JPWO2020112563A5 - - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority benefit from U.S. Provisional Patent Application No. 62/771,482, filed November 26, 2018, the entire contents of which are incorporated herein by reference. contained within.

Inclusion by reference of materials in the ASCII text file This application includes the sequence listing contained in the following ASCII text file submitted at the same time: File name: NKT0028WO_ST25.txt; created on November 22, 2019, size 9.4KB. Incorporated by reference.

Background Malignant tumors are the result of uncontrolled proliferation of cells within the body. Similarly, many diseases or infections result from dysregulation of normal tissue growth and cell death. Although surgical methods or drug therapy have long been used as front-line treatments, immunotherapy is a new option for treating cancerous or diseased tissue.

Overview Traditional anticancer drug therapy has relied on surgical approaches, radiation therapy, chemotherapy, or a combination of these methods. Similarly, many diseases or infections are treated with traditional drug therapy, but recent advances in the development of biologics are changing treatment methods. Once research has elucidated the mechanisms behind certain types of cancer, this has been used to develop targeted cancer treatments. Targeted therapy is a cancer treatment that uses specific drugs to suppress or prevent cancer cell growth by targeting specific genes or proteins found in cancer cells or cells that help cancer grow (such as blood vessel cells). It is. Recently, genetic engineering has been used to develop ways to harness specific aspects of the immune system to fight cancer. In some cases, a patient's own immune cells are modified to specifically eradicate the patient's cancer type. As detailed below, various types of immune cells can be used, such as T cells or natural killer (NK cells).

In some embodiments, methods for simultaneous expansion of multiple populations of immune cells are provided. In one embodiment, two, three, four or more different cell types are (i) co-propagated together or (ii) co-propagated in parallel and then combined. In some embodiments, the NK cells and T cells have an NK cell:T cell ratio of about 5:1, about 10:1, about 20:1, e.g., 5:1, 7:1, 10:1, Grown together or separately and combined in a ratio of 12:1, 15:1, 17:1, 20:1 or any ratio therebetween. In another embodiment, there are at least two times more NK cells than T cells. These ratios (more NK cells than T cells) are particularly advantageous in some embodiments, in conjunction with long-term cytotoxicity from T cells (due to more proliferated NK cells and their cytotoxic activity). ) enabling robust acute anti-tumor responses. In some embodiments, not only T cell proliferation is suppressed, but the inhibitory effect of NK cells on T cells modulates T cell cytokine release compared to a T cell only approach. In T cell-only therapy, various cytokines released from T cells act in an autocrine manner to upregulate T cell proliferation and activity. If proliferation and activation are left unchecked, neurotoxicity, "target/non-target" recognition, anaphylaxis, and even cytokine release syndrome (CRS), a potentially life-threatening systemic disease seen after administration of antibodies, adoptive T-cell therapy, can occur. It may cause various side effects such as ``inflammatory reactions''). In some embodiments, T cells still release certain cytokines, but rather than causing T cell stimulation (and release of additional cytokines that can cause CRS), these cytokines actively stimulate NK cells. It acts to stimulate. Thus, in some responses, NK cells weaken the ability of T cells to self-stimulate by inducing proliferation (and more cytotoxicity). Therefore, the presence of NK cells reduces T cell proliferative and cytokine releasing activity. Thus, in some embodiments, the combination of NK cells and T cells provides a more effective and safer cancer immunotherapy product. In some embodiments, the T cells include one, two, three or more of the following subtypes: cytotoxic T cells, helper T cells, NKT cells, or gamma delta T cells. In one aspect, the collective total of the plurality of subtypes is the ratio described herein (including, but not limited to, 5-10:1, 10-15:1, 15-20:1). ) is calculated. In another aspect, one sum of subtypes is calculated for the ratios described herein (including, but not limited to, 5:1).

In some embodiments, the first cell type is in a ratio of about 5 to 10:1, such as 5:1, 6:1, 7:1, 8:1, 9:1 and 10:1. combined with cell types. In some embodiments, the first cell type is in a ratio of about 11 to 15:1, such as 11:1, 12:1, 13:1, 14:1 and 15:1 to the second cell type. combined. In some embodiments, the first cell type is in a ratio of about 16 to 20:1, such as 16:1, 17:1, 18:1, 19:1 and 20:1 to the second cell type. combined. In another embodiment, there is at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more of cell type 1 than cell type 2. In another embodiment, a third cell type is also included. In one embodiment, cell type 1 includes NK cells and cell type 2 includes T cells. In other embodiments, other cell types are used.

Accordingly, the present invention provides a method of generating a mixed population of immune cells (eg, engineered immune cells) having specific relative proportions of subpopulations of the mixed population. In some embodiments, these methods allow for the generation of cell populations that provide robust cytotoxic effects against target cells, such as tumor cells, while certain subpopulations of genetically modified immune cells are more Reduces the potential for adverse immune effects that can occur if present in high amounts. In some embodiments, a method of generating a mixed population of immune cells comprising at least two subpopulations of genetically modified immune cells, comprising: isolating a population of mononuclear cells from a blood sample, the method comprising: the mononuclear cell population comprising at least a first and a second immune cell subpopulation, wherein the first subpopulation of immune cells is isolated from the isolated mononuclear cells; culturing a subpopulation of the first immune cell subpopulation in a culture vessel with a population of feeder cells, wherein the feeder cells contain at least one cell for stimulating proliferation of natural killer (NK) cells within the first immune cell subpopulation. isolating a second immune cell subpopulation from said isolated mononuclear cells; in a culture vessel with at least one molecule that stimulates the proliferation of T cells within said second immune cell subpopulation to expand a population of T cells; A portion of cytotoxic NK cells and a portion of cytotoxic T cells that have been genetically engineered to express proteins or drugs that improve toxicity and/or reduce side effects. generating a mixed population of genetically modified immune cells. In some embodiments, the combination of two (or more) expanded genetically modified immune cells is performed in vitro, while in other embodiments, the combination is performed in vivo (e.g., the expanded cell types by concomitant or sequential administration).

In some embodiments, the at least one molecule for stimulating NK cells is selected from 4-1BB ligand (4-1BBL), interleukin 15 (IL-15), and combinations thereof. In some embodiments, the molecule for stimulating NK cell proliferation comprises a combination of 4-1BBL and IL15. In some embodiments, interleukin 15 (IL-15) is membrane bound to the feeder cell. In some embodiments, culturing the first subpopulation of isolated immune cells comprises adding one or more of soluble interleukin 2, soluble interleukin 12, soluble interleukin 18, or a combination thereof to the culture. further comprising adding. In some embodiments, the at least one molecule for stimulating T cell proliferation comprises one or more antibodies to CD3 or antibodies to CD28. In some embodiments, at least one molecule for stimulating T cell proliferation comprises an anti-CD3 antibody. In a further embodiment, a mixture of anti-CD3 and anti-CD28 antibodies is used. In some embodiments, at least one molecule for stimulating T cell proliferation comprises an anti-CD28 antibody. Depending on the embodiment, antibodies against CD3 optionally include OKT3 antibodies. In some embodiments, the OKT3 antibody, other anti-CD3 antibody, or anti-CD28 antibody is expressed by feeder cells that are co-cultured with a second subpopulation of immune cells. In further embodiments, the antibody or other stimulating molecule is attached to a solid support. Solid supports include culture vessels, biocompatible beads containing labels, or superparamagnetic beads, depending on the embodiment. Thus, in some embodiments, the stimulatory molecule is provided by being expressed by feeder cells, while in some embodiments cell-free exansion is used (e.g., the stimulatory molecule is (added to the culture medium or otherwise attached to a solid support that is not a feeder cell).

In some embodiments, the method provides the expanded NK cell population with a genetically modified receptor configured to bind to a target expressed by a cancer cell and, upon binding, induce cytotoxic activity against the cancer cell. and introducing the encoding nucleic acid to generate genetically modified cytotoxic NK cells. Similarly, in some embodiments, the method provides an expanded T cell population with genetically modified cells configured to bind to a target expressed by a cancer cell and, upon binding, induce cytotoxic activity against the cancer cell. The method further includes introducing a nucleic acid encoding a receptor to produce a genetically modified cytotoxic cell. In some embodiments, the NK cells and T cells are transduced with the same genetically modified receptor, and in other embodiments, the NK cells and T cells are transduced with different genetically modified receptors. In some embodiments, when additional subtypes of cells are used in a mixed population, they may be engineered to express the same genetically modified receptors as NK cells and/or T cells, or may be engineered to express different genetically modified receptors. It may be designed to express a receptor type. In some embodiments, the nucleic acid introduced into the expanded cells encodes a chimeric antigen receptor (CAR). CARs can be receptors for a variety of targets expressed by tumor cells, including those specifically expressed by tumor cells as well as those overexpressed by tumor cells. (Thereby limiting the targeting of native cells with markers). In some embodiments, the CAR is CD19, CD123, BCMA, galactin, Ral-B, FLT3, CD70, DLL3, CD5, GUCY2C, EGFR, KREMEN2, PSMA, ALPPL2, CLDN6, CLDN18, GPR143, GRM8, LPAR3, GD2 , ADAM12, LECT1 or TMEM186. In some embodiments, the CAR is a receptor for one or more NKG2D ligands, including, but not limited to, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and/or ULBP6. In some embodiments, the genetically modified cells contain different cells, including in some embodiments a mixture of NK cells and T cells that are collectively directed against two, three, four or more different tumor targets. Constructed to express a CAR for a target. In some embodiments, the CAR is a receptor for CD19. In some embodiments, each subpopulation of the mixed population expresses an anti-CD19 CAR. In some embodiments, the anti-CD19 CAR comprises an OX40 costimulatory domain and a CD3ζ signaling domain operably linked to an anti-CD19 scFv. In some embodiments, the anti-CD19 CAR is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identical to SEQ ID NO: 1. encoded by a nucleic acid with a In some embodiments, the anti-CD19 CAR is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% Contains amino acid sequences that have sequence identity.

In some embodiments, the genetically modified cytotoxic NK cells and the genetically modified cytotoxic T cells are provided in a manner that (i) the genetically modified NK cells are enhanced against cancer cells as compared to the genetically modified NK cells alone; (ii) the genetically modified cytotoxic T cells are capable of increasing the number of proliferating genetically modified NK cells; and/or (iii) the genetically modified T cells are the only genetically modified T cells. When combined in a ratio that shows decreased cytokine release compared to the case of The ratio used can be tailored to a given patient, tumor type, severity or stage of tumor progression, or can be based on other diagnostic or prognostic factors. In some embodiments, the ratio of genetically modified NK cells to genetically modified T cells is about 5:1. In other embodiments, other ratios are used. For example, in some embodiments, the ratio of the first cell type to the second cell type is about 2:1, about 4:1, about 6:1, about 8:1, about 10:1, about 12 :1, about 14:1, about 16:1, about 18:1, about 20:1 or more (or any ratio there between).

In some embodiments, the blood sample from which immune cells are isolated is a peripheral blood sample. In further embodiments, the blood sample is a cord blood sample. In some embodiments, the mixed population of genetically modified immune cells comprises about 1x10 ^to about ^1x10 genetically modified cytotoxic NK cells and about ^1x10 to about ^1x10 genetically modified cytotoxic T cells. Contains. As described herein, various embodiments enable two or more immune cell types to perform effective cytotoxicity against target tumor cells, reducing or eliminating the risk of adverse immune effects, including CRS. It is combined in the ratio of In some embodiments, the mixed population of genetically modified immune cells exhibits a rapid cytotoxic effect on tumor cells, followed by an enhanced persistent cytotoxic effect on tumor cells. In some embodiments, the NK portion of the mixed population of genetically modified immune cells has a longer duration of persistent cytotoxic effect on tumor cells compared to NK cells or T cells alone.

In some embodiments, a mixed population of genetically modified cytotoxic immune cells is provided, which is a first subpopulation of immune cells comprising NK cells, wherein the NK cells are targets expressed by cancer cells. is designed to express a genetically modified receptor configured to bind to cancer cells and induce cytotoxic activity against cancer cells upon binding, wherein the first subpopulation has approximately 1×10 ⁸ to The second subpopulation contains immune cells, including approximately 1x10 ¹⁰ NK cells, where T cells bind to targets expressed by cancer cells and, upon binding, act against cancer cells. The second subpopulation contains about 1 x 10 ⁴ to about 1 x 10 ⁶ T cells, and the genetically engineered NK cells are engineered to express genetically modified receptors configured to induce cytotoxic activity. and mixed populations of genetically modified T cells have greater cytotoxicity against cancer cells than either NK cells alone or T cells alone at equivalent effector-to-target ratios at a given effector-to-target cell ratio. shows.

As described above, in some embodiments, the genetically modified receptors include, for example, CD19, CD123, galactin, Ral-B, FLT3, CD70, DLL3, CD5, GUCY2C, EGFR, KREMEN2, PSMA, ALPPL2, CLDN6, CLDN18. , GPR143, GRM8, LPAR3, GD2, ADAM12, LECT1 or TMEM186. In some embodiments, each subpopulation of the mixed population expresses an anti-CD19 CAR. In some embodiments, the anti-CD19 CAR comprises an OX40 costimulatory domain and a CD3ζ signaling domain operably linked to an anti-CD19 scFv. In some embodiments, the anti-CD19 CAR is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identical to SEQ ID NO: 1. encoded by a nucleic acid with a In some embodiments, the anti-CD19 CAR is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identical to SEQ ID NO:2. It contains an amino acid sequence with a specific character.

Additionally, the present invention provides a method for expanding a mixed population of immune cells comprising at least two subpopulations of immune cells, comprising: isolating a population of mononuclear cells from a blood sample; dividing the population into at least a first subpart and a second subpart, culturing the first subpart of isolated mononuclear cells with a first population of feeder cells in a culture vessel containing a first medium; wherein the first population of feeder cells expresses at least one molecule for stimulating proliferation of natural killer (NK) cells within the first subpart of the isolated mononuclear cells; and/or the first medium comprises at least one molecule for stimulating proliferation of natural killer (NK) cells within the first subpart of the isolated mononuclear cells, wherein the at least one molecule is selected from 4-1BB ligand (4-1BBL), interleukin 15 (IL-15) and combinations thereof, thereby generating a proliferating population of NK cells, secondary generation of isolated mononuclear cells. culturing a subpart of the mononuclear cells with a second population of feeder cells in a culture vessel containing a second culture medium, wherein the second population of feeder cells is a second subpart of the isolated mononuclear cells. and/or the second culture medium expresses at least one molecule for stimulating proliferation of one or more subpopulations of T cells within a second subpopulation of T cells within the isolated mononuclear cells. at least one molecule for stimulating the proliferation of one or more subpopulations of cells, thereby generating a proliferating population of one or more subpopulations of T cells, and at least a portion of the proliferating population of NK cells. A method is provided comprising combining at least a portion of a proliferating population of one or more subpopulations of cells to produce a mixed population of immune cells comprising at least two subpopulations of immune cells.

In some embodiments, the mixed population includes a higher proportion of NK cells compared to the total proportion of one or more subpopulations of T cells. In some embodiments, the one or more subpopulations of T cells include gamma-delta T cells.

The invention provides mixed populations of immune cells produced by the methods described herein. In some embodiments, a mixed population of immune cells is configured for use in treating a tumor, whether the tumor is a solid tumor or a suspended tumor. The invention also provides the use of a mixed population of immune cells described herein for the treatment of cancer. In some embodiments, the use of a mixed population of immune cells in the manufacture of a medicament for the treatment of cancer is provided.

Also, in some embodiments, a combination of genetically modified NK cells and genetically modified T cells is provided, wherein the ratio of NK cells to T cells is at least 5:1. In some embodiments, a combination of genetically modified NK cells and genetically modified T cells is provided, where the ratio of NK cells to T cells is at least 10:1. In some embodiments, a combination of genetically modified NK cells and genetically modified T cells is provided, wherein the ratio of NK cells to T cells is at least 12:1. In some embodiments, a combination of genetically modified NK cells and genetically modified T cells is provided, wherein the ratio of NK cells to T cells is at least 15:1. In some embodiments, a combination of genetically modified NK cells and genetically modified T cells is provided, wherein the ratio of NK cells to T cells is at least 20:1.

In some embodiments, the combination of cells at or about the indicated ratios induces enhanced cytotoxic activity of the genetically modified NK cells against the target tumor. In some embodiments, the combination of cells at or about the indicated ratios provides enhanced persistence of cytotoxic activity of the genetically modified NK cells against the target tumor compared to the genetically modified NK cells alone. induce sex. In some embodiments, the combination of cells at or about the indicated ratios induces decreased cytokine release by genetically modified T cells compared to genetically modified T cells alone. In some embodiments, the combination of cells at or about the indicated ratios induces decreased proliferation of genetically modified T cells compared to genetically modified T cells alone.

In some embodiments, methods of treating cancer using the mixed populations of cells described herein are provided. As described herein, in some embodiments, cell combination is achieved by mixing cell types in the desired ratio after expansion and before delivery to the patient. In some embodiments, the combination is performed by administration (eg, the required number of cells from each cell type is administered so that the desired ratio is achieved in vivo). In some embodiments, the method of treatment further comprises administering IL2. Embodiments involving producing the resulting combination in vivo may be accomplished by administering the first cell type first, or by administering the second cell type first. Good too. In some embodiments, administration of a first type of genetically modified immune cell can provide an improved microenvironment for a second type (or third type, etc.) of immune cells.

