IL-3R-targeting chimeric antigen receptor expressing cells

Abstract

Providing CAR-T cells with an excellent balance of efficacy and safety. [Solution] The present invention provides a polynucleotide encoding a chimeric antigen receptor (CAR) protein having a target-binding domain that binds to an interleukin-3 receptor (IL-3R), a transmembrane domain, and an intracellular signaling domain, wherein the target-binding domain is interleukin-3 (IL-3). The present invention also provides a vector containing the above polynucleotide, and genetically modified cells into which the above polypeptide or vector has been introduced.

Description

[Technical Field]

[0001] The present invention relates to polynucleotides encoding chimeric antigen receptors and vectors containing them, which are useful in the field of adoptive immunotherapy, as well as genetically modified cells expressing chimeric antigen receptors and methods for producing them. [Background technology]

[0002] In recent years, there has been a series of new approvals or expanded indications for small molecule drugs for the treatment of acute myeloid leukemia (AML). While these small molecule drugs significantly improve treatment options for AML and contribute to extending prognosis, drug resistance is inevitable with long-term use. Therefore, the development of novel modalities with alternative cytotoxic mechanisms that can contribute to improving the long-term prognosis of AML is desired. Immunotherapy using chimeric antigen receptor (CAR)-expressing T cells is one of the most promising next-generation cancer treatments for hematological malignancies, and its practical application in AML is eagerly awaited. However, although the development of CAR-T cells targeting various AML-related antigens has progressed, there are still no products in the world that are close to practical application (Non-Patent Literature 1).

[0003] CD123 (IL-3Rα), the α subunit of the interleukin-3 receptor (IL-3R), is an antigen expressed in 78-89% of AML cells and is also known to be expressed in AML stem cells. The development of CAR-T cells targeting CD123 (CD123 CAR-T cells) is actively underway. For example, Mardiros et al. reported that T cells expressing a CAR (scFv-type CAR) containing anti-CD123 scFv (26292 or 32716) as a target-binding domain delayed the progression of leukemia in mice (Non-Patent Literature 2, Patent Literature 1). However, because CD123 is widely expressed not only in AML cells but also in normal cells such as hematopoietic progenitor cells, lymphoid progenitor cells, myeloid progenitor cells, and mature leukocytes, it has been difficult to create CD123 CAR-T cells that strike a balance between efficacy and safety.

[0004] On the other hand, the present inventors have reported a CAR (ligand-type CAR) design technology that uses a receptor ligand instead of a conventional scFv in the target binding domain of the CAR as a method for producing CAR-T cells targeting a receptor (Patent Document 2).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] An object of the present invention is to provide CAR-T cells with an excellent balance of efficacy and safety.

Means for Solving the Problems

[0008] IL-3R is composed of an α-subunit, CD123, and a β-subunit, CD131. CD123 and CD131 exist as monomers in normal cells during the quiescent phase, but they constitutively bind to tumor cells, forming the CD123 / CD131 complex. Therefore, CARs targeting the CD123 / CD131 complex are considered to have lower off-tumor toxicity (toxicity resulting from recognition of normal tissue) and higher safety compared to CARs targeting CD123. IL-3, the ligand for IL-3R, has high affinity for the CD123 / CD131 complex but low affinity for CD123 alone (Broughton et al., Immunological Reviews, 2012, vol. 250:277-302). Therefore, CARs using IL-3 as the target-binding domain can specifically target the CD123 / CD131 complex.

[0009] Therefore, the inventors designed a CAR using IL-3, a ligand for IL-3R, as the target-binding domain, and found that cells expressing this CAR exhibit an antitumor effect against IL-3R-expressing tumors. The inventors further found that cells expressing a CAR using IL-3 with a substitution of lysine (K) to another amino acid at the position corresponding to the 105th amino acid of the amino acid sequence shown in SEQ ID NO: 6 exhibit a remarkable antitumor effect, but have low toxicity to normal cells and a wide safety margin.

[0010] In other words, the present invention encompasses the following: [1] A polynucleotide encoding a chimeric antigen receptor (CAR) protein having a target-binding domain that binds to an interleukin-3 receptor (IL-3R), a transmembrane domain, and an intracellular signaling domain, wherein the target-binding domain is interleukin-3 (IL-3). [2] The polynucleotide described in [1], wherein IL-3 has a substitution of lysine (K) with another amino acid at the position corresponding to the 105th amino acid of the amino acid sequence shown in SEQ ID NO: 6. [3] The polynucleotide according to [1] or [2], wherein IL-3 comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 6 or 7. [4] A vector containing a polynucleotide described in any one of [1] to [3]. [5] Genetically modified cells into which any one of the polynucleotides described in [1] to [3] or the vector described in [4] has been introduced. [6] A method for producing CAR protein-expressing cells, comprising introducing a polynucleotide described in any one of [1] to [3] or a vector described in [4] into cells. A therapeutic agent for diseases involving IL-3R-expressing cells, including the cells described in [7] and [5]. A pharmaceutical composition comprising the therapeutic agent described in [8] [7] and a pharmaceutically acceptable carrier. [9] The therapeutic agent according to [7] or the composition according to [8], wherein the disease involving IL-3R-expressing cells is selected from the group consisting of acute myeloid leukemia, chronic myeloid leukemia, hairy cell leukemia, B-cell lineage acute lymphoblastic leukemia, and blastic plasmacytoid dendritic cell neoplasm. A kit for generating CAR protein-expressing cells that target IL-3R-expressing cells, including the vector described in

[10] [4]. [Effects of the Invention]

[0011] The present invention provides genetically modified cells that bind to target cells expressing IL-3R and exert an antitumor effect. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 is a vector map showing the structure of a CAR expression plasmid. [Figure 2]Figure 2 shows flow cytometry data indicating the CAR expression rate on Day 16 in cell populations introduced with wtIL-3-CAR, IL-3_k17Rs6Hn109V-CAR, IL-3_h84Af26Mk105D-CAR, IL-3_n30Rn109W-CAR, IL-3_n30R-CAR, IL-3_k105W-CAR, or IL-3_h84Af26M-CAR. [Figure 3] Figure 3 is a graph showing (A) the total number of cells from Day 0 to 16 and (B) the number of CAR-T cells on Day 16 in cell populations into which wtIL-3-CAR, IL-3_k17Rs6Hn109V-CAR, IL-3_h84Af26Mk105D-CAR, IL-3_n30R-CAR, IL-3_k105W-CAR, or IL-3_h84Af26M-CAR was introduced. [Figure 4] Figure 4 is a graph showing the number of viable tumor cells after co-culturing control T cells, wtIL-3-CAR-T cells, IL-3_k17Rs6Hn109V-CAR-T cells, IL-3_h84Af26Mk105D-CAR-T cells, or IL-3_k105W-CAR-T cells (effectors) with tumor cells (targets) for 4 days at E:T ratios of 1:1, 1:5, and 1:10. [Figure 5] Figure 5 shows flow cytometry data indicating CAR expression rates on Day 14 in control T cells, cell populations introduced with IL-3_k105W-CAR, or cell populations introduced with anti-CD123 scFv-CAR. [Figure 6] Figure 6 is a graph showing (A) the total number of cells from Day 0 to 14 and (B) the number of CAR-T cells on Day 14 in a cell population into which IL-3_k105W-CAR-T cells or anti-CD123 scFv-CAR-T cells were introduced. [Figure 7]Figure 7 is a graph showing the number of viable tumor cells when control T cells, IL-3_k105W-CAR-T cells, or anti-CD123 scFv-CAR-T cells (effectors) were co-cultured with tumor cells MV4-11 (target) for 4 days at various E:T ratios. (A) Effectors derived from donor 1 were used. (B) Effectors derived from donor 2 were used. (C) Effectors derived from donor 3 were used. In the figure, * (asterisk) and ** indicate that there is a significant difference in the number of viable tumor cells between IL-3_k105W-CAR-T cells and anti-CD123 scFv-CAR-T cells based on the results of the t-test (p < 0.05 and p < 0.01, respectively). ns indicates that there is no significant difference in the number of viable tumor cells between IL-3_k105W-CAR-T cells and anti-CD123 scFv-CAR-T cells. [Figure 8] Figure 8 is a graph showing the number of colonies when control T cells, IL-3_k105W-CAR-T cells, or anti-CD123 scFv-CAR-T cells (effectors) were co-cultured with normal CD34+ bone marrow cells (targets) at various E:T ratios for 4 days, and then cultured in hematopoietic progenitor cell colony measurement medium. (A) Effectors derived from donor 1 were used. (B) Effectors derived from donor 2 were used. (C) Effectors derived from donor 3 were used. In the figure, * and ** indicate a significant difference in the number of colonies between IL-3_k105W-CAR-T cells and anti-CD123 scFv-CAR-T cells based on t-test results (p < 0.05 and p < 0.01, respectively). ns indicates no significant difference in the number of colonies between IL-3_k105W-CAR-T cells and anti-CD123 scFv-CAR-T cells. [Figure 9] Figure 9 is a graph showing the cytotoxic activity of IL-3_k105W-CAR-T cells and anti-CD123 scFv-CAR-T cells against tumor cells (MV4-11) at various E:T ratios. (A) Cytotoxic activity of effectors derived from donor 1. (B) Cytotoxic activity of effectors derived from donor 2. (C) Cytotoxic activity of effectors derived from donor 3. [Figure 10]Figure 10 is a graph showing the cytotoxic activity of IL-3_k105W-CAR-T cells and anti-CD123 scFv-CAR-T cells against normal cells (CD34+ myeloid cells) at various E:T ratios. (A) Cytotoxic activity of effectors derived from donor 1. (B) Cytotoxic activity of effectors derived from donor 2. (C) Cytotoxic activity of effectors derived from donor 3. [Figure 11] Figure 11 shows luminescence imaging images of tumor volume at 5, 9, 12, 16, 21, 28, and 35 days after tumor administration in immunodeficient mice that were intravenously administered tumor cells THP-1, and then intravenously administered control T cells, wtIL-3-CAR-T cells, IL-3_k105W-CAR-T cells, anti-CD123 scFv-CAR-T cells, or PBS to the mice 7 days after tumor administration. [Figure 12] Figure 12 is a graph showing the bioluminescence intensity at 5, 9, 12, 16, 21, 28, and 35 days after tumor administration in mice administered with control T cells, wtIL-3-CAR-T cells, IL-3_k105W-CAR-T cells, or anti-CD123 scFv-CAR-T cells, as quantified in the luminescence imaging images of Figure 11. [Figure 13] Figure 13 is a graph showing the bioluminescence intensity 35 days after tumor administration in mice administered IL-3_k105W-CAR-T cells or anti-CD123 scFv-CAR-T cells, as quantified in the luminescence imaging images of Figure 11. [Modes for carrying out the invention]

