EA048895B1 - MULTIPLEX GENOME EDITING OF IMMUNE CELLS TO ENHANCE FUNCTIONALITY AND RESISTANCE TO SUPPRESSIVE ENVIRONMENTS - Google Patents

Description

State of the art

This application claims the benefit of U.S. Provisional Patent Application Serial No. 62/772,406, filed November 28, 2018, which is incorporated herein by reference in its entirety.

1. Field of technology.

The present invention generally relates to the field of immunology, cell biology, molecular biology and medicine. More specifically, the invention relates to multiplex editing of immune cells and methods for using it.

2. Description of the related area.

Cellular immunotherapy holds great promise for the treatment of cancer. However, most immunotherapeutic approaches used alone have limited efficacy against most malignancies, particularly solid tumors. Reasons for this limited efficacy include decreased expression of tumor antigens on the surface of tumor cells, which reduces their detection by the immune system; expression of ligands for inhibitory receptors such as PD1, NKG2A, TIGIT, or CISH that induce immune cell inactivation; and induction of cells (e.g., regulatory T cells or myeloid-derived suppressor cells) in the microenvironment that secrete substances such as transforming growth factor β (TGFβ) and adenosine that suppress the immune response and promote cell proliferation and tumor survival. Thus, there is an unmet need for improved cellular immunotherapies.

Essence

The invention provides compositions and methods that relate to immunotherapy of a malignant tumor, in particular, including engineered immune cells. Particular embodiments relate to certain immune cells that have been modified by human hands so that they do not express or have reduced expression of one, two or more genes, and in particular cases, cells with such modification/modifications also express one or more heterologous proteins, including non-natural proteins, such as antigen receptors. The invention also relates to methods for producing non-natural immune cells. In some cases, the introduction of a heterologous antigen receptor occurs in the genomic locus of a gene, the expression of which is reduced or eliminated.

In one embodiment, the present invention relates to an in vitro method for disrupting at least two genes in an immune cell, wherein the at least two genes are selected from the group consisting of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7, and a combination thereof. In particular aspects, three, four, five, six or more genes are disrupted. In particular aspects, the disruption of two or more genes occurs simultaneously, such as in the same step of the method. The method may include introducing guide RNA (gRNA) for each gene into an immune cell.

The method may include knockdown of specific combinations of genes, such as, for example, the following: (a) NKG2A and CISH, (b) NKG2A and TGFBRII, (c) CISH and TGFBRII, (d) TIGIT and FOXO1, (e) TIGIT and TGFBRII, (f) CD96 and FOXO1, (g) CD96 and TGFBRII, (h) FOXO1 and TGFBRII, (i) CD96 and TIGIT, (j) CISH and TIGIT, (k) TIM3 and CISH, (l) TIM3 and TGFBRII, (m) FOXO1 and TGFBRII, (n) TIM3 and TIGIT, (o) SIGLEC7 and CISH, (p) SIGLEC7 and TGFBRII, (q) CD47 and CISH, (r) CD47 and TGFBRII, (s) SIRPA and CISH, (t) SIRPA and TGFBRII, (u) CD47 and TIGIT, (v) CD47 and SIRPA, (w) A2AR and CISH, (x) A2AR and TGFBRII, (y) ADAM17 and CISH, (z) TGFBRII and ADAM17, (a) A2AR and TIGIT, (b) SHP1 and CISH, (c) CISH and TGFBRII, (d) SHP1 and TGFBRII, (e) SHP1 and TIGIT, or (f) SHP1 and TIM3. The method may comprise knockdown of (1) NKG2A, CISH, and TGFBRII, (2) TIGIT, FOXO1, and TGFBRII, (3) TGFBRII, CD96, and TIGIT, (4) TGFBR2, CISH, and TIGIT, (5) TIM3, CISH, and TGFBRII, (6) CD96, FOXO1, and TGFBRII, (7) TGFBRII, TIM3, and TIGIT, (8) SIGLEC7, CISH, and TGFBRII, (9) CD47, CISH, and TGFBRII, (10) SIRPA, CISH, and TGFBRII, (11) TGFBRII, CD47, and TIGIT, (12) TGFBRII, CD47, and SIRPA, (13) A2AR, CISH, and TGFBRII, (14) TGFBRII, CISH, and ADAM17, (15) TGFBRII, TIM3 and TIGIT, (16) TGFBRII, A2AR and TIGIT, (17) SHP1, CISH and TGFBRII, (18) TGFBRII, CISH and SHP1, (19) TGFBRII, SHP1 and TIGIT, or (20) TGFBRII, SHP1 and TIM3. Any of the above subgroups can be combined with a second subgroup as described above. For example, any one of the subgroups aj1 can be combined with any one or more of the other subgroups a-j1, any one or more of the subgroups a-j1 can be combined with any one or more of the other subgroups 1-23, or any one or more of the subgroups 1-23 can be combined with any one or more of the other subgroups 1-23.

In some aspects, the method further comprises introducing into the cell an RNA-guided endonuclease, such as Cas9. Introducing the RNA-guided endonuclease may comprise introducing into the immune cell a nucleic acid, such as mRNA, encoding the RNA-guided endonuclease.

In certain aspects, an immune cell is a T cell, an NK cell, a B cell, a macrophage, an NK T cell, or a stem cell. In alternative cases, an immune cell is not

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A T cell, including a CAR T cell. In some aspects, the immune cell is engineered to express one or more chimeric antigen receptors (CARs) and/or one or more T cell receptors (TCRs). The immune cell may be virus-specific, such as a virus-specific T cell. The T cell may be a regulatory T cell. The B cell may be a regulatory B cell. In some aspects, the stem cell is a mesenchymal stem cell (MSC) or an induced pluripotent stem cell (iPS). In particular aspects, the T cell is a CD8+ T cell, a CD4+ T cell, or a gamma delta T cell. The immune cell may be isolated from peripheral blood, cord blood, bone marrow, or a mixture thereof. In some aspects, cord blood is pooled from 2 or more separate units of cord blood.

In some aspects, the introducing step comprises transfection or transduction. For example, introducing comprises electroporation, which can be performed more than once, such as two or three rounds of electroporation. In some aspects, a first group of CRISPR gRNAs is introduced in a first round of electroporation, and a second group of CRISPR gRNAs is introduced in a second round of electroporation. In particular cases, the first group of CRISPR gRNAs is different from the second group of CRISPR gRNAs. In particular aspects, the first group and/or the second group of CRISPR gRNAs include 1, 2, 3, or 4 or more CRISPR gRNAs. In some aspects, two CRISPR gRNAs are introduced in a first round of electroporation, and two different CRISPR gRNAs are introduced in a second round of electroporation. In particular embodiments, a group of CRISPR gRNAs comprises a group of gRNAs, at least two of which target different genes; in alternative embodiments, each group of gRNAs targets different genes.

In specific aspects, the method comprises disrupting NKG2A, CD47, TGFβR2, and CISH; NKG2A, CISH, TGFβR2, and ADORA2; NKG2A, TGFβR2, and CISH; TIGIT, CD96, CISH, and ADORA2; or ADAM17, TGFβR2, NKG2A, and SHP1.

In some aspects, the disruption results in increased antitumor cytotoxicity, in vivo proliferation, in vivo persistence, and/or improved immune cell function. In particular aspects, the immune cell has increased secretion of IFN-γ, CD107, and/or TNFu compared to a cell lacking the modification(s). In some aspects, the immune cell has increased production of perforin and/or granzyme B compared to a cell lacking the modification(s).

In further aspects, the method further comprises introducing the CAR and/or TCR into the immune cell, such as introducing a nucleic acid encoding the CAR and/or TCR into the immune cell. In some aspects, the nucleic acid is part of an expression vector, such as a retroviral vector. In certain aspects, the vector is an adenovirus-associated vector, such as AAV6. In some aspects, the vector further comprises an inhibitory gene sequence, such as an inhibitory gene sequence selected from the group consisting of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7, and combinations thereof. In particular aspects, the vector comprises a guide RNA for an inhibitory gene. The CAR can be flanked by homology arms for the inhibitory gene. In some aspects, introduction of a vector comprising a CAR sequence results in the insertion of the CAR into an inhibitory gene locus, such as an exon of an inhibitory gene, in an immune cell such that the CAR is under the control of an endogenous promoter of the inhibitory gene. In particular aspects, introduction of the vector further disrupts expression of the inhibitory gene.

In another embodiment, there is provided an immune cell, such as an immune cell of the disclosed embodiments, with impaired expression of at least two genes in an immune cell, produced by at least a step comprising introducing a CRISPR guide RNA (gRNA) for each gene into said immune cell, wherein the at least two genes are selected from the group consisting of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7 and a combination thereof. In some aspects, three, four, five, six or more genes are disrupted.

In certain aspects, the immune cell is a T cell, an NK cell, a B cell, a macrophage, an NK-T cell, or a stem cell. In some aspects, the immune cell is engineered to express a chimeric antigen receptor (CAR) and/or a T cell receptor (TCR). The immune cell can be virus-specific, such as a virus-specific T cell. The T cell can be a regulatory T cell. The B cell can be a regulatory B cell. In some aspects, the stem cell is a mesenchymal stem cell (MSC) or an induced pluripotent stem cell (iPS). In particular aspects, the T cell is a CD8+ T cell, a CD4+ T cell, or a gamma delta T cell. The immune cell may be isolated from peripheral blood, cord blood, bone marrow, or a mixture thereof. In some aspects, cord blood is pooled from 2 or more separate cord blood units.

In specific aspects, the method includes disrupting specific groups of genes, such as

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NKG2A, CD47, TGFβR2 and CISH; NKG2A, CISH, TGFβR2 and ADORA2; NKG2A, TGFβR2 and CISH; TIGIT, CD96, CISH and ADORA2; or ADAM17, TGFβR2, NKG2A and SHP1.

In some aspects, the disruption results in increased antitumor cytotoxicity, in vivo proliferation, in vivo persistence, and/or improved immune cell function. In particular aspects, the immune cell has increased secretion of IFN-γ, CD107, and/or TNFu. In some aspects, the immune cell has increased production of perforin and/or granzyme B.

In some aspects, the cell is engineered to express a CAR and/or a TCR. The CAR can be inserted into an endogenous inhibitory gene locus of the cell, such as an inhibitory gene locus selected from the group consisting of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7, and combinations thereof. In some aspects, the CAR is under the control of an endogenous inhibitory gene promoter. In certain aspects, CAR is inserted into the inhibitory gene locus via CRISPR-mediated gene editing.

In some aspects, the CAR comprises an antigen-binding domain selected from the group consisting of F(ab') ₂ , Fab', Fab, Fv, and scFv. In certain aspects, the CAR targets one or more tumor-associated antigens selected from the group consisting of CD19, CD319 (CS1), ROR1, CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD5, CD123, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11R alpha, kappa chain, lambda chain, CSPG4, ERBB2, WT-1, TRAIL/DR4, VEGFR2, CD33, CD47, CLL1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, and combinations thereof. In particular aspects, the CAR comprises at least one signaling domain selected from the group consisting of CD3ξ, CD28, OX40/CD134, 4-1BB/CD137, FcεRIγ, ICOS/CD278, ILRB/CD122, IL-2RG/CD132, DAP12, CD70, CD40, and combinations thereof. In some aspects, the immune cell comprises one or more heterologous cytokines, such as one or more of IL-7, IL-2, IL-15, IL-12, IL-18, and IL-21. In certain aspects, CAR also contains a suicide gene, such as membrane-bound non-secreted mutant TNF-alpha or inducible caspase 9.

The present document further provides an expression vector encoding at least one CAR and/or TCR, at least one inhibitory gene sequence, and at least one gRNA. In some aspects, the inhibitory gene sequence is derived from an inhibitory gene selected from the group consisting of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, and CD7. In particular aspects, the gRNA is specific for an inhibitory gene. In some aspects, the vector is a viral vector, such as an AAV vector. The CAR may be flanked by homologous arms for an inhibitory gene. Also provided herein is a host cell, such as a cell of the embodiments, engineered to express the vector of the embodiments. In aspects, the cell is a T cell, an NK cell, a B cell, or a stem cell.

The present document also provides a pharmaceutical composition comprising a population of immune cells of the disclosed embodiments. In another embodiment, a composition comprising a population of cells of the disclosed embodiments is provided for the treatment of an immune disorder, an infectious disease, and/or a malignant tumor.

In a further embodiment, a method of treating a disease or disorder in an individual is provided, comprising administering to the individual an effective amount of immune cells of the disclosed embodiments. In some aspects, the disease or disorder is an infectious disease, a malignancy such as a solid malignancy or a hematological malignancy, or a disorder associated with the immune system. The disorder associated with the immune system can be an autoimmune disorder, a graft-versus-host disease, an allograft rejection, or, for example, an inflammatory condition. In some aspects, the disorder associated with the immune system is an inflammatory condition, and the immune cells have substantially no glucocorticoid receptor expression. In certain aspects, the immune cells are autologous or allogeneic with respect to the recipient individual.

In further aspects, the method also includes administering at least a second therapeutic agent to the individual receiving the immune cells. In some aspects, the at least second therapeutic agent includes chemotherapy, immunotherapy, surgery, radiation therapy, hormonal therapy, or biotherapy. In certain aspects, the immune cells and/or at least the second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, transdermally, subcutaneously, regionally, or by direct injection.

- 3 048895 or perfusion.

In another embodiment, a method for engineering an immune cell to express a CAR is provided, comprising using a CRISPR gRNA to insert the CAR into an inhibitory gene locus of the immune cell. In some aspects, the CAR is encoded by an expression vector, such as a retroviral vector, a plasmid, a lentiviral vector, an adenoviral vector, an adenovirus associated viral vector, etc. In certain aspects, the viral vector is an adenovirus associated vector, such as AAV6.

In some aspects, the vector further comprises an inhibitory gene sequence, such as an inhibitory gene sequence selected from the group consisting of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7, and combinations thereof. In some aspects, the CRISPR gRNA is for an inhibitory gene. In certain aspects, the CAR is flanked by homology arms for the inhibitory gene. In certain aspects, the CAR is inserted into the locus of the inhibitory gene in any part of the gene, such as an exon of the inhibitory gene. The CAR may be under the control of an endogenous promoter of the inhibitory gene. In certain aspects, the CAR disrupts the expression of the inhibitory gene.

In some aspects, the CAR targets one or more tumor-associated antigens selected from the group consisting of CD19, CD319 (CS1), ROR1, CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD5, CD123, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11R alpha, kappa chain, lambda chain, CSPG4, ERBB2, WT-1, TRAIL/DR4, VEGFR2, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, and combinations thereof. In particular aspects, the CAR comprises at least one signaling domain selected from the group consisting of CD3ξ, CD28, OX40/CD134, 4-1BB/CD137, FcεRIγ, ICOS/CD278, ILRB/CD122, IL-2RG/CD132, DAP12, CD70, and CD40. In some aspects, the vector encoding the CAR also encodes a cytokine, such as IL-7, IL-2, IL-15, IL-12, IL-18, IL-21, or a combination thereof. In alternative cases, the cytokine is in a vector separate from the vector encoding the CAR. In certain aspects, the expression construct that encodes the CAR also contains a suicide gene, such as inducible caspase 9 or a membrane-bound non-secreted mutant of TNF-alpha.

Furthermore, the present document provides an immune cell with at least one CAR inserted into an inhibitory gene of an immune cell, such as an immune cell produced by the present methods. The present document also provides a composition comprising a population of immune cells from embodiments of the present invention, such as a population of T cells, B cells, NK cells, NK-T cells, macrophages, stem cells, a mixture thereof, etc.

In another embodiment, a composition is provided comprising a population of cells from the embodiments, and in certain embodiments, the population is used to treat any disease, including at least one immune-related disorder, infectious disease, and/or cancer.

In a further embodiment, a method of treating a disease or disorder in an individual is provided, comprising administering to the individual an effective amount of immune cells of the disclosed embodiments. In some aspects, the disease or disorder is an infectious disease, a malignancy such as a solid malignancy or a hematological malignancy, or an immune system-related disorder. The immune system-related disorder may be an autoimmune disorder, a graft-versus-host disease, an allograft rejection, and/or an inflammatory condition in some cases. In some aspects, the immune system-related disorder is an inflammatory condition, and the immune cells have substantially no glucocorticoid receptor expression. In certain aspects, the immune cells are autologous or allogeneic to the recipient individual.

In further aspects, the method further comprises administering to the individual at least a second therapeutic agent. In some aspects, the at least second therapeutic agent comprises chemotherapy, immunotherapy, surgery, radiation therapy, hormonal therapy, or biotherapy. In certain aspects, the immune cells and/or at least the second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, transdermally, subcutaneously, regionally, or by direct injection or perfusion. The immune cells and at least the second therapeutic agent may be administered at the same time or at different times, and when administered at different times or at the same time, but not in the same formulation, they may be administered by the same or different routes.

Other objects, features and advantages of the present invention will become apparent from

- 4 048895 of the following detailed description. It should be understood, however, that the detailed description and specific examples, indicating specific embodiments of the invention, are given only by way of illustration, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Brief description of the drawings

The following drawings form a part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Fig. 1. CRISPR/Cas9 mediates efficient disruption of multiple genes (NKG2A, CD47, TGFBR2, and CISH) in NK cells. In this set of genes, NKG2A and CD47 were knocked down in the first round of electroporation, while CISH and TGFBR2 were targeted in the second round of electroporation. The knockout efficiency was successfully confirmed by PCR and flow cytometry for both rounds of electroporation. Red (peaks on the right) and blue (peaks on the left) histograms in the flow panels represent protein expression before and after CRISPR knockout, respectively.

Fig. 2. Validation of multiplex gene editing in NK cells using a different gene set (TIGIT (T), CD96 (C), CISH (CH), Adenosine (A)). In this gene set, TIGIT and CD96 were knocked down in the first round of electroporation, while CISH and Adenosine were targeted in the second round of electroporation. The knockout efficiency was successfully confirmed by PCR and flow cytometry for both rounds of electroporation. Red (peaks on the right) and blue (peaks on the left) histograms in the flow panels represent protein expression before and after CRISPR knockout, respectively.

Fig. 3. Disruption of multiple genes (NKG2A, CD47, TGFBR2, and CISH) in NK cells results in increased functionality against tumor cell targets. Secretion of IFN-γ, INFa, and CD107 was enhanced after stimulation with target cell lines. Flow cytometric analysis of IFN-γ, TNFo, and CD 107 production was performed with different NK cells (edited versus Cas9 alone) co-stimulated with target cell lines for 5 h in the presence of Brefeldin A.

Fig. 4A, 4B. Disruption of multiple genes (NKG2A, CD47, TGFBR2, and CISH) in NK cells results in enhanced antitumor cytotoxicity. (Fig. 4A) The cytotoxic activity of gene-edited NK cells compared to Cas9-only NK cells was measured by ^51Cr release assay against K562 cells (Fig. 4B). After 30 min of treatment with recombinant TGF-β (50 ng/ml), pSMAD activity was measured by flow cytometry. Addition of exogenous TGF-β failed to induce pSMAD activation in CAR-NK knockout cells.

Fig. 5. NK cells lose CD 16 and CD62L expression upon cytokine stimulation or target recognition as shown by CyTOF analysis.

