Novel population of marrow-infiltrating lymphocytes comprising BCMA-specific chimeric antigen receptor and method for preparing same

Abstract

The present invention relates to: a novel population of marrow-infiltrating lymphocytes (MILs), comprising a chimeric antigen receptor comprising a BCMA-specific binding domain; and a method for preparing same. Specifically, the present invention provides a novel population of MILs which is specific to target cells due to BCMA CAR and has strong cytotoxicity against the target cells, and a method for culturing MILs, which can provide same. A novel population of MILs prepared by the method can be used as a new cancer immunotherapy due to the high anticancer effect thereof.

Description

Novel bone marrow infiltrating lymphocyte population comprising BCMA-specific chimeric antigen receptor and method for producing the same

[0001] The present invention relates to a novel population of marrow-infiltrating lymphocytes (MILs) comprising a chimeric antigen receptor specific for BCMA (B-cell maturation antigen) and a method for producing the same. Specifically, the present invention provides a novel population of MILs comprising a BCMA-specific chimeric antigen receptor that specifically binds to target cells expressing BCMA and has potent cytotoxicity, and a method for culturing MILs capable of producing the same. The MILs population can be used as a novel immuno-cancer therapy due to its high anticancer effect.

[0002] Multiple myeloma (MM) remains a challenging disease to treat, despite the development of several effective treatments, including proteasome inhibitors, immunomodulators, and monoclonal antibodies. Complete cure is rare, and most patients are refractory to treatment or experience relapse. Therefore, the development of new treatments for multiple myeloma that can overcome these challenges is urgent.

[0003] Adoptive T-cell therapy, also known as cellular immunotherapy, has made significant progress in the treatment of multiple myeloma over the past decade, with several licensed chimeric antigen receptor (CAR)-T cell therapies. However, current adoptive immunotherapy relies on peripheral blood lymphocytes (PBLs) as a T-cell source. PBLs lack intrinsic tumor specificity, making them susceptible to tumor evasion and antigen loss, a major drawback of PBL-generated CAR-T cells. One approach to overcome this drawback and enhance the tumor specificity of adoptive T-cell therapy is the use of tumor-infiltrating lymphocytes (TILs), polyclonal memory T cells targeting multiple tumor-associated antigens obtained from solid tumors in cancer patients. However, TILs are not suitable for all solid tumor patients. For example, TILs therapy for solid tumors is impractical for various reasons, including the lack of TILs in patients with so-called cold tumors with low immunogenicity, the long expansion time, and the high cost of requiring high concentrations of IL-2.

[0004]

[0005] [Prior Art Literature]

[0006] [Non-patent literature]

[0007] (Non-patent Document 1) Badalamenti G, Fanale D, Incorvaia L, Barraco N, Listμ A, Maragliano R, et al. Role of tumor-infiltrating lymphocytes in patients with solid tumors: Can a drop dig a stone? Cell Immunol. 2019;343:103753.

[0008] (Non-patent Document 2) Kimberly AN IM. Marrow Infiltrating Lymphocytes: Their Role in Adoptive Immunotherapy. The Cancer Journal. 2015;21(6):501-5. doi: 10.1097 / PPO.0000000000000159.

[0009] (Non-patent Document 3) Sponaas AM YR, Rustad EH, Standal T, Thoresen AS, Vo CD, et al. PD1 is expressed on exhausted T cells as well as virus specific memory CD8+ T cells in the bone marrow of myeloma patients. Oncotarget. 2018;9(62):32024-32035. doi: 10.18632 / oncotarget.25882.

[0010]

[0011] BCMA stands for B-cell maturation antigen, also known as CD269 or TNFRSF17. It is selectively expressed on B cells and plays a role in cell survival and proliferation. BCMA has been found to be expressed in several cancers involving B cells, including malignant B cells, multiple myeloma, and Hodgkin's lymphoma.

[0012] The present invention provides a method for treating cancers involving BCMA expression, such as multiple myeloma, by targeting BCMA.

[0013] The inventors of the present invention have completed the present invention by generating an ex vivo expanded and activated MIL (eMIL) isolated from a patient, which expresses a chimeric antigen receptor that specifically binds to BCMA, and evaluating the immunological characteristics and cytotoxicity of such CAR-expressing eMIL to demonstrate its superiority.

[0014] The present invention provides a population of MILs expressing an antigen-binding site, preferably a chimeric antigen receptor, that specifically binds to a cancer antigen, preferably BCMA, a method for producing the same, and a therapeutic use thereof. The therapeutic use of the present invention is preferably the treatment of multiple myeloma.

[0015] Bone marrow infiltrating lymphocytes comprising a BCMA-specific chimeric antigen receptor according to the present invention have excellent targeting efficiency by including two nanobodies specific for different epitopes of BCMA, and exhibit excellent anticancer effects against cancer, particularly multiple myeloma, by effectively inducing bone marrow infiltrating lymphocytes into target cells expressing BCMA. In addition, bone marrow infiltrating lymphocytes comprising a BCMA-specific chimeric antigen receptor according to the present invention exhibit excellent anticancer effects against CD8 + T cm (central memory T cells, central memory T cells) have a high proportion, and this high proportion of CD8 + T cm Silver can help bone marrow-infiltrating lymphocytes exhibit long-lasting cytotoxicity in the body. Compared to MILs that do not contain a chimeric antigen receptor, BCMA CAR-MILs comprising a chimeric antigen receptor according to the present invention exhibit increased cytotoxicity, such as an increased CD107a ratio, and thus can be effectively used for the prevention or treatment of cancer. Furthermore, CAR-MILs according to the present invention exhibit superior immuno-oncology capabilities, such as cytotoxicity against cancer cells, compared to other types of T cells containing the same CAR.

