EA046022B1 - ANTIGENSPECIFIC IMMUNE EFFECTOR CELLS - Google Patents

Description

This application claims benefit from U.S. Provisional Patent Application Serial No. 62/486,875, filed April 18, 2017, the entire contents of which are incorporated herein by reference.

State of the art

1. Field of technology.

The present invention generally relates to the field of molecular biology. More particularly, it relates to methods and compositions relating to antigen-specific immune effector cells such as T cells and NK cells.

2. Description of the prior art.

Despite technological advances in diagnostics and treatment options available to patients diagnosed with cancer, the prognosis is often still poor and many patients cannot be cured. Immunotherapy offers hope of effective yet targeted treatment for patients diagnosed with various tumors, with the potential to destroy malignant tumor cells without damaging normal tissue. Theoretically, T cells of the immune system are able to recognize sets of proteins specific to tumor cells and mediate their destruction through various effector mechanisms. Adoptive T-cell therapy attempts to harness and enhance the ability of a patient's own T cells to destroy tumors, and then return these effectors to the patient in a state in which they effectively eliminate residual tumor without damaging healthy tissue. Although this approach is not new to the field of oncoimmunology, many shortcomings in the clinical application of adoptive T-cell therapy prevent the full application of this approach in the treatment of cancer.

Current adoptive T-cell therapies are limited by the lack of patients and tumor-specific T cells, including their low abundance in the body, their inability to overcome a number of tumor immune evasion mechanisms, and their short lifespan. It is difficult to isolate and expand the usually low numbers of T cells that respond to the desired antigen. Therefore, there is an unmet need for therapeutically satisfactory and functional antigen-specific immune cells for effective use in immunotherapy.

Brief description of the invention

In a first embodiment, the present invention provides a method for producing antigen-specific effector T cells and/or NK cells, comprising engineering pluripotent stem cells (PSC) to express a chimeric antigen receptor (CAR), thereby obtaining CAR-PSC; differentiation or direct reprogramming of CAR-PSCs into CD34' hematopoietic progenitor cells (HPCs); further differentiation of CD34+ HPCs into T cells and/or NK cells; and expansion of T cells and/or NK cells. In some aspects, expansion includes co-culture with antigen-specific target cells, thereby obtaining antigen-specific effector T cells and/or NK cells.

In certain aspects, PSCs engineered to express CARs are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). In particular, iPSCs are reprogrammed from somatic cells such as T cells.

In some aspects, the step of differentiating CAR-PSCs into CD34+ HPCs involves conducting directed differentiation. In certain aspects, directed differentiation includes the formation of embryoid bodies (EB) in the presence of blebbistatin, an inhibitor of GSK-3, FGF2 and VEGF; bringing EB into contact with BMP4, VEGF and FGF2 to stimulate mesoderm induction; and differentiating EBs in the presence of Flt-3 ligand, IL-3, SCF and TPO, thereby obtaining HPCs. In some aspects, differentiation is carried out in media substantially free or free of BMP4. In other aspects, differentiation is carried out in the presence of BMP4. In particular aspects, the GSK-3 inhibitor is CHTR99021. In some aspects, differentiation further includes the presence of IL-11, cAMP and/or VEGF.

In some aspects, directed differentiation involves culturing single PSCs on an amine-coated surface in the presence of blebbistatin, BMP4, VEGF, and bFGF; initiation of differentiation by bringing PSCs into contact with BMP4, VEGF and FGF2; and further differentiating the PSC in the presence of Flt-3 ligand, IL-3, IL-6, SCF, TPO and heparin, thereby obtaining HPC, where the method does not involve the formation of EBs.

In particular aspects, the method includes culturing cells under certain conditions without a feeder, for example, throughout the entire method. In some aspects, PSCs are substantially transgene-free or transgene-free. In private aspects, PSCs are human. In certain aspects, the T cells are CD4+ T cells, CD8+ T cells, cytotoxic T cells, regulatory T cells, natural killer T cells, naïve T cells, memory T cells, and/or gamma delta cells.

In certain aspects, PSCs engineered to express CAR are further engineered to express ERG/ETV2, GATA2, and HOXA9, for example, under the control of a single inducible

- 1 046022 promoters. In some aspects, the method further includes constructing a PSC to express

HMGA2, MYCN, NR4A2, SOX17, TFEC, MEIS1 and/or HOXA4. Thus, in some aspects, the differentiation step of CAR-PSCs into CD34+ HPCs involves the induction of expression of ERG/ETV2, GATA2, and

HOXA9 for a period of time sufficient to produce HPCs and cease induction of expression before further differentiation of HPCs into T cells and/or NK cells.

In particular aspects, the step of differentiating CAR-PSCs into CD34+ HPCs further includes selecting cells expressing CD34 and/or CD43 prior to differentiation into antigen-specific T cells and/or NK cells. In particular aspects, selection includes magnetically activated cell sorting (MACS). In some aspects, cells expressing CD34 and/or CD43 comprise at least 25, 30, 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90% or more of the total cell population. In some aspects, the MAC step to select CD34 and/or CD43 positive cells is not performed.

In particular aspects, at least 2, 3, 4, 5, 10, 15, 20 or more percent of the HPCs express CAR. In some aspects, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 percent or more of the NPCs express CAR.

In some aspects, differentiation of HPCs into T cells and/or NK cells involves culturing HPCs on a surface coated with retronectin and Notch DLL-4 in the presence of ascorbic acid and nicotinamide under hypoxic conditions. In some aspects, the culture further contains SCF, FLT-3 ligand, TPO and IL-7, and optionally contains a GSK inhibitor (eg, CHIR99021), IL-2 and/or IL-12. In certain aspects, the expansion step further includes culturing antigen-specific T cells in the presence of anti-CD3 and anti-IL-2 antibodies. In additional aspects, the expansion step further includes culturing antigen-specific T cells in the presence of an antibody to CD3, IL-2, IL-15, and IL-21. In some aspects, the expansion step includes culturing antigen-specific T cells in the presence of an anti-CD3 antibody, FLT3 ligand, IL-7, IL-2, and/or IL-15, and optionally further includes SCF, TPO, and/or IL-21. In particular aspects, at least 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more than a percent of differentiated CD34' NPCs are CD3+CAR+ T cells. In some aspects, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50 percent or more of differentiated CD34+ HPCs are CD3-CAR+ NK cells. In some aspects, at least 2 percent of the expanded CD34+ HPCs are CD3+CAR+ T cells. In particular, at least 10% of expanded CD34+ HPCs are CD3-CAR+ NK cells.

In certain aspects, CARs and antigen-specific target cells target the same antigen. In one particular aspect, the antigen is CD19. In particular aspects, the antigen-specific target cells are tumor cells. In some aspects, the antigen-specific target cells are human. In particular aspects, the antigen-specific target cells are HLA class I negative. In some aspects, at least 5, such as 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, or more percent of the antigen-specific effector T cells exhibit cytotoxic activity against target cells. In certain aspects, at least 10, such as 15, 20, 25, 30, 35, 40, 45, 50, or more percent of the antigen-specific effector NK cells exhibit cytotoxic activity against target cells.

In some aspects, the CAR is encoded by DNA integrated into the PSC genome. In certain aspects, the CAR comprises an intracellular signaling domain, a transmembrane domain, and an extracellular domain containing an antigen binding region. In particular aspects, the intracellular signaling domains comprise CD3Z and CD28. In some aspects, the antigen binding region is F(ab')2, Fab', Fab, Fv, or scFv.

In certain aspects, PSCs are HLA homozygous. In some aspects, HLA homozygous PSCs are homozygous for one or more alleles of the HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, or HLA-DQ loci. In certain aspects, HLA homozygous PSCs are homozygous for two of the alleles of the HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, or HLA-DQ loci. In particular, HLA-homozygous PSCs are homozygous for HLA-A and HLA-B. In certain aspects, HLA homozygous PSCs are homozygous for HLA-A, HLA-B and HLA-C.

In yet another embodiment, a method for producing antigen-specific effector T cells and/or NK cells is provided, comprising engineering a PSC to express a CAR, thereby producing a CAR-PSC; culturing CAR-PSCs in the presence of blebbistatin, an inhibitor of GSK-3, FGF2 and VEGF, thereby obtaining EBs; bringing EB into contact with BMP4, VEGF and FCF2 to stimulate mesoderm induction; differentiation of EBs in the presence of Flt-3 ligand, BMP4, IL-3, SCF and TPO, thereby obtaining CD34 ⁺ HPCs; further differentiation of CD34 ⁺ HPCs into T cells and/or NK cells; and expansion of T cells and/or NK cells, where expansion includes co-culture with antigen-specific target cells thereby obtaining antigen-specific effector T cells and/or NK cells. In particular aspects, the GSK-3 inhibitor is CHIR99021.

In yet another embodiment, there is provided a method for producing antigen-specific effector T cells and/or NK cells, comprising engineering PSCs to express a CAR, thereby producing CAR-PSCs; culturing single CAR-PSCs on an amine-coated surface in the presence of blebbistatin, BMP4, VEGF, and initiating differentiation by bringing the CAR-PSCs into contact with BMP4, VEGF and FGF2; further differentiation of CAR-PSCs in the presence of Flt-3 ligand, IL-3, IL-6, SCF, TPO and heparin, thereby obtaining CD34+ HPCs; differentiation of CD34' HPCs into T cells and/or NK cells; and expansion of T cells and/or NK cells, where the expansion includes co-culture with antigen-specific target cells thereby obtaining antigen-specific effector T cells and/or NK cells, where the method does not include the formation of EBs.

In a further embodiment, a method for producing antigen-specific effector T cells and/or NK cells is provided, comprising engineering PSCs to express CARs, thereby producing CAR-PSCs; differentiation of CAR-PSCs into CD34+ HPCs; CD34+CD43+ HPC selection; further differentiation of CD34+CD43+ HPCs into T cells and/or NK cells; and expansion of T cells and/or NK cells, where expansion includes co-culture with antigen-specific target cells thereby obtaining antigen-specific effector T cells and/or NK cells.

In another embodiment, a population of antigen-specific effector T cells and/or NK cells is provided, obtained in accordance with the embodiments described above. The present application also provides a pharmaceutical composition comprising antigen-specific effector T cells and/or NK cells obtained in accordance with the embodiments described herein. Further provided herein is a composition comprising antigen-specific effector T cells and/or NK cells obtained in accordance with embodiments for treating cancer in a subject. The present application also provides the use of antigen-specific effector T cells and/or NK cells obtained in accordance with embodiments for the treatment of cancer.

In yet another embodiment, a method of treating cancer in a subject is provided, comprising administering to the subject an effective amount of antigen-specific effector T cells and/or NK cells obtained in accordance with the embodiments described herein. In some aspects, the cancer expresses a tumor antigen, and antigen-specific effector T cells and/or NK cells are targeted to the tumor antigen. In particular aspects, the subject is a person.

Other objects, features and advantages of the present invention will become apparent from the following detailed description of the invention. However, it should be understood that the detailed description of the invention and specific examples indicating preferred embodiments of the invention are provided for illustrative purposes only, as various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art from this detailed description of the invention.

Brief description of graphic materials

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

Fig. 1A-1G. Fig. 1A is a schematic representation of methods for generating T cells and NK cells from PSCs, including direct programming methods and directed differentiation methods. Fig. 1B is a diagram representing the differentiation of hPCS into T and NK cells in the absence of feeder and serum. Abbreviations: CHIR - GSK-3 inhibitor CHIR99021; HDM, serum-free hematopoietic differentiation medium; TCDM, T cell differentiation medium; TCEM, T cell expansion medium; HPC, hematopoietic progenitor cells; Optional stimulant components are marked with an asterisk (*). Fig. 1C - efficiency of obtaining CD34+CD43+ NPCs from PSCs through directed differentiation or direct programming. Fig. 1D - flow cytometric analysis of cultures for differentiation into T/NK. Fig. 1E - yield of different cell populations during differentiation. Fig. 1F - phenotype of PSC-derived T cells. Fig. 1G - expansion of PSC-derived T cells. Immobilized anti-CD3 (iCD3) antibodies are designed to expand PSC-derived T cells (bar graph). T cells proliferating in expansion culture acquire the characteristic morphology of irregularly shaped lymphoblasts (photo). T cells after 2 weeks of expansion acquire CD56 and CD8 expression (flow cytometry dot plots).

Fig. 2 - CAR expression in PSC-derived T/NK cells. CAR expression at differentiation stages was assessed by flow cytometry using L protein staining. CD3+ T cells were co-stained with a lambda chain mouse mAb to human CD3 (clone SP342). Expression of CAR E11 PSCs and E11-derived HPCs and T cells is shown.

Fig. 3A, B - CD19 CAR-mediated in vitro cytotoxicity in PSC-derived T/NK cells. Fig. 3A - in vitro cytotoxicity assay using luciferase-expressing CD19+

- 3,046,022 Daudi and Raji target cells. Fig. 3B - Cytolytic potential of CAR-T cells by real-time target cell counting using the Incucyte S3 Live Cell Assay System (Essen

Bioscience).

Fig. 4 - CD19 CAR-induced in vitro cytokine production in PSC-derived T/NK cells.

Fig. 5A, 5B - in vivo oncolytic potential of PSC-derived CAR-T/NK cells. Fig. 5A tumor progression in mice was monitored using in vivo bioluminescence imaging. Fig. 5B - survival curves for different groups of mice receiving either T (1C) or NK (A16) cells derived from PSCs.

Description of Illustrative Embodiments

In certain embodiments, the present invention provides highly efficient methods for generating antigen-specific immune effector cells from human pluripotent stem cells (PSCs) engineered to express an antigen receptor, such as a chimeric antigen receptor (CAR), referred to herein as CAR-PSC. Immune effector cells produced by the present methods may include, but are not limited to, T cells, NK cells, and iNKT cells.

PSCs can be generated by reprogramming (eg, retroviral or episomal methods) the original T cell population to generate T cell-derived iPSCs (TiPSCs). T cells can be isolated from various sources, such as a blood sample. The initial T cell population may retain characteristic T cell receptor (TCR) gene rearrangements and may be HLA homozygous cells (ie, homozygous for MHC class I and II genes). Accordingly, iPSCs can be derived from cells isolated from HLA homozygous subjects, referred to herein as HLA superdonors.

CAR-PSCs can then be differentiated or programmed to generate CD34' hematopoietic progenitor cells (HPCs). This can be achieved by directed differentiation using a combination of cytokines (eg, SCF, TPO, FLT-3, IL-6, IL-3 and heparin) (eg, described in application PCT/US2016/057899, incorporated herein in its entirety by links). In an alternative method, CAR-PSCs can be differentiated into CD34+ HPCs using direct programming using an expression construct encoding ETV2/ERG, GATA2, and HOXA9 (i.e., EGH) (e.g., described in application PCT/US2016/057893, incorporated herein in its entirety application via link). These EGH-PSCs can be further engineered to express HMGA2, MYCN, NR4A2, SOX17, TFEC, MEIS1, and/or HOXA4 for long-term transplantability.

Finally, CD34+ CAR-HPCs can be differentiated into CD3+ T cells or CD56+CD3 ⁻ NK cells. The optimal time during HPC differentiation (eg, days 7 to 11) for lymphoid potential may be days 7 to 11, as identified by expression of CD34 and CD43. For example, HPCs with increased lymphoid potential can be isolated by sorting fractions of cells positive for two or more of the markers CD144, CD34, CD45 and CD7.

An exemplary method for differentiating T cells involves using RetroNectin and DLL-4 as a feederless matrix. T cell differentiation can be further enhanced by using ascorbic acid to enhance efficiency and maturation, and by culturing under hypoxic conditions.

In addition, T cells and/or NK cells can be expanded by co-culture with antigen-specific target cells (eg, tumor cells) during the differentiation process. This method was found to increase the cytotoxic activity of T cells and NK cells against target cells, which was demonstrated, inter alia, by decreased tumor growth and increased survival of mice injected with tumor cells.

Thus, the methods of the present invention can provide unlimited numbers of antigen-specific immune effector cells, such as T cells and NK cells, for a wide range of applications, such as stable in vivo transplantation, in vitro compound screening, and studies of hematological disease mechanisms and damage.

Definitions.

As used herein, the term essentially free with respect to a particular component is used to mean that none of the specified components have been specifically formulated in the composition and/or are present only as a contaminant or in trace amounts. Thus, the total amount of said component due to any unintentional contamination of the composition is well 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.

In the context of a non-descriptive description, the use of the singular may mean one or more. In the claim(s) of this application, when used in conjunction with the word containing, the singular may mean one or more than one.

The term or in the claims is used to mean and/or, unless explicitly stated, that

- 4 046022 it refers only to alternatives, or the alternatives are mutually exclusive, although the disclosure supports a definition that refers only to alternatives and and/or. As used herein, the term other may mean at least second or more.

Throughout this application, the term is roughly used to indicate that the value includes the inherent error variation due to the instrument, method used to determine the value, or variation that exists among study subjects.

The term exogenous when used in relation to a protein, gene, nucleic acid or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid or polynucleotide that has been introduced into the cell or organism by artificial or natural means; or in relation to a cell, the term refers to a cell that has been isolated and subsequently introduced into other cells or into an organism by artificial or natural means. An exogenous nucleic acid may originate from another organism or cell, or it may represent one or more additional copies of a nucleic acid that is naturally present in the organism or cell. An exogenous cell can come from another organism or from the same organism. By way of non-limiting example, an exogenous nucleic acid is a nucleic acid that is located at a location on the chromosome different from where it would be found in natural cells, or is otherwise flanked by a nucleic acid sequence different from that found in nature.

By expression construct or expression cassette is meant a nucleic acid molecule capable of directing transcription. The construct includes at least one or more transcriptional control elements (such as promoters, enhancers, or functionally equivalent structures) that direct gene expression in one or more desired cell types, tissues, or organs. Additional elements may also be included, such as a transcription termination signal.

A vector or construct (sometimes called a gene delivery system or gene transfer carrier) refers to a macromolecule or complex of molecules containing a polynucleotide to be delivered to a host cell in vitro or in vivo.

A plasmid, a common type of vector, is an extrachromosomal DNA molecule, separate from chromosomal DNA, capable of replicating independently of chromosomal DNA. In some cases it is round and double-stranded.

An origin of replication (ori) or origin of replication is a DNA sequence, for example in a lymphotropic herpes virus, that, when present on a plasmid in a cell, is capable of maintaining associated sequences in the plasmid and/or a site at or near the site where DNA synthesis begins. As an example, the ori for EBV (Epstein-Barr virus) includes FR (20 imperfect copies of a 30 bp repeat) and preferably DS (double-stranded) sequences; however, other sites in EBV bind EBNA-1, for example, Rep* sequences can replace DS as the origin of replication (Kirshmaier and Sugden, 1998). Thus, the EBV origin of replication includes FR, DS, or Rep* sequences, or any functionally equivalent sequences derived from them through nucleic acid modifications or synthetic combinations. For example, a genetically engineered EBV origin of replication, for example engineered by insertion or mutation of individual elements, as specifically described in Lindner et al., 2008, may also be used in the present invention.

