Mutant IL-15 Compositions and Methods Thereof

Abstract

The present application provides modified immune cells that express mutant IL-15 polypeptides. In some embodiments, the modified immune cells further comprise an engineered receptor, such as a chimeric antigen receptor (CAR). The present application also provides methods and pharmaceutical compositions for cancer treatment using the modified immune cells described herein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to International Patent Applications No. PCT / CN2021 / 105481 and No. PCT / CN2021 / 105484, filed on July 9, 2021, the contents of which are incorporated by reference in their entireties herein.

[0002] Sequence description The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: P11200-PCT.220708.Sequence listing.xml, Recorded: July 8, 2022, Size: 102,176 bytes).

[0003] Field The present application relates to modified immune cells that express IL-15 polypeptides and methods of use thereof to treat diseases or conditions, such as cancer. [Background technology]

[0004] background Chimeric antigen receptor (CAR) T cells are cells that have been modified to produce engineered T cell receptors to induce immune responses. For example, CAR-T cells can be engineered to more effectively recognize cancer cells for improved cancer treatment. An alternative approach to CAR-T cell therapy is the use of natural killer (NK) cells, which are immune cells that kill target cells (e.g., tumor cells) by spontaneous cytotoxic activity that is independent of tumor antigens. Thus, CAR-NK cells may be engineered to target diverse antigens, enhance targeting to solid tumors, and achieve an overall effective antitumor response. Despite the success of CAR-NK cell therapy, these methods often suffer from a higher incidence of cytokine production (e.g., cytokine storm) that can cause damage at the site of injury. There is still a need for highly effective cell-based cancer immunotherapy.

[0005] Interleukin 15 (IL-15) is a cytokine that plays a role in the development and regulation of the immune system. In particular, IL-15 induces the proliferation, function, and development of CD8+ T cells, natural killer (NK) cells, killer T cells, B cells, intestinal intraepithelial lymphocytes (IELs), and antigen-presenting cells (APCs). Studies have shown that IL-15 is a potent activator of proinflammatory signaling in eukaryotic cells. IL-15 stimulates the production of proinflammatory cytokines and chemokines in a number of innate immune and non-immune cells, including dendritic cells (DCs), NK cells, epithelial cells, and lymph node stromal cells. IL-15 acts on cells in both lymphoid and non-lymphoid compartments (Van Belle and Grooten, Arch Immunol Ther Exp (2005) 53:115). Given its important role in the immune system, IL-15 administration has been used to enhance immune responses. Conversely, inhibitors of IL-15 activity can attenuate autoimmune and other undesirable immune responses (Waldmann, TA, 2006, Nature Rev. Immunol. 6:595-601). Engineered immune cells, such as T cells and NK cells expressing CARs, can be armed with IL-15 to provide enhanced anti-tumor activity. See, e.g., US9,629,877 and US10,428,305.

[0006] The disclosures of all publications, patents, patent applications, and published patent applications referenced herein are hereby incorporated by reference in their entirety. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] US9,629,877 [Patent Document 2] US10,428,305 [Non-patent literature]

[0008] [Non-Patent Document 1] Van Belle and Grooten, Arch Immunol Ther Exp(2005) 53:115 [Non-Patent Document 2] Waldmann, TA, 2006, Nature Rev. Immunol. 6:595-601 Summary of the Invention

[0009] overview The present application provides modified immune cells that express mutant IL-15 polypeptides and methods of their use to treat diseases or conditions, such as cancer.

[0010] One aspect of the application provides modified immune cells comprising a first heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising one or more amino acid substitutions at positions 8 and / or 62, wherein the numbering of the amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the IL-15 polypeptide comprises an amino acid residue at position 62 selected from the group consisting of glycine (G), isoleucine (I), glutamine (Q), valine (V), proline (P), leucine (L), alanine (A), serine (S), and tyrosine (Y). In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7.

[0011] In some embodiments of any one of the above modified immune cells, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the IL-15 polypeptide comprises a glutamic acid (E) amino acid residue at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:5.

[0012] One aspect of the application provides modified immune cells comprising a first heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising one or more amino acid substitutions at position 3 and / or position 25, wherein the numbering of the amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the IL-15 polypeptide comprises a tyrosine (Y) amino acid residue at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:78. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:78.

[0013] In some embodiments of any one of the above modified immune cells, the IL-15 polypeptide comprises an amino acid substitution at position 25. In some embodiments, the IL-15 polypeptide comprises a phenylalanine (F) amino acid residue at position 25. In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79.

[0014] One aspect of the application provides a modified immune cell comprising a first heterologous nucleic acid sequence encoding an IL-15 polypeptide, wherein the IL-15 polypeptide induces secretion of a proinflammatory cytokine by the modified immune cell at a level at least 50% lower than the secretion of a proinflammatory cytokine by a modified immune cell comprising a heterologous nucleic acid sequence encoding a wild-type IL-15 polypeptide. In some embodiments, the proinflammatory cytokine is IFNγ, TNFα, and / or GM-CSF. In some embodiments, the application provides a modified immune cell comprising a first heterologous nucleic acid sequence encoding an IL-15 polypeptide, wherein the first heterologous nucleic acid sequence enhances anti-tumor activity of the modified immune cell.

[0015] In some embodiments of any one of the above modified immune cells, the one or more amino acid substitutions reduce the affinity of the IL-15 polypeptide for IL-15Rβ compared to an IL-15 polypeptide that does not contain the one or more amino acid substitutions (e.g., a wild-type IL-15 polypeptide).

[0016] In some embodiments of any one of the above modified immune cells, the IL-15 polypeptide is secreted.

[0017] In some embodiments of any one of the modified immune cells above, the IL-15 polypeptide is membrane-bound. In some embodiments, the IL-15 polypeptide is membrane-bound via a glycosylphosphatidylinositol (GPI) anchor peptide sequence. In some embodiments, the GPI anchor peptide sequence is attached to a GPI linker. In some embodiments, the IL-15 polypeptide is membrane-bound via a transmembrane domain. In some embodiments, the IL-15 polypeptide is membrane-bound via a membrane anchor domain.

[0018] In some embodiments of any one of the above modified immune cells, the IL-15 polypeptide is a fusion protein comprising an IL-15 fragment fused to a second polypeptide fragment. In some embodiments, the second polypeptide fragment is selected from the group consisting of IL-15Rα, the extracellular domain of IL-15Rα, the sushi domain of IL-15Rα, the transmembrane domain of IL-15Rα, IL-15Rβ, the common gamma chain (γc), and combinations thereof. In some embodiments, the second polypeptide fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-55. In some embodiments of any one of the above modified immune cells, the IL-15 polypeptide comprises: (a) an antigen binding domain; (b) an IL-15 fragment; (c) a transmembrane domain; and (d) an intracellular domain.

[0019] In some embodiments of any one of the modified immune cells above, the modified immune cell comprises a second heterologous nucleic acid sequence encoding an engineered receptor. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR). In some embodiments, the CAR is a BCMA CAR, a CD19 CAR, or a GPC3 CAR. In some embodiments, the engineered receptor is a modified T cell receptor (TCR). In some embodiments, the engineered receptor is a T cell antigen conjugate (TAC) receptor. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter. In some embodiments, the first nucleic acid and the second nucleic acid are operably linked to separate promoters.

[0020] In some embodiments of any one of the above engineered immune cells, the engineered immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer (NK) cell, a NK cell, an iNK-T cell, a NK-T-like cell, an αβ T cell, and a γδ T cell. In some embodiments, the engineered immune cell is a NK cell. In some embodiments, the engineered immune cell is a cytotoxic T cell.

[0021] In some embodiments of any one of the above modified immune cells, the modified immune cells have reduced toxicity in vivo when administered to an individual compared to modified immune cells comprising a heterologous nucleic acid encoding a wild-type IL-15 polypeptide. In some embodiments, the modified immune cells have improved safety in vivo when administered to an individual compared to modified immune cells comprising a heterologous nucleic acid encoding a wild-type IL-15 polypeptide. In some embodiments, the modified immune cells have improved anti-tumor activity compared to modified immune cells comprising a heterologous nucleic acid encoding a wild-type IL-15 polypeptide.

[0022] One aspect of the application provides a method of making an engineered immune cell, the method comprising introducing into a precursor immune cell a first nucleic acid sequence encoding an IL-15 polypeptide comprising one or more amino acid substitutions at positions 8 and / or 62, wherein the numbering of the amino acid residue positions is according to SEQ ID NO:1.

[0023] One aspect of the application provides a method of making an engineered immune cell, the method comprising introducing into a precursor immune cell a first nucleic acid sequence encoding an IL-15 polypeptide comprising one or more amino acid substitutions at positions 3 and / or 25, wherein the numbering of the amino acid residue positions is according to SEQ ID NO:1.

[0024] In some embodiments of any one of the above methods of production, the precursor immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, NK cells, NK-T cells, iNK-T cells, NK-T-like cells, αβT cells, and γδT cells. In some embodiments, the precursor immune cells comprise an engineered receptor. In some embodiments of any one of the above methods of production, the method further comprises introducing a second nucleic acid encoding the engineered receptor into the precursor immune cells. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR), a modified T cell receptor (TCR), or a T cell antigen conjugate (TAC) receptor.

[0025] In some embodiments of any one of the above methods of making, the first nucleic acid sequence and the second nucleic acid sequence are present in the same vector.In some embodiments, the vector is a viral vector.In some embodiments, the viral vector is selected from the group consisting of adenovirus vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, herpes simplex virus vector, and derivatives thereof.

[0026] In some embodiments of any one of the above methods of making, the method further comprises isolating or enriching immune cells that comprise the first and / or second nucleic acid sequence.

[0027] Also provided is an altered immune cell produced by a method according to any one of the above production methods.

[0028] There is further provided a pharmaceutical composition comprising a modified immune cell according to any one of the above modified immune cells and a pharma- ceutically acceptable carrier.

[0029] Another aspect of the present application provides a method of treating cancer in an individual, comprising administering to the individual an effective amount of a pharmaceutical composition according to any one of the above pharmaceutical compositions. In some embodiments, the disease is cancer. In some embodiments, the individual has a low tumor burden. In some embodiments, the method does not result in a cytokine storm in the individual. In some embodiments, the individual is a human.

[0030] Another aspect of the application provides a method of reducing a cytokine storm in an individual receiving treatment with immune cells comprising an engineered receptor, the method comprising: (a) introducing into said immune cells a heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising one or more amino acid substitutions at positions 8 and / or 62, where the numbering of amino acid residue positions is according to SEQ ID NO:1, thereby providing a modified immune cell; and (b) administering to said individual an effective amount of said modified immune cell.

[0031] A further aspect of the application provides an engineered IL-15 polypeptide comprising the amino acid substitutions D8E and / or T62G, where the numbering of the amino acid residue positions is according to SEQ ID NO: 1. In some embodiments, the engineered IL-15 polypeptide comprises an amino acid sequence having at least about 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 7.

[0032] Another aspect of the application provides a method of enhancing anti-tumor activity of an immune cell comprising an engineered receptor, the method comprising: (a) introducing into said immune cell a heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising one or more amino acid substitutions at positions 3 and / or 25, thereby providing a modified immune cell, wherein the numbering of amino acid residue positions is according to SEQ ID NO:1; and (b) administering to said individual an effective amount of said modified immune cell.

[0033] A further aspect of the application provides an engineered IL-15 polypeptide comprising the amino acid substitutions V3Y and / or L25F, where the numbering of the amino acid residue positions is according to SEQ ID NO: 1. In some embodiments, the engineered IL-15 polypeptide comprises an amino acid sequence having at least about 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 78 and 79.

[0034] Compositions, methods of use, kits, and articles of manufacture comprising any one of the modified immune cells are also provided. [Brief description of the drawings]

[0035] [Figure 1A] Figure 1 shows the in vitro cytotoxic effect of NK cells expressing eight selected mutant constructs of BCMA CAR armed with secreted IL-15 (i.e., BCMA CAR-NK armed with sIL-15 m1-m8) against BCMA positive target cells, NCI-H929. Figure 2 shows the in vitro short-term (4 h) cytotoxicity of BCMA CAR-NK cells armed with mutant sIL-15 against target cells. Non-transduced NK cells (i.e., "UnNK") served as a control in this experiment. [Figure 1B] Figure 1 shows the in vitro cytotoxic effect of NK cells expressing eight selected mutant constructs of BCMA CAR armed with secreted IL-15 (i.e., BCMA CAR-NK armed with sIL-15 ml-m8) against BCMA positive target cells, NCI-H929. Figure 2 shows the in vitro cytotoxicity of BCMA CAR-NK cells armed with mutant sIL-15 against target cells (i.e., fraction of tumor cells relative to total cell number) in eight antigen stimulation experiments. Non-transduced NK cells (i.e., "UnNK") served as a control in this experiment. [Figure 1C] Figure 1 shows the in vitro cytotoxic effect of NK cells expressing eight selected mutant constructs of BCMA CAR armed with secreted IL-15 (i.e., BCMA CAR-NK armed with sIL-15 ml-m8) against BCMA-positive target cells, NCI-H929. Non-transduced NK cells (i.e., "UnNK") served as a control in this experiment. The mutant sIL-15 armed BCMA CAR-NK cells were also evaluated for fold proliferation during stimulation with tumor cells.

[0036] [Figure 2A]Figure 2 shows the in vivo antitumor efficacy of CAR-NK cells armed with wild-type secreted IL-15 (i.e., "sIL-15 wt") against BCMA-positive target cells, NCI-H929, in an NCG mouse model bearing multiple myeloma tumor xenografts (NCI-H929-luc model). Figures 2A-B show the antitumor activity (Figure 2A) and survival (Figure 2B) of mice treated with sIL-15 wt-armed NK cells, sIL-15 wt-armed CD19 CAR-NK cells, and sIL-15 wt-armed BCMA CAR-NK cells in mice. The combination of untransduced NK cells and intraperitoneal administration of hIL-15 (i.e., "UnNK i.v. + IL-15 i.v."), HBSS (- / -) vehicle control (i.e., "vehicle i.v."), and tumor-free NCG mice served as controls in this experiment. [Figure 2B] Figure 2 shows the in vivo antitumor efficacy of CAR-NK cells armed with wild-type secreted IL-15 (i.e., "sIL-15 wt") against BCMA-positive target cells, NCI-H929, in an NCG mouse model bearing multiple myeloma tumor xenografts (NCI-H929-luc model). Figures 2A-B show the antitumor activity (Figure 2A) and survival (Figure 2B) of mice treated with sIL-15 wt-armed NK cells, sIL-15 wt-armed CD19 CAR-NK cells, and sIL-15 wt-armed BCMA CAR-NK cells in mice. The combination of untransduced NK cells and intraperitoneal administration of hIL-15 (i.e., "UnNK i.v. + IL-15 i.v."), HBSS (- / -) vehicle control (i.e., "vehicle i.v."), and tumor-free NCG mice served as controls in this experiment.

[0037] [Figure 3A]Figure 1 shows the in vivo evaluation of BCMA CAR-NK cells armed with mutant secreted IL-15 against BCMA positive target cells, NCI-H929, in an NCG mouse model bearing multiple myeloma tumor xenografts (NCI-H929-luc model). A shows the antitumor efficacy of BCMA CAR-NK cells armed with mutant IL-15, and B shows the bioluminescence imaging (BLI) representation of the antitumor efficacy of BCMA CAR-NK cells armed with mutant IL-15 in mouse peripheral blood. HBSS (- / -) vehicle control (i.e., "vehicle") and tumor-free NCG mice served as controls in this experiment. [Figure 3B] Figure 1 shows the in vivo evaluation of BCMA CAR-NK cells armed with mutant secreted IL-15 against BCMA positive target cells, NCI-H929, in an NCG mouse model bearing multiple myeloma tumor xenografts (NCI-H929-luc model). A shows the antitumor efficacy of BCMA CAR-NK cells armed with mutant IL-15, and B shows the bioluminescence imaging (BLI) representation of the antitumor efficacy of BCMA CAR-NK cells armed with mutant IL-15 in mouse peripheral blood. HBSS (- / -) vehicle control (i.e., "vehicle") and tumor-free NCG mice served as controls in this experiment. [Figure 3C] Figure 1 shows the in vivo evaluation of BCMA CAR-NK cells armed with mutant secreted IL-15 against BCMA positive target cells, NCI-H929, in a multiple myeloma tumor xenograft-bearing NCG mouse model (NCI-H929-luc model). IFN-γ secretion in mouse plasma is shown. HBSS(- / -) vehicle control (i.e., "vehicle") and tumor-free NCG mice served as controls in this experiment.

[0038] [Figure 4A]Figure 1 shows the in vivo evaluation of BCMA CAR-NK cells armed with mutant secreted IL-15 and wild-type membrane-bound IL-15 against BCMA positive target cells, NCI-H929, in a high tumor burden NCG mouse model (NCI-H929-luc model). A shows anti-tumor efficacy and B shows PK of BCMA CAR in mouse peripheral blood. The combination of non-transduced NK cells and intraperitoneal administration of hIL-15 (i.e., "UnNK i.v. + IL-15 i.p.") served as a control in this experiment. [Figure 4B] Figure 1 shows the in vivo evaluation of BCMA CAR-NK cells armed with mutant secreted IL-15 and wild-type membrane-bound IL-15 against BCMA positive target cells, NCI-H929, in a high tumor burden NCG mouse model (NCI-H929-luc model). A shows anti-tumor efficacy and B shows PK of BCMA CAR in mouse peripheral blood. The combination of non-transduced NK cells and intraperitoneal administration of hIL-15 (i.e., "UnNK i.v. + IL-15 i.p.") served as a control in this experiment. [Figure 4C] Figure 1 shows the in vivo evaluation of BCMA CAR-NK cells armed with mutant secreted IL-15 and wild type membrane bound IL-15 against BCMA positive target cells, NCI-H929, in a NCG mouse model with high tumor burden (NCI-H929-luc model). C-D show BLI and survival curves of mice treated with BCMA CAR-NK cells armed with mutant IL-15. The combination of non-transduced NK cells and intraperitoneal administration of hIL-15 (i.e. "UnNK i.v. + IL-15 i.p.") served as a control in this experiment. [Figure 4D]Figure 1 shows the in vivo evaluation of BCMA CAR-NK cells armed with mutant secreted IL-15 and wild type membrane bound IL-15 against BCMA positive target cells, NCI-H929, in a NCG mouse model with high tumor burden (NCI-H929-luc model). C-D show BLI and survival curves of mice treated with BCMA CAR-NK cells armed with mutant IL-15. The combination of non-transduced NK cells and intraperitoneal administration of hIL-15 (i.e. "UnNK i.v. + IL-15 i.p.") served as a control in this experiment. [Figure 4E] Figure 1 shows the in vivo evaluation of BCMA CAR-NK cells armed with mutant secreted IL-15 and wild-type membrane-bound IL-15 against BCMA positive target cells, NCI-H929, in a NCG mouse model with high tumor burden (NCI-H929-luc model). Figures EG show pro-inflammatory cytokine levels in mouse plasma, including IFN-γ (E), TNF-α (F), and GM-CSF (G), consistent with the toxicity observations. The combination of non-transduced NK cells and intraperitoneal administration of hIL-15 (i.e., "UnNK i.v. + IL-15 i.p.") served as a control in this experiment. [Figure 4F] Figure 1 shows the in vivo evaluation of BCMA CAR-NK cells armed with mutant secreted IL-15 and wild-type membrane-bound IL-15 against BCMA positive target cells, NCI-H929, in a NCG mouse model with high tumor burden (NCI-H929-luc model). Figures EG show pro-inflammatory cytokine levels in mouse plasma, including IFN-γ (E), TNF-α (F), and GM-CSF (G), consistent with the toxicity observations. The combination of non-transduced NK cells and intraperitoneal administration of hIL-15 (i.e., "UnNK i.v. + IL-15 i.p.") served as a control in this experiment. [Figure 4G]Figure 1 shows the in vivo evaluation of BCMA CAR-NK cells armed with mutant secreted IL-15 and wild-type membrane-bound IL-15 against BCMA positive target cells, NCI-H929, in a NCG mouse model with high tumor burden (NCI-H929-luc model). Figures EG show pro-inflammatory cytokine levels in mouse plasma, including IFN-γ (E), TNF-α (F), and GM-CSF (G), consistent with the toxicity observations. The combination of non-transduced NK cells and intraperitoneal administration of hIL-15 (i.e., "UnNK i.v. + IL-15 i.p.") served as a control in this experiment.

[0039] [Figure 5A] Figure 5 shows the in vitro cytotoxic effect of NK cells expressing BCMA CAR armed with membrane-bound mutant IL-15 (i.e., membrane-bound IL-15 m6) against BCMA-positive target cells, NCI-H929. Figure 5A shows the short-term (4 h) in vitro cytotoxicity of BCMA CAR-NK cells armed with sIL-15 wt and BCMA CAR-NK cells armed with membrane-bound IL-15 m6 against target cells. Figure 5B shows the long-term in vitro cytotoxicity (i.e., fraction of tumor cells relative to total cell number) of BCMA CAR-NK cells armed with sIL-15 wt and BCMA CAR-NK cells armed with membrane-bound IL-15 m6 against target cells in four antigen stimulation experiments. Non-transduced NK cells (i.e., "UnNK") served as a control in this experiment. [Figure 5B]Figure 5 shows the in vitro cytotoxic effect of NK cells expressing BCMA CAR armed with membrane-bound mutant IL-15 (i.e., membrane-bound IL-15 m6) against BCMA-positive target cells, NCI-H929. Figure 5A shows the short-term (4 h) in vitro cytotoxicity of BCMA CAR-NK cells armed with sIL-15 wt and BCMA CAR-NK cells armed with membrane-bound IL-15 m6 against target cells. Figure 5B shows the long-term in vitro cytotoxicity (i.e., fraction of tumor cells relative to total cell number) of BCMA CAR-NK cells armed with sIL-15 wt and BCMA CAR-NK cells armed with membrane-bound IL-15 m6 against target cells in four antigen stimulation experiments. Non-transduced NK cells (i.e., "UnNK") served as a control in this experiment.

[0040] [Figure 6A] Figure 1 shows the in vitro cytotoxic effect of NK cells expressing BCMA CAR armed with membrane-bound mutant IL-15 (i.e., membrane-bound IL-15 m4) against BCMA positive target cells, NCI-H929. Figure 2 shows the in vitro short-term (4 hour) cytotoxicity of BCMA CAR-NK cells armed with sIL-15 wt and BCMA CAR-NK cells armed with membrane-bound IL-15 m4 against target cells. Non-transduced NK cells (i.e., "UnNK") served as a control in this experiment. [Figure 6B] Figure 1 shows the in vitro cytotoxic effect of NK cells expressing BCMA CAR armed with membrane-bound mutant IL-15 (i.e., membrane-bound IL-15 m4) against BCMA positive target cells, NCI-H929. Figure 2 shows the in vitro long-term cytotoxicity of BCMA CAR-NK cells armed with sIL-15 wt and BCMA CAR-NK cells armed with membrane-bound IL-15 m4 against target cells (i.e., fraction of tumor cells relative to total cell number) in seven antigen stimulation experiments. Non-transduced NK cells (i.e., "UnNK") served as a control in this experiment. [Figure 6C]Figure 1 shows the in vitro cytotoxic effect of NK cells expressing BCMA CAR armed with membrane-bound mutant IL-15 (i.e., membrane-bound IL-15 m4) against BCMA-positive target cells, NCI-H929. Non-transduced NK cells (i.e., "UnNK") served as a control in this experiment. During stimulation with tumor cells, sIL-15 wt-armed BCMA CAR-NK cells and membrane-bound IL-15 m4-armed BCMA CAR-NK cells were also evaluated for fold expansion.