[0016] Figure 1 is a schematic diagram showing a method for manufacturing a CAR-MILs group and the composition of a CAR according to the present invention.

[0017] Figure 2 is a flow cytometry graph showing the CAR expression rate of a CAR-MILs population of the present invention over time after transfection of the CAR according to the present invention into MILs.

[0018] Figure 3 is a line graph showing the CAR expression rate of a CAR-MILs population of the present invention over time after transfection of the CAR according to the present invention into MILs.

[0019] Figure 4 shows the CD3 expression within the CAR-MILs population over time after transduction of the CAR according to the present invention into MILs. + CD56 - T cells and cytokine-induced killer cells (CD3) + CD56 + This is the result of flow cytometry analysis showing the change in the ratio of T cells.

[0020] Figure 5 shows the CD3 expression within the CAR-MILs population over time after transduction of the CAR according to the present invention into MILs. + CD56 - T cells and cytokine-induced killer cells (CD3) + CD56 + This is a bar graph showing changes in T cells.

[0021] Figure 6 is a graph showing the expansion by proliferation of a CAR-MILs population over time after transduction of a CAR according to the present invention into MILs.

[0022] Figure 7 shows the CD8 expression within the CAR-MILs population over time after transduction of the CAR according to the present invention into MILs. + and CD4 + This is the result of flow cytometry analysis showing the change in the proportion of T cells.

[0023] Figure 8 shows the CD8 expression within the CAR-MILs population over time after transduction of the CAR according to the present invention into MILs.+ and CD4 + This is a bar graph showing T cell changes.

[0024] Figure 9 shows the number of naive T cells (CD62L) in the CAR-MILs population over time after transduction of the CAR according to the present invention into MILs. + CD45RA + cells), central memory T cells (central memory T cells; CD62L + CD45RA - cells), T emra cells (CD62L - CD45RA + cells), effector memory T cells (effector memory T cells; CD62L - CD45RA - This is a graph showing the results of flow cytometry analysis showing the change in the ratio of cells.

[0025] Figure 10 shows the number of T cells (CD62L) in the CAR-MILs population over time after transduction of the CAR according to the present invention into MILs. + CD45RA + cells), central memory T cells (central memory T cells; CD62L + CD45RA - cells), T emra cells (CD62L - CD45RA + cells), effector memory T cells (effector memory T cells; CD62L - CD45RA - This is a bar graph showing the change in the ratio of cells.

[0026] Figure 11 is a graph showing the results of flow cytometry analysis showing changes in the expression ratio of immune checkpoint molecules on the surface of a CAR-MIL population over time after transducing a CAR according to the present invention into MILs.

[0027] Figure 12 is a bar graph showing changes in the expression ratio of immune checkpoint molecules on the surface of a CAR-MIL population over time after transduction of a CAR according to the present invention into MILs.

[0028] Figure 13 shows the CD4 expression within the CAR-MILs population over time after transduction of the CAR according to the present invention into MILs. + CD25 + Foxp3 + Regulatory T cells (T reg ), CD8 + CD25 + Foxp3 + Regulatory T cells (T reg ), and CD4 + CD8 + CD25 + Foxp3 + Regulatory T cells (T reg ) is a graph showing the change in the ratio of flow cytometry results.

[0029] Figure 14 shows the CD4 expression within the CAR-MILs population over time after transduction of the CAR according to the present invention into MILs. + CD25 + Foxp3 + Regulatory T cells (T reg ), CD8 + CD25 + Foxp3 + Regulatory T cells (T reg ), and CD4 + CD8 + CD25 + Foxp3 + Regulatory T cells (T reg ) is a bar graph showing the change in the ratio.

[0030] Figure 15 is a graph showing the CD107a expression ratio on CAR-MILs when co-cultured with cancer cell lines or cancer cells isolated from patients on days 9, 14, and 19 after transducing CARs according to the present invention into MILs.

[0031] Figure 16 is a graph showing the cell death rate of cancer cells when co-cultured with a cancer cell line or cancer cells isolated from a patient on day 14 and day 19 after transducing a CAR according to the present invention into MILs.

[0032] Figure 17 is a graph showing that when a CAR-MILs population cultured in vitro for 14 days after transducing a CAR according to the present invention into MILs is co-cultured with autologous cancer cells isolated from a patient or the RPMI8226 cancer cell line, the target cancer cells are killed by the CAR-MILs population over time, thereby reducing the number of cancer cells.

[0033] Hereinafter, the present invention and embodiments will be described in detail with reference to the attached drawings so that those skilled in the art can easily implement the invention. However, the present invention may be implemented in various forms and is not limited to the embodiments described herein.

[0034] The immune cells of the present invention may refer to cells of hematopoietic origin that are functionally involved in the initiation and / or execution of innate and / or adaptive immune responses. The immune cells of the present invention may be derived from stem cells. The stem cells may be adult stem cells, non-human embryonic stem cells, non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, pluripotent stem cells, or hematopoietic stem cells.

[0035] The above immune cells may be, but are not limited to, leukocytes, neutrophils, eosinophils, basophils, monocytes, lymphocytes, T cells, cytotoxic T cells, natural killer T cells, dendritic cells, or a combination thereof.

[0036] As used herein, the term "marrow infiltrating lymphocytes (also referred to as "MILs" or "MILs") refers to lymphocytes derived from the bone marrow. As used herein, the term "eMILs" refers to MILs activated by incubation with anti-CD3 and anti-CD28 antibodies in the presence of interleukin. As used herein, the term "MILs" may refer to eMILs, depending on the context.

[0037] The term "activation" as used herein with respect to lymphocytes means that lymphocytes are initiated to proliferate and differentiate by antigen-specific receptors or mediators such as interleukins, and their anti-cancer function is increased.

