Novel population of marrow-infiltrating lymphocytes and method for producing the same

Abstract

The present invention relates to a novel population of marrow-infiltrating lymphocytes (MILs) and a method for producing same. Specifically, the present invention provides a MIL cultivation method that enables the production of a novel MIL population with strong and long-lasting cytotoxicity against target cells. The novel population of MILs produced 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 and method for producing the same

[0001] The present invention relates to a novel population of marrow-infiltrating lymphocytes (MILs) and a method for producing the same. Specifically, the present invention provides a method for culturing MILs capable of providing a novel population of MILs with potent and long-lasting cytotoxicity against target cells. The novel population of MILs produced by the method can be used as a novel immuno-oncology therapy due to its high anticancer effect.

[0002]

[0003] 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.

[0004] 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 escape 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, TIL 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.

[0005] Marrow-infiltrating lymphocytes (MILs), which are attracting attention as a novel T cell source for adoptive T cell therapy for solid tumors, have been reported to possess specificity for target cancer cells. Unlike TILs, MILs can be easily obtained from any multiple myeloma patient using a simple procedure and can be rapidly expanded. Therefore, MILs can be used as a source for generating multiple myeloma-specific T cells in adoptive T cell therapy.

[0006] The inventors of the present invention have conducted various studies on adoptive T cell therapy, particularly on a source of T cells that can be rapidly produced and have high tumor specificity, and as a result, have generated ex vivo expanded and activated MIL (eMIL) from multiple myeloma patients and evaluated the immunological characteristics and cytotoxicity of such eMIL, thereby proving its superiority and thereby completing the present invention.

[0007]

[0008] [Prior Art Literature]

[0009] [Non-patent literature]

[0010] (Non-patent Document 0001) Cowan AJ, Green DJ, Kwok M, Lee S, Coffey DG, Holmberg LA, et al. Diagnosis and Management of Multiple Myeloma: A Review. Jama. 2022;327(5):464-77.

[0011] (Non-patent document 0002) Kyle RA RS. Treatment of Multiple Myeloma: A Comprehensive Review. Clin Lymphoma Myeloma. 2009;9(4):278-88. doi: 10.3816 / CLM.2009.n.056.

[0012] (Non-patent document 0003) Bonini C, Mondino A. Adoptive T-cell therapy for cancer: The era of engineered T cells. Eur J Immunol. 2015;45(9):2457-69.

[0013]

[0014] The present invention aims to provide a method for producing a bone marrow infiltrating lymphocyte population that can be rapidly expanded in vitro and is active against tumors.

[0015] The present invention aims to provide a bone marrow infiltrating lymphocyte population active against tumors.

[0016] The present invention aims to provide a composition comprising a bone marrow infiltrating lymphocyte population.

[0017] The present invention aims to provide a method for treating a subject with multiple myeloma, comprising administering a population of bone marrow infiltrating lymphocytes.

[0018]

[0019] The present invention provides a method for culturing a population of bone marrow infiltrating lymphocytes (MIL), comprising the steps of: (a) isolating MIL from the bone marrow of a patient with multiple myeloma; and (b) culturing the isolated MIL by contacting it with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, IL-7, and IL-15.

[0020] The method according to the present invention has one feature that the concentration of IL-2 used in step (b) is less than 200 IU / mL.

[0021] The period of culturing MIL in step (b) of the method according to the present invention may be 14 to 21 days.

[0022] The period of culturing MIL in step (b) of the method according to the present invention may be 14 days.

[0023] The method according to the present invention is that the cultured MIL population obtained in step (b) is CD8 compared to the isolated MIL obtained in step (a). + The proportion of central memory T cells may have increased.

[0024] The method according to the present invention is that the cultured MIL population obtained in step (b) is CD8 compared to the isolated MIL obtained in step (a). + The proportion of central memory T cells increases, and CD4 + T cells (CD4 +The T cell) ratio may be reduced.

[0025] The method according to the present invention may be such that the cultured MIL population obtained in step (b) has a reduced proportion of regulatory T cells and myeloid-derived suppressor cells compared to the isolated MIL obtained in step (a).