[0013] The present invention will be described in detail below.

[0014] The present invention provides a polynucleotide encoding a chimeric antigen receptor (CAR) protein having a target-binding domain that binds to the interleukin-3 receptor (IL-3R), a transmembrane domain, and an intracellular signaling domain.

[0015] In this specification, “chimeric antigen receptor (CAR)” refers to a modified receptor whose target specificity can be transplanted into cells such as T cells (e.g., naive T cells, stem cell memory T cells, central memory T cells, effector memory T cells, or combinations thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors.

[0016] In this specification, “encode” means, as commonly used in the art, that a given nucleotide sequence encodes the amino acid sequence information of a given protein or (poly)peptide, and in this specification, both sense strands and antisense strands are used in the context of “encode.”

[0017] As used herein, “polynucleotide” includes, but is not limited to, natural or synthetic DNA and RNA, such as genomic DNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (nuclear small RNA), snoRNA (nucleolar small RNA), miRNA (microRNA), and / or tRNA.

[0018] In the present invention, the CAR protein has a target-binding domain that binds to IL-3R, a transmembrane domain, and an intracellular signaling domain.

[0019] In this specification, "domain" refers to a region within a polypeptide that folds into a specific structure independently of other regions.

[0020] Interleukin-3 (IL-3) is a cytokine that regulates the production and function of hematopoietic and immune cells. The IL-3 receptor, IL-3R, is composed of an α-subunit, CD123, and a β-subunit, CD131. CD123 is a transmembrane glycoprotein that provides specificity to IL-3. CD131, on the other hand, is a subunit common to the GM-CSF, IL-3, and IL-5 receptors and is an essential transmembrane glycoprotein for signal transduction. The amino acid sequence of the CD123 protein is registered, for example, as Uniprot accession number P26951. The amino acid sequence of the human CD131 protein is registered, for example, as Uniprot accession number P32927. When IL-3 binds to IL-3R, the JAK / STAT5, PI-3K, and MAPK signaling pathways are activated.

[0021] IL-3R is known to be expressed in a wide range of tumors. Examples of IL-3R-expressing tumors include, but are not limited to, hematopoietic malignancies, such as leukemias including acute myeloid leukemia, chronic myeloid leukemia, hairy cell leukemia, B-cell lineage acute lymphoblastic leukemia, and blastic plasmacytoid dendritic cell neoplasm. Examples of IL-3R-expressing tumor cell lines include MV4-11, THP-1, and Kasumi-1.

[0022] In the present invention, the target-binding domain that binds to IL-3R exhibits binding affinity to IL-3R (preferably its extracellular ligand-binding domain), enabling an immune response against target cells expressing IL-3R on their cell surface. The target-binding domain that binds to IL-3R preferably has high affinity for the CD123 / CD131 complex (e.g., Kd value of 500 pM or less) but low affinity for CD123 alone (e.g., Kd value of 50 nM or more). In the present invention, the target-binding domain that binds to IL-3R is, for example, IL-3. The IL-3 used as the target-binding domain may be derived from any mammal, including humans, livestock (horses, cattle, sheep, goats, pigs, etc.), companion animals (dogs, cats, rabbits, etc.), and experimental animals (mice, rats, monkeys, etc.), but is preferably derived from humans.

[0023] In the present invention, the IL-3 used as the target-binding domain may be wild-type IL-3 or an IL-3 mutant. Examples of wild-type IL-3 include polypeptides containing or consisting of the amino acid sequence shown in SEQ ID NO: 6 or 7. Examples of IL-3 mutants include IL-3 mutants having a substitution of lysine (K) with another amino acid at the position corresponding to the 105th amino acid in the amino acid sequence shown in SEQ ID NO: 6. Examples of substitutions for lysine (K) at position 105 of the amino acid sequence shown in Sequence ID No. 6 include substitution with alanine (A), isoleucine (I), leucine (L), methionine (M), valine (V), phenylalanine (F), tryptophan (W), tyrosine (Y), asparagine (N), cysteine ​​(C), glutamine (Q), serine (S), threonine (T), aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), glycine (G), or proline (P). Preferably, the substitution is with phenylalanine (F), tryptophan (W), or tyrosine (Y), and more preferably with tryptophan (W) (hereinafter sometimes referred to as "K105W"). IL-3 variants may be polypeptides that contain, or consist of, an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in, for example, SEQ ID NO: 6 or 7, and that retain the ability to bind to IL-3R. IL-3 variants may also be polypeptides that contain, or consist of, the amino acid sequence shown in, for example, SEQ ID NO: 17.

[0024] The binding ability of the target-binding domain to IL-3R is intended to be such that the Kd value is, for example, 100 nM or less, preferably 10 nM or less, more preferably 5 nM or less, and even more preferably 500 pM or less. This binding ability may be relatively weak compared to the binding ability between the antigen and the antibody.

[0025] As mentioned above, Mardiros et al. have developed a CAR that uses anti-CD123 scFv as its target-binding domain. However, no CARs that use IL-3 as their target-binding domain have been reported to date.

[0026] Conventional anti-CD123 scFv is thought to bind not only to the CD123 / CD131 complex present in tumor cells, but also to the CD123 monomer present in the quiescent phase of normal cells. Therefore, cells expressing CARs that use conventional anti-CD123 scFv as the target-binding domain are thought to have high off-tumor toxicity (toxicity resulting from recognition of normal tissue). In contrast, IL-3 has high affinity for the CD123 / CD131 complex present in tumor cells, but low affinity for CD123 alone present in the quiescent phase of normal cells (Broughton et al., Immunological Reviews, 2012, vol. 250:277-302). Therefore, cells expressing CARs that use IL-3 as the target-binding domain are thought to have lower off-tumor toxicity and higher safety compared to cells expressing CARs that use anti-CD123 scFv as the target-binding domain.

[0027] As shown in Examples 1-3 below, cells expressing CARs that use IL-3 as the target-binding domain exhibit antitumor effects against IL-3R-expressing tumors. Furthermore, as shown in Examples 1 and 3 below, cells expressing CARs that use IL-3 with the amino acid substitution K105W as the target-binding domain exhibit significantly higher antitumor effects compared to cells expressing CARs that use wild-type IL-2 as the target-binding domain. Moreover, as shown in Examples 1-3 below, cells expressing CARs that use IL-3 with the amino acid substitution K105W as the target-binding domain also exhibit lower expression of fatigue markers (PD-1 and LAG3) compared to cells expressing CARs that use anti-CD123 scFv as the target-binding domain, demonstrating high antitumor efficacy while exhibiting low toxicity to normal cells and a wide safety margin.

[0028] In the present invention, the CAR protein may optionally include an "extracellular spacer domain" between the extracellular target-binding domain and the transmembrane domain. The extracellular spacer domain is preferably a sequence that promotes the binding of the CAR to the antigen and enhances intracellular signaling. For example, an antibody Fc fragment, or a fragment or derivative thereof, an antibody hinge region, or a fragment or derivative thereof, an antibody CH2 region, an antibody CH3 region, an artificial spacer sequence, or a combination thereof can be used.

[0029] In one aspect of the present invention, the extracellular spacer domain can be (i) the hinge, CH2, and CH3 region of IgG4, (ii) the hinge region of IgG4, (iii) the hinge and CH2 region of IgG4, (iv) the hinge region of CD8a, (v) the hinge, CH2, and CH3 region of IgG1, (vi) the hinge region of IgG1, or (vii) the hinge and CH2 region of IgG1, or a combination thereof. For example, the IgG1 hinge region can preferably have the following amino acid sequence (SEQ ID NO: 1), but is not limited thereto. TIFF2026168857000001.tif1495

[0030] Furthermore, IgG1 having the amino acid sequence shown in SEQ ID NO: 2 can be suitably used as the CH2 region, and IgG1 having the amino acid sequence shown in SEQ ID NO: 3 can be suitably used as the CH3 region.

