TW202246492A - Systems and methods for enhanced immunotherapies - Google Patents

Abstract

The present disclosure describes systems and methods for immunotherapies. Immune cells can be engineered to exhibit enhanced half-life as compared to control cell (e.g., a non-engineered immune cell). Immune cells can be engineered to exhibit enhanced proliferation as compared to a control cell. Immune cells can be engineered to effectively and specifically target diseased cells (e.g., cancer cells) that a control cell otherwise is insufficient or unable to target. The engineered Immune cells disclosed herein can be engineered ex vivo, in vitro, and in some cases, in vivo. The engineered Immune cells that are prepared ex vivo or in vitro can be administered to a subject in need thereof to treat a disease (e.g., myeloma or solid tumors). The engineered Immune cells can be autologous to the subject. Alternatively, the engineered immune cells can be allogeneic to the subject.

Description

Systems and methods for enhanced immunotherapy

Cancer (eg, neoplasm, tumor) is the leading cause of death worldwide, accounting for approximately 10 million deaths per year. Cancer continues to place an increasing health, economic and emotional burden on individuals, families, communities and nations. Increased knowledge of cancer biology (eg, cancer immunobiology in particular) and genetic engineering has facilitated the development of adoptive cell therapies (eg, cellular immunotherapy) with the goal of treating or controlling many different cancers.

The present disclosure provides methods and systems for treating cancer. Some aspects of the present disclosure provide engineered immune cells, such as engineered natural killer (NK) cells, and methods for their use in treating cancer, such as hematological malignancies or solid tumors.

In one aspect, there is provided an engineered cell comprising: (i) CD16 variants for enhanced CD16 signaling compared to control cells; (ii) heterologous IL-15, heterologous IL-15 receptor or a fragment thereof, and (iii) a chimeric antigen receptor comprising an antigen binding portion capable of binding CD19.

In some embodiments, the antigen binding portion comprises a heavy chain variable region comprising an amino acid sequence at least 80% identical to SEQ ID NO. 16 and a light chain variable region, the light chain variable region comprising The variable region comprises an amino acid sequence at least 80% identical to SEQ ID NO. 17.

In some embodiments, engineered cells comprise stem cells, progenitor cells, and specialized cells. In some embodiments, stem cells comprise isolated stem cells and induced stem cells. In some embodiments, stem cells comprise embryonic stem cells and induced pluripotent stem cells. In some embodiments, progenitor cells comprise isolated progenitor cells and induced progenitor cells. In some embodiments, the progenitor cells comprise hematopoietic stem cells, myeloid progenitor cells, megakaryoline progenitor cells, erythroid progenitor cells, and lymphoid progenitor cells. In some embodiments, specific cells comprise isolated specific cells and induced specific cells. In some embodiments, specific cells comprise T cells, B cells and NK cells. In some embodiments, NK cells are derived from isolated stem cells or induced stem cells. In some embodiments, NK cells are derived from isolated progenitor cells or induced progenitor cells.

In some embodiments, the heterologous IL-15 comprises secreted IL-15 and membrane-bound IL-15.

In some embodiments, the heterologous IL-15 is secreted IL-15 comprising an amino acid sequence at least 80% identical to SEQ ID NO. 24. In some embodiments, the membrane-bound IL-15 comprises at least a portion of IL-15 and at least a portion of an IL-15 receptor.

In some embodiments, at least a portion of IL-15 and at least a portion of said IL-15 receptor are linked by a linker. In some embodiments, the linker is selected from (GGGGS)n, (EGKSSGSGSESKST)n, and (EGKSSGSGSESKST)n(GGGGS)n. In some embodiments, n is any integer selected from 1-10. In some embodiments, the linker comprises an amino acid sequence that is at least 80% identical to any one selected from the group consisting of SEQ ID NO. 18-20. In some embodiments, the membrane-bound IL-15 further comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an amino acid sequence at least 80% identical to SEQ ID NO. 21. In some embodiments, the membrane-bound IL-15 further comprises an extracellular signal peptide. In some embodiments, the extracellular signal peptide comprises amino acids at least 80% identical to SEQ ID NO. 22. In some embodiments, the membrane-bound IL-15 comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs. 13-15.

In some embodiments, the CD16 variant is heterologous to the cell. In some embodiments, the CD16 variant comprises F158V (F176V). In some embodiments, the CD16 variant further comprises S197P. In some embodiments, the CD16 variant comprises an amino acid sequence at least 80% identical to SEQ ID NO. 23.

In some embodiments, the chimeric antigen receptor further comprises a hinge domain. In some embodiments, the hinge domain comprises an amino acid sequence derived from CD3D, CD3E, CD3G, CD3c CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor polypeptide.

In some embodiments, the chimeric antigen receptor further comprises a transmembrane domain. In some embodiments, the transmembrane domain comprises an amino acid sequence derived from CD3D, CD3E, CD3G, CD3c CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40 , ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D or T cell receptor polypeptide .

In some embodiments, the chimeric antigen receptor further comprises a co-stimulatory domain. In some embodiments, the co-stimulatory domain comprises an amino acid sequence derived from CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA- 4 or NKG2D or any combination thereof.

In some embodiments, the chimeric antigen receptor further comprises a signaling domain. In some embodiments, the signaling domain comprises an amino acid sequence derived from CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C , NKG2D or any combination thereof.

In another aspect, there is provided a polynucleotide comprising: (i) a first sequence encoding a CD16 variant for enhanced CD16 signaling, (ii) a second sequence encoding a heterologous IL-15, and (iii) a third sequence encoding a chimeric antigen receptor comprising an antigen binding portion capable of binding CD19. In some embodiments, the first sequence encoding a CD16 variant comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO. 27.

In some embodiments, the second sequence encoding heterologous IL-15 comprises a nucleic acid sequence encoding secreted IL-15 or a nucleic acid sequence encoding membrane-bound IL-15. In some embodiments, the nucleic acid sequence encoding secreted IL-15 comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO.30. In some embodiments, the nucleic acid sequence encoding membrane-bound IL-15 comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO. 28 or 29.

In some embodiments, the third sequence comprises: (a) a nucleic acid sequence encoding the heavy chain variable region of the antigen binding portion, said nucleic acid sequence having at least 80% identity with SEQ ID NO 25; and (b) encoding an antigen The nucleic acid sequence of the light chain variable region of the binding portion, said nucleic acid sequence having at least 80% identity to SEQ ID NO 26.

In another aspect, there is provided an engineered cell comprising: (i) a CD16 variant for enhanced CD16 signaling compared to a control cell; (ii) a heterologous IL-15, wherein the heterologous IL- 15 is a membrane-bound IL-15 comprising an amino acid sequence at least 95% identical to any one selected from SEQ ID No. 13-15; and (iii) a chimeric antigen comprising an antigen-binding portion capable of binding to CD19 receptor.

In some embodiments, engineered cells comprise stem cells, progenitor cells, and specialized cells. In some embodiments, stem cells comprise isolated stem cells and induced stem cells. In some embodiments, stem cells comprise embryonic stem cells and induced pluripotent stem cells. In some embodiments, progenitor cells comprise isolated progenitor cells and induced progenitor cells. In some embodiments, the progenitor cells comprise hematopoietic stem cells, myeloid progenitor cells, megakaryoline progenitor cells, erythroid progenitor cells, and lymphoid progenitor cells. In some embodiments, specific cells comprise isolated specific cells and induced specific cells. In some embodiments, specific cells comprise T cells, B cells and NK cells. In some embodiments, NK cells are derived from isolated stem cells or induced stem cells. In some embodiments, NK cells are derived from isolated progenitor cells or induced progenitor cells.

In some embodiments, the CD16 variant is heterologous to the cell. In some embodiments, the CD16 variant comprises F158V (F176V). In some embodiments, the CD16 variant further comprises S197P. In some embodiments, the CD16 variant comprises an amino acid sequence at least 80% identical to SEQ ID NO. 23.

In some embodiments, the antigen binding portion is an antibody selected from Fab, Fab', F(ab')2, scFv, and sdAb. In some embodiments, the antigen binding portion comprises a heavy chain variable region comprising an amino acid sequence at least 80% identical to SEQ ID NO. 16 and a light chain variable region, the light chain variable region comprising The variable region comprises an amino acid sequence at least 80% identical to SEQ ID NO. 17.

In some embodiments, the chimeric antigen receptor further comprises a hinge domain. In some embodiments, the hinge domain comprises an amino acid sequence derived from CD3D, CD3E, CD3G, CD3c CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor polypeptide.

In some embodiments, the chimeric antigen receptor further comprises a transmembrane domain. In some embodiments, the transmembrane domain comprises an amino acid sequence derived from CD3D, CD3E, CD3G, CD3c CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40 , ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D or T cell receptor polypeptide .

In some embodiments, the chimeric antigen receptor further comprises a co-stimulatory domain. In some embodiments, the co-stimulatory domain comprises an amino acid sequence derived from CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA- 4 or NKG2D or any combination thereof.

In some embodiments, the chimeric antigen receptor further comprises a signaling domain. In some embodiments, the signaling domain comprises an amino acid sequence derived from CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C , NKG2D or any combination thereof.

In another aspect, a polynucleotide is provided comprising: (i) a first sequence encoding a CD16 variant for enhanced CD16 signaling; (ii) a second sequence encoding membrane-bound IL-15, Wherein said second sequence comprises a nucleic acid sequence having at least 95% identity with SEQ ID NO.28 or 29; and (iii) a third sequence encoding a chimeric antigen receptor, said chimeric antigen receptor comprising Antigen-binding portion of CD19.

In another aspect, an engineered cell is provided, comprising heterologous IL-15, wherein the heterologous IL-15 is membrane-bound IL-15, and the membrane-bound IL-15 comprises a compound selected from SEQ ID No. . an amino acid sequence having at least 95% identity to any of 13-15.

In one aspect, the present disclosure provides an engineered NK cell comprising: (1) secreted IL-15 heterologous to the engineered NK cell; and (2) one or more of the following: (i) for A CD16 variant with enhanced CD16 signaling compared to a control cell, wherein the CD16 variant is heterologous to the cell, or (ii) a chimeric polypeptide receptor comprising an antigen-binding portion capable of binding an antigen, wherein the antigen Not CD19, where engineered cells lack heterologous IL-15R. In some embodiments, the engineered NK cells further comprise a heterologous polynucleotide encoding secreted IL-15. In some embodiments, engineered NK cells are derived from isolated stem cells or induced stem cells.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise a CD16 variant. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cell comprises a chimeric polypeptide receptor. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cell comprises both (i) a CD16 variant and (ii) a chimeric polypeptide receptor. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cell further comprises a heterologous polynucleotide encoding secreted IL-15.

In some embodiments of any of the engineered NK cells disclosed herein, the antigen comprises one or more members selected from the group consisting of: BCMA, CD20, CD22, CD30, CD33, CD38, CD70, ĸ, Lewis ± Y, NKG2D ligand, ROR1, NY-ESO-1, NY-ESO-2, MART-1 and gp100. In some embodiments of any of the engineered NK cells disclosed herein, the antigen comprises an NKG2D ligand selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

In one aspect, the present disclosure provides an engineered NK cell derived from an isolated or induced stem cell comprising secreted IL-15 heterologous to the engineered NK cell, wherein the engineered cell lacks the heterologous IL-15R . In some embodiments, the engineered NK cells further comprise a heterologous polynucleotide encoding secreted IL-15.

In one aspect, the present disclosure provides engineered NK cells comprising membrane-bound IL-15 heterologous to the engineered NK cells, wherein the engineered NK cells further comprise one or more of the following: (a) is not IL- A heterologous cytokine of 15, (b) reduced expression or activity of an endogenous immunomodulatory polypeptide, wherein the endogenous immunomodulatory polypeptide is not B2M, or (c) a safety switch (or inducible cell death moiety). In some embodiments, engineered NK cells are derived from isolated stem cells or induced stem cells.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise a heterologous cytokine other than IL-15. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression or activity of an endogenous immunomodulatory polypeptide, wherein the endogenous immunomodulatory polypeptide is not B2M. In some embodiments of any of the engineered NK cells disclosed herein, wherein the engineered NK cell comprises a safety switch. In some embodiments of any one of the engineered NK cells disclosed herein, wherein the engineered NK cells comprise two or more of (a) to (c). In some embodiments of any one of the engineered NK cells disclosed herein, wherein the engineered NK cells comprise (a) to (c).

In some embodiments of any of the engineered NK cells disclosed herein, the membrane-bound IL-15 is part of a chimeric membrane receptor comprising IL-15 and at least a portion of an IL-15R. In some embodiments of any of the engineered NK cells disclosed herein, the chimeric membrane receptor comprises a sequence selected from the group consisting of: (i) four or more adjacent repeats of SEQ ID NO. 9 , (ii) SEQ ID NO.10, (iii) SEQ ID NO.11, (iv) combination of SEQ ID NO.9 and SEQ ID NO.11, (v) SEQ ID NO.12, (vi) SEQ ID NO. 13 and (vii) SEQ ID NO. 14).

In one aspect, the present disclosure provides an engineered NK cell comprising a CD16 variant comprising at least a portion of CD16 and at least a portion of CD64 for enhanced CD16 signaling in the engineered NK cell, wherein said engineered NK cells exhibit reduced expression or activity of an endogenous immunomodulatory polypeptide. In some embodiments, the engineered NK cells comprise heterologous IL-15 or fragments thereof. In some embodiments, the engineered NK cells comprise a receptor comprising a heterologous IL-15R or a fragment thereof. In some embodiments, engineered NK cells are derived from isolated stem cells or induced stem cells.

In one aspect, the present disclosure provides an engineered NK cell comprising reduced activity of endogenous IL-17 signaling, wherein the engineered NK cell is derived from an isolated stem cell or an induced stem cell. In some embodiments, the engineered NK cells further comprise (i) heterologous IL-15 or fragments thereof and/or (ii) heterologous IL-15R or fragments thereof. In some embodiments, engineered NK cells are derived from isolated stem cells or induced stem cells.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression of endogenous IL-17 or endogenous IL-17R. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression of endogenous IL-17 and endogenous IL-17R. In some embodiments of any of the engineered NK cells disclosed herein, the endogenous IL-17 comprises IL-17A. In some embodiments of any of the engineered NK cells disclosed herein, the endogenous IL-17 comprises IL-17F. In some embodiments of any of the engineered NK cells disclosed herein, the endogenous IL-17R comprises IL-17RA, IL-17RB, IL-17RC, IL-17RD, or IL-17RE. In some embodiments of any of the engineered NK cells disclosed herein, the endogenous IL-17R comprises IL-17RA.

In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cell exhibits an enhanced expression profile of NK cell markers without reduced expression of endogenous IL-17 compared to control cells or activity. In some embodiments of any of the engineered NK cells disclosed herein, the NK cell marker comprises human CD57 or killer immunoglobulin-like receptor (KIR).

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells exhibit enhanced survival in the presence of tumor cells, without reducing endogenous IL-17 signaling, compared to control cells conduction activity.

In one aspect, the present disclosure provides engineered NK cells comprising a heterologous STAT, wherein the engineered NK cells are derived from isolated stem cells or induced stem cells. In some embodiments, the engineered NK cells further comprise heterologous IL-15 or a fragment thereof. In some embodiments, the engineered NK cells further comprise a heterologous IL-15R or a fragment thereof.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells exhibit enhanced survival in the presence of tumor cells compared to control cells without the heterologous STAT. In some embodiments of any of the engineered NK cells disclosed herein, the heterologous STAT comprises STAT1, STAT2, STAT3, STAT4, STAT3, STAT4, STAT5A, STAT5B, or STAT6. In some embodiments of any of the engineered NK cells disclosed herein, the heterologous STAT comprises STAT3. In some embodiments of any of the engineered NK cells disclosed herein, the heterologous STAT comprises STAT5B.

In one aspect, the present disclosure provides engineered NK cells comprising (1) reduced expression or activity of an endogenous killer cell immunoglobulin-like receptor (KIR) and (2) one or more of : (a) a chimeric polypeptide receptor comprising an antigen-binding portion capable of binding to an antigen, (b) a heterologous cytokine, (c) a CD16 variant for enhanced CD16 signaling compared to control cells, wherein CD16 The variant is heterologous to the engineered NK cell, (d) a heterologous immunomodulatory polypeptide, or (e) reduced expression or activity of an endogenous immunomodulatory polypeptide. In some embodiments, the engineered NK cells further comprise (i) heterologous IL-15 or fragments thereof and/or (ii) heterologous IL-15R or fragments thereof. In some embodiments, engineered NK cells are derived from isolated stem cells or induced stem cells.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cell comprises a chimeric polypeptide receptor. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise heterologous cytokines. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise a CD16 variant. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise a heterologous immunomodulatory polypeptide. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression or activity of an endogenous immunomodulatory polypeptide. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cells comprise two or more of (a) to (e). In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cells comprise three or more of (a) to (e). In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cells comprise four or more of (a) to (e). In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise all of (a) to (e).

In some embodiments of any of the engineered NK cells disclosed herein, the KIR comprises KIR2D. In some embodiments of any of the engineered NK cells disclosed herein, the KIR2D comprises KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, or KIR2DS5. In some embodiments of any of the engineered NK cells disclosed herein, the KIR comprises KIR3D. In some embodiments of any of the engineered NK cells disclosed herein, the KIR3D comprises KIR3DL1, KIR3DL2, KIR3DL3, or KIR3DS1.

In one aspect, the present disclosure provides an engineered NK cell comprising reduced expression or activity of one or more of: (i) endogenous CD94, (ii) endogenous CD96, (iii) endogenous TGFβ receptor, or (iv) endogenous SHIP2, wherein the engineered cells are derived from isolated stem cells or induced stem cells. In some embodiments, the engineered NK cells further comprise (i) heterologous IL-15 or fragments thereof and/or (ii) heterologous IL-15R or fragments thereof. In some embodiments, engineered NK cells are derived from isolated stem cells or induced stem cells.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression or activity of endogenous CD94. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression or activity of endogenous CD96. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression or activity of endogenous TGFβ receptors. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression or activity of endogenous SHIP2. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression or activity of two or more of (i) to (iv). In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression or activity of three or more of (i) to (iv). In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression or activity of all of (i) to (iv).

In one aspect, the present disclosure provides an engineered NK cell comprising reduced expression or activity of one or more of: (i) endogenous CD80, (ii) endogenous CD86, (iii) endogenous ICOSL, (iv) endogenous CD40L, (v) endogenous MICA or MICB, or (vi) endogenous NKG2DL, wherein the engineered cells are derived from isolated or induced stem cells. In some embodiments, the engineered NK cells further comprise (i) heterologous IL-15 or fragments thereof and/or (ii) heterologous IL-15R or fragments thereof.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression or activity of endogenous CD80. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression or activity of endogenous CD86. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression or activity of endogenous ICOSL. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression or activity of endogenous CD40L. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression or activity of endogenous MICA or MICB. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression or activity of endogenous NKG2D. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression or activity of two or more of (i) to (vi). In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression or activity of three or more of (i) to (vi). In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression or activity of four or more of (i) to (vi). In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression or activity of five or more of (i) to (vi). In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise reduced expression or activity of (i) to (vi).

In one aspect, the present disclosure provides an engineered NK cell comprising (1) reduced expression or activity of endogenous ICAM1 and (2) one or more of: (a) comprising an antigen-binding Chimeric polypeptide receptors for antigen binding portions, (b) heterologous cytokines, or (c) CD16 variants for enhanced CD16 signaling compared to control cells, wherein the CD16 variants are heterologous to engineered NK cells source. In some embodiments, the engineered NK cells further comprise (i) heterologous IL-15 or fragments thereof and/or (ii) heterologous IL-15R or fragments thereof. In some embodiments, engineered NK cells are derived from isolated stem cells or induced stem cells.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cell comprises a chimeric polypeptide receptor. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise heterologous cytokines. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise a CD16 variant. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cells comprise two or more of (a) to (c). In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise all of (a) to (c).

In one aspect, the present disclosure provides an engineered NK cell comprising reduced expression or activity of endogenous ICAM1, wherein the engineered cell is derived from an induced stem cell. In some embodiments, the engineered NK cells further comprise (i) heterologous IL-15 or fragments thereof and/or (ii) heterologous IL-15R or fragments thereof. In some embodiments, engineered NK cells are derived from isolated stem cells or induced stem cells.

In one aspect, the present disclosure provides an engineered NK cell comprising one or more of: (i) heterologous PD-L2, or (ii) heterologous TGF-β. In some embodiments, the engineered NK cells further comprise (i) heterologous IL-15 or fragments thereof and/or (ii) heterologous IL-15R or fragments thereof. In some embodiments, engineered NK cells are derived from isolated stem cells or induced stem cells.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise allogeneic PD-L2. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise heterologous TGF-β. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise (i) heterologous PD-L2 and (ii) heterologous TGF-β.

In one aspect, the present disclosure provides an engineered NK cell comprising one or more of the following: (i) heterologous CCL21, (ii) heterologous IL-10, (iii) heterologous CD46, (iv) heterologous Source CD55, or (v) heterologous CD59, wherein the engineered cells are derived from isolated stem cells or induced stem cells. In some embodiments, the engineered NK cells further comprise (i) heterologous IL-15 or fragments thereof and/or (ii) heterologous IL-15R or fragments thereof.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise heterologous CCL21. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise heterologous IL-10. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise allogeneic CD46. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise heterologous CD55. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise allogeneic CD59. In some embodiments of any one of the engineered NK cells disclosed herein, the engineered NK cells comprise two or more of (i) to (v). In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise three or more of (i) to (v). In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise four or more of (i) to (v). In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise all of (i) to (v).

In one aspect, the present disclosure provides an engineered NK cell derived from an induced stem cell comprising a heterologous cytokine other than IL-15. In some embodiments, the heterologous cytokine other than IL-15 comprises IL-21.

In one aspect, the present disclosure provides an engineered NK cell derived from an induced stem cell comprising heterologous IL-21. In some embodiments, the engineered NK cells further comprise (i) heterologous IL-15 or fragments thereof and/or (ii) heterologous IL-15R or fragments thereof.

In one aspect, the present disclosure provides an engineered NK cell comprising a chimeric polypeptide receptor comprising an antigen binding portion capable of specifically binding CD38, wherein the engineered NK cell is derived from an isolated embryo Stem cells or induced stem cells. In some embodiments, the engineered NK cells further comprise (i) heterologous IL-15 or fragments thereof and/or (ii) heterologous IL-15R or fragments thereof.

In one aspect, the disclosure provides engineered NK cells comprising a chimeric polypeptide receptor comprising a CD8 transmembrane domain and one or more of: (i) 2B4 signaling domain and (ii) DAP10 signaling domain. In some embodiments, the engineered NK cells further comprise (i) heterologous IL-15 or fragments thereof and/or (ii) heterologous IL-15R or fragments thereof. In some embodiments, engineered NK cells are derived from isolated stem cells or induced stem cells.

In some embodiments of any of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises a 2B4 signaling domain. In some embodiments of any of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises a DAP10 signaling domain. In some embodiments of any of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises (i) a 2B4 signaling domain and (ii) a DAP10 signaling domain.

In one aspect, the present disclosure provides an engineered NK cell derived from an isolated stem cell or an induced stem cell comprising a chimeric polypeptide receptor comprising one or more of the following: (i) CD8 transmembrane domain, (ii) 2B4 signaling domain or (iii) DAP10 signaling domain. In some embodiments, the engineered NK cells further comprise (i) heterologous IL-15 or fragments thereof and/or (ii) heterologous IL-15R or fragments thereof.

In some embodiments of any of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises a CD8 transmembrane domain. In some embodiments of any of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises a 2B4 signaling domain. In some embodiments of any of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises a DAP10 signaling domain. In some embodiments of any of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises two or more of (i) to (iii). In some embodiments of any of the engineered NK cells disclosed herein, the chimeric polypeptide receptor comprises all of (i) to (iii).

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells exhibit reduced expression or activity of endogenous CD38. Alternatively, in some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells have unmodified expression or activity of endogenous CD38.

In some embodiments of any of the engineered NK cells disclosed herein, the heterologous IL-15 or fragment thereof is secreted by the engineered NK cells. Alternatively, in some embodiments of any of the engineered NK cells disclosed herein, the heterologous IL-15 or fragment thereof is membrane bound.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cell comprises a chimeric polypeptide receptor comprising an antigen binding portion capable of binding an antigen. In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cell comprises an additional chimeric polypeptide receptor comprising an antigen binding portion capable of binding a different antigen. In some embodiments of any of the engineered NK cells disclosed herein, the antigen binding portion of the chimeric polypeptide receptor is a multispecific binding portion capable of specifically binding two or more different antigens. In some embodiments of any of the engineered NK cells disclosed herein, the antigen comprises one or more members selected from the group consisting of BCMA, CD19, CD20, CD22, CD30, CD33, CD38, CD70, ĸ , Lewis Y, NKG2D ligand, ROR1, NY-ESO-1, NY-ESO-2, MART-1, and gp100. In some embodiments of any of the engineered NK cells disclosed herein, the antigen comprises an NKG2D ligand selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cell comprises a safety switch capable of causing death of the engineered NK cell. In some embodiments of any of the engineered NK cells disclosed herein, the safety switch comprises a protein selected from the group consisting of caspase (e.g., caspase 3, 7, or 9), thymidine kinase, cytosine deaminase, modified EGFR One or more members of the group consisting of and B cell CD20.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise heterologous cytokines. In some embodiments of any of the engineered NK cells disclosed herein, the heterologous cytokine comprises one or more selected from the group consisting of IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21 member. In some embodiments of any of the engineered NK cells disclosed herein, the heterologous cytokine is not IL15.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise a heterologous immunomodulatory polypeptide. In some embodiments of any of the engineered NK cells disclosed herein, the heterologous immunomodulatory polypeptide comprises a protein selected from the group consisting of HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-β, CCL21, IL10, One or more members of the group consisting of CD46, CD55 and CD59.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells exhibit reduced expression or activity of an endogenous immunomodulatory polypeptide. In some embodiments of any of the engineered NK cells disclosed herein, the endogenous immunomodulatory polypeptide comprises an immune checkpoint inhibitor or a modulator of hypoimmunity. In some embodiments of any of the engineered NK cells disclosed herein, the immune checkpoint inhibitor comprises a protein selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, IT, CD96, LAG3, TIGIT, One or more members of the group consisting of TGFβ receptor and 2B4. In some embodiments of any of the engineered NK cells disclosed herein, the immune checkpoint inhibitor comprises SHIP2. In some embodiments of any of the engineered NK cells disclosed herein, the hypoimmune modulator comprises a protein selected from the group consisting of B2M, CIITA, TAP1, TAP2, tap-related protein, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, One or more of the group consisting of CD40L, ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-β, CCL21, IL10, CD46, CD55 and CD59 members.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells comprise CD16 variants for enhanced CD16 signaling compared to control cells, wherein the CD16 variants are heterologous to the engineered NK cells of. In some embodiments of any of the engineered NK cells disclosed herein, the CD16 variant comprises a sequence selected from the group consisting of SEQ ID NO. 1-8.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells exhibit enhanced cytotoxicity against the target cells compared to control cells.

In some embodiments of any of the engineered NK cells disclosed herein, the engineered NK cells induce a reduced immune response in the host cell compared to control cells. In some embodiments of any of the engineered NK cells disclosed herein, the host cell is an immune cell.

In some embodiments of any of the engineered NK cells disclosed herein, the isolated stem cells comprise embryonic stem cells. In some embodiments of any of the engineered NK cells disclosed herein, the induced stem cells comprise induced pluripotent stem cells.

In one aspect, the present disclosure provides a method comprising: (1) obtaining a cell from a subject; and (2) producing an engineered NK cell of any one of the engineered NK cells disclosed herein from the cell.

In one aspect, the present disclosure provides a method comprising administering to a subject in need thereof a population of NK cells comprising engineered NK cells of any one of the engineered NK cells disclosed herein. In some embodiments, the method further comprises administering a separate therapeutic agent to the object. In some embodiments, the sole therapeutic agent is a chemotherapeutic agent.

In one aspect, the present disclosure provides a composition comprising an engineered NK cell of any one of the engineered NK cells disclosed herein.

In one aspect, the present disclosure provides a composition comprising: (1) an engineered immune cell comprising one or more members comprising (i) heterologous IL-15 or Fragments thereof, (ii) CD16 variants for enhanced CD16 signaling compared to control cells, wherein the CD16 variants are heterologous to engineered immune cells, or (iii) comprising an antigen-binding portion capable of binding an antigen a chimeric polypeptide receptor; and (2) a separate therapeutic agent capable of binding to CD20. In some embodiments, engineered NK cells are derived from isolated stem cells or induced stem cells. In some embodiments, the isolated stem cells comprise embryonic stem cells. In some embodiments, the induced stem cells comprise induced pluripotent stem cells.

In some embodiments of any of the compositions disclosed herein, the engineered immune cells comprise heterologous IL-15. In some embodiments, heterologous IL-15 or fragments thereof are secreted by engineered NK cells. In some embodiments, the heterologous IL-15 or fragment thereof is membrane bound.

In some embodiments of any of the compositions disclosed herein, the engineered immune cell comprises a CD16 variant. In some embodiments, the CD16 variant comprises a sequence selected from the group consisting of SEQ ID NO. 1-8.

In some embodiments of any of the compositions disclosed herein, the engineered immune cell comprises a chimeric polypeptide receptor.

In some embodiments of any of the compositions disclosed herein, the engineered immune cells comprise two or more of (i) to (iii). In some embodiments of any of the compositions disclosed herein, the engineered immune cells comprise (i) to (iii).

In some embodiments of any of the compositions disclosed herein, the engineered immune cells comprise engineered NK cells. In some embodiments of any of the compositions disclosed herein, the engineered immune cells comprise engineered T cells.

In some embodiments of any of the compositions disclosed herein, the engineered NK cells exhibit reduced expression or activity of endogenous CD38. In some embodiments of any of the compositions disclosed herein, the engineered NK cells have unmodified expression or activity of endogenous CD38.

In some embodiments of any of the compositions disclosed herein, the antigen binding portion of the chimeric polypeptide receptor is a multispecific binding portion capable of specifically binding two or more different antigens. In some embodiments of any of the compositions disclosed herein, the antigen comprises one or more members selected from the group consisting of BCMA, CD19, CD20, CD22, CD30, CD33, CD38, CD70, ĸ, Lewis ± Y, NKG2D ligand, ROR1, NY-ESO-1, NY-ESO-2, MART-1 and gp100. In some embodiments of any of the compositions disclosed herein, the antigen comprises an NKG2D ligand selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

In some embodiments of any of the compositions disclosed herein, the engineered NK cells further comprise a safety switch capable of causing death of the engineered NK cells. In some embodiments of any of the compositions disclosed herein, the safety switch comprises a protein selected from the group consisting of caspase (e.g., caspase 3, 7, or 9), thymidine kinase, cytosine deaminase, modified EGFR, and B One or more members of the group consisting of cells CD20.

In some embodiments of any of the compositions disclosed herein, the engineered NK cells further comprise a heterologous cytokine. In some embodiments of any of the compositions disclosed herein, the heterologous cytokine comprises one or more members selected from the group consisting of IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, and IL21. In some embodiments of any of the compositions disclosed herein, the heterologous cytokine is not IL15.