[0041] [Figure 7A] Figure 7 shows the in vivo evaluation of BCMA CAR-NK cells armed with wild-type secreted IL-15 (i.e., "sIL-15 wt") and mutant membrane-bound IL-15 against BCMA-positive target cells, NCI-H929, in a NCG mouse model with high tumor burden (NCI-H929-luc model). Figure 7A shows the PK of BCMA CAR in mouse peripheral blood. Figure 7B shows the survival curves of mice treated with BCMA CAR-NK cells armed with sIL-15 wt and with membrane-bound mutant IL-15 (i.e., mb-4 IL-15 m6 and mb-5 IL-15 m6). The combination of non-transduced NK cells and intraperitoneal administration of hIL-15 (i.e., "UnNK i.v. + IL-15 i.p.") served as a control in this experiment. [Figure 7B]Figure 7 shows the in vivo evaluation of BCMA CAR-NK cells armed with wild-type secreted IL-15 (i.e., "sIL-15 wt") and mutant membrane-bound IL-15 against BCMA-positive target cells, NCI-H929, in a NCG mouse model with high tumor burden (NCI-H929-luc model). Figure 7A shows the PK of BCMA CAR in mouse peripheral blood. Figure 7B shows the survival curves of mice treated with BCMA CAR-NK cells armed with sIL-15 wt and with membrane-bound mutant IL-15 (i.e., mb-4 IL-15 m6 and mb-5 IL-15 m6). The combination of non-transduced NK cells and intraperitoneal administration of hIL-15 (i.e., "UnNK i.v. + IL-15 i.p.") served as a control in this experiment.

[0042] [Figure 8A] Figure 8A shows the in vitro cytotoxicity of sIL-15 m17-armed GPC3 CAR-NK cells against Huh7 / Luc cells in short-term (Figure 8A) and long-term (Figure 8B) cell killing assays. sIL-15 m17-armed GPC3 CAR-NK cells showed stronger anti-tumor potency against Huh7 / Luc cells compared to sIL-15 wt-armed GPC3 CAR-NK cells after R2 in short-term and long-term killing assays. "UnNK" means untransduced NK cells. R0, R2, R4, and R5 in Figure 8B mean the number of stimulations with target cells. [Figure 8B] Figure 8A shows the in vitro cytotoxicity of sIL-15 m17-armed GPC3 CAR-NK cells against Huh7 / Luc cells in short-term (Figure 8A) and long-term (Figure 8B) cell killing assays. sIL-15 m17-armed GPC3 CAR-NK cells showed stronger anti-tumor potency against Huh7 / Luc cells compared to sIL-15 wt-armed GPC3 CAR-NK cells after R2 in short-term and long-term killing assays. "UnNK" means untransduced NK cells. R0, R2, R4, and R5 in Figure 8B mean the number of stimulations with target cells.

[0043] [Figure 9A] Figure 9A shows the in vitro cytotoxicity of sIL-15 m18-armed GPC3 CAR-NK cells against Huh7 / Luc cells in short-term (Figure 9A) and long-term (Figure 9B) cell killing assays. sIL-15 m18-armed GPC3 CAR-NK cells showed stronger anti-tumor potency against Huh7 / Luc cells compared to sIL-15 wt-armed GPC3 CAR-NK cells after R2 in short-term and long-term killing assays. "UnNK" means untransduced NK cells. R0, R2, R4, and R6 in Figure 9B mean the number of stimulations with target cells. [Figure 9B] Figure 9A shows the in vitro cytotoxicity of sIL-15 m18-armed GPC3 CAR-NK cells against Huh7 / Luc cells in short-term (Figure 9A) and long-term (Figure 9B) cell killing assays. sIL-15 m18-armed GPC3 CAR-NK cells showed stronger anti-tumor potency against Huh7 / Luc cells compared to sIL-15 wt-armed GPC3 CAR-NK cells after R2 in short-term and long-term killing assays. "UnNK" means untransduced NK cells. R0, R2, R4, and R6 in Figure 9B mean the number of stimulations with target cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Detailed Description The present application provides modified immune cells expressing mutant IL-15 polypeptides, which have potent tumor lytic activity and improved safety profile compared to modified immune cells expressing wild-type IL-15 polypeptides. In some embodiments, the mutant IL-15 polypeptides have reduced (i.e., weaker) binding affinity to IL-15β. In some embodiments, the IL-15 polypeptides comprise one or more amino acid substitutions at positions 8 and / or 62, where the numbering of the amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptides comprise one or more amino acid substitutions at positions 3 and / or 25, where the numbering of the amino acid residue positions is according to SEQ ID NO:1. The IL-15 polypeptides can be membrane-bound molecules or can be secreted from the modified immune cells. In some embodiments, the modified immune cells are natural killer (NK) cells and further express a chimeric antigen receptor (CAR) that specifically recognizes a target antigen of interest.

[0045] The engineered immune cells described herein are based at least in part on the discovery that wild-type IL-15-armed CAR NK cells result in toxic excessive cytokine secretion in subjects treated with IL-15-armed CAR NK cells. Compared to wild-type IL-15, mutant IL-15 polypeptides with reduced binding affinity to IL15-Rβ can attenuate cytokine secretion by immune cells armed with such IL-15 polypeptides, but at the same time attenuate the anti-tumor activity of the immune cells. Engineered immune cells (e.g., CAR NK cells) expressing mutant IL-15 polypeptides (e.g., D8E, T62G, V3Y, and L25F mutants) described herein retain or enhance potent anti-tumor efficacy without inducing overproduction of inflammatory cytokine(s), e.g., cytokine storm, in treated subjects.

[0046] Thus, one aspect of the application provides modified immune cells (e.g., NK cells or T cells) comprising a heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising one or more amino acid substitutions at positions 8, 62, 3, and / or 25, where the numbering of the amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptide comprises a D8E substitution. In some embodiments, the IL-15 polypeptide comprises a T62G substitution. In some embodiments, the IL-15 polypeptide comprises a V3Y substitution. In some embodiments, the IL-15 polypeptide comprises a L25F substitution. In some embodiments, the IL-15 polypeptide is secreted by the modified immune cell. In some embodiments, the IL-15 polypeptide is attached to the cell membrane of the modified immune cell via a GPI linker. In some embodiments, the IL-15 polypeptide comprises a transmembrane domain or a membrane anchor domain. In some embodiments, the modified immune cells further comprise an engineered receptor, such as a chimeric antigen receptor, a modified T cell receptor, or a T cell antigen conjugate (TAC) receptor.

[0047] Also provided are compositions (e.g., pharmaceutical compositions), kits, and articles of manufacture comprising the modified immune cells, as well as methods of treating a disease or condition (e.g., cancer) using the modified immune cells described herein.

[0048] I. Definition As used herein, "treatment" or "treating" is an effort to achieve a beneficial or desired outcome, including a clinical outcome. In accordance with the principles of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: reducing one or more symptoms caused by a disease, attenuating the extent of the disease, stabilizing the disease (e.g., preventing or slowing the progression of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or slowing the progression of the disease, improving the condition of the disease, causing remission (partial or complete) of the disease, reducing the dose of one or more other drugs required to treat the disease, slowing the progression of the disease, increasing the quality of life, and / or extending survival. Reduction of the pathological consequences of a disease (e.g., cancer) is also encompassed by "treatment". The methods of the present application contemplate any one or more of these aspects of treatment.

[0049] The term "prevention" and similar terms, such as "prevented," "preventing," and the like, refer to an approach that prevents, inhibits, or reduces the likelihood of recurrence of a disease or condition, such as cancer. It also refers to delaying the recurrence of a disease or condition, or delaying the recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar terms also include reducing the intensity, effect, symptoms, and / or burden of a disease or condition prior to the recurrence of the disease or condition.

[0050] As used herein, "delaying" the onset of cancer means to postpone, prevent, delay, prevent, stabilize, and / or prolong the onset of the disease. This delay may vary in length of time depending on the history of the disease and / or the individual being treated. A method of "delaying" the onset of cancer is one that reduces the probability of disease onset within a given time frame and / or reduces the extent of disease within a given time frame when compared to the absence of the method. Such comparisons are usually based on clinical trials using a statistically significant number of individuals. Onset of cancer may be detectable using standard methods, including but not limited to computerized axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Onset may also refer to the progression of cancer, which may be initially undetectable, and includes onset, recurrence, and onset.

[0051] The term "effective amount" as used herein refers to an amount of an agent or combination of agents sufficient to treat a particular disorder, condition, or disease, e.g., to improve, alleviate, relieve, and / or delay one or more of its symptoms. With respect to cancer, an effective amount includes an amount sufficient to shrink a tumor and / or reduce the rate of tumor growth (e.g., inhibit tumor growth), or prevent or delay other undesirable cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay the onset of a disease. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount may be administered in one or more administrations. An effective amount of a drug or composition may (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) inhibit, slow, slow to some extent, and preferably stop, cancer cell invasion into peripheral organs, (iv) inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis, (v) inhibit tumor growth, (vi) prevent or delay the onset and / or recurrence of tumors, and / or (vii) relieve to some extent one or more of the symptoms associated with cancer.

[0052] As used herein, an "individual" or "subject" refers to a mammal, including, but not limited to, a human, cow, horse, cat, dog, rodent, or primate. In some embodiments, an individual is a human.

[0053] An "isolated" nucleic acid refers to a nucleic acid molecule that is separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains a nucleic acid molecule, but in which the nucleic acid molecule is present extrachromosomally or in a chromosomal location different from its natural chromosomal location.

[0054] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term encompasses vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which it is introduced. Certain vectors are capable of inducing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0055] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which heterologous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with heterologous nucleic acid. This cell includes the primary subject cell and its progeny.

[0056] With respect to the polypeptide sequences identified herein, "percent amino acid sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the polypeptide sequence being compared after the sequences are aligned, and any conservative substitutions are considered as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine the appropriate parameters for evaluating alignment, including any algorithms required to obtain maximum alignment over the entire length of the sequences being compared. However, for the purposes herein, % amino acid sequence identity values ​​are generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0057] As used herein, "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that transfers one or more antigen specificities to a cell, such as a T cell. CARs are also known as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." In some embodiments, a CAR comprises the extracellular variable domain of an antibody specific for a tumor antigen and the intracellular signaling domain of a T cell receptor or other receptor, e.g., one or more costimulatory domains. "CAR-T" refers to a T cell expressing a CAR. "CAR-NK" refers to a NK cell expressing a CAR. As used herein, "BCMA CAR" refers to a CAR that specifically recognizes BCMA, "CD19 CAR" refers to a CAR that specifically recognizes CD19, and "GPC3 CAR" refers to a CAR that specifically recognizes GPC3.

[0058] As used herein, "T cell receptor" or "TCR" refers to an endogenous or modified T cell receptor that comprises an extracellular antigen-binding domain that binds to a specific antigenic peptide bound to an MHC molecule. In some embodiments, the TCR comprises a TCR alpha and a TCR beta polypeptide chain. In some embodiments, the TCR comprises a TCR gamma and a TCR delta polypeptide chain. In some embodiments, the TCR specifically binds to a tumor antigen. "TCR-T" refers to a T cell that expresses a recombinant TCR.

[0059] As used herein, "T cell antigen conjugate receptor" or "TAC receptor" refers to an engineered receptor that contains an extracellular antigen binding domain that binds to a specific antigen and a T cell receptor (TCR) binding domain, a transmembrane domain, and an intracellular domain of a co-receptor molecule. The TAC receptor utilizes the endogenous TCR of T cells that express the TAC receptor to elicit an antigen-specific T cell response against target cells.

[0060] The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. The term antibody includes, but is not limited to, fragments capable of binding to antigen, such as Fv, single chain Fv (scFv), Fab, Fab', and (Fab')2. The term antibody includes conventional four-chain antibodies, and single domain antibodies, such as heavy chain only antibodies or fragments thereof, such as V H Includes H.

[0061] As used herein, the terms "bind", "specifically bind" or "specific for" refer to a measurable and reproducible interaction, e.g., binding between a target and an antibody that determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that binds or specifically binds to a target (which may be an epitope) is an antibody that binds to the target with higher affinity, avidity, more readily, and / or for a longer period of time than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd value) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding can include, but does not require, exclusive binding.

[0062] The term "cell" encompasses the primary subject cell and its progeny.

[0063] It will be understood that the embodiments of the present disclosure described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0064] Reference herein to "about" a value or parameter encompasses (and describes) variations that are directed to the value or parameter itself. For example, a statement that refers to "about X" includes the statement of "X."

[0065] As used herein, a reference to not being a value or parameter generally means and describes a value or parameter "other than." For example, the method is not used to treat cancer of type X means that the method is used to treat cancers of types other than X.

[0066] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0067] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0068] It will be understood that certain features of the present disclosure that are described in separate embodiments for clarity may be provided in combination in a single embodiment. Conversely, various features of the present disclosure that are described in a single embodiment for brevity may be provided separately or in any suitable subcombination. All combinations of the embodiments relating to modified immune cells and methods of treatment described herein are specifically embraced by the application and disclosed herein as if every combination was individually and expressly disclosed. In addition, all subcombinations of modified immune cells listed in the embodiments describing such variables are also specifically embraced by the application and disclosed herein as if every such subcombination of proteins was individually and expressly disclosed herein.

[0069] II. Engineered Immune Cells One aspect of the present application provides modified immune cells comprising a heterologous nucleic acid sequence encoding an IL-15 polypeptide, comprising a mutant IL-15 having one or more amino acid substitutions compared to wild-type IL-15, wherein the IL-15 polypeptide, when expressed, is capable of binding to an IL-15 receptor. In some embodiments, the mutant IL-15 has a reduced binding affinity to the IL-15 receptor compared to wild-type IL-15. In some embodiments, the mutant IL-15 has a reduced binding affinity for IL-15Rβ compared to wild-type IL-15, for example, at least about 10% reduced, 20% reduced, 30% reduced, 40% reduced, 50% reduced, 60% reduced, 70% reduced, 80% reduced, 90% reduced, 12-fold reduced, 14-fold reduced, 16-fold reduced, 20-fold reduced, 25-fold reduced, 30-fold reduced, 40-fold reduced, or more reduced binding affinity. In some embodiments, the mutant IL-15 has a reduced binding affinity for IL-15Rβ compared to wild-type IL-15, for example, about 10-fold to about 20-fold reduced binding affinity. In some embodiments, the mutant IL-15 induces secretion by the engineered immune cells of any one of a reduced level of inflammatory cytokines (e.g., IFN-γ, TNF-α, and / or GM-CSF) compared to wild-type IL-15, e.g., at least about 10%, 20% reduced, 30% reduced, 40% reduced, 50% reduced, 60% reduced, 70% reduced, 80% reduced, 90% reduced, 2-fold reduced, 5-fold reduced, 10-fold reduced, 20-fold reduced, 50-fold reduced, 100-fold reduced, 200-fold reduced, 500-fold reduced, 1000-fold reduced, or more reduced levels compared to wild-type IL-15. In some embodiments, the engineered immune cells have reduced toxicity in vivo when administered to an individual compared to engineered immune cells comprising a heterologous nucleic acid encoding a wild-type IL-15 polypeptide.In some embodiments, the modified immune cells have improved in vivo safety when administered to an individual compared to modified immune cells comprising a heterologous nucleic acid encoding a wild-type IL-15 polypeptide. In some embodiments, an individual administered the modified immune cells does not exhibit a cytokine storm. In some embodiments, modified immune cells comprising a heterologous nucleic acid sequence encoding an IL-15 polypeptide, including a mutant IL-15 having one or more amino acid substitutions compared to wild-type IL-15, have enhanced anti-tumor activity compared to modified immune cells comprising a heterologous nucleic acid encoding a wild-type IL-15 polypeptide. In some embodiments, the IL-15 polypeptide is secreted. In some embodiments, the IL-15 polypeptide is membrane-bound. In some embodiments, the modified immune cells further comprise an engineered receptor, such as a chimeric antigen receptor (CAR), an engineered TCR, or a T-cell antigen conjugate (TAC) receptor. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor-infiltrating T cells, and dendritic cell (DC)-activated T cells.

[0070] In some embodiments, modified immune cells are provided that comprise a heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising an amino acid substitution at position 62, wherein the numbering of the amino acid residue positions is according to SEQ ID NO: 1. In some embodiments, the IL-15 polypeptide comprises an amino acid residue at position 62 selected from the group consisting of glycine (G), isoleucine (I), glutamine (Q), valine (V), proline (P), leucine (L), alanine (A), serine (S), and tyrosine (Y). In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-8 and 11-17. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-8 and 11-17. In some embodiments, the modified immune cell is selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor-infiltrating T cells, and DC-activated T cells.

[0071] In some embodiments, modified immune cells are provided that comprise a heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising a G at position 62, where the numbering of the amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptide comprises a T62G substitution. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the modified immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor infiltrating T cells, and DC-activated T cells.

[0072] In some embodiments, modified immune cells are provided that comprise a heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising an amino acid substitution at position 8, where the numbering of the amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptide comprises a glutamic acid (E) at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:5. In some embodiments, the modified immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor infiltrating T cells, and DC-activated T cells.

[0073] In some embodiments, modified immune cells are provided that comprise a heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising an amino acid substitution at position 3, wherein the numbering of the amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptide comprises a tyrosine (Y) at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:78. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:78. In some embodiments, the modified immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor infiltrating T cells, and DC-activated T cells.

[0074] In some embodiments, modified immune cells are provided that comprise a heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising an amino acid substitution at position 25, where the numbering of the amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptide comprises a glutamic acid (E) or a phenylalanine (F) at position 25. In some embodiments, the amino acid substitution at position 25 is L25E. In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:79. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:79. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor-infiltrating T cells, and DC-activated T cells.

[0075] In some embodiments, modified immune cells are provided that comprise a heterologous nucleic acid sequence encoding a secreted IL-15 polypeptide comprising one or more amino acid substitutions at positions 8, 62, 3, and / or 25, where the numbering of the amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptide has a reduced binding affinity to an IL-15 receptor (e.g., IL-15Rα and / or IL-15Rβ / γc) compared to a wild-type IL-15 polypeptide. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78.In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25. In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F. In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at both positions 8 and 62. In some embodiments, the IL-15 polypeptide is a fusion protein comprising an IL-15 fragment fused to the extracellular domain of IL-15Rα or the sushi domain of IL-15Rα. In some embodiments, the IL-15 polypeptide is a fusion protein comprising the amino acid sequence of SEQ ID NO: 57 or 58. In some embodiments, the modified immune cell is selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor-infiltrating T cells, and DC-activated T cells.

[0076] In some embodiments, modified immune cells are provided that comprise a heterologous nucleic acid sequence encoding a membrane-bound IL-15 polypeptide comprising one or more amino acid substitutions at positions 8, 62, 3, and / or 25, wherein the numbering of amino acid residue positions is according to SEQ ID NO:1, and wherein the IL-15 polypeptide comprises a glycosylphosphatidylinositol (GPI) anchor peptide sequence. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25.In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F. In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at both positions 8 and 62. In some embodiments, the GPI anchor peptide sequence is attached to a GPI linker. In some embodiments, the GPI anchor peptide sequence is located at the C-terminus of the IL-15 polypeptide. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor-infiltrating T cells, and DC-activated T cells.

[0077] In some embodiments, modified immune cells are provided that comprise a heterologous nucleic acid sequence encoding an IL-15 polypeptide that is a fusion protein comprising an IL-15 fragment fused to a second polypeptide fragment, wherein the IL-15 fragment comprises one or more amino acid substitutions at positions 8, 62, 3, and / or 25, where the numbering of amino acid residue positions is according to SEQ ID NO: 1. In some embodiments, the IL-15 fragment comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 fragment comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:7 (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, the IL-15 fragment comprises SEQ ID NO:7. In some embodiments, the IL-15 fragment comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 fragment comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:5 (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, the IL-15 fragment comprises SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25. In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F.In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79. In some embodiments, the IL-15 fragment comprises amino acid substitutions at positions 8 and 62. In some embodiments, the second polypeptide fragment is selected from the group consisting of IL-15Rα, the extracellular domain of IL-15Rα, the sushi domain of IL-15Rα, the transmembrane domain of IL-15Rα, IL-15Rβ, the common gamma chain (γc), an engineered receptor (e.g., a CAR, a TCR, or a TAC), and combinations thereof. In some embodiments, the second polypeptide fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-55. In some embodiments, the IL-15 fragment is fused to the second polypeptide fragment via a peptide linker. In some embodiments, the IL-15 polypeptide described herein above comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 57-64, 76, and 77. In some embodiments, the modified immune cell is selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβT cells, γδT cells, tumor-infiltrating T cells, and DC-activated T cells.

[0078] In some embodiments, modified immune cells are provided that comprise a heterologous nucleic acid sequence encoding a membrane-bound IL-15 polypeptide comprising one or more amino acid substitutions at positions 8, 62, 3, and / or 25, wherein the numbering of amino acid residue positions is according to SEQ ID NO:1, and wherein the IL-15 polypeptide comprises a transmembrane domain. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25. In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F.In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 79 (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at both positions 8 and 62. In some embodiments, the transmembrane domain is the transmembrane domain of IL-15Rα. In some embodiments, the IL-15 polypeptide further comprises an intracellular domain. In some embodiments, the IL-15 polypeptide comprises: (a) an antigen binding domain; (b) an IL-15 fragment; (c) a transmembrane domain; and (d) an intracellular domain. In some embodiments, the antigen binding domain is at the N-terminus of the IL-15 fragment. In some embodiments, the antigen binding domain is at the C-terminus of the IL-15 fragment. In some embodiments, the transmembrane domain is a CD4, CD3, CD8α, or CD28 transmembrane domain. In some embodiments, the IL-15 polypeptide further comprises a hinge domain, e.g., a hinge domain derived from CD8. In some embodiments, the intracellular domain comprises a primary intracellular signaling domain, e.g., an intracellular signaling domain of CD3ζ. In some embodiments, the intracellular domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, DAP10, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 65, 66, or 75. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor-infiltrating T cells, and DC-activated T cells.

[0079] In some embodiments, modified immune cells are provided that comprise a heterologous nucleic acid sequence encoding a membrane-bound IL-15 polypeptide comprising one or more amino acid substitutions at positions 8, 62, 3, and / or 25, wherein the numbering of amino acid residue positions is according to SEQ ID NO:1, and wherein the IL-15 polypeptide comprises a membrane anchor domain. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25. In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F.In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 79 (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at both positions 8 and 62. In some embodiments, the membrane anchor domain is derived from a molecule selected from the group consisting of IL-15Rα, the transmembrane domain of IL-15Rα, IL-15Rβ, the extracellular domain of IL-15Rα, the sushi domain of IL-15Rα, the extracellular domain of IL-15Rβ, the common gamma chain (γc), and combinations thereof. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 59-64, 76, and 77. In some embodiments, the engineered immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer (NK) cell, an NK-T cell, an iNK-T cell, an NK-T-like cell, an αβ T cell, a γδ T cell, a tumor-infiltrating T cell, and a DC-activated T cell.

[0080] In some embodiments, modified immune cells (e.g., NK cells or T cells) are provided that include a first heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising one or more amino acid substitutions at positions 8, 62, 3, and / or 25, where the numbering of amino acid residue positions is according to SEQ ID NO:1, and a second heterologous nucleic acid sequence encoding an engineered receptor. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25.In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F. In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at both positions 8 and 62. In some embodiments, the engineered receptor is a CAR, e.g., a BCMA CAR, a CD19 CAR, or a GPC3 CAR. In some embodiments, the engineered receptor is an engineered TCR. In some embodiments, the engineered receptor is a TAC receptor. In some embodiments, the first and second nucleic acid sequences are present on the same vector or separate vectors. In some embodiments, the first and second nucleic acid sequences are operably linked to the same promoter or separate promoters. In some embodiments, the modified immune cell is selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβT cells, γδT cells, tumor-infiltrating T cells, and DC-activated T cells.

