阅读说明：本技术 重组免疫细胞、制备方法和使用方法 (Recombinant immune cells, methods of making, and methods of use ) 是由 吴建明 布鲁斯·沃尔切克 罗伯特·哈里森·赫尔西克 李云芳 赫曼特·库马尔·米什拉 于 2018-10-26 设计创作，主要内容包括：重组免疫细胞表达异源IgG Fc受体。在一些实施方式中,异源IgG Fc受体可以是嵌合IgG Fc受体。通常,嵌合IgG Fc受体包括细胞外结构域、跨膜结构域和细胞内结构域。细胞外结构域通常包括足以与IgG Fc区域结合的CD64的部分。嵌合IgG Fc受体的细胞内结构域包括足以在IgG Fc区域与细胞外结构域结合时允许免疫受体酪氨酸基活化基序(ITAM)启动细胞信号传导的Fc受体的部分。(The recombinant immune cells express heterologous IgG Fc receptors. In some embodiments, the heterologous IgG Fc receptor can be a chimeric IgG Fc receptor. Typically, chimeric IgG Fc receptors include an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain typically includes a portion of CD64 sufficient to bind to the IgG Fc region. The intracellular domain of the chimeric IgG Fc receptor includes a portion of the Fc receptor sufficient to allow the immune receptor tyrosine-based activation motif (ITAM) to initiate cell signaling when the IgG Fc region binds to the extracellular domain.)

1. A chimeric IgG Fc receptor comprising:

an extracellular domain comprising a portion of CD64 sufficient to bind to an IgG Fc region;

a transmembrane domain; and

an intracellular domain comprising a portion of an Fc receptor immunoreceptor tyrosine-based activation motif (ITAM) sufficient to initiate cell signaling upon binding of an IgG Fc region to the extracellular domain.

2. The chimeric IgG Fc receptor of claim 1, wherein the intracellular domain comprises at least a portion of the intracellular domain of CD 16A.

3. The chimeric IgG Fc receptor of claim 1, wherein said intracellular domain comprises at least a portion of an intracellular domain of CD27, CD28, CD134(OX40), CD137(4-1BB), FcR1, NKG2D, CD244(2B4), FcR γ, DAP10, DAP12, or CD3 ζ.

4. The chimeric IgG Fc receptor of any preceding claim, wherein the extracellular domain comprises a CD16A cleavage site.

5. The chimeric IgG Fc receptor of any preceding claim, wherein the intracellular domain comprises a signaling domain.

6. A polynucleotide encoding the chimeric receptor of any preceding claim.

7. A recombinant cell comprising the polynucleotide of claim 6.

8. A recombinant cell expressing the IgG Fc chimeric receptor of any one of claims 1-5.

9. The recombinant cell of claim 8, wherein the recombinant cell is a Natural Killer (NK) cell.

10. A recombinant Natural Killer (NK) cell comprising a polynucleotide encoding CD 64.

11. Recombinant cells, including Natural Killer (NK) cells genetically modified to express CD 64.

12. A method of killing tumor cells, the method comprising:

contacting the tumor cell with an antibody that specifically binds to the tumor cell; and

contacting the tumor cell with the recombinant cell of any one of claims 7-11 under conditions effective for the recombinant cell to kill the tumor cell.

13. A method of treating a subject having a tumor, the method comprising:

administering to the subject an antibody that specifically binds to cells of the tumor; and

administering to the subject a composition comprising the recombinant cell of any one of claims 7-11 under conditions effective for the recombinant cell to kill cells of the tumor.

14. A composition, comprising:

the recombinant cell of any one of claims 7-11; and

an antibody that binds to the chimeric receptor.

15. A method of treating a subject having a tumor, the method comprising:

administering to the subject the composition of claim 14, wherein the antibody specifically binds to cells of the tumor.

Disclosure of Invention

The present disclosure describes, in one aspect, immune cells expressing heterologous IgG Fc receptors.

In some embodiments, the heterologous IgG Fc receptor can be a chimeric IgG Fc receptor. Typically, chimeric IgG Fc receptors include an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain typically includes a portion of CD64 sufficient to bind to the IgG Fc region. The intracellular domain of the chimeric IgG Fc receptor includes a portion of the Fc receptor immunoreceptor tyrosine-based activation motif (ITAM) sufficient to initiate cell signaling when the IgG Fc region binds to the extracellular domain.

In some of these embodiments, the intracellular domain comprises at least a portion of the intracellular domain of CD 16A. In other embodiments, the intracellular domain may comprise at least a portion of the intracellular domain of CD27, CD28, CD134(OX40), CD137(4-1BB), FcR γ, or CD3 ζ.

In some embodiments, the chimeric IgG Fc receptor may include a CD16A extracellular cleavage site. In some embodiments, the extracellular domain of the chimeric IgG Fc receptor may lack a CD16A extracellular cleavage site.

In some embodiments, the heterologous IgG Fc receptor can include an IgG Fc receptor that is not naturally expressed by the immune cell. In some of these embodiments, the immune cell can be a Natural Killer (NK) cell genetically modified to express CD 64.

In another aspect, the disclosure describes a polynucleotide encoding any embodiment of the heterologous IgG Fc receptors outlined above.

In another aspect, the present disclosure describes an immune cell genetically modified to include a polynucleotide encoding any embodiment of the heterologous IgG Fc receptor outlined above.

In another aspect, the disclosure describes a method of killing tumor cells. In general, the method comprises contacting a tumor cell with an antibody that specifically binds to the tumor cell, and contacting the tumor cell with any of the embodiments of recombinant immune cells outlined above under conditions wherein the recombinant immune cells effectively kill the tumor cell.

In another aspect, the disclosure describes a method of treating a subject having a tumor. In general, the method comprises administering to the subject an antibody that specifically binds to a cell of the tumor, and administering to the subject a composition comprising any embodiment of the recombinant immune cell outlined above under conditions in which the recombinant immune cell is effective to kill the cell of the tumor.

In another aspect, the present disclosure describes a composition comprising a complex formed between a therapeutic antibody and any embodiment of the recombinant immune cell outlined above, wherein the heterologous IgG Fc receptor binds to the Fc portion of the therapeutic antibody.

In another aspect, the disclosure describes a method of treating a subject having a tumor. In general, the method includes administering to the subject any embodiment of the composition just outlined, wherein the therapeutic antibody specifically binds to a cell of the tumor.

The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description more particularly exemplifies illustrative embodiments. Guidance is provided throughout the application in various places through a series of examples, which can be used in various combinations. In each case, the listed series are used only as representative groups and should not be interpreted as exclusive series.

Drawings

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FIG. 1 antibody dependent cell mediated cytotoxicity (ADCC).

FIG. 2 wild type CD16A, wild type CD64 and CD64/16A chimeric constructs. The scissors and dashed lines shown for CD16A represent extracellular proteolytic sites for ectodomain shedding.

