Novel bone marrow infiltrating lymphocyte population, pharmaceutical composition comprising same, and use thereof

Abstract

The present invention relates to a novel marrow infiltrating lymphocytes (MILs) population comprising a chimeric antigen receptor containing a B cell maturation antigen (BCMA) specific binding domain, and a method for preparing the same, and more particularly, to a novel marrow infiltrating lymphocytes (MILs) population comprising a chimeric antigen receptor containing a B cell maturation antigen (BCMA) specific binding domain. Specifically, the present invention provides a novel MILs group having characteristics of target cell specificity due to BCMA CAR and strong cytotoxicity to target cells, and a MILs culture method capable of providing the novel MILs group, the novel MILs group being useful as a novel immune anti-cancer therapy due to a high anti-cancer effect of the novel MILs group prepared by the method.

Description

Technical Field

[0001] This invention relates to a novel population of marrow-infiltrating lymphocytes (MILs) containing a B-cell maturation antigen (BCMA)-specific chimeric antigen receptor and a method for preparing the same. Specifically, this invention provides a novel MIL population containing a BCMA-specific chimeric antigen receptor and a method for culturing the MILs. The novel MIL population specifically binds to target cells expressing BCMA and exhibits strong cytotoxicity. Due to its high anticancer effect, this MIL population can be used as a novel immunotherapy for cancer treatment. Background Technology

[0002] Despite the development of various effective treatments, including proteasome inhibitors, immunomodulatory agents, and monoclonal antibodies, multiple myeloma (MM) remains a difficult disease to treat effectively. Cure is extremely rare, and the majority of MM patients do not respond to treatment or experience relapse. Therefore, there is a need to develop new treatment methods for MM that can overcome these challenges.

[0003] Over the past decade, adoptive T-cell therapy, known as cellular immunotherapy, has made significant progress in the treatment of multiple myeloma through several licensed chimeric antigen receptor (CAR)-T-cell therapies. However, current adoptive immunotherapies use peripheral blood lymphocytes (PBLs) as the T-cell source, which suffers from drawbacks such as tumor escape and antigen loss due to the lack of inherent tumor specificity. This is one of the main disadvantages of CAR-T cells derived from PBLs. One approach to overcome these drawbacks and improve the tumor specificity of adoptive T-cell therapy is to use a variety of tumor-infiltrating lymphocytes (TILs) containing polyclonal memory T cells obtained from solid tumors in cancer patients. These polyclonal memory T cells target tumor-associated antigens. However, TILs are not suitable for all patients with solid tumors. For example, TIL therapy is not practical for solid tumors because patients with so-called cold tumors with low immunogenicity do not have TILs, the expansion time is long and high concentrations of IL-2 are required, and the cost is high.

[0004] Existing technical documents

[0005] Non-patent literature

[0006] (Non-patent document 1) Badalamenti G, Fanale D, Incorvaia L, Barraco N, ListμA, 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.

[0007] (Non-patent document 2) Kimberly AN IM.Marrow Infiltrating Lymphocytes:TheirRole in Adoptive Immunotherapy.The Cancer Journal.2015;21(6):501-5.doi:10.1097 / PPO.0000000000000159.

[0008] (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. Summary of the Invention

[0009] The problem the invention aims to solve

[0010] BCMA is an abbreviation for B-cell maturation antigen, also known as CD269, TNFRSF17, etc. It is selectively expressed in B cells and participates in cell survival and proliferation. BCMA has been shown to be expressed in various cancers containing B cells (such as malignant B-cell lymphoma, multiple myeloma, and Hodgkin's lymphoma).

[0011] The purpose of this invention is to provide a method for treating carcinomas (e.g., multiple myeloma) that express BCMA by targeting BCMA.

[0012] The inventors of this invention completed the invention by generating a chimeric antigen receptor MIL that specifically binds to BCMA (the MIL being an ex vivo expanded and activated MIL, eMIL, isolated from a patient and expanded and activated in vitro), and by evaluating the immunological properties and cytotoxicity of this CAR-expressing eMIL.

[0013] The purpose of this invention is to provide a group of MILs expressing an antigen-binding site (preferably a chimeric antigen receptor) that specifically binds to a cancer antigen (preferably BCMA), a method for preparing the same, and its therapeutic use. The preferred therapeutic use of this invention is for the treatment of multiple myeloma.

[0014] means for solving problems

[0015] On one hand, the isolated bone marrow infiltrating lymphocyte population according to the present invention comprises: a chimeric antigen receptor containing a binding domain that specifically binds to B cell maturation antigens.

[0016] On the other hand, the pharmaceutical composition according to the invention comprises isolated bone marrow-infiltrating lymphocyte populations as described above.

[0017] On the other hand, the method for treating a patient with multiple myeloma according to the present invention includes the step of administering to the patient the isolated bone marrow-infiltrating lymphocyte population as described above.

[0018] In another aspect, the use of the bone marrow infiltrating lymphocyte population isolated as described above according to the present invention in the preparation of a medicament for treating cancer (multiple myeloma).

[0019] Invention Effects

[0020] The bone marrow-infiltrating lymphocytes of this invention, containing a BCMA-specific chimeric antigen receptor, utilize two nanobodies specific to different epitopes of BCMA, resulting in excellent targeting efficiency. This allows for the effective induction of bone marrow-infiltrating lymphocytes into BCMA-expressing target cells, thereby exhibiting superior anti-cancer effects against cancer, particularly multiple myeloma. Furthermore, the bone marrow-infiltrating lymphocytes of this invention containing a BCMA-specific chimeric antigen receptor have a high ratio of CD8+. + T cm (central memory T cells), a high proportion of CD8 cells + T cmThis can help bone marrow-infiltrating lymphocytes maintain sustained in vivo cytotoxicity. Compared to MILs that do not contain chimeric antigen receptors, the BCMACAR-MILs of the present invention, which contain chimeric antigen receptors, exhibit increased cytotoxicity (such as an increased CD107a ratio), and therefore can be effectively used for the prevention or treatment of cancer. In addition, the CAR-MILs of the present invention have superior immunomodulatory and anticancer capabilities compared to other types of T cells containing the same CAR, for example, cytotoxicity against cancer cells. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the preparation method of the CAR-MILs group and the structure of the CAR according to the present invention.