[0026] The method according to the present invention may be such that the cultured MIL population obtained in step (b) has reduced TIM3 and CD73 expression levels compared to the isolated MIL obtained in step (a).

[0027] In the method according to the present invention, the T memory stem cell population of the isolated bone marrow infiltrating lymphocyte population obtained in step (a) can be substantially completely differentiated into memory T cells after 5 days of culture or thereafter.

[0028] The present invention is a separated MIL population, which has increased CD8 + T cell and central memory T cell ratio, reduced CD4 + Provides an isolated MIL population characterized by a high proportion of T cells, regulatory T cells and myeloid-derived suppressor cells, and low expression levels of TIM3 and CD73.

[0029] The MIL population according to the present invention is CD138 compared to the isolated peripheral blood lymphocytes population. + A higher CD107a expression ratio, i.e., higher cytotoxicity, may be exhibited on primary multiple myeloma cells.

[0030] The present invention provides a composition comprising a MIL population prepared according to the present invention.

[0031] The composition according to the present invention may be a composition for use in the treatment of a subject having multiple myeloma.

[0032] The present invention provides a method for treating a subject with multiple myeloma, comprising administering a population of MILs prepared according to the present invention.

[0033]

[0034] A critical requirement for adoptive T-cell therapy is obtaining sufficient numbers of myeloma-specific T cells. Typically, peripheral blood lymphocytes have been used as a source of T cells for adoptive T-cell therapy due to the ease of obtaining large numbers of lymphocytes. However, peripheral blood lymphocytes obtained this way may have low tumor specificity. In multiple myeloma, the bone marrow is the site of disease initiation and progression and is a secondary lymphoid organ rich in T-cell markers and antigen-presenting cells. The abundance of antigen-presenting cells in the bone marrow allows for continuous exposure to malignant plasma cells, ultimately enriching and maintaining myeloma T cells with polyclonal antigen specificity compared to peripheral blood lymphocytes. According to the present invention, bone marrow-derived infiltrating lymphocytes can be successfully expanded and activated over a relatively short period of time.

[0035] In addition, the bone marrow infiltrating lymphocyte population expanded by the method of the present invention exhibits increased cytotoxicity compared to expanded peripheral blood lymphocytes. This is particularly true for CD138 cells in autologous patients. + This was more evident in experiments on primary cells.

[0036] The persistence of transferred T cells is also an important factor in achieving long-term efficacy in patients with multiple myeloma receiving adoptive T cell therapy. Chimeric antigen receptor T cells have recently emerged as a powerful immunotherapy for patients with multiple myeloma, but their response duration is relatively short at 8.8 months. The bone marrow-infiltrating lymphocyte cell population expanded by the method of the present invention is CD8 + It is characterized by a high proportion of Tcm, and these CD8 + An increase in the Tcm ratio suggests that bone marrow-infiltrating lymphocytes may have long-lasting cytotoxicity in the body.

[0037] The bone marrow-infiltrating lymphocyte population provided by the present invention is tumor antigen-specific and enriched for Tcm, resulting in long-term persistence. These characteristics make the bone marrow-infiltrating lymphocyte population provided by the present invention useful for immunotherapy and a promising platform for chimeric antigen receptor T cell therapy, replacing peripheral blood lymphocytes.

[0038]

[0039] Figure 1 is a schematic diagram showing a method for manufacturing an in vitro expanded and activated MIL.

[0040] Figure 2 is a graph showing the increase in MIL over time.

[0041] Figure 3 is a graph showing the expansion ratio of MIL.

[0042] Figure 4 shows CD8 over time. + and CD4 + This is a graph showing the results of T cell flow cytometry analysis.

[0043] Figure 5 shows CD8 over time. + and CD4 + This is a graph showing changes in T cells.

[0044] Figure 6 is a graph showing the increase in central memory T cells over time after in vitro MIL culture.

[0045] Figure 7 is a graph showing changes in central memory T cells over time after in vitro MIL culture.

