Reducing the killing of immune cells expressing nkg2d-based receptors

Abstract

The present invention relates to the field of immunotherapy, more specifically to the manufacture of cells for adoptive cell therapy. Provided herein are methods of preventing and / or reducing a killing agent in the production process of such cells, in particular cells expressing a chimeric NKG2D receptor. Also provided are cells and compositions comprising the cells that prevent and / or reduce a killing agent.

Description

Technical Field

[0001] The present invention relates to the field of immunotherapy, and more specifically to the production of cells for adoptive cell therapy. Provided herein are methods for preventing and / or reducing the production of such cells, particularly cells expressing the NKG2D receptor. Also provided are cells and compositions comprising cells that prevent and / or reduce the production of such cells. Background Art

[0002] Improvements in our understanding of the immune system have led to the development of a number of immune-directed therapies that are currently providing objective clinical responses in patients with advanced cancer. One of these approaches is chimeric antigen receptor (CAR) T cells, in which a patient’s T cells are genetically modified to express a tumor-targeting CAR and then returned to the patient in large numbers (1). This type of adoptive cell therapy has achieved a level of validation with objective clinical responses in patients with hematologic malignancies and is being further explored to treat a wider range of cancer indications (2-7). The concept of CARs has also expanded beyond T cells, with multiple cell types, including natural killer cells, being explored (8).

[0003] CARs are modular protein receptors that consist of a target binding domain connected to a structural domain, which typically includes an extracellular spacer domain and a transmembrane region fused to an intracellular signaling domain. Following ligand binding, downstream signaling initiated from the CAR activates the effector function of T cells, driving direct tumor cell killing and immune cytokine production.

[0004] A common problem with CAR T production is self-killing (or cannibalism). This phenomenon occurs when the CAR target is expressed on a T cell population. It is well known that T cell killing is a mechanism for maintaining T cell homeostasis (13). However, in therapeutic terms, T cell killing prevents the ability to generate the desired CAR T cell population for clinical application. This is particularly relevant in cases where the target itself (such as CD7 (14) or CD5 (15)) is selected for T cell lineage specificity to achieve T cell leukemia targeting. However, the problem is not limited to CAR T cell therapy. T cells with high affinity TCRs specific for the target protein (BIRC5) suffer from killing due to expression of the target antigen (16, 17). Similarly, NK cells expressing NK receptor ligands have also been shown to suffer from killing (24).

[0005] In summary, the use of artificial cell surface chimeric antigen receptor (CAR) constructs to give immune cells (such as T or NK cells) with predetermined target specificity provides a method for hypothetically targeting any tumor cell. The success of this method depends largely on the profile of the target antigen itself, most of which are not specific to tumors, but may be expressed on non-tumor cells. In some cases, the target antigen can be constitutively or transiently expressed on immune cells, which means that CAR-modified cells can commit suicide or kill.

[0006] Here, the focus is on CAR T cells engineered specifically for stress ligands targeting the natural killer group 2D (NKG2D) protein. Fusion of the full-length NKG2D sequence to the cytoplasmic domain of CD3ζ generates a CAR construct that results in activation of the CD3ζ domain by the T cell upon ligand binding by the NKG2D extracellular domain (18). NKG2D has eight known ligands, including the major histocompatibility complex class I-related A and B genes (MICA and MICB, respectively) and the UL16 binding protein (ULBP) family (ULBP1-6) (19). Expression of these NKG2D ligands is known to be induced under “stress” conditions, such as cell injury, infection, oxidative or thermal stress, or malignant transformation, resulting in a variety of human tumors expressing NKG2D ligands, highlighting the attractiveness of NKG2D as a receptor to be exploited in the context of CARs (19, 20). Indeed, mouse T cells bearing a mouse NKG2D CAR effectively eradicated a range of established hematologic and solid tumors in a syngeneic model system, demonstrating proof of concept for this approach (18).

[0007] Human T cells carrying the human NKG2D CAR (also known as NKR-2) can function as effector cells against a variety of tumor cells in vitro and can challenge established human tumors in the NSG mouse model (21). However, increasing the production of NKR-2 cells to generate higher cell numbers for clinical application has proven to be problematic due to the fact that activated T cells transiently express NKG2D ligands, a T cell killer identified as the cause.

[0008] Therefore, CARs consisting of a fusion of NKG2D protein and CD3ζ (NKR-2) give T cells a wide range of specificity for NKG2D ligands. However, T cells transiently express these ligands during activation, so NKR-2 T cells are killed, which greatly hinders the ability to use NKG2D as a therapy. In addition, in order to use therapy in the clinic, a large amount of upgrading and cryopreservation are required to deliver the required dosing schedule. It is well known that cryopreservation and freeze-thaw cycles can put pressure on cells, resulting in increased expression of stress-induced proteins (such as NKG2D ligands). In fact, it has been observed that both scale-up and cryopreservation of T cells expressing chimeric NKG2D receptors result in poor cell yields, which is probably due to suicide or killing agents.

[0009] Therefore, preventing or reducing the production of killer agents in these cells would be advantageous because it would not only increase cell yields but also reduce manufacturing costs and improve the therapeutic efficacy of these cells—in short, it would make the upscaling and cryopreservation required for clinical applications feasible from a practical and commercial perspective. SUMMARY OF THE INVENTION

[0011] Modified immune cells expressing the NKG2D protein fused to CD3ζ, specifically chimeric antigen receptor (CAR) T cells (NKG2D-CAR T cells), are specific for stress-induced ligands expressed in blood and solid cancers. However, these stress ligands can also be transiently expressed by activated immune cells or T cells, meaning that NKG2D-based immune cells may undergo suicide (killing) during the cell manufacturing process or during freeze-thaw cycles before infusion into patients.

[0012] One object of the present invention is to provide methods for reducing and / or preventing killer agents during the production of immune cells expressing chimeric NKG2D receptors, including functional inhibition of NKG2D signaling during cell production. Another object of the present invention is to provide methods for reducing and / or preventing killer molecules during freezing and / or thawing of frozen immune cells expressing chimeric NKG2D receptors, including functional inhibition of NKG2D signaling during freezing and / or thawing of cells.

[0013] Targeted inhibition of NKG2D expression, NKG2D ligand expression, or inhibition of enzyme function, particularly PI3K function, may overcome target-driven CAR T cell killing. It is specifically envisioned that functional inhibition of NKG2D signaling may be achieved through one or more of the following:

[0014] - Permanently or temporarily inhibit one or more NKG2D ligands of said immune cells;

[0015] - Transient inhibition of chimeric NKG2D receptors;

[0016] - Transient inhibition of downstream signaling of the chimeric NKG2D receptor.

[0017] According to embodiments envisaging permanent inhibition of one or more NKG2D ligands, this can be achieved in particular by gene knockout. To this end, gene editing technologies can be used, including but not limited to Crispr / Cas, TALEN, ZFN, meganucleases, MegaTAL nucleases.

[0018] According to a specific embodiment that envisions transient inhibition of downstream signaling, this is a transient inhibition of PI3K signaling. According to another specific embodiment, a wide range of PI3K inhibitors can be used to inhibit PI3K signaling. An example of such an inhibitor is LY294002. Another suitable inhibitor is idelalisib (Cal-101).

[0019] According to particular embodiments, functional inhibition of the receptor or ligand can be achieved using inhibitory RNA (eg, shRNA or siRNA) or antibodies directed against the NKG2D receptor or one or more of its ligands.

[0020] According to another specific embodiment, functional inhibition is achieved at the receptor level and is accomplished by inhibitory RNA or antibodies against NKG2D receptors. According to one aspect, antibodies against NKG2D receptors are used. In a specific embodiment of this aspect, the antibody against NKG2D receptors is an antibody that binds to the receptor without activating the chimeric receptor (i.e., a blocking or antagonistic antibody). According to the most specific embodiment, the antibody against NKG2D receptors is the commercially available 1D11 antibody (named after the clone from which it was isolated).

[0021] According to alternative specific embodiments, functional inhibition is achieved at the ligand level and is accomplished by inhibitory RNA or antibodies against one or more NKG2D ligands. It is particularly contemplated that the ligands are one or both of MICA and MICB. According to one aspect, shRNA directed against one or more NKG2D ligands is used.

[0022] According to certain embodiments, the immune cells to be produced are cells for adoptive cell transfer, such as T cells, NK cells, NKT cells, stem cells or iPSCs.

[0023] In another aspect, cells are provided that are less susceptible to killing agents when manufactured for adoptive transfer. According to this aspect, engineered immune cells are provided that comprise a nucleic acid molecule encoding a chimeric NKG2D receptor and at least one of:

[0024] - one or more endogenous genes encoding NKG2D ligands that have been engineered to be inactivated;

[0025] - One or more shRNAs directed against the chimeric NKG2D receptor and / or one or more NKG2D ligands.

[0026] In these immune cells, the NKG2D ligand is selected from the group consisting of: MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 and ULBP6. Particularly envisaged ligands are MICA and / or MICB.

[0027] According to another specific embodiment, an engineered immune cell is provided, comprising a nucleic acid molecule encoding a chimeric NKG2D receptor and at least one of:

[0028] - one or more endogenous genes encoding NKG2D ligands that have been engineered to be inactivated;

[0029] - One or more shRNAs directed against one or more NKG2D ligands.

[0030] According to another aspect, a composition comprising an immune cell comprising a nucleic acid molecule encoding a chimeric NKG2D receptor and

[0031] a) the cell further comprises

[0032] - one or more endogenous genes encoding NKG2D ligands that have been engineered to be inactivated;

[0033] - one or more shRNAs against the chimeric NKG2D receptor and / or one or more NKG2D ligands;

[0034] and / or

[0035] b) the composition further comprises

[0036] - one or more antibodies against the chimeric NKG2D receptor and / or one or more NKG2D ligands;

[0037] - Inhibitors of downstream signaling of the NKG2D receptor, in particular PI3K inhibitors.

[0038] According to a specific embodiment of this aspect, there is provided a composition comprising an immune cell comprising a nucleic acid molecule encoding a chimeric NKG2D receptor, the composition further comprising

[0039] - one or more antibodies against the chimeric NKG2D receptor and / or one or more NKG2D ligands; and / or

[0040] - Inhibitors of downstream signaling of the NKG2D receptor, in particular PI3K inhibitors.

[0041] According to another specific embodiment, a composition comprising an immune cell comprising a nucleic acid molecule encoding a chimeric NKG2D receptor is provided, wherein the composition further comprises

[0042] - one or more antibodies against the chimeric NKG2D receptor; and / or

[0043] - PI3K inhibitors, particularly LY294002 or idelalisib.

[0044] According to yet another aspect, there is provided an engineered immune cell or composition as described herein for use as a medicament.

[0045] They are particularly suitable for the treatment of cancer.

[0046] This is equivalent to stating that a method of treating cancer is provided, comprising administering an engineered immune cell comprising a nucleic acid molecule encoding a chimeric NKG2D receptor and at least one of:

[0047] - one or more endogenous genes encoding NKG2D ligands that have been engineered to be inactivated;

[0048] - One or more shRNAs directed against the chimeric NKG2D receptor and / or one or more NKG2D ligands.

[0049] To those who need it.

[0050] Likewise, a method of treating cancer is provided, comprising administering to a subject in need thereof a composition, wherein the composition comprises an immune cell comprising a nucleic acid molecule encoding a chimeric NKG2D receptor and

[0051] a) The cell further contains

[0052] - one or more endogenous genes encoding NKG2D ligands that have been engineered to be inactivated;

[0053] - one or more shRNAs against the chimeric NKG2D receptor and / or one or more NKG2D ligands;

[0054] and / or

[0055] b) the composition further comprises

[0056] - one or more antibodies against the chimeric NKG2D receptor and / or one or more NKG2D ligands;

[0057] -An inhibitor of downstream signaling of the NKG2D receptor, particularly a PI3K inhibitor.

[0058] Treatment can be autologous (the subject receives cells from his or her own body) or allogeneic (the immune cells come from a donor who is not the subject). BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 : Flow cytometric characterization of PBMC, tCD19, and NKG2D-CAR T cells in a representative NKR-2 T cell process. Monocytes purified from blood using density gradients were analyzed before (PBMC) and after (tCD19 and NKR-2 T cells) NKR-2 T cell processing. After collection, cells were gated for lymphocytes on SSC / FSC. Lymphocytes were then gated on CD3. All CD3-positive cells are then displayed as follows: (A) CD4 / CD8 distribution. (B) Surface expression of NKG2D (CD314). (C) Memory phenotype. CD62L and CD45RA were used to distinguish naive (CD62L+CD45RA+), central memory (CD62L+CD45RA-), effector memory (CD62L-CD45RA-), and terminally differentiated T cells expressing CD45RA (CD62L-CD45RA+). (D) Exhausted phenotype was determined by staining for CD223 (Lag-3) and CD279 (PD-1). One third of a representative donor is shown.