In some embodiments, methods are provided for co-expansion of multiple immune cell populations, such as for co-expansion of NK cells and T cells. Also provided are immune cell populations comprising mixtures of two or more immune cell subpopulations, and methods and uses of such compositions for the treatment of cancer and other causes of disease or damaged tissue. In some embodiments, a mixed cell population of NK cells and T cells advantageously has dual effects on target cells, e.g., a rapid phase of NK cell-induced cytotoxic effects and a consequence of T cell activation. as well as a longer period of tumor control. Advantageously, in some embodiments, the two types of cells can be produced together in a single process and administered in combination with each other. In some embodiments, a first part of the expansion process produces NK cells, then T cells, and a second part of the expansion process combines the resulting NK cell population and T cell population. , to achieve the desired ratio of NK:T cells. In a further embodiment, NK cells and T cells are co-cultured and the methodology is adjusted so that the desired ratio of one cell type to the other cell type is achieved after the combined expansion culture process. (as well as for any third, fourth or more cell types included in the mixture).

In some embodiments, both NK cells and T cells (and optionally additional immune cell types) can be expanded and introduced using the optimized construct within defined parameters. The resulting composite cell population advantageously exhibits synergistic effects on target cells representing optimized activity of each of the constituent immune cell subpopulations.

According to some embodiments, a method of expanding a mixed population of immune cells comprising at least two subpopulations of immune cells, the method comprising: isolating a population of mononuclear cells from a blood sample; culturing the cells in a culture vessel with a population of feeder cells, the feeder cells being configured to express at least one molecule for stimulating the proliferation of NK cells; co-cultivating the cells with the feeder cells; exposing a mononuclear cell to at least one molecule for stimulating proliferation of T cells within the isolated mononuclear cell, and co-culturing with said feeder cell at least one molecule for stimulating proliferation of said T cells; culturing mononuclear cells exposed to one molecule for a period sufficient to allow proliferation of both NK cell and T cell subpopulations, thereby forming at least two immune cell subpopulations. A method is provided for generating a mixed population of immune cells comprising: In some embodiments, the isolated mononuclear cell population comprises at least one cell, such as, for example, NK cells and one or more T cell subtypes (e.g., cytotoxic T cells, NKT cells, gamma delta T cells, etc.). It is characterized by containing two different types of immune cells.

In some embodiments, the methods described herein enrich isolated mononuclear cells to increase the relative proportion of NK cells compared to the isolated mononuclear cell population. It may further include converting. In some embodiments, the methods described herein deplete the isolated mononuclear cells to reduce the relative proportion of T cells compared to the isolated mononuclear cell population. It may further include:

In some embodiments, the at least one molecule for stimulating proliferation of natural killer (NK) cells within isolated mononuclear cells comprises 4-1BB ligand (4-1BBL), interleukin 15 (IL- 15) or a combination thereof. In some embodiments, other stimulating molecules may be used, including other interleukins. In some embodiments, in addition to or in place of expression of a stimulatory molecule by feeder cells, such stimulatory molecule is added to the culture medium. In some embodiments, the at least one molecule for stimulating NK cell proliferation comprises a combination of 4-1BBL and IL15. In some embodiments, interleukin 15 (IL-15) is membrane bound to the feeder cell.

In some embodiments, the at least one molecule for stimulating T cell proliferation comprises an antibody that interacts with a portion of the T cell receptor on the T cell. In some embodiments, the antibody used is an antibody against CD3 or against CD28. In some embodiments, bispecific antibodies (eg, antibodies that target both CD3 and CD28) are used. In some embodiments, the antibody comprises an OKT3 antibody. In some embodiments, CD3, CD28 and/or OKT3 antibodies are expressed by feeder cells. In further embodiments, CD3, CD28 and/or OKT3 antibodies are provided in culture medium.

In some embodiments, at least one molecule for stimulating T cell proliferation comprises an anti-CD3 antibody. In some embodiments, at least one molecule for stimulating T cell proliferation comprises an anti-CD28 antibody. In some embodiments, the at least one molecule for stimulating T cell proliferation comprises a mixture of anti-CD3 and anti-CD28 antibodies. In some embodiments, the antibody is attached to a solid support. In some embodiments, the solid support comprises a culture vessel, a biocompatible bead containing a label, or a superparamagnetic bead.

In some embodiments, exposing mononuclear cells co-cultured with feeder cells to at least one molecule for stimulating proliferation of T cells within the isolated mononuclear cells comprises This is done at the same time as the cells are co-cultured. However, in some embodiments, the exposure is done sequentially (eg, before or after co-cultivation). For example, in some embodiments, exposing mononuclear cells co-cultured with feeder cells to at least one molecule for stimulating proliferation of T cells within the isolated mononuclear cells comprises This is done at a later point in time than the cells are co-cultured with feeder cells. In some embodiments, the slower the time that mononuclear cells co-cultured with feeder cells are exposed to at least one molecule for stimulating T cell proliferation, the greater the extent of T cell proliferation from the mononuclear cell population. is inversely correlated with Thus, in some embodiments, the timing of exposure is used to modulate the extent of T cell (or T cell subtype) proliferation in the resulting mixed cell population.

In some embodiments, the ratio of feeder cells to mononuclear cells ranges from about 10:1 to 1:10. In some embodiments, the ratio of feeder cells to mononuclear cells is about 1:1. Other ratios are used in some embodiments, for example, the ratio of feeder cells: mononuclear cells is 1000:1, 500:1, 250:1, 100:1, 50:1, or the listed ratios. and any other ratio between and including it.

In some embodiments, the blood sample is a peripheral blood sample. In some embodiments, the blood sample is a cord blood sample. In some embodiments, the blood sample is from fetal-maternal tissue, such as placenta. In some embodiments, the source is used to generate a mixed immune cell population that is homologous to the recipient. In some embodiments, the recipient is a donor. In some embodiments, the blood sample and/or expanded mixed population is stored (eg, frozen) until ready for delivery to a recipient.

In some embodiments, the mixed population of immune cells has a ratio of NK cells to T cells (including various T cell subpopulations) ranging from about 100:1 to about 1:100. In some embodiments, approximately equal proportions of NK cells and T cells are used, such that the mixed population of immune cells has a ratio of NK cells to T cells of about 1:1.

In some embodiments, the mixed population of immune cells, where the ratio of NK cells to T cells is about 1:1 to about 100:1, is more rapid against tumor cells than NK cells alone or T cells alone. It shows a sustained cytotoxic effect on tumor cells. In some such embodiments, the mixed population of immune cells having a ratio of NK cells to T cells of between about 1:1 and about 1:100, as compared to NK cells alone or T cells alone, Shows a longer lasting cytotoxic effect on cells.

In some embodiments, the invention also provides a population of feeder cells configured to stimulate proliferation of at least two subpopulations of immune cells from a mononuclear cell population, wherein the feeder cells expresses at least one signal for stimulating proliferation of NK cells and at least one signal for stimulating proliferation of T cells (or a subpopulation of T cells), wherein the proliferation of NK cells at least one signal for NK cell proliferation is selected from 4-1BB ligand (4-1BBL), interleukin 15 (IL-15) and combinations thereof, wherein the at least one signal for NK cell proliferation is , molecules that interact with the T cell receptor CD3, and optionally molecules that interact with CD28 on T cells.

The invention also provides the use of such a population of feeder cells to generate a mixed cell population comprising NK cells and T cells. In some embodiments, the percentage of NK cells in the mixed cell population is greater than about 10%, and the percentage of T cells in the mixed cell population is greater than about 30%. In some embodiments, the percentage of NK cells in the mixed cell population and the percentage of T cells in the mixed cell population are each between about 40% and 60% of the total.

In some embodiments, a mixed population of NK cells and T cells (including subpopulations of T cells) is also provided, wherein the percentage of NK cells in the mixed cell population is greater than about 10% of the total, and the mixed cell population The proportion of T cells in the population is greater than about 30% of the total. In some embodiments, the percentage of NK cells in the mixed cell population and the percentage of T cells in the mixed cell population are each between about 40% and 60% of the total. In some embodiments, the mixed cell population exhibits bi-phasic cytotoxic kinetics toward target cells. In some such embodiments, the biphasic cytotoxicity kinetics comprises a rapid and intense initial cytotoxicity phase followed by a less intense cytotoxicity phase. In some embodiments, the target cells include tumor tissue. In some embodiments, the tumor tissue is a solid tumor, while in further embodiments, the tumor tissue is a suspended tumor (eg, a hematological tumor).

The invention also provides the use of a mixed population of NK cells and T cells for the treatment of cancer and/or in the manufacture of a medicament for the treatment of cancer.

Some additional aspects provided herein relate to methods for expanding a mixed population of immune cells comprising at least two subpopulations of immune cells, the method comprising: growing a population of mononuclear cells from a blood sample; isolating a population of mononuclear cells, wherein the population of isolated mononuclear cells comprises at least two different types of immune cells, and a culture vessel containing a culture medium containing the isolated mononuclear cells with a population of feeder cells. wherein the culturing is carried out for a sufficient period of time to allow expansion of both the NK cell subpopulation and the T cell subpopulation, thereby comprising at least two immune cell subpopulations. Involves generating a mixed population of immune cells.

In some embodiments, the feeder cells express at least one molecule for stimulating proliferation of natural killer (NK) cells within the isolated mononuclear cells, and/or the culture medium is isolated. and at least one molecule for stimulating the proliferation of natural killer (NK) cells within mononuclear cells. In some embodiments, the at least one molecule is selected from 4-1BB ligand (4-1BBL), interleukin 15 (IL-15), and combinations thereof. In some embodiments, the feeder cells express at least one molecule for stimulating the proliferation of T cells or one or more subpopulations of T cells within the isolated mononuclear cells and/or comprises at least one molecule for stimulating proliferation of T cells or one or more subpopulations of T cells within the isolated mononuclear cells. In some embodiments, the one or more subpopulations of T cells are cytotoxic T cells, natural killer T cells (NKT cells), effector T cells, helper T cells, memory T cells, regulatory T cells, gamma delta cells. one or more of T cells, mucosa-associated invariant T cells. In some embodiments, the blood sample is a cord blood sample.

The invention also provides a method for expanding a mixed population of immune cells comprising at least two subpopulations of immune cells, the method comprising: isolating a population of mononuclear cells from a blood sample; The population of isolated mononuclear cells comprises at least two different types of immune cells, dividing the population of isolated mononuclear cells into at least a first subpart and a second subpart, culturing a first subpart of isolated mononuclear cells with a first population of feeder cells in a culture vessel containing a first medium; culturing with a second population of feeder cells in a culture vessel containing a culture medium, combining at least a portion of the expanded population of NK cells with at least a portion of the expanded one or more subpopulations of T cells; A method is provided that includes generating a mixed population of immune cells that includes at least two subpopulations of immune cells.

In some embodiments, the first population of feeder cells expresses at least one molecule for stimulating proliferation of natural killer (NK) cells within the first subpart of isolated mononuclear cells; and/or the first culture medium comprises at least one molecule for stimulating proliferation of natural killer (NK) cells within the first subpart of the isolated mononuclear cells. In some embodiments, the at least one molecule is selected from 4-1BB ligand (4-1BBL), interleukin 15 (IL-15), and combinations thereof, and the second population of feeder cells expresses , and/or the second culture medium comprises at least one molecule for stimulating proliferation of one or more subpopulations of T cells within the second subpart of the isolated mononuclear cells.

In some embodiments, the mixed population comprises a higher proportion of NK cells compared to the total proportion of one or more subpopulations of T cells. In some embodiments, the one or more subpopulations of T cells include gamma-delta T cells.

Also provided in some embodiments is a composition or use thereof in the treatment of cancerous tumors according to any one of the embodiments described herein.

The following description of the drawings relates to tests and results that represent non-limiting aspects of the invention described herein.
Figures 1A-1D are schematic diagrams of donor cell composition from four healthy donors. FIG. 1A shows the approximate percentage breakdown of T cells, NK cells and other cells in a blood sample drawn from a first donor. Figures IB, 1C and ID show corresponding data from three additional donors. FIG. 2 shows a schematic process flow diagram by which various K562 clones effective for co-expansion of NK cells and T cells can be identified according to certain embodiments described herein. Briefly, the pooled K562 population is limited diluted and a selected number of individual clones are tested for their ability to expand both NK cells and T cells in culture. Blood samples from four donors shown in Figures 1A-1D are used. The feeder cell line (and clone) used for co-expansion of NK cells and T cells was 41BBL, a modified K562 cell genetically engineered to express membrane-bound IL-15 and membrane-bound anti-CD3 scFv OKT3. be. This feeder clone is treated with mitomycin C, and 5×10 ⁵ cells per well are seeded into a culture vessel. On day 0 of culture, donor peripheral blood mononuclear cells are seeded in the same wells as feeder clones at a density of 5 x ¹⁰ cells per well. Interleukin-2 (IL-2) was added at 40 units/mL on day 0 of co-culture, this was repeated on day 4 of co-culture, and again 400 units/mL of IL-2 was added on day 6 of co-culture. Add. Cells are grown from day 7 to day 13 of co-culture, and the ratio of NK cells to T cells is assessed during this period. Co-culture days 14 to 20 were used to determine variables that were adjusted early in the co-culture process, such as the introduction of secondary stimuli to supplement the stimulus provided by the engineered K562 cells. Then, determine the final cell population based on the ratio of NK cells and T cells.

Figures 3A-3B show data related to the fold increase in NK or T cell proliferation obtained from 4 donors after 7 days of culture with 24 different K562 clones. Figure 3A shows that certain K562 clones have similar growth efficiencies when using a native genetically modified K562-stimulated feeder cell line (e.g., a mixed population of genetically modified K562 cells rather than a population consisting of a single replicating clone). It shows what was shown. Figure 3B shows that each clone tested has the ability to strongly stimulate T cell proliferation. Figures 4A-4B show data related to cell composition obtained from 4 different donors after 7 days of growth culture with 24 different K562 clones. Figure 4A shows NK cells present in the expanded cell population after 7 days in culture from each of the four donors when expanded in each of the 24 individual cloned K562 populations used as feeder cells. Shows data on the percentage of Figure 4B shows the corresponding data on the percentage of T cells shown after 7 days of culture. These data indicate that, in general, the cell composition obtained after 7 days of culture reflects the initial (eg starting point) NK:T cell ratio. In some embodiments, described in more detail below, the methods described herein reduce the incoming ratio of NK cells:T cells or, by modification of the method, NK cells and/or T cells. This allowed for differential expansion of cells to manipulate any desired final NK cell to T cell ratio. FIG. 5 schematically depicts an expansion protocol according to some embodiments described herein in which a source of supplemental stimulation is added to the culture at variable time points. Similar to Figure 2, Figure 5 shows a timeline of the three stages of the expansion protocol. Again at time 0, a low dose of IL-2 is added to the culture, this is repeated on day 4, and then again (with a high dose of IL-2) on day 6. The schematic diagram in Figure 5 shows the addition of supplemental stimulation sources for T cell proliferation (e.g., CD3/CD28 beads) or no addition (“no beads,” control) during the first 6 days of culture. Shows the matrix to be explained. FACS analysis is performed over time at several steps during co-culture to assess the relative proliferation and transduction efficiency of NK and T cells.

Figures 6A-6B present data related to NK cell and T cell proliferation upon application of supplemental proliferation stimuli. Figure 6A shows the changes in NK cells after 7 days of culture assessed in the presence and absence of beads and the day CD3/CD28 beads (replenishment stimulus for T cells) were added to the co-culture. Figure 6B shows the corresponding data on T cell proliferation. Figures 7A-7D demonstrate the ability to simultaneously transduce both NK cells and T cells with a vector containing an exogenous gene for expression, and the transduction Data relating to the ability of both cell types to maintain expression of the expressed genes are presented. Figures 7A and 7B show the transduction efficiency of each cell type in four donors 3 days after transfection, as measured by flow cytometry. Figures 7C and 7D show maintenance of expression 7 days after introduction. Figures 8A-8D present data on changes in the ratio of NK cells to T cells with longer co-culture expansion times. Figures 9A-9C are schematic representations of various genetically modified cells for evaluating mixed populations of NK/T cells in vitro and/or in vivo. Figure 10 shows data regarding the cytotoxic effects of NK cells, T cells and mixed NK cell+T cell populations in re-exposure assays. FIG. 11 shows data regarding the evaluation of immune cell populations according to embodiments described herein in an in vitro 3-D culture environment.

