Method of induction of immune cells into a memory-like state

Abstract

The present invention pertains to the field of cellular immunotherapy. More precisely, it is directed to therapeutic interventions against cancer, immunodeficiency, infectious diseases, and autoimmune conditions utilizing human memory-like cytotoxic lymphocytes, including T cells, Natural Killer (NK) cells, Natural Killer T (NKT) cells, monocytes and other relevant cell types. Disclosed are methods for the isolation and induction of these cells into a memory-like state, which can find application in therapeutic contexts as well as in non-therapeutic contexts, such as in research endeavors.

Description

[0001] METHOD OF INDUCTION OF IMMUNE CELLS INTO A MEMORY-LIKE STATE

[0002] FIELD OF THE INVENTION

[0003] The present invention pertains to the field of gene and cellular immunotherapy. More precisely, it is directed to therapeutic interventions against cancer, immunodeficiency, infectious diseases, and autoimmune conditions utilizing human memory-like cytotoxic lymphocytes, including T cells, Natural Killer (NK) cells, Natural Killer T (NKT) cells, monocytes and other relevant cell types. Disclosed are methods for the isolation and induction of these cells into a memory-like state, which can find application in therapeutic contexts as well as in non-therapeutic contexts, such as in research endeavors.

[0004] BACKGROUND OF THE INVENTION

[0005] Human cytotoxic and phagocytic lymphocytes constitute vital components of the immune system, equipped with the capacity to eliminate diseased cells within the human body either through the directed delivery of cytotoxic granules or by leveraging phagocytic function1. The cytotoxic subset of lymphocytes encompasses various immune cell types, including Natural Killer (NK) cells, Natural Killer T (NKT) cells, and cytotoxic T cells such as CD8+ T cells, and gamma-delta T cells, among others. Monocytes and macrophages exemplify human phagocytic cells, adept at selectively engulfing and degrading other cells. While both cytotoxic and phagocytic immune cell types share the common goal of removing target cells from the body, they exhibit significant differences in their targeting mechanisms and interactions with respective target cells. T cells, distinguished by a unique somatically rearranged T-cell receptor (TCR), selectively recognize foreign antigen peptides within the context of major histocompatibility complex (MHC) proteins. In contrast, NK cells, monocytes, and macrophages utilize a set of germline-encoded receptors to engage ligands on the surface of target cells or to recognize antibody-opsonized targets. Regardless of the specific targeting mechanism employed, the ensuing activation signals during surface interactions lead to the release of cytotoxic granules or activation of phagocytic activity, accompanied by other effector phenotypes such as cell proliferation and cytokine secretion. The utility of cytotoxic and phagocytic lymphocytes in eliminating diseased cells from patients has found applications in medical treatments across several diseases. An illustrative example is the U.S. Food and Drug Administration (FDA) approval of chimeric antigen receptor-T (CAR-T) cells for the treatment of several hematologic malignancies2. CAR-T cells function by redirecting T cells to selectively eliminate cancer cells in a targetdependent manner. This innovative approach has recently been extended to the realm of autoimmune diseases, notably in the treatment of systemic lupus erythematosus3. In this context, the eradication of autoreactive B cells demonstrated a curative effect in patients treated with CD19-targeted CAR-T cells. In the field of infectious diseases, CAR-T cells have been employed to target HIV surface proteins, in an effort to eradicate all HIV- infected cells from the patient's body — an exceptionally challenging task4. Similarly, efforts have been undertaken to leverage NK cells engineered to express CD19-targeted CARs for the targeted elimination of cancer cells5. Notably, recent advancements have extended CAR-engineering to macrophages, demonstrating their efficacy in phagocytosis and the eradication of HER2-positive cancer cells6. The strategic utilization of cytotoxic and phagocytic cells in eliminating diseased cells from the body holds immense therapeutic potential, offering promising benefits to patients with conditions such as cancer, autoimmune diseases, and infectious diseases.

[0006] Historically, scientific understanding of immunological memory was based broadly on the immune system's capacity to recall and respond to infection re-challenge rapidly and effectively. At a molecular level, the foundation of immunological memory resides in the specialized T- and B-cell receptors, selected to recognize foreign antigens the organism has been previously exposed to. However, the function of memory T cells extends beyond merely encoding for antigen-specific TCRs as this specialized type of immune cell is also preprogrammed for robust activation, proliferation, and cytokine secretion7. These distinctive phenotypes emerge from broad chromatin changes and gene expression patterns, enabling memory T cells to persist in the body for many years until they are reactivated through TCR engagement. Upon reactivation, these cells rapidly transition into potent effector cells, marked by increased cytotoxicity, proliferative capacity, and cytokine secretion, thereby activating other immune cell types. Phenotypes analogous to those of memory T cells have been observed in other immune cell types. Notably, specific subtypes of naturally occurring NK cells in individuals infected with human cytomegalovirus (HCMV) exhibit the remarkable capacity to persist over extended periods, undergo rapid expansion, and efficiently eliminate HCMV-infected cells upon encounter8. Because of these attributes, these NK cells have been termed “adaptive” or "memory-like". Subsequent research revealed that treating human NK cells with a combination of cytokines, namely interleukin- 12 (IL- 12), interleukin- 15 (IL- 15), and interleukin- 18 (IL- 18), can induce a state that partially mirrors the memory-like features of naturally occurring adaptive NK cells and memory T cells9 10. This cell state, referred to as cytokine-induced memory-like (CIML) NK cells, has demonstrated significant efficacy in clinical applications. Notably, in several acute myeloid leukemia (AML) clinical trials, CIML NK cells displayed exceptional potency, leading to several complete responses11 12. Whether cytokine stimulation can endow other cytotoxic and phagocytic immune cells with memory-like phenotype is not well understood.

[0007] Despite their ability to acquire a memory-like phenotype, expand within patients' bodies, and effectively target cancer cells, the manufacturing of CIML NK cells faces significant challenges due to their over-activation and subsequent apoptosis.

[0008] Given the significant medical applications of CIML NK cells, and potentially other cytokine-induced memory-like immune cell types, there is a pressing need to enhance their manufacturing process so as to prevent apoptosis while ensuring the acquisition of a memory-like phenotype.

[0009] SUMMARY OF THE INVENTION

[0010] Several decades ago, it was reported that the combined treatment of NK cells with IL-2 and IL- 12, or IL- 15 and IL- 12, resulted in excellent activation of NK cells but also induced their apoptosis, that was partially dependent on TNF-alpha13. The equivalent effects of IL- 2 and IL- 15 arise from their shared binding to the common gamma chain (IL-2RG) and IL- 2RB receptors, differing only in the high-affinity binding receptor (IL-2RA for IL-2). Recent data on CIML NK cell manufacturing, utilizing a combination of IL- 12, IL- 15, and IL- 18 treatment, revealed that the apoptosis of activated CIML NK cells may also be linked to aberrant mitochondrial dynamics14. This increased apoptosis observed in CIML NK cells is not attributable to the action of individual cytokines, as IL-12, IL-2, and IL-15 individually promote NK cell survival15. In vivo studies further support this, as mice treated with combinations of IL- 12 and IL-2, or IL- 12 and IL- 15, experienced septic shock and mortality due to NK cell activation. Conversely, treatment with individual cytokines showed no systemic toxicities16.

[0011] The present inventors have conducted thorough experimentation in order to solve the apoptosis problem described above. Surprisingly, they discovered that contacting immune cells with certain cytokines in a distinct order provided unexpected improvements in the cell survival rate of said immune cells, whilst maintaining the required activation into the memory-like state.

[0012] Accordingly, the present invention provides a method of producing cytokine-induced memory-like (CIML) immune cells, said method comprising:

[0013] (a) isolating immune cells from a biological sample,

[0014] (b) contacting the immune cells with a first composition that comprises IL- 12 or a functional equivalent thereof, but does not comprise:

[0015] (i) IL-2 or a functional equivalent thereof,

[0016] (ii) IL- 15 or a functional equivalent thereof, and

[0017] (iii) IL-21 or a functional equivalent thereof;

[0018] (c) washing the immune cells to remove the first composition;

[0019] (d) subsequently contacting the immune cells with a second composition that comprises IL-2 or a functional equivalent thereof, but not IL- 12 or a functional equivalent thereof, thereby producing cytokine-induced memory-like (CIML) immune cells.

[0020] The present invention also provides a cytokine-induced memory-like (CIML) immune cell produced by the method described herein. The present invention also provides a population of cytokine-induced memory-like (CIML) immune cells wherein more than 20% of the cells in the population are CD25-positive, more than 30% of the cells in the population are CD71 -positive, and more than 70% of the cells in the population are CD69-positive.

[0021] The present invention also provides a pharmaceutical composition comprising the CIML immune cell as described herein, or the population of CIML cells as described herein, and a pharmaceutically acceptable carrier.

[0022] The present invention also provides a method of treating an individual in need thereof with a CIML immune cell, comprising administering to the individual a therapeutically effective amount of the CIML immune cell as described herein, the population of CIML immune cells as described herein, or the pharmaceutical composition as described herein.

[0023] The present invention also provides a method of treating an individual in need thereof with a CIML immune cell, comprising administering to the individual a therapeutically effective amount of the CIML immune cell, wherein the CIML immune cell is produced according to the methods as described herein.

[0024] The present invention also provides a composition comprising IL-2 or a functional equivalent thereof, IL- 15 or a functional equivalent thereof, and IL- 18 or a functional equivalent thereof.

[0025] The present invention also provides a composition comprising IL- 12 or a functional equivalent thereof, IL- 15 or a functional equivalent thereof, and IL- 18 or a functional equivalent thereof.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The detailed description of preferred embodiments of the invention provided herein is best comprehended when studied in conjunction with the accompanying drawings. The drawings depict presently preferred embodiments intended to elucidate the features of the invention. However, it is essential to note that the scope of the invention is not confined to the exact configurations and instrumentalities exemplified in the drawings.

[0028] Figure 1., comprising Figures 1A-C, is a series of images showing the messenger RNA expression of select cytokine receptors in various populations of human immune cell types, demonstrating that cytokine-induced memory-like cell state may be possible for cell types other than Natural Killer cells. Population-averaged gene expression data for purified human immune cells was acquired from the ImmGen Human Cell Atlas dataset. This dataset was generated using ultra-low-input RNA-sequencing using peripheral blood mononuclear cells from two healthy donors. The data is publicly accessible and can be found at immgen.org.

[0029] Figure 2., comprising Figures 2A-F, is a series of images showing schematic representations of cytokine receptor signaling mechanisms, specifically for IL- 12 (Figure 2A), IL- 18 (Figure 2B), IL-2 (Figure 2C-E), IL- 15 (Figure 2C-E), IL-21 (Figure 2F), and IL-7 (Figure 2G) cytokine stimulation. Of importance, the images illustrate that IL-2 and IL- 15 signal through the same set of signaling receptors, notably IL-2RB and IL-2RG (common gamma chain) thereby having an equivalent effect when administered to cells.

[0030] Figure 3., comprising Figures 3A-D, is a series of images demonstrating that simultaneous stimulation of NK or NKT cells with IL- 12, IL- 15, and IL- 18 results in apoptosis regardless of the blood source. In this experimental setup (Figure 3A) two sources of blood were used: 1. LRS cone shipped over-night on ice, and 2. Whole blood isolated and delivered on ice immediately following the blood draw. Each blood product was isolated from two different donors and the viability of purified NK / NKT cells was evaluated immediately after purification (day 0, Figure 3B), one day after stimulation (day 1, Figure 3C) or three days after stimulation (day 3, Figure 3D). As a control, purified NK / NKT cells were treated only with low-dose IL- 15 (2 ng / mL).