[0038] The term “expansion” as used herein means an increase in the number of cells by proliferation.

[0039] Bone marrow-infiltrating lymphocytes (TILs) differ significantly from peripheral blood lymphocytes (PBLs) as well as tumor-infiltrating lymphocytes (TILs). The bone marrow microenvironment represents a special immunological niche due to the abundance of antigen-presenting cells. The presence of these antigen-presenting cells maintains a higher level of central memory cells, such as those found in the bone marrow compartment, for antigen processing and presentation (Li JM et al J Immunol. 2009 Dec 15;183(12):7799-809). These TILs express markers of memory T cells, such as CD45RO. + and CD62L +, and have more memory cells than those found in the PBL population (Noonan K et al Clin Cancer Res. 2012 Mar 1;18(5):1426-34). Furthermore, MILs exhibit distinct properties from tumor-infiltrating lymphocytes (TILs) of hematological malignancies due to their ability to persistently prime memory cells to antigen (Beckhove P et al J Clin Invest. 2004 Jul 1;114 (1):67-76, Castiglioni P et al 6 J Immunol 2008;180:4956-4964). For example, unlike TILs, MILs can be harvested from whole patients and expanded ex vivo (Noonan, K et al. Sci. Transl Med. 2015 May 20;7(288):288ra78). TILs are found in only about 50% of patients, and only about 25% of patients contain expandable TILs. Furthermore, in contrast to PBLs, MILs possess a broad repertoire of endogenous antigens, accounting for their unique tumor specificity, which is not seen in PBLs (Noonan et al Clin Cancer Res).

[0040] The source of MILs in the CAR-MIL population according to the present invention can be obtained from patients with any of a number of cancer types, including hematological malignancies and solid tumors, preferably patients with multiple myeloma. Preferably, the source can be bone marrow cells with increased tumor specificity compared to peripheral blood.

[0041] The "chimeric antigen receptor (CAR)" of the present invention refers to a protein comprising an intracellular signaling domain and an extracellular binding domain. The chimeric antigen receptor (CAR) is composed of a binding domain capable of recognizing an antigen, a signal sequence, a hinge region, a transmembrane domain, and an intracellular signaling domain, which target tumor-associated antigens specific to the binding domain, thereby enabling immune cells to specifically attack cancer cells.

[0042] The inventors of the present invention have prepared a CAR comprising a BCMA dual-epitope binding nanobody and a CAR-MILs population comprising the CAR using bone marrow-derived mononuclear cells (BMMNC) of multiple myeloma patients showing high transduction efficiency and a strong anti-myeloma immune response, and have completed the present invention by proving that the CAR-MILs population is an innovative immunotherapeutic approach for multiple myeloma.

[0043] The "BCMA (B-cell maturation antigen; UniProt ID: Q02223)" of the present invention is an abbreviation for B-cell maturation antigen, and is also referred to as CD269, TNFRSF17, etc. They are selectively expressed in B cells and are involved in cell survival and proliferation.

[0044] Chimeric antigen receptor according to the present invention

[0045] The binding domain included in the chimeric antigen receptor of the present invention specifically recognizes BCMA, and the chimeric antigen receptor of the present invention may have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1.

[0046] Since the chimeric antigen receptor of the present invention specifically recognizes BCMA, it is also referred to as BCMA CAR.

[0047] The “nanobody” of the present invention refers to a single-domain antibody (sdAb), which has high stability and tissue penetration due to its small molecular weight derived from a single domain.

[0048] Nanobodies are the smallest stable antibody molecules known to date. They have high affinity for antigens and, due to their small size, can strongly penetrate tumor tissues and the blood-brain barrier, while exhibiting low toxicity and immunogenicity.

[0049] The binding domain included in the chimeric antigen receptor of the present invention comprises nanobodies VHH01 and VHH02 specific for BCMA, wherein the nanobodies may have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequences of SEQ ID NOs: 8 and 9, respectively.

[0050] The binding domain included in the chimeric antigen receptor of the present invention may include a VHH01 nanobody comprising the amino acid sequence of SEQ ID NO: 8 and a VHH02 nanobody comprising the amino acid sequence of SEQ ID NO: 9.

[0051] The nanobodies VHH01 and VHH02, which enable dual epitope binding to BCMA, have high specificity, and when VHH01 and VHH02 are included in a CAR, BCMA CAR-expressing T cells can be induced even in cancer cells expressing low levels of the target antigen. Dual nanobody CARs can exhibit greater activity than single nanobody CARs and can efficiently eliminate target tumor cells.

[0052] The above VHH01 and VHH02 may comprise a CDR having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequences of SEQ ID NOs: 2 to 7.

[0053] The above VHH01 and VHH02 may include CDR1 of SEQ ID NO: 2 or 3, CDR2 of SEQ ID NO: 4 or 5, and CDR3 of SEQ ID NO: 6 or 7.

[0054] The above VHH01 may include CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 4, and CDR3 of SEQ ID NO: 6, and the above VHH02 may include CDR1 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 7.

[0055] The chimeric antigen receptor of the present invention may further comprise at least one selected from the group consisting of a signal sequence, a hinge and transmembrane domain (TM), and an intracellular domain (intracellular signaling domain) together with a binding domain that specifically binds to BCMA.

[0056] The hinge and transmembrane domains included in the chimeric antigen receptor of the present invention are preferably, but not limited to, hinge and transmembrane domains derived from CD8α, and may preferably include the amino acid sequence of SEQ ID NO: 10, and may have at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 10.

[0057] The intracellular signaling domain according to the present invention is a portion located inside the cell membrane of an immune cell, i.e., in the cytoplasm, and refers to a portion that activates the immune response of an immune cell when an antibody bound to an extracellular domain binds to a target antigen.