Fig. 6. ADAM17 knockout in NK cells prevents CD16 and CD62L shedding.

Fig. 7. ADAM17 knockout in NK cells improves ADCC and cytotoxicity towards K562 targets.

Fig. 8. FACS-based screening of SHP1 knockout efficiency in NK cells after 72 h.

Fig. 9. SHP1 disruption in NK cells results in enhanced antitumor efficacy. NK cells were cocultured with K562 or Raji cells at a 1:1 ratio for 4 h. After incubation, cells were stained with annexin V, and live and dead cells were analyzed. K562 cells are sensitive to killing by NK cells, while Raji cells are resistant to killing by NK cells.

Fig. 10A, B. Disruption of SHP1 in NK cells results in enhanced antitumor efficacy (Fig. 10A). NK cells were co-cultured with K562 or Raji cells at a 2:1 ratio for 5 h (Fig. 10B). Shown are the percentages of lysis at different effector:target ratios, the percentage of IFNy, TNFo, and CD107a, and the percentage of live or dead cells.

Fig. 11. SHP1 disruption in NK-CAR cells results in enhanced antitumor efficacy as assessed by apoptosis assay.

Fig. 12A-C. (Fig. 12A) FACS-based assessment of NKG2A knockout efficiency at day 7. (Fig. 12B) NKG2A disruption in expanded NK cells results in enhanced antitumor efficacy. (Fig. 12C) NKG2A disruption in NK-CAR cells results in enhanced antitumor efficacy against Raji targets.

Fig. 13. The approach was validated with another set of genes - TIGIT (T), CD96 (C), CISH (CH), and Adenosine (ADORA2A) (A). For this set, the genes TIGIT and CD96 were knocked down in one set of NK cells during the first round of electroporation. CISH and Adenosine (ADORA2A) were targeted for the second round of knockout in TIGIT and CD96 KO cells. The knockout efficiency was successfully confirmed by PCR and flow cytometry for both rounds of electroporation. Red (peaks on the right) and blue (peaks on the left) histograms in the flow panels represent protein expression before and after CRISPR knockout, respectively.

Fig. 14. Disruption of multiple genes in NK cells results in enhanced antitumor activity

- 5 048895 efficiency. To assess this, multiple gene knockout cells (NKG2A, CISH, TGFBRII and Adenosine (ADORA2A)) and cells electroporated with Cas9 alone were used as controls for K562 cells (NK-sensitive) and Raji cells (NK-resistant) for 5 h. NK cell function was assessed by flow cytometry and an increase in TNFo, IFNy and CD107a was observed in KO cells upon stimulation of the target cell line.

Fig. 15. Disruption of multiple genes in NK cells results in enhanced antitumor efficacy. To assess this, multiple gene knockout cells (NKG2A, CISH, TGFBRII) and cells electroporated with Cas9 alone were used as controls. NKG2A expression was confirmed by flow cytometry. Addition of exogenous TGF-β failed to induce pSMAD activation in knockout CAR-NK cells.

Fig. 16. Disruption of multiple genes (NKG2A, TGFβR2 and CISH) in NK-CAR cells results in enhanced antitumor efficacy.

Fig. 17. TGFβR2 knockout protects NK-CAR cells from the suppressive effects of TGFβ.

Fig. 18. Multiplexed gene editing is reproducible with different NKCAR constructs and against different targets.

Fig. 19. Multiplex gene editing of multiple inhibitory genes maintains NK architecture and protects NK cells from TFGβ-induced exhaustion.

Description of Illustrative Embodiments

In certain embodiments, the present invention relates to a novel approach using CRISPR-Cas9 technology to simultaneously knockdown (or knockout) two or more genes (e.g., genes (such as those listed in Table 1) including the TGFβR2 receptor, NKG2A, TIGIT, and/or CISH) in human immune cells (e.g., T cells, NK cells, CAR-transduced T cells, or CAR-transduced NK cells). The immune cells may be derived from peripheral blood or cord blood, or a combination thereof.

The present studies have demonstrated that downregulation of these proteins correlates with improved in vivo function, proliferation, persistence, and cytotoxicity of T cells and NK cells. This strategy also protects T cells, NK cells, NK T cells, and iNKT cells from the immunosuppressive tumor microenvironment, which is primarily driven by TGFβ and adenosine. Thus, the present methods can be used to enhance the efficacy of various adoptive cell therapy products (e.g., NK cells, T cells such as virus-specific T cells and regulatory T cells, B cells such as regulatory B cells, CAR-transduced NK cells, CAR-T cells, and TCR-engineered T and NK cells, iNKT cells, NKT cells). Adoptive cell therapy products can be used to treat a variety of diseases, ranging from malignancies (e.g., hematological or solid malignancies) to infectious diseases and immune disorders.

In certain embodiments, the immune cells express at least one CAR, and there have been numerous advances in CAR engineering over the past several years. In fact, CAR-CD19 has shown impressive clinical results in patients with B-cell leukemia and lymphoma, leading to the FDA approval of two CAR T products in the past year. While CAR-transduced T cells have led the way in the past several years, a battery of preclinical studies, as well as a Phase I/II CAR NK study conducted by applicants, have also demonstrated the efficacy of CAR-NK cells against cancer. Despite the advances in CAR engineering, CARs are still primarily transduced into T cells or NK cells using viral vectors that randomly integrate into cellular DNA and can lead to clonal expansion, oncogenic transformation, altered gene transcription, or transcriptional silencing. Thus, it would be useful to find a way to target CAR insertion to a specific DNA locus.

Thus, in one embodiment, the present invention provides methods for inserting a CAR into a specific genetic locus, such as an inhibitory gene or checkpoint protein locus, using CRISPR/Cas9. Inserting a CAR into a gene locus can also be used to simultaneously disrupt gene expression, while optionally also placing it under the control of the promoter of that gene, if desired. In particular, the present methods can direct the insertion of a CAR into an inhibitory gene locus, such as the genes listed in Table 1, including, but not limited to, NKG2A, CISH, PD-1, TIGIT, TIM3, SHP1, or TGFβR2, such as using an AAV6 vector and CRISPR/Cas9 technology. Inserting a CAR into an inhibitory gene locus can disrupt the inhibitory effects of a checkpoint molecule (for example), while allowing CAR expression to be under the regulation of the checkpoint promoter and elevated in the tumor microenvironment. This is suitable for the application of CAR therapy in solid tumors, where increased expression of checkpoint molecules may negatively affect the success of CAR therapy. Thus, additional methods for obtaining adoptive cell therapies such as T cells, B cells, NK, NKT or iNKT cells are proposed using the CAR insertion method, which may have an improved safety profile.

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Definitions

As used herein, substantially free of a particular component means that none of said components have been intentionally formulated into the composition and/or are present only as an impurity or in minor amounts. The amount of said component resulting from any unintentional contamination of the overall composition is thus significantly below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of said component can be detected by standard analytical methods.

As used herein, the singular article "a", "an", or "an" may mean one or more. As used herein in a claim/claims, when used in combination with the word "comprising", the singular articles "an" or "an" may mean one or more than one. As used herein, "another" may mean at least a second or more. Furthermore, the terms "having", "including", "comprising", and "consisting" are interchangeable and one skilled in the art will recognize that the terms are open-ended terms. In particular embodiments, aspects of the invention may consist essentially of or consist of one or more sequences of the invention, for example. Some embodiments of the invention may consist of or consist essentially of one or more elements, steps of methods and/or processes of the invention. It is intended that any method or composition described herein may be implemented in relation to any other method or composition described herein. The scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of material, means, methods and steps described in the description. As used herein, the terms or and and/or are used to describe multiple components in combination or exclusive of each other. For example, x, y, and/or z may refer to only x, only y, only z, x, y and z, (x and y) or z, x or (y and z) or x or y or z. In particular, it is contemplated that x, y or z may be specifically excluded from an embodiment.

The use of the term or in the claims is intended to mean and/or unless it is explicitly stated that it refers only to alternatives or the alternatives are mutually exclusive, although the invention supports a definition that refers only to alternatives and and/or. As used herein, another may mean at least a second or more. The terms about, essentially and approximately mean essentially the stated value plus or minus 5%.

Reference throughout this specification to one of the embodiments, an embodiment, a particular embodiment, a related embodiment, a specific embodiment, an additional embodiment, or a further embodiment, or combinations thereof, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the above phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

An immune disorder, immune-related disorder, or immune-mediated disorder refers to a disorder in which the immune response plays a key role in the development or progression of the disease. Immune-mediated disorders include autoimmune disorders, allograft rejection, graft-versus-host disease, and inflammatory and allergic conditions.

An immune response is a response of an immune cell, such as a B cell or a T cell or an innate immune cell, to a stimulus. In one embodiment, the response is specific to a particular antigen (antigen-specific response).

As used herein, the term inhibitory gene refers to a gene whose gene product is directly or indirectly detrimental to the activity, proliferation, and/or persistence of one or more types of immune cells.

An autoimmune disease refers to a condition in which the immune system produces an immune response (e.g., a B-cell or T-cell response) against an antigen that is part of a healthy host (i.e., an autoantigen), with subsequent tissue damage. The autoantigen may originate from a host cell or may originate from a commensal organism, such as microorganisms (known as commensal organisms) that commonly colonize mucous membranes.

As used herein, the term engineered refers to an entity produced by the hand of man, including a cell, nucleic acid, polypeptide, vector, etc. In at least some cases, an engineered object is synthetic and contains elements that are not naturally occurring or configured in the manner in which they are

- 7 048895 are used in the invention.

Treatment or cure of a disease or condition refers to the implementation of a protocol, which may include the administration of one or more drugs to a patient, with the goal of alleviating the signs or symptoms of the disease. Desirable effects of treatment include a decrease in the rate of disease progression, improvement or alleviation of the disease, remission, or an improvement in prognosis. Alleviation may occur before the onset of signs or symptoms of the disease or condition, as well as after their onset. Thus, treatment or cure may include the prevention or avoidance of the disease or undesirable condition. In addition, treatment or cure does not require complete alleviation of signs or symptoms, does not require a cure, and in particular includes protocols that have only a minor effect on the patient.

The term therapeutic benefit or therapeutically effective as used throughout this application refers to anything that promotes or improves the well-being of an individual with respect to the medical treatment of the condition. This includes, but is not limited to, reducing the frequency or severity of signs or symptoms of the disease. For example, treating a cancer may include, for example, reducing the size of the tumor, reducing the invasiveness of the tumor, reducing the rate of growth of the cancer, or preventing metastasis. Treating a cancer may also refer to prolonging the survival of an individual with the cancer.

The individual and the patient are human or non-human, such as primates, mammals, and vertebrates. In possible embodiments, the individual is a human.

As used herein, a mammal is a suitable subject for the method of the present invention. The animal may be any representative of the higher class of vertebrates Mammalia, including humans, characterized by viviparity, body hair, and mammary glands in women that secrete milk to feed their young. In addition, mammals are characterized by their ability to maintain a constant body temperature despite changing climatic conditions. Examples of mammals include humans, cats, dogs, cows, mice, rats, horses, goats, sheep, and chimpanzees. Mammals may be referred to as patients, or subjects, or individuals.

The phrases pharmaceutically or pharmacologically acceptable refer to molecular structures and compositions that do not cause adverse, allergic or other undesirable reactions when administered to an animal, such as a human, in need thereof. The preparation of a pharmaceutical composition that includes an additional active ingredient will be understood by those skilled in the art in light of the present invention. In addition, for animals (e.g., humans), it should be understood that the preparations must meet the standards of sterility, pyrogenicity, general safety and purity as required by the FDA's Office of Biological Standards.

As used herein, a pharmaceutically acceptable carrier includes any and all aqueous vehicles (e.g., water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non-aqueous vehicles (e.g., propylene glycol, polyethylene glycol, vegetable oil, injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonicity agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, colorants, nutrient and fluid replenishing agents, such as combinations thereof that are known to one of skill in the art. areas. The pH and the exact concentration of the various components in the pharmaceutical composition are adjusted in accordance with known parameters.

As used herein, gene disruption refers to the elimination or reduction of the expression of one or more gene products encoded by the genes in a cell, compared to the level of expression of the gene product in the absence of disruption. Typical gene products include mRNA and protein products encoded by the gene. The disruption is in some cases temporary or reversible, and in other cases irreversible. The disruption is in some cases for a functional or full-length protein or mRNA, although a truncated or non-functional product may be obtained. In some embodiments herein, gene activity or function, as opposed to expression, is impaired. Gene disruption is generally caused by artificial means, i.e., by adding or introducing a compound, molecule, complex, or composition, and/or by disrupting the nucleic acid of the gene or associated with the gene, such as at the DNA level. Examples of methods for gene disruption include silencing

- 8 048 895 expression, knockdown, knockout and/or disruption methods of genes such as gene editing. Examples include antisense technologies such as RNAi, siRNA, shRNA, and/or ribozymes, which generally result in a transient reduction in expression, as well as gene editing methods that result in targeted inactivation or disruption of genes, such as by inducing breaks and/or homologous recombination. Examples include insertions, mutations, and deletions. Disruption typically results in suppression and/or complete absence of expression of the normal or wild-type product encoded by the gene. Examples of such gene disruptions are insertions, frameshifts and missense mutations, deletions, knockin and knockout of a gene or part of a gene, including deletions of the entire gene. Such disruptions may occur in the coding region, such as in one or more exons, resulting in the inability to produce a full-length product, a functional product, or any product, such as by insertion of a stop codon. Such disruptions may also occur as a result of disruption of a promoter or enhancer or other region affecting transcriptional activation, to prevent transcription of genes. Gene disruption includes gene targeting, including targeted gene inactivation by homologous recombination.

Multiplexed gene editing

In some embodiments, the present invention relates to multiplex gene editing of any type of immune cell. CRISPR is one example that can be used to disrupt the expression of two or more genes, such as 3, 4, 5, 6, 7, 8, 9, 10 or more genes in an immune cell. The genes can be selected from the genes listed in Table 1, such as NK cell receptor A (NKG2A), sialic acid-binding Ig-like lectin 7 (SIGLEC-7, CD328), lymphocyte activating protein 3 (LAG3), T cell immunoglobulin, mucin family member 3 (TIM3, CD366, HAVCR2), cytokine-inducible SH2-containing protein (CISH, CIS-1, SOCS), forkhead box protein O1 (FOXO1), transforming growth factor beta receptor 2 (TGFβR2), T cell immunoreceptor with Ig and ITIM domains (TIGIT), CD96, adenosine receptor 2A (ADORA2), nuclear receptor subfamily 3 group C member 1 (NR3C1), programmed cell death 1 (PD1), programmed cell death 1 ligand 1 (PDL-1), 2 programmed cell death 1 (PDL-2), CD47, signal regulatory protein alpha (SIRPA), SH2 domain-containing inositol 5-phosphatase 1 (SHIP1), ADAM metallopeptidase domain 17 (ADAM17), ribosomal protein S6 (RPS6), eukaryotic initiation factor 4E binding protein 1 (4EBP1), CD25, CD40, interleukin 21 receptor (IL21R), intercellular adhesion molecule 1 (ICAM1), CD95, CD80, CD86, interleukin 21 receptor (IL10R), CD5, CD7, or there may be other inhibitory genes. Gene editing allows for the simultaneous disruption of expression of multiple genes.

In some embodiments, the disruption of genes is carried out by a disruption in a gene, such as a knockout, insertion, missense or frameshift mutation, such as a biallelic frameshift mutation, deletion of all or part of a gene, such as one or more exons or parts thereof, thus, and/or knockin. For example, the disruption can be carried out using sequence-specific or targeted nucleases, including DNA-binding targeted nucleases, such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), and RNA-directed nucleases, such as CRISPR-associated nuclease (Cas), specifically designed to target a gene sequence or a part thereof.

In some embodiments, the disruption is temporary or reversible, such that gene expression is restored at a later time. In other embodiments, the disruption is not reversible or temporary, such as being permanent.

In some embodiments, gene disruption is performed by inducing one or more double-strand breaks and/or one or more single-strand breaks in a gene, typically in a targeted manner. In some embodiments, double-strand or single-strand breaks are produced by a nuclease, such as an endonuclease, such as a gene-targeting nuclease. In some aspects, breaks are induced in a coding region of a gene, such as an exon. For example, in some embodiments, induction occurs near the N-terminal portion of the coding region, such as in the first exon, in the second exon, or in a subsequent exon.

A guide RNA and a CRISPR enzyme or mRNA encoding a CRISPR enzyme may be introduced into an immune cell. In some aspects, 1, 2, 3, 4, 5 or more guide RNAs are introduced into the cell at the same time. For example, one, two or three guide RNAs may be introduced into the cell during the first electroporation, and then one, two or three additional guide RNAs may be introduced during the second electroporation, etc.

In some embodiments, gene disruption is performed using antisense methods such as RNA interference (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), and/or using ribozymes to selectively silence or repress gene expression. siRNA technology is RNAi that utilizes a double-stranded RNA molecule having a sequence homologous to the nucleotide sequence of an mRNA that is transcribed from a gene and a sequence

- 9 048895 complementary to a nucleotide sequence. The miRNA is primarily homologous/complementary to a single region of mRNA that is transcribed from a gene, or may be an miRNA that includes multiple RNA molecules that are homologous/complementary to different regions. In some aspects, the miRNA includes a polycistronic construct.

In some embodiments, the disruption is achieved using a DNA-targeting molecule, such as a DNA-binding protein or a DNA-binding nucleic acid, or a complex, compound, or composition comprising them, that specifically binds or hybridizes to a gene. In some embodiments, the DNA-targeting molecule comprises a DNA-binding domain, such as a zinc finger protein (ZFP) DNA-binding domain, a transcription activator-like protein (TAL) or TAL effector (TALE) DNA-binding domain, a clustered regularly interspaced short palindromic repeats (CRISPR) DNA-binding domain, or a meganuclease DNA-binding domain. The binding domains of zinc fingers, TALEs, and CRISPR systems can be engineered to bind to a predetermined nucleotide sequence, such as by engineering (changing one or more amino acids) the recognition helix region of a natural zinc finger protein or TALE protein. Engineered DNA-binding proteins (zinc fingers or TALEs) are proteins that are not naturally occurring. Rational design criteria include the use of substitution rules and computerized algorithms to process information in a database storing information on existing ZFP and/or TALE designs and linkage data.

In the case of CRISPR-mediated disruption, the guide RNA and endonuclease can be introduced into immune cells by any method known in the art to provide delivery into cells or subcellular compartments, and agents/chemicals and/or molecules (proteins and nucleic acids) that can be used include, but are not limited to, liposomal delivery vehicles, polymeric carriers, chemical carriers, lipoplexes, polyplexes, dendrimers, nanoparticles, emulsion, natural endocytosis or the phagocytosis pathway, and physical methods such as electroporation. In particular aspects, electroporation is used to introduce the guide RNA and endonuclease, or a nucleic acid encoding an endonuclease.

In one example, a specific method, a method for CRISPR knockout of multiple genes may include isolating immune cells, such as NK cells, from cord blood or peripheral blood. NK cells may be isolated and seeded onto plates with irradiated feeder cells at a ratio of 1:2, as one example. The cells may then be electroporated with gRNA and Cas9 in the presence of IL-2 at a concentration of 200 IU/mL. The medium may be replaced after 24 hours, as one example. After 1-3 days, the NK cells are isolated to remove feeder cells and then transduced with a CAR construct. The NK cells may then be subjected to a second CRISPR Cas9 knockout for additional gene(s). Following electroporation, the NK cells may be co-seeded with feeder cells for 5-9 days.