[0031] Figure 4., comprising Figures 4A-E, is a series of images demonstrating that splitting stimulation of NK / NKT cells with IL- 12 and IL- 15 into two separate steps preserves their viability regardless of the cell source. Two different sources of blood were used in this experimental setup (Figure 4A): 1. Frozen peripheral blood mononuclear cells (PBMCs) isolated from one-day old LRS cone, and 2. Frozen PBMCs isolated from same-day fresh whole blood. Frozen PBMCs were thawed, and NK / NKT cells were purified using negative magnetic bead selection. Purified NK / NKT cells were stimulated using the following conditions: A. Purified NK / NKT cells were maintained in low-dose IL- 15 (2 ng / mL) as a control condition; B. Purified NK / NKT cells were stimulated with 10 ng / mL IL- 12 and 50 ng / mL IL- 18 for 6 hours, washed, rested for 10 hours in media without cytokines, and then stimulated with 20 ng / mL of IL- 15 for 3 days; C. Purified NK / NKT cells were stimulated with 10 ng / mL IL-12 and 50 ng / mL IL-18 for 6 hours, washed, and immediately stimulated with 20 ng / mL of IL- 15 for 3 days; D. Purified NK / NKT cells were stimulated with 10 ng / mL IL- 12 and 50 ng / mL IL- 18 for 16 hours, washed, rested for 10 hours in media without cytokines, and then stimulated with 20 ng / mL of IL-15 for 3 days; E. Purified NK / NKT cells were stimulated with 10 ng / mL IL-12 and 50 ng / mL IL-18 for 16 hours, washed, and immediately stimulated with 20 ng / mL of IL- 15 for 3 days. The viability of purified NK / NKT cells was evaluated immediately after purification (day 0, Figure 4B), one day after stimulation (day 1 , Figure 4C) or three days after stimulation (day 3, Figure 4D). Figure 4E shows graph plots summarizing the viability data for each condition.

[0032] Figure 5., comprising Figures 5A-E, is a series of images demonstrating that purified NK / NKT cells stimulated with two split steps comprised of IL- 12 and IL- 18 in the first step, and IL- 15 in the second step can persist and expand in vivo (Figure 5A-B). The mouse strain used in this experiment is huIL-15-Tg NSG mouse strain that expresses low dose of human IL- 15 cytokine. Human NK cells showed persistence in this mouse strain and enhanced expansion with intraperitoneal supplementation of human recombinant IL-2. After 12 days human NK cells were identified in the peripheral blood (Figure 5C), spleen (Figure 5D), and bone marrow (Figure 5D).

[0033] Figure 6., comprising Figures 6A-N, is a series of images demonstrating the optimization of the split stimulated method. NK / NKT cells were purified from same-day fresh whole blood from two healthy donors, and stimulated at various cytokine combinations, concentrations of IL- 18, and timing of the first IL- 12 step (Figure 6A). After five days, stimulated NK / NKT cells were evaluated using viability analysis and expression of surface markers that are associated with NK cell activation and memory-like phenotype (Figure 6B-N).

[0034] Figure 7., comprising Figures 7A-F., is a series of images demonstrating that the optimized stimulation of CIML NK / NKT cells (Figure 7A) generates a cell type that is functionally equivalent (Figure 7B-F) to standard CIML NK cells as evaluated by surface marker expression and ability to kill various cancer cell lines in vitro. However, optimized CIML NK / NKT cells have increased viability relative to the standard CIML NK stimulation (Figure 7B).

[0035] Figure 8., comprising Figures 8A-E, is a series of images demonstrating that optimized CIML NK / NKT cells have the same ability to persist and expand in vivo as CIML NK / NKT cells generated using the standard method.

[0036] Figure 9., comprising Figures 9A-9B, is a series of images demonstrating that human CD3+ T cells stimulated using the optimized cytokine-induced memory-like (CIML) protocol exhibit enhanced alloreactivity compared to T cells stimulated with conventional cytokine stimulation. Figure 9A provides a schematic illustrating the isolation and stimulation process for T cells. Figure 9B shows the results of a cytotoxicity assay comparing conventional and CIML T cells co-cultured with the Raji cell line.

[0037] Figure 10., comprising Figures 10A-10D, is a series of images detailing the isolation process and CIML stimulation of a cellular product containing both T cells and NK cells. Figure 10A shows CD45RA expression in healthy donor peripheral blood mononuclear cells (PBMCs), indicating that the CD45RA compartment includes B cells, T cells, and NK cells. Figure 10B presents a schematic of the isolation and optimized CIML stimulation process for T and NK cells. Figure 10C depicts the depletion of CD 19+ B cells and the enrichment of CD45RA+ cells in the isolated cell product. Figure 10D illustrates the improved viability of the CIML-stimulated T and NK cell product.

[0038] DETAILED DESCRIPTION All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0039] Definitions

[0040] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.

[0041] It is to be understood that different applications of the disclosed invention may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0042] The term “about” or “around” when referring to a value refers to that value but within a reasonable degree of scientific error. Optionally, a value is “about x” or “around x” if it is within 10%, within 5%, or within 1% of x.

[0043] In addition, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes “cells”, and the like.

[0044] In general, the terms “comprising” or “comprises” are intended to mean including but not limited to. For example, the phrase “. . .a first composition that comprises IL-12” should be interpreted to mean that the first composition comprises at least IL- 12, but the first composition may also comprise further components, such as further cytokines or equivalents. In some embodiments of the invention, the words “comprising” or “comprises” are replaced with the phrases “consisting of' or “consists of The phrases “consisting of” or “consists of’ are intended to be limiting. For example, the phrase “. . .a first composition that consists of IL-12” should be interpreted to mean that the first composition comprises at least IL- 12, but no further components.

[0045] In some embodiments of the invention, the word “comprising ” is replaced with the phrase “consisting essentially of”. The term “consisting essentially of” means that specific further components can be present, namely those not materially affecting the essential characteristics of the subject matter.

[0046] The term “cytokine” is widely recognized in the field and encompasses a diverse group of proteins pivotal in cellular signaling. Within the cytokine family, notable members include various interleukins, interferons, chemokines, lymphokines, and tumor necrosis factors. Examples of specific cytokines encompass interleukin-2 (IL- 2), interleukin- 12 (IL-12), interleukin- 15 (IL-15), interleukin- 18 (IL-18), and interleukin-21 (IL-21). Cytokines exert their effects by binding to specific cytokine receptors, which are multi-chain receptors. Upon binding with their respective cytokines, these receptors initiate an intracellular signaling cascade, resulting in a change of the cell's functional state. Notably, cytokine receptors can also be activated through the use of antibodies and various fusion proteins, thereby facilitating functional binding analogous to that achieved through direct cytokine binding.

[0047] The term "memory-like" signifies a cellular state, specifically in an immune cell, marked by heightened cytotoxicity, proliferative capacity, and other effector functions, such as cytokine secretion or surface marker expression, in comparison to the non-activated cell state.

[0048] Furthermore, the term "cytokine-induced memory-like" pertains to a cellular state, particularly within an immune cell, that has undergone cytokine treatment. This state is characterized by increased cytotoxicity, proliferative capacity, and other effector functions, including cytokine secretion or surface marker expression, relative to the non-activated cell state. As an illustrative example, a cytokine-induced memory-like T cell refers to a T cell treated with cytokines, demonstrating heightened effector functions upon activation.

[0049] The phrase “cytokine-induced memory-like state” or “CIML” is intended to mean a trained immunity, or activated state achieved by immune cells in response to stimulation by cytokines. CIML immune cells have undergone transcriptional, epigenetic and metabolic reprogramming and have enhanced effector functions and increased persistence. Examples of CIML immune cells include T cells, CD4+ T cells, CD8+ T cells, y6 T cells, NK cells, NKT cells, dendritic cells, monocytes and macrophages. The term “cytokine-induced memory-like” or, equivalently, “CIML” in reference to the immune cells described herein, means having a “memory” or “memory-like” phenotype and produced using a priming agent.

[0050] The phrase "immune cell" or "immune effector cell" refers to a cell that may be part of the innate or adaptive immune system and executes a particular effector function such as alpha-beta T cells, NK cells (including memory-like NKs, ML NKs, and CIML NKs), NKT cells (including iNKT cells), B cells, innate lymphoid cells (ILC), cytokine induced killer (CIK) cells, lymphokine activated killer (LAK) cells, gamma-delta T cells, mesenchymal stem cells or mesenchymal stromal cells (MSC), monocytes and macrophages. Preferred immune cells are cells with cytotoxic effector function such as alpha-beta T cells, NK cells (including memory-like NKs, ML NKs, and CIML NKs), NKT cells (including iNKT cells), ILC, CIK cells, LAK cells or gamma-delta T cells.

[0051] " Effector function" means a specialized function of a cell, e.g., in an NK cell an effector function may be cytolytic activity or helper activity including the secretion of cytokines. Examples of immune cells include T cells, CD4+ T cells, CD8+ T cells, yS T cells, NK cells, NKT cells, dendritic cells, monocytes and macrophages.

[0052] Natural killer (NK) cells constitute a group of innate immune cells, which are often characterized as cytotoxic lymphocytes that exhibit antibody-dependent cellular cytotoxicity via target-directed release of granzyme, granulysin and perforin. Most NK cells have a specific cell surface marker profile (e.g., CD3-, CD56+, CD16+) in addition to a collection of various activating and inhibitory receptors. While more recently NK cells have become a significant component of certain cancer treatments, generation of significant quantities of NK cells (and especially autologous NK cells) has been a significant obstacle as the fraction of NK cells in whole blood is relatively low.

[0053] The phrase “cytokine-induced memory-like NK cell” or “ML NK cell” refers to a NK cell derived from an NK cell which has been activated ex vivo with at least one cytokine and maintains an enhanced memory-like function after challenge in the absence of the same cytokines. As used herein, the term “CIML NK cell” refers to a NK cell derived from an NK cell which has been activated with at least one cytokine and exhibits enhanced activation and interferon-gamma responses.

[0054] The term “isolating” is intended to mean separating a desired entity or entities from a sample or biological sample.

[0055] The phrase “biological sample” is intended to mean a sample taken from a human or animal subject, or a sample taken from a cell culture or cell preparation. Examples of biological samples suitable to be used in the invention are biological samples that comprise immune cells. In an embodiment of the invention, the biological sample is derived from a biological fluid. In a preferred embodiment, the biological fluid is whole blood, peripheral blood, Leukocyte Reduction System cone, a leukapheresis product or cord blood.

[0056] The term “contacting” is intended to mean exposing an entity to another entity for a period of time, such that the two entities interact, either directly or indirectly. The term “contacting” can be used interchangeably with the term “exposing” herein.

[0057] The phrase “functional equivalent” is intended to mean an entity that has equivalent function to a named entity, or a substantially similar function to the named entity such that the effects of the named entity and the functional equivalent are essentially the same. A functional equivalent of a cytokine may include functional fragments thereof, and / or fusion proteins comprising functional fragments thereof, and / or antibodies or antibody fragments with cognate binding sites that have an equivalent biological effect as a cytokine, or other cytokines.

[0058] The term “IL-12” refers to Interleukin- 12, a heterodimeric cytokine composed of the p35 and p40 subunits. It is produced by antigen-presenting cells and plays a critical role in host defense against intracellular microbial infection and control of malignancy via its ability to stimulate both innate and adaptive immune effector cells. Functional equivalents of IL- 12 include IL-12 fusion proteins, single-chain IL-12 fusion proteins, IL-12 receptor agonists, IL-12 receptor agonist antibodies, IL-12 mimetic peptides, recombinant IL-12-Fc fusion, and non-secreting(ns) IL-12. A schematic representation of the IL-12 receptor signaling mechanism is shown in Figure 2A.

