CXCL12 receptor-expressing chimeric antigen receptor (CAR)-T cells

Abstract

We provide CAR-T cell therapy for AML with a poor prognosis. [Solution] T cells are provided that co-express a chimeric antigen receptor (CAR) protein and a CXCL12 receptor protein on their cell membrane, wherein the chimeric antigen receptor (CAR) protein targets CD25 (IL-2 receptor α chain) or CD19. Also provided are a drug and a pharmaceutical composition that exhibit antitumor activity containing the T cells. Furthermore, a method is provided for the treatment and / or prevention of recurrence of neoplastic diseases in non-human subjects, comprising the step of administering T cells co-expressing a chimeric antigen receptor (CAR) protein targeting CD25 (IL-2 receptor α chain) or CD19 and a CXCL12 receptor protein on their cell membrane to a non-human subject.

Description

[Technical Field]

[0001] The present invention relates to improved chimeric antigen receptor (CAR)-T cell therapy. More specifically, the present invention relates to modified cells co-expressing a chimeric antigen receptor and the CXCL12 receptor protein, an antitumor agent containing said cells, and a pharmaceutical composition having antitumor activity. The present invention further relates to a method for treating neoplastic diseases using modified cells co-expressing a chimeric antigen receptor and the CXCL12 receptor protein. [Background technology]

[0002] Chimeric antigen receptor (CAR)-T cell therapy is adoptive immunotherapy that uses T cells (CAR-T cells) that express CARs that target specific antigens expressed on the surface of cells such as tumor cells. CAR-T cells are designed to recognize the target antigen and thereby attack cells that express the target antigen.

[0003] In recent years, CD19-targeting CAR-T cells have emerged as a promising treatment option for relapsed / refractory B-cell acute lymphoblastic leukemia (B-ALL) and diffuse large B-cell lymphoma (DLBCL) (Non-patent documents 1-3), and it is known that even a single dose can produce high therapeutic efficacy.

[0004] Since then, much effort has been made to evaluate whether CAR-T cell therapy can be applied to other hematological malignancies and solid tumors. For example, among hematological malignancies, acute myeloid leukemia (AML) is a disease that is clinically very difficult to cure, and in order to extend the survival of AML patients, CAR-T cells targeting molecules such as CD33 (Non-Patent Literature 4), CD123 (Non-Patent Literature 5), and the tumor-associated antigen Lewis-Y (TAA-LeY) (Non-Patent Literature 6) have been developed and are currently undergoing clinical evaluation. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Grupp , SA , Kalos , M , Barrett , D , Aplenc , R , Porter , DL , Rheingold , SR , Teachey , DT , Chew , A , Hauck , B , Wright , JF , et al. (2013). Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med 368, 1509–1518. 10.1056 / NOMoa1215134.

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[0006] There are several major obstacles in the development of CAR-T cells against AML. First, due to the similarity in gene expression between AML cells and normal hematopoietic stem / progenitor cells, it is difficult to find a suitable cell surface molecule as a target for CAR-T cells.

[0007] Second, the depletion of CD19+ B lymphocytes can be managed by intravenous immunoglobulin replacement. For example, the aforementioned CD33 is a molecule expressed on hematopoietic stem cells / progenitor cells (HSPC) and neutrophils, and possible adverse effects on normal myeloid cells such as neutrophils can cause potentially fatal febrile neutropenia and opportunistic infections caused by fungi and bacteria.

[0008] Finally, while CD19 is expressed on all B-ALL cells in B-ALL patients, the expression of cell surface molecules in AML is highly heterogeneous both among and within patients. [Means for Solving the Problem]

[0009] The present inventors performed global transcriptome analysis using leukemia-initiating cells (LIC) from clinically aggressive AML patients, normal CD34+CD38-CD45RA- hematopoietic stem cells (HSC) and neutrophils, and also analyzed the gene expression of patient-derived AML-initiating cells whose ability to develop AML in vivo has been demonstrated in NOD / SCID / Il2rgKO (NSG) xenograft assays.

[0010] As a result, CD25 (IL-2 receptor α chain, IL2RA) was identified as a cell surface molecule with therapeutic potential in acute myeloid leukemia (AML) (Saito et al., STM 2010). In addition to being reported as a poor prognostic marker for AML (Gonen et al blood 2012), CD25 / IL2RA is also expressed in other hematological malignancies including chronic myeloid leukemia (CML), adult T-cell leukemia / lymphoma, and Hodgkin lymphoma. However, since CD25 expression is also observed in subpopulations of regulatory T cells and activated T cells, it is not easy to evaluate whether CD25 / IL2RA is suitable as a target molecule for CAR-T cell therapy.

[0011] To develop CAR-T cell therapy for poor-prognosis AML, the present inventors constructed a lentiviral vector comprising a TCR signal sequence and a Fab antigen recognition site for human CD25 / IL2RA antigen. CD25-CAR lentiviral particles were introduced into cord blood-derived human T cells, resulting in 2×10 7 expansion of 6 or more CD25 CAR-T cells was achieved in vitro.

[0012] When the in vitro targeting efficiency of CD25 CAR-T cells against primary cultured cells of poor-prognosis AML was evaluated, cytotoxicity was observed only when CD25-expressing AML cells were used as targets, and was not observed in CD25-deficient leukemia.

[0013] Since the antigen-dependent cytotoxicity of CD25 CAR-T cells was confirmed, in vivo therapeutic experiments for AML were then established using NOD / SCID / IL2rgKO (NSG) patient-derived xenograft (PDX) animals. 5×10 were administered to three mice transplanted with AML cells (CD33-positive blood cells) from an AML patient (U390#2) 6A single infusion of CD25-targeted CAR-T cells resulted in a reduction of patient-derived leukemia cells in the peripheral blood (PB) of one out of three PDX mice. Four weeks after infusion, observation of three sacrificial mice revealed that the therapeutic effect was insufficient to eliminate invasive AML cells from the bone marrow of the recipient mice. HE staining demonstrated that the majority of the bone marrow cells were patient-derived AML cells.

[0014] To overcome the challenge that eradicating human AML cells is not optimal, the inventors investigated inducing CD25 CAR-T cells to the bone marrow by further modifying human T cells to express CXCR4, a known homing receptor, with the aim of more efficiently directing CAR-T cells to the bone marrow.

[0015] As a result, flow cytometry analysis of peripheral blood cells collected from mice administered with CAR-T cells showed that CD25 CAR-T cells expressing CXCR4 proliferated more efficiently in vivo than CD25 CAR-T cells that did not express CXCR4. Consistent with their high frequency and number, CXCR4-expressing CD25 CAR-T cells were able to eradicate human AML cells in peripheral blood, spleen, bone marrow, and liver. In long-term observational experiments, human CXCR4-expressing CD25 CAR-T cells remained in the bone marrow of treated mice for more than four months, preventing AML recurrence in recipient mice. In NSG mice transplanted to possess normal human innate and acquired immunity, no serious adverse effects of CXCR4-expressing CD25 CAR-T cells on normal human immune cells, such as unexpected proliferation of CAR-T cells, graft-versus-host disease, or severe inflammatory responses, were observed.

[0016] Based on the above findings, further investigation led the inventors to confirm that the improvement in CAR-T cell therapeutic effect through co-expression of CXCR4 also occurs in the case of CAR-T cells targeting other molecules, thus completing the present invention.