Table 1

Genes for multiplex editing or CAR knockin. Examples of knockin loci are shown.

NK cells, T cells, or MSC cells NKG2A Exon 4 SIGLEC-7 Exon 1 LAG3 Exon 1 TIM3 Exon 2 CISH Exon 5 FOXO1 Exon 1 TGFBR2 Exon 5 TIGIT Exon 2 CD96 Exon 2 ADORA2 Exon 2 NR3C1 Exon 2 PD1 Exon 1 PDL-1 Exon 3 PDL-2 Exon 3 CD47 Exon 2 SIRPA Exon 2

- 10 048895

SHIP1 Exon 1 ADAM17 Exon 1 B2M Exon 2 CD16 B cells or T cells RPSS6 Exon 2 4EBP1 Exon 4 CD25 Exon 3 CD40 Exon 3 IL21R Exon 1 ICAM1 Exon 4 CD95 Exon 2 CD80 Exon 3 CD86 Exon 1 IL10R Exon 3 CD5 CD7 Exon 2

Table 2

Examples of gRNA sequences for gene knockout

CISH (Exon 4) AGGCCACATAGTGCTGCACA (gRNA 1); SEQ ID NO:1 TGTACAGCAGTGGCTGGTGG (gRNA 2); SEQ ID NO:2 NKG2A (Exon 4) AACAACTATCGTTACCACAG; SEQ ID NO:3 A2AR (Exon 3) CTCCTCGGTGTACATCACGG (gRNA 1); SEQ ID NO:4 AGTAGTTGGTGACGTTCTGC (gRNA 2); SEQ ID NO:5 TIGIT (Exon 3) ACCCTGATGGGACGTACACT; SEQ ID NO:6 CD96 (Exon 2) AGGCACAGTAGAAGCCGTAT; SEQ ID NO:7 TIM3 (Exon 2) AGACGGGCACGAGGTTCCCT; SEQ ID NO:8 SHP1 (Exon 4) TCACGCACAAGAAACGTCCA; SEQ ID NO:9 PD1 (Exon 2) CCCCTTCGGTCACCCACGAGC; SEQ ID NO: 10 PDL1 (Exon 3) ATTTACTGTCACGGTTCCCA; SEQ ID NO:11 PDL2 (Exon 3) CCCCATAGATGATTATGCAT; SEQ ID NO: 12 TGFBR2 (Exon 5) GACGGCTGAGGAGCGGAAGA (gRNA 1); SEQ ID NO:13 TGTGGAGGTGAGCAATCCCC (gRNA 2); SEQ ID NO:14

In some embodiments, the immune cells of the present invention are modified to alter the expression of two or more genes. In some embodiments, the alteration of gene expression is accomplished by introducing a disruption into the gene, such as a knockout, an insertion, a missense mutation, or a frameshift mutation, such as a biallelic frameshift mutation, a deletion of all or part of the gene, such as one or more exons or a portion thereof, and/or a knockin. In particular embodiments, the altered gene expression can be accomplished by a sequence-specific or targeted nuclease, including a DNA-binding targeted nuclease, such as an RNA-guided nuclease, such as a CRISPR-linked nuclease (Cas), specifically designed to target a gene sequence or a portion thereof.

In some embodiments, the alteration of gene expression, activity and/or function is accomplished by disrupting the gene. In some aspects, the gene is modified such that its expression is reduced by at least 10, 20, 30 or 40%, generally at least or about 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% compared to expression in the absence of gene modification or in the absence of components introduced to effect the modification.

- 11 048895

In some embodiments, the disruption is temporary or reversible, such that gene expression is restored later if desired. In other embodiments, the disruption is not reversible or temporary, such as being permanent.

In some embodiments, the gene alteration is performed by inducing one or more double-strand breaks and/or one or more single-strand breaks in the gene, typically in a targeted manner. In some embodiments, the double-strand or single-strand breaks are produced by a nuclease, such as an endonuclease, such as a gene-targeting nuclease. In some aspects, the breaks are induced in a coding region of the gene, such as an exon. For example, in some embodiments, the induction occurs near the N-terminal portion of the coding region, such as in the first exon, in the second exon, or in a subsequent exon.

In some aspects, double-strand or single-strand breaks are repaired by a cellular repair process, such as non-homologous end joining (NHEJ) or homology-directed repair (HDR). In some aspects, the repair process is error-prone and results in gene disruption, such as a frameshift mutation, such as a biallelic frameshift mutation, which can result in a complete gene knockout. For example, in some aspects, the disruption includes inducing a deletion, mutation, and/or insertion. In some embodiments, the disruption results in the presence of a premature stop codon. In some aspects, the presence of an insertion, deletion, translocation, frameshift mutation, and/or premature stop codon results in impaired gene expression, activity, and/or function.

In some embodiments, the alteration is carried out using one or more DNA-binding nucleic acids, such as the alteration is carried out via an RNA-directed endonuclease (RGEN). For example, the alteration can be carried out using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. In general, the CRISPR system collectively refers to transcripts and other elements involved in the expression or directing the activity of CRISPR-associated (Cas) genes, including a Cas gene encoding sequence, a tracr (transactivating CRISPR) sequence (e.g., tracpRNA or active partial tracpRNA), a tracr-mate sequence (encompassing a direct repeat and a tracpRNA-processed partial direct repeat with respect to an endogenous CRISPR system), a guide sequence (also referred to as a spacer with respect to an endogenous CRISPR system), and/or other transcripts from a CRISPR locus.

A CRISPR/Cas nuclease or CRISPR/Cas nuclease system may include a noncoding RNA molecule (guide) that sequence-specifically binds to DNA and a Cas protein (e.g., Cas9) with nuclease functionality (e.g., two nuclease domains). One or more elements of the CRISPR system may be derived from a type I, type II, or type III CRISPR system, such as from a specific organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes.

In some aspects, a Cas nuclease and a gRNA (including a fusion of a cpRNA specific for a target sequence and a fixed tracpRNA) are introduced into a cell. Generally, target sites at the 5' end of the gRNA direct the nuclease to a target site, such as a gene, using complementary base pairing. The target site can be selected based on its location immediately 5' of a protospacer adjacent motif (PAM) sequence, typically NGG or NAG. In this regard, the gRNA is targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to match the target DNA sequence. Generally, a CRISPR system is characterized by elements that facilitate the formation of a CRISPR complex at a target sequence site. Typically, the target sequence is essentially a sequence for which a complementary guide sequence is designed, where hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Complete complementarity is not necessarily required, as long as the complementarity is sufficient to induce hybridization and promote the formation of a CRISPR complex.

The CRISPR system can induce double-strand breaks (DSBs) at a target site, followed by disruptions or alterations as described herein. In other embodiments, Cas9 variants that are considered nicks are used to induce a single strand break at a target site. Paired nicks can be used, for example, to increase specificity, each directed by a pair of different gRNAs that target sequences such that when nicks are introduced simultaneously, a 5' overhang is formed. In other embodiments, a catalytically inactive Cas9 is fused to a heterologous effector domain, such as a transcriptional repressor or activator, to affect gene expression.

The target sequence may include any polynucleotide, such as polynucleotides

- 12 048895

DNA or RNA. The target sequence may be located in the nucleus or cytoplasm of the cell, such as within a cell organelle. In general, a sequence or template that can be used for recombination at a target locus containing the target sequence is referred to as an editing template or an editing polynucleotide or an editing sequence. In some aspects, an exogenous template polynucleotide may be referred to as an editing template. In some aspects, the recombination is homologous recombination.

Typically, for the endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and combined with one or more Cas proteins) results in cleavage of one or both strands within the target sequence or adjacent to the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs). A tracr sequence, which may include or consist of all or a portion of a wild-type tracr sequence (e.g., about or about more than 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridizing at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to a guide sequence. The tracr sequence has sufficient complementarity with the tracr mate sequence to hybridize and participate in the formation of a CRISPR complex, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity over the length of the tracr mate sequence when optimally aligned.

One or more vectors that direct the expression of one or more CRISPR system elements can be introduced into a cell such that expression of the CRISPR system elements directs the formation of a CRISPR complex at one or more target sites. The components can also be delivered to the cells as proteins and/or RNA. For example, the Cas enzyme, the guide sequence associated with the tracr-mate sequence, and each tracr sequence could be operably linked to separate regulatory elements in separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined in a single vector with one or more additional vectors providing any CRISPR system components not included in the first vector. The vector can contain one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a cloning site). In some embodiments, the one or more insertion sites are located upstream and/or downstream of one or more sequence elements of the one or more vectors. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences within the cell.

The vector may contain a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof. These enzymes are known; For example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

The CRISPR enzyme may be Cas9 (e.g., from S. pyogenes or S. pneumonia). The CRISPR enzyme may direct cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within a sequence complementary to the target sequence. The vector may encode a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, an aspartate to alanine substitution (D10A) in the catalytic domain of RuvC I Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves one strand). In some embodiments, the Cas9 nickase can be used in combination with a guide sequence(s), such as two guide sequences that target the sense and antisense strands of the target DNA, respectively. This combination allows both strands to be cut and used to induce NHEJ or HDR.

In some embodiments, the enzyme-coding sequence encoding the CRISPR enzyme is codon optimized for expression, in particular, in cells, such as eukaryotic cells. The eukaryotic cells may be cells of or derived from a particular organism, such as mammals, including as

- 13 048895 non-limiting examples human, mouse, rat, rabbit, dog, or non-human primate. Generally, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in host cells of interest by replacing at least one codon of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell, while maintaining the native amino acid sequence. Different species show a particular bias for certain codons for a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which in turn is believed to depend, in part, on the properties of the codons being translated and the availability of a particular transfer RNA (tRNA) molecule. The prevalence of selected tRNAs in a cell generally reflects the codons most frequently used in peptide synthesis. Thus, genes can be adapted for optimal gene expression in a given organism based on codon optimization.

In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize to the target sequence and to direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its target sequence when optimally aligned using a suitable alignment algorithm is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more.

An optimal alignment can be determined using any suitable sequence alignment algorithm, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, Burrows-Wheeler transform-based algorithms (e.g., the Burrows-Wheeler aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

The CRISPR enzyme may be part of a fusion protein comprising one or more heterologous protein domains. The CRISPR enzyme fusion protein may include any additional sequence, optionally a linker sequence between any two domains. Examples of protein domains that may be fused to the CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). The CRISPR enzyme may be fused to a gene sequence encoding a protein or protein fragment that binds DNA molecules or binds other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may be part of a fusion protein comprising a CRISPR enzyme are described in US 20110059502, incorporated herein by reference.

Insertion of CAR and/or TCR into the inhibitory gene locus.

In some embodiments, the present invention relates to inserting a CAR and/or TCR into a specific gene locus in immune cells. The CAR and/or TCR can be inserted into an inhibitory gene locus, such as a gene selected from the group consisting of NKG2A, Siglec 7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, adenosine receptor 2A, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPa, SHIP1, ADAM17, pS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, CD7, and combinations thereof.

The insertion of one or more CARs and/or TCRs according to any of the methods described herein may be site-specific. For example, one or more CARs and/or TCRs may be inserted immediately adjacent to or near a promoter. In another example, one or more transgenes may be inserted adjacent to, near, or within an exon of a gene (e.g., an inhibitory gene). Such insertions may be used to knock out a CAR and/or TCR while disrupting gene expression. In another example, one or more CARs and/or TCRs may be inserted at

- 14 048895 adjacent to, near or within an intron of a gene. The CAR and/or TCR can be delivered using an adeno-associated virus (AAV)-based viral vector and integrated into the target genomic site. In some cases, an rAAV vector can be used to directly deliver the transgene to a specific site. For example, in some cases, the CAR and/or TCR can be integrated into at least a portion of NKG2A, Siglec 7, lAg3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, adenosine receptor 2A, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPa, SHIP1, ADAM17, pS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, CD5, or the CD7 gene using an rAAV or AAV vector.

Modification of a target locus of a cell can be achieved by introducing DNA into cells wherein the DNA is homologous to the target locus. The DNA can include a marker gene that allows selection of cells containing the integrated construct. Complementary DNA in the targeting vector can recombine with chromosomal DNA at the target locus. The marker gene can be flanked by complementary DNA sequences, a 3' recombination arm and a 5' recombination arm. Multiple loci can be targeted in a cell. For example, transgenes with recombination arms specific for one or more target loci can be introduced simultaneously so that multiple genomic modifications occur in a single step. The homology arms can be from about 0.2 kb to about 5 kb in length, such as from about 0.2 kb, 0.4 kb, or 1.5 kb. 0.6 kb, 0.8 kb, 1.0 kb, 1.2 kb, 1.4 kb, 1.6 kb, 1.8 kb, 2.0 kb, 2.2 kb, 2.4 kb, 2.6 kb, 2.8 kb, 3.0 kb, 3.2 kb, 3.4 kb, 3.6 kb, 3.8 kb, 4.0 kb, 4.2 kb, 4.4 kb, 4.6 kb, 4.8 kb, up to approximately 5.0 kb in length, for example.

In one method, the guide RNA can be designed to target a region of the inhibitory gene locus, such as adjacent to a promoter, exon, or intron of the gene. The guide RNA can target the 5' end of an exon, such as the first, second, or third exon of the inhibitory gene. The guide RNA can be included in a repair template of an AAV vector. The AAV vector can encode a self-cleaving peptide 2A, such as a P2A peptide, followed by CAR cDNA. The CAR cassette and the guide RNA sequence can be flanked by homology arms to the inhibitory gene. The AAV vector and Cas9, such as Cas9 mRNA, can then be introduced into the immune cell, such as by electroporation.

Immune cells.

Some embodiments of the present invention relate to immune cells that are engineered to have a knockout of multiple genes and/or to have a CAR knockin at an inhibitory gene locus. The immune cells can be T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or gamma delta T cells), NK cells, invariant NK cells, NKT cells, B cells, stem cells (e.g., mesenchymal stem cells (MSC) or induced pluripotent stem (iPS) cells). The immune cells can be virus-specific, express a CAR and/or a TCR. In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils and/or basophils. The present document also provides methods for obtaining and constructing immune cells, as well as methods for using and administering cells for adoptive cell therapy, in which case the cells can be autologous or allogeneic. Thus, immune cells can be used as immunotherapy, for example, to target malignant cells.

Immune cells can be isolated from individuals, particularly humans. Immune cells can be obtained from an individual of interest, such as an individual suspected of having a particular disease or condition, an individual suspected of being predisposed to a particular disease or condition, or an individual undergoing therapy for a particular disease or condition. condition. Immune cells can be obtained from any location in an individual, including, but not limited to, blood, cord blood, spleen, thymus, lymph nodes, and bone marrow. The isolated immune cells can be used immediately, or they can be stored for a period of time, such as by freezing.

Immune cells can be enriched/purified from any tissue in which they are found, including, but not limited to, blood (including blood collected by blood banks or blood from a blood bank), spleen, bone marrow, tissues removed and/or exposed during surgical procedures, and tissues obtained by biopsy. Tissues/organs from which immune cells are enriched, isolated and/or purified can be isolated from both living and non-living individuals, wherein non-living individuals are organ donors. In particular embodiments, immune cells are isolated from blood, such as peripheral blood or cord blood, or a mixture thereof. In some aspects, immune cells isolated from cord blood have enhanced immunomodulatory capacity, such as measured by suppression of CD4 positive or CD8 positive T cells. In specific aspects, immune cells are isolated from pooled blood, particularly pooled umbilical cord blood, to enhance immunomodulatory capacity. The pooled blood may be from 2 or more sources, such as 3, 4, 5, 6, 7,

- 15 048895

8, 9, 10 or more sources (eg donors).

The immune cell population may be obtained from an individual in need of therapy or suffering from a disease associated with decreased immune cell activity. Thus, the cells may be autologous to the individual in need of therapy. Alternatively, the immune cell population may be obtained from a donor, especially a histocompatible donor. The immune cell population may be obtained from peripheral blood, cord blood, bone marrow, spleen, or any other organ/tissue in which immune cells are present in the individual or donor. The immune cells may be isolated from a plurality of individuals and/or donors, such as from pooled cord blood.

When the immune cell population is obtained from a donor other than the individual, the donor is preferably allogeneic, provided that the cells obtained are compatible with the subject in the sense that they can be administered to the individual. Allogeneic donor cells may or may not be human leukocyte antigen (HLA) compatible.

T cells.

In some embodiments, the immune cells are T cells. Several major approaches to obtaining, activating, and expanding functional anti-tumor effector cells have been described over the past two decades. These include: autologous cells, such as tumor infiltrating lymphocytes (TILs); T cells activated ex vivo using autologous DCs, lymphocytes, artificial antigen-presenting cells (APCs), or beads coated with T cell ligands and activating antibodies, or cells isolated by capturing the membrane of target cells; allogeneic cells naturally expressing the host anti-tumor T cell receptor (TCR); and non-tumor-specific autologous or allogeneic cells genetically reprogrammed or redirected to express a tumor-reactive TCR or chimeric TCR molecules exhibiting antibody-like tumor recognition capability, known as T bodies. These approaches have resulted in a variety of protocols for T cell production and immunization that can be used in the methods described herein.

In some embodiments, the T cells are obtained from blood, bone marrow, lymph, umbilical cord, or lymphoid organs. In some aspects, the cells are human cells. The cells are typically primary cells, such as cells isolated directly from an individual and/or isolated from an individual and frozen. In some embodiments, the cells comprise one or more subsets of T cells or other cells, such as total populations of T cells, CD4 ⁺ cells, CD8 ⁺ cells, and subsets thereof, such as those defined by function, activation state, maturity, differentiation potential, expansion, recycling, localization, and/or persistence, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or differentiation grade. With respect to an individual to be treated, the cells may be allogeneic and/or autologous. In some aspects, as in standard technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from an individual, preparing, processing, culturing, and/or engineering them as described herein, and reintroducing them into the same patient before or after cryopreservation.

Among the T cell subtypes and subpopulations (e.g. CD4+ and/or CD8+ T cells) are naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes such as stem cell memory T (TSCM), central memory T cells (TCM), effector memory T cells (TEM) or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, natural and adaptive regulatory T (Treg) cells, helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.

In some embodiments, one or more T cell populations are enriched or depleted in cells that are positive for a particular marker, such as surface markers, or negative for a particular marker. In some cases, such markers are markers that are absent or expressed at relatively low levels in certain T cell populations (e.g., non-memory cells), but are present or expressed at relatively higher levels in some other T cell populations (e.g., memory cells).

In some embodiments, T cells are separated from the PBMC sample by negative selection for markers, such as CD 14, expressed on non-T cells, such as B cells, monocytes, or other leukocytes. In some aspects, the CD4 ⁺ or CD8 ⁺ selection step is used,

- 16 048895 to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations may be further divided into subpopulations by positive or negative selection for markers expressed or expressed at relatively higher levels in one or more subpopulations of naive, memory, and/or effector T cells.