FIG. 12A shows a study setup designed to identify effective ratios of chimeric antigen receptor-containing NK cells and chimeric antigen receptor-containing T cells as combination therapy against target tumor cells. Figure 12B shows cytotoxicity measured by IncuCyte assay (loss of signal indicates cytotoxic effect on target cells). Boxes have been added to identify the E:T and NK:T ratios that exhibit the greatest cytotoxicity against Nalm6 target cells. FIG. 13 shows data regarding cytotoxicity of the indicated control NK-only, T-only, or NK+T constructs against target tumor cells at the first stage and at a second re-exposure of tumor cells. Figures 14A-14B relate to studies that determine the effect of NK cells and T cells each on the cell number of the other cell type. Figure 14A shows the results of evaluating NK cell numbers when NK CAR cells were present at three different NK:Nalm6 ratios and T cells were present at the indicated ratios (X-axis). FIG. 14B shows the results of evaluating T cell numbers when T CAR cells were present at six different T:Nalm6 ratios and NK cells were present at the indicated ratios (X-axis). Testing was performed after 7 days of co-culture of NK cells and T cells (expressing a non-limiting example of anti-CD19 CAR (designated 19-1)) with Nalm6 cells. Figures 15A-15F show data on NK cell proliferation in the presence of T cells with chimeric antigen receptors. Figure 15A shows proliferation of NK cells (containing anti-CD19 CAR and present in a 1:1 ratio with target Nalm6 cells) in the absence of T cells. FIG. 15B shows the proliferation of NK19-1 cells when T cells with CAR of the same non-limiting example are present at a T:Nalm6 ratio of 1:16. Figure 15C shows proliferation of NK19-1 cells when T cells with the same non-limiting example of CAR are present at a T:Nalm6 ratio of 1:8. Figure 15D shows proliferation of NK19-1 cells when T cells with the same non-limiting example of CAR are present at a T:Nalm6 ratio of 1:4. FIG. 15E shows proliferation of NK19-1 cells when T cells with the same non-limiting example of CAR are present at a T:Nalm6 ratio of 1:2. Figure 15F shows additional NK cell proliferation data (variable T cell numbers) at two different E:T ratios of NK cells.

Figures 16A-16D show data on T cell proliferation in the presence of NK cells with anti-CD19 CAR. Figure 16A shows proliferation of T cells (with anti-CD19 CAR and present with target Nalm6 cells in a 1:2 ratio) in the absence of NK cells. Figure 16B shows the proliferation of T19-1 cells when NK cells with the same non-limiting example of CAR are present at a 1:1 NK:Nalm6 ratio. Figure 16C shows the proliferation of T19-1 cells when NK cells with the same non-limiting example of CAR are present at a 2:1 NK:Nalm6 ratio. Figure 16D shows additional T cell proliferation data (variable NK cell numbers) at various E:T ratios of NK cells. Figures 17A-17C are schematic diagrams summarizing the interactions of NK cells and T cells regarding proliferation. FIG. 17A is a schematic diagram of NK cell proliferation when only tumor cells are present. FIG. 17B is a schematic diagram of T cell proliferation when only tumor cells are present. Figure 17C shows a schematic representation of NK and T cell proliferation when both are present with tumor cells. Figures 18A-18L show data related to cytokine release by mixed populations of NK cells and T cells. Shown are GM-CSF (18A), IL6 (18B), IL4 (18C), IL10 (18D), IFNg (18E), IL2 (18F), TNFa (18G), IL21 (18H), IL5 (18I), IL13 (18J), IL9 (18K) and IL22 (18L).

FIG. 19 is a schematic representation of an in vivo tumor xenograft model used to assess the cytotoxicity of NK cells expanded according to embodiments described herein. Figures 20A-20E show bioluminescence and survival data reflecting tumor burden in mice receiving the indicated treatments. Figure 20A shows that NK cells and T Animals receiving cell combinations (at the indicated doses) are shown. All groups in Figure 20A received the indicated treatments on day 3. Figure 20B shows mice treated with combinations of NK cells and T cells at the indicated doses, with the group in the left column receiving NK cells on day 3 and T cells on day 4; the group in the left column receiving T cells on day 4; The group received T cells on the third day and NK cells on the fourth day. Figures 20A-20B show data up to day 31 after tumor cell implantation. Figure 20C shows data for selected groups (groups with surviving mice) from day 43 to day 121 after implantation of tumor cells at the indicated timings and cell doses. Figure 20D shows survival data for each experimental group. Figure 20E shows survival data for selected experimental groups (of those shown in Figure 20D).

Detailed Description Cancer is the abnormal growth of cells. Many types of cancer exist and can appear as solid tumors or as floating cells (eg, blood cancers). Until recently, cancer treatments included chemotherapy, radiation therapy and/or surgery. More recently, cancer-specific features have become clearer, making it easier to identify cancers, with relevance not only to diagnosis but also to the growing field of cancer immunotherapy. Cancer immunotherapy utilizes the defense mechanisms of various cells of the immune system to interact with and reduce or eliminate cancer cells. This method can not only be used to target and treat cancer, but also to treat other indications where cells may be infected, for example cells infected with bacteria or viruses. . In some embodiments, the immune cells are genetically engineered to be more effective against damaged or diseased tissues/cells. In some embodiments, as described in more detail herein, the expansion of immune cells, whether for administration to a patient in their native form, to increase the number of immune cells for use in therapy; Whether for administration as a mixture of cells in their native form or for subsequent genetic manipulation of the cells.

Immune Cells for Expansion and Use in Immunotherapy In some embodiments, cells of the immune system are harvested and expanded by the methods described herein, and the cells have a cytotoxic effect on specific target cells. Your abilities will be used. In some embodiments, the harvested/expanded cells are genetically engineered to have an enhanced cytotoxic effect on target cells, such as tumor cells. In some embodiments, white blood cells (leukocytes) are used because of their natural function of protecting the body from abnormal cell growth and infection. There are various types of leukocytes that play specific roles in the human immune system and are therefore a preferred starting point for the cell manipulations described herein. White blood cells include granulocytes and agranulocytes (depending on the presence or absence of granules in the cytoplasm). Granulocytes include basophils, eosinophils, neutrophils and mast cells. Agranulocytes include lymphocytes and monocytes.

Monocytes for Immunotherapy Monocytes are a subtype of white blood cells. Monocytes can differentiate into macrophages and myeloid dendritic cells. Monocytes are involved in the adaptive immune system and perform the main functions of phagocytosis, antigen presentation and cytokine production. Phagocytosis is the process of engulfing cellular material or whole cells and then digesting and destroying the engulfed cellular material. In some embodiments, monocytes are used in conjunction with one or more additional genetically engineered cells described herein.

Lymphocytes for Immunotherapy Lymphocytes, the other major subtypes of white blood cells, include T cells (cell-mediated, cytotoxic, adaptive immunity), natural killer cells (cell-mediated, cytotoxic, innate immune) and B cells (humoral, antibody-driven adaptive immunity). Although B cells are genetically engineered according to some embodiments described herein, some embodiments also relate to engineered T cells or engineered NK cells (some In embodiments, a mixture of T cells and NK cells is used).

T Cells for Immunotherapy T cells are distinguished from other lymphocyte subtypes (eg, B cells or NK cells) based on the presence of T cell receptors on the cell surface. T cells have a wide variety of subtypes, including effector T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, mucosa-associated invariant T cells, and gamma delta T cells. It can be classified into In some embodiments, particular subtypes of T cells are genetically engineered. In some embodiments, a mixed pool of T cell subtypes is genetically engineered. In some embodiments, there is no particular selection of the type of T cell that is genetically engineered to express the cytotoxic receptor complex described herein. In some embodiments, certain techniques are used to promote expansion/harvesting of T cells with a particular marker profile, such as the use of cytokine stimulation. For example, in some embodiments, activation of certain human T cells, eg, CD4 ⁺ T cells, CD8 ⁺ T cells, is achieved by using CD3 and/or CD28 as stimulatory molecules. In some embodiments, the method of treating or treating cancer or infection comprises administering a therapeutically effective amount of T cells alone or in combination with NK cells and/or other immune cells described herein. Provide a way to prevent it. In some embodiments, the T cells are genetically engineered to have enhanced cytotoxic effects on target cells. In some embodiments, the T cell is an autologous cell, while in some embodiments the T cell is an allogeneic cell.

NK Cells for Immunotherapy Some embodiments include administering a therapeutically effective amount of NK cells described herein alone or in combination with T cells and/or other immune cells. Methods of treating or preventing cancer or infectious diseases are provided. In some embodiments, NK cells are genetically engineered to have an enhanced cytotoxic effect on target cells. In some embodiments, the NK cell is an autologous cell, while in some embodiments the NK cell is an allogeneic cell. In some embodiments, NK cells are preferred due to their relatively high natural cytotoxicity. In some embodiments, the combinations of cells described herein (e.g., NK cells and T cells) interact synergistically to target cells (as compared to either NK cells alone or T cells alone). For example, it would be unexpectedly beneficial to provide more effective activity against tumor cells or other diseased cells.

Hematopoietic Stem Cells for Cancer Immunotherapy In some embodiments, hematopoietic stem cells (HSC) are used in the immunotherapy described herein. In some embodiments, the cells are genetically engineered to express homing sites and/or cytotoxic receptor complexes. In some embodiments, HSCs are used to take advantage of their engraftment capacity for long-term blood cell production, which can be used to target anti-cancer effectors, e.g., to counteract cancer remission. Can be a continuous source of cells. In some embodiments, this continued production helps offset allergies or depletion of other cell types due to, for example, the tumor microenvironment. In some embodiments, allogeneic hematopoietic stem cells are used, while in some embodiments, autologous hematopoietic stem cells are used. In some embodiments, HSCs are used in combination with one or more additional immune cell types described herein.

Feeder Cells for Immune Cell Expansion In some embodiments, a population of feeder cells (clonally (either populations or mixed populations).

In some embodiments, the cell line is used in co-culture with a population of immune cells to be expanded. Such cell lines are referred to herein as "stimulator cells" or "feeder cells." In some embodiments, the entire population of immune cells is to be expanded, while in some embodiments a selected subpopulation or subpopulations of immune cells are preferentially expanded. For example, in some embodiments, NK cells are preferentially expanded relative to other immune cell subpopulations. In some embodiments, two or more subpopulations are expanded together, in some embodiments simultaneously. In some embodiments, as discussed in more detail below, it is not necessary for the two or more subpopulations to be expanded to the same extent as each other. Rather, in some embodiments, one subpopulation can be grown more or less than the other so that the desired ratio of Population 1 to Population 2 is obtained at the end of the expansion period.

In some embodiments, the feeder cells are wild-type cells, but in some embodiments, the feeder cells are particularly active in immune cell proliferation and/or activation, particularly in the presence of one or more additional stimulatory signals. Genetically modified to suit the condition. As detailed below, various cell lines have been adapted to genetic modifications that can result in surface expression of specific molecules that stimulate NK activation. Certain cell types with low, restricted, or otherwise absent expression of major histocompatibility complex (MHC) I molecules may have an inhibitory effect on NK cells. As such, it is particularly useful for the simultaneous expansion of NK cells and T cells (and/or other mononuclear cells as desired, depending on the embodiment). In some embodiments, K562 cells are used, while in some embodiments K562 cells, Wilms tumor cell line HFWT, endometrial tumor cell line HHUA, melanoma cell line HMV-II, hepatoblastoma cell line HuH -6, small cell lung cancer cell line Lu-130 or Lu-134-A, neuroblastoma cell line NB19 or NB69, embryonic carcinoma testicular cell line NEC14, cervical cancer cell line TCO-2, and neuroblastoma cells The tumor cell line TNB1 is used.

In some embodiments, the cells need not be completely devoid of MHC I expression, but may express MHC I molecules at lower levels than wild-type cells. For example, in some embodiments, if wild-type cells express MHC at a level of X, the cell line used is less than 95% of X, less than 90% of The MHC may be expressed at an expression level of less than 70% of X, less than 50% of X, less than 25% of X, and any expression level between (and including) these values. In some embodiments, the stimulator cell is immortalized, eg, a cancer cell line. However, in some embodiments, the stimulator cells are primary cells. In some embodiments, the feeder cell is also a cell with reduced (or deleted) MHC II expression. In some embodiments, other cell lines (or clonal lines) capable of expressing MHC class I molecules can be used in conjunction with genetic modification of those cells to reduce or knock out MHC I expression. Such genetic modifications include blocking antibodies, interfering ligands (e.g., competitive inhibitors, non-competitive inhibitors, etc.) as well as various gene editing methods to prevent and/or reduce the expression of MHC I molecules. This can be accomplished by using techniques (eg, the Crispr/Cas-9 system), inhibitory RNA (eg, siRNA), or other molecular methods.

As described in more detail below, certain stimulatory molecules are optionally genetically engineered to be expressed by the feeder cells, and such stimulatory molecules are adapted to promote immune cell proliferation and/or activation. act. Stimulatory molecules can be expressed by feeder cells in some embodiments, but can also be provided separately. In some embodiments, the stimulating molecule can be added directly to the culture medium, with the advantage that its concentration can be easily assessed and replenished. In some embodiments, molecules include additives, such as metals, glasses, plastics, polymeric materials, particles (e.g., beads or microspheres), and/or lipids (either natural or synthetic), or stimuli. The molecule is provided immobilized or otherwise attached to some other material to which it can be attached.

Certain molecules promote proliferation, survival and/or activation of immune cells, and in some embodiments, promote proliferation, survival and/or activation of specific subpopulations of immune cells. Depending on the embodiment, the stimulatory molecule or molecules can be expressed on the surface of feeder cells used to expand the immune population (or populations). In further embodiments, one or more stimulating molecules are used to supplement the cell culture medium, either directly or attached to any additive (eg, beads). In some embodiments, the immune cell population is expanded relatively homogeneously (eg, no particular subpopulation is preferentially expanded). However, in some embodiments, more than one subpopulation is expanded. Depending on the embodiment, the two subpopulations may or may not be expanded to the same extent as each other. In some embodiments, they may be expanded to the same or similar relative extent, but at least in part with respect to the absolute number of cells of the resulting expanded immune cell population. The numbers differ based on the different starting numbers of cells in each of the two subpopulations. In some such embodiments, desired subpopulations are selectively isolated for further use, optionally before or after expansion of all immune cell populations (e.g., NK cells are separated from T cells). or vice versa).

In some embodiments, interleukin 15 (IL15) is used to promote proliferation of immune cells, such as NK cells. In some embodiments, IL15 is membrane bound on feeder cells (referred to herein as "mbIL15"). In some embodiments, IL15 is membrane bound by being bound or complexed to a transmembrane molecule or integral membrane protein. In some embodiments, the transmembrane domain of CD8α is used. In some embodiments, wild-type (eg, full-length) IL15 is expressed on or by the feeder cells. In some embodiments, IL15 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homology with full-length IL15. In some embodiments, a truncated form of IL15 is used. Further details about this and other constructs related to immune cell proliferation are described in PCT Application No. PCT/SG2018/050138, filed on March 27, 2018, the entire content of which is cited below. is hereby incorporated by reference.

In some embodiments, 4-1BB ligand (4-1BBL) is used to promote immune cell proliferation. 4-1BBL has an extracellular domain that interacts with its receptor, 4-1BB, on T cells, thereby providing costimulatory signals to T cells for survival, proliferation, and differentiation. In some embodiments, 4-1BBL is membrane bound on the feeder cell by being bound or complexed to a transmembrane molecule or integral membrane protein. In some embodiments, wild-type (eg, full-length) 4-1BBL is expressed on or by feeder cells. In some embodiments, 4-1BBL has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homology with full-length 4-1BBL. In some embodiments, a truncated form of 4-1BBL is used. In some embodiments, 4-1BBL is expressed or provided in culture other than by expression on feeder cells. In some embodiments, 4-1BBL is expressed on the surface of feeder cells (including by a clonal population of feeder cells) in combination with mbIL-15. In some embodiments, the combination of stimulatory signals provides a stimulus that induces proliferation of NK cells in the starting sample of immune cells or peripheral blood mononuclear cells.

As described herein, in some embodiments, a supplemental proliferative stimulus is also provided that functions to further enhance the proliferation of one or more subpopulations of immune cells. For example, in some embodiments, a supplemental stimulus is provided that enhances proliferation expansion of NK cells. In some embodiments, the supplemental stimulus functions to enhance T cell proliferation (while another stimulating molecule or molecules induces NK cell proliferation). In some embodiments, supplemental stimulation can be achieved with a single additional stimulation molecule. However, in some embodiments, complementary stimulation is provided by two or more stimulating molecules acting in concert with each other.

For example, in some embodiments, one or more cytokines are added to the medium in which NK cells and T cells are cultured. In some embodiments, IL12, IL18 or IL21 (or combinations thereof) are added. In some embodiments, the cytokine(s) are added by genetically engineering the feeder cells to express the cytokine(s). In some embodiments, membrane-bound cytokines are used; in other embodiments, feeder cells engineered to express soluble cytokines are used. In some embodiments, one or more soluble cytokines are added to the culture medium at the beginning of NK cell and T cell expansion, or at various times during.

In some embodiments, anti-CD3 antibodies are used to promote immune cell proliferation. In some embodiments, the anti-CD3 antibody is membrane bound to the feeder cell. In some embodiments, full-length anti-CD3 antibodies are expressed on feeder cells. However, in some embodiments, the anti-CD3 antibody comprises a single chain fragment variable region (scFv) fragment. Depending on the embodiment, antibodies can be monoclonal or polyclonal. In some embodiments, the anti-CD3 antibodies are various antigenic fragments and/or fusions selected from Fab', F(ab')2, single domain antibodies (e.g., bispecific antibodies, nanobodies). including. In some embodiments, the antibody is at least 70%, at least 75%, at least 80%, at least 85% of the group consisting of muromonab-CD3, otelixizumab, teplizumab, and bicilizumab, or one or more of muromonab-CD3, otelixizumab, teplizumab, and bicilizumab. %, at least 90%, at least 95% homology (or functional equivalence). In some embodiments, the anti-CD3 antibody is provided conjugated to additional materials that are added to the culture medium when feeder cells are co-cultured with the immune cells to be expanded. In some embodiments, this additional material is provided on the feeder cell's surface or via secretion in addition to the expression of the anti-CD3 antibody by the feeder cell.