[0017] In other words, embodiments of the present invention are as follows. 1. Cells that co-express chimeric antigen receptor (CAR) protein and CXCL12 receptor protein on the cell membrane. 2. The cell described in item 1 above, wherein the CXCL12 receptor is CXCR4. 3. The cell according to 1 or 2 above, wherein the chimeric antigen receptor (CAR) protein targets a cell surface antigen selected from CD25 (IL-2 receptor α chain), CD19, and CD7. 4. A T cell, which is one of the cells described in 1-3 above. 5. A drug exhibiting antitumor activity that contains any of the cells described in 1 to 4 above. 6. The agents described in item 5 above, used in combination with further antitumor agents and / or antitumor treatments. 7. A pharmaceutical composition containing the drug described in item 5 above and a pharmaceutically acceptable carrier. 8. The agent described in item 5 or 6 above, or the pharmaceutical composition described in item 7 above, for the treatment and / or prevention of relapse of a neoplastic disease selected from the group consisting of acute myeloid leukemia (AML), adult T-cell leukemia, B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), mixed-plasmid acute leukemia (MPAL), chronic myeloid leukemia (CML), Hodgkin lymphoma, and non-Hodgkin lymphoma. 9. A method for producing cells according to any one of items 1 to 4 above, comprising introducing a first polynucleotide encoding a chimeric antigen receptor (CAR) protein and a second polynucleotide encoding a CXCL12 receptor protein into cells. 10. The method according to item 9 above, wherein the chimeric antigen receptor (CAR) protein targets a cell surface antigen selected from CD25 (IL-2 receptor α chain), CD19, and CD7. 11. The method according to 9 or 10, wherein the first polynucleotide and the second polynucleotide are introduced into a cell by the same or different vectors. 12. A method for treating and / or preventing recurrence of a neoplastic disease in a subject, comprising the step of administering cells that co-express a chimeric antigen receptor (CAR) protein targeting CD25 (IL-2 receptor α chain) and a CXCL12 receptor protein on their cell membrane. 13. The method according to item 12 above, wherein the chimeric antigen receptor (CAR) protein targets a cell surface antigen selected from CD25 (IL-2 receptor α chain), CD19, and CD7. 14. The method described in 12 or 13 above, wherein the neoplastic disease is selected from the group consisting of acute myeloid leukemia (AML), adult T-cell leukemia, B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), mixed-plasmid acute leukemia (MPAL), chronic myeloid leukemia (CML), Hodgkin lymphoma, and non-Hodgkin lymphoma. 15. A method described in any of items 12 to 14 above, which involves administering a single dose to the above cells. 16. 10 per kg of the subject's body weight 4 ~10 9 A method according to any one of items 12 to 15 above, comprising administering the above-mentioned cells in a range of 1. 17. A method according to any one of 12 to 16 above, further comprising the step of measuring the expression level of the target cell surface antigen in the tumor cells of the subject before administration of the cells. 18. A method according to any one of items 12 to 17 above, further comprising the step of evaluating the therapeutic effect after administration of cells. 19. The method described in 18 above, wherein the therapeutic effect is evaluated by one or more indicators selected from (i) a reduction in tumor cells in the blood, (ii) a reduction in tumor cells in the bone marrow, (iii) a reduction in tumor cells in the spleen, and (iv) suppression of tumor cell infiltration into the liver in the subject. 20. Cells for use in the treatment and / or prevention of cancer recurrence, co-expressing a chimeric antigen receptor (CAR) protein and a CXCL12 receptor protein on the cell membrane. 21. The cell described in 20 above, wherein the chimeric antigen receptor (CAR) protein targets a cell surface antigen selected from CD25 (IL-2 receptor α chain), CD19, and CD7. 22. The cells described in 20 or 21 above, wherein the cancer is a neoplastic disease selected from the group consisting of acute myeloid leukemia (AML), adult T-cell leukemia, B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), mixed-plasmid acute leukemia (MPAL), chronic myeloid leukemia (CML), Hodgkin lymphoma, and non-Hodgkin lymphoma. 23. A T cell, which is one of the cells described in any of items 20-22 above. This specification includes the disclosures of U.S. Provisional Patent Application No. 63 / 107027, filed on 29 October 2020, which forms the basis of the priority claim of this application. [Effects of the Invention]

[0018] The present invention provides an extremely effective treatment and / or recurrence prevention method for neoplastic diseases that have a poor prognosis and are difficult to cure. CXCR4-expressing CAR-T cell therapy is a promising treatment strategy for neoplastic diseases with a poor prognosis. [Brief explanation of the drawing]