[0022] Figure 2 This is a flow cytometry diagram showing the change in CAR expression rate over time in the CAR-MILs population after transducing the CAR of the present invention into MILs.

[0023] Figure 3 This is a linear graph showing the change in CAR expression rate of the CAR-MILs population of the present invention over time after the CAR of the present invention is transduced into MILs.

[0024] Figure 4 This illustrates the CD3 group in the CAR-MILs cluster after the CAR of the present invention is transduced into MILs. + CD56 - T cells and cytokine-induced killer cells (CD3+) + CD56 + Flow cytometry analysis results showing the change in the ratio of T cells over time.

[0025] Figure 5 This illustrates the CD3 group in the CAR-MILs cluster after the CAR of the present invention is transduced into MILs. + CD56 - T cells and cytokine-induced killer cells (CD3+) + CD56 + A bar chart showing the changes in T cells over time.

[0026] Figure 6 This diagram illustrates the expansion of the CAR-MILs population over time after the CAR of the present invention is transduced into MILs.

[0027] Figure 7This illustrates the CD8 group in the CAR-MILs cluster after the CAR of the present invention is transduced into MILs. + and CD4 + Flow cytometry analysis results showing the change in the ratio of T cells over time.

[0028] Figure 8 This illustrates the CD8 group in the CAR-MILs cluster after the CAR of the present invention is transduced into MILs. + and CD4 + A bar chart showing the changes in T cells over time.

[0029] Figure 9 This illustrates the naive T cells (CD62L) in the CAR-MIL population after transducing the CAR of the present invention into MILs. + CD45RA + Central memory T cells (CD62L+CD45RA) - (cells), T emra Cells (CD62L) - CD45RA + (cells) and effector memory T cells (CD62L) - CD45RA - A flow cytometry analysis result showing the change in the ratio of cells over time.

[0030] Figure 10 This illustrates the initial T cells (CD62L) in the CAR-MILs population after transducing the CAR of the present invention into MILs. + CD45RA + CD62L cells, central memory T cells (CD62L) + CD45RA - Temra cells (CD62L cells) - CD45RA + (cells) and effector memory T cells (CD62L) - CD45RA - A bar chart showing how the ratio of cells changes over time.

[0031] Figure 11 This is a flow cytometry analysis result showing the change in the expression ratio of immune checkpoint molecules on the surface of CAR-MILs over time after the CAR of the present invention was transduced into MILs.

[0032] Figure 12 This is a bar graph showing the change over time in the expression ratio of immune checkpoint molecules on the surface of CAR-MILs after the CAR of the present invention is transduced into MILs.

[0033] Figure 13 This illustrates the CD4 group in the CAR-MILs cluster after the CAR of the present invention is transduced into MILs. + CD25 + FoxP3 + Regulatory T cells (T cells) reg CD8 + CD25 + FoxP3 + Regulatory T cells (T cells) reg ) and CD4 + CD8 + CD25 + FoxP3 + Regulatory T cells (T cells) reg The graph shows the flow cytometry results of the ratio of ) changing over time.

[0034] Figure 14 This illustrates the CD4 group in the CAR-MILs cluster after the CAR of the present invention is transduced into MILs. + CD25 + FoxP3 + Regulatory T cells (T cells) reg CD8 + CD25 + FoxP3 + Regulatory T cells (T cells) reg ) and CD4 + CD8 + CD25 + FoxP3 + Regulatory T cells (T cells) reg A bar chart showing how the ratio of ( ) changes over time.

[0035] Figure 15 This is a graph showing the CD107a expression rate on CAR-MILs when the CAR-MIL population of the present invention was co-cultured with cancer cell lines or cancer cells isolated from patients on days 9, 14, and 19 after transduction of the CAR to MILs.

[0036] Figure 16 This is a graph showing the apoptosis rate of cancer cells when the CAR-MIL population of the present invention is co-cultured with cancer cell lines or cancer cells isolated from patients on days 14 and 19 after transduction of the CAR into MILs.

[0037] Figure 17This diagram illustrates how, after transducing the CAR of the present invention into MILs, the target cancer cells are killed by the CAR-MILs population over time when the CAR-MILs population cultured in vitro for 14 days is co-cultured with autologous cancer cells or the RPMI8226 cancer cell line isolated from the patient, resulting in a reduction of cancer cells. Detailed Implementation

[0038] The present invention and its embodiments will now be described in detail with reference to the accompanying drawings to enable those skilled in the art to readily implement the invention. However, the present invention can be implemented in various forms and is not limited to the embodiments described herein.

[0039] The immune cells of this invention can refer to hematopoietic cells that are functionally included in the initiation and / or execution of innate and / or acquired immune responses. The immune cells of this invention can be derived from stem cells. Stem cells can 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.

[0040] The immune cells may be leukocytes, neutrophils, eosinophils, basophils, monocytes, lymphocytes, T cells, cytotoxic T cells, natural killer T cells, dendritic cells, or combinations thereof, but are not limited thereto.