FIG. 3 NK92 cells expressing wild-type CD16A or CD 64/16A. (A) NK92-CD64/16A cells were stained with anti-CD 16, anti-CD 64, or control antibodies. (B) NK92-CD16A cells were stained with anti-CD 16, anti-CD 64, or control antibodies. (C) NK92-CD64/16A, NK92-CD16A or NK92 parental cells were incubated with trastuzumab (trastuzumab) and then with anti-human IgG-APC secondary antibody, or with anti-human IgG-APC secondary antibody alone (control). All antibody staining levels were determined by flow cytometry.

Figure 4. NK92 cells expressing CD64/CD16A induced higher levels of ADCC compared to wild type CD 16A. (A) Standard ADCC assays were performed in which trastuzumab (herceptin) was included in the assay. (B) Standard ADCC assays were performed in which NK92-CD64/CD16A and NK92-CD16A cells were pre-incubated with trastuzumab, followed by mAb wash-off, and effector cells were then incubated with SKOV-3 target cells.

FIG. 5 flow cytometry data comparing phenotypic markers expressed by iNK-CD64/CD16A and iNK-pKT2 cells.

FIG. 6. Bar graph and data showing that iNK-CD64/CD16A cells induced higher levels of ADCC compared to iNK-CD16A cells using SKOV-3 target cells.

FIG. 7 bar graph and data showing that iNK-CD64/CD16A, but not iNK-CD16A cells, can be preloaded with therapeutic mAb and mediate ADCC.

Figure 8 bar graph and data showing that iNK-CD64/CD16A cells induced higher levels of ADCC compared to iNK-CD16A cells using MA148 target cells.

FIG. 9 amino acid sequence of an exemplary CD64/CD16A chimeric IgG Fc receptor (SEQ ID NO: 1).

FIG. 10 amino acid sequence of CD64 IgG Fc receptor (SEQ ID NO: 2).

FIG. 11 amino acid sequence of an exemplary CD16A-CD 28-BB-zeta chain chimeric IgG Fc receptor (SEQ ID NO: 3).

FIG. 12 is an amino acid sequence of an exemplary CD 16A-BB-zeta chain chimeric IgG Fc receptor (SEQ ID NO: 4).

FIG. 13 NK92 cells expressing CD 64/16A. (A) Schematic representation of the cell membrane forms of CD16A, CD64, and CD 64/16A. As shown, CD16A underwent ectodomain shedding by ADAM17 at a membrane proximal position, which was not present in CD64 and CD 64/16A. (B) NK92 parental cells, NK92-CD16A cells and NK92-CD64/16A cells were stained with anti-CD 16, anti-CD 64 or isotype matched negative control mAb and examined by flow cytometry. (C) NK92-CD16A and NK92-CD64/16A cells were incubated with SKOV-3 cells in the presence or absence of trastuzumab (5 μ g/ml) at 37 ℃ (E: T ═ 1:1) for 2 hours. NK92-CD16A and NK92-CD64/16A cells were then stained with anti-CD 16 mAb or anti-CD 64mAb, respectively, and examined by flow cytometry. Nonspecific antibody labeling was determined using appropriate isotype negative control mabs. Data are representative of at least three independent experiments.

Figure 14 CD64/16A promotes target cell conjugation, ADCC and IFN γ production. (A) eGFP expressing NK92-CD64/16A cells and CellTrace Violet labeled SKOV-3 cells were mixed at an E: T ratio of 1:2 in the presence or absence of trastuzumab (5. mu.g/ml), incubated at 37 ℃ for 60 minutes, fixed, and then analyzed by flow cytometry. Representative data for at least three independent experiments are shown. (B) NK92-CD64/16A cells were incubated with SKOV-3 cells (E: T ═ 20:1) and trastuzumab (tras.) at the indicated concentrations (left panel), or with SKOV-3 cells at the indicated E: T ratios in the presence or absence of trastuzumab (5 μ g/ml) (right panel) for 2 hours at 37 ℃. Data are expressed as% specific release and show mean ± SD of three independent experiments. Statistical significance was expressed as p <0.05, p < 0.01. (C) NK92-CD64/16A cells were incubated with SKOV-3 cells (E: T ═ 20:1) as indicated, in the presence or absence of trastuzumab (5 μ g/ml) and anti-CD 64mAb10.1 (10 μ g/ml), for 2 hours at 37 ℃. Data are expressed as% specific release and show mean ± SD of three independent experiments. Statistical significance was expressed as p < 0.01. (D) NK92-CD64/16A cells were incubated with SKOV-3 cells (E: T ═ 1:1) in the presence or absence of trastuzumab (5 μ g/ml) for 2 hours at 37 ℃. Secreted IFN γ levels were quantified by ELISA. Data are shown as the average of two independent experiments.

FIG. 15 CD64/16A was attached to soluble tumor targeting mAb and IgG fusion protein. (A) The relative expression levels of CD16A and CD64/16A on NK92 cells were determined by cell staining with anti-CD 16 and anti-CD 64 mAbs (black bars), respectively, or isotype-matched negative control antibodies (gray bars). The bar graph shows the Mean Fluorescence Intensity (MFI). + -. SD of three independent experiments. Representative flow cytometry data are displayed as histogram overlays. The dashed histogram shows CD64 staining of NK92-CD64/16A cells, the orange filled histogram shows CD16A staining of NK92-CD16A cells, and the green filled histogram shows isotype control antibody staining of NK92-CD16A cells. (B) NK92-CD16A and NK92-CD64/16A cells were incubated at 37 ℃ for 2 hours in the presence or absence of trastuzumab (5 μ g/ml), washed, stained with fluorophore-conjugated anti-human secondary antibody, and analyzed by flow cytometry. Data are representative of at least three independent experiments. (C) NK92-CD64/16A cells were incubated with cetuximab (cetuximab) or rituximab (rituximab) (5 μ g/ml each), washed, and then stained with fluorophore-conjugated anti-human secondary antibodies. Controls represent cells stained with anti-human secondary antibody only. NK92-CD64/16A cells were also incubated with L-selectin/Fc (5. mu.g/ml), washed, and then stained with fluorophore-conjugated anti-L-selectin mAb. NK92 cells lacked expression of endogenous L-selectin (data not shown). All staining was analyzed by flow cytometry. Data shown are representative of three independent experiments. (D) NK92-CD16A and NK92-CD64/16A cells were incubated in the presence or absence of trastuzumab (5. mu.g/ml), washed, and exposed to SKOV-3 cells at 37 ℃ for 2 hours at the indicated E: T cell ratio. Data are shown as mean ± SD of three independent experiments. Statistical significance was expressed as p <0.01, p < 0.001. bd is below the limit of detection (i.e., < spontaneous release of negative control cells). (E) NK92-CD16A and NK92-CD64/16A cells were incubated with SKOV-3 cells (E: T ═ 10:1) in the presence or absence of trastuzumab (5 μ g/ml) as indicated for 2 hours at 37 ℃. Data are shown as mean ± SD of three independent experiments. Statistical significance was expressed as p < 0.01.