In some embodiments, anti-CD28 antibodies are used to promote immune cell proliferation. In some embodiments, the anti-CD28 antibody is membrane bound on feeder cells. In some embodiments, full-length anti-CD28 antibodies are expressed on feeder cells. However, in some embodiments, the anti-CD28 antibody comprises a single chain fragment variable region (scFv) fragment. In some embodiments, the antibody is monoclonal or polyclonal. In some embodiments, the anti-CD28 antibodies are various antigenic fragments and/or fusions selected from Fab', F(ab')2, single domain antibodies (e.g., bispecific antibodies, nanobodies). including. In some embodiments, the antibody has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% homology (or functional equivalence) with one or more of Seralizumab and Seralizumab. selected from the group consisting of antibodies (or fragments) having In some embodiments, anti-CD28 antibodies are provided attached to additional materials that are added to the culture medium when co-cultivating the feeder cells with the immune cells to be expanded. In some embodiments, this additional material is provided on the feeder cell's surface or via secretion in addition to the expression of the anti-CD28 antibody by the feeder cell.

In some embodiments, anti-CD3 and anti-CD28 antibodies are provided together to synergistically enhance T cell proliferation. In some embodiments, anti-CD3 and anti-CD28 antibodies are provided attached to a solid support such as beads or other geometric shapes or other additional materials. In some embodiments, the population of beads has a portion bound to an anti-CD3 antibody and another portion bound to an anti-CD28 antibody, while in some embodiments the antibody is attached to the beads (or other material). ) both exist above.

Depending on the embodiment, and depending on the stimulatory molecule(s) of interest, the stimulatory molecule may be introduced at a specific point in the course of co-culture with the immune cell population, or multiple immune cell subpopulations ( for example, may be added to the culture or expressed by feeder cells). For example, rather than being constitutively expressed during co-culture, one or more stimulatory molecules may be under the control of an inducible or otherwise regulatable promoter. In this way, the desired stimulatory molecule(s) can be expressed at specific points in the proliferation and activation protocol by adding trigger molecules or stimulants to the co-culture at desired times. For example, in some embodiments it may be desirable to preferentially expand NK cells first, followed by T cells. Thus, in the early stages of proliferation, molecules that stimulate NK cells can be "turned on", and in later stages, molecules that stimulate T cells can be "turned on". As mentioned above, "on" can be the result of regulated expression of the stimulatory molecule or simply the result of the addition of the stimulatory molecule to the culture. As used herein, the terms "inducible promoter" and "regulatable promoter" shall be given their ordinary meanings and shall also be understood to mean that transcriptional activity is inhibited by the presence of certain biological or non-biological factors. shall mean a promoter that is regulated (eg, stimulated or inhibited) by. As used herein, the term "trigger molecule" or "trigger stimulus" shall be used in its conventional sense, including inducible promoters such as alcohols, tetracyclines, steroids, metals and other compounds, and elevated or low incubation temperatures. or means a chemical or physical substance or condition that acts on a regulatable promoter. Furthermore, regulatable expression of stimulatory molecules can also be utilized to reduce and/or eliminate expression of specific stimulatory molecules during the culture process. Such embodiments can improve immune cells, such as NK cells, by, for example, providing specific stimulatory signals at some point during the activation and proliferation process when NK cells are particularly susceptible to such signaling. Preferential proliferation of specific subpopulations can be promoted. In some embodiments, such an approach may result in unexpectedly robust activation and proliferation of NK cells. In still additional embodiments, the period of NK cell proliferation is extended, ultimately resulting in a larger population of activated NK cells for use in cancer immunotherapy, for example. Similarly, in some embodiments, another signal can be provided to expand a second (or additional) desired subpopulation of immune cells, such as T cells. As a result, in some embodiments, the resulting expanded cell population comprises a mixture of NK cells and T cells expanded to a different extent than the relative amounts of NK:T cells present in the donor. obtain.

In some embodiments, within a given population of feeder cells, certain individual cells within the population expand two or more desired immune cells, such as simultaneously expanding both NK cells and T cells. Particularly suitable for Identification of such individuals (eg, clones) is discussed in more detail below. In some embodiments, the identified clones are replicated such that a clonal population of feeder cells is provided, with each individual having the ability to simultaneously expand two or more types of immune cells, such as NK cells and T cells. , share the beneficial properties of the original parent cell. Thus, in some embodiments, a clonal population of feeder cells is used to expand one or more subpopulations of immune cells together, optionally simultaneously (eg, NK cells and T cells, etc.).

Donor Materials and Processing In some embodiments, methods for expanding multiple populations of immune cells are particularly advantageous because they provide sufficient flexibility to account for variations in donor cell populations. As shown in Figures 1A to 1D, the relative distribution of NK cells and T cells to other cells in baseline blood samples can vary considerably. In normal peripheral blood, the relative abundance of T cells ranges from about 10 to about 25%, while the normal T cell abundance ranges from about 2 to about 5% (e.g., https: See //www.bio-rad-antibodies.com/static/2017/flow/flow-cytometry-cell-frequency.pdf). Figure 1A shows those percentages from donor 1, where T cells are approximately 12.5%, NK cells are approximately 12.5%, and the remaining approximately 75% is composed of other cell types. Donor 2, shown in Figure 1B, had approximately 35% T cells and only approximately 5% NK cells. Donor 3 shown in Figure 1C had approximately 20% T cells and approximately 10% NK cells. Similarly, donor 4, shown in Figure ID, had approximately 25% T cells and approximately 5% NK cells. These sample data show that donor-to-donor variation is considerable and sometimes outside the "normal" expected range of NK and T cells.

As can be seen from the above data, in some embodiments the cultivation method or process can be adjusted to accommodate this variation in starting material. For example, in some embodiments, the methods and processes described herein can achieve a desired NK:T cell ratio even when starting with substantially different initial NK in the T cell population. In some embodiments, the percentage of NK cells provided as starting material can range from about 0.5% to about 35% of the peripheral blood mononuclear cells of the blood sample, including about 0.5% to about 35% of the peripheral blood mononuclear cells of the blood sample. 5% to about 1%, about 1% to about 2%, about 2% to about 4%, about 3% to about 5%, about 2% to about 5%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, and any explicitly stated values (including endpoints). ) includes any proportion of NK cells in the starting sample between In some embodiments, the percentage of NK cells in the starting sample ranges from about 2% to about 5%. Similarly, the percentage of T cells present in a blood sample may also vary. For example, in some embodiments, the percentage of T cells provided in the starting sample ranges from about 5% to about 30%, including about 5% to about 10%, about 10% to about 15%. % of T cells in the starting sample between about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, and explicitly stated values (including endpoints). Any percentage is included.

In some embodiments, due to the significant degree of proliferation that occurs during the practice of the methods and processes described herein, variations in starting materials have minimal impact on the final mixed cell population. In other words, in some embodiments, the cell populations may differ in absolute numbers of cells compared to each other, yet the resulting NK cell numbers, T cell numbers, and/or other desired The numbers of immune cell subpopulations are expanded to a significant degree so that they are clinically relevant.

As described in more detail below, the methods described herein are advantageous despite potentially significant differences in the relative abundance of NK cells versus T cells in the starting blood sample. and the process involves the coordinated expansion of both cell types (including optionally tunable additional immune cell subpopulations) to achieve the desired proportions in the final mixed population of NK cells and T cells. enable. For example, in some embodiments, the ratio of NK cells to T cells in the ultimate mixed population of cells can range from about 100:1 to about 1:100, depending on the embodiment.

Combined Expansion of Immune Cells As described above, in some embodiments, multiple subpopulations of immune cells are expanded in culture with each other. Depending on the embodiment, a single expansion protocol is used to grow multiple cell populations sequentially (e.g., initial expansion of NK cells followed by expansion of T cells) or in parallel (e.g., expansion of NK cells and T cells). (simultaneous co-culture). A single expansion protocol according to some embodiments preferentially expands one cell type over the other, e.g., even when both NK cells and T cells are present in culture at the same time. It may include multiple growth phases directed to.

As mentioned above, various types of feeder cells can be used to induce proliferation of multiple types of desired immune cells. The feeder cells used, in some embodiments, have an overall stimulatory pro-proliferative effect, but individual cells within the population potentially have varying properties compared to other members of the feeder cell population. It may be composed of a mixed population of feeder cells, including a population of feeder cells. However, in some embodiments, feeder cells with desirable properties for stimulating proliferation of multiple immune cell subpopulations are identified and clonally expanded, and the resulting clonal population of feeder cells is Used for complex proliferation of cell types. In some embodiments, two, three, four (or more) types of immune cells can be grown together. In some embodiments, the co-expanded immune cells include NK cells, cytotoxic T cells, natural killer T cells (NKT cells), effector T cells, helper T cells, memory T cells, regulatory T cells, gamma cells. delta T cells, mucosa-associated invariant T cells and/or various types of NK cell subpopulations (e.g., NK cells with stronger activating receptor signaling compared to inhibitory receptor signaling; or 2 or more of the following. A non-limiting aspect of the approach for co-proliferation is illustrated schematically in FIG. 2 and explained in more detail in the Examples below.

Depending on the embodiment, feeder cells (either a mixed population or a clonal population) are seeded into a culture vessel. The seeding density varies depending on the embodiment, and ranges, for example, from about 0.5×10 ⁵ cells/cm ² to about 5×10 ⁷ cells/cm ² , including about 1.0×10 ⁵ cells/cm 2 . ² to about 1.5×10 ⁵ cells/cm ² , about 1.5×10 ⁵ cells/cm ² to about 5×10 ⁵ cells/cm ² , about 5×10 ⁵ cells/cm ² to about 1.0 ×10 ⁶ cells/cm ² , about 1.0 × 10 ⁶ cells/cm ² to about 5 × 10 ⁶ cells/cm ² , about 5 × 10 ⁶ cells/cm ² to about 1.0 × 10 ⁷ cells/cm ² , from about 1.0×10 ⁷ cells/cm ² to about 5.0×10 ⁷ cells/cm ² , and any seeding density therebetween (including endpoints). In some embodiments, higher or lower initial seeding densities can be used depending on the expected number of immune cells that will be cultured with the feeder cells. In some embodiments, an initial seeding density of about 1 x ¹⁰ cells per ^cm2 is used.

In some embodiments, feeder cells may be pretreated with one or more agents to reduce the rate of proliferation of the feeder cells themselves. For example, in some embodiments, feeder cells are irradiated prior to culturing with the immune cell population to be expanded. In some embodiments, instead of or as a supplement to radiation, feeder cells are treated with an anti-proliferative agent, such as mitomycin C, for example.

As described above, in some embodiments certain individual cells within a larger population of feeder cells have properties desirable for the proliferation of more than one immune cell subtype. In some embodiments, the feeder cells used to expand the immune cell population are clonally derived populations of feeder cells. In other words, individual clones identified as having properties desirable for the proliferation of one or more types of immune cells are themselves clonally expanded so that the resulting population all share those beneficial properties. become. In some embodiments, exposing immune cells to be expanded to a clonal population of feeder cells can advantageously reduce variability in the final expanded immune cell population. As a result, in some embodiments greater product consistency is achieved.

Also, as described above, in some embodiments, the growth culture medium can be supplemented with one or more stimulating molecules. In some embodiments, such molecules promote the general health of the feeder cell layer, stimulate the expanded immune cell population, and improve the health of the immune cell population before, during, or after expansion. or some combination of these to work. In some embodiments, interleukin-2 (IL2) is used to supplement the culture medium. In some embodiments, the concentration of interleukin 2 may vary over the period of time during which immune cell proliferation takes place. The IL2 concentration is about 10 units/mL to about 20 units/mL, about 20 units/mL to about 40 units/mL, about 40 units/mL to about 60 units/mL, about 60 units/mL to about 80 units/mL. mL, about 80 units/mL to about 100 units/mL, about 100 units/mL to about 150 units/mL, about 150 units/mL to about 200 units/mL, about 200 units/mL to about 300 units/mL, about 300 units/mL to about 400 units/mL, about 400 units/mL to about 500 units/mL, about 500 units/mL to about 750 units/mL, about 750 units/mL to about 1000 units/mL, and these It can range from about 10 Units/mL to about 1000 Units/mL, including any concentration therebetween (including endpoints). Stimulation with IL-2 can be repeated multiple times per expansion protocol, according to certain embodiments. Furthermore, not only restimulation can be performed, but stimulation at various concentrations can be performed at different times during the expansion protocol. In some embodiments, other interleukins or other stimulatory molecules can be used in the processes described herein.

In some embodiments, the period of cell expansion is within the range of about 4 days to about 20 days. In some embodiments, different immune cell subpopulations proliferate at different rates depending on the period of culture with feeder cells. For example, certain cell types proliferate more rapidly at early time points, while other cell types proliferate more rapidly at later time points. Thus, if a proportion of different immune cell subtypes within the final immune cell population is desired, the timing of expansion can be adjusted accordingly. In some embodiments, growth conditions are employed for about 4-6 days, about 6-8 days, about 8-10 days, about 10-14 days, or about 14-21 days. In some embodiments, increasing the expansion culture time can increase the overall absolute number of proliferating cells. However, in some embodiments, a preliminary enrichment step is performed such that the starting number of each proliferating immune cell subtype is greater than that in the original collected sample, and the proliferation time is increased. It can be shortened in an adjustable manner.

In some embodiments, the ratio of feeder cells to expanded immune cells can be adjusted depending on the final desired result and the composition of the expanded immune cell population. For example, in some embodiments, a ratio of feeder cells:"target" cells of about 5:1 is used. In some embodiments, a ratio of 1:1 is used, and in further embodiments, about 1:10, 1:20, 1:50, 1:100, 1:1,000, 1:10,000, 1: 50,000, 1:100,000, 100,000:1, 50,000:1, 10,000:1, 1,000:1, 100:1, 50:1, 20:1, 10:1, and all ratio ranges and endpoints therebetween listed. In some embodiments, a combination of cell types is used (e.g., a combination of K562 and one or more additional cell types) such that NK cell activation and/or proliferation is independent of any single cell type. greater than could be achieved when used alone (e.g., as a result of synergistic effects between cell types). In some such embodiments, MHC I expression need not necessarily be reduced and/or deleted in each of the cell lines used in the combination. In some embodiments, the relative frequency of one cell type in combination with another cell type can be varied to maximize proliferation and activation of a desired immune cell population. For example, when using two feeder cell populations, the relative frequencies are 1:10, 1:20, 1:50, 1:100, 1:1,000, 1:10,000, 1:50,000, 1 :100,000, 100,000:1, 50,000:1, 10,000:1, 1,000:1, 100:1, 50:1, 20:1, 10:1, and those listed. may include all ratio ranges and endpoints between.

Similarly, in certain embodiments, adjusting the initial ratio of immune cell subtypes relative to each other, given that multiple immune cell subpopulations are expanded in combination, according to some embodiments described herein. Can be done. For example, as mentioned above, the relative abundance of NK cells to T cells in a given blood sample can vary between donors. Thus, in some embodiments, a desired input ratio of cell type 1 to cell type 2 is achieved when one cell type is in relative excess compared to the other cell type in a particular sample. In order to do so, an enrichment step or a depletion step can be carried out. For example, and without limitation, consider a blood sample drawn from a donor in which T cells make up about 25% of the collected cell population and NK cells make up about 5% of the collected cell population. Try. In such embodiments, a preliminary expansion step can be used to increase the number of NK cells such that the absolute number of NK cells is approximately equivalent to the absolute number of T cells from the collected blood sample. NK cells and T cells are then cultured with feeder cells at equivalent NK cell and T cell numbers to induce proliferation and ultimately adjust the ratio of NK cells to T cells in the proliferating cell population to the desired ratio. For example, it can be set to 1:1. Another approach is to deplete large populations of T cells to reduce T cell numbers to roughly equivalent numbers of NK cells, and then transfer equal numbers of NK cells and T cells to feeder cells, as described herein. can be co-cultured with

Similar to adjusting the ratio of feeder cells to immune cells to be expanded, the relative proportions of different types of immune cells to be expanded within a given culture can be adjusted. For example, the ratio of NK cells to T cells at the initial stage of a given culture may be about 10,000:1, about 7500:1, about 5000:1, about 2500:1, about 2000:1, about 1500:1, about 1000:1, about 750:1, about 500:1, about 250:1, about 100:1, about 75:10, about 50:1, about 25:1, about 15:1, about 10:1, about 5:1, about 1:1, about 1:5, about 1:10, about 1:25, about 1:50, about 1:75, about 1:100, about 1:250, about 1:500, about 1:750, about 1:1000, about 1:1500, about 1:2000, about 1:2500, about 1:5000, about 1:7500, about 1:10,000, and between the listed The range can be from about 10,000:1 to about 1:10,000 cells, including all ratios and endpoints.