[0046] Figure 8 is a graph showing differentiation of memory T stem cells (T memory stem cells; CD62L+ CD95+ cells) into central memory T cells (CD62L+ CD45RA- cells) within naive T cells (CD62L+ CD45RA+ cells) after in vitro MIL culture.

[0047] Figure 9 is a graph showing changes in the ratio of memory T stem cells (T memory stem cells; CD62L+ CD95+ cells) and central memory T cells (CD62L+ CD45RA- cells) over time after in vitro MIL culture.

[0048] Figure 10 is a graph showing the change in the expression ratio of immune checkpoint molecules on the surface of proliferated MILs after in vitro MIL culture.

[0049] Figure 11 is a graph showing the decrease in the expression rate of immune checkpoint molecules on the surface of proliferated MILs after in vitro MIL culture.

[0050] Figure 12 is a graph showing the change in the expression ratio of immune checkpoint molecules expressed on the surface of target cancer cells after co-culturing cancer cells and in vitro-proliferated MILs.

[0051] Figure 13 is a graph showing the change in the expression ratio of immune checkpoint molecules expressed on the surface of target cancer cells after co-culturing cancer cells and in vitro-proliferated MILs.

[0052] Figure 14 is a graph showing the decrease in the proportion of regulatory T cells (Tregs) in proliferated MILs over time after in vitro MIL culture.

[0053] Figure 15 is a graph showing the decrease in the percentage of myeloid-derived suppressor cells (MDSCs) in proliferated MILs over time after in vitro culture of MILs.

[0054] Figure 16 is a graph showing changes in IFN-γ production by stimulation of target cancer cells by MILs grown in vitro and increases in IFN-γ production by stimulation of autologous cancer cells isolated from a patient.

[0055] Figure 17 is a graph showing CD107a expression analysis showing a significant increase in cytotoxicity against autologous cancer cells isolated from patients with ex vivo-proliferated MILs.

[0056] Figure 18 is a graph showing changes in CD107a expression, indicating a significant increase in cytotoxicity against autologous cancer cells isolated from patients with ex vivo-proliferated MILs.

[0057] Figure 19 is a graph showing a significant increase in cytotoxicity of MILs grown in vitro for 14 days against target cancer cells.

[0058] Figure 20 is a graph showing the increase in cytotoxicity of MILs grown in vitro for 21 days against target cancer cells.

[0059] Figure 21 is a graph showing the percentage of surviving cancer cells after 20 hours of co-culture to confirm the cytotoxicity of MILs grown in vitro for 14 days against the U226 cancer cell line.

[0060] Figure 22 is a graph showing the percentage of surviving cancer cells after 20 hours of co-culture to confirm the cytotoxicity of MILs grown in vitro for 21 days against the U226 cancer cell line.

[0061] Figure 23 is a graph showing the percentage of surviving cancer cells after 20 hours of co-culture to confirm the cytotoxicity of MILs grown in vitro for 21 days against the RPMI8226 cancer cell line.

[0062]

[0063] 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 and embodiments described herein.

[0064] 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.

[0065] 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.

[0066] As used herein, the term "marrow infiltrating lymphocyte (MIL)" refers to lymphocytes derived from the bone marrow. In this specification, the term "MIL" may refer to eMIL depending on the context. Marrow infiltrating lymphocytes have distinguishing features from peripheral blood lymphocytes 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 allows for the maintenance of higher levels of central memory cells, such as those found in the bone marrow compartment, for processing and presenting antigens (Li JM et al J Immunol. 2009 Dec 15;183(12):7799-809). These MILs express markers of memory T cells, such as CD45RO. + and CD62L +, and have more memory cells than those found in peripheral blood lymphocyte populations (Noonan K et al Clin Cancer Res. 2012 Mar 1;18(5):1426-34). Furthermore, MILs are not just the “TILs” of hematological malignancies because of their ability to persistently prime memory cells to antigens (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). Unlike TILs, MILs can be harvested and expanded from any patient (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 contrast to peripheral blood lymphocytes, MILs possess a broad endogenous antigen repertoire, accounting for their unique tumor specificity that is not seen in PBLs (Noonan et al Clin Cancer Res).