[0058] The intracellular domain of the chimeric antigen receptor of the present invention may include two or more intracellular signaling domains. The intracellular signaling domain is preferably a 4-1BB-derived intracellular signaling domain linked to a CD3ζ-derived intracellular signaling domain, but is not limited thereto. Preferably, the 4-1BB-derived intracellular signaling domain may include an amino acid sequence of SEQ ID NO: 11, and the CD3ζ-derived intracellular signaling domain may include an amino acid sequence of SEQ ID NO: 12, and may have at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

[0059] The CAR-MIL population according to the present invention is not activated in vitro and does not contain a chimeric antigen receptor that specifically binds to a B-cell maturation antigen, compared to a MIL population (in other words, a MIL population just isolated from a patient). + T cells (CD8 + T cell) ratio increases, and CD4 + T cells (CD4 + It may be a group with a reduced T cell ratio.

[0060] As used herein, "just isolated" means that the MIL population isolated from the patient has not been expanded and activated ex vivo.

[0061] CAR-MILs according to the present invention may be a population having a reduced proportion of regulatory T cells compared to a MIL population (in other words, a MIL population freshly isolated from a patient) that is not activated in vitro and does not contain a chimeric antigen receptor that specifically binds to a B-cell maturation antigen.

[0062] CAR-MILs according to the present invention are not activated in vitro and do not contain a chimeric antigen receptor that specifically binds to B-cell maturation antigen, compared to a population of MILs (in other words, a population of MILs just isolated from a patient) that are CD62L + CD45RA - It may be a group with an increased proportion of central memory T cells.

[0063] CAR-MILs according to the present invention may be a population with a reduced level of PD-1 (Programmed cell death protein 1) expression compared to a MIL population (in other words, a MIL population just isolated from a patient) that is not activated in vitro and does not contain a chimeric antigen receptor that specifically binds to a B-cell maturation antigen.

[0064] CAR-MILs according to the present invention have increased CD8 compared to MIL populations (in other words, MIL populations freshly isolated from patients) that are not activated in vitro and do not contain chimeric antigen receptors that specifically bind to B-cell maturation antigens. + T cell and central memory T cell ratios, reduced CD4 + It may indicate a high T cell and regulatory T cell ratio, and a low PD-1 expression level.

[0065] CAR-MILs according to the present invention are not activated in vitro and are CD138 compared to isolated PBLs or MILs populations that do not contain a chimeric antigen receptor that specifically binds to B-cell maturation antigen. + They can exhibit superior cytotoxicity against primary multiple myeloma cells with a higher CD107a expression ratio. CAR-MILs according to the present invention also exhibit superior immuno-oncology capabilities, such as cytotoxicity against cancer cells, compared to other types of T cells containing the same CAR.

[0066] The proportions of different types of immune cells that make up an immune cell population, such as MILs, can be determined using conventional experimental techniques, such as flow cytometry. Different types of immune cells can be distinguished by the presence of uniquely expressed proteins on their cell surfaces.

[0067] The term "differentiation" as used herein refers to a developmental process that allows cells to become specialized for a particular function, for example, by acquiring morphological features and / or functions.

[0068] As used herein, the term "substantially fully differentiated" means that the cells have finally differentiated into mature, fully differentiated cells, and means that at least 90%, at least 95%, or 100% of the cells are differentiated.

[0069] The term "memory T cell" as used herein refers to a type of T cell, which is an antigen-specific T cell that remains for a long period of time even after the antigen has been removed, and is characterized by the presence of CD4 or CD8 proteins on the cell surface, and is a cell that rapidly converts into an effector T cell and responds to the antigen when re-exposed to a specific antigen.

[0070] The term "regulatory T cell" as used herein refers to a type of T cell that performs the function of maintaining homeostasis and self-tolerance by suppressing immune responses, and is characterized by the presence of one or more proteins selected from CD4 or CD8 and CD25 and Foxp3 proteins on the cell surface, for example, T reg It is also referred to as .

[0071] The term "central memory T cell" as used herein refers to a T cell that has a high self-renewal capacity and is mainly present in the lymph nodes and peripheral circulation, and is characterized by, for example, the presence of CD62L protein on the cell surface but absence of CD45RA protein on the cell surface, and T cm It is also referred to as .

[0072] The term "CD4" as used herein + "This means that CD4 (cluster of differentiation 4), a glycoprotein that acts as a co-receptor for the T-cell receptor (TCR), is present on the surface of the cell.

[0073] The term "CD8" as used herein + "This means that CD8 (cluster of differentiation 8), a glycoprotein that acts as a co-receptor of the T-cell receptor (TCR), is present on the surface of the cell.

[0074] The term "CD138" as used herein + "means that the CD138 protein, also referred to as syndecan-1, is present on the surface of the cell. CD138 is a member of the syndecan proteoglycan family, functions as a receptor for the extracellular matrix, is involved in cell differentiation, and shows increased expression in various types of cancer.

[0075] The term "CD107a" as used herein refers to CD107a protein, also known as lysosome-associated membrane glycoprotein 1, also known as LAMP-1, which is a protein involved in degranulation of immune cells.

[0076] The superscript “+” used herein indicates the presence of a protein displayed on the surface of the cell.

[0077] The subscript “-“ used in this specification means that the protein displayed on the surface of the cell is not present.

[0078] Method for manufacturing eMIL including BCMA CAR according to the present invention

[0079] The present invention provides a method for preparing an isolated bone marrow infiltrating lymphocyte population comprising a BCMA CAR, and a bone marrow infiltrating lymphocyte population prepared by such a method.

