Immune cell modification for toxicity reduction and use thereof in adoptive cell therapy

Abstract

An immune cell population comprising modified immune cells having reduced inflammatory properties wherein such modified immune cells may have reduced yield of one or more inflammatory cytokines (e.g., interleukin 2) and / or express one or more inflammatory cytokines (e.g., interleukin 2). The present invention relates to one or more antagonists of interleukin-6 (e.g., interleukin-6). Also provided herein are methods of producing such immune cell populations including the modified immune cells and methods of using such immune cell populations in cell therapy (e.g., for the treatment of cancer, infectious diseases, or immune diseases).

Description

[0001] Related Applications

[0002] This application is a divisional application based on a patent application with an application date of March 13, 2019, a priority date of March 14, 2018, an application number of 201980025881.3, and an invention name of "Immune cell modification for reducing toxicity and its use in adoptive cell therapy". Background Art

[0003] Adoptive cell transfer therapy is a type of immunotherapy involving the ex vivo expansion of autologous or allogeneic immune cells and the subsequent infusion into the patient. Immune cells can be modified in vitro to specifically target malignant cells. The prospects of adoptive cell transfer therapy are usually limited by toxicity (e.g., cytokine-related toxicity). For example, adoptive cell transfer immunotherapy may trigger non-physiological increases in cytokine levels (cytokine release syndrome), which may lead to recipient death (see, e.g., Morgan et al., Molecular Therapy 18 (4): 843-851, 2010).

[0004] Therefore, the development of methods to reduce the toxicity associated with adoptive cell transfer immunotherapy while maintaining efficacy is of great interest. Summary of the invention

[0005] The present disclosure is based at least in part on methods for developing inflammatory properties of immune cells used in adoptive immune cell therapy and toxicity associated with adoptive immune cell therapy. Such methods may include knocking out endogenous genes encoding inflammatory proteins, knocking in antagonists and immunosuppressive cytokines of such inflammatory proteins, or combinations thereof. Therefore, one aspect of the present disclosure is characterized in that an immune cell population includes: (i) a first plurality of modified immune cells, wherein the first plurality of modified immune cells produce one or more inflammatory proteins at reduced levels compared to wild-type immune cells of the same type under the same conditions; and (ii) a second plurality of modified immune cells, wherein the second plurality of modified immune cells express one or more antagonists of the one or more inflammatory proteins and / or express one or more immunosuppressive cytokines. In some embodiments, in each of the first plurality of modified immune cells, at least one endogenous allele of the one or more inflammatory proteins is knocked out. In some embodiments, the first plurality of modified immune cells, the second plurality of modified immune cells, or both are T cells or natural killer cells.

[0006] In certain embodiments, the inflammatory protein includes one or more inflammatory cytokines or their soluble receptors, one or more inflammatory growth factors, one or more cytotoxic molecules or combinations thereof. Exemplary inflammatory cytokines or their soluble receptors include, but are not limited to, IL2, IL1α, IL1β, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, sIL-1RI, sIL-2Rα, sIL6R, IFNα, IFNβ, IFNγ, MIPα, MIPβ, CSF1, LIF, G-CSF, GM-CSF, CXCL10, CCL5, eotaxin, TNF, MCP1, MIG, RAGE, CRP, angiopoietin-2, and VWF. Exemplary inflammatory growth factors include, but are not limited to, TGFα, VEGF, EGF, HGF, and FGF. Exemplary cytotoxic molecules include, but are not limited to, perforin, granzymes, and ferritin.

[0007] In some embodiments, the second plurality of modified immune cells comprises one or more exogenous nucleic acids encoding the one or more antagonists of one or more inflammatory proteins and / or one or more immunosuppressive cytokines. At least one of the exogenous nucleic acids can be incorporated into the genome of the first plurality of modified immune cells.

[0008] In some embodiments, the one or more antagonists of the one or more inflammatory proteins introduced into any modified immune cell of the second plurality of modified immune cells comprise a soluble receptor for the inflammatory protein and / or an antibody specific for the inflammatory protein.

[0009] In other embodiments, an immunosuppressive cytokine is introduced into the second plurality of modified immune cells. Exemplary immunosuppressive cytokines include, but are not limited to, TGFβ, IL-4, IL-10, IL-13, IL-33, IL-35, and IL-37.

[0010] In some embodiments, the first plurality of modified immune cells and the second plurality of immune cells of any of the immune cell populations described herein may be of the same type. Alternatively or additionally, the first plurality of modified immune cells overlaps with the second plurality of immune cells.

[0011] On the other hand, the present disclosure provides a kind of immune cell group, it includes a first plurality of modified immune cells, and the first plurality of modified immune cells produce less interleukin 2 (IL-2) relative to the wild-type immune cells of the same type under the same conditions.For example, in each cell of the first plurality of modified immune cells, at least one endogenous IL-2 allele is knocked out.In some cases, the first plurality of modified immune cells, when activated, produce less IL-2 relative to the wild-type immune cells activated under the same conditions.

[0012] In some cases, the total IL-2 production of the immune cell population is about 30-95% less than that of the wild-type counterpart under the same conditions. For example, the total IL-2 production of the immune cell population is about 50% of the total IL-2 production of the wild-type counterpart.

[0013] In some embodiments, the immune cell population described above may further include a second plurality of modified immune cells, wherein the second plurality of modified immune cells produce reduced levels of one or more inflammatory proteins as described herein when compared to wild-type immune cells of the same type. For example, the total level of the one or more inflammatory proteins produced by the immune cell population is at least 10% lower than the total level of the wild-type counterpart under the same conditions. In some examples, in each cell of the second plurality of modified immune cells, at least one endogenous allele of the one or more inflammatory proteins is knocked out.

[0014] In some embodiments, the first plurality of modified immune cells and the second plurality of modified immune cells are of the same type. In other embodiments, the first plurality of modified immune cells and the second plurality of modified immune cells overlap.

[0015] Alternatively or in addition, the immune cell population described above may further include a third plurality of modified immune cells, wherein the third plurality of modified immune cells express one or more exogenous antagonists of the one or more inflammatory proteins or one or more immunosuppressive cytokines as described herein. In some specific instances, the one or more inflammatory proteins include IL6, and the antagonist of IL6 is tocilizumab or sirukumab (sirukumab) or an antigen-binding fragment thereof (e.g., a single-chain antibody fragment (scFv) derived from tocilizumab or sirukumab).

[0016] The third plurality of modified immune cells may include one or more exogenous nucleic acids encoding the one or more antagonists of the one or more cytokines. At least one of the exogenous nucleic acids may be incorporated into the genome of the third plurality of modified immune cells. In some examples, the third plurality of modified immune cells and the first plurality of modified immune cells, the second plurality of modified immune cells, or both are of the same type. In some examples, the third plurality of modified immune cells overlap with the first plurality of modified immune cells, the second plurality of modified immune cells, or both.

[0017] In any or immune cell population described herein, the immune cell may be a T cell, a NK cell, a dendritic cell, a macrophage, a B cell, a neutrophil, an eosinophil, a basophil, a mast cell, a myeloid-derived suppressor cell, a mesenchymal stem cell, a precursor thereof, or a combination thereof. In some embodiments, the immune cell may be further modified to express a chimeric antigen receptor (CAR) and / or an exogenous T cell receptor. CAR may include an extracellular ligand binding domain, a transmembrane domain, and one or more intracellular signaling domains. In some examples, the extracellular ligand binding domain may include a single-chain antibody fragment specific for a cell surface protein, an extracellular domain of a cytokine receptor, or an extracellular domain of a co-stimulatory receptor. Alternatively or in addition, the one or more intracellular signaling domains may include (i) a signaling domain of CD3ζ and (ii) one or more signaling domains from one or more co-stimulatory proteins or cytokine receptors. In other examples, the CAR can include one or more stimulatory domains (e.g., co-stimulatory domains) from CD28, 4-1BB, 2B4, KIR, CD27, OX40, ICOS, MYD88, IL2 receptor and / or SynNotch.

[0018] In yet another embodiment, the present disclosure provides a method of producing a modified immune cell population with reduced inflammatory properties, the method comprising: (i) providing an immune cell population; and (ii) modifying the immune cells to thereby reduce IL-2 production. This method may further include (iii) modifying the immune cells to reduce the production of one or more inflammatory proteins as described herein, and optionally (iv) introducing one or more nucleic acids encoding one or more antagonists of one or more inflammatory proteins as described herein and / or one or more immunosuppressive cytokines into the immune cells, wherein the one or more nucleic acids are operably linked to one or more promoters for expressing the one or more antagonists in the immune cells.

[0019] Alternatively, provided herein is a method for producing a modified immune cell population with reduced inflammatory properties, the method comprising: (i) providing an immune cell population; (ii) modifying the immune cells to reduce the production of one or more inflammatory proteins as described herein; and (iii) introducing one or more nucleic acids encoding one or more antagonists of one or more inflammatory proteins as described herein and / or one or more immunosuppressive cytokines into the immune cells, wherein the one or more nucleic acids are operably linked to one or more promoters for expressing the one or more antagonists in the immune cells.

[0020] In any of the methods described herein, the modification step includes gene editing at an endogenous allele of a target protein (e.g., IL-2 or an inflammatory protein) of the immune cell. The gene editing may include clustered regularly spaced short palindromic repeats (CRISPR), transcription activator-like effector nucleases (TALENs) or zinc finger nucleases (ZFNs), endonucleases, large-range nucleases, mega-TALS, or a combination thereof. In some cases, IL-2 production can be reduced by about 30-95% (e.g., about 50%) by gene.

[0021] In any method described herein, the immune cell can be a T cell, a NK cell, a dendritic cell, a macrophage, a B cell, a neutrophil, an eosinophil, a basophil, a mast cell, a myeloid-derived suppressor cell, a precursor thereof, or a combination thereof. Any immune cell can further express a chimeric antigen receptor (CAR) and / or an exogenous T cell receptor also described herein.

[0022] Any immune cell population produced by the methods described herein is also within the scope of the present disclosure.

[0023] Further, the present invention provides a method of cell therapy, comprising administering any immune cell population described herein to a subject in need thereof. The subject may be a human patient suffering from a target disease described herein (e.g., cancer, infectious disease, or immune disorder).

[0024] Also within the scope of the present disclosure are immune cell populations as described herein for use in treating a target disease also as described herein, and the use of such immune cell populations in the preparation of a medicament for treating a target disease.

[0025] The details of one or more embodiments of the invention are set forth in the following description. Other features and advantages of the invention will become apparent from the following drawings and detailed description of several embodiments, and also from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1Schematic diagram of an exemplary scheme for using gene editing to neutralize inflammatory cytokine pathways. The left figure shows a combination of knock-in gene cassettes and knock-out gene editing (Scheme I). The right figure depicts a one-step strategy equivalent to Scheme I by simultaneously integrating transgenic knock-in and gene disruption (Scheme II).

[0027] Figure 2 Contains a graph showing the efficiency of anti-IL6R scFv (derived from tocilizumab) expressed from Jurkat E6-1 cells in blocking IL6-IL6R signaling. The anti-IL6R scFv cassette was knocked in. The negative control for comparison was IL6 reporter cells treated with culture medium. Anti-IL6 antibody was used as a positive control.

[0028] Figure 3 is a graph showing the gene editing efficiency of various sgRNA candidates targeting the human GM-CSF gene in Jurkat cells using the CRISPR / CAS9 system compared to control cells (no modification).

[0029] Figure 4 is a graph showing the gene editing efficiency of various sgRNA candidates targeting human IL-2 in Jurkat cells using the CRISPR / CAS9 system compared to control cells (no modification).

[0030] Figure 5 is a graph showing the gene editing efficiency of various sgRNA candidates targeting the human TNF gene in Jurkat cells using the CRISPR / CAS9 system compared to control cells (no modification).

[0031] Figures 6A-6D is a graph showing changes in body weight, survival rate, the presence of blood Nalm6 leukemia cells and human T cells in NSG mice injected with Nalm6 leukemia cells alone (CTRL, n=4) or subsequently treated with CD19 / IL6 / IL1RA TCR-CART cells (1 in the figure, n=5) or anti-CD19 / IL6 / IL1RA TCR / GM-CSF-CART cells (2 in the figure, n=6).

[0032] Figure 7 is a graph showing the cell expansion rate of T cells with reduced IL-2 production after gene editing with sgRNAs 1, 3, and 5 targeting IL-2 in response to anti-CD3 / CD28 stimulation. T cells modified with Cas9 alone were used as a control. DETAILED DESCRIPTION

[0033] Adoptive cell transfer immunotherapy relies on immune cell activation and cytokine secretion to eliminate disease cells. However, the systemic overproduction of cytokines causes safety issues and may be fatal to recipients sometimes. Morgan et al., "Molecular Therapy" 18 (4): 843-851, 2010. The present disclosure is intended to partially overcome this limitation by developing immune cells with reduced inflammatory properties. As described herein, this can be achieved by knocking out one or more cytokines or their receptors in immune cells to reduce the undesirable cytokines (e.g., inflammatory cytokines) produced by the immune cells to be transplanted to the recipient after activation and / or by knocking out one or more cytokine antagonists of immune cells, which can be applied to the immune cells of the recipient and neutralize the inflammatory signaling mediated by the host immune cells of the recipient after activation. The combination of knock-in and knock-out design will reduce the output and / or signaling of undesirable cytokines (e.g., inflammatory cytokines) of both the immune cells and host immune cells used for adoptive therapy, thereby significantly reducing the toxicity caused by the adoptive therapy of immune cells due to cytokine crisis.

[0034] In some instances, interleukin 2 (IL-2) can be knocked out in immune cells for adoptive immune cell therapy. IL-2 has been characterized as a T cell mitogen that is essential for T cell proliferation / activation. (See, e.g., Morgan et al., Science, 193(4257): 1007-8, 1976; Smith, Science, 240(4856): 1169-76, 1988). Surprisingly, studies described herein show that a reduction in IL2 production in immune cells (e.g., T cells) inhibits cell hyperproliferation while maintaining the healthy propagation rate of these immune cells in vitro. These results indicate that IL-2 may be a target cytokine for knocking out to reduce toxicity associated with adoptive cell therapy.

[0035] Modified immune cells as described herein, including knockout of one or more inflammatory proteins (e.g., inflammatory cytokines or soluble receptors thereof, inflammatory growth factors, or cytotoxic molecules), knock-in of one or more antagonists of inflammatory proteins or immunosuppressive cytokines, or a combination thereof, will be beneficial in limiting cytokine-associated toxicity in adoptive cell transfer immunotherapy and will be useful for effectively treating diseases including cancer, infectious diseases, and immune disorders with fewer side effects.

[0036] Thus, provided herein are immune cell populations, methods of producing such cell populations, and their use for reducing inflammation associated with adoptive immune cell therapy, the immune cell populations comprising modified immune cells having reduced inflammatory properties as described herein.

[0037] I. Modified immune cells

[0038] One aspect of the present disclosure provides a modified immune cell with reduced inflammatory properties compared to wild-type immune cells of the same type. Such modified immune cells may include knocking out one or more inflammatory proteins (e.g., inflammatory cytokines or their soluble receptors, inflammatory growth factors or cytotoxic molecules), knocking in an antagonist or a combination thereof of one or more inflammatory proteins or immunosuppressive cytokines. Wild-type cells refer to cells that do not have such knock-in and knock-out modifications.

[0039] (i) Inflammatory proteins and immunosuppressive cytokines

[0040] As used herein, an inflammatory protein refers to a protein that can directly or indirectly promote inflammation. The inflammatory protein can be an inflammatory cytokine or a soluble receptor of an inflammatory cytokine, an inflammatory growth factor, or a cytotoxic molecule.

[0041] In some embodiments, the inflammatory protein is an inflammatory cytokine or a soluble receptor for an inflammatory cytokine that promotes inflammation. In some cases, such inflammatory cytokines are produced by and participate in the upregulation of inflammatory responses. In nature, inflammatory cytokines are typically secreted by immune cells, such as helper T cells, macrophages, or some other types of immune cells.