[0031] In a preferred embodiment, the extracellular spacer domain may be the hinge, CH2, and CH3 regions of human IgG1 or a portion thereof.

[0032] In addition, in preferred embodiments, the extracellular spacer domain can be used as (i) the hinge region of human IgG1 alone (SEQ ID NO: 1), (ii) a combination of the hinge region of human IgG1 (SEQ ID NO: 1), the CH2 region (SEQ ID NO: 2), and the CH3 region (SEQ ID NO: 3), (iii) a combination of the hinge region of human IgG1 (SEQ ID NO: 1) and the CH3 region (SEQ ID NO: 3), (iv) the CH3 region alone (SEQ ID NO: 3), or (v) a combination of the CH2 region (SEQ ID NO: 2) and the CH3 region (SEQ ID NO: 3).

[0033] In one aspect of the present invention, a spacer sequence represented by formula (G4S)n can be used as an artificial spacer sequence for use in the extracellular spacer domain. In the formula, n is 1 to 10, preferably n=3. Spacers having such a spacer sequence are sometimes called peptide linkers. Peptide linkers that are suitably used in the art can be used as appropriate in the present invention. In this case, the composition and chain length of the peptide linker can be selected appropriately within a range that does not impair the function of the resulting CAR protein.

[0034] The extracellular spacer domain is not particularly limited, but can be appropriately selected from those exemplified above, or further modified based on common technical knowledge in the art, and used for the present invention. For example, as the extracellular spacer domain, a polypeptide containing the hinge region of human IgG1 (SEQ ID NO: 1) and a spacer sequence represented by (G4S)n, such as the polypeptide shown in SEQ ID NO: 25, can be suitably used.

[0035] The extracellular spacer domain can be expressed in host cells by inserting the nucleotide sequences encoding the amino acid sequences of each domain into a vector, so that it can be located between the target-binding domain and the transmembrane domain. Alternatively, the extracellular spacer domain can be modified using a pre-prepared plasmid CAR protein encoding polynucleotide as a template.

[0036] Modification of the extracellular spacer domain is useful, for example, when considering the effects on CAR gene expression rates in host cells of CAR-T cells into which CAR-encoding polynucleotides have been introduced, signal transduction, cellular senescence, tumor distribution, antigen recognition, or in vivo activity.

[0037] In the present invention, the CAR protein comprises an extracellular domain including a target-binding domain and optionally an extracellular spacer domain, a transmembrane domain, and an intracellular domain including an intracellular signaling domain and optionally a costimulatory domain. As is well known in the art, the "transmembrane domain" is a domain that has affinity for the lipid bilayer constituting the cell membrane, in contrast to the extracellular and intracellular domains, which are both hydrophilic domains.

[0038] The transmembrane domain is not particularly limited, as long as the CAR protein can reside on the cell membrane and does not impair the function of the target-binding domain and the intracellular signaling domain. However, a polypeptide derived from the same protein as the co-stimulatory domain, described later, may also function as the transmembrane domain. Examples of transmembrane domains that can be used include CD28, CD3ε, CD8α, CD3, CD4, or 4-1BB. For example, human CD28 (Uniprot No.: P10747 (153-179)) can be used as the transmembrane domain. Specifically, a protein having an amino acid sequence encoded by the nucleotide sequence NCBI Accession No.: NM_006139.3 (679-759) can be suitably used as the transmembrane domain.

[0039] CAR proteins may contain a "costimulatory domain." The costimulatory domain specifically binds to a costimulatory ligand, thereby mediating a cellular costimulatory response, such as CAR-T cell proliferation, cytokine production, functional differentiation, or target cell death, but are not limited to these. Examples of costimulatory domains include CD27, CD28, 4-1BB (CD137), CD134 (OX40), Dap10, CD2, CD5, CD30, CD40, PD-1, ICAM-1, LFA-1 (CD11a / CD18), TNFR-1, TNFR-II, Fas, and Lck. For example, the costimulatory domain could be human CD28 (Uniprot No.: P10747 (180-220)) or 4-1BB (GenBank: U03397.1). Specifically, a co-stimulatory domain having an amino acid sequence encoded by the nucleotide sequence of NCBI Accession No.: NM_006139.3 (760-882) can be suitably used.

[0040] When using transmembrane domains and co-stimulatory domains derived from human CD28, for example, those having the amino acid sequence shown in SEQ ID NO: 4 can be used.

[0041] CAR proteins contain an "intracellular signaling domain." This intracellular signaling domain transmits signals necessary for the effector function of immune cells. Examples of intracellular signaling domains include human CD3ζ chains, FcγRIII, FcεRI, the cytoplasmic terminal of an Fc receptor, a cytoplasmic receptor having an immunoreceptor tyrosine activation motif (ITAM), or a combination thereof. For example, the intracellular signaling domain can be a human CD3ζ chain (e.g., nucleotides 299-637 of NCBI Accession No. NM_000734.3). Specifically, a sequence having the amino acid sequence shown in SEQ ID NO. 5 can be suitably used as the intracellular signaling domain.

[0042] To promote the secretion of CAR proteins, the N-terminus of the protein appropriately includes a leader sequence that guides the protein's translocation during or after translation. Examples of useful leader sequences that can be suitably used in the present invention include, but are not limited to, human immunoglobulin (Ig) heavy chain signal peptides, CD8α signal peptides, or human GM-CSF receptor α signal peptides. For example, Ig heavy chain signal peptides derived from IgG1, IgG2, IgG3, IgA1, and IgM can be suitably used. As for the leader sequence, for example, one having the amino acid sequence shown in SEQ ID NO: 23 can be used.

[0043] The target polynucleotides can be easily prepared according to conventional methods. The base sequences encoding the amino acid sequences can be obtained from NCBI RefSeq IDs or GenBank Accession numbers indicating the amino acid sequences of each domain, and the polynucleotides of the present invention can be prepared using standard molecular biological and / or chemical procedures. For example, nucleic acids can be synthesized based on these base sequences, and the polynucleotides of the present invention can be prepared by combining DNA fragments obtained from a cDNA library using polymerase chain reaction (PCR).

[0044] Therefore, polynucleotides encoding CAR proteins can be created by linking polynucleotides encoding each of the domains mentioned above, and genetically modified cells can be created by introducing these polynucleotides into appropriate cells. Furthermore, CAR proteins can also be created by using a polynucleotide encoding an existing CAR protein, whose components other than the target-binding domain are identical, as a template, and rearranging the target-binding domain according to conventional methods.

[0045] Furthermore, depending on the purpose, one or more domains, such as the extracellular spacer domain, can be modified using polynucleotides encoding existing CAR proteins as templates, for example, by inverse PCR (iPCR). Techniques for modifying the extracellular spacer domain are described, for example, in Oncoimmunology, 2016, Vol.5, No.12, e1253656.

[0046] The method for introducing polynucleotides to create genetically modified cells is not particularly limited and any commonly used method is acceptable. When using a vector for introduction, the vectors that can be used are not particularly limited, but examples include lentiviral vectors, retroviral vectors, foam virus vectors, and adeno-associated virus vectors. Furthermore, the introduction of polynucleotides can be carried out by non-viral methods using transposons.For the transposon method, plasmid transposons can be used, and the sleeping beauty transposon system (e.g., Huang X, Guo H, et al. Mol Ther. 2008; 16: 580-9; Singh H, Manuri PR, et al. Cancer Res. 2008; 68: 2961-71; Deniger DC, Yu J, et al. PLoS One. 2015; 10: e0128151; Singh H, Moyes JS, et al. Cancer Gene Ther. 2015; 22: 95-100; Hou X, Du Y, et al. Cancer Biol Ther. 2015; 16: 8-16; Singh H, Huls H, et al. Immunol Rev. 2014; 257: 181-90; and Maiti SN, Huls H, et al. J) Immunother. 2013; 36: 112-23) or the piggyBac transposon system (Nakazawa Y, Huye LE, et al. J Immunother. 2009; 32: 826-36;Galvan DL, Nakazawa Y, et al. J Immunother. 2009; 32: 837-44;Nakazawa Mol Ther. 2011; 19: 2239-48;Saha S, Nakazawa Y, et al. J Vis Exp. 2012; (69): e4235;Nakazawa Y, Saha S, et al. J Immunother. 2013; 36: 3-10; Saito S, The methods described in Nakazawa Y, et al. Cytotherapy. 2014; 16: 1257-69; and Nakazawa et al. Journal of Hematology & Oncology (2016) 9:27 are suitable for use.

[0047] When using the piggyBac transposon system, typically, a plasmid containing the gene encoding the piggyBac transposase (hereinafter referred to as the piggyBac plasmid) and a plasmid having a structure in which a polynucleotide encoding the CAR protein is flanked by reversed piggyBac repeat sequences are introduced (transfected). Various methods can be used for transfection, such as electroporation, nucleofection, lipofection, and calcium phosphate methods. Both plasmids may contain a poly(A) addition reader sequence, a reporter gene, a selection marker gene, an enhancer sequence, etc.