[0019] [Figure 1]A: This shows the percentage of CD25-expressing cells in the bone marrow of PDX (Patient-Derived Xenograft) mice, which were created by transplanting AML cells derived from three patients with different CD25 expression levels into immunodeficient mice. From left to right: CD25-negative, moderate expression, and high expression. The reference figure shows the survival curves for CD25-negative and CD25-positive AML patients, respectively, reported in 2012. CD25 expression has been reported to be associated with a poor prognosis of AML (Gonen et al., Blood 2012). B: This shows the CD25 positivity rate in 84 AML patients measured by flow cytometry. Approximately 30% of AML patients were CD25-positive, including both high and moderate expression levels. C: This shows the results of evaluating CD25 expression in bone marrow cells of humanized mice, which were created by transplanting human hematopoietic stem cells derived from umbilical cord blood into immunodeficient mice. CD25 is a marker for regulatory T cells, and CD25 expression was observed in some activated T cells. In contrast, normal monocytes (CD14+), granulocytes (CD15+), and CD34+CD38- stem cells do not express CD25. [Figure 2] A: An example of a CAR-T construct targeting CD25 is shown. This is a second-generation CAR with the scFV of an anti-CD25 monoclonal antibody as the target-binding domain, and possessing a CD8a-derived leader sequence, hinge, transmembrane domain, and intracellular domains derived from 4-1BB and CD3ζ. B: This shows the in vitro cytotoxic activity of CD25 CAR-T cells against CD25-positive AML cells (U390, U346) and CD25-negative ALL cells (U328). The CAR-T cells targeted CD25-expressing AML cells (U390 and U346) but did not target CD25-negative leukemia cells (U328), and showed CD25-dependent cytotoxic activity. [Figure 3]This study presents the results of a flow cytometry analysis of human CD45-positive cells in PDX mice (#1, #2, #3) transplanted with human AML cells. Peripheral blood samples were collected weekly for four weeks, and CD33 and CD3 expression were used as indicators. All mice showed an increase in T cells (CD3+) and a decrease in AML cells (CD33+), although some AML cells remained in the peripheral blood after four weeks. While mouse #2 showed a good therapeutic response, the study revealed variability in the efficacy of CD25-targeted CAR-T cell therapy. [Figure 4] This image shows the results of HE staining of bone marrow sections after dissection four weeks following administration of 5 × 10⁶ CD25 CAR-T cells to PDX mice transplanted with AML cells. While various cell types, such as granulocytes and myelocytes, are observed in the bone marrow of immunodeficient mice (normal NSG), the bone marrow of AML PDX mice was found to be filled with leukemic blasts, indicating that the therapeutic effect of CD25 CAR-T cells was insufficient. Left: low magnification, Right: high magnification. [Figure 5-1] The results of flow cytometry of CD45-positive cells in peripheral blood two weeks after administration of CXCR4-expressing CD25 CAR-T cells to PDX mice transplanted with human AML cells (U390) are shown, compared to the case without CAR-T cell administration (untreated) and the case with CD25 CAR-T cell administration. The injected CXCR4-expressing CD25 CAR-T cells proliferated in vivo. [Figure 5-2] The results of flow cytometry of CD45-positive cells in peripheral blood four weeks after administration of CXCR4-expressing CD25 CAR-T cells to PDX mice transplanted with human AML cells (U390) are shown, compared to the case without CAR-T cell administration (untreated) and the case with CD25 CAR-T cell administration. In the case with CD25 CAR-T cell administration, almost all of the human cells were residual leukemia cells, whereas in the case with CXCR4-expressing CD25 CAR-T cell administration, CD33-positive leukemia (AML) cells were completely eliminated. [Figure 5-3]This report shows the flow cytometry results of CD45-positive cells in peripheral blood four months after administration of CXCR4-expressing CD25 CAR-T cells to PDX mice transplanted with human AML cells (U390). No AML cells were detected in the peripheral blood, and the effect of eliminating target AML cells within four weeks of administration was maintained for more than four months. After the elimination of target cells, the frequency and absolute number of CXCR4 CD25 CAR-T cells gradually decreased, and mouse CD45-positive cells accounted for the majority of peripheral blood cells. [Figure 6] This report shows the frequency (proportion) of AML cells, mouse leukocytes, and CAR-T cells in the peripheral blood cells of PDX mice transplanted with human AML cells. In mice without CAR-T cell administration (2 mice), most of the cells in the peripheral blood were human AML cells from immediately after AML cell transplantation until 4 weeks later. However, in mice injected with CD25 CAR-T cells (2 mice), one mouse showed a decrease in AML cells and a corresponding increase in CAR-T cells, while the other mouse did not show a decrease in AML cells. In contrast, in mice administered with CXCR4-expressing CD25 CAR-T cells (3 mice), AML cells decreased significantly, and AML cell-derived cells were completely eliminated from the circulating blood 3 weeks after CAR-T cell administration. The proportion of CAR-T cells increased while AML cells remained, but then gradually decreased. On the other hand, normal mouse leukocytes increased, and 14 weeks after CAR-T cell administration, more than 90% of the peripheral blood cells were mouse leukocytes. [Figure 7] These are photographs of the spleen and bone marrow of PDX mice transplanted with human AML cells, one treated with CXCR4-expressing CD25 CAR-T cells and the other untreated. In the untreated mice, splenomegaly and bone marrow whitening were observed, but in the mice treated with CXCR4-expressing CD25 CAR-T cells, a decrease in spleen size and reddening of the bone marrow, indicating the presence of red blood cells, were observed, confirming a significant therapeutic effect against AML. [Figure 8]This image shows HE staining results of bone marrow tissue from mice (U390 transplanted PDX mice) 4 weeks after administration of CXCR4-expressing CD25 CAR-T cells. It was confirmed that all normal leukocytes, erythrocytes, and megakaryocytes (which produce platelets) in the mice recovered after eradication of AML cells by CAR-T cells. The left image is low magnification, and the right image is high magnification. [Figure 9-1] This shows the results of flow cytometry analysis of bone marrow cells in PDX mice transplanted with human AML cells (U390), with and without treatment (untreated). The results for the untreated and CD25 CAR-T cell-treated samples are from 4 weeks after administration, and the results for the CXCR4-expressing CD25 CAR-T cell-treated sample are from 4 months after administration. [Figure 9-2] This report presents the results of flow cytometry analysis of bone marrow cells in PDX mice transplanted with human AML cells (U390), comparing those administered with CD25 CAR-T cells or CXCR4-expressing CD25 CAR-T cells and those that were not treated. The results for the untreated and CD25 CAR-T cell-treated samples are from 4 weeks after administration, while the results for the CXCR4-expressing CD25 CAR-T cell-treated sample are from 4 months after administration. Complete elimination of AML cells and recovery of normal mouse leukocytes (mCD45) were observed only in the group treated with CXCR4-expressing CD25 CAR-T cells. Complete elimination of AML cells and the presence of CAR-T cells were maintained for more than 140 days after CAR-T cell injection. [Figure 9-3] This paper presents the results of flow cytometry analysis of bone marrow cells in PDX mice transplanted with human AML cells (U390), comparing those administered with CD25 CAR-T cells or CXCR4-expressing CD25 CAR-T cells and those that were not treated. The results for the untreated and CD25 CAR-T cell-administered samples are from 4 weeks after administration, while the results for the CXCR4-expressing CD25 CAR-T cell-administered samples are from 4 months after administration. Recovery of mouse erythrocytes (Ter119) was observed only in the group treated with CXCR4-expressing CD25 CAR-T cells. [Figure 10]The images show the results of sacrificing AML-transplanted mice (right) that received a single dose of CXCR4-expressing CD25 CAR-T cells, and untransplanted mice (left), 150 days after administration, and staining their bone marrow tissue with HE staining or anti-CD34 monoclonal antibody. The eradication effect of AML cells was maintained even 5 months after CAR-T cell injection. [Figure 11-1] This graph shows the results of examining the percentage of leukemia cells (% chimerism) in spleen cells, bone marrow cells, and liver cells of PDX mice, which were created by transplanting cells from four different AML cases. Several weeks after administration of CD25 CAR-T or CXCR4-expressing CD25 CAR-T cells, the mice were sacrificed. Each dot represents one recipient mouse. In the spleen, administration of CXCR4-expressing CD25 CAR-T cells effectively eliminated leukemia cells in a significant number of recipient mice. In the bone marrow and liver, while CD25 CAR-T (CAR-T) administration resulted in a high frequency of residual leukemia cells in many mice, administration of CXCR4-expressing CD25 CAR-T cells (CXCR4 CAR-T) effectively eliminated leukemia cells with a considerable probability. NT: No administration. [Figure 11-2] The following shows the results of flow cytometry analysis of bone marrow cells obtained by sacrificing PDX mice, which were created by transplanting AML-derived cells, after several weeks of administration of CD25 CAR-T or CXCR4-expressing CD25 CAR-T cells. When CXCR4-expressing CD25 CAR-T cells were administered, leukemia cells disappeared, and subsequently, the number of CAR-T cells decreased without excessive activation, with normal white blood cells becoming the majority. [Figure 12] The results of flow cytometry analysis of liver cells 4 weeks after administration of CD25 CAR-T cells or CXCR4-expressing CD25 CAR-T cells to PDX mice transplanted with human AML cells (U300) are shown. In the liver, administration of CXCR4-expressing CD25 CAR-T cells resulted in an increase in T cells and complete disappearance of AML cells (confirmed in 4 out of 5 mice), demonstrating a significantly stronger therapeutic effect compared to administration of CD25 CAR-T cells. [Figure 13-1] The left side shows the percentage of leukemia cells in the peripheral blood of PDX mice transplanted with CD19-positive B-cell mixed-phenotype acute leukemia (MPAL, B / myeloid, U211) cells, after treatment with small molecule drugs and subsequent administration of CXCR4-expressing CD19 CAR-T cells. While the administration of small molecule drugs makes it appear as though leukemia cells have almost completely disappeared from the peripheral blood, they actually remain in the bone marrow. [Figure 13-2] After administration of small molecule therapeutic agents, administration of CXCR4-expressing CD19 CAR-T cells reduced the remaining MPAL cells from 10 days post-administration to 0.4% by 23 days, and completely disappeared by 32 and 39 days. [Figure 14] This shows the effect of CXCR4-expressing CD19 CAR-T (CXCR4 CD19 CAR-T) therapy in a xenograft model mouse using the TL1 cell line for Burkitt lymphoma. The left side shows the flow cytometry results from the bone marrow and liver of mice that were not administered CAR-T cells. The majority of human CD45-positive cells are CD19-positive, a B cell marker, and lymphoma cells make up the majority in both the bone marrow and liver. On the other hand, in mice administered CXCR4 CD19 CAR-T (right side), the majority of human CD45-positive cells in both the bone marrow and liver are CAR-T cells (CD3-positive), and lymphoma cells have almost completely disappeared. [Modes for carrying out the invention]

[0020] In one embodiment, the present invention provides cells that co-express a chimeric antigen receptor (CAR) protein and a CXCL12 receptor protein on the cell membrane.

[0021] <Chimera antigen receptor (CAR) protein> In this specification, "chimeric antigen receptor (CAR)" refers to a modified receptor whose target specificity can be transferred to cells. Hereinafter, in this specification, chimeric antigen receptors or chimeric antigen receptor proteins may be referred to as "CAR".

[0022] Here, the cells to which target specificity is transplanted by CAR are not limited, but for example, T cells can be suitably used. As T cells, for example, naive T cells, central memory T cells, effector memory T cells, or combinations thereof can be used. Furthermore, the T cells may be inflammatory T lymphocytes, cytotoxic T lymphocytes, or helper T lymphocytes. Cytotoxic T lymphocytes can be used in particular. In one embodiment, the T cells are selected from CD4+ T lymphocytes and CD8+ T lymphocytes. In a particular embodiment, the T cells modified according to the present invention are human T cells.

[0023] T cells can be obtained from a subject, for example, a human patient, by various non-limiting methods prior to the proliferation and genetic recombination of the cells of the present invention. T cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from an infection site, ascites, pleural fluid, spleen tissue, and tumors. In one embodiment, any number of T cell lines that are available and known to those skilled in the art can be used. In another embodiment, the cells may be obtained from a healthy donor or from a patient diagnosed with cancer. In another embodiment, the cells are part of a mixed population of cells exhibiting different phenotypic features. For convenience, cells modified to express a chimeric antigen receptor are referred to as "CAR-T cells" herein.

[0024] The structure of chimeric antigen receptor (CAR) proteins is well known in this field, and the CAR used in the present invention can utilize the structure of a commonly used CAR. Specifically, the CAR used in the present invention has a target-binding domain that specifically binds to a target molecule, a transmembrane domain, and an intracellular signaling domain. Here, "domain" refers to a region within a polypeptide that folds into a specific structure independently of other regions.

[0025] In this invention, the molecules targeted by CAR are antigens expressed on the surface of tumor cells, such as differentiation cluster molecules, e.g., CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138; tumor-associated surface antigens, e.g., ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), Lewis-Y (TAA-LeY), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disiaroganglioside GD2, ductal epithelial mucin, gp36, TAG-72, sphingoglycolipids, glioma-associated antigens, β-human chorionic gonadotropin, α-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostase-specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostain, PSMA, survivorbin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, major histocompatibility complex (MHC) molecule, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, and A1 domain (TnC A1) and fibroblast-associated protein (fap);Cell lineage-specific antigens or tissue-specific antigens, such as CD3, CD4, CD7, CD8, CD24, CD25 (IL-2 receptor α chain), CD32, CD33, CD34, CD123, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), CD180, GM-CSF, cytokine receptors, endoglin, major histocompatibility complex (MHC) molecules, lymphoblastic leukemia antigens, such as TNFRSF17 (UNIPROT Q02223), SLAMF7, GPRC5D, FKBP11, KAMP3, ITGA8, PRAME, and FCRL5, can be appropriately selected.