[0061] Figure 2 Mean fluorescence intensity (MFI) of CD314. AB) CD3 after harvest of non-transduced, mock-transduced, or NKR2-transduced T cells. + CD4 + or CD3 + CD8 + MFI of CD314 in T cells. Shown are the mean values ​​for non-transduced cells (n=9), mock CD19-transduced cells (n=11), and NKR2-transduced T cells (n=16). A two-tailed unpaired t-test with Welch's correction was used to assess significance (***P=0.0003, ****P<0.0001). C) Mean fluorescence intensity values ​​for non-transduced cells, mock CD19-transduced cells, or NKR2-transduced T cells from at least nine different donors. Shown are the MFI and standard error of the mean for CD4 and CD8 T cells.

[0062] Figure 3NKR-2 T cells recognize chronic myeloid leukemia (K562) and pancreatic cancer (PANC-1) but exhibit a cytotoxic effect due to NKG2DL expression. (A) T cells from healthy donors were transduced with tCD19 vectors (tCD19 T cells) or NKR-2 T cell vectors (NKG2D-CAR T cells) and cultured with the indicated cell lines or alone (-). After overnight co-culture, IFN-γ secretion (ng / ml) was quantified by ELISA. Each data point represents the mean of replicate wells from an independent experiment. Figures shown are (N=3). (B) NKR-2 T cells exhibit cytolytic activity. PANC-1 cells were co-cultured with thawed NKG2D-CAR T cells at an E:T ratio of 1:1. Alamar blue signal was determined after 20 hours. Percent lysis was determined by comparison with absorbance of untreated cancer cells (PANC-1). Data shown are mean ± SD of N=3 independent T cell donors. -: PANC-1 cells only; NKR-2 T cells: coculture of NKR-2 T cells and PANC-1 cells; tCD19: control tCD19 T cells cocultured with PANC-1 cells. (C) Transduced T cells were cultured in complete X-vivo (100 IU / mL IL-2) for 4 days. T cells were analyzed 96 hours after seeding to determine fold expansion relative to the initial cell seeding density. Data shown are the mean ± SD of N = 3 independent T cell donors. (D) Representative coculture experiments (two-thirds). GFP-positive T cells were cultured alone (mock GFP) or cocultured with NKR-2 T cells generated from the same donor (mock GFP + NKR-2 T cells). GFP positivity was analyzed by flow cytometry at the start of culture (T = 0 h) or after 24 hours of culture (T = 24 h). (EF) PBMCs were activated with 40 ng / mL anti-CD3 and 100 IU / mL IL-2 and cocultured for 8 days according to the normal manufacturing protocol. Cell samples were collected every two days and analyzed for NKG2D ligand expression as either RNA (E) or protein on the cell surface (F). Statistical significance was assessed using a two-tailed unpaired t-test. p < 0.05 was considered significant (*), and p < 0.01 (**). For qPCR and flow cytometry comparisons, a paired two-tailed t-test was used.

[0063] Figure 4NKR-2 T cells exhibit NKG2D-mediated killing, which can be inhibited by NKG2D-blocking antibodies or PI3K inhibitors. (A) MFI of NKG2D expression on NKR-2 T cells treated or untreated with increasing concentrations of LY294002. Data shown are the mean ± SD of N = 3 independent donors. (B) Transduced T cells were cultured in complete X-vivo (100 IU / ml IL-2) supplemented or not with increasing concentrations of LY294002 for expansion. T cells were analyzed for fold expansion relative to the initial cell seeding density 96 hours after plating. Data shown are the mean ± SD of N = 3 independent donors. (C) Cell viability after cryopreservation. NKR-2 T cells were generated in the presence of increasing concentrations of LY294002. After production, cells were harvested, washed, and prepared for cryopreservation. After cryopreservation, cells were thawed in a water bath and resuspended in 5% plasma / human serum albumin (HSA). Cell viability was assessed directly after thawing (T0h) or after 6 hours in plasma / HSA5% (T6h) (N=715 3) at 4°C. (D) NKR-2 T cells were produced in the presence of increasing concentrations of LY294002. After production, cells were harvested, washed, transferred to Plasmalyte / HSA1% (50×106NKR-2 T cells / ml) and stored at 4°C for 48 hours. After 48 hours, cell viability was assessed using trypan blue staining (N=3) and cell number was normalized at the time of cryopreservation. (E) tCD19 T cells were co-cultured with NKR-2 T cells produced by the same donor in the presence of increasing concentrations of blocking Ab (from 0(-) to 10μg / ml). After 44 hours of incubation, flow cytometric analysis of CD19 positivity and viability was obtained. The data were normalized with the CD19 positivity of a mock cultured without NKR-2T cells (N=3). (F) Thawed NKR-2 T cells were co-cultured with PANC-1 cells or K562 cells at a 1:1 ratio in the presence of CD314 blocking antibody, isotype control, or no antibody. After 24 hours of incubation, supernatants were collected and IFN-γ was measured (N=3). (G) Thawed NKR-2 T cells were cultured for 24 hours in the presence of isotype control, and CD314 blocking Ab (or no Ab) and IFN-γ levels were measured. The data shown are the mean ± SD of N=3 independent donors. Statistical significance was assessed using a two-tailed unpaired t-test. p < 0.05 was considered significant (*), p < 0.01 (**).

[0064] Figure 5 : MFI of NKG2D expression on NKR-2 T cells treated or untreated with increasing concentrations of LY294002.

[0065] Figure 6 : Fold expansion of tCD19 cells in the presence or absence of PI3K inhibitors

[0066] Figure 7 NKR-2 T cells generated with PI3K inhibitors produced large amounts of IFN-γ (A) and displayed an increased memory phenotype (B)

[0067] Figure 8 : Fold expansion of NKR-2 T cells with and without antibody blockade

[0068] Figure 9Antibody blockade of NKR-2 T cells adaptively restores the CD4 / CD8 ratio. (A) Cytolytic activity kinetics, one-third representative killing assay. Control tCD19 T cells or NKR-2 T cells treated with the PI3K inhibitor LY294002 or a blocking Ab were cultured in the presence of NucLight-positive PANC-1. PANC-1 viability was assessed every 2 hours using the IncuCyte S3 device. (B) CD4 / CD8 distribution at harvest. During the expansion phase, NKR-2 T cells were cultured with 5 μM LY294002 or 5 μg / mL of blocking Ab for 96 hours, harvested, and CD4 and CD8 populations measured by flow cytometry. Data shown are the mean ± SD of N = 4 independent T cell donors relative to the control tCD19 ratio. (C) CD4 / CD8 distribution at harvest, with delayed addition of blocking antibody. During the expansion phase (days 4 to 8), NKR-2 T cells were treated directly with 5 μM blocked Ab (day 4) or 48 hours later (day 6). At harvest (day 8), T cells were harvested and analyzed for CD4 and CD8 populations by flow cytometry. The data shown are the mean ± SD of N = 3 independent T cell donors relative to the control tCD19 CD4 / CD8 ratio. (D) Expansion comparison. NKR-2 T cells were cultured for 8 or 10 days in the presence of LY or blocking Ab (added on day 4 or day 6). The multiple expansion of T cells relative to the initial cell seeding density was analyzed at harvest. (E) Potency determination by IFN-γ secretion. Thawed NKR-2 T cells treated by the two methods were co-cultured in the presence of PANC-1 cells. After 44 hours of co-culture, IFN-γ secretion was measured by ELISA. The data shown are the mean ± SD of N = 4 independent T cell donors. (F) Cytolytic activity kinetics, one-third of which are representative killing assays. Thawed control tCD19 T cells or NKR-2 T cells treated with PI3Ki or blocking antibodies were cultured in the presence of NucLight-positive PANC-1 cells. PANC-1 viability was assessed every 2 hours using the IncuCyte S3 device. Statistical significance was assessed using a two-tailed unpaired t-test. P < 0.05 was considered significant (*), P < 0.01 (**), and P < 0.001 (***).

[0069] Figure 10 Idelalisib (Cal101) vs. NKG2D blocking antibody. Comparison of cell expansion (A), viability (B), and potency measured by IFNγ secretion after co-culture with K562 cells (C) cultured in the presence of a blocking NKG2D antibody or 5 μM Cal101.

[0070] Figure 11 Expression of NKG2D ligands on the surface of CD4+ (A) and CD8+ (B) T cells

[0071] Figure 12 Co-expression of MICA / B targeting shRNA reduced the killing

[0072] Figure 13 .Reducing the killing agent can increase the killing power of cancer cells. Detailed Description of the Invention

[0074] definition

[0075] The present invention will be described with respect to particular embodiments and with reference to certain drawings, but the invention is not limited thereto but only by the claims. Any reference signs in the claims should not be construed as limiting the scope. The drawings described are schematic and non-restrictive. In the drawings, the dimensions of some elements may be exaggerated and not drawn to scale for illustrative purposes. When the term "comprising" is used in this specification and claims, it does not exclude other elements or steps. When referring to a singular noun such as "a" or "an", the indefinite article or definite article is used and, unless otherwise stated, the word includes the plural form of the noun. "Substantially consisting of" means that any of the listed elements must be included, elements that will have a substantial effect on the basic and novel characteristics of the listed elements are excluded, and other elements may be included optionally. "Consisting of" means all elements except the listed elements. Embodiments defined by each of these terms are within the scope of the present invention.

[0076] Furthermore, the terms first, second, third, etc. in the description and claims are used to distinguish between similar elements and not necessarily to describe a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances, and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0077] The following terms or definitions are provided solely to aid in understanding the present invention.

[0078] Unless specifically defined herein, all terms used herein have the same meaning as those of ordinary skill in the art. Practitioners are particularly directed to Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, New York (2012); and Ausubel et al., Modern Protocols in Molecular Biology (through Supplement 114), John Wiley & Sons, New York (2016) for prior art definitions and terminology. The definitions provided herein should not be construed as having a scope less than that understood by one of ordinary skill in the art.

[0079] As used herein, the term "killer" refers to the killing of cells by genetically identical cells, most particularly immune cells.

[0080] As used herein, the term "reducing and / or preventing a killer" refers to reducing the occurrence of a killer in a cell population compared to a suitable control cell population (typically, but not necessarily, the same cell population in which NKG2D ligand inhibition does not occur). The reduction can be expressed as a percentage reduction compared to a control, for example, the killer is reduced by 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or even 100%. The reduction of the killer can also be assessed by increasing the yield or number of the final cells (because after killing fewer cells, more cells can survive and can reproduce). Therefore, it can be assessed by increasing the cell yield by 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100% or even more than 100%. Importantly, it can also be demonstrated by an increase in antigen-specific cytokine production (e.g., interferon gamma secretion) and / or in the case of T cells, a T cell memory phenotype (CD62L + / CD45RA - ) can be assessed by increasing the frequency of memory T cells. While these measures may not be directly related to absolute cell yield, an increase in antigen-specific cytokine production means more therapeutically active cells at the end of the production process, which equates to less killer in the therapeutic cells. The same is true for an increase in the frequency of memory T cells. Thus, a reduction in killer can be assessed by an increase in antigen-specific cytokine production of 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, or even greater than 100%. Alternatively, a reduction in killer can be achieved by increasing the frequency of memory T cells by 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, or even greater than 100%.

[0081] Measures to reduce insecticides can be taken before the killing process begins (usually preferred because it is more effective) or during the killing process. Prevention of killing refers to measures taken before the killing process begins. According to specific embodiments, absolute prevention of killing means that no NKG2D-induced killing occurs (ideally, no killing occurs at all), which is equivalent to a 100% reduction in killing.

[0082] The term "immune cell" as used herein refers to a cell that is part of the immune system (which can be an adaptive immune system or an innate immune system). Particularly envisioned immune cells include leukocytes (leukocytes), including lymphocytes, monocytes, macrophages, and dendritic cells. Particularly envisioned lymphocytes include T cells, NK cells, and B cells, with T cells being the most particularly envisioned. The immune cells used herein are typically immune cells manufactured for adoptive cell transfer (autologous transfer or allogeneic transfer). In the case of adoptive transfer, please note that immune cells will typically be primary cells (i.e., cells that are directly isolated from human or animal tissue, rather than or only cultured for a short period of time), rather than cell lines (i.e., cells that are continuously passaged for a long time and obtain homogenous genotype and phenotypic characteristics). According to a specific embodiment, the immune cells are primary cells. According to alternative specific embodiments, the immune cells are not cells from cell lines.