In some embodiments of any of the compositions disclosed herein, the engineered NK cells further comprise a heterologous immunomodulatory polypeptide. In some embodiments of any of the compositions disclosed herein, the heterologous immunomodulatory polypeptide comprises a protein selected from the group consisting of HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-β, CCL21, IL10, CD46, One or more members of the group consisting of CD55 and CD59.

In some embodiments of any of the compositions disclosed herein, the engineered NK cells exhibit reduced expression or activity of an endogenous immunomodulatory polypeptide. In some embodiments of any of the compositions disclosed herein, the endogenous immunomodulatory polypeptide comprises an immune checkpoint inhibitor or a modulator of hypoimmunity.

In some embodiments of any of the compositions disclosed herein, the immune checkpoint inhibitor comprises a receptor selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, IT, CD96, LAG3, TIGIT, TGFβ One or more members of the group consisting of body and 2B4. In some embodiments of any of the compositions disclosed herein, the immune checkpoint inhibitor comprises SHIP2. In some embodiments of any of the compositions disclosed herein, the hypoimmune modulator comprises a protein selected from the group consisting of B2M, CIITA, TAP1, TAP2, tap-related protein, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, One or more members of the group consisting of ICAM1, MICA, MICB, ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-β, CCL21, IL10, CD46, CD55 and CD59 .

In some embodiments of any of the compositions disclosed herein, the engineered NK cells exhibit enhanced cytotoxicity against target cells compared to control cells. In some embodiments of any of the compositions disclosed herein, the engineered NK cells induce a reduced immune response in the host cells compared to control cells.

In some embodiments of any of the compositions disclosed herein, the host cell is an immune cell.

In some embodiments of any of the compositions disclosed herein, the engineered NK cell further comprises a receptor comprising a heterologous IL-15R or a fragment thereof.

In one aspect, the present disclosure provides a method comprising: administering a subject composition of any one of the compositions disclosed herein to an object in need thereof. In some embodiments, the sole therapeutic agent includes a chemotherapeutic agent.

Other aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of the disclosure are shown and described. As will be realized, the disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and descriptions are to be regarded as illustrative and not restrictive. [incorporated by reference]

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and/or take precedence over any such conflicting material.

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that these embodiments are provided by way of example only. Numerous variations, changes and substitutions will occur to those skilled in the art without departing from the teachings of the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

As used in the specification and claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "chimeric transmembrane receptor" includes a plurality of chimeric transmembrane receptors.

The terms "about" or "approximately" generally mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value was measured or determined, i.e., measuring System limitations. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with regard to biological systems or processes, the term may mean within an order of magnitude of a value, preferably within 5 times the value, more preferably within 2 times the value. Where specific values are described in this application and claims, unless otherwise indicated, the term "about" should be assumed to mean within an acceptable error range for the specific value.

The use of an alternative (eg, "or") should be understood to mean either, both, or any combination of the alternatives. The term "and/or" should be understood to mean one or both of the alternatives.

The term "cell" generally refers to a biological cell. A cell can be the basic structural, functional and/or biological unit of a living organism. A cell can be derived from any organism having one or more cells. Some non-limiting examples include: prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotic organisms, protozoan cells, cells from plants (e.g., from plant crops, fruits, vegetables, grains, Soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugar cane, squash, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, lycopodium, hornworts, liverworts , moss cells), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh et al), seaweed (e.g. kelp), fungal cells (e.g. yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g. Drosophila, cnidaria, echinoderms, nematodes, etc.), cells from vertebrates Cells from animals (e.g., fish, amphibians, reptiles, birds, mammals), from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, human etc.) cells, etc. Sometimes cells are not derived from natural organisms (for example, cells can be made synthetically, sometimes called artificial cells).

As used interchangeably herein, the terms "reprogramming", "dedifferentiation", "increasing cell potential" or "increasing developmental potential" generally refer to methods of increasing cell potential or dedifferentiating cells to a less differentiated state. For example, a cell with increased cellular potential has more developmental plasticity (ie, can differentiate into more cell types) than the same cell in a non-reprogrammed state. In other words, a reprogrammed cell is a cell at a lower state of differentiation than the same cell in a non-reprogrammed state.

The term "differentiation" generally refers to the process by which unspecialized ("uncommitted") or less specialized cells acquire the characteristics of specialized cells (eg, immune cells). A differentiated or differentiation-induced cell is a cell that occupies a more specialized ("committed") position in a cell lineage. The term "committed" generally refers to a cell that has progressed to a point in the differentiation pathway where it would normally continue to differentiate into a particular cell type or subclass of cell type and would not normally be able to differentiate to a different cell type or revert to a less differentiated cell type.

The term "pluripotent" generally refers to the ability of a cell to form all lineages of a subject or somatic cell (ie, an embryonic body). For example, embryonic stem cells are a type of pluripotent stem cell capable of forming cells from each of the three germ layers (ectoderm, mesoderm and endoderm). Pluripotency can be a continuum of developmental potential, ranging from incomplete or partially pluripotent cells (such as ectodermal stem cells), which cannot give rise to complete organisms, to more primitive, more potent cells, which are able to give rise to Whole organisms (such as embryonic stem cells)).

The term "induced pluripotent stem cells" (iPSCs) generally refers to stem cells derived from differentiated cells (e.g., differentiated adult, neonatal, or fetal cells) that have been induced or altered (i.e., reprogrammed ) are cells capable of differentiating into tissues of all three germ layers or dermis (mesoderm, endoderm and ectoderm). The resulting iPSCs do not refer to cells found in nature. In some cases, iPSCs can be engineered to differentiate directly into committed cells (eg, natural killer (NK) cells). In some cases, iPSCs can be engineered to first differentiate into tissue-specific stem cells (eg, hematopoietic stem cells (HSCs)), which can then be further induced to differentiate into committed cells (eg, NK cells).

The term "embryonic stem cell" (ESC) generally refers to the naturally occurring pluripotent stem cells of the inner cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent, producing all derivatives of the three main germ layers (ectoderm, endoderm, and mesoderm) during development. In some cases, ESCs can be engineered to differentiate directly into committed cells (eg, NK cells). In some cases, ESCs can be engineered to first differentiate into tissue-specific stem cells (eg, HSCs), which can then be further induced to differentiate into committed cells (eg, NK cells).

The term "isolated stem cell" generally refers to any type of stem cell disclosed herein (eg, ESC, HSC, mesenchymal stem cell (MSC), etc.) isolated from a multicellular organism. For example, HSCs can be isolated from the body of a mammal (eg, a human). In another example, embryonic stem cells can be isolated from embryos.

The term "isolated" generally refers to a cell or population of cells that has been separated from its original environment. For example, the new environment of the isolated cell is substantially free of at least one component found in the environment in which the "unisolated" reference cell exists. An isolated cell can be a cell that has been removed from some or all components found in its natural environment, such as a cell isolated from a tissue or biopsy sample. The term also includes cells that have had at least one, some or all components removed from cells found in non-naturally occurring environments, such as cells isolated from cell culture or cell suspensions. Thus, an isolated cell is partly or completely separated from at least one component (including other substances, cells or populations of cells) found in nature or grown, stored or subsisted in a non-naturally occurring environment.

As used interchangeably herein, the terms "hematopoietic stem and progenitor cells," "hematopoietic stem cells," "hematopoietic progenitor cells," or "hematopoietic precursor cells" generally refer to cells that are committed to the hematopoietic lineage but are capable of further hematopoietic differentiation (e.g. differentiate into NK cells), and include multipotent hematopoietic stem cells (hemoblasts), myeloid progenitors, megakaryoline progenitors, erythroid progenitors, and lymphoid progenitors. Hematopoietic stem and progenitor cells (HSCs) are pluripotent stem cells that give rise to all blood cell types, including the myeloid lineage (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes , megakaryocytes/platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NK cells). In some instances, HSCs may be CD34+ hematopoietic cells capable of giving rise to mature myeloid and lymphoid cell types, including T cells, NK cells, and B cells.

The term "immune cell" generally refers to differentiated hematopoietic cells. Non-limiting examples of immune cells may include NK cells, T cells, monocytes, innate lymphocytes, tumor infiltrating lymphocytes, macrophages, granulocytes, and the like.

The term "NK cells" or "natural killer cells" generally refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). In some instances, the NK cell, which is phenotypically CD3- and CD56+, expresses at least one of NKG2C and CD57 (e.g., NKG2C, CD57, or both to the same or different extent), and optionally expresses CD16, But lack expression of one or more of: PLZF, SYK, FceRγ and EAT-2. In some instances, the isolated subpopulation of CD56+ NK cells can exhibit expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIRs, NKG2A, and/or DNAM-1.

As used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. A nucleotide may be the monomeric unit of a nucleic acid sequence such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The term nucleotide may include ribonucleoside triphosphates (adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP)), and deoxyribonucleoside triphosphate , such as dATP, dCTP, dITP, dUTP, dGTP, dTTP or derivatives thereof. Such derivatives may include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP and nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. The term nucleotide as used herein may denote dideoxyribonucleoside triphosphate (ddNTP) and derivatives thereof. Illustrative examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled by well known techniques. Labeling can also be done with quantum dots. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzymatic labels. Fluorescent labels for nucleotides can include, but are not limited to, luciferin, 5-carboxyluciferin (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyluciferin ( JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'Dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanine and 5-(2'-Aminoethyl)aminonaphthalene- 1-sulfonic acid (EDANS). Specific examples of fluorescently labeled nucleotides may include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [ JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP; FluoroLink Deoxynucleotide, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP from Amersham, Arlington Heights, Ill.; available from Boehringer Mannheim, Indianapolis , Luciferin-15-dATP, Luciferin-12-dUTP, Tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, Luciferin-12-ddUTP, Luciferin-12 from Ind. -UTP and luciferin-15-2'-dATP; and chromosomally labeled nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY available from Molecular Probes, Eugene, Oreg. -TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, Luciferin -12-UTP, Luciferin-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine Rhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP and Texas Red-12-dUTP. Nucleotides can also be labeled or labeled by chemical modification. The chemically modified mononucleotide can be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs may include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin -14-dCTP) and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

The terms "polynucleotide", "oligonucleotide" or "nucleic acid" are used interchangeably herein and generally refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleosides Acids, or analogs thereof, whether in single-, double-, or multiple-chain form. A polynucleotide can be exogenous or endogenous to the cell. Polynucleotides can be present in a cell-free environment. A polynucleotide may be a gene or a fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. A polynucleotide may include one or more analogs (eg, altered backbones, sugars, or nucleobases). Modifications to the nucleotide structure, if present, can be imparted before or after polymer assembly. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, heterologous nucleic acid, morpholino, locked nucleic acid, diol nucleic acid, threose nucleic acid, dideoxynucleotide, cordycepin, 7 - deaza-GTP, fluorophores (such as rhodamine or luciferin linked to sugars), thiol-containing nucleotides, nucleotides linked to biotin, fluorescent base analogs, CpG islands, formazan -7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, braidin and wyoside. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, locus(s) defined from linkage analysis, exons, introns, messenger RNA (mRNA), translocation RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, Vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides may be interrupted by non-nucleotide components.

The term "gene" generally refers to a nucleic acid encoding an RNA transcript (eg, DNA such as genomic DNA and cDNA) and its corresponding nucleotide sequence. As used herein with reference to genomic DNA, the term includes intervening non-coding regions as well as regulatory regions, and may include both 5' and 3' ends. In some uses, the term encompasses transcribed sequences, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons and introns. In some genes, the transcribed region will include an "open reading frame" that encodes a polypeptide. In some uses of the term, a "gene" includes only the coding sequence necessary to encode a polypeptide (eg, an "open reading frame" or "coding region"). In some instances, a gene does not encode a polypeptide, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some instances, the term "gene" includes not only transcribed sequences, but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers and promoters. A gene may refer to an "endogenous gene" or native gene in its natural location in the genome of an organism. A gene may refer to a "foreign gene" or a non-native gene. A non-native gene may refer to a gene that is not normally present in the host organism but which is introduced into the host organism by gene transfer. A non-native gene can also refer to a gene that is not in its natural location in the genome of an organism. A non-native gene can also refer to a naturally occurring nucleic acid or polypeptide sequence (eg, a non-native sequence) that includes mutations, insertions, and/or deletions.

The term "expression" generally refers to the process or processes by which a polynucleotide is transcribed (e.g., into mRNA or other RNA transcript) from a DNA template and/or the transcribed mRNA is subsequently translated into a peptide, polypeptide or protein process. Transcripts and encoded polypeptides may collectively be referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression can include splicing of mRNA in eukaryotic cells. With respect to expression, "up-regulation" generally refers to an increased expression level of a polynucleotide (e.g., RNA such as mRNA) and/or polypeptide sequence relative to its expression level in the wild-type state, while "down-regulation" generally refers to Reduced expression levels of a polynucleotide (eg, RNA such as mRNA) and/or polypeptide sequence relative to its expression in the wild-type state. Expression of the transfected gene can occur transiently or stably in the cell. During "transient expression," the transfected gene is not transferred to daughter cells during cell division. Since its expression is restricted to transfected cells, gene expression is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selective advantage on the transfected cells. Such a selective advantage may be resistance to a certain toxin presented to the cell.

As used interchangeably herein, the terms "peptide", "polypeptide" or "protein" generally refer to a polymer of at least two amino acid residues linked by peptide bond(s). The term does not denote a specific length of the polymer, nor is it intended to imply or distinguish whether the peptide was produced using recombinant techniques, chemical or enzymatic synthesis, or occurs naturally. The term applies to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, polymers may be interrupted by non-amino acids. The term includes amino acid chains of any length, including full-length proteins, and proteins with or without secondary and/or tertiary structure (eg, domains). The term also encompasses amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation and any other manipulation, such as conjugation with a labeling component. As used herein, the term "amino acid" generally refers to natural and unnatural amino acids, including but not limited to modified amino acids and amino acid analogs. Modified amino acids can include natural amino acids and unnatural amino acids that have been chemically modified to include groups or chemical moieties that do not naturally occur on amino acids. Amino acid analogs may refer to amino acid derivatives. The term "amino acid" includes both D-amino acids and L-amino acids.

As used herein with respect to polypeptides, the term "derivative", "variant" or "fragment" generally refers to, for example, by amino acid sequence, structure (e.g., secondary and/or tertiary), activity (e.g., enzymatic activity) ) and/or a polypeptide functionally related to the wild-type polypeptide. Derivatives, variants, and fragments of a polypeptide may include one or more amino acid changes (eg, mutations, insertions, and deletions), truncations, modifications, or combinations thereof, compared to the wild-type polypeptide.

As used herein with respect to polypeptide molecules (e.g., proteins), the terms "engineered", "chimeric" or "recombinant" generally refer to genetic engineering techniques applied to a nucleic acid encoding a polypeptide molecule and to expressing the polypeptide molecule. A polypeptide molecule having a heterologous amino acid sequence or an altered amino acid sequence in a cell or organism. As used herein with respect to polynucleotide molecules (e.g., DNA or RNA molecules), the term "engineered" or "recombinant" generally refers to a polynucleotide having a heterologous nucleic acid sequence or an altered nucleic acid sequence as a result of the application of genetic engineering techniques Acid molecule. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning techniques; transfection, transformation, and other gene transfer techniques; homologous recombination; site-directed mutagenesis; and gene fusion. In some cases, engineered or recombinant polynucleotides (eg, genomic DNA sequences) can be modified or altered in part by gene editing.

The term "gene editing portion" generally refers to a portion that can edit a nucleic acid sequence, whether exogenous or endogenous to the cell comprising the nucleic acid sequence. In some embodiments, the gene editing moiety regulates the expression of a gene by editing a nucleic acid sequence. In some cases, gene editing moieties can regulate gene expression by editing genomic DNA sequences. In some cases, gene editing moieties can regulate gene expression by editing mRNA templates. In some cases, editing a nucleic acid sequence can alter the underlying template for gene expression.

Alternatively or additionally, the gene editing moiety may be capable of modulating the production of DNA (e.g., chromosomal DNA or cDNA) by specifically binding to a target sequence operably coupled to the gene (or a target sequence within the gene). mRNA to regulate gene expression or activity. In some cases, the gene editing portion may recruit or contain at least one transcription factor, which binds to a specific DNA sequence, thereby controlling the rate of transcription of genetic information from DNA to mRNA. The gene-editing moiety itself can bind to the DNA and regulate transcription through physical barriers, such as preventing the assembly of proteins such as RNA polymerase and other related proteins on the DNA template. Gene editing moieties can regulate gene expression at the translational level, for example, by modulating protein production from mRNA templates. In some cases, gene editing moieties can regulate gene expression by affecting the stability of mRNA transcripts.

The term "antibody" generally refers to a protein binding molecule with immunoglobulin-like function. The term antibody includes antibodies (eg, monoclonal and polyclonal antibodies), as well as derivatives, variants and fragments thereof. Antibodies include, but are not limited to, immunoglobulins (Ig) of different classes (ie, IgA, IgG, IgM, IgD, and IgE) and subclasses (eg, IgGl, IgG2, etc.). Derivatives, variants or fragments thereof may refer to functional derivatives or fragments that retain (eg, complete and/or partial) binding specificity of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), minibody, diabody, and single domain antibody ("sdAb"). " or "Nanobodies" or "Camelid antibodies"). The term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered or chemically conjugated. Examples of optimized antibodies include affinity matured antibodies. Examples of engineered antibodies include Fc-optimized antibodies (eg, antibodies optimized in fragment crystallizable regions) and multispecific antibodies (eg, bispecific antibodies).

The term "chimeric polypeptide receptor" generally refers to a non-native polypeptide receptor that includes one or more antigen-binding moieties, each antigen-binding moiety capable of binding a specific antigen. Chimeric polypeptide receptors may be monospecific (ie, capable of binding one type of specific antigen). Alternatively, chimeric polypeptide receptors may be multispecific (ie, capable of binding two or more different types of specific antigens). Chimeric polypeptide receptors can be monovalent (ie, include a single antigen-binding portion). Alternatively, chimeric polypeptide receptors can be multivalent (ie, include multiple antigen-binding moieties). In some cases, a chimeric polypeptide receptor can comprise a T cell receptor (TCR) fusion protein (TFP) or a chimeric antigen receptor (CAR).

The term "antigen binding domain" generally refers to a construct that exhibits preferential binding to a particular target antigen. An antigen binding domain may be a polypeptide construct such as an antibody, a modification thereof, a fragment thereof, or a combination thereof. The antigen binding domain can be any antibody disclosed herein or a functional variant thereof. Non-limiting examples of antigen binding domains can include murine antibodies, human antibodies, humanized antibodies, camelid Ig, shark heavy-chain-only antibody (VNAR), Ig NAR, chimeric antibodies, recombinant Antibodies, or antibody fragments thereof. Non-limiting examples of antibody fragments include Fab, Fab', F(ab)'2, F(ab)'3, Fv, single chain antigen binding fragment (scFv), (scFv)2, disulfide bond stabilized Fv (dsFv), minibodies, diabodies, triabodies, tetrabodies, single-domain antigen-binding fragments (sdAbs, nanobodies), recombinant heavy chain-only antibodies (VHH) and whole antibody binding specificity maintained other antibody fragments.

The term "safety switch" generally refers to an engineered polypeptide construct designed to prevent potential toxicity or other adverse effects of a cell therapy. When expressed in a cell, a safety switch can induce host cell death, thereby inactivating the activity of cells in the host (eg, within an object). Therefore, the safety switch can be the suicide part. In some cases, a cell can be programmed to express a suicide moiety at a certain stage of its life cycle (eg, time-programmed). In some cases, expression of the suicide moiety in the cell can be conditional or inducible. In some examples, conditional regulation (eg, expression) of a suicide moiety can include control by small molecule-mediated post-translational initiation and tissue-specific and/or temporal transcriptional regulation. Thus, the safety switch could be part of the inducible suicide. Safety switches can mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional genetic regulation, and/or antibody-mediated depletion. In some cases, a safety switch can be initiated by an exogenous molecule (e.g., a drug or prodrug) that, when activated, triggers apoptosis and/or cell death of a cell (e.g., an engineered NK cell disclosed herein) .

The term "immunomodulatory polypeptide" generally refers to a polypeptide construct (e.g., protein, antibody, membrane-bound polypeptide, secreted polypeptide, cleavable polypeptide, non-cleavable polypeptide) capable of modulating or controlling one or more properties of immune cells (such as NK cells). peptides, etc.). One or more properties of an immune cell can include differentiation of the immune cell, morphology of the immune cell, expression of polynucleotide or polypeptide constructs within the immune cell, or activity of the immune cell (e.g., engineered NK cells against diseased cells (e.g., cancer cells) ) cytotoxic activity). The immunomodulatory polypeptide may be endogenous to the host cell. Alternatively or additionally, the immunomodulatory polypeptide may be heterologous to the host cell. In some instances, control of one or more properties of immune cells can be mediated by down-regulating expression (eg, inhibition, knockdown, or knockout) of an immunomodulatory polypeptide. Alternatively or additionally, control of one or more properties of immune cells can be mediated by upregulation of expression of an immunomodulatory polypeptide (eg, upregulation of an endogenous gene or knock-in of a heterologous gene encoding an immunomodulatory polypeptide). In another alternative or in addition, control of one or more properties of an immune cell may be mediated by maintaining expression of an immunomodulatory polypeptide for a period of time longer than the native or normal expression profile of the immunomodulatory polypeptide in the host cell. In some instances, an immunomodulatory polypeptide can include a hypoimmune modulator. In some instances, an immune modulating polypeptide can include an immune checkpoint inhibitor.

The term "modulator of hypoimmunity" generally refers to a polypeptide construct in a cell wherein there is either enhanced expression (e.g., by knock-in of a heterologous gene) or decreased expression (e.g., by Knockout or knockdown of an endogenous gene) can help the cell reduce or avoid an immune response (eg, an immune attack, such as adaptive immune rejection) from the host's body after administration to the host's body. In some cases, cells (e.g., engineered NK cells disclosed herein) can be modified to exhibit enhanced expression or reduced expression of a modulator of immunity, so that the cells can be used after a second or further infusion of the cells into the host (i.e., receptors) can evade host immune attack. Thus, the cell (i) will not be rejected by the host's immune system and/or (ii) will be more aggressively rejected by the host's immune system compared to a control cell without enhanced expression or reduced expression of the immunomodulator. Slow rate rejection. Cells exhibiting low enhanced expression or reduced expression of a modulator of immunity can be said to exhibit "hypoimmunity" or "immune privilege."

The term "immune checkpoint inhibitor" generally refers to a group of molecules present on the cell surface of immune cells (eg, T cells, myeloid cells, NK cells, B cells, etc.) Targeting the immune response of cells such as cancer cells (ie, an anti-cancer or anti-tumor immune response) modulates the cellular immune response. Target cells may express receptors or ligands for immune checkpoint inhibitors present on the surface of immune cells to engage the immune checkpoint inhibitor and downregulate or suppress the immune cell's immune response to the target cell. Thus, in some cases, down-regulating or inhibiting the expression of immune checkpoint inhibitors in immune cells can enhance or prolong the immune response of immune cells to target cells.

The term "immune response" generally refers to a T-cell-mediated and/or B-cell-mediated immune response from a host's immune system to an object (eg, a foreign body). Examples of immune responses include T cell responses such as cytokine production and cellular cytotoxicity. In some cases, the immune response can be achieved indirectly through T cell priming, such as antibody production (humoral response) and cytokine responsive cells such as macrophages.

The term "enhanced expression", "increased expression" or "upregulated expression" generally refers to the production of a moiety of interest (e.g., a polynucleotide or polypeptide) to a level higher than that of the moiety of interest in a host strain (e.g., a host cell). level of normal expression. The normal expression level can be substantially zero (or null) or higher than zero. Portions of interest may include endogenous genes or polypeptide constructs of the host strain. A portion of interest may include a heterologous gene or polypeptide construct introduced into the host strain or introduced into the host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked in (KI) into the genome of a host strain for enhanced expression of the polypeptide of interest in the host strain.

The term "enhanced activity", "increased activity" or "up-regulated activity" generally refers to the modification of the activity of the moiety of interest (e.g., polynucleotide or polypeptide) to a level higher than that sensed in the host strain (e.g., host cell). The level of the normal activity level of the part of interest. A normal activity level may be substantially zero (or null) or above zero. Portions of interest may include polypeptide constructs of the host strain. A portion of interest may include a heterologous polypeptide construct introduced into a host strain or introduced into a host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked in (KI) into the genome of a host strain for enhanced activity of the polypeptide of interest in the host strain.

The terms "reduced expression", "decreased expression" or "down-regulated expression" generally refer to production of a moiety of interest (e.g., a polynucleotide or polypeptide) to a level lower than that of the moiety of interest in a host strain (e.g., a host cell). level of normal expression. Normal expression levels are above zero. Portions of interest may include endogenous genes or polypeptide constructs of the host strain. In some cases, a moiety of interest can be knocked out or knocked down in the host strain. In some examples, reduced expression of the moiety of interest can include complete suppression of such expression in the host strain.

The term "reduced activity", "reduced activity" or "down-regulated activity" generally refers to the modification of the activity of the moiety of interest (e.g., polynucleotide or polypeptide) to be lower than that in the host strain (e.g., host cell). The level of the normal activity level of the part of interest. Normal activity levels are above zero. Portions of interest may include endogenous genes or polypeptide constructs of the host strain. In some cases, a moiety of interest can be knocked out or knocked down in the host strain. In some examples, reduced activity of a moiety of interest can include complete inhibition of such activity in a host strain.

As used interchangeably herein, the term "item", "individual" or "patient" generally refers to a vertebrate, preferably a mammal, such as a human. Mammals include, but are not limited to, murines, apes, humans, farm animals, sport animals, and pets. Also included are tissues, cells and progeny of biological entities obtained in vivo or cultured in vitro.

The terms "therapy" or "treatment" generally refer to means of obtaining a beneficial or desired result, including but not limited to therapeutic and/or prophylactic benefits. For example, treatment can include administration of a system or population of cells disclosed herein. A therapeutic benefit refers to any therapeutically relevant improvement or effect on one or more diseases, conditions or symptoms being treated. For prophylactic benefit, compositions may be administered to subjects at risk of developing a particular disease, condition or symptom, or to subjects reporting one or more physiological symptoms of a disease, even though the disease, condition or symptom may not have manifested.

The term "effective amount" or "therapeutically effective amount" generally refers to the amount of a composition, for example, the amount of a composition comprising immune cells, such as lymphocytes (e.g., T lymphocytes and/or NK cells) of the disclosed system, This amount is sufficient to produce the desired activity when administered to an article in need thereof. In the context of the present disclosure, the term "therapeutically effective" generally refers to an amount of the composition sufficient to delay manifestation, prevent progression, alleviate or alleviate at least one symptom of a disorder treated by the methods of the present disclosure. A． _ overview

T cells are part of the adaptive immune system and can be primed to recognize specific threats by recognizing immune proteins (ie, antigens) on the surface of foreign cells. In contrast, NK cells are part of the innate immune response and can respond to various objects considered "non-self". Generally, unlike T cells, NK cells can attack their target cells without sensitization (ie, antigen-specific sensitization) to eliminate foreign substances.

Unmodified NK cells derived from a subject (eg, HSCs derived from a subject) can be cultured and expanded ex vivo, and then administered to a subject as a therapy to attack its target cells (eg, cancer cells). However, NK cell-based therapies may be limited due to their short ex vivo or in vivo half-life and/or poor proliferation. In addition, unmodified NK cells may not effectively target more difficult-to-treat cancers, such as myeloma or solid tumors. Furthermore, the ex vivo production of NK cells based on blood-derived stem cells (eg, HSCs) can generate a limited supply of NK cells for effective adoptive immunotherapy.

Therefore, there remains a large unmet need for NK cells that are sourced and engineered to exhibit, for example, enhanced proliferation, half-life and cytotoxic activity against target cells. B． _ engineered immune cells

The present disclosure describes systems and methods for immunotherapy. Immune cells can be engineered to exhibit increased half-life compared to control cells (eg, non-engineered immune cells). Immune cells can be engineered to exhibit enhanced proliferation compared to control cells. Immune cells can be engineered to efficiently and specifically target diseased cells (eg, cancer cells) that are insufficiently or untargetable by control cells. The engineered immune cells disclosed herein can be engineered ex vivo, in vitro and in some cases in vivo. Engineered immune cells prepared ex vivo or in vitro can be administered to a subject in need to treat a disease (eg, myeloma or solid tumor). The engineered immune cells can be autologous to the subject. Alternatively, the engineered immune cells can be allogeneic to the subject.

In some cases, engineered immune cells (eg, engineered NK cells) disclosed herein can be derived from isolated stem cells (eg, isolated ESCs). In some cases, engineered immune cells disclosed herein can be derived from induced stem cells (eg, iPSCs).

In some cases, the stem cells (eg, isolated stem cells, induced stem cells) disclosed herein can be autologous cells or derived from autologous cells. Autologous cells can be obtained from a subject having or suspected of having a condition. Alternatively, autologous cells may be obtained from the subject before the subject is found to have the condition. In some cases, autologous cells may be allogeneic cells, such as universal stem cells that have reduced immunogenicity, reduced numbers, or do not require immunosuppressive drugs. Autologous cells can be obtained from healthy donors.

In some cases, engineered immune cells (eg, engineered NK cells) can be autologous cells. Engineered immune cells can be obtained from a subject having the condition or suspected of having the condition. Alternatively, engineered immune cells can be obtained from the subject before the subject is found to have the condition. In some cases, engineered immune cells can be allogeneic cells, eg, for general allogeneic immunotherapy with reduced immunogenicity and requiring reduced or no immunosuppressive drugs. Engineered immune cells can be obtained from healthy donors.

In some aspects, T cells can be engineered to exhibit increased half-life compared to control cells (eg, non-engineered T cells). T cells can be engineered to exhibit enhanced proliferation compared to control cells. T cells can be engineered to efficiently and specifically target diseased cells (eg, cancer cells) that are insufficiently or untargetable by control cells. The engineered T cells disclosed herein can be engineered ex vivo, in vitro and in some cases in vivo. Engineered T cells prepared ex vivo or in vitro can be administered to a subject in need to treat a disease (eg, myeloma or solid tumor). The engineered T cells can be autologous to the subject. Alternatively, the engineered immune cells can be allogeneic to the subject.

In some aspects, NK cells can be engineered to exhibit an increased half-life compared to control cells (eg, non-engineered NK cells). NK cells can be engineered to exhibit enhanced proliferation compared to control cells. NK cells can be engineered to efficiently and specifically target diseased cells (eg, cancer cells) that are insufficiently or untargetable by control cells. The engineered NK cells disclosed herein can be engineered ex vivo, in vitro and in some cases in vivo. The engineered NK cells prepared ex vivo or in vitro can be administered to a subject in need to treat a disease (eg, myeloma or solid tumor). The engineered NK cells can be autologous to the subject. Alternatively, the engineered NK cells can be allogeneic to the subject.

In one aspect, the disclosure provides engineered immune cells (eg, engineered NK cells). Engineered immune cells can comprise cytokines (eg, secreted cytokines) that are heterologous to the immune cells. In some instances, a heterologous cytokine can comprise a heterologous interleukin (IL) (eg, a heterologous secreted IL-15). Engineered immune cells may further comprise one or both of: (i) CD16 variants for enhanced CD16 signaling compared to control cells and (ii) chimeric polypeptide receptors comprising an antigen-binding portion capable of binding antigen body. In some examples, the antigen is not CD19. Thus, an antigen binding portion may not, and need not, exhibit any specific binding to CD19, but rather specific binding to an antigen (eg, one or more antigens) other than CD19.