[0026] Preferably, the molecules targeted by CAR in the present invention are antigens that are significantly or remarkably expressed in tumor cells compared to other cells, and are not limited to, but include, for example, CD7, CD19, CD20, GD2, CD22, CD25 (IL-2 receptor α chain), CD30, CD33, CD44, CD96, CD123, CD180, CEA, Her2 / neu, MUC1, MUC4, MUC6, EGFR, PRAME, VEGFR2, GM-CSFR, IL-11Rα, IL-13α2, Lewis-Y (TAA-LeY), etc.

[0027] More preferably, the molecules targeted by CAR in the present invention are antigens that are significantly or remarkably expressed in specific hematological tumor cells compared to other cells, and are not limited to, but include, for example, CD7, CD19, CD25 (IL-2 receptor α chain), CD33, CD123, tumor-associated antigen Lewis-Y (TAA-LeY), etc.

[0028] For example, CD7, CD25, CD96, CD123, PRAME, and CD180 can be targeted for the treatment and / or prevention of relapse in acute myeloid leukemia (AML). For example, CD19, CD20, CD32, and CD180 can be targeted for the treatment and / or prevention of relapse in B-cell acute lymphoblastic leukemia (B-ALL) and mixed-phenotype acute leukemia (MPAL). For example, CD7 can be targeted for the treatment and / or prevention of relapse in T-cell acute lymphoblastic leukemia (T-ALL), MPAL, and chronic myeloid leukemia (CML).

[0029] In this invention, one molecule may be selected as the target molecule of the CAR, but multiple CAR-T cells targeting different molecules can also be used to target multiple molecules. Furthermore, to target multiple molecules, CARs targeting each molecule can be introduced into the same cell.

[0030] Particularly preferred in the present invention, the CAR targets a cell surface antigen selected from CD25 (IL-2 receptor α chain), CD19, and CD7, which are expressed in hematological tumor cells.

[0031] CD25 (IL-2 receptor α chain) has previously been reported as a marker of poor prognosis in AML. CD25 has been reported to be overexpressed in AML-initiating cells compared to normal D34+CD38-hematopoietic stem cells / progenitor cells (HSPCs) (Saito et al., Science Translational Medicine 2010).

[0032] Information such as the gene sequence of CD25 is listed as Gene ID: 3559 in databases such as the National Center for Biotechnology Information (NCBI), and can be obtained as needed.

[0033] CD19 is known to be expressed in diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, and B-cell acute lymphoblastic leukemia (B-ALL), but not in non-hematopoietic cells, myeloid cells, erythrocytes, and T cells. The inventors of this invention have also confirmed the expression of CD19 in B-cell mixed-phenotype acute leukemia (MPAL).

[0034] Information such as the gene sequence of CD19 is listed as Gene ID: 930 in databases such as NCBI, and can be obtained as needed.

[0035] CD7 is a 40 kDa type I transmembrane glycoprotein expressed on thymocytes and mature T cells, and is also used as a marker for T-cell acute lymphoblastic leukemia (T-ALL). The inventors have also confirmed the expression of CD7 in T-cell mixed-phenotype acute leukemia (MPAL) and chronic myeloid leukemia (CML).

[0036] Information such as the gene sequence of CD7 is listed as Gene ID: 924 in databases such as NCBI, and can be obtained as needed.

[0037] The target-binding domain of a CAR protein may include, for example, a single-chain antibody (scFv) fragment containing the heavy chain (H chain) variable region and the light chain (L chain) variable region of a monoclonal antibody that specifically binds to the target molecule described above. Those skilled in the art can easily prepare monoclonal antibodies and scFv fragments usable for CARs against specific antigens, or they can be commercially available. For example, suitable target-binding domains for the present invention include scFv fragments that specifically bind to CD25, CD19, or CD7.

[0038] Alternatively, the target-binding domain may be a ligand that specifically binds to the target molecule, such as an affibody, a ligand-binding domain derived from a native receptor, such as a soluble protein / peptide ligand or peptide of the receptor on tumor cells.

[0039] CAR proteins may, in some cases, contain an "extracellular spacer domain" between the extracellular target-binding domain and the membrane-binding domain. The extracellular spacer domain should preferably be a sequence that promotes the binding of the CAR to the target molecule 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.

[0040] CAR proteins comprise 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.

[0041] The "transmembrane domain" is a domain that has affinity for the lipid bilayer that constitutes the cell membrane, in contrast to the extracellular and intracellular domains, which are both hydrophilic domains. 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, polypeptides derived from the same protein as the co-stimulatory domain, which will be described later, may also function as transmembrane domains. Examples of transmembrane domains that can be used include CD28, CD3ε, CD8α, CD3, CD4, or 4-1BB.

[0042] CAR proteins may contain a "co-stimulatory domain." The co-stimulatory domain specifically binds to a co-stimulatory ligand, thereby mediating a cellular co-stimulatory response, such as CAR-T cell proliferation, cytokine production, functional differentiation, or target cell death, but are not limited to these. Examples of co-stimulatory domains include CD27, CD28, 4-1BB (CD137), CD134 (OX40), Dap10, CD27, CD2, CD5, CD30, CD40, PD-1, ICAM-1, LFA-1 (CD11a / CD18), TNFR-1, TNFR-II, Fas, and Lck. For example, the co-stimulatory domain could be human 4-1BB (GenBank:U03397.1).

[0043] 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).

[0044] In first-generation CARs, the signaling domain originated from the cytoplasmic region of CD3ζ or the Fc receptor γ chain. While first-generation CARs have been shown to direct T cell cytotoxicity, they were considered insufficient for long-term proliferation and antitumor activity in vivo. To improve CAR-T cell viability and increase proliferation, signaling domains derived from costimulatory molecules such as CD28, OX-40 (CD134), and 4-1BB (CD137) are now used alone (second generation) or in combination (third generation).

[0045] Figure 2A shows an example of a CAR construct that can be suitably used in the present invention. In this example, the target binding domain is an scFv fragment targeting CD25. In the present invention, the target binding domain of the CAR may be an scFv fragment targeting CD19. Further, in the present invention, the target binding domain of the CAR may be an scFv fragment targeting CD7.

[0046] Methods for obtaining monoclonal antibodies against a target are known in the art, and a person skilled in the art can obtain a monoclonal antibody using a selected target as an antigen; monoclonal antibodies can also be obtained as commercially available products. A monoclonal antibody or an scFv fragment thereof can be synthesized based on common technical knowledge in the art, and the base sequence of a gene encoding these can also be obtained and synthesized.

[0047] <CXCL12 receptor protein> The cell of the present invention is characterized by expressing CAR on the cell surface and also expressing a CXCL12 receptor protein.

[0048] CXC chemokine ligand 12 (CXCL12) is a low-molecular-weight protein of 89 amino acids, also called SDF (Stromal Cell-Derived Factor)-1, and belongs to the CXC chemokine family. CXCL12 is a potent chemoattractant for lymphocytes, and it has been suggested that it plays an important role in angiogenesis by, for example, mobilizing endothelial progenitor cells from bone marrow. CXC motif chemokine receptor type 4 (CXCR4, Fusin, CD184), which is known as a receptor for CXCL12, is a 7-transmembrane G protein-coupled receptor, and its expression in neutrophils, monocytes, dendritic cells, NK cells, B cells, T cells, and platelets is known.

[0049] CXCL12 / SDF1 is a chemokine ligand that promotes the homing and migration of human and mouse hematopoietic cells or immune cells to multiple organs, including the bone marrow and liver. While not theoretically bound, it is thought that co-expression of a CXCL12 receptor, such as CXCR4, allows CAR-T cells to home to the bone marrow more efficiently than CAR-T cells that do not express CXCR4. The CXCL12 receptor that can be suitably used in this invention is CXCR4.

[0050] Human CXCR4 is a protein consisting of 360 amino acids encoded by the CXCR4 gene. The base sequence of the gene and the amino acid sequence of the protein are listed in databases such as the National Center for Biotechnology Information (NCBI) as Gene ID: 7852 and Accession No: CAA12166, and the gene and protein can be obtained based on this information.

[0051] The inventors have found that CAR-T cells expressing the CXCL12 receptor protein exhibit significantly superior cytotoxic activity against target cells compared to CAR-T cells that do not express the CXCL12 receptor protein. The expression of the CXCL12 receptor protein on cells, such as CXCR4, may be the entire CXCL12 receptor protein, or it may be a portion of the protein, such as a functional fragment, that specifically binds to CXCL12 and enhances the cytotoxic activity of CAR-T cells.