[0083] The phrase "chimeric NKG2D receptor" used herein refers to a non-naturally occurring receptor with specificity for an NKG2D ligand. It is chimeric because the binding portion is fused to one or more different portions (including at least one signaling portion), wherein at least one portion is from a different source (e.g., a different protein) than the binding portion. Examples of particular envisions of chimeric NKG2D receptors include NKG2D CAR, i.e., a chimeric antigen receptor with a binding portion derived from an NKG2D receptor. Such NKG2D CARs have been described, for example, in WO2006 / 036445 and WO2014 / 117121. Also included in the definition of chimeric NKG2D receptors herein are CARs with a binding portion that can recognize one or more NKG2D ligands not derived from NKG2D receptors (e.g., antibodies to one or more NKG2D ligands) or antibody-like portions (e.g., scFv, VHH, sdAb, etc.). It is then typically fused to a signaling moiety that transduces a signal in immune cells, particularly T cells (eg, the CD3 zeta chain or the Fcε receptor gamma chain).

[0084] As used herein, the term "functional inhibition of NKG2D signaling" refers to interference with the function of the NKG2D gene product (i.e., the product of the chimeric NKG2D receptor gene) at the DNA level (by inhibiting the formation of the NKG2D gene product or one or more of its ligands, i.e., by preventing or interfering with transcription), at the RNA level (by neutralizing or stabilizing mRNA to prevent or interfere with translation - the mRNA may also be a chimeric NKG2D receptor and / or one or more NKG2D ligands), or at the protein level (by neutralizing or inhibiting a chimeric NKG2D protein and / or one or more of its ligands). Neutralization at the protein level can be achieved at the cell surface (e.g., by inhibiting receptor-ligand interactions) or before the protein is expressed on the surface (e.g., by retaining the protein in an intracellular organelle). Typically, the ultimate functional effect of inhibiting NKG2D-induced signaling is to inhibit the activation of immune cells through signals generated by the chimeric NKG2D receptor, although this can be achieved indirectly (e.g., at the DNA level, or by inhibiting one or more ligands).

[0085] Functional inhibition of NKG2D signaling does not necessarily imply complete ablation of NKG2D-induced signaling, although this is conceivable. It is well known, particularly with antisense RNA and siRNA, but also with antibodies, that inhibition is often partial rather than complete. However, reducing the levels of functional NKG2D gene products or NKG2D ligands may have beneficial effects even in the absence of complete inhibition—particularly because killing agents are often cell density-dependent, thus reduced availability of functional ligands or receptors may result from lower cell densities. (Note that given the large number of cells required for future clinical trials, reducing the actual cell density by using culture dilutions is not feasible for generating suitable products.)

[0086] Thus, according to specific embodiments, the inhibition will result in a 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or up to a 100% reduction in the level of a functional chimeric NKG2D receptor gene product or one or more NKG2D ligands. Methods of measuring the level of a functional NKG2D receptor gene product or ligand are known to those of skill in the art, and measurements can be made before and after addition of an inhibitor to assess a reduction in the level of a functional gene product.

[0087] As used herein, the term "transient inhibition" means that the inhibition is temporary (or regulated in time), and the function of the chimeric NKG2D receptor is restored at a later time point. Typically, NKG2D-induced signal transduction will be inhibited during the production of immune cells (i.e., before they are used as a drug), and once the cells are administered to the patient, signal transduction capacity will be restored. This is because NKG2D-mediated signaling is important for therapeutic efficacy.

[0088] The term "NKG2D ligand" used in this application refers to the human genes MICA (Gene ID: 100507436), MICB (Gene ID: 4277), ULBP1 (Gene ID: 80329), ULBP2 (Gene ID: 80328), ULBP3 (Gene ID: 79465), ULBP4 or RAET1E (Gene ID: 135250), ULBP5 or RAET1G (Gene ID: 353091), ULBP6 or RAET1L (Gene ID: 154064) and their gene products (or related homologs when cells from other species are used).

[0089] The present application is the first to show that the killing of immune cells expressing chimeric NKG2D receptors can be prevented or reduced by inhibiting the function of the receptor (by inhibiting the receptor or one or more of its ligands, or both). This reduction in killing increases cell yield and reduces the cost of treatment, thereby promoting the availability of treatment in clinical settings.

[0090] The inhibitory effect can be transient (during the in vitro manufacturing process of the immune cells, rather than when administered to the patient, because the cells require the chimeric NKG2D receptor for their therapeutic immune cell function) or permanent (when only the ligand in the immune cell is inhibited, as this does not interfere with therapeutic efficacy).

[0091] Therefore, the present invention provides methods for reducing and / or preventing killer molecules during the production of immune cells expressing chimeric NKG2D receptors, including functionally inhibiting NKG2D signaling during the production of cells. These production methods are performed in vitro or ex vivo. Similarly, methods for reducing and / or preventing microbicidal activity during freeze / thaw cycles of immune cells expressing chimeric NKG2D receptors are provided, including functionally inhibiting NKG2D signaling during freeze / thaw cycles. These freeze / thaw methods can also be performed in vitro or ex vivo.

[0092] It is worth noting that, although NKG2D is the most intensively studied receptor involved in recognizing inducible self-antigens, it is not the only receptor in this family that recognizes stress-induced ligands (or induced self-antigens or markers of abnormal self, all used as equivalents herein). Other receptors capable of binding inducible self-antigens are NKG2C, NKG2E, NKG2F, NKG2H (e.g., NKG2D, all CD94 molecules) or natural cytotoxicity receptors (NCRs), such as NKp46, NKp30, and NKp44, and it is envisioned that the methods and compositions can also be applied mutatis mutandis to such chimeric receptors. Therefore, regardless of the position of NKG2D used in the application, this also applies to NKG2C, NKG2E, NKG2F, NKG2H, NKp46, NKp30, and NKp44. Note that the ligands for NKG2C, E, F, and H are class I non-classical MHC glycoproteins (HLA-E in humans).

[0093] These methods are in vitro methods (because they occur simultaneously with the production of the cells).

[0094] Functional inhibition of NKG2D signaling is achieved by one or more of the following:

[0095] - Permanently or temporarily inhibit one or more NKG2D ligands of said immune cells;

[0096] - Transient inhibition of chimeric NKG2D receptors;

[0097] - Transient inhibition of downstream signaling of the chimeric NKG2D receptor.

[0098] Inhibition can occur in a variety of ways: contact inhibition (competitive or noncompetitive), inhibition by interfering with the expression of the ligand or receptor, interference with the localization of the ligand or receptor (e.g., to prevent migration to the cell surface), inhibition by binding to the ligand or receptor or by inhibiting the interaction between the two, and inhibition of downstream signaling, to name a few.

[0099] Permanent inhibition of one or more NKG2D ligands is usually achieved by gene knockout. Indeed, gene editing has been shown to be a potential approach to specifically eliminate target antigen expression in engineered T cells (14). However, given that eight different ligands may be expressed, gene editing techniques to eliminate all of these polymorphic targets present challenges, and therefore it is particularly important to envision situations where only one or a few ligands need to be permanently inactivated. If more ligands are expressed, one of the alternative strategies may be more suitable for controlling the killing agent. The present inventors have determined that of the eight NKG2D ligands, MICA and MICB are primarily expressed in CD4 T cells. + and CD8 +MICA and MICB are expressed on the cell surface of human T cells. Thus, according to certain embodiments, inhibition of MICA and MICB is specifically envisaged.

[0100] Typically, functional inhibition can be achieved at three levels. First, at the DNA level, for example by removing or destroying a gene (typically an NKG2D ligand gene) in the immune cell, or preventing transcription from occurring (in both cases, preventing the synthesis of the gene product). Second, at the RNA level, for example, by preventing effective translation from occurring - this may be by destabilizing the mRNA so that it is degraded before translation occurs on the transcript, or by hybridizing with the mRNA. Third, at the protein level, for example, by binding to the protein, inhibiting its function, retaining the protein in a different cellular location, and / or marking the protein for degradation.

[0101] If inhibition is to be achieved at the DNA level, gene therapy can be used to knock out or destroy genes to complete. Because this typically results in permanent inhibition, it is particularly conceivable to inhibit NKG2D ligands in immune cells. As used herein, "knockout" can be gene knockout or genes can be knocked out by mutation (e.g., point mutation, insertion, deletion, frameshift or missense mutation) by techniques known in the art, including but not limited to retroviral gene transfer. Another method by which genes can be knocked out is to use engineered nucleases. Examples of such engineered nucleases include but are not limited to large-range nucleases, zinc finger nucleases, TALENs, megaTALs and CRISPR nucleases.

[0102] Meganucleases, commonly found in microbial species, possess a unique property: they possess very long recognition sequences (>14 bp) that allow them to create site-specific double-strand breaks in nucleic acids. This makes them naturally very specific for their target sequences, and through mutagenesis and high-throughput screening, hybrid meganuclease variants that recognize unique sequences can be generated. In contrast to meganucleases, the concept behind ZFN and TALEN technologies is based on nonspecific DNA-cleaving enzymes that can then be linked to peptides that recognize specific DNA sequences, such as zinc fingers and transcription activator-like effectors (TALEs). Zinc finger nucleases (ZFNs) are artificial restriction enzymes created by fusing zinc finger DNA-binding domains to DNA-cleaving domains. The zinc finger domains can be engineered to target desired DNA sequences, enabling zinc finger nucleases to target unique sequences within complex genomes. By exploiting endogenous DNA repair mechanisms, these agents can be used to precisely alter the genomes of higher organisms. TALENs function similarly to zinc fingers but rely on transcription activator-like effectors (TALEs) for DNA recognition. TALEs are found within repetitive sequences, with a one-to-one ratio of recognition between amino acids and recognized nucleotide pairs. Because TALEs occur in a repeating pattern, different combinations can be tried to create a wide variety of sequence specificities.

[0103] MegaTALs are derived from a combination of two different classes of DNA-targeting enzymes. Meganucleases (also known as homing endonucleases) are single peptide chains that have the advantage of having both DNA recognition and nuclease functions in the same domain. However, the recognition of meganucleases' targets is difficult to modify, and they typically have reduced specificity and lower target cleavage efficiency compared to other genome-targeting endonucleases. Transcription activator-like (TAL) effectors are DNA recognition proteins that have been linked to separate DNA endonuclease domains to achieve target DNA double-strand breaks. Compared to meganucleases, TALs are easy to engineer to target specific DNA sequences. Current platforms rely on a pair of TAL effectors, each coupled to a non-specific DNA cleavage domain, where DNA cleavage only occurs when the two TAL effectors bind to their respective sequences and the two endonuclease domains dimerize to cut the DNA. However, TAL effector nucleases can cause off-target activity, are much greater than meganucleases, and require the delivery of two separate proteins. megaTAL is a unification of TAL effectors and meganucleases.

[0104] CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats / Crispr-associated protein) is a genome editing technology that uses a modified version of a prokaryotic defense mechanism to permanently modify genes within an organism. By delivering a Cas (usually Cas9) nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at the desired location, thereby removing existing genes and / or adding new genes.

[0105] This immune response is typically achieved by permanently suppressing one or more NKG2D ligand genes through genetic knockout. This immune response is typically also achieved in immune cells where the NKG2D receptor is present. One or more NKG2D ligand genes may mean suppressing any combination of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6; thus, it may mean knocking out one, two, three, four, five, six, seven, or eight genes.

[0106] In addition to permanent inhibition of NKG2D ligands, functional inhibition of NKG2D signaling can also be achieved through transient inhibition, which can be inhibition of one or more NKG2D ligands on immune cells, inhibition of chimeric NKG2D receptors, or inhibition of downstream signaling.

[0107] The timeframe for transient inhibition will generally coincide with the manufacturing timeframe. The manufacturing of immune cells expressing chimeric NKG2D receptors involves several steps, and protocols can vary, but essentially, they will always include a transduction step (in which the chimeric NKG2D receptor is introduced into isolated immune cells), an expansion step (in which the cells are cultured and their numbers increased), and a harvesting step (in which the cells are isolated and reconstituted or concentrated prior to patient administration or cryopreservation). Transient inhibition specifically refers to inhibition during the expansion step, as this is when killing is most likely to occur, but transient inhibition can occur throughout the manufacturing process (from or even before the transduction step until or after the harvesting step). Typically, when an external inhibitor (e.g., an antibody) is used, it will be removed during the harvesting / reconstitution process. Therefore, the method can include a step to deactivate the inhibitor used. However, in other cases, the inhibitor may be transient (e.g., due to a short half-life) or under the control of an inducible promoter that is no longer active after the manufacturing process, thus eliminating the need for any active steps to terminate the inhibition of functional NKG2D signaling. In a typical setting, transient inhibition will entail inhibition from the transduction step to the administration / infusion step, but shorter or longer time frames are also envisioned.

[0108] One form of transient inhibition is transient gene inactivation. Transient gene inactivation can be achieved, for example, by expressing antisense RNA in immune cells or by administering antisense RNA to the cells. Antisense constructs can be delivered, for example, as expression plasmids that, when transcribed in cells, produce RNA that is complementary to at least a unique portion of the target mRNA (here, the mRNA of an NKG2D ligand or chimeric NKG2D receptor).