A gene, polynucleotide, coding region, sequence, segment, fragment or transgene that encodes a particular protein is a nucleic acid molecule that is transcribed and, optionally, also translated into a gene product, such as a polypeptide, in vitro or in vivo when placed under the control of appropriate regulatory sequences. The coding region may be present in cDNA form of genomic DNA or RNA. When present in the form of DNA, the nucleic acid molecule can be single-stranded (that is, the sense strand) or double-stranded. The boundaries of the coding region are defined by a start codon at the 5'-(amino) end and a translation stop codon at the 3'-(carboxy) end. The gene may include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. The transcription termination sequence is typically 3' to the gene sequence.

The term control elements collectively refers to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (IRES), enhancers, splice boundaries, and the like, which collectively mediate replication, transcription, post-transcriptional processing and translation of the coding sequence in the recipient cell. The presence of all of these control elements is not necessary, provided that the selected coding sequence is capable of replication, transcription and translation in the appropriate host cell.

- 5 046022

The term promoter is used herein in its usual sense to denote a nucleotide region containing a DNA regulatory sequence, where the regulatory sequence is derived from a gene capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. It may contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind to initiate specific transcription of a nucleic acid sequence. The phrases operably located, operably linked, under transcriptional control and transcriptionally controlled mean that the promoter is in the correct functional position and/or orientation relative to the nucleic acid sequence to control the initiation of transcription and/or expression of that sequence.

By enhancer is meant a nucleic acid sequence that, when located in the vicinity of a promoter, confers increased transcriptional activity compared to transcriptional activity due to the promoter alone in the absence of the enhancer domain.

By operably linked or co-expressed with respect to nucleic acid molecules, it is meant that two or more nucleic acid molecules (eg, a nucleic acid molecule to be transcribed, a promoter, and an enhancer element) are linked so as to permit transcription of the nucleic acid molecule. Functionally linked or co-expressed with respect to peptide and/or polypeptide molecules means that two or more peptide and/or polypeptide molecules are linked in such a way that a single polypeptide chain is formed, that is, a fusion polypeptide having at least one property of each peptide and/or polypeptide /or the polypeptide component of the fusion molecule. The fusion polypeptide is preferably chimeric, that is, composed of heterologous molecules.

Homology refers to the percentage of identity between two polynucleotides or two polypeptides. The correspondence between the first and second sequences can be determined by methods known in the art. For example, homology can be determined by directly comparing the sequence information of two polypeptide molecules by aligning the sequence information and using available computer programs. Alternatively, homology can be determined by hybridizing the polynucleotides under conditions that promote the formation of stable duplexes between homologous regions, followed by digestion with single-strand-specific nuclease(s) and determining the size of the digested fragments. Two DNA sequences or two polypeptide sequences are substantially homologous to each other when at least about 80%, preferably at least about 90%, and most preferably at least about 95% of the nucleotides or amino acids, respectively, are the same over a specified portion of the molecular length , as determined using the methods described above.

The term cell in this application is used in its broadest sense in the art and refers to a living organism that is the structural unit of tissue of a multicellular organism, surrounded by a membrane structure that isolates it from the environment, has the ability to reproduce itself and has genetic information and a mechanism for its expression . The cells used in the present application may be natural cells or artificially modified cells (eg, fusion cells, genetically modified cells, etc.).

The term stem cell as used herein refers to a cell that, under suitable conditions, is capable of differentiating into a wide range of specialized cell types and, under other suitable conditions, is capable of self-renewal and maintenance in a substantially undifferentiated pluripotent state. The term stem cell also covers pluripotent cell, multipotent cell, precursor cell and progenitor cell. Exemplary human stem cells may be derived from hematopoietic or mesenchymal stem cells derived from bone marrow tissue, embryonic stem cells derived from fetal tissue, or embryonic germ cells derived from fetal genital tissue. Exemplary pluripotent stem cells can also be derived from somatic cells by reprogramming them into a pluripotent state through the expression of certain transcription factors associated with pluripotency; these cells are called induced pluripotent stem cells or iPSC or iPS cells.

An embryonic stem (ES) cell is an undifferentiated pluripotent cell, derived from an early stage embryo, such as the inner cell mass at the blastocyst stage, or produced by artificial means (such as nuclear transfer), capable of giving rise to any type of differentiated cell in the embryo or an adult organism, including germ cells (such as sperm and eggs).

Induced pluripotent stem cells (iPSC or iPS cells) are cells obtained by reprogramming a somatic cell through expression or induction

- 6 046022 expression of a combination of factors (in this application called reprogramming factors). iPS cells can be generated using fetal, postnatal, neonatal, juvenile or adult somatic cells. In certain embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, Oct4 (sometimes called Oct 3/4), Sox2, c-Myc, Klf4, Nanog, and Lin28. In some embodiments, somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, at least four reprogramming factors, at least five reprogramming factors, at least six reprogramming factors, or at least seven reprogramming factors to reprogram a somatic cell into a pluripotent stem cell.

Hematopoietic progenitor cells or hematopoietic precursor cells refer to cells that are of the hematopoietic lineage but are capable of further hematopoietic differentiation and include hematopoietic stem cells, multipotential hematopoietic stem cells, common myeloid progenitors, megakaryocyte progenitors, red blood cell progenitors, and lymphoid progenitors. Hematopoietic stem cells (HSC) are multipotent stem cells that give rise to all types of blood cells, including myeloid (monocytes and macrophages, granulocytes (neutrophils, basophils, eosinophils and mast cells), red blood cells, megakaryocytes/platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NK cells) (see, for example, Doulatov et al., 2012; Notta et al., 2015). The term multilymphoid progenitor (MLP) describes any progenitor that gives rise to all lymphoid lineages (B, T and NK cells), but may or may not have other (myeloid) potentials (Doulatov et al., 2010) and is CD45RA7CD10 7CD7 ^- . Any B, T, or NK cell precursor may be called MLP. Common myeloid progenitor (CMP) refers to the common myeloid progenitor defined by the expression of CD45+/CD.317CD4.3 ^7CD.34 cells that can give rise to granulocytes, monocytes, megakaryocytes and erythrocytes. Hematopoietic progenitor cells can express CD34. Hematopoietic progenitor cells may co-express CD133 and be negative for CD38 expression. Hematopoietic progenitor cells include CD347CD45' hematopoietic progenitor cells and CD34 ⁺ /CD45 ⁺ /CD43 ⁺ hematopoietic progenitor cells. CD34 ⁺ /CD43 ⁺ /CD45 ⁺ hematopoietic progenitor cells may be highly enriched in myeloid progenitors. Hematopoietic cells also include various subpopulations of primitive hematopoietic cells, including: CD34-/CD133 7CD38 ^- (primitive hematopoietic progenitor cells), CD43(+)CD235a(+)CD41a(+/-) (erythro-megakaryopoietic), lin( -)CD34(+)CD43(+)CD45(-) (multipotent) and lin(-)CD34(+)CD43(+)CD45(+) (myeloid-deformed) cells, CD133+/ALDH+ (aldehyde dehydrogenase). It is contemplated that any of these types of primitive hematopoietic cells or hematopoietic progenitor cells can be converted into iPS cells as described herein. In some aspects, the cells may include mast cells, Langerhans cells, osteoclasts, NK cells, T cells, CIK-T cells, or other subtypes of T cells, NK cells, and B cells.

As used herein, the term immune cell(s) refers to cells of the immune system, including, but not limited to, T cells, NK cells, T/NK cells, dendritic cells, macrophages, B cells, neutrophils, red blood cells, monocytes, basophils, neutrophils, mast cells, eosinphils and any combination thereof.

T cell activator or a condition that activates a T cell refers to a stimulus that activates T cells and includes antigens that may be presented on antigen presenting cells or other surfaces; polyclonal activators that bind to multiple T cell receptor (TCR) complexes regardless of specificity and include lectins such as concanavalin A (Con-A) and phytohemagglutinin (PHA) and agents such as antibodies that specifically bind to invariant scaffolds epitopes on TCR or CD3 proteins; and superantigens, which stimulate significant numbers of T cells and include, for example, enterotoxins such as staphylococcal enterotoxins.

The terms T lymphocyte and T cell are used interchangeably and refer to a cell that expresses a TCR capable of recognizing an antigen when displayed on the surface of an antigen-presenting cell or matrix along with one or more MHC molecules or one or more non-classical MHC molecules.

The term T cell refers to T lymphocytes as defined in the art and is intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cells may be CD4 ⁺ T cells, CD8 ⁺ T cells, CD4 ⁺ CD8 ⁺ T cells, or CD4 ^- CD8 ^- cells. T cells may also be T helper cells, such as T helper 1 (TH1) or T helper 2 (TH2) cells, or TH17 cells, as well as cytotoxic T cells, regulatory T cells, natural killer T cells cells, naive T cells, memory T cells or gamma delta T cells (Wilson et al., 2009; Wynn, 2005; Ladi et al., 2006). T-7046022 cells that differ from each other by at least one marker, such as CD4, are referred to herein as T cell subsets.

CD4+ T cells refer to a subset of T cells that express CD4 on their surface and are associated with cell-mediated immune responses. They are characterized by secretion profiles after stimulation, which may include the secretion of cytokines such as IFN-gamma, TNF-alpha, IL-2, IL-4 and IL-10. CD4 are 55 kDa glycoproteins originally identified as differentiation antigens on T lymphocytes but also found on other cells including monocytes/macrophages. CD4 antigens are members of the immunoglobulin supergene family and participate as associative recognition elements in MHC (major histocompatibility complex) class II-restricted immune responses. On T lymphocytes they define a subpopulation of helpers/inducers.

CD8+ T cells belong to a subset of T cells that express CD8 on their surface, are MHC class I-restricted, and function as cytotoxic T cells. CD8 molecules are differentiation antigens found on thymocytes and cytotoxic and suppressor T lymphocytes. CD8 antigens are members of the immunoglobulin supergene family and are associative recognition elements in major histocompatibility complex class I-restricted interactions.

A pluripotent stem cell refers to a stem cell that has the potential to differentiate into all the cells that make up one or more tissues or organs, or, preferably, into any of the three germ layers: endoderm (epithelium of the stomach, gastrointestinal tract, lungs), mesoderm ( muscles, bones, blood, genitourinary system) or ectoderm (epidermal tissues and nervous system).

As used herein, the term somatic cell refers to any cell other than germ cells, such as an egg, sperm, or the like, that does not directly pass on its DNA to the next generation. Typically, somatic cells have limited or no pluripotency. Somatic cells used in this application may be natural or genetically modified.

Programming is a process that changes the type of offspring a cell can produce. For example, a cell is programmed if it has been modified so that it can produce progeny of at least one new cell type, either in culture or in vivo, compared to what it could have produced under the same conditions without the programming. This means that, after sufficient proliferation, a measurable proportion of progeny is observed that has the phenotypic characteristics of the new cell type, if such progeny essentially could not have been produced before programming; alternatively, the proportion having characteristics of the new cell type is markedly greater than before programming. This process includes differentiation, dedifferentiation and transdifferentiation.

Differentiation is the process by which a less specialized cell becomes a more specialized cell type. Dedifferentiation is a cellular process in which a partially or fully differentiated cell reverts to an earlier stage of development, such as pluripotency or multipotency. Transdifferentiation is the process of converting one differentiated cell type into another differentiated cell type. Typically, transdifferentiation by programming occurs without the cells passing through an intermediate stage of pluripotency, that is, cells are programmed directly from one differentiated cell type to another differentiated cell type. Under certain conditions, the proportion of progeny with characteristics of the new cell type may be at least about 1%, 5%, 25%, or more in order of increasing preference.

Reprogramming is a process that gives a cell a markedly increased ability to produce progeny of at least one new cell type, both in culture and in vivo, compared to the ability it would have under the same conditions without reprogramming. In particular, reprogramming is the process that endows a somatic cell with pluripotent potential. This means that, after sufficient proliferation, a measurable proportion of progeny is observed to have the phenotypic characteristics of the new cell type, if such progeny essentially could not have been generated prior to reprogramming; or the proportion that has the characteristics of the new cell type is noticeably larger than before the reprogramming. Under certain conditions, the proportion of progeny with characteristics of the new cell type may be at least about 1%, 5%, 25%, or more in order of increasing preference.

The term forward programming refers to the programming of a multipotent or pluripotent cell, as opposed to a differentiated somatic cell that does not have pluripotency, by providing the multipotent or pluripotent cell with one or more specific lineage-defining genes or gene products. For example, direct programming may describe the process of programming ESCs or iPSCs into hematopoietic progenitor cells

- 8 046022 or other progenitor cells, or into hematopoietic cells or other differentiated somatic cells.

As used herein, the term subject or subject in need refers to a mammal, preferably a human, male or female, of any age, in need of a cell or tissue transplant. Typically, a subject (also referred to herein as a recipient) requires a cell or tissue transplant due to a disorder or pathological or undesirable condition, process, or syndrome, or physical, morphological or physiological abnormality that is treatable by cell or tissue transplantation.

As used herein, gene disruption refers to the elimination or reduction of expression of one or more gene products encoded by a specified gene in a cell compared to the level of expression of the gene product in the absence of the disruption. Examples of gene products include mRNA and protein products encoded by the gene. The disorder in some cases is temporary or reversible, and in other cases it is permanent. The disorder in some cases relates to a functional or full-length protein or mRNA, despite the fact that 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 typically caused by artificial means, that is, the addition or introduction of a compound, molecule, complex or composition, and/or disruption of the nucleic acid of the gene or associated with the gene, for example, at the DNA level. Examples of gene disruption methods include gene silencing, knockdown, knockout and/or gene disruption methods such as gene editing. Examples include antisense technologies such as RNAi, siRNA, small hairpin RNAs and/or ribozymes, which typically result in a transient reduction in expression, as well as gene editing techniques that result in inactivation or disruption of the target gene, e.g. breaks and/or homologous recombination. Examples include insertions, mutations, and deletions. Disturbances usually result in suppression and/or complete absence of expression of the normal or wild-type product encoded by the gene. Examples of such gene disorders are insertions, frameshift and missense mutations, deletions, knockins and knockouts of a gene or part of a gene, including deletions of an entire gene. Such disruptions may occur in a coding region, for example, in one or more exons, resulting in failure to produce a full-length product, a functional product, or any product, for example, by insertion of a stop codon. Such disorders may also occur due to disturbances in a promoter or enhancer, or other region that affects transcriptional activation to prevent transcription of a gene. Gene disruption involves targeting genes, including targeted gene inactivation through homologous recombination.

A Notch ligand is a protein capable of binding to the Notch receptor polypeptide present in the membrane of a number of different mammalian cells, such as hematopoietic stem cells. Notch receptors that have been identified in human cells include Notch-1, Notch-2, Notch-3 and Notch-4. Notch ligands typically have a DSL domain (D-Delta, S-Serrate, and L-Lag2) containing 20 to 22 amino acids at the amino terminus and 3 to 8 EGF-like repeats (Furie and Furie, 1988; Knust et al., 1987 ; Suzuki et al., 1987) on the extracellular surface.

Superdonors in this application refer to individuals homozygous for certain MHC class I and II genes. These homozygous individuals can serve as superdonors, and their cells, including tissues and other materials containing their cells, can be transplanted into individuals homozygous or heterozygous for the haplotype. The superdonor may be homozygous for the locus/alleles of the ULA-A, HLA-B, HLA-C, HLA-DR, HLA-DP or HLA-DQ loci, respectively.

As used herein, the term chimeric antigen receptors (CARs) can refer, for example, to artificial T cell receptors, chimeric T cell receptors, or chimeric immunoreceptors, and includes engineered receptors that impart artificial specificity to a particular immune effector cell. CARs can be used to impart monoclonal antibody specificity to a T cell, allowing the generation of large numbers of specific T cells, for example for use in adoptive cell therapy. In certain embodiments, the CARs direct the cell's specificity to, for example, a tumor-associated antigen. In some embodiments, the CARs comprise an intracellular activation domain, a transmembrane domain, and an extracellular domain containing a tumor-associated antigen-binding region. In particular, CARs include single chain variable fragment (scFv) fusions derived from monoclonal antibodies fused to a CD3 zeta transmembrane domain and endodomain. The specificity of other CAR constructs may come from receptor ligands (eg, peptides) or from pattern recognition receptors such as dectins. In some cases, the location of the antigen recognition domain can be modified to reduce activation-induced cell death. In some cases, CARs contain domains for additional co-stimulatory signaling, such as CD3Z, FcR, CD27, CD28, CD137, DAP10 and/or OX40. In some cases, molecules, including co-stimulatory molecules, may be co-expressed with CARs.

- 9 046022 reporter genes for imaging (eg, for positron emission tomography), gene products that remove T cells when prodrugs are added, homing receptors, chemokines, chemokine receptors, cytokines and cytokine receptors.

The term antigen presenting cells (APC) refers to a class of cells capable of presenting one or more antigens in the form of a peptide-MHC complex that is recognized by specific effector cells of the immune system, and thus inducing an effective cellular immune response against the presented antigen or antigens. APCs can be intact whole cells such as macrophages, B cells, endothelial cells, activated T cells and dendritic cells; or other molecules of natural or synthetic origin, such as purified class I MHC molecules complexed with β2-microglobulin. Although many cell types may be capable of presenting antigens on their cell surface for recognition by T cells, only dendritic cells have the ability to present antigens in quantities effective to activate naïve T cells for cytotoxic T lymphocyte (CTL) responses.

Pluripotent stem cells.

In certain embodiments, the pluripotent stem cells are engineered to express an antigen receptor, such as a CAR. Pluripotent stem cells can be stem cells, including, but not limited to, induced pluripotent stem cells and embryonic stem cells. In particular, the pluripotent stem cells used herein are human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) capable of long-term proliferation in vitro while maintaining the ability to differentiate into all cell types of the body, including hematopoietic cells. progenitor cells according to the present invention.

Embryonic stem cells.

In certain aspects, pluripotent stem cells are ESCs. ES cells are derived from the inner cell mass of blastocysts and are highly differentiated in vitro. ES cells can be isolated by removing the outer trophectoderm layer of the developing embryo and then culturing the inner mass cells on a feeder layer of non-growing cells. The reseeded cells can continue to proliferate and produce new colonies of ES cells, which can be removed, dissociated, reseeded and allowed to grow. This process of subculturing undifferentiated ES cells can be repeated several times to obtain cell lines containing undifferentiated ES cells (US Pat. Nos. 5,843,780; 6,200,806; 7,029,913). ES cells have the potential to proliferate while maintaining their pluripotency. For example, ES cells are suitable for studies on cells and genes that control cell differentiation. The pluripotency of ES cells, combined with genetic manipulation and selection, can be exploited for in vivo gene analytical studies through the generation of transgenic, chimeric and knockout mice.