[0052] For example, CAR-T cells expressing CXCR4 and targeting CD25 (hereinafter sometimes referred to as CXCR4 CD25 CAR-T in this specification) showed excellent antitumor activity against acute myeloid leukemia (AML). Therefore, one embodiment of the cells of the present invention is a cell that co-expresses CXCR4 and a CAR targeting CD25.

[0053] Furthermore, CAR-T cells expressing CXCR4 and targeting CD19 (hereinafter sometimes referred to as CXCR4 CD19 CAR-T in this specification) showed excellent antitumor activity against CD19-positive acute lymphoblastic leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), B-cell mixed-phenotype acute leukemia (MPAL), and Burkitt lymphoma. Therefore, one embodiment of the cells of the present invention is a cell that co-expresses CXCR4 and a CAR targeting CD19.

[0054] Currently, ALL is treated with small molecule drugs such as BIRC inhibitors (e.g., AZD5582), BCL-2 inhibitors (e.g., venetoclax), and steroids, either alone or in combination. However, complete cure is not always possible with such small molecule drugs alone. The inventors confirmed that when mice transplanted with human ALL cells were treated with small molecule drugs alone, leukemia cells appeared to disappear from the peripheral blood, but in reality, leukemia cells remained in the bone marrow, potentially causing relapse. In contrast, administration of CXCR4-expressing CD19 CAR-T cells completely eliminated the ALL cells that had increased (relapsed) after small molecule treatment.

[0055] CAR-T cells expressing CXCR4 and targeting CD7 (hereinafter sometimes referred to as CXCR4 CD7 CAR-T in this specification) show remarkable therapeutic effects against CD7-positive T-cell acute lymphoblastic leukemia (T-ALL), T-cell mixed-phenotype acute leukemia (MPAL), and chronic myeloid leukemia (CML). Therefore, one embodiment of the cells of the present invention is a cell that co-expresses CXCR4 and a CD7-targeting CAR.

[0056] As described above, CAR-T cells expressing the CXCL12 receptor protein have excellent therapeutic and / or recurrence-preventive effects against tumor cells. Accordingly, the present invention also provides a drug containing the cells of the present invention described above that exhibits antitumor activity against tumor cells.

[0057] The diseases targeted for treatment / recurrence prevention by the agents of the present invention are neoplastic diseases, particularly hematological malignancies. Examples of hematological malignancies, though not limited to them, include leukemia and malignant lymphoma.

[0058] Leukemia is classified into myeloid leukemia and lymphoid leukemia. Myeloid leukemia includes acute myeloid leukemia (AML), acute promyelocytic leukemia, chronic myeloid leukemia (CML), and myelodysplastic syndrome. Lymphoid leukemia includes acute lymphoblastic leukemia (ALL) (e.g., B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), mixed-phenotype acute leukemia (MPAL)), chronic lymphocytic leukemia (CLL), and adult T-cell leukemia / lymphoma.

[0059] Malignant lymphomas include Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL), and non-Hodgkin lymphomas are classified into B-cell, T-cell, NK-cell, etc. Examples of B-cell malignant lymphomas include diffuse large B-cell lymphoma, Burkitt lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, mucosa-associated lymphoid tissue type extranodal marginal zone lymphoma (MALT lymphoma), nodal marginal zone lymphoma, follicular lymphoma, and mantle cell lymphoma. Examples of T-cell malignant lymphomas include peripheral T-cell lymphoma (unspecified type), enteropathy-associated T-cell lymphoma, anaplastic large cell lymphoma, hepatosplenic T-cell lymphoma, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma (nasal type), angioimmunoblastic T-cell lymphoma (large granular lymphocytic T-cell leukemia), adult T-cell leukemia / lymphoma, mycosis fungoides / Sézary syndrome, primary cutaneous anaplastic large cell lymphoma, and rapidly progressive NK-cell leukemia.

[0060] The diseases targeted for treatment / prevention by the agents of the present invention are neoplastic diseases selected from the group consisting of, for example, acute myeloid leukemia (AML), adult T-cell leukemia, B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), mixed-plasmid acute leukemia (MPAL), chronic myeloid leukemia (CML), Hodgkin lymphoma, and non-Hodgkin lymphoma.

[0061] The agent of the present invention is an anticancer agent for the above-mentioned neoplastic diseases. The agent of the present invention, namely CAR-T cells, may be used alone, or, depending on the type of cancer being treated and various conditions such as the progression of symptoms, and at the discretion of the physician, it may be used in combination with further antitumor agents and / or antitumor treatments with different mechanisms of action.

[0062] Further antitumor agents, while not particularly limited, include, for example, antimetabolites, platinum-based drugs, microtubule inhibitors, topoisomerase inhibitors, molecularly targeted drugs, and steroids.

[0063] Examples of antimetabolites include 5-fluorouracil (5-FU), trifluridine, fludarabine (or its active metabolite, fludarabine nucleoside), cytarabine, gemcitabine, decitabine, guadecitabine, or azacitidine. Examples of platinum-based drugs include cisplatin, oxaliplatin, or carboplatin. Examples of microtubule inhibitors include paclitaxel, docetaxel, vinblastine, vincristine, vindesine, vinorelbine, and eribulin. Examples of topoisomerase inhibitors include irinotecan and etoposide.

[0064] Examples of molecularly targeted drugs include CSF1R inhibitors, TIE2 inhibitors, TRKB inhibitors, ATR inhibitors, Chk1 inhibitors, HSP90 inhibitors, PARP inhibitors, EGFR inhibitors, Her2 inhibitors, VEGFR inhibitors, PDGFR inhibitors, MET inhibitors, AXL inhibitors, RET inhibitors, FLT3 inhibitors, KIT inhibitors, HCK inhibitors, BIRC inhibitors (e.g., AZD5582), or BCL-2 inhibitors (e.g., venetoclax). HCK inhibitors and BCL-2 inhibitors may be those disclosed in, for example, Japanese Patent Application Publication No. 2019-529423. Examples of steroids include dexamethasone. On the other hand, other anti-tumor treatments include surgery and radiation therapy.

[0065] While not limited thereto, it has been confirmed that the agents of the present invention can exert a more robust therapeutic effect against leukemia when combined with BIRC inhibitors and / or BCL-2 inhibitors. Accordingly, one aspect of the present invention includes a combination therapy comprising an agent containing CAR-T cells expressing CXCL12 receptor protein and a BIRC inhibitor and / or a BCL-2 inhibitor. For example, cells co-expressing the chimeric antigen receptor (CAR) protein of the present invention and the CXCL12 receptor protein on their cell membrane can be administered to patients with hematological malignancies in combination with a BIRC inhibitor (e.g., AZD5582). Furthermore, cells co-expressing the chimeric antigen receptor (CAR) protein of the present invention and the CXCL12 receptor protein on their cell membrane can be administered to patients with hematological malignancies in combination with a BCL-2 inhibitor (e.g., venetoclax).

[0066] The agent containing CAR-T cells expressing the CXCL12 receptor protein of the present invention can be administered sequentially or separately, simultaneously with the further antitumor agents and / or antitumor treatments described above. In one embodiment, the further antitumor agents and / or antitumor treatments can be administered simultaneously with the administration of the agent of the present invention. In one embodiment, the further antitumor agents and / or antitumor treatments can be administered after the administration of the agent of the present invention. In one embodiment, the further antitumor agents and / or antitumor treatments can be administered before the administration of the agent of the present invention. In one embodiment, the further antitumor agents and / or antitumor treatments can be administered simultaneously with the administration of the agent of the present invention. In another embodiment, the agent of the present invention can be administered if recurrence occurs after treatment with antitumor agents and / or antitumor treatments.

[0067] The present invention also provides a pharmaceutical composition containing the above-described agent of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition may contain the agent of the present invention alone or in combination with other active ingredients as an active ingredient. The pharmaceutical composition of the present invention is intended for the treatment and / or prevention of recurrence of the above-described neoplastic diseases. For example, the pharmaceutical composition of the present invention may be for the treatment and / or prevention of recurrence of neoplastic diseases selected from the group consisting of acute myeloid leukemia (AML), adult T-cell leukemia, B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), mixed-phenotype acute leukemia (MPAL), chronic myeloid leukemia (CML), Hodgkin lymphoma, and non-Hodgkin lymphoma.

[0068] As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic carrier that is necessary or preferable to add for the formulation and administration of a pharmaceutical composition without impairing the activity of the active ingredient. For example, it may be an aqueous medium such as water or physiological saline, and may appropriately contain excipients, isotonic agents, buffers, stabilizers, gelling agents, etc.