[0059] The term “IL-2” refers to Interleukin-2, an interleukin, a type of cytokine signaling molecule in the immune system. It is a 15.5-16 kDa protein that regulates the activities of white blood cells (leukocytes, often lymphocytes) that are responsible for immunity. Functional equivalents of IL-2 include Aldesleukin, Neoleukin 2 / 15, IL-2 mimics, recombinant fusions of IL-2 and diphtheria toxin (denileukin diftitox), IL-2 receptor agonists, IL-2 ic (IL-2 combined with a monoclonal antibody directed to IL-2), and IL-2 cytokine fused IL-2RA (CD25) ectodomain.

[0060] The term “IL-15” refers to Interleukin- 15, a pleiotropic cytokine with a broad range of biological functions in many diverse cell types. It plays a major role in the development of inflammatory and protective immune responses to microbial invaders and parasites by modulating immune cells of both the innate and adaptive immune systems. It is understood that IL-15 can act on its own or as a heterodimer with IL-15RA, trans-presented on the cell surface or in a soluble form. Functional equivalents of IL- 15 include IL- 15 receptor agonists, IL- 15 receptor agonist antibodies, IL-15 mimics and recombinant IL- 15 fusion proteins such as IL- 15 fused to IL-15RA.

[0061] Figures 2C, 2D and 2E provide schematic representations of IL-2 and IL- 15 cytokine receptor signaling mechanisms. IL-2 and IL- 15 are considered to be functionally equivalent in the methods of the present invention. Thus, in the methods of the present invention, IL-2 is considered to be a functional equivalent of IL- 15, and IL- 15 is considered to be a functional equivalent of IL-2.

[0062] The term “washing" is intended to mean exposing an entity to a solution to the extent that undesired components are removed from the entity to a substantial degree, such that the undesired components do not have an unwanted effect, or detrimental effect, on the entity.

[0063] The term “IL-18" refers to Interleukin- 18, a member of the IL-1 family of cytokines. IL-18 is synthesized as an inactive precursor requiring processing by caspase- 1 into an active cytokine. The IL- 18 precursor is constitutively present in nearly all cells in healthy humans and animals. The activity of IL- 18 is balanced by the presence of a high affinity, naturally occurring IL-18 binding protein (IL-18BP). Functional equivalents of IL-18 include IL-18 receptor agonists, IL-18 receptor agonist antibodies, IL-18 mimics and recombinant IL-18 fusion proteins. A schematic representation of the IL- 12 receptor signaling mechanism is shown in Figure 2B.

[0064] The term “IL-21" refers to Interleukin -21, a type I cytokine produced by T cells and natural killer T cells that has pleiotropic actions on a wide range of immune and non- immune cell types. IL-21 is produced mainly by CD4 + T cells and NKT cells, but it is also produced by CD8 + T cells. Functional equivalents of IL-21 include IL-21 receptor agonists, IL-21 receptor agonist antibodies, IL-21 mimics and recombinant IL-21 fusion proteins. A schematic representation of the IL-21 receptor signaling mechanism is shown in Figure 2F.

[0065] The term “IL-7' refers to Interleukin -7, a cytokine produced by stomal cells. IL-7 contributes to host defense by regulating the development and homeostasis of immune cells, including T lymphocytes, B lymphocytes, and natural killer (NK) cells. Functional equivalents of IL-7 include IL-7 receptor agonists, IL-7 receptor agonist antibodies, IL-7 mimics and recombinant IL-7 fusion proteins. A schematic representation of the IL-7 receptor signaling mechanism is shown in Figure 2G. Due to variations in cytokine manufacturing processes, which encompass diverse storage conditions, biological sources, and other influencing factors, the concentrations of cytokines expressed in weight / volume units may exhibit disparities among commercial vendors. To address this challenge, the National Institute for Biological Standards and Control has instituted the definition of international unit (U) activity standards for various human cytokines. For instance, in the case of human IL-2, the international unit activity is determined through the CTLL-2 mouse cytotoxic T cell activity assay, calibrated with the second international standard for human IL-2 (NIBSC code 86 / 500). Similarly, with regard to human IL- 15, an activity of approximately 25x10A6 lU / mg is ascribed to 1 ng / mL, calibrated using the reference standard for human IL-15 (NIBSC code 95 / 554). Furthermore, for human IL- 12, an activity of around 9xlOA6 HJ / mg is associated with 1 ng / mL, calibrated with the standard for human IL- 12 (NIBSC code 95 / 544). These defined international unit activities serve as standardized benchmarks to mitigate discrepancies in cytokine concentrations arising from various manufacturing processes.

[0066] When characterizing a cell as positive for a specific surface marker, such as "CD69- positive" or "CD25 -positive" it signifies that the corresponding protein is present on the outer membrane or surface of the cell. Therefore, a population of cells that is “30% CD69- positive” signifies that 30% of cells in the population express CD69 on their surface, whereas 70% of cells in the population lack the expression of CD69. This determination is typically made through known techniques like flow cytometry, which involves the use of fluorescently labeled antibodies designed to bind selectively to the designated marker. To validate the specificity of the antibody binding, an isotype-control antibody labeled with the same fluorescent dye is employed as a negative control.

[0067] Method of induction of CIML state

[0068] The present inventors have developed an improved method for producing cytokine-induced memory-like (CIML) immune cells, said method comprising:

[0069] (a) isolating immune cells from a biological sample,

[0070] (b) contacting the immune cells with a first composition that comprises IL- 12 or a functional equivalent thereof, but does not comprise: (i) IL-2 or a functional equivalent thereof,

[0071] (ii) IL- 15 or a functional equivalent thereof, and

[0072] (iii) IL-21 or a functional equivalent thereof;

[0073] (c) washing the immune cells to remove the first composition;

[0074] (d) subsequently contacting the immune cells with a second composition that comprises IL-2 or a functional equivalent thereof, but not IL- 12 or a functional equivalent thereof, thereby producing cytokine-induced memory-like (CIML) immune cells.

[0075] The present inventors have developed an improved method for producing cytokine-induced memory-like (CIML) immune cells, said method comprising:

[0076] (a) isolating immune cells from a biological sample,

[0077] (b) contacting the immune cells with a first composition that comprises IL- 12 or a functional equivalent thereof, but does not comprise:

[0078] (i) IL-2 or a functional equivalent thereof,

[0079] (ii) IL- 15 or a functional equivalent thereof, and

[0080] (iii) IL-21 or a functional equivalent thereof;

[0081] (c) washing the immune cells to remove the first composition;

[0082] (d) subsequently contacting the immune cells with a second composition that comprises IL- 15 or a functional equivalent thereof, but not IL- 12 or a functional equivalent thereof, thereby producing cytokine-induced memory-like (CIML) immune cells.

[0083] Said method of the invention may be an in vivo, ex vivo, or in vitro method. Said method of the invention is associated with the isolation, stimulation and expansion of CIML immune cells. Said method of the invention is associated with increasing the number of CIML immune cells in a biological sample.

[0084] In an embodiment of the invention, the immune cells are contacted with a first composition that comprises IL- 12 or a functional equivalent thereof, but does not comprise:

[0085] (i) IL-2 or a functional equivalent thereof,

[0086] (ii) IL- 15 or a functional equivalent thereof, and (iii) IL-21 or a functional equivalent thereof.

[0087] In an embodiment of the invention, the immune cells are contacted with a first composition that comprises IL- 12 or a functional equivalent thereof, but does not comprise:

[0088] (i) IL-2 or a functional equivalent thereof,

[0089] (ii) IL-7 or a functional equivalent thereof,

[0090] (iii) IL- 15 or a functional equivalent thereof, and

[0091] (iv) IL-21 or a functional equivalent thereof.

[0092] In a preferred embodiment the first composition further comprises IL- 18 or a functional equivalent thereof.

[0093] In an embodiment of the invention, the immune cells are contacted with a second composition that comprises IL-2 or a functional equivalent thereof, but does not comprise IL- 12 or a functional equivalent thereof.

[0094] In an embodiment of the invention, the immune cells are contacted with a second composition that comprises IL- 15 or a functional equivalent thereof, but does not comprise IL- 12 or a functional equivalent thereof.

[0095] In an embodiment of the invention, the second composition comprises IL- 15 or a functional equivalent thereof and IL-7 or a functional equivalent thereof, but does not comprise IL- 12 or a functional equivalent thereof. In a preferred embodiment the second composition comprises IL- 15 or a functional equivalent thereof, and / or IL-7 or a functional equivalent thereof, and / or IL- 18 or a functional equivalent thereof and / or IL-21 or a functional equivalent thereof. In a preferred embodiment the second composition comprises IL- 15 or a functional equivalent thereof, IL-7 or a functional equivalent thereof and IL- 18 or a functional equivalent thereof. In a preferred embodiment the second composition comprises IL- 15 or a functional equivalent thereof, IL-7 or a functional equivalent thereof, IL- 18 or a functional equivalent thereof and IL-21 or a functional equivalent thereof. In a preferred embodiment the second composition comprises IL-2 or a functional equivalent thereof, and further comprises IL- 15 or a functional equivalent thereof. In a preferred embodiment the second composition comprises IL-2 or a functional equivalent thereof, and / or IL- 15 or a functional equivalent thereof, and / or IL-7 or a functional equivalent thereof. In a preferred embodiment the second composition comprises IL-2 or a functional equivalent thereof, and / or IL- 15 or a functional equivalent thereof, and further comprises IL- 18 or a functional equivalent thereof. In a preferred embodiment the second composition comprises IL-2 or a functional equivalent thereof, and / or IL- 15 or a functional equivalent thereof, and / or IL-7 or a functional equivalent thereof, and further comprises IL- 18 or a functional equivalent thereof. In a preferred embodiment the second composition comprises IL-2 or a functional equivalent thereof, and / or IL- 15 or a functional equivalent thereof, and further comprises IL- 18 or a functional equivalent thereof and / or IL-21 or a functional equivalent thereof. In a preferred embodiment the second composition comprises IL-2 or a functional equivalent thereof, and / or IL- 15 or a functional equivalent thereof, and / or IL-7 or a functional equivalent thereof, and further comprises IL- 18 or a functional equivalent thereof and / or IL-21 or a functional equivalent thereof. In a preferred embodiment of the invention, the second composition comprises IL-2 or a functional equivalent thereof, IL- 15 or a functional equivalent thereof, and IL- 18 or a functional equivalent thereof. In a preferred embodiment of the invention, the second composition comprises IL-2 or a functional equivalent thereof, IL- 15 or a functional equivalent thereof, IL-7 or a functional equivalent thereof, and IL- 18 or a functional equivalent thereof. In a preferred embodiment of the invention, the second composition comprises IL-2 or a functional equivalent thereof, IL- 15 or a functional equivalent thereof, IL- 18 or a functional equivalent thereof, and IL-21 or a functional equivalent thereof. In a preferred embodiment of the invention, the second composition comprises IL-2 or a functional equivalent thereof, IL- 15 or a functional equivalent thereof, IL-7 or a functional equivalent thereof, IL- 18 or a functional equivalent thereof, and IL-21 or a functional equivalent thereof.

[0096] In a preferred embodiment of the invention the duration of the contacting step with the first composition is about 1 minute to about 24 hours. In a preferred embodiment, the duration of the contacting step the first composition is about 1 minute to about 23 hours, about 1 minute to about 22 hours, about 1 minute to about 21 hours, about 1 minute to about 20 hours, about 1 minute to about 19 hours, about 1 minute to about 18 hours, about 1 minute to about 17 hours, about 1 minute to about 16 hours, about 1 minute to about 15 hours, about 1 minute to about 14 hours, about 1 minute to about 13 hours, about 1 minute to about 12 hours, about 1 minute to about 11 hours, about 1 minute to about 10 hours, about 1 minute to about 9 hours, about 1 minute to about 8 hours, about 1 minute to about 7 hours, about 1 minute to about 6 hours. In an especially preferred embodiment, the duration of the contacting step with the first composition is about 1 minute to about 6 hours.