In some embodiments, the CD8+ T cells are further enriched or depleted for naive cells, central memory cells, effector memory cells, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the relevant subpopulation. In some embodiments, enrichment for central memory T cells (TCM) is performed to improve efficacy, such as to increase survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such subpopulations.

In some embodiments, the T cells are autologous T cells. In this method, tumor samples are obtained from patients and a single cell suspension is prepared. The single cell suspension can be prepared by any suitable method, such as mechanically (disaggregation of the tumor using, for example, a softMACS™ dissociator, Miltenyi Biotec, Auburn, CA) or enzymatically (for example, collagenase or DNase). Enzymatically digested single cell tumor suspensions are cultured in interleukin-2 (IL-2).

The cultured T cells can be pooled and rapidly expanded. Rapid expansion provides an increase in the number of antigen-specific T cells by at least about 50 times (e.g., 50, 60, 70, 80, 90, or 100 times or more) over a period of about 10 to about 14 days. More preferably, rapid expansion provides an increase by at least about 200 times (e.g., 200, 300, 400, 500, 600, 700, 800, 900 or more) over a period of about 10 to about 14 days.

Expansion can be accomplished by any of a number of methods known in the art. For example, T cells can be rapidly expanded using non-specific T cell receptor stimulation in the presence of feeder lymphocytes and either interleukin-2 (IL-2) or interleukin-15 (IL-15), with IL-2 being preferred. The non-specific T cell receptor stimulus can include approximately 30 ng/ml OKT3, a monoclonal antibody to mouse CD3 (available from Ortho-McNeil®, Raritan, N.J.). Alternatively, T cells can be rapidly expanded by stimulating peripheral blood mononuclear cells (PBMC) in vitro with one or more cancer antigens (including antigenic portions thereof, such as an epitope(s), or a cell), which may optionally be expressed by a vector, such as human leukocyte antigen A2 (HLA-A2) binding peptide, in the presence of a T cell growth factor such as 300 IU/ml IL-2 or IL-15, with IL-2 being preferred. The in vitro induced T cells are rapidly expanded by restimulation with the same cancer antigen(s) presented on HLA-A2-expressing antigen-presenting cells. Alternatively, T cells can be restimulated with irradiated autologous lymphocytes or irradiated HLA-A2+ allogeneic lymphocytes and IL-2, for example.

Autologous T cells can be modified to express a T cell growth factor that promotes the growth and activation of autologous T cells. Suitable T cell growth factors include, for example, interleukin (IL)-2, IL-7, IL-15, and IL-12. Suitable modification methods are known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In certain aspects, the modified autologous T cells express high levels of T cell growth factor. Coding sequences for T-cell growth factors such as IL-12 are readily available in the art, as are promoters whose functional linkage to the T-cell growth factor coding sequence facilitates high levels of expression.

NK cells.

In some embodiments, the immune cells are natural killer (NK) cells. NK cells are a subset of lymphocytes that exhibit spontaneous cytotoxicity against a variety of tumor cells, virus-infected cells, and certain normal cells in the bone marrow and thymus. NK cells differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus. NK cells can be detected using specific surface markers such as CD16, CD56, and CD8 in humans. NK cells do not express T cell antigen receptors, the pan T marker CD3, or surface immunoglobulin B cell receptors.

In certain embodiments, the NK cells are derived from human peripheral blood mononuclear cells (PBMC), unstimulated leukapheresis products (PBSC), human embryonic stem cells (hESC), iPSC, bone marrow, or umbilical cord blood using methods well known in the art. In particular, for

- 17 048895 NK cell production utilizes cord blood cells. In certain aspects, NK cells are isolated and expanded using a previously described ex vivo NK cell expansion method (Spanholtz et al., 2011; Shah et al., 2013). In this method, blood mononuclear cells are isolated by Ficoll density gradient centrifugation and cultured in a bioreactor with IL-2 and artificial antigen-presenting cells (iAPCs). After 7 days, the cell culture is depleted of any CD3-expressing cells and re-cultured for an additional 7 days. The cells are again depleted of CD3 and characterized to determine the percentage of CD56 ⁺ /CD3- cells or NK cells. In other ways, cord blood cells are used to generate NK cells by isolating CD34 ⁺ cells and differentiating them into CD56 ⁺ /CD3 cells by culturing in medium containing SCF, IL-7, IL-15, and IL-2.

In particular embodiments, the NK cells are expanded at some point during their production. In particular cases, expanding the NK cells comprises: stimulating cord blood mononuclear cells (MNCs) in the presence of antigen-presenting cells (APCs) and IL-2; and re-stimulating the cells with APCs to obtain expanded NK cells, wherein, at least in some cases, the method is carried out in a bioreactor. The stimulating step may direct MNCs to the NK cells. The re-stimulating step may or may not include the presence of IL-2. In particular aspects, the method does not include removing or adding any components of the medium during the stimulating step. In particular aspects, the method is carried out for less than 15 days, such as 14 days.

In a particular embodiment, NK cells are expanded by an ex vivo expansion method comprising: (a) obtaining a starting population of mononuclear cells (MNCs) from umbilical cord blood; (b) stimulating the MNCs in the presence of antigen-presenting cells (APCs) and IL-2; and (c) restimulating the cells with APCs to obtain expanded NK cells, wherein the method is carried out in a bioreactor and in accordance with good manufacturing practice (GMP) requirements. The stimulation in step (b) may direct the MNCs to the NK cells. Step (c) may or may not include the presence of IL-2. In particular aspects, the method does not include removing or adding any media components during step (b). In particular aspects, the method is carried out for less than 15 days, such as 14 days.

In some aspects, the method further comprises depleting cells positive for one or more specific markers, such as CD3, for example. In certain aspects, the depletion step is performed between steps (b) and (c). In some aspects, the cells are removed from the CD3 depletion bioreactor and placed in the bioreactor for step (c).

In certain aspects, obtaining a starting population of PNCs from cord blood comprises thawing the cord blood in the presence of dextran, human serum albumin (HSA), DNase, and/or magnesium chloride. In particular aspects, obtaining a starting population of PNCs from cord blood comprises thawing the cord blood in the presence of dextran and/or DNase. In particular aspects, the cord blood is washed in the presence of 5-20% dextran, such as 10% dextran. In certain aspects, the cord blood is suspended in the presence of magnesium chloride, such as at a concentration of 100-300 mM, in particular 200 mM. In some aspects, obtaining comprises performing Ficoll density gradient centrifugation to obtain mononuclear cells (PNCs).

In certain aspects, the bioreactor is a gas permeable bioreactor. In certain aspects, the gas permeable bioreactor is a G-Rex100M or a G-Rex100. In some aspects, the stimulation of step (b) is carried out in 3-5 L of medium, such as 3, 3.5, 4, 4.5, or 5 L.

In some aspects, the APCs are gamma irradiated. In certain aspects, the APCs are engineered to express membrane-bound IL-21 (mbIL-21). In particular aspects, the APCs are engineered to express IL-21, IL-15, and/or IL-2. In some aspects, the PBMCs and APCs are cultured in a 1:2 ratio. In some aspects, the IL-2 concentration is 50-200 IU/mL, such as 100 IU/mL. In particular aspects, IL-2 is replenished every 2-3 days.

In certain aspects, step (b) is performed for 6-8 days, such as 7 days. In some aspects, step (c) is performed for 6-8 days, such as 7 days. In some aspects, step (c) does not involve cell separation. In certain aspects, the cells are given IL-2 twice in step (c), and in certain cases, no other media components are added or removed in step (c).

In some aspects, the method includes using 3, 4, 5, or 6 bioreactors. In particular aspects, the method includes using less than 10 bioreactors.

In specific aspects, NK cells are expanded at least 500-fold, 800-fold, 1000-fold, 1200-fold, 1500-fold, 2000-fold, 2500-fold, 3000-fold, or 5000-fold. In particular, culturing NK cells in a bioreactor results in a greater than 1000-fold increase in NK cells compared to a static liquid culture.

In certain aspects, the method does not involve human leukocyte antigen (HLA) matching. In some aspects, the initial NK cell population is not obtained from a haploidentical donor.

In some respects, expanded NK cells have enhanced antitumor activity compared to NK cells expanded from peripheral blood. In certain

- 18 048 895 aspects, the expanded NK cells have a higher expression of one or more cell cycle genes, one or more cell division genes and/or one or more DNA replication genes compared to NK cells expanded from peripheral blood. In some aspects, the expanded NK cells have a higher proliferative capacity compared to expanded NK cells from peripheral blood. In some aspects, the expanded NK cells do not exhibit exhaustion. In certain aspects, exhaustion is determined by measuring the expression of perforin, granzyme, CD57, KLRG1 and/or PD1. In some aspects, the expanded NK cells have a high expression of perforin and/or granzyme. In certain aspects, the expanded NK cells have a low level of expression of CD57, KLRG1, and/or PD1 or no expression thereof.

In some aspects, the expanded NK cells comprise a clinically significant dose. In certain aspects, the cord blood is frozen cord blood. In certain aspects, the frozen cord blood has been tested for one or more infectious diseases, such as hepatitis A, hepatitis B, hepatitis C, Trypanosoma cruzi, HIV, human T-lymphotropic virus, syphilis, Zika virus, etc. In some aspects, the cord blood is a collection of cord blood, such as 3, 4, 5, 6, 7, or 8 individual cord blood units.

In some respects, NK cells are not autologous, such as with respect to the recipient individual. In certain respects, NK cells are not allogeneic, such as with respect to the recipient individual.

In some aspects, the APCs are universal antigen presenting cells (uAPCs). In certain aspects, the uAPCs are engineered to express (1) CD48 and/or CS1 (CD319), (2) membrane-bound interleukin-21 (mbIL-21), and (3) 41BB ligand (41BBL). In some aspects, the uAPCs express CD48. In certain aspects, the uAPCs express CS1. In specific aspects, the uAPCs express CD48 and CS1. In some aspects, the uAPCs have substantially no expression of endogenous HLA class I, II, and/or CD1d molecules. In certain aspects, the uAPCs express ICAM-1 (CD54) and/or LFA-3 (CD58). In particular, uAPCs are further defined as iAPCs derived from a leukemia cell, such as K562 cells.

Stem cells.

In some embodiments, the immune cells of the present invention may be stem cells, such as induced pluripotent stem cells (PSCs), mesenchymal stem cells (MSCs), or hematopoietic stem cells (HSCs).

The pluripotent stem cells used herein may be induced pluripotent stem (iPS) cells, commonly abbreviated as iPS cells or iPSCs. With the exception of germ cells, any cell may be used as a starting point for iPSCs. For example, cell types may be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, liver cells, or gastric cells. There is no limitation on the degree of cell differentiation or the age of the animal from which the cells are collected; even undifferentiated progenitor cells (including somatic stem cells) and terminally differentiated mature cells may be used as sources of somatic cells in the methods described herein.

Somatic cells can be reprogrammed to produce iPS cells using methods known to those skilled in the art. Generally, nuclear reprogramming factors are used to produce pluripotent stem cells from a somatic cell. In some embodiments, at least three, or at least four of Klf4, c-Myc, Oct3/4, Sox2, Nanog, and Lin28 are used. In other embodiments, Oct3/4, Sox2, cMyc, and Klf4, or Oct3/4, Sox2, Nanog, and Lin28 are used.

Once produced, the iPS can be cultured in a medium sufficient to maintain pluripotency. In certain embodiments, undefined conditions can be used; for example, pluripotent cells can be cultured on fibroblast feeder cells or a medium that has been exposed to fibroblast feeder cells to maintain the stem cells in an undifferentiated state. In some embodiments, the cell is cultured in the presence of mouse embryonic fibroblasts that have been treated with radiation or an antibiotic to stop cell division as feeder cells. Alternatively, pluripotent cells can be cultured and maintained in a substantially undifferentiated state using a defined, feeder cell-independent culture system, such as TESR™ medium or E8™/Essential 8™ medium.

Genetically engineered antigen receptors.

The immune cells of the present invention can be genetically engineered to express antigen receptors, such as engineered TCRs, CARs, chimeric cytokine receptors, chemokine receptors, combinations thereof, etc. For example, the immune cells are modified to express CARs and/or TCRs that have antigen specificity for a cancer antigen. Multiple CARs and/or TCRs, such as for different

- 19,048,895 antigens can be added to immune cells. In some aspects, the immune cells are engineered to express a CAR or TCR by inserting the CAR or TCR into an inhibitory gene locus using CRISPR.

Suitable modification methods are known in the art. See, for example, Sambrook and Ausubel, supra. For example, cells can be transduced to express a TCR having antigen specificity for a cancer antigen using the transduction methods described in Heemskerk et al., 2008 and Johnson et al., 2009.

Electroporation of RNA encoding full-length TCR α and β chains (or γ and δ) can be used as an alternative to overcome long-term autoreactivity problems caused by pairing of retrovirally transduced and endogenous TCR chains. Even if such alternative pairing takes place in a transient transfection strategy, the resulting autoreactive T cells will likely lose this autoreactivity over time because the introduced TCR α and β chains are expressed only transiently. When expression of the introduced TCR α and β chains diminishes, only normal autologous T cells remain. This is not the case when full-length TCR chains are introduced by stable retroviral transduction, in which the introduced TCR chains never disappear, causing persistent autoreactivity in the patient.

In some embodiments, the cells contain one or more nucleic acids introduced by genetic engineering that encode one or more antigen receptors, and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., not normally present in the cell or a sample obtained from the cell, such as acids obtained from another organism or cell that, for example, are not normally found in the engineered cell and/or the organism from which such a cell is obtained. In some embodiments, the nucleic acids are not natural, for example, the nucleic acid does not occur in nature (e.g., chimeric).

In some embodiments, the CAR comprises an extracellular antigen recognition domain that specifically binds to an antigen. In some embodiments, the antigen is a protein expressed on the surface of cells. In some embodiments, the CAR is a TCR-like CAR, and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein that, like the TCR, is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule.

Examples of antigen receptors, including CARs and recombinant TCRs, as well as methods for constructing and introducing the receptors into cells, include those described, for example, in International Patent Application Publications WO 200014257, WO 2013126726, WO 2012/129514, WO 2014031687, WO 2013/166321, WO 2013/071154, WO 2013/123061, US Patent Application Publications US 2002131960, US 2013287748, US 20130149337, US Patent Nos. 6,451995, 7,446190, 8,252592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application EP 2,537,416, and/or the examples described in Sadelain et al., 2013; Davila et al., 2013; Turtle et al., 2012; Wu et al., 2012. In some aspects, the genetically engineered antigen receptors include CARs as described in U.S. Patent No. 7,446,190 and the receptors described in International Patent Application Publication No. WO/2014055668 A1.

Chimeric antigen receptors.

In some embodiments, a CAR comprises: a) one or more intracellular signaling domains, b) a transmembrane domain, and c) an extracellular domain comprising an antigen-binding region.

In some embodiments, engineered antigen receptors comprise CARs, including activating or stimulatory CARs, costimulatory CARs (see WO 2014/055668), and/or inhibitory CARs (iCARs, see Fedorov et al., 2013). CARs generally comprise an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some aspects via linkers and/or transmembrane domain(s). Such molecules typically mimic or mimic a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone.

Particular embodiments of the present invention relate to the use of nucleic acids, including nucleic acids encoding an antigen-specific CAR polypeptide, including a CAR that has been humanized to reduce immunogenicity (hCAR), comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising one or more signaling motifs. In certain embodiments, the CAR may recognize an epitope comprising a shared space between one or more antigens. In certain embodiments, the binding region may comprise the complementarity determining regions of a monoclonal antibody, the variable regions of a monoclonal antibody, and/or antigen-binding fragments thereof. In another embodiment, the

- 20 048895 specificity comes from the peptide (eg, cytokine) that binds to the receptor.

It is contemplated that human CAR nucleic acids may be human genes used to enhance cellular immunotherapy for human patients. In a specific embodiment, the invention includes a full-length CAR cDNA or coding region. The antigen-binding regions or domain may comprise a fragment of the V _H and V _L chains of a single-chain variable fragment (scFv) derived from a particular human monoclonal antibody, such as those described in U.S. Patent 7,109,304, incorporated herein by reference. The fragment may also be any number of different antigen-binding domains of an antibody specific for a human antigen. In a more specific embodiment, the fragment is an antigen-specific scFv encoded by a sequence that is optimized to use human codons for expression in a human cell.

The configuration may be multidimensional, such as a diabody or multimers. Multimers are most likely formed by cross-pairing of the variable portion of the light and heavy chains into a diabody. The hinge portion of the construct may have several alternatives, ranging from complete deletion, to retention of the first cysteine, to replacement of serine with proline, to truncation to the first cysteine. The Fc portion may be deleted. Any protein that is stable and/or dimerizes may serve this purpose. It is possible to use only one of the Fc domains, such as the CH2 or CH3 domain from human immunoglobulin. It is also possible to use the hinge region, CH2 and CH3 of human immunoglobulin, which has been modified to improve dimerization. It is also possible to use only the hinge portion of immunoglobulin. It is also possible to use portions of CD8alpha.

In some embodiments, the CAR nucleic acid comprises a sequence encoding other costimulatory receptors, such as a transmembrane domain and a modified CD28 domain for intracellular signaling. Other costimulatory receptors include, but are not limited to, one or more of CD28, CD27, OX-40 (CD134), DAP10, DAP12, and 4-1BB (CD137). In addition to the primary signal initiated by CD3Z, the additional signal provided by the human costimulatory receptor inserted into the human CAR is important for the full activation of NK cells and can help improve in vivo persistence and therapeutic success of adoptive immunotherapy.

In some embodiments, a CAR is engineered to be specific for a particular antigen (or marker or ligand), such as an antigen expressed on a particular cell type targeted by adoptive therapy, such as a cancer marker, and/or an antigen designed to elicit a diminished response, such as an antigen expressed on a normal or healthy cell type. Thus, a CAR typically includes in its extracellular portion one or more antigen-binding molecules, such as one or more antigen-binding fragments, domains, or portions, or one or more variable domains of an antibody, and/or antibody molecules. In some embodiments, a CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the heavy chain variable domain (VH) and the light chain variable domain (VL) of a monoclonal antibody (mAb).

In certain embodiments of the chimeric antigen receptor, the antigen-specific portion of the receptor (which may be referred to as the extracellular domain including the antigen-binding region) comprises a binding domain for a tumor-associated antigen or a binding domain for a pathogen-specific antigen. Antigens include carbohydrate antigens recognized by pattern recognition receptors such as Dectin-1. The tumor-associated antigen may be of any kind, provided that it is expressed on the cell surface of tumor cells. Exemplary embodiments of tumor-associated antigens include CD19, CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, MUC-1, CD56, EGFR, c-Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma-associated antigen, mutated p53, mutated ras, etc. In certain embodiments, the CAR may be co-expressed with a cytokine to improve persistence at low levels of tumor-associated antigen. For example, the CAR may be co-expressed with one or more cytokines, such as IL-7, IL-2, IL-15, IL-12, IL-18, IL-21, or a combination thereof.

The open reading frame sequence encoding the chimeric receptor may be obtained from a genomic DNA source, a cDNA source, or may be synthesized (e.g., via PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, since introns have been found to stabilize mRNA. In addition, there may be an additional advantage in using endogenous or exogenous noncoding regions to stabilize mRNA.