FIG. 16 production of iNK cells expressing CD64/CD 16A. The ipscs were transduced to stably express CD64/16A, differentiated into NK cells, and then expanded using K562-mbIL21-41BBL feeder layer cells. iNK-CD64/16A cells and freshly isolated Peripheral Blood (PB) NK cells enriched from adult peripheral blood were stained for CD56, CD3 and various inhibitory and activating receptors, as indicated. CD64/16A expression was determined by staining cells with anti-CD 64 mAb. Representative data for at least three independent experiments are shown.

FIG. 17. iNK-CD64/16A cells showed enhanced ADCC compared to iNK-pKT2 control cells. (A) NK cells derived from ipscs transduced with empty vector (iNK-pKT2) or CD64/16A (iNK-CD64/16A) were stained for CD56, CD64, and CD16A, as indicated. (B) iNK-pKT2 and iNK-CD64/16A cells were incubated with SKOV-3 cells (E: T ═ 10:1) in the presence or absence of trastuzumab (5. mu.g/ml), functional blocking anti-CD 16 mAb 3G8 (5. mu.g/ml) and functional blocking anti-CD 64mAb10.1 (5. mu.g/ml) for 2 hours at 37 ℃ as indicated. Data are shown as mean ± SD of three independent experiments. Statistical significance was expressed as p < 0.001; p < 0.0001. (C) iNK-pKT2 and iNK-CD64/16A cells were incubated in the presence or absence of trastuzumab (5. mu.g/ml), washed, and exposed to SKOV-3 cells (E: T ═ 10:1) for 2 hours at 37 ℃. Data are shown as mean ± SD of three independent experiments. Statistical significance was expressed as p < 0.001.

FIG. 18 sequence alignment of canine CD16A (SEQ ID NO: 5), canine CD64sp (SEQ ID NO: 25) and human CD16A (SEQ ID NO: 6).

FIG. 19 sequence alignment of canine CD64(SEQ ID NO: 7) and human CD64(SEQ ID NO: 8).

Figure 20. NK92 cells expressing wild-type human CD64 mediate ADCC. (A) NK92-CD64 cells were stained with an isotype-matched negative control mAb or an anti-CD 64mAb (clone 10.1) and examined by flow cytometry. (B) NK92-CD64 cells and SKOV-3 cells (in the indicated E: T ratio) in the presence or absence of trastuzumab (tras.) (5. mu.g/ml) at 37 ℃ temperature for 2 hours. Representative data for at least three independent experiments are shown.

Detailed Description

The present disclosure describes recombinant immune cells, methods of making recombinant immune cells, and methods of using recombinant immune cells. Typically, recombinant immune cells are genetically modified to include a heterologous IgG Fc receptor. In some cases, a heterologous IgGFc receptor may be a chimeric receptor engineered to include domains from two or more receptors. In other embodiments, the heterologous may be an IgG Fc receptor that is not naturally expressed by the immune cell. Generally, recombinant immune cells provide a sustained cytotoxic immune response against a target (e.g., tumor cells) that is targeted for killing by immune cells because the target binds therapeutic antibodies recognized by IgG Fc receptors.

The cell-mediated immune defense mechanism involves the engagement of Fc receptors expressed by leukocytes with antibodies attached to target cells, thereby killing the target cells. This process is called antibody-dependent cell-mediated cytotoxicity (ADCC). Therapeutic monoclonal antibodies (mabs) have been generated against a variety of tumor antigens and have been tested in clinical trials for the treatment of infectious diseases, chronic diseases, and cancers, including, for example, AML, breast cancer, ovarian cancer, gastric cancer, neuroblastoma, and lymphoma. Many clinically successful mabs use ADCC as the mechanism of action. However, limitations of antibody therapy are the development of patient resistance and anergy in certain malignancies.

The present disclosure describes methods for enhancing the interaction of Fc receptors with therapeutic antibodies. The method involves a chimeric receptor comprising a CD16A domain and a CD64 domain.

CD16A (Fc γ RIIIA) is an IgG Fc receptor expressed by human Natural Killer (NK) cells, a population of cytotoxic lymphocytes, and is its only means of recognizing IgG bound to tumor cells or virus-infected cells. CD16A is a potent activating receptor that induces ADCC by NK cells (fig. 1). The CD16A transmembrane region is responsible for attachment to the CD3 ζ and/or FcR γ chain (FcR γ) comprising an immunoreceptor tyrosine-based activation motif (fig. 2; fig. 13A), and the CD16A cytoplasmic domain interacts with intracellular molecules critical to receptor function. CD16A is a low affinity Fc γ R with limited ability to bind to target cells coated with therapeutic mabs. CD16A also undergoes rapid down-regulation of expression upon cell activation, thereby significantly reducing its cell surface density and affinity for IgG. CD16A down-regulation occurs at an extracellular site proximal to the plasma membrane via a proteolytic event and is referred to as ectodomain shedding. The location of this cleavage site has been reported (Jing et al, 2015. public science library Integrated services (PLoS One) 10: e0121788) and is shown schematically in FIGS. 2 and 13A.

CD64(Fc γ RI) is another IgG Fc receptor and is expressed by monocytes, macrophages and activated neutrophils. CD64 is a high affinity IgG receptor. This receptor does not undergo ectodomain shedding after cell activation, nor does it naturally transduce the signal for ADCC in NK cells.

The present disclosure describes chimeric Fc γ rs comprising a CD16A domain and a CD64 domain. The chimeric receptor includes an extracellular region of human CD64 and a cytoplasmic region of human CD16A, exemplary embodiments of which are schematically illustrated in fig. 2 and 13A as CD 64/16A. In various embodiments, the chimeric CD64/CD16A receptor may comprise a CD64 transmembrane region or a CD16A transmembrane region. The CD64/16A construct has been engineered to lack a CD16A extracellular cleavage site and thus be less susceptible to ectodomain shedding (fig. 2; fig. 13A), but to include at least a portion of the intracellular region of CD16A involved in intracellular signaling.

Furthermore, although described herein in the context of exemplary embodiments in which the CD64 domain and CD16A comprise the amino acid sequences of human CD64 and human CD16A, respectively, the chimeric fcyrs described herein can include amino acid sequences that are or are derived from any suitable CD64 or CD16A naturally expressed by any species. Fig. 18 and 19 provide amino acid sequence alignments of human and canine amino acid sequences of CD16A (fig. 18) and CD64 (fig. 19).