[0042] Exemplary inflammatory cytokines or their soluble receptors include interleukin 1α (IL1α), interleukin 1β (IL1β), interleukin 2 (IL-2), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9 (IL-9), interleukin (IL-12), interleukin 15 (IL-15), interleukin 17 (IL-17), interleukin 18 (IL-18), interleukin 21 (IL-21), interleukin 23 (IL-23), sIL-1RI, sIL-2Rα, soluble IL-6 receptor (sIL6R), interferon α (IFNα), interferon β (IFNβ), IFNβ), interferon gamma (IFNγ), macrophage inflammatory proteins (e.g., MIPα and MIPβ), macrophage colony stimulating factor 1 (CSF1), leukemia inhibitory factor (LIF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), CXC motif chemokine ligand 10 (CXCL10), chemokine (CC motif) ligand 5 (CCL5), eotaxin, tumor necrosis factor (TNF), monocyte chemoattractant protein 1 (MCP1), monokine induced by gamma interferon (MIG), receptor for advanced glycation end products (RAGE), C-reactive protein (CRP), angiopoietin-2, and von Willebrand factor (VWF).

[0043] IL1α is also known as erythropoietin 1 and is a member of the interleukin 1 family of cytokines. This cytokine is produced by activated macrophages, epithelial cells, endothelial cells, and macrophages and signals through the interleukin-1 receptor. Human IL1α (e.g., GenBank accession number NP_000566.3) is encoded by the IL1A gene.

[0044] IL1β is a member of the interleukin 1 family of cytokines and carries out signal transduction through the IL1B receptor. IL1β has been involved in promoting inflammation in various diseases including type 1 diabetes and rheumatoid arthritis. For example, IL1β signal transduction can promote inflammatory pain hypersensitivity by inducing cyclooxygenase-2 (PTGS2 / COX2) expression. Human IL1β (e.g., Gene Bank Accession No. NP_000567.1) is encoded by the IL1B gene.

[0045] The interleukin 1 type I receptor (also known as IL-1RI) is encoded by the human IL1R1 gene. sIL-1RI refers to a soluble form of the IL-1RI receptor. Exemplary human IL-1RI sequences are provided under GenBank Accession Nos. NP_001307910.1, NP_001275635.1, NP_001307912.1, NP_001307913.1, NP_001307914.1, and NP_001307915.1.

[0046] IL-2 is a member of the IL-2 family of cytokines and plays a key role in stimulating lymphocyte proliferation (e.g., T cells and B cells) (see, e.g., Morgan et al., Science, 193(4257):1007-8, 1976; Smith, Science, 240(4856):1169-76, 1988; and Mingari et al., Nature, 312(5995):641-3, 1984). IL-2 is mainly produced by T cells (e.g., CD4+ and CD8+ T cells) after activation of T cells by antigen, and has also been reported to be expressed by dendritic cells and mast cells (see, e.g., Granucci et al., Nature Immunol., 2(9):882-8, 2001; and Hershko et al., Immunity, 35(4):562-71, 2011).

[0047] IL-2 receptor (IL-2R) complex, IL-2 can act on a variety of cell types to trigger immune responses. Taniguchi et al., Cell, 73(1):5-8, 1993. CD122 and CD132 form a dimeric IL-2 receptor (IL-2R), while the trimeric IL-2R also contains CD25. Human IL-2 (e.g., Gene Bank Accession No.: NP_000577.2) is encoded by the IL-2 gene.

[0048] Soluble IL2 receptor alpha subunit (sIL2Ra) is a soluble form of CD25. sIL2Ra is normally secreted by activated T cells and B cells (see, e.g., Rubin et al., J Immunol., 135(5):3172-7, 1985). Elevated sIL2Ra levels have been associated with hematological malignancies and autoimmune disorders (see, e.g., Rubin et al., Ann Intern Med., 113(8):619-27, 1990). In one example, sIL2Ra is human sIL2Ra.

[0049] IL-5 is a member of a discrete cytokine family that also includes granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3. IL-5 signals through the interleukin-5 receptor and can stimulate B cell growth. An exemplary human IL-5 sequence is provided under GenBank Accession No. NP_000870.1.

[0050] IL-6 is a pleiotropic cytokine that is usually produced by antigen presenting cells (including dendritic cells), macrophages and B cells in response to tissue damage and infection and has additional effects in regulating cell growth and differentiation (see, for example, Kamimura et al., Rev Physiol Biochem Pharmacol. 149: 1-38, 2003). IL6 is involved in inducing B cell differentiation, promoting acute phase protein synthesis in hepatocytes, enhancing the growth of plasma cells and myeloma fusion cells, and having interferon antiviral activity. In view of the various functions of IL6, IL6 is also referred to as B cell stimulating factor 2 (BSF2), hepatocyte-stimulating factor (HSF), hybridoma growth factor (HGF) and IFN-β2 (Kishimoto, Blood 74 (1): 1-10, 1989). As an example, the amino acid sequence of human IL6 is provided under Gene Bank Accession No. NP_000591.1.

[0051] IL-6 is signaled by a complex including the membrane glycoprotein gp130 and the IL-6 receptor (IL6R) (see, e.g., Hibi et al., Cell, 63(6): 1149-57, 1990). IL-6 binds to IL6R on target cells to promote gp130 homodimerization and subsequent signal transduction. As used herein, IL6R includes both membrane-bound and soluble forms of IL6R (sIL6R). When bound to IL-6, soluble IL6R (sIL6R) acts as an agonist and can also promote gp130 dimerization and signal transduction. Transsignaling may occur, whereby sIL-6R secretion by a specific cell type induces only cells expressing gp130 to respond to IL-6 (see, e.g., Taga et al., Annu Rev Immunol., 15:797-819, 1997; and Rose-John et al., Biochem J., 300(Pt2):281-90, 1994). In one example, sIL6R includes the extracellular domain of human IL6R (see, e.g., Peters et al., J Exp Med., 183(4):1399-406, 1996).

[0052] IL-7 is a cytokine involved in the development of B cells and T cells. IL7 can be used as a pre-pro-B cell growth stimulating factor by forming a heterodimer with hepatocyte growth factor. Exemplary human IL-7 sequences are provided under Gene Bank Accession Nos. NP_000871.1, NP_001186815.1, NP_001186816.1, and NP_001186817.1.

[0053] IL-8 is a chemoattractant cytokine and is secreted by a variety of cell types including leukocytes, monocytes and endothelial cells (see, e.g., Baggiolini et al., J. Clin. Invest. 84: 1045-1049, 1989). Although the primary target cell of IL-8 is neutrophils, it has been reported that IL-8 can also act as a chemokine for other cell types including basophils and T cells. IL-8 can signal through two G protein-coupled receptors, human CXCR1 and CXCR2 (see, e.g., Wu et al., Science 261(5117): 101-3, 1993; and Damaj et al., Journal of Biological Chemistry, 271(22): 12783-9, 1996). As an example, the human CXCL8 gene encodes at least two IL-8 isoforms. Human IL-8 isoform 1 is longer in length than isoform 2 and is typically expressed in non-immune cells, whereas isoform 2 is primarily expressed in monocytes and macrophages. An exemplary human IL-8 isoform 2 is provided under GenBank Accession No. NP_001341769.1.

[0054] IL-9 is a cytokine that is considered a regulator of hematopoietic cells and can stimulate cell proliferation. IL-9 signals through the interleukin 9 receptor and can activate signal transduction and activation (STAT) proteins. An exemplary human IL-9 sequence is provided under the gene bank accession number NP_000581.1.

[0055] IL-12 is a member of the interleukin 12 family of cytokines, which includes IL-12, IL-23, IL-27 and IL-35. In response to antigens, IL-12 can be produced by neutrophils, dendritic cells and macrophages. IL12 mainly acts on naive CD4+T cells. This cytokine consists of two subunits. As a heterodimer, IL-12 is composed of IL-12 subunit α (IL-12p35) and IL-12 subunit β (IL-12p40). In humans, each subunit is encoded by a different gene. For example, human IL-12 subunit α (e.g., GenBank Accession Nos. NP_000873.2, NP_001341511.1, and NP_001341512.1) is encoded by the IL12A gene, and human IL-12 subunit β (e.g., GenBank Accession No. NP_002178.2) is encoded by the IL12B gene.

[0056] IL-15 is a member of the 4-α helical bundle family of cytokines, and has been implicated in promoting differentiation and proliferation of B cells, T cells and natural killer cells (see, e.g., Mishra et al., Clinical Cancer Research (Clin Cancer Res.), 20 (8): 2044-50, 2014). IL-15 can act on antigen presenting dendritic cells, macrophages and monocytes by binding to heterotrimeric IL-15 receptor complexes. The IL-15 receptor complex has three subunits, which include IL15Rα, IL-2Rβ and IL-2Rγ (see, e.g., Mishra et al., Clinical Cancer Research, 20 (8): 2044-50, 2014). The IL15Rα subunit has a high affinity for IL-15 and exists in soluble and membrane forms. As an example, the amino acid sequence of human IL-15 is provided in Gene Bank Accession No. NP_751915.1.

[0057] Interleukin 17 (also known as IL17A, CTLA8 or IL-17) is a member of the IL17 family of cytokines, Gu et al., Cytokine, 64 (2): 477-85, 2013. IL-17 is mainly produced by helper T cells 17 (Th17 cells). Signaling is carried out through the heterodimeric receptor complex of IL17RA / IL17RC, and IL-17 can induce stromal cells to express inflammatory cytokines and chemokines (see, for example, Toy et al., Journal of Immunology, 177 (1): 36-9, 2006). As an example, the human IL17A gene encodes IL-17 (e.g., Gene Bank Accession No. NP_002181.1).

[0058] IL-18 is a member of the interleukin 1 superfamily of cytokines. IL-18 is produced by various cells including macrophages and signals through the interleukin-18 receptor. Exemplary human IL-18 sequences are provided under GenBank Accession Nos. NP_001553.1 and NP_001230140.1.

[0059] IL-21 is a member of the common γ chain family of cytokines, which includes IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. IL-21 is produced by activated CD4+T cells, and can act on T cells, B cells and natural killer cells by interacting with IL-21 receptors. Exemplary human IL-21 sequences are provided under gene bank accession numbers NP_068575.1 and NP_001193935.1.

[0060] IL-23 is a heterodimeric cytokine composed of IL12β subunit (IL-12p40) subunit and IL23A (IL-23p19) subunit. IL-23 is signaled by the IL23 receptor composed of IL12β1 subunit (IL12RB1) and IL23 specific subunit (IL23R). Similar to IL-12, IL-23 can participate in STAT4 signaling, but IL23 mainly acts on memory CD4+T cells. An exemplary human IL-23A sequence is provided under Gene Bank Accession No. NP_057668.1. Interferon (IFN) is a family of cytokines related to the innate immunity of host cells and resistance to viral infection. There are at least two types of interferons (see, for example, Le Page et al., "Review of Immunogenetics (Rev.Immunogenet.)", 2 (3): 374-86, 2000). Type I interferons (including IFNα and IFNβ) are usually produced by cells infected with viruses. In contrast, type II interferons (e.g., IFNγ) are typically produced by macrophages, T cells, and natural killer cells (NK). For example, the human genes IFNA1, IFNB1, and IFNG encode IFNα (e.g., GenBank Accession No. NP_076918.1), IFNβ (e.g., GenBank Accession No. NP_002167.1), and IFNγ (e.g., GenBank Accession No. NP_000610.2), respectively.

[0061] Macrophage inflammatory protein (MIP or also known as MIP-1CC protein) is a chemotactic cytokine, and there are at least four members of this subfamily. Members of the MIP family include CCL3 (MIP-1α), CCL4 (MIP-1β), CCL9 (MIP-1δ or previously also known as CCL10) and CCL15 (MIP-1γ) (see, for example, Maurer et al., Int J Biochem Cell Biol., 36(10):1882-6, 2004). These proteins can be produced by different cell types including macrophages, dendritic cells and lymphocytes (Driscoll, Exp Lung Res., 20(6):473-90, 1994). MIPs signal through G protein-coupled cell surface receptors including CCR1, CCR3 and CCR5. These receptors are usually expressed by monocytes / macrophages and lymphocytes. Exemplary MIPs include, but are not limited to, human CCL3 (MIP-1α) (eg, GenBank Accession No. NP_002974.1), human CCL4 (MIP-1β) (eg, GenBank Accession No. NP_002975.1), and human CCL15 (MIP-1γ) (eg, GenBank Accession No. NP_116741.2).

[0062] CSF1 (also known as macrophage colony stimulating factor or M-CSF) is a cytokine involved in the differentiation of hematopoietic stem cells into macrophages. CSF1 binds to the colony stimulating factor 1 receptor. Exemplary human CSF1 sequences are provided under GenBank Accession Nos. AAA52117.1, NP_000748.3, NP_757349.1, and NP_757350.1.

[0063] LIF is a pleiotropic cytokine involved in hematopoietic differentiation in bone marrow and normal leukemic cells, neuronal cell differentiation, and kidney development. LIF acts on the LIF receptor (LIFR-α). Exemplary LIF sequences are provided under GenBank Accession Nos. NP_002300.1 and NP_001244064.1.

[0064] G-CSF is a cytokine involved in the production, function and differentiation of granulocytes. G-CSF is produced by a variety of cells including endothelial cells and macrophages. G-CSF binds to the G-CSF receptor. Exemplary G-CSF sequences are provided under GenBank Accession Nos. NP_000750.1, NP_757373.1, NP_757374.2 and NP_001171618.1.

[0065] Granulocyte-macrophage colony stimulating factor (GM-CSF) is a member of the colony stimulating factor superfamily and is expressed by many different immune cells including macrophages, mast cells, natural killer cells and T cells. As a cytokine, GM-CSF can act as a hematopoietic growth factor (Metcalf, "Nature", 339 (6219): 27-30, 1989). GM-CSF can promote the growth and differentiation of various hematopoietic precursor cells including granulocytes, eosinophils, erythrocytes and macrophages by activating the GM-CSF receptor (GM-CSFR) (see, for example, Martinez-Moczygemba et al., "Journal of Allergy and Clinical Immunology (J Allergy Clin Immunol.)" 112 (4): 653-65, 2003). As an example, human GM-CSF (e.g., Gene Bank Accession No.: NP_000749.2) is encoded by the CSF2 gene.

[0066] CXCL10 (also known as interferon gamma-induced protein 10 or IP-10) is a cytokine secreted by various cell types (such as fibroblasts, endothelial cells and monocytes) in response to interferon gamma. CXCL10 has been implicated in chemotaxis and T cells that adhere to endothelial cells and acts on the chemokine receptor CXCR3. An exemplary human CXCL10 sequence is provided under GenBank Accession No. NP_001556.2.

[0067] CCL5 (also known as D17S136E, RANTES, SCYA5, SIS-δ, SISd, TCP228, eoCP or CC motif chemokine ligand 5) is a chemoattractant for blood monocytes, memory T helper cells and eosinophils. CCL5 can bind to the chemokine receptor CCR5. Exemplary human CCL5 sequences are provided under GenBank Accession Nos. NP_002976.2 and NP_001265665.1.

[0068] Eotaxin forms the CC chemokine subfamily of eotaxin. The members of this family include CCL11 (eotaxin-1), CCL24 (eotaxin-2) and CCL26 (eotaxin-3). Exemplary human eotaxin is provided under gene bank accession numbers CCL11 (NP_002977.1), (NP_002977.1), CCL24 (NP_002982.2) and CCL26 (NP_006063.1).

[0069] Tumor necrosis factor (TNF, such as TNFα) is a member of the tumor necrosis factor superfamily of cytokines. TNF is mainly secreted by macrophages and activated T cells, and can signal through two different receptors (TNFR1 and TNFR2) to regulate various cell functions, including apoptosis, cell survival and differentiation (Liu, "Cell Research (Cell Res.)", 15 (1): 24-7, 2005). As an example, human TNFα (e.g., Gene Bank Accession No.: NP_000585.2) is encoded by the TNF gene.

[0070] Monocyte chemoattractant protein 1 (MCP1 or CCL2) is a member of the chemokine family of cytokines. MCP1 can serve as a chemoattractant, and after infection or injury, signal transduction is performed by G protein-coupled receptor CCR2 to recruit dendritic cells, memory T cells and monocytes (see, e.g., Deshmane et al., "Interferon Cytokine Research Journal (JInterferon Cytokine Res.)", 29 (6): 313-26, 2009). In an example, MCP1 is human MCP1 (e.g., Gene Bank Accession No. NP_002973.1).

[0071] MIG (also known as CXCL9, CMK, Humig, MIG, SCYB9, crg-10 or CXC motif chemokine ligand 9) is a member of the CXC chemokine family. MIG is a chemoattractant for T cells, and its expression is upregulated by IFNγ. An exemplary human MIG sequence is provided under GenBank Accession No. NP_002407.1.