[0081] In some embodiments, modified immune cells (e.g., NK cells or T cells) are provided that include a first heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising one or more amino acid substitutions at positions 8, 62, 3, and / or 25, where the numbering of amino acid residue positions is according to SEQ ID NO:1, and a second heterologous nucleic acid sequence encoding an engineered receptor. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25.In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F. In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at both positions 8 and 62. In some embodiments, the engineered receptor is a CAR, e.g., a BCMA CAR, a CD19 CAR, or a GPC3 CAR. In some embodiments, the engineered receptor is an engineered TCR. In some embodiments, the engineered receptor is a TAC receptor. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present on the same vector or on separate vectors. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter or separate promoters. In some embodiments, the modified immune cell is selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor-infiltrating T cells, and DC-activated T cells. In some embodiments, the modified immune cell comprises a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-38, 42-49, 83, and 84.

[0082] In some embodiments, modified immune cells (e.g., NK cells or T cells) are provided that comprise a first heterologous nucleic acid sequence encoding a membrane-bound IL-15 polypeptide comprising a GPI anchor peptide sequence, the IL-15 polypeptide comprising one or more amino acid substitutions at positions 8, 62, 3, and / or 25, where the numbering of amino acid residue positions is according to SEQ ID NO:1, and a second heterologous nucleic acid sequence encoding an engineered receptor. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78.In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25. In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F. In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at both positions 8 and 62. In some embodiments, the GPI anchor peptide sequence is attached to a GPI linker. In some embodiments, the GPI anchor peptide sequence is located at the C-terminus of the IL-15 polypeptide. In some embodiments, the engineered receptor is a CAR, such as a BCMA CAR, a CD19 CAR, or a GPC3 CAR. In some embodiments, the engineered receptor is an engineered TCR. In some embodiments, the engineered receptor is a TAC receptor. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present on the same vector or on separate vectors. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter or to separate promoters. In some embodiments, the modified immune cell is selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor infiltrating T cells, and DC-activated T cells.

[0083] In some embodiments, modified immune cells (e.g., NK cells or T cells) are provided that comprise a first heterologous nucleic acid sequence encoding a membrane-bound IL-15 polypeptide comprising a transmembrane domain, the IL-15 polypeptide comprising one or more amino acid substitutions at positions 8, 62, 3, and / or 25, where the numbering of amino acid residue positions is according to SEQ ID NO: 1, and a second heterologous nucleic acid sequence encoding an engineered receptor. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78.In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25. In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F. In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at both positions 8 and 62. In some embodiments, the transmembrane domain is the transmembrane domain of IL-15Rα. In some embodiments, the IL-15 polypeptide further comprises an intracellular domain. In some embodiments, the engineered receptor is a CAR, such as a BCMA CAR, a CD19 CAR, or a GPC3 CAR. In some embodiments, the engineered receptor is an engineered TCR. In some embodiments, the engineered receptor is a TAC receptor. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present on the same vector or on separate vectors. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter or to separate promoters. In some embodiments, the modified immune cell is selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor infiltrating T cells, and DC-activated T cells.

[0084] In some embodiments, modified immune cells (e.g., NK cells or T cells) are provided that comprise a heterologous nucleic acid sequence encoding an engineered receptor comprising: (a) an antigen binding domain; (b) an IL-15 fragment; (c) a transmembrane domain; and (d) an intracellular domain, wherein the IL-15 fragment comprises one or more amino acid substitutions at positions 8, 62, 3, and / or 25, where the numbering of the amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25.In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F. In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 79 (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at both positions 8 and 62. In some embodiments, the antigen binding domain is at the N-terminus of the IL-15 fragment. In some embodiments, the antigen binding domain is at the C-terminus of the IL-15 fragment. In some embodiments, the transmembrane domain is a CD4, CD3, CD8α, or CD28 transmembrane domain. In some embodiments, the IL-15 polypeptide further comprises a hinge domain, e.g., a hinge domain derived from CD8α. In some embodiments, the intracellular domain comprises a primary intracellular signaling domain, e.g., the intracellular signaling domain of CD3ζ. In some embodiments, the intracellular domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, DAP10, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the intracellular domain comprises a costimulatory signaling domain of 4-1BB and a primary intracellular signaling domain of CD3ζ. In some embodiments, the antigen binding domain specifically binds BCMA, CD19, or GPC3. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor-infiltrating T cells, and DC-activated T cells.In some embodiments, the modified immune cell comprises a heterologous nucleic acid sequence encoding an engineered receptor comprising SEQ ID NO: 65, 66, or 75.

[0085] In some embodiments, modified immune cells (e.g., NK cells or T cells) are provided that comprise a first heterologous nucleic acid sequence encoding a membrane-bound IL-15 polypeptide comprising a membrane anchor domain, the IL-15 polypeptide comprising one or more amino acid substitutions at positions 8, 62, 3, and / or 25, where the numbering of amino acid residue positions is according to SEQ ID NO:1, and a second heterologous nucleic acid sequence encoding an engineered receptor. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78.In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25. In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F. In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at both positions 8 and 62. In some embodiments, the membrane anchor domain is derived from a molecule selected from the group consisting of IL-15Rα, the transmembrane domain of IL-15Rα, IL-15Rβ, the extracellular domain of IL-15Rα, the sushi domain of IL-15Rα, the extracellular domain of IL-15Rβ, the common gamma chain (γc), and combinations thereof. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 59-64, 76, and 77. In some embodiments, the engineered receptor is a CAR, such as a BCMA CAR, a CD19 CAR, or a GPC3 CAR. In some embodiments, the engineered receptor is an engineered TCR. In some embodiments, the engineered receptor is a TAC receptor. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present on the same vector or separate vectors. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter or separate promoters. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor-infiltrating T cells, and DC-activated T cells.

[0086] In some embodiments, modified immune cells (e.g., NK cells or T cells) are provided that comprise a first heterologous nucleic acid sequence encoding an IL-15 polypeptide that is a fusion protein comprising an IL-15 fragment fused to a second polypeptide fragment, the IL-15 polypeptide comprising one or more amino acid substitutions at positions 8, 62, 3, and / or 25, where the numbering of amino acid residue positions is according to SEQ ID NO:1, and a second heterologous nucleic acid sequence encoding an engineered receptor. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78.In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25. In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F. In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at both positions 8 and 62. In some embodiments, the second polypeptide fragment is selected from the group consisting of IL-15Rα, the extracellular domain of IL-15Rα, the sushi domain of IL-15Rα, the transmembrane domain of IL-15Rα, IL-15Rβ, the common gamma chain (γc), an engineered receptor (e.g., a CAR, a TCR, or a TAC), and combinations thereof. In some embodiments, the second polypeptide fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-55. In some embodiments, the IL-15 fragment is fused to the second polypeptide fragment via a peptide linker. In some embodiments, the IL-15 polypeptide described herein above comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 57-64, 76, and 77. In some embodiments, the engineered receptor is a CAR, e.g., a BCMA CAR, a CD19 CAR, or a GPC3 CAR. In some embodiments, the engineered receptor is an engineered TCR. In some embodiments, the engineered receptor is a TAC receptor. In some embodiments, the first and second nucleic acid sequences are present on the same vector or on separate vectors. In some embodiments, the first and second nucleic acid sequences are operably linked to the same promoter or separate promoters.In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor-infiltrating T cells, and DC-activated T cells.

[0087] In some embodiments, a CAR-expressing immune cell (e.g., a CAR-NK cell or a CAR-T cell) is provided that comprises a heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising one or more amino acid substitutions at positions 8, 62, 3, and / or 25, where the numbering of the amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25. In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F.In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 79 (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at both positions 8 and 62. In some embodiments, the IL-15 polypeptide is secreted. In some embodiments, the IL-15 polypeptide is membrane bound. In some embodiments, the IL-15 polypeptide comprises a GPI anchor peptide sequence. In some embodiments, the IL-15 polypeptide comprises a transmembrane domain. In some embodiments, the IL-15 polypeptide comprises a membrane anchor domain. In some embodiments, the IL-15 polypeptide is a fusion protein comprising an IL-15 fragment fused to a second polypeptide fragment. In some embodiments, the second polypeptide fragment is selected from the group consisting of IL-15Rα, the extracellular domain of IL-15Rα, the sushi domain of IL-15Rα, the transmembrane domain of IL-15Rα, IL-15Rβ, the common gamma chain (γc), an engineered receptor (e.g., a CAR, a TCR, or a TAC), and combinations thereof. In some embodiments, the engineered immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer (NK) cell, an NK-T cell, an iNK-T cell, an NK-T-like cell, an αβ T cell, a γδ T cell, a tumor-infiltrating T cell, and a DC-activated T cell.

[0088] In some embodiments, a CAR-expressing immune cell (e.g., a CAR-NK cell or a CAR-T cell) is provided that comprises a heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising one or more amino acid substitutions at positions 8, 62, 3, and / or 25, where the numbering of the amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25. In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F.In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 79 (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at both positions 8 and 62. In some embodiments, the IL-15 polypeptide is secreted. In some embodiments, the IL-15 polypeptide is membrane bound. In some embodiments, the IL-15 polypeptide comprises a GPI anchor peptide sequence. In some embodiments, the IL-15 polypeptide comprises a transmembrane domain. In some embodiments, the IL-15 polypeptide comprises a membrane anchor domain. In some embodiments, the IL-15 polypeptide is a fusion protein comprising an IL-15 fragment fused to a second polypeptide fragment. In some embodiments, the second polypeptide fragment is selected from the group consisting of IL-15Rα, the extracellular domain of IL-15Rα, the sushi domain of IL-15Rα, the transmembrane domain of IL-15Rα, IL-15Rβ, the common gamma chain (γc), an engineered receptor (e.g., a CAR, a TCR, or a TAC), and combinations thereof. In some embodiments, the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer (NK) cell, an NK-T cell, an iNK-T cell, an NK-T-like cell, an αβ T cell, a γδ T cell, a tumor-infiltrating T cell, and a DC-activated T cell.

[0089] In some embodiments, a TAC-expressing immune cell (e.g., a TAC-T cell) is provided that comprises a heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising one or more amino acid substitutions at positions 8, 62, 3, and / or 25, where the numbering of the amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25. In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F.In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 79 (e.g., at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at both positions 8 and 62. In some embodiments, the IL-15 polypeptide is secreted. In some embodiments, the IL-15 polypeptide is membrane bound. In some embodiments, the IL-15 polypeptide comprises a GPI anchor peptide sequence. In some embodiments, the IL-15 polypeptide comprises a transmembrane domain. In some embodiments, the IL-15 polypeptide comprises a membrane anchor domain. In some embodiments, the IL-15 polypeptide is a fusion protein comprising an IL-15 fragment fused to a second polypeptide fragment. In some embodiments, the second polypeptide fragment is selected from the group consisting of IL-15Rα, the extracellular domain of IL-15Rα, the sushi domain of IL-15Rα, the transmembrane domain of IL-15Rα, IL-15Rβ, the common gamma chain (γc), an engineered receptor (e.g., a CAR, a TCR, or a TAC), and combinations thereof. In some embodiments, the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer (NK) cell, an NK-T cell, an iNK-T cell, an NK-T-like cell, an αβ T cell, a γδ T cell, a tumor-infiltrating T cell, and a DC-activated T cell.

[0090] In some embodiments, a CAR-expressing immune cell (e.g., a CAR-NK cell or a CAR-T cell) is provided that comprises a heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising a T62G substitution, where the numbering of the amino acid residue positions is according to SEQ ID NO: 1. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 7. In some embodiments, the CAR is a BCMA CAR, a CD19 CAR, or a GPC3 CAR. In some embodiments, the immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor-infiltrating T cells, and DC-activated T cells.

[0091] In some embodiments, a CAR-expressing immune cell (e.g., a CAR-NK cell or a CAR-T cell) is provided that comprises a heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising a D8E substitution, where the numbering of the amino acid residue positions is according to SEQ ID NO: 1. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 5. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 5. In some embodiments, the CAR is a BCMA CAR, a CD19 CAR, or a GPC3 CAR. In some embodiments, the immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor-infiltrating T cells, and DC-activated T cells.

[0092] In some embodiments, a CAR-expressing immune cell (e.g., a CAR-NK cell or a CAR-T cell) is provided that comprises a heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising a V3Y substitution, where the numbering of the amino acid residue positions is according to SEQ ID NO: 1. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78. In some embodiments, the CAR is a BCMA CAR, a CD19 CAR, or a GPC3 CAR. In some embodiments, the immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor-infiltrating T cells, and DC-activated T cells.

[0093] In some embodiments, a CAR-expressing immune cell (e.g., a CAR-NK cell or a CAR-T cell) is provided that comprises a heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising an L25F substitution, where the numbering of the amino acid residue positions is according to SEQ ID NO: 1. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 85% (e.g., at least about any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79. In some embodiments, the CAR is a BCMA CAR, a CD19 CAR, or a GPC3 CAR. In some embodiments, the immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor-infiltrating T cells, and DC-activated T cells.

[0094] In some embodiments, modified immune cells (e.g., NK cells or T cells) are provided that comprise a heterologous nucleic acid encoding an IL-15 polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 57-66 and 75-77. In some embodiments, the IL-15 polypeptide is a CAR comprising an IL-15 fragment. In some embodiments, the modified immune cell further comprises a second heterologous nucleic acid encoding a chimeric antigen receptor (CAR). In some embodiments, the CAR is a BCMA CAR, a CD19 CAR, or a GPC3 CAR. In some embodiments, the IL-15 polypeptide is secreted from the modified immune cell. In some embodiments, the IL-15 polypeptide is membrane-bound. In some embodiments, the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer (NK) cell, a NK-T cell, an iNK-T cell, a NK-T-like cell, an αβ T cell, a γδ T cell, a tumor-infiltrating T cell, and a DC-activated T cell.

[0095] immune cells The modified immune cells may be derived from a variety of cell types and cell sources. Cells derived from any mammalian species are contemplated herein, including, but not limited to, mouse, rat, guinea pig, rabbit, dog, monkey, and human. In some embodiments, the modified immune cells are human cells. In some embodiments, the modified immune cells are allogeneic to the recipient individual (i.e., from the same species but a different donor). In some embodiments, the modified immune cells are autologous (i.e., the donor and recipient are the same). In some embodiments, the modified immune cells are syngeneic (i.e., the donor and recipient are different individuals but are identical twins).

[0096] In some embodiments, the modified immune cells are derived from primary cells. In some embodiments, the modified immune cells are primary cells isolated from an individual. In some embodiments, the modified immune cells are expanded (e.g., expanded and / or differentiated) from primary cells isolated from an individual. In some embodiments, the primary cells are hematopoietic. In some embodiments, the primary cells are harvested from the thymus. In some embodiments, the primary cells are harvested from lymph or lymph nodes (e.g., tumor-draining lymph nodes). In some embodiments, the primary cells are harvested from the spleen. In some embodiments, the primary cells are harvested from bone marrow. In some embodiments, the primary cells are harvested from blood, such as peripheral blood. In some embodiments, the primary cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, the primary cells are derived from plasma. In some embodiments, the primary cells are derived from a tumor. In some embodiments, the primary cells are harvested from the mucosal immune system. In some embodiments, the primary cells are obtained from a biopsy sample.

[0097] In some embodiments, the modified immune cells are derived from a cell line. In some embodiments, the modified immune cells are harvested from a commercially available cell line. In some embodiments, the modified immune cells are a cell line established from primary cells isolated from an individual. In some embodiments, the modified immune cells are expanded (e.g., expanded and / or differentiated) from a cell line. In some embodiments, the cell line is mortal. In some embodiments, the cell line is immortalized. In some embodiments, the cell line is a tumor cell line, e.g., a leukemia cell line or a lymphoma cell line. In some embodiments, the cell line is a cell line derived from PBMCs. In some embodiments, the cell line is a stem cell line. In some embodiments, the cell line is selected from the group consisting of HEK293-6E cells, NK-92 cells, and Jurkat cells.

[0098] Exemplary immune cells useful for the present application include, but are not limited to, dendritic cells (including immature and mature dendritic cells), T lymphocytes (e.g., naive T cells, effector T cells, memory T cells, cytotoxic T lymphocytes, helper T cells, natural-killer T cells, Treg cells, tumor-infiltrating lymphocytes (TILs), and lymphokine-activated killer (LAK) cells), B cells, natural killer (NK) cells, monocytes, macrophages, neutrophils, granulocytes, and combinations thereof. Subpopulations of immune cells may be defined by the presence or absence of one or more cell surface markers known in the art (e.g., CD3, CD4, CD8, CD19, CD20, CD11c, CD123, CD56, CD34, CD14, CD33, etc.). When the pharmaceutical composition comprises more than one type of modified immune cell, the modified immune cell can be a specific subpopulation of an immune cell type, a combination of subpopulations of an immune cell type, or a combination of two or more immune cell types. In some embodiments, the immune cell is in a homogeneous cell population. In some embodiments, the immune cell is in a heterogeneous cell population in which the immune cells have been expanded. In some embodiments, the modified immune cell is a lymphocyte. In some embodiments, the modified immune cell is not a lymphocyte. In some embodiments, the modified immune cell is suitable for adoptive immunotherapy. In some embodiments, the modified immune cell is a PBMC. In some embodiments, the modified immune cell is an immune cell derived from a PBMC. In some embodiments, the modified immune cell is a T cell. In some embodiments, the modified immune cell is a CD4 + In some embodiments, the engineered immune cells are CD8 T cells. + In some embodiments, the modified immune cell is a T cell. In some embodiments, the modified immune cell is a B cell. In some embodiments, the modified immune cell is a NK cell.

[0099] In some embodiments, the modified immune cells are derived from stem cells. In some embodiments, the stem cells are totipotent stem cells. In some embodiments, the stem cells are pluripotent stem cells. In some embodiments, the stem cells are unipotent stem cells. In some embodiments, the stem cells are progenitor cells. In some embodiments, the stem cells are embryonic stem cells. In some embodiments, the stem cells are hematopoietic stem cells. In some embodiments, the stem cells are mesenchymal stem cells. In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs).

[0100] The modified immune cells can comprise any number (e.g., any of 1, 2, 3, 4, 5, 10, 50, 100, 1000, or more) of heterologous nucleic acid sequences (including first and second nucleic acid sequences). In some embodiments, the modified immune cells comprise a single copy of the first and / or second heterologous nucleic acid sequence. In some embodiments, the modified immune cells comprise multiple copies of the first and / or second heterologous nucleic acid sequence. In some embodiments, the modified immune cells further comprise at least one additional heterologous nucleic acid sequence, e.g., a heterologous nucleic acid sequence encoding an immunomodulatory agent, e.g., a cytokine, chemokine, and / or an immune checkpoint inhibitor.

[0101] The nucleic acid(s) comprising the heterologous nucleic acid sequence(s) described herein may be transiently or stably incorporated into the modified immune cell. In some embodiments, the nucleic acid(s) are transiently expressed in the modified immune cell. For example, the nucleic acid(s) may be present in the nucleus of the modified immune cell as an extrachromosomal array. The nucleic acid(s) may be introduced into the modified immune cell using any transfection or transduction method known in the art, including viral or non-viral methods. Exemplary non-viral transfection methods include, but are not limited to, chemical-based transfection, such as the use of calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethyleneimine); non-chemical methods, such as electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, hydrodynamic transfer, or transposons; particle-based methods, such as the use of gene guns, magnetofection or magnetically assisted transfection, microprojectile guns; and hybrid methods, such as nucleofection.

[0102] In some embodiments, the heterologous nucleic acid sequence(s) are present in the genome of the modified immune cell. For example, the nucleic acid(s) comprising the heterologous nucleic acid sequence(s) can be integrated into the genome of the modified immune cell by any method known in the art, including, but not limited to, virally mediated integration, random integration, homologous recombination, and site-specific integration methods, such as the use of site-specific recombinases or integrases, transposases, transcription activator-like effector nucleases (TALEN®), CRISPR / Cas9, and zinc finger nucleases. In some embodiments, the heterologous nucleic acid sequence(s) are integrated into specifically designed loci in the genome of the modified immune cell. In some embodiments, the heterologous nucleic acid sequence(s) are integrated into integration hotspots in the genome of the modified immune cell. In some embodiments, the heterologous nucleic acid (sequence) is integrated into random loci in the genome of the modified immune cell. When multiple copies of a heterologous nucleic acid sequence(s) are present in a single modified immune cell, the heterologous nucleic acid sequence may be integrated at multiple loci in the genome of the modified immune cell.

[0103] IL-15 Polypeptides The modified immune cells described herein express mutant IL-15 polypeptides. The present application also provides IL-15 polypeptides and compositions thereof. In some embodiments, the IL-15 polypeptides provided herein provide potent anti-tumor effects without causing increased levels of inflammatory cytokines (e.g., cytokine storm).

[0104] In some embodiments, the IL-15 polypeptide is a full-length IL-15 molecule. In some embodiments, the IL-15 polypeptide comprises a functional portion of an IL-15 molecule. In some embodiments, the IL-15 polypeptide is a human IL-15 polypeptide. In some embodiments, the IL-15 polypeptide has a single chain. In some embodiments, the IL-15 polypeptide has two or more chains.

[0105] In some embodiments, the IL-15 polypeptides described herein can bind to the trimeric IL-15R (IL-15 receptor) complex. The IL-15 receptor consists of three polypeptides: the species-specific IL-15 ("IL-15Rα"), the IL-2 / IL-15Rβ ("IL-15Rβ"), and a common gamma chain ("γc") shared with various cytokines. In some embodiments, the IL-15 polypeptides can bind to the α chain ("IL-15Rα"), the common beta chain ("IL-15Rβ"), and / or the common gamma chain ("IL-15Rγc"). In some embodiments, the IL-15 polypeptides can bind to IL-15Rα. In some embodiments, the IL-15 polypeptides can bind to IL-15Rβ. In some embodiments, the IL-15 polypeptides can bind to IL-15Rβ / γc.

[0106] In some embodiments, the IL-15 polypeptide has a binding affinity for IL-15Rα equivalent to the wild-type IL-15 polypeptide. In some embodiments, the IL-15 polypeptide has a reduced binding affinity for IL-15β compared to the wild-type IL-15 polypeptide. An exemplary wild-type IL-15 polypeptide has the amino acid sequence of SEQ ID NO:1. SEQ ID NO:1 Wild type human IL-15 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0107] In some embodiments, the IL-15 polypeptide is about 10 -9 , 10 -10 , or 10 -12 In some embodiments, the IL-15 polypeptide binds to IL-15Rα with a KD of about 10 or more. -7 , 10 -8 , or 10 -9 It binds to IL-15Rβ with a KD equal to or greater than either of the above.

[0108] In some embodiments, the IL-15 polypeptide has a reduced binding affinity for IL-15Rβ compared to a wild-type IL-15 polypeptide, e.g., at least about a 10% reduced, 20% reduced, 30% reduced, 40% reduced, 2-fold reduced, 4-fold reduced, 6-fold reduced, 8-fold reduced, 10-fold reduced, 12-fold reduced, 14-fold reduced, 16-fold reduced, 18-fold reduced, 20-fold reduced, 30-fold reduced, 50-fold reduced, or more reduced binding affinity. In some embodiments, the IL-15 polypeptide has a reduced binding affinity for IL-15Rβ compared to a wild-type IL-15 polypeptide, e.g., any of about 50-fold or more reduced, 30-fold or more reduced, 20-fold or more reduced, 18-fold or more reduced, 16-fold or more reduced, 14-fold or more reduced, 12-fold or more reduced, 10-fold or more reduced, 8-fold or more reduced, 6-fold or more reduced, 4-fold or more reduced, 50% or less reduced, 40% or less reduced, 30% or less reduced, 20% or less reduced, 10% or less reduced, or less than reduced binding affinity. In some embodiments, the IL-15 polypeptide has a reduced binding affinity for IL-15Rβ compared to a wild-type IL-15 polypeptide, for example, any of about 10%-50% reduced, 2- to 10-fold reduced, 10- to 20-fold reduced, 20- to 40-fold reduced, 10- to 40-fold reduced, 10- to 50-fold reduced, 14- to 40-fold reduced, or 2- to 50-fold reduced binding affinity.