As used herein, an amino acid sequence of a domain is "derived from" an amino acid sequence of a reference polypeptide if the amino acid sequence of the domain has a specified amount of sequence similarity and/or sequence identity compared to the amino acid sequence of the reference polypeptide. Sequence similarity can be determined by aligning the residues of two polypeptides (e.g., the domain amino acid sequence and the amino acid sequence of a reference CD16A or CD64 polypeptide) to optimize the number of identical amino acids along the length of their sequences. In order to optimize the number of identical amino acids, gaps in either or both sequences are allowed in making the alignment, although the amino acids in each sequence must still maintain their correct order.

Pairwise comparative analysis of amino acid sequences can be performed using the BESTFIT algorithm in the GCG software package (version 10.2, Madison WI). Alternatively, the polypeptides may be compared using the BLASTP program of the BLAST 2 search algorithm, as described by Tatiana et al (FEMS Microbiol Lett,174,247-250(1999)), and available at the National Center for Biotechnology Information (NCBI)) website. Default values for all BLAST 2 search parameters may be used, including matrix (matrix) ═ BLOSUM 62; open gap penalty (open gap penalty) is 11, extended gap penalty (extension gap penalty) is 1, gap x _ drop is 50, expect (expect) is 10, font size (word) is 3, and filter on.

A domain's amino acid sequence is "derived" from the amino acid sequence of a reference polypeptide if the amino acid sequence of the domain has a specified degree of amino acid sequence "identity" or amino acid sequence "similarity". Amino acid sequence identity refers to the presence of identical amino acids. Amino acid sequence similarity refers to the presence of not only identical amino acids, but also conservative substitutions. Conservative substitutions of amino acids may be selected from other members of the class to which the substituted amino acid belongs. For example, it is well known in the field of protein biochemistry that an amino acid belonging to a group of amino acids having a specific size or characteristic (such as charge, hydrophobicity, and hydrophilicity) can be substituted for another amino acid without altering the activity of the protein, particularly in regions of the protein not directly related to biological activity. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Conservative substitutions include, for example, Lys to Arg, and vice versa, to retain a positive charge; glu is replaced with Asp, and vice versa, to maintain a negative charge; ser to Thr to maintain free-OH; and substitution of Gln to Asn to maintain free-NH₂. Likewise, biologically active classes of polypeptides comprising deletions or additions of one or more contiguous or noncontiguous amino acids that do not abrogate the functional activity of the polypeptide are also contemplated(iii) an analog.

An amino acid sequence of a CD16A domain or a CD64 domain is "derived from" a reference amino acid sequence if the domain amino acid sequence has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence similarity to the reference amino acid sequence.

An amino acid sequence of a CD16A domain or a CD64 domain is "derived from" a reference amino acid sequence if the domain amino acid sequence has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the reference amino acid sequence.

To determine whether a domain amino acid sequence is "derived from" a particular reference amino acid sequence, exemplary suitable reference polypeptides include the corresponding domains of human CD16A (SEQ ID NO: 6), canine CD16A (SEQ ID NO: 5), human CD64(SEQ ID NO: 8), canine CD64(SEQ ID NO: 7), canine CD64sp (SEQ ID NO: 25), or any of the constructs listed in Table 1.

Furthermore, although described herein in the context of the exemplary embodiments shown in fig. 2 and 13A, chimeric receptors can be designed to include different points of fusion between CD64 and CD16A shown in fig. 2 and 13A, to include a modified functional motif in the CD16A cleavage region, CD64 or CD16A region, and/or to add other signaling domains, such as, for example, signaling domains of CD27, CD28, CD134(OX40), CD137(4-1BB), FcR γ, or CD3 ζ. Thus, the chimeric receptor can be designed to increase proliferation of NK cells or other effector cells, increase survival of NK cells or other effector cells, increase potency of NK cells or other effector cells, and/or reduce NK cell depletion in vivo.

In certain embodiments, the chimeric Fc γ R may be modified to include a cytoplasmic domain or a signaling domain that confers additional functions to the chimeric receptor. For example, chimeric Fc γ rs may include a functional portion of CD28 that transduces signals involved in T cell proliferation, survival, and cytokine production. As another example, a chimeric Fc γ R may include a functional portion of 4-1BB, which contributes to clonal expansion, survival, and development of hematopoietic cells. As another example, a chimeric Fc γ R may compriseAs another example, the chimeric Fc γ R may include a functional portion of the cytoplasmic domain of DAP10, which includes a YxxM motif that specifically activates cellular cytotoxicity, cellular survival, and proliferation of NK and T cells, as another example, the chimeric Fc γ R may include a functional portion of the cytoplasmic domain of DAP10, which includes a phosphatidylinositol 3-kinase-dependent signaling pathway that specifically activates cellular cytotoxicity, cellular survival, and proliferation of NK and T cells, as another example, the chimeric Fc γ R may include a functional portion of the cytoplasmic domain of DAP12, which includes an ITAM motif that triggers cellular cytotoxicity, cellular survival, and proliferation of DAP12, as another example, the chimeric Fc γ R may include a functional portion of the transmembrane domain of DAP D (or CD314), which specifically links the functional portion of the transmembrane domain of NK 2, and survival domain to a constitutive signal of DAP 34, and proliferation of T cells, as well as a cytoplasmic receptor B receptor, a cytoplasmic receptor B, which specifically links the cellular proliferation of DAP 3629 to a cell proliferation receptor, a cytoplasmic receptor B, a chimeric Fc 5, aThe site is linked to the FcR gamma chain (FcR γ) to mediate the most efficient degranulation signaling in myeloid cells that triggers anaphylaxis, which can be exploited for cancer therapy using recombinant high affinity IgG Fc receptors.

Table 1 lists exemplary constructs that include exemplary cytoplasmic domain and/or signaling domain modifications.

TABLE 1

EC: an extracellular domain; TM: a transmembrane domain; CY: a cytoplasmic domain; SD: a signaling domain.

64: CD64, high affinity IgG Fc receptor Fc γ RI; mutCD 64: a high affinity IgG Fc receptor Fc γ R1 with cytoplasmic mutations that result in higher levels of cytokine production and degranulation; 16A: C16A, low affinity IgG Fc receptor Fc γ RIIIA; - -: no signaling domain; 28: CD28, a costimulatory receptor for cell proliferation and activation; BB: 4.1BB or CD 137;chain or CD 247; FcR γ: the FcR gamma chain; d10: DAP10 signaling linker; d12: DAP12 signaling linker; FceR: high affinity IgE Fc receptors; 16PKC^-: having a mutation at the PKC phosphorylation site to disrupt the CD16A cytoplasmic domain of cytokine production mediated by CD 16A; G2D: NKG2D or CD 314; 2B 4: NKR2B4 or CD 244.