[0097] In a preferred embodiment of the invention the immune cells are washed at least once to remove the first composition. In a preferred embodiment of the invention the immune cells are washed once, twice, three times, four times, or five times to remove the first composition. In a preferred embodiment of the invention the immune cells are washed three times to remove the first composition.

[0098] In a preferred embodiment of the invention the duration of the contacting step with the second composition is at least about 6 hours. In a preferred embodiment of the invention the duration of the contacting step with the second composition is at least about 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours or 72 hours.

[0099] In a preferred embodiment of the invention the duration of the contacting step with the second composition is no more than about 72 hours. In an especially preferred embodiment of the invention the duration of the contacting step with the second composition is no more than about 36 hours. In a further embodiment of the invention, the immune cells are washed at least once to remove the second composition. In a preferred embodiment of the invention the immune cells are washed once, twice, three times, four times, or five times to remove the second composition. In a preferred embodiment of the invention the immune cells are washed three times to remove the second composition.

[0100] The methods of the invention may also encompass additional steps subsequent to the contacting step with the second composition. For example, the methods of the invention encompass washing the immune cells after the contacting step with the second composition to remove the second composition, then subsequently contacting the immune cells with the first composition, then washing the immune cells to remove the first composition, then subsequently contacting the immune cells with the second composition. The methods of the invention may thus encompass multiple rounds of the following steps:

[0101] (a) contacting immune cells with the first composition;

[0102] (b) washing the immune cells to remove the first composition;

[0103] (c) contacting the immune cells with the second composition; and

[0104] (d) washing the immune cells to remove the second composition.

[0105] The duration of the contacting steps and the duration and number of wash steps for each round are as set out in the disclosure herein.

[0106] In an embodiment of the invention, the duration of the contacting step with the second composition is more than 72 hours. When the duration of the contacting step with the second composition is more than 72 hours, it is envisaged that the method further comprises washing the immune cells after the contacting step with the second composition to remove the second composition, then subsequently contacting the immune cells with the first composition, then washing the immune cells to remove the first composition, then subsequently contacting the immune cells with the second composition. The embodiment of the invention may thus encompass multiple rounds of the following steps:

[0107] (a) contacting immune cells with the first composition;

[0108] (b) washing the immune cells to remove the first composition;

[0109] (c) contacting the immune cells with the second composition; and (d) washing the immune cells to remove the second composition.

[0110] The duration of the contacting steps and the duration and number of wash steps for each round are as set out in the disclosure herein.

[0111] In a preferred embodiment of the invention the concentration of IL- 12 in the first composition may be about 3 ng / ml to about 30 ng / ml. In a preferred embodiment of the invention the concentration of IL- 12 in the first composition may be about 3 ng / mL, 4 ng / mL, 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 16, about 17 ng / ml, about 18 ng / ml, about 19 ng / ml, about 20 ng / ml, about 21 ng / ml, about 22 ng / ml, about 23 ng / ml, about 24 ng / ml, about 25 ng / ml, about 26, about 27 ng / ml, about 28 ng / ml, about 29 ng / ml or about 30 ng / ml. In a preferred embodiment of the invention, the concentration of IL- 12 in the first composition may be about 20 ng / ml.

[0112] In a preferred embodiment of the invention, the concentration of IL-18 in the first composition may be about 1 to about 100 ng / ml. In a preferred embodiment of the invention, the concentration of IL- 18 in the first composition may be about 5 to about 90 ng / ml, about 10 to about 80 ng / ml, about 15 to about 75 ng / ml, about 20 to about 60 ng / ml, or about 25 ng / ml. In a preferred embodiment of the invention, the concentration of IL- 18 in the first composition may be about 25 ng / ml.

[0113] In a preferred embodiment of the invention the concentration of IL-2 in the second composition may be about 3 U / ml to about 300 U / ml. In a preferred embodiment of the invention, the concentration of IL-2 in the second composition may be about 10 U / ml to about 200 U / ml, may be about 50 U / ml to about 150 U / ml, or about 100 U / ml.

[0114] In a preferred embodiment of the invention the concentration of IL- 15 in the second composition may be about 2 ng / ml to about 50 ng / ml. In a preferred embodiment of the invention, the concentration of IL- 15 in the second composition may be about 5 ng / ml to about 20 ng / ml, may be about 7 ng / ml to about 15 ng / ml, or about 5 ng / ml or about 10 ng / ml. In a preferred embodiment of the invention the concentration of IL-7 in the second composition may be about 2 ng / ml to about 50 ng / ml. In a preferred embodiment of the invention, the concentration of IL-7 in the second composition may be about 5 ng / ml to about 20 ng / ml, may be about 7 ng / ml to about 15 ng / ml, or about 5 ng / ml or about 10 ng / ml.

[0115] In a preferred embodiment of the invention, the concentration of IL- 18 in the second composition may be about 20 to about 100 ng / ml. In a preferred embodiment of the invention, the concentration of IL- 18 in the second composition may be about 25 to about 90 ng / ml, about 40 to about 80 ng / ml, about 50 to about 75 ng / ml, about 50 to about 60 ng / ml, or about 50 ng / ml.

[0116] In a preferred embodiment of the invention, the concentration of IL-21 in the second composition may be about 2 to about 100 ng / ml. In a preferred embodiment of the invention, the concentration of IL-21 in the second composition may be about 10 to about 50 ng / ml, about 15 to about 30 ng / ml, or about 20 ng / ml.

[0117] In a preferred embodiment of the invention, the method further comprises a rest step preceding the step of contacting the immune cells with the second composition. It is understood that during the rest step the immune cells are not in contact with a stimulating cytokine and the duration of the rest step can be determined by the person skilled in the art in view of their common general knowledge. In a preferred embodiment of the invention, the rest step is about 1 minute to 10 hours in duration. In a preferred embodiment of the invention, the rest step is about 1 hour to 10 hours in duration, about 2 hours to 10 hours in duration, about 3 hours to 10 hours in duration, about 4 hours to 10 hours in duration, about 5 hours to 10 hours in duration, about 6 hours to 10 hours in duration, about 7 hours to 10 hours in duration, about 8 hours to 10 hours in duration, about 9 hours to 10 hours in duration, or about 10 hours in duration.

[0118] In a preferred embodiment of the invention, the method further comprises expanding the CIML immune cells produced by the method of the invention. It is understood that the conditions for expansion can be determined by the person skilled in the art in view of their common general knowledge.

[0119] In a preferred embodiment of the invention, the immune cells are isolated from the biological sample that comprises at least one of the following cell types: T cells, CD4+ T cells, CD8+ T cells, 78 T cells, NK cells, NKT cells, dendritic cells, monocytes and macrophages.

[0120] In a preferred embodiment of the invention the method produces at least one of the following cell types: cytokine-induced memory-like T cells, cytokine-induced memorylike CD4+ T cells, cytokine-induced memory-like CD8+ T cells, cytokine-induced memory-like yS T cells, cytokine-induced memory-like NK cells, cytokine-induced memory-like NKT cells, cytokine-induced memory-like dendritic cells, cytokine-induced memory-like monocytes and cytokine-induced memory-like macrophages. In a preferred embodiment of the invention the method produces more than one of the cell types set out above.

[0121] In a preferred embodiment of the invention the method is for the production of cytokine- induced memory-like (CIML) NK cells. In a preferred embodiment of the invention the method is for the production of cytokine-induced memory-like (CIML) T cells.

[0122] In an embodiment of the invention, more than one type of CIML immune cell is produced by the method of the invention. In a preferred embodiment of the invention the method produces at least two of the following cell types: cytokine-induced memory-like T cells, cytokine-induced memory-like CD4+ T cells, cytokine-induced memory-like CD8+ T cells, cytokine-induced memory-like 78 T cells, cytokine-induced memory-like NK cells, cytokine-induced memory-like NKT cells, cytokine-induced memory-like dendritic cells, cytokine-induced memory-like monocytes and cytokine-induced memory-like macrophages. In an embodiment of the invention, the cytokine-induced memory-like (CIML) immune cells may include cytokine-induced memory-like T cells, cytokine-induced memory-like CD4+ T cells, cytokine-induced memory-like CD8+ T cells, cytokine-induced memorylike y8 T cells, cytokine-induced memory-like NK cells, cytokine-induced memory-like NKT cells, cytokine-induced memory-like dendritic cells, cytokine-induced memory-like monocytes and cytokine-induced memory-like macrophages.

[0123] In an embodiment of the invention, the cytokine-induced memory-like (CIML) immune cells may include CD45RA+T and NK cells.

[0124] In a preferred embodiment of the invention, the method further comprises modification of the CIML immune cells produced by the method of the invention. In a preferred embodiment, the method comprises transducing the CIML immune cells with a vector. In a preferred embodiment, the vector is a viral vector. In a preferred embodiment, the viral vector is a lentiviral vector, alpha retroviral vector or a gamma retroviral vector. In a preferred embodiment, the viral vector comprises a viral vector envelope that is pseudotyped with baboon endogenous retrovirus envelope glycoprotein (BaEV), VSV-G, RD114, GalV, H / F, G / F, or COCV envelope glycoproteins. In a preferred embodiment of the invention, the vector encodes for a chimeric antigen receptor (CAR), CD16A protein, or a modified CD16A protein. In an embodiment of the invention, the viral vector is packaged within a liposome, lipid nanoparticle, virus, virus-like particle or is delivered to the cell by microcarrier-mediated delivery.

[0125] Compositions

[0126] The present invention also encompasses a composition comprising IL-2 or a functional equivalent thereof, IL- 15 or a functional equivalent thereof, and IL- 18 or a functional equivalent thereof. In a preferred embodiment the composition further comprises IL-7 or a functional equivalent thereof. In a preferred embodiment the composition further comprises IL-21 or a functional equivalent thereof. In said compositions of the invention, IL-2 or a functional equivalent thereof, IL- 15 or a functional equivalent thereof, IL- 18 or a functional equivalent thereof, IL-7 or a functional equivalent thereof, and IL-21 or a functional equivalent thereof may be present in the concentrations as set out herein. S

[0127] The present invention also encompasses a composition comprising IL- 12 or a functional equivalent thereof, IL- 15 or a functional equivalent thereof, and IL- 18 or a functional equivalent thereof. In a preferred embodiment the composition further comprises IL-7 or a functional equivalent thereof. In a preferred embodiment the composition further comprises IL-21 or a functional equivalent thereof. In said compositions of the invention, IL- 12 or a functional equivalent thereof, IL- 15 or a functional equivalent thereof, IL- 18 or a functional equivalent thereof, IL-7 or a functional equivalent thereof, and IL-21 or a functional equivalent thereof may be present in the concentrations as set out herein.

[0128] CIML cells

[0129] The present invention encompasses cytokine-induced memory-like (CIML) immune cells produced by the methods of the invention. The present invention encompasses cytokine- induced memory-like (CIML) immune cells producible by the methods of the invention. The present invention encompasses cytokine induced memory-like (CIML) immune cells produced by the methods of the invention wherein more than 20% of the cells in the population are CD25-positive, more than 30% of the cells in the population are CD71- positive, and more than 70% of the cells in the population are CD69-positive. In a preferred embodiment of the invention, the CIML immune cells are NK cells or T cells. In a preferred embodiment of the invention, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% of the cells in the population are CD25-positive, at least 30%, at least 40%, at least 50%, at least 60% or at least 70% of the cells in the population are CD71 -positive, and at least 70%, at least 75%, at least 80%, at least 85% or at least 90% of the cells in the population are CD69-positive.