In another aspect, the disclosure provides engineered immune cells (eg, engineered NK cells). Engineered immune cells can comprise cytokines (eg, secreted cytokines) that are heterologous to the immune cells. In some instances, a heterologous cytokine can comprise a heterologous interleukin (IL) (eg, a heterologous secreted IL-15). Engineered immune cells may further comprise one or both of: (i) CD16 variants for enhanced CD16 signaling compared to control cells and (ii) chimeric polypeptide receptors comprising an antigen binding portion capable of binding CD19 body.

The engineered immune cells disclosed herein (e.g., engineered NK cells) may comprise heterologous receptors that are the corresponding receptors for the heterologous cytokines disclosed herein (e.g., heterologous IL-15 for heterologous IL-15). 15 receptor (IL-15R, eg, IL-15α or IL-15β)). Alternatively, engineered immune cells may not and need not contain any heterologous receptors that are corresponding receptors for heterologous cytokines. For example, an engineered immune cell comprising a heterologous IL (eg, IL-15) lacks a heterologous receptor for the heterologous IL (eg, IL-15R).

In one aspect, the disclosure provides engineered immune cells (eg, engineered NK cells). Engineered immune cells can comprise cytokines (eg, secreted cytokines) that are heterologous to the immune cells. A heterologous cytokine can also comprise a heterologous interleukin (IL) (eg, a heterologous secreted IL-15). Engineered immune cells may and need not contain heterologous receptors (eg, heterologous IL-15R) that are corresponding receptors for heterologous cytokines.

The heterologous cytokines disclosed herein (eg, heterologous IL) can be of the same species as the engineered immune cells (eg, engineered NK cells). For example, both heterologous cytokines and engineered immune cells can be of human origin. Alternatively, the heterologous cytokine can be of a different species than the engineered immune cells.

A heterologous cytokine (eg, heterologous IL) disclosed herein can be introduced into engineered immune cells (eg, engineered NK cells) by contacting the engineered immune cell with a heterologous polynucleotide encoding the heterologous cytokine. Heterologous polynucleotides can be integrated into chromosomes (eg, nuclear chromosomes) of engineered immune cells. Alternatively, the heterologous polynucleotide may not and need not be integrated into the chromosome of the engineered immune cell. In one example, mRNAs encoding heterologous cytokines can be introduced (or inserted) into engineered immune cells. In some embodiments, the heterologous polynucleotide is codon optimized to improve expression of the heterologous cytokine.

In some cases, a heterologous cytokine disclosed herein can be a heterologous IL. The heterologous IL disclosed herein may comprise at least 1, 2, 3, 4, 5 or more different types of heterologous IL. The heterologous IL disclosed herein may comprise up to 5, 4, 3 or 2 different types of heterologous IL. Alternatively, the heterologous IL can be a single type of heterologous IL. Non-limiting examples of heterologous IL may include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- -10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22 , IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL -35 and IL-36. In some examples, the heterologous IL can comprise one or more members selected from the group consisting of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and functional modifications thereof. For example, an engineered immune cell (eg, engineered NK cell) disclosed herein can comprise at least a portion of heterologous human IL-15 (or the gene encoding it).

In some instances, the heterologous cytokine is membrane-bound IL-15 comprising at least a portion of IL-15 and IL-15R. In some embodiments, at least a portion of the IL-15 and IL-15R of the membrane-bound IL-15 are linked by a linker. Any suitable linker known in the art may be used in membrane-bound IL-15. In some embodiments, the linker is selected from the group consisting of (GGGGS)n, (EGKSSGSGSESKST)n, and (EGKSSGSGSESKST)n(GGGGS)n, and n can be any integer selected from 1-10. In some embodiments, the linker is (GGGGS)n, and n can be any integer selected from 1-10. In some embodiments, the linker is selected from GGGGS, (GGGGS)2, (GGGGS)3, (GGGGS)4, (GGGGS)5, or (GGGGS)6. In some embodiments, the linker is (EGKSSGSGSESKST)n, and n can be any integer selected from 1-10. In some embodiments, the linker is selected from the group consisting of EGKSSGSGSESKST, (EGKSSGSGSESKST)2 or (EGKSSGSGSESKST)3. In some embodiments, the linker is (EGKSSGSGSESKST)n(GGGGS)n, and n can be any integer selected from 1-10. In some embodiments, the linker is EGKSSGSGSESKSTGGGGS or (EGKSSGSGSESKST)2GGGGS. In some embodiments, the linker comprises an amino acid sequence selected from SEQ ID No. 18-20.

In some embodiments, the membrane-bound IL-15 further comprises an intracellular signaling domain. Any suitable intracellular signaling domain known in the art may be used in the membrane-bound IL-15 of the present application. In some embodiments, membrane-bound IL-15 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity. In some embodiments, the membrane-bound IL-15 comprises the amino acid sequence of SEQ ID NO. 21.

In some embodiments, the membrane-bound IL-15 further comprises an extracellular signal peptide. Any suitable extracellular signal peptide known in the art can be used in the membrane-bound IL-15 of the present application. In some embodiments, the membrane-bound IL-15 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity. In some embodiments, the membrane-bound IL-15 comprises the amino acid sequence of SEQ ID NO. 22.

In some embodiments of any one of the engineered NK cells disclosed herein, the membrane-bound IL-15 comprises an amino acid sequence that is at least 70%, at least 75% identical to any one of SEQ ID NOs. 13-15 %, at least 80%, at least 85%, at least 90, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical. In some embodiments of any one of the engineered NK cells disclosed herein, the membrane-bound IL-15 comprises an amino acid sequence selected from any one of SEQ ID NOs. 13-15.

The heterologous cytokines (eg, heterologous IL) disclosed herein can be secreted cytokines. Alternatively, heterologous cytokines may not and need not be secreted by engineered immune cells. In this case, for example, heterologous cytokines can be bound to the cell surface of engineered immune cells.

In some instances, a heterologous cytokine (eg, heterologous IL) disclosed herein can be a secreted cytokine. Expression cassettes encoding heterologous cytokines can be introduced into engineered immune cells. The expression cassette may further encode additional heterologous polypeptides, such as heterologous receptors. Within the expression cassette, a first polynucleotide sequence encoding a heterologous cytokine and a second polynucleotide sequence encoding an additional heterologous polypeptide (e.g., a heterologous receptor) can be cleaved by a polynucleotide encoding a cleavage linker. Linkers are coupled to each other. In some examples, the heterologous receptor can be the corresponding receptor for a heterologous cytokine (eg, heterologous IL-15α or IL-15β of heterologous IL-15). Alternatively, the expression cassette may not and need not encode any other heterologous polypeptide other than a heterologous cytokine. In some embodiments, the heterologous polynucleotide is codon optimized to improve expression of the heterologous polypeptide.

A cleavable linker as disclosed herein may comprise a self-cleaving peptide, eg a self-cleaving 2A peptide. Self-cleaving peptides can be found in members of the picornaviridae virus family, including aphthviruses, such as foot-and-mouth disease virus (FMDV), equine rhinitis virus (ERAV), thosea asigna virus ) (TaV) and porcine Tyshin virus-1 (PTV-1), as well as heart viruses such as Theileria virus (eg Theileria murine encephalomyelitis) and encephalomyocarditis virus. Non-limiting examples of self-cleaving 2A peptides may include "F2A", "E2A", "P2A", "T2A" and functional variants thereof.

In some cases, a heterologous cytokine (eg, heterologous IL) disclosed herein can bind to the cell surface of an engineered immune cell (eg, engineered NK cell). In some examples, engineered immune cells can be genetically modified to couple a heterologous polynucleotide sequence encoding a heterologous cytokine to a gene encoding an endogenous transmembrane protein of the engineered immune cell. In one example, an endogenous transmembrane protein can be the corresponding receptor for a heterologous cytokine (eg, heterologous IL-15α or IL-15β of heterologous IL-15). In some examples, an expression cassette encoding a heterologous fusion polypeptide comprising (i) a heterologous cytokine coupled to (ii) a heterologous receptor can be introduced into engineered immune cells. Here, the heterologous cytokine may not and need not be cleavable from the heterologous receptor. Non-limiting examples of heterologous receptors may include corresponding receptors for heterologous cytokines (e.g., heterologous IL-15α or IL-15β for heterologous IL-15), or different receptors, such as a common gamma chain (γ _C ) receptors or modifications thereof.

The expression cassettes disclosed herein can be integrated into the genome of engineered cells (eg, engineered NK cells) through the action of the gene editing moieties disclosed herein. Alternatively, the expression cassette may not and need not be integrated into the genome of the engineered cell.

The engineered immune cells disclosed herein (e.g., engineered NK cells) can exhibit the expression of heterologous cytokines (e.g., heterologous IL, such as heterologous IL-15) and/or heterologous receptors (e.g., heterologous IL-15) disclosed herein. Enhanced signaling of the endogenous signaling pathway of heterologous IL receptors, such as heterologous IL-15R). Enhanced signaling of the endogenous signaling pathways disclosed herein can be determined by a number of methods, including but not limited to: (i) downstream signaling proteins (e.g., for IL-15/IL-15R, JAK3, STAT3, STAT5, etc. ) or (ii) downstream genes by Western blot or polymerase chain reaction (PCR) techniques (e.g., for IL-15/IL-15R, Mcl1, Cdk4/6, Mki67, Tnf, Gzmb, Gzmc, Ifng etc.) expression.

In some cases, in engineered immune cells of the present disclosure induced by heterologous cytokines and/or heterologous receptors (e.g., by heterologous cytokines and/or heterologous receptors disclosed herein, such as IL -15/IL-15R-induced) enhanced signaling of endogenous signaling pathways can be characterized by an increase in phosphorylation of downstream signaling proteins to at least or at most about 0.1-fold, at least or at most about 0.2-fold compared to control cells , at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, At least or at most about 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or At most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most About 5,000 times, or at least or at most about 10,000 times.

In some cases, in engineered immune cells of the present disclosure induced by heterologous cytokines and/or heterologous receptors (e.g., by heterologous cytokines and/or heterologous receptors disclosed herein, such as IL -15/IL-15R-induced) enhanced signaling of endogenous signaling pathways can be characterized by an increase in the expression of downstream genes by at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most, compared to control cells At most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most About 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 7 times 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times , or at least or at most about 10,000 times.

Enhanced CD16 signaling (eg, constitutively enabled signaling of CD16) of engineered immune cells (eg, engineered NK cells) disclosed herein can be achieved by having non-cleavable CD16 variants in the subject cells. CD16 (eg, CD16a) is a transmembrane protein expressed by immune cells (eg, NK cells) that binds to monomeric IgG attached to target cells to prime immune cells and promote antibody-dependent cell-mediated cytotoxicity (ADCC) . In non-engineered immune cells, binding between CD16 and monomeric IgG induces cleavage of CD16 protein at the cleavage site near the transmembrane domain, thereby modulating the cell surface density of CD16 upon immune cell priming. Therefore, endogenous CD16 of engineered immune cells can be modified to enhance their signaling. Alternatively, enhanced signaling variants of CD16 can be artificially introduced into engineered immune cells.

In some cases, the endogenous gene encoding CD16 of engineered immune cells can be genetically modified in its extracellular domain (e.g., F176V) by the action of the gene editing moieties disclosed herein, thereby modifying CD16 exhibits higher binding affinity to its targets (eg, monomeric IgG). In some cases, a heterologous gene encoding such modified CD16 can be introduced into the cell.

In some cases, the endogenous gene encoding CD16 of the engineered immune cells can be genetically modified by the action of the gene editing moieties disclosed herein such that the modified CD16 is non-cleavable and can induce enhanced CD16 signaling. In some examples, a cleavage site (e.g., positions 195-198) in the membrane proximal region (positions 189-212) of CD16 can be modified or eliminated (e.g., a CD16 S197P variant that is a non-cleavable CD16 variant) . In some cases, a heterologous gene encoding such modified CD16 can be introduced into the cell.

In some cases, a heterologous gene encoding a heterologous CD16 variant (i) that exhibits a higher sensitivity to its target (e.g., monomeric IgG) can be introduced into cells (i.e., hnCD16). binding affinity, and (ii) are non-cleavable. In some examples, the heterologous CD16 variant can be a modified CD16 including, for example, F176V and S197P, as disclosed herein. In some examples, the heterologous CD variant can be a fusion receptor protein comprising (i) at least a portion of CD16 with an inactivated cleavage site and (ii) a different cell surface protein, such as a glycoprotein (eg, CD64). The extracellular domain, which exhibits enhanced binding to the target (eg, monomeric IgG) compared to unmodified CD16.

In some instances, the heterologous CD16 variant comprises an amino acid sequence at least 70% identical to SEQ ID NO. 23. In some instances, the heterologous CD16 variant comprises an amino acid sequence at least 75% identical to SEQ ID NO. 23. In some instances, the heterologous CD16 variant comprises an amino acid sequence at least 80% identical to SEQ ID NO. 23. In some instances, the heterologous CD16 variant comprises an amino acid sequence at least 85% identical to SEQ ID NO. 23. In some instances, the heterologous CD16 variant comprises an amino acid sequence at least 90% identical to SEQ ID NO. 23. In some cases, the heterologous CD16 variant comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO. 23. In some instances, the heterologous CD16 variant comprises the amino acid sequence of SEQ ID NO. 23.

The heterologous genes disclosed herein can be integrated into the genome of engineered cells (eg, engineered NK cells) through the action of the gene editing moiety disclosed herein. Alternatively, the heterologous gene may not and need not be integrated into the genome of the engineered cell.

Enhanced CD16 signaling of the engineered immune cells (e.g., engineered NK cells) disclosed herein can be determined by a variety of methods, including but not limited to (i) phosphorylation of downstream signaling proteins (e.g., SHP-1 ) by Western blotting or (ii) expression of downstream genes (such as CD25, IFN-γ, TNF, etc.) by Western blot or PCR techniques.

In some cases, the CD16 signaling of the engineered immune cells of the disclosure (e.g., engineered NK cells comprising hnCD16) can be at least or at most about 0.1 times, at least or at most about 0.2 times, at least or at most about 0.2 times, the CD16 signaling of control cells. At least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least Or at most about 1 times, at least or at most about 2 times, at least or at most about 4 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or At most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most About 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times larger.

In some instances, enhanced CD16 signaling of engineered immune cells of the disclosure (e.g., engineered NK cells comprising hnCD16) can be characterized by increased phosphorylation of downstream signaling proteins by at least or at most About 0.1 times, at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.5 times 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times , at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, At least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times.

In some cases, enhanced CD16 signaling of engineered immune cells of the disclosure (e.g., engineered NK cells comprising hnCD16) can be characterized by an increase in the expression of downstream genes by at least or at most about 0.1-fold compared to control cells , at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, At least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or At most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most About 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times.

In some instances, CD16 signaling of engineered immune cells of the disclosure (e.g., engineered NK cells comprising hnCD16) can be prolonged to (e.g., longer than CD16 signaling initiated) compared to CD16 signaling of control cells duration) at least or at most about 0.1 times, at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times , at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, At least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times.

In one aspect, the disclosure provides engineered immune cells (eg, engineered NK cells). An engineered immune cell can comprise a heterologous cytokine disclosed herein, wherein the heterologous cytokine binds to the membrane (eg, plasma membrane) of the engineered immune cell. In some instances, a heterologous cytokine can comprise a heterologous IL as disclosed herein (eg, heterologous IL-15). Engineered immune cells may further comprise one, two, or all of the following: (a) different heterologous cytokines (e.g., the heterologous cytokines disclosed herein, in addition to the heterologous cytokines that bind to the membrane of the subject cell) , (b) reduced expression or activity of an endogenous immunomodulatory polypeptide, and (c) a safety switch. In some examples, the endogenous immunomodulatory polypeptide is not B2M. Thus, an endogenous immunomodulator can be, for example, a polypeptide other than B2M.

Engineered immune cells (e.g., engineered NK cells) can comprise different heterologous cytokines and one or both of: (b) reduced expression of endogenous immunomodulatory polypeptides (e.g., non-B2M polypeptides) or active, and (c) a safety switch. The engineered immune cells comprise reduced expression or activity of an endogenous immunomodulatory polypeptide (eg, non-B2M polypeptide) and one or both of: (a) different heterologous cytokines, and (c) a safety switch. The engineered immune cells contain a safety switch and one or both of (a) different heterologous cytokines, and (b) reduced expression or activity of endogenous immunomodulatory polypeptides (eg, non-B2M polypeptides). Engineered immune cells comprise all of (a), (b) and (c).

As disclosed herein, engineered immune cells (eg, engineered NK cells) can exhibit enhanced signaling of endogenous signaling pathways involving heterologous cytokines compared to control cells.

The expression or activity of endogenous immunomodulatory polypeptides can be reduced in engineered immune cells (eg, engineered NK cells), eg, by the action of the gene editing moieties disclosed herein.

Reduced expression or activity of endogenous immunomodulatory polypeptides in engineered immune cells disclosed herein (e.g., engineered NK cells) can be determined by a variety of methods, including but not limited to (i) downstream signaling proteins (e.g., for PD1/ PDL1 signaling, phosphorylation or dephosphorylation of SHP2, Igα/β, Syk, etc.); or (ii) expression of endogenous immunomodulatory polypeptides (eg, PD1) by Western blot or PCR techniques.

In some instances, the reduced expression of an endogenous immunomodulatory polypeptide in an engineered immune cell disclosed herein (e.g., an engineered NK cell) can be characterized by expression of an endogenous immunomodulatory polypeptide (e.g., PD1 ) as compared to a control cell Reduced to at least or at most about 1/0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, At least or at most about 1/0.7, at least or at most about 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/3, at least or At most about 1/4, at least or at most about 1/5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/ 70, at least or at most about 1/80, at least or at most about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, Or at least or at most about 1/10,000.

In some cases, reduced activity of endogenous immunomodulatory polypeptides in engineered immune cells disclosed herein (e.g., engineered NK cells) can be characterized by downstream signaling proteins (e.g., for PD1/PDL1 signaling) as compared to control cells. conduction, SHP2) phosphorylation is reduced to at least or at most about 1/0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most About 1/3, at least or at most about 1/4, at least or at most about 1/5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1 /9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60 , at least or at most about 1/70, at least or at most about 1/80, at least or at most about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least Or at most about 1/5,000, or at least or at most about 1/10,000.

In some cases, reduced activity of endogenous immunomodulatory polypeptides in engineered immune cells disclosed herein (e.g., engineered NK cells) can be characterized by downstream target signaling proteins (e.g., for PD1/PDL1 ) as compared to control cells. Signaling, phosphorylation of Igα/β or Syk) is increased by at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most about 0.3-fold, at least or at most about 0.4-fold, at least or at most about 0.5-fold, at least Or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or At most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most About 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times. A downstream target signaling protein may be a protein that is normally inhibited by the functional signaling pathway of an endogenous immunomodulatory polypeptide.

In one aspect, the disclosure provides engineered immune cells (eg, engineered NK cells). Engineered immune cells may comprise CD16 variants as disclosed herein for enhanced CD16 signaling in engineered NK cells. In some cases, a CD16 variant (eg, a heterologous CD16 variant) can comprise at least a portion of CD16 and at least a portion of CD64 (eg, hnCD16 disclosed herein). As disclosed herein, engineered immune cells can further comprise reduced expression or activity of endogenous immunomodulatory polypeptides compared to control cells.

As disclosed herein, engineered immune cells (eg, engineered NK cells) can exhibit enhanced CD16 signaling compared to control cells.

In one aspect, the disclosure provides engineered immune cells (eg, engineered NK cells). Engineered immune cells can comprise reduced activity of endogenous cytokine signaling (eg, endogenous IL signaling, eg, endogenous IL-17 signaling). Engineered immune cells can be derived from isolated stem cells (eg, isolated ESCs). Alternatively, engineered immune cells can be derived from induced stem cells (eg, iPSCs).

In some instances, engineered NK cells can be treated with inhibitors of endogenous cytokine signaling (eg, small molecule inhibitors). In some cases, engineered NK cells can include reduced expression of endogenous IL-17 or its endogenous receptor (eg, by indel or transgenic mutation, by transient or permanent suppression, etc.). In some instances, engineered NK cells can include reduced expression of endogenous IL-17. In some instances, engineered NK cells can include reduced expression of endogenous IL-17R. In some instances, engineered NK cells can include reduced expression of endogenous IL-17 and endogenous IL-17R.

In some instances, an endogenous cytokine disclosed herein can be endogenous IL. The endogenous IL disclosed herein may comprise at least 1, 2, 3, 4, 5 or more different types of endogenous IL. The endogenous IL disclosed herein may comprise up to 5, 4, 3 or 2 different types of endogenous IL. Alternatively, the endogenous IL may be a single type of endogenous IL. Non-limiting examples of endogenous ILs may include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- -10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22 , IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL -35 and IL-36. In some examples, the endogenous IL can be IL-17. Non-limiting examples of endogenous IL-17 can include IL-17A, IL-17F, and natural mutations thereof. For example, engineered immune cells (eg, engineered NK cells) disclosed herein can exhibit reduced expression or activity of IL-17A or IL-17F.

In some cases, an endogenous gene encoding an endogenous cytokine disclosed herein (eg, endogenous IL, such as IL-17) can be modified through the action of the gene editing moiety disclosed herein.

The endogenous receptor can be the corresponding receptor for any cytokine disclosed herein (eg, the corresponding receptor for any IL disclosed herein). In some cases, the endogenous receptor can be the corresponding receptor for IL (eg, IL-17R for IL-7 signaling). Non-limiting examples of IL-17R can include IL-17RA, IL-17RB, IL-17RC, IL-17RD, IL-17RE, and variants thereof. In one example, the endogenous IL-17R comprises IL-17RA.

In some instances, reduced expression or activity of an endogenous cytokine disclosed herein (e.g., endogenous IL, such as IL-17) or its endogenous receptor can be determined by a variety of methods, including but not limited to: (i) downstream Phosphorylation of signaling proteins (e.g. for IL-17, PI3K, Act1, MAP3K, MEK1/2, MKK3/6, MKK4/7, MKK3/6, ERK, p38, JNK, etc.) or (ii) expression of downstream genes (by Western blot or PCT technique). In some examples, genes downstream of IL cytokines such as IL-17 may include chemokines (e.g., CXCL1, CXCL2, CXCL8, CXCL9, CXCL10, CCL2, CCL20, etc.), cytokines (e.g., IL-6, TNFa, G-CSF, GM-CSF, etc.), acute phase response molecules (eg, SAA, CRP, lipocalin 2/24p3, etc.) and/or enzymes (eg, metalloproteases, eg, MMP1, MMP3, MMP9, MMP13).

In some instances, reduced expression or activity of an endogenous cytokine (e.g., endogenous IL, such as IL-17) or its endogenous receptor in engineered immune cells (e.g., engineered NK cells) disclosed herein can characterize For, compared with control cells, the phosphorylation of downstream signaling proteins of endogenous cytokines is reduced to at least or at most about 1/0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1/0.8, at least or at most about 1/0.9, at least or at most about 1/0. 1. At least or at most about 1/2, at least or at most about 1/3, at least or at most about 1/4, at least or at most about 1/5, at least or at most about 1/6, at least or at most about 1/7, At least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or At most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000.

In some instances, reduced expression or activity of an endogenous cytokine (e.g., endogenous IL, such as IL-17) or its endogenous receptor in engineered immune cells (e.g., engineered NK cells) disclosed herein can characterize is, compared with control cells, the expression of downstream genes of endogenous cytokines is reduced to at least or at most about 1/0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4 , at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/3, at least or at most about 1/4, at least or at most about 1/5, at least or at most about 1/6, at least or at most about 1/7, at least or at most About 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1 /50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most about 1/90, at least or at most about 1/100, at least or at most about 1/500 , at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000.

In some instances, the engineered cytokines disclosed herein are compared to control cells that do not exhibit reduced activity of endogenous cytokine signaling (e.g., endogenous IL signaling, such as endogenous IL-17 signaling) disclosed herein. The engineered immune cells (eg, engineered NK cells) can exhibit an enhanced expression profile of specific cellular markers (eg, NK cell markers) for committed immune cells. For example, non-limiting examples of specific cellular markers committed to NK cells may include CD57 or killer immunoglobulin-like receptor (KIR). KIRs can include KIR2D and/or KIR3D. KIR2D can comprise KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4 and/or KIR2DS5. KIR3Ds can include KIR3DL1, KIR3DL2, KIR3DL3 and/or KIR3DS1.

As disclosed herein, enhanced expression profiles of specific cellular markers for committed immune cells (eg, CD57 or KIRs for NK cells) can be determined by a variety of methods including, but not limited to, Western blot or PCR techniques.

In some cases, the engineered immune cells of the disclosure may express at least or at most about 0.1-fold, or at most about 0.1-fold, a specific cellular marker for committed immune cells (e.g., NK cell CD57 or KIR) in the engineered immune cells of the disclosure, At least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least Or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 4 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or At most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most About 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times higher, or at least or at most about 10,000 times higher.

In one aspect, the disclosure provides engineered immune cells (eg, engineered NK cells). Engineered immune cells may contain one or both of: (i) heterologous transcription factors (e.g., heterologous STATs), (ii) endogenous cytokine signaling (e.g., endogenous IL signaling disclosed herein ), and (iii) reduced expression or activity of an endogenous enzyme (eg, a ligase such as CBL-B). Engineered immune cells can be derived from isolated stem cells (eg, isolated ESCs). Alternatively, engineered immune cells can be derived from induced stem cells (eg, iPSCs).

The heterologous transcription factors may include at least 1, 2, 3, 4, 5 or more different types of heterologous transcription factors. Heterologous transcription factors may include up to 5, 4, 3 or 2 different types of transcription factors. Alternatively, the heterologous transcription factors can have a single type of transcription factor. Transcription factors may be involved in the immune activity, proliferation, apoptosis and/or differentiation of engineered immune cells. In some cases, the heterologous transcription factor of engineered immune cells (eg, engineered NK cells) can be a STAT. Non-limiting examples of STATs may include STAT1, STAT2, STAT3, STAT4, STAT3, STAT4, STAT5A, STAT5B, STAT6, and modifications thereof. In one example, STAT may include STAT3. In another example, STAT may include STAT5B.

In some instances, compared to control cells without reduced activity of (i) heterologous transcription factors (e.g., heterologous STATs) or (ii) endogenous cytokine signaling (e.g., endogenous IL-17 signaling) , the engineered immune cells (eg, engineered NK cells) disclosed herein can exhibit enhanced survival in the presence of tumor cells. In some cases, the engineered immune cells may survive at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most about 0.3-fold, at least or at most about 0.4-fold, at least or at most about 0.4-fold, at least or at most about 0.4-fold, At least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or At most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most About 80 times, at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times longer.

In one aspect, the disclosure provides engineered immune cells (eg, engineered NK cells). Engineered immune cells can exhibit reduced expression or activity of specific endogenous cell markers (eg, NK cell markers such as KIRs) disclosed herein for committed immune cells compared to control cells. In some examples, the specific endogenous cell marker is a KIR. The engineered immune cell may further comprise one or more of: (a) a chimeric polypeptide receptor comprising an antigen binding portion capable of binding an antigen as disclosed herein, (b) a heterologous cytokine as disclosed herein (e.g. , heterologous IL, such as IL-15), (c) CD16 variants for enhanced CD16 signaling compared to control cells, wherein the CD16 variants are heterologous to engineered NK cells as disclosed herein; (d) an immunomodulatory polypeptide disclosed herein, wherein the immunomodulatory polypeptide is heterologous to the engineered immune cell, and (e) reduced expression or activity of an endogenous immunomodulatory polypeptide as disclosed herein.

Engineered immune cells can comprise chimeric polypeptide receptors and one or more (eg, 1, 2, 3, or 4) of: (b) heterologous cytokines, (c) for enhanced CD16 signaling Transduced CD16 variants, (d) heterologous immunomodulatory agents, and (e) reduced expression or activity of endogenous immunomodulatory polypeptides.

Engineered immune cells can comprise heterologous cytokines and one or more (eg, 1, 2, 3, or 4) of: (a) chimeric polypeptide receptors, (c) for enhanced CD16 signaling Transduced CD16 variants, (d) heterologous immunomodulatory agents, and (e) reduced expression or activity of endogenous immunomodulatory polypeptides.

Engineered immune cells may comprise CD16 variants for enhanced CD16 signaling and one or more (eg, 1, 2, 3, or 4) of: (a) a chimeric polypeptide receptor, (b ) a heterologous cytokine, (d) a heterologous immunomodulator, and (e) reduced expression or activity of an endogenous immunomodulatory polypeptide.

Engineered immune cells can comprise a heterologous immune modulator and one or more (eg, 1, 2, 3, or 4) of: (a) chimeric polypeptide receptors, (b) heterologous cytokines, (c) CD16 variants for enhanced CD16 signaling, and (e) reduced expression or activity of endogenous immunomodulatory polypeptides.

Engineered immune cells can comprise reduced expression or activity of an endogenous immunomodulatory polypeptide and one or more (eg, 1, 2, 3, or 4) of: (a) a chimeric polypeptide receptor, (b ) heterologous cytokines, (c) CD16 variants for enhanced CD16 signaling, and (d) heterologous immune modulators.

Reduced expression or activity of specific endogenous cellular markers for committed immune cells disclosed herein, such as KIRs for NK cells, can be determined by a number of methods including, but not limited to, Western blot or PCR techniques.

In some cases, the expression of endogenous cell markers (eg, KIRs of NK cells) specific for committed immune cells in the engineered immune cells of the present disclosure can be at least or at most about 1/3 of the expression thereof by control cells. 0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, At least or at most about 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or At most about 1/5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/ 80, at least or at most about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 Low.

In one aspect, the disclosure provides engineered immune cells (eg, engineered NK cells). Engineered immune cells can exhibit reduced expression or activity of one or more endogenous immune checkpoint inhibitors (eg, CD94, CD96, TGFβ receptor, SHIP2, etc.). In some instances, engineered immune cells may exhibit reduced expression or activity of one or more of: (i) endogenous CD94, (ii) endogenous CD96, (iii) endogenous TGFβ receptor, and (iv) ) endogenous SHIP (eg SHIP2).

In some instances, the engineered immune cell can exhibit reduced expression or activity of endogenous CD94, and also exhibit reduced expression or activity of one or more (e.g., 1, 2, or all) of the following: ( ii) endogenous CD96, (iii) endogenous TGFβ receptor and (iv) endogenous SHIP (eg SHIP2).

In some instances, the engineered immune cell can exhibit reduced expression or activity of endogenous CD96, and also exhibit reduced expression or activity of one or more (e.g., 1, 2, or all) of the following: ( i) endogenous CD94, (iii) endogenous TGFβ receptor and (iv) endogenous SHIP (eg SHIP2).

In some instances, engineered immune cells may exhibit reduced expression or activity of endogenous TGFβ receptors and also exhibit reduced expression or activity of one or more (e.g., 1, 2, or all) of : (i) endogenous CD94, (ii) endogenous CD96 and (iv) endogenous SHIP (eg SHIP2).

In some instances, engineered immune cells may exhibit reduced expression or activity of an endogenous SHIP (e.g., SHIP2) and also exhibit reduced expression of one or more (e.g., 1, 2, or all) of Or active: (i) endogenous CD94, (ii) endogenous CD96 and (iii) endogenous TGFβ receptor.