[0067] The source of MIL for use in the method for culturing a MIL population according to the present invention can be obtained from a patient with any of several types of cancer, including hematological malignancies and solid tumors, preferably a patient with multiple myeloma. Preferably, the source can be obtained from bone marrow, which has increased tumor specificity compared to peripheral blood.

[0068] For isolated MIL cells, bone marrow cells can be activated and expanded in vitro to generate activated MILs by performing a method such as contacting them with anti-CD3 and anti-CD28 antibodies, wherein the contact can be performed using, for example, anti-CD3 and anti-CD28 antibody-coated magnetic beads. The process of contacting MIL cells with anti-CD3 and anti-CD20 antibodies is performed in the presence of IL-2, IL-7, and / or IL-15, and is preferably performed in an environment in which all of IL-2, IL-7, and IL-15 are present.

[0069] The step of culturing the isolated MIL cells in the present invention is preferably performed in the presence of a low concentration of IL-2, preferably less than 200 IU / mL, more preferably less than 100 IU / mL. When the concentration of IL-2 is 200 IU / mL or more, central memory CD8 cells are present in the MIL cell population. + The proportion of T cells may be reduced. The step of culturing the isolated MIL cells in the present invention may be performed in the presence of, for example, 100 IU / mL of IL-2, 10 ng / mL of IL-7, and 10 U / mL of IL-15.

[0070] The step of culturing the MIL cells isolated in the present invention can be performed for 14 to 21 days, and is preferably performed for 14 days. The inventors of the present invention have found that central memory CD8 cells are produced on the 14th day after culturing the MIL cells isolated according to the method of the present invention. + It was confirmed that the proportion of T cells reached 80%. In this way, central memory CD8 + A MIL cell population with a high proportion of T cells has not been previously described.

[0071] The MIL culture method according to the present invention is CD8 compared to the MIL population just isolated from the patient. + Central memory T cells (CD8 +The proportion of central memory T cells increases, and CD4 + T cells (CD4 + A population of MIL cells with a reduced T cell ratio can be provided. For example, a population of MIL cultured by the method according to the present invention can have a CD8 + The proportion of central memory T cells may be greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, or greater than 80%, and CD4 + The proportion of T cells may be less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%.

[0072] The MIL culture method according to the present invention can provide a MIL cell population with a reduced proportion of regulatory T cells and myeloid-derived suppressor cells compared to a MIL population just isolated from a patient. For example, a MIL population cultured by the method according to the present invention can have a proportion of regulatory T cells of 15% or less, 10% or less, 5% or less, or 1% or less, and a proportion of myeloid-derived suppressor cells of 15% or less, 10% or less, 5% or less, or 1% or less.

[0073] The MIL culture method according to the present invention can provide a MIL cell population with reduced TIM3 and CD73 expression levels compared to a MIL population just isolated from a patient. For example, a MIL cell population cultured using the method according to the present invention can have a TIM3 expression rate of 30% or less, 20% or less, or 10% or less, and a CD73 expression rate of 40% or less, 30% or less, 20% or less, or 10% or less.

[0074] The MIL culture method according to the present invention can provide a MIL cell population that is substantially completely differentiated into central memory T cells after 5 days or thereafter from a T memory stem cell population of a MIL population just isolated from a patient.

[0075] The isolated bone marrow infiltrating lymphocyte population according to the present invention has increased CD8 + T cell and central memory T cell ratios, reduced CD4 + Characterized by a proportion of T cells, regulatory T cells and myeloid-derived suppressor cells, and low expression levels of TIM3 and CD73, for example, an increased CD8 compared to a population of MILs just isolated from the patient. + T cell and central memory T cell ratios, reduced CD4 + It is characterized by a high proportion of T cells, regulatory T cells and myeloid-derived suppressor cells, and low expression levels of TIM3 and CD73.

[0076] The term "differentiation" as used herein refers to the developmental process by which cells become specialized for a particular function, for example, acquiring morphological features and / or functions.

[0077] 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.