The CD64/16A chimeric receptor may be encoded by a cDNA that can be transcribed and translated from an expression vector introduced into a host cell to produce a recombinant cell. The host cell may comprise a suitable leukocyte-like cell or primary leukocyte. Suitable leukocyte-like cells include, but are not limited to, hematopoietic cell lines or induced pluripotent stem cells. Suitable primary leukocytes include, but are not limited to, NK cells, monocytes, macrophages, neutrophils, or T lymphocytes. Expression of CD64/16A by genetically engineered leukocytes in the presence of native and therapeutic antibodies can enhance effector function of recombinant cell killing of target cells (e.g., tumor cells and virus-infected cells) as compared to unmodified host cells. Because the CD64/16A chimeric receptor binds IgG with high affinity, the therapeutic mAb can also be attached to effector cells expressing the construct prior to its administration to a patient. Thus, CD64/16A with the therapeutic mAb attached would provide a targeting element for the effector cells to direct them to the cancer site.

NK92 cells are a human NK cell line lacking endogenous CD16A expression. Generating NK cells expressing wild-type CD16A, wild-type CD64, or the exemplary chimeric receptor CD64/16A in a stable manner. NK92 cells expressing CD64/16A were stained with anti-CD 64mAb, but not with anti-CD 16 mAb (fig. 3A). NK92-CD16A cells were stained with anti-CD 16 mAb, but not with anti-CD 64mAb (fig. 3B). NK92 cells expressing CD64 could be stained with anti-CD 64mAb, but not with isotype-matched negative control mAb (fig. 20A).

Figure 3C shows the ability of NK92 cells expressing CD64/16A or CD16A to bind trastuzumab, a therapeutic mAb specific for HER2/EGFR2 overexpressed by certain malignancies. Untransduced NK92 cells, NK92-CD64/16A cells and NK92-CD16A cells were incubated with trastuzumab (5 μ g/ml) for 2 hours at room temperature, washed to remove unbound antibody, incubated with fluorophore-conjugated anti-human IgG secondary antibody, and then examined by flow cytometry. Compared with NK92-CD16A cells and NK92 cells, NK92-hCD64/16A cells bound to trastuzumab at a much higher level.

Figure 4 presents data showing that the exemplary chimeric receptor CD64/16A confers ADCC effector function on NK92 cells. NK92 cells expressing CD64/16A or wild-type CD16A were incubated with the human ovarian cancer cell line SKOV-3(20:1 ratio) in the presence or absence of trastuzumab (0.005. mu.g/ml, 0.05. mu.g/ml, 0.5. mu.g/ml or 5. mu.g/ml) at various concentrations. NK92 cells expressing CD64/16A or wild-type CD16A exhibited SKOV-3 cytotoxicity in the presence of trastuzumab. At all trastuzumab concentrations examined, CD 64/16A-expressing NK92 cells had higher target cell killing levels than NK92-CD16A cells (fig. 4A). NK92 cells expressing either CD64 also showed SKOV-3 cytotoxicity in the presence of trastuzumab (fig. 20B). In addition, NK92-CD64/16A and NK92-CD16A cells were pretreated with trastuzumab at 5 μ g/ml or 10 μ g/ml for 2 hours, washed to remove unbound antibody, and then incubated with SKOV-3 cells. In this assay, NK92-CD64/16A cells showed significantly enhanced killing of target cells compared to NK92-CD16A cells (fig. 4B).

CD64/16A is also expressed in ipscs, and these cells then differentiate into NK cells (referred to herein as iNK cells). As shown in figure 5, the expression of several NK cell markers was compared for iNK cells transduced with CD64/16A or empty vector (pKT2) as a control. iNK-CD64/16A and iNK-pKT2 cells are CD56⁺And CD3^-Indicating that they are indeed NK cells. They also expressed similar levels of various NK cell markers. iNK-CD64/16A and iNK-pKT2 cells were found to express similar levels of CD16A, while only iNK-CD64/16A was stained with anti-CD 64mAb (FIG. 5).

ADCC of iNK-CD64/16A and iNK-pKT2 cells was evaluated as described above for NK92 cells. iNK-CD64/16A and iNK-pKT2 cells were incubated with SKOV-3 cells at a ratio of 10:1 in the presence or absence of trastuzumab (5. mu.g/ml). Compared to iNK-pKT2 cells, iNK-CD64/16A cells showed increased SKOV-3 cytotoxicity and higher levels of ADCC in the presence of trastuzumab (FIG. 6).

Although iNK-CD64/16A and iNK-pKT2 cells expressed similar levels of CD16A (FIG. 5), the functional blocking anti-CD 16A mAb 3G8 only blocked ADCC of iNK-pKT2 cells (FIG. 6). In contrast, functional blocking anti-CD 64mAb10.1 only blocked ADCC of iNK-CD64/16A cells (FIG. 6). iNK-CD64/16A and iNK-pKT2 cells were also pretreated with trastuzumab at 10. mu.g/ml for 2 hours, washed to remove unbound antibody, and then incubated with SKOV-3 cells. In this assay, iNK-CD64/16A cells showed significantly enhanced killing of target cells compared to iNK-pKT2 cells (FIG. 7). MA148 is a human ovarian cancer cell line that expresses significantly lower levels of HER2 compared to SKOV-3 cells. ADCC was assessed iNK-CD64/16A and iNK-pKT2 cells when exposed to MA148 cells at various rates, with or without trastuzumab (5 μ g/ml). Again, in the presence of trastuzumab, iNK-CD64/16A cells exhibited significantly higher tumor cell killing levels than iNK-pKT2 cells at all effector to target cell ratios (fig. 8).

The chimeric receptor CD64/16A (FIG. 2; FIG. 13A) was stably expressed in the human NK cell line NK 92. These cells lack endogenous Fc γ R, but transduced cells expressing exogenous CD16A can mediate ADCC. As shown in fig. 13B, the anti-CD 64mAb stained NK92 cells expressing CD64/16A, but not parental NK92 cells or NK92 cells expressing CD 16A. anti-CD 16 mAb stained NK92 cells expressing CD16A, but not NK92 cells expressing CD64/16A or parental NK92 cells (fig. 13B). Upon NK cell activation, CD16A undergoes ectodomain shedding by ADAM17, resulting in its expression being rapidly down-regulated. CD16A and its isoform, CD16B, on neutrophils are cleaved by ADAM17, and this occurs in the extracellular region proximal to the cell membrane. The ADAM17 cleavage region of CD16A was absent from CD64 or CD64/16A (FIG. 13A). CD16A experienced a > 50% reduction in expression after NK92 stimulation by ADCC, whereas CD64/16A showed little to no downregulation (fig. 13C).