[0109] A faster method for inhibiting gene expression is based on the use of shorter antisense oligomers, which are composed of DNA or other synthetic structure types, such as phosphothioesters, 2'-O-alkyl ribonucleotide chimeras, locked nucleic acids (LNA), peptide nucleic acids (PNA) or morpholinos. Except RNA oligomers, PNA and morpholinos, all other antisense oligomers play a role in eukaryotic cells by the target cleavage mechanism mediated by RNase H. PNA and morpholinos bind to complementary DNA and RNA targets with high affinity and specificity, and therefore work by simple spatial blockade of the RNA translation mechanism, and seem to be completely resistant to nuclease attacks." antisense oligomers" refer to antisense molecules or antigene agents comprising an oligomer of at least about 10 nucleotides in length. In embodiments, antisense oligomers comprise at least 15, 18, 20, 25, 30, 35, 40 or 50 nucleotides. Antisense methods relate to the design of oligonucleotides (DNA or RNA or derivatives thereof) that are complementary to the mRNA encoded by the polynucleotide sequence of FMR1. Antisense RNA can be introduced into cells to inhibit the translation of complementary mRNA by pairing with it and physically hindering the translation mechanism. Therefore, the effect is stoichiometric. Absolute complementarity, although preferred, is not required. As referred to herein, a sequence that is "complementary" to a portion of RNA refers to a sequence that has sufficient complementarity to be able to hybridize with the RNA to form a stable duplex; in the case of a double-stranded antisense polynucleotide sequence, a single strand of double-stranded DNA can be tested, or the formation of a triplex can be detected. The ability to hybridize will depend on the degree of complementarity and the length of the antisense polynucleotide sequence. Generally, the longer the hybrid polynucleotide sequence, the more base mismatches there are with the RNA it may contain, and still form a stable duplex (as the case may be). Those skilled in the art can determine the permissible degree of mismatch by determining the melting point of the hybrid complex using standard procedures. Oligomers that are complementary to the 5' end of the message, such as the 5' untranslated region (UTR) up to and including the AUG translation start codon, should be most effective in inhibiting translation. However, it has recently been shown that sequences complementary to mRNA 3'UTR can also effectively inhibit the translation of mRNA (Wagner, R. (1994) Nature 372, 333-335). Therefore, oligomers complementary to the 5', 3'UTR or non-coding region of the target gene can be used in antisense methods to inhibit the translation of the endogenous mRNA encoded by the target gene. The oligomer complementary to the 5'UTR of the mRNA should include the complement of the AUG start codon. Antisense oligomers complementary to the mRNA coding region are translation inhibitors with lower efficiency, but can be used according to the present invention. Whether designed to hybridize to the 5', 3' or non-coding region of the mRNA, the length of the antisense oligomer should be at least 10 nucleotides, and the length of the preferred oligomer is 15 to about 50 nucleotides.In certain embodiments, the length of the oligomer is at least 15 nucleotides, at least 18 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 35 nucleotides, at least 40 nucleotides or at least 50 nucleotides. A related method uses ribozymes instead of antisense RNA. Ribozymes are catalytic RNA molecules with enzyme-like cleavage properties that can be designed to target specific RNA sequences. It has been reported that inactivation of target genes, including time- and tissue-specific gene inactivation, has been successfully achieved using ribozymes in mice, zebrafish and fruit flies. RNA interference (RNAi) is a form of post-transcriptional gene silencing. The phenomenon of RNA interference was first observed and described in Caenorhabditis elegans, which shows that exogenous double-stranded RNA (dsRNA) effectively destroys the activity of genes containing homologous sequences by inducing the mechanism of rapid degradation of target RNA. Several reports describe the same catalytic phenomenon in other organisms, including experiments demonstrating spatial and / or temporal control of gene inactivation, including plants (Arabidopsis thaliana), protozoa (Trypanosoma bruceii), invertebrates (Drosophila melanogaster), and vertebrates (Danio rerio and African clawed frogs (Xenopus laevis). Mediators of sequence-specific messenger RNA degradation are small interfering RNAs (siRNAs) produced by ribonuclease III cleavage of longer dsRNAs. Typically, siRNAs are between 20 and 25 nucleotides in length (Elbashir et al. (2001) Nature 411, 494-498). siRNAs typically contain a sequence-specific messenger RNA that is expressed by a standard Watson Crick base pairing interaction (hereinafter referred to as "base pairing") anneals a sense RNA strand and a complementary antisense RNA strand. The sense strand comprises a nucleic acid sequence identical to the target sequence contained in the target mRNA. The sense and antisense strands of the present siRNA may comprise two complementary single-stranded RNA molecules, or may comprise a single molecule in which two complementary portions are base-paired and covalently linked by a single-stranded "hairpin" region (commonly referred to as shRNA). The term "isolated" refers to alteration or removal from a natural state by human intervention. For example, rather than "isolating" an siRNA naturally present in a living animal, one "isolates" a synthetic siRNA or an siRNA partially or completely separated from coexisting substances in the natural state. The isolated siRNA may exist in a substantially purified form, or may exist in a non-natural environment, such as a cell to which the siRNA has been delivered.

[0110] The siRNA of the present invention can include partially purified RNA, substantially pure RNA, synthetic RNA or recombinantly produced RNA, as well as RNA that is modified from natural RNA by addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations can include the addition of non-nucleotide material to, for example, the ends of the siRNA or one or more internal nucleotides of the siRNA, including modifications that render the siRNA resistant to nuclease digestion.

[0111] One or both strands of the siRNA of the present invention may also comprise a 3' overhang. A "3' overhang" refers to at least one unpaired nucleotide extending from the 3' end of an RNA strand. Thus, in one embodiment, the siRNA of the present invention comprises at least one 3' overhang of 1 to about 6 nucleotides (including ribonucleotides or deoxynucleotides), preferably about 1 to about 5 nucleotides in length, more preferably about 1 to about 4 nucleotides in length, and particularly preferably about 1 to about 4 nucleotides in length.

[0112] In embodiments where both chains of the siRNA molecule comprise a 3' overhang, the length of the overhang may be the same or different for each chain. In a most preferred embodiment, the 3' overhang is present on both chains of the siRNA and is two nucleotides in length. In order to enhance the stability of the siRNA of the present invention, the 3' overhang may also be stabilized to prevent degradation. In one embodiment, the overhang is stabilized by comprising purine nucleotides, such as adenosine or guanosine nucleotides.

[0113] Alternatively, substitution of pyrimidine nucleotides with modified analogs is tolerated, for example, substitution of uridine nucleotides in 3' overhangs with 2' deoxythymidine, without affecting the efficiency of RNAi degradation. In particular, the absence of the 2' hydroxyl group in 2' deoxythymidine significantly enhances the nuclease resistance of 3' overhangs in tissue culture medium.

[0114] siRNA can be obtained using a variety of techniques known to those skilled in the art. For example, siRNA can be produced by chemical synthesis or recombinant methods known in the art. Preferably, siRNA of the present invention is chemically synthesized using appropriately protected ribonucleoside phosphoramidites and conventional DNA / RNA synthesizers. siRNA can be synthesized into two independent complementary RNA molecules or into a single RNA molecule with two complementary regions. Commercial suppliers of synthetic RNA molecules or synthetic reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colorado, USA), Pierce Chemical (part of Perbio Science, Rockford, Illinois, USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Massachusetts, USA) and Cruachem (Glasgow, UK).

[0115] Alternatively, any suitable promoter can also be used to express siRNA from a recombinant circular or linear DNA plasmid. Suitable promoters for expressing siRNA of the present invention from plasmids include, for example, U6 or H1 RNA pol III promoter sequences and cytomegalovirus promoters. The selection of other suitable promoters is within the skill of the art. The recombinant plasmid of the present invention can also include an inducible or regulatable promoter for expressing siRNA in a specific tissue or in a specific intracellular environment. The siRNA expressed from the recombinant plasmid can be separated from a cultured cell expression system by standard techniques, or can be expressed intracellularly, for example, in mammary tissue or neurons.

[0116] The siRNA of the present invention can also be expressed intracellularly from a recombinant viral vector. The recombinant viral vector comprises a sequence encoding the siRNA of the present invention and any suitable promoter for expressing the siRNA sequence. Suitable promoters include, for example, the U6 or H1 RNA pol III promoter sequences and the cytomegalovirus promoter. The selection of other suitable promoters is within the skill of the art. The recombinant viral vector of the present invention can also comprise an inducible or regulatable promoter for expressing the siRNA in tissues where the tumor is located.

[0117] As used herein, an "effective amount" of siRNA is an amount sufficient to cause RNAi-mediated degradation of the target mRNA, or an amount sufficient to reduce NKG2D signaling. RNAi-mediated degradation of the target mRNA can be detected by measuring the level of the target mRNA or protein in the subject's cells using standard techniques for isolating and quantifying mRNA or protein as described above.

[0118] It has been shown that the morpholino antisense oligonucleotides in zebrafish and frogs overcome the limitations of RNase H competent antisense oligonucleotides, which have many non-specific effects due to the non-target specific cleavage of other mRNA molecules caused by other low-level RNase H strict requirements. Therefore, Morpholino oligomers represent an important class of new antisense molecules. The oligomers of the present invention can be synthesized by standard methods known in the art. For example, phosphorothioate oligomers can be synthesized by the methods of Stein et al. (1988) Nucleic Acids Res.16,3209-3021, and methyl phosphonate oligomers can be prepared by using controlled pore glass polymer carriers (Sarin et al. (1988) Proc. Natl. Acad. Sci. USA.85,7448-7451). Morpholino oligomers can be synthesized by the methods of Summerton and Weller U.S. Patent Nos. 5,217,866 and 5,185,444.

[0119] Inhibition, especially transient inhibition, can also be achieved by inhibitors at the protein level, typical examples of which are antibodies against chimeric NKG2D receptors, or antibodies against one or more NKG2D ligands.

[0120] The term "antibody" refers to an antibody characterized by being specific for an NKG2D receptor, an NKG2D ligand, or any functional derivative thereof, preferably a monoclonal antibody; an antibody or antigen-binding fragment of the F(ab')2, F(ab) or single-chain Fv type, or any type of recombinant antibody derived therefrom. These antibodies of the present invention, including specific polyclonal antisera prepared against the target protein or any functional derivative thereof, have no cross-reactivity with other proteins. For example, the monoclonal antibodies of the present invention can be produced by any hybridoma readily formed according to classical methods from spleen cells and myeloma cell lines of animals (particularly mice or rats immunized against the target protein or any functional derivative thereof), and selected based on the ability of the hybridoma to produce monoclonal antibodies that recognize the target protein or any functional derivative thereof, which monoclonal antibodies were initially used for animal immunization. The monoclonal antibodies according to this embodiment of the present invention can be humanized forms of mouse monoclonal antibodies prepared by recombinant DNA technology, distinct from mouse and / or human genomic DNA sequences encoding the H and L chains or cDNA clones encoding the H and L chains. Alternatively, the monoclonal antibodies according to this embodiment of the present invention can be human monoclonal antibodies. Such human monoclonal antibodies are prepared, for example, by repopulating human peripheral blood lymphocytes (PBL) from severe combined immunodeficiency (SCID) mice as described in PCT / EP99 / 03605, or by using transgenic non-human animals capable of producing human antibodies as described in U.S. Patent No. 5,545,806. Fragments derived from these monoclonal antibodies, such as Fab, F(ab)'2 and scFv ("single-chain variable fragment"), constitute part of the present invention as long as they retain their original binding properties. Such fragments are typically produced by enzymatic digestion, for example with papain, pepsin or other proteases. It is well known to those skilled in the art that monoclonal antibodies or their fragments can be modified for various uses. The antibodies involved in the present invention can be labeled with appropriate markers of the enzymatic, fluorescent or radioactive type. In a specific embodiment, the antibodies directed against the target protein or its functional fragment are derived from camels. Camelid antibodies are fully described in WO94 / 25591, WO94 / 04678 and WO97 / 49805.

[0121] Other NKG2D signaling inhibitors at the protein level include, but are not limited to, peptide inhibitors of NKG2D ligands or chimeric receptors, peptide-aptamer inhibitors of NKG2D ligands or chimeric NKG2D receptors (Tomai et al., J Biol Chem. 2006), and protein interferons or Pept-Ins TM Methods described in WO2007 / 071789 or WO2012 / 123419, incorporated herein by reference.

[0122] Another approach to inhibition at the protein level is to interfere with secretory transport, thereby preventing the receptor and / or ligand from being transported to the cell membrane. Typically, this is a temporary form of inhibition, and normal cellular localization can be restored after providing the appropriate signal to the cell. An exemplary method based on this principle is the RUSH (Retention Using Selective Hooks) system (Boncompani et al., Nature Methods 2012 and WO2010142785). This is particularly envisioned for transient inhibition of chimeric NKG2D receptors.