[0072] Receptor for advanced glycation end products (RAGE or AGER) belongs to the immunoglobulin superfamily of cell surface receptors. RAGE can bind to a variety of ligands comprising advanced glycation end products and is related to innate immunity. Exemplary human RAGE proteins are provided under Gene Bank Accession Nos. NP_001127.1, NP_001193858.1, NP_001193861.1, NP_001193863.1, NP_001193865.1, NP_001193869.1, NP_751947.1 and NP_001193883.1.

[0073] C-reactive protein (CRP) is a member of the pentraxin family, which also includes serum amyloid P component (SAP). The circulating level of acute phase protein CRP usually rises in response to tissue damage, infection and inflammation. There are multiple CRP ligands, but CRP has the highest affinity for ligands containing phosphorylcholine, which are usually found on the surface of necrotic cells and apoptotic cells. CRP combined with lysophospholipids on the surface of damaged and apoptotic cells recruits C1q and subsequently activates the complement system (see, for example, Thompson et al., Structure February 15; 7 (2): 169-77, 1999; Pepys et al., Journal of Clinical Research 111 (12): 1805-1812, 2003). As an example, the amino acid sequence of human CRP can be found under Gene Bank Accession No. NP_000558.2.

[0074] Angiopoietin-2 (Ang2) is a member of the angiopoietin family, which includes angiopoietin-1, angiopoietin-2 and angiopoietin-4. Ang2 has been implicated in the regulation of endothelial cell junctions, vascular destabilization and increased vascular permeability (see, e.g., Zheng et al., Genes Dev., 28(14): 1592-603, 2014; and Ziegler et al., Journal of Clinical Research pii: 66549, 2013). As a Tie2 receptor ligand, Ang2 can also regulate vascular remodeling by regulating Tie2 signaling (see, e.g., Thurston et al., Cold Spring Harb Perspect Med., 2(9): a006550, 2012). As an example, the amino acid sequence of human Ang2 can be found under Gene Bank Accession No. NP_001138.1.

[0075] Von Willebrand factor (VMF) is a glycoprotein involved in hemostasis regulation. This protein is usually present in plasma, subendothelial connective tissue and platelet alpha particles. In order to mediate hemostasis, VWF can promote platelet adhesion to subendothelial connective tissue, and can also bind factor VIII, which is a coagulation factor (blood clotting factor) (Sadler, "Annu Rev Biochem. " 67:395-424,1998). VWF deficiency can cause von Willebrand disease, which is an inherited bleeding disorder. As an example, the amino acid sequence of people VMF can be found under gene bank accession number NP_000543.2.

[0076] In some embodiments, the inflammatory protein is an inflammatory growth factor. As used herein, an inflammatory growth factor is a growth factor that plays some role (directly or indirectly) in the inflammatory signaling pathway. In some cases, the inflammatory growth factor can interact with one or more cytokines to promote inflammation. Exemplary inflammatory growth factors include transforming growth factor alpha (TGFα), vascular endothelial factor (VEGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and fibroblast growth factor (FGF).

[0077] TGF alpha is a growth factor involved in cell proliferation, cell development and differentiation. It can carry out signal transduction through the epidermal growth factor receptor (EGFR). Exemplary human TGF alpha sequences are provided under GenBank Accession Nos. NP_003227.1, NP_001093161.1, NP_001295087.1 and NP_001295088.1.

[0078] VEGF (also known as vascular permeability factor or VPF) is a family of proteins involved in angiogenesis. In mammals, there are at least five VEGF family members, including VEGF-A, placental growth factor (PGF), VEGF-B, VEGF-C and VEGF-D. An exemplary human VEGF sequence is provided under GenBank Accession No. NP_001165097.1.

[0079] EGF is a growth factor that promotes cell growth, survival and differentiation. EGF is a ligand for the receptor EGFR. Exemplary human EGF sequences are provided under Gene Bank Accession Nos. NP_001954.2, NP_001171601.1, NP_001171602.1 and NP_001343950.1.

[0080] HGF is a growth factor involved in the regulation of cell movement, cell growth and morphogenesis. HGF binds to the c-MET receptor and is produced by mesenchymal cells. Exemplary human HGF sequences are provided under Gene Bank Accession Nos. NP_000592.3, NP_001010931.1, NP_001010932.1, NP_001010933.1 and NP_001010934.1.

[0081] FGF is a family of signal transduction molecules related to the various processes that comprise development. In humans, there are at least 22 members of the FGF family. For example, human FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, and FGF10 bind to FGF receptors. Exemplary human FGFs are provided under Gene Bank Accession Nos. NP_000791.1, NP_001997.5, and NP_005238.1.

[0082] In some embodiments, the inflammatory protein is a cytotoxic molecule. As used herein, a cytotoxic molecule is a molecule secreted by immune cells such as macrophages to kill disease cells. Exemplary cytotoxic molecules include perforin, granzyme, and ferritin.

[0083] Perforin is a cytolytic protein localized in the granules of cytotoxic T lymphocytes and natural killer cells. When secreting cytolytic granules, perforin binds to the membrane of the target cell and forms holes in the plasma membrane (see, e.g., Liu et al., Journal of Immunology 156(9):3292-300, 1996). In humans, perforin (e.g., GenBank Accession No. NP_001076585.1) is encoded by the PRF1 gene.

[0084] Granzymes are serine proteases secreted by cytotoxic T cells and natural killer cell granules, and are involved in inducing apoptosis in target cells (e.g., cells that have been infected with pathogens or have become malignant). In humans, there are at least five types of granzymes (see, e.g., Bots et al., J. Cell Sci., 119(Pt 24): 5011-4, 2006). Human genes GZMA, GZMB, GZMH, GZMK, and GZMM encode granzyme A (e.g., gene bank accession number NP_006135.1), granzyme B (e.g., gene bank accession number NP_004122.2), granzyme H (e.g., gene bank accession number NP_219491.1), granzyme K (e.g., gene bank accession number NP_002095.1), and granzyme M (e.g., gene bank accession number NP_005308.1), respectively.

[0085] Ferritin is a globular protein involved in iron storage. In animals, ferritin is a complex of ferritin light chain (FTL) and ferritin heavy chain (FTH) subunits. Although there are usually 24 subunits in a given ferritin complex, the ratio of FTL to FTH varies from cell type to cell type. FTH has been implicated in the regulation of hematopoiesis, hepatocyte apoptosis, cell differentiation and immune function (see, e.g., Recalcati et al., "Journal of Autoimmunity (J Autoimmun.)" 2008 30 (1-2): 84-9, 2008). An exemplary sequence of human FTH is provided in Gene Bank Accession No. NP_002023.2. An exemplary sequence of human FTL is provided in Gene Bank Accession No. NP_000137.2. In a variety of malignant tumors comprising cancer, the level of circulating ferritin is usually increased (see, e.g., Hazard et al., "Nature" 265 (5596): 755-6, 1977).

[0086] As used herein, immunosuppressive cytokines are anti-inflammatory cytokines. Anti-inflammatory cytokines include TGFβ, IL-4, IL-10, IL-13, IL-33, IL-35 and IL-37.

[0087] There are at least 30 members in the TGFβ family, including TGFβ1, TGFβ2 and TGFβ3. TGFβ has been involved in inhibiting macrophage and Th1 cell activity by inhibiting cytokine production. Exemplary TGFs include human TGFβ1 (e.g., Gene Bank Accession No. NP_000651.3), human TGFβ2 (e.g., Gene Bank Accession No. NP_001129071.1) and human TGFβ3 (e.g., Gene Bank Accession No. NP_003230.1).

[0088] IL-4 is a cytokine that has been implicated in the inhibition of tumor necrosis factor (TNF)-α and IL-1β production by lipopolysaccharide (LPS) activated human monocytes. Exemplary human IL4 sequences are provided under GenBank Accession Nos. NP_000580.1, NP_758858.1, and NP_001341919.1.

[0089] IL-10 is a pleiotropic cytokine involved in immunomodulation and inflammation. IL-10 is mainly produced by monocytes, but can also be produced by lymphocytes, and can regulate B cell survival, antibody production and proliferation. IL-10 can act on a receptor complex consisting of two IL-10 receptor 1 and two IL-10 receptor 2 proteins. An exemplary human IL-10 sequence is provided under Gene Bank Accession No. NP_000563.1.

[0090] IL-13 is an immunomodulatory cytokine that has been implicated in inhibiting the production of inflammatory cytokines in LPS-activated human monocytes. Exemplary human IL-13 sequences are provided under Gene Bank Accession Nos. NP_002179.2 and NP_001341920.1. IL-33 belongs to the interleukin 1 superfamily of cytokines, which also includes IL-1α, IL-1β, IL-1RA and IL-18. IL-33 binds to the IL1RL1 / ST2 receptor. IL-33 has previously been shown to have an anti-inflammatory effect in adipose tissue inflammation, and has been shown to have a protective effect in cardiovascular disease. Exemplary human IL-33 sequences are provided under GenBank Accession Nos. NP_001300974.1, NP_001186569.1, NP_001186570.1, NP_001300975.1, NP_001300977.1, and NP_001340731.1.

[0091] IL-35 is an IL-12 family cytokine. IL-35 is mainly produced by regulatory T cells and is involved in the suppression of the immune system. IL-35 is a heterodimeric protein consisting of IL-27β and IL12α subunits. Human IL-27β (e.g., gene bank accession number NP_005746.2) is encoded by the EBI3 gene. As mentioned above, human IL12α subunits are encoded by the IL12A gene.

[0092] IL-37 is a member of the IL-37 family of cytokines and is involved in the reduction of inflammation. IL-37 can inhibit the production of many pro-inflammatory cytokines, including IL-8, IL-6 and TNFα. Exemplary human IL-37 sequences are provided under GenBank Accession Nos. NP_055254.2, NP_775294.1, NP_775295.1, NP_775296.1 and NP_775297.1.

[0093] (ii) Knockout modification

[0094] In some embodiments, the modified immune cells described herein may include one or more "knockout" modifications targeting one or more endogenous inflammatory proteins as described herein. Knockout modification refers to any type of genetic modification to a host cell (e.g., an immune cell as described herein), compared to a wild-type host cell (i.e., a wild-type counterpart) of the same type lacking such genetic modification, the knockout modification results in a reduction in the yield of a target endogenous protein (e.g., a cytokine or other target protein as described herein). The yield level of the protein of interest of the cell group of interest can be compared to the yield level of the protein of interest of a control cell group (e.g., a modified immune cell group as described herein relative to a wild-type counterpart group) to determine the yield of the protein of interest as described herein, which is measured using the same number of cells under the same conditions (e.g., by using the same assay of the same experimental conditions). In some cases, when activated (e.g., with an antigen, a receptor agonist, or with cytokine stimulation) compared to a wild-type counterpart activated under the same conditions, the modified immune cell can therefore produce a lower level of target cytokines. Alternatively, the level of cytokine production can be measured without activating immune cells. In some cases, it may not be possible to detect the level of a specific cytokine produced by modified cells by conventional assays. Cytokine levels can be measured using conventional methods known in the art. For example, ELISA-type assays using cytokine-specific antibodies can be suitable for measuring cytokine levels.

[0095] Knockout modification can comprise gene editing of at least one endogenous allele of target inflammatory protein, including but not limited to insertion, deletion or replacement in the coding region of endogenous allele or non-coding regulatory region of endogenous allele, to disrupt the expression of target cytokine. As used herein, endogenous allele is a gene allele naturally found in a cell (i.e., natural to a cell).

[0096] Alternatively, the knockout modification can include the introduction of exogenous nucleic acids (e.g., antisense oligonucleotides, such as interfering RNA) that inhibit the expression of target inflammatory proteins as described herein. Exogenous nucleic acids refer to nucleic acids that are not produced by host cells before modification and are delivered to host cells by transfection methods (e.g., methods described herein).

[0097] Target inflammatory proteins subjected to knockout modification as described herein include, but are not limited to, inflammatory cytokines or soluble receptors thereof (e.g., IL2, IL1α, IL1β, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, sIL-1RI, sIL-2Rα, sIL6R, IFNα, IFNβ, IFNγ, MIPα, MIPβ, CSF1, LIF, G-CSF, GM-CSF, CXCL10, CCL5, eotaxin, TNF, MCP1, MIG, RAGE, CRP, angiopoietin-2, and VWF), inflammatory growth factors (e.g., TGFα, VEGF, EGF, HGF, and FGF), and cytotoxic molecules (e.g., perforin, granzymes, and ferritin).

[0098] One or more target cell factors / proteins in the target cell factor / protein as described above of the modified immune cell of knockout modification as described herein can be knocked out.In some cases, the endogenous gene encoded by the target cell factor / protein is subjected to gene editing in the coding region or non-coding regulatory region, so that the expression of the target cell factor / protein from the endogenous gene is destroyed, thereby reducing the output level of the modified immune cell to this target cell factor / protein.In other cases, the coding region of the target cell factor / protein can be used to have specific exogenous antisense nucleic acid (for example, the suitable region of the mRNA encoded by the target cell factor / protein) to modify immune cell, to reduce the expression of the target cell factor / protein.

[0099] The modified immune cells can contain one type of genetic modification as described herein or a combination of different genetic modifications (e.g., a combination of disruption of an endogenous gene for one target cytokine / protein and delivery of an exogenous antisense nucleic acid to downregulate expression of another target cytokine / protein).

[0100] (iii) Knock-in modification

[0101] In other embodiments, modified immune cells as described herein may include one or more "knock-in" modifications to express immunosuppressive cytokines or to express antagonists targeting one or more inflammatory proteins (target proteins), such as inflammatory proteins produced by the immune system of the recipient of adoptive immune cell therapy. Target proteins include inflammatory cytokines, their soluble receptors, inflammatory growth factors and cytotoxic molecules. Knock-in modifications may include delivering exogenous nucleic acids encoding immunosuppressive cytokines and / or cytokine antagonists to host cells (e.g., immune cells as described herein). Exogenous nucleic acids are effectively connected to suitable promoters so that encoded proteins (e.g., cytokine antagonists and / or immunosuppressive cytokines) can be expressed in host cells. In some cases, exogenous nucleic acids encoding antagonists and / or immunosuppressive cytokines may be integrated into the genome of the host cell. In other cases, exogenous nucleic acids may remain outside the chromosome (not integrated into the genome).

[0102] Examples of target inflammatory proteins include, but are not limited to, inflammatory cytokines or their soluble receptors (e.g., IL2, IL1α, IL1β, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, sIL-1RI, sIL-2Rα, sIL6R, IFNα, IFNβ, IFNγ, MIPα, MIPβ, CSF1, LIF, G-CSF, GM-CSF, CXCL10, CCL5, eotaxin, TNF, MCP1, MIG, RAGE, CRP, angiopoietin-2, and VWF), inflammatory growth factors (e.g., TGFα, VEGF, EGF, HGF, and FGF), and cytotoxic molecules (e.g., perforin, granzymes, and ferritin).

[0103] The modified immune cells comprising one or more knock-in modifications may include one or more exogenous nucleic acids (e.g., exogenous expression cassettes) for expressing an immunosuppressive cytokine and / or an antagonist of one or more target inflammatory proteins as described herein. For the purposes of this disclosure, it will be expressly understood that the term "antagonist" encompasses all previously identified terms, titles, and functional states and characteristics, whereby the target protein itself, the biological activity of the target protein, or the result of the biological activity is substantially ineffective, reduced, or neutralized to any meaningful extent (e.g., at least 20%, 50%, 70%, 85%, 90% or more).