To evaluate the function of CD64/16A, the ability of CD64/16A to elicit E: T conjugation, induce ADCC, and stimulate cytokine production after NK cells engage antibody-bound tumor cells was examined. NK cells form stable conjugates with target cells before releasing their granular contents. The conjugation of NK92-CD64/16A cells and SKOV-3 cells was examined using a two-color flow cytometer. SKOV-3 cells are an ovarian cancer cell line expressing HER2, and this assay was performed in the absence or presence of the anti-HER 2 therapeutic mAb trastuzumab. The bicistronic vector containing CD64/16A also expressed eGFP and its fluorescence was used to identify NK92 cells. SKOV-3 cells were labeled with the fluorescent dye CellTrace Violet. T-conjugation results in a two-color event, which is assessed by flow cytometry. Incubation of NK92-CD64/16A cells with SKOV-3 cells resulted in very low levels of conjugation after initial exposure, which increased after 60 minutes of exposure (fig. 14A). However, conjugation of NK92-CD64/16A cells and SKOV-3 was greatly enhanced in the presence of trastuzumab (fig. 14A). This enhancement of conjugation corresponds to a higher level of target cell killing. As shown in FIG. 14B, NK92-CD64/16A cells caused by SKOV-3 cell cytotoxicity changes according to trastuzumab concentration and E: T ratio. To confirm the role of CD64/16A in inducing target cell killing, ADCC assays were performed in the presence and absence of anti-cd64mab 10.1 (which blocks IgG binding) (fig. 14C). Cytokine production is also induced during ADCC, while NK cells are the major producers of IFN γ. NK92-CD64/16A cells exposed to SKOV-3 cells and trastuzumab produced significantly higher levels of IFN γ than SKOV-3 cells alone (fig. 14D). Taken together, the above findings demonstrate that the CD64 component of the recombinant receptor engages tumor-bound antibodies, and that the CD16A component promotes intracellular signaling leading to degranulation and cytokine production.

CD64 is distinguished from other Fc γ R members by its unique third extracellular domain, which contributes to its high affinity and stable binding to soluble monomeric IgG. Comparison of NK92 cells expressing higher affinity variants of CD64/16A or CD16A-176V for their ability to capture soluble therapeutic mAbs. The examined NK92 cell transducer expressed similar levels of CD64/16A and CD16A (FIG. 15A). NK92 cell transductants were incubated with trastuzumab for 2 hours, excess antibody was washed off, stained with fluorophore-conjugated anti-human IgG antibody, and then evaluated by flow cytometry. As shown in figure 15B, NK92-CD64/16A cells captured significantly higher levels of trastuzumab (8.1 doubling plus ± 1.3, mean of three independent experiments ± SD) compared to NK92-CD16A cells. In addition, NK92-CD64/16A cells efficiently captured the tumor targeting mabs cetuximab and rituximab, as well as the fusion protein L-selectin/Fc (fig. 15C).

NK92-CD64/16A cells with captured tumor targeting mAb were tested to determine if the cells mediated ADCC. For this assay, equal numbers of NK92-CD64/16A and NK92-CD16A cells were incubated with the same concentration of soluble trastuzumab, washed and exposed to SKOV-3 cells. Target cell killing of NK92-CD64/16A cells with captured trastuzumab was significantly higher than that of NK92-CD64/16A cells alone, and was superior to NK92-CD16A cells with or without trastuzumab treatment at all E: T ratios examined (fig. 15D). In contrast, SKOV-3 cytotoxicity was not significantly different by NK92-CD16A and NK92-CD64/16A cells in the presence of trastuzumab and without being washed away (fig. 15E), indicating equivalent cytotoxicity of both transductants. Taken together, these findings indicate that CD 64/16A-expressing NK92 cells can stably bind to soluble anti-tumor mabs and IgG fusion proteins, and that they can act as targeting elements to kill tumor cells.

Expression and function of CD64/16A in iPSC-derived NK cells

Undifferentiated iPSCs were transduced with the Sleeping Beauty transposon plasmid for nonrandom gene insertion and stable expression to express CD 64/16A. Ipscs were differentiated into hematopoietic cells and then into iNK cells as described in example 2. Generating CD34⁺CD43⁺CD45⁺Cells, further differentiated into iNK cells, and these cells were expanded for analysis using recombinant IL-2 and aapcs. CD56⁺CD3^-Is a marker phenotype for human NK cells, and these cells constitute the majority of our differentiated cell population (fig. 16). The expression of activating and inhibitory receptors on iNK cells was also assessed and compared to expression by peripheral blood NK cells. Certain receptors, such as CD16A, are expressed by a similar proportion of the two NK cell populations. However, expanded iNK cells lacked the expression of inhibitory KIR receptors KIR2DL2/3, KIR2DL1, and KIR3DL1, as well as certain activating receptors (NKp46 and NKG2D) (fig. 16). Another difference compared to peripheral blood NK cells was that iNK cells were stained with anti-CD 64mAb (FIG. 16), demonstrating expression of CD 64/16A.

To assess the function of CD64/16A in iNK cells, iNK cells derived from ipscs transduced with PKT2 empty vector or PKT2-CD64/16A were compared. Both iNK cell populations expressed the above-mentioned NK cell markers at similar levels and ratios (data not shown), including CD16A (fig. 17A), but only iNK-CD64/16A cells were stained with anti-CD 64mAb (fig. 17A). Both iNK transductants were demonstrated to have increased SKOV-3 cell killing in the presence of trastuzumab, but iNK-CD64/16A cells mediated significantly higher levels of ADCC compared to iNK-pKT2 control cells (fig. 17B). anti-CD 16 functional blocking mAb 3G8 effectively inhibited ADCC mediated by iNK-pKT2 cells (fig. 17B), but anti-CD 64mAb10.1 did not. In contrast, 10.1 blocked ADCC mediated by iNK-CD64/16A cells, but 3G8 did not (FIG. 17B). These findings indicate that iNK cells are cytolytic effectors that engage tumor cells in response to binding of CD16A and CD64/16A to antibodies.

In addition, iNK-CD64/16A and iNK-PKT2 cells were treated with soluble trastuzumab, excess antibody was washed away, and cells were exposed to SKOV-3 cells. Under these conditions, ADCC mediated by iNK-CD64/16A cells was surprisingly higher compared to iNK-pKT2 cells (fig. 17C), and it was also established that CD64/16A can capture soluble anti-tumor mabs as targeting elements for tumor cell killing.

Taken together, the data show that CD64/16A binds the therapeutic mAb with higher affinity than CD 16A. Moreover, CD64/16A expressed in NK92 and iNK cells confers the cells the ability to mediate higher levels of ADCC compared to NK92 and iNK cells expressing wild-type or endogenous CD16A, respectively. NK cells expressing CD64/16A facilitated cell-to-antibody binding tumor cell conjugation, cytotoxicity, and IFN γ production, demonstrating the function of both components of recombinant Fc γ R. NK92 and iNK cells expressing CD64/16A can be preloaded with therapeutic mabs before they are exposed to target cells. Cells expressing chimeric receptors and preloaded with therapeutic antibodies may allow for modification of engineered NK cells with different therapeutic mabs targeting elements of multiple types of cancer. Finally, CD64/16A lacks the ADAM17 cleavage region present in CD16A and does not undergo the same level of down-regulation of expression during ADCC.