It is assumed that the chimeric construct can be introduced into immune cells in the form of naked DNA or in a suitable vector. Methods for stable transfection of cells by electroporation with

- 21 048895 using naked DNA are known in the art. See, for example, U.S. Patent No. 6,410,319. Naked DNA generally refers to DNA encoding a chimeric receptor and contained in a plasmid expression vector in the correct orientation for expression.

Alternatively, a viral vector (e.g., a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector) can be used to introduce the chimeric construct into immune cells. Suitable vectors for use in the method of the present invention do not replicate in immune cells. A large number of virus-based vectors are known, wherein the number of virus copies maintained in the cell is low enough to maintain cell viability, such as, for example, vectors based on HIV, SV40, EBV, HSV, or BPV.

In some aspects, the antigen-specific binding or recognition component is associated with one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR includes a transmembrane domain fused to an extracellular domain of the CAR. In one embodiment, a transmembrane domain is used that is naturally associated with one of the CAR domains. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid association of such domains with transmembrane domains of the same or another surface membrane protein in order to minimize interactions with other members of the receptor complex.

The transmembrane domain in some embodiments is derived from a natural or synthetic source. If the source is natural, the domain in some aspects is derived from any membrane-associated protein or transmembrane protein. Transmembrane regions include those derived from T cell receptor alpha, beta or zeta chain molecules, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 gamma, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154, ICOS/CD278, GITR/CD357, NKG2D and DAP. Alternatively, the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan, and valine will be located at each end of the synthetic transmembrane domain.

In certain embodiments, the platform technologies described herein for genetically modifying immune cells, such as NK cells, include (i) non-viral gene transfer using an electroporation device (e.g., a nucleofector), (ii) CARs that signal through endodomains (e.g., CD28/CD3-Z, CD137/CD3-Z, or other combinations), (iii) CARs with variable lengths of extracellular domains connecting the antigen recognition domain to the cell surface, and, in some cases, (iv) artificial antigen-presenting cells (iAPCs) derived from K562 to enable robust and quantitative expansion of CAR ⁺ immune cells (Singh et al., 2008; Singh et al., 2011).

T cell receptor (TCR)

In some embodiments, genetically engineered antigen receptors include recombinant TCRs and/or TCRs cloned from natural T cells. A T cell receptor or TCR refers to a molecule that comprises variable chains a and β (also known as TCRα and TCRβ, respectively) or variable chains γ and δ (also known as TCRy and TCRδ, respectively) and that is capable of specifically binding to an antigenic peptide associated with an MHC receptor. In some embodiments, the TCR is in the αβ form.

Typically, TCRs that exist in αβ and γδ forms are generally structurally similar, but the T cells that express them may have different anatomical locations or functions. A TCR may be on the cell surface or in a soluble form. Generally, a TCR is on the surface of T cells (or T lymphocytes), where it is primarily responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, a TCR may also comprise a constant domain, a transmembrane domain, and/or a short cytoplasmic tail (see, e.g., Janeway et al, 1997). For example, in some aspects, each TCR chain may comprise one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. Unless otherwise specified, the term TCR should be understood to encompass functional fragments of the TCR. The term also includes intact or full-length TCR, including TCR in the αβ or γδ form.

Thus, for the purposes of this document, reference to a TCR includes any TCR or functional fragment, such as an antigen-binding portion of a TCR, that binds to a specific antigen peptide associated with an MHC molecule, i.e., an MHC-peptide complex. An antigen-binding portion or antigen-binding fragment of a TCR, which may be used interchangeably, refers to a molecule that contains part of the structural domains of a TCR but that binds an antigen (e.g., an MHC-peptide complex) to which the entire TCR binds. In some

- In 22 048 895 cases, the antigen-binding portion contains variable domains of the TCR, such as the variable chain α and variable chain β of the TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex, where, in general, each chain contains three chain complementarity-determining regions.

In some embodiments, the variable domains of the TCR chains associate to form loops or complementarity determining regions (CDRs), similar to immunoglobulins, that confer antigen recognition and determine peptide specificity by forming a binding site for the TCR molecule and determining peptide specificity. Typically, like immunoglobulins, the CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3 is the primary CDR responsible for recognizing processed antigen, although CDR1 of the alpha chain has also been shown to interact with the N-terminal portion of the antigenic peptide, while CDR1 of the beta chain interacts with the C-terminal portion of the peptide. CDR2 is believed to recognize the MHC molecule. In some embodiments, the β-chain variable region may comprise an additional hypervariable region (HV4).

In some embodiments, the TCR chains comprise a constant domain. For example, like immunoglobulins, the extracellular portion of a TCR chain (e.g., α-chain, β-chain) can comprise two immunoglobulin domains, a variable domain (e.g., Va or Vp; typically amino acids 1 to 116 based on Kabat numbering, Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 ^th ed) at the N-terminus and one constant domain (e.g., the α-chain constant domain or Ca, typically amino acids 117 to 259 based on Kabat, the β-chain constant domain or Cp, typically amino acids 117 to 295 based on Kabat) adjacent to the cell membrane. For example, in some cases, the extracellular portion of the TCR, formed by two chains, contains two membrane-proximal constant domains and two membrane-distal variable domains containing the CDRs. The constant domain of the TCR contains short junctional sequences in which a cysteine residue forms a disulfide bond, forming a linkage between the two chains. In some embodiments, the TCR may have additional cysteine residues in each of the α and β chains, such that the TCR contains two disulfide bonds in the constant domains.

In some embodiments, the TCR chains may comprise a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chains comprise a cytoplasmic tail. In some cases, the structure allows the TCR to bind to other molecules, such as CD3. For example, the TCR comprises constant domains with a transmembrane region that can anchor the protein in the cell membrane and bind to invariant subunits of the CD3 signaling apparatus or complex.

Basically, CD3 is a multiprotein complex that may contain three different chains (γ, δ, and ε) in mammals and a ζ chain. For example, in mammals, the complex may contain a CD3y chain, a CD3δ chain, two CD3ε chains, and a homodimer of the CD3Z chain. The CD3y, CD3δ, and CD3ε chains are closely related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of the CD3y, CD3δ, and CD3ε chains are negatively charged, a characteristic that allows these chains to associate with the positively charged chains of the T cell receptor. The intracellular tails of the CD3y, CD3δ, and CD3ε chains contain a single conserved motif known as the immunoreceptor tyrosine-based activation motif or ITAM, whereas each CD3Z chain has three motifs. ITAMs mainly contribute to the signaling ability of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in the propagation of the signal from the TCR to the cell. CD3 and ζ chains together with the TCR form the so-called T-cell receptor complex.

In some embodiments, the TCR may be a heterodimer of two chains, α and β (or optionally γ and δ), or may be a single-chain TCR construct. In some embodiments, the TCR is a heterodimer comprising two separate chains (α and β chains or γ and δ chains) that are linked, for example, by a disulfide bond or disulfide bonds. In some embodiments, a TCR for a target antigen (e.g., a cancer antigen) is identified and introduced into cells. In some embodiments, a nucleic acid encoding the TCR can be obtained from a variety of sources, such as by amplifying publicly available TCR DNA sequences using polymerase chain reaction (PCR). In some embodiments, the TCR is obtained from a biological source, such as a cell such as a T cell (e.g., a cytotoxic T cell), from T cell hybridomas, or another publicly available source. In some embodiments, the T cells can be derived from cells isolated in vivo. In some embodiments, a high-affinity T cell clone can be isolated from a patient and the TCR isolated. In some embodiments, the T cells can be a cultured T cell hybridoma or a T cell clone. In some embodiments, the TCR clone for the target antigen was generated in transgenic mice engineered to

- 23 048 895 with genes of the human immune system (e.g., the human leukocyte antigen or HLA system). See, e.g., tumor antigens (see, e.g., Parkhurst et al., 2009 and Cohen et al., 2005). In some embodiments, phage display is used to isolate a TCR against a target antigen (see, e.g., Varela-Rohena et al., 2008 and Li, 2005). In some embodiments, the TCR or an antigen-binding portion thereof can be synthetically produced based on knowledge of the TCR sequence.

Antigen presenting cells.

Antigen-presenting cells, which include macrophages, B lymphocytes, and dendritic cells, vary in the expression of a particular MHC molecule. APCs internalize antigen and reexpress a portion of that antigen along with the MHC molecule on their outer cell membrane. MHC is a large genetic complex with multiple loci. The MHC loci encode two major classes of membrane MHC molecules, designated MHC class I and class II. Helper T lymphocytes primarily recognize antigen bound to MHC class II molecules, and cytotoxic T lymphocytes recognize antigen bound to MHC class I molecules. In humans, MHC is referred to as the HLA complex and in mice as the H-2 complex.

In some cases, iAPCs are suitable for preparing the therapeutic compositions and cell therapy products of the embodiments. For general guidance regarding the preparation and use of antigen-presenting systems, see, e.g., U.S. Patent Nos. 6,225,042, 6,355,479, 6,362,001, and 6,790,662; U.S. Patent Application Publication Nos. 2009/0017000 and 2009/0004142; and International Publication No. WO2007/103009.

The iAPC systems may comprise at least one exogenous accessory molecule. Any suitable number and combination of accessory molecules may be used. The accessory molecule may be selected from accessory molecules such as costimulatory molecules and adhesion molecules. Exemplary costimulatory molecules include CD86, CD64 (FcyRI), 41BB ligand, and IL-21. Adhesion molecules may include binding glycoproteins such as selectins, transmembrane binding glycoproteins such as integrins, calcium-dependent proteins such as cadherins, and single-pass transmembrane immunoglobulin (1g) superfamily proteins such as intercellular adhesion molecules (ICAMs), which facilitate, for example, cell-cell or cell-matrix contact. Exemplary adhesion molecules include LFA-3 and ICAMs such as ICAM-1. Techniques, methods, and reagents useful for selecting, cloning, producing, and expressing exemplary accessory molecules, including costimulatory molecules and adhesion molecules, are disclosed, for example, in U.S. Patent Nos. 6,225,042, 6,355,479, and 6,362,001.

Antigens.

Antigens targeted by genetically engineered antigen receptors include antigens expressed in the context of a disease, condition, or cell type targeted by adoptive cell therapy. Diseases and conditions include proliferative, neoplastic, malignant tumors and disorders, including cancers and tumors, including hematological malignancies, malignancies of the immune system such as lymphomas, leukemias, and/or myelomas such as B-, T-, and myeloid leukemias, lymphomas, and multiple myelomas. In some embodiments, the antigen is selectively expressed or overexpressed on cells of the disease or condition, such as tumor or pathogenic cells, compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or expressed on engineered cells.

Any suitable antigen can be a target in the present method. The antigen can be associated with certain malignant cells, but not associated with non-malignant cells, in some cases. Examples of antigens include, but are not limited to, antigenic molecules from infectious agents, autoantigens, tumor/malignancy-associated antigens, and tumor neoantigens (Linnemann et al., 2015). In some aspects, the antigens include NY-ESO, EGFRvIII, Muc-1, Her2, CA-125, WT-1, Mage-A3, Mage-A4, Mage-A10, TRAIL/DR4, and CEA. In some aspects, antigens for two or more antigen receptors include, but are not limited to, CD19, EBNA, WT1, CD123, NY-ESO, EGFRvIII, MUC1, HER2, CA-125, WT1, Mage-A3, Mage-A4, Mage-A10, TRAIL/DR4, and/or CEA. The sequences of these antigens are known in the art, for example, in the GenBank® database: CD19 (Accession number NG_007275.1), EBNA (Accession number NG_002392.2), WT1 (Accession number NG_009272.1), CD123 (Accession number NC_000023.11), NY-ESO (Accession number NC_000023.11), EGFRvIII (Accession number NG_007726.3), MUC1 (Accession number NG_029383.1), HER2 (Accession number NG_007503.1), Ca-125 (Accession number NG_055257.1), WT1 (Accession number NG_009272.1), Mage-A3 (Accession number NG_013244.1), Mage-A4 (Accession number NG_013245.1), Mage-A10 (Accession number NC_000023.11), TRAIL/DR4 (Accession number NC_000003.12) and/or CEA (Accession number NC_000019.10).

Tumor-associated antigens, as examples, can be obtained from malignant tumors of the prostate gland, breast gland, colon, lung,

- 24 048895 pancreas, kidney, from mesothelioma, from malignant tumors of the ovary, liver, brain, bone, stomach, spleen, testicle, neck, anus, gallbladder, thyroid gland, or from melanoma. Examples of tumor-associated antigens or antigens derived from tumor cells include MAGE 1, 3, and MAGE 4 (or other MAGE antigens such as those described in International Patent Publication No. WO99/40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO 1); SAGE; and HAGE or GAGE. These are non-limiting examples of tumor antigens that are expressed in a wide range of tumor types, such as melanoma, lung carcinoma, sarcoma, and bladder carcinoma. See, for example, U.S. Patent No. 6,544,518. Tumor-associated antigens of prostate cancer include, for example, prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostatic acid phosphates, NKX3.1, and six-transmembrane epithelial antigen of the prostate (STEAP).

Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. Additionally, a tumor antigen may be an autopeptide hormone such as full-length gonadotropin-releasing hormone (GnRH), a short peptide of 10 amino acids useful in the treatment of many malignancies.

Tumor antigens include tumor antigens derived from malignant tumors that are characterized by the expression of a tumor-associated antigen, such as HER2/neu expression. Tumor-associated antigens of interest include lineage-specific tumor antigens, such as the melanocyte-melanoma lineage antigens MART-1/Melan-A, gp100, gp75, mda-7, tyrosinase, and tyrosinase-related protein. Illustrative tumor-associated antigens include, but are not limited to, tumor antigens derived from or comprising any one or more of p53, Ras, c-Myc, cytoplasmic serine/threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGEA4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MART-1, MC1R, Gp100, PSA, PSM, tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, CypB, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide 3-kinase (PI3K), TRK receptors, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, catenin/m, caspase-8/m, CEA, CDK4/m, ELF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin/m, RAGE, SART-2, TRP-2/INT2, 707-AP, annexin II, CDC27/m, TPI/mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6/AML, LDLR/FUT, Pml/RAR, tumor-associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR) (particularly EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor (VEGFR)), cytoplasmic tyrosine kinases (e.g., src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS and STATE, hypoxia-inducible factors (e.g., HIF1 and HIF-2), nuclear factor kappa B (NF-K), Notch receptors (e.g., Notch 1-4), c-Met, mammalian targets of rapamycin (mTOR), WNT, extracellular signal-regulated kinases (ERK) and their regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22alpha, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL/AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl GM1, mesothelin, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, LRRN1, and idiotype.

Antigens may include epitope regions or epitope peptides derived from genes mutated in tumor cells or from genes transcribed at different levels in tumor cells compared with normal cells, such as telomerase enzymes, survivin, mesothelin, mutated ras, bcr/abl rearrangement, Her2/neu, mutated or wild-type p53, cytochrome P450 1B1, and abnormally expressed intronic sequences such as N-acetylglucosaminyl transferase-V; clonal rearrangements of immunoglobulin genes generating unique idiotypes in myeloma and B-cell lymphomas; tumor antigens that include epitope regions or epitope peptides derived from oncoviral processes, such as the E6 and E7 proteins of human papillomavirus; the LMP2 protein of Epstein-Barr virus; non-mutated oncofetal proteins with tumor-selective expression, such as carcinoembryonic antigen and alpha-fetoprotein.

In other embodiments, the antigen is obtained or derived from a pathogenic microorganism or from an opportunistic microorganism (also referred to herein as an infectious disease microorganism), such as a virus, fungus, parasite, and bacterium. In some embodiments, antigens obtained from such a microorganism include full-length proteins.

- 25 048895

Examples of pathogenic organisms whose antigens are contemplated for use in the method described herein include human immunodeficiency virus (HIV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza A, B, and C viruses, vesicular stomatitis virus (VSV), polyomavirus (e.g., BK virus and JC virus), adenovirus, Staphylococcus species including methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus species including Streptococcus pneumoniae. As will be appreciated by one of skill in the art, proteins obtained from these and other pathogenic microorganisms for use as an antigen as described herein, and the nucleotide sequences encoding the proteins, can be identified in publications and in publicly available databases such as GENBANK®, SWISS-PROT® and TREMBL®.

Human immunodeficiency virus (HIV)-derived antigens include any of the structural proteins of the HIV virion (e.g., gp120, gp41, p17, p24), protease, reverse transcriptase, or the HIV proteins encoded by tat, rev, nef, vif, vpr, and vpu.

Antigens derived from herpes simplex virus (e.g., HSV1 and HSV2) include, but are not limited to, proteins expressed from the late genes of HSV. The group of late genes predominantly encodes proteins that form the virion particle. Such proteins include five proteins from the (UL) that form the viral capsid: UL6, UL18, UL35, UL38 and the major capsid protein UL19, UL45 and UL27, each of which can be used as an antigen described herein. Other exemplary HSV proteins contemplated for use as antigens herein include ICP27 (H1, H2) proteins, glycoprotein B (gB) and glycoprotein D (gD) proteins. The HSV genome includes at least 74 genes, each of which encodes a protein that can potentially be used as an antigen.

Cytomegalovirus (CMV)-derived antigens include CMV structural proteins, viral antigens expressed during the pre-early and early phases of viral replication, glycoproteins I and III, capsid protein, envelope protein, lower matrix protein pp65 (ppUL83), p52 (ppUL44), IE1 and 1E2 (UL123 and UL122), protein products of the UL128-UL150 gene cluster (Rykman, et al., 2006), envelope glycoprotein B (gB), gH, gN, and pp150. As will be appreciated by those skilled in the art, the CMV proteins for use as antigens described herein can be identified in publicly available databases such as GENBANK®, SWISS-PROT® and TREMBL® (see, for example, Bennekov et al., 2004 ; Loewendorf et al., 2010; Marschall et al., 2009).

Antigens derived from Epstein-Barr virus (EBV) that are contemplated for use in certain embodiments include the EBV lytic proteins gp350 and gp110, EBV proteins produced during the latent cycle of infection, including Epstein-Barr nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA leader protein (EBNA-LP), and latent membrane proteins (LMP)-1, LMP-2A, and LMP-2B (see, e.g., Lockey et al., 2008).

Antigens derived from respiratory syncytial virus (RSV) contemplated for use herein include any of eleven proteins encoded by the RSV gene, or antigenic fragments thereof: NS1, NS2, N (nucleocapsid protein), M (matrix protein), SH, G, and F (viral envelope proteins), M2 (second matrix protein), M2-1 (elongation factor), M2-2 (transcription regulation), RNA polymerase, and phosphoprotein P.

Vesicular stomatitis virus (VSV)-derived antigens that are being considered for use include any of the major proteins encoded by the VSV gene and its antigenic fragments: large protein (L), glycoprotein (G), nucleoprotein (N), phosphoprotein (P), and matrix protein (M) (see, e.g., Rieder et al., 1999).

Antigens derived from influenza virus that are contemplated for use in certain embodiments include hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix proteins M1 and M2, NS1, NS2 (NEP), PA, PB1, PB1-F2, and PB2.