In some cases, reduced expression or activity of an immune checkpoint inhibitor (e.g., CD94, CD96, TGFβ receptor, SHIP2, etc.) in an engineered immune cell of the disclosure (e.g., an engineered NK cell) can be a response to a control cell. Its expression is at least or at most about 1/0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0. 0.6, at least or at most about 1/0.7, at least or at most about 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1/5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or At most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/1/ 5,000, or at least or at most about 1/10,000 lower.

In some cases, the reduced expression or activity of endogenous CD94 in engineered immune cells of the present disclosure (e.g., engineered NK cells) can be at least or at most about 1/0.1, at least or at most, of its expression by control cells About 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1 /0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1/5 , at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most About 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 lower.

In some cases, the reduced expression or activity of endogenous CD96 in engineered immune cells of the present disclosure (e.g., engineered NK cells) can be at least or at most about 1/0.1, at least or at most, of its expression by control cells About 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1 /0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1/5 , at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most About 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 lower.

In some cases, the reduced expression or activity of endogenous TGFβ receptors in engineered immune cells of the disclosure (e.g., engineered NK cells) can be at least or at most about 1/0.1, at least about 1/0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most About 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1 /5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20 , at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least Or at least about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 lower.

In some cases, the reduced expression or activity of an endogenous SHIP (e.g., SHIP2) in an engineered immune cell of the disclosure (e.g., an engineered NK cell) can be at least or at most about 1/0.1 of its expression by a control cell , at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most About 1/5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1 /20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80 , at least or at most about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 lower .

In one aspect, the disclosure provides engineered immune cells (eg, engineered NK cells). As disclosed herein, engineered immune cells can exhibit reduced expression or activity of endogenous immunomodulatory polypeptides. In some instances, the endogenous immunomodulatory polypeptide comprises one or more hypoimmune modulators. In some instances, the engineered immune cells exhibit reduced expression or activity from one or more of the following hypoimmune modulators: (i) endogenous CD80, (ii) endogenous CD86, (iii) endogenous Source ICOSL, (iv) endogenous CD40L, (v) endogenous MICA or MICB or (vi) endogenous NKG2DL. Engineered immune cells can be derived from isolated stem cells (eg, isolated ESCs). Alternatively, engineered immune cells can be derived from induced stem cells (eg, iPSCs).

In some cases, as disclosed herein, endogenous modulators of hypoimmunity (e.g., CD80, CD86, CD80, CD86, The reduced expression or activity of ICOSL, CD40L, MICA, MICB, NKG2DL, etc.) can be at least or at most about 1/0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, At least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1/0.8, at least or at most about 1/0.9, at least or At most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1/5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/ 40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most about 1/90, at least or at most about 1/100, At least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 lower.

In some cases, the reduced expression or activity of endogenous CD80 in engineered immune cells of the present disclosure (e.g., engineered NK cells) can be at least or at most about 1/0.1, at least or at most, of its expression by control cells About 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1 /0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1/5 , at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most About 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 lower.

In some cases, the reduced expression or activity of endogenous CD86 in engineered immune cells of the present disclosure (e.g., engineered NK cells) can be at least or at most about 1/0.1, at least or at most, of its expression by control cells About 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1 /0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1/5 , at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most About 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 lower.

In some cases, the reduced expression or activity of endogenous ICOSL in engineered immune cells of the present disclosure (e.g., engineered NK cells) can be at least or at most about 1/0.1, at least or at most, of its expression by control cells About 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1 /0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1/5 , at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most About 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 lower.

In some instances, the reduced expression or activity of the endogenous hypoimmunity modulator CD40L in engineered immune cells of the present disclosure (e.g., engineered NK cells) can be at least or at most about 1/3 of its expression by control cells. 0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, At least or at most about 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or At most about 1/5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/ 80, at least or at most about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 Low.

In some cases, the reduced expression or activity of endogenous MICA or MICB in an engineered immune cell of the disclosure (e.g., an engineered NK cell) can be at least or at most about 1/0.1, at least about 1/0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most About 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1 /5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20 , at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least Or at least about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 lower.

In some cases, the reduced expression or activity of endogenous NKG2DL in an engineered immune cell of the disclosure (e.g., an engineered NK cell) can be at least or at most about 1/0.1, at least or at most, of its expression by a control cell About 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7, at least or at most about 1 /0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1/5 , at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most About 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/10,000 lower.

In one aspect, the disclosure provides engineered immune cells (eg, engineered NK cells). As disclosed herein, engineered immune cells can exhibit reduced expression or activity of endogenous immunomodulatory polypeptides. In some instances, the endogenous immunomodulatory polypeptide comprises a hypoimmune modulator (eg, ICAM1). The engineered immune cell further comprises one or more of: (a) a chimeric polypeptide receptor comprising an antigen binding portion capable of binding an antigen as disclosed herein, (b) a heterologous cytokine (e.g., a heterologous cytokine) as disclosed herein. IL, such as IL-15), and (c) CD16 variants for enhanced CD16 signaling compared to control cells.

In some cases, engineered immune cells (e.g., engineered NK cells) comprise a chimeric polypeptide receptor disclosed herein and one or both of: (b) a heterologous cytokine as disclosed herein (e.g., a heterologous IL, such as IL-15) and (c) CD16 variants for enhanced CD16 signaling.

In some cases, engineered immune cells (e.g., engineered NK cells) comprise a heterologous cytokine disclosed herein (e.g., a heterologous IL, such as IL-15) and one or both of: (a) Chimeric polypeptide receptors, and (c) CD16 variants for enhanced D16 signaling.

In some instances, engineered immune cells (e.g., engineered NK cells) comprise a CD16 variant for enhanced CD16 signaling and one or both of: (a) a chimeric polypeptide receptor as disclosed herein , and (b) a heterologous cytokine (eg, a heterologous IL, such as IL-15) as disclosed herein.

In some cases, as disclosed herein, reduced expression or activity of endogenous ICAM1 in engineered immune cells of the present disclosure (e.g., engineered NK cells) may be an increase in response to control cells as determined by Western blot or PCT techniques. Its expression is at least or at most about 1/0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0. 0.6, at least or at most about 1/0.7, at least or at most about 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/4, at least or at most about 1/4, at least or at most about 1/5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or At most about 1/10, at least or at most about 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1/80, at least or at most about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/1/ 5,000, or at least or at most about 1/10,000 lower.

In one aspect, the disclosure provides engineered immune cells (eg, engineered NK cells). As disclosed herein, engineered immune cells can exhibit reduced expression or activity of endogenous immunomodulatory polypeptides. In some instances, the endogenous immunomodulatory polypeptide comprises a hypoimmune modulator (eg, ICAM1). Engineered immune cells can be derived from induced stem cells (eg, iPSCs).

In some cases, as disclosed herein, the reduced expression or activity of endogenous ICAM1 in engineered immune cells of the present disclosure (eg, engineered NK cells) can be lower than its expression by control cells.

In one aspect, the disclosure provides engineered immune cells (eg, engineered NK cells). An engineered immune cell can comprise an immunomodulatory polypeptide disclosed herein, wherein the immunomodulatory polypeptide is heterologous to the engineered immune cell. In some instances, the immunomodulatory polypeptide comprises a hypoimmune modulator. The hypoimmune modulator can be PDL2. Alternatively or additionally, the hypoimmune modulator may be TGF-β.

In one aspect, the disclosure provides engineered immune cells (eg, engineered NK cells). An engineered immune cell can comprise an immunomodulatory polypeptide disclosed herein, wherein the immunomodulatory polypeptide is heterologous to the engineered immune cell. In some instances, the immunomodulatory polypeptide comprises a hypoimmune modulator. The hypoimmunity modulator may comprise one or more members of: (i) heterologous CCL21, (ii) heterologous IL-10, (iii) heterologous CD46, (iv) heterologous CD55, and (v) ) heterologous CD59. Engineered immune cells can be derived from isolated stem cells (eg, isolated ESCs). Alternatively, engineered immune cells can be derived from induced stem cells (eg, iPSCs).

In some cases, engineered immune cells (e.g., engineered NK cells) disclosed herein can include heterologous CCL21 and one or more (e.g., 1, 2, 3, or all) of the following: (ii) heterologous IL-10, (iii) heterologous CD46, (iv) heterologous CD55, and (v) heterologous CD59.

In some cases, engineered immune cells (e.g., engineered NK cells) disclosed herein can include heterologous IL-10 and one or more (e.g., 1, 2, 3, or all) of the following: (i) Heterologous CCL21, (iii) heterologous CD46, (iv) heterologous CD55, and (v) heterologous CD59.

In some cases, engineered immune cells (e.g., engineered NK cells) disclosed herein can include heterologous CD46 and one or more (e.g., 1, 2, 3, or all) of the following: (i) heterologous CCL21, (ii) heterologous IL-10, (iv) heterologous CD55, and (v) heterologous CD59.

In some cases, engineered immune cells (e.g., engineered NK cells) disclosed herein can include heterologous CD55 and one or more (e.g., 1, 2, 3, or all) of the following: (i) heterologous CCL21, (ii) heterologous IL-10, (iii) heterologous CD46, and (v) heterologous CD59.

In some cases, engineered immune cells (e.g., engineered NK cells) disclosed herein can include heterologous CD59 and one or more (e.g., 1, 2, 3, or all) of the following: (i) heterologous CCL21, (ii) heterologous IL-10, (iii) heterologous CD46, and (iv) heterologous CD55.

In one aspect, the disclosure provides engineered immune cells (eg, engineered NK cells). Engineered immune cells may comprise a heterologous cytokine other than IL-15 disclosed herein (eg, heterologous IL). In some examples, the heterologous cytokine comprises IL-21 or a variant thereof. Engineered immune cells can be derived from induced stem cells (eg, iPSCs).

In some cases, the control cells can be immune cells, such as NK cells, which are used for comparison purposes. In some cases, control cells can be cells that do not contain a heterologous cytokine (eg, IL-15). In some cases, the control cell can be a cell that does not contain a CD16 variant for enhanced CD16 signaling. In some cases, a control cell can be a cell comprising a chimeric polypeptide receptor comprising an antigen-binding portion capable of binding an antigen. In some cases, the control cell can be a cell comprising a heterologous IL-15R. In some cases, a control cell can be a cell that does not contain a membrane-bound heterologous cytokine (eg, IL-15). In some instances, a control cell can be a cell that does not exhibit reduced expression or activity of an endogenous immunomodulatory polypeptide. In some instances, a control cell can be a cell that does not exhibit reduced expression or activity of an endogenous cytokine (eg, IL-17) or its receptor (eg, IL-17R). In some cases, a control cell can be a cell that does not contain a heterologous transcription factor (eg, STAT). In some cases, a control cell can be a cell that does not exhibit reduced expression or activity of a specific endogenous cell marker (eg, a NK cell marker, such as a KIR) for committed immune cells. In some cases, a control cell can be a cell that does not contain a heterologous immunomodulatory polypeptide. In some instances, a control cell can be a cell that does not exhibit reduced expression or activity of one or more of: endogenous CD94, endogenous CD96, endogenous TGFβ receptor, or endogenous SHIP2. In some cases, a control cell may be a cell that does not exhibit reduced expression or activity of one or more of: endogenous CD80, endogenous CD86, endogenous ICOSL, endogenous CD40L, endogenous MICA or MICB or endogenous NKG2DL. In some instances, a control cell can be a cell that does not exhibit reduced expression or activity of ICAM1. In some cases, the control cells can be cells that do not contain heterologous PDL2 or heterologous TGFβ. In some cases, a control cell can be a cell that does not comprise one or more of: allogeneic CCL21, allogeneic IL-10, allogeneic CD46, allogeneic CD55, or allogeneic CD59. In some cases, control cells can be cells that do not contain allogeneic IL-21. In some cases, a control cell may be a cell not derived from a cell line. In some cases, control cells can be cells not derived from isolated ESCs. In some cases, the control cells can be cells not derived from iPSCs. C. _ Additional aspects of engineered immune cells

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cells can include a heterologous cytokine disclosed herein (eg, heterologous IL, such as IL-15). In some cases, engineered immune cells (eg, engineered NK cells) include heterologous receptors that are corresponding receptors for heterologous cytokines (eg, heterologous IL-15R), as disclosed herein. Accordingly, engineered immune cells (e.g., engineered NK cells) can exhibit induction by heterologous cytokines and/or heterologous receptors disclosed herein (e.g., by heterologous cytokines and/or heterologous receptors such as IL -15/IL-15R induced) enhanced signaling of the endogenous signaling pathway.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cell can exhibit reduced expression or activity of endogenous CD38 as compared to a control cell. Such engineered immune cells can be used to treat subjects with or suspected of having a leukocyte cancer, such as multiple myeloma (MM).

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the expression or activity of endogenous CD38 of the engineered immune cell may and need not be modified. Such engineered immune cells can be used to treat a subject having or suspected of having a disease (eg, cancer, tumor) other than multiple myeloma.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the engineered immune cell can include heterologous IL-15 or a fragment thereof, and the heterologous IL-15 or fragment thereof can be obtained from Engineered immune cell secretion.

In some embodiments, the secreted heterologous IL-15 comprises an amino acid sequence at least 70% identical to SEQ ID NO. 24. In some embodiments, the secreted heterologous IL-15 comprises an amino acid sequence at least 75% identical to SEQ ID NO. 24. In some embodiments, the secreted heterologous IL-15 comprises an amino acid sequence at least 80% identical to SEQ ID NO. 24. In some embodiments, the secreted heterologous IL-15 comprises an amino acid sequence at least 85% identical to SEQ ID NO. 24. In some embodiments, the secreted heterologous IL-15 comprises an amino acid sequence at least 90% identical to SEQ ID NO. 24. In some embodiments, the secreted heterologous IL-15 comprises an amino acid sequence at least 95% identical to SEQ ID NO. 24. In some embodiments, the secreted heterologous IL-15 comprises an amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO. 24. In some embodiments, the secreted heterologous IL-15 comprises the amino acid sequence of SEQ ID NO. 24.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the engineered immune cell can include heterologous IL-15 or a fragment thereof, and the heterologous IL-15 or fragment thereof can bind to the cell surface membrane of engineered immune cells.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the engineered immune cell can comprise at least one chimeric polypeptide receptor comprising an antigen-binding portion capable of binding an antigen, as in provided in this disclosure. In some examples, engineered immune cells can include multiple different chimeric polypeptide receptors to specifically bind multiple different antigens. In some examples, engineered immune cells can include at least one chimeric polypeptide receptor that includes multiple antigen binding portions to specifically bind multiple different antigens.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cell can include a safety switch capable of affecting the death of the engineered immune cell. Engineered immune cells can include genes encoding safety switches by the effect of gene editing in part (eg, integrated into the genome of the immune cell), as disclosed herein. In some cases, prodrugs can be introduced into engineered immune cells (e.g., administered to an object comprising engineered immune cells) and can be passed through the safety switch molecule in the event of an adverse event or when adaptive immunotherapy is no longer needed. to activate the prodrug to kill the subject immune cells. In some cases, the safety switch may comprise a protein selected from the group consisting of caspases (e.g., caspase 3, 7, or 9), thymidine kinase, cytosine deaminase, modified EGFR, B cell CD20, and functional variants thereof. One or more members of . In some cases, a safety switch can be initiated by an initiator (e.g., a small molecule or protein, such as an antibody) for post-translational, temporal, and/or site-specific regulation of death (or exhaustion) of the subject engineered immune cells . Non-limiting examples of safety switches and their initiators may include caspase 9 (or caspase 3 or 7) and AP1903; thymidine kinase (TK) and ganciclovir (GCV); and cytosine deaminase (CD) and 5-fluorocytosine (5-FC). Alternatively or additionally, a modified epidermal growth factor receptor (EGFR) comprising an epitope recognized by an antibody (e.g., an anti-EGFR Ab, such as cetuximab) can be used for depletion engineering when the subject cells are exposed to the antibody immune cells. In some cases, an engineered immune cell (e.g., an engineered NK cell) disclosed herein may comprise a protein selected from the group consisting of caspase 9 (caspase 3 or 7), thymidine kinase, cytosine deaminase, modified EGFR and safety switch proteins in CD20.3 of B cells.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cell can include a heterologous immune receptor polypeptide. The immunomodulatory polypeptide may include one or more members selected from HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-β, CCL21, IL10, CD46, CD55 and CD59.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cell can exhibit reduced expression or activity of an endogenous immunomodulatory polypeptide, as disclosed herein. Endogenous immunomodulatory polypeptides include immune checkpoint inhibitors or hypoimmunity modulators (or both).

In some cases, the immune checkpoint inhibitors disclosed herein can include an immune checkpoint inhibitor selected from the group consisting of PD1, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, NKG2D, TIGIT, CD96, LAG3, TIGIT, TGFβ receptor, and 2B4 one or more members. In some instances, the immune checkpoint inhibitor can include SHIP2.

In some cases, a hypoimmune modulator disclosed herein may comprise a protein selected from B2M, CIITA, TAP1, TAP2, tap-related protein, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB One or more members of , ULBP1, HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-β, CCL21, IL10, CD46, CD55 and CD59.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the engineered immune cell can comprise a CD16 variant for enhanced CD16 signaling compared to a control cell, wherein CD16 variants are heterologous to engineered immune cells.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cells can exhibit enhanced cytotoxicity against target cells compared to control cells. In some cases, an engineered immune cell disclosed herein may exhibit cytotoxicity (e.g., in vitro, ex vivo, or in vivo) toward a target cell or target cell population, as determined, for example, by tracking changes in the number of the target cell population At least or at most about 0.1 times, at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times the cytotoxicity of control cells , at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, At least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or At most about 100 times larger, at least or at most about 500 times larger, at least or at most about 1,000 times larger, at least or at most about 5,000 times larger, or at least or at most about 10,000 times larger.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the engineered immune cells can induce T cells from isolated immune cells (e.g., isolated T cells in vitro and and/or B cells, or the immune response of the host after administration of the engineered immune cells to the host). In some cases, as by, for example, measuring (i) the change in the number of the initial population of engineered immune cells after exposure to the isolated immune cells, or (ii) the number of isolated immune cells after exposure to the initial population of engineered immune cells The engineered immune cells disclosed herein can reduce the immune response from the isolated immune cells by at least or at most about 1 compared to the immune response from the isolated immune cells when exposed to control cells, as determined by changes in cytokine release. /0.1, at least or at most about 1/0.2, at least or at most about 1/0.3, at least or at most about 1/0.4, at least or at most about 1/0.5, at least or at most about 1/0.6, at least or at most about 1/0.7 , at least or at most about 1/0.8, at least or at most about 1/0.9, at least or at most about 1/1, at least or at most about 1/2, at least or at most about 1/3, at least or at most about 1/4, at least or at most about 1/5, at least or at most about 1/6, at least or at most about 1/7, at least or at most about 1/8, at least or at most about 1/9, at least or at most about 1/10, at least or at most About 1/20, at least or at most about 1/30, at least or at most about 1/40, at least or at most about 1/50, at least or at most about 1/60, at least or at most about 1/70, at least or at most about 1 /80, at least or at most about 1/90, at least or at most about 1/100, at least or at most about 1/500, at least or at most about 1/1,000, at least or at most about 1/5,000, or at least or at most about 1/1/ 10,000.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the engineered immune cells can be compared to control cells after exposure to isolated immune cells (e.g., isolated T cells in vitro). and/or B cells, or when the engineered immune cells are administered to the host, the host's immune cells) exhibit increased half-life. In some cases, when exposed to isolated immune cells (e.g., in vitro or in vivo), the half-life of the engineered immune cells can be At least or at most about 0.1 times, at least or at most about 0.2 times, at least or at most about 0.3 times, at least or at most about 0.4 times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most the half-life of the control cells About 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times, at least or at most about 3 times, at least or at most about 4 times, at least or at most about 3 times 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, at least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at most about 80 times, at least or at most about 90 times, at least or at most about 100 times , at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cells can achieve an enhancement of a function or pathological condition of a body tissue of a subject as compared to a control cell. In some cases, treatment with engineered immune cells can achieve at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most about 0.3-fold, at least or at most about 0.4-fold times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times , at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, At least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times Enhancement of the function or pathological condition of body tissues.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cells can achieve delayed degradation of a functional or pathological condition of a body tissue of the subject as compared to control cells. In some cases, treatment with engineered immune cells can achieve at least or at most about 0.1-fold, at least or at most about 0.2-fold, at least or at most about 0.3-fold, at least or at most about 0.4-fold times, at least or at most about 0.5 times, at least or at most about 0.6 times, at least or at most about 0.7 times, at least or at most about 0.8 times, at least or at most about 0.9 times, at least or at most about 1 times, at least or at most about 2 times , at least or at most about 3 times, at least or at most about 4 times, at least or at most about 5 times, at least or at most about 6 times, at least or at most about 7 times, at least or at most about 8 times, at least or at most about 9 times, At least or at most about 10 times, at least or at most about 20 times, at least or at most about 30 times, at least or at most about 40 times, at least or at most about 50 times, at least or at most about 60 times, at least or at most about 70 times, at least or at least or at most about 90 times, at least or at most about 100 times, at least or at most about 500 times, at least or at most about 1,000 times, at least or at most about 5,000 times, or at least or at most about 10,000 times Delayed degeneration of body tissues in functional or pathological conditions.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the bodily tissue may comprise a tissue selected from the group consisting of blood, plasma, serum, urine, perilymph, feces, saliva, semen, amniotic fluid, Cerebrospinal fluid, bile, sweat, tears, sputum, synovial fluid, vomit, bones, heart, thymus, arteries, blood vessels, lungs, muscles, stomach, intestines, liver, pancreas, spleen, kidneys, gallbladder, thyroid, adrenals, One or more members of breast, ovary, prostate, testis, skin, fat, eye, brain, infected tissue, diseased tissue, malignant tissue, calcified tissue, and healthy tissue.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, the engineered immune cell can induce an immune response against the target cell. Targets can be, for example, diseased cells, cancer cells, tumor cells, and the like.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, the heterologous gene can be operably associated with constitutive, inducible, temporal, tissue-specific and/or cellular Type-specific promoter coupling (eg, for knock-in). Non-limiting examples of promoters of interest may include CMV, EF1a, PGK, CAG, and UBC. Non-limiting examples of insertion sites may include AAVS1, CCR5, ROSA26, collagen, HTRP, H11, B2M, GAPDH, TCR, RUNX1, TAP1, TAP2, tap-related protein, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3 and TIGIT.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, one or more of the following endogenous genes may exhibit reduced expression or activity to enhance the performance of the engineered immune cells in the tumor microenvironment (ie, tumor microenvironment genes or "TME"). In some instances, having reduced expression or activity of the TME can enhance the immune activity of the engineered immune cell against the target cell. In some instances, the TME gene can be an immune checkpoint inhibitor. Non-limiting examples of TMEs may include: NKG2A, NKG2D, PD1, CTLA4, LAG3, TIM3, TIGIT, KIR2D, CD94, CD96, TGFβ receptor, 2B4, and SHIP2.

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, one or more heterologous genes (e.g., knocked in) may be expressed for enhanced function, e.g., CD137, CD80, CD86 , DAP10 (eg, with or without point mutations).

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, may exhibit reduced expression or activity of one or more of the following endogenous genes for, e.g., low immunity: B2M, CIITA, TAP1, TAP2, tap-related proteins, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, and NKG2DL (eg, ULBP1).

In some embodiments of any of the engineered immune cells (e.g., engineered NK cells) disclosed herein, one or more heterologous genes (e.g., knocked in) may be expressed for, e.g., hypoimmunity: HLA-E, CD47 , CD113, PDL1, PDL2, A2AR, HLA-G, TGF-β, CCL21, IL10, CD46, CD55, and CD59.

In some embodiments of any of the engineered immune cells (eg, engineered NK cells) disclosed herein, one or more heterologous genes (eg, knocked in): CD3, CD4, CD80, 41BBL, and CD131 may be expressed. D. _ chimeric antigen receptor

Engineered immune cells of the present disclosure (e.g., engineered NK cells) can include chimeric polypeptide receptors disclosed herein (e.g., at least 1, 2, 3, 4, 5 or more different types of chimeric polypeptide receptors) . Engineered immune cells can be engineered to express chimeric polypeptide receptors temporarily or permanently. In some cases, recombinant chimeric polypeptide receptors can be delivered to engineered immune cells by, for example, liposomes and incorporated into engineered immune cells by membrane fusion. In some cases, a heterologous polynucleotide construct (eg, DNA or RNA) encoding a chimeric polypeptide receptor can be delivered to engineered immune cells. The heterologous polynucleotide construct (i.e., gene) encoding the heterologous polynucleotide construct may be incorporated into the chromosome (i.e., chromosomal gene) of the engineered immune cell, or may not or need not be integrated into In the chromosomes of the engineered immune cells disclosed herein.

A chimeric polypeptide receptor can include a T cell receptor fusion protein (TFP). The term "T cell receptor fusion protein" or "TFP" generally refers to a recombinant polypeptide construct comprising (i) one or more antigen binding moieties (e.g., monospecific or multispecific) , (ii) at least a portion of the TCR extracellular domain, (iii) at least a portion of the TCR transmembrane domain, and (iv) at least a portion of the TCR intracellular domain.

In some cases, the endogenous T cell receptor (TCR) of the engineered immune cells (eg, engineered NK cells) of the present disclosure can be inactivated. In some examples, the function of the endogenous TCR of the engineered immune cell can be inhibited by an inhibitor. In some examples, a gene encoding a subunit of an endogenous TCR can be inactivated (eg, edited by the action of the gene editing moieties disclosed herein), such that the endogenous TCR is inactivated. The gene encoding an endogenous TCR subunit may be one or more of the following: TCRα, TCRβ, CD3ε, CD3δ, CD3γ, and CD3ζ.

Chimeric polypeptide receptors can include chimeric antigen receptors (CARs). The term "chimeric antigen receptor" or "CAR" generally refers to a recombinant polypeptide construct that includes at least an extracellular antigen-binding domain, a transmembrane domain, and includes a functional signaling structure derived from a stimulatory molecule. domain (also referred to herein as an "intracellular or intrinsic signaling domain"). In some instances, the stimulatory molecule can be the zeta chain associated with the T cell receptor complex. In some cases, the intracellular signaling domain also includes one or more functional signaling domains derived from at least one co-stimulatory molecule. In some instances, co-stimulatory molecules can include 4-1BB (ie, CD137), CD27, and/or CD28. In one aspect, the CAR includes an optional leader sequence at the amino terminus (N-terminus) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally removed from the antigen recognition domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane. cutting.

The CAR can be a first-, second-, third-, or fourth-generation CAR system, a functional variant thereof, or any combination thereof. First-generation CARs (e.g., CD19R or CD19CAR) include: an antigen-binding domain specific for a specific antigen (e.g., an antibody or antigen-binding fragment thereof, such as scFv, Fab fragment, VHH domain, or the VH of a heavy-chain antibody only) domain), a transmembrane domain derived from a self-regulatory immune receptor (such as a transmembrane domain from a CD28 receptor), and a signaling domain derived from a self-regulatory immune receptor (such as one or more (such as Three) ITAM domains derived from the intracellular region of the CD3ζ receptor or FcεRIγ). Second-generation CARs work by adding costimulatory domains (e.g., derived from costimulatory receptors that co-act with T cell receptors (e.g., CD28, CD137/4-1BB, and CD134/OX40) to the intracellular signaling domain portion of the CAR. body) to modify first-generation CARs, which eliminates the need to administer cofactors (such as IL-2) with the first-generation CARs. Third-generation CARs add multiple co-stimulatory domains to the intracellular signaling domain portion of the CAR (eg, CD3ζ-CD28-OX40 or CD3ζ-CD28-41BB). Fourth-generation CARs initiate cytokines (e.g., IL-12, IL-23, or IL-27) by adding the intracellular signaling portion of the CAR (e.g., between one or more co-stimulatory domains and the CD3ζ ITAM domain). ) or modify second- or third-generation CARs under the control of a CAR-inducible promoter (eg, the NFAT/IL-2 minimal promoter). In some cases, the CAR may be a new generation CAR system other than the first, second, third, or fourth generation CAR systems disclosed herein.

The hinge domain (e.g., the linker between the extracellular antigen-binding domain and the transmembrane domain) of the CAR in the engineered immune cells disclosed herein (e.g., engineered NK cells) may include CD3D, CD3E, CD3G, CD3c, CD4 , CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12 , IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D or the full length or at least a portion of the native or modified transmembrane region of a T cell receptor polypeptide.

The transmembrane domain of the CAR in the engineered immune cells disclosed herein (eg, engineered NK cells) may include CD3D, CD3E, CD3G, CD3c, CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4 -1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D or T The full length or at least a portion of a native or modified transmembrane region of a cell receptor polypeptide.

The hinge domain and the transmembrane domain of the CARs disclosed herein (eg, for engineering immune cells, eg, engineered NK cells) can be derived from the same protein (eg, CD8). Alternatively, the hinge and transmembrane domains of the CARs disclosed herein can be derived from different proteins.

The signaling domain of the CAR may comprise at least or at most about 1 signaling domain, at least or at most about 2 signaling domains, at least or at most about 3 signaling domains, at least or at most about 4 signaling domains domain, at least or at most about 5 signaling domains, at least or at most about 6 signaling domains, at least or at most about 7 signaling domains, at least or at most about 8 signaling domains, at least or at most about 9 signaling domains or at least or at most about 10 signaling domains.

The signaling domain (e.g., the signaling peptide of the intracellular signaling domain) of the CAR in the engineered immune cells disclosed herein (e.g., engineered NK cells) may comprise CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB ), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, NKG2D or the full length or at least a portion of a polypeptide of any combination thereof.

Alternatively or in addition (i.e., co-stimulatory domains), the signaling domain CAR in engineered immune cells (e.g., engineered NK cells) disclosed herein may comprise CD27, CD28, 4-1BB, OX40 , ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4 or NKG2D or any combination of the full length or at least a portion of the polypeptide.

In some cases, an engineered immune cell disclosed herein (eg, an engineered NK cell) includes a chimeric polypeptide receptor (eg, a CAR) that includes at least a CD8 transmembrane domain and one or more of Two: (i) 2B4 signaling domain and (ii) DAP10 signaling domain. In some cases, an engineered cell disclosed herein (e.g., an engineered NK cell) includes a chimeric polypeptide receptor (e.g., TFP or CAR) that includes at least (i) a CD8 transmembrane domain, (ii) 2B4 signaling domain and (iii) DAP10 signaling domain. The 2B4 signaling domain may be flanked by a CD8 transmembrane domain and a DAP10 signaling domain. Alternatively, the DAP10 signaling domain can be flanked by a CD8 transmembrane domain and a 2B4 signaling domain. In some cases, the chimeric polypeptide receptors disclosed herein can further comprise an additional signaling domain derived from CD3ζ.

The antigen (ie, target antigen) of the antigen-binding portion of a chimeric polypeptide receptor (eg, TFP or CAR) disclosed herein can be a cell surface marker, a secreted marker, or an intracellular marker.