CD64/16A was shown to play a role in two NK cell platforms, the NK92 cell line and primary NK cells derived from iPSC. NK92 cells lack inhibitory KIR receptors and exhibit high levels of natural cytotoxicity compared to other NK cell lines derived from patients. NK92 cells have been widely used to express modified genes to direct their cytolytic effector function, have been evaluated in preclinical studies, and are being clinically tested in cancer patients. ipscs are also well suited for genetic engineering and can differentiate into NK cells expressing various receptors to direct their effector functions. The iNK cells generated in this study lacked several inhibitory and activating receptors as indicators of immature state. In some applications, therapeutic iNK cells lacking inhibitory receptors and certain activating receptors may allow for more direct and/or more efficient tumor cell killing by engineered receptors.

iNK express endogenous CD16A and mediate ADCC, so they are cytotoxic effector cells. A single NK cell can kill multiple tumor cells in different ways. This includes a sequential process of contact and degranulation, known as continuous killing, and localized dispersion of its particulate content that kills surrounding tumor cells, known as bystander killing. Inhibitory CD16A shedding has been reported to delay NK cell detachment from target cells and to reduce continuous NK cell killing in vitro. NK cells expressing CD64/16A may be less efficient in continuous killing and more efficient in bystander killing due to the CD64 module and its lack of ectodomain shedding.

NK cells expressing CD64/16A as a therapeutic agent in combination with a therapeutic antibody have several potential advantages. Modification of CD64/16A expressing NK cells with an antibody can reduce the dose of therapeutic antibody administered to a patient. Fusion proteins containing the human IgG Fc region, such as L-selectin/Fc, can also be captured by CD64/16A and this can provide additional options for directing tissue and tumor antigen targeting of engineered NK cells. The NK92 and iNK cell platforms for adoptive cell therapy can also be easily genetically modified at the clonal level and expanded to clinical-scale cell numbers to produce engineered NK cells with improved effector activity as therapeutic agents for off-the-shelf cancer immunotherapy.

In some embodiments, iPSC-derived NK cells expressing CD64/16A may exhibit increased in vivo anti-cancer activity in the presence of a therapeutic mAb. For example, NOD/SCID/yc stably engineered to express firefly luciferase may be used^-/-(NSG) mouse and human cancer cell lines were used for bioluminescence imaging to test the activity of iNK cells against cancer cells. SKOV-3 and MA148 ovarian cancer cell lines can be used as modelsAnd (4) forming an in vivo target. NSG female mice can be sublethally irradiated and then injected intraperitoneally with tumor cells for bioluminescent imaging to quantify tumor growth or regression. Mice were administered IL-2 and/or IL-15 every other day for 4 weeks to promote NK cell survival in vivo. The therapeutic antibody (e.g., trastuzumab) can be administered, e.g., once per week for 4 weeks. Tumor growth/regression can be monitored by, for example, bioluminescence imaging and weighing of the mice. Mice can also be bled (e.g., once a week) to quantify human NK cell viability, and the expression/cell surface levels of various effector function markers can be further assessed by FACS. Evidence of metastasis can be determined, for example, by harvesting the internal organ and examining the metastasis of the internal organ.

In some embodiments, the number of cells in the therapeutic composition is at least 0.1x10 per dose⁵Individual cell, at least 1x10⁵Individual cell, at least 5x10⁵Individual cell, at least 1x10⁶Individual cell, at least 5x10⁶Individual cell, at least 1x10⁷Individual cell, at least 5x10⁷Individual cell, at least 1x10⁸Individual cell, at least 5x10⁸Individual cell, at least 1x10⁹Individual cell or at least 5x10⁹In some embodiments, the number of cells in the therapeutic composition is about 0.1 × 10 per dose⁵To about 1 × 10⁶(ii) individual cells; about 0.5x10 per dose⁶To about 1X10⁷About 0.5 × 10 per dose⁷To about 1 × 10⁸About 0.5 × 10 per dose⁸To about 1 × 10⁹(ii) individual cells; about 1x10 per dose⁹To about 5x10⁹About 0.5 × 10 per dose⁹To about 8 × 10⁹(ii) individual cells; about 3x10 per dose⁹To about 3X10¹⁰Individual cells, or any range in between. Typically, for a 60kg patient, 1x10⁸Individual cells/dose can be converted to 1.67x10⁶Individual cells/kg.

In one embodiment, the number of cells in the therapeutic composition is the number of immune cells in a portion or single cord blood, or at least0.1x10⁵At least 0.5x10 cells/kg body weight⁵At least 1x10 cells/kg body weight⁵At least 5x10 cells/kg body weight⁵At least 10x10 cells/kg body weight⁵At least 0.75x10 cells/kg body weight⁶At least 1.25x10 cells/kg body weight⁶At least 1.5x10 cells/kg body weight⁶At least 1.75x10 cells/kg body weight⁶At least 2x10 cells/kg body weight⁶At least 2.5x10 cells/kg body weight⁶At least 3x10 cells/kg body weight⁶At least 4x10 cells/kg body weight⁶At least 5x10 cells/kg body weight⁶At least 10x10 cells/kg body weight⁶At least 15x10 cells/kg body weight⁶At least 20x10 cells/kg body weight⁶At least 25x10 cells/kg body weight⁶At least 30x10 cells/kg body weight⁶1x10 cells/kg body weight⁸Individual cells/kg body weight, 5x10⁸Individual cells/kg body weight or 1x10⁹One cell/kg body weight.

In one illustrative embodiment, the cells provided to the subject are in an effective amount of at least 2 × 10⁶Individual cell/kg, at least 3 × 10⁶Individual cell/kg, at least 4 × 10⁶Individual cell/kg, at least 5 × 10⁶Individual cell/kg, at least 6x10⁶Individual cell/kg, at least 7x10⁶Individual cell/kg, at least 8x10⁶At least 9x10 cells/kg⁶Individual cell/kg or at least 10x10⁶One or more cells/kg, including all cells at intermediate doses.

In another illustrative embodiment, the effective amount of cells provided to the subject is about 2 × 10⁶Individual cell/kg, about 3 × 10⁶Individual cell/kg, about 4 × 10⁶Individual cell/kg, about 5 × 10⁶Individual cell/kg, about 6X10⁶Individual cell/kg, about 7X10⁶Individual cell/kg, about 8X10⁶Individual cell/kg, about 9X10⁶Individual cell/kg or about 10x10⁶One or more cells/kg, including all cells at intermediate doses.

