAR / mbIL-15-CD7 LNP-administered group showed around 30% CAR-positive rate. 32% of mbIL-15 positive cells were observed only in the CD19 CAR / mbIL-15-CD7 LNP-administered group.

[0392] These results indicate that CD19 CAR-LNP induces the expression of functional CAR gene in human T cells engrafted into mice and LNP encapsulating both of the CD19 CAR and mbIL-15 induces the expression of CD19 CAR and mbIL-15 respectively, demonstrating anti-tumor activity of CD19 CAR against tumors expressing CD19 and the enhancement of anti-tumor efficacy of CD19 CAR by the expression of mbIL-15.

[0393] T cell phenotype analysis after LNP administrationIn this study, mice were sacrificed at day 21 and spleens were collected and analyzed by flow cytometry to see the IL-15 armoring effect on T cell phenotype. Using cell suspensions prepared from spleen, the percentage of CD62L+CD45RO+ central memory T cells (Tcm), CD62L-CD45RO+ effector T cells (Teffe), CD62L-CD45RO- effector memory T cells (Tem), CD62L+CD45RO-CD95+ stem cell-like memory T cells (Tscm) and CD62L+CD45RO+CD95- naive T cells (Tn) were calculated by flow cytometry. Dead cells were excluded, and human CD3-positive cell fraction was defined as human T cells.

[0394] As shown in Figure 11D, in human T cells in the mouse spleen at day 21, the CD19 CAR / mbIL-15-CD7 LNP-administered group demonstrated the increase in Tcm and Tscm population compared to CD19 CAR-CD7 LNP-administered group.

[0395] These results indicate that co-expression of mbIL-15 and CD19 CAR by LNP administration leads to the change of T cell phenotype in the later phase in which tumor antigen was almost diminished, suggesting the contribution to the durable and stronger anti-tumor efficacy by CD19 CAR / mbIL-15-CD7 LNP group compared to the CD19 CAR-CD7 LNP group.

[0396] The lipid nanoparticle of the present invention can selectively introduce CAR or exogenous TCR into CD7-expressing cells such as T cells, NK cells, and the like in vivo. Therefore, the present invention is extremely useful as a novel in vivo cancer therapy based on a new concept.This application is based on a US provisional patent application No. 63 / 737,399 filed on December 20, 2024, the contents of which are incorporated in full herein.