[0109] In some embodiments, the IL-15 polypeptide induces secretion by the engineered immune cells of reduced levels of inflammatory cytokines compared to a wild-type IL-15 polypeptide, e.g., at least about a 10% reduced, 20% reduced, 30% reduced, 40% reduced, 2-fold reduced, 4-fold reduced, 6-fold reduced, 8-fold reduced, 10-fold reduced, 12-fold reduced, 14-fold reduced, 16-fold reduced, 18-fold reduced, 20-fold reduced, 30-fold reduced, 50-fold reduced, 100-fold reduced, 200-fold reduced, 500-fold reduced, 1000-fold reduced, or more reduced levels of inflammatory cytokines. In some embodiments, the IL-15 polypeptide induces secretion by the engineered immune cells of reduced levels of inflammatory cytokines compared to a wild-type IL-15 polypeptide, e.g., about or greater than 1000-fold reduced, 500-fold reduced, 200-fold reduced, 100-fold reduced, 50-fold reduced, 30-fold reduced, 20-fold reduced, 18-fold reduced, 16-fold reduced, 14-fold reduced, 12-fold reduced, 10-fold reduced, 8-fold reduced, 6-fold reduced, 4-fold reduced, 50% or less reduced, 40% or less reduced, 30% or less reduced, 20% or less reduced, 10% or less reduced, or less than or equal to reduced levels of inflammatory cytokines. In some embodiments, the IL-15 polypeptide induces secretion by the modified immune cells of reduced levels of inflammatory cytokines compared to a wild-type IL-15 polypeptide, e.g., either about 10%-50% reduced, 2-1000 fold reduced, 2-50 fold reduced, 50-100 fold reduced, 100-1000 fold reduced, 50-500 fold reduced, 10-100 fold reduced, 10-50 fold reduced, or 50-200 fold reduced levels of inflammatory cytokines.Exemplary inflammatory cytokines include, but are not limited to, IFN-γ, TNF-α, and GM-CSF. In some embodiments, the secretion level of the inflammatory cytokine is measured by an immunoserological test, such as an enzyme-linked immunosorbent assay (ELISA), a chemiluminescence immunoassay (CIA), or flow cytometry. In some embodiments, the secretion level of the inflammatory cytokine is measured in a cell-based assay. In some embodiments, the secretion level of the inflammatory cytokine is measured in vivo.

[0110] In some embodiments, the IL-15 polypeptide does not induce a cytokine storm in a subject administered an IL-15 polypeptide or modified immune cells comprising an IL-15 polypeptide. A "cytokine storm" occurs when a large number of inflammatory cytokines are produced at a higher than normal frequency. In some embodiments, the overproduction of cytokines can cause cytotoxicity by recruitment of other immune cells. In some embodiments, the inflammatory cytokines produced during a cytokine storm include members of the IL-20 family, IL-1α, IL-1β, IL-6, IL-33 LIF, IFN-γ, OSM, CNTF, TNF-α, TGF-β, GM-CSF, IL-11, IL-12, IL-17, IL-18, IL-8, and other inflammatory cytokines known in the art. In some embodiments, the inflammatory cytokines produced during a cytokine storm include IFN-γ, TNF-α, and GM-CSF. In some embodiments, a cytokine storm can be measured by any cytokine measurement technique known in the art. In some embodiments, the cytokine storm is measured by ELISA, CIA, or flow cytometry.

[0111] In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at one or more positions selected from the group consisting of 3, 8, 23, 25, 26, 58, 61, 62, and 89, where the numbering of the amino acid residue positions is according to SEQ ID NO: 1. Exemplary mutant IL-15 polypeptide sequences and their corresponding mutations are listed in Table 1 below.

[0112] [Table 1] TIFF2024527593000002.tif204165TIFF2024527593000003.tif123165

[0113] In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62, where the numbering of the amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptide comprises a hydrophobic amino acid residue at position 62. In some embodiments, the IL-15 polypeptide comprises an amino acid residue having a short side chain at position 62. In some embodiments, the IL-15 polypeptide comprises an amino acid residue at position 62 selected from the group consisting of glycine (G), isoleucine (I), glutamine (Q), valine (V), proline (P), leucine (L), alanine (A), serine (S), and tyrosine (Y). In some embodiments, the IL-15 polypeptide comprises an amino acid substitution selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the IL-15 polypeptide comprises a T62G substitution. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 85% (e.g., at least about any of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, and 11-17. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, and 11-17. In some embodiments, the IL-15 polypeptide comprises a single amino acid substitution described herein. In some embodiments, the IL-15 polypeptide comprises two or more amino acid substitutions described herein.

[0114] In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8, where the numbering of the amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptide comprises an acidic amino acid residue at position 8. In some embodiments, the IL-15 polypeptide comprises an E at position 8. In some embodiments, the IL-15 polypeptide comprises an uncharged amino acid residue at position 8. In some embodiments, the IL-15 polypeptide comprises a D8E substitution. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 85% (e.g., at least about any of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises a single amino acid substitution as described herein. In some embodiments, the IL-15 polypeptide comprises two or more amino acid substitutions described herein. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8 and an amino acid substitution at position 62. In some embodiments, the IL-15 polypeptide comprises a D8E and a T62G substitution.

[0115] In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3, where the numbering of the amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptide comprises a Y at position 3. In some embodiments, the IL-15 polypeptide comprises a V3Y substitution. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 85% (e.g., at least about any of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:78. In some embodiments, the IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO:78. In some embodiments, the IL-15 polypeptide comprises a single amino acid substitution as described herein. In some embodiments, the IL-15 polypeptide comprises two or more amino acid substitutions as described herein.

[0116] In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25, where the numbering of the amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptide comprises an F at position 25. In some embodiments, the IL-15 polypeptide comprises an L25F substitution. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 85% (e.g., at least about any of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:79. In some embodiments, the IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO:79. In some embodiments, the IL-15 polypeptide comprises a single amino acid substitution as described herein. In some embodiments, the IL-15 polypeptide comprises two or more amino acid substitutions as described herein.

[0117] In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at both positions 8 and 62. In some embodiments, the IL-15 polypeptide comprises a D8E and a T62G substitution. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at both positions 8 and 3. In some embodiments, the IL-15 polypeptide comprises a D8E and a V3Y substitution. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at both positions 8 and 25. In some embodiments, the IL-15 polypeptide comprises a D8E and a L25F substitution. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at both positions 62 and 3. In some embodiments, the IL-15 polypeptide comprises a T62G and a V3Y substitution. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at both positions 62 and 25. In some embodiments, the IL-15 polypeptide comprises a T62G and a L25F substitution. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at both positions 3 and 25. In some embodiments, the IL-15 polypeptide comprises a V3Y and an L25F substitution. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at positions 8, 62, and 3. In some embodiments, the IL-15 polypeptide comprises a D8E, a T62G, and a V3Y substitution. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at positions 8, 62, and 25. In some embodiments, the IL-15 polypeptide comprises a D8E, a T62G, and a L25F substitution. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at positions 8, 3, and 25. In some embodiments, the IL-15 polypeptide comprises a D8E, a V3Y, and a L25F substitution. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at positions 62, 3, and 25. In some embodiments, the IL-15 polypeptide comprises T62G, V3Y, and L25F substitutions. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at positions 8, 62, 3, and 25. In some embodiments, the IL-15 polypeptide comprises D8E, T62G, V3Y, and L25F substitutions.

[0118] In some embodiments, the IL-15 polypeptide is secreted from the modified immune cells. In some embodiments, the IL-15 polypeptide comprises an IL-15 fragment fused to the extracellular domain of IL-15Rα. In some embodiments, the IL-15 polypeptide comprises an IL-15 fragment fused to the sushi domain of IL-15Rα. In some embodiments, the IL-15 polypeptide comprises a signal peptide (also referred to herein as "SP"). Signal peptides (also known as "leader sequences") are usually inserted at the N-terminus of a protein immediately following the initiating methionine. The signal peptide may be cleaved upon transport of the IL-15 polypeptide from the modified immune cells, thereby resulting in the mature protein. Signal peptides may be natural or synthetic, and may be heterologous or homologous to the protein to which they are bound. The choice of signal peptides is wide and available to the skilled artisan, for example in the leader sequence online database maintained by the Department of Biochemistry, National University of Singapore. See Choo et al., BMC Bioinformatics, 6:249 (2005), and PCT Publication No. WO2006 / 081430. Exemplary signal peptide sequences include, but are not limited to, SEQ ID NOs:71-74. In some embodiments, the IL-15 polypeptide comprises a propeptide. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:70.

[0119] In some embodiments, the IL-15 polypeptide is a fusion protein comprising an IL-15 fragment fused to a second polypeptide fragment, in some embodiments, the second polypeptide fragment is selected from the group consisting of IL-15Rα, the extracellular domain of IL-15Rα, the sushi domain of IL-15Rα, the transmembrane domain of IL-15Rα, IL-15Rβ, the common gamma chain (γc), an engineered receptor (e.g., CAR, TCR, or TAC), and combinations thereof.

[0120] Exemplary IL-15 fusion polypeptides are listed below in Table 2. It will be understood that fusion proteins comprising any of the IL-15 muteins described herein are contemplated herein.

[0121] [Table 2]

[0122] In some embodiments, the IL-15 polypeptide is a fusion protein comprising an IL-15 fragment fused to the IL-15 receptor (IL-15R), a subunit thereof, or a portion thereof. In some embodiments, the IL-15 polypeptide comprises an IL-15 fragment fused to IL-15Rα. In some embodiments, the IL-15Rα is a full-length IL-15Rα molecule. In some embodiments, the IL-15Rα is a soluble form of IL-15Rα (e.g., sIL-15R or sIL-15Rα). In some embodiments, the IL-15Rα is the extracellular domain of a naturally occurring IL-15Rα molecule. In some embodiments, the IL-15R comprises a truncated or deleted cytoplasmic domain and a transmembrane domain, but retains a functional domain (e.g., a region of the IL-15R required to retain IL-15 binding activity). In some embodiments, the shortest region of IL-15R that retains IL-15 binding activity is the 65 amino acid sequence spanning the sushi domain of IL-15Rα. A "sushi domain" or "short consensus repeat" or "type 1 glycoprotein motif" is a common structural motif that promotes protein-protein interactions, where the motif contains four cysteines that form two disulfide bonds. In some embodiments, the IL-15Rα is the sushi domain of a naturally occurring IL-15Rα molecule. In some embodiments, the IL-15 polypeptide comprises an IL-15 fragment fused to the transmembrane domain of IL-15Rα. In some embodiments, the IL-15 polypeptide comprises an IL-15 fragment fused to the sushi domain and transmembrane domain of IL-15Rα.

[0123] Human IL-15Rα and fragments thereof are shown below. SEQ ID NO:50 (IL-15Rα extracellular domain) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT SEQ ID NO:51 (IL-15Rα sushi domain) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR SEQ ID NO:54 (full length IL-15Rα) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVP GSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL SEQ ID NO:55 (IL-15Rα transmembrane domain) VAISTSTVLLCGLSAVSLLACYL

[0124] In some embodiments, the IL-15 polypeptide comprises an IL-15 fragment fused to IL-15Rβ. In some embodiments, the IL-15Rβ is a full-length IL-15Rβ molecule. In some embodiments, the IL-15Rβ comprises the amino acid sequence of SEQ ID NO:52. SEQ ID NO:52 (full length IL15Rβ) AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQA SHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKF FSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYC TFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[0125] In some embodiments, the IL-15 polypeptide is an IL-15 fragment fused to γc. In some embodiments, γc is a full-length γc molecule. In some embodiments, γc comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the IL-15 polypeptide comprises an IL-15 fragment fused to IL-15Rβ, and the modified immune cell further comprises a heterologous nucleic acid sequence encoding γc. SEQ ID NO:53 (γc, common gamma chain) MLKPSLPFTSLLFLQLPLLGVGLNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMNCTWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEIHLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQLELNWNNRFLNHCLE HLVQYRTDWDHSWTEQSVDYRHKFSLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET

[0126] In some embodiments, the IL-15 polypeptide is membrane-bound. In some embodiments, the IL-15 polypeptide comprises a glycosylphosphatidylinositol (GPI) anchor peptide sequence. In some embodiments, the IL-15 polypeptide comprises a GPI anchor polypeptide sequence at the C-terminus. GPI anchor polypeptide sequences are known in the art and include, but are not limited to, the GPI anchor sequences of human LFA3, CD44, CD59, and human Fc gamma receptor III (CD16b). See Kueng et al., J Virol, 2007, 81(16):8666-8676. In some embodiments, the GPI anchor peptide sequence is linked to a GPI linker.

[0127] In some embodiments, the IL-15 polypeptide comprises an IL-15 fragment fused to a membrane anchor domain. In some embodiments, the membrane anchor domain comprises a sequence that can insert into a phospholipid bilayer (e.g., an amino acid residue having a hydrophobic side chain that interacts with a fatty acyl group of a membrane phospholipid). In some embodiments, the membrane anchor domain comprises a positively charged amino acid sequence. In some embodiments, the membrane anchor domain comprises a lipid.

[0128] In some embodiments, the IL-15 polypeptide comprises a transmembrane domain that can be directly or indirectly fused to an IL-15 fragment. The transmembrane domain can be derived from either natural or synthetic sources. As used herein, "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane, preferably in a eukaryotic cell membrane. The transmembrane domain that can be adapted for use in the IL-15 polypeptide described herein can be obtained from a naturally occurring protein. Alternatively, it can be a synthetic non-natural protein segment, for example, a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

[0129] Transmembrane domains are classified based on the three-dimensional structure of the transmembrane domain. For example, the transmembrane domain may form an alpha helix, a complex of two or more alpha helices, a beta barrel, or any other stable structure that can span the phospholipid bilayer of a cell. Furthermore, transmembrane domains may be further or alternatively classified based on the topology of the transmembrane domain, including the number of times the transmembrane domain passes across the membrane and the orientation of the protein. For example, a single-pass transmembrane protein passes through the cell membrane once, and a multi-pass transmembrane protein passes through the cell membrane at least twice (e.g., two, three, four, five, six, seven, or more times). Membrane proteins may be defined as type I, type II, or type III depending on their termini relative to the interior and exterior of the cell and the topology of the transmembrane segment(s). Type I membrane proteins have a single transmembrane region and are oriented such that the N-terminus of the protein is present on the extracellular side of the lipid bilayer of a cell and the C-terminus of the protein is present on the cytoplasmic side. Type II membrane proteins also have a single transmembrane region, but are oriented such that the C-terminus of the protein is on the extracellular side of the cell's lipid bilayer and the N-terminus of the protein is on the cytoplasmic side. Type III membrane proteins have multiple transmembrane segments and can be further subclassified based on the number of transmembrane segments and the location of the N- and C-termini.

[0130] In some embodiments, the transmembrane domain of the IL-15 polypeptides described herein is derived from a type I single-pass membrane protein. In some embodiments, transmembrane domains from multiple-pass membrane proteins may also be adapted for use in the IL-15 polypeptides described herein. A multiple-pass membrane protein may comprise a complex (at least two, three, four, five, six, seven, or more) alpha-helical or beta-sheet structure. Preferably, the N-terminus and C-terminus of the multiple-pass membrane protein are on opposite sides of the lipid bilayer, e.g., the N-terminus of the protein is on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is on the extracellular side.

[0131] In some embodiments, the transmembrane domain of the IL-15 polypeptide is selected from the group consisting of the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD16 0, CD19, IL-2Rβ, IL-2Rγ, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, IT GAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT In some embodiments, the transmembrane domain is selected from the transmembrane domains of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, 4-1BB, CD80, CD86, CD152, and PD1. In some embodiments, the transmembrane domain is derived from CD8α. In some embodiments, the transmembrane domain is derived from IL-15Rα.

[0132] The transmembrane domain used in the IL-15 polypeptides described herein may comprise at least a portion of a non-naturally occurring synthetic protein segment. In some embodiments, the transmembrane domain is a non-naturally occurring synthetic alpha-helix or beta-sheet. In some embodiments, the protein segment is at least about 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 amino acids, or more. Examples of synthetic transmembrane domains are known in the art (e.g., U.S. Pat. No. 7,052,906 B1 and PCT Publication No. WO 2000 / 032776 A2, the relevant disclosures of which are incorporated herein by reference).

[0133] The transmembrane domain may include a transmembrane region and a cytoplasmic region that is C-terminal to the transmembrane domain. The cytoplasmic region of the transmembrane domain may include three or more amino acids, and in some embodiments, serves to position the transmembrane domain in a lipid bilayer. In some embodiments, one or more cysteine ​​residues are present in the transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine ​​residues are present in the cytoplasmic region of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain includes positively charged amino acids. In some embodiments, the cytoplasmic region of the transmembrane domain includes the amino acids arginine, serine, and lysine.

[0134] In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the transmembrane domain of the IL-15 polypeptide comprises an artificial hydrophobic sequence. For example, a triplet of phenylalanine, tryptophan, and valine may be present at the C-terminus of the transmembrane domain. In some embodiments, the transmembrane region comprises predominantly hydrophobic amino acid residues, e.g., alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a poly-leucine-alanine sequence. The hydropathy or hydrophobic or hydrophilic properties of a protein or protein segment can be assessed by any method known in the art, for example, hydropathy analysis by the Kyte-Doolittle method.

[0135] The IL-15 polypeptide may include a hinge region between the IL-15 fragment and the transmembrane domain. A hinge region is an amino acid segment that is usually found between two domains of a protein and may provide flexibility to the protein, allowing one or both of the domains to move relative to each other. Any amino acid sequence that provides such flexibility and allows the movement of the IL-15 fragment in the IL-15 polypeptide relative to the transmembrane domain may be used.

[0136] The hinge region can comprise any of about 10 to 100 amino acids, e.g., about 15 to 75 amino acids, 20 to 50 amino acids, or 30 to 60 amino acids. In some embodiments, the hinge region can be at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids in length.

[0137] In some embodiments, the hinge region is a naturally occurring protein hinge region. Any protein hinge region known in the art to contain a hinge region can be adapted for use in the IL-15 polypeptides described herein. In some embodiments, the hinge region is at least a portion of a naturally occurring protein hinge region, conferring flexibility to the IL-15 polypeptide. In some embodiments, the hinge region is derived from CD8α. In some embodiments, the hinge region is a portion of the hinge region of CD8α, e.g., a fragment comprising at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge region of CD8α.

[0138] Hinge regions of antibodies, e.g., IgG, IgA, IgM, IgE, or IgD antibodies, are also adaptable for use in the IL-15 polypeptides described herein. In some embodiments, the hinge region is a hinge region that connects constant domains CH1 and CH2 of an antibody. In some embodiments, the hinge region is of an antibody and includes the hinge region of the antibody and one or more constant regions of the antibody. In some embodiments, the hinge region includes the hinge region of the antibody and the CH3 constant region of the antibody. In some embodiments, the hinge region includes the hinge region of the antibody and the CH2 and CH3 constant regions of the antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region includes the hinge region and the CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and CH3 constant region of an IgG1 antibody.

[0139] Non-naturally occurring peptides may also be used as hinge regions of IL-15 polypeptides, in some embodiments, the hinge region is a peptide linker, such as a (GxS)n linker, where x and n can independently be integers between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.

[0140] In some embodiments, the IL-15 polypeptide further comprises an intracellular domain, such as an intracellular signaling domain, hi some embodiments, the IL-15 polypeptide comprises (a) an IL-15 fragment; (b) a transmembrane domain; and (c) an intracellular domain.

[0141] In some embodiments, the IL-15 polypeptide is an engineered receptor comprising: (a) an antigen binding domain; (b) an IL-15 fragment; (c) a transmembrane domain; and (d) an intracellular domain. In some embodiments, the IL-15 polypeptide comprises two or more antigen binding domains. In some embodiments, the IL-15 polypeptide is a monospecific engineered receptor. In some embodiments, the IL-15 polypeptide is a bispecific engineered receptor. In some embodiments, the IL-15 polypeptide is a multispecific engineered receptor. In some embodiments, the IL-15 polypeptide is a multivalent, e.g., bivalent, engineered receptor. In some embodiments, the IL-15 polypeptide is a bi-epitopic engineered receptor. The antigen binding domain may be at the N-terminus or C-terminus of the IL-15 fragment. In some embodiments, the antigen binding domain is fused to the IL-15 fragment via a peptide linker. Exemplary engineered receptors include, but are not limited to, CAR, TCR, and TAC. The IL-15 polypeptide may include any component of the engineered receptors described in the subsection below, "Engineered Receptors."

[0142] In some embodiments, the intracellular domain comprises a costimulatory signaling domain. As used herein, the term "costimulatory signaling domain" refers to at least a portion of a protein that mediates signaling within a cell to elicit an immune response, such as an effector function. The costimulatory signaling domain of the IL-15 polypeptide described herein can be a cytoplasmic signaling domain from a costimulatory protein that transmits a signal and regulates responses mediated by immune cells, such as T cells, NK cells, DCs, lymph node (LN) stromal cells, macrophages, neutrophils, or eosinophils. The "costimulatory signaling domain" can be the cytoplasmic portion of a costimulatory molecule. The term "costimulatory molecule" refers to the cognate binding partner on an immune cell (e.g., T cell) that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the immune cell, such as, but not limited to, proliferation and survival.

[0143] In some embodiments, the intracellular domain comprises a single costimulatory signaling domain. In some embodiments, the intracellular domain comprises two or more (e.g., about any of two, three, four, or more) costimulatory signaling domains. In some embodiments, the intracellular domain comprises two or more of the same costimulatory signaling domains, for example, two copies of the costimulatory signaling domain of CD28. In some embodiments, the intracellular domain comprises two or more costimulatory signaling domains from different costimulatory proteins, for example, any two or more costimulatory proteins described herein. In some embodiments, the one or more costimulatory signaling domains are fused to each other via any peptide linker. The one or more costimulatory signaling domains can be arranged in any suitable order. Multiple costimulatory signaling domains can provide additive or synergistic stimulatory effects.

[0144] Activation of a costimulatory signaling domain in a host cell (e.g., an immune cell) can induce increased or decreased cytokine production and secretion in the cell, enhanced or attenuated phagocytic properties, proliferation, differentiation, survival, and / or cytotoxicity. The type(s) of costimulatory signaling domain are selected based on factors such as the type of immune cell in which the IL-15 polypeptide is expressed (e.g., T cells, NK cells, DCs, stromal cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function. Examples of costimulatory signaling domains for use in IL-15 polypeptides include, but are not limited to, members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6); members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFFR / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, lymphotoxin-α / TNF-β, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-α, and TNF RII / TNFRSF1B); members of the SLAM family (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150);and any other costimulatory molecule, such as the cytoplasmic signaling domain of a costimulatory protein, including CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA class I, HLA-DR, Ikaros, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLPR, lymphocyte function-associated antigen-1 (LFA-1), and NKG2C;

[0145] In some embodiments, the one or more costimulatory signaling domains are selected from the group consisting of a ligand that specifically binds to CD27, CD28, 4-1BB (i.e., CD137), OX40, DAP10, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0146] In some embodiments, the intracellular domain in the IL-15 polypeptide comprises a costimulatory signaling domain derived from CD28. In some embodiments, the intracellular domain in the IL-15 polypeptide comprises a costimulatory signaling domain derived from 4-1BB (i.e., CD137). In some embodiments, the intracellular domain in the IL-15 polypeptide comprises a costimulatory signaling domain derived from OX40. In some embodiments, the intracellular domain in the IL-15 polypeptide comprises a costimulatory signaling domain derived from DAP10. In some embodiments, the intracellular domain in the IL-15 polypeptide comprises a costimulatory signaling domain derived from CD27.

[0147] Variants of any of the costimulatory signaling domains described herein are also within the scope of the present disclosure, such that the costimulatory signaling domain can modulate the immune response of an immune cell. In some embodiments, the costimulatory signaling domain comprises up to 10 (e.g., 1, 2, 3, 4, 5, or 8) amino acid residue mutations compared to the wild-type counterpart. Such costimulatory signaling domains comprising one or more amino acid mutations may be referred to as variants. Mutations of amino acid residues in the costimulatory signaling domain may result in increased signaling and enhanced stimulation of the immune response compared to a costimulatory signaling domain that does not comprise the mutation. Mutations of amino acid residues in the costimulatory signaling domain may result in decreased signaling and reduced stimulation of the immune response compared to a costimulatory signaling domain that does not comprise the mutation.