[0041] The term "marrow infiltrating lymphocyte (also referred to as "MILs" or "MILs")" as used in this specification refers to lymphocytes derived from the bone marrow. In this specification, the term "eMILs" refers to MILs activated by contact with and culture of anti-CD3 and anti-CD28 antibodies under the influence of interleukins. In this specification, the term "MILs" may mean eMILs depending on the context.

[0042] The term "activation" used in this manual, which relates to lymphocytes, refers to the process by which lymphocytes begin to proliferate and differentiate through mediators (such as antigen-specific receptors or interleukins), thereby increasing their anti-cancer function.

[0043] The term "expansion" as used in this manual refers to increasing the number of cells through proliferation.

[0044] Bone marrow infiltrating lymphocytes are not only distinctly different from peripheral blood lymphocytes (referred to as “PBLs”), but also distinctly different from tumor-infiltrating lymphocytes (TILs). The bone marrow microenvironment, due to its abundance of antigen-presenting cells, constitutes a specialized immunological site. The presence of these antigen-presenting cells can maintain central memory cells with higher levels of antigen processing and presentation, as found in the bone marrow region (Li JM et al J Immunol. 2009 Dec 15; 183(12):7799-809). These MILs express memory T cell markers (e.g., CD45RO). + and CD62L + MILs contain more memory cells than PBLs (Noonan K et al. Clin Cancer Res. 2012 Mar 1; 18(5):1426-34). Furthermore, MILs possess the ability to persistently priming memory cells with antigens, thus exhibiting characteristics distinct from tumor-infiltrating lymphocytes (TILs) of hematologic malignancies (Beckhove P et al. J Clin Invest. 2004 Jul 1; 114(1):67-76, Castiglioni P et al. 6J Immunol 2008; 180:4956-4964). For example, unlike TILs, MILs are available from all patients and can be expanded in vitro (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. In addition, unlike PBLs, MILs have a broad repertoire of endogenous antigens that are not expressed in PBLs, representing inherent tumor specificity (Noonan et al Clin Cancer Res).

[0045] The MILs of the CAR-MILs group of the present invention can be obtained from patients with any type of cancer including hematologic malignancies and solid tumors, preferably from patients with multiple myeloma. Preferably, they can be obtained from bone marrow cells with increased tumor specificity compared to peripheral blood.

[0046] The "chimeric antigen receptor (CAR)" of this invention refers to a protein comprising an intracellular signal transduction domain and an extracellular binding domain. A chimeric antigen receptor (CAR) consists of an antigen-recognizing binding domain, a signaling sequence, a hinge region, a transmembrane domain, and an intracellular signal transduction domain. These components enable immune cells to target tumor-associated antigens specific to the binding domain, thereby specifically attacking cancer cells.

[0047] The inventors of this invention used bone marrow-derived mononuclear cells (BMMNCs) from multiple myeloma patients, which exhibited high conduction efficiency and potent anti-myeloma immune responses, to prepare CARs containing BCMA dual epitope-binding nanobodies and a group of CAR-MILs containing said CARs. The invention was completed by demonstrating that said CAR-MILs represent an innovative immunotherapy approach for multiple myeloma.

[0048] The "BCMA (B-cell maturation antigen; UniProt ID: Q02223)" of this invention is an abbreviation for B-cell maturation antigen, also known as CD269 and TNFRSF17, etc. These are selectively expressed in B cells and participate in cell survival and proliferation.

[0049] The chimeric antigen receptor of the present invention

[0050] The chimeric antigen receptor of the present invention contains a binding domain that 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.

[0051] The chimeric antigen receptor of this invention specifically recognizes BCMA, and is therefore also called BCMA CAR.

[0052] The "nanobody" of this invention refers to a single-domain antibody (sdAb), and due to its small molecular weight based on a single structural domain, it has high stability and tissue permeability.

[0053] Nanobodies are the smallest and most stable antibody molecules known to date. They have a high affinity for antigens and, due to their small size, can not only penetrate tumor tissues but also cross the blood-brain barrier. They also have low toxicity and low immunogenicity.

[0054] The chimeric antigen receptor of the present invention includes binding domains comprising VHH01 and VHH02 of BCMA-specific nanobodies, and 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 NO.8 and SEQ ID NO.9, respectively.

[0055] The chimeric antigen receptor of the present invention contains a binding domain that may contain a VHH01 nanobody containing the amino acid sequence of SEQ ID NO.8 and a VHH02 nanobody containing the amino acid sequence of SEQ ID NO.9.

[0056] The nanobodies VHH01 and VHH02, which can achieve dual epitope binding to BCMA, exhibit high specificity. Therefore, when the CAR contains VHH01 and VHH02, it can induce BCMA-expressing T cells even in cancer cells expressing low levels of the target antigen. Compared with mono-nanobody CARs, dual-nanobody CARs show stronger activity and can effectively remove target tumor cells.

[0057] The VHH01 and VHH02 may contain 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 NO.2 to SEQ ID NO.7.

[0058] The VHH01 and VHH02 may include CDR1 of SEQ ID NO.2 or SEQ ID NO.3, CDR2 of SEQ ID NO.4 or SEQ ID NO.5, and CDR3 of SEQ ID NO.6 or SEQ ID NO.7.

[0059] VHH01 may include CDR1 of SEQ ID NO.2, CDR2 of SEQ ID NO.4 and CDR3 of SEQ ID NO.6, and VHH02 may include CDR1 of SEQ ID NO.3, CDR2 of SEQ ID NO.5 and CDR3 of SEQ ID NO.7.

[0060] In addition to the binding domain that specifically binds to BCMA, the chimeric antigen receptor of the present invention may also include one or more of the following groups: signal sequence, hinge, transmembrane domain (TM), and intracellular domain (intracellular signal transduction domain).