[0080] The method comprises the steps of isolating marrow infiltrating lymphocytes from the bone marrow of a cancer patient, culturing the isolated marrow infiltrating lymphocytes by contacting them with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, transducing the isolated marrow infiltrating lymphocytes with a polynucleotide expressing a chimeric antigen receptor comprising a binding domain that specifically binds to a B-cell maturation antigen, and culturing the isolated marrow infiltrating lymphocytes in the presence of IL-2, IL-7, IL-15 and IL-21.

[0081] The chimeric antigen receptor, i.e., BCMA CAR, comprising a binding domain that specifically binds to a B-cell maturation antigen used in the above method may be as described in the section “Chimeric antigen receptor according to the present invention” above.

[0082] Specifically, the BCMA CAR used in the method according to the present invention may have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1.

[0083] The binding domain included in the BCMA CAR used in the method according to the present invention comprises nanobodies VHH01 and VHH02 specific for BCMA, wherein the nanobodies may have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequences of SEQ ID NOs: 8 and 9, respectively.

[0084] The binding domain included in the chimeric antigen receptor used in the method according to the present invention may include a VHH01 nanobody comprising the amino acid sequence of SEQ ID NO: 8 and a VHH02 nanobody comprising the amino acid sequence of SEQ ID NO: 9.

[0085] The VHH01 and VHH02 used in the method according to the present invention may include CDR1 of SEQ ID NO: 2 or 3, CDR2 of SEQ ID NO: 4 or 5, and CDR3 of SEQ ID NO: 6 or 7.

[0086] The VHH01 used in the method according to the present invention may include CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 4, and CDR3 of SEQ ID NO: 6, and the VHH02 may include CDR1 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 7.

[0087] The BCMA CAR used in the method according to the present invention may additionally include at least one selected from the group consisting of a signal peptide (SP), a hinge and transmembrane domain (TM), and an intracellular domain (intracellular signaling domain) together with a binding domain that specifically binds to BCMA.

[0088] The hinge and transmembrane domains are preferably, but not limited to, hinge and transmembrane domains derived from CD8α, and may preferably include the amino acid sequence of SEQ ID NO: 10, and may have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 10.

[0089] The intracellular domain of the BCMA CAR used in the method according to the present invention may include two or more intracellular signaling domains. The intracellular signaling domain is preferably a 4-1BB-derived intracellular signaling domain linked to a CD3ζ-derived intracellular signaling domain, but is not limited thereto. Preferably, the 4-1BB-derived intracellular signaling domain may include an amino acid sequence of SEQ ID NO: 11, and the CD3ζ-derived intracellular signaling domain may include an amino acid sequence of SEQ ID NO: 12, and may have at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

[0090] The present invention also relates to an eMIL manufactured by the above-described manufacturing method, i.e., an eMIL that expresses a BCMA CAR on its cell surface. As used herein, the term "eMIL" comprising a BCMA CAR means that the eMIL expresses a BCMA CAR on its cell surface.

[0091] The CAR-MIL population prepared according to the method of the present invention has a higher CD8 than the MIL population freshly isolated from the patient. + T cells (CD8 + T cell) ratio increases, and CD4 + T cells (CD4 + It may be a group with a reduced T cell ratio.

[0092] CAR-MILs manufactured according to the method of the present invention may be a population with a reduced proportion of regulatory T cells compared to a population of MILs just isolated from a patient.

[0093] CAR-MILs manufactured according to the method of the present invention have higher CD62L levels compared to a population of MILs just isolated from a patient. + CD45RA - It may be a group with an increased proportion of central memory T cells.

[0094] CAR-MILs manufactured according to the method of the present invention may be a group with a reduced level of PD-1 (Programmed cell death protein 1) expression compared to a group of MILs just isolated from a patient.

[0095] CAR-MILs manufactured according to the method of the present invention have increased CD8 + T cell and central memory T cell ratios, reduced CD4 + It may indicate a high T cell and regulatory T cell ratio, and a low PD-1 expression level.

[0096] CAR-MILs prepared according to the method of the present invention have a higher CD138 expression compared to isolated peripheral blood lymphocytes or isolated bone marrow infiltrating lymphocyte populations that do not express the chimeric antigen receptor. + It can exhibit superior cytotoxicity with a higher CD107a expression ratio against primary multiple myeloma cells.

[0097] The term "cancer" as used herein may be specifically, but is not limited to, one or more selected from the group consisting of brain tumor, cervical cancer, ovarian cancer, prostate cancer, lung cancer, bile duct cancer, kidney cancer, stomach cancer, liver cancer, retinoblastoma, choriocarcinoma, small intestine cancer, colon cancer and rectal cancer, non-small cell lung cancer, gastric adenocarcinoma, acute lymphoblastic leukemia, acute myeloid leukemia, breast cancer, bone sarcoma, bladder cancer, anaplastic astrocytoma, and multiple myeloma.

[0098] The term "multiple myeloma (MM)" as used herein refers to a type of blood cancer that occurs when plasma cells in the bone marrow abnormally differentiate and proliferate.

[0099] The term "prevention" as used herein means any action that inhibits or delays the onset of a disease by administering a composition, and "treatment" means any action that improves or beneficially changes the symptoms of a subject suspected of or suffering from a disease by administering a composition.

[0100] The dosage of the CAR-MILs cell population according to the present invention can be appropriately selected by those skilled in the art depending on the condition, weight, disease, formulation, route and period of administration of the subject.

[0101] The CAR-MIL cell population according to the present invention can be administered via any conventional route to reach the target tissue. Examples include, but are not limited to, intravenous administration, subcutaneous administration, and intravenous administration.

[0102] The term “combination administration” as used herein means administering two or more types of active ingredients simultaneously or sequentially.

[0103] The CAR-MIL cell population according to the present invention may be administered in combination with other anticancer agents appropriately selected by those skilled in the art. For example, it may be administered in combination with an immune checkpoint inhibitor, but is not limited thereto.