[0148] In some embodiments, the intracellular domain of the IL-15 polypeptide further comprises a primary intracellular signaling domain, for example, the intracellular signaling domain of CD3ζ.

[0149] In some embodiments, the membrane-bound IL-15 polypeptide further comprises a signal peptide that targets the IL-15 polypeptide to the secretory pathway of the cell (e.g., the ER) and allows for incorporation and anchoring of the IL-15 polypeptide into the lipid bilayer of the host cell. Signal peptides that are suitable for use in the transmembrane IL-15 polypeptides described herein will be apparent to one of skill in the art, including signal sequences of naturally occurring proteins or non-natural synthetic signal sequences. In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, IL-3, and IgG1 heavy chain. In some embodiments, the signal peptide is derived from CD8α.

[0150] In some embodiments, the IL-15 polypeptide comprises an IL-15 fragment and a GPI anchor peptide sequence, wherein the IL-15 fragment comprises an amino acid substitution at positions 8, 62, 3, and / or 25, where the numbering of amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:5 (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:78 (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25. In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F. In some embodiments, the amino acid substitution at position 25 is L25F.In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at both positions 8 and 62. In some embodiments, the GPI anchor peptide sequence is attached to a GPI linker. In some embodiments, the GPI anchor peptide sequence is located at the C-terminus of the IL-15 polypeptide.

[0151] In some embodiments, the IL-15 polypeptide comprises an IL-15 fragment and a transmembrane domain, wherein the IL-15 fragment comprises an amino acid substitution at positions 8, 62, 3, and / or 25, where the numbering of amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:5 (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:78 (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25. In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F. In some embodiments, the amino acid substitution at position 25 is L25F.In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at both positions 8 and 62. In some embodiments, the transmembrane domain is the transmembrane domain of IL-15Rα.

[0152] In some embodiments, the IL-15 polypeptide comprises an IL-15 fragment, a transmembrane domain, and an intracellular domain, wherein the IL-15 fragment comprises an amino acid substitution at positions 8, 62, 3, and / or 25, where the numbering of amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:5 (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:78 (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25. In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F. In some embodiments, the amino acid substitution at position 25 is L25F.In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at both positions 8 and 62. In some embodiments, the transmembrane domain is the transmembrane domain of CD4, CD3, CD8α, or CD28. In some embodiments, the IL-15 polypeptide further comprises a hinge domain, e.g., a hinge domain derived from CD8. In some embodiments, the intracellular domain comprises a primary intracellular signaling domain, e.g., the intracellular signaling domain of CD3ζ. In some embodiments, the intracellular domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, DAP10, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the intracellular domain comprises the costimulatory signaling domain of 4-1BB and the primary intracellular signaling domain of CD3zeta.

[0153] In some embodiments, the IL-15 polypeptide comprises an IL-15 fragment, a transmembrane domain, and a costimulatory signaling domain, wherein the IL-15 fragment comprises an amino acid substitution at positions 8, 62, 3, and / or 25, where the numbering of amino acid residue positions is according to SEQ ID NO:1. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:5 (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO:78 (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25. In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F.In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at both positions 8 and 62. In some embodiments, the transmembrane domain is the transmembrane domain of CD4, CD3, CD8α, or CD28. In some embodiments, the IL-15 polypeptide further comprises a hinge domain, e.g., a hinge domain derived from CD8. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, DAP10, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof.

[0154] The IL-15 polypeptide may include one or more peptide linkers disposed between different domains. For example, the IL-15 fragment and the second polypeptide fragment may be fused to each other via a peptide bond or peptide linker. The peptide linkers connecting the different domains may be the same or different. Each peptide linker may be individually optimized. The peptide linker may be of any suitable length. In some embodiments, the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50 amino acids in length or longer. In some embodiments, the peptide linker is about 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 amino acids in length or less, or any of the following: In some embodiments, the length of the peptide linker is about 1 to about 10 amino acids, about 1 to about 20 amino acids, about 1 to about 30 amino acids, about 5 to about 15 amino acids, about 10 to about 25 amino acids, about 5 to about 30 amino acids, about 10 to about 30 amino acids in length, about 30 to about 50 amino acids, or about 1 to about 50 amino acids.

[0155] The peptide linker may have a naturally occurring or non-naturally occurring sequence. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n (SEQ ID NO:67), (GSGGS) n (SEQ ID NO: 68), and (GGGS) n (SEQ ID NO:69), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. In some embodiments, the peptide linker has the amino acid sequence of SEQ ID NO:40 or 41.

[0156] In some embodiments, the sequence encoding the IL-15 polypeptide lacks some or all of the sequence upstream of the putative start codon. In some embodiments, the IL-15 polypeptide may contain certain amino acid mutations that do not affect (e.g., functionally affect) the binding of IL-15 to IL-15R. In some embodiments, the IL-15 polypeptide contains nucleotide changes (e.g., nucleotide substitutions, deletions, and / or additions). In some embodiments, the nucleotide changes occur in the mature IL-15 sequence to generate a mutant IL-15 polypeptide. The nucleotide changes may result in improved substrate specificity and function (e.g., anti-tumor effect) of the IL-15 polypeptide without overproduction of inflammatory cytokines.

[0157] Further description of IL-15 and IL-15 interactions is generally known in the art, see, e.g., US9,389,236, US10,464,993, US9,629,877, EP1777294A1, US10,428,305, US7,998,736, US9,303,080, and US9,931,377, which are incorporated herein by reference.

[0158] In some embodiments, the IL-15 polypeptide comprises an amino acid sequence variant of an IL-15 polypeptide described herein. For example, an amino acid sequence variant may be desired to modulate the binding affinity and / or other biological properties of the IL-15 polypeptide. An amino acid sequence variant of an IL-15 polypeptide may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the IL-15 polypeptide or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the IL-15 polypeptide. Any combination of deletions, insertions, and substitutions may be made to arrive at a final construct, provided that the final construct has the desired characteristics, e.g., TLR binding activity and / or pro-inflammatory activity.

[0159] In some embodiments, the IL-15 polypeptide comprises one or more (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20 or more amino acids) conservative substitutions compared to the sequence of any of the IL-15 polypeptides described herein. In some embodiments, the IL-15 polypeptide comprises at least about 80% sequence identity, e.g., at least about 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of any of the IL-15 polypeptides described herein. Like the IL-15 polypeptides described herein, the IL-15 polypeptide variants have similar anti-tumor activity and reduced toxicity.

[0160] Conservative substitutions are shown in Table A below. [Table A]

[0161] Amino acids can be divided into different classes according to common side chain properties: a. Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; b. Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c. Acidic: Asp, Glu; d. Basic: His, Lys, Arg; e. Residues that affect chain orientation: Gly, Pro; f. Aromatics: Trp, Tyr, Phe.

[0162] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0163] Those skilled in the art will understand that any suitable method can be used to generate mutations in the gene of interest, including mutagenesis, polymerase chain reaction, homologous recombination, or any other genetic engineering techniques known to those skilled in the art.Mutation can involve a single nucleotide (e.g., point mutation involving the removal, addition, or substitution of a single nucleotide base in a DNA sequence), or can involve the insertion or deletion of multiple nucleotides.Mutation can occur spontaneously as a result of events such as errors in the fidelity of DNA replication, or can be induced after exposure to chemical or physical mutagenic agents.Mutation can be site-specific by using specific targeting methods well known to those skilled in the art.

[0164] A useful method for identifying residues or regions of a polypeptide that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues, e.g., arg, asp, his, lys, and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the polypeptide drug with its target (e.g., IL-15 variant and IL-15 receptor) is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. The variants can be screened to determine whether they have the desired properties.

[0165] Amino acid sequence insertions include amino- and / or carboxy-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.

[0166] In some embodiments, a peptide tag (usually a short peptide sequence capable of being recognized by available antisera or chemical compounds) may be included for subsequent expression and trafficking of the IL-15 polypeptide. Many types of tag peptides may be used in the IL-15 polypeptides described herein, including but not limited to PK tags, FLAG octapeptides, MYC tags, HIS tags (usually a stretch of 4-10 histidine residues), and E tags (US 6,686,152). The tag peptide(s) may be independently located at the N-terminus of the protein, the C-terminus of the protein, internally, or at any of these locations if several tags are used. Tag peptides may be detected by immunodetection assays using anti-tag antibodies.

[0167] Engineered receptors Any of the above modified immune cells may further express an engineered receptor. Exemplary engineered receptors include, but are not limited to, CAR, engineered TCR, and TAC receptors. In some embodiments, the engineered receptor comprises an extracellular domain that specifically binds an antigen (e.g., a tumor antigen), a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain and / or a costimulatory domain. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain of a TCR co-receptor. In some embodiments, the engineered receptor is encoded by a heterologous nucleic acid sequence that encodes an IL-15 polypeptide. In some embodiments, the engineered receptor is encoded by a second heterologous nucleic acid operably linked to a promoter (e.g., a constitutive promoter or an inducible promoter). In some embodiments, the engineered receptor is introduced into the modified immune cell by passing the cell through a microfluidic system, such as CELL SQUEEZE® (see, e.g., U.S. Patent Publication No. 20140287509), while simultaneously inserting the protein into the cell membrane. The engineered receptor may enhance the function of the engineered immune cell, for example, by targeting the engineered immune cell, by transmitting a signal, and / or by enhancing the cytotoxicity of the engineered immune cell. In some embodiments, the engineered immune cell does not express an engineered receptor, e.g., a CAR, TCR, or TAC receptor.

[0168] In some embodiments, the engineered receptor comprises one or more specific binding domains that target at least one tumor antigen, and one or more intracellular effector domains, such as one or more primary intracellular signaling domains and / or costimulatory domains.

[0169] In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR). Many chimeric antigen receptors are known in the art and can be suitable for the modified immune cells of the present application. For example, by utilizing the antigen-binding fragment of an antibody molecule or an antibody variable domain, CARs with specificity for any cell surface marker can also be constructed. Any method for making CARs can be used herein. See, e.g., US6,410,319, US7,446,191, US7,514,537, US9765342B2, WO2002 / 077029, WO2015 / 142675, US2010 / 065818, US2010 / 025177, US2007 / 059298, WO2017025038A1, and Berger C. et al., J. Clinical Investigation 118:1 294-308 (2008), which are incorporated by reference herein. In some embodiments, the engineered immune cells are CAR-T cells.

[0170] The CAR of the present application comprises an extracellular domain comprising at least one targeting domain that specifically binds to at least one tumor antigen, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain generates a signal that promotes an immune effector function of a cell bearing the CAR, e.g., a CAR-T cell. An "immune effector function or immune effector response" refers, for example, to a function or response of an immune effector cell that enhances or promotes an immune attack on a target cell. For example, an immune effector function or response can refer to a property of a T cell or NK cell that promotes killing or inhibiting growth or proliferation of a target cell. For example, examples of immune effector functions in CAR-T cells include cytolytic activity (e.g., antibody-dependent cellular cytotoxicity or ADCC) and helper activity (e.g., secretion of cytokines). In some embodiments, a CAR has an intracellular signaling domain with attenuated immune effector function. In some embodiments, the CAR has an intracellular signaling domain that has no more than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less of an immune effector function (e.g., cytolytic function against a target cell) compared to a CAR with full-length and wild-type CD3ζ, and optionally one or more costimulatory domains. In some embodiments, the intracellular signaling domain generates a signal that promotes the proliferation and / or survival of a cell containing the CAR. In some embodiments, the CAR comprises one or more intracellular signaling domains selected from the signaling domains of CD28, CD137, CD3, CD27, CD40, ICOS, GITR, and OX40. The signaling domain of a naturally occurring molecule can include the entire intracellular (i.e., cytoplasmic) portion of the molecule, or the entire naturally occurring intracellular signaling domain, or a fragment or derivative thereof.

[0171] In some embodiments, the intracellular signaling domain of the CAR comprises a primary intracellular signaling domain. "Primary intracellular signaling domain" refers to a cytoplasmic signaling sequence that acts stimulatorily to induce immune effector function. In some embodiments, the primary intracellular signaling domain comprises a signaling motif known as an immune receptor tyrosine-based activation motif or ITAM. In some embodiments, the primary intracellular signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3zeta, CD3gamma, CD3delta, CD3epsilon, common FcRgamma (FCER1G), FcRbeta (FcεRib), CD79a, CD79b, FcγRIIa, DAP10, and DAP12. In some embodiments, the primary intracellular signaling domain comprises a non-functional or attenuated signaling domain of a protein selected from the group consisting of CD3zeta, CD3gamma, CD3delta, CD3epsilon, common FcRgamma (FCER1G), FcRbeta (FcεRib), CD79a, CD79b, FcγRIIa, DAP10, and DAP12. The non-functional or attenuated signaling domain can be a mutant signaling domain with a point mutation, insertion, or deletion that attenuates or eliminates one or more immune effector functions, such as cytolytic or helper activity, including antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the CAR comprises a non-functional or attenuated CD3zeta (i.e., CD3zeta or CD3z) signaling domain. In some embodiments, the intracellular signaling domain does not comprise a primary intracellular signaling domain. An attenuated primary intracellular signaling domain may elicit no more than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less immune effector function (e.g., cytolytic function against target cells) compared to a CAR having the same structure but a wild-type primary intracellular signaling domain.

[0172] In some embodiments, the intracellular signaling domain of the CAR comprises one or more (e.g., any one, two, three, or more) costimulatory domains. A "costimulatory domain" can be the intracellular portion of a costimulatory molecule. The term "costimulatory molecule" refers to a cognate binding partner on an immune cell (e.g., T cell) that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the immune cell, such as, but not limited to, proliferation and survival. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. Costimulatory molecules can be found in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, These include ligands that specifically bind to ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83.

[0173] In some embodiments, the CAR comprises a single costimulatory domain. In some embodiments, the CAR comprises two or more costimulatory domains. In some embodiments, the intracellular signaling domain comprises a functional primary intracellular signaling domain and one or more costimulatory domains. In some embodiments, the CAR does not comprise a functional primary intracellular signaling domain (e.g., CD3ζ). In some embodiments, the CAR comprises an intracellular signaling domain that consists of, or essentially consists of, one or more costimulatory domains. In some embodiments, the CAR comprises an intracellular signaling domain that consists of, or essentially consists of, a non-functional or attenuated primary intracellular signaling domain (e.g., mutant CD3ζ) and one or more costimulatory domains. Upon binding of the targeting domain to a tumor antigen, the costimulatory domain of the CAR can transmit signals for enhanced proliferation, survival, and differentiation of engineered immune cells (e.g., T cells) bearing the CAR, and can inhibit activation-induced cell death. In some embodiments, the one or more costimulatory signaling domains are derived from one or more molecules selected from the group consisting of CD27, CD28, 4-1BB (i.e., CD137), OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds CD83.

[0174] In some embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling domain derived from CD28. In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of CD3zeta and a costimulatory signaling domain of CD28. In some embodiments, the intracellular signaling domain in the chimeric receptor of the present application comprises a costimulatory signaling domain derived from 4-1BB (i.e., CD137). In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of CD3zeta and a costimulatory signaling domain of 4-1BB.

[0175] In some embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling domain of CD28 and a costimulatory signaling domain of 4-1BB. In some embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of CD3zeta, a costimulatory signaling domain of CD28, and a costimulatory signaling domain of 4-1BB. In some embodiments, the intracellular signaling domain comprises a polypeptide that comprises, from N-terminus to C-terminus, a costimulatory signaling domain of CD28, a costimulatory signaling domain of 4-1BB, and a cytoplasmic signaling domain of CD3zeta.

[0176] In some embodiments, the targeting domain of the CAR is an antibody or antibody fragment, such as an scFv, Fv, Fab, (Fab')2, single domain antibody (sdAb), or VFv. HIn some embodiments, the targeting domain of the CAR is an extracellular portion of a ligand or receptor that specifically binds to a tumor antigen. In some embodiments, one or more targeting domains of the CAR specifically bind to a single tumor antigen. In some embodiments, the CAR is a bispecific or multispecific CAR with targeting domains that bind to two or more tumor antigens. In some embodiments, the tumor antigen is selected from the group consisting of GPC3, CD19, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, CD20, CD22, CD33, CD38, CEA, EGFR (e.g., EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, and other tumor antigens of clinical significance, and combinations thereof. In some embodiments, the CAR specifically binds to a target antigen selected from the group consisting of BCMA, NY-ESO-1, VEGFR2, MAGE-A3, AFP, CD4, CD19, CD20, CD22, CD30, CD33, CD38, CD70, CD123, CEA, EGFR (e.g., EGFRvIII), GD2, GPC-2, GPC3, CLDN18.2, HER2, LILRB4, IL-13Rα2, IGF1R, mesothelin, PSMA, ROR1, WT1, NKG2D, CLL1, TGFaRII, TGFbRII, CCR5, CXCR4, CCR4, an HPV-associated antigen, and an EBV-associated antigen (e.g., LMP1 or LMP2).

[0177] In some embodiments, the CAR is a BCMA CAR. B-cell maturation antigen (BCMA), also known as CD269, belongs to the tumor necrosis factor receptor superfamily (Thompson et al., J. Exp. Medicine, 192(1):129-135, 2000). Human BCMA is expressed in plasma cells and can bind to B-cell activating factor (BAFF) and proliferation-inducing ligand (APRIL) (e.g., Mackay et al., 2003 and Kalled et al., Immunological Review, 204:43-54, 2005). BCMA can be used as an immunotherapeutic agent for various cancers, for example, as a target antigen for CAR-T cells. Thus, anti-BCMA antibodies (e.g., BCMA single domain antibodies) can be used in combination with cellular immunotherapy using CAR-T cells to enhance the cytotoxic effect against tumor cells. A wide variety of antigen binding domain sequences can be used as the targeting domain of BCMA CAR. See, e.g., WO2017 / 025038, which is incorporated herein in its entirety. In some embodiments, the BCMA CAR comprises, from N-terminus to C-terminus, a CD8 signal peptide (SP), an anti-BCMA sdAb, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB intracellular costimulatory domain, and a CD3ζ intracellular signaling domain. In some embodiments, the BCMA CAR comprises, from N-terminus to C-terminus, a CD8α signal peptide, a first anti-BCMA VHH, an optional linker, a second anti-BCMA VHH, a CD8α hinge, a CD8α transmembrane domain, a 4-1BB intracellular costimulatory domain, and a CD3ζ intracellular signaling domain. In some embodiments, the anti-BCMA VHH-VHH domain comprises the amino acid sequence of SEQ ID NO:23. In some embodiments, the BCMA CAR comprises the amino acid sequence of SEQ ID NO:26.

[0178] In some embodiments, the CAR is a CD19 CAR. CD19 is a B cell surface protein that is expressed throughout B cell development and is therefore expressed on almost all B cell malignancies, including several types of leukemia and many non-Hodgkin's lymphomas (Scheuermann RH and Racila E. Leuk Lymphoma. 1995; 18(5-6): 385-397). The near ubiquitous expression and specificity for a single cell lineage make CD19 an attractive target for CAR-modified T cell therapy. A wide variety of antigen binding domain sequences can be used as the targeting domain of the CD19 CAR. See, for example, WO2012 / 079000, which is incorporated herein in its entirety. In some embodiments, the CD19 CAR comprises, from N-terminus to C-terminus, a CD8α signal peptide, a CD19 scFv, a CD8α hinge, a CD8α transmembrane domain, a 4-1BB intracellular costimulatory domain, and a CD3ζ intracellular signaling domain. In some embodiments, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the CD19 CAR comprises the amino acid sequence of SEQ ID NO: 27.

[0179] In some embodiments, the CAR is a GPC3 CAR. Glypican-3 (GPC3) belongs to the glypican family, a group of heparan sulfate proteoglycans that are attached to the cell surface via a glycosylphosphatidylinositol anchor. GPC3 is highly expressed in a variety of pediatric solid embryonal tumors, including the majority of hepatoblastomas, Wilms' tumors, rhabdomyosarcomas, certain germ cell tumor subtypes, and a minority of rhabdomyosarcomas. A wide variety of antigen-binding domain sequences can be used as the targeting domain of the GPC3 CAR. See, for example, WO2016 / 049459, which is incorporated herein in its entirety. In some embodiments, the GPC3 CAR comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, a GPC3 scFv, a CD8α hinge, a CD8α transmembrane domain, a 4-1BB intracellular costimulatory domain, and a CD3ζ intracellular signaling domain. In some embodiments, the anti-GPC3 scFv comprises the amino acid sequence of SEQ ID NO:80. In some embodiments, the GPC3 CAR comprises the amino acid sequence of SEQ ID NO:81.

[0180] In some embodiments, the transmembrane domain of the CAR is selected from the group consisting of the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160 , CD19, IL-2Rβ, IL-2Rγ, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, IT GAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The CAR comprises a transmembrane domain selected from the transmembrane domains of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In some embodiments, the transmembrane domain of the CAR is the transmembrane domain of CD4, CD3, CD8α, or CD28. In some embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of CD8α.

[0181] In some embodiments, the extracellular domain is linked to the transmembrane domain by a hinge region. In one embodiment, the hinge region comprises the hinge region of CD8α.

[0182] In some embodiments, the CAR comprises a signal peptide, such as CD8αSP.

[0183] In some embodiments, the engineered receptor is a modified T cell receptor. In some embodiments, the engineered TCR is specific for a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of GPC3, CD19, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, VEGFR2, MAGE-A3, CD20, CD22, CD33, CD38, CEA, EGFR (e.g., EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, and other tumor antigens of clinical significance. In some embodiments, the tumor antigen is derived from an intracellular protein of a tumor cell. Numerous TCRs specific for tumor antigens (including tumor-associated antigens) have been described, including, for example, TCRs for NY-ESO-1 cancer testis antigen, p53 tumor suppressor antigen, melanoma (e.g., MARTI, gp100), leukemia (e.g., WT1, non-major histocompatibility antigen), and breast cancer (e.g., HER2, NY-BR1). Any TCR known in the art may be used in the present application. In some embodiments, the TCR has enhanced affinity for the tumor antigen. Exemplary TCRs and methods for introducing TCRs into immune cells are described, for example, in US5830755 and Kessels et al. Immunotherapy through TCR gene transfer. Nat. Immunol. 2, 957-961 (2001). In some embodiments, the modified immune cell is a TCR-T cell.

[0184] The TCR receptor complex is an octameric complex formed by the variable TCR receptor α and β chains (γ and δ chains in the case of γδ T cells) and three dimeric signaling modules, CD3δ / ε, CD3γ / ε, and CD247 (T cell surface glycoprotein CD3ζ chain) ζ / ζ or ζ / η. Ionic residues in the transmembrane domains of each subunit form a network of polar interactions that hold the complex together. The TCR complex functions to activate a signaling cascade in T cells.

[0185] In some embodiments, the engineered receptor is an engineered TCR that includes one or more T cell receptor (TCR) fusion proteins (TFPs). Exemplary TFPs are described, for example, in US20170166622A1, which is incorporated herein by reference. In some embodiments, the TFP includes an extracellular domain of a TCR subunit, including an extracellular domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, a functional fragment thereof, and an amino acid sequence thereof having at least one, but not more than 20 modifications, or a portion thereof. In some embodiments, the TFP includes a transmembrane domain, including a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, a functional fragment thereof, and an amino acid sequence thereof having at least one, but not more than 20 modifications. In some embodiments, the TFP comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of TCR alpha chain, TCR beta chain, TCR zeta chain, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.

[0186] In some embodiments, the TFP comprises a TCR subunit comprising at least a portion of the TCR extracellular domain, and a TCR intracellular domain that comprises a stimulatory domain derived from the intracellular signaling domain of CD3ε; and an antigen binding domain, wherein the TCR subunit and the antigen binding domain are operably linked and the TFP is incorporated into the TCR when expressed in a T cell.