[0061] The hinge and transmembrane domains included in the chimeric antigen receptor of the present invention are preferably derived from CD8α, but are not limited thereto. Preferably, they may 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.

[0062] The intracellular signal transduction domain of the present invention is located on the inner side of the cell membrane of immune cells (i.e., the cytoplasm). It refers to the site that can activate the immune response of immune cells when the antibody bound to the extracellular domain binds to the target antigen.

[0063] The intracellular domain of the chimeric antigen receptor of the present invention may comprise two or more intracellular signal transduction domains. The intracellular signal transduction domains are preferably a combination of an intracellular signal transduction domain derived from 4-1BB and an intracellular signal transduction domain derived from CD3ζ, but are not limited thereto. Preferably, the intracellular signal transduction domain derived from 4-1BB may comprise the amino acid sequence of SEQ ID NO. 11, and the intracellular signal transduction domain derived from CD3ζ may comprise the amino acid sequence of SEQ ID NO. 12, and they may be sequences having 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. 11 or SEQ ID NO. 12.

[0064] In contrast to in vitro inactive MIL populations that do not contain chimeric antigen receptors that specifically bind to B cell maturation antigens (in other words, MIL populations just isolated from a patient), the CAR-MIL populations of the present invention can be CD8+. + T cells (CD8) + Increased T cell ratio, CD4 + T cells (CD4) + A group with a decreased T cell ratio.

[0065] In this specification, "newly isolated" refers to MIL clusters isolated from the patient that have not yet been amplified and activated in vitro.

[0066] Compared to the in vitro inactive MIL population that does not contain chimeric antigen receptors that specifically bind to B cell mature antigens (in other words, the MIL population just isolated from the patient), the CAR-MILs of the present invention can be a population with a reduced regulatory T cell ratio.

[0067] In contrast to in vitro inactive MIL populations that do not contain chimeric antigen receptors that specifically bind to B cell maturation antigens (in other words, MIL populations just isolated from a patient), the CAR-MILs of the present invention can be CD62L. + CD45RA - A population with an increased ratio of central memory T cells.

[0068] Compared to the in vitro inactive MIL populations that do not contain chimeric antigen receptors that specifically bind to B cell mature antigens (in other words, the MIL populations just isolated from the patient), the CAR-MILs of the present invention can be a population with reduced expression levels of programmed cell death protein 1 (PD-1).

[0069] Compared to in vitro inactive MIL populations that do not contain chimeric antigen receptors that specifically bind to B cell maturation antigens (in other words, MIL populations just isolated from a patient), the CAR-MILs of the present invention can exhibit increased CD8+. + The ratio of T cells and central memory T cells, and the reduction in CD4+ + The ratio of T cells and regulatory T cells, as well as low PD-1 expression levels.

[0070] Compared to isolated PBLs or MILs that are not activated in vitro and do not contain chimeric antigen receptors that specifically bind to B cell maturation antigens, the CAR-MILs of the present invention can target CD138. + Primary multiple myeloma cells exhibit superior cytotoxicity with a higher CD107a expression rate. Furthermore, compared to other types of T cells containing the same CAR, the CAR-MILs of this invention possess superior immunomodulatory and anticancer capabilities, such as cytotoxicity against cancer cells.

[0071] The ratio of different types of immune cells that make up an immune cell population (such as MILs) can be determined using routine experimental techniques such as flow cytometry. Different types of immune cells can be distinguished by the presence of multiple proteins that are uniquely expressed on their cell surface.

[0072] As used in this specification, the term "differentiation" refers to the process by which cells specialize for a particular function, such as the developmental process of acquiring morphological features and / or functions.

[0073] The term “substantially fully differentiated” as used in this specification means that the cell matures and eventually differentiates into a fully differentiated cell, and that the cell differentiation is 90% or more, 95% or more, or 100% complete.

[0074] The term "memory T cell" as used in this specification refers to a type of T cell that is an antigen-specific T cell that can persist for a long time after the antigen has been removed. For example, memory T cells also have the characteristic of having CD4 or CD8 proteins on their cell surface, and they are cells that can rapidly transform into effector T cells and respond to the antigen when they are re-exposed to a specific antigen.

[0075] As used in this specification, the term "regulatory T cell" refers to a type of T cell that performs functions such as maintaining homeostasis and self-tolerance by suppressing the immune response. This type of T cell is characterized by having one or more proteins selected from CD4 or CD8, and the presence of CD25 and Foxp3 proteins on its cell surface. It is also referred to as a T cell. reg .

[0076] The term "central memory T cell" as used in this specification refers to T cells with high self-regenerative capacity, primarily found in lymph nodes and the peripheral circulatory system, characterized by the presence of CD62L protein on the cell surface but not CD45RA protein, and is also known as T cells. cm .

[0077] The term "CD4" used in this specification + "CD4" refers to the glycoprotein CD4, which acts as a co-receptor for the T-cell receptor (TCR), and exists on the cell surface.

[0078] The term "CD8" used in this specification + "" refers to the presence of cluster of differentiation 8 (CD8), a glycoprotein that acts as a co-receptor for the T-cell receptor (TCR), on the cell surface.

[0079] The term "CD138" is used in this specification. +"This refers to the CD138 protein, also known as syndecan-1, which is present on the cell surface. CD138 is a member of the syndecanproteoglycan family, which acts as a receptor for the extracellular matrix, participates in cell differentiation, and shows increased expression in various cancers."

[0080] The term "CD107a" as used in this specification refers to the CD107a protein, which is also known as LAMP-1, a lysosome-associated membrane glycoprotein 1 involved in immune cell degranulation.

[0081] The superscript "+" used in this specification indicates the presence of the indicated protein on the cell surface.