Claims

1.A lipid nanoparticle comprising the following (a) to (c), and having a ligand on its surface that targets an immunocyte:(a) a nucleic acid encoding a chimeric antigen receptor or an exogenous T cell receptor;(b) a cationic lipid; and(c) a non-cationic lipid,wherein the ligand is a polypeptide comprising a binding domain for CD7.2.The lipid nanoparticle according to claim 1, wherein the chimeric antigen receptor or exogenous T cell receptor is a polypeptide comprising a binding domain for glypican-3 (GPC3) or a polypeptide comprising a binding domain for CD19.3.The lipid nanoparticle according to claim 1 or 2, wherein the nucleic acid is encoding a chimeric antigen receptor, and the chimeric antigen receptor comprises the following (i) to (iv):(i) an extracellular antigen binding domain;(ii) a transmembrane domain;(iii) a co-stimulatory domain; and(iv) an intracellular signal transduction domain,wherein the extracellular antigen binding domain is a binding domain for GPC3 or a binding domain for CD19.4.The lipid nanoparticle according to claim 3, wherein the extracellular antigen binding domain is a binding domain comprisingheavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1,heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2,heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3,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, andlight chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6,a binding domain comprisingheavy 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,heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 9,light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 10,light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 11, andlight chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 12, ora binding domain comprisingheavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13,heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14,heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15,light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16,light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17, andlight chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 18.5.The lipid nanoparticle according to claim 3, wherein the extracellular antigen binding domain isa binding domain comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 19 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 20,a binding domain comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 22, ora binding domain comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 24.6.The lipid nanoparticle according to claim 3, wherein the extracellular antigen binding domain is scFv.7.The lipid nanoparticle according to claim 6, wherein the scFv isa polypeptide comprising the amino acid sequence shown in SEQ ID NO: 25,a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 26, ora polypeptide comprising the amino acid sequence shown in SEQ ID NO: 27.8.The lipid nanoparticle according to claim 3, wherein the transmembrane domain is a T cell receptor (TCR) alpha chain, a TCR beta chain, a TCR zeta chain, CD28, a CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.9.The lipid nanoparticle according to claim 3, wherein the co-stimulatory domain is OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1(CD11a / CD18), ICOS(CD278), DAP10, DAP12, 4-1BB(CD137), or a combination thereof.10.The lipid nanoparticle according to claim 3, wherein the intracellular signal transduction domain is CD3 zeta, a CD3 zeta variant, 4-1BB, CD28, CD134, CD137, Lck, DAP10, ICOS, or a combination thereof.11.The lipid nanoparticle according to claim 3, wherein the chimeric antigen receptor further comprises a hinge domain linking the extracellular antigen binding domain and the transmembrane domain.12.The lipid nanoparticle according to claim 3, wherein the combination of the transmembrane domain, the co-stimulatory domain, and the intracellular signal transduction domain is CD28-CD28-CD3 zeta or a CD28-CD28-CD3 zeta variant.13.The lipid nanoparticle according to claim 1 or 2, wherein the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 28, the amino acid sequence shown in SEQ ID NO: 29, or the amino acid sequence shown in SEQ ID NO: 30.14.The lipid nanoparticle according to claim 1 or 2, wherein the ligand comprises a binding domain comprisingheavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 34,heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 35,heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 36,light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 37,light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 38, andlight chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 39.15.The lipid nanoparticle according to claim 14, wherein the binding domain isa binding domain comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 40 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 41.16.The lipid nanoparticle according to claim 14, wherein the ligand is Fab.17.The lipid nanoparticle according to claim 1 or 2, wherein the ligand comprises the amino acid sequence shown in SEQ ID NO: 42 and 43.18.The lipid nanoparticle according to claim 1 or 2, wherein the immunocyte comprises a T cell and / or an NK cell.19.The lipid nanoparticle according to claim 1 or 2, wherein the immunocyte comprises a T cell and an NK cell.20.A lipid nanoparticle comprising the following (a) to (c), for promoting MHC class I molecule-dependent and MHC class I molecule-independent cell killing by a T cell and / or an NK cell:(a) a nucleic acid encoding a chimeric antigen receptor or an exogenous T cell receptor;(b) a cationic lipid; and(c) a non-cationic lipid,wherein a surface of the lipid nanoparticle has a ligand that targets an immunocyte, wherein the ligand is a polypeptide comprising a binding domain for CD7.21.The lipid nanoparticle according to claim 20, wherein the chimeric antigen receptor is a polypeptide comprising a binding domain for GPC3, or a polypeptide comprising a binding domain for CD19.22.A medicament comprising the lipid nanoparticle according to claim 1 or 2.23.The medicament according to claim 22, wherein the medicament is a prophylactic or therapeutic drug for cancer.24.The medicament according to claim 22, wherein the medicament introduces a chimeric antigen receptor gene or an exogenous T cell receptor gene into an in vivo immunocyte to induce an expression thereof.25.The medicament according to claim 22, wherein the medicament introduces a chimeric antigen receptor gene or an exogenous T cell receptor gene into an in vivo T cell and / or an NK cell to induce an expression thereof.26.The medicament according to claim 22, wherein the medicament introduces a chimeric antigen receptor gene or an exogenous T cell receptor gene into an in vivo T cell and an NK cell to induce an expression thereof.27.A method for introducing a chimeric antigen receptor or an exogenous T cell receptor into an in vivo immunocyte of a mammal to induce an expression thereof, comprising administering the lipid nanoparticle according to claim 1 or 2 to the mammal.28.A method for introducing a chimeric antigen receptor or an exogenous T cell receptor into an in vivo T cell and / or an NK cell of a mammal to induce an expression thereof, comprising administering the lipid nanoparticle according to claim 1 or 2 to the mammal.29.A method for introducing a chimeric antigen receptor or an exogenous T cell receptor into an in vivo T cell and an NK cell of a mammal to induce an expression thereof, comprising administering the lipid nanoparticle according to claim 1 or 2 to the mammal.30.A method for preventing or treating cancer in a mammal, comprising administering the lipid nanoparticle according to claim 1 or 2 to the mammal.31.The lipid nanoparticle according to claim 1 or 2 for use in the prophylaxis or treatment of cancer.32.Use of the lipid nanoparticle according to claim 1 or 2 in the manufacture of an agent for the prophylaxis or treatment of cancer.33.The lipid nanoparticle according to claim 1 or 2 for use in the induction of an expression of a chimeric antigen receptor or an exogenous T cell receptor.

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