[0048] The equipment used for electroporation is not limited to those mentioned above, but examples include the 4D-Nucleofector (Lonza Japan Co., Ltd.), NEPA21 (Neppageen Co., Ltd.), and Maxcyte GT (Maxcyte, Inc.), and can be operated according to their respective instruction manuals. Using the above method, for example, 1 × 10⁻¹⁶ 6 ~2×10 7 It is possible to introduce genes into cells within a certain range.

[0049] The cells into which the above polynucleotides are introduced can be mammalian cells, such as humans, or non-human mammalian cells such as monkeys, mice, rats, pigs, cattle, or dogs. Preferably, the cells into which the above polynucleotides are introduced are those that release cytotoxic proteins (such as perforin or granzyme). Specifically, the cells into which the above polynucleotides are introduced can be immune cells (e.g., T cells, NK cells, NK-T cells, macrophages) or immune cell progenitor cells (e.g., hematopoietic stem cells, lymphoid progenitor cells, myeloid progenitor cells). Furthermore, cells capable of differentiating into these cells, such as ES cells or iPS cells, can also be used. T cells include CD8-positive T cells, CD4-positive T cells, regulatory T cells, cytotoxic T cells, and tumor-infiltrating lymphocytes. The cells into which the above polynucleotides are introduced may be those collected from living organisms, those cultured on a large scale, or those established as cell lines. Cells into which the above polynucleotides can be introduced include, for example, cell populations containing T cells, such as peripheral blood mononuclear cells (PBMCs). When transplanting cells expressing the manufactured CAR or cells differentiated from such cells into a living organism, it is desirable to introduce the nucleic acid into the organism itself or cells collected from a similar organism.

[0050] Polynucleotides are introduced into the genetically modified cells of the present invention, and as T cells used in adoptive immunotherapy, T cells that are expected to have a sustained antitumor effect, such as stem cell memory T cells, can be used. Stem cell memory T cells can be easily identified by following conventional methods, for example, according to the description in Yang Xu, et al. Blood. 2014; 123:3750-3759.

[0051] In one embodiment, stem cell memory T cells are, for example, CD45R0 - CD62L + CD45RA + and CCR7 + T cells can be used.

[0052] The present invention also provides a vector comprising the polynucleotide described above.

[0053] The present invention also provides genetically modified cells into which the polynucleotides or vectors of the present invention described above have been introduced. The genetically modified cells of the present invention can express CAR proteins that bind to IL-3R-expressing cells on the cell membrane.

[0054] The present invention further provides a method for producing CAR protein-expressing cells, comprising introducing the polynucleotide or vector of the present invention into cells.

[0055] The culture and amplification of CAR protein-expressing cells are not particularly limited, but for example, after introducing polynucleotides encoding CAR proteins into cells as described above, non-specific or CAR-specific stimuli can be applied to the cells for purposes such as activating the CAR protein-expressing cells. The method of cell stimulation is not limited, but for example, non-specific cell stimulation can be performed using anti-CD3 antibodies and / or anti-CD28 antibodies, and CAR-specific stimulation can be performed using artificial antigen-presenting cells (aAPCs) that express antigen molecules or co-stimulatory factors that bind to CAR in tumor cell lines such as K562. The culture conditions here are not particularly limited, but for example, culturing at 37°C and 5% CO2 for 1 to 15 days or 1 to 10 days is preferable.

[0056] In the method of the present invention described above, introduction into cells is preferably carried out by the transposon method, although this is not limited to that method. Furthermore, as the transposon method, either the transposon system described above or any other system suitable for the present invention may be used, and is not particularly limited, but the method of the present invention can be preferably carried out by using the piggyBac method.

[0057] Another embodiment of nonspecific stimulation involves stimulating a cell population containing T cells with one or more viral peptide antigens, and then using cells whose viral replication ability has been inactivated by conventional methods as feeder cells to promote the activation of cells into which CAR has been introduced. The viral peptide antigens used may include, for example, an AdV antigen peptide mixture, a CMV antigen peptide mixture, or an EBV antigen peptide mixture, or a combination thereof.

[0058] Another embodiment of the specific stimulation involves co-culturing immature dendritic cells with CAR-expressing cells, as described in International Publication No. 2020 / 085480 and Japanese Patent Publication No. 2025-17424, by culturing PBMCs derived from the same individual from which the CAR-expressing cells originate in a medium containing human IL-4 and GM-CSF. As the immature dendritic cells, immature dendritic cells pulsed with a fusion protein of the CAR target antigen and the antibody's Fc domain polypeptide may be used. Furthermore, CAR-expressing cells may be cultured in the presence of the fusion protein before the above co-culturing, or the above co-culturing may be performed in the presence of the fusion protein.

[0059] Furthermore, to increase cell viability / proliferation, the cells can be cultured in the presence of one or more cytokines, and it is preferable to culture them in the presence of cytokines such as IL-7 and IL-15.

[0060] The present invention further provides a kit for producing CAR protein-expressing cells that target IL-3R-expressing cells, comprising the vector of the present invention described above. The kit of the present invention may appropriately include reagents, buffers, reaction vessels, instructions for use, etc., necessary for producing CAR protein-expressing cells. The kit of the present invention can be suitably used to produce the genetically modified cells of the present invention described above.

[0061] The cells of this invention are activated by signaling within the cell through a receptor-specific immune response to target cells expressing IL-3R on their surface. The activation of CAR-expressing cells varies depending on the host cell type and the intracellular domain of the CAR, but can be confirmed using indicators such as cytokine release, improved cell proliferation rate, and changes in cell surface molecules. The release of cytotoxic proteins (such as perforin and granzyme) leads to the destruction of cells expressing the receptor.

[0062] The genetically modified cells of the present invention can be used as a therapeutic agent for diseases involving IL-3R-expressing cells (for example, cancers that express IL-3R). Therefore, the present invention provides a therapeutic agent for diseases involving IL-3R-expressing cells, which includes the genetically modified cells of the present invention. Diseases that are expected to be treated with the therapeutic agent of the present invention are not limited to diseases that are sensitive to the cells, but include, for example, diseases related to cells that express IL-3R on their cell membranes, such as IL-3R-expressing tumors. Examples of IL-3R-expressing tumors include hematopoietic tumors, such as leukemias such as acute myeloid leukemia, chronic myeloid leukemia, hairy cell leukemia, B-cell lineage acute lymphoblastic leukemia, and blastic plasmacytoid dendritic cell tumors.

[0063] One embodiment of the therapeutic agent of the present invention is an anticancer agent against IL-3R expressing tumor cells, such as the tumors described above. The therapeutic agent or anticancer agent of the present invention can be used alone, or it can be used in combination with agents and / or therapies of different mechanisms.

[0064] Accordingly, the therapeutic agent of the present invention can be made into the form of a pharmaceutical composition, either alone or in combination with other active ingredients. The therapeutic agent or pharmaceutical composition of the present invention can be administered locally or systemically, and the administration form is not limited, but for example, intravenous administration is preferred. In addition to the therapeutic agent of the present invention and other active ingredients, the pharmaceutical composition may contain carriers, excipients, buffers, stabilizers and the like commonly used in the art depending on the administration form. In one aspect of the present invention, the pharmaceutical composition may comprise, for example, the therapeutic agent of the present invention and a pharmaceutically acceptable carrier.

[0065] The dosage of the therapeutic agent of the present invention varies depending on the patient's body weight, age, severity of the disease and the like, and is not particularly limited, but for example, 10 4 ~10 10 CAR-positive cell count / kg body weight, or 10 6 ~10 8 CAR-positive cell count / kg body weight, can be administered once to several times a day, every 2 days, every 3 days, every week, every 2 weeks, every month, every 2 months, or every 3 months.

[0066] The administration targets of the therapeutic agent or pharmaceutical composition of the present invention include any mammals including humans, domestic animals (horses, cattle, sheep, goats, pigs, etc.), pet animals (dogs, cats, rabbits, etc.), experimental animals (mice, rats, monkeys, etc.), etc., and humans are preferred.

[0067] The CAR expression rate (CAR-positive CD3-positive cell rate) of the cells contained in the therapeutic agent or pharmaceutical composition of the present invention can be, for example, 10% or more, 20% or more, or 30% or more.

[0068] The present invention further provides a method for treating a disease associated with IL-3R-expressing cells, comprising administering a therapeutically effective amount of the above-described therapeutic agent or pharmaceutical composition of the present invention to a patient. The therapeutically effective amount and administration regimen can be appropriately determined in consideration of various factors as described above. [Examples]

[0069] The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

[0070] [Simulation of CAR expression plasmid] We designed IL-3R CARs containing wild-type IL-3 or IL-3 variants as target-binding domains. Additionally, as a reference, we designed a CD123 CAR containing a single-chain antibody (scFv) against CD123 as a target-binding domain. The target-binding domains of each CAR are shown in the table below.

[0071] [Table 1]

[0072] In Table 1, all amino acid substitution positions are the same as those in SEQ ID NO: 6.