[0130] The present invention encompasses a population of cytokine induced memory-like (CIML) immune cells wherein more than 20% of the cells in the population are CD25-positive, more than 30% of the cells in the population are CD71 -positive, and more than 70% of the cells in the population are CD69-positive. In a preferred embodiment of the invention, the CIML immune cells are NK cells or T cells. In a preferred embodiment of the invention, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% of the cells in the population are CD25-positive, at least 30%, at least 40%, at least 50%, at least 60% or at least 70% of the cells in the population are CD71 -positive, and at least 70%, at least 75%, at least 80%, at least 85% or at least 90% of the cells in the population are CD69- positive.

[0131] In a preferred embodiment, the CIML immune cells of the invention or the population of CIML immune cells of the invention further comprise a vector. In a preferred embodiment, the vector is a viral vector. In a preferred embodiment, the viral vector is a lentiviral vector, alpha retroviral vector or a gamma retroviral vector. In a preferred embodiment, the viral vector comprises a viral vector envelope that is pseudotyped with baboon endogenous retrovirus envelope glycoprotein (BaEV), VSV-G, RD114, GalV, H / F, G / F, or COCV envelope glycoproteins. In a preferred embodiment of the invention, the vector encodes for a chimeric antigen receptor (CAR), CD16A protein, or a modified CD16A protein. In an embodiment of the invention, the viral vector is packaged within a liposome, lipid nanoparticle, virus, virus-like particle or is delivered to the cell by microcarrier-mediated delivery.

[0132] Pharmaceutical compositions

[0133] Also disclosed is a pharmaceutical composition comprising a CIML immune cell of the invention, or the population of CIML immune cells of the invention, and a pharmaceutically acceptable carrier. Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. The solution should be RNAse free. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

[0134] Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.

[0135] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.

[0136] Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0137] Treatment applications

[0138] The CIML immune cells disclosed herein can be used in medicine. The present invention encompasses a method of treating an individual in need thereof with a CIML immune cell, comprising administering to the individual a therapeutically effective amount of the CIML immune cell of the invention, or the CIML immune cells produced, or producible, according to the methods of the invention, or the population of CIML immune cells of the invention, or the pharmaceutical composition of the invention.

[0139] The present invention encompasses a CIML immune cell of the invention or produced, or producible, according to the methods of the invention, or population of CIML immune cells of the invention, or the pharmaceutical composition of the invention, for use in a method of treating an individual in need thereof with a CIML immune cell, comprising administering to the individual a therapeutically effective amount of the CIML immune cell of the invention, the population of CIML immune cells of the invention, or the pharmaceutical composition of the invention.

[0140] The present invention encompasses a CIML immune cell of the invention or produced, or producible, according to the methods of the invention, or population of CIML immune cells of the invention, or the pharmaceutical composition of the invention, for use in the manufacture of a medicament for use in a method of treating an individual in need thereof with a CIML immune cell, comprising administering to the individual a therapeutically effective amount of the CIML immune cell of the invention, the population of CIML immune cells of the invention, or the pharmaceutical composition of the invention.

[0141] The CIML immune cells disclosed herein can be used in the treatment or prevention of progression of proliferative diseases such as cancers and myelodysplastic syndromes. The cancer may be a hematologic malignancy or a solid tumor. Hematologic malignancies include leukemias, lymphomas, multiple myeloma, and subtypes thereof. Lymphomas can be classified in various ways, often based on the underlying type of malignant cell, including Hodgkin’s lymphoma (often cancers of Reed-Sternberg cells, but also sometimes originating in B cells; all other lymphomas are non-Hodgkin’ s lymphomas), nonHodgkin’s lymphomas, B-cell lymphomas, T-cell lymphomas, mantle cell lymphomas, Burkitt’s lymphoma, follicular lymphoma, and others as defined herein and known in the art. Myelodysplastic syndromes comprise a group of diseases affecting immature leukocytes and / or hematopoietic stem cells (HSCs); MDS may progress to AML.

[0142] B-cell lymphomas include, but are not limited to, diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), and others as defined herein and known in the art. T-cell lymphomas include T-cell acute lymphoblastic leukemia / lymphoma (T-ALL), peripheral T-cell lymphoma (PTCL), T-cell chronic lymphocytic leukemia (T-CLL), Sezary syndrome, and others as defined herein and known in the art.

[0143] Leukemias include acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (sometimes classified as a lymphoma), and others as defined herein and known in the art.

[0144] Plasma cell malignancies include lymphoplasmacytic lymphoma, plasmacytoma, and multiple myeloma.

[0145] Solid tumors include renal carcinoma, melanomas, neuroblastomas, gliomas or carcinomas such as tumors of the brain, head and neck, breast, lung (e.g., non-small cell lung cancer, NSCLC), reproductive tract (e.g., ovary), upper digestive tract, pancreas, liver, renal system (e.g., kidneys), bladder, prostate and colorectum.

[0146] Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing, or at risk of progressing to a later stage of, cancer. A determination of the need for treatment will typically be assessed by a history, physical exam, or diagnostic tests consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans, or other animals such as chickens. For example, the subject can be a human subject. Generally, a safe and effective amount of a therapy is, for example, an amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects.

[0147] According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration. Where the product is a cell therapy, the mode of administration will likely be via injection or infusion.

[0148] The therapeutic methods of the invention also encompass use of a CIML immune cell that is derived from an autologous cell of an individual to be treated.

[0149] EXAMPLES

[0150] EXAMPLE 1 : Materials and Methods

[0151] Isolation of Natural Killer (NK) cells and Natural Killer T (NKT) cells from healthy donors. NK and NKT cell isolation was performed using negative magnetic bead selection from one-day-old Leukocyte Reduction System (LRS) cone blood and same-day fresh Whole Blood. The LRS cone blood, approximately 8 mL in total volume, was flushed from the cone, pelleted using centrifugation and resuspended with 8 mL of Buffer A (Phosphate- Buffered Saline (PBS) supplemented with 2% (v / v) Fetal Bovine Serum (FBS) and 1 unit / mL Heparin, pH 7.4. Similarly, the fresh whole blood sample (150 mL total volume) was divided into 50 mL tubes and diluted 1:1 (v / v) with Buffer A. Peripheral Blood Mononuclear Cells (PBMCs) were separated by centrifugation using a density gradient medium, Lymphoprep (STEMCELL Technologies). Following Lymphoprep separation, PBMCs were resuspended at a density 5xlOA7 cells / mL in Buffer B (PBS, 2% FBS, and 1 mM EDTA, pH 7.4). Subsequently, human NK and NKT cells were isolated from PBMCs utilizing the EasySep Human NK Cell Isolation Kit (STEMCELL) and the Easy 50 EasySep Magnet (STEMCELL) according to manufacturer instructions. The isolated cells were then resuspended at a density of 2-3xlOA6 cells / mL in complete NK MACS media. The complete NK MACS media composition included NK MACS basal medium (Miltenyi Biotec), 10% Human AB serum (Sigma- Aldrich), 1% NK MACS supplement (part of the NK MACS basal medium kit), 100 pM nicotinamide (Sigma-Aldrich), and 1% Penicillin / Streptomycin (ThermoFisher Scientific) and various cytokines were added as indicated in the figure legends.

[0152] NK and NKT cell isolation from healthy donor leukopaks was achieved using CD56 positive magnetic bead selection. The leukapheresis blood underwent an initial wash using Buffer B (PBS, 2 mM EDTA, 0.5% FBS, pH 7.4). Subsequently, NK and NKT cells were directly isolated from the sample through positive magnetic selection using the StraightFrom Leukopak REAlease CD56 MicroBead Kit (Miltenyi Biotec) and the MultiMACS Cell 24 Separator Plus (Miltenyi Biotec) according to manufacturer instructions. The CD56+ cells were labeled with StraightFrom Leukopak REAlease CD56-Biotin (REAlease Biotin Complex). Following labeling, StraightFrom Leukopak REAlease Anti-Biotin MicroBeads were introduced to bind to the REAlease Biotin Complex. The resulting cell suspension was loaded onto a Multi-24 Column Block and placed in the magnetic field of the MultiMACS Cell 24 Separator Plus (Miltenyi Biotec). In magnetic field, the magnetically labeled NK and NKT cells were retained within the columns. Upon removal from the magnetic field, the columns were subjected to elution using the REAlease Bead release reagent, facilitating the release of NK and NKT cells while simultaneously removing the MicroBeads from the cells. Finally, the isolated cells were washed and resuspended at a density of 2xlOA6 cells / mL in complete NK MACS media and various recombinant cytokines were supplemented as indicated in the figure legends.

[0153] Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs) from Whole Blood of Healthy Donors.

[0154] Fresh whole blood samples from healthy donors were obtained from Research Blood Components (Watertown, MA). The whole blood was diluted 1 :1 with Dulbecco's Phosphate-Buffered Saline (DPBS) supplemented with 2% (v / v) heat- inactivated fetal bovine serum (FBS, Gibco). The diluted blood was gently layered over a Lymphoprep medium in SepMate-50 tubes (STEMCELL Technologies) and centrifuged at 1 ,200xg for 20 minutes at room temperature to isolate peripheral blood mononuclear cells (PBMCs). Following centrifugation, the plasma and PBMC layers were carefully transferred to a fresh 50 mL tube. The cells were pelleted by centrifugation at 600xg for 5 minutes, the supernatant was discarded, and the cell pellet was washed once with DPBS / 2% FBS buffer. The cells were centrifuged again and resuspended in 10 mL of pre-warmed complete RPMI medium. The total number of PBMCs was counted using Tuerk’s solution (Sigma- Aldrich, catalog number 1092770100), and a small volume of the PBMC suspension was used for flow cytometry to determine the percentage and viability of T cells, B cells, NK cells, and monocytes.

[0155] After counting, the cells were pelleted by centrifugation at 600xg for 5 minutes, and the supernatant was discarded. The cell pellet was then resuspended in CryoStor CS 10 solution (BioLife Solutions, catalog number 210502) at a concentration of 50 million PBMCs per mL. The cell suspension was aliquoted into cryovials, frozen in a controlled-rate freezer, and stored in a liquid nitrogen container until further use. For flow cytometry, the following antibodies were used: Brilliant Violet 605 anti-human CD45 (clone 2D1, BioLegend), FITC anti-human CD3 (clone OKT3, BioLegend), APC anti-human CD19 (clone HIB19, BioLegend), PE / Cyanine7 anti-human CD14 (clone S18004B, BioLegend), APC antihuman CD45RA (clone HI100, BioLegend) and PE anti-human CD56 (clone QA17A16, BioLegend). DAPI stain (4',6-diamidino-2-phenylindole, Thermo Fisher, catalog number DI 306) was used to evaluate cell viability. Prior to antibody staining, Fc receptors were blocked using Human TruStain FcX (BioLegend, catalog number 422301).

[0156] Isolation of T cells from healthy donors

[0157] Frozen peripheral blood mononuclear cells (PBMCs) stored in cryovials were thawed by placing the cryovial in a 37°C water bath for 2 minutes. The cell suspension was then transferred to a clean centrifuge tube, diluted 1:10 with pre-warmed complete RPMI, and centrifuged at 600xg for 5 minutes at room temperature. After centrifugation, the supernatant was completely aspirated, and the cell pellet was resuspended in 5 mL of complete RPMI. Viable cells were counted at this stage. To isolate T cells, CD3 MicroBeads and the MultiMACS Cell24 Separator (Miltenyi Biotec) were used. The PBMCs were centrifuged again at 600xg for 5 minutes and resuspended at a concentration of lOOxlO6cells / mL in cold Wash Buffer (Dulbecco’s phosphate-buffered saline without calcium and magnesium, pH 7.2, supplemented with 2 mM EDTA and 2% (v / v) heat-inactivated fetal bovine serum). CD3 MicroBeads (200 pL per lOOxlO6cells) were added to the suspension, mixed thoroughly, and incubated in the cold room for 15 minutes.