Methods for producing murine ES cells are well known. In one method, a preimplantation blastocyst from mouse line 129 is treated with a mouse antiserum to remove trophectoderm, and the inner cell mass is cultured on a feeder cell layer of chemically inactivated mouse embryonic fibroblasts in a medium containing fetal calf serum. Developing colonies of undifferentiated ES cells are subcultured on mouse embryonic fibroblast feeder layers in the presence of fetal calf serum to obtain ES cell populations. In some methods, murine ES cells can be grown in the absence of a feeder layer by adding the cytokine leukemia inhibitory factor (LLF) to the culture medium containing serum. In other methods, murine ES cells can be grown in serum-free medium in the presence of bone morphogenetic protein and LLF.

Human ES cells may be derived from or derived from a mammalian embryo at the zygote or blastocyst stage, produced by sperm-egg fusion, nuclear transfer, pathogenesis, or chromatin reprogramming and subsequent incorporation of the reprogrammed chromatin into the plasma membrane to produce an embryonic cell by previously described methods (Thomson and Marshall , 1998; Reubinoff et al., 2000). In one method, human blastocysts are exposed to anti-human serum and trophectoderm cells are lysed and removed from the inner cell mass cultured on a mouse embryonic fibroblast feeder layer. In addition, clusters of cells derived from the inner cell mass are chemically or mechanically dissociated, subcultured, and colonies with undifferentiated morphology are selected with a micropipette, dissociated, and subcultured. In some methods, human ES cells can be grown without serum by culturing the ES cells on a fibroblast feeder layer in the presence of basic fibroblast growth factor. In other methods, human ES cells can be grown without a feeder cell layer by culturing the cells on a protein matrix, such as MATRIGELTM or laminin, in the presence of conditioned medium containing basic fibroblast growth factor (Xu et al., 2001).

ES cells can also be derived from other organisms, including rhesus monkey and marmoset, by previously described methods (Thomson, and Marshall, 1998; Thomson et al., 1995; Thomson and Odorico, 2000; US Patent No. 5,843,780) , as well as from immortalized mouse and human cell lines. For example, immortalized human ES cell lines include MAOI, MA09, ACT-4, HI, H7, H9, H13, H14 and ACT30. As a further example, immortalized murine ES cell lines include the CGR8 cell line immortalized from the inner cell mass of murine 129 embryos, and CGR8 cell cultures can be grown in the presence of LLF without feeder layers.

ES stem cells can be detected by protein markers including Oct4 transcription factor, alkaline phosphatase (AP), SSEA-1 germline antigen, SSEA-3 stage-specific germline antigen, SSEA-4 stage-specific germline antigen, NANOG transcription factor, tumor rejection antigen 1-60 (TRA-1-60), tumor rejection antigen 1-81 (TRA-1-81), SOX2 or REX1.

Induced pluripotent stem cells.

In other aspects, the pluripotent stem cells used herein are induced pluripotent stem cells (iPS), commonly abbreviated to iPS cells or iPSC. Induction of pluripotency was initially accomplished in 2006 using mouse cells (Yamanaka et al. 2006) and in 2007 using human cells (Yu et al. 2007; Takahashi et al. 2007) by reprogramming somatic cells through the introduction of transcription factors associated with pluripotency. The use of iPSCs circumvents most of the ethical and practical issues associated with large-scale clinical use of ES cells, and patients with autologous iPSC-derived grafts may not require lifelong immunosuppressive treatment to prevent graft rejection.

With the exception of germ cells, any cell can be used as a starting point for iPSC generation. For example, the cell types may be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, liver cells, or gastric cells. T cells can also be used as a source of somatic cells for reprogramming (US Patent No. 8741648). There are no restrictions on the degree of differentiation of the cells or on the age of the animal from which the cells are obtained; even undifferentiated progenitor cells (including somatic stem cells) and, finally, differentiated mature cells can be used as sources of somatic cells in the methods disclosed herein.

Somatic cells can be reprogrammed to produce iPSCs using methods known to one of ordinary skill in the art. Induced pluripotent stem cells can be readily obtained by one skilled in the art, see, for example, US Patent Application Published No. 20090246875, US Patent Application Published No. 2010/0210014; US Patent Application Published No. 20120276636; US Patent No. 8058065; US Patent No. 8129187; US Patent No. 8268620; PCT Application Publication No. WO 2007/069666 A1 and US Patent No. 8,268,620, all of which are incorporated herein by reference. As a rule, nuclear reprogramming factors are used to obtain pluripotent stem cells from somatic cells. In some embodiments, at least three or at least four of Klf4, cMyc, Oct3/4, Sox2, Nanog, and Lin28 are used. In other embodiments, Oct3/4, Sox2, c-Myc and Klf4 are used.

Mouse and human cDNA sequences of these nuclear reprogramming agents are available by reference to the NCBI identification numbers mentioned in WO 2007/069666 and US Pat. No. 8,183,038, incorporated herein by reference. Methods of administering one or more reprogramming agents or nucleic acids encoding these reprogramming agents are known in the art and are disclosed, for example, in US Pat. Nos. 8,268,620, 8,691,574, 8,741,648, 8,546,140, U.S. Patent Published No. 8,900,871, and U.S. Pat. No. 8071369, both of which are incorporated herein by reference.

Once obtained, iPSCs can be cultured in a medium sufficient to maintain pluripotency. iPSCs can be used with a variety of media and techniques developed for culturing pluripotent stem cells, more specifically embryonic stem cells, as described in US Patent No. 7,442,548 and US Patent Application Published No. 2003/0211603. In the case of mouse cells, culture is carried out with the addition of leukemia inhibitory factor (LIF) to the normal medium as a differentiation suppressive factor. In the case of human cells, the addition of basic fibroblast growth factor (bFGF) instead of LIF is desirable. Other methods for culturing and maintaining iPSCs, as known to one skilled in the art, can be used with the present methods.

In some embodiments, undefined terms may be used; for example, pluripotent cells can be cultured on fibroblast feeder cells or media that has been exposed to fibroblast feeder cells to maintain the stem cells in an undifferentiated state. In some embodiments, the cell is cultured

- 11 046022 ut with the simultaneous presence of mouse embryonic fibroblasts, treated with radiation or an antibiotic to stop cell division, as feeding cells. Alternatively, pluripotent cells can be cultured and maintained in an essentially undifferentiated state using a chemically defined feeder-independent culture system such as TESR™ medium (Ludwig et al., 2006a; Ludwig et al., 2006b) or E8™/Essential 8™ (Chen et al., 2011).

Plasmids have been designed with a number of goals in mind, such as achieving regulated high copy numbers and preventing potential causes of plasmid instability in bacteria, and providing a means for selecting plasmids compatible with use in mammalian cells, including human cells. Particular attention was paid to the dual requirements of plasmids for use in human cells. First, they are suitable for maintenance and fermentation in E. coli, so that large quantities of DNA can be obtained and purified. Secondly, they are safe and suitable for use in humans and animals. The first requirement requires the availability of high copy number plasmids that can be selected relatively easily and stably maintained during bacterial fermentation. The second requirement requires attention to elements such as selectable markers and other coding sequences. In some embodiments, the plasmids encoding the marker consist of: (1) a high copy number origin of replication, (2) a selectable marker, such as, but not limited to, the peo gene for selection for antibiotic sensitivity using kanamycin, (3 ) transcription termination sequences, including a tyrosinase enhancer, and (4) multicloning sites for inclusion of various nucleic acid cassettes; and (5) a nucleic acid sequence encoding a marker operably linked to the tyrosinase promoter. There are a variety of plasmid vectors known in the art suitable for inducing a protein-encoding nucleic acid. These include, but are not limited to, the vectors disclosed in US Pat. No. 6,103,470; US Patent No. 7598364; US Patent No. 7,989,425 and US Patent No. 6,416,998, incorporated herein by reference.

The episomal gene delivery system may be a plasmid, an Epstein-Barr virus (EBV) episomal vector (US Pat. No. 8,546,140), a yeast-based vector, an adenovirus-based vector, a simian virus 40 (SV40)-based episomal vector, a bovine papillomavirus (BPV) or lentiviral vector. The viral gene delivery system may be an RNA- or DNA-based viral vector.

1. Cells for obtaining iPSCs.

Certain embodiments of the present invention relate to a starting population of somatic cells (eg, blood cells or skin cells) reprogrammed into iPSCs. The blood cell population may include peripheral blood mononuclear cells (MH11K), whole blood or fractions thereof containing mixed populations, spleen cells, bone marrow cells, tumor-infiltrating lymphocytes, cells obtained by leukapheresis, biopsies, and lymph nodes, e.g. , draining the tumor. Eligible donors include immunized donors, non-immunized (naive) donors, treated and untreated donors. A treated donor is a donor who has been exposed to one or more biological response modifiers. The untreated donor was not exposed to one or more biological response modifiers.

In some aspects, the blood cell population includes T cells. The T cells may be a purified population of T cells or, alternatively, the T cells may be present in a population with another cell type, such as B cells and/or other peripheral blood cells. The T cells may be a purified population of a T cell subset, such as CD4' T cells, or they may be a T cell population comprising different T cell subpopulations. In yet another embodiment, the T cells are clones of T cells that have been maintained in culture for extended periods of time. T cell clones can be transformed to varying degrees. In a specific embodiment, the T cells are a clone of T cells that proliferate indefinitely in culture.

In some aspects, the T cells are primary T cells. The term primary T cells is intended to include T cells obtained from an individual, as opposed to T cells that have been maintained in culture for extended periods of time. Thus, primary T cells are, in particular, peripheral blood T cells obtained from a subject. The primary T cell population may consist primarily of one subpopulation of T cells. Alternatively, the primary T cell population may be composed of different T cell subpopulations.

The T cells can be taken from previously stored blood samples, from a healthy individual or, alternatively, from an individual suffering from a condition. The condition may be an infectious disease, such as a condition resulting from a viral infection, a bacterial infection, or an infection by any other microorganism, or a hyperproliferative disease, such as a cancer, such as melanoma. In a specific embodiment, the T-12046022 cells are derived from an individual infected with human immunodeficiency virus (HIV). In yet another embodiment, the T cells are derived from a subject suffering from or susceptible to an autoimmune disease or T cell pathology. T cells can be of human origin, murine origin, or originate from any other mammalian species.

Methods for obtaining cell populations including T cells are well known in the art. For example, peripheral blood mononuclear cells (PBMCs) can be obtained in accordance with methods known in the art. Examples of such methods are set forth in the examples section and discussed by Kim et al. (1992); Biswas et al. (1990); Biswas et al. (1991).

In some aspects, the source population of blood cells includes hematopoietic stem cells (HSC). HSCs are usually found in the bone marrow but can be induced to enter the blood, a process called mobilization used clinically to collect large numbers of HSCs from peripheral blood. One preferred mobilizing agent is granulocyte colony-stimulating factor (G-CSF). CD34+ hematopoietic stem cells or progenitors circulating in the peripheral blood can be collected using apheresis techniques either in a non-mobilized state or after mobilization after administration of external hematopoietic growth factors such as C-GSF. The number of stem or progenitor cells collected after mobilization is greater than the number obtained after apheresis in the non-mobilized state. In some aspects, the source of the cell population is a subject whose cells have not been mobilized by externally applied factors since there is no need to enrich hematopoietic stem or progenitor cells.

Methods for obtaining hematopoietic progenitor cells from cell populations are also well known in the art. Hematopoietic progenitor cells can be expanded using various cytokines such as hSCF, hFLT3 and/or IL-3 (Akkina et al., 1996), or CD34+ cells can be enriched using MACS or FACS. As mentioned above, negative selection methods can also be used to enrich for CD34+ cells.

Cell populations for use in the methods described herein may be mammalian cells, such as human cells, non-human primate cells, rodent cells (eg, mouse or rat), bovine cells, sheep cells, porcine cells, equine cells. cages, sheep cages, dog cages and cat cages, or a mixture of these. Non-human primate cells include rhesus monkey cells. The cells can be obtained from an animal, such as a human, or they can be taken from cell lines. If the cells are obtained from an animal, they can be used as such, for example, as undivided cells (ie a mixed population); they may have been first immortalized in culture, for example by transformation; or they may have been subjected to preliminary purification methods. For example, a population of cells can be modified by positive or negative selection based on the expression of cell surface markers; stimulated by one or more antigens in vitro or in vivo; treated with one or more biological response modifiers in vitro or in vivo; or subjected to a combination of any or all of the above. In an illustrative embodiment, a population of cells is subjected to negative selection to deplete non-T cell cells and/or specific T cell subpopulations. Negative selection can be carried out based on the cell surface expression of various molecules, including B cell markers such as CD19 and CD20; monocyte marker CD14; NK cell marker CD56. Alternatively, the cell population may be subjected to negative selection to deplete non-CD34+ hematopoietic cells and/or specific hematopoietic cell subpopulations. Negative selection can be carried out based on the expression on the cell surface of various molecules, such as a mixture of antibodies (for example, CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, CD235a and CD41 (for example, for megakaryocyte lineage cells) , which can be used to separate other cell types, for example by MACS or column separation.

It is also possible to obtain a sample of cells from a subject and then enrich it with the desired cell type. For example, PBMCs and/or CD34+ hematopoietic cells can be isolated from blood, as described herein. Countercurrent centrifugation (elutriation) can be used to enrich T cells from PBMCs. Cells can also be isolated from other cells using various methods, such as isolation and/or activation with an antibody that binds to an epitope on the cell surface of the desired cell type, for example, some T cell isolation kits use conjugated antibody beads to activate the cells, and then enable column separation with the same beads. Another suitable method involves negative selection using antibodies to cell surface markers to selectively enrich for a particular cell type without activating the cell by interaction with the receptor.

Bone marrow cells can be obtained from the iliac crest, femur, tibia, spine, rib, or other medullary cavities. Bone marrow can be removed from the patient and isolated using various separation and washing techniques. Famous

- 13 046022 the procedure for isolating bone marrow cells includes the following stages: a) dividing the bone marrow suspension into three fractions by centrifugation and collecting the intermediate fraction or buffy platelet layer; b) centrifuging the buffy platelet fraction from step (a) again in a separation fluid, usually Ficoll (trade mark of Pharmacia Fine Chemicals AB), and collecting the intermediate fraction containing bone marrow cells; and c) washing the collected fraction from step (b) to isolate suitable bone marrow cells for retransplantation.

If it is necessary to use a cell population enriched in T cells, such cell populations can be obtained from a mixed cell population by leukapheresis and mechanical apheresis using a continuous flow cell separator. For example, T cells can be isolated from the buffy platelet layer by any known method, including Ficoll-Hypaque™ gradient separation, Percoll gradient separation, or elutriation.

In certain aspects, T cells are activated by agents that bind to T cell receptors, triggering a signaling cascade for T cell activation. For example, an anti-CD3 antibody may be used. To expand T cells to a significant number and proliferative state, a cytokine such as IL-2 can also be used for reprogramming. In a separate aspect, both anti-CD3 and anti-CD28 antibodies can be used to activate T cells by costimulation. In an alternative aspect, cross-linking of an anti-CD3 antibody, such as an anti-CD3 antibody coupled to a plate, may be used. If a soluble anti-CD3 antibody is used to activate T cells in PBMCs, it can bind to APCs in PBMCs, which then present the antibody to the T cells. If soluble anti-CD3 antibody alone is used in a population of purified T cells, anergy will be achieved for the above reasons. A particular embodiment involves culturing T cells in the presence of anti-CD3 (OKT3) antibody and IL-2, which is advantageous and convenient in that there is no need to use expensive and difficult to use beads or plate-associated antibody; following the addition of OKT3 and IL-2, the cellular environment of PBMCs promotes T cell activation. T cells then outnumber other cell types in the PBMC culture due to selective expansion.

In certain aspects, the starting blood cell population includes lymphoblastoid cells, such as lymphoblastoid cell lines (LCLs). The production of LCL is known in the art, for example, it is carried out by infecting B cells with Epstein-Barr virus (EBV) (Frisan et al., 2001).

2. Reprogramming of somatic cells.

In some embodiments, the original cell population (eg, T cells) is reprogrammed into iPSCs, for example, by methods described in US Patent Application Publication No. 2014/0315304, incorporated herein by reference in its entirety. In certain aspects of the present invention, reprogramming factors are expressed from expression cassettes contained in one or more vectors, such as an integrating vector or an episomal vector. In an additional aspect, reprogramming proteins can be introduced directly into somatic cells by protein transduction.

One skilled in the art will have sufficient knowledge to construct the vector using standard recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996, both of which are incorporated herein by reference). Vectors include, but are not limited to, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YAC), such as retroviral vectors (e.g., those derived from Moloney murine leukemia virus (MoMLV) vectors, MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g. derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenoviral (Ad) vectors, including forms competent to replication, replication-defective and eviscerated forms, adeno-associated virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papillomavirus vectors, Epstein-Barr virus vectors, herpes virus vectors, vaccinia virus vectors, virus vectors Harvey's murine sarcoma, mouse mammary tumor virus vectors, Rous sarcoma virus vectors.

Viral vectors.

In certain aspects of the present invention, viral vectors may be provided. When creating recombinant viral vectors, nonessential genes are usually replaced by the genome or coding sequence for a heterologous (or non-native) protein. A viral vector is a type of expression construct that uses viral sequences to introduce nucleic acid and possibly proteins into a cell. The ability of individual viruses to infect or enter cells by receptor-mediated endocytosis, and to integrate into the genomes of host cells and stably and efficiently express viral genes, has made them attractive candidates for the transfer of foreign nucleic acids into cells (eg, 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.

- 14 046022

Retroviruses are promising as gene delivery vectors due to their ability to integrate their genes into the host genome, carry large amounts of foreign genetic material, infect a wide range of cell species and types, and be packaged into specialized cell lines (Miller, 1992).

To construct a retroviral vector, a nucleic acid is inserted into the viral genome in place of specific viral sequences to produce a replication-defective virus. To obtain virions, a packaging cell line is constructed that contains the gag, pol and env genes, but does not contain LTR and packaging components (Mann et al., 1983). When a recombinant plasmid containing cDNA, along with retroviral LTRs and packaging sequences, is introduced into a dedicated cell line (for example, by calcium phosphate precipitation), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture medium (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The medium containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are capable of infecting a wide range of cell types. However, integration and stable expression require host cell division (Paskind et al., 1975).