[0073] Each CAR contains the target-binding domain described above, as well as the leader sequence, spacer, CD28, and CD3ζ shown in the table below.

[0074] [Table 2]

[0075] The nucleotide sequence encoding the IL-3R CAR described above was inserted between the 5'IR and 3'IR segments of the pIRII plasmid to construct an IL-3R CAR expression plasmid. Similarly, the nucleotide sequence encoding the CD123 CAR described above was inserted between the 5'IR and 3'IR segments of the pIRII plasmid to construct a CD123 CAR expression plasmid. The vector maps of the constructed CAR expression plasmids are shown in Figure 1.

[0076] [CAR-T cell generation] IL-3R CAR-T cells were generated by culturing PBMCs (Primary Biomedical Cells) into which IL-3R CAR or CD123 CAR had been introduced, while applying three types of stimuli (first to third stimuli). The specific generation method is as follows.

[0077] (1) Separation and preparation of PBMC Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy volunteer donors using Ficoll-PaquePlus (GE Healthcare, Chicago, IL). The isolated PBMCs were either used in the subsequent process on the same day or frozen for storage. Frozen PBMCs were thawed before use and ALyS was added with 5% artificial serum (animal-free; Cell Science & Technology Institute, Japan). TM Recovery culture was performed for 2 hours in 705 culture medium (Cell Science and Technology Institute Inc., Miyagi, Japan).

[0078] (2) Introduction of CAR gene (Day 0) PBMC15~20×10 separated and adjusted as described in (1) above 6 For each cell, add 7.5 μg of the IL-3R CAR expression plasmid or CD123 CAR expression plasmid prepared in the above [CAR expression plasmid preparation], 7.5 μg of the pCMV-piggyBac transposase plasmid (Matthew H Wilson et al., Molecular Therapy, 2007; 15(1): 139-145), and Supplement 1 to a P3 Primary Cell Nucleofector. TM 100 μl of Solution (Lonza, Basel, Switzerland) was added, and electroporation was performed using a 4D-Nucleofector (program FL-115) (Day 0).

[0079] The resulting transgenic cells were treated with recombinant human IL-7 (final concentration 10 ng / mL) (Miltenyi Biotec, Bergisch Gladbach, Germany), recombinant human IL-15 (final concentration 5 ng / mL) (Miltenyi Biotec, Bergisch Gladbach, Germany), and ALyS with 5% artificial serum. TM The cells were cultured in 705 culture medium using 24-well plates (Day 0-1). The culture environment was 37°C and 5% CO2.

[0080] (3) Preparation of immature dendritic cells (Day 0-2) PBMC 0.8~1.2×10 separated and adjusted in (1) above 6 Add human IL-4 (final concentration 10 ng / mL) and human GM-CSF (final concentration 10 ng / mL) (Miltenyi Biotec, Bergisch Gladbach, Germany) to 3 ml of ALyS TM 24-well G-REX in 705 culture medium R Culture was performed using (Wilson Wolf, Saint Paul, MN). Alternatively, 3-6 × 10¹⁶ PBMCs isolated and prepared in (1) above were used. 6 Add human IL-4 (final concentration 10 ng / mL) and human GM-CSF (final concentration 10 ng / mL) (Miltenyi Biotec, Bergisch Gladbach, Germany) to 10 ml of AlyS TM 6-well G-REX in 705 culture medium R The cells were cultured using (Wilson Wolf, Saint Paul, MN). The culture environment was 37°C and 5% CO2.

[0081] (4) First stimulus: Stimulation at CD123-Fc (Day 1-3) D-PBS (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and human CD123-Fc (ACROBiosystems, Delaware, Newark, USA) were added to a non-treated 24-well plate or a non-treated 48-well plate to a final concentration of 2 μg / mL of CD123-Fc, and the plates were left to stand overnight in a refrigerator to prepare CD123-Fc solid-phase plates (Day 0-1).

[0082] On Day 1, the CD123-Fc-containing solution was removed from the prepared CD123-Fc-immobilized plate to remove any excess CD123-Fc that had not been immobilized. The transgenic cells cultured in (2) above were transferred to the CD123-Fc-immobilized plate and cultured (Days 1-3). When transferring the transgenic cells from the 24-well plate in (2) above to the CD123-Fc-immobilized 24-well plate, the entire cell solution from one well was transferred to one well. When transferring the transgenic cells from the 24-well plate in (2) above to the CD123-Fc-immobilized 48-well plate, the cell solution was divided and transferred as appropriate.

[0083] (5) Second stimulation: Stimulation with CD123-Fc pulse iDC (Day 3~) On Day 2, the 24-well G-REX mentioned in (3) above R Discard 2.5 mL of the supernatant from each well, and add 0.5 mL of the remaining solution to human CD123-F C (Final concentration 2 μg / mL) was added. Alternatively, the 6-well G-REX from (3) above was added. R The supernatant (7 mL) was discarded, and human CD123-Fc (final concentration 2 μg / mL) was added to the remaining 3 mL. Subsequently, the cells were cultured at 37°C and 5% CO2 for more than 12 hours to obtain CD123-Fc pulsed immature dendritic cells (iDCs).

[0084] On Day 3, transfer the genetically modified cells cultured in (4) above to G-REX containing CD123-Fc pulse iDC. R In addition, AlyS contains 5% artificial serum.TM Culture medium 705 was added, recombinant human IL-7 (final concentration 10 ng / mL), and recombinant human IL-15 (final concentration 5 ng / mL), and cultivation was continued (Day 3 onwards). The culture environment was 37°C and 5% CO2. During the culture period, half of the culture medium was replaced every 3-4 days, and recombinant human IL-7 and recombinant human IL-15 were added to reach final concentrations of 10 ng / mL and 5 ng / mL, respectively.

[0085] (6) Third stimulus: Stimulation at CD123-Fc (Day 3~) In (5) above, the third stimulus was performed by not removing the iDC culture medium when mixing the gene-transformed cells with the iDCs, and by introducing the suspended CD123-Fc in the culture medium into the subsequent culture (final concentration 0.2 μg / mL).

[0086] (7) Production of control T cells (activated T cells, ATCs) PBMC isolated in (1) above 1.5 × 10 6 Each piece contains 5 ng / mL IL-15 and 5% artificial serum ALiS TM The cells were suspended in 705 culture medium and seeded onto 24-well non-treated plates coated with anti-CD3 and anti-CD28 antibodies (Miltenyi Biotec, Auburn, CA). Culturing was started at 37°C under 5% CO2 (Day 0). On the second day after initiation of culturing (Day 2), the cells were transferred to 24-well treated plates and culturing was continued. The culture medium was changed every 3-4 days during the culturing period.

[0087] [Evaluation of cell count, CAR expression rate, and CAR-T cell count] The total number of cultured cells, CAR expression rate, and number of CAR-T cells in the above-described [CAR-T cell production] were evaluated. The total number of cells was measured using trypan blue staining and an automated cell counter model R1 (Olympus Corporation, Tokyo, Japan). Trypan blue staining was performed by equal-volume dilution of the cell saturation with trypan blue. CAR expression rate was analyzed by flow cytometry. Specifically, to detect CAR-expressing cells, the recovered cells were stained with either a PE-labeled anti-human IL-3 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) or a CD123-Fc and R-PE-labeled anti-human IgG antibody (Jackson ImmunoResearch Inc. West Grove, PA, USA; AffiniPure F(ab')2Fragment Goat Anti-Human IgG (H+L)-R-PE). To detect T cells, an APC-labeled anti-CD3 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) was used. Next, the stained cells were subjected to a flow cytometer (BD FACSCantoII or FACSLyric, BD Biosciences San Jose, CA), and the obtained data was analyzed using Flowjo (BD Biosciences San Jose, CA). The percentage of CAR-positive CD3-positive cells was measured as the CAR expression rate. The number of CAR-T cells was calculated by multiplying the total number of cells by the CAR expression rate.

[0088] <Example 1. Production of IL-3R CAR-T cells and evaluation of in vitro antitumor effect> 1-1. Production of CAR-T cells Following the method described in [CAR-T cell production] above, IL-3R CAR was introduced into PBMCs (Day 0), and the gene-transformed cells were cultured while applying three types of stimuli (first to third stimuli), and control T cells were also cultured.

[0089] Cultured cells were harvested on days 8, 10, 14, and 16, and the total cell count, CAR expression rate, and CAR-T cell count were evaluated according to the method described in [Evaluation of cell count, CAR expression rate, and CAR-T cell count] above.

[0090] The results are shown in Figures 2 and 3. When any of the seven types of IL-3R CARs (wtIL-3-CAR, IL-3_k17Rs6Hn109V-CAR, IL-3_h84Af26Mk105D-CAR, IL-3_n30Rn109W-CAR, IL-3_n30R-CAR, IL-3_k105W-CAR, IL-3_h84Af26M-CAR) were introduced, T cells expressing IL-3R CARs (IL-3R CAR-T cells) were generated (Figures 1 and 2). In particular, when IL3_k105W-CAR was introduced, the CAR expression rate increased by approximately 1.5 times compared to when wtIL3-CAR was introduced (Figure 2), and the number of CAR-T cells increased by approximately 8 times (Figure 3B).