Typical viral antigens also include, but are not limited to, adenovirus polypeptides, alphavirus polypeptides, calicivirus polypeptides (e.g., calicivirus capsid antigen), coronavirus polypeptides, distemper virus polypeptides, Ebola virus polypeptides, enterovirus polypeptides, flavivirus polypeptides, hepatitis A (AE) virus polypeptides (hepatitis B core or surface antigen, E1 or E2 glycoproteins, hepatitis C virus core or nonstructural proteins), herpes virus polypeptides (including herpes simplex virus or varicella zoster virus glycoprotein), infectious peritonitis virus polypeptides, leukemia virus polypeptides, Marburg virus polypeptides, orthomyxovirus polypeptides, papillomavirus polypeptides, parainfluenza virus polypeptides (e.g., hemagglutinin and neuraminidase polypeptides), paramyxovirus, parvovirus polypeptides, pestivirus polypeptides, picornavirus polypeptides (e.g., poliovirus capsid polypeptide), smallpox virus polypeptides (e.g., vaccinia virus polypeptide), rabies virus polypeptides (e.g., rabies virus glycoprotein G), reovirus polypeptides, retrovirus polypeptides, and rotavirus polypeptides.

In some embodiments, the antigen may be a bacterial antigen. In

- 26 048895 In certain embodiments, the bacterial antigen of interest may be a secreted polypeptide. In other certain embodiments, bacterial antigens include antigens that have a portion or portions of a polypeptide presented on the outer cell surface of the bacteria.

Antigens derived from Staphylococcus species, including methicillin-resistant Staphylococcus aureus (MRSA), that have been considered for use include virulence regulators such as the Agr, Sar, and Sae systems, the Arl system, Sar homologues (Rot, MgrA, SarS, SarR, SarT, SarU, SarV, SarX, SarZ, and TcaR), the Srr system, and TRAP. Other Staphylococcus proteins that may serve as antigens include the Clp, HtrA, MsrR, aconitase, CcpA, SvrA, Msa, CfvA, and CfvB proteins (see, e.g., Staphylococcus: Molecular Genetics, 2008 Caister Academic Press, Ed. Jodi Lindsay). The genomes of two Staphylococcus aureus species (N315 and Mu50) have been sequenced and are publicly available, for example in PATRIC (PATRIC: The VBI PathoSystems Resource Integration Center, Snyder et al., 2007). As will be appreciated by those skilled in the art, Staphylococcus proteins for use as antigens can also be identified in other publicly available databases such as GenBank®, Swiss-Prot® and TrEMBL®.

Antigens derived from Streptococcus pneumoniae that are contemplated for use in certain embodiments include pneumolysin, PspA, choline binding protein A (CbpA), NanA, NanB, SpnHL, PavA, LytA, Pht, and pilin proteins (RrgA; RrgB; RrgC). Streptococcus pneumoniae antigenic proteins are also known in the art and can be used as an antigen in some embodiments (see, e.g., Zysk et al., 2000). The complete genomic sequence of a virulent strain of Streptococcus pneumoniae has been sequenced, and as one of skill in the art will appreciate, S. pneumoniae proteins for use herein can also be identified in other publicly available databases such as GENBANK®, SWISS-PROT®, and TREMBL®. Proteins of particular interest for the antigens of the present invention include virulence factors and proteins predicted to be found on the surface of pneumococci (see, e.g., Frolet et al., 2010).

Examples of bacterial antigens that can be used as antigens include, but are not limited to, Actinomyces polypeptides, Bacillus polypeptides, Bacteroides polypeptides, Bordetella polypeptides, Bartonella polypeptides, Borrelia polypeptides (e.g., B. burgdorferi OspA), Brucella polypeptides, Campylobacter polypeptides, Capnocytophaga polypeptides, Chlamydia polypeptides, Corynebacterium polypeptides, Coxiella polypeptides, Dermatophilus polypeptides, Enterococcus polypeptides, Ehrlichia polypeptides, Escherichia polypeptides, Francisella polypeptides, Fusobacterium polypeptides, Haemobartonella polypeptides, Haemophilus polypeptides (e.g., H. influenzae outer membrane protein type b), Helicobacter polypeptides, Klebsiella polypeptides, L-form bacterial polypeptides, Leptospira polypeptides, Listeria polypeptides, Mycobacteria polypeptides, Mycoplasma polypeptides, Neisseria polypeptides, Neorickettsia polypeptides, Nocardia polypeptides, Pasteurella polypeptides, Peptococcus polypeptides, Peptostreptococcus polypeptides, Pneumococcus polypeptides (i.e., S. pneumoniae polypeptides) (see description herein), Proteus polypeptides, Pseudomonas polypeptides, Rickettsia polypeptides, Rochalimaea polypeptides, Salmonella polypeptides, Shigella polypeptides, Staphylococcus polypeptides, group A streptococcus polypeptides (e.g., M proteins of S. pyogenes), group B streptococcus polypeptides (S. agalactiae), Treponema polypeptides, and Yersinia polypeptides (e.g., F1 and V antigens of Y pestis).

Examples of fungal antigens include, but are not limited to, Absidia polypeptides, Acremonium polypeptides, Alternaria polypeptides, Aspergillus polypeptides, Basidiobolus polypeptides, Bipolaris polypeptides, Blastomyces polypeptides, Candida polypeptides, Coccidioides polypeptides, Conidiobolus polypeptides, Cryptococcus polypeptides, Curvalaria polypeptides, Epidermophyton polypeptides, Exophiala polypeptides, Geotrichum polypeptides, Histoplasma polypeptides, Madurella polypeptides, Malassezia polypeptides, Microsporum polypeptides, Moniliella polypeptides, Mortierella polypeptides, Mucor polypeptides, Paecilomyces polypeptides, Penicillium polypeptides, Phialemonium polypeptides, Phialophora polypeptides, Prototheca polypeptides, Pseudallescheria polypeptides, Pseudomicrodochium, Pythium polypeptides, Rhinosporidium polypeptides, Rhizopus polypeptides, Scolecobasidium polypeptides, Sporothrix polypeptides, Stemphylium polypeptides, Trichophyton polypeptides, Trichosporon polypeptides, and Xylohypha polypeptides.

Examples of protozoan parasite antigens include, but are not limited to, Babesia polypeptides, Balantidium polypeptides, Besnoitia polypeptides, Cryptosporidium polypeptides, Eimeria polypeptides, Encephalitozoon polypeptides, Entamoeba polypeptides, Giardia polypeptides, Hammondia polypeptides, Hepatozoon polypeptides, Isospora polypeptides, Leishmania polypeptides, Microsporidia polypeptides, Neospora polypeptides, Nosema polypeptides, Pentatrichomonas polypeptides, Plasmodium polypeptides. Examples of helminth parasite antigens include, but are not limited to, Acanthocheilonema polypeptides, Aelurostrongylus polypeptides, Ancylostoma polypeptides, Angiostrongylus polypeptides, Ascaris polypeptides, Brugia polypeptides,

- 27 048895 Bunostomum polypeptides, Capillaria polypeptides, Chabertia polypeptides, Cooperia polypeptides, Crenosoma polypeptides, Dictyocaulus polypeptides, Dioctophyme polypeptides, Dipetalonema polypeptides, Diphyllobothrium polypeptides, Diplydium polypeptides, Dirofilaria polypeptides, Dracunculus polypeptides, Enterobius polypeptides, Filaroides polypeptides, Haemonchus polypeptides, Lagochilascaris polypeptides, Loa polypeptides, Mansonella polypeptides, Muellerius polypeptides, Nanophyetus polypeptides, Necator polypeptides, Nematodirus polypeptides, Oesophagostomum polypeptides, Onchocerca polypeptides, Opisthorchis polypeptides, Ostertagia polypeptides, Parafilaria polypeptides, Paragonimus polypeptides, Parascaris, Physaloptera polypeptides, Protostrongylus polypeptides, Setaria polypeptides, Spirocerca polypeptides, Spirometra polypeptides, Stephanofilaria polypeptides, Strongyloides polypeptides, Strongylus polypeptides, Thelazia polypeptides, Toxascaris polypeptides, Toxocara polypeptides, Trichinella polypeptides, Trichostrongylus polypeptides, Trichuris polypeptides, Uncinaria polypeptides, and Wuchereria polypeptides (e.g., P. falciparum circumsporozoite protein (PfCSP)), sporozoite surface protein 2 (PfSSP2), liver stage antigen carboxyl terminus 1 (PfLSA1 c-term), and secreted protein 1 (PfExp1), Pneumocystis polypeptides, Sarcocystis polypeptides, Schistosoma polypeptides, Theileria polypeptides, Toxoplasma and Trypanosoma polypeptides.

Examples of ectoparasite antigens include, but are not limited to, polypeptides (including antigens as well as allergens) from fleas; ticks, including hard and soft ticks; flies such as midges, mosquitoes, sand flies, black flies, horse flies, cow flies, deer flies, tsetse flies, common stingers, myiasis flies, and biting midges; ants; spiders, lice; mites; and true bugs such as bed bugs and triatomine bugs.

Suicidal genes.

In some cases, any of the cells of the present invention are modified to produce one or more agents other than heterologous cytokines, engineered receptors, etc. In particular embodiments, cells such as NK cells are engineered to contain one or more suicide genes, and as used herein, the term suicide gene is defined as a gene that, when administered as a prodrug, causes the gene product to convert to a compound that kills its host cell. In some cases, NK cell therapy may be subject to exposure to one or more suicide genes of any type when the individual receiving NK cell therapy and/or who has received NK cell therapy exhibits one or more symptoms of one or more adverse effects such as cytokine release syndrome, neurotoxicity, anaphylaxis/allergy, and/or on-target/off-target tumor toxicity (as examples) or the therapy is considered to be at risk of developing one or more symptoms, including in the near future. Use of a suicide gene may be part of a planned therapy protocol or may be used only when there is a recognized need for its use. In some cases, cell therapy is discontinued by use of the agent/agents targeting the suicide gene or gene product because the therapy is no longer needed.

Examples of suicide genes include engineered mutant polypeptides of nonsecreted (including membrane-bound) tumor necrosis factor (TNF)-alpha (see PCT/US19/62009, which is incorporated herein by reference in its entirety), and these can be targeted by delivery using an antibody that binds the mutant TNF-alpha. Examples of suicide gene/prodrug combinations that can be used are herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidylate kinase (Tdk:Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. One can use the E. coli purine nucleoside phosphorylase, the so-called suicide gene, which converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine. Other suicide genes include, as examples, CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 (CYP) enzymes, carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), ribosyltransferase enzyme (XGRTP), methionine-α,γ-lyase (MET), and thymidine phosphorylase (TP).

Delivery methods.

One skilled in the art will be well-versed in constructing vectors using standard recombinant techniques (see, e.g., Sambrook et al., 2001 and Ausubel et al., 1996, both incorporated herein by reference) for expressing the antigen receptors of the present invention. Vectors include, but are not limited to, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g., those derived from Moloney murine leukemia virus (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., those derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenoviral (Ad) vectors, including replication-competent, replication-restricted, and eviscerated forms thereof,

- 28 048895 adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papillomavirus vectors, Epstein-Barr virus vectors, herpes virus vectors, vaccinia virus vectors, Harvey sarcoma virus vectors in mice, murine mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, vesicular stomatitis virus vectors, Maraba virus vectors and adenovirus group B vectors (Enadenotusirev).

In particular embodiments, the vector is a multicistronic vector as described in PCT/US19/62014, which is incorporated herein by reference in its entirety. In such cases, a single vector may encode a CAR or TCR (and the expression construct may be configured in a modular format allowing for replacement of portions of the CAR or TCR), a suicide gene, and one or more cytokines.

Viral vectors.

In certain aspects, the present invention relates to viral vectors encoding an antigen receptor. In the production of recombinant viral vectors, genes that are not essential are typically replaced by a gene or coding sequence for a heterologous (or non-native) protein. A viral vector is a type of expression construct that uses a viral sequence to introduce nucleic acid and possibly proteins into a cell. The ability of some viruses to infect cells or enter cells via receptor-mediated endocytosis and integrate into host cell genomes and stably and efficiently express viral genes has made them attractive candidates for the transfer of foreign nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of viral vectors that can be used to deliver nucleic acid according to certain aspects of the present invention are described below.

Lentiviruses are complex retroviruses that, in addition to the normal retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. Lentiviral vectors are well known in the art (see, e.g., U.S. Patents 6,013,516 and 5,994,136).

Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for gene transfer and expression of nucleic acid sequences both in vivo and ex vivo. For example, a recombinant lentivirus capable of infecting a non-dividing cell, wherein a suitable host cell is transfected with two or more vectors carrying packaging functions, namely gag, pol and env, as well as rev and tat, is described in U.S. Patent 5,994,136, incorporated herein by reference.

Regulatory elements.

Expression cassettes included in vectors suitable for the present invention specifically comprise (in the 5'-3' direction) a eukaryotic transcriptional promoter operably linked to a protein-coding sequence, splicing signals including intervening sequences, and a transcription termination/polyadenylation sequence. Promoters and enhancers that control transcription of protein-coding genes in eukaryotic cells are composed of multiple genetic elements. The cellular machinery is capable of assembling and integrating the regulatory information conveyed by each element, allowing different genes to evolve under separate, often complex patterns of transcriptional regulation. A promoter used in relation to the present invention includes constitutive, inducible, and tissue-specific promoters.

Promoters/Enhancers.

The expression constructs provided herein contain a promoter for driving expression of the antigen receptor. The promoter essentially consists of a sequence that functions to mark the position of the start site for RNA synthesis. The best known example of this promoter is the TATA box, but in some promoters that lack a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the SV40 late gene promoter, a separate element lying above the start site itself helps to fix the initiation site. Additional promoter elements regulate the frequency of transcription initiation. They are typically located in the region of 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. To place a coding sequence under the control of a promoter, the 5' end of the transcription initiation site of the transcriptional reading frame must be positioned downstream (i.e., 3' of) the selected promoter. The upstream promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.

The distance between promoter elements is often flexible, so that promoter function is maintained when the elements are flipped or moved relative to each other. In the tk promoter, the distance between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, it appears that individual elements can function either together or independently to activate transcription. A promoter can

- 29 048895 to use or not to use in combination with an enhancer, which denotes a cis-acting regulatory sequence involved in the activation of transcription of a nucleic acid sequence.

The promoter may be a promoter naturally associated with a nucleic acid sequence, which may be obtained by isolating 5 non-coding sequences located upstream of the coding segment and/or exon. Such a promoter may be referred to as endogenous. Similarly, the enhancer may be an enhancer naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, and promoters or enhancers that are not natural, i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. For example, promoters that are most widely used in recombinant DNA construction include the β-lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. In addition to producing the nucleic acid sequences of promoters and enhancers synthetically, the sequences can be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions described herein. In addition, it is proposed that control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, etc. may also be used.

Naturally, it will be important to use a promoter and/or enhancer that effectively directs expression of the DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. Those skilled in the art of molecular biology are generally familiar with the use of combinations of promoters, enhancers, and cell types for protein expression (see, e.g., Sambrook et al. 1989, incorporated herein by reference). The promoters used may be constitutive, tissue-specific, inducible, and/or useful under appropriate conditions for driving high levels of expression of the introduced DNA segment, as this is advantageous in large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

Additionally, any promoter/enhancer combination (according to, for example, the EPDB eukaryotic promoter database, via the Internet at epd.isb-sib.ch/) can also be used to drive expression. Another possible embodiment is the use of a T3, T7 or SP6 cytoplasmic expression system. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.

Non-limiting examples of promoters include early or late viral promoters, such as the early or late SV40 promoters, the immediate early cytomegalovirus (CMV) promoters, the early Rous sarcoma virus (RSV) promoters; eukaryotic cell promoters, such as, for example, the beta-actin promoter, the GADPH promoter, the metallothionein promoter; and serially linked response element promoters, such as the cyclic AMP response element (cre) promoters, the serum response element (sre) promoter, the phorbol ester (TPA) promoter, and the response element (tre) promoters adjacent to the minimal TATA box. It is also possible to use a mammary tumor promoter (e.g., the minimal growth hormone promoter described in GenBank®, accession number X05244, nucleotides 283-341) or a mouse mammary tumor promoter (available from the ATCC, catalog number ATCC 45007). In some embodiments, the promoter is CMV IE, dectin-1, dectin-2, human CD11c, F4/80, SM22, RSV, SV40, Ad MLP, beta-actin, MHC class I or MHC class II promoter, however, any other promoter suitable for driving expression of a therapeutic gene is applicable to the practice of the present invention.

In certain aspects, the methods of the invention also relate to enhancer sequences, i.e., nucleic acid sequences that increase the activity of promoters and that have the potential to act in cis orientation, and regardless of their orientation, even at relatively large distances (up to several kilobases from the target promoter). However, the function of enhancers is not necessarily limited to such large distances, since they can also act in close proximity to a given promoter.

- 30 048895

Initiation signals and associated expression.

A specific initiation signal can also be used in the expression constructs of the present invention for efficient translation of the coding sequences. These signals include the ATG initiation codon or adjacent sequences. It may be necessary to provide exogenous translation control signals, including the ATG initiation codon. One skilled in the art will easily be able to determine this and provide the necessary signals. It is well known that the initiation codon must be in frame with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translation control signals and initiation codons can be either natural or synthetic. Expression efficiency can be increased by including appropriate enhancer elements for transcription.

In some embodiments, internal ribosome entry site (IRES) elements are used to create multigene, or polycistronic, transcripts. IRES elements are able to bypass the ribosome scanning model of $-methylated cap dependent translation and initiate translation at internal sites. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described, as well as an IRES transcript from mammals. IRES elements can be linked into heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. The IRES element ensures that each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed from a single promoter/enhancer to transcribe a single transcript.

Additionally, certain elements of the 2A sequence can be used to create linked or co-expression of genes in the constructs of the present invention. For example, a cleavage sequence can be used to co-express genes by linking open reading frames to form a single cistron. An example of a cleavage sequence is F2A (viral 2A) or a 2A-like sequence (e.g., Thea asigna virus 2A; T2A).

Replication origin sites.

In order to propagate the vector in a host cell, it may contain one or more origins of replication (often called ori), such as a nucleic acid sequence corresponding to, for example, the EBV oriP as described above, or a genetically engineered oriP with similar or enhanced programmed function, which is a specific nucleic acid sequence at which replication is initiated. Alternatively, an origin of replication from another virus that replicates extrachromosomally, as described above, or an autonomously replicating sequence (ARS) may be used.

Selection and markers for screening.

In some embodiments, cells comprising a construct of the present invention may be identified in vitro or in vivo by incorporating a marker into an expression vector. Such markers would impart an identifiable change to the cell, allowing for easy identification of cells comprising the expression vector. Generally, a selectable marker is a marker that confers a property that allows for selection. A positive selectable marker is a marker whose presence allows for selection for the marker, and a negative selectable marker is a marker whose presence prevents selection for the marker. An example of a positive selectable marker is a drug resistance marker.

Typically, the inclusion of a drug selectable marker helps in cloning and identifying transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are suitable as selectable markers. In addition to markers that confer a phenotype that allows differentiation of transformants based on the state realized, other types of markers are also considered, including markers for screening such as GFP, which is based on a colorimetric assay. Alternative screening enzymes can be used as negative selectable markers, such as thymidine kinase (tk) of the herpes simplex virus or chloramphenicol acetyltransferase (CAT). The skilled artisan will also be familiar with the use of immunological markers, possibly in combination with FACS analysis. The marker used is not considered important, provided that it is capable of being expressed simultaneously with the nucleic acid encoding the gene product. Other examples of selection and selectable markers are well known to those skilled in the art.