[0123] Small molecule inhibitors, such as small organic molecules and other drug candidates can be obtained from, for example, combinatorial and natural product libraries.

[0124] Particularly envisioned for transient inhibition of functional NKG2D signaling is inhibition of downstream signals through chimeric NKG2D receptors. It is demonstrated herein that a significant portion of the observed killer agents are mediated through the PI3K signaling pathway induced by NKG2D, which is the primary signaling pathway of the NKG2D / DAP10 complex. Therefore, effective inhibition of PI3K signaling is a functional inhibition of NKG2D signaling because it reduces the functional effects of ligand-receptor binding. Therefore, transient inhibition of downstream signaling can be achieved by transient inhibition of PI3K signals. Exemplary inhibitors include commercially available PI3K inhibitors. A particularly anticipated inhibitor is the wide-ranging PI3K inhibitor LY294002. Another particularly envisioned inhibitor is Cal101 (Idelarisib). Other examples include, for example, Copanlisib, Taselisib, Buparlisib, Duvelisib, Alpelisib, and Umbralisib.

[0125] The methods described herein are applicable to the preparation of immune cells expressing NKG2D. Typically, immune cell preparation occurs when cells are prepared or cultured for adoptive transfer. This can be an autologous adoptive transfer (where the subject receives their own modified and / or expanded cells) or an allogeneic adoptive transplant (where the subject receives cells from a different individual).

[0126] Many different types of immune cells are used for adoptive therapy and are therefore envisioned for use in the methods described herein. Examples of immune cells include, but are not limited to, T cells, NK cells, NKT cells, lymphocytes, stem cells, or iPSCs. The latter two are not immune cells themselves, but can be used for adoptive cell transfer to carry out immunotherapy (see Jiang et al., Cell Mol Immunol 2014; Themeli et al., Cell Stem Cell 2015). Typically, although manufacturing starts with stem cells or iPSCs (or can even start from the dedifferentiation step of immune cells to iPSCs), manufacturing will need to differentiate into immune cells before administration. Since the method of the present invention relates to a manufacturing process (i.e., a step before administration), the stem cells and iPSCs used in the manufacture of the immune cells for adoptive transfer are considered to be immune cells herein. According to a specific embodiment, the stem cells envisioned in the method do not involve the step of destroying human embryos.

[0127] Particularly contemplated cells for use in the methods of the invention are T cells and NK cells.

[0128] According to another aspect, engineered immune cells are provided in which killing agents are reduced. These cells are characterized by functional inhibition of intracellular NKG2D signaling, such as by knocking out an NKG2D ligand, permanently or temporarily inhibiting an NKG2D ligand, or transiently inhibiting a chimeric receptor (e.g., by expressing a transient inhibitor, transiently expressing an inhibitor, or temporarily retaining a receptor or ligand).

[0129] Thus, these cells comprise a nucleic acid molecule encoding a chimeric NKG2D receptor and at least one of:

[0130] - one or more endogenous genes encoding NKG2D ligands that have been engineered to be inactivated;

[0131] - one or more inhibitors against the chimeric NKG2D receptor and / or one or more NKG2D ligands;

[0132] - a binding tag fused to the chimeric NKG2D receptor and / or to one or more NKG2D ligands.

[0133] Binding tags (eg, streptavidin) can be used in methods such as RUSH (Retention using Selective Hooks) (Boncompain et al., Nature Methods 2012 and WO2010142785) and are particularly envisioned for transient inhibition of chimeric NKG2D receptors.

[0134] Particularly contemplated cells include a nucleic acid molecule encoding a chimeric NKG2D receptor and at least one of:

[0135] - one or more endogenous genes encoding NKG2D ligands that have been engineered to be inactivated;

[0136] - one or more inhibitors directed against the chimeric NKG2D receptor and / or one or more NKG2D ligands.

[0137] Inactivation of the endogenous gene can be performed as described above, for example using genome editing, engineered nucleases such as CRISPR / Cas, TALEN, zinc finger nucleases, meganucleases, MegaTAL nucleases or other suitable methods (including but not limited to Cre / Lox or Flp / FRT-based systems). Although not a prerequisite, in most cases the inactivated endogenous gene will be permanently inactivated (i.e., there is no obvious reversal of inactivation). This is why this approach is particularly suitable for the inactivation of ligands (immune cells do not need the ligand for immunotherapy) and for the inactivation of NKG2D receptors, because the ligand is required for the receptor in immunotherapy, so this approach is particularly rare.

[0138] Cells containing inhibitors will typically contain a plasmid encoding the inhibitor, as this is the most convenient way to ensure that the inhibitor is contained in the cell. Therefore, it is particularly envisioned that the inhibitor can be expressed from a plasmid. Although this can be accomplished with antibodies or peptides, it is most particularly envisioned that nucleic acid inhibitors, such as RNA interference techniques, such as siRNA or shRNA. Inhibitors (e.g., RNA inhibitors) can be directed against chimeric NKG2D receptors and / or against one or more NKG2D ligands. These NKG2D ligands are selected from MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

[0139] These cells can be provided in the form of themselves or in a composition. Such a composition can also be provided, wherein NKG2D signal transduction is inhibited by adding an (external) inhibitor to the cell, the inhibitor being absorbed by the cell, or exerting its inhibitory effect outside the cell. Therefore, according to another aspect, a composition of immune cells expressing chimeric NKG2D receptors and also comprising an inhibitor is provided. These cells can include one or more endogenous genes encoding NKG2D ligands that have been engineered to be inactivatable; and / or one or more inhibitors for chimeric NKG2D receptors and / or one or more NKG2D ligands encoded as nucleic acids in the cell, and / or binding tags NKG2D ligands fused to the chimeric NKG2D receptor and / or one or more fusions ("internal inhibitors", as described above).

[0140] Additionally or alternatively, the composition may comprise one or more inhibitors to the chimeric NKG2D receptor and / or one or more NKG2D ligands (not encoded as nucleic acid in the cell); and / or inhibitors of downstream signaling of the chimeric NKG2D receptor ("external inhibitors").

[0141] Particularly contemplated inhibitors of the chimeric NKG2D receptor and / or one or more NKG2D ligands include antibodies directed against these proteins. Particularly contemplated inhibitors of downstream signaling of the chimeric NKG2D receptor include PI3K inhibitors.

[0142] In both cases, the inhibitor may be contained within the cell, or the composition may comprise the immune cell and the inhibitor as separate components, i.e., outside the cell. Note that even if the composition can be provided as a separate component, the inhibitor may be taken up by the immune cell. For example, downstream signaling inhibitors (e.g., PI3K inhibitors) are typically small molecules that are readily taken up by cells. Antibodies may or may not be taken up by cells, but since the interaction between the NKG2D receptor and its ligand occurs outside the cell, cellular uptake is not a prerequisite for inhibition, e.g., competitive inhibitors may act outside the cell.

[0143] According to another aspect, there is provided an engineered immune cell as described herein or a composition for use as a medicine. According to another aspect, there is provided an engineered immune cell as described herein or a composition for use in treating a disease characterized by expression of NKG2D ligands. It is well known that NKG2D ligands (such as ligands of MICA, MICB or RAET1 / ULBP families) are induced autoantigens, i.e., they are cellular ligands expressed under abnormal conditions or cell stress conditions, most particularly expressed in cells that are stressed (e.g., inflamed), transformed or infected. Therefore, cells or compositions are provided for treating a disease (e.g., viral, bacterial, fungal infection) selected from an inflammatory disease, cancer or infection. Since cell therapy is very expensive, it is particularly envisioned to be used for life-threatening diseases. Therefore, most particularly, cells and compositions as described herein are provided for treating cancer. In principle, all cancers can be treated, including but not limited to bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, glioblastoma, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, gastric cancer and thyroid cancer; the most specifically envisioned cancers include leukemia (including AML), multiple myeloma, bladder cancer, breast cancer, colorectal cancer, ovarian cancer and pancreatic cancer. This is because these seven cancers generally have high expression of NKG2D ligands.

[0144] Providing cells and compositions for treatment is equivalent to providing methods of treating disease, comprising the step of administering these cells or compositions to a subject in need thereof. Thus, methods of treating inflammatory diseases, cancer or infection are provided, comprising administering cells

[0145] Particularly contemplated are methods of treating cancer in a subject in need thereof, the method comprising the step of administering to the subject an engineered immune cell comprising a non-natural nucleic acid molecule encoding a chimeric NKG2D receptor and at least one of:

[0146] - one or more endogenous genes encoding NKG2D ligands that have been engineered to be inactivated;

[0147] - one or more inhibitors against the chimeric NKG2D receptor and / or one or more NKG2D ligands;

[0148] - a binding tag fused to the chimeric NKG2D receptor and / or to one or more NKG2D ligands.

[0149] Even more particularly envisioned are methods of treating cancer in a subject in need thereof comprising the step of administering to said subject an engineered immune cell comprising a non-natural nucleic acid molecule encoding a chimeric NKG2D receptor and at least one:

[0150] - one or more endogenous genes encoding NKG2D ligands that have been engineered to be inactivated;

[0151] - one or more inhibitors directed against the chimeric NKG2D receptor and / or one or more NKG2D ligands.

[0152] Likewise, provided are methods of treating cancer in a subject in need thereof, comprising the step of administering to the subject a composition, wherein the composition comprises immune cells expressing a chimeric NKG2D receptor and one or more of:

[0153] - one or more endogenous genes encoding NKG2D ligands that have been engineered to be inactivated; and / or

[0154] - one or more inhibitors of the chimeric NKG2D receptor and / or one or more NKG2D ligands encoded as nucleic acids in the cell, and / or

[0155] - a binding tag fused to a chimeric NKG2D receptor and / or one or more NKG2D ligands; and / or

[0156] - one or more inhibitors of the chimeric NKG2D receptor and / or one or more NKG2D ligands (not encoded as nucleic acid in the cell); and / or

[0157] -Inhibitors of chimeric NKG2D receptor downstream signaling.

[0158] The immune cells may be autologous to the subject to which the cells are administered, or may be allogeneic, ie, derived from a different subject.

[0159] It should be understood that although specific embodiments, specific configurations, and materials and / or molecules have been discussed herein with respect to the cells and methods according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of the invention. The following examples are provided to better illustrate specific embodiments and should not be construed as limiting the present application. The present application is limited only by the claims. Example

[0160] introduce

[0161] Given that a plethora of NKG2D ligands can be expressed in T cells, gene editing to eliminate all ligand expression is not considered the most effective option. Therefore, alternative strategies to control the killing agent have been investigated to facilitate the delivery of NKG2D-targeted CAR T-cell therapies. Two different approaches have been explored: using signaling inhibitors or antibody-based approaches. Both result in inhibition of the killing agent, albeit to varying degrees.

[0162] The inclusion of a phosphoinositide-3-kinase inhibitor (LY294002) weakens the effect of the killing agent and provides a universal method for generating NKR-2CAR T cells. The use of PI3K inhibitors further enhances the efficacy of NKR-2 drive and converts the cells into a memory phenotype. Target-specific methods involving CAR autoantibody blockade lead to further increases in NKR-2CAR T cell production, reduced efficacy, and changes in the CD4 / CD8 ratio. By delaying the addition of blocking antibodies, these factors can be successfully skewed in vitro to enhance efficacy and change cell phenotypes. Despite differences in methods, inhibitor- or antibody-based methods still produce NKR-2CAR T cells with highly similar phenotypes and in vivo activity. Finally, although it is impractical to inhibit all NKG2D ligands, inhibiting the two most important ligands by transient (shRNA) or permanent (Crispr / Cas) methods also produces NKR-2CAR T cells with highly similar phenotypes.

[0163] These results suggest that target-driven killing can be overcome using different approaches that could enable the development of T-cell therapies where self-expression of target ligands is a limiting factor.

[0164] Materials and methods

[0165] Antibodies and flow cytometry

[0166] Cells were fluorescently labeled with CD3 (BD, 345766), CD4 (BD, 345809), CD8 (BD, 345772), CD314 (BD, 558071), CD45RA (BD, 550855), CD62L (BD, 555544), CD279 (eBioscience, 12-2799-42), CD19 (BD, 345791), CD223 (eBioscience, 25-2239-41), MICA / B (R&D Systems, FAB13001G-100), MICB (R&D Systems, FAB1599G), ULBP1 (R&D Systems, FAB1380C), ULBP2 / 5 / 6 (R&D Systems, FAB1298A), ULBP3 (R&D Systems, FAB1517P), ULBP4 (R&D Systems: FAB6285A), and the corresponding isotype according to the standard protocol. Briefly, cells were harvested and resuspended in a buffer containing DPBS (Life Technologies, A1285801) supplemented with 5% human serum albumin (Octapharma, 68209-633-02) and 0.01% NaN 3 (Sigma, S2002). Cells were incubated with antibodies at 4 ° C for 30 minutes, washed with PBS, and then analyzed on a Guava easyCyte 6HT cytometer (Millipore). All antibodies were titrated before use in the experiment. Viable cells were selected based on FSC / SSC. In all cases, unlabeled controls and isotype controls were used. FlowJo v10 was used for analysis.