[0187] In some embodiments, the TFP comprises a TCR subunit comprising at least a portion of the TCR extracellular domain, and a TCR intracellular domain that comprises a stimulatory domain derived from the intracellular signaling domain of CD3γ; and an antigen binding domain, wherein the TCR subunit and the antigen binding domain are operably linked and the TFP is incorporated into the TCR when expressed in a T cell.

[0188] In some embodiments, the TFP comprises a TCR subunit comprising at least a portion of the TCR extracellular domain, and a TCR intracellular domain that comprises a stimulatory domain derived from the intracellular signaling domain of CD3δ; and an antigen binding domain, wherein the TCR subunit and the antigen binding domain are operably linked and the TFP is incorporated into the TCR when expressed in a T cell.

[0189] In some embodiments, the TFP comprises a TCR subunit comprising at least a portion of the TCR extracellular domain, and a TCR intracellular domain that comprises a stimulatory domain derived from the intracellular signaling domain of TCRα; and an antigen binding domain, wherein the TCR subunit and the antigen binding domain are operably linked, and the TFP is incorporated into the TCR when expressed in a T cell.

[0190] In some embodiments, the TFP comprises a TCR subunit comprising at least a portion of the TCR extracellular domain, and a TCR intracellular domain that comprises a stimulatory domain derived from the intracellular signaling domain of TCRβ; and an antigen binding domain, wherein the TCR subunit and the antigen binding domain are operably linked, and the TFP is incorporated into the TCR when expressed in a T cell.

[0191] In some embodiments, the engineered receptor is a T cell antigen conjugate (TAC) receptor. Exemplary TAC receptors are described, for example, in US20160368964A1, which is incorporated herein by reference. In some embodiments, the TAC comprises a targeting domain, a TCR binding domain that specifically binds to a protein associated with the TCR complex, and a T cell receptor signaling domain. In some embodiments, the targeting domain is an antibody fragment, e.g., an scFv or V that specifically binds to a tumor antigen. H H. In some embodiments, the targeting domain is a designed ankyrin repeat (DARPin) polypeptide. In some embodiments, the tumor antigen is selected from the group consisting of GPC3, CD19, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, VEGFR2, MAGE-A3, CD20, CD22, CD33, CD38, CEA, EGFR (e.g., EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, and other tumor antigens of clinical significance. In some embodiments, the protein associated with the TCR complex is CD3, e.g., CD3ε. In some embodiments, the TCR binding domain is a single chain antibody, e.g., scFv or V H H. In some embodiments, the TCR binding domain is derived from UCHT1. In some embodiments, the TAC receptor comprises a cytoplasmic domain and a transmembrane domain. In some embodiments, the T cell receptor signaling domain comprises a cytoplasmic domain derived from a TCR co-receptor. Exemplary TCR co-receptors include, but are not limited to, CD4, CD8, CD28, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD 154. In some embodiments, the TAC receptor comprises a transmembrane domain and a cytoplasmic domain derived from CD4. In some embodiments, the TAC receptor comprises a transmembrane domain and a cytoplasmic domain derived from CD8 (e.g., CD8α).

[0192] T cell coreceptors are expressed as membrane proteins on T cells. They can result in the stabilization of the TCR / peptide / MHC complex and promote signal transduction. Two subtypes of T cell coreceptors, CD4 and CD8, show strong specificity for certain MHC classes. The CD4 coreceptor can only stabilize the TCR / MHCII complex, whereas the CD8 coreceptor can only stabilize the TCR / MHC1 complex. Differential expression of CD4 and CD8 on different T cell types results in different functional subpopulations of T cells. CD8+ T cells are cytotoxic T cells.

[0193] CD4 is a glycoprotein expressed on the surface of immune cells such as helper T cells, monocytes, macrophages, and dendritic cells. CD4 has four immunoglobulin domains (D1-D4) exposed on the extracellular cell surface. CD4 contains a special amino acid sequence in its short cytoplasmic / intracellular tail that allows the CD4 tail to recruit and interact with the tyrosine kinase Lck. When the TCR complex and CD4 each bind to different regions of the MHCII molecule, the proximity of the TCR complex and CD4 allows Lck to bind to the cytoplasmic tail of CD4 and tyrosine phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domain of CD3, thereby amplifying the signal generated by the TCR.

[0194] CD8 is a glycoprotein that is either a homodimer (rarely seen) composed of two α chains, each containing an immunoglobulin variable (IgV)-like extracellular domain and an intracellular tail linked to the membrane by a thin stalk, or a heterodimer (more common) composed of one α and one β chain. CD8 is expressed primarily on the surface of cytotoxic T cells, but can also be found on natural killer cells, cortical thymocytes, and dendritic cells. The CD8 cytoplasmic tail interacts with Lck, which phosphorylates the cytoplasmic CD3 and ζ chains of the TCR complex upon binding of the TCR to its specific antigen. Tyrosine phosphorylation of the cytoplasmic CD3 and ζ chains initiates a phosphorylation cascade that ultimately results in gene transcription.

[0195] In some embodiments, the modified immune cells express any combination of two or more engineered receptors, e.g., CAR, TCR, TAC receptors.

[0196] In some embodiments, the engineered receptor (e.g., CAR, TCR, or TAC) expressed by modified immune cells targets one or more tumor antigens. Tumor antigens are proteins produced by tumor cells that can induce immune responses, particularly immune responses mediated by T cells. The selection of the antigens targeted in the present disclosure depends on the specific type of cancer to be treated. Exemplary tumor antigens include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, HER2 / neu, survivin, and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0197] In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express a number of proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-associated molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins become the actual tumor-specific immunoglobulin antigens that are unique to each individual tumor. B-cell differentiation antigens, such as CD19, CD20, and CD37, are other candidates for target antigens in B-cell lymphomas.

[0198] In some embodiments, the tumor antigen is a tumor specific antigen (TSA) or tumor associated antigen (TAA). TSA is unique to tumor cells and does not occur in other cells in the body. TAA associated antigens are not unique to tumor cells and are also expressed in normal cells under conditions that cannot induce a state of immune tolerance to the antigen. Expression of antigens in tumors can occur under conditions that allow the immune system to respond to the antigen. TAA can be an antigen that is expressed in normal cells during fetal development, when the immune system is immature and cannot respond, or it can be an antigen that is usually present at very low levels in normal cells, but is expressed at very high levels in tumor cells.

[0199] Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed fetal antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG-72, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, and BTAA. , CA125, CA15-3\CA27.29\BCAA, CA195, CA242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0200] nucleic acid The modified immune cells described herein contain one or more heterologous nucleic acid sequence(s) encoding any of the IL-15 polypeptides and / or engineered receptors described herein.

[0201] Another aspect of the application provides an isolated nucleic acid comprising a nucleic acid sequence encoding any of the IL-15 polypeptides described herein. In some embodiments, an isolated nucleic acid comprising a nucleic acid sequence encoding any of the engineered receptors described herein is provided. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is linear. In some embodiments, the nucleic acid is circular.

[0202] The nucleic acid sequence encoding an IL-15 polypeptide and / or the nucleic acid encoding an engineered receptor may be operably linked to one or more regulatory sequences. Exemplary regulatory sequences that control transcription and / or translation of the coding sequence are known in the art and may include, but are not limited to, promoters, additional elements for proper initiation, regulation, and / or termination of transcription (e.g., polyA transcription termination sequences), mRNA transport (e.g., nuclear localization signal sequences), processing (e.g., splicing signals), stability (e.g., introns and non-coding 5' and 3' sequences), translation (e.g., initiation methionine, tripartite leader sequence, IRES ribosome binding site, signal peptide, etc.), and insertion sites for introducing inserts into viral vectors. In some embodiments, the regulatory sequences are promoters, transcription enhancers, and / or sequences that allow for proper expression of the IL-15 polypeptide and / or the engineered receptor.

[0203] The term "regulatory sequence" or "control sequence" refers to a DNA sequence that influences the expression of an operably linked coding sequence. The nature of such regulatory sequences varies depending on the host organism. In prokaryotes, regulatory sequences generally include promoters, ribosome binding sites, and terminators. In eukaryotes, regulatory sequences include promoters, terminators, and in some cases, enhancers, transactivators, or transcription factors.

[0204] The term "operably linked" refers to a juxtaposition in a relationship permitting the components so described to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the regulatory sequences.

[0205] As used herein, a "promoter" or "promoter region" refers to a segment of DNA or RNA that controls the transcription of DNA or RNA to which it is operably linked. A promoter region contains specific sequences involved in RNA polymerase recognition, binding, and transcription initiation. In addition, a promoter contains sequences that regulate RNA polymerase recognition, binding, and transcription initiation activity (i.e., binding of one or more transcription factors). These sequences can be cis-acting or responsive to trans-acting factors. Promoters can be constitutive or regulated, depending on the nature of the regulation. Regulated promoters can be inducible or environmentally responsive (e.g., responsive to cues such as pH, anaerobic conditions, osmolytes, temperature, light, or cell density). Many such promoter sequences are known in the art. See, e.g., U.S. Patent Nos. 4,980,285; 5,631,150; 5,707,928; 5,759,828; 5,888,783; 5,919,670, and Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989).

[0206] In some embodiments, the nucleic acid sequence encoding the IL-15 polypeptide is operably linked to a first promoter. In some embodiments, the nucleic acid sequence encoding the engineered receptor is operably linked to a second promoter. In some embodiments, the nucleic acid sequence encoding the IL-15 polypeptide and the nucleic acid sequence encoding the engineered receptor are operably linked to the same promoter. In some embodiments, the nucleic acid sequence encoding the IL-15 polypeptide and the nucleic acid sequence encoding the engineered receptor are operably linked to separate promoters.

[0207] In some embodiments, the promoter is an endogenous promoter. For example, a nucleic acid encoding an IL-15 polypeptide and / or an engineered receptor can be knocked into the genome of the modified immune cell downstream of an endogenous promoter using any method known in the art, such as CRISPR / Cas9 technology. In some embodiments, the endogenous promoter is a promoter of an abundant protein, such as β-actin. In some embodiments, the endogenous promoter is an inducible promoter, e.g., inducible by an endogenous activation signal of the modified immune cell. In some embodiments, when the modified immune cell is a T cell, the promoter is a T cell activation-dependent promoter (e.g., an IL-2 promoter, an NFAT promoter, or an NFκB promoter). In some embodiments, the promoter is a heterologous promoter.

[0208] A variety of promoters for gene expression in mammalian cells have been explored, and any of the promoters known in the art may be used in the present application. Promoters may be broadly categorized as constitutive promoters or regulated promoters, such as inducible promoters. In some embodiments, the heterologous nucleic acid sequence encoding the IL-15 polypeptide and / or engineered receptor is operably linked to a constitutive promoter. In some embodiments, the heterologous nucleic acid sequence encoding the IL-15 polypeptide and / or engineered receptor is operably linked to an inducible promoter. In some embodiments, the constitutive promoter is operably linked to the nucleic acid sequence encoding the IL-15 polypeptide, and the inducible promoter is operably linked to the nucleic acid sequence encoding the engineered receptor. In some embodiments, the constitutive promoter is operably linked to the nucleic acid sequence encoding the engineered receptor, and the inducible promoter is operably linked to the nucleic acid sequence encoding the IL-15 polypeptide. In some embodiments, a first inducible promoter is operably linked to the nucleic acid sequence encoding the IL-15 polypeptide, and a second inducible promoter is operably linked to the nucleic acid sequence encoding the engineered receptor. In some embodiments, the first inducible promoter is inducible by a first induction condition and the second inducible promoter is inducible by a second induction condition. In some embodiments, the first induction condition is the same as the second induction condition. In some embodiments, the first inducible promoter and the second inducible promoter are induced simultaneously. In some embodiments, the first inducible promoter and the second inducible promoter are induced sequentially, for example, the first inducible promoter is induced before the second inducible promoter or the first inducible promoter is induced after the second inducible promoter.

[0209] A constitutive promoter allows a heterologous gene (also referred to as a transgene) to be constitutively expressed in a host cell. Exemplary constitutive promoters contemplated herein include, but are not limited to, cytomegalovirus (CMV) promoter, human elongation factor-1 alpha (hEF1 alpha), ubiquitin C promoter (UbiC), phosphoglycerokinase promoter (PGK), simian virus 40 early promoter (SV40), and chicken β-actin promoter (CAGG) linked to the CMV early enhancer. The efficiency of such constitutive promoters in driving transgene expression has been widely compared in a large number of studies. In some embodiments, the promoter is a hEF1 alpha promoter.

[0210] In some embodiments, the promoter is an inducible promoter. Inducible promoters belong to the category of regulated promoters. Inducible promoters can be induced by one or more conditions, such as physical conditions, the microenvironment of the modified immune cell, or the physiological state of the modified immune cell, an inducer (e.g., an inducer), or a combination thereof. In some embodiments, the induction condition does not induce the expression of an endogenous gene in the modified immune cell and / or an endogenous gene in a subject administered the pharmaceutical composition. In some embodiments, the induction condition is selected from the group consisting of: an inducer, irradiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox conditions, tumor environment, and activation state of the modified immune cell.

[0211] In some embodiments, the promoter is induced by an inducer. In some embodiments, the inducer is a small molecule, such as a chemical compound. In some embodiments, the small molecule is selected from the group consisting of doxycycline, tetracycline, alcohol, metals, or steroids. Chemically inducible promoters are the most widely explored. Such promoters include promoters whose transcriptional activity is regulated by the presence or absence of small chemical molecules, such as doxycycline, tetracycline, alcohol, steroids, metals, and other compounds. The doxycycline-inducible system using reverse tetracycline-controlled transactivator (rtTA) and tetracycline-responsive element promoter (TRE) is currently the most established system. WO9429442 describes the tight control of gene expression in eukaryotic cells by tetracycline-responsive promoters. WO9601313 discloses tetracycline-controlled transcriptional modulators. Additionally, Tet technologies, such as the Tet-on system, are described, for example, on the TetSystems.com website. Any of the known chemically regulated promoters can be used to drive expression of the therapeutic proteins of the present application.

[0212] In some embodiments, the inducer is a polypeptide, such as a growth factor, a hormone, or a ligand for a cell surface receptor, e.g., a polypeptide that specifically binds to a tumor antigen. In some embodiments, the polypeptide is expressed by the modified immune cell. In some embodiments, the polypeptide is encoded by a nucleic acid in a heterologous nucleic acid. Many polypeptide inducers are also known in the art and may be suitable for use in the present application. For example, ecdysone receptor-based gene switches, progesterone receptor-based gene switches, and estrogen receptor-based gene switches belong to gene switches that use transactivators derived from steroid receptors (e.g., WO9637609 and WO9738117).

[0213] In some embodiments, the inducer comprises both a small molecule component and one or more polypeptides. For example, inducible promoters that depend on the dimerization of polypeptides are known in the art and may be suitable for use in the present application. The first small molecule CID system developed in 1993 used FK1012, a derivative of the drug FK506, to induce homodimerization of FKBP. By using a similar strategy, Wu et al. successfully generated dose-programmable CAR-T cells in an ON-switch mode by using rapalog / FKPB-FRB* and gibberellin / GID1-GAI dimerization-dependent gene switches (C.-Y.Wu et al., Science 350, aab4077(2015)). Other dimerization-dependent switch systems include coumermycin / GyrB-GyrB (Nature 383(6596):178-81) and HaXS / Snap-tag-HaloTag (Chemistry and Biology 20(4):549-57).

[0214] In some embodiments, the promoter is a light-inducible promoter and the induction condition is light. Light-inducible promoters for regulating gene expression in mammalian cells are also well known in the art (see, for example, Science 332,1565-1568 (2011); Nat. Methods 9,266-269 (2012); Nature 500:472-476 (2013); Nature Neuroscience 18:1202-1212 (2015)). Such gene regulation systems can be broadly classified into two categories based on (1) DNA binding or (2) regulation of the recruitment of transcription activation domains to proteins bound to DNA. For example, a blue light-controlled synthetic mammalian transcription system based on melanopsin has been developed and tested in mammalian cells, which responds to blue light (480 nm) and induces an increase in intracellular calcium that leads to the translocation of NFAT mediated by calcineurin. More recently, Motta-Mena et al. described a novel inducible gene expression system developed from the naturally occurring EL222 transcription factor that confers high-level blue-light-sensitive control of transcription initiation in human cell lines and zebrafish embryos (Nat. Chem. Biol. 10(3):196-202 (2014)). Furthermore, the red-light-induced interaction of the Arabidopsis thaliana photoreceptor phytochrome B (PhyB) with phytochrome interacting factor 6 (PIF6) was exploited for red-light-induced gene expression regulation. Furthermore, an ultraviolet B (UVB)-inducible gene expression system was also developed and proved effective in target gene transcription in mammalian cells (Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, Fourth Edition CRC Press, Jan. 2013). th , Chapter 25 of 2015). Any of the light-inducible promoters described herein may be used to drive expression of the therapeutic protein in this application.

[0215] In some embodiments, the promoter is a light-inducible promoter that is induced by a combination of a light-inducible molecule and light. For example, a photocleavable photocaged group on a chemical inducer keeps the inducer inactive unless the photocaged group is removed by irradiation or other means. Such light-inducible molecules include small molecule compounds, oligonucleotides, and proteins. For example, caged ecdysone, caged IPTG for use with the lac operon, caged toyocamycin for ribozyme-mediated gene expression, caged doxycycline for use with the Tet-on system, and caged dorapalogues for light-mediated FKBP / FRB dimerization have been developed (see, e.g., Curr Opin Chem Biol. 16(3-4):292-299 (2012)).

[0216] In some embodiments, the promoter is a radiation-inducible promoter and the inducing condition is radiation, such as ionizing radiation. Radiation-inducible promoters are also known in the art to control transgene expression. Changes in gene expression occur upon irradiation of cells. For example, a group of genes known as "immediate early genes" can respond immediately to ionizing radiation. Exemplary immediate early genes include, but are not limited to, Erg-1, p21 / WAF-1, GADD45α, t-PA, c-Fos, c-Jun, NF-κB, and AP1. Immediate early genes contain radiation response elements within their promoter regions. The consensus sequence CC(A / T)6GG has been found within the Erg-1 promoter and is referred to as the serum response element or known as the CArG element. The combination of radiation-inducible promoters and transgenes has been intensively studied and has proven to be efficient in terms of therapeutic effects. See, for example, Cancer Biol Ther. 6(7):1005-12 (2007) and Chapter 25 of Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, Fourth Edition CRC Press, Jan. 20 th ,2015.

[0217] In some embodiments, the promoter is a heat-inducible promoter and the inducing condition is heat. Heat-inducible promoters driving transgene expression have also been widely studied in the art. Heat shock or stress proteins (HSPs), including Hsp90, Hsp70, Hsp60, Hsp40, Hsp10, etc., play an important role in protecting cells under heat or other physical and chemical stress. Several heat-inducible promoters have been attempted in preclinical trials, including heat shock protein (HSP) promoters and growth arrest and DNA damage (GADD) 153 promoters. The promoter of the human hsp70B gene, first described in 1985, is considered to be one of the most efficient heat-inducible promoters. Huang et al. reported that after introduction of hsp70B-EGFP, hsp70B-TNFα, and hsp70B-IL12 coding sequences, tumor cells showed very high transgene expression upon heat treatment, but in the absence of heat treatment, no transgene expression was detected. And tumor growth was significantly delayed in vivo in the IL12 transgene + heat-treated mice (Cancer Res. 60:3435 (2000)). Another group of scientists linked the HSV-tk suicide gene to the hsp70B promoter and tested the system in nude mice bearing mouse mammary tumors. Mice injected with the hsp70B-HSVtk coding sequence into their tumors and heat-treated showed tumor regression and significant survival rates compared to non-heat-treated controls (Hum. Gene Ther. 11:2453 (2000)). Additional heat-inducible promoters known in the art are described in, for example, Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, Fourth Edition CRC Press, Jan. 20 th , 2015, Chapter 25. Any of the heat-inducible promoters discussed herein can be used to drive expression of the therapeutic proteins of the present application.

[0218] In some embodiments, the promoter is induced by redox conditions. Exemplary promoters that are induced by redox conditions include inducible promoters and hypoxia-inducible promoters. For example, Post DE et al. have developed a hypoxia-inducible factor (HIF)-responsive promoter that specifically and strongly induces transgene expression in HIF-active tumor cells (Gene Ther.8:1801-1807(2001); Cancer Res.67:6872-6881(2007)).

[0219] In some embodiments, the promoter is induced by a physiological state, such as an endogenous activation signal of the modified immune cell. In some embodiments, when the modified immune cell is a T cell, the promoter is a T cell activation-dependent promoter induced by an endogenous activation signal of the modified T cell. In some embodiments, the modified T cell is activated by an inducer, such as phorbol myristate acetate (PMA), ionomycin, or phytohemagglutinin. In some embodiments, the modified T cell is activated by recognition of a tumor antigen on a tumor cell via an engineered receptor (e.g., CAR, TCR, or TAC). In some embodiments, the T cell activation-dependent promoter is an IL-2 promoter. In some embodiments, the T cell activation-dependent promoter is an NFAT promoter. In some embodiments, the T cell activation-dependent promoter is an NFκB promoter.

[0220] The heterologous nucleic acid sequence(s) described herein may be present in a heterologous gene expression cassette that includes one or more protein coding sequences and, optionally, one or more promoters. In some embodiments, the heterologous gene expression cassette includes a single protein coding sequence. In some embodiments, the heterologous gene expression cassette includes two or more protein coding sequences driven by a single promoter (i.e., polycistronic). In some embodiments, the heterologous gene expression cassette further includes one or more regulatory sequences (e.g., 5'UTR, 3'UTR, enhancer sequences, IRES, transcription termination sequences), recombination sites, one or more selectable markers (e.g., antibiotic resistance genes, reporter genes, etc.), signal sequences, or combinations thereof.

[0221] In some embodiments, a vector is provided that includes any of the nucleic acids encoding an IL-15 polypeptide and / or engineered receptor described herein. In some embodiments, a vector is provided that includes a first nucleic acid sequence encoding any of the IL-15 polypeptides described herein and a second nucleic acid sequence encoding any of the engineered receptors described herein. In some embodiments, the first nucleic acid sequence encoding an IL-15 polypeptide is fused to the second nucleic acid sequence encoding the engineered receptor via a self-cleavable linker, e.g., a third nucleic acid sequence encoding a P2A, T2A, E2A, or F2A peptide. In some embodiments, the P2A sequence is GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 28). In some embodiments, a composition is provided that includes a first vector that includes a first nucleic acid sequence encoding any of the IL-15 polypeptides described herein and a second vector that includes a second nucleic acid sequence encoding any of the engineered receptors described herein.

[0222] In some embodiments, a vector is provided that comprises a first nucleic acid sequence encoding a CAR (e.g., a BCMA CAR, a CD19 CAR, or a GPC3 CAR) and a second nucleic acid sequence encoding an IL-15 polypeptide (e.g., a secreted or membrane-bound IL-15 polypeptide), wherein the first nucleic acid sequence is fused to the second nucleic acid sequence via a third nucleic acid sequence encoding a self-cleavable linker, such as P2A. In some embodiments, the vector comprises a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-39, 42-49, 57-66, 75-77, and 82-84.

[0223] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. In general, a suitable vector contains an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers. The term "vector" should also be construed to include non-plasmid and non-viral compounds that facilitate the introduction of nucleic acid into cells, such as polylysine compounds, liposomes, etc.

[0224] In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, retrovirus vectors, vaccinia vectors, herpes simplex virus vectors, and their derivatives. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.

[0225] A number of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged into retroviral particles using techniques known in the art. Recombinant viruses can then be isolated and delivered to in vivo or ex vivo modified immune cells. A number of retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors can be packaged using protocols known in the art. The resulting lentiviral vectors can be used to transduce mammalian cells (e.g., human T cells) using methods known in the art.