Other methods of nucleic acid delivery.

In addition to viral delivery of nucleic acids encoding an antigen receptor, the present invention further contemplates additional methods of recombinantly delivering genes into a given host cell.

- 31 048895

To introduce a nucleic acid, such as DNA or RNA, into the immune cells of the present invention, any suitable method for delivering nucleic acid to transform a cell, as described herein or as known to one of skill in the art, can be used. Such methods include, but are not limited to, direct DNA delivery, such as ex vivo transfection, by injection, including microinjection; by electroporation; by calcium phosphate precipitation; using DEAE-dextran followed by polyethylene glycol; direct ultrasound loading; liposome-mediated transfection and receptor-mediated transfection; by microparticle bombardment; by mixing with silicon carbide fibers; Agrobacterium-mediated transformation; by desiccation/inhibition-mediated DNA uptake, and by any combination of such methods. By using such methods, organelle(s), cell(s), tissue(s) or organism(s) can be stably or transiently transformed.

Treatment methods.

Embodiments of the present invention include methods for treating an individual for a cancer, any type of infection, and any immune disorder. The individual may use the method of treatment of the invention as an initial treatment or after or during another treatment. Immunotherapy methods may be tailored to the needs of an individual with a cancer based on the type or stage of the cancer, and, to a lesser extent, in some cases, the immunotherapy may be changed during the course of treatment of the individual.

In specific cases, the treatment methods are as follows: (1) Adoptive cell therapy with T or NK cells (expanded ex vivo or expressing CAR or TCR) for the treatment of cancer patients with any type of hematological malignancy, (2) Adoptive cell therapy with T or NK cells (expanded ex vivo or expressing CAR or TCR) for the treatment of cancer patients with any type of solid malignancy, (3) Adoptive cell therapy with Tregs and regulatory B cells (expanded ex vivo or expressing CAR or TCR) for the treatment of patients with immune disorders, (4) Adoptive cell therapy with T or NK cells (expanded ex vivo or expressing CAR or TCR) for the treatment of patients with infectious diseases. The present invention demonstrates for the first time that knockdown/knockout of multiple genes in human NK cells results in improved cell functionality and resistance to the tumor microenvironment. In specific embodiments, this has a direct impact on patient care using a novel immunotherapeutic approach that enhances the function of the patient's own immune cells or adoptively transferred immune cells. Embodiments provide a novel approach for generating highly functional T, NK and B cells (both ex vivo expanded and engineered with CAR or TCR) for immunotherapy. This includes targeting malignancies, both hematological and solid tumors (NK cells and T cells, as well as CAR T cells and CAR NK cells), autoimmune and alloimmune disorders (B cells, regulatory B cells and regulatory T cells) and treating infections (for pathogen-specific T cells).

In some embodiments, the present invention relates to methods of immunotherapy comprising administering an effective amount of immune cells of the present invention. In one embodiment, a medical disease or disorder is treated by transferring an immune population of NK cells that elicits an immune response. In certain embodiments, the present invention treats a cancer or infection by transferring an immune cell population that elicits an immune response. The present document provides methods of treating or delaying the progression of a cancer in an individual comprising administering to the individual an effective amount of an antigen-specific cell therapy. The present methods can be used to treat immune disorders, solid cancers, hematological cancers, viral infections.

Tumors for which the present methods of treatment are suitable include any type of malignant cells, such as those found in a solid tumor or a hematological tumor. Examples of solid tumors include, but are not limited to, a tumor of an organ selected from the group consisting of the pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Examples of hematological tumors include tumors of bone marrow, T- or B-cells, malignancies, leukemias, lymphomas, blastomas, myelomas, and the like. Additional examples of malignant tumors that can be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma), peritoneal cancer, gastric cancer, or stomach cancer (including gastrointestinal cancer and

- 32 048895 malignant tumor of the gastrointestinal stromal tract), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, prostate cancer, cancer of the female external genitalia, thyroid cancer, various types of head and neck cancer, and melanoma.

Malignant tumor may specifically refer to, but is not limited to, the following histologic types: malignant neoplasia, carcinoma, undifferentiated carcinoma, giant cell and spindle cell carcinoma, small cell carcinoma, papillary carcinoma, squamous cell carcinoma, lymphoepithelial carcinoma, basal cell carcinoma, pilomatrix carcinoma; transitional cell carcinoma, papillary transitional cell carcinoma, adenocarcinoma, malignant gastrinoma, cholangiocarcinoma, hepatocellular carcinoma, combined hepatocellular carcinoma and cholangiocarcinoma, trabecular adenocarcinoma, adenoid cystic carcinoma, adenocarcinoma in adenomatous polyp, adenocarcinoma, familial polyposis coli, solid carcinoma, malignant carcinoid tumor, bronchioloalveolar adenocarcinoma, papillary adenocarcinoma, chromophobe carcinoma, acidophilic carcinoma, oxyphilic adenocarcinoma, basophilic carcinoma, clear cell adenocarcinoma, granular cell carcinoma, follicular adenocarcinoma, papillary and follicular adenocarcinoma, nonencapsulating sclerosing carcinoma, adrenal cortex carcinoma, endometrioid carcinoma, adnexal carcinoma, apocrine adenocarcinoma, sebaceous adenocarcinoma, ceruminous adenocarcinoma, mucoepidermoid carcinoma, cystadenocarcinoma; papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, mucinous cystadenocarcinoma, mucinous adenocarcinoma, signet ring cell carcinoma, infiltrating ductal carcinoma, medullary carcinoma, lobular carcinoma, inflammatory carcinoma, Paget's disease of the breast, acinar cell carcinoma, adenosquamous carcinoma, adenocarcinoma with squamous cell metaplasia, malignant thymoma, malignant ovarian stromal tumor, malignant thecoma, malignant granulosa cell tumor, malignant androblastoma, Sertoli cell carcinoma, malignant Leydig cell tumor, malignant lipid cell tumor, malignant paraganglioma, malignant extramammary paraganglioma, pheochromocytoma, glomangiosarcoma, malignant melanoma, amelanotic melanoma, superficial spreading melanoma, malignant lentigo melanoma, acral lentiginous melanoma, nodular melanoma, malignant melanoma in giant pigmented nevi, epithelioid cell melanoma, malignant blue nevus, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, stromal sarcoma, malignant mixed tumor, mixed Müllerian tumor, nephroblastoma, hepatoblastoma, carcinosarcoma, malignant mesenchymoma, malignant Brenner tumor, malignant phyllodes tumor, synovial sarcoma, malignant mesothelioma, dysgerminoma, embryonal carcinoma, malignant teratoma, malignant struma of ovary, choriocarcinoma, malignant mesonephroma, hemangiosarcoma, malignant hemangioendothelioma, Kaposi's sarcoma, malignant hemangiopericytoma, lymphangiosarcoma, osteosarcoma, juxtacortical osteosarcoma, chondrosarcoma, malignant chondroblastoma, mesenchymal chondrosarcoma, giant cell tumor of bone, Ewing's sarcoma, malignant odontogenic tumor, ameloblastic odontosarcoma, malignant ameloblastoma, ameloblastic fibrosarcoma, malignant pinealoma, chordoma, malignant glioma, ependymoma, astrocytoma, protoplasmic astrocytoma, fibrillary astrocytoma, astroblastoma, glioblastoma, oligodendroglioma, oligodendroblastoma, primitive neuroectodermal tumor, cerebellar sarcoma, ganglioneuroblastoma, neuroblastoma, retinoblastoma, olfactory neurogenic tumor, malignant meningioma, neurofibrosarcoma, malignant neurilemoma, malignant granular cell tumor, malignant lymphoma, Hodgkin's disease, paragranuloma, malignant small lymphocytic lymphoma, malignant diffuse large cell lymphoma, malignant follicular lymphoma, mycosis fungoides, other specified non-Hodgkin's lymphomas, B-cell lymphoma, poorly differentiated/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediately differentiated/follicular NHL, intermediate-grade diffuse NHL, well-differentiated immunoblastic NHL, lymphoblastic well-differentiated NHL, well-differentiated small cell non-dissected NHL, massive NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative disease of the small intestine, leukemia, lymphoid leukemia, plasma cell leukemia, erythroleukemia, cellular lymphosarcoma leukemia, myelogenous leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, myeloid sarcoma, hairy cell leukemia, chronic lymphocytic leukemia (CLL),

- 33 048895 acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML) and chronic myelogenous leukemia.

Particular embodiments relate to methods of treating leukemia. Leukemia is a malignant tumor of the blood or bone marrow, and is characterized by abnormal proliferation (production by multiplication) of blood cells, usually white blood cells (leukocytes). It is part of a large group of diseases called hematologic neoplasias. Leukemia is a broad term that covers a range of diseases. Leukemia is clinically and pathologically divided into acute and chronic forms.

In some embodiments of the present invention, immune cells are delivered to a subject in need thereof, such as a subject having a cancer or infection. The cells then enhance the human immune system to attack the corresponding cancer or pathogenic cells. In some cases, the human is given one or more doses of immune cells. In cases where two or more doses of immune cells are administered to the subject, the duration between administrations should be sufficient to allow time for dissemination in the subject, and in particular embodiments, the duration between doses is 1, 2, 3, 4, 5, 6, 7 or more days.

In particular embodiments, the present invention provides methods for treating or preventing an immune-mediated disorder. In one embodiment, the individual has an autoimmune disease. Non-limiting examples of autoimmune diseases include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune adrenal diseases, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac spate dermatitis, chronic fatigue and immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutination syndrome, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA-mediated neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, immune-mediated diabetes mellitus or type 1 diabetes, myasthenia gravis, nephrotic syndrome (eg, minimal change disease, focal glomerulosclerosis, or meningeal nephropathy), pemphigus vulgaris, pernicious anemia, periarteritis nodosa, polychondria, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff person syndrome, systemic lupus erythematosus, lupus erythematosus, ulcerative colitis, uveitis, vasculitides (such as periarteritis nodosa, Takayasu's syndrome, temporal arteritis/giant cell arteritis, or vasculitis in dermatitis herpetiformis), vitiligo, and Wegener's granulomatosis. Thus, some examples of an autoimmune disease that can be treated using the methods described herein include, but are not limited to, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus type I, Crohn's disease; ulcerative colitis, myasthenia gravis, glomerulonephritis, ankylosing spondylitis, vasculitis, or psoriasis. The individual may also have an allergic disorder such as asthma.

In another embodiment, the individual is a recipient of an organ or stem cell transplant, and the immune cells are used to prevent and/or treat rejection. In specific embodiments, the individual develops or is at risk of developing a graft-versus-host disease. GVHD is a possible complication of any transplant that uses or contains stem cells from a related or unrelated donor. There are two types of GVHD: acute and chronic. Acute GVHD occurs within the first three months after transplantation. Signs of acute GVHD include a reddish skin rash on the arms and legs that may spread and become more severe, with peeling or blistering of the skin. Acute GVHD may also affect the stomach and intestines, in which case cramping, nausea, and diarrhea are present. Yellowing of the skin and eyes (jaundice) indicates that acute GVHD has affected the liver. Chronic GVHD is classified according to its severity: stage/grade 1 - mild; stage/grade 4 - severe. Chronic GVHD develops three months or later after transplantation. The symptoms of chronic GVHD are similar to those of acute GVHD, but in addition, chronic GVHD may also involve the mucous glands of the eyes, the salivary glands in the mouth, and the glands that lubricate the lining of the stomach and intestines. Any of the immune cell populations described herein can be used. Examples of a transplanted organ include a solid organ graft such as kidney, liver, skin, pancreas, lung, and/or heart, or a cellular graft such as islets,

- 34 048895 hepatocytes, myoblasts, bone marrow, hematopoietic or other stem cells. The transplant may be a composite graft, such as facial tissue. The immune cells may be administered prior to transplantation, concurrently with transplantation, or after transplantation. In some embodiments, the immune cells are administered prior to transplantation, such as at least 1 hour, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 1 month prior to transplantation. In one specific, non-limiting example, the administration of a therapeutically effective amount of immune cells occurs 3-5 days prior to transplantation.

In some embodiments, non-myeloablative lymphodepleting chemotherapy may be administered to the individual prior to the immune cell therapy. The non-myeloablative lymphodepleting chemotherapy may be any suitable such therapy that may be administered by any suitable route. Non-myeloablative lymphodepleting chemotherapy may include, for example, administration of cyclophosphamide and fludarabine, particularly if the cancer is melanoma, which may be metastatic. An exemplary route of administration of cyclophosphamide and fludarabine is intravenous. Likewise, any suitable dose of cyclophosphamide and fludarabine may be administered. In certain aspects, about 60 mg/kg of cyclophosphamide is administered for two days, followed by about 25 mg/m2 ^of fludarabine is administered for five days.

In some embodiments, a growth factor that promotes the growth and activation of immune cells is administered to the individual either simultaneously with the immune cells or later to the immune cells. The immune growth factor can be any suitable growth factor that promotes the growth and activation of immune cells. Examples of suitable immune cell growth factors include interleukin (IL)-2, IL-7, IL-12, IL-15, IL-18, and IL-21, which can be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL-2.

Therapeutically effective amounts of immune cells can be administered by a number of routes, including parenteral administration, such as intravenous, intraperitoneal, intramuscular, intrathoracic, intra-articular injection or infusion.

A therapeutically effective amount of immune cells for use in adoptive cell therapy is that amount that achieves the desired effect in an individual when treated. For example, this may be the amount of immune cells needed to inhibit the development of, or to induce regression of, an autoimmune or alloimmune disease, or that is able to alleviate symptoms caused by an autoimmune disease, such as pain and inflammation. It may be the amount needed to relieve symptoms associated with inflammation, such as pain, swelling, and fever. It may also be the amount needed to reduce or prevent rejection of a transplanted organ.

The immune cell population may be administered in treatment regimens appropriate to the disease, such as a single dose or more doses over one to several days to improve the disease state, or intermittent doses over a long period of time to suppress disease progression and prevent disease recurrence. The exact dose to be used in the formulation will also depend on the route of administration, the severity of the disease or disorder, and should be determined in accordance with the judgment of the practitioner and the circumstances of each patient. The therapeutically effective amount of immune cells will depend on the individual being treated, the severity and type of disease, and the route of administration. In some embodiments, doses that can be used to treat humans range from at least 3.8x10 ⁴ , at least 3.8x10 ⁵ , at least 3.8x10 ⁶ , at least 3.8x10 ⁷ , at least 3.8x10 ⁸ , at least 3.8x10 ⁹ , or at least 3.8x10 ¹⁰ immune cells/m ² . In a certain embodiment, the dose used in treating humans ranges from about 3.8x10 ⁹ to about 3.8x10 ¹⁰ immune cells/m ² . In additional embodiments, a therapeutically effective amount of immune cells may range from about ^5x106 cells/kg body weight to about ^7.5x108 cells/kg body weight, such as from about ^2x107 cells to about ^5x108 cells/kg body weight, or from about ^5x107 cells to about ^2x108 cells/kg body weight. The exact amount of immune cells can be readily determined by one of skill in the art based on the age, weight, sex, and physiological state of the individual. Effective doses can be extrapolated from dose-response curves obtained from in vitro test systems or animal models.

Immune cells may be administered in combination with one or more other therapeutic agents to treat an immune-mediated disorder. Combination therapies may include one or more antimicrobial agents (e.g., antibiotics, antivirals, and antifungals), antineoplastic agents (e.g.,

- 35 048895 fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressive agents (eg, azathioprine or glucocorticoids such as dexamethasone or prednisone), anti-inflammatory agents (eg, glucocorticoids such as hydrocortisone, dexamethasone, or prednisone, or non-steroidal anti-inflammatory drugs such as acetylsalicylic acid, ibuprofen, or naproxen sodium), cytokines (eg, interleukin-10 or transforming growth factor-beta), hormones (eg, estrogen), or vaccines. Additionally, immunosuppressive or tolerogenic agents may be administered, including, but not limited to, calcineurin inhibitors (e.g., cyclosporine and tacrolimus); mTOR inhibitors (e.g., rapamycin); mycophenolate mofetil, antibodies (e.g., those that recognize CD3, CD4, CD40, CD154, CD45, IVIG, or B cells); chemotherapeutic agents (e.g., methotrexate, treosulfan, busulfan); radiation; or chemokines, interleukins, or inhibitors thereof (e.g., BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors). Such additional pharmaceutical agents may be administered before, during, or after the administration of immune cells, depending on the desired effect. This administration of the cells and agent may occur by the same route or by different routes, and either at the same site or at a different site.

Pharmaceutical compositions.

The present document also provides pharmaceutical compositions and formulations comprising immune cells (e.g., T cells, B cells, or NK cells) and a pharmaceutically acceptable carrier.

The pharmaceutical compositions and formulations described herein can be prepared by mixing the active ingredients (such as an antibody or a polypeptide) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22 ^nd edition, 2012) in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complex compounds (e.g., Zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG). Examples of pharmaceutically acceptable carriers herein further include agents for dispersing drugs in the extracellular space such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), for example, soluble hyaluronidase glycoproteins PH-20 such as rHuPH20 (HYLENEX®, Baxter International). Specific examples of sHASEGPs and methods of use involving rHuPH20 are described in U.S. Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

Methods of combined treatment.

In certain embodiments, the compositions and methods of the present embodiments comprise a population of immune cells in combination with at least one additional therapy. The additional therapy may be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the above. The additional therapy may be in the form of adjuvant or neoadjuvant therapy.

In some embodiments, the adjunctive therapy is the administration of a small molecule enzyme inhibitor or an antimetastatic agent. In some embodiments, the adjunctive therapy is the administration of side effect limiting agents (e.g., agents designed to reduce the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the adjunctive therapy is radiation therapy. In some embodiments, the adjunctive therapy is surgery. In some embodiments, the adjunctive therapy is a combination of radiation therapy and surgery. In some embodiments, the adjunctive therapy is gamma irradiation. In some embodiments, the adjunctive therapy

- 36 048895 is a therapy targeting the PBK/AKT/mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and/or a chemopreventive agent. The additional therapy may be one or more chemotherapeutic agents known in the art.

The immune cell therapy can be administered before, during, after or in various combinations associated with additional cancer therapy, such as immune checkpoint therapy. The administrations can be at intervals ranging from simultaneous to minutes, to days, to weeks. In embodiments where the immune cell therapy is provided to the patient separately from the additional therapeutic agent, it can generally be ensured that no significant period of time elapses between the times of each delivery, so that the two compounds can still have an advantageous combined effect on the patient. In such cases, it is contemplated that it is possible to provide the patient with antibody therapy and anti-cancer therapy within about 12 to 24 or 72 hours of each other, and more specifically within about 6-12 hours of each other. In some situations it may be desirable to significantly prolong the treatment period, where the interval between the respective administrations ranges from several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8).