[0104] The present invention will be described in more detail through the following examples; however, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0105] [Manufacturing Example 1]

[0106] CAR-MILs production

[0107]

[0108] CAR-MILs were prepared using the same method as in Fig. 1. Specifically, bone marrow mononuclear cells ("BMMNCs") were isolated from the bone marrow of patients with multiple myeloma using density gradient centrifugation with Ficoll-Hypaque (d = 1.077, Lymphoprep™ Axis-Shield, Oslo, Norway). BMMNCs were then co-cultured with anti-CD3 / CD28 antibody-coated beads in RPMI 1640 medium containing 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, 4 mmol / L L-glutamine, and 200 U / mL recombinant human IL-2 in 24-well plates for 2 days. Thereafter, BMMNCs were transduced with a BCMA CAR having the nucleic acid sequence of SEQ ID NO: 13 in Table 4 below, and cultured in a medium containing 10 ng / mL IL-7, 10 U / mL IL-15, and 5 ng / mL IL-21 added to the RPMI medium. The medium was replaced every 2-3 days, and the CAR-MILs were continuously cultured for up to 21 days. The BCMA CAR comprises two nanobodies designated as VHH01 and VHH02, and the CDR sequences of the nanobodies are as shown in Table 2 below. The above BCMA CAR comprises VHH01 comprising an amino acid sequence of SEQ ID NO: 8, VHH02 comprising an amino acid sequence of SEQ ID NO: 9, a (G4S)5 linker, a hinge and transmembrane domain of CD8α comprising an amino acid sequence of SEQ ID NO: 10, an intracellular signaling domain of 4-1BB comprising an amino acid sequence of SEQ ID NO: 11, and an intracellular signaling domain of CD3ζ comprising an amino acid sequence of SEQ ID NO: 12, the sequences of which are as shown in Table 3 below.

[0109]

[0110]

[0111]

[0112]

[0113]

[0114] [Example 1]

[0115] Immune population analysis of CAR-MILs

[0116]

[0117] To analyze the immune cell population of CAR-MILs, CAR-MILs manufactured by the method of Manufacturing Example 1 above were treated with fluorescently labeled monoclonal antibodies, samples were collected using a flow cytometer, and the expression of cell surface markers was analyzed to confirm the composition of the immune cell population.

[0118] Specifically, CD3 + cells, NK cells (Natural killer cells), CIK cells (cytokine-induced killer cells), regulatory T cells (T reg ; regulatory T cells), CD4 + T cells and CD8 + To analyze T cells, etc., the composition of immune cell populations was confirmed using surface monoclonal antibodies such as CD3-FITC, CD56-PE-Cy7, CD4-PE, CD8-Amyan, CD62L-PE-Cy7, CD45RA, PD-1-Pacific blue, TIGIT-APC, TIM3-PerCP-Cy5.5, and CD73-APC-Cy7.

[0119] First, in order to confirm the transduction efficiency of the CAR according to the present invention in BCMA CAR-MILs, CAR-MILs (2 x 10 5Cells were washed with FACS buffer, stained with biotinylated human BCMA and Live / Dead-AmCyan (Invitrogen, Carlsbad, CA), and treated with Fc blocker. After washing twice, the cells were incubated with PE streptavidin antibody at room temperature for 30 minutes. The prepared samples were then analyzed using a flow cytometer (BD FACS Canto II, Becton Dickinson, Mountain View, CA, USA) to collect data, and the expression of cell surface markers was analyzed using Flow Jo software (TreeStar, San Carlos, CA, USA) to confirm the transduction efficiency of BCMA CAR-MILs.

[0120] The composition of the immune cell population was also performed in the same manner as above, changing the monoclonal antibody.

[0121] As a result, as shown in FIGS. 2 and 3, CAR-MILs showed a continuous increase in CAR expression rate for 21 days, and the expression rate of BCMA CAR on day 9 of culture was approximately 31.1% compared to day 0 of culture, and on day 21, it was 35.4%, showing an excellent expression rate due to the small size of the nanobody.

[0122] As shown in FIGS. 4 and 5, the BCMA CAR-MILs of the present invention induce cytokine-induced killer cells (CD3 + CD56 + ) was confirmed to contain a small number of them.

[0123] In addition, as shown in FIG. 6, the BCMA CAR-MILs of the present invention exhibit a high proliferation rate of about 400 times or more on the 19th day after transduction in vitro, and it was proven that they can be effectively used for cancer treatment due to such excellent proliferation rate.

[0124] In addition, as shown in FIGS. 7 and 8, after transducing the CAR according to the present invention, CD8 + The proportion of T cells continues to increase, and CD4 + We confirmed that T cells exhibiting cytotoxicity increased as the proportion of T cells decreased.

[0125] In addition, as shown in FIGS. 9 and 10, after transducing the CAR according to the present invention, T cm Central memory T cells (CD62L), also called + CD45RA - ) significantly increases the ratio of T cells and T emra (CD62L - CD45RA + ) confirmed that there were almost no groups of cells.

[0126] Additionally, as shown in FIGS. 11 and 12, it was confirmed that the CAR-MILs of the present invention can overcome the immune suppression or immune evasion mechanism of cancer cells by expressing immune checkpoint proteins such as PD-1 at significantly lower levels.

[0127]

[0128] [Example 2]

[0129] Analysis of Treg cell proportions in the CAR-MILs population

[0130]

[0131] The composition of the immune cell population of CAR-MILs was analyzed as in Example 1. As a result, as shown in Figures 13 and 14, CD4 + CD25 + Foxp3 + Regulatory T cells (T reg ) and CD8 + CD25 + Foxp3 + Regulatory T cells (T reg ) was confirmed to have a decreasing expression rate.