Lentiviruses are complex retroviruses that, in addition to the usual retroviral genes gag, pol and env, contain other genes with regulatory or structural function. Lentiviral vectors are well known in the art (see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; US Pat. Nos. 6,013,516 and 5,994,136).

Recombinant lentiviral vectors are capable of infecting nondividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, a recombinant lentivirus capable of infecting a non-dividing cell where 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 US Pat. No. 5,994,136, incorporated herein application via link.

Episomal vectors.

Certain aspects of the present invention may also provide for the use of extrachromosomal (ie, episomal) plasmid- or liposome-based vectors. Such episomal vectors may include, for example, oriP-based vectors and/or vectors encoding an EBNA-1 derivative. These vectors can allow large DNA fragments to be introduced into cells and maintained outside chromosomes, replicate once per cell cycle, efficiently divide into daughter cells, and induce virtually no immune response.

In particular, EBNA-1, the only viral protein required for replication of the oriP-based expression vector, does not induce a cellular immune response because it has evolved an efficient mechanism to bypass the processing required to present its antigens on MHC class I molecules (Levitskaya et al ., 1997). In addition, EBNA-1 can act trans to enhance cloned gene expression, inducing up to 100-fold increases in cloned gene expression in some cell lines (Langle-Rouault et al., 1998; Evans et al., 1997). Finally, the production of such oriP-based expression vectors is inexpensive.

Other extrachromosomal vectors include other lymphotropic herpes virus-based vectors. Lymphotropic herpes virus is a herpes virus that replicates in a lymphoblast (eg, human B lymphoblast) and becomes a plasmid during part of its natural life cycle. Herpes simplex virus (HSV) is not a lymphotropic herpes virus. Examples of lymphotropic herpes viruses include, but are not limited to, EBV, Kaposi's sarcoma virus (KSHV); simian herpes virus (HS) and Marek's disease virus (MDV). Other sources of episome-based vectors such as yeast ARS (autonomous replicating sequence), adenovirus, SV40 or BPV are also being considered.

One skilled in the art will have sufficient knowledge to construct the vector using standard recombinant techniques (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994, both of which are incorporated herein by reference).

Vectors may also contain other components or functional properties that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties to target cells. Such other components include, for example, components that affect cell binding or targeting (including components that mediate cell type or tissue type specific binding); components affecting the uptake of vector nucleic acid by the cell; components that affect the localization of the polynucleotide in the cell after uptake (such as agents that mediate nuclear localization); and components affecting the expression of the polynucleotide.

Such components may also include markers, such as detectable and/or selectable markers, which can be used to detect or select cells that have taken up and express the nucleic acid delivered by the vector. Such components may be provided as a natural feature of the vector (for example, when using certain viral vectors,

- 15 046022 having components or functional properties that provide binding and absorption), or the vectors can be modified to provide such functional properties. A large number of such vectors are known and commonly available in the art. When a vector is maintained in a host cell, it can be stably replicated by the cells during mitosis as a free-standing structure, incorporated into the genome of the host cell, or maintained in the nucleus or cytoplasm of the host cell.

Transposon based system.

In certain aspects, a transposon-transposase system may be used to deliver programming factors. For example, the transposon-transposase system may be the well-known Sleeping Beauty, the Frog Prince transposon-transposase system (see, for example, EP1507865 for a description of the latter), or the TTAA-specific PiggyBac transposon system.

Transposons are DNA sequences that can move to different positions in the genome of an individual cell; this process is called transposition. In the process, they can cause mutations and change the amount of DNA in the genome. Transposons have also been known as jumping genes and are examples of mobile genetic elements.

There are many mobile genetic elements, and they can be classified depending on the mechanism of their transposition. Class I mobile genetic elements, or retrotransposons, copy themselves by first being transcribed into RNA, then transcribed back into DNA by reverse transcriptase, and then inserted into another position in the genome. Class II mobile genetic elements move directly from one position to another using a transposase to cut and paste themselves into the genome.

In particular embodiments, the constructs (eg, multilineage constructs) of the present invention utilize the PiggyBac expression system. PiggyBac (PB) DNA transposons are mobilized by a cut-and-paste mechanism whereby a transposase enzyme (PB transposase), encoded by the transposon itself, excises and reintegrates the transposon at other sites in the genome. PB transposase specifically recognizes PB inverted terminal repeats (ITRs) flanking the transposon; it binds to these sequences and catalyzes transposon excision. RV is then integrated relatively randomly into TTAA sites throughout the genome. To create gene trap mutations (or adapted to produce transgenic animals), the transposase is delivered in trans on a single plasmid and co-transfected with a plasmid containing a donor transposon, a recombinant transposon containing a gene trap flanked by transposase binding sites (ITRs). The transposase will catalyze the excision of the transposon from the plasmid and its subsequent integration into the genome. Integration in the coding region will capture elements required for expression of the gene trap. RV has several ideal properties: (1) it is selectively inserted into genes (from 50 to 67% of insertions affect genes) (2) it does not exhibit local abrupt changes (wide genome coverage) (3) it is not sensitive to inhibition of overproduction, in which increased levels of transposase cause decreased transposition 4) it is neatly excised from the donor site, leaving no trace, unlike Sleeping Beauty.

Regulatory elements.

The expression cassettes included in the reprogramming vectors used in the present invention preferably contain (in the 5'-3' direction) a eukaryotic transcriptional promoter operably linked to a protein coding sequence, splicing signals including introns, and a transcription termination/polyadenylation sequence.

(i) Promoter/enhancers.

The expression constructs proposed herein contain a promoter to control the expression of programming genes. A promoter usually contains a sequence that functions as the location of the site that initiates RNA synthesis. Its best known example is the TATA box, but for some promoters that lack a TATA box, such as the mammalian terminal deoxynucleotidyl transferase gene promoter and the SV40 late gene promoter, a discrete element spanning the initiation site helps anchor the initiation site. Additional promoter elements regulate the frequency of transcription initiation. As a rule, they are located in the region of 30-110 bp. in the 3'-5' direction from the initiation site, although it has been shown that a number of promoters also contain functional elements in the 5'-3' direction from the initiation site. To bring the coding sequence under the control of a promoter, the 5' end of the transcription start site of the transcriptional reading frame is positioned in the 5'-3' direction (ie, 3'-) of the selected promoter. The promoter located in the 3'-5' direction stimulates DNA transcription and promotes the expression of the encoded RNA.

The spacing between promoter elements is often flexible so that promoter function is maintained when elements are inverted 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. Apparently, depending on the promoter, individual elements can function jointly or independently to activate transcription. A promoter may or may not be used in conjunction with an enhancer, which is a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

The promoter may be a promoter naturally associated with a nucleic acid sequence, which can be achieved by isolating 5' non-coding sequences located in the 3'-5' direction from the coding segment and/or exon. Such a promoter can be called endogenous. Likewise, an enhancer may be an enhancer naturally associated with a nucleic acid sequence located in the 5'-3' or 3'-5' direction 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 the nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other virus, or prokaryotic or eukaryotic cell, and promoters or enhancers not found in nature, that is, containing different elements of different regions of transcription regulation, and/or mutations that alter expression. For example, promoters most commonly used in recombinant DNA constructs include the β-lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. In addition to the synthetic route for obtaining promoter and enhancer nucleic acid sequences, sequences can be obtained using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (see US Patent Nos. 4,683,202 and 5,928,906 , each of which is incorporated herein by reference). In addition, it is contemplated that control sequences directing the transcription and/or expression of sequences within organelles that do not have nuclei, such as mitochondria, chloroplasts, and the like, may also be used.

Naturally, it is important to use a promoter and/or enhancer that effectively drives 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 will generally know the combinations of promoters, enhancers, and cell types used for protein expression (see, for example, Sambrook et al. 1989, incorporated herein by reference). The promoters used may be constitutive, tissue specific, inducible and/or suitable under appropriate conditions for directing high levels of expression of the introduced DNA segment, which is advantageous in the large scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

In addition, any promoter/enhancer combination (eg, according to the eukaryotic promoter database EPDB) can also be used to drive expression. Another possible embodiment relates to the use of a T3, T7 or SP6 cytoplasmic expression system. Eukaryotic cells can support cytoplasmic transcription of individual bacterial promoters if the appropriate bacterial polymerase is provided, either as part of a delivery complex or as an additional genetic expression construct.

Non-limiting examples of promoters include early or late viral promoters, such as SV40 early or late promoters, cytomegalovirus (CMV) immediate early promoters, Rous sarcoma virus (RSV) early promoters; promoters of eukaryotic cells, such as, for example, the beta-actin promoter (Ng, 1989; Quitsche et al., 1989), the GADPH promoter (Alexander et al., 1988, Ercolani et al., 1988), the metallothionein promoter (Karin et al. ., 1989; Richards et al., 1984); and concatenated response element promoters such as cyclic AMP response element (cre) promoters, serum response element (sre) promoter, phorbol ester (TPA) promoter, and response element (tre) promoters near the minimal TATA box. It is also possible to use human growth hormone promoter sequences (eg, the minimal human growth hormone promoter described in Genbank, accession number X05244, nucleotide 283-341) or the mouse mammary tumor promoter (available from ATCC, ATCC catalog number 45007).

Tissue-specific expression of transgenes may be desirable as a means of identifying resulting hematopoietic cells and progenitors, especially for reporter gene expression in hematopoietic cells and hematopoietic cell progenitors resulting from programming. To increase both specificity and activity, the use of cis-acting regulatory elements is provided. For example, a hematopoietic cell-specific promoter may be used. Many such hematopoietic cell-specific promoters are known in the art, for example, the hematopoietic gene promoters shown in Table 1. 1.

- 17 046022

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

Many hematopoietic cell promoter and enhancer sequences have been identified that may be suitable for use in the present methods. See, for example, US Pat. No. 5,556,954; US Patent Application No. 20020055144; US patent application 20090148425.

(ii) Initiation signals and associated expression.

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

In certain embodiments, internal ribosome entry site (IRES) elements are used to create multigene or polycistronic mRNAs. IRES elements are able to bypass the ribosome scanning model of 5'-terminal methylated cap-dependent translation and initiate translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (poliomyelitis and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well as IRES from mammalian mRNA (Macejak and Sarnow, 1991). IRES elements can be associated with heterologous open reading frames. Multiple open reading frames can be transcribed together, with each one separated by an IRES, creating polycistronic mRNAs. Thanks to the LRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single mRNA (see US Pat. Nos. 5,925,565 and 5,935,819, each of which is incorporated herein by reference).

In addition, certain 2A sequence elements can be used to create associated or co-expression of programming genes in the constructs of the present invention. For example, cleavage sequences can be used to co-express genes by linking open reading frames to form a single cistron. An example of a cleavage sequence is the F2A sequence (foot-and-mouth disease virus 2A) or a 2A-like sequence (eg, Thosea asigna virus 2A; T2A). In particular embodiments, the F2A cleavage peptide is used to couple gene expression in a multilineage construct.

Replication start points.

For propagation of the vector in a host cell, it may contain one or more origins of replication (often called oris), for example, a nucleic acid sequence corresponding to EBV oriP, as described above, or a genetically engineered oriP with similar or enhanced programming function, which represents the specific nucleic acid sequence at which replication begins. Alternatively, the origin of replication of another extrachromosomally replicating virus, as described above, or an autonomously replicating sequence (ARS) can be used.

Selectable markers and markers for screening.

In certain embodiments, cells containing the nucleic acid construct can be identified in vitro or in vivo by including a marker in an expression vector. Such markers impart an identifiable change to the cell, allowing cells containing the expression vector to be easily identified. Typically, a selectable marker is a marker that confers a property that allows selection. A positive selection marker is a marker whose presence allows selection, and a negative selection marker is a marker whose presence prevents selection. An example of a positive selection marker is a drug resistance marker.

Typically, the inclusion of a selectable marker to select for drug sensitivity aids in cloning and identification of transformants, for example, useful selectable markers are genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol. In addition to markers that impart a phenotype that makes it possible to distinguish transformants based on the implementation of certain conditions, other types of mars are also considered

- 18 046022 cores, including screening markers such as GFP (green fluorescent protein), based on colorimetric analysis. Alternatively, screening enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) can be used as negative selection markers. One skilled in the art will also know how to use immunological markers, possibly in combination with FACS analysis. The marker is not considered to be important provided that it is capable of being expressed with the nucleic acid encoding the gene product. Additional examples of selectable markers and screening markers are well known to one skilled in the art.

To introduce a nucleic acid, such as DNA or RNA, into pluripotent stem cells to be programmed into hematopoietic progenitor cells according to the present invention, any suitable methods for delivering the nucleic acid to transform the cell can be used, as described herein or as known to one skilled in the art. this field of technology. Such methods include, but are not limited to, direct delivery of DNA, for example, by ex vivo transfection (Wilson et al., 1989, Nabel et al., 1989), by injection (US Patent Nos. 5994624, 5981274, 5945100, 5780448, 5736524, 5702932, 5656610, 5589466 and 5580859, each of which is incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; US Pat. No. 5789215, incorporated herein by reference); by electroporation (US Pat. No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al., 1986; Potter et al., 1984); by precipitation of calcium phosphate (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct sound loading (Fechheimer et al., 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991) and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988); by bombardment with microparticles (PCT application No. WO 94/09699 and 95/06128; US patent No. 5610042; 5322783 5563055, 5550318, 5538877 and 5538880, incorporated herein by reference); by mixing with silicon carbide fibers (Kaeppler et al., 1990; US Pat. Nos. 5,302,523 and 5,464,765, incorporated herein by reference); by Agrobacterium-mediated transformation (US Patent Nos. 5,591,616 and 5,563,055, each of which is incorporated herein by reference); by desiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985), and any combination of such methods. Through the use of such technologies, organelle(s), cell(s), tissue(s) or organism(s) can be permanently or temporarily transformed.

MHC haplotype selection.

The major histocompatibility complex (MHC) is the leading cause of immune system rejection of allogeneic organ transplants. There are three main MHC class I haplotypes (A, B and C) and three main MHC class II haplotypes (DR, DP and DQ). HLA loci are highly polymorphic and distributed over 4 Mb on chromosome 6. The ability to haplotype HLA genes in this region is clinically important because this region is associated with autoimmune and infectious diseases, and the compatibility of donor and recipient HLA haplotypes can influence the clinical outcomes of transplantation. HLAs corresponding to MHC class I present peptides from the inside of the cell, and HLAs corresponding to URUC class II present antigens from the outside of the cell to T lymphocytes. Incompatibility of MHC haplotypes between the graft and the host causes an immune response against the graft and leads to its rejection. Thus, the patient may be treated with an immunosuppressant to prevent rejection. HLA-matched stem cell lines may help overcome the risk of immune system rejection.

Because of the importance of HLA in transplantation, HLA loci are routinely typed by serology and PCR to identify favorable donor-recipient pairs. Serological detection of HLA class I and II antigens can be performed using a complement-mediated lymphocytotoxicity test with purified T or B lymphocytes. This procedure is primarily used to select the HLA-A and -B loci. Molecular tissue typing can often be more accurate than serological testing. Low-resolution molecular methods such as SSOP (sequence-specific oligonucleotide probe) methods, in which PCR products are tested against a series of oligonucleotide probes, can be used to identify HLA antigens, and these methods are currently the most common methods used for HLA class II typing. High-resolution methods, such as SSP (sequence specific primer) methods, which use allele-specific PCR amplification primers, allow the identification of specific MHC alleles.

The compatibility of MHC donor and recipient increases significantly if the donor cells are HLA homozygous, that is, they contain identical alleles for each antigen-presenting protein. Most individuals are heterozygous for the MHC class I and II genes, but some individuals are homozygous for these genes. These homozygous individuals can serve as superdonors, and grafts derived from their cells can be transplanted into all individuals homozygous or heterozygous for a given haplotype. In addition, if homozygous donor cells have a haplotype that is common in the population, these cells may find use in transplant therapy for a large number of individuals.

Accordingly, in some embodiments, the PSCs used in the present methods can be obtained from somatic cells of the subject being treated or another subject with the same or substantially the same HLA type as the patient. In one case, the major HLAs (eg, the three major loci HLA-A, HLA-B, and HLA-DR) of the donor are identical to the major HLAs of the recipient. In some cases, the somatic cell donor may be a superdonor; thus, PSCs derived from a homozygous MHC superdonor can be used to generate HPCs and then immune cells such as T cells. Thus, immune effector cells obtained from a superdonor can be transplanted into subjects homozygous or heterozygous for a given haplotype. For example, immune cells can be homozygous for two HLA alleles, such as HLA-A and HLA-B. As such, immune cells obtained from superdonors can be used in the methods disclosed herein to produce immune cells that can potentially match a large number of potential recipients.

Genetically engineered antigen receptors.

PSCs can be genetically engineered to express antigen receptors such as engineered TCRs or CARs. For example, PSCs (eg, autologous or allogeneic) are modified to express a TCR or CAR that has antigenic specificity for a cancer antigen.

Suitable modification methods are known in the art. See, for example, the source Sambrook and Ausubel mentioned above. For example, cells can be transduced to express a TCR that is antigen specific for a cancer antigen using transduction methods described in Heemskerk et al. Hum Gene Ther. 19:496-510 (2008) and Johnson et al. Blood 114:535-46 (2009).

Electroporation of RNA encoding full-length α and β (or γ and δ) TCR chains 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 occurs in a transient transfection strategy, autoreactive T cells that can possibly be generated in this way will lose this autoreactivity after some time, since the introduced TCR α and β chains are only transiently expressed. When the expression of the introduced TCR α and β chain is reduced, only normal autologous T cells remain. Whereas in the case where full-length TCR chains are introduced by stable transduction with retroviruses, the introduced TCR chains will never disappear, causing permanent autoreactivity in the patient.

In some embodiments, the cells contain one or more genetically engineered nucleic acids that encode one or more antigen receptors, and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, that is, not typically present in the cell or sample derived from the cell, for example, they are derived from another organism or cell that, for example, is not typically present in the engineered cell and/or organism from which such a cell was obtained. In some embodiments, the nucleic acids are not naturally occurring, e.g., a non-naturally occurring nucleic acid (eg, 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 an intracellular protein peptide antigen that, like a TCR, is recognized on the surface of a cell 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 receptors into cells include those described, for example, in international patent application publications Nos. WO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/0711 54, WO2013 /123061, US Patent Application Publications Nos. US2002131960, US2013287748, US20130149337, US Patent Nos. 6451995, 7446190, 8252592, 8339645, 8398282, 7446179, 6410319, 707 0995, 7265209, 7354762, 7446191, 8324353 and 8479118, and European patent application No. EP2537416, and/or described in Sadelain et al., Cancer Discov. April 2013; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, genetically engineered antigen receptors include CARs, as described in US Pat. No. 7,446,190, and described in International Patent Application Publication No. WO/2014055668 Al.