[0167] Plasmid and vector production

[0168] Chimeric NKG2D (chNKG2D) constructs were prepared as previously described (Zhang, Barber, & Sentman, 2006) and cloned into the Mo-MLV-based nucleocapsid viral vector SFG between the NcoI and XhoI restriction sites. The pSFGGFP plasmid and pSFG htCD19.1 (encoding a truncated form of human CD19 (tCD19)) were a gift from Celdara Medical LLC (Lebanon, NH, USA). PG2-293 packaging cells were transiently transfected with the relevant plasmids and the VSV-G envelope plasmid. The retroviral suspension produced in PG2-293 cells was used to spin-transduce PG13 cells to obtain a stable producer cell line. After culture confluence, vector particles for transduction of human T lymphocytes were harvested from PG13 stable producer cells. Vector titers were measured using the Retro-XTM qRT-PCR titration kit (Life Technologies, CL 631453).

[0169] NKG2D-CAR T cell production

[0170] Peripheral blood mononuclear cells (PBMC) are separated from the whole blood of healthy donors (ImmuneHealth, CHU, Tivoli) by ficoll density gradient (VWR, 17-5442-03) according to standard procedures. In brief, whole blood is diluted 3 times with DPBS, then carefully added to the polysucrose layer in 50ml tube. Test tube is centrifuged with 500g, and the middle layer is carefully removed. PBMC is subsequently washed 3 times with DPBS, harvested and subsequently activated in X-Vivo 15 culture medium (Westburg, BE02-061Q) containing 5% human serum (Access Biologicals, 515-HI), and supplemented with 40ng / ml OKT3 (Miltenyi, 170-076-124) and 100IU / ml IL-2 (Miltenyi, 170-076-146). Cells are maintained at 37°C, 5% CO2 in an incubator for two days. The cells were then harvested and plated in 24-well plates (1 × 10 6 Cells were transduced with different viral vectors in 24-well plates (100 cells / well) and incubated for 2 days. Cells were then harvested from the 24-well plates, washed with HBSS (Westburg, BE10-543F), and transferred to G-Rex containers (Wilson Wolf, 80040S) for a further 4 days of expansion in X-Vivo 15 containing complete serum and IL-2 or as described in the text. At the end of the expansion phase, cells were harvested and used accordingly.

[0171] Cell lines and culture reagents

[0172] The chronic myeloid leukemia cell line K562 and the pancreatic cancer cell line PANC-1 were purchased from ATCC and maintained in IMDM (Westburg, LO BE12-722F) or DMEM (Westburg, LO BE12-604F), respectively, containing 10% FBS (Gibco, 16140071) and 1% penicillin / streptomycin (ThermoFisher Scientific, 15140122) until use. The PI3K inhibitor LY294002 was purchased from Selleck Chemicals (S1105). Inhibitory antibodies against NKG2D or corresponding isoforms were purchased from BioLegend (inhibitory antibody (Ultraleaf CD314) clone: ​​1D11, ImTec Diagnostics NV, 320814).

[0173] Cell lysis assay

[0174] Adherent PANC-1 cells were cultured in a 1:1 ratio in a flat-bottom 96-well plate in X-Vivo 15 medium containing 5% human serum (HS) without phenol red for 20 hours in the presence or absence of thawed NKR-2 T cells or tCD19-transduced cells. T cells were washed, and the remaining adherent PANC-1 cells were labeled with alamarBlue (ThermoFisher Scientific, DAL1025) for 4 hours. Viable cells were measured using fluorescence at 530 nm using a SpectraMax M2 (Molecular Devices), and relative cytolytic activity was calculated.

[0175] Cytokine release assay

[0176] Fresh NKR-2 T cells and / or control tCD19 cells were incubated with K562 or PANC-1 cells at a 1:1 ratio in X-Vivo 15 containing 5% HS. After 24 hours of incubation, supernatants were collected and IFN-γ was measured by ELISA (R&D Systems, SIF50) according to the manufacturer's protocol. As a positive control, cells were activated with PMA (Sigma-Aldrich, P8139-5MG) and ionomycin (Sigma-Aldrich, I0634-1MG). To assess the background level of activation, cells were not stimulated.

[0177] Antibody inhibition assay

[0178] NKR-2 T cells were incubated with 1 μg / mL of NKG2D blocking antibody, isotype control, or no antibody for 24 hours, and NKR-2 T cell-mediated IFN-γ secretion was measured. Similarly, NKR-2 cells were co-cultured with cancer cells in the presence of antibody, and cytokine secretion was measured.

[0179] RNA extraction and qPCR

[0180] PBMCs were stimulated with 40 ng / mL OKT3 and IL-2 (100 IU / mL) for 2 days, transduced and cultured with 40 ng / mL OKT3 and IL-2 (100 IU / mL) for another two days, and then expanded in the presence of IL-2 (100 IU / mL) until day 8 or day 10, as described in detail herein. Total RNA was isolated every two days using the RNeasy Mini Kit (Qiagen, 74104). Quantitative PCR reactions were performed using pre-designed TaqMan Gene Expression Assays (Hs04187752_mH, Hs01026642_m1, Hs00607609_mH, Hs00906262_m1, Hs00741286_m1, Hs01584111_mH, Hs04194671_s1, Hs00360941_m1, ThermoFisher Scientific) with NKG2D ligands and Light Cycler 480 RNA Master Mix (Roche, 04991885001). Relative expression was based on in-house designed primers (5′-GACGGCGAGCCCTTGG-3′ and 5′-GCACGAAAATTTTCTGCTGTCTT-3′) and probe (5′TEX615-TCTCCTTTGAGCTGTTTGCAGACAAGGT-3′BHQ TM ) obtained housekeeping gene cyclophilin. Results are expressed as fold induction compared to day 0 (calculated as 2^-ΔΔCT). All gene expression assays were tested on different cancer cell lines known to express ligands.

[0181] Animal studies

[0182] All in vivo experiments were performed at Voxcan (Marcy l'Etoile-France). Briefly, NOD / scid IL2rgnull (NSG) mice were irradiated 24 h before tumor injection (day -1). On day 0, 5 × 10 6THP-1-luc-GFP cells / mouse were transplanted with PBS. On day 7, THP-1-luc-GFP positive mice were divided into four treatment groups: (i) control received a single IV injection of vehicle (200 μl HBSS); (ii) mock tCD19 received a single IV injection of 10×10 6 Simulated tCD19 T cells (200 μL); (iii) NKR-2 LY single injection 10x10 6 NKR-2-LYT cells (200 μl); (iv) a single intravenous injection of 10x10 6 NKR-2 optimized antibody (200 μl). Tumor progression was assessed by bioluminescence imaging on days 4, 8, 15, 22, 28, and 35. Similarly, the body weight of each animal was measured three times a week starting from day 6.

[0183] Statistical analysis

[0184] Statistical significance was assessed using unpaired, paired two-tailed t-test, or the nonparametric Mann-Whitney U test, where applicable. Statistical significance was considered when p < 0.05.

[0185] Example 1. NKR-2 CAR T cells experience killing agents that drive the phenotype of engineered T cell populations and amplification.

[0186] After transduction and in vitro culture, in the absence of a control killing agent, the NKR-2 T cell population displayed a predominantly CD8 + T cell subset composition (truncated CD19 (tCD19), Figure 1 A). NKG2D expression was not restricted to NKR-2 T cells but was also clearly visible on control tCD19 T cells, although engagement of endogenous NKG2D failed to produce a therapeutic response in CAR T cells (32). However, the relative cell surface expression of NKG2D was highly increased in the NKR-2 T cell population, indicating that transduction of T cells with the CAR construct ( Figure 1 B) The mean fluorescence intensity of NKG2D (CD314) in CD4 + and CD8 + The expression of CAR in these two subsets was significantly higher in the NKR-2 T cell populations. Figure 2 AC).

[0187] Interestingly, the NKR-2 T cell population had a reduced relative frequency of naive cells (defined as double positive CD45RA+ and CD62L+ cells) compared to the tCD19 control T cell population, which was suggestive of an increased effector cell phenotype ( Figure 1 C-1D), CD279 (PD-1), and CD223 (Lag-3). After co-culture with cancer cell lines, NKR-2 T cells exhibited high levels of target cell-induced IFN-γ secretion ( Figure 3 A) and cytolytic activity ( Figure 3 B), confirming the functionality of NKR-2 T cells. However, the yield / fold expansion of NKR-2 T cells during culture and at harvest ( Figure 3 C) and viability (data not shown) were consistently reduced.

[0188] Expression of NKG2D ligands has been demonstrated in T cells during mitogenic activation (22), and it is well known that natural killer cells undergo cytotoxic killing due to the engagement of the NKG2D receptor (23, 24). Together, this raised the question of whether NKR-2 T cells were undergoing cytotoxic killing after transduction, which in turn led to low cell yield and viability, skewed CD4 / CD8 ratio, and enhanced effector memory differentiation. To test this, T cells from a donor were transduced with an eGFP expression vector and mixed with NKR-2 T cells generated from the same donor to explore whether general T cell killing occurred. Twenty-four hours later, there was a significant loss of eGFP T cells from the NKR-2 T cell co-culture, implying that NKR-2 T cells can kill autologous T cells ( Figure 3 D).

[0189] To understand the expression profile of NKG2D ligands in activated T cells, three healthy donors were used as a source of T cells, activated without transduction, and qPCR analysis was performed to examine the dynamics of NKG2D ligand mRNA expression profiles ( Figure 3 E). Rapid increases in MICA and ULBP2 mRNA levels were detected within two days of T cell activation. However, while ULBP2 mRNA levels remained high, MICA rapidly decreased to baseline within two days. ULBP3 mRNA gradually increased throughout the time course, while there was no relative increase in transcripts for MICB, ULBP5, ULBP1, or ULBP6. In contrast, ULBP4 mRNA increased slightly on day 4 but then returned to baseline levels. At the cell surface protein level, MICA / B followed a similar expression pattern to the corresponding mRNA, with a transient increase in MICA on day 2 followed by a gradual decrease ( Figure 3F). Unfortunately, no suitable antibodies are available to detect individual ULBP2 / 5 and 6 proteins. However, the immunoreactivity observed with antibodies that recognize all three family members is most likely due to ULBP2 based on mRNA expression profile, which was the only one to increase after day 2. Although highly induced at the mRNA 295 level, no cell surface ULBP3 was detected. ULBP4+ cells reached a peak of positivity on day 6 and then decreased to baseline by day 8, following the mRNA expression pattern (delayed by two days). Figure 3 F). These observations are reflected in the parallel kinetics of mean fluorescence intensity (Table 1).

[0190] Table 1. Mean fluorescence intensities (MFI) of all ligands from three different donors and their corresponding SDs after the manufacturing process (samples were analyzed on days 0, 2, 4, 6, and 8).

[0191]

[0192] Together, these data suggest that T cells modulate expression of NKG2D ligands upon mitogenic activation with MICA, ULBP4, and putatively ULBP2, being the predominant NKG2D ligands at the protein level, albeit with different expression kinetics.

[0193] Example 2. PI3K inhibition improves the viability of NKR-2 T cells during cryopreservation and drives NKR-2 antigen expression. Increased production of specific cytokines and an enhanced memory phenotype.

[0194] Upon ligand binding, NKG2D and its associated DAP10 initiate signaling through the PI3K pathway in a manner similar to CD28 (25, 26). Therefore, we questioned whether inhibition of PI3K signaling could abrogate NKR-2-mediated killing in T cell culture. To this end, increasing concentrations of LY294002, a broad PI3K inhibitor, were added to the transduction and expansion stages of NKR-2 production.

[0195] The addition of LY294002 resulted in several observations. First, cell surface expression of NKR-2T on NKR-2 T cells was reduced in a dose-dependent manner, reaching the level of control tCD19 T cells at 10 μM ( Figure 4 A and Figure 5 This reduction was reversible, as removal of LY from the culture resulted in an increase in NKG2D expression to the level of untreated NKR-2 T cells (data not shown). However, the inhibitors did not significantly improve cell yield, suggesting that the killing agent was not fully controlled during the culture process or that the PI3K inhibitor had a deleterious effect on proliferation ( Figure 3B). To evaluate whether LY294002 has an anti-proliferative effect, control tCD19 T cells were treated with a PI3K inhibitor during culture. These control T cells showed a significantly reduced proliferation capacity compared to untreated tCD19 cells ( Figure 6 ).