[0132] Therefore, the immune cell population of CAR-MILs manufactured by the method of the present invention includes immune suppressor cells (T reg ) was confirmed to contain almost no

[0133]

[0134] [Example 3]

[0135] Cytotoxicity analysis of CAR-MILs

[0136]

[0137] To determine whether CAR-MILs effectively kill multiple myeloma cells, we examined the functional differences between CAR-MILs and activated MILs (eMILs) and activated peripheral blood lymphocytes (ePBLs). Specifically, lactate dehydrogenase (LDH) assays, kinetic analyses, and CD107a degranulation assays were performed to evaluate the killing capacity of CAR-MILs.

[0138]

[0139] 3-1. CD107a degranulation analysis of CAR-MILs

[0140] CD107a degranulation assay was performed as follows: 5Х10 4 CAR-MILs of 5X10 in 96-well U-bottom plates 4 Dog target cells (K562, U266, RPMI8226, ARH77, and IM9 and CD138 isolated from patients + Cultured with or without target cells (multiple myeloma cells). 5Х10 4 Dog target cells (K562, U266, RPMI8226, ARH77, IM9, and CD138 +Cells were cultured with 5 μL PE-conjugated anti-human CD107a antibody in 96-well U-bottom plates with or without primary multiple myeloma cells. After 1 h, Monensin and brefeldin A (BD Biosciences) were added and incubated for an additional 4 h. Cells were then harvested after staining with anti-human CD3 antibody.

[0141] As shown in Figure 15, CAR-MILs obtained on the 9th day, CAR-MILs obtained on the 14th day, and CAR-MILs obtained on the 21st day after transduction of the CAR according to the present invention had higher CD107a levels than ePBL and eMIL at an E (Effector cell; CAR-MILs):T (Target cell; target cell) ratio of 1:1. + It was confirmed that it exhibited higher cell killing activity against cancer cells by showing a positive group ratio.

[0142]

[0143] 3-2. Tumor-specific cytotoxicity LDH analysis of CAR-MILs

[0144] LDH analysis was performed as follows: Target cells (K562, U266, RPMI8226, and CD138 + Multiple myeloma cells) were cultured in 100 μL of RPMI medium with 2 μg of anti-human HLA-A, B, or C antibody (clone: ​​W6 / 32, Biolegend, USA). 6 The target cells were added and cultured for 20 minutes, then washed by centrifugation. Afterwards, the target cells were cultured with CAR-MIL at a ratio of 1:1 in a Costar 96-well plate (Corning, USA) at 5% CO2, 37 oC for 6 h. Finally, the supernatant was collected to measure the concentration of LDH, a cytosolic enzyme released during cell lysis, and the concentration of LDH was measured using the CytoTox 96 non-radioactive cytotoxicity assay (Promega, USA), and the cell lysis rate was calculated according to the manufacturer's protocol.

[0145] CAR-MILs, eMILs, and ePBLs were treated with target leukemia (K562) cells that did not express BCMA or target myeloma cells that expressed BCMA (U266, RPMI8226, and CD138 + As a result of calculating the cell lysis rate after culturing with primary multiple myeloma cells, as shown in Figure 16, CAR-MILs showed no difference in cytotoxicity against K562 cells that do not express BCMA, which were used as a negative control, but showed significantly increased cytotoxicity against U266 and RPMI8226 cells that overexpress BCMA.

[0146]

[0147] 3-3. Analysis of tumor-specific cytotoxicity kinetics of CAR-MILs

[0148] To evaluate the cytotoxicity of BCMA CAR-MILs against cancer cells in real time, the IncuCyte® Cytotoxicity Assay (Satorius) was performed. RPMI8226 target cells and CD138+ primary multiple myeloma cells isolated from multiple myeloma patients were plated at a density of 1 x 10 in 96-well plates coated with poly-L-ornithine solution (Sigma-Aldrich). 4Cells (per 100 μL) were plated, and BCMA CAR-MILs were added at a 1:1 effector cell:target cell (E:T) ratio. IncuCyte® Cytotox Green Reagent (Satorius) was added at a 1:1000 dilution (100 nL) to the mixed cells, and the cell plate was placed in the Incucyte® Live-Cell Analysis System (Satorius) and incubated at 37°C for 30 minutes. The number of apoptotic cells identified by IncuCyte® Cytotox Green Reagent was monitored with the IncuCyte Live Cell Analysis System, and cytotoxicity was confirmed by capturing fluorescent images every 2 hours for 24 hours.

[0149] As shown in Fig. 17, it was confirmed that the green bodies displayed by IncuCyte®Cytotox Green Reagent, which specifically binds to apoptotic cells, increased significantly and were continuously maintained in the BCMA CAR-MIL treatment group, and thus it was confirmed that the CAR-MILs of the present invention consistently exhibited higher cell killing activity compared to ePBL and eMIL.

[0150]

[0151] Finally, as demonstrated in Examples 3-1 to 3-3, the CAR-MILs of the present invention were confirmed to exhibit superior cytotoxicity against target cancer cells compared to MILs and PBLs that do not express CARs. Furthermore, the functional differences between CAR-T and CAR-MIL were tested, and it was confirmed that CAR-MIL exhibited superior cytotoxicity against cancer cells.

Claims

1. Isolated marrow infiltrating lymphocytes (MILs) containing a chimeric antigen receptor (CAR) containing a binding domain that specifically binds to B-cell maturation antigen (BCMA).

2. A population of isolated bone marrow infiltrating lymphocytes comprising a chimeric receptor further comprising at least one selected from the group consisting of a signal sequence, a hinge, a linker, a transmembrane domain (TM), and an intracellular domain, in accordance with claim 1.