[0091] 1-2. Evaluation of in vitro antitumor effects The antitumor effects of four of the seven types of IL-3R CAR-T cells produced in 1-1 (wtIL-3-CAR-T cells, IL-3_k17Rs6Hn109V-CAR-T cells, IL-3_h84Af26Mk105D-CAR-T cells, and IL-3_k105W-CAR-T cells) were evaluated by co-culture testing.

[0092] Specifically, cells cultured from Day 14 to 16 in 1-1 were collected. The collected IL-3R CAR-T cells or control T cells (effectors) were co-cultured with the acute myeloid leukemia cell line MV4-11 (ATCC) (target) in effector:target ratios (E:T ratio) of 1:1, 1:5, and 1:10 in 10% FBS and penicillin / streptomycin-containing RPMI for 4 days. It was confirmed that MV4-11 expresses IL-3R.

[0093] After culturing was complete, the cells were harvested and stained with PE-labeled anti-CD33 antibody (BioLegend, California, San Diego, US) and APC-labeled anti-CD3 antibody (Miltenyi Biotec, Bergisch-Gladbach, Germany). The stained cells were subjected to flow cytometry using BD FACSLyric (BD Biosciences San Jose, CA), and the obtained data was analyzed using Flowjo to determine the number of viable tumor cells.

[0094] The results are shown in Figure 4. All four types of IL-3R CAR-T cells (wtIL-3-CAR-T cells, IL-3_k17Rs6Hn109V-CAR-T cells, IL-3_h84Af26Mk105D-CAR-T cells, and IL-3_k105W-CAR-T cells) reduced the number of tumor cells compared to control T cells. In particular, IL-3_k105W-CAR-T cells significantly reduced the number of tumor cells compared to wtIL-3-CAR-T cells.

[0095] <Example 2. Production of IL-3_k105W-CAR-T cells and anti-CD123 scFv-CAR-T cells, and comparison of in vitro antitumor effects and safety> 2-1. Production of CAR-T cells Following the method described in [CAR-T cell production] above, IL-3_k105W-CAR or anti-CD123 scFv-CAR was introduced into PBMCs (Day 0), and the transgenic cells were cultured while applying three types of stimulation (first to third stimulation), and control T cells were also cultured.

[0096] Cultured cells were harvested on Days 8, 10, and 14, and the total cell count, CAR expression rate, and CAR-T cell count were evaluated according to the method described in [Evaluation of Cell Count, CAR Expression Rate, and CAR-T Cell Count] above.

[0097] The results are shown in Figures 5 and 6. The CAR expression rate and CAR-T cell number when IL-3_k105W-CAR was introduced were similar to those when anti-CD123 scFv-CAR was introduced.

[0098] 2-2. Immunophenotypic Analysis The expression of PD1 and LAG3, and the expression of CD8 and CD4 in the IL-3_k105W-CAR-T cells and anti-CD123 scFv-CAR-T cells generated in 2-1 were analyzed by flow cytometry. Specifically, cells cultured from Day 14 to 16 in 2-1 were harvested, and the harvested cells were stained using the following reagents.

[0099] Detection of CAR-expressing cells: CD123-Fc and R-PE-labeled anti-IgG antibodies (Jackson ImmunoResearch Inc. West Grove, PA, USA; AffiniPure F(ab')2Fragment Goat Anti-Human IgG (H+L)-R-PE) T cell detection: APC-labeled anti-CD3 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) Detection of PD-1: BV421-labeled anti-PD-1 antibody (BD Biosciences San Jose, CA) LAG3 detection: BV421-labeled anti-LAG3 antibody (BD Biosciences San Jose, CA) CD4 detection: PacificBlue-labeled anti-CD4 antibody (Biolegend, San Diego, CA) CD8 detection: APCcy7-labeled anti-CD8 antibody (Biolegend, San Diego, CA)

[0100] Next, the stained cells were subjected to a flow cytometer (FACSLyric, BD Biosciences San Jose, CA), and the obtained data was analyzed using Flowjo, BD Biosciences San Jose, CA.

[0101] As a result, PD1 in IL-3_k105W-CAR-T cells + cell percentage, LAG3 + The cell percentages were 9.5% and 7.0%, respectively, and PD1 in anti-CD123 scFv-CAR-T cells + cell percentage, LAG3 + The cell proportions were 18.5% and 24.9%, respectively. Therefore, IL-3_k105W-CAR-T cells had significantly lower positivity rates for the fatigue markers PD1 and LAG3 than anti-CD123 scFv-CAR-T cells.

[0102] Furthermore, CD4 in IL-3_k105W-CAR-T cells - CD8 - cell percentage, CD4 - CD8 + cell percentage, CD4 + CD8 - cell percentage, CD4 + CD8 + The cell percentages were 1.14%, 58.6%, 37.7%, and 2.58%, respectively, and CD4 in anti-CD123 scFv-CAR-T cells. - CD8 - cell percentage, CD4 - CD8 + cell percentage, CD4 + CD8 - cell percentage, CD4 + CD8 + The cell percentages were 0.6%, 45.5%, 49.0%, and 4.88%, respectively. Therefore, IL-3_k105W-CAR-T cells have greater cytotoxic activity than anti-CD123 scFv-CAR-T cells. + The proportion of cells was high.

[0103] 2-3. Evaluation of in vitro antitumor effects The antitumor effects of IL-3_k105W-CAR-T cells and anti-CD123 scFv-CAR-T cells prepared in 2-1 above were evaluated by co-culture testing.

[0104] Specifically, cells derived from three donors (Donor 1, Donor 2, Donor 3) that had been cultured until Day 16 in 2-1 above were collected. The collected IL-3_k105W-CAR-T cells, anti-CD123 scFv-CAR-T cells, or control T cells (effectors) were co-cultured with the acute myeloid leukemia cell line MV4-11 (ATCC) (target) for 4 days in 10% FBS and penicillin / streptomycin-containing RPMI at various effector:target ratios (E:T ratio). The number of tumor cells used in co-culture was 1 × 10⁶ 5 ~2.5×10 5 There was one.

[0105] After culturing was complete, the cells were harvested and stained with PE-labeled anti-CD33 antibody (BioLegend, California, San Diego, US) and APC-labeled anti-CD3 antibody (Miltenyi Biotec, Bergisch-Gladbach, Germany). The stained cells were subjected to flow cytometry using BD FACSLyric (BD Biosciences San Jose, CA), and the obtained data was analyzed using Flowjo to determine the number of viable tumor cells.

[0106] The results are shown in Figures 7A-C. When control T cells were co-cultured with tumor cells (MV4-11), the number of viable tumor cells did not decrease in an effector dose-dependent manner. In contrast, when IL-3_k105W-CAR-T cells or anti-CD123 scFv-CAR-T cells were co-cultured with tumor cells, the number of viable tumor cells decreased in an effector dose-dependent manner. The number of viable tumor cells when IL-3_k105W-CAR-T cells were co-cultured with tumor cells was significantly lower or no significant difference compared to the number of viable tumor cells when anti-CD123 scFv-CAR-T cells were co-cultured with tumor cells. Therefore, it was shown that IL-3_k105W-CAR-T cells have a higher antitumor effect than anti-CD123 scFv-CAR-T cells.

[0107] 2-4. Toxicity Assessment The toxicity of IL-3_k105W-CAR-T cells and anti-CD123 scFv-CAR-T cells, prepared in 2-1 above, to normal cells was evaluated by co-culture testing.

[0108] Specifically, the IL-3_k105W-CAR-T cells, anti-CD123 scFv-CAR-T cells, and control T cells (effectors) derived from three donors (Donor 1, Donor 2, Donor 3) that were cultured and cryopreserved from Day 14 to 16 in 2-1 above were thawed the day before the co-culture test and underwent recovery culture. The thawed effectors and CD34 + Bone marrow cells (LONZA) (target) were co-cultured for 2 days at various effector:target ratios (E:T ratio). The culture medium used was RPMI medium containing 10% FBS-containing penicillin / streptomycin-containing DNase (final concentration 7.1 μg / mL) (STEMCELL TECHNOLOGIES, Vancouver, BC) / IL-3 (final concentration 10 ng / mL) / SCF (final concentration 10 ng / mL) / TPO (final concentration 10 ng / mL) (Miltenyi Biotec, Bergisch-Gladbach, Germany). The number of bone marrow cells used in co-culture was 500 cells / well.

[0109] Subsequently, the co-cultured cells were mixed with 5 mL of MethoCult H4434 classic (STEMCELL TECHNOLOGIES, Vancouver, BC), a hematopoietic progenitor cell colony measurement medium, and 1.1 mL was seeded into a Smart dish 6-well plate (STEMCELL TECHNOLOGIES, Vancouver, BC) to initiate culture. The culture environment was 37°C and 5% CO2. Colony counts were measured using STEMVISION (STEMCELL TECHNOLOGIES, Vancouver, BC) on days 15 and 16 from the start of culture.