[0226] In some embodiments, the vector is a non-viral vector, such as a plasmid, or an episomal expression vector.

[0227] In some embodiments, the vector is an expression vector. An "expression vector" is a construct that can be used to transform a selected host, resulting in the expression of a coding sequence in the selected host. An expression vector can be, for example, a cloning vector, a binary vector, or an integrative vector. Expression includes transcription of a nucleic acid molecule, preferably into a translatable mRNA. Regulatory elements that ensure expression in eukaryotic cells are known to those skilled in the art. For eukaryotic cells, an expression vector usually includes a promoter that ensures initiation of transcription, and optionally a polyA signal that ensures termination of transcription and stabilization of the transcript. Examples of regulatory elements that allow expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast, or the CMV promoter, SV40 promoter, RSV promoter (Rous sarcoma virus), CMV enhancer, SV40 enhancer, or globin intron in mammalian and other animal cells. Furthermore, depending on the expression system used, leader sequences (i.e., signal peptides) capable of directing the polypeptide to an intracellular compartment or secreting it into the medium can be added to the coding sequence of the described nucleic acid sequence and are well known in the art. The leader sequence(s) are assembled at an appropriate stage with translation initiation and termination sequences and, preferably, a leader sequence capable of directing secretion of the translated protein or a portion thereof into the periplasmic space or into the extracellular medium. Optionally, the nucleic acid sequence can encode a fusion protein containing an N-terminal identification peptide that provides a desired property, for example, stabilization or simplification of purification of the expressed recombinant product. Suitable expression vectors are known in the art, for example the Okayama-Berg cDNA expression vectors pcDV1 (Pharmacia), pEF-Neo, pCDM8, pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pEF-DHFR and pEF-ADA (Raum et al., Cancer Immunol Immunother (2001) 50(3), 141-150), or pSPORT1 (GIBCO BRL).

[0228] Preparation method The present application also provides methods of preparing any of the modified immune cells described herein.

[0229] In some embodiments, a method of making an engineered immune cell is provided, the method comprising introducing into a precursor immune cell a first nucleic acid sequence encoding any of the IL-15 polypeptides described herein. In some embodiments, the precursor immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer (NK) cell, an NK-T cell, an iNK-T cell, an NK-T-like cell, an αβ T cell, and a γδ T cell. In some embodiments, the precursor immune cell is a cytotoxic T cell. In some embodiments, the precursor immune cell is a γδ T cell. In some embodiments, the precursor immune cell is a tumor-infiltrating T cell or a DC-activated T cell. In some embodiments, the precursor immune cell comprises any of the engineered receptors described herein. In some embodiments, the method further comprises introducing into the precursor immune cell a second nucleic acid encoding any of the engineered receptors described herein. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR). In some embodiments, the engineered receptor is a modified T cell receptor (TCR). In some embodiments, the engineered receptor is a T cell antigen conjugate (TAC) receptor. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to separate promoters. In some embodiments, the first nucleic acid and the second nucleic acid are present in the same vector. In some embodiments, the first nucleic acid and the second nucleic acid are present in separate vectors. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a herpes simplex viral vector, and derivatives thereof. In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is an episomal expression vector. In some embodiments, the method further comprises isolating or enriching immune cells comprising the first nucleic acid sequence and / or the second nucleic acid sequence.In some embodiments, the method further comprises formulating the modified immune cells with at least one pharma- ceutically acceptable carrier.

[0230] In some embodiments, an isolated host cell is provided that comprises any of the nucleic acids or vectors described herein. The host cell may be useful for expressing or cloning an IL-15 polypeptide and / or engineered receptor, a nucleic acid or vector encoding an IL-15 polypeptide and / or engineered receptor. Suitable host cells may include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells, such as mammalian cells. In some embodiments, the host cell comprises a first vector encoding a first polypeptide and a second vector encoding a second polypeptide. In some embodiments, the host cell comprises a single vector that comprises the isolated nucleic acid encoding the first polypeptide and the second polypeptide.

[0231] Precursor immune cells may be prepared using a variety of methods known in the art. For example, primary immune cells such as T cells may be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, immune cells (e.g., T cells) may be obtained from a unit of blood collected from an individual using a number of techniques known in the art, such as FICOLL™ separation. In some embodiments, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS) or a washing solution lacking divalent cations such as calcium and magnesium. As will be readily appreciated by those of skill in the art, the washing step may be accomplished by methods known to those of skill in the art, for example, by using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processing machine, a Baxter CytoMate, or a Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be centrifuged using, for example, Ca 2+ Contains no Mg 2+ The cells may be resuspended in a variety of biocompatible buffers, such as free PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0232] In some embodiments, primary T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by PERCOLL™ gradient centrifugation or counterflow centrifugal elutriation. Specific subpopulations of T cells, such as CD3 + , CD28 + , CD4 +, CD8 + , CD45RA, and CD45RO cells can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 (i.e., 3x28) conjugated beads, e.g., DYNABEADS® M-450 CD3 / CD28 T, for a period of time sufficient for positive selection of the desired T cells.

[0233] In some embodiments, the T cell population can be further enriched by negative selection using a combination of antibodies directed against surface markers unique to the cells being negatively selected. For example, one method includes cell sorting and / or cell selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the cells being negatively selected. For example, negative selection can be used to enrich the T cell population for CD4 + Monoclonal antibody cocktails for enriching cells typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, CD4 + , CD25 + , CD62L hi , G.I.T.R. + , and FoxP3 + It may be desirable to enrich for or positively select regulatory T cells that typically express . Alternatively, in certain embodiments, regulatory T cells are depleted by anti-CD25 conjugated beads or other similar selection methods.

[0234] Methods for introducing vectors or nucleic acids into host cells (e.g., precursor immune cells) are known in the art. The vectors or nucleic acids can be introduced into the host cells by physical, chemical, or biological methods.

[0235] Physical methods for introducing vector(s) or nucleic acid(s) into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, the vector is introduced into the cell by electroporation.

[0236] Biological methods for introducing a vector(s) or nucleic acid(s) into a host cell include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian, e.g., human, cells.

[0237] Chemical means for introducing the vector(s) or nucleic acid(s) into a host cell include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro is a liposome (e.g., an artificial membrane vesicle).

[0238] In some embodiments, the transduced or transfected precursor immune cells are expanded ex vivo after introduction of the heterologous nucleic acid(s). In some embodiments, the transduced or transfected precursor immune cells are cultured for at least about any of 1, 2, 3, 4, 5, 6, 7, 10, 12, or 14 days to expand. In some embodiments, the transduced or transfected precursor immune cells are cultured for no more than about any of 1, 2, 3, 4, 5, 6, 7, 10, 12, or 14 days. In some embodiments, the transduced or transfected precursor immune cells are further evaluated or screened to select modified immune cells.

[0239] Reporter genes can be used to identify potentially transfected cells and to evaluate the function of regulatory sequences. In general, reporter genes are genes that are not present in or expressed by recipient organisms or tissues and that code for a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. The expression of the reporter gene is tested at a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes can include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., FEBS Letters 479:79-82(2000)).

[0240] Other methods for confirming the presence of heterologous nucleic acid(s) in precursor immune cells include, for example, molecular biological assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; biochemical assays, such as detecting the presence or absence of specific peptides by immunological methods (e.g., ELISA and Western blot).

[0241] III. Treatment Methods One aspect of the present application relates to a method of treating a disease or condition (e.g., cancer) in an individual, comprising administering to the individual an effective amount of any of the modified immune cells described herein. The present application contemplates the modified immune cells, which may be administered either alone or in any combination with another therapy, and in at least some aspects, with a pharma- ceutically acceptable carrier or excipient. In some embodiments, prior to administration, the modified immune cells may be combined with suitable pharmaceutical carriers and excipients well known in the art.

[0242] In some embodiments, a method of treating cancer (e.g., a solid cancer) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition comprising modified immune cells (e.g., NK cells) and a pharma- ceutically acceptable carrier, wherein the modified immune cells comprise a first heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising one or more amino acid substitutions at positions 8, 62, 3, and / or 25, wherein the numbering of the amino acid residue positions is according to SEQ ID NO: 1. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:78.In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25. In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F. In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises amino acid substitutions at both positions 8 and 62. In some embodiments, the IL-15 polypeptide is secreted. In some embodiments, the IL-15 polypeptide is membrane bound. In some embodiments, the modified immune cell further comprises an engineered receptor, such as a chimeric antigen receptor (CAR), an engineered TCR, or a T cell antigen conjugate (TAC) receptor. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present on the same vector or separate vectors. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter or separate promoters. In some embodiments, the modified immune cell is selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor infiltrating T cells, and DC-activated T cells.

[0243] In some embodiments, a method of treating cancer (e.g., a solid cancer) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition comprising modified immune cells (e.g., NK cells) and a pharma- ceutically acceptable carrier, wherein the modified immune cells comprise a first heterologous nucleic acid sequence encoding an IL-15 polypeptide that is a fusion protein comprising an IL-15 fragment and a second polypeptide fragment, wherein the IL-15 polypeptide comprises one or more amino acid substitutions at positions 8, 62, 3, and / or 25, wherein the numbering of amino acid residue positions is according to SEQ ID NO: 1. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:78.In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25. In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F. In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at both positions 8 and 62. In some embodiments, the second polypeptide fragment is selected from the group consisting of IL-15Rα, the extracellular domain of IL-15Rα, the sushi domain of IL-15Rα, the transmembrane domain of IL-15Rα, IL-15Rβ, the common gamma chain (γc), an engineered receptor (e.g., CAR, TCR, or TAC), and combinations thereof. In some embodiments, the modified immune cell further comprises a second heterologous nucleic acid sequence encoding an engineered receptor, such as a CAR, an engineered TCR, or a TAC receptor. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present on the same vector or separate vectors. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter or separate promoters. In some embodiments, the engineered immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) T cells, iNK-T cells, NK-T-like cells, αβ T cells, γδ T cells, tumor-infiltrating T cells, and DC-activated T cells.

[0244] In some embodiments, a method of treating cancer (e.g., a solid cancer) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition comprising modified immune cells (e.g., NK cells) and a pharma- ceutically acceptable carrier, wherein the modified immune cells comprise a first heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising a transmembrane domain, wherein the IL-15 polypeptide comprises one or more amino acid substitutions at positions 8, 62, 3, and / or 25, wherein the numbering of amino acid residue positions is according to SEQ ID NO: 1. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 62. In some embodiments, the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y. In some embodiments, the amino acid substitution at position 62 is T62G. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO:7. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 8. In some embodiments, the amino acid substitution at position 8 is D8E. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:5. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 3. In some embodiments, the amino acid substitution at position 3 is V3Y. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO:78.In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 78. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at position 25. In some embodiments, the amino acid substitution at position 25 is selected from the group consisting of L25E and L25F. In some embodiments, the amino acid substitution at position 25 is L25F. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 79. In some embodiments, the IL-15 polypeptide comprises an amino acid substitution at both positions 8 and 62. In some embodiments, the transmembrane domain is the transmembrane domain of IL-15Rα. In some embodiments, the IL-15 polypeptide further comprises an intracellular domain. In some embodiments, the IL-15 polypeptide comprises: (a) an antigen binding domain; (b) an IL-15 fragment; (c) a transmembrane domain; and (d) an intracellular domain. In some embodiments, the antigen binding domain is at the N-terminus of the IL-15 fragment. In some embodiments, the antigen binding domain is at the C-terminus of the IL-15 fragment. In some embodiments, the transmembrane domain is the transmembrane domain of CD4, CD3, CD8α, or CD28. In some embodiments, the IL-15 polypeptide further comprises a hinge domain, e.g., a hinge domain derived from CD8. In some embodiments, the intracellular domain comprises a primary intracellular signaling domain, e.g., the intracellular signaling domain of CD3ζ. In some embodiments, the intracellular domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, DAP10, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof.In some embodiments, the modified immune cell further comprises a second heterologous nucleic acid sequence encoding an engineered receptor, such as a CAR, an engineered TCR, or a TAC receptor. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present on the same vector or separate vectors. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter or separate promoters. In some embodiments, the modified immune cell is selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK-T cells, iNK-T cells, NK-T-like cells, αβT cells, γδT cells, tumor-infiltrating T cells, and DC-activated T cells.

[0245] In some embodiments, the method of treating cancer has one or more of the following biological effects: (1) killing cancer cells; (2) inhibiting the proliferation of cancer cells; (3) inducing a redistribution of peripheral T cells; (4) inducing an immune response in tumors; (5) reducing tumor size; (6) alleviating one or more symptoms in an individual with cancer; (7) inhibiting tumor metastasis; (8) prolonging survival; (9) increasing the time to cancer progression; (10) preventing, inhibiting, or reducing the likelihood of cancer recurrence; (11) improving the quality of life of an individual; (12) promoting T cell infiltration in tumors, and (13) reducing the occurrence of metastases (e.g., metastases to lymph nodes) or reducing an existing tumor metastatic burden. In some embodiments, the method achieves a tumor cell death rate of at least about any of 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the method reduces tumor size by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the method inhibits metastasis by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the method extends survival of an individual by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24 months, or longer. In some embodiments, the method extends time to cancer progression by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24 months, or longer.

[0246] The methods described herein are suitable for treating a variety of cancers, including both solid and liquid cancers. The methods are applicable to any stage of cancer, including early stage, non-metastatic, primary, advanced, locally advanced, metastatic, or in remission. The methods described herein can be used as a first line of therapy, a second line of therapy, a third line of therapy, or a combination therapy with other types of cancer therapy known in the art, such as chemotherapy, surgery, hormone therapy, radiation, gene therapy, immunotherapy (e.g., T-cell therapy), bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radiofrequency ablation, etc., as an adjuvant or neoadjuvant therapy (i.e., the methods can be performed before a primary / definitive therapy). In some embodiments, the methods are used to treat individuals who have been previously treated. In some embodiments, the cancer was refractory to the previous treatment. In some embodiments, the methods are used to treat individuals who have not been previously treated.

[0247] In some embodiments, the individual has a low tumor burden. The tumor burden of solid tumors can be measured according to the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 guidelines. See Eisenhauer EA et al., European Journal of Cancer 45(2009) 228-247. For example, tumor burden can be assessed at baseline of treatment for measurable tumors based on: (1) neoplastic lesions (e.g., by CT scan, caliper measurements on clinical examination, and / or chest X-ray), and (2) malignant lymph nodes. For example, tumor burden of solid tumors can be quantified as the sum of the diameters of five target lesions, with up to two per organ. Tumor burden of liquid tumors can be measured as the sum of the products of the diameters of up to six index lesions assessed by a radiologist according to the Cheson 2007 criteria. See Cheson BD et al., J.Clin.Oncol.,2007;25(5):579-586. In some embodiments, an individual with low tumor burden has about 4×10 3 , 3×10 3 , 2×103 , 1×10 3 , 5×10 2 , 2×10 2 , 1×10 2 mm 2 have a tumor burden of either 0.1% or less.

[0248] In some embodiments, the individual does not exhibit grade 3 or grade 4 adverse side effects after receiving the treatment. Grading of adverse events is according to the Common Terminology Criteria for Adverse Events v3.0 (CTCAE). In some embodiments, the individual does not exhibit cytokine storm after receiving the treatment.

[0249] The effective amount of modified immune cells administered in the methods described herein depends on a number of factors, such as the particular type and stage of cancer being treated, the route of administration, the activity of the IL-15 polypeptide and / or engineered receptor. An appropriate dosing regimen can be determined by a physician based on clinical factors, including the patient's size, body surface area, age, the particular compound being administered, sex, time and route of administration, health status, and other drugs being administered concomitantly. In some embodiments, the effective amount of the pharmaceutical composition is below a level that induces a toxicological effect (i.e., an effect that exceeds a clinically acceptable level of toxicity) when the pharmaceutical composition is administered to an individual, or at a level at which potential side effects can be controlled or tolerated. In some embodiments, the effective amount of the pharmaceutical composition is about 10 5 ~about 10 10 The modified immune cell comprises

[0250] In some embodiments, the pharmaceutical composition is administered once (e.g., bolus injection). In some embodiments, the pharmaceutical composition is administered multiple times (e.g., either two, three, four, five, six, or more times). In the case of multiple administrations, they may be performed by the same route or different routes and may be administered at the same site or different sites. The pharmaceutical composition may be administered at any suitable frequency, such as daily to once a year. Optimal dosages and treatment regimens for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0251] In some embodiments, the individual being treated is a mammal. Examples of mammals include, but are not limited to, humans, monkeys, rats, mice, hamsters, guinea pigs, dogs, cats, rabbits, pigs, sheep, goats, horses, cows, etc. In some embodiments, the individual is a human.

[0252] Pharmaceutical Compositions Further provided by the present application is a pharmaceutical composition comprising any of the modified immune cells described herein and an optional pharma- ceutically acceptable carrier.

[0253] The pharmaceutical compositions of the present application can include any number of modified immune cells. In some embodiments, the pharmaceutical compositions include a single copy of the modified immune cells. In some embodiments, the pharmaceutical compositions include at least about 1 copy, 10 copies, 100 copies, 1000 copies, 10 4 Copy, 10 5 Copy, 10 6 Copy, 10 7 Copy, 10 8 In some embodiments, the pharmaceutical composition comprises either one or more copies of the modified immune cell. In some embodiments, the pharmaceutical composition comprises a single type of modified immune cell. In some embodiments, the pharmaceutical composition comprises at least two types of modified immune cells, where the different types of modified immune cells differ in cell source, cell type, expressed chimeric receptor, and / or promoter, etc.

[0254] As used herein, "carrier" includes pharma- ceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or individuals exposed at the dosages and concentrations used. In many cases, physiologically acceptable carriers are pH-buffered aqueous solutions. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used.

[0255] Pharmaceutical compositions containing such carriers can be formulated by known conventional methods. Solvent or diluent is preferably isotonic, hypotonic or slightly hypertonic, and has relatively low ionic strength. Representative examples include sterilized water, physiological saline (e.g., sodium chloride), Ringer's solution, glucose, trehalose or saccharose solution, Hank's solution, and other physiologically balanced salt solutions (see, for example, the latest edition of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams & Wilkins).

[0256] The pharmaceutical compositions described herein may be administered by any suitable route. In some embodiments, the pharmaceutical compositions may be administered parenterally, transdermally (into the dermis), intraluminally, intraarterially (into an artery), intramuscularly (into a muscle), intrathecally, or intravenously. In some embodiments, the pharmaceutical compositions are administered subcutaneously (under the skin). In some embodiments, the pharmaceutical compositions are administered intravenously. In some embodiments, the pharmaceutical compositions are administered to an individual by infusion or injection. In some embodiments, the pharmaceutical compositions are administered directly to a target site, for example, by biolistic delivery to an internal or external target site, or by catheter directly to a site in an artery. In some embodiments, the pharmaceutical compositions are administered locally, for example, within a tumor. Administration may use a conventional syringe and needle, or any compound or device available in the art that can facilitate or improve delivery of an active agent(s) to a subject.

[0257] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases and the like. In addition, the pharmaceutical compositions of the present disclosure may include proteinaceous carriers, such as, for example, serum albumin or immunoglobulin, preferably of human origin. Various virus formulations are available in the art, either in frozen, liquid, or lyophilized form (e.g., WO98 / 02522, WO01 / 66137, WO03 / 053463, WO2007 / 056847, and WO2008 / 114021, etc.). Solid compositions (e.g., dry powder compositions or lyophilized compositions) can be obtained by processes including vacuum drying and lyophilization (see, e.g., WO2014 / 053571). It is envisioned that the pharmaceutical compositions of the present disclosure may contain, in addition to the modified immune cells described herein, further biologically active substances depending on the intended use of the pharmaceutical composition.

[0258] In some embodiments, the pharmaceutical composition is preferably buffered for human use. Suitable buffers include, but are not limited to, phosphate buffers (e.g., PBS), bicarbonate buffers, and / or Tris buffers capable of maintaining a physiological pH or a slightly basic pH (e.g., about pH 7 to about pH 9). In some embodiments, the pharmaceutical composition may also be made isotonic with blood by the addition of a suitable osmotic agent, such as glycerol.

[0259] In some embodiments, the pharmaceutical composition is contained in a single-use vial, e.g., a sealed single-use vial. In some embodiments, the pharmaceutical composition is contained in a multi-use vial. In some embodiments, the pharmaceutical composition is contained in a bulk container.

[0260] In some embodiments, pharmaceutical compositions must meet certain criteria for administration to an individual. For example, the U.S. Food and Drug Administration has issued regulatory guidelines setting standards for cell-based immunotherapy products, including 21 CFR 610 and 21 CFR 610.13. Methods for evaluating the appearance, identity, purity, safety, and / or efficacy of pharmaceutical compositions are known in the art. In some embodiments, the pharmaceutical composition is substantially free of foreign proteins that can cause allergic effects, such as proteins of animal origin used in cell culture other than the engineered immune cells. In some embodiments, "substantially free" means less than about 10%, less than 5%, less than 1%, less than 0.1%, less than 0.01%, less than 0.001%, less than 1 ppm, or less of the total volume or weight of the pharmaceutical composition. In some embodiments, the pharmaceutical composition is prepared in a GMP-level facility. In some embodiments, the pharmaceutical composition contains less than about 5 EU / kg body weight / hr endotoxin for parenteral administration. In some embodiments, for intravenous administration, at least about 70% of the modified immune cells in the pharmaceutical composition are viable. In some embodiments, the pharmaceutical composition exhibits a "no growth" result when evaluated using the 14-day direct inoculation test method described in the United States Pharmacopeia (USP). In some embodiments, prior to administration of the pharmaceutical composition, a sample containing both the modified immune cells and the pharma- ceutically acceptable excipients must be taken for sterility testing approximately 48-72 hours prior to final harvest (or contemporaneous with the final refeed of the culture). In some embodiments, the pharmaceutical composition is free of mycoplasma contamination. In some embodiments, the pharmaceutical composition is free of detectable microbial agents. In some embodiments, the pharmaceutical composition is free of infectious disease agents, such as HIV type I, HIV type II, HBV, HCV, human T-lymphotropic virus type I, and human T-lymphotropic virus type II.

[0261] IV. KITS AND ARTICLES OF MANUFACTURE Kits, formulations, and articles of manufacture comprising the modified immune cells or any of the compositions (e.g., pharmaceutical compositions) described herein are also provided. In some embodiments, kits are provided that comprise any of the pharmaceutical compositions described herein, and preferably provide instructions for their use. In some embodiments, the kits further comprise, in addition to the modified immune cells, a second cancer treatment, e.g., chemotherapy, hormonal therapy, and / or immunotherapy. The kit(s) may be tailored to the particular cancer of an individual and may include the respective second cancer treatment for that individual.

[0262] The kit may include one or more additional components that allow for the growth or induction of the modified immune cells, such as containers, reagents, culture media, inducers, cytokines, buffers, antibodies, etc. The kit may also include a device for local administration of the pharmaceutical composition to the tumor site (e.g., intratumoral injection).

[0263] The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. The kits may optionally provide additional components, such as buffers and instructional information. Thus, the present application also provides articles of manufacture that include vials (e.g., sealed vials), bottles, jars, flexible packaging, and the like. Some components of the kits may be packaged in aqueous media or in lyophilized form.

[0264] The article of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials, such as glass or plastic. Typically, the container holds a composition effective for treating a disease or disorder (e.g., cancer) described herein and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). The label or package insert indicates that the composition is used to treat a particular condition in an individual. The label or package insert further includes instructions for administering the composition to an individual. The label may indicate how to reconstitute and / or how to use. The container that holds the pharmaceutical composition may be a multi-use vial that allows for repeated administration (e.g., 2-6 administrations) of the reconstituted formulation. Package insert refers to instructions for use that are customarily included in commercial packaging of therapeutic products, including information on indications, usage, dosage, administration, contraindications, and / or warnings regarding the use of such therapeutic products. Additionally, the article of manufacture may further comprise a second container containing a pharma- ceutically acceptable buffer, such as sterile water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture or kit may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0265] The kit or article of manufacture may include a number of unit doses of the pharmaceutical composition and instructions for use packaged in an amount sufficient for storage and use in a pharmacy, such as a hospital pharmacy or compounding pharmacy.