3. A population of isolated bone marrow infiltrating lymphocytes comprising a nanobody comprising a binding domain that specifically binds to the B cell maturation antigen according to claim 1, a CDR1 of SEQ ID NO: 2 or 3, a CDR2 of SEQ ID NO: 4 or 5, and a CDR3 of SEQ ID NO: 6 or 7.

4. A population of isolated bone marrow infiltrating lymphocytes according to claim 1, wherein the binding domain that specifically binds to the B cell maturation antigen comprises a nanobody comprising CDR1 of SEQ ID NO: 2, CDR2 of SEQ ID NO: 4, and CDR3 of SEQ ID NO: 6, and a nanobody comprising CDR1 of SEQ ID NO: 3, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO:

7.

5. A population of isolated bone marrow infiltrating lymphocytes according to claim 1, wherein the binding domain that specifically binds to the B cell maturation antigen comprises a nanobody comprising an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 8 and 9.

6. A population of isolated bone marrow infiltrating lymphocytes in the first paragraph, wherein the binding domain that specifically binds to the B cell maturation antigen comprises a nanobody comprising the amino acid sequence of SEQ ID NO: 8 and a nanobody comprising the amino acid sequence of SEQ ID NO:

9.

7. A population of isolated bone marrow infiltrating lymphocytes according to claim 1, wherein the chimeric antigen receptor comprises the amino acid sequence of sequence number 1.

8. A population of isolated bone marrow infiltrating lymphocytes in the second paragraph, wherein the hinge and transmembrane domains comprise the hinge and transmembrane domains of CD8α.

9. A population of isolated bone marrow infiltrating lymphocytes in accordance with claim 8, wherein the hinge and transmembrane domains of CD8α comprise the amino acid sequence of SEQ ID NO:

10.

10. A population of isolated bone marrow infiltrating lymphocytes in the second paragraph, wherein the intracellular domain comprises an intracellular signal region sequence selected from the group consisting of 4-1BB and CD3ζ, or a sequence selected from the group consisting of a combination thereof.

11. A population of isolated bone marrow infiltrating lymphocytes in claim 10, wherein the intracellular signaling region sequence of 4-1BB comprises an amino acid sequence of SEQ ID NO: 11, and the intracellular signaling region sequence of CD3ζ comprises an amino acid sequence of SEQ ID NO:

12.

12. In the first paragraph, compared to a population of isolated bone marrow infiltrating lymphocytes that are not activated in vitro and do not contain a chimeric antigen receptor that specifically binds to a B-cell maturation antigen, CD8 + T cells (CD8 + T cell) ratio increases, and CD4 + T cells (CD4 + A population of isolated bone marrow-infiltrating lymphocytes with a reduced proportion of T cells.

13. In the first paragraph, an isolated bone marrow infiltrating lymphocyte population having a reduced proportion of regulatory T cells compared to an isolated bone marrow infiltrating lymphocyte population that is not activated in vitro and does not contain a chimeric antigen receptor that specifically binds to a B-cell maturation antigen.

14. A population of isolated bone marrow infiltrating lymphocytes having an increased proportion of central memory T cells compared to a population of isolated bone marrow infiltrating lymphocytes that are not activated in vitro and do not contain a chimeric antigen receptor that specifically binds to a B-cell maturation antigen, in accordance with claim 1.

15. In the first paragraph, an isolated bone marrow infiltrating lymphocyte population having a reduced level of PD-1 (Programmed cell death protein 1) expression compared to an isolated bone marrow infiltrating lymphocyte population that is not activated in vitro and does not contain a chimeric antigen receptor that specifically binds to a B-cell maturation antigen.

16. In the first paragraph, compared to a population of isolated bone marrow infiltrating lymphocytes that are not activated in vitro and do not contain a chimeric antigen receptor that specifically binds to a B-cell maturation antigen, increased CD8 + T cells, and central memory T cell proportions, reduced CD4 + Isolated bone marrow infiltrating lymphocyte populations showing a high proportion of T cells, regulatory T cells, and low expression levels of PD-1.

17. In the first paragraph, CD138 is compared to a population of isolated peripheral blood lymphocytes or bone marrow infiltrating lymphocytes that are not activated in vitro and do not contain a chimeric antigen receptor that specifically binds to a B-cell maturation antigen. + An isolated bone marrow infiltrating lymphocyte population exhibiting superior cytotoxicity with a higher CD107a expression ratio against primary multiple myeloma cells.

18. In the first paragraph, the separated bone marrow infiltrating lymphocytes (a) a step of isolating marrow infiltration lymphocytes (MILs) from the bone marrow of a cancer patient; (b) a step of culturing bone marrow infiltrating lymphocytes obtained from step (a) by contacting them with anti-CD3 and anti-CD28 antibodies in the presence of IL-2; (c) transducing a polynucleotide expressing a chimeric antigen receptor comprising a binding domain that specifically binds to a B-cell maturation antigen into the bone marrow infiltrating lymphocytes obtained from step (b); and (d) culturing the bone marrow infiltrating lymphocytes obtained from step (c) in the presence of IL-2, IL-7, IL-15 and IL-21; A population of isolated bone marrow infiltrating lymphocytes obtained by a method including:

19. A population of isolated bone marrow infiltrating lymphocytes in claim 18, wherein the cancer is multiple myeloma.

20. A pharmaceutical composition comprising a population of isolated bone marrow infiltrating lymphocytes according to any one of claims 1 to 19.

21. In claim 20, the composition is a pharmaceutical composition for use in the treatment of a subject having cancer.

22. A pharmaceutical composition according to claim 21, wherein the cancer is multiple myeloma.

23. A method for treating a subject with multiple myeloma, comprising administering to a subject in need of treatment a population of isolated bone marrow infiltrating lymphocytes according to any one of claims 1 to 19.

24. A method according to claim 23, wherein the cancer is multiple myeloma.

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