[0110] The results are shown in Figures 8A-C. IL-3_k105W-CAR-T cells or anti-CD123 scFv-CAR-T cells and normal cells (CD34 +When co-cultured with bone marrow cells, the number of colonies decreased in an effector dose-dependent manner. However, the number of colonies when IL-3_k105W-CAR-T cells were co-cultured with normal cells was significantly higher or no significant difference compared to the number of colonies when anti-CD123 scFv-CAR-T cells were co-cultured with normal cells. Therefore, it was shown that IL-3_k105W-CAR-T cells have lower toxicity to normal cells compared to anti-CD123 scFv-CAR-T cells. Note that control T cells and normal cells (CD34) were used. + Co-culturing with bone marrow cells was performed only at an E:T ratio of 20:1; it was not performed at any other E:T ratio.

[0111] 2-5. Evaluation of Safety Margin Based on the number of viable tumor cells measured in steps 2-3 above, the cytotoxic activity of IL-3_k105W-CAR-T cells and anti-CD123 scFv-CAR-T cells against tumor cells (MV4-11) was calculated using the following formula. Cytotoxic activity against tumor cells (%) = 100 - (Number of viable tumor cells when CAR-T cells and tumor cells are co-cultured / Number of viable tumor cells when tumor cells are cultured alone) × 100 Furthermore, from the number of colonies measured in steps 2-4 above, normal cells (CD34) of IL-3_k105W-CAR-T cells and anti-CD123 scFv-CAR-T cells were identified. + The cytotoxic activity against bone marrow cells was calculated. Cytotoxic activity against normal cells (%) = 100 - (Number of colonies when CAR-T cells and normal cells are co-cultured / Number of colonies when normal cells are cultured alone) × 100

[0112] Figure 9 shows a graph with the E:T ratio on the horizontal axis and cytotoxic activity against tumor cells on the vertical axis. Figure 10 shows a graph with the E:T ratio on the horizontal axis and cytotoxic activity against normal cells on the vertical axis.

[0113] Using the statistical analysis software Prism (GraphPad, Boston), the ED50 (50% effective dose) was calculated from the cytotoxic activity of IL-3_k105W-CAR-T cells and anti-CD123 scFv-CAR-T cells against tumor cells (MV4-11), and the cytotoxic activity of IL-3_k105W-CAR-T cells and anti-CD123 scFv-CAR-T cells against normal cells (CD34) was also calculated. + The LD50 (50% lethal dose) was calculated from the cytotoxic activity against bone marrow cells. The safety margin (LD50 / ED50) was calculated from the ED50 and LD50 values. The results are shown in the table below.

[0114] [Table 3]

[0115] In all donors, IL-3_k105W-CAR-T cells were shown to have a wider safety margin than anti-CD123 scFv-CAR-T cells (Table 3).

[0116] <Example 3. Comparison of in vivo antitumor effects of wtIL-3-CAR-T cells, IL-3_k105W-CAR-T cells, and anti-CD123 scFv-CAR-T cells> Following the method described in [CAR-T cell production] above, wtIL-3-CAR, IL-3_k105W-CAR, or anti-CD123 scFv-CAR was introduced into PBMCs (Day 0). The transgenic cells were cultured while applying three types of stimuli (1st to 3rd stimuli), and control T cells were also cultured. The cultured cells were harvested on Days 14-16. The obtained wtIL-3-CAR-T cells, IL-3_k105W-CAR-T cells, anti-CD123 scFv-CAR-T cells, and control T cells were used in the following experiments.

[0117] Immunodeficient mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ) (The Jackson Laboratory Bar Harbor) contains 5×10⁻¹⁰ human monocytic leukemia cell lines THP-1 (ATCC) expressing luciferase. 6 The cells were administered intravenously. It was confirmed that THP-1 expresses IL-3R. Five days after tumor administration, 300 μg of D-luciferin sodium (OZ Biosciences) dissolved in D-PBS was administered intraperitoneally to mice, and in vivo imaging was performed using IVIS Lumina LT (PerkinElmer Shelton, CT) to quantify the tumor burden. The mice were divided into groups so that the average tumor burden was approximately the same. Seven days after tumor administration, 5 × 10⁶ wtIL-3-CAR-T cells, IL-3_k105W-CAR-T cells, anti-CD123 scFv-CAR-T cells, or control T cells were administered. 6 D-luciferin sodium (OZ Biosciences) dissolved in D-PBS was administered intravenously to mice on days 9, 12, 16, 21, 28, and 35 after tumor administration. 300 μg of D-luciferin sodium dissolved in D-PBS was administered intraperitoneally to mice, and in vivo imaging was performed using IVIS Lumina LT (PerkinElmer Shelton, CT) to quantify the tumor volume.

[0118] The results are shown in Figures 11-13. Mice administered with wtIL-3-CAR-T cells, IL-3_k105W-CAR-T cells, or anti-CD123 scFv-CAR-T cells had lower tumor loads compared to mice administered with control T cells or PBS (Figure 11). In particular, mice administered with IL-3_k105W-CAR-T cells had the lowest tumor loads (Figures 12 and 13). Therefore, it was shown that wtIL-3-CAR-T cells and IL-3_k105W-CAR-T cells have antitumor effects, and that IL-3_k105W-CAR-T cells, in particular, have a remarkable antitumor effect.