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1. Chimeric antigen receptors.

In some embodiments, the CAR comprises: a) an intracellular signaling domain, b) a transmembrane domain, and c) an extracellular domain containing an antigen binding region.

In some embodiments, the engineered antigen receptors comprise CARs, including activating or stimulatory CARs, costimulatory CARs (see WO2014/055668) and/or inhibitory CARs (iCARs, see Fedorov et al., 2013). CARs typically 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 substantially 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.

Certain 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), containing an intracellular signaling domain, a transmembrane domain, and an extracellular domain containing one or more signaling motifs . In certain embodiments, the CAR may recognize an epitope comprising a region common to one or more antigens. In certain embodiments, the binding region may comprise complementarity determining regions of the monoclonal antibody, variable regions of the monoclonal antibody, and/or antigen binding fragments thereof. In yet another embodiment, this specificity is derived from a peptide (eg, a cytokine) that binds to the receptor.

It is proposed that human CAR nucleic acids may represent human genes used to enhance cellular immunotherapy in humans. In a specific embodiment, the invention includes a full-length CAR cDNA or coding region. The antigen binding regions or domain may comprise a portion of the VH and VL region of a single chain variable fragment (scFv) derived from a particular human monoclonal antibody, such as those described in US Pat. No. 7,109,304, incorporated herein by reference. The fragment may also be any number of different antigen binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragment is an antigen-specific scFv encoded by a sequence optimized to use a human codon for expression in human cells.

The configuration may be multimeric, such as a diabody or multimers. Most likely, multimers are formed by cross-pairing of the variable portion of the light and heavy chains in a diabody. The hinge portion of the construct can have several alternatives, from being completely removed to retaining the first cysteine, to substituting a proline rather than a serine, to being truncated down to the first cysteine. The Fc portion may be removed. Any protein that is stable and/or dimerizes can serve this purpose. Only one of the Fc domains can be used, for example the CH2 or CH3 domain from human immunoglobulin. The hinge, the CH2 and CH3 region of human immunoglobulin modified to improve dimerization, can also be used. It is also possible to use only the hinge portion of the immunoglobulin. CD8 alpha parts can also be used.

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

In some embodiments, the CAR is designed with specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type that is to be targeted by adoptive therapy, such as a cancer marker, and/or an antigen intended to induce mitigation response, such as an antigen expressed on normal or non-cancerous cells. Thus, a CAR in its extracellular portion typically contains one or more antigen binding molecules, such as one or more antigen binding fragments, domains or portions, or one or more antibody variable domains and/or antibody molecules. In some embodiments, the CAR includes an antigen binding portion or portions of an antibody molecule, such as a single chain antibody fragment (scFv) derived from the heavy (VH) and light (VL) chain variable regions 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 an extracellular domain containing an antigen-binding region) comprises a tumor-associated antigen or a pathogen-specific antigen-binding domain. Antigens include carbohydrate antigens recognized by pattern recognition receptors,

- 21 046022 such as Dectin-1. The tumor-associated antigen can be of any type, provided that it is expressed on the cell surface of tumor cells. Examples of embodiments of tumor-associated antigens include CD19, CD20, carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, CD56, EGFR, c-Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma-associated antigen, mutated p53, mutated ras and so on.

The open reading frame sequence encoding the chimeric receptor may be obtained from a genomic DNA source, a cDNA source, or may be synthesized (eg, by PCR), or combinations thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination of both, since introns have been found to stabilize the mRNA. In addition, it may also be advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA.

It is envisaged that the chimeric construct can be introduced into immune cells as naked DNA or in a suitable vector. Methods for stably transfecting cells by electroporation using naked DNA are known in the art. See, for example, US Pat. No. 6,410,319. Naked DNA generally refers to DNA encoding a chimeric receptor contained in a plasmid expression vector in the correct orientation for expression.

Alternatively, a viral vector (eg, 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 accordance with the method of the present invention do not replicate in immune cells. A large number of virus-based vectors are known where the number of copies of the virus maintained in the cell is low enough to maintain cell viability, such as, for example, HIV, SV40, EBV, HSV or BPV-based vectors.

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 domains in the CAR. In some cases, the transmembrane domain is selected or modified by amino acid substitutions to avoid binding of such domains to the transmembrane domains of the same or other membrane surface proteins to minimize interactions with other members of the receptor complex.

The transmembrane domain, in some embodiments, is derived from either a natural or synthetic source. If the source is natural, the domain is in some aspects derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., include at least the transmembrane region(s)) T cell receptor alpha, beta, or zeta chain, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45 molecules, 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 contains predominantly hydrophobic residues such as leucine and valine. In some aspects, at each end of the synthetic transmembrane domain is a triplet of phenylalanine, tryptophan, and valine.

In certain embodiments, platform technologies disclosed herein for genetic modification of immune cells, such as NK cells, include (i) non-viral gene transfer using an electroporation device (eg, a nucleofector), (ii) CARs signaling through endodomains (e.g., CD28/CD3-Z, CD137/CD3-Z, or other combinations), (iii) CARs with varying lengths of extracellular domains connecting the antigen recognition domain to the cell surface, and, in some cases, (iv) artificial antigen presenting cells (aAPCs) ) derived from K562 to be able to reliably and quantitatively expand CAR' immune cells (Singh et al., 2008; Singh et al., 2011).

2. T-cell receptor (TCR).

In some embodiments, the genetically engineered antigen receptors include recombinant TCRs and/or TCRs cloned from natural T cells. T cell receptor or TCR refers to a molecule containing the α and β chain variable regions (also known as TCRa and TCRe, respectively) or the γ and δ chain variable regions (also known as TCRy and TCRδ, respectively), and is capable of specifically bind to the antigenic peptide associated with the MHC receptor. In some embodiments, the TCR is in the αβ form.

In general, TCRs, existing in αβ and γδ forms, are usually structurally similar, but the T cells that express them may have different anatomical locations or functions. The TCR may be present on the cell surface or in soluble form. Typically, the TCR is present on the surface of T cells (or T lymphocytes), where it is usually responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, the TCR may also comprise a constant domain, a transmembrane domain, and/or a short cytoplasmic tail (see, for example, Janeway et al, Immunobiology: The Immune System in Health and Disease, ^3rd Ed., Current Biology Publications, p 433, 1997). For example, in some aspects, each TCR chain may have 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 CD3 complex proteins involved in mediating signal transduction. Unless otherwise indicated, the term TCR should be understood to cover its functional fragments. The term also covers intact or full-length TCRs, including TCRs in the αβ form or in the γδ form.

Thus, for purposes of this application, reference to a TCR includes any TCR or functional fragment, such as an antigen-binding portion of a TCR, that binds to a specific antigenic peptide associated with a MHC molecule, that is, an MHC-peptide complex. The terms antigen binding portion or antigen binding fragment of a TCR, which can be used interchangeably, refer to a molecule containing a region of TCR structural domains but binding an antigen (eg, MHC-peptide complex) to which the full-length TCR binds. In some cases, the antigen binding portion contains TCR variable domains, such as the α chain variable domain and the TCR β chain variable domain, sufficient to form a binding site for binding to a particular MHC-peptide complex, for example, where typically each chain contains three complementarity determining regions .

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

In some embodiments, the TCR chains comprise a constant domain. For example, as with immunoglobulins, the extracellular portion of the TCR chains (eg, α-chain, β-chain) may contain two immunoglobulin domains, a variable domain (eg, Va or _Vp ; typically amino acids 1 to 116 according to the Kabat numbering system, see Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, ^5th ed.) at the N-terminus and one constant domain (for example, constant domain a β-chain or C _a , usually amino acids 117 to 259 according to Kabat, β-chain constant domain or Cp, usually amino acids 117 to 295 according to 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 CDRs. The constant domain of the TCR domain contains short connecting sequences in which a cysteine residue forms a disulfide bond, linking the two chains. In some embodiments, the TCR may have an additional cysteine residue on 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, TCR chains contain a cytoplasmic tail. In some cases, the structure allows the TCR to associate with other molecules such as CD3. For example, a TCR containing constant domains with a transmembrane region can anchor the protein to the cell membrane and bind to invariant subunits of the signaling machinery or CD3 complex.

Typically, CD3 is a multiprotein complex that can have 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 CD3Z chains. The CD3y, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily, containing a single immunoglobulin domain. The transmembrane regions of the CD3y, CD3a, and CD3ε chains are negatively charged, a characteristic that allows these chains to associate with the positively charged T-cell receptor chains. The intracellular tails of the CD3y, CD3δ, and CD3ε chains each contain one conserved motif known as the immunoreceptor tyrosine-based activating motif or ITAM, whereas each CD3Z chain has three. Typically, ITAMs are involved in the signaling ability of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in propagating the signal from the TCR into the cell. The 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 with two chains α and β (or optionally γ and δ), or it may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer containing two distinct 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 (eg, a cancer antigen) is identified and introduced into cells. In some embodiments, the nucleic acid encoding the TCR can be obtained from various sources, such as polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences. In some embodiments, the TCR is obtained from a biological source, such as cells such as T cells (eg, cytotoxic T cells), T cell hybridomas, or other publicly available sources. In some embodiments, T cells may 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 can be isolated. In some embodiments, the T cells may be a cultured T cell hybridoma or clone. In some embodiments, the TCR clone for the target antigen is derived from transgenic mice engineered with human immune system genes (eg, human leukocyte antigen or HLA system). In some embodiments, phage display is used to isolate a TCR against a target antigen. In some embodiments, a TCR or an antigen-binding portion thereof may be synthesized using knowledge of the TCR sequence.

3. Antigen-presenting cells.

APCs, which include macrophages, B lymphocytes, and dendritic cells, differ in the expression of a specific MHC molecule. APCs take up antigen and re-express part of that antigen along with the MHC molecule on their outer cell membrane. MHC is a large genetic complex with several loci. The MHC loci encode two major classes of membrane MHC molecules, called class I and class II MHCs. T-helper lymphocytes usually recognize antigen associated with MHC class II molecules, and T-cytotoxic lymphocytes recognize antigen associated with MHC class I molecules. In humans, MHC is called the HLA complex, and in mice it is called the H-2 complex.

In some cases, aAPCs are useful for the preparation of therapeutic compositions and cell therapy products according to embodiments. For general guidance regarding the preparation and use of antigen presenting systems, see, for example, US Pat. Nos. 6,225,042, 6,355,479, 6,362,001, and 6,790,662.

AAPC systems may contain at least one exogenous accessory molecule. Any suitable amount and combination of auxiliary molecules may be used. The auxiliary molecule may be selected from auxiliary molecules such as co-stimulatory molecules and adhesion molecules. Examples of co-stimulatory molecules include CD86, CD64 (FcyI), 41BB ligand and IL-21. Adhesion molecules may include carbohydrate-binding glycoproteins such as selectins, transmembrane binding glycoproteins such as integrins, calcium-dependent proteins such as cadherins, and single-pass transmembrane immunoglobulin (Ig) superfamily proteins such as intercellular adhesion molecules (ICAMs), which promote, for example, contact between cells or between a cell and a matrix. Examples of adhesion molecules include LFA-3 and ICAMs such as ICAM-1. Methods, methods, and reagents suitable for selecting, cloning, producing, and expressing illustrative accessory molecules, including co-stimulatory molecules and adhesion molecules, are described, for example, in U.S. Patent Nos. 6,225,042, 6,355,479, and 6,362,001.

4. Antigens.

Antigens targeted by genetically engineered antigen receptors include those expressed in the context of the disease, condition, or cell type to be targeted by adoptive cell therapy. Diseases and conditions include proliferative, neoplastic and malignant diseases and disorders, including cancers and tumors, including hematological cancers, immune system cancers such as lymphomas, leukemias and/or myelomas such as B cell, T cell 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 may find use in the present method. Examples of antigens include, but are not limited to, antigenic molecules from infectious agents, auto/self antigens, tumor/cancer associated antigens, and tumor neoantigens (Linnemann et al., 2015).

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Tumor-associated antigens may originate from prostate, breast, colorectal, lung, pancreatic, kidney, mesothelioma, ovarian cancer, or melanoma. Examples of tumor-associated antigens or antigens derived from tumor cells include MAGE 1, 3 and MAGE 4 (or other MAGE antigens); PRAME; BAGE; RAGE, Lage (also known as NY-ESO-1); SAGE; and HAGE or GAGE. These non-limiting examples of tumor antigens are expressed in a wide range of tumor types, such as melanoma, lung cancer, sarcoma and bladder cancer. See, for example, US Pat. No. 6,544,518. Antigens associated with prostate cancer include, for example, prostate specific membrane antigen (PSMA), prostate specific antigen (PSA), prostate acid phosphate, NKX3.1 and six-transmembrane epithelial prostate antigen (STEAP).

Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and cryptin. In addition, the tumor antigen may be a self-peptide hormone, such as full-length gonadotropin-releasing hormone (GnRH), a short peptide of 10 amino acids in length useful for the treatment of many types of cancer.

Tumor antigens include tumor antigens derived from cancers characterized by the expression of tumor-associated antigens, such as HER-/neu expression. Tumor-associated antigens of interest include lineage-specific tumor antigens such as melanocyte-melanoma lineage antigens MART-1/Melan-A, gp100, gp75, mda-7, tyrosinase and tyrosinase-related protein. Examples of tumor-associated antigens include, but are not limited to, tumor antigens derived from or containing 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, MAGE-A4, 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, Cyp-B, 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, CDK-4/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-ab1, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 /AML, LDLR/FUT, Pml/RAR, tumor-associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (eg, epidermal growth factor receptor (EGFR) (particularly EGFRvIII), platelet-derived growth factor receptor (PDGFR), factor receptor vascular endothelial growth factor (VEGFR)), cytoplasmic tyrosine kinases (e.g. src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and transcriptional activators STAT3, STATS and STATE, hypoxia-inducible factors (e.g. HTF -1 and HTF-2), nuclear factor kappa bi (NF-B), Notch receptors (eg, Notch 1-4), c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinases (ERK) and their regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma antigen 5T4, SM22-alpha, 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 hybrid 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, TLE2, 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 in tumor cells at different levels compared to normal cells, such as the enzyme telomerase, survivin, mesothelin, mutated ras, bcr rearrangement abl, Her2/neu, mutated or wild-type p53, cytochrome P450 1B1 and pathologically expressed intronic sequences such as N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes generating unique idiotypes in myeloma and B-cell lymphomas; tumor antigens including epitope regions or epitope peptides derived from oncoviral processes, such as human papillomavirus E6 and E7 proteins; Epstein-Barr virus protein LMP2; unmutated oncofetal proteins with selective tumor expression, such as carcinoembryonic antigen and alpha-fetoprotein.

In other embodiments, the antigen is derived from 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 certain embodiments, antigens derived from such a microorganism include full-length proteins.

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Immune effector cells.

Hematopoietic progenitor cells.

PSCs of the present invention engineered to express an antigen receptor such as a CAR can be differentiated into HPCs by methods known in the art. In one method, CAR-PSCs are differentiated into CD34' HPCs through directed differentiation. In yet another method, CAR-PSCs are differentiated into CD34+ HPCs through direct programming.

1. Directed differentiation.

Certain embodiments of the present invention relate to the differentiation of CARPSCs into LPCs. CAR-PSCs can be differentiated into HPCs by methods known in the art, such as those described in US Pat. No. 8,372,642, incorporated herein by reference. In one method, combinations of BMP4, VEGF, Flt3 ligand, IL-3 and GM-CSF can be used to stimulate differentiation into hematopoietic cells. In certain embodiments, sequentially exposing cell cultures to a first medium to produce PSC differentiation, a second medium comprising BMP4, VEGF, and FGF, followed by culturing in a third medium comprising Flt3 ligand, SCF, TPO, IL-3, and IL-6 , can mediate the differentiation of pluripotent cells into HPCs and hematopoietic cells. A second chemically defined medium may also contain heparin. In addition, the inclusion of FGF-2 (50 ng/ml) in a medium containing BMP4 and VEGF can increase the efficiency of formation of hematopoietic progenitor cells from pluripotent cells. In addition, the inclusion of a glycogen synthase kinase-3 (GSK3) inhibitor (eg, CHIR99021, BIO, and SB-216763) in the chemically defined first medium can further enhance HPC production.

In general, differentiation of pluripotent cells into hematopoietic progenitor cells can be accomplished using defined or undefined conditions. It will be understood that certain conditions are generally preferred in embodiments where the resulting cells are intended for administration to a human. Hematopoietic stem cells can be derived from pluripotent stem cells under certain conditions (eg, using TeSR medium), and hematopoietic cells can be derived from embryoid bodies derived from pluripotent cells. In other embodiments, pluripotent cells can be co-cultured on OP9 cells or mouse embryonic fibroblasts and then differentiated.

Pluripotent cells can be allowed to form embryoid bodies or aggregates as part of the differentiation process. The formation of embryoid bodies (EBs) or clusters of growing cells to induce differentiation usually involves in vitro aggregation of human pluripotent stem cells into EBs and allows spontaneous and random differentiation of human pluripotent stem cells into various tissue types forming endoderm, ectoderm and mesoderm. Thus, three-dimensional EBs can be used to obtain a certain fraction of hematopoietic cells and endothelial cells.

EVs can be formed using the following procedure. Undifferentiated iPSCs adapted to feeder-free growth on MATRIGEL-coated plates can be harvested once confluent by treatment with 0.5 M EDTA for approximately 8-10 min at room temperature. EDTA is aspirated after incubation, and EBs can be generated by collecting cells in SFD containing rock inhibitor or blebbistatin. The medium can be replaced the next day with EB1 differentiation medium containing different cytokine compositions. Cells are seeded at a density of 0.25-0.5 million cells per ml to promote aggregate formation.

To stimulate aggregate formation, cells can be transferred to low-attachment plates for overnight incubation in serum-free differentiation medium (SFD) consisting of 75% IMDM (Gibco), 25% modified Ham's medium F12 (Cellgro) supplemented with 0. 05% N2 and B-27 without RA (retinoic acid) additives, 200 mM l-glutamine, 0.05 mg/ml magnesium salt of ascorbic acid-2-phosphate (Asc 2-P) (WAKO) and 4.5x10 ^-4 MTG. The next day, cells can be collected from each well and centrifuged. The cells can then be resuspended in EB differentiation medium consisting of SFD minimal medium supplemented with approximately 50 ng/ml bone morphogenetic factor (BMP4), approximately 50 ng/ml vascular endothelial growth factor (VEGF) and 50 ng/ml zb FGF during the first four days of differentiation. Cells are fed halfway every 48 hours. On the fifth day of differentiation, the medium is replaced with a second medium consisting of SFD medium supplemented with 50 ng/ml stem cell factor (SCF), approximately 50 ng/ml Flt-3 ligand (Flt-3L), 50 ng /ml interleukin-6 (IL-6), 50 ng/ml interleukin-3 (IL-3), 50 ng/ml thrombopoietin (TPO). Cells are fed halfway every 48 h with fresh differentiation media. Media changes are carried out by centrifuging the differentiation cultures at 300 g for 5 minutes and aspirating half the volume from the differentiation cultures and replacing it with fresh medium. In certain embodiments, the EB differentiation medium may include BMP4 (eg, about 50 ng/ml), VEGF (eg, about 50 ng/ml), and optionally FGF-2 (eg, about 25-75 ng /ml or approximately 50 ng/ml). The supernatant can be aspirated and replaced with fresh differentiation medium. Alternatively, the cells can be fed half every two days with fresh medium. Cells can be collected at different times during differentiation.