[0104] In some embodiments, the cytokine antagonists described herein can be antibodies specific to the target protein, antibodies specific to the receptor of the target protein, or antibodies to auxiliary proteins involved in the complex of the cytokine and its receptor. Such antibodies (antagonistic antibodies) also interfere with the binding of the target cytokine to the cognate receptor on its immune cell, thereby inhibiting the cell signaling mediated by the target protein. Antagonistic antibodies within the scope of the present disclosure include but are not limited to antibodies that can bind to the activity of inflammatory proteins and neutralize the activity of inflammatory proteins (e.g., inflammatory cytokines, including IL2, IL1α, IL1β, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, sIL-1RI, sIL-2Rα, sIL6R, IFNα, IFNβ, IFNγ , MIPα, MIPβ, CSF1, LIF, G-CSF, GM-CSF, CXCL10, CCL5, eotaxin, TNF, MCP1, MIG, RAGE, CRP, angiopoietin-2 and VWF, inflammatory growth factors, including TGFα, VEGF, EGF, HGF and FGF and cytotoxic molecules, including perforin, granzyme and ferritin) and antibodies capable of binding to receptors of any target protein where applicable. Exemplary antagonistic antibodies include clazakizumab, olokizumab, siltuximab, sirukumab (anti-IL6R), tocilizumab (anti-IL6R) and sarilumab (anti-IL6R) or antigen-binding fragments thereof, genetically modified forms thereof (e.g., scFv derived from a reference antibody). The genetically modified form of a known antibody (eg, a scFv antibody) comprises the same heavy and light chain complementarity determining regions as the reference antibody, and optionally comprises the same heavy and light chain variable regions as the reference antibody.

[0105] As used herein, antibodies (used interchangeably in the plural form) are immunoglobulin molecules that can specifically bind to a target protein (e.g., IL6 and other proteins described herein) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" not only covers complete (i.e., full-length) antibodies, but also covers antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chains (scFv), mutants thereof, fusion proteins including antibody portions, humanized antibodies, chimeric antibodies, double antibodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configurations of immunoglobulin molecules including antigen recognition sites of desired specificity, the modified configurations comprising glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies include antibodies of any class, such as IgD, IgE, IgG, IgA, or IgM (or its subclasses), and antibodies do not need to be of any particular class. According to the antibody amino acid sequence of its heavy chain constant domain, immunoglobulins can be divided into different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0106] In certain embodiments, antibodies as described herein can specifically bind to a target protein or its receptor.Antibodies that "specifically bind" (used interchangeably herein) to a target or epitope are well-known terms in the art, and methods for determining such specific binding are also well-known in the art.If a molecule reacts or associates with a specific target antigen more frequently, more quickly, longer lastingly, and / or with greater affinity than an alternative target, the molecule is referred to as exhibiting "specific binding".If an antibody is combined with a greater affinity, avidity is higher, more easily, and / or with greater duration than it is with other substances, the antibody is "specifically bound" to a target cytokine.For example, an antibody specifically (or preferentially) bound to an IL6 epitope is an antibody that binds to this IL6 epitope with greater affinity, avidity is higher, more easily, and / or with greater duration than it is with other IL6 epitopes.By reading this definition, it should also be understood that, for example, an antibody specifically bound to a first target antigen may or may not specifically bind to or preferentially bind to a second target antigen. Thus, "specific binding" or "preferential binding" does not necessarily require (although it can encompass) exclusive binding. Typically, but not necessarily, reference to binding means preferential binding.

[0107] In some embodiments, the antagonist antibodies of the target protein as described herein have a suitable binding affinity for the target protein (e.g., IL6 or GM-CSF) or an antigenic epitope thereof. As used herein, "binding affinity" refers to the apparent association constant or KA. KA is the reciprocal of the dissociation constant (KD). The antagonist antibodies described herein may have a binding affinity (KD) of at least 10 for the target antigen or antigenic epitope. -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 M or lower. An increased binding affinity corresponds to a decreased KD. Higher affinity binding of an antibody to a first antibody relative to a second antibody can be indicated by a higher KA (or a smaller numerical KD) for binding to the first antibody than the KA (or numerical KD) for binding to the second antibody. In this case, the antibody is specific for a first antigen (e.g., a first protein in a first conformation or a mimetic thereof) relative to a second antigen (e.g., the same first protein in a second conformation or a mimetic thereof; or a second protein). In some embodiments, the antagonist antibodies described herein have a higher binding affinity (higher KA or smaller KD) for a target protein in a mature form than the binding affinity for the target protein in a precursor form or another protein (e.g., an inflammatory protein in the same family as the target protein). The difference in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 37.5-fold, 50-fold, 70-fold, 80-fold, 91-fold, 100-fold, 500-fold, 1000-fold, 10,000-fold, or 10 5 times.

[0108] Binding affinity (or binding specificity) can be determined by various methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance or spectroscopy (e.g., using fluorescence measurement). Exemplary conditions for assessing binding affinity are in HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20). These techniques can be used to measure the concentration of bound binding protein based on the target protein concentration. The concentration of bound binding protein ([Bound]) is generally related to the concentration of free target protein ([Free]) by the following equation:

[0109] [Bound] = [Free] / (Kd + [Free])

[0110] Although, precise determination of KA is not always necessary, as sometimes activity in a functional assay (e.g., in vitro or in vivo) is sufficient to obtain a qualitative measure of affinity, obtain a qualitative measure of affinity, or obtain an inference of affinity, such as determined using methods such as ELISA or FACS analysis, which is proportional to KA and can therefore be used for comparison, such as determining whether a higher affinity is, for example, 2-fold higher.

[0111] In some examples, the antagonist antibodies described herein are human antibodies or humanized antibodies.Alternatively or additionally, the antagonist antibody is a single chain antibody (scFv).

[0112] In other embodiments, the inflammatory protein antagonist described herein can be a soluble receptor capable of binding to a target protein. Such soluble receptors can include the extracellular domain of the receptor of the target protein, but lack the transmembrane domain of the receptor so that the soluble receptor is not displayed on the cell surface. Soluble receptors can compete with cell surface receptors to bind to the target protein, thereby inhibiting the signal transduction mediated by the target protein. Exemplary soluble receptors for the present disclosure include soluble IL-6 receptor (IL6R), soluble IL1 receptor type II (sIL-1RII), soluble gp130, soluble RAGE, soluble IL15Rα (sIL-15Rα, see, for example, Mortier et al., Journal of Immunology 173 (3): 1681-8, 2004), and soluble CCR2 receptors (see, for example, Izhak et al., Journal of Immunology, 183 (1): 732-9, 2009). Interleukin-1 receptor type 2 (also known as IL-1RII) is encoded by the IL1R2 gene of people, and is considered to be a decoy receptor, and acts as an inhibitor of its ligand. Exemplary human IL-1RII sequences are provided under gene bank accession numbers NP_004624.1 and NP_001248348.1. As used herein, sIL-1RII refers to the soluble form of IL-1RII receptor. Modified immune cells including knock-in modifications include one or more exogenous nucleic acids encoded for one or more inflammatory protein antagonists as described herein. In some cases, exogenous nucleic acids include expression cassettes, wherein the coding sequence is operably connected to a suitable promoter (acting in immune cells to drive the expression of antagonists). Optionally, the expression cassette may include one or more regulatory elements, such as enhancers, polyA regulatory sequences, etc. In some instances, the expression cassette may be a part of a viral vector (e.g., a retroviral vector or an AAV vector) for delivering exogenous nucleic acids to immune cells and / or for integrating them into the host genome.

[0113] (iv) Cell populations comprising modified immune cells

[0114] Also provided herein is an immune cell group, which includes modified cells with knock-in modification, knock-out modification or a combination thereof as described herein.Immune cells can be T cells, NK cells, dendritic cells, macrophages, B cells, neutrophils, eosinophils, basophils, mast cells, myeloid-derived suppressor cells, mesenchymal stem cells, precursors thereof or a combination thereof.In some cases, immune cell groups are modified to express chimeric antigen receptors (CAR), which can be specific to antigens of disease cells, such as tumor antigens.Typical CAR constructs can include extracellular antigen binding fragments (e.g., scFv fragments), transmembrane domains, costimulatory signaling domains and CD3ζ that can bind to antigens of interest (e.g., tumor antigens).This CAR construct can further contain a hinge domain.

[0115] Alternatively or additionally, the immune cells can be further modified to express exogenous cytokines, chimeric antigen receptors (e.g., chimeric synNotch receptors, chimeric immune receptors, chimeric co-stimulatory receptors, chimeric killer cell immunoglobulin-like receptors (KIRs)), exogenous T cell receptors (TCRs), and / or have endogenous TCRs knocked out. A chimeric antigen receptor (CAR) generally includes (i) an extracellular ligand binding domain (e.g., an scFv fragment or an extracellular domain of a receptor (e.g., a cytokine receptor, a co-stimulatory receptor (e.g., CD28) or a checkpoint receptor (e.g., PD-1)) capable of binding to a target protein; (ii) a transmembrane domain for anchoring the CAR to the cell membrane; and (iii) one or more intracellular signaling domains for signal transduction, such as the signaling domains of CD3z, KIR, 2B4, CD28, 4-1BB, CD27, OX40, ICOS, MYD88, IL2 receptor, SynNotch, etc. A chimeric cytokine receptor refers to a CAR comprising an extracellular domain of a cytokine receptor. A chimeric co-stimulatory receptor refers to a CAR comprising an extracellular domain of a co-stimulatory receptor. A chimeric KIR receptor and a chimeric SynNotch receptor refer to CARs comprising signaling domains from KIR and SynNotch, respectively.

[0116] In some embodiments, the population of immune cells described herein may include at least (i) a first plurality of modified immune cells comprising one or more of the knockout modifications described herein; and (ii) a second plurality of modified immune cells comprising one or more of the knockout modifications also described herein.

[0117] The first plurality of modified immune cells may produce a reduced level of one or more of the cytokines / proteins described herein in conjunction with the knockout modification, such as IL2, IL1α, IL1β, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, sIL-1RI, sIL-2Rα, sIL6R, IFNα, IFNβ, IFNγ, MIPα, MIPβ, CSF1, LIF, G-CSF, GM-CSF, CXCL10, CCL5, eotaxin, TNF, MCP1, MIG, RAGE, CRP, angiopoietin-2, VWF, TGFα, VEGF, EGF, HGF and FGF, perforin, granzyme and ferritin. Some or all members of the first plurality of modified immune cells may have a knockout modification to reduce the yield of the inflammatory protein of interest. Alternatively, some or all members of the first plurality may have more than one knockout modification to reduce the production of more than one cytokine of interest. Different members of the first plurality of modified immune cells may have different knockout modifications or different combinations of knockout modifications. For example, one member may have a knockout of IL-2, another member may have a knockout of GM-CSF, and a third member may have both. All members of the first plurality of modified immune cells collectively have all knockout modifications of interest such that the production levels of all cytokines of interest are reduced.

[0118] The second plurality of modified immune cells can express one or more antagonists of one or more target proteins as described herein in conjunction with the knock-in modification, such as an antagonist of IL2, IL1α, IL1β, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, sIL-1RI, sIL-2Rα, sIL6R, IFNα, IFNβ, IFNγ, MIPα, MIPβ, CSF1, LIF, G-CSF, GM-CSF, CXCL10, CCL5, eotaxin, TNF, MCP1, MIG, RAGE, CRP, angiopoietin-2, VWF, TGFα, VEGF, EGF, HGF, FGF, perforin, granzyme, ferritin, or a combination thereof. Some or all members of the second plurality of modified immune cells may have a knock-in modification, i.e., carry an exogenous nucleic acid, and the exogenous nucleic acid may be designed to express a cytokine antagonist of concern, a kind of immunosuppressive cytokine of concern, a variety of antagonists of concern, a variety of immunosuppressive cytokines of concern, or a combination thereof. Alternatively, some or all members of the second plurality may have more than one knock-in modification, i.e., carry more than one exogenous nucleic acid, and each exogenous nucleic acid may be designed to express a cytokine antagonist, an immunosuppressive cytokine, more than one cytokine antagonist, more than one immunosuppressive cytokine or a combination thereof. Different members of the second plurality of modified immune cells may have different knock-in modifications or different knock-in modification combinations. For example, a member may have the knock-in of an IL6 antagonist, another member may have the knock-in of an IL15 antagonist, and a third member may have both. All members of the second plurality of modified immune cells may have all knock-in modifications of concern collectively, so that all antagonists of concern are expressed.

[0119] In an immune cell population comprising a first plurality of modified immune cells and a second plurality of modified immune cells, the total level of one or more cytokines / proteins in such cytokines / proteins produced thereby is reduced by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%) relative to the total level of one or more of the same cytokines / proteins produced by a wild-type counterpart (with the same number of cells) as determined under the same conditions (e.g., using the same assay and the same experimental conditions). The total level of cytokine / protein production can represent the production level of cytokines / proteins of immune cells when activated. Alternatively, the total level of cytokine / protein production can represent the production level of cytokines / proteins of deactivated immune cells. In some cases, the level of a specific cytokine produced by an immune cell population may not be detected by conventional assays.

[0120] Further, the immune cell population may produce one or more cytokine antagonists at appropriate levels for inhibiting signal transduction mediated by the target cytokine at a meaningful level, thereby reducing or eliminating side effects caused by such cytokines in adoptive immune cell therapy.

[0121] As described herein, the first multiple modified immune cells and the second multiple modified immune cells in the immune cell group can be the same immune cell type (for example, T cells or NK cells). In some cases, the first multiple modified immune cells and the second multiple modified immune cells can overlap, that is, have members with knock-in modification and knock-out modification. In other cases, the first multiple modified immune cells and the second multiple modified immune cells do not have overlapping members.

[0122] In another embodiment, there is provided herein a kind of immune cell group, it includes a plurality of modified immune cells of knockout modification with IL-2.In some cases, the total level of IL-2 produced by this immune cell group can be reduced by at least 10% (for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or higher) relative to the wild-type counterpart as determined under the same conditions.For example, the total level of IL-2 produced by immune cell group as described herein can be low about 30-95% compared with the wild-type counterpart as determined under the same conditions.In a specific example, the total level of IL-2 produced by immune cell group can be about 50% of the total level of IL-2 of wild-type counterpart.

[0123] The immune cell group may further include modified cells with additional knockout modifications and / or knock-in modifications as described herein, such that the total level of knockout cytokines / proteins is reduced relative to wild-type counterparts, and / or the immune cell group expresses a suitable level of cytokine antagonists. In some cases, the immune cell group further includes modified cells with GM-CSF knockout, TNFα knockout, and / or IL6 antagonist knock-in. Similarly, members of the immune cell group may have one or more knock-in modifications and / or knock-out modifications in the knock-in modifications and / or knock-out modifications, and different members may have different knock-in modifications / knock-out modifications or different knock-in modifications / knock-out modifications. All members of the immune cell group collectively have all knock-in modifications / knock-out modifications of interest.

[0124] II. Methods for preparing modified immune cells

[0125] Any knock-in modification and knock-out modification can be introduced into suitable immune cells by conventional methods and / or methods described herein. Typically, such methods will involve delivering genetic material into suitable immune cells to down-regulate the expression of target endogenous inflammatory proteins, to express cytokine antagonists of interest, or to express immunosuppressive cytokines of interest.

[0126] (i) Knockout modification

[0127] Any method known in the art for downregulating endogenous gene expression in a host cell may be used to reduce production levels of the target endogenous cytokines / proteins described herein.

[0128] In some cases, gene editing methods can be performed to modify the endogenous alleles of the gene of the target cytokine / protein (e.g., in the coding region or non-coding regulatory region), thereby reducing the expression of the target endogenous cytokine. The gene editing method may involve the use of an endonuclease capable of cutting the target region in the endogenous allele. In the absence of the template nucleic acid, non-homologous end joining can repair double-strand breaks in the genome, and mutations (e.g., insertions, deletions, and / or frameshifts) are introduced into the target site. Gene editing methods are generally classified based on the type of endonuclease involved in producing double-strand breaks in the target nucleic acid. Examples include, but are not limited to, clustered regularly spaced short palindromic repeats (CRISPR) / endonuclease systems, nucleases based on transcription activator-like effectors (TALENs), zinc finger nucleases (ZFNs), endonucleases (e.g., ARC homing endonucleases), large-range nucleases (e.g., mega-TALs), or combinations thereof.

[0129] The cutting of the gene region can include cutting one or two chains at the position of the target allele by an endonuclease. In certain embodiments, the cutting event can be followed by repairing the cut target polynucleotide by homologous recombination with an exogenous template polynucleotide, thereby causing an insertion, deletion or substitution of one or more nucleotides of the target nucleotide sequence. Such gene editing can result in decreased transcription of a target gene (e.g., IL2, IL1α, IL1β, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, sIL-1RI, sIL-2Rα, sIL6R, IFNα, IFNβ, IFNγ, MIPα, MIPβ, CSF1, LIF, G-CSF, GM-CSF, CXCL10, CCL5, eotaxin, TNF, MCP1, MIG, RAGE, CRP, angiopoietin-2, VWF, TGFα, VEGF, EGF, HGF, FGF, perforin, granzyme, ferritin, or a combination thereof).