[0069] The drug or pharmaceutical composition of the present invention can be administered locally or systemically, and the form of administration is not limited, but for example, in the case of treating leukemia, intravenous administration is preferred. The dosage of the drug of the present invention varies depending on the patient's weight, age, the severity of the disease, etc., and is not particularly limited, but for example, 10 per kg of the subject's weight. 4 ~10 10 pieces, or 10 4 ~10 9 The above-mentioned cells within a range of 10 can be administered. The drug or pharmaceutical composition of the present invention can be administered as a single dose. Alternatively, the drug or pharmaceutical composition of the present invention can be administered multiple times, once to several times a day, every two days, every three days, every week, every two weeks, every month, every two months, or every three months.

[0070] <Method for producing cells> The present invention also provides a method for producing the cells described above, comprising introducing a first polynucleotide encoding a chimeric antigen receptor (CAR) protein and a second polynucleotide encoding a CXCL12 receptor protein into the cells.

[0071] The target polynucleotide can be easily prepared by conventional methods. For polynucleotides encoding chimeric antigen receptor (CAR) proteins, the base sequences encoding each amino acid sequence can be obtained from NCBI RefSeq IDs or GenBank Accession numbers indicating the amino acid sequences of each domain (target-binding domain, extracellular spacer domain, transmembrane domain, costimulatory domain, and intracellular signaling domain). The first polynucleotide of the present invention can then be prepared by linking the polynucleotides encoding each domain using standard molecular biological and / or chemical procedures. For example, nucleic acids can be synthesized based on these base sequences, and the polynucleotide of the present invention can also be prepared by combining DNA fragments obtained from a cDNA library using polymerase chain reaction (PCR).

[0072] The second polynucleotide encoding the CXCL12 receptor protein can also be obtained from the NCBI RefSeq ID or GenBank Accession number indicating its amino acid sequence, and the second polynucleotide of the present invention can be prepared using standard molecular biological and / or chemical procedures.

[0073] The first and second polynucleotides described above can be introduced into cells, such as T cells, by any suitable method known in the art. Suitable methods for introducing nucleic acid molecules into cells include, but are not limited to, stable transformation methods in which the polynucleotide is integrated into the cellular genome, transient transformation methods in which the polynucleotide is not integrated into the cellular genome, and virus-mediated methods. For example, they may be introduced into cells by recombinant viral vectors (e.g., retroviruses, lentiviruses, adenoviruses), liposomes, etc. Transient transformation methods include, for example, microinjection, electroporation, or particle bombardment.

[0074] The polynucleotide introduced into the cell may be DNA or RNA. In one embodiment, the nucleic acid molecule introduced is DNA. In another embodiment, the nucleic acid molecule introduced into the cell is RNA, in particular mRNA encoding a CAR protein or a CXCL12 receptor protein.

[0075] For example, chimeric antigen receptor (CAR) proteins may target cell surface antigens selected from CD25 (IL-2 receptor α chain), CD19, and CD7.

[0076] The first polynucleotide and the second polynucleotide can be introduced into the cell by a single, identical vector, or they can be introduced into the cell by separate vectors.

[0077] <Treatment method> The present invention also provides a method for treating tumors, comprising administering the above-described agent or pharmaceutical composition of the present invention to a patient in a therapeutically effective dose. The therapeutically effective dose and administration regimen can be appropriately determined considering the type and stage of the target disease, the patient's age, etc. The subjects (patients) treated by the method of the present invention are mammals, such as mice, rats, dogs, cats, rabbits, cattle, horses, sheep, goats, monkeys, and humans. Preferably, the subjects treated by the method of the present invention are human patients. Alternatively, the subjects may be non-human mammals transplanted with human tumor cells, or non-human animals with neoplastic diseases.

[0078] The present invention provides a method for treating and / or preventing recurrence of neoplastic diseases in a subject, comprising the step of administering cells that co-express a CD25 (IL-2 receptor α chain) targeting chimeric antigen receptor (CAR) protein and a CXCL12 receptor protein on their cell membrane.

[0079] Chimeric antigen receptor (CAR) proteins may, but are not limited to, target cell surface antigens selected from, for example, CD25 (IL-2 receptor α chain), CD19, and CD7.

[0080] The neoplastic disease targeted for treatment and / or recurrence prevention in the method of the present invention may be any of the above-mentioned neoplastic diseases, and may be a disease selected from the group consisting of, for example, acute myeloid leukemia (AML), adult T-cell leukemia, B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), mixed-plasmid acute leukemia (MPAL), chronic myeloid leukemia (CML), Hodgkin lymphoma, and non-Hodgkin lymphoma.

[0081] The method of the present invention may involve a single administration to the above-mentioned cells. Alternatively, the method of the present invention may involve multiple administrations to the above-mentioned cells. Furthermore, the method of the present invention may be carried out in combination with the agent or pharmaceutical composition of the present invention, as described above, with further therapeutic agents, such as further antitumor agents and / or antitumor treatments. In the method of the present invention, 10 per kg of the subject's body weight 4 to 10 9 cells within the above range can be administered in a single dose or multiple doses.

[0082] In addition, the method of the present invention may further comprise a step of measuring the expression level of a target cell surface antigen in tumor cells of the subject before administration of the cells. For example, in one embodiment, the method of the present invention may comprise a step of measuring the expression level of CD25 in tumor cells of a subject before administration of CXCR4-expressing CD25 CAR-T cells. In another embodiment, the method of the present invention may comprise a step of measuring the expression level of CD19 in tumor cells of a subject before administration of CXCR4-expressing CD19 CAR-T cells. In still another embodiment, the method of the present invention may comprise a step of measuring the expression level of CD7 in tumor cells of a subject before administration of CXCR4-expressing CD7 CAR-T cells.

[0083] The method of the present invention may also further comprise a step of evaluating the therapeutic effect after administration of CXCR4-expressing CAR-T cells. Here, the therapeutic effect can be evaluated by one or more indicators selected from (i) reduction of tumor cells in blood, (ii) reduction of tumor cells in bone marrow, (iii) reduction of tumor cells in spleen, and (iv) inhibition of infiltration of tumor cells into the liver in the subject.

[0084] <Use for treatment and / or prevention of recurrence of cancer> The present invention also provides a cell that co-expresses a chimeric antigen receptor (CAR) protein and a CXCL12 receptor protein on the cell membrane for use in the treatment and / or prevention of recurrence of cancer.

[0085] For the cell, for example, the chimeric antigen receptor (CAR) protein expressed on the surface thereof targets a cell surface antigen selected from CD25 (IL-2 receptor α chain), CD19 and CD7.

[0086] The cancer described above may be any of the neoplastic diseases selected from the above list, for example, neoplastic diseases selected from the group consisting of acute myeloid leukemia (AML), adult T-cell leukemia, B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), mixed-plasmid acute leukemia (MPAL), chronic myeloid leukemia (CML), Hodgkin lymphoma, and non-Hodgkin lymphoma. The cells may be any of the above cells, but T cells are preferred. [Examples]

[0087] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0088] The lentiviral vector was constructed as follows. Using pHR_SFFV (Addgene plasmid # 79121, provided by Dr. Wendell Lim) as the lentiviral vector, the transgenes were packaged into the lentiviral vector by transfecting 293T cells with CD25-CAR (pHR_SFFV CD25CAR), mouse CXCR4 (pHR_SFFV mCXCR4), and packaging plasmids (pCMVR, pL2, pMD2.G-VSV-G, pAdV(Promega)) using JetPEI transfection reagent (Polyplus).

[0089] Four days after transfection, the viral supernatant was collected and centrifuged using Vivaspin 200 (merck). After centrifuging the concentrated viral supernatant overnight, the supernatant was discarded, and the pellet was lysed in T cell culture medium.

[0090] The culture and lentiviral infection of primary human T cells were performed using the following procedure. Human samples were collected from AML patients after obtaining informed consent. Umbilical cord blood was obtained from healthy donors at the Chubu Umbilical Cord Blood Bank (Aichi Prefecture).

[0091] Mononuclear cells from bone marrow and umbilical cord blood of AML patients were isolated by density gradient centrifugation. CD34 + Cells and CD34 - Cells were isolated using anti-human CD34 immunomagnetic beads by autoMACS (Miltenyi). T cells were isolated using CD34 by autoMACS with Pan T cells isolation kit (Miltenyi). - T cells were concentrated from the population. After isolation, they were cultured in X-Vivo15 (Lonza), 5% fetal bovine serum, 10 mM N-acetyl-L-cysteine ​​(Sigma-Aldrich #A9165), and 55 mM 2-mercaptoethanol (Jang hwan Cho 2018 Cell).