HPCs can be cultured from pluripotent stem cells using a chemically defined medium. Methods for differentiating pluripotent cells into hematopoietic CD34+ stem cells using chemically defined media are described, for example, in US patent application 12/715136, incorporated by reference in its entirety. It is intended that these methods may be used in the present invention.

For example, a chemically defined medium can be used to induce differentiation into hematopoietic CD34+ cells. The chemically defined medium may contain growth factors BMP4, VEGF, Flt3 ligand, IL-3 and/or GM-CSF. Pluripotent cells can be cultured in a chemically defined first medium containing BMP4, VEGF and optionally FGF-2, followed by culture in a second medium containing either (Flt3 ligand, IL-3 and GM-CSF) or ( Flt3, IL-3, IL-6 and TPO). The first and second media may also contain one or more of SCF, IL-6, G-CSF, EPO, FGF-2 and/or TPO. Conditions of significant hypoxia (eg, less than 20% O2) may further promote hematopoietic or endothelial differentiation.

Cells can be substantially separated from each other by mechanical or enzymatic means (eg, using trypsin or TrypLE™). A ROCK inhibitor (eg, H1152 or Y-27632) may also be included in the medium. It is understood that these approaches can be automated using, for example, robotic automation.

In certain embodiments, conditions of significant hypoxia can be used to stimulate the differentiation of pluripotent cells into hematopoietic progenitor cells. As will be understood by one skilled in the art, an oxygen content in the atmosphere of less than about 20.8% would be considered hypoxic. Human cells in culture can grow in an atmosphere with a reduced oxygen content compared to the surrounding air. This relative hypoxia can be achieved by reducing the oxygen content in the atmosphere in contact with the culture media. Embryonic cells typically develop in vivo under conditions of reduced oxygen, typically from about 1% to about 6% oxygen in the atmosphere, with carbon dioxide at ambient levels. Without being bound by any particular theory, it is believed that hypoxic conditions may mimic an aspect of certain conditions of embryonic development. As shown in the examples below, hypoxic conditions can be used in certain embodiments to promote further differentiation of pluripotent cells, such as iPSC or hESC, into a more differentiated cell type, such as HPC.

The following hypoxic conditions can be used to stimulate the differentiation of pluripotent cells into hematopoietic progenitor cells. In certain embodiments, an atmospheric oxygen content of less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14% less than about 13%, less less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, about 5%, about 4%, about 3% , about 2% or about 1%, can be used to stimulate differentiation into hematopoietic progenitor cells. In some embodiments, the hypoxic atmosphere contains approximately 5% oxygen gas.

Regardless of the specific medium used in any given expansion of hematopoietic progenitor cells, at least one cytokine is preferably added to the medium used at a concentration of from about 0.1 ng/ml to about 500 ng/ml, more often from 10 ng/ml up to 100 ng/ml.

Suitable cytokines include, but are not limited to, c-kit ligand (KL) (also called steel factor (StI), mast cell growth factor (MGF) and stem cell factor (SCF)), IL-6, G-CSF, IL- 3, GM-CSF, IL-1a, IL-11 MIP-1a, LIF, c-mpl/TPO ligand and flk2/flk3 ligand (Flt2L or Flt3L). In particular, the culture will include at least one of SCF, Flt3L and TPO. More specifically, the culture will include SCF, Flt3L and TPO.

In one embodiment, the cytokines are contained in the media and are replenished by perfusion of the medium. Alternatively, when using a bioreactor system, cytokines can be added separately, without perfusion of the medium, as a concentrated solution through separate inlets. When cytokines are added without perfusion, they are typically added to bioreactors as a 10x to 100x solution in an amount equal to one tenth to 1/100th the volume, with fresh cytokines added approximately every 2 to 4 days. Additionally, fresh concentrated cytokines can also be added individually to complement the cytokines in the perfused medium.

In some embodiments, HPCs exhibit methyl-CpG binding protein 2 (MeCP2) disruption and are cultured under conditions that promote myeloid differentiation or lymphoid differentiation. In some aspects, HPCs express nonfunctional MeCP2 that does not substantially bind to methylated DNA. In certain aspects, HPCs do not express MeCP2 at levels sufficient to affect MeCP2 DNA binding activity. In particular, MeCP2 does not function due to a truncation or mutation in the MeCP2 gene. In some aspects, generating HPCs exhibiting MeCP2 disruption involves contacting the HPCs with siRNA, small hairpin RNA, or a small molecule inhibitor of MeCP2.

(i) An example of a 3D differentiation method.

An example method for differentiating PSCs into HPCs involves maintenance under feeder-free conditions, for example, on MATRIGEL™ or vitronectin-coated plates in Essential 8 (E8) media. Aggregates are prepared from PSCs, particularly those that are subconfluent, e.g. <80% confluent), at a density of 0.5-1 million cells per ml in Essential 3 (E3) medium (e.g. containing only 3 of the 8 components of E8 medium: DMEM/minimal medium F12, ascorbic acid (e.g. 100-500 µM), magnesium 2phosphate and sodium selenite) supplemented with 50 ng/ml FGF2, 50 ng/ml VEGF, 2 µM CHIR99021 (GSK-3 inhibitor) and blebbistatin (inhibitor myosin-II) (eg 2-10 µM, eg 10 µM)). Aggregate formation and subsequent steps are carried out during 24-hour cultivation in ultra-low attachment (ULA) flasks with constant stirring.

The resulting cellular aggregates (i.e., EBs) are then transferred to serum-free differentiation medium (e.g., 50% IMDM, 50% Ham's F12 medium, 100 μg/ml polyvinyl alcohol, 100 μg/ml recombinant human serum albumin, 1x nonessential amino acid supplement (Invitrogen), 0.1x Chemically Specified Lipid Supplement (Invitrogen), 125 µM Magnesium Ascorbic Acid-2-Phosphate, 0.25 µM Linoleic Acid, Micronutrient Supplements A (0.3x), B (0.2x) and C (0.1x) (Corning), 5 mM sodium chloride, 100 μM monothioglycerol, 20 μM ethanolamine, 100 ng/ml heparin and 10 ng/ml IGF1) with the addition of hematopoietic mesoderm-inducing cytokines: 25 ng/ml BMP4, 50 mg/ml VEGF and 50 ng/ml FGF2. Cultivation is continued, for example, for 4 days, with a complete change of medium on the second day.

To support differentiation and expansion of hematopoietic CD34' progenitors, cell aggregates are then transferred to serum-free differentiation medium (as above) supplemented with hematopoietic support cytokines, e.g., 50 ng/ml SCF, 20 mg/ml TPO, 10 ng/ml FLT3L, 20 ng/ml IL-3 and 25 ng/ml BMP4. Cultivation is continued, for example, for 4 days, with a complete change of medium on the second day.

Cultures are harvested after the differentiation process, for example after 9 days. A suspension of individual cells is obtained by digestion of differentiated cell aggregates, for example, with accutase. Isolated CD34+ cells, such as those isolated by MACS, are then plated into T/NK differentiation cultures or cryopreserved for later use within 1 hour of isolation.

(i) An example of a 2D differentiation method.

In an alternative example of the method, PSCs are subjected to a 2D differentiation technique to produce HPCs. First, PSCs are acclimated to hypoxic conditions, for example, for 5-10 passages, in the absence of a feeder, for example, coated with MATRJGEL™ or vitronectin in Essential 8 (E8) medium. PSCs are separated from each other and plated on 6-well plates coated with PureCoat Amine (Corning Inc.) at a density of 25,000/cm in the presence of serum-free, chemically defined medium (SFD) supplemented with 5 μM blebbistatin (e.g., 2-10 μM, for example 10 µM). SFD minimal media may contain 75% IMDM (Invitrogen 12200-069) (with glutamine and 25 mM HEPES+P/S), 25% Ham's F12 medium (Mediatech 10-080-CV), 0.5% N2 supplement (Invitrogen 17502 -048), 1% B27 supplement without retinoic acid (Invitrogen 12587-010, 0.05% BSA, 50 µg/ml ascorbic acid and 4.5 x 10-4 M monothioglycerol with added 50 ng/ml BMP-4, VEGF and bFGF.

Induction of hematopoietic differentiation begins on day 1 by culturing, for example, in SFD minimal medium containing 75% IMDM (Invitrogen 12200-069) (with glutamine and 25 mM HEPES+P/S), 25% Ham's F12 medium (Mediatech 10 -080-CV), 0.5% N2 Supplement (Invitrogen 17502-048), 1% B27 Supplement Without Retinoic Acid (Invitrogen 12587-010), 0.05% BSA, 50 mcg/ml Ascorbic Acid, and 4.5 x 10-4 M monothioglycerol with the addition of 50 ng/ml BMP4, VEGF and bFGF. On day 2, the medium is aspirated and the cells are placed in fresh EB1 medium (e.g. SFD minimal medium containing 75% IMDM (Invitrogen 12200-069) (with glutamine and 25 mM HEPES+P/S), 25% Ham's F12 medium (Mediatech 10 -080-CV), 0.5% N2 supplement (Invitrogen 17502-048), 1% B27 supplement without retinoic acid (Invitrogen 12587-010, 0.05% BSA, 50 mcg/ml ascorbic acid and 4.5 x 10 -4 M monothioglycerol with the addition of 50 ng/ml BMP-4, VEGF and bFGF, for another 48 hours.

On days 5-10, the media is aspirated and the cells are placed in EB2 medium for the next 48 hours. EB2 medium may contain fresh SFD minimal medium containing 75% IMDM (Invitrogen 12200-069) (with glutamine and 25 mM HEPES+P /S), 25% Ham's F12 Medium (Mediatech 10-080-CV), 0.5% N2 Supplement (Invitrogen 17502-048), 1% Retinoic Acid Free B27 Supplement (Invitrogen 12587-010, 0.05% BSA, 50 µg/ml ascorbic acid and 4.5 x 10-4 M monothioglycerol with the addition of 50 ng/ml Flt-3 ligand, IL-3, IL-6, SCF and TPO, each at a concentration of 50 ng/ml, and 5000 units /ml heparin Cells were harvested on days 7, 8, 9, 10 of differentiation using TrypLE and stained for the presence of HPC and lymphoid progenitor markers.

2. Direct programming.

Certain embodiments of the present invention relate to the production of HPCs by directly programming CAR-PSCs through expression of a combination of programming genes important for hematopoietic cell differentiation/function. In one method, the PSC is modified to express at least three hematopoietic progenitor programming genes, such as an ETS gene (eg, ETC2 or ERG), a hematopoietic development gene (eg, GATA2), and a homeobox gene (eg, HOXA9), as described in the application. PCT/US2016/057893, which is incorporated herein by reference in its entirety. In particular aspects, the ETV2/ERG, GATA2 and HOXA9 genes are co-expressed by a single vector, such as the PiggyBac inducible vector, using a bidirectional Tight promoter, which is transfected into CAR-PSCs.

In addition, EGH-CAR-PSCs can be further modified to express additional genes for long-term transplantation potential. Examples of genes include HMGA2, MYCN, NR4A2, SOX17, TFEC, MEIS1, HOXA4, ZNF414, KLF4, ZNF131, BCL2, ETV6, ZNF350 and/or RBAK. For example, PSCs can be transfected with one or more vectors to express HMGA2, MYCN, NR4A2, SOX17, TFEC, MEIS1 and HOXA4.

Preferably, the ETV2/GAT2/HOXA9 genes are expressed only for a period of time sufficient to directly program PSCs into hematopoietic progenitor cells. Accordingly, genes for programming hematopoietic progenitors may be under the control of an inducible promoter. Thus, expression of hematopoietic progenitor programming genes can be induced in PSCs for a period of time sufficient to allow direct programming into multilineage hematopoietic progenitor cells. The time period can be from about 1 day to about 20 days, such as about 3, 4, 5, 6, 7, 8, 9 or 10 days. Alternatively, hematopoietic progenitor programming genes can be introduced into PSCs via an episomal vector. Thus, hematopoietic progenitor programming genes may be transiently expressed in PSCs.

Differentiation of lymphoid cells.

HPCs can then be further differentiated into lymphoid cells, including T cells, NK cells, and T/NK cells. In some aspects, HPCs during differentiation are isolated at day 712, for example, days 8-11, to differentiate into lymphoid cells. NPCs at this stage can be identified by the expression of CD34 and CD43. In addition, HPCs with lymphoid potential can express CD144, DLL4, CD7, and CD235 at low levels, which decrease at day 11, indicating that a certain threshold level of expression of these markers is required for primary cells to differentiate into lymphoid cells in the presence of DLL4.

In some aspects, NPCs isolated on days 7-11, eg, day 7, day 8, day 9, day 10, or day 11 of the differentiation process, can be differentiated into lymphoid cells such as T and NK cells. In some aspects, the timing of the origin of lymphoid progenitors coincides with the decline in hematoendothelial progenitors and the emergence of erythroid progenitors during HPC differentiation. In particular aspects, NPCs on day 9 may have increased efficiency in generating T cells. NPCs capable of lymphoid differentiation can be isolated and/or identified by the expression of certain markers. For example, cells with surface expression of CD34 and/or CD43, in particular those expressing both CD34 and CD43, can be selected for lymphoid differentiation, for example, using MACS sorting.

Additional markers for detecting lymphoid progenitors include DLL4, CD144, CD31, CD34, ^CD43b . CD45 ^lo/- , CD235, CD7, Flk-1, APNLR. In particular, the presence of populations double positive for CD34/CD7, CD235/CD7, DLL4/CD34, DLL4/CD31, DLL4/CD144 or CD34/CD43 is used to identify lymphoid progenitors ¹⁰ . The expression of CD144 on NPCs co-occurs with CD31, CD34 and DLL4. The expression of CD7 on NPCs co-occurs with CD235, CD34 and CD43. Therefore, HPCs coexpressing CD144 and CD7 exhibit lymphoid-potential uptake progenitors expressing membrane-bound notch ligand (DLL4) along with hematoendothelial markers and provide a phenotypic signature for emerging lymphoid progenitors capable of generating hematopoiesis-defining lineages in vitro. In particular, HPCs can be further sorted into cells with increased lymphoid potential by sorting for surface markers, including CD31, CD34, CD144, CD43, CD45, CD6, CD335, Flk-1 and DLL4. In some aspects, fractions of HPCs positive for CCD31, CD34, CD144, CD43, CD45, CD6, CD335, Flk-1, and DLL4 differentiate into lymphoid cells. In particular aspects, CD144/CD7-positive HPC fractions differentiate into lymphoid cells.

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NPCs can be cultured under certain conditions without a feeder for lymphoid differentiation. The culture medium may contain one or more matrix components such as RetroNectin, fibronectin or RGD peptide. Without being limited to any particular theory, it is believed that the matrix component may provide a solid support for the growth of embryonic stem cells. In certain embodiments, the matrix component may be applied to the culture surface and be in contact with the culture medium prior to seeding cells into the medium. For example, cells can be cultured in a chemically defined medium (eg, TeSR medium) on fibronectin or MATRIGEL™ coated plates until the cells are mechanically separated into clumps or single cells and induced to differentiate into hematopoietic progenitor cells.

Various matrix components can be used to culture pluripotent cells, including collagen (eg, collagen IV), laminin, vitronectin, MATRIGEL™, gelatin, polylysine, thrombospondin (eg, TSP-1, -2, -3, -4 and/or - 5) and/or PRONECTIN-F™. In certain embodiments, the use of MATRIGEL™, collagen IV, or laminin alone with cells previously cultured using TeSR may be avoided due to possible adverse effects on cell viability; however, these compositions can be effectively used in combination with other matrix components. Combinations of these matrix components may provide additional benefits for promoting cell growth and cell viability. In certain embodiments, 1, 2, 3, 4, 5, 6 or more of the above matrix components may be used to culture cells, for example, prior to hematopoietic differentiation.

In some embodiments, ascorbic acid may be used to enhance lymphoid differentiation. About 10 μM to about 1 mM ascorbic acid, for example, 100 to 500 μM, for example, about 50 μM to about 100 μM, for example, about 95 μM, can be added to a chemically defined medium. The ascorbic acid may be selected from various ascorbates, for example, magnesium salt of ascorbic acid phosphate. In some embodiments, nicotinamide (eg, nicotinic acid) can be used to enhance lymphoid differentiation, for example, at a concentration of from about 0.1 mM to about 5 mM.

In some aspects, HPCs differentiate into lymphoid cells, such as T cells, by altering endogenous Notch ligand activity by administering a substance that increases Notch ligand production in a subject. The method also includes culturing cells in a medium comprising an effective amount of notch ligand and one or more cytokines selected from the group consisting of IL7, IL-15, SCF, Flt-3 and IL-3. In some particular embodiments, the medium may further include IL-6. In some embodiments, the notch ligand is notch delta ligand 4 (DLL4), such as a DLL4:Fc chimera.

A Notch ligand is selected that stimulates and supports differentiation and proliferation of cells of the T cell lineage. The Notch ligand may be of human origin or may be derived from other species, including mammalian species such as rodent, dog, cat, pig, sheep, cow, goat and primates. Specific examples of Notch ligands include the delta family. The delta family includes delta-1 (Genbank ID AF003522, Homo sapiens), delta-3 (Genbank ID AF084576, Rattus norvegicus), delta-like 1 (Genbank ID NM-005618 and NP-005609, Homo sapiens; ID Genbank X80903, 148324, M. musculus), delta-like 3 (Genbank ID NM-053666, N446118, Rattus norvegicus), delta-4 (Genbank ID AF273454, BAB18580, Mus musculus; Genbank ID AF279305, AAF8 1912, Homo sapiens) and delta-like 4 (Genbank ID Q9NR61, AAF76427, AF253468, NM-019074, Homo sapiens; Genbank ID NM-019454, mus musculus). Notch ligands are commercially available or can be produced by recombinant DNA methods and purified to varying degrees.