[0130] The reduction level of the target endogenous cytokine / protein in the immune cell group can be adjusted by the level of the gene editing event introduced into the cell group. For example, a large amount of one or more gene editing components introduced into the host immune cell group will cause most of the host immune cells to have edited target endogenous alleles. In this way, the total production level of the target endogenous cytokine / protein will be reduced to a high level. Alternatively, a small amount of one or more gene editing components introduced into the host immune cell group will cause a small part of the host immune cells to have edited target endogenous alleles. In this way, the total production level of the target endogenous cytokine / protein will be reduced to a low level. Therefore, controlling the amount of one or more gene editing components to be delivered to the cell group can control the total reduction level of the target endogenous cytokine / protein. As known to those skilled in the art, other suitable methods may also be applicable to controlling the reduction level of the target cytokine / protein.

[0131] In some cases, the genetic modification of immune cells as described herein is performed using clustered regularly spaced short palindromic repeats (CRISPR) / endonuclease technology known in the art. The CRISPR / endonuclease system has been applicable to prokaryotic cells and eukaryotic cells. Gene editing using CRISPR generally depends on the expression of at least two components: a guide RNA sequence and an endonuclease (e.g., comprising Cpf1 and Cas9) that recognize a target nucleic acid sequence. The guide RNA helps guide the endonuclease to the target site, which generally contains a nucleotide sequence complementary (partially or completely) to the gRNA or a portion thereof. In some cases, the guide RNA is a two-piece RNA complex including a prototype spacer fragment and a scaffold RNA fragment complementary to the target nucleic acid sequence. In some cases, scaffold RNA is needed to help recruit the endonuclease to the target site. In some cases, the guide RNA is a single guide RNA (sgRNA) including both a prototype spacer sequence and a scaffold RNA sequence. An exemplary sequence of a scaffold RNA can be: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 31). Once at the target site, the endonuclease can generate a double-strand break. It is known to those skilled in the art that the nucleotide sequence of an RNA molecule contains the residue "U". The corresponding DNA sequence of any RNA sequence disclosed herein is also within the scope of the present disclosure. Such a DNA sequence will contain a "T" instead of a "U" in the corresponding RNA sequence.

[0132] The target nucleic acid for use with the CRISPR system is flanked on the 3' side by a protospacer adjacent motif (PAM), which can interact with the endonuclease and further participate in targeting the endonuclease activity to the target nucleic acid. It is generally believed that the PAM sequence of the flanking target nucleic acid depends on the endonuclease and the source from which the endonuclease is derived. For example, for the Cas9 endonuclease derived from Streptococcus pyogenes, the PAM sequence is NGG. For the Cas9 endonuclease derived from Staphylococcus aureus, the PAM sequence is NNGRRT. For the Cas9 endonuclease derived from Neisseria meningitidis, the PAM sequence is NNNNGATT. For the Cas9 endonuclease derived from Streptococcus thermophilus, the PAM sequence is NNAGAA. For the Cas9 endonuclease derived from Treponema denticola, the PAM sequence is NAAAC. For the Cpf1 nuclease, the PAM sequence is TTN.

[0133] The CRISPR / endonuclease system hybridized with the target sequence in the endogenous cytokine locus can be used to knock out the cytokine of interest.GRNA hybridizes (partially or completely complementary) with the target nucleic acid sequence (for example, the endogenous locus of cytokine) in the host cell genome.The length of the gRNA or its part hybridized with the target nucleic acid can be between 15-25 nucleotides, 18-22 nucleotides or 19-21 nucleotides.In some cases, the length of the gRNA sequence hybridized with the target nucleic acid is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.In some instances, the length of the gRNA sequence hybridized with the target nucleic acid is between 10-30 or between 15-25 nucleotides.

[0134] In some examples, the gRNA sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or at least 100% complementary to the target nucleic acid (see also U.S. Pat. No. 8,697,359, which is incorporated by reference for its teachings of complementarity of gRNA sequences to target polynucleotide sequences). It has been demonstrated that mismatches between the CRISPR guide sequence and the target nucleic acid near the 3' end of the target nucleic acid can eliminate nuclease cleavage activity (see, e.g., Upadhyay et al., Genes Genome Genetics (2013) 3(12):2233-2238). In some examples, the gRNA sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary to the 3' end of the target nucleic acid (e.g., the last 5, 6, 7, 8, 9, or 10 nucleotides of the 3' end of the target nucleic acid).

[0135] The "percent identity" of two nucleic acids is determined using the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87:2264-68, 1990, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-77, 1993. Such algorithms are incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol., 215:403-10, 1990. BLAST nucleotide searches can be performed with the NBLAST program (score = 100, word length - 12) to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. When there is a gap between two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0136] In some cases, the amount of one or more guide RNAs or sgRNAs introduced into a population of cells increases the population's sensitivity to one or more inflammatory proteins (e.g., IL2, IL1α, IL1β, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, sIL-1RI, sIL-2Rα, sIL6R, IFNα, IFNβ ...α, IL1α, IL1β, IL1β, IL-5, IL-6, IL-7, γ, MIPα, MIPβ, CSF1, LIF, G-CSF, GM-CSF, CXCL10, CCL5, eotaxin, TNF, MCP1, MIG, RAGE, CRP, angiopoietin-2, VWF, TGFα, VEGF, EGF, HGF, FGF, perforin, granzyme, ferritin, or a combination thereof) is reduced by at least 10% (at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%). For example, the amount of sgRNA in the CRISPR / CAS system targeting the IL-2 locus can result in a total population producing about 30-95% less IL-2. In one example, IL-2 production is reduced by about 50%. The CRISPR / endonuclease system can optionally be used to modify the endogenous GM-CSF locus and / or the TNF locus to reduce the production of these cytokines.

[0137] Various CRISPR / endonuclease systems are known in the art, and modifications are regular, and many references describe rules and parameters for guiding the design of CRISPR / endonuclease systems (e.g., including Cas9 target selection tools). See, for example, Hsu et al., Cell, 157(6):1262-78, 2014.

[0138] In some cases, TALEN technology known in the art is used to carry out genetic modification of immune cells as described herein. TALEN is an engineered restriction enzyme that can specifically bind to and cut a desired target DNA molecule. TALEN generally contains a transcription activator-like effector (TALE) DNA binding domain fused to a DNA cleavage domain. The DNA binding domain can contain a highly conserved 33-34 amino acid sequence, wherein there are two divergent amino acid RVDs (repeat variable dipeptide motifs) at positions 12 and 13. The RVD motif determines the binding specificity to the nucleic acid sequence and can be engineered according to methods known to those skilled in the art to specifically bind to a desired DNA sequence (see, e.g., Juillerat et al., Scientific Reports, 5:8150, 2015; Miller et al., Nature Biotechnology 29(2):143–8, 2011; Zhang et al., Nature Biotechnology 29(2):149–53, 2011; Geiβler et al., PLoS One ONE) 6(5):e19509, 2011; Boch, Nature Biotechnology 29(2):135–6, 2011; Boch et al., Science 326(5959):1509–12, 2009; and Moscou et al., Science 326(5959):1501, 2009. The DNA cleavage domain can be derived from the FokI endonuclease, which is active in many different cell types. The FokI domain acts as a dimer, so that sites in the target genome with proper orientation and spacing require two constructs with unique DNA binding domains. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between the two separate TALEN binding sites appear to be important parameters for achieving high activity levels. See, e.g., Miller et al., Nature Biotechnology 29:143-8, 2011.

[0139] TALENs specific for sequences in a target gene of interest (e.g., IL2, IL1α, IL1β, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, sIL-1RI, sIL-2Rα, sIL6R, IFNα, IFNβ, IFNγ, MIPα, MIPβ, CSF1, LIF, G-CSF, GM-CSF, CXCL10, CCL5, eotaxin, TNF, MCP1, MIG, RAGE, CRP, angiopoietin-2, VWF, TGFα, VEGF, EGF, HGF, FGF, perforin, granzyme, ferritin, or a combination thereof) can be constructed using any method known in the art, including various schemes using modular components. See, for example, Zhang et al., Nature Biotechnology 29, 2011: 149-53; and Geibler et al., PLoS One 6: e19509, 2011.

[0140] TALENs specific for the target gene of interest can be used inside cells to generate double-strand breaks (DSBs). If the repair mechanism improperly repairs the break by non-homologous end joining, mutations can be introduced at the break site. For example, inappropriate repair can introduce frameshift mutations.

[0141] In some examples, zinc finger nucleases (ZFNs) known in the art can be used to generate modified immune cell populations as described herein. Zinc finger nucleases (ZFNs) are restriction enzymes comprising an engineered zinc finger DNA binding domain connected to the catalytic domain of type II endonuclease FokI. The zinc finger DNA binding domain of each ZFN targets the connected FokI endonuclease to a specific site in the genome. Since FokI only acts as a dimer, a pair of ZFNs are usually engineered to bind to the homologous target "half site" sequence on the relative DNA strand. The target "half site" sequence is usually spaced apart so that a catalytically active FokI dimer can be formed between them. After FokI domain dimerization, a DNA double-strand break is generated between the ZFN half sites. As described above, non-homologous end joining can introduce mutations, and homology-directed repair can be used to introduce exogenous nucleic acids.

[0142] Many gene editing systems using ZFNs and considerations for designing ZFNs have been described; see, for example, Segal et al., Proc. Natl. Acad. Sci. USA 96(6):2758-63 (1999); Dreier B et al., J. Mol. Biol. 303(4):489-502, 2000; Liu Q et al., J. Biol. Chem. 277(6):3850-6, 2002; Dreier et al., J. Biol. Chem. 280(42):35588-97, 2005; and Dreier et al., J. Biol. Chem. 276(31):29466-78, 2001.

[0143] Any method known in the art can also be used to introduce a meganuclease (or homing endonuclease) to genetically engineer any modified cell described herein, wherein the meganuclease is a sequence-specific endonuclease that recognizes a long DNA target (usually between 14 and 40 base pairs). There are at least six meganuclease families, and the at least six meganuclease families are generally classified based on structural motifs, including LAGLIDADG, GIY-YIG, HNH, His-Cys box, PD-(D / E)XK and Vsr samples. Non-limiting examples of meganucleases include PI-SceI, I-CreI and I-TevI.

[0144] Various gene editing systems using meganucleases (including modified meganucleases) have been described in the art; see, for example, Steentoft et al., Glycobiology 24(8):663-80, 2014; Belfort and Bonocora, Methods Mol. Biol. 1123:1-26, 2014; Hafez and Hausner, Genome 55(8):553-69, (2012); and a review of the references cited herein.

[0145] Hybrid nucleases comprising MegaTAL can also be used. MegaTAL is a fusion of a TALE DNA binding domain with a catalytically active meganuclease. Such nucleases utilize the DNA binding specificity of TALE and the sequence cleavage specificity of a meganuclease. See, e.g., Boissel et al., NAR, 42: 2591-2601, 2014.

[0146] Alternatively, any knockout modification can be achieved using antisense oligonucleotides or ribozymes by methods known in the art.Antisense oligonucleotides specific for a target cytokine / protein refer to oligonucleotides that are complementary or partially complementary to a target region of an endogenous gene for a cytokine or an mRNA encoding such an endogenous gene.

[0147] Antisense oligonucleotides can include small interfering RNA (siRNA or RNAi), which can downregulate the expression of target cytokines by RNA interference. RNA interference (RNAi) is a process in which dsRNA guides the homology sequence-specific degradation of messenger RNA. In mammalian cells, RNAi can be triggered by 21 nucleotide duplexes of small interfering RNA (siRNA) without activating the host interferon response. The dsRNA used in the method disclosed herein can be siRNA (containing two separated and complementary RNA chains) or short hairpin RNA (i.e., RNA chains forming a tight hairpin structure), both of which can be designed based on the sequence of the target gene. Alternatively, it can be microRNA.

[0148] Any antisense oligonucleotide or expression cassette for producing such antisense oligonucleotide can be delivered to immune cells by conventional methods to downregulate the production of one or more target cytokines / proteins as described herein.

[0149] (ii) Knock-in modification

[0150] In order to produce the knock-in of one or more cytokine antagonists described herein, the coding sequence of any antagonist and / or immunosuppressive cytokine described herein can be cloned into a suitable expression vector (e.g., including but not limited to a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated vector, a PiggyBac transposon vector, and a SleepingBeauty transposon vector), and introduced into a host immune cell using conventional recombinant techniques. Sambrook et al., Molecular Cloning: Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press. As a result, the modified immune cells of the present disclosure may include one or more exogenous nucleic acids encoding at least one cytokine antagonist or at least one immunosuppressive cytokine. In some cases, the coding sequence of one or more antagonists and / or one or more immunosuppressive cytokines is integrated into the genome of the cell. In some cases, the coding sequence of one or more antagonists is not integrated into the genome of the cell.

[0151] The exogenous nucleic acid including the coding sequence of the cytokine antagonist or immunosuppressive cytokine of interest may further include a suitable promoter, which may be operably connected to the coding sequence. As used herein, a promoter refers to a nucleotide sequence (site) to which an RNA polymerase on a nucleic acid can bind, to initiate transcription of the coding DNA (e.g., for a cytokine antagonist) into mRNA, which is then translated into the corresponding protein (i.e., gene expression). When the promoter is in the correct functional position and toward the transcription initiation and expression (to produce the corresponding protein molecule) of the coding sequence relative to the coding sequence, the promoter is considered to be "operably connected" to the coding sequence. In some cases, the promoter described herein may be constitutive, which initiates transcription independent of other regulatory factors. In some cases, the promoter described herein may be inducible, which depends on the regulatory factor used for transcription. Exemplary promoters include, but are not limited to, ubiquitin, RSV, CMV, EF1α, and PGK1. In one example, one or more nucleic acids encoding one or more antagonists of one or more inflammatory cytokines as described herein, operably linked to one or more suitable promoters, can be introduced into immune cells by conventional methods to drive expression of the one or more antagonists.

[0152] In addition, the exogenous nucleic acids described herein may further contain, for example, some or all of the following: selectable marker genes, such as the neomycin gene for selecting stable or transient transfectants in mammalian cells; enhancer / promoter sequences from the immediate early genes of human CMV for high-level transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma replication origin and ColE1 for appropriate episomal replication; universal multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable methods for generating vectors containing transgenes are well known and available in the art. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press.

[0153] (iii) Preparation of immune cell populations including modified immune cells

[0154] An immune cell population comprising any modified immune cell described herein or a combination thereof can be prepared by introducing one or more knockout modifications in the knockout modifications, one or more knockin modifications in the knockin modifications, or a combination thereof into a host immune cell population. Knockin modifications and knockout modifications can be introduced into host cells in any order.

[0155] In some cases, one or more modifications are introduced into the host cell in a sequential manner without separation and / or enrichment of modified cells after the previous modification event and before the next modification event. In that case, the resulting immune cell population can be heterogeneous, comprising cells with different modifications or different combinations of modifications. Such immune cell populations may also include unmodified immune cells. The level of each modification event occurring in the immune cell population can be controlled by the amount of genetic material inducing such modifications relative to the total number of host immune cells. See also discussion above.

[0156] In other cases, after the first modification event and before the second modification event, the modified immune cells can be isolated and enriched. This method will result in the generation of a substantially homogeneous immune cell population having all knock-in modifications and / or knock-out modifications introduced into the cells.

[0157] In some examples, one or more knock-in modifications and one or more knock-out modifications are introduced into the host immune cells separately. For example, the knock-out modification is performed by gene editing to knock out the endogenous gene of the target cytokine, and the knock-in modification is performed by delivering a separate exogenous expression cassette for producing one or more cytokine antagonists into the host immune cells. Figure 1 An exemplary schematic diagram is provided in the left figure of .

[0158] Alternatively, if Figure 1 As shown in the right figure of , knock-in modification and knock-out modification can be performed simultaneously. For example, an exogenous nucleic acid encoding one or more cytokine antagonists can be introduced by a homology donor template together with the above-mentioned gene editing system to knock in one or more cytokine antagonists of interest and simultaneously knock out endogenous gene alleles to reduce the yield of endogenous cytokines / proteins as described herein. When using the CRISPR / nuclease system, the guide RNA can be designed to target the endogenous locus of the cytokine of interest, and at this time, an expression cassette for producing one or more cytokine antagonists of interest is introduced.