[0119] Sequence ID 1: Amino acid sequence of hinge-spacer-hinge EPKSCDKTHTCPPCDPAEPKSPDKTHTCP SEQ ID NO: 2 Amino acid sequence of the CH2 region of IgG1 PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK SEQ ID NO: 3 Amino acid sequence of the CH3 region of IgG1 GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Amino acid sequence of SEQ ID NO: 4 CD28 FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS Amino acid sequence of SEQ ID NO: 5 CD3ζ RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Amino acid sequence of wtIL-3 (without signal peptide) (SEQ ID NO: 6) SWVNCSNMIDEIITHLKQPPLPLLDFNNLNGEDQDILMENNLRRPNLEAFNRAVKSLQNASAIESILKNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLKTLENAQAQQTTLSLAIF Amino acid sequence of wtIL-3 (with signal peptide) (SEQ ID NO: 7) MSRLPVLLLLQLLVRPGLQAPMTQTTPLKTSWVNCSNMIDEIITHLKQPPLPLLDFNNLNGEDQDILMENNLRRPNLEAFNRAVKSLQNASAIESILKNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLKTLENAQAQQTTLSLAIF Sequence ID 8: The nucleotide sequence encoding wtIL-3 atgagccgcctgcccgtcctgctcctgctccaactcctggtccgccccggactccaagctcccatgacccagacaacgcccttgaagacaagctgggttaactgctctaacatg atcgatgaaattataacacacttaaagcagccacctttgcctttgctggacttcaacaacctcaatggggaagaccaagacattctgatggaaaataaccttcgaaggccaaac ctggaggcattcaacagggctgtcaagagtttacagaacgcatcagcaattgagagcattcttaaaaatctcctgccatgtctgccctggccacggccgcacccacgcgacat ccaatccatatcaaggacggtgactggaatgaattccggaggaaactgacgttctatctgaaaaccccttgagaatgcgcaggctcaacagacgactttgagcctcgcgatcttt Amino acid sequence of SEQ ID NO: 9 IL-3_k17Rs6Hn109V MSRLPVLLLLQLLVRPGLQAPMTQTTPLKTSWVNCHNMIDEIITHLRQPPLPLLDFNNLNGEDQDILMENNLRRPNLEAFNRAVKSLQNASAIESILKNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLKTLEVAQAQQTTLSLAIF The base sequence encoding SEQ ID NO: 10 IL-3_k17Rs6Hn109V atgagccgcctgcccgtcctgctcctgctccaactcctggtccgccccggactccaagctcccatgacccagacaacgcccttgaagacaagctgggttaactgccataacatg atcgatgaaattataacacacttaagacagccacctttgcctttgctggacttcaacaacctcaatggggaagaccaagacattctgatggaaaataaccttcgaaggccaaac ctggaggcattcaacagggctgtcaagagtttacagaacgcatcagcaattgagagcattcttaaaaatctcctgccatgtctgccctggccacggccgcacccacgcgacat ccaatccatatcaaggacggtgactggaatgaattccggaggaaactgacgttctatctgaaaaccccttgaggttgcgcaggctcaacagacgactttgagcctcgcgatcttt Amino acid sequence of SEQ ID NO: 11 IL-3_h84Af26Mk105D MSRLPVLLLLQLLVRPGLQAPMTQTTPLKTSWVNCSNMIDEIITHLKQPPLPLLDMNNLNGEDQDILMENNLRRPNLEAFNRAVKSLQNASAIESILKNLLPCLPLATAAPTRAPIHIKDGDWNEFRRKLTFYLDTLENAQAQQTTLSLAIF The base sequence encoding SEQ ID NO: 12 IL-3_h84Af26Mk105D atgagccgcctgcccgtcctgctcctgctccaactcctggtccgccccggactccaagctcccatgacccagacaacgcccttgaagacaagctgggttaactgctctaacatg atcgatgaaattataacacacttaaagcagccacctttgcctttgctggacatgaacaacctcaatggggaagaccaagacattctgatggaaaataaccttcgaaggccaaac ctggaggcattcaacagggctgtcaagagtttacagaacgcatcagcaattgagagcattcttaaaaatctcctgccatgtctgcccctggccacggccgcacccacgcgagct ccaatccatatcaaggacggtgactggaatgaattccggaggaaactgacgttctatctggatacccttgagaatgcgcaggctcaacagacgactttgagcctcgcgatcttt Amino acid sequence of SEQ ID NO: 13 IL-3_n30Rn109W MSRLPVLLLLQLLVRPGLQAPMTQTTPLKTSWVNCSNMIDEIITHLKQPPLPLLDFNNLRGEDQDILMENNLRRPNLEAFNRAVKSLQNASAIESILKNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLKTLEWAQAQQTTLSLAIF The base sequence encoding SEQ ID NO: 14 IL-3_n30Rn109W atgagccgcctgcccgtcctgctcctgctccaactcctggtccgccccggactccaagctcccatgacccagacaacgcccttgaagacaagctgggttaactgctctaacatg atcgatgaaattataacacacttaaagcagccacctttgcctttgctggacttcaacaacctcagaggggaagaccaagacattctgatggaaaataaccttcgaaggccaaac ctggaggcattcaacagggctgtcaagagtttacagaacgcatcagcaattgagagcattcttaaaaatctcctgccatgtctgccctggccacggccgcacccacgcgacat ccaatccatatcaaggacggtgactggaatgaattccggaggaaactgacgttctatctgaaaacccttgagtgggcgcaggctcaacagacgactttgagcctcgcgatcttt Amino acid sequence of SEQ ID NO: 15 IL-3_n30R MSRLPVLLLLQLLVRPGLQAPMTQTTPLKTSWVNCSNMIDEIITHLKQPPLPLLDFNLRGEDQDILMENNLRRPNLEAFNRAVKSLQNASAIESILKNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLKTLENAQAQQTTLSLAIF The base sequence encoding SEQ ID NO: 16 IL-3_n30R atgagccgcctgcccgtcctgctcctgctccaactcctggtccgccccggactccaagctcccatgacccagacaacgcccttgaagacaagctgggttaactgctctaacatg atcgatgaaattataacacacttaaagcagccacctttgcctttgctggacttcaacaacctcagaggggaagaccaagacattctgatggaaaataaccttcgaaggccaaac ctggaggcattcaacagggctgtcaagagtttacagaacgcatcagcaattgagagcattcttaaaaatctcctgccatgtctgccctggccacggccgcacccacgcgacat ccaatccatatcaaggacggtgactggaatgaattccggaggaaactgacgttctatctgaaaaccccttgagaatgcgcaggctcaacagacgactttgagcctcgcgatcttt Amino acid sequence of SEQ ID NO: 17 IL-3_k105W MSRLPVLLLLQLLVRPGLQAPMTQTTPLKTSWVNCSNMIDEIITHLKQPPLPLLDFNNLNGEDQDILMENNLRRPNLEAFNRAVKSLQNASAIESILKNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLWTLENAQAQQTTLSLAIF The base sequence encoding SEQ ID NO: 18 IL-3_k105W atgagccgcctgcccgtcctgctcctgctccaactcctggtccgccccggactccaagctcccatgacccagacaacgcccttgaagacaagctgggttaactgctctaacatg atcgatgaaattataacacacttaaagcagccacctttgcctttgctggacttcaacaacctcaatggggaagaccaagacattctgatggaaaataaccttcgaaggccaaac ctggaggcattcaacagggctgtcaagagtttacagaacgcatcagcaattgagagcattcttaaaaatctcctgccatgtctgccctggccacggccgcacccacgcgacat ccaatccatatcaaggacggtgactggaatgaattccggaggaaactgacgttctatctgtggacccttgagaatgcgcaggctcaacagacgactttgagcctcgcgatcttt Amino acid sequence of SEQ ID NO: 19 IL-3_h84Af26M MSRLPVLLLLQLLVRPGLQAPMTQTTPLKTSWVNCSNMIDEIITHLKQPPLPLLDMNNLNGEDQDILMENNLRRPNLEAFNRAVKSLQNASAIESILKNLLPCLPLATAAPTRAPIHIKDGDWNEFRRKLTFYLKTLENAQAQQTTLSLAIF The base sequence encoding SEQ ID NO: 20 IL-3_h84Af26M atgagccgcctgcccgtcctgctcctgctccaactcctggtccgccccggactccaagctcccatgacccagacaacgcccttgaagacaagctgggttaactgctctaacatg atcgatgaaattataacacacttaaagcagccacctttgcctttgctggacatgaacaacctcaatggggaagaccaagacattctgatggaaaataaccttcgaaggccaaac ctggaggcattcaacagggctgtcaagagtttacagaacgcatcagcaattgagagcattcttaaaaatctcctgccatgtctgcccctggccacggccgcacccacgcgagct ccaatccatatcaaggacggtgactggaatgaattccggaggaaactgacgttctatctgaaaaccccttgagaatgcgcaggctcaacagacgactttgagcctcgcgatcttt Amino acid sequence of SEQ ID NO: 21 Anti-CD123 scFv MLLLVTSLLLCELPHPAFLLIPQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWVKQRPDQGLEWIGRIDPYDSETHYNQKFKDKAILTVDKSSSTAYMQLSSLTSEDSAVYYCARGNWDDYWGQ GTTLTVSSGGGGSGGGGSGGGSDVQITQSPSYLAASPGETITINCRASKSISKDLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNKYPYTFGGGTKLEIK Sequence ID 22: The base sequence encoding anti-CD123 scFv atgctgctgctggtgaccagcctgctgctgtgcgagctgccccacccgcctttctgctgatcccccaggtgcagctgcagcagccgggcgcggaac tggtgcgcccgggcgcgagcgtgaaactgagctgcaaagcgagcggctatacctttaccagctattggatgaactgggtgaaacagcgcccggatca gggcctggaatggattggccgcattgatccgtatgatagcgaaacccattataaccagaaatttaaagataaagcgattctgaccgtggataaaagcagcagcaccgcgtatatgcagctgagcagcctgaccagcgaagatagcgcggtgtattattgcgcgcgcggcaactgggatgattattggggccagg gcaccaccctgaccgtgagcagcggcggcggcggcagcggcggcggcggcagcggcggcggcggcagcgatgtgcagattaccccagagcccgagcta tctggcggcgagcccgggcgaaaccattaccattaactgccgcgcgagcaaaagcattagcaaagatctggcgtggtatcaggaaaaaccgggcaaa accaacaaactgctgatttatagcggcagcaccctgcagagcggcattccgagccgcttttagcggcagcggcagcggcaccgattttaccctgaccattagcagcctggaaccggaagattttgcgatgtattattgccagcagcataacaaatatccgtataccttggcggcggcaccaaactggaaattaaa SEQ ID NO: 23 Amino acid sequence of the leader sequence MEFGLSWLFLVAILKGVQC Sequence ID 24: Base sequence encoding the reader sequence ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGC Sequence ID 25: Amino acid sequence of the spacer. EPKSCDKTHTCPPCDPAEPKSPDKTHTCPGGGGSGGGGSGGGGSKDPK Sequence ID 26: Base sequence encoding the spacer GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGTGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCAGGTGGTGGTGGATCCGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTAAAGATCCCAAA The base sequence encoding Sequence ID No. 27, CD28. TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC The base sequence encoding sequence number 28 CD3ζ AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAG GCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAAGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCTAA Sequence ID 29 CMV promoter nucleotide sequence TCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTGCGATCGCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATC SEQ ID NO: 30 Nucleotide sequence of SV40pA GACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAAC

Claims

1. A polynucleotide encoding a chimeric antigen receptor (CAR) protein having a target-binding domain that binds to the interleukin-3 receptor (IL-3R), a transmembrane domain, and an intracellular signaling domain, wherein the target-binding domain is interleukin-3 (IL-3).

2. The polynucleotide according to claim 1, wherein IL-3 has a substitution of lysine (K) with another amino acid at the position corresponding to the 105th amino acid of the amino acid sequence shown in SEQ ID NO:

6.

3. The polynucleotide according to claim 1, wherein IL-3 comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 6 or 7.

4. A vector comprising the polynucleotide described in claim 1.

5. Genetically modified cells into which the polynucleotide described in any one of claims 1 to 3, or the vector described in claim 4, has been introduced.

6. A method for producing CAR protein-expressing cells, comprising introducing a polynucleotide according to any one of claims 1 to 3, or a vector according to claim 4, into cells.

7. A therapeutic agent for diseases involving IL-3R-expressing cells, comprising the cells described in claim 5.

8. A pharmaceutical composition comprising the therapeutic agent described in claim 7 and a pharmaceutically acceptable carrier.

9. The therapeutic agent according to claim 7, wherein the disease involving IL-3R-expressing cells is selected from the group consisting of acute myeloid leukemia, chronic myeloid leukemia, hairy cell leukemia, B-cell lineage acute lymphoblastic leukemia, and blastic plasmacytoid dendritic cell neoplasm.

10. The composition according to claim 8, wherein the disease involving IL-3R-expressing cells is selected from the group consisting of acute myeloid leukemia, chronic myeloid leukemia, hairy cell leukemia, B-cell lineage acute lymphoblastic leukemia, and blastic plasmacytoid dendritic cell neoplasm.

11. A kit for producing CAR protein-expressing cells that target IL-3R-expressing cells, comprising the vector described in claim 4.

Images