[0078] 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 which, when re-exposed to a specific antigen, quickly converts into an effector T cell and responds to the antigen.

[0079] 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 T reg It is also referred to as .

[0080] The term "T memory stem cell" as used herein refers to a type of memory T cell, which has the characteristics of long lifespan, continuous self-renewal, rapid differentiation into effector T cells, and resistance to cell death. scm It is also referred to as .

[0081] The term "myeloid-derived suppressor cell" as used herein refers to a type of immature myeloid cell that suppresses both innate and adaptive immune responses, also referred to as MDSC.

[0082] The term "central memory T cell" as used herein has a high self-renewal capacity and is present primarily in lymph nodes and the peripheral circulation.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] The dosage of the MIL cell population according to the present invention can be appropriately selected by a person skilled in the art depending on the condition, weight, disease, formulation, route and period of administration of the subject.

[0089] The 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.

[0090] The term “combination administration” as used herein means administering two or more types of active ingredients simultaneously or sequentially.

[0091] The MIL cell population according to 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.

[0092] The present invention may be administered in combination with, but is not limited to, an immune checkpoint inhibitor that inhibits TIGIT, an immune checkpoint protein whose amount is confirmed to increase on the surface of tumor cells upon contact with a population of MIL cells cultured according to the present invention, such as tiragolumab.

[0093] According to the present invention, the MIL cell population cultured in accordance with the present invention may be administered in combination with an immune checkpoint inhibitor that inhibits CD73, an immune checkpoint protein whose amount has been confirmed to increase on the surface of tumor cells upon contact, such as, but not limited to, oleclumab.

[0094] 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.

[0095]

[0096] [Manufacturing Example 1]

[0097] Production of ex vivo expanded and activated MILs (eMILs)

[0098]

[0099] Ex vivo expanded and activated MIL (eMIL) was prepared using the same method as in Figure 1. Specifically, bone marrow mononuclear cells (BMMNCs) from the bone marrow of multiple myeloma patients were isolated using density gradient centrifugation with Ficoll-Hypaque (d = 1.077, Lymphoprep®; Axis-Shield, Oslo, Norway). Then, BMMNCs were co-cultured with anti-CD3 / CD28 antibody-coated beads in 24-well plates in RPMI 1640 medium containing 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, 4 mmol / L L-glutamine, 100 U / mL recombinant human IL-2, 10 ng / mL IL-7, and 10 U / mL IL-15. The medium was replaced every 2-3 days, and eMIL was continuously cultured for up to 21 days. As shown in Figures 2 and 3, eMIL continuously increased for 21 days, and the median expansion rate on day 14 of culture compared to day 0 of culture was 55.7%, and the median expansion rate on day 21 was 374.3, showing a significant increase.

[0100]

[0101] [Example 1]

[0102] Immune population analysis of ex vivo expanded and activated MILs (eMILs)

[0103]

[0104] To analyze the immune cell population of eMIL, the eMIL manufactured by the method of Manufacturing Example 1 was treated with a fluorescently labeled monoclonal antibody, a sample was collected using a flow cytometer, and the expression of cell surface markers was analyzed to confirm the composition of the immune cell population.

[0105] Specifically, CD3 +cells, NK cells (Natural killer cells), CIK cells (cytokine-induced killer cells), MDSC (myeloid-derived suppressor cells), regulatory T cells (T reg ; regulatory T cells), CD4 + T cells, CD8 + T cells, naive T cells, central memory T cells (T cm ), TEMRA cells (terminally differentiated effector T cells), T em To analyze cells (effector memory T cells), etc., eMIL (2 x 10 5 Cells were washed with FACS buffer and treated with Fc blocker. Afterwards, they were stained with surface monoclonal antibodies such as CD3-FITC, CD56-PE-Cy7, CD4-PE, CD8-Amyan, CD62L-PE-Cy7, CD45RA, CD33-Pacific blue, CD11b-Amyan, and PD-1-Pacific for 30 minutes at 4°C, and then washed and permeabilized with FACSTM Permeabilizing Solution 2 (BD Bioscience) for 30 minutes at room temperature. The prepared samples were collected on a BD FACS Canto II (Becton Dickinson, Mountain View, CA, USA), and the expression of cell surface markers was analyzed using Flow Jo software (TreeStar, San Carlos, CA, USA) to confirm the composition of immune cell populations.