[0159] Alternatively, exogenous nucleic acids (e.g., expression cassettes encoding cytokine antagonists) can be introduced into cells along with TALENs. This process can be used to introduce DNA fragments into target genes of interest and / or introduce defects into endogenous genes, thereby reducing the expression of the target gene, and at this time introduce exogenous nucleic acids for expressing one or more cytokine antagonists.

[0160] Immune cell groups can be further modified to express exogenous cytokines, chimeric antigen receptors (CARs) as described herein, such as chimeric cytokine receptors, chimeric synNotch receptors, chimeric immune receptors, chimeric costimulatory receptors, chimeric killer cell immunoglobulin-like receptors (KIRs), and / or exogenous T cell receptors. This can be done before, after, or simultaneously with knock-in modification and / or knock-out modification. Such receptors can be cloned and integrated into any suitable expression vector using conventional recombinant techniques. Considerations for the design of chimeric antigen receptors are also known in the art. See, for example, Sadelain et al., Cancer Discov., 3(4): 388-98, 2013.

[0161] III. Therapeutic Applications

[0162] Any immune cell population including modified immune cells as described herein can be used for adoptive immune cell therapy for treating target diseases such as leukemia or lymphoma. Due to the knock-in and knock-out modifications introduced into immune cells, it is expected that the therapeutic use of such modifications will reduce the cytotoxicity associated with conventional adoptive immune cell therapy (reducing the inflammatory cytokine production and / or signaling of both the immune cells used in adoptive immune cell therapy and the recipient's endogenous immune cells, which can be activated by the infused immune cells), while achieving the same or better therapeutic effect.

[0163] In order to practice the treatment methods described herein, an effective amount of immune cell groups (including any modified immune cells as described herein) can be administered to a subject in need of treatment by a suitable route (e.g., intravenous infusion). Immune cell groups can be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition before administration, which is also within the scope of the present disclosure. Immune cells can be autologous for a subject, that is, immune cells are obtained from a subject in need of treatment, and are modified to reduce the expression of one or more target cytokines / proteins, such as described herein, to express one or more cytokine antagonists described herein, to express CAR constructs and / or exogenous TCRs or a combination thereof. The resulting modified immune cells can then be administered to the same subject. Compared with the administration of non-autologous cells, the administration of autologous cells to a subject can result in a reduction in the rejection of immune cells. Alternatively, immune cells can be allogeneic cells, that is, the cells are obtained from a first subject, modified as described herein and applied to a second subject different from the first subject but belonging to the same species. For example, allogeneic immune cells can be derived from human donors and applied to human recipients different from the donor.

[0164] The subject to be treated can be a mammal (e.g., a human, mouse, pig, cow, rat, dog, guinea pig, rabbit, hamster, cat, goat, sheep, or monkey). The subject may be suffering from cancer, an infectious disease, or an immune disorder. Exemplary cancers include, but are not limited to, hematologic malignancies (e.g., B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, and multiple myeloma). Exemplary infectious diseases include, but are not limited to, human immunodeficiency virus (HIV) infection, Epstein-Barr virus (EBV) infection, human papillomavirus (HPV) infection, dengue virus infection, malaria, sepsis, and Escherichia coli (E. coli) infection. Exemplary immune disorders include, but are not limited to, autoimmune diseases such as rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, and vasculitis.

[0165] As used herein, the term "effective amount" refers to the amount of each active agent required for the treatment effect of the subject, either alone or in combination with one or more active agents. As recognized by those skilled in the art, the effective amount varies according to the specific condition treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex and weight, treatment duration, route of administration, excipient use, co-use with other active agents (if any), and similar factors within the knowledge and expertise of health practitioners. The amount to be administered depends on the subject to be treated, including the ability of the individual immune system to produce a cell-mediated immune response, for example. The precise amount of the active ingredient to be administered depends on the judgment of the practitioner. However, those skilled in the art can easily determine a suitable dosage range.

[0166] As used herein, the term "treat" refers to the application or administration of a composition comprising one or more active agents to a subject suffering from a target disease, a symptom of a target disease, or a susceptibility to a target disease, with the purpose of curing, rehabilitating, alleviating, mitigating, altering, remedying, ameliorating, improving, or affecting the disease, a symptom of a disease, or a susceptibility to a disease.

[0167] Immune cell populations including modified immune cells as described herein can be used in combination with other types of therapies for cancer, such as chemotherapy, surgery, radiation, gene therapy, etc. Such therapies can be administered simultaneously or sequentially (in any order) with the immunotherapy described herein. When co-administered with additional therapeutic agents, the appropriate therapeutically effective dose of each agent can be reduced due to additive or synergistic effects.

[0168] Non-limiting examples of other anti-cancer therapeutics that can be used in combination with the modified immune cells described herein include, but are not limited to, immune checkpoint inhibitors (e.g., PDL1, PD1 and CTLA4 inhibitors), anti-angiogenic agents (e.g., TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitors of metalloproteinases, prolactin, angiostatin, endostatin, bFGF soluble receptor, transforming growth factor β, interferon α, soluble KDR and FLT-1 receptors, and placenta proliferator-activated protein kinase); VEGF antagonists (e.g., anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments); chemotherapeutic compounds.Exemplary chemotherapeutic compounds include pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine); purine analogs (e.g., fludarabine); folate antagonists (e.g., mercaptopurine and thioguanine); antiproliferative or antimitotic agents, such as vinca alkaloids. alkaloids; microtubule disrupting agents such as taxanes (e.g., paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones and vinorelbine, and epidipodophyllotoxin; DNA damaging agents (e.g., actinomycin, amsacrine, anthracycline, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, carbosporin, chloramphenicol ... (including but not limited to oxaliplatin, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethyhnelamine, oxaliplatin, ifosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide, and etoposide).

[0169] In some embodiments, radiation or radiation and chemotherapy are used in combination with a population of cells comprising the modified immune cells described herein. Additional useful agents and therapies can be found in Physician's Desk Reference, Supplement 59th Edition, (2005), Thomson PDR, Montvale NJ; Gennaro et al., eds. Remington's The Science and Practice of Pharmacy, Supplement 20th Edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., eds. Harrison's Principles of Internal Medicine, Supplement 15th Edition, (2001), McGraw Hill, NY; Berkow et al., eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories. Research Laboratories, Rahway NJ.

[0170] IV. Kits for therapeutic use or preparation of modified immune cells

[0171] The present disclosure also provides kits for any target disease described herein involving the immune cell populations described herein, and kits for preparing modified immune cells as described herein.

[0172] As described herein, the kit for therapeutic use may include one or more containers including an immune cell group, which may be formulated into a pharmaceutical composition. Immune cell groups include any modified immune cells described herein or a combination thereof. Immune cell groups (such as T lymphocytes, NK cells, and other cells described herein) may further express CAR constructs and / or exogenous TCR as described herein.

[0173] In some embodiments, the kit may additionally include instructions for using an immune cell population in any of the methods described herein. The included instructions may include a description of administering an immune cell population or a pharmaceutical composition comprising the immune cell population to a subject to achieve a desired activity in the subject. The kit may further include a description of selecting a subject suitable for treatment based on identifying whether the subject needs treatment. In some embodiments, the instructions include a description of administering an immune cell population or a pharmaceutical composition comprising the immune cell population to a subject in need of treatment.

[0174] The instructions related to the use of the pharmaceutical composition of the immune cell group or the immune cell group as described herein generally include information about the dosage, dosage regimen and route of administration for the intended treatment. The container can be a unit dose, bulk packaging (e.g., multi-dose packaging) or a subunit dose. The instructions provided in the kit of the present disclosure are generally written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical composition is used to treat a disease or condition of a subject, delay the onset of a disease or condition of a subject and / or alleviate a disease or condition of a subject.

[0175] The kit provided herein adopts suitable packaging. Suitable packaging includes but is not limited to vials, bottles, jars, soft packaging, etc. It is also envisioned that the packaging used in combination with a specific device (such as an inhaler, a nasal application device or an infusion device) is used. The kit can have a sterile import port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle). The container can also have a sterile import port. At least one active agent in the pharmaceutical composition is an immune cell population (for example, T lymphocytes or NK cells) including any modified immune cells or a combination thereof.

[0176] The kit may optionally provide additional components such as buffers and interpretative information. Typically, the kit includes a container and a label or one or more package inserts on or associated with the container. In some embodiments, the disclosure provides an article comprising the contents of the kit described above.

[0177] Also provided here is a kit for preparing modified immune cells as described herein. This kit may include one or more containers, each containing reagents for introducing knock-in modifications and / or knock-out modifications into immune cells. For example, the kit may contain one or more components of a gene editing system for performing one or more knock-out modifications as described herein. Alternatively or in addition, the kit may include one or more exogenous nucleic acids for expressing cytokine antagonists as described herein, and reagents for delivering exogenous nucleic acids to host immune cells. This kit may further include instructions for performing desired modifications to host immune cells.

[0178] V. General Technology

[0179] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are fully explained in the following literature, such as: Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1989) Academic Press; Animal Cell Culture (RI Freshney, ed. 1987); Introduction to Cell and Tissue Culture (JPather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (JP Cellis, ed., 1989) Academic Press; Procedures (A. Doyle, JB Griffiths and DG Newell, eds., 1993-8) John Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FMAusubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (John Wiley & Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 1994); =Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical approach, Volumes I and II (D. N. Glover, ed. 1985); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins, eds. 1985); Transcription and Translation (B. D. Hames & S. J. Higgins, eds. 1984); Animal Cell Culture (R. I. Freshney, ed. 1986); Immobilized Cells and Enzymes (I. R. L. Press, 1986); and B. Perbal, A Practical Guide to Molecular Cloning. practical Guide To Molecular Cloning (1984); FMAusubel et al., (eds.).

[0180] The application of the present disclosure is not limited to the construction details and component arrangements set forth in the specification of this article or shown in the accompanying drawings. The present disclosure can have other embodiments and can be practiced or carried out in different ways. Moreover, the wording and terminology used herein are for illustrative purposes and should not be considered to be restrictive. The use of "comprising", "including" or "having", "containing", "involving" and its variations herein is intended to cover the items listed thereafter and their equivalents and other items. Also as used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural references.

[0181] The present invention provides the following implementation modes:

[0182] Item 1. An immune cell population comprising a first plurality of modified immune cells, wherein the first plurality of modified immune cells produce less interleukin 2 (IL-2) relative to wild-type immune cells of the same type under the same conditions.

[0183] Item 2. A population of immune cells according to Item 1, wherein the first plurality of modified immune cells, when activated, produce less IL-2 relative to the wild-type immune cells activated under the same conditions.

[0184] Item 3. An immune cell population according to Item 1 or Item 2, wherein the total IL-2 production of the immune cell population is about 30-95% less than that of the wild-type counterpart under the same conditions.

[0185] Item 4. An immune cell population according to Item 3, wherein the total IL-2 production of the immune cell population is approximately 50% of the total IL-2 production of the wild-type counterpart.

[0186] Item 5. A population of immune cells according to any one of Items 1 to 4, wherein in each cell of the first plurality of modified immune cells, at least one endogenous IL-2 allele is knocked out.

[0187] Item 6. An immune cell population according to any one of Items 1 to 5, further comprising a second plurality of modified immune cells, wherein the second plurality of modified immune cells produce reduced levels of one or more inflammatory proteins relative to wild-type immune cells of the same type, wherein the inflammatory proteins include one or more inflammatory cytokines or their soluble receptors, one or more inflammatory growth factors, one or more cytotoxic molecules or a combination thereof.

[0188] Item 7. An immune cell population according to Item 6, wherein the one or more inflammatory cytokines or their soluble receptors are selected from the group consisting of IL1α, IL1β, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, sIL-1RI, sIL-2Rα, sIL6R, IFNα, IFNβ, IFNγ, MIPα, MIPβ, CSF1, LIF, G-CSF, GM-CSF, CXCL10, CCL5, eotaxin, TNF, MCP1, MIG, RAGE, CRP, angiopoietin-2 and VWF.

[0189] Item 8. The immune cell population according to Item 6, wherein the one or more inflammatory growth factors are selected from the group consisting of: TGFα, VEGF, EGF, HGF and FGF

[0190] Item 9. An immune cell population according to Item 6, wherein the one or more cytotoxic molecules are selected from the group consisting of perforin, granzyme and ferritin.

[0191] Item 10. An immune cell population according to any one of Items 6 to 9, wherein the total level of the one or more inflammatory proteins produced by the immune cell population is at least 10% lower than the total level of the wild-type counterpart under the same conditions.

[0192] Item 11. A population of immune cells according to any one of Items 6 to 10, wherein in each cell of the second plurality of modified immune cells, at least one endogenous allele of the one or more inflammatory proteins is knocked out.

[0193] Item 12. A population of immune cells according to any one of Items 6 to 11, wherein the first plurality of modified immune cells and the second plurality of modified immune cells are of the same type.

[0194] Item 13. A population of immune cells according to Item 12, wherein the first plurality of modified immune cells and the second plurality of modified immune cells overlap.

[0195] Item 14. An immune cell population according to any one of Items 1 to 13, further comprising a third plurality of modified immune cells, wherein the third plurality of modified immune cells express one or more exogenous antagonists of the one or more inflammatory proteins and / or one or more immunosuppressive cytokines.

[0196] Item 15. An immune cell population according to Item 14, wherein the one or more immunosuppressive cytokines are selected from the group consisting of: TGFβ, IL-4, IL-10, IL-13, IL-33, IL-35 and IL-37.

[0197] Item 16. An immune cell population according to Item 14, wherein the one or more exogenous antagonists of the one or more inflammatory proteins include soluble receptors for the inflammatory proteins and / or antibodies specific to the inflammatory proteins.

[0198] Item 17. An immune cell population according to Item 14, wherein the one or more inflammatory proteins include IL6, and the antagonist of IL6 is tocilizumab, sirukumab, sarilumab, siltuximab, olokizumab, clazakizumab or an antigen-binding fragment thereof.

[0199] Item 18. An immune cell population according to Item 17, wherein the antagonist is a single-chain antibody fragment (scFv).

[0200] Item 19. An immune cell population according to Item 18, wherein the scFv comprises the amino acid sequence of SEQ ID NO:22.

[0201] Item 20. A population of immune cells according to any one of Items 14 to 19, wherein the third plurality of modified immune cells comprises one or more exogenous nucleic acids encoding the one or more exogenous antagonists of the one or more inflammatory proteins and / or the one or more immunosuppressive cytokines.

[0202] Item 21. A population of immune cells according to Item 20, wherein at least one of the exogenous nucleic acids is incorporated into the genome of the third plurality of modified immune cells.

[0203] Item 22. A population of immune cells according to any one of Items 14 to 21, wherein the third plurality of modified immune cells are of the same type as the first plurality of modified immune cells, the second plurality of modified immune cells, or both.

[0204] Item 23. A population of immune cells according to Item 22, wherein the third plurality of modified immune cells overlaps with the first plurality of modified immune cells, the second plurality of modified immune cells, or both.

[0205] Item 24. An immune cell population according to any one of Items 1 to 23, wherein the immune cells are T cells, NK cells, dendritic cells, macrophages, B cells, neutrophils, eosinophils, basophils, mast cells, myeloid-derived suppressor cells, mesenchymal stem cells, their precursors or a combination thereof.

[0206] Item 25. An immune cell population according to any one of Items 1 to 24, wherein the immune cells express a chimeric antigen receptor (CAR) and / or an exogenous T cell receptor, wherein the CAR comprises an extracellular ligand binding domain, a transmembrane domain, and one or more intracellular signaling domains, and wherein optionally, the endogenous TCR of the immune cells is knocked out.

[0207] Item 26. An immune cell population according to Item 25, wherein the extracellular ligand binding domain comprises a single-chain antibody fragment specific for a cell surface protein, an extracellular domain of a cytokine receptor, or an extracellular domain of a co-stimulatory receptor.

[0208] Item 27. An immune cell population according to Item 26, wherein the one or more intracellular signaling domains include (i) a signaling domain of CD3ζ and / or (ii) one or more signaling domains from one or more co-stimulatory proteins or cytokine receptors.

[0209] Item 28. An immune cell population according to Item 27, wherein the co-stimulatory protein or cytokine receptor is selected from the group consisting of: CD28, 4-1BB, 2B4, KIR, CD27, OX40, ICOS, MYD88, IL2 receptor and SynNotch.