The method further includes the step of maintaining HPC cells in culture as described above for a period of time sufficient to produce differentiated NK cells. In some embodiments, differentiated NK cells appear in cultures along with T cells, however, NK cells may stop proliferating after week 6. Typically, determination of the increase in the number of NK cells and/or their differentiation state is assessed using conventional methods known to those skilled in the art. For example, cultured cells can be monitored by flow cytometry for NK cell development by staining the cells with anti-CD56 antibodies and anti-CD3 antibodies. An anti-CD3 antibody such as OKT3 may be present at a concentration of 0.5 to 2 μg. Cells that are CD567CD3 ^- . will indicate differentiated NK cells.

In particular aspects, lymphoid differentiation into CD3+ T cells or CD56+CD3 ^- NK cells is carried out through 2D cultivation under hypoxic conditions on retro-coated plates

- 30 046022 nectin and DLL4. In particular aspects, the treated non-tissue culture plate can be coated with a chimera of DLL4:Fc and RetroNectin (fibronectin fragment CH-296; Takara Shuzo, Japan) for use in lymphoid differentiation of NPCs. Differentiation may include a first period of differentiation into T/NK cells, followed by a second period of T or NK cell expansion. The first differentiation period can be from about 1 week to about 2 weeks, and the second multiplication period can also be from about 1 week to about 2 weeks. Thus, the entire period of lymphoid differentiation and proliferation may be approximately 2-4 weeks.

In some embodiments, the lymphoid differentiation process may include co-culture with antigen-specific target cells, such as antigen-specific tumor cells, to enhance the cytotoxic activity of T cells and NK cells. In particular aspects, the expansion period during lymphoid differentiation may include co-culture with antigen-specific target cells. For example, CD19 CAR T cells or NK cells can be co-cultured with CD19+ tumor cells (eg, CO19+ Daudi cells) for an expansion period, for example, one to three weeks, particularly two weeks. In some aspects, the propagation period may further include the addition of cytokines such as IL-2, IL-15 and/or IL-21, particularly IL-2, to enhance propagation. The cytokine concentration can be optimized, for example, to 10 to 50 μM.

Cell culture.

In certain embodiments, conditions of significant hypoxia may be used to promote differentiation of HPCs into myeloid or lymphoid lineages. In certain embodiments, an atmospheric oxygen content of less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14% less than about 13%, less less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, about 5%, about 4%, about 3% , about 2% or about 1%, can be used to stimulate differentiation into hematopoietic progenitor cells. In some embodiments, the hypoxic atmosphere contains approximately 5% oxygen gas.

As described herein, one or more culture media with a specific chemical composition can be effectively used to stimulate the differentiation of HPC into myeloid and lymphoid lineages; in particular, eliminating animal products such as whey and mouse feeders may reduce the risks associated with cellular exposure to animal products and produce cells that can be more safely administered to humans. Because traditional stem cell culture development relied on serum products and murine feeder beds to differentiate stem cells into different cell types, these traditional techniques limited the scale on which differentiation could be carried out, increased biological variability and potential contamination, and greatly hampered the use of ES cells in translational therapies. , in which they might otherwise be useful.

Typically, the cells of the present invention are cultured in a culture medium, which is a nutrient-rich buffered solution capable of supporting cell growth. Culture media suitable for the isolation, expansion and differentiation of pluripotent stem cells into hematopoietic progenitor cells and hematopoietic cells according to the method described herein include, but are not limited to, Dulbecco's modified Eagle's medium (DMEM), DMEM/F-15, RPMI 1640, Iscove's modified Dulbecco's media (IMDM), and Opti-MEM SFM (Invitrogen Inc.). Chemically defined media includes minimum essential media such as Iscove's Modified Dulbecco's Medium (IMDM) (Gibco) supplemented with human serum albumin, ExCyte human lipoprotein, transferrin, insulin, vitamins, essential and non-essential amino acids, sodium pyruvate, glutamine, and mitogens are also suitable. As used herein, the term mitogen refers to an agent that stimulates cell division. The agent may be a chemical, usually some form of protein, that stimulates cell division, causing mitosis. In one embodiment, serum-free media, such as those described in US Pat. No. 08/464,599 and WO 96/39487, and complete media, as described in US Pat. No. 5,486,359, are contemplated for use in the methods of the present invention. In some embodiments, culture media medium supplemented with 10% fetal bovine serum (FBS), human autologous serum, human AB serum, or platelet-rich plasma supplemented with heparin (2 U/ml).

Immune cells can be obtained by culturing pluripotent stem cells or hematopoietic progenitor cells in a medium under conditions that increase intracellular

- 31 046022 level of factors sufficient to stimulate the differentiation of cells into myeloid or lymphoid lineages. The medium may also contain one or more hematopoietic cell differentiation and maturation agents, such as various types of growth factors. These agents may either help induce cells to adopt a more mature phenotype, or preferentially promote the survival of mature cells, or a combination of both. Differentiation and maturation agents may include soluble growth factors (peptide hormones, cytokines, ligand-receptor complexes, and other compounds) capable of stimulating the growth of cells of the hematopoietic cell line. Non-limiting examples of such agents include, but are not limited to, hematopoietic or endothelial growth factors such as fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), stem cell factor (SCF), thrombopoietin (TPO), FLT-3 ligand ( FLT3L), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-9 (IL-9) or granulocyte colony-stimulating factor (G-CSF), or isoforms or variants thereof.

Application of antigen-specific immune cells.

The antigen-specific immune effector cells provided by the methods and compositions of the individual aspects can be used in a variety of applications. These include, but are not limited to, in vivo cell transplantation or implantation; screening of cytotoxic compounds, carcinogens, growth factors/mutagen regulation, pharmaceutical compounds, etc. in vitro; elucidation of the mechanism of hematological diseases and injuries; studying the mechanism of action of drugs and/or growth factors; diagnosis and monitoring of cancer in a patient; gene therapy; and the production of bioactive products, to name a few.

Screening of Test Compounds

The immune cells of the present invention can be used to screen for factors (such as solvents, small molecule drugs, peptides and polynucleotides) or environmental conditions (such as culture conditions or manipulation) that affect the characteristics of the lymphoid cells provided herein.

Specific screening applications of the present invention relate to the testing of pharmaceutical compounds in drug research. The reader is referred to general information in the standard textbook In vitro Methods in Pharmaceutical Research, Academic Press, 1997, and US Patent No. 5,030,015. In certain aspects, myeloid and lymphoid cells serve as test cells for routine drug screening and toxicity assays previously performed on hematopoietic cells and precursors in a young culture. Assessing the activity of candidate pharmaceutical compounds typically involves combining hematopoietic cells or progenitors as proposed in certain aspects with the candidate compound, determining any change in morphology, marker phenotype, or metabolic activity of cells associated with the compound (compared to untreated cells or cells treated with an inert compound) and then relating the effect of the compound to the observed change. Screening may be performed either because the compound is intended to have a pharmacological effect on hematopoietic cells or progenitors, or because the compound intended to have some other effect may have an undesirable effect on hematopoietic cells or progenitors. Two or more drugs can be tested in combination (by combining with cells either simultaneously or sequentially) to detect possible drug-drug interaction effects.

Adoptive cell therapy

In some embodiments, the present invention relates to methods of immunotherapy comprising administering an effective amount of immune cells according to the present invention. In one embodiment, a medical disease or disorder is treated by transferring a population of immune cells that induce an immune response. In certain embodiments of the present invention, cancer or infection is treated by transferring a population of immune cells that produce an immune response. The present application provides methods for treating or delaying the progression of 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 and viral infections.

Tumors for which the present treatment methods are suitable include any type of malignant cell, such as those present in a solid tumor or hematologic tumor. Examples of solid tumors may include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate and breast. Examples of hematological tumors include bone marrow tumors, Tili B-cell leukemias, leukemias, lymphomas, blastomas, myelomas and the like. Additional examples of cancers treatable using the methods provided herein include, but are not limited to, lung cancer (including small cell carcinoma

- 32 046022 lung, non-small cell lung cancer, lung adenocarcinoma and squamous cell lung carcinoma), peritoneal cancer, stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, cancer liver, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer and melanoma.

Cancer may, for example, have the following histological type, although it is not limited to the above: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; 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; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial coli-polyposis; solid carcinoma; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophilic carcinoma; oxyphilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenal cortex carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrative ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extrathoracic paraganglioma malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant lentigo melanoma; acral lentiginous melanomas; nodular melanomas; malignant melanoma in giant pigmented nevi; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; Müllerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; Brenner tumor, malignant; phyllodes cystosarcoma, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; ovarian goiter, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other characterized non-Hodgkin's lymphomas; B cell lymphoma; non-Hodgkin's lymphoma (NHL) low-grade/follicular; small lymphocytic (SL) NHL; Moderate/follicular NHL; diffuse NHL of moderate malignancy; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; small cell NHL with undissected nuclei of high grade; massive NHL disease; mantle cell lymphoma; AIDS-associated lymphoma; Waldenström's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative diseases of the small intestine; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; leukemia from lymphosarcoma cells; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic

- 33 046022 leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia.

To determine the suitability of the cells provided herein for therapeutic use, the cells may first be tested in a suitable animal model. At one level, cells are assessed by their ability to survive and maintain their phenotype in vivo. The cells provided herein are administered to immunodeficient animals (such as NOG mice or animals rendered immunodeficient by chemical exposure or radiation) at a site amenable to further observation, such as under the renal capsule, into the spleen, into the liver lobule, or into the bone marrow. Tissues are collected after several days to several weeks or more, and assessed whether the original cell types, such as red blood cells, are still present. This can be accomplished by labeling the injected cells with a detectable label (such as green fluorescent protein or β-galactosidase); or by measuring a constitutive marker specific to the injected human cells. If the cells provided herein are tested in a rodent model, the presence and phenotype of the introduced cells can be assessed by immunohistochemistry or ELISA using anti-human antibodies, or by real-time PNR analysis using primers and hybridization conditions that provide amplification, specific for human polynucleotide sequences. Suitable markers for assessing gene expression at the mRNA or protein level are presented elsewhere in this application.

Immune cells produced by the methods of the present invention can be tested in various animal models for their ability to treat hematological disorders and injuries. For example, a mouse model of sickle cell disease or a T/B cell-deficient Rag-2 knockout mouse may be particularly suitable animal models for testing the myeloid and lymphoid cells described herein.

Immune cells provided in certain aspects of the present invention that demonstrate desirable functional characteristics or efficacy in animal models may also be suitable for direct administration to humans in need thereof. For hemostasis purposes, cells can be injected anywhere that has sufficient access to the blood circulation. Hematopoietic cells or their precursors can also be delivered to the site of injury or disease.

The cells provided in certain aspects of the present invention can be used to treat any subject in need thereof. Human conditions that may be suitable for such therapy include various anemias and hemoglobinopathies, as well as diseases characterized by a decrease in the number of hematopoietic cells (such as, for example, myelodysplastic syndrome, myelofibrosis, neutropenia, agranulocytosis, Glanzmann's thrombasthenia, thrombocytopenia and acquired immunodeficiency syndrome). For human therapy, the dose is typically about 10 ⁹ to 10 ¹² cells and typically about 5 x 10 ⁹ to 5 x 10 ¹⁰ cells, depending on the body weight of the subject, the nature and severity of the lesion, and the replicative capacity of the cells administered. The final responsibility for determining the treatment regimen and appropriate dose rests with the attending physician.

Therapeutically effective amounts of immune cells can be administered by several routes, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intrasternal or 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 the subject being treated. For example, it may be the number of immune cells needed to inhibit the development or regression of an autoimmune or alloimmune disease, or to reduce symptoms caused by an autoimmune disease, such as pain and inflammation. It may also 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 population of immune cells can be administered in treatment regimens appropriate to the disease, for example, in one or more doses over one to several days to improve the disease, or in intermittent doses over an extended period of time to inhibit disease progression and prevent disease relapse. The exact dose to be used in the drug will also depend on the route of administration and the severity of the disease or disorder, and should be selected according to the judgment of the practitioner and the specifics of each patient. The therapeutically effective number of immune cells will depend on the subject being treated, the severity and type of lesion, and the route of administration. In some embodiments, dosages that may be used in treating humans range from at least 3.8 x 10 ⁴ , at least 3.8 x 10 ⁵ , at least 3.8 x 10 ⁶ , at least 3.8 x 10 ⁷ , at least at least 3.8x10 ⁸ , at least 3.8x10 ⁹ or at least 3.8x10 ¹⁰ immune cells/m ² . In a particular embodiment, the dose used in treating humans is from about 3.8 x 10 ⁹ to about 3.8 x 10 ¹⁰ immune cells/m ² . In additional embodiments, the therapeutically effective number of immune cells can be from about 5 x 10 ⁶ cells per

- 34 046022 kg body weight to approximately 7.5 x 10 ⁸ cells per 1 kg body weight, for example, from approximately 2 x 10 ⁷ cells to approximately 5 x 10 ⁸ cells per 1 kg of body weight, or from approximately 5 x 10 ⁷ cells to approximately 2 x 10 ⁸ cells per 1 kg of body weight. One skilled in the art can easily determine the exact number of immune cells based on the age, body weight, gender and physiological condition of the subject. 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-related disorder. Combination therapy may include, but is not limited to, one or more antimicrobial agents (eg, antibiotics, antivirals, and antifungals), antineoplastic agents (eg, fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressive agents (eg, , fludarabine, etoposide, doxorubicin or vincristine), immunosuppressants (for example, azathioprine or glucocorticoids such as dexamethasone or prednisone), anti-inflammatory drugs (for example, glucocorticoids such as hydrocortisone, dexamethasone or prednisone, or non-steroidal anti-inflammatory drugs such as acetylsalicylic acid, ibuprofen or naproxena sodium), cytokines (eg, interleukin-10 or transforming growth factor beta), hormones (eg, estrogen), or a vaccine. In addition, immunosuppressive or tolerogenic agents may be administered, including, but not limited to, calcineurin inhibitors (eg, cyclosporine and tacrolimus); mTOR inhibitors (eg rapamycin); mycophenolate mofetil, antibodies (eg, recognizing CD3, CD4, CD40, CD154, CD45, IVIG or B cells); chemotherapeutic agents (for example, methotrexate, treosulfan, busulfan); irradiation; or chemokines, interleukins or their inhibitors (eg, BAFF, IL-2, anti-IL-2R, IL-4, JAK kinase inhibitors). Such additional pharmaceutical agents may be administered before, during, or after administration of the immune cells, depending on the desired effect. Such administration of cells and agent may be carried out by the same route or by different routes, and at the same site or at a different site.

Pharmaceutical compositions.

The present application also provides pharmaceutical compositions and preparations containing immune cells (eg, T cells or NK cells) and a pharmaceutically acceptable carrier. Administration may be autologous or non-autologous. For example, T cells and/or NK cells and compositions containing them can be obtained from one subject and administered to the same subject or another compatible subject. The immune cells of the present invention can be administered by local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When administered, a therapeutic composition of the present invention is typically provided in a unit dosage form for injection (eg, solution, suspension, emulsion).

Pharmaceutical compositions and preparations as described herein can be prepared by admixing active ingredients (such as an antibody or polypeptide) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences ^22nd edition, 2012), in the form of lyophilized preparations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include, but are not limited to, buffers such as phosphate, citrate and other organic salts; antioxidants including ascorbic acid and methionine; preservatives (eg, octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (containing less than 10 residues) polypeptides; 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; complexing agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal-containing complexes (for example, Zn-protein complexes) and/or nonionic surfactants, for example, polyethylene glycol (PEG). Examples of pharmaceutically acceptable carriers herein further include agents for intercellular drug dispersal, e.g., soluble neutral active hyaluronidase glycoproteins (sHASEGP), e.g., soluble human hyaluronidase glycoproteins PH-20, e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.). Some examples of sHASEGP and their uses, including rHuPH20, are described in US Patent Application Publications Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

- 35 046022

Examples

The following examples are included to demonstrate embodiments of the invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples are techniques that have been found by the inventors to function adequately in the practice of the present invention and, thus, may be considered preferred methods for implementing the same. However, it will be clear to those skilled in the art in light of this disclosure that many changes can be made to the specific embodiments described without departing from the spirit and scope of the invention to achieve the same or similar results.

Example 1: Generation of PSC-derived T/NK cells expressing anti-CD19 CAR.

Two separate methods were used to generate pluripotent stem cells expressing chimeric antigen receptor (CAR). In one method, transgene-free PSCs are generated from T cells using retroviral vectors to produce 1C cells (US20160257939; incorporated herein by reference in their entirety) or episomal vectors to produce E11 cells. In the second method, PSCs were transfected with the PiggyBac expression vector encoding the hematopoietic programming genes ETV2/ERG, GATA2 and HOXA9 (designed H1 ESCs with introduced DOX-inducible hematopoietic programming genes ETV2-GATA2-HOXA9) to obtain A16 cells. PSCs generated by both methods were then genetically modified to constitutively express a second-generation chimeric antigen receptor (CAR) to human CD19, consisting of an scFv domain derived from the human CD19-binding mAb FMC63, a CD28 co-stimulatory domain, and a CD3Z signaling domain.

Unmodified and CAR-modified PSCs were differentiated into CD34' T/NK progenitors by cytokine-directed differentiation (1C, E11) or EGH-induced programming (A16) (Fig. 1A-B). Isolated CD34+ progenitors were further differentiated into CD3+ T- (1C, E11) and CD3-CD56+ NK cells (A16) using a 4-week hypoxic culture on DLL4/retronectin-coated plates in StemSpan SFEM (Stem Cell Technologies) supplemented with ascorbic acid. magnesium acid and phosphate (0.25 mM), nicotinamide (2 mM) and cytokines (SCF, TPO, FLT3L, IL-7, IL-2). Mitomycin C-treated Daudi cells (a line of CD19+ Blymphoblastoid cells negative for HLA class I, deficient in β2-microglobulin) were added to parallel T-/NK cell cultures during the last 2 weeks of differentiation as a source of antigen (CD19)-specific activation CAR. The generated T/NK cells were designated as follows: 1C- or E11-derived T cells - T, CAR-T, CAR-T/Ag (Daudi co-culture); A16-derived NK cells - NK, CAR-NK, CAR-NK/Ag (Daudi co-culture).

CAR expression at differentiation stages was assessed by flow cytometry using protein L staining. CD3 ⁺ T cells were co-stained with a lambda chain mouse mAb to human CD3 (clone SP34-2). After differentiation into T/M<-cells, CAR expression was significantly suppressed, but CAR-positive T and NK cells could be selectively expanded after culture activation with CAR-specific antigen (Ag; co-culture with CD19' Daudi cells) .

The cytotoxic function of PSC-derived T (1C) and NK (A16) cells was assessed using an in vitro cytotoxicity assay using CD19' luciferase-expressing Daudi and Raji target cells. Targets were incubated with T/NK effectors for 24 h at a 1:1 ratio, and luciferase activity was quantified in culture lysates using the Steady-Glo Luciferase Assay System (Promega). The percentage of cytotoxicity was calculated using the formula: (1- (ET/T)) x 100, where ET is the culture of effectors + targets, T is targets only.