[0158] After incubation, the cells were washed twice with cold Wash Buffer and resuspended at lOOxlO6cells / mL. The suspension was applied to a pre-equilibrated LS column (Miltenyi Biotec) using 1 LS column per lOOxlO6cells. The column was washed with 15 mL of Wash Buffer, and the CD3+ T cells were eluted and washed-off the column with additional Wash Buffer with magnetic field turned off. The eluted cells were centrifuged at 600xg for 5 minutes and resuspended either in complete NK MACS medium or other medium such as TexMACS or Human Plasma-Like Medium (Miltenyi Biotec, catalog number 130-097-196 or Gibco, catalog number A4899101). The purity and viability of the isolated T cells were assessed using flow cytometry. Following isolation, T cells were activated with the indicated cytokines.

[0159] Isolation of CD45RA-positive T and NK cells from healthy donors

[0160] Frozen peripheral blood mononuclear cells (PBMCs) stored in cryovials were thawed by placing the cryovial in a 37°C water bath for 2 minutes. The cell suspension was then transferred to a clean centrifuge tube, diluted 1:10 with pre-warmed complete RPMI, and centrifuged at 600xg for 5 minutes at room temperature. After centrifugation, the supernatant was completely aspirated, and the cell pellet was resuspended in 5 mL of complete RPMI. Viable cells were counted at this stage. To isolate CD45RA+ cells, CD 19- and CD45RA- MicroBeads and the MultiMACS Cell24 Separator (Miltenyi Biotec) were used. The PBMCs were centrifuged again at 600xg for 5 minutes and resuspended at a concentration of lOOxlO6cells / mL in cold Wash Buffer (Dulbecco’s phosphate-buffered saline without calcium and magnesium, pH 7.2, supplemented with 2 mM EDTA and 2% (v / v) heat- inactivated fetal bovine serum). Human CD19 MicroBeads (200 pL per lOOxlO6cells) were added to the suspension, mixed thoroughly, and incubated in the cold room for 15 minutes. After incubation, the cells were washed twice with cold Wash Buffer and resuspended at lOOxlO6cells / mL. The suspension was applied to a pre-equilibrated LD column (Miltenyi Biotec) using 1 LD column per lOOxlO6cells. The flowthrough was collected and the column was washed with 15 mL of Wash Buffer, The B cell-depleted cell suspension was collected and centrifuged at 600xg for 5 minutes. The cell pellet was resuspended at a concentration of lOOxlO6cells / mL in cold Wash Buffer and human CD45RA MicroBeads (200 pL per 100x106cells) were added to the suspension, mixed thoroughly, and incubated in the cold room for 15 minutes. The suspension was applied to a pre-equilibrated LS column (Miltenyi Biotec) using 1 LS column per lOOxlO6cells. The column was washed with 15 mL of Wash Buffer, and the CD45RA+ cells were eluted and washed-off the column with additional Wash Buffer with magnetic field turned off. The eluted cells were centrifuged at 600xg for 5 minutes and resuspended either in complete NK MACS medium. The purity and viability of the isolated CD45RA+ cells were assessed using flow cytometry. Following isolation, cells were activated with the indicated cytokines.

[0161] Immunophenotyping. For cell surface antibody staining of NK and NKT cells, 50,000- 100,000 cells were washed and resuspended in 200 pL of FACS buffer (PBS supplemented with 2% FBS). 5 pL of Human TruStain (BioLegend) was added to the sample and incubated for 5 min at room temperature to block non-specific binding of IgG antibodies. Cells were incubated with antibodies (2 pL each) on ice for 15-20 minutes, washed twice with 1 mL of FACS buffer, and after the final wash resuspended with 200 pL of FACS buffer supplemented with DAPI (1 pg / mL) and placed on ice before flow cytometry analysis. The Flow Cytometry data was analyzed using Flow-Jo software (BD Biosciences).

[0162] The following antibodies were used to determine the purity of NK and NKT cells: Brilliant Violet 605 anti-human CD45 (clone 2D1 , BioLegend), FITC anti-human CD3 (clone OKT3, BioLegend), APC anti-human CD19 (clone HIB19, BioLegend) and PE anti-human CD56 (clone QA17A16, BioLegend). Live NK cells were defined as DAPI", CD45+, CD56+, CD 19’ , CD3 and live NKT cells were defined as DAPI’, CD45+, CD56+, CD19 , CD3+.

[0163] The following antibodies were used for the analysis of NK and NKT cell phenotype: FITC anti-human CD25 (clone BC96, Biolegend), PE anti-human CD71 (clone CY1G4, Biolegend), FITC anti-human NKp46 (clone 9E2, Biolegend), PE anti -human CD 16 (clone 3G8, Cell Signaling Technology), FITC anti-human CD70 (clone 113-16, Biolegend), PE anti-human NKG2D (clone 1D11, Biolegend), APC anti-human CD94 (clone DX22, Biolegend), Brilliant Violet 605 anti-human 4-1BB (clone 4B4-1, Biolegend), APC antihuman CD69 (clone FN50, Biolegend), PE anti-human NKG2C (clone S19005E, Biolegend), APC anti-human NKp44 (clone P44-8, Biolegend), APC anti-human TRAIL (clone RIK-2, Biolegend).

[0164] For the analysis NK and NKT cell viability, cells were washed twice with Annexin V Buffer (10 mM HEPES-NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCh), and resuspended with 200 pL Annexin V Buffer supplemented with APC Annexin V (2 pL, BioLegend) and DAPI. Live cells were defined as Annexin V, DAPL.

[0165] Manufacturing of cytokine-induced memory-like (CIML) NK and NKT cells using the standard protocol. Freshly isolated population of human NK and NKT cells was resuspended at 2xl06cells / mL in complete NK MACS media and placed in a sterile, tissue culture-treated 6-well polystyrene plate at 2 mL total volume per well. The media was then supplemented with IL-12 (10 ng / mL, Miltenyi Biotec), IL-15 (50 ng / mL, Miltenyi Biotec), and IL- 18 (50 ng / mL, BioLegend), and the plate was placed in a 37°C tissue culture incubator for 16 hours. After 16 hours, the cells were washed 3 times with phosphate - buffered saline and resuspended in NK MACS media supplemented with either 300 U / mL IL-2 or 10 ng / mL IL- 15 at 2xl06cells / mL and placed in a fresh 6-well plate.

[0166] Manufacturing of cytokine-induced memory-like (CIML) NK and NKT cells using the optimized protocol. Freshly isolated population of human NK and NKT cells was resuspended at 2xl06cells / mL in complete NK MACS media and placed in a sterile, tissue culture-treated 6-well polystyrene plate at 2 mL total volume per well. The media was then supplemented with 20 ng / mL IL- 12 and 25 ng / mL IL- 18 and the plate was placed in a 37°C tissue culture incubator for 6 hours. After 6 hours, the cells were washed three times with PBS to remove the cytokines and then resuspended at 2xl06cells / mL in complete NK MACS media and placed in a fresh, sterile, tissue culture-treated 6-well polystyrene plate at 2 mL total volume per well. The media was then supplemented with 5 ng / mL IL- 15 and 50 ng / mL IL-18 and the plate was placed in a 37°C tissue culture incubator for 72 hours.

[0167] Manufacturing of cytokine-induced memory-like (CIML) T cells using the optimized CIML protocol.

[0168] Freshly isolated population of human T cells was resuspended at 2xl06cells / mL in complete NK MACS media and placed in a sterile, tissue culture-treated 6-well polystyrene plate at 2 mL total volume per well. The media was then supplemented with 20 ng / mL IL- 12 and 25 ng / mL IL- 18 and the plate was placed in a 37°C tissue culture incubator for 6 hours. After 6 hours, the cells were washed three times with PBS supplemented with 5% (w / v) human serum albumin to remove the cytokines and then resuspended at 2xl06cells / mL in complete NK MACS media and placed in a fresh, sterile, tissue culture-treated 6-well polystyrene plate at 2 mL total volume per well. The media was then supplemented with 10 ng / mL IL- 15, 10 ng / mL IL-7 and 50 ng / mL IL-18 and the plate was placed in a 37°C tissue culture incubator for 72 hours. The viability and phenotype of cells was at that point evaluated using the methods described above.

[0169] Manufacturing of cytokine-induced memory-like (CIML) CD45RA+ T and NK cells using the optimized CIML protocol.

[0170] Freshly isolated population of B cell-depleted CD45RA+ cells was resuspended at 2xl06cells / mL in complete NK MACS media and placed in a sterile, tissue culture-treated 6-well polystyrene plate at 2 mL total volume per well. The media was then supplemented with 20 ng / mL IL- 12 and 25 ng / mL IL- 18 and the plate was placed in a 37°C tissue culture incubator for 6 hours. After 6 hours, the cells were washed three times with PBS supplemented with 5% (w / v) human serum albumin to remove the cytokines and then resuspended at 2xl06cells / mL in complete NK MACS media and placed in a fresh, sterile, tissue culture-treated 6-well polystyrene plate at 2 mL total volume per well. The media was then supplemented with 10 ng / mL IL- 15, 10 ng / mL IL-7, 10 ng / mL IL-21 and 50 ng / mL IL- 18 and the plate was placed in a 37°C tissue culture incubator for 72 hours. The viability and phenotype of cells was at that point evaluated using the methods described above.

[0171] NK and NKT cell expansion and persistence in vivo. Experiments involving mice were performed in accordance with approved IACUC protocol. The injected human NK cells were manufactured for the experiment as described above. In all experiments, male NOD.Cg- Prkdcsc,dIL2rgtmlWjITg(IL-15)lSz / SzJ mice (Jackson Laboratories), referred to as huIL-15 Tg NSG, of 6-12 weeks of age were used. On day 0, mice were injected intravenously (i.v.) with human NK cells resuspended in USP-grade PBS. Additionally, where indicated, mice received supplemental intraperitoneal (i.p.) injections of human recombinant IL-2 (75,000 U / injection) every 3 days. All mice were weighed, monitored and body condition (BC) scored every 3 days. Where indicated, peripheral blood was collected via the submandibular vein for evaluation of NK cell persistence. 100 pL of blood was collected, red blood cells were lysed, and remaining cells were stained for the presence of surface antigens. At endpoint, all mice were sacrificed, and cardiac blood, spleen and bone marrow were harvested and processed. All samples were stained with the following antibodies: Brilliant Violet 605 anti-human CD45 (BioLegend), APC anti-human CD3 (BioLegend), and PE antihuman CD56 (BioLegend), as described above, then analyzed by flow cytometry on the Attune NxT Flow Cytometer (ThermoFisher Scientific). Data was analyzed with the use of FlowJo Software (BD Biosciences).

[0172] T and NK cell cytotoxicity assay using suspension cell lines. T, NK and NKT cells were purified and stimulated with cytokines as described above. Nalm6 (acute lymphoblastic leukemia), Raji (Burkitt lymphoma) and OCI-AML3 (acute myeloid leukemia) cell lines were obtained from the American Type Culture Collection (ATCC) and transduced with lentiviral particles harboring Luciferase-P2A-mScarlet transgene. Transduced cancer cells were purified using flow-cytometry assisted sorting of mScarlethlcells, and used in subsequent experiments. For cytotoxicity experiments using Nalm6, Raji and OCLAML3 cell lines, 100,000 cancer cells / well were seeded in a 96- well round-bottom plate in complete NK MACS media supplemented with 2 ng / mL IL- 15 in 100 uL volume, or where indicated seeded in complete NK MACS media without added cytokines. CIML NK and NKT cells were added to each well at indicated effector: target ratio (for 1 : 1 100,000 effector cells, etc.) in a 100 uL volume in complete NK MACS media without cytokines or supplemented with 2 ng / mL IL-15. The plate was centrifuged at 300xg for 1 minute to allow cells to settle to the bottom of the well, and the plate was then placed in a 37°C tissue culture incubator for 16 hours. After 16 hours, the cells were washed twice using Annexin V Buffer and resuspended in 200 pL of Annexin V Buffer supplemented with APC Annexin V, DAPI, and Precision Count Beads (5,000 beads per well, BioLegend). The percentage of live cancer cells was determined using flow cytometry, live cells defined as DAPTAnnexinV mScarlet+single cells, normalized to the number of counting beads per well, and divided by the number of cancer cells incubated without T, NK and NKT cells.