[0106] As a result, as shown in Figures 4 and 5, most of the CD3 on the first day of culture (day 0) + Most of the MILs have CD4 + It was composed of T cells (55.2%) and CD8 + T cells were confirmed to be 39%. CD4 on days 14 and 21 of culture+ The proportion of T cells was 31% and 18.4%, respectively, and CD8 on the 14th day of culture. + The proportion of T cells was 59.8% on day 21, and 74.6% on day 21.

[0107] Additionally, as shown in Figures 6 to 7, on the first day of culture (day 0), BMMNC showed T em (CD62L - CD45RA - ) 60%, naive T cells (CD62L + CD45RA + ) 12%, T cm (CD62L + CD45RA - ) 10%, and 18% of TEMRA (CD62L - CD45RA + ) was confirmed to contain T. Afterwards, on the 14th day of culture, cm The proportion of T increased to 80%, while em It was confirmed that the ratio was 20%, and on the 21st day of culture, T cm The ratio is 60% on average, T em The proportion was 40%. Moreover, it was confirmed that there were no populations of naive T cells and TEMRA cells on days 14 and 21 of culture.

[0108] As shown in Figures 8 to 9, BMMNCs produced T memory stem cells (T) on the first day of culture (day 0). scm ; CD62L + CD45RA + CD95 + ) was 13.5%, and using the new MIL culture technique, T scm The ratio increased to almost 100% on the 5th day, and from the 7th day of culture, T scm The cell population is T cm was completely differentiated. In addition, compared to 14 days of culture, on day 21, T cm This decrease was confirmed, and it was confirmed that the optimal time for obtaining eMIL was the 14th day of culture.

[0109] Additionally, as shown in Figures 10 and 11, on day 14 of culture, CD3 + T cells showed low expression levels of PD-1 and TIGIT (1.2% and 4.8%, respectively), but high expression levels of TIM3 and CD73 (21% and 33%, respectively).

[0110] In addition, as shown in Figures 12 and 13, it was confirmed that the expression of TIGIT and CD73 in cancer cell lines (U266, ARH77, IM9, RPMI8226, and K562) significantly increased when co-cultured with eMIL. In the absence of eMIL, all cancer cells showed negative expression of TIGIT, and most cancer cells (U266, ARH77, IM9, and K562) showed negative expression of CD73.

[0111] After co-culture, the expression of TIGIT and CD73 checkpoint molecules was increased in eMIL and cancer cells, but not in CD8 + T cm We confirmed that eMIL with a high cell ratio was successfully generated from BMMNC of multiple myeloma patients.

[0112]

[0113] [Example 2]

[0114] Analysis of MDSC and Treg cell ratios in the eMIL group

[0115]

[0116] Immune population analysis of eMIL was performed as in Example 1. As a result, as shown in Figure 14, CD3 + CD4 + CD25 + Foxp3 + Regulatory T cells (T reg ) expression ratio decreased dramatically on the 14th and 21st days of culture, and as shown in Figure 15, CD3 - CD33 + CD11b +The proportion of myeloid-derived suppressor cells (MDSCs) also decreased.

[0117] Therefore, the immune population of eMIL manufactured by the method of the present invention includes immunosuppressive cells (T reg It was confirmed that it contains almost no MDSCs.

[0118]

[0119] [Example 3]

[0120] Cytotoxicity analysis of eMIL

[0121]

[0122] To determine whether eMIL effectively kills multiple myeloma cells, we examined the functional differences between eMIL and activated peripheral blood lymphocytes (ePBLs). Specifically, we performed IFN-γ ELISA, LDH assay, IncuCyte assay, and CD107a degranulation assay to evaluate the killing capacity of eMIL.