[0082] The subscript "-" in this instruction manual indicates that the protein shown is not present on the cell surface.

[0083] The present invention provides a method for preparing eMIL containing BCMA CAR.

[0084] This invention provides a method for preparing isolated bone marrow infiltrating lymphocyte populations containing BCMA CAR, and the bone marrow infiltrating lymphocyte populations prepared by the method.

[0085] The method includes the following steps: isolating marrowinfiltration lymphocytes from the bone marrow of a cancer patient; culturing the isolated marrowinfiltration lymphocytes in the presence of IL-2 with anti-CD3 and anti-CD28 antibodies; transducing the isolated marrowinfiltration lymphocytes with a polynucleotide expressing a chimeric antigen receptor containing a binding domain that specifically binds to B cell maturation antigens; and culturing the isolated marrowinfiltration lymphocytes in the presence of IL-2, IL-7, IL-15, and IL-21.

[0086] The chimeric antigen receptor (i.e., BCMA CAR) used in the method, which includes a binding domain that specifically binds to B cell maturation antigens, can be the technology described in the "Chimeric Antigen Receptor of the Invention" section.

[0087] Specifically, the BCMACAR used in the method 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.

[0088] The binding domains contained in the BCMA CAR used in the method of the present invention comprise BCMA-specific nanobodies VHH01 and VHH02, and 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 NO.8 and SEQ ID NO.9, respectively.

[0089] The chimeric antigen receptor used in the method of the present invention contains a binding domain that may contain a VHH01 nanobody containing the amino acid sequence of SEQ ID NO. 8 and a VHH02 nanobody containing the amino acid sequence of SEQ ID NO. 9.

[0090] The VHH01 and VHH02 used in the method of the present invention may include CDR1 of SEQ ID NO.2 or SEQ ID NO.3, CDR2 of SEQ ID NO.4 or SEQ ID NO.5, and CDR3 of SEQ ID NO.6 or SEQ ID NO.7.

[0091] The VHH01 used in the method of 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.

[0092] In addition to the binding domain that specifically binds to BCMA, the BCMACAR used in the method of the present invention may also include one or more of the following groups: signal peptide (SP), hinge, transmembrane domain (TM), and intracellular domain (intracellular signal transduction domain).

[0093] The hinge and transmembrane domain are preferably derived from CD8α, but are not limited thereto. Preferably, they may contain 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.

[0094] The intracellular domain of the BCMA CAR used in the method of the present invention may contain two or more intracellular signal transduction domains. Preferably, the intracellular signal transduction domains are a domain derived from 4-1BB linked with an intracellular signal transduction domain derived from CD3ζ, but are not limited thereto. Preferably, the intracellular signal transduction domain derived from 4-1BB may contain the amino acid sequence of SEQ ID NO. 11, and the intracellular signal transduction domain derived from CD3ζ may contain the amino acid sequence of SEQ ID NO. 12, 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 sequences of SEQ ID NO. 11 or SEQ ID NO. 12.

[0095] This invention relates to an eMIL prepared by the above-described preparation method, namely, an eMIL expressing BCMACAR on its cell surface. In this specification, eMIL containing BCMACAR refers to the expression of BCMACAR on the cell surface of the eMIL.

[0096] Compared to MIL clusters just isolated from a patient, the CAR-MIL clusters prepared according to the method of the present invention can be CD8. + T cells (CD8) + Increased T cell ratio, CD4 + T cells (CD4) + A group with a decreased T cell ratio.

[0097] Compared to MIL populations just isolated from a patient, CAR-MILs prepared according to the method of the present invention can be populations with a reduced ratio of regulatory T cells.

[0098] Compared to MIL clusters just isolated from a patient, CAR-MILs prepared according to the method of the present invention can be CD62L. + CD45RA - A population with an increased ratio of central memory T cells.

[0099] Compared to MIL clusters just isolated from patients, CAR-MILs prepared according to the method of the present invention can be a cluster with reduced expression levels of programmed cell death protein 1 (PD-1).

[0100] CAR-MILs prepared according to the method of the present invention can exhibit increased CD8. + The ratio of T cells and central memory T cells, and the reduction in CD4+ +The ratio of T cells and regulatory T cells, as well as low PD-1 expression levels.

[0101] Compared to isolated peripheral blood lymphocytes or isolated bone marrow infiltrating lymphocyte populations that do not express chimeric antigen receptors, CAR-MILs prepared according to the method of the present invention can target CD138. + Primary multiple myeloma cells exhibit superior cytotoxicity with a higher CD107a expression rate.

[0102] The term "cancer" as used in this specification may specifically include, but is not limited to, one or more of the following: brain tumors, cervical cancer, ovarian cancer, prostate cancer, lung cancer, bile duct cancer, kidney cancer, gastric cancer, liver cancer, retinoblastoma, choriocarcinoma, small bowel cancer, colorectal cancer and rectal cancer, non-small cell lung cancer, gastric adenocarcinoma, acute lymphoblastic leukemia, acute myeloid leukemia, breast cancer, osteosarcoma, bladder cancer, anaplastic astrocytoma and multiple myeloma.

[0103] As used in this manual, the term "multiple myeloma (MM)" refers to a blood cancer caused by the abnormal differentiation and proliferation of plasma cells in the bone marrow.

[0104] As used in this specification, the term "prevention" refers to all actions that suppress or delay the onset of a disease by applying the composition, and "treatment" refers to all actions that improve or beneficially change the symptoms of a suspected disease or an individual with the disease by applying the composition.

[0105] The dosage of the CAR-MILs cell population of the present invention can be appropriately selected by those skilled in the art based on the subject's condition, weight, disease, dosage form, route of administration, and time of administration.