Non-limiting examples of antigens (i.e., target antigens) of the antigen-binding portion of a chimeric polypeptide receptor (e.g., TFP or CAR) disclosed herein may include ADGRE2, carbonic anhydrase IX (CA1X), CCRI, CCR4, carcinoembryonic Antigen (CEA), CD3ζ, CD5, CD8, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD133, CD138, CD269 (BCMA ), CD S, CLEC12A, antigens (e.g., cell surface antigens) of cytomegalovirus (CMV)-infected cells, epithelial glycoprotein 2 (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), EGFRvIII, receptor tyrosine protein kinase erb-B2, 3, 4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor a , ganglioside G2 (GD2), ganglioside G3 (GD3), gp100, human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), ICAM-1, integration IL-13 receptor subunit α-2 (IL-13Rα2), κ light chain, kinase insertion domain receptor (KDR), κ, Lewis A (CA19.9), Lewis Y (LeY ), L1 cell adhesion molecule (L1-CAM), LILRB2, MART-1, melanoma antigen family A 1 (MAGE-A1), MICA/B, mucin 1 (Muc-1), mucin 16 (Muc- 16), mesothelin (MSLN), NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, NY-ESO-2, carcinoembryonic antigen (h5T4), PRAIVIE, prostate stem cell antigen (PSCA ), PRAME prostate-specific membrane antigen (PSMA), ROR1, tumor-associated glycoprotein 72 (TAG-72), TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT- 1) and various pathogen antigens (eg, pathogen antigens derived from viruses, bacteria, fungi, parasites, and protozoa capable of causing disease). In some examples, the pathogen antigen is derived from HIV, HBV, EBV, HPV, Lasse virus, influenza virus, or coronavirus.

Other examples of antigens for the antigen binding portion of the chimeric polypeptide receptors disclosed herein may include: 1-40-beta-amyloid, 4-1BB, 5AC, 5T4, Activin receptor-like kinase 1, ACVR2B, adenocarcinoma Antigen, AGS-22M6, alpha-fetoprotein, angiopoietin 2, angiopoietin 3, anthrax toxin, AOC3 (VAP-1), B7-H3, Bacillus anthracis, BAFF, beta-amyloid, B lymphoma cells , C242 antigen, C5, CA-125, domestic dog (Canis lupus familiaris) IL31, carbonic anhydrase 9 (CA-IX), cardiac myosin, CCL11 (eotaxin-1), CCR4, CCR5 , CD11, CD18, CD125, CD140a, CD147 (basicin), CD15, CD152, CD154 (CD40L), CD19, CD2, CD20, CD200, CD22, CD221, CD25 (α of IL-2 receptor chain), CD27, CD274, CD28, CD3, CD3ε, CD30, CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44 v6, CD5, CD51, CD52, CD56, CD6, CD70, CD74, CD79B, CD80, CEA, CEA-associated antigen, CFD, ch4D5, CLDN18.2, Clostridium difficile, agglutination factor A, CSF1R, CSF2, CTLA-4, C-X-C chemokine receptor type 4, cytomegalovirus, cytomegalovirus glycoprotein B. Dabigatran, DLL4, DPP4, DR5, E. coli Shiga toxin type 1, E. coli Shiga toxin type 2, EGFL7, EGFR, endotoxin, EpCAM, episialin, ERBB3, E. coli, F protein of respiratory syncytial virus, FAP, fibrin II beta chain, fibronectin extra domain B, folate hydrolase, folate receptor 1, folate receptor alpha, Frizzled receptor, gangliosides GD2, GD2, GD3 Ganglioside, Glypican 3, GMCSF receptor alpha chain, GPNMB, Growth differentiation factor 8, GUCY2C, Hemagglutinin, Hepatitis B surface antigen, Hepatitis B virus, HER1, HER2/neu, HER3, HGF, HHGFR, Histone Complex, HIV-1, HLA-DR, HNGF, Hsp90, Human Scatter Factor Receptor Kinase, Human TNF, Human Beta Amyloid, ICAM-1 (CD54), IFN-α, IFN-γ, IgE, IgE Fc region, IGF-1 receptor, IGF-1, IGHE, IL17A, IL17F, IL20, IL-12, IL-13, IL-1 7. IL-1β, IL-22, IL-23, IL-31RA, IL-4, IL-5, IL-6, IL-6 receptor, IL-9, ILGF2, influenza A hemagglutinin, A Influenza virus hemagglutinin, insulin-like growth factor I receptor, integrin α4β7, integrin α4, integrin α5β1, integrin α7β7, integrin αIIbβ3, integrin αvβ3, interferon α/β receptor, interferon γ Inducible protein, ITGA2, ITGB2 (CD18), KIR2D, Lewis-Y antigen, LFA-1 (CD11a), LINGO-1, lipoteichoic acid, LOXL2, L-selectin (CD62L), LTA, MCP-1, Mesothelin, MIF, MS4A1, MSLN, MUC1, mucin CanAg, myelin-associated glycoprotein, myostatin, NCA-90 (granulocyte antigen), neuronal apoptosis-regulating protease 1, NGF, N-hydroxyethyl Acylneuraminic acid, NOGO-A, Notch receptor, NRP1, Oryctolagus cuniculus, OX-40, oxLDL, PCSK9, PD-1, PDCD1, PDGF-Rα, sodium phosphate cotransporter, phosphatidylserine, Platelet-derived growth factor receptor beta, prostate cancer cells, Pseudomonas aeruginosa, rabies virus glycoprotein, RANKL, respiratory syncytial virus, RHD, rhesus factor, RON, RTN4, Sclerostin, SDC1, Selectin P, SLAMF7, SOST, sphingosine-1-phosphate, Staphylococcus aureus, STEAP1, TAG-72, T cell receptor, TEM1, tenascin C, TFPI, TGF-β1, TGF-β2, TGF- β, TNF-α, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, tumor-specific glycosylation of MUC1, tumor-associated calcium signal transducer 2, TWEAK receptor, TYRP1 (glycoprotein 75), VEGFA , VEGFR1, VEGFR2, vimentin and VWF.

Other examples of antigens for the antigen binding portion of the chimeric polypeptide receptors disclosed herein may include: 707-AP, biotinylated molecules, a-actinin-4, abl-bcr alb-b3 (b2a2), abl- bcr alb-b4 (b3a2), adipophilin, AFP, AIM-2, annexin II, ART-4, BAGE, b-catenin, bcr-abl, bcr-abl p190 (e1a2), bcr-abl p210 (b2a2), bcr-abl p210 (b3a2), BING-4, CAG-3, CAIX, CAMEL, caspase-8, CD171, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD44v7/8, CDC27, CDK-4, CEA, CLCA2, Cyp-B, DAM-10, DAM-6, DEK-CAN, EGFRvIII, EGP-2, EGP-40, ELF2, Ep-CAM, EphA2, EphA3, erb-B2, erb-B3, erb-B4, ES-ESO-1a, ETV6/AML, FBP, fetal acetylcholine receptor, FGF-5, FN, G250, GAGE-1, GAGE-2, GAGE- 3. GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GD2, GD3, GnT-V, Gp100, gp75, Her-2, HLA-A*0201-R170I, HMW-MAA, HSP70-2 M, HST-2 (FGF6), HST-2/neu, hTERT, iCE, IL-11Rα, IL-13Rα2, KDR, KIAA0205, K-RAS, L1-cell adhesion molecule, LAGE-1, LDLR /FUT, Lewis Y, MAGE-1, MAGE-10, MAGE-12, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A6 , MAGE-B1, MAGE-B2, malic enzyme, mammaglobin-A (Mammaglobin-A), MART-1/Melan-A, MART-2, MC1R, M-CSF, mesothelin, MUC1, MUC16, MUC2, MUM-1, MUM-2, MUM-3, myosin, NA88-A, Neo-PAP, NKG2D, NPM/ALK, N-RAS, NY-ESO-1, OA1, OGT, carcinoembryonic antigen ( h5T4), OS-9, P polypeptide, P15, P53, PRAME, PSA, PSCA, PSMA, PTPRK , RAGE, ROR1, RU1, RU2, SART-1, SART-2, SART-3, SOX10, SSX-2, Survivin (Survivin), Survivin-2B, SYT/SSX, TAG-72, TEL/AML1, TGFaRII, TGFbRII, TP1, TRAG-3, TRG, TRP-1, TRP-2, TRP-2/INT2, TRP-2-6b, tyrosinase, VEGF-R2, WT1, α-folate receptor, and κ - light chain.

Other examples of antigens of the antigen binding portion of the chimeric polypeptide receptors disclosed herein may include antibodies, fragments or variants thereof. Such antibodies may be natural antibodies (eg, naturally secreted by immune cells of a subject, such as B cells), synthetic antibodies, or modified antibodies. In some cases, the antigen of the antigen binding portion of a chimeric polypeptide receptor disclosed herein may comprise an Fc domain of an antibody from the group consisting of: 20-(74)-(74) (milatuzumab (milatuzumab; veltuzumab), 20-2b-2b, 3F8, 74-(20)-(20) (milatuzumab; veltuzumab) , 8H9, A33, AB-16B5, abavolumab (abagovomab), abciximab (abciximab), abituzumab (abituzumab), lintuzumab (zlintuzumab)), aktomumab (actoxumab), adalimumab, ADC-1013, ADCT-301, ADCT-402, adecatumumab, aducanumab, afelimomab ), AFM13, afutuzumab, AGEN1884, AGS15E, AGS-16C3F, AGS67E, alacizumab pegol, ALD518, alemtuzumab, aletuzumab (alirocumab), altumomab pentetate, amatuximab, AMG 228, AMG 820, anatumomab mafenatox, Leixing-anetuzumab ( anetumab ravtansine), anifrolumab, anrukinzumab, APN301, APN311, apolizumab, APX003/SIM-BD0801 (sevacizumab), APX005M, aspirin Arcitumomab, ARX788, ascrinvacumab, aselizumab, ASG-15ME, atezolizumab, atinumab, ATL101, atlizumab (also known as tocilizumab), attolimumab, avelumab, B-701, bapineuzumab, baciximab Anti (basiliximab), bavituximab (bavituximab), BAY1129980, BA Y1187982, betumomab, begelomab, belimumab, benralizumab, bertilimumab Besilesomab, Betalutin (177Lu-tetraxetan-tetulomab), bevacizumab, BEVZ92 (bevacizumab biosimilar), bezlotoxumab, BGB-A317, BHQ880, BI 836880, BI-505, biciromab, bimagrumab, bimekizumab, bivatuzumab mertansine, BIW-8962, blinatumomab, blosozumab, BMS-936559, BMS-986012, BMS-986016, BMS-986148, BMS-986178, BNC101, bococizumab, brentuximab vedotin, BrevaRex, briakinumab, brodalumab, brodalumab Brolucizumab, brontictuzumab, C2-2b-2b, canakinumab, cantuzumab mertansine, cantuzumab ravtansine ), Caplacizumab, caprumab pendetide, carlumab, catumaxomab, CBR96-doxorubicin Immunoconjugates, CBT124 (bevacizumab), CC-90002, CDX-014, CDX-1401, cedelizumab, certolizumab pegol, cetuximab Monoclonal antibody (cetuximab), CGEN-15001T, CGEN-15022, CGEN-15029, CGEN-15049, CGEN-15052, CGEN-15092, Ch.14.18, Citatuzumab bogatox, Cetuzumab (cixutumumab), clarizumab (clazakizumab), clenoliximab, clivatuzumab tetraxetan, CM-24, codrituzumab, coltuximab ravtansine, conatumumab ), Concizumab, Cotara (iodine I-131 derlotuximab biotin), cR6261, crenezumab, DA-3111 (trastuzumab ( trastuzumab biosimilars), dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, dapirolizumab pegol, Daratumumab, Daratumumab Enhanze (daratumumab), Darleukin, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, depa Depatuxizumab, Depatuxizumab mafodotin, derlotuximab biotin, detumomab, DI-B4, Denutuximab (dinutuximab), diridavumab, DKN-01, DMOT4039A, dorlimomab aritox, trazitumab, DS-1123, DS-8895, dugortuzumab Duligotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edobacomab ), edrecolomab, efalizumab, efungumab, eldelumab, elgemtumab, efalizumab Elotuzumab, elsilimomab, emactuzumab, emibetuzumab, enakinumab avatuzumab, enfortumab vedotin, enlimomab pegol, enoblituzumab, enokizumab, enoticumab, ensituximab, western apilimomab ( epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, FF-21101, FGFR2 antibody-drug conjugate, Fibromun, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, fontolizumab, foralumab, foravirumab, FPA144 , fresolimumab, FS102, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, plus Gavilimomab, gemtuzumab ozogamicin, Gerilimzumab, gevokizumab, girentuximab, glembatumumab vedotin, GNR-006, GNR-011, golimumab, gomiliximab, GSK2849330, GSK2857916, GSK317943998, 1lk 18K322A MAb, hu3S193, Hu8F4, HuL2G7, HuMab-5B1, ibalizumab, ibritumomab tiuxetan, icrucumab, idarucizumab , IGN002, IGN523, igovomab (igovomab), IMAB362, IMAB362 (claudiximab), imalumumab (imaluma b), IMC-CS4, IMC-D11, imciromab, imgatuzumab, IMGN529, IMMU-102 (yttrium Y-90 epratuzumab tetraxetan), IMMU-114, ImmuTune IMP701 antagonistic antibody, INCAGN1876, inclacumab, INCSHR1210 , indatuximab ravtansine, indusatumab vedotin, infliximab, inolimomab, inotuzumab ozogamicin, intetumumab, Ipafricept, IPH4102, ipilimumab, ipilimumab ( iratumumab), isatuximab, istiratumab, itolizumab, ixekizumab, JNJ-56022473, JNJ-61610588, Keleximab Monoclonal antibody (keliximab), KTN3379, L19IL2/L19TNF, Labetuzumab, Labetuzumab Govitecan, LAG525, lambrolizumab, lampalizumab, L-DOS47, lemalesomab, lenzilumab, lerdelimumab, Leukotuximab, lexatumumab, libivirumab, lifastuzumab vedotin, ligelizumab, lilotomab satetraxetan, lintuzumab, lirilumab, LKZ145, lodelcizumab, lokivetmab, lorvotuzumab mertansine, lucatumumab, lulizumab (lulizumab pegol), lumiliximab, lumretuzumab, LY3164530, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, MB311, MCS-110, MEDI0562, MEDI-0639, MEDI0680, MEDI- 3617, MEDI-551 (inebilizumab), M EDI-565, MEDI6469, mepolizumab, metelimumab, MGB453, MGD006/S80880, MGD007, MGD009, MGD011, milatuzumab, milatuzumab-SN-38, minretumomab, mirvetuximab soravtansine , mitumomab, MK-4166, MM-111, MM-151, MM-302, mogamulizumab, MOR202, MOR208, MORAb-066, morolimumab, motavizumab, motuximab Moxetumomab pasudotox, muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natal Natalizumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, NOV-10, obiltoxaximab ), obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, OMP-131R10, OMP-305B83, entuzumab (onartuzumab), ontuxizumab, opicinumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab ), otlertuzumab, OX002/MEN1309, oxelumab, ozanezumab, ozoralizumab, pagibaximab ), palivizumab, panitumumab, panko mab), PankoMab-GEX, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, PAT-SC1, PAT-SM6, pembrolizumab, pemtumomab, Perakizumab, pertuzumab, pexelizumab, PF-05082566 (utomilumab), PF-06647263, PF-06671008, PF-06801591, pidilizumab, pinatuzumab vedotin , pintumomab, placulumab, polatuzumab vedotin, ponezumab, priliximab, pritoxaximab, pritumumab, PRO 140, Proxinium, PSMA ADC, ranibizumab Quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, regavirumab, REGN1400, REGN2810/SAR439684, reslizumab, RFM-203, RG7356, RG7386, RG7802, RG7813, RG7841, RG7876, RG7888, RG7986, rilotumumab, rinucumab, rituximab, RM-1929, RO7009789, robatumumab, roledumab, romosozumab, rontalizumab, Rovelizumab, ruplizumab, sacituzumab govitecan, samalizumab, SAR408701, SAR566658, sarilumab, SAT 012, satumomab pendetide, SCT200, SCT400, SEA-CD40, secukinumab, seribantumab, setoxaximab, sevirumab, SGN-GN-CD19A CD19B, SGN-CD33A, SGN-CD70A, SGN-LIV1A, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab , stamulumab, sulesomab, suvizumab, SYD985, SYM004 (futuximab and modotuximab), Sym015, TAB08, tabalumab, tacatuzumab tetraxetan), tadocizumab, talizumab, tanezumab, tanibirumab, taplitumomab paptox, tarextumab, TB-403, tefibazumab, Teleukin , telimomab aritox, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, Tetulomab (tetulomab), TG-1303, TGN1412, thorium-227-epratuzumab conjugate, ticilimumab, tigatuzumab, tiratuzumab Tildrakizumab, Tisotumab vedotin, TNX-650, tocilizumab, toralizumab, tosatoxumab, tositumomab, Tovetumab, tralokinumab, trastuzumab, trastuzumab emtansine, TRBS07, TRC105, tregalizumab, tremelimumab, trevogrumab, TRPH 011, TRX518, TSR-042, TTI-200.7, tucotuzumab celmoleukin, tuviruma b. U3-1565, U3-1784, ublituximab, ulocuplumab, urelumab, urtoxazumab, ustekinumab, vadastuximab Talirine, Vitin-vandotuzumab (vandortuzumab vedotin), vantictumab, vanucizumab, vapaliximab, varlilumab, vatelizumab, VB6-845, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, YYB -101, zalutumumab, zanolimumab, zatuximab, ziralimumab, and zolimomab aritox.

In some cases, an engineered immune cell (e.g., an engineered NK cell) disclosed herein can include a chimeric polypeptide receptor (e.g., TFP or CAR) that includes an antigen-binding domain and that binds an antigen The domain may be capable of specifically and preferentially binding an antigen comprising a ligand selected from BCMA, CD20, CD22, CD30, CD33, CD38, CD70, kappa, Lewis Y, NKG2D, ROR1, NY-ESO-1, One or more members of NY-ESO-2, MART-1 and gp100. Non-limiting examples of NKG2D ligands include one or more members selected from the group consisting of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.

In some cases, an engineered immune cell (e.g., an engineered NK cell) disclosed herein can comprise a chimeric polypeptide receptor (e.g., TFP or CAR) that comprises an antigen binding domain and that binds an antigen A domain may be capable of specifically and preferentially binding CD19. Any suitable antigen binding domain capable of specifically and preferentially binding to CD19 may be used in the chimeric polypeptide receptors of the present application. In some embodiments, the antigen binding domain is selected from Fab, Fab', F(ab')2, scFv and sdAb. In some embodiments, the antigen binding domain capable of specifically and preferentially binding CD19 comprises a heavy chain variable region, a light chain variable region, or a combination thereof.

In some embodiments, the heavy chain variable region capable of specifically and preferentially binding to the antigen binding domain of CD19 comprises at least 70%, at least 75%, at least 80%, at least 85%, at least , an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO.16.

In some embodiments, the light chain variable region capable of specifically and preferentially binding to the antigen binding domain of CD19 comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90. An amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical. In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO. 17.

In some cases, engineered immune cells disclosed herein (e.g., engineered NK cells) can include a chimeric polypeptide receptor (e.g., TFP or CAR) that includes an antigen-binding receptor capable of specifically binding a target cell antigen. domain, and the engineered immune cell may exhibit reduced expression or activity of an endogenous gene encoding the same antigen of the chimeric polypeptide receptor. Thus, populations of engineered immune cells can avoid targeting and killing each other, eg, when administered to a subject in need.

In some cases, an engineered immune cell (e.g., an engineered NK cell) disclosed herein can include a chimeric polypeptide receptor (e.g., TFP or CAR) that includes an antigen-binding domain and that binds an antigen A domain may be capable of specifically and preferentially binding CD38. In some instances, the endogenous gene encoding CD38 of the engineered immune cell can be modified to achieve reduced expression or activity of endogenous CD38. In some instances, a subject engineered immune cell comprising a chimeric polypeptide receptor for CD38 may be capable of targeting and effecting the death (or degradation) of plasma cells.

In some cases, an engineered immune cell (e.g., an engineered NK cell) disclosed herein can include a chimeric polypeptide receptor (e.g., TFP or CAR) that includes an antigen-binding domain and that binds an antigen A domain may be capable of specifically and preferentially binding CD38. In some examples, the engineered immune cells are engineered NK cells derived from isolated ESCs or induced stem cells (eg, iPSCs). In some instances, the endogenous gene encoding CD38 of the engineered immune cell can be modified to achieve reduced expression or activity of endogenous CD38. E. _ stem cell

Any of the engineered immune cells (eg, engineered NK cells) disclosed herein can be derived from isolated stem cells (eg, ESCs) or induced stem cells (iPSCs). Isolated stem cells or induced stem cells can be modified (eg, genetically modified) to generate engineered immune cells.

In some cases, the pluripotency of a stem cell (eg, ESC or iPSC) can be determined in part by assessing the pluripotency characteristics of the cell. Pluripotency characteristics may include, but are not limited to: (i) pluripotent stem cell morphology; (ii) unlimited self-renewal potential; (iii) pluripotent stem cell markers, including but not limited to SSEA1 (mouse only), SSEA3/4 , SSEA5, TRA1-60/81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133/prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30 and/or expression of CD50; (iv) ability to differentiate into all three somatic lineages (ectoderm, mesoderm, and endoderm); (v) teratoma formation consisting of all three somatic lineages; and (vi) formation by Formation of embryoid bodies composed of cells of three somatic lineages.

In some cases, stem cells (eg, ESCs or iPSCs) can be genetically modified to generate (eg, induce differentiation into) CD34+ hematopoietic stem cells. Stem cells can be genetically modified to express any of the heterologous polypeptides disclosed herein (eg, cytokines, receptors, etc.) before, after, or during induced differentiation of hematopoietic stem cells. Stem cells can be genetically modified to reduce the expression or activity of any of the endogenous genes or polypeptides (eg, cytokines, receptors, etc.) disclosed herein before, after, or during induced hematopoietic stem cell differentiation. In some instances, such genetically modified CD34+ hematopoietic stem cells are any of the engineered immune cells of the disclosure or a source thereof.

In some examples, stem cells disclosed herein can be cultured in media with ROCKi (Y-27632) (e.g., at about 10 micromolar (μM)), SCF (e.g., at about 40 nanogram/milliliter (ng/mL)) , VEGF (for example, in a medium of about 20 ng/mL) and BMP-4 (for example, in a medium of about 20 ng/mL) in APEL medium to differentiate into CD34+ hematopoietic stem cells.

In some cases, CD34+ hematopoietic stem cells can be induced (eg, genetically modified with one or more characteristics of any of the engineered immune cells of the disclosure) to differentiate into committed immune cells, such as T cells or NK cells. Thus, in some cases, the induced differentiation process produces any of the engineered NK cells of the disclosure.

In some examples, IL-3 (e.g., about 5 ng/mL), IL-7 (e.g., about 20 ng/mL), IL-15 (e.g., about 10 ng/mL), SCF (e.g., about 20 ng/mL) and Flt3L (for example, about 10 ng/mL), the genetically modified CD34+ hematopoietic stem cells were cultured to differentiate into CD45+ NK cells.

In some cases, CD45+ NK cells can be expanded in culture using a gas permeable rapid expansion (G-Rex) platform, eg, in media including IL-2, mbIL-21 aAPC.

In some cases, the iPSC-derived NK cells disclosed herein can be cultured with one or more heterologous cytokines including Il-2, IL-15, or IL-21. In some cases, the iPSC-derived NK cells disclosed herein can be cultured (eg, for cell expansion) with one or more heterologous cytokines selected from IL-2, IL-15, and IL-21. In some cases, the iPSC-derived NK cells disclosed herein can be combined with two or more heterologous cytokines selected from IL-2, IL-15, and IL-21 (e.g., IL-2 and IL-15, IL-21 -2 and IL-21, or IL-15 and IL-21) were cultured together simultaneously or sequentially in any order. In some cases, the iPSC-derived NK cells disclosed herein can be cultured with all of II-2, IL-15, and IL-21 simultaneously or sequentially in any order. F. _ Gene editing or delivery of genetic material

Gene editing moieties disclosed herein may include CRISPR-associated polypeptides (Cas), zinc finger nucleases (ZFNs), zinc finger-associated gene regulatory polypeptides, transcriptional initiation factor-like effector nucleases (TALENs), transcriptional initiation factor-like effector-associated genes Regulatory polypeptides, meganucleases, natural master transcription factors, epigenetic modifying enzymes, recombinases, flippases, transposases, RNA binding proteins (RBPs), Argonaute proteins, any derivatives thereof, any variants thereof, or any fragment. In some embodiments, the actuator moiety includes a Cas protein, and the system further includes a guide RNA (gRNA) complexed with the Cas protein. In some embodiments, the actuator moiety comprises a RBP complexed with a gRNA capable of forming a complex with the Cas protein. In some embodiments, the gRNA includes a targeting segment that exhibits at least 80% sequence identity to the target polynucleotide. In some embodiments, the Cas protein substantially lacks DNA cleavage activity.

In some cases, suitable gene editing moieties include CRISPR-associated (Cas) proteins or Cas nucleases, including Type I CRISPR-associated (Cas) polypeptides, Type II CRISPR-associated (Cas) polypeptides, Type III CRISPR-associated (Cas) polypeptides, Type III CRISPR-associated (Cas) polypeptides, (Cas) polypeptides, Type IV CRISPR-associated (Cas) polypeptides, Type V CRISPR-associated (Cas) polypeptides, and Type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFNs); transcriptional initiation factor-like effector nucleic acids Enzymes (TALENs); meganucleases; RNA-binding proteins (RBPs); CRISPR-associated RNA-binding proteins; recombinases; flippases; transposases; (aAgo) and eukaryotic Argonaute (eAgo)); any derivatives thereof, any variants thereof; and any fragments thereof.

Non-limiting examples of Cas proteins include: c2c1, C2c2, c2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, Cas1O, Cas1Od, CasF, CasG, CasH, Cpf1, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1 , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx1O, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1 , Csf2, Csf3, Csf4 and Cul966 and homologues or modified forms thereof.

In some cases, the gene editing moieties disclosed herein can be fused with additional functional moieties (e.g., to form fusion moieties), and non-limiting examples of the functions of the additional functional moieties can include: methyltransferase activity, demethylation Base enzyme activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, unwinding Enzyme activity, photolyase activity or glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristylation activity, remodeling activity, protease activity, oxidoreductase activity, transferase activity, Hydrolase activity, lyase activity, isomerase activity, synthase activity, synthetase activity and demyristylation activity. For example, a fusion protein can be a fusion of a Cas protein with part of an effector or repressor function.

Alternatively or additionally, gene editing (eg knock-in) or delivery of heterologous genetic material can be achieved. Other viral and non-viral based gene transfer methods can be used to introduce nucleic acids into host cells (eg, stem cells, hematopoietic stem cells, etc. disclosed herein). Such methods can be used to administer nucleic acids encoding polypeptide molecules of the present disclosure to cells in culture (or in a host organism). Viral vector delivery systems can include DNA and RNA viruses, which can have episomal or integrated genomes after delivery to cells. Non-viral vector delivery systems can include DNA plasmids, RNA (eg, transcripts of the vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes.

Systems based on RNA or DNA viruses can be used to target specific cells and transport viral payloads to the cell's nucleus. Viral vectors can be used to treat cells in vitro, and the modified cells can optionally be administered (ex vivo). Alternatively, the viral vector can be administered directly (in vivo) to the subject. Viral-based systems can include retroviral, lentiviral, adenoviral, adeno-associated viral and herpes simplex virus vectors for gene transfer. Integration in the host genome can occur by retroviral, lentiviral, and adeno-associated viral gene transfer methods, which can result in long-term expression of the inserted transgene.

Methods of non-viral delivery of nucleic acids may include lipofection, nucleofection, microinjection, biolistic, virosomes, liposomes, immunoliposomes, polycation or lipid-nucleic acid conjugates, naked DNA, artificial virions, and Reagent Enhanced Absorption of DNA. Cationic and neutral lipids for lipofection can be recognized using efficient receptors suitable for polynucleotides.

Alternatively or additionally, antisense oligonucleotides can be used to inhibit or silence target gene expression. Non-limiting examples of antisense oligonucleotides can include short hairpin RNA (shRNA), microRNA (miRNA), and small interfering RNA (siRNA). G. _ combination therapy

The engineered immune cells (eg, engineered NK cells) of the disclosure can be combined with co-therapeutic agents to treat a subject in need. In some cases, the engineered immune cells can be administered to the subject before, simultaneously with, or after the co-therapeutic agent is administered to the subject.

In one aspect, the present disclosure provides a composition comprising (a) any of the engineered immune cells disclosed herein (e.g., engineered NK cells) and (b) a combination therapeutic agent (i.e., a single therapeutic agent) ( For example, an antibody, such as an anti-CD20 antibody or an anti-PD1 antibody). In some cases, engineered immune cells may include one or more of: (i) a heterologous cytokine disclosed herein (eg, heterologous IL, such as IL-15), (ii) a heterologous cytokine disclosed herein for enhancing and (iii) a chimeric polypeptide receptor disclosed herein comprising an antigen-binding portion capable of binding an antigen. In some examples, the combination therapy includes an anti-CD20 antibody.

In some cases, engineered immune cells may include a heterologous cytokine disclosed herein (eg, IL-15) and one or both of: (ii) a CD16 variant for enhanced CD16 signaling, and (iii) ) A chimeric polypeptide receptor comprising an antigen binding portion.

In some cases, engineered immune cells can include CD16 variants for enhanced CD16 signaling and one or both of: (i) a heterologous cytokine (e.g., IL-15), and (iii) include Chimeric Polypeptide Receptors of Antigen Binding Portions.

In some cases, the engineered immune cell can include a chimeric polypeptide receptor that includes an antigen binding portion and one or both of: (i) a heterologous cytokine (e.g., IL-15), and (ii) CD16 variants for enhanced CD16 signaling.

Non-limiting examples of combination therapeutic agents may include: cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, agents used in radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for the treatment of cancer, For example, anti-CD20 antibodies, anti-PD1 antibodies (e.g. pembrolizumab) platelet-derived growth factor inhibitors (e.g. GLEEVEC™ (imatinib mesylate)), COX-2 inhibitors (e.g. celecoxib), Interferons, cytokines, antagonists (eg, neutralizing antibodies) that bind to one or more of the following targets PDGFR-β, BlyS, APRIL, BCMA receptors, TRAIL/Apo2, other bioactive agents and organic chemical agents, etc. .

The term "cytotoxic agent" generally refers to a substance that inhibits or prevents cellular function and/or causes cellular destruction. Non-limiting examples of cytotoxic agents can include: radioisotopes (e.g., radioisotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and Lu), chemotherapeutic agents such as methotrexate, doxorubicin Adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other embedded agents, enzymes and fragments thereof (such as nucleolytic enzymes), antibiotics and toxins (such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin).