[0173] NK cell cytotoxicity assay using adherent cell lines.

[0174] 786-0 (clear cell renal carcinoma), ACHN (renal adenocarcinoma), and A-498 (renal carcinoma) cell lines were obtained from the American Type Culture Collection (ATCC) and transduced with lentiviral particles harboring Luciferase-P2A-mScarlet transgene. Transduced cancer cells were purified using flow-cytometry assisted sorting of mScarlet111expressing cells and used in subsequent experiments. 50,000 cancer cells / well were seeded in a 96-well flat-bottom tissue culture plate in 200 uL of RPMI media supplemented with 10% FBS, 1% penicillin / streptomycin, and 2 mM 1-Glutamine. The cells were grown for 48 hours and allowed to reach confluency, at which point the media was changed to 100 pL of complete NK MACS media without added cytokines or supplemented with 2 ng / mL IL- 15. CIML T, NK and NKT cells were added to each well at indicated effector: target ratio (for 1:1 200,000 effector cells were used, number determined in previous experiments) in a 100 uL volume in complete NK MACS media without cytokines or supplemented with 2 ng / mL IL-15, and the plate was placed in a 37°C tissue culture incubator for 16 hours. After 16 hours, the cells were lifted from the well by pipetting and transferred to a round-bottom 96- well plate. The cells were washed twice with PBS, and trypsinized for 5 min at 37°C to achieve single-cell suspension. After trypsinization, the cells were washed again using Annexin V Buffer and resuspended in 200 pL of Annexin V Buffer supplemented with APC Annexin V, DAPI, and Precision Count Beads (5,000 beads per well, BioLegend). The percentage of live cancer cells was determined using flow cytometry. Live cells were defined as DAPrAnnexinV mScarlet+single cells, normalized to the number of counting beads per well, and divided by the number of cancer cells incubated without NK cells.

[0175] Production of lentiviral vector particles pseudotyped with BaEV-Rless envelope

[0176] The adherent 293T human embryonic kidney cell line (ATCC, catalog number CRL-3216) was trypsinized, washed once with phosphate-buffered saline (PBS) to remove trypsin, and transferred to FreeStyle 293 Expression Medium (Gibco, catalog number 12-338-018) supplemented with 100 U / mL penicillin, 100 pg / mL streptomycin, 2% (v / v) heat- inactivated fetal bovine serum, and 33 pM phenol red (complete FreeStyle medium). The cells were adjusted to a density of 0.5xl06cells / mL and expanded in the presence of anti-clumping agent (ACA, used at a 2,000x dilution, Gibco, catalog number 0010057 AE) at 37°C in a tissue-culture shaker with 8% CO2 (shaking at 120 revolutions per minute). Cell cultures were expanded until they reached a maximum density of 3xl06cells / mL, at which point they were split into fresh complete FreeStyle medium at 0.5xl06cells / mL and continued to expand with ACA. Viability and media color were monitored every 2-3 days, with media exchanges performed as necessary. Cells were expanded using this protocol for at least one week prior to transfection with PEL

[0177] On the day of transfection, cells were counted, centrifuged at 600xg for 5 minutes, washed twice with Dulbecco’s PBS (DPBS) to remove residual ACA, resuspended in fresh prewarmed complete FreeStyle medium without ACA at a density of 1.5xl06cells / mL, and returned to the 37°C shaker. DNA plasmids were mixed at the following molar ratios: 1 mol of psPAX2, 1.2 mol of the transfer plasmid, and 0.5 mol of the BaEV-Rless envelope plasmid (pMD2.G in which VSV-G is replaced with BaEV-Rless), using 1.5 pg of total plasmid DNA per million cells. Opti-MEM was added to the plasmid mixture up to 5% of the total cell suspension volume, and the DNA mixture was resuspended thoroughly. In a separate tube, PEI (at a 1 : 1 w / w ratio relative to total plasmid DNA) was resuspended in Opti-MEM, also at 5% of the total cell suspension volume, and incubated for 5 minutes at room temperature. The PEI solution was then combined with the DNA mixture, gently mixed, and incubated at room temperature for 10 minutes to allow for complex formation. The cell suspension was removed from the 37°C shaker, and the PEI / DN A complex mixture was added dropwise to the cells. After 16 hours, the cells were centrifuged, and the medium was replaced with an equal volume of fresh, pre-warmed complete FreeStyle medium supplemented with ACA.

[0178] Forty-eight hours post-transfection, cells were centrifuged, and the supernatant containing lentiviral particles was harvested and stored at 4°C. The cells were resuspended in an equal volume of fresh, pre-warmed complete FreeStyle medium supplemented with ACA. Seventy-two hours post-transfection, the cells were again centrifuged, and the supernatant was collected. Both supernatants were pooled and filtered through a Sartolab 0.45 pm PES filtration unit with diatomaceous earth as a filtration aid (Sartorius). Lentiviral particles were concentrated by centrifugation at 10,000xg for 16 hours at 4°C. After centrifugation, the supernatant was discarded, and the pellet containing lentiviral particles was resuspended in 1 mL of lentiviral vector resuspension buffer (100 mM NaCl, 1% w / v sucrose, 1% w / v mannitol, 20 mM Tris-HCl pH 7.3). The resuspended lentiviral particles were aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C until use. The Jurkat cell line (Clone E6-1, ATCC catalog number TIB- 152) was cultured in RPMI 1640 Medium (ATCC modification, Gibco catalog number A1049101) supplemented with 100 U / mL penicillin, 100 g / mL streptomycin, and 10% (v / v) heat-inactivated fetal bovine serum (complete RPMI). During the expansion phase, cells were maintained in a 37°C CO2 tissue-culture incubator and split every 2-3 days, ensuring a maximum density of approximately 3x10A6 cells / mL. Cells were not used beyond 30 passages. To determine the infectious titer of BaEV-Rless-pseudotyped lentiviral particles, Jurkat cells were centrifuged at 600xg for 5 minutes and resuspended in fresh, pre-warmed complete RPMI at a density of 1x106cells / mL. A 2 mL aliquot of the cell suspension (2xlOA6 cells) was transferred to each well of a 6- well tissue-culture plate (Corning catalog number 3516). Various volumes of the lentiviral vector (LVV) particle suspension were added to the wells in a dilution series, typically 5 pL, 1 pL, 0.1 pL, 0.05 pL, and 0.025 pL, with one well left untreated to serve as a negative control. Cells were transduced using spinoculation at l,000xg for 1 hour at 37°C, after which the plate was returned to the 37°C CO2 incubator. The following day, 1.5 mL of medium was aspirated from each well and replaced with 1.5 mL of fresh, pre- warmed complete RPMI. Forty-eight hours post-transduction, cells from each well were transferred to a 96-well round-bottom plate, washed once with FACS buffer (Dullbeco’s phosphate- buffered saline supplemented with 5% v / v heat-inactivated fetal bovine serum), and resuspended in 200 pL of FACS buffer containing DAPI. Flow cytometry was used to determine the percentage of transduced Jurkat cells by gating on live singlets and comparing fluorescent cell populations to the negative control. The infectious titer was calculated only for groups where 5-20% of cells were fluorescently positive. The titer was determined by multiplying the percentage of transduced cells by the total starting number of cells (2xlOA6) and dividing by the volume of lenti virus used.

[0179] EXAMPLE 2: Standard CIML Stimulation Results in NK cell apoptosis

[0180] To assess the viability of human CIML NK and NKT cells derived from various blood sources, LRS cones were shipped overnight to the research facility, and fresh whole blood (WB) was delivered on the same day from two distinct healthy donors (refer to Figure 3A for the experimental flow-chart). The purity and viability of NK and NKT cells were determined immediately after isolation for each sample (refer to Figure 3B). Subsequently, the cells underwent cytokine-induced stimulation to induce a memory-like state using the standard protocol (simultaneous IL-12, IL-15, and IL-18, followed by a wash step and high- dose IL-2 expansion media), or they were placed in low-dose IL-15 media to sustain cell viability. The viability of both standard CIML NK / NKT cells and conventional NK / NKT cells (maintained in low-dose IL- 15) was analyzed at 24 hours (refer to Figure 3C) and 72 hours (refer to Figure 3D) post-cytokine stimulation. By day 3, all CIML samples (both LRS and WB donors) exhibited a significant decrease in viability relative to samples maintained in low-dose IL-15, with fresh WB donors experiencing a more pronounced drop. These findings indicate that standard CIML stimulation leads to substantial apoptosis of NK and NKT cells, irrespective of the blood source and across different donors.

[0181] EXAMPLE 3: Splitting IL- 12 and IL- 15 exposure maintains NK cell viability.

[0182] To investigate the hypothesis that preserving NK cell viability can be achieved by separately stimulating NK cells with IL- 12 and IL- 15, we utilized NK cells isolated from two different blood sources (LRS cones and whole blood), each from two separate donors. The hypothesis was tested under five different conditions to stimulate isolated NK cells, as outlined in the experimental flowchart in Figure 4A. Under condition A, cell viability was maintained with low-dose IL- 15, without IL- 12 stimulation. In conditions B and C, NK cells were stimulated with IL-12 (10 ng / mL) and IL-18 (50 ng / mL) for 6 hours, washed, and either rested in media for 10 hours before being stimulated with 20 ng / mL IL- 15 (condition B) or immediately stimulated with 20 ng / mL IL- 15 (condition C). Similarly, in conditions D and E, NK cells were stimulated with IL-12 and IL-18 for 16 hours, washed, and either rested in media for 10 hours before stimulation with 20 ng / mL IL- 15 (condition D) or immediately stimulated with 20 ng / mL IL- 15 (condition E). Purity and viability were analyzed immediately after NK cell isolation (refer to Figure 4B), 24 hours after cytokine stimulation (refer to Figure 4C), and 72 hours after cytokine stimulation (refer to Figure 4D). Irrespective of the blood source, the viability of NK cells across conditions and donors did not significantly decrease relative to low-dose IL- 15 treatment (refer to Figure 4E). This suggests that the split exposure of IL- 12 and IL- 15 into two separate steps maintains NK cell viability.

[0183] To assess the ability of NK cells stimulated with IL-12 and IL-15 in two separate steps to persist and expand in vivo, huIL15-Tg NSG mice were engrafted with stimulated NK cells and either left untreated or supplemented with recombinant human IL-2 via intraperitoneal injection (refer to Figure 5A for the experimental flowchart and Figure 5B for the purity and viability of the injected NK cells). Mice supplemented with IL-2 demonstrated a significant expansion of peripheral blood human NK cells compared to mice not supplemented with cytokine (refer to Figure 5C). Furthermore, mice supplemented with IL- 2 exhibited a significant distribution of human NK cells into the bone marrow and spleen, with an increased frequency relative to mice not supplemented with IL-2 (refer to Figure 5D). Throughout the experimental duration, none of the mice experienced weight loss, indicating that neither NK cells nor IL-2 supplementation was toxic to the mice (refer to Figure 5E). This suggests that NK cells stimulated with IL-12 and IL-15 in two separate steps can persist and expand in vivo, especially in the presence of IL-2 cytokine supplementation.