[0266] Exemplary embodiments Embodiment 1. A modified immune cell comprising a first heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising one or more amino acid substitutions at positions 8 and / or 62, wherein the numbering of the amino acid residue positions is according to SEQ ID NO:1.

[0267] Embodiment 2 The modified immune cell of embodiment 1, wherein said IL-15 polypeptide comprises an amino acid substitution at position 62.

[0268] Embodiment 3. The modified immune cell of embodiment 2, wherein said IL-15 polypeptide comprises an amino acid residue at position 62 selected from the group consisting of glycine (G), isoleucine (I), glutamine (Q), valine (V), proline (P), leucine (L), alanine (A), serine (S), and tyrosine (Y).

[0269] Embodiment 4. The modified immune cell of embodiment 2 or 3, wherein the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y.

[0270] Embodiment 5. The modified immune cell of embodiment 4, wherein said amino acid substitution at position 62 is T62G.

[0271] Embodiment 6. The modified immune cell of any one of embodiments 2 to 5, wherein the IL-15 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:7.

[0272] Embodiment 7 The modified immune cell of any one of the preceding embodiments, wherein said IL-15 polypeptide comprises an amino acid substitution at position 8.

[0273] Embodiment 8 The modified immune cell of embodiment 7, wherein said IL-15 polypeptide comprises a glutamic acid (E) amino acid residue at position 8.

[0274] Embodiment 9. The modified immune cell of embodiment 8, wherein said amino acid substitution at position 8 is D8E.

[0275] Embodiment 10. The modified immune cell of embodiment 8 or 9, wherein the IL-15 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:5.

[0276] Embodiment 11. A modified immune cell comprising a first heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising one or more amino acid substitutions at positions 3 and / or 25, wherein the numbering of the amino acid residue positions is according to SEQ ID NO:1.

[0277] Embodiment 12. The modified immune cell of embodiment 11, wherein said amino acid substitution at position 3 is V3Y and / or said amino acid substitution at position 25 is L25F.

[0278] Embodiment 13. The modified immune cell of embodiment 12, wherein the IL-15 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 78 or 79.

[0279] Embodiment 14 The modified immune cell of any one of the preceding embodiments, wherein said one or more amino acid substitutions reduce the affinity of said IL-15 polypeptide for IL-15Rβ compared to an IL-15 polypeptide that does not comprise said one or more amino acid substitutions.

[0280] Embodiment 15. A modified immune cell comprising a first heterologous nucleic acid sequence encoding an IL-15 polypeptide, wherein said IL-15 polypeptide induces secretion of a proinflammatory cytokine by said modified immune cell at a level that is at least 50% lower than the secretion of a proinflammatory cytokine by a modified immune cell comprising a heterologous nucleic acid sequence encoding a wild-type IL-15 polypeptide.

[0281] Embodiment 16 The modified immune cell of any one of the preceding embodiments, wherein said IL-15 polypeptide is secreted.

[0282] Embodiment 17. The modified immune cell of any one of embodiments 1 to 16, wherein the IL-15 polypeptide is membrane-bound.

[0283] Embodiment 18 The modified immune cell of embodiment 17, wherein the IL-15 polypeptide comprises a glycosylphosphatidylinositol (GPI) anchor peptide sequence.

[0284] Embodiment 19 The modified immune cell of embodiment 17, wherein the IL-15 polypeptide comprises a transmembrane domain.

[0285] Embodiment 20 The modified immune cell of embodiment 17, wherein the IL-15 polypeptide comprises a membrane anchor domain.

[0286] Embodiment 21 The modified immune cell of any one of the preceding embodiments, wherein said IL-15 polypeptide is a fusion protein comprising an IL-15 fragment fused to a second polypeptide fragment.

[0287] Embodiment 22. The modified immune cell of embodiment 21, wherein the second polypeptide fragment is selected from the group consisting of IL-15Rα, the extracellular domain of IL-15Rα, the sushi domain of IL-15Rα, the transmembrane domain of IL-15Rα, IL-15Rβ, and the common gamma chain (γc).

[0288] Embodiment 23. The modified immune cell of embodiment 22, wherein the second polypeptide fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-55.

[0289] Embodiment 24. The modified immune cell of embodiment 17, wherein the IL-15 polypeptide comprises: (a) an antigen-binding domain; (b) an IL-15 fragment; (c) a transmembrane domain; and (d) an intracellular domain.

[0290] Embodiment 25 The modified immune cell of any one of embodiments 1 to 23, wherein the modified immune cell comprises a second heterologous nucleic acid sequence encoding an engineered receptor.

[0291] Embodiment 26 The modified immune cell of embodiment 25, wherein the engineered receptor is a chimeric antigen receptor (CAR).

[0292] Embodiment 27. The modified immune cell of embodiment 26, wherein the CAR is a BCMA CAR, a CD19 CAR, or a GPC3 CAR.

[0293] Embodiment 28 The modified immune cell of embodiment 25, wherein the engineered receptor is a modified T cell receptor (TCR).

[0294] Embodiment 29. The modified immune cell of embodiment 25, wherein the engineered receptor is a T cell antigen conjugate (TAC) receptor.

[0295] Embodiment 30. The modified immune cell of any one of embodiments 25 to 29, wherein the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter.

[0296] Embodiment 31. The modified immune cell of any one of embodiments 25 to 29, wherein the first nucleic acid and the second nucleic acid are operably linked to separate promoters.

[0297] Embodiment 32. The modified immune cell of any one of the preceding embodiments, wherein the modified immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer (NK) cell, a NK cell, an iNK-T cell, a NK-T-like cell, an αβ T cell, and a γδ T cell.

[0298] Embodiment 33 The modified immune cell of embodiment 32, wherein the modified immune cell is a NK cell.

[0299] Embodiment 34 The modified immune cell of embodiment 32, wherein the modified immune cell is a cytotoxic T cell.

[0300] Embodiment 35 The modified immune cell of any one of the preceding embodiments, wherein the modified immune cell has reduced toxicity in vivo when administered to an individual compared to a modified immune cell that does not comprise the first heterologous nucleic acid encoding the IL-15 polypeptide.

[0301] Embodiment 36. A method of producing an engineered immune cell, comprising introducing into a precursor immune cell a first nucleic acid sequence encoding an IL-15 polypeptide comprising one or more amino acid substitutions at positions 8 and / or 62, wherein the numbering of the amino acid residue positions is according to SEQ ID NO:1.

[0302] Embodiment 37. The method of embodiment 36, wherein the precursor immune cells are selected from the group consisting of cytotoxic T cells, helper T cells, NK cells, NK-T cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

[0303] Embodiment 38 The method of embodiment 36 or 37, wherein the precursor immune cells comprise an engineered receptor.

[0304] Embodiment 39 The method of embodiment 36 or 37, further comprising introducing into said precursor immune cells a second nucleic acid encoding an engineered receptor.

[0305] Embodiment 40. The method of embodiment 38 or 39, wherein the engineered receptor is a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), or a T cell antigen conjugate (TAC) receptor.

[0306] Embodiment 41 The method of embodiment 39 or 40, wherein the first nucleic acid sequence and the second nucleic acid sequence are present in the same vector.

[0307] Embodiment 42 The method of embodiment 41, wherein the vector is a viral vector.

[0308] Embodiment 43. The method of embodiment 42, wherein the viral vector is selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a herpes simplex viral vector, and derivatives thereof.

[0309] Embodiment 44. The method of any one of embodiments 36 to 43, further comprising isolating or enriching immune cells comprising the first nucleic acid sequence and / or the second nucleic acid sequence.

[0310] Embodiment 45. A modified immune cell produced by the method according to any one of embodiments 36 to 44.

[0311] Embodiment 46. A pharmaceutical composition comprising the modified immune cells according to embodiments 1 to 35 and 45, and a pharma- ceutically acceptable carrier.

[0312] Embodiment 47. A method for treating a disease in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of embodiment 46.

[0313] Embodiment 48 The method of embodiment 47, wherein the disease is cancer.

[0314] Embodiment 49 The method of embodiment 48, wherein the individual has a low tumor burden.

[0315] Embodiment 50. The method of any one of embodiments 47 to 49, which does not result in a cytokine storm in the individual.

[0316] Embodiment 51. The method of any one of embodiments 47 to 50, wherein the individual is a human.

[0317] Embodiment 52. A method of reducing a cytokine storm in an individual receiving treatment with immune cells comprising an engineered receptor, comprising: (a) introducing into said immune cell a heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising one or more amino acid substitutions at positions 8 and / or 62, wherein the numbering of amino acid residue positions is according to SEQ ID NO:1, thereby providing an altered immune cell; and (b) administering to the individual an effective amount of the modified immune cells. The method comprising:

[0318] Embodiment 53. An engineered IL-15 polypeptide comprising the amino acid substitutions D8E, T62G, V3Y, and / or L25F, wherein the numbering of the amino acid residue positions is according to SEQ ID NO:1.

[0319] Embodiment 54. The engineered IL-15 polypeptide of embodiment 53, comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 7, 78, and 79. EXAMPLES

[0320] The following examples are intended merely to illustrate the present disclosure and therefore should not be construed as limiting the present disclosure in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

[0321] Example 1: Preparation of CAR-NK cells expressing exogenously introduced wild-type or mutant IL-15 This example shows the construction of exemplary armed CAR-NK cells expressing exogenously introduced IL-15. Specifically, this example shows the construction of BCMA CAR-NK cells armed with wild-type or mutant IL-15 and CD19 CAR-NK cells armed with wild-type or mutant IL-15.

[0322] 1.1. Construction of chimeric antigen receptor (CAR) To construct a BCMA CAR or a CD19 CAR, a CAR scaffold sequence encoding a CAR scaffold polypeptide comprising, from N-terminus to C-terminus, a CD8α hinge domain (SEQ ID NO: 18), a CD8α transmembrane domain (SEQ ID NO: 19), a CD137 costimulatory signaling domain (SEQ ID NO: 20), a CD3ζ primary intracellular signaling domain (SEQ ID NO: 21), and an IL-15 arming design sequence (i.e., one sequence selected from SEQ ID NO: 2 to SEQ ID NO: 17, and SEQ ID NO: 78 and SEQ ID NO: 79) was chemically synthesized and cloned downstream of a constitutive hEF1α promoter for in vitro transcription of a pre-modified retroviral vector (MSCV vector) and operably linked thereto. Retroviral supernatants were transiently produced as exemplified in Blood (2006) 108(12):3890-3897. The sequences of exemplary CAR constructs are shown below. SEQ ID NO: 26 BCMA CAR amino acid sequence MALPVTALLLPLALLLHAARPAVQLVESGGGLVQAGDSLRLTCTASGRAFSTYFMAWFRQAPGKEREFVAGIAWSGGSTAYADSVKGRFTISRDNAKNTVYLQMNSLKSEDTAVYYCASRG IEVEEFGAWGQGTQVTVSSGGGGSQVQLEESGGGSVQAGGSLRLSCAYTYSTYSNYYMGWFREAPGKARTSVAIISSDTTITYKDAVKGRFTISKDNAKNTLYLQMNSLKPEDSAMYRCAAW TSDWSVAYWGQGTQVTVSSTSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 29 BCMA CAR amino acid sequence armed with sIL-15 wt MALPVTALLLPLALLLHAARPAVQLVESGGGLVQAGDSLRLTCTASGRAFSTYFMAWFRQAPGKEREFVAGIAWSGGSTAYADSVKGRFTISRDNAKNTVYLQMNSLKSEDTAVYYCASRGIEVEEFGAWGQGTQVTVSSGGGGSQVQLEESGGGSVQAGGSLRLSC AYTYSTYSNYYMGWFREAPGKARTSVAIISSDTTITYKDAVKGRFTISKDNAKNTLYLQMNSLKPEDSAMYRCAAWTSDWSVAYWGQGTQVTVSSTSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKR GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDV EENPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0323] 1.2. Construction of CAR-natural killer (NK) cells NK proliferation Human peripheral blood mononuclear cells (PBMCs) were purchased from HemaCare Corporation. PBMCs were thawed and cultured with K562 genetically modified to express membrane-bound IL-15 and 4-1BB ligand (i.e., K562-mb15-41BBL). Cells were expanded using stem cell growth medium (SCGM; Cell Genix, Freiburg) supplemented with 50 IU of IL-2 / 1 mL of culture medium. After 7 days of culture, cells were harvested and purified using anti-CD3 Dynabeads (Miltenyi, Cat. No.: 11365D). The resulting NK cells were cultured and expanded in SCGM medium supplemented with 50 IU of IL-2 / 1 mL of culture medium.

[0324] Viral transduction into NK cells Harvest NK cells and incubate at 0.25 x 10 6 The NK cells were suspended in 2 mL of RPMI-1640 medium at a concentration of 100 µg / mL. Retroviral supernatant was added to the NK cells and the cells were incubated overnight at 37 °C. After incubation, the cells were pelleted by centrifugation and the medium was replaced with fresh SCGM containing 200 IU of IL-2 / mL culture medium. The transduced NK cells were cultured and used for experiments after 12–20 days of expansion.

[0325] Example 2: In vitro screening of rationally designed mutant IL-15-armed CAR NK cells This example shows comparable in vitro anti-tumor activity of CAR-NK cells armed with mutant secreted IL-15 (i.e., "sIL-15 m6" or "sIL-15 m4") compared to CAR-NK cells armed with wild-type secreted IL-15 (i.e., "sIL-15 wt").

[0326] Affinity measurement of IL-15 muteins for IL-15Rα and IL-2Rβ The IL-15 receptor consists of three polypeptides: the species-specific IL-15Rα ("IL-15Rα"), the common IL-2Rβ / IL-15Rβ ("IL-15Rβ" or "IL-2Rβ"), and the common gamma chain ("γC" or "gC"). The binding domains of IL-15 responsible for binding of IL-15 to the IL-15α and β receptors were analyzed. Several single residue mutations were made, and in vitro binding affinity of each mutant IL-15 polypeptide to IL-15Rα and IL-15Rβ was performed.

[0327] Briefly, HEK293 cells were pelleted and the crude IL-15 mutein supernatant was used for affinity measurements by surface plasmon resonance (SPR). Experiments were performed on a Biacore T200 SPR biosensor (GE Healthcare) at room temperature. Anti-Avi tag sensor chips were prepared at 25°C with a running buffer of 10 mM HEPES, 150 mM NaCI, 3 mM EDTA, and 0.005% (v / v) Tween-20 (pH 7.4). All surfaces of a Biacore CM5 sensor chip were activated with a 1:1 (v / v) mixture of 400 mM EDC and 100 mM NHS for 7 min at a flow rate of 10 μL / min. Anti-Avi reagent (Genscript, Cat. No. A00674-200) was diluted to 30 μg / mL in 10 mM sodium acetate, pH 5.0, and injected over all flow cells at 10 μL / min for 7 min. All flow cells were blocked with 1 M ethanolamine·HCl, pH 8.5, at 10 μL / min for 7 min.

[0328] Affinity measurements of all mutein supernatants were performed on a Biacore T200 at 25° C. using a running buffer (pH 7.4) of 10 mM HEPES, 150 mM NaCI, 3 mM EDTA, and 0.005% (v / v) Tween-20. Avi-tagged IL-15 muteins were captured on flow cells 2, 3, and 4 at a flow rate of 10 μL / min for 30 s. Flow cell 1 was used as the reference surface. After capture of the IL-15 muteins, the analytes (human IL-15Rα protein in the concentration range of 0.15625 nM to 1280 nM or human IL-2Rβ in the concentration range of 0.625 nM to 1280 nM) were injected on all flow cells for 100 s at a flow rate of 30 μL / min. After each analyte injection, dissociation was monitored for 600 s (i.e., when the analyte was human IL-15Rα protein) or 300 s (i.e., when the analyte was human IL-2Rβ protein), followed by regeneration of all flow cells with three 15 s injections of 10 mM glycine·HCl (pH 2.0). For double referencing, buffer cycle sensorgrams were collected (see, e.g., Myszka DG. 1999. Journal of molecular recognition 12:279-284). For kinetic analysis, double-referenced sensorgrams were globally fitted to a simple 1:1 Langmuir binding model using Biacore T200 Evaluation Software version 3.0.

[0329] Kinetic and affinity parameters of exemplary muteins are shown in Tables 3-4 below.

[0330] [Table 3]

[0331] [Table 4]

[0332] Screening of CAR-NK cells armed with IL-15 muteins Several variants were selected for expression in armed CAR-NK cells, namely, m1, m2, m3, m4, m5, m6, m7, m8, m17, and m18, which contain the amino acid substitutions A23L, L25E, Y26G, D8E, D61E, T62G, T62I, E89K, V3Y, and L25F, respectively, relative to human wild-type IL-15 (SEQ ID NO: 1). Table 1 shows the amino acid sequences of the IL-15 muteins. Table 5 shows the construction of IL-15 armed CAR-NK cells expressing wild-type and mutant sIL-15. CAR-NK cells were generated using the method described in Example 1.

[0333] [Table 5] TIFF2024527593000009.tif190165TIFF2024527593000010.tif123165

[0334] CAR-NK cells armed with mutant IL-15 were tested for in vitro cytotoxicity in short-term (Figure 1A) and long-term (Figure 1B) cell killing assays. Figure 1A shows in vitro short-term (4 h) killing of BCMA-positive targets, NCI-H929 cells, by BCMA CAR-NK cells armed with mutant IL-15 (E:T = 1:1, 0.25:1, 0.0625:1). All BCMA CAR-NK cells armed with mutant IL-15 (e.g., m1-m8) showed potent antitumor efficacy in short-term killing assays compared to untransduced NK cell (i.e., "UnNK") controls. The percentage of cytotoxicity against target cells was calculated by 7-AAD+%: 7-AAD+ cells / target cells x 100%.

[0335] FIG. 1B shows the in vitro long-term killing of BCMA CAR-NK cells armed with mutant IL-15 (e.g., m4-m8) against NCI-H929 cells (E:T=1:4). For each stimulation, tumor cells were added to NK cells for co-culture for about 24-48 hours, and after co-culture, the cells were harvested for further analysis by flow cytometry. Antigen stimulation was repeated a total of eight times. As shown in FIG. 1B, BCMA CAR-NK cells armed with mutant IL-15 m4, m5, m6, and m7 showed better anti-tumor efficacy compared to BCMA CAR-NK cells armed with sIL-15 wt. During stimulation with tumor cells, BCMA CAR-NK armed with mutant IL-15 m4 and m6 further showed better proliferation (FIG. 1C). NK cells expressing wild type hIL-15 (hIL-15-P2A-EGFP, SEQ ID NO:56) were used as a control.

[0336] Example 3: In vivo toxicity of BCMA CAR-NK cells armed with wild-type IL-15 This example shows that in an NCG mouse model bearing multiple myeloma tumor xenografts (NCI-H929-luc model), toxicity is observed in CAR-NK cells armed with wild-type secreted IL-15 (i.e., "sIL-15 wt"), independent of BCMA targeting and CAR expression.

[0337] BCMA CAR constructs were prepared as described in Example 1. To generate tumor xenografts, NCG mice were inoculated with NCI-H929-Luc cells (1×10 6 Tumor-implanted mice were injected intravenously with 100 ng / mouse of a BCMA-positive multiple myeloma cell line, #ATCC CRL-9068™, transduced with luciferase. 10-14 days later, tumor-implanted mice were treated with CD19 CAR-NK cells armed with sIL-15 wt, BCMA CAR-NK cells armed with sIL-1...

Claims

A modified immune cell comprising a first heterologous nucleic acid sequence encoding an IL-15 polypeptide comprising one or more amino acid substitutions at positions 62, 8, 3, and / or 25, wherein the numbering of the amino acid residue positions follows SEQ ID NO: 1, said modified immune cell. **Claim 2** The modified immune cell according to claim 1, wherein the IL-15 polypeptide comprises: (1) an amino acid substitution at position 62; wherein the amino acid substitution at position 62 is selected from the group consisting of T62G, T62I, T62Q, T62V, T62P, T62L, T62A, T62S, and T62Y; (2) an amino acid substitution at position 8; wherein the amino acid substitution at position 8 is D8E; (3) an amino acid substitution at position 3; wherein the amino acid substitution at position 3 is V3Y; (4) an amino acid substitution at position 25; wherein the amino acid substitution at position 25 is L25F. **Claim 3** The modified immune cell according to claim 2, wherein the amino acid substitution at position 62 is T62G, and wherein the IL-15 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

7. **Claim 4** The modified immune cell according to claim 2, wherein the IL-15 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 5, 78, or 79. **Claim 5** The modified immune cell according to claim 1, wherein the one or more amino acid substitutions reduce the affinity of the IL-15 polypeptide for IL-15Rβ as compared to an IL-15 polypeptide that does not comprise the one or more amino acid substitutions. **Claim 6**: The modified immune cell according to claim 1, wherein the modified immune cell secretes inflammatory cytokines at a level at least 50% lower than the secretion of inflammatory cytokines by a modified immune cell containing a heterologous nucleic acid sequence encoding a wild-type IL-15 polypeptide, and contains a first heterologous nucleic acid sequence encoding an IL-15 polypeptide that induces the secretion of inflammatory cytokines by the modified immune cell. **Claim 7** The modified immune cell according to claim 1, wherein the IL-15 polypeptide is a secreted type. **Claim 8** The modified immune cell according to claim 1, wherein the IL-15 polypeptide is a membrane-bound type. **Claim 9** The modified immune cell according to claim 8, wherein the IL-15 polypeptide contains a glycosylphosphatidylinositol (GPI) anchor peptide sequence, a transmembrane domain, or a membrane anchor domain. **Claim 10** The modified immune cell according to claim 1, wherein the IL-15 polypeptide is a fusion protein containing an IL-15 fragment fused to a second polypeptide fragment. **Claim 11** The modified immune cell according to claim 10, wherein the second polypeptide fragment is selected from the group consisting of IL-15Rα, the extracellular domain of IL-15Rα, the sushi domain of IL-15Rα, the transmembrane domain of IL-15Rα, IL-15Rβ, the common γ chain (γc), and combinations thereof. **Claim 12** The modified immune cell according to claim 11, wherein the second polypeptide fragment contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 50 to 55. **Claim 13** The modified immune cell according to claim 8, wherein the IL-15 polypeptide contains (a) an antigen-binding domain; (b) an IL-15 fragment; (c) a transmembrane domain; and (d) an intracellular domain. **Claim 14** The modified immune cell according to claim 1, wherein the modified immune cell comprises a second heterologous nucleic acid sequence encoding a genetically engineered receptor.

15. The modified immune cell according to claim 14, wherein the genetically engineered receptor is a chimeric antigen receptor (CAR), a modified T cell receptor (TCR), or a T cell antigen conjugate (TAC) receptor.

16. The modified immune cell according to claim 1, wherein the modified immune cell is selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) cells, NK cells, iNK-T cells, NK-T-like cells, αβ T cells, and γδ T cells.

17. The modified immune cell according to claim 1, wherein when the modified immune cell is administered to an individual, it has reduced toxicity in vivo compared to a modified immune cell that does not contain the first heterologous nucleic acid encoding the IL-15 polypeptide.

18. A pharmaceutical composition comprising the modified immune cell according to claim 1 and a pharmaceutically acceptable carrier.

19. Use of the pharmaceutical composition according to claim 18 in the manufacture of a medicament for treating a disease in an individual.

20. A genetically engineered IL-15 polypeptide comprising the amino acid substitutions D8E, T62G, V3Y, and / or L25F, wherein the numbering of the amino acid residue positions follows SEQ ID NO:

1.

21. The genetically engineered IL-15 polypeptide according to claim 20, comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 7, 78, and 79.

22. A nucleic acid comprising a nucleic acid sequence encoding the IL-15 polypeptide according to claim 20.

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