Non-limiting examples of chemotherapeutic agents may include: alkylating agents such as thiotegua and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and pipoxafam; aziridine Pyridines such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimines and methylmelamines including hexamethylmelamine, triethylenemelamine , triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone )); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analog topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolamine lectin, and 9-aminocamptothecin); bryostatin; callystatin; CC- 1065 (including its adolaisine, kizellesine and bizelesine synthetic analogues); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (specifically nostocin 1 and candocin 8); dolastatin; duocarmycin (including synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; (pancratistatin); sarcodictyn; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, clophosphamide, estramustine, ifosfamide amine, dichloromethyldiethylamine, methoxymustine hydrochloride, melphalan, new enbixing, benzyl mustard cholesterol, prednimustine, cyclophosphamide, uramustine; nitroso Ureas such as carmustine, chlorurecin, formustine, lomustine, nimustine and ranimnustine; antibiotics such as enediyne antibiotics; danemycin ( dynemicins), including danemycin A; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclarithromycin (aclacinomysins), actinomycin, athramycin, azaserine, bleomycins, cactinomycin, carabicin, carmine, carcinophilus Carzinophilin, Chromomycin dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholinodoxorubicin, cyanogen Morpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, ethorubicin, idarubicin, macillomycin, silk Mitomycins such as mitomycin C, mycophenolic acid, nogamycin, olivine, pelomycin, potfiromycin, puromycin, quelamycin , rhodorubicin, streptoglobin, streptozotocin, tubercidin, ubenimex, netastatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folate analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercapto Purines, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azuridine, carmofur, arabinose Cytidine, dideoxyuridine, doxifluridine, enoxitabine, floxuridine; androgens such as calusterone, drostanolone propionate, cyclic Thiandrol, metandrostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trolosteine; folic acid supplements such as folinic acid; aceglatone; Aidophosphamide glycoside; aminolevulinic acid; eniluracil; amsacridine; bestrabucil; bisantrene; edatraxate; defofamine; Demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguanidine hydrazone; mitoxantrone; mopidanmol; Nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Pr oducts, Eugene, Oreg.); razoxane; rhizocin; ,2"-Trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine )); Urethane (urethan); Vindesine (ELDISINE®, FILDESIN®); Dacarbazine; Mannomustine; Dibromomannitol; Dibromodulcitol; Neo (gacytosine); arabinoside ("Ara-C"); thiotepa; taxanes, such as taxanes, including TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE without Cremophor ™, albumin-engineered nanoparticles of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.) and TAXOTERE® docetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine ( GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosf amide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; formyltetrahydrofolate; vinorelbine (NAVELBINE®); daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid ; capecitabine (XELODA®); any of the above pharmaceutically acceptable salts, acids or derivatives; and combinations of two or more of the above, such as CHOP (cyclophosphamide, doxorubicin, vinca Neospine and Prednisolone Combination Therapy), and FOLFOX (short for Oxaliplatin (ELOXATIN™) Combination of 5-FU and Leucovorin). Additional chemotherapeutic agents include cytotoxic agents useful as antibody drug conjugates such as the maytansinoids (eg DM1 ) and the auristatins MMAE and MMAF.

Examples of chemotherapeutic agents may also include "antihormonal agents" or "endocrine therapeutics" that act to modulate, reduce, block or inhibit the action of hormones that can promote cancer growth, and are usually in systemic form, or systemic therapy. They may be hormones themselves. Examples include antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), EVISTA® raloxifene, droloxifene, 4- Hydroxytamoxifen, travoxifene, raloxifene hydrochloride (keoxifene), LY117018, onapristone, and FARESTON® toremifene; antiprogestins; estrogen receptor down-regulators (ERDs) ; agents that suppress or shut down ovarian function, such as luteinizing hormone-releasing hormone (LHRH) agonists such as LUPRON® and ELIGARD) leuprolide acetate, goserelin acetate, buserelin acetate, and triptorelin (tripterelin); other antiandrogens such as flutamide, nilutamide, and bicalutamide; and aromatase inhibitors that inhibit aromatase (which regulates estrogen production in the adrenal gland), For example, 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® trazole and ARIMIDEX® anastrozole. Additionally, this definition of a chemotherapeutic agent includes bisphosphonates such as clodronate (e.g. BONEFOS® or OSTAC®), DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid/zoledronic acid salt, FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, or ACTONEL® risedronate; and troxacitabine (1,3-di oxolane nucleoside cytosine analogs); antisense oligonucleotides, especially those that inhibit genes in signaling pathways associated with abnormal cell proliferation, such as PKC-α, Raf, H-Ras, and epidermal growth factor Antisense oligonucleotides expressed by the receptor (EGFR); vaccines such as THERATOPE® vaccine and gene therapy vaccines such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; LURTOTECAN® topoisomerase 1 inhibitors; ABARELIX® rmRH; lapatinib ditosylate (a small molecule inhibitor of ErbB-2 and EGFR dual tyrosine kinases, also known as GW572016); and any of the pharmaceutically acceptable salts, acids, or derivatives of the foregoing.

Examples of chemotherapeutic agents may also include antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec), pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab ( Bexxar, Corixia) and an antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth). Other humanized monoclonal antibodies that have therapeutic potential as agents in combination with the compounds of the invention include: apratuzumab, atezolizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cilizumab, Tocilizumab pegylated, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, feMzumab, fontolizumab, gemtuzumab ozo Mixing, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motuzumab, motovizumab, natalizumab, niger Tocilizumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, peckizumab, ralivizumab, ranibizumab, reslivizumab, reslivizumab, resyvizumab, Rovetuzumab, ruplizumab, cilostuzumab, siplizumab, sortuzumab, tacatuzumab tetraxetan, tacatuzumab, tacatuzumab, tefibazumab, tocilizumab, toralizumab , tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and anti-interleukin 12 (ABT-874/J695, Wyeth Research and Abbott Laboratories) (this is a genetically modified to recognize interleukin 12 p40 recombinant full-length IgG1λ antibody with only human sequences of the protein).

Examples of chemotherapeutic agents may also include "tyrosine kinase inhibitors" such as EGFR targeting agents (e.g. small molecules, antibodies, etc.); small molecule HER2 tyrosine kinase inhibitors such as TAK165 (available from Takeda); CP -724,714 (oral selective inhibitors of the ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual HER inhibitors such as EKB-569 that preferentially binds EGFR but inhibits cells overexpressing both HER2 and EGFR (available from Wyeth); lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such as canertinib (canertinib) (CI-1033; Pharmacia); Raf-1 inhibitors, such as the antisense reagent ISIS-5132 (available from ISIS Pharmaceuticals) that inhibits Raf-1 signaling; non-HER-targeted TK inhibitors, such as formazan Imatinib sulfonate (GLEEVEC®, available from Glaxo SmithKline); multitargeted tyrosine kinase inhibitors such as sunitinib (SUTENT®, available from Pfizer); VEGF receptor tyrosine kinase inhibitors , such as vatalanib (PTK787/ZK222584, available from Novartis/Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, such as PD 153035, 4-( 3-Chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H - pyrrolo[2,3-d]pyrimidines; curcumin (diferulic methane, 4,5-bis(4-fluoroanilino)phthalimide); tyrosine containing nitrothiophene moiety Tyrphostins; PD-0183805 (Warner-Lamber); antisense molecules (eg, those that bind to HER-encoding nucleic acids); quinoxalines (US Pat. No. 5,804,396); tryphostins (U.S. Patent No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-HER inhibitors such as CI-1033 (Pfizer); Affinitac (ISIS3521; Isis/Lilly); Ni (GLEEVEC®); PKI166 (Novartis); G W2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Sematinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); Mycin (sirolimus, RAPAMUNE®).

Examples of chemotherapeutic agents may also include: dexamethasone, interferon, colchicine, chlorpheniramine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol , amifostine, arsenic trioxide, asparaginase, live BCG, bevacizimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, Denileukin, dextropropylimide, epoetin alfa, erlotinib, filgrastim, histrelin acetate, imomolide Anti (ibritumomab), interferon α-2a, interferon α-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, o Preleukin, palivermin, pamidronate, pegasin, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, Porfimer sodium, quinacrine, rasburicase, sargragrastim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate and zoledronic acid, and pharmaceutically acceptable salts thereof.

Examples of chemotherapeutic agents may also include hydrocortisone, hydrocortisone acetate, cortisone acetate, ticortisone pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide , budesonide, desonide, fluocinolone, fluocinolone, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocorolone, hydrocortisone-17-butyl hydrocortisone-17-pentanoate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone- 17-butyrate, clobetasol-17-propionate, fluocorone hexanoate, flucorone pivalate, and flupredidine acetate: Immunoselective anti-inflammatory peptides (ImSAIDs) such as Phenylalanine-glutamine-glycine (FEG) and its D-isomer form (feG) (IMULAN BioTherapeutics, LLC); antirheumatic agents such as azathioprine, cyclosporine (cyclosporine A ), D-penicillamine, gold salts, hydroxychloroquine, leflunomideminocycline, sulfasalazine, tumor necrosis factor alpha (TNFα) blockers (such as etanercept (ENBREL®)), Infliximab (REMICADE®), adalimumab (HUMIRA®), beselizumab (CIMZIA®), golimumab (SIMPONI®), interleukin 1 (IL-1) inhibitor Blockers (such as anakinra (KINERET®)), T cell co-stimulation blockers (such as abatacept (ORENCIA®)), interleukin 6 (IL-6) blockers (such as tocilizumab anti (ACTEMERA®)); interleukin 13 (IL-13) blockers (e.g. lebrikizumab); interferon alpha (IFN) blockers (e.g. rontalizumab); β7 integrin blockers blockers (e.g. rhuMAbβ7); IgE pathway blockers (e.g. anti-M1 Prime); secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa/β2 blockers (e.g. anti-lymphotoxin alpha (LTa )); miscellaneous investigational agents (e.g., thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyltransferase inhibitors (L-739749, L-744832)); polyphenols , such as quercetin, resveratrol, picatanol, epigallocatechin gallate, theaflavins, flavanols, proanthocyanidins, betulinic acid and its derivatives; phagocytosis inhibitors, such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; acetylcamptothecin, scopolamine Lectins and 9-aminocamptothecin); podophyllotoxin; tegafur (UFTOR AL®); bexarotene (TARGRETIN®); bisphosphonates such as clodronate (eg, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, azole Ledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or Risedronate (ACTONEL®); and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine; guarifosin, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteasome inhibitors (e.g., PS341); CCI-779; tipifamib (R11577); orafenib, ABT510; oblimersen sodium) (GENASENSE®); picogenin; farnesyltransferase inhibitors, such as lonafamib (SCH 6636, SARASAR™); and any pharmaceutically acceptable salt, acid, or derivative thereof; or more combinations.

The term "growth inhibitory agent" generally refers to a compound or composition that inhibits the growth and/or proliferation of cells (eg, cells whose growth is dependent on PD-L1 expression) in vitro or in vivo. A growth inhibitory agent may be one that significantly reduces the percentage of cells in S phase. Non-limiting examples of growth inhibitory agents include agents that block cell cycle progression (outside of S phase), such as agents that induce G1 arrest and M phase arrest. Classical M-phase blockers include vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as the anthracycline doxorubicin ((8S-cis)- 10-[(3-Amino-2,3,6-trideoxy-α-L-lyxosyl-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6 ,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthonaphthalenedione), epirubicin, daunorubicin, etoposide and bleomycin . Those agents that block G1 also spill over into S-phase arrest, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, nitrogen mustard, cisplatin, methotrexate, 5-fluorouracil, and Cytarabine (Ara-C). Taxanes (paclitaxel and docetaxel) are anticancer drugs derived from taxanes. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote microtubule assembly of tubulin dimers and stabilize microtubules by preventing depolymerization, which leads to inhibition of mitosis of cells. H. _ Instructions

Engineered immune cells (eg, engineered NK cells) of the disclosure can be used (eg, administered) to treat a subject in need. A subject may have or may be suspected of having a condition such as a disease (eg, cancer, tumor, tissue degeneration, fibrosis, etc.). Cells (eg, stem cells or committed adult cells) can be obtained from a subject, and such cells can be cultured ex vivo and genetically modified to produce any of the subject engineered immune cells (eg, any engineered NK cell) disclosed herein. The engineered immune cells can then be administered to the subject for adaptive immunotherapy.

At least or at most about 1 dose, at least or at most about 2 doses, at least or at most about 3 doses, at least or at most about 4 doses, at least or at most about 5 doses, at least or at most about 6 doses, At least or at most about 7 doses, at least or at most about 8 doses, at least or at most about 9 doses, or at least or at most about 10 doses of the population of engineered immune cells (e.g., engineered NK cells) of the present disclosure The object is treated (eg administered).

In one aspect, the present disclosure provides a method comprising: (a) obtaining a cell from a subject; and (b) producing any of the engineered immune cells disclosed herein (eg, engineered NK cells) from the cell. In some cases, the cells obtained from the article are ESCs. In some cases, cells (eg, fibroblasts, eg, adult dermal fibroblasts) obtained from the subject are modified and converted into iPSCs.

In one aspect, the present disclosure provides a method comprising administering to a subject in need thereof a population of NK cells comprising any of the engineered immune cells (eg, engineered NK cells) disclosed herein. In some cases, the method can further comprise administering to the subject a co-therapeutic agent (eg, a chemotherapeutic agent, an anti-CD20 antibody, etc.).

In one aspect, the present disclosure provides a method comprising administering any one of the compositions disclosed herein to a subject in need thereof. In some cases, a composition can include (i) any of the engineered immune cells disclosed herein (eg, engineered NK cells), and (ii) a combination therapeutic agent (eg, a chemotherapeutic agent, an anti-CD20 antibody, etc.).

Any of the methods disclosed herein can be used to treat a target cell, target tissue, target condition, or target disease of a subject.

The disease of interest may be a viral, bacterial, and/or parasitic infection; an inflammatory and/or autoimmune disease; or a neoplasm, such as cancer and/or tumor.

Target cells can be diseased cells. Diseased cells may have altered metabolism, gene expression, and/or morphological characteristics. Diseased cells can be cancer cells, diabetic cells and apoptotic cells. A diseased cell can be a cell from a diseased object. Exemplary diseases may include blood disorders, cancer, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, heart disease, and the like.

A variety of target cells can be killed using any of the engineered immune cells (eg, engineered NK cells) disclosed herein. Target cells can include a variety of cell types. Target cells can be in vitro. Target cells can be in vivo. Target cells can be ex vivo. Target cells can be isolated cells. A target cell may be a cell in an organism. A target cell can be an organism. Target cells can be cells in cell culture. The target cell can be one of a collection of cells. Target cells can be mammalian cells or derived from mammalian cells. The target cell can be a rodent cell or be derived from a rodent cell. Target cells can be human cells or derived from human cells. Target cells can be prokaryotic cells or derived from prokaryotic cells. The target cell may be a bacterial cell or may be derived from a bacterial cell. The target cell may be an archaeal cell or be derived from an archaeal cell. Target cells can be eukaryotic cells or derived from eukaryotic cells. Target cells can be pluripotent stem cells. The target cell can be a plant cell or be derived from a plant cell. Target cells can be animal cells or derived from animal cells. The target cell may be an invertebrate cell or be derived from an invertebrate cell. The target cell can be a vertebrate cell or be derived from a vertebrate cell. The target cell may be a microbial cell or be derived from a microbial cell. The target cell may be a fungal cell or be derived from a fungal cell. Target cells can be from specific organs or tissues.

Target cells can be stem cells or progenitor cells. Target cells can include stem cells (eg, adult stem cells, embryonic stem cells, induced pluripotent stem (iPS) cells) and progenitor cells (eg, cardiac progenitor cells, neural progenitor cells, etc.). Target cells can include mammalian stem cells and progenitor cells, including rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, and the like. Clonal cells can include progeny of the cells. Target cells can include target nucleic acids. Target cells can be in living organisms. Target cells can be genetically modified cells. A target cell can be a host cell.

Target cells may be totipotent stem cells, however, in some embodiments of the present disclosure, the term "cell" may be used but may not denote totipotent stem cells. The target cell may be a plant cell, but in some embodiments of the present disclosure, the term "cell" may be used but may not refer to a plant cell. Target cells can be pluripotent cells. For example, a target cell may be a multipotent hematopoietic cell that can differentiate into other cells in the hematopoietic cell lineage, but may not be able to differentiate into any other non-hematopoietic cells. Target cells may be capable of developing into whole organisms. A target cell may or may not be able to develop into a whole organism. Target cells can be whole organisms.

Target cells can be primary cells. For example, a culture of primary cells may be passaged 0, 1, 2, 4, 5, 10, 15 or more times. A cell may be a unicellular organism. Cells can be grown in culture.

Target cells can be diseased cells. Diseased cells may have altered metabolism, gene expression, and/or morphological characteristics. Diseased cells can be cancer cells, diabetic cells and apoptotic cells. A diseased cell can be a cell from a diseased object. Exemplary diseases may include blood disorders, cancer, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, heart disease, and the like.

If the target cells are primary cells, they can be harvested from the individual by any means. For example, leukocytes can be harvested by apheresis, leukapheresis, density gradient separation, and the like. Cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be harvested by biopsy. Appropriate solutions may be used to disperse or suspend harvested cells. Such a solution may typically be a balanced salt solution (e.g., physiological saline, phosphate-buffered saline (PBS), Hank's balanced salt solution, etc.), conveniently supplemented with fetal bovine serum or other naturally occurring factors and low concentrations of acceptable buffer. Buffers may include HEPES, phosphate buffer, lactate buffer, and the like. Cells can be used immediately, or they can be stored (eg, by freezing). Frozen cells can be thawed and reused. Cells can be frozen in DMSO, serum, media buffer (eg, 10% DMSO, 50% serum, 40% buffered media), and/or some other such common solution for preserving cells at freezing temperatures.

Non-limiting examples of cells that may be target cells include, but are not limited to: lymphoid cells such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, helper T cells), natural killer cells , cytokine-induced killer (CIK) cells (see for example US20080241194); myeloid cells such as granulocytes (basophils, eosinophils, neutrophils/multilobed neutrophils), Monocytes/macrophages, erythrocytes (reticulocytes), mast cells, platelets/megakaryocytes, dendritic cells; cells from the endocrine system, including thyroid (thyroid epithelium, parafollicular cells), parathyroid (parathyroid chief cells, eosinophils), adrenal (chromaffin cells), pineal (pineal cells) cells; cells of the nervous system, including glial cells (astrocytes, microglia), giant cell neurosecretory cells, stellate cells, Bertscher cells, and pituitary gland (gonadotrophs, corticotropin-secreting cells, thyrotrophs, somatotrophs, prolactin-secreting cells); cells of the respiratory system, Including lung cells (type I pneumocytes, type II pneumocytes), Clara cells, goblet cells, dust cells; cells of the circulatory system, including cardiomyocytes, pericytes; cells of the digestive system, including stomach (gastric chief cells, parietal cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, S cells; enteroendocrine cells, including enterochromaffin cells, APUD cells, liver (hepatocytes, Kupffer cells), cartilage/bone/muscle; bone cells, including osteoblasts, osteocytes, osteoclasts, teeth (cementoblasts, ameloblasts); chondrocytes, including chondroblasts, chondrocytes; skin cells , including trichocytes, keratinocytes, melanocytes (nevus cells); muscle cells, including myocytes; urinary system cells, including podocytes, juxtaglomerular cells, and mesangial/renal cells Extraglomerular mesangial cells, renal proximal tubule brush border cells, macula densa cells; reproductive system cells including spermatozoa, Sertoli cells, Leydig cells, eggs; and other cells including adipocytes, fibroblasts cells, tenocytes, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), keratinocytes of fingernails and toenails, nail bed basal cells (stem cells), medullary hair shaft cells, cortical hair shaft cells, surface Hair shaft cells, epidermal root sheath cells, Huxley layer root sheath cells, Henle layer root sheath cells, outer root sheath cells, hair matrix cells (stem cells), moisture stratified barrier epithelial cells, (cornea, tongue, oral cavity, Surface epithelium of stratified squamous epithelium (of esophagus, anal canal, distal urethra, and vagina), basal cells (stem cells) of epithelium (of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina), urethra Epithelial cells (lining the bladder and urethra), exocrine epithelial cells, salivary gland mucous cells (polysaccharide-rich secretions), salivary gland serous cells (glycoproteinase-rich secretion), von Ebner gland cells in the tongue (taste bud wash), mammary gland cells (milk secretion), lacrimal gland cells (tear secretion), cerumen gland cells in the ear (wax secretion Eccrine cells), eccrine dark cells (glycoprotein secretions), eccrine clear cells (small molecule secretions), apocrine sweat gland cells (odourous secretions, sensitive to sex hormones), eyelid mole gland cells (specialized sweat glands ), sebocytes (lipid-rich sebum secretion), nasal Bowman's glands (olfactory epithelium washings), duodenal Bruner's glands (enzymes and alkaline mucus), seminal vesicles (secrete semen components, including for sperm motility fructose), prostate cells (secretion of seminal fluid), bulbar urethral gland cells (mucus secretion), Bartholin cells (vaginal lubricating secretion), Littley gland cells (mucus secretion), endometrial cells (carbohydrate secretions), isolated goblet cells of the respiratory and gastrointestinal tracts (mucus secretions), mucous cells of the gastric mucosa (mucus secretions), gastric gland zymogen cells (pepsinogen ), pancreatic acinar cells (bicarbonate and digestive enzyme secretion), intestinal Paneth cells (lysozyme secretion), lung type II pneumocytes (surfactant secretion), lung Clara cells, hormone secretion cells, anterior pituitary cells, somatotroph cells, prolactin hormone secreting cells, thyrotroph cells, gonadotroph cells, corticotroph cells, middle pituitary cells, magnocellular neurosecretory cells, intestinal and respiratory tract cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid principal cells, eosinophils, adrenal cells, chromaffin cells, Leydig cells, follicular endometrial cells, luteal cells of ruptured follicles, Granular luteal cells, alveolar luteal cells, juxtaglomerular cells (renin secretion), macula densa cells of the kidney, metabolic and storage cells, barrier function cells (lung, intestine, exocrine glands, and genitourinary tract), kidney, Type I pneumocytes (lined air spaces in the lungs), pancreatic duct cells (alveolar heart cells), non-striated duct cells (of sweat glands, salivary glands, breast, etc.), duct cells (of seminal vesicles, prostate, etc.), duct cells that close internal body cavities Epithelial cells, ciliated cells with propulsion function, extracellular matrix secreting cells, contractile cells; skeletal muscle cells, stem cells, cardiomyocytes, blood and immune system cells, erythrocytes (red blood cells), megakaryocytes (platelet precursors), monocytes , connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglia (in central nervous system), blood and immune system ( Various types) of neutrophils, eosinophils, basophils, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells , reticulocytes, stem cells and committed progenitor cells, pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory sensor cells , autonomic neuronal cells, sensory organs and peripheral neuronal support cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelium, germ cells, oogonia/oocytes , spermatids, spermatocytes, spermatogonia (spermatocyte stem cells), spermatozoa, feeder cells, follicular cells, Sertoli cells (in the testis), thymic epithelial cells, mesenchymal cells, and mesenchymal kidney cells.

Of particular interest are cancer cells. In some embodiments, the target cells are cancer cells. Non-limiting examples of cancer cells include cancer cells including: acanthoma, acinar cell carcinoma, acoustic neuroma, acral melanoma, acral hidradenoma, acute eosinophilic leukemia, acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, enamel epithelioma, adenocarcinoma, adenoid cyst Sexual carcinoma, adenoma, odontogenic adenomatoid tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft tissue sarcoma, ameloblastic fibroma, anal Carcinoma, anaplastic large cell lymphoma, anaplastic thyroid carcinoma, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix carcinoma, astrocytoma, atypical teratoid rhabdoid tumor, basal Cell carcinoma, basaloid carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, biliary tract carcinoma, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brenner's tumor, bronchial neoplasm, bronchioloalveolar carcinoma, brown tumor, Burkitt's lymphoma, carcinoma of unknown primary (cancer), carcinoid tumor, carcinoma, carcinoma in situ, penile carcinoma, unknown primary Carcinoma, carcinosarcoma, giant lymph node hyperplasia, central nervous system embryonal tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, cholangiocarcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, Choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disease, chronic neutrophil leukemia, clear cell neoplasm, colon cancer, colorectal cancer, craniopharynx cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma protuberans, dermoid cyst, hyperplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, embryonal carcinoma , endometrial sinus tumor, endometrial cancer, endometrial uterine cancer, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymal blastoma, ependymoma, epithelioid sarcoma, erythroleukemia , Esophageal cancer, Olfactory neuroblastoma, Ewing family tumors, Ewing family sarcoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic cholangiocarcinoma, extramammary Paget's disease, fallopian tube cancer , Fetus in Fetus, Fibroma, Fibrosarcoma, Follicular Lymphoma, Follicular Thyroid Cancer, Gallbladder Cancer, Gallbladder Cancer, Ganglioglioma, Gangliocytoma, Gastric Cancer, Gastric Lymphoma, Gastrointestinal Cancer, Gastrointestinal Tract carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell tumor, germ cell tumor, gestational choriocarcinoma, gestational trophoblastic tumor, giant cell tumor of bone, glioblastoma multiforme, glioma , glioma, glomus tumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, head and neck cancer, heart cancer, hemangioblastoma, hemangiopericytoma , angiosarcoma, hematologic malignancies Tumors, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast-ovarian cancer syndrome, Hodgkin's lymphoma, Hodgkin's lymphoma, hypopharyngeal carcinoma, hypothalamic glioma, inflammatory breast cancer, eye Intrinsic melanoma, islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, kidney cancer, Kratskin's tumor, Krucken Berger's tumor, laryngeal cancer, laryngeal cancer, lentiginous melanoma, leukemia, leukaemia, lip and mouth cancer, liposarcoma, lung cancer, luteinoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoid leukemia , lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, malignant salamander Newt tumor, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid carcinoma, medulloblastoma, medulloblastoma, medullary epithelioma, melanoma, melanoma, meninges tumor, Merkel cell carcinoma, mesothelioma, mesothelioma, occult primary metastatic squamous neck carcinoma, metastatic urothelial carcinoma, mixed Müllerian tumor, monocytic leukemia, oral cancer, mucinous Sexual neoplasms, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma, mycosis fungoides, mycosis fungoides, myelodysplastic disease, myelodysplastic syndrome, myeloid leukemia, myeloid sarcoma, Myeloproliferative disease, myxoma, nasal cavity carcinoma, nasopharyngeal carcinoma, nasopharyngeal carcinoma, neoplasm, neuronoma, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgies Gold lymphoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, eye tumors, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, oral cancer , oral cavity cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, breast Paget's disease, lung superior sulcus tumor, pancreatic cancer, pancreatic cancer , papillary thyroid carcinoma, papillomatosis, paraganglioma, paranasal sinus carcinoma, parathyroid carcinoma, penile carcinoma, perivascular epithelioid cell tumor, pharyngeal carcinoma, pheochromocytoma, pineal differentiation Solid tumors, pinealoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasmacytoma, pleuropulmonary blastoma, multiple embryonal tumor, precursor T lymphoblastic lymphoma, primary central nervous system Lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal carcinoma, primitive neuroectodermal tumor, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, Respiratory cancer involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, sacrococcygeal teratoma, salivary gland carcinoma, sarcoma, schwannoma, sebaceous gland carcinoma, secondary neoplasms, seminoma, serous adenoma, branch Aid - Stromal cell tumor, sex cord stromal cell tumor, Sezary syndrome, signet ring cell carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small bowel cancer, soft tissue sarcoma , somatostatoma, sooty warts, spinal cord tumors, spinal cord tumors, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, supratentorial primitive neuroectodermal tumor, surface epithelial stromal tumor, Synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, end-stage lymphoma, testicular cancer, alveoli Cancer of the larynx, thymus, thymoma, thyroid, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal carcinoma, urethral carcinoma, genitourinary tract neoplasms, uterine sarcoma, uveal melanoma, vaginal carcinoma, Verner Morrison Syndrome, Verrucous Carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenström's Macroglobulinemia, Warthin's Tumor, Wilms' Tumor, and combinations thereof. In some embodiments, the targeted cancer cells represent a subpopulation within a population of cancer cells, such as cancer stem cells. In some embodiments, the cancer is of the hematopoietic lineage, such as lymphoma. The antigen may be a tumor-associated antigen.

In some instances, a target cell (eg, a B cell) disclosed herein is associated or suspected of being associated with an autoimmune disease. A subject treated with any one of the engineered immune cells (eg, engineered NK cells) of the present disclosure may have or may be suspected of having an autoimmune disease.

Non-limiting examples of autoimmune diseases may include: acute diffuse encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, allergic asthma, allergic Rhinitis, alopecia areata, amyloidosis, ankylosing spondylitis, antibody-mediated transplant rejection, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, Autoimmune autonomic dysfunction, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immune deficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune pancreatitis, autoimmune Retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal and neuronal neuropathy, Barrow's disease, Behcet's disease, bullous pemphigoid, Cardiomyopathy, giant lymphadenopathy, celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss Syndrome, Cicatricial Pemphigoid/Benign Mucosal Pemphigoid, Crohn's Disease, Cogans Syndrome, Cold Agglutinin Disease, Congenital Heart Block, Coxsackie's Myocarditis, CREST Disease, Idiopathic Mixed Cryoglobulinemia, demyelinating neuropathy, dermatitis herpetiformis, dermatomyositis, Devick's disease (neuromyelitis optica), discoid lupus, Dresler syndrome, endometriosis , eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), glomerulonephritis, pulmonary Hemorrhage-nephritic syndrome, granulomatosis with polyangiitis (GPA), Graves' disease, Guillain-Bali syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, allergic purpura, herpes of pregnancy, hypogamma Proteinemia, hypergammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunomodulatory lipoproteins, inclusion body myositis, inflammatory bowel disease, insulin-dependent Diabetes mellitus (type 1), interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, woody conjunctivitis , linear IgA disease (LAD), lupus (SLE), Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), monoclonal gammopathy of undetermined significance (MGUS), Murren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's), neutropenia, ocular cicatricial pemphigoid, optic neuritis , recurrent rheumatic disease, PANDAS (pediatric autoimmune neuropsychiatric disease associated with streptococcal bacteria), paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonage-Turner syndrome, pars plana (peripheral uveitis), pemphigus, peripheral nerve Lesions, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyinflammation nodosa, autoimmune polyglandular syndrome types I, II, and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, gangrenous pus Dermatosis, pure red cell aplasia, Raynaud's phenomenon, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis , sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff man syndrome, subacute bacterial endocarditis (SBE), Susak's syndrome, Sympathetic ophthalmia, Taurus arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease ( UCTD), uveitis, vasculitis, vesicular bullous dermatosis, vitiligo, Waldenström macroglobulinemia (WM), and Wegener's granulomatosis (Granuloma with polyangiitis (GPA)).

In some instances, the autoimmune disease comprises one or more members selected from the group consisting of rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus (lupus or SLE), myasthenia gravis, multiple Sclerosis, scleroderma, Addison's disease, bullous pemphigoid, pemphigus vulgaris, Guillain-Barré syndrome, Sjogren's syndrome, dermatomyositis, thrombotic thrombocytopenic purpura, hypergamma Proteinemia, monoclonal gammopathy of undetermined significance (MGUS), Waldenström macroglobulinemia (WM), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), Hashimoto's encephalopathy (HE), Hashimoto's Thyroiditis, Graves' disease, Wegener's granulomatosis, and antibody-mediated transplant rejection (eg, for tissue transplants, such as kidney transplants). In examples, the autoimmune disease may be type 1 diabetes, lupus or rheumatoid arthritis.

In some instances, the target disease is acute myeloid leukemia (AML). For example, any engineered immune cell (for example, engineered NK cell) disclosed herein can be administered to an object in need to treat AML, and the engineered immune cell includes one or more of the following: (i) comprising: A chimeric polypeptide receptor of an antigen-binding domain that binds an antigen disclosed herein (eg, CD33), (ii) a heterologous cytokine disclosed herein (eg, IL-15), and (iii) a method disclosed herein for enhancing CD16 variants of CD16 signaling.

In some instances, the target disease is non-Hodgkin's lymphoma (NHL).

In some instances, the target disease is chronic lymphocytic leukemia (CLL).