While unmodified T/NK cells did not have any detectable activity, both CAR-expressing T (1C) and NK cells (A16) demonstrated cytotoxicity toward CD19+ targets. CAR-dependent cytotoxic activity was significantly enhanced after 2 weeks of co-culture of T/NK with CD19 ⁺ (Daudi) target cells (CAR/Ag variant) ( Fig. 3A ).

The cytolytic potential of CAR-T cells was confirmed by real-time target cell counting using the Incucyte S3 Live Cell Assay System (Essen Bioscience). GFP-expressing CD19 ⁺ target Raji cells were incubated with unmodified and CAR-transfected PSC-derived (E11 TiPSC) T cells at a 1:1 ratio. Time-lapse imaging and counting of GFP ⁺ Raji cells was performed for 9 hours. A significant decrease in viable (GFP ⁺ ) Raji cells was detected in culture with CAR-T cells only (Figure 3B).

CAR-T cells derived from 2 weeks of T/NK differentiation followed by 1 week of anti-CD3 antibody-induced expansion of a PSC-derived (E11 TiPSC) CD34 ⁺ HPC T cell culture were incubated for 24 hours with murine mastocytoma cells P815 transfected with human CD19 antigen (CD19 ⁺ P815) and non-transfected P815 to assess CAR-induced and constitutive cytokine production, respectively. Cytokines were measured in culture supernatants using the LEGENDplex Flow Cytometry Multiplex Cytokine Assay (BioLegend) (Figure 4). CAR-T cells showed specific CAR-induced production of LFNy, TNFa, IL-2, IL-13 and GM-CSF. The cytotoxic properties of PSC-derived CAR-T cells were also confirmed by the constitutive secretion of granzyme B.

To test the efficacy of CAR-T cells in vivo, 8-week-old NSG mice were injected intraperitoneally (i.p.) with 5 x 10 ⁴ luciferase-expressing Raji cells. The next day (day 1), T cells (derived from 1C) and NK cells (derived from A16) were injected (i.p.) at a dose of 10 ⁷ /mouse. T/NK injections were repeated on days 3 and 5. During T/NK injections (days 1–5), all mice were additionally injected (i.p.) with a combination of the cytokines IL-2 and IL-15 (both at a dose of 500 ng/mouse). Tumor progression in mice was monitored using in vivo bioluminescence imaging every 2 weeks (Fig. 5A). Anesthetized mice injected (i.p.) with 150 mg/kg InVivo-Glo luciferin (Promega) were analyzed within 15 min of luciferin administration using a Pearl Trilogy in vivo imaging device (LiCor). Survival curves for different groups of mice treated with PSC-derived T (1C) or NK cells (A16) are shown in Fig. 5V.

Tumors detected after 2 weeks were significantly reduced, and the average survival of mice was prolonged by ~2-fold with a single course of treatment (3 injections at 2-day intervals) with CAR-expressing PSC-derived T or NK cells. Oncolytic potential in vivo can be significantly improved by pre-coculturing CAR-expressing PSC-derived T or NK cells with antigen-positive tumor cells in vitro.

Example 2. Methods of directed differentiation.

Differentiation of PCS into CD34 ⁺ lymphohematopoietic progenitors: The T cell-derived PSCs from Example 1 (TiPSC 1C and E11, derived from peripheral blood T cells by retroviral and episomal reprogramming, respectively) were differentiated into CD34' hematopoietic progenitors by suspension culture of aggregates. PSCs were maintained under feeder-free conditions in 6-well plates coated with Matrigel™ or vitronectin in Essential 8 (E8) medium. Aggregates were prepared from subconfluent PSCs (<80% confluent) at a density of 0.5 million cells per ml in E8 medium supplemented with 2 μM CHIR99021 (GSK-3 inhibitor) and 5 μM blebbistatin (myosin II inhibitor). The formation of aggregates was carried out during 6 h of cultivation in ultra-low attachment (ULA) flasks with continuous stirring on a shaking platform at 15-20 rpm (including all subsequent cultivation stages).

The culture with preformed cell aggregates was gradually transferred to serum-free hematopoietic differentiation medium (HDM: 50% LMDM, 50% Ham's F12 medium, 100 μg/ml polyvinyl alcohol, 100 μg/ml recombinant human serum albumin, 1x essential amino acid supplement (Invitrogen) , 0.1x chemically defined lipid supplement (Invitrogen), 125 µM magnesium ascorbic acid-2-phosphate, 0.25 µM linoleic acid, trace element supplements A (0.3x), B (0.2x) and C ( 0, 1x) (Corning), 5 mM sodium chloride, 100 μM monothioglycerol, 20 μM ethanolamine, 100 ng/ml heparin and 10 ng/ml IGF1) supplemented with 2 μM CHIR99021, 50 ng/ml VEGF and 50 ng/ml FGF2 by adding equal volumes after 6 and 24 hours. On day 2, cell aggregates were precipitated by sedimentation for 15 minutes, the medium was aspirated and the cultures were transferred to HDM with the addition of cytokines that induce hematopoietic mesoderm - 25 ng/ml BMP4, 50 mg/ml VEGF and 50 ng/ml FGF2. Cultivation was continued for 3 days with a complete change of medium daily.

To support differentiation and proliferation of hematopoietic CD34+ progenitors, cell aggregates were further transferred into HDM with the addition of hematopoietic supporting cytokines - 50 ng/ml SCF, 50 ng/ml TPO, 20 ng/ml FLT3L and 20 ng/ml IL-3. Cultivation was continued for 3-5 days with a complete change of medium daily. To improve the transition process into hematopoietic cells, the following enhancing components can be added: LL-11 (5-20 ng/ml), 8BrcAMP (100-300 μM) and/or VEGF (20-50 ng/ml).

HPCs, identified in suspension as floating individual cells or small clusters, were collected after a 2+3+3-5 day differentiation process (total of 8-10 days). HPCs were isolated by filtering the entire differentiation culture through 70- and 30-μm cell sieves (Corning). HCPs collected from the filtrate by centrifugation were washed once in HDM and resuspended in TCDM (T cell differentiation medium). To enrich HPCs for T/NK differentiation potential, isolation of the CD34 ⁺ HPC fraction can be performed by magnetically activated cell sorting (MACS) using direct CD34 microbeads (Myltenyi Biotec) according to the manufacturer's recommendations. HPCs were plated onto T/NK differentiation cultures or cryopreserved for later use within 1 h of isolation.

T/NK Differentiation Cultures: For T/NK differentiation, treated plastic nontissue culture plates were coated with Notch ligand hDLL4-Fc chimeric protein and retronectin diluted in PBS (0.5 μg/cm each). Before cell seeding, the coating solution was aspirated, the plates were washed once with PBS and filled with 0.25 ml/cm ² T-cell differentiation medium (TCDM), consisting of StemSpan SFEM (Stem Cell Technologies), GlutaMax (1/100), ascorbic acid and magnesium phosphate (250 μM), nicotinamide (2 mM) and cytokines SCF, TPO, FLT3L and IL7 (50 ng/ml each). PSC-derived NPCs were seeded at a density of 5000 cells/cm and cultured under hypoxic conditions (5% O ₂ ) in a CO ₂ incubator for 2 weeks with the addition of an equal volume of TCDM culture on day 3 and replacement of half the culture volume every other day. All differentiated cells were collected by gentle resuspension and collection of unattached cells, followed by collection of poorly attached cells for 5–10 min of PBS-EDTA (0.5 mM) treatment. To continue the differentiation process and improve T/NK cell yield, T cell differentiation can be repeated by replating the collected cells into freshly prepared DLL4/retronectin plates at a density of 10,000 cells/cm until the desired T/NK cell yield is achieved. cells. The efficiency of T/NK differentiation can also be improved by adding the following enhancing components to TCDM: CHIR99021 (1-3 μM), IL-2 (2-10 ng/ml), IL-12 (10-50 ng/ml).

T cell expansion cultures: For T cell expansion, treated non-tissue culture plates were coated with anti-CD3 mAb (clone OKT3), hDLL4-Fc, and retronectin diluted in PBS (0.5 μg/cm each). Before cell seeding, the coating solution was aspirated, the plates were washed once with PBS and filled with 0.25 ml/cm T cell expansion medium (TCDM) consisting of StemSpan SFEM (Stem Cell Technologies), Glutamax (1/100), ascorbic acid magnesium acid and phosphate (250 μM), nicotinamide (2 mM) and cytokines SCF, TPO, FLT3L, IL7 (50 ng/ml each) and IL2, IL15 (10 ng/ml each). IL-21 (5-20 ng/ml) can also be added to improve reproduction. Cells collected from T/NK differentiation cultures were seeded at a density of 10,000 cells/cm and cultured under hypoxic conditions (5% O2) in a CO2 incubator for up to 2 weeks with the addition of an equal volume of TCEM culture on day 3 and replacing half the volume of culture every other day. Expanded T cells were collected by gentle resuspension and collection of unattached cells.

Example 3. Characteristics of the method of directed differentiation.

PSCs were first differentiated into CD34+ NPCs in suspension culture with cell aggregates through sequential steps of WNT-induced differentiation preparation, mesoderm induction, and NPC differentiation over 8–10 days ( Fig. 1B ). The NPC fraction was isolated by filtration. MAC sorting for CD34+ HPCs was not performed, although it can be performed using direct paramagnetic CD34 beads (Myltenyi Biotec) if desired. NPCs were then transferred to human DLL4-Fc ⁺ retronectin-coated plates to differentiate into T/NKs for 24 weeks. T cells can be further expanded for 1-2 weeks in culture on plates coated with anti-CD3 mAb (clone OKT3).

PSC-derived (1C TiPSC) CD34+ cells, after 2 weeks of T/NK differentiation, formed a typical lymphoid cell population defined by low FSC/SSC parameters (Fig. 1D, left dot plot). This lymphoid population contained primarily CD3 ⁺ T and CD56 + CD3 ⁻ NK cells ( Fig. 1D , middle dot plot). The T cell population included CD4 ⁺ and CD8 ⁺ single- and double-positive cells, as well as a significant proportion of double-negative cells ( Fig. 1D , right dot plot).

The yields of each corresponding cell type were calculated as the ratio of the obtained absolute numbers of cells to the initial ones at each stage of cell production. For example, a yield of 1.5 CD34+HPC indicates that an average of 1.5 (resulting amount) CD34 ⁺ HPC can be obtained from 1 (initial amount) PSC. Accordingly, a yield of 102 T cells indicates that 102 (resulting number) T cells can be obtained from 1 (initial number) CD34 ⁺ HPC (Fig. 1E).

PSC-derived (1C TiPSC) T cells (CD3 ⁺ ), differentiated and expanded for 4 weeks, expressed α/β TCR (not γ/δ or invariant Va24 NKT TCR) and typical T cell markers CD5, CD27, and CD7 (Fig. .1F). They also expressed markers associated with cytotoxic T/NK cells (CD161, CD94) and activation markers (CD69).

Immobilized anti-CD3 (iCD3) antibodies were minimally necessary and sufficient to achieve expansion of PSC-derived T cells ( Fig. 1G , bar graph). Stimulation in solution with anti-CD3 and anti-CD28 mAbs (sCD3, sCD28) was not effective either alone, in combination (sCD3 ⁺ sCD28), or when added to iCD3 (iCD3 ⁺ sCD28). T cells proliferating in expansion culture acquire the characteristic irregular lymphoblast morphology (Figure 1G, photograph). In contrast to the relatively heterogeneous population of parent cells, cells collected after 2 weeks of T cell expansion were essentially pure CD3 ⁺ T cells that also expressed CD56 and acquired CD8 expression ( Fig. 1G , flow cytometry dot plots). Thus, the directed differentiation method makes it possible to efficiently obtain T cells.

All methods disclosed and claimed herein can be obtained and performed without undue experimentation in light of the present disclosure. Although compositions and methods with

- 38 046022 according to the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the methods, as well as the steps or sequence of steps of the methods described herein, may include variations without departing from the concept, spirit and scope inventions. More specifically, it will be apparent that certain agents that are both chemically and physiologically related can be replaced by the agents described herein and the same or similar results will be achieved. All such like 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 by the appended claims.

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Claims (4)

US Patent No. 8372642 US Patent No. 8741648 US Patent Application Publication No. 20020055144 US Patent Application Publication No. 20050260186 US Patent Application Publication No. 20060104968 US Patent Application Publication No. 20090148425 US Patent Application Publication No. 20090246875 US Patent Application Publication No. 2010/0210014 Publication US patent application no. 20120276636 US Patent Application Publication No. 2014/0315304 US Patent Application Publication No. 20160257939 Wilson et al., Nature Reviews Immunology, 9: 91-105, 2009 Wilson et al., Science, 244:1344-1346, 1989. Wong et al., Gene, 10:8794, 1980. Wynn, Nature Immunology, 6:1069-1070, 2005. Xu et al., Nat. Biotechnol., 19:971-974, 2001. Yamanaka et al., Cell, 131(5):861–72, 2007. CLAIM 1. A method for producing antigen-specific effector T cells and/or NK cells, comprising the steps of: (1) engineering pluripotent stem cells (PSC) to express a chimeric antigen receptor (CAR), thereby obtaining CAR-PSC; (2) differentiation of CAR-PSCs into CD34+ hematopoietic progenitor cells (HPCs); (3) further differentiation of CD34+ HPCs into T cells and/or NK cells; and (4) expansion of T cells and/or NK cells, including co-culture with antigen-specific target cells, thereby obtaining antigen-specific effector T cells; and /or NK cells, where step (2) involves conducting directed differentiation, including: (i) formation of embryoid bodies (EB) in the presence of blebbistatin, an inhibitor of GSK-3, FGF2 and VEGF; (ii) contacting EBs with BMP-4, VEGF and FGF2 to stimulate mesoderm induction; and (iii) differentiating EBs in the presence of Flt-3 ligand, IL-3, SCF and TPO, thereby producing NPCs. 2. Method according to claim 1, where: (a) PSCs in step (1) are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), where optionally the iPSCs are reprogrammed from T cells; (b) The T cells are CD4' T cells, CD8' T cells, cytotoxic T cells, regulatory T cells, natural killer T cells, naïve T cells, memory T cells, or gammadelta T cells ; and/or (c) differentiation according to step (2iii) includes: (A) EB differentiation occurs essentially in the absence of BMP4; or (B) EB differentiation carried out in the presence of IL-11, cAMP and/or VEGF. 3. The method according to claim 1 or 2, wherein the PSCs are substantially free of transgenes, optionally where the PSCs are human. 4. Method according to claim 1, where: (a) the PSC in step (1) further comprises engineering the PSC to express ERG/ETV2, GATA2 and HOXA9 under the control of a single inducible promoter, optionally further comprising engineering the PSC to express HMGA2, MYCN, NR4A2, SOX17, TFEC, MEIS1 and HOXA4, optionally, wherein the programming according to step (2) further includes inducing expression of ERG/ETV2, GATA2 and HOXA9 for a period of time sufficient to obtain HPC, and stopping the induction of expression before the start of step (3); - 42 046022 (b) the method involves culturing cells under certain conditions without a feeder; (c) step (2) further comprises selecting cells expressing CD34 and CD43 prior to differentiation into antigen-specific T cells and/or NK cells, optionally wherein: (i) selection involves performing magnetically activated cell sorting (MACS); (ii) cells expressing CD34 and CD43 constitute at least 35% of the total cell population; or (iii) cells expressing CD34 and CD43 constitute at least 65% of the total cell population; (d) differentiation according to step (3) involves culturing HPC on a surface coated with retronectin and Notch DLL-4, in the presence of ascorbic acid and nicotinamide under hypoxic conditions, optionally where: (i) the culture further comprises SCF, FLT-3 ligand, TPO and IL-7, optionally wherein the culture further contains a GSK-3 inhibitor, IL-2 and/or IL-12; (ii) at least 1.5% of differentiated CD34' NPCs are CD3+CAR+ T cells; (iii) at least 2% of differentiated CD34' HPCs are CD3 CAR' NK cells; or (iv) at least 10% of the expanded CD34' HPCs are CD3 CAR' NK cells; and/or (f) expansion according to step (4) further includes culturing antigen-specific T cells in the presence of: (i) antibodies to CD3, IL-2 and IL-15, optionally, wherein at least 2% of the expanded CD34+ HPCs are CD3 ^' CAR ^' T cells; (ii) antibodies to CD3, IL-2, IL-15 and IL-21 or (iii) antibodies to CD3, FLT3 ligand, IL-7, IL-2, IL-15 and/or IL-21, optionally, additionally including one or two additional components selected from the group consisting of SCF and SRW. 5. Method according to claim 1, where: (a) at least 5% of HPCs express CAR; (b) at least 10% of HPCs express CAR; or (c) at least 15% of HPCs express CAR. 6. Method according to claim 1, where: (a) the CAR and the antigen-specific target cells are directed to the same antigen, optionally where the antigen is CD19; (b) the antigen-specific target cells are tumor cells; or (c) the antigen-specific target cells are human, optionally where the antigen-specific target cells are HLA class I negative, optionally where at least 5% of the antigen-specific effector T cells express cytotoxic activity against target cells; or at least 40% of antigen-specific effector NK cells exhibit cytotoxic activity against target cells. 7. Method according to claim 1, where: (a) CAR is encoded by DNA integrated into the PSC genome; (b) The CAR comprises an intracellular signaling domain, a transmembrane domain, and an extracellular domain containing an antigen binding region, optionally where: (i) intracellular signaling domains contain CD3Z and CD28; and/or (ii) the antigen binding region is F(ab')2, Fab', Fab, Fv or scFv; (c) PSCs are HLA homozygous, optionally where: (i) HLA-homozygous PSCs are homozygous for one or more alleles of the HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP or HLA-DQ loci; or (ii) the HLA-homozygous PSCs are homozygous for two of the alleles of the HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP or HLA-DQ loci, optionally where the HLA-homozygous PSCs are HLA homozygous -A and HLA-B; or (iii) HLA homozygous PSCs are homozygous for HLA-A, HLA-B and HLA-C. 8. A method for obtaining antigen-specific effector T cells and/or NK cells, including the steps: (1) constructing PSCs to express CARs, thereby obtaining CAR-PSCs; (2) culturing CAR-PSCs in the presence of blebbistatin, an inhibitor of GSK-3, FGF2 and VEGF, thereby generating EBs; (3) bringing EB into contact with BMP-4, VEGF and FGF2 to stimulate mesoderm induction; (4) differentiation of EBs in the presence of Flt-3 ligand, IL-3, SCF and TPO, thereby obtaining CD34 ^' NPC; -