[0092] T cells were stimulated with 25 μl of Human T-activator CD3 / CD28 DynaBeades (Thermo Scientific #11132D), and 1 × 10⁶ cells were added to 1 ml of culture medium. 6 The cells were individually separated. The next day, T cells were placed in a 96-well plate at a rate of 1 x 10⁶ per well for lentiviral infection. 5 The cells were plated at a density of 1 cells / 100 μl. 16-100 μl of CAR lentivirus was added to 100 μl of T cell suspension using vectofusin-1 (10 μg / ml, Miltenyi) and cultured for 1 day. Half of the culture medium containing the infected T cells was removed and the same amount of fresh medium was added. Two days after infection, the expression of 25CAR in the infected T cells was analyzed using CD25-FC fusion protein, and the expression of mCXCR4 was analyzed using anti-mCXCR4 antibody. After confirming the expression of surface proteins, CD25 CAR-T cells were injected into AML model mice via the posterior orbital sinus.

[0093] Flow cytometry was performed by labeling cells with monoclonal antibodies (CD45, CD3, CD4, CD8, CD25, CD33, mouse CD45) and analyzing them using FACSAria III or FACSCanto II (BD Biosciences).

[0094] [Reference Example 1: Expression of CD25 / IL2RA on the surface of normal and malignant human hematopoietic cells] CD25 (IL-2Rα chain) is found in AML-affected cells and normal CD34 + CD38 - It has been identified as one of the genes that are expressed differently in hematopoietic stem cells / progenitor cells (Saito, et al. 2010 Sci Trans Med). Furthermore, CD25 expression in AML has been reported as a poor prognostic factor (Gonen, et al. 2012 Blood, Nguyen et al. Cancer Research 2020). In AML, which has diverse genetic abnormalities, the degree of CD25 expression may vary from case to case.

[0095] To investigate heterogeneity of CD25 expression in cells from AML patients, we used CD33 from 84 AML-derived cells. + The cell surface expression of CD25 in leukemia blasts was analyzed using flow cytometry.

[0096] As a result, 57 cases were CD25-negative, 14 cases showed moderate CD25 expression, and 13 cases showed high CD25 expression. CD25 was expressed to varying degrees in patient-derived AML cells in 27 out of 84 cases (32.2%) (Figures 1A and 1B).

[0097] Furthermore, analysis of CD25 expression in immature and mature human hematopoietic cells revealed that, consistent with our previous report (Saito, et al 2010 Sci Trans Med), the frequency of CD25-expressing cells was similar to that of hematopoietic stem cells / progenitor cells (CD34). + C38 - ) and bone marrow-erythroid progenitor cells (CD34 + CD38 + ) was low (Figure 1C). Also, monocytes (CD14 + ), granulocytes (CD15 + ), and NK cells (CD56 + CD25 protein expression in all of these cells was less than 5%, and human T cells (CD3 +In this study, 8.8% were found to express CD25 (Figure 1C).

[0098] [Reference Example 2: Cytotoxic activity of CAR-T cells targeting CD25] To generate CAR-T cells targeting the CD25 antigen, we designed a lentiviral vector containing an anti-IL2RA / CD25 protein single-chain variable fragment (scFv) and intracellular CD3z and CD137(4-1BB) signaling domains (Figure 2A) (Michael C. Milone, et al. 2009 Mol Therapy 17(8): 1453-64).

[0099] Next, we evaluated the in vitro killing effect of CAR-T cells against patient leukemia cells with varying degrees of CD25 expression. For this purpose, we constructed in vitro T cell-mediated cytotoxicity assays using patient AML samples with and without CD25 expression.

[0100] When cultured with CD25CAR-T cells for 3 days, the survival of CD25-positive primary AML cells (U390, U346) was significantly impaired, and the absolute number of AML cells decreased, but no such changes were observed in nonspecific activated T cells (U328) (Figure 2B). Since AML cell death did not occur when using CD25-negative AML cells as targets, or activated T cells without CAR as effectors, it was shown that AML cell death occurs via the recognition of CD25 by CAR-T cells.

[0101] [Reference Example 3: CD25 CAR-T cells cannot eradicate CD25-positive AML in vivo.] Since antigen-specific cytotoxicity of CAR-T cells against AML in CD25-expressing patients was observed in vitro, the in vivo therapeutic effect of CAR-T cells was evaluated.

[0102] NSG mice transplanted with patient-derived AML cells were created as follows: NOD.Cg-Prkdc scid Il2rgtmlWjl / Sz (NOD-SCID -IL2rg null ) The mice were obtained from Jackson Laboratory using NOD.Cg-Prkdc scid We obtained a strain (NOD-SCID) (Shultz et al. 2005 J immunol) that had been crossbred with a complete null mutation at the Il2rg locus.

[0103] NOD-SCID-IL2rg null Newborn mice were irradiated whole-body with 150 cGy of radiation, followed by intravenous infusion of human AML cells. To prepare AML transplant recipients, 10 cells were administered to each recipient. 3 ~10 5 Each recipient was injected with AML transplant cells (Ishikawa et al. 2007 nature biotechnol). To create normal human HSC transplant recipients, 10 cells were injected into each recipient. 4 Individual 7AAD-lineage (hCD3 / hCD4 / hCD8)-hCD34+hCD38- CB cells were injected (Ishikawa et al. 2005 Blood). Transplantation of human peripheral blood cells was evaluated by post-orbital phlebotomy.

[0104] After confirming the presence of AML cells in the peripheral blood of NSG mice transplanted with AML cells, 5 × 10 CD25 CAR-T cells were used. 6 A single dose of the drug was administered, and peripheral blood was collected weekly for four weeks and analyzed using a flow cytometer. As shown in Figure 3, all mice showed an increase in the proportion of CAR-T cells (CD3+) in human CD45 and a decrease in the proportion of AML (hCD45+CD33+CD25+), but even after four weeks, hCD45 + hCD33 + AML blasts remained. Furthermore, there was variability in the effectiveness, with some mice showing a strong effect (like mouse #2) and others showing a weaker effect (like mice #1 and #3).

[0105] Furthermore, when mice were dissected four weeks after administration of CD25 CAR-T cells and the bone marrow was stained with HE, as shown in Figure 4, various types of cells such as granulocytes and myelocytes were observed in the bone marrow of immunodeficient mice (normal NSG), but in the bone marrow of AML PDX mice, even after administration of CD25 CAR-T cells, the majority of bone marrow cells were AML cells, and erythrocytes, megakaryocytes, and normal leukocytes were very few.

[0106] [Example 1: Effect of CXCR4-expressing CD25 CAR-T cells 1] Because the therapeutic effect of CD25 CAR-T cells in bone marrow is not optimal, we designed a CAR construct that would allow CAR-T cells to better home in the bone marrow. To generate CXCR4-expressing CD25 CAR-T cells, we transfected 293 T cells with both the pHR_SFFV_mCXCR4 vector and the pHR_SFFV_CD25CAR vector.

[0107] After administering CD25 CAR-T cells or CXCR4-expressing CD25 CAR-T cells to PDX mice transplanted with AML cells, peripheral blood was collected weekly, and cells were separated by flow cytometry to examine the proportions of AML cells, mouse leukocytes, and CAR-T cells. The results showed a steady decrease in human AML cells over time during the 4-week observation period, and complete elimination of human AML 4 weeks after injection (Figures 5-1 to 5-3, Figure 6).

[0108] Treatment with CXCR4-expressing CD25 CAR-T cells resulted in complete elimination of CD33-positive leukemia cells within 4 weeks, and this effect was maintained for more than 4 months. Following the reduction in leukemia cells, CAR-T cells also decreased, with mouse CD45 cells becoming the majority of peripheral blood cells. In other words, this study demonstrated that cells co-expressing CXCR4 were more effective in the in vivo eradication of human AML cells by CD25 CAR-T cells.

[0109] [Example 2: Effects of CXCR4-expressing CD25 CAR-T cells (Part 2)] In Example 1, the spleen and bone marrow of PDX mice administered CXCR4-expressing CD25 CAR-T cells were removed and visually observed in comparison to mice that did not receive the cells. As shown in Figure 7, the mice administered CXCR4-expressing CD25 CAR-T cells showed a reduction in spleen size to normal levels and reddening of the bone marrow, indicating the recovery of erythroid cells.

[0110] Figure 8 shows the results of HE staining of bone marrow tissue from mice (U390 transplanted PDX mice) 4 weeks after administration of CXCR4-expressing CD25 CAR-T cells. It was confirmed that all normal leukocytes, erythrocytes, and megakaryocytes that produce platelets in the mice were restored after eradication of AML cells by CAR-T cells.