In some instances, the disease of interest is B-cell leukemia (BCL). For example, any engineered immune cell disclosed herein (for example, engineered NK cell) may be administered to an object in need to treat BCL, and the engineered immune cell includes one or more of the following: (i) A chimeric polypeptide receptor comprising an antigen binding domain capable of binding CD19, (ii) a heterologous cytokine disclosed herein (e.g., IL-15), and (iii) a CD16 disclosed herein for enhanced CD16 signaling Variants.

In some instances, the target disease is non-small cell lung cancer (NSCLC).

In some instances, target cells form tumors (ie, solid tumors). Tumors treated with the methods herein can result in stable tumor growth (eg, one or more tumors that do not increase in size by more than 1%, 5%, 10%, 15%, or 20%, and/or do not metastasize). In some instances, the tumor remains stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks. In some instances, the tumor remains stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months. In some instances, the tumor remains stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years. In some instances, the size of the tumor or the number of tumor cells is reduced by at least about 5%, 10%, 15%, 20%, 25, 30%, 35%, 40%, 45%, 50%, 55%, 60% , 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some instances, tumors were completely eliminated or reduced below detection levels. In some instances, the subject remains tumor-free (eg, in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks after treatment. In some instances, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months after treatment. In some instances, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years after treatment. [Implementation] Example 1 : Engineered NK cells

Table 1 shows examples of engineered NK cells with and without genetic modification, and possible functional and therapeutic indications. Examples of therapeutic indications may include acute myeloid leukemia (AML), multiple myeloma (MM), myelodysplastic syndrome (MDS), B-cell leukemia, T-cell leukemia, solid tumors, and blood cancers. Table 1 serial number genetic modification Function Treatment Indications 1 No Non-engineered sNK AML, MM, MDS 2 aCD19-CAR + IL15 + hnCD16 Target Specificity + Persistence + ADCC B cell leukemia +/- anti-CD20 3 aCD19-CAR + IL15 + hnCD16 + i-caspase 9 Target Specificity + Persistence + ADCC + Safety Switch B cell leukemia +/- anti-CD20 4 hnCD16+IL15 ADCC + Persistence Solid Tumor + Anti-PD-1 5 hnCD16-CD64+IL15 ADCC + Persistence Solid Tumor + Anti-PD-1 6 hnCD16+IL15+CD38-KO ADCC + Persistence Multiple myeloma-/+ anti-CD38 7 aBCMA-CAR+hnCD16+IL15+CD38-KO Target Specificity + Persistence + ADCC Multiple myeloma-/+ anti-CD38 8 CAR + IL15 + hnCD16 + TME gene Target specificity + persistence + ADCC + enhanced activity Leukemia, Solid Tumors 9 CAR + IL15 + hnCD16 + TME gene + low immunity gene (for re-administration) Target specificity + persistence + ADCC + enhanced activity + low immunity (for re-dosing) Leukemia, Solid Tumors 10 CAR + IL15 + hnCD16 + TME gene + low immunity gene (for re-administration) + icaspase 9 Target specificity + persistence + ADCC + enhanced activity + low immunity (for re-dosing) + safety switch Leukemia, Solid Tumors Example 3 : Enhanced CD16 Signaling

To improve adaptive immunotherapy, NK cells can be engineered to exhibit enhanced CD16 signaling. Exemplary hnCD16 amino acid sequence:

SEQ ID NO. 1 : MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWFHVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK

SEQ IN NO. 23 MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFVIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVPTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK工程化 NK細胞：

NK92 cells were engineered to exhibit enhanced CD16 signaling. NK92 cells were engineered to express CD64/CD16A fusion protein (SEQ ID NO. 1) and CD16 variant (SEQ ID NO. 24) (ie hnCD16).

The resulting hnCD16 NK92 cells were validated by identifying enhanced expression of both CD16 (e.g., by anti-CD16-PE antibody) and CD64 (e.g., by anti-CD64-APC/AF700 antibody) using fluorescence-activated cell sorting (FACS), As shown in Figure 1A. Wild-type (WT) NK92 cells were used as controls.

The hnCD16 construct sequence may comprise "FHVS" (SEQ ID NO. 2). The hnCD16 construct sequence may comprise "WFHVS" (SEQ ID NO. 3). The hnCD16 construct sequence may comprise "FHVSF" (SEQ ID NO. 4). The hnCD16 construct sequence may comprise "WFHVSF" (SEQ ID NO. 5). The hnCD16 construct sequence may comprise "VWFHVSFC" (SEQ ID NO. 6). The hnCD16 construct sequence may comprise "PVWFHVSFCL" (SEQ ID NO. 7). The hnCD16 construct sequence may comprise "TPVWFHVSFCLV" (SEQ ID NO. 8). Enhanced Targeting:

hnCD16 Nk92 cells were cultured alone (unstimulated, control), or in the presence of K562 cells capable of priming NK cells (K562) or phorbol 12-myristate 13-acetate (PMA) for priming CD16 and induces its cleavage. The data showed that hnCD16 NK92 cells were highly resistant to priming-induced CD16a cleavage compared to peripheral blood (PB) NK cells as controls (Fig. 1B). For example, for hnCD16 NK92 cells, treatment with PMA slightly decreased the percentage of CD16+ cells from 92% to 85%, while the same treatment decreased the percentage of CD16+ cells from 96% to 25% (Figure 1B). Persistence of hnCD16 in hnCD16 NK92 cells was also confirmed using anti-CD64 antibody (Fig. 1C). In addition, it was observed that hnCD16 NK92 cells did not downregulate endogenous CD16 expression upon stimulation (eg, K652 or PMA) (Fig. ID and IE). Enhanced Antibody Combination Therapy

Target cells (Raji cells) were treated with (i) hnCD16 NK92 cells and (ii) anti-CD20 antibody or hIgG as controls. Data show that the combination of hnCD16 NK92 cells with anti-CD20 antibody induces increased target cell death (e.g., at least in part Death mediated by ADCC) (Figure 1F and 1G). Example 4 : Enhanced Survival and Persistence of NK Cells

To improve the durability of adaptive immunotherapy, NK cells can be engineered to contain at least (i) heterologous transcription factors (such as STATs) and (ii) endogenous cytokine receptors (such as endogenous IL receptors, For example, decreased expression or activity of IL-17R). Having a combination of (i) and (ii) in engineered NK cells can synergistically improve the persistence of engineered NK cells compared to having only one or neither of (i) and (ii). Engineered NK cells:

NK cells were generated from isolated ESCs or iPSCs. NK cells are engineered to express a heterologous STAT (eg, STAT3 and/or STAT5B). Genes encoding heterologous STATs are incorporated into the genome of NK cells by viral transduction or by the action of the gene editing moieties disclosed herein. NK cells were also engineered to exhibit reduced expression or activity of endogenous IL-17R (ie STAT3 ⁺ IL-17R ⁻ NK cells). NK cells with either (i) heterologous STATs and (ii) reduced expression or activity of IL-17R or unengineered NK cells were used as controls. In Vitro Survival and Persistence:

In the absence of exogenous cytokines, the engineered STAT3 ⁺ IL ^- 17R- NK cells can be cultured in vitro to evaluate the viability and growth (or proliferation ability) of the engineered STAT3 ⁺ IL ^- 17R- NK cells. NK cells were cultured for 3-6 weeks in medium without addition of exogenous cytokines. Compared with control cells, engineered STAT3 ⁺ IL ^- 17R- NK cells exhibited significantly higher NK cell numbers, indicating enhanced survival and persistence of engineered STAT3 ⁺ IL ^- 17R- NK cells in vitro. Pharmacokinetics (PK) in vivo :

Engineered STAT3 ⁺ IL ^- 17R− NK cells can be administered in NCG mice with a Raji xenograft model. NCG mice are triple immunodeficient and lack functional/mature T, B and NK cells, and have reduced macrophage and dendritic cell function to accommodate a xenograft model. Engineered STAT3 ⁺ IL-17R ^- NK cells and control cells were administered to respective Raji xenograft model mice via intravenous (IV) tail vein injection at a dose of approximately 1 x 10 ⁶ cells per animal. From about 7 days to about 28 days after infusion, mice injected with engineered STAT3 ⁺ IL ^- 17R- NK cells showed higher NK cell concentration in peripheral blood, indicating the effect of engineered STAT3 ⁺ IL ^- 17R- NK cells Enhanced in vivo survival and persistence. Embodiment 5 : engineered anti- CD19 NK cells

NK92 cells were engineered to express an anti-CD19 CAR and then cultured in the presence of CD19+ Raji cells to evaluate the targeting of Raji cells by engineered anti-CD19 NK cells. Wild-type (WT) NK92 cells were used as controls. Anti-CD19 CAR NK cells exhibited enhanced cytotoxicity against Raji cells (as determined by reduced numbers of surviving Raji cells) compared to controls (Figure 2A and 2B). Furthermore, anti-CD19 CAR NK cells exhibited enhanced expression of endogenous CD107a (indicative of cytotoxic granule release) compared to controls when cultured in the presence of Raji cells (Figure 2C and 2D). Furthermore, anti-CD19 CAR NK cells exhibited enhanced cytokine production (eg, IFN-γ and/or TNF-α production) compared to controls when cultured in the presence of Raji cells (Fig. 2E-2G). Embodiment 6 : Engineering hIL15 NK cells Engineering NK cells:

NK92 cells were engineered with (i) hIL-15 knock-in or (ii) hIL-15-hIL15R fusion polypeptide knock-in. Two variants of hIL-15-hIL15R fusion polypeptides were tested. The first variant (i.e., hIL15-IL15Ra fusion-1 or "fus1") was designed with a linker between hIL-15 and hIL15R comprising one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8 or more repeats) "GGGGS" (SEQ ID NO. 9), eg "GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS" (SEQ ID NO. 10). The second variant (i.e., hIL15-IL15Ra fusion-2 or "fus2") was designed with a linker between hIL-15 and hIL15R comprising one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8 or more repeats) "GGGGS" (SEQ ID NO.9) and one or more (for example, at least 1, 2, 3, 4, 5, 6, 7 , 8 or more repeats) "EGKSSGSGSESKST" (SEQ ID NO. 11), for example "EGKSSGSGSESKSTEGKSSGSGSESKSTGGGGS" (SEQ ID NO. 12). NK92 cells knocked in with either of the hIL-15-hIL15R fusion polypeptide variants were positive for hIL-15 (Fig. 3A).

In addition, engineered NK92 cells expressing either variant of the hIL-15-hIL15R fusion polypeptide for enhanced IL-15 signaling exhibit longer persistence. Western blot analysis showed that, compared with secreted IL-15 (IL15), IL-15-stimulated Increased phosphorylation of STAT5 (Fig. 3B). hIL15-IL15Ra 融合型 -1 序列(SEQ ID NO. 13)： MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL hIL15-IL15Ra 融合型 -2 序列(SEQ ID NO.14)： MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS EGKSSGSGSESKSTEGKSSGSGSESKSTGGGGS GITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL實施例 7 ： 體外 aCD19-CAR 、 hnCD16 和 IL -15 functional analysis. aCD19-CAR

As shown in Figure 4A, aCD19-CAR containing humanized anti-CD19 scFv, human CD8a hinge, CD8a transmembrane domain, 2B4 co-stimulatory and CD3ζ signaling domain was designed, wherein the anti-CD19 CAR used in aCD19-CAR The VH and VL sequences of the scFv are shown in SEQ ID NO.16 and SEQ IN NO.17.

NK92 cells were engineered to express aCD19-CAR and then cultured in the presence of K562 (CD19-negative) and K562-CD19 (CD19-positive) cells to evaluate the targeting of engineered aCD19-CAR NK cells to K562-CD19 cells. Wild-type (WT) NK92 cells were used as controls.

FACS analysis in Figure 6A shows the expression of aCD19-CAR at the cell surface of NK-CAR NK92 cells. Figure 6B shows that aCD19-CAR NK92 cells exhibited CD19 antigen-specific killing of K562-CD19 cells at effector:target (E:T) ratios of 0.2:1 and 1:1. Figure 6C shows that aCD19-CAR NK92 cells exhibit higher IFN upon antigen-specific stimulation of K562-CD19 cells compared to WT-NK92 cells at E:T ratios of 0.2:1 and 1:1 -γ generated. (* indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001 (Student's t-test)). hnCD16

As shown in Figure 4B, hnCD16 (SEQ ID NO. 23) was designed with a variant of human CD16 with F158V and S197P mutations. Then, NK92 cells were engineered to express hnCD16 to enhance CD16 signaling. The resulting NK92 cells were highly resistant to priming-induced CD16a cleavage and had excellent in vitro ADCC against CD19-expressing Raji cells.

Specifically, as shown in FIG. 7A , FACS analysis showed the expression of hnCD16 (human CD16 variant with F158V and S197P mutations) at the cell surface of NK92 cells. Figure 7B shows that hnCD16-expressing NK92 cells are highly resistant to priming-induced CD16a cleavage, and PMA/ionomycin was used as a positive control for stimulation. Figure 7C shows that hnCD16 expressing NK92 cells showed superior in vitro ADCC against CD19 expressing Raji cells at different E:T ratios. IL-15

Figure 4C exemplarily shows a design denoted IL-15-RF (membrane-bound heterologous IL-15) comprising IL-15 and the IL15 receptor. NK92 cells were engineered to express IL-15-RF (hlL15-IL15Ra fusion-1 as described in Example 6, SEQ ID No. 13).

As shown in Figure 8A, IL-15-RF was expressed and anchored on the surface of NK92 cells. The results of Western blot analysis in Figure 8B also showed enhanced phosphorylation of STAT5 and STAT3 stimulated by IL-15, indicating that in NK92 cells expressing IL-15-RF and secreted IL-15 (SEQ ID NO. 24) Initiation of the downstream pathway of IL-15. WT NK92 cells added with exogenous IL-15 were used as positive control. Embodiment 8 . Design of NK-019 constructs containing aCD19-CAR , hnCD16 , and IL-15RF , and expression of NK-019 constructs in NK92 cells.

An exemplary design of the NK-019 construct comprising aCD19-CAR, hnCD16 and IL-15 is shown in Figure 5A. Then, NK92 cells were transfected with three different NK-019 constructs: (1) NK-019w and NK-019 constructs comprising aCD19-CAR, hnCD16 and secreted IL-15 (SEQ ID No. 25); (2) NK-019wf1: NK-019 construct comprising aCD19-CAR, hnCD16 and hIL15-IL15Ra fusion-1 (SEQ ID No. 13); (3) NK-019wf2: NK-019 construct comprising aCD19-CAR, hnCD16 and hIL15-IL15Ra fusion-2 (SEQ ID No.14).

After transfection, the expressions of aCD19-CAR, hnCD16 and IL-15 components in NK92 cells were detected by FAC, respectively. As shown in Figure 9, the aCD19-CAR, hnCD16 and IL-15 components of the NK019 construct were successfully expressed simultaneously on NK92 cells. Embodiment 9 . Efficacy of NK cells expressing NK-019 against CD19 expressing target cells.

In efficacy assays, K562 (CD19 negative) and K562-CD19 (CD19 positive) cells were used as target cells.

Specifically, NK92 cells were transfected with NK-019w, NK-019wf1, and NK-019wf2, and then cultured in the presence of K562 (CD19-negative) and K562-CD19 (CD19-positive) cells to evaluate the effect of engineered NK cells on K562-CD19 Cell targeting. Wild-type (WT) NK92 cells were used as controls. As shown in Figure 10, NK92 cells transfected with different NK-019 constructs all showed hCD19-antigen-specific cytolytic ability to K562-CD19 cells, among which NK92 cells transfected with NK-019w showed the highest cell lytic activity.

Furthermore, as shown in Figure 11A, when cultured in the presence of K562 cells, NK92 cells transfected with NK-019w, NK-019wf1 and NK-019wf2 exhibited enhanced expression of endogenous CD107a compared to controls, Indicates the release of cytotoxic granules by NK cells. Furthermore, as shown in FIG. 11B , NK92 cells transfected with NK-019w, NK-019wf1 and NK-019wf2 exhibited enhanced IFN-γ production compared to controls when cultured in the presence of K562 cells. Embodiment 10 . Antitumor activity of NK-019 NK92 cells in the Raji-NCG mouse model .

NCG mice were irradiated with 100 cGy on day -2 and injected intravenously with luciferase-expressing Raji-Luc cells ( ^5x105 cells/mouse) on day -1. On day 0, mice were divided into five groups, four of which ^were injected intravenously (5x10 cells/mouse) with NK92 cells, and cells expressing NK-019w (w-NK019-NK92), NK-019wf1 (wf1- NK92 cells of NK019-NK92) and NK-019wf2 (wf2-NK019-NK92), as shown in Figure 12A.

On days 7, 14, 21, and 28, tumor cell expansion in mice was visualized by in vivo imaging and analyzed based on fluorescence intensity. As shown in Figures 12B and 12C, at day 12, NK92 cells expressing NK-019w, NK-019wf1, and NK-019wf2 significantly inhibited the expansion of CD19-positive tumors in NCG mice, indicating that NK92 cells of NK-019 In vivo antitumor activity. Embodiment 11 . Expression of NK-019 in iPSCs and differentiation of iPSCs into iNK cells.

An exemplary NK-019 construct comprising aCD19-CAR, hnCD16 and IL-15 flanked by PiggyBac transposon ITRs was used as a donor vector for transgene integration. As shown in Figures 13A and 13B, after gene editing and clonal derivation, the expression of aCD19-CAR, hnCD16 and IL-15 was detected on the cell surface of iPSC monoclonals. SSEA-4 is a pluripotency marker for iPSCs. Figure 13A shows the expression of SSEA-4 on gene-edited iPSCs, indicating the pluripotency of the edited iPSC cells.

The ability of gene-edited iPSCs to differentiate into iNK cells was further evaluated. As shown in Figure 14A, at day 7, NK-019w-expressing iPSCs were differentiated into CD34+ iHSCs. On day 34, CD34+ iHSCs were further differentiated into mature iNK cells (CD45+ and CD56+), consistently expressing aCD19-CAR, hnCD16 and IL-15RF on the cell surface, as shown in Figure 14B.

While preferred embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that these embodiments are provided by way of example only. It is intended that the invention not be limited to the specific examples provided in the specification. While the invention has been described with reference to the foregoing specification, the descriptions and illustrations of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it is to be understood that all aspects of the invention are not limited to the specific descriptions, configurations or relative proportions set forth herein which are subject to various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. Accordingly, it is contemplated that the present invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. sequence listing SEQ ID NO. sequence 1 MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWKDKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWFHVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK 2 FHVS 3 wxya 4 FHVSF 5 WFHVSF 6 VWFHVSFC 7 PVWFHVSFCL 8 TPVWFHVSFCLV 9 GGGGS 10 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 11 EGKSSGSGSESKST 12 EGKSSGSGSESKSTEGKSSGSGSESKSTGGGGS 13 MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL 14 MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSEGKSSGSGSESKSTEGKSSGSGSESKSTGGGGSGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL 15 MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSEGKSSGSGSESKSTEGKSSGSGSESKSTEGKSSGSGSESKSTGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL 16 QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS 17 DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK 18 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 19 EGKSSGSGSESKSTEGKSSGSGSESKSTGGGGS 20 EGKSSGSGSESKSTEGKSSGSGSESKSTEGKSSGSGSESKST twenty one KSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL twenty two MRISKPHLRSISIQCYLCLLLLNSHFLTEAGIHVFILGCFSAGLPKTEA twenty three MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFVIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVPTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK twenty four MRISKPHLRSISIQCYLCLLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 25 CAGGTGCAGCTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGTCCTCAGTGAAGATTTCCTGCAAGGCTTCTGGCTATGCATTCAGTAGCTACTGGATGAACTGGGTGAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGACAGATTTGGCCTGGAGATGGTGATACTAACTACAATGGAAAGTTCAAGGGTAAAGCCACTCTGACTGCAGACGAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTAGCATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGACGGGAGACTACGACGGTAGGCCGTTATTACTATGCTATGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCC 26 GATATCCAGCTGACCCAGTCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTATTTGAACTGGTACCAACAGATTCCAGGACAGCCACCCAAACTCCTCATCTATGATGCATCCAATCTAGTTTCTGGGATCCCACCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGAAGGTGGATGCTGCAACCTATCACTGTCAGCAAAGTACTGAGGATCCGTGGACGTTCGGTGGAGGGACCAAGCTCGAGATCAA 27 ATGTGGCAGCTGCTGCTGCCTACCGCTCTGCTGCTGCTGGTTTCCGCTGGAATGAGGACAGAGGACCTGCCTAAGGCCGTGGTGTTTCTGGAGCCTCAGTGGTACAGAGTGCTGGAGAAGGACTCCGTGACACTGAAGTGCCAGGGCGCTTACAGCCCCGAAGATAACTCCACCCAGTGGTTCCACAATGAGAGCCTGATCTCCAGCCAGGCCAGCTCTTACTTCATCGATGCCGCCACAGTGGATGATAGCGGCGAGTACAGGTGTCAGACCAATCTGAGCACACTGTCCGATCCCGTGCAGCTGGAGGTGCATATCGGCTGGCTGCTGCTGCAGGCTCCAAGATGGGTGTTTAAGGAGGAGGACCCTATCCACCTGAGGTGCCACAGCTGGAAGAATACCGCCCTGCACAAGGTGACATACCTGCAGAATGGCAAGGGCAGAAAGTACTTTCACCACAATAGCGATTTCGTGATCCCTAAGGCCACCCTGAAGGATAGCGGCAGCTACTTCTGTAGGGGCCTGTTCGGCAGCAAGAATGTGTCCAGCGAGACAGTGAATATCACAATCACCCAGGGCCTGGCCGTGCCAACAATCTCTAGCTTCTTCCCTCCCGGCTACCAGGTGAGCTTCTGCCTGGTTATGGTGCTGCTGTTTGCCGTGGACACCGGCCTGTACTTTAGCGTGAAAACTAATATTCGGAGCAGCACCAGAGATTGGAAGGACCACAAGTTTAAGTGGAGAAAGGACCCTCAGGACAAG 28 ATGAGAATTTCGAAACCACATTTGAGAAGCATTTCCATCCAGTGCTACTTGTGTTTACTTCTAAACAGTCATTTTCTAACTGAAGCTGGCATTCATGTGTTCATTTTGGGCTGTTTCTCCGCCGGGCTTCCTAAAACCGAAGCCAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGCGATGTTCACCCCTCCTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAATTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGCATTCATGATACAGTAGAAAATCTGATCATCCTGGCAAACAACAGTTTGAGCTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGCGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCTGGTGGTGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGGTGGCGGTGGCTCGGGCGGAGGTGGGTCGGGTGGCGGCGGATCAGGCATCACCTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCCGGGAGAGATACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAATGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGCATTAGAGATCCTGCCCTGGTTCACCAACGGCCAGCGCCACCCTCCACCGTAACGACGGCAGGGGTGACCCCACAGCCAGAGAGCCTCTCCCCTTCTGGAAAAGAGCCCGCAGCTTCATCTCCCTCCTCAAACAACACAGCGGCCACAACAGCAGCTATTGTCCCGGGCTCCCAACTGATGCCTTCAAAATCACCTTCCACCGGAACCACAGAGATAAGCAGTCATGAGTCCT CCCACGGCACCCCCTCTCAAACAACCGCCAAGAACTGGGAACTCACAGCATCCGCCTCCCACCAACCGCCCGGCGTGTATCCACAGGGCCACAGCGACACCACTGTGGCTATCTCCACGTCCACTGTCCTGCTGTGTGGGCTGAGCGCTGTGTCTCTCCTGGCATGCTACCTCAAGTCAAGGCAAACTCCCCCGCTGGCCTCCGTTGAAATGGAAGCCATGGAGGCTCTGCCGGTGACTTGGGGGACCAGCAGCAGAGATGAGGACTTGGAAAACTGCTCTCACCACCTA 29 ATGAGAATTTCGAAACCACATTTGAGAAGCATTTCCATCCAGTGCTACTTGTGTTTACTTCTAAACAGTCATTTTCTAACTGAAGCTGGCATTCATGTGTTCATTTTGGGCTGTTTCTCCGCCGGGCTTCCTAAAACCGAAGCCAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGCGATGTTCACCCCTCCTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAATTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGCATTCATGATACAGTAGAAAATCTGATCATCCTGGCAAACAACAGTTTGAGCTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGCGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCTGAAGGAAAATCTTCTGGATCTGGCTCCGAAAGCAAATCGACCGAGGGCAAATCATCCGGCTCCGGCTCGGAGTCTAAATCTACGGGTGGCGGCGGATCAGGCATCACCTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCCGGGAGAGATACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAATGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGCATTAGAGATCCTGCCCTGGTTCACCAACGGCCAGCGCCACCCTCCACCGTAACGACGGCAGGGGTGACCCCACAGCCAGAGAGCCTCTCCCCTTCTGGAAAAGAGCCCGCAGCTTCATCTCCCTCCTCAAACAACACAGCGGCCACAACAGCAGCTATTGTCCCGGGCTCCCAACTGATGCCTTCAAAATCACCTTCCACCGGAACCACAGAGATAAGCAGTC ATGAGTCCTCCCACGGCACCCCCTCTCAAACAACCGCCAAGAACTGGGAACTCACAGCATCCGCCTCCCACCAACCGCCCGGCGTGTATCCACAGGGCCACAGCGACACCACTGTGGCTATCTCCACGTCCACTGTCCTGCTGTGTGGGCTGAGCGCTGTGTCTCTCCTGGCATGCTACCTCAAGTCAAGGCAAACTCCCCCGCTGGCCTCCGTTGAAATGGAAGCCATGGAGGCTCTGCCGGTGACTTGGGGGACCAGCAGCAGAGATGAGGACTTGGAAAACTGCTCTCACCACCTA 30 ATGAGAATTTCGAAACCACATTTGAGAAGCATTTCCATCCAGTGCTACTTGTGTTTACTTCTAAACAGTCATTTTCTAACTGAAGCTGGCATTCATGTGTTCATTTTGGGCTGTTTCTCCGCCGGGCTTCCTAAAACCGAAGCCAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGCGATGTTCACCCCTCCTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAATTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGCATTCATGATACAGTAGAAAATCTGATCATCCTGGCAAACAACAGTTTGAGCTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGCGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCT

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention may be better understood by reference to the following detailed description of illustrative embodiments in which the principles of the invention are utilized, together with the accompanying drawings (also referred to herein as "the Figures") in which:

Figures 1A-1G show engineered NK cells comprising CD16 variants for enhanced CD16 signaling;

Figures 2A-2G show engineered NK cells comprising a chimeric antigen receptor for CD19; and

Figures 3A and 3B show engineered T cells containing allogeneic human IL-15.

4A - 4C show the design of aCD19-CAR, hnCD16 and IL-15RF.

Figures 5A-5B show the design of NK-019 constructs and engineered NK cells expressing NK-019 constructs;

Figures 6A-6C show enhanced NK cell activity conferred by aCD19-CAR;

Figures 7A-7C show enhanced ADCC conferred by hnCD16;

Figures 8A-8B show enhanced IL-15 signaling conferred by IL-15-RF;

Figure 9 shows the expression of NK-019 constructs in NK92 cells;

Figure 10 shows the CD19-specific cytolytic ability of NK-019 NK92 cells to K562-CD19 cells;

Figure 11A shows enhanced CD107a expression in NK-019 NK92 cells after human CD19+ stimulation, and Figure 11B shows enhanced interferon-γ production by NK-019 Nk92 cells after human CD19+ stimulation;

Figures 12A-12C show the antitumor activity of NK-019 NK92 cells in the Raji-NCG mouse model;

Figures 13A-13B show expression of NK-019 constructs in iPSCs; and

Figures 14A-14B show the differentiation of NK-019 iPSCs into NK-019 iNK cells.