As described in more detail below, in some embodiments, preferential proliferation of one of the multiple types of immune cells being cultured in combination with other types of immune cells as well as feeder cells is achieved. Use a supplemental stimulus that is intended to induce. In some embodiments, the supplemental stimulus is added at the beginning of the proliferation phase and can be considered essentially part of the original stimulus environment. In a further embodiment, the introduction of the co-stimulus is delayed, e.g. a first phase of proliferation of a particular cell type is followed by a second phase of proliferation of a different cell type, the latter being induced by the introduction of the co-stimulus. can do. In some embodiments, the supplementary step is performed at about 3 days, about 4 days, about 5 days, about 7 days, about 10 days, about 14 days after the start of the co-culture, or at any time between after the start of the co-culture. A stimulus can be administered. In other words, supplemental stimulation is applied after approximately 0% of the expansion protocol is completed, after approximately 10% of the expansion protocol is completed, after approximately 25% of the expansion protocol is completed, and after approximately 50% of the expansion protocol is completed. or after approximately 75% of the expansion protocol is complete.

In some embodiments, as discussed in more detail below, it is not necessary for the two or more subpopulations to be expanded to the same extent as each other. Rather, in some embodiments, one subpopulation can be expanded more or less than the other subpopulations such that a desired ratio of Population 1 to Population 2 is obtained at the end of the expansion period. Desired ratios after expansion include, for example, about 10,000:1, about 7500:1, about 5000:1, about 2500:1, about 2000:1, about 1500:1, about 1000:1, about 750:1. 1, about 500:1, about 250:1, about 100:1, about 75:10, about 50:1, about 25:1, about 15:1, about 10:1, about 5:1, about 1: 1, about 1:5, about 1:10, about 1:25, about 1:50, about 1:75, about 1:100, about 1:250, about 1:500, about 1:750, about 1: 1000, about 1:1500, about 1:2000, about 1:2500, about 1:5000, about 1:7500, about 1:10,000, and all ratios and endpoints therebetween recited. , the ratio of NK cells to T cells can range from about 10,000:1 to about 1:10,000 cells.

Genetic Manipulation of Expanded Cells In some embodiments, expanded immune cells can be administered to a subject in need of immunotherapy without further modification. In some embodiments, the expanded immune cell population is mixed with a pharmaceutically acceptable carrier prior to administration. As detailed below, administration can be by any of a variety of routes and can be in an autologous or allogeneic setting. In some embodiments, the expanded cells are genetically engineered to enhance their cytotoxic effect on target tumor cells. For example, NK cells and/or T cells are genetically engineered to express chimeric receptors that enhance the ability of said NK cells and/or T cells to recognize, for example, tumor cells and/or to exert a cytotoxic effect. can do. Cells may be of a variety of types, including those described in detail in PCT Patent Application No. PCT/US2018/024650, filed March 27, 2018, the entire contents of which are incorporated herein by reference. It can be genetically engineered in different ways.

Cytotoxic Receptor Complexes A variety of cytotoxic receptors can be expressed by the NK/T cells described herein. In some embodiments, the receptor comprises a chimeric antigen receptor (CAR). In some embodiments, the CAR is directed against one or more of the target tumor markers described herein. In some embodiments, a CAR is encoded by a particular polynucleotide encoding an amino acid sequence. In some embodiments, polynucleotides encoding anti-CD19 moieties/CD8 hinge/CD8TM/OX40/CD3ζ chimeric antigen receptor complexes are provided. The polynucleotide comprises or consists of an anti-CD19 scFv, a CD8a hinge, a CD8a transmembrane domain, an OX40 domain and a CD3ζ domain. In some embodiments, the chimeric antigen receptor further comprises mbIL15. In such embodiments, the polynucleotide comprises or consists of an anti-CD19 scFv, CD8a hinge, CD8a transmembrane domain, OX40 domain, CD3zeta domain, 2A cleavage site and mbIL-15 domain, as described herein. Become. In some embodiments, the receptor complex is encoded by a nucleic acid molecule having the sequence of SEQ ID NO:1. In some embodiments, the nucleic acid sequence encoding the NK19 chimeric antigen receptor is at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% different from SEQ ID NO: 1. % or at least about 99% sequence identity, homology and/or functional equivalence. In some embodiments, the chimeric receptor comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the NK19 chimeric antigen receptor is at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% different from SEQ ID NO:2. % sequence identity, homology and/or functional equivalence. In some embodiments, the CD19 scFv does not include a Flag tag. In some embodiments, humanized and/or codon-optimized CARs are used. Further examples of tumor binding constructs that may be used in accordance with embodiments described herein include PCT Patent Application No. PCT/US2018/024650 filed March 27, 2018, United States Provisional Patent Application No. PCT/US2018/024650 filed March 5, 2019 No. 62/814,180, U.S. Provisional Patent Application No. 62/895,910 filed September 4, 2019, U.S. Provisional Patent Application No. 62/932,165 filed November 7, 2019, and No. 62/924,967, filed October 23, 2013, each of which is incorporated herein by reference in its entirety.

ADMINISTRATION AND DOSAGE Herein, a method of treating a subject with cancer, comprising administering to the subject a composition comprising an expanded mixed population of immune cells (e.g., a mixture of NK cells and T cells). Further provided is a method comprising: In some embodiments, the proliferating cells are genetically engineered to express a cytotoxic receptor complex (eg, a chimeric receptor or chimeric antigen receptor). Also provided is the use of such genetically modified immune cells to treat cancer. In certain embodiments, treatment of a subject with an expanded mixed cell population described herein achieves, for example, one, two, three, four or more of the following effects: (i) (ii) reduce the duration of symptoms associated with a disease; (iii) protect against progression of a disease or symptoms associated therewith; (iv) reduce the severity of a disease or symptoms associated therewith; (iv) reduce the severity of a disease or symptoms associated therewith; (v) protection against the occurrence or development of symptoms associated with the disease; (vi) protection against recurrence of symptoms associated with the disease; (vii) reduction in subject hospitalization; (viii) reduction in length of hospital stay; (ix) prolong the survival of subjects with the disease; (x) reduce the number of symptoms associated with the disease; (xi) enhance, improve, supplement, complement or enhance the preventive or therapeutic effects of another treatment; xii) Biphasic (e.g. immediate and long-acting) cytotoxic effects on target cells. Each of these comparisons is for example to a different treatment for the disease, including cell-based immunotherapy for the disease using cells that do not express the constructs described herein.

Administration can be by a variety of routes including, but not limited to, intravenous, intraarterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal and/or local delivery to the affected tissue. The dosage of the expanded mixed population of immune cells can be readily determined for a given subject based on the subject's body weight, the type and condition of the disease, and the desired aggressiveness of the treatment. , depending on the embodiment, ranges from about 10 ⁵ cells per kg to about 10 ¹² cells per kg (e.g., 10 ⁵ -10 ⁷ , 10 ⁷ -10 ¹⁰ , 10 ¹⁰ -10 ¹² and overlapping ranges therein). . In some embodiments, the mixed population of immune cells has genetically engineered proportions of various cell types. For example, in a mixed population of NK cells and T cells, the NK:T cell ratio may be about 1000:1, about 750:1, about 500:1, about 250:1, about 100:1, about 75:1, about 50:1, about 25:1, about 10:1, about 5:1, about 2:1, about 1:1, about 1:2, about 1:5, about 1:10, about 1:25, A range of about 1:50, about 1:75, about 1:100, about 1:250, about 1:500, about 1:750 or about 1:1000 (or any ratio therebetween listed) It can be. In some embodiments, the ratio of NK cells to T cells is expressed in terms of target cell number. For example, in some embodiments, the NK:target cell ratio is about 1:1, 1:2, 1:4, 1:8, or 1:10, and the T cell:target cell ratio is about 1. :2, 1:4, 1:8 or 1:10. In some embodiments, a combination of these ratios is used, eg, 1:4 NK cells:target cells and 1:8 T cells:target cells.

Similarly, in some embodiments, three (or more) different types or subtypes of cells can be used in a mixed population. The ratio therein ranges from about 1:1:1, where the three subtypes of cells are approximately equal to each other, to about 1000:1:1, or about 1:1, where one cell type predominates over the other. : The range can be up to 1000. Similarly, depending on the embodiment, intermediate ratios may be used, such as a ratio of about 10:5:1, or about 1:5:10, or about 5:1:10. Additionally, subpopulations can be combined in a range of ratios with each other. For example, if a population consists of two cell types, predetermined parameters are used to determine the population size of the first cell type, and this is used to determine the population size of the second (or third, fourth, etc.) cell type. population size can be determined. As a non-limiting example, the cytotoxicity of NK cells to a target cell population can be calculated to determine the number of NK cells to be added to the population, which can be used in subsequent calculations to calculate the number of T cells to be added to the population. used as input value. If a particularly effective population of NK cells is used, NK cells may make up, for example, 25% of the mixed population, with gamma delta T cells making up the remainder (if a third or fourth cell type is added) The rest will be less).

In some embodiments, dose escalation regimens are also provided. In some embodiments, for example, a range of immune cells (eg, NK cells and T cells) between about 1 x 10 ⁶ cells/kg and about 1 x 10 ⁸ cells/kg is administered. Depending on the embodiment, various types of cancer can be treated. In some embodiments, hepatocellular carcinoma is treated. Additional aspects provided herein include the treatment or prevention of the following non-limiting examples of cancer, including acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenal Cortical cancer, Kaposi's sarcoma, lymphoma, gastrointestinal cancer, appendiceal cancer, central nervous system cancer, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone tumor, brain tumor (astrocytoma, spinal cord tumor, brainstem glioma, glioblastoma) breast cancer, bronchial tumors, Burkitt's lymphoma, cervical cancer, colorectal cancer, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative disease, ductal cancer, endometrial cancer, esophageal cancer, gastric cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell leukemia, renal cell carcinoma, leukemia, oral cancer , nasopharyngeal cancer, liver cancer, lung cancer (including but not limited to non-small cell lung cancer (NSCLC) and small cell lung cancer), pancreatic cancer, intestinal cancer, lymphoma, melanoma, eye cancer, ovarian cancer, pancreatic cancer, Includes, but is not limited to, prostate cancer, pituitary cancer, uterine cancer and vaginal cancer.

As discussed in more detail herein, various genetically modified cells can be used to target a number of different tumor types. Depending on the embodiment, solid or free-floating tumors can be targeted. In some embodiments, the genetically modified cells described herein are used to treat hematologic tumors. For example, in some embodiments, acute myeloid leukemia (AML)
) cells are targeted. AML results from clonal proliferation of blasts in the bone marrow, blood or other tissues. AML accounts for approximately 3% of all cancers, and the incidence of AML increases with age. For patients under 65 years of age, complete remission is achieved in approximately 70-80% of patients, and the overall survival rate is approximately 35%. In patients over 65 years of age, the number decreases, with 40-60% of cases remitting and overall survival decreasing to about 5%. Shown in the immunohistochemistry panel of Figure 2 is a bone marrow aspirate that stained positively for leukemic blasts.

A variety of markers can be targeted using the genetically modified cells described herein. CD19, CD123, NY-ESO, BCMA, etc. are expressed in many cancers. In some embodiments, particularly in AML, CD123 and NKG2D ligand are upregulated. In certain patients (eg, AML patients), surface expression of NKG2D ligands (including, but not limited to, MICA, MICB, ULPB1, ULPB2, ULPB3) is upregulated. Similarly, in some of these patients, a significant proportion (eg, about 80% or more) of blast cells, leukemic stem cells, and endothelial cells are CD123 positive. Accordingly, in some embodiments, immune cells are genetically engineered to target NKG2D ligands and/or CD123.

The following list includes non-limiting examples of antigens that can be bound by the binding sites/activation sites described herein: Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic Antigen (CEA), CD123, CD19, Claudin 6, NY-ESO, GD-2, GD-3, Dectin-1, Cancer Antigen-125 (CA-125), CA19-9, Calretinin, MUC-1, Upper Encapsular membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), total cystic disease fluid protein (GCDFP-15), HMB-45 antigen, high molecular weight melanoma-associated antigen (HMW-MAA), protein melan A (melanoma antigen recognized by T lymphocytes; MART-1), Myo-Dol, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, dimer of pyruvate kinase isoenzyme type M2 (tumor M2-PK), Abnormal ras protein, abnormal p53 protein, fuc-GMl, GM2 (Embryonic tumor antigen-immunogenicity-1; OFA-I-1); GD2 (OFA-I-2), GM3, GD3, α-actinin -4, Bage-1, BCR-ABL, BCR-Abl fusion protein, β-catenin, CA15-3 (CA27.29/BCAA), CA195, CA242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, Coa-1, dek-can fusion protein, EBNA, EF2, Epstein Van virus antigen, ETV6-AML1 fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, 3 , neo-PAP, myosin class I, OS-9, pml-RARa fusion protein, PTPRK, triose phosphate isomerase, Gage3,4,5,6,7, GnTV, Herv-K-mel, Lage-1, NA-88 , NY-Eso-l/Lage-2, SP17, SSX-2, TRP2-Int2, gplOO (Pmel17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, RAGE, GAGE-1, GAGE-2, pl5 (58), RAGE, SCP-1, Hom/Mel-40, PRAME, HER-2/neu, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, human papillomavirus (HPV) Antigens E6 and E7, TSP-180, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72-4, CA19-9, CA72-4, CAM17.1, NuMa, 13-catenin, Mum -1, pi6, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68/KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD22, CD27, CD30, CD70, EGFRvIII ( epidermal growth factor receptor variant III), sperm protein 17 (Spl7), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma alternative reading frame protein), Trp-p8, STEAP1 (prostatic acid phosphatase), Transmembrane epithelial antigen 1), integrin ανβ3 (CD61), galactin, Ral-B, FLT3, CD70, DLL#, CD5, GUCY2C, EGFR, KREMEN2, PSMA, ALPPL2, CLDN6, CLDN18, GPR143, GRM8, LPAR3, GD2 , ADAM12, LECT1, TMEM186, etc.

EXAMPLES The following is a non-limiting description of the test methods and materials used in the examples below.

Example 1 - K562 Clone Sequencing As noted above, in some embodiments, feeder cell lines are used to expand both NK cells and T cells from donor blood samples. This example was performed to distinguish from a population of modified K562.

In view of the above, in some embodiments, preliminary steps are performed to adjust the relative proportions of particular cell types within a population of immune cells isolated from a donor. For example, in some embodiments, a portion of T cells can be selectively removed from the initial sample to prevent T cells from overwhelmingly outnumbering NK cells during the expansion process. In some embodiments, any ratio of NK cells to T cells can be achieved by such preliminary steps. For example, in some embodiments, a 1:1 NK:T cell ratio is used at the initiation of expansion. In further embodiments, NK cells can be enriched with a starting material pool for T cells, if desired. For example, in some embodiments, this includes about 1000:1, about 750:1, about 500:1, about 250:1, about 100:1, about 75:1, about 50:1, about 25: A starting ratio of NK:T cells of about 1000:1 to about 10:1 is used, including 1, about 10:1, or any ratio therebetween as explicitly stated.

As shown in Figure 2, limiting dilution was applied to a population of K562 cells to generate a smaller number of individual K562 clones for screening. Starting K562 cells were genetically engineered to express 4-1BBL as well as membrane-bound IL15. In some embodiments, K562 cells can be further modified to express one or more additional stimulatory molecules, such as, for example, IL12 and/or IL18. In a further embodiment, the K562 cells need not be modified, but are, if desired, and the culture medium is supplemented with one or more such additional stimulating molecules. Detailed information regarding such K562 cells is described in PCT Application No. PCT/SG2018/050138 filed on March 27, 2018, the entire content of which is incorporated herein by reference. In this particular example, which also applies to other embodiments, the K562-41BBL-mbIL15 clone was also genetically engineered to express the OKT3 antibody single chain fragment variable (scFv), a murine monoclonal antibody of the immunoglobulin IgG2a isotype. It was done. This antibody targets CD3, which is part of a multimolecular complex that exists only on mature T cells. The interaction between T cells and OKT3 activates T cells. Since the mouse OKT3 antibody reacts with an epitope on the epsilon subunit of the human CD3 complex, it can enhance the ability of K562 cells, which stimulate NK cells, to stimulate T cells. By cloning at limited dilutions, different subclones can be identified that may express different amounts of the introduced genes (here mbIL15, 4-1BBL and scFv OKT3). In particular, variability in the expression of genes introduced into feeder cells can result in different characteristics with respect to immune cell proliferation. In other words, a given clone can more robustly (eg, efficiently) expand NK cells than T cells. In some embodiments, a given clone is capable of more robustly (eg, efficiently) expanding T cells than NK cells. In yet another aspect, a given clone may result in a proliferating population of immune cells that are generally healthier, able to survive longer, more effective against target cells, and the like. Identification of clones with specific growth characteristics is useful in tailoring the final composition of mixed cell populations. For example, if you know that a particular clone proliferates NK cells twice as fast as T cells and you want to form a mixed cell population at a ratio of approximately 2:1, use this clone to form a starting population. It can be grown to its desired ratio. In some embodiments, two or more clones can be used to independently expand immune cells, and the final mixed cell population can be a combination of independently expanded immune cell populations. In such approaches, given clones can be selected based on their ability to preferentially proliferate particular immune cell types. For example, in the case of a mixed population of NK cells, cytotoxic T cells, and gamma delta T cells, three clones can be utilized to expand one of these three populations, and the selected clone selected for having beneficial properties for propagating one of the types.