[0196] As expected, NKR-2 T cells treated with the PI3K inhibitor showed increased cell viability after cryopreservation or after storage at 4°C for 48 hours ( Figure 3 C, 3D). NKR-2 T cells generated by PI3K inhibitors produced a large amount of IFN-γ in a LY294002 dose-dependent manner ( Figure 7 A). Finally, NKR-2 T cells cultured with LY294002 also appear to have increased CD62L + / CD45RA - Phenotype ( Figure 7 B), which is consistent with previous work using this inhibitor to modulate the memory phenotype of T cells (27,28).

[0197] Overall, the addition of a PI3K inhibitor had a beneficial effect on the viability of NKR-2 T cells, which is attractive for therapeutic applications.

[0198] Example 3. Antibody-mediated NKG2D blockade prevents NKR-2 CAR T cell killing.

[0199] Initial experiments that included an anti-NKG2D antibody (clone 1D11) during NKR-2 T cell culture demonstrated that NKR-2 T cell yields at the end of culture were comparable to control T cells (2.6-fold expansion of NKR-2 T cells and 13.8-fold expansion of NKR-2 T cells with antibody blocking capacity ( Figure 8 This suggests that antibody blockade can eliminate NKG2D-targeted killing. Dose titration experiments showed that antibody concentrations of 2.5 μg / mL and above can protect tCD19 T cells 358 from NKR-2 T cell-targeted killing ( Figure 4 E). Anti-NKG2D antibodies can also effectively block the release of IFN-γ during target cell engagement ( Figure 4 F), thus confirming the specificity of NKR-2 T cells. The effective killing of killer agents by anti-NKG2D antibodies was further supported by the fact that the IFN-γ release observed during the generation of NKR-2 T cells, which may be caused by T cell-killing killer agents, was significantly reduced by the addition of blocking antibodies ( Figure 4G). Because mouse blocking antibodies may cause toxicity through antibody-dependent cell-mediated cytotoxicity (ADCC), extensive washing steps were implemented after harvest. IgG ELISA and flow cytometry experiments showed that no contaminating antibodies were detected in the supernatant or on the cell surface after harvest (data not shown). To assess ADCC, NK cells were co-cultured with autologous NKR-2 cells in the presence of 5 μg / mL Ab in the absence of evidence of ADCC (data not shown). Together, these data indicate that the addition of anti-NKG2D blocking antibodies controls NKR-2 T cell CAR-driven killing.

[0200] Example 4. Antibody-mediated NKG2D blockade and PI3K effects on NKG2D signaling in the production of NKR-2 expressing cells The inhibition of conduction is functionally equivalent.

[0201] Adaptation of the Ab blocking process during in vitro NKR-2 cell expansion resulted in improved yields with equivalent in vitro and in vivo activity.

[0202] Comparison between NKR-2 T cells generated using the Ab and PI3K inhibitor procedures revealed different cytolytic kinetics, implying that the Ab procedure altered T cell characteristics ( Figure 9 A). When comparing the two processes, the main difference observed was the CD4 / CD8 ratio, which was consistently skewed towards the CD8 population at day 8 after addition of the PI3K inhibitor. Interestingly, blocking NKR-2 T cells rescued the CD4+ population, suggesting that the observed skewed CD4 / CD8 ratio is cytotoxic agent dependent ( Figure 9 B) The most likely explanation for the difference in ratios is either a relative increase in the ratio due to proliferation of CD4 T cells or depletion of CD4 T cells by CD8 T cells.

[0203] We hypothesized that the enhanced lytic activity of NKR-2 T cells generated by PI3K inhibitors might be due to a lower CD4 / CD8 ratio. To address this issue, we adapted the blocking antibody process by adding the blocking antibody on day 6 rather than immediately after transduction (day 4). This modification resulted in the generation of NKR-2 T cells with a CD4 / CD8 ratio similar to that of cells generated by PI3K inhibitors ( Figure 9 C), while maintaining comparable fold expansion to control T cells ( Figure 9 D). Subsequently, although there were subtle differences in some parameters between the two processes, such as expression of the activation marker CD25 and memory phenotype (data not shown), functional cytokine secretion and cytolytic activity of NKR-2 T cells against target cancer cells were comparable between the two processes ( Figure 9 E, F).

[0204] In preliminary in vivo experiments using NOD SCID mice, NKR-2 cells generated by the LY and Ab process showed similar antitumor activity in an established acute myeloid leukemia (THP-1) tumor model (8 days after infusion, as observed by bioluminescence; tCD19: 5.76E10 + / - 4.46E10; NKR-2 LY: 7.15E08 + / - 1.01E09; NKR-2 Ab: 6.43E08 + / - 1.25E08; data not shown). A one-way ANOVA with Tukey's post hoc test showed significant differences between LY and tCD19 control cells (p: 0.02), while Ab-generated NKR-2 compared to tCD19 (p = 0.03). No differences were observed between the LY and Ab groups (p = 0.95). In addition, similar engraftment was observed after 24 h in both the NKR-2LY and Ab groups (LY group: 1.838 + / - 1.07%; Ab group: 1.792 + / - 0.56%; data not shown), indicating that no significant difference was detected in the short-term engraftment between the two groups.

[0205] Taken together, these combined data demonstrate that NKR-2 T cells generated using an adaptive blocking antibody procedure exhibit similar short-term engraftment and potency to cells generated using a PI3K inhibitor.

[0206] Effects on NKR2 production are PI3K-mediated and not due to specific inhibitors

[0207] To further confirm whether the effects of LY294002 were indeed related to its PI3K inhibitory activity, several other PI3K inhibitors, including wortmannin and CAL-101 (idelalisib), were also tested. Figure 10 Representative data for CAL-101 are shown in . Figure 10 Cal-101 ​​behaved similarly to the blocking antibody in terms of cell viability (A) and cell viability (B). Like other PI3K inhibitors, cells appeared to produce more interferon ( Figure 10 C), which may help improve efficacy.

[0208] Downstream inhibitors of the PI3K pathway (e.g., the glycogen synthase kinase 3β inhibitor TWS119 or the mTOR inhibitor rapamycin) were also tested. The results obtained were similar (data not shown), although inhibition of PI3K appeared to result in cells with suboptimal properties, with increased proliferation compared to cells without NKG2D signaling inhibition. This may be due, at least in part, to the toxicity of, for example, rapamycin.

[0209] Example 5. Inhibition of NKG2D ligands also leads to improvements in cell yield and cytolytic activity

[0210] NKG2D is known to bind to eight different stress-induced ligands (NKG2DLs) that are ubiquitous in tumors but absent in healthy tissues. We aimed to identify key NKG2DLs expressed on T cells after activation. PBMCs were activated with OKT3 and anti-CD3 antibodies on day 0. Expression of eight NKG2DLs was assessed on the surface of CD4+ and CD8+ T cells every other day. Figure 11 ). After activation, MICA / B and MICB are upregulated on the cell surface of CD4 and CD8 T cells, with expression reaching a peak 2-4 days after activation. Subsequently, expression decreases until day 10. ULBP1 and ULBP2 are expressed at low levels, while ULBP2 is restricted to CD4+ T cells ( Figure 11 There is little evidence that other ligands are expressed on T cells.

[0211] Parallel studies identified MICA and MICB as the primary stimulators of NKG2D CARs (data not shown), leading us to propose that MICA and MICB are the primary NKG2DLs responsible for T cell killing.

[0212] We then explored specific targeting of MICA and MICB with single shRNAs, which was feasible due to the high sequence similarity. Primary T cells were transduced with different shRNAs and MICA and MICB protein expression was assessed. This screen identified two shRNAs that reduced cell surface expression of MICA and MICB (data not shown). Next, we designed a retroviral vector encoding an NKG2D CAR and co-expressing the candidate shRNAs. We evaluated the level of killing in T cells engineered with an NKG2D-based CAR or co-expressing the shRNAs by cell expansion ( Figure 12 Engineering of a single retroviral vector encoding both the NKG2D CAR and the shRNA generated T cells that exhibited significantly reduced killing potency in vitro compared to cells without the shRNA ( Figure 12 ) and increased the expansion rate of NKG2D CAR T cells, bringing it close to that of control T cells.

[0213] We then evaluated the in vitro antitumor efficacy of NKG2D-based CAR T cells with and without shRNA targeting MICA / B. Cells lacking shRNA showed specific killing of AML HL-60 cells at different effector to target (E:T) ratios. However, co-expression of MICA / B shRNA #2 or #4 improved cancer cell killing, especially at lower E:T ratios ( Figure 13A). 24 hours after co-culture, shRNA expression increased T cell recovery, and the reduced killing ability may be due to the reduction of killing agents ( Figure 13 B).

[0214] In conclusion, knocking out NKG2D ligands in T cells showed the same improved manufacturing outcomes as NKG2D inhibition or PI3K inhibition.

[0215] Similar data were obtained when MICA and MICB were permanently disabled using Crispr / Cas (data not shown). A notable difference is that shRNA inhibition can be accomplished in a transient manner (e.g., only during the manufacturing process), whereas gene knockout (here using Crispr / Cas) is permanent. Depending on the context, this may or may not be desirable.

[0216] discuss

[0217] The recent approvals of CD19 CAR T cell therapy in B-cell acute lymphoblastic leukemia (bALL) and diffuse large B-cell lymphoma (DLBCL) provide strong clinical validation for this approach and provide impetus for the development of CAR T cell therapies beyond CD19+ B malignancies. Target selection is crucial for therapeutic success. To date, the identification of tumor-exclusive antigens has been challenging. A recent bioinformatics study combining proteomic and genomic approaches in acute myeloid leukemia (AML) indicated that there are no tumor-specific cell surface antigens and that antibody-based CAR T targeting of AML may require complex combinatorial targeting strategies (29). Therefore, the majority of target antigens tested to date are tumor-associated antigens, where expression of the target may also be present on normal healthy cells. Examples include CD19 in B19 malignancies (3, 4), CD123 in AML (30), CD7 (14), and CEA in a range of solid tumors (31). However, this poses a problem in situations where the target antigen may be permanently or transiently expressed on T cells, as CAR-engineered T cells are likely to subsequently target self and other antigens in culture, resulting in T cell killing, effectively translating into reduced or even zero cell yield.

[0218] Gene editing now offers a clinically relevant approach to prevent the expression of specific proteins, thereby enabling the expansion of CAR T cells that might otherwise be exposed to killer agents. (14) However, the multi-target specificity of NKG2D-based CARs means that the apparent potential for gene editing to eliminate eight different proteins in primary T cells and effectively express CAR constructs is nonetheless challenging to implement clinically. Therefore, this paper proposes alternative strategies to avoid killer agents during cell culture, thereby enabling the generation and delivery of NKG2D-focused CAR T cell therapies.

[0219] In these embodiments, PI3K inhibitors provide a general method for controlling killer agents by reducing NKG2D expression on the cell surface. To our knowledge, this is the first report of such an observation. The mechanism by which PI3K inhibitor treatment causes loss of cell surface NKG2D is currently unknown. It is known that NKG2D cell surface localization is mediated by its association with DAP10 (Upsahw et al., 2006). One hypothesis for the loss of cell surface NKG2D may be that DAP10 is affected by PI3K inhibitor treatment (e.g., reducing RNA levels, inhibiting transcription, and even inhibiting post-translational modifications (e.g., glycosylation), which are necessary for DAP10 to bind to NKG2D (Park YP et al., 2011, Blood)). These may ultimately lead to preventing the NKG2D-DAP10 complex from being expressed on the cell surface, thereby inhibiting the killer agent.However, PI3K inhibition is also associated with decreased cell proliferation (Aagaard-Tillery KM, Jelinek DF. Phosphatidylinositol 3-kinase activation in normal human B lymphocytes. J Immunol. 1996; 156: 4543-4554.11. Fruman DA, Snapper SB, Yballe CM, et al. Impaired B cell development and proliferation in the absence of phosphoinositide 3-kinase p85alpha. Science. 1999; 283: 393-397.12. Shi J, Cinek T, Truitt KE, Imboden JB. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, blocks antigen-mediated, but not CD3 monoclonal antibody-induced, activation of murine CD4 T cells. J Immunol.1997;158:4688-4695.13.Truitt KE,Shi J,Gibson S,Segal LG,Mills GB,Imboden JB.CD28 delivers costimulatory signals independently of its association with phosphatidylinositol 3-kinase.J Immunol.1995;155:4702-4710.), Therefore, in cases where relatively low doses of CAR T cells are required, this inhibitor approach provides an effective solution to control T cell killing agents, but in cases where large numbers of cells are required, the use of this approach is more limited.