[0210] Item 29. An immune cell population comprising:

[0211] (i) a first plurality of modified immune cells that produce reduced levels of one or more inflammatory proteins compared to wild-type immune cells of the same type under the same conditions; and

[0212] (ii) a second plurality of modified immune cells expressing one or more antagonists of the one or more inflammatory proteins and / or expressing one or more immunosuppressive cytokines.

[0213] Item 30. An immune cell population according to Item 29, wherein the inflammatory proteins include one or more inflammatory cytokines or soluble receptors thereof, one or more inflammatory growth factors, one or more cytotoxic molecules or a combination thereof.

[0214] Item 31. An immune cell population according to Item 30, wherein the one or more inflammatory cytokines or their soluble receptors are selected from the group consisting of IL2, IL1α, IL1β, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, sIL-1RI, sIL-2Rα, sIL6R, IFNα, IFNβ, IFNγ, MIPα, MIPβ, CSF1, LIF, G-CSF, GM-CSF, CXCL10, CCL5, eotaxin, TNF, MCP1, MIG, RAGE, CRP, angiopoietin-2 and VWF.

[0215] Item 32. An immune cell population according to any one of Items 30 to 31, wherein the one or more inflammatory growth factors are selected from the group consisting of: TGFα, VEGF, EGF, HGF and FGF.

[0216] Item 33. An immune cell population according to any one of Items 30 to 32, wherein the one or more cytotoxic molecules are selected from the group consisting of perforin, granzyme and ferritin.

[0217] Item 34. An immune cell population according to any one of Items 29 to 33, wherein the one or more immunosuppressive cytokines are selected from the group consisting of: TGFβ, IL-4, IL-10, IL-13, IL-33, IL-35 and IL-37.

[0218] Item 35. A population of immune cells according to any one of Items 29 to 34, wherein the first plurality of modified immune cells and the second plurality of immune cells are of the same type.

[0219] Item 36. A population of immune cells according to Item 35, wherein the first plurality of modified immune cells overlaps with the second plurality of immune cells.

[0220] Item 37. A population of immune cells according to any one of Items 29 to 36, wherein the first plurality of modified immune cells, the second plurality of modified immune cells, or both express a chimeric antigen receptor (CAR) and / or an exogenous T cell receptor, wherein the CAR comprises an extracellular ligand binding domain, a transmembrane domain, and one or more intracellular signaling domains, and wherein optionally, the endogenous TCR of the immune cells is knocked out.

[0221] Item 38. An immune cell population according to Item 37, wherein the extracellular ligand binding domain comprises a single-chain antibody fragment specific for a cell surface protein, an extracellular domain of a cytokine receptor, or an extracellular domain of a co-stimulatory receptor.

[0222] Item 39. An immune cell population according to Item 37 or Item 38, wherein the one or more intracellular signaling domains include (i) a signaling domain of CD3ζ and / or (ii) one or more signaling domains from one or more co-stimulatory proteins or cytokine receptors.

[0223] Item 40. An immune cell population according to any one of Items 37 to 39, wherein the co-stimulatory protein or cytokine receptor is selected from the group consisting of: CD28, 4-1BB, 2B4, KIR, CD27, OX40, ICOS, MYD88, IL2 receptor and SynNotch.

[0224] Item 41. An immune cell population according to any one of Items 29 to 40, wherein the first plurality of modified immune cells, the second plurality of modified immune cells, or both are T cells, NK cells, dendritic cells, macrophages, B cells, neutrophils, eosinophils, basophils, mast cells, myeloid-derived suppressor cells, mesenchymal stem cells, or precursors thereof.

[0225] Item 42. A population of immune cells according to any one of Items 29 to 41, wherein in each cell of the first plurality of modified immune cells, at least one endogenous allele of the one or more inflammatory proteins is knocked out.

[0226] Item 43. An immune cell population according to any one of Items 29 to 42, wherein the one or more antagonists of the one or more inflammatory proteins comprise soluble receptors for the inflammatory proteins and / or antibodies specific to the inflammatory proteins.

[0227] Item 44. A population of immune cells according to any one of Items 29 to 43, wherein the second plurality of modified immune cells comprises one or more exogenous nucleic acids encoding one or more antagonists of one or more inflammatory proteins and / or one or more immunosuppressive cytokines.

[0228] Item 45. A population of immune cells according to Item 44, wherein at least one of the exogenous nucleic acids is incorporated into the genome of the second plurality of modified immune cells.

[0229] Item 46. A method of producing a modified immune cell population with reduced inflammatory properties, the method comprising:

[0230] (i) providing an immune cell population; and

[0231] (ii) modifying the immune cell to thereby reduce IL-2 production.

[0232] Item 47. A method according to Item 46, further comprising (iii) modifying the immune cells to reduce the production of one or more inflammatory proteins, and optionally (iv) introducing one or more nucleic acids encoding one or more antagonists of one or more inflammatory proteins and / or one or more immunosuppressive cytokines into the immune cells, wherein the one or more nucleic acids are operably linked to one or more promoters for expressing the one or more antagonists and / or the one or more immunosuppressive cytokines in the immune cells.

[0233] Item 48. A method of producing a modified immune cell population having reduced inflammatory properties, the method comprising:

[0234] (i) providing immune cell populations;

[0235] (ii) modifying the immune cell to reduce production of one or more inflammatory proteins; and

[0236] (iii) incorporating into the immune cells one or more nucleic acids encoding one or more antagonists of one or more inflammatory proteins and / or one or more immunosuppressive cytokines, wherein the one or more nucleic acids are operably linked to one or more promoters for expressing the one or more antagonists and / or the one or more immunosuppressive cytokines in the immune cells.

[0237] Item 49. A method according to any one of Items 46 to 48, wherein the modification step comprises performing gene editing at an endogenous allele of IL-2 of the immune cell or an endogenous allele of the one or more inflammatory proteins.

[0238] Item 50. A method according to Item 49, wherein the gene editing comprises clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nuclease (TALEN) or zinc finger nuclease (ZFN), optionally an ARC homing endonuclease, a large-range nuclease, a mega-TALS or a combination thereof.

[0239] Item 51. A method according to any one of Items 46 to 50, wherein the IL-2 production is reduced by about 30-95%.

[0240] Item 52. A method according to Item 51, wherein the IL-2 production is reduced by about 50%.

[0241] Item 53. A method according to any one of Items 48 to 50, wherein the one or more inflammatory proteins include one or more inflammatory cytokines or soluble receptors thereof, one or more inflammatory growth factors, one or more cytotoxic molecules, or a combination thereof.

[0242] Item 54. A method according to Item 53, wherein the one or more inflammatory cytokines or their soluble receptors are selected from the group consisting of IL1α, IL1β, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, sIL-1RI, sIL-2Rα, sIL6R, IFNα, IFNβ, IFNγ, MIPα, MIPβ, CSF1, LIF, G-CSF, GM-CSF, CXCL10, CCL5, eotaxin, TNF, MCP1, MIG, RAGE, CRP, angiopoietin-2 and VWF.

[0243] Item 55. A method according to Item 53 or 54, wherein the one or more inflammatory growth factors are selected from the group consisting of: TGFα, VEGF, EGF, HGF and FGF.

[0244] Item 56. A method according to any one of Items 53 to 55, wherein the one or more cytotoxic molecules are selected from the group consisting of perforin, granzymes and ferritin.

[0245] Item 57. A method according to any one of Items 53 to 56, wherein the one or more antagonists of the one or more inflammatory proteins comprise a soluble receptor for the inflammatory protein and / or an antibody specific for the inflammatory protein.

[0246] Item 58. A method according to any one of Items 46 to 57, wherein the immune cells are T cells, NK cells, dendritic cells, macrophages, B cells, neutrophils, eosinophils, basophils, mast cells, myeloid-derived suppressor cells, mesenchymal stem cells, their precursors or a combination thereof.

[0247] Item 59. A method according to any one of Items 46 to 58, wherein the immune cell expresses a chimeric antigen receptor (CAR) and / or an exogenous T cell receptor, wherein the CAR comprises an extracellular ligand binding domain, a transmembrane domain, and one or more intracellular signaling domains, and wherein optionally, the endogenous TCR of the immune cell is knocked out.

[0248] Item 60. A method according to Item 59, wherein the extracellular ligand binding domain comprises a single-chain antibody fragment specific for a cell surface protein, an extracellular domain of a cytokine receptor, or an extracellular domain of a co-stimulatory receptor.

[0249] Item 61. A method according to any one of Items 59 to 60, wherein the one or more intracellular signaling domains include (i) a signaling domain of CD3ζ and / or (ii) one or more signaling domains from one or more co-stimulatory proteins or cytokine receptors.

[0250] Item 62. A method according to Item 61, wherein the co-stimulatory protein or cytokine receptor is selected from the group consisting of: CD28, 4-1BB, 2B4, KIR, CD27, OX40, ICOS, MYD88, IL2 receptor and SynNotch.

[0251] Item 63. An immune cell population prepared by the method according to any one of Items 46 to 62.

[0252] Item 64. A method of cell therapy, comprising administering to a subject in need thereof a population of immune cells according to any one of Items 1 to 45 and 63.

[0253] Item 65. A method according to Item 64, wherein the subject is a human patient suffering from cancer, an infectious disease or an immune disorder.

[0254] Without further elaboration, it is believed that those skilled in the art will be able to make the best use of the present invention based on the above description. Therefore, the following specific examples should be interpreted as being merely illustrative and not limiting the remainder of the present disclosure in any way. For the purposes or themes mentioned herein, all publications cited herein are incorporated by reference.

[0255] Example 1. Expression of anti-IL6R antibodies in T cells carrying a knock-in gene expression cassette successfully inhibited IL6 signaling.

[0256] The third generation self-deactivation (SIN) lentiviral vector encoding the single-chain variable fragment (scFv) antibody of tocilizumab derived from targeting IL6R was produced by Lipofectamine 2000 transfection. The obtained lentiviral vector was then applied to the rotational transduction (2500rpm for 90 minutes at room temperature) of Jurkat E6-1 cells (acute T cell leukemia). Amplification and cultivation of transduced cells to collect the supernatant containing secreted scFv were used to assess the use of HEK-Blue IL-6 cells (Invivogen) to block human IL6 pathway signaling. HEK-Blue IL-6 reporter cells were used because they can produce secreted embryonic alkaline phosphatase (SEAP) when human IL6 is stimulated. The light absorbance of the converted substrate Quanti-Blue (Invivogen) was measured by a spectrophotometer at a wavelength of 650nm to quantify SEAP output.

[0257] like Figure 2 As shown, different dilutions of supernatant from Jurkat T cells with a knock-in gene cassette encoding the tocilizumab scFv were tested. Supernatants from these cells inhibited IL6 activity by binding to IL6R expressed on reporter cells. For example, even at a 1 / 8 dilution of the supernatant, there was a reduction in IL6 pathway signaling ( Figure 2 ). These results indicate that the tocilizumab scFv encoded by the knock-in gene cassette is functional and effectively blocks IL6-IL6R signaling.

[0258] Example 2. Knockout of endogenous cytokine genes.

[0259] GM-CSF, IL2 and TNF knockouts were produced using Thermo Scientific GeneArt Crispr / Cas9 editing kits. Each sgRNA includes a spacer sequence for targeting the gene site to be edited and a scaffold RNA with a sequence of SEQ ID NO:31. The spacer sequence was designed by targeting a 19-20nt sequence (alternatively 17-20nt) before the PAM sequence in exon I of the human GM-CSF, IL2 and TNF gene loci. sgRNA is synthesized by in vitro transcription by GeneArt sgRNA synthesis kits of Thermo Scientific. TrueCut Cas9 protein v2 of Thermo Scientific is combined with sgRNA to form a ribonucleoprotein (RNP) complex and introduced into Jurkat cells by a BTX ECM830 electroporator. About 7 days after electroporation, the successful destruction of gene expression was verified by ELISA kits (BD Biosciences and R&D systems). For ELISA, engineered Jurkat cells were activated overnight by PMA / mycin, and supernatants were collected for analysis of cytokine production. Figure 3-5 Shown are the percent cytokine reduction for each cytokine tested relative to wild-type Jurkat cells with normal secretion of the cytokine.

[0260] Eight sgRNAs targeting human GM-CSF were tested in Jurkat cells. Most of the sgRNAs tested resulted in some decrease in GM-CSF levels ( Figure 3 For example, sgRNA candidates 3-6 showed a reduction of GM-CSF levels by more than 40% ( Figure 3 ). The sequences in human GM-CSF targeted by sgRNA1-8 are shown by double-stranded sequences respectively:

[0261] 5'-GCTGCAGAGCCTGCTGCTCT-3'(SEQ ID NO:1)

[0262] 3'-CGACGTCTCGGACGACGAGA-5'(SEQ ID NO:74)

[0263] 5'-GGAGCATGTGAATGCCATCC-3'(SEQ ID NO:2)

[0264] 3'-CCTCGTACACTTACGGTAGG-5'(SEQ ID NO:75)

[0265] 5'-GCATGTGAATGCCATCCAGG-3'(SEQ ID NO:3)

[0266] 3'-CGTACACTTACGGTAGGTCC-5'(SEQ ID NO:76)

[0267] 5'-GAGACGCCGGGCCTCCTGGA-3'(SEQ ID NO:4)

[0268] 3'-CTCTGCGGCCCGGAGGACCT-5'(SEQ ID NO:77)

[0269] 5'-GATGGCATTCACATGCTCCC-3'(SEQ ID NO:5)

[0270] 3'-CTACCGTAAGTGTACGAGGG-5'(SEQ ID NO:78)

[0271] 5'-GCTCCCAGGGCTGCGTGCTG-3'(SEQ ID NO:6)

[0272] 3'-CGAGGGTCCCGACGCACGAC-5'(SEQ ID NO:79)

[0273] 5'-GCGTGCTGGGGCTGGGCGAG-3'(SEQ ID NO:7)

[0274] 3'-CGCACGACCCCGACCCGCTC-5'(SEQ ID NO:80)

[0275] 5'-GCTGGGGCTGGGCGAGCGGG-3'(SEQ ID NO:8)

[0276] 3'-CGACCCCGACCCGCTCGCCC-5'(SEQ ID NO:81)

[0277] An exemplary protospacer sequence in a gRNA (e.g., sgRNA) for targeting the above-mentioned human GM-CSF site and an exemplary sgRNA sequence containing the same are provided below (respectively): sgRNA1 spacer:

[0278] GCUGCAGAGCCUGCUGCUCU(SEQ ID NO:32)

[0279] sgRNA1 full sequence:

[0280] GCUGCAGAGCCUGCUGCUCU GUUUUAGAGCUAGAAAUAGCAAGUUAA

[0281] AAUAAGGCUAGUCCGUUAUCAACUUGAAAAGUGGCACCGAGUCCGGUG CUUUU(SEQ ID NO:33)

[0282] sgRNA2 spacer:

[0283] GGAGCAUGUGAAUGCCAUCC(SEQ ID NO:34)

[0284] sgRNA2 full sequence:

[0285] GGAGCAUGUGAAUGCCAUCC GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 35) sgRNA3 spacer:

[0286] GCAUGUGAAUGCCAUCCAGG(SEQ ID NO:36)

[0287] sgRNA3 full sequence:

[0288] GCAUGUGAAUGCCAUCCAGG GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 37) sgRNA4 spacer:

[0289] GAGACGCCGGGCCUCCUGGA(SEQ ID NO:38)

[0290] sgRNA4 full sequence:

[0291] GAGACGCCGGGCCUCCUGGA GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 39) sgRNA5 spacer:

[0292] GAUGGCAUUCACAUGCUCCC(SEQ ID NO:40)

[0293] sgRNA5 full sequence:

[0294] GAUGGCAUUCACAUGCUCCC GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 41) sgRNA6 spacer:

[0295] GCUCCCAGGGCUGCGUGCUG(SEQ ID NO:42)

[0296] sgRNA6 full sequence:

[0297] GCUCCCAGGGCUGCGUGCUG GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 43) sgRNA7 spacer:

[0298] GCGUGCUGGGGCUGGGCGAG(SEQ ID NO:44)

[0299] sgRNA7 full sequence:

[0300] GCGUGCUGGGGCUGGGCGAG GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 45) sgRNA8 spacer:

[0301] GCUGGGGCUGGGCGAGCGGG(SEQ ID NO:46)

[0302] sgRNA8 full sequence:

[0303] GCUGGGGCUGGGCGAGCGGG GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAGUGGCACCGAGUCCGGUGCUUUU(SEQ ID NO:47)

[0304] Similarly, five sgRNAs targeting human IL2 were also tested in Jurkat cells. Figure 4 As shown, sgRNA candidates 1, 3, and 5 showed more than 20% reduction in IL2 levels, with sgRNA candidates 1 and 5 showing more than 40% reduction in the cytokine.