[0106] The CAR-MILs cell population of the present invention can reach target tissues through all conventional routes of administration. For example, it can be administered via intravenous, subcutaneous, or intraperitoneal administration, but is not limited thereto.

[0107] The term "combined administration" as used in this instruction manual refers to the simultaneous or sequential administration of two or more active ingredients.

[0108] The CAR-MILs cell population of the present invention can be administered in combination with other anticancer agents appropriately selected by those skilled in the art. For example, it can be administered in combination with immune checkpoint inhibitors, but is not limited thereto.

[0109] The present invention will be described in more detail below through embodiments, but the following embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0110] [Preparation Example 1]

[0111] Preparation of CAR-MILs

[0112] pass Figure 1 CAR-MILs were prepared using the method shown. Specifically, CAR-MILs were prepared by using Ficoll-Hypaque (d = 1.077, Lymphoprep...). TM Bone marrow mononuclear cells (“BMMNCs”) were isolated from the bone marrow of multiple myeloma patients using density gradient centrifugation (Axis-Shield, Oslo, Norway). BMMNCs were then co-cultured for 2 days in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, 100 μg / mL streptomycin, 4 mmol / L L-glutamine, and 200 U / mL recombinant human IL-2 with anti-CD3 / CD28 antibody-coated magnetic beads (Dyna beads) in 24-well plates. BCMACARs with the nucleic acid sequence of SEQ ID NO. 13 (as shown in Table 4) were then transduced into BMMNCs and cultured in RPMI medium containing 10 ng / mL IL-7, 10 U / mL IL-15, and 5 ng / mL IL-21. The medium was changed every 2–3 days, and the CAR-MILs were cultured continuously for 21 days. The BCMACAR comprises two nanobodies, designated VHH01 and VHH02, and the CDR sequences of the nanobodies are shown in Table 2 below. The BCMACAR contains VHH01 containing the amino acid sequence of SEQ ID NO. 8, VHH02 containing the amino acid sequence of SEQ ID NO. 9, a (G4S)5 linker, a hinge and transmembrane domain of CD8α containing the amino acid sequence of SEQ ID NO. 10, an intracellular signal transduction domain of 4-1BB containing the amino acid sequence of SEQ ID NO. 11, and an intracellular signal transduction domain of CD3ζ containing the amino acid sequence of SEQ ID NO. 12, wherein the above sequences are shown in Table 3 below.

[0113] Table 1

[0114]

[0115] Table 2

[0116]

[0117] Table 3

[0118]

[0119] Table 4

[0120]

[0121] [Example 1]

[0122] Immune population analysis of CAR-MILs

[0123] To analyze the immune cell population of CAR-MILs, the CAR-MILs prepared by the method described in Preparation Example 1 were treated with fluorescently labeled monoclonal antibodies. After collecting the samples using a flow cytometer, the expression of cell surface markers was analyzed, thereby confirming the composition of the immune cell population.

[0124] Specifically, in order to analyze CD3 + Cells, NK cells (natural killer cells), CIK cells (cytokine-induced killer cells), regulatory T cells (T cells) reg (regulatory T cells), CD4 + T cells and CD8 + The composition of the immune cell population 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) on T cells, etc.

[0125] First, in BCMA CAR-MILs, to confirm the transduction efficiency of the CAR of the present invention, the CAR-MILs (2 × 10⁻⁶) were washed with FACS buffer. 5After cell incubation, the cells were stained with biotinylated human BCMA and Live / Dead-AmCyan (Invitrogen, Carlsbad, CA) and treated with an 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 cytometry system (BD FACS Canto II, Becton Dickinson, Mountain View, CA, USA), and data were collected. The expression of cell surface markers was analyzed using FlowJo software (TreeStar, San Carlos, CA, USA) to confirm the transduction efficiency of BCMA CAR-MILs.

[0126] The composition of the immune cell population is also carried out in the same way as described above, by replacing the monoclonal antibody.

[0127] like Figure 2 and Figure 3 As shown, the results indicate that the CAR expression rate of CAR-MILs continued to increase for 21 days. Compared with day 0 of culture, the expression rate of BCMA CAR was approximately 31.1% on day 9 of culture and 35.4% on day 21, demonstrating excellent expression rate based on the small size of nanobodies.

[0128] like Figure 4 and Figure 5 As shown, it was confirmed that the BCMA CAR-MILs of the present invention contain a small amount of cytokine-induced killer cells (CD3+). + CD56 + ).

[0129] In addition, such as Figure 6 As shown, the BCMA CAR-MILs of the present invention exhibited a proliferation rate of more than 400 times on day 19 after in vitro transduction, and due to such an excellent proliferation rate, it has been confirmed that they can be effectively used to treat cancer.

[0130] In addition, such as Figure 7 and Figure 8 As shown, after transducing the CAR of this invention, CD8 + The ratio of T cells continued to increase, and cells exhibiting cytotoxicity (such as CD4+) were confirmed. + The ratio of T cells decreased, while the number of T cells increased.

[0131] In addition, such as Figure 9 and Figure 10 As shown, after transducing the CAR of this invention, it is also referred to as T. cm Central memory T cells (CD62L) + CD45RA - The ratio increased significantly, and it was confirmed that there were almost no naïve T cells and T cells. emra (CD62L - CD45RA + (A group of cells)

[0132] In addition, such as Figure 11 and Figure 12 As shown, it was confirmed that the immune checkpoint proteins (such as PD-1) of the CAR-MILs of the present invention are expressed at significantly low levels, thus overcoming the immunosuppression or immune escape mechanisms of cancer cells.