Various combinations can be used. For the example below, immune cell therapy is labeled A and cancer therapy is labeled B:

A/B/A, B/A/B, B/B/A, A/A/B, A/B/B, B/A/A, A/B/B/B, B/A/B/B, B/B/B/A, B/B/A/B, A/A/B/B, A/B/A/B, A/B/B/A, B/B/A/A, B/A/B/A, B/A/A/B, A/A/A/B, B/A/A/A, A/B/A/A, A/A/B/A.

When administering any compound or therapy according to the present embodiments, the patient must follow the general protocols for administering such compounds, taking into account the toxicity of the agents, if any. Thus, in some embodiments, there is a toxicity monitoring step that can be attributed to combination therapy.

Chemotherapy.

A wide range of chemotherapeutic agents can be used in accordance with the present embodiments. The term chemotherapy refers to the use of drugs to treat a malignant tumor. A chemotherapeutic agent is used to refer to a compound or composition that is administered to treat a malignant tumor. These agents or drugs are classified by the mode of their activity in the cell, such as whether they affect the cell cycle and at what stage they affect the cell cycle. Alternatively, an agent can be characterized based on its ability to directly cross-link DNA, intercalate into DNA, or cause chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoride, triethylenethiophosphoride, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyine; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichine, phenesterine, prednimustine, trofosfamide, and uramustine; nitrosoureas such as carmustine, chlorzotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (eg, calicheamicin, especially calicheamicin gamma I and calicheamicin omega I1); dynemicin, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and also neocarzinostatin chromophore and related chromoproteins chromophores of enediyne antibiotics, aclacinomysins, actinomycin, autarnicin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnicin, olivomycins, peplomycin, potfiromycin, puromycin, quellamicin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; adrenal suppressants such as mitotane and trilostane; folate supplements such as frolinic acid;

- 37 048895 aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bizantrene; edatraxate; defofamine; demecolcine; diaziquone; elformitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxan; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anhydrine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosin; arabinoside (Ara-C); cyclophosphamide; taxoids such as paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (eg, CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylchylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, protein farnesyl transferase inhibitors, transplatin, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

Radiation therapy

Other factors that induce DNA damage and are commonly used include the well-known γ-rays, x-rays, and/or targeted delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors such as microwaves, proton beam irradiation, and UV irradiation are also being considered. All of these factors are likely to affect a wide range of DNA damage, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. Dose ranges for x-rays vary from daily doses of 50 to 200 roentgens over a long period of time (3 to 4 weeks) to single doses of 2,000 to 6,000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the tumor cells.

Immunotherapy

It is understood by those skilled in the art that additional immunotherapy can be used in combination or in conjunction with the methods of the embodiments. In relation to the treatment of malignant tumors, immunotherapeutic agents generally rely on the use of immune effector cells and molecules to target and destroy malignant cells. Rituximab (RITUXAN®) is such an example. The immune effector can be, for example, an antibody specific for a marker on the surface of a tumor cell. The antibody itself can serve as an effector of the therapy or it can recruit other cells to actually affect the destruction of the cells. The antibody can also be conjugated to a drug or toxin (chemotherapeutic drug, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that directly or indirectly interacts with the target tumor cell. Various effector cells include cytotoxic T cells and NK cells.

Antibody-drug conjugates (ADCs) contain monoclonal antibodies (MAbs) that are covalently linked to cell-killing drugs and can be used in combination therapies. This approach combines the high specificity of MAbs for their antigen targets with highly potent cytotoxic drugs to produce armed MAbs that deliver the payload (drug) to tumor cells with elevated antigen levels. Targeted drug delivery also minimizes exposure of the drug to normal tissues, resulting in reduced toxicity and an improved therapeutic index. Examples of ADCs include ADCETRIS® (brentuximab vedotin) and KADCYLA® (trastuzumab emtansine, or T-DM1).

In one aspect of immunotherapy, the tumor cell must carry some marker that is targetable, i.e., not present on most other cells. There are many tumor markers, and any of them may be suitable for targeting with respect to the present embodiments. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialylated Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is the combination of antitumor effects with immunostimulatory effects. There are also immunostimulatory molecules including cytokines such as IL-2, IL-4, IL-12, GM-CSF, IFN-gamma, chemokines such as MIP-1, MCP-1, IL-8 and growth factors such as FLT3 ligand.

Examples of immunotherapy include immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds; cytokine therapy, such as interferons α, β, and γ, IL-1, GM-CSF, and TNF; gene therapy, such as TNF, IL-1, IL-2, and p53; and

- 38 048895 monoclonal antibodies, such as anti-CD20, anti-GM2 ganglioside, and anti-p185. It is contemplated that one or more anti-cancer drugs may be used with the antibody therapy described herein.

In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints either increase a signal (e.g., costimulatory molecules) or decrease a signal. Immune checkpoint inhibitors that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD 152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene 3 (LAG3), programmed death protein 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V domain of Ig suppressor of T cell activation (VISTA). Specifically, immune checkpoint inhibitors target the PD-1 and/or CTLA-4 axis.

Immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of the ligand or receptors, or in particular antibodies such as human antibodies. Known inhibitors of immune checkpoint proteins or their analogues may be used, in particular chimerized, humanized or human forms of antibodies. As is known to those skilled in the art, alternative and/or equivalent names may be used for certain antibodies mentioned in the present invention. Such alternative and/or equivalent names are interchangeable with respect to the present invention. For example, it is known that lambrolizumab is also known under the alternative and equivalent names MK-3475 and pembrolizumab.

In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a particular aspect, the ligand binding partners of PD-1 are PDL1 and/or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partners. In a particular aspect, the binding partners of PDL1 are PD-1 and/or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partners. In a particular aspect, the binding partner of PDL2 is PD-1. The antagonist may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an immunoglobulin Fc region sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody that may be used. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH900475, is an example of an anti-PD-1 antibody. CT-011, also known as hBAT or hBAT-1, is also an anti-PD-1 antibody. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor.

Another immune checkpoint that can be targeted by the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA4 has GeneBank accession number L15006. CTLA-4 is located on the surface of T cells and acts as a switch when it binds to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of T helper cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T cell costimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2, respectively, on antigen-presenting cells. CTLA4 transmits an inhibitory signal to T cells, while CD28 transmits a stimulatory signal. Intracellular CTLA4 is also expressed in regulatory T cells and may be important for their function. Activation of T cells via the T cell receptor and CD28 results in increased expression of CTLA-4, an inhibitory receptor for B7 molecules.

In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

Antibodies to human CTLA-4 (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be produced using methods

- 39 048 895 well known in the art. Alternatively, well known anti-CTLA-4 antibodies can be used. An example of an anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof. In other embodiments, the antibody comprises the CDRs or VRs of the heavy and light chains of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and/or binds to the same epitope on CTLA-4 as the aforementioned antibodies. In another embodiment, the antibody has at least about 90% amino acid sequence identity with the above antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

Surgical intervention

Approximately 60% of people with cancer will undergo some type of surgery, which includes preventive, diagnostic or staging, curative and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised and/or destroyed, and such surgery can be used in combination with other therapies, such as the treatment of the present embodiments, chemotherapy, radiation therapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery and microscope-guided surgery (Mohs surgery).

After some or all of the cancer cells, tissue, or tumor has been removed, a cavity may form in the body. Treatment may be by perfusion, direct injection, or local application of additional anticancer therapy to the affected area. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may also have different dosages.

Other means

It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to enhance the therapeutic efficacy of the treatment. These additional agents include agents that affect upregulation of cellular receptors and gap junctions, cytostatics and differentiation agents, cell adhesion inhibitors, agents that sensitize hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of gap junctions may enhance the anti-hyperproliferative effects on a neighboring hyperproliferative cell population. In other embodiments, cytostatics or differentiation agents may be used in combination with certain aspects of the present embodiments to enhance the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors are intended to enhance the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that sensitize a hyperproliferative cell to apoptosis, such as the c225 antibody, may be used in combination with certain aspects of the present embodiments to enhance treatment efficacy.

Industrial products or kits

The present document also provides an article of manufacture or a kit comprising immune cells. The article of manufacture or kit may further comprise a package insert comprising instructions for using the immune cells to treat or delay the progression of a cancer in an individual or to enhance immune function in an individual having a cancer. Any of the antigen-specific immune cells described herein may be included in an article of manufacture or kits. Suitable containers include, for example, bottles, vials, bags and syringes. The container may be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or a metal alloy (e.g., stainless steel or hastelloy). In some embodiments, the container comprises a composition, and a label on or associated with the container may indicate instructions for use. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and a package insert with instructions for use. In some embodiments, the article of manufacture further includes one or more other agents (e.g., a chemotherapeutic agent and an antineoplastic agent). Suitable containers for one or more agents

- 40 048895 include, for example, bottles, vials, bags and syringes.

Examples

The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art will appreciate that the methods disclosed in the following examples represent methods discovered by the inventor for the proper operation of the practice of the invention, and thus may be considered as preferred methods for its practice. However, those skilled in the art will appreciate, in light of the present invention, that many changes can be made to the specific embodiments described and a like or similar result can be obtained without departing from the spirit and scope of the invention.

Example 1. Multiplex gene editing.

To test the efficiency of simultaneous disruption of multiple genes in immune cells such as T cells and NK cells, several studies were conducted testing disruption of different combinations of genes using CRISPR. In the first study, CRISPR/Cas9 was used to disrupt the expression of NKG2A, CD47, TGFBR2, and CISH in NK cells. In this set, NKG2A and CD47 genes were knocked out in the first round of electroporation, and CISH and TGFBR2 were targeted in the second round of electroporation. The knockout efficiency was successfully confirmed by PCR and flow cytometry for both rounds of electroporation (Fig. 1).

The multigene disruption mode was confirmed in additional gene sets including TiGiT, CD96, CISH, adenosine (Fig. 2) and NKG2A, CD47, TGFBR2 and CISH (Fig. 3). Disruption of multiple genes was found to result in gain of functionality against tumor cell targets. Flow cytometric analysis of IFN-γ, TNFa and CD107 production was performed with different NK cells (edited versus Cas9 alone) co-stimulated with target cell lines for 5 h in the presence of Brefeldin A. Following stimulation with target cell lines, secretion of IFN-γ, TNFu and CD107 was enhanced (Fig. 3).

This increased functionality was confirmed by the disruption of NKG2A, CD47, TGFBR2, and CISH in NK cells, which showed enhanced antitumor cytotoxicity (Fig. 4A) as measured by the 51Cr release assay toward K562 cells. Furthermore, after 30 min of treatment with recombinant TGF-β (50 ng/ml), pSMAD activity was measured by flow cytometry (Fig. 4B). It was also observed that NK cells lost CD16 and CD62L expression upon cytokine stimulation or target recognition (Fig. 5), and ADAM17 knockout in NK cells prevented CD16 and CD62L rejection (Fig. 6) and improved ADCC and cytotoxicity toward K562 targets (Fig. 7).

Further studies showed that disruption of SHP1 in NK cells resulted in enhanced antitumor efficacy (Figs. 9 and 10). NK cells were co-cultured with K562/Raji cells at a 1:1 ratio for 4 h. After incubation, cells were stained with annexin V and live and dead cells were analyzed. K562 cells were sensitive to NK cell killing, while Raji cells were resistant to NK cell killing. Furthermore, disruption of NKG2A in NKCAR cells resulted in enhanced antitumor efficacy against Raji targets (Fig. 12).

The present approach was further validated with additional gene sets - TIGIT, CD96, CISH, and ADENOSINE, as well as NKG2A, CISH, TGFBRII, and ADENOSINE. NK cell function was assessed by flow cytometry and an increase in TNFa, IFNy, and CD107a was observed in cells after stimulation with the target cell line (Fig. 13-14).

Thus, the present methods can be used to simultaneously disrupt the expression of multiple genes in immune cells to enhance their functionality.

Example 2. Methods.

Preformation of crRNA-Cas9 complexes and electroporation of 1 or 2:crRNAs overlapping adjacent regions were designed and used for each gene. Reactions with 1 μg cas9 (PNA Bio) and 500 μg crRNA (sum of all crRNAs) were performed for each gene and incubated on ice for 20 min. After 20 min, 250,000 NK cells were resuspended in T-buffer* (included with the Invitrogen Neon Electroporation Kit, total volume including RNP complex and cells should be 14 μl) and electroporated with a 10 μl electroporation tip using the Neon Transfection System. Electroporation conditions were 1600 V, 10 ms, and 3 pulses* for NK cells. Cells were then added to a culture plate with APC (1 NK: 2 APC), SCGM medium (preferably without antibiotics), 200 IU/ml IL2 and left to recover in an incubator at 37°C.

Precomplexation of cpRNA and electroporation: The cpRNA and tracpRNA duplex were mixed by pipetting and centrifuging. The mixture was incubated at 95°C for 5 min in a thermal cycler and then allowed to cool to room temperature on the benchtop.

Table 3

Duplex crRNA and tracpRNA

Volume Concentration Volume Concentration cpPHK #1 2.2 µl 100 µM cpPHK #2 2.2 µl 100 µM

- 41 048895

tracpPHK 2.2 µl 100 µM tracpPHK 2.2 µl 100 µM IDTE Buffer 5.6 µl IDTE Buffer 5.6 µl total volume 10 µl 44 µM total volume 10 µl 44 µM

The initial concentration of cpRNA and tracpRNA is 100 μM. The final concentration after mixing them at an equimolar concentration is 44 μM.

Table 4

Cas9 nuclease

Volume Alt-R S.p. Cas9 Nuclease 3NLS (61 µM) 3 µl T-buffer 7 µl total volume 10 µl final concentration 18 µM

Table 5

Combination of cpRNA:tracpRNA and Cas9 nuclease mix

Volume cpRNA duplex #1:tracpRNA (Step 1) 2 µl Cas9 (Stage 2) 2 µl total volume 4 µl

The cpRNA:tracpRNA duplex was combined with the Cas9 nuclease mixture by pipetting and incubated at room temperature for 15 min. The mixture was then combined with cpRNA.

Table 6 cpRNA #1 and cpRNA #2

Volume cpRNA #1+tracpRNA+cas9 (Step 3) 2.25 µl cpRNA #2+tracpRNA+cas9 (Step 3) 2.25 µl total volume 4.5 µl

Electroporation was performed by first preparing culture plates with APC (1 NK:2 APC), SCGM medium (preferably without antibiotic), and 200 IU/ml IL-2. Prepare 250,000 cells per well, resuspended in 7.5 μl T-buffer immediately before use. Electroporation conditions were 1600 V, 10 ms, and 3 pulses*. Cells were then added to the culture plate and allowed to recover in a 37°C incubator.

NK cell expansion: Isolate NK cells from cord blood or peripheral blood using Miltenyi NK Cell Isolation Kit (130-092-657). Plate NK cells with feeder cells at a 1:2 ratio (1 NK cell and 2 feeder cells) in the presence of IL-2 (200 IU/ml) in SCGM medium. Change medium after 24 hours with IL-2. On day 4, re-isolate NK cells using NK Cell Isolation Kit to remove feeder cells or wait until day 7 until all feeder cells are gone. Transduce with chimeric antigen receptor or epitoporate for Crispr-Cas9.

All methods disclosed and claimed herein can be made and performed without undue experimentation in light of the present invention. Although the compositions and methods of the present invention have been described with respect to preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the methods and to the steps or sequence of steps in the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of the invention as defined in the appended claims.

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Claims (16)

U.S. Patent No. 9,175,268 U.S. Patent Publication No. 2010/0210014 U.S. Patent Publication No. 12/478,154 U.S. Patent Publication No. 2002131960 U.S. Patent Publication No. 2003/0211603 U.S. Patent Publication No. 2005/0260186 U.S. Patent Publication No. 2006/0104968 U.S. Patent Publication No. 2009/0004142 U.S. Patent Publication No. 2009/0017000 U.S. Patent Publication No. 2009/0246875 U.S. Patent Publication No. 2011/0104125 U.S. Patent Publication No. 2011/0301073 U.S. Patent Publication US Patent Publication No. 20110008369 US Patent Publication No. 2012/0276636 US Patent Publication No. 2013/0315884 US Patent Publication No. 20130149337 US Patent Publication No. 2013287748 US Patent Publication No. 2014/0120622 US Patent Publication No. 2014022021 US Patent Publication No. 20140294898 Varela-Rohena et al. Nat Med. 14: 1390-1395, 2008. Wang et al. J Immunother. 35(9):689-701, 2012. Wu et al., Adv. Cancer Res., 90: 127-56, 2003. Wu et al., Cancer, 18(2): 160-75, 2012. Yamanaka et al., Cell, 131(5):861–72, 2007. Yu et al., Science, 318:1917-1920, 2007. Zysk et al., Infect. Immun. 68(6):3740-43, 2000. CLAUSE OF THE INVENTION 1. An in vitro method for disrupting at least three genes in a natural killer cell, wherein said at least three genes are selected from the group consisting of (1) NKG2A, CISH and TGFBRII, (2) TIGIT, FOXO1 and TGFBRII, (3) TGFBRII, CD96 and TIGIT, (4) TGFBRII, CISH and TIGIT, (5) TIM3, CISH and TGFBRII, (6) CD96, FOXO1 and TGFBRII, (7) TGFBRII, TIM3 and TIGIT, (8) SIGLEC7, CISH and TGFBRII, (9) CD47, CISH and TGFBRII, (10) SIRPA, CISH and TGFBRII, (11) TGFBRII, CD47 and TIGIT, (12) TGFBRII, CD47 and SIRPA, (13) A2AR, CISH and TGFBRII, (14) TGFBRII, CISH and ADAM17, (15) TGFBRII, TIM3 and TIGIT, (16) TGFBRII, A2AR and TIGIT, (17) SHP1, CISH and TGFBRII, (18) TGFBRII, CISH and SHP1, (19) TGFBRII, SHP1 and TIGIT and (20) TGFBRII, SHP1 and TIM3. 2. The method of claim 1, wherein the natural killer cell is engineered to express a chimeric antigen receptor (CAR) and/or a T cell receptor (TCR). 3. The method according to claim 1, wherein the natural killer cell is virus-specific. 4. The method according to claim 1 or 2, wherein the natural killer cell is isolated from umbilical cord blood. 5. The method according to claim 4, wherein the umbilical cord blood is combined from two or more individual units of umbilical cord blood. 6. The method according to any one of claims 1, 2, 4 and 5, further comprising introducing a CAR or TCR into said natural killer cell. 7. The method of claim 6, wherein the administering comprises administering a nucleic acid encoding said CAR or TCR into said natural killer cell. 8. The method according to claim 7, wherein the nucleic acid is in an expression vector. 9. The method according to claim 8, wherein the vector additionally comprises one or more inhibitory gene sequences. 10. The method according to claim 9, wherein the vector further comprises a guide RNA for said inhibitory gene. 11. The method of claim 10, wherein the CAR is flanked by homologous arms for said inhibitory genes. 12. The method of claim 11, wherein the introduction of a vector containing a CAR sequence results in the insertion of the CAR into the locus of an inhibitory gene in said immune cell. 13. The method according to claim 12, wherein the CAR is inserted into an exon of said inhibitory gene. 14. The method according to claim 12, wherein the CAR is under the control of an endogenous promoter of an inhibitory gene. 15. The method according to claim 12, wherein the introduction of the vector additionally disrupts the expression of said inhibitory gene. 16. A natural killer cell with impaired expression of at least three genes in the cell -