[0305] The sequences in human IL2 targeted by sgRNA1-5 are shown by double-stranded sequences, respectively:

[0306] 5'-GACTTAGTGCAATGCAAGAC-3'(SEQ ID NO:9)

[0307] 3'-CTGAATCACGTTACGTTCTG-5'(SEQ ID NO:82)

[0308] 5'-GATTTACAGATGATTTTGAA-3'(SEQ ID NO:10)

[0309] 3'-CTAAATGTCTACTAAAACTT-5'(SEQ ID NO:83)

[0310] 5'-AAGAAAACACAGCTACAAC-3'(SEQ ID NO:11)

[0311] 3'-TTCTTTTGTGTCGATGTTG-5'(SEQ ID NO:84)

[0312] 5'-CAACTGGAGCATTTACTGC-3'(SEQ ID NO:12)

[0313] 3'-GTTGACCTCGTAAATGACG-5'(SEQ ID NO:85)

[0314] 5'-TCTTTGTAGAACTTGAAGT-3'(SEQ ID NO:13)

[0315] 3'-AGAAACATCTTGAACTTCA-5'(SEQ ID NO:86)

[0316] An exemplary protospacer sequence in a gRNA (e.g., sgRNA) for targeting the above-mentioned human IL2 site and an exemplary sgRNA sequence containing the same are provided below (respectively): sgRNA1 spacer:

[0317] GACUUAAGUGCAAUGCAAGAC(SEQ ID NO:48)

[0318] sgRNA1 full sequence:

[0319] GACUUAGUGCAAUGCAAGAC GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 49) sgRNA2 spacer:

[0320] GAUUUACAGAUGAUUUUGAA(SEQ ID NO:50)

[0321] sgRNA2 full sequence:

[0322] GAUUUACAGAUGAUUUUGAA GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 51) sgRNA3 spacer:

[0323] AAGAAAACACAGCUACAAC(SEQ ID NO:52)

[0324] sgRNA3 full sequence

[0325] AAGAAAACACAGCUACAAC GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 53) sgRNA4 spacer:

[0326] CAACUGGAGCAUUUACUGC(SEQ ID NO:54)

[0327] sgRNA4 full sequence:

[0328] CAACUGGAGCAUUUACUGCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 55) sgRNA5 spacer:

[0329] UCUUUGUAGAACUUGAAGU(SEQ ID NO:56)

[0330] sgRNA5 full sequence:

[0331] UCUUUGUAGAACUUGAAGU GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUU(SEQ ID NO:57)

[0332] To reduce TNF production in Jurkat cells, eight sgRNAs targeting human TNF were characterized. Figure 5 As shown, all eight sgRNAs reduced TNF production. For example, sgRNA candidates 1-6 and 8 reduced TNF cytokine production by more than 70%.

[0333] The sequences in human TNF targeted by sgRNAs 1-8 are shown by double-stranded sequences, respectively:

[0334] 5'-GAGCACTGAAAGCATGATCC-3'(SEQ ID NO:14)

[0335] 3'-CTCGTGACTTTCGTACTAGG-5'(SEQ ID NO:87)

[0336] 5'-GGACGTGGAGCTGGCCGAGG-3'(SEQ ID NO:15)

[0337] 3'-CCTGCACCTCGACCGGCTCC-5'(SEQ ID NO:88)

[0338] 5'-GAGGCGCTCCCCAAGAAGAC-3'(SEQ ID NO:16)

[0339] 3'-CTCCGCGAGGGGTTCTTCTG-5'(SEQ ID NO:89)

[0340] 5'-GGGGGGCCCCAGGGCTCCAGG-3'(SEQ ID NO:17)

[0341] 3'-CCCCCGGGGTCCCGAGGTCC-5'(SEQ ID NO:90)

[0342] 5'-GCTGAGGAACAAGCACCGCC-3'(SEQ ID NO:18)

[0343] 3'-CGACTCCTTGTTCGTGGCGG-5'(SEQ ID NO:91)

[0344] 5'-GGCGCCTGCCACGATCAGGA-3'(SEQ ID NO:19)

[0345] 3'-CCGCGGACGGTGCTAGTCCT-5'(SEQ ID NO:92)

[0346] 5'-GTGCAGCAGGCAGAAGAGCG-3'(SEQ ID NO:20)

[0347] 3'-CACGTCGTCCGTCTTCTCGC-5'(SEQ ID NO:93)

[0348] 5'-GGAGTGATCGGCCCCCAGA-3'(SEQ ID NO:21)

[0349] 3'-CCTCACTAGCCGGGGGTCT-5'(SEQ ID NO:94)

[0350] An exemplary protospacer sequence in a gRNA (e.g., sgRNA) for targeting the above-mentioned human TNF site and an exemplary sgRNA sequence containing the sequence are provided below (respectively): sgRNA1 spacer:

[0351] GAGCACUGAAAGCAUGAUCC(SEQ ID NO:58)

[0352] sgRNA1 full sequence:

[0353] GAGCACUGAAAGCAUGAUCC GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 59) sgRNA2 spacer:

[0354] GGACGUGGAGCUGGCCGAGG(SEQ ID NO:60)

[0355] sgRNA2 full sequence:

[0356] GGACGUGGAGCUGGCCGAGG GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 61) sgRNA 3 spacer:

[0357] GAGGCGCUCCCCAAGAAGAC(SEQ ID NO:62)

[0358] sgRNA3 full sequence:

[0359] GAGGCGCUCCCCAAGAAGAC GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 63) sgRNA4 spacer:

[0360] GGGGGCCCCAGGGCUCCAGG(SEQ ID NO:64)

[0361] sgRNA 4 full sequence:

[0362] GGGGGCCCCAGGGCUCCAGG GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 65) sgRNA5 spacer:

[0363] GCUGAGGAACAAGCACCGCC(SEQ ID NO:66)

[0364] sgRNA 5 full sequence:

[0365] GCUGAGGAACAAGCACCGCC GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 67) sgRNA6 spacer:

[0366] GGCGCCUGCCACGAUCAGGA(SEQ ID NO:68)

[0367] sgRNA6 full sequence:

[0368] GGCGCCUGCCACGAUCAGGA GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 69) sgRNA7 spacer:

[0369] GUGCAGCAGGCAGAAGAGCG(SEQ ID NO:70)

[0370] sgRNA7 full sequence:

[0371] GUGCAGCAGGCAGAAGAGCG GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 71) sgRNA8 spacer:

[0372] GGAGUGAUCGGCCCCCAGA(SEQ ID NO:72)

[0373] sgRNA8 full sequence:

[0374] GGAGUGAUCGGCCCCCAGA GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUU(SEQ ID NO:73)

[0375] Example 3. GM-CSF knockout and IL6 blocker / IL-1 blocker-secreting anti-CD19 CART cells exert potent cytotoxicity against Nalm6 leukemia cells in vivo

[0376] Human T cells were transduced with a lentiviral vector encoding (i) anti-CD19 CAR and (ii) anti-IL6 scFv antibodies, and (iii) IL1RA. The CD8 leader sequence was located before the anti-IL6 scFv. The nucleotide sequence encoding the T2A peptide was located between the coding sequences of (i) and (ii), and the nucleotide sequence encoding the P2A peptide was located between the coding sequences of (ii) and (iii). There was a human growth hormone signal sequence between P2A and (iii).

[0377] The anti-CD19 CAR contains the CD8 leader sequence, anti-CD19 scFv fragment, CD8 hinge domain, CD8 transmembrane domain, 4-1BB co-stimulatory domain and CD3ζ domain from N-terminus to C-terminus.

[0378] Exemplary amino acid sequences of the domains of the anti-IL6 scFv, IL-1RA, and anti-CD19 CAR used in this particular example are provided below:

[0379] Anti-IL-6 scFv antibody (SEQ ID NO: 22):

[0380] EIVLTQSPATLSLSPGERATLSCSASISVSYMYWYQQKPGQAPRLLIYDMSNLASGI

[0381] PARFSGSGSGTDFTLTISSLEPEDFAVYYCMQWSGYPYTFGGGTKVEIKGGGGSGGG

[0382] GSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSPFAMSWVRQAPGKGLEWVAK

[0383] ISPGGSWTYYSDTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLWGYYALD

[0384] IWGQGTTVTVSS

[0385] Human growth hormone leader sequence (SEQ ID NO: 23):

[0386] MATGSRTSLLLAFGLLCLPWLQEGSA IL-1RA (SEQ ID NO:24):

[0387] RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALF

[0388] LGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESA

[0389] ACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE

[0390] CD8 leader sequence (SEQ ID NO:25):

[0391] MALPVTALLLPLALLLHAARP

[0392] Anti-CD19 scFv (SEQ ID NO: 26):

[0393] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSG

[0394] VPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGG

[0395] GGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLG

[0396] VIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYA

[0397] MDYWGQGTSVTVSS

[0398] CD8 hinge domain (SEQ ID NO: 27):

[0399] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD CD8 transmembrane domain (SEQ ID NO: 28):

[0400] IYIWAPLAGTCGVLLLSLVITLYC

[0401] 4-1BB co-stimulatory domain (SEQ ID NO: 29):

[0402] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD3z (SEQ ID NO:30):

[0403] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG

[0404] LYNELQKDKMAEAAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0405] Six to eight-week-old NGS mice (Jackson Labs) were injected intravenously with 1×10 6Six days later, mice were injected with 2×10 GFP+Nalm6 leukemia cells expressing anti-CD19 chimeric antigen receptor (CAR), anti-IL-6 scFv antibody of SEQ ID NO: 22, and IL-1RA of SEQ ID NO: 24. 6 T cells (anti-CD19 / IL6 / IL1 TCR-CART cells; 1 in Figure 6, n=5) or T cells expressing anti-CD19 CAR, IL-6 blocker and IL-1RA and knocking out the endogenous GM-CSF gene (anti-CD19 / IL6 / IL1 TCR- / GM-CSF-CART cells, 2 in Figure 6, n=6). Mice that did not receive CART cells were used as controls (CTRL in Figure 6, n=4).

[0406] The body weight, survival rate, and the number of GFP+Nalm6 leukemic cells and CD45+CD3- T cells in the blood of the mice were monitored by Trucount beads (BD Biosciences). Fig. 6A As shown, mice implanted with Nalm6 leukemia cells and subsequently treated with anti-CD19 / IL6 / IL1RA TCR-CART cells (Group 1) or anti-CD19 / IL6 / IL1RA TCR- / GM-CSF-CART cells (Group 2) were able to maintain their body weight 44 days after injection, while control mice lost weight rapidly after 10 days. Three mice died of leukemia, and one mouse was sacrificed due to obvious disease in the control group. Similarly, Figure 6B The survival curve shown indicates that 100% of the mice in Groups 1 and 2 survived, whereas no mice in the control group survived after day 20.

[0407] To evaluate the cytotoxic effect of CART cells on Nalm6 leukemia cells, the remaining GFP+Nalm6 cells in mice were measured. Figure 6C As shown, throughout the experiment, Nalm6 leukemia cells proliferated in the blood of control mice. In contrast, few or no Nalm6 leukemia cells were detected in mice treated with CART cells (Groups 1 and 2). These results indicate that both anti-CD19 / IL6 / IL1RA TCR-CART cells and anti-CD19 / IL6 / IL1RA TCR- / GM-CSF-CART cells are able to kill Nalm6 leukemia cells in vivo.

[0408] In addition, to evaluate the nature of the injected human T cells, the amount of CD45+CD3- T cells in the blood of the treated mice was measured over time. In both Group 1 and Group 2, the level of human T cells present in the blood decreased over time, indicating that GM-CSF KO by gene editing did not transform T cells into leukemia-like cells. Fig.6D .

[0409] Example 4: Effect of IL-2 knockout on T cell expansion

[0410] T cells showing reduced IL-2 production after gene editing with sgRNAs 1, 3, and 5 targeting IL-2 were restimulated with anti-CD3 / CD28 beads. The sequences of sgRNAs 1, 3, and 5 are provided in Example 2 above. Subsequent T cell expansion was analyzed. T cells with electroporated Cas9 alone were included as a control. No exogenous IL-2 was added during restimulation.

[0411] The results obtained from this study (such as Figure 7 (shown) indicates that compared with the control group, T cells with IL-2 knockout are still able to expand, but at a slightly lower rate. This result indicates that T cells with IL-2 knockout are still able to proliferate in vitro, and T cells proliferate at a lower rate indicating that IL-2 knockout will effectively inhibit the toxicity induced by T cell hyperproliferation.

[0412] Other embodiments

[0413] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature used for the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is merely an example of a general series of equivalent or similar features.

[0414] Through the above description, those skilled in the art can easily determine the essential characteristics of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention to make it suitable for various uses and conditions. Therefore, other embodiments are also within the scope of the claims.

Claims

1. An immune cell population comprising genetically engineered immune cells expressing immunosuppressive cytokines encoded by exogenous nucleic acids.

2. The immune cell population according to claim 1, wherein the immunosuppressive cytokine is selected from the group consisting of TGFβ, IL-4, IL-10, IL-13, IL-33, IL-35 and IL-37.

3. The immune cell population according to claim 1, wherein the genetically engineered immune cells further produce reduced levels of one or more inflammatory proteins compared to wild-type immune cells of the same type under the same conditions.

4. The immune cell population according to claim 3, wherein the one or more inflammatory proteins are selected from IL2,IL1α,IL1β,IL-5,IL-6,IL-7,IL-8,IL-9,IL-12,IL-15,IL-17,IL-18, IL-21, IL-23, sIL-1RI, sIL-2Rα, sIL6R, IFNα, IFNβ, IFNγ, MIPα, MIPβ, CSF1, LIF, G-CSF, GM-CSF, CXCL10, CCL5, eotaxin, TNF, MCP1, MIG, RAGE, CRP, angiopoietin-2 and VWF.

5. The immune cell population of claim 3, wherein the genetically engineered immune cells express one or more antibodies targeting the one or more inflammatory proteins, thereby reducing the level of inflammatory proteins in the immune cells. The immune cell population according to claim 5 , wherein the one or more antibodies comprise anti-IL6R antibody or anti-IL6 antibody.

7. The immune cell population of claim 3, wherein at least one endogenous allele of the one or more inflammatory proteins is knocked out in the immune cells, thereby reducing the level of the encoded inflammatory protein in the immune cells. 8 . The immune cell population according to claim 7 , wherein the immune cells have a knocked-out endogenous GM-CSF gene, a knocked-out endogenous IL-2 gene or a knocked-out TNFα gene.

9. The immune cell population of claim 1, wherein the immune cells further express a chimeric antigen receptor (CAR) or an exogenous T cell receptor.

10. The immune cell population according to claim 1, wherein the immune cells include T cells, NK cells, dendritic cells, macrophages, B cells, neutrophils, eosinophils, basophils, mast cells, myeloid-derived suppressor cells, mesenchymal stem cells or precursors thereof.

11. Use of the immune cell population according to any one of claims 1 to 10 in the preparation of a medicament for adoptive cell therapy in a subject.

12. The use according to claim 11, wherein the subject is a human patient suffering from cancer, infectious disease or immune disorder.

13. A method for producing the immune cell population according to any one of claims 1 to 10, the method comprising: (i) providing a plurality of immune cells, (ii) introducing an exogenous nucleic acid encoding the immunosuppressive cytokine into the immune cell; and (iii) modifying the immune cell to reduce production of the one or more inflammatory proteins; Thereby, the immune cell population described in any one of claims 1 to 9 is produced.

14. The method of claim 13, wherein step (iii) is performed by (a) knocking out one or more endogenous genes encoding the one or more inflammatory proteins, or (b) introducing a nucleic acid into the immune cell, the nucleic acid encoding one or more antagonists of the one or more inflammatory proteins.

15. The method of claim 14, wherein the one or more antagonists are one or more antibodies.

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