[0184] EXAMPLE 4: Optimization of IL-12 / 15 split stimulation method with peripheral blood NK cells.

[0185] To identify the optimal method for generating CIML NK cells, an optimization screen was conducted on NK cells isolated from fresh whole blood obtained from two healthy donors (refer to Figure 6A for the experimental flowchart of the screening method). The screening involved four steps. In the first step, IL- 12 (20 ng / mL) and variable concentrations of IL- 18 (0, 1, 25, 50, 75, or 100 ng / mL) were added to the media, and cells were stimulated for varying durations (6, 9, 12, and 24 hours). In the second step, IL-12 and IL-18 were washed off. In the third step, cells were stimulated either with IL- 15 (5 ng / mL) alone or a combination of IL-15 (5 ng / mL) and IL-18 (50 ng / mL) for 72 hours. In the fourth step, extra IL- 15 was added to the media to promote NK cell survival for an additional 48 hours. Following the fourth step, the stimulated NK cells were analyzed for viability and surface marker expression (refer to Figure 6B-N). NK cells that were stimulated with IL- 12 (20 ng / mL) and IL-18 (25 ng / mL) for 6 hours, washed, and then stimulated with IL-15 (5 ng / mL) and IL- 18 (50 ng / mL) exhibited optimal viability and surface marker expression associated with a memory -like phenotype.

[0186] EXAMPLE 5: Phenotypic comparison of CIML NK cells generated using standard and optimized protocol. To assess whether CIML NK and NKT cells generated using an optimized protocol can replicate the characteristics of cells generated using the standard protocol, NK and NKT cells were isolated from a fresh Leukapheresis blood sample of a healthy donor and cytokine-stimulated as outlined in the experimental flowchart (refer to Figure 7A). The viability and surface marker expression of Optimized CIML NKs were compared with Standard CIML NKs after 5 days (refer to Figure 7B-D). Optimized CIML NK cells closely resembled standard CIML NK cells across all markers, except for CD25, which was constitutively expressed on optimized CIML NK cells. Moreover, optimized CIML NK cells exhibited significantly improved viability compared to standard CIML NK cells (refer to Figure 7B). These optimized CIML NK cells also replicated the cytotoxic activity against various cancer cell lines (refer to Figure 7E) and demonstrated the ability to be transduced with BaEV-pseudotyped lentiviral particles (refer to Figure 7F), similar to standard CIML NK cells.

[0187] To examine potential phenotypic differences between CIML NK cells generated using the optimized and standard protocols, huIL-15-Tg NSG mice were injected with each cell type, and cell distribution was analyzed 18 days after engraftment (refer to Figure 8A for the experimental flowchart). Throughout the study, all injected mice maintained normal body weight, indicating that neither standard nor optimized CIML NK cells exhibited toxicity (refer to Figure 8B). Both standard and optimized CIML NK cells were detected in the peripheral blood, bone marrow, and spleen of huIL-15-Tg NSG mice at equal frequencies, providing substantial evidence that optimized CIML NK cells can closely replicate the distribution pattern observed with standard CIML NK cells (refer to Figure 8C-E). This suggests that the phenotypic characteristics of the two cell types are comparable in vivo.

[0188] EXAMPLE 6. CIML T cells exhibit enhanced alloreactivity.

[0189] An optimized cytokine-induced memory-like (CIML) stimulation protocol was developed to activate human peripheral blood natural killer (NK) cells while maintaining cell viability. To determine whether this protocol also enhances the activation of CD3+ T cells, T cells were isolated from a healthy donor and stimulated using either a conventional cytokine stimulation protocol (combined IL-7 and IL-15) or the optimized CIML protocol (see Figure 9A). T cell activation was assessed in an alloreactivity assay (Figure 9B), in which conventional or CIML T cells were co-cultured with the Raji cell line for 24 hours. After co-culture, cytotoxic activity was evaluated by flow cytometry. Results demonstrated that CIML T cells exhibited enhanced alloreactivity compared to conventional T cells at all tested effector- to-target ratios, indicating that the CIML stimulation protocol improves T cell receptor (TCR) activation of CD3+ T cells.

[0190] EXAMPLE 7. Optimized CIML Stimulation of CD45RA+ T and NK Cells Enhances Viability of the Cellular Product.

[0191] The optimized CIML stimulation protocol was further evaluated for its ability to enhance the viability of a cellular product containing both NK and T cells. To generate a cellular product, NK and T cells were simultaneously isolated from the peripheral blood of healthy donors using CD45RA, a marker expressed on approximately 60% of CD45+ cells in both PBMCs from fresh whole blood and frozen PBMCs that have been thawed immediately prior to analysis (Figure 10A, left graph).

[0192] Analysis of PBMCs from fresh whole blood revealed that the CD45RA+ compartment primarily consists of B cells, NK cells, and T cells, with minimal contributions from other cell types (Figure 10A, middle graph). In contrast, freeze-thawed PBMCs showed an increased percentage of cells lacking CD 19, CD3, or CD56 expression, likely due to the loss of CD3 marker on T cells during the freeze-thaw process (Figure 10A, right graph). To isolate a product composed of T and NK cells, B cells were depleted from the PBMC mixture using CD19 microbeads, followed by positive selection of CD45RA+ cells using CD45RA microbeads (Figure 10B). This process produced a cell product with over 95% CD45RA+ cells and minimal contamination by CD19+ B cells (Figure 10C).

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Claims

CLAIMS1. A method of producing cytokine-induced memory-like (CIML) immune cells, said method comprising:(a) isolating immune cells from a biological sample,(b) contacting the immune cells with a first composition that comprises IL- 12 or a functional equivalent thereof, but does not comprise:(i) IL-2 or a functional equivalent thereof,(ii) IL- 15 or a functional equivalent thereof, and(iii) IL-21 or a functional equivalent thereof;(c) washing the immune cells to remove the first composition;(d) subsequently contacting the immune cells with a second composition that comprises IL-2 or a functional equivalent thereof, but not IL- 12 or a functional equivalent thereof, thereby producing cytokine-induced memory-like (CIML) immune cells.

2. The method of claim 1 , wherein the first composition further comprises IL- 18 or a functional equivalent thereof.

3. The method of claim 1, wherein the first composition does not comprise IL-7 or a functional equivalent thereof.

4. The method of any one of the preceding claims, wherein the second composition further comprises IL- 15 or a functional equivalent thereof.

5. The method of any one of the preceding claims, wherein the second composition further comprises IL- 18 or a functional equivalent thereof.

6. The method of any one of the preceding claims, wherein the second composition further comprises IL-21 or a functional equivalent thereof.

7. The method of any one of the preceding claims, wherein the second composition further comprises IL-7 or a functional equivalent thereof.

8. The method of any one of the preceding claims, wherein the duration of the step (b) contacting step is about 1 minute to about 24 hours.

9. The method of any one of the preceding claims, wherein the duration of the step (b) contacting step is about 1 minute to about 6 hours.

10. The method of any one of the preceding claims, wherein the duration of the step (d) contacting step is at least about 6 hours.

11. The method of claim 10, wherein the duration of the step (d) contacting step is no more than about 72 hours.

12. The method of claim 11 , wherein the duration of the step (d) contacting step is no more than about 36 hours.

13. The method of any one of the preceding claims, further comprising a rest step preceding the step (d) contacting step.

14. The method of any one of the preceding claims, wherein the biological sample is derived from a biological fluid.

15. The method of claim 14, wherein the biological fluid is whole blood, peripheral blood, Leukocyte Reduction System cone, a leukapheresis product or cord blood.

16. The method of any one of the preceding claims, wherein the immune cells isolated from the biological sample comprise at least one of the following cell types: T cells, CD4+ T cells, CD8+ T cells, yS T cells, NK cells, NKT cells, dendritic cells, monocytes and macrophages.

17. The method of claim 16, wherein at least one of the following cell types: cytokine- induced memory-like T cells, cytokine-induced memory-like CD4+ T cells, cytokine- induced memory-like CD8+ T cells, cytokine-induced memory-like y8 T cells, cytokine- induced memory-like NK cells, cytokine-induced memory-like NKT cells, cytokine- induced memory-like dendritic cells, cytokine-induced memory-like monocytes and cytokine-induced memory-like macrophages, are produced by the method.

18. The method of any one of the preceding claims, further comprising expanding the cytokine-induced memory-like (CIML) immune cells.

19. The method of any one of the preceding claims, further comprising transducing the cytokine-induced memory-like (CIML) immune cells with a vector.

20. The method of claim 19, wherein the vector is a viral vector.

21. The method of claim 20, wherein the viral vector is a lentiviral vector, alpha retroviral vector or a gamma retroviral vector.

22. The method of claim 21 , wherein the viral vector comprises a viral vector envelope that is pseudotyped with baboon endogenous retrovirus envelope glycoprotein (BaEV), VSV-G, RD114, GalV, H / F, G / F, or COCV envelope glycoproteins.

23. The method of any one of claims 19 to 22, wherein the vector is packaged within a liposome, lipid nanoparticle, virus, virus-like particle or is delivered by microcarrier- mediated delivery.

24. A cytokine-induced memory-like (CIML) immune cell produced by the method of any one of the preceding claims.

25. A population of cytokine induced memory-like (CIML) immune cells wherein more than 20% of the cells in the population are CD25 -positive, more than 30% of thecells in the population are CD71 -positive, and more than 70% of the cells in the population are CD69-positive.

26. The population of CIML immune cells of claim 25, comprising the vector as described in any one of claims 19 to 23.

27. A pharmaceutical composition comprising the CIML immune cell of claim 24, or the population of CIML immune cells of claim 25 or claim 26, and a pharmaceutically acceptable carrier.

28. The CIML immune cell of claim 24, or the population of CIML immune cells of claim 25 or claim 26, or the pharmaceutical composition of claim 27, for use in medicine.

29. A method of treating an individual in need thereof with a CIML immune cell, comprising administering to the individual a therapeutically effective amount of the CIML immune cell of claim 24, the population of CIML immune cells of claim 25 or claim 26, or the pharmaceutical composition of claim 27.

30. A method of treating an individual in need thereof with a CIML immune cell, comprising administering to the individual a therapeutically effective amount of the CIML immune cell, wherein the CIML immune cell is produced according to the method of any one of claims 1 to 23.

31. The method of claim 29 or claim 30, wherein the individual to be treated has cancer.

32. The method of claim 31 , wherein the cancer is hematologic.

33. The method of claim 32, wherein the hematologic cancer is selected from leukemia, lymphoma, multiple myeloma, a plasma cell malignancy and myelodysplastic syndrome.

34. The method of claim 33, wherein the hematologic cancer is a B-cell lymphoma or a T-cell lymphoma.

35. The method of claim 31 , wherein the cancer is a solid tumor.

36. The method of claim 35, wherein the solid tumor is selected from a renal carcinoma, a melanoma, a neuroblastoma, a glioma, a sarcoma and a carcinoma.

37. The method of any one of claims 29 to 36, wherein the CIML immune cell is derived from an autologous cell of the individual.

38. A composition comprising IL-2 or a functional equivalent thereof, IL-15 or a functional equivalent thereof, and IL- 18 or a functional equivalent thereof.

39. The composition of claim 38, wherein the composition further comprises IL-7 or a functional equivalent thereof.

40. The composition of claim 38 or claim 39, wherein the composition further comprises IL-21 or a functional equivalent thereof.

40. A composition comprising IL- 12 or a functional equivalent thereof, IL- 15 or a functional equivalent thereof, and IL- 18 or a functional equivalent thereof.

41. The composition of claim 40, wherein the composition further comprises IL-7 or a functional equivalent thereof.

42. The composition of claim 40 or claim 41, wherein the composition further comprises IL-21 or a functional equivalent thereof.

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