In another illustrative embodiment, the effective amount of cells provided to the subject is about 2x10⁶Individual cell/kg to about 10x10⁶Individual cell/kg, about 3X10⁶Individual cell/kg to about 10x10⁶Individual cell/kg, about 4X10⁶Individual cell/kg to about 10x10⁶Individual cell/kg, about 5X10⁶Individual cell/kg to about 10x10⁶Individual cell/kg, 2X10⁶Individual cell/kg to about 6x10⁶Individual cell/kg, 2X10⁶Individual cell/kg to about 7x10⁶Individual cell/kg, 2X10⁶Individual cell/kg to about 8x10⁶Individual cell/kg, 3X10⁶Individual cell/kg to about 6x10⁶Individual cell/kg, 3X10⁶Individual cell/kg to about 7x10⁶Individual cell/kg, 3X10⁶Individual cell/kg to about 8x10⁶Individual cell/kg, 4X10⁶Individual cell/kg to about 6x10⁶Individual cell/kg, 4X10⁶Individual cell/kg to about 7x10⁶Individual cell/kg, 4X10⁶Individual cell/kg to about 8x10⁶Individual cell/kg, 5X10⁶Individual cell/kg to about 6x10⁶Individual cell/kg, 5X10⁶Individual cell/kg to about 7x10⁶Individual cell/kg, 5X10⁶Individual cell/kg to about 8x10⁶Individual cell/kg or 6x10⁶Individual cell/kg to about 8x10⁶Individual cells/kg, including all cells at intermediate doses.

Certain variations in dosage will necessarily occur depending on the condition of the subject to be treated. In any event, the person responsible for administration will determine the appropriate dosage to be appropriate for the individual subject.

In some embodiments, the therapeutic use of the cell is a single dose therapy. In some embodiments, the therapeutic use of the derived hematopoietic lineage cells is a multi-dose therapy. In some embodiments, the multiple dose therapy is one dose per day, one dose per 3 days, one dose per 7 days, one dose per 10 days, one dose per 15 days, one dose per 20 days, one dose per 25 days, one dose per 30 days, one dose per 35 days, one dose per 40 days, one dose per 45 days, or one dose per 50 days, or one dose on any number of days in between.

Compositions comprising the cell populations described herein can be sterile and can be suitable and ready for administration to a human patient (i.e., can be administered without any further processing). A cell-based composition ready for administration means that the composition does not require any further processing or manipulation prior to transplantation or administration to a subject. In some embodiments, such a cell population may comprise an isolated cell population that is expanded and/or modulated with one or more reagents prior to administration.

In certain embodiments, the primary and co-stimulatory signals for the therapeutic cells may be provided by different protocols. For example, the agent providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in "cis" form) or coupled to a separate surface (i.e., in "trans" form). Alternatively, one agent may be coupled to the surface while the other agent is in solution. In one embodiment, the agent that provides the co-stimulatory signal may be bound to the cell surface, and the agent that provides the primary activation signal is in solution or coupled to the surface. In certain embodiments, both reagents may be in solution. In another embodiment, the reagent may be in a soluble form and then cross-linked to a surface, such as Fc receptor expressing cells or antibodies or other binding agents to be bound to the reagent, such as artificial antigen presenting cells (aapcs) as disclosed in U.S. patent application publication nos. 20040101519 and 20060034810, which are designed to activate and expand T lymphocytes.

Therapeutic compositions suitable for administration to a patient may include one or more pharmaceutically acceptable carriers (additives) and/or diluents (e.g., pharmaceutically acceptable media, such as cell culture media) or other pharmaceutically acceptable components. The pharmaceutically acceptable carrier and/or diluent will depend, in part, on the particular composition being administered and the particular method used to administer the therapeutic composition. Thus, a variety of suitable formulations of therapeutic compositions are well known to those of skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 17 th edition 1985, the disclosure of which is incorporated herein by reference in its entirety).

In particular embodiments, a therapeutic cell composition having a cell population isolated as described herein further has a pharmaceutically acceptable cell culture medium, or a pharmaceutically acceptable carrier and/or diluent. Therapeutic compositions comprising the cell populations disclosed herein can be administered separately by intravenous, intraperitoneal, enteral or intratracheal administration, or in combination with other suitable compounds to achieve the desired therapeutic goal.

These pharmaceutically acceptable carriers and/or diluents may be present in an amount sufficient to maintain the pH of the therapeutic composition between about 3 to about 10. Thus, the buffer may comprise as much as about 5% by weight of the total composition. Electrolytes such as, but not limited to, sodium chloride and potassium chloride may also be included in the therapeutic composition. In one aspect, the therapeutic composition has a pH in the range of about 4 to about 10. Alternatively, the pH of the therapeutic composition is in the range of about 5 to about 9, about 6 to about 9, or about 6.5 to about 8. In another embodiment, the therapeutic composition comprises a buffer having a pH in one of the pH ranges. In another embodiment, the therapeutic composition has a pH of about 7. Alternatively, the pH of the therapeutic composition is in the range of about 6.8 to about 7.4. In yet another embodiment, the therapeutic composition has a pH of about 7.4.

The present disclosure also provides for the use of a pharmaceutically acceptable cell culture medium in particular compositions and/or cultures of cells described herein. Such compositions are suitable for administration to a human subject. In general, any medium that supports the maintenance, growth, and/or health of iPSC-derived immune cells is suitable for use as a pharmaceutical cell culture medium. In a particular embodiment, the pharmaceutically acceptable cell culture medium is serum-free and/or feeder-free medium. In various embodiments, the serum-free medium is animal component-free, and may optionally be protein-free. Optionally, the culture medium may comprise a biopharmaceutically acceptable recombinant protein. An animal component-free medium is a medium in which the components are derived from a non-animal source. The recombinant protein replaces a natural animal protein in an animal component-free medium, and the nutrient components are obtained from synthetic, plant, or microbial sources. In contrast, protein-free medium is defined as being substantially free of proteins. It will be understood by those of ordinary skill in the art that the above examples of media are illustrative and are in no way limiting of the formulation of suitable media, and that there are many alternative suitable media known and available to those of skill in the art.

In the preceding description and appended claims, the term "and/or" means one or all of the listed elements, or a combination of any two or more of the listed elements; the terms "comprises," "comprising," and variations thereof, are to be construed as open-ended, i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one; and the recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

In the foregoing description, certain embodiments have been described in isolation for the sake of clarity. Certain embodiments may include combinations of compatible features described herein in connection with one or more embodiments, unless it is otherwise expressly stated that the features of a particular embodiment are incompatible with the features of another embodiment.

For any of the methods disclosed herein that include discrete steps, the steps may be performed in any order that is practicable. Also, any combination of two or more steps may be performed simultaneously, as appropriate.

The invention is illustrated by the following examples. It is to be understood that the specific examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as described herein.