Although 24 clones were identified and clonally expanded in this example (each clone was clonally expanded for 3 weeks), larger numbers of clones can easily be prepared. After clonal expansion, K562 cells (pretreated with mitomycin C) were plated at 5 x ¹⁰ cells per well (24-well plates). Gas permeable membrane culture vessels were used in this study (G-Rex, Wilson Wolf, ST Paul Minnesota). In other embodiments, standard culture vessels can also be used. 5×10 ⁵ immune cells (NK cells and T cells) from each of the 4 donors were plated on day 0, and each sample was plated with the clonally expanded K562 population. 40 U/mL of IL-2 was added to the culture solution on days 0 and 4, and a high concentration of 400 U/mL of IL-2 was added on the 6th day of co-culture. Co-culture was carried out for a total of 20 days.

Figures 3A-3B show data related to the proliferation of NK cells (Figure 3A) and T cells (Figure 3B). Together these data indicate that both NK cells and T cells can be expanded using the clonally derived K562 cell population as feeder cells. As shown in Figure 3A, certain clones showed particularly effective NK cell proliferation. In particular, FIG. 3A shows that clone K562 feeder cells induce NK cell proliferation ranging from about 15-fold to about 90-fold, depending on the clone and donor used. Figure 3B shows that T cell proliferation was strongly stimulated by all K562 clones tested, with expansions ranging from approximately 50-fold to approximately 350-fold depending on the clone and donor tested. T cell doubling time was approximately 24 hours or less for all donors. These data demonstrate that it is not only possible to expand both NK cells and T cells in the same culture, but also that robust expansion of individual cell types can occur.

Figures 4A-4B show data when a mixed population of NK and T cells from each of four donors was expanded over 7 days using each of 24 K562 clones as feeder cells. There is. In short, the composition of the proliferated cell population largely reflects the ratio of NK cells to T cells. As shown in Figure 4A, post-expanded NK cells ranged from approximately 2% to approximately 23% of the cells in the parental sample (e.g., compared to the same percentage from the original donor sample shown in Figures 1A-1D). to roughly compare the total number of cells present in the total population after expansion). Figure 4B shows similar data for T cells, with percentages ranging from about 65% to about 95% of the parental population. In general, the respective NK:T cell ratios after expansion reflect the relative ratios from the original samples (eg, those of FIGS. 1A-1D). Therefore, in some embodiments, the starting NK cell to T cell ratio can be used as a guideline for the final NK cell to T cell ratio that occurs after expansion. As described above, in certain embodiments, initial adjustment of cell ratios before initiating proliferation can be used to direct the final ratios of different cell types to each other after proliferation is complete. Furthermore, as detailed below, changes in the method can be performed during expansion to further fine-tune the proportion of cells generated after expansion.

Example 2 - Dual Modality Expansion of Mixed Cell Populations
As mentioned above, NK cells and T cells can be expanded together in culture to obtain a ratio of cell types that approximately reflects the starting ratio of cells from the initial blood sample. This example was performed to implement a dual modality expansion protocol that allows for adjustment of the relative proliferation rates of NK cell and T cell subpopulations.

Figure 5 provides an overview of the study design performed. The expansion timeline and initial seeding of cells is substantially similar to that described above, but cultures are exposed to supplemental growth stimuli at various times after initiation of co-culture, or as described above. (as a control). The schematic diagram in Figure 5 shows that supplementary growth stimuli were added in the matrix covering the first 6 days of co-culture. In this example, as described above, the supplemental growth stimulation includes adding CD3/CD28 Dynabeads to the culture medium to stimulate T cell proliferation. In further embodiments, these (or other) supplemental growth stimuli can be added - for example, one or more antibodies can be added directly to stimulate T cell proliferation. Cultures are supplemented with IL-2 at various time points, as shown in the schematic, and FACS analysis is used to assess growth factors and NK:T cell ratios, as well as transduction efficiency during and after expansion. .

Figures 6A-6B show data related to the proliferation of NK cells (Figure 6A) and T cells (Figure 6B) measured after 7 days of culture, with CD3/CD28 polystyrene beads (Fig. Beads coated with anti-CD3 antibody and anti-CD28 antibody) were added as a supplementary proliferation stimulus for T cells. In other words, the column on day 2 represents the number of NK cells or T cells when CD3/CD28 polystyrene beads were added on day 2 and proliferation was measured on day 7 (5 days of incubation with beads). It is a relative amount of growth. Figure 6A shows that initially adding CD3/CD28 polystyrene beads had no negative effect on NK cell proliferation. As shown in the "no beads" data point, the fold change expansion was comparable to that shown in the data in Figure 3A. Addition of CD3/CD28 polystyrene beads between day 0 (start of co-culture) and day 6 of culture did not result in any significant change in NK cell proliferation in any of the four donors. Each population still expanded approximately 15-fold to peak approximately 85-90 times. Furthermore, NK cell proliferation was relatively consistent over time within a given donor. For example, donor 669 consistently showed an approximately 40-fold increase in NK cells in the absence of beads or when beads were added at any time within the first 6 days of culture. In some embodiments, the addition of CD3/CD28 polystyrene beads may actually enhance NK cell proliferation rather than simply inhibiting the process.

Figure 6B shows relevant data regarding T cell proliferation when adding CD3/CD28 polystyrene beads as a supplemental proliferation stimulus. In contrast to NK cells, T cell proliferation differs markedly depending on the presence or absence of CD3/CD28 polystyrene beads and the timing of addition of CD3/CD28 polystyrene beads. As shown in the "No Beads" section, in the absence of CD3/CD28 polystyrene beads, little to no T cell proliferation occurred when measured on day 7. As can be seen from the general shape of the curves in Figure 6B, T cells proliferated more robustly when CD3/CD28 polystyrene beads were added early in the culture process. For example, addition of CD3/CD28 polystyrene beads at the beginning of co-culture with NK cells and K562 feeder cells resulted in a nearly 100-fold increase in T cells in all donors. This induction was reduced to an approximately 75-fold increase when beads were added after 1 day of culture. Relative proliferation continued to decline with later additions of beads. Thus, in some embodiments, the supplemental growth stimulus is provided at an early point in time, e.g., at the beginning of co-culture, within about 6 hours of co-culture, within about 12 hours of co-culture, about 18 hours of co-culture. Maximal T cell proliferation occurs when present within or within about 24 hours of co-culture. In some embodiments, the relative proliferation of T cells is fine-tuned based on when to provide supplemental proliferation stimulation to achieve the desired NK:T cell ratio in the final expanded population. be able to. In other words, if the desired final population has a large proportion of T cells compared to NK cells, supplemental T cell proliferation stimulation can be provided relatively early in the co-culture process. Alternatively, if the desired final cell population is more evenly distributed between NK cells and T cells, the addition of supplemental T cell proliferation stimuli can be delayed until later in the co-culture process. . Thus, depending on the embodiment, the culture conditions and relative timing of including (or excluding) supplemental stimuli for expanding T cells can be fine-tuned to increase the number of cells in the final expanded immune cell population. The final ratio can be controlled.

As described above, in some embodiments, the expanded population of immune cells (e.g., NK cells and T cells) is provided on an "as is" basis and/or after incorporation into a pharmaceutically acceptable carrier. It can be administered to a subject in need of treatment. However, in some embodiments, the cells are genetically engineered with chimeric constructs to increase cytotoxicity toward target cells (e.g., PCT Patent Application No. PCT/US2018 filed March 27, 2018). 024650, the entire contents of which are incorporated herein by reference). To assess the capacity of the expanded NK and T cell populations, tests were performed to determine their ability to transduce cells.

A gamma retrovirus encoding green fluorescent protein (GFP) was introduced into co-proliferated NK cells and T cells. Cells were transduced on day 7 and transduction efficiency was measured by FACS on days 10 and 14. The data are shown in Figures 7A to 7D. Figure 7A shows the presence or absence of CD3/CD28 polystyrene beads (“no beads” control) or the time of bead addition relative to the start of co-culture with T cells and K562 feeder cells in cultures at day 10. The ratio of GFP-positive NK cells to total NK cells is shown for each donor. As shown, neither the presence or absence of beads nor the timing of their addition seemed to have a significant effect on the efficiency of NK cell introduction on day 10. Overall, for all four donors, transduction efficiency ranged from about 80% to about 95% (compared to the starting population of NK cells in the original blood sample collected). Similarly, T cells were also easily transduced on day 10, as shown in Figure 7B. Although the transduction efficiency at day 10 appears to be slightly lower compared to NK cells, for all four donors the efficiency ranged from approximately 25% to approximately 65% (original blood samples collected). compared to the starting population of NK cells).

Analysis of transduction efficiency up to 14 days after the start of co-culture shows that the stability of transgene expression in both NK and T cells after transfection is dependent on initial donor variability and the presence or absence of CD3/CD28 beads or CD3 /CD28 beads were shown to be maintained until at least the second week of culture, regardless of the timing of addition. These data are shown in Figure 7C (for NK cells) and Figure 7D (for T cells). Figure 7C shows that for all donors, regardless of the addition or timing of addition of CD3/CD28 beads to expand T cells, transduction efficiency remains between approximately 78% and 95%. Similar to T cells at day 10 of co-culture, at day 14 T cell transduction rates ranged from about 30% to about 70% for all four donors. Together, these data demonstrate that not only do both NK cells and T cells proliferate readily in the presence of each other, but that each cell type is susceptible to transduction, indicating that target cells It has been shown that cells can be genetically engineered to enhance their cell-promoting effects.

As mentioned above, the NK:T cell ratio can be adjusted based on the conditions used during the expansion period (also to adjust the starting cell population ratio or after expansion). (You may also take specific steps.) To examine how the ratio of NK/T cells changes depending on the timing of addition of CD3/CD28 beads, the ratio of each cell type in the final culture medium was measured on days 7 and 14, and the results are shown in FIGS. 8A to 8D. Figure 8A shows NK cell count data at day 7 (“no beads control”, or each donor cell population was co-cultured with K562-41BB-mbIL15 cells and CD3/CD28 beads were added at various time points). added). Consistent with the relative proliferation of each cell type, Figure 8A shows an interesting trend in which the proportion of NK cells in the final culture increases as CD3/CD28 beads are added later in the expansion period. Interestingly, as shown in Figure 8C, the proportion of NK cells in the final culture continued to increase at day 14, with some donors having almost 100% of the cells present in the initial sample as NK cells. Met. This is also noteworthy in that the evaluation on day 14 was post-transduction. This indicates that NK cells do not lose their proliferative capacity as a result of transduction with exogenous vectors, whereas T cells may require earlier stimulation or become exhausted during the culture period. It shows.

In contrast to NK cells, addition of CD3/CD28 beads during the culture period reduces the proportion of T cells. T cell populations exposed to beads later in the co-culture period show a decrease in the overall T cell percentage. This is probably due to the shortened period that these T cell populations were exposed to the stimulatory effects of CD3/CD28 beads. The data in FIG. 8D also shows that the percentage of T cells decreases during the second week of culture, as represented by the data on day 14. These data show that the T cell percentage at day 14 peaked at approximately 70% of the parental cell number, but fell to as low as 1-2% (e.g., CD3/CD28 For samples where beads were added on day 4, 5 or 6 of co-culture). This may reflect the effect of a shorter period of stimulation of T cells and/or the removal of a large number of stimulatory beads during the transduction protocol. It may also reflect T cell exhaustion. However, when these data are combined, it can be seen that during the two week co-culture period, NK cells tend to increase and T cells tend to decrease. Knowing this general behavior, it is possible to adjust culture conditions to optimize the rate of increase in NK cells and the rate of decrease in T cells in order to obtain a mixed cell population after expansion with the desired properties. can.

Taken together, this test data (similar to Example 1) indicates that NK cells and T cells can be successfully co-proliferated according to some embodiments described herein. According to some embodiments, NK cells and T cells are obtained by co-cultivating the two immune cell populations with K562 cells, which in some embodiments are modified to express 4-1BBL and/or mbIL15. They can be co-propagated at the same time. In yet additional embodiments, the K562 cells are further modified to present specific T cell stimulatory signals, such as, for example, antibodies or other molecules that interact with T cells and stimulate their proliferation. In some embodiments, K562 cells are engineered to express OKT3 antibodies. Alternatively, or in addition, according to some embodiments, separate T cell stimulation is provided. For example, according to some embodiments, anti-CD3 antibodies and/or anti-CD28 antibodies are provided to stimulate T cell proliferation. Thus, these data indicate that there are multiple different ways in which NK cells and T cells can be co-proliferated. Advantageously, the choice of method used can be adjusted depending on the desired ratio of NK cells to T cells in the final expanded immune cell population. For example, the data described herein shows that the K562.41BBL.mbIL15.scFv-OKT3 clone is able to expand NK cells, while also reliably expanding T cells. Such an approach is therefore suitable when a high T cell:NK cell ratio is desired. Additionally, certain K562 clones have been confirmed to yield high NK ratios after expansion. Therefore, in certain embodiments, such clones can be used when it is desired to have a more balanced ratio of NK cells and T cells. Additionally, if you want more control over the final NK/T cell ratio, rather than incorporating T cell stimulation into the feeder cell line, you can use a feeder cell line that is genetically engineered to preferentially proliferate NK cells. Can be combined with another complementary T cell proliferation stimulus. According to some embodiments, this can be accomplished by combining feeder cells, such as K562.41BBL.mbIL15 feeder cells, with, for example, CD3/CD28 T cell stimulation beads. The timing of introducing additional T cell stimulation can also be used to fine-tune the final ratio of NK cells to T cells. For example, higher NK:T ratios can be achieved by introducing T cell stimulation later in the co-culture process, whereas higher T:NK ratios can be achieved by introducing separate T cell stimulation earlier in the co-culture process. This can be achieved by doing. As such, the expansion methods described herein are advantageously flexible with respect to the various starting materials and adjustment of the procedure, and ultimately result in a composite cell with the desired ratio between the various cell types in the resulting expanded population. Immune cell populations can be obtained.

Example 3 - Model System for Evaluating Mixed Immune Cell Populations As can be appreciated, the development of the novel mixed cell populations described herein, performed by various aspects of the methods described herein, includes: Innovative evaluation panels may be required. Accordingly, in some embodiments, certain model systems are provided for assessing one or more of survival, proliferation, persistence, intravasation, and/or efficacy (e.g., cytotoxicity) of mixed immune cell populations. Ru.

Figures 9A-9C schematically depict the generation of various custom cell types to evaluate the efficacy of composite immune cell populations. Daudi cells are B-lymphoblastic floating cells derived from patients with Burkitt's lymphoma. As shown in Figure 9A, lentiviral constructs were used to express various reporters such as green fluorescent protein (GFP), nuclear red (NucRed), and CD19 (lacking the intracellular domain) coupled to firefly luciferase (fflucCD19). can be transfected into cells. As shown in Figure 9B, Raji cells, another human B lymphocyte suspension cell line, are genetically engineered in a similar manner. FIG. 9B is a schematic diagram for introducing CD19 (lacking an intracellular domain) and luciferase into HL60 cells, a human myeloblast suspension cell line. These NucRed and GFP constructs were used for in vitro testing, and the firefly luciferase constructs were used for in vivo testing. Each CD19 construct (lacking the intracellular domain) is genetically engineered such that tumor cells that do not normally express CD19 actually express CD19, and exert a cytotoxic effect on target cells that express CD19. can be targeted by genetically engineered NK cells and T cells. We also performed a functional evaluation using a kinetic killing assay to determine the timeframe in which NK cells and T cells each induce cytotoxic effects on target cells. .

HL60 target cells expressing CD19 were used in re-exposure assays to evaluate the cytotoxic effects of NK cells alone, T cells alone, or a combination of NK+T cells. On day 0, NK, T, and NK+T cell populations were exposed to 20,000 CD19-expressing HL60 cells. On day 4, these cell populations were re-exposed with an additional 10,000 CD19-expressing HL60 cells. Target-only cells were cultured under the same conditions but washed with PBS before adding target cells a second time (as a control). The resulting data are shown in Figure 10, where the NucRed numbers represent the number of live CD19-expressing HL60 cells counted per well.

As seen from the NK (CAR19) curve, NK cells targeting CD19 exhibit rapid initial cell death of the target cells. However, after re-exposure on day 4, the number of surviving HL60 cells increased steadily. In contrast, the T cell curve (T(CAR19)) shows a slower rate of initial cell death. T cells eliminated most targets, but in contrast to the rapid effect on NK cells, T cells took until day 4 to eliminate the first bolus of CD19-expressing HL60 cells. Even after exposure to additional HL60 cells on day 4, T cells maintained a stable cytotoxic effect on CD19-expressing HL60 cells.