[0111] Furthermore, flow cytometry analysis of bone marrow cells revealed, consistent with the above results, that only when CXCR4-expressing CD25 CAR-T cells were administered did complete elimination of leukemia cells and recovery of normal cells such as mouse erythrocytes (Ter119) and leukocytes (mCD45) occur (Figures 9-1 to 9-3). The eradication effect of CXCR4-expressing CD25 CAR-T cells on AML cells in the bone marrow was maintained even 5 months after CAR-T cell injection (Figure 10).

[0112] In the liver, an increase in T cells and complete disappearance of leukemia cells were observed only when CXCR4-expressing CD25 CAR-T cells were administered, demonstrating a significantly stronger therapeutic effect compared to when CD25 CAR-T cells were administered (Figures 11-1 to 11-2, Figure 12).

[0113] Furthermore, histological examination confirmed the absence of GVHD or other pathological inflammation in the liver, intestines, and skin of mice administered CXCR4-expressing CD25 CAR-T cells (data not shown). In two further cases, significantly superior cell death of human AML cells was observed with CXCR4-expressing CD25 CAR-T cells. In two of the three cases examined, AML cell infiltration into the recipient's liver tissue was confirmed. The liver is an organ that shows strong expression of CXCL-12 / SDF1, and it was considered that the co-expression of CXCR4 in CD25 CAR-T cells had a potent therapeutic effect against human AML cells infiltrating the recipient animals' liver tissue.

[0114] As described above, 5 × 10 6 A single infusion of CXCR4-expressing CD25 CAR-T cells may be sufficient to achieve a long-term therapeutic response in invasive AML expressing CD25.

[0115] [Example 3: Effect of CXCR4-expressing CD19 CAR-T cells 1] PDX mice transplanted with CD19-positive B-cell mixed-phenotype acute leukemia (MPAL, B / myeloid, U211) cells were treated with the BIRC inhibitor AZD5582 0.5 mg / kg / day (intraperitoneal administration), the BCL-2 inhibitor (venetoclax) 30 mg / kg / day (oral administration), and dexamethasone (DEX) 30 mg / kg / day (intraperitoneal administration). As shown in Figure 13-1, after 10 doses, the leukemia cells (MPAL) in the peripheral blood decreased compared to pre-treatment levels (from 37.8% to 0.9%), but leukemia cells still remained. These cells were also found in the bone marrow (data not shown). The presence of residual leukocytes can cause relapse, therefore, CXCR4-expressing CD19 CAR-T cells (5 × 10) after treatment with molecular targeted drugs. 6 Individual doses were administered.

[0116] As a result, as shown in Figure 13-2, MPAL cells showing relapse were present on day 10 after CAR-T cell administration, but this decreased to 0.4% on day 23, and by day 32 and day 39, MPAL cells had completely disappeared.

[0117] [Example 4: Effects of CXCR4-expressing CD19 CAR-T cells (Part 2)] Burkitt lymphoma, a high-grade B-cell lymphoma, is treated with multi-drug chemotherapy. However, in cases that are resistant to treatment or relapse, even with high-dose chemotherapy combined with autologous hematopoietic stem cell transplantation or allogeneic hematopoietic stem cell transplantation, the 5-year survival rate is reported to be less than 50%, indicating a poor prognosis (J Oncol Pract. 2018 Nov;14(11):665-671).

[0118] In this example, using the TL1 cell line for Burkitt lymphoma (obtained from the Medical Cell Resource Center, Institute of Development, Aging and Cancer, Tohoku University), a xenografted Burkitt lymphoma model mouse was created in the same manner as in Example 1, and 5 × 10⁶ CAR-T cells expressing CXCR4 and having binding specificity to CD19 (CXCR4-expressing CD19 CAR-T) cells were produced. 6 The effects of individual administration were examined.

[0119] The left side of Figure 14 shows the flow cytometry results from the bone marrow (BM) and liver of mice that were not administered CAR-T cells. It can be seen that the majority of human CD45-positive cells are also positive for the B cell marker CD19, and that lymphoma cells make up a large proportion of both the bone marrow and liver.

[0120] In contrast, in mice administered CXCR4-expressing CD19 CAR-T cells (right), it was found that in both the bone marrow and liver, the majority of human CD45-positive cells were CAR-T cells (CD3-positive), and lymphoma cells were almost completely eliminated. [Industrial applicability]

[0121] The present invention provides an extremely effective treatment and / or recurrence prevention method for neoplastic diseases that have a poor prognosis and are difficult to cure. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. Cells that co-express chimeric antigen receptor (CAR) protein and CXCL12 receptor protein on their cell membrane.

2. The cell according to claim 1, wherein the CXCL12 receptor is CXCR4.

3. The cell according to claim 1 or 2, wherein the chimeric antigen receptor (CAR) protein targets a cell surface antigen selected from CD25 (IL-2 receptor α chain), CD19, and CD7.

4. A cell according to any one of claims 1 to 3, which is a T cell.

5. A drug exhibiting antitumor activity, comprising cells according to any one of claims 1 to 4.

6. The agent according to claim 5, which is used in combination with further antitumor agents and / or antitumor treatments.

7. A pharmaceutical composition comprising the agent described in claim 5 and a pharmaceutically acceptable carrier.

8. The agent according to claim 5 or 6 or the pharmaceutical composition according to claim 7 for the treatment and / or prevention of relapse of a neoplastic disease selected from the group consisting of acute myeloid leukemia (AML), adult T-cell leukemia, B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), mixed-plasmid acute leukemia (MPAL), chronic myeloid leukemia (CML), Hodgkin lymphoma, and non-Hodgkin lymphoma.

9. A method for producing cells according to any one of claims 1 to 4, comprising introducing a first polynucleotide encoding a chimeric antigen receptor (CAR) protein and a second polynucleotide encoding a CXCL12 receptor protein into cells.

10. The method according to claim 9, wherein the chimeric antigen receptor (CAR) protein targets a cell surface antigen selected from CD25 (IL-2 receptor α chain), CD19, and CD7.

11. The method according to claim 9 or 10, wherein the first polynucleotide and the second polynucleotide are introduced into the cell by the same or different vectors.

12. A method for treating and / or preventing recurrence of a neoplastic disease in a subject, comprising the step of administering cells that co-express a chimeric antigen receptor (CAR) protein targeting CD25 (IL-2 receptor α chain) and a CXCL12 receptor protein on their cell membrane.

13. The method according to claim 12, wherein the chimeric antigen receptor (CAR) protein targets a cell surface antigen selected from CD25 (IL-2 receptor α chain), CD19, and CD7.

14. The method according to claim 12 or 13, wherein the neoplastic disease is selected from the group consisting of acute myeloid leukemia (AML), adult T-cell leukemia, B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), mixed-plasmid acute leukemia (MPAL), chronic myeloid leukemia (CML), Hodgkin lymphoma, and non-Hodgkin lymphoma.

15. The method according to any one of claims 12 to 14, wherein the above cells are administered as a single dose.

16. 10 per kg of body weight 4 ~10 9 The method according to any one of claims 12 to 15, wherein the above-mentioned cells in a range of 100 are administered.

17. The method according to any one of claims 12 to 16, further comprising the step of measuring the expression level of the target cell surface antigen in the tumor cells of the subject before administration of the cells.

18. The method according to any one of claims 12 to 17, further comprising the step of evaluating the therapeutic effect after administration of cells.

19. The method according to claim 18, wherein the therapeutic effect is evaluated by one or more indicators selected from (i) a reduction in tumor cells in the blood, (ii) a reduction in tumor cells in the bone marrow, (iii) a reduction in tumor cells in the spleen, and (iv) suppression of tumor cell infiltration into the liver in the subject.

20. Cells for use in the treatment and / or prevention of cancer recurrence, co-expressing a chimeric antigen receptor (CAR) protein and a CXCL12 receptor protein on the cell membrane.

21. The cell according to claim 20, wherein the chimeric antigen receptor (CAR) protein targets a cell surface antigen selected from CD25 (IL-2 receptor α chain), CD19, and CD7.

22. The cell according to claim 20 or 21, wherein the cancer is a neoplastic disease selected from the group consisting of acute myeloid leukemia (AML), adult T-cell leukemia, B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), mixed-plasmid acute leukemia (MPAL), chronic myeloid leukemia (CML), Hodgkin lymphoma, and non-Hodgkin lymphoma.

23. A cell according to any one of claims 20 to 22, which is a T cell.

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