[0133] [Example 2]

[0134] CAR-MILs group T reg Analysis of cell ratio

[0135] As in Example 1, the composition of the immune cell population of CAR-MILs was analyzed. Figure 13 and Figure 14 As shown, the results confirm CD4 + CD25 + FoxP3 + Regulatory T cells (T cells) reg ) and CD8 + CD25 + FoxP3 + Regulatory T cells (T cells) reg The expression rate of ) decreased.

[0136] Therefore, it was confirmed that the immune cell population of CAR-MILs prepared by the method of the present invention contains almost no immunosuppressive cells (T cells). reg ).

[0137] [Example 3]

[0138] Cytotoxicity analysis of CAR-MILs

[0139] To confirm whether CAR-MILs can effectively kill multiple myeloma cells, the experiment examined the functional differences between CAR-MILs and activated MILs (eMILs) and activated peripheral blood lymphocytes (ePBLs). Specifically, to evaluate the killing ability of CAR-MILs, lactate dehydrogenase (LDH) analysis, kinetic analysis, and CD107a degranulation analysis were performed.

[0140] 3-1. CD107a degranulation analysis of CAR-MILs

[0141] Degranulation analysis of CD107a was performed as follows. 5 × 10⁻⁶ 4 One CAR-MIL is placed in a 96-hole U-bottom plate with 5×10 4 Target cells (K562, U266, RPMI8226, ARH77, IM9, and CD138 isolated from the patient) + Multiple myeloma cells can be cultured together or without target cells. The culture may contain 5 × 10⁻⁶ cells. 4 Target cells (K562, U266, RPMI8226, ARH77, IM9 and CD138) + Primary multiple myeloma cells were cultured in 96-well U-shaped plates with 5 μL of PE-conjugated anti-human CD107a antibody. After 1 hour, monensin and brefeldin A (BD Biosciences) were added, and the cells were cultured for an additional 4 hours. Cells were then obtained after staining with anti-human CD3 antibody.

[0142] like Figure 15 As shown, at an E (effector cell) : T (target cell) 1:1 ratio, compared with ePBL and eMIL, the CAR-MILs obtained on day 9, day 14, and day 21 after transduction of the CAR of this invention all exhibited higher CD107a levels. + The positive group ratio confirmed that it exhibited higher cell-killing activity against cancer cells.

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

[0144] LDH analysis was performed as follows. For target cells (K562, U266, RPMI8226, and CD138)... + For multiple myeloma cells, 10 6Target cells were placed in 100 μL of RPMI medium containing 2 μg of anti-human HLA-A, HLA-B, or HLA-C antibody (clone: ​​W6 / 32, Biolegend, USA) and cultured for 20 minutes, followed by washing by centrifugation. Then, the target cells were co-cultured with CAR-MIL at a 1:1 ratio in Costa 96-well plates (Corning, USA) for 6 hours at 37°C and 5% CO2. Finally, to determine the concentration of the cytoplasmic enzyme LDH released during cell lysis, the supernatant was collected, and the LDH concentration was measured using the CytoTox 96 non-radioactive cytotoxicity assay (Promega, USA). The cell lysis rate was calculated according to the manufacturer's recommended protocol.

[0145] CAR-MILs, eMILs, and ePBLs were combined with target leukemia (K562) cells that do not express BCMA or target myeloma cells (U266, RPMI8226, and CD138) that express BCMA. + The results of calculating the cell lysis rate after co-culturing primary multiple myeloma cells, such as... Figure 16 As shown, CAR-MILs did not show a difference in cytotoxicity against K562 cells that did not express BCMA, which served as a negative control, but showed a significant increase in cytotoxicity against U266 and RPMI8226 cells that overexpressed BCMA.

[0146] 3-3. Tumor-specific cytotoxicity kinetics of CAR-MILs

[0147] To evaluate the cytotoxicity of BCMA CAR-MILs against cancer cells in real time, the following was performed: Cytotoxicity Assay (Satorius). RPMI8226 and CD138 isolated from multiple myeloma patients were used as target cells. + Primary multiple myeloma cells at a rate of 1×10 4 Cells (per 100 μL) were seeded into 96-well plates coated with poly-L-ornithine solution (Sigma-Aldrich), in which BCMA CAR-MILs were added at a 1:1 effector cell:target cell (E:T) ratio. In the cells mixed in this manner, a 1:1000 dilution (100 nL) was added... After applying Cytotox Green Reagent (Sartorius), place the cell plate into... In a Sartorius Live-Cell Analysis System, the reaction was carried out at 37°C for 30 minutes. This was for analysis using... The number of apoptotic cells confirmed by the cytotoxic green reagent was monitored using the IncuCyte live cell analysis system, and cytotoxicity was confirmed by capturing fluorescence images every 2 hours over a continuous 24-hour period.

[0148] like Figure 17 As shown, in the BCMACAR-MIL treatment group, it was confirmed that the virus was transmitted through specific binding to apoptotic cells. The green cells shown by the cytotoxic green reagent increased significantly and remained so, thus confirming that the CAR-MILs of the present invention exhibited a sustained higher cell-killing activity compared to ePBL and eMIL.

[0149] Finally, as shown in Examples 3-1 to 3-3, compared with MILs and PBLs that do not express CAR, the CAR-MILs of the present invention were confirmed to exhibit superior cytotoxicity against target cancer cells. Furthermore, the experiments confirmed the functional differences between CAR-T and CAR-MILs, confirming that CAR-MILs exhibit superior cytotoxicity against cancer cells.

[0150] Sequence Directory

[0151] Attached is an electronic copy of the sequence catalog.

Claims

1. An isolated population of bone marrow-infiltrating lymphocytes, comprising: Chimeric antigen receptors containing a binding domain that specifically binds to B cell maturation antigens.