             <![CDATA[<110> HANGZHOU QIHAN BIOTECHNOLOGY CO., LTD.)]]> <![CDATA[<120> SYSTEMS AND METHODS FOR BOOSTED IMMUNOTHERAPY ]]> <![CDATA[<130> 54809-722.851]]> <![CDATA[<140> TW 111106847]]> <![CDATA[<141> 2022-02-24]]> <![CDATA [<150> PCT/CN2021/077693]]> <![CDATA[<151> 2021-02-24]]> <![CDATA[<160> 42 ]]> <![CDATA[<170> PatentIn 3.5 version]]> <![CDATA[<210> 1]]> <![CDATA[<211> 338]]> <![CDATA[<212> PRT]]> <![CDATA[<213> artificial sequence ]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequences: Synthetic Peptides]]> <![CDATA[<400> 1]]> Met Trp Phe Leu Thr Thr Leu Leu Leu Trp Val Pro Val Asp Gly Gln 1 5 10 15 Val Asp Thr Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val Ser 20 25 30 Val Phe Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45 Pro Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55 60 Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser 65 70 75 80 Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile 85 90 95 Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg 100 105 110 Val Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys 115 120 125 Asp Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys Ala Phe 130 135 140 Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn Ile 145 150 155 160 Ser His Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg Tyr 165 170 175 Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185 190 Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val 195 200 205 Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220 Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn 225 230 235 240 Thr Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser Gly 245 250 255 Leu Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val Leu Lys Arg 260 265 270 Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Leu Gln Leu Pro Thr Pro 275 280 285 Val Trp Phe His Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala 290 295 300 Val Asp Thr Gly Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser 305 310 315 320 Thr Arg Asp Trp Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln 325 330 335 Asp Lys <![CDATA[<210> 2]]> <![CDATA[<211> 4]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> < ![CDATA[<220>]]> <![CDATA[<223> Description of artificial sequence: synthetic peptide]]> <![CDATA[<400> 2]]> Phe His Val Ser 1 <![CDATA[ <210> 3]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[< 220>]]> <![CDATA[<223> Description of artificial sequence: synthetic peptide]]> <![CDATA[<400> 3]]> Trp Phe His Val Ser 1 5 <![CDATA[<210> 4]]> <![CDATA[<211> 5]]> <![ CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]] > <![CDATA[<400> 4]]> Phe His Val Ser Phe 1 5 <![CDATA[<210> 5]]> <![CDATA[<211> 6]]> <![CDATA[< 212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]]> <! [CDATA[<400> 5]]> Trp Phe His Val Ser Phe 1 5 <![CDATA[<210> 6]]> <![CDATA[<211> 8]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]]> <![CDATA [<400> 6]]> Val Trp Phe His Val Ser Phe Cys 1 5 <![CDATA[<210> 7]]> <![CDATA[<211> 10]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]]> <![CDATA [<400> 7]]> Pro Val Trp Phe His Val Ser Phe Cys Leu 1 5 10 <![CDATA[<210> 8]]> <![CDATA[<211> 12]]> <![CDATA[ <212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]]> < ![CDATA[<400> 8]]> Thr Pro Val Trp Phe His Val Ser Phe Cys Leu Val 1 5 10 <![CDATA[<210> 9]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> < ![CDATA[<220>]]> <![CDATA[<223> Description of artificial sequence: synthetic peptide]]> <![CDATA[<400> 9]]> Gly Gly Gly Gly Ser 1 5 <![ CDATA[<210> 10]]> <![CDATA[<211> 30]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA [<220>]]> <![CDATA[<223> Description of artificial sequences: synthetic peptides]]> <![CDATA[<400> 10]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 30 <![CDATA[<210> 11]]> <![CDATA[<211> 14]]> <! [CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide] ]> <![CDATA[<400> 11]]> Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr 1 5 10 <![CDATA[<210> 12]]> <![CDATA[<211 > 33]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Artificial Description of sequence: synthetic polypeptide]]> <![CDATA[<400> 12]]> Glu Gly Lys Ser Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Glu Gly 1 5 10 15 Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Gly Gly Gly Gly 20 25 30 Ser <![CDATA[<210> 13]]> <![CDATA[<211> 430]]> <![CDATA[<212> PRT]]> <![ CDATA[<213> artificial sequence]] > <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequences: Synthetic Peptides]]> <![CDATA[<400> 13]]> Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr 1 5 10 15 Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His 20 25 30 Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala 35 40 45 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 50 55 60 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 65 70 75 80 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 85 90 95 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 100 105 110 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Ser Ser Asn Gly Asn Val 115 120 125 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 130 135 140 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 145 150 155 160 Thr Ser Gly Gly Gly Gly Ser Gly Gl y Gly Gly Ser Gly Gly Gly Gly 165 170 175 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 180 185 190 Gly Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp 195 200 205 Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser 210 215 220 Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu 225 230 235 240 Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys 245 250 255 Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr 260 265 270 Val Thr Thr Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser 275 280 285 Gly Lys Glu Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala 290 295 300 Thr Thr Ala Ala Ile Val Pro Gly Ser Gln Le u Met Pro Ser Lys Ser 305 310 315 320 Pro Ser Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly 325 330 335 Thr Pro Ser Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala 340 345 350 Ser His Gln Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr 355 360 365 Val Ala Ile Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val 370 375 380 Ser Leu Leu Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala 385 390 395 400 Ser Val Glu Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr 405 410 415 Ser Ser Arg Asp Glu Asp Leu Glu Asn Cys Ser His His Leu 420 425 430 <![CDATA[<210> 14]]> <![CDATA[<211> 433]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>] ]> <![CDATA[<223> Description of artificial sequences: synthetic peptides]]> <![CDATA[ <400> 14]]> Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr 1 5 10 15 Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His 20 25 30 Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala 35 40 45 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 50 55 60 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 65 70 75 80 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 85 90 95 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 100 105 110 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 115 120 125 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 130 135 140 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 145 150 155 160 Thr Ser Glu Gly Lys Ser Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr 165 170 175 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Gly Gly 180 185 190 Gly Gly Ser Gly Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala 195 200 205 Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile 210 215 220 Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu 225 230 235 240 Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser 245 250 255 Leu Lys Cys Ile Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro 260 265 270 Pro Ser Thr Val Thr Thr Ala Gly Val Thr Pro Gln Pro Glu Ser Leu 275 280 285 Ser Pro Ser Gly Lys Glu Pro Ala Ala Ser Ser Ser Pro Ser Ser Asn Asn 290 295 300 Thr Ala Ala Thr Thr Ala Ala Ile Val Pro Gly Ser Gln Leu Met Pro 305 310 315 320 Ser Lys Ser Pro Ser Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser 325 330 335 Ser His Gly Thr Pro Ser Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr 340 345 350 Ala Ser Ala Ser His Gln Pro Pro Gly Val Tyr Pro Gln Gly His Ser 355 360 365 Asp Thr Thr Val Ala Ile Ser Thr Ser Thr Val Leu Leu Cys Gly Leu 370 375 380 Ser Ala Val Ser Leu Leu Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro 385 390 395 400 Pro Leu Ala Ser Val Glu Met Glu Ala Met Glu Ala Leu Pro Val Thr 405 410 415 Trp Gly Thr Ser Ser Arg Asp Glu Asp Leu Glu Asn Cys Ser His His 420 425 430 Leu <![CDATA[<210> 15]]> < ![CDATA[<211> 442]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![ CDATA[<223> Description of artificial sequence: synthetic peptide]]> <![CDATA[<400> 15]]> Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr 1 5 10 15 Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His 20 25 30 Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala 35 40 45 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 50 55 60 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 65 70 75 80 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 85 90 95 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 100 105 110 Asn Leu Ile Ile Leu Ala Asn Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 115 120 125 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 130 135 140 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 145 150 155 160 Thr Ser Glu Gly Lys Ser Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr 165 170 175 Glu Gly Lys Ser Ser Gly Ser Gly Se r Glu Ser Lys Ser Thr Glu Gly 180 185 190 Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Gly Ile Thr Cys 195 200 205 Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr 210 215 220 Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg 225 230 235 240 Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr 245 250 255 Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro 260 265 270 Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val Thr Thr Ala 275 280 285 Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly Lys Glu Pro 290 295 300 Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala Ala 305 310 315 320 Ile Val Pro Gly Ser Gl n Leu Met Pro Ser Lys Ser Pro Ser Thr Gly 325 330 335 Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr Pro Ser Gln 340 345 350 Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser His Gln Pro 355 360 365 Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val Ala Ile Ser 370 375 380 Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val Ser Leu Leu Ala 385 390 395 400 Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser Val Glu Met 405 410 415 Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr Ser Ser Arg Asp 420 425 430 Glu Asp Leu Glu Asn Cys Ser His His Leu 435 440 <![CDATA[<210> 16 ]]> <![CDATA[<211> 124]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]] > <![CDATA[<223> Description of Artificial Sequences: Synthetic Peptides]]> <![CDATA[<400> 16]]> Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gly Gly Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Arg Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <![CDATA[<210 > 17]]> <![CDATA[<211> 111]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Description of artificial sequence: synthetic peptide]]> <![CDATA[<400> 17]]> Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Asp Val Tyr Asp 20 25 30 Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <![CDATA[<210> 18]]> <![CDATA[<211> 30]]> <![CDATA[< 212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]]> <! [CDATA[<400> 18]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 30 <![ CDATA[<210> 19]]> <![CDATA[<211> 33]]> <![CDATA[<212> PRT]]> <![ CDATA[<213> artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> description of artificial sequence: synthetic peptide]]> <![CDATA[<400> 19]] > Glu Gly Lys Ser Ser Gly Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Glu Gly 1 5 10 15 Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Gly Gly Gly Gly 20 25 30 Ser <![CDATA[<210> 20 ]]> <![CDATA[<211> 42]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]] > <![CDATA[<223>Description of artificial sequence: synthetic peptide]]> <![CDATA[<400> 20]]> Glu Gly Lys Ser Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Glu Gly 1 5 10 15 Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Glu Gly Lys Ser 20 25 30 Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr 35 40 <![CDATA[<210> 21]]> <![CDATA[< 211> 39]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequences: Synthetic Peptides]]> <![CDATA[<400> 21]]> Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser Val Glu Met Glu Ala Met 1 5 10 15 Glu Ala Leu Pro Val Thr Trp Gly Thr Ser Ser Arg Asp Glu Asp Leu 20 25 30 Glu Asn Cys Ser His His Leu 35 <![CDATA[<210> 22]]> <![CDATA[<211> 48]]> <![CDATA[<212 > PRT]]> <![CDATA[< 213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]]> <![CDATA[<400> 22]]> Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr 1 5 10 15 Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His 20 25 30 Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala 35 40 45 <![CDATA[<210> 23]]> <![CDATA[<211> 254]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]]> <![CDATA[<400> 23]]> Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Leu Val Ser Ala 1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr 65 70 75 80 Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Val Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190 Gly Leu Ala Val Pro Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln 195 200 205 Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220 Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp 225 230 235 240 Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 245 250 <![CD ATA[<210> 24]]> <![CDATA[<211> 162]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA [<220>]]> <![CDATA[<223> Description of Artificial Sequences: Synthetic Peptides]]> <![CDATA[<400> 24]]> Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr 1 5 10 15 Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His 20 25 30 Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala 35 40 45 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 50 55 60 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 65 70 75 80 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 85 90 95 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 100 105 110 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Ser Asn Gly Asn Val 115 120 125 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 130 135 140 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 145 150 155 160 Thr Ser <![CDATA[<210> 25]]> <![CDATA[<211> 372]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of artificial sequences: synthetic polynucleotides]]> <![CDATA[<400> 25]]> caggtgcagc tgcagcagtc tggggctgag ctggtgaggc ctgggtcctc agtgaagatt 60 tcctgcaagg cttctggcta tgcattcagt agctactgga tgaactgggt gaagcagagg 120 cctggacagg gtcttgagtg gattggacag atttggcctg gagatggtga tactaactac 180 aatggaaagt tcaagggtaa agccactctg actgcagacg aatcctccag cacagcctac 240 atgcaactca gcagcctagc atctgaggac tctgcggtct atttctgtgc aagacgggag 300 actacgacgg taggccgtta ttactatgct atggactact ggggccaagg gaccacggtc 360 accgtctcct cc 372 <![CDATA[<210 > 26]]> <![CDATA[<211> 332]]> <![CDATA[<212> DNA]]> <![CDATA[<213> artificial sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> Description of artificial sequences: synthetic polynucleotides]]> <![CDATA[<400> 26]]> gatatccagc tgacccagtc tccagcttct ttggctgtgt ctctagggca gagggccacc 60 atctcctgca aggccagcca aagtgttgat tatgatggtg atagttatt 1gg0gaact caacagattc caggacagcc acccaaactc ctcatctatg atgca tccaa tctagtttct 180 gggatcccac ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat 240 cctgtggaga aggtggatgc tgcaacctat cactgtcagc aaagtactga ggatccgtgg 300 acgttcggtg gagggaccaa gctcgagatc aa 332 <![CDATA[<210> 27]]> <![CDATA[<211> 762]]> <![CDATA [<212> DNA]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Polynucleotide ]]> <![CDATA[<400> 27]]> atgtggcagc tgctgctgcc taccgctctg ctgctgctgg tttccgctgg aatgaggaca 60 gaggacctgc ctaaggccgt ggtgtttctg gagcctcagt ggtacagagt gctggagaag 120 gactccgtga cactgaagtg ccagggcgct tacagccccg aagataactc cacccagtgg 180 ttccacaatg agagcctgat ctccagccag gccagctctt acttcatcga tgccgccaca 240 gtggatgata gcggcgagta caggtgtcag accaatctga gcacactgtc cgatcccgtg 300 cagctggagg tgcatatcgg ctggctgctg ctgcaggctc caagatgggt gtttaaggag 360 gaggacccta tccacctgag gtgccacagc tggaagaata ccgccctgca caaggtgaca 420 tacctgcaga atggcaaggg cagaaagtac tttcaccaca atagcgattt cgtgatccct 480 aaggccaccc tgaaggatag cggcagctac ttctgtaggg gcctgttcgg cagcaagaat 540 gtgtccagcg agacagtgaa tatcacaatc acccagggcc tggccgtgcc aacaatctct 600 agcttcttcc ctcccggcta ccaggtgagc ttctgcctgg ttatggtgct gctgtttgcc 660 gtggacaccg gcctgtactt tagcgtgaaa actaatattc ggagcagcac cagagattgg 720 aaggaccaca agtttaagtg gagaaaggac cctcaggaca ag 762 <![CDATA[<210> 28]]> <![CDATA[<211> 1290]]> <![CDATA[<212> DNA]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Artificial Sequence Description: Synthesis聚核苷酸]]> <![CDATA[<400> 28]]> atgagaattt cgaaaccaca tttgagaagc atttccatcc agtgctactt gtgtttactt 60 ctaaacagtc attttctaac tgaagctggc attcatgtgt tcattttggg ctgtttctcc 120 gccgggcttc ctaaaaccga agccaactgg gtgaatgtaa taagtgattt gaaaaaaatt 180 gaagatctta ttcaatctat gcatattgat gctactttat atacggaaag cgatgttcac 240 ccctcctgca aagtaacagc aatgaagtgc tttctcttgg aattacaagt tatttcactt 300 gagtccggag atgcaagcat tcatgataca gtagaaaatc tgatcatcct ggcaaacaac 360 agtttgagct ctaatgggaa tgtaacagaa tctggatgca aagaatgcga ggaactggag 420 gaaaaaaata ttaaagaatt tttgcagagt tttgtacata ttgtccaaat gttcatcaac 480 acttctggtg gtggcggttc aggcgga ggt ggctctggcg gtggcggatc gggtggcggt 540 ggctcgggcg gaggtgggtc gggtggcggc ggatcaggca tcacctgccc tccccccatg 600 tccgtggaac acgcagacat ctgggtcaag agctacagct tgtactcccg ggagagatac 660 atttgtaact ctggtttcaa gcgtaaagcc ggcacgtcca gcctgacgga atgcgtgttg 720 aacaaggcca cgaatgtcgc ccactggaca acccccagtc tcaaatgcat tagagatcct 780 gccctggttc accaacggcc agcgccaccc tccaccgtaa cgacggcagg ggtgacccca 840 cagccagaga gcctctcccc ttctggaaaa gagcccgcag cttcatctcc ctcctcaaac 900 aacacagcgg ccacaacagc agctattgtc ccgggctccc aactgatgcc ttcaaaatca 960 ccttccaccg gaaccacaga gataagcagt catgagtcct cccacggcac cccctctcaa 1020 acaaccgcca agaactggga actcacagca tccgcctccc accaaccgcc cggcgtgtat 1080 ccacagggcc acagcgacac cactgtggct atctccacgt ccactgtcct gctgtgtggg 1140 ctgagcgctg tgtctctcct ggcatgctac ctcaagtcaa ggcaaactcc cccgctggcc 1200 tccgttgaaa tggaagccat ggaggctctg ccggtgactt gggggaccag cagcagagat 1260 gaggacttgg aaaactgctc tcaccaccta 1290 <![CDATA[<210 > 29]]> <![CDATA[<211> 1299]]> <![CDATA[<212> DNA]]> <![CDATA[< 213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223>Description of artificial sequence: synthetic polynucleotide]]> <![CDATA[<400> 29]] > atgagaattt cgaaaccaca tttgagaagc atttccatcc agtgctactt gtgtttactt 60 ctaaacagtc attttctaac tgaagctggc attcatgtgt tcattttggg ctgtttctcc 120 gccgggcttc ctaaaaccga agccaactgg gtgaatgtaa taagtgattt gaaaaaaatt 180 gaagatctta ttcaatctat gcatattgat gctactttat atacggaaag cgatgttcac 240 ccctcctgca aagtaacagc aatgaagtgc tttctcttgg aattacaagt tatttcactt 300 gagtccggag atgcaagcat tcatgataca gtagaaaatc tgatcatcct ggcaaacaac 360 agtttgagct ctaatgggaa tgtaacagaa tctggatgca aagaatgcga ggaactggag 420 gaaaaaaata ttaaagaatt tttgcagagt tttgtacata ttgtccaaat gttcatcaac 480 acttctgaag gaaaatcttc tggatctggc tccgaaagca aatcgaccga gggcaaatca 540 tccggctccg gctcggagtc taaatctacg ggtggcggcg gatcaggcat cacctgccct 600 ccccccatgt ccgtggaaca cgcagacatc tgggtcaaga gctacagctt gtactcccgg 660 gagagataca tttgtaactc tggtttcaag cgtaaagccg gcacgtccag cctgacggaa 720 tgcgtgttga acaaggccac gaatgtcgcc cactggacaa cccccagtct caaatgcatt 780 agagat cctg ccctggttca ccaacggcca gcgccaccct ccaccgtaac gacggcaggg 840 gtgaccccac agccagagag cctctcccct tctggaaaag agcccgcagc ttcatctccc 900 tcctcaaaca acacagcggc cacaacagca gctattgtcc cgggctccca actgatgcct 960 tcaaaatcac cttccaccgg aaccacagag ataagcagtc atgagtcctc ccacggcacc 1020 ccctctcaaa caaccgccaa gaactgggaa ctcacagcat ccgcctccca ccaaccgccc 1080 ggcgtgtatc cacagggcca cagcgacacc actgtggcta tctccacgtc cactgtcctg 1140 ctgtgtgggc tgagcgctgt gtctctcctg gcatgctacc tcaagtcaag gcaaactccc 1200 ccgctggcct ccgttgaaat ggaagccatg gaggctctgc cggtgacttg ggggaccagc 1260 agcagagatg aggacttgga aaactgctct caccaccta 1299 <![CDATA[<210> 30]]> <![CDATA[<211> 486]]> <![CDATA[<212> DNA]]> <![ CDATA[<213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223>Description of artificial sequence: synthetic polynucleotide]]> <![CDATA[<400> 30]]> atgagaattt cgaaaccaca tttgagaagc atttccatcc agtgctactt gtgtttactt 60 ctaaacagtc attttctaac tgaagctggc attcatgtgt tcattttggg ctgtttctcc 120 gccgggcttc ctaaaaccga agccaactgg gtgaatgtaa taagtgattt gaaaaaaatt 180 gaagatctta ttcaatctat gc atattgat gctactttat atacggaaag cgatgttcac 240 ccctcctgca aagtaacagc aatgaagtgc tttctcttgg aattacaagt tatttcactt 300 gagtccggag atgcaagcat tcatgataca gtagaaaatc tgatcatcct ggcaaacaac 360 agtttgagct ctaatgggaa tgtaacagaa tctggatgca aagaatgcga ggaactggag 420 gaaaaaaata ttaaagaatt tttgcagagt tttgtacata ttgtccaaat gttcatcaac 480 acttct 486 <![CDATA[<210> 31]]> <![ CDATA[<211> 19]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[ <223> Description of artificial sequence: synthetic peptide]]> <![CDATA[<400> 31]]> Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Gly Gly 1 5 10 15 Gly Gly Ser <! [CDATA[<210> 32]]> <![CDATA[<211> 50]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![ CDATA[<220>]]> <![CDATA[<223> Description of artificial sequence: synthetic peptide]]> <![CDATA[<220>]]> <![CDATA[<221> SITE]]> < ![CDATA[<222> (1)..(50)]]> <![CDATA[<223> This sequence can contain 1 to 10 "Gly Gly Gly Gly Ser" repeating units]]> <![CDATA [<400> 32]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 35 40 45 Gly Ser 50 <![CDATA[<210> 33]]> <![CDATA[<211> 140]]> <! [CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide] ]> <![CDATA[<220>]]> <![CDATA[<221> SITE]]> <![CDATA[<222> (1)..(140)]]> <![CDATA[< 223> This sequence can contain 1 to 10 repeating units of "Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr"]]> <![CDATA[<400> 33]]> Glu Gly Lys Ser Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Glu Gly 1 5 10 15 Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Glu Gly Lys Ser 20 25 30 Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Glu Gly Lys Ser Ser Gly 35 40 45 Ser Gly Ser Glu Ser Lys Ser Thr Glu Gly Lys Ser Ser Gly Ser Gly 50 55 60 Ser Glu Ser Lys Ser Thr Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu 65 70 75 80 Ser Lys Ser Thr Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys 85 90 95 Ser Thr Glu Gly Lys Ser Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr 100 105 110 Glu Gly Lys Ser Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Glu Gly 115 120 125 Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr 130 135 140 <![CDATA[<210> 34]]> <![CDATA[<211> 190]]> <! [CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide] ]> <![CDATA[<220>]]> <![CDATA[<221> SITE]]> <![CDATA[<222> (1)..(140)]]> <![CDATA[< 223> This region can contain 1 to 10 repeating units of "Glu Gly Lys Ser Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr"]]> <![CDATA[<220>]]> <![CDATA[<221> SITE]]> <![CDATA[<222> (141)..(190)]]> <![CDATA[<223> This region can contain 1 to 10 "Gly Gly Gly Gly Ser" repeating units]] > <![CDATA[<400> 34]]> Glu Gly Lys Ser Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Glu Gly 1 5 10 15 Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Glu Gly Lys Ser 20 25 30 Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Glu Gly Lys Ser Ser Ser Gly 35 40 45 Ser Gly Ser Glu Ser Lys Ser Thr Glu Gly Lys Ser Ser Ser Gly Ser Gly 50 55 60 Ser Glu Ser Lys Ser Thr Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu 65 70 75 80 Ser Lys Ser Thr Glu Gly L ys Ser Ser Gly Ser Gly Ser Glu Ser Lys 85 90 95 Ser Thr Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr 100 105 110 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Glu Gly 115 120 125 Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 165 170 175 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 180 185 190 <![CDATA[<210> 35]]> <![CDATA[<211> 10]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Artificial Sequence Description: Synthesis peptide]]> <![CDATA[<400> 35]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <![CDATA[<210> 36]]> <![CDATA[<211> 15 ]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <! [CDATA[<220>]]> <![CDATA[<223> Description of artificial sequence: synthetic peptide]]> <![CDATA[<400> 36]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <![CDATA[<210> 37]]> <![CDATA[<211> 20]]> <![CDATA[<212> PRT]]> <![CDATA[< 213> Artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223>Description of artificial sequence: synthetic peptide]]> <![CDATA[<400> 37]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <![CDATA[<210> 38]]> <![CDATA[<211> 25]]> <![ CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]] > <![CDATA[<400> 38]]> Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 <![CDATA[< 210> 39]]> <![CDATA[<211> 28]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial sequence]]> <![CDATA[<220 >]]> <![CDATA[<223> Description of artificial sequence: synthetic peptide]]> <![CDATA[<400> 39]]> Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Glu Gly 1 5 10 15 Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr 20 25 <![CDATA[<210> 40]]> <![CDATA[<211> 5]]> <![CDATA[<212> PR T]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Description of Artificial Sequence: Synthetic Peptide]]> <![CDATA [<220> ]]> <![CDATA[<223> For detailed description of substitutions and preferred embodiments, see submitted specifications]]> <![CDATA[<400> 40]]> Gly Gly Gly Gly Ser 1 5 <![CDATA[<210> 41]]> <![CDATA[<211> 14]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence] ]> <![CDATA[<220>]]> <![CDATA[<223> Description of artificial sequence: synthetic peptide]]> <![CDATA[<220> ]]> <![ CDATA[<223> See submitted specification for detailed description of substitutions and preferred embodiments]]> <![CDATA[<400> 41]]> Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr 1 5 10 <![CDATA[<210> 42]]> <![CDATA[<211> 19]]> <![CDATA[<212> PRT]]> <![CDATA[<213> artificial sequence] ]> <![CDATA[<220>]]> <![CDATA[<223> Description of artificial sequence: synthetic peptide]]> <![CDATA[<220> ]]> <![CDATA[<223> For detailed descriptions of alternative and preferred embodiments, see submitted specifications]]> <![CDATA[<400> 42]]> Glu Gly Lys Ser Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr Gly Gly 1 5 10 15 Gly Gly Ser
      

Claims (67)

An engineered cell comprising: (i) CD16 variants for enhanced CD16 signaling compared to control cells, (ii) heterologous IL-15, heterologous IL-15 receptor or a fragment thereof, and (iii) A chimeric antigen receptor comprising an antigen binding portion capable of binding to CD19. The engineered cell of claim 1, wherein the antigen-binding portion comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises amino acids with at least 80% identity to SEQ ID NO.16 Sequence, the light chain variable region comprises an amino acid sequence having at least 80% identity with SEQ ID NO.17. The engineered cells according to claim 1 or 2, wherein said engineered cells include stem cells, progenitor cells and specific cells. The engineered cell according to claim 3, wherein the stem cells include isolated stem cells and induced stem cells. The engineered cell according to claim 3, wherein said stem cells include embryonic stem cells and induced pluripotent stem cells. The engineered cell according to claim 3, wherein said progenitor cells include isolated progenitor cells and induced progenitor cells. The engineered cell according to claim 3, wherein said progenitor cells include hematopoietic stem cells, myeloid progenitor cells, megakaryotic progenitor cells, erythroid progenitor cells and lymphoid progenitor cells. The engineered cell according to claim 3, wherein said specific cells include isolated specific cells and induced specific cells. The engineered cell according to claim 3, wherein said specific cells include T cells, B cells and NK cells. The engineered cell according to claim 9, wherein the NK cells are derived from isolated stem cells or induced stem cells. The engineered cell according to claim 9, wherein the NK cells are derived from isolated progenitor cells or induced progenitor cells. The engineered cell according to any one of claims 1 to 11, wherein the heterologous IL-15 comprises secreted IL-15 and membrane-bound IL-15. The engineered cell according to claim 12, wherein the heterologous IL-15 is a secreted IL-15 comprising an amino acid sequence at least 80% identical to SEQ ID NO.24. The engineered cell according to claim 12, wherein the membrane-bound IL-15 comprises at least a part of IL-15 and at least a part of IL-15 receptor. The engineered cell according to claim 13, wherein at least a part of the IL-15 and at least a part of the IL-15 receptor are linked through a linker. The engineered cell according to claim 15, wherein the linker is selected from (GGGGS)n, (EGKSSGSGSESKST)n and (EGKSSGSGSESKST)n(GGGGS)n. The engineered cell according to claim 16, wherein said n is any integer selected from 1-10. The engineered cell according to any one of claims 15 to 17, wherein the linker comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NO.18-20. The engineered cell according to any one of claims 12 to 18, wherein the membrane-bound IL-15 further comprises an intracellular signaling domain. The engineered cell according to claim 19, wherein the intracellular signaling domain comprises amino acids having at least 80% identity to SEQ ID NO.21. The engineered cell according to any one of claims 12 to 20, wherein the membrane-bound IL-15 further comprises an extracellular signal peptide. The engineered cell according to claim 21, wherein the extracellular signal peptide comprises an amino acid having at least 80% identity with SEQ ID NO.22. The engineered cell according to any one of claims 12 to 22, wherein the membrane-bound IL-15 comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NO.13-15. The engineered cell according to any one of claims 1 to 23, wherein said CD16 variant is heterologous to said cell. The engineered cell according to any one of claims 1 to 24, wherein the CD16 variant comprises F158V (F176V). The engineered cell according to claim 25, wherein the CD16 variant further comprises S197P. The engineered cell according to any one of claims 1 to 26, wherein said CD16 variant comprises an amino acid sequence having at least 80% identity to SEQ ID NO.23. The engineered cell according to any one of claims 1 to 27, wherein the chimeric antigen receptor further comprises a hinge domain. The engineered cell according to claim 28, wherein the hinge domain comprises a compound derived from CD3D, CD3E, CD3G, CD3c CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS , ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D or amino acids of T cell receptor polypeptide sequence. The engineered cell according to any one of claims 1 to 29, wherein the chimeric antigen receptor further comprises a transmembrane domain. The engineered cell according to claim 30, wherein the transmembrane domain comprises CD3D, CD3E, CD3G, CD3c CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D or T cell receptor polypeptide amino acid sequence. The engineered cell according to any one of claims 1 to 31, wherein the chimeric antigen receptor further comprises a co-stimulatory domain. The engineered cell according to claim 32, wherein the co-stimulatory domain comprises CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4 or the amino acid sequence of NKG2D or any combination thereof. The engineered cell according to any one of claims 1 to 33, wherein the chimeric antigen receptor further comprises a signaling domain. The engineered cell as claimed in claim 34, wherein the signaling domain comprises a protein derived from CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, The amino acid sequence of NKG2D or any combination thereof. A polynucleotide comprising: (i) a first sequence encoding a CD16 variant for enhanced CD16 signaling, (ii) a second sequence encoding heterologous IL-15, and (iii) a third sequence encoding a chimeric antigen receptor comprising an antigen binding portion capable of binding to CD19. The polynucleotide according to claim 36, wherein said first sequence encoding said CD16 variant comprises a nucleic acid sequence having at least 80% identity with SEQ ID NO.27. The polynucleotide according to claim 36 or 37, wherein the second sequence encoding the heterologous IL-15 comprises a nucleic acid sequence encoding secreted IL-15 or a nucleic acid sequence encoding membrane-bound IL-15. The polynucleotide according to claim 38, wherein the nucleic acid sequence encoding the secreted IL-15 comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO.30. The polynucleotide according to claim 38, wherein the nucleic acid sequence encoding membrane-bound IL-15 comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO.28 or 29. The polynucleotide according to any one of claims 36 to 40, wherein the third sequence comprises: (a) the nucleic acid sequence of the heavy chain variable region of coding antigen binding part, described nucleic acid sequence has at least 80% identity with SEQ ID NO 25, and (b) a nucleic acid sequence encoding the light chain variable region of the antigen binding portion, said nucleic acid sequence having at least 80% identity to SEQ ID NO 26. An engineered cell comprising: (i) CD16 variants for enhanced CD16 signaling compared to control cells, (ii) heterologous IL-15, wherein said heterologous IL-15 is membrane-bound IL-15, said membrane-bound IL-15 comprises any one selected from SEQ ID No.13-15 having at least 95% identical amino acid sequences, and (iii) A chimeric antigen receptor comprising an antigen binding portion capable of binding to CD19. The engineered cell according to claim 42, wherein said engineered cell includes stem cells, progenitor cells and specialized cells. The engineered cell according to claim 43, wherein said stem cells include isolated stem cells and induced stem cells. The engineered cell according to claim 43, wherein said stem cells include embryonic stem cells and induced pluripotent stem cells. The engineered cell according to claim 43, wherein said progenitor cells include isolated progenitor cells and induced progenitor cells. The engineered cell according to claim 43, wherein said progenitor cells include hematopoietic stem cells, myeloid progenitor cells, megakaryoline progenitor cells, erythroid progenitor cells and lymphoid progenitor cells. The engineered cell according to claim 43, wherein the specific cells include isolated specific cells and induced specific cells. The engineered cell according to claim 43, wherein said specific cells include T cells, B cells and NK cells. The engineered cell according to claim 49, wherein the NK cells are derived from isolated stem cells or induced stem cells. The engineered cell according to claim 49, wherein the NK cells are derived from isolated progenitor cells or induced progenitor cells. The engineered cell according to any one of claims 42 to 51, wherein said CD16 variant is heterologous to said cell. The engineered cell according to claim 52, wherein said CD16 variant comprises F158V (F176V). The engineered cell according to claim 53, wherein the CD16 variant further comprises S197P. The engineered cell according to any one of claims 42 to 54, wherein said CD16 variant comprises an amino acid sequence having at least 80% identity to SEQ ID NO.23. The engineered cell according to any one of claims 42 to 55, wherein the antigen binding portion is an antibody selected from Fab, Fab', F(ab')2, scFv and sdAb. The engineered cell according to any one of claims 42 to 56, wherein the antigen-binding portion comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises at least An amino acid sequence of 80% identity, said light chain variable region comprising an amino acid sequence having at least 80% identity to SEQ ID NO.17. The engineered cell according to any one of claims 42 to 57, wherein said chimeric antigen receptor further comprises a hinge domain. The engineered cell according to claim 58, wherein the hinge domain comprises CD3D, CD3E, CD3G, CD3c CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS , ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D or amino acids of T cell receptor polypeptide sequence. The engineered cell according to any one of claims 42 to 59, wherein the chimeric antigen receptor further comprises a transmembrane domain. The engineered cell according to claim 60, wherein the transmembrane domain comprises CD3D, CD3E, CD3G, CD3c CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D or T cell receptor polypeptide amino acid sequence. The engineered cell according to any one of claims 42 to 61, wherein said chimeric antigen receptor further comprises a co-stimulatory domain. The engineered cell according to claim 62, wherein the co-stimulatory domain comprises CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4 or the amino acid sequence of NKG2D or any combination thereof. The engineered cell according to any one of claims 42 to 63, wherein the chimeric antigen receptor further comprises a signaling domain. The engineered cell as claimed in claim 64, wherein the signaling domain comprises a protein derived from CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB), IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, The amino acid sequence of NKG2D or any combination thereof. A polynucleotide comprising: (i) a first sequence encoding a CD16 variant for enhanced CD16 signaling, (ii) a second sequence encoding membrane-bound IL-15, wherein said second sequence comprises a nucleic acid sequence with at least 95% identity to SEQ ID NO.28 or 29, and (iii) a third sequence encoding a chimeric antigen receptor comprising an antigen binding portion capable of binding to CD19. An engineered cell, comprising heterologous IL-15, wherein the heterologous IL-15 is membrane-bound IL-15, and the membrane-bound IL-15 comprises and is selected from any of SEQ ID No.13-15 An amino acid sequence that is at least 95% identical.

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