[0123]

[0124] 3-1. Tumor-specific cytotoxicity of eMIL IFN-γ ELISA analysis

[0125] IFN-γ ELISA analysis was performed as follows: 5Х10 4 eMIL and ePBL of 5X10 in 96-well U-bottom plates 4 The cells were cultured with or without target cells (K562, U266, RPMI8226, ARH77, and IM9). After 24 h, the supernatants were collected, and IFN-γ production was measured using an ELISA kit (BD Biosciences) according to the manufacturer's protocol. The production was determined by analyzing each sample in triplicate and calculating the average absorbance for each standard and sample analysis set.

[0126] As shown in Figure 16, on day 14 of culture, eMIL showed a slightly increased level of IFN-γ production for cancer cell lines (K562, U266, ARH77, and IM9) compared to the ePBL control, whereas CD138 isolated from multiple myeloma patients + The level of IFN-γ production in eMIL from autologous primary tumor cells was significantly increased compared to the ePBL control group.

[0127]

[0128] 3-2. IncuCyte analysis of tumor-specific cytotoxicity of eMIL and CD107a degranulation analysis

[0129] IncuCyte analysis was performed as follows. Multiple myeloma cell lines (U266 and RPMI8226) were transduced with green fluorescent protein (GFP)-lentivirus, and GFP-positive multiple myeloma cells were sorted by FACS. For kinetic analysis of tumor cell death, each GFP-transduced cell line (U266 and RPMI8226) was pre-stained with 2 μg of anti-human HLA-A, B, or C antibody (clone: ​​W6 / 32, Biolegend). 10 cells were then added to 100 μL of RPMI medium. 6 After adding the dog cells and culturing them for 20 minutes, they were washed by centrifugation. Each GFP-transduced cell line was seeded at 1X10 per well in a 96-well U-bottom plate. 4 After individual aliquots of cells at a concentration of 1:1, eMIL was added the following day at an effector:target (E:T) ratio of 1:1. The number of viable target cells was monitored by hourly fluorescence imaging over 72 h using the IncuCyte Live Cell Analysis System (Satorius). The number of viable cells was quantified using IncuCyte S3 software (Satorius) and normalized to the number of viable cells compared to the control group containing only target cells without eMIL treatment.

[0130] CD107a degranulation assay was performed as follows: 5Х10 4 eMIL and ePBL of 5X10 in 96-well U-bottom plates 4 Target cells (K562, U266, RPMI8226, ARH77, and IM9 and CD138 isolated from patients + Cultured with or without target cells (multiple myeloma cells). 5Х10 4 Target cells (K562, U266, RPMI8226, ARH77, IM9, and CD138 + Cells were cultured in 96-well U-bottom plates with or without primary multiple myeloma cells (5 μL PE-conjugated anti-human CD107a antibody). 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.

[0131] As shown in Figures 17 and 18, on day 14 of culture, eMIL isolated CD138 directly from the patient + Higher CD107a on multiple myeloma cells + Although a positive population could be identified, this was not the case in the target cell line. In contrast, ePBLs isolated from cancer cell lines and primary CD138 cells from patients + Lower CD107a when co-cultured with multiple myeloma cells + It represents a positive group.

[0132]

[0133] 3-3. Tumor-specific cytotoxicity LDH analysis of eMIL

[0134] LDH analysis was performed as follows. Target cells (K562, U266, RPMI8226, ARH77, IM9, 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 After adding the target cells and culturing them for 20 minutes, they were washed by centrifugation. Afterwards, the target cells were cultured with eMIL at a 1:1 ratio in a Costar 96-well plate (Corning, USA) at 5% CO2, 37 o C 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.

[0135] eMIL and ePBL were used in target leukemia (K562) and target myeloma cells (ARH77, U266, RPMI8226, IM9, and CD138 + After culturing with primary multiple myeloma cells, the cell lysis rate was calculated, as shown in Figures 19 to 23, eMIL in all cases, especially CD138 isolated from the patient. + It showed greater cytotoxicity against primary multiple myeloma cells. In particular, blocking CD138 with MHC class I antibodies + It was confirmed that the cytotoxic response of activated eMIL against primary multiple myeloma cells was completely abolished.