2. The isolated bone marrow infiltrating lymphocyte population according to claim 1, wherein, The antigen-chimeric receptor further comprises one or more selected from the group consisting of a signal sequence, a hinge, a linker and a transmembrane domain, and an intracellular domain.

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

7.

4. The isolated bone marrow infiltrating lymphocyte population according to claim 1, wherein, The binding domain that specifically binds to B cell maturation antigen includes a nanobody, wherein the nanobody is 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. The isolated bone marrow infiltrating lymphocyte population according to claim 1, wherein, The binding domain that specifically binds to B cell maturation antigen comprises a nanobody, the nanobody comprising an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO. 8 and SEQ ID NO.

9.

6. The isolated bone marrow infiltrating lymphocyte population according to claim 1, wherein, The binding domain that specifically binds to B cell maturation antigen includes a nanobody, wherein the nanobody is a nanobody containing the amino acid sequence of SEQ ID NO.8 and a nanobody containing the amino acid sequence of SEQ ID NO.

9.

7. The isolated bone marrow infiltrating lymphocyte population according to claim 1, wherein, The chimeric antigen receptor contains the amino acid sequence of SEQ ID NO.

1.

8. The isolated bone marrow infiltrating lymphocyte population according to claim 2, wherein, The hinge and transmembrane structural domain includes the hinge and transmembrane structural domain of CD8α.

9. The isolated bone marrow infiltrating lymphocyte population according to claim 8, wherein, The hinge and transmembrane domain of the CD8α contain the amino acid sequence of SEQ ID NO.

10.

10. The isolated bone marrow infiltrating lymphocyte population according to claim 2, wherein, The intracellular domain comprises sequences selected from the group consisting of 4-1BB intracellular signaling region sequences, CD3ζ intracellular signaling region sequences, and combinations thereof.

11. The isolated bone marrow infiltrating lymphocyte population according to claim 10, wherein, The intracellular signaling region sequence of the 4-1BB contains the amino acid sequence of SEQ ID NO.11, and the intracellular signaling region sequence of the CD3ζ contains the amino acid sequence of SEQ ID NO.

12.

12. The isolated bone marrow infiltrating lymphocyte population according to claim 1, wherein, Compared to isolated bone marrow infiltrating lymphocyte populations that are not activated in vitro and do not contain chimeric antigen receptors that specifically bind to B cell maturation antigens, CD8 + The T cell ratio increased, and CD4 + The T cell ratio is reduced.

13. The isolated bone marrow infiltrating lymphocyte population according to claim 1, wherein, The ratio of regulatory T cells was reduced compared to isolated bone marrow infiltrating lymphocyte populations that were not activated in vitro and did not contain chimeric antigen receptors that specifically bind to B cell maturation antigens.

14. The isolated bone marrow infiltrating lymphocyte population according to claim 1, wherein, The ratio of central memory T cells was increased compared to isolated bone marrow infiltrating lymphocyte populations that were not activated in vitro and did not contain chimeric antigen receptors that specifically bind to B cell mature antigens.

15. The isolated bone marrow infiltrating lymphocyte population according to claim 1, wherein, PD-1 expression levels were reduced compared to isolated bone marrow infiltrating lymphocyte populations that were not activated in vitro and did not contain chimeric antigen receptors that specifically bind to B cell maturation antigens.

16. The isolated bone marrow infiltrating lymphocyte population according to claim 1, wherein, Compared to isolated bone marrow infiltrating lymphocyte populations that were not activated in vitro and did not contain chimeric antigen receptors that specifically bind to B cell maturation antigens, they exhibited increased CD8+. + The ratio of T cells and central memory T cells, and the reduction in CD4+ + The ratio of T cells and regulatory T cells, as well as low PD-1 expression levels.

17. The isolated bone marrow infiltrating lymphocyte population according to claim 1, wherein, Compared to isolated peripheral blood lymphocytes or bone marrow infiltrating lymphocyte populations that are not activated in vitro and do not contain chimeric antigen receptors that specifically bind to B cell maturation antigens, CD138 + Primary multiple myeloma cells exhibit superior cytotoxicity with a higher CD107a expression rate.

18. The isolated bone marrow infiltrating lymphocyte population according to claim 1, wherein, The isolated bone marrow-infiltrating lymphocytes were obtained by a method comprising the following steps: Step a: Isolate bone marrow-infiltrating lymphocytes from the bone marrow of cancer patients; Step b: In the presence of IL-2, the bone marrow infiltrating lymphocytes obtained in step a are cultured in contact with anti-CD3 antibody and anti-CD28 antibody. Step c, transducing a polynucleotide expressing a chimeric antigen receptor containing a binding domain that specifically binds to B cell maturation antigens into the bone marrow-infiltrating lymphocytes obtained in step b. as well as Step d: Culture the bone marrow infiltrating lymphocytes obtained in step c in the presence of IL-2, IL-7, IL-15 and IL-21.

19. The isolated bone marrow infiltrating lymphocyte population according to claim 18, wherein, The cancer in question is multiple myeloma.

20. A pharmaceutical composition, wherein, The pharmaceutical composition comprises the isolated bone marrow infiltrating lymphocyte population as described in any one of claims 1 to 19.

21. The pharmaceutical composition according to claim 20, wherein, The composition is used to treat patients with cancer.

22. The pharmaceutical composition according to claim 21, wherein, The cancer in question is multiple myeloma.

23. Use of an isolated bone marrow infiltrating lymphocyte population according to any one of claims 1 to 19 in the preparation of a medicament for treating cancer.

24. The use according to claim 23, wherein, The cancer in question is multiple myeloma.

Images