[0220] Another approach to inhibit killer molecules during the production of NKR-2 T cells is to use specific blocking antibodies during the expansion phase. This enables control of T cell killer molecules and expansion of T cells to the same level as control tCD19 T cells. This approach depends largely on the antibody used, as it requires blocking the killer agent without inducing CAR activation (as shown by the greatly reduced levels of cytokine production during culture in the absence of target antigen). The addition of specific blocking antibodies provides a solution for large-scale expansion of NKR-2 T cells.

[0221] A major concern regarding the blocking antibody process is the potential for antibody modification of expanded NKR-2 CAR T cells, leading to rapid elimination of the cells following infusion through antibody-dependent clearance mechanisms. However, analyses clearly demonstrated that NKR-2 cells lacking the anti-CD314 antibody coating and undergoing re-expression analysis indicated that antibody binding appears to result in the loss of NKG2D and the NKR-2 CAR from the cell surface. Following binding to the target ligand, endogenous NKG2D has been shown to be rapidly internalized as a mechanism for controlling NK cell activation. The observations here suggest that, in the context of NKR-2, the CAR also appears to be internalized. This is advantageous from the perspective of adoptive T cell therapy because specific methods for removing bound antibodies do not need to be developed.

[0222] In addition to the observed differences between the courses and the effects of PI3K inhibitors, these preclinical data provide the first evidence that NKR-2 is an effective therapy in a mouse model of AML. Furthermore, these results are consistent with other studies in which the lack of pretreatment and multiple injections of NKR-2 were the criteria for tumor eradication and long-term survival in treated mice (Zhang et al. Cancer Res. 2007; 67(22): 11029-36; Barber et al., Gene Ther. 2011; 18(5): 509-16).

[0223] Furthermore, although knocking out or ablation of all NKG2D ligands is not considered feasible or feasible, similar improvements in yield have been achieved by inhibiting the two most common ligands in T cells via shRNA or CRISPR, suggesting that even partial inhibition of NKG2D signaling can improve killing.

[0224] Taken together, this work demonstrates that T cell killers can be administered through general approaches (acting on downstream signaling) such as PI3K inhibition or receptor-specific approaches such as blocking antibodies or elimination of receptor ligands. In particular, PI3K inhibitor and blocking antibody approaches can be used to generate immune cell products with reduced levels of killing, and each approach has potential advantages for generating T cell products where other approaches, such as gene editing to eliminate targets in T cell populations, are challenging or are not currently feasible or desirable.

[0225] References

[0226] 1. Fesnak AD, June CH, Levine BL (2016) Engineered T cells: the promise and challenges of cancer immunotherapy. Nat Rev Cancer.16:566-81.doi:10.1038 / nrc.2016.97

[0227] 2.Brenner MK(2017)Next Steps in the CAR Journey of a ThousandMiles.Mol Ther.25:2226-7.doi:10.1016 / j.ymthe.2017.09.013

[0228] 3. Davila ML, Brentjens RJ (2016) CD19-Targeted CAR T cells as novelcancer immunotherapy for relapsed or refractory B-cell acute lymphoblasticleukemia. Clin Adv Hematol Oncol. 14:802-8.

[0229] 4.Kochenderfer JN, Somerville RPT, Lu T et al. (2017) Long-DurationComplete Remissions of Diffuse Large B Cell Lymphoma after Anti-CD19 ChimericAntigen Receptor T Cell Therapy. Mol Ther.25:2245-53.doi:10.1016 / j.ymthe.2017.07.004

[0230] 5.Park JH,Geyer MB,Brentjens RJ(2016)CD19-targeted CAR T-celltherapeutics for hematologic malignancies:interpreting clinical outcomes todate.Blood.127:3312-20.doi:10.1182 / blood-2016-02-629063

[0231] 6.Rossig C(2017)CAR T cell immunotherapy in hematology andbeyond.Clin Immunol.doi:10.1016 / j.clim.2017.09.016

[0232] 7.Turtle CJ,Hay KA,Hanafi LA et al.(2017)Durable Molecular Remissionsin Chronic Lymphocytic Leukemia Treated With CD19-Specific Chimeric AntigenReceptor-Modified T Cells After Failure of Ibrutinib.J Clin Oncol.35:3010-20.doi:10.1200 / JCO.2017.72.8519

[0233] 8.Rezvani K,Rouce R,Liu E,Shpall E(2017)Engineering Natural KillerCells for Cancer Immunotherapy.Mol Ther.25:1769-81.doi:10.1016 / j.ymthe.2017.06.012

[0234] 9.Adair PR,Kim YC,Zhang AH,Yoon J,Scott DW(2017)Human Tregs MadeAntigen Specific by Gene Modification:The Power to Treat Autoimmunity andAntidrug Antibodies with Precision.Front Immunol.8:1117.doi:10.3389 / fimmu.2017.01117

[0235] 10.Guest RD,Kirillova N,Mowbray S et al.(2014)Definition andapplication of good manufacturing process-compliant production of CEA-specific chimeric antigen receptor expressing T-cells for phase I / IIclinicaltrial.Cancer Immunol Immunother.63:133-45.doi:10.1007 / s00262-013-1492-9

[0236] 11.Levine BL,Miskin J,Wonnacott K,Keir C(2017)Global Manufacturing ofCAR T Cell Therapy.Mol Ther Methods Clin Dev.4:92-101.doi:10.1016 / j.omtm.2016.12.006

[0237] 12.Wang X,Riviere I(2016)Clinical manufacturing of CAR T cells:foundation of a promising therapy.Mol Ther Oncolytics.3:16015.doi:10.1038 / mto.2016.15

[0238] 13.Callard RE,Stark J,Yates AJ(2003)Fratricide:a mechanism forTmemory-cell homeostasis.Trends Immunol.24:370-5.

[0239] 14.Gomes-Silva D,Srinivasan M,Sharma S et al.(2017)CD7-edited Tcellsexpressing a CD7-specific CAR for the therapy of T-cellmalignancies.Blood.130:285-96.doi:10.1182 / blood-2017-01-761320

[0240] 15.Mamonkin M,Rouce RH,Tashiro H,Brenner MK(2015)AT-cell-directedchimeric antigen receptor for the selective treatment of T-cellmalignancies.Blood.126:983-92.doi:10.1182 / blood-2015-02-629527

[0241] 16.Leisegang M,Wilde S,Spranger S,Milosevic S,Frankenberger B,UckertW,Schendel DJ(2010)MHC-restricted fratricide of human lymphocytes expressingsurvivin-specific transgenic T cell receptors.J Clin Invest.120:3869-77.doi:10.1172 / JCI43437

[0242] 17.Schendel DJ,Frankenberger B(2013)Limitations for TCR gene therapyby MHC-restricted fratricide and TCR-mediated hematopoietic stem celltoxicity.Oncoimmunology.2:e22410.doi:10.4161 / onci.22410

[0243] 18.Sentman CL,Meehan KR(2014)NKG2D CARs as cell therapy forcancer.Cancer J.20:156-9.doi:10.1097 / PPO.0000000000000029

[0244] 19.Lanier LL(2015)NKG2D Receptor and Its Ligands in HostDefense.Cancer Immunol Res.3:575-82.doi:10.1158 / 2326-6066.CIR-15-0098

[0245] 20.Spear P,Wu MR,Sentman ML,Sentman CL(2013)NKG2D ligands astherapeutic targets.Cancer Immun.13:8.

[0246] 21.Demoulin B,Cook WJ,Murad J,Graber DJ,Sentman ML,Lonez C,Gilham DE,Sentman CL,Agaugue S(2017)Exploiting natural killer group 2D receptors forCAR T-cell therapy.Future Oncol.13:1593-605.doi:10.2217 / fon-2017-0102

[0247] 22.Molinero LL,Fuertes MB,Rabinovich GA,Fainboim L,Zwirner NW(2002)Activation-induced expression of MICA on T lymphocytes involves engagement ofCD3 and CD28.J Leukoc Biol.71:791-7.

[0248] 23.Madera S,Rapp M,Firth MA,Beilke JN,Lanier LL,Sun JC(2016)Type IIFN promotes NK cell expansion during viral infection by protecting NK cellsagainst fratricide.J Exp Med.213:225-33.doi:10.1084 / jem.20150712

[0249] 24.Nakamura K,Nakayama M,Kawano M,Amagai R,Ishii T,Harigae H,Ogasawara K(2013)Fratricide of natural killer cells dressed with tumor-derived NKG2D ligand.Proc Natl Acad Sci US A.110:9421–6.doi:10.1073 / pnas.1300140110

[0250] 25.Smith-Garvin JE,Koretzky GA,Jordan MS(2009)T cell activation.AnnuRev Immunol.27:591–619.doi:10.1146 / annurev.immunol.021908.132706

[0251] 26.Upshaw JL,Arneson LN,Schoon RA,Dick CJ,Billadeau DD,Leibson PJ(2006)NKG2D-mediated signaling requires a DAP10-bound Grb2-Vav1 intermediateand phosphatidylinositol-3-kinase in human natural killer cells.NatImmunol.7:524-32.doi:10.1038 / ni1325

[0252] 27.Abu Eid R,Ahmad S,Lin Y et al.(2017)Enhanced Therapeutic Efficacyand Memory of Tumor-Specific CD8 T Cells by Ex Vivo PI3K-deltaInhibition.Cancer Res.77:4135-45.doi:10.1158 / 0008-5472.CAN-16-1925

[0253] 28.Perkins MR,Grande S,Hamel A et al.(2015)Manufacturing an EnhancedCAR T Cell Product By Inhibition of the PI3K / Akt Pathway During T CellExpansion Results in Improved In Vivo Efficacy of Anti-BCMA CAR TCells.Blood.126:1893.

[0254] 29.Perna F,Berman SH,Soni RK,Mansilla-Soto J,Eyquem J,Hamieh M,Hendrickson RC,Brennan CW,Sadelain M(2017)Integrating Proteomics andTranscriptomics for Systematic Combinatorial Chimeric Antigen ReceptorTherapy of AML.Cancer Cell.32:506-19 e5.doi:10.1016 / j.ccell.2017.09.004

[0255] 30.Mardiros A,Dos Santos C,McDonald T et al.(2013)T cells expressingCD123-specific chimeric antigen receptors exhibit specific cytolytic effectorfunctions and antitumor effects against human acute myeloidleukemia.Blood.122:3138-48.doi:10.1182 / blood-2012-12-474056

[0256] 31.Thistlethwaite FC,Gilham DE,Guest RD et al.(2017)The clinicalefficacy of first-generation carcinoembryonic antigen(CEACAM5)-specific CAR Tcells is limited by poor persistence and transient pre-conditioning-dependentrespiratory toxicity.Cancer Immunol Immunother.66:1425-36.doi:10.1007 / s00262-017-2034-7

[0257] 32. Barber A, Sentman CL. NKG2D receptor regulates human effector T-cellcytokine production. Blood(2011)117:6571–81.doi:10.1182 / blood-2011-01-329417 Sequence Listing <110> Celiad AG <120> Agents that reduce the killing of immune cells expressing NKG2D-based receptors <130> P49682WO <140> PCT / EP2018 / 083661 <141> 2018-12-05 <150> EP17205560.0 <151> 2017-12-05 <160> 3 <170> BiSSAP 1.3.6 <210> 1 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Cyclophilin Primer 1 <400> 1 gacggcgagc ccttgg 16 <210> 2 <211> twenty three <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Cyclophilin Primer 2 <400> 2 gcacgaaaat tttctgctgt ctt 23 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Cyclophilin Probe <400> 3 tctcctttga gctgtttgca gacaaggt 28

Claims

1. A method for reducing and / or preventing autokilling during the manufacture of immune cells expressing a chimeric NKG2D receptor, comprising functionally inhibiting NKG2D signaling during the manufacture of said cells; wherein said functional inhibition of NKG2D signaling is achieved by one or more of the following: - Temporarily inhibiting one or more NKG2D ligands MICA and / or MICB of said immune cells using shRNA or antibodies against one or more NKG2D ligands; - transient inhibition of the chimeric NKG2D receptor; - Transient inhibition of downstream signaling of the chimeric NKG2D receptor by transiently inhibiting PI3K signaling using the PI3K inhibitor LY294002.

2. The method of claim 1, wherein the immune cells are cells for adoptive cell transfer.

3. An engineered immune cell comprising a nucleic acid molecule encoding a chimeric NKG2D receptor and one or more shRNAs targeting the chimeric NKG2D receptor and / or one or more NKG2D ligands; wherein the NKG2D ligand is selected from MICA and MICB.

4. An immune cell composition comprising a nucleic acid molecule encoding a chimeric NKG2D receptor, wherein the cell further comprises: - one or more shRNAs directed against a chimeric NKG2D receptor and / or one or more NKG2D ligands; wherein said NKG2D ligands are selected from MICA and MICB; And / or the composition further comprises -Inhibitors of downstream signaling of the NKG2D receptor. The composition according to claim 4 , wherein the inhibitor of downstream signaling of the NKG2D receptor is a PI3K inhibitor.

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