[0136] As shown in Figures 24 and 25, the cytotoxicity of eMIL against multiple myeloma cells was higher on day 14 compared to day 21 of culture, and according to the data, it was confirmed that the optimal time to harvest eMIL using the new MIL culture technique was on day 14 of culture.

[0137]

[0138] Finally, as shown in Examples 3-1 to 3-3, it was confirmed that eMIL was successfully manufactured and operated in BMMNC of multiple myeloma patients according to the manufacturing method of the present invention.

Claims

1. (a) a step of isolating marrow infiltration lymphocytes (MILs) from the bone marrow of a patient with multiple myeloma; and (b) a step of culturing the separated bone marrow infiltrating lymphocytes by contacting them with anti-CD3 and anti-CD28 antibodies in the presence of IL-2, IL-7 and IL-15; A method for culturing a population of isolated bone marrow infiltrating lymphocytes, comprising:

2. A method for culturing a population of isolated bone marrow infiltrating lymphocytes, characterized in that the concentration of IL-2 in the first paragraph is less than 200 IU / mL.

3. A method for culturing a population of isolated bone marrow infiltrating lymphocytes, characterized in that in step (b), bone marrow infiltrating lymphocytes are cultured for 14 to 21 days in paragraph 1 or 2.

4. A method for culturing a population of isolated bone marrow infiltrating lymphocytes, characterized in that the bone marrow infiltrating lymphocytes in step (b) are cultured for 14 days in paragraph 1 or 2.

5. In the first or second paragraph, the cultured bone marrow infiltrating lymphocyte population obtained from step (b) has a higher CD8 level than the isolated bone marrow infiltrating lymphocyte population obtained from step (a). + A method for culturing a population of isolated bone marrow infiltrating lymphocytes, characterized by an increased proportion of central memory T cells.

6. In the first or second paragraph, the cultured bone marrow infiltrating lymphocyte population obtained from step (b) has a higher CD8 than the isolated bone marrow infiltrating lymphocyte population obtained from step (a). + Central memory T cells (CD8 + The proportion of central memory T cells increases, and CD4 + T cells (CD4 + A method for culturing a population of isolated bone marrow infiltrating lymphocytes, characterized by a reduced proportion of T cells.

7. A method for culturing a population of isolated bone marrow infiltrating lymphocytes, wherein the population of cultured bone marrow infiltrating lymphocytes obtained from step (b) has a reduced proportion of regulatory T cells and myeloid-derived suppressor cells compared to the population of isolated MILs obtained from step (a), in paragraph 1 or 2.

8. A method for culturing a population of isolated bone marrow infiltrating lymphocytes, wherein the cultured population of bone marrow infiltrating lymphocytes obtained from step (b) has a decreased level of TIM3 and CD73 expression compared to the population of isolated bone marrow infiltrating lymphocytes obtained from step (a), in paragraph 1 or 2.

9. A method for culturing a population of isolated bone marrow infiltrating lymphocytes according to claim 1 or 2, characterized in that the population of T memory stem cells of the population of isolated bone marrow infiltrating lymphocytes obtained from step (a) is substantially completely differentiated into central memory T cells 5 days after or after the culture according to step (b).

10. As a separate bone marrow infiltrating lymphocyte population, CD8 + T cell and central memory T cell ratios, decreased CD4 + An isolated bone marrow infiltrating lymphocyte population characterized by high T cell, regulatory T cell and myeloid-derived suppressor cell ratios, and low expression levels of TIM3 and CD73.

11. In clause 10, CD138 compared to the isolated peripheral blood lymphocytes population + An isolated bone marrow infiltrating lymphocyte population characterized by a higher CD107a expression ratio on primary multiple myeloma cells.

12. A composition comprising a population of isolated bone marrow infiltrating lymphocytes according to claim 10 or 11.

13. A composition according to claim 12, wherein the composition is for use in the treatment of a subject having multiple myeloma.

14. A method for treating a subject having multiple myeloma, comprising administering a population of isolated bone marrow infiltrating lymphocytes according to claim 10 or 11.

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