The NK+T cell population was generated by replacing 50% of the population of NK(CAR19) cells with T(CAR19) cells. When first exposed with HL60 cells expressing CD19, the NK cell portion of the population retained the ability to induce rapid cell death of target cells. However, in contrast to the NK-only population, re-exposure with CD19-expressing HL60 cells on day 4 exerted a long-term cytotoxic effect on T cells, and the overall cytotoxic effect after re-exposure was The results were similar to those obtained with the T cell-only population. As shown in this study, when the total number of effector cells (NK, T or NK+T) is held constant, NK+T (1:1 ratio) has a more rapid cell killing effect than T(CAR19) alone. , showed a more sustained cell killing effect than NK (CAR19) alone.

Thus, the NK+ T cell population advantageously retained a rapid and sustained cell killing effect. This "mixed" cytotoxicity offers several advantages in cancer immunotherapy. Many cancers are known to be adaptive and have "camouflage" mechanisms to avoid detection by the immune system. The rapid cell death effect of NK cells in mixed NK+T populations helps to eliminate cancer cells before such camouflage effects take effect. Furthermore, even if cancer cells are quickly killed, a small number of cells may remain and the tumor may recur. Therefore, the T cell portion of the NK+ T cell population can induce long-term cytotoxic effects and reduce the risk of recurrence. Although this study demonstrated a 1:1 effector cell ratio (NK:T) as described above, in some embodiments other ratios are achieved with the methods described herein. be able to. In some embodiments, the final ratio can be tailored to the particular therapeutic situation. For example, slow growing tumors may be optimally treated with lower NK:T ratios, which may also bias cytotoxicity kinetics towards slower but longer duration anti-cancer effects. In contrast, for aggressive tumors, a higher NK:T ratio may be desired, as the initial cytotoxic kinetics may be faster to achieve rapid destruction of tumor cells and/or tumor clearance. This is because the remaining part of the tumor is “cleaned up” due to the prolonged T cell dynamics. Similarly, in some embodiments, various ratios of NK+ T cell populations can be administered at different times during patient treatment. For example, a high ratio of cytotoxic NK:T cell population may be administered at the beginning of administration to suppress initial tumor growth, and then a lower ratio may be administered to maintain the effect and suppress tumor growth in the long term. NK: capable of transitioning into the T cell population (like a drug loading dose followed by a maintenance dose).

Example 4 - Functional evaluation in three-dimensional culture In order to more easily reproduce the natural environment of some tumors, particularly solid tumors, intratumoral infiltration was modeled and the tumor microenvironment was modeled with mixed immune cell populations. A 3-D culture system was developed to test how the cells react and perform.

FIG. 11 is a schematic diagram of one such model environment. VersaGel® (Cypre, Inc. San Francisco, CA) is a hydrogel that can be used to encapsulate cells and has optical properties suitable for downstream optical imaging to analyze cells. . VersaGel (registered trademark) was placed in the center of a 96-well culture dish, and 10,000 CD19-expressing HL60 cells were seeded onto the gel. The top left of Figure 11 shows the seeded cells on day 0, with cells marking the edge of the gel in the central area of the well, and a circular area outside where normal culture wells would be. Please be careful. The bottom left panel shows a representative culture well on day 8, and the middle inset shows the generation of spheroids. The left part of the inset shows that spheroid generation is confined to the area of VersaGel®, the dotted line indicates the divide between the gel and normal culture medium, and the right part shows that the spheroid generation is limited to the area of VersaGel®, and the right part shows that HL60 cells normal cell culture growth. The upper right part of Figure 11 shows short exposure visualization of HL60 spheroids expressing NucRed (labeled/arrow). Longer exposure (bottom left) shows a single NucRed-expressing HL60 cell outside the VersaGel® boundary and thus not forming a spheroid.

In some embodiments, spheroids recreate a 3D tumor microenvironment and allow testing how immune cells, such as mixed populations of NK+ T cells, function in the recreated tumor microenvironment. For example, this approach can be used to test the ability of NK+ T cell populations to infiltrate tumors and exert cytotoxic effects deep within the tumor, rather than just acting on surface cells.

In some embodiments, 3D tumor spheroids are exposed to various ratios of NK+ T cell populations and tumor cell viability is measured, e.g., in the case of CD19-expressing NucRed-expressing HL60 (or other) tumor cells, the red channel fluorescence signal Evaluate by detection. In some embodiments, a mixed population of NK+T cells can exhibit enhanced cytotoxic activity compared to either NK cells alone or T cells alone. Similarly, in some embodiments, a mixed population of NK+T cells may exhibit enhanced tumor invasion compared to either NK cells alone or T cells alone. In some embodiments, the ratio of NK cells to T cells is altered to alter the kinetics of cytotoxicity, achieving an initial rapid onset of cytotoxic effect followed by a long-term cytotoxic effect, as described above. Alternatively, a ratio can be implemented that results in a slower initial phase and a more prolonged cytotoxic phase (based on T cells).

Example 5 - Evaluation of NK cell and T cell combination therapy To further evaluate the beneficial interactions in NK cell and T cell combination therapy, further studies were performed both in vitro and in vivo. As described above, and as further explained based on the experimental data below, in some embodiments, NK cells and T cells synergistically interact with each other with respect to cell proliferation and their cytotoxic effects on target tumor cells. It was found that they interact. As outlined in the study below, NK cell and T cell interactions were evaluated in terms of cytotoxicity (using a matrix assay) and persistence of cytotoxicity (using a tumor re-exposure assay). Ta. Furthermore, we also conducted tests to elucidate the cellular mechanisms that lead to the synergistic effects of NK cells and T cells. Cytokine profiles of NK cells and T cells were also evaluated as well as in vivo antitumor efficacy in a xenograft mouse model.

The aim of this study was that the combination of NK cells and T cells demonstrated efficient tumor killing activity, increased persistence of activity and improved cytokine release, with improved patient safety (e.g. The aim was to determine whether a reduction in the risk of cytokine release syndrome (cytokine release profile) can be achieved in terms of cytokine release syndrome.

FIG. 12A shows various concentrations of NK cells and T cells expressing anti-CD19 CAR (as a non-limiting example of a chimeric antigen receptor) and the resulting cell response to Nalm6 tumor cells (as an example of a target cancer type). A schematic matrix is shown for evaluating injury effects. For the CAR construct used in this study, which is a non-limiting example of a CAR that can be used for NK cells and T cells used in the embodiments described herein, the anti-CD19 conjugate is FMC63 scFv. In some embodiments, the conjugate is humanized. Although in the studies described herein the CAR has a Flag tag, it can be appreciated that according to some embodiments, for example, in clinical constructs, no tag is used. In some embodiments, the CAR includes an OX40 costimulatory domain and a CD3ζ stimulatory domain. In some embodiments, the CAR-encoding nucleic acid further encodes an IL15 domain. In some embodiments, IL15 is expressed separately by NK cells and/or T cells as a membrane-bound polypeptide. As shown in the figure, T cells expressing this construct are designated as T19-1 and NK cells expressing this construct are designated as NK19-1. FIG. 12A shows various T19-1 and Nalm6 ratios on the X-axis of the matrix (shown as T19-1=0, or 1:1, 1:2, 1:4 or 1:8). The Y-axis shows various NK19-1 to Nalm6 ratios (denoted as NK19-1=0, or 1:1, 1:2, 1:4, 1:8 or 1:16). By tracing the intersection of the X and Y axes, it is possible to confirm the ratio of NK cells and T cells to Nalm6 target cells. Nalm6 target cells are CD19-positive B-cell precursor leukemia cell lines that also stably express NucRed, so they can be used as an indicator of Nalm6 cell survival (therefore, NK/T cell cytotoxicity). (correlated with gender).

FIG. 12B shows test results in which a decrease in red fluorescent signal correlates with stronger cytotoxicity against Nalm6 tumor cells. As can be seen from the data, as the E:T ratio decreases (for either NK cells or T cells), Nalm6 cell viability increases. For example, see the case where T19-1:Nalm6 is 1:8 and NK19-1=0 (top right pair of circular images). At this E:T, T cells were unable to produce significant cytotoxic effects on Nalm6 target cells. Similarly, even in the presence of both NK and T cells, Nalm6 cells continued to proliferate at low concentrations (bottom right of 1:8 T19-1 and 1:16 NK19-1). (see pair of circular images). However, at many other E:T ratios, the combination of NK cells and T cells worked together to exert strong antitumor effects. As can be seen from this result, when either NK19-1 or T19-1 cells are present at an E:T ratio of 1:1, Nalm6 proliferation is almost constant, regardless of the ratio of other effector cells to tumor cells. Completely blocked. Similar results were seen when either NK cells or T cells were present in a 1:2 ratio. These 1:1 E:T ratios were gradually reduced (using each effector cell type alone). Remarkably, proliferation of Nalm6 cells was observed when NK19-1 cells were used alone and when T19-1 cells were used alone at an E:T ratio of 1:2. However, when T cells and NK cells were used in combination at an E:T ratio of 1:2, resulting in an effector:target ratio of 1:1, Nalm6 proliferation was no longer observed. These results indicate that NK cells and T cells act synergistically with each other. Neither cell type alone could suppress Nalm6 proliferation at a ratio of 1:2, but when combined at a concentration that was "ineffective" alone, the cytotoxic effect was enhanced by the combination of NK + T cells ("(NK + T): Nalm6=1:1” (see the image in the dashed line frame). The synergy between NK cells and T cells was more pronounced when the two cell types were combined at a 1:4 effector to target ratio, respectively. Both NK cells and T cells allowed significant proliferation of Nalm6 when used alone at 1:4. However, when NK cells and T cells were used together at an E:T ratio of 1:4, the overall ratio was 1:2, and Nalm6 proliferation was significantly suppressed compared to when either cell was used alone. It was found that cell damage was enhanced. Again, these data indicate that combination therapy using both NK cells and T cells allows the two immune cell types to act synergistically with each other, increasing the cytotoxic effect on target cells. .

Figure 13 presents additional data related to the enhanced ability of mixed populations of NK cells and T cells to act cytotoxicly on target tumor cells. The data shown in Figure 13 shows the proliferation of Nalm6 tumor cells in a re-exposure model with T cells or NK cells alone, as well as a combination of NK cells and T cells. The first exposure to tumor cells was at time point zero of the study, and the second exposure occurred 7 days later. As can be seen, only the targeted tumor cells are significantly proliferating during the first 7 days and the second re-exposure. When T cells were used alone (in this non-limiting study, T cells expressed anti-CD19 CAR), continued proliferation of Nalm6 cells was seen during the first 7 days. The second exposure caused a rapid increase in target cell proliferation, but the level remained approximately constant for the duration of the study. NK cells expressing anti-CD19 CAR according to some embodiments described herein effectively controlled proliferation during the initial exposure period. There was a slight delay in target cell proliferation immediately after the second exposure, but thereafter target tumor cells proliferated significantly. Notably, however, the combination of NK cells and T cells controlled target cell proliferation during the first and second exposure periods and effectively inhibited target cell proliferation throughout the duration of the study. I prevented it. These data indicate that the combination of NK cells and T cells not only increases cytotoxicity (as seen in the results above), but also increases the persistence of cytotoxicity. According to some embodiments, this increased persistence not only allows for a longer duration of cytotoxicity against target cells, but also allows shorter dosing intervals and reduces potential side effects. advantageous to

Further studies were performed to elucidate some of the mechanisms when using a combination of NK cells and T cells. It is noted that the combination of NK cells and T cells results in at least a qualitative increase in immune cell numbers, leading to the investigation of whether increased cell numbers mediate the observed increased persistence. Furthermore, we also aimed to clarify whether the increase in cell number was due to an increase in NK cells, T cells, or a combination thereof. Figures 14A-14B show cell count data collected using a combination of fluorescent cytotoxicity assay (as used in the studies described above) and cell phenotyping by FACS. These data were collected on day 7 after mixing immune and target cells. Figure 14A shows NK cell numbers using the indicated NK:target (Nalm6) ratios (traces) at the indicated T:target ratios (X-axis). Again, as a non-limiting aspect, both NK cells and T cells in this study were genetically engineered to express anti-CD19 CAR (19-1). As shown in the bottom trace (black circle), when the input number of NK cells was zero, no NK cells were detected at any T cell:target ratio. In contrast, when the NK cell:Nalm6 ratio is 1:4, as the T cell:Nalm6 ratio decreases (e.g., more T cells are present), the number of NK cells detected (black squares ) has increased. The number of NK cells increased nonlinearly when the T cell:Nalm6 ratio increased from 1:8 to 1:4 compared to 1:2. This general pattern was also detected when the NK cell:Nalm6 ratio was low (1:2, filled triangles). It is noted that in the absence of T cells, regardless of the starting number of NK cells, NK cell proliferation was limited (see T19-1=0). When T cells were present at a 1:16 ratio, NK cell numbers (indicating NK cell proliferation) changed little when NK:Nalm6 was started at a 1:2 or 1:4 ratio. However, when the relative concentration of T cells was high, a higher degree of NK cell proliferation was induced in the 1:2 NK:Nalm6 sample. FIG. 14B shows the corresponding data related to measuring T cell numbers in the presence of various concentrations of NK cells. Each trace relates to one showing the ratio of T cells to Nalm6 target tumor cells, with the X-axis showing various concentrations of NK cells:Nalm6 target tumor cells. NK cells had little effect on T cell numbers, except for two traces when the T cell:Nalm6 ratio was 1:4 or 1:2. However, in the presence of NK cells with a NK cell:Nalm6 ratio of 1:4 or 1:2, the final T cell number was significantly reduced (see closed inverted triangle and closed diamond traces).

Additional investigations were performed to monitor whether changes in cell number were due to changes in cell proliferation. An approach was used to monitor changes in fluorescence with increasing cell number. In this method, a fluorescent dye is added to cells at time point zero (using ThermoFisher's CellTrace™ Violet Cell Proliferation Kit Violet). With each mitosis of the parent cell, the fluorescent signal is divided into the two cells and therefore decreases synchronously. Similarly, during the second mitosis, each of the two first generation daughter cells gives rise to two granddaughter cells, and the relative fluorescence signal decreases accordingly. Therefore, in this assay, a decrease in fluorescent signal corresponds to proliferation of labeled cells. In these studies, both NK cells and T cells were genetically engineered to express non-limiting aspects of anti-CD19CAR, allowing them to exert cytotoxic effects against Nalm6 target cells, as described above. After gene introduction, NK cells and T cells were co-cultured, and cell proliferation fluorescence analysis was performed. Nalm6 cells were exposed to these NK/T cells and incubated for 6 days. Cells were then stained with CD56-PE dye and CD3-APC dye, and FACS analysis was performed using gates on CD56 ⁺ CD3 ⁻ or CD56 ⁻ CD3 ⁺ cells (NK cells or T cells, respectively) to determine whether NK cells or T cells was detected. The resulting data from FACS analysis is shown in Figures 15A-15E. Each of Figures 15A-15E shows NK cell numbers with increased numbers of T cells (decreased T cell:Nalm6 ratio) and a 1:1 NK19-1:Nalm6 ratio. As can be seen, as the number of T cells increases, the slope of the CD56 ⁺ CD3 ⁻ gated curve becomes wider, indicating an increasing number of proliferating NK cells. The data are summarized in Figure 15F, showing the curves when the NK19-1:Nalm6 ratio is 1:1 (raw data in Figures 15A-15E) and the curves when the NK19-1:Nalm6 ratio is 2:1. Two curves showing cell proliferation are shown. This summary data shows that NK cell proliferation synchronously increases in response to the presence of T cells as NK cells increase.

Corresponding data are shown in Figures 16A-16D for T cells. Figures 16A-16C show T cell numbers in the presence of successively increased NK cell numbers (NK = 0 in A, NK:Nalm6 = 1:1 in B, NK:Nalm6 = 2:1 in C). Measure. The T cell:Nalm6 ratio was 1:2. In contrast to the curves shown in Figure 15, the signal associated with proliferating T cells decreases continuously as the number of NK cells increases. These data are summarized in Figure 16D and show that as the ratio of NK cells to target cells increases, the proportion of proliferating T cells consistently decreases. Most notable is the substantial reduction detected using the most dilute T cell concentration (1:16 T19-1:Nalm6).

Without being bound by theory, this study is based on data relating to an increase in the number of T cells promoting an increase in the number of proliferating NK cells and data showing that an increase in the number of NK cells leads to a decrease in the number of proliferating T cells. We developed the schematic model shown in Figures 17A-17C. Figure 17A shows proliferation of NK cells alone in the presence of target tumor cells and the resulting restricted proliferation of NK cells expressing CAR. FIG. 17B shows the corresponding schematic model when T cells exhibit strong proliferation when alone in the presence of target tumor cells. Figure 17C schematically depicts NK cell proliferation in the presence of T cells as well as target tumor cells. As shown schematically, at least some of the proliferation signals that would otherwise cause proliferation of T cells have a positive impact on NK cells. As a result, NK cells (which by themselves had only limited proliferation) proliferate. This shows a more limited degree of proliferation in the presence of NK cells, in contrast to the strong proliferation that T cells exhibit when cultured alone with target tumor cells. Taking these data together, it appears that the positive influence of T cells on NK cell proliferation begins when a threshold population or concentration of NK cells is present. Accordingly, in some embodiments using mixed populations of NK cells and T cells, the ratio of NK cells to T cells is adjusted such that a beneficial effect on NK cell proliferation is achieved. Thus, as described above, in accordance with some embodiments described herein, a mixed population of NK cells and T cells can be combined to provide enhanced cytotoxicity against target tumor cells, as well as enhanced persistence. One or both of these can be generated, at least in part, from enhanced T cell-based NK cell proliferation.

To further elucidate the mechanisms that play a role in the enhanced and sustained cytotoxicity of mixed populations of NK cells and T cells, cytokine profile analysis was performed and the results are shown in Figures 18A-18L. Data was collected using cells from two different donors. For each donor, a mixed population of NK cells (either NK = 0, NK:Nalm6 = 1:4, NK:Nalm6 = 1:2) was used to determine whether Nalm6 cells alone (T = 0) or the presence of T cells were used. Below, an analysis of cytokine production by NK cells and T cells was measured when the T:Nalm6 ratio was either 1:2, 1:4, 1:8, or 1:16. The evaluation was performed on the 5th day after the start of co-culture of immune cells and targets. In general, the data in Figures 18A-18L show a decrease in the production of selected cytokines that corresponds to a decrease in the amount of T cells and, importantly, is associated with potential side effects from T cell therapy such as cytokine release syndrome. GM-CSF and cytokines such as IL6. For example, Figure 18A shows GM-CSF concentrations released from T cells from two different donors. These data indicate that when the T cell:target ratio is low, less GM-CSF is produced by the cells and CRS is less likely. Although this trend is similar in the presence of NK cells, Figure 18A shows that increasing the number of NK cells in the co-culture increases the level of GM-CSF that accumulates in the system, regardless of the starting T cell concentration. Indicates a decrease. Another notable trend is shown in Figure 18E. This figure shows that increasing the number of NK cells does not reduce IFNg production, a cytokine important in inducing cytotoxicity to target cells. In other words, the cell combinations used in some embodiments described herein are not self-limiting in that the presence of NK cells along with T cells disrupts the activity of one cell. Rather, as described herein, these two types of cells work in conjunction with each other to produce a synergistic cytotoxic effect on target cells. Figure 18F shows the production of IL2. In the first donor, there was little IL-2 production by T cells, regardless of the number of NK cells present. In the second donor, IL-2 production was detected by T cells only in the absence of NK cells, but production did not correlate with dose dependence. The presence of NK cells may alter the extent of secretion by specific T cells, alter the overall level of T cells contributing to cytokine accumulation, or act as a sink for the binding of secreted cytokines. (thus affecting NK cell proliferation or activity). It is also possible that other cytokines produced by T cells, in addition to the cytokines tested here, contribute directly or indirectly to NK cell proliferation and/or persistence. Advantageously, in some embodiments, the reduction in cytokine production when using NK cells and T cells in combination coordinately reverses the risk of cytokine release syndrome, while at the same time enhancing the cytotoxicity of NK cells. and induce persistence.

To further evaluate the enhanced cytotoxicity and persistence by mixed populations of NK and T cells, we used a xenograft model in which NSG mice were implanted with Nalm6-targeted tumor cells. Nalm6 cells are genetically engineered to express GFP as well as firefly luciferase, the latter of which can detect tumor burden in transplanted mice by measuring bioluminescence. Figure 19 shows a schematic study plan for the xenograft model. Mice were injected with a combination of NK and T cells genetically engineered to express non-limiting examples of anti-CD19 CARs (19-1). Tumor cells were injected on day 0, and NK cells and T cells were injected alone or in combination on day 3 (Figure 20A). In some groups, NK cells were administered on day 3 followed by T cells on day 4, or T cells were administered on day 3 followed by NK cells on day 4 ( Figure 20B). Bioluminescence imaging was performed at the intervals shown in Figure 19. The bioluminescence results are shown in Figures 20A-20C, where the data is shown separated by day post-implantation, and the indicated cell doses and order of administration. Figure 20A shows PBS, 2.5 x 10 ⁶ NK cells expressing anti-CD19 CAR 19-1 construct, 2.5 x 10 ⁶ T cells expressing anti-CD19 CAR 19-1 construct, or 5x 10 ⁵ cells, a combination of NK cells and T cells (both 2.5 x 10 ⁶ cells), or a combination of NK cells and T cells (2.5 x 10 ⁶ NK cells and 5 x 10 ⁵ T cells) Luminescence data from the second day to the 31st day from the start of the test are shown for the test group administered with. FIG. 20B is data for the same time frame for the test group with staggered administration of NK cells and T cells on days 3 and 4. The two columns on the left show the results of the test group that received NK cells on day 3 and T cells on day 4, and the two columns on the right show the results of the test group that received T cells on day 3 and T cells on day 4. The results of the test group that received NK cells on day 1 are shown. Each group received both NK cells and T cells at a dose of ^2.5x10 cells (high dose) and a group receiving NK cells at a dose of ^2.5x10 cells and T cells at a dose of ^5x10 cells. (low dose).

It is noted that over the first 30 days of the study, when the cell combination was administered together, only NK cells inhibited tumor growth more effectively compared to the PBS control. In accordance with some embodiments described herein, the combination of high doses of NK cells and low doses of T cells (far right column of FIG. 20A) compared to NK cells alone (for the same dose of NK cells) , showing markedly improved tumor control. This treatment group also shows improved tumor control compared to T cells alone (at the same dose of T cells). In some embodiments, the NK:T cell ratio not only enables tumor control, but also achieves the same in conjunction with decreased cytokine release and reduced risk of CRS. Both high dose T cells alone and high dose NK/T cells showed tumor control over 30 days.

The next most effective treatment in terms of preventing an increase in tumor burden was a treatment regimen in which a starting dose of NK cells was administered followed by a lower dose of T cells the next day (see second column of Figure 20B). ). The next most effective treatment was when T cells alone were administered at low doses, which significantly prevented tumor growth for almost 3 weeks. The next most effective combination was a combination of T cells and NK cells, in which T cells were administered at a low dose first, followed by NK cells one day later. The next most effective treatment was when NK cells and low doses of T cells were administered on the same day. Again, the study is aimed at treatment groups that have some degree of tumor progression within the first 30 days. However, from the first 30 days of data, in accordance with some embodiments, a higher ratio of NK cells compared to T cells, such as about 5:1, about 10:1 or about 20:1, depending on the embodiment. The ratio was confirmed to result in robust acute and intermediate cytotoxicity. As mentioned above, while a small number of T cells can provide sufficient NK-cytotoxicity and stimulation of NK-proliferation, it is not so abundant that NK cells cannot suppress T-cell proliferation to some extent. As a result, T cell-induced cytokine release can be reduced (thus, harmful inflammatory effects can be reduced).

The other groups tested inhibited tumor growth beyond that point, as shown in Figure 20C. Without being bound by theory, these data suggest that there is probably a more robust initial response in these groups, thereby facilitating long-term control, and/or some early inhibition of T cell proliferation or We suggest that there may be other inhibitions that confer a longer active lifespan before exhaustion. High doses of T cells and NK cells were administered for the first 74 days, T cells on day 3 and NK cells on day 4 suppressed tumor growth for approximately 65 days, and 74 days after tumor implantation. A significant tumor burden was finally identified in the eye. Administration of high doses of T cells alone was also able to significantly slow tumor progression, with only minimal tumor growth detected at day 74. The two most effective treatments overall were those that combined NK cells and T cells. When NK cells were administered on the third day, followed by a high dose of T cells on the fourth day, almost no tumors were detected on the 74th day, and 80% of the mice survived. Similarly, when both NK cells and T cells were administered in large doses on day 3, 100% of the mice in the treatment group survived, with only a few tumors detected on day 74 after tumor implantation. The effect was even greater. This trend continued even after 81-121 days after tumor transplantation, and even if NK cells were administered after T cells (high dose), and even higher doses of T cells were administered, the same effect was observed. It was possible to suppress tumor growth. In particular, the combination of NK cells and T cells controlled tumor growth more strongly, with the most effective control seen when the combination of NK cells and T cells (high dose) was administered on the same day. Figure 20D shows the survival curves of all groups together and reflects the bioluminescence data described above. Figure 20E shows only selected groups to more clearly demonstrate the enhanced tumor control over 4 months seen with the combination of NK cells and T cells. In particular, tumor control is shown to be significantly improved when T cells are administered after NK cells and when NK cells and T cells are administered together. Taken together, these data indicate that the combination of NK cells and T cells is a highly effective cancer treatment. According to some embodiments, by varying the concentration of NK cells and T cells, the cytotoxicity as well as the persistence of the combination product can be fine-tuned to achieve long-term tumor growth control. . Similarly, according to some embodiments, the combination of NK cells and T cells does not necessarily need to be administered simultaneously to achieve high efficacy. According to some embodiments, one of the two cell types can be administered at an earlier time point and the other cell type at a later time point. This allows one cell type, such as NK cells, to exert an effect at an early time point that is later synergistically complemented by the cytotoxic and/or proliferative effects of a second cell type, such as T cells. That will happen. For example, depending on the type of tumor to be treated, the administration of a first cell type may enhance and/or maximize one or more properties of a second cell type in a beneficial environment (e.g., an engineered tumor microenvironment). The order of administration may be varied to produce . In other words, in some embodiments, the sequential administration of the first cell type and the second cell type allows the first cell type to be used for optimal cytotoxicity by the second cell type. Prepare the environment. Furthermore, as provided in some embodiments described herein, mixed populations of NK cells and T cells can be co-administered to obtain significantly effective anti-tumor effects.

It is contemplated that various combinations or subcombinations of the specific features and aspects of the embodiments described above may be made and still constitute one or more of the inventions. Furthermore, any description herein of any specific feature, aspect, method, characteristic, quality, attribute, element, etc. with reference to one aspect may be used in connection with all other aspects described herein. Can be done. Accordingly, it should be understood that various features and aspects of the disclosed embodiments can be combined or substituted with each other to form various embodiments of the disclosed invention. Therefore, it is intended that the scope of the invention described herein should not be limited by the specific disclosed embodiments set forth above. In addition, while the invention is susceptible to various modifications and alternative forms, specific examples thereof have been shown in the drawings and are herein described in detail. However, the invention is not limited to the particular forms or methods disclosed; on the contrary, the invention covers all aspects falling within the spirit and scope of the various described embodiments and the appended claims. It is to be understood that modifications, equivalents, and substitutions of . The methods disclosed herein do not need to be performed in the order presented. The methods disclosed herein include certain actions performed by a performer, but may also include explicitly or implicitly directing those actions to a third party. For example, an act such as "administering an expanded NK cell population" includes "instructing administration of an expanded NK cell population." Additionally, where a feature or aspect of the present disclosure is described in terms of the Markush Group, those skilled in the art will appreciate that the disclosure is thereby also described in terms of any individual member or subgroup of members of the Markush Group. can recognize that there is

The ranges described herein also include any and all overlaps, subranges and combinations thereof. Terms such as “up to”, “at least”, “greater than”, “less than”, “between”, etc. Including the number of Numbers preceded by terms such as "about" or "approximately" are inclusive of the recited number. For example, "90%" includes "90%". In some embodiments, at least 95% homology includes 96%, 97%, 98%, 99% and 100% homology to the reference sequence. Furthermore, when a sequence is disclosed as "comprising" a nucleotide or amino acid sequence, such reference does not include, unless specified otherwise, that the sequence "comprises," "consists of," or "essentially consists of" the described sequence. It shall also include "consisting of".

Articles such as "a," "an," and "the" mean one or more, unless the contrary indicates or is clear from the context. As used herein and in the claims, the phrase "and/or" should be understood to mean "either or both" of the elements so conjoined. Multiple elements listed with "and/or" should be construed in the same manner, ie, meaning "one or more" of the elements so conjoined. Other elements may be optionally present in addition to the elements specifically specified by "and/or". As used herein and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when used in a list of elements, "or" or "and/or" shall be construed as inclusive, i.e., including at least one, optionally more than one, of the list of elements. , shall optionally contain additional unlisted elements. Only terms that clearly indicate the opposite, such as “only one of” or “actely one of”, mean to include exactly one element of a list of numbers or elements. do. Accordingly, a claim containing "or" between one or more members of a group includes, unless there are indications to the contrary, one, two or more, or all of the group members are present or employed in a given product or process. or other relevance. Embodiments are provided in which exactly one member of a group is present, employed, or otherwise associated with a given product or process. Embodiments are provided in which multiple or all members of a group are present, employed, or otherwise related in a given product or process. Any one or more claims may be amended to explicitly exclude any aspect, aspect, feature, element or property or combination thereof. Any one or more claims may be amended to exclude any agent, composition, amount, dosage, route of administration, cell type, target, cell marker, antigen, targeting site, or combinations thereof. good.

Any titles or subheadings used herein are for organizational purposes and should not be used to limit the scope of the embodiments described herein.

Claims (18)

A method for producing a mixed population of genetically modified immune cells comprising at least two subpopulations of genetically modified immune cells, the method comprising:
isolating a mononuclear cell population from a blood sample, wherein the isolated mononuclear cell population comprises at least a first immune cell subpopulation and a second immune cell subpopulation. ;
isolating a first immune cell subpopulation from the isolated mononuclear cells;
a step of culturing the isolated first immune cell subpopulation in a culture vessel together with a feeder cell population, wherein the feeder cells are a natural killer (natural killer) within the first immune cell subpopulation; NK) configured to express at least one molecule for stimulating cell proliferation, and said at least one molecule is 4-1BB ligand (4-1BBL), interleukin 15 (IL-15); and a combination thereof, thereby producing a proliferating population of NK cells;
isolating a second immune cell subpopulation from the isolated mononuclear cells;
culturing a second immune cell subpopulation in a culture vessel with at least one molecule for stimulating proliferation of T cells within the second immune cell subpopulation, the at least one molecule for stimulating the T cells comprises one or more antibodies to CD3 or antibodies to CD28, thereby generating a proliferating population of T cells;
A nucleic acid encoding a genetically modified receptor configured to bind to CD19 expressed by cancer cells and induce cytotoxic activity against cancer cells upon binding is introduced into an expanded NK cell population to generating modified cytotoxic NK cells;
A nucleic acid encoding a genetically modified receptor configured to bind to CD19 expressed by cancer cells and induce cytotoxic activity against cancer cells upon binding is introduced into an expanded T cell population to produce genetically modified cells. generating toxic T cells;
generating a mixed population of genetically modified immune cells by combining a portion of the genetically modified cytotoxic NK cells and a portion of the genetically modified cytotoxic T cells, wherein the genetically modified cells the number of toxic NK cells being combined in a ratio at least 5 times greater than the number of genetically modified cytotoxic T cells ;
including methods. 2. The method of claim 1, wherein the at least one molecule for stimulating NK cell proliferation comprises a combination of 4-1BBL and interleukin 15 (IL-15). 3. The method of claim 2, wherein the IL-15 is membrane bound to the feeder cells. Culturing the isolated first immune cell subpopulation further comprises adding one or more of soluble interleukin 2, soluble interleukin 12, soluble interleukin 18, or a combination thereof to the culture. 4. A method according to any one of claims 1 to 3, comprising: 5. Method according to any one of claims 1 to 4, characterized in that both NK cells and T cells are transformed with a nucleic acid encoding the same chimeric antigen receptor. 5. The method according to any one of claims 1 to 4, wherein the NK cells and T cells are transformed with nucleic acids encoding different chimeric antigen receptors. 6. The method of claim 5 , wherein the chimeric antigen receptor comprises an OX40 costimulatory domain and a CD3ζ signaling domain operably linked to an anti-CD19 scFv. 8. The method of claim 5 or 7 , wherein the chimeric antigen receptor is encoded by a nucleic acid having at least 95% sequence identity to SEQ ID NO:1. 8. The method of claim 5 or 7, wherein the chimeric antigen receptor comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:2. 10. The method of any one of claims 1 to 9 , wherein the at least one molecule for stimulating T cell proliferation comprises an anti- CD3 antibody . 11. The method of any one of claims 1 to 10, wherein the at least one molecule for stimulating T cell proliferation comprises an anti-CD28 antibody. 12. A method according to any one of claims 1 to 11, wherein the blood sample is a peripheral blood sample. 12. A method according to any one of claims 1 to 11, wherein the blood sample is an umbilical cord blood sample. The mixed population of genetically modified immune cells contains 1 x 10 ⁸ to 1 x 10 ¹⁰ genetically modified cytotoxic NK cells and 1 x 10 ⁶ to 1 x 10 ⁸ genetically modified cytotoxic T cells. 14. The method according to any one of claims 1 to 13, comprising a cell. A mixed population of genetically modified immune cells exhibits (i) a rapid cytotoxic effect on tumor cells, followed by an enhanced and persistent cytotoxic effect on tumor cells, compared to NK cells alone or T cells alone; 15. The method of claim 14, wherein the method exhibits or (ii) exerts a sustained cytotoxic effect on tumor cells for a longer period of time compared to NK cells alone or T cells alone. 16. A mixed population of immune cells produced by a method according to any one of claims 1 to 15 . 17. A mixed population of immune cells according to claim 16 for use in the treatment of tumors. 18. Use of a mixed population of immune cells according to claim 16 or 17 in the manufacture of a medicament for the treatment of cancer.